Department of Medicine, University of Essen, Essen, Germany
Correspondence and offprint requests to: Dr Martin C. Michel, Nephrology Laboratory IG1, Klinikum, Hufelandstr. 55, D-45122 Essen, Germany.
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Abstract |
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Methods. Genomic DNA was isolated from 147 hypertensive patients and digested with DraI. Genotypes at the 2A-adrenoceptor were identified by restriction fragment length polymorphism. Genotype at each locus was related to blood pressure, family history of hypertension and various clinical chemistry parameters.
Results. The 2A-adrenoceptor polymorphism was not significantly associated with blood pressure or a family history of hypertension. Patients with the d allele of the
2A-adrenoceptor had significantly lower HbA1 (5.6 vs 6.2%, P=0.0344) and HbA1c (3.4 vs 3.9%, P=0.0237) and total cholesterol (212 vs 229 mg/dl, P=0.0333) than those without. Similar trends, which failed to reach statistical significance, were seen for glucose, triglycerides and LDL cholesterol.
Conclusions. We propose that alleles at the 2A-adrenoceptor locus might contribute to interindividual differences in the regulation of human lipid and glucose metabolism.
Keywords: 2A-adrenoceptor; cholesterol; gene polymorphism; HbA1
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Introduction |
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During recent years, we have been interested in hereditary effects on 2-adrenoceptor expression which may relate to the pathophysiology of hypertension and the frequently associated metabolic syndrome [7]. While studies comparing platelet
2-adrenoceptor density between normotensive and hypertensive subjects have yielded conflicting results [8], three studies consistently have reported that subjects with a family history of hypertension have higher platelet
2-adrenoceptor expression than those without; this was found in both normotensive and hypertensive subjects and could already be detected in children [911]. In this respect, a `gene doseresponse curve' may exist, as children with two essential hypertensive parents have more platelet
2-adrenoceptors than those with one hypertensive parent who in turn have more than those with two normotensive parents [11]. Moreover, the variance of platelet
2-adrenoceptor density in children with one hypertensive parent is greater than in children with two normotensive parents [10], as would be expected since only half of these children should have inherited the respective genes. A genetic component of
2-adrenoceptor regulation has also been suggested in rat studies on renal
2-adrenoceptor expression (for review, see [8]). Thus, the expression of
2-adrenoceptors clearly involves a hereditary component and this may relate to the pathogenesis of essential hypertension.
Three subtypes of 2-adrenoceptors exist in man which are encoded by distinct genes located on human chromosomes 10, 2 and 4, and are designated
2A,
2B and
2C, respectively [12]. The above studies on a hereditary component in the regulation of human
2-adrenoceptors at the protein level have been performed using platelets, which express a homogeneous population of
2A-adrenoceptors [1214]. A cDNA encoding the human platelet
2A-adrenoceptor was cloned originally by Kobilka et al. [15]. The corresponding gene is located in the region q24q26 of human chromosome 10 [12] and does not contain introns [16]. Using a 5.5 kb fragment of the
2A-adrenoceptor gene as the probe, two alleles of the human
2A-adrenoceptor gene can be distinguished by restriction fragment length polymorphism (RFLP) upon digestion of genomic DNA by the endonuclease DraI, while a range of 47 other restriction enzymes did not detect polymorphic restriction fragments [17]. The frequencies of the two alleles appear to be consistent across various Caucasian populations from the US [1719] and Australia [20], while African Americans appear to have a different allele distribution [18,19].
Therefore, the present study was designed to answer two questions: firstly, are alleles at the 2A-adrenoceptor locus as detected by DraI RFLP associated with a family history of hypertension? Second, do such alleles correlate with parameters which are regulated physiologically by
2A-adrenoceptors, e.g. those of lipid and glucose metabolism? Thus, we have determined the genotype at the
2A-adrenoceptor locus in 147 subjects and tested for a possible association of the alleles at this locus with a family history of hypertension and with several metabolic parameters. These subjects were a subgroup of the participants of the HANE study, a multicentre intervention trial on the treatment of essential hypertension [21]. Thus, our study is based solely on hypertensive subjects. This is justified because several previous studies have demonstrated that family history is a better predictor of the hereditary component of
2A-adrenoceptor expression than the absence or presence of hypertension [911].
