Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

Chicoric acid mitigates impaired insulin sensitivity by improving mitochondrial function

Mitochondrial dysfunction is associated with insulin resistance. Although chicoric acid (CA) is known to have beneficial effects on insulin sensitivity, the involvement of mitochondrial function has not been elucidated yet. Here, we investigated the effect of CA on insulin resistance and mitochondrial dysfunction. In palmitate-induced insulin-resistant C2C12 myotubes, CA improved impaired glucose uptake and insulin signaling pathways, along with enhanced mitochondrial membrane potential and oxygen consumption. CA treatment in diet-induced obese mice ameliorated glucose tolerance and increased insulin sensitivity. CA treatment also recovered the dysregulated expression of glucose metabolism-related genes in the high-fat-fed mice. CA significantly increased the mitochondrial DNA content, citrate synthase, and ATP content, as well as the expression of genes related to mitochondrial biogenesis and oxidative phosphorylation in the liver and skeletal muscle in high-fat- fed obese mice. These findings suggested that CA attenuates insulin resistance and promotes insulin sensitivity by enhancing mitochondrial function.     

Divergent effects of GLP-1 analogs exendin-4 and exendin-9 on the expression of myosin heavy chain isoforms in C2C12 myotubes

Exendin 1-39 amide (Ex-4) and its truncated form exendin 9-39 amide (Ex-9) are peptides of non-mammalian nature, which act as an agonist and antagonist, respectively, of the glucagon-like peptide-1 (GLP-1) receptor in mammals. GLP-1 is an intestinal peptide that plays an important role in the regulation of glucose metabolism and glucose uptake in skeletal muscle; however, the effects of its two analogs (Ex-4 and Ex-9) on myofiber properties are still unclear. Here, we report the effects of Ex-4 and Ex-9 alone or in combination on the myosin heavy chain (MyHC) type composition and the glucose uptake capacity in differentiated C2C12 myotubes. Neither Ex-4 nor Ex-9 altered basal glucose uptake, whereas Ex-9 significantly increased insulin-stimulated glucose uptake, suggesting enhanced insulin sensitivity. The mRNA expression of MyHC I and 2A as well as the percentage of MyHC I protein was remarkably increased in Ex-9-treated myotubes. In contrast, Ex-4, alone or in combination with Ex-9, caused a significant reduction in MyHC 2A mRNA expression and the percentage of MyHC I protein. Consistent with the MyHC type switching peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α expression in myotubes was remarkably increased by Ex-9 yet was significantly inhibited by Ex-4. In addition, intracellular concentrations of free Ca²⁺ were increased in all treatment groups, but only Ex-9-treated myotubes showed higher calcineurin A protein content. Taken together, our data suggest that Ex-9 promotes oxidative differentiation in myotubes to improve cell insulin sensitivity, probably through calcineurin and PGC-1α mediated pathways.     

Remodeling of Oxidative Energy Metabolism by Galactose Improves Glucose Handling and Metabolic Switching in Human Skeletal Muscle Cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [¹⁴C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [¹⁴C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.     

Palmitate-induced Down-regulation of Sortilin and Impaired GLUT4 Trafficking in C2C12 Myotubes

Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKCθ and TNF-α-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKCθ activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor γ agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKCθ states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKCθ-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor γ agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.     

Huang-Qi San ameliorates hyperlipidemia with obesity rats via activating brown adipocytes and converting white adipocytes into brown-like adipocytes

