Dissociation of hepatic steatosis and insulin resistance in mice overexpressing DGAT in the liver

Hepatic steatosis, the accumulation of lipids in the liver, is widely believed to result in insulin resistance. To test the causal relationship between hepatic steatosis and insulin resistance, we generated mice that overexpress acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step of triacylglycerol (TG) biosynthesis, in the liver (Liv-DGAT2 mice). Liv-DGAT2 mice developed hepatic steatosis, with increased amounts of TG, diacylglycerol, ceramides, and unsaturated long-chain fatty acyl-CoAs in the liver. However, they had no abnormalities in plasma glucose and insulin levels, glucose and insulin tolerance, rates of glucose infusion and hepatic glucose production during hyperinsulinemic-euglycemic clamp studies, or activities of insulin-stimulated signaling proteins in the liver. DGAT1 overexpression in the liver also failed to induce glucose or insulin intolerance. Our results indicate that DGAT-mediated lipid accumulation in the liver is insufficient to cause insulin resistance and show that hepatic steatosis can occur independently of insulin resistance.     

Mitochondrial dysfunction and inhibition of myoblast differentiation in mice with high-fat-diet-induced pre-diabetes

Pre-diabetes is characterized by impaired glucose tolerance (IGT) and/or impaired fasting glucose. Impairment of skeletal muscle function is closely associated with the progression of diabetes. However, the entire pathological characteristics and mechanisms of pre-diabetes in skeletal muscle remain fully unknown. Here, we established a mouse model of pre-diabetes, in which 6-week-old male C57BL6/J mice were fed either normal diet or high-fat diet (HFD) for 8 or 16 weeks. Both non-fasting and fasting glucose levels and the results of glucose and insulin tolerance tests showed that mice fed an 8-week HFD developed pre-diabetes with IGT; whereas mice fed a 16-week HFD presented with impaired fasting glucose and impaired glucose tolerance (IFG-IGT). Mice at both stages of pre-diabetes displayed decreased numbers of mitochondria in skeletal muscle. Moreover, IFG-IGT mice exhibited decreased mitochondrial membrane potential and ATP production in skeletal muscle and muscle degeneration characterized by a shift in muscle fibers from predominantly oxidative type I to glycolytic type II. Western blotting and histological analysis confirmed that myoblast differentiation was only inhibited in IFG-IGT mice. For primary skeletal muscle satellite cells, inhibition of differentiation was observed in palmitic acid-induced insulin resistance model. Moreover, enhanced myoblast differentiation increased glucose uptake and insulin sensitivity. These findings indicate that pre-diabetes result in mitochondrial dysfunction and inhibition of myoblast differentiation in skeletal muscle. Therefore, interventions that enhance myoblast differentiation may improve insulin resistance of diabetes at the earlier stage.     

ApoA5 knockdown improves whole-body insulin sensitivity in high-fat-fed mice by reducing ectopic lipid content

ApoA5 has a critical role in the regulation of plasma TG concentrations. In order to determine whether ApoA5 also impacts ectopic lipid deposition in liver and skeletal muscle, as well as tissue insulin sensitivity, we treated mice with an antisense oligonucleotide (ASO) to decrease hepatic expression of ApoA5. ASO treatment reduced ApoA5 protein expression in liver by 60–70%. ApoA5 ASO-treated mice displayed approximately 3-fold higher plasma TG concentrations, which were associated with decreased plasma TG clearance. Furthermore, ApoA5 ASO-treated mice fed a high-fat diet (HFD) exhibited reduced liver and skeletal muscle TG uptake and reduced liver and muscle TG and diacylglycerol (DAG) content. HFD-fed ApoA5 ASO-treated mice were protected from HFD-induced insulin resistance, as assessed by hyperinsulinemic-euglycemic clamps. This protection could be attributed to increases in both hepatic and peripheral insulin responsiveness associated with decreased DAG activation of protein kinase C (PKC)-ε and PKCθ in liver and muscle, respectively, and increased insulin-stimulated AKT2 pho­sphory­lation in these tissues. In summary, these studies demonstrate a novel role for ApoA5 as a modulator of susceptibility to diet-induced liver and muscle insulin resistance through regulation of ectopic lipid accumulation in liver and skeletal muscle.     

