The Effects Of Hyperinsulinemia And Hyperglycaemica In Type 2 Diabetes Mellitus On Human And Mouse Pancreatic Stellate Cell Function And Proliferation

Epidemiological studies support strong links between obesity,diabetes and pancreatitis and pancreatic adenocarcinoma(PDAC).One meta-analysis showed that the relative risks(RR)for type 2 diabetes mellitus(T2DM)patients with different durations(1 year,1-4 years 5-9 years,more than 10 years)to develop PD AC were 5.38,1.95,1.49 and 1.47,separately.T2DM is associated with insulin resistance,hyperglycemia and hyperinsulinemia,the latter due to increased insulin secretion by pancreatic beta-cells.The mechanisms leading T2DM patients to develop PDAC is not fully understood.Most research on insulin in PDAC and other cancer types has focused on the mechanisms underlying cellular growth and survival in cancer cells,while their effects on stromal cells have received less attention.The histopathological characteristic of PDAC is fibrosis which mainly due to the activation of pancreatic stellate cell(PaSC).We reported that high fat diet-induced PDAC progression in mice is associated with hyperinsulinemia,hyperglycemia and PaSC activation.The present study examines the effects of high concentrations of insulin and glucose on mouse and human PaSC growth,fibrosing responses and the underlying singnaling pathway.Part One:Type 2 diabetes and Long-term feeding with high fat,high calorie diets are associated with islet fibrosis and activation of pancreatic stellate cellsObjective:To evaluate the PaSC activation and fibrosis response in the pancreas of T2DM patients and high fat,high calorie diets(HFCD)feeding mice.Methods:(1)Formalin-fixed and paraffin-embedded(FFPE)human pancreatic specimens were obtained from cadaveric tissues from organ donors at City of Hope(Duarte,CA).For this study,pancreatic tissue specimens from a total of 6 organ donors were analyzed.Three of the donors had no history of diabetes and displayed normal pancreas histology,and the other three donors had a previous history of T2DM.The study was performed in accordance with regulations and IRB protocols approved by the Institutional Review Boards of the City of Hope and Cedars-Sinai Medical Center.Serial sections(4 ?m thick)from FFPE tissues were either stained using Masson's trichrome to evaluate the extent of collagen deposition,or analyzed by immunofluorescence for insulin and a-smooth muscle actin(a-SMA)to visualize pancreatic P cells and activated PaSC.(2)C57BL/6J male mice at 6 weeks of age were randomly placed either on a HFCD or a control diet(CD)and animals were sacrificed at 3,6,9 and 12 months.Mouse pancreas tissues were fixed in 4%buffered formaldehyde,embedded in paraffin,sectioned(4 ?m thick),and stained using Sirius red to measure the extent of collagen deposition and double immunofluorescence staining was performed for insulin and a-SMA to detect pancreatic ? cells and activated PaSC.Results:(1)We found that non-diabetic control pancreas appears normal,with bundles of collagen fibers mainly restricted to perivascular regions.On the other hand,pancreas sections from T2DM donors showed extensive collagen deposition,not only in perivascular regions,but also within islets and in a periacinar areas in the vicinity of affected islets.Pancreatic collagen deposition was significantly increased,by about 3-fold in donors with T2DM compared to controls.Moreover,we investigated that,from the double immunofluorescence staining,a-SMA immunofluorescence was mainly restricted to perivascular regions in normal controls.However,islets from T2DM patients were fibrotic and displayed abundant ?-SMA+ activated PaSC within the islets and in surrounding periacinar areas.(2)From the Sirius red staning,we observed that mice fed CD had collagen around vascular elements but not islets.On the oher hand,pancreas from mice fed HFCD displayed abundant collagen I deposition around islets and adjacent acini.Furthermore,the collagen deposition associated with the presence of activated PaSC in the vicinity of islets was progressive by the time course.In accordance with these data,we identified numerous activated PaSC within islets of HFCD-fed mice.Conclusions:Our data provide new evidence indicating that obesity and T2DM create an environment conducive to PaSC activation and fibrogenesis and also provide a hypothesis foundation for the further study of the hyperglycemia and hyperinsulinemia effects on human and mouse PaSC perliferation,fibrosing responses,and underlying signaling pathway.Part Two:Isolation and identification of human and mouse pancreatic stellate cell and the effects of high concentrations of insulin and glucose on pancreatic stllalte cell proliferation and function Objective:To isolate and identify the PaSC from mouse and human pancreas and examine the expression of Insulin receptor(IR)and insulin-like growth factor receptor(IGF-1R)in PaSC.Furthermore,evaluate the effect of high concentrations of insulin and glucose on the PaSC cell activation,proliferation and fibrosing response.Method:(1)We isolated and identified mPaSC by using immunofluorescence staining for PaSC markers(a-SMA,Desmin,Vimentin),and extracellular matrix(ECM)production(collagen I,collagen III,fibronectin).(2)We performed immunofluorescence staining,qPCR and western Blot to examine whether PaSC expressed IR and IGF-1R,and whether high concentrations of insulin modulate IR/IGF-1R expression.(3)We next examined whether the high concentrations of insulin may accelerate the differentiation of quiescent PaSC into the myofibroblast phenotype.