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Fatty acids and steroid hormone action
PROSTAGLANDINSLEUKOTRIENES AND ESSENTIALFATTYACIDS Prostaglandins Leukotrienes and Essential Fatty Acids (1995) 52, 185-190 © Pearson Professional Ltd 1995
Fatty Acids and Steroid Hormone Action E. A. Nunez, M. Haourigui, M. E. Martin and C. Benassayag Laboratoire de Biochimie Endocrinienne, Facultd de Mddecine Xavier Bichat, Universit( Denis Diderot-Paris 7, B.P. 416, 75870 Paris Cedex 18, France (Reprint requests to EAN)
contradicted by authors who have denied their occurrence in vivo (9). We will describe some of our recent results showing that the data obtained in vitro are actually confirmed under in vivo conditions. Lastly, we will use these data to develop a model showing the regulatory loop between lipase activity, fatty acids per se, fatty acid metabolites, and the biological activity of steroids on cell multiplication and on cell differentiation. It is particularly important to mention that NEFAs are potent modulators or regulators of many of the enzymes involved in the membrane peptide signalling (10). The studies carried out in our laboratory (11, 12) and presented in this issue (by us and Dr Sumida) clearly indicate that NEFAs are the regulators of the two mechanisms by which steroid (nuclear) and peptide signals (membrane) are transferred, and that NEFAs regulate the cross talk between these two signalling pathways involved in cell multiplication or differentiation.
INTRODUCTION
Epidemiological and experimental nutritional studies have shown that there is a link between lipids, endocrinology and cancer (1). These studies have been confirmed by the demonstration that the multiplication of estrogen-dependent cells, such as MCF7 or breast cancer epithelial cells, can be either positively or negatively influenced by non-esterified fatty acids (NEFAs), particularly unsaturated NEFAs (UFAs) (1, 2). Many studies have shown that changes in fipid nutrition induce changes in the reproductive functions (3-5). Our interest in this field was triggered by our in vitro observations that unsaturated and polyunsaturated unesterified fatty acids (UFAs) inhibit the binding of natural estrogens (6) to murine (estrophilic) alphafetoprotein (AFP). AFP is produced during fetal and postnatal life and by hepatocellular carcinoma cells and yolk sac-derived tumors (7). The function of this protein is still not known, but it is becoming more and more evident that AFP has several biological actions, which depend on the surrounding conditions (7). It is now clear that these biological functions depend on the fatty acids, growth factors, steroids in the environment. Besides several other functions, AFP regulates the multiplication or differentiation of steroid-dependent cells by a mechanism which is not yet elucidated. Recent results suggest that the regulating effect of AFP on fatty acid concentrations and metabolism are particularly important for the way this takes place (7). Thus, it is important to elucidate the endocrine role of NEFAs to understand one of the biological roles of AFP. It is clear also from various in vitro studies that the binding, electrophoretic, antigenic properties (and by consequence probably the biological properties) of different plasma steroid binding proteins like corticosteroid binding globulin are positively or negatively influenced by NEFAs (8). This review summarizes the results obtained in vitro (and at the molecular level) showing how NEFAs modulate or regulate the various steps in the transfer of steroidborne information. Some of these results have been
IN VITRO STUDIES S H O W I N G M O D U L A T I O N BY NEFAS OF STEROID M E T A B O L I S M
Many recent in vitro studies (12) have shown that NEFAs, without any metabolic transformation, are able to modulate various steps in the transfer of steroid information (Fig. 1). Recently new data have been obtained showing: Influence of NEFAs on gonadotropin secretion
The main results describing this action have been recently reviewed (13). Arachidonic acid (AA) and/or its metabolites mediate GnRH-stimulated gonadotropin release. It seems probable that AA acts on the initial phase of exocytosis of the stored LH. PKC, phospholipase A2, diacyl glycerol lipase, the influx of calcium, the cyclooxygenase and lipoxygenase pathways seem to be also involved in this action. The relative importance of these pathways involved in the stimulation of LH activity seems to depend on the type of LH secretion (basal, stimulated, glycosylated LH). Further work is needed to amplify these data. 185
186
Prostaglandins Leukotrienes and Essential Fatty Acids
Gn RH
Cholesterol exogenousend°gen°us (lipoproteins)
~
GS
~ I Steroid IO} n Metabolism I ~"
HRE
otI
.//sz LS
OorO (PKC...)
' P (S.B.P., AFP, ...) cell mult.
GS : Gonadostimulin
S : Steroid P : Steroid binding protein S.B.P. : Sex steroid binding protein LS : Lipoidal steroid PKC : Phospho protein kinase Fig.
1
Cell diff.
AFP : Alphafetoprotein Px : Protein O : Non esterified fatty acids "per se" O : Non esterified fatty acids metabolites TF : Transcription factor R : Receptor HRE : Hormone responsive element
Role of NEFA on the various steps in the transfer of steroid information leading to the expression of a biological function.
