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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 (~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 (~70%, P < 0.001) independent of diet composition and despite normal insulin-stimulated regulation of insulin receptor substrate-l-associated phosphatidylinositol 3-kinase, Akt, GSK-3, and glycogen synthase. Interestingly, exercise resulted in a sustained reduction (~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. AMP-activated protein kinase; acetyl coenzyme A carboxylase; intramuscular triacylglycerol; Akt; diet

Women exhibit an enhanced capability for lipid metabolism during endurance exercise compared with men. The underlying regulatory mechanisms behind this sex-related difference are not well understood but may comprise signaling through a myocyte enhancer factor 2 (MEF2) regulatory pathway. The primary purpose of this study, therefore, was to investigate the protein signaling of MEF2 regulatory pathway components at rest and during 90 min of bicycling exercise at 60% [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]in healthy, moderately trained men (n = 8) and women (n = 9) to elucidate the potential role of these proteins in substrate utilization during exercise. A secondary purpose was to screen for mRNA expression of MEF2 isoforms and myogenic regulatory factor (MRF) family members of transcription factors at rest and during exercise. Muscle biopsies were obtained before and immediately after exercise. Nuclear AMP-activated protein kinase-[alpha] ([alpha]AMPK) [Thr.sup.172] (P < 0.001), histone deacetylase 5 (HDAC5) [Ser.sup.498] (P < 0.001), and MEF2 Thr (P < 0.01) phosphorylation increased with exercise. No significant sex differences were observed at rest or during exercise. At rest, no significant sex differences were observed in mRNA expression of the measured transcription factors, mRNA for transcription factors MyoD, myogenin, MRF4, MEF2A, MEF2C, MEF2D, and peroxisome proliferator-activated receptor-3, coactivator Is (PGCI[alpha]) were significantly upregulated by exercise. Of these, MEF2A mRNA increased 25% specifically in women (P < 0.05), whereas MEF2D mRNA tended to increase in men (P = 0.11). Although minor sex differences in mRNA expression were observed, the main finding of the present study was the implication of a joint signaling action of AMPK, HDAC5, and PGCI[alpha] on MEF2 in the immediate regulatory response to endurance exercise. This signaling response was independent of sex. skeletal muscle; myogenic regulatory factor; AMP-activated protein kinase; histone deacetylase 5; peroxisome proliferator-activated receptor-[gamma] coactivator 1[alpha]

Women have been shown to use more intramuscular triacylglycerol (IMTG) during exercise than men. To investigate whether this could be due to sex-specific regulation of hormone-sensitive lipase (HSL) and to use sex comparison as a model to gain further insight into HSL regulation, nine women and eight men performed bicycle exercise (90 min, 60% [Vo.sub.2 peak]), and skeletal muscle HSL expression, phosphorylation, and activity were determined. Supporting previous findings, basal IMTG content (P < 0.001) and net IMTG decrease during exercise (P < 0.01) were higher in women than in men and correlated significantly (r = 0.72, P = 0.001). Muscle HSL mRNA (80%, P = 0.11) and protein content (50%, P < 0.05) were higher in women than in men. HSL total activity increased during exercise (47%, P < 0.05) but did not differ between sexes. Accordingly, HSL specific activity (HSL activity per HSL protein content) increased during exercise (62%, P < 0.05) and was generally higher in men than in women (82%, P < 0.05). A similar pattern was observed for HSL [Ser.sup.659] phosphorylation, suggesting a role in regulation of HSL activity. Likewise, plasma epinephrine increased during exercise (P < 0.05) and was higher in men than in women during the end of the exercise bout (P < 0.05). We conclude that, although HSL expression and [Ser.sup.659] phosphorylation in skeletal muscle during exercise is sex specific, total muscle HSL activity measured in vitro was similar between sexes. The higher basal IMTG content in women compared with men is therefore the best candidate to explain the higher IMTG net hydrolysis during exercise in women. intramuscular triacylglycerol; epinephrine; extracellular signal-regulated kinase

