In this study, the rat type 2 diabetes mellitus (T2DM) model was established through tail vein injection with low dose of streptozotocin (STZ) and high fat diet for 8 weeks, and then treated with Jiaotai Pill. The oral glucose tolerance test (OGTT), fasting serum insulin (FINS), free fatty acid(FFA) levels and blood lipid were assayed. HOMA-IR was calculated. Pancreatic pathology was performed. And pancreatic triglyceride (TG) content was examined by the lipid extraction method. Pancreatic islet cell apoptosis were detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). According to the results, the model group showed abnormal OGTT, increased FINS, HOMA-IR, FFA, lipid disorder, obvious fat accumulation and significantly increased TG content in pancreatic tissues, and enhanced pancreatic islet cell apoptosis. Compared with the model group, the Jiaotai Pill group displayed improved OGTT, reduced FINS, HOMA-IR, FFA, recovered lipid disorder, decreased fat accumulation and significantly declined TG content in pancreatic tissues, and lowered pancreatic islet cell apoptosis. In summary, Jiaotai pill could effectively treat type 2 diabetes in rats. Its mechanism may be related to the reduction in pancreatic fat accumulation and islet cell apoptosis.
Animals ; Apoptosis ; drug effects ; Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; physiopathology ; Drugs, Chinese Herbal ; administration & dosage ; Fats ; metabolism ; Glucose Tolerance Test ; Humans ; Islets of Langerhans ; cytology ; drug effects ; Male ; Pancreas ; drug effects ; metabolism ; Rats ; Rats, Wistar

BACKGROUNDSuppressor of cytokine signaling (SOCS) proteins are inhibitors of cytokine signaling pathway involved in negative feedback loops. Although SOCS1 is an important intracellular suppressor of apoptosis in a variety of cell types, its role in cytokine-induced pancreatic β-cell apoptosis remains unclear. The present study investigated potential effects of SOCS1 on the cytokine-induced pancreatic β-cell apoptosis.

METHODSAfter successfully transfected with SOCS1/pEGFP-C1 or pEGFP-C1 plasmids to overexpress SOCS1, RINm5F (rat insulinoma cell line) cells were exposed to cytokines, interferon (IFN)-γ alone, IFN-γ+interleukin (IL)-1β, IFN-β+IL-1β+tumor necrosis factor (TNF)-α respectively. Pancreatic β-cell apoptosis was assessed by using MTT, FACS, and caspase-3 activity assays. Protein phosphorylation of Janus kinase 2 (JAK2) and signal transducers and activators of transcription 1 (STAT1) were verified by Western blotting and mRNA expression of inducible nitric oxide synthase (iNOS), NF-κB and Fas were analyzed by RT-PCR.

RESULTSOverexpression of SOCS1 in RINm5F cells was shown to attenuate IFN-γ alone, IFN-γ+IL-1β and IFN-γ+TNF-α+IL-1β mediated apoptosis. Phosphorylation of JAK2 and STAT1 significantly decreased in RINm5F cells which overexpressed SOCS1 protein. Overexpression of SOCS1 significantly suppressed cytokine-induced iNOS mRNA levels.

CONCLUSIONOverexpression of SOCS1 protects pancreatic islets from cytokine-induced cell apoptosis via the JAK2/STAT1 pathway.

Animals ; Apoptosis ; drug effects ; genetics ; Blotting, Western ; Cell Line ; Cytokines ; pharmacology ; Interferon-gamma ; pharmacology ; Interleukin-1 ; pharmacology ; Islets of Langerhans ; cytology ; drug effects ; Janus Kinase 2 ; metabolism ; Phosphorylation ; drug effects ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; STAT1 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; drug effects ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

The protective effects of Da Chai Hu Granules (DCHKL) on islet cells which were incubated with 4 mmol x L(-1) alloxan (AXN) were studied. The viability of islet cells were measured with MTT. Insulin released into medium and in islets was detected by radioimmunoassay. Cell apoptosis rate was determined by flow cytometry. The expression of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax in islet cells were measured with RT-PCR (reverse transcription polymerase chain reaction). Serum containing DCHKL can promote the activity of islet cells significantly (P < 0.01). Basal insulin secretion and high glucose-stimulated insulin secretion increased significantly (P < 0.01). Serum containing DCHKL can inhibit apoptosis of islet cells, the ratio of apoptosis was decreased. Serum containing DCHKL increased expression of Bcl-2 mRNA and decreased expression of Bax mRNA. DCHKL can significantly promote proliferation of islet cells and increase the amount of basal secretion of pancreatic islet cells and high glucose-stimulated insulin secretion. The expression of Bcl-2 increased significantly. The expression of Bax decreased significantly. DCHKL have a protective effect on the islet cells.
Alloxan ; toxicity ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Insulin ; metabolism ; secretion ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Plants, Medicinal ; chemistry ; Protective Agents ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; genetics ; metabolism

OBJECTIVETo study the signaling pathways associated with lipopolysaccharide (LPS)-induced inflammation in islet micro-endothelial cells (IMECs) and the mechanism of pravastatin intervention.

