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The mechanistic target of rapamycin complex (mTORC) regulates protein synthesis and can be activated by branched-chain amino acids (BCAAs). mTORC has also been implicated in the regulation of mitochondrial metabolism and BCAA catabolism. Some speculate that mTORC overactivation by BCAAs may contribute to insulin resistance. The present experiments assessed the effect of mTORC activation on myotube metabolism and insulin sensitivity using the mTORC agonist MHY1485, which does not share structural similarities with BCAAs. METHODS: C2C12 myotubes were treated with MHY1485 or DMSO control both with and without rapamycin. Gene expression was assessed using qRT-PCR and insulin sensitivity and protein expression by western blot. Glycolytic and mitochondrial metabolism were measured by extracellular acidification rate and oxygen consumption. Mitochondrial and lipid content were analyzed by fluorescent staining. Liquid chromatography-mass spectrometry was used to assess extracellular BCAAs. RESULTS: Rapamycin reduced p-mTORC expression, mitochondrial content, and mitochondrial function. Surprisingly, MHY1485 did not alter p-mTORC expression or cell metabolism. Neither treatment altered indicators of BCAA metabolism or extracellular BCAA content. CONCLUSION: Collectively, inhibition of mTORC via rapamycin reduces myotube metabolism and mitochondrial content but not BCAA metabolism. The lack of p-mTORC activation by MHY1485 is a limitation of these experiments and warrants additional investigation.
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Mitocondrias , Fibras Musculares Esqueléticas , Sirolimus , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Animales , Ratones , Sirolimus/farmacología , Línea Celular , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Aminoácidos de Cadena Ramificada/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Resistencia a la Insulina , Serina-Treonina Quinasas TOR/metabolismo , NaftiridinasRESUMEN
Branched-chain amino acids (BCAA) are correlated with severity of insulin resistance, which may partially result from mitochondrial dysfunction. Mitochondrial dysfunction is also common during insulin resistance and is regulated in part by altered mitochondrial fusion and fission (mitochondrial dynamics). To assess the effect of BCAA on mitochondrial dynamics during insulin resistance, the present study examined the effect of BCAA on mitochondrial function and indicators of mitochondrial dynamics in a myotube model of insulin resistance. C2C12 myotubes were treated with stock DMEM or DMEM with additional BCAA at 0.2 mM, 2 mM, or 20 mM to achieve a continuum of concentrations ranging from physiologically attainable to supraphysiological (BCAA overload) both with and without hyperinsulinemia-mediated insulin resistance. qRT-PCR and Western blot were used to measure gene and protein expression of targets associated with mitochondrial dynamics. Mitochondrial function was assessed by oxygen consumption, and mitochondrial content was measured using mitochondrial-specific staining. Insulin resistance reduced mitochondrial function, peroxisome proliferator-activated receptor gamma coactivator 1-alpha mRNA, and citrate synthase expression mRNA, but not protein expression. Excess BCAA at 20 mM also independently reduced mitochondrial function in insulin-sensitive cells. BCAA did not alter indicators of mitochondrial dynamics at the mRNA or protein level, while insulin resistance reduced mitochondrial fission protein 1 mRNA, but not protein expression. Collectively, BCAA at excessively high levels or coupled with insulin resistances reduce mitochondrial function and content but do not appear to alter mitochondrial dynamics under the tested conditions.
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Fructose is a commonly consumed monosaccharide implicated in developing several metabolic diseases. Previously, elevated branched-chain amino acids (BCAA) have been correlated with the severity of insulin resistance. Most recently, the effect of fructose consumption on the downregulation of BCAA catabolic enzymes was observed. Thus, this mechanistic study investigated the effects of physiologically attainable levels of fructose, both with and without concurrent insulin resistance, in a myotube model of skeletal muscle. METHODS: C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. RESULTS: Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. DISCUSSION: Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.