BackgroundBrown adipose tissue (BAT) activation is a promising therapeutic target to treat hyperlipidemia with obesity. Huang-Qi San (HQS), an traditional Chinese medicine, can ameliorate hyperlipidemia with obesity, but its mechanism of action (MOA) is not understood.PurposeTo articulate the MOA for HQS with animal models.MethodsThe main chemical constituents of HQS were identified by high-performance liquid chromatography (HPLC) based assay. Hyperlipidemia with obesity rat models induced by high-fat diet were employed in the study. The levels of the fasting plasma glucose (FPG), triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were measured to evaluate the ability of HQS to ameliorate hyperlipidemia with obesity. Pathological analyses of organs were conducted with Oil Red O staining, hematoxylin-eosin (H&E) staining and transmission electron microscopy. The expression of mRNAs related to thermogenic genes, fatty acid oxidation-related genes and mitochondria biogenic genes were examined by quantitative real-time PCR. The protein expressions of uncoupling protein 1 (UCP1) were investigated by immunohistochemistry and western blot. Simultaneously, the protein expression of PR domain containing 16 (PRDM16), ATP synthase F1 subunit alpha (ATP5A) was detected by western blot.ResultsHQS ameliorates metabolic disorder, lipid ectopic deposition, obesity and maintained glucose homeostasis in hyperlipidemia with obesity rats. HQS can significantly increase the number of mitochondria and reduced the size of the intracellular lipid droplets in BAT, and increase the expression of BAT activation-related genes (UCP1, PGC1α, PGC1β, Prdm16, CD137, TBX1, CPT1a, PPARα, Tfam, NRF1 and NRF2) in vivo. Furthermore, UCP1, PRDM16 and ATP5A proteins of BAT were increased.ConclusionHQS can activate BAT and browning of S-WAT (subcutaneous white adipose tissue) through activating the PRDM16/PGC1α/UCP1 pathway, augmenting mitochondrial biogenesis and fatty acid oxidation to increase thermogenesis and energy expenditure, resulting in a significant amelioration of hyperlipidemia with obesity. Therefore, HQS is an effective therapeutic medicine for the treatment of hyperlipidemia with obesity.     

Phenethyl isothiocyanate stimulates glucose uptake through the Akt pathway in C2C12 myotubes

Phenethyl isothiocyanate (PEITC) is an aromatic isothiocyanate present in cruciferous vegetables. Several studies have shown that isothiocyanates regulate various intracellular signaling pathways, and thereby show anti-inflammatory and detoxifying activities. However, little is known about the effects of PEITC on glucose metabolism. In this study, we examined whether PEITC promotes glucose utilization in mouse skeletal muscle cells, C2C12 myotubes. PEITC induced glucose uptake, glucose transporter 4 (Glut4) translocation to the plasma membrane, and activation of Akt and ERK in C2C12 cells. Inhibition of Akt suppressed PEITC-induced Glut4 translocation and glucose uptake, whereas ERK inhibition did not. Furthermore, PEITC increased phosphorylation of ErbB2 and ErbB3. Treatment with a pan-ErbB inhibitor reduced Akt activation and the subsequent glucose uptake induced by PEITC. These results indicate that PEITC promotes glucose utilization through the ErbB/Akt pathway in C2C12 myotubes. PEITC may therefore serve as a dietary constituent with beneficial effects on the carbohydrate metabolism.

Abbreviations: PEITC: phenethyl isothiocyanate; Glut4: glucose transporter 4; PI3K: phosphatidylinositide 3-kinase; Nrf2: erythroid?2-related factor; ARE: antioxidant response element; HO?1: heme oxygenase?1; NRG: neuregulin     

Mitochondrial DNA Damage via Augmented Oxidative Stress Regulates Endoplasmic Reticulum Stress and Autophagy: Crosstalk,Links and Signaling

Saturated free fatty acids (FFAs) have been implicated in the increase of oxidative stress, mitochondrial dysfunction, endoplasmic reticulum (ER) stress, autophagy, and insulin resistance (IR) observed in skeletal muscle. Previously, we have shown that palmitate-induced mitochondrial DNA (mtDNA) damage triggers mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) production, apoptosis and IR in L6 myotubes. The present study showed that mitochondrial overexpression of human 8-oxoguanine DNA glycosylase/AP lyase (hOGG1) decreased palmitate-induced carbonylation of proteins in mitochondria. Additionally, we found that protection of mtDNA from palmitate-induced damage significantly diminished markers of both ER stress and autophagy in L6 myotubes. Moreover, we observed that the addition of ROS scavenger, N-acetylcystein (NAC), to palmitate diminished both ER stress and autophagy markers mimicking the effect of mitochondrial overexpression of hOGG1. This is the first study to show that mtDNA damage is upstream of palmitate-induced ER stress and autophagy in skeletal muscle cells.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

The Effects of Propionate and Valerate on Insulin Responsiveness for Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myotubes via G Protein-Coupled Receptor 41

Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gα_i/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes.     

