| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Type 2 Diabetes Drug Hunting Team (Y.L., M.D.M., K.A.O., K.W.S., A.R.-M.), Atherosclerosis Drug Hunting Team (W.R.B.), and Discovery Pathology (S.K.S., J.M.S.), Lilly Research Laboratories, Indianapolis, Indiana 46285; Deltagen, Inc. (S.K.), San Carlos, California 94070; and Isis Pharmaceuticals (B.P.M., S.F.M., S.B.), Carlsbad, California 92008
Address all correspondence and requests for reprints to: Anne Reifel Miller, Ph.D., Type 2 Diabetes Drug Hunting Team, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285. E-mail: a.r.miller{at}lilly.com.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
agonists stimulate endogenous adiponectin production (1). Adiponectin has been indicated in several pharmacological mechanisms, including those to treat diabetes, inflammation, and atherosclerosis (2, 3). Mice with a disruption of the adiponectin gene are more susceptible to insulin resistance induced by high calorie intake (4, 5). Two receptors for adiponectin, AdipoR1 and AdipoR2, were recently cloned. AdipoR1 is ubiquitously expressed, most abundantly in skeletal muscle in both humans and mice. AdipoR2 is predominantly expressed in liver in mouse and liver and muscle in humans. AdipoRs are indicated in the activation of AMP-activated kinase (AMPK) and regulation of muscle fatty acid oxidation and hepatic gluconeogenesis (6). Factors such as insulin, nuclear receptors, and fasting/refeeding states have been indicated in the regulated expression of AdipoRs in mice (7, 8, 9). The expression patterns of AdipoR1 and AdipoR2 in liver, muscle, and fat are differentially displayed in genetically modified animal models of obesity and diabetes, such as ob/ob and db/db mice (7, 10). In healthy human subjects, AdipoR1 expression in myotubes correlates positively with plasma insulin, triglyceride, and cholesterol levels whereas AdipoR2 expression correlates only with triglyceride levels (11). Genetic studies have suggested that lower mRNA levels and genetic variations in both AdipoRs are associated with insulin resistance and/or type 2 diabetes (T2D) in human subjects (12, 13, 14). The precise signal transduction pathways and the overall in vivo function of AdipoRs are largely unknown. We generated AdipoR2 null mice and monitored lipid and glucose metabolism in these mice on either standard chow diet (Std) or high-fat chow diet (HF) feeding. Our study demonstrates that AdipoR2 is a pivotal gene in the development of diet-induced obesity and T2D and may shed more light on the potential use of AdipoR2 as a drug target in the treatment of metabolic diseases.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-antibody was custom-made by Zymed (San Francisco, CA). NuPAGE Bis-Tris Mini-Gel/MOPS system and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA). Lysing Matrix D shaker tubes were purchased from Q-Biogene (Irvine, CA). RNase-free DNase set and RNeasy mini RNA preparation kits were purchased from QIAGEN (Valencia, CA). Ribogreen was from Molecular Probes (Eugene, OR). TaqMan PCR Master Mix and high-yield cDNA archive system were purchased from Applied Biosystems (ABI, Foster City, CA).
Disruption of the AdipoR2 gene
AdipoR2-deficient mice were generated by Deltagen, Inc. (San Carlos, CA). A 6.93-kb IRES-lacZ reporter and neomycin resistance cassette (IRES-lacZ-neo) was subcloned into a 3.7-kb fragment isolated from a mouse genomic phage library, such that 187 bp coding for the protein were replaced by IRES-LacZ-neo. The targeting vector was linearized and electroporated into 129/OlaHsd mouse embryonic (ES) stem cells. Colonies that gave rise to the correct homologous recombination were confirmed by Southern blot analysis using a probe adjacent to the 5' region of homology. Male chimeric mice were generated by injection of the targeted ES cells into C57BL/6J blastocysts, and germline transmission was confirmed by PCR analysis.
