Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment

  Udgivelsesdato: 2008-FebOBJECTIVE: Insulin resistance in skeletal muscle is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the molecular mechanisms underlying skeletal muscle insulin resistance and the insulin-sensitizing effect of thiazolidinediones in PCOS in vivo are less well characterized. RESEARCH DESIGN AND METHODS: We determined molecular mediators of insulin signaling to glucose transport in skeletal muscle biopsies of 24 PCOS patients and 14 matched control subjects metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry, and we examined the effect of 16 weeks of treatment with pioglitazone in PCOS patients. RESULTS: Impaired insulin-mediated total (R(d)) oxidative and nonoxidative glucose disposal (NOGD) was paralleled by reduced insulin-stimulated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation in muscle of PCOS patients. Akt phosphorylation at Ser473 and Thr308 correlated positively with R(d) and NOGD in the insulin-stimulated state. Serum free testosterone was inversely related to insulin-stimulated R(d) and NOGD in PCOS. Importantly, the pioglitazone-mediated improvement in insulin-stimulated glucose metabolism, which did not fully reach normal levels, was accompanied by normalization of insulin-mediated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation. AMPK activity and phosphorylation were similar in the two groups and did not respond to pioglitazone in PCOS patients. CONCLUSIONS: Impaired insulin signaling through Akt and AS160 in part explains insulin resistance at the molecular level in skeletal muscle in PCOS, and the ability of pioglitazone to enhance insulin sensitivity involves improved signaling through Akt and AS160. Moreover, our data provide correlative evidence that hyperandrogenism in PCOS may contribute to insulin resistanc

Expression of fibroblast growth factor-21 in muscle is associated with lipodystrophy, insulin resistance and lipid disturbances in patients with HIV

BACKGROUND: Fibroblast growth factor (FGF)-21 is a novel regulator of glucose and lipid metabolism. Recently, increased FGF-21 mRNA expression in muscle was found in patients with type 2 diabetes, but the role for FGF-21 in muscle is not well understood. Patients with HIV-infection and lipodystrophy are characterised by various degree of lipid-driven insulin resistance. We hypothesized that muscle FGF-21 mRNA would be altered in HIV patients with lipodystrophy. DESIGN: Twenty-five HIV-infected men with lipodystrophy (LD) and 15 age-matched healthy controls, received an oral glucose tolerance test and a euglycemic-hyperinsulinemic clamp (50 mU/m2/min) combined with 6,6-H2 glucose infusion. Muscle biopsies were obtained and FGF-21 mRNA and glycogen synthase (GS) activity were measured. RESULTS: Subjects with HIV were insulin resistant compared with non-HIV subjects. Compared to controls, HIV subjects demonstrated a twofold increase of plasma FGF-21 from 70.4±56.8 pg/ml vs 109.1±71.8 pg/ml, respectively (p = 0.04) and an eight-fold increase in muscular FGF-21 mRNA expression (p = 0.001). Muscle FGF-21 mRNA correlated inversely with the rate of disappearance of glucose during insulin clamp (r = -0.54, p = 0.0009), and the GS fractional velocity in muscle (r = -0.39, p = 0.03), and directly with fasting insulin (r = 0.50, p = 0.0022), HOMA-IR (r = 0.47, p = 0.004), triglycerides (r = 0.60. P = 0.0001), waist-to-hip ratio (r = 0.51, p = 0.0001) and limb fat mass (-0.46, p = 0.004), but not to plasma FGF-21. CONCLUSION: FGF-21 mRNA is increased in skeletal muscle in HIV patients and correlates to whole-body (primarily reflecting muscle) insulin resistance, but not to plasma FGF-21. Those findings add to the evidence that FGF-21 is a myokine and may suggest that muscle FGF-21 is working in a local manner

PL - 026 Mismatch between skeletal muscle glucose delivery, interstitial concentration and membrane permeability may limit insulin sensitivity after exercise

