Aldosterone Is Not Associated With Metabolic and Microvascular Insulin Sensitivity in Abdominally Obese Men

Aldosterone Is Not Associated With Metabolic and Microvascular Insulin Sensitivity in Abdominally... Abstract Context Impaired insulin-mediated muscle microvascular recruitment (IMMR) may add to the development of insulin resistance and hypertension. Increased aldosterone levels have been linked to these obesity-related complications in severely to morbidly obese individuals and to impaired microvascular function in experimental studies. Objectives To investigate whether aldosterone levels are associated with IMMR, insulin sensitivity, and blood pressure in lean and moderately abdominally obese men, and to study the effect of weight loss. Design, Setting, Participants, Intervention, Main Outcome Measures In 25 lean and 53 abdominally obese men, 24-hour blood pressure measurement was performed, and aldosterone levels were measured using ultra-performance liquid chromatography tandem mass spectrometry. Insulin sensitivity was assessed by determining whole-body glucose disposal during a hyperinsulinemic clamp. IMMR in forearm skeletal muscle was measured with contrast-enhanced ultrasonography. These assessments were repeated in the abdominally obese men following an 8-week weight loss or weight stable period. Results Sodium excretion and aldosterone levels were similar in lean and abdominally obese participants, but sodium excretion was inversely associated with aldosterone concentration only in the lean individuals [lean, β/100 mmol sodium excretion (adjusted for age and urinary potassium excretion) = −0.481 (95% confidence interval, −0.949 to −0.013); abdominally obese, β/100 mmol sodium excretion = −0.081 (95% confidence interval, −0.433 to 0.271); P for interaction = 0.02]. Aldosterone was not associated with IMMR, insulin sensitivity, or blood pressure and was unaffected by weight loss. Conclusion In moderately abdominally obese men, the inverse relationship between sodium excretion and aldosterone concentration is less than that in lean men but does not translate into higher aldosterone levels. The absolute aldosterone level does not explain differences in microvascular and metabolic insulin sensitivity and blood pressure between lean and moderately abdominally obese men. Obesity is accompanied by impaired insulin-mediated microvascular dilatation (1, 2). A reduced ability of insulin to dilate microvessels can add to the development of obesity-associated hypertension by increasing peripheral vascular resistance (3) and to the development of metabolic insulin resistance by impeding insulin-stimulated glucose disposal (4–7). In contrast, weight loss has been associated with amelioration of microvascular dysfunction, including the microvascular response to insulin (5, 8–10), and the improvement of skeletal muscle microvascular function was an independent determinant of the increase in insulin-induced glucose uptake (5, 9). The molecular basis of these phenomena has not been fully elucidated. Over the years, evidence has accumulated suggesting involvement of aldosterone in the pathogenesis of obesity-related hypertension and insulin resistance. Higher aldosterone levels have been observed in severely to morbidly obese individuals, in parallel with higher blood pressure (11–13), which might be a consequence of aldosterone synthesis in visceral adipose tissue (12, 14). In addition, aldosterone has been reported to correlate with insulin resistance in normotensive, overweight individuals (15) and to predict for the development of insulin resistance in a general population (16). In contrast, the aldosterone concentration has been found to decrease with weight loss in severely to morbidly obese (11–13, 17) and hypertensive obese individuals (18, 19), and this was accompanied, not only by reductions in blood pressure (11–13, 17–19), but also by improvement in insulin sensitivity (12). Several experimental studies have shown that aldosterone interferes with microvascular function (20–22) and, more specifically, vascular insulin signaling (23), and mineralocorticoid receptor blockade improved insulin-mediated aortic dilatation in female mice fed a Western diet (24) and coronary microvascular function in individuals with type 2 diabetes (25). However, mediators of the effect of excess weight on (risk factors for) cardiovascular disease are not necessarily similar in severely to morbidly obese and overweight to moderately obese individuals, which might also apply to aldosterone (26). Whether aldosterone levels are increased in a less advanced stage of obesity and whether they are associated with impaired insulin-mediated muscle microvascular dilatation and, thus, reduced insulin-stimulated glucose uptake and higher blood pressure in humans has not been studied. Given the previously reported association of visceral obesity with elevated plasma aldosterone concentration (12), we hypothesized that increased aldosterone levels in abdominally, but not morbidly, obese men would contribute to the development of microvascular and, therefore, metabolic insulin resistance and higher blood pressure. In addition, we expected these abnormalities to be reversible by weight loss. Therefore, the aims of the present study were to assess the association of aldosterone levels with insulin-mediated muscle microvascular recruitment (IMMR), whole-body glucose disposal, and blood pressure in abdominally obese, compared with lean men, and to determine the effect of weight loss on these variables in the abdominally obese men. Materials and Methods Study population Apparently healthy men were recruited via advertisements in local newspapers or among participants of previous investigations. Ultimately, 53 abdominally obese and 25 lean white men were enrolled in our randomized controlled trial with blinded analyses. Participants were aged 18 to 65 years, nonsmoking, nondiabetic, and free of cardiovascular disease and had a waist circumference <94 cm (lean) or 102 to 110 cm (abdominally obese) and had had a stable body weight for ≥3 months. The exclusion criteria were fasting plasma glucose >7.0 mmol/L, hemoglobin A1c >6.5%, serum total cholesterol >8.0 mmol/L, serum triglycerides >4.5 mmol/L, systolic blood pressure (SBP) >160 mm Hg, and the use of medication affecting the blood pressure, lipid profile, or glucose metabolism. All participants gave written informed consent. The local ethics committee approved the study, which was performed in accordance with the Declaration of Helsinki and registered at ClinicalTrials.gov (NCT01675401). Study design Abdominally obese men were randomly assigned in a 1:1 ratio to either an 8-week weight loss program or maintenance of their habitual diet and were studied before and after the 8-week period. Lean men were studied at baseline only. Randomization was performed by an independent investigator using block randomization with variable block sizes and stratification for age <50 and >50 years, because the effect of weight loss on microvascular and metabolic insulin sensitivity and blood pressure could differ with age. An independent investigator revealed the allocation to the participant and research team on completion of all baseline measurements. The weight loss program was designed to induce a ∼10% reduction in body weight and consisted of 4 to 5 weeks of a very low calorie diet providing 2.1 MJ/day (Modifast; Novartis Nutrition, Breda, The Netherlands), 1 to 2 weeks of an energy-restricted diet providing 4.2 MJ/day, a weight stable phase of 2 weeks, and weekly dietary counseling. During the 8-week period, the control group was also monitored to avoid fluctuations in weight. Both groups were instructed not to alter their exercise pattern throughout the study. At baseline (both lean and abdominally obese men) and after the 8-week period (abdominally obese men only), 24-hour urine samples were collected for assessment of sodium, potassium, and creatinine excretion, and 24-hour ambulatory blood pressure measurements were performed (Mobilograph; New Generation, I.E.M., Stolberg, Germany). Blood pressure was measured on the nondominant arm every 15 minutes during the day and every 30 minutes during the night. The measurements were conducted in a temperature-controlled room (24° ± 0.5°C) after a 12-hour overnight fast with the participants in the supine position. The men were instructed to refrain from alcohol and meals rich in lipids for a period of 24 hours before each study day and to refrain from strenuous physical exercise for a period of 48 hours before each study day. After insertion of two intravenous catheters and a 30-minute acclimatization period with the participants in the supine position, blood samples were taken for determination of glucose, hemoglobin A1c, overall lipid profile, and aldosterone concentrations, and the microvascular blood volume of the forearm skeletal muscle was measured at baseline. Assessment of insulin sensitivity We determined metabolic insulin sensitivity using a modified version of the hyperinsulinemic, euglycemic clamp technique, as described by DeFronzo et al. (27). In brief, insulin (Novorapid; Novo Nordisk, Bagsvaerd, Denmark) was administered in a primed continuous manner at a rate of 1 mU/kg/min for 180 minutes. Isoglycemia was maintained by adjusting the rate of a 20% d-glucose