Erik A. Richter

Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

1 Age‐associated loss of skeletal muscle mass and strength can partly be counteracted by resistance training, causing a net synthesis of muscular proteins. Protein synthesis is influenced synergistically by postexercise amino acid supplementation, but the importance of the timing of protein intake remains unresolved. 2 The study investigated the importance of immediate (P0) or delayed (P2) intake of an oral protein supplement upon muscle hypertrophy and strength over a period of resistance training in elderly males. 3 Thirteen men (age, 74 ± 1 years; body mass index (BMI), 25 ± 1 kg m−2 (means ± S.E.M.)) completed a 12 week resistance training programme (3 times per week) receiving oral protein in liquid form (10 g protein, 7 g carbohydrate, 3 g fat) immediately after (P0) or 2 h after (P2) each training session. Muscle hypertrophy was evaluated by magnetic resonance imaging (MRI) and from muscle biopsies and muscle strength was determined using dynamic and isokinetic strength measurements. Body composition was determined from dual‐energy X‐ray absorptiometry (DEXA) and food records were obtained over 4 days. The plasma insulin response to protein supplementation was also determined. 4 In response to training, the cross‐sectional area of m. quadriceps femoris (54.6 ± 0.5 to 58.3 ± 0.5 cm2) and mean fibre area (4047 ± 320 to 5019 ± 615 μm2) increased in the P0 group, whereas no significant increase was observed in P2. For P0 both dynamic and isokinetic strength increased, by 46 and 15 %, respectively (P < 0.05), whereas P2 only improved in dynamic strength, by 36 % (P < 0.05). No differences in glucose or insulin response were observed between protein intake at 0 and 2 h postexercise. 5 We conclude that early intake of an oral protein supplement after resistance training is important for the development of hypertrophy in skeletal muscle of elderly men in response to resistance training.

The AMP-activated protein kinase α2 catalytic subunit controls whole-body insulin sensitivity

AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKalpha2 catalytic subunit gene was inactivated. AMPKalpha2(-/-) mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKalpha2(-/-) pancreatic islets, glucose- and L-arginine-stimulated insulin secretion were not affected. AMPKalpha2(-/-) mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKalpha2(-/-) muscles. These data indicate that AMPKalpha2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKalpha2(-/-) mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKalpha2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity.

Exercise, GLUT4, and Skeletal Muscle Glucose Uptake

Glucose is an important fuel for contracting muscle, and normal glucose metabolism is vital for health. Glucose enters the muscle cell via facilitated diffusion through the GLUT4 glucose transporter which translocates from intracellular storage depots to the plasma membrane and T-tubules upon muscle contraction. Here we discuss the current understanding of how exercise-induced muscle glucose uptake is regulated. We briefly discuss the role of glucose supply and metabolism and concentrate on GLUT4 translocation and the molecular signaling that sets this in motion during muscle contractions. Contraction-induced molecular signaling is complex and involves a variety of signaling molecules including AMPK, Ca(2+), and NOS in the proximal part of the signaling cascade as well as GTPases, Rab, and SNARE proteins and cytoskeletal components in the distal part. While acute regulation of muscle glucose uptake relies on GLUT4 translocation, glucose uptake also depends on muscle GLUT4 expression which is increased following exercise. AMPK and CaMKII are key signaling kinases that appear to regulate GLUT4 expression via the HDAC4/5-MEF2 axis and MEF2-GEF interactions resulting in nuclear export of HDAC4/5 in turn leading to histone hyperacetylation on the GLUT4 promoter and increased GLUT4 transcription. Exercise training is the most potent stimulus to increase skeletal muscle GLUT4 expression, an effect that may partly contribute to improved insulin action and glucose disposal and enhanced muscle glycogen storage following exercise training in health and disease.

Isoform-specific and exercise intensity-dependent activation of 5'-AMP-activated protein kinase in human skeletal muscle.

