Flemming Dela

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

•  Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects. •  The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein, and complex I–IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres). •  Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content. •  mtDNA was found to be a poor biomarker of mitochondrial content. •  Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.

Circulating levels of TNF-alpha and IL-6-relation to truncal fat mass and muscle mass in healthy elderly individuals and in patients with type-2 diabetes

The purpose of the current study was to test the hypothesis that an altered fat distribution in elderly healthy subjects and in patients with type-2 diabetes contributes to high circulating levels of interleukin (IL)-6 and tumor necrotic factor (TNF)-alpha, which secondly is related to lower muscle mass. Twenty young controls, (20-35 yr), 20 healthy elderly subjects (65-80 yr) and 16 elderly patients with type 2 diabetes (65-80 yr) were included in a cross sectional study. Plasma levels of TNF-alpha and IL-6 were measured after an overnight fast. Dual-energy X-ray absorptiometry and total body potassium counting measured truncal fat, appendicular skeletal muscle mass (ASM) and body cell mass (BCM), respectively. TNF-alpha, IL-6 and the relative truncal fat mass were higher in elderly compared with young controls. ASM was lower in diabetic men than in young controls and BCM was lower in elderly men compared with young men. TNF-alpha and IL-6 were correlated with the absolute as well as the relative truncal fat mass in univariate regression analyses. Similar results were found in multivariate linear regression analyses after adjusting for the effect of age and gender. TNF-alpha was related to lower ASM and BCM in elderly men both in a univariate regression analysis and a multivariate regression analysis. In conclusion, high plasma levels of TNF-alpha and IL-6 in elderly healthy people and in patients with type 2 diabetes are associated with increased truncal fat mass, suggesting that cytokines are partly derived from this adipose tissue bed. Furthermore, TNF-alpha was related to lower ASM and BCM, suggesting that TNF-alpha contributes to sarcopenia in ageing.

Insulin-Stimulated Muscle Glucose Clearance in Patients With NIDDM: Effects of One-Legged Physical Training

Physical training increases insulin action in skeletal muscle in healthy men. In non-insulin-dependent diabetes mellitus (NIDDM), only minor improvements in whole-body insulin action are seen. We studied the effect of training on insulin-mediated glucose clearance rates (GCRs) in the whole body and in leg muscle in seven patients with NIDDM and in eight healthy control subjects. One-legged training was performed for 10 weeks. GCR in whole body and in both legs were measured before, the day after, and 6 days after training by hyperinsulinemic (28, 88, and 480 mU · min−1 · m−2), isoglycemic clamps combined with the leg balance technique. On the 5th day of detraining, one bout of exercise was performed with the nontraining leg. Muscle biopsies were obtained before and after training. Whole-body GCRs were always lower (P < 0.05) in NIDDM patients compared with control subjects and increased (P < 0.05) in response to training. In untrained muscle, GCR was lower (P < 0.05) in NIDDM patients (13 ± 4, 91 ± 9, and 148 ± 12 ml/min) compared with control subjects (56 ± 12, 126 ± 14, and 180 ± 14 ml/min). It Increased (P < 0.05) in both groups in response to training (43 ± 10, 144 ± 17, and 205 ± 24 [NIDDM patients] and 84 ± 10, 212 ± 20, and 249 ± 16 ml/min [control subjects]). Acute exercise did not increase leg GCR. In NIDDM patients, the effect of training was lost after 6 days, while the effect lasted longer in control subjects. Training increased (P < 0.05) muscle lactate production and glucose storage as well as glycogen synthase (GS) mRNA in both groups. We conclude that training increases insulin action in skeletal muscle in control subjects and NIDDM patients, and in NIDDM patients normal values may be obtained. The increase in trained muscle cannot fully account for the increase in whole-body GCR. Improvements in GCR involve enhancement of insulin-mediated increase in muscle blood flow and the ability to extract glucose. They are accompanied by enhanced nonoxidative glucose disposal and increases in GS mRNA. The improvements in insulin action are short-lived.

