Lack of effect of vitamin D administration during pregnancy and early life on diabetes incidence in the non-obese diabetic mouse.

BACKGROUND AND AIMS: Several studies have suggested that vitamin D supplementation in early life may reduce the risk of developing type 1 diabetes in later life. The non-obese diabetic (NOD) mouse is a model of spontaneous type 1 diabetes currently used for testing hypothesis/compounds aimed at disease prevention. In this study, we tested the effect of vitamin D (16 IU by gavage) on diabetes incidence in NOD/Ba mice treated from conception with olive oil containing vitamin D via maternal dosing up to 10 weeks of age and followed up until 32 weeks of age. METHODS: Twelve breeding pairs were administered olive oil containing vitamin D during pregnancy, 15 days following the birth of the pups and for the next 10 weeks subsequently. The same breeding pairs were bred again after a clearance period of 15 days using a control solution to produce a control litter. This control group received a control solution for the same period of time. Diabetes incidence, degree of insulitis, and insulin content in the pancreas were investigated in the two groups. RESULTS: 12 vitamin D-treated NOD mice developed diabetes compared to 15 animals in the control group (Log rank test p = 0.899, NS). There were no significant differences between the groups in diabetes incidence, time of onset of the disease, degree of insulitis, or the insulin content in the pancreas. CONCLUSION: Vitamin D administered in utero and in the early stages of life at the dosage used does not change the incidence of diabetes or modify the disease process that leads to beta cell destruction in the NOD mouse.

Reduced cumulative incidence of diabetes but not insulitis following administration of chimeric human IL-15-murine IgG2b in NOD mice.

BACKGROUND: It has been recently demonstrated that apoptosis is involved in beta-cell destruction in the NOD mouse model of diabetes. The aim of the present study was to investigate whether IL-15, a cytokine involved in the modulation of the apoptotic process, is capable of modifying the natural history of diabetes and/or insulitis in pre-diabetic NOD mice. The rationale for the use of IL-15-IgG2b recombinant cytokine is related to its long half-life (28 +/- 4 h). METHODS: At 10 weeks of age, 2 groups of 24 female mice were treated with single or multiple i.p. doses of IL-15-IgG2b respectively. As control, 2 groups of 24 age- and litter-matched female mice were injected intra-peritoneally with single or multiple doses of IgG2b immunoglobulin. RESULTS: Diabetes incidence at 33 weeks of age was lower in the group of mice treated with multiple doses than in the control group (p = 0.03). The cumulative incidence of diabetes at 33 weeks of age between single-dose treated mice and the control group was similar. No significant differences in the calculated index of insulitis were observed in all treated and control mice. CONCLUSIONS: We conclude that IL-15-IgG2b reduces the cumulative incidence of diabetes, without affecting the extent and severity of the insulitis process. Considering this and the well-defined anti-apoptotic effects of IL-15, we suggest that the reduction of diabetes incidence could be due to a down-regulation of beta-cell apoptosis.

A wheat-based, diabetes-promoting diet induces a Th1-type cytokine bias in the gut of NOD mice.

Dietary antigens are candidate environmental factors in the pathogenesis of type 1 diabetes. In the non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, cereal-based diets promote disease development, whereas the diets based on hydrolysed proteins or non-diabetogenic proteins are protective. The hypothesis that diabetogenic diets modulate the cytokine balance in the gut was tested. NOD mice were fed with NTP-2000 (mainly a wheat-based milk-free diet) or Prosobee (a semi-purified hypoallergenic diet based on soy protein isolate) or Prosobee plus casein (milk protein fraction). The mRNA levels of IFN-gamma, IL-10, TNF-alpha, TGF-beta, and inducible NO synthase in the small intestine and the Peyer's patches were determined by semi-quantitative RT-PCR. Mice fed on the cereal-based NTP-2000 diet expressed higher levels of the Th1-type and pro-inflammatory markers IFN-gamma, TNF-alpha, and inducible NO synthase mRNA compared to the Prosobee-fed animals. The expression of the counterregulatory cytokines IL-10 and TGF-beta was unaffected. This resulted in a significant bias of the intestinal cytokine balance towards T helper cell type 1 after feeding NTP-2000. The cytokine mRNA levels in the gut-associated Peyer's patches were not affected. Thus, modulation of gut immunoreactivity by diet may contribute to disease development in NOD mice.

