Christian Frøsig

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.

Effects of Endurance Exercise Training on Insulin Signaling in Human Skeletal Muscle Interactions at the Level of Phosphatidylinositol 3-Kinase, Akt, and AS160

The purpose of this study was to investigate the mechanisms explaining improved insulin-stimulated glucose uptake after exercise training in human skeletal muscle. Eight healthy men performed 3 weeks of one-legged knee extensor endurance exercise training. Fifteen hours after the last exercise bout, insulin-stimulated glucose uptake was ∼60% higher (P < 0.01) in the trained compared with the untrained leg during a hyperinsulinemic-euglycemic clamp. Muscle biopsies were obtained before and after training as well as after 10 and 120 min of insulin stimulation in both legs. Protein content of Akt1/2 (55 ± 17%, P < 0.05), AS160 (25 ± 8%, P = 0.08), GLUT4 (52 ± 19%, P < 0.001), hexokinase 2 (HK2) (197 ± 40%, P < 0.001), and insulin-responsive aminopeptidase (65 ± 15%, P < 0.001) increased in muscle in response to training. During hyperinsulinemia, activities of insulin receptor substrate-1 (IRS-1)–associated phosphatidylinositol 3-kinase (PI3-K) (P < 0.005), Akt1 (P < 0.05), Akt2 (P < 0.005), and glycogen synthase (GS) (percent I-form, P < 0.05) increased similarly in both trained and untrained muscle, consistent with increased phosphorylation of Akt Thr308, Akt Ser473, AS160, glycogen synthase kinase (GSK)-3α Ser21, and GSK-3β Ser9 and decreased phosphorylation of GS site 3a+b (all P < 0.005). Interestingly, training improved insulin action on thigh blood flow, and, furthermore, in both basal and insulin-stimulated muscle tissue, activities of Akt1 and GS and phosphorylation of AS160 increased with training (all P < 0.05). In contrast, training reduced IRS-1–associated PI3-K activity (P < 0.05) in both basal and insulin-stimulated muscle tissue. Our findings do not support generally improved insulin signaling after endurance training; rather it seems that improved insulin-stimulated glucose uptake may result from hemodynamic adaptations as well as increased cellular protein content of individual insulin signaling components and molecules involved in glucose transport and metabolism.

Impaired Insulin-Stimulated Phosphorylation of Akt and AS160 in Skeletal Muscle of Women With Polycystic Ovary Syndrome Is Reversed by Pioglitazone Treatment

OBJECTIVE— Insulin resistance in skeletal muscle is a major risk factor for type 2 diabetes in women with polycystic ovary syndrome (PCOS). However, the molecular mechanisms underlying skeletal muscle insulin resistance and the insulin-sensitizing effect of thiazolidinediones in PCOS in vivo are less well characterized. RESEARCH DESIGN AND METHODS— We determined molecular mediators of insulin signaling to glucose transport in skeletal muscle biopsies of 24 PCOS patients and 14 matched control subjects metabolically characterized by euglycemic-hyperinsulinemic clamps and indirect calorimetry, and we examined the effect of 16 weeks of treatment with pioglitazone in PCOS patients. RESULTS— Impaired insulin-mediated total (Rd) oxidative and nonoxidative glucose disposal (NOGD) was paralleled by reduced insulin-stimulated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation in muscle of PCOS patients. Akt phosphorylation at Ser473 and Thr308 correlated positively with Rd and NOGD in the insulin-stimulated state. Serum free testosterone was inversely related to insulin-stimulated Rd and NOGD in PCOS. Importantly, the pioglitazone-mediated improvement in insulin-stimulated glucose metabolism, which did not fully reach normal levels, was accompanied by normalization of insulin-mediated Akt phosphorylation at Ser473 and Thr308 and AS160 phosphorylation. AMPK activity and phosphorylation were similar in the two groups and did not respond to pioglitazone in PCOS patients. CONCLUSIONS— Impaired insulin signaling through Akt and AS160 in part explains insulin resistance at the molecular level in skeletal muscle in PCOS, and the ability of pioglitazone to enhance insulin sensitivity involves improved signaling through Akt and AS160. Moreover, our data provide correlative evidence that hyperandrogenism in PCOS may contribute to insulin resistance.

Improved insulin sensitivity after exercise: focus on insulin signaling.

After a single bout of exercise, the ability of insulin to stimulate glucose uptake is markedly improved locally in the previously active muscles. This makes exercise a potent stimulus counteracting insulin resistance characterizing type 2 diabetes (T2D). It is believed that at least part of the mechanism relates to an improved ability of insulin to stimulate translocation of glucose transporters (GLUT4) to the muscle membrane after exercise. How this is accomplished is still unclear; however, an obvious possibility is that exercise interacts with the insulin signaling pathway to GLUT4 translocation allowing for a more potent insulin response. Parallel to unraveling of the insulin signaling cascade, this has been investigated within the past 25 years. Reviewing existing studies clearly indicates that improved insulin action can occur independent of interactions with proximal insulin signaling. In contrast, more recent observations indicate that interactions exist at the distal signaling level of AS160 and atypical protein kinase C (aPKC). Although the functional interpretation is lacking, these novel observations may present a breakthrough in understanding the beneficial interplay between exercise and insulin action.

