Peter Kovács

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.

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 features of newly established congenic diabetes-prone BB.SHR rat strains

The well-known association of hypertension and diabetes mellitus and the lack of suitable animal models to study diabetic hypertension prompted us to transfer 4 chromosomal regions with quantitative trait loci (QTLs) for blood pressure of the spontaneously hypertensive SHR rat onto the genetic background of the diabetes-prone and normotensive BB/OK rat. Four congenic strains developed are named as BB. Sa (Chr.1), BB.Bp2 (Chr.18), BB.1K (Chr.20) and BB.Xs (Chr.X). Because the systolic blood pressure is significantly elevated in all congenics, renal related traits were investigated in serum and urine. Comparing BB/OK and their congenic derivatives, significant differences were found in all serum and in 7 out of 8 urine constituents studied. Most significant differences were found between BB/OK and BB.Bp2 rats. Significant differences were also found between the different congenic strains indicating that each congenic strain has its own phenotype and that each chromosomal region contains most probably further QTLs for some of the traits studied.

Metabolic variability among disease-resistant inbred rat strains and in comparison with wild rats (Rattus norvegicus).

1. Inbreeding and optimization of environmental conditions for laboratory rats may have led to the survival of mutants with metabolic aberrations but without evident disease phenotype. Therefore, in the present study, we compared metabolic traits between so‐called disease‐resistant inbred rat strains Dark Agouti (DA), Brown Norway (BN), Lewis (LEW), Wistar‐Kyoto (WKY), Fischer 344 (F344) and wild rats (Rattus norvegicus).

Mapping of quantitative trait loci for body weight on chromosomes 1 and 4 in the rat

Quantitative trait loci (QTLs) affecting body weight were investigated in the backcross population derived from diabetic BB/OK and spontaneously hypertensive rat (SHR). The F1 hybrids were backcrossed onto BB/OK rats, and QTL analysis was performed with the resulting backcross population on chromosomes 1, 3, 4, 10, 13 and 18. According to the stringent threshold for a lod score of 3.0, markers on chromosomes 1 and 4 were found to be linked with body weight. The QTL with a peak lod score (3.3) on chromosome 1 for a male population was located within the region flanked by loci Igf2 and D1Mgh12. On chromosome 4, linkage between the body weight and the region around the Npy locus was observed (lod score 3.1). The existence of the QTL on chromosome 4 affecting body weight was confirmed by congenic BB.LL rats, carrying chromosomal region of SHR (D4Mit6‐Npy‐Spr) on the genetic background of the BB/OK rat.

Comparison of genetic variability at microsatellite loci in wild rats and inbred rat strains (Rattus norvegicus)

Genetic polymorphism of the major histocompatibility complex (Cramer et al. 1988; Giinther 1979) and of some classical biochemical markers (Bender et al. 1985; Cramer et at. 1988; Erikkson et al. 1976; Yamada et at. 1993) has been studied in wild rats. However, the overall degree of genetic variability in wild rat populations remains unclear. This prompted us to analyze 11 male and 9 female rats of three different regions of Germany (north, northeast, south) with the aid of 58 microsatellite markers. Five wild rats were captured in Rostock (north, R1-R5), three in Greifswald (northeast, G1-G3), 10 in an industrial pig farm (RU 1-RU 10) that is about 30 km distant from Greifswald, and two in a farm near Munich (south, B1,B2). To compare the alleles in the wild rat with those of laboratory rats, nine classical inbred strains were also analyzed. Most inbred rat strains, BB/OK, LEW, DA, WOK.IW, WOK.1A, NEDH, were bred in our own animal facility and have been described elsewhere (K16ting and Vogt 1991; K16ting et at. 1995a, 1995b). SHR and BN rats were commercially obtained from Mollegaard Breeding & Research Centre Ltd, Denmark. HTG rats, described recently (Klimes et at. 1995), were bred at the Slovak Academy of Sciences, Institute of Experimental Endocrinology, Bratislava, Slovak Republic. For the genetic analysis, samples of genomic DNA were extracted from the liver. Fifty-eight microsatellites were amplified by PCR as recently described (Klrting et al. 1995a). Table 1 shows the allele distribution at 58 loci located on 17 chromosomes (columns 1-3) in 9 classical inbred rat strains tested (column 4) and 20 wild rats (column 5). The alleles of wild rats were subdivided into those found only in wild rats (new, column 6) and those found in inbred but not in wild rats (column 7), respectively. Furthermore, the heterozygosity per locus is given in total (column 8) and separated for male (column 9) and female wild rats (column 10). The total number of alleles in inbred and wild rats was not significantly different (204 vs. 197). However, the number and identity of the alleles at most loci differed between wild and inbred rats. There were only 5 out of 58 loci (Mtlpb, Spr, Ar, Pflcfbl, Alb) at which the same number and the same alleles were found in both wild and inbred rats. The majority of loci in wild rats were characterized by both new and some already known alleles. There were 15 loci at which all known alleles from inbred rats plus some new alleles were detected. Five new alleles were found at the Inhbabl locus, three at the Ncam, two at the Grl, and one was found at each of the Secr, Igk, Fabpl, Mycs, Cryg, Glutb,

