Search

Skeletal muscle is a highly adaptable tissue and remodels in response to exercise training. Using short RNA sequencing, we determine the miRNA profile of skeletal muscle from healthy male volunteers before and after a 14-day aerobic exercise training regime. Among the exercise training-responsive miRNAs identified, miR-19b-3p was selected for further validation. Overexpression of miR-19b-3p in human skeletal muscle cells increases insulin signaling, glucose uptake, and maximal oxygen consumption, recapitulating the adaptive response to aerobic exercise training. Overexpression of miR-19b-3p in mouse flexor digitorum brevis muscle enhances contraction-induced glucose uptake, indicating that miR-19b-3p exerts control on exercise training-induced adaptations in skeletal muscle. Potential targets of miR-19b-3p that are reduced after aerobic exercise training include KIF13A, MAPK6, RNF11, and VPS37A. Amongst these, RNF11 silencing potentiates glucose uptake in human skeletal muscle cells. Collectively, we identify miR-19b-3p as an aerobic exercise training-induced miRNA that regulates skeletal muscle glucose metabolism., (© 2021. The Author(s).)

We aimed to determine whether interrupting prolonged sitting improves glycemic control and the metabolic profile of free-living adults with obesity. Sixteen sedentary individuals {10 women/6 men; median [interquartile range (IQR)] age 50 (44-53) yr, body mass index (BMI) 32 (32-35.8) kg/m 2 } were fitted with continuous glucose and activity monitors for 4 wk. After a 1-wk baseline period, participants were randomized into habitual lifestyle (Control) or frequent activity breaks from sitting (FABS) intervention groups. Each day, between 0800 and 1800 h, FABS received smartwatch notifications to break sitting with 3 min of low-to-moderate-intensity physical activity every 30 min. Glycemic control was assessed by oral glucose tolerance test (OGTT) and continuous glucose monitoring. Blood samples and vastus lateralis biopsies were taken for assessment of clinical chemistry and the skeletal muscle lipidome, respectively. Compared with baseline, FABS increased median steps by 744 [IQR (483-951)] and walking time by 10.4 [IQR (2.2-24.6)] min/day. Other indices of activity/sedentary behavior were unchanged. Glucose tolerance and average 24-h glucose curves were also unaffected. However, mean (±SD) fasting glucose levels [-0.34 (±0.37) mmol/L] and daily glucose variation [%CV; -2% (±2.2%)] reduced in FABS, suggesting a modest benefit for glycemic control that was most robust at higher volumes of daily activity. Clinical chemistry and the skeletal muscle lipidome were largely unperturbed, although two long-chain triglycerides increased 1.25-fold in FABS, postintervention. All parameters remained stable in control. Under free-living conditions, FABS lowered fasting glucose and glucose variability. Larger volumes of activity breaks from sitting may be required to promote greater health benefits. NEW & NOTEWORTHY Under free-living conditions, breaking sitting modestly increased activity behavior. Breaking sitting was insufficient to modulate glucose tolerance or the skeletal muscle lipidome. Activity breaks reduced fasting blood glucose levels and daily glucose variation compared with baseline, with a tendency to also decrease fasting LDLc. This intervention may represent the minimal dose for breaking sedentary behavior, with larger volumes of activity possibly required to promote greater health benefits.

Diurnal oscillations in energy metabolism are linked to the activity of biological clocks and contribute to whole-body glucose homeostasis. Postprandially, skeletal muscle takes up approximately 80% of circulatory glucose and hence is a key organ in maintenance of glucose homeostasis. Dysregulation of molecular clock components in skeletal muscle disrupts whole-body glucose homeostasis. Next to light-dark cycles, nonphotic cues such as nutrient intake and physical activity are also potent cues to (re)set (dys)regulated clocks. Physical exercise is one of the most potent ways to improve myocellular insulin sensitivity. Given the role of the biological clock in glucose homeostasis and the power of exercise to improve insulin sensitivity, one can hypothesize that there might be an optimal time for exercise to maximally improve insulin sensitivity and glucose homeostasis. In this review, we aim to summarize the available information related to the interaction of diurnal rhythm, glucose homeostasis, and physical exercise as a nonphotic cue to correct dysregulation of human glucose metabolism., (© 2020 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society (TOS).)

During exercise, skeletal muscles release cytokines, peptides, and metabolites that exert autocrine, paracrine, or endocrine effects on glucose homeostasis. In this study, we investigated the effects of secreted protein acidic and rich in cysteine (SPARC), an exercise-responsive myokine, on glucose metabolism in human and mouse skeletal muscle. SPARC-knockout mice showed impaired systemic metabolism and reduced phosphorylation of AMPK and protein kinase B in skeletal muscle. Treatment of SPARC-knockout mice with recombinant SPARC improved glucose tolerance and concomitantly activated AMPK in skeletal muscle. These effects were dependent on AMPK-γ3 because SPARC treatment enhanced skeletal muscle glucose uptake in wild-type mice but not in AMPK-γ3-knockout mice. SPARC strongly interacted with the voltage-dependent calcium channel, and inhibition of calcium-dependent signaling prevented SPARC-induced AMPK phosphorylation in human and mouse myotubes. Finally, chronic SPARC treatment improved systemic glucose tolerance and AMPK signaling in skeletal muscle of high-fat diet-induced obese mice, highlighting the efficacy of SPARC treatment in the management of metabolic diseases. Thus, our findings suggest that SPARC treatment mimics the effects of exercise on glucose tolerance by enhancing AMPK-dependent glucose uptake in skeletal muscle.-Aoi, W., Hirano, N., Lassiter, D. G., Björnholm, M., Chibalin, A. V., Sakuma, K., Tanimura, Y., Mizushima, K., Takagi, T., Naito, Y., Zierath, J. R., Krook, A. Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP-activated protein kinase activation.

