Relationships Among Plasma Aldosterone, High-Density Lipoprotein Cholesterol, and Insulin in Humans
Abstract To investigate the pathogenesis of hypertension in patients with obesity and insulin resistance and to explore the role of plasma lipids, we studied 30 subjects at the end of 7 days of low (20 mEq/d) then high (200 mEq/d) sodium diets. Glucose and insulin tolerance tests were performed at the end of each week and blood and urine collected for measurements of plasma aldosterone, renin activity, electrolytes, insulin, and lipoproteins. There was a strong negative correlation between plasma aldosterone and high-density lipoprotein cholesterol during both diets. There were weaker positive correlations between plasma aldosterone and insulin or triglycerides. When the aldosterone-renin ratio was the dependent variable and the correlation controlled for serum potassium, the inverse relationship with high-density lipoprotein cholesterol and the positive correlation with insulin remained, but only during the high salt diet. Subjects were divided into three groups based on high-density lipoprotein cholesterol. Subjects with the lowest high-density lipoprotein cholesterol levels showed the highest aldosterone, plasma triglycerides, body mass index, and waist-to-hip ratio. Those subjects also demonstrated the greatest resistance to insulin action on glucose and plasma unesterified fatty acids. There was a weak direct correlation between plasma aldosterone and systolic blood pressure during the high salt diet. These data suggest that high aldosterone levels may be a link between dyslipidemia, insulin resistance, and hypertension, a relationship made more evident by high salt intake.
A low level of plasma high-density lipoprotein cholesterol (HDL-C) is one component of a cluster of coronary disease risk factors that also includes abdominal obesity, hypertension, hyperinsulinemia, and insulin resistance.1 The causal relationships among these abnormalities, especially the pathogenesis of hypertension, are not understood. In the work reported here, we examined plasma renin activity (PRA), aldosterone levels, and other blood chemistries during high and low dietary salt intake in 30 human subjects with widely different coronary disease risk. Our goal was to assess the possible role of plasma lipids in the regulation of the renin-angiotensin-aldosterone axis. This search was stimulated by our observation that unesterified fatty acids can inhibit aldosterone secretion in vitro.2 We did not detect a relationship between fatty acids and aldosterone, but we found an unexpected inverse correlation between plasma HDL-C and aldosterone. We also saw less consistent and less powerful direct correlations of plasma aldosterone with insulin and triglyceride levels. These statistical relationships suggest pathogenetic links among plasma lipoproteins, insulin, and aldosterone, substances that have been regarded until now as independent contributors to cardiovascular regulation and coronary disease risk.
Thirty subjects, 23 men and 7 women, were recruited from the Hypertension Clinic of the Medical College of Wisconsin and by advertisement. Volunteers signed an informed consent document after listening to an explanation of the study, all parts of which had been approved by the Human Research Review Committee. All subjects underwent a medical history, physical examination, and laboratory tests to exclude health problems other than the risk factors under study. Anthropometric measurements and calculations were obtained according to published techniques.3 4
Volunteers were interviewed by a nutritionist to determine their usual diet. Individualized isocaloric diets were designed using the nutritionist III diet analysis software (N-Squared Computing). The diet was controlled for Na+ (20 mmol/d), K+ (65 mmol/d), Ca2+ (20 mmol/d), and Mg2+ (12 mmol/d). The caloric composition of the diet was 45% to 50% carbohydrate, 35% to 40% fat, and 15% protein. During the high salt period, the same diet was supplemented with eighteen 600-mg NaCl tablets daily (184 mmol/d added). Volunteers came to the hospital on alternate days to obtain all food and beverages from the Clinical Research Center (CRC) kitchen.
After 5 days on the low salt diet, subjects began a 24-hour urine collection at 7 am, fasted overnight, and then reported to the CRC at 7 am the following morning to terminate the urine collection. After weight and blood pressure were measured, a 20-gauge plastic catheter was inserted into a dorsal hand vein in preparation for an oral glucose tolerance test. With the subject supine, the hand with the indwelling catheter was placed in a box heated to 70°C to arterialize venous blood.5 Beginning 20 minutes later, three aliquots of blood were drawn at 10-minute intervals for glucose and insulin. Aliquots of the final sample were also prepared for blood counts and chemistries, PRA, plasma lipids, aldosterone, and norepinephrine determinations. Seventy-five grams of glucose in water was ingested over 2 minutes and blood drawn at 30, 60, 90, and 120 minutes.
