BioBreeding Diabetes Resistant Rat Research Articles

Impact of metabolic substrate modification on myocardial efficiency in a rat model of obesity and diabetes

Abstract Background Congenic leptin receptor deficient rat generated by introgression of the Koletsky leptin receptor mutation into BioBreeding Diabetes Resistant rat (BBDR.lepr−/−) is a novel animal model combining obesity, systemic insulin resistance and diabetes. Systemic insulin resistance is associated with reduced myocardial glucose utilization, but its effect on myocardial external efficiency, i.e. the ability of the myocardium to convert energy into external stroke work, remains uncertain. Purpose To characterize cardiac energy metabolism and function in BBDR.lepr−/− rats and to study the effect of dipeptidyl peptidase 4 (DPP-4) inhibitor linagliptin in this model. Methods Cardiac phenotype was evaluated in six-month-old male BBDR.lepr−/− rats (n=11) and age-matched male non-diabetic lean control littermates (BBDR.lepr+/− or BBDR.lepr+/+ rats, n=14). Of these, 7 BBDR.lepr−/− rats and 6 controls underwent cardiac ultrasound, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography/computed tomography (PET/CT), and [11C]acetate PET in order to evaluate cardiac structure and function as well as glucose and oxidative metabolism. In the remaining rats, fatty acid metabolism was evaluated by [18F]fluorothia-6-heptadecanoic acid ([18F]FTHA) PET/CT. In the linagliptin intervention study, 25 BBDR.lepr−/− male rats were randomly divided into control group (n=11) that received regular chow diet and linagliptin group (n=14) that received linagliptin (10mg/kg/d) mixed in the chow diet for three months. After the intervention, the rats underwent cardiac ultrasound, [18F]FDG PET/CT, and [11C]acetate PET. Results Compared with controls, BBDR.lepr−/− rats showed increased left ventricle (LV) mass (∼40%, p>0.001) and higher systolic blood pressure (∼10%, p=0.02). However, fractional shortening and cardiac output were similar in both groups. Myocardial fractional uptake rate of glucose measured with [18F]FDG PET was significantly reduced (∼86%, p=0.004) (Fig. 1A, E), whereas myocardial fatty acid uptake measured by [18F]FTHA PET was not significantly increased (free fatty acid (FFA) corrected standardized uptake value (SUV) ∼21%, p=0.54) (Fig. 1B) in BBDR.lepr−/− compared to controls. Myocardial oxygen consumption assessed by [11C]acetate PET was similar in both groups (Fig. 1C, E), but LV work per gram of myocardium was reduced (∼28%, p=0.001) resulting in reduced myocardial external efficiency (∼21%, p=0.03) (Fig. 1D) in BBDR.lepr−/− compared to controls. Treatment with linagliptin significantly enhanced myocardial fractional uptake rate of glucose (∼166%, p=0.006) (Fig. 2A, C), but had no effect on efficiency of cardiac work (Fig. 2B). Conclusions Obese and diabetic BBDR.lepr−/− rats demonstrate LV hypertrophy and markedly reduced myocardial glucose utilization associated with impaired myocardial external efficiency despite normal LV systolic function. Enhancement of myocardial glucose uptake by linagliptin did not improve efficiency of cardiac work. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): IMI-SUMMIT

Diabetes complications in congenic leptin receptor deficient BBDR.cg‐lepr.cp rats (1072.6)

Diabetes is a chronic disease that is associated with devastating complications. Here, we characterize micro‐ and macrovascular changes in a novel congenic BBDR.cg‐lepr.cp rat line, generated by introgression of the Koletsky leptin receptor mutation onto BioBreeding Diabetes Resistant rats. Interestingly, two subgroups of male BBDR.cg‐lepr.cp rats could be distinguished based on their blood glucose (BG), one severely diabetic (D) and one with moderately (M) elevated BG. Both groups were obese and showed significant enlargement of liver, heart and kidneys when compared to control littermates. Serum insulin, leptin, triglycerides and cholesterol were increased in all BBDR.cg‐lepr.cp rats when compared to controls, but with significant differences between groups; triglycerides and cholesterol were higher in D rats, leptin and insulin were higher in M rats. Albumin/creatinine ratio was dramatically increased and histological examination of kidneys revealed dilated and filled tubuli and loss of glomerular podocin expression, this was more pronounced in D rats when compared to M rats. All BBDR.cg‐lepr.cp rats had significantly higher systolic blood pressure, but surprisingly only M rats had increased aortic mRNA expression of endothelial activation and inflammatory markers. Collectively, our results point to different underlying factors for micro‐ and macrovascular disease in diabetes.Grant Funding Source: Supported by VR 2011‐3900; the SUMMIT consortium (IMI‐2008/115006), Påhlsson and Diabetes Found.

