PURPOSE: In non-excitable cells, which include parotid and pancreatic acinar cells, Ca(2+) entry is triggered via a mechanism known as capacitative Ca(2+) entry, or store-operated Ca(2+) entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca(2+) stores, which acts as an important factor triggering Ca(2+) entry. However, both the mechanism of store-mediated Ca(2+) entry and the molecular identity of store-operated Ca(2+) channel (SOCC) remain uncertain. MATERIALS AND METHODS: In the present study we investigated the Ca(2+) entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system. RESULTS: Treatment with thapsigargin (Tg), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase, in an extracellular Ca(2+) free state, and subsequent exposure to a high external calcium state evoked Ca(2+) entry, while treatment with lanthanum, a non-specific blocker of plasma Ca(2+) channel, completely blocked Tg-induced Ca(2+) entry. Microfluorometric imaging showed that Tg-induced Ca(2+) entry started at a basal membrane, not a apical membrane. CONCLUSION: These results suggest that Ca2+ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.
Animals ; Calcium/*metabolism ; Calcium Channels/drug effects/metabolism ; Cells, Cultured ; Endoplasmic Reticulum/drug effects/*metabolism ; Mice ; Mice, Inbred ICR ; Microscopy, Fluorescence ; Pancreas/cytology/drug effects/*metabolism ; Parotid Gland/cytology/drug effects/*metabolism ; Thapsigargin/pharmacology

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

BACKGROUND/AIMS: Pancreatic acini of streptozotocin (STZ)-induced diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion has been closely related to the intracellular calcium concentration ([Ca2+]i) of the acinar cell. In the present study, sequential changes of the intracellular calcium signal which probably underlie the altered enzyme secretion in response to CCK-8 were investigated using pancreatic acini from diabetic rats. METHODS: Diabetic rats were prepared by single intravenous injection of STZ (70 mg/kg). Stimulating experiments with CCK-8 were performed 7 days later. Pancreatic acini were isolated by collagenase digestion. Amylase release and [Ca2+]i were measured by colorimethod and calcium imaging, respectively. The geometry of intracellular calcium signal was analyzed. RESULTS: Normal acini exhibited concentration-dependent [Ca2+]i increase and regular oscillatory calcium signal on CCK-8 stimulation. Amylase release was also concentration-dependent. However, diabetic acini showed significantly less [Ca2+]i increase, prolonged time to peak [Ca2+]i, decreased calcium spikes number, and decreased amylase release compared with normal acini. The decreased [Ca2+]i in diabetic acini was restored significantly by insulin treatment. CONCLUSIONS: Relatively decreased amylase release in diabetic pancreatic acini in response to CCK, appears to be associated with altered calcium signal due to insulin deficiency.
Amylases/*secretion ; Animals ; Calcium Signaling/*drug effects ; Diabetes Mellitus, Experimental/*physiopathology ; Pancreas/cytology/metabolism/*secretion ; Rats ; Rats, Sprague-Dawley ; Sincalide/*pharmacology

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

Dexamethasone converts pluripotent pancreatic AR42J cells into exocrine cells expressing digestive enzymes. In order to address molecular mechanism of this differentiation, we have investigated the role of mitogen-activated protein (MAP) kinase pathway and gene expressions of p21(waf1/cip1)and nuclear oncogenes (c-fos and c-myc) during AR42J cell differentiation. Dexamethasone markedly increased the intracellular and secreted amylase contents as well as its mRNA level. However, cell growth and DNA content were significantly decreased. With these phenotypic changes, AR42J cells induced transient mRNA expression of p21(waf1/cip1)gene, which reached maximal level by 6 h and then declined gradually toward basal state. In contrast to p21(waf1/cip1), c-fos gene expression was transiently inhibited by 6 h and then recovered to basal level by 24 h. Increased c-myc expression detected after 3 h, peaked by 12 h, and remained elevated during the rest of observation. Dexamethasone inhibited epidermal growth factor-induced phosphorylation of extracellular signal regulated kinase. Inhibition of MAP kinase pathway by PD98059 resulted in further elevation of the dexamethasone-induced amylase mRNA and p21(waf1/cip1)gene expression. These results suggest that p21(waf1/cip1)and nuclear oncogenes are involved in dexamethasone-induced differentiation and inhibition of MAP kinase pathway accelerates the conversion of undifferentiated AR42J cells into amylase-secreting exocrine cells.
Amylases/genetics ; Animals ; Cell Differentiation/*drug effects ; Cell Division/drug effects ; Cell Line, Tumor ; Cyclins/genetics/*metabolism ; Dexamethasone/*pharmacology ; Gene Expression Regulation/drug effects ; Genes, fos/genetics ; Genes, myc/genetics ; MAP Kinase Signaling System/*drug effects ; Mitogen-Activated Protein Kinases/*metabolism ; Pancreas/cytology/*drug effects/enzymology/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Support, Non-U.S. Gov't

OBJECTIVETo study the inhibition effect of grape procyanidin (GPC) on the cell apoptosis and injury of proliferation induced by radiation.

