OBJECTIVE To investigate the effects of heme oxygenase-1 (HO-1) gene on human islets in vitro, and to explore the potential value of gene therapy in clinical islet transplantation. METHODS Adenovirus vector carrying human HO-1 gene (Ad-HO-1) or EGPF (Ad-EGFP) were established respectively. Human cadaveric pancreases were isolated, purified, cultured, and divided into 3 groups to be transfected with Ad-HO-1, Ad-EGFP or blank vector. Human tumor necrosis factor and cyclohexamide (CHX) were added into the culture fluid of the pancreatic islets. 48 hours later the pancreatic islets were digested into single cells. Flow cytometry was used to detect the apoptosis. Glucose of the concentration of 16.7 mmol/L was added into the culture fluid of the 3 groups of islet cells. After 1-hour co-incubation radioimmunochemistry was used to detect the level of insulin in the supernatant. RESULTS After stimulation of glucose the insulin concentration in the supernatant of the Ad-HO-1 group was 270 mIU/L +/- 89 mIU/L, significantly higher than those of the Ad-EGFP group (189 mIU/L +/- 88 mIU/L) and control group (182 mIU/L +/- 59 mIU/L, both P < 0.05). The apoptotic ratio of the Ad-HO-1 group was 63.1% +/- 10.9%, significantly lower than that of the control group (90.9% +/- 11.3%, P < 0.01) after treatment with TNFalpha and CHX. CONCLUSION Transfection of Ad-HO-1 into human islets improves anti-apoptotic function in cultured human islets and promotes insulin release of human pancreatic islets.
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.
Key words:
Islet cells; Adipocytes; IKKβ; a-lipoic acid
Objective To in ve stigate islet autoantibodies in relation to clinical characteristics and islet β cell function in patients with diabetes. Methods Using cohort study, the general clinical and biochemical characteristics w ere investigated in 733 hospitalized diabetic patients. Serum C peptide and seru m antibodies to islet cell cytoplasmic antigens (ICA), glutamic acid decarboxyla se (GADA) and protein tyrosine phosphatase (IA-2A) were determined by immunoflu orescence method or commercial kits. Results Tw enty cases (34.5%) showed positive serum GADA and 13 cases (22.4%) positive ICA in 58 patients with acute onset and ketoacidosis, who were diagnosed as type 1 d iabetes. Serum IA-2A was determined in 22 of these 58 patients and 6 (27.3%) ca ses showed positive results: among 675 patients diagnosed as type 2 diabetes, 91 cases (13.5%) showed at least one antibody positive, 68 (10.1%) were found GADA positive and 18 (2.7%) ICA positive and 23 (8.7%) IA-2A positive (263 sera wer e tested). According to positive or negative serum antibodies and types of onset of the disease, the patients were divided into three groups, type 1 diabetes (w ith acute onset), latent autoimmune diabetes in adults (LADA) and type 2 diabete s. The prevalences of autoantibodies were almost the same in LADA patients with different durations of diabetes. However, the prevalence became lower in type 1 diabetes patients after 2 years. Fasting and postprandial C peptide levels fell gradually with advancing course of the disease. The falling speed was the fastes t in type 1 diabetes patients and the slowest in type 2 diabetes patients among the three groups. Stepwise regression analysis showed that GADA was a risk facto r for declining of fasting and postprandial C peptide levels. Co nclusion There are differences in autoantibody prevalences amon g type 1 diabetes, LADA and type 2 diabetes patients. Combined detection of the autoantibodies would be helpful for diagnosis. LADA patients have slow β cell d estruction and show higher serum C peptide levels even many years after diagnosi s of diabetes, and protecting their residual β cells might slow down the progre ssion of diabetes.
Background Islet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation. Methods Cadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI=20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI=20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-α (rTNFα) and cycloheximide (CHX) for 48 hours. Results Adenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 ± 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 ± 89.37) mIU/L and (175.95 ± 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P<0.05). After treatment with rTNFα and CHX the apoptotic ratio of islet cells was (63.09 ± 10.86)% in the HO-1 group, significantly lower than (90.86 ± 11.25)% in the control group (P<0.05). Conclusions Transduction of human islets with Ad-HO-1 can protect against TNF-α and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.