Intracellular mechanisms regulating fat oxidation were investigated in human skeletal muscle during exercise. Eight young, healthy, moderately trained men performed bicycle exercise (60 min, 65% peak [O.sub.2] consumption) on two occasions, where they ingested either 1) a high-carbohydrate diet (H-CHO) or 2) a low-carbohydrate diet (L-CHO) before exercise to alter muscle glycogen content as well as to induce, respectively, low and high rates of fat oxidation. Leg fat oxidation was 122% higher during exercise in L-CHO than in H-CHO (P < 0.001). In keeping with this, the activity of [[alpha].sub.2]-AMP-activated protein kinase ([[alpha].sub.2]-AMPK) was increased twice as much in L-CHO as in H-CHO (P < 0.01) at 60 min of exercise. However, acetyl-CoA carboxylase (ACC)[beta] [Ser.sup.221] phosphorylation was increased to the same extent (6-fold) under the two conditions. The concentration of malonyl-CoA was reduced 13% by exercise in both conditions (P < 0.05). Pyruvate dehydrogenase activity was higher during exercise in H-CHO than in L-CHO (P < 0.01). In H-CHO only, the concentrations of acetyl-CoA and acetylcarnitine were increased (P < 0.001), and the concentration of free carnitine was decreased (P < 0.01), by exercise. The data suggest that a decrease in the concentration of malonyl-CoA, secondary to [[alpha].sub.2]-AMPK activation and ACC inhibition (by phosphorylation), contributes to the increase in fat oxidation observed at the onset of exercise regardless of muscle glycogen levels. They also suggest that, with high muscle glycogen, the availability of free carnitine may limit fat oxidation during exercise, due to its increased use for acetylcarnitine formation. acetylcarnitine; acetyl coenzyme A; pyruvate dehydrogenase activity; adenosine 5'-monosphosphate-activated protein kinase; acetyl coenzyme A carboxylase

Regulation of plasma long-chain fatty acid oxidation in relation to uptake in human skeletal muscle during exercise. Am J Physiol Endocrinol Metab 287: E696-E705, 2004. First published June 8, 2004; 10.1152/ajpendo.00001.2004.--In the present study, we investigated possible sites of regulation of long-chain fatty acid (LCFA) oxidation in contracting human skeletal muscle. Leg plasma LCFA kinetics were determined in eight healthy men during bicycling (60 min, 65% peak oxygen uptake) with either high (H-FOX) or low (L-FOX) leg tat oxidation (H-FOX: 1,098 [+ or -] 140; L-FOX: 494 [+ or -] 84 [micro]mol FA/min, P < 0.001), which was achieved by manipulating preexercise muscle glycogen (H-FOX: 197 [+ or -] 21; L-FOX: 504 [+ or -] 25 mmol/kg dry wt, P < 0.001). Several blood metabolites and hormones were kept nearly similar between trials by allocating a preexercise meal and infusing glucose intravenously during exercise. During exercise, leg plasma LCFA fractional extraction was identical between trials (H-FOX: 17.8 [+ or -] 1.6; L-FOX: 18.2 [+ or -] 1.8%, not significant), suggesting similar LCFA transport capacity in muscle. On the contrary, leg plasma LCFA oxidation was 99% higher in H-FOX than in L-FOX (421 [+ or -] 47 vs. 212 [+ or -] 37 [micro]mol/min, P < 0.001). Probably due to the slightly higher (P < 0.01) plasma LCFA concentration in H-FOX than in L-FOX, leg plasma LCFA uptake was nonsignificantly (P = 0.17) higher (25%) in H-FOX than in L-FOX, yet the fraction of plasma LCFA uptake oxidized was 61% higher (P < 0.05) in H-FOX than in L-FOX. Accordingly, the muscle content of several lipid-binding proteins did not differ significantly between trials, although fatty acid translocase/CD36 and caveolin-1 were elevated (P < 0.05) by the high-intensity exercise and dietary manipulation allocated on the day before the experimental trial. The present data suggest that, in contracting human skeletal muscle with different fat oxidation rates achieved by manipulating preexcrcise glycogen content, transsar-colemmal transport is not limiting plasma LCFA oxidation. Rather, the latter seems to be limited by intracellular regulatory mechanisms. plasma membrane fatty acid-binding protein; cytosolic fatty acid-binding protein; fatty acid translocase; caveolin

