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

Rivera, Caroline N.; Watne, Rachel M.; Brown, Zoe; Mitchell, Samantha A.; Wommack, Andrew J.; Vaughan, Roger A.

doi:10.1007/s00726-022-03224-7

Cited by 7 publications

(5 citation statements)

References 32 publications

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“…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 …”

Section: Discussioncontrasting

confidence: 96%

“…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. Effect of BT2 at 5 µM for 24 h with and without insulin resistance on protein expression of pBCKDHa (Ser293).…”

Section: Discussionmentioning

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: 96%

Section: Discussionmentioning

confidence: 99%

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 …”

Section: Discussioncontrasting

confidence: 89%

“…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. First, the use of a nonspecific DNA hypomethylating agent is a clear limitation.…”

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

confidence: 93%

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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“…In line with these observations, Lian et al showed activation of myotube 5′-AMP-activated protein kinase (AMPK), a master regulator of energetics which activates PGC-1 α [ 20 ], appears to increase BCAA catabolic enzyme expression and/or activity [ 21 ]. Importantly, however, activation of BCAA catabolism via AMPK activators, such as AICAR during cell culture experiments, might be dependent on concentration and glucose availability [ 22 ], and may reduce BCAA catabolism despite an upregulation of BCAA catabolic enzymes, which may accompany metabolic stress as seen with atrophy [ 23 , 24 ]. Collectively, these observations agree with others demonstrating exercise (another activator of PPAR β / δ , AMPK, and PGC-1 α ) increases BCAA metabolism [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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 β/δ (PPARβ/δ). The present report examined the effects of the PPARβ/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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Effect of AMPK activation and glucose availability on myotube LAT1 expression and BCAA utilization

Cited by 7 publications

References 32 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

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

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