The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin-releasing hormone receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance and glucose metabolism. We investigated a modified UCN2 peptide as a potential therapeutic agent for the treatment of obesity and insulin resistance, with a specific focus on skeletal muscle. High-fat-fed mice (C57BL/6J) were injected daily with a PEGylated UCN2 peptide (compound A) at 0.3 mg/kg subcutaneously for 14 days. Compound A reduced body weight, food intake, whole-body fat mass, and intramuscular triglycerides compared with vehicle-treated controls. Furthermore, whole-body glucose tolerance was improved by compound A treatment, with increased insulin-stimulated Akt phosphorylation at Ser 473 and Thr 308 in skeletal muscle, concomitant with increased glucose transport into extensor digitorum longus and gastrocnemius muscle. Mechanistically, this is linked to a direct effect on skeletal muscle because ex vivo exposure of soleus muscle from chow-fed lean mice to compound A increased glucose transport and insulin signaling. Moreover, exposure of GLUT4-Myc-labeled L6 myoblasts to compound A increased GLUT4 trafficking. Our results demonstrate that modified UCN2 peptides may be efficacious in the treatment of type 2 diabetes by acting as an insulin sensitizer in skeletal muscle., (© 2019 by the American Diabetes Association.)

Objective: Forkhead box class O (FOXO) transcription factors regulate whole body energy metabolism, skeletal muscle mass, and substrate switching. FOXO1 and FOXO3 are highly abundant transcription factors, but their precise role in skeletal muscle metabolism has not been fully elucidated., Methods: To elucidate the role of FOXO in skeletal muscle, dominant negative (dn) constructs for FOXO1 (FOXO1dn) or FOXO3 (FOXO3dn) were transfected by electroporation into mouse tibialis anterior muscle and glucose uptake, signal transduction, and gene expression profiles were assessed after an oral glucose tolerance test. Results were compared against contralateral control transfected muscle., Results: FOXO1dn and FOXO3dn attenuated glucose uptake (35%, p < 0.01 and 20%, p < 0.05), GLUT4 protein (40%, p < 0.05 and 10%, p < 0.05), and subunits of the oxidative phosphorylation cascade. Intramuscular glycogen content was decreased (20%, p < 0.05) by FOXO3dn, but not FOXO1dn. Transcriptomic analysis revealed major pathways affected by FOXO1dn or FOXO3dn revolve around metabolism and inflammation. FOXO1dn increased Akt protein (140%, p < 0.001), p-Akt Ser473 (720%, p < 0.05) and p-Akt Thr308 (570%, p < 0.01), whereas FOXO3dn was without effect. FOXO1dn and FOXO3dn increased mTOR protein content (170% and 190%, p < 0.05), and p-p70S6K Thr389 (420%, p < 0.01 and 300%, p < 0.01), while p-mTOR Ser2448 (500%, p < 0.01), was only increased by FOXO1dn. Chemokines and immune cell markers were robustly upregulated in skeletal muscle following the FOXOdn transfections, but not after control transfection., Conclusions: FOXO1 and FOXO3 regulate glucose metabolism and markers of inflammation in skeletal muscle, implicating transcriptional control governing "immunometabolic" dynamics., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

MicroRNAs have emerged as important regulators of glucose and lipid metabolism in several tissues; however, their role in skeletal muscle remains poorly characterized. We determined the effects of the miR-29 family on glucose metabolism, lipid metabolism, and insulin responsiveness in skeletal muscle. We provide evidence that miR-29a and miR-29c are increased in skeletal muscle from patients with type 2 diabetes and are decreased following endurance training in healthy young men and in rats. In primary human skeletal muscle cells, inhibition and overexpression strategies demonstrate that miR-29a and miR-29c regulate glucose uptake and insulin-stimulated glucose metabolism. We identified that miR-29 overexpression attenuates insulin signaling and expression of insulin receptor substrate 1 and phosphoinositide 3-kinase. Moreover, miR-29 overexpression reduces hexokinase 2 expression and activity. Conversely, overexpression of miR-29 by electroporation of mouse tibialis anterior muscle decreased glucose uptake and glycogen content in vivo, concomitant with decreased abundance of GLUT4. We also provide evidence that fatty acid oxidation is negatively regulated by miR-29 overexpression, potentially through the regulation of peroxisome proliferator-activated receptor γ coactivator-1α expression. Collectively, we reveal that miR-29 acts as an important regulator of insulin-stimulated glucose metabolism and lipid oxidation, with relevance to human physiology and type 2 diabetes., (© 2017 by the American Diabetes Association.)

