AbstractThe overall goal of this proposal is to determine the role of glyoxalase 1 (GLO1) in the pathogenesis of obesityType 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Greater than 34 million Americans havediabetes and another 88 million are considered pre-diabetic. This is largely attributed to the prevalence ofobesity with 72% of American adults currently classified as overweight or obese. Among the comorbiditiesassociated with T2D over 70% of patients have NAFLD. Epidemiological studies have linked high fructoseconsumption with obesity T2D and NAFLD. This proposal identifies GLO1 as pro-NAFLD/obesogenic gene.Using CRISPR-Cas9 we have generated GLO1 knockout mice. When fed a high-fat high sucrose diet thesemice display significantly blunted weight gain restored glucose tolerance and reduced hepatic steatosiscompared to wild-type counterparts. GLO1 is a ubiquitously expressed enzyme that detoxifies the glycolytic by-product methylglyoxal (MGO). When GLO1 activity is disrupted MGO levels increase resulting in long-livedprotein post-translational modifications. We have shown that MGO serves as a metabolic sensor for nutrient fluxregulating glycolytic output and transcriptional responses to sugar. Thus we hypothesize that GLO1 is a pro-NAFLD/obesogenic gene reducing MGO and removing the brakes on metabolism. We will test this hypothesisby addressing the following three Specific Aims: In Specific Aim 1 we will quantify the impact of Glo1 on whole-body energetics and hepatic lipid metabolism using a 16-week chow- or high-fat high-sucrose diet. We willquantify lean vs. fat mass energy expenditure and total activity. Hepatic fatty acid oxidation and mitochondrialrespiration will be quantified in primary hepatocytes. Lastly lipogeneic genes will be assessed using RNA-seq.In Specific Aim 2 we will determine the impact of Glo1 on intestinal fructose metabolism. The intestine is aprimary site of fructose metabolism. Stable isotope labeling via 13C6-fructose oral gavage will be used to quantifyintestinal hepatic muscle adipose and circulating fructose metabolites. This approach will quantitativelydetermine how Glo1 regulates intestinal carbohydrate metabolism a previously unexplored area of research.Finally in Specific Aim 3 we will confirm that MGO-derived histone PTMs regulate transcriptional responses tometabolism in vivo. Site-specific canonical and MGO-derived PTMs will be quantified in each tissue/cohort.Putative reader domains will be identified for MG-H1 histone modifications. Lastly liver intestine muscleadipose and pancreas will be subjected to RNA-seq and ATAC-seq. This approach will determine the globalepigenomic landscape across multiple tissues in a physiologically relevant model for obesity T2D and NAFLD.Collectively this proposal will combine mechanistic biochemistry with a multi-omics approach to determine themechanisms by which GLO1 propagates disease progression.