Cesar Osorio-Fuentealba

Electrical Stimuli Release ATP to Increase GLUT4 Translocation and Glucose Uptake via PI3Kγ-Akt-AS160 in Skeletal Muscle Cells

Skeletal muscle glucose uptake in response to exercise is preserved in insulin-resistant conditions, but the signals involved are debated. ATP is released from skeletal muscle by contractile activity and can autocrinely signal through purinergic receptors, and we hypothesized it may influence glucose uptake. Electrical stimulation, ATP, and insulin each increased fluorescent 2-NBD-Glucose (2-NBDG) uptake in primary myotubes, but only electrical stimulation and ATP-dependent 2-NBDG uptake were inhibited by adenosine-phosphate phosphatase and by purinergic receptor blockade (suramin). Electrical stimulation transiently elevated extracellular ATP and caused Akt phosphorylation that was additive to insulin and inhibited by suramin. Exogenous ATP transiently activated Akt and, inhibiting phosphatidylinositol 3-kinase (PI3K) or Akt as well as dominant-negative Akt mutant, reduced ATP-dependent 2-NBDG uptake and Akt phosphorylation. ATP-dependent 2-NBDG uptake was also inhibited by the G protein βγ subunit-interacting peptide βark-ct and by the phosphatidylinositol 3-kinase-γ (PI3Kγ) inhibitor AS605240. ATP caused translocation of GLUT4myc-eGFP to the cell surface, mechanistically mediated by increased exocytosis involving AS160/Rab8A reduced by dominant-negative Akt or PI3Kγ kinase-dead mutants, and potentiated by myristoylated PI3Kγ. ATP stimulated 2-NBDG uptake in normal and insulin-resistant adult muscle fibers, resembling the reported effect of exercise. Hence, the ATP-induced pathway may be tapped to bypass insulin resistance.

Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

Insulin reduces the velocity of mobile Myo1c-positive GLUT4 vesicles beneath the muscle cell plasma membrane as visualized by total internal reflection fluorescence microscopy. Binding of vesicle-bound Myo1c to actin filaments underlies Myo1cs participation in GLUT4 vesicle tethering for subsequent productive docking and fusion of GLUT4 vesicles with the plasma membrane.

Insulin elicits a ROS-activated and an IP3-dependent Ca2+ release, which both impinge on GLUT4 translocation

ABSTRACT Insulin signaling includes generation of low levels of H2O2; however, its origin and contribution to insulin-stimulated glucose transport are unknown. We tested the impact of H2O2 on insulin-dependent glucose transport and GLUT4 translocation in skeletal muscle cells. H2O2 increased the translocation of GLUT4 with an exofacial Myc-epitope tag between the first and second transmembrane domains (GLUT4myc), an effect additive to that of insulin. The anti-oxidants N-acetyl L-cysteine and Trolox, the p47phox–NOX2 NADPH oxidase inhibitory peptide gp91-ds-tat or p47phox knockdown each reduced insulin-dependent GLUT4myc translocation. Importantly, gp91-ds-tat suppressed insulin-dependent H2O2 production. A ryanodine receptor (RyR) channel agonist stimulated GLUT4myc translocation and insulin stimulated RyR1-mediated Ca2+ release by promoting RyR1 S-glutathionylation. This pathway acts in parallel to insulin-mediated stimulation of inositol-1,4,5-trisphosphate (IP3)-activated Ca2+ channels, in response to activation of phosphatidylinositol 3-kinase and its downstream target phospholipase C, resulting in Ca2+ transfer to the mitochondria. An inhibitor of IP3 receptors, Xestospongin B, reduced both insulin-dependent IP3 production and GLUT4myc translocation. We propose that, in addition to the canonical &agr;,&bgr; phosphatidylinositol 3-kinase to Akt pathway, insulin engages both RyR-mediated Ca2+ release and IP3-receptor-mediated mitochondrial Ca2+ uptake, and that these signals jointly stimulate glucose uptake.

IGF-1 Induces IP3-dependent Calcium Signal Involved in the Regulation of Myostatin Gene Expression Mediated by NFAT During Myoblast Differentiation

