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We recorded arterial pressure (BP) and heart rate (HR) in type-1 diabetic rats vs. controls for >6 months. Diabetic rats (DIAB) were maintained on insulin from the day glucose >250 mg/dl ("Day 0"). Weight was similar between groups until ~3 weeks before Day 0 when the weight in DIAB transiently lagged the controls (CONT); this difference was maintained throughout the study, but both groups otherwise gained weight in parallel. Plasma glucose attained 371 ± 109 (SD) mg/dl by day 1 in DIAB. Mean BP was similar across groups, and declined through the initial 4-6 months in both the CONT (at -0.06 ± 0.04 mmHg/day) and in the DIAB (at -0.14 ± 0.21 mmHg/day; NS vs. CONT). HR in the CONT (Month 1: 341 ± 13 bpm) exceeded DIAB (325 ± 25 bpm) through ~6 months after Day 0, and also decreased progressively over this period in CONT (-0.19 ± 0.14 bpm/day) and DIAB (-0.29 ± 0.23 bpm/day; NS vs. CONT) before leveling. The BP power within 0.35-0.45 Hz changed during the 90 min before vs. after the transition from dark to light, and light to dark; there were no between group differences. The slope of the log-log linear portion of the BP power spectrum between 1.0/h and 1/min was similar across groups, and increased in both from month 1 to month 6. Regulatory mechanisms maintain similar profiles in BP and HR in diabetic vs. control animals through the initial half year of the disease., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aims: The Gimap gene family has been shown to be integral to T cell survival and development. A frameshift mutation in Gimap5, one of seven members of the Gimap family, results in lymphopenia and is a prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat. While not contributing to lymphopenia, the Gimap family members proximal to Gimap5, encompassed within the Iddm39 quantitative trait locus (QTL), have been implicated in T1D. We hypothesized that expression of the Gimap family members within the Iddm39 QTL, during thymocyte development as well as in peripheral T and B cells contribute to T1D., Main Methods: Cell sorted subpopulations were analyzed by quantitative real time (qRT) PCR., Key Findings: Gimap4 expression was reduced in DR.(lyp/lyp) rat double negative, double positive and CD8 single positive (SP) thymocytes while expression of Gimap8, Gimap6, and Gimap7 was reduced only in CD8 SP thymocytes. Interestingly, expression of the entire Gimap gene family was reduced in DR.(lyp/lyp) rat peripheral T cells compared to non-lymphopenic, non-diabetic DR.(+/+) rats. With the exception of Gimap6, the Gimap family genes were not expressed in B cells from spleen and mesenteric lymph node (MLN). Expression of Gimap9 was only detected in hematopoietic cells of non B cell lineage such as macrophage, dendritic or NK cells., Significance: These results suggest that lack of the Gimap5 protein in the DR.(lyp/lyp) congenic rat was associated with impaired expression of the entire family of Gimap genes and may regulate T cell homeostasis in the peripheral lymphoid organs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Lactobacillus johnsonii (Ljo) N6.2 has been shown to mitigate the development of type 1 diabetes when administered to diabetes-prone rats. The specific mechanisms underlying this observed response remain under investigation. The objective of this study was to assess the effect of Ljo N6.2 on mucosal inflammatory response using differentiated Caco-2 monolayers. The mRNA expression levels of CCL20, CXCL8, and CXCL10 chemokines were determined by qRT-PCR. Ljo at 10(11) CFU/L induced a strong response in all chemokines examined. To assess the specific host-signaling pathways involved, we performed RT-PCR amplification of Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors. TLR7 and TLR9 expression levels were induced 4.2- and 9-fold, respectively, whereas other TLR and nucleotide-binding oligomerization domain receptors were not modified. A similar effect was observed in Caco-2 monolayers treated with Ljo cell-free extract or purified nucleic acids (NA). Increased levels of IFN type 1 and IFN regulators Stat1 and IRF7 followed the upregulation of TLR9. Activation of TLR9 was also evidenced by increased Frizzled 5 expression in Ljo-treated Caco-2 cells and an increase in the number of Paneth cells in Ljo-fed, diabetes-prone rats. These results are in agreement with the polarizing-tolerizing mechanism recently described in which the apical stimulation of TLR9 in intestinal epithelial cells leads to a higher state of immunologic alertness. Furthermore, these results suggest that live probiotics could be, in the future, replaced with select cellular components.

B(io) B(reedding)/O(ttawa) K(alsburg) rats spontaneously develop insulin-dependent type 1 diabetes. Days before BB/OK rats become diabetic, their body seems to be flabby which may be attributed to loss of subcutaneous fat. However, the rats are normoglycemic and manifest 3-4 days later. This observation prompted us to search for possibilities to avoid the loss of adipose tissue. BB/OK rats were subcutaneously grafted with visceral adipose tissue. In total, 34 (71%) out of 48 male and 23 (49%) out of 47 female BB/OK rats grafted with adipose tissue developed type 1 diabetes so that significantly more females than males were protected from diabetes development (p=0.03). In the control group, 17 (85%) out of 20 male and 20 (95%) out of 21 female BB/OK rats were diabetic. Adipose tissue transplantation can protect BB/OK rats from type 1 diabetes development in a sex specific manner. One could conclude that the manipulations have influenced fat accumulation and/or fat metabolism which prevent type 1 diabetes development in about 50% of BB/OK rats. This idea is supported by the finding that a mutation in the leptin receptor of NOD mice suppresses type 1 diabetes progression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have recently found that 3T3-L1 adipocytes secrete microvesicles, known as adipocyte-derived microvesicles (ADMs), with angiogenic activity. In this study, we found that ADMs contain RNA without typical 28S and 18S ribosomal RNA inside the vesicles. Microarray analysis revealed that ADMs contain approximately 7000 mRNAs and 140 microRNAs. Most of transcripts for adipocyte-specific and dominant genes were contained in the ADMs, and their abundance was mostly correlated with that in the donor cells. Abundance of adipocyte-related microRNAs was also mostly correlated with that in the donor cells. ADMs mediated transport of adiponectin and resistin gene transcripts into RAW264.7 macrophages. Moreover, adipocyte-specific gene transcripts such as adiponectin, resistin, and PPARgamma2 were found in microvesicles isolated from rat serum. Thus, ADM might play a role as a novel intercellular communication tool by transporting RNA in paracrine and possibly endocrine manners., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

In this animal study, three topical antibacterial dressings, Acticoat, chlorhexidine acetate 0.5% and silver sulfadiazine 1%, were compared in the treatment of Acinetobacter baumannii contamination of burns. All treatments were effective and prevented the organism invading the muscle and causing systemic infection, so there were significant differences between the results of the treatment groups and the control group. Mean eschar concentrations did not differ significantly between the silver sulfadiazine and chlorhexidine acetate groups, but there were significant differences between these and the Acticoat group, indicating that Acticoat eliminated A. baumannii from the tissues more effectively.

