Insulin-stimulated Muscle Glucose Transport Research Articles

β-catenin regulates muscle glucose transport via actin remodelling and M-cadherin binding

ObjectiveSkeletal muscle glucose disposal following a meal is mediated through insulin-stimulated movement of the GLUT4-containing vesicles to the cell surface. The highly conserved scaffold-protein β-catenin is an emerging regulator of vesicle trafficking in other tissues. Here, we investigated the involvement of β-catenin in skeletal muscle insulin-stimulated glucose transport. MethodsGlucose homeostasis and transport was investigated in inducible muscle specific β-catenin knockout (BCAT-mKO) mice. The effect of β-catenin deletion and mutation of β-catenin serine 552 on signal transduction, glucose uptake and protein–protein interactions were determined in L6-G4-myc cells, and β-catenin insulin-responsive binding partners were identified via immunoprecipitation coupled to label-free proteomics. ResultsSkeletal muscle specific deletion of β-catenin impaired whole-body insulin sensitivity and insulin-stimulated glucose uptake into muscle independent of canonical Wnt signalling. In response to insulin, β-catenin was phosphorylated at serine 552 in an Akt-dependent manner, and in L6-G4-myc cells, mutation of β-cateninS552 impaired insulin-induced actin-polymerisation, resulting in attenuated insulin-induced glucose transport and GLUT4 translocation. β-catenin was found to interact with M-cadherin in an insulin-dependent β-cateninS552-phosphorylation dependent manner, and loss of M-cadherin in L6-G4-myc cells attenuated insulin-induced actin-polymerisation and glucose transport. ConclusionsOur data suggest that β-catenin is a novel mediator of glucose transport in skeletal muscle and may contribute to insulin-induced actin-cytoskeleton remodelling to support GLUT4 translocation.

297-OR: ß-Catenin Facilitates Skeletal Muscle Insulin-Stimulated Glucose Transport through the Regulation of Actin Remodeling

Skeletal muscle insulin-resistance is a major risk factor for type 2 diabetes. During the early stages of insulin-resistance the proximal insulin-signalling proteins are activated by insulin despite GLUT4 vesicle translocation and glucose transport being impaired. There is growing evidence that the dual function protein β-catenin can support vesicle trafficking, and is dysregulated in insulin resistance. Here we investigate the potential role of β-catenin in facilitating skeletal muscle glucose transport. We report that novel adult-induced skeletal muscle-specific β-catenin knockout mice are insulin resistant and have impaired skeletal muscle glucose transport. Furthermore, the mutation of β-cateninS552 in muscle cells impairs insulin-stimulated GLUT4 translocation and glucose uptake, an effect that appears to be independent of β-catenin’s WNT-signalling transcriptional role. Using immunoprecipitation coupled to proteomics we identify M-cadherin as an insulin-sensitive binding partner of β-catenin, which we confirmed to be required for normal insulin-stimulated glucose transport. Consistent with the role of cadherins in regulating actin filament organization, β-cateninS552 mutant muscle cells have impaired insulin-stimulated actin remodelling. This suggests that insulin-mediated phosphorylation of β-cateninS552 promotes the binding of β-catenin to M-cadherin which then provides support for actin polymerisation at the plasma membrane, a critical step for GLUT4 trafficking. We therefore propose that the dysregulation of β-catenin may play a role insulin resistance independent of proximal insulin signalling. Disclosure S.W. Masson: None. T.L. Merry: None. Funding Rutherford Discovery Fellowship (RDF-15-UOA-020)

Maternal Obesity and Western-Style Diet Impair Fetal and Juvenile Offspring Skeletal Muscle Insulin-Stimulated Glucose Transport in Nonhuman Primates.

