Hormonal regulation of lipolysis

Lipases play a key role in regulation of whole body energy homeostasis. Control of lipolysis is important for energy partitioning and balance and maintains the size of fat depots in the body. Dysregulation of lipolytic activities affects lipid absorption, mobilization and transport, and is causative for lipid-related diseases. The release of free fatty acids is dependent on the lipolysis of stored triacylglycerol which is tightly controlled by neural regulation and several hormones to meet energy demands. Molecular mechanisms of this regulation are poorly understood but post-translational modification, protein interactions, protein localizations and access of lipases to their substrates appear to play a major role. Phosphorylation of hormone sensitive lipase, the lipolysis regulator perilipin 1 and more recently also adipose triglyceride lipase (ATGL) have been described on various sometimes controversial sites by different sometimes unknown kinases. Regulations and functions of described sites, however, are poorly understood. Moreover, no information is yet available on regulation of other important players, such as monoglyceride lipase or the ATGL regulators comparative gene identification 58 (CGI-58/ABHD-5) and G0/G1 switch gene 2. We have established an analytical platform for activity-based discovery, detection, profiling and imaging of lipolytic enzymes in intact cells and tissues. In our activity-based proteomic screens we have repeatedly detected active isoforms of lipases suggesting that they are posttranslationally modified. Moreover, we have recently discovered a novel post translational modification of CGI-58 by protein kinase A. Previous work by others and us points thus towards the existence of lipolytic complexes and post translational modification of the involved proteins, warranting a comprehensive screen for novel regulators of lipolysis. The major objective of the proposed project "Hormonal regulation of lipolysis" is to provide a better understanding of the regulation of lipid homeostasis. We will perform in vitro kinase assays to search for novel substrates of known kinases among known lipolytic proteins and protein regulators, investigate the (phospho)proteomic effects of hormonal regulation on lipolytic complexes in murine and human tissues, and functionally analyze selected novel potential regulators of lipolysis (i.e. novel phosphosites, including our novel CGI-58 phosphosite, and/or novel interacting proteins). Although many protein functions appear to be conserved in humans and mice there is growing evidence for important interspecies differences, especially in complex diseases caused by genetic and environmental factors, which cannot be easily reproduced in model organisms. While activity-based probes were already successfully employed to identify novel lipolytic enzymes in mouse liver and adipose tissue homogenates, this study will for the first time shed light on the lipolytic proteome of human adipose tissues in situ directly at enzymatic activity level. Combined with standard quantitative (phospho)proteomic profiling relevant regulators of lipolytic activities will be identified and might offer entry points for future therapies.

The project has made a significant contribution to our understanding of the posttranslational control of lipid homeostasis. This process plays an important role in energy partitioning and balance and maintains the size of fat depots in the body. Its dysregulation is causative for lipid related diseases, such as type 2 diabetes, atherosclerosis, musculoskeletal disorders, and certain types of cancers. A better knowledge of the involved regulatory molecular mechanisms will enable better prevention and treatment strategies of these diseases in the future. In mammals energy is stored mainly in the form of triacylglycerols and mobilized during periods of starvation and increased energy demand by lipolysis. The main sites for long and short term intracellular triacylglycerol storage are lipid droplets (LDs) in adipose tissue and the liver, respectively. LDs consist of a neutral lipid core surrounded by a phospholipid monolayer which is coated with LD-associated proteins including proteins with roles in signaling, cytoskeletal organization, intracellular trafficking and lipid metabolism. Key proteins involved in lipid mobilization include the intracellular lipases, namely adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and monoacylglycerol lipase (MGL), and their regulators: perilipins, G0/G1 switch gene 2 (G0S2) and comparative gene identification 58 (CGI-58/ABHD5). ATGL catalyzes the initial and rate limiting step of triacylglycerol hydrolysis which is dependent upon activation of ATGL by CGI-58. Mutations of the ATGL activator CGI-58 have been associated with ChanarinDorfman syndrome, a neutral lipid storage disease characterized by intracellular accumulation of triacylglycerols in most human tissues. The release of fatty acids is dependent on lipolysis of stored triacylglycerols which is tightly controlled by several hormones to meet energy demands while avoiding lipotoxicity. Molecular mechanisms of this regulation are very complex and poorly understood but post- translational modification, protein interactions, protein localizations and access of lipases to their substrates have all been implicated to play crucial roles. We found protein isoforms of lipases and CGI-58 in activity-based proteomic screenings and identified tyrosine 330 as a highly nucleophilic residue which is modified by fatty acid ester mimicking probes. The site was indeed only recently shown to be involved in inhibition of lipolysis by binding to fatty acyl CoA under conditions of high intracellular fatty acid concentrations. We moreover recently identified serine-239 of CGI-58 to be a novel substrate of protein kinase A and confirmed the occurrence of phosphorylated CGI-58 in adipose tissue. The mechanism by which phosphorylation of CGI-58 regulates lipolysis appears to involve the regulation of its subcellular localization by affecting its interaction with perilipin 1. We could show that site specific mutation of serine-239 to alanine, which inhibits its phosphorylation, interferes with the subcellular location of CGI-58. This may inhibit its interaction with ATGL, which is required for its activation. Thus CGI-58 may act as an energy sensor and a hub for regulation of lipid metabolism by beta-adrenergic signaling.