Neuropeptide contents in the duodenum of non-obese diabetic mice

Twelve pre-diabetic and 12 diabetic female NOD mice aged 22–24 weeks were studied. As controls, 12 female BALB/cJ of the same age and sex were used. The duodenal content of several neuropeptides, namely vasoactive intestinal polypeptide (VIP), neurotensin, neuropeptide Y (NPY), galanin, gastrin-releasing peptide (GRP) and enkephalin was determined by radioimmunoassay of tissue extracts. The VIP content in duodenal extracts from both pre-diabetic and diabetic NOD mice was significantly higher than that of the controls. The enkephalin content of the duodenum of diabetic mice was significantly higher than that of the controls, while no significant difference was found between the controls and pre-diabetic mice. There was no statistically significant difference between controls and NOD mice regarding the duodenal content of neurotensin, NPY, galanin or GRP. It has been suggested that the high duodenal content of VIP appears to be primary to the onset of diabetes and that the high enkephalin content may be attributable to the diabetic state. The changes in the duodenal content of VIP and enkephalin reported here in an animal model for diabetes type I might be of relevance for the gastrointestinal complications occurring in human diabetes. Received: 6 August 1997 / Accepted in revised form: 14 November 1997     

Insulin and glucagon in spontaneously diabetic non-obese mice

Summary To investigate the rôle of glucagon in the development of diabetes mellitus, spontaneously diabetic non-obese mice were studied before (group 1) and after the onset of diabetes mellitus (group 2). In group 1, fasting blood glucose and insulin in plasma and pancreas did not differ significantly, while plasma glucagon was elevated (48.9±10.4 versus control 18.6±6.0pmol/l). In group 2, the insulin content of plasma and the pancreas were markedly reduced, whereas plasma glucagon was elevated (180.9±59.1 pmol/l). When diabetic mice were treated with insulin for 4 weeks (group 3), plasma glucagon was markedly reduced compared with that of group 2 (30.3±9.0 pmol/l). In group 1, glucagon and glucagon-like immunoreactivity of the intestine were reduced. The glucagon content of the intestine was elevated in group 2. Group 3 elicited increased contents of gastric glucagon as well as intestinal glucagon-like immunoreactivity. We conclude that, in addition to insulin deficiency, hypersecretion of glucagon might contribute to the development and clinical course of diabetes mellitus in the non-obese diabetic mouse.     

Gangliosides in vivo reduce diabetes incidence in non-obese diabetic mice

Summary Non-obese diabetic mice were treated daily with a mixture of ganliosides from day 30 until day 250 of life or until the mice became diabetic. Ganglioside treatment reduced diabetes incidence from 80–90% to 47% and from 20–30% to zero in female and male mice respectively. Gangliosides did not affect the frequency of perivasculitis. It is concluded that gangliosides can reduce diabetes incidence in non-obese diabetic mice.Deceased     

In utero undernutrition reduces diabetes incidence in non-obese diabetic mice

AIMS/HYPOTHESIS: Observational studies in humans suggest that low birthweight may decrease the risk of type 1 diabetes, but the mechanism is unknown. We hypothesised that antenatal undernutrition would decrease the incidence of type 1 diabetes in non-obese diabetic (NOD) mice. MATERIALS AND METHODS: A 40% restriction of energy intake was applied to pregnant NOD dams from day 12.5 to day 18.5 of gestation, resulting in intrauterine growth retardation of offspring. All mice were fed a standard diet after weaning. Control and undernourished female offspring were followed to assess diabetes incidence. Male NOD mice were treated with cyclophosphamide to accelerate development of diabetes. Glucose homeostasis, body composition and pancreatic histology were compared in control and undernourished offspring. RESULTS: Mean birthweight was lower in undernourished than in control mice (p = 0.00003). At 24 weeks of age, the cumulative incidence of spontaneous diabetes in female mice was 73% in control and 48% in undernourished mice (p = 0.003). In cyclophosphamide-treated male mice, antenatal undernutrition also tended to reduce the development of diabetes (p = 0.058). Maternal leptin levels were lower in undernourished dams on day 18.5 of pregnancy (p = 0.039), while postnatal leptin levels were significantly higher in undernourished offspring at 4, 20 and 27 weeks of life (p < 0.05). Beta cell mass was similar in both groups (control = 0.4 mg; undernourished = 0.54 mg; p = 0.24). Histological evidence of apoptosis at 20 weeks was greater in control than in undernourished mice (control = 6.3 +/- 1.4%; undernourished = 4.2 +/- 0.3%, p = 0.05). CONCLUSIONS/INTERPRETATION: Antenatal undernutrition reduces the incidence of diabetes in NOD mice, perhaps via alterations in apoptosis.     

