Impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients

Insulin resistance is the best predictor for the development of diabetes in offspring of type 2 diabetic patients, but the mechanism responsible for it remains unknown. Recent studies have demonstrated increased intramyocellular lipid, decreased mitochondrial ATP synthesis, and decreased mitochondrial density in the muscle of lean, insulin-resistant offspring of type 2 diabetic patients. These data suggest an important role for mitochondrial dysfunction in the pathogenesis of type 2 diabetes. To further explore this hypothesis, we assessed rates of substrate oxidation in the muscle of these same individuals using (13)C magnetic resonance spectroscopy (MRS). Young, lean, insulin-resistant offspring of type 2 diabetic patients and insulin-sensitive control subjects underwent (13)C MRS studies to noninvasively assess rates of substrate oxidation in muscle by monitoring the incorporation of (13)C label into C(4) glutamate during a [2-(13)C]acetate infusion. Using this approach, we found that rates of muscle mitochondrial substrate oxidation were decreased by 30% in lean, insulin-resistant offspring (59.8 +/- 5.1 nmol x g(-1) x min(-1), P = 0.02) compared with insulin-sensitive control subjects (96.1 +/- 16.3 nmol x g(-1) x min(-1)). These data support the hypothesis that insulin resistance in skeletal muscle of insulin-resistant offspring is associated with dysregulation of intramyocellular fatty acid metabolism, possibly because of an inherited defect in the activity of mitochondrial oxidative phosphorylation.     

Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects.

Insulin resistance plays an important role in the pathogenesis of type 2 diabetes; however, the multiple mechanisms causing insulin resistance are not yet fully understood. The aim of this study was to explore the possible contribution of intramyocellular lipid content in the pathogenesis of skeletal muscle insulin resistance. We compared insulin-resistant and insulin-sensitive subjects. To meet stringent matching criteria for other known confounders of insulin resistance, these individuals were selected from an extensively metabolically characterized group of 280 first-degree relatives of type 2 diabetic subjects. Some 13 lean insulin-resistant and 13 lean insulin-sensitive subjects were matched for sex, age, BMI, percent body fat, physical fitness, and waist-to-hip ratio. Insulin sensitivity was determined by the hyperinsulinemic-euglycemic clamp method (for insulin-resistant subjects, glucose metabolic clearance rate [MCR] was 5.77+/-0.28 ml x kg(-1) x min(-1) [mean +/- SE]; for insulin-sensitive subjects, MCR was 10.15+/-0.7 ml x kg(-1) x min(-1); P<0.002). Proton magnetic resonance spectroscopy (MRS) was used to measure intramyocellular lipid content (IMCL) in both groups. MRS studies demonstrated that in soleus muscle, IMCL was increased by 84% (11.8+/-1.6 vs. 6.4+/-0.59 arbitrary units; P = 0.008 ), and in tibialis anterior muscle, IMCL was increased by 57% (3.26+/-0.36 vs. 2.08+/-0.3 arbitrary units; P = 0.017) in the insulin-resistant offspring, whereas the extramyocellular lipid content and total muscle lipid content were not statistically different between the two groups. These data demonstrate that in these well-matched groups of lean subjects, IMCL is increased in insulin-resistant offspring of type 2 diabetic subjects when compared with an insulin-sensitive group matched for age, BMI, body fat distribution, percent body fat, and degree of physical fitness. These results indicate that increased IMCL represents an early abnormality in the pathogenesis of insulin resistance and suggest that increased IMCL may contribute to the defective glucose uptake in skeletal muscle in insulin-resistant subjects.     

Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1H-13C nuclear magnetic resonance spectroscopy assessment in offspring of type 2 diabetic parents.

