Key points• The discovery of microRNAs (miRNAs) has established new mechanisms that control health, but little is known about the regulation of skeletal muscle miRNAs in response to exercise.• This study investigated components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and 206), and several miRNAs dysregulated in muscle myopathies, and showed that 3 h following an acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and miR-181a were all increased, while miR-9, -23a, -23b and -31 were decreased.• Short-term training increased miR-1 and miR-29b, while miR-31 remained decreased.• Negative correlations were observed between miR-9 and HDAC4 protein, miR-31 and HDAC4protein and between miR-31 and NRF1 protein, 3 h after exercise.• miR-31 binding to the HDAC4 and NRF1 3 untranslated region (UTR) reduced luciferase reporter activity.• Exercise rapidly and transiently regulates several miRNA species potentially involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis.Abstract The identification of microRNAs (miRNAs) has established new mechanisms that control skeletal muscle adaptation to exercise. The present study investigated the mRNA regulation of components of the miRNA biogenesis pathway (Drosha, Dicer and Exportin-5), muscle enriched miRNAs, (miR-1, -133a, -133b and -206), and several miRNAs dysregulated in muscle myopathies (miR-9, -23, -29, -31 and -181). Measurements were made in muscle biopsies from nine healthy untrained males at rest, 3 h following an acute bout of moderate-intensity endurance cycling and following 10 days of endurance training. Bioinformatics analysis was used to predict potential miRNA targets. In the 3 h period following the acute exercise bout, Drosha, Dicer and Exportin-5, as well as miR-1, -133a, -133-b and -181a were all increased. In contrast miR-9, -23a, -23b and -31 were decreased. Short-term training increased miR-1 and -29b, while miR-31 remained decreased. Negative correlations were observed between miR-9 and HDAC4 protein (r = −0.71; P = 0.04), miR-31 and HDAC4 protein (r = −0.87; P = 0.026) and miR-31 and NRF1 protein (r = −0.77; P = 0.01) 3 h following exercise. miR-31 binding to the HDAC4 and NRF1 3 untranslated region (UTR) reduced luciferase reporter activity. Exercise rapidly and transiently regulates several miRNA species in muscle. Several of these miRNAs may be involved in the regulation of skeletal muscle regeneration, gene transcription and mitochondrial biogenesis. Identifying endurance exercise-mediated stress signals regulating skeletal muscle miRNAs, as well as validating their targets and regulatory pathways post exercise, will advance our understanding of their potential role/s in human health.

Aims/hypothesis The 5′-AMP-activated protein kinase (AMPK) pathway is intact in type 2 diabetic patients and is seen as a target for diabetes treatment. In this study, we aimed to assess the impact of the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) on both glucose and fatty acid metabolism in vivo in type 2 diabetic patients.Methods Stable isotope methodology and blood and muscle biopsy sampling were applied to assess blood glucose and fatty acid kinetics following continuous i.v. infusion of AICAR (0.75 mg kg −1 min −1 ) and/or NaCl (0.9%) in ten male type 2 diabetic patients (age 64±2 years; BMI 28±1 kg/m 2 ). Results Plasma glucose rate of appearance (R a ) was reduced following AICAR administration, while plasma glucose rate of disappearance (R d ) was similar in the AICAR and control test. Consequently, blood glucose disposal (R d expressed as a percentage of R a ) was increased following AICAR infusion (p<0.001). Accordingly, a greater decline in plasma glucose concentration was observed following AICAR infusion (p<0.001). Plasma NEFA R a and R d were both significantly reduced in response to AICAR infusion, and were accompanied by a significant decline in plasma NEFA concentration. Although AMPK phosphorylation in skeletal muscle was not increased, we observed a significant increase in acetyl-CoA carboxylase phosphorylation (p<0.001). Conclusions/interpretation The i.v. administration of AICAR reduces hepatic glucose output, thereby lowering blood glucose concentrations in vivo in type 2 diabetic patients. Furthermore, AICAR administration stimulates hepatic fatty acid oxidation and/or inhibits whole body lipolysis, thereby reducing plasma NEFA concentration.

