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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2383
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 7 2761-2769
Copyright © 2006 by The Endocrine Society

Leptin Interferes with Adrenocorticotropin/3',5'-Cyclic Adenosine Monophosphate (cAMP) Signaling, Possibly through a Janus Kinase 2-Phosphatidylinositol 3-Kinase/Akt-Phosphodiesterase 3-cAMP Pathway, to Down-Regulate Cholesterol Side-Chain Cleavage Cytochrome P450 Enzyme in Human Adrenocortical NCI-H295 Cell Line

Hao-Ting Hsu1, Yuan-Ching Chang1, Yi-Ning Chiu, Chien-Liang Liu, King-Jen Chang and Ing-Cherng Guo

Department of Veterinary Medicine (H.-T.H., Y.-C.C., Y.-N.C., I.-C.G.), College of Bio-Resources and Agriculture, and Department of Surgery (K.-J.C.), National Taiwan University Hospital, National Taiwan University, Taipei 10617, Taiwan; Department of Surgery (Y.-C.C., C.-L.L.), Mackay Memorial Hospital, Taipei 10449, Taiwan; and Mackay Medicine, Nursing and Management College (Y.-C.C., C.-L.L.), Taipei 11260, Taiwan

Address all correspondence and requests for reprints to: Ing-Cherng Guo, Department of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, Taipei 10617, Taiwan. E-mail: iguo{at}ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism.

Objective: We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line.

Methods: Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system.

Results: Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between –1.7 and –1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3-kinase/Akt (wortmannin) as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including nonspecific 3-isobutyl-1-methylxanthine and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-nonhydrolyzable N6-monobutyryl-cAMP.

Conclusions: Leptin interferes with ACTH/cAMP signaling, possibly through a cAMP-degrading mechanism involving activation of JAK2, phosphatidylinositol 3-kinase, and PDE3, to down-regulate P450scc expression and consequent adrenal steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPERADRENOCORTICISM WAS COMMONLY observed in both leptin-deficient ob/ob mice and long-form leptin receptor (OBRb)-mutated db/db mice (1, 2); moreover, long-term supplement of leptin decreased circulating corticosterone levels in ob/ob mice but not db/db mice (3). An inverse correlation between serum levels of cortisol and leptin also existed in human subjects (4). These observations revealed that leptin-liganded OBRb signaling negatively controls adrenal steroidogenesis. In addition, leptin suppressed stress-induced release of CRH from rat hypothalamus to indirectly inhibit plasma levels of ACTH and corticosterone (5). On the other hand, glucocorticoids stimulated the leptin secretion of adipocytes (6). Taken together, these earlier observations introduced adipose tissues and leptin into the hypothalamic-pituitary-adrenal (H-P-A) axis. To constitute part of the H-P-A axis, CRH/ACTH-induced adrenal glucocorticoids stimulate adipose tissues to secrete leptin, which in turn exerts a long-loop feedback on hypothalamic CRH release to down-regulate adrenal steroidogenesis.

Expressions of leptin receptors (OBR) detected in cells or tissues of the adrenal cortex from mice (7), rats (8, 9), bovines (10), and humans (11, 12, 13) enabled adrenocortical cells to accept leptin signaling. A recent study of neurotoxin-damaged female rats showed that a decease of adrenal OBR expression causes adrenals to become refractory to leptin inhibition and hypersensitive to ACTH stimulation (14), implying that leptin may also have a direct interaction with adrenal cortex to modulate activation of the H-P-A axis. Therefore, in addition to the endocrine mode, a possible paracrine mode emerges from the facts that adrenals are usually embedded in adipose mass and both adrenal glucocorticoids and adipocytes-secreted leptin reciprocally regulate each other (6, 14). Accumulated evidence showing the direct effect of leptin on adrenocortical cells is controversial. The initial studies by Malendowicz et al. (8, 9, 15) found that leptin had no effect on ACTH-stimulated steroid secretion but elicited ACTH-like secretagogue effect in dispersed rat adrenocortical cells. On the contrary, Bornstein and his colleagues (11, 13, 16, 17) showed leptin inhibited basal and ACTH-stimulated adrenal steroid release in primary adrenocortical cells from bovines and humans and the NCI-H295 cell line. Similar leptin inhibition was also found in cells from a human adrenocortical adenoma (18). The distinct response of rats from that of bovines and humans might be attributed to a possible species selectivity of leptin effect. However, Pralong and colleagues observed the leptin inhibition of ACTH-stimulated steroid secretion in primary adrenal cells from humans (19) as well as rats (19, 20).