Insulin resistance is the best prediction factor for the clinical onset of type 2 diabetes. It was suggested that intramuscular triglyceride store may be a primary pathogenic factor for its development. To test this hypothesis, 14 young lean offspring of type 2 diabetic parents, a model of in vivo insulin resistance with increased risk to develop diabetes, and 14 healthy subjects matched for anthropomorphic parameters and life habits were studied with 1) euglycemic-hyperinsulinemic clamp to assess whole body insulin sensitivity, 2) localized 1H nuclear magnetic resonance (NMR) spectroscopy of the soleus (higher content of fiber type I, insulin sensitive) and tibialis anterior (higher content of fiber type IIb, less insulin sensitive) muscles to assess intramyocellular triglyceride content, 3) 13C NMR of the calf subcutaneous adipose tissue to assess composition in saturated/unsaturated carbons of triglyceride fatty acid chains, and 4) dual X-ray energy absorption to assess body composition. Offspring of diabetic parents, notwithstanding normal fat content and distribution, were characterized by insulin resistance and increased intramyocellular triglyceride content in the soleus (P < 0.01) but not in the tibialis anterior (P = 0.19), but showed a normal content of saturated/unsaturated carbons in the fatty acid chain of subcutaneous adipocytes. Stepwise regression analysis selected intramyocellular triglyceride soleus content and plasma free fatty acid levels as the main predictors of whole body insulin sensitivity. In conclusion, 1H and 13C NMR spectroscopy revealed intramyocellular abnormalities of lipid metabolism associated with whole body insulin resistance in subjects at high risk of developing diabetes, and might be useful tools for noninvasively monitoring these alterations in diabetes and prediabetic states.     

Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects

Increased accumulation of fatty acids and their derivatives can impair insulin-stimulated glucose disposal by skeletal muscle. To characterize the nature of the defects in lipid metabolism and to evaluate the effects of thiazolidinedione treatment, we analyzed the levels of triacylglycerol, long-chain fatty acyl-coA, malonyl-CoA, fatty acid oxidation, AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), malonyl-CoA decarboxylase, and fatty acid transport proteins in muscle biopsies from nondiabetic lean, obese, and type 2 subjects before and after an euglycemic-hyperinsulinemic clamp as well as pre-and post-3-month rosiglitazone treatment. We observed that low AMPK and high ACC activities resulted in elevation of malonyl-CoA levels and lower fatty acid oxidation rates. These conditions, along with the basal higher expression levels of fatty acid transporters, led accumulation of long-chain fatty acyl-coA and triacylglycerol in insulin-resistant muscle. During the insulin infusion, muscle fatty acid oxidation was reduced to a greater extent in the lean compared with the insulin-resistant subjects. In contrast, isolated muscle mitochondria from the type 2 subjects exhibited a greater rate of fatty acid oxidation compared with the lean group. All of these abnormalities in the type 2 diabetic group were reversed by rosiglitazone treatment. In conclusion, these studies have shown that elevated malonyl-CoA levels and decreased fatty acid oxidation are key abnormalities in insulin-resistant muscle, and, in type 2 diabetic patients, thiazolidinedione treatment can reverse these abnormalities.     

Activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 is defective in muscle in type 2 diabetes and impaired glucose tolerance: amelioration by rosiglitazone and exercise

Insulin resistance in type 2 diabetes is partly due to impaired glucose transport in skeletal muscle. Atypical protein kinase C (aPKC) and protein kinase B (PKB), operating downstream of phosphatidylinositol (PI) 3-kinase and its lipid product, PI-3,4,5-(PO(4))(3) (PIP(3)), apparently mediate insulin effects on glucose transport. We examined these signaling factors during hyperinsulinemic-euglycemic clamp studies in nondiabetic subjects, subjects with impaired glucose tolerance (IGT), and type 2 diabetic subjects. In nondiabetic control subjects, insulin provoked twofold increases in muscle aPKC activity. In both IGT and diabetes, aPKC activation was markedly (70-80%) diminished, most likely reflecting impaired activation of insulin receptor substrate (IRS)-1-dependent PI 3-kinase and decreased ability of PIP(3) to directly activate aPKCs; additionally, muscle PKC-zeta levels were diminished by 40%. PKB activation was diminished in patients with IGT but not significantly in diabetic patients. The insulin sensitizer rosiglitazone improved insulin-stimulated IRS-1-dependent PI 3-kinase and aPKC activation, as well as glucose disposal rates. Bicycle exercise, which activates aPKCs and stimulates glucose transport independently of PI 3-kinase, activated aPKCs comparably to insulin in nondiabetic subjects and better than insulin in diabetic patients. Defective aPKC activation contributes to skeletal muscle insulin resistance in IGT and type 2 diabetes, rosiglitazone improves insulin-stimulated aPKC activation, and exercise directly activates aPKCs in diabetic muscle.     

