Lyon Normotensive Rats Research Articles

Systems biology with high-throughput sequencing reveals genetic mechanisms underlying the metabolic syndrome in the Lyon hypertensive rat.

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon hypertensive and Lyon normotensive rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, an F2 intercross between Lyon hypertensive and Lyon normotensive was comprehensively studied. Multidimensional data were obtained including genotypes of 1536 single-nucleotide polymorphisms, 23 physiological traits, and >150 billion nucleotides of RNA-seq reads from the livers of F2 intercross offspring and parental rats. Phenotypic and expression quantitative trait loci (eQTL) were mapped. Application of systems biology methods identified 17 candidate MetS genes. Several putative causal cis-eQTL were identified corresponding with phenotypic QTL loci. We found an eQTL hotspot on rat chromosome 17 that is causally associated with multiple MetS-related traits and found RGD1562963, a gene regulated in cis by this eQTL hotspot, as the most likely eQTL driver gene directly affected by genetic variation between Lyon hypertensive and Lyon normotensive rats. Our study sheds light on the intricate pathogenesis of MetS and demonstrates that systems biology with high-throughput sequencing is a powerful method to study the pathogenesis of complex genetic diseases.

Genomic structure of nucleotide diversity among Lyon rat models of metabolic syndrome

BackgroundThe metabolic syndrome (MetS), a complex disorder involving hypertension, obesity, dyslipidemia and insulin resistance, is a major risk factor for heart disease, stroke, and diabetes. The Lyon Hypertensive (LH), Lyon Normotensive (LN) and Lyon Low-pressure (LL) rats are inbred strains simultaneously derived from a common outbred Sprague Dawley colony by selection for high, normal, and low blood pressure, respectively. Further studies found that LH is a MetS susceptible strain, while LN is resistant and LL has an intermediate phenotype. Whole genome sequencing determined that, while the strains are phenotypically divergent, they are nearly 98% similar at the nucleotide level. Using the sequence of the three strains, we applied an approach that harnesses the distribution of Observed Strain Differences (OSD), or nucleotide diversity, to distinguish genomic regions of identity-by-descent (IBD) from those with divergent ancestry between the three strains. This information was then used to fine-map QTL identified in a cross between LH and LN rats in order to identify candidate genes causing the phenotypes.ResultsWe identified haplotypes that, in total, contain at least 95% of the identifiable polymorphisms between the Lyon strains that are likely of differing ancestral origin. By intersecting the identified haplotype blocks with Quantitative Trait Loci (QTL) previously identified in a cross between LH and LN strains, the candidate QTL regions have been narrowed by 78%. Because the genome sequence has been determined, we were further able to identify putative functional variants in genes that are candidates for causing the QTL.ConclusionsWhole genome sequence analysis between the LH, LN, and LL strains identified the haplotype structure of these three strains and identified candidate genes with sequence variants predicted to affect gene function. This approach, merged with additional integrative genetics approaches, will likely lead to novel mechanisms underlying complex disease and provide new drug targets and therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-197) contains supplementary material, which is available to authorized users.

Abstract 18436: Systems Biology Identifies a Gene Driving Expression of Metabolic Syndrome in Lyon Rats

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including obesity, hypertension, dyslipidemia, and insulin resistance. The Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats are models of MetS sensitivity and resistance, respectively. To identify genetic loci influencing phenotypes underlying MetS, an F2 intercross between LH and LN was studied to identify physiological quantitative trait loci (pQTL) contributing to plasma lipid and leptin levels, blood pressure, and body weight/adiposity. We performed mRNA-seq in F2 rats to identify the loci regulating gene expression, or expression (e)QTL, followed by gene expression network analyses. Systems biology methods were applied to identify candidate MetS genes, including genes previously shown to be MetS-related. We found an eQTL hotspot on RNO17, which was also located within pQTLs for MetS-related traits. The genes regulated by this eQTL hotspot were mainly in two co-expression network modules (a mitochondria related module and a gene regulation related module) and causally affect many MetS-related traits. Multiple evidences strongly and consistently support RGD1562963, a gene regulated in cis by this eQTL hotspot as the eQTL driver gene directly affected by genetic variation between LH and LN rats. Our study sheds light on the intricate pathogenesis of MetS and that systems biology with high-throughput sequencing is a powerful method to study the etiology of complex multifactorial diseases.

