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Molecular Endocrinology, doi:10.1210/me.2005-0266
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Molecular Endocrinology 20 (5): 1153-1163
Copyright © 2006 by The Endocrine Society

Dehydroepiandrosterone Mimics Acute Actions of Insulin to Stimulate Production of Both Nitric Oxide and Endothelin 1 via Distinct Phosphatidylinositol 3-Kinase- and Mitogen-Activated Protein Kinase-Dependent Pathways in Vascular Endothelium

Gloria Formoso, Hui Chen, Jeong-a Kim, Monica Montagnani, Agostino Consoli and Michael J. Quon

Diabetes Unit (G.F., H.C., J.-a.K., M.J.Q.), National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, Maryland 20892; Department of Pharmacology and Human Physiology (M.M.), University of Bari Medical School, 70124 Bari, Italy; and Department of Medicine and Aging Sciences (A.C.), University of Chieti, 66100 Chieti, Italy

Address all correspondence and requests for reprints to: Michael J. Quon, M.D., Ph.D., Chief, Diabetes Unit, National Center for Complementary and Alternative Medicine, National Institutes of Health, Building 10, Room 6C-205, 10 Center Drive MSC 1632, Bethesda, Maryland 20892-1632. E-mail: quonm{at}nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dehydroepiandrosterone (DHEA) is an adrenal steroid and nutritional supplement that may improve insulin sensitivity. Although steroid hormones classically act by regulating transcription, they may also signal through cell surface receptors to mediate nongenomic actions. Because DHEA may augment insulin sensitivity, we hypothesized that DHEA mimics vascular actions of insulin to acutely activate signaling pathways in endothelium-mediating production of nitric oxide (NO) and endothelin 1 (ET-1). Treatment of bovine aortic endothelial cells with either insulin or DHEA (100 nM, 5 min) stimulated significant increases in NO production (assessed with NO-selective fluorescent dye diaminofluorescein 2). These responses were abolished by pretreatment of cells with L-NAME (nitro-L-arginine methyl ester; NO synthase inhibitor) or wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor]. Under similar conditions, insulin- or DHEA-stimulated phosphorylation of Akt (Ser473) and endothelial nitric oxide synthase (Ser1179) was inhibited by pretreatment of cells with wortmannin (but not MAPK kinase inhibitor PD98059). Acute DHEA treatment also caused phosphorylation of MAPK (Thr202/Tyr204) that was inhibitable by PD98059 (but not wortmannin). DHEA treatment of bovine aortic endothelial cells (100 nM, 5 min) stimulated a 2-fold increase in ET-1 secretion that was abolished by pretreatment of cells with PD98059 (but not wortmannin). We conclude that DHEA has acute, nongenomic actions in endothelium to stimulate production of the vasodilator NO via PI 3-kinase-dependent pathways and secretion of the vasoconstrictor ET-1 via MAPK-dependent pathways. Altering the balance between PI 3-kinase- and MAPK-dependent signaling in vascular endothelium may determine whether DHEA has beneficial or harmful effects relevant to the pathophysiology of diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DEHYDROEPIANDROSTERONE (DHEA) and its precursor DHEA sulfate (DHEA-S) are the most abundant circulating steroid hormones in humans (1). Circulating levels of DHEA decline with age (2), and some epidemiological studies suggest that decreased DHEA levels increase cardiovascular risk (3). An inverse relationship between DHEA levels and fasting insulin concentrations is also present in humans (4). Moreover, in healthy men, oral DHEA supplementation (1600 mg/d for 4 wk) reduces body fat and serum low-density lipoprotein cholesterol levels while increasing muscle mass (5). In addition, supplementation with DHEA for 2 wk significantly increases flow-mediated dilation in brachial arteries of healthy postmenopausal women (6), and DHEA replacement (50 mg daily for 12 wk) in hypoadrenal women improves insulin sensitivity (7). Taken together, these studies suggest that DHEA mediates increased insulin sensitivity. Interestingly, acute hyperinsulinemia in humans causes a decline in serum DHEA-S concentrations (8, 9). Thus, determination of insulin and DHEA levels may involve reciprocal regulation by those hormones. DHEA is available in the United States without prescription as a nutritional supplement that is touted for its putative antiaging properties and beneficial effects on metabolic and cardiovascular health (10, 11). However, molecular mechanisms mediating DHEA action are poorly understood.

