The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reaction were used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

The antidiabetic SGLT2 inhibitor canagliflozin reduces mitochondrial metabolism in a model of skeletal muscle insulin resistance.

AIMS: Sodium-glucose cotransporter 2 (SGLT2) inhibitors such as canagliflozin (CANA) have emerged as an effective adjuvant therapy in the management of diabetes, however, past observations suggest CANA may alter skeletal muscle mass and function. The purpose of this work was to investigate the effects of CANA on skeletal muscle metabolism both with and without insulin resistance. METHODS: C2C12 myotubes were treated with CANA with or without insulin resistance. Western blot and qRT-PCR were used to assess protein and gene expression, respectively. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Mitochondrial, nuclei and lipid content were measured using fluorescent staining and microscopy. RESULTS: CANA decreased mitochondrial function and glycolytic metabolism as did insulin resistance, however, these changes occurred without significant alterations in gene expression associated with each pathway. Additionally, while insulin resistance reduced insulin-stimulated pAkt expression, CANA had no significant effect on insulin sensitivity. CONCLUSIONS: CANA appears to reduce mitochondrial and glycolytic metabolism without altering gene expression governing these pathways, suggesting a reduction in substrate may be responsible for lower metabolism.

5-Aza-2'-deoxycytidine-mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity.

Type 2 diabetes is characterized by elevated blood glucose and reduced insulin sensitivity in target tissues. Moreover, reduced mitochondrial metabolism and expressional profile of genes governing mitochondrial metabolism (such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α]) are also reduced during insulin resistance. Epigenetic regulation via DNA methylation of genes including PGC-1α may contribute to diminished mitochondrial capacity, while hypomethylation of PGC-1α (such as that invoked by exercise) has been associated with increased PGC-1α expression and favorable metabolic outcomes. The purpose of the present report is to characterize the effects of DNA hypomethylation on myotube metabolism and expression of several related metabolic targets. C2C12 myotubes were treated with 5-Aza-2'-deoxycytidine (5-Aza) for either 24 or 72 h both with and without hyperinsulinemic-induced insulin resistance. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via quantitative real time polymerase chain reaction and western blot analysis, respectively. Though expression of PGC-1α and other related targets remained unaltered, insulin resistance and 5-Aza treatment significantly reduced mitochondrial metabolism. Similarly, peak glycolytic metabolism was diminished by 5-Aza-treated cells, while basal glycolytic metabolism was unaltered. 5-Aza also reduced the expression of branched-chain amino acid (BCAA) catabolic components, however BCAA utilization was enhanced during insulin resistance with 5-Aza treatment. Together the present work provides proof-of-concept evidence of the potential role of DNA methylation in the regulation of mitochondrial metabolism and the potential interactions with insulin resistance in a model of skeletal muscle.

Physiological 4-phenylbutyrate promotes mitochondrial biogenesis and metabolism in C2C12 myotubes.

Type 2 diabetes is characterized by elevated circulating blood metabolites such as glucose, insulin, and branched chain amino acids (BCAA), which often coincide with reduced mitochondrial function. 4-Phenylbutyrate (PBA), an ammonia scavenger, has been shown to activate BCAA metabolism, resolve endoplasmic reticulum (ER) stress, and rescue BCAA-mediated insulin resistance. To determine the effect of PBA on the altered metabolic phenotype featured in type 2 diabetes, the present study investigated the effect of PBA on various metabolic parameters including mitochondrial metabolism and mitochondrial biogenesis. C2C12 myotubes were treated with PBA at 0.5 mM (representing physiologically attainable blood concentrations) or 10 mM (representing physiologically unattainable/proof-of-concept levels) for up to 24 h. Mitochondrial and glycolytic metabolism were assessed via oxygen consumption and extracellular acidification rate, respectively. Mitochondrial content, lipid content, and ER stress were measured by fluorescent staining. Metabolic gene expression was measured by qRT-PCR. Both doses of PBA increased expression of indicators of mitochondrial biogenesis, though only PBA at 0.5 mM increased mitochondrial function and content while 10 mM PBA reduced mitochondrial function and content. PBA at 0.5 mM also rescued reduced mitochondrial function during insulin resistance, though PBA also caused a reduced insulin stimulated pAkt expression during insulin resistance. PBA treatment also increased extracellular BCAA accumulation during insulin resistance despite unchanged pBCKDH expression. Taken together, PBA may increase mitochondrial biogenesis, content, and function in a dose-dependent fashion which may have implications for prevention or treatment of metabolic disease such as insulin resistance.

