The cardiovascular effects of salidroside in the Goto-Kakizaki diabetic rat model.

Many factors, including hyperglycemia, hypertension, obesity, dyslipidemia, and a sedentary lifestyle, contribute to a high prevalence of cardiovascular disease. Specific vascular impairment treatments in the context of diabetes and vascular risk need to be improved. Salidroside is the primary active component of Rhodiola rosea and has documented antioxidative, cardioprotective, and vasculoprotective properties. The aim of this study was to test the hypothesis that salidroside has protective effects against hyperglycemia, hypertension, and vasodilation impairment in the Goto-Kakizaki (GK) rat model of diabetes. We evaluated cardiovascular parameters (e.g., daytime/nighttime systolic and diastolic blood pressure, heart rate, and activity), metabolic parameters (e.g., body weight, food and water consumption, serum fructosamine level, glucose tolerance), eNOS / phospho-eNOS expression level and in vitro vascular reactivity of aorta and second-order mesenteric arteries in Wistar-Kyoto (control) and GK (diabetic) rats treated with salidroside (40 mg/kg) or placebo (water) for 5 weeks. GK rats showed hypertension, marked glucose intolerance, and impaired endothelium-dependent and endothelium-independent vasodilation capacity. Salidroside showed beneficial effects on endothelial and non-endothelial vasodilation and likely acts on the endothelium and smooth muscle cells through the soluble guanylyl cyclase pathway. Despite its vascular effects, salidroside had no effect on blood pressure and heart rate in GK and control rats, it did not improve glucose metabolism or limit hypertension in the GK model of type 2 diabetes.

Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes.

AIMS/HYPOTHESIS: MicroRNAs regulate a broad range of biological mechanisms. To investigate the relationship between microRNA expression and type 2 diabetes, we compared global microRNA expression in insulin target tissues from three inbred rat strains that differ in diabetes susceptibility. METHODS: Using microarrays, we measured the expression of 283 microRNAs in adipose, liver and muscle tissue from hyperglycaemic (Goto-Kakizaki), intermediate glycaemic (Wistar Kyoto) and normoglycaemic (Brown Norway) rats (n = 5 for each strain). Expression was compared across strains and validated using quantitative RT-PCR. Furthermore, microRNA expression variation in adipose tissue was investigated in 3T3-L1 adipocytes exposed to hyperglycaemic conditions. RESULTS: We found 29 significantly differentiated microRNAs (p(adjusted) < 0.05): nine in adipose tissue, 18 in liver and two in muscle. Of these, five microRNAs had expression patterns that correlated with the strain-specific glycaemic phenotype. MiR-222 (p(adjusted) = 0.0005) and miR-27a (p(adjusted) = 0.006) were upregulated in adipose tissue; miR-195 (p(adjusted) = 0.006) and miR-103 (p(adjusted) = 0.04) were upregulated in liver; and miR-10b (p(adjusted) = 0.004) was downregulated in muscle. Exposure of 3T3-L1 adipocytes to increased glucose concentration upregulated the expression of miR-222 (p = 0.008), miR-27a (p = 0.02) and the previously reported miR-29a (p = 0.02). Predicted target genes of these differentially expressed microRNAs are involved in pathways relevant to type 2 diabetes. CONCLUSION: The expression patterns of miR-222, miR-27a, miR-195, miR-103 and miR-10b varied with hyperglycaemia, suggesting a role for these microRNAs in the pathophysiology of type 2 diabetes, as modelled by the Gyoto-Kakizaki rat. We observed similar patterns of expression of miR-222, miR-27a and miR-29a in adipocytes as a response to increased glucose levels, which supports our hypothesis that altered expression of microRNAs accompanies primary events related to the pathogenesis of type 2 diabetes.

Subtle metabolic and liver gene transcriptional changes underlie diet-induced fatty liver susceptibility in insulin-resistant mice.

