(211d) Application of 13C Flux Analysis to Identify Molecular Disease Mechanisms: From Cells to Whole Animals

¹³C metabolic flux analysis (MFA) is the gold standard approach for quantifying rates of biochemical reactions inside living cells. It has been widely applied to debottleneck the metabolism of industrial host organisms, but it is now being increasingly used to investigate metabolic phenotypes of human disease models. As an example of the latter, my group has applied ¹³C MFA to quantitatively map the metabolic alterations that occur in liver cells exposed to toxic levels of free fatty acids (FFAs). In humans, abnormal hepatic lipid accumulation caused by FFA overload leads to a condition known as nonalcoholic fatty liver disease (NAFLD), which is closely associated with obesity and type-2 diabetes and is estimated to affect up to 30% of the U.S. population. Unfortunately, the factors that control disease severity are poorly understood, and therapeutic strategies for preventing or reversing NAFLD are limited.

Based on ¹³C MFA studies of cultured hepatocytes and hepatic cell lines, we have uncovered a novel lipotoxicity mechanism by which saturated FFAs promote abnormal cell metabolism and oxidative stress in liver cells. This process involves dysregulation of intracellular calcium homeostasis that subsequently promotes increased mitochondrial metabolism and accumulation of toxic reactive oxygen species. Surprisingly, ¹³C MFA revealed that these metabolic alterations were not driven by increased FFA oxidation, but instead were fueled by increased entry of glutamate carbon into the citric acid cycle (CAC). I will discuss these findings as well as my lab’s ongoing work to develop an in vivo ²H/¹³C MFA approach to simultaneously assess multiple gluconeogenic, CAC, and anaplerotic fluxes in the livers of conscious, unstressed mice. Our goal is to establish a scalable in vivo MFA platform that can be used to examine liver metabolic phenotypes in mouse models of NAFLD and to test specific hypotheses suggested by our in vitro studies.