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Objective To test whether glucagon like peptide-1 (GLP-1) would regulate the expression of visfatin in adipocytes,and to explore the mechanism of this effect.Methods Fully differentiated 3T3-L1 adipocytes were treated with GLP-1.Total RNA was extracted for analyzing the level of visfatin mRNA by quantitative RT-PCR.The media were collected for measuring the level of visfatin protein by enzyme linked immuno-assay (ELISA).In order to test the involvement of PKA pathway,the adipocytes were pretreated with a specific pharmacological PKA inhibitor H89 for 30 min before GLP-1 was added.Results GLP-1 increased visfatin expression in a time-and dose-dependent manner.The level of visfatin significantly increased at the concentration of 10 10 mol/L GLP-1 (P＜0.05),and reached the peak at 10-9 mol/L (P＜0.01).After incubation for 18 hours,GLP-1 dominantly increased the level of visfatin (P＜0.05).Inhibition of PKA pathway by H89 partially blocked the effect of GLP-1 on visfatin expression.Conclusions GLP-1 may enhance the expression of visfatin in 3T3-L1 adipocyte via the PKA pathway,which might contribute to the improvement in glucose homeostasis.
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ObjectiveTo compare the yield rate of rats islets between different collagenase digestion groups.MethodsThe SD rats were randomly divided into two groups as following by using random digits table:collagenase P group (pancreas digested by 1 mg/ml collagenase P) and type Ⅴ collagenase group (pancreas digested by 1 mg/ml type Ⅴ collagenase).After pancreas digestion,rat islet cells in two groups were culture,purified and stained with DTZ.The mean islet number and islet equivalent (IEQ) before and after purification were measured under an inverted microscope.The viability of purified islets was assessed by fluorescence staining of aridine orange (AO) and propidium iodide (PI) under the fluorescence microscopy.After purification and culture for two days,islets function was evaluated by insulin releasing tests in the two groups.ResultsBefore purification,there was no significant difference in the islets number obtained from the pancreas between two groups (P＞0.05),but there was significant difference in the IEQ (P＜0.05).After purification,the islets number in type Ⅴcollagenase group and collagenase P group was (485 ± 113)/pancrease and (643 ± 82)/pancrease,and IEQ was (674 ± 157)/pancreas and (989 ± 126)/pancreas,respectively (P＜0.05).Islet viability in type Ⅴcollagenase group and collagenase P group was (96.13 ±1.13) ％ and (96.38 ± 0.92) ％ respectively (P＞0.05).The results of insulin releasing tests revealed there was no significant difference in islet function stimulated by hypoglycemia and hyperglycemia between two groups (P＞0.05).ConclusionTwo types of collagenase are suitable for the islets digestion in rats.The stability of digestion and yield rate of purified islets in collagenase P group are higher than in type Ⅴ collagenase group.
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To investigate the effects and the mechanism of visfatin on MIN6 cell signaling pathway and apoptosis induced by palmitate.Human recombinant visfatin promotes protein kinase B (Akt) and extracellularsignal regulating kinase (ERK)1/2 phosphorylation in dose-and time-dependent manner,and prevents MIN6 cell from apoptosis induced by palmitate (P<0.05 or P<0.01).The activation of Akt and ERK1/2 signaling pathway may be one of the molecular mechanisms of visfatin.
