Ornella Russo

Genetic Variation in the Renin–Angiotensin System and Abdominal Adiposity in Men: The Olivetti Prospective Heart Study

Context Angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and angiotensin II receptor type I (AT2R1) are expressed in adipose tissue, but their role in obesity is unknown. Common polymorphisms involve the ACE gene on chromosome 17 (ACE I/D), the AGT gene on chromosome 1, and the AT2R1 receptor gene on chromosome 3. Contribution Among 959 adult Italian men, DD homozygosity in the ACE gene was associated with overweight (odds ratio, 1.82 [95% CI, 1.16 to 2.87]) and abdominal obesity (odds ratio, 1.76 [CI, 1.06 to 2.90]) compared with the genotypes I/D and II. It was also associated with increases in weight over 20 years. Polymorphisms of AGT and AT2R1 were unrelated to measures of body size. Implications These results suggest that the reninangiotensin system plays a role in the development of obesity. The Editors Human obesity is caused by the interaction of genetic predisposition and many environmental and lifestyle factors (1). Although the chromosomal location of a few putative major genes for human obesity has been identified (2-5), the presence of a greater number of minor genes involved in the process of adipogenesis or in the regulation of adipocyte metabolism probably engenders susceptibility to obesity (6). Polymorphism in several obesity candidate genes has been the subject of intensive investigation, but little attention has been paid to the genes encoding for components of the reninangiotensin system. The products of these genes (angiotensinogen [AGT], angiotensin-converting enzyme [ACE], and angiotensin II type 1 [AT2R1] and type II [AT2R2] receptors) are expressed in the adipose tissue in animal models as well as in humans (7-10). Recent experimental studies suggest that adipose tissue in the reninangiotensin system plays a role in adipocyte growth and differentiation through angiotensin II (11, 12). In addition, epidemiologic studies have reported associations between AGT plasma levels (13, 14), plasma renin activity (15, 16), plasma ACE activity (17), and body mass index (BMI). We investigated the relationship of overweight, obesity, and body fat distribution to three common polymorphisms of the reninangiotensin system: intron 16 of the ACE gene on chromosome 17 (18), the M235T polymorphism of the AGT gene in exon 2 on chromosome 1 (19), and the A-to-C polymorphism in the 3-untranslated region at nucleotide 1166 of the AT2R1 gene on chromosome 3 (20). Because the reninangiotensin system plays a fundamental role in blood pressure regulation and in vascular and cardiac modifications, these polymorphisms have been studied with regard to hypertension and cardiovascular disease. Associations have been reported between the AGT M235T and the AT2R1 A1166C variants and hypertension (19-24), as well as among ACE I/D polymorphism, insulin sensitivity (25-27), and the risk for coronary heart and cerebrovascular disease (28, 29). In adult men who attended the 19941995 follow-up examination of the Olivetti Prospective Heart Study, we examined associations between these polymorphic variants and overweight or obesity, body fat distribution, and related metabolic and hemodynamic variables. We also reported longitudinal findings for a subset of participants who were first examined in 1975 and had been followed for 20 years. Methods Study Sample and Procedures We used the DNA bank and the database of the Olivetti Prospective Heart Study, an epidemiologic investigation of cardiovascular risk factors in men working at the Olivetti factories in southern Italy. The procedures of the Olivetti Prospective Heart Study, which began in 1975, have been described in detail elsewhere (30). Between May 1994 and December 1995, we examined 1075 men 25 to 75 years of age. The participants were seen in the morning, after fasting, in a quiet room at the medical center of the Olivetti factories in Pozzuoli and Marcianise, suburbs of Naples, Italy. We obtained anthropometric measurements, performed blood tests, and administered a fixed-sequence questionnaire that assessed demographic information and medical history. Genotyping of the three polymorphisms of the reninangiotensin system was possible in 959 participants. A group of 457 men seen at the 19941995 follow-up visit of the Olivetti Prospective Heart Study had also been examined in 1975; of these, 143 reported being under dietary restriction for various reasons at follow-up examination. Since the Olivetti Prospective Heart Study aims to evaluate spontaneous changes in body mass and blood pressure, this subgroup was excluded from the main analysis. The local ethics committee approved the study protocol, and participants gave informed consent. Anthropometric Measurements Body weight and height were measured on a standard beam-balance scale with an attached ruler. Body weight was measured to the nearest 0.1 kg, and height was measured to the nearest 1 