Endocrinology Recent Issues

Does Finger Fat Produce Sex Differences in Second to Fourth Digit Ratios?

Wallen, K. — Tue, 20 Oct 2009 10:02:54 PDT

Accelerated Cartilage Resorption by Chondroclasts during Bone Fracture Healing in Osteoprotegerin-Deficient Mice

Tue, 20 Oct 2009 10:02:54 PDT

Receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG), a decoy receptor of RANKL, maintain bone mass by regulating the differentiation of osteoclasts, which are bone-resorbing cells. Endochondral bone ossification and bone fracture healing involve cartilage resorption, a less well-understood process that is needed for replacement of cartilage by bone. Here we describe the role of OPG produced by chondrocytes in chondroclastogenesis. Fracture healing in OPG^–/– mice showed faster union of the fractured bone, faster resorption of the cartilaginous callus, and an increased number of chondroclasts at the chondroosseous junctions compared with that in wild-type littermates. When a cultured pellet of OPG^–/– chondrocytes was transplanted beneath the kidney capsule, the pellet recruited many chondroclasts. The pellet showed the ability to induce tartrate-resistant acid phosphatase-positive multinucleated cells from RAW 264.7 cells in vitro. Finally, OPG^–/– chondrocytes (but not wild-type chondrocytes) cultured with spleen cells induced many tartrate-resistant acid phosphatase-positive multinucleated cells. The expression of RANKL and OPG in chondrocytes was regulated by several osteotropic factors including 1,25-dihydroxyvitamin D₃, PTHrP, IL-1, and TNF-. Thus, local OPG produced by chondrocytes probably controls cartilage resorption as a negative regulator for chondrocyte-dependent chondroclastogenesis.

Bone Formation Regulates Circulating Concentrations of Fibroblast Growth Factor 23

Samadfam, R., Richard, C., Nguyen-Yamamoto, L., Bolivar, I., Goltzman, D. — Tue, 20 Oct 2009 10:02:54 PDT

We examined the role of bone remodeling in the regulation of circulating concentrations of FGF23 using mouse models manifesting differing degrees of coupled and uncoupled bone turnover. Administration of the antiresorptive agent osteoprotegerin produced a profound reduction in bone resorption and formation in male and oophorectomized female mice, accompanied by an increase in serum levels of fibroblast growth factor 23 (FGF23) and a reduction in circulating 1,25-dihydroxyvitamin D [1,25(OH)₂D]. In contrast, exogenous PTH(1-34) administration increased bone turnover and reduced circulating FGF23. In 1,25(OH)₂D-deficient, 25-hydroxyvitamin D 1-hydroxylase null mice on a high-calcium diet, endogenous PTH was elevated, bone formation but not resorption was increased, and serum FGF23 was virtually undetectable; on a rescue diet, serum calcium was normalized, PTH levels were reduced, bone formation was reduced, and serum FGF23 levels increased. After PTH treatment of wild-type mice, gene expression of dentin matrix protein 1 (DMP1) in bone was increased, whereas gene expression of FGF23 was reduced. In vitro studies in the osteoblastic cell line UMR-106 showed that externally added DMP1 could inhibit FGF23 gene expression and production stimulated by 1,25(OH)₂D₃. The results show that osteoblastic bone formation is a potent modulator of FGF23 production and release into the circulation, suggest that the biological consequences on mineral homeostasis of circulating FGF23 may also be dependent on the prevailing rate of bone turnover, and provide evidence that DMP1 may be a direct negative regulator of FGF23 production in osteoblastic cells.

Progesterone Receptor Membrane Component-1 Regulates the Development and Cisplatin Sensitivity of Human Ovarian Tumors in Athymic Nude Mice

Peluso, J. J., Gawkowska, A., Liu, X., Shioda, T., Pru, J. K. — Tue, 20 Oct 2009 10:02:54 PDT

To determine whether progesterone receptor membrane component 1 (PGRMC1) regulates the development and cisplatin (CDDP)-sensitivity of human ovarian tumors, PGRMC1 was depleted from a human ovarian cancer cell line, dsRed-SKOV-3 cells, using a short hairpin RNA knockdown approach. Compared with parental dsRed-SKOV-3 cells, the PGRMC1-deplete cells grew slower in vitro and did not show progesterone’s (P4) antiapoptotic effect. In fact, P4 induced apoptosis in PGRMC1-deplete cells in a dose-dependent manner. When transplanted into the peritoneum of athymic nude mice, parental dsRed-SKOV-3 cells developed numerous tumors, which were classified as either typical or oxyphilic clear cell tumors. CDDP increased the percentage of apoptotic nuclei in typical clear cell tumors and P4 attenuated CDDP-induced apoptosis. In contrast, the percentage of apoptotic nuclei in oxyphilic clear cell tumors was low (≤1%) and was not significantly affected by CDDP and/or P4. Compared with tumors derived from parental dsRed SKOV-3 cells, PGRMC1-deplete tumors: 1) developed in fewer mice, 2) formed less frequently, 3) appeared smaller, and 4) resulted in fewer oxyphilic clear cell tumors. These PGRMC1-deplete tumors were not responsive to CDDP’s apoptotic effects. The failure to respond to CDDP could be due to their poorly developed microvasculature system as judged by percentage of CD31-stained endothelial cells and/or their increased expression of ATP-binding cassette transporters, which are involved in drug resistance. Taken together, these findings indicate that PGRMC1 plays an essential role in the development and CDDP sensitivity of human ovarian tumors.

{beta}-Cell-Specific Overexpression of Glutathione Peroxidase Preserves Intranuclear MafA and Reverses Diabetes in db/db Mice

Tue, 20 Oct 2009 10:02:54 PDT

Chronic hyperglycemia causes oxidative stress, which contributes to damage in various tissues and cells, including pancreatic β-cells. The expression levels of antioxidant enzymes in the islet are low compared with other tissues, rendering the β-cell more susceptible to damage caused by hyperglycemia. The aim of this study was to investigate whether increasing levels of endogenous glutathione peroxidase-1 (GPx-1), specifically in β-cells, can protect them against the adverse effects of chronic hyperglycemia and assess mechanisms that may be involved. C57BLKS/J mice overexpressing the antioxidant enzyme GPx-1 only in pancreatic β-cells were generated. The biological effectiveness of the overexpressed GPx-1 transgene was documented when β-cells of transgenic mice were protected from streptozotocin. The transgene was then introgressed into the β-cells of db/db mice. Without use of hypoglycemic agents, hyperglycemia in db/db-GPx(+) mice was initially ameliorated compared with db/db-GPx(–) animals and then substantially reversed by 20 wk of age. β-Cell volume and insulin granulation and immunostaining were greater in db/db-GPx(+) animals compared with db/db-GPx(–) animals. Importantly, the loss of intranuclear musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) that was observed in nontransgenic db/db mice was prevented by GPx-1 overexpression, making this a likely mechanism for the improved glycemic control. These studies demonstrate that enhancement of intrinsic antioxidant defenses of the β-cell protects it against deterioration during hyperglycemia.

Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice

Tue, 20 Oct 2009 10:02:54 PDT

Insulin deficiency in type 1 diabetes leads to disruptions in glucose, lipid, and ketone metabolism with resultant hyperglycemia, hyperlipidemia, and ketonemia. Exogenous insulin and hepatic insulin gene therapy cannot mimic the robust glucose-stimulated insulin secretion (GSIS) from native pancreatic islets. Gene therapy of streptozotocin-diabetic mice with neurogenin 3 (Ngn3) and betacellulin (Btc) leads to the induction of periportal oval cell-derived neo-islets that exhibit GSIS. We hence hypothesized that this gene therapy regimen may lead to a complete correction of the glucose and lipid metabolic abnormalities associated with insulin deficiency; we further hypothesized that the neo-islets formed in response to Ngn3-Btc gene delivery may display an ultrastructure and transcription profile similar to that of pancreatic islets. We injected streptozotocin-diabetic mice with helper-dependent adenoviral vectors carrying Ngn3 and Btc, which restored GSIS and reversed hyperglycemia in these animals. The treatment also normalized hepatic glucose secretion and reversed ketonemia. Furthermore, it restored hepatic glycogen content and reinstated hepatic lipogenesis-related gene transcripts back to nondiabetic levels. By transmission electron microscopy, the neo-islets displayed electron-dense granules that were similar in appearance to those in pancreatic islets. Finally, using RNA obtained by laser capture microdissection of the periportal neo-islets and normal pancreatic islets, we found that the neo-islets and pancreatic islets exhibited a very similar transcription profile on microarray-based transcriptome analysis. Taken together, this indicates that Ngn3-Btc gene therapy corrects the underlying dysregulated glucose and lipid metabolism in insulin-deficient diabetic mice by inducing neo-islets in the liver that are similar to pancreatic islets in structure and gene expression profile.

Phosphatidyl Inositol 3-Kinase Signaling in Hypothalamic Proopiomelanocortin Neurons Contributes to the Regulation of Glucose Homeostasis

Tue, 20 Oct 2009 10:02:54 PDT

Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85 alone induced resistance to diet-induced obesity. In contrast, deletion of the p110 catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

Impaired Skeletal Muscle {beta}-Adrenergic Activation and Lipolysis Are Associated with Whole-Body Insulin Resistance in Rats Bred for Low Intrinsic Exercise Capacity

Tue, 20 Oct 2009 10:02:54 PDT

Rats selectively bred for high endurance running capacity (HCR) have higher insulin sensitivity and improved metabolic health compared with those bred for low endurance capacity (LCR). We investigated several skeletal muscle characteristics, in vitro and in vivo, that could contribute to the metabolic phenotypes observed in sedentary LCR and HCR rats. After 16 generations of selective breeding, HCR had approximately 400% higher running capacity (P < 0.001), improved insulin sensitivity (P < 0.001), and lower fasting plasma glucose and triglycerides (P < 0.05) compared with LCR. Skeletal muscle ceramide and diacylglycerol content, basal AMP-activated protein kinase (AMPK) activity, and basal lipolysis were similar between LCR and HCR. However, the stimulation of lipolysis in response to 10 µm isoproterenol was 70% higher in HCR (P = 0.004). Impaired isoproterenol sensitivity in LCR was associated with lower basal triacylglycerol lipase activity, Ser660 phosphorylation of HSL, and β2-adrenergic receptor protein content in skeletal muscle. Expression of the orphan nuclear receptor Nur77, which is induced by β-adrenergic signaling and is associated with insulin sensitivity, was lower in LCR (P < 0.05). Muscle protein content of Nur77 target genes, including uncoupling protein 3, fatty acid translocase/CD36, and the AMPK 3 subunit were also lower in LCR (P < 0.05). Our investigation associates whole-body insulin resistance with impaired β-adrenergic response and reduced expression of genes that are critical regulators of glucose and lipid metabolism in skeletal muscle. We identify impaired β-adrenergic signal transduction as a potential mechanism for impaired metabolic health after artificial selection for low intrinsic exercise capacity.

Short-Term Regulation of Tumor Necrosis Factor-{alpha}-Induced Lipolysis in 3T3-L1 Adipocytes Is Mediated through the Inducible Nitric Oxide Synthase/Nitric Oxide-Dependent Pathway

Lien, C.-C., Au, L.-C., Tsai, Y.-L., Ho, L.-T., Juan, C.-C. — Tue, 20 Oct 2009 10:02:54 PDT

TNF- has several effects on adipocytes that may be related to the development of type 2 diabetes in obese subjects. Many studies demonstrated that long-term treatment with TNF- increases lipolysis in adipocytes. However, the short-term (<4 h) effects of TNF- on lipolysis have not been well investigated. The aim of this study was to investigate the short-term regulatory mechanism of TNF--induced lipolysis in 3T3-L1 adipocytes. Well-differentiated 3T3-L1 adipocytes were used. Lipolysis was determined by measuring glycerol release. Expression of inducible nitric oxide (iNOS) and nitric oxide (NO) production were measured, respectively, by Western blots and the Griess reagent. A selective iNOS inhibitor (s-ethylisothiourea · HBr), an adenylyl cyclase inhibitor (SQ22536), and a guanylyl cyclase inhibitor (LY83583) were used to investigate the involvement of iNOS, cAMP, and cGMP in TNF--induced lipolysis. Transient transfection with iNOS short hairpin RNA was performed to confirm the involvement of iNOS in TNF--induced lipolysis. Phosphorylation of hormone-sensitive lipase (HSL) was measured by immunoprecipitation and Western blotting. Results showed that short-term TNF- treatment significantly increased lipolysis, iNOS expression, and NO production in a time- and dose-dependent manner. Furthermore, treatment with the NO donor S-nitroso-N-acetylpenicillamine also stimulated lipolysis and HSL phosphorylation in 3T3-L1 adipocytes. Moreover, pretreatment with inhibitors of iNOS and guanylate cyclase, but not an adenylate cyclase inhibitor, abolished TNF--induced lipolysis and HSL phosphorylation. Suppression of TNF--induced iNOS expression using short hairpin RNA significantly reduced TNF--induced lipolysis. In conclusion, short-term TNF- treatment induces lipolysis in 3T3-L1 adipocytes by increasing iNOS expression and NO production, which activates the guanylyl cyclase/cGMP-dependent pathway and induces phosphorylation of HSL.

Changes in Hypothalamically Mediated Acute-Phase Inflammatory Responses to Lipopolysaccharide in Diet-Induced Obese Rats

Pohl, J., Woodside, B., Luheshi, G. N. — Tue, 20 Oct 2009 10:02:54 PDT

Recent evidence suggests that inflammation may be a common underlying cause of many obesity-associated conditions. To test whether obesity changes the response to inflammation, we investigated its effects on the acute phase of the inflammatory response to an endogenous pathogen, lipopolysaccharide (LPS). Diet-induced obese male Wistar rats exhibited an increased and prolonged fever response to LPS (100 µg/kg) relative to lean rats. LPS-treated obese rats also showed a greater increase in circulating TNF-, IL-6, and IL-1 receptor antagonist within the first 8 h after LPS injection. LPS induced an increase in circulating leptin only in obese rats with no effect in lean rats. Analysis of expression of pyrogenic signaling in the hypothalamus demonstrated that obese rats show a greater increase in IL-1β peaking at 2 h after LPS injection and suppressor of cytokine signaling 3 and IL-6 peaking at the 8-h time point. LPS-treated obese rats showed a significantly higher expression of IL-1 receptor antagonist in white adipose tissue (WAT) than lean rats, and WAT from obese rats incubated in LPS-supplemented medium (100 ng/ml) secreted a significantly higher level of IL-6. Overall, these results suggest that diet-induced obesity induces changes in the inflammatory response rendering the obese rats more responsive to the effects of LPS. These data also support the hypothesis that qualitative changes in WAT associated with obesity may contribute to these effects.

Central Nesfatin-1 Reduces Dark-Phase Food Intake and Gastric Emptying in Rats: Differential Role of Corticotropin-Releasing Factor2 Receptor

Tue, 20 Oct 2009 10:02:54 PDT

Nesfatin-1, derived from nucleobindin2, is expressed in the hypothalamus and reported in one study to reduce food intake (FI) in rats. To characterize the central anorexigenic action of nesfatin-1 and whether gastric emptying (GE) is altered, we injected nesfatin-1 into the lateral brain ventricle (intracerebroventricular, icv) or fourth ventricle (4v) in chronically cannulated rats or into the cisterna magna (intracisternal, ic) under short anesthesia and compared with ip injection. Nesfatin-1 (0.05 µg/rat, icv) decreased 2–3 h and 3–6 h dark-phase FI by 87 and 45%, respectively, whereas ip administration (2 µg/rat) had no effect. The corticotropin-releasing factor (CRF)₁/CRF₂ antagonist astressin-B or the CRF₂ antagonist astressin₂-B abolished icv nesfatin-1’s anorexigenic action, whereas an astressin₂-B analog, devoid of CRF-receptor binding affinity, did not. Nesfatin-1 icv induced a dose-dependent reduction of GE by 26 and 43% that was not modified by icv astressin₂-B. Nesfatin-1 into the 4v (0.05 µg/rat) or ic (0.5 µg/rat) decreased cumulative dark-phase FI by 29 and 60% at 1 h and by 41 and 37% between 3 and 5 h, respectively. This effect was neither altered by ic astressin₂-B nor associated with changes in GE. Cholecystokinin (ip) induced Fos expression in 43% of nesfatin-1 neurons in the paraventricular hypothalamic nucleus and 24% of those in the nucleus tractus solitarius. These data indicate that nesfatin-1 acts centrally to reduce dark phase FI through CRF₂-receptor-dependent pathways after forebrain injection and CRF₂-receptor-independent pathways after hindbrain injection. Activation of nesfatin-1 neurons by cholecystokinin at sites regulating food intake may suggest a role in gut peptide satiation effect.

In Vivo Characterization of High Basal Signaling from the Ghrelin Receptor

Tue, 20 Oct 2009 10:02:55 PDT

The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg¹, D-Phe⁵, D-Trp^7,9, Leu¹¹]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution. Food intake and body weight were significantly decreased in the group of rats treated with the inverse agonist, as compared with the groups treated with the control peptide or the vehicle. In the hypothalamus, the expression of neuropeptide Y and uncoupling protein 2 was decreased by the inverse agonist. In a hypothalamic cell line that endogenously expresses the ghrelin receptor, we observed high basal activity of the cAMP response element binding protein, an important signaling transduction pathway for appetite regulation. The activation was further increased by ghrelin administration and decreased by administration of the inverse agonist. It is suggested that the high constitutive signaling activity is important for the in vivo function of the ghrelin receptor in the control of food intake and body weight.

Fibroblast Growth Factor 21-Deficient Mice Demonstrate Impaired Adaptation to Ketosis

Badman, M. K., Koester, A., Flier, J. S., Kharitonenkov, A., Maratos-Flier, E. — Tue, 20 Oct 2009 10:02:55 PDT

Fibroblast growth factor 21 (FGF21) is a key metabolic regulator. Expressed primarily in liver and adipose tissue, FGF21 is induced via peroxisome proliferator-activated receptor (PPAR) pathways during states requiring increased fatty acid oxidation including fasting and consumption of a ketogenic diet. To test the hypothesis that FGF21 is a physiological regulator that plays a role in lipid oxidation, we generated mice with targeted disruption of the Fgf21 locus (FGF21 knockout). Mice lacking FGF21 had mild weight gain and slightly impaired glucose homeostasis, indicating a role in long-term energy homeostasis. Furthermore, FGF21KO mice tolerated a 24-h fast, indicating that FGF21 is not essential in the early stages of starvation. In contrast to wild-type animals in which feeding KD leads to dramatic weight loss, FGF21KO mice fed KD gained weight, developed hepatosteatosis, and showed marked impairments in ketogenesis and glucose control. This confirms the physiological importance of FGF21 in the adaptation to KD feeding. At a molecular level, these effects were accompanied by lower levels of expression of PGC1 and PGC1β in FGF21KO mice, strongly implicating these key transcriptional regulators in the action of FGF21. Furthermore, within the liver, the maturation of the lipogenic transcription factor sterol regulatory element-binding protein-1c was increased in FGF21KO mice, implicating posttranscriptional events in the maladaptation of FGF21KO mice to KD. These data reinforce the role of FGF21 is a critical regulator of long-term energy balance and metabolism. Mice lacking FGF21 cannot respond appropriately to a ketogenic diet, resulting in an impaired ability to mobilize and utilize lipids.

Sex Steroid-Dependent and -Independent Action of Hydroxysteroid (17{beta}) Dehydrogenase 2: Evidence from Transgenic Female Mice

Tue, 20 Oct 2009 10:02:55 PDT

We have recently generated transgenic (TG) mice overexpressing human hydroxysteroid (17β) dehydrogenase 2 enzyme (HSD17B2TG mice) under the ubiquitous chicken β-actin promoter. As shown in the present study, the HSD17B2TG female mice presented with slower gain of body weight as compared with the wild-type (WT) littermates and suffered from ovarian dysfunction and mammary gland hyperplasia associated with increased expression of multiple pregnancy-associated genes. The macroscopic phenotype observed in the mammary gland was likely to be dependent on the increased progesterone and prolactin secretion, and a normal histological appearance was observed in HSD17B2TG mammary gland transplanted into a WT host. However, a significant suppression of several known estrogen target genes in the HSD17B2TG mammary transplants in WT females was observed, suggesting that HSD17B2 modulates estrogen action in vivo. Interestingly, the growth retardation of HSD17B2TG females was not efficiently rescued in the bi-TG mice expressing both HSD17B2 and HSD17B1 enzymes, and the bi-TG mice presented with certain masculinized phenotypes, including lack of nipples and closed vagina, recently reported for HSD17B1TG females. The present data suggest that HSD17B2 expression affects both sex steroid-independent and steroid-dependent pathways.

Modulation of Vitamin D Receptor Activity by the Corepressor Hairless: Differential Effects of Hairless Isoforms

Malloy, P. J., Wang, J., Jensen, K., Feldman, D. — Tue, 20 Oct 2009 10:02:55 PDT

The vitamin D receptor (VDR) and its corepressor Hairless (HR) are thought to regulate key steps in the hair cycle because mutations in VDR or HR cause alopecia in humans and mice. Many mammalian cells express two major HR isoforms due to alternative splicing of exon 17. HR isoform-a encodes an 1189-amino acid protein (full-length HR), and isoform-b encodes an 1134-amino acid protein (HR1072-1126). We demonstrated that both HR isoforms are expressed in primary human keratinocytes and in the human keratinocyte cell line HaCaT. In transfected COS-7 cells, the full-length HR repressed VDR-mediated transactivation. In contrast, HR1072-1126 failed to suppress and even stimulated VDR-mediated transactivation. In coimmunoprecipitation, both HR isoforms interacted with the VDR, but only the full-length HR interacted with histone deacetylase 1 (HDAC1). Alanine mutagenesis of two conserved glutamic acids residues (E1100A/E1101A) encoded by exon 17 completely eliminated HR corepressor activity and interactions with HDAC1. When the two HR isoforms were coexpressed in COS-7 cells, the corepressor activity of the full-length HR was not antagonized by the HR1072-1126 isoform. When transfected into HaCaT cells, the full-length HR inhibited endogenous CYP24A1 basal gene expression as well as 1,25-dihydroxyvitamin D₃-stimulated CYP24A1 expression. HR1072-1126 failed to suppress basal or 1,25-dihydroxyvitamin D₃-stimulated CYP24A1 gene expression. In conclusion, we have demonstrated that both HR isoforms are expressed in keratinocytes and that the HR1072-1126 isoform lacks corepressor activity and is unable to bind HDACs. HR1072-1126 may function as a coactivator in some settings by inhibiting HDAC recruitment to the VDR transcriptional complex.

Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

Rivers, C., Flynn, A., Qian, X., Matthews, L., Lightman, S., Ray, D., Norman, M. — Tue, 20 Oct 2009 10:02:55 PDT

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GR donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GR and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression.

Activity of Human Pregnancy Insulin-Like Growth Factor Binding Protein-3: Determination by Reconstituting Recombinant Complexes

Yan, X., Payet, L. D., Baxter, R. C., Firth, S. M. — Tue, 20 Oct 2009 10:02:55 PDT

During pregnancy, IGF binding protein-3 (IGFBP-3) is completely proteolyzed to fragments with low affinities for IGFs but appears to transport IGFs normally in high-molecular-mass complexes. We previously reported that synthetic isolated amino- and carboxyl-terminal domains of IGFBP-3 cooperate to bind IGFs, and we investigated whether this is the mechanism whereby proteolyzed IGFBP-3 fragments bind IGFs normally in pregnancy serum. Two fragments of IGFBP-3 have been isolated from pregnancy serum, one with the same N-terminal sequence as intact IGFBP-3 (GASSG) and the other with an N-terminal sequence ¹⁶⁰KVDYE. Recombinant forms of these proteins, IGFBP-3^1-159 and IGFBP-3^160-264, have been synthesized and characterized, demonstrating that although the fragments individually have greatly reduced affinity for IGF complex formation, when combined they cooperate to form complexes with IGF with or without the acid-labile subunit, inhibit IGF transport across endothelial cell monolayers and inhibit IGF-I-induced IGF type I receptor phosphorylation. It is proposed that proteolysis of IGFBP-3 into two discrete complementary fragments does not significantly increase IGF bioavailability, consistent with previous findings that proteolyzed IGFBP-3 in pregnancy serum is functionally normal and circulates as part of the IGF ternary complexes.

Colony-Stimulating Factor-1 (CSF-1) Directly Inhibits Receptor Activator of Nuclear Factor-{kappa}B Ligand (RANKL) Expression by Osteoblasts

Wittrant, Y., Gorin, Y., Mohan, S., Wagner, B., Abboud-Werner, S. L. — Tue, 20 Oct 2009 10:02:55 PDT

Colony-stimulating factor-1 (CSF-1), released by osteoblasts, stimulates the proliferation of osteoclast progenitors via the c-fms receptor (CSF-1R) and, in combination with receptor activator of nuclear factor-B ligand (RANKL), leads to the formation of mature osteoclasts. Whether the CSF-1R is expressed by osteoblasts and mediates specific biological effects in osteoblasts has not been explored. Wild-type primary calvaria osteoblasts (OB) were analyzed for CSF-1R expression (RT-PCR and Western blot) and functionality (immunocomplex kinase assay). OB were serum starved for 24 h, and the effect of CSF-1 (0–100 ng/ml) on OB biological activities was determined at 48 h. In wild-type mouse bone marrow cultures, CSF-1 was tested for its effect on RANKL mRNA and osteoclast formation. Because ROS influence osteoblast RANKL expression, studies analyzed the effect of CSF-1 on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and Nox1 and Nox4 proteins. Results indicate that OB express CSF-1R mRNA and protein and that CSF-1R could be phosphorylated in the presence of CSF-1. In osteoblasts, CSF-1 decreased RANKL mRNA in a dose- and time-dependent manner. Incubation of bone marrow cultures with CSF-1 resulted in a significant decline in tartrate-resistant acid phosphatase (TRACP) activity and CTR expression. RANKL-decreased expression by CSF-1 was correlated with a decrease of NADPH oxidase activity as well as Nox1 and Nox4 protein levels. These findings provide the first evidence that osteoblasts express CSF-1R and are a target for CSF-1 ligand. CSF-1-mediated inhibition of RANKL expression on osteoblasts may provide an important mechanism for coupling bone formation/resorption and preventing excessive osteoclastogenesis during normal skeletal growth.

Phosphatidylinositol 3 Kinase/Akt Signal Relay Cooperates with Smad in Bone Morphogenetic Protein-2-Induced Colony Stimulating Factor-1 (CSF-1) Expression and Osteoclast Differentiation

Mandal, C. C., Ghosh Choudhury, G., Ghosh-Choudhury, N. — Tue, 20 Oct 2009 10:02:55 PDT

Murine spleen cells produce mature osteoclasts when cocultured with osteoblastic cells. Colony-stimulating factor (CSF)-1 is the growth factor required for differentiating the monocyte-macrophage precursor cells into preosteoclasts. Bone morphogenic protein (BMP) signaling in osteoblasts regulates bone mass in mice, suggesting a role of BMP in osteoclastogenesis along with osteoblast activity. The intracellular signal transduction cross talk regulating the osteoblastic production of CSF-1 as a mechanism of BMP-induced osteoclastogenesis is described in this report. We have recently described the involvement of Smad 1/5 in BMP-2-induced CSF-1 expression and osteoclast formation. In this study, using the pharmacological inhibitors and the adenovirus (Ad) vectors expressing dominant-negative (DN) phosphatidylinositol 3 kinase (PI3K), the PI3K-signaling inhibitor, phosphatase and tensin homolog deleted in chromosome 10 (PTEN) or DN Akt kinase in the in vitro coculture assay, we show an essential role of the lipid kinase cascade in BMP-2-mediated multinucleated osteoclast formation and CSF-1 mRNA expression, transcription, and secretion. Inhibition of PI3K/Akt signaling blocked the binding of Smads 1/5 to the CSF-1 BMP-responsive element present in the CSF-1 promoter, resulting in attenuation of Smad-dependent CSF-1 transcription. Furthermore, PI3K inhibition and DN Akt prevented association of the transcriptional coactivator, CREB (cAMP response element binding protein) binding protein (CBP), with Smads 1/5. Together, these data for the first time demonstrate that PI3K-dependent Akt activation regulates BMP-2-induced CSF-1 expression and provides a mechanism for osteoblastic cell-assisted osteoclast differentiation.

Maternal High-Fat Diet Promotes Body Length Increases and Insulin Insensitivity in Second-Generation Mice

Dunn, G. A., Bale, T. L. — Tue, 20 Oct 2009 10:02:55 PDT

Maternal obesity and diet consumption during pregnancy have been linked to offspring adiposity, cardiovascular disease, and impaired glucose metabolism. Furthermore, nutrition during development is clearly linked to somatic growth. However, few studies have examined whether phenotypes derived from maternal high-fat diet exposure can be passed to subsequent generations and by what mechanisms this may occur. Here we report the novel finding of a significant body length increase that persisted across at least two generations of offspring in response to maternal high-fat diet exposure. This phenotype is not attributable to altered intrauterine conditions or maternal feeding behavior because maternal and paternal lineages were able to transmit the effect, supporting a true epigenetic manner of inheritance. We also detected a heritable feature of reduced insulin sensitivity across two generations. Alterations in the GH secretagogue receptor (GHSR), the GHSR transcriptional repressor AF5q31, plasma IGF-I concentrations, and IGF-binding protein-3 (IGFBP3) suggest a contribution of the GH axis. These studies provide evidence that the heritability of body length and glucose homeostasis are modulated by maternal diet across multiple generations, providing a mechanism where length can increase rapidly in concert with caloric availability.

Selective Estrogen Receptor Modulators Decrease Reactive Astrogliosis in the Injured Brain: Effects of Aging and Prolonged Depletion of Ovarian Hormones

Tue, 20 Oct 2009 10:02:55 PDT

After brain injury, astrocytes acquire a reactive phenotype characterized by a series of morphological and molecular modifications, including the expression of the cytoskeletal protein vimentin. Previous studies have shown that estradiol down-regulates reactive astrogliosis. In this study we assessed whether raloxifene and tamoxifen, two selective estrogen receptor modulators, have effects similar to estradiol in astrocytes. We also assessed whether aging and the timing of estrogenic therapy after ovariectomy influence the action of the estrogenic compounds. Four groups of animals were studied: 1) young rats, ovariectomized at 2 months of age; 2) middle-aged rats, ovariectomized at 8 months of age; 3) aged rats, ovariectomized at 18 months of age; and 4) aged rats, ovariectomized at 2 months and sham operated at 18 months of age. Fifteen days after ovariectomy or sham surgery, animals received a stab wound brain injury and the treatment with the estrogenic compounds. The number of vimentin-immunoreactive astrocytes after injury was significantly higher in the hippocampus of aged rats after a long-term ovariectomy compared with aged animals after a short-term ovariectomy and middle-aged rats. In addition, reactive astrocytes were more numerous in the two groups of aged animals than in young animals. Despite these differences, the estrogenic compounds reduced reactive astrogliosis in all animal groups. These findings indicate that estradiol, raloxifene, and tamoxifen are potential candidates for the control of astrogliosis in young and older individuals and after a prolonged depletion of ovarian hormones.

The Mammalian Target of Rapamycin as Novel Central Regulator of Puberty Onset via Modulation of Hypothalamic Kiss1 System

Tue, 20 Oct 2009 10:02:55 PDT

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that operates as sensor of cellular energy status and effector for its coupling to cell growth and proliferation. At the hypothalamic arcuate nucleus, mTOR signaling has been recently proposed as transducer for leptin effects on energy homeostasis and food intake. However, whether central mTOR also participates in metabolic regulation of fertility remains unexplored. We provide herein evidence for the involvement of mTOR in the control of puberty onset and LH secretion, likely via modulation of hypothalamic expression of Kiss1. Acute activation of mTOR by l-leucine stimulated LH secretion in pubertal female rats, whereas chronic l-leucine infusion partially rescued the state of hypogonadotropism induced by food restriction. Conversely, blockade of central mTOR signaling by rapamycin caused inhibition of the gonadotropic axis at puberty, with significantly delayed vaginal opening, decreased LH and estradiol levels, and ovarian and uterine atrophy. Inactivation of mTOR also blunted the positive effects of leptin on puberty onset in food-restricted females. Yet the GnRH/LH system retained their ability to respond to ovariectomy and kisspeptin-10 after sustained blockade of mTOR, ruling out the possibility of unspecific disruption of GnRH function by rapamycin. Finally, mTOR inactivation evoked a significant decrease of Kiss1 expression at the hypothalamus, with dramatic suppression of Kiss1 mRNA levels at the arcuate nucleus. Altogether our results unveil the role of central mTOR signaling in the control of puberty onset and gonadotropin secretion, a phenomenon that involves the regulation of Kiss1 and may contribute to the functional coupling between energy balance and gonadal activation and function.

Global Disturbances in Autonomic Function Yield Cardiovascular Instability and Hypertension in the Chromogranin A Null Mouse

Gayen, J. R., Gu, Y., O'Connor, D. T., Mahata, S. K. — Tue, 20 Oct 2009 10:02:55 PDT

We reported previously that chromogranin A (Chga) knockout (KO) mice are hypertensive and hyperadrenergic. Here we sought to determine the basis of such alterations by probing physiological, biochemical, and pharmacological responses to perturbations of the autonomic nervous system. In the conscious state, KO mice had substantially elevated basal high blood pressure (BP) and heart rate (HR); immobilization stress caused increments in systolic BP and HR in both wild-type (WT) and KO mice, with higher maxima but blunted increments in the KO state. Catestatin (CST; CHGA_352–372) selectively diminished stress-induced increments in BP and HR in KO mice, implicating CST as an antihypertensive peptide, even in stressful conditions. Heightened plasma catecholamines in KO mice returned to WT level after CST. Stress caused further increments in catecholamines in WT mice but no change in KO mice. KO mice displayed diminished baroreflex sensitivity in response to either phenylephrine or sodium nitroprusside, accounting for exaggerated pressor and depressor responses to these compounds; baroreceptor function was normalized by CST. To probe the relative roles of endogenous/basal sympathetic vs. parasympathetic tone in control of BP and HR, we used the muscarinic-cholinergic antagonist atropine or the β-adrenergic antagonist propranolol; HR and BP responses to each antagonist were exaggerated in KO animals. We conclude that ablation of Chga expression results in global disturbances in autonomic function, both sympathetic and parasympathetic, that can be abrogated (or rescued), at least in part, by replacement of CST. The results point to mechanisms whereby CHGA and its CST fragment act to control cardiovascular homeostasis.