Abstract 518: Systems Biology Approach to Identify Genetic Mechanisms Underlying the Metabolic Syndrome in the LH Rat

The metabolic syndrome (MetS) is a collection of co-occurring complex disorders including hypertension, obesity, dyslipidemia, and insulin resistance. The Lyon Hypertensive (LH) and Lyon Normotensive (LN) rats are models of MetS sensitivity and resistance, respectively. To identify genetic determinants and mechanisms underlying MetS, rats from an F2 intercross between LH and LN were studied. Multi-dimensional data were obtained including SNP genotypes, physiological traits including blood pressure, plasma lipid and leptin levels, and body weight/adiposity, and more than 150 billion nucleotides of RNA-Seq reads from the livers of F2 (n=36), LH (n=6), and LN (n=6) individuals. We identified 17 pQTLs (physiological quantitative trait loci) and 1200 eQTLs (gene expression quantitative trait loci) at genome-wide p<0.05. Systems biology methods were applied to prioritize candidate MetS genes, including genes previously shown to be MetS-related. We identified an eQTL peak on rat chromosome 17 (RNO17) shared by large number of genes; RNO17 also has pQTL for MetS-related traits. Network analysis in these genes identified two major co-regulation modules involving mitochondria and gene regulation; genes in the two modules causally affect multiple MetS traits. A gene, RGD1562963, closest to the eQTL peak was identified as the eQTL driver gene. It is directly affected by genetic variation between LH and LN rats, affects downstream genes in the eQTL cluster, and is correlated with MetS phenotypes. Our study sheds light on the intricate pathogenesis of MetS and proved that systems biology with high-throughput sequencing is a powerful method to study the etiology of complicated diseases.

Abstract 651: Genetic Mapping Of Traits And Transcriptome In The Lyon Hypertensive Rat

The metabolic syndrome (MetS) is characterized by obesity, dyslipidemia, hypertension, and insulin resistance and is a major risk factor for cardiovascular disease. Using the Lyon Hypertensive and Lyon Normotensive rats as models of the metabolic syndrome, we aim to determine both physiological and transcriptional regulation underlying the syndrome. To identify genetic loci influencing phenotypes underlying MetS, an F2 intercross between LH and LN with 150 offspring was studied to identify physiological quantitative trait loci (pQTL) contributing to plasma lipid levels, blood pressure, and body weight/adiposity. We performed RNA-seq in livers from 36 selected rats from the intercross to identify the mapping of gene expression, or expression (e)QTL. Linkage analyses identified pQTL and eQTL with significant logarithm of odds (lod) scores, whose genome-wide significance was determined by permutation testing. We identified 17 pQTL on 5 rat chromosomes and 75 on 16 chromosomes. It has been determined that LH chromosome 17 contributes to multiple features of MetS; furthermore, an eQTL on rat chromosome 17 was identified that regulates the expression of multiple genes in trans, meaning the regulated gene falls outside the QTL interval or on another chromosome. We hypothesize these genes share a common transcriptional regulator which is affected by genetic variation on chromosome 17. eQTLs are likely to influence precursor pathways and cofactors of the metabolic syndrome. Identifying these in the LH rat could lead to translational studies to determine their role in the human MetS.

Visfatin is associated with lipid metabolic abnormalities in Lyon hypertensive rats

1. Visfatin (also known as nicotinamide phosphoribosyltransferase or Nampt) is a novel adipokine associated with metabolic abnormalities in obesity and diabetes. The aim of the present study was to determine whether visfatin levels in the circulation and visfatin expression in fat tissues are altered in Lyon hypertensive (LH) rats, a hypertensive strain with numerous metabolic abnormalities, and, if so, to explore the possible correlations between blood visfatin levels and the metabolic abnormalities in LH rats. 2. Male 18-20-week-old LH rats and their control, Lyon normotensive (LN) rats, were used. Blood pressure was recorded in conscious, unrestrained rats. Fat tissue weight was determined. Serum visfatin concentrations were determined by enzyme immunoassay, whereas visfatin expression in fat tissues was determined by western blotting. Relationships between two parameters were evaluated by linear regression analysis. 3. The LH rats had significantly higher bodyweight and fat tissue weight compared with LN rats. Serum lipid levels of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and triglyceride (TG) were all significantly higher in LH rats than in LN rats. Moreover, serum visfatin levels were higher in LH rats than in LN rats and were positively correlated with bodyweight, fat tissue weight and serum lipid levels (i.e. TC, HDL-C, LDL-C, TG and the TG:HDL-C ratio). There were no significant differences in serum glucose and insulin concentrations, or in the whole-body insulin resistance index, between LN and LH rats, and serum visfatin levels were not correlated with any of these parameters. Visfatin expression in visceral fat tissue, but not in subcutaneous fat tissue, was markedly upregulated in LH compared with LN rats. 4. In conclusion, the results of the present study demonstrate that serum visfatin levels in LH rats are elevated and are associated with lipid metabolic abnormalities.

