Cellular and Biochemical Pathways of Adipose Metabolism and Thermogenesis - PROJECT SUMMARY Our overall goal is to better understand mechanisms of energy expenditure in mammalian systems, with a special emphasis on adipose thermogenesis. An improved understanding of these processes is likely to allow better ways to control metabolic disorders of positive energy balance, such as obesity, diabetes and certain cancers. Previous efforts in our lab, continuing into this grant cycle, have advanced work on a new thermogenic biochemical cycle that we termed the futile creatine cycle (FCC). This pathway involves the formation and dissipation of phosphocreatine and utilizes 4 major genes: GATM, the creatine transporter, CKB and TNAP. The hydrolysis of phosphocreatine is a critical step and, using biochemical and genetic methods, we identified this enzyme as TNAP. This well-known phosphatase was shown previously (in other cell types) to be a cell surface and secreted protein; in thermogenic fat cells, TNAP was largely localized to mitochondria. This altered localization is one of the key steps in the FCC. One key Aim in this new proposal will determine the cellular and biochemical pathways by which TNAP becomes localized to mitochondria. Preliminary data strongly suggests that this pathway involves “lipid rafts” on the cell surface or in the endoplasmic reticulum. Experiments disrupting lipid rafts either chemically or genetically will allow the identification of the complete set of proteins and protein modifications that depend on this pathway. Protein identification will be accomplished by two different methods of protein Mass Spectrometry, “top down” and “bottom up”. These data, in turn, will lead to both gain and loss of function experiments in cells and in mice; these will critically test the roles of various factors in the pathways of TNAP localization. Importantly, these data will potentially identify a “raft to mitochondria” pathway that may be used by other proteins whose localization in mitochondria is also poorly understood. Another key Aim of this proposal is to understand how the FCC co-exists with the classical UCP1 pathway, given that they both transform energy contained within the same mitochondrial membrane potential into heat. We show in preliminary studies, using two distinct sets of data, that RNAs for the FCC and UCP1 pathways are differentially expressed in at least 2 distinct subtypes of beige cells. We will characterize both subtypes of cells in vivo, first using immunocytochemistry. In addition, we will characterize the RNA and protein signatures of these cellular sub- types, using mitochondrial proteomics and bulk RNA sequencing of immortalized cell subtype clones that are in hand. We will also do functional analyses of these clones by respirometry after genetic ablation of molecules that potentially control TNAP localization and/or other aspects of energy expenditure. Bulk RNA data should also identify cell surface proteins that can potentially be used to sort cells isolated directly from animals and allow us to follow when and how these cellular subtypes diverge during development. Lastly, RNA data from this Aim should allow us to predict and identify the transcription factors that give the cells their identity as UCP1-beige or FCC-beige. Again, these hypotheses will be tested in vivo with gain and loss of function transgenesis in mice.
