In vitro effects of insulin on aldosterone production in rat zona glomerulosa cells

Life Sciences, Vol. 50, pp. 1781-1787 Printed

Pergamon P r e s s

in t h e U S A

IN VITRO EFFECTS OF INSULIN ON A L D O S T E R O N E P R O D U C T I O N IN RAT ZONA G L O M E R U L O S A CELLS Dan Petrasek,

Gerard Jensen*,

Michael Tuck,

and Naftali Stern**

Division of Endocrinology, Sepulveda V.A.M.C. and U.C.L.A. School of Medicine, Division 111E, Sepulveda V.A.M.C., Sepulveda, CA 91343, U.S.A. (Received in final form March 26, 1992) Summary Though long standing diabetes mellitus is frequently a c c o m p a n i e d by hypoaldosteronism, the role of insulin in this setting has never been clearly established. In the p r e s e n t study we have examined the direct effects of insulin on aldosterone p r o d u c t i o n in rat zona g l o m e r u l o s a cells in vitro. Insulin is shown to d i r e c t l y stimulate a l d o s t e r o n e p r o d u c t i o n in a dose d e p e n d e n t manner, and to a t t e n u a t e a n g i o t e n s i n II m e d i a t e d a l d o s t e r o n e production, without affecting angiotensin II receptor binding kinetics. Insulin had no effect on aldosterone p r o d u c t i o n m e d i a t e d by the other p h y s i o l o g i c a l stimuli (K÷ and ACTH). These data suggest a possible interaction between insulin and angiotensin II in the r e g u l a t i o n of a l d o s t e r o n e secretion. The characterization of insulin actions has been the object of study in a variety of cell systems (1,2). In several recent studies (3-5) there has been considerable emphasis on the possible roles of inositol phosphates, d i a c y l g l y c e r o l and Ca 2÷ as mediators of insulin actions. These mediators are also involved in the m e c h a n i s m of action of other peptide hormones, including a n g i o t e n s i n II. The present study provides first evidence for a cell system in which both insulin and a n g i o t e n s i n II are agonists, possibly sharing some intracellular pathway. This possibility is suggested by a novel finding for insulin, in that the hormone behaves as an agonist in its own right, and as an a n g i o t e n s i n II antagonist in terms of aldosterone production. Methods Male Sprague-Dawley rats (B.K., Fremont, CA, USA) weighing 275-350 g were held in our animal facility for a minimum of 7 days prior to experiment, and allowed free access to tap water and food (Purina Rat Chow, Ralson Purina, St. Louis, MO, USA). Zona g l o m e r u l o s a cells were prepared as previously d e s c r i b e d (6) with minor modifications. Briefly, adrenals from d e c a p i t a t e d rats were decapsulated, and the capsular portions were p r e i n c u b a t e d in a shaking water bath at 37°C for 50 minutes at 50 Present address: *Department of Chemistry, U n i v e r s i t y of Southern California, Los Angeles, California, 90089-0482 Corresponding address: ~ D e p a r t m e n t of Endocrinology, SouraskyT e l - A v i v Medical Center, Ichilov Hospital, 6 Weitzman Street, Tel-Aviv 64239, Israel 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights r e s e r v e d .

