Evaluation of T-cell receptor CD3+γδ in gestational diabetes mellitus

Few studies have shown a significant increase of D3⁺ T-cell receptor (TCR) γδ in the early phases of type 1 diabetes. We wished to determine if CD3⁺ TCR γδ is involved in the pathogenesis of gestational diabetes mellitus (GDM). We studied 29 GDM patients and 21 normal pregnant women. Lymphocyte subpopulations (CD3⁺ TCR αδ, CD3⁺ TCR γδ), islet cell antibodies (ICA), glutamic acid decarboxylase antibodies (GAD) and protein tyrosine phosphatase antibodies (IA₂-Ab) were evaluated in all patients. The percentage of CD3⁺ TCR γδ was significantly higher in GDM women than in the control group (5.1 ± 2.9% vs. 3.7 ± 1.7%, p < 0.05%). No abnormalities of the other lymphocyte subpopulations were found. All subjects were negative for ICA; 2 GDM patients wer positive for GAD, but no relationship was found between GAD positivity and CD3⁺γδ levels in these 2 patients. Further follow-up studies of these patients are required to verify if the CD3⁺ TCR γδ receptor is a useful marker for diabetes development. Received: 3 June 2000 / Accepted in revised form: 3 March 2001     

The DPP4 inhibitor linagliptin delays the onset of diabetes and preserves β-cell mass in non-obese diabetic mice

Recent data indicate that dipeptidyl peptidase 4 (DPP4) inhibitors have anti-inflammatory and β-cell-sparing effects in animal models of type 1 diabetes. To evaluate the effects of the DPP4 inhibitor linagliptin on β-cell mass and insulinitis, we examined the progression of diabetes (blood glucose >11?mmol/l) in non-obese diabetic (NOD) mice with terminal stereological assessment of cellular pancreatic changes. Female NOD mice were fed a normal chow diet or a diet containing linagliptin 0.083?g/kg chow for 60 days. At study end, the incidence of diabetes in linagliptin-treated mice was reduced by almost 50% compared with vehicle (10 of 31 mice vs 18 of 30 mice, P=0.021). The total islet mass and total β-cell mass, identified by insulin immunoreactivity, were greater in non-diabetic linagliptin-treated mice compared with non-diabetic vehicle-treated mice (P<0.01 for both) but were greatly reduced in diabetic mice irrespective of treatment. No changes were seen in the α, δ and γ endocrine cell pool. Moreover, the total mass of lymphocyte insulinitis was significantly reduced in linagliptin-treated mice compared with vehicle. The data indicate that linagliptin treatment delays the onset of diabetes in NOD mice by protecting β-cell mass.     

Reg (regenerating) gene overexpression in islets from non-obese diabetic mice with accelerated diabetes: role of IFNβ

Aims/hypothesis The expression of IFNβ in beta cells results in accelerated type 1 diabetes. The REG family of beta cell proliferation factors have been described as autoantigens in autoimmune diabetes. The aim of this study was to determine the effect of IFNβ on Reg expression, and the implications of this in terms of autoimmunity. Methods Reg gene expression was determined in islets from non-obese diabetic (NOD) RIP-HuIFNβ mice by cDNA microarray, quantitative real-time PCR and immunohistochemistry. The effect of IFNβ on Reg1 and Reg2 expression was assessed in the NOD insulinoma cell line NIT-1. IL-6, known to induce Reg expression, was measured in the insulitis microenvironment. Morphological studies were carried out to determine islet enlargement in this model. Results Reg2 was upregulated in islets from the NOD RIP-HuIFNβ mice at the onset of the autoimmune attack. IFNβ upregulates Reg1 and Reg2 genes in NIT-1 cells. The expression of Il6 was increased in islets from transgenic mice and in NIT-1 cells exposed to HuIFNβ. Moreover, islets from transgenic mice were enlarged compared with those from wild-type mice. Conclusions/interpretation Reg overexpression correlates well with the acceleration of diabetes in this model. The upregulation of Reg suggests that islets try to improve hyperglycaemia by regenerating the cells lost in the autoimmune attack. Reg expression is regulated by several factors such as inflammation. Therefore, the overexpression of an IFNβ-induced autoantigen (REG) in the islets during inflammation might contribute to the premature onset of diabetes.     