We investigated molecular changes in the response to insulin in prostates of spontaneously developed (Bio Breeding) and streptozotocin (STZ)-induced diabetic rats that received sufficient amounts (euglycemic group), or suboptimal doses (hyperglycemic group) of insulin for 32 weeks, using Affymetrix GeneChip analysis of gene expression. Alterations in gene expression levels identified by microarray analysis, having potential biological relevance to prostate growth, were verified by real-time reverse transcription polymerase chain reaction (RT-PCR). A significant decrease in the weight of ventral prostate was observed in the hyperglycemic STZ-induced but not spontaneously developed diabetic group. Microarray analysis revealed that gene expression profiles were distinctly different in each region of the prostate, and that hyperglycemic diabetes in spontaneously developed and STZ-diabetic rats was associated with differential changes in the prostatic expression levels of 856 genes, of which 35 were related to cell growth, proliferation and death. RT-PCR data verified significant differences in the mRNA expression levels of Igfbp6, Tieg, and Clu between euglycemic and hyperglycemic groups, whereas expression levels of these genes in control and euglycemic diabetic groups were not significantly different. In ventral prostate, the mRNA expression levels of Igfbp6 and Tieg were significantly higher in the hyperglycemic STZ-induced diabetic than in the hyperglycemic spontaneously diabetic BBDP/Wor rats. Our data demonstrate that the diabetes induced by STZ in the BBDR/Wor rats affects prostate growth and the molecular response to insulin differently than that observed in BBDP/Wor rats that develop diabetes spontaneously.

Anticipating the future use of arginine to enhance fetal and neonatal growth as well as to treat diabetes and obesity, we performed studies in pigs, rats, and sheep to determine the pharmacokinetics of orally or i.v. administered arginine and the safety of its chronic supplementation. Our results indicate that all 3 species rapidly catabolized the supplemental arginine. The elevated circulating concentrations of arginine generally returned to baseline levels within 4-5 h after administration, with the rates varying with the age and physiological status of the animals. The clearance of arginine was greater in pregnant than in nonpregnant animals, in young than in adult animals, in lean than in obese animals, and in type-1 diabetic than in nondiabetic animals. I.v. administration of arginine-HCl to pregnant ewes (at least 0.081 g arginine.kg body weight-1.d-1) did not result in any undesirable treatment-related effect. Neonatal pigs, growing-finishing pigs, pregnant pigs, and adult rats tolerated large amounts of chronic supplemental arginine (e.g. 0.62, 0.32, 0.21, and 2.14 g.kg body weight-1.d-1, respectively) administered via enteral diets without the appearance of any adverse effect. On the basis of the comparative studies and a consideration of species differences in food intake per kilogram body weight, we estimate that a 70-kg human subject should be able to tolerate long-term parenteral and enteral supplemental doses of 6 and 15 g/d arginine, respectively, in addition to a basal amount of arginine (4-6 g/d) from regular diets.

Excitotoxic glutamate release occurs in several neurological disorders. One source is derived from the hydrolysis of the neuropeptide N-acetyl aspartyl glutamate (NAAG) by glutamate carboxypeptidase II (GCPII, also known as NAALADase). Drugs that attenuate glutamate transmission have been shown to relieve neuropathic pain, however side effects have limited their clinical use. It appears that GCPII is exclusively recruited to provide a glutamate source in hyperglutamatergic, excitotoxic conditions and therefore would be devoid of such side effects. Here we report on the therapeutic effects of an orally bio-available GCP II inhibitor on established painful and sensory neuropathy in the spontaneously diabetic BB/Wor rat. It significantly improved hyperalgesia, nerve conduction velocity and underlying myelinated fiber atrophy. The data suggest that GCP II inhibition may provide a meaningful and effective approach to the treatment of painful diabetic neuropathy.

Costimulatory signals regulate T-cell activation. To investigate the role of costimulation in autoimmunity and transplantation, we studied the BB rat model of type 1 diabetes. Diabetes-prone BB (BBDP) rats spontaneously develop disease when 55-120 days of age. We observed that two anti-CD28 monoclonal antibodies (mAb) with different functional activities completely prevented diabetes in BBDP rats. Anti-CD154 mAb delayed diabetes, whereas treatment with CTLA4-Ig or anti-CD80 mAb accelerated disease. Anti-CD86 or anti-CD134L mAbs had no effect. Diabetes resistant BB (BBDR) rats are disease-free, but >95% of them develop diabetes after treatment with polyinosinic-polycytidylic acid and an mAb that depletes Treg cells. In the induced BBDR model, anti-CD154 mAb delayed onset of diabetes, whereas CTLA4-Ig, anti-CD134L or either of the anti-CD28 mAbs had little or no effect. In contrast, blockade of the CD134-CD134L pathway was highly effective for preventing autoimmune recurrence against syngeneic islet grafts in diabetic BBDR hosts. Blockade of the CD40-CD154 pathway was also effective, but less so. These data suggest that the effectiveness of costimulation blockade in the treatment of type 1 diabetes is dependent on both the costimulatory pathway targeted and the mechanism of induction, stage, intensity and duration of the pathogenic process.

Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5-70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-beta1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-beta1) cDNA or antisense TGF-beta1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin-angiotensin system activation, TGF-beta1 expression, and PKC-beta1 signaling.

BB rats lose >50% of their islet sympathetic nerve terminals soon after diabetes onset, markedly impairing the glucagon response to activation of these nerves. In this study, we sought evidence that this degree of disease-induced nerve terminal damage affected their neuronal cell bodies. Increased galanin expression was used as a marker of the change of phenotype that occurs in neuronal cell bodies when their axons are severely damaged. The celiac ganglion (CG) was analyzed because it is a major source of the sympathetic nerves that project to the pancreatic islets. But we first needed to determine if damaging nerve terminals could increase galanin expression in this ganglion and, if so, when that expression was maximal. Severe, global nerve terminal damage produced a dramatic increase of CG galanin expression which was maximal 5 days later. We next determined if a global, but partial, nerve terminal loss would also increase galanin expression and found a significant increase of galanin mRNA and its peptide in the CG. Finally, we determined if the disease-induced, partial and islet-selective loss of nerve terminals seen in BB diabetic rats was sufficient to increase galanin: we, again, found a significant increase of galanin mRNA and its peptide in their CG. These increases did not occur in their superior cervical ganglia. We conclude that the selective damage to islet sympathetic nerve terminals seen in BB diabetic rats, rather than the systemic factors of diabetic hyperglycemia or insulin deficiency, causes the increased galanin expression observed in the CG of this animal model of type 1 diabetes.

