PROJECT SUMMARY/ABSTRACT Nutrient fuel flexibility, the ability of mitochondria to switch oxidation fuel source for energy production, is a key adaptation against fluctuations in nutrient supply. Adaptive resistance to lipid utilization has been linked to diseases including diabetes, non-alcoholic fatty liver disease and cardiomyopathy but the mechanisms underlying lipid (in)flexibility remain understudied. O-GlcNAcylation is a nutrient-sensitive protein modification that is reversibly attached by the OGT enzyme and removed by OGA. Decreased OGA activity leads to a fuel shift away from lipid oxidation in mice through an unknown mechanism. However, there is a lipid droplet-enriched OGA isoform (sOGA) that is well-positioned to detect fatty acid supply/demand and to act on O-GlcNAcylated targets through mitochondrial interactions or nuclear signaling pathways. Furthermore, a diabetogenic polymorphism in the OGA gene was found near the splice junction, which regulates production of sOGA. Recently in the lab, a metabolomics analysis of mouse tissue revealed a novel correlation between OGA loss and acylcarnitine deficiency, a requisite metabolite pool in the uptake of long-chain fatty acids into mitochondria through the carnitine shuttle. Therefore, the central hypothesis of this proposal is that OGA is required for the upregulation of carnitine-dependent mitochondrial lipid oxidation and that sOGA, rather than the canonical lOGA, is the predominant mediator. The goal of the research plan is to characterize OGA regulation of carnitine shuttle flux through the modification of shuttle proteins (1), the supply of carnitine (2) and/or the nutrient-sensitive transcription of critical proteins (3). All specific aims rely on germline OGA knockout (KO) mice/fibroblasts and/or cells transfected with GFP- or HA-tagged sOGA or lOGA expressing plasmids as models. Aim 1 focuses on sOGA modification of carnitine shuttle proteins using mitochondrial oxidation rate, immunofluorescent (IF) imaging and OGA immunoprecipitation (IP) + mass spectrometry/western blot to evaluate nutrient fuel flexibility, OGA/target co-localization and isoform-specific OGA targets. Aim 2 tests O-GlcNAc suppression of OCTN2 transporter-mediated carnitine uptake through qPCR/blotting for OCTN2 expression/protein, a tritiated carnitine uptake assay and an OGA KO rescue attempt through in vivo carnitine supplementation. Aim 3 posits PLIN5 deglycosylation is required for its phosphorylation, nuclear translocation and activation of PPAR𝝰, a transcriptional regulator of carnitine homeostasis and lipid oxidation. This aim tracks PPAR𝝰 activity using an in-house plasmid luciferase detector and IF, IP and blotting to characterize phospho-dead/mimetic and O-GlcNAc-dead PLIN5 proteins, generated through site-directed mutagenesis. Experiments will be conducted in the labs and core facilities of the NIDDK Laboratory of Cellular and Molecular Biology under the sponsorship of Dr. John A. Hanover. The trai...