DNA methylation is altered by environmental factors. We hypothesized that DNA methylation is altered in skeletal muscle in response to either insulin or glucose exposure. We performed a genome-wide DNA methylation analysis in muscle from healthy men before and after insulin exposure. DNA methylation of selected genes was determined in muscle from healthy men and men with type 2 diabetes before and after a glucose tolerance test. Insulin altered DNA methylation in the 3' untranslated region of the calcium pump ATP2A3 gene. Insulin increased DNA methylation in the gene body of DAPK3 , a gene involved in cell proliferation, apoptosis, and autophagy. DAPK3 methylation was reduced in patients with type 2 diabetes. Carbohydrate ingestion reduced DAPK3 DNA methylation in healthy men and men with type 2 diabetes, suggesting glucose may play a role. Supporting this, DAPK3 DNA methylation was inversely correlated with the 2-h glucose concentration. Whereas glucose incorporation to glycogen was unaltered by small interfering RNA against DAPK3 , palmitate oxidation was increased. In conclusion, insulin and glucose exposure acutely alter the DNA methylation profile of skeletal muscle, indicating that DNA methylation constitutes a rapidly adaptive epigenetic mark. Furthermore, insulin and glucose modulate DAPK3 DNA methylation in a reciprocal manner, suggesting a feedback loop in the control of the epigenome., (© 2017 by the American Diabetes Association.)

MicroRNAs have been described as important regulators of skeletal muscle differentiation and development, but the role of microRNAs in glucose and lipid metabolism is less well established. Here we review the microRNAs involved in insulin resistance and glucose metabolism, as well as microRNAs regulating lipid metabolism and mitochondrial functions in skeletal muscle, with an emphasis on metabolic disorders such as type 2 diabetes and the adaptive response to exercise training. Finally, we raise some methodological considerations for studying microRNAs, as well as challenges investigators may face when elucidating the direct role of microRNAs in the regulation of glucose and lipid metabolism in skeletal muscle. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez., (Copyright © 2016 Elsevier B.V. All rights reserved.)

miRNAs regulate protein abundance and control diverse aspects of cellular processes and biological functions in metabolic diseases, such as obesity and type 2 diabetes (T2D). Let (lethal)-7 miRNAs specifically targets genes associated with T2D and have been implicated in the regulation of peripheral glucose metabolism, yet the direct regulators of let-7 miRNA expression are unknown. In the present study, we report on a putative promoter region for the let-7a-1, let-7f-1 and let-7d gene cluster on chromosome 9 and characterize the promoter activity of this novel area. We show that promoter activity and let-7 miRNA expression is dynamically regulated in response to different factors including serum, glucose, tumour necrosis factor (TNF)-α and caffeine. These findings will contribute to understanding the interaction between precise promoter elements to control the transcription and translation of let-7 miRNA genes., (© 2015 Authors; published by Portland Press Limited.)

Background: Roux-en-Y gastric bypass (RYGB) surgery rapidly increases whole body insulin sensitivity, with changes in several organs including skeletal muscle. Objectives were to determine whether improvements in insulin action in skeletal muscle may occur directly at the level of the myocyte or secondarily from changes in systemic factors associated with weight loss. Myotubes were derived before and after RYGB surgery. The setting was Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden., Methods: Eight patients (body mass index (BMI) 41.8 kg/m(2); age 41 yr) underwent RYGB surgery. Before and 6 months after RYGB surgery, skeletal muscle biopsies were collected from vastus lateralis muscle. Satellite cells derived from skeletal muscle biopsies were propagated in vitro as myoblasts and differentiated into myotubes., Results: Expression of myogenic markers is increased in myoblasts derived from biopsies taken 6 months after bypass surgery, compared with their respective presurgery condition. Furthermore, glycogen synthesis, tyrosine phosphorylation of insulin receptor (IRS)-1-Tyr612 and Interleukin (IL)-8 secretion were increased, while fatty acid oxidation and circulating IL8 levels remain unaltered. Myotubes derived from muscle biopsies obtained after RYGB surgery displayed increased insulin-stimulated phosphorylation of protein kinase B (PKB)-Thr308 and proline-rich Akt substrate of 40 kDa (PRAS40)-Thr246., Conclusions: RYGB surgery is accompanied by enhanced glucose metabolism and insulin signaling, altered IL8 secretion and changes in mRNA levels and myogenic markers in cultured skeletal muscle cells. Thus, RYGB surgery involves intrinsic reprogramming of skeletal muscle to increase peripheral insulin sensitivity and glucose metabolism., (Copyright © 2015 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