A multi-centre, blinded international trial of the effect of A(1) and A(2) beta-casein variants on diabetes incidence in two rodent models of spontaneous Type I diabetes.

AIMS/HYPOTHESIS: The diabetes-inducing potential of cows' milk is still debated and there is no consensus on the diabetogenicity of individual milk proteins. A(1)-beta-casein has been associated with increased diabetes frequency in ecological studies and in NOD mice. Our aim was to ascertain whether A(1)-beta-casein was more diabetogenic than A(2) and to test the diabetogenicity of a milk-free diet in animals representing different forms of spontaneous Type I (insulin-dependent) diabetes mellitus. METHODS: Defined diets were coded and shipped to laboratories in New Zealand (NOD/NZ), Canada (BB) and the UK (NOD/Ba). Base diets were Pregestimil (PG) and ProSobee (PS). Purified fractions of whole casein (WC), A(1)or A(2)-beta-casein were added at 10%. A milk-free, wheat-predominant, NTP-2000 diet was the control. Animals were fed from weaning up to 150 or 250 days, and insulitis, diabetes frequency and expression of pancreatic cytokines were assessed. RESULTS: Diabetes incidence was highest in three locations in animals fed NTP-2000. PG and PS diets were protective except for NOD/Ba mice fed PG+WC where incidence was similar to NTP-2000. A(1) and A(2) diets were protective in both models, but A(1) beta-casein was slightly more diabetogenic in PS-fed BB rats. The New Zealand study was confounded by an infection. CONCLUSION/INTERPRETATION: A milk-free, wheat-predominant diet was highly diabetogenic in three widely separate locations in both animal models. A previous result that A(1) beta-casein was more diabetogenic than A(2) beta-casein in NOD mice was not confirmed; both beta-casein variants were protective in BB rats and NOD mice. Whole Casein promoted diabetes in NOD/Ba but protected BB showing that unique diabetes haplotypes react differently to dietary proteins. A(1)- was more diabetogenic than A(2)-beta-casein only in PS-fed BB rats. Neither the analysis of insulitis nor of pancreatic cytokine gene expression showed a difference between A(1) or A(2) beta-casein fed animals. Milk caseins are unlikely to be exclusive promoters of Type I diabetes, but could enhance the outcome of diabetes in some cases. Other diet components such as wheat could be more important promoters of Type I diabetes.

Adrenalitis in the non-obese diabetic mouse.

The non-obese diabetic (NOD) mouse is a model of spontaneous type-1 diabetes used in the field of diabetes research. This study looked at the adrenal glands of NOD and control mice both indirectly in vivo for hormone secretion, and directly in vitro for histological examination. Adrenal glands were taken from NOD mice, of both sexes, at different ages and corticosterone and adrenocorticotropic hormone (ACTH) plasma levels evaluated by radioimmunoassay. There was evidence of lymphocytic infiltration of the adrenal glands, which however, was not accompanied by changes in corticosterone levels. There was a reduction in ACTH levels with age (R2 = 0.98). Mice from other strains (TFW, CBA and Balb/c) showed no lymphocytic infiltration in the adrenal glands and had lower levels of corticosterone than NOD mice of similar ages, but the differences were not significant. In conclusion, since the NOD mouse shows histological signs of adrenalitis, thyroiditis, sialitis and parathyroiditis, this animal can be regarded as a model to investigate mechanisms involved in diffuse lymphocytic infiltration of peripheral endocrine glands (polyendocrine autoimmunity). In addition, if diabetes in the NOD mouse is the result of a polyendocrine disorder rather than a process specific for diabetes, then this finding may have implications for attempts to prevent type-1 diabetes in humans.

Intradermal administration of GAD & evaluation of diabetes incidence in mice: possible relevance for skin tests in humans.

