The Effect Of Hypothalamus BCL6 On Peripheral Glucose And Lipid Metabolism And Energy Balance And Mechanism

Objective: To investigate the effects of BCL6 in mediobasal hypothalamus(MBH)on glucose and lipid metabolism and energy balance and its possible mechanismMethods: Eight-week-old C57BL/6J mice were divided into 4 groups with 6 mice in each group.They were fed normal diet(ND)and high-fat diet(HFD)for 12 weeks,respectively.Then,C57 mice in 4 groups underwent stereotactic surgery,and were injected with adenovirus Ad-GFP or Ad-BCL6 in the third ventricle of the hypothalamus.After two days,the body weight,feeding,rectal temperature,oxygen consumption and RER of the mice were measured.Insulin tolerance test(ITT)and glucose tolerance test(GTT)were measured simultaneous ly.The morphological changes of liver,WAT and BAT were observed by hematoxylin-eosin(HE)staining.The liver of mice was stained with oil red O to observe the lipid deposition.The expression of uncoupled protein 1(UCP1)was detected by western-blot and immunohistochemical staining.After leptin stimulation,the hypothalamus of mice were taken and the expressions of p-m TOR/m TOR,p-FOXO1/FOXO1,p-ERK/ERK,p-STAT3/STAT3 and POMC were detected by western-blot.The expression of p-STAT3 and POMC in hypothalamus was detected by immunofluorescence staining.Ad-BCL6 and sh RNA-BCL6 plasmid were transfected into N2 A cells,respectively.N2 A cells were modeled with glucosamine.After leptin stimulation,the expressions of p-m TOR/m TOR,p-FOXO1/FOXO1,p-ERK/ERK,p-STAT3/STAT3 and POMC were detected by western-blot.Results: After the overexpression of BCL6 in the MBH region of hypothalamus in C57 mice,mice on the high-fat diet showed weight gain and increased food intake.The oxygen consumption,RER and rectal temperature of mice decreased.Insulin sensitivity was reduced and insulin resistance occured.After hypothalamic overexpression of BCL6,hepatic steatosis was induced with high fat diet and lipid deposition in liver was increased.The volume of WAT was increased.The cavitation of BAT was obvious,and the expression of UCP1 was decreased.When BCL6 was overexpressed in the MBH region of hypothalamus,the expression level of p-STAT3 and POMC in the ARC region were decreased under the stimulation of leptin.However,under normal diet,there were no significant differences in body weight and food intake.Under high fat diet,the expression of p-m TOR/m TOR,p-ERK/ERK and p-FOXO1/FOXO1 in the hypothalamus of mice overexpressing BCL6 in the MBH region of the hypothalamus did not change,but the expression of p-STAT3 and POMC were decreased.Under normal diet,the protein expressions of p-m TOR/m TOR,p-ERK/ERK,p-FOXO1/FOXO1,p-STAT3 and POMC in BCL6-overexpressed mice did not change.After N2 A cells were transfected with Ad-BCL6,the protein expressions of p-m TOR/m TOR,p-ERK/ERK and p-Foxo1/Foxo1 were not changed,while p-STAT3 and POMC were decreased.After sh RNA-BCL6 plasmid transfection,the expression of p-m TOR/m TOR,p-ERK/ERK and p-Foxo1/Foxo1 did not change,while p-STAT3 and POMC were increased.Conclusion: Overexpression of BCL6 in the MBH region of hypothalamus can decrease the glucose metabolism and energy balance.The possible mechanism is that BCL6 inhib ites the STAT3-POMC signaling pathway in hypothalamus.Objective: To further verify the regulation of hypothalamic BCL6 in glucose and lipid metabolism and energy balance,and explore whether BCL6 plays a role by regulating the expression of STAT3-POMC.Methods: Obrb-Cre and POMC-Cre mice were selected to use AAV-DIO-BCL6 virus expressed by Cre enzyme.The adeno-associated virus AAV-DIO-GFP or AAV-DIO-BCL6 were injected into the hypothalamic nucleus,and they were given a normal diet(ND)and a high fat diet for 12 weeks.The weight,food intake and rectal temperature of mice were measured.Simultaneously glucose tolerance test(GTT)and insulin tolerance test(ITT)were conducted.The liver,WAT and BAT of mice were removed.The morphological changes of liver,WAT and BAT of mice were observed with hematoxylin-eosin(HE)staining.The liver of mice was stained with Oil Red O to observe the fat deposits.Western-blot and immunohistochemical staining were performed on BAT to detect the expression of uncoupling protein 1(UCP1).Immunofluorescence staining was used to detect the expression changes of factors such as p-STAT3 and POMC in the arcuate nucleus(ARC)area of the hypothalamus.Results: After BCL6 was overexpressed in POMC neurons in the ARC area of the hypothalamus,the food intake and the weight of mice were increased.The rectal temperature of mice was decreased.The insulin tolerance and the insulin sens itivity were decreased.After overexpression of BCL6,fatty degeneration of the liver was occurred under high-fat diet and lipid deposition increased.The volume of WAT was increased.The cavitation of BAT was obvious,and the expression of UCP1 was decreased.Under the stimulation of leptin,the expression level of p-STAT3 and POMC in the ARC area were reduced.Conclusion: The investigation elucidates that overexpression of BCL6 in POMC neurons in the ARC area of the hypothalamus reduced peripheral energy metabolism and energy balance in mice.The possible mechanism is that BCL6 inhibits the signaling pathway of hypothalamic STAT3-POMC.Objective: To explore how hypothalamic BCL6 regulates glucose and lipid metabolism and energy balance by inhibiting STAT3-POMC.Methods: The UCSC database was used to check the 2000 bp sequence upstream of the STAT3 promoter,and the Jaspar database was used to predict the possible binding sites of BCL6 in the STAT3 promoter region,and point mutations on the predicted possible binding sites of the STAT3 promoter region were performed.2000 bp full-length luciferase reporter plasmid upstream of the STAT3 promoter and a mutant plasmid with the predicted binding site were constructed.The full-length luciferase reporter plasmid and mutant plasmid were co-transfected with the BCL6 overexpression plasmid into HEK293 T cells for luciferase reporter experiment.At the same time,the corresponding primers of the binding site predicted by the STAT3 promoter were constructed,and Ad-BCL6 was transfected into the model N2 A cells,and Chromatin immunoprecipitation(CHIP)was performed 48 hours later.Results: The Jaspar database predicted that BCL6 has binding sites at-492~-442 and-952~-965 upstream of the STAT3 promoter.Luciferase experiments showed that BCL6 has an inhibitory effect on the full length of the STAT3 promoter and the-442~-492 mutant promoter activity of STAT3.BCL6 has no inhibitory effect on the mutation activity of STAT3-952~-965 mutant promoter.CHIP assay detected that the primers at binding sites-952 ~-965 of STAT3 promoter could specifically amplify the fragment,but the primers at binding sites-442 ~-492 of STAT3 promoter could not amplify the fragment.Electrophoresis of the amplified products also s howed that binding sites of STAT3 promoter-952 ~-965 were present,while binding sites of STAT3 promoter-442 ~-492 were not present.Conclusion: BCL6 in the hypothalamus regulates glucose and lipid metabolism and energy balance by binding to STAT3 promoter-952~-965 and inhibiting STAT3 transcription,resulting in reduced expression of POMC.