Some chronic effects of DHEA supplementation may be due to testosterone and estrogen because these sex steroids are derived from DHEA (12). Steroid hormones classically act by regulating transcription through specific nuclear receptors. However, both estrogen and corticosteroids also have rapid, nongenomic actions in vascular endothelium to activate endothelial nitric oxide synthase (eNOS) by a PI 3-kinase-dependent mechanism involving phosphorylation of eNOS at Ser1179 by Akt (13, 14). Intriguingly, this is similar to signaling mechanisms regulating vasodilator actions of insulin (15, 16, 17, 18, 19, 20). Recent studies in both cells and animals suggest that DHEA may have acute nongenomic actions that mimic both metabolic and vascular actions of insulin. For example, acute DHEA treatment of adipocytes stimulates translocation of the insulin-responsive glucose transporter GLUT 4 to the cell surface with resultant increases in glucose uptake (21, 22, 23). Consistent with these findings, DHEA treatment of diabetic rats for 2 wk increases insulin-stimulated glucose uptake in vivo (22). With respect to vascular actions of DHEA, displaceable binding of labeled DHEA to plasma membranes of bovine aortic endothelial cells (BAECs) suggests that specific cell surface DHEA receptors are present in vascular endothelium (24, 25). Moreover, acute DHEA treatment of endothelial cells may stimulate production of the vasodilator nitric oxide (NO) (24, 25, 26). However, these previous studies inferred production of NO using indirect methods. We previously reported that insulin directly stimulates production of NO from vascular endothelium via PI 3-kinase-dependent mechanisms (15, 16, 17, 18), which leads to increased blood flow. This vasodilator action of insulin serves to significantly augment insulin-stimulated glucose uptake in vivo by increasing delivery of both glucose and insulin to target tissues (19, 20). On the other hand, insulin also stimulates secretion of the potent vasoconstrictor endothelin 1 (ET-1) from vascular endothelium in humans (27). This vasoconstrictor action of insulin, mediated by MAPK-dependent pathways, may contribute to insulin resistance and hypertension (28). Indeed, in insulin-resistant states, PI 3-kinase pathways are impaired whereas MAPK pathways are enhanced. This may result in imbalance between beneficial and pathological actions of insulin in the vasculature (19, 20, 29). In the present study, we evaluate acute, nongenomic actions of DHEA to mimic effects of insulin to stimulate production of both NO and ET-1 via distinct PI 3-kinase- and MAPK-dependent pathways, respectively, in vascular endothelium.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DHEA Acutely Stimulates Production of NO in Endothelial Cells
We used the NO-specific fluorescent dye diaminofluorescein 2 (DAF-2) to evaluate acute effects of DHEA treatment (100 nM for 0, 1, 3, 5, and 30 min) to stimulate production of NO in BAECs. After 1-min stimulation with DHEA, we observed a significant increase in green fluorescence in BAECs indicative of NO production (Fig. 1Go). This response was maximal after 5 min and declined to basal levels after 30 min. To verify that the increase in green fluorescence in response to DHEA treatment specifically reflected NO production, we compared results from BAECs treated with insulin or DHEA (100 nM, 5 min) in the absence or presence of L-NAME (nitro-L-arginine methyl ester; NOS inhibitor). Reassuringly, acute treatment with either insulin or DHEA resulted in a significant increase in green fluorescence that was completely blocked by pretreatment with L-NAME (Fig. 2Go). Because insulin-stimulated production of eNOS depends on activation of PI 3-kinase (15, 16), we used the PI 3-kinase inhibitor wortmannin to determine whether DHEA-stimulated production of NO also requires PI 3-kinase (Fig. 3Go). As expected, insulin-stimulated production of NO was blocked by pretreatment of BAECs with wortmannin whereas the response to lysophosphatidic acid (LPA, an agonist that is PI 3-kinase independent) was unaffected. Importantly, acute DHEA-stimulated production of NO in BAECs was blocked by pretreatment of cells with wortmannin. Taken together, these results indicate that, similar to insulin, DHEA acutely stimulates production of NO in vascular endothelial cells in a time-dependent and PI 3-kinase-dependent manner.


Figure 1
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  		Fig. 1. DHEA Acutely Stimulates Production of NO in Vascular Endothelial Cells

BAECs were serum-starved and loaded with DAF-2 DA as described in Materials and Methods before treatment with DHEA (100 nM) for 0, 1, 3, 5, and 30 min. After DHEA treatment, cells were fixed in 2% paraformaldehyde for 5 min at 4 C and then viewed using an epifluorescent microscope. A, Emission of green light (510 nm) from cells excited by light at 480 nm is indicative of NO production. A representative time course experiment is shown for experiments that were repeated independently three times. B, Phase contrast view of cells corresponding to images shown in panel A.

 

Figure 2
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  		Fig. 2. Inhibition of Nitric Oxide Synthase Blocks Both Insulin- and DHEA-Stimulated Production of NO in BAECs

Cells were serum starved and loaded with DAF-2 DA as described in Materials and Methods before treatment with either insulin (100 nM) or DHEA (100 nM) for 5 min. In some groups of cells, L-NAME (100 µM) was added 30 min before cells were loaded with DAF-2 DA. After insulin or DHEA treatment, cells were fixed in 2% paraformaldehyde for 5 min at 4 C and then viewed using an epifluorescent microscope. A, Emission of green light (510 nm) from cells excited by light at 480 nm is indicative of NO production. A representative set of experiments is shown for experiments that were repeated independently five times. B, Phase contrast view of cells corresponding to images shown in panel A.

 

Figure 3
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  		Fig. 3. Acute Stimulation of NO Production by DHEA in BAEC Requires PI 3-Kinase Activity

Cells were serum starved and loaded with DAF-2 DA as described in Materials and Methods before treatment with either insulin (100 nM, 5 min), LPA (5 µM, 1.5 min), or DHEA (100 nM, 5 min). In some groups of cells, the PI 3-kinase inhibitor wortmannin (100 nM) was added 30 min before cells were loaded with DAF-2 DA. After insulin, LPA, or DHEA treatment, cells were fixed in 2% paraformaldehyde for 5 min at 4 C and then viewed using an epifluorescent microscope. A, Emission of green light (510 nm) from cells excited by light at 480 nm is indicative of NO production. A representative set of experiments is shown for experiments that were repeated independently three times. B, Phase contrast view of cells corresponding to images shown in panel A.