AdipoR2-deficient mice
Heterozygous AdipoR2+/– mice were backcrossed with C57BL/6 for eight generations. Male wild-type (WT) and homozygous (AdipoR2–/–) littermates were obtained by Het x Het breeding. Animals (four to five per cage) were maintained on a 12-h light, 12-h dark cycle and received house water ad libitum. All animals were maintained according to either all state and federal regulations and the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals or the Institutional Animal Use and Care Committee of Eli Lilly and Co. and the National Institutes of Health Guide for the Use and Care of Laboratory Animals depending on the housing locations.
Diets for AdipoR2-deficient mice
WT and AdipoR2–/– mice were randomized by body weight and were assigned into either Std (2018; Teklad, Madison, WI) or HF (03307; Teklad) groups at 4–5 wk of age. The nutrient compositions (percentage of kilocalories) of the Std are 16% from fat, 61% from carbohydrate, and 23% from protein and of the HF are 59% from fat, 26% from carbohydrate, and 15% from protein. Mice were kept on diets ad libitum continuously for 22 and 27 wk for two independent studies, respectively.
The ob/ob mice
C57BL/6J-Lepob/Lepob mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were maintained on a 12-h light, 12-h dark cycle and received house water and Diet 5015 (Purina Lab Diet, Richmond, IN) ad libitum. The experiment was initiated at 6–8 wk of age. All animals were kept in accordance with the Institutional American Association for the Accreditation of Laboratory Animal Care guidelines.
Antisense oligo (ASO) treatment
The ob/ob mice were administered with saline or the indicated ASOs (25 mg/kg) twice weekly via sc injection for 4 wk. The following ASOs were administered to ob/ob mice: AdipoR2, 5'-TGGCTCGTTCATGGGATACC-3'; phosphatase and tensin homolog deleted on chromosome 10, 5'-CTGCTAGCCTCTGGATTTGA-3'; TNF--induced adipose-related protein, 5'-GGCAAGGAAGTGATTCCCAA-3'.
Physiological studies
Body weight was recorded weekly for 22 wk, and food intake was measured weekly for 12 continuous weeks. Plasma glucose, triglyceride, and cholesterol levels were measured periodically using a Hitachi 912 clinical chemistry analyzer (Roche Diagnostics, Indianapolis, IN). Plasma glucose levels in ob/ob mice were determined on an Olympus AS 400e clinical analyzer. Plasma insulin levels were analyzed with LINCO adipokine multiplex by Linco Diagnostics (St. Charles, MO). Plasma adiponectin levels were determined using a mouse adiponectin ELISA kit (R&D Systems, Minneapolis, MN).
Lipoprotein distribution
Lipoproteins were separated by fast protein liquid chromatography, and cholesterol was quantitated with an in-line detection system as previously described (15).
Insulin tolerance test
Mice were fasted for 6 h, and then insulin (1.0 U/kg Humulin R, Lilly) was administrated by ip injection. The tail vein was bled at 0, 15, 30, and 60 min, and blood glucose levels were measured in triplicate with Accu-Chek Advantage glucometers (Roche).
Oral glucose tolerance test
Mice were fasted for 16 h, the tail vein was bled at 0, 30, 60, and 120 min after an oral bolus of glucose (2 g/kg). Blood glucose levels were measured in triplicate with Accu-Chek Advantage glucometers (Roche).
Phosphorylation of insulin receptor and PKB in vivo
Mice were injected iv with 1.0 U/kg insulin or insulin diluent after 6 h fasting. Livers were removed 1.5 min after injection and were collected in liquid nitrogen. Tissue (10 mg) was homogenized and extracted with 1 ml buffer B (16) in a Lysing Matrix D shaker tube. Liver homogenates were centrifuged twice at 14,000 rpm for 15 min at 4 C. Proteins were resolved in 4–12% gradient NuPAGE Bis-Tris SDS-PAGE and transferred onto nitrocellulose membrane, followed by immunoblotting with antibodies as indicated. Densitometry was performed using Scion Image, and bar graphs are presented as mean + SEM.
Immunohistochemistry
Pancreata were fixed in 4% paraformaldehyde and embedded in paraffin. Pancreatic sections (5 µm) were immunostained for insulin using the automated Ventana Discovery XT staining module (Ventana Medical Systems, Tucson, AZ).