The effect of insulin resistance on extracellular BCAA accumulation and SLC25A44 expression in a myotube model of skeletal muscle insulin resistance.

Insulin resistance is often accompanied by elevated circulating branched-chain amino acids (BCAA). We investigated the effects of insulin resistance on the mitochondrial BCAA transporter, SLC25A44, using a myotube model of insulin resistance. Insulin sensitivity and SLC25A44 expression were assessed via Western blot. Liquid chromatography-mass spectrometry was used to evaluate extracellular BCAA media content. Insulin resistance reduced pAkt activation following insulin stimulation but did not alter SLC25A44 expression. Under select conditions, insulin resistance led to the accumulation of extracellular BCAA.

The Selective LAT1 Inhibitor JPH203 Enhances Mitochondrial Metabolism and Content in Insulin-Sensitive and Insulin-Resistant C2C12 Myotubes.

Population data have shown an association between higher circulating branched-chain amino acids (BCAA) and the severity of insulin resistance in people with diabetes. While several studies have assessed BCAA metabolism as a potential target for regulation, less attention has been paid to the role of L-type amino acid transporter 1 (LAT1), the primary transporter of BCAA in skeletal muscle. The aim of this study was to assess the impact of JPH203 (JPH), a LAT1 inhibitor, on myotube metabolism in both insulin-sensitive and insulin-resistant myotubes. C2C12 myotubes were treated with or without 1 µM or 2 µM JPH for 24 h with or without insulin resistance. Western blot and qRT-PCR were used to assess protein content and gene expression, respectively. Mitochondrial and glycolytic metabolism were measured via Seahorse Assay, and fluorescent staining was used to measure mitochondrial content. BCAA media content was quantified using liquid chromatography-mass spectrometry. JPH at 1 µM (but not 2 µM) increased mitochondrial metabolism and content without inducing changes in mRNA expression of transcripts associated with mitochondrial biogenesis or mitochondrial dynamics. Along with increased mitochondrial function, 1µM treatment also reduced extracellular leucine and valine. JPH at 2 µM reduced pAkt signaling and increased extracellular accumulation of isoleucine without inducing changes in BCAA metabolic genes. Collectively, JPH may increase mitochondrial function independent of the mitochondrial biogenic transcription pathway; however, high doses may reduce insulin signaling.

PPARß/δ Agonism with GW501516 Increases Myotube PGC-1α Content and Reduces BCAA Media Content Independent of Changes in BCAA Catabolic Enzyme Expression.

Background: Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism with reduced expression of genes governing metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, increased circulating BCAA in diabetics may be partially explained by reduced PGC-1α expression. PGC-1α functions in-part through interactions with peroxisome proliferator-activated receptor ß/δ (PPARß/δ). The present report examined the effects of the PPARß/δ agonism on cell metabolism and related gene/protein expression of cultured myotubes, with a primary emphasis on determining the effects of GW on BCAA disposal and catabolic enzyme expression. Methods: C2C12 myotubes were treated with GW501516 (GW) for up to 24 hours. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Media BCAA content was assessed via liquid chromatography-mass spectrometry (LC/MS). Results: GW significantly increased PGC-1α protein expression, mitochondrial content, and mitochondrial function. GW also significantly reduced BCAA content within culture media following 24-hour treatment; however, expression of BCAA catabolic enzymes/transporter was unchanged. Conclusion: These data confirm the ability of GW to increase muscle PGC-1α content and decrease BCAA media content without affecting BCAA catabolic enzymes/transporter. These findings suggest heightened BCAA uptake (and possibly metabolism) may occur without substantial changes in the protein levels of related cell machinery.

Insulin resistance promotes extracellular BCAA accumulation without altering LAT1 content, independent of prior BCAA treatment in a myotube model of skeletal muscle.

PURPOSE: Type 2 diabetes is characterized by reduced insulin sensitivity which correlates with increased circulating BCAA. These experiments investigated the effects of insulin resistance with and without excess BCAA on myotube insulin sensitivity and L-type amino acid transporter-1 (LAT1). METHODS: C2C12 myotubes were treated with or without excess BCAA for 1 or 6 days, both with and without insulin resistance. Western blot was used to assess insulin sensitivity and LAT1 content. Liquid chromatography-mass spectrometry was used to evaluate BCAA media content. RESULTS: Insulin resistance was associated with significantly increased extracellular BCAA accumulation independent of LAT1 content. Conversely, prior BCAA treatment was not associated with extracellular BCAA accumulation regardless of level of insulin sensitivity. CONCLUSION: These data suggest insulin resistance, but not BCAA treatment, promotes extracellular BCAA accumulation independent of changes in LAT1 content, implicating insulin resistance as a causal agent of extracellular BCAA accumulation.