cm; participants wore light indoor clothing and no shoes. Body mass index was calculated as weight in kilograms divided by the square of the height in meters. At the 19941995 examination, but not at the 1975 examination, waist and arm circumferences were also measured. Waist circumference was measured at the umbilicus level as participants stood erect with abdomens relaxed, arms at their sides, and feet together. After the acromion was marked with each participants arm flexed at a 90-degree angle, arm circumference was measured at the midpoint between the acromion and the olecranon with the arm relaxed and hanging just away from the side of the body. The measurements were obtained to the nearest 0.1 cm with a flexible plastic measuring tape. Overweight was defined as a BMI greater than or equal to 27 kg/m2, obesity was defined as a BMI greater than or equal to 30 kg/m2, and abdominal adiposity was defined as a waist circumference greater than 1.00 m. Blood Pressure Measurement Blood pressure was measured after the participant had been sitting upright for at least 10 minutes. Systolic and diastolic (phase V) blood pressure was measured three 2 minutes apart with a random-zero sphygmomanometer (Gelman Hawksley Ltd., Sussex, United Kingdom). The first reading for each type of pressure was discarded, and the average of the second two readings was recorded. Hypertension was defined as a systolic blood pressure 140 mm Hg or greater, a diastolic blood pressure 90 mm Hg or greater, or both, or as current use of antihypertensive drugs. Biochemical Assays After blood pressure was measured, a fasting venous blood sample was taken in the seated position without stasis. The blood specimens were immediately subjected to centrifugation and stored at 70 C until analyzed. Glucose levels were measured by using an automated method (Cobas-Mira, Roche, Italy), and serum insulin concentration was measured by using radioimmunoassay (Insuline Lisophase, Technogenetics, Milan, Italy). Insulin resistance was estimated by homeostasis model assessment using the following formula, as described by Matthews and colleagues (31): fasting serum insulin level (U/mL) fasting serum glucose level (mmol/L)/22.5. Gene Polymorphisms in the ReninAngiotensin System Genomic DNA was isolated from leukocytes with a nonenzymatic, salting-out procedure (32). The ACE I/D polymorphism in intron 16 was typed by using the method of Rigat and associates (33). To address the possibility of mistyping ID heterozygotes as DD homozygotes because of the preferential amplification of the smaller D allele, all samples typed as DD homozygotes were subjected to a second, independent polymerase chain reaction with a primer pair that permits amplification only in the presence of the I allele; this was done by using the method described by Lindpaintner and coworkers (34). The T235 allele of the ATG gene was detected by using the method of Russ and colleagues (35), and the A1166C polymorphism of the AT2R1 gene was tested as described elsewhere (36). Allelic frequencies were estimated by using gene counting, and genotype distribution was tested for HardyWeinberg equilibrium by using chi-square analysis. Statistical Analysis Analysis of variance was used to evaluate differences in quantitative variables according to genotype; analysis of covariance was performed to account for confounders. A nonparametric test (KruskalWallis) was used for variables that were not normally distributed. The interaction between the effects of gene polymorphism and age on the anthropometric indexes and blood pressure was tested by using multiple linear regression analysis. The association of categorical variables with gene polymorphisms was tested by using logistic regression analysis and is expressed as odds ratios and 95% CIs. Results are expressed as the mean SD or as the mean SE, as specified. Two-sided P values and 95% CIs were used to test the statistical significance of between-group differences. Statistical analysis was performed by using SPSS statistical software, version 10.0 (SPSS, Inc., Chicago, Illinois). Role of the Funding Source The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Table 1 summarizes the main characteristics of the participants of the Olivetti Prospective Heart Study at the 19941995 examination. Nine hundred fifty-nine participants were tested for ACE, AGT, and AT2R1 polymorphism. For the ACE I/D polymorphism, 40% (n = 385) had the DD genotype, 45% (n = 431) had the ID genotype, and 15% (n = 143) had the II genotype. For the AGT polymorphism, 31% (n = 297) had the M235M genotype, 48% (n = 460) had the M235T genotype, and 21% (n = 202) had the T235T genotype. For the AT2R1 polymorphism, 54% (n = 518) had the A1166A genotype, 39% (n = 377) had the A1166C genotype, and 7% (n = 64) had the C1166C genotype. All three polymorphisms were in HardyWeinberg equilibrium, showing that the study sample excluded selection pressure for the genotypes under investigation. Table 1. Characteristics of