Low-grade inflammation associated with type 2 diabetes (T2DM) is postulated to exacerbate insulin resistance. We report that serum levels, as well as IL-13 secreted from cultured skeletal muscle, are reduced in T2DM vs. normal glucose-tolerant (NGT) subjects. IL-13 exposure increases skeletal muscle glucose uptake, oxidation, and glycogen synthesis via an Akt-dependent mechanism. Expression of microRNA let-7a and let-7d, which are direct translational repressors of the IL-13 gene, was increased in skeletal muscle from T2DM patients. Overexpression of let-7a and let-7d in cultured myotubes reduced IL-13 secretion. Furthermore, basal glycogen synthesis was reduced in cultured myotubes exposed to an IL-13-neutralizing antibody. Thus, IL-13 is synthesized and released by skeletal muscle through a mechanism involving let-7, and this effect is attenuated in skeletal muscle from insulin-resistant T2DM patients. In conclusion, IL-13 plays an autocrine role in skeletal muscle to increase glucose uptake and metabolism, suggesting a role in glucose homeostasis in metabolic disease.

Interleukin-6 (IL-6) has a dual role in modulating insulin sensitivity, with evidence for this cytokine as both an enhancer and inhibitor of insulin action. We determined the effect of IL-6 exposure on glucose and lipid metabolism in cultured myotubes established from people with normal glucose tolerance or type 2 diabetes. Acute IL-6 exposure increased glycogen synthesis, glucose uptake, and signal transducer and activator of transcription 3 (STAT3) phosphorylation in cultured myotubes from normal glucose tolerant subjects. However, in type 2 diabetic patients, IL-6 was without effect on glucose metabolism and STAT3 signaling, concomitant with increased suppressor of cytokine signaling 3 (SOCS3) expression. IL-6 increased fatty acid oxidation in myotubes from type 2 diabetic and normal glucose tolerant subjects. Expression of IL-6, IL-6 receptor (IL-6R), or glycoprotein 130, as well as IL-6 secretion, was unaltered between cultured myotubes from normal glucose tolerant or type 2 diabetic subjects. Circulating serum IL-6 concentration was unaltered between normal glucose tolerant and type 2 diabetic subjects. In summary, skeletal muscle cells from type 2 diabetic patients display selective IL-6 resistance for glucose rather than lipid metabolism. In conclusion, IL-6 appears to play a differential role in regulating metabolism in type 2 diabetic patients compared with normal glucose tolerant subjects.

The incidence and prevalence of type 1, insulin dependent, diabetes is increasing worldwide, spurring efforts to develop and improve therapeutic modalities to improve clinical outcomes for patients. Patients with type 1 diabetes are absolutely dependent on exogenous insulin replacement. Despite advances with novel rapid-acting and intermediate-acting insulin analogues, the net result of exogenous delivery is non-physiologic with respect to both timing and the circulating insulin concentrations achieved. This leads to periods of hyperglycaemia and hypoglycaemia, both of which contribute negatively to overall clinical outcome. Thus, better understanding of optimal insulin regimens is of clinical relevance for patients with type 1 diabetes.

Objective: Endothelin (ET)-1 is a vasoconstrictor and proinflammatory peptide that may interfere with glucose uptake. Our objective was to investigate whether exogenous ET-1 affects glucose uptake in the forearm of individuals with insulin resistance and in cultured human skeletal muscle cells., Research Design and Methods: Nine male subjects (aged 61 ± 3 years) with insulin resistance (M value <5.5 mg/kg/min or a homeostasis model assessment of insulin resistance index >2.5) participated in a protocol using saline infusion followed by ET-1 infusion (20 pmol/min) for 2 h into the brachial artery. Forearm blood flow (FBF), endothelium-dependent vasodilatation, and endothelium-independent vasodilatation were assessed. Molecular signaling and glucose uptake were determined in cultured skeletal muscle cells., Results: ET-1 decreased forearm glucose uptake (FGU) by 39% (P < 0.05) after the 2-h infusion. ET-1 reduced basal FBF by 36% after the 2-h infusion (P < 0.05) and impaired both endothelium-dependent vasodilatation (P < 0.01) and endothelium-independent vasodilatation (P < 0.05). ET(A) and ET(B) receptor expression was detected on cultured skeletal muscle cells. One-hour ET-1 incubation increased glucose uptake in cells from healthy control subjects but not from type 2 diabetic patients. Incubation with ET-1 for 24 h reduced glucose uptake in cells from healthy subjects. ET-1 decreased insulin-stimulated Akt phosphorylation and increased phosphorylation of insulin receptor substrate-1 serine 636., Conclusions: ET-1 not only induces vascular dysfunction but also acutely impairs FGU in individuals with insulin resistance and in skeletal muscle cells from type 2 diabetic subjects. These findings suggest that ET-1 may contribute to the development of insulin resistance in skeletal muscle in humans.