Renin-angiotensin system in two genetically normotensive strains of lyon rats

Compared to the Lyon normotensive (LN) controls, adult Lyon hypertensive rats (LH) exhibit a renin-angiotensin system (RAS) dependent hypertension despite a low renin secretion. This discrepancy could be explained by the elevated slow pressor response to angiotensin II (AII) found in LH rats compared to LN controls. To evaluate more precisely the pathophysiological importance of this increased response, the present work aimed at determining whether the characteristics of the RAS were identical in LN and low blood pressure (LL) rats, the other normotensive control strain simultaneously selected with LH rats. Plasma and kidney renin and prorenin were measured in 11-week-old LN and LL rats. Aortic blood pressure (BP) was recorded at 15 weeks of age in freely moving rats of both strains either untreated or having received an angiotensin converting enzyme inhibitor, perindopril (3 mg/kg/day orally) since the age of 3 weeks. Acute dose-response curves were constructed for AII and norepinephrine (NE). The long-term pressor effects of AII (200 ng/kg/min) and NE (1000 ng/kg/min) were measured after chronic infusions in perindopril-treated LN and LL rats. LN and LL rats exhibited similar mean BP level before (114 ± 2 and 117 ± 2 mm Hg, respectively) and after perindopril treatment (91 ± 3 and 93 ± 1 mm Hg, respectively). Plasma and kidney renin and prorenin were decreased in LL rats. In acute conditions, LL rats exhibited an unspecific hypersensitivity to AII and NE. Chronically given AII exerted a greater pressor effect in LL than in LN rats after 4 weeks (113 ± 3 v 97 ± 5 mm Hg in LL and LN rats respectively, P < .05) and, even more, after 8 weeks of infusion (144 ± 9 v 124 ± 4 mm Hg in LL and LN rats respectively, P < .05). The NE was devoid of chronic pressor effects. In conclusion, 1) the increased slow pressor response to AII may not be a critical pathogenetic factor in the development of hypertension, as it also exists in normotensive LL rats; 2) LN and LL rats have the same normal BP despite marked differences in their RAS, thus suggesting that there could be several forms of normotension as known for hypertension; and 3) the simple comparison between one genetically hypertensive strain and one single normotensive control strain does not allow one to conclude that a phenotypic difference is of pathophysiological significance.

Polymorphisms in the carboxy-terminus of the epithelial sodium channel in rat models for hypertension.