1 5′‐AMP‐activated protein kinase (AMPK) has been suggested to play a key role in the regulation of metabolism in skeletal muscle. AMPK is activated in treadmill‐exercised and electrically stimulated rodent muscles. Whether AMPK is activated during exercise in humans is unknown. 2 We investigated the degree of activation and deactivation of α‐isoforms of AMPK during and after exercise. Healthy human subjects performed bicycle exercise on two separate occasions at either a low (∼50% maximum rate of O2 uptake (V̇O2,max) for 90 min) or a high (∼75% V̇O2,max for 60 min) intensity. Biopsies from the vastus lateralis muscle were obtained before and immediately after exercise, and after 3 h of recovery. 3 We observed a 3‐ to 4‐fold activation of the α2‐AMPK isoform immediately after high intensity exercise, whereas no activation was observed after low intensity exercise. The activation of α2‐AMPK was totally reversed 3 h after exercise. In contrast, α1‐AMPK was not activated during either of the two exercise trials. 4 The in vitro AMP dependency of α2‐AMPK was significantly greater than that of α1‐AMPK (∼3‐ vs.∼2‐fold). 5 We conclude that in humans activation of α2‐AMPK during exercise is dependent upon exercise intensity. The stable activation of α2‐AMPK, presumably due to the activation of an upstream AMPK kinase, is compatible with a role for this kinase complex in the regulation of skeletal muscle metabolism during exercise, whereas the lack of stable α1‐AMPK activation makes this kinase complex a less likely candidate.

AMPK and the biochemistry of exercise: implications for human health and disease.

AMPK (AMP-activated protein kinase) is a phylogenetically conserved fuel-sensing enzyme that is present in all mammalian cells. During exercise, it is activated in skeletal muscle in humans, and at least in rodents, also in adipose tissue, liver and perhaps other organs by events that increase the AMP/ATP ratio. When activated, AMPK stimulates energy-generating processes such as glucose uptake and fatty acid oxidation and decreases energy-consuming processes such as protein and lipid synthesis. Exercise is perhaps the most powerful physiological activator of AMPK and a unique model for studying its many physiological roles. In addition, it improves the metabolic status of rodents with a metabolic syndrome phenotype, as does treatment with AMPK-activating agents; it is therefore tempting to attribute the therapeutic benefits of regular physical activity to activation of AMPK. Here we review the acute and chronic effects of exercise on AMPK activity in skeletal muscle and other tissues. We also discuss the potential role of AMPK activation in mediating the prevention and treatment by exercise of specific disorders associated with the metabolic syndrome, including Type 2 diabetes and Alzheimers disease.

AMP-activated protein kinase (AMPK) β1β2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise

AMP-activated protein kinase (AMPK) β1 or β2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle, α2β2γ3-containing heterotrimers predominate. However, compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK α2, β2, and γ3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism, because these models have normal mitochondrial content, contraction-stimulated glucose uptake, and insulin sensitivity. In the current study, we generated mice lacking both AMPK β1 and β2 isoforms in skeletal muscle (β1β2M-KO). β1β2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly, young β1β2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake, findings that were not apparent in mice with single mutations or deletions in muscle α, β, or γ subunits.

Oral creatine supplementation facilitates the rehabilitation of disuse atrophy and alters the expression of muscle myogenic factors in humans

1 We investigated the effect of oral creatine supplementation during leg immobilization and rehabilitation on muscle volume and function, and on myogenic transcription factor expression in human subjects. 2 A double‐blind trial was performed in young healthy volunteers (n=22). A cast was used to immobilize the right leg for 2 weeks. Thereafter the subjects participated in a knee‐extension rehabilitation programme (3 sessions week−1, 10 weeks). Half of the subjects received creatine monohydrate (CR; from 20 g down to 5 g daily), whilst the others ingested placebo (P; maltodextrin). 3 Before and after immobilization, and after 3 and 10 weeks of rehabilitation training, the cross‐sectional area (CSA) of the quadriceps muscle was assessed by NMR imaging. In addition, an isokinetic dynamometer was used to measure maximal knee‐extension power (Wmax), and needle biopsy samples taken from the vastus lateralis muscle were examined to asses expression of the myogenic transcription factors MyoD, myogenin, Myf5, and MRF4, and muscle fibre diameters. 4 Immobilization decreased quadriceps muscle CSA (∼10 %) and Wmax (∼25 %) by the same magnitude in both groups. During rehabilitation, CSA and Wmax recovered at a faster rate in CR than in P (P < 0.05 for both parameters). Immobilization changed myogenic factor protein expression in neither P nor CR. However, after rehabilitation myogenin protein expression was increased in P but not in CR (P < 0.05), whilst MRF4 protein expression was increased in CR but not in P (P < 0.05). In addition, the change in MRF4 expression was correlated with the change in mean muscle fibre diameter (r=0.73, P < 0.05). 5 It is concluded that oral creatine supplementation stimulates muscle hypertrophy during rehabilitative strength training. This effect may be mediated by a creatine‐induced change in MRF4 and myogenin expression.