Physical Training Increases Muscle GLUT4 Protein and mRNA in Patients With NIDDM

Patients with non-insulin-dependent diabetes mellitus (NIDDM) exhibit insulin resistance and decreased glucose transport in skeletal muscle. Total content of muscle GLUT4 protein is not affected by NIDDM, whereas GLUT4 mRNA content is reported, variously, to be unaffected or increased. Physical training is recommended in the treatment of NIDDM, but the effect of training on muscle GLUT4 protein and mRNA content is unknown. To clarify the effect of training in NIDDM, seven men with NIDDM (58 ± 2 years of age [mean ± SE]) and eight healthy men (59 ± 1 years of age) (control group) performed one-legged ergometer bicycle training for 9 weeks, 6 days/week, 30 min/day. Biopsies were obtained from the vastus lateralis leg muscle before and after training. GLUT4 protein analyses was performed along with analyses of muscle biopsies from five young (23 ± 1 years of age) (young group), healthy subjects who participated in a previously published identical study. In response to training, maximal oxygen uptake increased (Δ 3.3 ± 1.8 in NIDDM subjects and 4.5 ± 1.2 ml.min−1·kg−1 in control subjects [both P < 0.05]). Before training, GLUT4 protein content was similar in NIDDM, control, and young subjects (0.35 ± 0.02, 0.34 ± 0.03, and 0.41 ± 0.03 arbitrary units, respectively), and it increased (P < 0.05) in all groups during training (to 0.43 ± 0.03, 0.40 ± 0.03, and 0.57 ± 0.08 arbitrary units, respectively). GLUT4 mRNA content was always lower in NIDDM compared with control subjects (P < 0.05) and increased in both groups (P < 0.05) during training (94 ± 6 to 122 ± 8 and 151 ± 5 to 170 ± 4 arbitrary units/10 μg total RNA, respectively). We conclude that muscle GLUT4 protein and mRNA increase in both NIDDM and control subjects in response to training. GLUT4 mRNA content is lower in NIDDM subjects compared with control subjects. GLUT4 protein content does not change with age.

Effects of preoperative oral carbohydrates and peptides on postoperative endocrine response, mobilization, nutrition and muscle function in abdominal surgery

Background: Surgery is succeeded by long‐lasting state of relative peripheral insulin resistance, which is reduced by giving glucose infusion or oral carbohydrate‐rich drinks immediate before operating instead of fasting. The aim of the present study was to investigate whether oral carbohydrate or carbohydrate with peptide drinks preoperatively instead of fasting would improve postoperative voluntary muscle strength, nutritional intake and ambulation, decrease postoperative fatigue, anxiety and discomfort, and reduce the endocrine response to surgery.

GLUT 4 and insulin receptor binding and kinase activity in trained human muscle.

1. Physical training enhances sensitivity and responsiveness of insulin‐mediated glucose uptake in human muscle. This study examines if this effect of physical training is due to increased insulin receptor function or increased total concentration of insulin‐recruitable glucose transporter protein (GLUT 4). 2. Seven healthy young subjects carried out single leg bicycle training for 10 weeks at 70% of one leg maximal oxygen uptake (VO2,max). Subsequently biopsies were taken from the vastus lateralis muscle of both legs. 3. Single leg VO2,max increased for the trained leg (46 +/‐ 3 to 52 +/‐ 2 ml min‐1 kg‐1 (means +/‐ S.E.M., P < 0.05), and cytochrome c oxidase activity was higher in this compared to the untrained leg (2.0 +/‐ 0.1 vs. 1.4 +/‐ 0.1 nmol s‐1 (mg muscle)‐1, P < 0.05). Insulin binding as well as basal‐ and insulin‐stimulated receptor kinase activity did not differ between trained and untrained muscle. The concentration of GLUT 4 protein was higher in the former (14.9 +/‐ 1.9 vs. 11.6 +/‐ 1.0 arbitrary units (micrograms protein)‐1 in crude membranes, P < 0.05). The training‐induced increase in GLUT 4 (26 +/‐ 11%) matched a previously reported increase in maximum insulin‐stimulated leg glucose uptake (25 +/‐ 7%) in the same subjects, and individual values of the two variables correlated (correlation coefficient (r) = 0.84, P < 0.05). 4. In conclusion, in human muscle training induces a local contraction‐dependent increase in GLUT 4 protein, which enhances the effect of insulin on glucose uptake. On the other hand, insulin receptor function in muscle is unlikely to be affected by training.

