Dennis L. Ball

Epoxy-Keto Derivative of Linoleic Acid Stimulates Aldosterone Secretion

Abstract—Plasma levels of aldosterone are not always predictable from the activity of renin and the concentration of potassium. Among the unexplained are elevated levels of aldosterone in some obese humans. Obesity is characterized by increased plasma fatty acids and oxidative stress. We postulated that oxidized fatty acids stimulate aldosteronogenesis. The most readily oxidized fatty acids are the polyunsaturated, and the most abundant of those is linoleic acid. We tested oxidized derivatives of linoleic acid for effects on rat adrenal cells. One derivative, 12,13-epoxy-9-keto-10(trans)-octadecenoic acid (EKODE), was particularly potent. EKODE stimulated aldosteronogenesis at concentrations from 0.5 to 5 &mgr;mol/L, and inhibited aldosteronogenesis at higher doses. EKODE’s stimulatory effect was most prominent when angiotensin and potassium effects were submaximal. The lipid’s mechanism of action was on the early pathway leading to pregnenolone; its action was inhibited by atrial natriuretic peptide. Plasma EKODE was measured by liquid chromatography/mass spectrometry. All human plasmas tested contained EKODE in concentrations ranging from 10−9 to 5×10−7 mol/L. In samples from 24 adults, levels of EKODE correlated directly with aldosterone (r =0.53, P =0.007). In the 12 blacks in that cohort, EKODE also correlated with body mass index and systolic pressure. Those other correlations were not seen in white subjects. The results suggest that oxidized derivatives of polyunsaturated fatty acids other than arachidonic are biologically active. Compounds like EKODE, derived from linoleic acid, may affect adrenal steroid production in humans and mediate some of the deleterious effects of obesity and oxidative stress, especially in blacks.

Relationships Among Plasma Aldosterone, High-Density Lipoprotein Cholesterol, and Insulin in Humans

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.

Fatty acid effects on angiotensin receptors

Saturated and unsaturated fatty acids were tested for their ability to inhibit specific binding of angiotensin to receptors in bovine adrenal glomerulosa and fasciculata cells, and homogenates of tissue from bovine adrenal and renal artery. Several naturally occurring fatty acids were inhibitory. The most potent fatty acids in the angiotensin-adrenal system were unsaturated C-18, C-20, and C-22 congeners, including oleic, linoleic, and arachidonic acids. These fatty acids inhibited angiotensin binding to adrenal glomerulosa cells by 50% at concentrations between 4 and 8 x 10-6 M. Inhibition by linoleic acid was predominantly competitive, reducing receptor affinity by ∼50% at the ID50 of the fatty acid. Renal artery binding sites were more sensitive than adrenal sites to linoleic, linolenic, and trans-vaccenic acids, but not to other fatty acids. Inhibition was not affected by indomethacin, nordihydroguaretic acid, or a guanosine triphosphate alogue. Fatty acids inhibiting the angiotensin-adrenal system had no effect on angiotensin antibody, adrenal receptors for atrial natriuretic factor, or myometrial receptors for bradykinin. Some fatty acids nonspecifically inhibited aldosterone production, blocking the response to angiotensin and substrate concentrations of corticosterone. This inhibition was apparently unrelated to effects on receptors. Albumin in the buffer partly neutralized the effects of added fatty acids. Pretreatment of fresh adrenal cells with albumin increased their ability to bind angiotensin. This was reversed by adding back the lipids adsorbed to the albumin in the wash. Results suggest that endogenous fatty acids can regulate angiotensin receptors.

Torsemide inhibits aldosterone secretion in vitro

Torsemide inhibited aldosterone secretion by adrenal cells from rats, cows, and guinea pigs stimulated in vitro by potassium, angiotensin, dibutyryl cyclic AMP, ACTH, or corticosterone. Inhibitory concentrations for adrenal cells (micromolar) were comparable with those reported to inhibit ion transport in isolated renal tubules. Inhibition of aldosterone secretion could reduce kaliuresis, and that may explain why torsemide causes less kaliuresis than other diuretics.

Effects of dietary gamma-linolenic acid on blood pressure and adrenal angiotensin receptors in hypertensive rats.

Abstract In a previous study, we showed that dietary gamma-linolenic acid (GLA), an omega-6 polyunsaturated fatty acid found in borage oil (BOR), attenuates the development of hypertension in young spontaneously hypertensive rats (SHR). The purpose of this study was to determine the effects of dietary GLA on established hypertension in adult rats, as well as its effects on components of the renin-angiotensin-aldosterone axis. For 5 weeks, male SHR (14–15 weeks old) were fed a basal fat-free diet to which 11% by weight of sesame oil (SES) or BOR was added. Systolic blood pressure (SBP), determined by the tail cuff method, and weight were measured weekly. Plasma renin activity (PRA), aldosterone (PA), and corticosterone (PC) levels were measured at the end of the dietary treatments. The adrenal glands were homogenized, and angiotensin II (ANG II) binding was measured and plotted according to Scatchard. Systolic blood pressure was 12 mmHg lower at Week 5 in SHR fed the BOR diet compared to SES-fed rats (P < 0.005). Weight gains were similar in both dietary groups. Plasma aldosterone was lower, PRA was higher, and the PA/PRA ratio was significantly lower (P < 0.05) in BOR-fed rats. Levels of PC were the same in both groups. The BOR-enriched diet reduced adrenal ANG II receptor density and affinity compared to the SES diet. Results suggest that BOR inhibits adrenal responsiveness to ANG II by an action on adrenal receptors. Our findings demonstrated that dietary GLA lowers SBP in adult SHR. This effect may be mediated, at least in part, by interference with the renin-angiotensin-aldosterone system at the level of adrenal ANG II receptors.

