BioBreeding Rats Research Articles

Clearance of apoptotic beta-cells is reduced in neonatal autoimmune diabetes-prone rats.

The kinetics of beta-cell death in neonatal diabetes-prone (BBdp) and diabetes-resistant (BBdr) BioBreeding rats was investigated using both direct (histochemical) and indirect (mathematical modelling) techniques. In both BBdp and BBdr rats, the incidence of TUNEL positive beta-cells increased until 10 days of age before declining. The number of apoptotic beta-cells was significantly higher in BBdp as compared to BBdr neonates from birth until 20 days of age (P<0.05). Using a mathematical model applied to the time course of beta-cell mass and replication rate, a wave of net beta-cell loss was detected between 10 and 20 days of age in both strains. In contrast to the observed difference in the incidence of TUNEL positive beta-cells, with the model-based approach we found no difference in the rate of beta-cell apoptosis between BBdp and BBdr rats prior to weaning. As the number of apoptotic cells present in a tissue depends on the rate at which cells die and the rate at which the apoptotic cell debris is cleared, we compared in vitro phagocytosis of apoptotic thymocytes by peritoneal macrophages from 2-week-old BBdp and BBdr rats. Macrophages from BBdp neonates engulfed significantly less apoptotic cells as compared to BBdr neonates (P<0.0005). Taken together, these findings suggest that there is impaired clearance of apoptotic beta-cells in diabetes-prone BB rats during the neonatal period.

Invertase, maltase, lactase, and peroxidase activities in duodenum of BB rats.

The development of immune-mediated diabetes in BB rats may involve a defect of the gastrointestinal tract (GI), as suggested by increased gut permeability. This study aimed at measuring invertase, maltase, lactase, and peroxidase activities in the duodenum of diabetesprone BioBreeding (BBdp) rats and control BioBreeding rats (BBc) given free access to NIH-07 diet up to the time of killing at 60 66 d of age. After washing the entire small intestine, the duodenal mucosa was scraped off in the first 5-cm segment from the pylorus and frozen in distilled water. Invertase, maltase, and lactase activities were measured by monitoring the conversion of [U-(14)C]sucrose, [U-(14)C]maltose, and [D-[1-(14)C]glucose] lactose to radioactive hexoses, which were phosphorylated in the presence of adenosine triphosphatase and yeast hexokinase and then separated from their precursor by ion-exchange chromatography. Peroxidase activity was measured by a spectrophotometric procedure. In the BBdp rats, the activity of invertase, maltase, and lactase averaged, respectively, 70.2 +/- 4.4, 81.2 +/- 4.3, and 75.7 +/- 4.1% (n = 16 and p < 0.001 in all cases) of the control values found in BBc rats of the same sex. Inversely, after exclusion of two female BBc rats with abnormally high plasma D-glucose concentration, the activity of peroxidase in the BBdp rats averaged 157.4 +/- 20.0% (n = 16; p < 0.02) of the mean control value recorded in BBc rats of the same sex (100.0 +/- 9.3%; n = 14). These findings are compatible with the view that a proinflammatory state of the GI associated with compromise function may precede the occurrence of pancreatic insulitis in BBdp rats and, possibly, human subjects with type 1 diabetes.

Glucagon-like peptide 1 content of intestinal tract in adult rats injected with streptozotocin either during neonatal period or 7 d before sacrifice.

