ChemBio Spotlight Week 3
Getting to the Heart of the Matter: The role of PKA in Plin5 regulated lipolysis in cardiac muscle
Lipid Droplets (LD) are cellular organelles present across many organisms. LDs play a crucial roll by storing triglycerides as an energy source for the cell and participating in a variety of cellular processes such as the generation of signaling molecules. Lipid mobilization from LDs is regulated by the association and phosphorylation of peripilin proteins which recruit and release lipases and cofactors necessary for triglyceride catabolism to the LD surface. LDs in adipose tissue, where triglycerides are frequently stored, are known to be under the regulation of peripilin 1 which is phosphorylated by protein kinase A (PKA), but there have been very few studies done on the regulation of peripilin 5 and LDs in cardiac muscle (CM). In CM the energy demand is continuously high and fatty acids are frequently lipolysed from LDs to meet this demand. An inability to break down cardiac LDs due to a lack of adipose triglyceride lipase (ATGL) or an overexpression of cardiac specific peripilin 5 (CM-Plin5) causes abnormal retention of lipids in the cell (a condition known as steatosis). However, there is a discrepancy in the effect of this cardiac steatosis that is not yet understood: in individuals lacking ATGL, steatosis results in heart dysfunction and shortened life span, but individuals with steatosis due to lack of CM-Plin5 have heart function and life spans compatible with individuals lacking steatosis entirely. Pollak et al. hypothesized that cardiac lipolysis was not constantly inhibited by cardiac Plin5 overexpression, as is the case for individuals lacking ATGL, but instead was somehow regulated. They sought to determine the currently unknown impact of PKA on the regulation of Plin5 as a potential explanation for this incomplete lipolytic barrier.
Several experiments get at the heart of their findings. An increase in cardiac triglyceride levels in CM-Plin5 transgenic mice in both fasted and non-fasted states as compared to wild type (WT) mice indicates that the overexpression of CM-Plin5 is causing the increased triglyceride level, rather than diet. Immunoblot and densitometric analysis of Plin5 protein levels in heart homogenates from fasted and non-fasted mice reveal no significant difference in the test groups, indicating that cardiac triglyceride breakdown is not controlled by CM-Plin5 protein expression levels. Incubation of CM-Plin5 and AGTL-deficient cardiac tissue with and without PKA displays a marked increase in fatty acid (FA) release from Plin5-enriched LDs as compared to the AGTL-deficient cardiac tissue, indicating that PKA incubation decreases the lipolytic barrier caused by Plin5 overexpression. Similar experiments in mouse models display consistent results. The authors show that PKA is active by examining immunoblot analysis in vitro in cardiac tissues and in vivo in mouse models which both display increased levels of phosphorylated versus non-phosphorylated hormone-sensitive lipase (HSL). The authors investigate Ser155 as a possible PKA phosphorylation site on Plin5 by performing a single amino acid mutation at the suspected phosphorylation site and observing a decrease in induced FA release as compared to non-mutated Plin5. Finally, the authors investigate literature suggestions that Plin5 overexpression may promote association of LDs and the mitochondria by measuring FAO in mitochondria and mitochondrial LD content from various CM-Plin5 mice and find that the mitochondria display increased content and impaired FAO capacity. The authors propose a mechanism for PKA phosphorylation regulation of TG breakdown from Plin5 LDs where a rise in cAMP activates PKA which phosphorylates Plin5 at Ser155, releasing CGI-58 which stimulates the hydrolytic activity of ATGL (Figure 7, Stimulated). This is in contrast to the LD in a basal, non-fasted state where cAMP is never elevated and Plin5 does not get phosphorylated (Figure 7, Basal).
Although it was previously known that Plin5 played some role in cardiac muscle LD lipolysis, Pollak et al. provides evidence for the role of PKA phosphorylation of Plin5 at Ser155 in the regulation of cardiac lipolysis and explains the discrepancy with cardiac steatosis and normal heart health and life span in individuals with Plin5 overexpression. In the future, investigation of other potential PKA phosphorylation sites on Plin5 and uncovering other molecules potentially at play in the regulation of cardiac LD lipolysis will be of primary interest.