Islet isolation
Mice were euthanized by CO2 followed by cervical dislocation. The common bile duct was cannulated with a 27-gauge needle, and the pancreas was inflated with 2.4 ml Hanks’ buffer containing 2% BSA and 1 mg/ml collagenase. The pancreas was removed and digested in Hanks’ buffer at 37 C for 4–5 min, and then islets were purified on a Histopaque-1100 gradient for 18 min at 750 x g. Histopaque-1100 was prepared by mixing Histopaque 1077 and Histopaque 1119 at a ratio of 1:1.2. Islets were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin for 2–4 h and then stored at –80 C until RNA isolation.
RNA isolation
Total RNA from liver, white adipose tissues, quadriceps, and islet of WT and AdipoR2–/– mice was extracted with TRIzol reagent in Lysing Matrix D shaker tubes. Total RNA from liver and white adipose tissue of ob/ob mice were homogenized in guanidinium isothiocyanate and extracted with cesium chloride gradient. All RNA samples were subjected to DNase I treatment and additional clean-up using the RNeasy mini RNA preparation kit.
Quantitative real-time PCR
cDNA was synthesized using the high-yield cDNA archive system. qRT-PCR was performed using an ABI 7900 Prism or an ABI 7700 sequence detection system. The assayed mRNA was normalized to the relative expression of 36B4 mRNA or to Ribogreen fluorescence as indicated. The sequences of primers and probes used in this research will be provided on request.
Statistical analysis
Data from WT and AdipoR2–/– mice were analyzed using JMP 5.1 software to perform two-way ANOVA test (genotype and diet), which was followed by Tukey HSD (honest significant difference) test for multiple comparisons. The null hypothesis was rejected at P < 0.05. Data from ob/ob mice treated with ASOs were analyzed using Excel spreadsheet to perform one-way ANOVA test, and Dunnett’s adjusted P values were calculated using the Splus Statistical package. The null hypothesis was rejected at P < 0.01.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Three sets of real-time PCR probes and primers detecting exon 3 (N-terminal, NT), exon 5, and exon 7 (C-terminal, CT) were used to verify the absence of full-length AdipoR2 mRNA (Fig. 1B). In both liver and sc fat, the probe to exon 5 detected no signal in AdipoR2–/– mice, whereas NT probe detected significantly higher signals in AdipoR2–/– mice on HF [knockout (KO)-HF] compared with the other three groups of mice (Fig. 1B, WT-Std, WT-HF, and KO-Std). The augmented signal indicates that AdipoR2 may be cross-regulated by genotype and diet at a transcriptional level. Signals detected by the CT probe in AdipoR2–/– mice were about 12–15% (liver) and 33% (fat) of WT mice (Fig. 1B). AdipoR1 mRNA levels increased 38% in livers of KO-HF mice compared with WT-HF mice (Fig. 1C), and no significant change was observed in sc fat, mesentery fat, skeletal muscle, or islet between genotypes (Fig. 1C).
|
). However, the rate of body weight gain for the KO-HF group was significantly lower than that of the WT-HF group beginning at the second week on diet and lasting until 15 wk on diet (Fig. 2A). Body weights were not different between the two Std groups (Fig. 2A). As expected, calorie intakes in both HF groups were higher than the two Std groups, yet rates of food consumption were not different between genotypes (Fig. 2B).