Abnormalities of renal sodium handling in the metabolic syndrome. Results of the Olivetti Heart Study.

Objective The mechanisms underlying high blood pressure in the framework of metabolic syndrome (MS) are not clarified: we thus analyzed the relationship of MS and its components to renal tubular sodium handling among participants of the Olivetti Heart Study, an epidemiological investigation of a representative sample of adult white male population in southern Italy. Methods Proximal (FPRNa) and distal (FDRNa) fractional sodium reabsorption were estimated by the clearance of exogenous lithium in 702 participants aged 25–75 years examined in 1994–1995. Blood pressure and relevant anthropometric and biochemical variables were also measured. The diagnosis of MS was based on modified National Cholesterol Education Program (NCEP)-Adult Treatment Panel III (ATP III) criteria. Results FPRNa, but not FDRNa, was directly associated with body mass index (BMI), waist circumference, diastolic pressure, serum triglyceride and uric acid, independently of age and of antihypertensive treatment. After adjustment for age, FPRNa, but not FDRNa, was significantly greater in individuals with MS, as compared to those without [77.6% (95% confidence interval = 76.7–80.1) versus 74.4% (73.7–75.1), P < 0.001]. A similar difference was observed after the exclusion of participants on current antihypertensive treatment (P = 0.018). In untreated individuals, a significant interaction was observed between obesity and insulin resistance as related to FPRNa (P = 0.002): the highest age-adjusted levels of FPRNa were detected in obese hypertensive and obese insulin-resistant participants. Conclusion In this sample of an adult male population, MS was associated with an increased rate of FPRNa. This finding is relevant to the pathophysiology of MS and possibly to the prevention of its cardiovascular and renal consequences.

Relationship of the Trp64Arg polymorphism of the beta3-adrenoceptor gene to central adiposity and high blood pressure : Interaction with age. Cross-sectional and longitudinal findings of the Olivetti Prospective Heart Study

