Search

The nicotinamide adenine dinucleotide-dependent deacetylase, sirtuin (SIRT)1, in skeletal muscle is reduced in insulin-resistant states. However, whether this is an initial mechanism responsible for mediating insulin resistance in human skeletal muscle remains to be investigated. Also, SIRT1 acts as a mitochondrial gene transcriptional regulator and is induced by a short-term, high-fat diet (HFD) in human skeletal muscle. Whether saturated or unsaturated fatty acids (FAs) in the diet are important for this is unknown. We subjected 17 healthy, young men to a eucaloric control (Con) diet and 1 of 2 hypercaloric [+75% energy (E%)] HFDs for 3 d enriched in either saturated (Sat) FA (79 E% fat; Sat) or unsaturated FA (78 E% fat; Unsat). After Sat, SIRT1 protein content and activity in skeletal muscle increased ( P < 0.05; ∼40%) while remaining unchanged after Unsat. Whole-body insulin sensitivity and insulin-stimulated leg glucose uptake were reduced ( P < 0.01; ∼20%) to a similar extent compared to Con after both HFDs. We demonstrate a novel FA type-dependent regulation of SIRT1 protein in human skeletal muscle. Moreover, regulation of SIRT1 does not seem to be an initiating factor responsible for mediating insulin resistance in human skeletal muscle.-Fritzen, A. M., Lundsgaard, A.-M., Jeppesen, J. F., Sjoberg, K. A., Hoeg, L. D., Deleuran, H. H., Wojtaszewski, J. F. P., Richter, E. A., Kiens, B. Fatty acid type-specific regulation of SIRT1 does not affect insulin sensitivity in human skeletal muscle.

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. Abstract 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

It is well known that exercise has a major impact on substrate metabolism for many hours after exercise. However, the regulatory mechanisms increasing lipid oxidation and facilitating glycogen resynthesis in the post-exercise period are unknown. To address this, substrate oxidation was measured after prolonged exercise and during the following 6 h post-exercise in 5´-AMP activated protein kinase (AMPK) α2 and α1 knock-out (KO) and wild-type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post-exercise recovery, a lower lipid oxidation (P < 0.05) and higher glucose oxidation were observed in AMPKα2 KO (respiratory exchange ratio (RER) = 0.84 ± 0.02) than in WT and AMPKα1 KO (average RER = 0.80 ± 0.01) without genotype differences in muscle malonyl-CoA or free-carnitine concentrations. A similar increase in muscle pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in WT and AMPKα2 KO was observed following exercise, which is consistent with AMPKα2 deficiency not affecting the exercise-induced activation of the PDK4 transcriptional regulators HDAC4 and SIRT1. Interestingly, PDK4 protein content increased (63%, P < 0.001) in WT but remained unchanged in AMPKα2 KO. In accordance with the lack of increase in PDK4 protein content, lower (P < 0.01) inhibitory pyruvate dehydrogenase (PDH)-E1α Ser(293) phosphorylation was observed in AMPKα2 KO muscle compared to WT. These findings indicate that AMPKα2 regulates muscle metabolism post-exercise through inhibition of the PDH complex and hence glucose oxidation, subsequently creating conditions for increased fatty acid oxidation.

Acute infusion of glucagon-like peptide-1 (GLP-1) has potent effects on blood flow distribution through the microcirculation in healthy humans and rats. A high fat diet induces impairments in insulin-mediated microvascular recruitment (MVR) and muscle glucose uptake, and here we examined whether this could be reversed by GLP-1. Using contrast-enhanced ultrasound, microvascular recruitment was assessed by continuous real-time imaging of gas-filled microbubbles in the microcirculation after acute (5 days) and prolonged (8 weeks) high fat diet (HF)-induced insulin resistance in rats. A euglycaemic hyperinsulinaemic clamp (3 mU min−1 kg−1), with or without a co-infusion of GLP-1 (100 pmol l−1), was performed in anaesthetized rats. Consumption of HF attenuated the insulin-mediated MVR in both 5 day and 8 week HF interventions which was associated with a 50% reduction in insulin-mediated glucose uptake compared to controls. Acute administration of GLP-1 restored the normal microvascular response by increasing the MVR after both 5 days and 8 weeks of HF intervention (P

New Findings What is the topic of this review? This report addresses novel mechanisms regulating the utilization of long-chain fatty acids, with emphasis on FAT/CD36 and lipolysis of intramuscular triacylglycerol in skeletal muscle during exercise and contractions. What advances does it highlight? Recent findings show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) collectively account for at least 98% of total triacylglycerol lipase activity in skeletal muscle during muscle contractions. The relative importance of HSL and ATGL for breakdown of intramuscular triacylglycerol during muscle contractions is discussed. Collectively, these findings contribute to the understanding of skeletal muscle lipid metabolism during exercise and muscle contractions. Exercise increases the utilization of lipids in muscle. The sources of lipids are long-chain fatty acids taken up from the plasma and fatty acids released from stores of intramuscular triacylglycerol by the action of intramuscular lipases. In the present review, we focus on the role of fatty acid binding proteins, particularly fatty acid translocase/cluster of differentiation 36 (FAT/CD36), in the exercise- and contraction-induced increase in uptake of long-chain fatty acids in muscle. The FAT/CD36 translocates from intracellular depots to the surface membrane upon initiation of exercise/muscle contractions. This occurs independently of AMP-activated protein kinase, and data suggest that Ca2+-related signalling is responsible. The FAT/CD36 has an important role; long-chain fatty acid uptake is markedly decreased in FAT/CD36 knockout mice during contractions/exercise compared with wild-type control mice. In skeletal muscle, 98% of the lipase activity is accounted for by adipose triglyceride lipase and hormone-sensitive lipase. Give that inhibition or knockout of hormone-sensitive lipase does not impair lipolysis in muscle during contraction, the data point to an important role of adipose triglyceride lipase in regulation of muscle lipolysis. Although the molecular regulation of the lipases in muscle is not understood, it is speculated that intramuscular lipolysis may be regulated in part by the availability of the plasma concentration of long-chain fatty acids.