The resting content and use of myocellular triacylglycerol (MCTG) during 90 min of submaximal exercise [60% of peak oxygen uptake (V[o.sub.2 peak])] were studied in 21 eumenorrheic female and 21 male subjects at different training levels [untrained (UT), moderately trained (MT), and endurance trained (END)]. Males and females were matched according to their V[o.sub.2peak] expressed relative to lean body mass, physical activity level, and training history. All subjects ingested the same controlled diet for 8 days, and all females were tested in the midfollicular phase of the menstrual cycle. Resting MCTG, measured with the muscle biopsy technique, averaged 48.4 [+ or -] 4.2, 48.5 [+ or -] 8.4, and 52.2 [+ or -] 5.8 mmol/kg dry wt in UT, MT, and END females, respectively, and 34.1 [+ or -] 4.9, 31.6 [+ or -] 3.3, and 38.4 [+ or -] 3.0 mmol/kg dry wt in UT, MT, and END males, respectively (P < 0.001, females vs. males in all groups). Exercise decreased MCTG content in the female subjects by an average of 25%, regardless of training status, whereas in the male groups MCTG content was unaffected by exercise. The arterial plasma insulin concentration was higher (P < 0.05) and the arterial plasma epinephrine concentration was lower (P < 0.05) in the females than in the males at rest and during exercise. MCTG use was correlated to the resting concentration of MCTG (P < 0.001). We conclude that resting content and use of MCTG during exercise are related to gender and furthermore are independent of training status. muscle substrate; training; triglycerides

Gender differences in substrate utilization during submaximal exercise in endurance-trained subjects. Am J Physiol Endocrinol Metab 282: E435-E447, 2002; 10.1152/ajpendo.00266.2001.--Substrate utilization across the leg during 90 min of bicycle exercise at 58% of peak oxygen uptake (V[O.sup.2 peak]) was studied in seven endurance-trained males and seven endurance-trained, eumenorrheic females by applying arteriovenous catheterization, stable isotopes, and muscle biopsies. The female and male groups were matched according to V[O.sup.2 peak] per kilogram of lean body mass, physical activity level, and training history of the subjects. All subjects consumed the same diet, well controlled in terms of nutrient composition as well as energy content, for 8 days preceding the experiment, and all females were tested in the midfollicular phase of the menstrual cycle. During exercise, respiratory exchange ratio (RER) and leg respiratory quotient (RQ) were similar in females and males. Myocellular triacylglycerol (TG) degradation was negligible in males but amounted to 12.4 [+ or -] 3.2 mmol/kg dry wt in females and corresponded to 25.0 [+ or -] 6.0 and 5.0 [+ or -] 7.3% of total oxygen uptake in females and males, respectively (P < 0.05). Utilization of plasma fatty acids (12.0 [+ or -] 2.5 and 9.6 [+ or -] 1.5%), blood glucose (13.6 [+ or -] 1.5 and 14.3 [+ or -] 1.5%), and glycogen (48.5 [+ or -] 4.9 and 42.8 [+ or -] 2.1%) were similar in females and males. Thus, in females, measured substrate oxidation accounted for 99% of the leg oxygen uptake, whereas in males 28% of leg oxygen uptake was unaccounted for in terms of measured oxidized lipid substrates. These findings may indicate that males utilized additional lipid sources, presumably very low density lipoprotein-TG or TG located between muscle fibers. On the basis of RER and leg RQ, it is concluded that no gender difference existed in the relative contribution from carbohydrate and lipids to the oxidative metabolism across the leg during submaximal exercise at the same relative workload. However, an effect of gender appears to occur in the utilization of the different lipid sources. [[sup.13]C]palmitate; plasma fatty acids; myocellular triacylglycerol; glucose; glycogen