Convergence of 3',5'-Cyclic Adenosine 5'-Monophosphate/Protein Kinase A and Glycogen Synthase Kinase-3{beta}/{beta}-Catenin Signaling in Corpus Luteum Progesterone Synthesis

Tue, 20 Oct 2009 10:02:55 PDT

Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3β and β-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3β at N-terminal Ser9 causing its inactivation and resulted in the accumulation of β-catenin. Overexpression of N-terminal truncated β-catenin (90 β-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3β (GSK-S9A) reduced β-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of β-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3β/β-catenin signaling may contribute to the acute increase in progesterone production in response to LH.

Colony-Stimulating Factor 2 (CSF-2) Improves Development and Posttransfer Survival of Bovine Embryos Produced in Vitro

Tue, 20 Oct 2009 10:02:55 PDT

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30–35 of gestation. Moreover, treatment with CSF2 from either d 1–7 or 5–7 after insemination reduced pregnancy loss after d 30–35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

Glucocorticoids Amplify Dibutyl Phthalate-Induced Disruption of Testosterone Production and Male Reproductive Development

Tue, 20 Oct 2009 10:02:55 PDT

Common male reproductive abnormalities including cryptorchidism, hypospadias, and low sperm counts may comprise a testicular dysgenesis syndrome (TDS), resulting from fetal testis dysfunction during a critical developmental period involving reduced androgen production/action. The recent increase in TDS prevalence suggests environmental/lifestyle factors may be etiologically important. The developing fetus is exposed to multimodal challenges, and we hypothesized that exposure to a combination of factors rather than single agents may be important in the pathogenesis of TDS. We experimentally induced fetal testis dysfunction in rats via treatment of pregnant females daily from embryonic day (e) 13.5 to e21.5 with vehicle, 100 or 500 mg/kg · d dibutyl phthalate (DBP), 0.1 mg/kg · d dexamethasone (Dex), or a combination of DBP + Dex. In adulthood, penile length/normality, testis weight/descent, prostate weight, and plasma testosterone levels were measured plus anogenital distance (AGD) as a measure of androgen action within the masculinization programming window. Intratesticular testosterone and steroidogenic enzyme gene expression were measured in fetal testes at e17.5. High-dose DBP reduced fetal intratesticular testosterone and steroidogenic gene expression; induced mild hypospadias (31%) and cryptorchidism (53%); and reduced penile length, AGD, and testis and prostate weight in adulthood. Dex alone had no effect except to reduce birth weight but amplified the adverse effects of 500 mg/kg · d DBP and exacerbated the effects of 100 mg/kg · d DBP. All adverse effects were highly correlated to AGD, emphasizing the etiological importance of the masculinization programming window. These findings suggest that exposure to common environmental chemicals in combination with, for example, maternal stress, may increase the risk of common male reproductive abnormalities, with implications for human populations.

In Pursuit of Leucine-Rich Repeat-Containing G Protein-Coupled Receptor-5 Regulation and Function in the Uterus

Sun, X., Jackson, L., Dey, S. K., Daikoku, T. — Tue, 20 Oct 2009 10:02:55 PDT

Leucine-rich repeat-containing G protein-coupled receptor (LGR)-5 is a recently identified marker of stem cells in adult intestinal epithelium and hair follicles. Because of this characteristic, we studied the status of Lgr5 expression in the mouse uterus under various conditions. Lgr5 is highly expressed in the uterine epithelium of immature mice and is dramatically down-regulated after the mice resume estrous cycles. Surprisingly, whereas its expression is up-regulated in uteri of ovariectomized mice, the expression is down-regulated by estrogen and progesterone via their cognate nuclear receptors, estrogen receptor- and progesterone receptor, respectively. Using a mouse endometrial cancer model, we also found that Lgr5 is highly expressed in the epithelium during the initial stages of tumorigenesis but is remarkably down-regulated in fully developed tumors. Lgr5 is a downstream target of Wnt signaling in the intestine. Genetic evidence shows that either excessive or absence of Wnt signaling dampens Lgr5 expression in the uterus. Collectively, our results show that Lgr5 expression in the mouse uterine epithelium is unique and dynamically regulated under various physiological and pathological states of the uterus, suggesting that this orphan receptor has important functions in uterine biology. However, identifying definitive uterine function of LGR5 will require further investigation using conditional deletion of uterine Lgr5 because systemic deletion of this gene is neonatally lethal.

Transcriptional Profiling of the Hormone-Responsive Stages of Spermatogenesis Reveals Cell-, Stage-, and Hormone-Specific Events

Tue, 20 Oct 2009 10:02:55 PDT

Spermatogenesis occurs within the highly complex seminiferous epithelium. This cyclic process is accompanied by dynamic stage-specific transcriptional changes and is driven by androgens and FSH by mechanisms that are unclear. Here we report the impact of acute androgen and FSH suppression on the transcriptional dynamics of the seminiferous epithelium. We used transcriptional profiling to compare the most hormone-sensitive seminiferous epithelial stages (VII and VIII) from control and hormone-suppressed adult rats, together with publicly available datasets to delineate stage- and cell-specific transcriptional changes. The analyses reveal that, in these stages, there was a hormone-responsive down-regulation of spermatogonial and Sertoli cell transcripts maximally expressed in the earlier spermatogenic stages (I–VI). Transcripts expressed in Sertoli cells from stage VII and beyond were both up- and down-regulated by hormone suppression, with lysosome function, immune system-related genes, and lipid metabolism predicted to be hormone responsive. Hormone-responsive genes with putative roles in integrin-mediated cell adhesion were also identified. In pachytene spermatocytes, there was an initiation of transcription likely important for the completion of meiosis. A transcriptional switch in round spermatids was observed, from a hormone-responsive down-regulation of transcripts expressed in steps 1–7 spermatids to a hormone-independent up-regulation of transcripts expressed in steps 8–11 and likely involved in spermatid differentiation and DNA compaction. This study points to the existence of hormone-responsive global transcriptional repressors in Sertoli cells, spermatogonia, and spermatids and reveals novel and diverse cell-specific responses of the seminiferous epithelium to hormone suppression.

Foxl2, a Forkhead Transcription Factor, Modulates Nonclassical Activity of the Estrogen Receptor-{alpha}

Kim, S.-Y., Weiss, J., Tong, M., Laronda, M. M., Lee, E.-J., Jameson, J. L. — Tue, 20 Oct 2009 10:02:55 PDT

Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)- signaling. We show that Foxl2 has no effect on classical ER-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ER signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ER, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ER rather than direct action at AP1 binding sites. First, ER is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ER and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ER signaling and thus modulation by ER/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ER in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ER stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ER and Foxl2 interact and that Foxl2 selectively suppresses ER-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ER and Foxl2 to regulate ovarian development and function.

Spatiotemporal Coupling of Focal Extracellular Matrix Degradation and Reconstruction in the Menstrual Human Endometrium

Tue, 20 Oct 2009 10:02:55 PDT

Coupling of focal degradation and renewal of the functional layer of menstrual endometrium is a key event of the female reproductive biology. The precise mechanisms by which the various endometrial cell populations control extracellular matrix (ECM) degradation in the functionalis while preserving the basalis and the respective contribution of basalis and functionalis in endometrium regeneration are still unclear. We therefore compared the transcriptome of stromal and glandular cells isolated by laser capture microdissection from the basalis as well as degraded and preserved areas of the functionalis in menstrual endometria. Data were validated by in situ hybridization. Expression profile of selected genes was further analyzed throughout the menstrual cycle, and their response to ovarian steroids withdrawal was studied in a mouse xenograft model. Immunohistochemistry confirmed the results at the protein level. Algorithms for sample clustering segregated biological samples according to cell type and tissue depth, indicating distinct gene expression profiles. Pairwise comparisons identified the greatest numbers of differentially expressed genes in the lysed functionalis when compared with the basalis. Strikingly, in addition to genes products associated with tissue degradation (matrix metalloproteinase and plasmin systems) and apoptosis, superficial lysed stroma was enriched in gene products associated with ECM biosynthesis (collagens and their processing enzymes). These results support the hypothesis that fragments of the functionalis participate in endometrial regeneration during late menstruation. Moreover, menstrual reflux of lysed fragments overexpressing ECM components and adhesion molecules could easily facilitate implantation of endometriotic lesions.

Comparison between Hippocampus-Synthesized and Circulation-Derived Sex Steroids in the Hippocampus

Tue, 20 Oct 2009 10:02:55 PDT

Estradiol (E2) and other sex steroids play essential roles in the modulation of synaptic plasticity and neuroprotection in the hippocampus. To clarify the mechanisms for these events, it is important to determine the respective role of circulating vs. locally produced sex steroids in the male hippocampus. Liquid chromatography-tandem mass spectrometry in combination with novel derivatization was employed to determine the concentration of sex steroids in adult male rat hippocampus. The hippocampal levels of 17β-E2, testosterone (T), and dihydrotestosterone (DHT) were 8.4, 16.9, and 6.6 nm, respectively, and these levels were significantly higher than circulating levels. The hippocampal estrone (E1) level was, in contrast, very low around 0.015 nm. After castration to deplete circulating high level T, hippocampal levels of T and DHT decreased considerably to 18 and 3%, respectively, whereas E2 level only slightly decreased to 83%. The strong reduction in hippocampal DHT resulting from castration implies that circulating T may be a main origin of DHT. In combination with results obtained from metabolism analysis of [³H]steroids, we suggest that male hippocampal E2 synthesis pathway may be androstenedione -> T -> E2 or dehydroepiandrosterone -> androstenediol -> T -> E2 but not androstenedione -> E1 -> E2.

The RAPID Method for Blood Processing Yields New Insight in Plasma Concentrations and Molecular Forms of Circulating Gut Peptides

Tue, 20 Oct 2009 10:02:55 PDT

The correct identification of circulating molecular forms and measurement of peptide levels in blood entails that the endocrine peptide being studied is stable and recovered in good yields during blood processing. However, it is not clear whether this is achieved in studies using standard blood processing. Therefore, we compared peptide concentration and form of 12 ¹²⁵I-labeled peptides using the standard procedure (EDTA-blood on ice) and a new method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls, and Dilution (RAPID). During standard processing there was at least 80% loss for calcitonin-gene-related peptide and cholecystokinin-58 (CCK-58) and more than 35% loss for amylin, insulin, peptide YY forms (PYY_(1–36) and PYY_(3–36)), and somatostatin-28. In contrast, the RAPID method significantly improved the recovery for 11 of 12 peptides (P < 0.05) and eliminated the breakdown of endocrine peptides occurring after standard processing as reflected in radically changed molecular forms for CCK-58, gastrin-releasing peptide, somatostatin-28, and ghrelin. For endogenous ghrelin, this led to an acyl/total ghrelin ratio of 1:5 instead of 1:19 by the standard method. These results show that the RAPID method enables accurate assessment of circulating gut peptide concentrations and forms such as CCK-58, acylated ghrelin, and somatostatin-28. Therefore, the RAPID method represents an efficacious means to detect circulating variations in peptide concentrations and form relevant to the understanding of physiological function of endocrine peptides.

Fingers as a Marker of Prenatal Androgen Exposure

Berenbaum, S. A., Bryk, K. K., Nowak, N., Quigley, C. A., Moffat, S. — Tue, 20 Oct 2009 10:02:55 PDT

Interest in biological substrates of sex-related variations in psychological and physiological characteristics has led to a search for biomarkers of prenatal hormone exposure that can be measured postnatally. There has been particular interest in digit ratio, the relative lengths of the second and fourth fingers (2D:4D), but its validity as a measure of prenatal androgen has not been established. We report the strongest evaluation of the value of 2D:4D as a biomarker for early androgen exposure. Individuals with 46,XY karyotype but no effective prenatal androgen exposure due to complete androgen insensitivity syndrome had digit ratios that were feminized: they were higher than those of typical men and similar to those of typical women. Nevertheless, the effect was modest in size, and there was considerable within-group variability and between-group overlap, indicating that digit ratio is not a good marker of individual differences in prenatal androgen exposure.

Sphingosine Kinase as a Regulator of Calcium Entry through Autocrine Sphingosine 1-Phosphate Signaling in Thyroid FRTL-5 Cells

Tue, 20 Oct 2009 10:02:55 PDT

Calcium entry is one of the main regulators of intracellular signaling. Here, we have described the importance of sphingosine, sphingosine kinase 1 (SK1), and sphingosine 1-phosphate (S1P) in regulating calcium entry in thyroid FRTL-5 cells. In cells incubated with the phosphatase inhibitor calyculin A, which evokes calcium entry without mobilizing sequestered intracellular calcium, sphingosine inhibited calcium entry in a concentration-dependent manner. Furthermore, inhibiting SK1 or the ATP-binding cassette ABCC1 multidrug transporter attenuated calcium entry. The addition of exogenous S1P restored calcium entry. Neither sphingosine nor inhibition of SK1 attenuated thapsigargin-evoked calcium entry. Blocking S1P receptor 2 or phospholipase C attenuated calcium entry, whereas blocking S1P receptor 3 did not. Overexpression of wild-type SK1, but not SK2, enhanced calyculin-evoked calcium entry compared with mock-transfected cells, whereas calcium entry was decreased in cells transfected with the dominant-negative G82D SK1 mutant. Exogenous S1P restored calcium entry in G82D cells. Our results suggest that the calcium entry pathway is blocked by sphingosine and that activation of SK1 and the production of S1P, through an autocrine mechanism, facilitate calcium entry through activation of S1P receptor 2. This is a novel mechanism by which the sphingosine-S1P rheostat regulates cellular calcium homeostasis.

T Helper Type 17 Immune Response Plays an Indispensable Role for Development of Iodine-Induced Autoimmune Thyroiditis in Nonobese Diabetic-H2h4 Mice

Tue, 20 Oct 2009 10:02:56 PDT

T helper type 1(Th1)/Th2 paradigm has been expanded by discovery of a novel effector T cell (T_eff) subset, Th17 cells, which produce a proinflammatory cytokine IL-17. Th17 cells have recently been shown to play a major role in numerous autoimmune diseases that had previously been thought to be Th1-dominant diseases. We here studied the significance of Th17 cells in iodine-induced autoimmune thyroiditis in nonobese diabetic-H2^h4 mice, a mouse model of Hashimoto’s thyroiditis in humans, which spontaneously develop antithyroglobulin autoantibodies and intrathyroidal lymphocyte infiltration when supplied with iodine in the drinking water. We observed increased numbers of Th1 and Th17 cells in spleen and accumulation of both types of T_eff in the thyroid glands of iodine-fed wild-type mice, indicating that Th17 cells as well as Th1 cells constitute thyroid lesions. Furthermore, the incidence and severity of intrathyroidal lymphocyte infiltration, and the titers of antithyroglobulin autoantibodies were markedly reduced in iodine-treated IL-17^–/– mice as compared with wild-type mice. Of interest, IL-17^+/– mice showed an intermediate phenotype. Therefore, the present study, together with a previous report demonstrating the importance of Th1, not Th2, immune response for developing thyroiditis using mice deficient for interferon- or IL-4, clearly indicates that both Th1 and Th17 cells are critical T_eff subsets for the pathogenesis of spontaneous autoimmune thyroiditis in nonobese diabetic-H2^h4 mice.

Residues K128, 132, and 134 in the Thyroid Hormone Receptor-{alpha} Are Essential for Receptor Acetylation and Activity

Sanchez-Pacheco, A., Martinez-Iglesias, O., Mendez-Pertuz, M., Aranda, A. — Tue, 20 Oct 2009 10:02:56 PDT

The thyroid hormone receptor (TR)- is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TR is posttranslationally modified by acetylation in response to its own ligand (T₃). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TR decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TR mutant lost the ability to transactivate even at high T₃ concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TR interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TR suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TR.

The Blood-Brain Barrier Thyroxine Transporter Organic Anion-Transporting Polypeptide 1c1 Displays Atypical Transport Kinetics

Westholm, D. E., Salo, D. R., Viken, K. J., Rumbley, J. N., Anderson, G. W. — Tue, 20 Oct 2009 10:02:56 PDT

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T₄ transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17β-glucuronide (E₂17βG). Intriguingly, published data suggest that E₂17βG inhibition of Oatp1c1-mediated T₄ transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T₄ and E₂17βG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T₄ and E₂17βG concentrations (100 pm to 300 nm and 27 nm to 200 µm, respectively). Eadie-Hofstee plots derived from these detailed T₄ and E₂17βG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T₄ and E₂17βG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T₄ and E₂17βG. T₄ and E₂17βG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E₂17βG transport more strongly than T₄ transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.

Evidence for a Homodimeric Structure of Human Monocarboxylate Transporter 8

Tue, 20 Oct 2009 10:02:56 PDT

The human monocarboxylate transporter 8 (hMCT8) protein mediates transport of thyroid hormone across the plasma membrane. Association of hMCT8 mutations with severe psychomotor retardation and disturbed thyroid hormone levels has established its physiological relevance, but little is still known about the basic properties of hMCT8. In this study we present evidence that hMCT8 does not form heterodimers with the ancillary proteins basigin, embigin, or neuroplastin, unlike other MCTs. In contrast, it is suggested that MCT8 exists as monomer and homodimer in transiently and stably transfected cells. Apparently hMCT8 forms stable dimers because the complex is resistant to denaturing conditions and dithiothreitol. Cotransfection of wild-type hMCT8 with a mutant lacking amino acids 267–360 resulted in formation of homo-and heterodimers of the variants, indicating that transmembrane domains 4–6 are not involved in the dimerization process. Furthermore, we explored the structural and functional role of the 10 Cys residues in hMCT8. All possible Cys>Ala mutants did not behave differently from wild-type hMCT8 in protein expression, cross-linking experiments with HgCl₂ and transport function. Our findings indicate that individual Cys residues are not important for the function of hMCT8 or suggest that hMCT8 has other yet-undiscovered functions in which cysteines play an essential role.

Knockdown of the Type 3 Iodothyronine Deiodinase (D3) Interacting Protein Peroxiredoxin 3 Decreases D3-Mediated Deiodination in Intact Cells

Tue, 20 Oct 2009 10:02:56 PDT

The type 3 iodothyronine deiodinase (D3) is the primary deiodinase that inactivates thyroid hormone. Immunoprecipitation of D3, followed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry, identified peroxiredoxin 3 (Prx3) as a D3-associated protein. This interaction was confirmed using reverse coimmunoprecipitation, in which pull-down of Prx3 resulted in D3 isolation, and by fluorescence resonance energy transfer between cyan fluorescent protein-D3 and yellow fluorescent protein-Prx3. Prx3 overexpression did not change D3 activity in transfected HEK 293 cells; however, Prx3 knockdown resulted in a 50% decrease in D3-mediated whole-cell deiodination. Notably, D3 activity of cell lysates with dithiothreitol as an exogenous reducing factor and D3 protein levels were not decreased with Prx3 knockdown, indicating that the observed reduction in whole-cell deiodination was not simply due to a decrease in D3 enzyme levels. Prx3 knockdown did not change D3’s affinity for T₃ because saturation of D3-mediated whole-cell deiodination occurred between 20 and 200 nm T₃ both with and without Prx3. Furthermore, the decrease in D3 activity in whole cells was not attributable to nonspecific oxidative stress because pretreatment with the antioxidant N-acetyl cysteine did not reverse the effects of Prx3 knockdown. Thioredoxin, the cofactor needed for Prx3 regeneration, supported D3 microsomal activity; however, Prx3 knockdown did not change D3 activity in this system. In conclusion, knockdown of Prx3 decreases D3 activity in whole cells, whereas absolute levels of D3 are unchanged, consistent with Prx3 playing a rate-limiting role in the regeneration of the D3 enzyme.

Paired Box Gene 8-Peroxisome Proliferator-Activated Receptor-{gamma} Fusion Protein and Loss of Phosphatase and Tensin Homolog Synergistically Cause Thyroid Hyperplasia in Transgenic Mice

Tue, 20 Oct 2009 10:02:56 PDT

Approximately 35% of follicular thyroid carcinomas and a small fraction of follicular adenomas are associated with a t(2;3)(q13;p25) chromosomal translocation that fuses paired box gene 8 (PAX8) with the peroxisome proliferator-activated receptor- gene (PPARG), resulting in expression of a PAX8-PPAR fusion protein, PPFP. The mechanism by which PPFP contributes to follicular thyroid neoplasia is poorly understood. Therefore, we have created mice with thyroid-specific expression of PPFP. At 1 yr of age, 25% of PPFP mice demonstrate mild thyroid hyperplasia. We bred these mice to mice with thyroid-specific single-allele deletion of the tumor suppressor Pten, denoted ThyPten^+/–. In humans, PTEN deletion is associated with follicular adenomas and carcinomas, and in mice, deletion of one Pten allele causes mild thyroid hyperplasia. We found that PPFP synergizes with ThyPten^+/– to cause marked thyroid hyperplasia, but carcinomas were not observed. AKT phosphorylation was increased as expected in the ThyPten^+/– thyroids, and also was increased in the PPFP thyroids and in human PPFP follicular cancers. Staining for the cell cycle marker Ki-67 was increased in the PPFP, ThyPten^+/–, and PPFP;ThyPten^+/– thyroids compared with wild-type thyroids. Several genes with increased expression in PPFP cancers also were found to be increased in the thyroids of PPFP mice. This transgenic mouse model of thyroidal PPFP expression exhibits properties similar to those of PPFP thyroid cancers. However, the mice develop thyroid hyperplasia, not carcinoma, suggesting that additional events are required to cause follicular thyroid cancer.

Hypercortisolemia Is Associated with Severity of Bone Loss and Depression in Hypothalamic Amenorrhea and Anorexia Nervosa

Tue, 20 Oct 2009 10:02:56 PDT

Phosphodiesterase 8B Gene Polymorphism Is Associated with Subclinical Hypothyroidism in Pregnancy

Tue, 20 Oct 2009 10:02:56 PDT

The Role of Insulin-Like Growth Factor (IGF) Binding Protein-2 in the Insulin-Mediated Decrease in IGF-I Bioactivity

Tue, 20 Oct 2009 10:02:56 PDT

The Association of Endogenous Sex Hormones, Adiposity, and Insulin Resistance with Incident Diabetes in Postmenopausal Women

Tue, 20 Oct 2009 10:02:56 PDT

Erratum

Tue, 20 Oct 2009 10:02:56 PDT

Absence of Estrogen Receptor-Related-{alpha} Increases Osteoblastic Differentiation and Cancellous Bone Mineral Density

Mon, 21 Sep 2009 10:02:24 PDT

The nuclear orphan receptor human estrogen receptor-related receptor (ERR)- is implicated in bone metabolism. We studied the effect of ERR silencing in human mesenchymal stem cells (hMSCs) during osteoblastogenesis. We found that ERR silencing led to an increase of bone sialoprotein and a decrease of osteopontin mRNA levels, suggesting enhanced osteoblastic differentiation. This was confirmed by an increased ability of hMSCs to deposit calcium. Concomitantly, knockdown of ERR inhibited adipogenesis, resulting in a decrease in adipocyte number and adipocyte marker gene expression. In line with a negative role of ERR in bone metabolism, we found that adult female and male ERR-deficient mice displayed a moderate increase in femoral cancellous bone volume and density. Osteoblast surface was increased and marrow fat volume decreased in these animals. Furthermore, ERR-deficient osteoblasts displayed increased differentiation properties in vitro in line with our observations in hMSCs. In summary, we identified a role for ERR in bone mass regulation by affecting osteoblastic differentiation.

Trefoil Factor-1 (TFF1) Enhances Oncogenicity of Mammary Carcinoma Cells

Mon, 21 Sep 2009 10:02:24 PDT

The functional role of autocrine trefoil factor-1 (TFF1) in mammary carcinoma has not been previously elucidated. Herein, we demonstrate that forced expression of TFF1 in mammary carcinoma cells resulted in increased total cell number as a consequence of increased cell proliferation and survival. Forced expression of TFF1 enhanced anchorage-independent growth and promoted scattered cell morphology with increased cell migration and invasion. Moreover, forced expression of TFF1 increased tumor size in xenograft models. Conversely, RNA interference-mediated depletion of TFF1 in mammary carcinoma cells significantly reduced anchorage-independent growth and migration. Furthermore, neutralization of secreted TFF1 protein by polyclonal antibody decreased mammary carcinoma cell viability in vitro and resulted in regression of mammary carcinoma xenografts. We have therefore demonstrated that TFF1 possesses oncogenic functions in mammary carcinoma cells. Functional antagonism of TFF1 can therefore be considered as a novel therapeutic strategy for mammary carcinoma.

Restoring Endocrine Response in Breast Cancer Cells by Inhibition of the Sphingosine Kinase-1 Signaling Pathway

Sukocheva, O., Wang, L., Verrier, E., Vadas, M. A., Xia, P. — Mon, 21 Sep 2009 10:02:25 PDT

We previously demonstrated that sphingosine kinase-1 (SphK1) is an important mediator in the cytoplasmic signaling of estrogens, including Ca²⁺ mobilization, ERK1/2 activation, and the epidermal growth factor receptor transactivation. Here we report for the first time that SphK1 activity is causally associated with endocrine resistance in MCF-7 human breast cancer cells. Enforced overexpression of human SphK1 in MCF-7 cells resulted in enhanced cell proliferation and resistance to tamoxifen-induced cell growth arrest and apoptosis. Tamoxifen-resistant (TamR) MCF-7 cells selected by prolonged exposure to 4-hydroxytamoxifen, exhibited higher levels in SphK1 expression and activity, compared with the control cells. Inhibition of SphK1 activity by either specific pharmaceutical inhibitors or the dominant-negative mutant SphK1^G82D restored the antiproliferative and proapoptotic effects of tamoxifen in the TamR cells. Furthermore, silencing of SphK1, but not SphK2, expression by the specific small interference RNA also restored the tamoxifen responsiveness in the TamR cells. Thus, blockade of the SphK1 signaling pathway may reprogram cellular responsiveness to tamoxifen and abrogate antiestrogen resistance in human breast cancer cells.

An Adiponectin-Like Molecule with Antidiabetic Properties

Mon, 21 Sep 2009 10:02:25 PDT

Adiponectin increases glucose transport, reduces inflammation, and controls vascular functions. Hence, we propose that treatment with a recombinant globular domain of adiponectin (rgAd110-244) has significant therapeutic potential to treat insulin resistance. Mice were fed for 3 months on a high-fat diet (HFD) to induce insulin resistance, diabetes, and moderate weight gain. The mice were first infused iv with different doses of rgAd110-244 (0.12, 0.4, and 1.2 µg/kg · min) for 5 h. Basal and insulin-sensitive glucose use rates were assessed by the use of a submaximal rate of insulin in the awake free-moving mouse. rgAd110-244 reduced, with dose dependence, epinephrine-induced hyperglycemia and HFD-induced insulin resistance by increasing whole-body glucose use (35% at the highest dose) and glycolysis rates. Similarly, the reduction of plasma free fatty acid concentrations by insulin was dramatically improved. Basal hepatic glucose production was unchanged by rgAd110-244 infusion. This acute rgAd110-244 treatment improved glucose homeostasis and was associated with an increased content of muscle phospho-Akt, glycogen synthase kinase-3β, and AMP-activated kinase. Second, HFD mice were chronically treated with sc rgAd110-244 injections (10, 30, and 100 µg/kg). Fasting glycemia and insulin-sensitive glucose use were improved by rgAd110-244 at the highest dose at completion of the treatment, with concomitant reduction in body weight gain. We here show for the first time that a recombinant adiponectin fragment (110-244 amino acids called rgAd110-244) is able to treat insulin-resistant diabetes. Our results strongly suggest further pharmacological investigation of rgAd110-244 with the objective of developing a new treatment of insulin-resistant diabetes.

The Power of Programming: International Conference on Development Origins of Health and Disease May 6-8, 2010, Munich, Germany

Mon, 21 Sep 2009 10:02:25 PDT

Hypothalamic Leptin Signaling Regulates Hepatic Insulin Sensitivity via a Neurocircuit Involving the Vagus Nerve

Mon, 21 Sep 2009 10:02:25 PDT

Recent evidence suggests that hormones such as insulin and leptin act in the hypothalamus to regulate energy balance and glucose metabolism. Here we show that in leptin receptor-deficient Koletsky (fa^k/fa^k) rats, adenovirally induced expression of leptin receptors in the area of the hypothalamic arcuate nucleus improved peripheral insulin sensitivity via enhanced suppression of hepatic glucose production, with no change of insulin-stimulated glucose uptake or disposal. This effect was associated with increased insulin signal transduction via phosphatidylinositol-3-OH kinase (as measured by pY-insulin receptor substrate-1 and pS-PKB/Akt) in liver, but not skeletal muscle, and with reduced hepatic expression of the gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate kinase. Moreover, the beneficial effects of hypothalamic leptin signaling on hepatic insulin sensitivity were blocked by selective hepatic vagotomy. We conclude that hypothalamic leptin action increases peripheral insulin sensitivity primarily via effects on the liver and that the mechanism underlying this effect is dependent on the hepatic branch of the vagus nerve.

Remission of Type 1 Diabetes after Anti-CD3 Antibody Treatment and Transplantation of Embryonic Pancreatic Precursors

Begum, S., Chen, W., Herold, K. C., Papaioannou, V. E. — Mon, 21 Sep 2009 10:02:25 PDT

Type 1 diabetes (T1D) mellitus is characterized by progressive autoimmune destruction of insulin producing β-cells of the pancreatic islets of Langerhans. Cure of the disease will require control of autoimmunity to halt the destruction of β-cells in the pancreas and restoration of β-cell mass. We have built on the success of preclinical and clinical trials of anti-CD3 antibody treatment in modulating the immune response of T1D by the induction of tolerance and combined this treatment, using the nonobese diabetic mouse model, with a transplantation approach using fetal pancreatic anlagen as a source of β-cell precursor or progenitor cells. Here we report that transplantation of pancreatic anlagen into diabetic nonobese diabetic mice rendered tolerant to the autoimmune process by treatment with anti-CD3 antibody resulted in long-term recovery from diabetes with restored metabolic control. Using a green fluorescent protein marker that made it possible to unequivocally identify the cells derived from the transplanted tissue, we show that the transplanted anlagen cells migrate to the host pancreas and provide a major source of insulin leading to restoration of normal glucose tolerance. Our results contrast with other studies that showed restoration of endogenous islets after infusion of spleen cells in mice treated with Freund’s complete adjuvant and suggest that pancreatic fetal tissue has a tropism for the pancreatic site. This study suggests a novel mechanism of β-cell restoration by the migration of precursor cells or their progeny to the host pancreas and highlights the feasibility of using pancreatic precursors in combination with immune modulation as a treatment to effect long-term remission of T1D.

Low- and High-Density Lipoproteins Modulate Function, Apoptosis, and Proliferation of Primary Human and Murine Pancreatic {beta}-Cells

Mon, 21 Sep 2009 10:02:25 PDT

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation, and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human β-cells, LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of β-cells from wild-type and LDL receptor-deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-1β and glucose-induced apoptosis. IL-1β-induced β-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A1 (the main protein component of HDL), or sphingosine-1-phosphate (a bioactive sphingolipid mostly carried by HDL). In murine β-cells, the protective effect of HDL against IL-1β-induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type 1. Our data show that both LDL and HDL affect function or survival of β-cells and raise the question whether dyslipidemia contributes to β-cell failure and hence the manifestation and progression of type 2 diabetes mellitus.

Insulin Receptor Substrate-2 in {beta}-Cells Decreases Diabetes in Nonobese Diabetic Mice

Mon, 21 Sep 2009 10:02:25 PDT

Insulin receptor substrate-2 (Irs2) integrates insulin-like signals with glucose and cAMP agonists to regulate β-cell growth, function, and survival. This study investigated whether increased Irs2 concentration in β-cells could reduce β-cell destruction and the incidence of type 1 diabetes in nonobese diabetic (NOD) mice. NOD mice were intercrossed with C57BL/6 mice overexpressing Irs2 specifically in β-cells to create NOD^Irs2 mice. After backcrossing NOD^Irs2 mice for 12 generations, glucose homeostasis and diabetes incidence were compared against NOD littermates. Compared with 12-wk-old NOD mice, the progression of severe insulitis was reduced and islet mass was increased in NOD^Irs2 mice. Moreover, the risk of diabetes decreased 50% in NOD^Irs2 mice until the experiment was terminated at 40 wk of age. Nondiabetic NOD^Irs2 mice displayed better glucose tolerance than nondiabetic NOD mice throughout the duration of the study and up to the age of 18 months. The effect of Irs2 to increase islet mass and improve glucose tolerance raised the possibility that NOD^Irs2 mice might have an increased capacity to respond to anti-CD3 antibody, which can induce remission of overt diabetes in some NOD mice. Anti-CD3 antibody injections restored glucose tolerance in newly diabetic NOD and NOD^Irs2 mice; however, anti-CD3-treated NOD^Irs2 mice were less likely than NOD mice to relapse during the experimental period because they displayed 10-fold greater β-cell mass and mitogenesis. In conclusion, increased Irs2 attenuated the progression of β-cell destruction, promoted β-cell mitogenesis, and reduced diabetes incidence in NOD^Irs2 mice.

Complex Regulation of Mammalian Target of Rapamycin Complex 1 in the Basomedial Hypothalamus by Leptin and Nutritional Status

Mon, 21 Sep 2009 10:02:25 PDT

The medial basal hypothalamus, including the arcuate nucleus (ARC) and the ventromedial hypothalamic nucleus (VMH), integrates signals of energy status to modulate metabolism and energy balance. Leptin and feeding regulate the mammalian target of rapamycin complex 1 (mTORC1) in the hypothalamus, and hypothalamic mTORC1 contributes to the control of feeding and energy balance. To determine the mechanisms by which leptin modulates mTORC1 in specific hypothalamic neurons, we immunohistochemically assessed the mTORC1-dependent phosphorylation of ribosomal protein S6 (pS6). In addition to confirming the modulation of ARC mTORC1 activity by acute leptin treatment, this analysis revealed the robust activation of mTORC1-dependent ARC pS6 in response to fasting and leptin deficiency in leptin receptor-expressing Agouti-related protein neurons. In contrast, fasting and leptin deficiency suppress VMH mTORC1 signaling. The appropriate regulation of ARC mTORC1 by mutant leptin receptor isoforms correlated with their ability to suppress the activity of Agouti-related protein neurons, suggesting the potential stimulation of mTORC1 by the neuronal activity. Indeed, fasting- and leptin deficiency-induced pS6-immunoreactivity (IR) extensively colocalized with c-Fos-IR in ARC and VMH neurons. Furthermore, ghrelin, which activates orexigenic ARC neurons, increased ARC mTORC1 activity and induced colocalized pS6- and c-Fos-IR. Thus, neuronal activity promotes mTORC1/pS6 in response to signals of energy deficit. In contrast, insulin, which activates mTORC1 via the phosphatidylinositol 3-kinase pathway, increased ARC and VMH pS6-IR in the absence of neuronal activation. The regulation of mTORC1 in the basomedial hypothalamus thus varies by cell and stimulus type, as opposed to responding in a uniform manner to nutritional and hormonal perturbations.