Muscle-specific IRS-1 Ser->Ala transgenic mice are protected from fat-induced insulin resistance in skeletal muscle

OBJECTIVE—Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo.RESEARCH DESIGN AND METHODS—To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser³⁰², Ser³⁰⁷, and Ser⁶¹² mutated to alanine (Tg IRS-1 Ser→Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo.RESULTS—Tg IRS-1 Ser→Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser→Ala mice displayed a significant increase in insulin-stimulated IRS-1–associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates.CONCLUSIONS—These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.Insulin resistance in skeletal muscle plays a major role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance in skeletal muscle are poorly understood (1). Reduced insulin-stimulated glucose transport activity exists with reduced insulin receptor substrate (IRS)-1–associated phosphatidylinositol 3-kinase (PI3-kinase) activity in patients with type 2 diabetes and the offspring of type 2 diabetic parents (2–5). Increased serine phosphorylation of IRS-1 has been suggested to be responsible for this phenomenon (6), and, consistent with this hypothesis, recent studies have demonstrated hyperserine phosphorylation of IRS-1 on Ser³⁰², Ser³⁰⁷, Ser⁶¹², and Ser⁶³⁶ in several insulin-resistant rodent models (7–10), as well as in lean insulin-resistant offspring of type 2 diabetic parents (11). Circulating factors that are increased in obese and inflammatory states, such as tumor necrosis factor-α, activate Ser/Thr kinases (12,13). Also, recent studies (14–18) have demonstrated a strong relationship between intramyocelullar lipid accumulation and insulin resistance in muscle independent of alterations in circulating adipocytokines. Intramyocellular fatty acid metabolites, such as diacylglycerol, have been postulated to activate a serine kinase cascade leading to increased serine phosphorylation of IRS-1. Furthermore, high-fat diet–induced insulin resistance has been abrogated in rodent models in which certain Ser/Thr kinases (c-Jun N-terminal kinase, inhibitor of nuclear factor κB kinase β subunit, S6 kinase-1, and protein kinase C-θ) were either knocked down or pharmacologically inhibited (8,9,19–21). However, it remains unknown whether increased IRS-1 serine phosphorylation plays a causative role in the pathogenesis of fat-induced insulin resistance in skeletal muscle or whether it is merely an associated phenomenon. To address this question, we generated IRS-1 Ser³⁰², Ser³⁰⁷, and Ser⁶¹² to Ala mutant–overexpression (Tg IRS-1 Ser→Ala) mice using a muscle-specific myosin light-chain-2 promoter and assessed insulin responsiveness in vivo by intraperitoneal glucose tolerance tests and hyperinsulinemic-euglycemic clamp studies.     

Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade.

To examine the mechanism by which free fatty acids (FFAs) induce insulin resistance in vivo, awake chronically catheterized rats underwent a hyperinsulinemic-euglycemic clamp with or without a 5-h preinfusion of lipid/heparin to raise plasma FFA concentrations. Increased plasma FFAs resulted in insulin resistance as reflected by a approximately 35% reduction in the glucose infusion rate (P < 0.05 vs. control). The insulin resistance was associated with a 40-50% reduction in 13C nuclear magnetic resonance (NMR)-determined rates of muscle glycogen synthesis (P < 0.01 vs. control) and muscle glucose oxidation (P < 0.01 vs. control), which in turn could be attributed to a approximately 25% reduction in glucose transport activity as assessed by 2-[1,2-3H]deoxyglucose uptake in vivo (P < 0.05 vs. control). This lipid-induced decrease in insulin-stimulated muscle glucose metabolism was associated with 1) a approximately 50% reduction in insulin-stimulated insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity (P < 0.05 vs. control), 2) a blunting in insulin-stimulated IRS-1 tyrosine phosphorylation (P < 0.05, lipid-infused versus glycerol-infused), and 3) a four-fold increase in membrane-bound, or active, protein kinase C (PKC) theta (P < 0.05 vs. control). We conclude that acute elevations of plasma FFA levels for 5 h induce skeletal muscle insulin resistance in vivo via a reduction in insulin-stimulated muscle glycogen synthesis and glucose oxidation that can be attributed to reduced glucose transport activity. These changes are associated with abnormalities in the insulin signaling cascade and may be mediated by FFA activation of PKC theta.     

Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats

To determine whether impaired Akt (protein kinase B or rac) activation contributes to insulin resistance in vivo, we examined the expression, phosphorylation, and kinase activities of Akt1 and Akt2 isoforms in insulin target tissues of insulin-resistant obese Zucker rats. In lean rats, insulin (10 U/kg i.v. x 2.5 min) stimulated Akt1 activity 6.2-, 8.8-, and 4.4-fold and Akt2 activity 5.4-, 9.3-, and 1.8-fold in muscle, liver, and adipose tissue, respectively. In obese rats, insulin-stimulated Akt1 activity decreased 30% in muscle and 21% in adipose tissue but increased 37% in liver compared with lean littermates. Insulin-stimulated Akt2 activity decreased 29% in muscle and 37% in liver but increased 24% in adipose tissue. Akt2 protein levels were reduced 56% in muscle and 35% in liver of obese rats, but Akt1 expression was unaltered. Phosphoinositide 3-kinase (PI3K) activity associated with insulin receptor substrate (IRS)-1 or phosphotyrosine was reduced 67-86% in tissues of obese rats because of lower IRS-1 protein levels and reduced insulin receptor and IRS-1 phosphorylation. In adipose tissue of obese rats, in spite of an 86% reduction in insulin-stimulated PI3K activity, activation of Akt2 was increased. Maximal insulin-stimulated (100 nmol/l) glucose transport was reduced 70% in isolated adipocytes, with a rightward shift in the insulin dose response for transport and for Akt1 stimulation but normal sensitivity for Akt2. These findings suggest that PI3K-dependent effects on glucose transport in adipocytes are not mediated primarily by Akt2. Akt1 and Akt2 activations by insulin have a similar time course and are maximal by 2.5 min in adipocytes of both lean and obese rats. We conclude that 1) activation of Akt1 and Akt2 in vivo is much less impaired than activation of PI3K in this insulin-resistant state, and 2) the mechanisms for divergent alterations in insulin action on Akt1 and Akt2 activities in tissues of insulin-resistant obese rats involve tissue- and isoform-specific changes in both expression and activation.     

Reversal of nonalcoholic hepatic steatosis, hepatic insulin resistance, and hyperglycemia by moderate weight reduction in patients with type 2 diabetes

To examine the mechanism by which moderate weight reduction improves basal and insulin-stimulated rates of glucose metabolism in patients with type 2 diabetes, we used (1)H magnetic resonance spectroscopy to assess intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) content in conjunction with hyperinsulinemic-euglycemic clamps using [6,6-(2)H(2)]glucose to assess rates of glucose production and insulin-stimulated peripheral glucose uptake. Eight obese patients with type 2 diabetes were studied before and after weight stabilization on a moderately hypocaloric very-low-fat diet (3%). The diabetic patients were markedly insulin resistant in both liver and muscle compared with the lean control subjects. These changes were associated with marked increases in IHL (12.2 +/- 3.4 vs. 0.6 +/- 0.1%; P = 0.02) and IMCL (2.0 +/- 0.3 vs. 1.2 +/- 0.1%; P = 0.02) compared with the control subjects. A weight loss of only approximately 8 kg resulted in normalization of fasting plasma glucose concentrations (8.8 +/- 0.5 vs. 6.4 +/- 0.3 mmol/l; P < 0.0005), rates of basal glucose production (193 +/- 7 vs. 153 +/- 10 mg/min; P < 0.0005), and the percentage suppression of hepatic glucose production during the clamp (29 +/- 22 vs. 99 +/- 3%; P = 0.003). These improvements in basal and insulin-stimulated hepatic glucose metabolism were associated with an 81 +/- 4% reduction in IHL (P = 0.0009) but no significant change in insulin-stimulated peripheral glucose uptake or IMCL (2.0 +/- 0.3 vs. 1.9 +/- 0.3%; P = 0.21). In conclusion, these data support the hypothesis that moderate weight loss normalizes fasting hyperglycemia in patients with poorly controlled type 2 diabetes by mobilizing a relatively small pool of IHL, which reverses hepatic insulin resistance and normalizes rates of basal glucose production, independent of any changes in insulin-stimulated peripheral glucose metabolism.     