Effects of α-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle

We tested the hypothesis that 5′AMP‐activated protein kinase (AMPK) plays an important role in regulating the acute, exercise‐induced activation of metabolic genes in skeletal muscle, which were dissected from whole‐body α2‐ and α1‐AMPK knockout (KO) and wild‐type (WT) mice at rest, after treadmill running (90 min), and in recovery. Running increased α1‐AMPK kinase activity, phosphorylation (P) of AMPK, and acetyl‐CoA carboxylase (ACC)β in α2‐WT and α2‐KO muscles and increased α2‐AMPK kinase activity in α2‐WT. In α2‐KO muscles, AMPK‐P and ACCβ‐P were markedly lower compared with α2‐WT. However, in α1‐WT and α1‐KO muscles, AMPK‐P and ACCβ‐P levels were identical at rest and increased similarly during exercise in the two genotypes. The α2‐KO decreased peroxisome‐proliferator‐activated receptor γ coactivator (PGC)‐1α, uncoupling protein‐3 (UCP3), and hexokinase II (HKII) transcription at rest but did not affect exercise‐induced transcription. Exercise increased the mRNA content of PGC‐1α, Forkhead box class O (FOXO)1, HKII, and pyruvate dehydrogenase kinase 4 (PDK4) similarly in α2‐WT and α2‐KO mice, whereas glucose transporter GLUT 4, carnitine palmitoyltransferase 1 (CPTI), lipoprotein lipase, and UCP3 mRNA were unchanged by exercise in both genotypes. CPTI mRNA was lower in α2‐KO muscles than in α2‐WT muscles at all time‐points. In α1‐WT and α1‐KO muscles, running increased the mRNA content of PGC‐1α and FOXOl similarly. The α2‐KO was associated with lower muscle adenosine 5′‐triphosphate content, and the inosine monophosphate content increased substantially at the end of exercise only in α2‐KO muscles. In addition, subcutaneous injection of 5‐aminoimidazole‐4‐carboxamide‐1‐β‐4‐ribofuranoside (AICAR) increased the mRNA content of PGC‐1α, HKII, FOXO1, PDK4, and UCP3, and α2‐KO abolished the AICAR‐induced increases in PGC‐1α and HKII mRNA. In conclusion, KO of the α2‐ but not the α1‐AMPK isoform markedly diminished AMPK activation during running. Nevertheless, exercise‐induced activation of the investigated genes in mouse skeletal muscle was not impaired in α1‐ or α2‐AMPK KO muscles. Although it cannot be ruled out that activation of the remaining α‐isoform is sufficient to increase gene activation during exercise, the present data do not support an essential role of AMPK in regulating exercise‐induced gene activation in skeletal muscle.

Utilization of skeletal muscle triacylglycerol during postexercise recovery in humans