Free fatty acid stimulated increase in LDL cholesterol uptake by cells The acylation of LDL by fatty acids (14) or the unmasking of a putative receptor (15) leads to the uptake and degradation of the lipoproteins by cells unable to synthesize LDL receptor. The two actions have been described for placental microvilli and fibroblast. The placenta uses the exogenous cholesterol obtained by these means for the intensive steroidogenesis which takes place during gestation (16). Action of NEFAs and their metabolites on steroidogenesis Various enzymes (5 ~ reductase (17), aromatase (18), 18 hydroxydehydrogenase (19), 5ene, 3 ~ hydroxysteroiddehydrogenase (20), 17 [~ hydroxysteroid-dehydrogenase (21)) of steroid metabolism are under the control of NEFAs. It is also important to remember that the NEFAs and their metabolites produced in the cyclooxygenase, lipoxygenase, or epoxygenase pathways can have a
different modulation effect on steroid metabolism or function. This is a particularly difficult problem to precisely describe, giving each UFA substrate and its various metabolites a precise role. One must realize that if inhibition of an enzyme which produces a fatty acid metabolite does in fact inhibit the biological effect of the UFA, it is not necessarily the metabolite which is responsible for the effect of the UFA. The increased concentration of the UFA itself can be responsible for this effect. It is clear from recent data that it is the accumulation of the free UFA (having a positive or negative biphasic effect) which is responsible for the inhibition or activation of a function and not the metabolite (22). UFAs may also regulate steroidogenesis in another rather complicated way. A recent study (23) showed that AA per se, via activation of PKC, exerts a negative effect (short-term) on the early synthesis of testosterone by LH-stimulated Leydig cell. But when PKC is down regulated by arachidonic acid (long-term), steroidogenesis is enhanced due to the action of arachidonic metabolites produced via the lipoxygenase pathway. These metabolites stimulate testosterone biosynthesis.
Fatty Acids and SteroidHormoneAction 187
Action of NEFAs on steroid binding by specific steroid binding proteins Numerous in vitro studies have shown that NEFAs can influence positively or negatively the binding of a steroid to its specific binding protein in the plasma (12). But other studies contradict this data and suggest that the NEFA pool has no influence on the binding of steroid in vivo (9). The discrepancies between these in vitro results can be explained by the different experimental conditions in which these studies have been performed (dilution, ratio between albumin or other specific steroid or fatty acid binding proteins, concentration in the milieu of other ligands, nutritional situation, etc.). It is also necessary to evaluate the in situ concentration of the fatty acid, steroid and binding protein at a precise point of the cell structure (12). The relative percentage of each factor at this precise point can greatly modify, positively or negatively, the binding properties of a specific steroid binding protein in the plasma or in the tissue.
Effect of NEFAs on specific steroid binding to tissue receptors Many studies have shown the positive or negative effects of UFAs on the binding of steroid hormones to the superfamily of steroid hormone receptors (12). Recently, we have found that the site of interaction of polyunsaturated fatty acids is on a fragment of the rat glucocorticoid receptor containing the hormone-binding domain and identified some sequences C-terminal of the DNA-binding domain (24). We do not yet know if this binding is direct or indirect via another fatty acid binding protein which may be bound to this region.
IN VIVO INFLUENCE OF NEFAS ON STEROID ACTION Changes in plasma NEFAs induced in vivo by heparin and their effect on the functional properties of ~-fetoprotein Previous in vitro studies have shown that UFAs induce conformational changes in rodent and human AFP (12). We have carried out a series of studies to determine whether such changes in the binding and immunological properties of rat AFP also occur in vivo (25). In these experiments, NEFA concentrations were increased in young male rats (15, 21 and 28 days old) by acute i.v. injection of heparin (200 UUKg). Plasma estrogens (estrone and estradiol) did not change after injection of heparin. But there were large increases in plasma NEFAs 10-20min post-heparin injection, with a return to normal 60 rain later. This transient rise in plasma NEFA caused a 50% drop (p < 0.001) in the binding of estradiol to AFP of 15, 21 and 28-day-old rats by reducing the number of binding
sites (p<0.001), leaving the affinity constant (Ka) unchanged. NEFAs extracted from post-heparin plasma induced similar changes in estradiol binding to purified rat AFP. The rise in plasma NEFAs caused a loss of AFP immunoreactivity, especially in 21 (p < 0.001), and 28day-old rats (p < 0.001), but not in 15-day-old rats. This age-dependent response correlated with the NEFAs/AFP ratio (44 in 15-day-old rats, 375 in 21-day-old rats, and 6000 in 28-day-old rats). One of the most striking findings to come out of these experiments is the reversible effect of the rise of NEFAs on the binding and immunological properties of AFP. This indicates that AFP can adapt to, and interact with the environmental changes which occur during ontogenesis, oncogenesis and in pathological situations.
