Type 2 diabetes (T2D) affects 1 in 6 veterans and is the leading cause of blindness, renal failure and non-traumatic loss of limb. Increased hepatic gluconeogenesis is the main cause of fasting hyperglycemia and contributes to postprandial hyperglycemia. Many attribute the increase in gluconeogenesis to increased transcription of phosphoenolpyruvate carboxykinase and glucose 6- phosphate. However, previous studies by this lab demonstrated that hyperglycemia develops in humans with T2D and rodents with hyperglycemia without increases in PEPCK mRNA or protein expression. Knockdown of PEPCK does not affect fasting glucose concentration or rates of glucose production. In short, PEPCK expression does not appreciably impact hepatic gluconeogenesis. In searching for alternate explanations accounting for the increases in gluconeogenesis, hepatic pyruvate carboxylase (PC) protein was observed to closely relate with HbA1c in humans (R=0.80, P<0.01). Hepatic PC protein content is also increased in chronically fat-fed rodents. Similar increases in PC protein expression are seen with prolonged fasting and ketogenic diets, conditions with increased β- oxidation. These changes occurred without changes in PC mRNA. Moreover, decreasing PC expression in a variety of rodent models demonstrated that PC expression, unlike PEPCK expression, regulates glucose production. Decreasing PC expression also improved multiple metabolic insults associated with overfeeding, including weight gain, hepatic steatosis, insulin resistance and hyperlipidemia. Preliminary data suggest that this increase in PC protein content is associated with an increase in lysine acetylation of PC with a reciprocal decrease in PC ubiquitination. The overarching hypothesis for these studies is that increased acetyl CoA promotes lysine acetylation of PC which decreases ubiquitination of PC leading to an increase in protein content and increases the gluconeogenic capacity of the liver. The studies described in this proposal will explore the underlying mechanisms for, and metabolic impact of, increased hepatic PC protein. The studies in Aim 1 will establish the mechanism of lysine acetylation of PC. Specifically, whether fatty acids are the source of the acetyl group attached to PC and whether increasing or decreasing lipid oxidation leads to similar changes in lysine acetylation and PC protein content using a variety of in vitro, in vivo rodent studies where we manipulate cellular lipolysis and acetyl CoA concentrations. In Aim 2, experiments will assess whether lysine acetylation occurs via a non-enzymatic process, identify the specific lysine sites are acetylated with HFF, determine the impact of specific sites on protein activity and stability using lysine mutants (using KQ mutants) and determine whether PC ubiquitination occurs within the mitochondria and impact protein stability. Finally, in Aim 3, we will translate these findings to humans. We will use human cell culture to establish whether lipol...