T-cell receptor Vβ8. 1 peptide reduces coxsackievirus-induced cardiopathology in aged mice

Viral myocarditis is an important cause of heart failure and cardiomyopathy. Immunosenescence, characterized by a dramatic reduction in immune responsiveness, can increase susceptibility to cardiopathology from viral infections. The T-cell receptor (TCR) Vβ 8.1 peptide, a 16-merpeptide, has shown immuno-regulating and immunostimulating effects in viral-induced immunodeficiency. In our study, 18-mo-old C57BI/6 female mice were treated twice with TCR Vβ8.1 peptide and 10 d before sacrifice were injected ip with coxsackievirus B3. Cardiac histopathology was assessed for lesion severity. Splenocyte cytokine production (interleukin-2,-4,-6, interferon-γ) and heart viral titers were determined. Our data suggest that immunosenescence suppressed both T helper (Th1) and Th2 cytokine production and that treatment with TCR Vβ8.1 peptide induced cytokine stimulation close to levels seen in young mice. Nontreated aged mice developed some degree of myocarditis (75% mild and 25% severe), whereas only 35% of the peptide-treated aged group developed cardiopathology, with 25% being mild and 10% severe. Heart tissue from nontreated aged mice infected with coxsackievirus had a higher viral titer than hearts of aged mice equally infected but treated with the peptide. In conclusion, TCR Vβ8.1 peptide induced immunoregulation, and inhibited or reduced coxsackievirus B3-induced, cardiopathology in aged mice.     

Anti-IL-7 receptor-α reverses established type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function

Genetic variation in the IL-7 receptor-α (IL-7R) gene is associated with susceptibility to human type 1 diabetes (T1D). Here we investigate the therapeutic efficacy and mechanism of IL-7Rα antibody in a mouse model of T1D. IL-7Rα antibody induces durable, complete remission in newly onset diabetic mice after only two to three injections. IL-7 increases, whereas IL-7Rα antibody therapy reduces, the IFN-γ-producing CD4(+) (T(H)1) and IFN-γ-producing CD8(+) T cells. Conversely, IL-7 decreases and IL-7Rα antibody enhances the inhibitory receptor Programmed Death 1 (PD-1) expression in the effector T cells. Programmed Death 1 blockade reversed the immune tolerance mediated by the IL-7Rα antibody therapy. Furthermore, IL-7Rα antibody therapy increases the frequency of regulatory T cells without affecting their suppressor activity. The durable efficacy and the multipronged tolerogenic mechanisms of IL-7Rα antibody therapy suggest a unique disease-modifying approach to T1D.     

Effect of arsenic trioxide on human hepatocarcinoma in nude mice

AIM:To study the effect of arsenic trioxide (As_2O_3) on humanhepatoma cell line 8EL-7402 in vivo.METHODS:Human hepatoma cell line BEL-7402 culturedin vitro was inoculated into nude mice and arsenic trioxide,5-Fu and saline were injected into abdominal cavity of thenude mice respectively.The volumes of tumor and generalconditions of the nude mice and structural changes of theliver and kidney were observed.Morphologic changes werestudied under electron microscope.Expression of AFP wasinvestigated by immunohistochemical method.RESULTS:As_2O_3 could inhibit the growth of tumon The tumorgrowth inhibitory rate in mice treated with 2.5 mg/kg As_2O_3was 53.42% on the tenth day.The tumor growth inhibitoryrate in mice treated with 5 mg/kg As_2O_3 was 79.280/0 on thefifth day and 96.58% on the tenth day respectively.As_2O_3 didnot damage the liver and kidney of nude mice,or affect theblood system.Typical apoptotic morphological changeswere found under electron microscope,and the change ofmitochondria was obvious.The expression rate of AFPdeclined after treatment.CONCLUSION:Arsenic trioxide can induce apoptosis ofhuman hepatoma cells,and inhibit proliferation of tumorwith no obvious side effects on liver and kidney.     