Skeletal muscle plays an important role in the regulation of energy homeostasis; therefore, the ability of skeletal muscle to adapt and alter metabolic gene expression in response to changes in physiological demands is critical for energy balance. Individuals with cervical spinal cord lesions are characterized by tetraplegia, impaired thermoregulation, and altered skeletal muscle morphology. We characterized skeletal muscle metabolic gene expression patterns, as well as protein content, in these individuals to assess the impact of spinal cord injury on critical determinants of skeletal muscle metabolism. Our results demonstrate that mRNA levels and protein expression of skeletal muscle genes essential for glucose storage are reduced, whereas expression of glycolytic genes is reciprocally increased in individuals with spinal cord injury. Furthermore, expression of genes essential for lipid oxidation is coordinately reduced in spinal cord injured subjects, consistent with a marked reduction of mitochondrial proteins. Thus spinal cord injury resulted in a profound and tightly coordinated change in skeletal muscle metabolic gene expression program that is associated with the aberrant metabolic features of the tissue.

Context: Expression of the vasoconstrictor and proinflammatory peptide endothelin (ET)-1 is increased in insulin-resistant (IR) subjects., Objective: The aim of this study was to investigate whether ET-1 regulates skeletal muscle glucose uptake in IR subjects in vivo and in cultured human skeletal muscle cells., Design and Participants: Eleven subjects participated in three protocols using brachial artery infusion of: A) BQ123 (10 nmol/min) and BQ788 (10 nmol/min) (ET(A) and ET(B) receptor antagonist, respectively), followed by coinfusion with insulin (0.05 mU/kg/min); B) insulin alone; and C) insulin followed by coinfusion with ET-1 (20 pmol/min)., Main Outcome Measures: Forearm blood flow (FBF) and forearm glucose uptake (FGU) were determined. Glucose uptake and molecular signaling were determined in cultured skeletal muscle cells., Results: ET(A)/ET(B) receptor blockade increased FGU by 63% (P < 0.05). Coadministration of insulin caused a further 2-fold increase in FGU (P < 0.001). ET(A)/ET(B) receptor blockade combined with insulin resulted in greater FGU than insulin infusion alone (P < 0.005). ET(A)/ET(B) receptor blockade increased FBF by 30% (P < 0.05), with a further 16% increase (P < 0.01) during insulin coinfusion. ET-1 decreased basal FBF by 35% without affecting FGU. ET-1 impaired basal and insulin-stimulated glucose uptake in cultured muscle cells (P < 0.01) via an effect that was prevented by ET(A)/ET(B) receptor blockade., Conclusion: ET(A)/ET(B) receptor blockade enhances basal and insulin-stimulated glucose uptake in IR subjects. ET-1 directly impairs glucose uptake in skeletal muscle cells via a receptor-dependent mechanism. These data suggest that ET-1 regulates glucose metabolism via receptor-dependent mechanisms in IR subjects.

Objective: Malonyl coenzyme A (CoA) decarboxylase (MCD) is a key enzyme responsible for malonyl-CoA turnover and functions in the control of the balance between lipid and glucose metabolism. We utilized RNA interference (siRNA)-based gene silencing to determine the direct role of MCD on metabolic responses in primary human skeletal muscle., Research Design and Methods: We used siRNA to silence MCD gene expression in cultured human myotubes from healthy volunteers (seven male and seven female) with no known metabolic disorders. Thereafter, we determined lipid and glucose metabolism and signal transduction under basal and insulin-stimulated conditions., Results: RNA interference-based silencing of MCD expression (75% reduction) increased malonyl-CoA levels twofold and shifted substrate utilization from lipid to glucose oxidation. RNA interference-based depletion of MCD reduced basal palmitate oxidation. In parallel with this reduction, palmitate uptake was decreased under basal (40%) and insulin-stimulated (49%) conditions compared with myotubes transfected with a scrambled sequence. MCD silencing increased basal and insulin-mediated glucose oxidation 1.4- and 2.6-fold, respectively, compared with myotubes transfected with a scrambled sequence. In addition, glucose transport and cell-surface GLUT4 content was increased. In contrast, insulin action on IRS-1 tyrosine phosphorylation, tyrosine-associated phosphatidylinositol (PI) 3-kinase activity, Akt, and glycogen synthase kinase (GSK) phosphorylation was unaltered between myotubes transfected with siRNA against MCD versus a scrambled sequence., Conclusions: These results provide evidence that MCD silencing suppresses lipid uptake and enhances glucose uptake in primary human myotubes. In conclusion, MCD expression plays a key reciprocal role in the balance between lipid and glucose metabolism.

The peroxisome proliferator-activated receptor (PPAR)delta has been implicated in the regulation of lipid metabolism in skeletal muscle. Furthermore, activation of PPARdelta has been proposed to improve insulin sensitivity and reduce glucose levels in animal models of type 2 diabetes. We recently demonstrated that the PPARdelta agonist GW501516 activates AMP-activated protein kinase (AMPK) and stimulates glucose uptake in skeletal muscle. However, the underlying mechanism remains to be clearly identified. In this study, we first confirmed that incubation of primary cultured human muscle cells with GW501516 induced AMPK phosphorylation and increased fatty acid transport and oxidation and glucose uptake. Using small interfering RNA, we have demonstrated that PPARdelta expression is required for the effect of GW501516 on the intracellular accumulation of fatty acids. Furthermore, we have shown that the subsequent increase in fatty acid oxidation induced by GW501516 is dependent on both PPARdelta and AMPK. Concomitant with these metabolic changes, we provide evidence that GW501516 increases the expression of key genes involved in lipid metabolism (FABP3, CPT1, and PDK4) by a PPARdelta-dependent mechanism. Finally, we have also demonstrated that the GW501516-mediated increase in glucose uptake requires AMPK but not PPARdelta. In conclusion, the PPARdelta agonist GW501516 promotes changes in lipid/glucose metabolism and gene expression in human skeletal muscle cells by PPARdelta- and AMPK-dependent and -independent mechanisms.

