Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact

Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic beta-cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of beta-cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.     

Mechanism of glucose intolerance in mice with dominant negative mutation of CEACAM1

Mice with liver-specific overexpression of dominant negative phosphorylation-defective S503A-CEACAM1 mutant (L-SACC1) developed chronic hyperinsulinemia resulting from blunted hepatic clearance of insulin, visceral obesity, and glucose intolerance. To determine the underlying mechanism of altered glucose homeostasis, a 2-h hyperinsulinemic euglycemic clamp was performed, and tissue-specific glucose and lipid metabolism was assessed in awake L-SACC1 and wild-type mice. Inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) caused insulin resistance in liver that was mostly due to increased expression of fatty acid synthase and lipid metabolism, resulting in elevated intrahepatic levels of triglyceride and long-chain acyl-CoAs. Whole body insulin resistance in the L-SACC1 mice was further attributed to defects in insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Insulin resistance in peripheral tissues was associated with significantly elevated intramuscular fat contents that may be secondary to increased whole body adiposity (assessed by (1)H-MRS) in the L-SACC1 mice. Overall, these results demonstrate that L-SACC1 is a mouse model in which chronic hyperinsulinemia acts as a cause, and not a consequence, of insulin resistance. Our findings further indicate the important role of CEACAM1 and hepatic insulin clearance in the pathogenesis of obesity and insulin resistance.     

Metabolic and therapeutic lessons from genetic manipulation of GLUT4

This review focuses on the effects of varying levels of GLUT4, the insulin-sensitive glucose transporter, on insulin sensitivity and whole body glucose homeostasis. Three mouse models are discussed including MLC-GLUT4 mice which overexpress GLUT4 specifically in skeletal muscle, GLUT4 null mice which express no GLUT4, and the MLC-GLUT4 null mice which express GLUT4 only in skeletal muscle. Overexpressing GLUT4 specifically in the skeletal muscle results in increased insulin sensitivity in the MLC-GLUT4 mice. In contrast, the GLUT4 null mice exhibit insulin intolerance accompanied by abnormalities in glucose and lipid metabolism. Restoring GLUT4 expression in skeletal muscle in the MLC-GLUT4 null mice results in normal glucose metabolism but continued abnormal lipid metabolism. The results of experiments using these mouse models demonstrates that modifying the expression of GLUT4 profoundly affects whole body insulin action and consequently glucose and lipid metabolism.     

Skeletal muscle reprogramming by activation of calcineurin improves insulin action on metabolic pathways

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow- versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-gamma co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.     

Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue

Several lines of evidence suggest that a functional relationship exists between caveolin-1 and insulin signaling. However, it remains unknown whether caveolin-1 is normally required for proper insulin receptor signaling in vivo. To address this issue, we examined the status of insulin receptor signaling in caveolin-1 (–/–)-deficient (Cav-1 null) mice. Here, we show that Cav-1 null mice placed on a high-fat diet for 9 mo develop postprandial hyperinsulinemia. An insulin tolerance test (ITT) revealed that young Cav-1 null mice on a normal chow diet are significantly unresponsive to insulin, compared with their wild-type counterparts. This insulin resistance is due to a primary defect in adipose tissue, as evidenced by drastically reduced insulin receptor protein levels (>90%), without any changes in insulin receptor mRNA levels. These data suggest that caveolin-1 acts as a molecular chaperone that is necessary for the proper stabilization of the insulin receptor in adipocytes in vivo. In support of this notion, we demonstrate that recombinant expression of caveolin-1 in Cav-1 null mouse embryo fibroblasts rescues insulin receptor protein expression. These data provide evidence that the lean body phenotype observed in the Cav-1 knockout mice is due, at least in part, to a defect in insulin-regulated lipogenesis. caveolae; caveolin; insulin signaling; protein stabilization; knockout mice     

Hormone-sensitive lipase knockout mice have increased hepatic insulin sensitivity and are protected from short-term diet-induced insulin resistance in skeletal muscle and heart