Effect of AMPK activation and glucose availability on myotube LAT1 expression and BCAA utilization.

Those with insulin resistance often display increased circulating branched-chain amino acids (BCAA), which has been largely attributable to reduced BCAA catabolic capacity. Metabolic stimuli such as exercise activates AMP-activated kinase (AMPK), which promotes the metabolism of BCAA and induction/activation of BCAA catabolic enzymes. Though much attention has been paid to BCAA catabolic machinery, few studies have assessed the effect of AMPK activation on the predominant BCAA transporter, L-type amino acid transporter 1 (LAT1). This study assessed the effect of AMPK activation on LAT1 expression via common chemical AMPK activators in a cell model of skeletal muscle. C2C12 myotubes were treated with either 1 mM AICAR, 1 mM Metformin, or filter-sterilized water (control) for 24 h with either low- (5 mM) or high-glucose (25 mM) media. LAT1 and pAMPK protein content were measured via western blot. BCAA media content was measured using liquid chromatography-mass spectrometry. AICAR treatment significantly increased pAMPK and reduced LAT1 expression. Collectively, pAMPK and LAT1 displayed a significant inverse relationship independent of glucose levels. During low-glucose experiments, AICAR-treated cells had higher BCAA media content compared to other groups, and an inverse relationship between LAT1 and BCAA media content was observed, however, these effects were not consistently observed during high-glucose conditions. Further investigation with AICAR with and without concurrent LAT1 inhibition (via JPH203) also revealed reduced BCAA utilization in AICAR-treated cells regardless of LAT1 inhibition (which also independently reduced BCAA utilization). pAMPK activation via AICAR (but not Metformin) may reduce LAT1 expression and BCAA uptake in a glucose-dependent manner.

Branched-Chain Amino Acids and Mitochondrial Biogenesis: An Overview and Mechanistic Summary.

Branched-chain amino acids (BCAA) are essential in the diet and promote several vital cell responses which may have benefits for health and athletic performance, as well as disease prevention. While BCAA are well-known for their ability to stimulate muscle protein synthesis, their effects on cell energetics are also becoming well-documented, but these receive less attention. In this review, much of the current evidence demonstrating BCAA ability (as individual amino acids or as part of dietary mixtures) to alter regulators of cellular energetics with an emphasis on mitochondrial biogenesis and related signaling is highlighted. Several studies have shown, both in vitro and in vivo, that BCAA (either individual or as a mixture) may promote signaling associated with increased mitochondrial biogenesis including the upregulation of master regulator of mitochondrial biogenesis peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), as well as numerous downstream targets and related function. However, sparse data in humans and the difficulty of controlling variables associated with feeding studies leave the physiological relevance of these findings unclear. Future well-controlled diet studies will be needed to assess if BCAA consumption is associated with increased mitochondrial biogenesis and improved metabolic outcomes in healthy and/or diseased human populations.

Excess glutamine does not alter myotube metabolism or insulin sensitivity.

Glutamine is an amino acid previously linked with improved skeletal muscle metabolism and insulin signaling, however, past observations often use cell culture models with only supraphysiological concentrations. Additionally, past reports have yet to simultaneously investigate both metabolic outcomes and insulin signaling. The present report utilized cell culture experiments and measured the effects of both physiological and supraphysiological levels of glutamine on myotube metabolism and insulin signaling/resistance. It was hypothesized the addition of glutamine at any level would increase cell metabolism and related gene expression, as well as improve insulin signaling versus respective control cells. C2C12 myotubes were treated with glutamine ranging from 0.25 mM-4 mM (or media control) for 24 h to capture a range of physiological and supraphysiological concentrations. qRT-PCR was used to measure metabolic gene expression. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Insulin sensitivity (indicated by pAkt:Akt) and metabolism following glucose/insulin infusion were also assessed. Glutamine treatment consistently increased mitochondrial and glycolytic metabolism versus true controls (cells treated with media void of glutamine), however, supraphysiological glutamine did not enhance metabolism beyond that of cells with physiological levels of glutamine. Neither physiological nor supraphysiological levels of glutamine altered insulin signaling regardless of insulin stimulation or insulin resistance when compared with respective controls. These data demonstrate excess glutamine does not appear to alter myotube metabolism or glucose disposal when base levels of glutamine are present. Moreover, glutamine does not appear to alter insulin sensitivity regardless of level of insulin resistance or presence of insulin stimulation.