The Human Lipodystrophy Gene Product Berardinelli-Seip Congenital Lipodystrophy 2/Seipin Plays a Key Role in Adipocyte Differentiation

Chen, W., Yechoor, V. K., Chang, B. H.-J., Li, M. V., March, K. L., Chan, L. — Mon, 21 Sep 2009 10:02:25 PDT

Mutations in the Berardinelli-Seip congenital lipodystrophy 2 gene (BSCL2) are the underlying defect in patients with congenital generalized lipodystrophy type 2. BSCL2 encodes a protein called seipin, whose function is largely unknown. In this study, we investigated the role of Bscl2 in the regulation of adipocyte differentiation. Bscl2 mRNA is highly up-regulated during standard hormone-induced adipogenesis in 3T3-L1 cells in vitro. However, this up-regulation does not occur during mesenchymal stem cell (C3H10T1/2 cells) commitment to the preadipocyte lineage. Knockdown of Bscl2 by short hairpin RNA in C3H10T1/2 cells has no effect on bone morphogenetic protein-4-induced preadipocyte commitment. However, knockdown in 3T3-L1 cells prevents adipogenesis induced by a standard hormone cocktail, but adipogenesis can be rescued by the addition of peroxisome proliferator-activated receptor- agonist pioglitazone at an early stage of differentiation. Interestingly, pioglitazone-induced differentiation in the absence of standard hormone is not associated with up-regulated Bscl2 expression. On the other hand, short hairpin RNA-knockdown of Bscl2 largely blocks pioglitazone-induced adipose differentiation. These experiments suggest that Bscl2 may be essential for normal adipogenesis; it works upstream or at the level of peroxisome proliferator-activated receptor-, enabling the latter to exert its full activity during adipogenesis. Loss of Bscl2 function thus interferes with the normal transcriptional cascade of adipogenesis during fat cell differentiation, resulting in near total loss of fat or lipodystrophy.

Central Ghrelin Regulates Peripheral Lipid Metabolism in a Growth Hormone-Independent Fashion

Mon, 21 Sep 2009 10:02:25 PDT

GH plays a major role in the regulation of lipid metabolism and alterations in GH axis elicit major changes in fat distribution and mobilization. For example, in patients with GH deficiency (GHD) or in mice lacking the GH receptor, the percentage of fat is increased. In addition to the direct actions of GH on lipid metabolism, current evidence indicates that ghrelin, a stomach-derived peptide hormone with potent GH secretagogue action, increases lipogenesis in white adipose tissue (WAT) through a hypothalamic-mediated mechanism. Still, the mechanism by which GH tone modulates ghrelin actions on WAT remains unclear. Here we investigated the effect of central ghrelin administration on lipid metabolism in lipogenic tissues (liver and WAT) in the absence of GH, by using a model for the study of GHD, namely the spontaneous dwarf rat, which shows increased body fat. Our data demonstrate that central chronic ghrelin administration regulates adipose lipid metabolism, mainly in a GH-independent fashion, as a result of increased mRNA, protein expression, and activity levels of fatty acid metabolism enzymes. On the contrary, central ghrelin regulates hepatic lipogenesis de novo in a GH-independent fashion but lipid mobilization in a GH-dependent fashion because carnitine palmitoyltransferase 1 was decreased only in wild-type Lewis rats. These findings suggest the existence of a new central nervous system-based neuroendocrine circuit, regulating metabolic homeostasis of adipose tissue. Understanding the molecular mechanism underlying the interplay between GH and ghrelin and their effects on lipid metabolism will provide new strategies for the design and development of suitable drugs for the treatment of GHD, obesity, and its comorbidities.

Vascular Insulin-Like Growth Factor-I Resistance and Diet-Induced Obesity

Mon, 21 Sep 2009 10:02:25 PDT

Obesity and type 2 diabetes mellitus are characterized by insulin resistance, reduced bioavailability of the antiatherosclerotic signaling molecule nitric oxide (NO), and accelerated atherosclerosis. IGF-I, the principal growth-stimulating peptide, which shares many of the effects of insulin, may, like insulin, also be involved in metabolic and vascular homeostasis. We examined the effects of IGF-I on NO bioavailability and the effect of obesity/type 2 diabetes mellitus on IGF-I actions at a whole-body level and in the vasculature. In aortic rings IGF-I blunted phenylephrine-mediated vasoconstriction and relaxed rings preconstricted with phenylephrine, an effect blocked by N^G-monomethyl l-arginine. IGF-I increased NO synthase activity to an extent similar to that seen with insulin and in-vivo IGF-I led to serine phosphorylation of endothelial NO synthase (eNOS). Mice rendered obese using a high-fat diet were less sensitive to the glucose-lowering effects of insulin and IGF-I. IGF-I increased aortic phospho-eNOS levels in lean mice, an effect that was blunted in obese mice. eNOS activity in aortae of lean mice increased 1.6-fold in response to IGF-I compared with obese mice. IGF-I-mediated vasorelaxation was blunted in obese mice. These data demonstrate that IGF-I increases eNOS phosphorylation in-vivo, increases eNOS activity, and leads to NO-dependent relaxation of conduit vessels. Obesity is associated with resistance to IGF-I at a whole-body level and in the endothelium. Vascular IGF-I resistance may represent a novel therapeutic target to prevent or slow the accelerated vasculopathy seen in humans with obesity or type 2 diabetes mellitus.

Gonadotropin-Releasing Hormone-Mediated Phosphorylation of Estrogen Receptor-{alpha} Contributes to fosB Expression in Mouse Gonadotrophs

Chen, J., An, B.-S., Cheng, L., Hammond, G. L., Leung, P. C. K. — Mon, 21 Sep 2009 10:02:25 PDT

Estrogen receptors (ERs) are activated by their ligands as well as signaling pathways that alter ER phosphorylation in response to peptide hormones and growth factors. In pituitary gonadotrophs, GnRHs act via the type I GnRH receptor (GnRHR). Both GnRH subtypes (GnRH-I and -II) activate an estrogen response element (ERE)-driven luciferase reporter gene in LβT2 mouse pituitary cells, and GnRH-I is most potent in this regard. Moreover, antide (a GnRH antagonist) and a GnRHR small interfering RNA (siRNA) abrogate this effect, whereas an ER antagonist (ICI 182,780) does not. The ER in LβT2 cells is phosphorylated at Ser¹¹⁸ in the nucleus and at Ser¹⁶⁷ in both nucleus and cytoplasm after GnRH treatments and coincided with increased ER binding to its coactivator, the p300/cAMP response element-binding protein-associated factor (PCAF). Moreover, siRNA-mediated knockdown of PCAF levels attenuated GnRH-induced ERE-luciferase transactivation in these cells. Most importantly, both GnRH subtypes robustly up-regulated expression of the immediate early response gene, fosB, whereas cotreatment with ER siRNA or PCAF siRNA attenuated this effect. This appears to occur at the transcriptional level because corecruitment of ER and PCAF to an ERE within the endogenous fosB promoter was increased by GnRH treatments, as shown by chromatin immunoprecipitation assays. These data demonstrate that GnRH-mediated phosphorylation of ER in mouse LβT2 pituitary cells results in its rapid association with PCAF and the transcriptional activation of fosB, and we demonstrate that this in turn likely activates other genes in pituitary cells including the FSH β-subunit gene.

17{beta}-Estradiol at Low Concentrations Acts through Distinct Pathways in Normal Versus Benign Prostatic Hyperplasia-Derived Prostate Stromal Cells

Park, I. I., Zhang, Q., Liu, V., Kozlowski, J. M., Zhang, J., Lee, C. — Mon, 21 Sep 2009 10:02:25 PDT

The aim of this study was to identify differential responses to low concentrations of 17β-estradiol (E2) in primary stromal cell cultures derived from either normal organ donors or benign prostatic hyperplasia or hypertrophy (BPH) specimens. Furthermore, we sought to identify the potential mechanism of E2 action in these cell types, through either a genomic or nongenomic mechanism. We initially treated stromal cells derived from five normal prostates or five BPH specimens with low concentrations of E2 (0.001–1.0 nm) and analyzed their growth response. To determine whether genomic or nongenomic pathways were involved, we performed studies using specific estrogen receptor antagonists to confirm transcriptional activity or MAPK inhibitors to confirm the involvement of rapid signaling. Results of these studies revealed a fundamental difference in the mechanism of the response to E2. In normal cells, we found that a nongenomic, rapid E2 signaling pathway is predominantly involved, mediated by G protein-coupled receptor-30 and the subsequent activation of ERK1/2. In BPH-derived prostate stromal cells, a genomic pathway is predominantly involved because the addition of ICI 182780 was sufficient to abrogate any estrogenic effects. In conclusion, prostate stromal cells respond to far lower concentrations of E2 than previously recognized or examined, and this response is mediated through two distinct mechanisms, depending on its origin. This may provide the basis for new insights into the causes of, and possible treatments for, BPH.

Activation of Phosphatidylinositol 3-Kinase/Protein Kinase B by Corticotropin-Releasing Factor in Human Monocytes

Chandras, C., Koutmani, Y., Kokkotou, E., Pothoulakis, C., Karalis, K. P. — Mon, 21 Sep 2009 10:02:25 PDT

Corticotropin-releasing factor (CRF) exerts proinflammatory effects in peripheral tissues, whereas the intracellular pathways mediating these effects have not been completely characterized yet. We have previously shown that CRF induces nuclear factor-B DNA-binding activity in mouse and human leukocytes. Here we demonstrate that in the human monocytic THP-1 cells, CRF activates the phosphatidylinositol 3-kinase (PI3K)/Akt and ERK1/2 pathways. These effects of CRF are mediated by corticotropin-releasing factor receptor 2 (CRF2), as suggested by their abolishment after treatment with the specific CRF2 antagonist, astressin 2B. The CRF-mediated PI3K/Akt activation induces cell survival as suggested by the stimulation of the antiapoptotic factor Bcl-2. ERK1/2 activation results in up-regulation of IL-8 expression, an effect inhibited by the CRF-induced activation of PI3K/Akt. These studies demonstrate novel effects of CRF in human monocytes mediated by the activation of PI3K/Akt. Moreover, they reveal pathway-specific effects of the CRF/CRF2 system in chemokine activation and cell survival that may be of importance for the development of novel therapeutics for inflammatory diseases.

Dissociation between Rat Hippocampal CA1 and Dentate Gyrus Cells in Their Response to Corticosterone: Effects on Calcium Channel Protein and Current

Mon, 21 Sep 2009 10:02:25 PDT

Stress and corticosterone affect, via glucocorticoid receptors, cellular physiology in the rodent brain. A well-documented example concerns corticosteroid effects on high-voltage activated (L type) calcium currents in the hippocampal CA1 area. We tested whether corticosterone also affects calcium currents in another hippocampal area that highly expresses glucocorticoid receptors, i.e. the dentate gyrus (DG). Remarkably, corticosterone (100 nm, given for 20 min, 1–4.5 hr before recording) did not change high-voltage activated calcium currents in the DG, whereas currents in the CA1 area of the same rats were increased. Follow-up studies revealed that no apparent dissociation between the two areas was observed with respect to transcriptional regulation of calcium channel subunits; thus, in both areas corticosterone increased mRNA levels of the calcium channel-β4 but not the () Ca_v1.2 subunit. At the protein level, however, β4 and Ca_v1.2 levels were significantly up-regulated by corticosterone in the CA1 but not the DG area. These data suggest that stress-induced elevations in the level of corticosterone result in a regionally differentiated physiological response that is not simply determined by the glucocorticoid receptor distribution and that the observed regional differentiation may be caused by a gene involved in the translational machinery or in mechanisms regulating mRNA or protein stability.

Fibroblast Growth Factor 21 Regulates Lipolysis in White Adipose Tissue But Is Not Required for Ketogenesis and Triglyceride Clearance in Liver

Mon, 21 Sep 2009 10:02:25 PDT

Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse functions. Fgf21, a unique member of the Fgf family, is expected to function as a metabolic regulator in an endocrine manner. Hepatic Fgf21 expression was increased by fasting. The phenotypes of hepatic Fgf21 transgenic or knockdown mice and high-fat, low-carbohydrate ketogenic diet-fed mice suggests that Fgf21 stimulates lipolysis in the white adipose tissue during normal feeding and is required for ketogenesis and triglyceride clearance in the liver during fasting. However, the physiological roles of Fgf21 remain unclear. To elucidate the physiological roles of Fgf21, we generated Fgf21 knockout (KO) mice by targeted disruption. Fgf21 KO mice were viable, fertile, and seemingly normal. Food intake, oxygen consumption, and energy expenditure were also essentially unchanged in Fgf21 KO mice. However, hypertrophy of adipocytes, decreased lipolysis in adipocytes, and decreased blood nonesterified fatty acid levels were observed when Fgf21 KO mice were fed normally. In contrast, increased lipolysis in adipocytes and increased blood nonesterified fatty acid levels were observed in Fgf21 KO mice by fasting for 24 h, indicating that Fgf21 stimulates lipolysis in the white adipose tissue during feeding but inhibits it during fasting. In contrast, unexpectedly, hepatic triglyceride levels were essentially unchanged in Fgf21 KO mice. In addition, ketogenesis in Fgf21 KO mice was not impaired by fasting for 24 h. The present results indicate that Fgf21 regulates lipolysis in adipocytes in response to the metabolic state but is not required for ketogenesis and triglyceride clearance in the liver.

Effects of Maternal Global Nutrient Restriction on Fetal Baboon Hepatic Insulin-Like Growth Factor System Genes and Gene Products

Mon, 21 Sep 2009 10:02:25 PDT

Knowledge of altered maternal nutrition effects on growth-regulating systems is critical to understanding normal and abnormal fetal development. There are many reports of hepatic fetal IGF system responses to maternal nutrient restriction (MNR) during pregnancy in rodents and sheep but none in nonhuman primates. We determined effects of MNR on the fetal baboon hepatic IGF system. Social groups of female baboons were fed ad libitum, controls, or 70% controls (MNR) from 0.16 to 0.5 gestation and fetuses delivered by cesarean section. Fetal liver tissue was analyzed for IGF-I, IGF-II, and IGF binding protein (IGFBP)-3 mRNA by in situ hybridization and quantitative RT-PCR and protein by immunohistochemistry (IHC); IGF-I receptor, IGF-II receptor by quantitative RT-PCR and IHC and IGFBP-1 by in situ hybridization and IHC. MNR did not alter fetal body or liver weight. Fetal hepatic glycogen staining increased with MNR. MNR reduced fetal hepatic IGF-I and IGF-II and increased IGFBP-1 mRNA and decreased IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor protein and increased protein for IGFBP-1 and IGFBP-3. MNR increased caspase-3, indicating apoptosis and decreased Akt staining, indicating decreased nutrient sensing. In conclusion, whereas fetal body and liver weights did not change in response to moderate MNR during the first half of baboon pregnancy, the major indices of function of the hepatic IGF system measured were all reduced.

Resistin Regulates Pituitary Somatotrope Cell Function through the Activation of Multiple Signaling Pathways

Mon, 21 Sep 2009 10:02:25 PDT

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues and organs, including the pituitary. However, there is no information on whether resistin, as described previously for other adipokines such as leptin and adiponectin, could regulate this gland. Likewise, the molecular basis of resistin actions remains largely unexplored. Here we show that administration of resistin to dispersed rat anterior pituitary cells increased GH release in both the short (4 h) and long (24 h) term, decreased mRNA levels of the receptor of the somatotrope regulator ghrelin, and increased free cytosolic Ca²⁺ concentration in single somatotropes. By means of a pharmacological approach, we found that the stimulatory action of resistin occurs through a Gs protein-dependent mechanism and that the adenylate cyclase/cAMP/protein kinase A pathway, the phosphatidylinositol 3-kinase/Akt pathway, protein kinase C, and extracellular Ca²⁺ entry through L-type voltage-sensitive Ca²⁺ channels are essential players in mediating the effects of resistin on somatotropes. Taken together, our results demonstrate for the first time a regulatory role for resistin on somatotrope function and provide novel insights on the intracellular mechanisms activated by this protein.

Impact of Melatonin and Molecular Clockwork Components on the Expression of Thyrotropin {beta}-Chain (Tshb) and the Tsh Receptor in the Mouse Pars Tuberalis

Unfried, C., Ansari, N., Yasuo, S., Korf, H.-W., von Gall, C. — Mon, 21 Sep 2009 10:02:25 PDT

Photoperiodic regulation of reproduction in birds and mammals involves thyrotropin β-chain (TSHb), which is secreted from the pars tuberalis (PT) and controls the expression of deiodinase type 2 and 3 in the ependymal cell layer of the infundibular recess (EC) via TSH receptors (TSHRs). To analyze the impact of melatonin and the molecular clockwork on the expression of Tshb and Tshr, we investigated melatonin-proficient C3H wild-type (WT), melatonin receptor 1-deficient (MT1-/-) or clockprotein PERIOD1-deficient (mPER1-/-) mice. Expression of Tshb and TSHb immunoreactivity in PT were low during day and high during the night in WT, high during the day and low during the night in mPER1-deficient, and equally high during the day and night in MT1-deficient mice. Melatonin injections into WT acutely suppressed Tshb expression. Transcription assays showed that the 5' upstream region of the Tshb gene could be controlled by clockproteins. Tshr levels in PT were low during the day and high during the night in WT and mPER1-deficient mice and equally low in MT1-deficient mice. Tshr expression in the EC did not show a day/night variation. Melatonin injections into WT acutely induced Tshr expression in PT but not in EC. TSH stimulation of hypothalamic slice cultures of WT induced phosphorylated cAMP response element-binding protein in PT and EC and deiodinase type 2 in the EC. Our data suggest that Tshb expression in PT is controlled by melatonin and the molecular clockwork and that melatonin activates Tshr expression in PT but not in EC. They also confirm the functional importance of TSHR in the PT and EC.

Estradiol Negative Feedback Regulation by Glutamatergic Afferents to A15 Dopaminergic Neurons: Variation with Season

Mon, 21 Sep 2009 10:02:25 PDT

It is now clear that seasonal breeding in ewes is due to an increase in response to estradiol (E₂) negative feedback in the nonbreeding season (anestrus) that is mediated by the A15 group of dopaminergic (DA) neurons. Because A15 cells do not contain estrogen receptors, we have postulated the presence of estrogen-responsive afferents and recently reported evidence that input from neurons containing -aminobutyric acid (GABA) contribute to the control of A15 activity by E₂. However, GABAergic afferents account for only a fraction of A15 synaptic input and do not appear to vary with season. We therefore investigated the possible role of stimulatory glutamatergic input to A15 neurons. In experiments 1 and 2, local administration into the A15 of either a N-methyl-d-aspartate (NMDA) receptor or a kainate/-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor antagonist stimulated episodic LH secretion in a dose-dependent manner in ovary-intact anestrous ewes. In experiment 3, we examined the number of glutamatergic close contacts onto A15 neurons using dual immunocytochemistry in tissue from E₂-treated ovariectomized anestrous and breeding season ewes. All A15 DA neurons were contacted by glutamatergic vesicles, and the number of close contacts was significantly higher in anestrus than the breeding season. Finally, using a triple-label immunocytochemistry procedure, we did not observe any colocalization of markers for GABA and glutamate in vesicles contacting A15 neurons. These results support the hypothesis that glutamatergic afferents actively stimulate A15 DA neurons in ovary-intact anestrous ewes and raise the possibility that alterations in this input may contribute to increased A15 neural activity during anestrus.

Mammalian Target of Rapamycin Is Activated in Association with Myometrial Proliferation during Pregnancy

Jaffer, S., Shynlova, O., Lye, S. — Mon, 21 Sep 2009 10:02:25 PDT

The adaptive growth of the uterus during gestation involves gradual changes in cellular phenotypes from the early proliferative to the intermediate synthetic phase of cellular hypertrophy, ending in the final contractile/labour phenotype. The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth and proliferation in many tissues. We hypothesized that mTOR was a mediator of hormone-initiated myometrial hyperplasia during gestation. The protein expression and phosphorylation levels of mTOR, its upstream regulators [insulin receptor substrate-1, phosphoinositide-3-kinase (PI3K), Akt], and downstream effectors [S6-kinase-1 (S6K1) and eI4FE-binding protein 1 (4EBP1)] were analyzed throughout normal pregnancy in rats. In addition, we used an ovariectomized (OVX) rat model to analyze the modulation of the mTOR pathway and proliferative activity of the uterine myocytes by estradiol alone and in combination with the mTOR-specific inhibitor rapamycin. Our results demonstrate that insulin receptor substrate-1 protein levels and the phosphorylated (activated) forms of PI3K, mTOR, and S6K1 were significantly up-regulated in the rat myometrium during the proliferative phase of pregnancy. Treatment of the OVX rats with estradiol caused a transient increase in IGF-I followed by an up-regulation of the PI3K/mTOR pathway, which became apparent by a cascade of phosphorylation reactions (P-P85, P-Akt, P-mTOR, P-S6K1, and P-4EBP1). Rapamycin blocked activation of P-mTOR, P-S6K1, and P-4EBP1 proteins and significantly reduced the number of proliferating cells in the myometrium of OVX rats. Our in vivo data demonstrate that estradiol was able to activate the PI3K/mTOR signaling pathway in uterine myocytes and suggest that this activation is responsible for the induction of myometrial hyperplasia during early gestation.

Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Zama, A. M., Uzumcu, M. — Mon, 21 Sep 2009 10:02:25 PDT

Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-β, which was down-regulated, whereas ER was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 µg/kg·d or 100 mg/kg·d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ER and ERβ genes in postnatal d 50–60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERβ promoter regions (P < 0.05), whereas the ER promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

Characterization of the Endocannabinoid System in Human Spermatozoa and Involvement of Transient Receptor Potential Vanilloid 1 Receptor in Their Fertilizing Ability

Mon, 21 Sep 2009 10:02:25 PDT

Human spermatozoa express type-1 cannabinoid receptor (CB1), whose activation by anandamide (AEA) affects motility and acrosome reaction (AR). In this study, we extended the characterization of the AEA-related endocannabinoid system in human spermatozoa, and we focused on the involvement of the AEA-binding vanilloid receptor (TRPV1) in their fertilizing ability. Protein expression was revealed for CB1 (~56 kDa), TRPV1 (~95 kDa), AEA-synthesizing phospholipase D (NAPE-PLD) (~46 kDa), and AEA-hydrolyzing enzyme [fatty acid amide hydrolase (FAAH), ~66 kDa]. Both AEA-binding receptors (CB1 and TRPV1) exhibited a functional binding activity; enzymatic activity was demonstrated for NAPE-PLD, FAAH, and the purported endocannabinoid membrane transporter (EMT). Immunoreactivity for CB1, NAPE-PLD, and FAAH was localized in the postacrosomal region and in the midpiece, whereas for TRPV1, it was restricted to the postacrosomal region. Capsazepine (CPZ), a selective antagonist of TRPV1, inhibited progesterone (P)-enhanced sperm/oocyte fusion, as evaluated by the hamster egg penetration test. This inhibition was due to a reduction of the P-induced AR rate above the spontaneous AR rate, which was instead increased. The sperm exposure to OMDM-1, a specific inhibitor of EMT, prevented the promoting effect of CPZ on spontaneous AR rate and restored the sperm responsiveness to P. No significant effects could be observed on sperm motility. In conclusion, this study provides unprecedented evidence that human spermatozoa exhibit a completely functional endocannabinoid system related to AEA and that the AEA-binding TRPV1 receptor could be involved in the sperm fertilizing ability.

Increased Mitogen-Activated Protein Kinase Kinase/Extracellularly Regulated Kinase Activity in Human Endometrial Stromal Fibroblasts of Women with Endometriosis Reduces 3',5'-Cyclic Adenosine 5'-Monophosphate Inhibition of Cyclin D1

Velarde, M. C., Aghajanova, L., Nezhat, C. R., Giudice, L. C. — Mon, 21 Sep 2009 10:02:25 PDT

Endometriosis is characterized by endometrial tissue growth outside the uterus, due primarily to survival, proliferation, and neoangiogenesis of eutopic endometrial cells and fragments refluxed into the peritoneal cavity during menses. Although various signaling molecules, including cAMP, regulate endometrial proliferation, survival, and embryonic receptivity in endometrium of women without endometriosis, the exact molecular signaling pathways in endometrium of women with disease remain unclear. Given the persistence of a proliferative profile and differential expression of genes associated with the MAPK signaling cascade in early secretory endometrium of women with endometriosis, we hypothesized that ERK1/2 activity influences cAMP regulation of the cell cycle. Here, we demonstrate that 8-Br-cAMP inhibits bromodeoxyuridine incorporation and cyclin D1 (CCND1) expression in cultured human endometrial stromal fibroblasts (hESF) from women without but not with endometriosis. Incubation with serum-containing or serum-free medium resulted in higher phospho-ERK1/2 levels in hESF of women with vs. without disease, independent of 8-Br-cAMP treatment. The MAPK kinase-1/2 inhibitor, U0126, fully restored cAMP down-regulation of CCND1, but not cAMP up-regulation of IGFBP1, in hESF of women with vs. without endometriosis. Immunohistochemistry demonstrated the highest phospho-ERK1/2 in the late-secretory epithelial and stromal cells in women without disease, in contrast to intense immunostaining in early-secretory epithelial and stromal cells in those with disease. These findings suggest that increased activation of ERK1/2 in endometrial cells from women with endometriosis may be responsible for persistent proliferative changes in secretory-phase endometrium.

14-3-3 Protein Regulates Cell Adhesion in the Seminiferous Epithelium of Rat Testes

Wong, E. W. P., Sun, S., Li, M. W. M., Lee, W. M., Cheng, C. Y. — Mon, 21 Sep 2009 10:02:25 PDT

Polarity proteins have been implicated in regulating and maintaining tight junction (TJ) and cell polarity in epithelia. Here we report 14-3-3, the homolog of Caenorhabditis elegans Par5 in mammalian cells, which is known to confer cell polarity at TJ, is found at the apical ectoplasmic specialization (ES), a testis-specific adherens junction type restricted to the Sertoli cell-elongating spermatid interface, in which TJ is absent. 14-3-3 was shown to play a critical role in conferring cell adhesion at the apical ES. A loss of 14-3-3 expression at the apical ES was detected in the seminiferous epithelium before spermiation. Involvement of 14-3-3 in Sertoli cell adhesion was confirmed by its knockdown by RNA interference in Sertoli cells cultured in vitro with established TJ permeability barrier that mimicked the blood-testis barrier (BTB) in vivo. Mislocalization of N-cadherin and zonula occludens-1, but not - and β-catenins, was observed after 14-3-3 knockdown in Sertoli cells, moving from the cell-cell interface to cytosol, indicating a disruption of cell adhesion. Studies by endocytosis assay illustrated that this loss of cell adhesion was mediated by an increase in the kinetics of endocytosis of N-cadherin and junctional adhesion molecule-A at the BTB, which may represent a general mechanism by which polarity proteins regulate cell adhesion. In summary, the testis is using 14-3-3 to regulate cell adhesion at the apical ES to facilitate spermiation and at the BTB to facilitate the transit of preleptotene spermatocytes at stages VIII–IX of the epithelial cycle. 14-3-3 may act as a molecular switch that coordinates these two cellular events in the seminiferous epithelium during spermatogenesis.

An in Vivo Study on Adjudin and Blood-Testis Barrier Dynamics

Kopera, I. A., Su, L., Bilinska, B., Cheng, C. Y., Mruk, D. D. — Mon, 21 Sep 2009 10:02:25 PDT

Adjudin is known to specifically affect Sertoli-germ cell adhesion, resulting in germ cell loss from the seminiferous epithelium and transient infertility. The apical ectoplasmic specialization (ES) was shown to be the primary target of adjudin because adhesion was unaffected in organs that lack this structure. Herein we expand previous findings by treating rat pups with adjudin, and we aimed to address two questions. First, can adjudin perturb germ cell adhesion in the seminiferous epithelium of testes in which the apical ES is not yet present? Second, can adjudin affect assembly of the blood-testis barrier (BTB) at 15–18 d of age? Interesting changes were noted when aged-matched testes from control and adjudin-treated rats were examined, including a delay in the appearance of developing germ cells as well as a delay in the formation of the tubule lumen. Immunoblotting using antibodies against BTB-constituent proteins indicated that formation of the BTB was affected in rat pups gavaged with adjudin. These results were corroborated by immunofluorescence microscopy, which showed profound changes in the cellular distribution of tight junction and basal ES proteins. Moreover, the BTB was shown to be compromised in 30-d-old rats when its integrity was assessed by a functional in vivo assay. By 45 d of age, however, the seminiferous epithelium of treated rats was indistinguishable from that of control rats. Collectively these results demonstrate that adjudin targets the apical ES as well as the basal ES and tight junction, which in turn delays assembly of the BTB.

Hsa-miR-222 Is Involved in Differentiation of Endometrial Stromal Cells in Vitro

Mon, 21 Sep 2009 10:02:25 PDT

Decidualization is a critical step during embryo implantation and characterized by the differentiation of endometrial stromal cells (ESCs) into decidual cells. Because miRNAs are important determinants of cellular fate specification, in this study, the miRNA expression in ESCs during in vitro decidualization was profiled by using a microarray. Significance analysis of microarrays revealed that 49 miRNA genes were differently (>2-fold) expressed between the noninduced ESCs and induced ESCs with a false discovery rate of 0. The expression variance of hsa-miR-222, 221, 143, 101, 30d, 30c, 181b, 27b, 29b, 507, and 23a was validated by using quantitative PCR (P < 0.05). Based on microRNA (miRNA) and mRNA expression variance and predicted target genes of miRNAs, a bioinformatic model of miRNAs controlling ESCs differentiation was formulated. Finally, we proved that down-regulation of has-miR-222 could decrease the number of cells in S phase during ESCs differentiation (P < 0.05). Antisense oligonucleotides of has-miR-222 could increase reporter gene expression by targeting the 3' untranslated regions of CDKN1C/p57kip2 mRNAs as well as increase CDKN1C/p57kip2 protein levels (P < 0.05). In conclusion, our results suggest that a subset of miRNAs play a key role in gene reprogramming during ESCs decidualization and that hsa-miR-222 participates in ESC differentiation by regulating ESCs terminally withdrawing from the cell cycle.

The Protein-Tyrosine Phosphatase, Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2, Is a Crucial Mediator of Exogenous Insulin-Like Growth Factor Signaling to Human Trophoblast

Forbes, K., West, G., Garside, R., Aplin, J. D., Westwood, M. — Mon, 21 Sep 2009 10:02:26 PDT

Adequate fetal growth depends on placental transfer of nutrients and gases from the mother; thus, as pregnancy progresses, the placenta must grow to meet the increasing demands of the developing fetus. IGFs control proliferation, differentiation, and survival of trophoblast in first-trimester placenta via intracellular tyrosine kinase signaling cascades, the activation of which is also regulated by tyrosine phosphatases. The protein-tyrosine phosphatase, Src homology-2 domain containing protein tyrosine phosphatase (SHP)-2, is crucial for mouse placental development and is known to mediate IGF actions in other systems. In this study we examined the role of SHP-2 in regulating IGF-mediated proliferation in human trophoblast. Immunohistochemical analysis demonstrated that SHP-2 is expressed strongly in cytotrophoblast and only weakly in syncytium. After small interfering RNA-mediated knockdown of SHP-2 in BeWo choriocarcinoma cells and human first-trimester placental explants, IGF-induced trophoblast proliferation, examined using immunohistochemical analysis of Ki67 and 5-bromo-2'-deoxyuridine incorporation, was significantly reduced (P < 0.05). Kinase activation assays suggested that SHP-2 interacts with the MAPK pathway to mediate these effects. Markers of trophoblast differentiation were elevated after SHP-2 knockdown. This study demonstrates a role for tyrosine phosphatases in human trophoblast and establishes SHP-2 as a component of the IGF signaling pathway that is required for normal placental growth.

Sertoli Cell Androgen Receptor DNA Binding Domain Is Essential for the Completion of Spermatogenesis

Mon, 21 Sep 2009 10:02:26 PDT

We examined the biological importance of Sertoli cell androgen receptor (AR) genomic interaction, using a Cre-loxP approach to selectively disrupt the AR DNA-binding domain (AR-DBD). Sertoli cell (SC)-specific transgenic Abpa or AMH promoters targeted Cre-mediated inframe excision of mouse Ar exon-3, encoding the AR-DBD second zinc-finger (ZF2), generating SC-specific mutant AR^ZF2 lines designated Abp.SCAR^ZF2 and AMH.SCAR^ZF2, respectively. Both SCAR^ZF2 lines produced infertile males exhibiting spermatogenic arrest, despite normal SC numbers and immunolocalized SC nuclear AR. Adult homozygous TgCre^(+/+) SCAR^ZF2 or double-TgCre^(+/–) Abp/AMH.SCAR^ZF2 males displayed equivalent small testes 30% of normal size, representing maximal Cre-loxP-disruption of Sertoli AR function. Hemizygous TgCre^(+/–) vs. homozygous TgCre^(+/+) Abp.SCAR^ZF2 testes were larger (47% normal size) with more postmeiotic development, indicating dose-dependent Cre-mediated disruption of SC-specific AR-DBD activity. SCAR^ZF2 males exhibited adult Leydig cell hypertrophy but normal serum testosterone levels. Sertoli cell-specific Rhox5 and Spinlw1 transcription, regulated by divergent or classical androgen-response elements, respectively, were both decreased in postnatal SCAR^ZF2 vs. control testes, demonstrating SC-specific AR-DBD function as early as postnatal d 5. However, Rhox5 expression declined dose-dependently, whereas Spinlw1 expression increased, in adult TgCre^(+/–) and TgCre^(+/+) SCAR^ZF2 testes, revealing differential temporal control for distinct AR-regulated transcripts. Androgen-repressed Ngfr was not up-regulated in SCAR^ZF2 testes, suggesting maintenance of a nonclassical mechanism independent of AR-DBD. Thus, our unique SCAR^ZF2 paradigm provided dose-dependent Cre-mediated disruption of testicular development and gene expression revealing that the AR-DBD is essential for SC function and postmeiotic spermatogenesis. Nongenomic or AR-DBD-independent pathways appear secondary or play no major independent role in SC function.