|
). At 8 wk on diet, fasted triglyceride levels in both HF groups were higher than that in their corresponding Std groups. Although triglyceride levels in WT-HF mice dropped dramatically, it was still significantly higher than that in KO-HF mice (Fig. 2C). For the remaining 14 wk on diet, changes in triglyceride levels among the four groups were rather mild although statistically different at some time points (Fig. 2C)
Total plasma cholesterol levels in both HF groups were significantly elevated compared with their corresponding Std groups, yet cholesterol levels in KO-HF mice were consistently lower than that in WT-HF mice in both fed and fasted conditions (Fig. 2D). No differences were seen between the two Std groups (Fig. 2D). Lipid fractionation from serum of fasted mice after 16 wk on diet demonstrated that HF feeding increased very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) peak areas. Additionally, it also revealed the appearance of a peak with an elution time slightly later than the LDL peak seen with Std feeding. This new peak is probably a cholesterol-enriched, apolipoprotein AI- and apolipoprotein E-containing lipoprotein subfraction, which can be called large HDL (lgHDL) (Fig. 2E). In KO-HF mice, all three peak areas (VLDL, LDL/lgHDL, and HDL) were smaller than those seen from WT-HF mice. In contrast, the two Std groups showed very similar profiles (Fig. 2E). Similar lipoprotein distribution patterns were seen in mice at 8 and 20 wk on diet (data not shown). Combined analysis of the three peak areas (VLDL, LDL/lgHDL, and HDL) at three time points (8, 16, and 20 wk on diet) demonstrated that the LDL/lgHDL peak was significantly smaller in the KO-HF group compared with WT-HF group (Fig. 2F). These findings suggest that AdipoR2–/– mice are partially yet specifically protected from HF-induced dyslipidemia.
AdipoR2 disruption diminishes HF-induced insulin resistance and lowers plasma glucose levels in ob/ob mice
Based on the positive impact of adiponectin on insulin sensitivity (2), we hypothesized that mice deficient in AdipoR2 would be more prone to HF-induced severe insulin resistance. To our surprise, oral glucose tolerance test (OGTT) performed on mice at 8 wk on diet showed that WT-HF mice were profoundly glucose intolerant, but KO-HF mice were only mildly glucose intolerant (Fig. 3A). Insulin tolerance test (ITT) conducted on mice at 14 wk on diet further confirmed that KO-HF mice developed less severe insulin resistance than WT-HF mice (Fig. 3B). The results from the ITT were duplicated in a second set of mice at 11 and 23 wk on diet (data not shown). Plasma adiponectin levels were not significantly different between the two Std groups (Fig. 3C), although adiponectin mRNA levels in sc fat of KO-Std mice were significantly lower than that in WT-Std mice (Fig. 3D). Interestingly, HF feeding increased plasma adiponectin levels in both genotypes at an early stage (4 wk on diet, Fig. 3C). As HF feeding continued, WT-HF mice showed a decline in plasma adiponectin levels as expected (Fig. 3C). Unexpectedly, plasma adiponectin levels in KO-HF mice kept ascending (Fig. 3C). On the contrary, adiponectin mRNA levels in sc or mesentery fat of KO-HF mice were not different from that of WT-HF mice (Fig. 3D).
|
, A and B). No difference was observed between the two Std groups (Fig. 4, A and B). Phosphorylation of PKB exhibited a trend of, but not a significant increase in, AdipoR2–/– mice (Fig. 4, A and C).
|
), we could not rule out the possibility that a translated intracellular NT fragment is present and functions as a constitutive active form of AdipoR2. To clarify this ambiguity, we introduced AdipoR2-ASO into ob/ob mice and monitored plasma glucose levels. Four weeks after ASO treatment, AdipoR2 mRNA expression was significantly suppressed in liver (
85%, Fig. 5A) and epididymal fat (70%, data not shown). Liver AdipoR1 mRNA was not affected by AdipoR2-ASO treatment (data not shown). Consistent with the insulin-sensitizing phenotype of AdipoR2–/– mice, fed plasma glucose levels were significantly reduced in ob/ob mice treated with AdipoR2-ASO (Fig. 5B). Phosphatase and tensin homolog deleted on chromosome 10-ASO and TNF--induced adipose-related protein-ASO served as positive and negative control, respectively (Fig. 5, A and B).
