Transplantation of purified islet cells in diabetic BB rats

Summary The ability to prepare purified islet Beta-cell aggregates was used to examine the survival of this cell type after allotransplantation in diabetic BB rats. The aggregates were intraportally implanted in numbers that were previously found to correct a streptozotocin-induced diabetic state in syngeneic or allogeneic Brown Norway recipients. When the grafts were prepared from RT1^u/l donors, which shared the MHC-class I antigen with the BB recipients (RT1^u/u), their implant sites became diffusely infiltrated by inflammatory cells and their metabolic function was completely lost within 5 weeks. MHC-class I incompatible islet Beta-cell allografts (RT1^n/n) exhibited a longer survival, in particular when combined with other islet endocrine cells and/or when covered by a 5-week cyclosporin treatment. In the latter combination, 10 of 12 BB rat recipients remained normoglycaemic over the 10-week observation period, their liver implants presenting a comparable insulin reserve and similarly discrete mononuclear cell infiltration as streptozotocin-diabetic Brown Norway rats receiving this treatment. However, administration of cyclosporin to diabetic BB rats was associated with a morbidity that was not observed in drug-treated streptozotocin-diabetic Brown Norway animals or in untreated diabetic BB rats. It is concluded that MHC-incompatible islet Beta cells can induce a long-term normalization in diabetic BB rats provided that they are implanted under conditions which allow allograft acceptance. The standardized preparation of purified islet Beta-cell grafts can help assessing the conditions for successful transplantations in diabetes with an autoimmune origin.     

Pre-diabetes in the spontaneously diabetic BB/E rat: pancreatic infiltration and islet cell proliferation

A cohort of BB/E rats derived from litters with a high and low incidence of IDDM was studied prospectively to examine the relationship between circulating autoantibodies, islet insulin secretion, pancreatic infiltration, and islet cell replication during the pre-diabetic period. Although a higher incidence of islet cell surface (ICSA) and insulin autoantibodies (IAA) was detected in the diabetes-prone than in the low diabetic-incidence BB/E rats there was no correlation between the two antibodies in individual animals. Moreover, ICSA, but not IAA, were associated with loss of first phase islet insulin release. Between 75 and 105 days of age the number of diabetes-prone rats with ICSA and impaired islet insulin secretory function increased. Over the same period, there was a concomitant increase in the proportion of diabetes-prone animals with pancreatic infiltration, and increased islet endocrine cell proliferation. All these interrelated phenomena were observed in diabetes-prone BB/E rats at a time when the animals were normoglycaemic.     

Apoptosis and disease progression in the spontaneously diabetic BB/S rat

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is an autoimmune disease culminating in pancreatic beta-cell destruction. A role for apoptosis in this destruction has been suggested, although controversy exists over the identity of the apoptotic cells and the time of onset of apoptosis. This study investigates the extent and timing of islet cell apoptosis in vivo in the spontaneously diabetic BB/S rat. Methods. Pancreatic biopsies were taken from 30 diabetes-prone and 6 diabetes-resistant BB/S rats matched for age. Animals were serially biopsied before, during and after development of diabetes and apoptotic cells analysed in serial sections. The diabetes-prone group included animals (n = 6) that had insulitis but did not develop diabetes. Results. Apoptosis was not detected in any pancreatic sections from diabetes resistant animals at any age investigated or from any animal before 50 days of age. By 68 days, apoptosis was, however, detectable in both the diabetes-prone group and in the group that had insulitus but did not develop diabetes and this correlated with a decrease in pancreatic insulin staining and a development of insulitis. There was a further increase in apoptosis in the diabetes-prone group at 85 days, which coincided with the time of onset of diabetes (84 days). In addition, there was a sixfold increase in intra-islet apoptosis between 68 and 85 days in the diabetes-prone group and at 85 days intra-islet apoptosis was threefold higher in the diabetes-prone group than in the group that had insulitus but did not develop diabetes. At 107 days, apoptosis (total and intra-islet) was higher in the group that had insulitus but did not develop diabetes (OND-DP) than in either the diabetes resistant (DR) or diabetes-prone (DP) groups. Conclusion/interpretation. We have shown significant islet cell apoptosis in the pancreas of diabetes-prone BB/S rats, which coincides with the appearance of insulitis and the onset of diabetes. We have also detected differences in the levels of apoptosis between diabetic and non-diabetic animals and suggest that such differences could be an important determinant of disease progression in this animal model of Type I diabetes. [Diabetologia (2001) 44: 320–324] Received: 15 May 2000 and in revised form: 31 October 2000     

