Endocrinology Current Issue

If I Only Had a Whole Brain: The Importance of Extrahypothalamic Areas in the Energy Balance Equation

Schneider, J. E. — Fri, 20 Nov 2009 10:02:55 PST

Lighting Up Neuronal Pathways: The Development of a Novel Transgenic Rat that Identifies Fos-Activated Neurons Using a Red Fluorescent Protein

Appleyard, S. M. — Fri, 20 Nov 2009 10:02:55 PST

Insulin-Regulated Glucagon-Like Peptide-1 Release from L Cells: Actin' Out

Thurmond, D. C. — Fri, 20 Nov 2009 10:02:55 PST

Urocortin: A Few Inflammatory Remarks

Davidson, S. M., Yellon, D. M. — Fri, 20 Nov 2009 10:02:55 PST

Smad1-Smad5 Ovarian Conditional Knockout Mice Develop a Disease Profile Similar to the Juvenile Form of Human Granulosa Cell Tumors

Middlebrook, B. S., Eldin, K., Li, X., Shivasankaran, S., Pangas, S. A. — Fri, 20 Nov 2009 10:02:55 PST

Granulosa cell tumors (GCTs) of the ovary are rare sex cord stromal tumors. Although generally indolent, GCTs recur, and if not diagnosed and treated in early stages, survival rates are significantly shortened. Very little is known regarding GCT etiology. Because of the low incidence of cases and lack of standard diagnostics, mouse models for granulosa cell tumors are a valuable tool for studying GCTs and provide models for developing diagnostic and treatment strategies. We recently developed a novel mouse model of metastatic granulosa cell tumors by genetic deletion of the bone morphogenetic protein signaling transcription factors (SMADs) in granulosa cells of the ovary. Histological and serum hormone analyses reveal that this mouse model most closely resembles the juvenile form of GCT. We further analyzed samples of human juvenile GCT (JGCT) for expression of anti-Müllerian hormone and activation of two major signaling pathways: TGFβ/SMAD2/3 and wingless-related mouse mammary tumor virus integration site (Wnt)/β-catenin. The TGFβ family is active in mouse Smad1-Smad5 double knockout tumors, and here we show that this pathway, but not the β-catenin pathway, is activated in samples of human JGCT. These data suggest that the SMAD family, possibly through disruption of SMAD1/5 or activation of SMAD2/3 may contribute to the pathogenesis of JGCT in humans.

Free Fatty Acids Induce a Proinflammatory Response in Islets via the Abundantly Expressed Interleukin-1 Receptor I

Fri, 20 Nov 2009 10:02:55 PST

Islets of patients with type 2 diabetes mellitus (T2DM) display features of an inflammatory process including elevated levels of the cytokine IL-1β, various chemokines, and macrophages. IL-1β is a master regulator of inflammation, and IL-1 receptor type I (IL-1RI) blockage improves glycemia and insulin secretion in humans with T2DM and in high-fat-fed mice pointing to a pivotal role of IL-1RI activity in intra-islet inflammation. Given the association of dyslipidemia and T2DM, we tested whether free fatty acids (FFA) promote the expression of proinflammatory factors in human and mouse islets and investigated a role for the IL-1RI in this response. A comparison of 22 mouse tissues revealed the highest IL-1RI expression levels in islets and MIN6 β-cells. FFA induced IL-1β, IL-6, and IL-8 in human islets and IL-1β and KC in mouse islets. Elevated glucose concentrations enhanced FFA-induced proinflammatory factors in human islets. Blocking the IL-1RI with the IL-1R antagonist (IL-1Ra) strongly inhibited FFA-mediated expression of proinflammatory factors in human and mouse islets. Antibody inhibition of IL-1β revealed that FFA stimulated IL-1RI activity via the induction of the receptor ligand. FFA-induced IL-1β and KC expression in mouse islets was completely dependent on the IL-1R/Toll-like receptor (TLR) docking protein Myd88 and partly dependent on TLR2 and -4. Activation of TLR2 in purified human β-cells and islets stimulated the expression of proinflammatory factors, and IL-1RI activity increased the TLR2 response in human islets. We conclude that FFA and TLR stimulation induce proinflammatory factors in islets and that IL-1RI engagement results in signal amplification.

Ubiquitin Carboxyl-Terminal Hydrolase L3 Promotes Insulin Signaling and Adipogenesis

Suzuki, M., Setsuie, R., Wada, K. — Fri, 20 Nov 2009 10:02:55 PST

Insulin is a potent adipogenic hormone that triggers the induction of a series of transcription factors and specific proteins governing the differentiation of preadipocytes into mature adipocytes. Here we report that ubiquitin carboxyl-terminal hydrolase (UCH)-L3, a deubiquitinating enzyme, promotes insulin signaling and adipogenesis. Uchl3^–/– mice had less visceral white adipose tissue compared with wild-type mice. In vitro adipogenesis experiments revealed that mouse embryonic fibroblasts (MEFs) and preadipocytes from Uchl3^–/– mice had impaired ability to differentiate into mature adipocytes than those from wild-type mice. This difference was diminished by removing insulin from the medium. RT-PCR analysis showed that insulin-regulated expression of srebp1c, fas, glut4, and adiponectin is impaired in Uchl3^–/– cells. The phosphorylation of insulin/IGF-I receptor, Akt, glycogen synthase kinase-3β, and FoxO1 was decreased in Uchl3^–/– MEFs treated with insulin. Moreover, ectopic expression of wild-type UCH-L3 restored the phosphorylation of insulin/IGF-I receptor and adipocyte differentiation in Uchl3^–/– MEFs. In contrast, hydrolase activity-deficient UCH-L3 did not enhance insulin signaling and the expression of glut4, fabp4, and adiponectin, resulting in impaired formation of large lipid droplets. These results suggest that UCH-L3 promotes adipogenesis by enhancing insulin signaling in a hydrolase activity-dependent manner.

Neurogenin 3-Specific Dipeptidyl Peptidase-2 Deficiency Causes Impaired Glucose Tolerance, Insulin Resistance, and Visceral Obesity

Fri, 20 Nov 2009 10:02:55 PST

The control of glucose metabolism is a complex process, and dysregulation at any level can cause impaired glucose tolerance and insulin resistance. These two defects are well-known characteristics associated with obesity and onset of type 2 diabetes. Here we introduce the N-terminal dipeptidase, DPP2, as a novel regulator of the glucose metabolism. We generated mice with a neurogenin 3 (NGN3)-specific DPP2 knockdown (kd) to explore a possible role of DPP2 in maintaining metabolic homeostasis. These mice spontaneously developed hyperinsulinemia, glucose intolerance, and insulin resistance by 4 months of age. In addition, we observed an increase in food intake in DPP2 kd mice, which was associated with a significant increase in adipose tissue mass and enhanced liver steatosis but no difference in body weight. In accordance with these findings, the mutant mice had a higher rate of respiratory exchange than the control littermates. This phenotype was exacerbated with age and when challenged with a high-fat diet. We report, for the first time, that DPP2 enzyme activity is essential for preventing hyperinsulinemia and maintaining glucose homeostasis. Interestingly, the phenotype of NGN3-DPP2 kd mice is opposite that of DPP4 knockout mice with regard to glucose metabolism, namely the former have normal glucagon-like peptide 1 levels but present with glucose intolerance, whereas the latter have increased glucagon-like peptide 1, which is accompanied by augmented glucose tolerance.

The Rho Guanosine 5'-Triphosphatase, Cell Division Cycle 42, Is Required for Insulin-Induced Actin Remodeling and Glucagon-Like Peptide-1 Secretion in the Intestinal Endocrine L Cell

Lim, G. E., Xu, M., Sun, J., Jin, T., Brubaker, P. L. — Fri, 20 Nov 2009 10:02:55 PST

Rho GTPases, such as cell division cycle 42 (Cdc42) and ras-related C3 botulinum toxin substrate 1 (Rac1), have been identified as regulators of F-actin dynamics and hormone release from endocrine cells; however, their role in secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1), from the enteroendocrine L cell is unknown. Insulin induced a 1.4-fold increase in L cell GLP-1 release; however, secretion was potentiated to 2.1-fold in the presence of the F-actin depolymerizing agent, latrunculin B, suggesting that F-actin functions as a permissive barrier. In murine GLUTag L cells, insulin stimulated F-actin depolymerization and Cdc42 activation simultaneously, and these events occurred prior to detectable increases in insulin-induced GLP-1 release. After insulin treatment, Cdc42-dependent p21-activated kinase-1 (PAK1) activation was also detected, and transfection of small-interfering RNA against Cdc42 or of dominant-negative Cdc42(T17N) impaired insulin-stimulated PAK1 activation, actin remodeling, and GLP-1 secretion. Overexpression of kinase-dead PAK1(K299R) or PAK1 small interfering RNA similarly attenuated insulin-induced GLP-1 secretion. Knockdown or inhibition of Cdc42 and PAK1 activities also prevented activation of MAPK/ERK (MEK)-1/2-ERK1/2 by insulin, which was previously identified as a critical pathway for insulin-regulated GLP-1 release. Taken together, these data identify a novel signaling pathway in the endocrine L cell, whereby Cdc42 regulates actin remodeling, activation of the cannonical 1/2-ERK1/2 pathway and PAK1, and GLP-1 secretion in response to insulin.