In vitro cell mediated reactivity to Glutamic Acid Decarboxylase (GAD) has been reported in man and in the non-obese diabetic (NOD) mouse. The demonstration of such reactivity in vivo using GAD in a simple intradermal skin test would be useful for mass screening of subjects at risk of Type 1 diabetes. Such a skin test could be simply applied to the forearm, then signs of local reaction would indicate patients at risk. However, in order to safely apply a skin test of this type it must be certain that administration of the antigen does not itself provoke or start the process leading to diabetes in susceptible individuals. In the present study the NOD mouse model was used. GAD and two peptides of GAD, which may have relevance to the disease process, were applied intradermally to these mice to determine whether a local reaction could be seen and to see if the diabetes rate was altered. Moreover, Balb/c mice, which can be considered to be at zero risk of developing the disease, were also injected with the same GAD and GAD peptides. No significant differences were seen in the diabetes incidence of the treatment groups compared to the control groups in either the NOD or Balb/c mice although a local swelling was seen in female NOD mice susceptible to diabetes after GAD administration in the footpad. We conclude that the administration of GAD and/or GAD peptides does not provoke or accelerate diabetes incidence in the NOD mouse and that an intradermal skin-test with GAD may be suitable for preliminary trials aimed at large scale screening of humans for their potential to develop type 1 diabetes.

Incomplete Freund's adjuvant reduces diabetes in the non-obese diabetic mouse.

As the study of type 1 diabetes moves towards preventive therapy, the role of adjuvants needs to be addressed. Incomplete Freund's adjuvant (IFA) is thought of as "immunologically inert" as, unlike complete FA (CFA), it has no components designed to provoke an immune response. We investigated the effect of IFA as an immunomodulator on the disease process leading to type 1 diabetes in the non-obese diabetic (NOD) mouse. 24 NOD mice were injected intradermally (i.d.) at 8 and 12 weeks of age with a 1:1 mixture of IFA and saline; 24 controls received saline alone. Splenocytes were tested against antigens thought to be involved in the disease process, namely insulin, a GAD peptide, a beta-casein peptide, a Glut-2 peptide and concanavalin A (ConA) as a non-specific antigen. In the IFA experiment diabetes incidence was 13% compared to 38% in the controls (p < 0.05). In vitro, splenocytes from IFA treated animals showed non-specific immunosuppression with ConA (p < 0.01), whereas the response to 1-casein and Glut-2 was raised in IFA treated animals with respect to controls. ELISA using supernatants from IFA treated animals, showed a typical Th2 cytokine pattern, whereas controls showed a Th1 pattern. In conclusion, IFA alone can reduce diabetes incidence in the NOD mouse apparently by modulating the immune response towards beta-cell related specific antigens. As IFA has been adopted as an adjuvant in preventive trials in the NOD mouse, this might have implications for the interpretation of previous and future results.

Diet can influence the ability of nicotinamide to prevent diabetes in the non-obese diabetic mouse: a preliminary study.

BACKGROUND: The non-obese diabetic (NOD) mouse is a widely used model of Type 1 diabetes mellitus (Type 1 DM), which displays many of the characteristics of the disease found in humans. Nicotinamide (NA) is currently being tested in large-scale, multi-centre human trials for the prevention of Type 1 DM in subjects considered 'at risk' of developing the disease. Human trial populations will certainly differ in their dietary patterns and alterations were made to the diet given to NOD mice to determine if this could alter the effect of NA administration on Type 1 DM incidence. METHODS: The effect of NA in the diet was examined, both with and without carbohydrate in the form of a sucrose supplement, on diabetes incidence and insulitis levels in the NOD mouse. The effects of NA and sucrose were each tested alone as well as in combination. RESULTS: Diabetes was unaltered using a low dose NA-supplemented diet (625 mg/kg diet). Diabetes incidence was also unaltered using unmodified diet together with drinking water supplemented with either 5% or 10% w/v sucrose or plain water for controls. However, with mice given NA-supplemented diet (625 mg/kg diet) together with sucrose-supplemented or plain water as previously, diabetes was reduced in the NA+10% sucrose group (p<0.001). Finally, a higher dose of NA was given in supplemented diet (1000 mg/kg). Again, neither sucrose nor NA alone altered the incidence of diabetes, but NA treatment combined with a 10% w/v sucrose-supplemented drinking water reduced diabetes incidence (p<0.001). No mice showed alterations in insulitis, blood-glucose or insulin levels with respect to controls. CONCLUSION: Altering dietary patterns using sucrose can affect the ability of NA to prevent diabetes in the NOD mouse. This finding may be relevant for human studies with NA aimed at preventing Type 1 DM and suggests that diet may need to be monitored or even controlled in these studies.