Insulin-like growth factors prevent cytokine-mediated cell death in isolated islets of Langerhans from pre-diabetic non-obese diabetic mice

Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) contribute to the initial stages of the autoimmune destruction of pancreatic beta cells. IL-1beta is released by activated macrophages resident within islets, and its cytotoxic actions include a stimulation of nitric oxide (NO) production and the initiation of apoptosis. Insulin-like growth factors (IGFs)-I and -II prevent apoptosis in non-islet tissues. This study investigated whether IGFs are cytoprotective for isolated islets of Langerhans from non-obese diabetic mice (NOD) mice exposed to cytokines. Pancreatic islets isolated from 5-6-week-old, pre-diabetic female NOD mice were cultured for 48 h before exposure to IL-1beta (1 ng/ml), TNF-alpha (5 ng/ml), IFN-gamma (5 ng/ml) or IGF-I or -II (100 ng/ml) for a further 48 h. The incidence of islet cell apoptosis was increased in the presence of each cytokine, but this was significantly reversed in the presence of IGF-I or -II (IL-1beta control 3.5+/-1.6%, IL-1beta 1 ng/ml 27.1+/-5.8%, IL-1beta+IGF-I 100 ng/ml 4.4+/-2.3%, P<0.05). The majority of apoptotic cells demonstrated immunoreactive glucose transporter 2 (GLUT-2), suggesting that they were beta cells. Islet cell viability was also assessed by trypan blue exclusion. Results suggested that apoptosis was the predominant cause of cell death following exposure to each of the cytokines. Co-incubation with either IGF-I or -II was protective against the cytotoxic effects of IL-1beta and TNF-alpha, but less so against the effect of IFN-gamma. Exposure to cytokines also reduced insulin release, and this was not reversed by incubation with IGFs. Immunohistochemistry showed that IGF-I was present in vivo in islets from pre-diabetic NOD mice which did not demonstrate insulitis, but not in islets with extensive immune infiltration. Similar results were seen for IGF-binding proteins (IGFBPs). These results suggest that IGFs protect pre-diabetic NOD mouse islets from the cytotoxic actions of IL-1beta, TNF-alpha and IFN-gamma by mechanisms which include a reduction in apoptosis.     

血糖与NOD小鼠肝细胞脂肪变性的关系

目的探讨非肥胖性糖尿病(None-obese diabetic NOD)小鼠的肝组织学改变及其与空腹血糖的关系.方法26只NOD小鼠,低脂低蛋白饮食饲养于屏障系统内.观察至发生显性糖尿病或9月龄时处死.分析肝细胞脂肪变性与空腹血糖的关系.结果实验结束时,11只小鼠空腹血糖水平始终正常,15只(57.7%)发生糖尿病.糖尿病组空腹血糖、糖化血红蛋白较血糖正常组显著升高,两组小鼠体重无显著差异.苏木精-伊红染色显示肝组织学改变基本正常者7例(26.9%),其余小鼠均有泡沫样和(或)大泡性肝细胞脂肪变性,8例(30.8%)达到脂肪肝的诊断标准.相关分析显示,随着NOD小鼠血糖水平升高,肝脂肪变性程度呈加剧趋势;糖尿病组肝脂肪变程度显著重于血糖正常组(秩和检验H=14.084,P＜0.001).脂肪肝组(n=8)血糖和糖化血红蛋白显著高于非脂肪肝组(n=17),分别为26.10±7.12对12.31±10.00mmol/L(t=3.057,P＜0.01)和6.90±1.66对3.91±2.11mmol/L(t=3.547,P＜0.01).结论NOD小鼠是研究1型糖尿病与脂肪肝相关性的良好模型,空腹血糖水平与NOD小鼠肝脂肪变性程度密切相关.     