Effect of macrophage migration inhibitory factor on insulin resistance and islet β-cell function in gestational diabetes mellitus

To study the level of macrophage migration inhibitory factor (MIF) in serum and the expression of MIF mRNA in abdominal subcutaneous adipose tissue,and to investigate its impact on insulin resistance and islet β-cell dysfunction in gestational diabetes mellitus (GDM).120 pregnancy women from the Affiliated Hospital of Qingdao University Medical College and Taian Central Hospital were enrolled,including 60 GDM women and 60 women with normal glucose tolerance (NGT).The serum MIF in GDM group was higher than that of NGT group [(3.58±1.02 vs 1.23±0.62) ng/ml,P<0.01].Multiple stepwise regression analysis showed that body mass index was an independent affective factor of the serum levels of MIF (r2 =0.516).The serum levels of MIF and the expressions of MIF mRNA in abdominal subcutaneous adipose tissue were significantly higher in GDM group than NGT group.MIF may contribute to insulin resistance and β-cell dysfunction in GDM.Body mass index seems to be an independent factor in affecting the serum levels of MIF.     

Effect of macrophage migration inhibitory factor on insulin resistance and islet β-cell function in gestational diabetes mellitus

To study the level of macrophage migration inhibitory factor (MIF) in serum and the expression of MIF mRNA in abdominal subcutaneous adipose tissue,and to investigate its impact on insulin resistance and islet β-cell dysfunction in gestational diabetes mellitus (GDM).120 pregnancy women from the Affiliated Hospital of Qingdao University Medical College and Taian Central Hospital were enrolled,including 60 GDM women and 60 women with normal glucose tolerance (NGT).The serum MIF in GDM group was higher than that of NGT group [(3.58±1.02 vs 1.23±0.62) ng/ml,P<0.01].Multiple stepwise regression analysis showed that body mass index was an independent affective factor of the serum levels of MIF (r2 =0.516).The serum levels of MIF and the expressions of MIF mRNA in abdominal subcutaneous adipose tissue were significantly higher in GDM group than NGT group.MIF may contribute to insulin resistance and β-cell dysfunction in GDM.Body mass index seems to be an independent factor in affecting the serum levels of MIF.     

Effect of an α-glycosidase inhibitor on experimentally-induced obesity in mice

Summary The effect of prolonged treatment with acarbose, an inhibitor of -glycosidase, has been studied in mice made obese and hyperinsulinaemic by goldthioglucose. After the onset of obesity, one month after goldthioglucose administration, mice were then treated, with or without a 10% sucrose supplement, for four months with acarbose, added to the diet at 50 mg/100 g food. When mice received a standard diet, acarbose had no effect on body weight, blood glucose or insulin levels. In contrast, in the control obese mice receiving a 10% sucrose-enriched diet, it decreased the body weight gain, and prevented the rise in glycaemia and insulinaemia. Basal (non insulin-stimulated) glucose uptake, which is decreased in isolated soleus muscle from untreated obese mice, returned to normal values under acarbose treatment. However, muscle insulin resistance was not improved in acarbose-treated obese mice at maximal and submaximal effective concentrations, despite a higher insulin binding in muscles of acarbose-treated obese than in control obese animals. Furthermore, insulin receptor autophosphorylation and tyrosine kinase activity were altered similarly in treated and untreated obese mice compared to lean mice.     

Effect of insulin and metformin on methylation and glycolipid metabolism of peroxisome proliferator-activated receptor γcoactivator-1A of rat offspring with gestational diabetes mellitus