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.

In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

Fatty acids (FAs) as fuel for energy utilization during exercise originate from different sources: FAs transported in the circulation either bound to albumin or as triacylglycerol (TG) carried by very low density lipoproteins and FAs from lipolysis of muscle TG stores. Despite a high rate of energy expenditure during high intensity exercise the total FA oxidation is suppressed to below that observed during moderate intensity exercise. Although this has been known for many years, the mechanisms behind this phenomenon are still not fully elucidated. A failure of adipose tissue to deliver sufficient FAs to exercising muscle has been proposed, but evidence is emerging that factors within the muscle might be of more importance. The high rate of glycolysis during high intensity exercise might be the ‘driving force’ via the increased production of acetyl-CoA, which in turn is trapped by carnitine. This will lead to decreased availability of free carnitine for long chain FA transport into mitochondria. This review summarizes our present view on how FA metabolism is regulated during exercise with a special focus on the limitations in FA oxidation in the transition from moderate to high intensity exercise in humans.

In modern societies, oversupply of calories leads to obesity and chronic metabolic stress, which may lead to development of disease. Oversupply of calories is often associated with elevated plasma lipid concentrations and accumulation of lipids in skeletal muscle leading to decreased insulin sensitivity. Consequently, enhanced fat oxidation might be beneficial in counteracting lipid accumulation. Exercise is the most effective way to increase fat oxidation, because it increases metabolic rate. Lipid metabolism can also be altered by dietary manipulations. Thus, a fat rich diet leads to increased potential for fat oxidation by increasing the content of several of the proteins in the fat oxidative pathway. The regulation of both exercise and diet induced lipid oxidation will be discussed in this review.

Aim: Mitochondrial dysfunction has been suggested to play a significant role in obesity and insulin resistance. The aim of the present study was to investigate if changes in obesity and insulin resistance were related to similar changes in mitochondrial capacity for hydrogenperoxide release in Zucker diabetic fatty rats and their lean littermates. Methods: Thirty-four rats were used in this study. Rats were either lean or obese Zucker rats killed at 5–6 (young) or 12–14 (adults) weeks of age. Mitochondria were isolated from soleus muscles; respiration and release of hydrogenperoxide were determined and related to citrate synthase activity to determine intrinsic mitochondrial function. Mitochondrial-specific super-oxide dismuthase (MnSOD) protein content was determined in isolated mitochondria and muscle homogenate. Catalase protein content was determined in muscle homogenate. Results: Young lean and obese rats had a higher mitochondrial respiration when using palmitoyl-l-carnitine as substrate compared with adult lean and obese rats. The obese strain had higher mitochondrial hydrogenperoxide release but only in the adult animals. In both lean and obese animals, increased age was associated with increased mitochondrial hydrogenperoxide release. MnSOD tended to be higher in the obese strain in the isolated mitochondria. Regardless of age, catalase protein content was significantly lower in the obese rats. Conclusions: This study shows that the augmented increase in obesity and insulin resistance seen in Zucker diabetic fatty rats is associated with increased capacity for mitochondrial hydrogenperoxide release.

AIM Glucose ingestion may improve exercise endurance, but it apparently also influences the transcription rate of several metabolic genes and it alters muscle metabolism during an acute exercise bout. Therefore, we investigated how chronic training responses are affected by glucose ingestion. METHODS In previously untrained males performance and various muscular adaptations were evaluated before and after 8 weeks of supervised endurance training conducted either with (n = 8; CHO group) or without (n = 7; placebo) glucose supplementation. RESULTS The two groups achieved similar improvements in maximal oxygen uptake and peak power output during incremental cycling (both parameters elevated by 17% on average) and both groups lost approximately 3 kg of fat mass during the 8 weeks of training. An equal reduction in respiratory exchange ratio (0.02 units) during submaximal exercise was observed in both groups. Beta-hydroxyacyl-CoA-dehydrogenase activity was increased in both groups, however, to a larger extent in the placebo group (45 +/- 11%) than CHO (23 +/- 9%, P < 0.05). GLUT-4 protein expression increased by 74 +/- 14% in the placebo group and 45 +/- 14% in CHO (both P < 0.05), while resting muscle glycogen increased (P < 0.05) to a larger extent in the placebo group (96 +/- 4%) than CHO (33 +/- 2%). CONCLUSION These results show that carbohydrate supplementation consumed during exercise training influences various muscular training adaptations, but improvements in cardiorespiratory fitness and reductions in fat mass are not affected.