Glucagon-like peptide 1 (GLP-1) content of the intestinal tract was recently found to be lower in diabetesprone BioBreeding (BBdp) rats than in the corresponding control animals (BBc rats), a finding compatible with the idea that an inflammatory intestinal state precedes insulitis in these diabetes-prone animals. This study aimed at measuring GLP-1 content of the intestinal tract both in another animal model of type 1 diabetes and in an animal model of type 2 diabetes. GLP-1 content of the jejunum, ileum, colon, and cecum was measured in male and female adult control rats and animals injected with streptozotocin (STZ) either during the neonatal period or 7 d before sacrifice. GLP-1 content of the intestinal tract was higher in type 1 diabetic rats than in control animals. Such was not the case in the type 2 diabetic rats. The findings recorded in the rats injected with STZ either during the neonatal period or later in life indicate that hyperglycemia and/or insulin deficiency do not cause a decrease in GLP-1 content of the intestinal tract. On the contrary, such a content may increase when the glucose intolerance and hypoinsulinemia are sufficiently pronounced, as was the case in the type 1 diabetic rats. These findings are thus compatible with the view that the decreased GLP-1 content of the intestinal tract in BBdp rats may result from intestinal inflammation.

The antibody response to bacteriophage is linked to the lymphopenia gene in congenic BioBreeding rats

Congenic BioBreeding (BB) rats, homozygous for the autosomal lymphopenia (Lyp) gene (Lyp/Lyp), heterozygous (Lyp/+), or wild-type (+/+), were immunized with the T cell-dependent bacteriophage ΦX174 to determine effects of Lyp on primary and secondary antibody responses. The primary ΦX174 antibody response did not differ between the three different genotypes. In contrast, the secondary immune response, expressed as the peak neutralizing titer, was markedly reduced in Lyp/Lyp (9.9±3.2; mean value±S.E.M. for seven rats) compared to both Lyp/+ (51±12; n=13; P=0.006) and +/+ (100±20; n=7; P=0.004) BB rats. We suggest that the secondary antibody response to the T cell-dependent neoantigen ΦX174 is linked in a recessive manner to genetic factor(s) in the Lyp gene region.

The antibody response to bacteriophage is linked to the lymphopenia gene in congenic BioBreeding rats.

Congenic BioBreeding (BB) rats, homozygous for the autosomal lymphopenia (Lyp) gene (Lyp/Lyp), heterozygous (Lyp/+), or wild-type (+/+), were immunized with the T cell-dependent bacteriophage PhiX174 to determine effects of Lyp on primary and secondary antibody responses. The primary PhiX174 antibody response did not differ between the three different genotypes. In contrast, the secondary immune response, expressed as the peak neutralizing titer, was markedly reduced in Lyp/Lyp (9.9+/-3.2; mean value+/-S.E.M. for seven rats) compared to both Lyp/+ (51+/-12; n=13; P=0.006) and +/+ (100+/-20; n=7; P=0.004) BB rats. We suggest that the secondary antibody response to the T cell-dependent neoantigen PhiX174 is linked in a recessive manner to genetic factor(s) in the Lyp gene region.

CD40/154 blockade and rejection of islet allografts in the streptozotocin and autoimmune diabetic rat.

Islet transplantation in children with autoimmune diabetes will require immunosuppression that has minimal toxicity and side-effects, and overcomes the barrier of autoimmunity. Since antibodies directed against the CD40/154 co-stimulatory pathway may meet these criteria, we have tested the ability of hamster antirat CD154 (AH.F5, Biogen) to prevent rejection of renal subcapsular islet allografts in streptozotocin (STZ) or autoimmune (AUTO) diabetic diabetes-resistant biobreeding (DRBB) rats. STZ diabetic rats that received anti-CD154 at 15 mg/kg per dose but not 10 mg/kg per dose did not have evidence of rejection until about 80-120 d post-transplantation, by which time antibody concentrations had returned to undetectable levels. Rats retreated with anti-CD154 before recurrence of diabetes had a prolonged period of disease-free survival. Most of these rats had recurrence following a spleen cell challenge. In contrast, AUTO diabetic DRBB rats treated with anti-CD154 had recurrence of diabetes between 7 and 12 d following transplantation of the Dark Agouti (DA) islets. In a separate set, AUTO diabetic rats that received a simultaneous islet isograft, islet allograft and thyroid allograft had focal accumulation of lymphocytes at the periphery of the isograft, while the islet and thyroid allografts had diffuse infiltration with lymphocytes and destruction of tissue with no residual staining for glucagon. Therefore, autoimmunity adds an additional barrier to islet allotransplantation that is not overcome with CD40/154 blockade in an animal model that closely parallels autoimmune diabetes in humans. The results indicate the importance of testing regimen of islet transplantation in animal models of autoimmune diabetes.