Impact of Simvastatin on Adipose Tissue: Pleiotropic Effects in Vivo

Khan, T., Hamilton, M. P., Mundy, D. I., Chua, S. C., Scherer, P. E. — Fri, 20 Nov 2009 10:02:55 PST

Statins belong to a class of drugs well known for their ability to reduce circulating low-density lipoprotein cholesterol. In addition to cholesterol lowering, they also exhibit potential antiinflammatory and antioxidant properties, suggesting that tissues other than liver may be targeted by statins to exert their beneficial metabolic effects. Adipocytes have received very little attention as a potential target of these drugs, possibly because adipocytes are not a major source of biosynthetic cholesterol. Here, we examine the effects of simvastatin on the secretory pathway, inflammation, and cellular metabolism of adipocytes as well as on whole-body insulin sensitivity. We find that statins have a selective effect on the secretion of the insulin-sensitizing adipokine adiponectin by reducing circulating levels of the high-molecular-weight form of adiponectin specifically with a concomitant increase in intracellular adiponectin levels. However, these effects on adiponectin do not translate into changes in metabolism or whole-body insulin sensitivity, potentially due to additional antiinflammatory properties of statins. In addition, ob/ob mice treated with statins have reduced adiposity and an altered ultrastructure of the plasma membrane with respect to caveolar histology. Our data demonstrate that statins have major effects on the cellular physiology of the adipocyte on multiple levels.

Poly(Adenosine 5'-Diphosphate-Ribose) Polymerase Inhibition Counteracts Multiple Manifestations of Experimental Type 1 Diabetic Nephropathy

Fri, 20 Nov 2009 10:02:55 PST

This study was aimed at evaluating the role for poly(ADP-ribose) polymerase (PARP) in early nephropathy associated with type 1 diabetes. Control and streptozotocin-diabetic rats were maintained with or without treatment with one of two structurally unrelated PARP inhibitors, 1,5-isoquinolinediol (ISO) and 10-(4-methyl-piperazin-1-ylmethyl)-2H-7-oxa-1,2-diaza-benzo[de] anthracen-3-one (GPI-15427), at 3 mg/kg^–1 · d^–1 ip and 30 mg/kg^–1 · d^–1, respectively, for 10 wk after the first 2 wk without treatment. PARP activity in the renal cortex was assessed by immunohistochemistry and Western blot analysis of poly(ADP-ribosyl)ated proteins. Variables of diabetic nephropathy in urine and renal cortex were evaluated by ELISA, Western blot analysis, immunohistochemistry, and colorimetry. Urinary albumin excretion was increased about 4-fold in diabetic rats, and this increase was prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-associated increase in poly(ADP-ribose) immunoreactivities in renal glomeruli and tubuli and poly(ADP-ribosyl)ated protein level. Renal concentrations of TGF-β₁, vascular endothelial growth factor, endothelin-1, TNF-, monocyte chemoattractant protein-1, lipid peroxidation products, and nitrotyrosine were increased in diabetic rats, and all these changes as well as an increase in urinary TNF- excretion were completely or partially prevented by ISO and GPI-15427. PARP inhibition counteracted diabetes-induced up-regulation of endothelin (B) receptor, podocyte loss, accumulation of collagen-1 (IY), periodic acid-Schiff-positive substances, fibronectin, and advanced glycation end-products in the renal cortex. In conclusion, PARP activation is implicated in multiple changes characteristic for early nephropathy associated with type 1 diabetes. These findings provide rationale for development and further studies of PARP inhibitors and PARP inhibitor-containing combination therapies.

Forkhead Box O1/Pancreatic and Duodenal Homeobox 1 Intracellular Translocation Is Regulated by c-Jun N-Terminal Kinase and Involved in Prostaglandin E2-Induced Pancreatic {beta}-Cell Dysfunction

Meng, Z., Lv, J., Luo, Y., Lin, Y., Zhu, Y., Nie, J., Yang, T., Sun, Y., Han, X. — Fri, 20 Nov 2009 10:02:55 PST

Prostaglandin E₂ (PGE₂) is a well-known mediator of β-cell dysfunction in both type 1 and type 2 diabetes. We recently reported that down-regulation of the Akt pathway activity is implicated in PGE₂-induced pancreatic β-cell dysfunction. The aim of this study was to further dissect the signaling pathway of this process in pancreatic β-cell line HIT-T15 cells and primary mouse islets. We found that PGE₂ time-dependently increased the c-Jun N-terminal kinase (JNK) pathway activity. JNK inhibition by the JNK-specific inhibitor SP600125 reversed PGE₂-inhibited glucose-stimulated insulin secretion (GSIS). PGE₂ induced dephosphorylation of Akt and FOXO1, leading to nuclear localization and transactivation of FOXO1. Activation of FOXO1 induced nuclear exclusion but had no obvious effect on the whole-cell protein level of pancreatic and duodenal homeobox 1 (PDX1). However, these effects were all attenuated by JNK inhibition. Furthermore, adenovirus-mediated overexpression of dominant-negative (DN)-FOXO1 abolished whereas constitutively active (CA)-FOXO1 mimicked the effects of PGE₂ on GSIS in isolated mouse islets. In addition, we demonstrated that DN-JNK1 but not DN-JNK2 or CA-Akt abolished the PGE₂-induced AP-1 luciferase reporter activity, whereas DN-JNK1 and CA-Akt but not DN-JNK2 reversed the effect of PGE₂ on FOXO1 transcriptional activity, and overexpression of DN-JNK1 rescued PGE₂-impaired GSIS in mouse islets. Our results revealed that activation of the JNK is involved in PGE₂-induced β-cell dysfunction. PGE₂-mediated JNK1 activation, through dephosphorylation of Akt and FOXO1, leads to nuclear accumulation of FOXO1 and nucleocytoplasmic shuttling of PDX1, finally resulting in defective GSIS in pancreatic β-cells.

Increased Tau Phosphorylation and Cleavage in Mouse Models of Type 1 and Type 2 Diabetes

Kim, B., Backus, C., Oh, S., Hayes, J. M., Feldman, E. L. — Fri, 20 Nov 2009 10:02:55 PST

As the population of the United States ages, the incidence of age-related neurodegenerative and systemic diseases including Alzheimer’s disease (AD) and diabetes is increasing rapidly. Multiple studies report that patients with diabetes have a 50–75% increased risk of developing AD compared with age- and gender-matched patients without diabetes. Abnormally phosphorylated tau is a major building block of neurofibrillary tangles, a classic neuropathological characteristic of AD. In addition, proteolytic tau cleavage promotes AD progression due to cleaved tau serving as a nucleation center for the pathological assembly of tau filaments. The current study examines tau modification in type 1 (streptozotocin-injected) and type 2 (db/db) mouse models of diabetes. Tau phosphorylation is increased in the cortex and hippocampus of db/db mice compared with db+ control mouse brain. Interestingly, there is an age-dependent increase in tau cleavage that is not observed in age-matched control db+ animals. Streptozotocin injection also increased tau phosphorylation; however, the increase was less significant compared with the type 2 mouse model, and more importantly, no tau cleavage was detected. Our results suggest tau modification caused by insulin dysfunction and hyperglycemia may contribute to the increased incidence of AD in diabetes. We hypothesize that type 1 and type 2 diabetes may contribute to AD through different mechanisms; in type 2 diabetes, hyperglycemia-mediated tau cleavage may be the key feature, whereas insulin deficiency may be the major contributing factor in type 1 diabetes.

Facilitative Glucose Transporter 9 Expression Affects Glucose Sensing in Pancreatic {beta}-Cells

Evans, S. A., Doblado, M., Chi, M. M., Corbett, J. A., Moley, K. H. — Fri, 20 Nov 2009 10:02:55 PST

Facilitative glucose transporters (GLUTs) including GLUT9, accelerate the facilitative diffusion of glucose across the plasma membrane. Studies in GLUT2-deficient mice suggested the existence of another GLUT in the mammalian β-cell responsible for glucose sensing. The objective of this study was to determine the expression and function of GLUT9 in murine and human β-cells. mRNA and protein expression levels were determined for both isoforms of GLUT9 in murine and human isolated islets as well as insulinoma cell lines (MIN6). Immunohistochemistry and subcellular localization were performed to localize the protein within the cell. Small interfering RNA knockdown of GLUT9 was used to determine the effect of this transporter, in the presence of GLUT2, on cell metabolism and insulin secretion in MIN6 and INS cells. In this report we demonstrate that GLUT9a and GLUT9b are expressed in pancreatic islets and that this expression localizes to insulin-containing β-cells. Subcellular localization studies indicate that mGLUT9b is found associated with the plasma membrane as well as in the high-density microsome fraction and low-density microsome fraction, whereas mGLUT9a appears to be located only in the high-density microsome and low-density microsome under basal conditions. Functionally GLUT9 appears to participate in the regulation of glucose-stimulated insulin secretion in addition to GLUT2. small interfering RNA knockdown of GLUT9 results in reduced cellular ATP levels that correlate with reductions in glucose-stimulated insulin secretion in MIN6 and INS cells. These studies confirm the expression of GLUT9a and GLUT9b in murine and human β-cells and suggest that GLUT9 may participate in glucose-sensing in β-cells.