AICAR stimulates mitochondrial biogenesis and BCAA catabolic enzyme expression in C2C12 myotubes.

Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism. Insulin resistant populations often exhibit reduced expression of genes governing mitochondrial metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Interestingly, PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, the consistently observed increased circulating levels of BCAA in diabetics may be partially explained by reduced PGC-1α expression. Conversely, PGC-1α upregulation appears to increase BCAA catabolism. PGC-1α activity is regulated by 5'-AMP-activated protein kinase (AMPK), however, only limited experimental data exists on the effect of AMPK activation in the regulation of BCAA catabolism. The present report examined the effects of the commonly used AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the metabolism and expression of several related targets (including BCAA catabolic enzymes) of cultured myotubes. C2C12 myotubes were treated with AICAR at 1 mM for up to 24 h. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via qRT-PCR and western blot, respectively. AICAR treatment significantly increased mitochondrial content and peak mitochondrial capacity. AICAR treatment also increased AMPK activation and mRNA expression of several regulators of mitochondrial biogenesis but reduced glycolytic metabolism and mRNA expression of several glycolytic enzymes. Interestingly, branched-chain alpha-keto acid dehydrogenase a (BCKDHa) protein was significantly increased following AICAR-treatment suggesting increased overall BCAA catabolic capacity in AICAR-treated cells. Together, these experiments demonstrate AICAR/AMPK activation can upregulate BCAA catabolic machinery in a model of skeletal muscle.

Excess branched-chain amino acids alter myotube metabolism and substrate preference which is worsened by concurrent insulin resistance.

PURPOSE: Branched-chain amino acids (BCAA) have been shown to enhance several cellular signaling pathways including protein synthesis and mitochondrial biogenesis, yet population data demonstrate a correlation between circulating BCAA and severity of insulin resistance which has been hypothesized to be, in part, a byproduct of BCAA inhibition of mitochondrial function. The purpose of this study is to examine the effect of a BCAA mixture on muscle metabolism and related gene expression in vitro. METHODS: C2C12 myotubes were treated with a BCAA mixture containing leucine:isoleucine:valine at a ratio of 2:1:1 at 0.2, 2, or 20 mM (based on leucine content) for 6 days. qRT-PCR was used to measure metabolic gene expression. Oxygen consumption and extracellular acidification were used to assess mitochondrial and glycolytic metabolism, respectively. Mitochondrial content was determined via mitochondrial-specific staining. RESULTS: Despite significantly elevated mitochondrial staining, 6-day BCAA treatment reduced basal mitochondrial metabolism at a supraphysiological concentration (20 mM) in both insulin sensitive and resistant cells. Peak mitochondrial capacity was also reduced in insulin-resistant (but not insulin sensitive) cells. Conversely, basal glycolytic metabolism was elevated following 20 mM BCAA treatment, regardless of insulin resistance. In addition, insulin-resistant cells treated with 20 mM BCAA exhibited reduced gene expression of Ppargc1a, Cytc, Atp5b, Glut4, and several glycolytic enzymes versus insulin sensitive cells treated with 20 mM BCAA. CONCLUSIONS: Collectively, these findings suggest BCAA at supraphysiologically high levels may negatively alter mitochondrial metabolism, and concurrent insulin resistance may also diminish peak mitochondrial capacity, as well as impede molecular adaptations that support a transition to a glycolytic preference/compensation.

Branched-chain amino acids at supraphysiological but not physiological levels reduce myotube insulin sensitivity.

