Study On Differentiation Direction Of Islet Stellate Cells And Its Decisive Factors

Part 1High-fat diet promotes differentiation of islet stellate cell into fibroblast-like cells Aims: Recent evidences have shown lipid metabolism disorder is closely associated with islet dysfunction in type 2 diabetes.Islet fibrosis caused by islet stellate cells(ISCs)differentiating into fibroblasts is one of the important pathophysiological mechanisms for islet failure.However,whether ISCs is related with islet dysfunction arising from obesity is not fully understood.This study intended to investigate the effect of high-fat on glucose homeostasis through regulating ISCs biological phenotypes on islet function in vivo and in vitro.Methods: Obese rat models were established by high-fat diet(HFD)for 4,8,12 and20 weeks,respectively.The body weight,visceral fat content,blood triglyceride and islet free fatty acid(FFA)of each group were dynamically measured.Glucose metabolism phenotypes were assessed by glucose/insulin tolerance tests.Pancreas sections were stained with H&E,masson,and immunohistochemical staining to evaluate islet morphology,islet fibrosis degree,and the expression of fibroblast-like ISCs markers(α-smooth muscle actin,α-SMA).After cultured ISCs were treated with300 μM palmitic acid(PA)for 48 h,72 h,and 96 h,expression of ISCs fibrotic differentiation markers(α-SMA,Collagen I(Col I),and Fibronectin(FN))at m RNA and protein levels were detected using quantitative PCR and western blot.Islet and fibroblast-like ISCs were co-cultured for different time periods,the effects of fibroblast-like ISCs on islet function and islet viability were examined by enzyme-linked immunosorbent and cell viability assay.Results: After 4-week HFD fed,body weight,visceral fat content,serum triglyceride,and islet FFA levels increased with the extension of feeding duration.HFD 20-week rats showed increased insulin resistance degree,glucose intolerance,disordered arrangement of cells,light-stained insulin area,reduced α cells proportion,extensive fibrotic areas,and increased percentage of α-SMA positive ISCs in islets.In vitro,PA treatment increased gene and protein expression of α-SMA,Col I and FN in ISCs.After islets co-cultured with fibrotic ISCs for 48 h,the supernatant insulin level was significantly higher than that in islets alone,while peak ratio of glucose-stimulated insulin secretion and islet viability exhibited the opposite tendency.Conclusions: HFD induced increased α-SMA expression of ISCs in islets,which lead to islet fibrosis;PA intervention promoted ISCs to differentiate into fibroblast-like cells,resulting in islet dysfunction and decreased islet vitality.Our study provides preliminary experimental data for the role of ISCs in the progression of islet fibrosis caused by high fat,suggesting differentiation of ISCs into fibroblast-like cells is a new underlying mechanism of islet impairment in T2 DM.Part 2Vitamin A inhibits differentiation of islet stellate cell into fibroblast-like cells Aims: Recent studies showed vitamin A(VA)and its derivatives play an essential role in insulin sensitivity and glucose homeostasis,contributing to the pathological process of diabetes.Islet stellate cells(ISCs)are VA-storing cells in islets.However,whether ISCs are involved in VA-related changes of islet function remains unclear.This study intends to explore whether and how VA affects glucose homeostasis by regulating biological phenotype of ISCs in vivo and in vitro.Methods: Six-week-old male C57BL/6 mice were randomly fed a VA-sufficient diet or a VA-deficient(VAD)diet for 6-and 12-weeks.Subsequently,a subclass of the VAD diet-fed mice for 12 weeks was switched to a VA-deficient rescued(VADR)diet for an additional 8 weeks.Body weight and random blood glucose levels were measured weekly.Serum and tissue VA levels were determined by high-performance liquid chromatography.Glucose metabolic phenotype,islet morphology,and ISCs fibrotic differentiation markers(α-SMA、Col I and FN)were assessed by glucose tolerance tests,immunofluorescence and immunohistochemistry staining.In vitro,ISCs fibrotic differentiation degree with retinol intervention in different