Abstract 566: Branched Chain Ketoacid Dehydrogenase Kinase Inhibition Alters Substrate Utilization and Gene Expression in Myocytes

Abstract

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type II diabetes and metabolic syndrome. Emerging data also suggest that impaired BCAA catabolism may play a role in heart failure progression. BCAA are catabolized via the branched chain ketoacid (BCKA) dehydrogenase enzyme complex (BCKDH). BCKD kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDHE1a subunit, and the phosphatase PPM1k dephosphorylates this same site to activate BCAA catabolism. Using an inhibitor of BCKDK (BT2), BCAA catabolism is increased in vivo. Here, we utilized metabolomics to evaluate the contribution of BCAA catabolism to substrate preference in heart and skeletal muscle. Surprisingly, BCKDK inhibition with BT2 had no effect on incorporation of glucose into TCA cycle intermediates in heart or skeletal muscle. Because others have recently shown that the primary site of BCAA catabolism is skeletal muscle, we knocked down BCKDK and PPM1k in human skeletal myocytes to further investigate how BCKDK loss or inhibition affects substrate utilization. Similar to our in vivo observations, knockdown of BCKDK and PPM1k had no effect on glucose and pyruvate utilization in a mitochondrial function assay. However, an increase in maximal respiration was observed after BCKDK knockdown when fatty acids were used. To evaluate the mechanisms underlying this increase we then performed RNAseq in these cells after BCKDK and PPM1K knockdown and observed changes in a number of genes that may explain these alterations in substrate utilization. Finally, we performed C13 BCAA metabolomics in human skeletal myocytes after BT2 treatment or knockdown of BCKDK and PPM1k. Using BT2, we observed a dose-responsive reduction in BCKA production from C13 BCAA by the muscle cells as expected; however, though BCKA production was increased after PPM1k was knocked down, we surprisingly did not observe a decrease in BCKA production after BCKDK knockdown. Collectively these data suggest that BCKDK inhibition may improve metabolism and cardiac function by altering substrate preference in skeletal myocytes.