METHODSIMECs exposed to LPS, SB203580, pravastatin, or SB203580+pravastatin were examined for cell apoptosis with Hoechst staining and flow cytometry and for expression levels of total-p38, photophosphorylation-p38 (p-p38) and iNOS with Western blotting.

RESULTSThe apoptosis rate and expression levels of total-p38, p-p38, iNOS in IMECs all increased after LPS exposure. Pravastatin, SB203580, and their combination significantly attenuated LPS-induced enhancement of cell apoptosis and total-p38, p-p38, and iNOS expressions in IMECs.

CONCLUSIONLPS-induced inflammatory toxicity in IMECs is associated with the activation of P38MAPK and iNOS/NO signaling pathways. Pravastatin can inhibit these pathways and suppress the apoptosis and necrosis of IMECs to relieve the cell inflammatory injuries.

Animals ; Apoptosis ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; Inflammation ; Islets of Langerhans ; blood supply ; MAP Kinase Signaling System ; drug effects ; Mice ; Nitric Oxide Synthase Type II ; metabolism ; Phosphorylation ; Pravastatin ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.
Animals ; Biological Markers/metabolism ; *Cell Culture Techniques ; *Cell Differentiation ; Cell Proliferation/drug effects ; Cell Separation ; Cells, Cultured ; Dermis/*cytology/drug effects ; Diabetes Mellitus, Experimental/*surgery ; Female ; Fibroblasts/*cytology/drug effects ; Genitalia, Female/*cytology ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/metabolism ; Homeodomain Proteins/metabolism ; Humans ; Insulin/pharmacology/secretion ; Insulin-Secreting Cells/*cytology/metabolism ; *Islets of Langerhans Transplantation ; Mesenchymal Stem Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Nude ; Niacinamide/pharmacology ; Recovery of Function ; SOXF Transcription Factors/metabolism ; Sodium Selenite/pharmacology ; Trans-Activators/metabolism ; Transferrin/pharmacology

OBJECTIVETo study the effects of in vitro inducement on the expression of SF1-G imprinted genes, Kcnq1 and Cdkn1c during the course of differentiation from mouse embryonic stem (ES) cells to islet-like cells.

METHODSMouse ES cells were induced to differentiate into islet-like cells in vitro. The expression of islet specific markers was tested by RT-PCR or immunofluorescence. RT-PCR/RFLP was used to test the imprinted genes parental expression in cells at different stages.

RESULTSIslet specific genes, such as Insulin, Glucagon, Somatostatin, IAPP and Glut2, were expressed in differentiated cells. The proteins of insulin, C-peptide and Somastatin were expressed in the final stage cells. Imprinted gene Kcnq1 and Cdkn1c were biallelicly expressed in islet-like cells.

CONCLUSIONSMouse ES cells can be successfully induced into islet-like cells in vitro. Gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells (causing loss of imprinting) during the in vitro inducement.

Animals ; Cell Differentiation ; drug effects ; Insulin ; Islets of Langerhans ; cytology ; Mice ; Mouse Embryonic Stem Cells ; Proteins ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells

OBJECTIVETo investigate the effect of potassium channel opener (diazoxide) on the islet cells apoptosis and bcl-2 and bax gene expressions in diabetic rats.

METHODSIslet cell apoptosis was induced by intraperitoneal injection of streptozocin (STZ). The rats were randomly allocated into normal control group (NC group), diabetes mellitus group (DM group), and diazoxide group (DIA group), all treated with diazoxide for 4 weeks. During and after the treatment, the general state, body weight, fasting plasma glucose (FPG), food intake, and oral glucose tolerance of the rats were assessed. The expressions of Bcl-2 and Bax in rat islet cells were measured by immunohistochemistry, and the cell apoptosis was analyzed by TUNEL assay.

RESULTSCompared with the NC group, the rats in the DM group showed significantly decreased body weight (P<0.05), increased blood glucose at o and 120 min after oral glucose administration, decreased expressions of Bcl-2 (P<0.01), increased expression of Bax (P<0.01), and increased islet cell apoptosis (P<0.05). Diazoxide treatment significantly decreased the body weight (P<0.05), decreased the blood glucose, increased Bcl-2 expression (P<0.01), decreased Bax expression (P<0.05), and reduced the islet cell apoptosis (P>0.05) of the diabetic rats.

CONCLUSIONBy causing potassium channel opening, diazoxide can obviously improve the oral glucose tolerance, reduce the body weight, and up-regulate Bcl-2 and down-regulate Bax expression in diabetic rats. Diazoxide can also reduce the apoptosis of the islet cells in diabetic rats.