Methods The association of the Trp64Arg polymorphism of the beta3-adrenoceptor (beta3-AR) gene with high blood pressure, central adiposity and other features of the metabolic syndrome was investigated in a large unselected sample of a white male working population in Southern Italy (n = 979). Results In the whole population, subjects heterozygous for the Trp64Arg mutation (11.2%) were not different from the homozygous Trp64Trp for any of the variables investigated. However, upon stratification for age, among men in the upper tertile of age (> 53 years), the Trp64Arg genotype was associated with higher waist : hip ratio (0.992 ± 0.021 versus 0.982 ± 0.037, P < 0.05), serum uric acid (6.34 ± 1.50 versus 5.75 ± 1.30 μmol/l, P < 0.05) and systolic blood pressure (144.3 ± 19.4 versus 136.9 ± 18.9 mmHg, P < 0.05) compared with the wild-type homozygotes. Accordingly, in the same age group, the carriers of Trp64Arg genotype were more often in the upper tertile of abdominal adiposity (69.7 versus 43.7%, P < 0.02) and serum uric acid (56.3 versus 34.8%, P < 0.02) and were more often hypertensive (68.6 versus 57.6%, P < 0.058) than the Trp64Trp homozygotes. No such differences were observed in younger age groups. No association was found with fasting serum insulin and the homeostasis model assessment (HOMA) index of insulin resistance. Furthermore, in a subgroup of 457 men for whom retrospective 20-year follow-up data were available, the variant genotype was associated with a higher probability of developing overweight (44.7 versus 27.0%, P < 0.05) and a trend to higher blood pressure (52.6 versus 38.4%, P = 0.09) over 20 years. Conclusion We conclude that the Trp64Arg variant of the beta3-AR receptor predicts a greater tendency to develop abdominal adiposity and high blood pressure with advancing age.

Circulating leptin levels predict the development of metabolic syndrome in middle-aged men: an 8-year follow-up study.

Background Because high circulating plasma leptin is associated with many features of the metabolic syndrome (MS), such as abdominal obesity, insulin resistance and high blood pressure (BP), we analysed the ability of plasma leptin concentration to predict the risk of developing MS in a prospective investigation of adult male participants of the Olivetti Heart Study (OHS). Methods and results Three hundred and sixty out of 907 men participating in the 1994–95 and 2002–04 OHS examinations (mean age at baseline 50.4 years, range 25–73 years) were free of MS at first visit according to NCEP-ATP III criteria (modified for the lack of high-density lipoprotein cholesterol measurement at baseline). During an average follow-up period of 8 years, there were 52 incident cases of MS (14.5%) due, in particular, to a rise in the prevalence of high BP (+42.4%), abdominal obesity (+16.4%) and impaired fasting glucose (IFG, +6.1%). In multivariate analyses, a one standard deviation difference in baseline plasma leptin concentration was associated with a 1.58-fold greater risk of developing MS (95% confidence interval = 1.10–2.30, P = 0.016) accounting for age, waist circumference, homeostatic assessment model index, smoking, alcohol consumption and physical activity. In particular, plasma leptin was positively associated with the risk of developing high BP (0.006) and IFG (0.014), after adjustment for confounders. Conclusion In this sample of an adult male population free of MS at baseline, circulating plasma leptin was a significant predictor of the risk of MS and, in particular, of its high BP and IFG components, independently of potential confounders.

High-Circulating Leptin Levels Are Associated with Greater Risk of Hypertension in Men Independently of Body Mass and Insulin Resistance: Results of an Eight-Year Follow-Up Study

BACKGROUND We previously reported a significant association between plasma leptin (LPT) concentration and blood pressure (BP), which was partly independent of serum insulin levels and insulin resistance. The aims of this study were to detect whether serum LPT levels predict the development of hypertension (HPT) in the 8-yr follow-up investigation of a sample of an adult male population (the Olivetti Heart Study), and to evaluate the role of body mass index (BMI) and insulin resistance in this putative association. PATIENTS AND METHODS The study population was made up of 489 untreated normotensive subjects examined in 1994-1995 (age: 50.1 +/- 6.7 yr; BMI: 26.3 +/- 2.8 kg/m(2); BP: 120 +/- 10/78 +/- 6 mm Hg; and homeostatic model assessment index: 2.1 +/- 1.6). RESULTS The HPT incidence over 8 yr was 35%. The participants with incident HPT had similar age but higher BMI (P < 0.001), serum LPT (P < 0.001), and BP (P < 0.01) at baseline. One sd positive difference in baseline serum LPT log was associated at univariate analysis with a 49% higher rate of HPT [95% confidence interval (CI) 22-83; P < 0.001]). In three different models of multivariable logistical regression analysis, LPT was respectively associated with a 41% greater risk to develop HPT (95% CI 15-74; P < 0.001) upon adjustment for age and baseline BP, with a 48% (95% CI 20-81) greater risk when adding the homeostatic assessment model index to the model, and with 33% greater risk (95% CI 6-67; P < 0.02) upon adjustment for BMI. CONCLUSIONS In this sample of originally normotensive men, circulating LPT level was a significant predictor of the risk to develop HPT over 8 yr, independently of BMI and insulin resistance.