The BBZ/Wor rat: clinical characteristics of the diabetic syndrome

Summary The BBZ/Wor rat is a model of obesity and autoimmune diabetes mellitus developed by crossing the BB/Wor and Zucker rats. We studied circulating glucose and insulin levels, islet morphology and lymphocyte subsets in lean and obese BBZ/Wor rats before and after the onset of diabetes, and studied the clinical course of diabetes in animals after interruption of exogenous insulin therapy. Lean BBZ/Wor rats developed insulin-dependent diabetes and died in ketoacidosis within 1 week after cessation of insulin injections. Diabetes also developed in obese rats, but these animals were not insulin-dependent and survived for months without insulin therapy. The islets of the lean diabetic rats revealed complete destruction of pancreatic beta cells and plasma insulin levels were virtually undetectable. In contrast, the islets of the obese rats revealed insulitis and substantial beta-cell loss, however autoimmune bete-cell destruction was incomplete, and residual beta cells were presumably responsible for the presence of measurable levels of plasma insulin and the long-term survival of obese diabetics without insulin therapy. Obese rats were hyperinsulinaemic, developed diabetes significantly earlier, and with a greater incidence than lean rats, suggesting a possible relationship between enhanced beta-cell metabolic activity and immune destruction. Obese males became diabetic more frequently and at an earlier age than obese females and lean rats of both sexes, suggesting a role for gender in the pathogenesis of diabetes. We conclude that the BBZ/Wor rat is a unique animal model for investigating the interaction of obesity, beta-cell metabolism, autoimmune insulitis and genetic predisposition to diabetes.     

Testicular Sertoli cells exert both protective and destructive effects on syngeneic islet grafts in non-obese diabetic mice

Abstract Aims/hypothesis. Testicular Sertoli cells protect allogeneic islet grafts from rejection after transplantation into animals with chemically induced diabetes. The aims of this study were to determine whether Sertoli cells can protect syngeneic islets from autoimmune destruction after transplantation into non-obese diabetic (NOD) mice and, if so, whether protection is due to Sertoli cell expression of Fas ligand (FasL), believed to be the mechanism that protects against allograft rejection.?Methods. We compared the survival of syngeneic islets transplanted under the renal capsule of non-obese diabetic mice, alone and together with purified Sertoli cells prepared from testes of newborn non-obese diabetic mice. Additionally, we examined the composition of the islet and Sertoli cell co-transplants by immunohistochemistry to determine whether islet graft survival correlated with Sertoli cell expression of Fas ligand.?Results. Sertoli cell doses of 1, 2 and 4 × 10⁶ cells produced a dose-dependent prolongation of median islet graft survival from 11 days (islets alone) to 32 days (islets + 4 × 10⁶ Sertoli cells); addition of 8 × 10⁶ Sertoli cells to the islet grafts decreased, however, median survival to 8 days. Immunohistochemical analysis of the islet and Sertoli cell co-transplants showed a correlation between Fas ligand expression by Sertoli cells and graft infiltration by neutrophilic leucocytes, leading to islet beta-cell destruction and diabetes recurrence.?Conclusion/interpretation. Sertoli cells exert opposing effects on survival of syngeneic islet grafts in non-obese diabetic mice: Fas ligand-dependent neutrophil infiltration and graft destruction, and Fas ligand-independent protection of the graft from autoimmune destruction. [Diabetologia (2000) 43: 474–480] Received: 13 December 1999 and in revised form: 20 January 2000     