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oscillations per minute under 95% 0=/5% C02 in modified K r e b s - R i n g e r b u f f e r (pH = 7.4, henceforth "buffer") containing 1.85 mg/ml L-glutamine, 1.25% vol/vol of both essential and none s s e n t i a l amino acids, and 4% bovine serum albumin (BSA). The K÷ concentration was 3.7 mM. Following this preincubation, cells were filtered through a double layer of gauze and c e n t r i f u g e d at 750 x g for 10 minutes at room temperture. The s u p e r n a t a n t was discarded, and the pellet c o n t a i n i n g cells w a s h e d twice in buffer. Cells were then r e s u s p e n e d in b u f f e r c o n t a i n i n g only 0.5% BSA, and p r e i n c u b a t e d a second time for 60 m i n u t e s in the o x y g e n a t e d shaking water bath at 37°C. F o l l o w i n g this second preincubation, the cells were r e c e n t r i f u g e d at 750 x g for 10 minutes at room temperature, the s u p e r n a t a n t discarded, and the pellet w a s h e d once in buffer with 4% BSA. For the d e t e r m i n a t i o n of a l d o s t e r o n e p r o d u c t i o n rate, cells were suspended in buffer c o n t a i n i n g 4% BSA to yield 75,000 to 100,000 cells/ml (94 ± 2% of which were zona glomerulosa cells, the rest being zona fasciculata cells). 2 ml aliquots of the cell s u s p e n s i o n were i n c u b a t e d in an oxygenated, shaking w a t e r bath at 37°C for 60 minutes in triplicate with graded concentrations of insulin (Humulin R, Eli Lilly and Co., Indianapolis, IN, USA), a n g i o t e n s i o n II, ACTH and/or KCI (in the latter case raised from the basal K ÷ c o n c e n t r a t i o n of 3.7 mM). A l d s t e r o n e content in the entire incubate volume (cells and media, which were frozen and stored at -20°C prior to assaying) were measured by radioimmunoassay (using antibodies from Endocrine Sciences, Tarzana, CA, USA). Angiotensin II receptor b i n d i n g experiments in the absence and p r e s e n c e of 5 ng/ml insulin were p e r f o r m e d as described (7) with minor modifications. Adrenal c a p s u l a r tissue o b t a i n e d from seven to eight rats per assay was h o m o g e n i z e d with a telfon glass homogenizer in 2 ml of ice cold 10 mM Tris buffer (pH = 7.4) c o n t a i n i n g 25 mM sucrose and I mM EDTA. Following centrifugation of the h o m o g e n a t e at 1500 x g for 10 m i n u t e s the pellet was d i s c a r d e d and s u p e r n a t a n t then r e c e n t r i f u g e d at 20,000 x g for 30 minutes. The resultant pellet was washed twice and finally r e s u s p e n d e d in 50 mM Tris buffer (pH = 7.4) containing 120 mM NaCI, 1 mM EDTA, 2 mM MgCI2, and 0.2% BSA. Membranes (240 to 300 ug of c a p s u l a r protein) were incubated in t r i p l i c a t e for 45 minutes at 22°C with 150,000 dpm of 1 2 S I - l a b e l l e d a n g i o t e n s i n II (New E n g l a n d Nuclear, Boston, MA, USA); specific activity = 1,880 uCi/ug) alone or in the presence of various c o n c e n t r a t i o n s of u n l a b e l l e d a n g i o t e n s i n II (3 x 10 -~° M to I x 10-6M). Bound ~2SI-labelled angiotenisn II was s e p a r a t e d from the free r a d i o l i g a n d by M i l l i p o r e filtrations through buffer presoaked glass fiber GF/C filters (Whatman Inc., Clifton, NJ, USA). N o n s p e c i f i c b i n d i n g was 5 ± I% of the total binding. The p r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d by the m e t h o d of Lowry et al. (8). Data were a s s e s s e d by analysis of variance. The p a i r e d - t - t e s t was used for specific b e t w e e n group analysis when the overall analysis showed a s i g n i f i c a n t b e t w e e n - g r o u p difference. Results Figure 1 shows the direct in vitro effects of insulin on a l d o s t e r o n e p r o d u c t i o n in freshly p r e p a r e d rat zona glomerulosa cells. Insulin significantly stimulated aldosterone production at c o n c e n t r a t i o n s ranging from 1 ng/ml to 100 ng/ml (5 mU/ml to 100 mU/ml) with a maximal response at 5 ng/ml (control: 8.49 ±