(4)Western Blot was processed to measure the effects of combined high insulin and high glucose concentrations on insulin-induced IR/IGF-1R signaling in imPaSC.(5)Finally,we observed the influence of high concentrations of glucose and insulin on regulation of fibrosing responses and PaSC proliferation.Results:(1)Isolated mouse primary PaSCs was fully activated after 5 days and was characterized by specific stellate cell markers(a-SMA,Desmin,Vimentin)matrix protein(collagen I,collagen III,fibronectin)and absence of stromal cell surface marker and cytoceratin 19(CK 19,a duct cell marker)verified the purity of isolated mouse PaSC.(2)PaSC co-express IR and IGF-1R receptors and mRNA levels of both IR and IGF-1R for quiescent and culture-activated primary mPaSC were similar.By RNA analysis techniques,we found that primary mPaSC expressed predominantly the IR-A rather than the IR-B isoform.Moreover,short-term treatment with insulin(25-100 nM)dose-dependently induced the downregulation of both IR and IGF-1R expression.However,this kind of downregulation were almost entirely restored to their control levels.(3)Insulin treatment did not induce activation of quiescent mPaSC,irrespective of the normal or high glucose concentration in the culture medium.However,insulin stimulation did promoted collagen and fibronectin expression levels in fully activated primary mPaSC.(4)Insulin-induced IR/IGF-1R phosphorylation(7.4-fold increasing than control with 100 nM Insulin)led to activation of downstream signaling cascades including MAPK/ERK and Akt/mTOR pathways.We found a rapid but transiently increased phosphorylation of ERK1/2 and SAPK/JNK in insulin treated cells.Compared to these effects,insulin activation of Akt/mTOR signaling was robust and sustained.Furthermore,cell lysates were prepared after 1,3,6 and 24 h insulin stimulation.As indicated by specific tyrosine phosphorylation,insulin induced IR/IGF-1R activation in cells cultured in both 5 mM and 25 mM glucose media,an effect that persisted for at least 24 h.Compared to 5 mM glucose,levels of p-IR/IGF-1R were slightly higher at various time points in cells treated with 25 mM glucose.Consistent with this enhanced receptor activation,significantly higher Akt and ERK1/2 activation levels were induced by insulin stimulation of cells cultured in 25 mM glucose than in 5 mM glucose media.(5)After 24 h incubation,high insulin,25 mM glucose,and in particular the combination of both promoted mRNA levels of the ECM proteins fibronectin and alpha-1 type I collagen(COL1A1).However,the combined treatment of insulin and 25 mM glucose did not affect mRNA levels of a-SMA.From the MTT(Thiazolyl Blue Tetrazolium Bromide)assay,we found that treatment with 100 nM insulin or 25 mM glucose alone induced a similar 1.8-fold elevation in cell proliferation compared to basal 5 mM glucose,while the combined treatment markedly enhanced imPaSC proliferation,by 2.7-fold.Conclusion:PaSC co-express IR and IGF-1R receptors and mRNA levels of both IR and IGF-1R for quiescent and culture-activated primary mPaSC were similar.Insulin-induced IR/IGF-1R phosphorylation led to activation of downstream signaling cascades including MAPK/ERK and Akt/mTOR pathways.High concentration of insulin,in particular combined with high glucose promoted the PaSC mRNA levels of the ECM proteins and enhanced cell proliferation.However,both insulin and glucose showed no effect on PaSC activation.Part Three:The role of mTOR in insulin-induced enhancement of proliferation and fibrosing responses in PaSCObjective:In our observation of part Two,AKT/mTOR pathway seemed much more acutely regulated by insulin than ERK1/2,so we chose to examine this pathway more extensively in PaSC.To elucidate the participation of Akt/mTOR signaling pathways on insulin-induced cell proliferation and upregulation of ECM proteins in PaSC,we used KU63794(KU)as a selective dual inhibitor of both mTOR complecx 1(mTORC1)and mTOR complecx 2(mTORC2).Methods:(1)PaSCs were pre-incubated with either KU(1?M)or fresh DMEM/F12 medium for 1h.(2)PaSCs were cultured with insulin(100 nM)or combined KU(1?M)for 15 min to 48 h.At the end of the culture period,involved signaling pathway was examined by western blot.(3)PaSCs were cultured with insulin(100 nM)or combined KU(1 ?M)for 48 h.At the end of the culture period,western blot was performed to evaluate the ECM proteins production.(4)MTT assay was processed to observe the PaSC cell proliferation after the culture period(48 h or 96 h)ended.Results:(1)KU(1 ?M)completely inhibited basal and insulin-induced phosphorylation of the mTORCl substrate P70S6K at Thr 389 in imPaSC,and this effect persisted for at least 48 h.Meanwhile,KU significantly diminished insulin-induced phosphorylation of AKT at Ser 473,a site regulated by mTORC2,but did not change ERK1/2 activation.(2)Consistent with its effects on Akt/mTOR pathway,KU was sufficient in blocking insulin-induced increasing of ECM proteins in imPaSC.The upregulation in protein levels of fibronectin,collagen I and P4HA2(a key endoplasmic reticulum enzyme required for proper collagen folding)induced by insulin were prevented by pre-incubation with KU(1 ?M).In contrast,protein levels of the PaSC activation marker a-SMA were neither enhanced with insulin nor reduced by KU.(3)KU(1 ?M)effectively inhibited insulin-induced imPaS C cell proliferation and similar effects were observed in primary activated hPSC and mPaSC.Conclusion:The mTOR blocking study supports a key role for mTOR in regulating the activated,pro-fibrosing phenotype and proliferation in PaSC.