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Aminoácidos de Cadena Ramificada , Fructosa , Resistencia a la Insulina , Fibras Musculares Esqueléticas , Animales , Fructosa/farmacología , Aminoácidos de Cadena Ramificada/metabolismo , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Línea Celular , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Glucólisis/efectos de los fármacos , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Consumo de Oxígeno/efectos de los fármacosRESUMEN
PURPOSE: Autophagy and heat shock protein (HSP) response are proteostatic systems involved in the acute and adaptive responses to exercise. These systems may upregulate sequentially following cellular stress including acute exercise, however, currently few data exist in humans. This study investigated the autophagic and HSP responses to acute intense lower body resistance exercise in peripheral blood mononuclear cells (PBMCs) with and without branched-chain amino acids (BCAA) supplementation. METHODS: Twenty resistance-trained males (22.3 ± 1.5 yr; 175.4 ± .7 cm; 86.4 ± 15.6 kg) performed a bout of intense lower body resistance exercise and markers of autophagy and HSP70 were measured immediately post- (IPE) and 2, 4, 24, 48, and 72 h post-exercise. Prior to resistance exercise, 10 subjects were randomly assigned to BCAA supplementation of 0.22 g/kg/d for 5 days pre-exercise and up to 72 h following exercise while the other 10 subjects consumed a placebo (PLCB). RESULTS: There were no difference in autophagy markers or HSP70 expression between BCAA and PLCB groups. LC3II protein expression was significantly lower 2 and 4 h post-exercise compared to pre-exercise. LC3II: I ratio was not different at any time point compared to pre-exercise. Protein expression of p62 was lower IPE, 2, and 4 h post-exercise and elevated 24 h post-exercise. HSP70 expression was elevated 48 and 72 h post-exercise. CONCLUSIONS: Autophagy and HSP70 are upregulated in PBMCs following intense resistance exercise with autophagy increasing initially post-exercise and HSP response in the latter period. Moreover, BCAA supplementation did not affect this response.
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Empowering the Participant Voice (EPV) is an NCATS-funded six-CTSA collaboration to develop, demonstrate, and disseminate a low-cost infrastructure for collecting timely feedback from research participants, fostering trust, and providing data for improving clinical translational research. EPV leverages the validated Research Participant Perception Survey (RPPS) and the popular REDCap electronic data-capture platform. This report describes the development of infrastructure designed to overcome identified institutional barriers to routinely collecting participant feedback using RPPS and demonstration use cases. Sites engaged local stakeholders iteratively, incorporating feedback about anticipated value and potential concerns into project design. The team defined common standards and operations, developed software, and produced a detailed planning and implementation Guide. By May 2023, 2,575 participants diverse in age, race, ethnicity, and sex had responded to approximately 13,850 survey invitations (18.6%); 29% of responses included free-text comments. EPV infrastructure enabled sites to routinely access local and multi-site research participant experience data on an interactive analytics dashboard. The EPV learning collaborative continues to test initiatives to improve survey reach and optimize infrastructure and process. Broad uptake of EPV will expand the evidence base, enable hypothesis generation, and drive research-on-research locally and nationally to enhance the clinical research enterprise.
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OBJECTIVES: To determine the agreement between HNNE and TIMP at TCA for preterm infants born <32+0 weeks' gestation, and to evaluate their correlation to PDMS-2 at 12-month corrected age (CA). METHODS: Infants born between November 2013 to June 2022 who had both HNNE and TIMP performed at TCA of 37+0-41+6 weeks gestation, and motor outcome assessed using the PDMS-2 at 12-month old were enrolled. The HNNE and 12-month PDMS-2 findings were categorized as optimal vs sub-optimal. TIMP was categorized as typical vs atypical. Cohen's kappa was used to determine the agreement between HNNE and TIMP. Sensitivity analysis and Receiver Operating Characteristic (ROC) curves were used to evaluate the predictive values of HNNE and TIMP on motor outcome at CA of 12-months. RESULTS: HNNE and TIMP done on 125 infants at TCA do not show reliable agreement. HNNE demonstrated slight and fair agreement with the 12-month Total Motor Quotient (TMQ) and Fine Motor Quotient (FMQ) of the PDMS-2 respectively. TIMP at TCA demonstrated fair agreement with all sub-domains of motor function on PDMS-2 at 12-months. In comparison with TIMP, HNNE at TCA is more sensitive at predicting suboptimal total, gross and fine motor outcomes at 12-month CA with sensitivity of 68.4 %, 51.9 %, and 83.3 % vs 44.4 %, 31.8 % and 53.3 % respectively. Atypical TIMP at TCA is more specific for suboptimal total, gross and fine motor outcomes at 12-month CA with specificity of 90.3 %, 89 % and 90.5 % respectively. Neurobehavioral assessments at TCA using HNNE and TIMP were predictive of suboptimal fine motor quotient at CA of 12-months with AUC of 0.760 (p = 0.011) and 0.718 (p = 0.032) respectively. The difference in AUC between the 2 instruments of 0.042 was not statistically significant (p = 0.741). CONCLUSIONS: While the HNNE and TIMP done at TCA did not demonstrate significant agreement, suboptimal HNNE and atypical TIMP at TCA were predictive of suboptimal FMQ on PDMS-2 at 12-month CA.
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Recien Nacido Prematuro , Lactante , Recién Nacido , Humanos , Embarazo , Femenino , Edad Gestacional , Examen NeurológicoRESUMEN
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.
