Anne-Marie Lundsgaard

Gender Differences in Skeletal Muscle Substrate Metabolism – Molecular Mechanisms and Insulin Sensitivity

It has become increasingly apparent that substrate metabolism is subject to gender-specific regulation, and the aim of this review is to outline the available evidence of molecular gender differences in glucose and lipid metabolism of skeletal muscle. Female sex has been suggested to have a favorable effect on glucose homeostasis, and the available evidence from hyperinsulinemic–euglycemic clamp studies is summarized to delineate whether there is a gender difference in whole-body insulin sensitivity and in particular insulin-stimulated glucose uptake of skeletal muscle. Whether an eventual higher insulin sensitivity of female skeletal muscle can be related to gender-specific regulation of molecular metabolism will be topic for discussion. Gender differences in muscle fiber type distribution and substrate availability to and in skeletal muscle are highly relevant for substrate metabolism in men and women. In particular, the molecular machinery for glucose and fatty acid oxidative and storage capacities in skeletal muscle and its implications for substrate utilization during metabolic situations of daily living are discussed, emphasizing their relevance for substrate choice in the fed and fasted state, and during periods of physical activity and recovery. Together, handling of carbohydrate and lipids and regulation of their utilization in skeletal muscle have implications for whole-body glucose homeostasis in men and women. 17-β estradiol is the most important female sex hormone, and the identification of estradiol receptors in skeletal muscle has opened for a role in regulation of substrate metabolism. Also, higher levels of circulating adipokines as adiponectin and leptin in women and their implications for muscle metabolism will be considered.

Regulation of autophagy in human skeletal muscle: effects of exercise, exercise training and insulin stimulation.

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.

Circulating FGF21 in humans is potently induced by short term overfeeding of carbohydrates

Objective Fibroblast-growth factor 21 (FGF21) is thought to be important in metabolic regulation. Recently, low protein diets have been shown to increase circulating FGF21 levels. However, when energy contribution from dietary protein is lowered, other macronutrients, such as carbohydrates, must be increased to meet eucaloric balance. This raises the possibility that intake of a diet rich in carbohydrates may induce an increase in plasma FGF21 levels per se. Here we studied the role of dietary carbohydrates on the levels of circulating FGF21 and concomitant physiologic effects by feeding healthy men a carbohydrate rich diet without reducing protein intake. Methods A diet enriched in carbohydrates (80 E% carbohydrate; CHO) and a eucaloric control diet (CON) were provided to nine healthy men for three days. The energy intake during the CHO diet was increased (+75% energy) to ensure similar dietary protein intake in CHO and CON. To control for the effect of caloric surplus, we similarly overfed (+75% energy) the same subjects for three days with a fat-rich diet (78 E% fat; FAT), consisting of primarily unsaturated fatty acids. The three diets were provided in random order. Results After CHO, plasma FGF21 concentration increased 8-fold compared to CON (329 ± 99 vs. 39 ± 9 pg ml−1, p < 0.05). In contrast, after FAT only a non-significant tendency (p = 0.073) to an increase in plasma FGF21 concentration was found. The increase in FGF21 concentration after CHO correlated closely (r = 0.88, p < 0.01) with increased leg glucose uptake (62%, p < 0.05) and increased hepatic glucose production (17%, p < 0.01), indicating increased glucose turnover. Plasma fatty acid (FA) concentration was decreased by 68% (p < 0.01), supported by reduced subcutaneous adipose tissue HSL Ser660 phosphorylation (p < 0.01) and perilipin 1 protein content (p < 0.01), pointing to a suppression of adipose tissue lipolysis. Concomitantly, a 146% increase in the plasma marker of hepatic de novo lipogenesis C16:1 n−7 FA (p < 0.01) was observed together with 101% increased plasma TG concentration (p < 0.001) in association with CHO intake and increased plasma FGF21 concentration. Conclusion Excess dietary carbohydrate, but not fat, led to markedly increased FGF21 secretion in humans, notably without protein restriction, and affected glucose and lipid homeostais.

New Nordic Diet-Induced Weight Loss Is Accompanied by Changes in Metabolism and AMPK Signaling in Adipose Tissue.