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Subjects and methods |
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2A-Adrenoceptor genotyping
Genomic DNA was isolated from 4 ml of EDTA-anticoagulated blood, which had been stored at -70°C, using an extraction procedure with phenol and chloroform/isoamylalcohol (24:1). Samples were stored at 4°C until enzymatic digestion. Approximately 1.5 µg of DNA were digested with the restriction enzyme DraI according to the enzyme manufacturer's instructions. The digests were separated on 0.6% agarose gels, and transferred onto nylon membranes according to Southern [23].
The 2A-adrenoceptor gene locus probe was prepared using random primer labelling [24] from the 5.5 kb genomic DNA fragment isolated by Kobilka et al. [15] which contains the whole 1350 bp coding region and 5'- and 3'-untranslated regions.
Non-specific DNA-binding sites on the nylon membrane were blocked by pre-hybridization for 2 h at 65°C in pre-hybridization buffer [4x SSPE (0.72 M NaCl, 40 mM NaH2PO4, 4 mM Na2EDTA), 6% polyethylenglycol 20 000, 0.5% SDS, 100 µg/ml salmon sperm DNA, 2x Denhardt solution (0.2 mg/ml Ficoll 400, 0.2 mg/ml polyvinylpyrrolidone, 0.2 mg/ml bovine serum albumin fraction V)]. Following short boiling, aliquots of the labelled probe (~106 c.p.m./ml of pre-hybridization buffer) were added to the nylon membranes. Hybridization was performed under constant agitation at 65°C overnight. Thereafter, the blots were washed using solutions of decreasing ionic strength starting with four times for 5 min with 2x SSC buffer at room temperature and a final stringency of 10 min with 1x SSC buffer at 65°C with 0.1% SDS being added to the SSC buffer. Thereafter, the blots were wrapped in foil and used for exposure of autoradiographic films for 35 days at -80°C.
Clinical chemistry
From each patient, a blood sample was obtained after an overnight fast. Plasma renin activity was determined in our laboratory using a commerically available radioimmunoassay (Sorin, Saluggia, Italy). The following serum parameters were determined in the laboratory of Drs W. Eicke and L. Röcker (Berlin, Germany): Na+, K+, Ca2+, Mg2+, inorganic phosphate, creatinine, uric acid, glucose, HbA1, HbA1c, total cholesterol, HDL cholesterol, triglycerides, GPT and alkaline phosphatase. Serum LDL cholesterol was calculated using the equation LDL cholesterol=total cholesterolHDL cholesterol(triglycerides/5).
For technical reasons, the measurement of serum parameters was done in two batches of similar size ~1 year apart. The mean values were similar between both batches for most parameters, but a statistically significant difference existed for HbA1 (7.2±0.2 vs 5.0±0.1%, P <0.0001), HbA1c (4.4±0.1 vs 3.2±0.1%, P <0.0001) and HDL (49.7±2.3 vs 44.0±1.7 mg/dl, P=0.0450). The frequency of genotypes at the 2A-adrenoceptor locus was similar in both batches (data not shown).
Chemicals
Proteinase K, Klenow enzyme and DraI were obtained from Boehringer Mannheim (Mannheim, Germany), Biodyne A nylon membranes from Pall Corp. (Portsmouth, UK), agarose from Bethesda Research Laboratories (Bethdesda, MD), hexanucleotides from Pharmacia Biotec (Freiburg, Germany) and [32P]dCTP from New England Nuclear (Dreieich, Germany).