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Resistencia a la Insulina , Insulina , Humanos , Aminoácidos de Cadena Ramificada/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas , Inhibidores de Proteínas Quinasas/farmacología , ProtonesRESUMEN
Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/metabolismo , ARN Interferente Pequeño/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ARN Bicatenario , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismoRESUMEN
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.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Fenilbutiratos , Humanos , Diabetes Mellitus Tipo 2/metabolismo , Biogénesis de Organelos , Línea Celular , Fibras Musculares Esqueléticas/metabolismo , Insulina/metabolismo , Aminoácidos de Cadena Ramificada/farmacología , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
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.
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Resistencia a la Insulina , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Canagliflozina/farmacología , Canagliflozina/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Músculo Esquelético/metabolismo , Mitocondrias/metabolismoRESUMEN
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.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Humanos , Resistencia a la Insulina/genética , Decitabina/farmacología , Metilación de ADN , Diabetes Mellitus Tipo 2/metabolismo , Epigénesis Genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Insulina/metabolismo , Aminoácidos de Cadena Ramificada/genética , Aminoácidos de Cadena Ramificada/metabolismo , ADN/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/farmacologíaRESUMEN
(1) Background: With increasing international travel and mass population displacement due to war, famine, climate change, and immigration, pathogens, such as Staphylococcus aureus (S. aureus), can also spread across borders. Methicillin-resistant S. aureus (MRSA) most commonly causes skin and soft tissue infections (SSTIs), as well as more invasive infections. One clonal strain, S. aureus USA300, originating in the United States, has spread worldwide. We hypothesized that S. aureus USA300 would still be the leading clonal strain among US-born compared to non-US-born residents, even though risk factors for SSTIs may be similar in these two populations (2) Methods: In this study, 421 participants presenting with SSTIs were enrolled from six community health centers (CHCs) in New York City. The prevalence, risk factors, and molecular characteristics for MRSA and specifically clonal strain USA300 were examined in relation to the patients' self-identified country of birth. (3) Results: Patients born in the US were more likely to have S. aureus SSTIs identified as MRSA USA300. While being male and sharing hygiene products with others were also significant risks for MRSA SSTI, we found exposure to animals, such as owning a pet or working at an animal facility, was specifically associated with risk for SSTIs caused by MRSA USA300. Latin American USA300 variant (LV USA300) was most common in participants born in Latin America. Spatial analysis showed that MRSA USA300 SSTI cases were more clustered together compared to other clonal types either from MRSA or methicillin-sensitive S. aureus (MSSA) SSTI cases. (4) Conclusions: Immigrants with S. aureus infections have unique risk factors and S. aureus molecular characteristics that may differ from US-born patients. Hence, it is important to identify birthplace in MRSA surveillance and monitoring. Spatial analysis may also capture additional information for surveillance that other methods do not.
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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.
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Aminoácidos de Cadena Ramificada , Resistencia a la Insulina , Humanos , Aminoácidos de Cadena Ramificada/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Insulina/metabolismoRESUMEN
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.
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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.
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Introduction: Clinical trials are a vital component of translational science, providing crucial information on the efficacy and safety of new interventions and forming the basis for regulatory approval and/or clinical adoption. At the same time, they are complex to design, conduct, monitor, and report successfully. Concerns over the last two decades about the quality of the design and the lack of completion and reporting of clinical trials, characterized as a lack of "informativeness," highlighted by the experience during the COVID-19 pandemic, have led to several initiatives to address the serious shortcomings of the United States clinical research enterprise. Methods and Results: Against this background, we detail the policies, procedures, and programs that we have developed in The Rockefeller University Center for Clinical and Translational Science (CCTS), supported by a Clinical and Translational Science Award (CTSA) program grant since 2006, to support the development, conduct, and reporting of informative clinical studies. Conclusions: We have focused on building a data-driven infrastructure to both assist individual investigators and bring translational science to each element of the clinical investigation process, with the goal of both generating new knowledge and accelerating the uptake of that knowledge into practice.
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The AutoScore framework can automatically generate data-driven clinical scores in various clinical applications. Here, we present a protocol for developing clinical scoring systems for binary, survival, and ordinal outcomes using the open-source AutoScore package. We describe steps for package installation, detailed data processing and checking, and variable ranking. We then explain how to iterate through steps for variable selection, score generation, fine-tuning, and evaluation to generate understandable and explainable scoring systems using data-driven evidence and clinical knowledge. For complete details on the use and execution of this protocol, please refer to Xie et al. (2020),1 Xie et al. (2022)2, Saffari et al. (2022)3 and the online tutorial https://nliulab.github.io/AutoScore/.
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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.
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Glucosa , Metformina , Aminoácidos de Cadena Ramificada/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Glucosa/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Ratones , AnimalesRESUMEN
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.