Congenic diabetes-prone BB.Sa and BB.Xs rats differ from their progenitor strain BB/OK in frequency and severity of insulin-dependent diabetes mellitus.

Two newly established congenic diabetes-prone BB rat strains designated BB.Sa and BB.Xs carrying a region of chromosome 1 (Sa-Lsn-Secr-Igf2-Tnt, 16 cM) and a region of chromosome X (DXMgh3-Mycs/Pfkb1-Ar, 36 cM) of the SHR rats, respectively, were studied to determine whether the transferred chromosomal regions influence diabetes frequency, age at onset, and clinical picture. Therefore, 4 complete litters of BB/OK (n = 43), BB.Sa (n = 45), and BB.Xs (n = 41) were observed for diabetes occurrence up to the age of 30 weeks. From these litters 6 diabetic males of each strain manifesting in an interval of 1 week were chosen to study body weight, blood glucose, insulin requirement to survive, and several diabetes-related serum constituents at onset of diabetes and after a diabetes duration of 150 days. The diabetes frequency was significantly lower in BB.Xs than in rats of the parental strain BB/OK, whereas comparable frequencies were found between BB/OK and BB.Sa rats. Obvious differences were observed 150 days after diabetes onset between BB/OK and both BB.Sa and BB.Xs rats. BB/OK rats were significantly heavier and needed significantly more insulin/100 g body weight than BB.Sa and BB.Xs rats. Comparisons of the serum constituents as lipids, proteins, and minerals revealed significant differences between diabetic BB/OK rats and their diabetic congenic derivatives in several traits studied at onset and after 150 days of insulin treatment. These results not only show the power of congenic lines in diabetes research, but indicate for the first time that there are genetic factors on chromosomes 1 and X influencing frequency and severity of diabetes in the BB/OK rat.     

Loci of the immune system are implicated in diabetes frequency and age at onset of diabetes in BB rats

The spontaneously diabetic BB rat is a well-established animal model in diabetes research developing an insulin-dependent type-1 diabetes mellitus closely resembling human diabetes. By several crossing studies using BB/OK rats it has been demonstrated that beside the MHC class-II genes of the RT1^u haplotype, Iddm1, and the lymphopenia, Iddm2, at least two additional non-MHC genes located on chromosomes 6 (Iddm4) and 18 (Iddm3) are involved in diabetes development. In addition, there are at least three genes located on chromosomes 6 (Dm1), 8 (Dm2) and 10 (Dm3) influencing the age at onset of diabetes. Comparing the homologous regions between rat and human, it is shown that most diabetogenic genes lie on human chromosomes near genes involved in immune processes providing human geneticist with new candidate regions for the analysis of diabetogenic non-MHC genes in human type-1 diabetes.     

Phenotypic selection: a successful strategy to fix major genes of hypertension

Spontaneously diabetic BB/OK rats are not genetically susceptible to develop diabetic complications as hypertension or nephropathy. Recently, we generated 5 congenic BB. SHR rat strains by transferring different chromosomal regions of the spontaneously hypertensive rat (SHR) onto the genetic background of BB/OK rats. Four out of 5 strains showed a weak increase of blood pressure (8 mmHg). This weak blood pressure effect indicated that the transferred regions fo not contain major genes for hypertension. That prompted us to choose the classical procedure of phenotypic selection to fix major genes causing hypertension in a BB/OK rat subline generated by cross of BB/OK and SHR and repeated backcrossing of animals with highest blood pressure onto normotensive BB/OK rats. After 7 backcrosses (N8), all backcross parents were genetically analysed with the aid of 259 microsatellites to identify loci causing blood pressure of 177 ± 10 mmHg in this BB/OK rat subline. The data revealed, that loci on chromosome 1, 14 and 18 were heterozygous until BC5, BC6 and BC7, respectively. Considering the relative stable high blood pressure during the backcross procedure, these loci might be of essential importance for the development of hypertension in the SHR.     