Effect of free fatty acids on the binding of glucocorticoids to corticosteroid binding globulin (CBG) and liver receptors during development in the rat (26, 27) The ontogenic patterns of NEFAs and CBG lead to specific NEFAs/CBG molar ratios at each stage of development. Plasma NEFAs are higher in young rats than in adult, while CBG is lower in immature rats. This gives a NEFAs/CBG molar ratio of 6000 in suckling rats (15 days), 2500 in weaning rats (21 days), 750 in prepubertal rats (28 days) and 360 in adult rats (60 days). We have examined the effect of heparin-induced lipolysis on the function of CBG at these four stages. The amounts of CBG in the plasma did not change, but CBG binding activity did. Thus the large increase in plasma NEFAs which occurs 10rain and 2 0 m in (p < 0.001) after lipolysis in 15-day- and 21-day-old rats were associated with a 50% drop (p < 0.001) in binding (C values L/g) of corticosterone (B) to immature rats CBG 60 min post-injection. The decrease in binding in 15-day-old rats is due to a reduction in the number of binding sites (n) (p < 0.001), with no change in the affinity constant (Ka). Both binding parameters changed (Ka was reduced and n increased) in 21-day-old rats. The increase in plasma NEFAs occurred later (20 and 60 rain p < 0.001) in 28-day- and 60-day-old rats. It was correlated with an increase in B binding to CBG (2-3fold p < 0.001). The increased B binding was due to an increase in Ka (p < 0.01), with no changes in n for 28day-old rats and to a 2-fold increase in n, without significant changes in Ka for adult rats. Such variations in CBG binding parameters strongly suggest that a rise in plasma NEFAs induces conformational changes in CBG. NEFAs extracted from post-heparin plasma, or a mixture of NEFAs with the same composition induced similar changes in B binding to prepurified immature and purified adult rat CBG. These opposing age-dependent CBG responses to lipolysis could be due to the NEFAs/CBG molar ratio and also to differences in CBG glycosylation. Our
188 Prostaglandins Leukotrienes and Essential Fatty Acids
results emphasize the importance of the NEFAs environment in modulating the endocrine function of the CBG during development. The endogenous NEFAs in the liver cytosol of immature rats was also doubled, 60 rain after heparin-induced lipolysis. The increased cytosol NEFAs, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor (GR). The decrease resulted from a significantly lower apparent K a for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between K, (r = -0.93) and n (r = -0.90) and the increased liver cytosol NEFAs content. Thus the higher plasma NEFAs induced in vivo by lipase activation or a standard NEFAs mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR. The liver content of endogenous NEFAs in mature rats was decreased (6%), particularly for the polyunsaturated fatty acids, 60 min after heparin-induced lipolysis. This decrease in non-esterified fatty acid contents was accompanied by a significant increase in dexamethasone binding (Ka enhanced, 1.5-fold, n no change).
Stimulation of glucocorticoid binding to human eorticosteroid binding globulin after post-prandial lipolysis of a high-fat meal (28) As shown in earlier in vitro studies for both rat and man (8, 12), and in vivo for the rat (26, 27), NEFAs promote changes in the molecular state of CBG which, depending on their concentration and degree of unsaturation, either enhance or reduce cortisol (F) binding. We studied the influence of the changing serum NEFAs concentration that occurred after the ingestion of fat-rich meals differing in the nature of fatty acids to determine whether such changes in CBG binding activity occur in vivo for man. 10 normolipidemic women volunteers (20-30 years old) ingested 60 g loads of different fats: tallow (46% saturated, 38% C 18:1), oleic-sunflower acid (76% C 18:1), sunflower (65% C 18:2), and a mixture of oils (42% C18:1 and 43% C18:2). Blood samples were collected before the fat load and after 2, 4, 6 and 8 h. The CBG concentration did not change post-prandially, but the CBG activity changed, and these changes differed according to the fat ingested. Tallow and sunflower-oleic fat loads caused a 2-fold rise in NEFAs at 6 and 8 h (p < 0.005 Dunnet's test) influencing mostly the saturated and monounsaturated fatty acids (p <0.005). Concomitantly, CBG binding indices significantly increased (p < 0.005), as a result of a 2-fold increase in affinity constant (Ka), while the number of binding sites (n) slightly decreased (30%). The ingestion of sunflower or mixed fats led to a moderate NEFAs rise 8 h after the meal (25%) (P < 0.01), inducing no significant changes in F binding to CBG. These results provide evidence that post-prandial increases in serum NEFAs can induce conformational and
functional changes in CBG in vivo, which may regulate tile availability of glucocorticoids and alter their biological impact. Thus, the serum concentrations of cortisol were high at the beginning of the experimental period (8:00 AM). After ingestion of sunflower or mixed fat they gradually decreased and were 2-fold lower at the end of the test (04:00 PM), in agreement with a circadian variation in plasma cortisol. By contrast, after the meals of tallow or sunflower-oleic oils, the diurnal rhythm of cortisol was lost. As a result, 8 h post-prandially, i.e at 04:00 PM, the level of cortisol was not significantly different from that of the morning.
DISCUSSION It is clear that the overwhelming evidence indicates that fatty acids do regulate in vivo at least some steps of the transfer of the steroid hormone message (Fig. 1). The global effect of NEFAs on a steroid-regulated function can be due to NEFAs affecting not only one but several steps. Thus, the change in the diurnal cortisol concentration observed in the women volunteers after a breakfast loaded with tallow or oleic-sunflower may be due to the enhanced binding capacity of cortisol by CBG, and/or the NEFA effect on ACTH production (29) and/or the diminution of cortisol catabolism (30). More work is needed to extend these in vivo observations to other steps in the transfer of the steroid message, such as receptor-mediated uptake of the plasma specific binding protein - steroid complex or specific protein transcription at the genomic level. It would also be interesting to perform new studies using other lipolytic procedures (e.g. lipolysis resulting from adrenergic stimulation) and using specific inhibitors of lipolytic enzymes. These experiments inducing lipolysis could be done on animals or humans after nutritional manipulation. It is particularly easy to alter the lipid profile of a mamalian organism by changing the quality of the fatty acid intake (saturated, unsaturated, series (n-3, n-6, etc.)) (4). All these studies have been performed in the blood stream. In fact, it would be particularly interesting to analyse the changes in FFA which occur in situ in normal arld some physiopathological situations (peroxisomal pathologies, diabetes, AIDS) in tissues (e.g. the seminal fluid where important variations in the concentration of UFAs are observed (31), placenta, liver, prostate). These experiments should be performed in real time using appropriate techniques taking into account the fact that this time could be very short. Nevertheless, results that have been obtained until now can be used to elaborate a model which incorporates a regulatory loop between lipase activity, fatty acids per se, fatty acid metabolism and steroids, acting on cell multiplication and differentiation (Fig. 2). We suggest that the shift between cell multiplication and cell differentiation depends on this balance between fatty acids and their metabolites, together with other factors like growth
Fatty Acids and Steroid Hormone Action
Lipoproteins] Lipids
Acknowledgements
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l
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~
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Model showing the regulatory loop between lipase activity, fatty acids per se, fatty acid metabolism and the biological activity of a steroid on cell multiplication and cell differentiation.