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.

We investigated if acute endurance-type exercise interacts with insulin-stimulated activation of atypical protein kinase C (aPKC) and insulin signalling to peptide chain elongation in human skeletal muscle. Four hours after acute one-legged exercise, insulin-induced glucose uptake was approximately 80% higher (N = 12, P < 0.05) in previously exercised muscle, measured during a euglycaemic-hyperinsulinaemic clamp (100 microU ml(-1)). Insulin increased (P < 0.05) both insulin receptor substrate (IRS)-1 and IRS-2 associated phosphatidylinositol (PI)-3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser(473) and Thr(308) in skeletal muscle. Interestingly, in response to prior exercise IRS-2-associated PI-3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. This coincided with correspondingly altered phosphorylation of the extracellular-regulated protein kinase 1/2 (ERK 1/2), p70S6 kinase (P70S6K), eukaryotic elongation factor 2 (eEF2) kinase and eEF2. aPKC was similarly activated by insulin in rested and exercised muscle, without detectable changes in aPKC Thr(410) phosphorylation. However, when adding phosphatidylinositol-3,4,5-triphosphate (PIP3), the signalling product of PI-3 kinase, to basal muscle homogenates, aPKC was more potently activated (P = 0.01) in previously exercised muscle. Collectively, this study shows that endurance-type exercise interacts with insulin signalling to peptide chain elongation. Although protein turnover was not evaluated, this suggests that capacity for protein synthesis after acute endurance-type exercise may be improved. Furthermore, endurance exercise increased the responsiveness of aPKC to PIP3 providing a possible link to improved insulin-stimulated glucose uptake after exercise.

The aim of the study was to examine local muscle metabolism in response to graded exercise when the involved muscle mass is too small to elicit marked hormonal changes and local blood flow restriction. Nine healthy overnight fasted male subjects performed knee extension exercise with both thighs kicking at 25% of maximal power (Wmax) for 45 min (23+/-1% of pulmonary) followed by 35 min of kicking with one thigh at 65% and the other at 85% W(max) (40+/-1% ). Primed constant infusion of [U-13C] palmitate and [2H5]glycerol was carried out. Blood was sampled from a femoral artery and both femoral veins, and thigh blood flow was determined by thermodilution. Muscle biopsies were obtained from m. vastus lateralis of both thighs. From rest through exercise at 25, 65 and 85% Wmax the thigh blood flow (0.3+/-0.1, 2.5+/-0.2, 3.5+/-0.2, 4.1+/-0.3 l min(-1)) and oxygen uptake (0.02+/-0.01, 0.27+/-0.03, 0.48+/-0.04, 0.55+/-0.05 l min(-1)) increased (P

Ca2+ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed. Furthermore, the effect of exercise duration and intensity on skeletal muscle CaMKII activity and phosphorylation of downstream targets was examined. Eight healthy men exercised at approximately 67% of peak pulmonary O2 uptake(VO2peak) with muscle samples taken at rest and after 1, 10, 30, 60 and 90 min of exercise. Ten other men exercised for three consecutive 10 min bouts at 35%, 60% and 85% VO2peak with muscle samples taken at rest, at the end of each interval and 30 min post-exercise. There was a rapid and transient increase in autonomous CaMKII activity and CaMKII phosphorylation at Thr287 in skeletal muscle during exercise. Furthermore, the phosphorylation of phospholamban (PLN) at Thr17, which was identified as a CaMKII substrate in skeletal muscle, was rapidly (< 1 min) increased by exercise, and remained phosphorylated 5-fold above basal level during 90 min of exercise. The phosphorylation of serum response factor at Ser103, a putative CaMKII substrate, was higher after 30 min of exercise. PLN phosphorylation at Thr17 was higher with increasing exercise intensities. These data indicate that CaMKII is the major multifunctional CaMK in skeletal muscle and its activation occurs rapidly and is sustained during continuous exercise, with the activation being greater during intense exercise.