This study systematically evaluated the conditions required for generating immature rat bone marrow-derived dendritic cells (BMDCs) and characterized their phenotype. The culture of Wistar rat bone marrow cells for 7 days in an optimal cytokine environment (granulocyte macrophage-colony stimulating factor (GM-CSF), 10 ng/ml; IL-4, 5 ng/ml) resulted in adherent and non-adherent cell populations, but only the adherent population predominantly expressed the rat DC marker OX62. Adherent OX62+ cells were immature, in that they expressed lower levels of CD86 and MHC class II and were more phagocytic than their non-adherent OX62+ counterparts. Adherent BMDCs constitutively produced low levels of IL-12 and nitric oxide (NO), levels of both of which were markedly increased following lipopolysaccharide (LPS) activation. Activation also increased the proportion of OX62+ cells expressing CD40, CD54 and CD86 and their intensity of expression, however, unlike murine BMDCs, it had no effect on CD80 and MHC class II expression. Although the proliferation of allogeneic Lewis splenocytes in response to immature resting and LPS-activated (mature) Wistar BMDCs was of a similar magnitude, levels of IL-12 after 5 days were significantly higher in cultures containing LPS-activated BMDCs and the IFN-gamma/IL-4 cytokine ratio differed markedly (2.35 vs. 6.66, respectively). This study systematically defines conditions for generating immature rat BMDC populations and demonstrates qualitative differences in the phenotype of immune responses induced by resting and LPS-activated BMDC populations.

The beta-cell loss seen in diabetes mellitus could be monitored clinically by positron emission tomography (PET) if imaging agents were sufficiently specific for beta-cells to overcome the high ratio of non-beta-cell to beta-cell tissue in pancreas. In this report, we present a screening assay for identifying beta-cell-specific compounds that is based on the relative accumulation and retention by islet, INS-1, and exocrine (PANC-1) cells of candidate molecules. Molecules thought to have a high affinity for beta-cells were tested and included glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone were the most specific, but the specificity ratios fell well below those needed to attain robust signal to background ratio as a PET imaging agent for quantifying beta-cell mass. In vivo tests of the biodistribution of glibenclamide and fluorodithizone in rats indicated that the compounds were not specifically associated with pancreas, bearing out the predictions of the in vitro screen.

Sitosterolemia is an autosomal recessive disorder caused by mutations in the ABCG5 or ABCG8 half-transporter genes. These mutations disrupt the mechanism that distinguishes between absorbed sterols and is most prominently characterized by hyperabsorption and impaired biliary elimination of dietary plant sterols. Sitosterolemia patients retain 15-20% of dietary plant sterols, whereas normal individuals absorb less than 1-5%. Normotensive Wistar Kyoto inbred (WKY inbred), spontaneously hypertensive rat (SHR), and stroke-prone spontaneously hypertensive rat (SHRSP) strains also display increased absorption and decreased elimination of dietary plant sterols. To determine if the genes responsible for sitosterolemia in humans are also responsible for phytosterolemia in rats, we sequenced the Abcg5 and Abcg8 genes in WKY inbred, SHR, and SHRSP rat strains. All three strains possessed a homozygous guanine-to-thymine transversion in exon 12 of the Abcg5 gene that results in the substitution of a conserved glycine residue for a cysteine amino acid in the extracellular loop between the fifth and sixth membrane-spanning domains of the ATP binding cassette half-transporter, sterolin-1. The identification of this naturally occurring mutation confirms that these rat strains are important animal models of sitosterolemia in which to study the mechanisms of sterol trafficking.

Animal models for autoimmune polyglandular syndrome that are either spontaneous or induced by manipulations are presented. In addition, recent cloning of the autoimmune regulator (AIRE) gene responsible for autoimmune polyglandular syndrome type I, and its homologue in mice, Aire, provided the opportunity to study the effect of this gene on autoimmune diseases by producing knockout mice. These animal models make it possible to perform experiments that cannot be performed in humans, which will increase our understanding of the cause and molecular mechanisms of the autoimmune diseases and lead to the development of effective methods for their prevention and intervention.

Aims/hypothesis: Hyperglutamatergic activity induced by ischemia is believed to underlie neuronal damage in a variety of neurological disorders, including neuropathic pain. Since ischemia is believed to be a prominent mechanism involved in diabetic polyneuropathy (DPN), we investigated the effect of the glutamate carboxypeptidase II (GCPII, EC #3.4-17.21; previously termed NAALADase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG to NAA and glutamate, on the development of DPN in type 1 diabetic BB/Wor rats., Methods: Diabetic animals were treated with 10 mg/kg/day i.p. of the selective GCPII inhibitor GPI-5232 from onset of diabetes for 6 months. Hyperalgesia to thermal stimulation and nerve conduction velocity (NCV) were measured monthly. The effect on structural DPN was assessed by scoring of single, teased myelinated fibers, myelinated fiber morphometry and ultrastructural examination of C-fibers at 6 months., Results: GCPII inhibition showed significant but partial effects on hyperalgesia (p<0.001), nerve conduction slowing (p<0.01) axonal and nodal structural changes (p<0.001), small myelinated fiber atrophy, and degenerative changes of C-fibers., Conclusions: GCPII inhibition has beneficial effects on hyperalgesia, nerve function, and structural degenerative changes in DPN, which are likely mediated by inhibition of ischemia-induced glutamate release.

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma, IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.

Streptozotocin (STZ)-induced diabetic rats have been associated with an impaired metabolic availability of vitamin A (retinol). This study was undertaken to investigate whether Biobreeding (BB) rats, in which diabetes mellitus resembling human type I diabetes develops spontaneously, respond the same way at the onset of diabetes. Weaning diabetes-prone (BBdp) and normal (BBn) BB rats consumed NIH-07 nonpurified diet ad libitum until 120 d of age. Plasma and hepatic concentrations of retinol and its carriers, retinol-binding protein (RBP) and transthyretin (TTR) were lower in diabetic BB (BBd) rats than in BBn rats. In parallel with RBP, the abundance of mRNA was lower in the liver of BBd rats. Furthermore, the status of zinc, an important factor for the synthesis of RBP, was also disturbed in BBd rats, as indicated by lower circulatory levels and greater urinary excretion. To determine whether the biochemical evidence of vitamin A deficiency in BBd rats could be reversed, BBdp rats were fed a diet supplemented with vitamin A either alone or in combination with zinc. None of these treatments increased plasma vitamin A concentration. The hepatic abundance of RBP mRNA was significantly greater, whereas circulatory RBP concentrations were unaffected by vitamin A plus zinc supplementation. Overall, these results suggest that impaired metabolic availability of vitamin A, possibly caused by its decreased transport from hepatic stores, is another metabolic derangement associated with type I diabetes.

