Hepatic Ketogenesis as a Novel Regulator of Liver Metabolism and Injury

Ketone body metabolism plays the fundamental metabolic role of generating alternative fuel sources, in the form of circulating ketone bodies, derived from breakdown of fatty acids, traditionally during states of carbohydrate depletion. Ketone bodies are produced in the mitochondria of the liver via ketogenesis, a process driven by activity of the fate-committing ketogenic enzyme, mitochondrial 3-hydroxymethylglutaryl-CoA synthase (HMGCS2). Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect nearly one billion individuals worldwide and approximately 30% of all adults in the United States. NAFLD is defined by pathological lipid accumulation in the liver, is strongly correlated with obesity and the metabolic syndrome, and can progress to the more severe non-alcoholic steatohepatitis (NASH) if left untreated. Despite this, the mechanisms driving hepatic steatosis and steatohepatitis are still unknown, and few therapies exist to address this spectrum of liver disorders. In particular, the role of mitochondrial metabolism, the central organelle in fatty acid oxidation, remains incompletely defined. Ketogenesis is positioned at a pivotal juncture in the hepatic mitochondria, amongst the tricarboxylic acid (TCA) cycle, gluconeogenesis, and fatty acid oxidation. In this dissertation, I utilize novel mouse models, mass spectrometry and nuclear magnetic resonance imaging of 13C-labeled substrate flux in vivo, and measures of mitochondrial morphology and function among other biochemical and systems physiology techniques to demonstrate a critical role for hepatic ketogenesis in regulating mitochondrial metabolism and liver injury in the context of the neonatal period, during feeding and fasting in adulthood, and in overnutrition. Using a novel murine model of HMGCS2-deficiency, I show that ketogenesis insufficient, fasting, adult mice are markedly steatotic with concomitant hyperglycemia. However, upon high-fat diet feeding, an inability to generate ketone bodies from fatty acids instead results in severe liver inflammation and injury, which is associated with a redirection of acetyl-CoA flux towards de novo lipogenesis (DNL) and sequestration of free coenzyme A, further disrupting mitochondrial metabolic pathways. I further demonstrate that this metabolic reprogramming in the ketogenesis insufficient context provokes shifts in the hepatic phospholipidome, as well as mitochondrial morphology and function. These studies implicate a significant role for hepatic ketogenesis in regulating complex metabolic pathways in the liver in a classically non-ketogenic, carbohydrate replete state, and establish it as a compelling target to better understand and address the metabolic dysfunction seen in the livers of obese and diabetic individuals

Determination of Glutathione Disulfide Levels in Biological Samples Using Thiol-disulfide Exchanging Agent, Dithiothreitol

A reverse-phase HPLC method incorporating dithiothreitol (DTT) reduction for quantitative determination of oxidized glutathione (GSSG) in biological samples is described here. This method is based on our previous enzymatic reduction technique that uses N-1-(pyrenyl) maleimide (NPM) as a derivatizing agent. In our earlier method, glutathione disulfide (GSSG) was measured by first reducing it to GSH with glutathione reductase (GR) in the presence of NADPH. However, this is a very costly and time-consuming technique. The method described here employs a common and inexpensive thiol-disulfide exchanging agent, DTT, for reduction of GSSG to GSH, followed by derivatization with NPM. The calibration curves are linear over a concentration range of 25-1250 nm (r2 \u3e 0.995). The coefficients of variations for intra-run precision and inter-run precision range from 0.49 to 5.10% with an accuracy range of 1.78-6.15%. The percentage of relative recovery ranges from 97.3 to 103.2%. This new method provides a simple, efficient, and cost-effective way of determining glutathione disulfide levels with a 2.5 nm limit of detection per 5 µL injection volume