Defective signaling through Akt-2 and -3 but not Akt-1 in insulin-resistant human skeletal muscle: potential role in insulin resistance

Recent evidence has shown that activation of phosphatidyinositol-3-kinase (PI3K) and Akt, necessary for insulin stimulation of glucose transport, is impaired in insulin resistance. It is unknown, however, which Akt isoform shows impaired activation in insulin resistance. Additionally, related growth factors (epidermal or platelet-derived vascular) also stimulate PI3K, but it is unknown whether production of 3,4,5 phosphatidyinositol is sufficient to stimulate glucose transport in insulin-resistant muscle. Moreover, these studies were performed in rodents, and little data exists from humans. Hence, we investigated the stimulation of PI3K and Akt-1, -2, and -3 by insulin and epidermal growth factors (EGFs) in skeletal muscles from lean and obese insulin-resistant humans. Insulin activated all Akt isoforms in lean muscles, whereas only Akt-1 was activated in obese muscles. Insulin receptor substrate (IRS)-1 was associated with PI3K activity, which is necessary for Akt activation by insulin, and was reduced in obese muscles, and this was accompanied by decreased IRS-1 expression. In contrast, insulin- or EGF-stimulated phosphotyrosine-associated PI3K activity was not different between lean and obese muscles. These results show that a defect in the ability of insulin to activate Akt-2 and -3 may explain the impaired insulin-stimulated glucose transport in insulin resistance. Additionally, these data also show that different upstream or downstream signals may regulate the activity of the various Akt isoforms.     

Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats

Activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofurano-side (AICAR) increases glucose transport in skeletal muscle via an insulin-independent pathway. To examine the effects of AMPK activation on skeletal muscle glucose transport activity and whole-body carbohydrate and lipid metabolism in an insulin-resistant rat model, awake obese Zuckerfa/fa rats (n = 26) and their lean (n = 23) littermates were infused for 90 min with AICAR, insulin, or saline. The insulin infusion rate (4 mU.kg(-1).min(-1)) was selected to match the glucose requirements during AICAR (bolus, 100 mg/kg; constant, 10 mg.kg(-1).min(-1)) isoglycemic clamps in the lean rats. The effects of these identical AICAR and insulin infusion rates were then examined in the obese Zucker rats. AICAR infusion increased muscle AMPK activity more than fivefold (P < 0.01 vs. control and insulin) in both lean and obese rats. Plasma triglycerides, fatty acid concentrations, and glycerol turnover, as assessed by [2-13C]glycerol, were all decreased in both lean and obese rats infused with AICAR (P < 0.05 vs. basal), whereas insulin had no effect on these parameters in the obese rats. Endogenous glucose production rates, measured by [U-13C]glucose, were suppressed by >50% during AICAR and insulin infusions in both lean and obese rats (P < 0.05 vs. basal). In lean rats, rates of whole-body glucose disposal increased by more than two-fold (P < 0.05 vs. basal) during both AICAR and insulin infusion; [3H]2-deoxy-D-glucose transport activity increased to a similar extent, by >2.2-fold (both P < 0.05 vs. control), in both soleus and red gastrocnemius muscles of lean rats infused with either AICAR or insulin. In the obese Zucker rats, neither AICAR nor insulin stimulated whole-body glucose disposal or soleus muscle glucose transport activity. However, AICAR increased glucose transport activity by approximately 2.4-fold (P < 0.05 vs. control) in the red gastrocnemius from obese rats, whereas insulin had no effect. In summary, acute infusion of AICAR in an insulin-resistant rat model activates skeletal muscle AMPK and increases glucose transport activity in red gastrocnemius muscle while suppressing endogenous glucose production and lipolysis. Because type 2 diabetes is characterized by diminished rates of insulin-stimulated glucose uptake as well as increased basal rates of endogenous glucose production and lipolysis, these results suggest that AICAR-related compounds may represent a new class of antidiabetic agents.     

