Part2Impairment Of Insulin Secretion Induced By Glucose In Response To Inflammatory Cytokines In The Mouse Islet

Part1The Method of Mouse Islet Isolation, Purification and IdentificationObjective To investigate the method of isolating and purifying the mouse islets.Methods The isolation of mouse pancreatic islets was carried out by stationary digestion after multi-point injection of collagenase solution. A discontinuous Histopaque solution (1119,1110,1080and1060) was applied to the purification of the islets. The specificity and activity of the islets was determined by dithizone (DTZ) and Trypan blue staining. The function was evaluated by the glucose stimulating insulin releasing test.Results After purification, each pancreas could yield (114±15) islets, with the average purity (77.12±3.23)%and viability>90%. Islet cell insulin release stimulation is good, about20islets in vitro culture medium insulin content in sugar-free, low(2.8mmol/L) and high glucose (22.2mmol/L) concentrations were(23.80±3.52)μIU/mL,(67.57±4.04)μIU/mL,(164.32±10.75)μIU/mL. Statistically significant difference between groups (F=322.36, P<0.05). The insulin from the islets fed with low concentration glucose was2.84times that with OmM concentration(t=-14.15, P<0.05). The insulin from the islets fed with high concentration glucose was2.43times that with low concentration(t=-14.59, P<0.05). The insulin from high concentration glucose was6.90times that with no glucose(t=-21.51, P＜0.05).Conclusion Multi-point injection perfusion by collagenase digestion of the pancreas, discontinuous density gradient Histopaque centrifugation and pick up by manual to take the purification of the islet, it could yield high purity, biology form complete islets. The islets could feel the different concentrations of glucose stimulation. So, it is a simple and efficient method of islet isolation and purification.. Part2Impairment of Insulin Secretion Induced by Glucose in Response to Inflammatory Cytokines in the Mouse IsletObjective To investigate the impairment of insulin secretion induced by glucose in response to inflammatory cytokines IL-1β and IFN-y in mouse islet.Methods The mouse islet were repaired overnight after isolation and purification (by the method of Part1), and then seeded in24-well dishes (20/well, total8wells) and added for48hours in complete culture medium. IL-1β (0.25ng/ml,2.5ng/ml) and/or IFN-γ (1OOu/ml,1000u/ml) was included into the culture medium for24hours. After24hours the islets were preincubated for30min in KRBH buffer (no glucose). KRBH was then discarded and replaced with fresh buffer containing22.2mM glucose for1hour. Supernatants were collected for insulin assays by radioimmunoassay.Results After24hours incubated with cytokines in mouse islet, the concentration of insulin for the control group is (40.61±0.74) uIU/ml, high glucose (22.2mmol/L) insulin release stimulation group (142.48±1.22) uIU/ml, difference between groups was statistically significant (t=-123.669, P<0.05); IL-1β (0.25ng/ml,2.5ng/ml) group in the high glucose (22.2mmol/L) stimulated insulin release is (113.83β5.65) ulU/ml and (91.01±2.62) ulU/ml, compared with pure glucose-stimulated insulin release were decreased by20%[(113.83±5.65)vs (142.48±1.22) ulU/ml, t=8.759, P＜0.05] and36%[(91.01±2.62)vs (142.48±1.22) ulU/ml, t=30.874, P＜0.05]; IFN-y (100u/ml,1000u/ml) group in the high glucose (22.2mmol/L) stimulation of insulin release is (116.09±6.11) ulU/ml and (71.78±3.51)uIU/ml, compared with pure glucose-stimulated insulin release decreased by19%[(116.09±6.11) vs (142.48±1.22) uIU/ml, t=7.336, P <0.05] and49%[(71.78±3.51) vs (142.48±1.22) uIU/ml, t=13.687, P＜0.05]; combination group (IL-1β2.5ng/ml＋IFN-γ1000u/ml) in high glucose (22.2mmol/L) stimulation of insulin secretion (31.77±2.18) uIU/ml was decreased more significantly, compared with IL-1β (2.5ng/ml) group decreased by65%[(31.77±2.18)vs(91.01±2.62) uIU/ml, t=30.103, P <0.05], compared with IFN-ygroup of high glucose-stimulated insulin release, decreased by55%[(31.77±2.18) vs(71.78±3.51) ulU/ml, t=-50.485, P<0.05]; there were significant differences among the three groups (F=329.098, P <0.05).Conclusion Glucose-induced insulin secretion was inhibited by inflammatory cytokines IL-1B, IFN-y or in cooperation in the mouse islet. Part3Activation of MAP Kinases Pathway Induced by Glucose Stimulation in the NIT-1Cell LineObjective To investigate the activation of MAP Kinases signal transduction pathway in the NIT-1cell line in response to glucose stimulation.Methods1.NIT-1cells were incubated in complete culture medium, and then were seeded in6-well dishes(lx105per well, total6wells) and incubated for48hours in complete culture medium then preincubated for0.5hours in the KRBH buffer(no glucose). KRBH was then discarded and replaced with fresh buffer containing0,5.5,11.1,16.7,22.2mM glucose incubated for30minutes. At the end of the incubations, cells were lysed on ice. The supernatants containing whole cell lysates were used for Western blotting.2. Cells were seeded in6-well dishes and incubated for48hours in complete culture medium then preincubated for0.5hours in KRBH buffer. KRBH was then discarded and replaced with fresh buffer containing16.7/11.1mM glucose for0,5,15,30,60,120minutes.At the end of the incubations, cells were lysed on ice. The supernatants containing whole cell lysates were used for Western blotting to investigate the phosphorylation level of ERK1/2, p38and JNK.Results1. The ERK1/2phosphorylation level was increased with glucose concentration compare to control group by glucose stimulation in the NIT-1cell line, it reaches a peak in response to16.7mM glucose stimulation; p38phosphorylation level reaches a peak in response to11.1mM glucose stimulation,16.7mM and22.2mM glucose stimulated p38phosphorylation levels is decreasd then.2. The ERK1/2phosphorylation level begin increased at15minitus, it reaches a peak in response to16.7mM glucose stimulation for30min glucose stimulation and gradually decreased as the stimulation time; the p38phosphorylation level reaches a peak in response to11.1mM glucose stimulation for60min glucose stimulation and gradual decline with stimulation time then.3. The JNK phosphorylation level by5.5,11.1,16.7and22.2mM glucose-stimulated has no change compared with OmM glucose stimulation in NIT-1cells.Conclusion Glucose stimulation can activate ERKl/, p38but had no effect of JNK pathway in the NIT-1cell line.