infusion based on the plasma glucose measurements performed at 5-minute intervals. Whole-body glucose disposal was estimated from the steady-state glucose infusion rate 90 to 150 minutes after initiation of insulin administration. The whole-body glucose disposal is expressed per kilogram body weight per unit of plasma insulin concentration (M/I value), thus correcting for variations in the steady-state insulin concentrations. For convenience, the M/I ratio was multiplied by 100. Evaluation of skeletal muscle microvascular function IMMR was assessed using contrast-enhanced ultrasonography, as described previously (5). In brief, the microvascular blood volume of forearm skeletal muscle was measured before and during hyperinsulinemia using a Toshiba Aplio XG ultrasound system (Toshiba, Otawara, Japan) during continuous intravenous administration of sulfur hexafluoride gas-filled microbubbles (SonoVue; Bracco Diagnostics, Amsterdam, The Netherlands). After a steady-state microbubble concentration was achieved (3 minutes), five real-time replenishment curves of 30 seconds were acquired. These replenishment curves were stored and analyzed offline in a blinded fashion after completion of the trial using the CHI-Q software (Toshiba). The replenishment curves were fitted to the exponential function [y = A(1 − e−βt)], where t is the time since the high mechanical index pulse, y is the video intensity at any given t, A is the plateau video intensity (representing the microvascular blood volume), and β is the microvascular flow velocity. IMMR was calculated as the relative increase in muscle microvascular blood volume during hyperinsulinemia. Measurement of subcutaneous and visceral fat volumes Subcutaneous and visceral fat volumes were measured using magnetic resonance imaging, as previously described (5). Blood and urine measurements Plasma glucose was determined using a YSI2300 glucose analyzer (YSI, Yellow Springs, OH). The serum insulin levels during the hyperinsulinemic clamp were measured using Mercodia Iso-Insulin enzyme-linked immunosorbent assay [Mercodia AB, Uppsala, Sweden; intra-assay coefficient of variation (CV), 2.8% to 3.2%; interassay CV, 3% to 3.9%]. Serum aldosterone was analyzed using ultra-performance liquid chromatography tandem mass spectrometry, as described previously (28), with minor modifications. In brief, 30 µL of 0.14 µmol/L D7-aldosterone in 50/50 methanol/water (volume-to-volume ratio; %) was added to 300 µL of serum. The samples were mixed and subsequently deproteinized with acetone. After centrifugation (10 minutes, 25°C, 14000 rpm), the supernatant was extracted with two volumes of 800 µL tert-butyl methyl ether at room temperature. The phases were separated by centrifugation (1 minute, 25°C, 4600 rpm), and the upper organic phase was transferred to a 4-mL glass vial and dried under nitrogen at 35°C. The dried residue was dissolved in 100 µL 35/65 methanol/water (volume-to-volume ratio; %). Finally, 10 µL was injected into the ultra-performance liquid chromatography tandem mass spectrometry for analysis. The intra-assay CVs ranged from 4.0% to 8.7% and the interassay CVs from 4.5% to 10.4%. The urinary sodium and potassium excretion were determined using the ion-selective electrode method. Statistical analysis Normally distributed variables are expressed as the mean ± standard deviation. The variables with a skewed distribution are displayed as the median and interquartile range, and natural logarithmic transformation was performed before further analyses (M/I value and aldosterone). Independent sample t tests were used to compare the groups at baseline and the differences in the anthropometric, metabolic, hemodynamic, and hormonal variables between the abdominally obese men after the weight loss or weight stable period. The aldosterone concentration in the lean and abdominally obese men was compared using analysis of covariance and in abdominally obese individuals before and after the weight loss or weight stable period by repeated measures analysis of covariance, with adjustment for age, mean arterial pressure (MAP) (in the comparison between the lean and abdominally obese men), and urinary sodium and potassium excretion. The relationships of, and changes in, the aldosterone levels with the (alterations in) IMMR, M/I value, and 24-hour ambulatory blood pressure were assessed using multiple linear regression with adjustment for age and differences in urinary sodium and potassium excretion. The associations of, and changes in, aldosterone concentration with the (alterations in) other anthropometric, metabolic, and hemodynamic variables are presented as Pearson correlation coefficients. Analyses were performed using the SPSS statistical software package, SPSS Statistics, version 20 (IBM, Armonk, NY). Two-tailed P values of < 0.05 were considered statistically significant. Results Cross-sectional analyses One of the 53 abdominally obese participants was excluded because of protocol violations. Data on urinary sodium and potassium excretion were available for 21 lean men and 46 abdominally obese men. The baseline characteristics of the lean and abdominally obese men are presented in Table 1. The lean men had significantly lower subcutaneous and visceral adipose tissue volumes, fasting plasma glucose levels, and SBP and diastolic blood pressure (DBP) compared with the abdominally obese men. The aldosterone levels, however, were similar in both groups. Urinary sodium excretion was consistent with the average salt intake of Dutch men (29). Table 1. Baseline Characteristics of Lean and Abdominally Obese Men Characteristic  Lean Men  Abdominally Obese Men  Participants, n  25  52  Age, y  54 (25–62)  52 (46–61)  BMI, kg/m2  23.3 ± 1.8a  30.1 ± 2.1  Waist circumference, cm  85 ± 6a  107 ± 4  Subcutaneous adipose tissue, L  1.45 ± 0.51a  3.06 ± 0.77  Visceral adipose tissue, L  0.89 ± 0.42a  2.36 ± 0.72  Fasting plasma glucose, mmol/L  5.35 ± 0.29a  5.64 ± 0.48  Hemoglobin, A1c, %  5.2 ± 0.4  5.3 ± 0.4  24-h SBP/DBP, mm Hg  118 ± 9/73 ± 9a  123 ± 9/80 ± 7  24-h MAP, mm Hg  93 ± 8a  100 ± 7  24-h Pulse pressure, mm Hg  45 ± 8  43 ± 7  Aldosterone, pmol/L  274 (178–512)  278 (169–392)  Urinary sodium excretion, mmol/24 h  153 ± 73  172 ± 64  Urinary potassium excretion, mmol/24 h  81 ± 30  83 ± 23  Urinary creatinine excretion, mmol/24 h  14 ± 3a  18 ± 3  Characteristic  Lean Men  Abdominally Obese Men  Participants, n  25  52  Age, y  54 (25–62)  52 (46–61)  BMI, kg/m2  23.3 ± 1.8a  30.1 ± 2.1  Waist circumference, cm  85 ± 6a  107 ± 4  Subcutaneous adipose tissue, L  1.45 ± 0.51a  3.06 ± 0.77  Visceral adipose tissue, L  0.89 ± 0.42a  2.36 ± 0.72  Fasting plasma glucose, mmol/L  5.35 ± 0.29a  5.64 ± 0.48  Hemoglobin, A1c, %  5.2 ± 0.4  5.3 ± 0.4  24-h SBP/DBP, mm Hg  118 ± 9/73 ± 9a  123 ± 9/80 ± 7  24-h MAP, mm Hg  93 ± 8a  100 ± 7  24-h Pulse pressure, mm Hg  45 ± 8  43 ± 7  Aldosterone, pmol/L  274 (178–512)  278 (169–392)  Urinary sodium excretion, mmol/24 h  153 ± 73  172 ± 64  Urinary potassium excretion, mmol/24 h  81 ± 30  83 ± 23  Urinary creatinine excretion, mmol/24 h  14 ± 3a  18 ± 3  Data presented as the mean ± standard deviation or median (interquartile range). a Lean vs abdominally obese, P ≤ 0.01. View Large Adjustment for age, MAP, and urinary sodium and potassium excretion yielded similar results with regard to aldosterone concentrations in the lean and abdominally obese participants (F1,61 = 0.026; P = 0.87; Fig. 1). Both the M/I value and the IMMR were higher in the lean than in the abdominally obese men (Table 2). The aldosterone levels were not associated with the IMMR, M/I value, or 24-hour SBP [natural logarithm (ln) IMMR, β = −1.713, P = 0.82; ln M/I value, β = −0.131, P = 0.29; SBP, β = −2.533, P = 0.14], which remained unchanged after adjustment for age and urinary sodium and potassium excretion (ln IMMR, β = −0.715, P = 0.93; ln M/I value, β = −0.100, P = 0.46; SBP, β = −1.815, P = 0.34). Supplemental Table 1 shows the correlations of aldosterone with the other anthropometric, metabolic, and hemodynamic variables. Aldosterone was not associated with either body mass index (BMI) or subcutaneous and visceral adipose tissue volumes but was inversely associated with DBP (r = −0.241, P = 0.04), which became nonsignificant after adjustment for confounders and for the 24-hour MAP (data not shown). These associations were similar in the lean and abdominally obese men (P for interaction all > 0.14), except for the associations with urinary sodium excretion. Figure 1. View largeDownload slide Baseline aldosterone levels in lean and obese individuals (analysis of covariance with adjustment for age, MAP, and urinary sodium and potassium excretion). Figure 1. View largeDownload slide Baseline aldosterone levels in lean and obese individuals (analysis of covariance with adjustment for age, MAP, and urinary sodium and potassium excretion). Table 2. Microvascular and Metabolic Insulin Sensitivity in Lean vs Abdominally Obese Men Variable  Lean Men  Abdominally Obese Men  Participants, n  25  52  M value, mg/kg/min  6.8 ± 1.8a  4.1 ± 1.3  M/I value, (mg/kg/min per mU/L) × 100  9.9 (6.7–12.1)a  4.4 (2.9–5.5)  IMMR, %  44 ± 41a  −3.5 ± 27  Variable  Lean Men  Abdominally Obese Men  Participants, n  25  52  M value, mg/kg/min  6.8 ± 1.8a  4.1 ± 1.3  M/I value, (mg/kg/min per mU/L) × 100  9.9 (6.7–12.1)a  