The utilization of muscle triacylglycerols was studied during and after prolonged bicycle ergometer exercise to exhaustion in eight healthy young men. Two days before exercise and in the postexercise recovery period, subjects were fed a carbohydrate-rich diet (65-70% of energy from carbohydrates). Exercise decreased muscle glycogen concentrations from 533 +/- 18 to 108 +/- 10 mmol/kg dry wt, whereas muscle triacylglycerol concentrations were unaffected (49 +/- 5 before vs. 49 +/- 8 mmol/kg dry wt after exercise). During the first 18 h after exercise, muscle glycogen concentrations were restored to 409 +/- 20 mmol/kg dry wt. In contrast, muscle triacylglycerol concentrations decreased (P < 0.05) to a nadir of 38 +/- 5 mmol/kg dry wt, and muscle lipoprotein lipase activity increased by 72% compared with values before exercise. Pulmonary respiratory exchange ratio values of 0.80-0.82 indicated a relatively high fractional lipid combustion despite the high carbohydrate intake. From 18 to 42 h of recovery, muscle glycogen synthesis was slow and muscle triacylglycerol concentrations and lipoprotein lipase activity were restored to the preexercise values. It is concluded that muscle triacylglycerol concentrations are not diminished during exhaustive glycogen-depleting exercise. However, in the postexercise recovery period, muscle glycogen resynthesis has high metabolic priority, resulting in postexercise lipid combustion despite a high carbohydrate intake. It is suggested that muscle triacylglycerols, and probably very low density lipoprotein triacylglycerols, are important in providing fuel for muscle metabolism in the postexercise recovery period.The utilization of muscle triacylglycerols was studied during and after prolonged bicycle ergometer exercise to exhaustion in eight healthy young men. Two days before exercise and in the postexercise recovery period, subjects were fed a carbohydrate-rich diet (65-70% of energy from carbohydrates). Exercise decreased muscle glycogen concentrations from 533 ± 18 to 108 ± 10 mmol/kg dry wt, whereas muscle triacylglycerol concentrations were unaffected (49 ± 5 before vs. 49 ± 8 mmol/kg dry wt after exercise). During the first 18 h after exercise, muscle glycogen concentrations were restored to 409 ± 20 mmol/kg dry wt. In contrast, muscle triacylglycerol concentrations decreased ( P < 0.05) to a nadir of 38 ± 5 mmol/kg dry wt, and muscle lipoprotein lipase activity increased by 72% compared with values before exercise. Pulmonary respiratory exchange ratio values of 0.80-0.82 indicated a relatively high fractional lipid combustion despite the high carbohydrate intake. From 18 to 42 h of recovery, muscle glycogen synthesis was slow and muscle triacylglycerol concentrations and lipoprotein lipase activity were restored to the preexercise values. It is concluded that muscle triacylglycerol concentrations are not diminished during exhaustive glycogen-depleting exercise. However, in the postexercise recovery period, muscle glycogen resynthesis has high metabolic priority, resulting in postexercise lipid combustion despite a high carbohydrate intake. It is suggested that muscle triacylglycerols, and probably very low density lipoprotein triacylglycerols, are important in providing fuel for muscle metabolism in the postexercise recovery period.

Muscle contractions induce interleukin-6 mRNA production in rat skeletal muscles

1 The present study explored the hypothesis that interleukin‐6 (IL‐6) might be locally produced in response to skeletal muscle contractions and whether the production might reflect the type of muscle contraction performed. Rats were anaesthetized and the calf muscles of one limb were stimulated electrically for concentric or eccentric contractions (4 × 10 contractions with 1 min of rest between the 4 series, 100 Hz). The contralateral muscles served as unstimulated controls. The mRNA levels for IL‐6, the glucose transport protein GLUT‐4 and β‐actin in the rat muscles (white and red gastrocnemius and soleus) were quantified by quantitative competitive RT‐PCR. 2 The IL‐6 mRNA level, measured 30 min after the stimulation, increased after both eccentric and concentric contractions and there were no significant differences in IL‐6 mRNA levels between the different muscle fibre types. No significant increase in IL‐6 mRNA level was seen in the unstimulated contralateral muscle fibres. 3 No increase in GLUT‐4 mRNA level was detected, indicating that the increase in IL‐6 mRNA level was not due to general changes in transcription. 4 We conclude that IL‐6 is locally produced after muscle contraction, with no significant differences between different muscle fibre types. This local production of IL‐6 is not due to general changes in transcription, since no changes in the level of GLUT‐4 mRNA were found. The fact that increased IL‐6 mRNA levels were seen after both concentric and eccentric contractions indicates that the production of IL‐6 is not solely due to muscle damage, seen primarily after eccentric exercise.