Lipid-Induced Insulin Resistance Affects Women Less Than Men and Is Not Accompanied by Inflammation or Impaired Proximal Insulin Signaling

OBJECTIVE We have previously shown that overnight fasted women have higher insulin-stimulated whole body and leg glucose uptake despite a higher intramyocellular triacylglycerol concentration than men. Women also express higher muscle mRNA levels of proteins related to lipid metabolism than men. We therefore hypothesized that women would be less prone to lipid-induced insulin resistance. RESEARCH DESIGN AND METHODS Insulin sensitivity of whole-body and leg glucose disposal was studied in 16 young well-matched healthy men and women infused with intralipid or saline for 7 h. Muscle biopsies were obtained before and during a euglycemic-hyperinsulinemic clamp (1.42 mU · kg−1 · min−1). RESULTS Intralipid infusion reduced whole-body glucose infusion rate by 26% in women and 38% in men (P < 0.05), and insulin-stimulated leg glucose uptake was reduced significantly less in women (45%) than men (60%) after intralipid infusion. Hepatic glucose production was decreased during the clamp similarly in women and men irrespective of intralipid infusion. Intralipid did not impair insulin or AMPK signaling in muscle and subcutaneous fat, did not cause accumulation of muscle lipid intermediates, and did not impair insulin-stimulated glycogen synthase activity in muscle or increase plasma concentrations of inflammatory cytokines. In vitro glucose transport in giant sarcolemmal vesicles was not decreased by acute exposure to fatty acids. Leg lactate release was increased and respiratory exchange ratio was decreased by intralipid. CONCLUSIONS Intralipid infusion causes less insulin resistance of muscle glucose uptake in women than in men. This insulin resistance is not due to decreased canonical insulin signaling, accumulation of lipid intermediates, inflammation, or direct inhibition of GLUT activity. Rather, a higher leg lactate release and lower glucose oxidation with intralipid infusion may suggest a metabolic feedback regulation of glucose metabolism.

Nitric oxide production is a proximal signaling event controlling exercise-induced mRNA expression in human skeletal muscle

Previous studies have described the magnitude and time course by which several genes are regu‐lated within exercising skeletal muscle. These include interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), heme oxygen‐ase‐1 (HO‐1), and heat shock protein‐72 (HSP72), which are involved in secondary signaling and preservation of intracellular environment. However, the primary signaling mechanisms coupling contraction to transcription are unknown. We hypothesized that exercise‐induced nitric oxide (NO) production is an important signaling event for IL‐6, IL‐8, HO‐1, and HSP72 expression in muscle. Twenty healthy males participated in the study. By realtime PCR, mRNA levels for 11 genes were determined in thigh muscle biopsies obtained 1) before and after 2 h knee extensor exercise without (control) and with con‐comitant NO synthase inhibition (nitro‐L‐arginine methyl ester, L‐NAME, 5 mgkg_1);or 2) before and after 2 h femoral artery infusion of the NO donor nitroglycerin (NTG, 1.5 μgkg_1min_1). L‐NAME caused marked reductions in exercise‐induced expression of 4 of 11 mRNAs including IL‐6, IL‐8, and HO‐1. IL‐6 protein release from the study leg to the circulation increased in the control but not in the L‐NAME trial. NTG infusion significantly augmented expression of the mRNAs attenuated by L‐NAME. These findings advance the novel concept that NO production contributes to regulation of gene expression in muscle during exercise. Subsequently, we sought evidence for involvement of AMP‐activated kinase or nuclear factor kappa B, but found none.—Steensberg, A., Keller, C., Hillig, T., Frosig, C., Wojtaszewski, J. F. P., Pedersen, B. K., Pilegaard, H., Sander, M. Nitric oxide production is a proximal signaling event controlling exercise‐induced mRNA expression in human skeletal muscle. FASEB J. 21, 2683–2694 (2007)

Effect of endurance exercise training on Ca2+–calmodulin-dependent protein kinase II expression and signalling in skeletal muscle of humans

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.

Exercise‐induced TBC1D1 Ser237 phosphorylation and 14‐3‐3 protein binding capacity in human skeletal muscle

TBC1D1 is a Rab‐GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise‐induced regulation of TBC1D1 Ser237 phosphorylation and 14‐3‐3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all‐out cycle exercise lasting either 30 s, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (∼70–230%, P < 0.005), with the greatest response observed after 20 min of cycling. Interestingly, capacity of TBC1D1 to bind 14‐3‐3 protein showed a similar pattern of regulation, increasing 60–250% (P < 0.001). Furthermore, recombinant 5′AMP‐activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14‐3‐3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock‐out and wild‐type mice were stimulated to contract in vitro. In wild‐type and α1 knock‐out mice, contractions resulted in a similar ∼100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock‐out mice were characterized by reduced protein content of TBC1D1 (∼50%, P < 0.001) as well as in basal and contraction‐stimulated (∼60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14‐3‐3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle.

AMP‐activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle

•  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.

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.