4.4 (2.9–5.5)  IMMR, %  44 ± 41a  −3.5 ± 27  Data presented as the mean ± standard deviation or median (interquartile range). Abbreviation: M value, whole body glucose disposal. a Lean vs abdominally obese, P ≤ 0.01. View Large Urinary sodium excretion was inversely associated with aldosterone levels only in the lean men (β per 100 mmol sodium excretion [adjusted for age and urinary potassium excretion] = −0.481 [95% confidence interval (CI), −0.949 to −0.013], P < 0.05; i.e., for each 100 mmol greater sodium excretion, the aldosterone levels were lower by 38% [95% CI, 1% to 61%]) and not in the abdominally obese men [β per 100 mmol sodium excretion (adjusted for age and urinary potassium excretion) = −0.081 (95% CI, −0.433 to 0.271), P = 0.65; i.e., for each 100 mmol greater sodium excretion, the aldosterone levels were lower by 8% (95% CI, −31 to 35); P for interaction = 0.02; Fig. 2]. Figure 2. View largeDownload slide Association of 24-hour urinary sodium excretion with aldosterone levels in lean (white circles) and abdominally obese (black circles) individuals. Regression coefficients adjusted for age and urinary potassium excretion. Figure 2. View largeDownload slide Association of 24-hour urinary sodium excretion with aldosterone levels in lean (white circles) and abdominally obese (black circles) individuals. Regression coefficients adjusted for age and urinary potassium excretion. Adjustment of the associations of aldosterone levels with IMMR, M/I value, and 24-hour SBP for urinary sodium excretion did not materially change the regression coefficients (Supplemental Table 2). Effects of weight loss Of the 53 abdominally obese men, 26 were allocated to the weight loss intervention and 27 to the weight stable (control) group. Two men discontinued the weight loss intervention, one because of noncompliance, and one for personal reasons; a third man who had been allocated to the weight loss program was excluded from the analyses because of protocol violations, leaving 23 men in the weight loss group. One man in the control group did not complete the study for a non–study-related reason. Ultimately, analyses were performed for 23 men in the weight loss group and 26 men in the weight stable group. Data on urinary sodium and potassium excretion at follow-up were available for 19 participants randomized to the weight loss intervention and 24 participants in the control group. The anthropometric, metabolic, hemodynamic, and hormonal variables at baseline and the effects of weight loss on these variables are shown in Table 3. Both groups were comparable with regard to baseline characteristics, with the exception of SBP, which was lower in the weight loss group (120 ± 9 vs 126 ± 8 mm Hg; P = 0.03). Table 3. Anthropometric, Metabolic, Hemodynamic, and Hormonal Variables in Abdominally Obese Men and Effects of Weight Loss Variable  Weight Loss Group (n = 23)   Weight Stable Group (n = 26)   Baseline  Δ  Baseline  Δ  Age, y  52 (47–62)  NA  52 (45–61)  NA  BMI, kg/m2  30.2 ± 1.5  −3.0 ± 0.8b  29.9 ± 2.5  0.1 ± 0.33  Waist circumference, cm  107 ± 3  −11 ± 2b  106 ± 4  0 ± 2  Subcutaneous adipose tissue, L  2.81 ± 0.65a  −0.34 ± 0.44  3.26 ± 0.78  −0.36 ± 0.53  Visceral adipose tissue, L  2.53 ± 0.78  −0.33 ± 0.49  2.19 ± 0.68  −0.29 ± 0.55  Fasting plasma glucose, mmol/L  5.49 ± 0.37  −0.21 ± 0.33c  5.75 ± 0.53  0.00 ± 0.28  Hemoglobin A1c, %  5.2 ± 0.3  −0.2 ± 0.3c  5.3 ± 0.4  −0.1 ± 0.2  M value, mg/kg/min  4.1 ± 1.3  1.3 ± 1.2b  4.0 ± 1.4  −0.1 ± 0.9  M/I value, (mg/kg/min per mU/L) × 100  3.4 (2.5–5.3)  2.6 ± 2.7b  4.8 (2.7–6.2)  −1.0 ± 2.4  IMMR, %  −5.0 ± 27  40 ± 49b  0.7 ± 28  −0.3 ± 28  24-h SBP/DBP, mm Hg  120 ± 9a/78 ± 8  −5±6b/−5 ± 5b  126 ± 8/82 ± 7  −1±6/−1 ± 5  24-h MAP, mm Hg  98 ± 8a  −5±5b  102 ± 7  −1±5  24-h Pulse pressure, mm Hg  42 ± 7  0 ± 3  44 ± 5  0 ± 4  Aldosterone, pmol/L  306 (187–394)  −61 ± 126  253 (156–422)  −30 ± 141  Urinary sodium excretion, mmol/24 h  163 ± 62  19 ± 80  183 ± 65  0.1 ± 87  Urinary potassium excretion, mmol/24 h  83 ± 20  −4.9 ± 24  85 ± 25  −5.3 ± 38  Urinary creatinine excretion, mmol/24 h  18 ± 3  0.4 ± 6.3  18 ± 4  −1.9 ± 3.6  Variable  Weight Loss Group (n = 23)   Weight Stable Group (n = 26)   Baseline  Δ  Baseline  Δ  Age, y  52 (47–62)  NA  52 (45–61)  NA  BMI, kg/m2  30.2 ± 1.5  −3.0 ± 0.8b  29.9 ± 2.5  0.1 ± 0.33  Waist circumference, cm  107 ± 3  −11 ± 2b  106 ± 4  0 ± 2  Subcutaneous adipose tissue, L  2.81 ± 0.65a  −0.34 ± 0.44  3.26 ± 0.78  −0.36 ± 0.53  Visceral adipose tissue, L  2.53 ± 0.78  −0.33 ± 0.49  2.19 ± 0.68  −0.29 ± 0.55  Fasting plasma glucose, mmol/L  5.49 ± 0.37  −0.21 ± 0.33c  5.75 ± 0.53  0.00 ± 0.28  Hemoglobin A1c, %  5.2 ± 0.3  −0.2 ± 0.3c  5.3 ± 0.4  −0.1 ± 0.2  M value, mg/kg/min  4.1 ± 1.3  1.3 ± 1.2b  4.0 ± 1.4  −0.1 ± 0.9  M/I value, (mg/kg/min per mU/L) × 100  3.4 (2.5–5.3)  2.6 ± 2.7b  4.8 (2.7–6.2)  −1.0 ± 2.4  IMMR, %  −5.0 ± 27  40 ± 49b  0.7 ± 28  −0.3 ± 28  24-h SBP/DBP, mm Hg  120 ± 9a/78 ± 8  −5±6b/−5 ± 5b  126 ± 8/82 ± 7  −1±6/−1 ± 5  24-h MAP, mm Hg  98 ± 8a  −5±5b  102 ± 7  −1±5  24-h Pulse pressure, mm Hg  42 ± 7  0 ± 3  44 ± 5  0 ± 4  Aldosterone, pmol/L  306 (187–394)  −61 ± 126  253 (156–422)  −30 ± 141  Urinary sodium excretion, mmol/24 h  163 ± 62  19 ± 80  183 ± 65  0.1 ± 87  Urinary potassium excretion, mmol/24 h  83 ± 20  −4.9 ± 24  85 ± 25  −5.3 ± 38  Urinary creatinine excretion, mmol/24 h  18 ± 3  0.4 ± 6.3  18 ± 4  −1.9 ± 3.6  Data are presented as means ± standard deviation or median (interquartile range). Abbreviations: M value, whole-body glucose disposal; NA, not applicable. a Weight loss group at baseline vs weight stable group at baseline, P < 0.05. b Change in weight loss group vs change in weight stable group, P ≤ 0.01. c Change in weight loss group vs change in weight stable group, P < 0.05. View Large In the control group, the waist circumference, M/I value, IMMR, and 24-hour ambulatory blood pressure were unchanged over time. In the weight loss group, the waist circumference decreased significantly by 11 ± 2.1 cm (P < 0.01), the M/I value increased by 2.6 ± 2.7 (mg/kg/min per mU/L) × 100 (P < 0.01), and IMMR increased by 40% ± 49% (P < 0.01). The SBP, DBP, and MAP decreased significantly (SBP, −5 ± 6 mm Hg, P < 0.01; DBP, −5 ± 5 mm Hg, P < 0.01; MAP, −5 ± 5 mm Hg, P < 0.01), but the pulse pressure was unaffected. The aldosterone levels remained unchanged as well. The absence of an effect of weight loss on aldosterone concentration was confirmed after adjustment for age and urinary sodium and potassium excretion at the follow-up assessments (F1,38 = 0.824, P = 0.37). Changes in the aldosterone concentration after weight loss were not related to alterations in IMMR, M/I value, or 24-hour SBP (∆IMMR, β = −0.020, P = 0.65; ∆M/I value, β = −0.001, P = 0.72; ∆24-hour SBP, β = 0.002, P = 0.73). The regression coefficients remained unchanged after adjustment for potential confounders (Fig. 3). The associations of the changes in aldosterone concentration with alterations in the other anthropometric, metabolic, and hemodynamic variables resulting from the weight loss intervention are shown in Supplemental Table 3. These associations were similar in the weight loss and weight stable groups (P for interaction > 0.11 for all). Figure 3. View largeDownload slide Associations of changes in aldosterone levels (∆aldosterone) after the weight loss (white squares) or weight stable period (black squares) with (A) alterations in IMMR (∆ IMMR), (B) alterations in M/I value (∆ M/I value), and (C) alterations in 24-hour SBP (∆ SBP). Regression coefficients adjusted for age and differences in urinary sodium and potassium excretion. Figure 3. View largeDownload slide Associations of changes in aldosterone levels (∆aldosterone) after the weight loss (white squares) or weight stable period (black squares) with (A) alterations in IMMR (∆ IMMR), (B) alterations in M/I value (∆ M/I value), and (C) alterations in 24-hour SBP (∆ SBP). Regression coefficients adjusted for age and differences in urinary sodium and potassium excretion. Additional analyses When we excluded participants with a BMI of 25 to 30 kg/m2 and thus men who would be classified as overweight on the basis of their BMI (n = 29), the comparison of aldosterone levels between the lean and obese men yielded similar results: BMI ≤25 kg/m2 (n = 21), 257 pmol/L (95% CI, 178 to 565), BMI ≥30 kg/m2, 288 pmol/L (95% CI, 174 to 394); P = 0.50. In the present study, the variation in ad libitum sodium intake was relatively large. To investigate whether the response of aldosterone levels to controlled variation in sodium ingestion in lean and abdominally obese men is comparable to the cross-sectional associations between ad libitum sodium intake and the corresponding aldosterone concentration in the present population (described previously), we determined the relationship between 24-hour urinary sodium excretion and aldosterone levels after a low-sodium diet (50 mmol sodium chloride/24 hours) and high-sodium diet (250 mmol sodium chloride/24 hours) for 7 days in randomized order in 20 lean and 20 abdominally obese individuals in a separate data set (the characteristics of this study population are presented in Supplemental Table 4). After the low- and high-sodium diets, the aldosterone levels were not statistically significantly different between the lean and abdominally obese