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R^-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R^-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R^-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.     

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.     

Improvement of insulin sensitivity by antagonism of the renin-angiotensin system

The reduced capacity of insulin to stimulate glucose transport into skeletal muscle, termed insulin resistance, is a primary defect leading to the development of prediabetes and overt type 2 diabetes. Although the etiology of this skeletal muscle insulin resistance is multifactorial, there is accumulating evidence that one contributor is overactivity of the renin-angiotensin system (RAS). Angiotensin II (ANG II) produced from this system can act on ANG II type 1 receptors both in the vascular endothelium and in myocytes, with an enhancement of the intracellular production of reactive oxygen species (ROS). Evidence from animal model and cultured skeletal muscle cell line studies indicates ANG II can induce insulin resistance. Chronic ANG II infusion into an insulin-sensitive rat produces a markedly insulin-resistant state that is associated with a negative impact of ROS on the skeletal muscle glucose transport system. ANG II treatment of L6 myocytes causes impaired insulin receptor substrate (IRS)-1-dependent insulin signaling that is accompanied by augmentation of NADPH oxidase-mediated ROS production. Further critical evidence has been obtained from the TG(mREN2)27 rat, a model of RAS overactivity and insulin resistance. The TG(mREN2)27 rat displays whole body and skeletal muscle insulin resistance that is associated with local oxidative stress and a significant reduction in the functionality of the insulin receptor (IR)/IRS-1-dependent insulin signaling. Treatment with a selective ANG II type 1 receptor antagonist leads to improvements in whole body insulin sensitivity, enhanced insulin-stimulated glucose transport in muscle, and reduced local oxidative stress. In addition, exercise training of TG(mREN2)27 rats enhances whole body and skeletal muscle insulin action. However, these metabolic improvements elicited by antagonism of ANG II action or exercise training are independent of upregulation of IR/IRS-1-dependent signaling. Collectively, these findings support targeting the RAS in the design of interventions to improve metabolic and cardiovascular function in conditions of insulin resistance associated with prediabetes and type 2 diabetes.     

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.     

Reduction in BACE1 decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice

Insulin resistance and impaired glucose homoeostasis are important indicators of Type?2 diabetes and are early risk factors of AD (Alzheimer's disease). An essential feature of AD pathology is the presence of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which regulates production of toxic amyloid peptides. However, whether BACE1 also plays a role in glucose homoeostasis is presently unknown. We have used transgenic mice to analyse the effects of loss of BACE1 on body weight, and lipid and glucose homoeostasis. BACE1-/- mice are lean, with decreased adiposity, higher energy expenditure, and improved glucose disposal and peripheral insulin sensitivity than wild-type littermates. BACE1-/- mice are also protected from diet-induced obesity. BACE1-deficient skeletal muscle and liver exhibit improved insulin sensitivity. In a skeletal muscle cell line, BACE1 inhibition increased glucose uptake and enhanced insulin sensitivity. The loss of BACE1 is associated with increased levels of UCP1 (uncoupling protein 1) in BAT (brown adipose tissue) and UCP2 and UCP3 mRNA in skeletal muscle, indicative of increased uncoupled respiration and metabolic inefficiency. Thus BACE1 levels may play a critical role in glucose and lipid homoeostasis in conditions of chronic nutrient excess. Therefore strategies that ameliorate BACE1 activity may be important novel approaches for the treatment of diabetes.     

