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 ?- 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 lipolysis and lipid oxidation regulated PC lysine acetylation and ubiquitination and identify the specific lysine residues that are involved. Moreover, we will assess hepatic PC lysine acetylation and protein content in a cohort of normoglycemic and diabetic patients undergoing bariatric surgery. This will allow us to establish the extent to which PC KAc and protein content regulate glycemia, insulin resistance and possibly even steatohepatitis. Together these studies will establish the mechanisms underlying the increases in PC protein content and establish how the increases in the gluconeogenic capacity of the liver and potentially contribute to the development of the metabolic syndrome and T2D.

Public Health Relevance

Type 2 diabetes (T2D) affects 1 in 6 veterans and is the leading cause of blindness, renal failure and non-traumatic loss of limb. One of the important changes that contributes to the mortality and morbidity of diabetes is the increased glucose production (gluconeogenesis) by the liver. The studies in this proposal focus on the role of pyruvate carboxylase, the first enzyme in this pathway. Specifically, the investigators hypothesize that conditions of fat excess will increase the amount of liver PC protein by tagging the protein with acetyl CoA, the end product of fat metabolism. The studies will explore the mechanisms whereby fat metabolism may alter the protein stability and assess whether these mechanisms are relevant to humans. These studies will provide new information on how diabetes may develop in obesity and inform the development of new therapies for diabetes.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Endocriniology A (ENDA)
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VA Connecticut Healthcare System
West Haven
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Gassaway, Brandon M; Petersen, Max C; Surovtseva, Yulia V et al. (2018) PKC? contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling. Proc Natl Acad Sci U S A 115:E8996-E9005
Samuel, Varman T; Shulman, Gerald I (2018) Nonalcoholic Fatty Liver Disease as a Nexus of Metabolic and Hepatic Diseases. Cell Metab 27:22-41
Vatner, Daniel F; Goedeke, Leigh; Camporez, Joao-Paulo G et al. (2018) Angptl8 antisense oligonucleotide improves adipose lipid metabolism and prevents diet-induced NAFLD and hepatic insulin resistance in rodents. Diabetologia 61:1435-1446
Lee, Hui-Young; Lee, Jae Sung; Alves, Tiago et al. (2017) Mitochondrial-Targeted Catalase Protects Against High-Fat Diet-Induced Muscle Insulin Resistance by Decreasing Intramuscular Lipid Accumulation. Diabetes 66:2072-2081
Ter Horst, Kasper W; Gilijamse, Pim W; Versteeg, Ruth I et al. (2017) Hepatic Diacylglycerol-Associated Protein Kinase C? Translocation Links Hepatic Steatosis to Hepatic Insulin Resistance in Humans. Cell Rep 19:1997-2004
Popov, Violeta B; Jornayvaz, Francois R; Akgul, Emin O et al. (2016) Second-generation antisense oligonucleotides against ?-catenin protect mice against diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. FASEB J 30:1207-17
Herman, Mark A; Samuel, Varman T (2016) The Sweet Path to Metabolic Demise: Fructose and Lipid Synthesis. Trends Endocrinol Metab 27:719-730
Samuel, Varman T; Shulman, Gerald I (2016) The pathogenesis of insulin resistance: integrating signaling pathways and substrate flux. J Clin Invest 126:12-22
Habtemichael, Estifanos N; Alcázar-Román, Abel; Rubin, Bradley R et al. (2015) Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle. J Biol Chem 290:14454-61
Pesta, Dominik H; Samuel, Varman T (2014) A high-protein diet for reducing body fat: mechanisms and possible caveats. Nutr Metab (Lond) 11:53

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