individuals. However, multivariate regression analyses adjusted for age, sex, and urinary potassium excretion showed that for each 100-mmol increase in urinary sodium excretion, aldosterone levels decreased more in the lean than in the abdominally obese participants (i.e., by 140 pmol/L; 95% CI, 289 to −10; P = 0.07 in lean individuals and by 52 pmol/L, 95% CI, 237 to −133; P = 0.56 in abdominally obese individuals; P for interaction = 0.07; Supplemental Fig. 1). Discussion The present study has shown that serum aldosterone levels are not higher in abdominally obese men, compared with lean men, under circumstances of ad libitum sodium intake and are not associated with microvascular and metabolic insulin sensitivity or blood pressure. Moreover, the serum aldosterone concentration was unaffected by a weight loss intervention in abdominally obese men, although we observed a relevant decline in both insulin resistance and blood pressure. Several previous investigations have reported elevated aldosterone levels in parallel with higher blood pressure in severely to morbidly obese participants (11–13). The excess aldosterone in these individuals could be at least partially derived from the visceral adipose tissue (12, 14). Therefore, in our study, in moderately abdominally obese men, the capacity of adipose tissue to produce aldosterone might have been insufficient to enable detection of elevations in aldosterone levels. Experimental studies have shown that aldosterone can impair microvascular function and vascular insulin signaling (20–23), which might predispose to the development of obesity-associated insulin resistance and hypertension (1–3, 7). Nevertheless, we could not establish an association of aldosterone levels with IMMR in skeletal muscle, insulin stimulated-glucose disposal, or blood pressure. This again could be explained by the absence of differences in the aldosterone concentration between the lean and abdominally obese men and, thus, the limited dispersion in aldosterone levels. It is also possible that the increase in aldosterone levels reported in severely to morbidly obese individuals reflects either an epiphenomenon that occurs later in the course of obesity and is not causally related to the development of insulin resistance and hypertension or is a consequence of renal microvascular dysfunction and subsequent renin–angiotensin–aldosterone system activation. However, observations of impaired insulin sensitivity in parallel with substantially elevated blood pressure in individuals with endogenously high exposure to aldosterone, such as patients with primary aldosteronism, which are both ameliorated by pharmacological or surgical treatment (30), suggest otherwise. In addition, the endothelial function in these individuals is more impaired than in patients with essential hypertension of equal severity (31). Therefore, our results do not preclude an effect of aldosterone on microvascular and, thus, metabolic insulin sensitivity and blood pressure but suggest that such an effect might become more prominent with an increasing severity of obesity and thus a greater extent of aldosterone excess. This also indicates that in the current stage of obesity, mechanisms other than aldosterone surplus must be responsible for the increase in blood pressure. Our data do not imply that regulation of aldosterone levels is completely normal in individuals with moderate abdominal obesity, although this does not immediately seem to affect IMMR, whole-body glucose disposal, and blood pressure. We found that the physiological inverse association of urinary sodium excretion with aldosterone levels was stronger in lean than abdominally obese individuals, pointing to a potential derangement in the abdominally obese group that might eventually add to the already existing increase in blood pressure. This was confirmed by our data from a separate experiment showing that after 7 days of low- and high-salt intake in randomized order, suppression of aldosterone levels by increasing sodium intake was impaired in the abdominally obese participants, consistent with previous findings (32). These observations are relevant given that aldosterone seems to be particularly detrimental to the endothelium in the presence of high sodium intake (33). Moreover, aldosterone production was found to correlate with insulin resistance in lean and overweight, normotensive individuals consuming a high-salt diet (15). With these observations, we cannot exclude that the absence of an association of higher aldosterone levels with microvascular and metabolic insulin resistance and higher blood pressure in the present investigation can be partially explained by a sodium intake in accordance with, and not greater than, the average ingestion of Dutch men (29). In contrast to our findings, other investigators have reported that weight loss in severely to morbidly obese individuals and hypertensive obese individuals is paralleled by reductions in aldosterone concentrations (11–13, 17–19), together with a decline in blood pressure (11–13, 17–19) and amelioration of insulin resistance (12). Although in the present study, insulin sensitivity was improved and blood pressure decreased substantially in the abdominally obese men randomized to the weight loss intervention compared with the control group, the aldosterone levels remained unchanged. Again, this might have been because our abdominally obese participants would be categorized as overweight to moderately obese instead of severely obese on the basis of their BMI, which might entail a limited ability of aldosterone overproduction, as outlined previously. One limitation of the present investigation was the use of an exclusively male study population, which affects the generalizability of the results. That the sodium and potassium intake was not standardized and was estimated from single 24-hour urine collections is another limitation. Theoretically, this could have led us to underestimate the true aldosterone levels in the abdominally obese individuals. This was, however, unlikely because, in a separate experiment, after 7 days of standardized low and high sodium intake, the aldosterone levels were also not statistically significantly different between the lean and abdominally obese participants. Nevertheless, the inverse association between urinary sodium excretion and serum aldosterone levels was less pronounced in the abdominally obese than in the lean participants in both the observational (Fig. 2) and the experimental (Supplemental Figure 1) portions of the present study. This suggests a subtle abnormality in the control of aldosterone concentration by sodium intake which, in this stage of obesity, does not affect the absolute aldosterone levels. However, it does not detract from our findings that the aldosterone levels, whether or not considering its regulation by sodium ingestion, were not substantially associated with microvascular or metabolic insulin sensitivity or blood pressure in either lean or moderately abdominally obese men. An important strength of our investigation was its design, which allowed us to study the association between aldosterone and obesity-related complications, including microvascular and metabolic insulin resistance and hypertension, in both a cross-sectional and longitudinal manner. In addition, we used the reference standard for assessment of insulin sensitivity and were able to measure microvascular function in skeletal muscle, which is the main peripheral location of insulin-mediated glucose uptake, thus allowing a thorough evaluation of the relationship between microvascular and metabolic insulin sensitivity. In conclusion, the results of the present study suggest that at an early stage of abdominal obesity, the absolute aldosterone levels are not elevated and do not contribute to the development of microvascular and metabolic insulin resistance and hypertension. However, the inverse relationship between sodium excretion and aldosterone concentration is less than that in lean men, although this does not immediately affect IMMR, insulin-stimulated glucose disposal, and blood pressure. It is possible that the role of aldosterone in this respect becomes more prominent with an increasing severity of obesity. Future research should be directed toward investigating the association of aldosterone with microvascular and metabolic insulin sensitivity and blood pressure in a study population with a broader range of abdominal obesity and one that also includes women. Moreover, it would be valuable to study the effect of mineralocorticoid receptor blockade on vascular and metabolic insulin signaling and blood pressure in these individuals as an alternative to weight loss for the amelioration of insulin resistance and hypertension, which is often difficult to achieve and sustain. Abbreviations: BMI body mass index CI confidence interval CV coefficient of variation DBP diastolic blood pressure IMMR insulin-mediated muscle microvascular recruitment ln natural logarithm MAP mean arterial pressure M/I value whole-body glucose disposal per kilogram body weight per unit of plasma insulin concentration SBP systolic blood pressure. Acknowledgments Financial Support: This work was supported by research grant CH001 from the Top Institute Food and Nutrition, a public–private partnership on precompetitive research in food and nutrition. The public partners were responsible for the study design, data collection and analysis, decision to publish, and preparation of the manuscript. Clinical Trial Information: ClinicalTrials.gov no. NCT01675401 (registered 19 February 2014). Disclosure Summary: The authors have nothing to disclose. References 1. de Jongh RT, Serné EH, IJzerman RG, de Vries G, Stehouwer CD. 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Endocrine Society
