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Research on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation.We compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data.After type 1 diabetes manifestation CD8(+) T cells, CD68(+) macrophages and CD4(+) T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed.With the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Udgivelsesdato: 2007-Mar AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes. MATERIALS AND METHODS: Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20. RESULTS: A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p

Aims/hypothesis Accumulating data suggest that the gut immune system plays a role in the development of type 1 diabetes. The intestinal flora is essential for the development of the (gut) immune system and the establishment of tolerance. It has been reported that oral administration of food and bacterial antigens early in life suppresses later development of diabetes in the Bio-Breeding diabetes-prone (BB-DP) rat. This study was designed to investigate the possible relationship between the development of diabetes and the composition of intestinal flora.Materials and methods The intestinal flora of BB-DP rats, a rat model for type 1 diabetes, was characterised long before the clinical onset of diabetes by fluorescent in situ hybridisation. In a separate experiment, BB-DP rats were treated with antibiotics and the effect on diabetes incidence and level of insulitis was analysed.Results We observed a difference in bacterial composition between rats that eventually did and those that did not develop diabetes. This difference was detectable long before clinical onset of the disease. Rats that did not develop diabetes at a later age displayed a lower amount of Bacteroides sp. Modulation of the intestinal flora through antibiotic treatment decreased the incidence and delayed the onset of diabetes. A combination of antibiotic treatment and a protective hydrolysed casein diet completely prevented diabetes in the BB-DP rat.Conclusions/interpretaion Our data suggest that the intestinal flora is involved in the development of type 1 diabetes. Factors influencing composition of the intestinal flora could be a target for therapeutic intervention.

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is characterized by selective destruction of the insulin producing beta cells. Interleukin-1β (IL-1β) modulates the beta-cell function, protein synthesis, energy production and causes apoptosis. We have previously shown changes in the expression of 82 out of 1 815 protein spots detected by two dimensional gel electrophoresis in IL-1β exposed diabetes prone Bio Breeding (BB-DP) rat islets of Langerhans in vitro. The aim of this study was to identify the proteins in these 82 spots by mass spectrometry and compare these changes with those seen in IL-1β exposed Wistar Furth (WF) rat islets. Methods. The 82 protein spots, that changed expression after IL-1β exposure, were all re-identified on preparative gels of 200 000 neonatal WF rat islets, cut out and subjected to mass spectrometry for identification. Results. Forty-five different proteins were identified from 51 spots and grouped according to function: (i) energy transduction and redox potentials; (ii) glycolytic and Krebs cycle enzymes; (iii) protein, DNA and RNA synthesis, chaperoning and protein folding; (iv) signal transduction, regulation, differentiation and apoptosis; (v) cellular defence; and (vi) other functions. Comparison of IL-1β exposed BB-DP and WF islets showed common changes in 14 proteins and several proteins influencing similar pathways, suggesting that similar routes in the two strains lead to beta-cell destruction. Conclusion/interpretation. We demonstrate that proteome analysis is a powerful tool to identify proteins and pathways in BB-DP rat islets exposed to IL-1β.

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]

Aims/hypothesis. Renal hypertrophy occurs early in diabetes mellitus and precedes the development of glomerulosclerosis and tubulointerstitial fibrosis. We have previously shown that cultured mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and undergo cellular hypertrophy. High glucose-mediated induction of p27Kip1, an inhibitor of cyclin-dependent kinases, is essential in this process. Further investigations have also shown that p27Kip1 and p21Cip1, other cyclin-dependent kinase inhibitors, are up regulated in the kidneys of mice with Type I (insulin-dependent) as well as Type II (non-insulin-dependent) diabetes mellitus. Our study was undertaken to test a potential effect of short-term treatment with the angiotensin-converting enzyme inhibitor enalapril on the glomerular expression of the cyclin-dependent kinase inhibitors p16INK4, p21Cip1, and p27Kip1 in BBdp rats, an autoimmune model of Type I diabetes.¶Methods. We evaluated p16INK4, p21Cip1, and p27Kip1 protein expression in isolated glomeruli by western blots. We also assessed p27Kip1 positive glomerular cells by immunohistochemistry.¶Results. Glomerular expression of all three cyclin-dependent kinase inhibitors were stimulated in BBdp rats compared with non-diabetic BBdr animals. Enalapril treatment for 3 weeks, started after the onset of diabetes, reduced the glomerular expression of p16INK4 and p27Kip1 but not of p21Cip1. Enalapril also prevented the increase in kidney weights observed in BBdp rats but had no effect on systolic blood pressure or glucose concentrations.¶Conclusion/interpretation. Our data show that enalapril attenuates the glomerular expression of cyclin-dependent kinase inhibitors in diabetes and suggest a molecular mechanism of how angiotensin-converting enzyme inhibitors prevent renal hypertrophy in diabetes. [Diabetologia (1999) 42: 1425–1432]

The impact of oral treatment with insulin on disease development was studied in diabetes prone BB rats. Because of the positive outcome of a prior study in non obese diabetic (NOD) mice, BB rats received insulin in combination with a bacterial adjuvant. Porcine insulin was given orally twice weekly from 35–100 days of age, the E. coli preparation OM-89 was fed on alternate days. Other groups received vehicle, the bacterial adjuvant, or insulin alone. Both insulin containing oral dosing regimens induced a transient non significant delay in diabetes onset. Insulin alone, however did not decrease the final diabetes incidence. Oral dosing with insulin plus adjuvant caused exacerbation of disease development as judged from the decreased survival rate in comparison with the insulin treated group (p < 0.05). Intra-islet infiltration also increased (p < 0.005) compared with the insulin or vehicle treated groups. The effect correlated with enhanced interferon gamma (IFNγ) and decreased interleukin 10 (IL-10) gene expression in the gut suggesting a shift towards proinflammatory T helper 1 (Th1) reactivity (p < 0.01). Although treatment with adjuvant alone also increased the degree of insulitis, an enhanced incidence of diabetes and a shift in cytokine expression was only seen in the group receiving insulin plus adjuvant. Taken together, the data suggest that treatment with a bacterial adjuvant and oral insulin may alter the gut immunoregulatory state such that disease promoting rather than protective immune responses are induced. [Diabetologia (1998) 41: 844–847]

In diabetes prone BB rat pancreas the Th1/ Th2 cytokine balance and the expression of inducible nitric oxide synthase (iNOS) was determined by mRNA analysis before and after the onset of insulitis. Specific mRNA was amplified by reverse transcriptase polymerase chain reaction, quantitated with radiolabelled probes by phosphoimaging and calibrated with the amount of co-amplified beta-actin mRNA. At 50 days of age, prior to recognizable insulitis, there was already significantly enhanced expression of both, Th1 and Th2 cytokines, and of iNOS mRNA, when compared to Wistar rat pancreas (p < 0.001). This supports the concept of an inconspicuous early phase of islet infiltration by single immunocytes, called single cell insulitis. At 70 days of age mononuclear infiltration of islets had begun and was associated with upregulation of interferon gamma (IFN gamma) and iNOS, but downregulation of interleukin-10 and transforming growth factor beta mRNA (p < 0.001). These findings correlate the onset of insulitis with a shift of the Th1/Th2 cytokine balance towards Th1 cell reactivity. Indeed there was a close correlation of the Th1/Th2 cytokine ratio but not of absolute IFN gamma mRNA levels with the insulitis score. Vaccination at day 50 with tetanus toxoid did not affect cytokine gene expression while diphtheria toxoid and even more strongly BCG administration induced a shift towards Th2 reactivity (p < 0.001) while iNOS mRNA was decreased (p < 0.01). Oral dosing with immunostimulatory components of Escherichia coli also changed the quality of inflammation. Oral lipopolysaccharide (LPS) from E. coli and OM-89, an endotoxin free extract containing immunostimulatory glycolipopeptides and heat shock protein (hsp) 65, both downregulated IFN gamma mRNA while only OM-89 in addition suppressed iNOS mRNA and enhanced Th2 cytokine gene expression (p < 0.001). We conclude that the onset of insulitis is associated with a shift towards Th1 cytokine and iNOS gene expression. Diphtheria toxoid and BCG vaccination stimulates Th2 reactivity but does not downregulate Th1. The latter can be achieved through oral administration of LPS or a glycopeptide fraction (OM-89) from E. coli.

The aim of the study was to investigate the distribution in the eye of sulphatide, an acid glycolipid which has previously been demonstrated in islets of Langerhans, nervous tissue and in kidney glomeruli of diabetic patients, and against which antibodies have been found in patients with newly diagnosed insulin-dependent diabetes mellitus. A specific monoclonal antibody, Sulph I, was used for detection of sulphatide by thin-layer chromatography, and light and electron microscope immunohistochemistry. A distinct, patchy staining was found in the choroid layer and the ciliary processes. The antigen was confirmed to be sulphatide and its concentration in human eyes was 30 nmol sulphatide/g wet tissue. By electron microscopy, anti-sulphatide choroid labelling was demonstrated in pericytes and in smooth muscle cells surrounding vessels. No Sulph I-negative pericytes were seen. Double labelling with Sulph I and anti-smooth muscle actin revealed that only pericytes in the eye contained sulphatide and not those in heart, lung, liver, adrenal, spleen, lymph node, thymus, or pancreatic tissue. Thus, sharing of the autoantigen sulphatide has been demonstrated between islets of Langerhans and pericytes in the choroid layer of the eye.

Aims/hypothesis. Environmental factors such as diet and bacterial antigens play an important role in the onset of Type 1 diabetes. Different self-antigens are suggested to play a role in the development of diabetes. Antibodies against the 60-kDa heat shock protein 60, which have a high homology to bacterial heat shock protein 65, have been found in the circulation at the onset of diabetes in humans and in pre-diabetic NOD-mice. One of the immunodominant epitopes in autoimmune diabetes is p277, a specific peptide of human heat shock protein 60 corresponding to positions 437-460. In this study we investigated whether neonatal oral administration of DiaPep277 (a synthetic peptide analogue of p277) affected the development of diabetes in the BioBreeding-Diabetes Prone (BB-DP) rat, and whether this could potentiate the effect of a protective hydrolysed casein-diet.Methods. BB-DP rats were orally inoculated once per day with placebo or DiaPep277 at days 4, 5, 6 and 7 of life. At the age of 21 days rats were weaned on to a conventional, cereal-based diet or on to the hydrolysed casein-diet.Results. The development of diabetes in animals receiving DiaPep277 in combination with the hydrolysed casein-diet was delayed by 17 days, and a relative reduction of the incidence by 64% was seen. Non-diabetic animals did not show any sign of insulitis.Conclusions/interpretation. Short-term neonatal feeding with p277 in early life, combined with diet adaptation, appears to provide a procedure to significantly reduce the development of Type 1 diabetes in later life.

Diabetes-prone BioBreeding (DPBB) rats were fed a diabetogenic, mainly plant-based rodent diet, Purina Chow 5001, or a diabetes-retardant, hydrolysed casein-based diet. The expression of MHC class I antigens on pancreatic beta cells occurred at around 25 days of age in Purina Chow-fed rats, and progressively increased with the length of time of feeding with the Purina diet. Most of the Purina Chow-fed DPBB rats revealed hyperexpression of MHC class I antigens on their pancreatic beta cells by 50 days of age. Approximately 92% of the hyperexpressed Purina Chow-fed DPBB rats developed severe insulitis and diabetes. In contrast, the majority of hydrolysed casein-fed DPBB rats did not show MHC class I antigen hyperexpression and these rats failed to develop insulitis or diabetes. Purina Chow-fed Wistar-Furth rats and diabetes-resistant BioBreeding (DRBB) rats showed only very weak background staining for MHC class I antigens on their beta cells. When Purina Chow-fed (DPBB rats were treated with silica to inhibit macrophage infiltration into the pancreatic islets, the hyperexpression of MHC class I antigens was seen even more clearly, as beta cells remained intact. MHC class II antigens were not detected on pancreatic beta cells from DPBB, DRBB or Wistar-Furth rats, regardless of their diet. On the basis of these observations, we concluded that hyperexpression of MHC class I antigens on pancreatic beta cells was mainly restricted to Purina Chow-fed DPBB rats and that suppression of non-macrophage-dependent MHC class I antigen hyperexpression on pancreatic beta cells by a hydrolysed caseinbased diet resulted in the prevention of insulitis and diabetes.

Gastric emptying was measured in normal and insulin-treated spontaneously diabetic BB rats using the retention of an acaloric methylcellulose gel containing phenol red delivered by gavage. Dye content in stomachs removed after killing 20 min later was determined spectroscopically, and was compared to that in rats killed immediately after gavage to assess emptying. Diabetic rats had a markedly greater gastric emptying (90.3 +/- 1.7% passed) compared to normal Harlan Sprague Dawley rats (49.1 +/- 4.7% passed; p0.001) and non-diabetic BB rats (61.1 +/- 9.2% passed; p0.001). The pancreatic beta-cell peptide, amylin, which is deficient in insulin-dependent diabetes mellitus, dose-dependently inhibited gastric emptying in both normal and diabetic rats. The ED50 of the response in normal rats measured by phenol red and novel [3-3H]glucose gavage techniques was approximately 0.4 microgram. This dose was estimated to increase plasma amylin concentration by a mean of approximately 20 pmol/l to concentrations within the range observed in vivo. It is proposed that amylin could participate in the physiological control of nutrient entry into the duodenum and that the accelerated gastric emptying seen in BB rats could be related to their lack of amylin secretion.

The aim of this study was to examine the influence of inhibition on angiotensin converting enzyme (ACE) of myocardial function and perfusion of the rat impaired by diabetes. Spontaneously diabetic rats were treated with the ACE-inhibitor captopril for 4 months. Cardiac performance was analysed in the isolated heart perfused at constant volume. Epicardial perfusion was determined by measuring changes in epicardial fluorescence after injection of a bolus of fluoresceinisothiocyanate-dextrane (3 kDa) as described previously. As compared to untreated diabetic controls, captopril prevented the increase of end diastolic pressure, coronary perfusion pressure and vascular resistance. The intravascular volume was enlarged and the epicardial perfusion rate increased in hearts of diabetic rats treated with captopril as compared to diabetic controls. Treatment of diabetic rats with the ACE-inhibitor captopril (1) increases the number of perfused capillaries, and (2) can partly prevent the development of cardiac dysfunction in diabetes. Together with morphological data demonstrating an inhibition of interstitial and perivascular fibrosis in hearts of diabetic rats treated with captopril, our data suggest that ACE-inhibition is cardioprotective in diabetes. These observations are also compatible with the assumption that an accelerated generation of angiotensin II may be involved in the pathophysiological chain of events leading to diabetic cardiopathy.

The diabetic syndrome in the DP-BB rat results from progressive beta-cell destruction by autoimmune responses. However, the initial events causing the autoimmune destruction of beta cells remain largely unknown. Our recent experimental results suggest that the delayed expression of a beta-cell-specific autoantigen may result in the initiation of beta-cellspecific autoimmunity. The present investigation was initiated to identify such an autoantigen. Islets were isolated from DP-BB rats of several different ages, and protein extracts from the membrane fraction of the islet preparations were immunoprecipitated with sera from diabetic DP-BB rats. We have found that a membrane-bound islet cell-specific 38 kDa autoantigen is not expressed early in the life of DP-BB rats, but is delayed-expressed by approximately 30 days of age, the time at which immunological effectors begin to recognize beta cells. In contrast, a 64 kDa islet cell protein is expressed from birth in DP-BB rats. On the basis of these observations, we suggest that delayed expression of a gene encoding for the membrane-bound islet cell-specific 38 kDa autoantigen may result in a breakdown of self-tolerance, leading to beta-cell-specific autoimmune IDDM in the BB rat. [Diabetologia (1994) 37: 460-465]

Inflammatory cells invading islets are thought to be mediators of islet destruction in spontaneous autoimmune diabetes mellitus. Thus methods were developed to isolate and characterize in situ islet inflammatory cells from 75-95-day-old prediabetic and diabetic BB rats. Islet inflammatory cells were structurally examined using single- and double-colour flow cytometry. Functional studies consisted of cytolytic assays using normal rat islet target cells and in situ islet or spleen effector cells. Structural data reveal natural killer cells to be the major cell population (70%) of total immune cells present in inflamed islets during prediabetes. At diabetes onset, the natural killer cell population remained at a high level (47%), but an increasing population of T cells (40%) was noted also. Analyses of T-cell subsets before and after diabetes onset revealed CD4+T cells as predominant (50-55% of total T cells) with double-negative (CD4-CD8-) T cells (25-30%) and CD8+T cells (15-20%) also present in significant quantities. Activated T cells accounted only for a minority of T cells (

A polyol-pathway-related mechanism has been invoked in the pathogenesis of murine and human diabetic peripheral neuropathy in which progressive axonal atrophy and axo-glial dysjunction constitute the cardinal structural abnormalities. We have previously reported similar neuroanatomical changes in the optic nerve of 6-month diabetic BB/W-rats. In the present study we demonstrate progression of axonal atrophy and axo-glial dysjunction in the optic nerve in 12-month diabetic BB/W-rats. These structural lesions showed highly significant correlations with the associated prolongation of the latencies of the visual evoked potentials, suggesting that axo-glial dysjunction and axonal atrophy are major determinants for impaired optic nerve function. As in peripheral nerve, the polyol-pathway is present in the optic nerve and is activated by hyperglycaemia and galactosaemia. In this study we further examined the treatment effect of the aldose reductase inhibitor ponalrestat, given from 3 weeks of diabetes and continued throughout the study protocol. This regimen resulted in complete prevention of axo-glial dysjunction, and had a significant ameliorating effect on visual evoked potential latencies, but had no effect on optic nerve axonal atrophy. This latter finding differs from the effect of aldose reductase inhibition on diabetic peripheral nerve and suggests that axonal atrophy of central nerve tracts in diabetes may be the consequence of other metabolic abnormalities or alternatively the present regimen was insufficient to protect central axons from the effects of an increased activity of the polyol pathway.

Inhibition of glucosaminyl N-deacetylase activity, a key enzyme in heparan sulphate sulphation, may be involved in the development of late diabetic vascular complications. We examined the effect of short- and long-term metabolic control on N-deacetylase activity in streptozotocin diabetic H and U rats. Spontaneously diabetic BB rats were included in parts of the study. Over a 3-week period blood glucose was maintained at predetermined levels (6-10 mmol/l or 10-20 mmol/l) by insulin treatment and then during the final 2 days rapidly reversed in half of each group. In the U rats, the hepatic N-deacetylase activity significantly decreased by 10-15% following short- and long-term poor metabolic control and the inhibition was entirely reversed by short-term good control. In the H rats a similar, not significant, effect was seen. BB rats in long-term poor control showed a 10% reduction in hepatic N-deacetylase activity (p = 0.003). Glomerular N-deacetylase activity was reduced in U rats after long-term poor control (p = 0.004) but not in H and BB rats. There was an overall correlation between urinary albumin excretion and glomerular N-deacetylase activity (r = -0.60, p0.0001). We conclude that diabetes-induced inhibition of hepatic N-deacetylase is not restricted to the streptozotocin diabetic model, and that short-term blood glucose control is of major importance. Genetic factors and tissue specificity influence the vulnerability of the enzyme. Finally, the study suggests an association between N-deacetylase activity and urinary albumin excretion.

The effect of the alpha-glucosidase inhibitor acarbose on postprandial hyperglycaemia was explored in the spontaneously diabetic BB/W-rat. Acarbose-treatment (5 mg.kg body weight-1.day-1) of diabetic BB/W-rats maintained on small doses of insulin, was associated with a 40% reduction in the 24-h glucose area compared to non-treated diabetic rats. Over a 4 month treatment period this reduction in cumulative hyperglycaemia resulted in a complete prevention of autonomic polyneuropathy as indicated by R-BAR values. The development of somatic polyneuropathy in the BB/W-rat was significantly attenuated by acarbose treatment with a partial prevention of the characteristic nerve conduction velocity slowing during the first 3 months of diabetes, but no longer at 4 months. Characteristic structural abnormalities associated with diabetes in this model, such as axonal atrophy and axo-glial dysjunction, were significantly but only partially prevented in rats treated with acarbose for a diabetes duration of 4 months. These data suggest that postprandial lowering of hyperglycaemia resulting in a decrease in cumulative hyperglycaemia retards the development of diabetic polyneuropathies in the BB/W-rat.

Alginate-polylysine microencapsulation has been proposed as a method of protecting transplanted pancreatic islets against immunological attack. Using this technique, prolonged graft survival has been reported in some diabetic animals. However, in the spontaneously diabetic insulin-dependent BB/E rat we found that intraperitoneal implantation of microencapsulated islets had only a short-lived effect on hyperglycaemia. Recovered microcapsules (both those implanted empty and containing islets) were surrounded by a foreign body type cellular overgrowth and, although many capsules remained intact, encapsulated islets were observed to be disintegrating. Loss of Beta cells was confirmed by immunohistology. Various polymer materials used in artificial membranes have been shown to activate macrophages involved in foreign body reactions and induce synthesis of interleukin-1 beta, a known Beta-cell toxin. Reduced secretion of insulin and progressive islet damage (indicated by a significant reduction in residual islet insulin and DNA content) were demonstrated when microencapsulated islets were incubated with interleukin-1 beta in vitro for 9 days. Similar effects were seen following exposure to a combination of gamma interferon and alpha tumour necrosis factor. Successful use of microencapsulation in islet transplantation depends upon the development of biocompatible membranes. The exclusion of smaller molecules, such as cytokines, which may be involved in foreign body mediated damage and microencapsulated islet graft rejection, could also be important.

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.

To prove whether a cell-mediated mechanism is responsible for maintaining long-term normoglycaemia in BB/OK rats with a proved immune attack (insulitis, reduced Beta-cell volume), we transferred lymphocytes obtained from those rats into normoglycaemic diabetes-prone BB/OK rats or into diabetic BB/OK rats receiving a simultaneous syngeneic islet graft. Our results show the presence of a lymphocyte population in the long-term normoglycaemic BB/OK rats, which is able to arrest pancreatic Beta-cell destruction in diabetes-prone BB/OK rats detected by a decreased diabetes incidence following single lymphocyte transfusion. Syngeneic islets were destroyed by recurrence of the autoimmune process when transplanted into diabetic BB/OK rats. Lymphocytes obtained from long-term normoglycaemic BB/OK rats were able to protect the syngeneic BB/OK islet graft from autoimmune destruction in diabetic BB/OK rats, whereas allogeneic islet destruction was not prevented. The phenotype of the effective lymphocyte population is not yet clear, but it is negative for RT6. We conclude that the mechanism responsible for maintaining normoglycaemia in long-term normoglycaemic BB/OK rats is cell mediated, because this property can be transferred to prevent autoimmune destruction of pancreatic Beta cells.

The aim of this study was to evaluate the nature and extent of neuronal loss in dorsal root ganglia (DRG) in diabetic polyneuropathy.We examined 10-month diabetic BioBreeding/Worcester (BB/Wor) rats with respect to DRG ultrastructure and morphometry, sural nerve morphometry, pro- and anti-apoptotic proteins, the expression of neurotrophic factors and their receptors, and sensory nerve functions.In diabetic rats, DRG neurons decreased to 73% of normal, owing to loss of substance P and calcitonin gene-related peptide-positive neurons. Levels of pro-apoptotic active caspase-3, Bax and low-affinity nerve growth factor (NGF) were increased in DRG. The concentration of anti-apoptotic heat shock protein (HSP) 70 in DRG was decreased, whereas concentrations of Bcl-xl and HSP27 were unaltered. Levels of poly(ADP-ribose) polymerase (PARP) and cleaved PARP were unaltered. Levels of NGF in sciatic nerve and concentrations of the high-affinity NGF receptor, insulin receptor and IGF-I receptor in DRG were significantly decreased. Sensory nerve conduction velocity decreased to 78% of normal. Hyperalgesia increased up to 6 months. Myelinated and unmyelinated fibre numbers of the sural nerve were significantly decreased in diabetic rats. DRG examinations revealed no evidence of apoptosis, mitochondrial changes or abnormalities of the endoplasmic reticulum. Instead, neurons demonstrated progressive vacuolar degenerative changes of the Golgi apparatus, with fragmentation and formation of large cytoplasmic vacuoles. These data show that sustained apoptotic stress is present in DRG of chronically diabetic BB/Wor rats, but fails to proceed to apoptotic cell death.Progressive DRG neuronal loss, particularly of small neurons, occurs in the type 1 diabetic BB/Wor rat. This is associated with neurotrophic withdrawal and progressive degeneration of the Golgi apparatus.

Type 1 diabetes is the result of an inflammatory T helper 1 (Th1) lymphocyte-mediated beta cell destructive process. The majority of diabetes-prone BioBreeding (BBdp) rats fed wheat protein-based diets, such as NTP-2000, develop type 1 diabetes and display a mild coeliac-like enteropathy. Mesenteric lymph nodes (MLNs), which drain the gut, are the major inductive site where dietary antigens are recognised in the gut-associated lymphoid tissue (GALT). We hypothesised that this compartment could be a site of abnormal wheat protein-induced Th1 cell activation.MLN cells were isolated from BBdp and BB control (BBc) rats that were fed NTP-2000 or a hydrolysed casein (HC)-based diet at ages that pre-date classic insulitis. The inflammatory status, phenotype and proliferation of these cells in response to wheat protein were determined.The expression ratio of T-bet : Gata3, master transcription factors for Th1 and Th2 cytokines, was increased in the MLN from NTP-2000-fed BBdp rats compared with that from BBc rats, mainly due to decreased Gata3 expression. CD3(+)CD4(+)IFN-gamma(+) T cells were more prevalent in the MLN of wheat-fed BBdp rats, but remained at control levels in BBdp rats fed a diabetes-retardant HC diet. BBdp MLN cells proliferated in response to wheat protein antigens in a specific, dose-dependent manner, and93% of cells were CD3(+)CD4(+) T cells. This proliferation was associated with a low proportion of CD4(+)CD25(+) T cells and a high proportion of dendritic cells in the MLN of BBdp rats.Before insulitis is established, the MLNs of wheat-fed BBdp rats contain an unusually high proportion of Th1 cells that proliferate specifically in response to wheat protein antigens.

Sir Winston Churchill summed up our position in this debate when he said that “Men stumble over the truth from time to time, but most pick themselves up and hurry off as if nothing happened”. To our thinking, we (the Type 1 diabetes research community) have stumbled both in terms of the way in which data derived from animal models of Type 1 diabetes have been handled, and in the manner in which the field continues to move forward without proper acknowledgement of what has been learned. Animal models such as the non-obese diabetic (NOD) mouse and the biobreeding (BB) rat develop immune-mediated disease with features resembling Type 1 diabetes in humans [1]. Although these animal models of autoimmune diabetes have proved to be valuable tools to study certain aspects of the disease process [2], they have also led to misconceptions and erroneous extrapolations, as well as false expectations with regard to the efficacy of immunotherapy. Hence, on a number of counts, we would argue that animal models have limited value when it comes to teaching us about Type 1 diabetes in humans. The immune system There are profound differences between the immune systems of mice and men. These have recently been summarised comprehensively by Mestas and Hughes, and include discrepancies in both innate and adaptive immunity [3]. Relevant examples of more than 80 known incompatibilities would include: balance of leucocyte subsets, defensins, toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and lambda5) and T cell (ZAP70 and common gamma-chain) signalling pathway components, Thy-1, gamma delta T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression. Given the breadth of these functional differences, these discrepancies surely limit the usefulness of mouse models in studying Type 1 diabetes. Therefore, such differences should be taken into account when animals are used as preclinical models of human disease. Nonetheless, they are generally ignored.

that animal models in general, and rodent models of Type 1 diabetes in particular, are imperfect reflections of human disease [1]. This is the inevitable consequence of an evolutionary separation of over 65 million years. While pointing out the numerous differences that distinguish human from rodent immune systems, Mestas and Hughes [2] note that “after all, most of us do not live with our noses a half-inch off the ground.” Clearly, divergent evolution in genera occupying different niches argues against using a mouse or rat model of spontaneous Type 1 diabetes as an exact blueprint for disease in humans. There are, however, sufficient similarities between the aetiopathogenesis of Type 1 diabetes in humans, mice and rats to justify the efforts devoted to animal research (Table 1). While there are significant differences between the various rodent models, this heterogeneity surely carries important lessons for understanding the potential heterogeneity underlying this complex disease in humans. The major rodent models of spontaneous Type 1 diabetes are the Nonobese Diabetic (NOD) mouse and

Type 1 diabetes mellitus is a multifactorial autoimmune disease characterised by selective destruction of beta cells in the islets of Langerhans. We have previously shown that IL-1 beta modulates beta cell function, causes beta cell death and induces expression changes in 82 out of 1815 protein spots detected by two-dimensional gel electrophoresis (2-DGE) in diabetes-prone bio-breeding (BB-DP) rat islets in vitro. The aim of this study was to describe the relevance of these proteins in the development of diabetes in vivo.Syngeneic neonatal islets ( n=200) were transplanted under the kidney capsule of 30-day-old BB-DP and control rats, removed to different time points after transplantation or at the onset of diabetes, and metabolically labelled with S(35)-methionine for 2-DGE. The 82 proteins were re-localised and followed. In addition, transplants were examined for expression of IL-1 beta mRNA by in situ hybridisation.All 82 proteins could be re-localised in all syngeneic transplants from BB-DP and control rats. A total of 60 of the 82 proteins were changed during development of diabetes. Of the 82 proteins, 32 were changed in expression at the onset of diabetes compared to non-diabetic BB-DP rats, and 25 of these were changed as by IL-1 beta in vitro. Highest expression of IL-1 beta mRNA was found at the onset of diabetes.IL-1 beta-induced protein expression changes in islets in vitro also occur in vivo and change in a complex pattern during the development of diabetes in the BB-DP rat. No single protein seems to be responsible for the development of diabetes, but rather the cumulative numbers of changes seem to interfere with the intracellular stability of the beta cell.

Our aim was to determine whether the divergent effects of chronic exposure to hyperglycaemia or hypoglycaemia on the glycaemic threshold for auditory brainstem dysfunction are reflected in the extra-cellular fluid (ECF) concentrations of glucose in the inferior colliculus during hypoglycaemia in the diabetic BB rat.Microdialysis was used to measure inferior colliculus ECF glucose concentrations under basal and hyperinsulinaemic (20 mU/kg.min) hypoglycaemic conditions.ECF glucose is increased under basal (hyperglycaemic) conditions and decreases during hypoglycaemia in both recurrently hypoglycaemic and chronically hyperglycaemic diabetic BB rats (to 0.5+/-0.1 and 0.8+/-0.2 mmol/L respectively), with no significant differences between groups. In both groups the plasma to ECF glucose ratio doubled during hypoglycaemia.Prior exposure to recurrent hypoglycaemia does not lead to increased ECF glucose concentrations in the inferior colliculus of diabetic BB rats. The resistance to impaired brainstem function seen in recurrently hypoglycaemic rats during hypoglycaemia cannot simply be attributed to increased blood-brain barrier glucose transport within this brain region.

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 1β-casein has been associated with increased diabetes frequency in ecological studies and in NOD mice. Our aim was to ascertain whether A 1 -β-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 -β-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 β-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 A1 β-casein was more diabetogenic than A2 β-casein in NOD mice was not confirmed; both β-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 -β-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 β-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. [Diabetologia (2002) 45:1240‐1246]

Aims/hypothesis. Insulin and C-peptide exert neuroprotective effects and are deficient in Type I (insulin-dependent) diabetes mellitus but not in Type II (non-insulin-dependent) diabetes mellitus. These studies were designed to test the preventive and interventional effects of C-peptide replacement on diabetic polyneuropathy in the Type I diabetic BB/Wor rat. Methods. Diabetic BB/Wor rats were replaced with rat C-peptide from onset of diabetes and between 5 and 8 months of diabetes. They were examined at 2 and 8 months and compared to non-C-peptide replaced BB/Wor rats, Type II diabetic (non-C-peptide deficient) BB/Z rats and non-diabetic control rats. Animals were monitored as to hyperglycaemia and nerve conduction velocity (NCV). Acute changes such as neural Na+/K+-ATPase and paranodal swelling were examined at 2 months, morphometric and teased fiber analyses were done at 8 months. Results. C-peptide replacement for 2 months in Type I diabetic rats prevented the acute NCV defect by 59 % (p < 0.005), the neural Na+/K+-ATPase defect by 55 % (p < 0.001) and acute paranodal swelling by 61 % (p < 0.001). Eight months of C-peptide replacement prevented the chronic nerve conduction defect by 71 % (p < 0.001) and totally prevented axoglial dysjunction (p < 0.001) and paranodal demyelination (p < 0.001). C-peptide treatment from 5 to 8 months showed a 13 % (p < 0.05) improvement in NCV, a 33 % (p < 0.05) improvement in axoglial dysjunction, normalization (p < 0.001) of paranodal demyelination, repair of axonal degeneration (p < 0.01), and a fourfold (p < 0.001) increase in nerve fibre regeneration. Conclusion/interpretation. C-peptide replacement of Type I BB/Wor-rats partially prevents acute and chronic metabolic, functional and structural changes that separate Type I diabetic polyneuropathy from its Type II counterpart suggesting that C-peptide deficiency plays a pathogenetic role in Type I diabetic polyneuropathy. [Diabetologia (2001) 44: 889–897]

Aims/hypothesis. Cyclophosphamide has been shown to augment the diabetic process in NOD mouse and BB rat models of Type I (insulin-dependent) diabetes mellitus. Because cyclophosphamide has, however, been shown to increase immunoregulatory cell activity, we examined if cyclophosphamide treatment increases immunoregulatory cell activity and inhibits the diabetic process in BB rats. Methods. The development of insulitis and diabetes was explored in BB rats treated with saline and cyclophosphamide (60 to 175 mg/kg body weight). Subsets of spleen cells were assessed by flow cytometry and cytokine gene expression by RT-PCR. To determine if cyclophosphamide induces immunoregulatory cell activity, the development of diabetes was assessed in BB rats injected with spleen cells from rats treated with saline and cyclophosphamide. Results. All dosages of cyclophosphamide decreased the development of diabetes. The degree of insulitis was lower in pancreata from 55-day-old rats treated with cyclophosphamide than those from controls. Cyclophosphamide caused no alterations in the numbers of NK cells, T-cell subsets, or RT6.1+ T cells. The adoptive transfer of spleen cells from cyclophosphamide-treated rats to BB rats inhibited the development of diabetes. Cyclophosphamide treatment decreased IL-12, IL-1β, IL-2, IFN-γ and TNF-α gene expressions in mononuclear spleen cells but IL-4 gene expression increased. Conclusion/interpretation. These findings show that cyclophosphamide treatment decreases the development of diabetes by inhibiting the development of insulitis. This inhibitory action of cyclophosphamide on the diabetic process seems to be mediated by the induction of immunoregulatory cell activity. The suppression of cytokines that promote Th1 cell differentiation by cyclophosphamide treatment could also play a part in the diabetes sparing effect of cyclophosphamide. [Diabetologia (2000) 43: 986–994]

Aims/hypothesis. We did experiments to explore the pathways putatively leading to Type I (insulin-dependent) diabetes mellitus, and their association with the MHC locus, the major genetic determinant of disease susceptibility.¶Methods. Normal MHC congenic rat strains that do not spontaneously develop diabetes or any other autoimmune syndrome were injected with the interferon-alpha inducer polyinosinic-polycytidylic acid (Poly IC).¶Results. Insulitis and diabetes developed only in strains expressing Class IIu genes and was independent of the Class I haplotype. Poly IC induced islet cell Class I hyperexpression, up regulation of pancreatic endothelial intercellular adhesion molecule-1 and vascular adhesion molecule-1 and a T-cell and macrophage infiltration of the pancreatic interstitium in all rat strains studied, including diabetes-resistant strains. Poly IC also induced the generation of diabetes-transferring spleen cells in most Class IIu haplotype rats, including the diabetes-resistant WF rat.¶Conclusion/Interpretation. The minimum requirements for autoimmune diabetes development in the rat include: RT1 Class IIu genes, a T-cell repertoire containing beta-cell autoreactive T cells and a triggering event which breaks tolerance by the local up regulation of pancreatic endothelial adhesion receptors. Even when all of the minimum requirements have, however, been met, most Class IIu rats do not develop diabetes in response to autoimmune stimuli. It is clear, nonetheless, that susceptibility to diabetes is widely distributed in the RT1 u rat. [Diabetologia (2000) 43: 890–898]

Aims/hypothesis. To examine the functional, metabolic and structural abnormalities in peripheral nerve in the spontaneously Type II (non-insulin-dependent) diabetic BBZDR/Wor rat and compare these data with those in the Type I (insulin-dependent) diabetic BB/Wor rat.¶Methods. Animals were examined at 6 and 14 months of diabetes. Nerve conduction velocity was measured longitudinally. Nerve polyols were analysed using gas liquid chromatography and Na/K+-ATPase activity was measured enzymatically. Light and electron microscopic techniques were used for nerve morphometry.¶Results. Diabetic BBZDR/Wor rats showed a slowly progressive nerve conduction defect that reached 17 % (p < 0.01) at 14 months. There was a decrease in Na+/K+-ATPase of 35 % (p < 0.05). Structurally, there were mild myelinated fibre atrophy (p < 0.05), mild or absent changes of the node of Ranvier, but significant (p < 0.001) segmental demyelination and Wallerian degeneration. These findings point to a more severe nerve conduction defect, severe myelinated fibre atrophy and profound nodal changes in Type I spontaneously diabetic BB/Wor rats maintained at the same hyperglycaemic concentrations.¶Conclusion/interpretation. We conclude that other factors, beside hyperglycaemia, are involved in the pathogenesis of the more severe Type I diabetic neuropathy which possibly involve insulin and C-peptide deficiencies. [Diabetologia (2000) 43: 786–793]

Following activation peritoneal macrophages from diabetes prone BB rats secreted strikingly higher amounts of tumour necrosis factor alpha than found for macrophages from diabetes resistant or normal Wistar rats. Enhanced tumour necrosis factor alpha production was detected prior to the occurrence of insulitis. Cultures of macrophages derived from precursor cells in diabetes prone BB rat bone marrow also showed upregulated tumour necrosis factor alpha secretion upon challenge with endotoxin and interferon gamma. Tumour necrosis factor alpha hypersecretion may contribute to autoimmune diabetes by affecting thymic and post-thymic T-cell maturation and by promoting pancreatic islet inflammation.

Type 1 (insulin-dependent) diabetes mellitus results from autoimmune cell-mediated destruction of pancreatic islet Beta cells. Since theophylline has suppressive effects on immune responses, we sought to determine if this agent might protect against destruction of islet Beta cells in diabetes-prone BB rats. Diabetes-prone rats were divided into four groups and treated, from age 30 to 125 days, with: (a) no treatment (control), (b) theophylline 2 mg.ml-1 added to the drinking water, (c) cyclosporine 5 mg.kg-1.day-1 administered by gavage, and (d) theophylline plus cyclosporine. By age 125 days, diabetes (glucosuria and serum glucose greater than 11 mmol.l-1) had appeared in 15 of 17 (88%) of control rats, 10 of 18 (56%) on theophylline, 6 of 17 (35%) on cyclosporine, and 1 of 17 (6%) on theophylline plus cyclosporine. Protection against diabetes by theophylline and cyclosporine was associated with preservation of pancreatic Beta cell mass (insulin content). The protective effects of combination therapy with theophylline and cyclosporine were achieved at very low serum concentrations of cyclosporine. These findings suggest that theophylline may be a useful adjunct in the immunosuppressive therapy of Type 1 diabetes.

Previous studies demonstrating reduced plasma concentrations of ascorbic acid (AA) in diabetes and interactions between this vitamin and biochemical mechanisms such as synthesis of structural proteins, oxidative stress, polyol pathway and non-enzymatic glycation of proteins suggest that disturbed AA metabolism may be important in the pathogenesis of diabetic microangiopathy. However, limited information is available on the concentration of AA in tissues which develop diabetic complications. This study demonstrates reduced renal but not sciatic nerve or plasma AA concentration in two animal models of insulin-dependent diabetes mellitus, namely the STZ-diabetic rat and the spontaneously diabetic BB rat. Decreased lens AA concentration was also observed in STZ-diabetic rats. Improvement of glycaemic control by insulin treatment (albeit insufficient to achieve normoglycaemia) partially corrected lens and renal AA concentration in STZ-diabetic rats. AA treatment increased kidney and lens AA concentrations of STZ-diabetic and non-diabetic rats and corrected the abnormalities observed for untreated diabetic rats. Sciatic nerve AA concentration was not increased by AA treatment in any group. Tissue ratios of dehydroascorbic acid (DHAA)/AA, one index of oxidative stress, were not different between the diabetic and non-diabetic groups and were unaltered by AA supplementation. AA treatment of STZ-diabetic rats had no effect on elevated tissue concentrations of glucose, sorbitol and fructose or reduced myo-inositol concentration. The effect of reduced tissue AA levels in diabetes on either collagen synthesis or ability to combat increased free radical production is not known. However, correction of abnormal kidney and lens AA concentrations in experimental diabetes by AA supplementation suggests that if AA does have a role in the development or progression of the renal and ocular complications of diabetes, this treatment could be beneficial. [Diabetologia (1998) 41: 516–523]

We examined the effects of acute supplementation with arginine in vitro on endothelium-dependent relaxation in aortic rings taken from female genetic, diabetes-prone BB rats. Sensitivity to norepinephrine-induced contraction was unaltered in rings of diabetic BB rats compared to rings from non-diabetic littermates. In precontracted rings, acetylcholine produced a concentration-dependent relaxation which was impaired by diabetes. This relaxation was blocked by L-nitroarginine in both control and diabetic rings. Addition of 3 mmol/l L-arginine (but not D-arginine) enhanced relaxation in diabetic rings similar to that seen in control rings without arginine. L-arginine had no effect on acetylcholine-induced relaxation in control rings. In contrast, relaxation-induced by nitroglycerin in diabetic rings without endothelium was not altered by L-arginine treatment. Thus, a defect in the utilization of arginine by nitric oxide synthase exists in the endothelium of the diabetic BB rat.

Weaning onto chow diets causes the highest incidence of diabetes in the BB rat. Changes in gut development and absorption of nutrients in the diabetes prone rat and the subsequent effect on pancreatic function may play a role in the ultimate development of the disease. BB diabetes prone (dp) and BB normal (n) dams were fed chow diets. Pups were killed at various ages ranging from 7 to 30 days. BBdp rats had higher small intestine and colon weights expressed per body weight at all ages (p < 0.0001). RNA content (mg/g) in the jejunum, ileum and colon was higher in the BBdp rats beginning at the critical period at 21 days and maintained at 24 days and 30 days (p < 0.0001). Proglucagon message decreased with age in both BBdp and BBn animals (p < 0.0001). Levels of proglucagon mRNA were higher in BBdp compared to BBn animals only in the ileum at 10 days (p < 0.01). Adjusting for total ileal and colonic RNA content resulted in BBdp animals having higher total colonic proglucagon mRNA at 21, 24 and 30 days (p < 0.0001). Plasma GLP-1(7–36) amide was more than doubled in BBdp compared to BBn animals (p < 0.0005) at 30 days. Expressing sodium-dependent D-glucose co-transporter (SGLT-1), GLUT2 and GLUT5 mRNA per total jejunal RNA shows increased transporter mRNA in BBdp compared to BBn rats at weaning (21 days) (p < 0.05). Radical differences exist between BBdp and BBn animals at ’critical periods' in both proglucagon and glucose transporter gene expression. These differences may help explain altered growth and diseases incidence between these two strains [Diabetologia (1997) 40: 871–878].

The lymphopenia gene (lyp ) on rat chromosome 4 is closely linked to autoimmune diabetes in the BioBreeding (BB) rat. Lyp controls the number of peripheral lymphocytes by reducing T cells of the RT6 + phenotype by almost 90 %. Following nine cycles of marker-assisted cross-intercross breeding we have developed congenic lyp/lyp, lyp/ + and + / + (wildtype) rats on the background of DR rats. Prediabetic and insulitis free lyp/lyp, lyp/ + and + / + rats were used to determine the effect of lyp on cytokine expression in the thymus. In situ hybridization of thymus cryosections showed that the interferon gamma (IFNγ) mRNA expression was highest in lyp/lyp rats and the hybridization signal was restricted to the medullary compartment. The frequency of IFNγ and interleukin (IL)-10 mRNA expressing cells in isolated thymocytes determined by quantitative image analysis, demonstrated an increased IFNγ:IL-10 ratio in thymocytes from lyp/lyp homozygotes compared to lyp/ + and + / + rats. This confirmed a lyp gene dose-dependent segregation of the IFNγhigh phenotype. Recombinant human glutamic acid decarboxylase (GAD65) increased the number of IFNγ and IL-10 mRNA expressing thymocytes after in vitro culture. We conclude that the quantitative ratio of cytokine producing thymocytes is associated with the lyp genotype. These potentially autoreactive thymocytes may explain the establishment of beta-cell directed autoimmunity in the BB rat despite peripheral lymphopenia. [Diabetologia (1997) 40: 786–792]

Blood glucose control plays a prominent role in the aetiology of diabetic complications. Recent data support the hypothesis that non-enzymatic pathways (glycation and oxidation) are involved in the pathogenesis of tissue damage in diabetes mellitus. In this study the level of pentosidine, a marker of glycation, and the intensity of collagen-linked fluorescence glycation (370/440 and 335/385 nm) and oxidation-related (356/460 and 390/460 nm), have been examined in spontaneously diabetic rats with good and poor glycaemic control. Pentosidine increased dramatically in rats with poor control, and slightly in those with good control. At the end of the study, after 6 months of diabetes, pentosidine levels were 13 ± 5 and 2.1 ± 0.5 pmol/mg collagen, respectively (control rats: 1.1 ± 0.1 pmol/mg collagen). A similar pattern was observed for both glycation or oxidation-related fluorescence. The group of rats with poor control always showed elevated average values when compared to rats with good control, with a relative increase of over 200 %. The results emphasize the role of good glycaemic control in preventing the growth of glycation or oxidation end-products in collagen. On comparison between the general mean level of all glycated haemoglobin and the mean pentosidine level of the three groups, a very good exponential correlation was found (r = 0.993, p < 0.001). The fluorescence values presented a less strong relationship, but a correlation with glycaemic control was still present. If the post-translational modifications of proteins play a leading role in the pathogenesis of complications it is possible to conclude that strict glycaemic control, obtained by accurate insulin therapy can prevent them by inhibiting the non-enzymatic modification of proteins and delaying their accumulation in collagen. The therapeutic implications are obvious. [Diabetologia (1996) 39: 1440–1447]

A differing individual expression of fructosyllysine-specific receptors has been found on the monocytes of 90 insulin-dependent diabetic patients and 101 healthy control subjects. The degree of receptor expression is neither age- nor sex-dependent; however, in the diabetic group it correlates significantly with the severity and age of onset of diabetic microangiopathy. To interpret the results of the human study, spontaneously diabetic and non-diabetic BB/OK rats were used to estimate tissue content of glucose-modified proteins and capillary basement membrane thickness in relation to the receptor expression on macrophages. In non-diabetic and diabetic rats no correlation was found between receptor expression and tissue content (i.e. artery, nerve) of fructosyllysine and fluorescent advanced glycation end products. However, animals which express the fructosyllysine receptor showed a greater increase in muscle capillary basement membrane thickness. There are indications that fructosyllysine receptor expression is positively associated with indices of diabetic complications such as microangiopathy and/or capillary basement membrane thickening.

