Impaired BCAA catabolism during adipocyte differentiation decreases glycolytic flux

Abstract

ObjectiveAdipocytes upregulate branched-chain amino acid (BCAA) catabolism during differentiation, but the downstream metabolic and biological impacts of this induction remain unclear. Here we sought to understand how BCAA catabolism supports intermediary metabolism and function in cultured adipocytes.MethodsUsing CRISPR/Cas9 gene-editing, we generated 3T3-L1 pre-adipocytes deficient in either branched-chain ketoacid dehydrogenase, subunit alpha (Bckdha) or acyl-CoA dehydrogenase 8 (Acad8), which predominantly metabolizes valine-derived isobutyryl-CoA, and examined their metabolism after differentiation. We performed stable-isotope tracing and mass spectrometry, developed a 13C metabolic flux analysis (13C-MFA) model incorporating central carbon metabolism and lipid biosynthesis, and performed RNA-sequencing to further explore functional changes.ResultsGeneration of polyclonal cultures deficient in Bckdha resulted in a 90%+ decrease in [U-13C6]leucine labeling in citrate in differentiated adipocytes (p<0.001). Lipid droplet accumulation and mRNA levels of key markers of adipocyte differentiation were unaffected by Bckdha deficiency; however, gene set enrichment analysis (GSEA) indicated adipogenesis and glycolysis were the primary downregulated pathways in these cells. Bckdha-deficient adipocytes had decreased glucose uptake (p<0.05), glycolytic flux (p<0.05), and increased glutamine contribution to the TCA cycle (p<0.05). These findings were supported by reduced lactate secretion (p<0.001) and oxygen consumption rates (p<0.05) and correlated with the transcriptional changes observed in adipocytes. Total de novo fatty acid synthesis flux was maintained but levels of odd-chain fatty acids and branched-chain fatty acids were reduced, as was the C16:1/C16:0 ratio. 13C-MFA confirmed these changes and highlighted the complex relationship between the TCA cycle, BCAA catabolism, and mitochondrial respiration. Finally, Acad8 deficiency resulted in far fewer differentially expressed genes compared to Bckdha deficiency and milder metabolic changes overall.ConclusionsThese findings highlight key metabolic and transcriptional changes that occur due to Bckdha deficiency in adipocytes and identify important links between BCAA catabolism, glycolytic flux, and respiration.HighlightsBckdha deficiency reduced glycolysis and lactate secretion in adipocytesPyruvate and glutamine contribution to TCA metabolism and acetyl-CoA increasedde novo fatty acid synthesis flux was maintained but fatty acid diversity decreasedRNA-seq data highlighted regulation of genes in glycolysis, adipogenesis, and EMTAcad8 deficiency resulted in fewer altered transcripts and milder metabolic changes

Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids

“…The addition of isotopically labeled iso-C16:0 BCFA to these Bckdha-KO adipocytes reliably confirms the structure of the observed peaks. We infected pre-adipocyte cultures with lentivirus delivering CRISPR-Cas and sgRNAs, confirming loss of Bckdha and BCAA catabolism after differentiation (26). These cells were treated with 100 mM 2 H 7 -iso-C16:0-BSA complex for 96 hours followed by lipid extraction and UHPLC-MS analysis.…”

“…3A, black peak). Notably, these acyl chains are presumed to all be straight chain considering the absence of BCFA production in the Bckdha-KO adipocyte model (26). Next, we examined TAG 48:0 species containing a tracerinduced mass shift due to 2 H 7 -iso-C16:0 incorporation (Fig.…”