Adipokines promote lipotoxicity in human skeletal muscle cells

Studies have shown the implication of specific adipokines or fatty acids (FA) in the pathogenesis of insulin resistance. However, the interplay of adipokines with FA remains poorly understood. This study aimed to investigate the combined effects of adipokines and low concentrations of palmitic acid (PA, 100 μmol/l) on skeletal muscle metabolism. Human skeletal muscle cells were incubated with adipocyte-conditioned medium (CM), PA or PA+CM, and FA transporter and FA metabolism were analysed. CM-incubation increased CD36 level (1.8 fold) and PA-uptake (1.4 fold). However, only co-application of PA+CM resulted in profound lipid accumulation (5.3 fold), 60% reduction of PA-oxidation and 3.5 fold increased diacylglycerol content. Our results support a novel role for adipokines in the pathogenesis of T2D by increasing the lipotoxic potential of PA, notably of low concentrations. This implies an increased lipotoxic risk already at an early stage of weight gain, when lipolysis has not yet contributed to increased plasma free FA levels.     

Insulin degradation by human skeletal muscle

Although previous studies from this and other laboratories have extensively characterized insulin degrading activity in animal tissues, little information has been available on insulin responsive human tissues. The present study describes the insulin degrading activity in skeletal muscle from normal human subjects. Fractionation of a sucrose homogenate of skeletal muscle demonstrated that 97% of the total neutral insulin degrading activity was in the 100 000 × g supernatant with no detectable glutathione-insulin transhydrogenase activity. The 100 000×g pellet contained 85% of the total acid protease activity and all the glutathione-insulin transhydrogenase activity. The soluble insulin degrading activity was purified 1400-fold by ammonium sulfate fractionation, molecular exclusion, ion-exchange and affinity chromatography. Enzymatic activity was determined by measuring an increase in trichloroacetic acid-soluble products of the ¹²⁵I-labeled hormone substrates. The purified enzyme showed marked proteolytic specificity for insulin with a K_m of 1.63·10^?7 M (±0.32) and was competitively inhibited by proinsulin and glucagon with K_i values of 2.1 · 10?⁶ M and 4.0 · 10^?6 M, respectively. This insulin protease exhibited a pH optimum between 7 and 8, a molecular weight of 120 000 and was capable of degrading glucagon. Inhibition studies demonstrated that a sulfhydryl group is essential for activity. Molecular exclusion chromatography of [¹²⁵I]insulin degraded products revealed a time-dependent increase in degradation products with molecular weights intermediate between intact insulin and iodotyrosine. These studies demonstrate that the major enzymatic system responsible for insulin degrading activity is a soluble cysteine protease capable of rapidly metabolizing insulin under physiologic conditions.     

Metabolic switching of human skeletal muscle cells in vitro

In this review we will focus on external factors that may modify energy metabolism in human skeletal muscle cells (myotubes) and the ability of the myotubes to switch between lipid and glucose oxidation. We describe the metabolic parameters suppressibility, adaptability and substrate-regulated flexibility, and show the influence of nutrients such as fatty acids and glucose (chronic hyperglycemia), and some pharmacological agents modifying nuclear receptors (PPAR and LXR), on these parameters in human myotubes. Possible cellular mechanisms for changes in these parameters will also be highlighted.     

Secretome profiling of primary human skeletal muscle cells

The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.     

Growth factors in skeletal muscle regeneration

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGFβs, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGFβ has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Glutathione peroxidase 3 mediates the antioxidant effect of peroxisome proliferator-activated receptor gamma in human skeletal muscle cells

Oxidative stress plays an important role in the pathogenesis of insulin resistance and type 2 diabetes mellitus and in diabetic vascular complications. Thiazolidinediones (TZDs), a class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, improve insulin sensitivity and are currently used for the treatment of type 2 diabetes mellitus. Here, we show that TZD prevents oxidative stress-induced insulin resistance in human skeletal muscle cells, as indicated by the increase in insulin-stimulated glucose uptake and insulin signaling. Importantly, TZD-mediated activation of PPARgamma induces gene expression of glutathione peroxidase 3 (GPx3), which reduces extracellular H(2)O(2) levels causing insulin resistance in skeletal muscle cells. Inhibition of GPx3 expression prevents the antioxidant effects of TZDs on insulin action in oxidative stress-induced insulin-resistant cells, suggesting that GPx3 is required for the regulation of PPARgamma-mediated antioxidant effects. Furthermore, reduced plasma GPx3 levels were found in patients with type 2 diabetes mellitus and in db/db/DIO mice. Collectively, these results suggest that the antioxidant effect of PPARgamma is exclusively mediated by GPx3 and further imply that GPx3 may be a therapeutic target for insulin resistance and diabetes mellitus.     

