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Aims/hypothesis Insulin-mediated glucose disposal rates (R d) are reduced in type 2 diabetic patients, a process in which intrinsic signalling defects are thought to be involved. Phosphorylation of TBC1 domain family, member 4 (TBC1D4) is at present the most distal insulin receptor signalling event linked to glucose transport. In this study, we examined insulin action on site-specific phosphorylation of TBC1D4 and the effect of exercise training on insulin action and signalling to TBC1D4 in skeletal muscle from type 2 diabetic patients. Methods During a 3 h euglycaemic-hyperinsulinaemic (80 mU min⁻¹ m⁻²) clamp, we obtained M. vastus lateralis biopsies from 13 obese type 2 diabetic and 13 obese, non-diabetic control individuals before and after 10 weeks of endurance exercise-training. Results Before training, reductions in insulin-stimulated R d, together with impaired insulin-stimulated glycogen synthase fractional velocity, Akt Thr³⁰⁸ phosphorylation and phosphorylation of TBC1D4 at Ser³¹⁸, Ser⁵⁸⁸ and Ser⁷⁵¹ were observed in skeletal muscle from diabetic patients. Interestingly, exercise-training normalised insulin-induced TBC1D4 phosphorylation in diabetic patients. This happened independently of increased TBC1D4 protein content, but exercise-training did not normalise Akt phosphorylation in diabetic patients. In both groups, training-induced improvements in insulin-stimulated R d (~20%) were associated with increased muscle protein content of Akt, TBC1D4, α2-AMP-activated kinase (AMPK), glycogen synthase, hexokinase II and GLUT4 (20-75%). Conclusions/interpretation Impaired insulin-induced site-specific TBC1D4 phosphorylation may contribute to skeletal muscle insulin resistance in type 2 diabetes. The mechanisms by which exercise-training improves insulin sensitivity in type 2 diabetes may involve augmented signalling of TBC1D4 and increased skeletal muscle content of key insulin signalling and effector proteins, e.g., Akt, TBC1D4, AMPK, glycogen synthase, GLUT4 and hexokinase II.

High fat feeding impairs skeletal muscle metabolic flexibility and induces insulin resistance, whereas exercise training exerts positive effects on substrate handling and improves insulin sensitivity. To identify the genomic mechanisms by which exercise ameliorates some of the deleterious effects of high fat feeding, we investigated the transcriptional and epigenetic response of human skeletal muscle to 9 days of a high-fat diet (HFD) alone (Sed-HFD) or in combination with resistance exercise (Ex-HFD), using genome-wide profiling of gene expression and DNA methylation. HFD markedly induced expression of immune and inflammatory genes, which was not attenuated by Ex. Conversely, Ex markedly remodelled expression of genes associated with muscle growth and structure. We detected marked DNA methylation changes following HFD alone and in combination with Ex. Among the genes that showed a significant association between DNA methylation and gene expression changes were PYGM, which was epigenetically regulated in both groups, and ANGPTL4, which was regulated only following Ex. In conclusion, while short-term Ex did not prevent a HFD-induced inflammatory response, it provoked a genomic response that may protect skeletal muscle from atrophy. These epigenetic adaptations provide mechanistic insight into the gene-specific regulation of inflammatory and metabolic processes in human skeletal muscle.

Aims/hypothesis We used oestrogen receptor-α (ERα) knockout (ERKO) and receptor-β (ERβ) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis. Methods Endogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic-hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding steroyl-CoA desaturase and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR. Results ERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose- and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemic-hyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release. Conclusions/interpretation We conclude that oestrogens, acting via ERα, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression.