The effect of altitude hypoxia on glucose homeostasis in men

1 Exposure to altitude hypoxia elicits changes in glucose homeostasis with increases in glucose and insulin concentrations within the first few days at altitude. Both increased and unchanged hepatic glucose production (HGP) have previously been reported in response to acute altitude hypoxia. Insulin action on glucose uptake has never been investigated during altitude hypoxia. 2 In eight healthy, sea level resident men (27 ± 1 years (mean ± S.E.M.); weight, 72 ± 2 kg; height, 182 ± 2 cm) hyperinsulinaemic (50 mU min−1 m−2), euglycaemic clamps were carried out at sea level, and subsequently on days 2 and 7 after a rapid passive ascent to an altitude of 4559 m. 3 Acute mountain sickness scores increased in the first days of altitude exposure, with a peak on day 2. Basal HGP did not change with the transition from sea level (2.2 ± 0.2 mg min− kg−1) to altitude (2.0 ± 0.1 and 2.1 ± 0.2 mg min−1 kg−1, days 2 and 7, respectively). Insulin‐stimulated glucose uptake rate was halved on day two compared with sea level (4.5 ± 0.6 and 9.8 ± 1.1 mg min−1 kg−1, respectively; P < 0.05), and was partly restored on day 7 (7.4 ± 1.4 mg min−1 kg−1; P < 0.05vs. day two and sea level). Concentrations of glucagon and growth hormone remained unchanged, whereas glucose, C‐peptide and cortisol increased on day 2. Noradrenaline concentrations increased during the stay at altitude, while adrenaline concentrations remained unchanged. In response to insulin infusion, catecholamines increased on day 2 (noradrenaline and adrenaline) and day 7 (adrenaline), but not at sea level. 4 In conclusion, insulin action decreases markedly in response to two days of altitude hypoxia, but improves with more prolonged exposure. HGP is always unchanged. The changes in insulin action may in part be explained by the changes in counter‐regulatory hormones.

5′AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes

Strength training enhances insulin sensitivity and represents an alternative to endurance training for patients with type 2 diabetes (T2DM). The 5′AMP‐activated protein kinase (AMPK) may mediate adaptations in skeletal muscle in response to exercise training; however, little is known about adaptations within the AMPK system itself. We investigated the effect of strength training and T2DM on the isoform expression and the heterotrimeric composition of the AMPK in human skeletal muscle. Ten patients with T2DM and seven healthy subjects strength trained (T) one leg for 6 weeks, while the other leg remained untrained (UT). Muscle biopsies were obtained before and after the training period. Basal AMPK activity and protein/mRNA expression of both catalytic (α1 and α2) and regulatory (β1, β2, γ1, γ2a, γ2b and γ3) AMPK isoforms were independent of T2DM, whereas the protein content of α1 (+16%), β2 (+14%) and γ1 (+29%) was higher and the γ3 content was lower (−48%) in trained compared with untrained muscle (all P < 0.01). The majority of α protein co‐immunoprecipitated with β2 and α2/β2 accounted for the majority of these complexes. γ3 was only associated with α2 and β2 subunits, and accounted for ∼20% of all α2/β2 complexes. The remaining α2/β2 and the α1/β2 complexes were associated with γ1. The trimer composition was unaffected by T2DM, whereas training induced a shift from γ3‐ to γ1‐containing trimers. The data question muscular AMPK as a primary cause of T2DM whereas the maintained function in patients with T2DM makes muscular AMPK an obvious therapeutic target. In human skeletal muscle only three of 12 possible AMPK trimer combinations exist, and the expression of the subunit isoforms is susceptible to moderate strength training, which may influence metabolism and improve energy homeostasis in trained muscle.