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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 I h of one-legged knee extensor exercise (~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 (--70%, P < 0.001) independent of diet com- position 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 (~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. [ABSTRACT FROM AUTHOR]

5′AMP-activated protein kinase (AMPK) is an energy sensor activated by perturbed cellular energy status such as during muscle contraction. Activated AMPK is thought to regulate several key metabolic pathways. We used sex comparison to investigate whether AMPK signalling in skeletal muscle regulates fat oxidation during exercise. Moderately trained women and men completed 90 min bicycle exercise at 60% . Both AMPK Thr172 phosphorylation and α2AMPK activity were increased by exercise in men (∼200%, P < 0.001) but not significantly in women. The sex difference in muscle AMPK activation with exercise was accompanied by an increase in muscle free AMP (∼164%, P < 0.01), free AMP/ATP ratio (159%, P < 0.05), and creatine (∼42%, P < 0.001) in men but not in women (NS), suggesting that lack of AMPK activation in women was due to better maintenance of muscle cellular energy balance compared with men. During exercise, fat oxidation per kg lean body mass was higher in women than in men ( P < 0.05). Regression analysis revealed that a higher proportion of type 1 muscle fibres (∼23%, P < 0.01) and a higher capillarization (∼23%, P < 0.05) in women than in men could partly explain the sex difference in α2AMPK activity ( r=−0.54, P < 0.05) and fat oxidation ( r= 0.64, P < 0.05) during exercise. On the other hand, fat oxidation appeared not to be regulated via AMPK. In conclusion, during prolonged submaximal exercise at 60% , higher fat oxidation in women cannot be explained by higher AMPK signalling but is accompanied by improved muscle cellular energy balance in women probably due to sex specific muscle morphology. [ABSTRACT FROM AUTHOR]

Hormone-sensitive lipase (HSL) catalyses the hydrolysis of myocellular triacylglycerol (MCTG), which is a potential energy source during exercise. Therefore, it is important to elucidate the regulation of HSL activity in human skeletal muscle during exercise. The main purpose of the present study was to investigate the role of 5′AMP-activated protein kinase (AMPK) in the regulation of muscle HSL activity and Ser565 phosphorylation (the presumed AMPK target site) in healthy, moderately trained men during 60 min bicycling (65% VO2 peak), αAMPK activity during exercise was manipulated by studying subjects with either low (LG) or high (HG) muscle glycogen content. HSL activity was distinguished from the activity of other neutral lipases by immunoinhibition of HSL using an anti-HSL antibody. During exercise a 62% higher (P< 0.01) α2AMPK activity in LG than in HG was paralleled by a similar difference (61%, P<0.01) in HSL Ser565 phosphorylation but without any difference between trials in HSL activity or MCTG hydrolysis. HSL activity was increased (117%, P < 0.05) at 30 min of exercise but not at 60 min of exercise. In both trials, HSL phosphorylation on Ser565 (a presumed PKA target site) was not increased by exercise despite a fourfold increase (P< 0.001) in plasma adrenaline. ERK1/2 phosphorylation was increased by exercise in both trials (P< 0.001) and was higher in LG than in HG both at rest and during exercise (P = 0.06). In conclusion, the present study suggests that AMPK phosphorylates HSL on Ser565 in human skeletal muscle during exercise with reduced muscle glycogen. Apparently, HSL Ser565phosphorylation by AMPK during exercise had no effect on HSL activity. Alternatively, other factors including ERK may have counterbalanced any effect of AMPK on HSL activity. [ABSTRACT FROM AUTHOR]