Losartan and ozagrel reverse retinal arteriolar constriction in non-obese diabetic mice

Objective: Reductions in retinal blood flow are observed early in diabetes. Venules may influence arteriolar constriction and flow; therefore, we hypothesized that diabetes would induce the constriction of arterioles that are in close proximity to venules, with the constriction mediated by thromboxane and angiotensin II. Methods: Using nonobese diabetic (NOD) mice, retinal measurements were performed three weeks following the age at which glucose levels exceeded 200 mg/dL, with accompanying experiments on age‐matched normoglycemic NOD mice. The measurements included retinal arteriolar diameters and red blood cell velocities and were repeated following an injection of the thromboxane synthase inhibitor, ozagrel. Mice were subdivided into equal groups and given drinking water with or without the angiotensin II receptor antagonist, losartan. Results: Retinal arterioles were constricted in hyperglycemic mice, with a significant reduction in flow. However, not all arterioles were equally affected; the vasoconstriction was limited to arterioles that were in closer proximity to venules. The arteriolar vasoconstriction (mean arteriolar diameters = 51 ± 1 vs. 61 ± 1 μ m in controls; p < 0.01) was eliminated by both ozagrel (61 ± 2 μ m) and losartan (63 ± 2 μ m). Conclusions: Venule‐dependent arteriolar vasoconstriction in NOD mice is mediated by thromboxane and/or angiotensin II.     

Ingested interferon α suppresses Type I diabetes in non-obese diabetic mice

Summary Type I diabetes mellitus is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The non- obese diabetic mouse is a model of the human autoimmune disease Type I diabetes [1–3]. We have previously shown that ingested type 1 interferon inhibits chronic relapsing experimental autoimmune encephalomyelitis and the adoptive transfer of experimental autoimmune encephalomyelites by T cells, and decreases both antigen-specific and mitogen-induced pro-inflammatory cytokine secretion in this disorder. We therefore tried to determine whether ingested murine interferon α inhibits insulinitis and suppresses Type I diabetes mellitus in non-obese diabetic mice. Murine interferon α, given daily, decreased islet inflammation and suppressed diabetes. It increased the concanavalin A and ionomycin plus myristic acid palmitic ester-induced production of interleukin 4 and 10 and interferon γ-secretion in spleen cells from treated mice. Adoptive transfer of unstimulated splenocytes secreting interleukin 4 and interleukin 10 from fed interferon α donors suppressed spontaneous diabetes mellitus in recipients. The protective effect of adoptively transferred unstimulated splenocytes shows the presence of ingested interferon α-activated regulatory splenic cell populations that may work via increased interleukin 4 or interleukin 10 production. Ingested interferon α administered during vulnerable periods in at-risk populations may potentially provide a continuous, convenient, non-toxic and effective treatment for Type I diabetes. [Diabetologia (1998) 41: 1227–1232] Received: 14 April 1998 and in revised form: 11 May 1998     

Dissecting autoimmune diabetes through genetic manipulation of non-obese diabetic mice

Type 1 diabetes results from a genetically and immunologically complex autoimmune process that is specifically directed against the pancreatic beta cells. Non-obese diabetic mice spontaneously develop a form of autoimmune diabetes closely resembling the disease in humans. This happens because, like human diabetic patients, non-obese diabetic mice have an unfortunate combination of apparently normal alleles at numerous loci associated with Type 1 diabetes. In isolation, each of these allelic variants affords a small degree of susceptibility to diabetes. In combination, however, they set in motion a series of immunological events that lead to islet inflammation and overt diabetes. Type 1 diabetes is associated with defects in self-tolerance and immunoregulation. It involves presentation of beta cell antigens to autoreactive T lymphocytes by professional antigen-presenting cells, the recruitment of antigen-activated T cells into pancreatic islets, and the differentiation of these antigen-activated lymphocytes into beta cell killers. Understanding the precise sequence of events in the pathogenesis of Type 1 diabetes has been, and remains, a challenging task. Much of our understanding of the immunology of the disease stems from studies of genetically engineered, non-obese diabetic mice. These mice provide reductionist systems, with which the contribution of individual cellular elements, molecules or genes to the disease process can be dissected. This review focuses on the lessons that have been learned through studies of these mice.Abbreviations APCs, antigen-presenting cells - 2m, beta-2 microglobulin - CD62L, L-selectin - CDR3, complementarity-determining region 3 - CTL, cytotoxic T lymphocyte - DC, dendritic cell - IA-2, insulinoma-associated protein 2 - ICA69, islet cell antigen 69 KDa - ICAM-1, intercellular adhesion molecule-1 - IFN-, interferon- - LFA-1, leucocyte function-associated antigen-1 - mAb, monoclonal antibody - NK, natural killer - MIP-1, macrophage inflammatory protein 1 - NOD, non-obese diabetic - 8.3-NOD, 8.3-TCR-transgenic NOD mice - PLN, pancreatic lymph node - rag, recombination-activating gene - RAG-2, recombination-activating gene 2 - RIP, rat insulin promoter - TCR, T cell receptor     