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Objective To explore the difference involved in the activation of inflammation pathway and the plasma level of inflammatory factors in the subjects with different sorts of insulin sensitivity. Methods The study was carried out in 38 women, consisting of obesity (n = 22 ) and control (n = 16 ) groups according to body mass index. The insulin sensitivity was assessed by homeostasis model assessment of insulin resistance (HOMAIR). Plasma concentrations of interleukin-6 (II-6) and IL-1β were determined by enzyme immunoassay. Western blot analysis was used to examine total protein expression and phosphorylation levels of IκB kinase (IKK) ,inhibitor of nuclear factor-κB ( IκB ) in peripheral blood leukcocytes. Electrophoretic mobility shift assay (EMSA)was used to detect the binding activity of NFκB. Results The levels of fasting plasma insulin[62.2 ( 20.0-127. 0) pmol/L vs 19. 15 ( 14. 2-47. 8 ) pmol/L, P<0. 01], HOMA-IR[2. 32 ( 0. 76-5.49 ) vs 0.70(0.53-1.7),P<0.0l], HbA1 C[(5.42±0. 45 ) % vs ( 5.08 ±0. 38) %, P<0. 05], triglyceride[( 1.75 ±0. 68 vs 1.22 ±0. 58 )mmol/L, P<0. 05], plasma IL-6[3. 15 (0. 03-22. 2) pg/ml vs 1.26 (0. 74-6.06 ) pg/ml, P<0. 01], and IL-1 β[6. 53 ( 0. 84-36 ) pg/ml vs 3. 16( 1.48-8. 86 ) pg/ml, P<0. 01]in obesity group were significantly higher than those in control group. Compared with control group, the levels of IKKo, IKKβ expression and IκBα serine phosphorylation in obesity group were markedly increased, while the expression of IκBα was significantly reduced. Accompanied with the degradation of IκBα protein, the binding activity of NFκB in obesity group was significantly increased. Conclusions The plasma levels of IL-6 and IL-1β were significantly raised in obesity group. The activation of IKK-IκB-NFκB pathway is closely associated with the genesis and development of insulin resistance in obese subjects.
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Objective To investigate the effects of differentiated 3T3-L1 adipoeytes on inflammation of rat islet cells,as well as the protective effect of a-lipoic acid on the inflammation in vitro.Methotis Rat islet cells were divided into three groups:the control group,the experimental co-culture system group(cocuhured with differentiated mature 3T3-LI adipoeytes)and the intervention group (cocuhured with mature 3T3-LI adipocytes containing 4 μg/ml a-lipoic acid).Insulin releasing lest wag performed for estinmting the function of islet cells in culture supernatant of difierent groups.At the same time,the expression level of IKKIβin islet cells Was detected by western blot and realtime PCR.Results There was significant decrease of insulin stimulation index (SI) in experimental co-culture system group compared with the control group and intervention group(1.0 ±0.1 vs 2.6±0.2,2.5±0.5 respectively;P<0.01),while,the mRNA(4.62±0.60 vs1.00±0.46 and 2.25±0.75;P<0.01)and protein expression of IKKβ were significandy increased in the experimental group as compared with the other two groups.Conclusions In the co-culture system of adipocytes/islet cells,impaired function of islet cells could be induced by IKKβ activation,IKKβ Was a key molecule in inflamnmtion signal pathway in islet cells and could be activated by 3T3-LI adipocytes.a-lipoic acid Was able to reverse the impaired function of islet cells by suppressing IKKβ expression.
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Visfatin was expressed in rat anti mouse islets,as well as in MIN6 cells. The visfatin expression was affected by various concentrations of environmental glucose (5.5 and 33.3 mmoL/L) and palmitate(0.5 mmol/L). As compared with low-level glucose medium (5.5 mmol/L, 1.0±0.11) , visfatin expression increased in media with high glucose and palmitate (1.32 ±0. 18, 1. 33±0. 15,1.72±0.27, all P<0. 05). The result suggests that visfatin seems to be involved in the regulation of insulin secretion.
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NOD mice were treated with pentoxifylline (FTX) to investigate the incidence of cyclophosphamide-accelerated diabetes, the apoptosis and the insulin expression of β-cells and expressions of Fas or FasL mRNA in both pancreas and spleen. The results showed that incidence of diabetes in PTX group was significantly lower compared with control group (P<0.05). The apoptosis of β-cells was decreased in PTX group with higher insulin expression level in islet cells. The expression of FasL mRNA in pancreas of PTX group was lower than that of control group (P<0.05), and there was no significant difference in Fas mRNA expression between two groups. Both Fas and FasL mRNA levels in spleen of PTX group were much higher than those of control group (P<0.05 or P<0.01).