Intramyocellular triacylglycerol (IMTG) has been suggested to represent an important substrate source during exercise. In the present study, IMTG utilization during exercise is assessed through the use of various methodologies. In addition, we identified differences in the use of intramyocellular lipids deposited in the immediate subsarcolemmal (SS) area and those stored in the more central region of the fiber. Contemporary stable isotope technology was applied in combination with muscle tissue sampling before and immediately after 3 h of moderate-intensity cycling exercise (62 ± 2% V̇o2 max) in eight well-trained male cyclists. Continuous infusions with [U-13C]palmitate and [6,6-2H2]glucose were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate whole body IMTG and glycogen use. Both immunohistochemical analyses of oil red O (ORO)-stained muscle cross sections and biochemical triacylglycerol (TG) extraction were performed to assess muscle lipid content. During exercise, plasma FFA, muscle (and/or lipoprotein)-derived TG, plasma glucose, and muscle glycogen oxidation contributed 24 ± 2, 22 ± 3, 11 ± 1, and 43 ± 3% to total energy expenditure, respectively. In accordance, a significant net decline in muscle lipid content was observed following exercise as assessed by ORO staining (67 ± 8%) and biochemical TG extraction (49 ± 8%), and a positive correlation was observed between methods ( r = 0.56; P < 0.05). Lipid depots located in the SS area were utilized to a greater extent than the more centrally located depots. This is the first study to show significant use of IMTG as a substrate source during exercise in healthy males via the concurrent implementation of three major methodologies. In addition, this study shows differences in resting subcellular intramyocellular lipid deposit distribution and in the subsequent net use of these deposits during exercise.

Aims/hypothesisImpaired regulation of lipolysis and accumulation of lipid intermediates may contribute to obesity-related insulin resistance and type 2 diabetes mellitus. We investigated insulin-mediated suppression of lipolysis in abdominal subcutaneous adipose tissue (AT) and skeletal muscle (SM) of obese men with normal glucose tolerance (NGT) and obese type 2 diabetic men.MethodsEleven NGT men and nine long-term diagnosed type 2 diabetic men (7 ± 1 years), matched for age (58 ± 2 vs 62 ± 2 years), BMI (31.4 ± 0.6 vs 30.5 ± 0.6 kg/m2) and (28.9 ± 1.5 vs 29.5 ± 2.4 ml kg−1 min−1) participated in this study. Interstitial glycerol concentrations in AT and SM were assessed using microdialysis during a 1 h basal period and a 6 h stepwise hyperinsulinaemic–euglycaemic clamp (8, 20 and 40 mU m−2 min−1). AT and SM biopsies were collected to investigate underlying mechanisms.ResultsHyperinsulinaemia suppressed interstitial SM glycerol concentrations less in men with type 2 diabetes (−7 ± 6%, −13 ± 9% and −27 ± 9%) compared with men with NGT (−21 ± 7%, −38 ± 8% and −53 ± 8%) (p = 0.014). This was accompanied by increased circulating fatty acid and glycerol concentrations, a lower glucose infusion rate (21.8 ± 3.1 vs 30.5 ± 2.0 μmol kg body weight−1 min−1; p < 0.05), higher hormone-sensitive lipase (HSL) serine 660 phosphorylation, increased saturated diacylglycerol (DAG) lipid species in the muscle membrane and increased protein kinase C (PKC) activation in type 2 diabetic men vs men with NGT. No significant differences in insulin-mediated reduction in AT interstitial glycerol were observed between groups.Conclusions/interpretationOur results suggest that a blunted insulin-mediated suppression of SM lipolysis may promote the accumulation of membrane saturated DAG, aggravating insulin resistance, at least partly mediated by PKC. This may represent an important mechanism involved in the progression of insulin resistance towards type 2 diabetes.Trial registration: ClinicalTrials.gov NCT01680133Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-2995-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fibertype-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63±4% of maximal O 2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg −1 h −1 ) or without (CON placebo trial; water only). Continuous infusions with [U-13 C] palmitate and [6,6-2 H 2 ] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (R a ) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38±19 and 57±22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA R a and subsequent plasma FFA concentrations were lower, resulting in a 34±12% reduction in plasma FFA oxidation rates during exercise (P<0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men.