Data analysis
A small number of patients were excluded from the analysis since their data indicated protocol violations, e.g. total cholesterol of >500 mg/dl or fasting glucose of >180 mg/dl. The statistical significance of differences between patients of differing genotypes was tested using Fisher's exact test for family history and unpaired two-tailed t-tests for the other parameters. All statistical calculations were performed using the InStat program (GraphPAD Software, San Diego, CA), and P <0.05 was considered significant. Corrections for multiple comparisons were not performed, and the resulting P-values are to be interpreted in a descriptive manner. All data are the mean±SEM of n subjects.
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Results |
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Among the parameters of lipid metabolism patients with a d allele of the 2A-adrenoceptor had significantly less total cholesterol than those without (212±8 vs 229±4 mg/dl, P=0.0333; Table 2
). Similarly, triglycerides and LDL were an average of 10 mg/dl and 13 mg/dl, respectively, lower in patients with a d allele than in those without, but these differences did not reach statistical significance (Table 2
), while HDL was similar in both genotypes (44.6±2.3 vs 47.5±1.8 mg/dl, P=0.3763). A batch-specific analysis of HDL levels did not reveal consistent differences between genotypes at any of the receptor loci (data not shown).
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Discussion |
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The observed frequencies of the two alleles at the 2A-adrenoceptor gene locus in the present study were very similar to those in three independent Caucasian populations from the US [1719] and one from Australia [20], indicating that a possible founder effect did not markedly affect allele distribution in our study. Since
-adrenoceptors may be involved in the hereditary component of essential hypertension [7], several groups of investigators have determined whether alleles of the human
2A-adrenoceptor gene as detected upon DraI digestion of genomic DNA might be associated with essential hypertension. While a significant association between genotype at the DraI site and hypertension in Caucasians from the US was reported in one study [19], two studies with Caucasian populations from Australia [20] and the US [18] have not detected significant differences of allele frequencies at this locus between normotensive and hypertensive subjects. Similarly, the present study has not detected an association of genotype at this locus and blood pressure within a hypertensive population based on Caucasian subjects from Austria, Germany and Switzerland. An altered frequency of the d allele was reported in hypertensive relative to normotensive African Americans in one [18] but not in another study [19]. Therefore, the results regarding an association between genotype at the
2A-adrenoceptor locus and hypertension are not conclusive at the moment, but the majority of studies in Caucasians including the present one have not detected such an association.
The above conflicting data are not very surprising since studies on altered platelet 2A-adrenoceptor protein expression in hypertensive patients have also yielded controversial results [8]. On the other hand, an association between family history of hypertension and increased platelet
2A-adrenoceptors has been reported consistently in normotensive and hypertensive populations [911]. Therefore, we have studied whether the genotypes at the
2A-adrenoceptor locus are associated with an altered prevalence of a family history of hypertension. However, we have not detected such an association, which is in line with two studies in Caucasian populations from Australia [20] and the US [18], which have investigated the combined effect of hypertension and family history thereof. Therefore, it appears that a family history of hypertension may be associated with increased platelet
2A-adrenoceptor expression but this is not reflected by the genotype defined by the DraI polymorphism.
Irrespective of a possible association of the 2A-adrenoceptor gene polymorphism with hypertension or a family history thereof, it may be clinically relevant. Thus, subjects with the d allele were reported to have an enhanced platelet aggregation in response to adrenaline, an increased heart rate response to lower body negative pressure, and a decreased sodium excretion in response to immersion in thermally neutral water [22], findings which are consistent with an enhanced
2A-adrenoceptor function. These data clearly indicate cardiovascular and renal consequences of the
2A-adrenoceptor gene polymorphism.
