TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1(Ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IR(Tyr960), both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(Ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IR(Tyr960), both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.     

Phosphoenolpyruvate carboxykinase overexpression selectively attenuates insulin signaling and hepatic insulin sensitivity in transgenic mice

The ability of insulin to suppress gluconeogenesis in type II diabetes mellitus is impaired; however, the cellular mechanisms for this insulin resistance remain poorly understood. To address this question, we generated transgenic (TG) mice overexpressing the phosphoenolpyruvate carboxykinase (PEPCK) gene under control of its own promoter. TG mice had increased basal hepatic glucose production (HGP), but normal levels of plasma free fatty acids (FFAs) and whole-body glucose disposal during a hyperinsulinemic-euglycemic clamp compared with wild-type controls. The steady-state levels of PEPCK and glucose-6-phosphatase mRNAs were elevated in livers of TG mice and were resistant to down-regulation by insulin. Conversely, GLUT2 and glucokinase mRNA levels were appropriately regulated by insulin, suggesting that insulin resistance is selective to gluconeogenic gene expression. Insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate (IRS)-1, and associated phosphatidylinositol 3-kinase were normal in TG mice, whereas IRS-2 protein and phosphorylation were down-regulated compared with control mice. These results establish that a modest (2-fold) increase in PEPCK gene expression in vivo is sufficient to increase HGP without affecting FFA concentrations. Furthermore, these results demonstrate that PEPCK overexpression results in a metabolic pattern that increases glucose-6-phosphatase mRNA and results in a selective decrease in IRS-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression. However, acute suppression of HGP and glycolytic gene expression remained intact, suggesting that FFA and/or IRS-1 signaling, in addition to reduced IRS-2, plays an important role in downstream insulin signal transduction pathways involved in control of gluconeogenesis and progression to type II diabetes mellitus.     

PGC-1 promotes insulin resistance in liver through PPAR-alpha-dependent induction of TRB-3

Insulin resistance is a major hallmark in the development of type 2 diabetes, which is characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in muscle. The nuclear hormone receptor coactivator PGC-1 (peroxisome proliferator-activated (PPAR)-gamma coactivator-1) has been implicated in the onset of type 2 diabetes. Hepatic PGC-1 expression is elevated in mouse models of this disease, where it promotes constitutive activation of gluconeogenesis and fatty acid oxidation through its association with the nuclear hormone receptors HNF-4 and PPAR-alpha, respectively. Here we show that PGC-1-deficient mice, generated by adenoviral delivery of PGC-1 RNA interference (RNAi) to the liver, experience fasting hypoglycemia. Hepatic insulin sensitivity was enhanced in PGC-1-deficient mice, reflecting in part the reduced expression of the mammalian tribbles homolog TRB-3, a fasting-inducible inhibitor of the serine-threonine kinase Akt/PKB (ref. 6). We show here that, in the liver, TRB-3 is a target for PPAR-alpha. Knockdown of hepatic TRB-3 expression improved glucose tolerance, whereas hepatic overexpression of TRB-3 reversed the insulin-sensitive phenotype of PGC-1-deficient mice. These results indicate a link between nuclear hormone receptor and insulin signaling pathways, and suggest a potential role for TRB-3 inhibitors in the treatment of type 2 diabetes.     

Re-examination of the role of suppressor of cytokine signaling 1 (SOCS1) in the regulation of toll-like receptor signaling