Melatonin Improves Glucose Homeostasis and Endothelial Vascular Function in High-Fat Diet-Fed Insulin-Resistant Mice

Fri, 20 Nov 2009 10:02:55 PST

Obesity and insulin resistance represent a problem of utmost clinical significance worldwide. Insulin-resistant states are characterized by the inability of insulin to induce proper signal transduction leading to defective glucose uptake in skeletal muscle tissue and impaired insulin-induced vasodilation. In various pathophysiological models, melatonin interacts with crucial molecules of the insulin signaling pathway, but its effects on glucose homeostasis are not known. In a diet-induced mouse model of insulin resistance and normal chow-fed control mice, we sought to assess the effects of an 8-wk oral treatment with melatonin on insulin and glucose tolerance and to understand underlying mechanisms. In high-fat diet-fed mice, but not in normal chow-fed control mice, melatonin significantly improved insulin sensitivity and glucose tolerance, as evidenced by a higher rate of glucose infusion to maintain euglycemia during hyperinsulinemic clamp studies and an attenuated hyperglycemic response to an ip glucose challenge. Regarding underlying mechanisms, we found that melatonin restored insulin-induced vasodilation to skeletal muscle, a major site of glucose utilization. This was due, at least in part, to the improvement of insulin signal transduction in the vasculature, as evidenced by increased insulin-induced phosphorylation of Akt and endoethelial nitric oxide synthase in aortas harvested from melatonin-treated high-fat diet-fed mice. In contrast, melatonin had no effect on the ability of insulin to promote glucose uptake in skeletal muscle tissue in vitro. These data demonstrate for the first time that in a diet-induced rodent model of insulin resistance, melatonin improves glucose homeostasis by restoring the vascular action of insulin.

Adiponectin Expression Is Induced by Vitamin E via a Peroxisome Proliferator-Activated Receptor {gamma}-Dependent Mechanism

Fri, 20 Nov 2009 10:02:55 PST

Adiponectin is a well-known adipokine secreted by adipocytes that presents insulin-sensitizing properties. The regulation of expression of this adipokine by micronutrients is largely unknown. We demonstrate here that adiponectin expression is induced in adipocytes after exposure to tocopherols via the peroxisome proliferator-activated receptor (PPAR) pathway. Vitamin E force feeding resulted in an induction of adiponectin in mice at both mRNA and protein levels. Adiponectin mRNA and protein secretion were also increased by vitamin E (- and -tocopherol) in 3T3-L1 cells, together with PPAR mRNA, independent of an antioxidant effect. In transient transfections, both - and -vitamers induced the luciferase gene reporter under the control of a human adiponectin promoter via a PPAR-responsive element. The induction of adiponectin by tocopherols seems to be PPAR dependent, because it was blocked by the specific antagonist GW9662. Finally, we showed that intracellular concentrations of a PPAR endogenous ligand, 15-deoxy-12,14-prostaglandin J2, increased after treatment with tocopherols in 3T3-L1 cells. In summary, vitamin E up-regulates adiponectin expression via a mechanism that implicates PPAR together with its endogenous ligand 15-deoxy-12,14-prostaglandin J2. The induction of adiponectin via an original molecular mechanism could be considered as the basis for the beneficial effect of vitamin E on insulin sensitivity.

Central Administration of Resveratrol Improves Diet-Induced Diabetes

Fri, 20 Nov 2009 10:02:55 PST

Resveratrol is a natural polyphenolic compound that activates nicotinamide adenosine dinucleotide-dependent deacetylase SIRT1. Resveratrol has recently been shown to exert potent antidiabetic actions when orally delivered to animal models of type 2 diabetes. However, the tissue(s) mediating these beneficial effects is unknown. Because SIRT1 is expressed in central nervous system (CNS) neurons known to control glucose and insulin homeostasis, we hypothesized that resveratrol antidiabetic effects are mediated by the brain. Here, we report that long-term intracerebroventricular infusion of resveratrol normalizes hyperglycemia and greatly improves hyperinsulinemia in diet-induced obese and diabetic mice. It is noteworthy that these effects are independent of changes in body weight, food intake, and circulating leptin levels. In addition, CNS resveratrol delivery improves hypothalamic nuclear factor-B inflammatory signaling by reducing acetylated-RelA/p65 and total RelA/p65 protein contents, and inhibitor of nuclear factor-B and IB kinase β mRNA levels. Furthermore, this treatment leads to reduced hepatic phosphoenolpyruvate carboxykinase 1 mRNA and protein levels and ameliorates pyruvate-induced hyperglycemia in this mouse model of type 2 diabetes. Collectively, our results unveiled a previously unrecognized key role for the CNS in mediating the antidiabetic actions of resveratrol.

Glucose Generates Coincident Insulin and Somatostatin Pulses and Antisynchronous Glucagon Pulses from Human Pancreatic Islets

Hellman, B., Salehi, A., Gylfe, E., Dansk, H., Grapengiesser, E. — Fri, 20 Nov 2009 10:02:55 PST

The kinetics of insulin, glucagon and somatostatin release was studied in human pancreatic islets. Batches of 10–15 islets were perifused and the hormones measured with RIA in 30-sec fractions. Increase of glucose from 3 to 20 mm resulted in a brief pulse of glucagon coinciding with suppression of basal insulin and somatostatin release. There was a subsequent drop of glucagon release concomitant with the appearance of a pronounced pulse of insulin and a slightly delayed pulse of somatostatin. Continued exposure to 20 mm glucose generated pulsatile release of the three hormones with 7- to 8-min periods accounting for 60–70% of the secreted amounts. Glucose caused pronounced stimulation of average insulin and somatostatin release. However, the nadirs between the glucagon pulses were lower than the secretion at 3 mm glucose, resulting in 18% suppression of average release. The repetitive glucagon pulses were antisynchronous to coincident pulses of insulin and somatostatin. The resulting greater than 20-fold variations of the insulin to glucagon ratio might be essential for minute-to-minute regulation of the hepatic glucose production.

Response to Carbohydrate and Fat Refeeding in the Expression of Genes Involved in Nutrient Partitioning and Metabolism: Striking Effects on Fibroblast Growth Factor-21 Induction

Sanchez, J., Palou, A., Pico, C. — Fri, 20 Nov 2009 10:02:55 PST

This study aimed to assess the effects of carbohydrate (CHO) and fat intake on the expression of key genes related with nutrient partitioning and metabolism in main tissues involved in energy metabolism (white adipose tissue, liver, and skeletal muscle). Rats were studied under different conditions: feeding state, 24 h fasting, and 12 h refeeding after 24 h fasting with isocaloric amounts of CHO or fat. Fat, but not CHO, refeeding was associated with an increase in serum and liver triglyceride content. Main changes in gene expression elicited by CHO compared with fat refeeding were: 1) higher expression levels of genes related with lipogenesis (PPAR2, ChREBP, FAS), glucose uptake and metabolism (GLUT4, HKII), fatty acid uptake (LPL, CD36), and lipolysis (ATGL, HSL) in white adipose tissue; 2) higher expression levels of genes related with lipogenesis (FAS, SCD1) but lower ones related with fatty acid uptake (CD36) and oxidation (PPAR, CPT1, PDK4) in liver; and 3) higher expression levels of GLUT4 but lower ones related with fatty acid oxidation (PDK4 and UCP3) in muscle. It is worth mentioning that both CHO and fat refeeding resulted in a robust increase in both hepatic mRNA and circulating levels of fibroblast growth factor-21, compared with fasted levels. In summary, these results, showing marked differences in gene expression after CHO and fat refeeding, can explain diet-associated differences in fuel handling and partitioning between tissues; in addition, a role of fibroblast growth factor-21 in metabolic adaptations, not only in the ketotic state but also to face an unbalanced nutritional situation, is suggested.

Hypothalamic and Hindbrain Melanocortin Receptors Contribute to the Feeding, Thermogenic, and Cardiovascular Action of Melanocortins

Skibicka, K. P., Grill, H. J. — Fri, 20 Nov 2009 10:02:55 PST

Forebrain ventricular delivery of melanocortin receptor (MC3/4R) agonist increases energy expenditure and decreases food intake (FI). Because forebrain ventricular delivery provides ligand to various anatomically distributed MC3/4R-bearing nuclei, it is unclear which of the receptor subpopulations contributes to the feeding suppression and the sympathetic-thermogenic effects observed. The literature indicates that reexpression of MC4R in the paraventricular nucleus (PVH) affects the feeding but not the energetic phenotype of the MC4R knockout, suggesting that divergent MC4R populations mediate energy expenditure (hindbrain) and FI (hypothalamus) effects of stimulation. Not consistent with this view are data indicating that PVH sympathetic projection neurons express MC4Rs and that feeding effects are induced from hindbrain MC4R sites. Therefore, we hypothesize an opposing perspective: that stimulation of anatomically diverse MC3/4R-bearing nuclei triggers energetic as well as feeding effects. To test this hypothesis, ventricle subthreshold doses of MC3/4R agonist (5 and 10 pmol) were applied in separate experiments to six hindbrain and hypothalamic sites; core temperature (Tc), heart rate (HR), spontaneous activity (SPA), and FI were measured in behaving rats. Nucleus tractus solitarius and PVH stimulation increased Tc, HR, and SPA and decreased FI. Rostral ventrolateral medulla, parabrachial nucleus, and retrochiasmatic area stimulation increased Tc, HR, but not SPA, and decreased FI. The response profile differed to some extent for each nucleus tested, suggesting differential output circuitries for the measured parameters. Data are consistent with the view that energetic and feeding responses are not controlled by regionally divergent MC3/4Rs and can be elicited from multiple, anatomically distributed MC3/4R populations.