AIMS/HYPOTHESIS: Complex changes in gene expression are associated with insulin resistance and non-alcoholic fatty liver disease (NAFLD) promoted by feeding a high-fat diet (HFD). We used functional genomic technologies to document molecular mechanisms associated with diet-induced NAFLD. MATERIALS AND METHODS: Male 129S6 mice were fed a diet containing 40% fat (high-fat diet, HFD) for 15 weeks. Glucose tolerance, in vivo insulin secretion, plasma lipid profile and adiposity were determined. Plasma metabonomics and liver transcriptomics were used to identify changes in gene expression associated with HFD-induced NAFLD. RESULTS: In HFD-fed mice, NAFLD and impaired glucose and lipid homeostasis were associated with increased hepatic transcription of genes involved in fatty acid uptake, intracellular transport, modification and elongation, whilst genes involved in beta-oxidation and lipoprotein secretion were, paradoxically, also upregulated. NAFLD developed despite strong and sustained downregulation of transcription of the gene encoding stearoyl-coenzyme A desaturase 1 (Scd1) and uncoordinated regulation of transcription of Scd1 and the gene encoding sterol regulatory element binding factor 1c (Srebf1c) transcription. Inflammatory mechanisms appeared to be stimulated by HFD. CONCLUSIONS/INTERPRETATION: Our results provide an accurate representation of subtle changes in metabolic and gene expression regulation underlying disease-promoting and compensatory mechanisms, collectively contributing to diet-induced insulin resistance and NAFLD. They suggest that proposed models of NAFLD pathogenesis can be enriched with novel diet-reactive genes and disease mechanisms.

Genetic control of plasma lipid levels in a cross derived from normoglycaemic Brown Norway and spontaneously diabetic Goto-Kakizaki rats.

AIMS/HYPOTHESIS: Dyslipidaemia is a main component of the insulin resistance syndrome. The inbred Goto-Kakizaki (GK) rat is a model of spontaneous type 2 diabetes and insulin resistance, which has been used to identify diabetes-related susceptibility loci in genetic crosses. The objective of our study was to test the genetic control of lipid metabolism in the GK rat and investigate a possible relationship with known genetic loci regulating glucose homeostasis in this strain. MATERIALS AND METHODS: Plasma concentration of triglycerides, phospholipids, total cholesterol, HDL, LDL and VLDL cholesterol were determined in a cohort of 151 hybrids of an F2 cross derived from GK and non-diabetic Brown Norway (BN) rats. Data from the genome-wide scan of the F2 hybrids were used to test for evidence of genetic linkage to the lipid quantitative traits. RESULTS: We identified statistically significant quantitative trait loci (QTLs) that control the level of plasma phospholipids and triglycerides (chromosome 1), LDL cholesterol (chromosome 3) and total and HDL cholesterol (chromosomes 1 and 5). These QTLs do not coincide with previously identified diabetes susceptibility loci in a similar cross. The significance of lipid QTLs mapped to chromosomes 1 and 5 is strongly influenced by sex. CONCLUSION/INTERPRETATION: We established that several genetic loci control the quantitative variations of plasma lipid variables in a GKxBN cross. They appear to be distinct from known GK diabetes QTLs, indicating that lipid metabolism and traits directly relevant to glucose and insulin regulation are controlled by different gene variants in this strain combination.

Enhanced insulin secretion and cholesterol metabolism in congenic strains of the spontaneously diabetic (Type 2) Goto Kakizaki rat are controlled by independent genetic loci in rat chromosome 8.