Infants born to mothers with obesity have a greater risk for childhood obesity and metabolic diseases; however, the underlying biological mechanisms remain poorly understood. We used a Japanese macaque model to investigate whether maternal obesity combined with a Western-style diet (WSD) impairs offspring muscle insulin action. Adult females were fed a control or WSD prior to and during pregnancy through lactation, and offspring subsequently weaned to a control or WSD. Muscle glucose uptake and signaling were measured ex vivo in fetal (n = 5-8/group) and juvenile (n = 8/group) offspring. In vivo signaling was evaluated after an insulin bolus just prior to weaning (n = 4-5/group). Maternal WSD reduced insulin-stimulated glucose uptake and impaired insulin signaling at the level of Akt phosphorylation in fetal muscle. In juvenile offspring, insulin-stimulated glucose uptake was similarly reduced by both maternal and postweaning WSD and corresponded to modest reductions in insulin-stimulated Akt phosphorylation relative to controls. We conclude that maternal WSD leads to a persistent decrease in offspring muscle insulin-stimulated glucose uptake even in the absence of increased offspring adiposity or markers of systemic insulin resistance. Switching offspring to a healthy diet did not reverse the effects of maternal WSD on muscle insulin action, suggesting earlier interventions may be warranted.

Autotaxin-LPA signaling contributes to obesity-induced insulin resistance in muscle and impairs mitochondrial metabolism

Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid (LPA). ATX-LPA signaling has been implicated in diet-induced obesity and systemic insulin resistance. However, it remains unclear whether the ATX-LPA pathway influences insulin function and energy metabolism in target tissues, particularly skeletal muscle, the major site of insulin-stimulated glucose disposal. The objective of this study was to test whether the ATX-LPA pathway impacts tissue insulin signaling and mitochondrial metabolism in skeletal muscle during obesity. Male mice with heterozygous ATX deficiency (ATX+/-) were protected from obesity, systemic insulin resistance, and cardiomyocyte dysfunction following high-fat high-sucrose (HFHS) feeding. HFHS-fed ATX+/- mice also had improved insulin-stimulated AKT phosphorylation in white adipose tissue, liver, heart, and skeletal muscle. Preserved insulin-stimulated glucose transport in muscle from HFHS-fed ATX+/- mice was associated with improved mitochondrial pyruvate oxidation in the absence of changes in fat oxidation and ectopic lipid accumulation. Similarly, incubation with LPA decreased insulin-stimulated AKT phosphorylation and mitochondrial energy metabolism in C2C12 myotubes at baseline and following palmitate-induced insulin resistance. Taken together, our results suggest that the ATX-LPA pathway contributes to obesity-induced insulin resistance in metabolically relevant tissues. Our data also suggest that LPA directly impairs skeletal muscle insulin signaling and mitochondrial function.

Metabolic Inflexibility Revisited—Muscle Substrate Oxidation Is Mechanistically Dissociated from Muscle Insulin Resistance in Rats

The Metabolic Inflexibility Hypothesis and Randle Hypothesis posit that alterations in muscle glucose and fat oxidation are key factors in the pathogenesis of obesity (lipid)-associated skeletal muscle insulin resistance. In order to test these hypotheses we used a stable isotope tracer ([U-13C]glucose) to measure the ratio of pyruvate dehydrogenase flux and citrate synthase flux (VPDH/VCS), a highly tissue-specific index for the proportional contribution of glucose oxidation to total mitochondrial oxidation in vivo. We found that insulin resistant rats fed a high fat diet (HFD) for 4 weeks did not have altered substrate (VPDH/VCS) oxidation in soleus muscle in the fasting state (5.8%±2.5% vs. 2.6%±1.0%). Hyperinsulinemic-euglycemic clamps increased relative glucose oxidation in both normal and insulin resistant rats, although this increase was blunted in insulin resistant rats (51.6±4.9% vs. 33.3±4.5%, p<0.05), which could be mostly attributed to an impairment in insulin-stimulated muscle glucose transport flux (124.4±18.7 vs. 69.3±11.3 nmol/(g•minute), p<0.05). Additionally, we found that an acute infusion of lipid during a clamp in normal rats significantly reduced relative glucose oxidation in soleus muscle (49.0±3.6% vs. 26.7±2.7%, p<0.001) without any effects on insulin-stimulated peripheral glucose metabolism (27.2±1.4 vs. 24.3±1.5 mg/(kg•minute), NS) or muscle glucose transport (143.8±22.4 vs. 138.1±18.3 nmol/(g•minute), NS). Conclusion: These data show that basal substrate preference in muscle is not altered in insulin resistant rats and that acute modulation of substrate oxidation in normal rats does not affect muscle insulin sensitivity, therefore challenging the Metabolic Inflexibility Hypothesis and Randle Hypothesis in the pathogenesis of lipid-induced muscle insulin resistance. Disclosure J.D. Song: None. R.J. Perry: None. A. Munk: None. Y. Zhang: None. D. Zhang: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Impairment of insulin-stimulated glucose transport and ERK activation by adipocyte-specific knockout of PKC-λ produces a phenotype characterized by diminished adiposity and enhanced insulin suppression of hepatic gluconeogenesis

