The Mechnism Of Grb14 Enhances The HSL-mediated Lipolysis In Liver Cells

Lipid is one of the most important energy storage for animals.Lipid droplets(LDs)are composed of different neutral lipids,phospholipids and lipid droplet related proteins.During fasting conditions or in absence of energy supply,triglyceride lipase(ATGL),hormone sensitive lipase(HSL)and monoglyceride lipase(MGL)decompose lipid droplets into fatty acids and glycerol as the energy for the body.Besides the lipolytic enzymes,various growth factors,hormones and transcription factors are also involved in lipolysis,which disturbance cause obesity,metabolic syndrome,non acoholic fatty liver disease,hyperinsulinemia,and cancer in the body.Growth factor receptor binding protein 14(Grb14)is an adaptor protein that has been shown to inhibit insulin receptor tyrosine kinase activity.However,it is not clear how Grb14 regulates lipolysis metabolism.Therefore,this study uses molecular biology techniques such as immunofluorescence,immunoprecipitation,triglycerides content and glycerol release test,in vitro protein purification,and real-time fluorescence quantitative PCR to explore the role of Grb14 in lipolysis metabolism in the protein and RNA levels.Our study aims to reveal the mechanism of Grb14 in lipolysis.The main conclusions are as follows:(1)Grb14 promotes the decomposition of lipid droplets in liver cells.The overexpression of Grb14 affects the triglycerides content in the cells and the release of glycerol in the culture medium: the triglycerides content was significantly reduced,glycerol was increased.The immunofluorescence results have shown that lipid droplets were significantly reduced in Grb14 overexpressed cells.It is speculated that overexpression of Grb14 promotes lipolysis in the cells.(2)Grb14 cooperates with HSL to promote breakdown of lipid droplets in liver cells.HSL,the earliest discovered lipase,participates in the hydrolysis of triglycerides and diglycerides,and cholesterol esters.Results showed that the N-terminus of Grb14 interacted with N-terminus of HSL.Through the determination of triglycerides and glycerol,we found that Grb14 and HSL synergistically promote the hydrolysis of triglycerides.The results of immunofluorescence experiment is consistent with biochemical test.The lipid droplet s content of cells expressing Grb14 or HSL was significantly reduced,while the lipid droplet content of cells co-expressing Grb14 and HSL disappeared utterly.Besides,overexpression of Grb14 in human derived liver cells(HL-7702)found that the m RNA level of lipolysis related genes such as ATGL,HSL,CGI-58,ADRB,ACOX and LPL were significantly up-regulated,knockdown Grb14 found that most of these genes were significantly down-regulated.These results showed that Grb14 promoted the lipolysis through HSL both in the RNA level and the protein level.(3)Grb14 facilitates lipid droplets decomposition by regulating HSL hydrolase activity and lipophagy.We found that Grb14 directly affects PKA-mediated HSL phosphorylation,thereby regulating lipolysis.Meanwhile,Grb14 promotes lipophagy in HL-7702 cells by affecting the protein level of autophagy markers and accumulation of autophagy substrate,which provides a novel idea about the decomposition of lipid droplets in liver cells.In summary,Grb14 acts as a new signaling node that regulates lipolysis by acting as an upstream element of HSL in the liver cells.