Exploring the molecular details on how leptin regulates adrenal steroidogenesis will provide insight into the leptin effect. The fact that ACTH-stimulated expressions of certain enzymes or proteins functioning in steroid biosynthesis are reduced by leptin makes the leptin inhibition of adrenal steroidogenesis more convincing. The ACTH-stimulated mRNA accumulations of three adrenal cytochrome enzymes essential for cortisol synthesis, including P450 cholesterol side-chain cleavage enzyme (P450scc), P450 21{alpha}-hydroxylase (P450c21), and P450 17{alpha}-hydroxylase, 17,20-lyase/desmolase (P45017{alpha}), were reduced by leptin in bovines (16, 17). Similar leptin inhibition was also found in ACTH-stimulated expressions of human P45017{alpha} mRNA (11, 13) as well as rat steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) (20, 21). StAR and PBR regulate delivery of cholesterol into mitochondria for subsequent steroid biosynthesis. Collectively, the above-mentioned ACTH/cAMP-responsive steroidogenic enzymes and proteins are negatively regulated by leptin. However, different from the bovine homolog and the members of steroidogenic P450 family, adrenal P450scc in humans and rats seemed nonresponsive to leptin (11, 13, 20). The unique response of P450scc to leptin is intriguing.

This paper investigated the signaling pathway and mechanism of leptin inhibition on human adrenal steroidogenesis. We demonstrated that leptin interfered with ACTH/cAMP signaling possibly through a cAMP-degrading mechanism involving activation of Janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and phosphodiesterase (PDE) 3 to down-regulate P450scc expression and subsequent steroid secretion. Our results suggest that all ACTH/cAMP-regulated adrenal steroidogenic enzymes and proteins could be negatively modulated by leptin via the proposed JAK2-PI3K/Akt-PDE3-cAMP pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and chemicals

Human adrenocortical NCI-H295 cells were cultured in DMEM/F-12 medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 1.2 mg/ml sodium bicarbonate, 100 U/ml penicillin G, and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO). Human recombinant leptin, ACTH, cholera toxin, wortmannin, Tyrphostin AG490, 3-isobutyl-1-methylxanthine (IBMX), SKF94836, and N6-monobutyryl-cAMP (N6-MB-cAMP) were purchased from Sigma.

RNA extraction and cDNA synthesis

Total RNA was extracted from NCI-H295 cells with Trizol reagent (Invitrogen) according to the instructions of the manufacturer. The isolated RNA was reversely transcribed into cDNA with the following protocol: 5 µg total RNA was denatured at 65 C for 20 min, cooled on ice, and incubated with a reverse transcriptase reaction mixture containing 10 U RNase inhibitor, 100 µM deoxynucleotide triphosphate, 1 µM oligo (poly-dT) (Roche, Mannheim, Germany), and 4 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) at 37 C for 90 min. The reverse transcription reaction was stopped at 72 C for 10 min to inactivate Moloney murine leukemia virus reverse transcriptase, denatured at 95 C for 5 min, and stored at 4 C.

PCR

The cDNA was further amplified by specific primer pairs, including forward OBRb (5'-CCA GAA ACG TTT GAG CAT CT-3') and reverse OBRb (5'-CAA AAG CAC ACC ACT CTC TC-3'), forward short-form OBR (OBRa) (5'-GAA GGA GTG GGA AAA CCA AAG-3') and reverse OBRa (5'-CCA CCA TAT GTT AAC TCT CAG-3'), forward P450scc (5'-CAG TGG CAC TTG TAT GAG ATG-3') and reverse P450scc (5'-TGG TCA TCT CTA GCT CAG CG-3') as well as forward glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-TCC CAT CAC CAT CTT CCA-3') and reverse GAPDH (5'-CAT CAC GCC ACA GTT TCC-3'). The PCR mixture contained cDNA, 100 µM deoxynucleotide triphosphate, 0.5 U Taq, and 0.5 µM forward and reverse primers in a final volume of 50 µl. The annealing temperatures for OBRa, OBRb, P450scc, and GAPDH were 55, 60, 57, and 60 C, respectively. Cycle numbers were 35 for OBR, 45 for P450scc, and 25 for GAPDH.

