New Advances in Our Understanding of the Control and Functions of Brown Adipose Tissue Thermogenesis

Abstract

Brown adipose tissue (BAT) generates heat in a process referred to as non-shivering thermogenesis (NST). The process is dependent on the proton leak activity of uncoupling protein1 (UCP1), a protein found in the mitochondrial inner membrane. Physiologically, NST is activated by environmental cold, and to a lesser extent, diet. NST is an energetically costly process, and thus activated BAT consumes remarkable amounts of fatty acids and glucose, which positively influences systemic metabolism. Studies in mice have shown that defective BAT activity can contribute to the development of obesity, and increased BAT activity can protect against obesity. In humans, amounts of BAT are highest in newborns, and atrophy with age. BAT is also a secretory organ that releases ‘batokines’ and other signaling molecules, some of which are in small extracellular vesicels (sEV). The latter may act on BAT itself in a autocrine fashion, or on other tissues in an endocrine fashion, which may also contribute to the systemic effect of BAT activity. The overall aim of my Ph.D. thesis was to elucidate molecular mechanisms controlling BAT activity and its regulatory effects on other tissues/cells. In my first project, the deacetylation control of BAT activity was studied in mice. Mitochondrial deacetylation is mainly mediated by Sirtuin 3 (SIRT3). Previous studies showed that cold increases the transcript level of SIRT3 in BAT, and that fasted Sirt3 knockout (Sirt3KO) mice are cold intolerant, suggesting a potential thermoregulatory role of SIRT3. However, the molecular mechanisms by which SIRT3 regulates BAT thermogenesis are not fully understood. Here, we examined functional links between SIRT3 and UCP1. To study this, wild-type (WT) and Sirt3KO mice were used to perform physiological, molecular, and proteomic analyses of BAT when it is activated by cold or by the β3-adrenergic agonist, CL316,243. Our findings indicated that the absence of SIRT3 in ad libitum fed mice led to impaired use of BAT lipid droplets, defective thermoregulation and decreased BAT mitochondrial respiration, without affecting the expression of UCP1. Label-free mass spectrometry revealed that the absence of SIRT3 increased the acetylation status of several BAT mitochondrial proteins including UCP1 and proteins involved in crucial pathways upstream of UCP1, such as the complexes of the electron transport chain (ETC) and acylcarnitine/fatty acid oxidation (FAO) metabolism. Therefore, we next examined the effect of hyperacetylated sites found in those BAT mitochondrial proteins on their functions. Mutagenesis work conducted in a cellular model revealed that SIRT3-regulated acetylation sites on UCP1 did not impact proton leak respiration when UCP1 was activated. However, analysis of acylcarnitines in the blood showed that the absence of SIRT3 resulted in a decrease in the levels of selected medium-chain and long-chain acylcarnitines. Additionally, functional analysis of ETC complexes in BAT mitochondria demonstrated that the absence of SIRT3 decreased the activities of complex I and complex II (CI and CII), which could impair the production of proton motive force, required for the activity of UCP1. Altogether our results indicate that SIRT3 regulates BAT thermogenesis indirectly by targeting proteins involved in crucial pathways upstream of UCP1. In my second project, we studied the metabolic response of BAT to hypoxia in naked mole rats (NMRs). NMRs are exceptionally tolerant to hypoxic environments; during acute hypoxia their body temperature is decreased nearly to the ambient temperature. The underlying mechanisms of these observations are not well understood. We hypothesized that BAT activity in NMRs is decreased during hypoxia. To study this, NMRs were exposed to normoxia (21% O2/1hr), hypoxia (7% O2/1hr), or hypoxia followed by recovery (21% O2/1hr). Thermal imaging, body temperature measurements, thermogenic protein and overall protein ubiquitination levels, and mitochondrial morphology analyses of BAT were conducted. In addition, the level of thermogenic proteins in iBAT from different species of mole rats were examined under normoxic or hypoxic conditions for comparative and evolutionary analyses. Our results illustrated that during acute hypoxia, NMRs had rapidly decreased their body temperature close to the ambient temperature. Also, thermal imaging showed that heat produced in interscapular BAT region in hypoxic NMRs was decreased regardless of the degree to which BAT was activated. Western blotting analysis revealed that levels of UCP1 and selected ETC proteins were markedly decreased in hypoxic NMRs. Also, UCP1 was decreased in some, but not all other species of mole rats that were studied. To probe possible mechanisms of these marked acute decreases in mitochondrial proteins, levels of ubiquitinated proteins in BAT were examined and found to be increased by hypoxia. Consistently, ultrastructural analyses of BAT by electron microscopy indicated abnormal mitochondrial morphology in BAT of hypoxic NMRs, including abnormal cristae and jagged membranes suggesting that mitophagy events may be induced during hypoxia. Further investigations are needed to examine the potential role of proteasome-mediated degradation or mitophagy mechanisms in BAT during hypoxia. Our results reveal that hypoxia diminished BAT thermogenesis in NMRs, at least in part, by targeting mitochondrial thermogenesis related proteins and by altering mitochondrial morphology. In my third project, the metabolic effects of BAT-derived sEV on skeletal muscle (SkM) cells were investigated. BAT releases sEV and its activation increases the release of sEV. However, the roles of activated BAT-derived sEV are not well understood. Generally, sEV are involved in interorgan communication and contain regulatory moleculaes that can be taken by recipient tissues or cells to regulate their functions. During cold exposure, BAT and skeletal muscle (SkM) produce NST and shivering, respectively, to maintain controlled body temperature. Defects in the thermogenic function in one of the tissue leads to increase the heat production in the other tissues, suggesting cross-talk mechanisms between BAT and SkM. Given the role of sEV, we questioned whether activated BAT-derived sEV effect the function of SkM cells. Thus, we investigate the metabolic effects of sEV released from acutely activated BAT, in vivo or in vitro, on SkM myoblast bioenergetics. Our findings demonstrated that BAT releases sEV and its acute activation does not significantly impact the size and concentration of sEV released in mouse plasma or conditioned medium. Surprisingly, bioenergetics analyses illustrated that neither plasma sEV from CL316,243-injected mice nor sEV from conditioned medium of CL316,243-treated BAT explants had impacts on C2C12 myoblasts bioenergetics following one day of treatment. To conclude, our findings demonstrate that sEV released from acutely activated BAT do not influence the bioenergetics of C2C12 myoblasts under normal metabolic conditions. Further investigations are needed to examine the effects of BAT-derived sEV on SkM cells under different metabolic conditions or time-frames. Overall, the findings reported in this thesis expand our understanding of the control of BAT and its systemic roles in thermoregulatory metabolism, which is important in the field of metabolic physiology.