Volunteers continued the low salt diet at home after their glucose tolerance test and returned to the CRC the following morning, after an overnight fast, for performance of an intravenous insulin tolerance test. Postvoid weight was again obtained, a venous catheter was placed in a hand vein, the hand was warmed, and arterialized venous blood was drawn at 10-minute intervals. After the third baseline sample, a bolus of human insulin was administered into the antecubital vein at a dose of 0.1 U/kg. Blood was drawn 3, 6, 9, 12, and 15 minutes after the insulin infusion. All samples were analyzed for insulin, glucose, and nonesterified fatty acids.
After the first insulin tolerance test, volunteers began a week of the same diet supplemented with salt tablets. They returned for repetition of glucose and insulin tolerance tests on days 13 and 14.
Blood Pressure Measurements
Blood pressures were measured at the time of screening for admission to the study and during CRC admissions. A mercury sphygmomanometer was used at the screening examination with cuffs of appropriate size. During CRC admissions, pressures were measured with a Dinamap 1846SX device (Criticon, Inc). Measurements were made in triplicate after the subjects had rested for 5 minutes seated but before insertion of intravenous catheters. Results are reported as the mean of three values.
Serum insulin and glucose levels and plasma norepinephrine and renin activities were measured as described previously.6 Aldosterone and cortisol concentrations were measured by radioimmunoassay using reagents supplied by Diagnostic Products Corp. Possible interference with the aldosterone radioimmunoassay by HDL or another substance in plasmas with low aldosterone values was ruled out by two approaches described in “Results.” HDL-C and other plasma lipids were measured using the Ektachem instrument (Eastman Kodak Co) in the clinical laboratory. Apolipoprotein A-I was measured by immunoturbidometric analysis using an antibody supplied by Atlantic Antibody Corp. Fatty acids in EDTA plasma were measured by the method described by Barash and Akov7 based on the radioactive nickel method of Ho.
All data are presented as mean±SEM. Analyses were performed with spss 6.0 software (SPSS, Inc). The area under the curve (AUC) for insulin during the oral glucose tolerance test and the rate of glucose disappearance during the insulin tolerance test (KITT) were calculated by standard methods.8 9 Correlations were tested using multiple linear regression analysis. Partial correlation coefficients were generated by controlling for different variables alone and serially. Data obtained during low and high salt diets were analyzed separately. In a second type of analysis, subjects were divided into three groups based on their HDL-C levels. Other differences among the three groups were tested with one-way ANOVA followed by repeated measures adjusted t tests. Within each group defined by HDL-C, the differences between high and low salt diets were tested by paired t tests. A value of P<.05 was accepted as statistically significant.
After we obtained the results described below, we measured the aldosterone levels in plasma samples from a previous cohort of subjects who had been studied under less rigorous dietary control to measure their hemodynamic and adrenergic responses. Lipoproteins and insulin had been measured in fresh serum from these subjects at the time of the experiment. We measured their plasma aldosterone levels in samples that had been stored frozen for 2 to 4 years.
Characteristics of the Subjects
Correlations With Plasma Aldosterone Levels
Aldosterone correlated directly with PRA during the low salt diet but not during the high salt diet (Table 2⇑). There was a weak correlation, not statistically significant, between aldosterone and serum potassium during the low salt diet, again not seen during the high salt diet. There was no correlation between plasma nonesterified fatty acids and aldosterone at the start of the insulin tolerance test, whether subjects were eating low salt or high salt diets.
The strongest correlation with aldosterone we observed in these subjects was an inverse relationship with HDL-C. This was equally robust during high and low salt diets (Fig 1⇑). There were weaker, direct correlations of plasma aldosterone with insulin and triglycerides.