 
DHEA Acutely Stimulates Phosphorylation of Akt at Ser473 and eNOS at Ser1179 by a PI 3-Kinase-Dependent and MAPK-Independent Mechanism
Insulin-stimulated production of NO requires PI 3-kinase-dependent activation of Akt that then directly phosphorylates and activates eNOS (17). We used phospho-specific antibodies to evaluate the ability of DHEA to acutely stimulate phosphorylation of Akt in BAECs in the absence or presence of wortmannin or the MAPK kinase (MEK) inhibitor PD98059. Both insulin and DHEA treatment significantly increased phosphorylation of Akt at Ser473 (Fig. 4Go, lanes 1–3). This response was completely blocked by pretreatment of cells with wortmannin (Fig. 4Go, lanes 4–6). Interestingly, MAPK blockade significantly increased phosphorylation of Akt in response to insulin (cf. Fig. 4Go, lanes 2 and 8) but did not significantly alter basal or DHEA-stimulated phosphorylation of Akt (cf. Fig. 4Go, lanes 7 and 9 with lanes 1 and 3). Thus, similar to insulin, DHEA stimulates phosphorylation (and presumably activation) of Akt. Because Akt can phosphorylate eNOS at Ser1179 (17), we next evaluated phosphorylation of eNOS at Ser1179 in response to insulin or DHEA treatment either in the absence or presence of wortmannin or PD98059 (Fig. 5Go). Consistent with results shown in Fig. 4Go, both insulin and DHEA acutely stimulated phosphorylation of eNOS at Ser1179 (Fig. 5Go, lanes 1–3). This response was blocked by pretreatment of cells with wortmannin (Fig. 5Go, lanes 4–6). Interestingly, MAPK blockade significantly increased phosphorylation of eNOS at Ser1179 under basal conditions (Fig. 5Go, cf. lanes 1 and 7) and tended to increase phosphorylation of eNOS in the presence of either insulin or DHEA stimulation (Fig. 5Go, cf. lanes 8–9 with lanes 2–3). We confirmed these results in intact cells by using immunofluorescence microscopy with phospho-specific antibodies against eNOSS1179 (Fig. 6Go). As expected, insulin stimulated a substantial increase in phosphorylation of eNOS at Ser1179 in BAECs. Similarly, acute DHEA treatment substantially increased phosphorylation of eNOS at Ser1179 when compared with untreated cells (Fig. 6Go, upper panels). Expression of eNOS in BAECs was not altered by acute treatment of cells with either insulin or DHEA (Fig. 6Go, lower panels). Thus, similar to insulin, DHEA stimulates phosphorylation of both Akt and eNOS using a PI 3-kinase-dependent, MAPK-independent mechanism.


Figure 4
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  		Fig. 4. Phosphorylation of Akt at Ser473 in Response to Acute DHEA or Insulin Treatment Requires Activation of PI 3-Kinase but not MAPK

BAECs were serum-starved overnight and then treated without or with insulin (100 nM) or DHEA (100 nM) for 5 min. Some groups of cells were pretreated with the PI 3-kinase inhibitor wortmannin (100 nM) or the MEK inhibitor PD98059 (25 µM) for 1 h. Cell lysates were then subjected to immunoblotting as described in Materials and Methods. A, Representative immunoblots obtained using anti-phospho-AktS473 antibody and anti-Akt antibody are shown. B, Scanning densitometry was used to quantify results from multiple independent experiments represented in panel A (mean ± SEM of seven independent experiments). Both insulin and DHEA acutely stimulated significant phosphorylation of Akt at Ser473 (P < 0.01). In the presence of wortmannin, neither insulin nor DHEA treatment significantly increased phosphorylation of Akt at Ser473 (P > 0.1). By contrast, in the presence of PD98059, both DHEA and insulin stimulated significant increases in phosphorylation of Akt at Ser473 (P < 0.0004). When compared with insulin-stimulated control cells, pretreatment with PD98059 significantly enhanced phosphorylation of Akt at Ser473 in response to insulin (P < 0.01). Ins, Insulin.

 

Figure 5
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  		Fig. 5. Phosphorylation of eNOS at Ser1179 in Response to Acute DHEA or Insulin Treatment Requires Activation of PI 3-Kinase But Not MAPK

BAECs were serum-starved overnight and then treated without or with insulin (100 nM) or DHEA (100 nM) for 5 min. Some groups of cells were pretreated with the PI 3-kinase inhibitor wortmannin (100 nM) or the MEK inhibitor PD98059 (25 µM) for 1 h. Cell lysates were then subjected to immunoblotting as described in Materials and Methods. A, Representative immunoblots obtained using anti-phospho-eNOS (phospho-Ser1179) antibody and anti-eNOS antibody are shown. B, Scanning densitometry was used to quantify results from multiple independent experiments represented in panel A (mean ± SEM of six independent experiments). Both insulin and DHEA acutely stimulated significant phosphorylation of eNOS at Ser1179 (P < 0.01). In the presence of wortmannin, neither insulin nor DHEA treatment significantly increased phosphorylation of eNOS at Ser1179 (P > 0.1). By contrast, in the presence of PD98059, both DHEA and insulin stimulated significant increases in phosphorylation of eNOS at Ser1179 (P < 0.002). Incubation with PD98059 alone resulted in a significant increase in phosphorylation of eNOS at Ser1179 when compared with untreated cells (P < 0.03). Ins, Insulin.