BB rat lyp mutation and Type 1 diabetes.

BioBreeding (BB) rats spontaneously develop an autoimmune diabetic syndrome similar to that observed in humans and NOD mice. One of the diabetes susceptibility loci maps to the lyp locus on chromosome 4. In this article we describe the consequences of the BB rat lyp mutation on T-cell homeostasis, repertoire and function, as well as its role in the pathogenesis of type I diabetes.

New therapeutic approaches to type 1 diabetes: from prevention to cellular or gene therapies.

New therapeutic approaches to type 1 diabetes: from prevention to cellular or gene therapies.

Increased numbers of caveolae in retinal endothelium and pericytes in hypertensive diabetic rats

Long-term clinical studies have now shown that tight control of blood pressure in type 2 diabetes reduces the risk of diabetes-related death and common diabetic complications, including diabetic retinopathy. However, the mechanisms by which hypertension enhances diabetic microvascular disease, especially diabetic retinopathy, are poorly understood. We developed an experimental model of hypertension in diabetic rats and studied the early ultrastructural changes in retinal capillaries under these conditions. Hypertension was induced in diabetic BioBreeding (BB) rats by unilateral nephrectomy, weekly subcutaneous mineralocorticoid and 0.9% oral saline. Serial blood pressures and ultrastructural features of retinal capillaries were recorded in four groups: normotensive Wistar rats, normotensive diabetic rats, hypertensive Wistar rats and hypertensive diabetic rats. A significant and sustained increase in systolic blood pressure occurred in both groups of nephrectomised rats. There was a significant increase in the number of caveolae (i) in both pericytes and endothelial cells in animals with hypertension and diabetes together compared with all other groups and (ii) in pericytes in animals with diabetes alone. The number of direct contacts between pericytes and endothelial cells was reduced in diabetic and hypertensive diabetic animals. Hypertension and diabetes had an interactive effect in producing retinal capillary basement membrane thickening. In the BB rat hypertension and diabetes have an interactive effect in increasing the number of caveolae in both endothelial cells and pericytes. We speculate that this may be a reflection of changes in calcium and nitric oxide metabolism in these animals.

Cellular and molecular pathogenic mechanisms of insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM), also known as type 1 diabetes, is an organ-specific autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells. The hypothesis that IDDM is an autoimmune disease has been considerably strengthened by the study of animal models such as the BioBreeding (BB) rat and the nonobese diabetic (NOD) mouse, both of which spontaneously develop a diabetic syndrome similar to human IDDM. Beta cell autoantigens, macrophages, dendritic cells, B lymphocytes, and T cells have been shown to be involved in the pathogenesis of autoimmune diabetes. Among the beta cell autoantigens identified, glutamic acid decarboxylase (GAD) has been extensively studied and is the best characterized. Beta cell-specific suppression of GAD expression in NOD mice results in the prevention of IDDM. Macrophages and/or dendritic cells are the first cell types to infiltrate the pancreatic islets. Macrophages play an essential role in the development and activation of beta cell-cytotoxic T cells. B lymphocytes play a role as antigen-presenting cells, and T cells have been shown to play a critical role as final effectors that kill beta cells. Cytokines secreted by immunocytes, including macrophages and T cells, may regulate the direction of the immune response toward Th1 or Th2 as well as cytotoxic effector cell or suppressor cell dominance. Beta cells are destroyed by apoptosis through Fas-Fas ligand and TNF-TNF receptor interactions and by granzymes and perforin released from cytotoxic effector T cells. Therefore, the activated macrophages and T cells, and cytokines secreted from these immunocytes, act synergistically to destroy beta cells, resulting in the development of autoimmune IDDM.

