Ingrid Klöting

Influence of diabetic metabolic state on fracture healing in spontaneously diabetic rats

Type 1 diabetes mellitus (DM) has been shown to alter the properties of bone and impair fracture healing in both humans and animals. The objective of this study was to examine changes in the histomorphometrical, histological and mechanical parameters of bone and remodeling during fracture healing, depending on the diabetic metabolic state in spontaneously diabetic BB/O(ttawa)K(arlsburg) rats, a rat strain that represents a close homology to DM in man.

IL-1β, IFN-γ and TNF-α increase vulnerability of pancreatic beta cells to autoimmune destruction

Abstract In the pathogenesis of type-1 diabetes insulin-producing β-cells are destroyed by cellular autoimmune processes. The locality of β-cell destruction is the inflamed pancreatic islet. During insulitis cytokines released from islet-infiltrating mononuclear cells affect β-cells at several levels. We investigated whether cytokine-induced β-cell destruction is associated with changes in the expression of the surface receptors intercellular adhesion molecule (ICAM)-1 and Fas. Islets from diabetes-prone and congenic diabetes-resistant BB rats were exposed to interleukin (IL)-1β alone or in combination with interferon (IFN)-γ plus tumour necrosis factor (TNF)-α. Cytokines decreased islet insulin content, suppressed glucose stimulated insulin secretion and generated enhanced amounts of nitric oxide and DNA-strand breaks. While no membrane alterations of IL-1β treated islets cells were detectable, the cytokine combination caused damage of cell membranes. Independent of diabetes susceptibility IL-1β treated islet β-cells expressed a significantly increased amount of ICAM-1 on their surfaces which was not further increased by IFN-γ+TNF-α. However, IL-1β induced Fas expression was significantly enhanced only on β-cells from diabetes-prone BB rats. From these results we suggest that IL-1β mediates the major stimulus for ICAM-1 induction which is possibly a necessary but not sufficient step in the process of β-cell destruction. Obviously, the additional enhancement of Fas expression on the surface of β-cells is important for destruction. The combined action of all three cytokines induced the expression of Fas on the β-cell surface independent of diabetes susceptibility, indicating that such a strong stimulus in vitro may induce processes different from the precise mechanisms of β-cell destruction in vivo.

Long-term arterial pressure in spontaneously hypertensive rats is set by the kidney.

Objectives We investigated whether arterial pressure in spontaneously hypertensive rats (SHR) can be normalized by a kidney graft from normotensive histocompatible donors. In addition, the effect of differential genetic predisposition to hypertension of recipients of an SHR kidney on the development of post-transplantation hypertension was studied. Methods SHR were transplanted with a kidney from congenic rats (BB.1K) homozygous for a 2 cM segment of SHR chromosome 20, including the major histocompatibility complex class Ia and class II genes. BB.1K and F1 hybrids (F1H, SHR × Wistar–Kyoto rats) were transplanted with an SHR kidney and the development of renal post-transplantation hypertension was monitored. Results Thirty days after renal transplantation, mean arterial pressure (MAP) was 116 ± 4 mmHg in SHR with a BB.1K kidney (n = 8) versus 168 ± 2 mmHg in sham-operated SHR (n = 10);P < 0.001. Cumulative renal sodium balance (mmol/100 g body weight) over 21 days after bilateral nephrectomy was 6.8 ± 0.6 in SHR with a BB.1K kidney versus 10.8 ± 1.6 in sham-operated SHR (P < 0.05). Within 60 days of transplantation, MAP increased in BB.1K and in F1H transplanted with an SHR kidney (n = 7 per group) by 38 ± 5 mmHg and 43 ± 8 mmHg, respectively. Conclusions In SHR, arterial pressure can be normalized by a kidney graft from normotensive donors. The genetic predisposition of the recipients to hypertension does not modify the rate and the extent of the arterial pressure rise induced by an SHR kidney graft.