Interleukin (IL)-6 is a proinflammatory cytokine shown to modify insulin sensitivity. Elevated plasma levels of IL-6 are observed in insulin-resistant states. Interestingly, plasma IL-6 levels also increase during exercise, with skeletal muscle being the predominant source. Thus, IL-6 has also been suggested to promote insulin-mediated glucose utilization. In this study, we determined the direct effects of IL-6 on glucose transport and signal transduction in human skeletal muscle. Skeletal muscle strips were prepared from vastus lateralis biopsies obtained from 22 healthy men. Muscle strips were incubated with or without IL-6 (120 ng/ml). We found that IL-6 increased glucose transport in human skeletal muscle 1.3-fold (P < 0.05). A 30-min pre-exposure to IL-6 did not affect insulin-stimulated glucose transport. IL-6 also increased skeletal muscle glucose incorporation into glycogen, as well as glucose oxidation (1.5- and 1.3-fold, respectively; P < 0.05). IL-6 increased phosphorylation of STAT3 (signal transducer and activator of transcription 3; P < 0.05), AMP-activated protein kinase (P = 0.063), and p38 mitogen-activated protein kinase (P < 0.05) and reduced phosphorylation of S6 ribosomal protein (P < 0.05). In contrast, phosphorylation of protein kinase B/Akt, AS160 (Akt substrate of 160 kDa), and GSK3alpha/beta (glycogen synthase kinase 3alpha/beta) as well as insulin receptor substrate 1-associated phosphatidylinositol 3-kinase activity remained unaltered. In conclusion, acute IL-6 exposure increases glucose metabolism in resting human skeletal muscle. Insulin-stimulated glucose transport and insulin signaling were unchanged after IL-6 exposure.

We identified signaling pathways by which IL-6 regulates skeletal muscle differentiation and metabolism. Primary human skeletal muscle cells were exposed to IL-6 (25 ng/ml either acutely or for several days), and small interfering RNA gene silencing was applied to measure glucose and fat metabolism. Chronic IL-6 exposure increased myotube fusion and formation and the mRNA expression of glucose transporter 4, peroxisome proliferator activated receptor (PPAR)alpha, PPARdelta, PPARgamma, PPARgamma coactivator 1, glycogen synthase, myocyte enhancer factor 2D, uncoupling protein 2, fatty acid transporter 4, and IL-6 (P < 0.05), whereas glucose transporter 1, CCAAT/enhancer-binding protein-alpha, and uncoupling protein 3 were decreased. IL-6 increased glucose incorporation into glycogen, glucose uptake, lactate production, and fatty acid uptake and oxidation, concomitant with increased phosphorylation of AMP-activated protein kinase (AMPK), signal transducer and activator of transcription 3, and ERK1/2. IL-6 also increased phosphatidylinositol (PI) 3-kinase activity (450%; P < 0.05), which was blunted by subsequent insulin-stimulation (P < 0.05). IL-6-mediated glucose metabolism was suppressed, but lipid metabolism was unaltered, by inhibition of PI3-kinase with LY294002. The small interfering RNA-directed depletion of AMPK reduced IL-6-mediated fatty acid oxidation and palmitate uptake but did not reduce glycogen synthesis. In summary, IL-6 increases glycogen synthesis via a PI3-kinase-dependent mechanism and enhances lipid oxidation via an AMPK-dependent mechanism in skeletal muscle. Thus, IL-6 directly promotes skeletal muscle differentiation and regulates muscle substrate utilization, promoting glycogen storage and lipid oxidation.

Activators of peroxisome proliferator-activated receptor (PPAR)gamma have been studied intensively for their insulin-sensitizing properties and antidiabetic effects. Recently, a specific PPARdelta activator (GW501516) was reported to attenuate plasma glucose and insulin levels when administered to genetically obese ob/ob mice. This study was performed to determine whether specific activation of PPARdelta has direct effects on insulin action in skeletal muscle. Specific activation of PPARdelta using two pharmacological agonists (GW501516 and GW0742) increased glucose uptake independently of insulin in differentiated C2C12 myotubes. In cultured primary human skeletal myotubes, GW501516 increased glucose uptake independently of insulin and enhanced subsequent insulin stimulation. PPARdelta agonists increased the respective phosphorylation and expression of AMP-activated protein kinase 1.9-fold (P < 0.05) and 1.8-fold (P < 0.05), of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (MAPK) 2.2-fold (P < 0.05) and 1.7-fold (P < 0.05), and of p38 MAPK 1.2-fold (P < 0.05) and 1.4-fold (P < 0.05). Basal and insulin-stimulated protein kinase B/Akt was unaltered in cells preexposed to PPARdelta agonists. Preincubation of myotubes with the p38 MAPK inhibitor SB203580 reduced insulin- and PPARdelta-mediated increase in glucose uptake, whereas the mitogen-activated protein kinase kinase inhibitor PD98059 was without effect. PPARdelta agonists reduced mRNA expression of PPARdelta, sterol regulatory element binding protein (SREBP)-1a, and SREBP-1c (P < 0.05). In contrast, mRNA expression of PPARgamma, PPARgamma coactivator 1, GLUT1, and GLUT4 was unaltered. Our results provide evidence to suggest that PPARdelta agonists increase glucose metabolism and promote gene regulatory responses in cultured human skeletal muscle. Moreover, we provide biological validation of PPARdelta as a potential target for antidiabetic therapy.