Tissue-specific knockout (KO) of atypical protein kinase C-λ (PKC-λ) impairs insulin-stimulated glucose transport in muscle (M) and lipid synthesis in liver (L), thereby producing insulin resistance in MλKO mice and insulin-hypersensitivity in LλKO mice. Here, we generated mice with KO of PKC-λ in adipocytes, i.e., AλKO mice. In isolated adipocytes of AλKO mice, insulin-stimulated aPKC activity and glucose transport were diminished, as were ERK levels and activity. Insulin-stimulated glucose transport and insulin activation of ERK in adipocytes of wild-type mice were similarly inhibited by acute inhibition of PKC-λ with a highly-specific chemical inhibitor. With impairments in glucose transport and ERK activation, AλKO mice had diminished adiposity and serum leptin levels. In addition, AλKO mice had normal glucose tolerance and insulin hypersensitivity owing to enhanced suppression of hepatic glucose output, which apparently reflected increases in Akt activity and FoxO1 phosphorylation, and subsequent decreases in expression of gluconeogenic phosphoenolpyruvate carboxykinase. We conclude that: PKC-λ is required for insulin-stimulated glucose transport and ERK signaling in mouse adipocytes; and diminution of these processes is attended by leanness and therefore hypoleptinemia. How these and perhaps other PKC-λ-dependent processes communicate to liver and improve insulin suppression of hepatic gluconeogenesis remains unclear.

ANG (1–7) reduces ANG II‐induced insulin resistance by increasing Akt phosphorylation via a Mas receptor‐dependent mechanism in rat skeletal muscle

Angiotensin II (ANG II) induces hypertension via the ANG II type 1 receptor, while angiotensin (1–7) (ANG (1–7)), a peptide derived primarily from ANG II, elicits an antihypertensive effect via the Mas receptor. Although a critical role of ANG II in the etiology of skeletal muscle insulin resistance is well documented, the role of the ANG (1–7)/Mas receptor axis in this context is poorly understood. Therefore, we determined whether ANG (1–7) is effective in ameliorating the negative effects of ANG II on insulin‐stimulated insulin signaling and glucose transport in isolated soleus muscle strips from lean Zucker rats. ANG II (500 nM for 2 hr) alone decreased insulin‐stimulated glucose transport by 45% (p<0.05). In the presence of 500–1000 nM ANG (1–7), insulin‐stimulated glucose transport in muscle exposed to ANG II improved by ~25% (p<0.05). Moreover, this ANG (1–7)‐mediated improvement in glucose transport was associated with increased Akt Ser467 phosphorylation (47%, p<0.05). The positive effects of ANG (1–7) on insulin‐stimulated glucose transport and Akt phosphorylation were completely blunted in presence of 1000 nM A779, a selective Mas receptor antagonist. The present study demonstrates that ANG (1–7), via a Mas receptor‐dependent mechanism, can partially prevent the inhibitory effect of ANG II on glucose transport in isolated mammalian skeletal muscle, associated with enhanced Akt phosphorylation.