Adoptive transfer of diabetes to and from old normoglycaemic BB rats

Summary Approximately 4% of diabetes-prone BB/ Mol rats escape overt diabetes which occurs in other rats between 56 and 130 days of age. The ability of preactivated spleen cells from older non-diabetic and from acutely diabetic rats to adoptively transfer diabetes into young diabetes-prone rats was compared, and it was found that they transferred disease with similar incidence and with overlapping onset times in the recipients. Old non-diabetic rats were themselves susceptible to diabetes adoptively transferred from acutely diabetic or from old non-diabetic donors. Lymphocytic insulitis and pancreatic insulin content in unmanipulated old non-diabetic rats were both intermediate between those seen in acutely diabetic and in diabetes-resistant rats. In vivo treatment with polyinosinic-polycytidylic acid induced diabetes with faster onset in old non-diabetic rats than in young diabetes-prone rats. Adoptive transfer of fresh, whole spleen cells from old non-diabetic rats did not protect young BB rats against spontaneous diabetes, while cells from diabetes-resistant rats did. Spleens from old non-diabetic rats contained significantly lower percentages of T cells than spleens from acutely diabetic rats but not lower than spleens from age-matched diabetic rats, suggesting that this reduction was age-related. Finally, spleens from both old non-diabetic and from acutely diabetic rats were negative for the regulatory RT6⁺ T-cell subset. It is concluded that quiescent beta-cell autoimmunity seen in a fraction of BB/ Mol rats can be reactivated upon non-antigen-specific immune stimulation.Abbreviations DP Diabetes-prone - DR diabetes-resistant - OND old non-diabetic - AD acutely diabetic - OD old diabetic - AT adoptive transfer - NK natural killer     

Studies on autoimmunity for initiation of Beta-cell destruction VIII. Pancreatic Beta-cell dependent autoantibody to a 38 kilodalton protein precedes the clinical onset of diabetes in BB rats

Summary Autoantibody to a rat islet cell-protein of 38 kilodalton was detectable at around 30 days of age in the sera of diabetes-prone Biobreeding (DP-BB) rats by both immunoprecipitation and differential Western blotting methods. Anti-38 kilodalton islet cell autoantibody was not, however, observed in the sera from 5- to 20-day-old DP-BB rats. Over 90% of DP-BB rats in which the antibody was detected, eventually developed Type 1 (insulin-dependent) diabetes mellitus. The antibody disappeared within 2 weeks after diabetes onset. However, it was preserved in the sera of DP-BB rats which had been treated with silica to prevent insulitis. The anti-38 kilodalton islet cell autoantibody was not detected in sera from control Wistar Furth (WF) rats. The autoantibody also cross-reacted with a rat insulinoma (RINm5F) cell protein of 38 kilodalton, but did not react with protein from mouse fibroblast (L-929 cells), rat pituitary cells (GH3 cells), or normal rat lymphocytes. The production of the autoantibody appears to be pancreatic Beta-cell dependent, since the autoantibody disappears after almost complete depletion of Beta cells, but is consistently present as long as Beta cells remain. Identification of the Beta-cell dependent anti-38 kilodalton islet cell autoantibody, which cross-reacts with a rat insulinoma cell protein of 38 kilodalton and precedes the onset of Type 1 diabetes in BB rats, will be invaluable for study of the molecular nature of a target islet cell autoantigen associated with the induction of autoimmunity in DP-BB rats.     

The effect of hyperglycaemia on pancreatic islets transplanted into rats beneath the kidney capsule

Summary The effect of hyperglycaemia on islet transplantation in the rat has been examined in two ways, using syngeneic transplantation of 400 islets to the kidney capsule and subsequent measurement of kidney insulin content as a measurement of B-cell survival. Firstly, islets were transplanted into either diabetic or normal rats, then 6 months later the composite kidney/islet graft was transplanted into a normal rat. The insulin content was measured after a further 6 months and was found to be significantly reduced in islets exposed to hyperglycaemia in the primary recipient. These findings are interpreted as showing that long-term exposure of islets to hyperglycaemia results in B-cell loss. In the second experiment 400 islets were transplanted into either a long-term (6 months) diabetic or a normal rat. Two weeks later the composite kidney/islet graft was transplanted into a normal rat. The insulin content was measured after a further 6 months and no significant difference was found, whether the primary recipient was diabetic or not. These results are interpreted as showing that islet graft implantation is not impaired in long-term diabetic rats.     

