Diabetic BB Rats Research Articles

Lymphocytic 2′,5′-O1igoadenylate Synthetase Is Insensitive to dsRNA and Interferon Stimulation in Autoimmune BB Rats

The interferon (IFN)-dependent 2',5'-oligoadenylate synthetase (2-5A synthetase), which produces 2',5'-oligoadenylates from ATP, was analyzed in homogenates of isolated peripheral blood lymphocytes (PBL) from BB and Sprague-Dawley rats, man, sheep, and beagle dog. In all the examined species, the 2-5A synthetase was expressed constitutively and showed sensitivity differences to poly(I:C) (synthetic dsRNA). The 2-5A synthetase activity in the absence of poly(I:C) was high in the BB and Sprague-Dawley rat where only 2-5A dimers were synthesized. With the notable exception of PBL homogenates from BB rats, increasing poly(I:C) concentrations resulted in an increased 2-5A synthetase activity leading to the production of higher 2-5A oligomers, predominantly the octamer. Diabetes-resistant, diabetes-prone, and diabetic BB rats were indistinguishable in that their 2-5A synthetase was insensitive to poly(I:C). Preincubation of PBL from BB and Sprague-Dawley rats with up to 1,000 U/ml rat IFN elicited a moderate increase of 60% in the activity level of 2-5A synthetase. In contrast, preincubation of human PBL with human IFN-alpha led as expected to a 300% increase in 2-5A activity. Thus, the BB rat was markedly different from the other species in producing only the biologically inactive 2-5A dimers and in having a high basal 2-5A synthetase activity, that was unaffected by poly(I:C). We believe that these factors per se or together may render the BB rat more susceptible to virus attacks and/or may create a background that will facilitate the development of autoimmune processes.

Islet cells but not thyrocytes are susceptible to lysis by NK cells

BB rats develop both pancreatic insulitis and lymphocytic thyroiditis, but whereas spontaneous autoimmune diabetes is common, hypothyroidism is rare. Splenic natural killer (NK) cells from acutely diabetic (AD) BB rats and from athymic nude rats are known to be cytotoxic to rat islet cells in vitro. To investigate possible differential tissue susceptibility to lysis by NK cells or their cytokines such as cytolysin (perforin) or NK cytotoxic factor (NKCF), we used an in vitro 51Cr-release assay to measure the cytotoxicity of splenocytes, cytolysin or NKCF against Wistar Furth (WF) and Fischer 344 (F-344) rat islet cells, and FRTL-5 F-344-derived and WRT Wistar-derived rat thyrocytes. The results demonstrated that spleen cells from AD-BB (RT1 u) rats and athymic F-344 nude (RT1 1) rats are cytotoxic to WF (RT1 u) islets and F-344 (RT1 1) islets, but not to FRTL-5 (RT1 1) or WRT (class IRT1 1) thyrocytes. WF and F-344 rat spleen cells were not cytotoxic to any of these cells. Thyrocytes are known to express class II molecules on their surface in chronic thyroiditis. We found that treatment of thyrocytes with interferon-gamma (IFN-γ) induced class II expression but did not increase the cytotoxicity of splenocytes against these cells. Cytolysin and NKCF were both cytotoxic to islets in a dose dependent manner, but FRTL-5 thyrocytes were resistant to killing by these cytokines. These findings suggest that islet cells and thyrocytes in vitro are differentially susceptible to lysis by NK cells.

Intrathymic Islet Transplantation in the Spontaneously Diabetic BB Rat

Recently it was demonstrated that pancreatic islet allografts transplanted to the thymus of rats made diabetic chemically are not rejected and induce specific unresponsiveness to subsequent extrathymic transplants. The authors report that the thymus can also serve as an effective islet transplantation site in spontaneously diabetic BB rats, in which autoimmunity and rejection can destroy islets. Intrathymic Lewis islet grafts consistently reversed hyperglycemia for more than 120 days in these rats, and in three of four recipients the grafts promoted subsequent survival of intraportal islets. In contrast intraportal islet allografts in naive BB hosts all failed rapidly. The authors also show that the immunologically privileged status of the thymus cannot prevent rejection of islet allografts in Wistar Furth (WF) rats sensitized with donor strain skin and that suppressor cells are not likely to contribute to the unresponsive state because adoptive transfer of spleen cells from WF rats bearing established intrathymic Lewis islets fails to prolong islet allograft survival in secondary hosts.

Increased NPY in BB diabetic rat pancreas

Increased NPY in BB diabetic rat pancreas

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

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.