Atypical Protein Kinase C Activity in the Hypothalamus Is Required for Lipopolysaccharide-Mediated Sickness Responses

Fri, 20 Nov 2009 10:02:55 PST

By activating the Toll-like receptor 4-nuclear factor-B signal transduction pathway, the bacterial endotoxin lipopolysaccharide (LPS) induces anorexia, weight loss, fever, and other components of the sickness response. By comparison, the hormones leptin and insulin cause anorexia without sickness via a central mechanism involving the phosphatidylinositol-3 kinase signaling pathway. In the current study, we investigated whether a common Toll-like receptor 4 and phosphatidylinositol-3 kinase signaling intermediate, atypical protein kinase C/ (aPKC), contributes to changes of energy balance induced by these stimuli. Immunohistochemistry analysis revealed that aPKC is expressed in the arcuate and paraventricular nuclei of the hypothalamus, key sites of leptin, insulin, and LPS action. Although administration of LPS, insulin, and leptin each acutely increased hypothalamic aPKC activity at doses that also reduce food intake, LPS treatment caused over 10-fold greater activation of hypothalamic a PKC signaling than that induced by leptin or insulin. Intracerebroventricular pretreatment with an aPKC inhibitor blocked anorexia induced by LPS but not insulin or leptin. Similarly, LPS-induced hypothalamic inflammation (as judged by induction of proinflammatory cytokine gene expression) and neuronal activation in the paraventricular nucleus (as judged by c-fos induction) were reduced by central aPKC inhibition. Although intracerebroventricular aPKC inhibitor administration also abolished LPS-induced fever, it had no effect on sickness-related hypoactivity or weight loss. We conclude that although hypothalamic aPKC signaling is not required for food intake inhibition by insulin or leptin, it plays a key role in inflammatory anorexia and fever induced by LPS.

Effects of TWEAK (TNF Superfamily Member 12) on Differentiation, Metabolism, and Secretory Function of Human Primary Preadipocytes and Adipocytes

Fri, 20 Nov 2009 10:02:55 PST

Expansion of adipose tissue mass by hypertrophy and hyperplasia is the hallmark of obesity. An automated cDNA screen was established to identify secreted human proteins with an inhibitory effect on adipocyte differentiation and, thereby, a potential inhibitory effect on adipose tissue growth. A member of the TNF superfamily, TNF-like weak inducer of apoptosis (TWEAK; TNF superfamily 12) was identified by means of high-throughput screening with the lipophilic dye Nile Red as an inhibitor of murine adipocyte differentiation and, subsequently, also of human adipocyte differentiation. TWEAK inhibited lipid deposition in a dose-dependent manner without causing cytotoxic effects. This inhibitory action was mimicked by an agonistic antibody of the TWEAK receptor. The TWEAK receptor (fibroblast growth factor inducible 14; CD266) was expressed on human primary preadipocytes and mature adipocytes. Knockdown of TWEAK receptor by short-hairpin RNA abolished the inhibitory effect of TWEAK on cell differentiation, demonstrating that the effects of TWEAK are mediated by its specific receptor. Inhibition of differentiation was the result of interference at an early step of transcriptional activation as assessed by decreased peroxisome proliferator-activated receptor-, CCAAT enhancer-binding protein (C/EBP), and CCAAT enhancer-binding protein β (C/EBPβ) mRNA expression. In contrast to TNF, basal and insulin-stimulated glucose uptake and lipolysis of terminally differentiated mature adipocytes and secretion of proinflammatory cytokines were not altered in the presence of TWEAK, and nuclear factor B activity was only weakly induced. We conclude from our findings that TWEAK and the corresponding agonistic antibody have the potential to prevent adipose tissue growth without adversely influencing central metabolic pathways or proinflammatory cytokine secretion in adipose tissue.

Curcumin Inhibits srebp-2 Expression in Activated Hepatic Stellate Cells in Vitro by Reducing the Activity of Specificity Protein-1

Kang, Q., Chen, A. — Fri, 20 Nov 2009 10:02:55 PST

Elevated levels of cholesterol/low-density lipoprotein (LDL) are a risk factor for the development of nonalcoholic steatohepatitis and its associated hepatic fibrosis. However, underlying mechanisms remain elusive. We previously reported that curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)- and stimulated its activity, leading to the inhibition of the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis. We recently showed that curcumin suppressed gene expression of LDL receptor in activated HSCs in vitro by repressing gene expression of the transcription factor sterol regulatory element binding protein-2 (SREBP-2), leading to the reduction in the level of intracellular cholesterol in HSCs and to the attenuation of the stimulatory effects of LDL on HSCs activation. The current study aimed at exploring molecular mechanisms by which curcumin inhibits srebp-2 expression in HSCs. Promoter deletion assays, mutagenesis assays, and EMSAs localize a specificity protein-1 (SP-1) binding GC-box in the srebp-2 promoter, which is responsible for enhancing the promoter activity and responding to curcumin in HSCs. Curcumin suppresses gene expression of SP-1 and reduces its trans-activation activity, which are mediated by the activation of PPAR. The inhibitory effect of curcumin on SP-1 binding to the GC-box is confirmed by chromatin immuno-precipitation. In summary, our results demonstrate that curcumin inhibits srebp-2 expression in cultured HSCs by activating PPAR and reducing the SP-1 activity, leading to the repression of ldlr expression. These results provide novel insights into molecular mechanisms by which curcumin inhibits LDL-induced HSC activation.

Mechanisms Involved in 3',5'-Cyclic Adenosine Monophosphate-Mediated Inhibition of the Ubiquitin-Proteasome System in Skeletal Muscle

Fri, 20 Nov 2009 10:02:56 PST

Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of β₂-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective β₂-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from β₂-AR knockout mice. The suppressive effect of β₂-agonist on atrogin-1 was not mediated by PGC-1 (peroxisome proliferator-activated receptor- coactivator 1 known to be induced by β₂-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1 knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of β₂-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

Inhibition of Oxygen-Induced Hypoxia-Inducible Factor-1{alpha} Degradation Unmasks Estradiol Induction of Vascular Endothelial Growth Factor Expression in ECC-1 Cancer Cells in Vitro

Molitoris, K. H., Kazi, A. A., Koos, R. D. — Fri, 20 Nov 2009 10:02:56 PST

Estradiol (E₂) rapidly and strongly induces vascular endothelial growth factor (VEGF) transcription in uterine endometrial epithelial cells in vivo. We have shown that this is mediated by both the estrogen receptor- and hypoxia-inducible factor (HIF)-1. By contrast, E₂ induces little or no VEGF expression in cultured breast or endometrial cancer cells, which lack HIF-1 due to the abnormally high concentration of oxygen (~20%) to which they are exposed. To test the hypothesis that restoring HIF-1 in cultured cells would restore the ability of E₂ to induce VEGF expression, we treated human endometrial cancer cells (ECC-1) with cobalt chloride (CoCl₂;100 µm), which prevents oxygen-induced HIF-1 degradation. HIF-1 was absent in untreated ECC-1 cells but detectable by 4 h after treatment with CoCl₂ alone, as was a significant increase in VEGF mRNA. E₂ plus CoCl₂ induced detectable HIF-1 expression at 2 h and an even higher level than that induced by CoCl₂ alone at 4 h; this HIF-1 was localized in the nuclei. This was accompanied by increasing VEGF expression, with the increase at 4 h severalfold higher than that induced by CoCl₂ alone and was concurrent with recruitment of both HIF-1 and estrogen receptor- to the VEGF promoter. These results confirm that HIF-1 plays an essential role in E₂-induced expression of VEGF. Through the induction of increased microvascular permeability and the consequent exudation of plasma growth factors, VEGF in turn may play an essential role in cancer cell proliferation in vivo.

Evolutionary History and Functional Characterization of Androgen Receptor Genes in Jawed Vertebrates

Ogino, Y., Katoh, H., Kuraku, S., Yamada, G. — Fri, 20 Nov 2009 10:02:56 PST

Vertebrates show diverse sexual characters in sexually attractive and reproductive organs, which are regulated by steroid hormones, particularly androgens. However, the evolutionary history of androgen receptor (AR) gene remains largely unknown on the basis of phylogenic and functional analyses. To elucidate the evolutionary history and functional diversification of AR genes in vertebrates, we cloned the AR cDNAs from a shark, basal ray-finned fishes (Actinopterygii), namely bichir and sturgeon (Acipenseriformes), and teleosts including a basal teleost, arowana (Osteoglossiformes). Molecular phylogenetic analysis revealed that the gene duplication event that gave rise to two different teleost ARs ( and β) likely occurred in the actinopterygian lineage leading to teleosts after the divergence of Acipenseriformes but before the split of Osteoglossiformes, which is compatible with the phylogenetic timing of teleost-specific genome duplication. Searching for AR genes in the medaka genome indicated that the teleost AR gene duplication has been associated with the duplication between chromosomes 10 and 14. Our functional analysis revealed that the shark AR activates the target gene via androgen response element by classical androgens. The teleost AR showed the unique intracellular localization with a significantly higher transactivating capacity than that by teleost ARβ. These findings indicate that the most ancient type of AR, as activated by the classical androgens as ligands, emerged before the Chondrichthyes-Osteichthyes split, and the AR gene was duplicated during the teleost-specific genome duplication event. We report here for the first time the accurate evolutionary history of AR gene and functional characterization of AR duplicates in teleost lineage.

