Mechanism Of Hypothalamic Energy Regulation Imbalance Caused By Seipin Deficiency

Objective:1.This study used a mouse obesity model induced by a high fat diet to explore the role of the lipid metabolism molecule Seipin in the hypothalamic energy metabolism imbalance.2.Using Seipin knockout mice to observe the effect of systemic metabolic imbalance on hypothalamic energy metabolism related neurons,reveal the regulatory mechanism of energy metabolism centers,and seek effective regulatory means to provide theoretical basis for the treatment of metabolic diseases.Method:1.Six week old male C57BL/6 mice of wild type（WT）were selected and divided into high fat diet group and normal diet group.They were fed for 8 weeks to establish a mouse obesity model induced by high fat diet.The body weight,random blood glucose,and blood lipids of each group of mice were recorded.The oxygen consumption（VO₂）,carbon dioxide production（VCO₂）,respiratory exchange rate（RER）,food intake,and caloric production of the mice were measured through metabolic cages.2.RT-PCR was used to detect the mRNA expression levels of AGRP,POMC,and Seipin in the hypothalamus of mice in each group;Western blot was used to detect the protein expression changes of agouti related neuropeptide（AGRP）,pro-opiomelanocortin（POMC）,and Seipin in the hypothalamus of mice in each group;Immunofluorescence staining was used to detect the changes in fluorescence intensity of AGRP,POMC,and Seipin in the hypothalamus of mice in each group.3.Using RT-PCR to detect mRNA level of the inflammatory related factor interleukin-1β（IL-1β）、tumor necrosis factorα（TNFα）、interleukin-6（IL-6）in the hypothalamus of mice in each group;The expression of glial fibrillary acidic protein（GFAP）,a marker of mature astrocytes,in the hypothalamus of mice in each group was observed by immunofluorescence staining.4.To construct a systemic knockout Seipin gene mouse,select 3-month-old WT and Seipin KO mice,record the weight of each group of mice,and measure their random blood glucose.The oxygen consumption（VO₂）,carbon dioxide production（VCO₂）,respiratory exchange rate（RER）,food intake,and heat production of the mice were measured using a metabolic cage.5.Three month old WT and Seipin-KO mice were selected and the mRNA levels of AGRP and POMC in the hypothalamus of each group of mice were detected by RT-PCR;Western blot was used to detect the protein expression changes of AGRP and POMC in the hypothalamus of mice in each group;Immunofluorescence staining was used to detect the levels of AGRP and POMC in the hypothalamus of mice in each group.6.Using RT-PCR to detect mRNA level of the inflammatory related factor IL-1β、TNFα、IL-6 in the hypothalamus of mice in each group;Immunofluorescence staining was used to detect the level of GFAP in the hypothalamus of mice in each group.7.Three month old WT,Seipin-KO,and Seipin-KO mice supplemented with rosiglitazone（ROSI）were selected to detect the level of GFAP in the hypothalamus of each group of mice using immunofluorescence staining;Using RT-PCR to detect mRNA level of the inflammatory related factor IL-1β、TNFα、IL-6 in the hypothalamus of mice in each group.8.Three month old WT,Seipin-KO,and Seipin-KO+ROSI mice were selected and Western blot was used to detect the protein expression of AGRP and POMC in the hypothalamus of each group of mice;RT-PCR was used to detect the mRNA levels of AGRP and POMC in the hypothalamus of mice in each group;Immunofluorescence staining was used to detect the levels of AGRP and POMC in the hypothalamus of mice in each group.Result:1.To detect the basic metabolism of mice fed a high fat diet（HFD）group and a normal fat diet（NFD）group for 8 weeks.The results showed that compared with the normal diet group,the body weight,blood sugar,and blood lipid levels of mice in the high fat diet group were significantly increased（P<0.05）.The metabolic cage test results showed that the mice in the high fat diet group had lower oxygen consumption（VO₂）,carbon dioxide production（VCO₂）,respiratory exchange rate（RER）,and calorie production than those in the normal diet group,while their calorie intake was higher than those in the normal diet group（P<0.05）.The above results indicate that mice on a high fat diet exhibit a positive energy balance.2.To detect the pathological characteristics of the hypothalamus of mice fed a high fat diet