Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.     

Protective effect of chrysin on mice with insulin resistance

AIM: To explore the effects of chrysin on insulin resistance (IRe) in a mouse model. METHODS: Male C57 mice were randomly divided into control group, IRe group, low-dose chrysin group (IRe+chrysin-low) and high-dose chrysin group (IRe+chrysin-high). After 24 weeks, the body weight, liver index and fat mass in all mice were detected. The blood glucose, insulin level and HOMA-IR were measured to determine the changes of the insulin resistance in the animals. The oxidative stress (SOD, GSH-Px and MDA) was also measured. The mRNA expression of insulin signaling pathway molecules (IR, IRS1, IRS2, Glut2 and Glut4) and inflammatory factors (TNF-α, IL-1β, IL-6 and NF-κB) was analyzed by real-time PCR. The protein levels of IRS1 and p65, and their phosphorylation were detected by Western blot. RESULTS: After 24-week intervention, the indicators in IRe group were higher than those in control group, including body fat deposition, serum glucose, serum insulin, HOMA-IR and liver oxidative stress (P<0.01), indicating that the model of insulin resistance was successfully established. Low dose and high dose of chrysin decreased the body weight, serum glucose, serum insulin and HOMA-IR in the IRe mice (P<0.05). The liver oxidative stress was also reduced in both groups (P<0.05). However, no statistical difference of the indexes between IRe+chrysin-low group and IRe+chrysin-high group was observed. Chrysin upregulated the mRNA expression of IR, IRS1, IRS2, Glut2 and Glut4 (P<0.05), and down-regulated the mRNA expression of various inflammatory factors. The inhibitory effect of chrysin on the mRNA expression of NF-κB was observed (P<0.05), especially in high dose group (P<0.05). It was confirmed that the effect of chrysin on liver IRe was related with the increase in the p-IRS1 levels and decrease in the p-p65 levels by Western blot. CONCLUSION: Chrysin inhibits obesity, hyperglycemia and hyperinsulinemia, and relieves insulin resistance and oxidative stress, which might be closely related to the regulation of insulin signaling pathway and the inhibition of inflammatory factor expression.     

Effect of glucocorticoid on PI-3K and p38 MAPK signaling pathways in 3T3-L1 adipocytes

AIM: To study the effects of dexamethasone (DEX) on the glucose transport system and the PI-3K/Akt and p38 MAPK insulin signaling pathways in 3T3-L1 adipocytes,and to investigate the possible mechanism in glucocorticoid induced insulin resistance. METHODS: The 3T3-L1 adipocytes were exposed to DEX for 48 h and incubated with 100 nmol/L insulin for additional 30 min. The glucose uptake was measured by detecting the glucose content in cell culture supernatants. Then expression and distribution of Glut4 was measured. The insulin signaling proteins Akt,phospho-Akt,p38MAPK and phospho-p38MAPK were also measured with Western blotting. RESULTS: DEX inhibited insulin stimulated glucose transport capacity in 3T3-L1 adipocytes. DEX did not alter the amount of Glut4 protein in total cell lysates but attenuated the insulin-stimulated Glut4 translocation to the plasma membrane. DEX significantly inhibited insulin stimulated phosphorylation of Akt and p38 MAPK. CONCLUSION: These results suggest that DEX alters insulin stimulated glucose transport capacity in 3T3-L1 adipocytes,which is mediated by attenuating insulin stimulated activation of PI3K-Akt and p38 MAPK pathways,and reducing insulin stimulated Glut4 translocation and transport activity. These may lead to insulin resistance in 3T3-L1 adipocytes.     

Mechanisms of PGF2α on the glucose-induced insulin secretion in NIT-1β cells

AIM:To investigate the cellular mechanisms by which PGF₂α promotes glucose-stimulated insulin secretion in NIT-1 beta cells. METHODS:Using the radioimmunoassay (RIA), the amount of the PGF₂α augmentation of glucose stimulated insulin secession was determined in different conditions, and the confocal laser scanning methods by Fluo-3AM as a fluorescent probe were used to analyze the changes of intracellular calcium in NIT-1β cells. RESULTS:At the lower glucose (0, 5.5 mmol/L), PGF₂α (5 μmol/L) failed to potentiate insulin secretion (P>0.05). However, in the presence of 16.5 mmol/L glucose, PGF₂α increased significantly in insulin secretion (P<0.05). Neither the AC inhibitor ddA nor the GC inhibitor Ly-83583 altered PGF₂α-potentiated insulin secretion in the presence of 16.5 mmol/L (P<0.05 or P<0.01). Otherwise, the PLC inhibitor U-73122 and the PKC blocker calphostin C both counteracted the insulinotropic of PGF₂α (P<0.01 or P<0.05). Moreover, exposure of the NIT-1β cells to 5 μmol/L PGF₂α induced a rapid increase of intracellular calcium (P<0.01). The inhibitor, ddA or Ly-83583 had no impact on PGF₂α-induced elevation of the intracellular calcium (P<0.01). Pretreatment of the cells with U-73122 completely prevented the calcium response induced by PGF₂α (P<0.01). CONCLUSION:Efects of PGF₂α was independent of cAMP or cGMP, potentiated glucose (16.5 mmol/L)-induced insulin secretion in NIT-1β cells through stimulation of phospholipase C, which subsequently mediated the elevation of intracellular calcium and activation of protein kinase C.     