Salicylate Prevents Virus-Induced Type 1 Diabetes in the BBDR Rat

Epidemiologic and clinical evidence suggests that virus infection plays an important role in human type 1 diabetes pathogenesis. We used the virus-inducible BioBreeding Diabetes Resistant (BBDR) rat to investigate the ability of sodium salicylate, a non-steroidal anti-inflammatory drug (NSAID), to modulate development of type 1 diabetes. BBDR rats treated with Kilham rat virus (KRV) and polyinosinic:polycytidylic acid (pIC, a TLR3 agonist) develop diabetes at nearly 100% incidence by ~2 weeks. We found distinct temporal profiles of the proinflammatory serum cytokines, IL-1β, IL-6, IFN-γ, IL-12, and haptoglobin (an acute phase protein) in KRV+pIC treated rats. Significant elevations of IL-1β and IL-12, coupled with sustained elevations of haptoglobin, were specific to KRV+pIC and not found in rats co-treated with pIC and H1, a non-diabetogenic virus. Salicylate administered concurrently with KRV+pIC inhibited the elevations in IL-1β, IL-6, IFN-γ and haptoglobin almost completely, and reduced IL-12 levels significantly. Salicylate prevented diabetes in a dose-dependent manner, and diabetes-free animals had no evidence of insulitis. Our data support an important role for innate immunity in virus-induced type 1 diabetes pathogenesis. The ability of salicylate to prevent diabetes in this robust animal model demonstrates its potential use to prevent or attenuate human autoimmune diabetes.

Variations in Rodent Models of Type 1 Diabetes: Islet Morphology

Type 1 diabetes (T1D) is characterized by hyperglycemia due to lost or damaged islet insulin-producing β-cells. Rodent models of T1D result in hyperglycemia, but with different forms of islet deterioration. This study focused on 1 toxin-induced and 2 autoimmune rodent models of T1D: BioBreeding Diabetes Resistant rats, nonobese diabetic mice, and Dark Agouti rats treated with streptozotocin. Immunochemistry was used to evaluate the insulin levels in the β-cells, cell composition, and insulitis. T1D caused complete or significant loss of β-cells in all animal models, while increasing numbers of α-cells. Lymphocytic infiltration was noted in and around islets early in the progression of autoimmune diabetes. The loss of lymphocytic infiltration coincided with the absence of β-cells. In all models, the remaining α- and δ-cells regrouped by relocating to the islet center. The resulting islets were smaller in size and irregularly shaped. Insulin injections subsequent to induction of toxin-induced diabetes significantly preserved β-cells and islet morphology. Diabetes in animal models is anatomically heterogeneous and involves important changes in numbers and location of the remaining α- and δ-cells. Comparisons with human pancreatic sections from healthy and diabetic donors showed similar morphological changes to the diabetic BBDR rat model.

Haptoglobin as an early serum biomarker of virus-induced autoimmune type 1 diabetes in biobreeding diabetes resistant and LEW1.WR1 rats

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11-21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.

Epidemiology of type 1 diabetes and what animal models teach us about the role of viruses in disease mechanisms

There is a consensus among epidemiologists that the worldwide incidence rate of type 1 diabetes has been rising in recent decades. The cause of this rise is unknown, but epidemiological studies suggest the involvement of environmental factors, and viral infections in particular. Data demonstrating a cause-and-effect relationship between microbial infections and type 1 diabetes and how viruses may cause disease in humans are currently lacking. However, new evidence from animal models supports the hypothesis that viruses induce disease via mechanisms linked with innate immune upregulation. In the BioBreeding Diabetes Resistant rat, infection with a parvovirus induces islet destruction via upregulation of the toll-like receptor 9 (TLR9) signaling pathway. Data from mouse models of diabetes implicate TLR2, TLR3, and TLR7 in the disease process. Understanding the link between environmental agents and innate immune pathways involved in early stages of diabetes may advance the design of immune interventions to prevent disease in genetically susceptible individuals.