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1.40, i n s u l i n 5 ng/ml: 22.55 ± 4.67 ng/106 cells, p<0.01). A l t h o u g h the 5 n g / m l d o s e a c h i e v e d the m a x i m a l r e s p o n s e , this was not significantly different from the 25 n g / m l a n d 100 n g / m l doses. Figure 2 demonstrates that aldosterone response to angiotensin II over a range of c o n c e n t r a t i o n s (10-SM-10-1°M) b o t h a l o n e and in c o m b i n a t i o n w i t h 5 n g / m l insulin. AS s e e n in the figure, i n s u l i n s i g n i f i c a n t l y a t t e n u a t e d the a n g i o t e n s i n II m e d i a t e d s t i m u l a t i o n of a l d o s t e r o n e p r o d u c t i o n at both 10 -8 M and 10 -9 M c o n c e n t r a t i o n s of a n g i o t e n s i n II. Both angiotensin II s t i m u l a t i o n of a l d o s t e r o n e p r o d u c t i o n and i n s u l i n attenuation thereof were m a x i m a l at the 10 -9 M c o n c e n t r a t i o n of a n g i o t e n s i n II (angiotensin II 10 -9 M: 75.1 ± 8.6 ng/106 cells, a n g i o t e n s i n II 10 -9 M + insulin 5 ng/ml: 52.3 ± 6.3 ng/106 cells, p < 0 . 0 1 ) . In contrast, when insulin at 5 n g / m l w a s c o i n c u b a t e d w i t h e i t h e r K ÷ or A C T H o v e r a w i d e r a n g e of c o n c e n t r a t i o n s , we could not observe any significant c h a n g e in a l d o s t e r o n e p r o d u c t i o n . T a b l e s I and 2, r e s p e c t i v e l y , d e p i c t the K ÷ and ACTH stimulated a l d o s t e r o n e p r o d u c t i o n b o t h alone and in c o m b i n a t i o n w i t h 5 n g / m l insulin. At all ACTH and K÷ concentrations e x c e p t 10 - 1 1 M ACTH, i n s u l i n a p p e a r e d to e n h a n c e a l d o s t e r o n e p r o d u c t i o n , though in no c a s e are the d i f f e r e n c e s s t a t i s t i c a l l y s i g n i f i c a n t .

Table

1:

E f f e c t of i n s u l i n on K ~ s t i m u l a t e d p r o d u c t i o n (ng/106 cells, n=3)

[K+]: No

insulin

Insulin

Table

2:

[ACTH]: No

Insulin

Insulin 5ng/ml

5ng/ml

aldosterone

5.7 mM

8.7 m M

10.7

50.9±17.6

213.9±20.2

177.9±20.1

88.1±33.3

254.6±40.4

184.5±30.1

E f f e c t of i n s u l i n on A C T H s t i m u l a t e d p r o d u c t i o n (ng/106 cells, n=3) 10-IOM

mM

aldosterone

10-12M

10-11M

10-9M

60.4±13.9

126.8±24.1

285.5±50.8

265.9±46.6

292.2±84.9

69.9±13.6

84.7~25.1

316.1±62.7

368.7±55.8

292.8±84.9

10-aM

To determine w h e t h e r i n h i b i t o r y e f f e c t of i n s u l i n is t a k i n g p l a c e at the a n g i o t e n s i n II membrane receptor, angiotensin II binding studies were performed (Table 3). No e v i d e n c e for a l t e r n a t i o n in a n g i o t e n s i n II r e c e p t o r a f f i n i t y or concentration could be d e m o n s t r a t e d in the p r e s e n c e of 5 n g / m l i n s u l i n (Table 3).

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Table 3:

Effects of Insulin on Aldosterone

Effect of insulin on a n g i o t e n s i n (n=3)

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II

Receptor Concentration (fmol/mg protein) No insulin Insulin 5ng/ml

receptor

binding

Kd (nM)

964±48

0.89±0.25

1035±32

0z75±0.17

Discussion Angiotensin II, K÷ and ACTH are the major known p h y s i o l o g i c a l regulators of aldosterone b i o s y n t h e s i s in m a m m a l i a n zona g l o m e r u l o s a cells. Several studies have alluded to the p o s s i b i l i t y that insulin may play a role in m e d i a t i n g a l d o s t e r o n e p r o d u c t i o n (10,11). Despite this implication, a direct effect of insulin on a l d o s t e r o n e production has not been tested until now. The primary finding in this study is that insulin directly affects a l d o s t e r o n e p r o d u c t i o n in rat zona g l o m e r u l o s a cells in a bimodal manner. Insulin was seen to both directly stimulate a l d o s t e r o n e p r o d u c t i o n and significantly attentuate angiotensin II m e d i a t e d aldosterone production. It is n o t e w o r t h y that the s t i m u l a t i n g c o n c e n t r a t i o n s of insulin seen here are similar to those shown by Olefsky (9) to stimulate 2 - d e o x y g l u c o s e uptake in rat adipocytes, whereas much higher insulin concentrations are g e n e r a l l y required to reproduce IGF effects (14). A l t h o u g h this is the first demonstration of a direct effect of insulin on a l d o s t e r o n e production, it is noteworthy that insulin receptors are d e m o n s t r a b l e in the adrenal cortex (12-14) and that there are precedents for an insulin role in s t e r o i d o g e n e s i s in other cell systems (15,16). 25-

T

zo:

J

,0~

INSULIN (nglml]

FIG. I In vitro aldosterone production by rat zona g l o m e r u l o s a cells during a 60 minute incubation in the presence of various c o n c e n t r a t i o n s of insulin. Results are means ± SEM of seven separate experiments (*p<0.05 compared to control (C);~*p<0.01 compared to control).

