Insulin-induced membrane permeability to glucose in human muscles at rest and following exercise.

KEY POINTS: Increased insulin action is an important component of the health benefits of exercise, but its regulation is complex and not fully elucidated. Previous studies of insulin-stimulated GLUT4 translocation to the skeletal muscle membrane found insufficient increases to explain the increases in glucose uptake. By determination of leg glucose uptake and interstitial muscle glucose concentration, insulin-induced muscle membrane permeability to glucose was calculated 4 h after one-legged knee-extensor exercise during a submaximal euglycaemic-hyperinsulinaemic clamp. It was found that during submaximal insulin stimulation, muscle membrane permeability to glucose in humans increases twice as much in previously exercised vs. rested muscle and outstrips the supply of glucose, which then becomes limiting for glucose uptake. This methodology can now be employed to determine muscle membrane permeability to glucose in people with diabetes, who have reduced insulin action, and in principle can also be used to determine membrane permeability to other substrates or metabolites. ABSTRACT: Increased insulin action is an important component of the health benefits of exercise, but the regulation of insulin action in vivo is complex and not fully elucidated. Previously determined increases in skeletal muscle insulin-stimulated GLUT4 translocation are inconsistent and mostly cannot explain the increases in insulin action in humans. Here we used leg glucose uptake (LGU) and interstitial muscle glucose concentration to calculate insulin-induced muscle membrane permeability to glucose, a variable not previously possible to quantify in humans. Muscle membrane permeability to glucose, measured 4 h after one-legged knee-extensor exercise, increased ∼17-fold during a submaximal euglycaemic-hyperinsulinaemic clamp in rested muscle (R) and ∼36-fold in exercised muscle (EX). Femoral arterial infusion of NG -monomethyl l-arginine acetate or ATP decreased and increased, respectively, leg blood flow (LBF) in both legs but did not affect membrane glucose permeability. Decreasing LBF reduced interstitial glucose concentrations to ∼2 mM in the exercised but only to ∼3.5 mM in non-exercised muscle and abrogated the augmented effect of insulin on LGU in the EX leg. Increasing LBF by ATP infusion increased LGU in both legs with uptake higher in the EX leg. We conclude that it is possible to measure functional muscle membrane permeability to glucose in humans and it increases twice as much in exercised vs. rested muscle during submaximal insulin stimulation. We also show that muscle perfusion is an important regulator of muscle glucose uptake when membrane permeability to glucose is high and we show that the capillary wall can be a significant barrier for glucose transport.

Regulation of autophagy in human skeletal muscle: effects of exercise, exercise training and insulin stimulation.

KEY POINTS: Regulation of autophagy in human muscle in many aspects differs from the majority of previous reports based on studies in cell systems and rodent muscle. An acute bout of exercise and insulin stimulation reduce human muscle autophagosome content. An acute bout of exercise regulates autophagy by a local contraction-induced mechanism. Exercise training increases the capacity for formation of autophagosomes in human muscle. AMPK activation during exercise seems insufficient to regulate autophagosome content in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy-inhibiting effect of insulin. Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one-legged exercise, one-legged exercise training and subsequent insulin stimulation in exercised and non-exercised human muscle. Acute one-legged exercise decreased (P<0.01) lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3) (∼ 50%) and the LC3-II/LC3-I ratio (∼ 60%) indicating that content of autophagosomes decreases with exercise in human muscle. The decrease in LC3-II/LC3-I ratio did not correlate with activation of 5'AMP activated protein kinase (AMPK) trimer complexes in human muscle. Consistently, pharmacological AMPK activation with 5-aminoimidazole-4-carboxamide riboside (AICAR) in mouse muscle did not affect the LC3-II/LC3-I ratio. Four hours after exercise, insulin further reduced (P<0.01) the LC3-II/LC3-I ratio (∼ 80%) in muscle of the exercised and non-exercised leg in humans. This coincided with increased Ser-757 phosphorylation of Unc51 like kinase 1 (ULK1), which is suggested as a mammalian target of rapamycin complex 1 (mTORC1) target. Accordingly, inhibition of mTOR signalling in mouse muscle prevented the ability of insulin to reduce the LC3-II/LC3-I ratio. In response to 3 weeks of one-legged exercise training, the LC3-II/LC3-I ratio decreased (P<0.05) in both trained and untrained muscle and this change was largely driven by an increase in LC3-I content. Taken together, acute exercise and insulin stimulation reduce muscle autophagosome content, while exercise training may increase the capacity for formation of autophagosomes in muscle. Moreover, AMPK activation during exercise may not be sufficient to regulate autophagy in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy-inhibiting effect of insulin.

AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle.

Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK α2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK α2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK α2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK α2 KD mice. In conclusion, functional α2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related.

Insulin Sensitization Following a Single Exercise Bout Is Uncoupled to Glycogen in Human Skeletal Muscle: A Meta-analysis of 13 Single-Center Human Studies.

Exercise profoundly influences glycemic control by enhancing muscle insulin sensitivity, thus promoting glucometabolic health. While prior glycogen breakdown so far has been deemed integral for muscle insulin sensitivity to be potentiated by exercise, the mechanisms underlying this phenomenon remain enigmatic. We have combined original data from 13 of our studies that investigated insulin action in skeletal muscle either under rested conditions or following a bout of one-legged knee extensor exercise in healthy young male individuals (n = 106). Insulin-stimulated glucose uptake was potentiated and occurred substantially faster in the prior contracted muscles. In this otherwise homogenous group of individuals, a remarkable biological diversity in the glucometabolic responses to insulin is apparent both in skeletal muscle and at the whole-body level. In contrast to the prevailing concept, our analyses reveal that insulin-stimulated muscle glucose uptake and the potentiation thereof by exercise are not associated with muscle glycogen synthase activity, muscle glycogen content, or degree of glycogen utilization during the preceding exercise bout. Our data further suggest that the phenomenon of improved insulin sensitivity in prior contracted muscle is not regulated in a homeostatic feedback manner from glycogen. Instead, we put forward the idea that this phenomenon is regulated by cellular allostatic mechanisms that elevate the muscle glycogen storage set point and enhance insulin sensitivity to promote the uptake of glucose toward faster glycogen resynthesis without development of glucose overload/toxicity or feedback inhibition.

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 5AMP-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.

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

OBJECTIVE: Evidence for AMP-activated protein kinase (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 a chronic lack of AMPK function. 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 by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter. 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 and 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, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of 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.

Nitric oxide production is a proximal signaling event controlling exercise-induced mRNA expression in human skeletal muscle.

Previous studies have described the magnitude and time course by which several genes are regulated within exercising skeletal muscle. These include interleukin-6 (IL-6), interleukin-8 (IL-8), heme oxygenase-1 (HO-1), and heat shock protein-72 (HSP72), which are involved in secondary signaling and preservation of intracellular environment. However, the primary signaling mechanisms coupling contraction to transcription are unknown. We hypothesized that exercise-induced nitric oxide (NO) production is an important signaling event for IL-6, IL-8, HO-1, and HSP72 expression in muscle. Twenty healthy males participated in the study. By real-time PCR, mRNA levels for 11 genes were determined in thigh muscle biopsies obtained 1) before and after 2 h knee extensor exercise without (control) and with concomitant NO synthase inhibition (nitro-L-arginine methyl ester, L-NAME, 5 mg x kg(-1)); or 2) before and after 2 h femoral artery infusion of the NO donor nitroglycerin (NTG, 1.5 microg x kg(-1) x min(-1)). L-NAME caused marked reductions in exercise-induced expression of 4 of 11 mRNAs including IL-6, IL-8, and HO-1. IL-6 protein release from the study leg to the circulation increased in the control but not in the L-NAME trial. NTG infusion significantly augmented expression of the mRNAs attenuated by L-NAME. These findings advance the novel concept that NO production contributes to regulation of gene expression in muscle during exercise. Subsequently, we sought evidence for involvement of AMP-activated kinase or nuclear factor kappa B, but found none.

