Kinetics of Low Abundant Intracellular Molecules, Including Receptors, Signaling Molecules, and Hormones in Human Health and Disease Using Heavy Water (2H2O) Labeling and Mass Spectrometry

My broad goals are to use new stable isotopic metabolic labeling techniques with tandem mass spectrometric analysis to measure in both preclinical models and human subjects, the synthesis and breakdown rates of low abundance proteins that play key roles in common metabolic disorders such as heart disease, diabetes, non-alcoholic fatty liver disease and counterregulatory hormones on muscle protein metabolism. First, I developed a mass spectrometry method to measure the turnover rates of the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9). We revealed that lower hepatic synthesis and secretion of PCSK9, an SREBP2 (sterol response element binding protein) target gene, results in longer hepatic LDLR t½ in response to cholesterol feeding in mice in the face of high intracellular cholesterol content. PCSK9 modulation opposes the canonical lowering of LDLR mRNA and synthesis by cholesterol surplus and preserves LDLR levels. Secondly, I developed a non-invasive technique using 2H2O labeling and mass spectrometry for measuring the synthesis of pancreatic beta cell insulin in circulation as a tool to monitor the progression of diabetes in human beings. The significance of this work is that a) we have revealed how long pancreatic beta cell insulin lives for, and b) the effect of prediabetes on insulin biogenesis. Our third objective was to develop a method to determine the kinetics of PNPLA3, Patatin-like phospholipase domain-containing protein 3. This gene single nucleotide polymorphisms confers susceptibility for non-alcoholic fatty liver disease (NAFLD) by accumulating PNPLA3 proteins on lipid droplets. To understand the kinetic basis of this genetic polymorphisms associated with NAFLD, we would like to determine how PNPLA3 turnover rates are altered in the liver in NAFLD patients with different PNPLA3 genetic backgrounds. Our fourth objective investigated how the treatment with acetyl-CoA carboxylase inhibitors (ACCi) increases plasma triglyceride (TG) concentrations in patients with non-alcoholic steatohepatitis (NASH), with variable results reported for concentrations of plasma apolipoprotein B (ApoB). We determined the effects of treatment with the ACCi, firsocostat in NASH on production and clearance rates of plasma LDL ApoB-containing particles. For the last part of this dissertation, I used stable isotopic labeling with 2H2O combined with mass spectrometry to measure the turnover rates of proteins across the proteome in response to glucocorticoid treatment and/or in a Pik3r1 (a glucocorticoid response gene) knockout mouse model. We revealed a suppressive effect on lowering protein turnover rates in muscle of Pik3r1 knockout mice when given dexamethasone. This dynamic proteomic study revealed how Pik3r1 regulates skeletal muscle protein metabolism. Collectively, these studies are anticipated to shed insight into the metabolic physiology of common metabolic diseases such as atherosclerotic cardiovascular disease (ASCVD), type 2 diabetes mellitus (T2D), non-alcoholic fatty liver disease, and muscle protein metabolism.

Advisor: Hellerstein, Marc