Objective:To discuss the effect of insulin and metformin on amethylation and glycolipid metabolism of peroxisome proliferator-activated receptor γ coactivator-1A(PPARGC1A) of rat offspring with gestational diabetes mellitus(GDM).Methods:A total of 45 pregnant rats received the intraperitoneal injection of streptozotocin to establish the pregnant rat model of GDM.A total of 21 pregnant rats with GDM were randomly divided into three groups,with 7ruts in each group,namely the insulin group,metformin group and control group.Rats in the insulin group received the abdominal subcutaneous injection of 1 mL/kg recombinant insulin glargine at 18:00 every day.Rats in the metformin group received the intragastric infusion of metformin hydrochloride at 18:00 every day,with the first dose of 300 mg/kg.The doses of two groups were adjusted every 3 d to maintain the blood glucose level at 2.65-7.62 mmol/L.Rats in the control group received the intragastric infusion of 1 mL normal saline at 18:00 every day.After the natural delivery of pregnant rats.10 offspring rats were randomly selected from each group.At birth,4 wk and 8 wk after the birth of offspring rats,the weight of offspring rats was measured.The blood glucose level of offspring rats was measured at 4wk and 8 wk,while the level of serum insulin,triglyceride and leptin was measured at 8 wk.Results:The weight of offspring rats at birth in the insulin group and metformin group was significantly lower than the one in the control group(P0.05),and there was no significant difference at 4 wk and 8 wk among three groups(P0.05).The fasting blood glucose and random blood glucose in the insulin group and metformin group at 4 wk and 8 wk were all significantly lower than ones in the control group(P0.05);there was no significant difference between the insulin group and metformin group(P0.05).The expression of PPARGC1 A mRNA in the insulin group and metformin group was significantly higher and the methylation level of PPARGC1 A was significantly lower than the one in the control group(P0.05),but there was no significant difference between the insulin group and metformin group(P0.05).Insulin and leptin at 8 wk in the insulin group and metformin group were significantly higher,while triglyceride was significantly lower than the one in the control group(P0.05);triglyceride level of rats in the insulin group was significantly higher than the one in the metformin group(P0.05).There was no significant difference in insulin and leptin level of offspring rats between the insulin group and metformin group(P0.05).Conclusions:GDM can induce the methylation of PPARGC1 A of offspring rats to reduce the expression of PPARGC1 A mRNA and then cause the disorder of glycolipid metabolism when the offspring rats grow up;the insulin or metformin in the treatment of pregnant rats with GDM can reduce the methylation level of PPARGC1 A and thus improve the abnormal glycolipid metabolism of offspring rats.     

Effects of chronic administration of alogliptin on the development of diabetes and β-cell function in high fat diet/streptozotocin diabetic mice

Aim: Alogliptin is a potent and highly selective dipeptidyl peptidase‐4 (DPP‐4) inhibitor. The aim of this study was to determine its effects on glucose control and pancreas islet function and to identify the underlying molecular mechanisms after chronic administration, in a non‐genetic mouse model of type 2 diabetes. Methods: Alogliptin (5, 15 and 45 mg/kg) was orally administered to high fat diet/streptozotocin (HFD/STZ) diabetic mice daily for 10 weeks. Postprandial and 6‐h fasting blood glucose levels, blood A1C level, oral glucose tolerance and pancreas insulin content were measured during or after the treatment period. Alogliptin plasma concentration was determined by an LC/MS/MS method. Islet morphology and architectural changes were evaluated with immunohistochemical analysis. Islet endocrine secretion ability was assessed by measuring insulin release from isolated islets which were challenged with 16 mM glucose and 30 mM potassium chloride, respectively. Gene expression profiles of the pancreas were analysed using the mouse diabetes RT² Profiler PCR array which contains 84 genes related to the onset, development and progression of diabetes. Results: Alogliptin showed dose‐dependent reduction of postprandial and fasting blood glucose levels and blood A1C levels. Glucose clearance ability and pancreas insulin content were both increased. Alogliptin significantly restored the β‐cell mass and islet morphology, thus preserving islet function of insulin secretion. Expression of 10 genes including Ins1 was significantly changed in the pancreas of diabetic mice. Chronic alogliptin treatment completely or partially reversed the abnormalities in gene expression. Conclusions: Chronic treatment of alogliptin improved glucose control and facilitated restoration of islet architecture and function in HFD/STZ diabetic mice. The gene expression profiles suggest that the underlying molecular mechanisms of β‐cell protection by alogliptin may involve alleviating endoplasmic reticulum burden and mitochondria oxidative stress, increasing β‐cell differentiation and proliferation, enhancing islet architecture remodelling and preserving islet function.     

Effect of NF-κB decoy on insulin resistance of adipocytes from patients with type 2 diabetes mellitus

Aim

This study aimed to investigate whether NF-κB contributes to insulin resistance in type 2 diabetes (T2DM).

