Control of uncoupling protein-1 in brown adipose tissue

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Brown adipose tissue (BAT) is a highly metabolic tissue found in most small mammals; hibernating mammals; newborn humans and at lower levels in adult humans. Its physiological role is thermogenesis for the purpose of thermoregulation in environmental cold. Brown adipocytes are rich with mitochondria, and therein, uncoupling protein-1 (UCP1) is high comprising ~10% of mitochondrial protein. When BAT is activated, UCP1 acts as a proton leak protein; protonmotive force decreases; and respiration increases dramatically. Indeed, the activation of BAT in mice doubles whole body energy expenditure in the absence of shivering. Despite the excitement surrounding the recent discovery that BAT is present in adult humans, we only have a rudimentary understanding of the mechanisms that turn BAT on and off. The purpose of this research is to elucidate these mechanisms, and to focus on the control of this unique protein in BAT, UCP1. Our NSERC funded research since 1995 has focused on BAT metabolism. Overall, the proposed research aims to elucidate novel biochemical mechanisms that activate UCP1 protein and to explore the role of glutathione redox status in the control of BAT thermogenesis. Our published findings show that reactive oxygen species (ROS) emission is normally high, and the glutathione redox ratio is low in BAT vs. skeletal muscle mitochondria. Markers of oxidative damage however are low in BAT. Our preliminary findings using mass spectrometry and studies in hibernoma cells demonstrate a role for deactylation activation of UCP1. Sirtuin-3 (SIRT3) is an NAD-dependent deacetylase in the mitochondrial matrix. SIRT3 is highly expressed in tissues enriched with mitochondria, including BAT. Its expression therein is high under conditions of fatty acid oxidation, i.e., during cold exposure. SIRT3 deacetylates and activates MnSOD, a matrix enzyme important in quenching superoxide; it also increases MnSOD transcription. Many questions remain regarding the control of UCP1 and the role of unique redox characteristics of BAT. The research aims are 1) Characterize SIRT3 deacetylation of UCP1 and other BAT proteins in cellular and mouse models; 2) Determine the degree to which SIRT3-mediated control of UCP1 contributes to BAT thermogenesis; and 3) Determine the roles of mitochondrial redox and ROS on SIRT3-dependent and -independent BAT thermogenesis. Approaches will include studies of BAT mitochondria, cells and proteins from various types of mice. E.g., SIRT3 knockouts, SIRT3 overexpressors and UCP1 knockouts, under different physiological conditions. Outcome determinations include whole body energetics (indirect calorimetry); levels of protein acetylation (mass spectrometry); mitochondrial content; oxygen consumption; redox ratios; ROS emission; and oxidative damage. Post-translational modification of UCP1 and other proteins will be probed. In vitro cellular models (e.g., HIB-1B cells) will be used to study the effects of mutating key SIRT3 residues on metabolic parameters. It is hypothesized that UCP1 is activated by deacetylation; that other mitochondrial proteins are coordinately activated by deacetylation (SDH, ICD ACADL and several OXPHOS proteins) and that these processes are activated by cold exposure. It is further hypothesized while ROS levels are high during uncoupled respiration in BAT, SIRT3 activation of MnSOD and other processes keep oxidative damage low, even as the glutathione redox ratio is relatively highly oxidized. It is anticipated that the proposed research will lead to an advanced understanding of the processes controlling BAT thermogenesis and mammalian thermoregulation. The proposed work will also provide a cutting-edge training in metabolic research to trainees.