Skeletal muscle PLIN proteins, ATGL and CGI-58, interactions at rest and following stimulated contraction

Abstract

Evidence indicates that skeletal muscle lipid droplet-associated proteins (PLINs) regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN1 is thought to regulate lipolysis by directly interacting with comparative gene identification-58 (CGI-58), an activator of adipose triglyceride lipase (ATGL). Upon lipolytic stimulation, PLIN1 is phosphorylated, releasing CGI-58 to fully activate ATGL and initiate triglyceride breakdown. The absence of PLIN1 in skeletal muscle leads us to believe that other PLIN family members undertake this role. Our purpose was to examine interactions between PLIN2, PLIN3, and PLIN5, with ATGL and its coactivator CGI-58 at rest and following contraction. Isolated rat solei were incubated for 30 min at rest or during 30 min of intermittent tetanic stimulation [150-ms volleys at 60 Hz with a train rate of 20 tetani/min (25°C)] to maximally stimulate intramuscular lipid breakdown. Results show that the interaction between ATGL and CGI-58 increased 128% following contraction (P ϭ 0.041). Further, ATGL interacts with PLIN2, PLIN3, and PLIN5 at rest and following contraction. The PLIN2-ATGL interaction decreased significantly by 21% following stimulation (P ϭ 0.013). Both PLIN3 and PLIN5 coprecipitated with CGI-58 at rest and following contraction, while there was no detectable interaction between PLIN2 and CGI-58 in either condition. Therefore, our findings indicate that in skeletal muscle, during contraction-induced muscle lipolysis, ATGL and CGI-58 strongly associate and that the PLIN proteins work together to regulate lipolysis, in part, by preventing ATGL and CGI-58 interactions at rest. adipocyte differentiation-related protein; adipophilin; OXPAT; MLDP; TIP47; ABHD5 FATTY ACIDS (FA) RELEASED from intramuscular triglycerides (IMTG) during lipolysis provide an important source of energy during muscle contraction. In skeletal muscle, IMTGs are packaged into lipid droplets that possess a unique coat of proteins associated with the surrounding phospholipid monolayer. This protein coat provides an interface for specific processes, such as transport, lipogenesis, and lipolysis (10, 34). Perilipins (PLINs) are the most recognized family of lipid droplet proteins and are the most likely to be involved in the regulation of lipogenesis and lipolysis in skeletal muscle (31).Our understanding of PLIN proteins in skeletal muscle is limited; however, studies in other tissues and in cell culture indicate that PLIN proteins are key regulators of lipid metabolism, as they appear to be directly involved with how cells and tissues store, mobilize, and utilize fatty acids (8,12,15,34,35,62). The PLIN family consists of five members, PLIN1

Piecing together the puzzle of perilipin proteins and skeletal muscle lipolysis

“…In isolated soleus muscle, HSL translocates to PLIN2-coated lipid droplets with either ␤-adrenergic stimulation or electrically stimulated contraction (Prats et al 2006). In addition, recent work in isolated rat soleus muscle has revealed that PLIN2 interacts with HSL, as well as with ATGL, and that this interaction is unchanged following stimulated contraction or epinephrine stimulation (MacPherson et al 2013a(MacPherson et al , 2013b. Together, this work indicates a role for PLIN2 in regulating the location of the lipases in relation to the lipid droplets.…”

“…Previous work with skeletal muscle contraction has shown a translocation of HSL towards PLIN2-coated lipid droplets (Prats et al 2006). Results from recent work (MacPherson et al 2013a(MacPherson et al , 2013b provide the first evidence of a physical interaction between PLIN2 and HSL; however, this interaction remains unchanged following isolated muscle contraction or epinephrine stimulation alone or in combination. This indicates that in skeletal muscle, PLIN2 may not be involved in recruiting HSL to the lipid droplet surface, or that once HSL has translocated to the lipid droplet, it does not interact with PLIN2.…”

“…Although PLIN1 is not expressed in skeletal muscle, evidence suggests that skeletal muscle lipolysis is regulated by PLIN protein interactions occurring on the lipid droplet surface in a manner similar to that observed in adipose tissue (Blanchette-Mackie et al 1995;Prats et al 2006;Brasaemle 2007;Ducharme and Bickel 2008;Peters et al 2012;MacPherson et al 2013a). Skeletal muscle is very distinct from adipose tissue given its higher metabolic rate, which is accelerated during exercise.…”

“…However, activation of PKA in cells with ectopic expression of PLIN5 and ATGL resulted in increased lipolysis (Wang et al 2011a). In skeletal muscle, PLIN5 is highly colocalized with ATGL and CGI-58 (Mason et al 2014a), and it is now known that PLIN5 interacts with both ATGL and CGI-58; however, this interaction remains unchanged in the face of lipolytic challenges (i.e., contraction and adrenergic stimulation) (MacPherson et al 2013a(MacPherson et al , 2013b. Further, in whole skeletal muscle, PLIN5 is serine phosphorylated; however, this phosphorylation state and interactions with CGI-58 and ATGL remain unchanged following either contractile or epinephrine stimulation (MacPherson et al 2013b).…”

Exercise and Regulation of Lipid Metabolism

“…While investigation of whether exercise induces alterations in these protein-protein interactions is in its infancy, several studies are yielding exciting insight into mechanisms responsible for the exercise-induced increase in ATGL activity. [47][48][49][50][51][52][53] HSL has greater specificity for DAGs (10-fold) and cholesteryl (5-fold) than TGs or monoacylglycerides. 17 Moreover, Hsl-deficient mice exhibit DAG accrual suggesting this enzyme has a primary role in DAG hydrolysis in vivo.…”