高血糖与非肥胖糖尿病小鼠肝脏病变的关系

目的观察非肥胖糖尿病(NOD)小鼠的肝脏损伤,探讨高血糖与肝脏病变的关系。方法53只雌性NOD小鼠随机分为普通饲料组(NC组,n=23)和高脂饲料组(HF组,n=30)。所有小鼠均在确诊为糖尿病后2周或观察至9月龄时处死。检测各组的肝指数、生化指标及肝脏组织学改变。结果NC组小鼠均无肝细胞脂肪变性发生,但糖尿病组(NC-DM组)肝指数、血糖、TG及ALT、AST均高于非糖尿病组(NC-ND组)小鼠(P0.05)。HF组小鼠均出现不同程度的肝细胞脂肪变性,糖尿病组(HF-DM)重于非糖尿病组(HF-ND)(P0.01),所有小鼠均表现为肝脏轻度非特异性炎症。DM组小鼠肝细胞胞质有糖原蓄积。结论NOD小鼠肝细胞脂肪变性与高脂饮食有关,高血糖可引起胞质糖原蓄积及肝酶学异常,并加剧肝细胞脂肪变性的程度。     

Th1细胞在NOD小鼠糖尿病早期的应用

目的评价Th1细胞对NOD小鼠糖尿病早期变化的作用。方法选择4 w、8 w和16 w的雌性NOD小鼠,采用流式细胞术测定脾Th1细胞,采用免疫组化法检测胰腺内Th1细胞。结果随年龄增加,雌性NOD小鼠脾和胰腺内Th1细胞逐渐增加。结论 Th1细胞参与NOD小鼠糖尿病早期的病理过程。     

Apoptosis in the adrenal gland of non-obese diabetic (NOD) mice.

Apoptosis appears to play an important role in the development of diabetes in the non-obese diabetic (NOD) mouse. Since the autoimmune process leading to the manifestation of insulin dependent diabetes mellitus (IDDM) can also affect the sympathochromaffin system, we analyzed the role of apoptosis and infiltration of the adrenal medulla as features of this autoimmune process in parallel with the development of diabetes. Prediabetic and diabetic NOD mice aged 3 to 30 weeks were studied and compared with control mice. Apoptosis was assessed by in situ end-labeling method and ultrastructural analysis. Adrenals were screened for lymphocytic infiltration by conventional hematoxylin-eosin staining. Chromaffin cells were characterized by immunohistochemical staining against synaptophysin and tyrosine hydroxylase. Apoptotic nuclei were detected in all mice studied at a very low level, mainly occuring within the connective tissue between medulla and cortex. The maximum score was achieved at 3 weeks (1.91+/-0.48 apoptotic cells/1000 counted cells; n = 4). There was no significant difference between NOD mice and control mice. No correlation could be found between blood glucose levels and apoptosis. On the ultrastructural level, apoptotic cells presented typical features of apoptosis, i.e. condensed nuclei and cytoplasm. Neither in NOD mice nor in controls lymphocytic infiltration or fibrosis of the adrenal was detected. Even NOD mice with overt diabetes did not exhibit morphological signs of medullitis. In summary, no signs of immune destruction of the adrenal medulla in NOD mice aged 3 to 30 weeks could be detected.     

