Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular and Molecular Physiology

First Advisor

Shulman, Gerald

Abstract

Type 2 diabetes (T2D) is a chronic condition that currently affects 463 million people worldwide and is expected to affect 700 million people by the year of 2045. People with T2D suffer from many downstream conditions, such as cardiovascular diseases and stroke, vision loss, renal function impairment and amputations due to limb extremity damages, and are also much more susceptible to severe COVID-19 conditions. This devastating disease not only inflicts severe personal suffering on the individuals affected but also inflicts a huge economic burden on the society. For example, the annual cost of diagnosed diabetes in United States is estimated to be $327B in 2017. Therefore, understanding the mechanisms underlying the pathogenesis of T2D is of great importance, and effective therapeutic inventions can then be developed to remediate the situation.Insulin resistance is the primary characteristic of T2D, where insulin fails to activate downstream signaling pathways, causing excessive glucose build-up in the circulation and tissues starved from critical nutrients. In the human body, liver serves as the center of numerous metabolic functions and is pivotal in regulating glucose homeostasis. Hepatic insulin resistance (HIR) significantly contributes to hyperglycemia via increased hepatic glucose output and decreased hepatic glucose storage. Understanding the mechanism of HIR is thus critical. Nonalcoholic fatty liver disease (NAFLD) is strongly associated with HIR, however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 (DGAT2) in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCε activation, and insulin receptor kinase (IRK)-T1160 phosphorylation resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCε KD ameliorated high-fat diet (HFD)-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCε-induced HIR by promoting IRK-T1160 phosphorylation. White adipose tissue (WAT) is also of great regulatory importance in glucose metabolism. Insulin-mediated suppression of WAT lipolysis is an important anabolic function that is dysregulated in the state of overnutrition. However, the mechanism of short-term HFD-induced WAT insulin resistance is poorly understood. Based on our studies in the liver and preliminary studies in the WAT, we hypothesize that a short-term HFD causes WAT insulin resistance through a similar mechanism. Increases in PM sn-1,2-DAG content, which promotes PKCε activation, impairs insulin signaling by phosphorylating IRK-T1160. To test this hypothesis, we assessed WAT insulin action in 7-day HFD-fed versus regular chow diet-fed rats during a hyperinsulinemic-euglycemic clamp. HFD feeding caused WAT insulin resistance, reflected by reductions in both insulin-mediated WAT glucose uptake and suppression of WAT lipolysis. These changes were specifically associated with increased PM sn-1,2-DAG content, increased PKCε activation and impaired insulin-stimulated IRK-Y1162 phosphorylation. In order to examine the role of IRK-T1160 phosphorylation in mediating lipid-induced WAT insulin resistance, we examined these same parameters in short-term HFD-fed IRKT1150A mice (IRK-T1150 is the mouse homolog of human IRK-T1160). Similar to the rat study, short-term HFD feeding induced WAT insulin resistance in WT control mice but failed to induce WAT insulin resistance in IRKT1150A mice. Taken together these data demonstrate that the PM sn-1,2-DAG - PKCε - IRK-T1160 phosphorylation pathway plays an important role in mediating lipid-induced hepatic and WAT insulin resistance and represents a potential therapeutic target to improve insulin sensitivity in the liver and WAT.

Download
COinS