factors (Fig. 3). The biphasic effect of UFAs on PKC (or other enzyme involved in membrane signal transduction) can be one of the crucial elements which determine this balance (23). The hypothesis may be further refined by introducing an action of the fatty acid binding proteins, like albumin, AFP and the genuine fatty acid binding proteins. We have recently speculated on this type of regulation for AFP (7). Taken together with the studies presented in this issue (11) on the influence of UFAs on glucocorticoiddependent gene transcription, it is clear that fatty acids are regulators of the two mechanisms by which steroid (intranuclear) and peptide signals (membrane) are transferred. Fatty acids regulate the cross-talk between these two signalling pathways and hence the shift from cell multiplication to cell differentiation.
Fatty acids binding pr ein~ (AFP, FABP, .....~t
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189
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3 Taking together various results (7, 23), we hypothesize that the shift between cell multiplication and cell differentiation depends on the balance between fatty acids per se and their metabolites together with other factors like growth factors. The hypothesis may be further refined by introducing an action of fatty acid binding proteins.
We are grateful to Dr C. Owen Parkes for help in preparing the manuscript and Mrs Paulette He Kuo Chu for secretarial assistance and Pierrette Neant for the documentation.
References 1. Nunez E A. Non-esterified fatty acids: role in the molecular events linking endocrinology and oncology via nutrition. Tumor Biol 1987; 8: 273-280. 2. Bandyopadhyay G K, Hwang S I, Imagawa W, Nandi S. Role of polyunsaturated fatty acids as signal transducers: amplification of signals from growth factor receptors by fatty acids in mammary epithelial cells. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 71-78. 3. Smith S S, Neuringer M, Ojeda S R. Essential fatty acid deficiency delays the onset of puberty in the female rat. Endocrinology 1989; 125: 1650-1659. 4. Zhang Z, Benson B, Logan J L. Dietary fish oil delays puberty in female rats. Biol Reprod 1992; 47: 998-1003. 5. Olsen S F, Hansen H S, Sorensen T I A, Jensen B, Secher N J, Sommer S. Intake of marine fat, rich in (n-3)polyunsaturated fatty acids, may increase birthweight by prolonging gestation. Lancet 1986; 16: 367-369. 6. Benassayag C, Vallette G, Delorme J, Savu L, Nunez E A, Jayle M F. Rat and human embryo and post-natal sera contain a potent endogenous competitor of estrogen-rat alpha-fetoprotein interactions. Steroids 1977; 30:771-785. 7. Nunez E A. Biological role of alpha-fetoprotein in the endocrinological field: data and hypotheses. Tumor Biol 1994; 15: 63-72. 8. Martin M E, Benassayag C, Nunez E A. Selective changes in binding and immunological properties of human corticosteroid binding globulin by free fatty acids. Endocrinology 1988; 123: 1178-1186. 9. Diver M J. The effect of free fatty acids on the in-vitro binding of testosterone in human plasma. J Endocrinol 1993; 136: 327-330. 10. Graber R, Sumida C, Nunez E A. Fatty acids and cell signal transduction. J Lipid Mediator 1994; 9:91-116. 11. Sumida C, Vallette G, Thobie N, Nunez E A. Fatty acids potentiate the glucocorticoid-dependent transcription from the MMTV-LTR promoter. Endocrine Society 76th Annual Meeting 1994; Abst. 1429. 12. Nunez E A, Free fatty acids as modulators of the steroid hormone message. Prostagland Leukot Essent Fatty Acids 1993; 48: 63-70. 13. Eberhardt, Kiesel L. Role of arachidonic acid and lipoxygenase products in the mechanism of gonadotropin secretion: an update. Prostaglandins Leukot Essent Fatty Acids 1992; 47: 239-246. 14. Alsat E, Mondon F, Rebourcet R et at. Identification of specific binding sites for acetylated low density lipoprotein in microvillus membranes from human placenta. Mol Cell Endocrinol 1985; 41: 229-235. 15. Bihain B E, Yen F T. Free fatty acids activate a highaffinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia. Biochemistry 1992; 31: 46284636. 16. Malassine A, Alsat E, Besse C, Rebourcet R, Cedard L. Acetylated low density lipoprotein endocytosis by human syncytiotrophoblast in culture. Placenta 1990; 11: 191-204. 17. Liang T, Liao S. Inhibition of steroid 5 c~-reductase by specific aliphatic unsaturated fatty acids. J Biochem 1992; 285: 557-562. 18. Boone D L, Currie W D, Leung P K C. Arachidonic acid and cell signalling in the ovary and placenta. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 79-87. 19. Goodfriend T L, Ball D L, Elliott M E et al. Fatty acids may regulate aldosterone secretion and mediate some of insulin's effects on blood pressure. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 43-50.
