Regulation of brown fat fuel utilization by the malate-aspartate shuttle - PROJECT SUMMARY Brown adipose tissue (BAT) in rodents and humans has a high capacity to oxidize nutrients and convert nutrient- derived energy into heat. During cold exposure or pharmacologic stimulation of β3-adrenergic receptor (β3AR), BAT takes up large amounts of diverse substrates (e.g. fatty acids, glucose and amino acids) from the circulation. However, it remains elusive how BAT orchestrates metabolic utilization of various substrates for heat production. The goal of this project is to elucidate the mechanism that coordinates complex substrate utilization in BAT in response to cold or β3AR stimulation. The malate-aspartate shuttle (MAS), which is composed of cytosolic and mitochondrial enzymes GOT1/2 and MDH1/2, is a biochemical system that facilitates the net transfer of cytosolic NADH produced in glycolysis to the mitochondrial electron transport chain (ETC) while regenerating NAD+ in the cytosol to maintain glycolysis. For this transfer, the MAS utilizes a subset of amino acids and metabolites as shuttling substrates. In the preliminary study, we found that, unlike in other tissues, Got1 expression is very low in BAT. However, cold exposure or pharmacological β3-adrenergic agonism markedly induces Got1 expression in BAT while other enzymes remain unchanged. Our preliminary study provides strong evidence for GOT1- dependent MAS activation and its impact on substrate utilization in BAT: i) Got1 overexpression in brown adipocytes increases cytosolic NADH oxidation along with an increase in mitochondrial respiration; ii) Transgenic overexpression of Got1 in BAT increases energy expenditure and improves cold tolerance in mice; and iii) Surprisingly, Got1 overexpression in BAT enhances fatty acid oxidation while Got1 deletion leads to a decline in cold-stimulated FA oxidation. In this project, we will test the hypothesis that cold-induced GOT1 acts as a molecular switch turning on the MAS, which in turn serves as a mechanism to coordinate cold-stimulated glycolysis, substrate shuttling, and FA oxidation in BAT to support thermogenesis. To test this hypothesis, we will use an integrative approach that combines animal physiology with cell and molecular techniques. In Aim 1, we will define the role of GOT1 in MAS activation by using stable isotope tracing, NADH biosensor Peredox, and high-resolution respirometry. In Aim 2, we will elucidate the mechanism by which GOT1 enhances fatty acid oxidation. In Aim 3, we will delineate the mechanism by which β3AR signaling increases GOT1 activity. The study outcomes will identify GOT1 as a critical node that links cold-stimulated β3AR signaling to the MAS, which coordinates glycolysis, amino acid utilization, and FA oxidation in BAT in response to cold.