Insulin secretion and islet endocrine cell content at onset and during the early stages of diabetes in the BB rat: effect of the level of glycemic control

Although it is agreed that autoimmune destruction of pancreatic islets in diabetic BB rats is rapid, reports of endocrine cell content of islets from BB diabetic rats at the time of onset of diabetes vary considerably. Because of the rapid onset of the disease (hours) and the attendant changes in islet morphology and insulin secretion, it was the aim of this study to compare islet beta-cell numbers to other islet endocrine cells as close to the time of onset of hyperglycemia as possible (within 12 h). As it has been reported that hyperglycemia renders the beta cell insensitive to glucose, the early effects of different levels of insulin therapy (well-controlled vs. poorly controlled glycemia) on islet morphology and insulin secretion were examined. When measured within 12 h of onset, insulin content of BB diabetic islets, measured by morphometric analysis or pancreatic extraction, was 60% of insulin content of control islets. Despite significant amounts of insulin remaining in the pancreas, 1-day diabetic rats exhibited fasting hyperglycemia and were glucose intolerant. The insulin response from the isolated perfused pancreas to glucose and the glucose-dependent insulinotropic hormone, gastric inhibitory polypeptide (GIP), was reduced by 95%. Islet content of other endocrine peptides, glucagon, somatostatin, and pancreatic polypeptide, was normal at onset and at 2 weeks post onset. A group of diabetic animals, maintained in a hyperglycemic state for 7 days with low doses of insulin, were compared with a group kept normoglycemic by appropriate insulin therapy. No insulin could be detected in islets of poorly controlled diabetics, while well-controlled animals had 30% of the normal islet insulin content.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in Bladder Function in the One Year Spontaneously Diabetic BB Rat

Micturition characteristics and in vitro urinary bladder function were investigated in insulin-treated spontaneously diabetic BB rats and age-matched non-diabetic controls one year after the onset of diabetes. BB rats weighed less than controls and were hyperglycemic. Diabetic rats consumed larger volumes of water and excreted larger volumes of urine than controls. The frequency of micturitions and the mean volumes of urine excreted per micturition were significantly increased in BB rats compared to age-matched controls.Associated with the micturition changes in the BB rats were significant increases in bladder body mass. Contractile responses of strips from bladder bodies and bases were measured in response to nerve stimulation, carbachol, phenylephrine, ATP, and KCl. No significant differences between controls and diabetics were found in the absolute contractile responses of bladder body strips to nerve stimulation, carbachol, ATP, or KCl. However, if the data were transformed to correct for the increases in tissue mass in the diabetics, there were significant decreases in the responses of bladder body strips from BB rats to carbachol, ATP, and KCl, but not to nerve stimulation. Even after transformation, there were no differences in the responses of bladder base strips to carbachol, phenylephrine, or KCl. The data indicate that significant changes in micturition characteristics are evident one year after the onset of diabetes in the spontaneously diabetic BB rat. These changes are slow in development, since they are absent six months after the onset of diabetes. The changes in micturition and bladder strip contractility are qualitatively similar to, but quantitatively modest in comparison with those caused by streptozotocin-induced diabetes mellitus. The quantitative differences are probably attributable to an ameliorative effect of the insulin received by the BB rat.

The Anomeric Malaise: A Manifestation of B-Cell Glucotoxicity

In non-insulin-dependent diabetic subjects and in various animal models of spontaneous or experimental chronic hyperglycaemia, the secretory response of the pancreatic B-cell to a rapid rise in extracellular D-glucose concentration is characterized by a paradoxical, early and transient fall in insulin output and/or an altered anomeric specificity. These two features of B-cell glucotoxicity may be accounted for by the accumulation of glycogen in the B-cell and the interference of changes in glycogenolysis with the hexose-induced increase in glycolytic flux. The inhibitory action of D-glucose upon glycogenolysis displaying alpha-stereospecificity, the metabolic and secretory response to alpha-D-glucose is expected to be more severely affected than that evoked by the beta-anomer. Such a preferential alteration of the response to alpha-D-glucose was indeed documented in diabetic subjects, BB rats, duct-ligated rabbits, and adult rats either injected with streptozotocin during the neonatal period or rendered hyperglycaemic by the repeated administration of diazoxide. In these experimental models, the attenuation, suppression or even reversal of the anomeric preference in insulin release appeared related to the severity and duration of the hyperglycaemic state. A clear distinction ought to be made between these features of B-cell glucotoxicity and other etiopathogenic factors of B-cell dysfunction, such as the long term deleterious effect of streptozotocin upon the activity of key mitochondrial dehydrogenases.