Congenic BB.SHR (D4Mit6-Npy-Spr) Rats: A New Aid to Dissect the Genetics of Obesity

Objective: The phenotypic characterization of congenic BB.LL rats recombining a segment of the SHR chromosome 4 (D4Mit6-Npy-Spr; 12 cM) into the BB/OK background indicated that these rats were not lymphopenic and did not develop diabetes, but they were significantly heavier (at 16 weeks of age) and showed higher serum triglycerides and total cholesterol concentration. Research Methods and Procedures: BB.LL rats were longitudinally studied for facets of metabolic syndrome (body mass index, blood glucose, serum lipids, insulin, leptin, and systolic and diastolic blood pressure) from 2 to 12 months of age. Results: In this study, it was shown that BB.LL are obese, hyperleptinemic, hyperinsulinemic, and dyslipidemic compared with their parental BB/OK rats. Discussion: It can be concluded that there is a gene(s) in the introgressed segment causing incomplete metabolic syndrome, because they do not develop hypertension and diabetes. To identify the gene(s), the introgressed chromosomal segment must be systematically whittled down to generate recombinants and new subcongenic lines carrying a much smaller segment of the SHR/Mol rat to increase the chance of identification of the appropriate gene(s).     

Congenic mapping of type 1 diabetes--protective gene(s) in an interval of 4 Mb on rat chromosome 6q32

Congenic BB.SHR rats introgressing a segment of SHR chromosome 6 onto BB/OK background showed a reduction of diabetes frequency by 72% compared with BB/OK. To identify underlying gene(s), the introgressed segment was shortened and the expression of seven genes (Yy1, Dlk1/Pref-1, Wd40 repeat, Cdc42, Rtl1, Traf3, and Tnfaip2) was studied in blood and spleen of non-diabetic BB/OK, BB.6S, and SHR males and females at an age of 30, 70, and 90 days. The phenotype of congenic sublines narrowed the diabetes-protective region to 4 Mb. The relative expression of Yy1 and Pref-1 in blood and of Pref-1 in spleen was significantly reduced by 50-90% in male and female BB.6S and SHR compared with BB/OK favouring Yy1 and Pref-1 as candidate genes. All other genes were differently expressed according to gender and strain.     

Alleles on Rat Chromosome 4 (D4Got41‐Fabp1/Tacr1) Regulate Subphenotypes of Obesity

Objective: The use of inbred animal models is an essential component of the genetic dissection of complex diseases. Because quantitative trait loci for serum triglycerides, total cholesterol, and body weight were mapped on chromosome 4 in a cross of BioBreeding/OttawaKarlsburg (BB/OK) and spontaneously hypertensive (SHR) rats, we established a congenic BB.SHR rat strain by introgressing a SHR segment of chromosome 4 (D4Got41‐Tacr1) into a BB/OK background. The phenotype of these BB.SHR rats (BB.4S) confirmed the quantitative trait loci. To discover whether the phenotype of BB.4S can only be attributed to the SHR segment per se, we established an additional congenic BB.WOKW strain by introgressing a similar segment of chromosome 4 (D4Got41‐Fabp1) of the Wistar Ottawa Karlsburg RT1^u rat into a BB/OK background, termed briefly BB.4W. Research Methods and Procedures: Male normoglycemic BB/OK (20), BB.4S (20), and BB.4W (16) rats were longitudinally studied for body weight, serum triglycerides, total and high‐density lipoprotein‐cholesterol, and glucose tolerance. At the end of the observation period (32 weeks), serum insulin, leptin, and adiposity index (AI) were determined. Results and Discussion: Congenic BB.4S and BB.4W were significantly heavier, and AI, serum triglycerides, and total cholesterol values were significantly elevated in BB.4S and BB.4W compared with BB/OK but more pronounced in BB.4S. The highest serum insulin was found in BB.4W and highest leptin in BB.4S. Because the body weight gain and AI were comparable between BB.4S and BB.4W, the obviously higher insulin levels in BB.4W and higher leptin values in BB.4S suggest that the two congenics most probably define two subphenotypes of obesity and provide the unique opportunity to study their genetics.     

Analysis of the rat Iddm14 diabetes susceptibility locus in multiple rat strains: identification of a susceptibility haplotype in the Tcrb-V locus

Iddm14 (formerly Iddm4) is a non-MHC-linked genetic locus associated with autoimmune diabetes. Its effects have been well-documented in BB-derived rats in which diabetes is either induced by immunologic perturbation or occurs spontaneously. The role of Iddm14 in non-BB rat strains is unknown. Our goal was to extend the analysis of Iddm14 in new diabetes-susceptible strains and to identify candidate genes in the rat Iddm14 diabetes susceptibility locus that are common to these multiple diabetic strains. To determine if Iddm14 is important in strains other than BB, we first genotyped a (LEW.1WR1 × WF)F2 cohort in which diabetes was induced by perturbation with polyinosinic:polycytidylic acid. We found that Iddm14 is a major determinant of diabetes susceptibility in LEW.1WR1 rats. We then used nucleotide sequencing to establish a strain distribution pattern of polymorphisms (insertions, deletions, and single nucleotide polymorphisms [SNPs]) that predicts susceptibility to diabetes in a panel of inbred and congenic rats. Using the positional information from the congenic strains and the new linkage data, we identified a susceptibility haplotype in the T-cell receptor Vβ chain (Tcrb-V) locus. This haplotype includes Tcrb-V13, which is identical in five susceptible strains but different in resistant WF and F344 rats. We conclude that Iddm14 is a powerful determinant of both spontaneous and induced autoimmune diabetes in multiple rat strains, and that Tcrb-V13 SNPs constitute a haplotype of gene elements that may be critical for autoimmune diabetes in rats.     