AIMS: Branched-chain amino acids (BCAA) are often emphasized in the diets of avid exercisers, yet population data demonstrates a correlation between circulating BCAA and insulin resistance. However, it is unclear if BCAA independently promote insulin resistance in otherwise healthy cells. The purpose of this study is to examine the effect of a BCAA mixture on muscle insulin signaling in vitro in both insulin resistant and sensitive cells. MATERIALS AND METHODS: C2C12 myotubes were treated with a BCAA mixture containing leucine:isoleucine:valine at a ratio of 2:1:1 at 0.2, 2, or 20 mM (based on leucine content) for either 30 min, 1 day, or 6 days. Western blot was used to assess insulin sensitivity of cells treated with BCAA both with and without concurrent insulin resistance, and, with and without insulin stimulation. RESULTS: BCAA treatment for 1 day significantly reduced basal, but not insulin-stimulated pAkt expression. BCAA treatment for 6 days resulted in significantly reduced basal insulin signaling in healthy cells and insulin-stimulated insulin signaling in insulin resistant (but not insulin sensitive) cells. CONCLUSION: Similar to previous observations demonstrating BCAA may correlate with insulin resistance during metabolically stressed conditions, we demonstrate excessively high BCAA exposure can negatively influence basal insulin signaling, as well as insulin sensitivity in insulin resistant myotubes. However, given the intentionally high concentrations of BCAA used in this study, the extent to which these observations translate to in vivo models is unclear and warrants further investigation.

Comparing the effects of palmitate, insulin, and palmitate-insulin co-treatment on myotube metabolism and insulin resistance.

Previous studies have shown various metabolic stressors such as saturated fatty acids (SFA) and excess insulin promote insulin resistance in metabolically meaningful cell types (such as skeletal muscle). Additionally, these stressors have been linked with suppressed mitochondrial metabolism, which is also a common characteristic of skeletal muscle of diabetics. This study characterized the individual and combined effects of excess lipid and excess insulin on myotube metabolism and related metabolic gene and protein expression. C2C12 myotubes were treated with either 500 µM palmitate (PAM), 100 nM insulin (IR), or both (PAM-IR). qRT-PCR and western blot were used to measure metabolic gene and protein expression, respectively. Oxygen consumption was used to measure mitochondrial metabolism. Glycolytic metabolism and insulin-mediated glucose uptake were measured via extracellular acidification rate. Cellular lipid and mitochondrial content were measured using Nile Red and NAO staining, respectively. IR and PAM-IR treatments led to reductions in p-Akt expression. IR treatment reduced insulin mediated glucose metabolism while PAM and PAM-IR treatment showed increases with concurrent reductions in mitochondrial metabolism. All three treatments showed suppression in mitochondrial metabolism. PAM and PAM-IR also showed increases in glycolytic metabolism. While PAM and PAM-IR significantly increased lipid content, expression of inflammatory and lipogenic proteins were unaltered. Lastly, PAM-IR reduced BCAT2 protein expression, a regulator of BCAA metabolism. Both stressors independently reduced insulin signaling, mitochondrial function, and cell metabolism, however, only PAM-IR co-treatment significantly reduced the expression of regulators of metabolism not seen with individual stressors, suggesting an additive effect of stressors on metabolic programming.

Free Fatty Acid-Induced Peptide YY Expression Is Dependent on TG Synthesis Rate and Xbp1 Splicing.

Gut-derived satiety hormones provide negative feedback to suppress food intake and maintain metabolic function in peripheral tissues. Despite the wealth of knowledge of the systemic effects of these hormones, very little is known concerning the mechanisms by which nutrients, such as dietary fats, can promote the expression of genes involved in L-cell hormone production. We have tested the role of various dietary fats and found that after hydrolysis into free fatty acids (FFA's), there is a differential response in the extent to which they induce PYY gene and protein production. The effect of FFA's also seems to relate to triglyceride (TG) re-esterification rate, with MUFA re-esterifying faster with lower PYY production. We have also found that there are differences in potency of FFA's based on their desaturation patterns in vitro. The potency effect of FFA's is influenced by the rate of TG re-esterification, such that the longer FFA's are in contact with L-cells, the more PYY they produce. We found that chronic consumption of high-fat diets enables the small intestine to re-esterify FFA's into TG faster and earlier which resulted in a blunted postprandial PYY response. Lastly, we found that FFA's induce X-box-binding protein-1 activation (Xbp1s) in L-cells and that adenoviral delivery of Xbp1s was sufficient to induce PYY gene expression. Taken together, the present work indicates that dietary fat can induce satiety, in part, prior to re-esterification. Chronic high-fat diet consumption increases the rate of re-esterification which diminishes satiety and may lead to increased food intake. Targeting intestinal TG synthesis may prove beneficial in restoring obesity-associated reductions in postprandial satiety.

Effect of valine on myotube insulin sensitivity and metabolism with and without insulin resistance.