The insulin sensitizing effects of PPAR-γ agonist are associated to changes in adiponectin index and adiponectin receptors in Zucker fatty rats

The adiponectin high molecular weight isoform (HMW-adp) and its relation with the other adiponectin isoforms (adiponectin index, SA), have been identified as essential for the adiponectin insulin sensitizing effects. The objective of this study is to gain further insight on the effect of the insulin sensitizing agents, PPAR-γ agonists, on the distribution of the adiponectin isoforms and the adiponectin receptors, adipoR1 and adipoR2 in an animal model of obesity and insulin resistance.To achieve the objective, Zucker fatty rats were treated with pioglitazone, rosiglitazone or placebo for six weeks. At the end of the treatment, total adiponectin, adiponectin isoforms and adiponectin receptors expression were measured. In order to see the possible relation with insulin sensitivity parameters, HOMA-IR, muscle insulin-stimulated glucose transport, muscle GLUT4 and plasma free fatty acids were also measured.The two glitazones improved insulin sensitivity and both muscle insulin-stimulated glucose transport and GLUT4 total content. Total plasma adiponectin and visceral fat HMW-adp were increased only by pioglitazone. On the other hand, both glitazones changed the distribution of adiponectin isoforms in plasma, leading to an increase in the SA of 21% by pioglitazone and 31% by rosiglitazone. Muscle adipoR1 expression was increased by both glitazones whereas liver adipoR2 expression was increased by rosiglitazone and tended to increase in the pioglitazone group.The insulin sensitizing action of glitazones is mediated, at least in part, by their effect on muscle insulin-stimulated glucose transport and by their direct influence on the adiponectin index and the adiponectin receptors expression.

Mice with AS160/TBC1D4-Thr649Ala Knockin Mutation Are Glucose Intolerant with Reduced Insulin Sensitivity and Altered GLUT4 Trafficking

SummaryAS160 has emerged as a key player in insulin-mediated glucose transport through controlling GLUT4 trafficking, which is thought to be regulated by insulin-stimulated phosphorylation of sites including the 14-3-3 binding phospho-Thr649 (equivalent to Thr642 in human AS160). To define physiological roles of AS160-Thr649 phosphorylation and 14-3-3 binding in glucose homeostasis, we substituted this residue by a nonphosphorylatable alanine by knockin mutation in mice. The mutant protein was expressed at normal levels, while insulin-stimulated AS160 binding to 14-3-3s was abolished in homozygous knockin mice. These animals displayed impaired glucose disposal and insulin sensitivity, which were associated with decreased glucose uptake in vivo. Insulin-stimulated glucose transport and cell surface GLUT4 content were reduced in isolated muscles, but not in adipocytes. These results provide genetic evidence that insulin-induced AS160-Thr649 phosphorylation and/or its binding to 14-3-3 play an important role in regulating whole-body glucose homeostasis, at least in part through regulating GLUT4 trafficking in muscle.

Is “fat-induced” muscle insulin resistance rapidly reversible?

Elevated plasma free fatty acids (FFA) cause insulin resistance and are thought to play a key role in mediating insulin resistance in patients with the metabolic syndrome (MTS) and type 2 diabetes mellitus (DM). Two experimental models used to study the mechanisms responsible for insulin resistance in patients are high-fat diet-fed rodents and administration of triglycerides and heparin to raise plasma FFA. As evidence that insulin resistance in high-fat diet-fed rats is due to high FFA, it has been reported that the insulin resistance is rapidly reversed by an overnight fast, a high-glucose meal, and an exercise bout. If true, these findings would invalidate the high-fat diet-fed rodent as a model for MTS or type 2 DM, because insulin resistance is not rapidly reversed by these treatments in patients. The purpose of this study was to determine whether diet-induced insulin resistance is, in fact, rapidly reversible. Incubation of muscles in vitro rapidly reversed insulin resistance induced by administration of triglycerides and heparin, but not by a high-fat diet. An overnight fast and a high-glucose meal were followed by a large increase in insulin-stimulated muscle glucose transport. However, these are adaptive responses, rather than reversals of insulin resistance, because they also occurred in muscles of insulin-sensitive, chow-fed control rats. Our results show that insulin resistance induced by high FFA, i.e., Randle glucose-fatty acid cycle, is transient. In contrast, the insulin resistance induced by a high-fat diet does not reverse rapidly.