Quantitative real-time PCR

The quantitative real-time PCR and the quantification of specific PCR products were performed by the LightCycler (Roche) according to the manufacturer’s instructions. Briefly, the reaction mixture contained cDNA, 0.5 µM primer pairs and 2x QuantiTest SYBR Green PCR master mix (QIAGEN, Valencia, CA) in a final volume of 10 µl. The amplification protocol was set at 95 C for 15 min and 55 cycles at 94 C for 15 sec, 53 C for 20 sec, and 72 C for 20 sec, followed by the melting curve determination between 65 and 95 C to make sure of the single specific product. The specific primer pairs are forward P450scc (5'-GAG ATG GCA CGC AAC CTG AAG-3') and reverse P450scc (5'-CTT AGT GTC TCC TTG ATG CTG GC-3') as well as forward GAPDH (5'-GCT GTA GCC AAA TTC GTT GTC-3') and reverse GAPDH (5'-GAT GAC ATC AAG AAG GTG GTG-3').

Hormone assay

Progesterone and cortisol were detected with an ELISA modified from the published protocols (22, 23). Briefly, equal volumes (50 µl) of cultured media and horseradish peroxidase-labeled progesterone or cortisol were added into the 0.1% gelatin-blocked 96-well assay plate coated with a monoclonal antibody against progesterone or cortisol and incubated at 37 C for 60 min. After washing with 0.1% Tween 20 in PBS, the color was developed in a mixture of 3.7 mM o-phenylenediamine in 0.003% H2O2 for 30 min and then stopped by 6 N sulfuric acid. The optical absorbance was measured by ELISA reader at 490 nm, and the hormonal concentrations were determined by comparing to a standard curve.

Western blotting

The Western blotting analysis was modified from a previous study (24). In brief, the cells were spun down, washed with PBS, and incubated in cell lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and a proteinase inhibitor cocktail] for 30 min. After a brief centrifugation, 50 µg of proteins from the supernatant were applied to 12% SDS-PAGE gel. The electrophoresed proteins were then electrotransferred onto a nitrocellulose membrane; probed with primary antibodies specific for P450scc (25), JAK2 (Santa Cruz Biotechnology, Santa Cruz, CA), phosphor-JAK2 (Upstate, Lake Placid, NY), Akt (BD Biosciences, Franklin Lakes, NJ), phosphor-Akt (Cell Signaling Technology, Beverly, MA), or ß-actin (Sigma); and subsequently bound with peroxidase-conjugated secondary antibodies (Jackson, Bar Harbor, ME). After reacting with a chemiluminescence substrate (Pharmacia, Uppsala, Sweden), the blot was exposed to X-film (Fuji, Tokyo, Japan) for autography.

Luciferase assay

The CYP11A1 promoter-driven firefly luciferase construct (400 ng/well) and cytomegalovirus (CMV) promoter-driven renilla luciferase construct (20 ng/well) were cotransfected into NCI-H295 cells, 80% confluent in 48-well plate, with lipofectamine (Invitrogen), and then 24 h later were treated with or without reagents for 24 h. Next the cells were harvested and subjected to the dual-luminescence reporter gene assay system (PerkinElmer, Norwalk, CT) according to the manufacturer’s instructions to analyze the CYP11A1 promoter activity.

Quantitative assay of cAMP levels

After NCI-H295 cells were treated with cholera toxin for 2 h or ACTH for 6 h, hydrochloric acid was added into the cell cultures to stop endogenous phosphodiesterase activity. Then the cellular cAMP levels were analyzed with the Correlate-EIA direct cAMP enzyme immunoassay kit (Assay Designs, Ann Arbor, MI) according to the manufacturer’s instructions.

Statistical analysis

Data were presented as mean ± SD. Statistical analyses were performed by Student’s t test. Statistical difference was indicated as 1, P < 0.05, or **, P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin interferes with ACTH/cholera toxin-induced adrenal steroidogenesis

Constitutive expressions of leptin receptors, OBRb and OBRa, in NCI-H295 cells were verified by RT-PCR (Fig. 1AGo), indicating the capability of cells to receive leptin signaling. Next we tested the response of NCI-H295 to a 24-h treatment of leptin (10, 100, 1000 ng/ml). At up to 10 times the physiological dose (1000 ng/ml), leptin failed to affect the basal secretions of progesterone and cortisol detected with ELISA (data not shown), whereas the physiological dose (100 ng/ml) of leptin significantly reduced the secretions of both steroids induced by 100 ng/ml ACTH or 50 ng/ml cholera toxin (P < 0.01) (Fig. 1BGo).