Endothelial cell injury has been implicated in the increased incidence of vascular disease associated with diabetes mellitus. In diabetic humans, elevated plasma von Willebrand Factor (vWF) has been interpreted as an indication of endothelial damage. In contrast, in an animal model of inherited insulin-dependent diabetes, the bio-breeding (BB) rat, plasma vWF levels did not differ from those in age-matched control rats during the first 7 months of diabetes although morphological evidence of mild aortic endothelial alteration or injury was observed. In the present study efforts have been made to define the endothelial alterations in BB diabetic rats compared to controls more precisely over this time period. Thus, adhesion molecules: intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were evaluated by in situ immunohistochemistry, vWF content was determined by biochemical analysis of aortic extracts and by quantitative immunohistochemistry, plasma vWF levels were measured by ELISA and vWF mRNA by RNAse protection assay. Neither age nor diabetic state significantly affected either the expression of adhesion molecules, or the levels of circulating vWF. Endothelial vWF content was significantly increased in the diabetic vessels, as observed by both approaches but the vWF mRNA content was not different from that in control vessels. Plasma plasminogen activator inhibitor (PAI-1) activity was significantly increased in diabetic animals. In conclusion, endothelial alterations in BB rats associated with diabetes, together with the raised plasma PAI-1 levels, promote the thrombogenic potential of the vessel wall, and are consistent with an increased risk for vascular disease.

The association between early exposure to cow's milk products in infancy and risk for insulin dependent diabetes mellitus (IDDM) is controversial. We examined whether the ingestion of cow's milk-based infant formula altered the expression of the diabetic syndrome in the BB/Wor rat, an animal model of IDDM. Pregnant BB/Wor dams were obtained from the NIH contract colony at the University of Massachusetts and housed under semi-barrier conditions. Rat pups were intubated with 1 to 2 ml of commercially available cow's milk-based infant formula (Enfamil or Nutramigen) or sham intubated (controls) daily from day 12 to day 25 of life. Pups were weaned at day 25 and monitored for glucosuria daily through 120 days of life. All rats including dams consumed a milk-free rat chow and acidified water ad libitum throughout the study. The mean age of disease onset was 4 to 10 days earlier in Nutramigen-fed and Enfamil-fed rats relative to controls (84+/-3, 78+/-2 and 88+/-4 days, respectively); the mean age of disease onset was significantly different between controls and Enfamil-fed animals (p<0.05). At 120 days, 60% (12/20) of control rats developed diabetes versus 100% of animals fed either type of infant formula prior to weaning (15/15:Enfamil-fed; 19/19:Nutramigen-fed) (p<0.05). These data indicate that direct, early ingestion of cow's milk-based formula was related to the expression of diabetes in the BB/Wor rat.

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 Ca2+ 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.

The spontaneously hypertensive rat (SHR/Mol) and the spontaneously diabetic BB/OK rat were crossed, and the F1 hybrids were backcrossed onto the BB/OK rat in order to search for quantitative trait loci (QTL) affecting serum total cholesterol and triglycerides on chromosomes 1, 3, 4, 10, 13, 18, and X. On chromosome 4 a QTL for triglyceride levels (lod score 3.3) was found within the region flanked by the D4Mit9 and Il-6 markers. Suggestive linkage (lod score 1.9) was found for total cholesterol on chromosome 4 at the Spr locus. Also, on chromosome 1 suggestive linkage for both investigated traits was found at marker D1Mit14 (lod score 1.9 for triglycerides, 2.1 for total cholesterol). The results of the study could contribute to the explanation of the genetic basis of lipid abnormalities, which are a common feature of pathological disorders such as coronary heart disease, hypertension, or non-insulin-dependent diabetes.

By several crossing studies it has been demonstrated that the MHC class-II genes of the RT1u haplotype, Iddm1, and the lymphopenia, Iddm2, are essential, but not sufficient for diabetes development in the BB rat. Using diabetic BB/OK and diabetes-resistant DA rats it has been shown that a third non-MHC gene, Iddm3, on chromosome 18 cosegregates with diabetes in the BB/OK rat subline. Because mapping results need not be consistent among different crosses, we genetically analysed a new cross population using diabetic BB/OK and diabetes-resistant SHR/Mol rats analysing 73 microsatellite markers. The genetic analysis of Iddm1 and Iddm2 homozygous [(BB/OK x SHR)F1 x BB/OK] first backcross hybrids (BC1) confirmed the action of Iddm3 and one predisposing non-MHC locus, Iddm4, near Ighe/D6Mgh2 on chromosome 6 and one protective locus, Iddm5r(esistance), detected around Igf2/Tnt on chromosome 1. From these novel findings it is concluded that the diabetogenic phenotype of the BB/OK rat subline is the result of the interaction of predisposing and protecting diabetes genes.

Type II collagen (CII) is a potent arthritogen in the BB rat. To determine whether a restricted group of T cells is involved in the pathogenesis of collagen-induced arthritis, lymphocytes from synovium, peripheral blood, and lymph nodes of arthritic rats were studied for T cell receptor (TCR) V beta gene usage using polymerase chain reaction (PCR). Oligoclonal TCR V beta usage was found only in synovium recovered day 2 post-arthritis onset, but not day 7; lymph node and peripheral blood T cells showed diverse TCR usage at both times. To determine whether T cell local clonal expansion occurred in synovium at day 2 of arthritis, cDNA for four TCR beta families was sequenced through VDJ regions. Strong selective expansion of TCR V beta 8.2, 4, and 17 was noted. Importantly, the dominant clonotype of V beta 8.2 was identical to that of a lymph node-derived T hybridoma specific for the immunodominant epitope in CII(181-210). Cells from synovium (day 2 postonset) analyzed by flow cytometry also showed V beta 8.2+ cell enrichment. These observations, plus finding that T cells from inflamed synovium respond to CII(181-201) in vitro, suggest the local recruitment and clonal expansion of some T cells families, possibly driven by autologous CII released during cartilage degradation.