Local Secretion of Urocortin 1 Promotes Microvascular Permeability during Lipopolysaccharide-Induced Inflammation

Fri, 20 Nov 2009 10:02:56 PST

Urocortin 1 (Ucn1) is a neuropeptide that regulates vascular tone and is implicated in both the vascular and immune cell-mediated responses to inflammation. The role of Ucn1 in regulating microvascular permeability has not been determined. We hypothesized that local Ucn1 release promotes microvascular permeability and that this effect augments the local gastrointestinal vascular response to lipopolysaccharide (LPS)-induced systemic inflammation. We measured hydraulic (L_p) and macromolecule permeability in mesenteric venules. We show that a continuous infusion of 10^–7 m Ucn1 in a postcapillary venule increased L_p 2-fold over baseline, as did LPS-induced inflammation. However, simultaneous infusion of Ucn1 and LPS markedly increased L_p by 7-fold. After local knockdown of Ucn1 using RNA interference, infusion of Ucn1 with LPS resulted in return to 2-fold increase, confirming that Ucn1 synergistically augments hydraulic permeability during inflammation. LPS and Ucn1 treatment also resulted in increased numbers of interstitial microspheres, which colocalized with CD31⁺ immune cells. Ucn1 activity is mediated through two receptor subtypes, CRH-R₁ and CRH-R₂. CRH-R₁ receptor blockade exacerbated, whereas CRH-R₂ receptor blockade decreased the LPS-induced increase in L_p. Finally, treatment with the c-JUN N-terminal kinase (JNK) antagonist SP600125 during infusion of LPS, but not Ucn1, decreased L_p. These findings suggest that Ucn1 increases microvascular permeability and acts synergistically with LPS to increase fluid and macromolecule losses during inflammation. Knockdown of endogenous Ucn1 during inflammation attenuates synergistic increases in L_p. Ucn1’s effect on L_p is partially mediated by the CRH-R₂ receptor and acts independently of the c-JUN N-terminal kinase signal transduction pathway.

The Spontaneous Ala147Thr Amino Acid Substitution within the Translocator Protein Influences Pregnenolone Production in Lymphomonocytes of Healthy Individuals

Fri, 20 Nov 2009 10:02:56 PST

The de novo production of steroids and neurosteroids begins in mitochondria by the conversion of cholesterol to pregnenolone through cytochrome P450 side-chain cleavage (CYP11A1) enzymatic activity. The C-terminal amino acid domain of the translocator protein (TSPO) has been demonstrated to bind cholesterol, thereby determining its mitochondrial translocation. The goal of the present study was to investigate the effect of the Ala147Thr single-nucleotide polymorphism localized in this TSPO region on pregnenolone production in healthy volunteers. Pregnenolone production was evaluated in a peripheral cell model, represented by circulating lymphomonocytes. First, CYP11A1 expression, both at mRNA and protein level, was demonstrated. Pregnenolone production varied among genotype groups. Comparison of pregnenolone mean values revealed that Thr147 homozygous or heterozygous individuals had significantly lower pregnenolone levels compared with Ala147 homozygous individuals. These findings suggested a dominant effect of the minor allelic variant Thr147 to produce this first metabolite of the steroidogenesis pathway. Interestingly, Ala147 homozygous individuals exhibited significant higher levels of circulating cholesterol-rich low-density lipoproteins with respect to heterozygous individuals. In conclusion, our results demonstrate that the Ala147Thr spontaneous amino acid substitution within TSPO is able to affect pregnenolone production; this should encourage further studies to investigate its potential role in polygenic dyslipidemias.

Cathepsin D Is the Primary Protease for the Generation of Adenohypophyseal Vasoinhibins: Cleavage Occurs within the Prolactin Secretory Granules

Fri, 20 Nov 2009 10:02:56 PST

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser¹⁴⁹, the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

Atrial Natriuretic Peptide Promotes Pancreatic Islet {beta}-Cell Growth and Akt/Foxo1a/Cyclin D2 Signaling

You, H., Laychock, S. G. — Fri, 20 Nov 2009 10:02:56 PST

The adult differentiated insulin-secreting pancreatic islet β-cell experiences slow growth. This study shows that atrial natriuretic peptide (ANP) stimulates cell proliferation and [³H]thymidine incorporation in INS-1E glucose-sensitive rat β-cell line cells and isolated rat islet DNA. In addition, cGMP, the second messenger of natriuretic peptide receptors (NPR) A and B, stimulated islet DNA biosynthesis. The NPR-A receptor was expressed in INS-1E cells and islets. ANP-stimulated INS-1E cell DNA biosynthesis was blocked by preincubation with LY294002 (50 µm), an inhibitor of phosphatidylinositol 3'-kinase (PI3K). An indicator of cell cycle progression, cyclin D2 mRNA was increased by 2- to 3-fold in ANP- or 8-Br-cGMP-treated INS-1E cells and islets, and these responses were inhibited by LY294002. ANP and 8-Br-cGMP stimulated the phosphorylation of Akt and Foxo1a in INS-1E cells and islets, and LY294002 inhibited these responses. In contrast, ANP reduced the levels of phospho-ERK in INS-1E cells. Pancreas duodenum homeobox-1 (PDX-1) is essential for pancreas development, insulin production, and glucose homeostasis, and ANP increased PDX-1 mRNA levels by 2- to 3-fold in INS-1E cells and islets. The levels of glucokinase mRNA in islets and INS-1E cells were also increased in response to ANP. The evidence suggests that pancreatic β-cell NPR-A stimulation results in activation of a growth-promoting signaling pathway that includes PI3K/Akt/Foxo1a/cyclin D2. These data support the conclusion that the activation of Akt by ANP or 8-Br-cGMP promotes cyclin D2, PDX-1, and glucokinase transcription by phosphorylating and restricting Foxo1a activity.

Effects of Maternal Dexamethasone Treatment in Early Pregnancy on Pituitary-Adrenal Axis in Fetal Sheep

Fri, 20 Nov 2009 10:02:56 PST

Fetal exposure to elevated levels of bioactive glucocorticoids early in gestation, as in suspected cases of congenital adrenal hyperplasia, may result in adverse neurological events. Fetal hypothalamic-pituitary-adrenal development and function may be involved. We investigated immediate and long-term effects of maternal dexamethasone (DEX) administration early in pregnancy on fetal growth and pituitary-adrenal activity in sheep. Pregnant ewes carrying singleton fetuses (total n = 119) were randomized to control (2 ml saline/ewe) or DEX-treated groups (im injections of 0.14 mg/kg ewe weight · 12 h) at 40–41 d gestation (dG). At 50, 100, 125, and 140 dG, fetal plasma and tissues were collected. DEX-exposed fetuses were lighter than controls at 100 dG (P < 0.05) but not at any other times. Fetal plasma ACTH levels and pituitary POMC and PC-1 mRNA levels were similar between groups. Fetal plasma cortisol levels were significantly reduced after DEX exposure in both male and female fetuses at 50 dG (P < 0.05), were similar at 100 and 125 dG, but were significantly higher than controls at 140 dG. At 140 dG, there was increased adrenal P450C₁₇ and 3β-HSD mRNA in female fetuses and reduced expression of ACTH-R mRNA in males. Fetal hepatic CBG mRNA levels mimicked plasma cortisol patterns. DEX exposure reduced CBG only in males at 50 dG (P < 0.05). Placental mRNA levels of 11β-HSD2 were increased after DEX in males (P < 0.05). Therefore, in sheep, early DEX may alter the developmental trajectory of the fetal hypothalamic-pituitary-adrenal axis, directly increasing fetal adrenal activation but not anterior pituitary function. In females, this effect may be attributed, in part, to increased fetal adrenal steroidogenic activity.

Growth Hormone-Releasing Peptide-2 Suppresses Vascular Oxidative Stress in ApoE-/- Mice But Does Not Reduce Atherosclerosis

Fri, 20 Nov 2009 10:02:56 PST

GH-releasing peptide-2 (GHRP-2) is a synthetic peptide that increases circulating GH and IGF-I levels. It also binds to CD36, a scavenger receptor for oxidized low-density lipoprotein (OxLDL), and may prevent cellular uptake of this proatherogenic complex. To determine its potential antiatherogenic effects, GHRP-2 (20 µg twice daily) was administered sc to ApoE^–/– mice for 12 wk. GHRP-2 increased circulating IGF-I 1.2- to 1.6-fold and decreased circulating interferon- by 66%. Although GHRP-2 did not alter atherosclerotic plaque area, it decreased aortic production of superoxide as assessed by dihydroethidium staining. GHRP-2 decreased aortic gene expression of 12/15-lipoxygenase by 92% and reduced the aortic expression of interferon- and macrophage migration inhibitory factor. In cultured aortic smooth muscle cells, GHRP-2 prevented the OxLDL-induced generation of peroxides, down-regulation of IGF-I receptor, and apoptosis. In macrophages, GHRP-2 reduced lipid accumulation with OxLDL exposure. In summary, GHRP-2 exerts antioxidant effects in vivo and in vitro but does not reduce plaque burden. The lack of an antiatherogenic effect may be due to GH-dependent effects in vivo, thereby blunting the effect of increased IGF-I.