It has been shown in some (Tarnopolsky et al. 1990; Phillips et al. 1993; Horton et al. 1998; Friedlander et al. 1998; Friedlander et al. 1999; Carter et al. 2001a; Venables et al. 2005) but not all (Burguera et al. 2000; Steffensen et al. 2002; Roepstorff et al. 2002; Riddell et al. 2003; Perreault et al. 2004) previous studies that relative fat oxidation during exercise is higher in women than in men. In those studies where no significant sex difference has been observed in relative fat oxidation during exercise, the tendency has still been in favour of higher relative fat oxidation in women than in men (Burguera et al. 2000; Steffensen et al. 2002; Roepstorff et al. 2002; Riddell et al. 2003; Perreault et al. 2004). Therefore, it seems reasonable to conclude that there is a small sex difference in relative fat oxidation during exercise, but that this sex difference is not always possible to detect in studies with a limited number of subjects. The mechanism(s) behind the higher relative fat oxidation seen during exercise in women compared with men are at present unknown. 5′AMP-activated protein kinase (AMPK) acts as an energy sensor in skeletal muscle (Hardie & Carling, 1997; Winder, 2001). Activation of AMPK is thought to increase energy producing pathways while switching off energy consuming pathways (Hardie & Carling, 1997; Winder, 2001). Exercise is one situation in which AMPK is activated in skeletal muscle (Fujii et al. 2000; Wojtaszewski et al. 2000), thereby phosphorylating and inactivating acetyl-CoA carboxylase β (ACCβ), which should lead to a decreased production of malonyl-CoA (Rasmussen & Winder, 1997; Winder, 1998). Malonyl-CoA is a known inhibitor of carnitine palmitoyl-CoA transferase I (CPT I), the enzyme catalysing the entry of long-chain fatty acyl-CoA into mitochondria for oxidation. It follows that a decrease in malonyl-CoA could have the potential to relieve inhibition of CPT I and allow for elevated fat oxidation, although measured concentrations of malonyl-CoA in skeletal muscle greatly exceed the IC50 of CPT I for malonyl-CoA (McGarry et al. 1983). In line with AMPK being involved in regulating fat oxidation, several rodent studies have shown increased AMPK activity and decreased ACC activity and malonyl-CoA concentration when going from rest to exercise, a transition characterized by markedly elevated fat oxidation (Winder & Hardie, 1996; Rasmussen & Winder, 1997; Winder, 1998). Thus, it is generally believed that AMPK signalling through the ACC–malonyl CoA–CPT I pathway is a very important mechanism to regulate fat oxidation in skeletal muscle during exercise. This contention is widespread despite recent human studies showing that high fat oxidation during exercise with low muscle glycogen or after short-term exercise training cannot be explained by increased signalling from AMPK via ACC to CPT I (McConell et al. 2005; Roepstorff et al. 2005a). Moreover, although some human studies have found a decrease in muscle malonyl-CoA content from rest to exercise (Dean et al. 2000; Roepstorff et al. 2005a), this has not been a consistent finding (Odland et al. 1996; Odland et al. 1998). Since women, compared with men, are thought to oxidize more fat during exercise, sex comparison would be a suitable model to further investigate whether AMPK regulates fat oxidation during exercise. If so, one would expect a more marked skeletal muscle AMPK activation by exercise in women than in men. In the present study, we investigated fat oxidation as well as skeletal muscle AMPK expression and activation during 90 min bicycle exercise at 60% VO2peak in women and men. It was found that fat oxidation during exercise was higher in women than in men and that AMPK Thr172 phosphorylation and α2AMPK activity during exercise were both higher in men than in women. These findings suggest that AMPK signalling is not a major regulator of fat oxidation during prolonged exercise in humans.

Protein synthesis in skeletal muscle is known to decrease during contractions but the underlying regulatory mechanisms are unknown. Here, the effect of exercise on skeletal muscle eukaryotic elongation factor 2 (eEF2) phosphorylation, a key component in protein translation machinery, was examined. Eight healthy men exercised on a cycle ergometer at a workload eliciting, similar to 67% peak pulmonary oxygen consumption ( (O2) peak) with skeletal muscle biopsies taken from the vastus lateralis muscle at rest as well as after 1, 10, 30, 60 and 90 min of exercise. In response to exercise, there was a rapid (i.e. < 1 min) 5- to 7-fold increase in eEF2 phosphorylation at Thr56 that was sustained for 90 min of continuous exercise. The in vitro activity of skeletal muscle eEF2 kinase was not altered by exercise indicating that the increased activity of eEF2 kinase to eEF2 is not mediated by covalent mechanisms. In support of this, the increase in AMPK activity was temporally unrelated to eEF2 phosphorylation. However, skeletal muscle eEF2 kinase was potently activated by Ca2+-calmodulin in vitro, suggesting that the higher eEF2 phosphorylation in working skeletal muscle is mediated by allosteric activation of eEF2 kinase by Ca2+ signalling via calmodulin. Given that eEF2 phosphorylation inhibits eEF2 activity and mRNA translation, these findings suggest that the inhibition of protein synthesis in contracting skeletal muscle is due to the Ca2+-induced stimulation of eEF2 kinase.

Atypical protein kinase C (aPKC) and extracellular signal-regulated kinase (ERK) are emerging as important signalling molecules in the regulation of metabolism and gene expression in skeletal muscle. Exercise is known to increase activity of aPKC and ERK in skeletal muscle but the effect of exercise intensity hereon has not been studied. Furthermore, the relationship between activity and phosphorylation of the two enzymes during exercise is unknown. Nine healthy young men exercised for 30 min on a bicycle ergometer on two occasions. One occasion consisted of three consecutive 10 min bouts of 35, 60 and 85% of peak pulmonary oxygen uptake V(O(2 peak)) and the second of one 30 min bout at 35% of V(O(2 peak)). Both trials also included 30 min recovery. Muscle biopsies were obtained from the vastus lateralis muscle before and after each exercise bout. Exercise increased muscle aPKC activity at 35% V(O(2 peak)), whereupon no further increase was observed at higher exercise intensities. Activation of aPKC was not accompanied by increased phosphorylation of aPKC Thr(410/403). ERK1/2 activity increased in a similar pattern to aPKC, reaching maximal activity at 35% V(O(2 peak)), whereas ERK1 Thr(202)/Tyr(204) and ERK2 Thr(183)/Tyr(185) phosphorylation increased with increasing exercise intensity. Thus, aPKC and ERK1/2 activity in muscle during exercise did not correspond to phosphorylation of sites on aPKC or ERK1/2, respectively, which are considered important for their activation. It is concluded that assessment of aPKC and ERK1/2 activity in muscle using phosphospecific antibodies did not reflect direct activity measurements on immunoprecipitated enzyme in vitro. Thus, estimation of enzyme activity during exercise by use of phosphospecific antibodies should not be performed uncritically. In addition, increase in muscle activity of aPKC or ERK1/2 during exercise is not closely related to energy demands of the muscle but may serve other regulatory or permissive functions in muscle.