Figure 1
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  		FIG. 1. Leptin interferes with ACTH/cAMP signaling on adrenal steroidogenesis. Total RNA extracted from NCI-H295 cells was analyzed by RT-PCR, and the specific OBR cDNA products, including the 609-bp OBRb and 386-bp OBRa, were visible on the agarose gel (A). The constitutive expressions of leptin receptors enable NCI-H295 to accept the signal from leptin. Cells were further treated with vehicle, leptin (L), ACTH, ACTH plus leptin, cholera toxin (CTX), or cholera toxin plus leptin for 24 h. The cultured media were collected and levels of progesterone and cortisol were determined by ELISA (B). The data calculated from five repeated experiments are expressed as the percentage of the vehicle group, which is set at 100%, and graphed with the mean ± SD value. The statistically significant difference between two indicated groups is labeled (**, P < 0.01). Leptin has no influence on basal adrenal steroid secretion but inhibits ACTH/cAMP signal-induced adrenal steroidogenesis.

 
Leptin down-regulates P450scc expression

Cytochrome P450scc encoded by CYP11A1 gene is the first and rate-limiting enzyme in adrenal steroidogenesis; we therefore monitored its response to leptin. After another 24-h treatment, the cholera toxin-stimulated expressions of P450scc protein and mRNA measured with Western blotting and RT-PCR, respectively, were concomitantly reduced by leptin (Fig. 2Go, A and B). To confirm the result of RT-PCR, the quantitative real-time PCR was carried out. We found that both ACTH- and cholera toxin-induced P450scc mRNA expressions were significantly deceased by leptin (P < 0.05 for ACTH; P < 0.01 for cholera toxin) (Fig. 2Go, C and D). To further investigate how leptin controls CYP11A1 transcription, NCI-H295 was separately transfected with a deletion clone harboring a 4.4-, 1.7-, or 1.5-kb CYP11A1 promoter in front of the reporter luciferase gene and then treated with ACTH or/and leptin. Twenty-four hours later, transfected cell extracts were subjected to luciferase activity assay to reflect the activities of tested promoter regions. Both ACTH and cholera toxin induced the activities of all clones except the one with the 1.5-kb CYP11A1 promoter; interestingly, leptin significantly suppressed their inductions (Fig. 3Go, A and B). These results indicate leptin negatively controls ACTH/cholera toxin-induced CYP11A1 promoter activity to inhibit P450scc expression. Curiously, the ACTH/cholera toxin-responsive region and leptin-responsive region were colocated within the same –1.7 kb/–1.5 kb fragment.


Figure 2
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  		FIG. 2. Leptin interferes with ACTH/cAMP signaling on P450scc expression. After cells were incubated with vehicle, leptin (L), cholera toxin (CTX), or cholera toxin plus leptin for 24 h, the cell lysates were subjected to Western blotting for monitoring the protein amounts of P450scc. The ß-actin was detected as the internal control (A). Simultaneously, their total RNA was analyzed with RT-PCR (B) or quantitative real-time PCR (C) to measure the accumulation of P450scc mRNA with GAPDH as the internal control. Likewise, the P450scc mRNA of cells treated with vehicle, leptin (L), ACTH, or ACTH plus leptin was measured with quantitative real-time PCR (D). The Western blotting and RT-PCR were repeated four and three times, respectively, with similar results. For real-time PCR, the data calculated from three repeated experiments are expressed as the percentage of the vehicle group, which is set at 100%, and graphed with the mean ± SD value. The statistically significant difference between two indicated groups is labeled (*, P < 0.05; **, P < 0.01). Well correlated with hormonal profile, ACTH- or cholera toxin-induced expressions of P450scc proteins and mRNA are reduced by leptin, suggesting that leptin inhibits ACTH/cAMP signal-induced adrenal steroidogenesis, possibly through down-regulation of protein and mRNA expressions of P450scc, the first and rate-limiting enzyme in steroid biosynthesis.

 

Figure 3
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  		FIG. 3. Leptin down-regulates ACTH/cAMP signal-induced CYP11A1 promoter activity via a cAMP-responsive region. NCI-H295 cells were transfected with the construct containing firefly luciferase driven by 4.4-, 1.7-, or 1.5-kb CYP11A1 promoter and then treated with vehicle, leptin (L), ACTH, ACTH plus leptin (A), cholera toxin (CTX), or cholera toxin plus leptin (B) for 24 h. A construct with CMV promoter-driven renilla luciferase was cotransfected as an internal control. After normalization with the internal control, the relative reporter luciferase activities calculated from five repeated experiments and compared with the vehicle group of phSCC4.4kb-Luc, which is set at 100%, are graphed with the mean ± SD value. The statistically significant difference between compared groups is labeled (*, P < 0.05; **, P < 0.01). ACTH and cholera toxin stimulate the activity of CYP11A1 promoter longer than 1.5 kb, suggesting that a cAMP-responsive region is located between –1.7 and –1.5 kb. Moreover, leptin diminishes cAMP responsiveness of that cAMP-responsive region.