Differential Endothelin Receptor Expression and Function in Rat Myometrial Cells and Leiomyoma ELT3 Cells

Raymond, M.-N., Robin, P., De Zen, F., Vilain, G., Tanfin, Z. — Mon, 21 Sep 2009 10:02:26 PDT

Uterine leiomyoma are the most common benign tumors of the myometrium. We previously identified endothelin (ET)-1 as a proliferative and antiapoptotic factor in Eker rat-derived leiomyoma (ELT3) cells. A major role of ETB receptor in the prosurvival effect was revealed. Here we investigated, in ELT3 and myometrial cells, the respective contribution of ETA and ETB in the proliferative effect of ET-1. In myometrial cells, binding experiments show that ETA is almost exclusively expressed and stimulates phospholipase C (PLC) activity and ERK1/2 phosphorylation and proliferation. In ELT3 cells, ETB is expressed at about the same level as ETA, and the two receptors are differently coupled to Gi protein. The ETB agonist, sarafotoxin S6c, stimulates PLC activity 60% less than ET-1 but is as potent as ET-1 to increase ERK1/2 phosphorylation and induce proliferation. However, the ability of ETA to activate ERK1/2 is observed after ETB desensitization. Although ETA and ETB antagonists partially reduce ET-1 stimulated PLC activity, they are without effect on ET-1-induced ERK1/2 phosphorylation and proliferation. Only the simultaneous use of ETA and ETB antagonists reduces ET-1-triggered ERK1/2 activation. These unconventional properties of ETRs may reveal the existence of functional ETA-ETB heterodimers. Finally, treatment of ELT3 cells with ETB but not ETA-directed small interfering RNA reduces the proliferative effect of ET-1. All the data obtained in ELT3 cells strengthen the relation between ETB overexpression, which decreases the ETA to ETB ratio, and the ability of leiomyoma cells to highly proliferate and resist apoptosis.

C-Type Natriuretic Peptide Forms in Pregnancy: Maternal Plasma Profiles during Ovine Gestation Correlate with Placental and Fetal Maturation

Mon, 21 Sep 2009 10:02:26 PDT

Circulating concentrations of C-type natriuretic peptide (CNP) and a related amino terminal fragment (NTproCNP) were measured at weekly intervals from preconception to 3 wk postpartum in ewes with twins (n = 8) and nonpregnant ewes (n = 8). In contrast to low and stable values in nonpregnant ewes (CNP, 0.75 ± 0.08; NTproCNP, 22 ± 2 pmol/liter), CNP forms increased abruptly at 40–50 d of gestation and rose to peak values (CNP, 31 ± 5, NTproCNP, 270 ± 16 pmol/liter) at about d 120. Approximately 7 d prepartum, the concentration of both CNP forms fell precipitously to preconception values immediately postpartum. In separate studies, circulating maternal CNP forms were positively related to fetal number at d 120. Consistent with a major contribution from the placenta to circulating levels, the concentrations of CNP forms were elevated in the placentome (cotyledon: CNP, 18 ± 4, NTproCNP, 52 ± 10 pmol/g; caruncle: CNP, 13 ± 3, NTproCNP, 31 ± 6 pmol/g) and much higher than those of intercaruncular uterine tissue (CNP, 0.19 ± 0.05, NTproCNP, 0.98 ± 0.2 pmol/g) in late-gestation ewes (P < 0.001, n = 4). These distinctive patterns of maternal plasma CNP forms, positive relation with fetal number, and greatly elevated protein concentrations in the placentome demonstrate the hormone’s strong relation to placental and fetal maturation. The findings provide a firm basis for future studies of the functional role of CNP in fetal-maternal welfare.

Inhibin B Is a More Potent Suppressor of Rat Follicle-Stimulating Hormone Release than Inhibin A in Vitro and in Vivo

Mon, 21 Sep 2009 10:02:26 PDT

Mature 31- and 34-kDa inhibin A and B negatively regulate the release of FSH from the anterior pituitary; however, a direct comparison of these hormones in vivo has not been undertaken. The bioactivities of highly purified preparations of recombinant human 31-kDa inhibin A and B were determined in rat pituitary cells in vitro, and in ovariectomized adult rats in vivo based on suppression of plasma FSH. The 31-kDa inhibin B was 4.2-fold more bioactive than inhibin A in vitro and 1.45 (1.01–2.79)-fold more bioactive in vivo than 31-kDa inhibin A. However, the corresponding relative binding affinities of 31-kDa inhibin B for betaglycan, betaglycan+activin type II receptor (ActRII)-A, and betaglycan+ActRIIB were lower (IC₅₀ 2200, 400, and 750 pm, respectively) compared with 31-kDa inhibin A (IC₅₀ 190, 80, and 290 pm, respectively). A 2.7- and 2.5-fold reduction in in vitro bioactivity was observed between the 31- and 34-kDa inhibin A and 31- and 34-kDa inhibin B, respectively, and these decreases in bioactivities were matched by a parallel reduction in binding to betaglycan and betaglycan+ActRIIA/B. It is concluded that the increased in vitro and in vivo bioactivities of 31-kDa inhibin B cannot be explained by a higher affinity to betaglycan or activin type II receptors; thus, additional factors mediate inhibin B’s action. In addition, similar reductions in in vitro bioactivity and betaglycan+ActRIIA/B binding between 31- and 34-kDa inhibins A and B are attributed to hindrance by the additional carbohydrate group at Asn³⁰² in the formation of a functional inhibin+betaglycan+ActRIIA/B complex.

Metformin Inhibits Aromatase via an Extracellular Signal-Regulated Kinase-Mediated Pathway

Rice, S., Pellatt, L., Ramanathan, K., Whitehead, S. A., Mason, H. D. — Mon, 21 Sep 2009 10:02:26 PDT

Metformin treatment, now widely prescribed in polycystic ovary syndrome, is aimed at correcting the associated insulin resistance, but it has also been shown to directly inhibit ovarian steroidogenesis. The mechanisms, however, by which metformin inhibits estradiol production in human granulosa cells remains unknown. Granulosa luteal cells were incubted with metformin, insulin, or combined metformin and insulin treatment, and aromatase mRNA expression was quantified using real-time RT-PCR. Enzyme activity was assessed by the conversion of ³H-androstenedione to estrone and estradiol. Metformin’s effect on the expression of specific untranslated first exon aromatase promoters was analyzed using semiquantitative PCR. The involvement of MAPK kinase (MEK)/ERK pathway was investigated by immunoblotting for aromatase, phosphorylated, and total ERK-1,2 from cells cultured as above with/without the MEK inhibitor PD98059. Metformin significantly inhibited basal and insulin-stimulated aromatase mRNA expression, with parallel results from the aromatase activity assay and protein assessment. This suppression was via down-regulation of aromatase promoter II, I.3, and 1.4 expression and was reversed by the addition of PD98059. Involvement of the ERK signaling pathway was demonstrated by the significant increase in phosphorylated ERK-1,2 with the combined metformin and insulin treatment. We have shown for the first time in human granulosa cells that metformin signficantly attenuated basal and insulin-stimulated P450 aromatase mRNA expression and activity, via silencing of key promoters. This occurred by activation of MEK/ERK pathway, which negatively regulated aromatase production. This is an important consideration given metformin’s widespread use in polycystic ovary syndrome and may further support a possible therapeutic indication in estrogen-dependent breast tumors.

Developmental Changes in Pituitary Adenylate Cyclase Activating Polypeptide Expression during the Perinatal Period: Possible Role in Fetal Gonadotroph Regulation

Moore, J. P., Villafuerte, B. C., Unick, C. A., Winters, S. J. — Mon, 21 Sep 2009 10:02:26 PDT

Normal reproductive functioning may require secretion of LH independently of FSH. Variation in GnRH pulse frequency and inhibin negative feedback are mechanisms for differential gonadotropin regulation; however, the first instance of differential regulation in rats is during fetal development, prior to the establishment of GnRH connections, when LH accumulates appreciably 2–4 d prior to FSH. Pituitary adenylate cyclase activating polypeptide (PACAP) can differentially regulate the gonadotropins in vitro by stimulating -subunit transcription, lengthening LHβ transcripts and decreasing FSHβ mRNA levels, probably through stimulation of follistatin transcription. These experiments are the first to examine whether PACAP influences gonadotroph function in perinatal pituitaries. In vivo, pituitary PACAP mRNA and peptide levels were high at embryonic d 19 and declined by 94 and 85%, respectively, after parturition. This was accompanied by a decrease of 65 and 96% in total follistatin and follistatin-288 mRNAs. These changes were temporally associated with a 20- and 6.5-fold rise in FSHβ and GnRH receptor mRNAs, respectively, with no significant increase in LHβ mRNA. In pituitary cell cultures from fetal and postnatal male rats, PACAP mRNA levels were likewise highest in fetal cultures in which the PACAP 6-38 antagonist decreased -subunit and increased FSHβ mRNA. PACAP 6-38 also reduced basal and GnRH-stimulated LH secretion with little effect on FSH. These data support the hypothesis that PACAP expressed at high levels in the fetal pituitary stimulates -subunit expression and LH secretion and restrains FSH synthesis relative to LH and that a decline in PACAP allows for the neonatal rise in FSH and GnRH receptor because follistatin is decreased.

Morphological Ultrasound Microimaging of Thyroid in Living Mice

Mon, 21 Sep 2009 10:02:26 PDT

The objective of the study was to explore high-frequency ultrasound (HFUS) for noninvasive microimaging of thyroid in living mice. Thyroid examination was performed by HFUS in 10 normal C57BL/6 mice, eight mice treated by propylthiouracil, and 22 Tg-TRK-T1 transgenic mice. The dimension of the gland and the presence of nodules were evaluated. Nodules were classified as malignant (hypoechogenicity, poorly defined margins, internal microcalcification, irregular shapes, and extra glandular extension) or not, and the findings were compared with histological data. Thyroid images were successfully obtained in all the animals analyzed. Normal thyroid reached a volume of 4.92 µl (range 2.11–4.92 µl). Mice with propylthiouracil-induced goiter showed diffuse thyroid enlargement (median volume 6.67 µl, range 4.09–8.82 µl). In 19 of 22 Tg-TRK-T1 mice (86%), HFUS identified a nodular process (the smallest detected nodule had a diameter of 0.46 mm). Eleven nodules were classified as malignant and eight as benign. Compared with histological analysis, HFUS showed a sensitivity of 100% in the detection of thyroid nodules and a specificity of 60% (two of the nodules identified by HFUS were not confirmed at the histology). The specificity and sensitivity of HFUS in predicting the malignancy of the thyroid nodules were 83 and 91%, respectively. Thus, HFUS is an accurate imaging modality that can potentially replace more invasive techniques, and, therefore, it represents a significant advancement in phenotypic assessment of mouse models of thyroid cancer.

A Novel Loss-of-Function Mutation, Gln459Arg, of the Calcium-Sensing Receptor Gene Associated with Apparent Autosomal Recessive Inheritance of Familial Hypocalciuric Hypercalcemia

Mon, 21 Sep 2009 10:02:26 PDT

Effects of Bariatric Surgical Weight Loss in Mothers on Intergenerational Transmission of Obesity

Mon, 21 Sep 2009 10:02:26 PDT

Subcutaneous Injection of Kisspeptin-54 Acutely Stimulates Gonadotropin Secretion in Women with Hypothalamic Amenorrhea, But Chronic Administration Causes Tachyphylaxis

Mon, 21 Sep 2009 10:02:26 PDT

The Impact of Exercise Training Compared to Caloric Restriction on Hepatic and Peripheral Insulin Resistance in Obesity

Mon, 21 Sep 2009 10:02:26 PDT

Glucocorticoid Production and Regulation in Thymus: Of Mice and Birds

Gomez-Sanchez, C. E. — Fri, 21 Aug 2009 10:03:34 PDT

New Evidence that an Epigenetic Mechanism Mediates Testosterone-Dependent Brain Masculinization

Baum, M. J. — Fri, 21 Aug 2009 10:03:34 PDT

Do Animal Models of Polycystic Ovary Syndrome Help to Understand Its Pathogenesis and Management? Yes, but Their Limitations Should be Recognized

Franks, S. — Fri, 21 Aug 2009 10:03:34 PDT

Evolution and Male Fertility: Lessons from the Insulin-Like Factor 6 Gene (Insl6)

Ivell, R., Grutzner, F. — Fri, 21 Aug 2009 10:03:34 PDT

Epigenetic Alterations in Human Prostate Cancers

Nelson, W. G., De Marzo, A. M., Yegnasubramanian, S. — Fri, 21 Aug 2009 10:03:34 PDT

Human prostate cancer cells carry a myriad of genome defects, including both genetic and epigenetic alterations. These changes, which can be maintained through mitosis, generate malignant phenotypes capable of selective growth, survival, invasion, and metastasis. During prostatic carcinogenesis, epigenetic changes arise earlier than genetic defects, linking the appearance of epigenetic alterations in some way to disease etiology. The most common genetic defect thus far described, leading to fusion transcripts between the androgen-regulated gene TMPRSS2 and genes from the ETS family of transcription factors, likely endows prostate cancer cells with the ability to co-opt androgen signaling, the major prostate differentiation pathway, to support the malignant phenotype. Whether epigenetic changes promote the appearance of TMPRSS2-ETS family fusion transcripts or collaborate with fusion transcript expression in the pathogenesis of prostate cancer has not been established. However, a growing list of epigenetic alterations has provided new opportunities for clinical tests that might aid in prostate cancer screening, detection, diagnosis, staging, and risk stratification. The epigenetic changes appear to be more attractive than genetic changes as prostate cancer biomarkers because epigenetic alterations are present in a greater fraction of prostate cancer cases than any of the known genetic defects. In addition, an emerging generation of assay strategies for detection of specific DNA sequences carrying ^5-meC, the major epigenetic genome mark, has pushed somatic epigenetic alterations to the forefront of molecular biomarker assay development for cancer. Finally, a growing portfolio of epigenetic drugs, capable of reversing the phenotypic consequences of somatic epigenetic defects, has entered clinical trials for prostate cancer in the search for a new rational therapy for the disease.

Minireview: Epigenetic Changes in Ovarian Cancer

Balch, C., Fang, F., Matei, D. E., Huang, T. H.-M., Nephew, K. P. — Fri, 21 Aug 2009 10:03:35 PDT

Epigenetic aberrations, including DNA methylation, histone modifications, and micro-RNA dysregulation, are now well established in the development and progression of ovarian cancer, and their gradual accumulation is associated with advancing disease stage and grade. Epigenetic aberrations are relatively stable, associated with distinct disease subtypes, and present in circulating serum, representing promising diagnostic, prognostic, and pharmacodynamic biomarkers. In contrast to DNA mutations and deletions, aberrant gene-repressive epigenetic modifications are potentially reversible by epigenetic therapies, including inhibitors of DNA methylation or histone-modifying enzymes. Although epigenetic monotherapies have not shown activity against solid tumors, including ovarian cancer, preclinical studies suggest they will be effective when used in combination with one another or with conventional chemotherapeutics, and combinatorial epigenetic therapy regiments are being examined in cancer clinical trials. A greater understanding of the role of epigenetics in ovarian neoplasia will provide for improved interventions against this devastating malignancy.

Matrix Extracellular Phosphoglycoprotein (MEPE) Is a New Bone Renal Hormone and Vascularization Modulator

David, V., Martin, A., Hedge, A.-M., Rowe, P. S. N. — Fri, 21 Aug 2009 10:03:35 PDT

Increased matrix extracellular phosphoglycoprotein (MEPE) expression occurs in several phosphate and bone-mineral metabolic disorders. To resolve whether MEPE plays a role, we created a murine model overexpressing MEPE protein (MEPE tgn) in bone. MEPE tgn mice displayed a growth and mineralization defect with altered bone-renal vascularization that persisted to adulthood. The growth mineralization defect was due to a decrease in bone remodeling, and MEPE tgn mice were resistant to diet-induced renal calcification. MEPE protein-derived urinary ASARM peptides and reduced urinary Ca X PO4 product mediated the suppressed renal calcification. Osteoblastic cells displayed reduced activity but normal differentiation. Osteoclastic precursors were unable to differentiate in the presence of osteoblasts. In the kidney, NPT2a up-regulation induced an increase in phosphate renal reabsorption, leading to hyperphosphatemia. We conclude MEPE and MEPE-phosphate-regulating gene with homologies to endopeptidases on the X chromosome (MEPE-PHEX) interactions are components to an age-diet-dependent pathway that regulates bone turnover and mineralization and suppresses renal calcification. This novel pathway also modulates bone-renal vascularization and bone turnover.

Chondrocyte-Specific Modulation of Cyp27b1 Expression Supports a Role for Local Synthesis of 1,25-Dihydroxyvitamin D3 in Growth Plate Development

Naja, R. P., Dardenne, O., Arabian, A., St. Arnaud, R. — Fri, 21 Aug 2009 10:03:35 PDT

The Cyp27b1 enzyme (25-hydroxyvitamin D-1-hydroxylase) that converts 25-hydroxyvitamin D into the active metabolite, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is expressed in kidney but also in other cell types such as chondrocytes. This suggests that local production of 1,25(OH)₂D₃ could play an important role in the differentiation of these cells. To test this hypothesis, we engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. Inactivation of both alleles of the Cyp27b1 gene led to decreased RANKL expression and reduced osteoclastogenesis, increased width of the hypertrophic zone of the growth plate at embryonic d 15.5, increased bone volume in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. The expression of the angiogenic marker VEGF was decreased, accompanied by decreased platelet/endothelial cell adhesion molecule-1 staining in the neonatal growth plate, suggesting a delay in vascularization. In parallel, we engineered strains of mice overexpressing a Cyp27b1 transgene in chondrocytes by coupling the Cyp27b1 cDNA to the collagen ₁(II) promoter. The transgenic mice showed a mirror image phenotype when compared with the tissue-specific inactivation, i.e. a reduction in the width of the hypertrophic zone of the embryonic growth plate, decreased bone volume in neonatal long bones, and inverse expression patterns of chondrocytic differentiation markers. These results support an intracrine role of 1,25(OH)₂D₃ in endochondral ossification and chondrocyte development in vivo.

Carcinogenic Effects of Exogenous and Endogenous Glucagon-Like Peptide-2 in Azoxymethane-Treated Mice

Iakoubov, R., Lauffer, L. M., Trivedi, S., Kim, Y.-I. J., Brubaker, P. L. — Fri, 21 Aug 2009 10:03:35 PDT

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent intestinotropic hormone that promotes intestinal growth, via increased intestinal proliferation and decreased apoptosis, as well as increases in nutrient absorption and barrier function. The long-acting analog h(Gly²)GLP-2[1-33] is currently being tested for treatment of short bowel syndrome and Crohn’s disease. However, the role of GLP-2 in colon carcinogenesis is controversial. To assess the intestinotropic effects of exogenous and endogenous GLP-2, C57BL6/J mice were injected with 1µg h(Gly²)GLP-2[1-33]; 30 or 60 ng hGLP-2[3-33], a GLP-2 receptor antagonist; or PBS (4 wk, twice a day, sc). Chronic h(Gly²)GLP-2[1-33] increased small intestinal weight/body weight (P < 0.001), villus height (P < 0.001), crypt depth (P < 0.001), and crypt cell proliferation, as measured by expression of the proliferative marker Ki67 (P < 0.05–0.01). In contrast, chronic hGLP-2[3-33] decreased small intestinal weight/body weight (P < 0.05) and colon weight/body weight (P < 0.05). To assess the carcinogenic effects of endogenous and exogenous GLP-2, separate mice were injected with azoxymethane (10 mg/kg, 4 wk, every 7 d, ip), followed by 1.5 µg h(Gly²)GLP-2[1-33], 30 ng hGLP-2[3-33], or PBS (4 wk, twice a day, sc) 2 or 12 wk thereafter. At 10 or 46 wk after azoxymethane treatment, the numbers of aberrant crypt foci increased with h(Gly²)GLP-2[1-33] (P < 0.001) and decreased with hGLP-2[3-33] (P < 0.01–0.05) treatment. Furthermore, mucin-depleted aberrant foci, consistent with progressive dysplasia, were almost exclusively present in h(Gly²)GLP-2[1-33]-treated mice (P < 0.01–0.001). Additionally, adenocarcinomas developed in h(Gly²)GLP-2[1-33]-treated mice but not in those receiving hGLP-2[3-33] or PBS. Taken together, these studies indicate that chronic treatment with GLP-2 enhances colon carcinogenesis, whereas antagonism of the GLP-2 receptor decreases dysplasia, with possible implications for human therapy.

Fibroblast Growth Factor 2 Reactivates G1 Checkpoint in SK-N-MC Cells via Regulation of p21, Inhibitor of Differentiation Genes (Id1-3), and Epithelium-Mesenchyme Transition-Like Events

Fri, 21 Aug 2009 10:03:35 PDT

We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.

The Glucose Transporter 2 Undergoes Plasma Membrane Endocytosis and Lysosomal Degradation in a Secretagogue-Dependent Manner

Hou, J. C., Williams, D., Vicogne, J., Pessin, J. E. — Fri, 21 Aug 2009 10:03:35 PDT

In β-cells of the pancreas, the glucose transporter (GLUT)-2 facilitative glucose transporter protein is localized to the plasma membrane and functions as part of the glucose sensing mechanism for the stimulation of insulin secretion. We observed that expressed GLUT2 protein in the cultured Min6B1 cell line undergoes enhanced endocytosis at high extracellular glucose concentrations that stimulate insulin secretion. Moreover, the internalized GLUT2 protein undergoes rapid degradation induced by chronic high-glucose or arginine stimulation but does not undergo plasma membrane recycling or accumulation in any microscopically apparent intracellular membrane compartment. The rapid degradation of GLUT2 was prevented by lysosomal inhibition (chloroquine) concomitant with the accumulation of GLUT2 in endomembrane structures. In contrast, neither endocytosis nor the lack of internal membrane localized GLUT2 remained completely unaffected by proteosomal inhibition (lactacystin) or an heat shock protein-90 inhibitor (geldanamycin). Moreover, the endocytosis and degradation of GLUT2 was specific for β-cells because expression of GLUT2 in 3T3L1 adipocytes remained cell surface localized and did not display a rapid rate of degradation. Together, these data demonstrate that hyperglycemia directly affects β-cell function and activates a trafficking pathway that results in the rapid endocytosis and degradation of the cell surface GLUT2 glucose transporter.

Inhibition of Forkhead Box O1 Protects Pancreatic {beta}-Cells against Dexamethasone-Induced Dysfunction

Fri, 21 Aug 2009 10:03:35 PDT

Forkhead Box O1 (FoxO1) is a key transcription regulator of insulin/IGF-I signaling pathway, and its activity can be increased by dexamethasone (DEX) in several cell types. However, the role of FoxO1 in DEX-induced pancreatic β-cell dysfunction has not been fully understood. Therefore, in this study, we investigated whether FoxO1 could mediate DEX-induced β-cell dysfunction and the possible underlying mechanisms in pancreatic β-cell line RINm5F cells and primary rat islet. We found that DEX markedly increased FoxO1 mRNA and protein expression and decreased FoxO1 phosphorylation through the Akt pathway, which resulted in an increase in active FoxO1 in RINm5F cells and isolated rat islets. Activated FoxO1 subsequently inhibited pancreatic duodenal homeobox-1 expression and induced nuclear exclusion of pancreatic duodenal homeobox-1. Knockdown of FoxO1 by RNA interference restored the expression of pancreatic duodenal homeobox-1 and prevented DEX-induced dysfunction of glucose-stimulated insulin secretion in rat islets. Together, the results of present study demonstrate that FoxO1 is integrally involved in DEX-induced inhibition of pancreatic duodenal homeobox-1 and glucose-stimulated insulin secretion dysfunction in pancreatic islet β-cells. Inhibition of FoxO1 can effectively protect β-cells against DEX-induced dysfunction.

Suppression of Peroxisome Proliferator-Activated Receptor {gamma}-Coactivator-1{alpha} Normalizes the Glucolipotoxicity-Induced Decreased BETA2/NeuroD Gene Transcription and Improved Glucose Tolerance in Diabetic Rats

Fri, 21 Aug 2009 10:03:35 PDT

Peroxisome proliferator-activated receptor -coactivator-1 (PGC-1) is significantly elevated in the islets of animal models of diabetes. However, the molecular mechanism has not been clarified. We investigated whether the suppression of PGC-1 expression protects against β-cell dysfunction in vivo and determined the mechanism of action of PGC-1 in β-cells. The studies were performed in glucolipotixicity-induced primary rat islets and INS-1 cells. In vitro and in vivo approaches using adenoviruses were used to evaluate the role of PGC-1 in glucolipotoxicity-associated β-cell dysfunction. The expression of PGC-1 in cultured β-cells increased gradually with glucolipotoxicity. The overexpression of PGC-1 also suppressed the expression of the insulin and β-cell E-box transcription factor (BETA2/NeuroD) genes, which was reversed by PGC-1 small interfering RNA (siRNA). BETA2/NeuroD, p300-enhanced BETA2/NeuroD, and insulin transcriptional activities were significantly suppressed by Ad-PGC-1 but were rescued by Ad-siPGC-1. PGC-1 binding at the glucocorticoid receptor site on the BETA2/NeuroD promoter increased in the presence of PGC-1. Ad-siPGC-1 injection through the celiac arteries of 90% pancreatectomized diabetic rats improved their glucose tolerance and maintained their fasting insulin levels. The suppression of PGC-1 expression protects the glucolipotoxicity-induced β-cell dysfunction in vivo and in vitro. A better understanding of the functions of molecules such as PGC-1, which play key roles in intracellular fuel regulation, could herald a new era of the treatment of patients with type 2 diabetes mellitus by providing protection from glucolipotoxicity, which is an important cause of the development and progression of the disease.

Fibroblast Growth Factor 21 Controls Glycemia via Regulation of Hepatic Glucose Flux and Insulin Sensitivity

Fri, 21 Aug 2009 10:03:35 PDT

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator shown to improve glycemic control. However, the molecular and functional mechanisms underlying FGF21-mediated improvements in glycemic control are not completely understood. We examined FGF21 effects on insulin sensitivity and glucose fluxes upon chronic (daily injection for 8 d) and acute (6 h infusion) administration in ob/+ and ob/ob mice. Results show that chronic FGF21 ameliorated fasting hyperglycemia in ob/ob mice via increased glucose disposal and improved hepatic insulin sensitivity. Acute FGF21 suppressed hepatic glucose production, increased liver glycogen, lowered glucagon, and improved glucose clearance in ob/+ mice. These effects were blunted in ob/ob mice. Neither chronic nor acute FGF21 altered skeletal muscle or adipose tissue glucose uptake in either genotype. In conclusion, FGF21 has potent glycemic effects caused by hepatic changes in glucose flux and improved insulin sensitivity. Thus, these studies define mechanisms underlying anti-hyperglycemic actions of FGF21 and support its therapeutic potential.

The Power of Programming: International Conference on Developmental Origins of Health and Disease May 6-10, 2010, Munich, Germany

Fri, 21 Aug 2009 10:03:35 PDT

Inhibition of Nuclear Factor-{kappa}B or Bax Prevents Endoplasmic Reticulum Stress- But Not Nitric Oxide-Mediated Apoptosis in INS-1E Cells

Fri, 21 Aug 2009 10:03:35 PDT

Accumulating evidence suggests that endoplasmic reticulum (ER) stress by mechanisms that include ER Ca²⁺ depletion via NO-dependent down-regulation of sarcoendoplasmic reticulum Ca²⁺ ATPase 2b (SERCA2b) contributes to β-cell death in type 1 diabetes. To clarify whether the molecular pathways elicited by NO and ER Ca²⁺ depletion differ, we here compare the direct effects of NO, in the form of the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), with the effects of SERCA2 inhibitor thapsigargin (TG) on MAPK, nuclear factor B (NFB), Bcl-2 proteins, ER stress, and apoptosis. Exposure of INS-1E cells to TG or SNAP caused caspase-3 cleavage and apoptosis. Both TG and SNAP induced activation of the proapoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP). However, other classical ER stress-induced markers such as up-regulation of ER chaperone Bip and alternative splicing of the transcription factor Xbp-1 were exclusively activated by TG. TG exposure caused NFB activation, as assessed by IB degradation and NFB DNA binding. Inhibition of NFB or the Bcl-2 family member Bax pathways protected β-cells against TG- but not SNAP-induced β-cell death. These data suggest that NO generation and direct SERCA2 inhibition cause two quantitative and qualitative different forms of ER stress. In contrast to NO, direct ER stress induced by SERCA inhibition causes activation of ER stress signaling pathways and elicit proapoptotic signaling via NFB and Bax.

Activation of Liver X Receptor Regulates Substrate Oxidation in White Adipocytes

Fri, 21 Aug 2009 10:03:35 PDT

Liver X receptors (LXRs) are nuclear receptors with established roles in cholesterol, lipid, and carbohydrate metabolism, although their function in adipocytes is not well characterized. Increased adipose tissue mass in obesity is associated with increased adipocyte lipolysis. Fatty acids (FA) generated by lipolysis can be oxidized by mitochondrial β-oxidation, reesterified, or released from the adipocyte. The latter results in higher circulating levels of free FAs, in turn causing obesity-related metabolic complications. However, mitochondrial β-oxidation can at least in part counteract an increased output of FA into circulation. In this study, we provide evidence that activation of LXRs up-regulates mitochondrial β-oxidation in both human and murine white adipocytes. We also show that the expression of a kinase regulating the cellular fuel switch, pyruvate dehydrogenase kinase 4 (PDK4), is up-regulated by the LXR agonist GW3965 in both in vitro differentiated human primary adipocytes and differentiated murine 3T3-L1 cells. Moreover, activation of LXR causes PDK4-dependent phosphorylation of the pyruvate dehydrogenase complex, thereby decreasing its activity and attenuating glucose oxidation. The specificity of the GW3965 effect on oxidation was confirmed by RNA interference targeting LXRs. We propose that LXR has an important role in the regulation of substrate oxidation and the switch between lipids and carbohydrates as cellular fuel in both human and murine white adipocytes.

Call for abstracts

Fri, 21 Aug 2009 10:03:35 PDT

The Effect of Angiotensin-Converting Enzyme Inhibition Using Captopril on Energy Balance and Glucose Homeostasis

Fri, 21 Aug 2009 10:03:35 PDT

Increasing evidence suggests that the renin-angiotensin-system contributes to the etiology of obesity. To evaluate the role of the renin-angiotensin-system in energy and glucose homeostasis, we examined body weight and composition, food intake, and glucose tolerance in rats given the angiotensin-converting enzyme inhibitor, captopril (~40 mg/kg · d). Rats given captopril weighed less than controls when fed a high-fat diet (369.3 ± 8.0 vs. 441.7 ± 8.5 g after 35 d; P < 0.001) or low-fat chow (320.1 ± 4.9 vs. 339.8 ± 5.1 g after 21 d; P < 0.0001). This difference was attributable to reductions in adipose mass gained on high-fat (23.8 ± 2.0 vs. 65.12 ± 8.4 g after 35 d; P < 0.0001) and low-fat diets (12.2 ± 0.7 vs. 17.3 ± 1.3 g after 21 d; P < 0.001). Rats given captopril ate significantly less [3110.3 ± 57.8 vs. 3592.4 ± 88.8 kcal (cumulative 35 d high fat diet intake); P < 0.001] despite increased in neuropeptide-Y mRNA expression in the arcuate nucleus of the hypothalamus and had improved glucose tolerance compared with free-fed controls. Comparisons with pair-fed controls indicated that decreases in diet-induced weight gain and adiposity and improved glucose tolerance were due, primarily, to decreased food intake. To determine whether captopril caused animals to defend a lower body weight, animals in both groups were fasted for 24 h and subsequently restricted to 20% of their intake for 2 d. When free food was returned, captopril and control rats returned to their respective body weights and elicited comparable hyperphagic responses. These results suggest that angiotensin-converting enzyme inhibition protects against the development of diet-induced obesity and glucose intolerance.

Receptors for Tumor Necrosis Factor-{alpha} Play a Protective Role against Obesity and Alter Adipose Tissue Macrophage Status

Pamir, N., McMillen, T. S., Kaiyala, K. J., Schwartz, M. W., LeBoeuf, R. C. — Fri, 21 Aug 2009 10:03:35 PDT

TNF- signals through two receptors, TNFR1 and TNFR2. Our goals were: 1) determine the role of TNFRs in obesity and metabolic disease and 2) investigate whether TNFRs contribute to the link between obesity and adipose tissue macrophage infiltration and polarization. R1^–/–R2^–/– (RKO) and wild-type (WT) mice were fed standard chow or a high-fat/high-sucrose diet (HFHS) over 14 wk. Body composition, food intake, and energy expenditure were measured. Oral glucose tolerance and insulin sensitivity tests assessed glucose homeostasis. Adipose tissue and systemic inflammatory status were evaluated by quantifying plasma adipokine levels and macrophage-specific gene expression in fat. RKO mice were heavier (10%) and fatter (18%) than WT controls at 4 wk of age and were 26% heavier and 50% fatter than WT after 14 wk of HFHS diet feeding. Age- and diet-adjusted 24-h oxygen consumption, activity, and respiratory exchange ratio were significantly reduced in RKO mice. Obese RKO mice were markedly insulin resistant, suggesting that intact TNFR signaling is not required for the effect of obesity to impair glucose metabolism. Adipose tissue from HFHS-fed RKO mice exhibited increased macrophage infiltration, but compared with WT mice, macrophage phenotypic markers featured a predominance of antiinflammatory M2 over proinflammatory M1 cells. TNFRs play a physiological role to limit body weight and adiposity by modestly increasing metabolic rate and fatty acid oxidation, and they are required for obesity-induced activation of adipose tissue macrophages. Despite these effects, TNFRs are not required for obesity-induced insulin resistance.