Small intestinal enteropathy in non-obese diabetic mice fed a diet containing wheat

Aims/hypothesis A deranged mucosal immune response and dietary factors may play an important role in the pathogenesis of type 1 diabetes. The aims of our work were to look for the presence of small intestinal enteropathy in non-obese diabetic (NOD) mice in relation to the presence of wheat proteins in the diet, and to assess their role in the risk of developing diabetes.Methods Female NOD mice were fed a standard or gluten-free diet or a gluten-free diet with the addition of wheat proteins (MGFD). Small intestine architecture, intraepithelial CD3⁺ infiltration, epithelial expression of H2-IA, mRNA for IFN-gamma and IL-4 were assessed.Results NOD mice fed a standard diet showed reduced villous height, increased intraepithelial infiltration by CD3⁺ cells and enhanced expression of H2-IA and IFN-gamma mRNA when compared with mice on the gluten-free diet. The cumulative diabetes incidence at 43 weeks of age was 65% in the latter and 97% in the former (p<0.01). Mice on MGFD also showed increased epithelial infiltration and a higher incidence of diabetes.Conclusions/interpretation Mice fed a wheat-containing diet showed a higher incidence of diabetes, signs of small intestinal enteropathy and higher mucosal levels of proinflammatory cytokines.     

The anti-inflammatory compound lisofylline prevents Type I diabetes in non-obese diabetic mice

AIMS/HYPOTHESIS: Pro-inflammatory cytokines are increased during the active stages of Type I (insulin-dependent) diabetes mellitus. The aim of this study was to investigate the applicability of using a new anti-inflammatory compound, Lisofylline, to prevent diabetes in non-obese diabetic (NOD) mice. Lisofylline has previously been shown to block Th1 cell differentiation and to reduce IL-1 beta-induced dysfunction in rat islets. METHODS: Lisofylline was added to isolated NOD islets in vitro, with or without IL-1 beta. Insulin secretion and DNA damage of the islets was assessed. Lisofylline was administered to female non-obese diabetic mice starting at 4, 7 and 17 weeks of age for 3 weeks. Cytokines and blood glucose concentrations were monitored. Histology and immunohistochemistry were carried out in pancreatic sections. Splenocytes isolated from donor mice were intravenously injected into immunodeficient NOD (NOD.scid) mice. RESULTS: In vitro, Lisofylline preserved beta-cell insulin secretion and inhibited DNA damage of islets in the presence of IL-1 beta. In vivo, Lisofylline suppressed IFN-gamma production, reduced the onset of insulitis and diabetes, and inhibited diabetes after transfer of splenocytes from Lisofylline-treated donors to NOD.scid recipients. However, cotransfer of splenocytes from both Lisofylline-treated and diabetic NOD donors did not suppress diabetes in recipient mice. CONCLUSION/INTERPRETATION: Lisofylline prevents the onset of autoimmune diabetes in NOD mice by a mechanism that does not seem to enhance the function of regulatory T cells, but could be associated with suppression of proinflammatory cytokines and reduction of cellular infiltration in islets. This study suggests that Lisofylline could have therapeutic benefits in preventing the onset of Type I diabetes.     

Diabetes preventive gluten-free diet decreases the number of caecal bacteria in non-obese diabetic mice

BACKGROUND: A gluten-free diet reduces the incidence of diabetes mellitus in non-obese diabetic (NOD) mice, but the mechanism is not known. The aim of this study was to examine the possible influence of the diet on the caecal bacterial flora, which may affect the intestinal physiology and mediate disease prevention. METHODS: Two groups of NOD mice from the age of 3 weeks were fed either a gluten-free diet or a standard diet. Each diabetic mouse, when diagnosed, along with a non-diabetic mouse from the same diet group and two non-diabetic mice from the alternate diet group were euthanized and sampled for classical bacteriological examination. RESULTS: Nine out of 19 (47%) standard-fed mice and 1 out of 19 (5%) gluten-free-fed mice developed diabetes (p < 0.01). Mice on the gluten-free diet had significantly fewer aerobically (p < 0.01) and microaerophilically (p < 0.001) cultivated bacteria in their intestines than standard-fed mice. Non-diabetic mice also had significantly fewer microa erophilic and anaerobic bacteria than diabetic mice (p < 0.05). These differences were primarily due to a difference in the Gram-positive flora. CONCLUSIONS: The gluten-free diet compared to the standard diet both qualitatively and quantitatively substantially altered the composition of the caecal bacterial flora in NOD mice. Although Gram-positive bacteria might influence the beta cells through certain digestive products, it is more likely to assume that any effect on diabetes incidence is immunological.     