The knowledge of genetic factors determining the age at onset of diabetes may help to delay the development of diabetes and its complications. This prompted us to use our well-characterized BB/OK rat whose age at onset of diabetes can change from 50 to more than 400 days for crossing studies to search for loci cosegregating with the age at onset. Fifty-nine diabetic first backcross hybrids resulting from crosses between diabetic BB/OK and diabetes-resistant DA and SHR rats were genotyped with PCR-analyzed microsatellite markers located on 21 chromosomes. Loci on chromosomes 6 (Ighe -D6Mgh2) and 8 (D8Mit2- Apoc3) were linked with the age at onset. Hybrids which were homozygous for the BB alleles developed significantly earlier diabetes than hybrids which were heterozygous The difference between the age at onset of heterozygous and homozygous hybrids reached a maximum at the loci Ighe on chromosome 6 (+ 32 days, p = 0.0018) and D8Mit2 on chromosome 8 (+ 28 days, p = 0.007). Candidate genes around the loci linked with the age at onset of diabetes are involved in the humoral and cellular immune response. For the first time, this study provides evidence that genetic factors can affect the age at onset of diabetes in the rat.

Alpha-interferon (IFN-alpha) is thought to be important in the pathogenesis of insulin dependent diabetes mellitus (IDDM). However, since potent inducers of IFN-alpha, viruses, have been shown to modulate immune function and autoimmunity, we investigated whether administration of recombinant IFN-alpha (rIFN-alpha) would inhibit the diabetic process in BB rats. The development of diabetes was significantly inhibited by injections of either 10(5) units or 4x10(5) units rIFN-alpha. rIFN-alpha 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-alpha 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-alpha administration. In vitro incubation with rIFN-alpha inhibited the Con A proliferative response of mononuclear splenocytes of BB rats but not of Sprague Dawley rats. These results document that rIFN-alpha 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.

Besides other factors, lymphopenia (ll) is essential for development of insulin-dependent diabetes mellitus in the BB rat. It is unknown which phenotypic consequence may have a non-lymphopenia. Therefore, the region containing the lymphopenia gene of the BB/OK rat was replaced with that of the non-lymphopenic (LL) and hypertensive rat, SHR (11 cM, D4Mit6-LL-Npy-Spr). The resulting congenic strain, BB.LL, did not develop diabetes up to an age of 30 weeks and was non-T-lymphopenic. The SHR region did not influence the blood pressure of the BB.LL rat, but influenced obviously the motor activity, body weight, serum lipids, and the 24h urine excretion of urea, sodium, and potassium.

In this study the spontaneously hypertensive rat (SHR/Mol) and the spontaneously diabetic BB/OK rat were crossed, and the F1 hybrids were backcrossed onto the BB/OK rat in order to search for cosegregation between blood pressure and loci on chromosomes 4 and 13. Cosegregation of microsatellites on chromosomes 4 (Spr, Npy, D4mit6, Il-6) and 13 (Atp1a2, D13Mit1, D13Uwm1) with blood pressure was evaluated using one-way analysis of variance. On chromosome 4 linkage of the Npy and D4Mit6 markers to systolic blood pressure was observed. A blood pressure QTL was also found on chromosome 13 within the renin locus. Surprisingly, alleles of the SHR strain at loci showing linkage to blood pressure on chromosome 4 and 13 promote lower blood pressure than the same alleles from the BB/OK strain. The transfer of D4Mit6 and renin locus from the SHR rat onto the genetic background of BB/OK rat will probably not lead to a model of diabetic hypertension, but the thorough characterisation of such congenics could contribute to the explanation of genetics and pathophysiology of hypertension in the SHR rat., (Copyright 1997 Academic Press.)

Nonenzymatic glycation of proteins and oxidative stress are considered independent factors important in the development of the complications of diabetes but may be interrelated by the process of autoxidative glycation. This pathway involves monosaccharide autoxidation to a reactive ketoaldehyde analogue and subsequent reaction with protein to form a ketoimine adduct. This study demonstrates that delta-gluconolactone (delta-GL), an oxidised analogue of glucose, is a potent glycating agent in vitro of haemoglobin present in blood samples from insulin-dependent diabetic and non-diabetic human subjects and from spontaneously diabetic, insulin-dependent BB/Edinburgh (BB/E) rats. The percentage glycated haemoglobin after incubation (37 degrees C, 5 h) with delta-GL (25 mmol/l) was significantly (P < 0.002) higher than that observed using an equimolar concentration of glucose. Intravenous administration of delta-GL (1 g/kg) to non-diabetic BB/E rats also significantly increased glycation of haemoglobin (6.0 +/- 0.1% vs 4.9 +/- 0.1%, P < 0.01) whereas intravenous injection of an identical dose of glucose had no significant effect (5.1 +/- 0.1% vs 5.0 +/- 0.2%). These results support the hypothesis that nonenzymatic glycation of proteins involves attachment by both native and oxidised monosaccharides. Further investigation of the interactions between diabetes-associated increases in oxidative stress and glycation on the development and progression of the vascular complications of diabetes is necessary.

Hypertension and diabetes mellitus are known to be frequently associated. The genetic dissection of diseases such as hypertension or diabetes mellitus is possible by using experimental crosses, which allow identification of loci influencing phenotypic traits (quantitative trait loci - QTLs). In this study the spontaneously hypertensive rat (SHR) and spontaneously diabetic, but normotensive rat (BB/OK) were crossed and the F2 population was analysed in order to search for QTLs on selected chromosomes (1, 10, 18) for blood pressure and some metabolic traits related to diabetes, renal function and hypertension. There were 3 regions found on chromosome 1 which showed linkage to blood pressure. The strongest evidence for linkage was observed between loci Igf2 and D1Mgh12. On chromosome 10 there was a QTL for blood pressure found between Ppy and Abp and on chromosome 18 there were three regions (Ttr-Grl, Tilp-Gja1, Olf-D18Mit9) with linkage to blood pressure. Since the 24 hr albumin and phosphate excretion correlated with blood pressure in F2 hybrids, the same regions were linked to both parameters. Region with linkage to serum concentrations of cholesterol (probably located beyond the terminal marker Ttr of the linkage group) were also found. The results of this study with a new F2(BB x SHR) population confirm the existence of previously described blood pressure loci (Sa and Bp2) and showed novel QTLs on chromosomes 1, 10 and 18.

We have previously reported that weekly administration of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), reduces the incidence of insulin-dependent diabetes mellitus (IDDM) in the BB Wistar rat, and this effect is likely due to immunosuppression by dCF. In the present study, we examined the effect of altering the dose and scheduling of dCF on prevention of IDDM in the BB rat. When rats were treated from day 25 of age with 2.5, 4, or 10 mg of dCF/kg/week, the percentage of diabetes-free animals at 120 days of age was 40, 60, and 80% respectively, compared with 10% for control animals, demonstrating increased protection against IDDM with increased dCF dose. Histological assessment of the pancreata from animals that became diabetic revealed a marked mononuclear infiltrate and a loss of positive staining for beta cell granules. In contrast, pancreata from animals that remained diabetes-free appeared normal. Protection against IDDM by dCF was time dependent and only occurred if treatments were initiated by day 30 of age. In addition, the protective effect persisted after drug withdrawal. Further studies are required to determine the optimum duration of therapy with dCF to prevent IDDM and to examine the immunological mechanism responsible for this effect.