Exposing isolated rat skeletal muscle to 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, a pharmacological activator of AMP-activated protein kinase (AMPK)] plus serum leads to a subsequent increase in insulin-stimulated glucose transport (Fisher JS, Gao J, Han DH, Holloszy JO, and Nolte LA. Am J Physiol Endocrinol Metab 282: E18-E23, 2002). Our goal was to determine whether preincubation of primary human skeletal muscle cells with human serum and AICAR (Serum+AICAR) would also induce a subsequent elevation in insulin-stimulated glucose uptake. Cells were preincubated for 1 h under 4 conditions: 1) without AICAR or serum (Control), 2) with serum, 3) with AICAR, or 4) with Serum+AICAR. Some cells were then collected for immunoblot analysis to assess phosphorylation of AMPK (pAMPK) and its substrate acetyl-CoA carboxylase (ACC). Other cells were incubated for an additional 4 h without AICAR or serum and then used to measure basal or insulin-stimulated 2-deoxyglucose (2-DG) uptake. Level of pAMPK was increased (P < 0.01) for myotubes exposed to Serum+AICAR vs. all other groups. Phosphorylated ACC (pACC) levels were higher for both Serum+AICAR (P < 0.05) and AICAR (P < 0.05) vs. Control and Serum groups. Basal (P < 0.05) and 1.2 nM insulin-stimulated (P < 0.005) 2-DG uptake was higher for Serum vs. all other preincubation conditions at equal insulin concentration. Regardless of insulin concentration (0, 1.2, or 18 nM), 2-DG was unaltered in cells preincubated with Serum+AICAR vs. Control cells. In contrast to results with isolated rat skeletal muscle, increasing the pAMPK and pACC in human myocytes via preincubation with Serum+AICAR was insufficient to lead to a subsequent enhancement in insulin-stimulated glucose uptake.

The molecular signaling mechanisms by which insulin leads to increased glucose transport and metabolism and gene expression are not completely elucidated. We have characterized the nature of insulin signaling defects in skeletal muscle from Type 2 diabetic patients. Insulin receptor substrate (IRS-1) phosphorylation, phosphatidylinositol (PI) 3-kinase activity, and glucose transport activity are impaired as a consequence of functional defects, whereas insulin receptor tyrosine phosphorylation, mitogen-activated protein kinase (MAPK) phosphorylation, and glycogen synthase activity are normal. Using biotinylated photoaffinity labeling, we have shown that reduced cell surface GLUT4 levels can explain glucose transport defects in skeletal muscle from Type 2 diabetic patients under insulin-stimulated conditions. Current work is focused on mechanisms behind insulin-dependent and insulin-independent regulation of glucose uptake. We have recently determined the independent effects of insulin and hypoxia/AICAR exposure on glucose transport and cell surface GLUT4 content in skeletal muscle from nondiabetic and Type 2 diabetic subjects. Hypoxia and AICAR increase glucose transport via an insulin-independent mechanism involving activation of 5'-AMP-activated kinase (AMPK). AMPK signaling is intact, because 5-aminoimidazole-4-carboxamide 1-beta-D-ribonucleoside (AICAR) increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation to a similar extent in Type 2 diabetic and nondiabetic subjects. However, AICAR responses on glucose uptake were impaired. Our studies highlight important AMPK-dependent and independent pathways in the regulation of GLUT4 and glucose transport activity in insulin resistant skeletal muscle. Understanding signaling mechanisms to downstream metabolic responses may provide valuable clues to a future therapy for Type 2 diabetes.

Using RNA interference (RNAi), we specifically down-regulate protein expression in differentiated human skeletal myotube cultures. Serum stimulation of myotubes increases glucose uptake. Using a sensitive photolabeling technique, we demonstrate that this increase in glucose uptake is accompanied by increased cell-surface content of glucose transporter (GLUT) 1. Using RNAi, we specifically reduce GLUT1 mRNA and protein expression, leading to inhibition of serum-mediated increase in glucose transport. Thus, we demonstrate the utility of RNAi in a primary human differentiated cell system, and apply this methodology to demonstrate that serum-mediated increase in glucose transport in human skeletal muscle cells is dependent on GLUT1.