Our data indicate that HbA1 and HbA1c as indicators of glucose metabolism and total cholesterol as an indicator of lipid metabolism are significantly lower in patients with the d allele. Other parameters of glucose and lipid metabolism (glucose, LDL cholesterol, triglycerides) were also lower in such patients but these differences failed to reach statistical significance. It may be argued that the detection of a statistically significant association of genotype at the 2A-adrenoceptor locus with three parameters may not be surprising in light of the number of parameters which were investigated. While this possibility cannot be excluded, we nevertheless consider the observed associations to be specific for several reasons. Firstly, the same significant association was seen consistently within both batches of patient samples. Second, a trend in the same direction was observed for other parameters of glucose and lipid metabolism, but did not reach statistical significance with the given number of subjects. Third, significant genotypephenotype associations were observed only for parameters which are regulated by
2A-adrenoceptors, while parameters which are considered to be unrelated to
2-adrenoceptors (e.g. uric acid, electrolytes and alkaline phosphatase) were not consistently different between the two groups. Thus, functional studies have shown that inhibition of lipolysis in human adipocytes [26] and inhibition of insulin release in mice [27] and rats [28] occur via an
2A-adrenoceptor. The
2A-adrenoceptor also is the dominant
2-adrenoceptor subtype in the human pancreas at the mRNA level [29]. Fourth, the observed assocations were not found in our population with polymorphisms of the AT1 angiotensin receptor (unpublished observations) or the Y1 neuropeptide Y receptor [30], although the latter is involved in the regulation of lipid and glucose metabolism in similar ways as the
2A-adrenoceptor [31]. Thus, we propose that alleles at the human
2A-adrenoceptor gene locus are associated specifically with alterations in parameters of lipid and glucose metabolism and might possibly play a modulatory role in the syndrome of insulin resistance. It might be argued that the observed differences between parameters in patients with and without the d allele are too small to be relevant. However, we feel that the observed size of the differences makes them more plausible as all of the above parameters are fine-tuned by a multitude of regulatory pathways. A very large alteration in any one of these parameters would indicate that
2A-adrenoceptors play a dominant role in its regulation, which is unlikely for all of the parameters investigated here.
The association of the polymorphism with enhanced platelet responsiveness to adrenaline [22] indicates that it may lead to enhanced 2A-adrenoceptor function. Since
2-adrenoceptor stimulation inhibits pancreatic insulin release [27,28], this should result in worsened carbohydrate metabolism, while the opposite was observed in the present study. Moreover, the d allele of the
2A-adrenoceptor is associated with enhanced platelet aggregation [22] and decreased cholesterol (present study), while previous studies have suggested that high cholesterol is associated with enhanced platelet aggregation in response to
2-adrenoceptor stimulation [32]. Elucidation of these apparent discrepancies will require further studies.
The associations between genotype at the 2A- adrenoceptor locus and the various cardiovascular, renal and metabolic parameters could have two causes: firstly, it is possible that the
2A-adrenoceptor gene locus is in linkage disequilibrium with a nearby locus involved in lipid and glucose metabolism. Secondly, it is possible that the alleles of the
2A-adrenoceptor gene somehow are related to the function of this receptor and thus indirectly might affect lipid and glucose metabolism. Although we have no definitive proof of this, we consider the first possibility unlikely for several reasons. Numerous gene alterations resulting in a disturbed lipid and/or glucose metabolism have been identified [33] but none of these loci is close to the
2A-adrenoceptor locus on chromosome 10 q24q26. Moreover, these alterations are usually associated with large changes in plasma lipids, and our study design has systematically excluded patients with clearly pathological plasma lipids or glucose levels. However, mutations in such genes with smaller effects on lipid or glucose metabolism might have contributed to the background `noise' in our population.
In conclusion, we have confirmed that two major genotypes of the human 2A-adrenoceptor gene can be detected using RFLP based on the DraI endonuclease. Our data and the majority of the published data indicate that these genotypes are not associated with alterations of blood pressure or family history of hypertension in Caucasian subjects. On the other hand, they are associated with alterations of cardiovascular and renal function [22] and alterations of some parameters of lipid and glucose metabolism which are known to be regulated by
2A-adrenoceptors. While the molecular basis of the associations remains to be studied, receptor polymorphisms might be important not only pathophysiologically; they might also contribute to interindividual differences in the response to therapeutically administered
2-adrenoceptor agonists and antagonists.
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Acknowledgments |
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Notes |
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References |
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