points was evaluated by quantitative PCR and western blot.Changes in ISCs phenotype and its effect on islets were assessed by lentiviral transduction and enzyme-linked immunosorbent assays in a co-culture system.Results: VAD mice displayed reduction of tissue VA levels and gene expression of VA signaling factors,including cellular retinol binding protein 1(CRBP1).VAD mice showed irregular shaped islets,glucose intolerance,loss of β cell mass,and upregulated expression of α-SMA,Col I,and FN in islets.Reintroduction of dietary VA restored pancreatic VA levels and endocrine hormone profiles,and decreased the proportion of α-SMA positive ISCs,as well as Col I and FN.In vitro,incubation with retinol decreased the expression of ISCs fibrotic differentiation markers(α-SMA,ColI,and FN)and increased gene expression of VA signaling factors.ISCs-interfering CRBP1 had the classical polygonal appearance similar to the one of quiescent ISCs,reduced protein expression of α-SMA,FN,and Col I,and lower proliferation and migration ability compared with ISCs-NC.After co-culturing with ISCs-interfering CRBP1,the insulin secretion ability and islets viability improved compared with those with ISCs-overexpressed CRBP1.Likewise,the concentration of caspase-3 in islets was significantly diminished in CRBP1 interfering exposure.Conclusions: VA is involved in the regulation of islet function by inhibiting the differentiation of ISCs into fibroblast-like cells.Restoring ISCs quiescence via CRBP1 inhibition could alleviate the impairment of islet function caused by fibroblast-like ISCs exposure.Therefore,blocking ISCs from differentiating into fibroblast-like cells and maintaining its resting state through regulation of VA levels may be a new strategy for diabetes treatment.Part 3The differentiation of islet stellate cell into insulin-producing cells Aims: Recent studies demonstrated that stellate cells could differentiate into a variety of specific parenchymal cells.Here,we will explore whether ISCs share properties of stem/progenitor cells and have the potential to be β-like cells.Methods: ISCs were isolated by density gradient centrifugation method and identified its biological characterization using Oil O staining.The expression of adult stem/progenitor markers in cultured ISCs were detected by reverse transcription polymerase chain reaction(RT-PCR)and immunofluorescence.ISCs were induced by endocrine differentiation medium.The endocrine differentiation phenotype was analyzed by RT-PCR,immunofluorescence,dithizone staining,electron microscopy and enzyme-linked immunosorbent assay.Results: Freshly isolated ISCs displayed a classical polygonal appearance containing lipid-loaded vesicles.With culturing,ISCs transformed into fibroblast-like cells.The gene expression of and the adult stem/progenitor cell markers(desmin,CK-19,GFAP,Vimentin)of cultured ISCs were increased in a time-dependent manner,while the markers responsible for β cell differentiation exhibited the opposite tendency.With stepwise induction of pancreatic specialized differentiation,differentiated ISCs gradually changed morphology from typically elongated fibroblast-shaped cells into the appearance of round cells leading to formation of islet clusters.These differentiated cells expressed multi-pancreatic β cell-specific markers and comprised C-peptide-positive cells.Most of C-peptide-positive cells co-expressed insulin and glucagon,as well as CK-19,GFAP,Vimentin.Insulin content in differentiated cells was detected.Glucose-stimulated insulin secretion of differentiated cell clusters tended to increase,nevertheless,no significant difference was found.Ultrastructural studies showed differentiated ISCs revealed the presence of extensive hypertrophy of the rough endoplasmic reticulum,interspersed electron-dense granules,abundant lysosomes in the cytoplasm,and fibers of collagen in the extracellular compartment.Conclusions: ISCs shared properties of stem/progenitor cells and could differentiate into insulin-producing cells in vitro.Therefore,it may lead to a new opinion for physiological role of ISCs and provide useful resource for future limited source of cells for diabetes mellitus therapy.