Associations of selenium status with cardiometabolic risk factors: An 8-year follow-up analysis of the Olivetti Heart Study

OBJECTIVE High selenium status has been associated with adverse cardiometabolic outcomes in selenium-replete populations such as the US. In populations with lower selenium status such as in Italy, there is little epidemiological evidence about the association of selenium with cardiometabolic risk factors. We therefore examined cross-sectional and prospective relationships of serum selenium concentrations with cardiometabolic risk factors including blood pressure, diabetes and blood lipids in the Olivetti Heart Study. METHODS The study population consisted of 445 adult male individuals for whom baseline serum selenium measurement and cardiometabolic risk factors at baseline (1994-1995) and follow-up examination (2002-2004: average follow-up=8 years) were available. Serum selenium was measured by atomic absorption spectrophotometry. RESULTS Average serum selenium concentration at baseline was 77.5 ± 18.4 μg/L. In cross-sectional analyses, serum selenium levels were positively associated with serum total cholesterol (p for trend <0.0001) and prevalent diabetes (p for trend <0.05). In prospective analysis, serum selenium at baseline was likewise a strong predictor of serum total cholesterol (p=0.002) and LDL-cholesterol (p=0.001) at follow-up, after adjustment for age, BMI, cigarette smoking, physical activity, and lipid-lowering medication. These associations, however, were no longer significant after additional adjustment for baseline blood lipids. Selenium at baseline did not predict changes in total cholesterol levels between the baseline and follow-up examinations [β-coefficient (± SE)= 0.09 ± 0.12 (p=0.46)]. CONCLUSION These findings corroborate previous cross-sectional associations of high selenium status with adverse blood lipid profile and diabetes. However, prospective analyses do not support the causality of these relations. Randomized and experimental evidence is necessary to clarify the mechanisms underlying the observed cross-sectional associations.

Interaction between the C( 344)T polymorphism of CYP11B2 and age in the regulation of blood pressure and plasma aldosterone levels: cross-sectional and longitudinal findings of the Olivetti Prospective Heart Study

Objective To study the interaction between the C(−344)T CYP11B2 polymorphism and known determinants (age, body mass and dietary sodium) of blood pressure and plasma aldosterone. Design Cross-sectional and longitudinal (1980–1995) survey of male workers in southern Italy. Setting Medical centre of the Olivetti factories. Participants In 1995, the C(−344)T polymorphism was characterized in 811 untreated men. A subgroup of 280 participants already seen in 1980 was the object of longitudinal analysis. Main outcome measures Blood pressure, demographic, anthropometric and biochemical variables (serum and urinary electrolytes and plasma aldosterone) and frequency of the C(−344)T polymorphism. Results In the whole population, there was no difference among genotypes for any of the variables examined. However, multiple regression showed a significant interaction between age (but not body mass or sodium intake) and genotype with regard to systolic (P = 0.03) and diastolic (P = 0.02) pressure variability independently of covariates. Diastolic pressure increased linearly with age in carriers of the T allele (TT, P < 0.001 and TC, P = 0.005), but not in CC homozygotes (P = 0.848). In T carriers – but not in CC homozygotes – blood pressure and serum potassium increased and plasma aldosterone and serum sodium decreased across quintiles of age (P < 0.001 for all trends). In the longitudinal study, diastolic pressure increased significantly over time only in T carriers (TC+TT: +2.6 ± 0.6, versus CC: −0.4 ± 1.5 mmHg, P = 0.04). Conclusion Inter-individual variation of blood pressure and plasma aldosterone is affected by the interaction of CYP11B2 C(−344)T polymorphism and ageing, thus supporting a role for this variant in mechanisms affecting blood pressure regulation.