Altered myocellular and abdominal fat partitioning predict disturbance in insulin action in HIV protease inhibitor-related lipodystrophy

HIV protease inhibitor-related lipodystrophy is characterized by peripheral fat loss, hyperlipidemia, and insulin resistance. Increased availability of lipid to muscle may be one of the mechanisms that induce insulin resistance. Regional fat, intramyocellular lipid (by (1)H-magnetic resonance spectroscopy), serum lipids, and insulin-stimulated glucose disposal (by hyperinsulinemic-euglycemic clamp) were quantified in 10 men who had HIV-1 infection with moderate to severe lipodystrophy and a control group of 10 nonlipodystrophic men who had HIV-1 infection and were na?ve to protease inhibitors to examine the effects of lipodystrophy on glucose and lipid metabolism. Lipodystrophic subjects showed lower insulin-stimulated glucose disposal than control subjects (P = 0.001) and had increased serum triglycerides (P = 0.03), less limb fat (P = 0.02), increased visceral fat as a proportion of total abdominal fat (P = 0.003), and increased intramyocellular lipid (1.90 +/- 0.15 vs. 1.23 +/- 0.16% of water resonance peak area; P = 0.007). In both groups combined, visceral fat related strongly to intramyocellular lipid (r = 0.83, P < 0.0001) and intramyocellular lipid related negatively to insulin-stimulated glucose disposal (r = -0.71, P = 0.0005). Fasting serum cholesterol and triglycerides related positively to intramyocellular lipid and visceral fat in lipodystrophic subjects only. The data indicate that lipodystrophy is associated with increased lipid content in muscle accompanying impaired insulin action. The results do not establish causation but emphasize the interrelationships among visceral fat, myocyte lipid, and insulin action.     

Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity

This study was conducted to investigate the possible involvement of protein kinase C (PKC) and serine/threonine phosphorylation of the insulin receptor in insulin resistance and/or obesity. Insulin receptor tyrosine kinase activity was depressed in muscle from obese insulin-resistant patients compared with lean insulin-responsive control subjects. Alkaline phosphatase treatment resulted in a significant 48% increase in in vitro insulin-stimulated receptor tyrosine kinase activity in obese but not lean muscle. To investigate the involvement of PKC in skeletal muscle insulin resistance and/or obesity, membrane-associated PKC activity and the protein content of various PKC isoforms were measured in human skeletal muscle from lean, insulin-responsive, and obese insulin-resistant patients. Membrane-associated PKC activity was not changed; however, PKC-beta protein content, assayed by Western blot analysis, was significantly higher, whereas PKC-theta, -eta, and -mu were significantly lower in muscle from obese patients compared with muscle from lean control subjects. Incubation of muscle strips with insulin significantly increased membrane-associated PKC activity in muscle from obese but not lean subjects. PKC-delta, -beta, and -theta were translocated from the cytosol to the membrane fraction in response to insulin treatment. These results suggest that in skeletal muscle from insulin-resistant obese patients, insulin receptor tyrosine kinase activity was reduced because of hyperphosphorylation on serine/threonine residues. Membrane-associated PKC-beta protein was elevated under basal conditions, and membrane-associated total PKC activity was increased under insulin-stimulated conditions in muscle from obese insulin-resistant patients. Thus, we postulate that the decreased tyrosine kinase activity of the insulin receptor may be caused by serine/threonine phosphorylation by PKC.     

