Role Of SREBP-1c In The Activation Of Islet Stellate Cells And Its Mediating Changes Of Islet Function

Aim: In our previous study,we successfully isolated islet stellate cell(ISC)from mouse,rat and human islets using the traditional tissue outgrowth method.We also explored the potential role of ISC in islet fibrosis during the process of type 2 diabetes mellitus.However,our previous study did not confirm the exact location and content of ISC in islets.Besides,ISC isolated by the outgrowth method might be affected by different kinds of hormones secreted by the islets.On this regard,we may not be able to get purified quiescent ISC(using the previous method),let alone comparing the differential gene expression with activated ISC.In the process of ISC activation,the loss of vitamin A containing lipid droplets is considered as a very important morphologic change.As a key regulator of lipogenesis,sterol regulatory element binding protein 1c(SREBP-1c)can effectively inhibit ISC’s homologous cell—hepatic stellate cell’s activation.Yet the role of SREBP-1c on ISC activation and ISC mediated islet function change has not been studied so far.Given these,this study aimed to(1)confirm the location and content of ISC in islets;(2)develop a method to isolate purified quiescent ISC,compare the biological characteristics of ISC isolated by the newly developed methods with the traditional outgrowth method;(3)elucidate the role of SREBP-1c on ISC biological characteristics;and(4)explore the functional difference among islets coated with different state of ISC.Methods: 1.Immunofluorescence and transmission electron microscope were used to observe the location of ISC in mouse,rat and human islets.They were also applied to explore the co-location of ISC with islet endothelial cell(IEC)and to study the ISC change in the islets cultured in vitro for different days.2.Quiescent ISC was isolated by the density gradient centrifugation and compared with traditional outgrowth method.Phase-contrast microscope imaging,oil red O staining and auto-fluorescence during UV light excitation were utilized to identify the quiescent ISC.Immunofluorescence staining and western blot were employed to detect the expression of α-SMA,desmin,vimentin,GFAP,Col-Ⅰ,Col-Ⅲ and fibronectin for the identification of activated ISC and also comparison for activation and ECM production with ISC isolated by the outgrowth method.Oil red O staining was applied to compare the lipid content change of ISC isolated by different method.Wound healing and transwell migration assay were employed for the migration characteristics.Cell viability was assessed by CCK8 assay.3.Newly isolated quiescent ISC,ISC separately cultured in vitro for 3d and7 d were collected for total RNA isolation.ISC activation,ECM synthesis,lipogenesis and inflammation related gene in the above 3 groups were compared using real-time PCR.The relation of SREBP-1c with α-SMA in ISC cultured in vitro for 3 days was explored by immunofluorescence.SREBP-1c overexpression in ISC was accomplished by lentivirus.Oil red O was applied to observe the effect of SREBP-1c overexpression on ISC lipid content change.ISC activation and ECM synthesis after SREBP-1c overexpression were evaluated by real-time PCR and western blot.Ki-67 fluorescence intensity and p-ERK content were used to assess the proliferation rate of ISC overexpressed SREBP-1c.Migration of transfected ISC was evaluated by wound healing assay and p-AKT content.4.Islets were separately coated by SREBP-1c transfected ISC and control virus transfected ISC,islets without coating were served as the control(con: islets without coating,co-ISC-SRE: islets coated with SREBP-1c overexpression ISC;co-ISC-con: islets coated with random sequence overexpressed ISC).Islets in different groups were separately cultured in medium containing different concentrations of glucose(5.5mM and 20 mM)for 24 h.Then the islets were incubated in KRB buffer containing 2mM glucose,20 mM glucose and 30 mM KCl,with the supernatant collected for the assessment of insulin and glucagon in order to evaluate the effect of ISC coating on islet function.Results: 1.Desmin positive ISC existed in mouse,rat and human islets.In mouse and rat,ISC was randomly distributed in the islets;while in human islets,it was majorly located at the boundary of islets.Co-localization analysis and transmission electron microscope revealed that ISC was mainly co-localized with islet endothelial cell(Nearly 50% desmin positive ISC co-localized with CD31 positive IEC).When extending the in vitro culturing time,there was a tremendous loss of IEC,whereas the ISC and ISC secreted ECM components were increased over time.Although the content of ISC in islets of high-fat diet mouse was not significantly higher than the normal control,ISC in islets of obesity mouse was more prone to aggregate.2.Density gradient centrifugation can successfully isolate lipid droplets containing quiescent ISC,and the isolated ISC which can be activated during the in vitro culture.ISC isolated by density gradient centrifugation method had a higher oil red positive cell percent during the in vitro culture time of 24 h,48h and 96 h compared with the ISC isolated by traditional outgrowth method.In addition,ISC isolated by this new method was less activated than the ISC isolated by the outgrowth method.The proliferation and migration rate in the ISC isolated by the new method were also lower than the control ISC.3.As with the in vitro culturing time extending,the mRNA related to ISC activation(α-SMA),the ECM component(Col-Ⅰ,Col-Ⅲand FN)as well as the inflammatory factor(IL-1α,CCL2 and CCL7)were significantly increased,while the mRNA related to lipogenesis(PPARγ and SREBP-1c)were decreased.Immunofluorescence staining of ISC cultured for 3d after isolation further proved the opposite tendency of SREBP-1c and α-SMA expression.Gain of function experiment revealed that ISC overexpressed with SREBP-1c had a lower activation rate and a reduced ECM synthesis.In ISC overexpressed with SREBP-1c lipid droplets re-formation can be observed by phase contrast microscopy and oil red O staining.But SREBP-1c overexpression had no effect on ISC proliferation and migration.4.Islets coated by different states of ISC or not were incubated in normal glucose concentration for 24 h then the islet function assessment result showed that the high glucose or potassium stimulated insulin release was the lowest in the co-ISC-con group.Whereas the con group had a highest value in all three groups and had a statistical significance compared with the co-ISC-con group.Besides,the co-ISC-con group had a highest reduction in glucagon release ratio.After the different groups of islets incubated in a high glucose concentration for 24 h,the islets in the co-ISC-con group had the highest insulin stimulation index and also the lowest glucagon release,which was significantly lower than the co-ISC-SRE group but similar to the situation in the con group.Conclusion: 1.ISC existed in islets and nearly half of ISC co-localized with islet endothelial cell.In the hight-fat diet mouse islets,ISC was more prone to aggregate.2.Quiescent ISC can be isolated by the density gradient centrifugation method,and ISC isolated by this method was lower in activation,collagen synthesis,proliferation and migration than the ISC isolated by the outgrowth method.3.SREBP-1c overexpression can inhibit ISC activation and ECM synthesis.Besides,it can also promote the reformation of lipid droplets,but SREBP-1c overexpression had no effect on ISC proliferation and migration.4.When the islet environmental glucose level is normal,ISC,no matter whether activated or not,had barely no effect on islet function.However,when the glucose concentration arises,activated ISC may drive islet β cell to secrete more insulin,and also change islet glucagon secretion.Yet,quiescent ISC may,to some extent,help to counteract this overcompensation.