Exercise Increases Human Skeletal Muscle Insulin Sensitivity via Coordinated Increases in Microvascular Perfusion and Molecular Signaling.

Insulin resistance is a major health risk, and although exercise clearly improves skeletal muscle insulin sensitivity, the mechanisms are unclear. Here we show that initiation of a euglycemic-hyperinsulinemic clamp 4 h after single-legged exercise in humans increased microvascular perfusion (determined by contrast-enhanced ultrasound) by 65% in the exercised leg and 25% in the rested leg (P < 0.05) and that leg glucose uptake increased 50% more (P < 0.05) in the exercised leg than in the rested leg. Importantly, infusion of the nitric oxide synthase inhibitor l-NG-monomethyl-l-arginine acetate (l-NMMA) into both femoral arteries reversed the insulin-stimulated increase in microvascular perfusion in both legs and abrogated the greater glucose uptake in the exercised compared with the rested leg. Skeletal muscle phosphorylation of TBC1D4 Ser318 and Ser704 and glycogen synthase activity were greater in the exercised leg before insulin and increased similarly in both legs during the clamp, and l-NMMA had no effect on these insulin-stimulated signaling pathways. Therefore, acute exercise increases insulin sensitivity of muscle by a coordinated increase in insulin-stimulated microvascular perfusion and molecular signaling at the level of TBC1D4 and glycogen synthase in muscle. This secures improved glucose delivery on the one hand and increased ability to take up and dispose of the delivered glucose on the other hand.

Skeletal muscle insulin signaling defects downstream of phosphatidylinositol 3-kinase at the level of Akt are associated with impaired nonoxidative glucose disposal in HIV lipodystrophy.

More than 40% of HIV-infected patients on highly active antiretroviral therapy (HAART) experience fat redistribution (lipodystrophy), a syndrome associated with insulin resistance primarily affecting insulin-stimulated nonoxidative glucose metabolism (NOGM(ins)). Skeletal muscle biopsies, obtained from 18 lipodystrophic nondiabetic patients (LIPO) and 18 nondiabetic patients without lipodystrophy (NONLIPO) before and during hyperinsulinemic (40 mU.m(-2).min(-1))-euglycemic clamps, were analyzed for insulin signaling effectors. All patients were on HAART. Both LIPO and NONLIPO patients were normoglycemic (4.9 +/- 0.1 and 4.8 +/- 0.1 mmol/l, respectively); however, NOGM(ins) was reduced by 49% in LIPO patients (P < 0.001). NOGM(ins) correlated positively with insulin-stimulated glycogen synthase activity (I-form, P < 0.001, n = 36). Glycogen synthase activity (I-form) correlated inversely with phosphorylation of glycogen synthase sites 2+2a (P < 0.001, n = 36) and sites 3a+b (P < 0.001, n = 36) during clamp. Incremental glycogen synthase-kinase-3alpha and -3beta phosphorylation was attenuated in LIPO patients (Ps < 0.05). Insulin-stimulated Akt Ser473 and Akt Thr308 phosphorylation was decreased in LIPO patients (P < 0.05), whereas insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity increased significantly (P < 0.001) and similarly (NS) in both groups during clamp. Thus, low glycogen synthase activity explained impaired NOGM(ins) in HIV lipodystrophy, and insulin signaling defects were downstream of PI 3-kinase at the level of Akt. These results suggest mechanisms for the insulin resistance greatly enhancing the risk of type 2 diabetes in HIV lipodystrophy.

Role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle.