Phenotypic selection: a successful strategy to fix major genes of hypertension.

Hypertension is dominantly inherited in cross hybrids between hypertensive SHR/Mol and normotensive BB/OK rats. We used these cross hybrids for repeated backcrossing of selected hypertensive animals onto normotensive BB/OK rats to fix high blood pressure and to generate a hypertensive and diabetic BB/OK rat subline. After 8 backcrosses, the backcross parents were genetically analysed with the aid of 259 microsatellite markers to identify SHR genes causing blood pressure of 177 +/- 10 mmHg in this BB/OK rat subline. Loci on chromosomes 1, 14 and 18 showed longest heterozygosity. These loci might contain major genes of the SHR rat causing hypertension in this BB/OK rat subline. This classical strategy seems to be most suitable to fix major genes of hypertension in particular and complex traits in general and therefore to generate new animal models.     

Congenic strain confirms putative quantitative trait locus for body weight in the rat

Quantitative trait loci (QTLs) affecting body weight were investigated in the backcross population derived from nondiabetic BB/OK and spontaneously hypertensive rat (SHR) strains. The F₁ hybrids were backcrossed onto SHR rats, and QTL analysis was performed separately with the resulting backcross populations for each sex on Chromosomes (Chrs) 1, 3, 4, 10, 13, and 18. The body weight was determined at the age of 14 weeks, and the statistical analysis was performed with MAPMAKER/QTL 1.1b computer program. According to the stringent threshold for a lod score of 3.0, markers on Chr 1 were found to be linked with body weight. The QTL with a peak lod score (5.1) on Chr 1 for a male population was located within markers Igf2 and D1Mgh12. In contrast, in the female population the body weight affecting QTL (lod = 5.7) on Chr 1 was located between the D1Mit3 and Lsn markers. The existence of QTLs on Chr 1 affecting body weight in the male population was confirmed by congenic BB.Sa rats, carrying chromosomal region of SHR (Sa-Igf2) on the genetic background of BB rat. Received: 14 July 1997 / Accepted: 22 December 1997     

Increased susceptibility to kidney injury by transfer of genomic segment from SHR onto Dahl S genetic background

The Dahl salt-sensitive (S) rat is a widely studied model of salt-sensitive hypertension and develops proteinuria, glomerulosclerosis, and renal interstitial fibrosis. An earlier genetic analysis using a population derived from the S and spontaneously hypertensive rat (SHR) identified eight genomic regions linked to renal injury in the S rat and one protective locus on chromosome 11. The "protective" locus in the S rat was replaced with the SHR genomic segment conferring "susceptibility" to kidney injury. The progression of kidney injury in the S.SHR(11) congenic strain was characterized in the present study. Groups of S and S.SHR(11) rats were followed for 12 wk on either a low-salt (0.3% NaCl) or high-salt (2% NaCl) diet. By week 12 (low-salt), S.SHR(11) demonstrated a significant decline in kidney function compared with the S. Blood pressure was significantly elevated in both strains on high salt. Despite similar blood pressure, the S.SHR(11) exhibited a more significant decline in kidney function compared with the S. The decline in S.SHR(11) kidney function was associated with more severe kidney injury including tubular loss, immune cell infiltration, and tubulointerstitial fibrosis compared with the S. Most prominently, the S.SHR(11) exhibited a high degree of medullary fibrosis and a significant increase in renal vascular medial hypertrophy. In summary, genetic modification of the S rat generated a model of accelerated renal disease that may provide a better system to study progression to renal failure as well as lead to the identification of genetic variants involved in kidney injury.     