Methods

Subcutaneous abdominal adipose tissue was obtained from T2DM patients and non-diabetic control subjects. Pre-adipocytes were cultured and differentiated into adipocytes in vitro. Upon insulin stimulation, IRS-1 tyrosine and AKT (Ser473) phosphorylation were examined by immunoprecipitation and immunoblotting, while levels of inflammatory mediators IL-6 and MCP-1, and the DNA-binding activity of NF-κB, were examined by ELISA and electrophoretic mobility shift assay (EMSA), respectively. NF-κB decoy molecules were introduced into T2DM adipocytes, and their effects on all these molecular events evaluated.

Results

Compared with cells from non-diabetic subjects, adipocytes from T2DM patients showed signs of insulin resistance, with significantly reduced IRS-1 tyrosine and AKT (Ser 473) phosphorylation levels in response to insulin stimulation. At the same time, T2DM cells displayed elevated levels of IL-6 and MCP-1, and NF-κB activity. Introduction of NF-κB decoy molecules significantly inhibited both IL-6 secretion and NF-κB activity, while enhancing insulin-stimulated IRS-1 tyrosine and AKT (Ser473) phosphorylation in T2DM adipocytes.

Conclusion

Abdominal subcutaneous fat cells from T2DM patients display signs of insulin resistance and microinflammatory status. NF-κB decoy molecules inhibited NF-κB overactivation and also partly reversed insulin resistance. These results provide evidence of a link between inflammation and insulin resistance in T2DM cells, suggesting a potential contribution of inflammation to the mechanism of insulin resistance.     

Effect of α2-adrenoceptor antagonist on platelet activation during insulin-induced hypoglycaemia in Type 2 (non-insulin-dependent) diabetes mellitus

Summary The role of epinephrine in platelet activation and the effect of an ₂-adrenoceptor antagonist, midaglizole, during insulin-induced hypoglycaemia in Type 2 (noninsulin-dependent) diabetes mellitus were examined. The action of midaglizole as a platelet ₂-antagonist was confirmed by in vitro studies using platelet-rich plasma and washed platelet suspension. Hypoglycaemia was induced by a bolus injection of short-acting insulin in 24 diabetic patients. They were divided into two groups, a control group (n=12) and an ₂-group (n=12), and midaglizole was administered orally 60 min before insulin injection in the latter. Blood glucose and plasma C-peptide levels were significantly decreased (p<0.005) by insulin injection in both groups. Counter-regulatory hormones, including epinephrine, and arginine vasopressin were similarly increased at the hypoglycaemic nadir compared with the levels at 0 min in both groups. Plasma -thromboglobulin was increased at the hypoglycaemic nadir (165.5±12.6ng/ml) compared with the level at 0 min (121.0±11.5, p<0.005) in the control group, whereas no significant increase was demonstrated in the ₂ group. These results suggest that plasma epinephrine plays an important role in platelet activation during hypoglycaemia in Type 2 diabetes mellitus, and that the platelet activation is prevented by ₂-adrenoceptor antagonist.     

Effect of TNF-α inhibition on urinary albumin excretion in experimental diabetic rats

The objective is to assess the effect of TNF-α inhibition on urinary albumin excretion in experimental diabetic rats. Male Wistar rats, 8-week-old, were categorized into four groups, which were the control (n = 9), diabetes (n = 9), infliximab-treated diabetes (n = 10), and FR167653-treated diabetes (n = 9) groups. Diabetes was induced by intraperitoneal injection of STZ (40 mg/kg). Thereafter, infliximab was injected intraperitoneally once a month (5.5 mg/kg) and FR167653 was administered orally by mixing with the rat chow (0.08%). The effects of infliximab and FR167653 on urinary albumin excretion were observed for 12 weeks. Body weight, blood sugar, 24-h urinary TNF-α, and 24-h urinary albumin/creatinine ratio (Ualb/Ucr) levels were determined at 1, 4, 8, and 12 weeks after the STZ-injection. Treatment of rats with STZ caused a significant loss of body weight, as well as polyuria and hyperglycemia within 1 week, while the urinary excretions of albumin and TNF-α were increased. Neither infliximab nor FR167653 affected body weight or blood sugar levels, whereas both decreased urinary albumin excretion, together with a modest decrease in the urinary excretion of TNF-α. These results suggest a role of TNF-α in the pathogenesis of diabetic nephropathy and show that TNF-α inhibition is a potential therapeutic strategy.