α-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption

Genetic variation in alpha-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of alpha-adducin in controlling Na/K pump activity, wild-type or mutated rat and human alpha-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, alpha-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.

Combination of Renin-Angiotensin System Polymorphisms Is Associated With Altered Renal Sodium Handling and Hypertension

Abstract—Genes of the renin-angiotensin–aldosterone system (RAAS) are natural candidates for sodium homeostasis and blood pressure regulation. To investigate the effect of a combination of polymorphisms of RAAS genes on renal sodium handling and blood pressure, 918 participants to the Olivetti Heart Study were genotyped for the following polymorphisms: I/D of angiotensin converting enzyme (ACE), M235T of angiotensinogen (AGT), A1166C of angiotensin II type-1 receptor (AT1R), and C-344T of aldosterone synthase (CYP11B2). The segmental renal sodium handling was evaluated by the fractional excretions of exogenous lithium (FE-Li), uric acid (FE-UA), and sodium (FE-Na). Twenty-eight carriers of triple homozygosity for M (AGT), A (AT1R), and C (CYP11B2) in the presence of the D allele of ACE (DD/ID) showed lower FE-Li (20.0%±5.9% versus 25.0%±7.5%; P =0.004; mean±sD), FE-UA (6.3%±2.0% versus 8.2%±2.7%; P =0.001), and FE-Na (0.96%±0.41% versus 1.22%±0.61%; P =0.004) as compared with all other allelic combinations (n=890), independently from age and body mass, suggesting an enhanced rate of proximal tubular sodium reabsorption. The carriers of the MM, AA, CC, DD/ID combination showed a substantially higher probability of being hypertensive (OR: 3.4 [(99% CI: 1.1 to 10.1]), independently of age and body mass. This relatively rare combination of allelic variants of candidate genes of the RAAS is associated with a significant alteration in proximal renal sodium handling and with higher risk of hypertension, suggesting that a combination of polymorphic variants at different candidate loci may affect phenotypic expression even in the absence of detectable effects of each variant at any single locus.

Excess dietary sodium and inadequate potassium intake in Italy: Results of the MINISAL study

OBJECTIVE As excess sodium and inadequate potassium intake are causally related to hypertension and cardiovascular disease, the MINISAL-GIRCSI Program aimed to provide reliable estimates of dietary sodium and potassium intake in representative samples of the Italian population. DESIGN AND METHODS Random samples of adult population were collected from 12 Italian regions, including 1168 men and 1112 women aged 35-79 yrs. Electrolyte intake was estimated from 24 hour urine collections and creatinine was measured to estimate the accuracy of the collection. Anthropometric indices were measured with standardised procedures. RESULTS The average sodium excretion was 189 mmol (or 10.9 g of salt/day) among men and 147 mmol (or 8.5 g) among women (range 27-472 and 36-471 mmol, respectively). Ninety-seven % of men and 87% of women had a consumption higher than the WHO recommended target of 5g/day. The 24 h average potassium excretion was 63 and 55 mmol, respectively (range 17-171 and 20-126 mmol), 96% of men and 99% of women having an intake lower than 100 mmol/day (European and American guideline recommendation). The mean sodium/potassium ratio was 3.1 and 2.8 respectively, i.e. over threefold greater than the desirable level of 0.85. The highest sodium intake was observed in Southern regions. Sodium and potassium excretion were both progressively higher the higher the BMI (p < 0.0001). CONCLUSIONS These MINISAL preliminary results indicate that in all the Italian regions thus far surveyed dietary sodium intake was largely higher and potassium intake lower than the recommended intakes. They also highlight the critical association between overweight and excess salt intake.