Prenatally Induced Changes in Muscle Structure and Metabolic Function Facilitate Exercise-Induced Obesity Prevention

Fri, 21 Aug 2009 10:03:35 PDT

Effective regulation of energy metabolism is vital for the maintenance of optimal health, and an inability to make these dynamic adjustments is a recognized cause of obesity and metabolic disorders. Epidemiological and experimental studies have highlighted the role of prenatal factors in the disease process, and it is now generally accepted that maternal nutrition during pregnancy significantly influences intrauterine development, shaping postnatal health. Consequences of impaired nutrition during fetal development include intrauterine growth restriction (IUGR) and subsequent obesity development in adult life. We have previously shown that prenatal undernutrition has a lasting effect on behavior, with IUGR offspring expressing a higher preference for voluntary exercise, and moderate daily exercise preventing obesity development. The present study investigated skeletal muscle structure in IUGR offspring and how moderate daily exercise drives changes in metabolic pathways that promote obesity prevention. Pregnant Wistar rats were either fed chow ad libitum or undernourished, generating control or IUGR offspring respectively. Although red muscle structure indicated higher oxidative capacity in IUGR offspring, obesity prevention was not due to increased fatty acid oxidation, indicated by decreased peroxisomal proliferator-activated receptor- coactivator 1 and carnitine-palmitoyltransferase 1 expression. In contrast, increased protein kinase C expression and glycogen content in white muscle of exercised IUGR offspring suggests an enhanced capacity for anaerobic utilization of glucose. Furthermore, exercise-induced lactate accumulation was effectively prevented by stimulation of a lactate shuttle, driven by the increases in monocarboxylate transporters-4 and -1 in white muscle. This enhanced metabolic flexibility in IUGR offspring may facilitate muscle contractile performance and therefore support moderate daily exercise for effective obesity prevention.

The Mediator Complex Subunit 1 Enhances Transcription of Genes Needed for Adrenal Androgen Production

Nakamura, Y., Xing, Y., Sasano, H., Rainey, W. E. — Fri, 21 Aug 2009 10:03:35 PDT

There are three enzymes involved in the biosynthesis of the adrenal androgen dehydroepiandrosterone (DHEA) sulfate. Cholesterol side-chain cleavage (CYP11A1) and 17-hydroxylase/17,20-lyase (CYP17) metabolize cholesterol into DHEA, whereas steroid sulfotransferase family 2A1 (SULT2A1) is responsible for conversion of DHEA to DHEA sulfate. We previously examined the mechanisms regulating CYP11A1, CYP17, and SULT2A1 transcription and found that each is regulated, in part, by the transcription factor GATA-6. Previous studies suggested that mediator complex subunit 1 (MED1, also called PPARBP or TRAP220) is a cofactor involved in not only the regulation of nuclear receptors but also the activation of GATA-6 transcription. Herein we demonstrated a role for MED1 in the regulation of CYP11A1, CYP17, and SULT2A1 transcription. Transient transfection assays with SULT2A1 deletion and mutation promoter constructs allowed the determination of specific the GATA-6 binding cis-regulatory elements necessary for transactivation of SULT2A1 transcription. Binding of MED1 and GATA-6 was confirmed by coimmunoprecipitation/Western analysis and chromatin immunoprecipitation assay. We demonstrated expression of MED1 mRNA and protein in the human adrenal and determined that knockdown of MED1 expression via specific small interfering RNA attenuated CYP11A1, CYP17, and SULT2A1 expression levels in H295R cells. In addition, we demonstrated that MED1 enhanced GATA-6 stimulated transcription of promoter constructs for each of these genes. Moreover, the activity of MED1 for SULT2A1 promoter was mediated by GATA-6 via the –190 GATA-binding site. These data support the hypothesis that MED1 and GATA-6 are key regulators of SULT2A1 expression, and they play important roles in adrenal androgen production.

The 14th International Thyroid Congress September 11-16, 2010 Palais des Congres, Paris, France

Fri, 21 Aug 2009 10:03:35 PDT

Biochemical Factors Governing the Steady-State Estrone/Estradiol Ratios Catalyzed by Human 17{beta}-Hydroxysteroid Dehydrogenases Types 1 and 2 in HEK-293 Cells

Fri, 21 Aug 2009 10:03:35 PDT

Human 17β-hydroxysteroid dehydrogenase types 1 and 2 (17βHSD1 and 17βHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17βHSD1 and 17βHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17βHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2'-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP⁺ ratio. The shift to E2 oxidation parallels changes in apparent K_m values for purified 17βHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17βHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17βHSD2, and these mutations lower the apparent K_m of recombinant 17βHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17βHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP⁺ ratios. In contrast, the preference of 17βHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17βHSD2 in intact cells.

Thymocyte-Synthesized Glucocorticoids Play a Role in Thymocyte Homeostasis and Are Down-Regulated by Adrenocorticotropic Hormone

Qiao, S., Okret, S., Jondal, M. — Fri, 21 Aug 2009 10:03:36 PDT

Thymocytes from adult mice synthesize glucocorticoids (GCs), and some data indicate a role for this hormone production in thymic homeostasis. Here we present further support for this view by showing that the dramatic increase in thymocyte number seen after adrenalectomy (ADX) does not correlate with the decrease in systemic GCs but rather with an ACTH-mediated down-regulation of GC synthesis in thymocytes. High ACTH concentrations caused by ADX in wild-type mice down-regulated CYP11B1 mRNA expression, encoding the last enzyme required for corticosterone synthesis and as a consequence reduced GC synthesis in thymocytes. This was not seen in IL-1β/IL-18 double-knockout mice unable to respond to ADX with high ACTH levels. However, if ADX IL-1β/IL-18 double-knockout mice were treated with ACTH, this led to a down-regulation of CYP11B1 and GC synthesis in thymocytes. In addition, in vivo treatment of mice with the CYP11B1 antagonist metyrapone, without affecting the systemic corticosterone level, increased thymocyte numbers and in vitro treatment of isolated thymocytes prevented thymocyte loss. Furthermore, in vitro experiments showed that both ACTH and its receptor-induced second-messenger molecule cAMP down-regulated mRNA expression of critical enzymes in GC steroidogenesis and GC synthesis in thymocytes. We conclude that thymocyte-produced GCs are important for the homeostasis of adult mouse thymocytes and that high ACTH level, in contrast to stimulating GC synthesis in the adrenal glands, has the opposite effect in thymocytes.

Distinct, Time-Dependent Effects of Voluntary Exercise on Circadian and Ultradian Rhythms and Stress Responses of Free Corticosterone in the Rat Hippocampus

Fri, 21 Aug 2009 10:03:36 PDT

Previous work has shown that allowing rats to voluntarily exercise in a running wheel for 4 wk modifies the hypothalamic-pituitary-adrenal axis and behavioral coping responses to stress. To investigate whether long-term voluntary exercise would also affect the free, biologically active fraction of corticosterone in the brain, we conducted an in vivo microdialysis study in the hippocampus of rats. We monitored both the baseline circadian and ultradian patterns of corticosterone in hippocampus dialysates over the diurnal cycle and the responses to forced swim and novelty stress at different stages of exercise. Exercise for 1 d, 2 d, or 1 wk did not affect baseline circadian and ultradian pulse parameters or stress-induced hippocampal free corticosterone concentrations suggesting that acute or short-term periods of exercise do not affect baseline and stress-induced hormone levels. Baseline hormone parameters in 4 wk exercised rats, however, showed significantly increased pulse amplitudes (+108%) and mean free corticosterone levels (+42%) between 1500 and 2100 h but not between 0900 and 1500 h. Surprisingly, although our previous work showed substantial changes in stress-evoked plasma (total) corticosterone responses in long-term exercised animals, no differences in stress-induced hippocampal free hormone responses could be observed between exercised and sedentary animals. This lack of differences was not caused by compensatory changes in plasma corticosteroid-binding-globulin binding levels in exercising rats. Thus, long-term exercising rats show anticipatory increases in glucocorticoid output before the start of the active phase. These rats also reveal the putative existence of a containment mechanism preventing overexposure of the brain to glucocorticoid hormones.

24-Dehydrocholesterol Reductase/Seladin-1: A Key Protein Differentially Involved in Adrenocorticotropin Effects Observed in Human and Rat Adrenal Cortex

Battista, M.-C., Roberge, C., Martinez, A., Gallo-Payet, N. — Fri, 21 Aug 2009 10:03:36 PDT

DHCR24 (24-dehydrocholesterol reductase), or seladin-1, is one of the most expressed genes in the adrenal gland. Because the rat and human adult adrenal cortex differ in their respective functional properties, the aim of the present study was to verify whether seladin-1 may be differentially involved in basal and ACTH-stimulated steroidogenesis and oxidative stress management. Seladin-1 expression was predominantly observed in both human and rat zona fasciculata, with a predominant cytoplasmic localization in human cells and a nucleo-cytoplasmic distribution in rat cells. In human fasciculata cells, localization of the protein was primarily associated with the endoplasmic reticulum. Although its expression was increased by ACTH, its intracellular localization was not altered by ACTH treatment (10 nm) or by the seladin-1 inhibitor U18666A (75 nm). Preincubation with U18666A did not modify the ACTH-induced increase in cortisol secretion but abolished the ACTH-induced increase in dehydroepiandrosterone secretion. In rat fasciculata cells, ACTH induced a massive redistribution of seladin-1 from the cytoplasm (cis-Golgi apparatus) to the nucleus, which was inhibited by preincubation with U18666A. Preincubation with U18666A also decreased ACTH-induced seladin-1 and 11β-hydroxylase protein expression as well as corticosterone production, increased ACTH-induced ROS production but decreased ACTH-induced expression of the detoxifying protein aldo-ketoreductase 1b7. Thus, protection against acutely elevated ACTH-induced oxidative stress in rat fasciculata cells is correlated with nuclear relocalization of seladin-1 and its effects on cellular detoxifying machinery. Altogether, these results indicate that seladin-1 expression and intracellular localization are correlated with both the intensity and nature of ACTH-induced steroidogenesis and resultant oxidative stress.

Soluble Corticotropin-Releasing Hormone Receptor 2{alpha} Splice Variant Is Efficiently Translated But Not Trafficked for Secretion

Evans, R. T., Seasholtz, A. F. — Fri, 21 Aug 2009 10:03:36 PDT

CRH directs the physiological and behavioral responses to stress. Its activity is mediated by CRH receptors (CRH-R) 1 and 2 and modulated by the CRH-binding protein. Aberrant regulation of this system has been associated with anxiety disorders and major depression, demonstrating the importance of understanding the regulation of CRH activity. An mRNA splice variant of CRH-R2 (sCRH-R2) was recently identified that encodes the receptor’s ligand-binding extracellular domain but terminates before the transmembrane domains. It was therefore predicted to serve as a secreted decoy receptor, mimicking the ability of CRH-binding protein to sequester free CRH. Although the splice variant contains a premature termination codon, predicting its degradation by nonsense-mediated RNA decay, cycloheximide experiments and polysome profiles demonstrated that sCRH-R2 mRNA escaped this regulation and was efficiently translated. However, the resulting protein was unable to serve as a decoy receptor because it failed to traffic for secretion because of an ineffective signal peptide and was ultimately subjected to proteosomal degradation. Several other truncated splice variants of G protein-coupled transmembrane receptors regulate the amount of full-length receptor expression through dimerization and misrouting; however, receptor binding assays and immunofluorescence of cells cotransfected with sCRH-R2 and CRH-R2 or CRH-R1 indicated that sCRH-R2 protein does not alter trafficking or binding of full-length CRH-R. Although sCRH-R2 protein does not appear to function as an intracellular or extracellular decoy receptor, the regulated unproductive splicing of CRH-R2 pre-mRNA to sCRH-R2 may selectively alter the cellular levels of full-length CRH-R2 mRNA and hence functional CRH-R2 receptor levels.

Male-Biased Effects of Gonadotropin-Releasing Hormone Neuron-Specific Deletion of the Phosphoinositide 3-Kinase Regulatory Subunit p85{alpha} on the Reproductive Axis

Acosta-Martinez, M., Luo, J., Elias, C., Wolfe, A., Levine, J. E. — Fri, 21 Aug 2009 10:03:36 PDT

GnRH neurosecretion is subject to regulation by insulin, IGF-I, leptin, and other neuroendocrine modulators whose effects may be conveyed by activation of phosphoinositide 3-kinase (PI3K)-mediated pathways. It is not known, however, whether any of these regulatory actions are exerted directly, via activation of PI3K in GnRH neurons, or whether they are primarily conveyed via effects on afferent circuitries governing GnRH neurosecretion. To investigate the role of PI3K signaling in GnRH neurons, we used conditional gene targeting to ablate expression of the major PI3K regulatory subunit, p85, in GnRH neurons. Combined in situ hybridization and immunohistochemistry confirmed reduction of p85 mRNA expression in GnRH neurons of GnRH-p85 knockout (KO) animals. Females of both genotypes exhibited estrous cyclicity and had comparable serum LH, estradiol-17β, and FSH levels. In male GnRH-p85KO mice, serum LH, testosterone, and sperm counts were significantly reduced compared with wild type. To investigate the role of the other major regulatory subunit, p85β, on the direct control of GnRH neuronal function, we generated mice with a GnRH-neuron-specific p85 deletion on a global βKO background. No additional reproductive effects in male or female mice were found, suggesting that p85β does not substitute p85 activity toward PI3K function in GnRH neurons. Our results suggest that p85, and thus PI3K activity, participates in the control of GnRH neuronal activity in male mice. The sex-specific phenotype in these mice raises the possibility that PI3K activation during early development may establish sex differences in GnRH neuronal function.

Estradiol-17{beta} Inhibits Gonadotropin-Releasing Hormone-Induced Ca2+ in Gonadotropes to Regulate Negative Feedback on Luteinizing Hormone Release

Fri, 21 Aug 2009 10:03:36 PDT

In pituitary gonadotropes, estrogens have biphasic actions to cause an initial negative feedback followed by a positive feedback on LH secretion, but the mechanisms involved are not clearly understood. To investigate the feedback effects of estrogen, we used mixed ovine pituitary cell cultures (48–72 h), which were treated with 10^–9 m estradiol-17β (E₂) or vehicle followed by a pulse of 10^–9 m GnRH. Medium was collected for LH assay and cells extracted to determine activation of MAPK (phosphorylated ERK-1/2). E₂ treatment for 5 min reduced GnRH-induced LH release and caused phosphorylation of ERK-1/2. E₂ alone also caused phosphorylation of ERK-1/2, similar to the response evoked by GnRH alone. GnRH increased cytoplasmic intracellular free calcium concentration ([Ca²⁺]_i) and this was abolished by 2 min pretreatment with E₂ or E-bovine serum albumen conjugate. Blockade of Ca²⁺ channels with nifedipine had no effect on the initial peak of GnRH-induced increase in [Ca²⁺]_i but reduced its duration by 27 ± 6%. Depletion of intracellular Ca²⁺ stores with thapsigargin prevented GnRH-induced increase in [Ca²⁺]_i. Thapsigargin (10^–7 m) or nifedipine (10^–5 m) pretreatment (15 min) of cells lowered GnRH-induced LH secretion by 30 ± 6 and 50% ± 4%, respectively. We conclude that inhibition of the GnRH-induced increase in [Ca²⁺]_i in gonadotropes by E₂ is a likely mechanism for the negative feedback effect of E₂ on LH secretion involving a rapid nongenomic effect of E₂. Activation of the MAPK pathway by E₂ may be the mechanism for the time-delayed positive feedback effect on LH secretion at the level of the gonadotrope.

Salt-Inducible Kinase 1 in the Rat Pinealocyte: Adrenergic Regulation and Role in Arylalkylamine N-Acetyltransferase Gene Transcription

Kanyo, R., Price, D. M., Chik, C. L., Ho, A. K. — Fri, 21 Aug 2009 10:03:36 PDT

The recognition of the basic leucine zipper domain in the regulation of transcriptional activity of cAMP response element-binding protein by salt-inducible kinase (SIK) prompted our investigation of the regulatory role of this kinase in the induction of Aa-nat and other cAMP-regulated genes in the rat pineal gland. Here we report Sik1 expression was induced by norepinephrine (NE) in rat pinealocytes primarily through activation of β-adrenergic receptors, with a minor contribution from activation of -adrenergic receptors. Treatments with dibutyryl cAMP, and to a lesser extent, agents that elevate intracellular Ca²⁺ mimicked the effect of NE on Sik1 expression. In parallel to the results of the pineal cell culture studies, a marked nocturnal induction of Sik1 transcription was found in whole-animal studies. Knockdown of Sik1 by short hairpin RNA amplified the NE-stimulated Aa-nat transcription and other adrenergic-regulated genes, including Mapk phosphatase 1, inducible cAMP repressor, and type 2 iodothyronine deiodinase in a time-dependent manner. In contrast, overexpressing Sik1 had an inhibitory effect on the NE induction of Aa-nat and other adrenergic-regulated genes. Together, our results indicate that the adrenergic induction of Sik1 in the rat pineal gland is primarily through the β-adrenergic receptor -> protein kinase A pathway. SIK1 appears to function as part of an endogenous repressive mechanism that regulates the peak and indirectly the duration of expression of Aa-nat and other cAMP-regulated genes. These findings support a role for SIK1 in framing the temporal expression profile of Aa-nat and other adrenergic-regulated genes in the rat pineal gland.

Regulation of Food Intake and Gonadotropin-Releasing Hormone/Luteinizing Hormone during Lactation: Role of Insulin and Leptin

Xu, J., Kirigiti, M. A., Grove, K. L., Smith, M. S. — Fri, 21 Aug 2009 10:03:36 PDT

Negative energy balance during lactation is reflected by low levels of insulin and leptin and is associated with chronic hyperphagia and suppressed GnRH/LH activity. We studied whether restoration of insulin and/or leptin to physiological levels would reverse the lactation-associated hyperphagia, changes in hypothalamic neuropeptide expression [increased neuropeptide Y (NPY) and agouti-related protein (AGRP) and decreased proopiomelanocortin (POMC), kisspeptin (Kiss1), and neurokinin B (NKB)] and suppression of LH. Ovariectomized lactating rats (eight pups) were treated for 48 h with sc minipumps containing saline, human insulin, or rat leptin. The arcuate nucleus (ARH) was analyzed for NPY, AGRP, POMC, Kiss1, and NKB mRNA expression; the dorsal medial hypothalamus (DMH) was analyzed for NPY mRNA. Insulin replacement reversed the increase in ARH NPY/AGRP mRNAs, partially recovered POMC, but had no effect on recovering Kiss1/NKB. Leptin replacement only affected POMC, which was fully recovered. Insulin/leptin dual replacement had similar effects as insulin replacement alone but with a slight increase in Kiss1/NKB. The lactation-induced increase in DMH NPY was unchanged after treatments. Restoration of insulin and/or leptin had no effect on food intake, body weight, serum glucose or serum LH. These results suggest that the negative energy balance of lactation is not required for the hyperphagic drive, although it is involved in the orexigenic changes in the ARH. The chronic hyperphagia of lactation is most likely sustained by the induction of NPY in the DMH. The negative energy balance also does not appear to be a necessary prerequisite for the suppression of GnRH/LH activity.

Epigenetic Control of Sexual Differentiation of the Bed Nucleus of the Stria Terminalis

Murray, E. K., Hien, A., de Vries, G. J., Forger, N. G. — Fri, 21 Aug 2009 10:03:36 PDT

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is larger in volume and contains more cells in male than female mice. These sex differences depend on testosterone and arise from a higher rate of cell death during early postnatal life in females. There is a delay of several days between the testosterone surge at birth and sexually dimorphic cell death in the BNSTp, suggesting that epigenetic mechanisms may be involved. We tested the hypothesis that chromatin remodeling plays a role in sexual differentiation of the BNSTp by manipulating the balance between histone acetylation and deacetylation using a histone deacetylase inhibitor. In the first experiment, a single injection of valproic acid (VPA) on the day of birth increased acetylation of histone H3 in the brain 24 h later. Next, males, females, and females treated neonatally with testosterone were administered VPA or saline on postnatal d 1 and 2 and killed at 21 d of age. VPA treatment did not influence volume or cell number of the BNSTp in control females but significantly reduced both parameters in males and testosterone-treated females. As a result, the sex differences were eliminated. VPA did not affect volume or cell number in the suprachiasmatic nucleus or the anterodorsal nucleus of the thalamus, which also did not differ between males and females. These findings suggest that a disruption in histone deacetylation may lead to long-term alterations in gene expression that block the masculinizing actions of testosterone in the BNSTp.

Transitional Versus Surgical Menopause in a Rodent Model: Etiology of Ovarian Hormone Loss Impacts Memory and the Acetylcholine System

Fri, 21 Aug 2009 10:03:36 PDT

Clinical research suggests that type of ovarian hormone loss at menopause influences cognition. Until recently ovariectomy (OVX) has been the primary rodent model to examine effects of ovarian hormone loss on cognition. This model limits evaluations to abrupt and complete ovarian hormone loss, modeling less than 13% of women who receive surgical menopause. The majority of women do not have their ovaries surgically removed and undergo transitional hormone loss via ovarian follicular depletion. 4-Vinylcyclohexene-diepoxide (VCD) produces gradual ovarian follicular depletion in the rodent, with hormone profiles more similar to naturally menopausal women vs. OVX. We directly compared VCD and OVX models to examine whether type of hormone loss (transitional vs. surgical) impacted cognition as assessed on a maze battery as well as the cholinergic system tested via scopolamine mnemonic challenge and brain acetylcholinesterase activity. Middle-aged rats received either sham surgery, OVX surgery, VCD, or VCD then OVX to assess effects of removal of residual ovarian output after transitional menopause and follicular depletion. VCD-induced transitional menopause impaired learning of a spatial recent memory task; surgical removal of residual ovarian hormones by OVX abolished this negative effect of transitional menopause. Furthermore, transitional menopause before OVX was better for memory than an abrupt loss of hormones via OVX only. Surgical ovarian hormone loss, regardless of menopause history, increased hippocampal acetylcholinesterase activity. Circulating gonadotropin and androstenedione levels were related to cognitive competence. Collectively, findings suggest that in the rat, initiation of transitional menopause before surgical ovary removal can benefit mnemonic function and could obviate some negative cognitive consequences of surgical menopause alone.

Retinoic Acid Stimulates 17{beta}-Estradiol and Testosterone Synthesis in Rat Hippocampal Slice Cultures

Fri, 21 Aug 2009 10:03:36 PDT

The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17β-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P450₁₇, P450 aromatase and estrogen receptor-β in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P450₁₇ were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P450₁₇ and a 2-fold increase in de novo synthesis of 17β-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P450₁₇ transcription via retinoid X receptor signaling.

Chronic Intermittent Hypoxia Induces 11{beta}-Hydroxysteroid Dehydrogenase in Rat Heart

Fri, 21 Aug 2009 10:03:36 PDT

Corticosteroids are known to not only regulate electrolyte homeostasis but also play a role in the cardiovascular system, including myocardial remodeling. Because transgenic mice that overexpress 11β-hydroxysteroid dehydrogenase (11HSD) type 2 in cardiomyocytes have been shown to spontaneously develop cardiac hypertrophy and fibrosis, we investigated whether changes in the cardiac metabolism of glucocorticoids accompany remodeling of the heart under physiological conditions. In the present study, glucocorticoid metabolism and 11HSD2 were explored in the hearts of rats exposed to chronic intermittent hypobaric hypoxia (CIH), which induces hypertrophy and fibrosis of the right and less of the left ventricle. We first demonstrated that adaptation to CIH led to a significant increase in 11HSD2 transcript levels and activity in the myocardium. In contrast, neither 11HSD1 activity and mRNA level nor the abundance of mineralocorticoid and glucocorticoid receptor mRNA were up-regulated. The adaptation to CIH also led to an increase of 11HSD2 mRNA in isolated cardiomyocytes, whereas 11HSD1, glucocorticoid receptor, and mineralocorticoid receptor mRNA levels were not changed in comparison with the cardiomyocytes of control normoxic rats. The changes in cardiac metabolism of glucocorticoids were accompanied by inflammatory responses. The expression levels of the proinflammatory markers cyclooxygenase-2 and osteopontin were significantly increased in both the myocardium and the cardiomyocytes isolated from rats exposed to CIH. These findings suggest that myocardial remodeling induced by CIH is associated with the up-regulation of cardiac 11HSD2. Consequently, local metabolism of glucocorticoids could indeed play a role in cardiac hypertrophy and fibrosis.

The p38 Mitogen-Activated Protein Kinase Regulates 11{beta}-Hydroxysteroid Dehydrogenase Type 2 (11{beta}-HSD2) Expression in Human Trophoblast Cells through Modulation of 11{beta}-HSD2 Messenger Ribonucleic Acid Stability

Sharma, A., Guan, H., Yang, K. — Fri, 21 Aug 2009 10:03:36 PDT

The placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2; encoded by the HSD11B2 gene) has emerged as a key player in controlling fetal development, but its regulation is incompletely understood. Here we identified p38 MAPK as an important regulator of placental 11β-HSD2. We showed that inhibition of p38 MAPK with the pharmacological inhibitor SB202190 led to an approximately 50% reduction in 11β-HSD2 activity, protein, and mRNA in primary human placental trophoblast cells. Furthermore, the effect of SB202190 was confirmed by the use of two additional p38 inhibitors, SB203580 and SB220025. In addition, SB202190 decreased the half-life of 11β-HSD2 mRNA without altering the HSD11B2 promoter activity, indicating that p38 MAPK regulates placental 11β-HSD2 expression through modulation of 11β-HSD2 mRNA stability. Importantly, small interfering RNA-mediated knockdown of p38 caused a 50% reduction in 11β-HSD2 activity, suggesting that p38 is the primary p38 isoform involved. Taken together, these findings suggest a novel pathway controlling placental 11β-HSD2 expression resulting from the activation of p38 MAPK. Given that p38 is abundantly expressed in the human placenta in which its function is largely unknown, our present study also reveals 11β-HSD2 as an important target through which p38 may regulate human placental function and consequently fetal growth and development.

Rosiglitazone Increases the Expression of Peroxisome Proliferator-Activated Receptor-{gamma} Target Genes in Adipose Tissue, Liver, and Skeletal Muscle in the Sheep Fetus in Late Gestation

Muhlhausler, B. S., Morrison, J. L., McMillen, I. C. — Fri, 21 Aug 2009 10:03:36 PDT

Exposure to maternal overnutrition increases the expression of peroxisome proliferator-activated receptor- (PPAR) in adipose tissue before birth, and it has been proposed that the precocial activation of PPAR target genes may lead to increased fat deposition in postnatal life. In this study, we determined the effect of intrafetal administration of a PPAR agonist, rosiglitazone, on PPAR target gene expression in fetal adipose tissue as well indirect actions of rosiglitazone on fetal liver and skeletal muscle. Osmotic pumps containing rosiglitazone (n = 7) or vehicle (15% ethanol, n = 7) were implanted into fetuses at 123–126 d gestation (term = 150 ± 3 d gestation). At 137–141 d gestation, tissues were collected and mRNA expression of PPAR, lipoprotein lipase (LPL), adiponectin, and glycerol-3-phosphate dehydrogenase (G3PDH) in adipose tissue, PPAR and PPAR-coactivator 1 (PGC1) in liver and muscle and phosphoenolpyruvate carboxykinase (PEPCK) in liver determined by quantitative real-time RT-PCR. Plasma insulin concentrations were lower in rosiglitazone-treated fetuses (P < 0.02). Rosiglitazone treatment resulted in increased expression of LPL and adiponectin mRNA (P < 0.01) in fetal adipose tissue. The expression of PPAR mRNA in liver (P < 0.05) and PGC1 mRNA (P < 0.02) in skeletal muscle were also increased by rosiglitazone treatment. Rosiglitazone treatment increased expression of PPAR target genes within fetal adipose tissue and also had direct or indirect actions on the fetal liver and muscle. The effects of activating PPAR in fetal adipose tissue mimic those induced by prenatal overnutrition, and it is therefore possible that activation of PPAR may be the initiating mechanism in the pathway from prenatal overnutrition to postnatal obesity.

Insulin-Like Growth Factor Binding Protein-1 in the Ruminant Uterus: Potential Endometrial Marker and Regulator of Conceptus Elongation

Fri, 21 Aug 2009 10:03:36 PDT

Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon- (IFNT), the pregnancy recognition signal that maintains ovarian progesterone (P4) production. These studies determined temporal and spatial alterations in IGF binding protein (IGFBP)-1 and IGFBP3 in the ovine and bovine uterus; effects of P4 and IFNT on their expression in the ovine uterus; and effects of IGFBP1 on ovine trophectoderm cell proliferation, migration, and attachment. IGFBP1 and IGFBP3 were studied because they are the only IGFBPs specifically expressed by the endometrial luminal epithelia in sheep. In sheep, IGFBP1 and IGFBP3 expression was coordinate with the period of conceptus elongation, whereas only IGFBP1 expression was coordinate with conceptus elongation in cattle. IGFBP1 mRNA in the ovine endometria was between 5- and 29-fold more abundant between d 12 and 16 of pregnancy compared with the estrous cycle and greater on d 16 of pregnancy than nonpregnancy in the bovine uterus. In sheep, P4 induced and IFNT stimulated expression of IGFBP1 but not IGFBP3; however, the effect of IFNT did not mimic the abundant increase observed in pregnant ewes. Therefore, IGFBP1 expression in the endometrium is regulated by another factor from the conceptus. IGFBP1 did not affect the proliferation of ovine trophectoderm cells in vitro but did stimulate their migration and mediate their attachment. These studies reveal that IGFBP1 is a common endometrial marker of conceptus elongation in sheep and cattle and most likely regulates conceptus elongation by stimulating migration and attachment of the trophectoderm.

Up-Regulation of {alpha}5-Integrin by E-Cadherin Loss in Hypoxia and Its Key Role in the Migration of Extravillous Trophoblast Cells during Early Implantation

Fri, 21 Aug 2009 10:03:36 PDT

During early pregnancy, cytotrophoblast cells differentiate into extravillous trophoblast (EVT) cells and invade the uterine spiral arteries. This physiological process is essential for the development of maternal-fetal circulation. Because EVT cells are exposed to a low-oxygen environment during this process, we investigated the role of hypoxia in the mechanism that regulates the invasive behavior of EVT cells. Real-time PCR and immunofluorescent analysis were performed to investigate how hypoxia influences the expression of E-cadherin in villous explants cultures and in trophoblast-derived BeWo cells. We determined that hypoxia induced E-cadherin down-regulation through Snail up-regulation in villous explant cultures. The influence of E-cadherin loss was examined by analyzing the expression of ₅-integrin and phosphorylated focal adhesion kinase (FAK) by Western blot and evaluating trophoblast invasion using a matrigel invasion assay. E-cadherin loss induced the up-regulation of ₅-integrin, which leads to the tyrosine phosphorylation of FAK, resulting in an increase in the invasive activity of EVT cells. An ₅-integrin neutralizing antibody inhibited the invasion of EVT cells by attenuating FAK tyrosine phosphorylation. Immunohistochemical analysis using clinical placental bed biopsies revealed that ₅-integrin was up-regulated and FAK tyrosine phosphorylated (Try⁸⁶¹) as EVT cells invade the uterine myometrium, whereas E-cadherin expression was down-regulated. These results suggest that ₅-integrin up-regulation induced by E-cadherin loss under hypoxia has a crucial role in regulating the migration of EVT cells. This finding should help us reach a better understanding of the pathogenesis of critical gestational diseases, such as preeclampsia.

The Uterine Placental Bed Renin-Angiotensin System in Normal and Preeclamptic Pregnancy

Fri, 21 Aug 2009 10:03:36 PDT

Previously, we demonstrated activation of the renin-angiotensin system in the fetal placental chorionic villi, but it is unknown whether the immediately adjacent area of the maternal uterine placental bed is regulated similarly. This study measured angiotensin peptides, renin-angiotensin system component mRNAs, and receptor binding in the fundus from nonpregnant subjects (n = 19) and in the uterine placental bed from normal (n = 20) and preeclamptic (n = 14) subjects. In the uterine placental bed from normal pregnant women, angiotensin II peptide levels and angiotensinogen, angiotensin-converting enzyme, angiotensin receptor type 1 (AT₁), AT₂, and Mas mRNA expression were lower as compared with the nonpregnant subjects. In preeclamptic uterine placental bed, angiotensin II peptide levels and renin and angiotensin-converting enzyme mRNA expression were significantly higher than normal pregnant subjects. The AT₂ receptor was the predominant receptor subtype in the nonpregnant fundus, whereas all angiotensin receptor binding was undetectable in normal and preeclamptic pregnant uterine placental bed compared with nonpregnant fundus. These findings suggest that the maternal uterine placental bed may play an endocrine role by producing angiotensin II, which acts in the adjacent placenta to vasoconstrict fetal chorionic villi vessels where we have shown previously that AT₁ receptors predominate. This would lead to decreased maternal-fetal oxygen exchange and fetal nutrition, a known characteristic of preeclampsia.

Chorionic Gonadotropin Regulates Prostaglandin E Synthase via a Phosphatidylinositol 3-Kinase-Extracellular Regulatory Kinase Pathway in a Human Endometrial Epithelial Cell Line: Implications for Endometrial Responses for Embryo Implantation

Banerjee, P., Sapru, K., Strakova, Z., Fazleabas, A. T. — Fri, 21 Aug 2009 10:03:36 PDT

Successful implantation necessitates modulation of the uterine environment by the embryo for a specific period of time during the menstrual cycle. Infusion of chorionic gonadotropin (CG) into the oviducts of baboons to mimic embryo transit induces a myriad of morphological, biochemical, and molecular changes in the endometrium. Endometrial epithelial cells from both baboons and humans when stimulated by CG in vitro, activates a cAMP-independent MAPK pathway leading to prostaglandin E₂ (PGE₂) synthesis. This study shows that in the human endometrial cell line, HES, CG, acting via its G-protein coupled receptor, phosphorylates protein kinase B, c-Raf, and ERK1/2 in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Furthermore, ERK1/2 phosphorylation is independent of the signaling paradigms of G_s, G_I, and epidermal growth factor receptor (EGFR) transactivation, typical of gonadal cells, indicating an alternative signaling pattern in the endometrium. After phosphorylation by CG, ERK1/2 translocates to the nucleus in a time-dependent manner. Downstream of ERK1/2, CG activates the nuclear transcription factor, Elk1, also in a PI3K-MAPK-dependent manner. Lastly, we show that in HES cells, this pathway regulates the expression of the microsomal enzyme PGE₂ synthase (mPTGES), a terminal prostanoid synthase responsible for PGE₂ synthesis. CG regulates the mPTGES promoter and also induces mPTGES synthesis in HES cells via the PI3K-ERK1/2 pathway. We suggest that this alternative PI3K-ERK-Elk pathway activated by CG regulates prostaglandin production by the endometrial epithelium and serves as an early trigger to prepare the endometrium for implantation.