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Copyright © 2018 Endocrine Society
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Abstract

Abstract Context Impaired insulin-mediated muscle microvascular recruitment (IMMR) may add to the development of insulin resistance and hypertension. Increased aldosterone levels have been linked to these obesity-related complications in severely to morbidly obese individuals and to impaired microvascular function in experimental studies. Objectives To investigate whether aldosterone levels are associated with IMMR, insulin sensitivity, and blood pressure in lean and moderately abdominally obese men, and to study the effect of weight loss. Design, Setting, Participants, Intervention, Main Outcome Measures In 25 lean and 53 abdominally obese men, 24-hour blood pressure measurement was performed, and aldosterone levels were measured using ultra-performance liquid chromatography tandem mass spectrometry. Insulin sensitivity was assessed by determining whole-body glucose disposal during a hyperinsulinemic clamp. IMMR in forearm skeletal muscle was measured with contrast-enhanced ultrasonography. These assessments were repeated in the abdominally obese men following an 8-week weight loss or weight stable period. Results Sodium excretion and aldosterone levels were similar in lean and abdominally obese participants, but sodium excretion was inversely associated with aldosterone concentration only in the lean individuals [lean, β/100 mmol sodium excretion (adjusted for age and urinary potassium excretion) = −0.481 (95% confidence interval, −0.949 to −0.013); abdominally obese, β/100 mmol sodium excretion = −0.081 (95% confidence interval, −0.433 to 0.271); P for interaction = 0.02]. Aldosterone was not associated with IMMR, insulin sensitivity, or blood pressure and was unaffected by weight loss. Conclusion In moderately abdominally obese men, the inverse relationship between sodium excretion and aldosterone concentration is less than that in lean men but does not translate into higher aldosterone levels. The absolute aldosterone level does not explain differences in microvascular and metabolic insulin sensitivity and blood pressure between lean and moderately abdominally obese men. Obesity is accompanied by impaired insulin-mediated microvascular dilatation (1, 2). A reduced ability of insulin to dilate microvessels can add to the development of obesity-associated hypertension by increasing peripheral vascular resistance (3) and to the development of metabolic insulin resistance by impeding insulin-stimulated glucose disposal (4–7). In contrast, weight loss has been associated with amelioration of microvascular dysfunction, including the microvascular response to insulin (5, 8–10), and the improvement of skeletal muscle microvascular function was an independent determinant of the increase in insulin-induced glucose uptake (5, 9). The molecular basis of these phenomena has not been fully elucidated. Over the years, evidence has accumulated suggesting involvement of aldosterone in the pathogenesis of obesity-related hypertension and insulin resistance. Higher aldosterone levels have been observed in severely to morbidly obese individuals, in parallel with higher blood pressure (11–13), which might be a consequence of aldosterone synthesis in visceral adipose tissue (12, 14). In addition, aldosterone has been reported to correlate with insulin resistance in normotensive, overweight individuals (15) and to predict for the development of insulin resistance in a general population (16). In contrast, the aldosterone concentration has been found to decrease with weight loss in severely to morbidly obese (11–13, 17) and hypertensive obese individuals (18, 19), and this was accompanied, not only by reductions in blood pressure (11–13, 17–19), but also by improvement in insulin sensitivity (12). Several experimental studies have shown that aldosterone interferes with microvascular function (20–22) and, more specifically, vascular insulin signaling (23), and mineralocorticoid receptor blockade improved insulin-mediated aortic dilatation in female mice fed a Western diet (24) and coronary microvascular function in individuals with type 2 diabetes (25). However, mediators of the effect of excess weight on (risk factors for) cardiovascular disease are not necessarily similar in severely to morbidly obese and overweight to moderately obese individuals, which might also apply to aldosterone (26). Whether aldosterone levels are increased in a less advanced stage of obesity and whether they are associated with impaired insulin-mediated muscle microvascular dilatation and, thus, reduced insulin-stimulated glucose uptake and higher blood pressure in humans has not been studied. Given the previously reported association of visceral obesity with elevated plasma aldosterone concentration (12), we hypothesized that increased aldosterone levels in abdominally, but not morbidly, obese men would contribute to the development of microvascular and, therefore, metabolic insulin resistance and higher blood pressure. In addition, we expected these abnormalities to be reversible by weight loss. Therefore, the aims of the present study were to assess the association of aldosterone levels with insulin-mediated muscle microvascular recruitment (IMMR), whole-body glucose disposal, and blood pressure in abdominally obese, compared with lean men, and to determine the effect of weight loss on these variables in the abdominally obese men. Materials and Methods Study population Apparently healthy men were recruited via advertisements in local newspapers or among participants of previous investigations. Ultimately, 53 abdominally obese and 25 lean white men were enrolled in our randomized controlled trial with blinded analyses. Participants were aged 18 to 65 years, nonsmoking, nondiabetic, and free of cardiovascular disease and had a waist circumference <94 cm (lean) or 102 to 110 cm (abdominally obese) and had had a stable body weight for ≥3 months. The exclusion criteria were fasting plasma glucose >7.0 mmol/L, hemoglobin A1c >6.5%, serum total cholesterol >8.0 mmol/L, serum triglycerides >4.5 mmol/L, systolic blood pressure (SBP) >160 mm Hg, and the use of medication affecting the blood pressure, lipid profile, or glucose metabolism. All participants gave written informed consent. The local ethics committee approved the study, which was performed in accordance with the Declaration of Helsinki and registered at ClinicalTrials.gov (NCT01675401). Study design Abdominally obese men were randomly assigned in a 1:1 ratio to either an 8-week weight loss program or maintenance of their habitual diet and were studied before and after the 8-week period. Lean men were studied at baseline only. Randomization was performed by an independent investigator using block randomization with variable block sizes and stratification for age <50 and >50 years, because the effect of weight loss on microvascular and metabolic insulin sensitivity and blood pressure could differ with age. An independent investigator revealed the allocation to the participant and research team on completion of all baseline measurements. The weight loss program was designed to induce a ∼10% reduction in body weight and consisted of 4 to 5 weeks of a very low calorie diet providing 2.1 MJ/day (Modifast; Novartis Nutrition, Breda, The Netherlands), 1 to 2 weeks of an energy-restricted diet providing 4.2 MJ/day, a weight stable phase of 2 weeks, and weekly dietary counseling. During the 8-week period, the control group was also monitored to avoid fluctuations in weight. Both groups were instructed not to alter their exercise pattern throughout the study. At baseline (both lean and abdominally obese men) and after the 8-week period (abdominally obese men only), 24-hour urine samples were collected for assessment of sodium, potassium, and creatinine excretion, and 24-hour ambulatory blood pressure measurements were performed (Mobilograph; New Generation, I.E.M., Stolberg, Germany). Blood pressure was measured on the nondominant arm every 15 minutes during the day and every 30 minutes during the night. The measurements were conducted in a temperature-controlled room (24° ± 0.5°C) after a 12-hour overnight fast with the participants in the supine position. The men were instructed to refrain from alcohol and meals rich in lipids for a period of 24 hours before each study day and to refrain from strenuous physical exercise for a period of 48 hours before each study day. After insertion of two intravenous catheters and a 30-minute acclimatization period with the participants in the supine position, blood samples were taken for determination of glucose, hemoglobin A1c, overall lipid profile, and aldosterone concentrations, and the microvascular blood volume of the forearm skeletal muscle was measured at baseline. Assessment