Uncarboxylated osteocalcin decreases insulin-stimulated glucose uptake without affecting insulin signaling and regulators of mitochondrial biogenesis in myotubes

Uncarboxylated osteocalcin (uOC) is a circulating bone matrix protein, which has previously been shown to regulate glucose uptake and systemic metabolism. However, the cellular mechanism by which uOC acts has yet to be elucidated. C2C12 mouse myotubes were treated for 72 h with uOC (1–100 ng/mL). Cellular metabolism was analyzed using oxygen consumption and extracellular acidification rate. Metabolic gene and protein expression were measured via quantitative real-time polymerase chain reaction and Western blot, respectively. Additionally, C2C12 myotubes were treated with 10 ng/mL uOC to examine glucose uptake and activation of insulin signaling with or without insulin resistance. Finally, cellular lipid content was measured via Oil Red O and Nile Red staining. uOC treatment resulted in dose-dependent alterations of oxygen consumption with little effect on regulators of mitochondrial metabolism. Basal expression of regulators of glucose uptake were unaffected by uOC treatment. However, insulin-stimulated glucose uptake was blunted by uOC treatment with no concurrent alterations in insulin signaling. While chronic insulin treatment resulted in suppressed activation of Akt, concurrent uOC treatment was unable to prevent these detrimental effects on insulin signaling. uOC treatment had no effect on markers of lipogenesis and cellular lipid content. These findings suggest that 72-h uOC treatment may alter oxygen consumption without effect on regulators of mitochondrial biogenesis. Additionally, uOC treatment suppressed insulin-stimulated glucose uptake in cultured myotubes but had little effect on insulin signaling or regulators of cellular metabolism and was unable to mitigate insulin resistance.     

2,6-Dimethoxy-1,4-benzoquinone increases skeletal muscle mass and performance by regulating AKT/mTOR signaling and mitochondrial function

Background2,6-Dimethoxy-1,4-benzoquinone (DMBQ), a natural phytochemical present in fermented wheat germ, has been reported to exert anti-cancer, anti-inflammatory, and anti-adipogenic effects. However, the effect of DMBQ on muscle hypertrophy and myoblast differentiation has not been elucidated.PurposeWe investigated the effect of DMBQ on skeletal muscle mass and muscle function and then determined the possible mechanism of DMBQ.MethodsTo examine myogenic differentiation and hypertrophy, confluent C2C12 cells were incubated in differentiation medium with or without various concentrations of DMBQ for 4 days. In animal experiments, C57BL/6 mice were fed DMBQ-containing AIN-93 diet for 7 weeks. Grip strength, treadmill, microscopic evaluation of muscle tissue, western blotting, and quantitative real-time PCR were performed.ResultsDMBQ significantly increased fusion index, myotube size, and the protein expression of myosin heavy chain (MHC). DMBQ increased the phosphorylation of protein kinase B (AKT) and p70 ribosomal protein S6 kinase (S6K), whereas the phosphorylation of these proteins was abolished by the phosphoinositide 3-kinase inhibitor LY294002 in C2C12 cells. In addition, DMBQ treatment increased peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), which programs mitochondrial biogenesis, protein levels compared with control C2C12 cells. DMBQ significantly increased maximal respiration and spare respiratory capacity in C2C12 cells. In animal experiments, DMBQ increased skeletal muscle weights and skeletal muscle fiber size compared with the control group values. In addition, the DMBQ group showed increased grip strength and running distance on an accelerating treadmill. The protein expression of total MHC, MHC1, MHC2A, and MHC2B in skeletal muscle was upregulated by DMBQ supplementation. We found that DMBQ increased the phosphorylation of AKT and mammalian target of rapamycin (mTOR), as well as downstream S6K and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in skeletal muscle. DMBQ also stimulated mRNA expression of PGC1α, accompanied by an increase in mitochondrial DNA content, oxidative phosphorylation (OXPHOS) proteins, and oxidative enzyme activity.ConclusionCollectively, DMBQ was shown to increase skeletal muscle mass and performance by regulating the AKT/mTOR signaling pathway and enhancing mitochondrial function, which might be useful for the treatment and prevention of skeletal muscle atrophy.     