|
), whereas the fasted glucose levels in KO-HF mice were significantly higher than the KO-Std group at 16 and 20 wk on diet in a gradually ascending pattern (Fig. 6A). Fasted glucose levels in KO-HF mice became significantly higher than that in WT-HF mice at 20 wk on diet (KO-HF 186 mg/dl vs. WT-HF 143 mg/dl). Accordingly, stabilized glucose levels in WT-HF mice were associated with significantly elevated plasma insulin levels (Fig. 6B). In contrast, the increase of insulin levels in KO-HF mice was far less dramatic (Fig. 6B). These findings implicate that ß-cells with a defect in the AdipoR2 gene are incapable of sufficient compensation even for moderate insulin resistance. No difference was seen on fasting glucose and insulin levels between the two Std groups (Fig. 6, A and B). Fed glucose levels were not different among the four groups (data not shown).
|
), suggesting very active ß-cell replication and neogenesis. In contrast, both KO-Std and KO-HF mice displayed images similar to the WT-Std group in terms of islet size and number. No considerable difference was detected in pancreatic immunostaining of glucagon, caspase-3, and Ki-67 among the four groups at this stage on diet (data not shown).
AdipoR2 disruption modifies genes involved in hepatic lipid and glucose metabolism
AdipoR2 is predominantly expressed in mouse liver, which is also the major organ involved in lipid and glucose metabolism. Therefore, hepatic genes were investigated to explore mechanisms responsible for the improved lipid profile and insulin sensitivity in AdipoR2–/– mice with HF feeding. Expression of fatty acid synthase (FAS) was significantly suppressed in KO-Std mice, but its expression in KO-HF mice was comparable to WT-HF mice (Fig. 7). Monoacylglycerol acyltransferase 1 (MGAT1) expression was transcriptionally up-regulated by HF feeding in both genotypes, yet the enhancement in AdipoR2–/– mice was considerably lower (Fig. 7). Acyl coenzyme A:cholesterol transferase (ACAT) catalyzes the esterification of cholesterol with fatty acids, allowing the storage of cholesterol esters in cytosolic lipid droplets and assembly of cholesterol esters in the neutral lipid core of lipoproteins (19). ACAT2, but not ACAT1, mRNA level was significantly reduced in livers of KO-Std mice compared with WT-Std mice (Fig. 7). In addition to the less impaired IR tyrosine phosphorylation (Fig. 4A), the improved glucose disposal in KO-HF mice may also be attributable to the reduction in gluconeogenesis. Glucose-6-phosphatase and phosphoenolpyruvate carboxylase were affected neither by diet nor by genotype (Fig. 7). An interesting finding was that expression of pyruvate dehydrogenase kinase 4 (PDK4) was significantly down-regulated in AdipoR2–/– mice, and it was not significantly enhanced by HF feeding as in WT mice (Fig. 7).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Adiponectin acts as an insulin sensitizer by enhancing fatty acid oxidation in skeletal muscle and suppressing hepatic glucose production in various animal models (20, 21, 22, 23). Adiponectin transgenic mice showed amelioration of insulin resistance and ß-cell degranulation (20, 21). Acute peripheral administration of adiponectin improves insulin sensitivity in normal c57BL/6, ob/ob, nonobese diabetic, streptozotocin-treated, and lipoatrophic mice (22, 23). In contrast, as mentioned in the introductory section, adiponectin-deficient mice develop severe insulin resistance triggered by HF feeding (4, 5). In view of the above evidence, we have hypothesized that mice lacking AdipoR2 would be more susceptible to HF-induced insulin resistance. To test this hypothesis, we studied glucose metabolism in animal models deficient in the AdipoR2 gene in two different ways. The AdipoR2–/– mice have an NT transcript but no full-length AdipoR2 present in all tissues, and the AdipoR2-ASO-treated ob/ob mice have only 20–30% full-length AdipoR2 remaining in liver and epididymal fat. To our surprise, our data showed that upon HF challenge, AdipoR2–/– mice were less insulin resistant as suggested by the improved ITT at both early and late stages, improved OGTT at early stage, and enhanced IR tyrosine phosphorylation. AdipoR2-ASO treatment also lowered plasma glucose levels in ob/ob mice. Our study, using two different approaches to disrupt AdipoR2 gene in both normal C57BL/6 and leptin-deficient diabetic animal models collectively showed that disruption of AdipoR2 gene improved insulin sensitivity.