METHODSThree indices including apoptosis rate, proliferation rate and expression of bcl-2 and bax protein were examined in the mice pancreas after taking different dose GPC by mouth and radiation by (60)Co-gamma ray once.

RESULTSThe cell proliferation and bcl-2 expression in high dose GPC group (3.16% +/- 0.13% and 49.8% respectively), were higher than those in radiation control group (0.64% +/- 0.11%, 29.7%), but the cell apoptosis rate and bax expression (19.8% and 55.0% respectively), were lower than those in radiation control group (35.6%, 85.7%). All the above differences were statistically significant (P < 0.05).

CONCLUSIONGPC has certain protective effect against the mice pancreatic cell apoptosis and the abnormal expression of bcl-2 and bax protein induced by (60)Co-gamma.

Animals ; Apoptosis ; drug effects ; radiation effects ; Biflavonoids ; pharmacology ; Catechin ; pharmacology ; Cell Proliferation ; Mice ; Mice, Inbred Strains ; Pancreas ; cytology ; drug effects ; radiation effects ; Proanthocyanidins ; pharmacology ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Vitis ; chemistry ; bcl-2-Associated X Protein ; metabolism

In order to construct the recombinant retrovirus vector of human ngn3 gene and its packaging cell line, we successfully amplified the open reading frame (ORF) of ngn3 gene from human fetal pancreatic tissue by RT-PCR. The PCR products of human ngn3 gene was subcloned into pMD18-T vectors and sequenced. Results showed that its sequence was fully consistent with the ngn3 gene published in GenBank(GenBank Accession No. BC126468). The correct fragment was digested by EcoR I and Hpa I from recombinant pMD18-T vector and inserted into the same restriction enzyme sites of retroviral vector pMSCV-neo. We got recombinant retrovirus vector pMSCV-ngn3, which was identified by double restriction enzyme digestion and then transfected into PT67 cells by lipofectamine 2000. We established the PT67-ngn3 packaging cell line by G418 selection, which was detected by RT-PCR and immunohistochemistry staining. The detection results showed that the Ngn3 expressed at the mRNA and protein level in the packaging cell line. RT-PCR detection and electronic microscope analysis showed that the recombinant retroviral vector pMSCV-ngn3 was packaged into infectious virus particles and released into the supernatant of the cells. These results demonstrated that a PT67-ngn3 packaging cell line was successfully established, and this could facilitate the study of differentiation of the human fetal pancreatic progenitor cells into insulin-producing cells by using the ngn3 gene.
Basic Helix-Loop-Helix Transcription Factors ; biosynthesis ; genetics ; Cell Differentiation ; drug effects ; Cell Line ; Cloning, Molecular ; Fetus ; Genetic Vectors ; genetics ; Humans ; Insulin-Secreting Cells ; cytology ; Molecular Sequence Data ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Open Reading Frames ; genetics ; Pancreas ; cytology ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Retroviridae ; genetics ; metabolism ; Stem Cells ; cytology ; Transfection

OBJECTIVETo investigate the effects of Pantago depressa var. montata. Extract (PDM) on the metabolisms of glucose and lipids in mice as well as the underlined mechanism.

METHODFasting serum glucose (FSG) in normal mice was determined after oral administration of PDM. The effects of PDM on diabetic mice induced by alloxan were investigated by observing the changes of glucose tolerance, the contents of FSG, glycosylated serum protein (GSP), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO) and the injured degree of pancreatic islet. Effects of PDM on the injured human umbilical vein endothelial cell lines (ECV304) induced by H2O2 were also investigated.

RESULTPDM showed no any significant effect on FSG in normal mice. However, in the mice with diabetes induced by alloxan PDM could remarkably decrease serum glucose tolerance, the contents of FSG, GSP, TC, TG and LDL-C and significantly increased the ratio of HDL-C/TC, the activity of SOD and the concentration of NO. The damage of pancreatic islet induced by alloxan was also significantly attenuated by PDM. Furthermore, PDM promoted the viability of injured ECV304, elevated SOD level and reduced the contents of MDA.

CONCLUSIONThe results in the present study suggest that PDM can significantly attenuate hyperlipidemia and hyperglycemia in diabetic mice, which are probably due to its effects of antioxidation and amelioration of damaged pancreatic islet induced by free radicals.

Animals ; Blood Glucose ; metabolism ; Blood Proteins ; Cell Survival ; drug effects ; Cells, Cultured ; Cholesterol ; blood ; Diabetes Mellitus, Experimental ; blood ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Glucose Tolerance Test ; Glycoproteins ; blood ; Humans ; Hypoglycemic Agents ; pharmacology ; Hypolipidemic Agents ; pharmacology ; Lipids ; blood ; Male ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred ICR ; Nitric Oxide ; blood ; Pancreas ; pathology ; Plantago ; chemistry ; Plants, Medicinal ; chemistry ; Superoxide Dismutase ; metabolism ; Umbilical Veins ; cytology