Homotypic cluster formation of dendritic cells, a close correlate of their state of maturation. Defects in the biobreeding diabetes-prone rat

Aggregation of dendritic cells (DCs) in homotypic clusters has been described in vivo in lymph and skin, and here we report studies on homotypic clustering of rat splenic (s) DCs in vitro. Wistar rat sDCs readily formed homotypic clusters in culture, which increased in number and size over time (with a peak at t = 3 h). Keeping the cells at higher densities or treatment with anti-CD43 induced more and larger homotypic clusters. After such enhanced clustering the DCs had increased their T cell stimulating capabilities in syngeneic mixed lymphocyte reaction, and had a higher expression of CD80 and CD86 (signs of maturation). Ag transfer from bovine serum albumin-fluorescein isothiocyanate-pulsed to unpulsed DCs was observed during clustering. Here we also show that sDCs of the biobreeding diabetes-prone (BB-DP) rat, a model of autoimmune diabetes/thyroiditis, formed fewer and smaller clusters than Wistar sDCs, and that DC-DC clustering resulted in only a modest maturation of the cells (as determined in syn MLR and by phenotyping). Anti-CD43 completely restored the clustering defect BB-DP DCs in vitro, yet T cell-stimulating capability was only restored to a limited extent. Ag transfer in BB-DP DC clusters was similar.

Thymic expression of insulin‐related genes in an animal model of autoimmune type 1 diabetes

Background Insulin and multiple other autoantigens have been implicated in the pathogenesis of autoimmune type 1 diabetes, but the origin of immunological self-reactivity specifically oriented against insulin-secreting islet β-cells remains obscure. The primary objective of the present study was to investigate the hypothesis that a defect in thymic central T-cell self-tolerance of the insulin hormone family could contribute to the pathophysiology of type 1 diabetes. This hypothesis was investigated in a classic animal model of type 1 diabetes, the Bio-Breeding (BB) rat. Methods The expression of the mammalian insulin-related genes (Ins, Igf1 and Igf2) was analysed in the thymus of inbred Wistar Furth rats (WF), diabetes-resistant BB (BBDR) and diabetes-prone BB (BBDP) rats. Results RT-PCR analyses of total RNA from WF, BBDP and BBDR thymi revealed that Igf1 and Ins mRNAs are present in 15/15 thymi from 2-day-old, 5-day-old and 5-week-old WF, BBDR and BBDP rats. In contrast, a complete absence of Igf2 mRNA was observed in more than 80% of BBDP thymi. The absence of detectable Igf2 transcripts in the thymus of BBDP rats is tissue-specific, since Igf2 mRNAs were detected in all BBDP brains and livers examined. Using a specific immunoradiometric assay, the concentration of thymic IGF-2 protein was significantly lower in BBDP than in BBDR rats (p<0.01). Conclusions The present study suggests an association between the emergence of autoimmune diabetes and a defect in Igf2 expression in the thymus of BBDP rats. This tissue-specific defect in gene expression could contribute both to the lymphopenia of these rats (by impaired T-cell development) and the absence of central T-cell self-tolerance of the insulin hormone family (by defective negative selection of self-reactive T-cells). Copyright © 2001 John Wiley & Sons, Ltd.

Thymic expression of insulin-related genes in an animal model of autoimmune type 1 diabetes.