Triacylglycerol stored in skeletal muscle (myocellular triacylglycerol, MCTG) serves as an energy depot, which can be utilized during exercise. However, knowledge about the regulation of triacylglycerol hydrolysis in skeletal muscle is very limited, especially when it comes to molecular mechanisms involved in regulation of the activity of hormone-sensitive lipase (HSL), the enzyme thought to catalyse triacylglycerol hydrolysis in skeletal muscle. Most information about the regulation of HSL activity stems from adipocytes, where the regulation of triacylglycerol hydrolysis by HSL has been quite extensively studied (Holm et al. 2000). For instance, in adipocytes adrenaline-stimulated HSL activity was shown to be mediated by cAMP-dependent protein kinase (PKA), which phosphorylated HSL on Ser563, Ser659 and Ser660 (numbering refers to the rat sequence) (Holm et al. 2000). On the other hand, inhibition of HSL activity may be mediated by 5′AMP-activated protein kinase (AMPK). Thus, bovine adipocyte HSL was phosphorylated in vitro on Ser565 by AMPK, which led to inhibition of subsequent phosphorylation by PKA on Ser563 (Garton et al. 1989). Correspondingly, incubation of isolated rat adipocytes with 5-aminoimidazole-4-carboxamide-riboside (AICAR), which activated AMPK, inhibited lipolysis stimulated by the β-adrenergic agonist isoprenaline (Sullivan et al. 1994; Corton et al. 1995). In rat skeletal muscle, HSL has only recently been detected and, furthermore, it was shown that HSL was activated during muscle contraction (Langfort et al. 2000). In human skeletal muscle total neutral lipase activation has been reported during exercise (Kjaer et al. 2000; Watt et al. 2003a) and it was recently confirmed that this exercise-induced increase in total neutral lipase activity could be ascribed to HSL (Watt et al. 2004b). However, the molecular mechanisms behind the activation of HSL in human skeletal muscle during exercise are still largely unknown but could be further elucidated by the investigation of site-specific HSL phosphorylation. AMPK is an important fuel gauge, which is activated during various types of cellular stress (Hayashi et al. 2000) including exercise (Wojtaszewski et al. 2000). Activation of AMPK accelerates energy-providing pathways and inhibits energy-consuming pathways (Hardie & Carling, 1997). While in adipocytes pharmacological activation of AMPK with AICAR inhibits the HSL activation induced by β-adrenergic agents (Corton et al. 1995; Sullivan et al. 1994), it seems difficult to reconcile this with the view that activation of AMPK would inhibit HSL activity in skeletal muscle during exercise since this would decrease energy provision from MCTG. Nevertheless, while the present study was in progress Watt et al. (2004b) published evidence that the increase in HSL activity in human skeletal muscle induced by exercise at 70% Vo2peak with normal muscle glycogen content and minimal α2AMPK activation was completely abolished when α2AMPK activation was high due to low muscle glycogen content. The authors also found that AICAR inhibited the HSL activation induced by adrenaline in L6 myotubes and therefore they concluded that β-adrenergic stimulation of HSL activity in skeletal muscle can be overridden by AMPK inhibition of HSL activity (Watt et al. 2004b). In the study by Watt et al. (2004b), the difference in HSL activation by exercise between the low glycogen and the control trials occurred in the face of different circulating glucose, fatty acid and adrenaline levels. Therefore, the isolated effect of AMPK on HSL activity during exercise was not readily deducible from that study (Watt et al. 2004b). Furthermore, it is still unknown whether AMPK can phosphorylate HSL on Ser565 in human skeletal muscle during exercise. Therefore, in the present study we investigated the effect of moderate intensity exercise on HSL activity and MCTG hydrolysis in human skeletal muscle during two trials with markedly different muscle AMPK activity but with minimal differences in circulating levels of several metabolites and hormones. To obtain new information on the mechanisms involved in regulation of HSL activity we also studied phosphorylation of Ser563 and Ser565 on HSL. The difference between trials in AMPK activity during exercise was achieved via alteration of pre-exercise muscle glycogen stores to low (LG) or high (HG) levels by preceding exercise and dietary manipulation as previously described (Derave et al. 2000; Wojtaszewski et al. 2003). Potential differences between LG and HG trials in adrenaline-stimulated phosphorylation of HSL on Ser563 by PKA could influence HSL Ser565 phosphorylation (Garton et al. 1989) and/or HSL activity. Therefore, subjects ingested a light pre-exercise meal 4.5 h before exercise and glucose was infused intravenously during exercise to keep blood glucose concentrations constant and similar between trials and thereby minimize the difference in plasma adrenaline concentrations which might otherwise be inherent in the present study design (Wojtaszewski et al. 2003). It was our hypothesis that in human skeletal muscle, AMPK activation during exercise in the LG trial would increase HSL Ser565 phosphorylation but without any direct effect of AMPK on HSL activity.