Innate Immune Pathways in Virus‐Induced Autoimmune Diabetes

We recently hypothesized that Toll-like receptor-induced innate upregulation by Kilham rat virus (KRV) in the BioBreeding diabetes-resistant (BBDR) rat model plays a key role in the mechanism of diabetes induction. To address this hypothesis, we analyzed innate immune signaling pathways upregulated by KRV in vitro and in vivo. We demonstrate that KRV activates the signal transducer and activator of transcription (STAT)-1 in spleen cells in vitro and that this activation can be blocked by TLR9 inhibition. We also show that KRV upregulates STAT-1 in pancreatic lymph nodes early after virus infection. Our data may implicate TLR9-induced STAT-1 signaling pathways in KRV-induced innate immune activation in the BBDR rat and raise the possibility that these pathways are involved in mediating autoimmune diabetes.

TLR9-Signaling Pathways Are Involved in Kilham Rat Virus-Induced Autoimmune Diabetes in the Biobreeding Diabetes-Resistant Rat

Viral infections are associated epidemiologically with the expression of type 1 diabetes in humans, but the mechanisms underlying this putative association are unknown. To investigate the role of viruses in diabetes, we used a model of viral induction of autoimmune diabetes in genetically susceptible biobreeding diabetes-resistant (BBDR) rats. BBDR rats do not develop diabetes in viral-Ab-free environments, but approximately 25% of animals infected with the parvovirus Kilham rat virus (KRV) develop autoimmune diabetes via a mechanism that does not involve beta cell infection. Using this model, we recently documented that TLR agonists synergize with KRV infection and increase disease penetrance. We now report that KRV itself activates innate immunity through TLR ligation. We show that KRV infection strongly stimulates BBDR splenocytes to produce the proinflammatory cytokines IL-6 and IL-12p40 but not TNF-alpha. KRV infection induces high levels of IL-12p40 by splenic B cells and Flt-3-ligand-induced bone marrow-derived dendritic cells (DCs) but only low levels of IL-12p40 production by thioglycolate-elicited peritoneal macrophages or GM-CSF plus IL-4-induced bone marrow-derived DCs. KRV-induced cytokine production is blocked by pharmacological inhibitors of protein kinase R and NF-kappaB. Genomic KRV DNA also induces BBDR splenocytes and Flt-3L-induced DCs from wild-type but not TLR9-deficient mice to produce IL-12p40; KRV-induced up-regulation of B lymphocytes can be blocked by TLR9 antagonists including inhibitory CpG and chloroquine. Administration of chloroquine to virus-infected BBDR rats decreases the incidence of diabetes and decreases blood levels of IL-12p40. Our data implicate the TLR9-signaling pathway in KRV-induced innate immune activation and autoimmune diabetes in the BBDR rat.

Rat Pancreatic Ganglioside Expression: Differences between a Model of Autoimmune Islet B Cell Destruction and a Normal Strain

Islet cell antibodies (ICA) bind antigens expressed in both human and rat pancreatic islets. Biochemical studies have shown that an ICA-autoantigen has the properties of a monosialo-ganglioside migrating between GM2 and GM1 standards (GM2-1). We therefore aimed to isolate and characterize gangliosides from whole pancreas and isolated islets of bio breeding diabetes-prone (BB-DP), bio breeding diabetes-resistant (BB-DR), and Wistar Furth (WF) rat strains. Gangliosides were characterized by TLC, HPLC, diode array analysis, and ganglioside-specific staining. ICA binding was studied by indirect immunostaining. The GM2-1 fraction was present in BB-DP, BB-DR, and WF rat pancreases (11, 17, and 9.5%, respectively, of total ganglioside content). Substantial differences were found in other fractions: in BB-DP pancreas, in addition to GM2-1, the main fractions were GM3 (49%), GD1a (12%), GT1b (5%), and a ganglioside migrating between GM1 and GD3 standards (23%), while in BB-DR pancreas the above components were 71, 5.5, 2, and 4.5%, respectively; in WF pancreas, the main fractions were GM3, GD3, GD1a, GT1b and a trisialoganglioside (GT*) migrating above the GT1b standard (42.7, 7, 20.2, 13.8, and 6.8, respectively). A different pattern of ganglioside expression was found in isolated islets of BBDP, BB-DR, and WF rats: the GM2-1 fraction represented, respectively, 29.1, 30.4, and 31.6% of total ganglioside content; GM3 51.1, 66, and 68.4%. A fraction migrating between GM1 and GD3 standards was present only in BB-DP and BB-DR islets (19.8 and 3.6%, respectively). ICA-positive human sera reacted with pancreas of all rat strains studied, with similar end-point titers. In conclusion, (1) the GM2-1 ganglioside, in the same way as a putative target antigen of ICA, is equally expressed in BB-DP, BB-DR, and WF rat pancreata; and (2) the GM1-GD3 is expressed in higher amounts in BB-DP than in BB-DR pancreas and islets and is absent in WF.