| Clinically,chronic diseases such as hyperglycemia,hyperlipidemia,and hypertension tend to appear concomitantly in middle-aged and elderly people.The concept of metabolic syndrome puts these chronic and metabolic symptoms as an integrated group of metabolic syndromes.These diseases are independent but closely related to each other in the meantime.Inasmuch,the adult treatment group guidelines of the American National Cholesterol Education Program clearly refer to diabetes and coronary heart disease as equal-risk diseases.In-depth exploration of the mechanism of crosstalk between various metabolic disorders in the metabolic syndrome has a very realistic and important significance for the prevention and treatment of the metabolic syndrome integrally.Proprotein convertase subtilisin kexin type 9(PCSK9)is a key gene that regulates lipid metabolism discovered in 2003.In recent years,increasing evidences suggested that PCSK9 is related to insulin production and secretion,and the relevance between PCSK9 and diabetogenesis has received great attention.In the previous study,we found that in DMEM containing different glucose concentrations,the expression levels of PCSK9 in HepG2 cells were significantly different,which suggested that PCSK9 may be a key gene linking glucose metabolism and lipid metabolism.Our work aims to explore whether the increase in PCSK9 expression by alternation of glucose concentration is caused by the effect of glucose on the glycosylation level and stability of sterol regulatory element binding protein cleavage activated protein(SCAP),in an attempt to provide new insights to association between metabolism of important energy substances involving carbohydrates and lipids.Objective To investigate whether glucose can increase the stability of SCAP by promoting SCAP N-glycosylation,thereby up-regulating the level of PCSK9 and participating in affecting cellular cholesterol metabolism.Methods(1)HepG2 cells were cultured in 25 T culture flasks with complete medium containing low-concentration glucose(containing5m M D-glucose)DMEM+10% fetal bovine serum until the confluence reached 70%～80%,and they were divided into 3 groups:Low-concentration glucose group(5m M D-glucose);medium-concentration glucose group(15m M D-glucose);IX high-concentration glucose group(25m M D-glucose).After 24 hours of culture,the cells were fully lysed with RIPA and total protein was extracted.Western blot was used to detect the expression level of PCSK9,low-density lipoprotein receptor,sterol regulatory element binding factor2(SREBP2),and SCAP.(2)Inoculate HepG2 cells in 12-well plates,change the serum-free medium after complete adherence and culture for12 hours and then process them in batches.The experimental groupings are the same as before.After 24 hours of incubation,cells are fixed with3% paraformaldehyde solution and stained with Oil Red O and hematoxylin,and observe the staining results under an inverted microscope.(3)HepG2 cells were cultured in 25 T culture flasks with complete medium containing low-concentration glucose(containing5m M D-glucose)DMEM+10% fetal bovine serum until the confluency reached 70%～80%,and the following groupings were performed:High-concentration glucose group(25m M D-glucose);low-concentration tunicamycin treatment group(25m M D-glucose + 0.4μg/m L tunicamycin);medium-concentration tunicamycin treatment group(25m M D-glucose + 0.8μg/m L Tunicamycin);high-concentration tunicamycin treatment group(25m M D-glucose + 1.2μg/m L tunicamycin).After 48 hours of incubation,the cells were fully lysed with RIPA,total protein was extracted and Western blot was used to detect the expression level of SCAP.(4)HepG2 cells were cultured in 25 T culture flasks with complete medium containing low-concentration glucose(containing 5m M D-glucose)DMEM+10% fetal bovine serum until the confluence reached 70%～80%,and then grouped as follows:Low-concentration glucose group(5m M D-glucose);medium-concentration glucose group(15m M D-glucose);high-concentration glucose group(25m M D-glucose);tunicamycin treatment group(25m M D-glucose + 0.8μg/m L tunicamycin).After 48 hours of culture,the cells were fully lysed with RIPA,total protein was extracted and Western blot was used to detect the expression level of PCSK9,low-density lipoprotein receptor(LDLR),SREBP2,and SCAP.(5)Inoculate HepG2 cells in 12-well plates,and group them as follows:low-concentration glucose group(5m M D-glucose);medium-concentration glucose group(15m M D-glucose);high-concentration glucose group(25m M D-glucose);Tunicamycin treatment group(25m M D-glucose + 0.8μg/m L tunicamycin),cultured for 48 hours,discarded the medium,and replaced with a serum-free medium containing 20μg/m L Di I-LDL with red fluorescent label After incubating for 4 hours,wash 3 times with 1×phosphate buffer(1×PBS)and fix with 3% formaldehyde PBS solution.Perform nuclear staining with DAPI stain for 10 minutes.Observe the red fluorescence and blue fluorescence under an inverted fluorescence microscope.Results As the glucose concentration increased,the expression of PCSK9 XI in HepG2 cells showed a significant increasing trend and it is the same with the expression of LDLR,SREBP2,SCAP increased,and the concentration of the diffuse shallow band above the main band of SCAP gradually increased;With the increase of glucose concentration,the area of HepG2 cells stained by Oil Red O stain also showed a gradually increasing trend;under different concentrations of tunicamycin treatment(0μg/m L,0.4μg/m L,0.8μg/m L,1.2μg/m L),SCAP expression and the concentration of the diffuse shallow band above the main band was significantly higher in 0.8μg/m L tunicamycin treatment group and1.2μg/m L tunicamycin treatment group and lower in glucose control group and 0.4μg/m L treatment group;the 0.8μg/m L tunicamycin treatment group SCAP expression and glycosylation level were significantly lower than the high glucose concentration control group,0.8μg/m L The expression levels of SREBP2,PCSK9 and their expression levels in the m L tunicamycin treatment group were also lower than those in the high glucose concentration control group;while the expression of LDLR in 0.8 μg/m L tunicamycin treatment group was lower than that of the high glucose concentration control group;the Di I-LDL red-stained area in the 0.8μg/m L tunicamycin treatment group had lower fluorescence intensity than the high glucose concentration control group.Conclusions 1.Glucose promotes SCAP glycosylation,up-regulates the expression of SREBP2,and then up-regulates the expression of PCSK9.Tunicamycin can inhibit the above effects by inhibiting SCAP glycosylation;2.The ability of glucose to regulate the lipid metabolism of HepG2 cells through the above pathways are counteracted probably because SREBP2 can simultaneously up-regulate the expression of LDLR. |
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