OBJECTIVETo investigate the effect of high glucose on mitochondrial respiratory chain function in INS-1 cells.

METHODSThe pancreatic beta cell line INS-1 was divided into the normal control (NC), high glucose (HG), and N-acetyl-L-cysteine (NAC) pretreatment groups, which were cultured for 72 h in the presence of 5.5 mmol/L glucose, 16.7 mmol/L glucose, and 16.7 mmol/L glucose with 1.0 mmol/L NAC, respectively. The activities of the enzyme complexes I and III of the respiratory chain in the cells were assessed with spectrophotometry, the ATP levels were examined using a luciferinluciferase kit, and insulin levels detected by radioimmunoassay.

RESULTSThe activities of the respiratory chain enzyme complexes I and III were 1.53-/+0.24 and 1.08-/+0.22 micromol.mg(-1).min(-1) in high glucose group, respectively, significantly lower than those in the normal control group (2.31-/+0.33 and 1.92-/+0.39 micromol.mg(-1).min(-1), P<0.01). ATP and insulin levels also decreased significantly in high glucose group as compared with those in the normal control group (P<0.01). The addition of NAC partially inhibited high glucose-induced decreases in the enzyme complex activities, ATP levels and insulin secretion (P<0.05).

CONCLUSIONThe respiratory chain function is positively correlated to insulin secretion in INS-1 cells, and exposure to high glucose causes impairment of the two enzyme complexes activities through oxidative stress, resulting in the mitochondrial respiratory chain dysfunction. High glucose-induced damages of the mitochondrial respiratory chain function can be partially inhibited by NAC.

Cell Respiration ; drug effects ; Cells, Cultured ; Glucose ; pharmacology ; Humans ; Insulin-Secreting Cells ; cytology ; physiology ; Mitochondria ; physiology ; Oxidative Stress ; drug effects

In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 microg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro.
Amnion ; cytology ; Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Humans ; Insulin-Secreting Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Pancreas ; physiology ; surgery ; Pancreatic Extracts ; pharmacology ; Rats ; Regeneration

BACKGROUNDIslet transplantation is an effective way of reversing type I diabetes. However, islet transplantation is hampered by issues such as immune rejection and shortage of donor islets. Mesenchymal stem cells can differentiate into insulin-producing cells. However, the potential of human umbilical cord mesenchymal stem cells (huMSCs) to become insulin-producing cells remains undetermined.

METHODSWe isolated and induced cultured huMSCs under islet cell culture conditions. The response of huMSCs were monitored under an inverted phase contrast microscope. Immunocytochemical and immunofluorescence staining methods were used to measure insulin and glucagon protein levels. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect gene expression of human insulin and PDX-1. Dithizone-staining was employed to determine the zinc contents in huMSCs. Insulin secretion was also evaluated through radioimmunoassay.

RESULTSHuMSCs induced by nicotinamide and β-mercaptoethanol or by neurogenic differentiation 1 gene (NeuroD1) transfection gradually changed morphology from typically elongated fibroblast-shaped cells to round cells. They had a tendency to form clusters. Immunocytochemical studies showed positive expression of human insulin and glucagon in these cells in response to induction. RT-PCR experiments found that huMSCs expressed insulin and PDX-1 genes following induction and dithizone stained the cytoplasm of huMSCs a brownish red color after induction. Insulin secretion in induced huMSCs was significantly elevated compared with the control group (t = 6.183, P < 0.05).

CONCLUSIONSHuMSCs are able to differentiate into insulin-producing cells in vitro. The potential use of huMSCs in β cell replacement therapy of diabetes needs to be studied further.

Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Cellular Reprogramming ; genetics ; physiology ; Female ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Cord ; cytology ; Wharton Jelly ; cytology

OBJECTIVETo investigate the differentiation of human amniotic epithelial cells (hAECs) into insulin secreting cells (ISCs) in vitro.

METHODSThe hAECs were isolated from human amnion by trypsin digestion, and the phenotype of the isolated cells were identified by flow cytometry and immunocytochemical staining. The hAECs at passage 3 were treated with nicotinamide and N2 supplement to investigate their differentiation into ISCs. At different times after differentiation, the expression of insulin and beta2 microglobulin (beta2-MG) was determined by immunocytochemical staining, while the content of insulin in supernatant from cultured hAECs was detected by radioimmunoassay and the expressions of insulin, pancreatic and duodenal homeobox factor-1 (PDX-1) mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS(1) hAECs expressed high percent of CD29, CD73, CD166 and CK19. (2) At 7, 14 and 21 days, the percentages of insulin-positive cells in induced groups were 74.00% +/- 1.73%, 75.33% +/- 1.15% (see symbol) 75.67% +/- 0.58% respectively, which were negative in control groups. (3) At 7, 14 and 21 days, contents of insulin in supernatant from induced groups were (328.47 +/- 3.22) microIU/ml, (332.26 +/- 1.22) microIU/ml and (329.68 +/- 2.57) microIU/ml respectively, they were significantly higher than those in control groups (All P < 0.01). (4) PDX-1 mRNA and beta2-MG were expressed before and after the induction of hAECs, but insulin mRNA was expressed only in the induced groups.