Suppressor of cytokine signaling 1 (SOCS1) is an obligate negative regulator of cytokine signaling and most importantly in vivo, signaling via the interferon-gamma (IFN-gamma) receptor. SOCS1, via its Src homology 2 domain, binds to phosphotyrosine residues in its targets, reducing the amplitude of signaling from cytokine receptors. SOCS1 is also implicated in blocking Toll-like receptor (TLR) signaling in macrophages activated by TLR agonists such as lipopolysaccharide (LPS), thus regulating multiple steps in the activation of innate immune responses. To rigorously test this, we isolated macrophages from Socs1-/- mice on multiple genetic backgrounds. We found no evidence that SOCS1 blocked TLR-activated pathways, endotoxin tolerance, or nitric oxide production. However, Socs1-/-;IFN-gamma-/- mice were extremely susceptible to LPS challenge, confirming previous findings. Because LPS induces IFN-beta production from macrophages, we tested whether SOCS1 regulates IFN-alpha/beta receptor signaling. We find that SOCS1 is required to inhibit IFN-alpha/beta receptor signaling in vitro. Furthermore, the absence of a single allele encoding TYK2, a JAK (Janus kinase) family member essential IFN-alpha/beta receptor signaling, rescued Socs1-/- mice from early lethality, even in the presence of IFN-gamma. We conclude that previous reports linking SOCS1 to TLR signaling are most likely due to effects on IFN-alpha/beta receptor signaling.     

SOCS-1 deficiency does not prevent diet-induced insulin resistance

Obesity is associated with inflammation and increased expression of suppressor of cytokine signaling (SOCS) proteins, which inhibit cytokine and insulin signaling. Thus, reducing SOCS expression could prevent the development of obesity-induced insulin resistance. Using SOCS-1 knockout mice, we investigated the contribution of SOCS-1 in the development of insulin resistance induced by a high-fat diet (HFD). SOCS-1 knockout mice on HFD gained 70% more weight, displayed a 2.3-fold increase in epididymal fat pads mass and increased hepatic lipid content. This was accompanied by increased mRNA expression of leptin and the macrophage marker CD68 in white adipose tissue and of SREBP1c and FAS in liver. HFD also induced hyperglycemia in SOCS-1 deficient mice with impairment of glucose and insulin tolerance tests. Thus, despite the role of SOCS proteins in obesity-related insulin resistance, SOCS-1 deficiency alone is not able to prevent insulin resistance induced by a diet rich in fat.     

Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes

Although elevation of the blood glucose level is a causal adverse effect of treatment with interferon (IFN), the precise underlying molecular mechanism is largely unknown. We examined the effects of type I and type II IFN (IFN-β and IFN-γ) on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. IFN-β suppressed insulin-induced tyrosine phosphorylation of IRS-1 without affecting its expression, whereas IFN-γ reduced both the protein level and tyrosine phosphorylation. Although both IFNs stimulated phosphorylation of STAT1 (at Tyr(701)) and STAT3 (at Tyr(705)) after treatment for 30 min, subsequent properties of induction of the SOCS isoform were different. IFN-β preferentially induced SOCS1 rather than SOCS3, whereas IFN-γ strongly induced SOCS3 expression alone. In addition, adenovirus-mediated overexpression of either SOCS1 or SOCS3 inhibited insulin-induced tyrosine phosphorylation of IRS-1, whereas the reduction of IRS-1 protein was observed only in SOCS3-expressed cells. Notably, IFN-β-induced SOCS1 expression and suppression of insulin-induced tyrosine phosphorylation of IRS-1 were attenuated by siRNA-mediated knockdown of STAT1. In contrast, adenovirus-mediated expression of a dominant-negative STAT3 (F-STAT3) attenuated IFN-γ-induced SOCS3 expression, reduction of IRS-1 protein, and suppression of insulin-induced glucose uptake but did not have any effect on the IFN-β-mediated SOCS1 expression and inhibition of insulin-induced glucose uptake. Interestingly, pretreatment of IFN-γ with IL-6 synergistically suppressed insulin signaling, even when IL-6 alone had no significant effect. These results indicate that type I and type II IFN induce insulin resistance by inducing distinct SOCS isoforms, and IL-6 synergistically augments IFN-γ-induced insulin resistance by potentiating STAT3-mediated SOCS3 induction in 3T3-L1 adipocytes.     

Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor. A potential mechanism for cytokine-mediated insulin resistance

Insulin resistance contributes to a number of metabolic disorders, including type II diabetes, hypertension, and atherosclerosis. Cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, and hormones, such as growth hormone, are known to cause insulin resistance, but the mechanisms by which they inhibit the cellular response to insulin have not been elucidated. One mechanism by which these agents could cause insulin resistance is by inducing the expression of cellular proteins that inhibit insulin receptor (IR) signaling. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling pathways, the expression of which is regulated by certain cytokines. SOCS proteins are therefore attractive candidates as mediators of cytokine-induced insulin resistance. We have found that SOCS-1 and SOCS-6 interact with the IR when expressed in human hepatoma cells (HepG2) or in rat hepatoma cells overexpressing the human IR. In SOCS-1-expressing cells, insulin treatment increases the extent of interaction with the IR, whereas in SOCS-6-expressing cells the association with the IR appears to require insulin treatment. SOCS-1 and SOCS-6 do not inhibit insulin-dependent IR autophosphorylation, but both proteins inhibit insulin-dependent activation of ERK1/2 and protein kinase B in vivo and IR-directed phosphorylation of IRS-1 in vitro. These results suggest that SOCS proteins may be inhibitors of IR signaling and could mediate cytokine-induced insulin resistance and contribute to the pathogenesis of type II diabetes.     

Increased bone mass, altered trabecular architecture and modified growth plate organization in the growing skeleton of SOCS2 deficient mice

Suppressor of cytokine signalling-2 (SOCS2) negatively regulates the signal transduction of several cytokines. Socs2(-/-) mice show increased longitudinal skeletal growth associated with deregulated GH/IGF-1 signalling. The present study examined the role of SOCS2 in endochondral ossification and trabecular and cortical bone formation, and investigated whether pro-inflammatory cytokines associated with pediatric chronic inflammatory disorders mediate their effects through SOCS2. Seven-week-old Socs2(-/-) mice were heavier (27%; P < 0.001) and longer (6%; P < 0.001) than wild-type mice. Socs2(-/-) tibiae were longer (8%; P < 0.001) and broader (18%; P < 0.001) than that of wild-type mice, and the Socs2(-/-) mice had wider growth plates (24%; P < 0.001) with wider proliferative and hypertrophic zones (10% (P < 0.05) and 14% (P < 0.001) respectively). Socs2(-/-) mice showed increased total cross-sectional bone area (16%: P < 0.001), coupled to increased total tissue area (17%; P < 0.05) compared to tibia from wild-type mice. Socs2(-/-) mice showed increased percent bone volume (101%; P < 0.001), trabecular number (82%; P < 0.001) and trabecular thickness (11%; P < 0.001), with associated decreases in trabecular separation (19%; P < 0.001). TNFalpha exposure to growth plate chondrocytes for 48 h increased SOCS2 protein expression. Growth of metatarsals from 1-day-old Socs2(-/-) and Socs2(+/+) mice, as well as expression of Aggrecan, Collagen Type II and Collagen Type X, were inhibited by TNFalpha, with no effect of genotype. Our data indicate that physiological levels of SOCS2 negatively regulate bone formation and endochondral growth. Our results further suggest that pro-inflammatory cytokines mediate their inhibitory effects on longitudinal bone growth through a mechanism that is independent of SOCS2.     

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1^−/−Ifng^−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng^−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1^−/−Ifng^−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng^−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.     

Impaired hepatic insulin signalling in PON2-deficient mice: a novel role for the PON2/apoE axis on the macrophage inflammatory response

Hepatic glucose metabolism is strongly influenced by oxidative stress and pro-inflammatory stimuli. PON2 (paraoxonase 2), an enzyme with undefined antioxidant properties, protects against atherosclerosis. PON2-deficient (PON2-def) mice have elevated hepatic oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages. In the present paper, we demonstrate that PON2 deficiency is associated with inhibitory insulin-mediated phosphorylation of hepatic IRS-1 (insulin receptor substrate-1). Unexpectedly, we observed a marked improvement in the hepatic IRS-1 phosphorylation state in PON2-def/apoE (apolipoprotein E)(-/-) mice, relative to apoE(-/-) mice. Factors secreted from activated macrophage cultures derived from PON2-def and PON2-def/apoE(-/-) mice are sufficient to modulate insulin signalling in cultured hepatocytes in a manner similar to that observed in vivo. We show that the protective effect on insulin signalling in PON2-def/apoE(-/-) mice is directly associated with altered production of macrophage pro-inflammatory mediators, but not elevated intracellular oxidative stress levels. We further present evidence that modulation of the macrophage inflammatory response in PON2-def/apoE(-/-) mice is mediated by a shift in the balance of NO and ONOO(-) (peroxynitrite) formation. Our results demonstrate that PON2 plays an important role in hepatic insulin signalling and underscores the influence of macrophage-mediated inflammatory response on hepatic insulin sensitivity.     