Comparative mapping of rat <Emphasis Type="Italic">Iddm4</Emphasis> to segments on HSA7 and MMU6

Iddm4 is one of several susceptibility genes that have been identified in the BB rat model of type 1 diabetes. The BB rat allele of this gene confers dominant predisposition to diabetes induction by immune perturbation in both the diabetes-prone and the diabetes-resistant substrains, whereas the Wistar Furth (WF) allele confers resistance. We have positioned the gene in a 2.8-cM region on rat Chromosome (Chr) 4, proximal to Lyp/Ian4l1. We have produced a radiation hybrid map of the Iddm4-region that includes a number of rat genes with their mouse and human orthologs. We present a comparative map of the rat Iddm4 region in rat, human, and mouse, assigning the gene to a 6.3-Mb segment between PTN and ZYX at 7q32 in the human genome, and to a 5.7-Mb segment between Ptn and Zyx in the mouse genome.     

Mapping a blood pressure quantitative trait locus to a 5.7-cM region in Dahl salt-sensitive rats

A region on rat Chromosome (Chr) 2 of the Dahl salt-sensitive rat (S) was shown previously to contain a quantitative trait locus (QTL) for blood pressure (BP). This was achieved first by linkage, followed by the use of congenic strains. A congenic strain, designated S.MNS-D2Mit6/Adh, contained a segment of Chr 2 from the Milan Normotensive (MNS) rat in the S genetic background. Since the region containing the QTL was roughly 80 cM in size, a further reduction was needed towards the positional or candidate gene cloning. Currently, two congenic substrains were made from the original strain S.MNS-D2Mit6/Adh. One of these two substrains showed a BP-lowering effect, whereas the other substrain did not. Deducing the segment not shared in the two substrains, the BP QTL has to be present in a chromosome region of roughly 5.7 cM between the marker D2Rat303 and the locus for the neutroendopeptidase gene (Nep). Nep is not included within the segment. This region does not seem to contain any candidate genes well known for the BP control. Thus, the final identification of the QTL will most likely lead to the discovery of a brand new gene for the BP regulation. Received: 14 December 2000 / Accepted: 18 January 2001     

Ex vivo gene transfer of viral interleukin-10 to BB rat islets: no protection after transplantation to diabetic BB rats

Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation.     

High-resolution mapping of the blood pressure QTL on chromosome 7 using Dahl rat congenic strains

It was previously shown using Dahl salt-sensitive (S) and salt-resistant (R) rats that a blood pressure quantitative trait locus (QTL) was present on rat chromosome 7. In the present work, this QTL was localized to a region less than 0.54 cM in size on the linkage map using a series of congenic strains. This region was contained in a single yeast artificial chromosome that was 220 kb long. This small segment still contained the primary candidate locus Cyp11b1 (11beta-hydroxylase), but the adjacent candidate genes Cyp11b2 (aldosterone synthase) and Cyp11b3 were ruled out. It is concluded that 11beta-hydroxylase, through its known genetic variants altering the production of 18-hydroxy-11-deoxy corticosterone, is very likely to account for the blood pressure QTL on chromosome 7 in the Dahl rat model of hypertension. This QTL accounts for about 23 mm Hg under the condition of 2% NaCl diet for 24 days.     

Utilization of marker-assisted congenics to map two blood pressure quantitative trait loci in Dahl rats

A broad Chromosome (Chr) 10 region of the Dahl salt-sensitive (S) rat was shown by linkage and the use of congenic strains to contain a blood pressure (BP) quantitative trait locus (QTL). To further narrow down the region harboring the QTL, four congenic strains carrying smaller segments were made by replacing various segments of the S rats with the homologous segments of the Lewis (LEW) rats. The construction of these congenic strains was facilitated by a genome-wide marker screening. One congenic strain, assigned as S.L4, showed a BP-lowering effect, and the region harboring a BP QTL, designated QTL1, is localized to a segment of about 15 cM. Two other strains, assigned as S.L2 and S.L5, contained an overlapping segment, and both showed a BP-lowering effect. In contrast, the fourth congenic strain, assigned as S.L1, contained a smaller and shared fragment with S.L2 and S.L5, but it did not have a BP-lowering effect. Deducing from the segment in common in S.L2 and S.L5, and not shared between S.L1 and both congenic strains S.L2 and S.L5, the region harboring a QTL, designated as QTL2, was narrowed to about 12 cM. The current work showed the general applicability of the `speed congenic' approach to map and fine-map BP QTL. Received: 1 February 2001 / Accepted: 29 March 2001     

SHR Y chromosome enhances the nocturnal blood pressure in socially interacting rats