AIMS/HYPOTHESIS: Genetic investigations in the spontaneously diabetic (Type 2) Goto Kakizaki (GK) rat have identified quantitative trait loci (QTL) for diabetes-related phenotypes. The aims of this study were to refine the chromosomal mapping of a QTL ( Nidd/gk5) identified in chromosome 8 of the GK rat and to define a pathophysiological profile of GK gene variants underlying the QTL effects in congenics. METHODS: Genetic linkage analysis was carried out with chromosome 8 markers genotyped in a GKxBN F2 intercross previously used to map diabetes QTL. Two congenic strains were designed to contain GK haplotypes in the region of Nidd/gk5 transferred onto a Brown Norway (BN) genetic background, and a broad spectrum of diabetes phenotypes were characterised in the animals. RESULTS: Results from QTL mapping suggest that variations in glucose-stimulated insulin secretion in vivo, and in body weight are controlled by different chromosome 8 loci (LOD3.53; p=0.0004 and LOD4.19; p=0.00007, respectively). Extensive physiological screening in male and female congenics at 12 and 24 weeks revealed the existence of GK variants at the locus Nidd/gk5, independently responsible for significantly enhanced insulin secretion and increased levels of plasma triglycerides, phospholipids and HDL, LDL and total cholesterol. Sequence polymorphisms detected between the BN and GK strains in genes encoding ApoAI, AIV, CIII and Lipc do not account for these effects. CONCLUSIONS/INTERPRETATION: We refined the localisation of the QTL Nidd/gk5 and its pathophysiological characteristics in congenic strains derived for the locus. These congenic strains provide novel models for testing the contribution of a subset of GK alleles on diabetes phenotypes and for identifying diabetes susceptibility genes.

Localization, cDNA sequence and genomic organization of the rat seipin gene (Bscl2) and sequence analysis in inbred rat models of Type 2 diabetes mellitus.

Mutations in the gene encoding seipin cause Berardinelli-Seip congenital lipodystrophy 2, with symptoms including near-absence of adipose tissue and altered glucose tolerance. Radiation hybrid analysis localized the seipin gene (Bscl2) in rat to a major quantitative trait locus in rat chromosome 1 linked to glucose intolerance in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. We determined the genomic organization of Bscl2 and screened coding exons and flanking intron sequences for mutations in GK, Wistar and Brown Norway rats, as well as in the Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rat. Two silent single nucleotide polymorphisms that were identified also were found in non-diabetic rat strains. We conclude that mutations in the gene for seipin are unlikely to contribute to diabetes in GK and OLETF rats.

Genetic dissection of region around the Sa gene on rat chromosome 1: evidence for multiple loci affecting blood pressure.

A region with a major effect on blood pressure (BP) is located on rat chromosome 1 in the vicinity of the Sa gene, a candidate gene for BP regulation. Previously, we observed a single linkage peak for BP in this region in second filial generation rats derived from a cross of the spontaneously hypertensive rat (SHR) with the Wistar-Kyoto rat (WKY), and we have reported the isolation of the region containing the BP effect in reciprocal congenic strains (WKY.SHR-Sa) and (SHR.WKY-Sa) derived from these animals. Here, we report the further genetic dissection of this region. Two congenic substrains each were derived from WKY.SHR-Sa (WISA1 and WISA2) and SHR.WKY-Sa (SISA1 and SISA2) by backcrossing to WKY and SHR, respectively. Although there was some overlap of the introgressed regions retained in the various substrains, the segments in WISA1 and SISA1 did not overlap. Furthermore, although the Sa allele in WISA1, WISA2, and SISA2 remained donor in origin, recombination in SISA1 reverted it back to the recipient (SHR) allele. Surprisingly, all 4 substrains demonstrated a highly significant BP difference compared with that of their respective parental strain, which was of a magnitude similar to those seen in the original congenic strains. The findings strongly indicate that there are at least 2 quantitative trait loci (QTLs) affecting BP in this region of rat chromosome 1. Furthermore, the BP effect seen in SISA1 indicates that at least a proportion of the BP effect of this region of rat chromosome 1 cannot be due to the Sa gene. SISA1 contains an introgressed segment of <3 cM, and this will facilitate the physical mapping of the BP QTL(s) located within it and the identification of the susceptibility-conferring genes. Our observations serve to illustrate the complexity of QTL dissection and the care needed to interpret findings from congenic studies.

Genetic influences on the end-stage effector phase of arthritis.