Interactions of impaired glucose transport and phosphorylation in skeletal muscle insulin resistance: a dose-response assessment using positron emission tomography

It has been postulated that glucose transport is the principal site of skeletal muscle insulin resistance in obesity and type 2 diabetes, though a distribution of control between glucose transport and phosphorylation has also been proposed. The current study examined whether the respective contributions of transport and phosphorylation to insulin resistance are modulated across a dose range of insulin stimulation. Rate constants for transport and phosphorylation in skeletal muscle were estimated using dynamic positron emission tomography (PET) imaging of 2-deoxy-2[18F]fluoro-D-glucose ([18F]FDG) during insulin infusions at three rates (0, 40, and 120 mU/m2 per min) in lean glucose-tolerant, obese glucose-tolerant, and obese type 2 diabetic subjects. Parallel studies of arteriovenous fractional extraction across the leg of [18F]FDG and [2-3H] glucose were performed to measure the "lumped constant" (LC) (i.e., the analog effect) for [18F]FDG to determine whether this value is affected by insulin dose or insulin resistance. The value of the LC was similar across insulin doses and groups. Leg glucose uptake (LGU) also provided a measure of skeletal muscle glucose metabolism independent of PET. [18F]FDG uptake determined by PET imaging strongly correlated with LGU across groups and across insulin doses (r = 0.81, P < 0.001). Likewise, LGU correlated with PET parameters of glucose transport (r = 0.67, P < 0.001) and glucose phosphorylation (r = 0.86, P < 0.001). Glucose transport increased in response to insulin in the lean and obese groups (P < 0.05), but did not increase significantly in the type 2 diabetic group. A dose-responsive pattern of stimulation of glucose phosphorylation was observed in all groups of subjects (P < 0.05); however, glucose phosphorylation was lower in both the obese and type 2 diabetic groups compared with the lean group at the moderate insulin dose (P < 0.05). These findings indicate an important interaction between transport and phosphorylation in the insulin resistance of obesity and type 2 diabetes.     

5-amino-imidazole carboxamide riboside increases glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes

AMP-activated protein kinase (AMPK) activation by AICAR (5-amino-imidazole carboxamide riboside) is correlated with increased glucose transport in rodent skeletal muscle via an insulin-independent pathway. We determined in vitro effects of insulin and/or AICAR exposure on glucose transport and cell-surface GLUT4 content in skeletal muscle from nondiabetic men and men with type 2 diabetes. AICAR increased glucose transport in a dose-dependent manner in healthy subjects. Insulin and AICAR increased glucose transport and cell-surface GLUT4 content to a similar extent in control subjects. In contrast, insulin- and AICAR-stimulated responses on glucose transport and cell-surface GLUT4 content were impaired in subjects with type 2 diabetes. Importantly, exposure of type 2 diabetic skeletal muscle to a combination of insulin and AICAR increased glucose transport and cell-surface GLUT4 content to levels achieved in control subjects. AICAR increased AMPK and acetyl-CoA carboxylase phosphorylation to a similar extent in skeletal muscle from subjects with type 2 diabetes and nondiabetic subjects. Our studies highlight the potential importance of AMPK-dependent pathways in the regulation of GLUT4 and glucose transport activity in insulin-resistant skeletal muscle. Activation of AMPK is an attractive strategy to enhance glucose transport through increased cell surface GLUT4 content in insulin-resistant skeletal muscle.     

Increased lipid availability impairs insulin-stimulated ATP synthesis in human skeletal muscle

Insulin resistance correlates with intramyocellular lipid content (IMCL) and plasma free fatty acids (FFAs) and was recently linked to mitochondrial dysfunction. We examined the underlying relationships by measuring skeletal muscle ATP synthase flux, glucose transport/phosphorylation, and IMCL in response to different plasma insulin and plasma FFA concentrations. Healthy men were studied twice during hyperinsulinemic-euglycemic clamps with (LIP) or without (CON) lipid infusion (plasma FFA: CON approximately 36 vs. LIP approximately 1,034 micromol/l, P < 0.001). ATP synthase flux, glucose-6-phosphate (G6P), and IMCL were determined before and during the clamp in calf muscle using (31)P and (1)H magnetic resonance spectroscopy. Plasma lipid elevation resulted in approximately 46% reduced whole-body glucose metabolism (180-360 min; P < 0.0001 vs. CON) and a 70% lower rise of G6P (P < 0.05 vs. CON) without significant changes in IMCL (LIP 117 +/- 12% vs. CON 93 +/- 3% of basal, P = 0.073). During the clamp, ATP synthase flux increased by approximately 60% under control conditions (P = 0.02 vs. baseline) and was 24% lower during lipid infusion (LIP 11.0 +/- 0.9 vs. CON 14.6 +/- 1.2 micromol . g muscle(-1) . min(-1), P < 0.05). Physiologically increased plasma FFA concentrations reduce insulin-stimulated muscle ATP synthase flux in parallel with induction of insulin resistance.     