Melanocortins May Stimulate Reproduction by Activating Orexin Neurons in the Dorsomedial Hypothalamus and Kisspeptin Neurons in the Preoptic Area of the Ewe

Backholer, K., Smith, J., Clarke, I. J. — Fri, 20 Nov 2009 10:02:56 PST

To further test the hypothesis that melanocortins stimulate the reproductive axis, we treated ewes with melanocortin agonist (MTII) in the luteal phase of the estrous cycle and during seasonal anestrus. Lateral ventricular infusion of MTII (10 µg/h) during the luteal phase increased LH secretion. Retrograde neuronal tracing in the brain showed few proopiomelanocortin or kisspeptin cells in the arcuate nucleus, but more than 70% of kisspeptin cells in the dorsolateral preoptic area (POA), projecting to the ventromedial POA in which GnRH cells are located. MTII infusion (20 h) was repeated in luteal phase ewes and brains were harvested to measure gene expression of preproorexin and kisspeptin. Expression of orexin in the dorsomedial hypothalamus and kisspeptin in the POA was up-regulated by MTII treatment and Kiss1 in the arcuate nucleus was down-regulated. Seasonally anestrous ewes were progesterone primed and then treated (lateral ventricular) with MTII (10 µg/h) or vehicle for 30 h, and blood samples were collected every 2 h from 4 h before infusion until 6 h afterward to monitor acute response in terms of LH levels. A rise in basal LH levels was seen, but samples collected around the time of the predicted LH surge did not indicate that an ovulatory event occurred. We conclude that melanocortins are positive regulators of the reproductive neuroendocrine system, but treatment with melanocortins does not fully overcome seasonal acyclicity. The stimulatory effect of melanocortin in the luteal phase of the estrous cycle may be via the activation of kisspeptin cells in the POA and/or orexin cells in the dorsomedial hypothalamus.

Gonadotropin-Releasing Hormone Neuroterminals and Their Microenvironment in the Median Eminence: Effects of Aging and Estradiol Treatment

Yin, W., Wu, D., Noel, M. L., Gore, A. C. — Fri, 20 Nov 2009 10:02:56 PST

The GnRH decapeptide controls reproductive function through its release from neuroendocrine terminals in the median eminence, a site where there is a convergence of numerous nerve terminals and glial cells. Previous work showed dynamic changes in the GnRH-glial-capillary network in the median eminence under different physiological conditions. Because aging in rats is associated with a diminution of GnRH release and responsiveness to estradiol feedback, we examined effects of age and estradiol treatment on these anatomical interactions. Rats were ovariectomized at young (4 months), middle-aged (11 months), or old (22–23 months) ages, allowed 4 wk to recover, and then treated with vehicle or estradiol for 72 h followed by perfusion. Immunofluorescence of GnRH was measured, and immunogold electron microscopic analyses were performed to study the ultrastructural properties of GnRH neuroterminals and their microenvironment. Although the GnRH immunofluorescent signal showed no significant changes with age and estradiol treatment, we found that the median eminence underwent both qualitative and quantitative structural changes with age, including a disorganization of cytoarchitecture with aging and a decrease in the apposition of GnRH neuroterminals to glia with age and estradiol treatment. Thus, although GnRH neurons can continue to synthesize and transport peptide, changes in the GnRH neuroterminal-glial-capillary machinery occur during reproductive senescence in a manner consistent with a disconnection of these elements and a potential dysregulation of GnRH neurosecretion.

Brain-Endocrine Interactions: A Microvascular Route in the Mediobasal Hypothalamus

Fri, 20 Nov 2009 10:02:56 PST

Blood-borne hormones acting in the mediobasal hypothalamus, like those controlling food intake, require relatively direct access to target chemosensory neurons of the arcuate nucleus (ARC). An anatomical substrate for this is a permeable microvasculature with fenestrated endothelial cells in the ARC, a system that has awaited comprehensive documentation. Here, the immunofluorescent detection of endothelial fenestral diaphragms in the rat ARC allowed us to quantitate permeable microvessels throughout its rostrocaudal extent. We have determined that permeable microvessels are part of the subependymal plexus irrigating exclusively the ventromedial (vm) ARC from the subadjacent neuroendocrine median eminence. Unexpectedly, permeable microvessels were concentrated proximal to the pituitary stalk. This marked topography strongly supports the functional importance of retrograde blood flow from the pituitary to the vmARC, therefore making a functional relationship between peripheral long-loop, pituitary short-loop, and neuroendocrine ultra-short loop feedback, altogether converging for integration in the vmARC (formerly known as the hypophysiotrophic area), thereby so pivotal as a multicompetent brain endocrinostat.

Expression of Guanylyl Cyclase (GC)-A and GC-B during Brain Development: Evidence for a Role of GC-B in Perinatal Neurogenesis

Fri, 20 Nov 2009 10:02:56 PST

Atrial (ANP) and C-type (CNP) natriuretic peptide generate physiological effects via selective activation of two closely related membrane receptors with guanylyl cyclase (GC) activity, known as GC-A and GC-B. As yet, however, the discrete roles for ANP/GC-A vs. CNP/GC-B signaling in many mammalian tissues are still poorly understood. We here used receptor affinity labeling and GC assays to characterize comparatively GC-A/GC-B expression and functional activity during rat brain development. The study revealed that GC-B predominates in the developing and GC-A in the adult brain, with regional differences each between cerebral cortex, cerebellum, and brain stem. Whereas GC-A levels nearly continuously increase between embryonal d 18 and adult, GC-B expression in brain is highest and widely distributed around postnatal d 1. The striking perinatal GC-B peak coincides with elevated expression of nestin, a marker protein for neural stem/progenitor cells. Immunohistochemical investigations revealed a cell body-restricted subcellular localization of GC-B and perinatal abundance of GC-B-expressing cells in regions high in nestin-expressing cells. However, and supported by examination of nestin-GFP transgenic mice, GC-B and nestin are not coexpressed in the same cells. Rather, GC-B⁺ cells are distinguished by expression of NeuN, an early marker of differentiating neurons. These findings suggest that GC-B⁺ cells represent neuronal fate-specific progeny of nestin⁺ progenitors and raise the attention to specific and pronounced activities of CNP/GC-B signaling during perinatal brain maturation. The absence of this activity may cause the neurological disorders observed in GC-B-deficient mice.

Kisspeptin Neurons in the Ovine Arcuate Nucleus and Preoptic Area Are Involved in the Preovulatory Luteinizing Hormone Surge

Smith, J. T., Li, Q., Pereira, A., Clarke, I. J. — Fri, 20 Nov 2009 10:02:56 PST

Kisspeptin is the product of the Kiss1 gene that regulates GnRH secretion. In sheep, Kiss1 mRNA-expressing cells are found in the preoptic area (POA) and arcuate nucleus (ARC), and expression is up-regulated in the caudal ARC during the periovulatory period. We hypothesized that kisspeptin neurons in the ARC are activated by estradiol-17β prior to the preovulatory LH surge. Ovariectomized ewes were treated as follows: 1) estradiol-17β implants (sc 2 wk) to cause tonic negative feedback; 2) vehicle (no estrogen negative or positive feedback); or 3) positive feedback/GnRH surge-inducing injection of estradiol-17β (50 µg iv). For groups 2 and 3, brains were collected 1 h after treatment and kisspeptin/Fos immunoreactivity was examined. In the caudal and mid-ARC, the percentage of kisspeptin cells that were Fos immunoreactive increased after acute estradiol treatment (group 3) over that seen in the other two groups. Kisspeptin/Fos colocalization was also quantified in ewes during the luteal and late-follicular phase of the estrous cycle, showing a trend toward an increase in colocalization in the late-follicular phase. Kisspeptin/Fos colocalization was similar in the POA across groups in both experiments. Analysis of Kiss1 mRNA by in situ hybridization revealed an increase in expression during the late-follicular phase in the caudal ARC and POA. These data suggest kisspeptin neurons located in the caudal extent of the ARC are involved in generating the positive feedback preovulatory GnRH/LH surge in the ewe, but there may also be a role for Kiss1-expressing cells in the POA.

Androgens Induce Dopaminergic Neurotoxicity via Caspase-3-Dependent Activation of Protein Kinase C{delta}

Cunningham, R. L., Giuffrida, A., Roberts, J. L. — Fri, 20 Nov 2009 10:02:56 PST

Aged men have a greater incidence of Parkinson’s disease (PD) than women. PD is a neurodegenerative condition associated with the loss of dopamine neurons in the nigrostriatal pathway. This study examined the neurotoxic effects of androgens in a dopaminergic cell line (N27 cells) and the downstream signaling pathways activated by androgens. Treatment of N27 cells with testosterone- and dihydrotestosterone-induced mitochondrial dysfunction, protein kinase C (PKC)- cleavage, and apoptosis in dopaminergic neuronal cells. Inhibition of caspase-3 prevented the cleavage of PKC from the full-length element to the catalytic fragment and apoptosis in N27 cells, suggesting that androgen-induced apoptosis is mediated by caspase-3-dependent activation of PKC. Androgen-induced apoptosis may be specific to dopamine neurons as evidenced by a lack of testosterone-induced apoptosis in GnRH neurons. These results support a neurotoxic consequence of testosterone on dopaminergic neurons and may provide insight into the gender bias found in PD.

Effect of RF-Amide-Related Peptide-3 on Luteinizing Hormone and Follicle-Stimulating Hormone Synthesis and Secretion in Ovine Pituitary Gonadotropes

Sari, I. P., Rao, A., Smith, J. T., Tilbrook, A. J., Clarke, I. J. — Fri, 20 Nov 2009 10:02:56 PST

GnRH provides the primary stimulus for the reproductive axis, but original work also revealed the existence of a gonadotropin-inhibitory hormone (GnIH) in birds. In mammals, GnIH properties are displayed by a hypothalamic dodecapeptide, which is a member of the RF-amide family, namely RF-amide-related peptide (RFRP)-3. This peptide inhibits GnRH-stimulated gonadotropin secretion from ovine pituitary cells in culture, but it is not known whether there are effects on gonadotropin synthesis. The aim of the present study was to determine the effects of RFRP-3 on the expression of genes for β-subunits of the gonadotropins in ovine pituitary cells from gonadectomized ewes and rams. Cells in primary culture were given GnRH or vehicle pulses every 8 h for 24 h with and without RFRP-3 treatment. GnRH stimulated LH and FSH secretion, which was reduced by RFRP-3. Quantitative real-time PCR revealed increased expression of LHβ and FSHβ subunit genes after GnRH treatment and a specific reduction in expression after RFRP-3 treatment. There was no effect on the expression of GH, proopiomelanocortin, or prolactin genes. Western blotting showed that GnRH stimulated phosphorylation of ERK (phospho-ERK-1/2), and this effect was abolished by RFRP-3. We conclude that RFRP-3 acts on the pituitary gonadotropes to inhibit synthesis of the gonadotropins, and this effect may be mediated by a reduction in the GnRH-stimulated second messenger phospho-ERK-1/2.

