To Explore The Role Of Rab Proteins In AMPK Phosphorylation Regulatory Network

Energy homeostasis is essential for maintaining normal metabolism of the body.If energy intake is greater than consumption,energy homeostasis will be destroyed,leading to the occurrence and development of metabolism-related diseases.AMPactivated protein kinase（AMPK）is a major regulator of energy homeostasis,it senses energy state and regulates the activity of multiple downstream target proteins to control metabolic process when its phosphorylation level is changed.This phosphorylation regulatory network is delicate and complex,but the specific mechanism needs to be further explored.Rab proteins are a type of small G proteins,mainly localized to the membrane of the endomembrane system of cells and are responsible for the formation and transportation of vesicles.However,a number of studies have shown that Rab proteins are also involved in regulating metabolic processes.Whether AMPK can regulate energy metabolism through Rab proteins remain unclear.In addition,it has not been confirmed whether Rab proteins regulate AMPK phosphorylation.The purpose of this study was to explore how AMPK functions through Rab proteins and whether Rab proteins regulate AMPK phosphorylation.We first constructed a glucose starvation system to explore whether AMPK,as the core of energy metabolism regulator,can sense energy deficiency and then be phosphorylated and activated.The results showed that AMPK can sense energy deficiency induced by decreased glucose concentration and is gradually phosphorylated and activated in HEK293 T and Hep G2 cells.Activated AMPK can further phosphorylate and activate downstream TBC1 domain family member 1（TBC1D1）.During this process,we found that,peroxisome proliferators-activated receptor γ（PPARγ）,as an important regulator of energy balance,gradually decreased according to the endogenous and exogenous expression levels.To further verify whether PPARγ is regulated by AMPK,we used AMPK inhibitor Compound C to reverse AMPK phosphorylation.The results showed that the expression level of PPARγ was gradually increased after AMPK phosphorylation was inhibited.Therefore,in order to explore how AMPK regulates the expression level of PPARγ,we detected whether AMPK downstream target protein TBC1D1 interacts with PPARγ on the basis of previous studies.Through protein purification and binding assay in vitro,we proved that TBC1D1 interacts with PPARγ.However,this effect was independent of TBC1D1 phosphorylation,suggesting that some other protein plays a role in the AMPK phosphorylation regulatory network.Considering that TBC1D1 is an upstream GTPases activating protein（GAP）of Rab proteins,we hypothesized that Rab proteins may play a role in AMPK regulation of PPARγ.Therefore,we overexpressed PPARγ and different concentrations of Rab family proteins in HEK 293 T cells,and screened Rab proteins interacting with PPARγ,and finally screened Rab2 A that might play a role in stabilizing PPARγ protein.Protein binding assay in vitro further proved the interaction between Rab2 A and PPARγ.To further explore the specific mechanism of Rab2 A stabilizing PPARγ,we overexpressed Rab2 A and PPARγ in HEK 293 T cells,and selected lysosomal autophagy inhibitor bafilomycin A1 and ubiquitin-proteasome inhibitor MG132.The results showed that Rab2 A could inhibit PPARγ degradation by ubiquitin-proteasome pathway.In addition,Rab proteins have two structural forms of GTP and GDP.To explore the structural forms of Rab2 A in stabilizing PPARγ,we constructed a western diet induced obesity mouse model and a genetic mouse model of TBC1D1 protein serine 231 inactivation mutation.Protein binding assay in vitro demonstrated that Rab2 A stabilized PPARγ in the form of GTP after phosphorylation of AMPK was blocked.These results suggest that Rab2 A plays a role in the phosphorylation regulatory network of AMPK by regulating PPARγ.PPARγ,as a regulatory factor of lipid metabolism,is involved in the processes of fat formation,triglyceride synthesis and lipid droplet formation.Therefore,in order to investigate whether the expression of PPARγ and lipid metabolism level will be changed with Rab2 A knockdown,we constructed Rab2 A knockdown mice which fed with a high-fat diet for 2 months.The results showed that the expression levels of proteins involved in lipid metabolism and cholesterol synthesis were significantly inhibited,and the lipid droplets in the liver of the mice were also significantly reduced.In addition,we detected some metabolic indexes in the blood of the mice and found that with Rab2 A knockdown,the levels of triglyceride and cholesterol in the blood of mice was decreased,while the levels of insulin,glucose and free fatty acids did not change significantly,suggesting that the knockdown of Rab2 A has a certain alleviating effect on obesity and non-alcoholic fatty liver diseases.To further explore whether the Rab family members regulate AMPK phosphorylation during energy deficiency,we overexpressed various Rab proteins in3T3-L1 cells,and used glucose starvation assay to explore the changes of AMPK phosphorylation,so as to screen Rab proteins that can regulate AMPK phosphorylation.The results showed that Rab5 C could inhibit AMPK phosphorylation.In conclusion,Rab2 A plays a role in the phosphorylation regulatory network of AMPK by regulating the expression of PPARγ,and when the phosphorylation function of AMPK is blocked under sufficient energy,Rab2 A stabilizes PPARγ in the form of GTP binding.Rab2 A may be a new target for the treatment of obesity and non-alcoholic fatty liver diseases.In addition,Rab5 C inhibited the phosphorylation of AMPK under glucose starvation.