Obesity and β-cell dysfunction due to oxidative stress impact the pathogenesis of type 2 diabetes mellitus. We co-cultured 3T3L1 adipocytes and islet cells in the presence or absence of the antioxidant α-lipoic acid (LA) and assayed the effects of the adipocytes and LA on the secretion of insulin by the islet cells and on the activities of factors involved in secretion and oxidative stress. At low glucose concentrations (2.8 mmol/l), the presence of adipocytes (co-culture) increased insulin secretion compared with islet cells cultured alone (control) and this increase was diminished by LA (co-culture plus LA). At high glucose concentrations (22 mmol/l), insulin secretion levels were similar for all islet groups, resulting in a restoration of the stimulation index in the presence of LA. The mRNA levels of the glucose-stimulated insulin secretion (GSIS) genes glucokinase, glucose transporter 2 and Kir6.2 were downregulated under co-culture and co-culture plus LA conditions. Protein and tyrosine phosphorylation levels of insulin receptor-β and insulin receptor substrate-1 were decreased under co-culture conditions and were restored by LA treatment. Cellular malondialdehyde levels increased in the co-cultured islets and this increase was blocked by LA. The mRNA levels of superoxide dismutase and catalase were reduced under co-culture conditions and these reductions were eliminated by the addition of LA. In conclusion, 3T3L1 adipocytes disturb insulin secretion and induce islet dysfunction. The effects may be mediated by multiple pathways, which include downregulation of GSIS gene expression, suppression of islet cell insulin signaling and the induction of oxidative stress. LA may protect islet cells via activation of islet cell insulin signaling and the mRNA expression of antioxidant enzymes.
To investigate the effects of immunosuppressive agents on insulin secretion of human islet cells in vitro.Human islet cells were isolated by the solution of the liberase and purified by Ficoll's density gradient centrifugation and then were exposed to various concentrations of four immunosuppressive agents for 24 hr respectively. Glucose-stimulated insulin secretion during subsequent static incubation was measured using the human insulin ELISA kit.Glucose-stimulated insulin secretion from human islet cells was significantly reduced after exposed to high concentrations of MMF and FK506 (both P < 0.05). No significant reduction in insulin secretion was observed from human islet cells after exposed to FTY720 and rapamycin (both P > 0.05).(1) High concentrations of MMF and FK506 have deleterious effects on insulin secretion in human islet cells. Low-dose FK506 + MMF is available for clinical use. (2) FTY720 and rapamycin have no adverse effects on insulin secretion in human islet cells. FTY720 and rapamycin may become useful immunosupressants for future clinical islet allotransplantation.
Islet transplantation represents a potential curative treatment for diabetes mellitus, but considerable transplanted islets are injured by rejection and other stimulating factors after transplantation. Therefore, reduction of damage of transplanted islets may be critical for successful islet transplantation. Pancreatic islets, as a cellular graft, are especially suited for reconstitution ex vivo, and an attractive strategy to protect islets is to use gene therapy to transduce islets prior to transplantation with factors that can inhibit the local immune attack or make islet cells more resistant to apoptosis. Gene transfer vectors and targeted gene are key strategies of gene therapy.
Objective To research the expression difference in angiotensin Ⅱ (AT Ⅱ) and angiotension-converting enzymes 2(ACE2) in mice pancreas between type 1 and 2 diabetes mellitus.Methods The protein expression of AT Ⅱ and ACE2 were measured by immunohistochemical and Western blotting in 12-week-old and 24-week-old C57b1/6 mice (n=10),nonobese-diabetes (NOD) mice (n=10) and type 2 diabetes model (db/db) mice (n=10).Results The expression of AT Ⅱ was increased significantly in 12-week-old and 24-week-old db/db mice pancreatic tissue.ACE2 were also found to be markedly upregulated in 12-week-old NOD pancreas but not in 24-week-old NOD pancreas;meanwhile the expression regulation of ACE2 was in the opposite way in db/db mice.Conclusions The expressions of ACE2 is increased in the early T1DM pancreas,but AT Ⅱ don't change significantly;however,the expression of AT Ⅱ is raised in early and late T2DM pancreas,and ACE2 is also found to be upregulated in advanced stage.
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