OBJECTIVETo investigate the differential potential of mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) into insulin-secreting cells and its inducing condition.

METHODSUCB nucleated cells (NCs) were isolated and cultured in Mesencult media. The obtained UCB MSC were purified by adherence method and expanded. Then they were induced with epidermal growth factor (EGF), B-mercaptoethanol and high concentration of glucose. The induced cells were identified by RT-PCR. Intracellular insulin was examined by immunocytochemistry. The quantity of insulin secretion and glucose-simulated insulin release were examined by chemiluminescence immunoassay. The induced cells were also transplanted into renal subcapsular space of STZ-induced hyperglycemic mice to observe the in vivo lowering effect on hyperglycemia.

RESULTSThe induced cells morphologically became round and were gathering into a mass. The expression of some genes related to pancreatic islet was found by RT-PCR. Chemiluminescence immunoassay showed insulin positivity and the cells secreted a low concentration of insulin [(0.37 +/- 0.06) mU/L]. The induced cells responded to high glucose challenge with a stimulation index of 1.76. After those cells grafted into renal sub-capsule there was an in vivo lowering effect on blood glucose level on STZ hyperglycemic mice.

CONCLUSIONMSCs from UCB can differentiated into insulin secreting cells.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Diabetes Mellitus, Experimental ; surgery ; Fetal Blood ; cytology ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; metabolism ; transplantation ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Mice ; Mice, Nude

OBJECTIVETo construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells.

METHODSThe sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.

RESULTSRT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase.

CONCLUSIONThe successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.

Cell Cycle ; drug effects ; Genetic Vectors ; genetics ; Glucose ; pharmacology ; Humans ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Parathyroid Hormone, Type 1 ; genetics ; metabolism

Insulin resistance and insulin secretion deficiency are main machanisms in inducing type 2 diabetes mellitus (T2DM), and mitochondria damage plays an important role in them. Research shows that autophagy is a self-protective mechanism of cells, which plays an important role in maintaining the normal structure and function of pancreatic β cells and improving insulin resistance. Previous studies show that traditional Chinese medicine can regulate cell autophagy to influence β cells and insulin resistance, type 2 diabetes mellitus and its complications. Thus this review will talk about the process of the relationship between autophagy and T2DM and the intervention effect of traditional Chinese medicine.
Animals ; Autophagy ; drug effects ; Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; physiopathology ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Insulin ; metabolism ; Insulin Resistance ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism

To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic beta-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic beta-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.
Apoptosis/*drug effects ; Cell Line ; Fatty Acids, Nonesterified/*pharmacology ; Glycogen Synthase Kinase 3/*metabolism ; Insulin-Secreting Cells/*cytology ; Oleic Acid/pharmacology ; Phosphorylation

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

OBJECTIVETo explore the effects of berberine on the pancreatic 13 cell apoptosis in rats with insulin resistance (IR).

METHODSIR Wistar rat model was established by feeding with high fructose diet. After 6-week treatment of berberine, oral glucose tolerance test (OGTT) was performed. Then fasting insulin level (Fins) was detected and insulin sensitivity index (ISI) calculated. The islet was isolated and purified. The pancreatic p3 cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). The apoptosis-related protein ASK1 and Caspase-12 expressions were examined by immunohistochemical assay.

RESULTSCompared with the normal group, the blood glucose at 0 and 1 h increased, the Fins increased and ISI decreased, the blood lipids were disarranged, the pancreatic beta cell apoptosis increased, and ASK1 and Caspase-12 protein expressions increased in IR rats. Compared with the model group, the blood glucose at 0 and 1 h and the Fins decreased, ISI increased, the disarranged blood lipids were improved, the pancreatic beta cell apoptosis decreased, and the ASK1 expression decreased, but with no obvious change in the Caspase-12 expressions in the berberine group.

CONCLUSIONSBerberine could alleviate IR state in IR rats and inhibit pancreatic 13 cell apoptosis. Its mechanism might be correlated with the inhibition of ASK1 protein expressions.

Animals ; Apoptosis ; drug effects ; Berberine ; pharmacology ; Blood Glucose ; analysis ; Caspase 12 ; metabolism ; Cell Line ; Insulin Resistance ; Insulin-Secreting Cells ; cytology ; drug effects ; MAP Kinase Kinase Kinase 5 ; metabolism ; Male ; Rats ; Rats, Wistar

OBJECTIVETo study the effect of Mudan Granule (MD) on the glucose metabolism and beta cell function in monosodium glutamate (MSG) induced obese mice with insulin resistance (IR).