Adipose triglyceride lipase regulation of skeletal muscle lipid metabolism and insulin responsiveness

Adipose triglyceride lipase (ATGL) is important for triglyceride (TG) metabolism in adipose tissue, and ATGL-null mice show increased adiposity. Given the apparent importance of ATGL in TG metabolism and the association of lipid deposition with insulin resistance, we examined the role of ATGL in regulating skeletal muscle lipid metabolism and insulin-stimulated glucose disposal. ATGL expression in myotubes was reduced by small interfering RNA and increased with a retrovirus encoding GFP-HA-ATGL. ATGL was also overexpressed in rats by in vivo electrotransfer. ATGL was down-regulated in skeletal muscle of obese, insulin-resistant mice and negatively correlated with intramyocellular TG levels. ATGL small interfering RNA in myotubes reduced TG hydrolase activity and increased TG content, whereas ATGL overexpression induced the reciprocal response, indicating that ATGL is an essential TG lipase in skeletal muscle. ATGL overexpression in myotubes increased the oxidation of fatty acid liberated from TG and diglyceride and ceramide contents. These responses in cells were largely recapitulated in rats overexpressing ATGL. When ATGL protein expression and TG hydrolase activity in obese, insulin-resistant rats were restored to levels observed in lean rats, TG content was reduced; however, the insulin resistance induced by the high-fat diet persisted. In conclusion, ATGL TG hydrolysis in skeletal muscle is a critical determinant of lipid metabolism and storage. Although ATGL content and TG hydrolase activity are decreased in obese, insulin-resistant phenotypes, overexpression does not rescue the condition, indicating reduced ATGL is unlikely to be a primary cause of obesity-associated insulin resistance.     

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.     

Aerobic exercise ameliorates insulin resistance in C57BL/6 J mice via activating Sestrin3

Skeletal muscle insulin resistance (IR) is closely linked to hyperglycemia and metabolic disorders. Regular exercise enhances insulin sensitivity in skeletal muscle, but its underlying mechanisms remain unknown. Sestrin3 (SESN3) is a stress-inducible protein that protects against obesity-induced hepatic steatosis and insulin resistance. Regular exercise training is known to increase SESN3 expression in skeletal muscle. The purpose of this study was to explore whether SESN3 mediates the metabolic effects of exercise in the mouse model of high-fat diet (HFD)-induced IR. SESN3^?/? mice exhibited severer body weight gain, ectopic lipid accumulation, and dysregulation of glucose metabolism after long-term HFD feeding compared with the wild-type (WT) mice. Moreover, we found that SESN3 deficiency weakened the effects of exercise on reducing serum insulin levels and improving glucose tolerance in mice. Exercise training increased pAKT-S473 and GLUT4 expression, accompanied by enhanced pmTOR-S2481 (an indicator of mTORC2 activity) in WT quadriceps that were less pronounced in SESN3^?/? mice. SESN3 overexpression in C2C12 myotubes further confirmed that SESN3 played an important role in skeletal muscle glucose metabolism. SESN3 overexpression increased the binding of Rictor to mTOR and pmTOR-S2481 in C2C12 myotubes. Moreover, SESN3 overexpression resulted in an elevation of glucose uptake and a concomitant increase of pAKT-S473 in C2C12 myotubes, whereas these effects were diminished by downregulation of mTORC2 activity. Taken together, SESN3 is a crucial protein in amplifying the beneficial effects of exercise on insulin sensitivity in skeletal muscle and systemic glucose levels. SESN3/mTORC2/AKT pathway mediated the effects of exercise on skeletal muscle insulin sensitivity.     

Insulin-induced activation of atypical protein kinase C, but not protein kinase B, is maintained in diabetic (ob/ob and Goto-Kakazaki) liver. Contrasting insulin signaling patterns in liver versus muscle define phenotypes of type 2 diabetic and high fat-induced insulin-resistant states

Insulin resistance in type 2 diabetes is characterized by defects in muscle glucose uptake and hepatic overproduction of both glucose and lipids. These hepatic defects are perplexing because insulin normally suppresses glucose production and increases lipid synthesis in the liver. To understand the mechanisms for these seemingly paradoxical defects, we examined the activation of atypical protein kinase C (aPKC) and protein kinase B (PKB), two key signaling factors that operate downstream of phosphatidylinositol 3-kinase and regulate various insulin-sensitive metabolic processes. Livers and muscles of three insulin-resistant rodent models were studied. In livers of type 2 diabetic non-obese Goto-Kakazaki rats and ob/ob-diabetic mice, the activation of PKB was impaired, whereas activation of aPKC was surprisingly maintained. In livers of non-diabetic high fatfed mice, the activation of both aPKC and PKB was maintained. In contrast to the maintenance of aPKC activation in the liver, insulin activation of aPKC was impaired in muscles of Goto-Kakazaki-diabetic rats and ob/ob-diabetic and non-diabetic high fat-fed mice. These findings suggest that, at least in these rodent models, (a) defects in aPKC activation contribute importantly to skeletal muscle insulin resistance observed in both high fat feeding and type 2 diabetes; (b) insulin signaling defects in muscle are not necessarily accompanied by similar defects in liver; (c) defects in hepatic PKB activation occur in association with, and probably contribute importantly to, the development of overt diabetes; and (d) maintenance of hepatic aPKC activation may explain the continued effectiveness of insulin for stimulating certain metabolic actions in the liver.     