DISPOSITION OF ORAL AND INTRAVENOUS PRAVASTATIN IN MRP2-DEFICIENT TR– RATS

The aim of this study was to characterize the role of the efflux transporter Mrp2 (Abcc2) in the pharmacokinetics of orally and intravenously administered pravastatin in rats. Eight Mrp2-deficient TR– rats and eight wild-type rats were given an oral dose of 20 mg/kg pravastatin. Four TR– animals and four wild-type animals were studied after intravenous administration of pravastatin (5 mg/kg). The TR– rats showed a 6.1-fold higher mean area under the plasma concentration-time curve (AUC) of pravastatin (p < 0.001) after oral administration and a 4.7-fold higher AUC (p < 0.01) after intravenous administration of pravastatin as compared with the wild-type animals. The mean systemic (total) clearance of pravastatin was 4.6-fold higher (39.2 versus 8.50 l/h/kg, p < 0.001) and the mean V 4.3-fold higher (14.1 versus 3.29 l/kg, p < 0.01) in the wild-type rats. The mean renal clearance of pravastatin in the TR– rats was 16.5-fold increased as compared with the wild-type animals (0.695 versus 0.042 l/h/kg, p < 0.05). The increased systemic exposure to oral pravastatin in the TR– rats was associated with a greater inhibitory effect on 3-hydroxy-3-methylglutaryl CoA reductase, as shown by smaller lathosterol to cholesterol concentration ratios. These results suggest that the reduced biliary pravastatin excretion in the Mrp2-deficient TR– rats is partly compensated for by increased urinary excretion of pravastatin. Furthermore, intestinal Mrp2 does not appear to play a major role in the oral absorption of pravastatin in normal rats.

Type 1 Diabetes in BioBreeding Rats Is Critically Linked to an Imbalance between Th17 and Regulatory T Cells and an Altered TCR Repertoire

Diabetes-prone BioBreeding (DP-BB) rats spontaneously develop type 1 diabetes mellitus (T1DM) on grounds of their MHC haplotype RT1u and a point mutation in the Gimap5 gene. In this study, we report that DP-BB rats exhibit an increasingly severe imbalance, in particular between Th17 and regulatory T (Treg) cells, within the first months of age. This can be assigned to an excess in effector T cells because neither the percentage nor the function of the Treg cells is compromised. Flow cytometric analysis of Vβ segment usage and CDR3 spectratyping further suggest that the disturbed repertoire of peripheral T cells may also contribute to the development of T1DM in DP-BB rats. Importantly, expansion of Treg cells in vivo by means of a CD28 superagonistic Ab as well as adoptive transfer of Treg cells efficiently interferes with the development of T1DM in DP-BB rats, whereas treatment with conventional Th cells does not afford protection. Using a newly generated strain of enhanced GFP transgenic rats, we could further demonstrate that the transferred Treg cells persist in the recipient rats for several months and partially correct the imbalance between Th17 and Treg cells. Thus, our data support the hypothesis that unchecked effector T cell action and a disturbed T cell repertoire contribute to the development of T1DM in DP-BB rats, which may also have implications for a better understanding of the human disease.

The polygenetically inherited metabolic syndrome of WOKW rats is associated with insulin resistance and altered gene expression in adipose tissue

Wistar Ottawa Karlsburg W (RT1u) rats (WOKW) develop a complete metabolic syndrome closely resembling the human disease. The aim of this study was to characterize the phenotype of adipose tissue in WOKW rats with regard to adipocyte metabolism, insulin resistance, and gene expression and thus to define the phenotype more precisely.

Diabetes and Hypertension in Rodent Models

As shown by ourselves and others, animals models closely resembling human complex diseases like IDDM in BB/OK and hypertension in SHR/Mol rats can be used to dissect a complex disease into discrete genetic factors as has been done for hypertension in (BB/OK x SHR/Mol) cross hybrids. Discrete genetic factors, so-called QTLs, were detected on chromosomes 1, 10, 18, 20, and X. To gain additional information about the physiologic effect of the mapped blood pressure QTLs, genetically defined regions of the SHR rat were transferred onto the genetic background of diabetes-prone BB/OK rats. Four new congenic BB.SHR rats named BB.Sa, BB.Bp2, BB.1K, and BB.Xs were generated and characterized telemetrically for blood pressure, heart rate, and motor activity. The data demonstrate clearly that each single blood pressure QTL of the SHR rat causes a significant increase of the systolic blood pressure and has a different influence on diastolic blood pressure, heart rate, and motor activity. The effects were modified differently by the diabetic state in BB.Sa, BB.Bp2, and BB.Xs rats carrying all diabetogenic genes of the BB/OK rats. The results demonstrate that these newly established congenic strains are a unique tool to study the physiological control of blood pressure by a single blood pressure QTL on the one hand and their interaction with hyperglycemia on the other. It is well within the bounds of possibility that diabetic congenics reflect the diabetic hypertension seen in diabetic patients. Because of the synteny conservation in gene order between different mammals, genes of the appropriate human region could therefore be candidate genes for hypertension in diabetics. Furthermore, these congenic strains can also be used to study interactions between a blood pressure QTL and various selected environmental conditions. In this way, one could learn which QTL can be influenced by environmental factors and to what extent. Another point is the study of gene interactions. Because congenics are genetically identical except for the defined transferred region, congenics can be crossed to investigate the interaction between two or three blood pressure QTLs selected by the investigator and not by nature. These QTL combinations can be studied in the nondiabetic as well as diabetic state. Although the advantage of congenic strains has been shown, the transferred chromosomal regions are too large to pinpoint the gene responsible for the phenotypic change. Therefore, regions on each chromosome must be systematically whittled down, which can be done by crossing the congenics with BB/OK rats and intercrossing their progeny to generate recombinants. These can then be used for the creation of new congenic lines carrying a much smaller region of the SHR/Mol rat. This has been started for the region on chromosome 1 spanning a 16-cM region from the Sa to the Igf2 gene. BB.Sa rats were therefore backcrossed onto BB/OK rats and the resulting progeny were intercrossed. The aim will be to create at least three new congenic BB.Sa rat strains homozygous for the SHR alleles of Sa, Lsn, or Igf2 genes. However, new problems will emerge with these new congenics. To genetically define small regions requires more dense polymorphic markers than are currently available. Dense polymorphic markers will also be necessary to split the other regions on chromosomes 10, 18, 20, and X. We expect that in the near future it will be possible using this approach to define small regions of < 0.5 cM. The recent progress in gene mapping in the rat gives hope that the use of such congenic lines will allow the identification and recovery of the blood pressure genes in the near future.