of insulin sensitivity We determined metabolic insulin sensitivity using a modified version of the hyperinsulinemic, euglycemic clamp technique, as described by DeFronzo et al. (27). In brief, insulin (Novorapid; Novo Nordisk, Bagsvaerd, Denmark) was administered in a primed continuous manner at a rate of 1 mU/kg/min for 180 minutes. Isoglycemia was maintained by adjusting the rate of a 20% d-glucose infusion based on the plasma glucose measurements performed at 5-minute intervals. Whole-body glucose disposal was estimated from the steady-state glucose infusion rate 90 to 150 minutes after initiation of insulin administration. The whole-body glucose disposal is expressed per kilogram body weight per unit of plasma insulin concentration (M/I value), thus correcting for variations in the steady-state insulin concentrations. For convenience, the M/I ratio was multiplied by 100. Evaluation of skeletal muscle microvascular function IMMR was assessed using contrast-enhanced ultrasonography, as described previously (5). In brief, the microvascular blood volume of forearm skeletal muscle was measured before and during hyperinsulinemia using a Toshiba Aplio XG ultrasound system (Toshiba, Otawara, Japan) during continuous intravenous administration of sulfur hexafluoride gas-filled microbubbles (SonoVue; Bracco Diagnostics, Amsterdam, The Netherlands). After a steady-state microbubble concentration was achieved (3 minutes), five real-time replenishment curves of 30 seconds were acquired. These replenishment curves were stored and analyzed offline in a blinded fashion after completion of the trial using the CHI-Q software (Toshiba). The replenishment curves were fitted to the exponential function [y = A(1 − e−βt)], where t is the time since the high mechanical index pulse, y is the video intensity at any given t, A is the plateau video intensity (representing the microvascular blood volume), and β is the microvascular flow velocity. IMMR was calculated as the relative increase in muscle microvascular blood volume during hyperinsulinemia. Measurement of subcutaneous and visceral fat volumes Subcutaneous and visceral fat volumes were measured using magnetic resonance imaging, as previously described (5). Blood and urine measurements Plasma glucose was determined using a YSI2300 glucose analyzer (YSI, Yellow Springs, OH). The serum insulin levels during the hyperinsulinemic clamp were measured using Mercodia Iso-Insulin enzyme-linked immunosorbent assay [Mercodia AB, Uppsala, Sweden; intra-assay coefficient of variation (CV), 2.8% to 3.2%; interassay CV, 3% to 3.9%]. Serum aldosterone was analyzed using ultra-performance liquid chromatography tandem mass spectrometry, as described previously (28), with minor modifications. In brief, 30 µL of 0.14 µmol/L D7-aldosterone in 50/50 methanol/water (volume-to-volume ratio; %) was added to 300 µL of serum. The samples were mixed and subsequently deproteinized with acetone. After centrifugation (10 minutes, 25°C, 14000 rpm), the supernatant was extracted with two volumes of 800 µL tert-butyl methyl ether at room temperature. The phases were separated by centrifugation (1 minute, 25°C, 4600 rpm), and the upper organic phase was transferred to a 4-mL glass vial and dried under nitrogen at 35°C. The dried residue was dissolved in 100 µL 35/65 methanol/water (volume-to-volume ratio; %). Finally, 10 µL was injected into the ultra-performance liquid chromatography tandem mass spectrometry for analysis. The intra-assay CVs ranged from 4.0% to 8.7% and the interassay CVs from 4.5% to 10.4%. The urinary sodium and potassium excretion were determined using the ion-selective electrode method. Statistical analysis Normally distributed variables are expressed as the mean ± standard deviation. The variables with a skewed distribution are displayed as the median and interquartile range, and natural logarithmic transformation was performed before further analyses (M/I value and aldosterone). Independent sample t tests were used to compare the groups at baseline and the differences in the anthropometric, metabolic, hemodynamic, and hormonal variables between the abdominally obese men after the weight loss or weight stable period. The aldosterone concentration in the lean and abdominally obese men was compared using analysis of covariance and in abdominally obese individuals before and after the weight loss or weight stable period by repeated measures analysis of covariance, with adjustment for age, mean arterial pressure (MAP) (in the comparison between the lean and abdominally obese men), and urinary sodium and potassium excretion. The relationships of, and changes in, the aldosterone levels with the (alterations in) IMMR, M/I value, and 24-hour ambulatory blood pressure were assessed using multiple linear regression with adjustment for age and differences in urinary sodium and potassium excretion. The associations of, and changes in, aldosterone concentration with the (alterations in) other anthropometric, metabolic, and hemodynamic variables are presented as Pearson correlation coefficients. Analyses were performed using the SPSS statistical software package, SPSS Statistics, version 20 (IBM, Armonk, NY). Two-tailed P values of < 0.05 were considered statistically significant. Results Cross-sectional analyses One of the 53 abdominally obese participants was excluded because of protocol violations. Data on urinary sodium and potassium excretion were available for 21 lean men and 46 abdominally obese men. The baseline characteristics of the lean and abdominally obese men are presented in Table 1. The lean men had significantly lower subcutaneous and visceral adipose tissue volumes, fasting plasma glucose levels, and SBP and diastolic blood pressure (DBP) compared with the abdominally obese men. The aldosterone levels, however, were similar in both groups. Urinary sodium excretion was consistent with the average salt intake of Dutch men (29). Table 1. Baseline Characteristics of Lean and Abdominally Obese Men Characteristic  Lean Men  Abdominally Obese Men  Participants, n  25  52  Age, y  54 (25–62)  52 (46–61)  BMI, kg/m2  23.3 ± 1.8a  30.1 ± 2.1  Waist circumference, cm  85 ± 6a  107 ± 4  Subcutaneous adipose tissue, L  1.45 ± 0.51a  3.06 ± 0.77  Visceral adipose tissue, L  0.89 ± 0.42a  2.36 ± 0.72  Fasting plasma glucose, mmol/L  5.35 ± 0.29a  5.64 ± 0.48  Hemoglobin, A1c, %  5.2 ± 0.4  5.3 ± 0.4  24-h SBP/DBP, mm Hg  118 ± 9/73 ± 9a  123 ± 9/80 ± 7  24-h MAP, mm Hg  93 ± 8a  100 ± 7  24-h Pulse pressure, mm Hg  45 ± 8  43 ± 7  Aldosterone, pmol/L  274 (178–512)  278 (169–392)  Urinary sodium excretion, mmol/24 h  153 ± 73  172 ± 64  Urinary potassium excretion, mmol/24 h  81 ± 30  83 ± 23  Urinary creatinine excretion, mmol/24 h  14 ± 3a  18 ± 3  Characteristic  Lean Men  Abdominally Obese Men  Participants, n  25  52  Age, y  54 (25–62)  52 (46–61)  BMI, kg/m2  23.3 ± 1.8a  30.1 ± 2.1  Waist circumference, cm  85 ± 6a  107 ± 4  Subcutaneous adipose tissue, L  1.45 ± 0.51a  3.06 ± 0.77  Visceral adipose tissue, L  0.89 ± 0.42a  2.36 ± 0.72  Fasting plasma glucose, mmol/L  5.35 ± 0.29a  5.64 ± 0.48  Hemoglobin, A1c, %  5.2 ± 0.4  5.3 ± 0.4  24-h SBP/DBP, mm Hg  118 ± 9/73 ± 9a  123 ± 9/80 ± 7  24-h MAP, mm Hg  93 ± 8a  100 ± 7  24-h Pulse pressure, mm Hg  45 ± 8  43 ± 7  Aldosterone, pmol/L  274 (178–512)  278 (169–392)  Urinary sodium excretion, mmol/24 h  153 ± 73  172 ± 64  Urinary potassium excretion, mmol/24 h  81 ± 30  83 ± 23  Urinary creatinine excretion, mmol/24 h  14 ± 3a  18 ± 3  Data presented as the mean ± standard deviation or median (interquartile range). a Lean vs abdominally obese, P ≤ 0.01. View Large Adjustment for age, MAP, and urinary sodium and potassium excretion yielded similar results with regard to aldosterone concentrations in the lean and abdominally obese participants (F1,61 = 0.026; P = 0.87; Fig. 1). Both the M/I value and the IMMR were higher in the lean than in the abdominally obese men (Table 2). The aldosterone levels were not associated with the IMMR, M/I value, or 24-hour SBP [natural logarithm (ln) IMMR, β = −1.713, P = 0.82; ln M/I value, β = −0.131, P = 0.29; SBP, β = −2.533, P = 0.14], which remained unchanged after adjustment for age and urinary sodium and potassium excretion (ln IMMR, β = −0.715, P = 0.93; ln M/I value, β = −0.100, P = 0.46; SBP, β = −1.815, P = 0.34). Supplemental Table 1 shows the correlations of aldosterone with the other anthropometric, metabolic, and hemodynamic variables. Aldosterone was not associated with either body mass index (BMI) or subcutaneous and visceral adipose tissue volumes but was inversely associated with DBP (r = −0.241, P = 0.04), which became nonsignificant after adjustment for confounders and for the 24-hour MAP (data not shown). These associations were similar in the lean and abdominally obese men (P for interaction all > 0.14), except for the associations with urinary sodium excretion. Figure 1. View largeDownload slide Baseline aldosterone levels in lean and obese individuals (analysis of covariance with adjustment for age, MAP, and urinary sodium and potassium excretion). Figure 1. View largeDownload slide Baseline aldosterone levels in lean and obese individuals (analysis of covariance with adjustment for age, MAP, and urinary sodium and potassium excretion). Table 2. Microvascular and Metabolic Insulin Sensitivity