Low density lipoprotein (LDL) receptor-related protein 6 (LRP6) regulates body fat and glucose homeostasis by modulating nutrient sensing pathways and mitochondrial energy expenditure

Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6(+/-) mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6(+/-) mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6(+/-) mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6(+/-) mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.     

SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.     

Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms

Insulin resistance is a pathophysiological component of type 2 diabetes and obesity and also occurs in states of stress, infection, and inflammation associated with an upregulation of cytokines. Here we show that in both obesity and lipopolysaccharide (LPS)-induced endotoxemia there is an increase in suppressor of cytokine signaling (SOCS) proteins, SOCS-1 and SOCS-3, in liver, muscle, and, to a lesser extent, fat. In concordance with these increases by LPS, tyrosine phosphorylation of the insulin receptor (IR) is partially impaired and phosphorylation of the insulin receptor substrate (IRS) proteins is almost completely suppressed. Direct overexpression of SOCS-3 in liver by adenoviral-mediated gene transfer markedly decreases tyrosine phosphorylation of both IRS-1 and IRS-2, while SOCS-1 overexpression preferentially inhibits IRS-2 phosphorylation. Neither affects IR phosphorylation, although both SOCS-1 and SOCS-3 bind to the insulin receptor in vivo in an insulin-dependent fashion. Experiments with cultured cells expressing mutant insulin receptors reveal that SOCS-3 binds to Tyr960 of IR, a key residue for the recognition of IRS-1 and IRS-2, whereas SOCS-1 binds to the domain in the catalytic loop essential for IRS-2 recognition in vitro. Moreover, overexpression of either SOCS-1 or SOCS-3 attenuates insulin-induced glycogen synthesis in L6 myotubes and activation of glucose uptake in 3T3L1 adipocytes. By contrast, a reduction of SOCS-1 or SOCS-3 by antisense treatment partially restores tumor necrosis factor alpha-induced downregulation of tyrosine phosphorylation of IRS proteins in 3T3L1 adipocytes. These data indicate that SOCS-1 and SOCS-3 act as negative regulators in insulin signaling and serve as one of the missing links between insulin resistance and cytokine signaling.     

Regulation of Insulin and Leptin Signaling by Muscle Suppressor of Cytokine Signaling 3 (SOCS3)

Skeletal muscle resistance to the key metabolic hormones, leptin and insulin, is an early defect in obesity. Suppressor of cytokine signaling 3 (SOCS3) is a major negative regulator of both leptin and insulin signaling, thereby implicating SOCS3 in the pathogenesis of obesity and associated metabolic abnormalities. Here, we demonstrate that SOCS3 mRNA expression is increased in murine skeletal muscle in the setting of diet-induced and genetic obesity, inflammation, and hyperlipidemia. To further evaluate the contribution of muscle SOCS3 to leptin and insulin resistance in obesity, we generated transgenic mice with muscle-specific overexpression of SOCS3 (MCK/SOCS3 mice). Despite similar body weight, MCK/SOCS3 mice develop impaired systemic and muscle-specific glucose homeostasis and insulin action based on glucose and insulin tolerance tests, hyperinsulinemic-euglycemic clamps, and insulin signaling studies. With regards to leptin action, MCK/SOCS3 mice exhibit suppressed basal and leptin-stimulated activity and phosphorylation of alpha2 AMP-activated protein kinase (α2AMPK) and its downstream target, acetyl-CoA carboxylase (ACC). Muscle SOCS3 overexpression also suppresses leptin-regulated genes involved in fatty acid oxidation and mitochondrial function. These studies demonstrate that SOC3 within skeletal muscle is a critical regulator of leptin and insulin action and that increased SOCS may mediate insulin and leptin resistance in obesity.     

