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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.

Objective: 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.

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. 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 L-NMMA 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.

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 four hours 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 leg glucose uptake increased 50% more (p<0.05) in the exercised leg than the rested leg. Importantly, infusion of the nitric oxide synthase inhibitor 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 Ser(318) and Ser(704) 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.

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 as well as in response to subsequent insulin stimulation in exercised and non-exercised human muscle. Acute one-legged exercise decreased (p<0.01) lipidation of 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 AMPK trimer complexes in human muscle. Consistently, pharmacological AMPK activation with 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 man. This coincided with increased Ser-757 phosphorylation of ULK1, which is suggested as an 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, insulin stimulation and acute exercise reduces 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 likely mediates the autophagy-inhibiting effect of insulin. This article is protected by copyright. All rights reserved.

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 a2AMPK 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 a2AMPK 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 a2AMPK in muscle. It is concluded that a2AMPK 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 a2AMPK activity does not increase susceptibility for insulin resistance in skeletal muscle.

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 and design 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 7h. Muscle biopsies were obtained before and during a euglycemic hyperinsulinemic (1.42 mU·kg(-1)·min(-1)) clamp. Results: Intralipid infusion reduced whole body glucose infusion rate 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. Conclusion: 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 glucose transporter activity. A higher leg lactate release and lower glucose oxidation with intralipid infusion may rather suggest a metabolic feed back regulation of glucose

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.

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.

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.

Strength training enhances insulin sensitivity and represents an alternative to endurance training for patients with type 2 diabetes (T2DM). The 5'AMP-activated protein kinase (AMPK) may mediate adaptations in skeletal muscle in response to exercise training; however, little is known about adaptations within the AMPK system itself. We investigated the effect of strength training and T2DM on the isoform expression and the heterotrimeric composition of the AMPK in human skeletal muscle. Ten patients with T2DM and seven healthy subjects strength trained (T) one leg for 6 weeks, while the other leg remained untrained (UT). Muscle biopsies were obtained before and after the training period. Basal AMPK activity and protein/mRNA expression of both catalytic (alpha1 and alpha2) and regulatory (beta1, beta2, gamma1, gamma2a, gamma2b and gamma3) AMPK isoforms were independent of T2DM, whereas the protein content of alpha1 (+16%), beta2 (+14%) and gamma1 (+29%) was higher and the gamma3 content was lower (-48%) in trained compared with untrained muscle (all P < 0.01). The majority of alpha protein co-immunoprecipitated with beta2 and alpha2/beta2 accounted for the majority of these complexes. gamma3 was only associated with alpha2 and beta2 subunits, and accounted for approximately 20% of all alpha2/beta2 complexes. The remaining alpha2/beta2 and the alpha1/beta2 complexes were associated with gamma1. The trimer composition was unaffected by T2DM, whereas training induced a shift from gamma3- to gamma1-containing trimers. The data question muscular AMPK as a primary cause of T2DM whereas the maintained function in patients with T2DM makes muscular AMPK an obvious therapeutic target. In human skeletal muscle only three of 12 possible AMPK trimer combinations exist, and the expression of the subunit isoforms is susceptible to moderate strength training, which may influence metabolism and improve energy homeostasis in trained muscle.

Udgivelsesdato: dec.