RT6 is a cell surface alloantigen that identifies a regulatory subset of peripheral T cells in the rat. Diabetes-prone BB rats are deficient in peripheral RT6+ T cells and develop spontaneous autoimmune insulin-dependent diabetes mellitus. Diabetes-resistant BB rats have normal numbers of RT6+ T cells, and insulin-dependent diabetes mellitus can be induced in these animals by in vivo depletion of peripheral RT6+ cells. Athymic rats are also severely deficient in peripheral RT6+ T cells. Although very different with respect to the peripheral RT6+ cell compartment, normal, athymic, and diabetes-prone BB rats all generate RT6+ intestinal epithelial lymphocytes (IELs). The goal of these studies was to analyze the ontogeny of RT6+ IELs and peripheral lymphoid cells by in situ immunohistochemistry. We observed the following. 1) RT6+ IELs appear before alpha(beta) T-cell-receptor- expressing IELs in diabetes-prone BB, diabetes-resistant BB, and athymic WAG rats. 2) In vivo depletion of peripheral RT6+ T cells in diabetes-resistant BB rats using a cytotoxic monoclonal antibody is not accompanied by depletion of RT6+ IELs. 3) A population of RT6+ T-cell-receptor-negative IELs is present in normal, euthymic diabetes-resistant BB rats, constitutes a larger percentage of the euthymic but lymphopenic diabetes-prone BB rat IEL population, and is the predominant IEL phenotype in athymic WAG rats. These results suggest that RT6+ cells are composed of both thymus-dependent and thymus-independent cell subsets that have different developmental characteristics and may differ in function.

Endothelial dysfunction is known to occur in chemically-induced animal models of diabetes. The BB diabetic rat is a genetic diabetes-prone model which more closely resembles Type I diabetes mellitus. In this study, we examined the role of superoxide anion radical and cyclooxygenase activity on endothelial dysfunction in aorta of the spontaneous diabetic BB rat. Vascular endothelial function was studied in vitro in aortic rings from 8-wk diabetic rats and age-matched nondiabetic littermates. There was no alteration in reactivity to norepinephrine as a result of diabetes. Relaxation to acetylcholine (but not nitroglycerin) was impaired in diabetic rings. Relaxation to acetylcholine was abolished by 100 microM L-nitroarginine but unaltered by an equimolar concentration of aminoguanidine (an inducible nitric oxide synthase inhibitor) in both control and diabetic rings. Incubation with 10 microM indomethacin did not alter relaxation to acetylcholine in either control or diabetic rings. In contrast, addition of 20 U/ml superoxide dismutase enhanced relaxation to acetylcholine in diabetic rings but had no effect on relaxation to acetylcholine in control rings. Thus, nitric oxide-mediated, endothelium-dependent relaxation is diminished in aortic rings of the genetic diabetic BB rat. Furthermore, superoxide anion radicals but not cyclooxygenase products play an important role in endothelial dysfunction in this genetic diabetic model.

The effect of feeding a diet that produces a high or low incidence of diabetes on immune abnormalities proposed to contribute to the pathogenesis of autoimmune-mediated diabetes was investigated. Diabetes-prone (BBdp) and nondiabetes-prone (BBn) BB rats (21 d) were fed for 21 d a nonpurified (high incidence) or purified (low diabetes incidence) diet. Compared with BBn, immune cells from spleen and mesenteric lymph nodes of BBdp rats demonstrated higher rates of metabolite production from glucose and glutamine, higher splenic cytotoxicity (lysis of 51Cr-labeled YAC-1 cells) and lower mitogen responses (3H thymidine uptake). Bypassing the membrane, using the mitogen phorbol myristate acetate + lonomycin, improved the BBdp mitogen response, suggesting a membrane defect. Immune cells from BBdp rats fed the purified, compared with the nonpurified, diet had lower rates of glutamine (spleen) and glucose (lymph nodes) metabolism and lower splenic cytotoxic activity. Although diet altered the proportion of T and B cells in lymph nodes of BBdp rats, it did not correct the abnormal lymphocyte distribution or effect mitogen responses. Feeding the purified, compared with nonpurified, diet to BBn rats altered energy metabolism by lymph node cells and resulted in lower splenic cytotoxic activity. Reduced splenic natural killer cell activity and decreased immune cell metabolism are unlikely the mechanism for the preventative effect of feeding a purified diet to BBdp rats but are indicative of reduced immune activity.

A novel neutrophil elastase inhibitor, ONO-5046, was administered to BB/DR rats and DBA/1 mice immunized with type II collagen to study its effect on the development of collagen-induced arthritis (CA), an experimental model of human rheumatoid arthritis. ONO-5046 reduced the incidence as well as the severity of CA in both rats and mice. This suppressive effect on severity was correlated with improvement of the histological findings, particularly with reduced destruction of the articular cartilage. These results indicate that neutrophil elastase may play an important role in the pathogenesis of CA.

In rats, RT6 and CD45RC are expressed by mature peripheral T cells. The underrepresentation of T cells expressing these markers in the T lymphocytopenic BB rat might therefore be a reflection of a relatively immature T cell population. With the use of Thy-1 as a marker for recent thymic migrants, it was demonstrated that BB rats indeed have a phenotypically less mature T cell population than age-matched control rats. However, this could not account for the reduced percentages of RT6+ and CD45RC+ T cells, as these were also decreased among mature Thy-1- T cells of BB rats. Although relatively overrepresented, absolute numbers of Thy-1+ T cells were reduced in BB rats. Absolute numbers of mature Thy-1- T cells were also reduced in BB rats, but to a much larger degree than would proportionally be expected. Our findings taken together led us to conclude that both reduced thymic output and a defect in peripheral expansion are involved in the T lymphocytopenia of BB rats.

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.