Liver-specific inducible nitric-oxide synthase expression is sufficient to cause hepatic insulin resistance and mild hyperglycemia in mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.     

Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.     

Suppressor of cytokine signaling-2 deficiency induces molecular and metabolic changes that partially overlap with growth hormone-dependent effects

Suppressor of cytokine signaling-2 (SOCS2)-deficient (SOCS2-/-) mice grow significantly larger than their littermates, suggesting that SOCS2 is important in the negative regulation of the actions of GH and/or IGF-I. The aim of this study was to identify genes and metabolic parameters that might contribute to the SOCS2-/- phenotype. We demonstrate that although SOCS2 deficiency induces significant changes in hepatic gene expression, only a fraction of these overlap with known GH-induced effects in the liver, suggesting that SOCS2 might be an important regulator of other growth factors and cytokines acting on the liver. However, an important role of GH and IGF-I in the phenotype of these animals was demonstrated by an overexpression of IGF-binding protein-3 mRNA in the liver and increased levels of circulating IGF-binding protein-3. Other GH-like effects included diminished serum triglycerides and down-regulation of lipoprotein lipase in adipose tissue. Interestingly, SOCS2-/- mice did not differ from their wild-type littermates in glucose or insulin tolerance tests, which is in contrast with the known diabetogenic effects of GH. Furthermore, there was no evidence of impaired insulin signaling in primary hepatocytes isolated from SOCS2-/- mice. Moreover, increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA was detected in skeletal muscle, which might contribute to normal glycemic control despite the apparent overactivity of the GH/IGF-I axis. Our data indicate that SOCS2 deficiency partially mimics a state of increased GH activity, but also results in changes that cannot be related to known GH effects.     

Protein-tyrosine phosphatase 1B deficiency reduces insulin resistance and the diabetic phenotype in mice with polygenic insulin resistance

Mice heterozygous for insulin receptor (IR) and IR substrate (IRS)-1 deficiency provide a model of polygenic type 2 diabetes in which early-onset, genetically programmed insulin resistance leads to diabetes. Protein-tyrosine phosphatase 1B (PTP1B) dephosphorylates tyrosine residues in IR and possibly IRS proteins, thereby inhibiting insulin signaling. Mice lacking PTP1B are lean and have increased insulin sensitivity. To determine whether PTP1B can modify polygenic insulin resistance, we crossed PTP1B-/- mice with mice with a double heterozygous deficiency of IR and IRS-1 alleles (DHet). DHet mice weighed slightly less than wild-type mice and exhibited severe insulin resistance and hyperglycemia, with approximately 35% of DHet males developing diabetes by 9-10 weeks of age. Body weight in DHet mice with PTP1B deficiency was similar to that in DHet mice. However, absence of PTP1B in DHet mice markedly improved glucose tolerance and insulin sensitivity at 10-11 weeks of age and reduced the incidence of diabetes and hyperplastic pancreatic islets at 6 months of age. Insulin-stimulated phosphorylation of IR, IRS proteins, Akt/protein kinase B, glycogen synthase kinase 3beta, and p70(S6K) was impaired in DHet mouse muscle and liver and was differentially improved by PTP1B deficiency. In addition, increased phosphoenolpyruvate carboxykinase expression in DHet mouse liver was reversed by PTP1B deficiency. In summary, PTP1B deficiency reduces insulin resistance and hyperglycemia without altering body weight in a model of polygenic type 2 diabetes. Thus, even in the setting of high genetic risk for diabetes, reducing PTP1B is partially protective, further demonstrating its attractiveness as a target for prevention and treatment of type 2 diabetes.