Disruption of protein folding in the endoplasmic reticulum—“ER stress”—is associated with many different
metabolic diseases, particularly those associated with obesity that affect between 22 and 30 percent of adults
in the U.S. Because of this exceptional disease burden, it is important to understand the factors that cause ER
stress during metabolic dysregulation. Yet the pathways by which metabolic activity and ER homeostasis are
coupled are poorly understood.
Mitochondria are central to metabolism, and the TCA cycle is the hub of this activity, accepting substrates
from glycolysis and fatty acid oxidation for catabolism, generating reducing equivalents for electron transport
and for the maintenance of cellular redox homeostasis, and providing building materials for the reductive
biosynthesis of lipids, glucose, and amino acids. Because of its centrality to so many processes, flux through
the TCA cycle is likely to affect many diverse cellular pathways, even those with no obvious direct connection.
This includes ER protein processing, which is sensitive to changes in redox state, amino acid availability, and
cellular lipid content.
In this proposal, we provide evidence for a previously unknown functional relationship between TCA cycle
activity and ER homeostasis in metabolically active cells, including hepatocytes, myocytes, and adipocytes,
that depends on production of NADPH by the TCA cycle and redox regulation of glutathione. This proposal is
designed to identify the basic mechanisms linking TCA-dependent NADPH production in the mitochondria to
homeostasis in the ER. Toward that end, we propose three specific aims: (1) Determine how NADPH
production and compartmentalization link nutrient flow to ER stress; (2) Determine how changes to
mitochondrial and cytosolic glutathione redox promote ER oxidation; and (3) Determine how TCA activity and
glutathione redox alter ER function. We will achieve these aims using a combination of genetic and
pharmacological tools to manipulate TCA cycle activity; cutting-edge biosensors to monitor changes in cellular
redox status; manipulation and analysis of ER-mitochondrial contacts; and molecular biology approaches to
manipulate and assess ER functionality. The outcome of this work will be a mechanistic understanding of how
metabolic activity alters ER function to contribute to disease.