We measured cortisol levels in the same plasmas and tested for correlations as above. There were no correlations between cortisol and HDL-C, insulin, triglycerides, or aldosterone (data not shown). After we had measured all of the parameters listed above and noticed the correlations, we rethawed 29 of the 30 plasma samples obtained after the high salt diet and measured apolipoprotein A-I by radioimmunoassay. The correlation between apolipoprotein A and aldosterone was almost identical to that for HDL-C and aldosterone (−.52 as opposed to −.58; P=.004 instead of .001, n=29).
To separate the effects of HDL-C, insulin, and tri- glycerides from the influence of renin and potassium, we used two statistical manipulations. In the first, partial correlations were sought using the aldosterone-renin ratio as dependent variable while controlling statistically for serum potassium. These results are listed in Table 3⇓. An inverse correlation between the aldosterone-renin ratio and HDL-C, independent of serum potassium, was again apparent during the high salt diet but not during the low salt diet. There was a weaker, direct correlation between the aldosterone-renin ratio and insulin, again seen only during the high salt diet. There was no statistically significant correlation between the aldosterone-renin ratio and triglycerides.
In the second approach, partial correlation coefficients were calculated controlling for renin. This again showed a highly significant inverse correlation between aldosterone and HDL-C during both diets, a direct correlation between aldosterone and the insulin response to oral glucose during both diets, and a direct correlation between aldosterone and plasma fasting insulin concentration significant during the high salt diet only.
To test for an effect of gender, we analyzed the data for men and women separately. The inverse correlation between aldosterone and HDL-C was approximately the same for men alone as for all subjects combined (−.51 versus −.58 during high salt diet; −.50 versus −.51 during low salt). Probability values for all four correlations were .014 or less. The inverse correlation for the seven women on a high salt diet (−.27) failed to reach statistical significance. The same relationships were found for the other correlations in Tables 2⇑ and 4⇓: significant correlations for men alone, but too few women for statistical significance.
Correlations With HDL-C
To test the hypothesis that HDL-C was a surrogate for another influence on aldosterone secretion, we sought correlations between HDL-C and all measured chemical and physiological variables. Table 4⇑ lists some of the results of this survey. There were negative correlations of HDL-C with insulin and triglycerides during both low and high salt diets. There were weak negative correlations with PRA and serum potassium seen only while subjects ate a low salt diet. This survey failed to expose a possible surrogate regulator of aldosterone aside from those found in the first examination of the data, described above.
Grouping Patients According to HDL-C
When the potential importance of HDL-C emerged from our search for aldosterone correlates, we examined the data in another way, categorizing patients based on HDL-C levels. We used cut points that divided our 30 subjects roughly into thirds. This division created groups in which several variables clustered with HDL-C. Tables 5⇓ and 6⇓ present the data.
As expected, subjects with low HDL-C levels were more obese and had higher waist-to-hip ratios than those with high HDL-C levels (Table 5⇑). The group with the highest HDL-C levels had the lowest blood pressures. The metabolic and hormonal characteristics of the three groups also differed (Table 6⇑). As predicted from the correlations, plasma aldosterone levels were relatively high in subjects with low HDL-C, regardless of diet. In addition, PRA and potassium were high in the low-HDL group, but only during the low salt diet. In accordance with the recognized clustering of coronary risk factors, subjects with low HDL-C had evidence of insulin resistance manifested as higher insulin levels before and during a glucose tolerance test.10
The sensitivity of subjects to the antilipolytic action of insulin was assessed by measuring the fall in plasma unesterified fatty acids during an insulin infusion. This decrement was smallest in subjects with the lowest HDL-C, who also showed the greatest resistance to the glucose-lowering effects of insulin and had the highest plasma aldosterone levels (Fig 2⇓).
Reliability of Radioimmunoassay for Aldosterone
We tested for interference by high HDL-C levels with the radioimmunoassay for aldosterone in two ways. In the first, we assayed mixtures of plasmas with widely different levels of HDL and aldosterone. For example, we tested a mixture composed of equal parts of a high-HDL/low-aldosterone sample and a low-HDL/high-aldosterone sample. If the sample with high HDL-C had contained a substance that interfered with aldosterone assays, the mixture should have given a result lower than the calculated mean. In six mixtures, the aldosterone assay was 107±3% of the calculated mean, showing no evidence of interference with the radioimmunoassay by plasma constituents in the low-aldosterone sample.