Immunosuppressive and protective effects of human α1-antitrypsin on pancreatic β-cell transplantation

AIM：To study the immunosuppressive and protective effects of human α1-antitrypsin (hAAT) on pancreatic β-cell transplantation. METHODS： An NIT-1 cell line (NIT-hAAT) was constructed, which can stably express the protein of hAAT. The BALB/c mice were intraperitoneally injected with NIT-1 and NIT-hAAT cell lines twice to induce cytotoxic T-lymphocytes (CTL). The apoptotic situation, the cytokine expression, and the mRNA expression of inflammatory factors were examined after mixed culture of CTL with NIT-1or NIT-hAAT cell line pretreated with mitomycin. Both cell lines were transferred into the left renal capsule of the diabetic mice to dynamically observe the changes of blood sugar and body weight, the serum levels of insulin and C-peptide, and the pathological changes of the transplanted sites. RESULTS： The results of extended CTL killing assay showed that the cytotoxic effect on NIT-hAAT cell acceptor mice was significantly reduced compared with NIT-1 cell acceptor mice. hAAT effectively reduced apoptosis, inhibited the mRNA expression of inflammatory factors IL-1β and IL-6, and adjusted the balance of Th1/Th2 cytokine expression. After NIT-hAAT was transplanted into the diabetic mice, blood glucose decreased obviously and maintained for 28 d. The serum levels of insulin and C-peptide increased obviously. The infiltration of the inflammatory cells in the transplanted sites significantly reduced. CONCLUSION：hAAT has the abilities of reducing cytotoxic effect of CTL on the β-cells, inhibiting inflammatory factor expression, and stopping short-term immunological rejection of the acceptor. hAAT has obvious immunosuppressive and protective effects on pancreatic β-cell transplantation for treatment of diabetes.     

Role of GPR40 mediates the effects of FFAs on lipoapoptosis in mouse β-cell line NIT-1

AIM: To evaluate the role of G protein-coupled receptor 40 (GPR40) mediates the effects of free fatty acids (FFAs) on lipoapoptosis in mouse β-cell line NIT-1 and the mechanisms involved in this process.METHODS: NIT-1 cells were supplemented with palmitate (500 μmol/L) or oleate (500 μmol/L) for 48 h, then apoptosis of the cells was determined by the methods of Hoechst 33342, TUNEL and flow cytometry (Annexin V/PI). The small interfering RNA technique was used to inhibit the expression of GPR40 in NIT-1 cell. The mock, control siRNA and GPR40 siRNA transfected cells were either supplemented with palmitate (500 μmol/L) or co-incubated with palmitate and oleate (500 μmol/L for each) for 48 h. The percentages of apoptotic cells were quantified. The expression of p-c-Jun, Bcl-2 and Bax were detected by Western blotting.RESULTS: Palmitate induced β cell lipoapoptosis, whereas oleate inhibited NIT-1 cells from palmitate-induced lipoapoptosis. No significant difference of the percentages of apoptotic cells was indicated among the mock, control siRNA and GPR40 siRNA transfected cells treated with palmitate (P>0.05). However, after co-incubated with palmitate and oleate (500 μmol/L for each) for 48 h, the percentage of apoptotic cells in GPR40 siRNA transfected cells was greater than that in mock (P<0.05), while the expression of p-c-Jun was decreased. The expressions of Bcl-2 and Bax were not affected.CONCLUSION: Palmitate induced β cell lipoapoptosis might not be mediated through GPR40, whereas oleate inhibits NIT-1 cells from palmitate-induced lipoapoptosis, which is mediated at least in part through GPR40, the change of c-Jun expression may play an role in this process, suggesting that GPR40 might be implicated in the control of β cell mass plasticity and GPR40 probably provides a link between obesity and type 2 diabetes.     

