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

Rivera, Caroline N.; Kamer, Madison M.; Rivera, Madison E.; Watne, Rachel M.; Macgowan, Trent C.; Wommack, Andrew J.; Vaughan, Roger A.

doi:10.1016/j.mce.2022.111800

Cited by 7 publications

(11 citation statements)

References 53 publications

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“…28 Thus, altered BCKDH phosphorylation appears to be one of several factors regulating BCAA metabolism. We also observed reduced BCAA media content during insulin resistance, which differs from prior observations by our group, which showed a substantial accumulation of extracellular BCAA in insulin-resistant groups 26 ; however, a recovery period following insulin exposure appears to be necessary for hyperinsulinemic-mediated accumulation of extracellular BCAA. 30 Thus, the lack of extracellular BCAA accumulation under the tested circumstances may be a limitation of the model.…”

Section: Discussioncontrasting

confidence: 98%

Section: Methodsmentioning

confidence: 99%

“…Chromatographic separation and quantification of leucine, isoleucine, and valine was performed using a Shimadzu Nexera UHPLC system equipped with a Phenomenex Kinetex C18 100 Å column (100 ×3 mm, 2.6 µm) kept at a temperature of 30°C connected to Shimadzu LCMS-8045 triple quadrupole mass spectrometer (Shimadzu) fitted with a DUIS ion source. 26 The source used nebulizer gas 2.0 L/min, drying gas 10.0 L/min, desolvation line temperature 200°C, and heat block temperature 200°C, with CID gas 230 kPa. The mobile phases of A (water with 0.1% formic acid) and B (methanol with 0.1% formic acid) were used at a flow rate of 0.4 mL/min for the following gradient method: 0 min, 20% B; 1.5 min, 20% B; 1.7 min, 40% B; 3.5 min, 40% B; 5 min, 65% B; 8 min, 65% B; followed by 4 A stock solution containing all BCAAs at a concentration of 8.0 mM was obtained by dissolving each amino acid in water/methanol solution (50:50, v/v) and kept at 4°C.…”

Section: Liquid Chromatography-mass Spectrometry (Lc-ms)mentioning

confidence: 99%

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

Rivera,

Smith,

Draper

et al. 2024

J of Cellular Biochemistry

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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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Section: Discussioncontrasting

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Section: Liquid Chromatography-mass Spectrometry (Lc-ms)mentioning

confidence: 99%

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

Rivera,

Smith,

Draper

et al. 2024

J of Cellular Biochemistry

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“…Surprisingly, 5-Aza appeared to enhance leucine and isoleucine disposal during insulin resistance. We also observed reduced BCAA media content during insulin resistance which seemed at odds with previous observations, 34 however in our previous report, insulin resistance was followed by a recovery period from the hyperinsulinemic conditions which appears to be necessary for hyperinsulinemic-mediated accumulation of extracellular BCAA. 35 Taken together, DNA methylation may regulate mitochondrial signaling/function in cultured myotubes, however, several mechanisms of action are unclear (in-part due to the somewhat unpredictable nature of 5-Aza DNA integration and the unknown initial DNA methylation status of each target), warranting the mention of important limitations of our work.…”

Section: -Aza-mediated Dna Hypomethylation and Insulin Resistance Sup...contrasting

confidence: 87%

“…Surprisingly, 5‐Aza appeared to enhance leucine and isoleucine disposal during insulin resistance. We also observed reduced BCAA media content during insulin resistance which seemed at odds with previous observations, 34 however in our previous report, insulin resistance was followed by a recovery period from the hyperinsulinemic conditions which appears to be necessary for hyperinsulinemic‐mediated accumulation of extracellular BCAA 35 …”

Section: Discussioncontrasting

confidence: 73%

5‐Aza‐2′‐deoxycytidine‐mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity

Rivera,

Kamer,

Cook

et al. 2023

Cell Biochemistry & Function

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

Rivera,

Watne,

Wommack

et al. 2023

Amino Acids

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

Cited by 7 publications

References 53 publications

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

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

5‐Aza‐2′‐deoxycytidine‐mediated DNA hypomethylation with and without concurrent insulin resistance suppresses myotube mitochondrial capacity

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

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