Development and Application of Q-Flux: Insights Into Liver and Renal Cortical Mitochondrial Metabolism

Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular and Molecular Physiology

First Advisor

Shulman, Gerald

Abstract

Mitochondria are membrane-bound organelles critical for generating the energy needed to power the body’s biochemical processes. The intricacies of mitochondria and the tricyclic acid (TCA) cycle contained within them were first recognized in the 1930s by Hans Krebs; in recent years, the field of mitochondrial metabolism has seen renewed interest, with focus shifting towards understanding how disrupted metabolism drives various diseases such as Type 2 Diabetes Mellitus, cancer, and neurodegeneration. Despite increased interest, there is a lack of advanced methods to study metabolism in detail. The aim of this dissertation is to develop a novel, widely implementable tool that measures the absolute rates of discreet enzymes and metabolic processes in vivo. To this end, I invented Q-Flux, a 13C5 glutamine-based metabolic flux analysis technique. Chapter 1 presents the equations that form the basis of Q-Flux and demonstrates its validity across various physiological contexts. While validating the Q-Flux method, I made several significant discoveries. For example, I showed that Complex II of the electron transport chain flows bidirectionally in vivo for the first time. I also quantitated the absolute rate of glutamine and branched-chain amino acid utilization for glucose production during hyperglucagonemia. During hyperinsulinemia, I found that reverse flux through Complex II in enhanced, the rate of TCA cycling is decreased, and glutamine catabolism is suppressed.

Having validated Q-Flux in the liver, I extended its application to the renal cortex in Chapter 2. The kidney’s role in glucose production, particularly in the context of Type 2 Diabetes, has long been debated, as methodological limitations have hindered a precise understanding. I built upon previous frameworks for estimating hepatic vs. renal glucose production while simultaneously using Q-Flux to determine the substrates that feed renal gluconeogenesis. I found that renal glucose production drives dysregulated whole-body glucose homeostasis in insulin resistance, and I discovered that glycerol, likely derived white adipose tissue lipolysis, is the key substrate that feeds this excess renal gluconeogenesis. I complemented these flux studies with extensive characterization of renal cortical insulin signaling and found that lipid-induced insulin resistance occurs in the renal cortex and is driven by the plasma membrane sn-1,2-DAG-PKCε-IRK1150 phosphorylation axis that has been previously described in liver and skeletal muscle; however, importantly, restoring insulin signaling within the renal cortex did not correct abnormal glycerol-driven renal gluconeogenesis, suggesting that this dysregulated process is extrarenal, largely driven by glycerol delivery secondary to excess lipolysis.

Q-Flux represents a major technological advance and is now being used in laboratories across the world to power scientific discovery. The discovery that renal glucose production from glycerol is a key driver of disrupted glucose homeostasis in insulin resistance represents a major finding and provides a promising target for future glucose lowering therapies.

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