 

Figure 6
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  		Fig. 6. Both DHEA and Insulin Acutely Stimulate Phosphorylation of eNOS at Ser1179 in BAECs

Cells were serum starved overnight and then stimulated without or with insulin (100 nM) or DHEA (100 nM) for 5 min. Cells were subsequently fixed with 4% paraformaldehyde and incubated with phospho-specific antibodies that detect eNOS phosphorylated at Ser1179 or antibodies against eNOS. Alexa Fluor 568-conjugated goat antirabbit IgG or Alexa Fluor 488-conjugated goat antimouse IgG were used as secondary antibodies. Results were visualized using an epifluorescent microscope as described in Materials and Methods.

 
Acute Actions of DHEA to Stimulate MAPK-Dependent Secretion of ET-1 in Vascular Endothelium
In addition to stimulating production of NO, insulin has MAPK-dependent vascular actions to stimulate ET-1 secretion in BAECs (28). To determine whether DHEA also mimics these vasoconstrictor actions of insulin, we evaluated the ability of DHEA to acutely stimulate phosphorylation of MAPK. Cell lysates from BAECs treated without or with insulin or DHEA in the absence or presence of wortmannin or PD98059 were immunoblotted with phospho-specific MAPK antibodies (Fig. 7Go). Both insulin and DHEA acutely stimulated phosphorylation of MAPK (Fig. 7Go, lanes 1–3). This response was blocked by pretreatment of cells with PD98059 (Fig. 7Go, lanes 7–9). As expected, PI 3-kinase blockade with wortmannin did not significantly inhibit either insulin- or DHEA-stimulated phosphorylation of MAPK (Fig. 7Go, lanes 4–6). However, in the basal state, incubation with wortmannin alone significantly increased phosphorylation of MAPK when compared with untreated cells (Fig. 7Go, cf. lanes 1 and 4). We next evaluated the ability of acute DHEA treatment to stimulate secretion of ET-1 by measuring ET-1 levels in conditioned media from untreated and treated cells. Acute insulin and DHEA treatment of BAECs both caused a 2-fold increase in ET-1 secretion (Fig. 8AGo). This was abolished by pretreatment of cells with PD98059 but not wortmannin. As expected, in these same cells, pretreatment of BAECs with PD98059 blocked phosphorylation of MAPK (but not Akt) whereas wortmannin blocked phosphorylation of Akt (but not MAPK) in response to either insulin or DHEA (Fig. 8BGo). As a control, we repeated these experiments using treatment with vehicle, DHEA, wortmannin, or PD98059 alone (Fig. 8Go, C and D). These experiments demonstrate that treatment with wortmannin or PD98059 alone does not alter ET-1 secretion. Note that the absolute basal ET-1 concentrations in Fig. 8Go, A and B, are approximately twice those shown in Fig. 8Go, panels C and D, because the cell density for these later experiments was approximately half of that used for Fig. 8Go, A and B. Thus, DHEA also mimics acute actions of insulin to stimulate phosphorylation of MAPK and enhance ET-1 secretion from vascular endothelium.


Figure 7
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  		Fig. 7. Both DHEA and Insulin Acutely Stimulate Phosphorylation of MAPK in BAECs

Cells were serum starved overnight and then treated without or with insulin (100 nM) or DHEA (100 nM) for 5 min. Some groups of cells were pretreated with the PI 3-kinase inhibitor wortmannin (100 nM) or the MEK inhibitor PD98059 (25 µM) for 1 h. Cell lysates were then subjected to immunoblotting as described in Materials and Methods. A, Representative immunoblots obtained using anti-phospho-p42/p44 MAPKT202/Y204 antibody and anti-MAPK antibody are shown. B, Scanning densitometry was used to quantify results from multiple independent experiments represented in panel A (mean ± SEM of nine independent experiments). Both insulin and DHEA acutely stimulated significant phosphorylation of MAPK (P < 0.001). Pretreatment with wortmannin did not significantly inhibit the effects of insulin or DHEA to stimulate phosphorylation of MAPK. However, incubation with wortmannin alone resulted in a significant increase in phosphorylation of MAPK when compared with untreated cells (P < 0.04). In the presence of PD98059, neither DHEA nor insulin treatment elicited a significant increase in MAPK phosphorylation (P > 0.1). Ins, Insulin.