Autonomic neuropathy in myocardium of spontaneous diabetic BB-rats

Autonomic neuropathy in myocardium of spontaneous diabetic BB-rats

Transplantation of purified islet cells in diabetic BB rats

The ability to prepare purified islet Beta-cell aggregates was used to examine the survival of this cell type after allotransplantation in diabetic BB rats. The aggregates were intraportally implanted in numbers that were previously found to correct a streptozotocin-induced diabetic state in syngeneic or allogeneic Brown Norway recipients. When the grafts were prepared from RT1u/l donors, which shared the MHC-class I antigen with the BB recipients (RT1u/u), their implant sites became diffusely infiltrated by inflammatory cells and their metabolic function was completely lost within 5 weeks. MHC-class I incompatible islet Beta-cell allografts (RT1n/n) exhibited a longer survival, in particular when combined with other islet endocrine cells and/or when covered by a 5-week cyclosporin treatment. In the latter combination, 10 of 12 BB rat recipients remained normoglycaemic over the 10-week observation period, their liver implants presenting a comparable insulin reserve and similarly discrete mononuclear cell infiltration as streptozotocin-diabetic Brown Norway rats receiving this treatment. However, administration of cyclosporin to diabetic BB rats was associated with a morbidity that was not observed in drug-treated streptozotocin-diabetic Brown Norway animals or in untreated diabetic BB rats. It is concluded that MHC-incompatible islet Beta cells can induce a long-term normalization in diabetic BB rats provided that they are implanted under conditions which allow allograft acceptance. The standardized preparation of purified islet Beta-cell grafts can help assessing the conditions for successful transplantations in diabetes with an autoimmune origin.

In vitro contractile responses of vasa deferentia from spontaneously diabetic BB rats.

1. The effects of diabetes of 6 months' duration on autonomic function of the vas deferens were examined in spontaneously diabetic BB rats and diabetes-resistant BB rats. 2. Diabetes caused significant decreases in rat and testes weights, but had no effects on the weights of vasa deferentia compared to diabetes-resistant controls. 3. The contractile responses of vasa deferentia to nerve stimulation and to carbachol were increased compared to controls. However, the contractile responses of vasa deferentia from diabetic BB rats to ATP, phenylephrine, and 120 mM KCl were unaltered. 4. The increased responses to carbachol were associated with increases in the muscarinic receptor density, but no changes were detected in the number of dihydropyridine binding sites. 5. The data indicate that, in contrast to vasa deferentia from streptozotocin-diabetic rats which exhibit decreases in responsiveness to nerve stimulation and a non-specific supersensitivity to contractile agents, vasa deferentia from diabetic BB rats have no changes in sensitivity, and increased contractile responses to only nerve stimulation and carbachol. These differences in urogenital sensitivity to diabetes may be related to the insulin treatment which diabetic BB rats require to keep them alive.

Elevated glutamine metabolism in splenocytes from spontaneously diabetic BB rats.

To investigate the metabolic fates of glutamine in splenocytes from the BB rat with spontaneous immunologically mediated insulin-dependent diabetes, freshly isolated cells were incubated in Krebs-Ringer Hepes buffer with 1.0 mM-[U-14C]glutamine and 0, 4 mM- or 15 mM-glucose. (1) The major products of glutamine metabolism in splenocytes from normal and diabetic rats were ammonia, glutamate, aspartate and CO2. (2) The addition of glucose increased (P less than 0.01) glutamate production, but decreased (P less than 0.01) aspartate and CO2 production from glutamine, as compared with the values obtained in the absence of glucose. However, there were no differences in these metabolites of glutamine at 4 mM- and 15 mM-glucose. (3) At all glucose concentrations used, the productions of ammonia, glutamate, aspartate and CO2 from glutamine were all markedly increased (P less than 0.01) in splenocytes from diabetic rats. (4) Potential ATP production from glutamine in the splenocytes was similar to that from glucose, and was increased in cells from the diabetic rat. (5) ATP concentrations were increased (P less than 0.01) in diabetic-rat splenocytes in the presence of glutamine with or without glucose. (6) Our results demonstrate that glutamine is an important energy substrate for splenocytes and suggest that the increased glutamine metabolism may be associated with the activation of certain subsets of splenocytes in the immunologically mediated diabetic syndrome.

