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

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Abstract

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.

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Abbreviations

3HIB:

3-Hydroxyisobutyrate

ACC:

Acetyl-CoA carboxylase

AMPK:

5′ Adenosine monophosphate-activated protein kinase

BCAA:

Branched-chain amino acid

BCAT:

Branched-chain aminotransferase

BCKDH:

Branched-chain alpha-keto acid dehydrogenase

BCKDK:

Branched-chain alpha-keto acid dehydrogenase kinase

CS:

Citrate synthase

ECAR:

Extracellular acidification rate

FCCP:

Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone

GLUT4:

Glucose transporter 4

HIBADH:

3-Hydroxyisobutyrate dehydrogenase

LDHa:

Lactate dehydrogenase A

NRF1/2:

Nuclear respiratory factor 1/2

OCR:

Oxygen consumption rate

PGC-1α:

Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha

PK:

Pyruvate kinase

Sirt1/3:

NAD+-dependent deacetylase sirtuin-1/3

TBP:

TATA Binding Protein

TFAM:

Mitochondrial transcription factor A

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Acknowledgements

Support for this work was provided by the Department of Exercise Science within the Congdon School of Health Sciences. We would like also to thank the Department of Physical Therapy (Congdon School of Health Sciences) for the use of shared lab space and equipment. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Exercise Science, High Point University, One University Parkway, High Point, NC, 27262-3598, USA

    Madison E. Rivera, Emily S. Lyon, Michele A. Johnson & Roger A. Vaughan

  2. School of Health Sciences, Lindenwood University, St. Charles, MO, 63301, USA

    Kyle L. Sunderland

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  1. Madison E. Rivera
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Contributions

MER conducted experiments and was lead author for the manuscript. ESL and MAJ conducted experiments and assisted with manuscript preparation. KLS assisted with manuscript preparation. RAV conceived the study, conducted and oversaw experiments, performed all statistical analyses, and oversaw manuscript preparation. All authors have read and approved the final manuscript. Authors and contributors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Correspondence to Roger A. Vaughan.

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11010_2020_3720_MOESM1_ESM.tif

Fig. S1 Effect of valine at 2 mM for 48 on basal and peak mitochondrial and glycolytic metabolism. (a) Effect of valine at 2.0 mM for 48 h on myotube oxygen consumption (O2 pMol/min) under basal and peak metabolic conditions with Seahorse profile at right. (b) Effect of valine at 2.0 mM for 48 h on myotube glycolytic metabolism (mpH/min) under basal and peak metabolic conditions with Seahorse profile at right. Notes: Metabolism was analyzed using a student’s t test using n = 46 per group from two independent experiments. Oxygen consumption was normalized to non-mitochondrial respiration.Supplementary file1 (TIF 104 kb)

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Rivera, M.E., Lyon, E.S., Johnson, M.A. et al. Effect of valine on myotube insulin sensitivity and metabolism with and without insulin resistance. Mol Cell Biochem 468, 169–183 (2020). https://doi.org/10.1007/s11010-020-03720-y

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