Troglitazone exhibits immunomodulatory activity on the cytokine production of activated human lymphocytes.

Troglitazone (TGL), a thiazolidinedione compound that improves the response of peripheral target tissue to insulin, also has anti-inflammatory properties, a potential means of protection from Type 1 (insulin dependent) diabetes. In order to test the ability of TGL to affect cytokine production, peripheral blood mononuclear cells from healthy donors were exposed to TGL in the presence or absence of a polyclonal activator (PHA) and the production of cytokines assayed. TGL enhanced PHA response, promoted secretion of the cytokines granulocyte and macrophage colony-stimulating factor and leukaemia inhibitory factor and inhibited tumour necrosis factor-alpha secretion, consistent with causing Th-2 differentiation in T-cells. These results suggest that TGL is capable of modulating cytokine production and could therefore influence Th1/Th2 differentiation.

Troglitazone prevents insulin dependent diabetes in the non-obese diabetic mouse.

Troglitazone has recently been introduced in the treatment of Type 2 diabetes. In addition to its anti-diabetic effects it acts as a perixosome proliferator activated receptor-gamma (PPAR-gamma) agonist and has anti-inflammatory properties by inhibiting macrophage tumour necrosis factor-alpha (TNF-alpha) secretion. It also inhibits the production of endothelial selectin (e-selectin). Troglitazone also reduces interleukin-1alpha induced nitric oxide production in pancreatic beta-cells, which may be relevant in preventing nitric oxide mediated damage to these cells in the Type 1 diabetes process. We tested troglitazone in the spontaneous model of autoimmune diabetes, the non-obese diabetic (NOD) mouse, to determine its effect on the disease process. When administered by gavage from weaning at a dose of 400 mg/kg body weight (n = 32), troglitazone reduced the incidence of diabetes by 16 weeks compared to controls (n = 32) in a pattern that was maintained up to the conclusion of the experiment at 31 weeks of age (p < 0.05). Insulitis was unaltered (index = 1.05 +/- 0.71 vs. 1.13 +/- 0.82, treated vs. controls, p = 0.78). The study was repeated using troglitazone in the diet of NOD mice (n = 24) to give a dose of approximately 200 mg/kg body weight in order to provide a more consistent level of troglitazone during the time course of the experiment. There was a reduction of diabetes incidence in this group but it did not reach significance. Insulin levels were reduced in gavage treated mice although such reduction did not reach significance (p < 0.07). We conclude that, in view of its effect on this model of autoimmune diabetes and because of its known function as an insulin sensitiser, troglitazone might be considered for potential use in those patients with Type 1 masquerading as Type 2 diabetes.

Metformin does not alter diabetes incidence in the NOD mouse.

In insulin-dependent (type 1) diabetes mellitus, increasing peripheral insulin sensitivity might be a useful approach in controlling the process leading to beta cell destruction by reducing insulin output and thereby reducing the antigenicity associated with its release. The aim of this study was to investigate whether the use of a biguanide, Metformin, which has been suggested to increase insulin sensitivity, was capable of modifying the natural history of diabetes in a model of type 1 diabetes, the non-obese diabetic (NOD) mouse. Using age-, sex- and litter-matched groups, three groups of 32 animals each were treated with Metformin in their drinking water at a high dose of 200 mg/kg body weight and at a low dose of 20 mg/kg body weight; the third group of mice acted as controls. Diabetes incidence at 30 weeks of age was similar in all groups. No significant differences in the calculated index of insulitis were observed in treated or control animals. We conclude that Metformin does not affect the disease process leading to clinical diabetes in this animal model.

CD95 and CD95-ligand expression in endocrine pancreas of NOD, NOR and BALB/c mice.