Osteopontin-induced ER stress results in insulin resistance in C2C12 myocytes

AIM: To investigate the potential role of osteopontin (OPN) in insulin resistance in C2C12 myocytes and its underlying mechanism. METHODS: C2C12 myoblasts were induced by low concentration of serum and insulin treatment to differentiate into myocytes. Western blot was performed to detect the protein abundance and phosphorylation using specific antibodies. Plasma membrane was isolated by centrifugation. Glucose uptake was measured by glucose uptake assay. RESULTS: OPN treatment suppressed insulin-stimulated protein kinase B (Akt) phosphorylation in a dose-and time-dependent manner, accompanied with decreased membrane translocation of glucose transporter type 4 (Glut4) and reduced glucose uptake. Neutralization of OPN specific receptor in skeletal muscle with CD44 antibody mitigated OPN-induced inhibitory impact on insulin action. Furthermore, OPN treatment resulted in endoplasmic reticulum (ER) stress and phosphorylation of c-Jun N-terminal kinase (JNK). Administration of ER stress inhibitor 4-phenylbutyrate (4-PBA) diminished the detrimental effects of OPN on JNK phosphorylation, Glut4 membrane translocation and glucose uptake. CONCLUSION: ER stress mediated OPN-induced insulin resistance in C2C12 myocytes.     

Effects of berberine on the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acids

AIM: To investigate the effects of berberine on the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acid. METHODS: The influence of berberine at different concentrations on NIT-1 cells cultured with or without high glucose and saturated fatty acid were determined and compared using MTT colorimeric assay. The cell apoptotic rate was also determined by flow cytometry assay and in situ TUNEL method. RESULTS: The effects of berberine at different concentrations on NIT-1 cells showed dose-dependent, low dose (≤5 μmol/L) had dispensable cytotoxicity; meanwhile, high dose showed distinct effects. On the other hand, low dose of berberine alleviated the apoptosis in NIT-1 cells induced by high glucose and saturated fatty acid, when adding berberine to cell medium. CONCLUSION: Berberine inhibited the apoptosis of NIT-1 cells induced by high glucose and saturated fatty acid.     

Effects of L-carnosine on NIT-1 islet cells

AIM: To investigate the protective effect of L-carnosine on insulin secretion, proliferation and apoptosis of β-cells impaired by high glucose. METHODS: NIT-1 cells were pre-treated with glucose at concentrations of 11.1 mmol/L (control level) and 33.3 mmol/L (high level) for 72 h, and then the cells were stimulated with various concentrations of glucose (0, 5 and 25 mmol/L) and/or L-carnosine (0, 1 and 20 mmol/L). The level of insulin in the medium was measured by radioimmunoassay. To detect the effect of L-carnosine on proliferation and apoptosis, NIT-1 cells were divided into 4 groups according to different culture conditions for 72 h: group C (with 11.1 mmol/L glucose), group H (with 33.3 mmol/L glucose), group H+A (with 33.3 mmol/L glucose+ 1 mmol/L L-carnosine) and group H+B (with 33.3 mmol/L glucose +20 mmol/L L-carnosine). Proliferous or apoptotic cells were identified by BrdU labeling and flow cytometry (labeling with annexin V-FITC/PI),respectively. Total RNA was extracted and the mRNA expression of caspase-3 and bcl-2 was measured by RT-PCR. The caspase-3 activity was also checked by fluorometric assay kit. RESULTS: The insulin in high-level glucose group was lower than that in control-level glucose group. L-carnosine at concentration of 20 mmol/L notably increased the insulin secretion of the cells pre-treated with glucose at control level or high level. The proliferation and apoptosis were both increased in group H compared with group C, but the total cell counts declined because the apoptotic rate was higher than the proliferation rate. L-carnosine at concentration of 1 mmol/L significantly increased the proliferation rate and decreased the apoptotic rate. The mRNA level of caspase-3 was decreased and the mRNA level of bcl-2 was increased after the cells were treated with L-carnosine at concentration of 1 mmol/L. L-carnosine at concentrations of both 1 mmol/L and 20 mmol/L significantly decreased the caspase-3 activity. CONCLUSION: L-carnosine at high level directly stimulates insulin secretion in NIT-1 cells, and L-carnosine at normal level promotes the cell proliferation and inhibits apoptosis induced by high concentration of glucose. Caspsase-3 and Bcl-2 may be partly involved in this process.     