Metabolic features in disease-resistant as well as in spontaneously hypertensive rats and newly established obese Wistar Ottawa Karlsburg inbred rats.

OBJECTIVE: Studies, comparing several disease-prone and disease-resistant rat strains to elucidate the extent and severity of syndromes resembling human diseases are lacking. Therefore we studied the inbred rat strains BB/OK, BN/Crl, LEW/K and WKY/Crl in comparison with SHR/Mol and WOKW/K rats as models of metabolic syndrome.DESIGN: Body weight and body mass index (BMI) were measured in 12 males of each strain at 14 weeks. In addition blood glucose, serum triglycerides, cholesterol, insulin and leptin were determined at 12, 13 and 14 weeks of age.RESULTS: In contrast to SHR animals, WOKW rats develop a severe metabolic syndrome including obesity, hyperleptinemia, hyperinsulinemia and dyslipidemia.CONCLUSION: We conclude that; (i) the choice of disease-resistant inbred rat strains as ‘healthy controls’ for a disease-prone strain has to be carefully evaluated; (ii) in comparison with SHR, WOKW rats develop most if not all facets of the metabolic syndrome described in human and (iii) as with the human disease the syndrome in rats is polygenic.

EFFECTS OF QUANTITATIVE TRAIT LOCI FOR LIPID PHENOTYPES IN THE RAT ARE INFLUENCED BY AGE

1. Previous study on the backcross hybrids derived from a cross of the spontaneously hypertensive rat (SHR/Mol) and the spontaneously diabetic BB/OK rat demonstrated the existence of quantitative trait loci (QTL) affecting lipid phenotypes on chromosome 4 and suggestive linkage of lipid phenotypes with markers on chromosome 1. Because the previous study was performed with backcross hybrids at 12 weeks of age and it is known that lipid phenotypes can show age‐related differences, in the present study, the effect of QTL (chromosome 1 and 4) on serum triglycerides and cholesterol was longitudinally analysed between 20 and 32 weeks of age in backcross hybrids.

Metabolic syndrome and aging in Wistar Ottawa Karlsburg W rats.

BACKGROUND AND OBJECTIVE: Comparative studies have shown that Wistar Ottawa Karlsburg W (RT1u) rats (WOKW) develop a nearly complete metabolic syndrome with obesity, moderate hypertension, dyslipidemia, hyperinsulinemia, and impaired glucose tolerance up to an age of 28 weeks. Because metabolic data thereafter are missing, WOKW and disease-resistant DA rats were studied for 12 months beginning at an age of 5 months.METHODS: Eighteen male inbred WOKW and DA rats were studied monthly from the 5th to the 17th month of life for traits of the metabolic syndrome such as body weight, body mass index (BMI), serum triglycerides, total cholesterol, leptin, insulin as well as glucose tolerance, 24 h excretion of urine total protein and creatinine including telemetric measurement of blood pressure in six males per each group.RESULTS: Except for serum total cholesterol, the measured values for most traits studied were significantly higher in WOKW than in DA rats at an age of 5 months. At an age of 17 months all traits were significantly elevated in WOKW compared with DA rats. WOKW rats were hypertensive, dyslipidemic, obese, glucose intolerant, hyperinsulinemic and proteinuric.CONCLUSION: Considering the phenotype of the WOKW rat described until now and the fact that the metabolic syndrome in this rat is polygenetically determined, the WOKW rat is the most suitable animal model to study the pathophysiology of the facets of the syndrome.