The effect of moderate exercise on postprandial glucose homeostasis in NIDDM patients

Summary The influence of exercise on glycaemia in the post-prandial state was studied for the first time in non-insulin-dependent diabetic (NIDDM) patients. Meal-induced glucose responses were followed for 8 h in 9 diet-treated patients with NIDDM. Subjects consumed a standardized breakfast and 4 h later a standardized lunch. They were studied in the resting state (control day (CD)) and on another day 45 min of bicycle exercise (53 ± 2 % V˙O2max (mean ± SEM)) was performed 45 min after breakfast (exercise day (ED)). On day 3 (diet day (DD)), the breakfast meal was reduced corresponding to the extra energy expenditure during the exercise period on ED. Responses were calculated as areas under the plasma concentration curve (AUC) during 4 h after either breakfast (B-AUC) or lunch (L-AUC). B-AUC for glucose was identical on ED (215 ± 63 mmol/l · 240 min) and DD (219 ± 60 mmol/l · 240 min) and on these days lower (p < 0.05) than on CD (453 ± 78 mmol/l · 240 min). L-AUC for glucose on CD, ED and DD did not differ significantly. B-AUCs for both insulin and C-peptide were also significantly lower on ED and DD as compared to CD (Insulin: 31337 ± 8682, 26092 ± 6457 and 47649 ± 15046 mmol/l · 240 min, respectively. C-peptide: 99 ± 19, 104 ± 26 and 195 ± 31 pmol/ml · 240 min, respectively). Rate of appearance (Ra) for glucose was unaffected by exercise whereas rate of disappearance (Rd) increased significantly. No differences in Ra or Rd were observed after lunch. In conclusion, postprandial exercise of moderate intensity decreases glycaemia and plasma insulin levels after breakfast in NIDDM patients, but this effect does not persist during and after the following lunch meal. Reduction of breakfast caloric intake has the same effect on postprandial glycaemia and insulin secretion as an equivalent exercise-induced increase in caloric expenditure. [Diabetologia (1997) 40: 447–453]

Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men

Physical inactivity is a risk factor for insulin resistance. We examined the effect of 9 days of bed rest on basal and insulin-stimulated expression of genes potentially involved in insulin action by applying hypothesis-generating microarray in parallel with candidate gene real-time PCR approaches in 20 healthy young men. Furthermore, we investigated whether bed rest affected DNA methylation in the promoter region of the peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) gene. Subjects were reexamined after 4 wk of retraining. We found that bed rest induced insulin resistance and altered the expression of more than 4,500 genes. These changes were only partly normalized after 4 wk of retraining. Pathway analyses revealed significant downregulation of 34 pathways, predominantly those of genes associated with mitochondrial function, including PPARGC1A. Despite induction of insulin resistance, bed rest resulted in a paradoxically increased response to acute insulin stimulation in the general expression of genes, particularly those involved in inflammation and endoplasmatic reticulum (ER) stress. Furthermore, bed rest changed gene expressions of several insulin resistance and diabetes candidate genes. We also observed a trend toward increased PPARGC1A DNA methylation after bed rest. We conclude that impaired expression of PPARGC1A and other genes involved in mitochondrial function as well as a paradoxically increased response to insulin of genes involved in inflammation and ER stress may contribute to the development of insulin resistance induced by bed rest. Lack of complete normalization of changes after 4 wk of retraining underscores the importance of maintaining a minimum of daily physical activity.