Resistin acutely impairs insulin-stimulated glucose transport in rodent muscle in the presence, but not absence, of palmitate

Resistin is a cytokine implicated in the development of insulin resistance. However, there has been little investigation of the effects of resistin on fatty acid (FA) metabolism and insulin response in skeletal muscle, a key tissue for glucose disposal. The purpose of the present study was to examine the role of altered FA metabolism as a cause of resistin's inhibition of insulin-stimulated glucose transport in muscle. Isolated rat soleus muscles were incubated acutely (2 h) in the presence or absence of 600 ng/ml resistin, with or without 2 mM palmitate. Resistin acutely impaired insulin-stimulated glucose transport and Akt phosphorylation, but only in the presence of palmitate, implicating a role for altered FA metabolism. This impairment of glucose transport induced by resistin plus palmitate could be pharmacologically rescued by the inclusion of aimidazole carboxamide ribonucleotide, a stimulator of AMP-activated protein kinase and FA oxidation, as well as inhibitors of ceramide synthesis (myriocin, fumonisin). However, to our surprise, resistin actually blunted the palmitate-induced increase in muscle ceramide content; as expected, ceramide content was significantly lowered by fumonisin. In summary, the acute impairment of insulin response by resistin was manifested only in the presence of high palmitate and was alleviated when FA metabolism was manipulated (increased oxidation, inhibited ceramide synthesis). Resistin's acute impairment of insulin response does not appear to require an absolute increase in ceramide content; however, reducing ceramide content alleviated the impairment in glucose transport and insulin signaling.

Combined thiazolidinedione–metformin treatment synergistically improves insulin signalling to insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase, atypical protein kinase C and protein kinase B/Akt in human diabetic muscle

Insulin-stimulated glucose transport in muscle is impaired in type 2 diabetes, presumably reflecting reduced activation of atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). As previously shown, reductions in aPKC activation are seen at sub-maximal and maximal insulin stimulation, reductions in PKB activation are best seen at sub-maximal insulin stimulation and aPKC reductions at maximal insulin are partly improved by thiazolidinedione or metformin treatment. However, effects of combined thiazolidinedione-metformin treatment on aPKC or PKB activation by sub-maximal and maximal insulin are unknown. Type 2 diabetic patients were examined before and 5 to 6 weeks after combined thiazolidinedione-metformin therapy for activation of muscle aPKC and PKBbeta and their upstream activators, the insulin receptor (IR) and IRS-1-associated phosphatidylinositol 3-kinase (PI3K), during euglycaemic-hyperinsulinaemic clamp studies conducted with sub-maximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin concentrations. Following combined thiazolidinedione-metformin therapy, increases in glucose disposal and increases in sub-maximal and maximal insulin-induced activities of all four muscle signalling factors, IR, IRS-1-dependent PI3K (IRS-1/PI3K), aPKC and PKBbeta, were observed. Increases in PKBbeta enzyme activity were accompanied by increases in phosphorylation of PKB and its substrate, AS160, which is needed for glucose transport. Despite improved aPKC activity, muscle aPKC levels, which are diminished in type 2 diabetes, were not altered. Combined thiazolidinedione-metformin treatment markedly improves sub-maximal and maximal insulin signalling to IR, IRS-1/PI3K, aPKC and PKBbeta in type 2 diabetic muscle. These improvements exceed those previously reported after treatment with either agent alone.