Insulin and multiple other autoantigens have been implicated in the pathogenesis of autoimmune type 1 diabetes, but the origin of immunological self-reactivity specifically oriented against insulin-secreting islet beta-cells remains obscure. The primary objective of the present study was to investigate the hypothesis that a defect in thymic central T-cell self-tolerance of the insulin hormone family could contribute to the pathophysiology of type 1 diabetes. This hypothesis was investigated in a classic animal model of type 1 diabetes, the Bio-Breeding (BB) rat. The expression of the mammalian insulin-related genes (Ins, Igf1 and Igf2) was analysed in the thymus of inbred Wistar Furth rats (WF), diabetes-resistant BB (BBDR) and diabetes-prone BB (BBDP) rats. RT-PCR analyses of total RNA from WF, BBDP and BBDR thymi revealed that Igf1 and Ins mRNAs are present in 15/15 thymi from 2-day-old, 5-day-old and 5-week-old WF, BBDR and BBDP rats. In contrast, a complete absence of Igf2 mRNA was observed in more than 80% of BBDP thymi. The absence of detectable Igf2 transcripts in the thymus of BBDP rats is tissue-specific, since Igf2 mRNAs were detected in all BBDP brains and livers examined. Using a specific immunoradiometric assay, the concentration of thymic IGF-2 protein was significantly lower in BBDP than in BBDR rats (p<0.01). The present study suggests an association between the emergence of autoimmune diabetes and a defect in Igf2 expression in the thymus of BBDP rats. This tissue-specific defect in gene expression could contribute both to the lymphopenia of these rats (by impaired T-cell development) and the absence of central T-cell self-tolerance of the insulin hormone family (by defective negative selection of self-reactive T-cells).

Hydrolysed Casein Diet Protects BB Rats from Developing Diabetes by Promoting Islet Neogenesis

Feeding diabetes-prone BioBreeding (BBdp) rats a hydrolysed-casein (HC)-based semi-purified diet results in two-to-three-fold fewer diabetes cases compared with feeding cereal-based diets such as NIH-07 (NIH). We showed previously that young NIH-fed BBdp rats had decreased islet area at a time when classic insulitis was minimal. Rats fed an HC diet maintained near normal islet area followed 3–4 weeks later by a deviation of the pancreas cytokine pattern from Th1 to Th2/Th3. This finding raised the possibility that BBdp rats were more susceptible to diet-induced changes in islet homeostasis. To investigate this possibility further, BBdp rats were fed an NIH or HC diet from days 23 to 45. Bouin's fixed sections of pancreas were stained with H&E or antibodies for insulin and glucagon. Cell proliferation nuclear antigen (PCNA) was used as a marker of cell proliferation and cells were stained for putative markers of islet neogenesis, cytokeratin 20 (CK20) and Bcl-2. Apoptotic bodies were recognized by morphological features and by TUNEL-positive staining. BBdp rats fed an HC diet had a significantly higher β-cell fraction than rats fed NIH, whereas α-cell fraction and β-cell size were not affected by diet or rat type. Apoptotic bodies of β-cells were rare and unaffected by diet. The number of PCNA+β-cells was not affected by diet. CK20 expression was localized in the ductular system and at the periphery of islets in rats aged 7 and 45 days. There were more CK20+islets in BBdp rats fed NIH than in those fed HC but the CK20 area fraction was unaffected by diet. Bcl-2 expression was scattered among ducts and central acinar cells. The number of extra-islet insulin+and glucagon+clusters (<four cells) was significantly higher in animals fed the HC diet compared with those fed NIH. Most of the insulin+clusters were also homeodomain-containing transcription factor pancreas duodenum homeobox gene-1 (PDX-1) positive. Glucagon+/PDX-1+clusters were rarely found. These data are consistent with a shift in pancreas homeostasis that maintains islet cell mass by increased islet neogenesis, a process that was enhanced in animals fed a diabetes-retardant diet.