Skeletal muscle damage and impaired regeneration due to LPL-mediated lipotoxicity

According to the concept of lipotoxicity, ectopic accumulation of lipids in non-adipose tissue induces pathological changes. The most prominent effects are seen in fatty liver disease, lipid cardiomyopathy, non-insulin-dependent diabetes mellitus, insulin resistance and skeletal muscle myopathy. We used the MCK(m)-hLPL mouse distinguished by skeletal and cardiac muscle-specific human lipoprotein lipase (hLPL) overexpression to investigate effects of lipid overload in skeletal muscle. We were intrigued to find that ectopic lipid accumulation induced proteasomal activity, apoptosis and skeletal muscle damage. In line with these findings we observed reduced Musculus gastrocnemius and Musculus quadriceps mass in transgenic animals, accompanied by severely impaired physical endurance. We suggest that muscle loss was aggravated by impaired muscle regeneration as evidenced by reduced cross-sectional area of regenerating myofibers after cardiotoxin-induced injury in MCK(m)-hLPL mice. Similarly, an almost complete loss of myogenic potential was observed in C2C12 murine myoblasts upon overexpression of LPL. Our findings directly link lipid overload to muscle damage, impaired regeneration and loss of performance. These findings support the concept of lipotoxicity and are a further step to explain pathological effects seen in muscle of obese patients, patients with the metabolic syndrome and patients with cancer-associated cachexia.     

A novel insulin sensitizer (S15511) enhances insulin-stimulated glucose uptake in rat skeletal muscles.

Type 2 diabetes is preceded by the presence of skeletal muscle insulin resistance, and drugs that increase insulin sensitivity in skeletal muscle prevent the disease. S15511 is an original compound with demonstrated effects on insulin sensitivity in animal models of insulin resistance. However, the mechanisms behind the insulin-sensitizing effect of S15511 are unknown. The aim of our study was to explore whether S15511 improves insulin sensitivity in skeletal muscles. Insulin sensitivity was assessed in skeletal muscles from S15511-treated rats by measuring intracellular insulin-signaling activity and insulin-stimulated glucose transport in isolated muscles. In addition, GLUT4 expression and glycogen levels were assessed after treatment. S15511 treatment was associated with an increase in insulin-stimulated glucose transport in type IIb fibers, while type I fibers were unaffected. The enhanced glucose transport was mirrored by a fiber type-specific increase in GLUT4 expression, while no improvement in insulin-signaling activity was observed. S15511 is a novel insulin sensitizer that is capable of improving glucose homeostasis in nondiabetic rats. The compound enhances skeletal muscle insulin sensitivity and specifically targets type IIb muscle fibers by increasing GLUT4 expression. Together these data show S15511 to be a potentially promising new drug in the treatment and prevention of type 2 diabetes.     