In the second test for assay interference, we measured aldosterone in lipid extracts made from plasmas with widely different aldosterone and HDL-C levels. The extraction procedure used ethyl acetate and heptane, solvents that denature lipoproteins and concentrate aldosterone. We compared the ratios of aldosterone in the high/low samples measured before and after extraction. In six comparisons, the ratios after extraction were, if anything, greater than those calculated from unextracted plasma, again showing that the differences between high and low aldosterone values were not caused by artifacts from plasma proteins.
Correlations With Blood Pressure
There was a weak correlation between systolic blood pressure and plasma aldosterone while subjects were eating a high salt diet (r=.39, P=.033). No correlation was observed between aldosterone and diastolic pressure during the high salt diet or either pressure during salt restriction. There was no correlation between HDL-C and blood pressure nor between insulin and blood pressure during either diet.
Analysis of Earlier Cohort
Data from a group of subjects studied 2 years earlier were reanalyzed to check the generalizability of our correlations with plasma aldosterone. Lipoprotein, renin, and insulin measurements had been performed on fresh blood specimens drawn at the time of the experiment. Plasma aldosterone was measured in plasma samples that had been frozen as long as 4 years. In 20 subjects studied on a high salt diet, the correlation coefficient between plasma aldosterone and HDL-C was −.42 (P=.06). There was no correlation between these variables during a low salt diet. Renin activities were available in 16 of these subjects, and the correlation coefficient of the aldosterone-renin ratio and HDL-C was −.32 (P=.23). In other words, the results from this earlier, smaller experiment are consistent with the aldosterone-HDL inverse correlation found in the more recent study, but they do not achieve statistical significance standing alone. In the earlier trial, there was a statistically significant direct correlation between insulin and aldosterone while subjects were eating a high salt diet (r=.63, p=.002).
This study was designed to identify metabolic and humoral components of a coronary risk factor cluster that could contribute to the elevated blood pressure observed in affected patients. The known metabolic components of the cluster include hyperinsulinemia, insulin resistance, and dyslipidemia with depressed HDL-C and elevated triglyceride levels.1
In another study, we report that aldosterone levels are relatively high in subjects with the coronary disease risk factor cluster.11 We now suggest that the risk factor determining aldosterone levels in this syndrome may be low HDL-C. The inverse correlation we observed between HDL-C and plasma aldosterone resembles the inverse correlation observed by Lind et al12 between HDL-C and urinary excretion of aldosterone.
The relationship between HDL-C and plasma aldosterone persisted whether the subjects’ dietary salt was low or high, although the mean aldosterone levels were widely different under these two conditions. The relationship was also seen when the effects of renin and potassium on aldosterone were excluded statistically, but when the data were handled that way, the relationship was statistically strongest during the high salt diet. These data, together with the finding by Lind et al,12 suggest that HDL-C modulates the sensitivity of the adrenal glomerulosa, making it less responsive to its classic stimuli.
Although the data suggest that HDLs inhibit adrenal aldosterone secretion, that effect would be opposite to some phenomena observed in vitro. Adrenal cortical cells bear receptors for lipoproteins, one function of which is assumed to be to supply cholesterol for steroid biosynthesis.13 Experiments with adrenal glomerulosa cells in vitro showed that exogenous HDL stimulated aldosterone biosynthesis.14 Of course, it is possible that very high HDL levels in vivo might do to the adrenal what they are purported to do to the arterial wall—remove cholesterol from the tissue. Another possibility is that HDL-C stimulates synthesis of a mineralocorticoid other than aldosterone. We measured only one other plasma steroid, and lipoprotein levels did not appear to affect cortisol secretion in our subjects.
We showed that the inverse correlation held for apolipoprotein A-I as well as for HDL-C. This raises the possibility that the lipoprotein moiety itself has a direct inhibitory effect on aldosterone secretion; HDL alters calcium channels in some cells, and intracellular calcium is involved in regulating aldosterone secretion.15 16 Finally, in the in vitro experiments cited above, HDL was prepared by centrifugation in a high salt density gradient, and that salt treatment may have removed the hypothetical adrenal inhibitor suggested by our results.