Population data have consistently demonstrated a correlation between circulating branched-chain amino acids (BCAA) and insulin resistance. Most recently valine catabolite, 3-hydroxyisobutyrate, has emerged as a potential cause of BCAA-mediated insulin resistance; however, it is unclear if valine independently promotes insulin resistance. It is also unclear if excess valine influences the ability of cells to degrade BCAA. Therefore, this study investigated the effect of valine on muscle insulin signaling and related metabolism in vitro. C2C12 myotubes were treated with varying concentrations (0.5 mM-2 mM) of valine for up to 48 h. qRT-PCR and western blot were used to measure metabolic gene and protein expression, respectively. Insulin sensitivity (indicated by pAkt:Akt), metabolic gene and protein expression, and cell metabolism were also measured following valine treatment both with and without varying levels of insulin resistance. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Valine did not alter regulators of mitochondrial biogenesis or glycolysis; however, valine reduced branched-chain alpha-keto acid dehydrogenase a (Bckdha) mRNA (but not protein) expression which was exacerbated by insulin resistance. Valine treatment had no effect on pAkt:Akt following either acute or 48-h treatment, regardless of insulin stimulation or varying levels of insulin resistance. In conclusion, despite consistent population data demonstrating a relationship between circulating BCAA (and related metabolites) and insulin resistance, valine does not appear to independently alter insulin sensitivity or worsen insulin resistance in the myotube model of skeletal muscle.

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.

Effect of metformin on myotube BCAA catabolism.

Metformin has antihyperglycemic properties and is a commonly prescribed drug for type II diabetes mellitus. Metformin functions in part by activating 5'-AMP-activated protein kinase, reducing hepatic gluconeogenesis and blood glucose. Metformin also upregulates peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α). Several population studies have shown levels of circulating branched-chain amino acids (BCAA) positively correlate with insulin resistance. Because BCAA catabolic enzyme content is regulated by PGC-1α, we hypothesized metformin may alter BCAA catabolism. Therefore, the purpose of this work was to investigate the effect of metformin at varying concentrations on myotube metabolism and related gene and protein expression. C2C12 myotubes were treated with metformin at 30 uM (physiological) or 2 mM (supraphysiological) for up to 24 hours. Metabolic gene expression was measured via quantitative real time polymerase chain reaction, protein expression was measured using Western blot, and mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Supraphysiological metformin upregulated PGC-1α mRNA expression along with related downstream targets, yet the reduced expression of electron transport chain components as well as basal and peak cell metabolism. Supraphysiological metformin also suppressed branched-chain aminotransferase 2 (BCAT2) and branched-chain-alpha-keto acid dehydrogenase E1a (BCKDHa) mRNA expression as well as BCAT2 protein expression and BCKDHa activity, which was accompanied by decreased Kruppel-like factor 15 protein expression. Physiological levels of metformin suppressed BCKDHa and cytochrome c oxidase mRNA expression at early time points (4-12 hours) but had no effect on any other outcomes. Together these data suggest metformin may suppress BCAA catabolic enzyme expression or activity, possibly reducing levels of circulating gluconeogenic substrates.

Leucine increases mitochondrial metabolism and lipid content without altering insulin signaling in myotubes.

Elevated circulating branched-chain amino acids (BCAA) such as leucine have been consistently correlated with increasing severity of insulin resistance across numerous populations. BCAA may promote insulin resistance through either mTOR-mediated suppression of insulin receptor substrate-1 or through the accumulation of toxic BCAA catabolites. Although the link between circulating BCAA and insulin resistance has been consistent, it has yet to be concluded if BCAA causally contribute to the development or worsening of insulin resistance. This work investigated the effect of leucine both with and without varying levels of insulin resistance on metabolism, metabolic gene expression, and insulin signaling. C2C12 myotubes were treated with and without varied concentrations of leucine up to 2 mM for 24 h both with and without varied levels of insulin resistance. Gene and protein expression were measured via qRT-PCR and Western blot, respectively. Mitochondrial metabolism was measured via O2 consumption. Leucine at 2 mM increased oxidative metabolism as well as gene expression of mitochondrial biogenesis, which was associated with increased cellular lipid content. Despite increased lipid content of leucine-treated cells, neither acute nor chronic leucine treatment at 2 mM affected insulin signaling in insulin sensitive, mildly insulin resistant, or severely insulin resistant cells. Similarly, leucine at lower concentrations (0.25 mM, 0.5 mM, and 1 mM) did not alter insulin signaling either, regardless of insulin resistance. Leucine appears to improve myotube oxidative metabolism and related metabolic gene expression. And despite increased lipid content of leucine-treated cells, leucine does not appear to alter insulin sensitivity either acutely or chronically, regardless of level of insulin resistance.