190 Prostaglarldins Leukotrieues and Essential Fatty Acids 20. Weiner M, Reiner J M. Control of placental 3~-hydroxyD5-steroid dehydrogenase: partial characterization of an endogenous inhibitor and an endogenous activator. Mech Age Dev 1978; 7: 445-453. 21. Blomquist C H, Lindeman N J, Hakanson E Y. Inactivation of soluble 1713hydroxysteroid dehydrogenase of human placenta by fatty acids. J Steroid Biochem 1985; 23: 357-363. 22. Lafrance M, Hansel W. Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum. PSEBM 1992; 201: 106-113. 23. Pilaf Lopez-Ruiz M, Cboi M S K, Rose M P, West A P, Cooke B A. Direct effect of arachidonic acid on protein kinase C and LH- Stimulated steroidogenesis in rat leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C. Endocrinology 1992; 130: 1122-1130. 24. Sumida C, Vallette G, Nunez E A. Interaction of unsaturated fatty acids with rat liver glucocorticoid receptors: studies to localize the site of interaction. Acta Endocrinol 1993; 129: 348-355. 25. Haourigui M, Thobie N, Martin M E, Benassayag C, Nunez E A. In vivo transient rise in plasma free fatty acids alters the functional properties of c~-fetoprotein. Biochim Biophys Acta 1992; 1125: 157-165.
26. Haourigui M, Martin M E, Thobie N, Benassayag C, Nunez E A. Stimulation of the binding properties of adult rat corticosteroid-binding globulin by a lipolysis-induced rise in plasma free fatty acids. Endocrinology 1993; 133: 183-191. 27. Haourigui M, Vallette G, Martin M E, Sumida C, Benassayag C, Nunez E A. In vivo effect of free fatty acids on the specific binding of glucocorticosteroids to corticosteroid binding globulin and liver receptors in immature rats. Steroids 1994; 59: 46-54. 28. Haourigui M, Sakr S, Thobie Net al. Postprandial stimulation of glucocorticoid binding activity of human CBG after fat ingestion. Fatty acids and Lipids from cell biology to human disease; 1st International Congress of the ISSFAL, Lugano 1993: pp. 90. 29. Widmaier E P, Rosen K, Abbott B. Free fatty acids activate the hypotbalamic-pituitary-adrenocortical axis in rats. Endocrinology 1992; 13l: 2313-2318. 30. Klein A, Bruser B, Robinson J B, Pinkerton P H, Malkin A. Effects of a non-viral fraction of acquired immunodeficiency syndrome plasma on the vulnerability of lymphocytes to cortisol. J Endocrinol 1987; 112: 259-264. 31. Felden F, Martin M E, Gueant J L, Benassayag C, Nunez E A. Free fatty acid-induced alterations in the steroidbinding properties of rat androgen-binding protein. Biochem Biophys Res Commun 1993; 190: 602-608.
Fatty Acids and Steroid Hormone Action E. A. Nunez, M. Haourigui, M. E. Martin and C. Benassayag Laboratoire de Biochimie Endocrinienne, Facultd de Mddecine Xavier Bichat, Universit( Denis Diderot-Paris 7, B.P. 416, 75870 Paris Cedex 18, France (Reprint requests to EAN)
contradicted by authors who have denied their occurrence in vivo (9). We will describe some of our recent results showing that the data obtained in vitro are actually confirmed under in vivo conditions. Lastly, we will use these data to develop a model showing the regulatory loop between lipase activity, fatty acids per se, fatty acid metabolites, and the biological activity of steroids on cell multiplication and on cell differentiation. It is particularly important to mention that NEFAs are potent modulators or regulators of many of the enzymes involved in the membrane peptide signalling (10). The studies carried out in our laboratory (11, 12) and presented in this issue (by us and Dr Sumida) clearly indicate that NEFAs are the regulators of the two mechanisms by which steroid (nuclear) and peptide signals (membrane) are transferred, and that NEFAs regulate the cross talk between these two signalling pathways involved in cell multiplication or differentiation.