Long-chain fatty acids (LCFA) are important sources of energy in contracting skeletal muscle: during the course of endurance exercise the contribution of LCFA in energy metabolism increases whereas when the intensity of exercise increases, the energy need is covered more and more by carbohydrates. Although this has been known for nearly 100 years, the mechanisms controlling fatty acid uptake and oxidation during various exercise modes are still not completely elucidated. Besides passive diffusion, data suggest that both membrane-associated and cytosolic fatty acid binding proteins are involved in the uptake of LCFA into skeletal muscle. However, data from human studies suggest that the regulation of fatty acid utilization in skeletal muscle during exercise lies mainly within the entrance into the mitochondria or metabolism within the mitochondria. Although possible compartmentalization within the cell makes definitive conclusions difficult, available evidence suggests that changes in malonyl CoA concentration in muscle do not play a major regulatory role in controlling LCFA oxidation during exercise in man. In contrast, it is suggested that the availability of free carnitine may play a major regulatory role in oxidation of LCFA during exercise.

5′-AMP-activated protein kinase (AMPK) is activated in skeletal muscle from rats during treadmill exercise and electrical stimulation in situ and in vitro (Winder & Hardie, 1996; Hutber, 1997; Hayashi et al. 1998). The in vivo regulation of AMPK is still unclear, but phosphorylation by an AMPK kinase and allosteric regulation by an increased concentration of AMP and a decreased concentration of creatine phosphate may all lead to increased muscle AMPK activity in response to contractile activity (Hardie et al. 1998; Winder & Hardie, 1999). Studies suggest that the activation of AMPK may lead to the increased fatty acid oxidation observed during and after exercise (Ruderman et al. 1999). However, artificial activation of AMPK by 5-aminoimidazole-4-carboxamide (AICA)-riboside, leading to increased fatty acid oxidation, also increases the rate of glucose uptake in perfused rat muscle (Merrill et al. 1997) and in isolated incubated muscle (Hayashi et al. 1998). Recently, it was confirmed that the effects of AICA-riboside on glucose transport involve recruitment of the glucose transporter GLUT4 to the plasma membrane (Kurth-Kraczek et al. 1999). In addition, two studies have shown a relationship between the activation of glucose transport and AMPK in isolated rat skeletal muscle when subjected to a range of metabolic stressors (e.g. hypoxia and dinitrophenol), electrical stimulation and AICA-riboside (Ihlemann et al. 1999; Hayashi et al. 2000a). Thus, a host of correlative data reported so far support a regulatory role for AMPK in glucose transport in response to contractile activity, but there are also data in slow-twitch rodent muscle showing dissociation between AMPK activation and contraction-induced glucose transport (Derave et al. 2000). AMPK is a heterotrimeric protein consisting of a catalytic (α) and two non-catalytic (β and γ) subunits (Hardie et al. 1998). Two isoforms of both the α- and β-subunits and several γ-subunit isoforms have been identified (Stapleton et al. 1996; Thornton et al. 1998; Cheung et al. 2000). In rat skeletal muscle both α1- and α2-isoforms are expressed (Gao et al. 1995; Stapleton et al. 1996), and stimulation with contraction or AICA-riboside induces an equal fold increase in the α1- and α2-AMPK activity (Hayashi et al. 2000a). In contrast, when rat hindlimb muscle is stimulated electrically in situ only α2-AMPK is activated (Vavvas et al. 1997). Although it has been shown that acetyl CoA carboxylase, a substrate for AMPK, is deactivated by exercise in humans (Dean et al. 2000), no reports of the effect of exercise on AMPK activity in human skeletal muscle have been published. Therefore, in the present study, the effects of low and high intensity bicycle exercise of equal energy expenditure on isoform-specific AMPK activity in human skeletal muscle were investigated. We demonstrate an exercise intensity-dependent activation of α2-AMPK, whereas α1-AMPK activity was unchanged. In addition, using in vitro kinase assays a marked AMP dependency of α2-AMPK activity was observed, whereas α1-AMPK activity was less AMP dependent.