Orthotopic transplant fetal rat pancreatic stem cells for treating diabetes mellitus

AIM: To observe the possibility of differentiation of fetal rat pancreatic stem cells into islet-like cell cluster by transplantation of fetal rat pancreatic stem cells into pancreatic parenchyma in diabetic SD rats. METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16. SRY DNA was examined to discriminate gender by fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult SD rats were divided into three groups including 10 pancreatic parenchymal orthotopic transplantation, 10 experimental controls and 10 normal controls. In orthotopic transplantation group, 1×10⁶ male fetal pancreatic stem cells per rat were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume of PBS was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodically. 8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assessed by Western blotting. RESULTS: 5 of 12 fetal rats were male according to FISH. After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry with 74.1% of PSCs expressed nestin. The orthotopic transplantation of PSCs led to stable reduction in hyperglycemia and increase in insulin level in serum (3 weeks after transplantation), culminating (5 weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipients pancreata 8 weeks post-transplantation. The expression levels of insulin mRNA and protein in the orthotopic transplanted recipients pancreata were higher than those in experimental control (P<0.05), and the expressions of PDX-1 mRNA and protein were also higher than those in normal control (P<0.05). CONCLUSION: When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat differentiates into islet-like cell cluster, gains comparable function with normal islets and reverses experimental diabetes.     

Effect of insulin on the SGK1 expression and extracellular matrix synthesis in human mesangial cells cultured in high glucose

AIM:To study the effect of insulin on the serum and glucocorticoid-inducible kinase 1 (SGK1) expression and extracellular matrix synthesis in human glomerular mesangial cells (HMC) cultured in high glucose. METHODS:The HMCs were cultured in the presence of 5.5 or 25 mmol/L glucose with or without 100 nmol/L insulin (i.e. NG, HG, NI and HI groups). 4 h latter, expressions of SGK1, insulin receptor substrate-1 (IRS1) and IRS2 in corresponding groups were detected by immunofluorescence or examined by Western blotting. The phosphorylation of IRS1 and IRS2 was measured by immunoprecipitation. 24 h latter, connective tissue growth factor(CTGF) and fibronectin (FN) were also examined by RT-PCR and ELISA, respectively. RESULTS:Compared with NG, the SGK1 protein expression in HG, NI and HI groups was significantly higher (P<0.01). High glucose mainly caused IRS2 protein and its phosphorylation level increase (P<0.01). When treated with 100 nmol/L insulin, IRS1 protein and its phosphorylation in HI group apparently elevated while slight inhibition of IRS2 protein expression and its phosphorylation were observed (HI vs HG, P<0.05). High glucose enhanced the expression of CTGF and FN, and insulin strengthened this effect.CONCLUSION:Insulin and high glucose up-regulate the expression of SGK1 in mesangial cells through different target molecular pathways and ultimately enhance ECM synthesis. The effect of insulin is highly associated with IRS1 signaling cascades.     

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect

AIM: To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1 (PDX-1) and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells (MSCs). METHODS: Recombinant vector containing PDX-1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells (MSCs) cultured in vitro. Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs. RESULTS: Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of transfection was above 40%. RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs. CONCLUSION: Recombinant vector containing PDX-1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.     

Administration of magnolol postpones development of hypertension in juvenile spontaneous hypertensive rats

AIM:To investigate the effects of magnolol (MAG) on blood pressure and aortic vasodilatation to insulin in juvenile spontaneous hypertensive rats (SHR). METHODS:Four-week-old male SHR and age-matched normotensive Wistar-Kyoto (WKY) control rats were used. SHR and WKY rats were randomized into 2 groups and treated daily by gavage with vehicle (distilled water) or MAG (100 mg·kg-1·d-1). After 3 weeks of treatment, blood pressure, aortic vasorelaxation, fasting glucose and plasma insulin levels, the expressions of PPARγ and TRB3, and insulin-stimulated Akt/endothelial nitric oxide synthase (eNOS) activation were measured. In vitro, human umbilical vein endothelial cells (HUVECs) were cultured in the medium containing glucose (25 mmol/L) and palmitate (500 μmol/L). RESULTS:Treatment of young SHR with MAG for 3 weeks decreased blood pressure, improved insulin-induced aortic vasodilation, and Akt and eNOS activation , increased PPARγ expression and decreased TRB3 expression. In cultured HUVECs, MAG incubation increased PPARγ exprssion, decreased TRB3 expression, and elevated insulin-induced phosphorylated Akt and eNOS levels and NO production, which were reversed by PPARγ antagonist. CONCLUSION: Treatment of young SHR with MAG at the prehypertensive stage decreases blood pressure via improving vascular insulin resistance that is at least partly attributable to up-regulation of PPARγ, down-regulation of TRB3 and consequently activation of Akt and eNOS in blood vessel .     