Objective The relationship between skeletal muscle perfusion, interstitial glucose concentration and sarcolemmal permeability to glucose in exercise-induced increases in muscle insulin sensitivity is not well established. A single bout of exercise increases skeletal muscle insulin sensitivity through coordinated increases in insulin-stimulated microvascular perfusion and insulin signalling  Reducing leg and muscle microvascular blood flow with local nitric oxide synthase (NOS) inhibition during a hyperinsulinaemic euglycaemic clamp reduces leg glucose uptake in a previously exercised, but not in a contralateral non-exercised leg, without affecting insulin signalling in either leg  (Sjoberg et al. 2017). Therefore, it is possible that the reduction in muscle perfusion decreases muscle interstitial glucose concentration to a point that limits skeletal muscle insulin-stimulated glucose uptake following exercise. We examined this using microdialysis of vastus lateralis muscle. Methods Ten healthy males (Age: 27±1 yr., Weight: 77.7±2.3 kg, BMI 23.9±0.5, VO2 peak: 50.7±1.5 ml·kg-1·min-1) performed 60 min of 1-legged knee extensor exercise at 80% of 1-legged peak work load with three 5 min intervals at 100% 1-legged peak work load. Participants then rested for 4 hours and catheters were inserted into the femoral artery and vein of both legs for subsequent measurement of leg glucose uptake and for femoral artery infusion of the NOS inhibitor NG-monomethyl L-arginine acetate (L-NMMA) and the vasodilator ATP. Catheters were also placed in antecubital veins for infusion of insulin and glucose. Three microdialysis catheters, with a semi-permeable membrane the length of 30 mm and a molecular cut-off at 20,000 dalton, were inserted into the vastus lateral muscle of both legs. Glucose and D-[6-3H(N)]glucose were added to the perfusate. Four hours after discontinuing the exercise a 225 minute euglycaemic hyperinsulinaemic clamp was initiated (insulin infusion 1.4 mU-1kg-1min). Ninety min into the clamp L-NMMA was infused at a constant rate (0.4 mg·kg-1 leg mass·min-1) into both femoral arteries for 45 min. The insulin infusion was maintained for another 90 min and during the last 45 min ATP (0.3 μmol∙ml-1) was infused locally into both femoral arteries at a rate of 200-350 μl∙min-1 to obtain a leg blood flow that was double the blood flow during insulin only infusion. A second control protocol was undertaken that was identical in regards to exercise and recovery but no insulin, L-NMMA or ATP was infused. Results During the clamp leg glucose uptake and leg blood flow were higher (P<0.05) in the previously exercised than the control leg whereas the interstitial glucose concentration decreased to lower (P<0.05) values in the exercised (~3.1mM) than the control (~4.8mM) leg. Estimated sarcolemmal glucose permeability was twice as high (P<0.05) in the exercised compared with the rested leg. The NOS inhibitor L-NMMA decreased LBF in both legs and interstitial glucose concentration dropped to ~2.3 mM in the exercised but only to ~3.7 mM in non-exercised muscle. This abrogated the augmented effect of insulin on LGU in the exercised leg while apparent sarcolemmal permeability to glucose remained unchanged with L-NMMA in both legs. Doubling leg blood flow by local infusion of ATP increased leg glucose uptake in both legs without any major change in interstitial glucose concentration or sarcolemmal permeability to glucose. Conclusions These findings suggest that during flow restriction due to L-NMMA, the interstitial glucose concentration becomes limiting for leg glucose uptake in exercised but not in non-exercised muscle. Therefore, the vasodilatory effect of insulin is an important component of the increased insulin sensitivity to stimulate glucose uptake following exercise by limiting the drop in the interstitial glucose concentration that occurs due to the increased sarcolemmal permeability to glucose. Reference Sjoberg, K. A., C. Frosig, R. Kjobsted, L. Sylow, M. Kleinert, A. C. Betik, C. S. Shaw, B. Kiens, J. F. P. Wojtaszewski, S. Rattigan, E. A. Richter, and G. K. McConell. Exercise Increases Human Skeletal Muscle Insulin Sensitivity via Coordinated Increases in Microvascular Perfusion and Molecular Signaling.  Diabetes  66: 1501-10, 2017

Inducible deletion of skeletal muscle AMPKα 1 reveals that AMPK is required for nucleotide balance but dispensable for muscle glucose uptake and fat oxidation during exercise

International audienceObjective: Current evidence for AMPK-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to chronic lack of AMPK function. In an attempt to study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice.Methods: Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated using the Cre/loxP system with the Cre driven by the human skeletal muscle actin (HSA) promotor.Results: During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed, muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole body substrate utilization as well as muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion AMPKα subunits in adult mice.Conclusions: Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise, but is dispensable for regulating muscle glucose uptake, FA oxidation and substrate utilization during exercise

Exercise-induced TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle

TBC1D1 is a Rab-GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise-induced regulation of TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all-out cycle exercise lasting either 30 s, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (∼70–230%, P < 0.005), with the greatest response observed after 20 min of cycling. Interestingly, capacity of TBC1D1 to bind 14-3-3 protein showed a similar pattern of regulation, increasing 60–250% (P < 0.001). Furthermore, recombinant 5′AMP-activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14-3-3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock-out and wild-type mice were stimulated to contract in vitro. In wild-type and α1 knock-out mice, contractions resulted in a similar ∼100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock-out mice were characterized by reduced protein content of TBC1D1 (∼50%, P < 0.001) as well as in basal and contraction-stimulated (∼60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14-3-3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle