Considering the major role of insulin signaling on fatty acid synthesis via stimulation of lipogenic enzymes, differential effects of insulin signaling on individual carbon fluxes for fatty acid synthesis have been investigated by comparing the individual lipogenic fluxes in WT and IRS-1 knockout (IRS-1 KO) brown adipocytes. Results from experiments on WT and IRS-1 KO cells incubated with [5-¹³C] glutamine were consistent with the existence of reductive carboxylation pathway. Analysis of isotopomer distribution of nine metabolites related to the lipogenic routes from glucose and glutamine in IRS-1 KO cells using [U-¹³C] glutamine as compared to that in WT cells indicated that flux through reductive carboxylation pathway was diminished while flux through conventional TCA cycle was stimulated due to absence of insulin signaling in IRS-1 KO cells. This observation was confirmed by quantitative estimation of individual lipogenic fluxes in IRS-1 KO cells and their comparison with fluxes in WT cells. Thus, these results suggest that glutamine’s substantial contribution to fatty acid synthesis can be directly manipulated by controlling the flux through reductive carboxylation of alpha-ketoglutarate to citrate using hormone (insulin). / Singapore-MIT Alliance (SMA)

<p dir="ltr">Adipose tissue consists of adipocytes that store energy within lipid droplets and are a central component of lipid metabolism. Mammals contain white, brown and beige adipocytes, which differ in their metabolic roles. White adipocytes store energy, in the form of triglycerides, within lipid droplets and predominantly take on an energy storage role. Brown and beige adipocytes promote energy expenditure and the dissipation of energy as heat through non-shivering thermogenesis. Since energy expenditure combats excess caloric intake and overeating, non-shivering thermogenesis has become heavily researched for its potential therapeutic use in combatting the continued increase in obesity and metabolic disorders worldwide.</p><p dir="ltr">In addition to ATP synthesis, mitochondria are required for a multitude of metabolic processes that maintain cellular homeostasis, including non-shivering thermogenesis. Brown and beige adipocyte mitochondria are specialized to perform non-shivering thermogenesis in response to an environmental stressor like cold exposure. Uncoupling protein 1 (UCP1) is uniquely characteristic of brown and beige adipocyte mitochondria, because it allows oxidative phosphorylation to be uncoupled from ATP synthesis. In order to enhance non-shivering thermogenesis, ongoing molecular characterization of brown adipose tissue (BAT) is being conducted to identify proteins that regulate mitochondrial function and UCP1 activity. In this study, I explored the function of LETM1-domain containing 1 (LETMD1), a novel mitochondrial inner membrane protein with unknown function in BAT. We generated a global (<i>Letmd1</i>^<em>KO</em>) and UCP1+ cell-specific <i>(Letmd1</i>^<em>UKO</em><i>) knockout</i><i> </i>mouse model to study the whole-body and cell-autonomous role of LETMD1 in BAT, respectively. Loss-of-function studies resulted in striking, BAT-specific phenotypic differences, including whitened BAT under thermoneutral, room temperature and cold exposure. Both knockout models were cold intolerant without access to food, and became hypothermic within a few hours of fasted cold exposure. Loss of normal mitochondria structure and cristae arrangement were also evident in knockout BAT, resulting in a decreased number of mitochondria and decreased number of cristae per mitochondrion. Mitochondrial DNA copy number was also significantly decreased in both knockout models. Abnormal mitochondria morphology was supported by increased reactive oxygen species (ROS) accumulation in both knockout models and the visualization of protein aggregates and mitophagy-like morphologies in <i>Letmd1</i>^<em>UKO</em><i> </i><i>mice specifically</i>. TurboID proximity labeling of brown adipocytes revealed enrichment of several respiratory chain complex proteins, mitochondrial ribosome proteins and mitochondrial protein import machinery. Moreover, the aggregation of misfolded nuclear-encoded mitochondrial proteins, including several respiratory chain and mitochondrial ribosome proteins, suggested that LETMD1 facilitates mitochondrial protein import and mitochondrial ribosome assembly, thereby compromising respiratory chain assembly and function during non-shivering thermogenesis. Overall, this study identifies LETMD1 as a novel regulator of brown adipocyte mitochondrial structure and thermogenic function and highlights the requirement of LETMD1 for mitochondrial biogenesis.</p>

During embryonic development, adipocytes emerge from microvasculature. Lineage-­‐tracing studies in mice have shown that adipocyte progenitors reside in the adipose tissue capillaries. However, the direct evidence of an association between adipocyte progenitors and vasculature in humans is lacking. A specific class of adipocytes (brown and beige/brite) expresses the uncoupling protein 1 (UCP1), which consumes glucose and fatty acids to generate heat. The abundance of UCP1- containing adipocytes correlates with a lean metabolically healthy phenotype in human. However, a causal relationship between the presence of these cells and metabolic benefits in human is not clear. In this thesis, I report human adipocyte progenitors proliferate in response to pro-angiogenic factors in association with adipose capillary networks in-vitro. The capillary-derived adipocytes transform from being UCP1-negative to positive upon adenylate cyclase activation, a defining feature of the brite/beige phenotype. Activated cells have denser, round mitochondria with UCP1 protein, and display uncoupled respiration. When implanted into NOD-scid IL2rgnull (NSG) mice, the adipocytes can form a vascularized fat pad that induces vascularization and becomes integrated into mouse circulatory system. In normal or high fat diet-fed NSG mice, activated brite/beige adipocytes enhance systemic glucose tolerance and improved hepatic steatosis, thus providing evidence for their potential therapeutic use. The adipocytes also express neuroendocrine and secretory factors such as Interleukin-33, proprotein convertase PCSK1 and proenkephalin PENK, which are correlated with human obesity. Finally, analyses on single-cell clones of capillary-sprout cells reveal the existence of diverse adipogenic progenitor populations. Further characterization of the clones will define the identifying features of the diverse adipocyte progenitor types that exist in human adipose tissue.