Periodic Reporting for period 4 - aCROBAT (Circadian Regulation Of Brown Adipose Thermogenesis) Reporting period: 2019-11-01 to 2020-10-31 Summary of the context and overall objectives of the project The healthcare burden posed by the obesity and diabetes pandemics is rapidly approaching a crippling point and clinical intervention strategies are desperately needed. Thermogenic adipose possesses a remarkable energy-dissipating and glucose-consuming capacity, yet strategies to exploit the anti-diabetes and anti-obesity therapeutic potential of this tissue have remained elusive. The chief obstacle has been devising translational methods that can stimulate sustained adipose thermogenesis with limited deleterious side effects. Thermogenic adipose is controlled by both environmental temperature and the body’s clock. Thus, our goal in aCROBAT was to discover and characterize novel factors within these well-conserved programs through which we can unlock the calorie-burning, glucose-consuming power of thermogenic adipose. We investigated both cell surface receptors and intracellular enzymes that we identified from unbiased big-datasets.Throughout the grant period, we found that (1) the metabolite NAD+ and its biosynthetic enzyme, NAMPT, are critical for the diet-dependent and circadian plasticity of adipose tissue, (2) brown adipose cardiolipin synthesis is a driver of adipose thermogenesis and whole body energy homeostasis, (3) lipolysis induced expression of the constitutively active G protein coupled receptor 3 regulates adipose thermogenesis, and (4) a novel hormone axis in adipose tissue that regulates energy expenditure and circadian feeding rhythm is a target for anti-obesity therapy. The novel hormone axis has led to a University of Copenhagen patent and a spinout company to explore the commercial and therapeutic potential. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have generated unbiased proteomic and transcriptional profiles for our discovery platform to identify key enzymes and receptors controlling brown fat activity. We have validated some of our top hits in fat cells and have made, or are in the process of making, mouse models to study the physiological function of these candidates. We published two manuscripts on key enzymes in circadian and cold control of brown adipose metabolism with several more papers in revision or review. Collectively, our findings from aCROBAT made major in-roads into the understanding of adipose biology and the connection to metabolic disease. In the first published study, we investigated an enzyme called NAMPT, which is involved in control of fat cell bioenergetic programs and circadian rhythm. We found that when we deleted Nampt only in fat tissue of mice, the mice did not become obese when given a diet high in fat. Even though the mice were fed a diet similar to eating only hamburgers, hot dogs, and pizza, they gained no more weight than mice fed a healthier, lower fat diet. Our research is the first to reveal that NAMPT is a critical circadian factor in expansion of fat tissue and is indispensable for the development of obesity. More broadly, NAMPT represents a novel paradigm by which mammals can specifically control consumption of dietary fat based on how much energy can be stored in the fat tissue.The goal of the second published study was to investigate which enzymes were most critical in mediating environmental control of brown fat activity. Combined proteomic and lipidomic analyses revealed that a single lipid species known as cardiolipins and their obligate synthase, CRLS1, were not only indispensable drivers of brown fat energy expenditure but affected metabolic homeostasis or balance throughout the body. Depleting cardiolipin in brown fat caused mice to become insulin resistant and diabetic. These results showed for the first time that a lipid in the mitochondria of brown fat was capable of modulating whole body insulin sensitivity. Human CRLS1 expression in fat tissue of healthy or diabetic individuals suggests that our findings are translatable. This study involved the generation of several new mouse models, human cells, tissue, and genetic association studies, and collaborative efforts between basic researchers and clinicians in Germany, the US, and Denmark.In the aCROBAT proposal, we outlined the goal of leveraging our basic research findings into bona fide innovation to fight metabolic disease. During the third reporting period we began realizing that goal when we spun out a new company, Embark Biotech, from the University of Copenhagen with the help of an ERC PoC grant. Embark is based on a novel receptor in brown adipose tissue that significantly increases calorie-burning and reduces bodyweight in obese mice. We made the discovery during the first stages of aCROBAT and have characterised the mechanism-of-action by generating tissue specific receptor knockout mice. In parallel, we are now optimising the pharmaco-kinetic properties of the target to develop a next-generation obesity therapeutic. Additionally, we have uncovered another receptor, GPR3, that plays a critical role in the activation of human brown adipose. Depleting this receptor in fat cells isolated from seven different patients showed decreased thermogenic gene expression in all cases. We developed gain and loss-of-function animal models to study a causal role for this receptor in thermogenic adipose physiology. We found that activation of this receptor is fully sufficient to drive the calorie-burning function of brown adipose even in the absence of sympathetic signalling. This receptor and its agonists will eventually form the basis for a second spinout company my group based on the ERC starting grant. In the meantime, we are finalizing the revision for the manuscript.Finally, we have continued on the work on the Nampt NAD+ biosynthetic pathway in adipose, focusing on the role of Nampt in the molecular clock. Previous dogma in the field argues that Nampt is an integral member of the clock in all cells. However, these studies were all performed in immortalised cel lines. We used our adipose specific and muscle specific Nampt KOs, combined with global gene profiling to uncover that Nampt dramatically affects the brown adipose clock but does not impinge on skeletal muscle rhythmicity at all. Moreover, Nampt knockdown has completely different effects on transcription in white adipose compared to brown. This work represents the first characterisation of Nampt's role in the molecular clock in an in vivo setting across different tissues. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) If we are successful in identifying one or more bona fide drug targets from the cell surface or within, than we anticipate an enormous socio-economic impact. There is a massive demand for potent anti-obesity and anti-diabetes drugs as the number of affected individuals sky-rockets higher and higher. We now have 2 viable candidates targeting different aspects of brown fat calorie-burning and glucose consuming functions. We also hope that our work will broaden the understanding of how environmental cues like temperature and light interact with the body’s molecular clock to control metabolism and maintain energy balance. At the conclusion of this ERC project, we have two publications and three more in revision or under review. The spinout company, which we established during this starting grant, has carried out studies up through nonhuman primates of our novel obesity drug and are moving towards clinical trials. Finally, all the big datasets generated throughout the ERC programme will be made freely available through open source publications and databases for the use of the scientific community. slide1.jpeg