Insulin resistance in skeletal muscle and heart plays a major role in the development of type 2 diabetes and diabetic heart failure and may be causally associated with altered lipid metabolism. Hormone-sensitive lipase (HSL) is a rate-determining enzyme in the hydrolysis of triglyceride in adipocytes, and HSL-deficient mice have reduced circulating fatty acids and are resistant to diet-induced obesity. To determine the metabolic role of HSL, we examined the changes in tissue-specific insulin action and glucose metabolism in vivo during hyperinsulinemic euglycemic clamps after 3 wk of high-fat or normal chow diet in awake, HSL-deficient (HSL-KO) mice. On normal diet, HSL-KO mice showed a twofold increase in hepatic insulin action but a 40% decrease in insulin-stimulated cardiac glucose uptake compared with wild-type littermates. High-fat feeding caused a similar increase in whole body fat mass in both groups of mice. Insulin-stimulated glucose uptake was reduced by 50-80% in skeletal muscle and heart of wild-type mice after high-fat feeding. In contrast, HSL-KO mice were protected from diet-induced insulin resistance in skeletal muscle and heart, and these effects were associated with reduced intramuscular triglyceride and fatty acyl-CoA levels in the fat-fed HSL-KO mice. Overall, these findings demonstrate the important role of HSL on skeletal muscle, heart, and liver glucose metabolism.     

Endothelium-dependent delivery of insulin to muscle interstitium

Insulin contributes to skeletal muscle glucose uptake by increasing blood flow and recruiting perfused capillaries. In this issue of Cell Metabolism, Kubota et al. (2011) show that deletion of IRS-2 in endothelial cells in mice causes impaired transcapillary insulin transport, decreased insulin-stimulated glucose uptake in muscle, and mild glucose intolerance.     

Muscle-specific Pparg deletion causes insulin resistance

Thiazolidinediones (TZDs) are insulin-sensitizing drugs and are potent agonists of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Although muscle is the major organ responsible for insulin-stimulated glucose disposal, PPAR-gamma is more highly expressed in adipose tissue than in muscle. To address this issue, we used the Cre-loxP system to knock out Pparg, the gene encoding PPAR-gamma, in mouse skeletal muscle. As early as 4 months of age, mice with targeted disruption of PPAR-gamma in muscle showed glucose intolerance and progressive insulin resistance. Using the hyperinsulinemic-euglycemic clamp technique, the in vivo insulin-stimulated glucose disposal rate (IS-GDR) was reduced by approximately 80% and was unchanged by 3 weeks of TZD treatment. These effects reveal a crucial role for muscle PPAR-gamma in the maintenance of skeletal muscle insulin action, the etiology of insulin resistance and the action of TZDs.     

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling

Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding insulin resistance and whether lactate-induced insulin resistance is accompanied by altered insulin signaling and/or insulin-stimulated glucose transport in skeletal muscle. Hyperinsulinemic euglycemic clamps were conducted for 6 h in conscious, overnight-fasted rats with or without lactate infusion (120 micromol x kg(-1) x min(-1)) during the final 3.5 h. Lactate infusion increased plasma lactate levels about fourfold. The elevation of plasma lactate had rapid effects to suppress insulin-stimulated glycolysis, which clearly preceded its effect to decrease insulin-stimulated glucose uptake. Both submaximal and maximal insulin-stimulated glucose transport decreased 25-30% (P < 0.05) in soleus but not in epitrochlearis muscles of lactate-infused rats. Lactate infusion did not alter insulin's ability to phosphorylate the insulin receptor, the insulin receptor substrate (IRS)-1, or IRS-2 but decreased insulin's ability to stimulate IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase activities and Akt/protein kinase B activity by 47, 75, and 55%, respectively (P < 0.05 for all). In conclusion, elevation of plasma lactate suppressed glycolysis before its effect on insulin-stimulated glucose uptake, consistent with the hypothesis that suppression of glucose metabolism could precede and cause insulin resistance. In addition, lactate-induced insulin resistance was associated with impaired insulin signaling and decreased insulin-stimulated glucose transport in skeletal muscle.     