Timing of the Ovarian Circadian Clock Is Regulated by Gonadotropins

Yoshikawa, T., Sellix, M., Pezuk, P., Menaker, M. — Fri, 21 Aug 2009 10:03:36 PDT

The timing of ovulation is critically important to the success of reproduction. Current thinking attributes the timing of ovulation to LH secretion by the pituitary, itself timed by signals from the hypothalamus. The discovery of an internal circadian timer in the ovary raises the possibility that ovulation is in fact timed by an interaction between clocks in the hypothalamus/pituitary and those in the ovary. We asked whether ovarian clocks were influenced by signals from the brain and pituitary. Ovaries of Period1-luciferase transgenic rats display circadian rhythms in vitro. To determine whether the phase of these rhythms is set by neural or endocrine signals, we surgically denervated or heterotopically transplanted ovaries with or without encapsulation in dialysis membranes. Animals’ light-dark cycles were phase advanced or delayed 6 h, and the resetting of the ovarian clock was tracked by culturing ovaries at intervals over the next 12 d. Resetting trajectories of control, surgically denervated, and encapsulated ovaries were similar, demonstrating that endocrine signals are sufficient to transmit phase information to the ovary. We next evaluated LH and FSH as potential endocrine signals. Using the phase of Per1-luc expression in granulosa cell cultures, we demonstrated that both of these pituitary hormones caused large phase shifts when applied to the cultured cells. We hypothesize that the ovarian circadian clock is entrained by hormonal signals from the pituitary and that ovulation depends, in part, on the phase in the ovarian circadian cycle at which these signals occur.

Inactivation of Insulin-Like Factor 6 Disrupts the Progression of Spermatogenesis at Late Meiotic Prophase

Fri, 21 Aug 2009 10:03:36 PDT

Insulin-like factor 6 (INSL6), a member of the insulin-like superfamily, is predominantly expressed in male germ cells. Expression of the Insl6 is first detected in mouse testis at postnatal d 15 when the first wave of spermatogenesis progresses to pachytene spermatocytes. To elucidate the role of INSL6 in germ cell development, we generated Insl6-deficient mice. The majority of the Insl6-deficient males on a hybrid genetic background exhibited impaired fertility, whereas females were fertile. The number of mature sperm and sperm motility were drastically reduced in the epididymis. The reduced sperm count could be due to apoptotic death of a significant number of developing germ cells. Analysis of germ cell development during the juvenile life showed an arrest of the first wave of spermatogenesis in late meiotic prophase. RNA analysis revealed a significant decrease in expression of late meiotic- and postmeiotic-specific marker genes, whereas expression of early meiotic-specific genes remains unaffected in the Insl6^–/– testes. These results demonstrate that INSL6 is required for the progression of spermatogenesis.

Adiponectin Attenuation of Endocrine Function within Human Term Trophoblast Cells

McDonald, E. A., Wolfe, M. W. — Fri, 21 Aug 2009 10:03:36 PDT

The hormone adiponectin has been shown to be important in maintaining insulin sensitivity throughout the body, whereas potential effects on the placenta have not been assessed. Pregnancy constitutes a unique physiological environment in which metabolism has a profound effect on the health of both the mother and the developing fetus. It is imperative that a delicate balance in glucose delivery be maintained between maternal tissues and the fetal/placental unit. Adiponectin’s role in regulating peripheral insulin responsiveness suggests it may be a factor in maintaining this balance during gestation as well. Examination of human cytotrophoblast cells revealed that mRNA for both adiponectin receptors, adipoR1 and adipoR2, are abundantly expressed at term. We were, however, unable to reliably detect mRNA for adiponectin in primary cytotrophoblasts. Expression of both receptors was maintained after induction of syncytium formation by exogenous epidermal growth factor treatment. Treatment of cytotrophoblasts with adiponectin resulted in a significant drop, as assessed by quantitative RT-PCR, in expression for a number of genes involved in the endocrine function of the placenta, including the chorionic gonadotropin subunits, placental lactogen, and some steroidogenic enzymes. Immunofluorescent staining for connexin 43 and desmoplakin in primary trophoblasts revealed that adiponectin does not inhibit syncytialization of trophoblast cells in culture. Taken together, these data describe a novel role for maternal adiponectin in regulating the placental environment. Determination of the effects of such adipokines on the maternal-fetal interface is increasingly important, because the incidence of pregnancies complicated by gestational diabetes remains a significant health problem in developed countries.

Targeted Gene Knockdown in Zebrafish Reveals Distinct Intraembryonic Functions for Insulin-Like Growth Factor II Signaling

White, Y. A. R., Kyle, J. T., Wood, A. W. — Fri, 21 Aug 2009 10:03:36 PDT

IGF-II is the predominant IGF ligand regulating prenatal growth in all vertebrates, including humans, but its central role in placental development has confounded efforts to fully elucidate its functions within the embryo. Here we use a nonplacental model vertebrate (zebrafish) to interrogate the intraembryonic functions of IGF-II signaling. The zebrafish genome contains two coorthologs of mammalian IGF2 (igf2a, igf2b), which exhibit distinct patterns of expression during embryogenesis. Expression of igf2a mRNA is restricted to the notochord, primarily during segmentation/neurulation. By contrast, igf2b mRNA is expressed in midline tissues adjacent to the notochord, with additional sites of expression in the ventral forebrain, and the pronephros. To identify their intraembryonic functions, we suppressed the expression of each gene with morpholino oligonucleotides. Knockdown of igf2a led to defects in dorsal midline development, characterized by delayed segmentation, notochord undulations, and ventral curvature. Similarly, suppression of igf2b led to defects in dorsal midline development but also induced ectopic fusion of the nephron primordia, and defects in ventral forebrain development. Subsequent onset of severe body edema in igf2b, but not igf2a morphants, further suggested a distinct role for igf2b in development of the embryonic kidney. Simultaneous knockdown of both genes increased the severity of dorsal midline defects, confirming a conserved role for both genes in dorsal midline development. Collectively, these data provide evidence that the zebrafish orthologs of IGF2 function in dorsal midline development during segmentation/neurulation, whereas one paralog, igf2b, has evolved additional, distinct functions during subsequent organogenesis.

Activator Protein-2 Impairs the Invasion of a Human Extravillous Trophoblast Cell Line

Fri, 21 Aug 2009 10:03:37 PDT

The reduced migration/invasion of extravillous trophoblasts (EVTs) is a key feature of the genesis of preeclampsia. We and others previously reported that transcriptional factors activator protein-2 (AP-2) and AP-2 act as suppressors of tumor invasion. The present study examined the expressions of AP-2 and AP-2 in preeclamptic placenta vs. control placenta and investigated their effect on the function of EVTs. The expressions of AP-2 and AP-2 were elevated in the preeclamptic placentas in comparison with the gestational age-matched control placentas. Their expressions also increased in EVTs of the preeclamptic placentas. Thereafter, we transfected AP-2 or AP-2 into human EVT cell line, HTR-8/SVneo. The overexpression of AP-2 or AP-2 decreased the migratory and invasive abilities in HTR-8/SVneo cells. This was followed by the reduction of protease activated receptor-1 and matrix metalloproteinases and a significant induction of plasminogen activator inhibitor-1 and the tissue inhibitor of metalloproteinase-1. AP-2 and AP-2 were weakly expressed in the cultured EVTs and HTR-8/SVneo cells, whereas they were induced by TNF-, which increases in preeclamptic placenta and impairs trophoblast invasion. In the presence of TNF-, the invasion of the HTR-8/SVneo cells was partially restored by a blocking of AP-2 induction using small interfering RNA of AP-2. The present data suggest that AP-2 may suppress trophoblast migration and invasion, thus leading to a shallow placentation in preeclampsia.

The Notch Target Gene Hes1 Regulates Cell Cycle Inhibitor Expression in the Developing Pituitary

Monahan, P., Rybak, S., Raetzman, L. T. — Fri, 21 Aug 2009 10:03:37 PDT

The pituitary is an endocrine gland responsible for the release of hormones, which regulate growth, metabolism, and reproduction. Diseases such as hypopituitarism or pituitary adenomas are able to disrupt pituitary function leading to suboptimal function of the entire endocrine system. Growth of the pituitary during development and adulthood is a tightly regulated process. Hairy and enhancer of split (HES1), a transcription factor whose expression is initiated by the Notch signaling pathway, is a repressor of cell cycle inhibitors. We hypothesize that with the loss of Hes1, pituitary progenitors are no longer maintained in a proliferative state, choosing instead to exit the cell cycle. To test this hypothesis, we examined the expression of cell cycle regulators in wild-type and Hes1-deficient pituitaries. Our studies indicate that in early pituitary development [embryonic day (e) 10.5], cells contained in the Rathke’s pouch of Hes1 mutants have decreased proliferation, indicated by changes in phosphohistone H3 expression. Furthermore, pituitaries lacking Hes1 have increased cell cycle exit, shown by significant increases in the cyclin-dependent kinase inhibitors, p27 and p57, from e10.5 to e14.5. Additionally, Hes1 mutant pituitaries have ectopic expression of p21 in Rathke’s pouch progenitors, an area coincident with increased cell death. These observations taken together indicate a role for HES1 in the control of cell cycle exit and in mediating the balance between proliferation and differentiation, allowing for the properly timed emergence of hormone secreting cell types.

Elevated Levels of Insulin-Like Growth Factor (IGF)-I in Serum Rescue the Severe Growth Retardation of IGF-I Null Mice

Wu, Y., Sun, H., Yakar, S., LeRoith, D. — Fri, 21 Aug 2009 10:03:37 PDT

IGF-I plays a vital role in growth and development and acts in an endocrine and an autocrine/paracrine fashion. The purpose of the current study was to clarify whether elevated levels of IGF-I in serum can rescue the severe growth retardation and organ development and function of igf-I null mice. To address that, we overexpressed a rat igf-I transgene specifically in the liver of igf-I null mice. We found that in the total absence of tissue IGF-I, elevated levels of IGF-I in serum can support normal body size at puberty and after puberty but are insufficient to fully support the female reproductive system (evident by irregular estrous cycle, impaired development of ovarian corpus luteum, reduced number of uterine glands and endometrial hypoplasia, all leading to decreased number of pregnancies and litter size). We conclude that most autocrine/paracrine actions of IGF-I that determine organ growth and function can be compensated by elevated levels of endocrine IGF-I. However, in mice, full compensatory responses are evident later in development, suggesting that autocrine/paracrine IGF-I is critical for neonatal development. Furthermore, we show that tissue IGF-I is necessary for the development of the female reproductive system and cannot be compensated by elevated levels of serum IGF-I.

Paracrine Signals from the Mouse Conceptus Are Not Required for the Normal Progression of Decidualization

Herington, J. L., Underwood, T., McConaha, M., Bany, B. M. — Fri, 21 Aug 2009 10:03:37 PDT

The purpose of this study was to determine whether the conceptus directs the formation of a tight- and adherens-dependent permeability barrier formed by the primary decidual zone and normal progression of decidual cell differentiation during embryo implantation. Four artificial models of decidualization were used, some apparently more physiological than others. The results show that both the formation of the permeability barrier and decidual cell differentiation of three of the artificial models were quite different from that of pregnant uteri. One artificial model of decidualization, namely pseudopregnant animals receiving concanavalin A-coated Sepharose bead transfers on d 2.5 of pseudopregnancy, better recapitulated the decidual changes that occur in the pregnant uterus undergoing decidualization. This included the formation of a primary decidual zone-like permeability barrier and decidual growth. This model also exhibited similar temporal changes of the expression of genes involved in decidualization that are markers of decidual cell differentiation. Overall, the results of this study indicate that some models of inducing decidualization artificially produce responses that are more similar to those occurring in the pregnant uterus, whereas others are quite different. More importantly, the results suggest that concanavalin A-coated Sepharose beads can provide an equivalent stimulus as the trophectoderm to cause the formation of the primary decidual zone permeability barrier.

Low Renal Mineralocorticoid Receptor Expression at Birth Contributes to Partial Aldosterone Resistance in Neonates

Fri, 21 Aug 2009 10:03:37 PDT

The human neonatal period is characterized by renal immaturity with impaired capacity to regulate water and sodium homeostasis, resembling partial aldosterone resistance. Because aldosterone effects are mediated by the mineralocorticoid receptor (MR), we postulated that this hormonal unresponsiveness could be related to low MR expression in the distal nephron. We measured aldosterone and renin levels in umbilical cord blood of healthy newborns. We used quantitative real-time PCR and immunohistochemistry to analyze the expression of MR and key players of the mineralocorticoid signaling pathway during human and mouse renal development. High aldosterone and renin levels were found at birth. MR mRNA was detected in mouse kidney at d 16 postcoitum, peaking at d 18 postcoitum, but its expression was surprisingly very low at birth, rising progressively afterward. Similar biphasic temporal expression was observed during human renal embryogenesis, with a transient expression between 15 and 24 wk of gestation but an undetectable immunoreactive MR in late gestational and neonatal kidneys. This cyclic MR expression was tightly correlated with the evolution of the 11β-hydroxysteroid dehydrogenase type 2 and the epithelial sodium channel -subunit. In contrast, glucocorticoid and vasopressin receptors and aquaporin 2 followed a progressive and sustained evolution during renal maturation. Our study provides the first evidence for a low renal MR expression level at birth, despite high aldosterone levels, which could account for compromised postnatal sodium handling. Elucidation of regulatory mechanisms governing MR expression should lead to new strategies for the management of sodium waste in preterms and neonates.

A Unique Rodent Model of Cardiometabolic Risk Associated with the Metabolic Syndrome and Polycystic Ovary Syndrome

Fri, 21 Aug 2009 10:03:37 PDT

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, oligo-/anovulation, and polycystic ovarian morphology and is a complex endocrine disorder that also presents with features of the metabolic syndrome, including obesity, insulin resistance, and dyslipidemia. These latter symptoms form cardiometabolic risk factors predisposing individuals to the development of type 2 diabetes and cardiovascular disease (CVD). To date, animal models to study PCOS in the context of the metabolic syndrome and CVD risk have been lacking. The aim of this study was to investigate the JCR:LA-cp rodent as an animal model of PCOS associated with the metabolic syndrome. Metabolic indices were measured at 6 and 12 wk, and reproductive parameters including ovarian morphology and estrous cyclicity were assessed at 12 wk or adulthood. At 6 wk of age, the cp/cp genotype of the JCR:LA-cp strain developed visceral obesity, insulin resistance, and dyslipidemia (hypertriglyceridemia and hypercholesterolemia) compared with control animals. Serum testosterone concentrations were not significantly different between groups at 6 wk of age. However, at 12 wk, the cp/cp genotype had higher serum testosterone concentrations, compared with control animals, and presented with oligoovulation, a decreased number of corpora lutea, and an increased number of total follicles, in particular atretic and cystic follicles. The cardiometabolic risk factors in the cp/cp animals were exacerbated at 12 wk including obesity, insulin resistance, and dyslipidemia. The results of this study demonstrate that the JCR:LA-cp rodent may be a useful PCOS-like model to study early mechanisms involved in the etiology of cardiometabolic risk factors in the context of both PCOS and the metabolic syndrome.

A Rat Model of Epilepsy in Women: A Tool to Study Physiological Interactions between Endocrine Systems and Seizures

Fri, 21 Aug 2009 10:03:37 PDT

Epilepsy in women is influenced by endocrine status and antiepileptic drugs, but without an animal model, the effects of endocrine variables and antiepileptic drugs cannot be easily dissociated from the influence of epilepsy itself. Animal models have had limited utility because experimentally induced seizures typically result in reproductive failure. This study was conducted to develop an improved animal model. The muscarinic convulsant pilocarpine was used to elicit status epilepticus (SE) in adult female Sprague Dawley rats. The selective estrogen receptor modulator raloxifene was administered 30 min before pilocarpine. An anticonvulsant barbiturate, pentobarbital, was injected 5–10 min after the onset of SE and at least once thereafter to minimize acute convulsions. Mortality, morbidity, estrous cyclicity, and the ultimate success of the procedure (i.e. induction of recurrent, spontaneous seizures) were monitored. The combination of raloxifene and pentobarbital led to significantly improved estrous cyclicity compared with previous methods. Animals treated with raloxifene and pentobarbital became epileptic, as defined by the recurrence of spontaneous convulsions in the weeks after SE. The results of this study provide an improved animal model to examine the interactions between seizures and ovarian hormone secretion. The results also suggest that treatment of SE with raloxifene may benefit women with SE.

Analysis of Hypertrophic Thyrotrophs in Pituitaries of Athyroid Pax8-/- Mice

Mittag, J., Friedrichsen, S., Strube, A., Heuer, H., Bauer, K. — Fri, 21 Aug 2009 10:03:37 PDT

Thyroid hormone is important for pituitary development and maintenance. We previously reported that in the Pax8^–/– mouse model of congenital hypothyroidism, lactotrophs are almost undetectable, whereas the thyrotrophs exhibit hyperplasia and hypertrophy. Because the latter might be caused by an overstimulation of thyrotrophs with TRH, we analyzed TRH-R1^–/–Pax8^–/– double-knockout mice, which miss a functional thyroid gland and the TRH transducing receptor-1 at pituitary target sites. Interestingly, in these double mutants, the hypertrophy and hyperplasia of the thyrotrophs still persist, suggesting that the phenotype is rather a direct consequence of the athyroidism of the animals. The increased expression of TSH in the Pax8^–/– mice was paralleled by a strongly up-regulated expression of deiodinase type 2 (Dio2) in thyrotrophic cells. Moreover, coexpression of TSH and Dio2 could also be demonstrated in the pituitary of wild-type mice, underlining the important role of this enzyme in the negative feedback regulation of TSH by thyroid hormone. As another consequence of the athyroidism in the mutant mice, tyrosine hydroxylase mRNA expression was found to be also highly up-regulated in thyrotrophic cells of the pituitaries from Pax8^–/– mice, whereas the transcript levels in the hypothalamus were not affected. Accordingly, tyrosine hydroxylase protein levels, enzyme activities, and ultimately dopamine concentrations were found to be strongly increased in the pituitaries of Pax8^–/– mice compared with wild-type animals. These findings may explain in part the reduced number of lactotrophs found in the pituitary of athyroid Pax8^–/– mice and suggest a novel paracrine regulatory mechanism of lactotroph activity.

A Thyroid Hormone Analog with Reduced Dependence on the Monocarboxylate Transporter 8 for Tissue Transport

Di Cosmo, C., Liao, X.-H., Dumitrescu, A. M., Weiss, R. E., Refetoff, S. — Fri, 21 Aug 2009 10:03:37 PDT

Mutations of the thyroid hormone (TH) cell membrane transporter MCT8, on chromosome-X, produce severe mental and neurological impairment in men. We generated a Mct8-deficient mouse (Mct8KO) manifesting the human thyroid phenotype. Although these mice have no neurological manifestations, they have decreased brain T₃ content and high deiodinase 2 (D2) activity, reflecting TH deprivation. In contrast and as in serum, liver T₃ content is high, resulting in increased deiodinase 1 (D1), suggesting that in this tissue TH entry is Mct8 independent. We tested the effect of 3,5-diiodothyropropionic acid (DITPA), a TH receptor agonist, for its dependence on Mct8 in Mct8KO and wild-type (Wt) mice tissues. After depletion of endogenous TH, mice were given three different doses of DITPA. Effects were compared with treatment with two doses of l-T₄. As expected, physiological doses of l-T₄ normalized serum TSH, brain D2, and liver D1 in Wt mice but not the Mct8KO mice. The higher dose of T₄ suppressed TSH in the Wt mice, normalized TSH and brain D2 in Mct8KO mice, but produced a thyrotoxic effect on liver D1 in both genotypes. In contrast DITPA produced similar effects on TSH, D2, and D1 in both Wt and Mct8KO mice. The higher dose fully normalized all measurements and other parameters of TH action. Thus, DITPA is relatively MCT8 independent for entry into the brain and corrects the TH deficit in Mct8KO mice without causing thyrotoxic effect in liver. The potential clinical utility of this analog to patients with MCT8 mutations requires further studies.

HHEX-IDE Polymorphism is Associated with Low Birth Weight in Offspring with A Family History of Type 1 Diabetes

Winkler, C., Illig, T., Koczwara, K., Bonifacio, E., Ziegler, A.-G. — Fri, 21 Aug 2009 10:03:37 PDT

Growth Hormone Excess Promotes Breast Cancer Chemoresistance

Fri, 21 Aug 2009 10:03:37 PDT

Evidence for a Specific Defect in Hippocampal Memory in Overt and Subclinical Hypothyroidism

Fri, 21 Aug 2009 10:03:37 PDT

Erratum

Fri, 21 Aug 2009 10:03:37 PDT

Unraveling Androgen Action in Muscle: Genetic Tools Probing Cellular Mechanisms

MacLean, H. E., Handelsman, D. J. — Tue, 21 Jul 2009 10:03:32 PDT

Corticotropin-Releasing Factor, Serotonin, and Sex: Keys to the Castle of Depressive Illness

Koenig, J. I. — Tue, 21 Jul 2009 10:03:32 PDT

Understanding Estrogen Action during Menopause

Iqbal, J., Zaidi, M. — Tue, 21 Jul 2009 10:03:32 PDT

Really Old Hormones Up to New Tricks: Glycoprotein Hormone Subunits May Have Roles in Development

Trudeau, V. L. — Tue, 21 Jul 2009 10:03:32 PDT

The Enhanced Hypercalcemic Response to 20-Epi-1,25-Dihydroxyvitamin D3 Results from a Selective and Prolonged Induction of Intestinal Calcium-Regulating Genes

Zella, L. A., Meyer, M. B., Nerenz, R. D., Pike, J. W. — Tue, 21 Jul 2009 10:03:32 PDT

20-Epi-1,25-dihydroxyvitamin D₃ (20-epi-1,25(OH)₂D₃) is a vitamin D analog that exhibits unique biologic properties. The mechanism(s) responsible for these activities remains unclear. Here we explore the ability of 20-epi-1,25(OH)₂D₃ to induce calcemic responses in mice in vivo and identify a potential mechanism. Surprisingly, the levels of calcemia induced at 24 h after single injections of equivalent doses of 1,25(OH)₂D₃ or 20-epi-1,25(OH)₂D₃ were similar, suggesting that both compounds were equal in both potency and efficacy. This similarity was also observed at genes involved in calcium homeostasis including, S100g (calbindin D9K), Trpv6, Cldn2 (claudin 2), Trpv5, and Tnfsf11 (Rankl) as well as Cyp24a1. Despite this, the activities of the two compounds at 48 h were strikingly different. Thus, whereas the activity of 1,25-dihydroxyvitamin D₃ declined at this time point, the response to 20-epi-1,25(OH)₂D₃ was increased. This unique profile was not due to an exaggerated induction of calcium regulating genes in the intestine, kidney, or bone but to a sustained action on these genes in the intestine. This conclusion was supported by studies using in vivo chromatin immunoprecipitation analysis, which revealed a prolonged presence of vitamin D receptor and RNA polymerase II at the Trpv6 and Cyp24a1 promoters and a sustained increase in histone 4 acetylation in these gene regions as well. We conclude that 20-epi-1,25(OH)₂D₃ displays superagonist properties largely as a result of its duration of action in the intestine. This action is likely due to a decrease in the rate of intestinal-specific degradation of the ligand rather than to an increase in the functional stability of the vitamin D receptor.

Pioglitazone Inhibits Toll-Like Receptor Expression and Activity in Human Monocytes and db/db Mice

Dasu, M. R., Park, S., Devaraj, S., Jialal, I. — Tue, 21 Jul 2009 10:03:33 PDT

Toll-like receptors (TLRs) are key innate immune sensors of endogenous damage signals and play an important role in inflammatory diseases like diabetes and atherosclerosis. Pioglitazone (PIO), a peroxisome proliferator-activated receptor (PPAR)- agonist, has been reported to be an antiinflammatory agent. Thus, in the present study, we examined the antiinflammatory effects of PIO on TLR2 and TLR4 expression in human monocytes exposed to Pam3CSK4 (Pam; TLR2 ligand) and purified lipopolysaccharide (LPS; TLR4 ligand) using flow cytometry and real-time RT-PCR. Monocytes were isolated from healthy human volunteers and pretreated with PIO (1 µm) followed by Pam (170 ng/ml) and LPS (160 ng/ml) challenge. PIO significantly decreased Pam- and LPS-induced TLR2 (–56%) and TLR4 (–78%) expression (P < 0.05). In addition, PIO decreased TLR ligand-induced nuclear factor-B activity (–63%), IL-1β (–50%), IL-6 (–52%), monocyte chemoattractant protein-1(–83%), and TNF- (–87%) compared with control. Next, PIO-treated db/db mice (n = 6/group) showed decreased TLR2 (–60%) and TLR4 (–45%) expression in peritoneal macrophages compared with vehicle control mice (P < 0.001) with associated decrease in MyD88-dependent signaling and nuclear factor-B activation. Data suggest that Pam- and LPS-induced TLR2 and TLR4 expression are inhibited by PIO in human monocytes and db/db mice. Thus, we define a novel pathway by which PIO could induce antiinflammatory effects.

Pioglitazone Acutely Reduces Insulin Secretion and Causes Metabolic Deceleration of the Pancreatic {beta}-Cell at Submaximal Glucose Concentrations

Tue, 21 Jul 2009 10:03:33 PDT

Thiazolidinediones (TZDs) have beneficial effects on glucose homeostasis via enhancement of insulin sensitivity and preservation of β-cell function. How TZDs preserve β-cells is uncertain, but it might involve direct effects via both peroxisome proliferator-activated receptor--dependent and -independent pathways. To gain insight into the independent pathway(s), we assessed the effects of short-term (≤90 min) exposure to pioglitazone (Pio) (10 to 50 µM) on glucose-induced insulin secretion (GIIS), AMP-activated protein kinase (AMPK) activation, and β-cell metabolism in INS 832/13 β-cells and rat islets. Pio caused a right shift in the dose-dependence of GIIS, such that insulin release was reduced at intermediate glucose but unaffected at either basal or maximal glucose concentrations. This was associated in INS 832/13 cells with alterations in energy metabolism, characterized by reduced glucose oxidation, mitochondrial membrane polarization, and ATP levels. Pio caused AMPK phosphorylation and its action on GIIS was reversed by the AMPK inhibitor compound C. Pio also reduced palmitate esterification into complex lipids and inhibited lipolysis. As for insulin secretion, the alterations in β-cell metabolic processes were mostly alleviated at elevated glucose. Similarly, the antidiabetic agents and AMPK activators metformin and berberine caused a right shift in the dose dependence of GIIS. In conclusion, Pio acutely reduces glucose oxidation, energy metabolism, and glycerolipid/fatty acid cycling of the β-cell at intermediate glucose concentrations. We suggest that AMPK activation and the metabolic deceleration of the β-cell caused by Pio contribute to its known effects to reduce hyperinsulinemia and preserve β-cell function and act as an antidiabetic agent.

Insulin and Insulin-Like Growth Factor-I Receptors Differentially Mediate Insulin-Stimulated Adhesion Molecule Production by Endothelial Cells

Li, G., Barrett, E. J., Ko, S.-H., Cao, W., Liu, Z. — Tue, 21 Jul 2009 10:03:33 PDT

Patients with type 2 diabetes are hyperinsulinemic and insulin resistant and develop premature atherosclerosis. High concentrations of insulin stimulate the production of adhesion molecules by endothelial cells (ECs). ECs express abundant IGF-I receptors as well as insulin receptors. Whether IGF-I receptors contribute to insulin-induced endothelial production of adhesion molecules is unknown. Bovine aortic ECs (BAECs) were incubated with insulin (100 nm) for 24 h. The cellular content of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was measured, and monocyte adhesion to ECs was quantified. Insulin increased both VCAM-1 (P < 0.001) and ICAM-1 (P < 0.0002) content, which was accompanied by an increased number of monocytes adherent to BAECs (P = 0.0001). Inhibition of either MAPK kinase-1 or p38 MAPK but not phosphatidylinositol 3-kinase abolished insulin-mediated production of adhesion molecules. Insulin receptor small interfering RNA knockdown abolished insulin-stimulated increases of ICAM-1 but not VCAM-1. Conversely, IGF-I receptor blockade with either a neutralizing antibody or specific small interfering RNA eliminated insulin-induced VCAM-1 but not ICAM-1 production. Blockade of signaling via either the insulin or IGF-I receptors decreased monocyte adherence to BAECs (P < 0.01 for each). We conclude that insulin and IGF-I receptors differentially mediate the production of adhesion molecules by ECs and monocyte adhesion onto the vascular endothelium in response to the hyperinsulinemic state. Dual-receptor activation may most effectively contribute to the pathogenesis of atherosclerotic disease in diabetes.

Insulin Stimulates the Expression of Carbohydrate Response Element Binding Protein (ChREBP) by Attenuating the Repressive Effect of Pit-1, Oct-1/Oct-2, and Unc-86 Homeodomain Protein Octamer Transcription Factor-1

Sirek, A. S., Liu, L., Naples, M., Adeli, K., Ng, D. S., Jin, T. — Tue, 21 Jul 2009 10:03:33 PDT

The carbohydrate response element binding protein (ChREBP) has been recognized as a key controller of hepatic lipogenesis. Whereas the function of ChREBP has been extensively investigated, mechanisms underlying its transcription remain largely unknown, although ChREBP production is elevated in a hyperinsulinemic mouse model. We located a conserved Pit-1, Oct-1/Oct-2, and Unc-86 (POU) protein binding site (ATGCTAAT) within the proximal promoter region of human ChREBP. This site interacts with the POU homeodomain protein octamer transcription factor-1 (Oct-1), as detected by gel shift and chromatin immunoprecipitation assays. Oct-1 cotransfection in the human HepG2 cell line repressed ChREBP promoter activity approximately 50–75% (P < 0.01 to P < 0.001), and this repression was dependent on the existence of the POU binding site. Furthermore, overexpression of Oct-1 repressed endogenous ChREBP mRNA and protein expression, whereas knockdown of Oct-1 expression, using a lentivirus-based small hairpin RNA approach, led to increased ChREBP mRNA and protein expression. In contrast, HepG2 cells treated with 10 or 100 nm insulin for 4 or 8 h resulted in an approximately 2-fold increase of ChREBP promoter activity (P < 0.05 to P < 0.01). Insulin (10 nm) also stimulated endogenous ChREBP expression in HepG2 and primary hamster hepatocytes. More importantly, we found that the stimulatory effect of insulin on ChREBP promoter activity was dependent on the presence of the POU binding site, and insulin treatment reduced Oct-1 expression levels. Our observations therefore identify Oct-1 as a transcriptional repressor of ChREBP and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

Caveolin-1 Loss of Function Accelerates Glucose Transporter 4 and Insulin Receptor Degradation in 3T3-L1 Adipocytes

Tue, 21 Jul 2009 10:03:33 PDT

Caveolae are a specialized type of lipid rafts that are stabilized by oligomers of caveolin protein. Caveolae are particularly enriched in adipocytes. Here we analyzed the effects of caveolin-1 knockdown and caveolae ablation on adipocyte function. To this end, we obtained several multiclonal mouse 3T3-L1 cell lines with a reduced expression of caveolin-1 (95% reduction) by a small interfering RNA approach using lentiviral vectors. Control cell lines were obtained by lentiviral infection with lentiviral vectors encoding appropriate scrambled RNAs. Caveolin-1 knockdown adipocytes showed a drastic reduction in the number of caveolae (95% decrease) and cholera toxin labeling was reorganized in dynamic plasma membrane microdomains. Caveolin-1 depletion caused a specific decrease in glucose transporter 4 (GLUT4) and insulin receptor protein levels. This reduction was not the result of a generalized defect in adipocyte differentiation or altered gene expression but was explained by faster degradation of these proteins. Caveolin-1 knockdown adipocytes showed reductions in insulin-stimulated glucose transport, insulin-triggered GLUT4 recruitment to the cell surface, and insulin receptor activation. In all, our data indicate that caveolin-1 loss of function reduces maximal insulin response through lowered stability and diminished expression of insulin receptors and GLUT4. We propose that caveolin-1/caveolae control insulin action in adipose cells.

Targeted Disruption of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Promotes Diet-Induced Hepatic Steatosis and Insulin Resistance

Tue, 21 Jul 2009 10:03:33 PDT

Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) is a cell adhesion molecule within the Ig superfamily. The Tyr-phosphorylated isoform of CC1 (CC1-L) plays an important metabolic role in the regulation of hepatic insulin clearance. In this report, we show that CC1-deficient (Cc1^–/–) mice are prone to hepatic steatosis, as revealed by significantly elevated hepatic triglyceride and both total and esterified cholesterol levels compared with age-matched wild-type controls. Cc1^–/– mice were also predisposed to lipid-induced hepatic steatosis and dysfunction as indicated by their greater susceptibility to store lipids and express elevated levels of enzymatic markers of liver damage after chronic feeding of a high-fat diet. Hepatic steatosis in the Cc1^–/– mice was linked to a significant increase in the expression of key lipogenic (fatty acid synthase, acetyl CoA carboxylase) and cholesterol synthetic (3-hydroxy-3-methylglutaryl-coenzyme A reductase) enzymes under the control of sterol regulatory element binding proteins-1c and -2 transcription factors. Cc1^–/– mice also exhibited impaired insulin clearance, glucose intolerance, liver insulin resistance, and elevated hepatic expression of the key gluconeogenic transcriptional activators peroxisome proliferator-activated receptor- coactivator-1 and Forkhead box O1. Lack of CC1 also exacerbated both glucose intolerance and hepatic insulin resistance induced by high-fat feeding, but insulin clearance was not further deteriorated in the high-fat-fed Cc1^–/– mice. In conclusion, our data indicate that CC1 is a key regulator of hepatic lipogenesis and that Cc1^–/– mice are predisposed to liver steatosis, leading to hepatic insulin resistance and liver damage, particularly when chronically exposed to dietary fat.

Hypothalamic Injection of Oxyntomodulin Suppresses Circulating Ghrelin-Like Immunoreactivity

Tue, 21 Jul 2009 10:03:33 PDT

Ghrelin is a gastric peptide that regulates appetite and GH secretion. Circulating ghrelin levels are elevated by fasting and suppressed postprandially. However, the mechanisms regulating circulating ghrelin levels are unclear. Oxyntomodulin is an anorexic peptide hormone released from L cells in the gut. We investigated the effects of intracerebroventricular (icv) administration of oxyntomodulin on circulating ghrelin levels. The icv administration of 1, 3, or 10 nmol oxyntomodulin reduced circulating acylated and total (acylated and des-acylated) ghrelin 60 min after icv injection. Administration of 1 nmol oxyntomodulin directly into the arcuate nucleus of the hypothalamus significantly reduced total and acylated ghrelin levels, and administration of 3 nmol oxyntomodulin into the lateral ventricle induced c-fos mRNA expression in arcuate nucleus neurons expressing the glucagon-like peptide-1 (GLP-1) receptor. In a final study, the reduction in total ghrelin observed after icv injection of 3 nmol oxyntomodulin was blocked by coadministration of the GLP-1 receptor antagonist exendin (9–39). These studies suggest oxyntomodulin reduces peripheral ghrelin levels via GLP-1 receptor-dependent hypothalamic pathways. Postprandial release of anorexic gut hormones may thus act centrally to contribute to the postprandial reduction in circulating ghrelin.

