Studies on the regulation of PKA-mediated lipolysis

Abstract

Lipolysis is a delicate process regulated by complex signaling cascades and sequential enzymatic activations. Among them, PKA signaling is known to be one of key pathways that influence lipolysis. Genetic studies on the roles of PKA in lipolysis with mouse models appear to have various limitations including complexity of PKA subunits and in vivo compensatory effects. In this study, I have used Caenorhabditis elegans and mammalian adipocytes as model systems to investigate PKA function and genes involved in PKA-mediated lipolysis. C. elegans and mammalian adipocytes are complementary systems to test PKA because PKA activity can be effectively modulated by knockdown or chemicals. In C. elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, I demonstrate that in intestinal cells of C. elegans, fasting increases cAMP levels, which activates PKA to stimulate lipolysis via Adipose TriGlyceride Lipase-1 (C05D11.7

ATGL-1) and a newly identified gene LIpid Droplet protein-1 (C25A1.12

LID-1). LID-1 modulates lipolysis by binding and recruiting ATGL-1 during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNAi resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. Moreover, ATGL-1 protein stability and LID-1 binding was augmented by PKA activation, eventually leading to increased lipolysis. In addition, through the ethylmethane sulfonate (EMS) mutagenesis screening in C. elegans, I have isolated three mutants that suppress PKA function. These mutant suppressed dumpy morphology and restored Nile Red-stained granules induced by kin-2 RNAi. Since the identification of suppressor genes has not been completed, further studies are required for the identification of the gene(s) of interest. In the study using mammalian adipocytes, combinatorial siRNA transfection have revealed that, among four regulatory subunits of PKA, RI and RII subunits are required for the inhibition of lipolysis and lipid droplet maintenance under basal state. Knockdown of RI and RII subunits increased glycerol release and decreased lipid droplets without any lipolytic stimulus. Interestingly, knockdown of regulatory subunits increased ATGL protein level, which would play a key role in PKA-mediated lipolysis. Furthermore, I observed that the expression of PKA RII subunit was decreased in high-fat diet (HFD) fed mice, indicating potential roles of PKA subunits in lipolysis and hyperlipidemia. Taken together, these data suggest the crucial role of PKA activity in lipid homeostasis with concerted action of lipase and lipid droplet protein.