PGC1alpha Pathway: Novel Intracellular and Extracellular Mediators

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Project Abstract The PGC1a pathway has emerged as the dominant pathway for the modulation of mitochondrial biogenesis and oxidative metabolism in most tissues. Deficiencies in this pathway have been associated with pathological conditions such as obesity, diabetes and various neurological and neuromuscular disorders. Studies of the regulatory mechanisms at work here is thus fundamental to our understanding of energy homeostasis and the development of drugs to treat these and other disorders. The previous cycle of this grant illustrated that PGC1a is subject to robust control at the level of translation of its mRNA. In this proposal we interrogate trans-acting RNA-binding factors that might participate in the regulation of PGC1a translation and identify a little-known RNA- binding factor, RBM43, that is dramatically regulated in opposition to PGC1a. It is expressed lower in thermogenic fat than in energy-storing white fat, and it is suppressed by cold temperatures. Importantly, experimental reduction of RBM43 by siRNA causes an increase in PGC1a translation and oxidative metbolism. Conversely, increased expression of RBM43 decreases ribosome occupancy of the PGC1a mRNA, consistent with an action on the transational machinery. Here we propose mechanistic, genetic and biological studies of RBM43. Mice with a global mutation in Rbm43 have already been made in our lab, and show increased PGC1a levels and activity in adipose tissues. The physiological effects of this mutation will be determined by challenging mice with cold exposure and high fat diets. Gene expression in adipose tissues will be determined by RNA-seq, while weight gain, body composition and glucose tolerance will be determined using standard methods, including the use of metabolic cages. Fat-selective knockouts will be made using a Cre recombinase driven by the adiponectin promoter. To understand the mechanism by which RBM43 acts, its direct RNA targets will be identified by photo-crosslinking and the RBM43 protein domains and RNA sequences responsible for these interactions will be mapped. The PGC1a pathway also affects cells and tissues distant from the site of PGC1a expression, potentially through myokine or adipokine secretion. To more thoroughly investigate polypeptides secreted under the influence of PGC1a, a new method for the isolation of interstitial fluids from muscle and fat was developed. These fluids provide an excellent substrate for the application of sensitive and quantitative mass spectrometry. Using this approach, we have identified dozens of potential new myokines and adipokines under the control of PGC1a. Of particular interest is prosaposin (PSAP), a CNS neurotrophic factor not known to be secreted by peripheral tissues. Recombinant PSAP has effects on iWAT cells to stimulate thermogenic gene expression. We will characterize these effects in greater detail using RNA-seq and respirometry; the Psap gene will also be ablated in an adipose-selective fashion to determine its roles in thermogenesis and adipose innervation. Together, these studies will give broader insights into the regulation of the PGC1a pathway and potentially provide “actionable” targets for therapeutic intervention in metabolic diseases and other disorders.