We examined the influence of hyperoxia on peak oxygen uptake (Vo 2peak ) and peripheral gas exchange during exercise with the quadriceps femoris muscle. Young, trained men In = 5) and women (n = 3) performed single-leg knee-extension exercise at 70% and 100% of maximum while inspiring normal air (NOX) or 60% O 2 (HIOX). Blood was sampled from the femoral vein of the exercising limb and from the contralateral artery. In comparison with NOX, hyperoxic arterial O 2 tension (P a o 2 ) increased from 13.5 ± 0.3 (x ± SE) to 41.6 ± 0.3 kPa, O 2 saturation (S a O 2 ) from 98 ± 0.1 to 100 ± 0.1%, and O 2 concentration (C a O 2 ) from 177 ± 4 to 186 ± 4 mL L -1 (all P < 0.01). Peak exercise femoral venous Po 2 (P v o 2 ) was also higher in HiOX (3.68 ± 0.06 vs. 3.39 ± 0.7 kPa; P < 0.05), indicating a higher O 2 diffusion driving pressure. HiOX femoral venous O 2 saturation averaged 36.8 ± 2.0% as opposed to 33.4 ± 1.5% in NOX (P < 0.05) and O 2 concentration 63 ± 6 vs. 55 ± 4 mL L -1 (P < 0.05). Peak exercise quadriceps blood flow (Q leg ), measured by the thermodilution technique, was lower in HiOX than in NOX, 6.4 ± 0.5 vs. 7.3 ± 0.9 L min -1 (P < 0.05); mean arterial blood pressure at inguinal height was similar in NOX and HiOX at 144 and 142 mmHg, respectively. O 2 delivery to the limb (O leq times C a O 2 ) was not significantly different in HiOX and NOX. Vo 2peak of the exercising limb averaged 890 mL min -1 in NOX and 801 mL min -1 in HiOX (n.s.) corresponding to 365 and 330 mL min -1 per kg active muscle, respectively. The Vo 2peak -to-P v o 2 ratio was lower (P < 0.05) in HiOX than in NOX suggesting a lower O 2 conductance. We conclude that the similar Vo 2peak values despite higher O 2 driving pressure in HiOX indicates a peripheral limitation for Vo 2peak . This may relate to saturation of the rate of O 2 turnover in the mitochondria during exercise with a small muscle group but can also be caused by tissue diffusion limitation related to lower O 2 conductance.

Summary. The concentration of endothelin-1 in forearm venous plasma was measured in 10 healthy men at rest and during ergometer bicycling at 65% of maximal aerobic capacity until exhaustion (96± 10 min, mean ± SE). A control group of 10 comparable subjects rested for 2 h. Mean plasma endothelin-1 concentration at rest was 2–0 ± 0–2 pg ml-1, n= 20. The concentration decreased significantly by 21% during the first 30 min of exercise, whereupon it increased so that the concentration after 60 min of exercise was no different from resting values. The change in endothelin concentration could not be explained by changes in plasma volume. Unspecific effects of catheterization or time could also not explain the change in endothelin-1, since in the 10 control subjects who did not exercise, plasma endothelin-1 did not change significantly over 120 min. It is concluded that the concentration of endothelin-1 in forearm venous plasma changes in a biphasic manner during prolonged exhaustive bicycle exercise in man. An initial decrease in concentration is followed by an increase restoring the concentration to resting values after 60 min exercise.

Summary. Postprandial glycaemia, hormonal response and energy expenditure (EE) were measured after two isocaloric carbohydrate-rich meals (69 energy-percent [E%] carbohydrate, 16 E% fat) rich in either mono- and disaccharides (47 E%, half of which sucrose) (MD) or polysaccharides (64 E%; P). Both meals were based on natural food items and were tested in a cross-over design in a group of sedentary (S) and trained (T) healthy, young subjects. Compared with the P meal blood glucose concentrations were significantly decreased and plasma insulin concentration significantly increased after the MD meal. A group difference was observed after the P meal with increased glucose and decreased insulin concentrations for T compared with S. Plasma norepinephrine concentrations were significantly increased after the MD meal compared with the P meal, but for S only. For both groups a significantly increased EE was observed 3 and 5 h after the MD meal compared with the P meal. However, no significant differences between the meals were observed in plasma insulin or catecholamine concentrations 3 and 5 h postprandially, when EE was measured. In conclusion, significant differences were observed in postprandial glucose and hormone responses as well as energy expenditure after two meals based on either mono- and disaccharide-rich or polysaccharide-rich food items. Physical fitness level influenced the glucose and hormonal responses but not the relative increases in postprandial energy expenditure.