The non-obese diabetic (NOD) mouse is widely used to study the pathogenesis of insulin-dependent diabetes mellitus. However, the mechanisms responsible for beta-cell destruction, in this model, are still poorly defined. The CD95/CD95L system among other effector systems has been implicated in beta-cell death. In this study we investigated in NOD, non-obese resistant (NOR) and Balb/c mice the expression of CD95 and CD95L in alpha and beta pancreatic cells by immunohistochemistry and immunofluorescence. We demonstrate that alpha cells in the islets of Langherans constitutively express CD95L forming a natural shield around beta cells.

Baclofen, a gamma-aminobutyric acid-b receptor agonist, delays diabetes onset in the non-obese diabetic mouse.

Glutamic acid decarboxylase (GAD) is the enzyme responsible for the synthesis of gamma-aminobutyric acid (GABA). GAD has been identified as a 64-kDa antigen expressed in pancreatic beta-cells, to which autoantibodies are generated prior to the onset of type 1 (insulin-dependent) diabetes mellitus. GAD may therefore be an initiating factor in beta-cell destruction. We administered baclofen, a GABA-B receptor agonist, to non-obese diabetic (NOD) mice in an attempt to down-regulate GAD expression and thereby reduce the incidence of diabetes. Twenty-four female NOD mice were given baclofen in their drinking water at a final dose of 50 mg/kg body weight daily from weaning to 30 weeks of age. Twenty-four sex- and litter-matched mice were used as controls. At 30 weeks there was no difference in the incidence of diabetes in the treated group compared with the controls. However, there was a significant delay in the onset of diabetes in the treated group (P < 0.001, parallelism test). The degree of insulitis and the GAD activity in the pancreas per mg of protein were unchanged by baclofen treatment with respect to controls. These results suggest that baclofen may be effective in delaying diabetes onset in NOD mice by stimulating GABA activity, as this neurotransmitter, localised in the islets, may modulate insulin secretion and the antigen expression associated with it.

Vitamin E delays diabetes onset in the non-obese diabetic mouse.

Vitamin E was administered to non-obese diabetic (NOD) mice to determine if the selective destruction of pancreatic beta cells leading to Type 1 (insulin dependent) diabetes mellitus could be halted by virtue of this vitamin's free oxygen radical scavenger activity. Two groups of NOD mice were treated from 3 weeks of age until 30 weeks of age with either diet supplemented with vitamin E or control diet. Diabetes incidence was recorded as well as the degree of lymphocytic infiltration of the pancreas (insulitis) in animals which did not develop diabetes. Vitamin E did not reduce the incidence of diabetes by 30 weeks of age, however it did significantly delay the onset of the disease (p < 0.01--parallelism test). There were no differences in the degree of insulitis with respect to control mice. We conclude that antioxidant therapy with Vitamin E delays diabetes onset in NOD mice without having an apparent effect on the autoimmune process.

Histological study of pancreatic beta-cell loss in relation to the insulitis process in the non-obese diabetic mouse.

Non-obese diabetic (NOD) mice spontaneously develop an autoimmune diabetes with higher incidence in females than in males. In order to elucidate possible factors involved in the different incidence of diabetes between male and female mice, we studied the progression of pancreatic beta-cell loss in relation to mononuclear cell infiltration of the pancreas (insulitis). We examined the pancreas of 76 NOD mice (39 males and 37 females) of different ages. The beta-cell content was assessed by immunoperoxidase staining of sections with an anti-insulin serum and the severity of insulitis was determined by haematoxylin staining of the same sections. A semi-quantitative criterion was used to grade both parameters. The results showed that females have a faster loss of beta-cell mass, which progressively decreases with the increase of severity of insulitis. In males, a medium to severe degree of insulitis is required before initial loss of beta cells occurs. Under the age of 10 weeks there was a significantly lower content of beta cells in females than males (2.84 +/- 0.03 vs 2.67 +/- 0.07; P = 0.02). Since we never observed a significant difference in the degree of mononuclear cell infiltration in age-matched males and females, these data support the hypothesis of weaker beta-cell resistance to immunological attack in female mice. Thus beta-cell sensitivity, in addition to immunological activity, is an important factor in the pathogenesis of insulin dependent diabetes.