Fatty acids may regulate aldosterone secretion and mediate some of insulin's effects on blood pressure.

Experiments in vitro and observation made in humans suggest that some unesterified fatty acids (FA) participate, as inhibitors, in the regulation of aldosterone secretion. Removal of FA from adrenal glomerulosa cells with albumin increases the responses to angiotensin II (AII) and dibutyryl cyclic AMP. Micromolar concentrations of some FA including arachidonic, oleic, linoleic, eicosapentaenoic, and docosahexaenoic inhibit aldosterone secretion by adrenal glomerulosa cells. Inhibition is specific--some acids like stearic are inactive, and the adrenal fasciculata is relatively resistant to inhibition. Oleic acid rapidly and reversibly inhibits aldosterone secretion by perfused dog adrenals. Observations in vivo suggest a reciprocal relationship between plasma levels of FA and aldosterone: insulin infusion into dogs lowers plasma FA and increases adrenal responsiveness to All; salt infusions into humans increase plasma FA as aldosterone falls; plasma FA are low in low-renin essential hypertension where adrenal responsiveness to All is high; plasma FA are inversely correlated with ratios of aldosterone to renin in black hypertensives; and plasma FA are high in some seriously ill patients whose aldosterone levels are inexplicably low. All receptors and the final step of aldosterone biosynthesis, oxidation at the 18 position, are the adrenal sites most sensitive to FA. Insulins antinatriuresis may be mediated in part by its ability to lower plasma FA and thereby enhance adrenal response to secretagogues.

OXIDIZED PRODUCTS OF LINOLEIC ACID STIMULATE ADRENAL STEROIDOGENESIS

Adrenal steroidogenesis is under complex control, and clinical observations suggest that not all regulators have been identified. We postulated that fatty acid oxidation products found in the diet or formed in the body could affect steroidogenesis. Linoleic acid is a prominent constituent of animal fat and is readily oxidized. We found that several products of linoleic acid oxidation affect production of aldosterone and corticosterone by isolated cells from rat adrenals. We characterized one linoleic acid derivative by gas chromatography/mass spectrometry. It is 12,13-epoxy-9-oxo-10(trans)-octadecenoic acid (“EKODE”). At concentrations between 1 and 30 µM, EKODE stimulated production of aldosterone by zona glomerulosa cells, but at concentrations above 50 µM, it was inhibitory. In zona fasciculata cells, EKODE stimulated corticosterone production at concentrations of 5 µM or greater, and there was no evidence of inhibition at high concentrations. Stimulation of steroidogenesis was observed after 15 min of incubation and continued for at least 2 hrs. The potential relevance of our findings to the hypertension of obesity is discussed.

Activation of the antioxidant response element by specific oxidized metabolites of linoleic acid

Linoleic acid is required for normal mammalian health and development, but is also prone to oxidation, yielding metabolites with biological effects. We screened linoleic acid, other fatty acids, and some of their derivatives and found that an epoxy-keto derivative of linoleic acid (but neither linoleic acid itself nor others of its oxidation products) strongly activates the antioxidant response element (ARE) in IMR-32 neuroblastoma cells and cerebro-cortical neurons. The active compound, 12,13-epoxy-9-keto-10(trans)-octadecenoic acid (EKODE), induces the expression of ARE-regulated cytoprotective genes such as NQO1 at the transcript and protein levels. EKODE requires transcription factor NRF2 and PI3-kinase for ARE activity. The results suggest that specific oxidation products of linoleic acid may initiate responses that lessen damage caused by oxidative stress.

Salt loads raise plasma fatty acids and lower insulin.

Some fatty acids are potent inhibitors of angiotensin binding and aldosterone production in adrenal glomerulosa cells and thereby may be involved in regulating salt and water balance. To study the possible regulation of fatty acids by salt, we measured the levels of unesterified fatty acids in plasma from patients subjected to extremes of dietary salt intake and saline infusion. Insulin and catecholamines, two known regulators of plasma fatty acids, also were measured. Infusion of 2 1 saline over 4 hours caused the levels of most unesterified fatty acids to rise. Total unesterified fatty acids rose 60-100%. A high salt diet caused a smaller rise in total unesterified fatty acids (approximately 33%). In both instances, oleic and palmitoleic acids showed the greatest proportionate increases, whereas stearic acid was relatively unaffected. When salt loads were administered by either intravenous or dietary routes, plasma insulin levels fell by approximately 50%. Plasma norepinephrine increased after saline infusion but not during a high salt diet Postsaline levels of fatty acids correlated inversely with postsaline levels of aldosterone, supporting a possible role for fatty acids as physiological regulators of the adrenal glomerulosa. A rise in plasma fatty acids and fall in insulin in response to salt loads could act in concert to increase sodium excretion, constituting a physiological mechanism contributing to salt and water balance.

Lead Increases Aldosterone Production by Rat Adrenal Cells

Exposure to lead has been postulated to contribute to elevated blood pressure in humans and has been shown to raise blood pressure in animals. The mechanism of action of lead on blood pressure is unknown. We fed lead to rats in their drinking water and then examined the production of aldosterone by their adrenal cells in vitro. We also measured excretion of aldosterone and corticosterone by intact rats stimulated with corticotropin, with and without lead treatment. At a dose (273 ppm) that raised blood levels to 30 to 40 micrograms/dL, comparable to blood levels in exposed humans, lead induced increased aldosterone secretion in vitro and in vivo. The effect of lead was most evident when cells or animals were stimulated with aldosterone secretagogues. Experiments in vitro indicate that exposure to lead in vivo increases activity of one or more steps in the late pathway of aldosterone biosynthesis. The results suggest that the hypertensive effect of lead involves relative hyperaldosteronism and may be most evident when secretion of this hormone is stimulated.