Alcohol dependence causes physical, social, and moral harms and currently represents an important public health concern. According to the World Health Organization (WHO), alcoholism is the third leading cause of death worldwide, after tobacco consumption and hypertension. Recent epidemiologic studies have shown a growing trend in alcohol abuse among adolescents, characterized by the consumption of large doses of alcohol over a short time period. Since brain development is an ongoing process during adolescence, short- and long-term brain damage associated with drinking behavior could lead to serious consequences for health and wellbeing. Accumulating evidence indicates that alcohol impairs the function of different components of the melanocortin system, a major player involved in the consolidation of addictive behaviors during adolescence and adulthood. Here, we hypothesize the possible implications of melanocortins and glial cells in the onset and progression of alcohol addiction. In particular, we propose that alcohol-induced decrease in α-MSH levels may trigger a cascade of glial inflammatory pathways that culminate in altered gliotransmission in the ventral tegmental area (VTA) and nucleus accumbens (NAc). The latter might potentiate dopaminergic drive in the NAc, contributing to increase the vulnerability to alcohol dependence and addiction in the adolescence and adulthood.
GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding-deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.
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.
In athletics, achieving peak performance during competitions is crucial. Warm-up strategies play a crucial role in optimizing the strength-speed performance of sprinters in athletics, especially tailored to the physiological demands of speed events. The need to balance flexibility, prevent injuries, and enhance power output makes the selection of an effective warm-up protocol essential. This narrative review examines different warm-up methods used by athletes and their effects on strength-speed in sprinters in athletics. The main findings indicate that Foam Rolling (FR), Isometric Exercises and Pre-Competitive Massages have no significant effects on sprint performance. Static stretching and prolonged Pre-Competitive Massages have negative impacts on strength and power. The Vibration Platform enhances step length, step rate and running velocity, jump height and total number of jumps performed in a 30-s period in non-experienced sprinters. Eccentric Exercise increases vertical force, Post-Activation Potentiation (PAP) demonstrates a reduction in 100-meter time and short-term improvement in vertical and horizontal jumps. Blood Flow Restriction (BFR) significantly improving jump height and flight time. Various warm-up methods have been identified, some focusing on flexibility, others potentially detrimental, and some enhancing strength and power. Implementing effective warm-ups, particularly those promoting strength and power, poses a challenge for coaches seeking reliable alternatives to boost performance.
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 stimulates glucose uptake in skeletal muscle fibers by promoting the translocation of Glut4 glucose transporters to the transverse tubule (TT) system. Cholesterol is highly enriched in TT, raising the possibility that modifying TT cholesterol content may affect insulin‐induced glucose transport. By feeding mice a high fat diet (HFD), we increased TT cholesterol content and modified insulin‐induced glucose uptake. Male C57BL/6J mice were fed a normal or a HFD for 8 weeks. Cholesterol levels in triads from HFD‐fed mice were 30% higher than in controls whereas insulin‐stimulated glucose uptake in fibers from HFD‐fed mice was lower than in controls. In FDB muscle, pre‐incubation with low doses of Methyl‐β‐cyclodextrin (MβCD), reported to extract membrane cholesterol, decreased Akt phosphorylation without altering the activity of AMPK, and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. MβCD restored insulin sensitivity in HFD‐fed mice, even after Akt or CaMKII inhibition and increased membrane GLUT4 content. Indinavir, a GLUT4 antagonist, inhibited the effect of MβCD. Our findings suggest that TT cholesterol content has a critical role in GLUT4 translocation and glucose transport and that partial cholesterol removal from muscle fibers may be a strategy to improve insulin resistance. Supported by ACT1111 and FONDECYT 3110105.
The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1W80A or Akt2W80A). Akt1W80A or Akt2W80A are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase.
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.
In skeletal muscle, cholesterol is enriched in the transverse tubule membranes (TT), where insulin‐promoted GLUT4 translocation occurs. To test the role of TT cholesterol content, we fed mice a high fat diet (HFD) and measured insulin triggered glucose uptake. Cholesterol levels were measured in TT‐enriched triad fractions isolated from skeletal muscle of male C57BL/6J mice fed either a normal or a HFD (60% fat) for 8 weeks. Isolated muscle fibers from flexor digitorum brevis were used for fluorescence glucose uptake determinations. Akt activity in myotubes was determined by Western blot analysis. Relative to controls, body weight, body fat content and metabolic variables including insulin resistance were higher in HFD fed mice. Cholesterol levels in triads from HFD‐fed mice were 30% higher than in controls. Preincubation of myotubes with Methyl‐β‐cyclodextrin to remove cholesterol partially inhibited Akt phosphorylation in controls, and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. Cholesterol removal restored insulin sensitivity in HFD‐fed mice, independently of Akt and CaMKII inhibition. Our findings indicate that cholesterol removal from muscle fibers may be a strategy for improving insulin resistance in HFD‐fed mice and suggest that TT cholesterol has a physiological role in glucose transport. FONDECYT 3110105, FONDAP 15010006.
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