Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

The effect of age and unilateral leg immobilization for 2 weeks on substrate utilization during moderate-intensity exercise in human skeletal muscle.

KEY POINTS: This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms. ABSTRACT: Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 20 ± 1 W (∼50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age- and immobilization-induced IMTG accumulation.

Hyperglycaemia normalises insulin action on glucose metabolism but not the impaired activation of AKT and glycogen synthase in the skeletal muscle of patients with type 2 diabetes.

AIMS/HYPOTHESIS: In type 2 diabetes, reduced insulin-stimulated glucose disposal, primarily glycogen synthesis, is associated with defective insulin activation of glycogen synthase (GS) in skeletal muscle. Hyperglycaemia may compensate for these defects, but to what extent it involves improved insulin signalling to glycogen synthesis remains to be clarified. METHODS: Whole-body glucose metabolism was studied in 12 patients with type 2 diabetes, and 10 lean and 10 obese non-diabetic controls by means of indirect calorimetry and tracers during a euglycaemic-hyperinsulinaemic clamp. The diabetic patients underwent a second isoglycaemic-hyperinsulinaemic clamp maintaining fasting hyperglycaemia. Muscle biopsies from m. vastus lateralis were obtained before and after the clamp for examination of GS and relevant insulin signalling components. RESULTS: During euglycaemia, insulin-stimulated glucose disposal, glucose oxidation and non-oxidative glucose metabolism were reduced in the diabetic group compared with both control groups (p < 0.05). This was associated with impaired insulin-stimulated GS and AKT2 activity, deficient dephosphorylation at GS sites 2 + 2a, and reduced Thr308 and Ser473 phosphorylation of AKT. When studied under hyperglycaemia, all variables of insulin-stimulated glucose metabolism were normalised compared with the weight-matched controls. However, insulin activation and dephosphorylation (site 2 + 2a) of GS as well as activation of AKT2 and phosphorylation at Thr308 and Ser473 remained impaired (p < 0.05). CONCLUSIONS/INTERPRETATIONS: These data confirm that hyperglycaemia compensates for decreased whole-body glucose disposal in type 2 diabetes. In contrast to previous less well-controlled studies, we provide evidence that the compensatory effect of hyperglycaemia in patients with type 2 diabetes does not involve normalisation of insulin action on GS or upstream signalling in skeletal muscle.

Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent.

The aim of this study was to investigate the molecular mechanisms regulating FA translocase CD36 (FAT/CD36) translocation and FA uptake in skeletal muscle during contractions. In one model, wild-type (WT) and AMP-dependent protein kinase kinase dead (AMPK KD) mice were exercised or extensor digitorum longus (EDL) and soleus (SOL) muscles were contracted, ex vivo. In separate studies, FAT/CD36 translocation and FA uptake in response to muscle contractions were investigated in the perfused rat hindlimb. Exercise induced a similar increase in skeletal muscle cell surface membrane FAT/CD36 content in WT (+34%) and AMPK KD (+37%) mice. In contrast, 5-aminoimidazole-4-carboxamide ribonucleoside only induced an increase in cell surface FAT/CD36 content in WT (+29%) mice. Furthermore, in the perfused rat hindlimb, muscle contraction induced a rapid (1 min, +15%) and sustained (10 min, +24%) FAT/CD36 relocation to cell surface membranes. The increase in cell surface FAT/CD36 protein content with muscle contractions was associated with increased FA uptake, both in EDL and SOL muscle from WT and AMPK KD mice and in the perfused rat hindlimb. This suggests that AMPK is not essential in regulation of FAT/CD36 translocation and FA uptake in skeletal muscle during contractions. However, AMPK could be important in regulation of FAT/CD36 distribution in other physiological situations.

Impaired insulin-induced site-specific phosphorylation of TBC1 domain family, member 4 (TBC1D4) in skeletal muscle of type 2 diabetes patients is restored by endurance exercise-training.

AIMS/HYPOTHESIS: Insulin-mediated glucose disposal rates (R(d)) are reduced in type 2 diabetic patients, a process in which intrinsic signalling defects are thought to be involved. Phosphorylation of TBC1 domain family, member 4 (TBC1D4) is at present the most distal insulin receptor signalling event linked to glucose transport. In this study, we examined insulin action on site-specific phosphorylation of TBC1D4 and the effect of exercise training on insulin action and signalling to TBC1D4 in skeletal muscle from type 2 diabetic patients. METHODS: During a 3 h euglycaemic-hyperinsulinaemic (80 mU min⁻¹ m⁻²) clamp, we obtained M. vastus lateralis biopsies from 13 obese type 2 diabetic and 13 obese, non-diabetic control individuals before and after 10 weeks of endurance exercise-training. RESULTS: Before training, reductions in insulin-stimulated R (d), together with impaired insulin-stimulated glycogen synthase fractional velocity, Akt Thr³°8 phosphorylation and phosphorylation of TBC1D4 at Ser³¹8, Ser588 and Ser75¹ were observed in skeletal muscle from diabetic patients. Interestingly, exercise-training normalised insulin-induced TBC1D4 phosphorylation in diabetic patients. This happened independently of increased TBC1D4 protein content, but exercise-training did not normalise Akt phosphorylation in diabetic patients. In both groups, training-induced improvements in insulin-stimulated R(d) (~20%) were associated with increased muscle protein content of Akt, TBC1D4, α2-AMP-activated kinase (AMPK), glycogen synthase, hexokinase II and GLUT4 (20-75%). CONCLUSIONS/INTERPRETATION: Impaired insulin-induced site-specific TBC1D4 phosphorylation may contribute to skeletal muscle insulin resistance in type 2 diabetes. The mechanisms by which exercise-training improves insulin sensitivity in type 2 diabetes may involve augmented signalling of TBC1D4 and increased skeletal muscle content of key insulin signalling and effector proteins, e.g., Akt, TBC1D4, AMPK, glycogen synthase, GLUT4 and hexokinase II.