Activin Signaling: Effects on Body Composition and Mitochondrial Energy Metabolism

Tue, 21 Jul 2009 10:03:33 PDT

Activin-βA and activin-βB (encoded by Inhba and Inhbb genes, respectively) are closely related TGF-β superfamily members that participate in a variety of biological processes. We previously generated mice with an insertion allele at the Inhba locus, Inhba^BK. In this allele, the sequence encoding the Inhba mature domain is replaced with that of Inhbb, rendering the gene product functionally hypomorphic. Homozygous (Inhba^BK/BK) and hemizygous (Inhba^BK/–) mice are smaller and leaner than their wild-type littermates, and many tissues are disproportionately small relative to total body weight. To determine the mechanisms that contribute to these phenomena, we investigated the metabolic consequences of the mutation. Although the growth of Inhba^BK mice is improved by providing a calorie-rich diet, diet-induced obesity, fatty liver, and insulin resistance (hallmarks of chronic caloric excess) do not develop, despite greater caloric intake than wild-type controls. Physiological, molecular, and biochemical analyses all revealed characteristics that are commonly associated with increased mitochondrial energy metabolism, with a corresponding up-regulation of several genes that reflect enhanced mitochondrial biogenesis and function. Oxygen consumption, an indirect measure of the metabolic rate, was markedly increased in Inhba^BK/BK mice, and polarographic analysis of liver mitochondria revealed an increase in ADP-independent oxygen consumption, consistent with constitutive uncoupling of the inner mitochondrial membrane. These findings establish a functional relationship between activin signaling and mitochondrial energy metabolism and further support the rationale to target this signaling pathway for the medical treatment of cachexia, obesity, and diabetes.

In Vivo and ex Vivo Regulation of Visfatin Production by Leptin in Human and Murine Adipose Tissue: Role of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Signaling Pathways

Tue, 21 Jul 2009 10:03:33 PDT

Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P < 0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10^–9 m, returning to baseline at leptin 10^–7 m. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P < 0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10^–7 m (P < 0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin receptor gene was knocked down using small interfering RNA, leptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin.

Growth Differentiation Factor 9 Enhances Activin A-Induced Inhibin B Production in Human Granulosa Cells

Shi, F.-T., Cheung, A. P., Leung, P. C. K. — Tue, 21 Jul 2009 10:03:33 PDT

Activin A or growth differentiation factor 9 (GDF9) alone can increase β_B-mRNA level in human granulosa-lutein cells from women undergoing in vitro fertilization, but their potential interactions and related cell signaling pathways involved are unknown. We therefore compared inhibin subunit and inhibin levels and activation of activin receptors (ACVRs) and Smad signaling pathway in these human granulosa-lutein cells with and without GDF9 and/or activin A treatment. Inhibin subunit (, β_A, β_B), ACVR, and Smad2/3/4/7 mRNA levels, inhibin A and B production, and Smad phosphorylation were assessed by real-time RT-PCR, ELISA, and immunoblotting, respectively. Data were analyzed by ANOVA followed by Tukey’s test. Activin A (1–50 ng/ml) or GDF9 (1–200 ng/ml) alone had only little stimulatory effects on - and β_A-mRNA levels. In contrast, GDF9 could stimulate β_B-subunit levels but to a lesser degree than the dose- and time-dependent effects of activin A. Compared with untreated cells, GDF9 pretreatment for 24 h significantly enhanced activin A-induced β_B-mRNA levels, inhibin B secretion, and Smad2/3 phosphorylation (effects attenuated by bone morphogenetic protein receptor 2 extracellular domain, a GDF9 antagonist); and induced ACVR2B/1B and Smad2/3 but reduced Smad7 (an inhibitory Smad) mRNA levels. We report here for the first time that GDF9 enhances cell response to activin A by modulating key components of the activin signaling pathway in regulating inhibin subunits and hence inhibin B production in human granulosa-lutein cells.

Cathepsin L Colocalizes with Chromogranin A in Chromaffin Vesicles to Generate Active Peptides

Tue, 21 Jul 2009 10:03:33 PDT

Chromogranin A (CgA), the major soluble protein in chromaffin granules, is proteolytically processed to generate biologically active peptides including the catecholamine release inhibitory peptide catestatin. Here we sought to determine whether cysteine protease cathepsin L (CTSL), a novel enzyme for proteolytic processing of neuropeptides, acts like the well-established serine proteases [prohormone convertase (PC)1/3 or PC2] to generate catestatin by proteolytic processing of CgA. We found that endogenous CTSL colocalizes with CgA in the secretory vesicles of primary rat chromaffin cells. Transfection of PC12 cells with an expression plasmid encoding CTSL directed expression of CTSL toward secretory vesicles. Deconvolution fluorescence microscopy suggested greater colocalization of CTSL with CgA than the lysosomal marker LGP110. The overexpression of CTSL in PC12 cells caused cleavage of full-length CgA. CTSL also cleaved CgA in vitro, in time- and dose-dependent fashion, and specificity of the process was documented through E64 (thiol reagent) inhibition. Mass spectrometry on CTSL-digested recombinant CgA identified a catestatin-region peptide, corresponding to CgA_360–373. The pool of peptides generated from the CTSL cleavage of CgA inhibited nicotine-induced catecholamine secretion from PC12 cells. CTSL processing in the catestatin region was diminished by naturally occurring catestatin variants, especially Pro370Leu and Gly364Ser. Among the CTSL-generated peptides, a subset matched those found in the catestatin region in vivo. These findings indicate that CgA can be a substrate for the cysteine protease CTSL both in vitro and in cella, and their colocalization within chromaffin granules in cella suggests the likelihood of an enzyme/substrate relationship in vivo.

Androgen Signaling in Myocytes Contributes to the Maintenance of Muscle Mass and Fiber Type Regulation But Not to Muscle Strength or Fatigue

Tue, 21 Jul 2009 10:03:33 PDT

Muscle frailty is considered a major cause of disability in the elderly and chronically ill. However, the exact role of androgen receptor (AR) signaling in muscle remains unclear. Therefore, a postmitotic myocyte-specific AR knockout (mARKO) mouse model was created and investigated together with a mouse model with ubiquitous AR deletion. Muscles from mARKO mice displayed a marked reduction in AR protein (60–88%). Interestingly, body weights and lean body mass were lower in mARKO vs. control mice (–8%). The weight of the highly androgen-sensitive musculus levator ani was significantly reduced (–46%), whereas the weights of other peripheral skeletal muscles were not or only slightly reduced. mARKO mice had lower intra-abdominal fat but did not demonstrate a cortical or trabecular bone phenotype, indicating that selective ablation of the AR in myocytes affected male body composition but not skeletal homeostasis. Furthermore, muscle contractile performance in mARKO mice did not differ from their controls. Myocyte-specific AR ablation resulted in a conversion of fast toward slow fibers, without affecting muscle strength or fatigue. Similar results were obtained in ubiquitous AR deletion, showing lower body weight, whereas some but not all muscle weights were reduced. The percent slow fibers was increased, but no changes in muscle strength or fatigue could be detected. Together, our findings show that myocyte AR signaling contributes to the maintenance of muscle mass and fiber type regulation but not to muscle strength or fatigue. The levator ani weight remains the most sensitive and specific marker of AR-mediated anabolic action on muscle.

Extralarge XL{alpha}s (XXL{alpha}s), a Variant of Stimulatory G Protein {alpha}-Subunit (Gs{alpha}), Is a Distinct, Membrane-Anchored GNAS Product that Can Mimic Gs{alpha}

Tue, 21 Jul 2009 10:03:33 PDT

GNAS gives rise to multiple imprinted gene products, including the -subunit of the stimulatory G protein (Gs) and its variant XLs. Based on genomic sequence, the translation of XLs begins from the middle of a long open reading frame, suggesting the existence of an N-terminally extended variant termed extralarge XLs (XXLs). Although XXLs, like Gs and XLs, would be affected by most disease-causing GNAS mutations, its authenticity and biological significance remained unknown. Here we identified a mouse cDNA clone that comprises the entire open reading frame encoding XXLs. Whereas XXLs mRNA was readily detected in mouse heart by RT-PCR, it appeared virtually absent in insulinoma-derived INS-1 cells. By Northern blots and RT-PCR, XXLs mRNA was detected primarily in the mouse brain, cerebellum, and spleen. Immunohistochemistry using a specific anti-XXLs antibody demonstrated XXLs protein in multiple brain areas, including dorsal hippocampus and cortex. In transfected cells, full-length human XXLs was localized to the plasma membrane and mediated isoproterenol- and cholera toxin-stimulated cAMP accumulation. XXLs-R844H, which bears a mutation analogous to that in the constitutively active Gs mutant Gs-R201H (gsp oncogene), displayed elevated basal signaling. However, unlike Gs-R201H, which mostly remains in the cytoplasm, both XXLs-R844H and a constitutively active XLs mutant localized to the plasma membrane. Hence, XXLs is a distinct GNAS product and can mimic Gs, but the constitutively active XXLs and Gs mutants differ from each other regarding subcellular targeting. Our findings suggest that XXLs deficiency or hyperactivity may contribute to the pathogenesis of diseases caused by GNAS mutations.

A Signaling Network in Phenylephrine-Induced Benign Prostatic Hyperplasia

Tue, 21 Jul 2009 10:03:33 PDT

Benign prostatic hyperplasia (BPH) is an age-related disease of unknown etiology characterized by prostatic enlargement and coinciding with distinctive alterations in tissue histomorphology. To identify the molecular mechanisms underlying the development of BPH, we conducted a DNA microarray study using a previously described animal model in which chronic (1)-adrenergic stimulation by repeated administration of phenylephrine evokes histomorphological changes in the rat prostate that resemble human BPH. Bioinformatic tools were applied to microarray data obtained from prostate tissue to construct a network model of potentially relevant signal transduction pathways. Significant involvement of inflammatory pathways was demonstrable, including evidence for activation of a TGF-β signaling cascade. The heterodimeric protein clusterin (apolipoprotein J) was also identified as a prominent node in the network. Responsiveness of TGF-β signaling and clusterin gene and protein expression were confirmed independently of the microarray data, verifying some components of the model. This is the first attempt to develop a comprehensive molecular network for histological BPH induced by adrenergic activation. The study also implicated clusterin as a novel biochemical target for therapy.

Leptin-Stimulated Endothelial Nitric-Oxide Synthase via an Adenosine 5'-Monophosphate-Activated Protein Kinase/Akt Signaling Pathway Is Attenuated by Interaction with C-Reactive Protein

Tue, 21 Jul 2009 10:03:33 PDT

The AMP-activated protein kinase (AMPK) lies upstream of Akt in the pathway leading to endothelial NO synthase (eNOS) activation. Whether leptin promotes eNOS activation via AMPK-dependent activation of Akt, and which of the two AMPK catalytic subunits is involved, remains unknown. Leptin resistance may be partly attributed to interaction between leptin and C-reactive protein (CRP). We hypothesized that leptin effect on eNOS activation in human aortic endothelial cells might be blunted by direct interaction with human recombinant CRP. Small interfering RNAs (siRNAs) were used to knock down expression of 1- or 2-AMPK in transient transfection assay to evaluate which is involved in this pathway and whether leptin effect on eNOS activation in human aortic endothelial cells might be blunted by direct interaction with human CRP. siRNA-mediated down-regulation of AMPK1, but not AMPK2, abolished leptin-induced Akt-Ser⁴⁷³ phosphorylation, eNOS-Ser¹¹⁷⁷ phosphorylation, eNOS activation, and cGMP accumulation. By contrast, siRNA-mediated knockdown of Akt1 did not affect AMPK1 phosphorylation, but it abolished leptin-induced phosphorylation of Akt-Ser⁴⁷³ and eNOS-Ser¹¹⁷⁷, suggesting that Akt functions downstream of AMPK1. Preincubation of leptin with human recombinant CRP impaired leptin-induced AMPK activation, eNOS-Ser¹¹⁷⁷ phosphorylation, eNOS activity, and intracellular cGMP accumulation. The data are consistent with a model implicating an AMPK1->Akt->eNOS pathway leading to NO production in response to leptin supporting the idea that interaction between leptin and CRP may have a role in impairing leptin effect on eNOS activation, suggesting a link between leptin resistance, low-grade inflammation, and endothelial dysfunction.

Differential Signaling Activation by Insulin and Insulin-Like Growth Factors I and II upon Binding to Insulin Receptor Isoform A

Sacco, A., Morcavallo, A., Pandini, G., Vigneri, R., Belfiore, A. — Tue, 21 Jul 2009 10:03:33 PDT

A variety of human malignancies overexpresses isoform A of the insulin receptor (IR-A) and produces IGFs (IGF-I and/or IGF-II). IR-A binds IGF-II with high affinity (although 4-fold lower than that for insulin), whereas it binds IGF-I with low affinity (approximately 30-fold lower than that for insulin). However, in engineered cells expressing only the IR-A, but not IGF-I receptor (R^–/IR-A cells), IGF-II is a more potent mitogen than insulin. Herein, we investigated downstream signaling of IGF-II, IGF-I, and insulin in R^–/IR-A cells to better understand their role in cell growth. We found that despite inducing a lower IR-A autophosphorylation than insulin, IGF-II was more potent than insulin for activating p70S6 kinase (p70S6K) and approximately equally potent in activating the early peaks of ERK1/2 and Akt. However, ERK1/2 activation persisted longer after IGF-II, whereas Akt activation persisted longer after insulin. Therefore, cells stimulated with IGF-II had a higher p70S6K/Akt activation ratio than cells stimulated with insulin. Remarkably, IGF-I also elicited a similar signaling pattern as IGF-II, despite inducing minimal IR-A autophosphorylation. ERK1/2 and protein kinase C seem to be involved in the preferential stimulation of p70S6K by IGFs. In conclusion, our study has identified a novel complex role of IR-A, which not only elicits a unique signaling pattern after IGF-II binding but also induces substantial downstream signaling upon binding to the low-affinity ligand IGF-I. These results underline the role of IR-A in physiology and disease.

In Vitro and in Vivo Effects of Adiponectin on Bone

Tue, 21 Jul 2009 10:03:33 PDT

Fat mass impacts on both bone turnover and bone density and is a critical risk factor for osteoporotic fractures. Adipocyte-derived hormones may contribute to this relationship, and adiponectin is a principal circulating adipokine. However, its effects on bone remain unclear. We have, therefore, investigated the direct effects of adiponectin on primary cultures of osteoblastic and osteoclastic cells in vitro and determined its integrated effects in vivo by characterizing the bone phenotype of adiponectin-deficient mice. Adiponectin was dose-dependently mitogenic to primary rat and human osteoblasts (~50% increase at 10 µg/ml) and markedly inhibited osteoclastogenesis at concentrations of 1 µg/ml or greater. It had no effect on osteoclastogenesis in RAW-264.7 cells or on bone resorption in isolated mature osteoclasts. In adiponectin knockout (AdKO) male C57BL/6J mice, trabecular bone volume and trabecular number (assessed by microcomputed tomography) were increased at 14 wk of age by 30% (P = 0.02) and 38% (P = 0.0009), respectively. Similar, nonsignificant trends were observed at 8 and 22 wk of age. Biomechanical testing showed lower bone fragility and reduced cortical hardness at 14 wk. We conclude that adiponectin stimulates osteoblast growth but inhibits osteoclastogenesis, probably via an effect on stromal cells. However, the AdKO mouse has increased bone mass, suggesting that adiponectin also has indirect effects on bone, possibly through modulating growth factor action or insulin sensitivity. Because adiponectin does influence bone mass in vivo, it is likely to be a contributor to the fat-bone relationship.

Insulin-Like Growth Factor Type 1 Receptor and Insulin Receptor Isoform Expression and Signaling in Mammary Epithelial Cells

Rowzee, A. M., Ludwig, D. L., Wood, T. L. — Tue, 21 Jul 2009 10:03:33 PDT

The insulin receptor (IR) isoforms and the IGF type 1 receptor (IGF-1R) share a high degree of structural homology but differ in ligand binding kinetics and functions. We developed a highly specific quantitative PCR assay to quantify and compare IR-A, IR-B, and IGF-1R expression within an RNA population. We determined receptor expression in primary murine mammary epithelial cells (MECs) during postnatal development. Both IR isoform mRNAs were 3- to 16-fold higher than IGF-1R expression at all developmental times. IR protein was also 3- to 10-fold higher than IGF-1R protein; however, significantly less IGF-1R was found in hybrid receptors at early (49%) vs. late (79%) pregnancy, indicating that the amount of hybrid receptor is developmentally regulated. Despite high IR expression, IGF ligands were more effective than insulin in stimulating the insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt pathway in acutely isolated MECs from virgin glands. Although approximately 40% of IR transcripts were the IGF-II-sensitive IR-A isoform, IGF-II failed to stimulate IR phosphorylation, and an IGF-1R-specific blocking antibody completely abrogated IGF-II-mediated Akt phosphorylation in the virgin MECs. Taken together, these data suggest that the IGF-1R is more active in signaling than the IR and is the predominant mediator of IGF actions in virgin MECs.

Endotoxin-Induced Growth Hormone Resistance in Skeletal Muscle

Chen, Y., Sood, S., Krishnamurthy, V. M. R., Rotwein, P., Rabkin, R. — Tue, 21 Jul 2009 10:03:33 PDT

Inflammation-induced skeletal muscle wasting is a serious clinical problem and arises in part because of resistance to GH-stimulated IGF-I expression. Although it is established that in the liver, resistance develops because of impaired signaling through the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) transduction pathway, together with a more distal defect in STAT5 DNA-binding activity, the situation in skeletal muscle is unclear. Accordingly, we set out to characterize the mechanisms behind the skeletal muscle resistance to GH in rats with acute inflammation induced by endotoxin. Endotoxin caused significant declines in GH-stimulated STAT5a/b phosphorylation and IGF-I gene expression, and this occurred despite a lack of change in signaling protein levels or phosphorylation of JAK2. In whole muscle, GH-stimulated phospho-STAT5a/b levels were reduced by half, and in the nucleus, phospho-STAT5b levels were similarly reduced. Furthermore, the binding of phosphorylated STAT5b to DNA was reduced and to a similar extent to the reduction in nuclear phosphorylated STAT5b. Interestingly, GH-induced androgen receptor gene expression was also suppressed. Thus, it appears that skeletal muscle resistance to GH-stimulated IGF-I expression in acute endotoxemia arises from a defect in STAT5b signaling, with a proportionate reduction in STAT5b DNA binding. Finally, it appears that resistance to GH-induced androgen receptor expression also develops and, together with the attenuated GH-induced IGF-I expression, likely plays an important role in the muscle wasting that arises in endotoxin-induced inflammation.

Regulation of Rapid Signal Transducer and Activator of Transcription-5 Phosphorylation in the Resting Cells of the Growth Plate and in the Liver by Growth Hormone and Feeding

Gevers, E. F., Hannah, M. J., Waters, M. J., Robinson, I. C. A. F. — Tue, 21 Jul 2009 10:03:33 PDT

GH has physiological functions in many tissues, but the cellular targets for direct effects of GH remain ill defined in complex tissues such as the growth plate in which the contribution of direct vs. indirect actions of GH remains controversial. The Janus kinase (Jak)-signal transducer and activator of transcription (STAT)-5 pathway is activated by GH, so we developed a method to visualize nuclear Stat5b and phosphorylated Stat5 in single cells in response to a pulse of GH. Hep2 cells did not show a Stat5 phosphorylation (pY-Stat5) response to GH except in cells transfected to express GH receptors. ATDC5 cells express GH receptors and showed GH-induced pY-Stat5 responses, which varied with their state of chondrocyte differentiation. In vivo, Stat5b^+ve nuclei were seen in the resting and prehypertrophic chondrocytes of the growth plate. After a single ip pulse of human GH or mouse GH, but not prolactin, pY-Stat5 responses were visible in cells in the resting zone and groove of Ranvier, 10–45 min later. Prehypertrophic chondrocytes showed no pY-Stat5 response to GH. GH target cells were also identified in other tissues, and a marked variability in spatiotemporal pY-Stat5 responses was evident. Endogenous hepatic pY-Stat5 was detected in mice with intact GH secretion but only during a GH pulse. Fasting and chronic exposure to GH attenuated the pY-Stat5 response to an acute GH injection. In conclusion, pY-Stat5 responses to GH vary in time and space, are sensitive to nutritional status, and may be inhibited by prior GH exposure. In the growth plate, our data provide direct in vivo support for an early role of GH to regulate the fate of immature chondrocytes.

Gastric Mammalian Target of Rapamycin Signaling Regulates Ghrelin Production and Food Intake

Tue, 21 Jul 2009 10:03:33 PDT

Ghrelin, a gastric hormone, provides a hunger signal to the central nervous system to stimulate food intake. Mammalian target of rapamycin (mTOR) is an intracellular fuel sensor critical for cellular energy homeostasis. Here we showed the reciprocal relationship of gastric mTOR signaling and ghrelin during changes in energy status. mTOR activity was down-regulated, whereas gastric preproghrelin and circulating ghrelin were increased by fasting. In db/db mice, gastric mTOR signaling was enhanced, whereas gastric preproghrelin and circulating ghrelin were decreased. Inhibition of the gastric mTOR signaling by rapamycin stimulated the expression of gastric preproghrelin and ghrelin mRNA and increased plasma ghrelin in both wild-type and db/db mice. Activation of the gastric mTOR signaling by l-leucine decreased the expression of gastric preproghrelin and the level of plasma ghrelin. Overexpression of mTOR attenuated ghrelin promoter activity, whereas inhibition of mTOR activity by overexpression of TSC1 or TSC2 increased its activity. Ghrelin receptor antagonist d-Lys-3-GH-releasing peptide-6 abolished the rapamycin-induced increment in food intake despite that plasma ghrelin remained elevated. mTOR is therefore a gastric fuel sensor whose activity is linked to the regulation of energy intake through ghrelin.

Computational and Functional Analysis of Growth Hormone (GH)-Regulated Genes Identifies the Transcriptional Repressor B-Cell Lymphoma 6 (Bc16) as a Participant in GH-Regulated Transcription

Tue, 21 Jul 2009 10:03:33 PDT

For insight into transcriptional mechanisms mediating physiological responses to GH, data mining was performed on a profile of GH-regulated genes induced or inhibited at different times in highly responsive 3T3-F442A adipocytes. Gene set enrichment analysis indicated that GH-regulated genes are enriched in pathways including phosphoinositide and insulin signaling and suggested that suppressor of cytokine signaling 2 (SOCS2) and phosphoinositide 3' kinase regulatory subunit p85 (Pik3r1) are important targets. Model-based Chinese restaurant clustering identified a group of genes highly regulated by GH at times consistent with its key physiological actions. This cluster included IGF-I, phosphoinositide 3' kinase p85, SOCS2, and cytokine-inducible SH2-containing protein. It also contains the most strongly repressed gene in the profile, B cell lymphoma 6 (Bcl6), a transcriptional repressor. Quantitative real-time PCR verified the strong decrease in Bcl6 mRNA after GH treatment and induction of the other genes in the cluster. Transcriptional network analysis of the genes implicated signal transducer and activator of transcription (Stat) 5 as hub regulating the most responsive genes, Igf1, Socs2, Cish, and Bcl6. Transcriptional activation analysis demonstrated that Bcl6 inhibits SOCS2-luciferase and blunts its stimulation by GH. Occupancy of endogenous Bcl6 on SOCS2 DNA decreased after GH treatment, whereas occupancy of Stat5 increased concomitantly. Thus, GH-mediated inhibition of Bcl6 expression may reverse the repression of SOCS2 and facilitate SOCS2 activation by GH. Together these analyses identify Bcl6 as a participant in GH-regulated gene expression and suggest an interplay between the repressor Bcl6 and the activator Stat5 in regulating genes, which contribute to GH responses.

Short Photoperiod-Induced Decrease of Histamine H3 Receptors Facilitates Activation of Hypothalamic Neurons in the Siberian Hamster

Tue, 21 Jul 2009 10:03:33 PDT

Nonhibernating seasonal mammals have adapted to temporal changes in food availability through behavioral and physiological mechanisms to store food and energy during times of predictable plenty and conserve energy during predicted shortage. Little is known, however, of the hypothalamic neuronal events that lead to a change in behavior or physiology. Here we show for the first time that a shift from long summer-like to short winter-like photoperiod, which induces physiological adaptation to winter in the Siberian hamster, including a body weight decrease of up to 30%, increases neuronal activity in the dorsomedial region of the arcuate nucleus (dmpARC) assessed by electrophysiological patch-clamping recording. Increased neuronal activity in short days is dependent on a photoperiod-driven down-regulation of H3 receptor expression and can be mimicked in long-day dmpARC neurons by the application of the H3 receptor antagonist, clobenproprit. Short-day activation of dmpARC neurons results in increased c-Fos expression. Tract tracing with the trans-synaptic retrograde tracer, pseudorabies virus, delivered into adipose tissue reveals a multisynaptic neuronal sympathetic outflow from dmpARC to white adipose tissue. These data strongly suggest that increased activity of dmpARC neurons, as a consequence of down-regulation of the histamine H3 receptor, contributes to the physiological adaptation of body weight regulation in seasonal photoperiod.

Circadian Regulation of Kiss1 Neurons: Implications for Timing the Preovulatory Gonadotropin-Releasing Hormone/Luteinizing Hormone Surge

Tue, 21 Jul 2009 10:03:33 PDT

The preovulatory GnRH/LH surge depends on the presence of estradiol (E₂) and is gated by a circadian oscillator in the suprachiasmatic nucleus (SCN) that causes the surge to occur within a specific temporal window. Although the mechanisms by which the clock times the LH surge are unclear, evidence suggests that the SCN is linked to GnRH neurons through a multisynaptic pathway that includes neurons in the anteroventral periventricular nucleus (AVPV). Recently, Kiss1 neurons in the AVPV have been implicated in the surge mechanism, suggesting that they may integrate circadian and E₂ signals to generate the LH surge. We tested whether Kiss1 neurons display circadian patterns of regulation in synchrony with the temporal pattern of LH secretion. Mice housed in 14 h light, 10 h dark were ovariectomized, given E₂ capsules (or nothing), and transferred into constant darkness. Two days later, the mice were killed at various times of day and their LH and Kiss1 levels assessed. In E₂-treated females, LH levels were low except during late subjective day (indicative of an LH surge). Similarly, AVPV Kiss1 expression and c-fos coexpression in Kiss1 neurons showed circadian patterns that peaked coincident with LH. These temporal changes in Kiss1 neurons occurred under steady-state E₂ and constant environmental conditions, suggesting that Kiss1 neurons are regulated by circadian signals. In the absence of E₂, animals displayed no circadian pattern in LH secretion or Kiss1 expression. Collectively, these findings suggest that the LH surge is controlled by AVPV Kiss1 neurons whose activity is gated by SCN signals in an E₂-dependent manner.

Androgen Regulates the Sexually Dimorphic Gastrin-Releasing Peptide System in the Lumbar Spinal Cord that Mediates Male Sexual Function

Tue, 21 Jul 2009 10:03:34 PDT

A collection of neurons in the upper lumbar spinal cord of male rats projects to the lower lumbar spinal cord, releasing gastrin-releasing peptide (GRP) onto somatic and autonomic centers known to regulate male sexual reflexes such as erection and ejaculation. Because these reflexes are androgen dependent, we asked whether manipulating levels of androgen in adult rats would affect GRP expression in this spinal center. We found that castration resulted, 28 d later, in a profound decrease in the expression of GRP in the spinal cord, as reflected in immunocytochemistry and competitive ELISA for the protein as well as real-time quantitative PCR for the transcript. These effects were prevented if the castrates were treated with testosterone propionate. Genetically male (XY) rats with the dysfunctional testicular feminization allele for the androgen receptor (AR) displayed GRP mRNA and protein levels in the spinal cord similar to those of females, indicating that androgen normally maintains the system through AR. We saw no effect of castration or the testicular feminization allele on expression of the receptor for GRP in the spinal cord, but castration did reduce expression of AR transcripts within the spinal cord as revealed by real-time quantitative PCR and Western blots. Taken together, these results suggest that androgen signaling plays a pivotal role in the regulation of GRP expression in male lumbar spinal cord. A greater understanding of how androgen modulates the spinal GRP system might lead to new therapeutic approaches to male sexual dysfunction.

The Effects of Cycling Levels of 17{beta}-Estradiol and Progesterone on the Magnitude of Temporomandibular Joint-Induced Nociception

Kramer, P. R., Bellinger, L. L. — Tue, 21 Jul 2009 10:03:34 PDT

A greater incidence of temporomandibular joint (TMJ) pain is reported in females, suggesting that gonadal hormones may play a role in this condition. However, the exact roles of 17β-estradiol (E2) and progesterone (P4) in TMJ pain are not completely known. Two experiments were performed to determine the separate roles of E2 and P4 in TMJ nociception at various stages of the estrous cycle. Ovariectomized (OVX) rats were cycled with physiological concentrations of E2 or P4. The E2-cycled rats then received bilateral TMJ injections of saline (SAL) or complete Freund’s adjuvant (CFA) on the morning of diestrus-2 (low E2 condition) or proestrus (high E2 condition). As a control, OVX rats (no ovarian E2 and no replacement) were injected with SAL or CFA. The TMJ nociception was measured using a validated novel method in which an increase in meal duration directly correlated to the intensity of deep TMJ nociception. In the E2 experiment, CFA injection, but not SAL, increased TMJ nociception in the OVX group, but the effect was less pronounced in diestrus-2 and even less in proestrus. In the P4 experiment, the rats receiving TMJ CFA in diestrus-2 (end of minor P4 surge) did not show an increase in TMJ nociception, whereas the rats injected in proestrus (major P4 surge), estrus (low P4), and metestrus (low P4) had similar increases in TMJ nociception. The hormones’ concentration did not affect TMJ IL-1β, IL-6, C-C motif ligand 20, or C-X-C motif ligand 2 or the trigeminal ganglia calcitonin gene-related peptide. The high physiological concentrations of E2 observed at proestrus and the low P4 concentrations observed at diestrus-2 attenuated or eliminated CFA-induced TMJ nociception. The results suggest that the cyclic estrous cycle concentrations of E2 and P4 can influence CFA-induced TMJ nociception in the rat.

Testosterone Programs Adult Social Behavior before and during, But Not after, Adolescence

Schulz, K. M., Zehr, J. L., Salas-Ramirez, K. Y., Sisk, C. L. — Tue, 21 Jul 2009 10:03:34 PDT

Whereas the adolescent brain is a major target for gonadal hormones, our understanding of hormonal influences on adolescent neural and behavioral development remains limited. These experiments investigated how variations in the timing of testosterone (T) exposure, relative to adolescence, alters the strength of steroid-sensitive neural circuits underlying social behavior in male Syrian hamsters. Experiment 1 simulated early, on-time, and late pubertal development by gonadectomizing males on postnatal d 10 and treating with SILASTIC brand T implants for 19 d before, during, or after adolescence. T treatment before or during, but not after, adolescence facilitated mating behavior in adulthood. In addition, preadolescent T treatments most effectively increased mating behavior overall, indicating that the timing of exposure to pubertal hormones contributes to individual differences in adult behavior. Experiment 2 examined the effects of preadolescent T treatment on behavior and brain regional volumes within the mating neural circuit of juvenile males (i.e. still preadolescent). Although preadolescent T treatment did not induce reproductive behavior in juvenile males, it did increase volumes of the bed nucleus of the stria terminalis, sexually dimorphic nucleus, posterodorsal medial amygdala, and posteroventral medial amygdala to adult-typical size. In contrast, juvenile anterodorsal medial amygdala and ventromedial hypothalamus volumes were not changed by preadolescent T treatment yet differed significantly in volume from adult controls, suggesting that further maturation of these brain regions during adolescence is required for the expression of male reproductive behavior. Thus, adolescent maturation of social behavior may involve both steroid-independent and -dependent processes, and adolescence marks the end of a postnatal period of sensitivity to steroid-dependent organization of the brain.

The Excitatory Peptide Kisspeptin Restores the Luteinizing Hormone Surge and Modulates Amino Acid Neurotransmission in the Medial Preoptic Area of Middle-Aged Rats

Tue, 21 Jul 2009 10:03:34 PDT

Reproductive success depends on a robust and appropriately timed preovulatory LH surge. The LH surge, in turn, requires ovarian steroid modulation of GnRH neuron activation by the neuropeptide kisspeptin and glutamate and -aminobutyric acid (GABA) neurotransmission in the medial preoptic area (mPOA). Middle-aged females exhibit reduced excitation of GnRH neurons and attenuated LH surges under estrogen-positive feedback conditions, in part, due to increased GABA and decreased glutamate neurotransmission in the mPOA. This study tested the hypothesis that altered kisspeptin regulation by ovarian steroids plays a role in age-related LH surge dysfunction. We demonstrate that middle-aged rats exhibiting delayed and attenuated LH surges have reduced levels of Kiss1 mRNA in the anterior hypothalamus under estrogen-positive feedback conditions. Kisspeptin application directly into the mPOA rescues total LH release and the LH surge amplitude in middle-aged rats and increases glutamate and decreases GABA release to levels seen in the mPOA of young females. Moreover, the N-methyl-d-aspartate receptor antagonist MK801 blocks kisspeptin reinstatement of the LH surge. These observations suggest that age-related LH surge dysfunction results, in part, from reduced kisspeptin drive under estrogen-positive feedback conditions and that kisspeptin regulates GnRH/LH release, in part, through modulation of mPOA glutamate and GABA release.