A dithiocarbamate analogue decreases intraislet cell infiltration and the incidence of diabetes mellitus in the genetic diabetes-prone BB rat

SUMMARY: Dithiocarbamates are a class of agents that have interesting biologic properties including the ability to limit the production and/or action of nitric oxide (NO). These agents are also potential immunosuppressant agents. Since immunosuppressant agents have been examined for remission of disease in clinical trials, we wanted to examine whether a dithiocarbamate analogue, NOX-200, might inhibit diabetogenesis in the genetic diabetes-prone BB rat model. Immunohistochemical analysis revealed inducible NO synthase (iNOS) gene expression in pancreatic islets of both normoglycemic and hyperglycemic diabetes-prone BB rats but not in diabetes-prone BB rats at the early age of 30 days or in diabetes-resistant BB rats. A qualitative decrease in immunostaining for iNOS was also observed in the pancreata of drug-treated animals. Long-term treatment with NOX-200, used alone or in combination with low-dose cyclosporine (CsA), significantly reduced the incidence of diabetes mellitus. In the subset of animals that became diabetic, NOX-200 did not alter either the time to onset of hyperglycemia or the level of hyperglycemia, insulinopenia, or lymphocytic cell infiltration into the pancreas. In contrast, in animals that did not develop hyperglycemia, treatment with NOX-200 decreased inflammatory cell infiltration into the pancreas equipotent to that seen using CsA. These studies demonstrate the potential therapeutic efficacy of dithiocarbamates to oppose the development of autoimmune insulin-dependent diabetes mellitus by limiting inflammatory cell activation/infiltration.     

Diabetic nephropathy and beta-cell replacement therapy

Whole pancreas transplantation can effectively restore endogenous insulin secretion in type 1 diabetes mellitus, and prevent, retard, or reverse diabetic complications. The effect of a simultaneous pancreas and kidney transplantation (SPKT) on diabetic complications is variable. These reports must be interpreted in the light of the fact that most recipients received a pancreas in combination with a kidney graft after having already had diabetes for over two decades. Nevertheless, the potential benefits should also be balanced against the risk of peroperative morbidity and the requirement of long-term immunosuppressive medication. Transplantation of a whole pancreas is currently the only reliable option to achieve long-term normoglycaemia. The success of pancreatic islets transplantation will ultimately depend on the longevity of pancreatic islets, requiring further development of immunosuppressive regimens which are not toxic to the islets and prevent recurrent autoimmune destruction of transplanted pancreatic beta-cells.     

Congenic BB.SHR rat provides evidence for effects of a chromosome 4 segment (D4Mit6-Npy approximately 1 cm) on total serum and lipoprotein lipid concentration and composition after feeding a high-fat, high-cholesterol diet

Congenic BB.SHR (previously referred to as BB.LL) rats were generated by transferring the segment of chromosome 4 flanked by the D4Mit6 and Spr loci from the spontaneously hypertensive rat (SHR/Mol) onto the genetic background of the diabetes-prone BB/OK rat. In this study, the influence of the above-mentioned region of chromosome 4 on triglyceride, cholesterol, and phospholipid phenotypes after a high-fat, high-cholesterol diet was examined by comparison of BB.SHR congenic rats with BB/OK rats. BB/OK and BB.SHR had comparable concentrations of basal and postdietary serum insulin, as well as of basal total serum triglycerides and had an identical body weight and food intake at the beginning of the test period. However, after 4 weeks on the test diet, BB.SHR rats were significantly heavier than BB/OK rats and had significantly higher food intake and lower total serum triglyceride concentrations. The basal serum leptin level was significantly lower, but postdietary serum leptin concentration did not show a significant difference between the 2 strains. Furthermore, significantly higher basal total serum cholesterol and phospholipid levels were observed in BB.SHR rats, but this difference disappeared after feeding the high-fat, high-cholesterol diet. Postdietary high-density lipoprotein (HDL)(2) cholesterol and phospholipid levels were significantly elevated in BB.SHR rats when compared with BB/OK rats. The 2 strains also differed slightly, but significantly, with respect to the other HDL phospholipid concentrations. In addition to previously described differences between BB/OK and BB.SHR rats, the results of this study clearly show the impact of genes, lying within the transferred segment, on serum lipid phenotypes after high-fat, high-cholesterol diet.     

Fas ligand-mediated mechanisms are involved in autoimmune destruction of islet beta cells in non-obese diabetic mice

Aims/hypothesis. A mechanism implicated in pancreatic islet beta-cell destruction in autoimmune diabetes is the binding of the Fas ligand (FasL) on T cells to Fas receptors on beta cells, causing their destruction. Evidence for this mechanism is, however, controversial. The aim of this study was to find whether the Fas ligand contributes to beta-cell death in autoimmune diabetes. Methods. We transplanted syngeneic islets under the renal capsule in non-obese diabetic (NOD) mice and treated the mice with a neutralizing monoclonal antibody to the Fas ligand. Survival of beta cells in islet grafts and phenotypes of graft-infiltrating cells were investigated. Results. We found 58 % (7 of 12) of mice treated with anti-Fas ligand antibody were normoglycaemic at 30 days after islet transplantation compared with none (0 of 9) of the mice treated with control antibody. Immunohistochemical analysis of islet grafts showed that infiltration of leucocytes (CD4⁺ T cells, CD8⁺ T cells, macrophages and neutrophils) and apoptosis of beta cells in the grafts was significantly decreased in mice treated with anti-Fas ligand antibody. Expression of proinflammatory cytokines (interleukin 1 alpha, tumour necrosis factor alpha and interferon gamma) was not different in islet grafts of mice treated with anti-Fas ligand and control antibodies. Conclusion/interpretation. These findings indicate that Fas ligand-mediated mechanisms play a major part in promoting leucocytic infiltration of islets and beta-cell destruction in autoimmune diabetes. [Diabetologia (2000) 43: 1149–1156] Received: 31 March 2000 and in revised form: 5 June 2000     

Implanted islets in the anterior chamber of the eye are prone to autoimmune attack in a mouse model of diabetes

Aims/hypothesis

Type 1 diabetes is an autoimmune disease resulting from the destruction of insulin-producing beta cells. Along with advances in generating replacement beta cells for treating diabetes, there is also increasing demand for non-invasive tools to evaluate the recurrence of autoimmune attack on transplanted tissue. Here, we examined the anterior chamber of the eye as a potential islet transplant site, and also evaluated whether in vivo imaging of the islets transplanted in the eye could enable real-time visualisation of autoimmune processes underway in the pancreas.

Methods

Syngeneic islet equivalents were transplanted into the eye or kidney capsule of streptozotocin-induced diabetic C57BL/6 mice to compare islet dose (25–125 islet equivalents) and function across transplant sites. Autoimmune attack of syngeneic islets was evaluated in the pancreas and eye tissues of NOD and NOD-severe combined immunodeficient (SCID) mice given diabetogenic splenocytes.

Results

Islet transplantation in the eye decreased fasting plasma glucose levels and increased weight gain and survival in an islet-dose-dependent manner. Even 50 islets in the eye reduced blood glucose levels, whereas ≥200 islets were required in the kidney for a similar effect. Autoimmune destruction of pancreatic islets in the eye mirrored that in the pancreas and could be visualised in real time by non-invasive imaging.

Conclusions/interpretation

We found that far fewer islets were required to restore normoglycaemia when transplanted into the anterior chamber of the eye vs the kidney capsule. However, our results suggest that islets are not protected against autoimmune attack in the eye, making this a suitable site for visualising autoimmune processes against transplanted tissue.     