Rethinking leptin and insulin action: therapeutic opportunities for diabetes

Leptin is an adipocyte-derived hormone that primarily acts in the hypothalamus and plays a key role in the regulation of food intake, body weight, energy expenditure and neuroendocrine function. Leptin has direct peripheral effects on several tissues, and it may be independently involved in insulin secretion and action besides its effects on body weight regulation. Basal plasma leptin and insulin concentrations correlate with each other. Insulin and glucose appear to increase leptin secretion. In turn, leptin increases peripheral insulin sensitivity while decreasing insulin secretion from pancreatic beta cells. Leptin increases skeletal muscle glucose uptake and oxidation, and suppresses hepatic glucose output. Effects of leptin on lipid metabolism might reduce lipotoxicity and therefore contribute to the improvement of hepatic, skeletal and whole body insulin sensitivity. Leptin is the first adipokine used in the treatment of hypoleptinemic clinical disorders. Although leptin therapy has limited success in common obesity, it has impressive effects in congenital leptin deficiency, lipoatrophic diabetes and syndromes of severe insulin resistance. Leptin has been reported to ameliorate hyperinsulinemia and diabetes in the clinical setting of congenital leptin deficiency. It also improves hyperglycemia, insulin resistance, hyperinsulinemia, dyslipidemia and hepatic steatosis in lipoatrophic diabetes. These promising results warrant clinical trials to test the hypothesis that leptin alone or with classical antidiabetic agents may potentially be beneficial in the treatment of hypoleptinemic non-obese individuals with glucose intolerance and diabetes. This review summarizes the clinical applications of leptin, particularly emphasizing the effects of leptin on glucose homeostasis.     

Disruption of the SH2-B gene causes age-dependent insulin resistance and glucose intolerance

Insulin regulates glucose homeostasis by binding and activating the insulin receptor, and defects in insulin responses (insulin resistance) induce type 2 diabetes. SH2-B, an Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein, binds via its SH2 domain to insulin receptor in response to insulin; however, its physiological role remains unclear. Here we show that SH2-B was expressed in the liver, skeletal muscle, and fat. Systemic deletion of SH2-B impaired insulin receptor activation and signaling in the liver, skeletal muscle, and fat, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and IRS2 and activation of the phosphatidylinositol 3-kinase/Akt and the Erk1/2 pathways. Consequently, SH2-B-/- knockout mice developed age-dependent hyperinsulinemia, hyperglycemia, and glucose intolerance. Moreover, SH2-B directly enhanced autophosphorylation of insulin receptor and tyrosine phosphorylation of IRS1 and IRS2 in an SH2 domain-dependent manner in cultured cells. Our data suggest that SH2-B is a physiological enhancer of insulin receptor activation and is required for maintaining normal insulin sensitivity and glucose homeostasis during aging.     

Muscle-specific Pten deletion protects against insulin resistance and diabetes

Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.     

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.     

Palmitate- and lipopolysaccharide-activated macrophages evoke contrasting insulin responses in muscle cells

Factors secreted by macrophages contribute to whole body insulin resistance, acting in part on adipose tissue. Muscle is the major tissue for glucose disposal, but how macrophage-derived factors impact skeletal muscle glucose uptake is unknown, or whether the macrophage environment influences this response. We hypothesized that conditioned medium from macrophages pretreated with palmitate or LPS would directly affect insulin action and glucose uptake in muscle cells. L6-GLUT4myc myoblasts were exposed to conditioned medium from RAW 264.7 macrophages pretreated with palmitate or LPS. Conditioned medium from palmitate-treated RAW 264.7 macrophages inhibited myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation while activating JNK p38 MAPK, decreasing IkappaBalpha, and elevating inflammation markers. Surprisingly, and opposite to its effects on adipose cells, conditioned medium from LPS-treated macrophages stimulated myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation without affecting stress kinases or inflammation indexes. This medium had markedly elevated IL-10 levels, and IL-10, alone, potentiated insulin action in myoblasts and partly reversed the insulin resistance imparted by medium from palmitate-treated macrophages. IL-10 neutralizing antibodies blunted the positive influence of LPS macrophage-conditioned medium. We conclude that myoblasts and adipocytes respond differently to cytokines. Furthermore, depending on their environment, macrophages negatively or positively influence muscle cells. Macrophages exposed to palmitate produce a mixture of proinflammatory cytokines that reduce insulin action in muscle cells; conversely, LPS-activated macrophages increase insulin action, likely via IL-10. Macrophages may be an integral element in glucose homeostasis in vivo, relaying effects of circulating factors to skeletal muscle.     