 

Figure 8
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  		Fig. 8. DHEA Acutely Stimulates Secretion of ET-1 from BAECs

Cells were serum starved overnight and then treated without or with insulin (100 nM) or DHEA (100 nM) for 5 min. Some groups of cells were pretreated with the PI 3-kinase inhibitor wortmannin (100 nM) or the MEK inhibitor PD98059 (25 µM) for 1 h. A, Conditioned media from untreated and treated cells were collected for measurement of ET-1 by ELISA. Both insulin and DHEA stimulated a significant 2-fold increase in ET-1 levels over basal (P < 0.001). These effects of insulin and DHEA were not inhibited by pretreatment with wortmannin but were completely blocked by pretreatment with PD98059. Results shown are the mean ± SEM for experiments that were repeated independently seven times in duplicate. B, Cell lysates from experiments described in panel A were subjected to immunoblotting for phospho-MAPKT202/Y204), MAPK, phospho-AktS473), and Akt. A set of representative immunoblots is shown from experiments that were repeated independently seven times. C, Conditioned media from untreated and treated cells were collected for measurement of ET-1 by ELISA. DHEA stimulated a significant 2-fold increase in ET-1 levels over basal (P < 0.001). Treatment with wort mannin or PD98059 alone did not have any effect on basal ET-1 levels. Results shown are the mean ± SEM for experiments that were repeated independently three times in duplicate. D, Cell lysates from experiments described in panel C were subjected to immunoblotting for phospho-MAPKT202/Y204), MAPK, phospho-AktS473), and Akt. A set of representative immunoblots is shown from experiments that were repeated independently three times. Ins, Insulin; PD, PD98059; Wort, wortmannin.

 
Acute DHEA-Stimulated Production of NO in BAECs Is Not Mediated by Activation of Insulin, Estrogen, Glucocorticoid, or Peroxisome Proliferator-Activated Receptor (PPAR)-{alpha} or -{gamma}
Potential mechanisms that may explain acute signaling effects of DHEA in BAECs include activation of heterologous receptors (e.g., receptors for insulin, estrogen, glucocorticoids, or PPARs) that are known to activate PI 3-kinase leading to phosphorylation and activation of eNOS. To help rule out these possibilities, we evaluated tyrosine phosphorylation of the insulin receptor in BAECs in response to insulin or DHEA treatment (Fig. 9AGo). Only insulin, but not DHEA treatment, resulted in autophosphorylation of the insulin receptor. Expression levels of the insulin receptor were not changed by acute treatment with either insulin or DHEA. Thus, acute actions of DHEA to stimulate production of NO are unlikely to involve activation of the insulin receptor. Similarly, acute effects of insulin and DHEA to stimulate production of NO were unaffected by pretreatment of BAECs with the estrogen receptor antagonist ICI 182,780. As expected, this inhibitor completely blocked acute estrogen-stimulated production of NO (Fig. 9BGo). The glucocorticoid receptor antagonist RU486 was effective at blocking effects of dexamethasone to increase phosphorylation of eNOS at Ser1179 without impairing the ability of DHEA to acutely phosphorylate eNOS (Fig. 9DGo). Finally, PPAR antagonist GW9662 was unable to block DHEA-stimulated phosphorylation of eNOS at concentrations that are known to be effective for blocking both PPAR-{alpha} and -{gamma} (Fig. 9DGo).


Figure 9
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  		Fig. 9. Acute DHEA-Stimulated Production of NO in BAECs Is Not Mediated by Activation of Insulin Receptors, Estrogen Receptors, Glucocorticoid Receptors, or PPAR-{alpha} or -{gamma}

A, Cells were serum starved overnight and then treated with vehicle control, insulin (100 nM), or DHEA (100 nM) for 5 min. Cell lysates were immunoblotted with antiphosphotyrosine and antiinsulin receptor (IR) antibodies. A representative blot is shown from experiments that were repeated independently three times. B, Cells were serum starved and loaded with DAF-2 DA as described in Materials and Methods before treatment with either insulin (100 nM), 17ß-estradiol (20 nM), or DHEA (100 nM) for 5 min. In some groups of cells, estrogen receptor antagonist ICI 182,780 (10 µM) was added 30 min before cells were loaded with DAF-2 DA. After insulin, 17ß-estradiol, or DHEA treatment, cells were fixed in 2% paraformaldehyde for 5 min at 4 C and then viewed using an epifluorescent microscope. Emission of green light (510 nm) from cells excited by light at 480 nm is indicative of NO production. A representative set of experiments is shown for experiments that were repeated independently three times. C, Phase contrast view of cells corresponding to images shown in panel A. D, Cells were serum starved overnight and then stimulated without or with DHEA (100 nM, 5 min) or dexamethasone (100 nM, 30 min) in the absence or presence of pretreatment with RU486 (10 µM, 1 h) or GW9662 (1 µM, 1 h). Cells were subsequently fixed with 3% paraformaldehyde and incubated with phospho-specific antibodies that detect eNOS phosphorylated at Ser1179 or antibodies against eNOS. Cy3-conjugated goat antirabbit IgG, or Cy3-conjugated goat antimouse IgG (AffiniPure; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used as secondary antibodies. Results were visualized using an epifluorescent microscope. Representative images are shown from experiments that were repeated independently three times. Dexa, Dexamethason; pY, phosphotyrosine.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Physiological roles for endogenous DHEA and therapeutic benefits of DHEA supplementation for the treatment of aging, neurodegeneration, insulin resistance, and cardiovascular diseases remain obscure and controversial despite intensive study for more than 50 yr (3, 11, 30). Because DHEA is a precursor for androgens and estrogens (12), putative biological effects of DHEA may be secondary to actions of these other steroid hormones. It remains possible that DHEA mediates its own classical steroid actions through transcriptional regulation independent from that of other sex steroids. However, a specific nuclear hormone receptor for DHEA has never been convincingly identified (31). It is now recognized that steroids, including estrogen, progesterone, and glucocorticoids, also mediate actions through cell surface receptors that acutely activate signal transduction pathways common to G protein-coupled receptors and receptor tyrosine kinases (14, 32, 33). Interestingly, specific binding of DHEA to the cell surface of endothelial cells has recently been reported (24). Moreover, DHEA acutely stimulates a variety of intracellular signal transduction pathways related to both metabolic and vascular physiology (21, 22, 23, 24, 25, 26). The insulin receptor is a tyrosine kinase that activates both PI 3-kinase- and MAPK-dependent pathways in metabolic and vascular tissues (20, 34). Recent studies suggest that DHEA has acute actions in adipose tissue related to glucose uptake as well as vascular actions in endothelium to stimulate production of NO. Taken together with previously published clinical and physiological data on metabolic and cardiovascular effects of DHEA, we hypothesized that DHEA may mimic acute vascular actions of insulin to both increase production of NO as well as increase secretion of ET-1 by distinct intracellular signaling pathways.