During induction of the autophagosomal degradation process, LC3-I is lipidated to LC3-II and associates to the cargo isolation membrane allowing for autophagosome formation. Lipidation of LC3 results in an increased LC3-II/LC3-I ratio, and this ratio is an often used marker for autophagy in various tissues, including skeletal muscle. From cell studies AMPK has been proposed to be necessary and sufficient for LC3 lipidation. The aim of the present study was to investigate the role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle. We observed an increase in the LC3-II/LC3-I ratio in skeletal muscle of AMPKα2 kinase-dead (KD) (p<0.001) and wild type (WT) (p<0.05) mice after 12h of fasting, which was greater (p<0.05) in AMPKα2 KD mice than in WT. The fasting-induced increase in the LC3-II/LC3-I ratio in both genotypes coincided with an initial decrease (p<0.01) in plasma insulin concentration, a subsequent decrease in muscle mTORC1 signaling and increased (p<0.05) levels of the autophagy-promoting proteins, FoxO3a and ULK1. Furthermore, a higher (p<0.01) LC3-II/LC3-I ratio was observed in old compared to young mice. We were not able to detect any change in LC3 lipidation with either in vivo treadmill exercise or in situ contractions. Collectively, these findings suggest that AMPKα2 is not necessary for induction of LC3 lipidation with fasting and aging. Furthermore, LC3 lipidation is increased in muscle lacking functional AMPKα2 during fasting and aging. Moreover, LC3 lipidation seems not to be a universal response to muscle contraction in mice.

AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD.

The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p < 0.01) and superoxide dismutase 2 (MnSOD; p < 0.05) protein abundance in quadriceps muscle of wild-type (WT; n = 13-15), but not AMPK α2 kinase dead (KD; n = 12-13) mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p = 0.051; n = 6). To further elucidate a role for AMPK in mediating these effects, we treated WT (n = 7-8) and AMPK α2 KD (n = 7-9) mice with 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). Four weeks of daily AICAR injections (500 mg/kg) resulted in AMPK-dependent increases in SIRT3 (p < 0.05) and MnSOD (p < 0.01) in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n = 9-10). Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p < 0.01) and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p < 0.05). Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training.

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.

Insulin signaling in skeletal muscle of HIV-infected patients in response to endurance and strength training.

Human immunodeficiency virus (HIV)-infected patients with lipodystrophy have decreased insulin-stimulated glucose uptake. Both endurance and resistance training improve insulin-stimulated glucose uptake in skeletal muscle of HIV-infected patients, but the mechanisms are unknown. This study aims to identify the molecular pathways involved in the beneficial effects of training on insulin-stimulated glucose uptake in skeletal muscle of HIV-infected patients. Eighteen sedentary male HIV-infected patients underwent a 16 week supervised training intervention, either resistance or strength training. Euglycemic-hyperinsulinemic clamps with muscle biopsies were performed before and after the training interventions. Fifteen age- and body mass index (BMI)-matched HIV-negative men served as a sedentary baseline group. Phosphorylation and total protein expression of insulin signaling molecules as well as glycogen synthase (GS) activity were analyzed in skeletal muscle biopsies in relation to insulin stimulation before and after training. HIV-infected patients had reduced basal and insulin-stimulated GS activity (%fractional velocity, [FV]) as well as impaired insulin-stimulated Akt(thr308) phosphorylation. Despite improving insulin-stimulated glucose uptake, neither endurance nor strength training changed the phosphorylation status of insulin signaling proteins or affected GS activity. However; endurance training markedly increased the total Akt protein expression, and both training modalities increased hexokinase II (HKII) protein. HIV-infected patients with lipodystrophy have decreased insulin-stimulated glucose uptake in skeletal muscle and defects in insulin-stimulated phosphorylation of Akt(thr308). Endurance and strength training increase insulin-stimulated glucose uptake in these patients, and the muscular training adaptation is associated with improved capacity for phosphorylation of glucose by HKII, rather than changes in markers of insulin signaling to glucose uptake or glycogen synthesis.

AMPK and insulin action--responses to ageing and high fat diet.