in Lean vs Abdominally Obese Men Variable  Lean Men  Abdominally Obese Men  Participants, n  25  52  M value, mg/kg/min  6.8 ± 1.8a  4.1 ± 1.3  M/I value, (mg/kg/min per mU/L) × 100  9.9 (6.7–12.1)a  4.4 (2.9–5.5)  IMMR, %  44 ± 41a  −3.5 ± 27  Variable  Lean Men  Abdominally Obese Men  Participants, n  25  52  M value, mg/kg/min  6.8 ± 1.8a  4.1 ± 1.3  M/I value, (mg/kg/min per mU/L) × 100  9.9 (6.7–12.1)a  4.4 (2.9–5.5)  IMMR, %  44 ± 41a  −3.5 ± 27  Data presented as the mean ± standard deviation or median (interquartile range). Abbreviation: M value, whole body glucose disposal. a Lean vs abdominally obese, P ≤ 0.01. View Large Urinary sodium excretion was inversely associated with aldosterone levels only in the lean men (β per 100 mmol sodium excretion [adjusted for age and urinary potassium excretion] = −0.481 [95% confidence interval (CI), −0.949 to −0.013], P < 0.05; i.e., for each 100 mmol greater sodium excretion, the aldosterone levels were lower by 38% [95% CI, 1% to 61%]) and not in the abdominally obese men [β per 100 mmol sodium excretion (adjusted for age and urinary potassium excretion) = −0.081 (95% CI, −0.433 to 0.271), P = 0.65; i.e., for each 100 mmol greater sodium excretion, the aldosterone levels were lower by 8% (95% CI, −31 to 35); P for interaction = 0.02; Fig. 2]. Figure 2. View largeDownload slide Association of 24-hour urinary sodium excretion with aldosterone levels in lean (white circles) and abdominally obese (black circles) individuals. Regression coefficients adjusted for age and urinary potassium excretion. Figure 2. View largeDownload slide Association of 24-hour urinary sodium excretion with aldosterone levels in lean (white circles) and abdominally obese (black circles) individuals. Regression coefficients adjusted for age and urinary potassium excretion. Adjustment of the associations of aldosterone levels with IMMR, M/I value, and 24-hour SBP for urinary sodium excretion did not materially change the regression coefficients (Supplemental Table 2). Effects of weight loss Of the 53 abdominally obese men, 26 were allocated to the weight loss intervention and 27 to the weight stable (control) group. Two men discontinued the weight loss intervention, one because of noncompliance, and one for personal reasons; a third man who had been allocated to the weight loss program was excluded from the analyses because of protocol violations, leaving 23 men in the weight loss group. One man in the control group did not complete the study for a non–study-related reason. Ultimately, analyses were performed for 23 men in the weight loss group and 26 men in the weight stable group. Data on urinary sodium and potassium excretion at follow-up were available for 19 participants randomized to the weight loss intervention and 24 participants in the control group. The anthropometric, metabolic, hemodynamic, and hormonal variables at baseline and the effects of weight loss on these variables are shown in Table 3. Both groups were comparable with regard to baseline characteristics, with the exception of SBP, which was lower in the weight loss group (120 ± 9 vs 126 ± 8 mm Hg; P = 0.03). Table 3. Anthropometric, Metabolic, Hemodynamic, and Hormonal Variables in Abdominally Obese Men and Effects of Weight Loss Variable  Weight Loss Group (n = 23)   Weight Stable Group (n = 26)   Baseline  Δ  Baseline  Δ  Age, y  52 (47–62)  NA  52 (45–61)  NA  BMI, kg/m2  30.2 ± 1.5  −3.0 ± 0.8b  29.9 ± 2.5  0.1 ± 0.33  Waist circumference, cm  107 ± 3  −11 ± 2b  106 ± 4  0 ± 2  Subcutaneous adipose tissue, L  2.81 ± 0.65a  −0.34 ± 0.44  3.26 ± 0.78  −0.36 ± 0.53  Visceral adipose tissue, L  2.53 ± 0.78  −0.33 ± 0.49  2.19 ± 0.68  −0.29 ± 0.55  Fasting plasma glucose, mmol/L  5.49 ± 0.37  −0.21 ± 0.33c  5.75 ± 0.53  0.00 ± 0.28  Hemoglobin A1c, %  5.2 ± 0.3  −0.2 ± 0.3c  5.3 ± 0.4  −0.1 ± 0.2  M value, mg/kg/min  4.1 ± 1.3  1.3 ± 1.2b  4.0 ± 1.4  −0.1 ± 0.9  M/I value, (mg/kg/min per mU/L) × 100  3.4 (2.5–5.3)  2.6 ± 2.7b  4.8 (2.7–6.2)  −1.0 ± 2.4  IMMR, %  −5.0 ± 27  40 ± 49b  0.7 ± 28  −0.3 ± 28  24-h SBP/DBP, mm Hg  120 ± 9a/78 ± 8  −5±6b/−5 ± 5b  126 ± 8/82 ± 7  −1±6/−1 ± 5  24-h MAP, mm Hg  98 ± 8a  −5±5b  102 ± 7  −1±5  24-h Pulse pressure, mm Hg  42 ± 7  0 ± 3  44 ± 5  0 ± 4  Aldosterone, pmol/L  306 (187–394)  −61 ± 126  253 (156–422)  −30 ± 141  Urinary sodium excretion, mmol/24 h  163 ± 62  19 ± 80  183 ± 65  0.1 ± 87  Urinary potassium excretion, mmol/24 h  83 ± 20  −4.9 ± 24  85 ± 25  −5.3 ± 38  Urinary creatinine excretion, mmol/24 h  18 ± 3  0.4 ± 6.3  18 ± 4  −1.9 ± 3.6  Variable  Weight Loss Group (n = 23)   Weight Stable Group (n = 26)   Baseline  Δ  Baseline  Δ  Age, y  52 (47–62)  NA  52 (45–61)  NA  BMI, kg/m2  30.2 ± 1.5  −3.0 ± 0.8b  29.9 ± 2.5  0.1 ± 0.33  Waist circumference, cm  107 ± 3  −11 ± 2b  106 ± 4  0 ± 2  Subcutaneous adipose tissue, L  2.81 ± 0.65a  −0.34 ± 0.44  3.26 ± 0.78  −0.36 ± 0.53  Visceral adipose tissue, L  2.53 ± 0.78  −0.33 ± 0.49  2.19 ± 0.68  −0.29 ± 0.55  Fasting plasma glucose, mmol/L  5.49 ± 0.37  −0.21 ± 0.33c  5.75 ± 0.53  0.00 ± 0.28  Hemoglobin A1c, %  5.2 ± 0.3  −0.2 ± 0.3c  5.3 ± 0.4  −0.1 ± 0.2  M value, mg/kg/min  4.1 ± 1.3  1.3 ± 1.2b  4.0 ± 1.4  −0.1 ± 0.9  M/I value, (mg/kg/min per mU/L) × 100  3.4 (2.5–5.3)  2.6 ± 2.7b  4.8 (2.7–6.2)  −1.0 ± 2.4  IMMR, %  −5.0 ± 27  40 ± 49b  0.7 ± 28  −0.3 ± 28  24-h SBP/DBP, mm Hg  120 ± 9a/78 ± 8  −5±6b/−5 ± 5b  126 ± 8/82 ± 7  −1±6/−1 ± 5  24-h MAP, mm Hg  98 ± 8a  −5±5b  102 ± 7  −1±5  24-h Pulse pressure, mm Hg  42 ± 7  0 ± 3  44 ± 5  0 ± 4  Aldosterone, pmol/L  306 (187–394)  −61 ± 126  253 (156–422)  −30 ± 141  Urinary sodium excretion, mmol/24 h  163 ± 62  19 ± 80  183 ± 65  0.1 ± 87  Urinary potassium excretion, mmol/24 h  83 ± 20  −4.9 ± 24  85 ± 25  −5.3 ± 38  Urinary creatinine excretion, mmol/24 h  18 ± 3  0.4 ± 6.3  18 ± 4  −1.9 ± 3.6  Data are presented as means ± standard deviation or median (interquartile range). Abbreviations: M value, whole-body glucose disposal; NA, not applicable. a Weight loss group at baseline vs weight stable group at baseline, P < 0.05. b Change in weight loss group vs change in weight stable group, P ≤ 0.01. c Change in weight loss group vs change in weight stable group, P < 0.05. View Large In the control group, the waist circumference, M/I value, IMMR, and 24-hour ambulatory blood pressure were unchanged over time. In the weight loss group, the waist circumference decreased significantly by 11 ± 2.1 cm (P < 0.01), the M/I value increased by 2.6 ± 2.7 (mg/kg/min per mU/L) × 100 (P < 0.01), and IMMR increased by 40% ± 49% (P < 0.01). The SBP, DBP, and MAP decreased significantly (SBP, −5 ± 6 mm Hg, P < 0.01; DBP, −5 ± 5 mm Hg, P < 0.01; MAP, −5 ± 5 mm Hg, P < 0.01), but the pulse pressure was unaffected. The aldosterone levels remained unchanged as well. The absence of an effect of weight loss on aldosterone concentration was confirmed after adjustment for age and urinary sodium and potassium excretion at the follow-up assessments (F1,38 = 0.824, P = 0.37). Changes in the aldosterone concentration after weight loss were not related to alterations in IMMR, M/I value, or 24-hour SBP (∆IMMR, β = −0.020, P = 0.65; ∆M/I value, β = −0.001, P = 0.72; ∆24-hour SBP, β = 0.002, P = 0.73). The regression coefficients remained unchanged after adjustment for potential confounders (Fig. 3). The associations of the changes in aldosterone concentration with alterations in the other anthropometric, metabolic, and hemodynamic variables resulting from the weight loss intervention are shown in Supplemental Table 3. These associations were similar in the weight loss and weight stable groups (P for interaction > 0.11 for all). Figure 3. View largeDownload slide Associations of changes in aldosterone levels (∆aldosterone) after the weight loss (white squares) or weight stable period (black squares) with (A) alterations in IMMR (∆ IMMR), (B) alterations in M/I value (∆ M/I value), and (C) alterations in 24-hour SBP (∆ SBP). Regression coefficients adjusted for age and differences in urinary sodium and potassium excretion. Figure 3. View largeDownload slide Associations of changes in aldosterone levels (∆aldosterone) after the weight loss (white squares) or weight stable period (black squares) with (A) alterations in IMMR (∆ IMMR), (B) alterations in M/I value (∆ M/I value), and (C) alterations in 24-hour SBP (∆ SBP). Regression coefficients adjusted for age and differences in urinary sodium and potassium excretion. Additional analyses When we excluded participants with a BMI of 25 to 30 kg/m2 and thus men who would be classified as overweight on the basis of their BMI (n = 29), the comparison of aldosterone levels between the lean and obese men yielded similar results: BMI ≤25 kg/m2 (n = 21), 257 pmol/L (95% CI, 178 to 565), BMI ≥30 kg/m2, 288 pmol/L (95% CI, 174 to 394); P = 0.50. In the present study, the variation in ad libitum sodium intake was relatively large. To investigate whether the response of aldosterone levels to controlled variation in sodium ingestion in lean and abdominally obese men is comparable to the cross-sectional associations between ad libitum sodium intake and the corresponding aldosterone concentration in the present population (described previously), we determined the relationship between 24-hour urinary sodium excretion and aldosterone levels after a low-sodium diet (50 mmol sodium