CONTEXT The molecular mechanisms behind diet-induced metabolic improvements remain to be studied. OBJECTIVE This study sought to investigate whether expression of proteins in skeletal muscle or adipose tissue could explain improvements in glucose and lipid homeostasis after weight loss. DESIGN Volunteers consumed a New Nordic Diet (NND) or an Average Danish Diet for 26 weeks in a controlled, free-living setting. SUBJECTS Sixty four moderately obese women and men (44 ± 2 y; body mass index, 31 ± 1 kg/m(2)). INTERVENTION Fasting blood samples and biopsies from the vastus lateralis muscle and subcutaneous abdominal adipose tissue (SCAT) were obtained at week 0 and 26. OUTCOME Gene and protein expressions were analyzed by real-time PCR and Western blotting. RESULTS Improved homeostasis homeostatic model of assessment-insulin resistance index and lowered plasma triacylglycerol concentration after NND coincided with molecular adaptations in SCAT but not in skeletal muscle. NND induced greater reduction in fat mass than ADD (-6 ± 1 kg and -2 ± 1 kg; P < .01). In SCAT this was associated with increased AMPK and acetyl-CoA carboxylase phosphorylation (P < .05). Concomitantly, NND induced up-regulation of Akt2 and Akt substrate of 160 kDa (P < .05) as well as fatty acid transport protein 4 and membrane associated fatty acid binding protein (P < .05). Indices of increased oxidative capacity were observed, as carnitine palmitoyl transferase 1 mRNA (P = .08) as well as citrate synthase (P = .1) and cytochrome c (P = .05) protein tended to increase. CONCLUSION NND-induced metabolic improvements were accompanied by increased AMPK signaling in SCAT, suggesting a role of AMPK in these adaptations. The concomitant up-regulation of key glucose and lipid-handling proteins suggests an improved metabolic capacity in adipose tissue after weight loss.

Adiponectin concentration is associated with muscle insulin sensitivity, AMPK phosphorylation, and ceramide content in skeletal muscles of men but not women.

Adiponectin is an adipokine that regulates metabolism and increases insulin sensitivity. Mechanisms behind this insulin-sensitizing effect have been investigated in rodents, but little is known in humans, especially in skeletal muscle. Women have higher serum concentrations of adiponectin than men and are generally more insulin sensitive in skeletal muscle than men. We show here that large differences exist between men and women with regard to apparent adiponectin regulation of insulin-stimulated glucose uptake in skeletal muscle. Serum adiponectin was significantly associated with leg glucose uptake in healthy, young, lean men, but the association was absent in women. In addition, serum adiponectin was significantly associated with AMP-activated protein kinase (AMPK) phosphorylation in skeletal muscles of men but not in women. Serum adiponectin was also significantly, negatively associated with skeletal muscle ceramide content in men only, and interestingly, ceramide content was negatively associated with adiponectin receptor 1 (AdipoR1) expression in skeletal muscles of men. Women had lower AdipoR1 expression in skeletal muscle and a lower percentage of glycolytic adiponectin-sensitive type 2 muscle fibers than men. These associations suggest that the insulin-sensitizing effect of adiponectin on human male skeletal muscles may be mediated via AdipoR1 to activation of AMPK, leading to lowering of ceramide content. The lower skeletal muscle AdipoR1 protein expression and lower expression of adiponectin-sensitive type 2 muscle fibers in women than in men may explain the apparent lesser sensitivity to adiponectin in women.

Role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle

During induction of the autophagosomal degradation process, LC3-I is lipidated to LC3-II and associates to the cargo isolation membrane allowing for autophagosome formation. Lipidation of LC3 results in an increased LC3-II/LC3-I ratio, and this ratio is an often used marker for autophagy in various tissues, including skeletal muscle. From cell studies AMPK has been proposed to be necessary and sufficient for LC3 lipidation. The aim of the present study was to investigate the role of AMPK in regulation of LC3 lipidation as a marker of autophagy in skeletal muscle. We observed an increase in the LC3-II/LC3-I ratio in skeletal muscle of AMPKα2 kinase-dead (KD) (p<0.001) and wild type (WT) (p<0.05) mice after 12h of fasting, which was greater (p<0.05) in AMPKα2 KD mice than in WT. The fasting-induced increase in the LC3-II/LC3-I ratio in both genotypes coincided with an initial decrease (p<0.01) in plasma insulin concentration, a subsequent decrease in muscle mTORC1 signaling and increased (p<0.05) levels of the autophagy-promoting proteins, FoxO3a and ULK1. Furthermore, a higher (p<0.01) LC3-II/LC3-I ratio was observed in old compared to young mice. We were not able to detect any change in LC3 lipidation with either in vivo treadmill exercise or in situ contractions. Collectively, these findings suggest that AMPKα2 is not necessary for induction of LC3 lipidation with fasting and aging. Furthermore, LC3 lipidation is increased in muscle lacking functional AMPKα2 during fasting and aging. Moreover, LC3 lipidation seems not to be a universal response to muscle contraction in mice.