INTRODUCTION
Epidemiological and experimental nutritional studies have shown that there is a link between lipids, endocrinology and cancer (1). These studies have been confirmed by the demonstration that the multiplication of estrogen-dependent cells, such as MCF7 or breast cancer epithelial cells, can be either positively or negatively influenced by non-esterified fatty acids (NEFAs), particularly unsaturated NEFAs (UFAs) (1, 2). Many studies have shown that changes in fipid nutrition induce changes in the reproductive functions (3-5). Our interest in this field was triggered by our in vitro observations that unsaturated and polyunsaturated unesterified fatty acids (UFAs) inhibit the binding of natural estrogens (6) to murine (estrophilic) alphafetoprotein (AFP). AFP is produced during fetal and postnatal life and by hepatocellular carcinoma cells and yolk sac-derived tumors (7). The function of this protein is still not known, but it is becoming more and more evident that AFP has several biological actions, which depend on the surrounding conditions (7). It is now clear that these biological functions depend on the fatty acids, growth factors, steroids in the environment. Besides several other functions, AFP regulates the multiplication or differentiation of steroid-dependent cells by a mechanism which is not yet elucidated. Recent results suggest that the regulating effect of AFP on fatty acid concentrations and metabolism are particularly important for the way this takes place (7). Thus, it is important to elucidate the endocrine role of NEFAs to understand one of the biological roles of AFP. It is clear also from various in vitro studies that the binding, electrophoretic, antigenic properties (and by consequence probably the biological properties) of different plasma steroid binding proteins like corticosteroid binding globulin are positively or negatively influenced by NEFAs (8). This review summarizes the results obtained in vitro (and at the molecular level) showing how NEFAs modulate or regulate the various steps in the transfer of steroidborne information. Some of these results have been
IN VITRO STUDIES S H O W I N G M O D U L A T I O N BY NEFAS OF STEROID M E T A B O L I S M
Many recent in vitro studies (12) have shown that NEFAs, without any metabolic transformation, are able to modulate various steps in the transfer of steroid information (Fig. 1). Recently new data have been obtained showing: Influence of NEFAs on gonadotropin secretion
The main results describing this action have been recently reviewed (13). Arachidonic acid (AA) and/or its metabolites mediate GnRH-stimulated gonadotropin release. It seems probable that AA acts on the initial phase of exocytosis of the stored LH. PKC, phospholipase A2, diacyl glycerol lipase, the influx of calcium, the cyclooxygenase and lipoxygenase pathways seem to be also involved in this action. The relative importance of these pathways involved in the stimulation of LH activity seems to depend on the type of LH secretion (basal, stimulated, glycosylated LH). Further work is needed to amplify these data. 185
186
Prostaglandins Leukotrienes and Essential Fatty Acids
Gn RH
Cholesterol exogenousend°gen°us (lipoproteins)
~
GS
~ I Steroid IO} n Metabolism I ~"
HRE
otI
.//sz LS
OorO (PKC...)
' P (S.B.P., AFP, ...) cell mult.
GS : Gonadostimulin
S : Steroid P : Steroid binding protein S.B.P. : Sex steroid binding protein LS : Lipoidal steroid PKC : Phospho protein kinase Fig.
1
Cell diff.
AFP : Alphafetoprotein Px : Protein O : Non esterified fatty acids "per se" O : Non esterified fatty acids metabolites TF : Transcription factor R : Receptor HRE : Hormone responsive element
Role of NEFA on the various steps in the transfer of steroid information leading to the expression of a biological function.
Free fatty acid stimulated increase in LDL cholesterol uptake by cells The acylation of LDL by fatty acids (14) or the unmasking of a putative receptor (15) leads to the uptake and degradation of the lipoproteins by cells unable to synthesize LDL receptor. The two actions have been described for placental microvilli and fibroblast. The placenta uses the exogenous cholesterol obtained by these means for the intensive steroidogenesis which takes place during gestation (16). Action of NEFAs and their metabolites on steroidogenesis Various enzymes (5 ~ reductase (17), aromatase (18), 18 hydroxydehydrogenase (19), 5ene, 3 ~ hydroxysteroiddehydrogenase (20), 17 [~ hydroxysteroid-dehydrogenase (21)) of steroid metabolism are under the control of NEFAs. It is also important to remember that the NEFAs and their metabolites produced in the cyclooxygenase, lipoxygenase, or epoxygenase pathways can have a
different modulation effect on steroid metabolism or function. This is a particularly difficult problem to precisely describe, giving each UFA substrate and its various metabolites a precise role. One must realize that if inhibition of an enzyme which produces a fatty acid metabolite does in fact inhibit the biological effect of the UFA, it is not necessarily the metabolite which is responsible for the effect of the UFA. The increased concentration of the UFA itself can be responsible for this effect. It is clear from recent data that it is the accumulation of the free UFA (having a positive or negative biphasic effect) which is responsible for the inhibition or activation of a function and not the metabolite (22). UFAs may also regulate steroidogenesis in another rather complicated way. A recent study (23) showed that AA per se, via activation of PKC, exerts a negative effect (short-term) on the early synthesis of testosterone by LH-stimulated Leydig cell. But when PKC is down regulated by arachidonic acid (long-term), steroidogenesis is enhanced due to the action of arachidonic metabolites produced via the lipoxygenase pathway. These metabolites stimulate testosterone biosynthesis.
Fatty Acids and SteroidHormoneAction 187
Action of NEFAs on steroid binding by specific steroid binding proteins Numerous in vitro studies have shown that NEFAs can influence positively or negatively the binding of a steroid to its specific binding protein in the plasma (12). But other studies contradict this data and suggest that the NEFA pool has no influence on the binding of steroid in vivo (9). The discrepancies between these in vitro results can be explained by the different experimental conditions in which these studies have been performed (dilution, ratio between albumin or other specific steroid or fatty acid binding proteins, concentration in the milieu of other ligands, nutritional situation, etc.). It is also necessary to evaluate the in situ concentration of the fatty acid, steroid and binding protein at a precise point of the cell structure (12). The relative percentage of each factor at this precise point can greatly modify, positively or negatively, the binding properties of a specific steroid binding protein in the plasma or in the tissue.