group and a normal diet group for 8 weeks.Western blot results showed that the protein level of POMC in the high fat diet group decreased,while the protein level of AGRP increased,while the protein level of Seipin decreased.The results of fluorescence quantitative PCR showed that the mRNA level of POMC in mice fed high fat diet decreased,while the mRNA level of AGRP increased,the mRNA level of Seipin decreased,and the mRNA level of the proinflammatory cytokine IL-1β、TNFα、IL-6 in mice fed high fat diet increased.Immunofluorescence staining results showed that in the hypothalamus of high-fat diet mice,the expression of appetite inhibiting POMC decreased,appetite promoting AGRP increased,Seipin decreased,and astrocyte marker GFAP increased.The above results show that changes in the expression of energy metabolism related neurons POMC and AGRP occur in the hypothalamus of mice fed a high fat diet,while decreased expression of Seipin is accompanied by inflammatory reactions.3.To detect the basic metabolism of WT and Seipin-KO mice aged 3 months.The results showed that compared with WT group,the body weight and blood glucose levels of3-month-old Seipin-KO mice were significantly increased（P<0.05）.The metabolic cage test results showed that the oxygen consumption（VO₂）,carbon dioxide production（VCO₂）,respiratory exchange rate（RER）,and caloric production of Seipin-KO mice were lower than those of WT mice,while the caloric intake was higher than that of WT mice（P<0.05）.All the above results suggest that there is a positive energy balance in 3-month-old Seipin-KO mice.4.Detect the pathological characteristics of the hypothalamus in WT and Seipin-KO mice aged 3 months.Western blot and RT-PCR results showed that the protein level of POMC in the high fat diet group decreased,while the protein and mRNA levels of AGRP increased,and the mRNA level of the proinflammatory cytokine IL-1β、TNFα、IL-6increased.The results of immunofluorescence staining showed that in the hypothalamus of mice fed a high fat diet,the expression of POMC decreased,the expression of AGRP increased,and the expression of GFAP increased.The above results suggest that in the hypothalamus of Seipin-KO mice,the expression of energy metabolism related neurons POMC and AGRP is altered,astrocytes are activated,and proinflammatory cytokines are increased.5.Detect the pathological characteristics of the hypothalamus in Seipin-KO mice after supplementation with rosiglitazone.Western blot and RT-PCR results showed that compared with mice in the Seipin-KO group,the protein and mRNA levels of POMC in the Seipin-KO+ROSI group increased,the protein and mRNA levels of AGRP decreased,and the mRNA level of the proinflammatory cytokine IL-1β、TNFα、IL-6 decreased;Immunofluorescence staining showed that the fluorescence intensity of astrocyte marker GFAP decreased.The above results showed that after ROSI supplementation,the expression of POMC and AGRP in the hypothalamus of Seipin-KO mice recovered,while the activity of astrocytes and the expression of pro-inflammatory factors decreased.6.To detect the basic metabolism of Seipin-KO mice after supplementation with rosiglitazone.The results showed that compared with the Seipin-KO group,the weight and blood glucose of the Seipin-KO+ROSI group mice decreased significantly（P<0.05）.The food intake of mice in the Seipin KO+ROSI group was lower than that of Seipin KO（P<0.05）.The above results indicate that the positive energy balance of Seipin-KO+ROSI mice is significantly improved.Conclusion:1.The expression of appetite inhibiting POMC in the hypothalamus of mice fed a high fat diet decreased,the expression of appetite promoting AGRP increased,and the expression of lipid metabolism molecule Seipin decreased,accompanied by inflammatory reactions.At the same time,the mice developed energy metabolism disorders.2.The expression of POMC in the hypothalamus of Seipin knockout mice decreased,while the expression of AGRP increased,accompanied by inflammatory reactions,while the mice developed energy metabolism disorders.3.Supplementing rosiglitazone,agonist of Seipin downstream molecule PPARγ,to Seipin knockout mice restored the expression of AGRP and POMC neurons and inhibited inflammatory reactions in the hypothalamus.Blood glucose levels,food intake,and body weight in mice returned to normal,improving energy metabolism disorders.