Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by thiazolidinediones (TZDs) improves insulin resistance by increasing insulin-stimulated glucose disposal in skeletal muscle. It remains debatable whether the effect of TZDs on muscle is direct or indirect via adipose tissue. We therefore generated mice with muscle-specific PPARgamma knockout (MuPPARgammaKO) using Cre/loxP recombination. Interestingly, MuPPARgammaKO mice developed excess adiposity despite reduced dietary intake. Although insulin-stimulated glucose uptake in muscle was not impaired, MuPPARgammaKO mice had whole-body insulin resistance with a 36% reduction (P

Berardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous genetic disease characterized by near absence of adipose tissue and severe insulin resistance. We have previously identified mutations in the seipin gene in a subset of our patients' cohort. Recently, disease-causing mutations in AGPAT2 have been reported in BSCL patients. In this study, we have performed mutation screening in AGPAT2 and the related AGPAT1 in patients with BSCL or other forms of lipodystrophy who have no detectable mutation in the seipin gene. We found 38 BSCL patients from 30 families with mutations in AGPAT2. Three of the known mutations were frequently found in our families. Of the eight new alterations, six are null mutations and two are missense mutations (Glu172Lys and Ala238Gly). All the patients harboring AGPAT2 mutations presented with typical features of BSCL. We did not find mutations in patients with other forms of lipodystrophies, including the syndromes of Lawrence, Dunnigan, and Barraquer-Simons, or with type A insulin resistance. In conclusion, mutations in the seipin gene and AGPAT2 are confined to the BSCL phenotype. Because we found mutations in 92 of the 94 BSCL patients studied, the seipin gene and AGPAT2 are the two major genes involved in the etiology of BSCL.

Caloric restriction has been shown to increase longevity in organisms ranging from yeast to mammals. In some organisms, this has been associated with a decreased fat mass and alterations in insulin/insulin-like growth factor 1 (IGF-1) pathways. To further explore these associations with enhanced longevity, we studied mice with a fat-specific insulin receptor knockout (FIRKO). These animals have reduced fat mass and are protected against age-related obesity and its subsequent metabolic abnormalities, although their food intake is normal. Both male and female FIRKO mice were found to have an increase in mean life-span of approximately 134 days (18%), with parallel increases in median and maximum life-spans. Thus, a reduction of fat mass without caloric restriction can be associated with increased longevity in mice, possibly through effects on insulin signaling.

A central component of type 2 diabetes and the metabolic syndrome is insulin resistance. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems. Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway provides a unique approach to determining the role of individual proteins in the molecular mechanism of insulin action and the pathogenesis of insulin resistance and diabetes. The role of the four major insulin receptor substrates (IRS1-4) in insulin and IGF-1 signaling have been examined by creating mice with targeted gene knockouts. Each produces a unique phenotype, indicating the complementary role of these signaling components. Combined heterozygous defects often produce synergistic or epistatic effects, although the final severity of the phenotype depends on the genetic background of the mice. Conditional knockouts of the insulin receptor have also been created using the Cre-lox system. These tissue specific knockouts have provide unique insights into the control of glucose homeostasis and the pathogenesis of type 2 diabetes, and have led to development of new hypotheses about the nature of the insulin action and development of diabetes.

Hepatocyte nuclear factors-3 (Foxa-1-3) are winged forkhead transcription factors that regulate gene expression in the liver and pancreatic islets and are required for normal metabolism. Here we show that Foxa-2 is expressed in preadipocytes and induced de novo in adipocytes of genetic and diet-induced rodent models of obesity. In preadipocytes Foxa-2 inhibits adipocyte differentiation by activating transcription of the Pref-1 gene. Foxa-2 and Pref-1 expression can be enhanced in primary preadipocytes by growth hormone, suggesting that the antiadipogenic activity of growth hormone is mediated by Foxa-2. In differentiated adipocytes Foxa-2 expression leads to induction of gene expression involved in glucose and fat metabolism, including glucose transporter-4, hexokinase-2, muscle-pyruvate kinase, hormone-sensitive lipase, and uncoupling proteins-2 and -3. Diet-induced obese mice with haploinsufficiency in Foxa-2 (Foxa-2+/-) develop increased adiposity compared with wild-type littermates as a result of decreased energy expenditure. Furthermore, adipocytes of these Foxa-2+/- mice exhibit defects in glucose uptake and metabolism. These data suggest that Foxa-2 plays an important role as a physiological regulator of adipocyte differentiation and metabolism.