The 5'-AMP-activated protein kinase (AMPK) is considered "a metabolic master-switch" in skeletal muscle reducing ATP- consuming processes whilst stimulating ATP regeneration. Within recent years, AMPK has also been proposed as a potential target to attenuate insulin resistance, although the exact role of AMPK is not well understood. Here we hypothesized that mice lacking α2AMPK activity in muscle would be more susceptible to develop insulin resistance associated with ageing alone or in combination with high fat diet. Young (∼4 month) or old (∼18 month) wild type and muscle specific α2AMPK kinase-dead mice on chow diet as well as old mice on 17 weeks of high fat diet were studied for whole body glucose homeostasis (OGTT, ITT and HOMA-IR), insulin signaling and insulin-stimulated glucose uptake in muscle. We demonstrate that high fat diet in old mice results in impaired glucose homeostasis and insulin stimulated glucose uptake in both the soleus and extensor digitorum longus muscle, coinciding with reduced insulin signaling at the level of Akt (pSer473 and pThr308), TBC1D1 (pThr590) and TBC1D4 (pThr642). In contrast to our hypothesis, the impact of ageing and high fat diet on insulin action was not worsened in mice lacking functional α2AMPK in muscle. It is concluded that α2AMPK deficiency in mouse skeletal muscle does not cause muscle insulin resistance in young and old mice and does not exacerbate obesity-induced insulin resistance in old mice suggesting that decreased α2AMPK activity does not increase susceptibility for insulin resistance in skeletal muscle.

Lipid-induced insulin resistance affects women less than men and is not accompanied by inflammation or impaired proximal insulin signaling.

OBJECTIVE: We have previously shown that overnight fasted women have higher insulin-stimulated whole body and leg glucose uptake despite a higher intramyocellular triacylglycerol concentration than men. Women also express higher muscle mRNA levels of proteins related to lipid metabolism than men. We therefore hypothesized that women would be less prone to lipid-induced insulin resistance. RESEARCH DESIGN AND METHODS: Insulin sensitivity of whole-body and leg glucose disposal was studied in 16 young well-matched healthy men and women infused with intralipid or saline for 7 h. Muscle biopsies were obtained before and during a euglycemic-hyperinsulinemic clamp (1.42 mU · kg⁻¹ · min⁻¹). RESULTS: Intralipid infusion reduced whole-body glucose infusion rate by 26% in women and 38% in men (P < 0.05), and insulin-stimulated leg glucose uptake was reduced significantly less in women (45%) than men (60%) after intralipid infusion. Hepatic glucose production was decreased during the clamp similarly in women and men irrespective of intralipid infusion. Intralipid did not impair insulin or AMPK signaling in muscle and subcutaneous fat, did not cause accumulation of muscle lipid intermediates, and did not impair insulin-stimulated glycogen synthase activity in muscle or increase plasma concentrations of inflammatory cytokines. In vitro glucose transport in giant sarcolemmal vesicles was not decreased by acute exposure to fatty acids. Leg lactate release was increased and respiratory exchange ratio was decreased by intralipid. CONCLUSIONS: Intralipid infusion causes less insulin resistance of muscle glucose uptake in women than in men. This insulin resistance is not due to decreased canonical insulin signaling, accumulation of lipid intermediates, inflammation, or direct inhibition of GLUT activity. Rather, a higher leg lactate release and lower glucose oxidation with intralipid infusion may suggest a metabolic feedback regulation of glucose metabolism.

Improved insulin sensitivity after exercise: focus on insulin signaling.