Transgenic mice with dominant negative PKC-theta in skeletal muscle: a new model of insulin resistance and obesity

Protein kinase C theta (PKC-theta) is the PKC isoform predominantly expressed in skeletal muscle, and it is supposed to mediate many signals necessary for muscle histogenesis and homeostasis, such as TGFbeta, nerve-dependent signals and insulin. To study the role of PKC-theta in these mechanisms we generated transgenic mice expressing a "kinase dead" mutant form of PKC-theta (PKC-thetaK/R), working as "dominant negative," specifically in skeletal muscle. These mice are viable and fertile, however, by the 6-7 months of age, they gain weight, mainly due to visceral fat deposition. Before the onset of obesity (4 months of age), they already show increased fasting and fed insulin levels and reduced insulin-sensitivity, as measured by ipITT, but normal glucose tolerance, as measured by ipGTT. After the 6-7 months of age, transgenic mice develop hyperinsulinemia in the fasting and fed state. The ipGTT revealed in the transgenic mice both hyperglycemia and hyperinsulinemia. At the molecular level, impaired activation of the IR/IRS/PI3K pathway and a significant decrease both in the levels and in insulin-stimulated activation of the serine/threonine kinase Akt were observed. Taken together these data demonstrate that over-expression of dominant negative PKC-theta in skeletal muscle causes obesity associated to insulin resistance, as demonstrated by defective receptor and post-receptorial activation of signaling cascade.     

Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle

Triglyceride accumulation in skeletal muscle contributes to insulin resistance in obesity. We recently showed that alpha-lipoic acid (ALA) reduces body weight and prevents the development of diabetes in diabetes-prone obese rats by reducing triglyceride accumulation in non-adipose tissues. AMP-activated protein kinase (AMPK) is a major regulator of cellular energy metabolism. We examined whether ALA lowers triglyceride accumulation in skeletal muscle by activating AMPK. Alpha2-AMPK activity was decreased in obese rats compared to control rats. Administration of ALA to obese rats increased insulin-stimulated glucose disposal in whole body and in skeletal muscle. ALA also increased fatty acid oxidation and activated AMPK in skeletal muscle. Adenovirus-mediated administration of dominant negative AMPK into skeletal muscle prevented the ALA-induced increases in fatty acid oxidation and insulin-stimulated glucose uptake. These results suggest that ALA-induced improvement of insulin sensitivity is mediated by activation of AMPK and reduced triglyceride accumulation in skeletal muscle.     

Overproduction of angiotensinogen from adipose tissue induces adipose inflammation, glucose intolerance, and insulin resistance

Although obesity is associated with overactivation of the white adipose tissue (WAT) renin-angiotensin system (RAS), a causal link between the latter and systemic insulin resistance is not established. We tested the hypothesis that overexpression of angiotensinogen (Agt) from WAT causes systemic insulin resistance via modulation of adipose inflammation. Glucose tolerance, systemic insulin sensitivity, and WAT inflammatory markers were analyzed in mice overexpressing Agt in the WAT (aP2-Agt mice). Proteomic studies and in vitro studies using 3T3-L1 adipocytes were performed to build a mechanistic framework. Male aP2-Agt mice exhibited glucose intolerance, insulin resistance, and lower insulin-stimulated glucose uptake by the skeletal muscle. The difference in glucose tolerance between genotypes was normalized by high-fat (HF) feeding, and was significantly improved by treatment with angiotensin-converting enzyme (ACE) inhibitor captopril. aP2-Agt mice also had higher monocyte chemotactic protein-1 (MCP-1) and lower interleukin-10 (IL-10) in the WAT, indicating adipose inflammation. Proteomic studies in WAT showed that they also had higher monoglyceride lipase (MGL) and glycerol-3-phosphate dehydrogenase levels. Treatment with angiotensin II (Ang II) increased MCP-1 and resistin secretion from adipocytes, which was prevented by cotreating with inhibitors of the nuclear factor-κB (NF-κB) pathway or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In conclusion, we show for the first time that adipose RAS overactivation causes glucose intolerance and systemic insulin resistance. The mechanisms appear to be via reduced skeletal muscle glucose uptake, at least in part due to Ang II-induced, NADPH oxidase and NFκB-dependent increases in WAT inflammation.     

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.