How heterozygous are wild rats (Rattus norvegicus)

Abstract Wild rats are the most heavily combatted mammalian pests in the world, but despite this they have never been exterminated. This presupposes an optimal function of the whole organism and is considered to depend largely on heterozygosity across many loci. However, until now no knowledge about the genetics of wild rats has been obtained using highly variable microsatellite markers, which can be efficiently analyzed by performing polymerase chain reaction (PCR). This prompted us to analyze genetically 20 wild rats from four different regions of Germany with the aid of 58 PCR microsatellite markers.

Wild rats (Rattus norvegicus) for mapping of disease-resistant genes

Summary Wild rat representing a disease-resistant phenotype and genotype, was used in a crossing study with spontaneously hypertensive rat (SHR) to search for quantitative trait loci (QTL) affecting blood pressure. Therefore, one male wild rat was crossed with SHR females and F1 hybrids were transferred in a pathogen free environment by wet-hysterectomy and backcrossed onto hypertensive SHR rats resulting in first backcross hybrids (BC1). Considering that the F1 hybrids are not uniform, as are the cross hybrids of inbred rat strains, we selected 72 BC1 progeny of one F1 female, which were characterised for systolic blood pressure, measured by tail cuff method and were genetically analysed using 200 microsatellites covering the whole genome. We found suggestive linkage of blood pressure to region on chromosome 2 flanked by D2Mit8 and Fgg loci (lod score 2.3). In addition, possible interaction between genes on chromosomes 7 and 3, X and 3, 14 and 3, 13 and 11 was described, indicating that blood pressure development in the SHR might be the result of interacting genes.

Congenic spontaneously diabetic hypertensive BB.SHR rats

Abstract ANIMAL MODELS for the study of the well-known association between diabetes and hypertension are currently unavailable. Because some genes causing hypertension in patients without diabetes may be also the same genes causing susceptibility of hypertension in diabetics, we used normotensive, but diabetes-prone BB/OK rats bred in our own animal facility and commercially obtained hypertensive, but diabetes-resistant SHR/Mol rats to establish a panel of congenic BB.SHR rat strains each carrying a single chromosome segment of the SHR rat known to be involved in the development of hypertension.

Phenotype and Genotype Comparison of Hereditary Hypertriglyceridemic (hHTG) and Brown‐Norway (BN) Rats Identification of Quantitative Trait Loci (QTLs) for the Insulin Resistance Syndrome

The hereditary hypertriglyceridemic (hHTG) rat manifesting hypertriglyceridemia, hyperinsulinemia, I insulin resistance,* glucose intolerance, mild hyperuri~emia,~ and hypertension4 provides a unique animal model for the human insulin resistance syndrome5 in which it may be possible to identify genetic factors predisposing to the syndrome. The classical method for identifying quantitative trait loci (QTLs) that influence traits in animal models is by cosegregation analysis. In this approach, the diseaseprone strain is crossed with a disease-resistant strain and FZ (or backcross) progeny are examined for cosegregation of genetic markers with quantitative differences in