After a single bout of exercise, the ability of insulin to stimulate glucose uptake is markedly improved locally in the previously active muscles. This makes exercise a potent stimulus counteracting insulin resistance characterizing type 2 diabetes (T2D). It is believed that at least part of the mechanism relates to an improved ability of insulin to stimulate translocation of glucose transporters (GLUT4) to the muscle membrane after exercise. How this is accomplished is still unclear; however, an obvious possibility is that exercise interacts with the insulin signaling pathway to GLUT4 translocation allowing for a more potent insulin response. Parallel to unraveling of the insulin signaling cascade, this has been investigated within the past 25 years. Reviewing existing studies clearly indicates that improved insulin action can occur independent of interactions with proximal insulin signaling. In contrast, more recent observations indicate that interactions exist at the distal signaling level of AS160 and atypical protein kinase C (aPKC). Although the functional interpretation is lacking, these novel observations may present a breakthrough in understanding the beneficial interplay between exercise and insulin action.

Reduced malonyl-CoA content in recovery from exercise correlates with improved insulin-stimulated glucose uptake in human skeletal muscle.

This study evaluated whether improved insulin-stimulated glucose uptake in recovery from acute exercise coincides with reduced malonyl-CoA (MCoA) content in human muscle. Furthermore, we investigated whether a high-fat diet [65 energy-% (Fat)] would alter the content of MCoA and insulin action compared with a high-carbohydrate diet [65 energy-% (CHO)]. After 4 days of isocaloric diet on two occasions (Fat/CHO), 12 male subjects performed 1 h of one-legged knee extensor exercise (approximately 80% peak workload). Four hours after exercise, insulin-stimulated glucose uptake was determined in both legs during a euglycemic-hyperinsulinemic clamp. Muscle biopsies were obtained in both legs before and after the clamp. Four hours after exercise, insulin-stimulated glucose uptake was improved (approximately 70%, P<0.001) independent of diet composition and despite normal insulin-stimulated regulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase, Akt, GSK-3, and glycogen synthase. Interestingly, exercise resulted in a sustained reduction (approximately 20%, P<0.05) in MCoA content 4 h after exercise that correlated (r=0.65, P<0.001) with improved insulin-stimulated glucose uptake. Four days of Fat diet resulted in an increased content of intramyocellular triacylglycerol (P<0.01) but did not influence muscle MCoA content or whole body insulin-stimulated glucose uptake. However, at the muscular level proximal insulin signaling and insulin-stimulated glucose uptake appeared to be compromised, although to a minor extent, by the Fat diet. Collectively, this study indicates that reduced muscle MCoA content in recovery from exercise may be part of the adaptive response leading to improved insulin action on glucose uptake after exercise in human muscle.

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

OBJECTIVE: 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 resistance.

Effect of endurance exercise training on Ca2+ calmodulin-dependent protein kinase II expression and signalling in skeletal muscle of humans.

Here the hypothesis that skeletal muscle Ca(2+)-calmodulin-dependent kinase II (CaMKII) expression and signalling would be modified by endurance training was tested. Eight healthy, young men completed 3 weeks of one-legged endurance exercise training with muscle samples taken from both legs before training and 15 h after the last exercise bout. Along with an approximately 40% increase in mitochondrial F(1)-ATP synthase expression, there was an approximately 1-fold increase in maximal CaMKII activity and CaMKII kinase isoform expression after training in the active leg only. Autonomous CaMKII activity and CaMKII autophosphorylation were increased to a similar extent. However, there was no change in alpha-CaMKII anchoring protein expression with training. Nor was there any change in expression or Thr(17) phosphorylation of the CaMKII substrate phospholamban with training. However, another CaMKII substrate, serum response factor (SRF), had an approximately 60% higher phosphorylation at Ser(103) after training, with no change in SRF expression. There were positive correlations between the increases in CaMKII expression and SRF phosphorylation as well as F(1)ATPase expression with training. After training, there was an increase in cyclic-AMP response element binding protein phosphorylation at Ser(133), but not expression, in muscle of both legs. Taken together, skeletal muscle CaMKII kinase isoform expression and SRF phosphorylation is higher with endurance-type exercise training, adaptations that are restricted to active muscle. This may contribute to greater Ca(2+) mediated regulation during exercise and the altered muscle phenotype with training.