Central P62 Regulates Peripheral Metabolic Balance In Energy-Related Neurons Through The Leptin-STAT3-POMC Pathway And Its Mechanism

PARTⅠ ROLE AND MECHANISM OF PERIPHERAL ENERGY METABOLISM AFTER SPECIFIC KNOCKOUT OF P62 IN CENTRAL OBRB NEURONSObjective: To investigate whether central p62 affects peripheral energy metabolism via ObRb neurons and its mechanisms using a hypothalamic ObRb neuron-specific knockout p62 mouse model.Methods: Eight-week-old ObRb-p62^+/+ and ObRb-p62 KO mice were given a general diet and high-fat feeding for 12 weeks,respectively.Body weight,food intake,anal temperature,respiratory quotient and exercise tolerance of the mice were measured.Insulin tolerance and glucose tolerance tests were performed.Oil red O staining was performed to observe changes in liver lipid droplets,and HE staining was performed to observe morphological changes in white fat,brown fat and liver.Immunohistochemical staining and Western blot were performed to observe the expression level of brown fat uncoupling protein 1.Tissue immunofluorescence staining was performed to detect changes in the expression of factors such as POMC and p-STAT3 in the ARC region of the hypothalamus.Results: Compared with the control group,ObRb-p62 KO mice showed no significant differences in food intake,accelerated body weight gain,reduced body oxygen consumption,reduced exercise tolerance,reduced insulin tolerance and sensitivity,marked fat vacuolation,larger fat,larger liver lipid droplets,marked fat deposition and reduced brown fat thermogenesis in both the general diet and high fat feeding conditions.leptin stimulation The number of POMC neurons was reduced and p-STAT3 expression was decreased.Conclusion: Specific knockout of p62 from hypothalamic ObRb neurons inhibited peripheral energy metabolism and homeostasis in mice,probably by regulating the central leptin-STAT3-POMC pathway.PARTⅡ THE EFFECT OF CENTRAL POMC NEURON SPECIFIC KNOCKOUT OF P62 ON ENERGY METABOLISM AND ITS MECHANISMObjective: To investigate whether central p62 affects peripheral energy metabolism via POMC neurons and its mechanisms using a mouse model with specific knockout of p62 in hypothalamic POMC neurons.Methods: Eight-week-old POMC-p62^+/+ and POMC-p62 KO mice were given a general diet and high-fat feeding for 12 weeks,respectively.Body weight,food intake,anal temperature,respiratory quotient and exercise tolerance of the mice were measured.Insulin tolerance and glucose tolerance tests were performed.Oil red O staining was performed to observe changes in liver lipid droplets,and HE staining was performed to observe morphological changes in white fat,brown fat and liver.Immunohistochemical staining and Western blot were performed to observe the expression level of brown fat uncoupling protein 1.Tissue immunofluorescence staining was performed to detect changes in the expression of factors such as POMC and p-STAT3 in the ARC region of the hypothalamus.Results: Compared with controls,POMC-p62 KO mice showed no significant differences in food intake,accelerated body weight gain,reduced body oxygen consumption,reduced exercise tolerance,reduced insulin tolerance and sensitivity,marked fat vacuolation,larger fat,larger liver lipid droplets,marked fat deposition and reduced brown fat thermogenesis in both the general diet and high fat feeding conditions.leptin stimulation The number of POMC neurons was reduced and p-STAT3 expression was decreased.Conclusion: Specific knockout of p62 from hypothalamic POMC neurons inhibited peripheral energy metabolism and homeostasis in mice,probably by regulating mainly the central leptin-STAT3-POMC pathway.PartⅢ VALIDATION OF CENTRAL P62 REGULATION OF ENERGY METABOLISM VIA THE LEPTIN-STAT3-POMC PATHWAYObjective: To verify whether central p62 regulates peripheral energy metabolism and homeostasis through leptin-STAT3-POMC using ob/ob mice with specific overexpression of p62 in the MBH region of the hypothalamus and leptin lateral ventricular infusionMethods: 8-week-old ob/ob mice were randomly divided into 2groups by digital method and nuclei were injected with adeno-associated virus AAV-GFP or AAV-p62 in the MBH region of the hypothalamus.15 days after recovery,acute and chronic infusions of leptin were administered to the lateral ventricles,and body weight,food intake,anal temperature,respiratory quotient and exercise tolerance were measured.Glucose tolerance and insulin tolerance tests were performed.Tissue immunofluorescence staining was performed to detect changes in the expression of POMC,p-STAT3 and other factors in the ARC region of the hypothalamus.Results: After overexpressing p62 in the MBH region of the ob/ob mouse hypothalamus,it can inhibit food intake and weight gain,increase energy consumption,and enhance insulin sensitivity and glucose tolerance.And compared with the AAV-p62/a CSF group,the AAV-p62/leptin group significantly reduced food intake and weight,increased energy consumption,increased insulin sensitivity and glucose tolerance.Conclusion: Central MBH-specific overexpression of p62 promotes peripheral energy metabolism in mice by a specific mechanism that regulates the central leptin-STAT3-POMC pathway.PARTⅣ IN VITRO STUDIES ON THE REGULATION OF ENERGY METABOLISM AND HOMEOSTASIS BY CENTRAL P62 AND EXPLORATION OF ITS MECHANISMSObjective: To explore the role of central p62 on energy metabolism and its preliminary mechanisms at the level of in vitro studies using mouse neuroma cells.Methods: Construct p62 overexpression and control plasmids,transfect N2 A cells,observe whether there is leptin stimulation,and detect the expression levels of p-STAT3,STAT3,p-FOXO1,FOXO1,POMC,and p62 by western blot;And use glucosamine to induce insulin resistance model,after treatment with STAT3 inhibitor,detect the expression level of POMC.The dual luciferase report experiment was used to explore the regulatory effect of p62 gene on the POMC promoter.Results: Through Western Blot experiment,it was found that compared with the control group,after overexpression of p62 plasmid,POMC expression level increased,p-STAT3/STAT3 level increased,FOXO1 and p-FOXO1 expression levels did not change significantly;and after leptin treatment The levels of P-STAT3 and POMC expression in the group were significantly higher than those in the group without leptin treatment.After stattic treatment,the expression level of POMC decreased.The analysis of the dual luciferase report experiment showed that compared with the empty plasmid,the POMC promoter activity was significantly increased after the p62 overexpression plasmid was added（p<0.01）.After stattic treatment,POMC promoter activity decreased（p<0.01）.Conclusion: Central p62 may regulate energy metabolism and balance mainly by promoting the phosphorylation of STAT3 and then increasing the expression of POMC.