Dietary effects on insulin and nutrient metabolism in mesenteric lymph node cells, splenocytes, and pancreatic islets of BB rats

The present studies were performed to determine if a protective diet has different effects on the metabolic activity or function of islet cells, as well as the metabolic activity of mesenteric lymph node (MLN) cells and spleen cells, from BioBreeding (BB) rats. Diabetes-prone BB (BBdp) rats and control non–diabetes-prone BB (BBc) rats were fed for about 20 days either a mainly plant-based diabetogenic diet, NIH-07 (NIH), or a protective semipurified diet with hydrolyzed casein (HC) as the amino acid source. At 6 to 8 weeks of age, BBdp rats had high plasma D-glucose and low insulin concentrations, low insulin content, and low metabolic and secretory responses to D-glucose in isolated pancreatic islets. Islet metabolism, as measured by accumulation of 14C-acidic metabolites, amino acids, and the ratio of D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was increased in control rats fed HC (P < .05); a similar trend in BBdp rats was not significant. Feeding the HC diet increased islet insulin content (P < .01) by 13% in BBdp and 23% in BBc rats; other metabolic and hormonal variables were unaffected. Compared with BBc rats, BBdp rats displayed higher rates of L-[U-14C]glutamine oxidation, D-[5-3H]glucose utilization, and D-[U-14C]glucose oxidation in MLN cells, but not in splenocytes. There was a dramatic decrease of L-[U-14C]glutamine oxidation in MLN cells from BBc and BBdp rats fed HC. Glycolysis was decreased in control rats. We conclude that the protection afforded by feeding BBdp rats a HC diet is associated with increased insulin in target β cells and downregulation of metabolic activity in gut-associated MLN cells. Metabolic activity in splenocytes, cells representative of the systemic immune system, was less affected. These data suggest that diet-induced metabolic changes occur in the islets and nearby cells of the gut immune system in the period before classic insulitis. Changes in the islets were smaller in comparison to the dramatic remodeling of nutrient catabolism in MLN cells. MLN downregulation may reflect baseline metabolic activity in the absence of diabetogenic (or other) food antigens and further highlights an important interaction between diabetogenic food antigens and the gut immune tissues.

Genetic dissection of a rat model for rheumatoid arthritis: significant gender influences on autosomal modifier loci.

Rheumatoid arthritis (RA) is a common, chronic, autoimmune, inflammatory disease that is influenced by genetic factors including gender. Many studies suggest that the genetic risk for RA is determined by the MHC, in particular class II alleles with a 'shared epitope' (SE), and multiple non-MHC loci. Other studies indicate that RA and other autoimmune diseases, in particular insulin-dependent diabetes mellitus (IDDM) and autoimmune thyroid disease (ATD), share genetic risk factors. Rat collagen-induced arthritis (CIA) is an experimental model with many features that resemble RA. The spontaneous diabetes-resistant bio-breeding rat, BB(DR), is of interest because it is susceptible to experimentally induced CIA, IDDM and ATD, and it has an SE in its MHC class II allele. To explore the genetics of CIA, including potential gender influences and the genetic relationships between CIA and other autoimmune diseases, we conducted a genome-wide scan for CIA regulatory loci in the F(2) progeny of BB(DR) and CIA-resistant BN rats. We identified 10 quantitative trait loci (QTLs), including 5 new ones (Cia15, Cia16*, Cia17, Cia18* and Cia19 on chromosomes 9, 10, 18 and two on the X chromosome, respectively), that regulated CIA severity. We also identified four QTLs, including two new ones (Ciaa4* and Ciaa5* on chromosomes 4 and 5, respectively), that regulated autoantibody titer to rat type II collagen. Many of these loci appeared to be gender influenced, and most co-localized with several other autoimmune trait loci. Our data support the view that multiple autoimmune diseases may share genetic risk factors, and suggest that many of these loci are gender influenced.

The Metabolic Availability of Vitamin A Is Decreased at the Onset of Diabetes in BB Rats

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.

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency.

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH(4)), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH(4) levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH(4) levels with sepiapterin increased NO production, suggesting that BH(4) deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH(4). GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH4), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH4 levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH4 levels with sepiapterin increased NO production, suggesting that BH4 deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH4. GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

Aortic endothelial cell von Willebrand factor content, and circulating plasminogen activator inhibitor-1 are increased, but expression of endothelial leukocyte adhesion molecules is unchanged in insulin-dependent diabetic BB rats

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.