Research topics

1.Genetic analysis of spontaneous hypertension.
2. Genetic analysis of other cardiovascular risk factors including dyslipidemia and insulin resistance.

Most significant results

Genetic isolation of a chromosome 4 region associated with hypertension, insulin resistance, and dyslipidemia in the spontaneously hypertensive rat: effects of CD36 deficiency

Clustering of metabolic cardiovascular risk factors is often observed in patients with essential hypertension. The spontaneously hypertensive rat (SHR) is the most widely studied model of essential hypertension that exhibits a number of abnormalities in carbohydrate and lipid metabolism. Recent linkage studies have suggested that quantitative trait loci (QTL) affecting blood pressure and resistance of isolated adipocytes to insulin and catecholamine action may be located on a single region of chromosome 4. To test this hypothesis and to investigate whether these QTL affect also systemic insulin resistance and dyslipidemia, we developed a new congenic strain by transferring a corresponding segment of chromosome 4 from the Brown Norway rat onto the SHR genetic background. The congenic strain (SHR-4) had significantly (p<0.05) decreased systolic blood pressure (SBP) (by telemetry), decreased insulin resistance, serum triglycerides, and free fatty acids.

Recently, this SHR-chromosome 4 congenic strain has been used to make a significant breakthrough in dissecting the genetic basis of impaired carbohydrate and lipid metabolism in the SHR. Fluorescently labeled cDNA probe, synthesized from poly(A) mRNA extracted from adipose tissue of SHR, BN and SHRchromosome 4 congenic rats was applied to microarray chips that contained approximately 10 000 individual rat cDNAs. Clones encoding rat CD36 showed reduced hybridization signals (greater than 90 %) for SHR compared to either BN or SHR-chromosome 4. Using RTPCR, the entire CD36 coding sequence of SHR, WKY and BN rats was determined and it was found that SHR cDNA contains multiple sequence variants, eleven of which predict amino acid changes. Seven of these mutations cluster in exon 6. Furthermore, the entire 3´-untranslated end of SHR CD36 was totally different when compared with BN or WKY rats. The CD36 was mapped on chromosome 4 by linkage analysis using a HinfI RFLP in the CD36 sequence. This RFLP revealed an unexpected band in WKY and BN rats which looked like heterozygotes. This finding, together with the large number of sequence variants detected in two closely related strains as SHR and WKY, suggested the possibility of a genomic duplication of CD36 in WKY and BN and of deletion in SHR. This hypothesis was confirmed by Southern blot analysis. Using probes from the region of exon 6 and from WKY 3´-untranslated end of CD36, only a single restriction fragment was observed in SHR genomic DNA. Additional bands in BN and WKY DNA nocfirmed the presence of at least one further copy of the CD36 gene in these strains.

To determine whether the SHR transcript results in production of a functional, mature protein, Western-blot analysis was performed on SHR microsomal pellets that were prepared to include the plasma membrane fraction in which CD36 normally resides. Plasma membranes from WKY and BN adipose tissue contained a substantial quantity of CD36, whereas no CD36 could be detected in SHR. A similar result was obtained in heart tissue. CD36 encodes a key protein involved in the cellular transport of fatty acids. Abnormalities in fatty acid metabolism have long been recognized as potential determinants of insulin resistance. Taken together, these findings provided an indirect evidence that CD36 is one of the major genes contributing to disordered carbohydrate and lipid metabolism in the SHR.

Transgenic rescue of defective CD36 ameliorates insulin resistance and circulating fatty acid levels in spontaneously hypertensive rats

To test the hypothesis that CD36 is one of the major genes contributing to disordered carbohydrate and lipid metabolism in the SHR, we used complementation analysis in novel SHR transgenic lines, SHR/Ola-TgN(EF1aCD36)10Ipcv and SHR/Ola-TgN(EF1aCD36)19Ipcv (hereafter referred to as SHR-TG10 and SHR-TG19) to directly investigate the role of mutant CD36 in the pathogenesis of hypertension and defective glucose and fatty acid metabolism. Transgenic expression of wild type CD36 in SHR harboring mutant CD36 induced significant alterations in fatty acid metabolism that were associated with improved glucose tolerance and insulin sensitivity. In male rats fed a high carbohydrate fructose diet, circulating levels of fatty acids in SHR-TG10 and SHR-TG19 expressing wildtype CD36 were significantly lower than in progenitor SHR harboring only mutant CD36 (p<0.05). In oral glucose tolerance tests, areas under the curves in both SHR-TG lines were significantly lower than in the SHR progenitor (p<0.001). Circulating levels of insulin were not significantly different among the strains. Insulin stimulated incorporation of glucose into diaphragmatic muscle was significantly greater in the two transgenic lines than in the SHR progenitor strain (p<0.01). Although the two transgenic lines showed comparable improvements in glucose tolerance and similar changes in fatty acid levels, only one of the lines showed reduction in blood pressure (p<0.005 by radiotelemetry). In all the tissues tested, expression of the CD36 transgene was greatest in the line that showed a reduction in blood pressure. The difference in transgene expression between the two lines was particularly striking in the kidney, with hypertension being attenuated in SHR-TG19 in which renal expression of CD36 was markedly increased. Thus, based on complementation analysis in two transgenic lines expressing wild-type CD36 on the genetic background of an SHR strain harboring the deletion variant of CD36, it can be concluded that defective CD36 can be a determinant of disordered fatty acid metabolism, glucose intolerance, and insulin resistance. In addition, hypertension was attenuated in the SHR transgenic line in which renal expression of CD36 was significantly increased. This raises the possibility that CD36 may act as a quantitative trait locus that regulates blood pressure by expression inside the kidney. Finally, the successful derivation of SHR-TG10 and SHR-TG19 should provide unique opportunities for investigating the mechanisms that influence risk factor clustering and susceptibility to target organ damage in hypertension (Pravenec et al., Nature Genetics 27:156-158, 2001). 

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