Obesity-Stimulated Aldosterone Release Is Not Related to an S1P-Dependent Mechanism
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235 3 S WERTH and others S1P and aldosterone release II 235:3 251–265 Research Obesity-stimulated aldosterone release is not related to an S1P-dependent mechanism Correspondence Stephan Werth1, Helge Müller-Fielitz1,2 and Walter Raasch1,2,3 should be addressed 1Institute of Experimental and Clinical Pharmacology and Toxicology, University of Lübeck, Lübeck, Germany to W Raasch 2CBBM (Center of Brain, Behavior and Metabolism), Lübeck, Germany Email 3DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Lübeck, Germany walter.raasch@pharma. uni-luebeck.de Abstract Aldosterone has been identified as an important factor in obesity-associated Key Words hypertension. Here, we investigated whether sphingosine-1-phosphate (S1P), which f renin–angiotensisn system has previously been linked to obesity, increases aldosterone release. S1P-induced f obesity aldosterone release was determined in NCI H295R cells in the presence of S1P receptor f steroids (S1PR) antagonists. In vivo release of S1P (100–300 µg/kgbw) was investigated in pithed, f hypertension lean Sprague Dawley (SD) rats, diet-obese spontaneous hypertensive rats (SHRs), as well f spingosine-1-phosphate as in lean or obese Zucker rats. Aldosterone secretion was increased in NCI H295R cells Endocrinology of by S1P, the selective S1PR1 agonist SEW2871 and the selective S1PR2 antagonist JTE013. Treatment with the S1PR1 antagonist W146 or fingolimod and the S1PR1/3 antagonist VPC02319 decreased baseline and/or S1P-stimulated aldosterone release. Compared to Journal saline-treated SD rats, plasma aldosterone increased by ~50 pg/mL after infusing S1P. Baseline levels of S1P and aldosterone were higher in obese than in lean SHRs. Adrenal S1PR expression did not differ between chow- or CD-fed rats that had the highest S1PR1 and lowest S1PR4 levels. S1P induced a short-lasting increase in plasma aldosterone in obese, but not in lean SHRs. However, 2-ANOVA did not demonstrate any difference between lean and obese rats. S1P-induced aldosterone release was also similar between obese and lean Zucker rats. We conclude that S1P is a local regulator of aldosterone production. S1PR1 agonism induces an increase in aldosterone secretion, while stimulating adrenal S1PR2 receptor suppresses aldosterone production. A significant role Journal of Endocrinology of S1P in influencing aldosterone secretion in states of obesity seems unlikely. (2017) 235, 251–265 Introduction The Framingham Offspring Study suggested that tissue of obese rats and humans (Giacchetti et al. 2002, increased abdominal fat mass accounts for 65–75% of Boustany et al. 2004, Engeli et al. 2005). Accordingly, the risk for hypertension (Garrison et al. 1987). Among ACE is upregulated in adipocytes (Bloem et al. 1995, other hormonal systems, the renin–angiotensin– Cooper et al. 1998, Karlsson et al. 1998), and obese aldosterone system (RAAS) has been established as a individuals demonstrate elevated circulating angiotensin major determinant of obesity-induced hypertension II (AngII) levels (Harte et al. 2005). In contrast, weight (Rahmouni et al. 2005). The RAAS is activated in the fat loss not only normalizes enzyme activity of the RAAS but http://joe.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-16-0550 Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 09:44:55PM via free access 10.1530/JOE-16-0550 Research S WERTH and others S1P and aldosterone release II 235:3 252 also attenuates blood pressure (Engeli et al. 2005). The ERK1/2-dependent upregulation of StAR activity (Krug et al. potency of AT1 receptor blockers to lower body weight 2007) and S1P induces both StAR mRNA expression and particularly in obese individuals and the findings that ERK1/2 activation (Brizuela et al. 2007, Lucki et al. 2012), body weight of double AT1A/AT1B receptor knockout mice we hypothesized that S1P acts as an MRF. The molecular is lower than in wild-type controls further strengthen mass of S1P is <50 kDa and it is heat-sensitive (>99°C), the functional cross-talk among weight, blood pressure both thought to be essential features of MRF (Lamounier- regulation and RAAS activity (Gembardt et al. 2008, Zepter et al. 2006). S1P is a bioactive sphingolipid and Müller-Fielitz et al. 2015). interacts with its particular Gi protein-coupled receptors, Aldosterone is the second key player of the RAAS. termed S1PR1-5 (Maceyka & Spiegel 2014, Proia & Hla Aldosterone production in zona glomerulosa (ZG) cells 2015). S1PRs are highly expressed in the human adrenal is regulated by distinct extracellular stimuli (potassium, gland (Wang et al. 2014). S1P interacts not only in cellular AngII, ACTH) via Ca2+ mobilization following cAMP- processes of immunity, inflammation and inflammatory dependent (ACTH) or -independent (AngII, potassium) disorders (Maceyka & Spiegel 2014, Proia & Hla 2015), but mechanisms. Aldosterone plasma levels positively is also involved in cardiovascular and metabolic functions correlate with fat mass in obesity (Lamounier-Zepter et al. (Bellini et al. 2015, Borodzicz et al. 2015). Interestingly, 2006, Krug & Ehrhart-Bornstein 2008) and with obesity- there is clear evidence that S1P levels are higher in plasma induced hypertension (Kidambi et al. 2007). The following and adipose tissue of obese subjects than in lean individuals findings indicate that aldosterone excess is not an (Samad et al. 2006, Blachnio-Zabielska et al. 2012a, epiphenomenon to higher AngII levels, but rather results Kowalski et al. 2013), thus supporting the hypothesis that from adrenal hypersensitivity to AngII among overweight S1P may indeed be the missing link between obesity and individuals: (1) AngII-mediated aldosterone secretion hypertension due to adrenal hypersensitization. Thus, was higher in obese than in lean individuals (Bentley- we hereby aimed to investigate whether S1P stimulates Lewis et al. 2007, Müller-Fielitz et al. 2012b); (2) adipocytes aldosterone release in a human adrenocortical cell line secrete mineralocorticoid-releasing factors (MRFs), which and, if so, which S1PR would be involved. Furthermore, Endocrinology themselves enhance aldosterone release independently of S1P-induced aldosterone release was expected to be higher of AngII (Ehrhart-Bornstein et al. 2003, 2004); (3) adipocytes in obese than in lean rats. sensitize adrenocortical cells to AngII via ERK1/2- dependent upregulation of steroidogenic acute regulatory Journal (StAR) protein (Krug et al. 2007); (4) ZG growth and the Materials and methods expression of aldosterone synthase and AT1B receptors were selectively heightened in obesity under concomitant Animals AngII stimulation, which further enhanced circulating aldosterone levels and augmented blood pressure The study was carried out according to the National response to AngII (Müller-Fielitz et al. 2012b); and (5) Institutes of Health guidelines for the care and use the rise in blood pressure after chronic AngII stimulation of laboratory animals. Animal care and experimental was diminished in adrenalectomized obese Zucker rats in procedures were approved by the ethics committee of the which plasma aldosterone levels could only be scarcely local regulatory authority. Male SD rats, SHRs and lean detected (Müller-Fielitz et al. 2012b). and obese Zucker rats were used in the experiments (all However, MRFs are still being identified , including from Charles River, Sulzberg, Germany). The animals were at least two that differ in heat sensitivity and molecular kept at room temperature with a 12-h/12-h dark (14:00– mass (< or >50 kDa) (Ehrhart-Bornstein et al. 2003, 2004). 02:00 h)/light (02:00–14:00 h) cycle. Based on observations that plasma LDL is increased in hypertensive obese patients, Ehrhart-Bornstein’s group Preparation of S1P stock solution also showed that LDL dramatically increased aldosterone release in human adrenocortical NCI H295R cells. Thus, S1P was obtained from Avanti Polar Lipids (Alabaster, aldosterone excess in hypertensive individuals may AL, USA). A methanolic S1P stock solution was prepared. partially result from elevated LDL levels. However, it S1P (0.5 mg/mL) was added to a methanol/water mixture was speculated that other MRFs may also be involved (95/5), heat-mixed (50°C) and sonicated until S1P (Lamounier-Zepter et al. 2006). Since the identified was suspended. Subsequently, 100 µL aliquots of this MRF was shown to increase aldosterone release via mixture were stored (−20°C) in glass tubes. For usage, http://joe.endocrinology-journals.org © 2017 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-16-0550 Printed in Great Britain Downloaded from Bioscientifica.com at 09/28/2021 09:44:55PM via free access PROOF ONLY Research S WERTH and others S1P and aldosterone release II 235:3 253 aliquots were warmed up to room temperature and the were performed without S1P antagonists. After 30-min methanol/water were evaporated under nitrogen flow. The preincubation, medium was removed and 500 µL fresh dry residue was dissolved in saline (0.9%), supplemented medium containing the appropriate S1P antagonist and with bovine serum albumin (BSA, 4 mg/mL). 10 µM S1P or vehicle was added. After 24 h, medium was collected and stored at −20°C until aldosterone analyses. Cells were washed twice with 500 µL PBS buffer and also Cell culture stored at −20°C until protein was quantified according In vitro assays of S1P-induced aldosterone release were to Lowry. performed by using the human adrenocortical carcinoma NCI H295R cell line. Only cells originating from the 4th Pithed rat preparation to 20th passage were used. In accordance with previous work, cells were grown in 20 mL Dulbecco’s Modified Eagle Studies on aldosterone response to S1P were performed in Medium/Ham’s F12 (DMEM/F12; from VWR International, a ‘pithed rat’ model, to only consider vascular reactivity Darmstadt, Germany) containing 15 mmol/L HEPES and to S1P for blood pressure effects. Rats were pithed at 2.5 mmol/L L-glutamine, supplemented with 1.125 g/L the beginning of the light cycle as previously described NaHCO3, insulin (66 nmol/L), hydrocortisone (10 nmol/L), (Raasch et al.