CONCLUSIONhAECs can differentiate into ISCs, having the potential application for therapy of type I diabetes.

Amnion ; cytology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; Flow Cytometry ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; RNA, Messenger ; genetics ; Trans-Activators ; metabolism ; beta 2-Microglobulin ; metabolism

BACKGROUNDPancreatic islet cell transplantation is an effective approach to treat type 1 diabetes. However, this therapy is not widely used because of the severe shortage of transplantable donor islets. This study investigated whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB) could be transdifferentiated into insulin producing cells in vitro and the role of extracellular matrix (ECM) gel in this procedure.

METHODSHuman UCB samples were collected and MSCs were isolated. MSCs specific marker proteins were analyzed by a flow cytometer. The capacities of osteoblast and adipocyte to differentiate were tested. Differentiation into islet like cell was induced by a 15-day protocol with or without ECM gel. Pancreatic characteristics were evaluated with immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Insulin content and release in response to glucose stimulation were detected with chemiluminescent immunoassay system.

RESULTSSixteen MSCs were isolated from 42 term human UCB units (38%). Human UCB-MSCs expressed MSCs specific markers and could be induced in vitro into osteoblast and adipocyte. Islet like cell clusters appeared about 9 days after pancreatic differentiation in the inducing system with ECM gel. The insulin positive cells accounted for (25.2 +/- 3.4)% of the induced cells. The induced cells expressed islet related genes and hormones, but were not very responsive to glucose challenge. When MSCs were induced without ECM gel, clusters formation and secretion of functional islet proteins could not be observed.

CONCLUSIONSHuman UCB-MSCs can differentiate into islet like cells in vitro and ECM gel plays an important role in pancreatic endocrine cell maturation and formation of three dimensional structures.

C-Peptide ; analysis ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Extracellular Matrix ; physiology ; Fetal Blood ; cytology ; Flow Cytometry ; Fluorescent Antibody Technique ; Glucagon ; analysis ; Humans ; Insulin ; analysis ; secretion ; Insulin-Secreting Cells ; cytology ; Mesenchymal Stromal Cells ; cytology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo explore the mechanisms of differentiation and development of pancreatic endocrine cells as well as pancreatic regeneration.

METHODSHuman embryonic pancreatic tissue at 7-14 weeks of gestation was collected. Diabetes mellitus rat model was induced with 65 mg/kg of streptozotocin. Insulin, glucagon, somatostatin, nestin, and cytokeratin 19 (CK19) of pancreatic tissues were observed by immunohistochemistry.

RESULTSAt 9 weeks of gestation, pancreatic epithelial cells began to co-express insulin, glucagon, somatostatin, and CK19 before migration. Islet cells gradually congregated along with the increase of aging, and at 14 weeks of gestation histological examination showed islet formation. At 12 weeks of gestation, nestin-positive cells could be seen in the pancreatic mesenchyme. During early embryogenesis, islet cells of pancreatic ducts co-expressed insulin, glucagon, and somatostatin. During pancreatic regeneration after damage, nestin expression of islet cells increased.

CONCLUSIONIn the early stage of embryogenesis, islet cells of primary pancreatic ducts can be differentiated to multipotential endocrine cells before migration. During tissue regeneration, pancreatic stem cells may differentiate and proliferate to form pancreatic islet.

Animals ; Cell Differentiation ; Diabetes Mellitus, Experimental ; chemically induced ; metabolism ; pathology ; Embryonic Development ; physiology ; Epithelial Cells ; cytology ; physiology ; Humans ; Insulin-Secreting Cells ; cytology ; physiology ; Islets of Langerhans ; cytology ; physiology ; Male ; Pancreas ; cytology ; embryology ; physiology ; Pancreatic Ducts ; cytology ; embryology ; physiology ; Rats ; Rats, Sprague-Dawley ; Regeneration ; physiology ; Stem Cells ; cytology ; metabolism ; physiology

BACKGROUNDIslet beta-cells are almost completely destroyed when patients with type 1 diabete are diagnosed. To date, insulin substitute therapy is still one of the main treatments. The cure of type 1 diabetes requires beta-cell regeneration from islet cell precursors and prevention of recurring autoimmunity. Therefore, beta-cell regeneration and proliferation emerge as a new research focus on therapy for type 1 diabetes. Islet beta-cell regeneration and development are controlled by many growth factors, especially insulin-like growth factor-1 (IGF-1).

