PLIN3 و PLIN5 ماهیچه های اسکلتی در حالت استراحت و پس از لیپولیز در طی تحریک آدرنرژیک یا کنتراکتیل سرین فسفوریله می باشند Skeletal muscle PLIN3 and PLIN5 are serine phosphorylated at rest and following lipolysis during adrenergic or contractile stimulation

Introduction Intramuscular triglycerides (IMTGs) represent an important energy source that can be mobilized during exercise through a combination of external hormonal (increased epinephrine) and internal metabolic signals (intracellular Ca2+ and metabolite concentrations). However, the exact mechanisms regulating IMTG breakdown during exercise are poorly understood. IMTGs are stored in metabolically active organelles known as lipid droplets that are encased by a phospholipid monolayer coated with a variety of proteins (Londos et al. 1999; Bartz et al. 2007). Evidence indicates that the regulation of skeletal muscle lipolysis is mediated by protein–protein interactions occurring on the lipid droplet surface (Prats et al. 2006; Macpherson et al. 2013). Specifically, a family of lipid droplet proteins, known as PLIN proteins, have emerged as likely candidates in mediating the hydrolysis of IMTGs (Brasaemle et al. 2004; Macpherson et al. 2013). To date, work investigating the role(s) of PLINs has focused on adipose tissue, however, recent investigations in skeletal muscle support a role for PLIN proteins in the regulation of IMTG degradation (Macpherson et al. 2012, 2013; Shaw et al. 2012; Shepherd et al. 2012, 2013). The PLIN family is composed of five members (PLIN1 through PLIN5) (Miura et al. 2002; Kimmel et al. 2010), each with a unique tissue distribution and potentially a unique role in cellular lipid metabolism (Wolins et al. 2006; Hsieh et al. 2012). PLIN1 is the only member of this family for which a specific role in regulating lipolysis has been determined, however, it is only expressed in adipose tissue. More specifically, in a basal state PLIN1 limits the activity of the rate-limiting lipase, adipose triglyceride (ATGL), by directly binding to its coactivator, CGI-58 (Brasaemle et al. 2000; Souza et al. 2002; Tansey et al. 2003; Miyoshi et al. 2006). Under lipolytic stimulation, initiated by catecholamines, it is believed that the protein kinase A (PKA)-dependent serine phosphorylation of PLIN1 initiates lipolysis by releasing CGI-58 and allowing it to bind to and activate ATGL (Egan et al. 1990; Granneman et al. 2007, 2009; Granneman and Moore 2008; Bezaire and Langin 2009). Further, phosphorylation of PLIN1 is required for hormone-sensitive lipases (HSL) recruitment to the lipid droplet through binding to PLIN1 (Wang et al. 2009). Skeletal muscle does not express PLIN1 and, thus far, similar roles for skeletal muscle PLIN proteins have yet to be determined. It has been suggested that PLIN2, PLIN3, and PLIN5 play a large role in regulating lipolysis in skeletal muscle (Macpherson et al. 2012, 2013; Peters et al. 2012; Shaw et al. 2012; Shepherd et al. 2012, 2013). PLIN2 is the predominant lipid droplet associated protein in skeletal muscle (Phillips et al. 2005) and PLIN5 is unique in that it is highly expressed in oxidative tissues (Wolins et al. 2006). Although knowledge of PLIN3 in skeletal muscle is scarce, recent work from our laboratory showed that ATGL interacts with PLIN2, PLIN3, and PLIN5 in isolated rat soleus muscle (Macpherson et al. 2013). These protein interactions suggest that PLIN2, PLIN3, and PLIN5 may have a role in the regulation of ATGL activity and therefore the initiation of skeletal muscle lipolysis. In adipose tissue, the reversible phosphorylation of PLIN1 is necessary for lipolytic activation (Su et al. 2003; Sztalryd et al. 2003; Marcinkiewicz et al. 2006; Miyoshi et al. 2006), however, the phosphorylation state of the remaining PLINs has yet to be investigated in skeletal muscle. A phosphorylation site has been identified on PLIN2 (serine 291) (Bartz et al. 2007), PLIN3 (serine 245) (Hickenbottom et al. 2004), and some evidence indicates that PLIN5 is a substrate for PKA phosphorylation (Wang et al. 2011). It is possible that the phosphorylation of PLIN2, PLIN3, or PLIN5 may be required to optimally position ATGL, CGI-58, and/or HSL for activation of lipolysis in skeletal muscle. Exercise leads to the activation of several skeletal muscle kinases, all of which may play into regulating the rate of lipolysis. Increased circulating epinephrine concentrations leads to the activation of PKA, while at the same time contraction increases intramuscular calcium levels and CaMK and ERK activation. The use of ATP during contraction also leads to increased levels of AMP, which activates AMPK. It is known that epinephrine and contraction activate skeletal muscle hormone-sensitive lipase (HSL) with additive effects, thus indicating that epinephrine and contraction activate HSL through different signaling mechanisms (Spriet et al. 1986; Hopp and Palmer 1990; Dyck and Bonen 1998; Donsmark et al. 2003, 2005; Langfort et al. 2003; Watt et al. 2003). Therefore, the purpose of this study was to examine the isolated and additive effects of epinephrine and contraction on skeletal muscle PLIN protein to ATGL and HSL interactions as well as phosphorylation status. A major objective of this study was to determine if PLIN2, PLIN3, and/or PLIN5 are phosphorylated and whether this changes during lipolysis. If so, this study aimed to separate adrenergic and/or contractile stimulation. A second objective of this study was to investigate the role of PLIN2, PLIN3, and PLIN5 in governing the accessibility of ATGL and HSL via direct protein–protein interactions during these perturbations. We hypothesized that skeletal muscle PLINto-lipase interactions are governed by PLIN phosphorylation status.