To investigate whether mutations in the C-terminus of the three subunits of the rat epithelial sodium channel (alphabetagamma-rENaC) contribute to the hypertensive phenotype in five rat models for essential hypertension. We sequenced the C-terminal regions of alpha-, beta- and gamma-rENaC genes in five different hypertensive rat strains [spontaneously hypertensive rats (SHR), Dahl salt-sensitive (SS/Jr) rats, Milan hypertensive (MHS) rats, Sabra hypertensive (SBH) rats and Lyon hypertensive rats (LHR)] and their normotensive controls [Wistar-Kyoto (WKY) rats, Dahl salt-resistant (SR/Jr) rats, Milan normotensive (MNS) rats, Sabra normotensive (SBN) rats and Lyon normotensive rats (LNR)]. Identified polymorphisms were tested for cosegregation with blood pressure as well as for increased epithelial sodium channel (ENaC) activity. Genomic DNA extracted from hypertensive and normotensive rat strains was amplified by the polymerase chain reaction and polymerase chain reaction fragments were sequenced. Cosegregation analysis was performed to test for correlations between blood pressure and different genotypes. The effects of a polymorphism on ENaC activity were assessed by functional expression in Xenopus laevis oocytes. The chromosomal location of the gene for gamma-ENaC was determined by linkage analysis in an F2 (MHS x MNS) population. We found no polymorphisms at the C-terminus of alpha- and beta-rENaC in the five rat models tested. We identified two polymorphisms at the C-terminus of the gamma-subunit, one leading to an amino acid change. Milan strains (MNS and MHS) were polymorphic for this mutation. By cosegregation analysis we could exclude the possibility that there was a correlation between blood pressure and this polymorphism. Functional expression of the polymorphism caused no increase in ENaC activity assessed by measurement of the amiloride-sensitive sodium current in Xenopus oocytes. The gene for the gamma-ENAC was located on rat chromosome 1. No polymorphisms at the C-terminus of the three subunits of the epithelial sodium channel cosegregating with blood pressure were detected in five different genetic rat models for hypertension. If an altered ENaC activity contributes to the pathogenesis of hypertension in these rats, it must thus arise from mutations in other parts of the protein, from mutations outside the coding region impairing the proper regulation of one of the subunits or from mutations in an ENaC-associated protein.

Major cardiovascular risk factors in Lyon hypertensive rats. A correlation analysis in a segregating population

To investigate the association of blood pressure with the other major cardiovascular risk factors in a large population of back-cross to Lyon hypertensive (LH) rats. Mean arterial pressure was recorded in male freely moving Lyon normotensive (LN), LH, F1 and backcross to LH rats aged 30 weeks. Plasma cholesterol, triglycerides, insulin, creatinine, urea, fibrinogen and haematocrit levels, and the insulin: glucose ratio were measured in 31-week-old rats and 24h albuminuria in 6-week-old rats. Adult LH rats exhibited a significant increase in plasma lipids, insulin, fibrinogen, creatinine, urea and haematocrit levels compared with LN rats. In young LH rats, at an age at which blood pressure is slightly increased, albuminuria was increased to a greater extent than expected from their blood pressure levels. In the adult back-cross to LH rats, only the plasma cholesterol level was associated with blood pressure. Moreover, the plasma cholesterol level was related to fibrinogen and haematocrit levels. Finally, in the same rats, albuminuria developed early in life was positively related to hypercholinesterolaemia measured later in life. Plasma cholesterol, fibrinogen, haematocrit levels and early albuminuria could act synergistically in the enhancement or the development, or both, of vascular and kidney damage in the LH rat. Most interestingly, the association between plasma cholesterol level and blood pressure indicates that, as in essential hypertension, hypercholesterolaemia is a major phenotype associated with hypertension in the LH rat.

Left ventricular weight but not blood pressure is associated with sex chromosomes in Lyon rats

To investigate, by studying reciprocal back-crosses to Lyon hypertensive (LH) rats, the involvement of sex chromosome loci in high blood pressure and heart weight in LH rats. Reciprocal F1 and F'1 generation male rats obtained from male LH x female Lyon normotensive (LN) and male LN x female LH crosses respectively, male back-cross rats obtained from male LH x female F1 or F'1 and from male F1 or F'1 x female LH crosses, and parental LH and LN male rats were studied at 29-31 weeks of age. Systolic, diastolic, mean and pulse pressures were measured beat to beat in unrestrained rats for 1 h. In addition, relative left and right ventricular weights were measured. The average blood pressures did not differ between F1 and F'1 rats or between the four populations of back-cross rats. On the contrary, the relative left ventricular weight was higher in F'1 than in F1 rats. As a role for loci on the Y chromosome could be discarded by comparison of the two populations of back-cross rats, which differ only in the origin of their Y chromosome, this increase in the relative left ventricular weight of F'1 rats was consistent with an effect of a locus on the LH X chromosome. This was supported by findings in the back-cross populations, those populations in which 100% of the rats carried an LH X chromosome having a significantly higher left ventricular weight than those in which only 50% of the rats carried such a chromosome. The estimate of the genetic determination was higher for left ventricular weight (41%) than for mean arterial pressure (19%). Although these two parameters were associated in the back-cross population, the origin of the X chromosome had no influence on this association. These findings indicate that, against the genetic background provided by the cross studied and at the age studied, the sex chromosomes of LH and LN rats do not contain loci at which alleles differentially influence blood pressure. They do, however, suggest that relative left ventricular weight in this model has a specific genetic determination, partly contributed by a locus on the X chromosome.