K/BxN T cell receptor transgenic mice are a model of inflammatory arthritis, most similar to rheumatoid arthritis, that is critically dependent on both T and B lymphocytes. Transfer of serum, or just immunoglobulins, from arthritic K/BxN animals into healthy recipients provokes arthritis efficiently, rapidly, and with high penetrance. We have explored the genetic heterogeneity in the response to serum transfer, thereby focussing on the end-stage effector phase of arthritis, leap-frogging the initiating events. Inbred mouse strains showed clear variability in their responses. A few were entirely refractory to disease induction, and those which did develop disease exhibited a range of severities. F1 analyses suggested that in most cases susceptibility was controlled in a polygenic additive fashion. One responder/nonresponder pair (C57Bl/6 x NOD) was studied in detail via a genome scan of F2 mice; supplementary information was provided by the examination of knock-out and congenic strains. Two genomic regions that are major, additive determinants of the rapidity and severity of K/BxN serum-transferred arthritis were highlighted. Concerning the first region, on proximal chromosome (chr)2, candidate assignment to the complement gene C5 was confirmed by both strain segregation analysis and functional data. Concerning the second, on distal chr1, coinciding with the Sle1 locus implicated in susceptibility to lupus-like autoimmune disease, a contribution by the fcgr2 candidate gene was excluded. Two other regions, on chr12 and chr18 may also contribute to susceptibility to serum-transferred arthritis, albeit to a more limited degree. The contributions of these loci are additive, but gene dosage effects at the C5 locus are such that it largely dominates. The clarity of these results argues that our focus on the terminal effector phase of arthritis in the K/BxN model will bear fruit.

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps.

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.

A dominant modifier of transgene methylation is mapped by QTL analysis to mouse chromosome 13.

The single-copy hepatitis B virus transgene in the E36 transgenic mouse strain undergoes methylation changes in a parent-of-origin, tissue, and strain-specific fashion. In a C57BL/6 background, the paternally transmitted transgene is methylated in 30% of cells, whereas it is methylated in more than 80% of cells in (BALB/c x C57BL/6) F1 mice. We established previously that several genetic factors were likely to contribute to the transgene methylation profile, some with demethylating and some with de novo methylating activities. Using quantitative trait loci (QTL) mapping, we have now localized one major modifier locus on chromosome 13 (Mod13), which explains a 30% increase in the methylation level of this transgene with no effect on the flanking endogenous sequences. No other QTL could be identified, except for a demethylating activity of low significance located on chromosome 12. Recombinant inbred mice containing a BALB/c allele of Mod13 were then used to show that the presence of Mod13 is sufficient to induce de novo methylation. A segregation between de novo methylation and repression of transgene expression was uncovered, suggesting that this genetic system is also useful for the identification of factors that interpret methylation patterns in the genome.

Rat chromosome 9 bears a major susceptibility locus for IgE response.

Injection of Brown Norway (BN) rats with gold salts provides a model to analyze the genetic control of the IgE response. A cohort of F2 progeny of susceptible BN and resistant LEW strains has been studied to carry out a genome-wide search for loci controlling the IgE response. Genome scanning identified two previously described loci, Atps1 and Atps2, and a new locus, Atps3. Atps1 linked to the MHC and Atps2 linked to the cytokine gene cluster that included the IL-4 region have been previously associated with serum IgE concentrations and with other Th2-dependent immune manifestations triggered by gold salts. The new interval, Atps3, identified on chromosome 9 (Lod score = 16), appears to play a major role in the control of the IgE response since it accounts for 31% of the genetic variance. Moreover, Atps3 is linked to anti-laminin antibody response and to glomerular immunoglobulin deposits. The identification and functional characterization of genes involved in these regions, particularly in Atps3, may shed light on the pathogenesis of atopic diseases in man.

Detailed comparative gene map of rat chromosome 1 with mouse and human genomes and physical mapping of an evolutionary chromosomal breakpoint.

We report the localization of 92 new gene-based markers assigned to rat chromosome 1 by linkage or radiation hybrid mapping. The markers were chosen to enrich gene mapping data in a region of the rat chromosome known to contain several of the principal quantitative trait loci in rodent models of human multifactorial disease. The composite map reported here provides map information on a total of 139 known genes, including 80 that have been localized in mouse and 109 that have been localized in human, and integrates the gene-based markers with anonymous microsatellites. The evolutionary breakpoints identifying 16 segments that are homologous regions in the human genome are defined. These data will facilitate genetic and comparative mapping studies and identification of novel candidate genes for the quantitative trait loci that have been localized to the region.