An elevated circulating sialic acid concentration is a risk factor for cardiovascular disease. Serum sialic acid levels are elevated in NIDDM but not in uncomplicated IDDM. To study why sialic acid is increased in some types of diabetes, we assayed plasma sialic acid in various animal models of diabetes: obese (ob/ob) mice, before and after streptozotocin treatment, neonatal streptozotocin-treated (nSTZ) rats, and diabetic BB rats during and after insulin treatment. In obese mice, which exhibit moderate hyperglycemia and marked hyperinsulinemia, plasma sialic acid was decreased by 45% (fed) and 42% (fasted), compared to lean controls. Fasting reduced plasma glucose and insulin but increased sialic acid in the obese and lean mice. There was a negative correlation (r = -0.84, P < 0.001) between log plasma insulin and sialic acid in the lean and obese mice. The plasma sialic acid:globulin ratio was reduced by 35% in obese mice vs. lean controls, indicating that there may be altered sialylation of glycoproteins in obese mice. Streptozotocin treatment of obese and lean mice reduced plasma insulin but increased sialic acid. In nSTZ rats, hyperglycemia was associated with mild hypoinsulinemia, but not significantly different from control animals, and sialic acid was not altered. In diabetic BB rats, plasma glucose rose from a mean of 4.9 to 23.5 mM 48 hr after insulin withdrawal but sialic acid did not change. We conclude that an elevated plasma sialic acid level is associated with marked insulin deficiency, rather than hyperglycemia per se. The magnitude and speed of this change in sialic acid varies between species.

The purpose of this study was to determine whether Glimepiride, an oral sulfonylurea drug, prevents the onset of diabetes in diabetic prone BB rats. S750181, a sulfonylurea drug that has minimal in vivo glucose metabolic effects, was also tested. In addition, the shortest period of sulfonylurea treatment required for prevention was determined. Eighty rats were studied for all treatment periods with 40 receiving a daily oral gavage dosage of glimepiride and 40 receiving a daily oral gavage dosage of vehicle solution. Diabetes onset was monitored by glycosuria and blood glucose levels. In study I, with a treatment period of 35-142 days of age, Glimepiride-treated rats showed a 32% incidence of diabetes, whereas control rats had a diabetes incidence of 55% (p < 0.04). In study II, with a treatment period of 60-140 days of age, Glimepiride-treated rats showed a 29% incidence of diabetes compared to 54% in controls (p < 0.03). Further, comparing the time of diabetes onset between the Glimepiride and control groups showed that Glimepiride delays diabetes onset (p < 0.02). In study III, with a treatment period of 60-100 days of age, Glimepiride-treated rats showed a 17% overall diabetes incidence at 170 days, whereas the controls were 43% (p < 0.01). In study IV, with a treatment period of 60-140 days of age, S750181-treated rats showed a 38% diabetes incidence and the control group showed a 43% diabetes incidence. There was no significant delaying or prevention effect observed in the S750181 group. To determine if Glimepiride affected autoimmune events, the severity of islet inflammation was examined. In study I, islet histology from total and nondiabetic animals indicated that Glimepiride-treated rats had a lower severity of islet inflammation than that of the control rats (p = 0.023). These studies show that a) Glimepiride has diabetes preventive effects, b) shorter treatment periods of only 40 days can be effective and c) Glimepiride decreases the severity of islet inflammation.

Abnormalities in the microenvironment of dorsal root ganglia (DRG) might play a role in the pathogenesis of sensory abnormalities in human diabetic neuropathy. We examined aspects of DRG microenvironment by measuring local blood flow and oxygen tension in the L4 dorsal root ganglia of female BB Wistar (BBW) diabetic rats with mild neuropathy. The findings were compared with concurrent measurements of local sciatic endoneurial blood flow and oxygen tension. Diabetic rats were treated with insulin and underwent electrophysiological, blood flow and oxygen tension measurements at either 7-11 or 17-23 weeks after the development of glycosuria. Nondiabetic female BB Wistar rats from the same colony served as controls. At both ages, BBW diabetic rats had significant abnormalities in sensory, but not motor conduction compared to nondiabetic controls. Sciatic endoneurial blood flow in the diabetic rats of both ages was similar to control values, but the older (17-23 week diabetic) BBW diabetic rats had a selective reduction in DRG blood flow. Sciatic endoneurial oxygen tensions were not significantly altered in the diabetic rats. DRG oxygen tension appeared lowered in younger (7-11 week diabetic) but not older (17-23 week diabetic) BBW rats. Our findings indicate that there are important changes in the DRG microenvironment of diabetic rats with selective sensory neuropathy.

We report an association between thymic epithelial defects and predisposition to autoimmunity. Diabetes-prone (DP) BB rats develop spontaneous hyperglycemia and are deficient in T cell subsets expressing the RT6 alloantigen. Diabetes resistant (DR) BB rats become diabetic if depleted of RT6+ T cells. The inciting immune system defects are unknown. We made the following observations: 1) Regions of thymic cortex and medulla devoid of thymic epithelium exist in DP-BB, DR-BB, and Lewis rats, all of which are susceptible to autoimmune disorders. Such defects were absent in eight normal rat strains. 2) Thymic epithelial defects are absent at birth, but present in BB rats at 4 weeks of age. 3) The genetic predisposition to thymic epithelial defects is an autosomal dominant trait. 4) The observation of thymic defects in (DP x WF)F1 rats led to the prediction that such animals, which never develop spontaneous autoimmunity, might be susceptible to its induction. Following depletion of RT6+ T cells we observed diabetes in 91%, and thyroiditis in 43%, of treated F1 animals (n = 23). Pancreatic insulitis was uniformly present. Because thymic epithelium participates in the positive and negative selection of developing thymocytes, we propose that thymic epithelial defects may play an important role in the predisposition of BB rats to autoimmunity.

Previously we demonstrated that in streptozotocin-induced or spontaneously diabetic BB rats (BB-SDR), low-Km cyclic AMP (cAMP), phosphodiesterase (PDE), and calmodulin (CaM) are decreased. Isolated fat cells of diabetic animals synthesized less CaM and contained reduced levels of CaM transcripts (Solomon SS, Palazzolo MR, Green SA, Raghow R. Biochem Biophys Res Commun 1990; 168: 1007-12). Treatment of diabetic animals with insulin restores CaM transcripts to normal. RNA was extracted from isolated hepatocytes from BB-SDR rats in primary tissue culture treated with insulin (from 2.8 x 10(4) to 1.4 x 10(6) microU/ml) for 48 hours, was immobilized on nitrocellulose, and was sequentially hybridized with radiolabeled probes for CaM, actin, and tubulin. Insulin stimulates steady state levels of mRNA for calmodulin > actin > tubulin. Furthermore, decreased steady state levels of CaM mRNA in hepatocytes from diabetic animals are restored to normal levels with in vitro insulin incubation. Data from nuclear transcription run-on assays demonstrate that insulin stimulates transcription of mRNA CaM by 80%. In addition, we observed RNA degradation in the untreated diabetic but not insulin-treated liver. These data support transcriptional as well as post-transcriptional effects of insulin on CaM mRNA. We postulate that in uncontrolled diabetes, elevations in levels of cAMP in tissue result in part from decreased activity of the apparently co-regulated PDE and CaM and that PDE inactivation in diabetes results from both insulin insufficiency and CaM down-regulation.