Acute Vascular Actions of DHEA to Stimulate Production of NO
Acute stimulation of BAECs with DHEA caused a significant time-dependent increase in NO production that was evident after 1 min. This required activation of PI 3-kinase (but not MAPK) and resulted in acute phosphorylation of Akt as well as phosphorylation of eNOS at its known Akt phosphorylation site. This is the first direct demonstration that DHEA can acutely stimulate phosphorylation of Akt and eNOS in vascular endothelium. Moreover, this is the first direct demonstration that DHEA stimulates production of NO in intact endothelial cells. Our results suggest that vascular actions of DHEA to stimulate activation of eNOS and increase production of NO are mediated by the PI 3-kinase/Akt/eNOS signaling pathway used by insulin, estrogen, and corticosteroids (13, 14, 15, 16, 17). Pretreatment of cells with the PI 3-kinase inhibitor wortmannin decreased basal eNOS phosphorylation (Fig. 5Go). This suggests that there is some signaling to eNOS even in the absence of ligands such as insulin or DHEA. However, we did not detect much of an effect of wortmannin pretreatment on basal NO production using the DAF-2 fluorescent dye (Fig. 3Go). This raises the possibility that there may not be a direct correspondence between eNOS phosphorylation and NO production in the absence of hormone stimulation. Alternatively, this may also reflect a difference in sensitivity between immunoblotting and the DAF-2 method. Interestingly, blocking MAPK signaling pathways with PD98059 significantly enhanced Akt phosphorylation in response to insulin but not to DHEA. However, treatment with this MEK inhibitor tended to enhance both basal as well as insulin and DHEA-stimulated phosphorylation eNOS. This suggests that other kinases downstream from PI 3-kinase (e.g. AMPK or SGK) may be also involved with DHEA-stimulated phosphorylation of eNOS. Indeed, adiponectin treatment acutely stimulates phosphorylation of both Akt and AMPK in BAECs. However, in the case of adiponectin, phosphorylation of eNOS at Ser1179 is due to AMPK rather than Akt (35). DHEA-stimulated production of NO occurs within minutes, and it correlates and depends on activation of both PI 3-kinase and Akt. These acute actions of DHEA are highly unlikely to be mediated by transcriptional regulation or derivatives of DHEA such as estrogen or testosterone.

Because DHEA may weakly bind to the estrogen or glucocorticoid receptor (see Ref. 31 for review), we used estrogen and glucocorticoid receptor antagonists to help rule out the possibility that these effects of DHEA are mediated by the estrogen receptor or by conversion of DHEA to estrogen. Although glucocorticoids also exhibit similar nongenomic actions in vascular endothelium (14), the time course of glucocorticoid-stimulated activation of eNOS is slower than what we observed with DHEA. Moreover, a previous study has shown that blockade of glucocorticoid receptors with RU486 does not inhibit acute DHEA-stimulated production of NO in endothelial cells (26). Results from our present study confirm these findings. Thus, it seems unlikely that glucocorticoid receptors are mediating the acute actions of DHEA we observe in vascular endothelial cells. Some actions of DHEA may be mediated by activation of PPAR-{alpha} (36). Interestingly, activation of PPAR-{alpha} by fenofibrate increases expression of eNOS in endothelial cells (37). However, it is unlikely that activation of PPAR-{alpha} by DHEA is mediating the acute activation of eNOS in endothelial cells that we observe because no acute activation of eNOS is found in response to fenofibrate (37), and RU486 blocks the effect of fenofibrate to increase eNOS expression (37) whereas RU486 does not inhibit acute effects of DHEA on NO production (26). When we pretreated BAECs with GW9662 at concentrations known to inhibit both PPAR-{alpha} and PPAR-{gamma}, this was unable to block the acute effect of DHEA to stimulate phosphorylation of eNOS at Ser1179. Thus, this vascular action of DHEA is unlikely to be mediated by PPARs. Finally, we demonstrated that DHEA treatment does not cause autophosphorylation of the insulin receptor. This suggests that acute vascular effects of DHEA are not mediated by activation of the insulin receptor (despite sharing similar downstream signaling pathways). Thus, DHEA joins the growing list of hormones, including insulin (16), estrogen (13), glucocorticoids (14), adiponectin (35), high-density lipoprotein (36), and leptin (37), that acutely activate eNOS in vascular endothelium by a PI 3-kinase-dependent signaling mechanism leading to phosphorylation of eNOS by Akt.