Apoptotic signaling induced by H2O2-mediated oxidative stress in differentiated C2C12 myotubes

AimsApoptotic signaling proteins were evaluated in postmitotic skeletal myotubes to test the hypothesis that oxidative stress induced by H₂O₂ activates both caspase-dependent and caspase-independent apoptotic proteins in differentiated C2C12 myotubes. We hypothesized that oxidative stress would decrease anti-apoptotic protein levels in C2C12 myotubes.Main methodsApoptotic regulatory factors and apoptosis-associated proteins including Bcl-2, Bax, Apaf-1, XIAP, ARC, cleaved PARP, p53, p21^Cip1/Waf1, c-Myc, HSP70, CuZnSOD, and MnSOD protein content were measured by immunoblots.Key findingsH₂O₂ induced apoptosis in myotubes as shown by DNA laddering and an elevation of apoptotic DNA fragmentation. Cell death ELISA showed increase in the extent of apoptotic DNA fragmentation following treatment with H₂O₂. Treatment with 4 mM of H₂O₂ for 24 or 96 h caused increase in Bax (56%, 227%), cytochrome c (282%, 701%), Smac/DIABLO (155%, 260%), caspase-3 protease activity (51%, 141%), and nuclear and cytosolic p53 (719%, 1581%) levels in the myotubes. As an estimate of the mitochondrial AIF release to the cytosol, AIF protein content measured in the mitochondria-free cytosolic fraction was elevated by 65% after 96 h treatment with 4 mM of H₂O₂. AIF measured in the nuclear protein fraction increased by 74% and 352% following treatment with 4 mM of H₂O₂ for 24 and 96 h, respectively. Bcl-2 declined in myotubes by 61% and 69% after 24 or 96 h of treatment in 4 mM H₂O₂, respectively.SignificanceThese findings indicate that both caspase-dependent and caspase-independent mechanisms are involved in coordinating the activation of apoptosis induced by H₂O₂ in differentiated myotubes.     

Metabolic effects of physiological levels of caffeine in myotubes

Caffeine has been shown to stimulate multiple major regulators of cell energetics including AMP-activated protein kinase (AMPK) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Additionally, caffeine induces peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitochondrial biogenesis. While caffeine enhances oxidative metabolism, experimental concentrations often exceed physiologically attainable concentrations through diet. This work measured the effects of low-level caffeine on cellular metabolism and gene expression in myotubes, as well as the dependence of caffeine’s effects on the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARβ/δ). C2C12 myotubes were treated with various doses of caffeine for up to 24 h. Gene and protein expression were measured via qRT-PCR and Western blot, respectively. Cellular metabolism was determined via oxygen consumption and extracellular acidification rate. Caffeine significantly induced regulators of mitochondrial biogenesis and oxidative metabolism. Mitochondrial staining was suppressed in PPARβ/δ-inhibited cells which was rescued by concurrent caffeine treatment. Caffeine-treated cells also displayed elevated peak oxidative metabolism which was partially abolished following PPARβ/δ inhibition. Similar to past observations, glucose uptake and GLUT4 content were elevated in caffeine-treated cells, however, glycolytic metabolism was unaltered following caffeine treatment. Physiological levels of caffeine appear to enhance cell metabolism through mechanisms partially dependent on PPARβ/δ.     

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate''s protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.     

Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes

Insulin resistance is a primary characteristic of type 2 diabetes. Several lines of evidence suggest that accumulation of free fatty acids in skeletal muscle may at least in part contribute to insulin resistance and may be linked to mitochondrial dysfunction, leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits protein kinase B (Akt) activity in response to insulin. However, the role of Bax and Bcl-2 in regulating palmitate-induced apoptosis has not been well studied. Therefore, the purpose of this study was to determine whether palmitate-induced apoptosis in C(2)C(12) myotubes is dependent on Bax to Bcl-2 binding. An additional purpose of this study was to determine whether the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate-treated myoblasts. Apoptotic signaling proteins were examined in C(2)C(12) myotubes treated overnight with palmitate. Bax to Bcl-2 binding was determined through a coimmunoprecipitation assay that was performed in myotubes after 2 h of serum starvation, followed by 10 min of serum reintroduction. This experiment evaluated whether temporal Akt activity coincided with Bax to Bcl-2 binding. Last, the contribution of Bax to palmitate-induced apoptosis was determined by treatment with Bax siRNA. Palmitate treatment increased apoptosis in C(2)C(12) myotubes as shown by a twofold increase in DNA fragmentation, an approximately fivefold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. In addition, there was a fourfold reduction in Bax to Bcl-2 binding with palmitate treatment, which mirrored the reduction in Akt(Ser473) phosphorylation. Furthermore, treatment of the C(2)C(12) myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax-mediated apoptosis in C(2)C(12) myotubes and that this effect corresponds to reductions in Akt(Ser473) phosphorylation.