METHODSRecombinant adenovirus encoding rat IGF-1 (rIGF-1) was constructed and transduced into rat beta-cells, RINm5F cells. Western blotting analysis and ELISA were used to detect rIGF-1 protein. Streptozotocin (STZ) was used to induce RINm5F cell destruction. The level of nitric oxide (NO) was detected in cell culture supernatants by the Griess reaction. Islet cell function was evaluated by glucose-stimulated insulin production. Flow cytometry analysis was further used to investigate the apoptosis of RINm5F cells. Thiaoollyl blue viability assay was applied to determine cell viability.

RESULTSThe recombined adenovirus-rIGF-1 was successfully constructed and the titer was 4.0 x 10(8) pfu/ml. The rIGF-1 protein was effectively expressed in the RINm5F cells and cell culture supernatants. rIGF-1 expression remarkably inhibited STZ-induced islet cell apoptosis and significantly decreased the level of NO. Furthermore, IGF-1 expression also significantly protected insulin secretion and cell proliferation in a time-dependent manner.

CONCLUSIONSOur study suggests that locally produced rIGF-I from RINm5F cells may be beneficial in maintaining beta-cell function, protecting beta-cells from the destruction of apoptosis factors and promoting beta-cell survival and proliferation. IGF-I might be considered as a candidate gene in gene therapy for type 1 diabetes. In addition, it appears that the apoptosis induced by STZ may be NO-dependent.

Adenoviridae ; genetics ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Proliferation ; Cell Survival ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; genetics ; physiology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Streptozocin ; pharmacology

Animals ; Exocytosis ; drug effects ; physiology ; Glucose ; pharmacology ; Insulin ; secretion ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; methods ; Potassium ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Vesicle-Associated Membrane Protein 2 ; genetics ; metabolism

AIMTo explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in murine MIN6 pancreatic beta-cells.

METHODSMIN6 cells were cultured in vitro. Cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin- V-PI staining. Nitric oxide levels were measured by Griess assay. Inducible nitric oxide synthase(iNOS) protein and NF-kappaBp65 fragment were detected by Western blot.

RESULTSExposure of 25 micromol/L t-BHP to MIN6 cells for 60 min, cell viability was reduced and the percentage of apoptosis was increased significantly. The levels of cytoplasmic iNOS protein and nitrite were elevated. Meanwhile, treatment with t-BHP resulted in nucleus NF-kappaBp65 fragment peaking at 20 min. Both L-NAME and N-Acetyl-l-cysteine (NAC) attenuated the elevated levels of nitrite and percentage of apoptosis due to t-BHP alone.

CONCLUSIONNF-kappa-iNOS-nitric oxide signalling pathway can mediated t-BHP induced apoptosis in MIN6 cells .

Animals ; Apoptosis ; Cell Line ; Insulin-Secreting Cells ; cytology ; Mice ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Oxidative Stress ; physiology ; Signal Transduction ; tert-Butylhydroperoxide ; pharmacology

The aim of this study was to assess the prevalence of diabetes and to study the effects of excess growth hormone (GH) on insulin sensitivity and beta-cell function in Korean acromegalic patients. One hundred and eighty-four acromegalic patients were analyzed to assess the prevalence of diabetes, and 52 naive acromegalic patients were enrolled in order to analyze insulin sensitivity and insulin secretion. Patients underwent a 75 g oral glucose tolerance test with measurements of GH, glucose, insulin, and C-peptide levels. The insulin sensitivity index and beta-cell function index were calculated and compared according to glucose status. Changes in the insulin sensitivity index and beta-cell function index were evaluated one to two months after surgery. Of the 184 patients, 17.4% were in the normal glucose tolerance (NGT) group, 45.1% were in the pre-diabetic group and 37.5% were in the diabetic group. The insulin sensitivity index (ISI0,120) was significantly higher and the HOMA-IR was lower in the NGT compared to the diabetic group (P = 0.001 and P = 0.037, respectively). The ISI0,120 and disposition index were significantly improved after tumor resection. Our findings suggest that both insulin sensitivity and beta-cell function are improved by tumor resection in acromegalic patients.
Acromegaly/*diagnosis/etiology/metabolism ; Adult ; Asian Continental Ancestry Group ; Blood Glucose/analysis ; C-Peptide/analysis ; Diabetes Mellitus/epidemiology ; Female ; Glucose Tolerance Test ; Human Growth Hormone/secretion ; Humans ; Insulin/blood/secretion ; *Insulin Resistance ; Insulin-Secreting Cells/cytology/*physiology ; Male ; Middle Aged ; Prediabetic State/epidemiology ; Republic of Korea