Palmitate acutely induces insulin resistance in isolated muscle from obese but not lean humans

Exposure to high fatty acids (FAs) induces whole body and skeletal muscle insulin resistance. The globular form of the adipokine, adiponectin (gAd), stimulates FA oxidation and improves insulin sensitivity; however, its ability to prevent lipid-induced insulin resistance in humans has not been tested. The purpose of this study was to determine 1) whether acute (4 h) exposure to 2 mM palmitate would impair insulin signaling and glucose transport in isolated human skeletal muscle, 2) whether muscle from obese humans is more susceptible to the effects of palmitate, and 3) whether the presence of 2 mM palmitate + 2.5 mug/ml gAd (P+gAd) could prevent the effects of palmitate. Insulin-stimulated (10 mU/ml) glucose transport was not different, relative to control, following exposure to palmitate (-10%) or P+gAd (-3%) in lean muscle. In obese muscle, the absolute increase in glucose transport from basal to insulin-stimulated conditions was significantly decreased following palmitate (-55%) and P+gAd (-36%) exposure (control vs. palmitate; control vs. P+gAd, P < 0.05). There was no difference in the absolute increase in glucose transport between palmitate and P+gAd, indicating that in the presence of palmitate, gAd did not improve glucose transport. The palmitate-induced reduction in insulin-stimulated glucose transport in muscle from obese individuals may have been due to reduced Ser Akt (control vs. palmitate; P+gAd, P < 0.05) and Akt substrate 160 (AS160) phosphorylation (control vs. palmitate; P+gAd, P < 0.05). FA oxidation was significantly increased in muscle of lean and obese individuals in the presence of gAd (P < 0.05), suggesting that the stimulatory effects of gAd on FA oxidation may not be sufficient to entirely prevent palmitate-induced insulin resistance in obese muscle.

Activation of the A1adenosine receptor increases insulin-stimulated glucose transport in isolated rat soleus muscle

The A1 adenosine receptor (A1AR) has been suggested to participate in insulin- and contraction-stimulated glucose transport in skeletal muscle, but the qualitative and quantitative nature of the effect are controversial. We sought to determine if A1AR is expressed in rat soleus muscle and then characterize its role in glucose transport in this muscle. A1AR mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. To examine the role of adenosine in 3-O-methylglucose transport, isolated muscles were exposed to adenosine deaminase and alpha,beta-methylene adenosine diphosphate to remove endogenous adenosine and were left unstimulated (basal) or stimulated with insulin. To assess the functional participation of A1AR in 3-O-methylglucose transport, muscles were incubated with A1-selective agonist and (or) antagonist in the absence of endogenous adenosine and with or without insulin. A1AR mRNA was expressed in soleus muscle and A1AR was present at the plasma membrane. Removal of endogenous adenosine reduced glucose transport in response to 100 microU/mL insulin (approximately 50%). The A1-selective agonist, N6-cyclopentyladenosine, increased submaximal (100 microU/mL) insulin-stimulated glucose transport in a dose-dependent manner (0.001-1.0 micromol/L). This stimulatory effect was inhibited by the A1-selective receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine in a concentration-dependent manner (0.001-1.0 micromol/L). However, neither activation nor inhibition of A1AR altered basal or maximal (10 mU/mL) insulin-stimulated glucose transport. Our results suggest that adenosine contributes approximately 50% to insulin-stimulated muscle glucose transport by activating the A1AR. This effect is limited to increasing insulin sensitivity, but not to either basal or maximal insulin-stimulated glucose uptake in rat soleus muscle.

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.