 
JAK2 and PI3K/Akt possibly mediate leptin signal

JAK2 and its potential downstream signal components, PI3K/Akt, frequently mediate intracellular leptin signaling in many tissues; thus, studies were done to explore their role in leptin-regulated steroidogenesis. After NCI-H295 was treated with leptin and/or ACTH in the absence or presence of either specific JAK2 inhibitor (AG490) or specific PI3K inhibitor (wortmannin) for 24 h, the steroid secretion was assayed. The administration of either AG490 or wortmannin blunted the significant inhibition by leptin of ACTH-induced cortisol secretion (Fig. 4AGo) as well as progesterone secretion (data not shown). Comparable results were observed from a similar experiment substituting cholera toxin for ACTH (Fig. 4BGo). We further examined the ability of leptin in activating JAK2 and Akt. Phosphorylated forms of JAK2 and Akt were rapidly increased by leptin within 5 min of treatment in NCI-H295 (Fig. 4Go, C and D). The results suggest that leptin down-regulates ACTH/cholera toxin-induced adrenal steroidogenesis, possibly via activation of JAK2 and PI3K/Akt.


Figure 4
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  		FIG. 4. JAK2 and PI3K/Akt may mediate leptin inhibition on ACTH/cAMP signal-induced adrenal steroidogenesis. NCI-H295 cells were incubated with vehicle, leptin (L), ACTH, ACTH plus leptin (A), cholera toxin (CTX), or cholera toxin plus leptin (B) in the absence or presence of specific JAK2 inhibitor AG490 (AG) or specific PI3K inhibitor wortmannin (W) for 24 h. The levels of cortisol secreted in the cultured media were detected by ELISA. The data calculated from five repeated experiments are expressed as the percentage of the vehicle group, which is set at 100%, and graphed with the mean ± SD value. The statistically significant difference (**, P < 0.01) or nonsignificant difference (NS) between compared groups is labeled. Either AG490 or wortmannin blocks leptin inhibition on ACTH- or cholera toxin-induced cortisol secretion, suggesting that both JAK2 and PI3K may mediate leptin signaling. Simultaneously the phosphorylations of JAK2 (C) and Akt (D) in NCI-H295 were detected with antibodies specific for phosphor-JAK2 (p-JAK2), total JAK2, phosphor-Akt (p-Akt), and total Akt after cells were incubated with leptin for a 2-h time course. The ß-actin was detected as the internal control. The phosphorylated forms of JAK2 and Akt are rapidly induced by leptin during the first 5-min incubation, suggesting that leptin activates JAK2 and PI3K/Akt signaling pathways.

 
Leptin signaling may involve PDE3

In the hypothalamus, PI3K mediates leptin signaling to regulate cAMP-hydrolyzing PDE3B (26). To determine whether PDE is involved in leptin-regulated steroidogenesis, the steroid secretion of NCI-H295 treated with leptin and/or ACTH in the absence or presence of either nonspecific PDE inhibitor (IBMX) or PDE3-selective inhibitor (SKF94836) for 24 h was assayed. Either IBMX or SKF94836 abrogated the significant inhibition by leptin on ACTH-induced cortisol secretion (Fig. 5AGo) as well as progesterone secretion (data not shown). Comparable results were observed from a similar experiment substituting cholera toxin for ACTH (Fig. 5BGo). Additionally, we used N6-MB-cAMP, a PDE-resistant cAMP analog, to test the role of PDE in leptin inhibition. Like cholera toxin, N6-MB-cAMP significantly stimulated cortisol secretion, but unlike cholera toxin, the stimulation by N6-MB-cAMP was resistant to leptin inhibition (Fig. 5CGo). A similar progesterone profile resulting from the same experiment was observed (data not shown). Subsequent analysis of CYP11A1 promoter activity also showed that leptin inhibited induction by cholera toxin but failed to affect induction by N6-MB-cAMP (Fig. 5DGo). The data suggest that leptin inhibition of ACTH/cholera toxin-induced adrenal steroidogenesis may involve PDE3 activity.