Animals ; Apoptosis ; drug effects ; Diabetes Mellitus, Experimental ; metabolism ; Diazoxide ; pharmacology ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the effect of heme oxygenase-1(HO-1) on proteins related to apoptosis in INS-1 cells with exposure to intermittent high glucose.

METHODSINS-1 cells cultured in vitro were divided into control group, persistent high glucose group (PHG), intermittent high glucose group (IHG), CoPP + intermittent high glucose group (CoPP+IHG), and ZnPP+ intermittent high glucose group (ZnPP+IHG). After 72 h of treatment with the corresponding protocols, the cells were examined for expressions of HO-1 protein by Western blotting and for expressions of Bax and Bcl-2 by immunocytochemistry.

RESULTSIn comparison with the control group, the cells in both PHG group and IHG group showed significantly increased expressions of HO-1 (P<0.01) and decreased Bcl-2/Bax ratios (P<0.05). The cells in CoPP+ IHG group exhibited a greater HO-1 protein expression but a lower Bcl-2/Bax ratio than those in IHG group (P<0.05) The ZnPP+IHG group demonstrated opposite changes in terms of HO-1, Bax and Bcl-2 expressions compared with the CoPP+IHG group.

CONCLUSIONIntermittent high glucose can lower Bcl-2/Bax ratio in INS-1 cells, and HO-1 may protect INS-1 cells against apoptosis possibly by up-regulating the Bcl-2/Bax ratio.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Glucose ; administration & dosage ; adverse effects ; Heme Oxygenase (Decyclizing) ; metabolism ; Islets of Langerhans ; cytology ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

The aim of this project is to establish a GLP-1 signaling pathway targeted cell model, for screening the new class of GLP-1 receptor agonists as anti-diabetic candidates. Firstly construct a recombined plasmid with multi-copied specific response element (RIP-CRE) regulated by GLP-1 signaling pathway and E-GFP reporter gene. Transient transfect this recombined plasmid into islet cell NIT-1, then detect the responsibility of transfected cell to GLP-1 analogue, Exendin 4. For secondly, use stable transfection and monocloning cell culture to obtain a GLP-1 signaling-specific cell line. It indicates that this cell model can response to Exendin 4, which response can be completely inhibited by GLP-1 receptor antagonist, Exendin 9-39, further showing GLP-1 receptor specific activity with a cAMP-PKA-independently mechanism. Establishment of this novel cell model can be used in high-throughput drug screening of peptides or small molecular GLP-1 analogues.
Animals ; Cell Line ; Cyclic AMP Response Element Modulator ; pharmacology ; Cyclic AMP-Dependent Protein Kinases ; antagonists & inhibitors ; Drug Delivery Systems ; Drug Evaluation, Preclinical ; Genes, Reporter ; Glucagon-Like Peptide-1 Receptor ; Green Fluorescent Proteins ; metabolism ; Hypoglycemic Agents ; agonists ; antagonists & inhibitors ; metabolism ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Isoquinolines ; pharmacology ; Peptide Fragments ; pharmacology ; Peptides ; antagonists & inhibitors ; pharmacology ; Plasmids ; Rats ; Receptors, Glucagon ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; Sulfonamides ; pharmacology ; Transfection ; Venoms ; pharmacology

AIMTo investigate the protective role of HO/CO systems in IL-1beta induced islest apoptosis and to explore the mechanisms of the protective effect of fructose-1, 6-disphosphate (FDP).

METHODSThe pancreases of the rats were removed to collect islets cells. The cells were incubated with IL-1beta with/or FDP. Cell activity, insulin secretion, HO-1 activity, CO content and apoptotic percentage were detected.

RESULTSHO-1 activity and CO content of the normal control group were low. IL-1beta induced a significant decrease of cell activity and insulin release, flow cytometry analysis showed that apoptotic percentage of islet cells remarkably increased following the addition of IL-1beta, FDP obviously improved the islets cellular activity damaged by IL-1beta, and basic amount of insulin secretion and stimulated by high glucose were improved (P < 0.01). Content of CO and activity of HO-1 were higher in the IL-1beta group than the normal control group (P < 0.05), and there were significant differences between the FDP groups and IL-1beta group. FDP decreased cell apoptotic percentage. Activities of HO-1 and content of CO were higher than that in the IL-1beta group (P < 0.01).

CONCLUSIONFDP can attenuate the IL-1beta induced apoptosis of cultured beta cells, the mechanism of which may be improved HO-1 activity and CO content.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Carbon Monoxide ; metabolism ; Cells, Cultured ; Female ; Fructosediphosphates ; pharmacology ; Heme Oxygenase (Decyclizing) ; metabolism ; physiology ; Insulin ; secretion ; Interleukin-1beta ; antagonists & inhibitors ; pharmacology ; Islets of Langerhans ; cytology ; Male ; Rats ; Rats, Wistar