Objective Epigenetic modifications contribute to the etiology of type 2 diabetes. Method We performed genome-wide methylome and transcriptome analysis in liver from severely obese men with or without type 2 diabetes and non-obese men to discover aberrant pathways underlying the development of insulin resistance. Results were validated by pyrosequencing. Result We identified hypomethylation of genes involved in hepatic glycolysis and insulin resistance, concomitant with increased mRNA expression and protein levels. Pyrosequencing revealed the CpG-site within ATF-motifs was hypomethylated in four of these genes in liver of severely obese non-diabetic and type 2 diabetic patients, suggesting epigenetic regulation of transcription by altered ATF-DNA binding. Conclusion Severely obese non-diabetic and type 2 diabetic patients have distinct alterations in the hepatic methylome and transcriptome, with hypomethylation of several genes controlling glucose metabolism within the ATF-motif regulatory site. Obesity appears to shift the epigenetic program of the liver towards increased glycolysis and lipogenesis, which may exacerbate the development of insulin resistance. [ABSTRACT FROM AUTHOR]

The article presents the results of a study on the role of fibroblast growth factor (FGF) 21 in metabolism of glucose in skeletal muscle of type 2 diabetics and overweight patients. The level of plasma FGF21 rose in diabetics and correlated with fasting insulin and body mass index (BMI). The direct effects of FGF21 on skeletal muscle glucose uptake are noted. It is also indicated that the level of serum FGF21 concentration was higher in diabetics in the highest tertile of fasting insulin and BMI.

The metabolic property of skeletal muscle is highly malleable and adapts to various physiological demands by shifting energy-substrate metabolism. Skeletal muscle metabolism has a significant impact on whole-body metabolism and substrate utilization. Glucose and lipids are the main oxidative fuel substrates in skeletal muscle, and their utilization is coordinated by complex regulatory mechanisms. In people with Type 2 diabetes, glucose uptake and lipid oxidation in skeletal muscle are impaired. These metabolic defects are coupled to impaired insulin signaling. Exercise increases glucose uptake and lipid oxidation by an insulin-independent mechanism. The AMP-activated protein kinase (AMPK) cascade is activated in response to metabolic stress and has therefore been implicated in the regulation of exercise-induced metabolic and gene regulatory responses. AMPK is a heterotrimeric complex composed of a catalytic α, and regulatory β and γ subunits. Selective regulation of AMPK in skeletal muscle may be chieved by targeting α1/β2/γ3 heterotrimeric complexes. Activation of AMPK enhances LUT4 translocation of glucose uptake in skeletal muscle from Type 2 diabetic patients and animal models of the disease by an insulin-independent mechanism. Transgenic overexpression of mutated forms of the AMPK γ3 subunit provide evidence that activation of AMPK promotes lipid oxidation and prevents the development of skeletal muscle insulin resistance. Thus, AMPK provides a molecular entry point into novel regulatory pathways to enhance lipid and glucose metabolism in an effort to prevent and treat skeletal muscle insulin resistance associated with Type 2 diabetes. [ABSTRACT FROM AUTHOR]

Investigates the activation of mitogen-activated protein kinase (MAPK) signaling cascades. Determination of the function of an elevation in the extracellular glucose concentration in skeletal muscle; Effect of high glucose on signal transduction to signal transducer; Effects of elevated glucose levels on extracellular signal-regulated protein kinase1/2 and p38 MAPK.

To determine whether defects in the insulin signal transduction pathway to glucose transport occur in a muscle fiber type-specific manner, post-receptor insulin-signaling events were assessed in oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skeletal muscle from Wistar or diabetic GK rats. In soleus muscle from GK rats, maximal insulin-stimulated (120 nmol/l) glucose transport was significantly decreased, compared with that of Wistar rats. In EDL muscle from GK rats, maximal insulin-stimulated glucose transport was normal, while the submaximal response was reduced compared with that of Wistar rats. We next treated diabetic GK rats with phlorizin for 4 weeks to determine whether restoration of glycemia would lead to improved insulin signal transduction. Phlorizin treatment of GK rats resulted in full restoration of insulin-stimulated glucose transport in soleus and EDL muscle. In soleus muscle from GK rats, submaximal and maximal insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity were markedly reduced, compared with that of Wistar rats, but only submaximal insulin-stimulated PI 3-kinase was restored after phlorizin treatment. In EDL muscle, insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI-3 kinase were not altered between GK and Wistar rats. Maximal insulin-stimulated Akt (protein kinase B) kinase activity is decreased in soleus muscle from GK rats and restored upon normalization of glycemia (Krook et al., Diabetes 46:2100-2114, 1997). Here, we show that in EDL muscle from GK rats, maximal insulin-stimulated Akt kinase activity is also impaired and restored to Wistar rat levels after phlorizin treatment. In conclusion, functional defects in IRS-1 and PI 3-kinase in skeletal muscle from diabetic GK rats are fiber-type-specific, with alterations observed in oxidative, but not glycolytic, muscle. Furthermore, regardless of muscle fiber type, downstream steps to PI 3-kinase (i.e., Akt and glucose transport) are sensitive to changes in the level of glycemia. [ABSTRACT FROM AUTHOR]