Figure 5
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  		FIG. 5. cAMP-degrading PDE3 possibly mediates leptin inhibition on ACTH/cAMP signal-induced adrenal steroidogenesis. NCI-H295 cells were incubated with vehicle, leptin (L), ACTH, ACTH plus leptin (A), cholera toxin (CTX), or cholera toxin plus leptin (B) in the absence or presence of nonspecific PDE inhibitor IBMX or PDE3-specific inhibitor SKF94836 (SKF) for 24 h, and then the levels of cortisol secreted in cultured media were analyzed with ELISA. Both PDE inhibitors blunt leptin inhibition on ACTH- or cholera toxin-induced cortisol secretion, suggesting that PDE3 is possibly involved in leptin signaling. Likewise, after transfection with phSCC1.7kb-Luc, a construct containing 1.7-kb CYP11A1 promoter-driven firefly luciferase, NCI-H295 cells were treated with vehicle, leptin (L), cholera toxin, cholera toxin plus leptin, N6-MB-cAMP, or N6-MB-cAMP plus leptin. A construct with CMV promoter-driven renilla luciferase was cotransfected as an internal control. The cortisol secretion in cultured media of transfected cells was detected by ELISA (C), and their cell extracts were subjected to luciferase activity assay (D). Leptin is not able to affect cortisol secretion and CYP11A1 promoter activity induced by PDE-resistant N6-MB-cAMP. The data calculated from five (A, B) or three (C, D) repeated experiments are expressed as the percentage of the vehicle group, which is set at 100%, and graphed with the mean ± SD value. The statistically significant difference (**, P < 0.01) or nonsignificant difference (NS) between compared groups is labeled. Leptin signaling and ACTH/cAMP signaling may converge on PDE3.

 
Leptin reduces ACTH/cholera toxin-induced cAMP synthesis to down-regulate CYP11A1 promoter activity, possibly through activation of JAK2, PI3K/Akt, and PDE3

The cAMP-degrading PDE3 plays a crucial role in leptin function; thus, the effect of leptin on cellular cAMP synthesis was studied. As an activator of cAMP synthesis, cholera toxin effectively stimulated cAMP levels in NCI-H295, but leptin significantly reduced its stimulation (P < 0.01) (Fig. 6AGo). Similar stimulation of cAMP levels by ACTH was also significantly decreased by leptin (P < 0.05) (Fig. 6BGo). Cotreatment with inhibitor of JAK2, PI3K/Akt, PDE or PDE3 completely prevented ACTH/cholera toxin-induced cAMP synthesis from leptin inhibition (Fig. 6Go, A and B).


Figure 6
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  		FIG. 6. Leptin reduces ACTH/cAMP signal-induced cAMP levels and CYP11A1 promoter activity, possibly through activation of JAK2, PI3K/Akt, and PDE3. NCI-H295 cells were treated with vehicle, leptin (L), cholera toxin (CTX), cholera toxin plus leptin, ACTH, or ACTH plus leptin in the absence or presence of specific inhibitors like AG490 (AG), wortmannin (W), IBMX, and SKF94836 (SKF). After a 2- (A) or 6-h (B) treatment, their cAMP levels were determined by ELISA. The data calculated from three repeated experiments are graphed with the mean ± SD value. Leptin reduces ACTH- or cholera toxin-induced cAMP levels, but all inhibitors block leptin inhibition. A 24-h experiment with the same treatment was performed in NCI-H295 cells cotransfected with phSCC1.7kb-Luc containing 1.7-kb CYP11A1 promoter-driven firefly luciferase and an internal control containing CMV promoter-driven renilla luciferase (C, D). After normalization with the internal control, the relative reporter luciferase activities calculated from five repeated experiments and compared with the vehicle group, which is set at 100%, are graphed with the mean ± SD value. The statistically significant difference (*, P < 0.05; **, P < 0.01) or nonsignificant difference (NS) between compared groups is labeled. All inhibitors prevent ACTH- or cholera toxin-induced CYP11A1 promoter activity from leptin inhibition. Leptin may trigger a cAMP-degrading mechanism through activation of JAK2, PI3K/Akt, and PDE3 to reduce ACTH- or cholera toxin-induced cAMP content and consequent CYP11A1 promoter activity.

