The Effect And Mechanism Of Hnscr On Glycolipid Metabolism Of The Body

Objuctive:1.To study the effect of Hnscr on insulin resistance and hepatic lipid deposition in HFD-induced obese mice.2.To investigate the effect of Hnscr expression levels in hypothalamic neural stem cells on insulin resistance and hepatic lipid deposition in mice under HFD-induced.3.To explore Hnscr regulates glycolipid metabolism through activation of NF-κB inflammatory pathway in ht NSCs.Method:1.We first found that the high-fat diet induction decreased Hnscr expression in the hypothalamus of mice in the adult male C57BL/6J wild-type mice of the same age after three months of normal chow diet(NCD)and high-fat diet(HFD)feeding.Subsequent extraction of primary hepatocytes from 6-month-old male wild-type C57BL/6J mice and Hnscr null C57BL/6J mice revealed that insulin resistance was increased in primary hepatocytes from Hnscr null mice.We also constructed NCD and HFD models in wild-type and Hnscr null mice,respectively.We further investigated the effects of Hnscr null on insulin resistance and hepatic lipid deposition in mice under HFD induction by performing the intraperitoneal glucose tolerance test(ip GTT)and intraperitoneal insulin tolerance test(ip ITT),measuring the levels of serum and liver triglyceride(TG),total cholesterol(TC),free fatty acid(FFA),serum alanine transaminase(ALT)and aspartate transaminase(AST)in each group of mice,and observing hematoxylin and eosin(H&E)staining and Oil Red staining of liver tissues.2.The groups of hypothalamic-specific Hnscr knockdown mice(AAV-Sox2-shHnscr group),hypothalamic-specific Hnscr gene overexpression mice(AAV-Sox2-promoter Hnscr group)and their control group(AAV-Sox2-Scramble)were constructed by injecting adeno-associated viruses(AAVs)into the mediobasal hypothalamic(MBH)locus of mice bilaterally,and further constructing a type 2diabetes model in the above mice to observe the effects of Hnscr expression levels in the hypothalamic neural stem cells(ht NSCs)on insulin resistance and liver lipid deposition in mice.3.Six-month-old male C57BL/6J Hnscr null and wild-type mice were obtained and executed after 1% pentobarbital sodium anesthesia.Then hypothalamus were taken to observe the effect of Hnscr knockout on the level of hypothalamic inflammation and the number of neural stem cells(NSCs).The effect of Hnscr deletion on inflammation levels was further observed in primary NSCs.In addition,we infected a highly transgenic strain of human embryonic kidney 293T(HEK293T,293T)with adenovirus(Adv)inserted with a temperature-sensitive gene of SV40 T-antigen to observe the effect of Hnscr overexpression on the level of cellular inflammation.Result:1.Hnscr expression was decreased in the hypothalamus of HFD male C57BL/6J wild-type mice compared to the NCD.In addition,insulin sensitivity was significantly reduced in the liver of Hnscr null mice.Further in the diet-induced models,a significant worsening of glucose tolerance and clearance could be observed in the NCD-Hnscr null compared to the NCD-WT.And in the absence of significant differences in body weight between the two groups,the TG levels of liver were significantly higher in the NCD-Hnscr null.These phenomena were more clearly observed in the HFD groups.Compared to the HFD-WT group,the HFD-Hnscr null group showed a significant deterioration in glucose tolerance and clearance,increased liver weight/body weight(LW/BW)ratio,increased liver TG and TC levels,and increased serum TG,TC,and ALT levels.2.The glucose tolerance and clearance worsened in the AAV-Sox2-shHnscr group compared with the control group;in contrast,the glucose tolerance and clearance improved in the AAV-Sox2-promoter Hnscr group.In addition,although the AAV-Sox2-promoter Hnscr group weighed slightly more than the control group,the LW/BW ratio was smaller and the weight of brown adipose tissue(BAT)was significantly heavier than that of the control group.H&E staining shows that Hnscr knockdown/overexpression in ht NSCs can exacerbate/reduce hepatic steatosis.3.In vivo experiments showed that the hypothalamic NF-κB phosphorylation level was increased in the Hnscr null mice compared with the wild-type mice;and the tissue immunofluorescence results indicated that the TNFα levels were increased in the Hnscr null mice,while the number of the neural stem cells was reduced.In vitro experiments showed that the inflammatory factors expression was increased in the ht NSCs of the Hnscr null mice.Furthermore,the inflammatory factors expression was decreased in the Hnscr overexpression 293 T cells.Although the WB assays revealed no significant changes in the NF-κB phosphorylation levels,the levels of the IKKβ phosphorylation in the Hnscr overexpression 293 T cells increased.Cellular immunofluorescence further observed that Hnscr overexpression downregulated the level of the NF-κB phosphorylation.Conclusion:Hnscr null exacerbated HFD-induced insulin resistance and liver lipid deposition in C57BL/6J mice,and further found that the expression level of Hnscr in the ht NSCs could affect HFD-induced insulin resistance.Through literature review,combined with existing studies that the pro-inflammatory pathway of IKKβ and downstream NF-κB mediated HFD-induced hypothalamic inflammation causing metabolic syndrome,we further verified in vivo and in vitro experiments that Hnscr regulation of glycolipid metabolism is associated with the NF-κB inflammatory pathway.5 Figures,22 Tables,and 50 references...