Lymphocyte vaccination protects prediabetic non-obese diabetic mice from developing diabetes mellitus

Summary In the therapeutic manoeuvre termed “lymphocyte vaccination”, activated lymphocytes capable of transferring an autoimmune disease are instead attenuated and given in vaccine form. We have previously shown that such a therapy administered to non-obese diabetic (NOD) mice at 6 weeks of age prevents diabetes mellitus. To assess whether this therapy has potential clinical relevance, in the present study lymphocyte vaccination was applied in NOD mice in 3 weekly doses commencing in the immediate prediabetic period (age 12 weeks), when insulitis is advanced and diabetes incipient. Of 30 NOD mice receiving active vaccine (composed of attenuated lymphocytes from diabetic NOD mice) 13 (43.3 %) remained non-diabetic to the age of 30 weeks, in comparison with 2 of 30 (6.7 %; p < 0.01) mice receiving a control vaccine (composed of attenuated lymphocytes from non-diabetic NOD/B10 mice) and 5 of 26 (19.2 %; p < 0.01) mice receiving saline carrier alone. Moreover, in an additional group of 10 NOD mice receiving active vaccine weekly between 12 and 30 weeks, 8 remained diabetes free at the end of the treatment. The most notable effect of the vaccine was that the delay in diabetes onset was accompanied by a reduction in insulitis and in some cases a complete absence of infiltrating lymphocytes at 30 weeks of age. Immunocytochemistry indicated that when present, islet infiltrating lymphocytes in non-diabetic mice that received active vaccine showed significantly reduced staining for interferon-γ, compared with the infiltrate seen in diabetic mice receiving the control vaccine or saline. This study demonstrates that the rapid progression to diabetes typically seen in 12-week-old NOD mice can be delayed by lymphocyte vaccination, supporting the possibility that a vaccine composed of attenuated autologous peripheral blood lymphocytes could be effective in high risk first degree relatives of patients with insulin dependent diabetes mellitus. [Diabetologia (1997) 40: 1388–1395] Received: 6 May 1997 and in revised form: 24 July 1997     

Gastric inhibitory polypeptide and the entero-insular axis in streptozotocin diabetic mice

The function of the entero-insular axis and abnormalities of circulating gastric inhibitory polypeptide (GIP) were examined in mice for 40 days after induction of streptozotocin diabetes. Compared with untreated controls, streptozotocin diabetic mice exhibited marked hyperglycaemia and hypoinsulinaemia, with impaired body weight gain, lipoatrophy, hyperphagia, intestinal hypertrophy, polydipsia and renal hypertrophy. Plasma GIP concentrations were elevated in fed but not fasted streptozotocin diabetic mice, and oral fat evoked a greater GIP response than control mice. In spite of marked hyperglycaemia, fat-stimulated GIP release did not raise plasma insulin in streptozotocin diabetic mice. Neither oral nor intraperitoneal glucose produced a significant insulin response in streptozotocin diabetic mice, although oral glucose resulted in a smaller change in glycaemia. The results indicate that streptozotocin diabetes in mice is associated with ineffectiveness of the entero-insular axis, despite elevated GIP concentrations, which are probably mediated through hyperphagia and defective feedback inhibition by insulin on intestinal K cells.     

Infection with Schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice

The spontaneous development of insulin dependent diabetes mellitus in non-obese diabetic (NOD) mice has been shown to be mediated by a Th1 response against beta cell antigens. It is known that in murine models of Schistosoma mansoni infection, egg production is associated with a switch from a Th1 to Th2 response. This subsequent dominance of a Th2 response in S.mansoni infected mice has been shown to influence the response to other infectious agents or antigens. We therefore determined whether infection with S.mansoni could influence the spontaneous incidence of insulin dependent diabetes mellitus (IDDM) in NOD mice. Infection with this helminth significantly reduced the spontaneous incidence of IDDM. IDDM was also prevented by injecting parasite eggs alone. Because until relatively recently humans might expect to succumb to a variety of infectious agents, the current freedom from infection might permit the expression of a genetic predisposition to autoimmune pathology and be responsible for the increased incidence of IDDM.