Mitochondrial dysfunction and lipotoxicity

Mitochondrial dysfunction in skeletal muscle has been suggested to underlie the development of insulin resistance and type 2 diabetes mellitus. Reduced mitochondrial capacity will contribute to the accumulation of lipid intermediates, desensitizing insulin signaling and leading to insulin resistance. Why mitochondrial function is reduced in the (pre-)diabetic state is, however, so far unknown. Although it is tempting to suggest that skeletal muscle insulin resistance may result from an inherited or acquired reduction in mitochondrial function in the pre-diabetic state, it cannot be excluded that mitochondrial dysfunction may in fact be the consequence of the insulin-resistant/diabetic state. Lipotoxicity, the deleterious effects of accumulating fatty acids in skeletal muscle cells, may lie at the basis of mitochondrial dysfunction: next to producing energy, mitochondria are also the major source of reactive oxygen species (ROS). Fatty acids accumulating in the vicinity of mitochondria are vulnerable to ROS-induced lipid peroxidation. Subsequently, these lipid peroxides could have lipotoxic effects on mtDNA, RNA and proteins of the mitochondrial machinery, leading to mitochondrial dysfunction. Indeed, increased lipid peroxidation has been reported in insulin resistant skeletal muscle and the mitochondrial uncoupling protein-3, which has been suggested to prevent lipid-induced mitochondrial damage, is reduced in subjects with an impaired glucose tolerance and in type 2 diabetic patients. These findings support the hypothesis that fat accumulation in skeletal muscle may precede the reduction in mitochondrial function that is observed in type 2 diabetes mellitus.     

Hyperglycemia, maturity-onset obesity, and insulin resistance in NONcNZO10/LtJ males, a new mouse model of type 2 diabetes

As a new mouse model of obesity-induced diabetes generated by combining quantitative trait loci from New Zealand Obese (NZO/HlLt) and Nonobese Nondiabetic (NON/LtJ) mice, NONcNZO10/LtJ (RCS10) male mice developed type 2 diabetes characterized by maturity onset obesity, hyperglycemia, and insulin resistance. To metabolically profile the progression to diabetes in preobese and obese states, a 2-h hyperinsulinemic euglycemic clamp was performed and organ-specific changes in insulin action were assessed in awake RCS10 and NON/LtJ (control) males at 8 and 13 wk of age. Prior to development of obesity and attendant increases in hepatic lipid content, 8-wk-old RCS10 mice developed insulin resistance in liver and skeletal muscle due to significant decreases in insulin-stimulated glucose uptake and GLUT4 expression in muscle. Transition to an obese and hyperglycemic state by 13 wk of age exacerbated insulin resistance in skeletal muscle, liver, and heart associated with organ-specific increases in lipid content. Thus, this polygenic mouse model of type 2 diabetes, wherein plasma insulin is only modestly elevated and obesity develops with maturity yet insulin action and glucose metabolism in skeletal muscle and liver are reduced at an early prediabetic age, should provide new insights into the etiology of type 2 diabetes.     

Lipid-induced Muscle Insulin Resistance Is Mediated by GGPPS via Modulation of the RhoA/Rho Kinase Signaling Pathway

Elevated circulating free fatty acid levels are important contributors to insulin resistance in the muscle and liver, but the underlying mechanisms require further elucidation. Here, we show that geranylgeranyl diphosphate synthase 1 (GGPPS), which is a branch point enzyme in the mevalonic acid pathway, promotes lipid-induced muscle insulin resistance through activation of the RhoA/Rho kinase signaling pathway. We have found that metabolic perturbation would increase GGPPS expression in the skeletal muscles of db/db mice and high fat diet-fed mice. To address the metabolic effects of GGPPS activity in skeletal muscle, we generated mice with specific GGPPS deletions in their skeletal muscle tissue. Heterozygous knock-out of GGPPS in the skeletal muscle improved systemic insulin sensitivity and glucose homeostasis in mice fed both normal chow and high fat diets. These metabolic alterations were accompanied by activated PI3K/Akt signaling and enhanced glucose uptake in the skeletal muscle. Further investigation showed that the free fatty acid-stimulated GGPPS expression in the skeletal muscle was able to enhance the geranylgeranylation of RhoA, which further induced the inhibitory phosphorylation of IRS-1 (Ser-307) by increasing Rho kinase activity. These results implicate a crucial role of the GGPPS/RhoA/Rho kinase/IRS-1 pathway in skeletal muscle, in which it mediates lipid-induced systemic insulin resistance in obese mice. Therefore, skeletal muscle GGPPS may represent a potential pharmacological target for the prevention and treatment of obesity-related type 2 diabetes.     

Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle

During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.