Mice double heterozygous (DH) for deletion of insulin receptor and insulin receptor substrate-1 are lean, insulin resistant, and have a phenotype that strongly depends on the genetic background of the mouse. On the C57BL/6 (B6) background, DH mice develop marked hyperinsulinemia and diabetes, whereas on the 129S6 background, DH mice exhibit only mild elevations of insulin and remain free of diabetes. F2 male mice created by an intercross between these two strains exhibit a 60% incidence of diabetes and a bell-shaped distribution of insulin levels as related to glucose, reminiscent of that in humans with type 2 diabetes. These mice also exhibit a wide range of leptin levels as related to body weight. A genome-wide scan of F2 mice reveals a quantitative trait locus (QTL) related to hyperinsulinemia on chromosome 14 (D14Mit55) with a peak logarithm of odds (LOD) score of 5.6, accounting for up to 69% of this trait. A QTL with a LOD score of 3.7 related to hyperleptinemia is present on chromosome 7 at D12Mit38 (a marker previously assigned to chromosome 12) in the area of the uncoupling protein 2/3 gene cluster. This locus also interacts synergistically with D14Mit55 in development of hyperinsulinemia and with a QTL on chromosome 12 (D12Mit231) related to hyperglycemia. These data demonstrate how multiple genetic modifiers can interact and influence the development of diabetes and the phenotype of animals with genetically programmed insulin resistance and provide evidence as to the location and nature of these genes.

To investigate the role of insulin receptor substrate-1 (IRS-1) and its downstream signaling in insulin-induced thermogenic differentiation of brown adipocytes, we have reconstituted IRS-1-deficient fetal brown adipocytes (IRS-1(-/-)) with wild-type IRS-1 (IRS-1(wt)). The lack of IRS-1 resulted in the inability of insulin to induce IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity and Akt phosphorylation in IRS-1(-/-) brown adipocytes. In addition, these cells showed an impairment in activating alpha-Akt, beta-Akt, and gamma-Akt isoforms upon insulin stimulation. Reconstitution of IRS-1(-/-) brown adipocytes with IRS-1(wt) restored the IRS-1/PI 3-kinase/Akt signaling pathway. Treatment of wild-type brown adipocytes with insulin for 24 h up-regulated uncoupling protein-1 (UCP-1) expression and transactivated the UCP-1 promoter; this effect was abolished in the absence of IRS-1 or in the presence of an Akt inhibitor and further recovered after IRS-1(wt) reconstitution. Neither UCP-2 nor UCP-3 was up-regulated by insulin in wild-type and IRS-1-deficient brown adipocytes. Insulin stimulated the expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and its DNA binding activity in wild-type brown adipocytes but not in IRS-1(-/-) cells. However, insulin stimulation of both C/EBPalpha expression and binding activity was restored after IRS-1(wt) reconstitution of deficient cells. Retrovirus-mediated expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma in IRS-1(-/-) brown adipocytes up-regulated UCP-1 protein content and transactivated UCP-1 promoter regardless of insulin stimulation. Both C/EBPalpha and peroxisome proliferator-activated receptor gamma reconstituted FAS mRNA expression, but only C/EBPalpha restored insulin sensitivity in the absence of IRS-1. Finally, reconstitution of IRS-1(-/-) brown adipocytes with the IRS-1 mutants IRS-1(Phe-895), which lacks IRS-1/growth factor receptor binding protein 2 binding but not IRS-1/p85-PI 3-kinase binding, or with IRS-1(Tyr-608/Tyr-628/Tyr-658), which only binds p85-PI 3-kinase, induced UCP-1 expression and transactivated the UCP-1 promoter. These data provide strong evidence for an essential role of IRS-1 through the PI 3-kinase/Akt signaling pathway inducing UCP-1 gene expression by insulin.

Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic beta cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican-American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. Although NRF-1 expression is decreased only in diabetic subjects, expression of both PPAR gamma coactivator 1-alpha and-beta (PGC1-alpha/PPARGC1 and PGC1-beta/PERC), coactivators of NRF-1 and PPAR gamma-dependent transcription, is decreased in both diabetic subjects and family history-positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRF-dependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.

Type 2 diabetes is a complex disease in which genetic and environmental factors interact to produce alterations in insulin action and insulin secretion, leading to hyperglycemia. To evaluate the influence of genetic background on development of diabetes in a genetically susceptible host, we generated mice that are double heterozygous (DH) for knockout of the insulin receptor and insulin receptor substrate-1 on three genetic backgrounds (C57BL/6 [B6], 129Sv, and DBA). Although DH mice on all backgrounds showed insulin resistance, their phenotypes were dramatically different. B6 DH mice exhibited marked hyperinsulinemia and massive islet hyperplasia and developed early hyperglycemia, with 85% overtly diabetic by 6 months. By contrast, 129Sv DH mice showed mild hyperinsulinemia and minimal islet hyperplasia, and