Glucose Promotes the Production of Interleukine-1{beta} and Cyclooxygenase-2 in Mesangial Cells via Enhanced (Pro)Renin Receptor Expression

Huang, J., Siragy, H. M. — Fri, 20 Nov 2009 10:02:56 PST

(Pro)renin receptor (PRR) is present in renal glomeruli, and its expression is up-regulated in diabetes. Similarly, renal inflammation is increased in the presence of hyperglycemia. The linkage between PRR and renal inflammation is not well established. We hypothesized that glucose-induced up-regulation of PRR leads to increased production of the proinflammatory factors IL-1β and cyclooxygenase-2 (COX-2). Studies were conducted in rat mesangial cells (RMCs) exposed to 30 mm d-glucose for 2 wk followed by PRR small interfering RNA knockdown, IL-1 receptor blockade with IL-1 receptor antagonist or angiotensin II type 1 receptor blockade with valsartan. The results showed that d-glucose treatment up-regulates prorenin, renin, angiotensin II, PRR, IL-1β, and COX-2 mRNA and protein expression and increases phosphorylation of ERK1/2, c-Jun N-terminal kinase, c-Jun, and nuclear factor-B (NF-B) p65 (serine 276,468 and 536), respectively. PRR small interfering RNA attenuated PRR, IL-1β, and COX-2 mRNA and protein expressions and significantly decreased angiotensin II production and phosphorylation of ERK1/2 and NF-B p65 associated with high glucose exposure. Similarly, IL-1 receptor antagonist significantly reduced COX-2 mRNA and protein expression induced by high glucose. COX-2 inhibition reduced high-glucose-induced PRR expression. We conclude that glucose induces the up-regulation of PRR and its ligands prorenin and renin, leading to increased IL-1β and COX-2 production via the angiotensin II-dependent pathway. It is also possible that PRR could enhance the production of these inflammatory cytokines through direct stimulation of ERK1/2-NF-B signaling cascade.

Growth Differentiation Factor-9 Mediates Follicle-Stimulating Hormone-Thyroid Hormone Interaction in the Regulation of Rat Preantral Follicular Development

Fri, 20 Nov 2009 10:02:56 PST

FSH regulates follicular growth in a stage-development fashion. Although preantral follicle stage is gonadotropin responsive, FSH is not required for preantral follicular growth. With the antrum, the follicles continue growing under the influence of FSH and become gonadotropin dependent. Although thyroid hormone is important for normal female reproductive function, its role and interaction with FSH in the regulation of preantral ovarian follicular growth is yet to be defined. In the present study, we have examined the action and interaction of FSH and T₃ in the regulation of the growth of preantral follicles, especially in their transition from preantral to early antral stage, using an established follicle culture system and evaluated the involvement of growth differentiation factor-9 (GDF-9) in this process in vitro. We have demonstrated that although T₃ alone had no effect on follicular development, it markedly enhanced FSH-induced preantral follicular growth. Although FSH alone significantly down-regulated FSH receptor (FSHR) mRNA abundance in the preantral follicles and T₃ alone was ineffective, expression of the message was significantly increased in the presence of both hormones. In addition, intra-oocyte injection of GDF-9 antisense oligonucleotides (GDF-9 morpholino) induced follicular cell apoptosis and suppressed follicular growth induced by FSH and T₃. These responses were attenuated by exogenous GDF-9. Our findings support the concept that thyroid hormone regulates ovarian follicular development through its direct action on the ovary and that promotes FSH-induced preantral follicular growth through up-regulation of FSHR, a mechanism dependent on the expression and action of oocyte-derived GDF-9.

In Utero Exposure to Di-(2-Ethylhexyl) Phthalate Decreases Mineralocorticoid Receptor Expression in the Adult Testis

Martinez-Arguelles, D. B., Culty, M., Zirkin, B. R., Papadopoulos, V. — Fri, 20 Nov 2009 10:02:56 PST

In utero exposure to di-(2-ethylhexyl) phthalate (DEHP) has been shown to result in decreased androgen formation by fetal and adult rat testes. In the fetus, decreased androgen is accompanied by the reduced expression of steroidogenic enzymes. The mechanism by which in utero exposure results in reduced androgen formation in the adult, however, is unknown. We hypothesized that deregulation of the nuclear steroid receptors might explain the effects of in utero DEHP exposure on adult testosterone production. To test this hypothesis, pregnant Sprague Dawley dams were gavaged with 100–950 mg DEHP per kilogram per day from gestational d 14–19, and testes were collected at gestational d 20 and postnatal days (PND) 3, 21, and 60. Among the nuclear receptors studied, the mineralocorticoid receptor (MR) mRNA and protein levels were reduced in PND60 interstitial Leydig cells, accompanied by reduced mRNA expression of MR-regulated genes. Methylation-sensitive PCR showed effects on the nuclear receptor subfamilies NR3A and -3C, but only MR was affected at PND60. Pyrosequencing of two CpG islands within the MR gene promoter revealed a loss of methylation in DEHP-treated animals that was correlated with reduced MR. Because MR activation is known to stimulate Leydig cell testosterone formation, and MR inhibition to be repressive, our results are consistent with the hypothesis that in utero exposure to DEHP leads to MR dysfunction and thus to depressed testosterone production in the adult. We suggest that decreased MR, possibly epigenetically mediated, is a novel mechanism by which phthalates may affect diverse functions later in life.

Small Ubiquitin-Like Modifier-2 Modification of Retinoic Acid Receptor-{alpha} Regulates Its Subcellular Localization and Transcriptional Activity

Zhu, L., Santos, N. C., Kim, K. H. — Fri, 20 Nov 2009 10:02:57 PST

The retinoic acid receptor- (Rara) gene is critical for germ cell development in the testis, as demonstrated by infertile Rara knockout male mice. The encoded protein for Rara (RARA) is expressed in both Sertoli cells and germ cells, but it is not always in the nucleus. Previously, all-trans retinoic acid (ATRA) was shown to increase the nuclear localization and transcriptional activity of RARA in Sertoli cells. Here, we identified a small ubiquitin-like modifier-2 (SUMO-2) modification as a novel posttranslational regulatory mechanism controlling the ATRA-dependent RARA subcellular localization and transcription. ATRA increased the SUMO-2 modification of RARA. In the presence of ATRA, lysine 166 (K166) and K171 of RARA were modified at a physiological concentration of SUMO-2, whereas in the absence of ATRA, K399 was the only site that was modified, but at a higher SUMO-2 concentration. However, K399 was critical for ATRA-controlled nuclear trafficking of RARA. In the presence of ATRA, a K399 mutation to arginine resulted in the cytoplasmic localization of K399R mutant, indicating that K166 and K171 sumoylations were inhibitory to nuclear localization. This may be due to SUMO/sentrin-specific peptidase 6 (SENP6) not being able to bind K399R mutant to desumoylate K166 and K171 in Sertoli cells, whereas it can bind RARA with intact K399. On the other hand, functional K166 and K171 sites for sumoylation were required for a full transcriptional activity, when K399 was intact. These results together suggest that both K166 and K171 sumoylation and desumoylation are critical for optimal RARA function.

The Cytokine Gene CXCL14 Restricts Human Trophoblast Cell Invasion by Suppressing Gelatinase Activity

Kuang, H., Chen, Q., Zhang, Y., Zhang, L., Peng, H., Ning, L., Cao, Y., Duan, E. — Fri, 20 Nov 2009 10:02:57 PST

Well-controlled trophoblast invasion into uterine decidua is a critical process for the normal development of placenta, which is tightly regulated by various factors produced within the trophoblast-endometrial microenvironment. CXCL14 is involved in tumor growth and metastasis, and its expression in placenta is temporally regulated during pregnancy. However, the role of CXCL14 in trophoblast function during human pregnancy is not clear. In this study, by using RT-PCR through human pregnancy, we found that CXCL14 was selectively expressed at early but not late pregnancy. Immunostaining revealed that CXCL14 proteins were strongly expressed in villous cytotrophoblasts and moderately in decidualized stromal cells but very weakly in syncytiotrophoblasts and extravillous trophoblasts. The effect of CXCL14 on trophoblast invasion were examined by using human villous explants cultured on Matrigel and further proved by invasion and migration assay of primary trophoblast cells and trophoblast cell line HTR-8/SVneo. Our data showed that CXCL14 significantly inhibited outgrowth of villous explant in vitro; this effect is due to suppression of trophoblast invasion and migration through regulating matrix metalloproteinases activities, whereas the trophoblast proliferation was not affected. Moreover, because a receptor for CXCL14 has not been identified, we performed further cell-specific CXCL14 binding activities with regard to different cell types within the maternal-fetal interface. Our data revealed that CXCL14 could specifically bind to trophoblast cells but not decidual cells from the maternal-fetal interface. These results suggest that CXCL14 plays an important role in regulating trophoblast invasion through an autocrine/paracrine manner during early pregnancy.