Enhanced 2-deoxy-D-glucose uptake and metabolism in splenocytes from diabetic and diabetes-prone BB rats. Further evidence to support prior in vivo activation

Glucose metabolism in splenocytes from the BB rat was studied for the presence of abnormalities in [14C] 2-deoxy-D-glucose (2-dGlc) uptake, [U-14C]glucose conversion to 14CO2, and the production of lactate and pyruvate. Cells were studied freshly isolated ("resting"), and following culture both unstimulated (control) and stimulated with concanavalin A (ConA) or phorbol myristate acetate (PMA) + ionomycin. Both resting and control cells from diabetic (BBd) and diabetes-prone (BBdp) rats transported more (p less than 0.05) 2-dGlc than did cells from nondiabetes-prone (BBn) rats. Consistent with prior in vivo activation, sustained in vitro, lactate production was higher (p less than 0.05) under control conditions in BBd and BBdp than in BBn cells. Lactate production increased less with ConA and PMA + ionomycin in both BBd and BBdp than in BBn cells. PMA + ionomycin increased 2-dGlc uptake as much in BBd and BBdp cells as in BBn cells. Elevated rates of pyruvate production were observed in BBd cells under resting, control, and (especially) ConA conditions, suggesting an abnormality in pyruvate conversion to lactate. Few changes were observed in 14CO2 production. The presence of similar abnormalities in BBdp cells to those of the BBd cells suggests that the diabetic state is not causal, and the absence of an in vitro effect of 15 mmol/liter glucose in BBn cells further tends to exclude hyperglycemia as a cause of these alterations.

Urinary Bladder Function 6 Months after the Onset of Diabetes in the Spontaneously Diabetic BB Rat

Urinary bladder function was examined in the spontaneously diabetic BB rat six months after the onset of diabetes. Diabetes caused significant decreases in rat weight and increases in bladder body weights and in vivo bladder capacities compared to age-matched controls, but no changes in the weights of bladder bases. The absolute contractile responses of urinary bladder body and base strips to nerve stimulation, carbachol, 5-hydroxytryptamine, ATP, phenylephrine, and KCl were unaltered by diabetes. However, when the data were corrected for tissue mass, there were slight but not significant decreases in contractile responses of strips from diabetic rats. There were increases in total muscarinic receptor numbers and calcium channel binding sites in bladder bodies from BB rats as a result of the increases in tissue mass. The data indicate that the six month-diabetic BB rat differs from the streptozotocin-diabetic rat in the sensitivity of the urinary bladder to the complications of diabetes, probably as a result of the insulin treatment required to keep BB rats alive.

Metabolic effects of IGF-I and insulin in spontaneously diabetic BB/w rats

To examine the influence of insulin-dependent diabetes on the metabolic response to insulin-like growth factor I (IGF-I), awake chronically catheterized diabetic and nondiabetic BB/w rats received IGF-I (5 micrograms.kg-1.min-1) or insulin (2 mU.kg-1.min-1) for 2 h while maintaining euglycemia. In nondiabetic rats, IGF-I and insulin produced similar twofold increases in glucose uptake, but insulin was more effective in reducing hepatic glucose production (90 +/- 15 vs. 5 +/- 11%; P less than 0.001) and beta-hydroxybutyrate levels (94 +/- 1 vs. 19 +/- 6%; P less than 0.001). In diabetic rats, insulin-stimulated glucose uptake was impaired (8.5 +/- 0.9 vs. 11.5 +/- 0.9 mg.kg-1.min-1 in nondiabetics; P less than 0.05). In contrast, IGF-I-stimulated glucose uptake was identical in diabetic and nondiabetic rats. Furthermore, IGF-I suppressed glucose production by 73% (P less than 0.01) and caused a greater lowering of beta-hydroxybutyrate levels (from 2.9 +/- 0.8 to 0.8 +/- 0.3 mumol/l) in diabetic rats. We conclude that 1) the capacity of IGF-I infusion to stimulate glucose uptake is maintained in spontaneously diabetic BB rats that are insulin resistant, and 2) IGF-I infusion suppresses elevated glucose production rates and plasma ketone concentrations in diabetic rats but is relatively ineffective in nondiabetic rats. Thus the metabolic responses to infused IGF-I do not appear to be diminished in diabetic rats with impaired responses to insulin.