METHODSMSG obese mice were induced by subcutaneous injecting MSG (4 g/kg for 7 successive days in neonatal ICR mice). Forty MSG mice with IR features were recruited and divided into four groups according to body weight, fasting blood glucose, triglyceride (TG), total cholesterol (TC), and the percentage of blood glucose decreased within 40 min in the IR test, i.e., the model group (Con), the low dose MD group, the high dose MD group, and the Metformin group (Met). Besides, another 10 ICR mice were recruited as the normal control group (Nor). The water solvent of 2.5 g/kg MD or 5 g/kg MD was respectively administered to mice in the low dose MD group and the high dose MD group. Metformin hydrochloride was given to mice in the Met group at 0.2 g/kg body weight. Equal dose solvent distilled water was administered to mice in the Nor group and the Con group by gastrogavage, once per day. All medication was lasted for 15 weeks. Insulin tolerance test (ITT) and oral glucose tolerance test (OGTT) were performed after 6 weeks of treatment. Beta cell function was assessed by hyperglycemic clamp technique. The morphological changes in the pancreas were evaluated by hematoxylin-eosin (HE) staining. Changes of iNOS, NF-kappaB p65, and p-NF-kappaB p65 in the pancreas were tested.

RESULTSCompared with the Nor group, the blood glucose level, AUC, and fasting blood insulin, ONOO-contents, iNOS activities, and the expression of iNOS, NF-kappaB p65 subunit, pNF-kappaB p65 subunit obviously increased; decreased percentage of blood glucose within 40 min in ITT, glucose infusion rate (GIR), Clamp 1 min insulin, and Max-Insulin obviously decreased in the Con group (P < 0.05, P < 0.01). Compared with the Con group, the aforesaid indices could be improved in the Met group (P < 0.05, P < 0.01). In the low dose MD group, AUC, iNOS activities, and the expression of iNOS and p-NF-kappaB p65 subunit obviously decreased; percentage of blood glucose within 40 min in ITT and GIR obviously increased (P < 0.05, P < 0.01). In the high dose MD group, AUC, ONOO-contents, iNOS activities, and the expression of iNOS, NF-kappaB p65 subunit, and p-NF-KB p65 subunit obviously decreased; percentage of blood glucose within 40 min in ITT, Max-Insulin, and GIR obviously increased (P < 0.05, P < 0.01).

CONCLUSIONMD could significantly improve IR and functional disorder of 3 cells in MSG obese mice, which might be associated with lowering inflammatory reaction in the pancreas.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Female ; Insulin Resistance ; Insulin-Secreting Cells ; drug effects ; metabolism ; Male ; Metformin ; pharmacology ; Mice ; Mice, Inbred ICR ; Mice, Obese ; Obesity ; chemically induced ; metabolism ; Pancreas ; cytology ; drug effects ; Sodium Glutamate

BACKGROUNDGlucagon-like peptide-1 (GLP-1) reduces fatty acid-induced beta-cell lipotoxicity in diabetes; however, the explicit mechanisms underlying this process are not fully understood. This study was designed to investigate the involvement of microRNA, which regulates gene expression by the sequence-specific inhibition of mRNA transcription in the GLP-1 mediation of beta-cell function.

METHODSThe cell viability and apoptosis were determined using an methyl thiazoleterazolium (MTT) assay and flow cytometry. The expression of genes involved in beta-cell function, including microRNA-34a and sirtuin 1, were investigated using real-time PCR. The underlying mechanisms of microRNA-34a were further explored using cell-transfection assays.

RESULTSA 24-hours incubation of INS-1 cells with palmitate significantly decreased cell viability, increased cell apoptosis and led to the activation of microRNA-34a and the suppression of sirtuin 1. A co-incubation with GLP-1 protected the cells against palmitate-induced toxicity in association with a reduction in palmitate-induced activation of microRNA-34a. Furthermore, palmitate-induced apoptosis was significantly increased in cells that were infected with microRNA-34a mimics and decreased in cells that were infected with microRNA-34a inhibitors.

CONCLUSIONMicroRNA-34a is involved in the mechanism of GLP-1 on the modulation of beta-cell growth and survival.

Animals ; Apoptosis ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Fatty Acids, Nonesterified ; toxicity ; Glucagon-Like Peptide 1 ; pharmacology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; MicroRNAs ; genetics ; metabolism ; Palmitic Acid ; pharmacology ; Rats ; Real-Time Polymerase Chain Reaction

OBJECTIVETo study the effect of Supplemented Taoren Chengqi decoction (STCD) on the secretion of insulin and proliferation of NIT-1.

METHODThe effect of STCD and the serum of rat after orally administrating of STCD on the secretion of insulin and proliferation of NIT-1 were studied. The proliferation of NIT-1 was measured by 3H-TdR incorporation and cell counting methods, while the secretion of insulin was measured from the cultured medium by the ultra sensitive rat insulin ELISIA kit.

RESULTBoth the STCD and the serum of rat after orally administrating of STCD significantly could increased the secretion of insulin and proliferation of NIT-1.

CONCLUSIONThe treatment of the diabetic patients by STCD might be through with its improvement of secretion of insulin and proliferation on pancreatic beta-cell.

Animals ; Cell Line ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Insulin ; secretion ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; secretion ; Male ; Mice ; Mice, Inbred NOD ; Mice, Transgenic ; Rats

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