Our objective was to test the hypothesis that nocturnal mean arterial pressure (MAP), heart rate (HR), and activity would be increased in 1) colony over individually caged rats and 2) the spontaneously hypertensive rat (SHR) Y chromosome strain (SHR/y colony) compared with Wistar-Kyoto (WKY) rats. MAP, HR, and activity were monitored using radiotelemetry. The nocturnal MAP rise expressed as the percentage change in MAP from light to dark was increased (P < 0.05) in the SHR/y colony. The SHR Y chromosome increased MAP in both the colony and caged groups compared with WKY (P < 0.001). The SHR/y colony animals spent 23% of a 24-h period at a MAP >120 mmHg, whereas the WKY colony animals spent 2% of a 24-h period in this range. The MAP of the SHR/y colony on clonidine was reduced (P < 0.001) to WKY baseline values. Activity but not HR was increased (P < 0.01) in the WKY and SHR/y colonies compared with caged animals. In conclusion, colony housing and the SHR Y chromosome increased MAP compared with individually caged housing.     

A missense mutation in the Abcg5 gene causes phytosterolemia in SHR,stroke-prone SHR,and WKY rats

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.     

BB rat diabetes susceptibility and body weight regulation genes colocalize on Chromosome 2

The genetic etiology of Type 1 (insulin-dependent) diabetes mellitus is complicated by the apparent presence of several diabetes susceptibility genetic regions. Type 1 diabetes in the inbred BioBreeding (BB) rat closely resembles the human disorder and was previously shown to involve two genes: the lymphopenia (lyp) region on Chromosome (Chr) 4 and RT1 ^u in the major histocompatibility complex (MHC) on Chr 20. In addition, a segregation analysis of an F₂ intercross between the diabetes-prone congenic BB DR ^lyp/lyp,u/u and F344^{+/+, lv/lv} rats indicated that at least one more genetic factor was responsible for Type 1 diabetes. In this study, we generated F₂N₂ progeny in a cross between non-diabetic F₂(DR ^lyp/lyp,u/u × F344) ^lyp/lyp,u/u and diabetic DR ^lyp/lyp,u/u rats. In a subsequent total genome scan, a third factor was mapped to the 21.3-cM region on Chr 2 between D2Mit14 and D2Mit15 (peak LOD score 4.7 with 67% penetrance). Interestingly, the homozygosity of the BB allele (b/b) for the Chr 2 region was significantly associated with a greater weight reduction after fasting than the homozygosity of the F344 allele (f/f, p < 0.008). In conclusion, the development of Type 1 diabetes in the congenic DR ^lyp/lyp rat is controlled by at least three genes: lymphopenia, MHC, and a third factor that may play a role in metabolism and body weight regulation. Received: December 1998 / Accepted: 10 May 1999     

Impaired post-thymic development of regulatory CD4+25+ T cells contributes to diabetes pathogenesis in BB rats

One of the BB rat diabetes (diabetes mellitus (DM)) susceptibility genes is an Ian5 mutation resulting in premature apoptosis of naive T cells. Impaired differentiation of regulatory T cells has been suggested as one possible mechanism through which this mutation contributes to antipancreatic autoimmunity. Using Ian5 congenic inbred rats (wild-type (non-lyp BB) and mutated (BB)), we assessed the development of BB regulatory CD8(-)4(+)25(+)T cells and their role in the pathogenesis of DM. BB rats have normal numbers of functional CD8(-)4(+)25(+)Foxp3(+) thymocytes. The proportion of CD25(+) cells among CD8(-)4(+) recent thymic emigrants is also normal while it is increased among more mature CD8(-)4(+) T cells. However, BB CD8(-)4(+)25(+)Foxp3(+) thymocytes fail to undergo homeostatic expansion and survive upon transfer to nude BB rats while Foxp3 expression is reduced in mature CD8(-)4(+)25(+) T cells suggesting that these cells are mostly activated cells. Consistent with this interpretation, peripheral BB CD8(-)4(+)25(+) T cells do not suppress anti-TCR-mediated activation of non-lyp BB CD8(-)4(+)25(-) T cells but rather stimulate it. Furthermore, adoptive transfer of unfractionated T cells from diabetic BB donors induces DM in 71% of the recipients while no DM occurred when donor T cells are depleted of CD8(-)4(+)25(+) cells. Adoptive transfer of 10(6) regulatory non-lyp BB CD8(-)4(+)25(+) T cells to young BB rats protects the recipients from DM. Taken together, these results demonstrate that the BB rat Ian5 mutation alters the survival and function of regulatory CD8(-)4(+)25(+) T cells at the post-thymic level, resulting in clonal expansion of diabetogenic T cells among peripheral CD8(-)4(+)25(+) cells.