Loss Of ER Ca²⁺ Sensor STIM1 In AgRP Neurons Confers Protection Against HFD-Induced Obesity

Objective: Obesity has become a serious public health problem,and the incidence of chronic diseases related to obesity has also increased significantly.Therefore,it is urgent to find new and effective interventions or therapeutic targets.In this study,we investigated the role of Stim1 in AgRP neurons of hypothalamus ARC in the development of nutritional obesity,and further studies the mechanism of Stim1 in weight regulation with conditional Stim1 knockout mouse model and genetic manipulations by AAV.And through this research,we want to provide a new target for the treatment of obesity.Methods: First,western blot and immunofluorescence were performed to detect the expression of Stim1 in hypothalamus.In vivo calcium imaging was used to confirm the inhibitory effect of BTP2 on SOCE.Then the HFD-fed mice which were inhibitited of SOCE in hypothalamus were used to measure body weight,fat mass,lean mass,the area of e WAT,GTT,the area of lipid droplets in liver,TC,food intake,the interscapular temperature,oxygen consumption and energy expenditure.AgRP neuron-specific Stim1 knockout mice(ASKO)were generated by crossing the Stim1 lox P/lox P mice with the AgRP-Cre mice.In vivo calcium imaging was also utilized to measure Ca2+ signals from AgRP neurons of AgRP-Cre and ASKO mice.The Stim1 lox P/lox P mice and ASKO mice were fed an HFD diet,and the body weight,food intake,thermogenisis and glucolipid metabolism were then monitored.Neuro2 a cells were infected with AAV-EF1a-DIO-Stim1 D76 A,and Stim1 D76 A expression effect on Ca2+ signals was measured by calcium imaging.Stim1 D76A-AAV was injected into the AgRP-Cre mice ARC to generate the AgRP neuron-specific Stim1 D76 A expressing mice.Body weight,food intake and energy expenditure were monitored after the animals had been placed on a chow-fed diet.Western blot was used to measure the protein expression levels of Neuro2 a cells which were treated with SOCE inhibitor,Stim1 knockdown plasmids or Stim1 D76 A expression plasmids.q RT-PCR was used to measure ribosomal m RNA and r RNA levels of Neuro2 a cells which were performed the same processing as WB.q RT-PCR and WB were used to detect the RNA and protein level of Oas3 and RNase L in Neuro2 a cells which were treated with the same processing.Oas3-AAV was injected bilaterally into the ARC of AgRP-Cre mice to generate the AgRP-specific Oas3 overexpressing mice,and AAV-Ctrl injected AgRP-Cre mice were used as control.These mice were fed with an HFD diet and the body weight,body compositions and food intake were measured.ns2-AAV and Ctrl-AAV were respectively injected bilaterally into the Arc nucleus of ASKO and Stim1 lox P/lox P mice.These mice were fed with an HFD diet and the body weight,body compositions and food intake were then monitored.Results: Stim1 protein was expressed in about 90% of neurons in the arcuate nucleus of the hypothalamus of mice.In vivo calcium imaging results showed that SOCE inhibition in hypothalamus led to decreased cellular calcium signal.Mice injected daily with BTP2 in the third ventricle decreased 1.5 g body weight(P<0.05)and 1 g fat mass(P=0.01)than the control group with HFD,improved glucose and lipid metabolism,decreased food intake,and increased heat production.Stim1 was knockout in AgRP neurons,the calcium signal intensity was lower than that of the control group under normal food,HFD 3d or HFD 7d fasting stimulation.In HFD-fed control and ASKO mice,the body weight of knockout group was significantly lower by about 10 g(P<0.05)compared with the control group,glucose and lipid metabolism was improved,energy intake was reduced and energy consumption was increased.Calcium imaging confirmed that expression of Stim1 D76 A increased Ca2+signal.The mice expressing Stim1 D76 A in AgRP neurons gained about 4-5 g body weight(P<0.05),and disturbed the balance of glucose and lipid metabolism,increased about 0.7g food intake(P<0.05),and significantly reduced energy consumption compared with the control group under the chow-fed diet.Neuro2 a cells were treated with SOCE inhibitor or Stim1 knockdown,the newly synthesized protein was reduced by 30-40%,ribosomal m RNA and r RNA levels were decreased,m RNA and protein levels of Oas3 were increased by about 0.5 times compared with the control group(P<0.05),m RNA and protein levels of RNase L were not significantly different.Neuro2 a cells expressed Stim1 D76 A,the newly synthesized protein increased by about 50%(P<0.05).The expression levels of ribosome m RNA and r RNA increased(P<0.05).The m RNA and protein levels of Oas3 decreased by about 50%(P<0.05),while the m RNA and protein levels of RNase L showed no significant difference.Mice that overexpressed Oas3 in AgRP neurons and were fed with HFD also reduced their body weight by about 10 g(P<0.05),body fat by about 6 g(P<0.05)and average daily food intake(P<0.05)compared with the control group.Mice that expressed ns2 in AgRP neurons in ASKO mice,which were fed with HFD,also had a significant increase in body weight of about 6 g(P<0.05),a body fat weight increase of about 4 g(P<0.05)and an average daily food intake(P<0.05)compared with ASKO mice which were injected with control virus.Conclusion: Stim1 expressed in AgRP neurons in the arcuate nucleus of hypothalamus plays an important role inregulating energy balance in mice which were fed with an HFD.It can regulate the energy balance of mice through the Oas3-RNase L signaling pathway which could influence the protein synthesis.