Tissue-specific effects of rosiglitazone and exercise in the treatment of lipid-induced insulin resistance

Both pharmacological intervention (i.e., thiazolidinediones [TZDs]) and lifestyle modification (i.e., exercise training) are clinically effective treatments for improving whole-body insulin sensitivity. However, the mechanism(s) by which these therapies reverse lipid-induced insulin resistance in skeletal muscle is unclear. We determined the effects of 4 weeks of rosiglitazone treatment and exercise training and their combined actions (rosiglitazone treatment and exercise training) on lipid and glucose metabolism in high-fat-fed rats. High-fat feeding resulted in decreased muscle insulin sensitivity, which was associated with increased rates of palmitate uptake and the accumulation of the fatty acid metabolites ceramide and diacylglycerol. Impairments in lipid metabolism were accompanied by defects in the Akt/AS160 signaling pathway. Exercise training, but not rosiglitazone treatment, reversed these impairments, resulting in improved insulin-stimulated glucose transport and increased rates of fatty acid oxidation in skeletal muscle. The improvements to glucose and lipid metabolism observed with exercise training were associated with increased AMP-activated protein kinase alpha1 activity; increased expression of Akt1, peroxisome proliferator-activated receptor gamma coactivator 1, and GLUT4; and a decrease in AS160 expression. In contrast, rosiglitazone treatment exacerbated lipid accumulation and decreased insulin-stimulated glucose transport in skeletal muscle. However, rosiglitazone, but not exercise training, increased adipose tissue GLUT4 and acetyl CoA carboxylase expression. Both exercise training and rosiglitazone decreased liver triacylglycerol content. Although both interventions can improve whole-body insulin sensitivity, our results show that they produce divergent effects on protein expression and triglyceride storage in different tissues. Accordingly, exercise training and rosiglitazone may act as complementary therapies for the treatment of insulin resistance.     

Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected].

To determine whether defects in the insulin signal transduction cascade are present in skeletal muscle from prediabetic individuals, we excised biopsies from eight glucose-intolerant male first-degree relatives of patients with type 2 diabetes (IGT relatives) and nine matched control subjects before and during a euglycemic-hyperinsulinemic clamp. IGT relatives were insulin-resistant in oxidative and nonoxidative pathways for glucose metabolism. In vivo insulin infusion increased skeletal muscle insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (P = 0.01) and phosphatidylinositide 3-kinase (PI 3-kinase) activity (phosphotyrosine and IRS-1 associated) in control subjects (P < 0.02) but not in IGT relatives (NS). The incremental increase in insulin action on IRS-1 tyrosine phosphorylation was lower in IGT relatives versus control subjects (P < 0.05). The incremental defects in signal transduction noted for IRS-1 and PI 3-kinase may be attributed to elevated basal phosphorylation/activity of these parameters, because absolute phosphorylation/activity under insulin-stimulated conditions was similar between IGT relatives and control subjects. Insulin increased Akt serine phosphorylation in control subjects and IGT relatives, with a tendency for reduced phosphorylation in IGT relatives (P = 0.12). In conclusion, aberrant phosphorylation/activity of IRS-1, PI 3-kinase, and Akt is observed in skeletal muscle from relatives of patients with type 2 diabetes with IGT. However, the elevated basal activity of these signaling intermediates and the lack of a strong correlation between these parameters to glucose metabolism suggests that other defects of insulin signal transduction and/or downstream components of glucose metabolism may play a greater role in the development of insulin resistance in skeletal muscle from relatives of patients with type 2 diabetes.     

Apelin treatment increases complete Fatty Acid oxidation, mitochondrial oxidative capacity, and biogenesis in muscle of insulin-resistant mice

Both acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)-induced obese and insulin-resistant mice treated by an apelin injection (0.1 μmol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondrial biogenesis and tighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement.