Diabetic BB Rats Research Articles

Increased gastrointestinal permeability is an early lesion in the spontaneously diabetic BB rat.

The BB rat spontaneously develops autoimmune diabetes. Feeding these animals a hydrolyzed casein diet significantly reduces the incidence of this disease, suggesting that a dietary antigen is involved in the pathogenesis of this disease. In other syndromes associated with luminal antigens, including celiac and Crohn's disease, increased intestinal permeability has been suggested to play an etiological role. Therefore, the objective of this study was to evaluate whether increased permeability was also present in BB rats before disease development. By measuring gastrointestinal permeability, in animals on a regular or hydrolyzed casein diet, we were able to demonstrate that increased gastric and small intestinal permeability appeared before the development of both insulitis and clinical diabetes. Although hydrolysis of dietary protein significantly reduced the incidence of diabetes, it did not alter the small intestinal permeability abnormality, suggesting that this is an early event. Increased permeability appears to have an early role in the genesis of several immunological diseases and may represent a common event in these diseases.

Chronic hypoglycemia and diabetes impair counterregulation induced by localized 2-deoxy-glucose perfusion of the ventromedial hypothalamus in rats.

Previous studies have demonstrated that the ventromedial hypothalamus (VMH) plays a critical role in sensing and responding to systemic hypoglycemia. To evaluate the mechanisms of defective counterregulation caused by iatrogenic hypoglycemia and diabetes per se, we delivered 2-deoxy-glucose (2-DG) via microdialysis into the VMH to produce localized cellular glucopenia in the absence of systemic hypoglycemia. Three groups of awake chronically catheterized rats were studied: 1) nondiabetic (with a mean daily glucose [MDG] of 6.9 mmol/l) BB control rats (n = 5); 2) chronically hypoglycemic nondiabetic (3-4 weeks, with an MDG of 2.7 mmol/l) BB rats (n = 5); and 3) moderately hyperglycemic insulin-treated diabetic (with an MDG of 12.4 mmol/l) BB rats (n = 8). In hypoglycemic rats, both glucagon and catecholamine responses to VMH glucopenia were markedly (77-93%) suppressed. In diabetic rats, VMH 2-DG perfusion was totally ineffective in stimulating glucagon release. The epinephrine response, but not the norepinephrine response, was also diminished by 38% in the diabetic group. We conclude that impaired counterregulation after chronic hypoglycemia may result from alterations of the VMH or its efferent pathways. In diabetes, the capacity of VMH glucopenia to activate the sympathoadrenal system is only modestly diminished; however, the communication between the VMH and the alpha-cell is totally interrupted.

Prevention of autoimmune recurrence and rejection by adenovirus-mediated CTLA4Ig gene transfer to the pancreatic graft in BB rat.

Type 1 diabetes is the result of a selective destruction of pancreatic islets by autoreactive T-cells. Therefore, in the context of islet or pancreas transplantation, newly transplanted beta-cells are threatened by both recurrent autoimmune and alloimmune responses in recipients with type 1 diabetes. In the present study, using spontaneously diabetic BB rats, we demonstrate that whereas isolated islets are susceptible to autoimmune recurrence and rejection, pancreaticoduodenal grafts are resistant to these biological processes. This resistance is mediated by lymphohematopoietic cells transplanted with the graft, since inactivation of these passenger cells by irradiation uniformly rendered the pancreaticoduodenal grafts susceptible to recurrent autoimmunity. We further studied the impact of local immunomodulation on autoimmune recurrence and rejection by ex vivo adenovirus-mediated CTLA4Ig gene transfer to pancreaticoduodenal grafts. Syngeneic DR-BB pancreaticoduodenal grafts transduced with AdmCTLA4Ig were rescued from recurrent autoimmunity. In fully histoincompatible LEW-->BB transplants, in which rejection and recurrence should be able to act synergistically, AdmCTLA4Ig transduced LEW-pancreaticoduodenal allografts enjoyed markedly prolonged survival in diabetic BB recipients. In situ reverse transcription-polymerase chain reaction revealed that transferred CTLA4Ig gene was strongly expressed in both endocrine and exocrine tissues on day 3. These results indicate the potential utility of local CD28-B7 costimulatory blockade for prevention of alloimmune and autoimmune destruction of pancreatic grafts in type 1 diabetic hosts.

Membrane abnormalities of vascular smooth muscle of mesenteric arteries of spontaneous diabetic BB rats.

Mesenteric arteries were isolated from the spontaneous diabetic BB rats, non diabetic BB rats and regular Wistar control rats. Gross morphology indicated that the mesenteric vascular bed of the control Wistar rats had a normal development of mesenteric fat pad around the vessels, while that of the diabetic BB rats showed drastically reduced perivascular fat pad, suggesting greater mobilization of fat for energy consumption in the hyperglycemic state of diabetes mellitus. The perivascular mesenteric fat pad of the non-diabetic BB rats was intermediate between those of the Wistar control and diabetic BB rats. The wet weight of the mesenteric arteries following removal of fat, vein and connective tissues was significantly greater in diabetic BB rats than in the corresponding controls. Microsomal membranes isolated from the mesenteric arteries of diabetic BB rats showed increased alkaline phosphatase and 5'-nucleotidase activities compared to those isolated from the two groups of non-diabetic control rats. Acid phosphatase activities were higher in both BB rat groups compared to the Wistar group. The total Ca2+ uptake by the microsomes of mesenteric arteries in the presence of ATP was not different among three experimental groups, but the ATP dependent active transport of Ca2+ was significantly increased and the passive Ca2+ binding was significantly reduced in diabetic group compared to the other two non-diabetic groups. Our results demonstrate that in the spontaneously diabetic BB rats, alterations in both structural and functional parameters may underline the vascular complications associated with type I diabetes mellitus in humans.

Intermittent Feeding and Fasting Reduces Diabetes Incidence in BB Rats

Food intake may be one of several factors which influence the risk of development of insulin dependent diabetes mellitus, but the influence of the pattern of food supply has not been studied previously. The aim of the present study was to investigate the effect of intermittent feeding and fasting upon diabetes in BB rats. This study included three groups. Group 1 served as control and included 77 animals, 79% became diabetic. In groups 2 and 3, after weaning, food but not water was withdrawn from the animals: 24 h twice a week in group 2; 24 h every second day in group 3. Group 2 included 40 BB rats, 50% (p < 0.004) became diabetic. Group 3 included 44 BB rats, 52% (p < 0.01) became diabetic. No differences were seen between sexes. Degree of insulitis was not influenced by changed food supply. Regarding blood glucose, no influence was seen among diabetic animals, among non-diabetic animals changed food supply reduced blood glucose values obtained at the end of the study. Intermittent feeding and fasting tended to reduce mean age at the time of diagnosis of diabetes, significance was reached only in female animals from group 3 compared to group 1. Body weight was obtained weekly. Intermittent feeding and fasting caused a reduced weight gain in group 2 as well as in group 3 compared to control animals; however, most pronounced in group 3 and also more pronounced among males compared to females. For pre-diabetic and non-diabetic animals comparable influence on body weight was seen. The main conclusion in the study is that intermittent feeding and fasting reduced diabetes incidence in BB rats.

Recombinant human platelet-activating factor acetylhydrolase reduces the frequency of diabetes in the diabetes-prone BB rat.

Platelet-activating factor (PAF) has been implicated in the development of type 1 diabetes. Our previous studies have suggested that PAF inhibitors reduce insulitis and the frequency of diabetes in BB rats. In this study, serum PAF levels were reduced to address the hypothesis that PAF is important for the development of insulitis. From the age of 35 days on, DP-BB rats were treated with human recombinant PAF acetylhydrolase (rPAF-AH), which efficiently inactivates PAF. Our data indicate that intraperitoneal injections of rPAF-AH reduce the incidence of diabetes in the DP-BB rat. Daily intraperitoneal injections of 6.0 mg/kg body wt rPAF-AH reduced the frequency of diabetes in saline-injected rats from 90% (27/30) to 57% (17/30) (P = 0.004). As found by morphometric analysis on pancreatic islets, DP-BB rats protected from diabetes had less severe degrees of insulitis in a dose-dependent manner. DP-BB rats protected by rPAF-AH also had a higher percentage of insulin-positive cells in pancreas sections compared with those from diabetic animals. We therefore speculated that the beta-cells were protected from insulitis by rPAF-AH.

Substance P binding in gastrointestinal tract of nondiabetic BB rat and changes in diabetic BB rat over time.

Our aim was to determine sites of substance P binding in the gut of the BB rat and examine changes in SP binding in the diabetic rat, over time. Specific binding of [125I]substance P was localized in sections of nondiabetic gut using emulsion autoradiography and quantitated in diabetic and nondiabetic gut using film autoradiography. High levels of SP binding were located in esophageal muscularis mucosa, circular muscle of the stomach and colon, deep muscular plexus, and in the circular muscle adjacent to the plexus in the ileum. The myenteric plexus demonstrated moderate to high levels of binding. Specific binding increased in the antrum and pylorus at three weeks and in the jejunum and distal colon at four weeks of diabetes but decreased in the distal ileum at two to four weeks of diabetes. Changes persisted at four to six months. These results contribute to understanding changes in the control of intestinal motility in diabetes.

Effects of recurrent hypoglycemia on brainstem function in diabetic BB rats: protective adaptation during acute hypoglycemia.

To determine whether antecedent recurrent hypoglycemia protects the brain from the adverse effects of a standardized hypoglycemic stimulus, we implanted electrodes in the inferior colliculi of diabetic rats to directly record inferior colliculi auditory-evoked potentials (ICEPs). Awake, chronically catheterized BB rats were studied after 2 weeks of insulin therapy designed to produce either chronic hyperglycemia (hyper-DM, glycated hemoglobin 7.6 +/- 0.4%) or recurrent hypoglycemia (hypo-DM, glycated hemoglobin 6.2 +/- 0.7%), and the results were compared with those observed in nondiabetic rats. When plasma glucose was lowered to and clamped at 2.8 mmol/l, the release of catecholamines was suppressed in the hypo-DM rats (epinephrine: 2.5 +/- 0.4 nmol/l) as compared with hyper-DM and the nondiabetic rats (9.3 +/- 2.3 and 32.7 +/- 6.1 nmol/l, respectively). ICEP latency was significantly delayed in hyper-DM and nondiabetic rats (P < 0.001), but it was unchanged in hypo-DM rats. A more pronounced reduction in plasma glucose (2.0 mmol/l), however, provoked a greater adrenergic response than that seen at 2.8 mmol/l and delayed ICEP latency by 23% in a separate group of hypo-DM animals. These data demonstrate that antecedent recurrent hypoglycemia attenuates the brainstem dysfunction associated with mild to moderate, but not severe, hypoglycemia in diabetic rats. This phenomenon may contribute to the alterations in hypoglycemia counterregulation seen in diabetic patients during intensive insulin therapy.

Effect of Diet on the Development of the Immune System in the BB Rat.

The incidence of autoimmune diabetes in the BB diabetic-prone (BBdp) rat can be substantially delayed or prevented by replacing cereal-based mixed ingredient (chow) rodent diets with semi-purified (SP) diets differing in protein sources. Replacing a chow diet with a casein-based SP diet early in life reduces the incidence of BB rat diabetes. Feeding soy-based SP diets results in lower diabetes rates than chow but higher than casein. The following study was conducted to determine the effect of early feeding of chow to casein and soy-based SP diets on the development of the immune system in the BB rat. BBdp and non-diabetes prone (BBn) rats were fed a defined chow (NIH) or one of a casein or soy SP diet, to the dams at least one week prior to birth and to the pups of 21-30 days of age. Pups were killed at 15 and 30 days of age and effects of early diet on several immune parameters were determined. At 30 days of age, all chow fed rats were heavier (p<0.05) than the rats of both SP diets. Although the BBdp rats in the chow and soy diet groups were significantly heavier (p<0.05) than BBn rats of the same diet groups, the body weights of casein-fed BBdp and BBn rats were similar. The BBdp animals had a lower (p<0.05) proportion of T cells than BBn rats at 30 days of age. However, the proportion of CD4 and CD8 cells did not differ significantly from BBn for the casein-fed rats. The BBdp animals had poor proliferative responses and their production of IL-2 was unchanged by diet, but the natural killer (NK) activity of the casein-fed rats was reduced (p<0.05) to a level not different from the BBn rats. Diet also altered the immune response of the BBn rats. Feeding a soy-based as compared to a casein-based diet reduced (p<0.05) T cell responses (proliferation and IL-2 production) but increased (p<0.05) parameters of the innate immune system (nitric oxide production and NK cell activity). The findings of this study have important implications in identifying an immune mechanism for dietary effects on the incidence of diabetes in the BB rat and are of potential interest in defining the role of dietary protein source on the development of the immune system.

Non-major histocompatibility complex-linked diabetes susceptibility loci on chromosomes 4 and 13 in a backcross of the DP-BB/Wor rat to the WF rat.

BB rats are used as models of autoimmune human IDDM. Genetic control of IDDM in both species is complex, including both major histocompatibility complex (MHC)-linked and non-MHC-linked genes. DP-BB rats develop IDDM spontaneously. Expression of disease in these animals requires homozygosity at the lyp locus, which causes lymphopenia. All genetic analyses of BB rat diabetes to date have backcrossed to the DP-BB strain or used (DP-BB x non-BB)F2 animals to ensure that a fraction of progeny are homozygous for lyp. Here we report the analysis of a backcross of the DP-BB rat to the histocompatible WF rat. Neither WF nor (WF x DP-BB)F1 animals develop spontaneous IDDM. However, 95% of (WF x DP-BB)F1 rats and a fraction of (WF x DP-BB) x WF backcross animals readily develop IDDM after treatment with polyinosinic:polycytidylic acid and a cytotoxic anti-RT6.1 monoclonal antibody. Using simple sequence length polymorphism analysis, we have mapped loci on chromosomes 4 and 13 that show significant linkage to IDDM expression and insulitis. The susceptibility locus on chromosome 4 is linked to, but not identical to, lyp. We propose a disease model for the BB rat that requires 1) the RT1u MHC haplotype for disease susceptibility, 2) a new locus on chromosome 4 for disease initiation (as measured by insulitis), 3) a new locus on chromosome 13 for disease progression in response to environmental perturbation, and 4) lyp for spontaneous expression of disease.

Effects of glucagon on glycogenolysis and gluconeogenesis are region-specific in periportal and perivenous hepatocytes

It has been established, mainly by histochemical and immunohistochemical studies, that liver cells are functionally heterogeneous, with periportal hepatocytes (PPHs) being predominantly gluconeogenic and perivenous hepatocytes (PVHs) being glycolytic. We therefore investigated the region-specific functional effects of glucagon on glycogenolysis and gluconeogenesis in isolated PPHs and PVHs prepared by the digitonin-collagenase method. BB rats, a model of insulin-dependent diabetes, were used to study the region-specific heterogeneity of gluconeogenesis in the diabetic state. Although glycogen content was not different between PVHs and PPHs in rats fed the normal diet, basal glucose release was 1.37 times greater in PVHs than in PPHs (P < .05). The increase in glucose release stimulated by 0.01 to 0.1 nmol/L glucagon was 1.52 times greater in PVHs than in PPHs (P < .05), whereas no differences were seen in response to 1 to 100 nmol/L glucagon. Glucose release from gluconeogenic substrates was 1.57 times greater in the PPHs than in the PVHs of fasted normal rats (P < .05), whereas the increase in gluconeogenesis produced by glucagon was not different between PPHs and PVHs. The glucagon-binding capacity, the cAMP release, and the increase in intracellular Ca 2+ stimulated by glucagon were not different between PPHs and PVHs in the fed or fasted states. Gluconeogenesis from gluconeogenic substrates was 1.52 times greater in the PPHs than in the PVHs of fasted nondiabetic BB rats (P < .05). After the development of diabetes, the gluconeogenic capacity in PVHs increased to the level observed in PPHs, but that in PPHs did not change. Thus there was no difference in gluconeogenesis between the PPHs and PVHs of diabetic BB rats. In both the PPHs and PVHs of diabetic BB rats, the 0.01 to 100 nmol/L glucagon-induced increase in gluconeogenesis was greater than that in PPHs from nondiabetic BB rats (2.30 and 3.07 times, P < .01, respectively). We conclude that PPHs and PVHs of normal rat liver express region-specific differences in their glycogenolytic and gluconeogenic responses to glucagon. In diabetic BB rats, the difference in the gluconeogenic capacity between PPHs and PVHs disappeared, whereas glucagon-induced gluconeogenesis was enhanced.

Aromatic hydroxylation in animal models of diabetes mellitus.

Although the involvement of oxidative stress is well documented in the diabetic state, the individual active oxygen species generated have not been demonstrated in animal models of diabetes currently used. Since streptozotocin-induced diabetes mellitus in animals still serves as an animal model of diabetes mellitus, but streptozotocin induces diabetes and generates oxidative stress per se, we decided to study whether aromatic hydroxylation reflecting hydroxyl radical attack was found in three animal models of diabetes mellitus without streptozotocin induction or in streptozotocin-induced diabetes only. For this purpose, we compared lipid peroxidation, aromatic hydroxylation of phenylalanine, glycoxidation in genetically determined diabetic mouse strains db/db and kk, and the diabetic BB rat to these parameters in the streptozotocin-treated rat. Kidney malondialdehyde concentrations, reflecting lipid peroxidation, pentosidine, and Nepsilon-caboxymethyllysine concentrations, reflecting glycoxidation, were significantly elevated in all diabetic groups as compared to their nondiabetic mates. Aromatic hydroxylation was significantly elevated in the streptozotocin-induced diabetic state exclusively. We conclude that biochemical, pathophysiological, and treatment studies in the streptozotocin model of diabetes mellitus may be confounded by the presence of products, reactions, and tissue damage generated by aromatic hydroxylation reflecting hydroxyl radical attack. We suggest it is not the diabetic state but streptozotocin that generates the hydroxyl radical, as reflected by aromatic hydroxylation in this model.

Serum Biopterin as a Marker of Immune Activation in the Spontaneously Diabetic BB Rat

Summary Tetrahydrobiopterin (BH4), is a pteridine product which is released by rodent macrophages and fibroblasts upon activation by cytokines. It has previously been reported as a potentially useful marker for monitoring immune activation during the prediabetic prodrome in the spontaneously diabetic BB rat. We have used serial serum BH4 measurements, first to confirm the hypothesis that serum BH4 is elevated as a result of immunostimulation, and second to monitor more closely the serum BH4 levels during the prediabetic prodrome in a cohort of diabetes prone BB rats. A modified biopterin radioimmunoassay protocol was used to establish a reference range of 25.5-80.1 nmolll (95% confidence limits) for 70 Wistar rats of mean age six months. When eight adult Wistar rats were treated with sic BCG there was a significant increase in serum BH4 level (p&lt;O.Ol) which returned to pre-treatment levels by day 16, coincident with the disappearance of visible signs of inflammation. A cohort of 26 diabetes prone BB rats, their diabetes resistant controls (n=20) and a cohort of Wistar controls (n=20) were then used to monitor serum BH4 levels from 30-120 days of age, at frequent intervals. The diabetes incidence in the diabetes prone animals was 100% with a mean age of onset of 80 days. The serum tetrahydrobiopterin level was significantly higher at day 30 in all three groups (p&lt; 0.05) than at any other time point. The serum BH4 level in the diabetes prone group was significantly higher at day 30 (p&lt;O.Ol ) and day 60 (p&lt;0.05) than in either control group. These results show that serum BH4 is a sensitive marker for immune activation following subcubaneous injection of BCG in the rat, and is detectable using RIA. Serum BH4 was maximum in this study at day 30, but the significantly higher levels at days 30 and 60 in the diabetes prone compared with the diabetes resistant animals suggest that the immune activation characteristic of prediabetes can be detected biochemically in blood samples.

Changes in the aortic endothelium and plasma von Willebrand factor levels during the onset and progression of insulin-dependent diabetes in BB rats

Endothelial injury has been implicated in the enhanced vascular disease associated with diabetes mellitus. In diabetic humans elevated plasma von Willebrand factor (vWF) levels have been interpreted as an indication of endothelial damage. Using the BB rat as a model of inherited insulin dependent-diabetes mellitus, plasma vWF and aortic endothelial ultrastructural alterations were examined during the first 7 months of diabetes. Total plasma vWF levels were determined by ELISA and vWF multimeric composition by electrophoresis. vWF was identified immunohistochemically. Following the onset of hyperglycemia, there were progressive alterations in aortic endothelial morphology, which were consistent with injury, and aortic intimal thickening was significantly greater in rats diabetic for 7 months compared to age-matched controls. Significant increases in the Weibel Palade (WP) body content of the endothelial cells were observed after 1 week and 2 months of diabetes, but not at later times. Endothelial alterations associated with the possible release of vWF appeared to involve fusion of WP bodies with other vacuoles rather than direct fusion with the cell membrane. Plasma vWF levels in diabetic rats were varied, but were not significantly different from those of control animals and did not correlate with either glucose or insulin levels. The multimeric composition of plasma vWF was also similar at all times in both diabetic and non-diabetic animals. From these observations, plasma vWF levels do not provide an indicator of the endothelial perturbations which occurs in diabetic rats.

Crosses between diabetic BB/OK and wild rats confirm that a third gene is essential for diabetes development.

Several crossing studies with diabetic BB rats have shown that in addition to the lymphopenia (Iddm1) and the MHC class II genes of the RT1U haplotype (Iddm2) there are further non-MHC genes essential for diabetes development. Because diabetes-resistant inbred rat strains may be homozygous for one of the diabetogenic non-MHC genes, masking the expression of diabetogenic genes and leading to an underestimation of the number of diabetogenic genes, we crossed wild and diabetic BB/OK rats. The F1 hybrids were backcrossed onto diabetic female (BC1W-F, n=97) and male BB/OK rats (BC1W-M, n=98) transferred to a specified-pathogen-free environment and studied for the frequency and age at onset of diabetes up to an age of 30 weeks. Comparing the results of these BC1 W hybrids with similarly derived hybrids using diabetes-resistant DA rats (BC1DA-F, n=113; BC1DA-M, n=216), the diabetes frequency in total was comparable indicating the action of three recessive genes. The percentage of diabetics in Iddm1 and Iddm2 homozygotes confirmed the existence of the third gene, Iddm3, but there were some sex differences; significantly more male than female BC1W-F and significantly more BC1DA-M than BC1DA-F males were diabetic. Regarding the age at onset, the BC1W-F hybrids manifested not only significantly earlier, but also more uniformly than BC1DA-F and BC1-M hybrids.

Antibodies to ICA512/IA-2 in Rodent Models of IDDM

Antibodies to ICA512/IA-2 are a well established marker of human IDDM and can be detected prior to and soon after the onset of insulin dependency. The non-obese diabetic (NOD) mouse and the diabetes-prone BB rat develop spontaneous diabetes as a consequence of T-cell mediated autoimmune destruction of islet β-cells, but the occurrence of autoantibodies is contro-versial. We tested sera from NOD mice and BB-rats for anti-ICA512 by a radioimmunoprecipitation assay (RIP). In sequential serum samples from 20 NOD mice, of which 15 developed diabetes, low levels of anti-ICA512 were demonstrable. Anti-ICA512 appeared close to the onset of hyperglycaemia and was usually transient. Non-diabetic NOD mice also produced anti-ICA512, but at a later age and at lower levels than the diabetic NOD mice. In a cross-sectional analysis of sera from BB rats, low levels of anti-ICA512 were present in 11/20 (55%) of non-diabetic-diabetes prone (DP) BB rats, 0/4 (0%) of diabetic DP BB rats, and 1/6 (17%) of diabetes-resistant BB rats. Anti-ICA512 was not detected in rats of other strains, including three Sprague-Dawley rats with streptozotocin-induced diabetes. In both NOD mice and BB rats the anti-ICA512 reactivity was directed to the cytoplasmic domain of the protein. The transient appearance of anti-ICA512 close to the onset of diabetes in NOD mice and the loss of these antibodies after diabetes onset is consistent with the occurrence of anti-ICA512 in human IDDM. Thus in both human IDDM and rodent models, anti-ICA512 is a marker of the impending onset of diabetes and disappears after diabetes onset.

Tissue ascorbic acid and polyol pathway metabolism in experimental 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 nonenzymatic 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.

Alpha interferon administration paradoxically inhibits the development of diabetes in BB rats

Alpha-interferon (IFN-α) is thought to be important in the pathogenesis of insulin dependent diabetes mellitus (IDDM). However, since potent inducers of IFN-α, viruses, have been shown to modulate immune function and autoimmunity, we investigated whether administration of recombinant IFN-α (rIFN-α) would inhibit the diabetic process in BB rats. The development of diabetes was significantly inhibited by injections of either 10 5 units or 4 × 10 5 units rIFN-α. rIFN-α was more effective in preventing disease when injections were initiated at an earlier age (28–30 days vs 35–40 days). Histologic examination revealed a markedly lower degree of insulitis in rIFN-α treated rats. The mean total peripheral WBC and differential count, T-cell subsets, peripheral blood NK cell number, splenic NK cell activity, and serum cytotoxic beta cell surface antibody levels were unaltered by rIFN-α administration. In vitro incubation with rIFN-α inhibited the Con A proliferative response of mononuclear splenocytes of BB rats but not of Sprague Dawley rats. These results document that rIFN-α treatment potently prevents diabetes by inhibiting the development of insulitis. This paradoxical diabetes sparing effect may have significant implications for the treatment and prevention of IDDM and towards the understanding the autoimmune process.

Immunological evidence for increased oxidative stress in diabetic rats.

The role of oxidative stress in aging and diabetes mellitus is currently under discussion. We previously showed age-dependent accumulations of fluorescent protein adducts with lipoperoxidative aldehydes, (malondialdehyde (MDA), and hydroxynonenal (HNE)) in rat skin collagen with diabetic BB rats exhibiting faster accumulation. Modified proteins have been shown to be immunogenic: antibody titres against rat serum albumin modified by MDA and HNE (MDA-RSA and HNE-RSA) or oxidized by reactive oxygen species were measured by ELISA as markers of oxidative damage in BB diabetic and non-diabetic rats. Each tested antibody titre was significantly higher in the diabetic than in the non-diabetic rats. A significant correlation existed between anti-MDA-RSA and anti-HNE-RSA antibody titers. Only the anti-HNE-RSA antibody titre increased significantly with age (p=0.052) in diabetic animals, while no titres increased significantly in non-diabetic animals. A major factor which correlated with the development of these antibodies was diabetes duration: this was significant (p=0.032) for anti-HNE-RSA antibody titre and slightly significant (p=0.05) for anti-MDA-RSA antibody titre. Thus, chronic hyperglycaemia is probably fundamental in the increase of oxidative stress. There is correlation between anti-aldehyde-RSA antibody titres and the corresponding aldehyde-related collagen-linked fluorescence: modified collagen may play a part in the observed immune response. Our data indicate a stronger immune response of diabetic rats against proteins modified by lipoperoxidative aldehydes and oxygen free radicals, and they support the hypothesis of increased oxidative damage in diabetes.

Preclinical studies of glimepiride.

The treatment of NIDDM is advancing on a number of fronts, ranging from the understanding of the mechanisms of action of existing agents to the development of new drugs. Glimepiride, the most recently U.S. approved sulfonylurea, is evaluated for its pancreatic and extrapancreatic activities in addition to safety, when compared with glibenclamide in preclinical studies. Glimepiride uniquely binds to the 65 kDa protein K(ATP) channel in pancreatic islet beta-cell membranes and directly stimulates insulin secretion. Glimepiride has a lower binding affinity and causes less insulin release than glibenclamide in normal dogs and humans. However, in various animal models, glimepiride produces a more rapid and pronounced blood glucose-decreasing activity than glibenclamide. In vivo studies indicate that glimepiride also significantly reduces HbA1c, blood glucose and fasting insulin levels via extrapancreatic activities. These insulin mimetic effects are supported by demonstration of stimulated lipogenesis in 3T3 adipocytes and glycogenesis in rat diaphragm, by more efficient translocation of GLTU4 in fat and skeletal muscle tissues, and by activation of key metabolic enzymes. The insulin sensitivity effects of glimepiride have been demonstrated in vivo by increased glucose disposal rates in euglycemic clamp studies and in vitro by increased sensitivity and responsiveness of insulin-induced glucose uptake. Moreover, glimepiride might stimulate insulin-mediated glucose utilization in hepatocytes. With exercise-induced hypoglycemia, a suppression of endogenous insulin secretion was observed for glimepiride only. Data accumulated from in vitro and animal studies suggest that glimepiride has the least cardiotoxic potential. Its relative activities in multiple cardiovascular studies do not correlate with its potency to lower blood glucose levels. Similar cardiovascular effects have been seen in human studies. In contrast to the lack of an acute action, chronic application of glimepiride to cultured cardiomyocytes was found to produce an approximate doubling of the basal glucose uptake rates by an insulin-independent pathway most probably involving increased protein expression of both GLUT1 and GLUT4. Like glibenclamide, glimepiride possibly has antiatherogenic activity by inhibiting platelet aggregation via suppression of arachidonic acid metabolism. Our recent studies on rats and humans indicated that glimepiride has immunomodulatory activity and this action appears to be related to lowering autoimmune responses rather than metabolic action. These studies have been extended to include glimepiride involvement with prevention of diabetes in BB rats using an islet transplantation model. Finally, sulfonylureas, including glimepiride, may be useful for treating and preventing NIDDM.