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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. 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. 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 FROM AUTHOR]

Key points NAD is a substrate for sirtuins (SIRTs), which regulate gene transcription in response to specific metabolic stresses., Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway., Using transgenic mouse models, we tested the hypothesis that skeletal muscle Nampt protein abundance would increase in response to metabolic stress in a manner dependent on the cellular nucleotide sensor, AMP-activated protein kinase (AMPK)., Exercise training, as well as repeated pharmacological activation of AMPK by 5-amino-1-β- d-ribofuranosyl-imidazole-4-carboxamide (AICAR), increased Nampt protein abundance. However, only the AICAR-mediated increase in Nampt protein abundance was dependent on AMPK., Our results suggest that cellular energy charge and nutrient sensing by SIRTs may be mechanistically related, and that Nampt may play a key role for cellular adaptation to metabolic stress., Abstract 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-β- 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. [ABSTRACT FROM AUTHOR]

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. In response to exercise, glucose uptake in muscle is increased by translocation of glucose transport proteins (GLUT4) from the cell interior to the cell surface. TBC1D1 is a recently identified protein believed to be involved in regulation of GLUT4 translocation in skeletal muscle. We show here that high-intensity cycle exercise in humans leads to regulation of TBC1D1, including increased phosphorylation of serine 237 and increased capacity of TBC1D1 to bind regulatory 14-3-3 proteins. We also provide evidence supporting the idea that the enzyme responsible for this regulation is AMPK, a kinase activated in response to AMP accumulation as observed during high-intensity exercise. This is the first study to demonstrate that TBC1D1 is regulated in response to exercise in human skeletal muscle, thus supporting a physiological role of TBC1D1 in regulation of glucose uptake during exercise. [ABSTRACT FROM AUTHOR]

Here the hypothesis that skeletal muscle Ca2+–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 ∼40% increase in mitochondrial F1-ATP synthase expression, there was an ∼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 α-CaMKII anchoring protein expression with training. Nor was there any change in expression or Thr17 phosphorylation of the CaMKII substrate phospholamban with training. However, another CaMKII substrate, serum response factor (SRF), had an ∼60% higher phosphorylation at Ser103 after training, with no change in SRF expression. There were positive correlations between the increases in CaMKII expression and SRF phosphorylation as well as F1ATPase expression with training. After training, there was an increase in cyclic-AMP response element binding protein phosphorylation at Ser133, 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 Ca2+ mediated regulation during exercise and the altered muscle phenotype with training. [ABSTRACT FROM AUTHOR]