UC San Diego

Metabolism is essential for the maintenance of cellular homeostasis as cells must either import or synthesize substrates for use in biosynthetic and energy generating reactions. Different cell types have different metabolic functions, and adipocyte metabolism changes dramatically during adipogenesis or in response to overnutrition/obesity. The metabolism of substrates such as glucose and fatty acids (FA) are well-studied in adipocytes, but evidence suggests that the metabolism of the essential branched-chain amino acids (BCAA - leucine, isoleucine, and valine) is particularly altered in obesity. The chapters of this dissertation are independent bodies of work that explore how BCAA metabolism changes in differentiating adipocytes, contributes to de novo lipogenesis (DNL), and regulates the fatty acid profile within the lipidome. Chapter 1, titled “Studying adipose tissue branched-chain amino acid metabolism in obesity and diabetes using stable-isotope tracing'” is a review of the relevant adipose- and BCAA-related literature and the utility of stable-isotope tracing and metabolic flux analysis (MFA) to the study adipocyte metabolism. Chapter 2, titled “Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis,” uses stable-isotope tracing and mass spectrometry to quantify the increase in the contribution of BCAA catabolism to the TCA cycle and DNL throughout differentiation. This chapter also details a vitamin B12 deficiency in typical adipocyte cell culture and demonstrates adipocytes' ability to reprogram metabolism to maintain BCAA levels in physiological media with lower amino acid concentrations. Chapter 3, titled “Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues,” is a broad characterization of branched-chain fatty acid (BCFA) synthesis in adipocytes and in mammals using a variety of stable-isotope tracers. We show that BCFAs are highly synthesized in adipose tissue due to expression of a critical protein in AT that facilitates their synthesis, are synthesized from intermediate metabolites in BCAA catabolism, and are decreased in a high-fat diet via a hypoxia-related mechanism. Chapter 4, titled “Altered branched-chain amino acid catabolism drives distinct changes in the TCA cycle and lipidome,” is currently being prepared for submission for publication. In this manuscript, we interrogate the metabolic and functional impacts of reduced BCAA catabolism through CRISPR/Cas9-mediated deficiency of Bckdha. While differentiation is unaffected by Bckdha deficiency, oxygen consumption rates are reduced, and relative glucose metabolism increases to support DNL. Furthermore, Bckdha deficiency decreased the levels of polyunsaturated fatty acid (PUFA)-containing sphingomyelin and increased the levels of PUFA-containing phosphatidylcholine. We hypothesize that the dramatic reduction of BCFAs and OCFAs in Bckdha deficient adipocytes is driving this FA reorganization phenotype. The functional impact of this change is unknown but membrane fluidity and permeability could be affected. Taken together, these collective studies demonstrate the importance of understanding the regulation and function of BCAA metabolism in adipocytes in the obese or insulin resistant state. These findings could impact biomarker development and improve our understanding of the progression of diabetes.