Leucine in the Presence of Inflammation Alters BCAA Metabolism in Human Myotubes

Abstract

RationaleMetabolomics studies suggest a positive association between fasting concentrations of circulating branched chain amino acids (BCAA) and the development of insulin resistance and type II diabetes. Whether BCAAs are a causative factor or a biomarker for the development of insulin resistance remains unclear. Proinflammatory cytokines (e.g., TNFα), which are commonly elevated in obesity, lower the expression of BCAA transport and oxidation genes and reduce leucine influx in differentiated adipocytes. As skeletal muscle accounts for ~65% of whole body BCAA transamination capacity in humans, the effect of inflammation on BCAA metabolism in skeletal muscle is important to understand, but remains unknown.ObjectiveTo test the hypothesis that TNFα will reduce BCAA uptake and metabolism in response to a leucine challenge in differentiated human skeletal muscle cells.MethodsPrimary human progenitor cells (myoblasts) were obtained from the vastus lateralis muscle of young males (n=6). Study 1: Sorted myoblasts were proliferated in growth media (GM) until ~80% confluent, then switched to differentiation media (DM). After 72 h of differentiation, 10 ng/mL of TNFα was added to the DM. Cells were incubated for an additional 48 h. Study 2: Same as Study 1 with the following modification: after 47 h of TNFα treatment, 0.5 mM of leucine (~blood leucine concentration post ingestion of 30g of protein) or 2 mM of leucine (supra‐physiologic dose) was added to the DM+TNFα. Cells from both studies were harvested after 48 h of DM+TNFα with or without 1 h of leucine. Control cells did not receive TNFα or leucine. Real‐time qPCR was used to measure mRNA abundance of genes associated with inflammation (NFκB, IL1β, IL6, TNFα), BCAA transport (Slc7a5), mitochondrial function (PCG1α), and the unfolded protein response (Hspa5). Immunoblotting was used to quantify protein levels of genes associated with BCAA transport (CD98), BCAA metabolism (Bcat2, Bckdhb), and protein synthesis (p70S6K). A colorimetric BCAA assay was used to measure cell culture media levels of BCAAs.ResultsTNFα treatment alone reduced mRNA levels of PCG1α (p<0.05) and increased mRNA levels of NFκB, IL1β, IL6, and endogenous TNFα (p<0.001). TNFα treatment also reduced BCAA levels in culture media (p<0.05), suggesting greater BCAA uptake. The leucine challenge (0.5 and 2 mM), in the presence of TNFα, increased mRNA levels of Slc7a5 and Hspa5 (p<0.01), reduced Bcat2 protein levels (p<0.05), and increased the ratio of phosphorylated p70S6K to total p70S6K (p<0.05). Bckdhb protein levels were lower with the 2mM leucine dose only (p<0.05). Protein levels of CD98 were not significantly different with either leucine dose (p=0.07).ConclusionsContrary to adipocytes and our hypothesis, TNFα increases BCAA uptake through upregulation of BCAA transport in differentiated myoblasts. Further, at a physiologically relevant leucine dose, in the presence of inflammation, protein synthesis and the unfolded protein response are also up‐regulated, while BCAA metabolism and mitochondrial biogenesis and function are down‐regulated. Whether elevated protein synthesis coupled with the unfolded protein response is problematic for the development of insulin resistance is unclear.Support or Funding InformationCornell Start‐up