High blood pressure and metabolic disorders are associated in the Lyon hypertensive rat

A large population of F2 rats, obtained from a cross between male Lyon hypertensive (LH) rats and female Lyon normotensive (LN) rats, was studied in order to assess the relationship between increased body weight, hyperlipidaemia and high blood pressure which characterize LH rats. Mean arterial pressure (MAP) was recorded in male, conscious, freely moving LH, LN, F1 and F2 rats aged 30 weeks. Plasma total cholesterol, high-density lipoprotein-, low-density lipoprotein- and very low-density lipoprotein-cholesterol, phospholipids, triglycerides, insulin and glucose were measured. In the F2 cohort it was observed that high MAP was a recessive trait that depends on several genes and was unrelated to body weight. The left ventricular weight, corrected for tibia length, was correlated with MAP. Plasma total and high-density lipoprotein-cholesterol and phospholipids concentrations were lower in the F1 rats than in the LN rats, suggesting an overdominance of the LN alleles. In the F2 rats MAP was related to total, high-density lipoprotein- and low-density lipoprotein-cholesterol. Plasma triglycerides, insulin and the insulin:glucose ratio, which were higher in the LH rats than in the LN rats, were also correlated with MAP in the F2 cohort. Using stepwise multiple regression analysis, MAP remained correlated with plasma total cholesterol, insulin and the insulin:glucose ratio, but not with triglycerides. Hypertension in LH rats is a recessive trait that is independent of body weight. In addition, the cosegregation of blood pressure with plasma cholesterol and, to a lesser degree, with insulin levels, which was observed in the present study provides the first direct evidence that these phenotypes are associated and are not due simply to genetic drift in the Lyon model.

Dietary Calcium, Vascular Reactivity, and Genetic Hypertension in the Lyon Rat Strain

In order to examine the relationship existing between dietary calcium and the development of hypertension, we developed a long-term study in the Lyon hypertensive rat strain (LH) and two control strains, the Lyon normotensive (LN) and low blood pressure rats (LL) given enriched (HCa, 2.5%), deprived (LCa, 0.03%) and normal (NCa, 0.6%) calcium diets. Evolution of body weight, systolic blood pressure (BP), plasma calcium and magnesium was monitored from 4 to 23 weeks of age. Total cardiovascular reactivity and contractile response of isolated aorta to norepinephrine were measured at 23 weeks of age. LH rats on HCa diet failed to develop hypertension (BP less than 150 mm Hg) whereas LH rats on LCa diet exhibited higher blood pressure levels than their controls fed the NCa diet. Moreover, in LN rats HCa diet slightly decreased BP whereas LCa had no effect. In the LL rats, on the contrary, only LCa diet slightly increased BP. In vivo responsiveness to NE was significantly higher in LH compared to LL and LN rats fed a NCa diet. HCa and LCa diets both induced a significant decrease in this response in LH rats. HCa diet increased the response in LN rats but decreased it in LL. In contrast, at the same age, the in vitro contractile response of isolated aorta to NE was significantly decreased in LH compared to LN and LL rats receiving NCa diet. Moreover in LH and LN rats on HCa diet the contractile response was markedly increased but no significant difference was observed with LCa diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific decrease of secretin/VIP-stimulated adenylate cyclase in the heart from the Lyon strain of hypertensive rats

The cardiac adenylate cyclase activity of genetically hypertensive rats from the Lyon strain (LH) was compared to that of Lyon normotensive rats (LN) and that of low blood pressure Lyon rats (LL). The major finding was a 30–35% decrease of secretin- and VIP-stimulated adenylate cyclase activity in cardiac membranes of LH rats that was already obvious in 5 week-old prehypertensive animals: this alteration was apparently specific for the cardiac secretin/VIP-stimulated adenylate cyclase activity, the same activity in membranes from brain, anterior pituitary, and liver being similar in LH, LN and LL rats. It is tempting to conclude that a selective alteration of functional cardiac secretin/VIP receptors in LH rats reflects a local hyperactivity of the sympathetic adrenergic system.