Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

Modifications of the interactions between endoplasmic reticulum (ER) and mitochondria, defined as mitochondria-associated membranes (MAMs), were recently shown to be involved in the control of hepatic insulin action and glucose homeostasis, but with conflicting results. Whereas skeletal muscle is the primary site of insulin-mediated glucose uptake and the main target for alterations in insulin-resistant states, the relevance of MAM integrity in muscle insulin resistance is unknown. Deciphering the importance of MAMs on muscle insulin signaling could help to clarify this controversy. Here, we show in skeletal muscle of different mice models of obesity and type 2 diabetes (T2D) a marked disruption of ER-mitochondria interactions as an early event preceding mitochondrial dysfunction and insulin resistance. Furthermore, in human myotubes, palmitate-induced insulin resistance is associated with a reduction of structural and functional ER-mitochondria interactions. Importantly, experimental increase of ER-mitochondria contacts in human myotubes prevents palmitate-induced alterations of insulin signaling and action, whereas disruption of MAM integrity alters the action of the hormone. Lastly, we found an association between altered insulin signaling and ER-mitochondria interactions in human myotubes from obese subjects with or without T2D compared with healthy lean subjects. Collectively, our data reveal a new role of MAM integrity in insulin action of skeletal muscle and highlight MAM disruption as an essential subcellular alteration associated with muscle insulin resistance in mice and humans. Therefore, reduced ER-mitochondria coupling could be a common alteration of several insulin-sensitive tissues playing a key role in altered glucose homeostasis in the context of obesity and T2D.

The endocrine-derived hormone fibroblast growth factor (FGF) 19 has recently emerged as a potential target for treating metabolic disease. Given that skeletal muscle is a key metabolic organ, we explored the role of FGF19 in that tissue. Here we report a novel function of FGF19 in regulating skeletal muscle mass through enlargement of muscle fiber size, and in protecting muscle from atrophy. Treatment with FGF19 causes skeletal muscle hypertrophy in mice, while physiological and pharmacological doses of FGF19 substantially increase the size of human myotubes in vitro. These effects were not elicited by FGF21, a closely related endocrine FGF member. Both in vitro and in vivo, FGF19 stimulates the phosphorylation of the extracellular-signal-regulated protein kinase 1/2 (ERK1/2) and the ribosomal protein S6 kinase (S6K1), an mTOR-dependent master regulator of muscle cell growth. Moreover, mice with a skeletal-muscle-specific genetic deficiency of β-Klotho (KLB), an obligate co-receptor for FGF15/19 (refs. 2,3), were unresponsive to the hypertrophic effect of FGF19. Finally, in mice, FGF19 ameliorates skeletal muscle atrophy induced by glucocorticoid treatment or obesity, as well as sarcopenia. Taken together, these findings provide evidence that the enterokine FGF19 is a novel factor in the regulation of skeletal muscle mass, and that it has therapeutic potential for the treatment of muscle wasting.

Obesity, through low-grade inflammation, can drive insulin resistance and type 2 diabetes. While infiltration of adipose tissue (AT) with mononuclear cells (MNCs) is well established in obesity, the functional consequences of these interactions are less understood. Herein, we cocultured human adipose-derived stem cells (ASCs) from obese individuals with MNCs and analyzed their reciprocal behavior. Presence of ASCs 1) enhanced interleukin (IL)-17A secretion by Th17 cells, 2) inhibited γ-interferon and tumor necrosis factor α secretion by Th1 cells, and 3) increased monocyte-mediated IL-1β secretion. IL-17A secretion also occurred in stromal vascular fractions issued from obese but not lean individuals. Th17 polarization mostly depended on physical contacts between ASCs and MNCs—with a contribution of intracellular adhesion molecule-1—and occurred through activation of the inflammasome and phosphatidylinositol 3-kinase pathways. ASCs favored STAT3 over STAT5 transcription factor binding on STAT binding sites within the IL-17A/F gene locus. Finally, conditioned media from activated ASC-MNC cocultures inhibited adipocyte differentiation mRNA markers and impaired insulin-mediated Akt phosphorylation and lipolysis inhibition. In conclusion, we report that obese- but not lean-derived ASCs induce Th17 promotion and monocyte activation. This proinflammatory environment, in turn, inhibits adipogenesis and adipocyte insulin response. The demonstration of an ASC-Th17-monocyte cell axis reveals a novel proinflammatory process taking place in AT during obesity and defines novel putative therapeutic targets.

ObjectiveCircadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion.MethodsWe established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock.ResultsBioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption.ConclusionsOur study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans.

Type 2 diabetic cardiomyopathy features Ca2+ signaling abnormalities, notably an altered mitochondrial Ca2+ handling. We here aimed to study if it might be due to a dysregulation of either the whole Ca2+ homeostasis, the reticulum–mitochondrial Ca2+ coupling, and/or the mitochondrial Ca2+ entry through the uniporter. Following a 16-week high-fat high-sucrose diet (HFHSD), mice developed cardiac insulin resistance, fibrosis, hypertrophy, lipid accumulation, and diastolic dysfunction when compared to standard diet. Ultrastructural and proteomic analyses of cardiac reticulum–mitochondria interface revealed tighter interactions not compatible with Ca2+ transport in HFHSD cardiomyocytes. Intramyocardial adenoviral injections of Ca2+ sensors were performed to measure Ca2+ fluxes in freshly isolated adult cardiomyocytes and to analyze the direct effects of in vivo type 2 diabetes on cardiomyocyte function. HFHSD resulted in a decreased IP3R–VDAC interaction and a reduced IP3-stimulated Ca2+ transfer to mitochondria, with no changes in reticular Ca2+ level, cytosolic Ca2+ transients, and mitochondrial Ca2+ uniporter function. Disruption of organelle Ca2+ exchange was associated with decreased mitochondrial bioenergetics and reduced cell contraction, which was rescued by an adenovirus-mediated expression of a reticulum-mitochondria linker. An 8-week diet reversal was able to restore cardiac insulin signaling, Ca2+ transfer, and cardiac function in HFHSD mice. Therefore, our study demonstrates that the reticulum–mitochondria Ca2+ miscoupling may play an early and reversible role in the development of diabetic cardiomyopathy by disrupting primarily the mitochondrial bioenergetics. A diet reversal, by counteracting the MAM-induced mitochondrial Ca2+ dysfunction, might contribute to restore normal cardiac function and prevent the exacerbation of diabetic cardiomyopathy.

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.lipid metabolism | circadian clock | human skeletal muscle | human primary myotubes | lipidomics C ircadian oscillations are daily cycles in behavior and physiology that are driven by the existence of underlying intrinsic biological clocks with near 24-h periods. This anticipatory mechanism has evolved to ensure that all aspects of behavior and physiology, including metabolic pathways, are temporally coordinated with daily cycles of rest-activity and feeding to provide the organism with an adaptive advantage (1). In mammals, circadian oscillations are driven by a master pacemaker, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which orchestrates subsidiary oscillators in peripheral organs via neuronal, endocrine, and metabolic signaling pathways (2, 3).Large-scale gene expression datasets suggest that, in mammals, the vast majority of circadian-gene expression is highly organ-specific (4-6). Key metabolic functions in peripheral organs are subject to daily oscillations, such as carbohydrate and lipid metabolism by the liver, skeletal muscle, and endocrine pancreas (7).Skeletal muscle is a major contributor of whole-body metabolism and is the main site of glucose uptake in the postprandial state (8). Therefore, perturbations in glucose sensing and metabolism in skeletal muscle are strongly associated with insulin resistance in type 2 diabetes (T2D) (9). Recent data support a fundamental role for the circadian muscle clock in the regulation of glucose uptake, with a significant re...