Effect of NEFAs on specific steroid binding to tissue receptors Many studies have shown the positive or negative effects of UFAs on the binding of steroid hormones to the superfamily of steroid hormone receptors (12). Recently, we have found that the site of interaction of polyunsaturated fatty acids is on a fragment of the rat glucocorticoid receptor containing the hormone-binding domain and identified some sequences C-terminal of the DNA-binding domain (24). We do not yet know if this binding is direct or indirect via another fatty acid binding protein which may be bound to this region.
IN VIVO INFLUENCE OF NEFAS ON STEROID ACTION Changes in plasma NEFAs induced in vivo by heparin and their effect on the functional properties of ~-fetoprotein Previous in vitro studies have shown that UFAs induce conformational changes in rodent and human AFP (12). We have carried out a series of studies to determine whether such changes in the binding and immunological properties of rat AFP also occur in vivo (25). In these experiments, NEFA concentrations were increased in young male rats (15, 21 and 28 days old) by acute i.v. injection of heparin (200 UUKg). Plasma estrogens (estrone and estradiol) did not change after injection of heparin. But there were large increases in plasma NEFAs 10-20min post-heparin injection, with a return to normal 60 rain later. This transient rise in plasma NEFA caused a 50% drop (p < 0.001) in the binding of estradiol to AFP of 15, 21 and 28-day-old rats by reducing the number of binding
sites (p<0.001), leaving the affinity constant (Ka) unchanged. NEFAs extracted from post-heparin plasma induced similar changes in estradiol binding to purified rat AFP. The rise in plasma NEFAs caused a loss of AFP immunoreactivity, especially in 21 (p < 0.001), and 28day-old rats (p < 0.001), but not in 15-day-old rats. This age-dependent response correlated with the NEFAs/AFP ratio (44 in 15-day-old rats, 375 in 21-day-old rats, and 6000 in 28-day-old rats). One of the most striking findings to come out of these experiments is the reversible effect of the rise of NEFAs on the binding and immunological properties of AFP. This indicates that AFP can adapt to, and interact with the environmental changes which occur during ontogenesis, oncogenesis and in pathological situations.
Effect of free fatty acids on the binding of glucocorticoids to corticosteroid binding globulin (CBG) and liver receptors during development in the rat (26, 27) The ontogenic patterns of NEFAs and CBG lead to specific NEFAs/CBG molar ratios at each stage of development. Plasma NEFAs are higher in young rats than in adult, while CBG is lower in immature rats. This gives a NEFAs/CBG molar ratio of 6000 in suckling rats (15 days), 2500 in weaning rats (21 days), 750 in prepubertal rats (28 days) and 360 in adult rats (60 days). We have examined the effect of heparin-induced lipolysis on the function of CBG at these four stages. The amounts of CBG in the plasma did not change, but CBG binding activity did. Thus the large increase in plasma NEFAs which occurs 10rain and 2 0 m in (p < 0.001) after lipolysis in 15-day- and 21-day-old rats were associated with a 50% drop (p < 0.001) in binding (C values L/g) of corticosterone (B) to immature rats CBG 60 min post-injection. The decrease in binding in 15-day-old rats is due to a reduction in the number of binding sites (n) (p < 0.001), with no change in the affinity constant (Ka). Both binding parameters changed (Ka was reduced and n increased) in 21-day-old rats. The increase in plasma NEFAs occurred later (20 and 60 rain p < 0.001) in 28-day- and 60-day-old rats. It was correlated with an increase in B binding to CBG (2-3fold p < 0.001). The increased B binding was due to an increase in Ka (p < 0.01), with no changes in n for 28day-old rats and to a 2-fold increase in n, without significant changes in Ka for adult rats. Such variations in CBG binding parameters strongly suggest that a rise in plasma NEFAs induces conformational changes in CBG. NEFAs extracted from post-heparin plasma, or a mixture of NEFAs with the same composition induced similar changes in B binding to prepurified immature and purified adult rat CBG. These opposing age-dependent CBG responses to lipolysis could be due to the NEFAs/CBG molar ratio and also to differences in CBG glycosylation. Our
188 Prostaglandins Leukotrienes and Essential Fatty Acids
results emphasize the importance of the NEFAs environment in modulating the endocrine function of the CBG during development. The endogenous NEFAs in the liver cytosol of immature rats was also doubled, 60 rain after heparin-induced lipolysis. The increased cytosol NEFAs, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor (GR). The decrease resulted from a significantly lower apparent K a for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between K, (r = -0.93) and n (r = -0.90) and the increased liver cytosol NEFAs content. Thus the higher plasma NEFAs induced in vivo by lipase activation or a standard NEFAs mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR. The liver content of endogenous NEFAs in mature rats was decreased (6%), particularly for the polyunsaturated fatty acids, 60 min after heparin-induced lipolysis. This decrease in non-esterified fatty acid contents was accompanied by a significant increase in dexamethasone binding (Ka enhanced, 1.5-fold, n no change).