Erythropoietin receptor in human skeletal muscle and the effects of acute and long-term injections with recombinant human erythropoietin on the skeletal muscle

The presence and potential physiological role of the erythropoietin receptor (Epo-R) were examined in human skeletal muscle. In this study we demonstrate that Epo-R is present in the endothelium, smooth muscle cells, and in fractions of the sarcolemma of skeletal muscle fibers. To study the potential effects of Epo in human skeletal muscle, two separate studies were conducted: one to study the acute effects of a single Epo injection on skeletal muscle gene expression and plasma hormones and another to study the effects of long-term (14 wk) Epo treatment on skeletal muscle structure. Subjects (n = 11) received a single Epo injection of 15,000 IU (double blinded, cross over, placebo). A single Epo injection reduced myoglobin and increased transferrin receptor and MRF-4 mRNA content within 10 h after injection. Plasma hormones remained unaltered. Capillarization and fiber hypertrophy was studied in subjects (n = 8) who received long-term Epo administration, and muscle biopsies were obtained before and after. Epo treatment did not alter mean fiber area (0.84 +/- 0.2 vs. 0.72 +/- 0.3 mm(2)), capillaries per fiber (4.3 +/- 0.5 vs. 4.4 +/- 1.3), or number of proliferating endothelial cells. In conclusion, the Epo-R is present in the vasculature and myocytes in human skeletal muscle, suggesting a role in both cell types. In accordance, a single injection of Epo regulates myoglobin, MRF-4, and transferrin receptor mRNA levels. However, in contrast to our hypothesis, prolonged Epo administration had no apparent effect on capillarization or muscle fiber hypertrophy.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

Thrombin stimulates dissociation and induction of HSP27 via p38 MAPK in vascular smooth muscle cells

We investigated the effects of thrombin on the induction of heat shock proteins (HSP) 70 and 27, and the mechanism behind the induction in aortic smooth muscle A10 cells. Thrombin increased the level of HSP27 but had little effect on the level of HSP70. Thrombin stimulated the accumulation of HSP27 dose dependently between 0.01 and 1 U/ml and cycloheximide reduced the accumulation. Thrombin stimulated an increase in the level of HSP27 mRNA and actinomycin D suppressed the thrombin-increased mRNA level. Thrombin induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The HSP27 accumulation by thrombin was reduced by SB-203580 and PD-169316 but not by SB-202474. SB-203580 and PD-169316 suppressed the thrombin-induced phosphorylation of p38 MAPK. SB-203580 reduced the thrombin-increased level of HSP27 mRNA. Dissociation of the aggregated HSP27 to the dissociated HSP27 was induced by thrombin. Dissociation was inhibited by SB-203580. Thrombin induced the phosphorylation of HSP27 and the phosphorylation was suppressed by SB-203580. These results indicate that thrombin stimulates not only the dissociation of HSP27 but also the induction of HSP27 via p38 MAPK activation in aortic smooth muscle cells.     

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.     

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.     

Expression of chemokine receptors in insulin-resistant human skeletal muscle cells.

Adipokines including chemokines are able to induce insulin resistance in human skeletal muscle cells, which may also be relevant for the observed link between obesity and diabetes. This study is aimed to analyze the expression of chemokine CC motif receptors (CCRs) in the insulin-resistant state in human skeletal muscle cells. Differentiated skeletal muscle cells were incubated for 24-72 hours with high concentrations of glucose and insulin (GI) or TNFalpha. In addition, myocytes were co-stimulated with monocyte chemotactic protein (MCP)-1 or adipocyte-conditioned medium (CM) and TNFalpha for 24 and 48 hours. Treatment with GI rapidly induced insulin resistance whereas TNFalpha impaired insulin signaling in a more chronic fashion (48-72 h). CM and MCP-1 also induced insulin resistance that was, however, not increased by co-stimulation with TNFalpha. Expression of CCR2 was decreased during differentiation but up-regulated in insulin-resistant myocytes after treatment with GI (24-72 h) and TNFalpha (72 h). Expression of CCR4 and CCR10 was down-regulated after treatment with TNFalpha, MCP-1, and CM. Our data show that the expression of CCR2, CCR4, and CCR10 is differentially regulated by different insulin resistance-inducing treatments in myotubes. However, we could not find a clear correlation between the level of insulin resistance and CCR expression in myotubes. In conclusion, we propose that upregulation of CCR2 in skeletal muscle does not represent a major step leading to muscle insulin resistance.     

Calcium currents in normal and dystrophic human skeletal muscle cells in culture

Human muscle cells obtained from biopsy specimens were grown in a primary culture system and electrophysiologically studied. Whole cell patch-clamp recordings revealed the presence of two types of calcium currents: (i) a low-threshold (-60 mV) one (ICa, T) with fast activation and inactivation kinetics (time-to-peak: 39 ms at -30 mV); and (ii) a high-threshold (-10 mV) one (ICa,L) with slower kinetics (time-to-peak: 550 ms at 20 mV). These two types of calcium currents could be also distinguished by their pharmacological characteristics since ICa,L was sensitive to the antagonist and agonist dihydropyridine derivatives contrary to ICa,T which was completely resistant to these compounds. These functional calcium channels existed both in normal and Duchenne dystrophic (DMD) human skeletal muscle cells in culture. We discuss a possible role of these two types of calcium channels in the myoplasmic calcium accumulation observed in the Duchenne muscular dystrophy.