Summary AMPK has emerged as a critical mechanism for salutary effects of polyphenols on lipid metabolic disorders in type 1 and type 2 diabetes. We demonstrate that AMPK interacts with and directly phosphorylates sterol regulatory element binding proteins (SREBP-1c and −2). Ser372 phosphorylation of SREBP-1c by AMPK is sufficient and necessary for inhibition of proteolytic processing and transcriptional activity of SREBP-1c in response to polyphenols and metformin. AMPK stimulates Ser372 phosphorylation, suppresses SREBP-1c cleavage and nuclear translocation, and represses SREBP-1c target gene expression in hepatocytes exposed to high glucose, leading to reduced lipogenesis and lipid accumulation. Hepatic activation of AMPK by the synthetic polyphenol S17834 protects against hepatic steatosis, hyperlipidemia, and accelerated atherosclerosis in diet-induced insulin resistant LDL receptor deficient mice in part through phosphorylation of SREBP-1c Ser372 and suppression of SREBP-1c and −2-dependent lipogenesis. AMPK-dependent phosphorylation of SREBP may offer novel therapeutic strategies to combat insulin resistance, dyslipidemia, and atherosclerosis.

Purpose Elevated lipogenesis regulated by sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor playing a central role in lipid metabolism, is a novel characteristic of glioblastoma (GBM). The aim of this study was to identify effective approaches to suppress GBM growth by inhibition of SREBP-1. As SREBP activation is negatively regulated by endoplasmic reticulum (ER) cholesterol, we sought to determine whether suppression of sterol O-acyltransferase (SOAT), a key enzyme converting ER cholesterol to cholesterol esters (CE) to store in lipid droplets (LDs), effectively suppressed SREBP-1 and blocked GBM growth. Experimental Design The presence of LDs in glioma patient tumor tissues was analyzed using immunofluorescence, immunohistochemistry and electronic microscopy. Western blotting and real-time PCR were performed to analyze protein levels and gene expression of GBM cells, respectively. Intracranial GBM xenografts were used to determine the effects of genetically silencing SOAT1 and SREBP on tumor growth. Results Our study unraveled that cholesterol esterification and LD formation are signature of GBM, and human patients with glioma possess elevated LDs that correlate with GBM progression and poor survival. We revealed that SOAT1 is highly expressed in GBM and functions as a key player in controlling the cholesterol esterification and storage in GBM. Targeting SOAT1 suppresses GBM growth and prolongs survival in xenograft models via inhibition of SREBP-1-regulated lipid synthesis. Conclusions Cholesterol esterification and storage in LDs are novel characteristics of GBM, and inhibiting SOAT1 to block cholesterol esterification is a promising therapeutic strategy to treat GBM by suppressing SREBP-1.

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.

SUMMARY Tumorigenesis is associated with increased glucose consumption and lipogenesis, but how these pathways are interlinked is unclear. Here, we delineate a pathway in which EGFR signaling, by increasing glucose uptake, promotes N-glycosylation of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and consequent activation of SREBP-1, an endoplasmic reticulum-bound transcription factor with central roles in lipid metabolism. Glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing movement of SCAP/SREBP to the Golgi and consequent proteolytic activation of SREBP. Xenograft studies reveal that blocking SCAP N-glycosylation ameliorates EGFRvIII-driven glioblastoma growth. Thus, SCAP acts as key glucose-responsive protein linking oncogenic signaling and fuel availability to SREBP-dependent lipogenesis. Targeting SCAP N-glycosylation may provide a promising means of treating malignancies and metabolic diseases.

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.

It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as "endocrine signals" during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube-exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.

Aims/hypothesisExosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route.MethodsExosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA).ResultsHP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues.Conclusions/interpretationExosomes act as ‘paracrine-like’ signals and modify muscle homeostasis during high-fat diets.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3337-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.