A Novel Synthetic Androgen Receptor Ligand, S42, Works as a Selective Androgen Receptor Modulator and Possesses Metabolic Effects with Little Impact on the Prostate

Fri, 20 Nov 2009 10:02:57 PST

We identified a novel synthetic steroid, S42, as a promising candidate of selective androgen receptor (AR) modulator. Results of the whole-cell binding assay using COS-7 cells exogenously expressing various steroid receptors indicated that S42 specifically binds to AR and progesterone receptor. When orchiectomized Sprague Dawley rats were administered with S42 for 3 wk, the muscle weight of the levator ani was increased as markedly as that induced by 5-dihydrotestosterone (DHT), but the weight of the prostate was not elevated at any doses in contrast to DHT. The plasma concentrations of gonadotropin and adiponectin, those down-regulated by DHT, were unaffected by S42. In addition, although the plasma triglyceride level was unaffected by DHT, it was significantly reduced by S42. This effect of S42 was associated with suppression of the SRBP-1c-mediated lipogenic and insulin-desensitizing pathway in the liver and visceral fat. Taken together, S42 works as an AR agonist in muscle and as an AR antagonist in the prostate, pituitary gland, and liver, accompanying beneficial potentials on lipid metabolism.

Evidence that Corticotropin-Releasing Hormone Modulates Myometrial Contractility during Human Pregnancy

Tyson, E. K., Smith, R., Read, M. — Fri, 20 Nov 2009 10:02:57 PST

As human pregnancy advances, CRH increases exponentially and is hypothesized to trigger the transition from myometrial quiescence to active contractions at labor. Paradoxically, CRH stimulates cAMP production, suggesting it should cause relaxation. To evaluate CRH as a mediator of quiescence, the effect of CRH on contractions in preterm and term myometria with concurrent progesterone (P4) was determined. In late gestation, we hypothesized that high concentrations of CRH down-regulate agonist-activated-cAMP relaxatory pathways and that increased phosphodiesterase (PDE) activity induces heterologous down-regulation of agonist-activated-cAMP pathways. CRH caused dose-dependent relaxation of spontaneously contracting myometrial strips of 31 ± 8% (mean ± sem; n = 12) and 35 ± 20% (n = 3) in term and preterm samples, respectively. CRH with P4 pretreatment caused a 40 ± 13% (n = 4) reduction in contractility, whereas in matched samples, CRH alone exerted a 26 ± 6% (n = 4) reduction, with a shift of CRH dose-response curves (P < 0.01, ANOVA). Pretreatment of strips with 10^–7 m CRH did not attenuate relaxation induced by subsequent CRH (n = 3) or salbutamol (β₂-agonist) treatment (n = 9). PDE inhibition by rolipram showed a 2.2- and 1.5-fold increase in maximal relaxation induced by CRH and salbutamol, respectively, with a shift of both dose-response curves (P < 0.05 and P < 0.01, ANOVA). In conclusion, CRH at physiological concentrations acts synergistically with P4 contributing to myometrial quiescence. P4 withdrawal may reduce CRH-mediated relaxation. Our functional model does not support homologous or heterologous down-regulation of agonist-stimulated-cAMP pathways by high CRH concentrations. PDE inhibition potentiates CRH and salbutamol-induced relaxation. Up-regulation of PDEs, through chronic cAMP elevation by CRH, could provide a mechanism for down-regulation of agonist-stimulated-cAMP pathways at term.

Transgenic Mice Expressing Green Fluorescent Protein under the Control of the Corticotropin-Releasing Hormone Promoter

Fri, 20 Nov 2009 10:02:57 PST

CRH is widely expressed in the brain and is of broad functional relevance to a number of physiological processes, including stress response, parturition, immune response, and ingestive behavior. To delineate further the organization of the central CRH network, we generated mice expressing green fluorescent protein (GFP) under the control of the CRH promoter, using bacterial artificial chromosome technology. Here we validate CRH-GFP transgene expression within specific brain regions and confirm the distribution of central GFP-producing cells to faithfully recapitulate that of CRH-expressing cells. Furthermore, we confirm the functional integrity of a population of GFP-producing cells by demonstrating their apposite responsiveness to nutritional status. We anticipate that this transgenic model will lend itself as a highly tractable tool for the investigation of CRH expression and function in discrete brain regions.

Robust Up-Regulation of Nuclear Red Fluorescent-Tagged Fos Marks Neuronal Activation in Green Fluorescent Vasopressin Neurons after Osmotic Stimulation in a Double-Transgenic Rat

Fri, 20 Nov 2009 10:02:57 PST

The up-regulation in the expression of mRNA or protein encoded by the c-fos gene is widely used as a marker of neuronal activation elicited by various stimuli. To facilitate the detection of activated neurons, we generated transgenic rats expressing a fusion gene consisting of c-fos coding sequences in frame with monomeric red fluorescent protein 1 (mRFP1) under the control of c-fos gene regulatory sequences (c-fos-mRFP1 rats). In c-fos-mRFP1 transgenic rats, 90 min after hypertonic saline ip administration, nuclear mRFP1 fluorescence was observed abundantly in brain regions known to be osmosensitive, namely the median preoptic nucleus, organum vasculosum lamina terminalis, supraoptic nucleus, paraventricular nucleus, and subfornical organ. Immunohistochemistry for Fos protein confirmed that the distribution of Fos-like immunoreactivity in nontransgenic rats was similar to those of mRFP1 fluorescence after ip administration of hypertonic saline in the transgenic rats. Several double-transgenic rats were obtained from matings between transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein fusion gene (AVP-eGFP rats) and c-fos-mRFP1 rats. In these double-transgenic rats, almost all eGFP neurons in the supraoptic nucleus and PVN expressed nuclear mRFP1 fluorescence 90 min after hypertonic saline administration. The c-fos-mRFP1 rats are a powerful tool that enables the facile identification of activated neurons in the nervous system. Furthermore, when combined with transgenes expressing another fluorophore under the control of cell-specific regulatory sequences, activation of specific neuronal cell types in response to physiological cues can be readily detected.

Thyroid Hormone Effects on Whole-Body Energy Homeostasis and Tissue-Specific Fatty Acid Uptake in Vivo

Fri, 20 Nov 2009 10:02:57 PST

The effects of thyroid hormone (TH) status on energy metabolism and tissue-specific substrate supply in vivo are incompletely understood. To study the effects of TH status on energy metabolism and tissue-specific fatty acid (FA) fluxes, we used metabolic cages as well as ¹⁴C-labeled FA and ³H-labeled triglyceride (TG) infusion in rats treated with methimazole and either 0 (hypothyroidism), 1.5 (euthyroidism), or 16.0 (thyrotoxicosis) µg per 100 g/d T₄ for 11 d. Thyrotoxicosis increased total energy expenditure by 38% (P = 0.02), resting energy expenditure by 61% (P = 0.002), and food intake by 18% (P = 0.004). Hypothyroidism tended to decrease total energy expenditure (10%; P = 0.064) and resting energy expenditure (12%; P = 0.025) but did not affect food intake. TH status did not affect spontaneous physical activity. Thyrotoxicosis increased fat oxidation (P = 0.006), whereas hypothyroidism decreased glucose oxidation (P = 0.035). Plasma FA concentration was increased in thyrotoxic but not hypothyroid rats. Thyrotoxicosis increased albumin-bound FA uptake in muscle and white adipose tissue (WAT), whereas hypothyroidism had no effect in any tissue studied, suggesting mass-driven albumin-bound FA uptake. During thyrotoxicosis, TG-derived FA uptake was increased in muscle and heart, unaffected in WAT, and decreased in brown adipose tissue. Conversely, during hypothyroidism TG-derived FA uptake was increased in WAT in association with increased lipoprotein lipase activity but unaffected in oxidative tissues and decreased in liver. In conclusion, TH status determines energy expenditure independently of spontaneous physical activity. The changes in whole-body lipid metabolism are accompanied by tissue-specific changes in TG-derived FA uptake in accordance with hyper- and hypometabolic states induced by thyrotoxicosis and hypothyroidism, respectively.

Cognition Is Not Modified by Large but Temporary Changes in Sex Hormones in Men

Young, L. A., Neiss, M. B., Samuels, M. H., Roselli, C. E., Janowsky, J. S. — Fri, 20 Nov 2009 10:02:57 PST

Polymorphisms Identified through Genome-Wide Association Studies and Their Associations with Type 2 Diabetes in Chinese, Malays, and Asian-Indians in Singapore

Fri, 20 Nov 2009 10:02:57 PST

Unraveling the Directional Link between Adiposity and Inflammation: A Bidirectional Mendelian Randomization Approach

Fri, 20 Nov 2009 10:02:57 PST

Associations between Body Composition, Circulating Interleukin-1 Receptor Antagonist, Osteocalcin, and Insulin Metabolism in Active Acromegaly

Fri, 20 Nov 2009 10:02:57 PST

Acute Tissue Injury Caused by Subcutaneous Fat Biopsies Produces Endoplasmic Reticulum Stress

Boden, G., Silviera, M., Smith, B., Cheung, P., Homko, C. — Fri, 20 Nov 2009 10:02:57 PST

Erratum

Fri, 20 Nov 2009 10:02:57 PST