Decreased levels of serum alpha-isoamylase prior to diabetes onset in BB rats

The development of insulin-dependent diabetes mellitus (IDDM) includes a prodrome of autoimmunity against pancreatic beta cells. The period of subclinical islet cell disease with altered beta-cell function may be prolonged. We have determined the serum pancreatic alpha-isoamylase in both young diabetes-prone (DP) and newly diabetic BB rats to test whether changes in the pancreas prior to IDDM are reflected by this enzyme, shown to be regulated by insulin. A prospective analysis of inbred BB rats (n = 28) that later developed diabetes showed that the alpha-isoamylase at the time of onset was reduced by 19% (p less than 0.02) compared with levels observed 1 week earlier and by 30% (p less than 0.01) compared with levels 2 weeks before onset. Furthermore, when compared to age-matched diabetes-resistant (DR) BB rats in a cross-sectional study, the DP BB rats investigated in groups at 20, 30, 40, 50, 60, and 70 days of age had significantly lower (p less than 0.01) serum alpha-isoamylase already from 50 days of age, which is 2-6 weeks prior to the expected onset of diabetes. Finally, in 70-day-old cofostered DP and DR male rats with identical body weight and rates of growth, the serum alpha-isoamylase was decreased in the DP yet nondiabetic (n = 8) rats compared with the DR (n = 8) rats (p less than 0.05). Reduced levels of serum alpha-isoamylase, therefore, may reflect loss of beta cells or beta-cell function in the pancreas of diabetes-prone but not yet diabetic BB rats.     

111Indium-labelled lymphocytes do not image or label the pancreas of the BB/W rat

Summary Autologous transfusions of ¹¹¹indium-labelled peripheral blood lymphocytes reportedly image the pancreas of patients with Type 1 (insulin-dependent) diabetes at the time of onset. We attempted to apply this technique to the spontaneously diabetic BB/W rat. First, acutely diabetic BB/W rats, diabetes-prone BB/W rats, diabetes-resistant W-line BB/ W rats, and Wistar Furth rats were given autologous transfusions of labelled peripheral blood lymphocytes. Radioactivity recovered from the pancreas was similar in all groups. No correlation was found between the intensity of imaging and the presence or intensity of insulitis. To decrease non-specific intravascular radioactivity, acutely diabetic, diabetes-prone, and W-line rats were perfused 48 h after autologous transfusion of labelled lymphocytes. Again, the intensity of recovered activity was similar in all groups, using both macroautoradiography and numerical counting techniques. A second set of experiments studied diabetes and insulitis induced by passive transfer of concanavalin A-treated splenic lymphocytes from acutely diabetic donors. Activated lymphocytes were labelled with ¹¹¹Indium and given to groups of diabetes-prone and diabetes-resistant rats. There were no differences in pancreatic localization 72–96 h after injection. Groups of diabetes-prone and diabetes-resistant rats were also given concanavalin A-activated lymphocytes and then challenged 2–10 days later with autologous transfusions of labelled peripheral blood lymphocytes. Again, no differences in organ labelling or imaging were detected. We conclude that the autologous transfusions of ¹¹¹indium-labelled lymphocytes do not label or image the pancreas of the BB/W rat.     

Normal insulin sensitivity during the phase of glucose intolerance but insulin resistance at the onset of diabetes in the spontaneously diabetic BB rat

Summary In diabetes-prone BB rats, 30 to 50% of animals undergo autoimmune destruction of the pancreatic B-cells leading to a short period of glucose intolerance, followed by an abrupt onset of diabetes. We have examined whether the glucose intolerance period and the onset of diabetes are associated with changes in insulin sensitivity, using the euglycaemic hyperinsulinaemic clamp coupled with [3-³H] glucose infusion. Glucose intolerant rats were detected by a transient glycosuria one hour after an oral glucose load performed every four days. Insulin sensitivity studied in these rats the day following their detection was normal. Other diabetes-prone BB rats were tested daily and studied on the first day of glycosuria. In the basal state, glucose production was increased in diabetic rats (11.3±1.1 vs 7.1±0.8mg·min^–1·kg^–1, p<0.05). Tissue glucose utilization was similar in diabetic and control rats (8.3±0.5 vs 7.1±0.8mg·min^–1·kg^–1) despite a three fold higher glycaemia in the diabetic rats. During the hyperinsulinaemic clamps, glycaemia was clamped at 6.1–6.6 mmol/l in diabetic and control rats. A decreased insulin sensitivity was observed in diabetic rats at submaximal (200 U/ml) and maximal (1500 U/ml) insulin concentrations for both inhibition of hepatic glucose production and stimulation of glucose utilization. No autoantibodies against insulin could be detected in the plasma of diabetic rats. Plasma concentrations of glucagon, catecholamines, ketone bodies and fatty acids were similar in control and diabetic rats during the clamp studies. Our results suggest that the decrease of basal insulin concentration is responsible for the insulin resistance in the diabetic BB rat at onset of diabetes, either directly or through the increased glycaemia.     