One plausible explanation for the inverse correlation between HDL-C and aldosterone is that the lipoprotein is a surrogate indicator of a third substance, or a physiological state, that affects the adrenal glomerulosa. Insulin might be that third substance. Next to HDL-C, the strongest correlations we found were between aldosterone and insulin. Insulin levels are high in patients whose HDL is low.17 Insulin infusion potentiates the stimulatory effects of angiotensin on aldosterone secretion in dogs, and insulin has been shown to stimulate aldosterone secretion in vitro.18 19 The weaker correlations with insulin might simply reflect the greater volatility of that parameter compared with HDL-C.
Another potential aldosterone regulator that emerges from our data is plasma triglycerides. Correlation coefficients for these circulating lipids were positive and approximately equal to those for insulin. There are no published data to indicate that triglycerides can stimulate aldosterone secretion directly.
We did not observe a correlation between aldosterone and plasma unesterified fatty acids, but none of the subjects had a fatty acid level that approached the concentration found to be inhibitory in vitro.2
Comparison of the two cohorts we examined suggests three reasons why an inverse correlation between HDL-C and plasma aldosterone has not been reported by other researchers. First, we saw a clearer relationship when subjects were examined during a high salt diet and PRA was low. Second, the diets of subjects in the current study were more nearly homogeneous in potassium content than those in the first study. Finally, the current study is larger than our earlier one, which may have permitted the correlation to achieve statistical significance. Although there are no other reports of a correlation between HDL-C and plasma aldosterone, it has been noted in the past that aldosterone is relatively high in obese subjects whose HDL-C may well have been low.11 20 21 22 23
The inverse correlation between HDL-C and aldosterone was stronger when subjects were eating a high salt compared with a low salt diet. The same was true for the direct correlation between insulin and aldosterone. These results suggest that subjects with low HDL-C and high insulin, most of whom bear the descriptor “insulin resistance syndrome” or “syndrome X,” can drive aldosterone secretion to relatively high levels by influences aside from the renin-angiotensin system. This hypothesis would suggest, in turn, that low-HDL/high-aldosterone subjects would have higher blood pressures than control subjects during a high salt diet, when renin was suppressed and other adrenal stimuli became relatively more important. We observed a direct correlation between systolic blood pressure and aldosterone during the high salt diet. The correlation did not hold for diastolic pressure nor for systolic or diastolic pressure during the low salt diet.
Our observation may link two separate lines of evidence about promoters of atherosclerotic cardiovascular disease. One is the well-known inverse correlation of HDL-C and coronary artery disease, widely presumed to reflect the ability of the lipoprotein to remove cholesterol from artery walls.24 The other comes from suggestions by Brunner et al25 and Weber and Villarreal26 that angiotensin and aldosterone exert direct deleterious effects on arteries and the heart. Our results would support a hypothesis that HDL protects against atherosclerosis in part by suppressing aldosterone secretion.
The data presented here, together with those from our previous studies, show that the risk factor cluster that includes hypertension, low HDL-C, insulin resistance, and abdominal obesity is also characterized by high circulating levels of aldosterone.11 This high steroid level may contribute to the elevated blood pressure and may also contribute to cardiovascular disease risk.
This work was supported by grants from the Department of Veterans Affairs and the National Institutes of Health (NHLBI R01-43164 and General Clinical Research Center grants M01-RR00058 to the Medical College of Wisconsin and M01-RR01070 to the Medical University of South Carolina). K.S. is the recipient of a Clinical Research Fellowship grant from the Medical University of South Carolina. Measurements of apolipoprotein A-I were performed by Dr Donald Wiebe, University of Wisconsin. The authors also thank the GCRC nursing and nutritional staff for their support and Susi Nehls for editorial assistance.
Reprint requests to Theodore L. Goodfriend, MD, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, WI 53705.
Presented in part at the National American Federation for Clinical Research meeting in Baltimore, Md, May 1994 (Clin Res. 1994;42:338A. Abstract.).
- Received March 15, 1994.
- Revision received July 19, 1994.
- Accepted August 16, 1994.
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