Differential aetiology and impact of phosphoinositide 3-kinase (PI3K) and Akt signalling in skeletal muscle on in vivo insulin action.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is a key factor in the development of type 2 diabetes and although some studies indicate that this could be partly attributed to reduced content and activity of various proximal and distal insulin signalling molecules, consensus is lacking. We therefore aimed to investigate the regulation of proximal insulin signalling in skeletal muscle and its effect on glucose metabolism in a large non-diabetic population. METHODS: We examined 184 non-diabetic twins with gold-standard techniques including the euglycaemic-hyperinsulinaemic clamp. Insulin signalling was evaluated at three key levels, i.e. the insulin receptor, IRS-1 and V-akt murine thymoma viral oncogene (Akt) levels, employing kinase assays and phospho-specific western blotting. RESULTS: Proximal insulin signalling was not associated with obesity, age or sex. However, birthweight was positively associated with IRS-1-associated phosphoinositide 3-kinase (PI3K; IRS-1-PI3K) activity (p = 0.04); maximal aerobic capacity (VO2(max)), paradoxically, was negatively associated with IRS-1-PI3K (p = 0.02) and Akt2 activity (p = 0.01). Additionally, we found low heritability estimates for most measures of insulin signalling activity. Glucose disposal was positively associated with Akt-308 phosphorylation (p < 0.001) and Akt2 activity (p = 0.05), but not with insulin receptor tyrosine kinase or IRS-1-PI3K activity. CONCLUSIONS/INTERPRETATION: With the exception of birthweight, 'classical' modifiers of insulin action, including genetics, age, sex, obesity and VO2(max) do not seem to mediate their most central effects on whole-body insulin sensitivity through modulation of proximal insulin signalling in skeletal muscle. We also demonstrated an association between Akt activity and in vivo insulin sensitivity, suggesting a role of Akt in control of in vivo insulin resistance and potentially in type 2 diabetes.

Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle.

5'AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and is regulated in muscle during exercise. We have previously established that only three of 12 possible AMPK alpha/beta/gamma-heterotrimers are present in human skeletal muscle. Previous studies describe discrepancies between total AMPK activity and regulation of its target acetyl-CoA-carboxylase (ACC)beta. Also, exercise training decreases expression of the regulatory gamma3 AMPK subunit and attenuates alpha2 AMPK activity during exercise. We hypothesize that these observations reflect a differential regulation of the AMPK heterotrimers. We provide evidence here that only the alpha2/beta2/gamma3 subunit is phosphorylated and activated during high-intensity exercise in vivo. The activity associated with the remaining two AMPK heterotrimers, alpha1/beta2/gamma1 and alpha2/beta2/gamma1, is either unchanged (20 min, 80% maximal oxygen uptake ) or decreased (30 or 120 s sprint-exercise). The differential activity of the heterotrimers leads to a total alpha-AMPK activity, that is decreased (30 s trial), unchanged (120 s trial) and increased (20 min trial). AMPK activity associated with the alpha2/beta2/gamma3 heterotrimer was strongly correlated to gamma3-associated alpha-Thr-172 AMPK phosphorylation (r(2) = 0.84, P < 0.001) and to ACCbeta Ser-221 phosphorylation (r(2) = 0.65, P < 0.001). These data single out the alpha2/beta2/gamma3 heterotrimer as an important actor in exercise-regulated AMPK signalling in human skeletal muscle, probably mediating phosphorylation of ACCbeta.

Transgenic models--a scientific tool to understand exercise-induced metabolism: the regulatory role of AMPK (5'-AMP-activated protein kinase) in glucose transport and glycogen synthase activity in skeletal muscle.

The AMPK (5'AMP-activated protein kinase) is becoming recognized as a critical regulator of energy metabolism. However, many of these effects in muscle metabolism have been ascribed to AMPK based on the use of the unspecific activator AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside). Using mouse models in which AMPK activity has been specifically blocked (kinase dead) or knocked out we and others have been able to conduct studies gaining more conclusive data on the role of AMPK in muscle metabolism. In this mini-review focus is on AMPK and its regulatory role for glucose transport and GS (glycogen synthase) activity in skeletal muscle, indicating that AMPK is a GS kinase in vivo which might influence GS activity during exercise and that AMPK is involved in AICAR/hypoxia-induced glucose transport but not or only partially in contraction-stimulated glucose transport.

Insulin signalling: effects of prior exercise.

After the discovery and clinical use of insulin for treatment of diabetes it became clear that some of the biological effect of insulin was dependent on the circumstances under which it was given. Relevant for this review is the notion that physical activity, in addition to its own direct metabolic effects also markedly affects the ability of insulin to stimulate a range of metabolic processes. More specifically, during and for a prolonged period after, exercise elicits effects on processes such as insulin-induced muscle glucose uptake and glucose metabolism which influence systemic glucose homeostasis. These phenomena are probably responsible for the improvement in glucose homeostasis and metabolic control that typically occurs with exercise in people with insulin resistance and probably contributes to the reduced risk for development of type 2 diabetes in individuals who engage in regular exercise. Here we focus on the influence of a single bout of exercise on the action of insulin on processes such as glucose uptake and glucose storage in skeletal muscle.