Previous studies in experimental diabetes have demonstrated cardiovascular abnormalities of the beta-adrenergic system and reduced adrenergically stimulated renal renin secretion. To examine the defect in the beta-adrenergic signal, glomerular cyclic adenosine monophosphate (cAMP) levels were measured in response to isoproterenol and other humoral agonists (coincubated with the phosphodiesterase inhibitor isomethylxanthine) in nondiabetic and diabetic BB/Wor rats. Basal (unstimulated) levels of glomerular cAMP did not differ between control and diabetic BB/Wor rats, nor did cAMP accumulation differ on incubation with the humoral agonists PGE2 and histamine. However, on incubation with varied concentrations of the nonselective beta-adrenergic agonist isoproterenol, control glomeruli demonstrated a twofold increase in cAMP while a negligible response was observed in diabetic glomeruli. Peak levels of cAMP were higher in control (192 +/- 24 pmol/mg protein) than in diabetic (141 +/- 8 pmol/mg protein) glomeruli (p < 0.01). No differences were observed on incubation with the adenylate cyclase stimulator forskolin. Measurement of glomerular beta-adrenoreceptors by coincubation with iodine 125-labeled cyanopindolol demonstrated no differences in either receptor number (Bmax) or affinity (KD). These data indicate that a specific defect in beta-adrenergic signalling exists in glomerular tissue from spontaneously diabetic rats. Because no decrease in forskolin-stimulated adenylate cyclase was observed, defective coupling of the receptor to its effector, perhaps through the guanine nucleotide stimulatory protein, may account for these observations.

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded polyribonucleotide that elicits immune responses analogous to those observed during viral infection. It is also known to modulate the expression of certain autoimmune disorders including diabetes mellitus in the BB rat and NOD mouse. The mechanism underlying these immunomodulatory effects is not known, but it could involve activation of vascular endothelium. We now report that parenteral poly I:C induces rat pancreatic endothelium to hyperexpress intercellular adhesion molecule 1 (CD54). This is accompanied by a perivascular recruitment of mononuclear cells to the exocrine pancreas. Corollary in vitro studies demonstrated that poly I:C is a potent activator of both rat and human endothelial cells in culture. It upregulates endothelial expression of several leukocyte adhesion molecules, stimulates the release of interleukin-6 and interleukin-8, and antagonizes interferon-gamma induction of major histocompatibility complex class II expression. We conclude that poly I:C activates endothelial cells to express surface molecules and cytokines in a pattern classically associated with leukocyte recruitment. These effects may in part contribute to the immunomodulatory effects of poly I:C in animal models of autoimmunity.

Hydroxyl radicals (.OH) may contribute to beta cell death. Because iron catalyzes .OH production, we examined whether administration of a novel, long-acting iron chelator, hydroxyethyl starch-deferoxamine (HES-DFO) could prevent diabetes in spontaneously diabetic biobreeding (BB) rats. In our colony, a peripheral lymphocyte count (PBLC) < 4200 mm3 has an 88% positive predictive value for onset of diabetes mellitus (DM). Rats with PBLC < 4200 mm3 were randomized at 6 weeks of age to receive 50 mg/kg of HES-DFO (a high molecular weight hydroxyethyl starch-conjugated derivative of deferoxamine) or equimolar hydroxyethyl starch (HES) alone given intraperitoneally three times weekly until DM or 120 days of age. Administration of HES significantly decreased the incidence of IDDM to 57% as compared with the incidence of 87% in the lymphopenic unmanipulated BB rats in the colony (p < 0.01). Administration of HES-DFO further significantly decreased the incidence of IDDM to 31% as compared with the lymphopenic unmanipulated rats (p < 0.01). When analyzed by sex, 3 of 17 (18%) HES-DFO-treated males developed DM, versus 10 of 17 (58%) of HES-treated males (p < 0.05, chi square); 8 of 19 (42%) of HES-DFO-treated females developed DM, versus 11 of 20 (55%) HES-treated females (p = NS). There were no differences between the groups in (1) mean time of onset of DM, (2) serum iron levels at study entry and completion, (3) weekly hematocrits, (4) total lymphocyte counts; and (5) weekly weight gains.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastric emptying of a suspended solid, digestible meal was examined after 30 to 90 days of diabetes in two strains of streptozotocin-treated rodents and in genetically diabetic BB/Wor rats. After a 14-hour overnight fast the animals were gavage-fed 1.0 gm of rat food suspended in water and were killed 30 minutes later. Ligatures were placed around the lower esophageal sphincter and the pylorus and the stomachs were removed intact. The stomachs were evacuated and the gastric contents and gastric remnant were separately dried and weighted. Both the streptozotocin-treated and BB/Wor rats showed significantly accelerated (1.5- to 2.2-fold) rates of gastric emptying of the meal compared with respective nondiabetic control animals. Streptozotocin-treated diabetic animals that received daily insulin supplementation or those that had undergone pancreas transplantation shortly after induction of diabetes showed a similar reduction in blood glucose levels and normal rates of gastric emptying. Diabetes had a significant but variable effect on gastric mass and body weight. This response was dependent on the strain of animal examined, whether or not insulin supplementation was administered, or whether the animal underwent pancreas transplantation. These observations indicate that early diabetes in rodents has a prokinetic effect on gastric emptying of a suspended solid digestible meal. Restoration of euglycemia by insulin treatment or pancreas transplantation is associated with return of the gastric emptying rate to normal. These changes in gastric emptying appear to be independent of the effects of diabetes on gastric mass.

Differences in the type and extent of diabetic glomerular injury occur in rats made diabetic with streptozotocin (STZ) and in a spontaneously diabetic rat model of the BB/Wor strain. The diabetes prone (BB/DP) rat develops no mesangial matrix expansion with diabetes, whereas the STZ-treated Sprague Dawley rat does. The present study was performed to determine if glomerular metalloprotease activities were decreased in these two rat models of diabetes. Rats were studied at 8 weeks following the onset of hyperglycemia. All diabetic rats were maintained moderately hyperglycemic (plasma glucose = 350 mg/dL) with daily injection of insulin. Glomerular preparations were made and metalloprotease activities were assayed by hydrolysis of gelatin. Metalloprotease activities were decreased by 40-50% in STZ-treated Sprague Dawley rats, but not in BB/DP rats or diabetes resistant rats (BD/DR--the control for the BB/DP) made diabetic by STZ-injection. These data suggest that the decrease in glomerular metalloprotease activity may contribute to mesangial matrix expansion in the diabetic Sprague Dawley rats.