Perilipin 5 in bridging lipolysis to energy catabolism

Abstract. The adult heart depends on fatty acids (FAs) as prime energy fuel albeit cardiomyocytes do not store triglycerides (TG) in large quantities. Nevertheless, FAs utilized for mitochondrial energy catabolism originate from TG transiently deposited within cardiac cells. Intracellular TG breakdown depends on adipose triglyceride lipase (ATGL) and its co-activator comparative gene identification-58 (CG-58). Humans harboring mutations in the ATGL gene develop severe cardiac steatosis and cardiomyopathy that can be lethal. Perilipin 5 (Plin5) is highly expressed in muscle and liver where the protein localizes to lipid droplets (LDs) to regulate TG deposition and mobilization. Plin5 binds CGI-58 to suppress lipolysis whereas energy deprivation leads to the release of CGI-58 to stimulate ATGL-mediated TG catabolism. Increased cellular levels of unbound FAs can be toxic and play a critical role in the development of heart disease. The dual binding of Plin5 to LDs and mitochondria led to the notion that this interplay could control FA flow to mitochondria thereby protecting cells from the local increase of non-esterified FAs and/or mitochondrial FA oversupply. Mitochondria exhibit high plasticity which is affected by fusion or fission of mitochondria. Recent findings suggest that mitochondrial fission and/or fusion are linked to mitochondrial energy catabolism and possibly lipolysis. Interestingly, overexpression of CGI-58, which is the most prominent binding partner of Plin5, induced mitochondrial fission and the autophagosomal degradation of mitochondria (mitophagy) in muscle cells suggesting that Plin5 interaction with CGI-58 may participate in the regulation of mitochondrial dynamics. In this project, we will investigate the functional role of Plin5 in physically linking cardiac lipolysis to mitochondrial FA supply and dynamics. To do so we will generate and overexpress mutant Plin5 in cardiac cell lines and explore the impact on mitochondrial energy catabolism and dynamics including mitochondrial fission/fusion and mitophagy. Finally, we will generate mice overexpressing mutant Plin5 and study the consequences on cardiac energy catabolism. The outcome of this protect will increase our understanding in energy catabolism of the healthy and diseased heart eventually leading to novel therapeutic strategies to counteract the development of heart disorders.

Intracellular triacylglycerol (TG) catabolism critically depends on the TG-hydrolytic activity of Adipose triglyceride lipase (ATGL) and the ATGL coactivator Comparative gene identification-58 (CGI-58). Previous studies demonstrated that Perilipin 5 (PLIN5) interacts with CGI-58 at the surface of lipid droplets (LDs) under non-stimulated conditions thereby maintaining TG catabolism low. Upon energy demand and ß-adrenergic stimulation, PLIN5 is phosphorylated at serine 155 by cAMP-dependent Proteinkinase A (PKA) thereby resolving the interaction of PLIN5 and CGI-58 thus allowing CGI-58 to stimulate the TG hydrolase activity of ATGL. To study the role of PLIN5 in cardiac energy metabolism, we previously generated and characterized transgenic mice with cardiac muscle (CM)-specific overexpression of PLIN5 (PLIN5-Tg). CM-specific PLIN5 overexpression provokes massive TG accumulation in the heart, which is compatible with normal heart function and life span in contrast to ATGL-deficiency, which causes lethal heart dysfunction in 3-month old mice. We hypothesized that TG hydrolysis is only transiently impaired in PLIN5-Tg mice and that PKA phosphorylation stimulates TG hydrolysis and fatty acid supply. In this project, we exchanged serine 155 by an alanine (PLIN5-S155A) and investigated the impact of disrupted PKA phosphorylation on heart energy metabolism in CM-specific PLIN5-S155A transgenic mice. Unexpectedly, the overall phenotype of PLIN5-S155A mice was virtually identical compared to PLIN5-Tg mice. Interestingly, we found several additional PLIN5 phosphorylation sites in vivo, which were partially increased in CM preparations derived from PLIN5-S155A mice. Moreover, we show that lowering lipolysis, as present in PLIN5/PLIN5-S155A-Tg mice, counteracts mitochondrial fission and may protect the mice from mitochondrial dysfunction as seen in ATGL deficiency. The increase in cardiac glucose utilization altered whole body energy homeostasis and protected PLIN5-Tg mice from high fat diet (HFD)-induced obesity and glucose intolerance. PLIN5 dually localizes to LDs and mitochondria and the coupling of LDs and lipolysis to mitochondria has been suggested to promote mitochondrial FA uptake and oxidation. To address this hypothesis, we generated various C-terminal PLIN5 deletiond and could show that solely lacking the last three AA of the C-terminus of PLIN5 abrogates the interaction with mitochondria but does not impact the interaction with CGI-58 and ATGL. Overexpression of this mutant had a similar impact on lipolysis and mitochondrial FA oxidation compared to cells overexpressing wild type PLIN5 suggesting that the interaction of LDs and mitochondrial is not a driver of mitochondrial FA catabolism. Nevertheless, we show that the coupling of LDs to mitochondria increases respiratory capacity and reduces mitochondrial stress.