The Effects And Mechanism Of Hypothalamus FSTL1 On Food Intake And Energy Metabolism

PART ONE EXPRESSION OF FSTL1 IN THE HYPOTHALAMUS AND ITS RELATIONSHIP WITH OBESITYObjective:Explore the relationship between the expression of FSTL1 in the hypothalamus and obesity.Methods:The expression and distribution of FSTL1 protein in the hypothalamus of 12-week-old C57BL/6J mice were detected by immunofluorescence.To explore the relationship between FSTL1 and obesity,the expression of FSTL1 protein in the hypothalamus of ob/ob,db/db mice and C57BL/6J mice fed with NCD or HFD for 12 weeks were detected by Western Blot.In order to study the effect of nutritional status on FSTL1 in the hypothalamus,12-week-old C57BL/6J mice were subjected to fasting and refeeding interventions,and the protein levels of hypothalamic FSTL1 under different nutritional status were detected.Results:Immunofluorescence results indicated that FSTL1 protein was expressed in ARC,VMH and DMH.The hypothalamic FSTL1 protein level of high-fat-fed C57BL/6J mice and db/db/,ob/ob mice decreased compared with the normal chow diet control group.Fasting promoted hypothalamic FSTL1 protein expression,and FSTL1 protein levels decreased after refeeding to be consistent with ad libitum fed state.Conclusion:FSTL1 is widely expressed in hypothalamus such as ARC,VMH and DMH and is related to obesity and nutritional status,suggesting that FSTL1 may be involved in the regulation of food intake and energy metabolism in the hypothalamus.PART TWO HYPOTHALAMUS FSTL1 REGULATION OF FOOD INTAKE AND PERIPHERAL ENERGY METABOLISM IN VIVO SECTION ONE EFFECTS OF HYPOTHALAMIC FSTL1 OVEREXPRESSION ON FOOD INTAKE AND ENERGY METABOLISM IN VIVOObjective:To explore the effect of hypothalamus overexpression of FSTL1 on food intake and energy metabolism.Methods:The 6-week-old C57BL/6J mice were randomly divided into 4 groups,injected with AAV-GFP and AAV-FSTL1 in the third ventricle,and fed with NCD or HFD for 12 weeks.Metabolic indexes such as food intake,body weight,rectal temperature,GTT,ITT were measured,RNA-seq was performed on the hypothalamus tissue of mice in each group.The effects of FSTL1 overexpression on the expression of AgRP,POMC and c-fos were detected by immunofluorescence after fasting and refeeding intervention,and the mRNA levels of POMC,AgRP and NPY were detected by RT-PCR.The hypothalamus protein was extracted in each group after insulin stimulation,and the effect of overexpression of FSTL1 on insulin signaling pathway was detected by Western blot.Results:Compared with the control group,the overexpression of FSTL1 in the hypothalamus in the HFD group resulted in a decrease in food intake,body weight,an increase in rectal temperature,and a decrease in the area under the curve of glucose tolerance and insulin tolerance.Hypothalamic FSTL1 overexpression decreased AgRP protein and mRNA levels and c-fos protein expression in the fasted state.The results of RNA-seq indicated that overexpression of FSTL1 effected the PI3K-AKT-FoxO1 pathway.The protein levels in PI3K-AKT-FoxO1 pathway were detected by Western blot,and the results indicated that overexpression of FSTL1 in the hypothalamus promoted the phosphorylation of AKT and FoxO1,and increased the nuclear export of FoxO1.Conclusion:Overexpression of FSTL1 in the hypothalamus may regulate food intake and energy metabolism through the AKT-FoxO1-AgRP signaling pathway.SECTION TWO EFFECTS OF FSTL1 OVEREXPRESSION IN AGRP NEURONS ON FOOD INTAKE AND ENERGY METABOLISMObjective:To explore the role of FSTL1 in the regulation of food intake and energy metabolism in AgRP neurons by overexpressing FSTL1 in AgRP neurons.Methods:6-week-old AgRP-cre mice were randomly divided into 4 groups,bilaterally injected with AAV-DIO-GFP or AAV-DIO-FSTL1 in ARC,and fed with NCD or HFD for 12 weeks.Body weight,rectal temperature,GTT,ITT,respiratory