The improved insulin sensitivity in KO-HF mice could directly result from the enhanced IR tyrosine phosphorylation in KO-HF mice. PKB phosphorylation showed only a trend of increased signal in AdipoR2–/– mice, and other signaling pathways under IR may play a more important role in leading to the improved glucose disposal in KO-HF mice. The less impaired glucose disposal in KO-HF mice may also be a result of decreased hepatic PDK4 expression. PDK4 plays a strategic and unique role in intermediary metabolism in liver by inactivating the pyruvate dehydrogenase complex (PDC), thus integrating gluconeogenesis and fatty acid synthesis through regulated pyruvate (24). Enhanced PDK4 expression in WT-HF mice presumably leads to enhanced gluconeogenesis due to accumulated pyruvate as a substrate. In contrast, the lower PDK4 expression in AdipoR2–/– mice would facilitate moving pyruvate into the citric acid cycle rather than gluconeogenesis, therefore allowing better glucose disposal compared with WT mice. The lack of phenotypic differences between the two genotypes on Std feeding indicates that the PDK/PDC system enables proper maintenance of metabolic endpoints (e.g. by adaptive changes of other PDK isoforms) when energy load is appropriate. However, excessive calorie load pushed the system out of capacity; hence, a phenotype developed.
The elevated adiponectin levels, alone or in combination with the enhanced AdipoR1 expression in liver, may contribute to the enhanced insulin sensitivity in KO-HF mice as well. Interestingly, genetic variants of AdipoR2 are associated with increased adiponectin levels and decreased triglyceride/VLDL levels in patients with metabolic syndrome (25). mRNA expression of AdipoR2, but not AdipoR1, in adipose tissue was specifically down-regulated in adiponectin transgenic mice and up-regulated in adiponectin-deficient mice (26). Such findings suggest a regulatory feedback loop by which adiponectin down-regulates the expression of AdipoR2 (26). Our results provided more evidence to suggest a feedback regulatory mechanism between AdipoR2 and plasma adiponectin levels. However, our findings cannot be interpreted by a simple negative feedback loop between AdipoR2 and adiponectin. On one hand, plasma adiponectin levels were elevated only in KO-HF mice, which was not accompanied by enhanced transcriptional expression of adiponectin gene in adipose tissue. Therefore, the elevation of plasma adiponectin levels in KO-HF vs. WT-HF mice may result from other regulatory mechanisms, such as secretion and clearance processes. On the other hand, the decreasing trend of plasma adiponectin levels in KO-Std mice vs. WT-Std mice (P < 0.05 at 4 and 8 wk; P < 0.01 at 22 wk on diet when the two Std groups were compared using a t test) intends to imply a positive correlation between AdipoR2 and adiponectin, which was transcriptionally down-regulated in sc fat of KO-Std mice. Taken together, our results suggest that the feedback regulation of adiponectin by AdipoR2 needs to be tightly associated with nutritional states.
It remains a mystery that disruption of AdipoR2, a receptor for adiponectin, resulted in a phenotype inconsistent with disruption of adiponectin in the sense of insulin sensitization. Animal models lacking AdipoR1 or both AdipoRs may bring some clarification to this field.
T2D is primarily characterized by insulin resistance and insufficient compensation of ß-cells. In the progression of T2D, decompensation of ß-cells is the key turning point where insulin resistance eventually becomes frank diabetes. Adiponectin is shown to counteract cytokine- and fatty-acid-induced apoptosis in the rat pancreatic ß-cell line INS-1 (27). However, adiponectin does not affect insulin secretion or basal/fatty-acid-induced ß-cell apoptosis in human islets (28). Our results showed that disruption of AdipoR2 did not result in any compensatory change of AdipoR1 expression in islet. Loss of AdipoR2 did not affect the ß-cell mass or insulin production when animals were on Std, suggesting that AdipoR2 does not play a critical role in the development and regular maintenance of ß-cell function in mice. However, disruption of AdipoR2 abolished the robust islet expansion in response to HF feeding, indicating that AdipoR2 is specifically involved in ß-cell replication and neogenesis in response to insulin resistance. Therefore, despite the improved peripheral insulin sensitivity, disruption of AdipoR2 resulted in significantly elevated fasting glucose levels in mice on HF at a longer time. Our data do not indicate an increase of ß-cell apoptosis in KO-HF mice at this stage; however, it is conceivable that apoptosis may be accelerated in a later stage, which may be mainly due to ß-cell exhaustion rather than a direct consequence of AdipoR2 loss.