5′‐AMP activated protein kinase α2 controls substrate metabolism during post‐exercise recovery via regulation of pyruvate dehydrogenase kinase 4

There is lower fat oxidation during post‐exercise recovery in mice lacking 5′‐AMP activated protein kinase α2 (AMPKα2). AMPKα2 is involved in post‐transcriptional and not transcriptional regulation of pyruvate dehydrogenase kinase 4 (PDK4) in muscle. Exercise‐induced AMPKα2 activity increases PDK4 protein content, in turn inhibiting pyruvate dehydrogenase activity and glucose oxidation. The mechanism for increased post‐exercise fat oxidation is by inhibition of carbohydrate oxidation allowing increased fat oxidation rather than by direct stimulation of fat oxidation.

Differential effects of glucagon‐like peptide‐1 on microvascular recruitment and glucose metabolism in short‐ and long‐term insulin resistance

Acute glucagon‐like peptide‐1 (GLP‐1) infusion reversed the high fat diet‐induced microvascular insulin resistance that occurred after both 5 days and 8 weeks of a high fat diet intervention. When GLP‐1 was co‐infused with insulin it had overt effects on whole body insulin sensitivity as well as insulin‐mediated skeletal muscle glucose uptake after 5 days of a high fat diet, but not after 8 weeks of high fat diet intervention. Acute GLP‐1 infusion did not have an additive effect to that of insulin on microvascular recruitment or skeletal muscle glucose uptake in the control group. Here we demonstrate that GLP‐1 potently increases the microvascular recruitment in rat skeletal muscle but does not increase glucose uptake in the fasting state. Thus, like insulin, GLP‐1 increased the microvascular recruitment but unlike insulin, GLP‐1 had no direct effect on skeletal muscle glucose uptake.

Opposite Regulation of Insulin Sensitivity by Dietary Lipid Versus Carbohydrate Excess

To understand the mechanisms in lipid-induced insulin resistance, a more physiological approach is to enhance fatty acid (FA) availability through the diet. Nine healthy men ingested two hypercaloric diets (in 75% excess of habitual caloric intake) for 3 days, enriched in unsaturated FA (78 energy % [E%] fat) (UNSAT) or carbohydrates (80 E% carbohydrate) (CHO) as well as a eucaloric control diet (CON). Compared with CON, the UNSAT diet reduced whole-body and leg glucose disposal during a hyperinsulinemic-euglycemic clamp, while decreasing hepatic glucose production. In muscle, diacylglycerol (DAG) and intramyocellular triacylglycerol were increased. The accumulated DAG was sn-1,3 DAG, which is known not to activate PKC, and insulin signaling was intact. UNSAT decreased PDH-E1α protein content and increased inhibitory PDH-E1α Ser300 phosphorylation and FA oxidation. CHO increased whole-body and leg insulin sensitivity, while increasing hepatic glucose production. After CHO, muscle PDH-E1α Ser300 phosphorylation was decreased, and glucose oxidation increased. After UNSAT, but not CHO, muscle glucose-6-phosphate content was 103% higher compared with CON during the clamp. Thus, PDH-E1α expression and covalent regulation, and hence the tricarboxylic acid cycle influx of pyruvate-derived acetyl-CoA relative to β-oxidation–derived acetyl-CoA, are suggested to impact on insulin-stimulated glucose uptake. Taken together, the oxidative metabolic fluxes of glucose and FA are powerful and opposite regulators of insulin action in muscle.

Molecular Regulation of Fatty Acid Oxidation in Skeletal Muscle during Aerobic Exercise

This review summarizes how fatty acid (FA) oxidation is regulated in skeletal muscle during exercise. From the available evidence it seems that acetyl-CoA availability in the mitochondrial matrix adjusts FA oxidation to exercise intensity and duration. This is executed at the step of mitochondrial fatty acyl import, as the extent of acetyl group sequestration by carnitine determines the availability of carnitine for the carnitine palmitoyltransferase 1 (CPT1) reaction. The rate of glycolysis seems therefore to be central to the amount of β-oxidation-derived acetyl-CoA that is oxidized in the tricarboxylic acid (TCA) cycle. FA oxidation during exercise is also determined by FA availability to mitochondria, dependent on trans-sarcolemmal FA uptake via cluster of differentiation 36/SR-B2 (CD36) and FAs mobilized from myocellular lipid droplets.