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Objective To observe the effect of three immunosuppressive agents on the function of human adult islets of langerhans in vitro, and explore the suitable immunosuppressive protocols for human islet transplantation.Methods In vitro, the isolated and purified islets were incubated with the immunosuppressive agents (mycophenolic acid, sirolimus, and FTY720) at different concentrations for 24 h, then stimulated by glucose at low ((2.8) mmol/L) and high ((16.7) mmol/L) levels. The (insulin) release after stimulations was detected using ELISA kit, and stimulation index was calculated.(Results) In control group (no agents exposure), the insulin release were ((7.37)?(1.74)) ng/ml at low (level) and ((15.15)?(5.39)) ng/ml at high level respectively, with stimulation index achieved to (2.06)?(0.46). Compared to control group, treatment with mycophenolic acid led to dramatic reduction of (insulin) release (P
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Objective To investigate the influence of pretr eatment with anti-B lymphocyte serum on the immunogenecity and endocrine functi on of islet preparations.Methods Porcine ICCs were pretre ated with anti-B lymphocyte serum which was produced using the mixed adult porc i ne B lymphocyte as antigen.The immunogenecity of islet was measured by mixed isl et lymphocyte culture (MILC) and SLA-DR positive cell number within the islet and the islet function was assayed with insulin release test.Results The cpm value of MILC of the group pretreated with anti-B lymphocy te serum and the MHCⅠ antigen and SLA-DR antigen positive cell numbers was mar kedly decreased in comparison with that of control group (P＜0.001).There wa s no significant difference in insulin release of additional different groups.[ WT5”HZ Conclusion The pretreatment of islet preparation with anti -B lymphocyte serum may decrease the immunogenecity of islet preparation,while there is no marked influence on its insulin release function.
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AIM: To investigate the intervention effect of pentoxifylline(PTX) on type 1 diabetes mellitus in non-obese diabetic(NOD)mice and explore its possible mechanism.METHODS: Eight-week-old NOD mice were treated with PTX to investigate the incidence of cyclophosphamide accelerating diabetes.The apoptosis of beta-cells was detected by TUNEL,the expressions of caspase-3 in islet of the NOD mice was checked by immunohistochemistry and the expressions of caspase-8 was determined by RT-PCR.RESULTS: The incidence of diabetes in PTX group was 40.63%,which was obviously lower than 69.70% in the control group(P
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Objective To lower immunogenesis of human adult islets,reduce the dose of immunosuppressors and promote wide development of transplantation of adult islets,isolated adult islets were in vitro pretreated.Methods Isolated adult islets were cultured at 24 ℃ for 2 days(24 ℃ group) or pretreated using MHC-II monoclonal antibody(monoclonal antibody and complements were added and cultivated for another day following one-day-24 ℃-culture,antibody group) as observation groups.Adult islets were cultured at 37 ℃ in CMRL 1066 medium plus 20 % fetal bovine serum for 2 days as control.The immunogenics of the islets was detected using lymphocyte-islet mixed culture(MILC) and immunohistochemical staining of HLA-DR and lymphocyte common antigen(LCA).The function and activity of the islets were identified using()~3H-leucine incorporation assay,insulin release test and in situ apoptosis assay.Results Compared with control,MILC stimulation index was remarkably lower in the antibody group(P(0.05)).The percentage of HLA-DR and LCA positive cells in the antibody groupand in the 24 ℃ group was much lower than that in control(All P
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Objective To investigate the influence of pretreatment with anti-B lymphocyte serum on the immunogenecity and endocrine function of islet preparations.Methods Porcine ICCs were pretreated with anti-B lymphocyte serum which was produced using the mixed adult porcine B lymphocyte as antigen.The immunogenecity of islet was measured by mixed islet lymphocyte culture (MILC) and SLA-DR positive cell number within the islet and the islet function was assayed with insulin release test.Results The cpm value of MILC of the group pretreated with anti-B lymphocyte serum and the MHCⅠ antigen and SLA-DR antigen positive cell numbers was markedly decreased in comparison with that of control group ( P
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The positive rate of autoantibodies was 29.7% in 1617 diabetic patients. The positive rate of monoautoantibody was lower than that of combined assay with 3 autoantibodies. Combined assay of multiple autoantibodies may enhance the positivity of early diagnosis of type 1 diabetes mellitus. Glutamic acid decarboxylase antibody (GADA) may be a better screening test for predicting insulin dependency, the prediction of GADA combined with protein tyrosine phosphatase antibody may approximate to 100%, whereas islet cell antibody assay may be needed in the adolescent patients.