T cells taken from normal rats treated with an exogenous source of bacterial superantigen in vivo specifically failed to proliferate following re-stimulation with the same superantigen in vitro. Responsiveness was restored following the addition of an exogenous source of interleukin-2 indicating that the T cells had been made functionally tolerant and not deleted. While staphylococcal enterotoxin treatment of normal rats virtually abolished T-cell proliferation to the same enterotoxin in vitro, T cells from similarly treated diabetes-prone Biobreeding (BB-DP) rats were markedly resistant to this in vivo effect. Responses in BB-DP rats were never reduced by more than 50% even when a 4 times more effective dose of enterotoxin was employed. The resistance of BB-DP peripheral T cells to staphylococcal enterotoxin-induced tolerance could not be attributed to differences in T-cell receptor V beta chain family usage of BB-DP vs normal T cells but was associated with qualitative differences in the way in which BB-DP T cells responded to staphylococcal enterotoxins in vitro. While under optimal stimulatory conditions BB-DP T-cell proliferative responses to staphylococcal enterotoxins appeared comparable to those from non-diabetes-prone animals, under superoptimal conditions BB-DP, but not diabetes-resistant, donor T-cell proliferative responses to staphylococcal enterotoxins could be blocked in vitro with antibodies to CD4 antigens. In addition, BB-DP T-cell proliferative responses were more sensitive to suboptimal staphylococcal enterotoxin doses in vitro. We discuss ways in which abnormal BB-DP T-cell responses to superantigens in general and resistance to staphylococcal enterotoxin-mediated tolerance induction in particular may play a role in the generation of a peripheral T-cell repertoire prone to autoimmunity.

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.

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.

Tetrahydrobiopterin (BH4) is a pteridine product which is released by rodent macrophages on ac-tivation by cytokines. We have used serial pancreatic biopsy, and measurement of serum biopterin at 30,60,90 and 120 days in the BB/S rat to relate histological change to macrophage activation during the course of pre-diabetes. Using immunohistochemistry, and an arbi-trary scoring system read blind and standardised against day 30, we found that pancreatic MHC class I, MHC class II and infiltrating macrophage staining were up-regulated in the BB/S diabetes-prone rats (n = 17) at day 60, markedly so at day 90, and less so at day 120. Staining for resident pancreatic macrophages remained unchanged throughout in diabetes prone, diabetes resis-tant and Wistar (n = 28) control animals. Serum biop-terin fell progressively and identically with age in BB diabetes resistant rats (n = 11) and Wistar controls. No change in weight gain or biopterin levels was observed in the biopsied animals. Mean serum biopterin levels in diabetes prone rats (of which 13 of 17 became diabetic at median 85 days) were the same as in diabetes resistant and Wistar rats at days 30,60 and 120, but showed a strik-ing and highly significant elevation (p > 0.001) atday 90. Although macrophages infiltrate the islet early in pre-diabetes, the timing of their activation is unknown. The rise in biopterin we observed is a potentially important immunological event which occurred late in the pro-gression of pre-diabetes. This acute terminal event has not been reported previously, and may modify current concepts concerning the tempo of cell destruction during pre-diabetes.

This study sought to determine the timecourse of development of reduced nerve laser Doppler flux in experimental diabetes and the effect on this anomaly of insulin treatment. In addition, we aimed to compare nerve laser Doppler flux in streptozotocin-and genetically-diabetic BB rat models. Sciatic nerve laser Doppler flux in diabetic rats was variable during the 2 days following streptozotocin injection; from day 4, when the measurement was 80% of control, fluxes fell steadily and formed a plateau at 40% of control values after 4 weeks of diabetes. In a second study, using rats with 4-week streptozotocin-diabetes, sciatic nerve laser Doppler flux was reduced to 44% of the value measured in control rats. Treatment of a parallel group of diabetic rats with insulin, by sustained release implants, prevented this ischaemia, so that nerve laser Doppler flux was 91% of controls. Nerve Doppler flux in BB rats with 6-week genetic diabetes was 57% of a control (non-diabetic) BB group. There were no differences in mean arterial pressures between control and diabetic rats in any of the studies. Heart rates of control and insulin-treated diabetic animals were higher than those of the untreated diabetic group; in the other studies heart rates of diabetic animals were numerically lower than controls, but not significantly so. These observations suggest that sciatic nerves of rats with short-term diabetes, whether induced with streptozotocin or of genetic origin, are markedly ischaemic and that this ischaemia in streptozotocindiabetes is evident within a week of diabetes onset, forms a plateau after 4 weeks and is maintained for at least 2 months. The findings also indicate that treatment of short-term diabetes with insulin can prevent nerve ischaemia.

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.

The BB/Wor diabetes-prone rat is an animal model of human insulin-dependent diabetes mellitus. In this model of spontaneous autoimmunity, natural killer cells are candidate cytotoxic effector cells, believed to be the mediators of beta-cell cytolysis in vivo. We therefore studied the effects of an anti-natural killer cell monoclonal antibody on the spontaneous development of diabetes in the BB/Wor rat. The 3.2.3 monoclonal antibody recognizes a molecular present on rat natural killer cells and selectively depletes these cells in vivo. Chronic treatment of diabetic-prone rats with 3.2.3 monoclonal antibody cleared circulating phenotypic natural killer cells, depleted in vitro spleen natural killer cell function, and profoundly reduced intra-islet accumulation of 3.2.3+ cells, but did not prevent or delay the onset of diabetes. These results indicate that natural killer cells are not necessary for the development of spontaneous diabetes in BB/Wor rats.

The role of the pancreatic microcirculation in the pathogenesis of Type 1 (insulin-dependent) diabetes mellitus remains poorly understood. Herein, a method is described for the ultrastructural investigation of the integrity of the pancreatic microvasculature. The method consists of histochemical detection and isolation of the islets followed by albumin and protein A-gold immunocytochemistry, whereby the distribution of endogenous albumin is used as a marker of endothelial integrity. This technique, applied to the study of spontaneously diabetic rats, reveals a selective increase in permeability of islet capillaries and post-capillary venules at the onset of diabetes, while acinar capillaries and arterioles remain intact. At 50 days of age, before the onset of diabetes, the microvasculature of diabetes-prone rats shows no alterations in permeability to albumin. When used in conjunction with morphometric analyses, this methodological approach may be useful for further studies in pathologic or experimental conditions involving the pancreatic microvasculature.