Controversy exists whether recruitment of a large muscle mass in dynamic exercise may outstrip the pumping capacity of the heart and require neurogenic vasoconstriction in exercising muscle to prevent a fall in arterial blood pressure. To elucidate this question, seven healthy young men cycled for 70 minutes at a work load of 55-60% VO2max. At 30 to 50 minutes, arm cranking was added and total work load increased to (mean +/- SE) 82 +/- 4% of VO2max. During leg exercise, leg blood flow average 6.15 +/- .511 minutes-1, mean arterial blood pressure 137 +/- 4 mmHg and leg conductance 42.3 +/- 2.2 ml minutes-1 mmHg-1. When arm cranking was added to leg cycling, leg blood flow did not change significantly, mean arterial blood pressure increased transiently to 147 +/- 5 mmHg and leg vascular conductance decreased transiently to 33.5 +/- 3.1 ml minutes-1 mmHg-1. Furthermore, arm cranking doubled leg noradrenaline spillover. When arm cranking was discontinued and leg cycling continued, leg blood flow was unchanged but mean arterial blood pressure decreased to values significantly below those measured in the first leg exercise period. Furthermore, leg vascular conductance increased transiently, and noradrenaline spillover decreased towards values measured during the first leg exercise period. It is concluded that addition of arm cranking to leg cycling increases leg noradrenaline spillover and decreases leg vascular conductance but leg blood flow remains unchanged because of a simultaneous increase in mean arterial blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Eight subjects performed one-legged, dynamic, knee-extensor exercise, first at 10 W followed by 10 min rest, then at an intense, exhaustive exercise load (65 W) lasting 3.2 min. After 60 min recovery, exercise was performed for 8-10 min each at 20, 30, 40 and 50 W. Measurements of pulmonary oxygen uptake, heart rate, blood pressure, leg blood flow, and femoral arterial-venous differences of oxygen content and lactate were performed as well as determination of ATP, creatine phosphate (CP) inosine monophosphate (IMP) and lactate concentrations on biopsy material from the quadriceps muscle before and immediately after the intense exercise, and at 3, 10 and 60 min into recovery. 2. Individual linear relations (r = 0.95-1.00) between the power outputs for submaximal exercise and oxygen uptakes (leg and pulmonary) were used to estimate the energy demand during intense exercise. Pulmonary and leg oxygen deficits determined as the difference between energy demand and oxygen uptake were 0.46 and 0.48 l (kg active muscle)-1, respectively. Limb and pulmonary oxygen debts (oxygen uptake during 60 min of recovery - pre-exercise oxygen uptake) were 0.55 and 1.65 l (kg active muscle)-1, respectively. 3. During the intense exercise, muscle [ATP] decreased by 30% and [CP] by 60% from resting concentrations of 6.2 and 22.4 mmol (kg wet wt)-1, respectively, and [IMP] increased to 1.1 mmol (kg wet wt)-1. Muscle [lactate] increased from 2 to 28.1 mmol (kg wet wt)-1, and the concomitant net lactate release was 14.8 mmol (kg wet wt)-1 or about 1/3 of the total net lactate production. During recovery 70% of the accumulated lactate was released to the blood, and the nucleotides and CP returned to about 40 and 85% of pre-exercise values at 3 and 10 min of recovery, respectively. 4. Total reduction in ATP and CP (and elevation of IMP) during the intense exercise amounted to 16.4 mmol ATP (kg wet wt)-1, which together with the lactate production accounted for 83.1 mmol ATP (kg wet wt)-1. In addition 6-8 mmol ATP (kg wet wt)-1 are made available related to accumulation of glycolytic intermediates including pyruvate (and alanine). Estimated leg oxygen deficit corresponded to an ATP production of 94.7 mmol ATP kg-1; this value included 3.1 mmol kg-1 related to unloading of HbO2 and MbO2.(ABSTRACT TRUNCATED AT 400 WORDS)

In a preceding prospective study an increased high-density lipoprotein (HDL)-cholesterol and apolipoprotein (apo) A-I concentration occurred in healthy, non-obese, initially sedentary men, aged 30--44 years, after 3 months of physical training. Dietary history data revealed an increase in the absolute amount of fat-intake. In the present study we investigated whether a changed fat intake would influence the levels of lipoproteins, especially the HDL fraction, in twenty-three regularly conditioning men, aged 30--44 years, with a maximal aerobic capacity of 53.4 ml/kg x min--1 +/- 0.8, mean +/- SE. Ten subjects participated in an experimental group and thirteen in a control group. The experimental group studied before, after 4 weeks on a fat-rich diet (fifty-four energy-% fat, twenty-nine energy-% carbohydrate) and after 4 weeks on a fat-poor diet (twenty-nine energy-% fat, fifty-three energy-% carbohydrate). The data reveal that these large dietary changes did not influence HDL-cholesterol concentrations (1.71 +/- 0.10, 1.69 +/- 0.12, 1.59 +/- 0.13 mmol/l, mean +/- SE, during ordinary, fat-rich and fat-poor diet, respectively) and only influenced apo A-I levels to a minor degree after the fat-poor diet (134 +/- 6.1, 134 +/- 7.1 and 123 +/- 4.7 arbitrary units, mean values +/- SE) during ordinary, fat-rich and fat-poor diet, respectively). No changes were observed in the controls before and after 8 weeks. During the study training habits and body weight were constant. These findings show that large variations in the intake of dietary neutral fat only to a minor extent influence the level of HDL-cholesterol and apo A-I in physically active subjects.

Previous studies have demonstrated an inverse relationship between plasma high density lipoproteins (HDL) cholesterol and coronary heart disease risk. In the present study we investigated prospectively the effect of a moderate physical conditioning programme on plasma lipids and lipoproteins, especially HDL-cholesterol and apolipoprotein A-I (apo A-I), the major apoprotein of HDL. Healthy, sedentary, middle-aged men were randomly selected and assigned either to a training group (n = 24, age 40 +/- 3.4, mean +/- SD) or to a control group (n = 13, age 39 +/- 5.0). Training consisted of various indoor and outdoor sports activities 45 min/day, 3 times/week for 12 weeks at an intensity of approximately 80% of measured maximal oxygen uptake (VO2 max). The trained subjects were studied at 4, 8 and 12 weeks. The training increased VO2 max by 12% (P less than 0.01). Increases were observed in both apo A-I (10%, P less than 0.02) and HDL-cholesterol (8%, P less than 0.02) after training, with significant increases already after 4 and 8 weeks, respectively. Furthermore, decreases in total plasma cholesterol (5%, P less than 0.004) and plasma triglycerides (26%, P less than 0.003) were found without changes in body weight, body composition, cigarette smoking, alcohol consumption or the percentage composition of dietary intake. Fasting serum-insulin concentrations decreased significantly during training. No changes were noted in the control group. The present study demonstrates prospectively that moderate physical training can increase HDL.