chloride/24 hours) and high-sodium diet (250 mmol sodium chloride/24 hours) for 7 days in randomized order in 20 lean and 20 abdominally obese individuals in a separate data set (the characteristics of this study population are presented in Supplemental Table 4). After the low- and high-sodium diets, the aldosterone levels were not statistically significantly different between the lean and abdominally obese individuals. However, multivariate regression analyses adjusted for age, sex, and urinary potassium excretion showed that for each 100-mmol increase in urinary sodium excretion, aldosterone levels decreased more in the lean than in the abdominally obese participants (i.e., by 140 pmol/L; 95% CI, 289 to −10; P = 0.07 in lean individuals and by 52 pmol/L, 95% CI, 237 to −133; P = 0.56 in abdominally obese individuals; P for interaction = 0.07; Supplemental Fig. 1). Discussion The present study has shown that serum aldosterone levels are not higher in abdominally obese men, compared with lean men, under circumstances of ad libitum sodium intake and are not associated with microvascular and metabolic insulin sensitivity or blood pressure. Moreover, the serum aldosterone concentration was unaffected by a weight loss intervention in abdominally obese men, although we observed a relevant decline in both insulin resistance and blood pressure. Several previous investigations have reported elevated aldosterone levels in parallel with higher blood pressure in severely to morbidly obese participants (11–13). The excess aldosterone in these individuals could be at least partially derived from the visceral adipose tissue (12, 14). Therefore, in our study, in moderately abdominally obese men, the capacity of adipose tissue to produce aldosterone might have been insufficient to enable detection of elevations in aldosterone levels. Experimental studies have shown that aldosterone can impair microvascular function and vascular insulin signaling (20–23), which might predispose to the development of obesity-associated insulin resistance and hypertension (1–3, 7). Nevertheless, we could not establish an association of aldosterone levels with IMMR in skeletal muscle, insulin stimulated-glucose disposal, or blood pressure. This again could be explained by the absence of differences in the aldosterone concentration between the lean and abdominally obese men and, thus, the limited dispersion in aldosterone levels. It is also possible that the increase in aldosterone levels reported in severely to morbidly obese individuals reflects either an epiphenomenon that occurs later in the course of obesity and is not causally related to the development of insulin resistance and hypertension or is a consequence of renal microvascular dysfunction and subsequent renin–angiotensin–aldosterone system activation. However, observations of impaired insulin sensitivity in parallel with substantially elevated blood pressure in individuals with endogenously high exposure to aldosterone, such as patients with primary aldosteronism, which are both ameliorated by pharmacological or surgical treatment (30), suggest otherwise. In addition, the endothelial function in these individuals is more impaired than in patients with essential hypertension of equal severity (31). Therefore, our results do not preclude an effect of aldosterone on microvascular and, thus, metabolic insulin sensitivity and blood pressure but suggest that such an effect might become more prominent with an increasing severity of obesity and thus a greater extent of aldosterone excess. This also indicates that in the current stage of obesity, mechanisms other than aldosterone surplus must be responsible for the increase in blood pressure. Our data do not imply that regulation of aldosterone levels is completely normal in individuals with moderate abdominal obesity, although this does not immediately seem to affect IMMR, whole-body glucose disposal, and blood pressure. We found that the physiological inverse association of urinary sodium excretion with aldosterone levels was stronger in lean than abdominally obese individuals, pointing to a potential derangement in the abdominally obese group that might eventually add to the already existing increase in blood pressure. This was confirmed by our data from a separate experiment showing that after 7 days of low- and high-salt intake in randomized order, suppression of aldosterone levels by increasing sodium intake was impaired in the abdominally obese participants, consistent with previous findings (32). These observations are relevant given that aldosterone seems to be particularly detrimental to the endothelium in the presence of high sodium intake (33). Moreover, aldosterone production was found to correlate with insulin resistance in lean and overweight, normotensive individuals consuming a high-salt diet (15). With these observations, we cannot exclude that the absence of an association of higher aldosterone levels with microvascular and metabolic insulin resistance and higher blood pressure in the present investigation can be partially explained by a sodium intake in accordance with, and not greater than, the average ingestion of Dutch men (29). In contrast to our findings, other investigators have reported that weight loss in severely to morbidly obese individuals and hypertensive obese individuals is paralleled by reductions in aldosterone concentrations (11–13, 17–19), together with a decline in blood pressure (11–13, 17–19) and amelioration of insulin resistance (12). Although in the present study, insulin sensitivity was improved and blood pressure decreased substantially in the abdominally obese men randomized to the weight loss intervention compared with the control group, the aldosterone levels remained unchanged. Again, this might have been because our abdominally obese participants would be categorized as overweight to moderately obese instead of severely obese on the basis of their BMI, which might entail a limited ability of aldosterone overproduction, as outlined previously. One limitation of the present investigation was the use of an exclusively male study population, which affects the generalizability of the results. That the sodium and potassium intake was not standardized and was estimated from single 24-hour urine collections is another limitation. Theoretically, this could have led us to underestimate the true aldosterone levels in the abdominally obese individuals. This was, however, unlikely because, in a separate experiment, after 7 days of standardized low and high sodium intake, the aldosterone levels were also not statistically significantly different between the lean and abdominally obese participants. Nevertheless, the inverse association between urinary sodium excretion and serum aldosterone levels was less pronounced in the abdominally obese than in the lean participants in both the observational (Fig. 2) and the experimental (Supplemental Figure 1) portions of the present study. This suggests a subtle abnormality in the control of aldosterone concentration by sodium intake which, in this stage of obesity, does not affect the absolute aldosterone levels. However, it does not detract from our findings that the aldosterone levels, whether or not considering its regulation by sodium ingestion, were not substantially associated with microvascular or metabolic insulin sensitivity or blood pressure in either lean or moderately abdominally obese men. An important strength of our investigation was its design, which allowed us to study the association between aldosterone and obesity-related complications, including microvascular and metabolic insulin resistance and hypertension, in both a cross-sectional and longitudinal manner. In addition, we used the reference standard for assessment of insulin sensitivity and were able to measure microvascular function in skeletal muscle, which is the main peripheral location of insulin-mediated glucose uptake, thus allowing a thorough evaluation of the relationship between microvascular and metabolic insulin sensitivity. In conclusion, the results of the present study suggest that at an early stage of abdominal obesity, the absolute aldosterone levels are not elevated and do not contribute to the development of microvascular and metabolic insulin resistance and hypertension. However, the inverse relationship between sodium excretion and aldosterone concentration is less than that in lean men, although this does not immediately affect IMMR, insulin-stimulated glucose disposal, and blood pressure. It is possible that the role of aldosterone in this respect becomes more prominent with an increasing severity of obesity. Future research should be directed toward investigating the association of aldosterone with microvascular and metabolic insulin sensitivity and blood pressure in a study population with a broader range of abdominal obesity and one that also includes women. Moreover, it would be valuable to study the effect of mineralocorticoid receptor blockade on vascular and metabolic insulin signaling and blood pressure in these individuals as an alternative to weight loss for the amelioration of insulin resistance and hypertension, which is often difficult to achieve and sustain. 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Feb 1, 2018

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