Stimulation of glucocorticoid binding to human eorticosteroid binding globulin after post-prandial lipolysis of a high-fat meal (28) As shown in earlier in vitro studies for both rat and man (8, 12), and in vivo for the rat (26, 27), NEFAs promote changes in the molecular state of CBG which, depending on their concentration and degree of unsaturation, either enhance or reduce cortisol (F) binding. We studied the influence of the changing serum NEFAs concentration that occurred after the ingestion of fat-rich meals differing in the nature of fatty acids to determine whether such changes in CBG binding activity occur in vivo for man. 10 normolipidemic women volunteers (20-30 years old) ingested 60 g loads of different fats: tallow (46% saturated, 38% C 18:1), oleic-sunflower acid (76% C 18:1), sunflower (65% C 18:2), and a mixture of oils (42% C18:1 and 43% C18:2). Blood samples were collected before the fat load and after 2, 4, 6 and 8 h. The CBG concentration did not change post-prandially, but the CBG activity changed, and these changes differed according to the fat ingested. Tallow and sunflower-oleic fat loads caused a 2-fold rise in NEFAs at 6 and 8 h (p < 0.005 Dunnet's test) influencing mostly the saturated and monounsaturated fatty acids (p <0.005). Concomitantly, CBG binding indices significantly increased (p < 0.005), as a result of a 2-fold increase in affinity constant (Ka), while the number of binding sites (n) slightly decreased (30%). The ingestion of sunflower or mixed fats led to a moderate NEFAs rise 8 h after the meal (25%) (P < 0.01), inducing no significant changes in F binding to CBG. These results provide evidence that post-prandial increases in serum NEFAs can induce conformational and
functional changes in CBG in vivo, which may regulate tile availability of glucocorticoids and alter their biological impact. Thus, the serum concentrations of cortisol were high at the beginning of the experimental period (8:00 AM). After ingestion of sunflower or mixed fat they gradually decreased and were 2-fold lower at the end of the test (04:00 PM), in agreement with a circadian variation in plasma cortisol. By contrast, after the meals of tallow or sunflower-oleic oils, the diurnal rhythm of cortisol was lost. As a result, 8 h post-prandially, i.e at 04:00 PM, the level of cortisol was not significantly different from that of the morning.
DISCUSSION It is clear that the overwhelming evidence indicates that fatty acids do regulate in vivo at least some steps of the transfer of the steroid hormone message (Fig. 1). The global effect of NEFAs on a steroid-regulated function can be due to NEFAs affecting not only one but several steps. Thus, the change in the diurnal cortisol concentration observed in the women volunteers after a breakfast loaded with tallow or oleic-sunflower may be due to the enhanced binding capacity of cortisol by CBG, and/or the NEFA effect on ACTH production (29) and/or the diminution of cortisol catabolism (30). More work is needed to extend these in vivo observations to other steps in the transfer of the steroid message, such as receptor-mediated uptake of the plasma specific binding protein - steroid complex or specific protein transcription at the genomic level. It would also be interesting to perform new studies using other lipolytic procedures (e.g. lipolysis resulting from adrenergic stimulation) and using specific inhibitors of lipolytic enzymes. These experiments inducing lipolysis could be done on animals or humans after nutritional manipulation. It is particularly easy to alter the lipid profile of a mamalian organism by changing the quality of the fatty acid intake (saturated, unsaturated, series (n-3, n-6, etc.)) (4). All these studies have been performed in the blood stream. In fact, it would be particularly interesting to analyse the changes in FFA which occur in situ in normal arld some physiopathological situations (peroxisomal pathologies, diabetes, AIDS) in tissues (e.g. the seminal fluid where important variations in the concentration of UFAs are observed (31), placenta, liver, prostate). These experiments should be performed in real time using appropriate techniques taking into account the fact that this time could be very short. Nevertheless, results that have been obtained until now can be used to elaborate a model which incorporates a regulatory loop between lipase activity, fatty acids per se, fatty acid metabolism and steroids, acting on cell multiplication and differentiation (Fig. 2). We suggest that the shift between cell multiplication and cell differentiation depends on this balance between fatty acids and their metabolites, together with other factors like growth
Fatty Acids and Steroid Hormone Action
Lipoproteins] Lipids
Acknowledgements
I ~
[
l
Lipases ,~
~
~
(Oe,,mu,,
--
I Metabolites Prostanoids
-- Oe,,d,,,)
I St ,o'ds I Fig.2
Model showing the regulatory loop between lipase activity, fatty acids per se, fatty acid metabolism and the biological activity of a steroid on cell multiplication and cell differentiation.
factors (Fig. 3). The biphasic effect of UFAs on PKC (or other enzyme involved in membrane signal transduction) can be one of the crucial elements which determine this balance (23). The hypothesis may be further refined by introducing an action of the fatty acid binding proteins, like albumin, AFP and the genuine fatty acid binding proteins. We have recently speculated on this type of regulation for AFP (7). Taken together with the studies presented in this issue (11) on the influence of UFAs on glucocorticoiddependent gene transcription, it is clear that fatty acids are regulators of the two mechanisms by which steroid (intranuclear) and peptide signals (membrane) are transferred. Fatty acids regulate the cross-talk between these two signalling pathways and hence the shift from cell multiplication to cell differentiation.
Fatty acids binding pr ein~ (AFP, FABP, .....~t
UFAs Metabolites ~k, V and Otherfactors
2
(steroidogenesis, 1
Cell differentiation
Fig.
189
?~,~1¢
i~,
UFAs Metabolites
and Otherfactors
[ Cellmultiplicati°n1
3 Taking together various results (7, 23), we hypothesize that the shift between cell multiplication and cell differentiation depends on the balance between fatty acids per se and their metabolites together with other factors like growth factors. The hypothesis may be further refined by introducing an action of fatty acid binding proteins.
We are grateful to Dr C. Owen Parkes for help in preparing the manuscript and Mrs Paulette He Kuo Chu for secretarial assistance and Pierrette Neant for the documentation.
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