Applicability of a "speed" congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2.

The identification of any quantitative trait locus (QTL) via a genome scan is only the first step toward the ultimate goal of gene identification. The next step is the production of congenic strains by which the existence of a QTL may be verified and the implicated chromosomal region be reduced to a size applicable to positional cloning of the causal gene. We used a speed congenic breeding protocol previously verified in mice for 2 blood pressure QTLs on rat chromosome 2. Four congenic strains were produced through introgression of various segments of chromosome 2 from Wistar-Kyoto rats from Glasgow colonies [WKY((Gla)) rats] into the recipient stroke-prone spontaneously hypertensive rats from Glasgow colonies [SHRSP((Gla))], and vice versa. The number of backcross generations required for each strain to achieve complete homozygosity at 83 background genetic markers in a "best" male varied between 3 and 4. Transfer of the region of rat chromosome 2 containing both QTLs from WKY((Gla)) into an SHRSP((Gla)) genetic background lowered both baseline and salt-loaded systolic blood pressure by approximately 20 and approximately 40 mm Hg in male congenic rats compared with the SHRSP parental strain (F=53.4, P<0.005; F=28.0, P< 0.0005, respectively). In contrast, control animals for stowaway heterozygosity presented no deviation from the blood pressure values recorded for the SHRSP((Gla)), indicating that if such heterozygosity exists, its effect on blood pressure is negligible. A reciprocal strategy in which 1 or both QTLs on rat chromosome 2 were transferred from SHRSP((Gla)) into a WKY((Gla)) genetic background resulted in statistically significant but smaller blood pressure increases for 1 of these QTLs. These results confirm the existence of blood pressure QTLs on rat chromosome 2 and demonstrate the applicability of a speed congenic strategy in the rat and emphasize the important role of the genetic background.

A gene map of the rat derived from linkage analysis and related regions in the mouse and human genomes.

We report the localization by linkage analysis in the rat genome of 148 new markers derived from 128 distinct known gene sequences, ESTs, and anonymous sequences selected in GenBank database on the basis of the presence of a repeated element. The composite linkage map of the rat contributed by our group integrates mapping information on a total of 370 different known genes, ESTs, and anonymous mouse or human sequences, and provides a valuable tool for comparative genome analysis. 206 and 254 homologous loci were identified in the mouse and human genomes respectively. Our linkage map, which combines both anonymous markers and gene markers, should facilitate the advancement of genetic studies for a wide variety of rat models characterized for complete phenotypes. The comparative genome mapping should define genetic regions in human likely to be homologous to susceptibility loci identified in rat and provide useful information for the identification of new potential candidates for genetic disorders.

Complete genome searches for quantitative trait loci controlling blood pressure and related traits in four segregating populations derived from Dahl hypertensive rats.

The Dahl salt-sensitive rat is one of the principal animal models of hereditary hypertension. Genome-wide searches were undertaken to detect quantitative trait loci (QTLs) that influence blood pressure, cardiac mass, and body weight in four F2 populations derived from Dahl salt-sensitive rats and different inbred normotensive control strains of rat. We detected three QTLs associated with one or more of the phenotypes, using a stringent statistical criterion for linkage (p < 0.00003). These included a novel QTL linked to blood pressure on rat Chromosome (Chr) 12, and another QTL on rat Chr 3 linked to body weight. A QTL on rat Chr 10 for which linkage to blood pressure has been described in other crosses was found to be a principal determinant of blood pressure and cardiac mass in some but not all of the crosses examined here. Three other regions showed evidence of linkage to these phenotypes with a less stringent statistical criterion of linkage at QTLs previously reported in other studies. As part of our study, microsatellite markers have been developed for three candidate genes for investigation in hypertension, and the genes have been localized by linkage mapping. These are: the rat Gs alpha subunit (Gnas) gene, the alpha-1B adrenergic receptor (Adra1b), and the Na+, K+-ATPase beta2 subunit (Atp1b2) gene.