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Effects of Insulin on Aldosterone

1785

140-

t20"o i0o~ 8o-

no insulin~ ' 4 , ~

~ 6o-

Z,n,u,i I

8 4oO J

'~ 2 0 -

i

io-IO

//

l

10-9

//

l

iO -e

ANGIOTENSIN 31" [M)

FIG.2 In vitro aldosterone production by rat zona g-~omerulosa cells during a 60 minute incubation in the presence of various concentrations of angiotensin II, both alone and in c o m b i n a t i o n with 5 ng/ml insulin. Results are means ± SEM of five separate experiments (*p<0.05 comparing 10 -s M angiotensin II ± 5 ng/ml insulin; **p<0.01 comparing 10 -9 M angiotensin II ± 5 ng/ml insulin). The effects of insulin in this study were noted with human insulin; its use in rat tissues thus merits consideration. There are two rat insulins, and all rats are homozygous for both (17). Rat insulin I and II have five and four amino acid differences from human sequence respectively (17). Though substitutions in these positions have been shown to cause changes in p r o i n s u l i n linkages and in the formation of zinc hexamers, they do not seem to affect receptor binding (18,19). C o m p a r i s o n of the binding of rat insulins and porcine insulin to rat adipocytes shows very similar activity, porcine insulin having actually slightly higher activity (19). At first glance the attenuating effect of insulin on a n g i o t e n s i n II m e d i a t e d steroidogenesis in zona g l o m e r u l o s a cells seemed paradoxical in view of the fact that both insulin and angiotensin II individually were observed to stimulate aldosterone production. Some investigators have suggested that insulin may enhance aldosterone production via a K ÷ pathway (10). It has long been recognized (20) that insulin lowers the plasma concentration of K ÷ by promoting the i n t r a c e l l u l a r entry of K ÷, possibly via a Na÷,K + pump (21). However, even if this mechanism is operative in zona g l o m e r u l o s a cells there is currently no evidence that an increase in i n t r a c e l l u l a r K ÷ pe T se will lead to an increase in aldosterone p r o d u c t i o n (22,23). I--t has been suggested that insulin activates diacylglyerol/protein kinase C system (3-5). However this

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appears to occur via a specific p h o s p h a l i p a s e C leading to the production of inositol glycan and a distinct a r a c h i n d o n i c aciddeplete diacylglycerol (24). These newly identified second messengers are distinct from the well established p h o s p h o i n o s i t i d e / C a 2÷ m e s s e n g e r system involved in a n g i o t e n s i n II-dependent steroidogenesis (24-29). However, insulin and a n g i o t e n s i n II could still be converging on some common element at a more distal site, so that insulin could alter a n g i o t e n s i n II's ability to m a x i m a l l y stimulate aldosterone production yet retain a modest capacity to independently stimulate a l d o s t e r o n e biosynthesis. Consistent with such a model is the observation that ACTH and K ÷ are not known to mediate aldosterone biosynthesis via an inositol p h o s p h a t e / d i a c y l g l y c e r o l pathway (24-27) and insulin did not affect their ability to stimulate aldosterone p r o d u c t i o n in this study. Finally, the selectivity of insulin in inhibiting only angiotensin II m e d i a t e d s t e r o i d o g e n e s i s could not be attributed to altered a n g i o t e n s i n II receptor binding kinetics, suggesting that the i n s u l i n / a n g i o t e n s i n II interaction likely takes place at a site distal to the a n g i o t e n s i n II membrane receptor. that the r e g u l a t i o n of a l d o s t e r o n e These results suggest in h y p e r i n s u l i n e m i c states via a s e c r e t i o n could be altered interaction between insulin and previously unrecognized a n g i o t e n s i n II. Acknowledgements This work was supported by a Veterans A d m i n i s t r a t i o n M.R.I.S. grant. The authors wish to thank Stephen Jensen for helpful discussions. The authors also wish to thank A n d r e w Tuck for his expert word processing assistance in p r e p a r a t i o n of this manuscript.

I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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