quotient and other metabolic indicators were measured.After 12 weeks of virus injection,each group was intervened by fasting and refeeding,and the effects of FSTL1 overexpression on the expressions of AgRP,POMC,FoxO1 and c-fos were observed by immunofluorescence staining.The morphology of the liver,iWAT and BAT was observed by HE staining;the lipid deposition in the liver was observed by Oil Red O staining;the expression of BAT UCP1 was detected by immunohistochemical staining.Results:After AgRP neurons specifically overexpressed FSTL1,the high-fat diet group showed decreased food intake and body weight,decreased area under the curve of glucose tolerance and insulin tolerance,increased oxygen consumption and lipid oxidation,and decreased RER and carbohydrate oxidation.In the FSTL1 overexpression group,liver lipid deposition was reduced,and BAT UCP1 expression was increased.Immunofluorescence staining showed that the expression of AgRP and c-fos decreased in the overexpression group during fasting state,and the nuclear export of FoxO1 increased.Lateral ventricle infusion of PI3K inhibitor can inhibit the effects of FSTL1 overexpression-induced food intake inhibition and improvement of blood metabolism.Conclusion:Overexpression of FSTL1 in AgRP neurons regulate food intake and energy metabolism through the AKT-FoxO1-AgRP signaling pathway.SECTION THREE FOXO1 MEDIATED REGULATION OF FOOD INTAKE AND ENERGY METABOLISM BY FSTL1 IN AGRP NEURONSObjective:To verify the mechanism of FSTL1 regulating food intake and energy metabolism by overexpressing FSTL1 in AgRP-specific FoxO1 knockout mice.Methods:6-week-old AgRP-cre and AgRP-specific FoxO1 knockout mice were randomly divided into 4 groups,bilaterally injected with AAV-DIO-GFP or AAV-DIO-FSTL1 in ARC,and fed with HFD for 12 weeks.Metabolic indicators such as food intake,body weight,rectal temperature,GTT,and ITT were measured.At 12 weeks after virus injection,the hypothalamus of mice was fasted and the AgRP protein was detected by immunofluorescence staining.The livers,iWAT and BAT were stained with HE and Oil Red O,and the expression of UCP1 in BAT was detected by immunohistochemistry.Results:Overexpression of FSTL1 in AgRP neurons resulted in decreased food intake,weight loss,increased thermogenesis and improved glucose metabolism,but overexpression of FSTL1 in AgRP-specific FoxO1 mice did not cause the above metabolic changes.Conclusion:Overexpression of FSTL1 in AgRP neurons regulates food intake and energy metabolism through FoxO1.PART THREE FSTL1 regulates food intake and energy metabolism through the AKT/FoxO1/AgRP pathwayObjective:To explore the mechanism of FSTL1 regulating food intake and energy metabolism in vitro.Methods:Overexpression of FSTL1 by lentivirus in SH-SY5Y and GT1-7 cell lines,or knockdown of FSTL1 by lentivirus in GT1-7 cells,combined with glucosamine and insulin treatment to explore the mechanism of FSTL1 regulation of metabolism;The cytoplasmic/nuclear proteins were extracted or cells were stained by immunofluorescence to observe the effect of FSTL1 overexpression/knockdown on the distribution of FoxO1.In addition,it was further verified whether FSTL1 functions by activating AKT through PI3K inhibitor intervention.The interaction of FSTL1 and AKT was verified by confocal colocalization and co-immunoprecipitation.Results:Overexpression of FSTL1 in SH-SY5Y and GT1-7 cells resulted in increased expression of p-AKT and p-FoxO1,which promoted FoxO1 nuclear export,and PI3K inhibitor could block the above effects.GT1-7 cells showed reduced expression of p-AKT,p-FoxO1,and the proportion of FoxO1 nuclear export after knockdown of FSTL1.Confocal colocalization and co-immunoprecipitation results suggested an interaction between FSTL1 and AKTConclusion:FSTL1 can affect the AKT-FoxO1-AgRP signaling pathway by interacting with AKT.