The reduced body weight of KO-HF mice was not due to reduction in food intake, because that was not different between genotypes. FAS is a central player in de novo lipogenesis (29). MGAT1 is another principal lipogenic enzyme that catalyzes the synthesis of diacylglycerol, a precursor of lipids such as triacylglycerol and phospholipids (30). The suppressed expression of FAS and MGAT1 indicate that lipogenesis was down-regulated in KO-HF mice. ACAT2 is the major ACAT isoform in liver and small intestine, ACAT2-deficient mice are completely resistant to HF/high-cholesterol diet-induced hypercholesterolemia, which proposes ACAT as a crucial gene in regulating dietary cholesterol absorption (31). It is possible that the intestinal expression of ACAT2 may also be suppressed in AdipoR2-deficient mice. Taken together, the suppression of FAS, MGAT1, and ACAT2 at the transcriptional level may serve as mechanisms leading to the lower body weight and improved lipid profile found in KO-HF mice.
Reviewing the presented data, we have noticed the following phenomena (see scheme): with proper calorie intake, deletion of AdipoR2 did not change lipid and glucose metabolic endpoints, implying properly orchestrated insulin sensitivity in peripheral tissues and insulin production by pancreatic ß-cells, whereas with excessive energy (fat) load, WT mice developed obesity, severe dyslipidemia, and extreme insulin resistance. Fortunately, the presence of AdipoR2 enabled ß-cells to compensate by remarkably amplified replication and neogenesis; therefore, WT mice maintained plasma glucose at a manageable level (although not perfectly normal). In the case of AdipoR2 disruption, AdipoR2–/– mice initially appreciated a beneficial outcome: less obesity, modest dyslipidemia, and moderate insulin resistance. However, the absence of AdipoR2 eradicated ß-cell replication and neogenesis specifically in response to insulin resistance, resulting in inadequate compensation even for the moderate peripheral insulin resistance; thus, AdipoR2–/– mice finally developed overt diabetes.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Disclosure Statement: The authors have nothing to disclose.
First Published Online October 26, 2006
Abbreviations: ACAT, Acyl coenzyme A:cholesterol transferase; AdipoR, adiponectin receptor; AMPK, AMP-activated kinase; ASO, antisense oligo; CT, C-terminal; FAS, fatty acid synthase; HDL, high-density lipoprotein; HF, high-fat chow diet; IR, insulin receptor; ITT, insulin tolerance test; KO, knockout; LDL, low-density lipoprotein; lgHDL, large HDL; MGAT1, monoacylglycerol acyltransferase 1; NT, N-terminal; OGTT, oral glucose tolerance test; PDC, pyruvate dehydrogenase complex; PDK4, pyruvate dehydrogenase kinase 4; PKB, protein kinase B; Std, standard chow diet; T2D, type 2 diabetes; VLDL, very-low-density lipoprotein; WT, wild type.
Received May 25, 2006.
Accepted for publication October 17, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
agonists. J Biol Chem 281:2654–2660, PPAR, and LXR. Biochem Biophys Res Commun 314:151–158[CrossRef][Medline]/ dual agonist with a unique in vitro profile and potent glucose and lipid effects in rodent models of type 2 diabetes and dyslipidemia. Mol Endocrinol 19:1593–1605This article has been cited by other articles:
 |
|
 |
|
  R. S. Ahima and M. A. Lazar Adipokines and the Peripheral and Neural Control of Energy Balance Mol. Endocrinol., May 1, 2008; 22(5): 1023 - 1031. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