Boon H, Kostovski E, Pirkmajer S, Song M, Lubarski I, Iversen PO, Hjeltnes N, Widegren U, Chibalin AV. Influence of chronic and acute spinal cord injury on skeletal muscle Na ϩ -K ϩ -ATPase and phospholemman expression in humans. Am J Physiol Endocrinol Metab 302: E864 -E871, 2012. First published January 24, 2012; doi:10.1152/ajpendo.00625.2011.-Na ϩ -K ϩ -ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na ϩ -K ϩ -ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na ϩ -K ϩ -ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na ϩ -K ϩ -ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an ablebodied control group. Chronic complete SCI was associated with a marked decrease in [ 3 H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the ␣ 1-, ␣2-, and ␤ 1-subunit of the Na ϩ -K ϩ -ATPase. In line with this finding, expression of the Na ϩ -K ϩ -ATPase ␣1-and ␣2-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na ϩ -K ϩ -ATPase and its regulatory proteins PLM and FXYD5. FXYD proteins; sodium pump; physical inactivity; paralysis THE Na ϩ -K ϩ -ATPase IS AN INTEGRAL MEMBRANE PROTEIN that is crucial for the maintenance of ion homeostasis, cell volume, and muscle contractibility. It is composed of two polypeptide subunits, a catalytic 112-kDa ␣-subunit (␣ 1 -, ␣ 2 -, and ␣ 3 -isoforms) and a 35-to 60-kDa glycosylated ␤-subunit (␤ 1 -, ␤ 2 -, and ␤ 3 -isoforms) (6, 35). The relative abundance of each isoform and Na ϩ -K ϩ -ATPase activity is regulated in a muscleand fiber type-specific manner (29,59,62

Aims/hypothesis Disturbances in substrate source metabolism and, more particularly, in fatty acid metabolism, play an important role in the aetiology and progression of type 2 diabetes. However, data on substrate source utilisation in type 2 diabetes are inconclusive. Methods [U-13 C]palmitate and [6,6-2 H 2 ]glucose tracers were used to assess plasma NEFA and glucose oxidation rates and to estimate the use of muscle-and/or lipoproteinderived triacylglycerol and muscle glycogen. Subjects were ten male patients who had a long-term (7±1 years) diagnosis of type 2 diabetes and were overweight, and ten matched healthy, male control subjects. Muscle biopsy samples were collected before and after exercise to assess muscle fibre type-specific intramyocellular lipid and glycogen content.

Using contemporary stable-isotope methodology and fluorescence microscopy, we assessed the impact of carbohydrate supplementation on whole-body and fibertype-specific intramyocellular triacylglycerol (IMTG) and glycogen use during prolonged endurance exercise. Ten endurance-trained male subjects were studied twice during 3 h of cycling at 63±4% of maximal O 2 uptake with either glucose ingestion (CHO trial; 0.7 g CHO kg −1 h −1 ) or without (CON placebo trial; water only). Continuous infusions with [U-13 C] palmitate and [6,6-2 H 2 ] glucose were applied to quantify plasma free fatty acids (FFA) and glucose oxidation rates and to estimate intramyocellular lipid and glycogen use. Before and after exercise, muscle biopsy samples were taken to quantify fiber-type-specific IMTG and glycogen content. Plasma glucose rate of appearance (R a ) and carbohydrate oxidation rates were substantially greater in the CHO vs CON trial. Carbohydrate supplementation resulted in a lower muscle glycogen use during the first hour of exercise in the CHO vs CON trial, resulting in a 38±19 and 57±22% decreased utilization in type I and II muscle-fiber glycogen content, respectively. In the CHO trial, both plasma FFA R a and subsequent plasma FFA concentrations were lower, resulting in a 34±12% reduction in plasma FFA oxidation rates during exercise (P<0.05). Carbohydrate intake did not augment IMTG utilization, as fluorescence microscopy revealed a 76 ± 21 and 78 ± 22% reduction in type I muscle-fiber lipid content in the CHO and CON trial, respectively. We conclude that carbohydrate supplementation during prolonged cycling exercise does not modulate IMTG use but spares muscle glycogen use during the initial stages of exercise in endurance-trained men.