Effects of miR-92a and miR-19b on insulin gene expression in mouse pancreatic β-cells

AIM To investigate the effect of microRNA-92a （miR-92a） and microRNA-19b （miR-19b） on the insulin expression in mouse pancreatic β-cells. METHODS The relative expression levels of endogenous miR-92a and miR-19b in mouse insulinoma MIN6 cells were detected by qPCR. The MIN6 cells were divided into control group， and experimental groups I and II， with 3 samples in each group， and transfected with negative control miRNA （NC）， miR-92a and miR-19b， respectively. The over-expression of the miRNAs was detected by qPCR. The morphological changes and viability of the cells were detected by optical microscopy and CCK8 assay， respectively. The expression of insulin was detected by qPCR， Western blot and immunofluorescence. The possible mechanisms of miR-92a and miR-19b regulating insulin expression were analyzed by bioinformatics prediction， dual-luciferase reporter assay， and Western blot. RESULTS Compared with the adult pancreatic progenitor cells， the expression of endogenous miR-92a and miR-19b in the MIN6 cells was decreased significantly （P<0.05）. Over-expression of miR-92a and miR-19b had no effect on the viability of MIN6 cells， but inhibited the expression of insulin at mRNA and protein levels. miR-19b significantly inhibited the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1 （P<0.05）. miR-92a had a fine-tuning effect on the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1. CONCLUSION miR-92a and miR-19b inhibit the insulin expression in mouse pancreatic β-cells.     

Metanephric cell microenvironment induces embryonic stem cells to differentiate toward renal cells

AIM: To explore the effects of metanephric cell microenvironment on inducing embryonic stem cells (ESCs) to differentiate toward renal cells.METHODS: Embryoid bodies (EBs) of D3 mouse embryonic stem cells were prepared by hanging drop culture, and the EBs were co-cultured indirectly with metanephric cells derived from E12.5 d mouse embryo. The EBs cell with spontaneous differentiation was used as the control. The proteins of Pax2 and WT-1 were analyzed by immunofluorescence assay. The mRNA expression of Pax2, WT-1, Lim1, Sall1, Emx2, GDNF, Wnt4, BMP7, Nephl, Nephrin, KSP and CD24 genes was detected by RT- PCR.RESULTS: The genes related to kidney development were expressed in the EBs cells after co-culture on day 3, and the mRNA expression of Pax2, WT-1, Emx2, GDNF, Nephl, Nephrin, KSP and CD24 was stronger than those in control group. Pax2 positive cells were found on day 3 in the co-cultured EBs cells, and the positive cells increased on day 5 and day 7. WT-1 protein positive cells were found in the co-cultured EBs cells on day 5. No Pax2 or WT-1 positive cell was observed in control group.CONCLUSION: Metanephric cell microenvironment promotes ESCs differentiation toward renal cells.     

Differentiation of mouse iPSCs into insulin-producing cells and experimental study on treating diabetes

AIM: To induce mouse induced pluripotent stem cells (iPSCs) to differentiate into insulin-producing cells (IPCs) by a new 3-step method, and to detect the efficiency and maturity for the treatment of diabetic mice. METHODS: We constructed iPSCs from mouse embryonic fibroblasts of male C57/C mouse by piggyBac transposon, then induced the iPSCs into IPCs by a 3-step method. The cell morphological change was traced by microscopy during the process of differentiation. The expression of mRNA and protein associated with islet β cell development was determined by real-time PCR and immunofluorescence staining. Flow cytometry was used to analysis the efficiency of differentiation. Insulin and C-peptide secretions of IPCs in response to glucose at high (25 mmol/L) or low (5.5 mmol/L) level were measured by ELISA. The IPCs were transplanted into the capsul of left kidney in the male C57/C diabetic mouse model. Blood glucose was continuously monitored for 28 day, serum insulin was tested by ELISA in different stages. The glucose tolerance test was performed on the 28th day, and the left kidney was excised. RESULTS: IPCs were obtained from mouse iPSCs by the 3-step method. The cells expressed the marker genes (Pdx1, Ngn3, Pax6 and Ins2) and proteins (Pdx1, Nkx6.1 and insulin) of β cells. The glucose stimulation induced the secretion of insulin and C-peptide. The efficiency of differentiation was 28% detected by flow cytometry. After transplantation of IPCs to the diabetic mice, the blood glucose was decreased to normal level on the 3rd day,and serum insulin level and the ability of regulating glucose were improved. IPCs were still alive after 28 d of transplantation by pathological observation. CONCLUSION: iPSCs is efficiently induced into IPCs by a 3-step method , and the induction time is shortened significantly. The hyperglycemia of diabetes mice is reversed after transplanting IPCs to same sex inbred strain mice.