Metformin and exercise reduce muscle FAT/CD36 and lipid accumulation and blunt the progression of high-fat diet-induced hyperglycemia

Derangements in skeletal muscle fatty acid (FA) metabolism associated with insulin resistance in obesity appear to involve decreased FA oxidation and increased accumulation of lipids such as ceramides and diacylglycerol (DAG). We investigated potential lipid-related mechanisms of metformin (Met) and/or exercise for blunting the progression of hyperglycemia/hyperinsulinemia and skeletal muscle insulin resistance in female Zucker diabetic fatty rats (ZDF), a high-fat (HF) diet-induced model of diabetes. Lean and ZDF rats consumed control or HF diet (48 kcal %fat) alone or with Met (500 mg/kg), with treadmill exercise, or with both exercise and Met interventions for 8 wk. HF-fed ZDF rats developed hyperglycemia (mean: 24.4 +/- 2.1 mM), impairments in muscle insulin-stimulated glucose transport, increases in the FA transporter FAT/CD36, and increases in total ceramide and DAG content. The development of hyperglycemia was significantly attenuated with all interventions, as was skeletal muscle FAT/CD36 abundance and ceramide and DAG content. Interestingly, improvements in insulin-stimulated glucose transport and increased GLUT4 transporter expression in isolated muscle were seen only in conditions that included exercise training. Reduced FA oxidation and increased triacylglycerol synthesis in isolated muscle were observed with all ZDF rats compared with lean rats (P < 0.01) and were unaltered by therapeutic intervention. However, exercise did induce modest increases in peroxisome proliferator-activated receptor-gamma coactivator-1alpha, citrate synthase, and beta-hydroxyacyl-CoA dehydrogenase activity. Thus reduction of skeletal muscle FAT/CD36 and content of ceramide and DAG may be important mechanisms by which exercise training blunts the progression of diet-induced insulin resistance in skeletal muscle.

Contrasting insulin dose-dependent defects in activation of atypical protein kinase C and protein kinase B/Akt in muscles of obese diabetic humans

Insulin-stimulated glucose transport in muscle is impaired in obesity and type 2 diabetes, but alterations in levels of relevant signalling factors, i.e. atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt), are still uncertain. Clamp studies using maximal insulin concentrations have revealed defects in activation of aPKC, but not PKB, in both obese non-diabetic and obese diabetic subjects. In contrast, clamp studies using submaximal insulin concentrations revealed defects in PKB activation/phosphorylation in obese non-diabetic and diabetic subjects, but changes in aPKC were not reported. The aim of this study was to test the hypothesis that dose-related effects of insulin may account for the reported differences in insulin signalling to PKB in diabetic muscle. We compared enzymatic activation of aPKC and PKB, and PKB phosphorylation (threonine-308 and serine-473) during hyperinsulinaemic-euglycaemic clamp studies using both submaximal (400-500 pmol/l) and maximal (1400 pmol/l) insulin levels in non-diabetic control and obese diabetic subjects. In lean control subjects, the submaximal insulin concentration increased aPKC activity and glucose disposal to approximately 50% of the maximal level and PKBbeta activity to 25% of the maximal level, but PKBalpha activity was not increased. In these subjects, phosphorylation of PKBalpha and PKBbeta was increased to near-maximal levels at submaximal insulin concentrations. In obese diabetic subjects, whereas aPKC activation was defective at submaximal and maximal insulin concentrations, PKBbeta activation and the phosphorylation of PKBbeta and PKBalpha were defective at submaximal, but not maximal, insulin concentrations. Defective PKBbeta activation/phosphorylation, seen on submaximal insulin stimulation in diabetic muscle, may largely reflect impaired activation of insulin signalling factors present in concentrations greater than those needed for full PKB activation/phosphorylation. Defective aPKC activation, seen at all insulin levels, appears to reflect, at least partly, an impaired action of distal factors needed for aPKC activation, or poor aPKC responsiveness.