Insulin prevents adoptive cell transfer of diabetes in the autoimmune non-obese diabetic mouse

Summary Early intensive insulin treatment is thought to improve subsequent Beta-cell function in Type 1 (insulin-dependent) diabetic patients. Prophylactic insulin administration also reduced diabetes incidence in diabetes-prone animals. To study the mechanisms by which these effects occur, we tested the ability of insulin therapy in the model of non-obese-diabetic mice, to prevent the penetration of committed T cells into the islets and subsequent Beta-cell destruction. Sublethally irradiated non-obese-diabetic males of 8 weeks of age were adoptively transferred with splenocytes from diabetic donors and treated with the maximum tolerable dosage of fast-acting insulin (0.5 U, twice daily) until 30 days after cell transfer. Diabetes incidence was compared to control animals injected with the same concentration of insulin diluent. After one month of treatment, the cumulative diabetes frequency was significantly less within the insulin-treated group (4 of 15, 26.6%) than in the control group (15 of 18, 83.3%; p <0.01). Pancreatic histological analysis of insulin-treated animals revealed a lower severity of insulitis and Beta-cell necrosis and a higher percentage of normal islets (46.6±10% vs 2.3±2%, p < 0.01), including five (33%) mice with no lesions. Immunoperoxydase staining of pancreatic sections indicated similar insulin and ganglioside staining of Beta cells from insulin-treated mice and control animals. Insulin-treated mice had comparable pancreatic insulin content to normal mice. Flow cytometry analysis of spleen cell populations indicated that insulin increased the number of Thy1,2⁺ and Lyt-2⁺ T cells. Although an effect at the Beta cell level cannot be definitely excluded, several lines of evidence suggest that insulin may influence the capacity of effector T cells to invade the islets and cause Beta-cell destruction. These effects may have potential interest for future immunointervention trials in Type 1 diabetic patients of recent onset.     

Preservation of GLUT 2 expression in islet beta cells of Kilham Rat Virus (KRV) — infected diabetes-resistant BB/Wor rats

Summary Loss of GLUT 2, the glucose transporter isoform of pancreatic beta cells, has been reported to accompany the onset and perhaps contribute to the pathogenesis, of insulin-dependent and non-insulin-dependent diabetes mellitus in BB/Wor and Zucker fatty rats. In this study we investigated the effect of Kilham Rat Virus infection on GLUT 2 expression in diabetes-resistant BB/Wor rats. Viral antibodyfree diabetes-resistant rats do not develop spontaneous diabetes, but inoculation with Kilham Rat Virus induces autoimmune beta-cell destruction and hyperglycaemia. Pancreas sections from normoglycaemic diabetes-resistant BB/Wor rats were obtained 5, 7 and 25 days after inoculation with Kilham Rat Virus and stained for GLUT 2 using a rabbit polyclonal antibody. At all time points, beta cells displayed GLUT 2 expression comparable to uninfected diabetes-resistant controls. Immunostained insulin content of the beta cells also remained unchanged. Sections were also examined from Kilham Rat Virus inoculated diabetes-resistant rats with lymphocytic insulitis or diabetes. GLUT 2 and insulin immunostaining were unchanged in non-diabetic rats with early insulitis. GLUT 2 beta-cell staining was variably reduced in diabetic rats with established insulitis and reduced beta-cell insulin immunostaining. Hence, the initial stages of Kilham Rat Virus-induced diabetes in diabetes-resistant rats are not accompanied by a significant reduction in GLUT 2 expression. These results suggest that the loss of GLUT 2 does not play a significant role in the aetiology of diabetes in the Kilham Rat Virus-infected diabetes-resistant BB/Wor rat.Abbreviations KRV Kilham Rat Virus - NHS normal horse serum - BB/Wor Biobreeding/Worcester rats