Our data are somewhat discordant with several previous studies that examined eNOS activity and NO production in response to acute DHEA treatment using indirect methods in isolated endothelial cell plasma membranes (24) or in conditioned media (25, 26). In those studies, increased production of nitrites in conditioned media and increased intracellular cGMP were observed after treatment for 5–30 min with DHEA. These effects were blocked by pertussis toxin (23) or PD98059 (25) but did not seem to depend on activation of PI 3-kinase or Akt. In our experiments, treatment of BAECs with PD98059, if anything, enhanced Akt and eNOS phosphorylation in response to insulin and DHEA. At the same time, treatment with this MEK inhibitor completely blocked DHEA-stimulated phosphorylation of MAPK and ET-1 secretion. It is possible that these discrepancies between previously published studies and our current study are due to differences in cell type, duration of stimulation with DHEA, and the reliance of those previous studies on indirect measures of NO production in conditioned media rather than direct measures of NO production in intact cells as in our current study.

Acute Vascular Actions of DHEA to Stimulate Secretion of ET-1
In the present study, we report, for the first time, that acute treatment of vascular endothelial cells resulted in activation of MAPK leading to novel vascular actions of DHEA to increase secretion of the potent vasoconstrictor ET-1. This was not dependent on activation of PI 3-kinase but required activation of MAPK. Interestingly, insulin-stimulated secretion of ET-1 is also independent of PI 3-kinase and dependent on activation of MAPK (28). Thus, DHEA mimics vascular actions of insulin to stimulate production of both vasodilator and vasoconstrictor substances in endothelium.

Pathophysiological Implications of Acute Vascular Actions of DHEA
The insulin receptor activates both PI 3-kinase- and MAPK-dependent signaling pathways (34). This is relevant to vascular biology because PI 3-kinase-dependent activation of eNOS leading to increased production of NO in endothelium has beneficial vascular effects to lower peripheral vascular resistance and inhibit platelet aggregation. By contrast, we recently demonstrated that insulin-stimulated MAPK signaling in endothelium promotes secretion of the vasoconstrictor ET-1 (28) and increases expression of cell adhesion molecules including vascular cell adhesion molecule 1 and E-selectin (29). Others have shown that MAPK-dependent pathways lead to activation of cation pumps (38). Taken together, these MAPK-dependent functions contribute to vasculopathy including increased peripheral vascular resistance, accelerated atherosclerosis, and increased plasma volume. In the present study, we demonstrated that DHEA, like insulin, stimulated production of NO as well as secretion of ET-1 via distinct intracellular signaling pathways (Fig. 10Go). Interestingly, we also recently found similar results with respect to adiponectin. That is, adiponectin stimulates both production of NO and secretion of ET-1 in vascular endothelium using pathways that are both overlapping and distinct from those used by insulin (35, 39). Thus, the net vasoactive effect of DHEA, insulin, and adiponectin may depend upon the balance between PI 3-kinase- and MAPK-dependent signaling. Data from the present study, as well as previous studies (28, 29), suggest that impairment in PI 3-kinase signaling enhances MAPK signaling. This is an important concept because metabolic insulin resistance is typically associated with selective impairment in PI 3-kinase signaling in the vasculature (40, 41). Thus, PI 3-kinase-specific insulin resistance in endothelium may predispose to imbalanced intracellular signaling pathways that favor prohypertensive and proatherogenic effects of insulin and DHEA. Conversely, under conditions of increased insulin sensitivity with relative impairment of MAPK pathways and enhancement of PI 3-kinase pathways, insulin and DHEA may have beneficial vascular effects. In summary, DHEA acutely activates both PI 3-kinase and MAPK-dependent pathways regulating production of NO and ET-1, respectively. Selective impairment of PI 3-kinase-dependent signaling under conditions of metabolic insulin resistance is predicted to cause imbalance between DHEA-stimulated production of NO and ET-1. These novel findings may help explain conflicting reports regarding putative beneficial effects of DHEA on metabolic and cardiovascular health in humans.


Figure 10
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  		Fig. 10. Acute Vasodilator and Vasoconstrictor Actions of DHEA Related to Production of NO and ET-1 May Be Mediated by Nontranscriptional Signaling Mechanisms

DHEA binding to putative membrane cell surface receptors leads to acute activation of both PI 3-kinase- and MAPK-dependent signaling. Activation of PI 3-kinase results in phosphorylation and activation of Akt that then directly phosphorylates eNOS, leading to increased production of NO. Activation of MAPK pathways mediates increased secretion of ET-1. P, Phosphorylation of protein.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
BAECs in primary culture (Cell Applications, San Diego, CA) were grown in Endothelial Growth Medium-Micro Vascular (Cambrex, Walkersville, MD) and used between passages 3 and 5 as previously described (17, 18). Cells were serum starved overnight with EBM (Endothelial Basal Medium) before initiation of experiments.

Measurement of NO Production in Fixed Cells
Production of NO was assessed using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA; Cayman Chemical, Ann Arbor, MI) as described elsewhere (42). Briefly, BAECs were grown to 95% confluence in chamber slides (Lab-Tek, Rochester, NY) and serum starved overnight in EBM supplemented with L-arginine (100 µM). Cells were then loaded with DAF-2 DA (final concentration, 3 µM) for 30 min at 37 C. After that, cells were rinsed three times with EBM at 37 C and kept in the dark. Cells were then treated with LPA (5 µM) for 1.5 min, insulin (100 nM), DHEA (100 nM), or 17ß-estradiol (20 nM) for 5 min. For time course experiments, cells were treated with DHEA for 0, 1, 3, 5, and 30 min. In some experiments, wortmannin (100 nM), L-NAME (100 µM), or ICI 182,780 (10 µM) was added to the media 30 min before loading with DAF-2 DA. After stimulation, cells were fixed in 2% paraformaldehyde (vol/vol) for 5 min at 4 C. Fixed cells were examined using an Olympus IX81 inverted microscope with attached charge-coupled device camera (Retiga Exi, Burnaby, British Columbia, Canada) using appropriate filters with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm. Images were captured using IP Labs Software (Scanalytics, Inc, Fairfax, VA).

Immunofluorescence Microscopy
BAECs were grown to 95% confluence in Lab-Tek chamber slides and serum starved overnight in EBM. Cells were then stimulated without or with insulin (100 nM) or DHEA (100 nM) for 5 min. After stimulation, cells were fixed with 4% paraformaldehyde at room temperature for 12 min, incubated with 0.5% Triton X-100 in PBS for 10 min, washed three times with PBS, and then blocked with 2% BSA in PBS for 1 h. This was followed by incubation with primary polyclonal antibodies against phospho-eNOSS1177 (Cell Signaling Technology, Beverly, MA) or eNOS (Transduction Laboratories, Inc., Lexington, KY) (1:200 in blocking solution) for 1 h at room temperature. The cells were then washed three times with PBS, followed by incubation with secondary antibodies for 1 h at room temperature (Alexa Fluor 568-conjugated goat antirabbit IgG or Alexa Fluor 488-conjugated goat antimouse IgG; Molecular Probes, Eugene, OR). Red and green immunofluorescence in the cells was evaluated using an Olympus IX81 microscope with the appropriate filters. Images were captured using an attached charge-coupled device camera in conjunction with IP Labs Software.

Immunoblotting
BAECs were grown in 60-mm dishes, serum starved overnight, and then treated with either insulin (100 nM) or DHEA (100 nM) for 5 min. In some experiments, wortmannin (100 nM) or PD98059 (25 µM) was added to cells 1 h before treatment with insulin or DHEA. Cell lysates were prepared using 300 µl of lysis buffer [100 mM NaCl, 20 mM HEPES (pH 7.9), 1% Triton X-100, 1 mM Na3VO4, 4 mM sodium pyrophosphate, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and complete protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN)]. Samples (50 µg total protein) were separated by 10% SDS-PAGE and immunoblotted with antibodies against eNOS (Transduction Laboratories), phospho-eNOSS1177, Akt, phospho-AktS473, p44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology), and phosphotyrosine (4G10; Upstate Biotechnology, Inc., Lake Placid, NY) according to standard methods. Blots were quantified by scanning densitometry (Molecular Dynamics, Inc., Sunnyvale, CA).

ELISA for ET-1
BAECs were grown in 60-mm dishes, serum starved overnight, and then media were replaced with fresh EBM at the beginning of the experiment. Cells were treated without or with wortmannin (100 nM) or PD98059 (25 µM) for 1 h and then stimulated without or with either insulin (100 nM) or DHEA (100 nM) for 5 min. Conditioned media (1 ml) was then collected from the dishes and split into two aliquots for determination of ET-1 concentrations using an ELISA microplate assay kit (Assay Designs, Inc., Ann Arbor, MI) according to the manufacturer’s instructions.

Statistics
Paired Student’s t tests were used where appropriate. P values <0.05 were considered to represent statistical significance.


   FOOTNOTES
 
This work was supported, in part, by a Research Award from the American Diabetes Association (to M.J.Q.) and by the Intramural Research Program, National Institutes of Health, National Center for Complementary and Alternative Medicine.

The authors have no disclosures.

First Published Online December 22, 2005

Abbreviations: BAECs, Bovine aortic endothelial cells; DAF-2, diaminofluorescein 2; DAF-2 DA, diaminofluorescein diacetate; DHEA, dehydroepiandrosterone; EBM, endothelial basal medium; eNOS, endothelial nitric oxide synthase; ET-1, endothelin 1; L-NAME, nitro-L-arginine methyl ester; LPA, lysophosphatidic acid; MEK, MAPK kinase; NO, nitric oxide; PI 3-kinase, phosphatidylinositol 3-kinase; PPAR, peroxisome proliferator-activated receptor.

Received for publication July 4, 2005. Accepted for publication December 13, 2005.


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