Atypical protein kinase C in insulin action and insulin resistance

It now seems clear that aPKC (atypical protein kinase C) isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. Moreover, there are marked defects in the activation of aPKCs under a variety of insulin-resistant conditions in humans, monkeys and rodents. In humans, defects in aPKC in muscle are seen in Type II diabetes and its precursors, obesity, the obesity-associated polycystic ovary syndrome and impaired glucose tolerance. These defects in muscle aPKC activation are due to both impaired activation of insulin receptor substrate-1-dependent PI3K (phosphoinositide 3-kinase) and the direct activation of aPKCs by the lipid product of PI3K, PI-3,4,5-(PO4)3. Although it is still uncertain which underlying defect comes first, the resultant defect in aPKC activation in muscle most certainly contributes significantly to the development of skeletal muscle insulin resistance. Of further note, unlike the seemingly ubiquitous presence of defective aPKC activation in skeletal muscle in insulin-resistant states, the activation of aPKC is normal or increased in livers of Type II diabetic and obese rodents. The maintenance of aPKC activation in the liver may explain how insulin-dependent lipid synthesis is maintained in these states, as aPKCs function mainly in the activation of enzymes important for lipid synthesis. Thus increased activation of liver aPKC in hyperinsulinaemic states may contribute significantly to the development of hyperlipidaemia in insulin-resistant states.

Cellular mechanism of insulin resistance: potential links with inflammation.

Insulin resistance is a pivotal feature in the pathogenesis of type 2 diabetes, and it may be detected 10-20 y before the clinical onset of hyperglycemia. Insulin resistance is due to the reduced ability of peripheral target tissues to respond properly to insulin stimulation. In particular, impaired insulin-stimulated muscle glycogen synthesis plays a significant role in insulin resistance. Glucose transport (GLUT4), phosphorylation (hexokinase) and storage (glycogen synthase) are the three potential rate-controlling steps regulating insulin-stimulated muscle glucose metabolism, and all three have been implicated as being the major defects responsible for causing insulin resistance in patients with type 2 diabetes. Using (13)C/(31)P magnetic resonance spectroscopy (MRS), we demonstrate that a defect in insulin-stimulated muscle glucose transport activity is the rate-controlling defect. Using a similar (13)C/(31)P MRS approach, we have also demonstrated that fatty acids cause insulin resistance in humans due to a decrease in insulin-stimulated muscle glucose transport activity, which could be attributed to reduced insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, a required step in insulin-stimulated glucose transport into muscle. Furthermore, we have recently proposed that this defect in insulin-stimulated muscle glucose transport activity may be due to the activation of a serine kinase cascade involving protein kinase C theta and IKK-beta, which are key downstream mediators of tissue inflammation. Finally, we propose that any perturbation that leads to an increase in intramyocellular lipid (fatty acid metabolites) content such as acquired or inherited defects in mitochondrial fatty acid oxidation, defects in adipocyte fat metabolism or simply increased fat delivery to muscle/liver due to increased energy intake will lead to insulin resistance through this final common pathway. Understanding these key cellular mechanisms of insulin resistance should help elucidate new targets for treating type 2 diabetes.

Epinephrine inhibits insulin-stimulated muscle glucose transport.

We recently demonstrated that epinephrine could inhibit the activation by insulin of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-kinase) in skeletal muscle (Hunt DG, Zhenping D, and Ivy JL. J Appl Physiol 92: 1285-1292, 2002). Activation of PI3-kinase is recognized as an essential step in the activation of muscle glucose transport by insulin. We therefore investigated the effect of epinephrine on insulin-stimulated glucose transport in both fast-twitch (epitrochlearis) and slow-twitch (soleus) muscle of the rat by using an isolated muscle preparation. Glucose transport was significantly increased in the epitrochlearis and soleus when incubated in 50 and 100 microU/ml insulin, respectively. Activation of glucose transport by 50 microU/ml insulin was inhibited by 24 nM epinephrine in both muscle types. This inhibition of glucose transport by epinephrine was accompanied by suppression of IRS-1-associated PI3-kinase activation. However, when muscles were incubated in 100 microU/ml insulin, 24 nM epinephrine was unable to inhibit IRS-1-associated PI3-kinase activation or glucose transport. Even when epinephrine concentration was increased to 500 nM, no attenuating effect was observed on glucose transport. Results of this study indicate that epinephrine is capable of inhibiting glucose transport activated by a moderate, but not a high, physiological insulin concentration. The inhibition of glucose transport by epinephrine appears to involve the inhibition of IRS-1-associated PI3-kinase activation.