 
We examined whether leptin regulates the activity of 1.7-kb CYP11A1 promoter, which contains a known cAMP-responsive region, through the cAMP-degrading pathway. After transfection with phSCC1.7kb-Luc, NCI-H295 was treated with leptin and/or cholera toxin in the absence or presence of distinct inhibitors for 24 h. The luciferase activity assay showed that any inhibitor of JAK2, PI3K/Akt, PDE, or PDE3 prohibited the inhibitory effect of leptin on cholera toxin- or ACTH-induced CYP11A1 promoter activity (Fig. 6Go, C and D). Taken together, these results reveal that leptin may activate a cAMP-degrading pathway to down-regulate CYP11A1 promoter activity, possibly through the –1.7 kb/–1.5 kb cAMP-responsive region.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that leptin directly inhibits ACTH/cholera toxin-induced but not basal adrenal steroidogenesis. Leptin interferes with ACTH/cAMP signaling through a proposed cAMP-degrading JAK2-PI3K/Akt-PDE3-cAMP pathway to down-regulate P450scc expression and consequential steroid secretion. These observations inspire us to deliberate on the influence of obesity on the physiological responses to stress, the metabolism of energy, and electrolytic balance.

Adrenal steroidogenic enzymes, including members of the cytochrome P450 family and hydroxysteroid dehydrogenase family and steroidogenic proteins like StAR and PBR are controlled by ACTH, mainly through the cAMP signaling pathway. We used P450scc as a representative model to study how leptin affects the ACTH/cAMP-regulated expressions of steroidogenic enzymes and proteins. Our in vitro studies have previously shown that cAMP signaling controls adrenal P450scc expression via a cAMP-responsive region, named U-CRS (upstream cAMP-responsive sequences) and located in 5'-flanking –1640/–1540 promoter region (25, 27, 28, 29). U-CRS with two cAMP response element-binding protein/activator protein 1-binding sites and a steroidogenic factor-1 binding site (30) functions in adrenal cells but not placental cells, depending on steroidogenic factor-1 expression (31). The essential role of U-CRS in ACTH/cAMP-regulated CYP11A1 promoter activity has been demonstrated in the mutagenic transgenic mouse model (32, 33). We tried to define leptin-responsive cis-elements, which were expected to mediate leptin inhibition on CYP11A1 transcription. With deletion experiments of CYP11A1 promoter, we showed that leptin and ACTH/cholera toxin act through the same –1.7 kb/–1.5 kb region, which contains the U-CRS. Most likely, signaling pathways of ACTH/cAMP and leptin converge on the U-CRS.

Similar to our observations, leptin inhibited the cAMP regulation of hepatic glucose metabolism (34, 35, 36) via a reduction of intracellular cAMP levels (36). Due to lacking an intrinsic tyrosine kinase domain, OBR needs the binding of JAK2 to mediate intracellular leptin signaling. The JAK2-associated OBRb is the full-length leptin receptor with intact elements that are required for subsequent signaling events. The binding of leptin to OBRb activates JAK2 to initiate three major signaling pathways such as ERK1/2 pathway, signal transducers and activators of transcription 3 pathway, and insulin receptor substrate (IRS)-dependent PI3K pathway (37). Among them, the IRS-dependent PI3K pathway is associated with the leptin-regulated reduction of intracellular cAMP content. It was first delineated that leptin triggers a signal pathway connecting PI3K-dependent activation of PDE3B with subsequent suppression of cAMP levels to inhibit glucagon-like peptide-1-stimulated insulin secretion in rat pancreatic islets (38). In rat primary hepatocytes, the glucagon-induced cAMP elevation was directly antagonized by leptin through the pathway involving stimulated binding of PI3K to IRS1 and IRS2 and activation of PI3K and Akt and a main cAMP-degrading enzyme, PI3K-dependent PDE3B (39). The proposed PI3K-PDE3B-cAMP pathway, possibly interacting with the JAK2-signal transducers and activators of transcription 3 pathway, also constituted a critical part of leptin signaling in the rat hypothalamic action on feeding (40). In good agreement with the above-mentioned studies, our experiments showed the activation of JAK2, PI3K/Akt, and PDE3 mediates leptin signaling to down-regulate ACTH/cAMP-induced human adrenal steroidogenesis. The present and previous studies support a leptin signaling pathway, wherein the PI3K plays a central role to link JAK2 with PI3K-dependent Akt and consequent PDE3B, leading to a decline of intracellular cAMP levels.