Skeletal muscle differentiation is a complex and highly regulated process characterized by cell cycle arrest, which is associated with morphological changes including myoblast alignment, elongation, and fusion into multinucleated myotubes. This is a balanced process dynamically coordinated by positive and negative signals such as the insulin‐like growth factor I (IGF‐1) and myostatin (MSTN), respectively. In this study, we report that the stimulation of skeletal myoblasts during differentiation with IGF‐1 induces a rapid and transient calcium increase from intracellular stores, which are principally mediated through the phospholipase C gamma (PLC γ)/inositol 1,4,5‐triphosphate (IP3)‐dependent signaling pathways. This response was completely blocked when myoblasts were incubated with LY294002 or transfected with the dominant‐negative p110 gamma, suggesting a fundamental role of phosphatidylinositol 3‐kinase (PI3K) in PLCγ activation. Additionally, we show that calcium released via IP3 and induced by IGF‐1 stimulates NFAT‐dependent gene transcription and nuclear translocation of the GFP‐labeled NFATc3 isoform. This activation was independent of extracellular calcium influx and calcium release mediated by ryanodine receptor (RyR). Finally, we examined mstn mRNA levels and mstn promoter activity in myoblasts stimulated with IGF‐1. We found a significant increase in mRNA contents and in reporter activity, which was inhibited by cyclosporin A, 11R‐VIVIT, and by inhibitors of the PI3Kγ, PLCγ, and IP3 receptor. Our results strongly suggest that IGF‐1 regulates myostatin transcription through the activation of the NFAT transcription factor in an IP3/calcium‐dependent manner. This is the first study to demonstrate a role of calcium‐dependent signaling pathways in the mRNA expression of myostatin. J. Cell. Physiol. 228: 1452–1463, 2013.

Testosterone increases GLUT4-dependent glucose uptake in cardiomyocytes

Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca2+ release and protein kinase activation and long‐term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2‐(N‐(7‐nitrobenz‐2‐oxa‐1, 3‐diazol‐4‐yl)amino)‐2‐deoxyglucose and [3H]2‐deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin‐bound testosterone‐induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4myc‐eGFP. Subsequently, testosterone increased GLUT4myc‐GFP exposure at the plasma membrane. Inhibition of Akt by the Akt‐inhibitor‐VIII had no effect. However, inhibition of Ca2+/calmodulin protein kinase (CaMKII) (KN‐93 and autocamtide‐2 related inhibitory peptide II) and AMP‐activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4myc‐eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4‐dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes. J. Cell. Physiol. 228: 2399–2407, 2013.

Hypoxia stimulates via separate pathways ERK phosphorylation and NF-κB activation in skeletal muscle cells in primary culture

Mammalian cells sense oxygen levels and respond to hypoxic conditions through the regulation of multiple signaling pathways and transcription factors. Here, we investigated the effects of hypoxia on the activity of two transcriptional regulators, ERK1/2 and NF-kappaB, in skeletal muscle cells in primary culture. We found that hypoxia significantly enhanced ERK1/2 phosphorylation and that it stimulated NF-kappaB-dependent gene transcription as well as nuclear translocation of a green fluorescent protein-labeled p65 NF-kappaB isoform. Phosphorylation of ERK1/2- and NF-kappaB-dependent transcription by hypoxia required calcium entry through L-type calcium channels. Calcium release from ryanodine-sensitive stores was also necessary for ERK1/2 activation but not for NF-kappaB-dependent-transcription. N-acetylcysteine, a general scavenger of reactive oxygen species, blocked hypoxia-induced ROS generation but did not affect the stimulation of ERK1/2 phosphorylation induced by hypoxia. In contrast, NF-kappaB activation was significantly inhibited by N-acetylcysteine and did not depend on ERK1/2 stimulation, as shown by the lack of effect of the upstream ERK inhibitor U-0126. These separate pathways of activation of ERK1/2 and NF-kappaB by hypoxia may contribute to muscle adaptation in response to hypoxic conditions.

Insulin-Dependent H2O2 Production Is Higher in Muscle Fibers of Mice Fed with a High-Fat Diet

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

The cholesterol-lowering agent methyl-β-cyclodextrin promotes glucose uptake via GLUT4 in adult muscle fibers and reduces insulin resistance in obese mice

Insulin stimulates glucose uptake in adult skeletal muscle by promoting the translocation of GLUT4 glucose transporters to the transverse tubule (T-tubule) membranes, which have particularly high cholesterol levels. We investigated whether T-tubule cholesterol content affects insulin-induced glucose transport. Feeding mice a high-fat diet (HFD) for 8 wk increased by 30% the T-tubule cholesterol content of triad-enriched vesicular fractions from muscle tissue compared with triads from control mice. Additionally, isolated muscle fibers (flexor digitorum brevis) from HFD-fed mice showed a 40% decrease in insulin-stimulated glucose uptake rates compared with fibers from control mice. In HFD-fed mice, four subcutaneous injections of MβCD, an agent reported to extract membrane cholesterol, improved their defective glucose tolerance test and normalized their high fasting glucose levels. The preincubation of isolated muscle fibers with relatively low concentrations of MβCD increased both basal and insulin-induced glucose uptake in fibers from controls or HFD-fed mice and decreased Akt phosphorylation without altering AMPK-mediated signaling. In fibers from HFD-fed mice, MβCD improved insulin sensitivity even after Akt or CaMK II inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, prevented the stimulatory effects of MβCD on glucose uptake. Addition of MβCD elicited ryanodine receptor-mediated calcium signals in isolated fibers, which were essential for glucose uptake. Our findings suggest that T-tubule cholesterol content exerts a critical regulatory role on insulin-stimulated GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may represent a useful strategy to counteract insulin resistance.