Aims/hypothesis TBC1 domain family, member 4 (TBC1D4; also known as AS160) is a cellular signalling intermediate to glucose transport regulated by insulin-dependent and -independent mechanisms. Skeletal muscle insulin sensitivity is increased after acute exercise by an unknown mechanism that does not involve modulation at proximal insulin signalling intermediates. We hypothesised that signalling through TBC1D4 is involved in this effect of exercise as it is a common signalling element for insulin and exercise. Methods Insulin-regulated glucose metabolism was evaluated in 12 healthy moderately trained young men 4 h after one-legged exercise at basal and during a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis biopsies were taken before and immediately after the clamp. Results Insulin stimulation increased glucose uptake in both legs, with greater effects (~80%, p < 0.01) in the previously exercised leg. TBC1D4 phosphorylation, assessed using the phospho-AKT (protein kinase B)substrate antibody and phospho- and site-specific antibodies targeting six phosphorylation sites on TBC1D4, increased at similar degrees to insulin stimulation in the previously exercised and rested legs (p < 0.01). However, TBC1D4 phosphorylation on Ser-318, Ser-341, Ser-588 and Ser-751 was higher in the previously exercised leg, both in the absence and in the presence of insulin (p < 0.01; Ser-588, p = 0.09; observed power = 0.39). 14-3-3 binding capacity for TBC1D4 increased equally (p < 0.01) in both legs during insulin stimulation. Conclusion/interpretation We provide evidence for site-specific phosphorylation of TBC1D4 in human skeletal muscle in response to physiological hyperinsulinaemia. The data support the idea that TBC1D4 is a nexus for insulin- and exercise-responsive signals that may mediate increased insulin action after exercise.

Exercise enhances insulin-stimulated glucose transport in skeletal muscle through changes in signal transduction and gene expression. The aim of this study was to assess the impact of acute and short-term exercise training on whole-body insulin-mediated glucose disposal and signal transduction along the canonical insulin signalling cascade. A euglycaemic-hyperinsulinaemic clamp, with vastus lateralis skeletal muscle biopsies, was performed at baseline and 16 h after an acute bout of exercise and short-term exercise training (7 days) in obese non-diabetic (n=7) and obese type 2 diabetic (n=8) subjects. Insulin-mediated glucose disposal was unchanged following acute exercise in both groups. Short-term exercise training increased insulin-mediated glucose disposal in obese type 2 diabetic (p<0.05), but not in obese non-diabetic subjects. Insulin activation of (1) IRS1, (2) IRS2, (3) phosphotyrosine-associated phosphatidylinositol-3 kinase activity and (4) the substrate of phosphorylated Akt, AS160, a functional Rab GTPase activating protein important for GLUT4 (now known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) translocation, was unchanged after acute or chronic exercise in either group. GLUT4 protein content was increased in obese type 2 diabetic subjects (p<0.05), but not in obese non-diabetic subjects following chronic exercise. Exercise training increased whole-body insulin-mediated glucose disposal in obese type 2 diabetic patients. These changes were independent of functional alterations in the insulin-signalling cascade and related to increased GLUT4 protein content.

Aims/hypothesis We investigated the direct effect of a nitric oxide donor (spermine NONOate) on glucose transport in isolated human skeletal muscle and L6 skeletal muscle cells. We hypothesised that pharmacological treatment of human skeletal muscle with N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. Methods Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 µmol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction was determined. Results In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p < 0.05), concomitant with increased cGMP levels (80-fold, p < 0.001). Phosphorylation of components of the canonical insulin signalling cascade was unaltered by spermine NONOate exposure, implicating an insulin-independent signalling mechanism. Consistent with this, spermine NONOate increased AMP-activated protein kinase (AMPK)-α1-associated activity (1.7-fold, p < 0.05). In L6 muscle cells, spermine NONOate increased glucose uptake (p < 0.01) and glycogen synthesis (p < 0.001), an effect that was in addition to that of insulin. Spermine NONOate also elicited a concomitant increase in AMPK and acetyl-CoA carboxylase phosphorylation. In the presence of the guanylate cyclase inhibitor LY-83583 (10 µmol/l), spermine NONOate had no effect on glycogen synthesis and AMPK-α1 phosphorylation. Conclusions/interpretation Pharmacological treatment of skeletal muscle with spermine NONOate increases glucose transport via insulin-independent signalling pathways involving increased intracellular cGMP levels and AMPK-α1-associated activity.

Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients. A Krook , M Björnholm , D Galuska , X J Jiang , R Fahlman , M G Myers, Jr , H Wallberg-Henriksson and J R Zierath Department of Clinical Physiology, Karolinska Hospital, Stockholm, Sweden. Abstract We characterized metabolic and mitogenic signaling pathways in isolated skeletal muscle from well-matched type 2 diabetic and control subjects. Time course studies of the insulin receptor, insulin receptor substrate (IRS)-1/2, and phosphatidylinositol (PI) 3-kinase revealed that signal transduction through this pathway was engaged between 4 and 40 min. Insulin-stimulated (0.6-60 nmol/l) tyrosine phosphorylation of the insulin receptor beta-subunit, mitogen-activated protein (MAP) kinase phosphorylation, and glycogen synthase activity were not altered in type 2 diabetic subjects. In contrast, insulin-stimulated tyrosine phosphorylation of IRS-1 and anti-phosphotyrosine-associated PI 3-kinase activity were reduced 40-55% in type 2 diabetic subjects at high insulin concentrations (2.4 and 60 nmol/l, respectively). Impaired glucose transport activity was noted at all insulin concentrations (0.6-60 nmol/l). Aberrant protein expression cannot account for these insulin-signaling defects because expression of insulin receptor, IRS-1, IRS-2, MAP kinase, or glycogen synthase was similar between type 2 diabetic and control subjects. In skeletal muscle from type 2 diabetic subjects, IRS-1 phosphorylation, PI 3-kinase activity, and glucose transport activity were impaired, whereas insulin receptor tyrosine phosphorylation, MAP kinase phosphorylation, and glycogen synthase activity were normal. Impaired insulin signal transduction in skeletal muscle from type 2 diabetic patients may partly account for reduced insulin-stimulated glucose transport; however, additional defects are likely to play a role.

p38 mitogen activated protein kinase (MAPK) is generally thought to facilitate signal transduction to genomic, rather than metabolic responses. However, recent evidence implicates a role for p38 MAPK in the regulation of glucose transport; a site of insulin resistance in Type 2 diabetes. Thus we determined p38 MAPK protein expression and phosphorylation in skeletal muscle from Type 2 diabetic patients and non-diabetic subjects. In vitro effects of insulin (120 nmol/l) or AICAR (1 mmol/l) on p38 MAPK expression and phosphorylation were determined in skeletal muscle from non-diabetic (n=6) and Type 2 diabetic (n=9) subjects. p38 MAPK protein expression was similar between Type 2 diabetic patients and non-diabetic subjects. Insulin exposure increased p38 MAPK phosphorylation in non-diabetic, but not in Type 2 diabetic patients. In contrast, basal phosphorylation of p38 MAPK was increased in skeletal muscle from Type 2 diabetic patients. Insulin increases p38 MAPK phosphorylation in skeletal muscle from non-diabetic subjects, but not in Type 2 diabetic patients. However, basal p38 MAPK phosphorylation is increased in skeletal muscle from Type 2 diabetic patients. Thus, aberrant p38 MAPK signalling might contribute to the pathogenesis of insulin resistance.

Conjugated linoleic acid (CLA) isomers have a number of beneficial health effects, as shown in biomedical studies with animal models. Previously, we reported that a mixture of CLA isomers improved glucose tolerance in ZDF rats and activated peroxisome proliferator-activated receptor (PPAR)-gamma response elements in vitro. Here, our aim was to elucidate the effect(s) of specific CLA isomers on whole-body glucose tolerance, insulin action in skeletal muscle, and expression of genes important in glucose and lipid metabolism. ZDF rats were fed either a control diet (CON), one of two CLA supplemented diets (1.5% CLA) containing differing isoforms of CLA (47% c9,t11; 47.9% c10,t12, 50:50; or 91% c9,t11, c9,t11 isomers), or were pair-fed CON diet to match the intake of 50:50. The 50:50 diet reduced adiposity and improved glucose tolerance compared with all other ZDF treatments. Insulin-stimulated glucose transport and glycogen synthase activity in skeletal muscle were improved with 50:50 compared with all other treatments. Neither phosphatidlyinositol 3-kinase activity nor Akt activity in muscle was affected by treatment. Uncoupling protein 2 in muscle and adipose tissue was upregulated by c9,t11 and 50:50 compared with ZDF controls. PPAR-gamma mRNA was downregulated in liver of c9,t11 and pair-fed ZDF rats. Thus, the improved glucose tolerance in 50:50 rats is attributable to, at least in part, improved insulin action in muscle, and CLA effects cannot be explained simply by reduced food intake.