The direct suppression of ACTH-stimulated corticoids secretion by leptin was commonly observed in primary adrenal cells dispersed from adult rats (19, 20), neonatal rats (21), bovines (16, 17), and humans (11, 13, 19), but diverse observations describing ACTH-like secretagogue effect of leptin on corticoids production in dispersed rat adrenocortical cells were also reported (8, 9, 15). The cause for the divergent observations of leptin effects is unclear but intriguing. The in vivo observations agreed with the inhibitory effect of leptin on adrenocortical functions. Leptin-deficient ob/ob mice with normal values of circulating ACTH were characterized as hyperadrenocorticism throughout their life (1), implying that the lack of leptin leads to increased ACTH responsiveness of adrenal glands. Maternal separation caused neonatal rats to have lower leptin but higher corticosterone levels in blood, revealing the negative correlation between secretions of leptin and corticosterone (21). Both StAR and PBR appear to be important mediators in leptin signaling on adrenal steroidogenesis because their ACTH-induced expressions were diminished by leptin in both adult (20) and neonatal rats (21). In addition to StAR and PBR, steroidogenic cytochrome P45017{alpha} and P450c21 enzymes are also potential mediators.

In primary adrenal cell cultures, the ACTH-stimulated mRNA levels of P45017{alpha} from bovines (16) and humans (11, 13) and P450c21 from bovines (17) were reduced by leptin. The role of P450scc was debatable because the inhibitory effect of leptin on the ACTH-stimulated adrenal P450scc was observed in bovines (17) but not in rats (20) and humans (11, 13). Our consistent observations from the levels of protein, mRNA, and promoter activity confirm the inhibitory effect of leptin on ACTH/cholera toxin-induced P450scc expression in human adrenocortical cells and indicate the important role of P450scc in leptin-regulated human adrenal steroidogenesis. Based on the cAMP-degrading mechanism underlying leptin action, we speculate that the expressions of all ACTH/cAMP-responsive steroidogenic enzymes and proteins should be potentially susceptible to the interference from leptin. The leptin inhibition of cAMP-induced expressions of 3ß-hydroxysteroid dehydrogenase and P450c21 was also seen in our studies (Hsu, H.-T., and I.-C. Guo, unpublished data). The inhibitory effect of leptin on basal steroidogenesis remains controversial. A previous report showed that leptin inhibited basal cortisol release in bovine primary cell cultures (16). However, we did not find any effect of leptin on basal adrenal steroidogenic system in a human adrenocortical NCI-H295 cell line. Results similar to ours were reported in primary cell cultures from rats (19, 20, 21) and humans (19). The cAMP-degrading mechanism may also be able to explain why leptin has insignificant influence on the basal adrenal steroidogenic system in our and other studies.

In this study, we elucidate the mechanism underlying the inhibitory effect of leptin on ACTH/cAMP-controlled adrenal steroidogenesis. The leptin signaling possibly activates a proposed JAK2-PI3K/Akt-PDE3-cAMP pathway to cross-talk with ACTH/cAMP signaling and consequently result in the reduction of intracellular cAMP content. In turn, the abated cAMP signal lessens adrenal steroidogenesis by down-regulating the expression of CYP11A1 gene, regarded as a typical model for all ACTH/cAMP-responsive steroidogenic genes.


   Footnotes
 
This work was supported by the National Taiwan University, Mackay Memorial Hospital, and National Science Council (NSC), Republic of China, Grants NSC-90-2320-B-002-158, and NSC 91-2313-B-002-387 (to I.-C.G.), NSC 92-2314-B-195-020, and NSC 93-2314-B-195-026 (to C.-L.L.), and NSC 91-2320-B-002-160 (to K.-J.C.).

Disclosure of potential conflicts of interest: H.-T.H., Y.-C.C., Y.-N.C., C.-L.L., K.-J.C., and I.-C.G. have nothing to declare.

First Published Online May 9, 2006

1 H.-T.H. and Y.-C.C. made equal contributions to this work. Back

Abbreviations: Akt, Protein kinase B; CMV, cytomegalovirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H-P-A, hypothalamic-pituitary-adrenal; IBMX, 3-isobutyl-1-methylxanthine; IRS, insulin receptor substrate; JAK2, Janus kinase 2; N6-MB-cAMP, N6-monobutyryl-cAMP; OBR, leptin receptor; OBRa, short-form OBR; OBRb, long-form OBR; PBR, peripheral-type benzodiazepine receptor; PDE, phosphodiesterase; PI3K, phosphatidylinositol 3-kinase; P450c21, P450 21{alpha}-hydroxylase; P45017{alpha}, P450 17{alpha}-hydroxylase, 17,20-lyase/desmolase; P450scc, P450 cholesterol side-chain cleavage enzyme; StAR, steroidogenic acute regulatory protein; U-CRS, upstream cAMP-responsive sequences.

Received November 1, 2005.

Accepted April 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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