Vitamin C and vitamin E status in the spontaneously diabetic BB rat before the onset of diabetes

Ascorbic acid (AA), dehydroascorbic acid (DHAA), and vitamin E were measured in tissues and plasma of 30 control and 30 spontaneously diabetic BioBreeding rats (BBdp) during development and before the onset of diabetes. At weaning, rats were fed an AIN-76 semisynthetic diet for 30, 64, or 113 days, after which plasma and tissues from 10 rats of each group were collected and analysed for AA, DHAA, and vitamin E. AA and DHAA levels were significantly increased in plasma and spleen of the diabetes-prone rats compared with those of the control group at 30 and 64 days, but the difference disappeared by 113 days. No differences were observed in liver, adrenals, thymus, and pancreas at any of the time periods. However, lower levels of vitamin E were observed in adrenal gland, thymus, and pancreas of the diabetes-prone rats. It is concluded that BBdp rats have an altered metablolism of AA, DHAA, and vitamin E, before the onset of diabetes. These changes could be due to genetic and physiological factors operating during development of this rat strain.

Milk Proteins in the Etiology of Insulin-Dependent Diabetes Mellitus (IDDM)

The etiology of insulin-dependent diabetes mellitus (IDDM) is multifactorial. The final cause of the disease, the specific destruction of the islet beta-cells, is the result of a cellular/humoral autoimmune process that operates in individuals with a particular genetic background in response to an external triggering factor(s). The most likely environmental triggers are virus infections and dietary factors. Among the latter group dietary proteins, mainly cow milk proteins, have been found to be important. Elimination of intact cow milk proteins from the diet significantly reduced the incidence of IDDM in the spontaneously diabetic BB rat, the elimination being most effective when it occurs during the pre-weaning period. Conversely, in newly discovered diabetics (both rats and children) increased levels of antibodies to cow milk proteins as compared with non-diabetic controls were found. These higher titres of antibodies were against beta-lactoglobulin and anti-bovine serum albumin. In further studies we found that antibodies to bovine serum albumin cross-react with a beta-cell membrane protein of Mr 69,000 and that this protein is likely induced by interferon. At the molecular level, a region of the bovine serum albumin has distinct homology to the beta-subunits of the MHC class II proteins Ia, DQ and DR, and antibodies raised against this bovine serum albumin region identified the same 69K beta cell membrane protein, in the same manner as antibodies to the third hypervariable region of DR-beta did. Our hypothesis is that bovine milk proteins (mainly bovine serum albumin) might be an important environmental factor providing specific peptides that share antigenic epitopes with host cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Biliary function in spontaneously diabetic BB rats

Biliary function in spontaneously diabetic BB rats

Vitamin D receptors and compensatory tissue growth in spontaneously diabetic BB rats.

Untreated diabetic (BB) rats exhibited compensatory intestinal growth which was associated with hyperplasia and was accompanied by an increase in unoccupied vitamin D receptors. Although vitamin D receptors were increased, low circulating 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] prevented amplification of the action of 1,25(OH)2D3, as evidenced by reductions in calbindin D-9K and alkaline phosphatase activity in the BB rat intestine compared to control. In the kidney, a lesser degree of compensatory growth was observed which was not associated with hyperplasia, and no significant effect of diabetes on vitamin D receptors or calbindin D-28K was observed. These studies suggest tissue-specific changes in 1,25(OH)2D3 metabolism during spontaneous diabetes which may be related to the hyperplasia which occurs during compensatory tissue growth.

Effects of Ginkgolide B, A Platelet-Activating Factor Inhibitor on Insulitis in the Spontaneously Diabetic Bb Rat

The BB rat spontaneously develops insulin-dependent diabetes mellitus (IDDM) in association with marked insulitis in the islet of Langerhans. Since platelet-activating factor (PAF-acether) is involved in allergic and inflammatory reactions, we tested a PAF antagonist, Ginkgolide B (BN 52021) for potential effects on islet inflammation and diabetes. Diabetes prone BB/Wor rats were treated daily from weaning at 25 days until 105 days of age with either saline (n = 30, controls), 10 (n = 25, low dose) or 20 (n = 30, high dose) mg/kg body weight of BN 52021. The overall incidence of IDDM was unaffected by treatment. Quantitative analysis of insulin area showed a dose-dependent protection of beta cells by Ginkgolide B, reflected in a 6- (low dose) to 8-fold (high dose) (P less than 0.01-0.005) increase in the insulin/glucagon cell ratio compared to the saline treated rats. Ginkgolide B reduced severe insulitis from 84% in the saline rats developing IDDM to 59% (n.s.) in the low and to 33% (P less than 0.001) in the high dose group. These data suggest that PAF inhibitors may prove useful in immunomodulator therapy of IDDM since beta cells are preserved.