Influence of Sex and Corticotropin-Releasing Factor Pathways as Determinants in Serotonin Sensitivity

McEuen, J. G., Semsar, K. A., Lim, M. A., Bale, T. L. — Tue, 21 Jul 2009 10:03:34 PDT

Stress sensitivity and sex are predictive factors in affective disorder susceptibility. Serotonin (5-HT) pathway recruitment by corticotropin-releasing factor (CRF) during stress is necessary in adaptive coping behaviors, but sex differences in such responses have not been investigated. Using selective 5-HT reuptake inhibitor (SSRI) administration to acutely elevate 5-HT in a genetic model of stress sensitivity, we examined behavioral and physiological responses in male and female stress-sensitive CRF receptor-2-deficient (R2KO) mice. Chronic SSRI treatment was used to confirm outcomes were specific to acute 5-HT elevation and not antidepressant efficacy. We hypothesized that R2KO mice would show a greater sensitivity to acute changes in 5-HT and that, because females typically are more stress sensitive, R2KO females would be the most responsive. Our results supported this hypothesis because females of both genotypes and R2KO males showed a greater sensitivity to an acute 10 mg/kg dose of citalopram in a tail suspension test, displaying decreased immobile time and increased latency to immobility. Furthermore, acute citalopram promoted significant anxiogenic-like effects that were specific to R2KO females in the elevated plus maze and light-dark box tests. Chronic citalopram did not produce these behavioral changes, supporting specificity to acute 5-HT modulation. Mechanistically, females had decreased hippocampal 5-HT transporter (SERT) levels, whereas R2KO mice showed reduced SERT in the prefrontal cortex, supporting a possible intersection of sex and genotype where R2KO females would have the lowest SERT to be blocked by the SSRI. This sensitivity to 5-HT-mediated anxiety in females may underlie a heightened vulnerability to stress-related affective disorders.

Enduring Influences of Peripubertal/Adolescent Stressors on Behavioral Response to Estradiol and Progesterone in Adult Female Mice

Laroche, J., Gasbarro, L., Herman, J. P., Blaustein, J. D. — Tue, 21 Jul 2009 10:03:34 PDT

Exposure to stressors during particular stages of development leads to acute and long-term physiological and behavioral changes. We have reported that shipping mice during the peripubertal/adolescent period results in decreased induction of feminine sexual behavior by estradiol and progesterone in adult female mice. To study further the factors involved in this decreased behavioral response, female mice were exposed to a variety of experimental stressors when 6 wk old. Effects of peripubertal/adolescent exposure to these stressors on acute plasma corticosterone levels and changes in body weight and adult behavioral response to estradiol and progesterone were assessed. Although restraint for three daily 3-h periods, 36-h food deprivation, or a multiple stressor regimen acutely increased plasma corticosterone levels and reduced body weight, only exposure to particular doses of the bacterial endotoxin lipopolysaccharide (LPS; 1–1.5 mg/kg body weight, doses that induced moderate levels of sickness behavior in these studies) resulted in reduced behavioral response to estradiol and progesterone in adulthood. Like the effects of shipping, the effects of LPS on adult feminine sexual behavior appear most robust when injected at 6 wk old and are limited to exposure during a vulnerable period at approximately 4–6 wk of age. Therefore, an immune stressor during the peripubertal/adolescent period, but not restraint, food restriction, or a combined stressor, has an enduring influence on behavioral response to estradiol and progesterone. This demonstrates that the decreased response to estradiol and progesterone is not a general response to all stressors during this developmental stage.

Role of the T-Type Calcium Channel CaV3.2 in the Chronotropic Action of Corticosteroids in Isolated Rat Ventricular Myocytes

Maturana, A., Lenglet, S., Python, M., Kuroda, S., Rossier, M. F. — Tue, 21 Jul 2009 10:03:34 PDT

The mineralocorticoid receptor is involved in the development of several cardiac dysfunctions, including lethal ventricular arrhythmias associated with heart failure or hyperaldosteronism, but the molecular mechanisms responsible for these effects remain to be clarified. Reexpression of low voltage-activated T-type calcium channels in ventricular myocytes together with other fetal genes during cardiac pathologies could confer automaticity to these cells and would represent a pro-arrhythmogenic condition if occurring in vivo. In the present study, we demonstrated that in isolated neonatal rat ventricular myocytes, corticosteroids selectively induced the expression of a particular isoform of T channel, Ca_V3.2/1H. This response was accompanied by an increase of the Ca_V3.2 T-type current, identified with the patch clamp technique by its sensitivity to nickel, and a concomitant acceleration of the myocyte spontaneous contractions. Silencing Ca_V3.2 expression markedly reduced the chronotropic response to steroids. Moreover, modulation of the frequency of cell contractions by different redox agents was independent of channel expression but involved a direct regulation of channel activity. Although oxidants increased both Ca_V3.2 current amplitude and beating frequency, they decreased L-type channel activity. Reducing agents had the opposite effect on these parameters. In conclusion, the acceleration of ventricular myocyte spontaneous contractions induced by corticosteroids in vitro appears dependent on the expression of the Ca_V3.2 T channel isoform and modulated by the redox potential of the cells. These results provide a molecular model that could explain the high incidence of arrhythmias observed in patients upon combination of inappropriate activation of the mineralocorticoid receptor and oxidative stress.

Cell-Specific Effects of Nitric Oxide Deficiency on Parathyroid Hormone-Related Peptide (PTHrP) Responsiveness and PTH1 Receptor Expression in Cardiovascular Cells

Tue, 21 Jul 2009 10:03:34 PDT

The missing influence of estrogen on endothelial nitric oxide (NO) synthase often forms the basis for a worsening of the cardiac risk profile for women in postmenopause. Various studies have shown that decreasing estrogen levels also directly effect the expression of PTHrP and TGFβ1. PTHrP is involved in the endothelium-dependent regulation of coronary resistance and cardiac function. The current study investigates to what extent chronic NO deficit affects the cardiac effects of PTHrP. NO deficit was achieved in female adult rats by feeding them the NO synthase inhibitor N-omega-nitro-l-arginine methyl ester over a period of 4 wk. Isolated hearts of the conditioned animals were investigated in Langendorff technique and perfused for 3 min with 100 nM PTHrP. The contraction behavior of isolated cardiomyocytes was registered in a cell-edge detection system. Hearts from untreated animals displayed a significant drop in left ventricular developed pressure and a pronounced increase in heart rate in consequence of short term PTHrP stimulation. In hearts from NO-deficient rats PTHrP no longer affected the inotropy and chronotropy. The vasodilating effect of PTHrP on coronary vessels was, however, independent of the NO level. These changes were accompanied by a differing expression of the PTH1 receptor. TGFβ1 was identified as an important mediator for the regulation of the PTH1 receptor in myocytic but not endothelial cells. These results indicate that chronic NO deficit down-regulates the PTH1 receptor in a TGFβ1-dependent way. These findings are important with respect to the relatively new therapy of postmenopausal osteoporosis with PTHrP analogs.

Estradiol Increases Guanosine 5'-Triphosphate Cyclohydrolase Expression Via the Nitric Oxide-Mediated Activation of Cyclic Adenosine 5'-Monophosphate Response Element Binding Protein

Sun, X., Kumar, S., Tian, J., Black, S. M. — Tue, 21 Jul 2009 10:03:34 PDT

A number of studies have demonstrated that estradiol can stimulate endothelial nitric oxide synthase expression and activity, resulting in enhanced nitric oxide (NO) generation. However, its effect on the NO synthase cofactor, tetrahydrobiopterin are less clear. Cellular tetrahydrobiopterin levels are regulated, at least in part, by GTP cyclohydrolase 1 (GCH1). Thus, the purpose of this study was to determine the effect of estradiol on GCH1 expression and the regulatory mechanisms in pulmonary arterial endothelial cells. Our data indicate that 17β-estradiol (E2) increases GCH1 transcription in a dose- and time-dependent manner, whereas estrogen receptor antagonism or NO synthase inhibition attenuated E2-stimulated GCH1 expression. Analysis of the GCH1 promoter fragment responsive to E2 revealed the presence of a cAMP response element, and we found that E2 triggers a rapid but transient elevation of phospho-cAMP response element-binding protein (CREB; <1 h) followed by a second sustained rise after 6 h. EMSA analysis revealed an increase in the binding of CREB during E2 treatment and mutation of the cAMP response element in the GCH1 promoter attenuated the E2-mediated increase in transcription. Furthermore, inhibition of the cAMP-dependent kinase, protein kinase A (PKA) completely abolished the E2-stimulated GCH1 promoter activity, whereas the stimulation of cAMP levels with forskolin increased GCH1 promoter activity, indicating the key role of cAMP in regulating GCH1 promoter activity. In conclusion, our results demonstrate that estradiol can modulate GCH1 expression via NO-mediated activation of CREB in pulmonary arterial endothelial cells. These findings provide new insight into the vascular protective effect of estradiol.

Chronic Treatment with the G Protein-Coupled Receptor 30 Agonist G-1 Decreases Blood Pressure in Ovariectomized mRen2.Lewis Rats

Tue, 21 Jul 2009 10:03:34 PDT

The mRen2.Lewis congenic strain is an estrogen-sensitive model of hypertension whereby estrogen depletion produces a significant and sustained increase in blood pressure. The recent identification of G protein-coupled receptor 30 (GPR30) as a third estrogen receptor isotype prompted us to test the hypothesis that this novel receptor exhibits beneficial cardiovascular actions in the hypertensive female mRen2.Lewis rat. Intact female, ovariectomized female (OVX), and male mRen2.Lewis rats were treated with the selective GPR30 agonist G-1 or vehicle via osmotic minipump for 2 wk. G-1 significantly reduced systolic blood pressure in OVX (178 ± 7 to 142 ± 10 mm Hg, P < 0.001, n = 8) but not intact female (144 ± 3 to 143 ± 5 mm Hg, P > 0.05, n = 5) or male mRen2.Lewis rats (207 ± 7 to 192 ± 5 mm Hg, P > 0.05, n = 7). G-1 did not alter uterine or body weight in OVX, suggesting activation of a receptor distinct from estrogen receptor- and -β. In isolated aortic rings from OVX, G-1 reduced constriction in response to angiotensin II. Vascular angiotensin-converting enzyme and angiotensin type 1 receptor mRNA were also lower, whereas angiotensin-converting enzyme-2 mRNA was increased. G-1 treatment in OVX was not associated with alterations in either endothelial nitric oxide synthase expression or acetylcholine-induced relaxation. Immunohistochemical staining for GPR30 was evident in both the intima and media of the aorta. We conclude that the novel estrogen receptor GPR30 may contribute to the beneficial cardiovascular actions of estrogen in female mRen2.Lewis rats through regulation of vascular components of the renin-angiotensin system.

Guanylyl Cyclase-A Inhibits Angiotensin II Type 2 Receptor-Mediated Pro-Hypertrophic Signaling in the Heart

Tue, 21 Jul 2009 10:03:34 PDT

Angiotensin II plays a key role in the development of cardiac hypertrophy. The contribution of the angiotensin II type 1 receptor (AT1) in angiotensin II-induced cardiac hypertrophy is well established, but the role of AT2 signaling remains controversial. Previously, we have shown that natriuretic peptide receptor/guanylyl cyclase-A (GCA) signaling protects the heart from hypertrophy at least in part by inhibiting AT1-mediated pro-hypertrophic signaling. Here, we investigated the role of AT2 in cardiac hypertrophy observed in mice lacking GCA. Real-time RT-PCR and immunoblotting approaches indicated that the cardiac AT2 gene was overexpressed in GCA-deficient mice. Mice lacking AT2 alone did not exhibit an abnormal cardiac phenotype. In contrast, GCA-deficiency-induced increases in heart to body weight ratio, cardiomyocyte cross-sectional area, and collagen accumulation as evidenced by van Gieson staining were attenuated when AT2 was absent. Furthermore, the up-regulated cardiac expression of hypertrophy-related genes in GCA-null animals was also suppressed. Pharmacological blockade of AT2 with PD123319 similarly attenuated cardiac hypertrophy in GCA-deficient mice. In addition, whereas the AT1 antagonist olmesartan attenuated cardiac hypertrophy in GCA-deficient mice, this treatment was without effect on cardiac hypertrophy in GCA/AT2-double null mice, notwithstanding its potent antihypertensive effect in these animals. These results suggest that the interplay of AT2 and AT1 may be important in the development of cardiac hypertrophy. Collectively, our findings support the assertion that GCA inhibits AT2-mediated pro-hypertrophic signaling in heart and offer new insights into endogenous cardioprotective mechanisms during disease pathogenesis.

Activation of the Lutropin/Choriogonadotropin Receptor Inhibits Apoptosis of Immature Leydig Cells in Primary Culture

Tai, P., Shiraishi, K., Ascoli, M. — Tue, 21 Jul 2009 10:03:34 PDT

We used proliferating primary cultures of immature rat Leydig cells expressing the recombinant human LH/choriogonadotropin (CG) receptor (LHR) to test the hypothesis that activation of this receptor inhibits apoptosis. We also compared the effects of LH/CG with epidermal growth factor (EGF) and IGF-I because these have been previously shown to stimulate proliferation and/or inhibit apoptosis in Leydig cells. Human CG (hCG), EGF, and IGF-I stimulated the phosphorylation of ERK1/2 and Akt in primary cultures of immature rat Leydig cells. These three hormones also robustly stimulated thymidine incorporation and inhibited drug-induced apoptosis. Using selective inhibitors of ERK1/2 (UO126) or Akt phosphorylation (LY294002), we show that the ERK1/2 and Akt cascades are both involved in the hCG- and EGF-dependent proliferation of Leydig cells, but only the ERK1/2 cascade is involved in their antiapoptotic actions. The same strategy showed that the proliferative and antiapoptotic actions of IGF-I are mediated entirely by the Akt pathway. These results show that activation of the LHR inhibits apoptosis in Leydig cells and that it does so through stimulation of the ERK1/2 pathway.

Brain-Derived Neurotrophic Factor Promotes Implantation and Subsequent Placental Development by Stimulating Trophoblast Cell Growth and Survival

Kawamura, K., Kawamura, N., Sato, W., Fukuda, J., Kumagai, J., Tanaka, T. — Tue, 21 Jul 2009 10:03:34 PDT

Successful implantation of the blastocyst and subsequent placental development is essential for reproduction. Expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5, together with their receptor, tyrosine kinase B (TrkB), in trophectoderm cells of blastocyst suggests their potential roles in implantation and placental development. Here we demonstrated that treatment with BDNF promoted blastocyst outgrowth, but not adhesion, in vitro and increased levels of the cell invasion marker matrix metalloproteinase-9 in cultured blastocysts through the phosphatidylinositol 3-kinase pathway. After implantation, BDNF and neurotrophin-4/5 proteins as well as TrkB were expressed in trophoblast cells and placentas during different stages of pregnancy. Both TrkB and its ligands were also expressed in decidual cells. Treatment of cultured trophoblast cells with the TrkB ectodomain, or a Trk receptor inhibitor K252a, suppressed cell growth as reflected by decreased proliferation and increased apoptosis, whereas an inactive plasma membrane nonpermeable K252b was ineffective. Studies using the specific inhibitors also indicated the importance of the phosphatidylinositol 3-kinase/Akt pathway in mediating the action of TrkB ligands. In vivo studies in pregnant mice further demonstrated that treatment with K252a, but not K252b, suppressed placental development accompanied by increases in trophoblast cell apoptosis and decreases in placental labyrinth zone at midgestation. In vivo K252a treatment also decreased fetal weight at late gestational stages. Our findings suggested important autocrine/paracrine roles of the BDNF/TrkB signaling system during implantation, subsequent placental development, and fetal growth by increasing trophoblast cell growth and survival.

Follicle-Stimulating Hormone (FSH) Transiently Blocks FSH Receptor Transcription by Increasing Inhibitor of Deoxyribonucleic Acid Binding/Differentiation-2 and Decreasing Upstream Stimulatory Factor Expression in Rat Sertoli Cells

Viswanathan, P., Wood, M. A., Walker, W. H. — Tue, 21 Jul 2009 10:03:34 PDT

FSH acts through the FSH receptor (FSHR) to modulate cell processes that are required to support developing spermatozoa. Within the testis, only Sertoli cells possess receptors for FSH and are the major targets for this regulator of spermatogenesis. FSH stimulation of Sertoli cells for 24–48 h is known to induce Fshr mRNA expression through an E-box motif (CACGTG) located 25 bp upstream of the transcription start site. In contrast, FSH stimulation for 8 h inhibits Fshr transcription. DNA-protein binding studies performed using nuclear extracts from Sertoli cells show that protein binding to the Fshr promoter E-box was reduced 68% after 6 h of FSH stimulation but increased 191% over basal levels after 48 h of stimulation. The proteins binding to the Fshr E-box were identified as upstream stimulatory factor (USF)-1 and -2. FSH stimulation transiently decreased USF1 levels and increased the expression of the inhibitor of DNA binding/differentiation (ID)-2 repressor protein with the same kinetics as the decreased USF/E-box interactions. Overexpression of ID2 resulted in a dose-dependent decrease in USF-driven Fshr promoter activity in the MSC-1 Sertoli cell line, and ID2 inhibited USF binding to the Fshr E-box. Together, these studies suggest that stimulation of Sertoli cells with FSH transiently decreases expression of the USF1 activator and induces accumulation of the ID2 repressor, to block USF binding to the Fshr promoter and delay activation of Fshr transcription. This FSH-regulated mechanism may explain the cyclical changes in Fshr expression that occurs in Sertoli cells in vivo.

Metalloproteinase-Dependent Shedding of Low-Density Lipoprotein Receptor-Related Protein-1 Ectodomain Decreases Endocytic Clearance of Endometrial Matrix Metalloproteinase-2 and -9 at Menstruation

Tue, 21 Jul 2009 10:03:34 PDT

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

Expression of Ectodermal Neural Cortex 1 and Its Association with Actin during the Ovulatory Process in the Rat

Tue, 21 Jul 2009 10:03:34 PDT

Ectodermal neural cortex (ENC) 1, a member of the kelch family of genes, is an actin-binding protein and plays a pivotal role in neuronal and adipocyte differentiation. The present study was designed to examine the gonadotropin regulation and action of ENC1 during the ovulatory process in immature rats. The levels of ENC1 mRNA and protein were stimulated by LH/human chorionic gonadotropin (hCG) within 3 h both in vivo and in vitro. In situ hybridization analysis revealed that ENC1 mRNA was localized not only in theca/interstitial cells but also in granulosa cells of preovulatory follicles but not of growing follicles in pregnant mare’s serum gonadotropin/hCG-treated ovaries. LH-induced ENC1 expression was suppressed by a high dose of protein kinase C inhibitor RO 31-8220 (10 µm) but not by low doses of RO 31-8220 (0.1–1.0 µm), suggesting the involvement of atypical protein kinase C. ENC1 was detected in both nucleus and cytoplasm that was increased by LH/hCG treatment. Both biochemical and morphological analysis revealed that LH/hCG treatment increased actin polymerization within 3 h in granulosa cells. Interestingly, ENC1 physically associated with actin and treatment with cytochalasin D, an actin-depolymerizing agent, abolished this association. Confocal microscopy further demonstrated the colocalization of ENC1 with filamentous actin (F-actin). The present study demonstrates that LH/hCG stimulates ENC1 expression and increases F-actin formation in granulosa cells. The present study further shows the physical association of ENC1 and F-actin, implicating the role of ENC1 in cytoskeletal reorganization during the differentiation of granulosa cells.

A Unique Human Chorionic Gonadotropin Antagonist Suppresses Ovarian Hyperstimulation Syndrome in Rats

Tue, 21 Jul 2009 10:03:34 PDT

Ovarian hyperstimulation syndrome (OHSS) is a complication of in vitro fertilization associated with physiological changes after hCG administration to induce final oocyte maturation. It presents as widespread increases in vascular permeability and, in rare cases, results in cycle cancellation, multi-organ dysfunction, and pregnancy termination. These physiological changes are due primarily to activation of the vascular endothelial growth factor (VEGF) system in response to exogenous human chorionic gonadotropin (hCG). An hCG antagonist (hCG-Ant) could attenuate these effects by competitively binding to the LH/CG receptor, thereby blocking LH activity in vivo. We expressed a form of hCG that lacks three of its four N-linked glycosylation sites and tested its efficacy as an antagonist. The hCG-Ant binds the LH receptor with an affinity similar to native hCG and inhibits cAMP response in vitro. In a rat model for ovarian stimulation, hCG-Ant dramatically reduces ovulation and steroid hormone production. In a well-established rat OHSS model, vascular permeability and vascular endothelial growth factor (VEGF) expression are dramatically reduced after hCG-Ant treatment. Finally, hCG-Ant does not appear to alter blastocyst development when given after hCG in mice. These studies demonstrate that removing specific glycosylation sites on native hCG can produce an hCG-Ant that is capable of binding without activating the LH receptor and blocking the actions of hCG. Thus hCG-Ant will be investigated as a potential therapy for OHSS.

Distinct Expression Patterns of Glycoprotein Hormone-{alpha}2 and -{beta}5 in a Basal Chordate Suggest Independent Developmental Functions

Dos Santos, S., Bardet, C., Bertrand, S., Escriva, H., Habert, D., Querat, B. — Tue, 21 Jul 2009 10:03:34 PDT

The vertebrate glycoprotein hormones (GpHs), gonadotropins and thyrotropin, are heterodimers composed of a common - and specific β-subunit. The recombinant heterodimer of two additional, structurally related proteins identified in vertebrate and protostome genomes, the glycoproteins-2 (GPA2) and-β5 (GPB5), was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However, differences in tissue distribution and expression levels of these proteins suggested that they might act as nonassociated factors, prompting further investigation on these proteins. In this study we show that GPA2 and GPB5 appeared with the emergence of bilateria and were maintained in most groups. These genes are tightly associated at the genomic level, an association, however, lost in tetrapods. Our structural and genomic environment comparison reinforces the hypothesis of their phylogenetic relationships with GpH- and -β. In contrast, the glycosylation status of GPA2 and GPB5 is highly variable further questioning heterodimer secretory efficiency and activity. As a first step toward understanding their function, we investigated the spatiotemporal expression of GPA2 and GPB5 genes at different developmental stages in a basal chordate, the amphioxus. Expression of GPB5 was essentially ubiquitous with an anteroposterior gradient in embryos. GPA2 embryonic and larvae expression was restricted to specific areas and, interestingly, partially overlapped that of a GpH receptor-related gene. In conclusion, we speculate that GPA2 and GPB5 have nondispensable and coordinated functions related to a novelty appeared with bilateria. These proteins would be active during embryonic development in a manner that does not require their heterodimerization.

Estradiol-17{beta}, Prostaglandin E2 (PGE2), and the PGE2 Receptor Are Involved in PGE2 Positive Feedback Loop in the Porcine Endometrium

Waclawik, A., Jabbour, H. N., Blitek, A., Ziecik, A. J. — Tue, 21 Jul 2009 10:03:34 PDT

Before implantation, the porcine endometrium and trophoblast synthesize elevated amounts of luteoprotective prostaglandin estradiol-17β (E₂) (PGE₂). We hypothesized that embryo signal, E₂, and PGE₂ modulate expression of key enzymes in PG synthesis: PG-endoperoxide synthase-2 (PTGS2), microsomal PGE synthase (mPGES-1), PGF synthase (PGFS), and PG 9-ketoreductase (CBR1) as well as PGE₂ receptor (PTGER2 and -4) expression and signaling within the endometrium. We determined the site of action of PGE₂ in endometrium during the estrous cycle and pregnancy. Endometrial tissue explants obtained from gilts (n = 6) on d 11–12 of the estrous cycle were treated with vehicle (control), PGE₂ (100 nM), E₂ (1–100 nm), or phorbol 12-myristate 13-acetate (100 nm, positive control). E₂ increased PGE₂ secretion through elevating expression of mPGES-1 mRNA and PTGS2 and mPGES-1 protein in endometrial explants. By contrast, E₂ decreased PGFS and CBR1 protein expression. E₂ also stimulated PTGER2 but not PTGER4 protein content. PGE₂ enhanced mPGES-1 and PTGER2 mRNA as well as PTGS2, mPGES-1, and PTGER2 protein expression. PGE₂ had no effect on PGFS, CBR1, and PTGER4 expression and PGF₂ release. Treatment of endometrial tissue with PGE₂ increased cAMP production. Cotreatment with PTGER2 antagonist (AH6809) but not PTGER4 antagonist (GW 627368X) inhibited significantly PGE₂-mediated cAMP production. PTGER2 protein was localized in luminal and glandular epithelium and blood vessels of endometrium and was significantly up-regulated on d 11–12 of pregnancy. Our results suggest that E₂ prevents luteolysis through enzymatic modification of PG synthesis and that E₂, PGE₂, and endometrial PTGER2 are involved in a PGE₂ positive feedback loop in porcine endometrium.

Progesterone Receptor A (PRA) and PRB-Independent Effects of Progesterone on Gonadotropin-Releasing Hormone Release

Tue, 21 Jul 2009 10:03:34 PDT

Progesterone’s (P4) negative feedback actions in the female reproductive axis are exerted in part by suppression of hypothalamic GnRH release. Here we show that P4 can inhibit GnRH release by a mechanism independent of a nuclear P4 receptor (PR_A/B). Injections of P4, but not vehicle, allopregnanolone, or dexamethasone, acutely suppressed LH levels in both wild-type and P4 receptor knockout ovariectomized mice; pituitary responsiveness to GnRH was retained during P4 treatment, indicating a hypothalamic action. Superfusion of GnRH-producing GT1-7 cells with medium containing 10^–7 m P4 produced a rapid reduction in GnRH release. Incubation with P4 (10^–9 to 10^–7 m) inhibited forskolin-stimulated cAMP accumulation; cotreatment with pertussis toxin prevented this effect. Treatment of GT1-7 cell membranes with P4 caused activation of an inhibitory G protein (G_i), as shown by immunoprecipitation with a G_i antibody of most of the increase in membrane-bound [³⁵S]GTP-S. Saturation binding analyses demonstrated the presence of a high affinity (K_d 5.85 nm), limited capacity (Bmax 62.2 nm) binding site for P4. RT-PCR analysis revealed the presence of mRNAs encoding both isoforms of the membrane P4 receptors, mPR and mPRβ. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPR siRNA attenuated specific P4 binding to GT1-7 cell membranes and reversed the P4 inhibition of cAMP accumulation. Taken together, our results suggest that negative feedback actions of P4 include rapid PR_A/B-independent effects on GnRH release that may in part be mediated by mPRs.

Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) Functions to Promote Uterine Decidual Angiogenesis during Early Pregnancy in the Mouse

Tue, 21 Jul 2009 10:03:34 PDT

Implantation of an embryo induces rapid proliferation and differentiation of uterine stromal cells, forming a new structure, the decidua. One salient feature of decidua formation is a marked increase in maternal angiogenesis. Vascular endothelial growth factor (VEGF)-dependent pathways are active in the ovary, uterus, and embryo, and inactivation of VEGF function in any of these structures might prevent normal pregnancy development. We hypothesized that decidual angiogenesis is regulated by VEGF acting through specific VEGF receptors (VEGFRs). To test this hypothesis, we developed a murine pregnancy model in which systemic administration of a receptor-blocking antibody would act specifically on uterine angiogenesis and not on ovarian or embryonic angiogenesis. In our model, ovarian function was replaced with exogenous progesterone, and blocking antibodies were administered prior to embryonic expression of VEGFRs. After administration of a single dose of the anti-VEGFR-2 antibody during the peri-implantation period, no embryos were detected on embryonic d 10.5. The pregnancy was disrupted because of a significant reduction in decidual angiogenesis, which under physiological conditions peaks on embryonic d 5.5 and 6.5. Inactivation of VEGFR-3 reduced angiogenesis in the primary decidual zone, whereas administration of VEGFR-1 blocking antibodies had no effect. Pregnancy was not disrupted after administration of anti-VEGFR-3 or anti-VEGFR-1 antibodies. Thus, the VEGF/VEGFR-2 pathway plays a key role in the maintenance of early pregnancy through its regulation of peri-implantation angiogenesis in the uterine decidua. This newly formed decidual vasculature serves as the first exchange apparatus for the developing embryo until the placenta becomes functionally active.

Theca-Specific Estrogen Receptor-{alpha} Knockout Mice Lose Fertility Prematurely

Tue, 21 Jul 2009 10:03:34 PDT

Estrogen receptor- (Esr1) mediates estrogen action in regulating at all levels of the hypothalamic-pituitary-ovarian axis. Whereas the importance of Esr1 in hypothalamus and pituitary has been demonstrated by loss of fertility in the neuron- and pituitary-specific Esr1 knockout mice, whether Esr1 plays a critical role in the ovary remains to be determined. In the ovary, Esr1 is mainly expressed in the theca/interstitial cells and germinal epithelium and thus is believed to mediate estrogen action in these cells. In this study, we assessed the importance of Esr1 in the ovarian theca cells in regulating female reproduction. The Cre-LoxP approach was used to selectively delete the Esr1 gene in the theca cells, and the reproductive consequence of the deletion was measured. Adolescent theca-specific Esr1 knockout (thEsr1KO) mice (<4 months of age) are fertile and cycling. However, they begin to display an erratic pattern of estrous cycles and become infertile before they reach the age of 6 months. The ovaries of thEsr1KOmice (≥4 months) have fewer corpora lutea but more antral follicles than the age-matching wild-type mice. The numbers of 17-hydroxylase-expressing cells are largely increased in the interstitium of the thEsr1KO mouse ovary. Interestingly, whereas basal levels of serum testosterone and FSH were mildly elevated, LH level was either markedly lower or undetectable in the thEsr1KO mice. When superstimulated by exogenous gonadotropins, thEsr1KO mice released significantly fewer oocytes that wild-type littermates and developed multiple hemorrhagic cysts. Taken together, this study demonstrates that theca Esr1 plays a critical role in regulating female reproduction.

The Progesterone Receptor Coactivator Hic-5 Is Involved in the Pathophysiology of Endometriosis

Aghajanova, L., Velarde, M. C., Giudice, L. C. — Tue, 21 Jul 2009 10:03:34 PDT

Endometriosis is an estrogen-dependent disorder primarily associated with pelvic pain and infertility in up to 10% of women of reproductive age. Recent studies suggest that resistance to progesterone action may contribute to the development and pathophysiology of this disorder. In this study we examined the in vivo and in vitro expression and function of one progesterone receptor (PR) coactivator, Hic-5, in human endometrium and endometrial stromal fibroblasts (hESFs) from 29 women with and 30 (control) women without endometriosis. Hic-5 was highly expressed in stromal, but not epithelial, cells in women without endometriosis, in a cycle-dependent manner. In contrast, Hic-5 expression was not regulated during the menstrual cycle in hESFs from women with endometriosis and was significantly reduced in hESFs from women with vs. without disease. Hic-5 mRNA expression throughout the cycle in endometrium from control women, but not those with endometriosis, correlated with expression of PR. Hic-5 mRNA in hESFs was significantly up-regulated in control but not endometriosis hESFs after treatment in vitro with 8-bromoadenosine-cAMP for 96 h but only modestly after 14 d of progesterone treatment. Hic-5 silencing did not influence cAMP-regulated gene expression but affected genes regulated solely by progesterone (e.g. DKK1 and calcitonin). Together the data suggest that the proposed progesterone resistance in endometrium from women with endometriosis derives, in part, from impaired expression of the PR coactivator, Hic-5, in endometrial tissue and cultured endometrial stromal fibroblasts.

Stromal Progesterone Receptors Mediate Induction of Indian Hedgehog (IHH) in Uterine Epithelium and Its Downstream Targets in Uterine Stroma

Tue, 21 Jul 2009 10:03:34 PDT

Uterine receptivity to embryo implantation depends on appropriate progesterone (P4) and estrogen stimulation. P4 rapidly stimulates production of the morphogen Indian hedgehog (IHH) in murine uterine epithelium as well as downstream molecules in the hedgehog pathway such as Patched homolog 1 (PTCH1) and nuclear receptor subfamily 2, group F, member 2 (NR2F2) in uterine stroma. Studies using IHH-null mice indicate that IHH is obligatory for the normal P4 response in the uterus. To determine whether IHH induction in uterine epithelium is mediated through P4 receptor (PR) in epithelium (E) and/or stroma (S), we produced tissue recombinants using uteri from neonatal PR knockout (ko) mice and wild-type (wt) mice containing PR in S and/or E or lacking PR altogether using a tissue recombinant methodology and assessed their response to P4. In tissue recombinants containing wt-S (wt-S + wt-E and wt-S + ko-E), P4 induced Ihh mRNA expression at 6 h that was 6-fold greater than in oil-treated controls (P < 0.05; n = 6) in both types of tissue recombinants despite the absence of epithelial PR in wt-S + ko-E grafts. Conversely, Ihh mRNA expression was unaffected by P4 in ko-S + ko-E and ko-S + wt-E grafts despite epithelial PR expression in the latter. Nr2f2 and Ptch1 mRNA expression was similar in that it was stimulated by P4 only in recombinants containing stromal PR. These results indicate that stromal PR is both necessary and sufficient for P4 stimulation of epithelial IHH as well as downstream events such as PTCH1 and NR2F2 increases in stroma.

Estrogens Augment Cell Surface TLR4 Expression on Murine Macrophages and Regulate Sepsis Susceptibility in Vivo

Rettew, J. A., Huet, Y. M., Marriott, I. — Tue, 21 Jul 2009 10:03:34 PDT

Gender-based differences exist in infectious disease susceptibility. In general, females generate more robust and potentially protective humoral and cell-mediated immune responses after antigenic challenge than their male counterparts. Furthermore, evidence is accumulating that sex may also influence the early perception of microbial challenges and the generation of inflammatory immune responses such as sepsis. These differences have previously been attributed to the actions of reproductive hormones. Whereas androgens have been shown to suppress acute host immune responses to bacterial endotoxin challenge, estrogens have been found to promote increased resistance to bacterial infections. However, the mechanisms by which estrogens exert immunoprotective effects have not been established. In this study, we investigated the in vivo effects of 17β-estradiol on endotoxin susceptibility in mice. Importantly, we have examined the actions of this female reproductive hormone on the expression of pattern recognition receptors that recognize bacterial endotoxin by key innate immune sentinel cells. We show that removal of endogenous estrogens decreases both pro- and antiinflammatory cytokine production, with a concomitant reduction in circulating levels of lipopolysaccharide-binding protein and cell surface expression of Toll-like receptor 4 on murine macrophages. Exogenous in vivo replacement of 17β-estradiol, but not progesterone, significantly elevates sera lipopolysaccharide-binding protein levels and cell surface expression of Toll-like receptor 4 and CD14 on macrophages. Furthermore, this effect corresponds with significantly higher inflammatory cytokine levels after in vivo lipopolysaccharide challenge and a marked increase in endotoxin-associated morbidity. Taken together, these data provide a potential mechanism underlying the immunoenhancing effects of estrogens.