Leptin Induces Growth Hormone Secretion from Peripheral Blood ...

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Endocrinology 144(12):5595–5603 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2003-0600

Leptin Induces Growth Hormone Secretion from Peripheral Blood Mononuclear Cells via a Protein Kinase C- and Nitric Oxide-Dependent Mechanism VISHWA DEEP DIXIT, MANFRED MIELENZ, DENNIS D. TAUB,

AND

NAHID PARVIZI

Institute of Animal Science Mariensee (V.D.D., M.M., N.P.), D-31535, Neustadt, Germany; and Laboratory of Immunology (D.D.T.), Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224 Leptin is a key mediator of signals regulating food intake and energy expenditure and exerts potent immunomodulatory effects. We investigated the mechanisms mediating the action of leptin on GH secretion from peripheral blood mononuclear cells (PBMCs). Using immunofluorescence microscopy, we demonstrated a polarized expression pattern of leptin receptor protein on the surface of mononuclear cells and constitutive expression of GH in PBMCs. Leptin exhibited a dosedependent stimulatory effect on GH secretion by PBMCs and also up-regulated the GH receptor gene expression. We did not observe any additive effects of leptin on GH secretion upon activation of cells with the plant mitogen phytohemagglutinin, unlike leptin, phytohemagglutinin exerted no effect on GH receptor mRNA expression. Leptin led to a nitric oxide (NO) synthase (NOS)-specific, dose-dependent increase in NO production from PBMCs because leptin-induced NO release

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EPTIN, THE Lep gene product, is a 16-kDa adipocytederived hormone that is a key mediator in the regulation of food intake and energy expenditure (1, 2). Leptin’s functions are quite pleiotropic, and it is implicated in a variety of cellular processes, including the modulation of immune cell function (3, 4). Leptin is structurally related to the long-chain helical cytokine family, which includes IL-2, IL12, and GH (5). The leptin receptor, which has sequence homology to members of the gp130 cytokine receptor superfamily, is widely distributed throughout the body, and its mRNA is known to be expressed in hematopoietic cells and lymphocytes (6, 7). The importance of leptin in immunity is evident in mice having a homozygous mutation in their leptin gene (Lep/Lep) or receptor. These mice have impaired T cell-mediated responses (8), and administration of leptin to ob/ob mice reverses the immunosuppressive effects of acute starvation (9). Leptin also exerts proinflammatory effects and has been implicated in stimulating type-1 cytokines in rodents (9). Moreover, a recent report gave impetus to the use of leptin as a therapeutic agent in obese subjects who were heterozygous for frameshift mutation in the Lep gene (10). In all of the subjects who demonstrated lower levels of circulating leptin, the number of CD4⫹ T cells, impaired prolifAbbreviations: GHR, GH receptor; HBSS, Hanks’ balanced salt solution; l-NAME, N␻-nitro-l-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; PBMC, peripheral blood mononuclear cells; PHA-M, phytohemagglutinin; PKC, protein kinase C; RT, reverse transcription; SNP, sodium nitroprusside.

was blocked by the addition of the NOS inhibitor N␻-Nitro-Larginine methyl ester and protein kinase C (PKC) inhibitor calphostin C. This leptin-induced GH secretion was dependent on both PKC and NO activation because the addition of PKC and NOS inhibitors inhibited leptin-induced GH production. Although the addition of sodium nitroprusside, a spontaneous liberator of NO, stimulated GH release from PBMCs, leptin had no additive or synergistic effect on sodium nitroprusside-induced GH production. Together, these findings demonstrate a unique action of leptin on immune cells via its ability to stimulate the GH production by blood mononuclear cells via PKC- and NO-dependent pathways. These data also support a probable role for local immune-derived GH in mediating some of the pleiotropic actions of leptin. (Endocrinology 144: 5595–5603, 2003)

erative potential, and aberrant cytokine synthesis was observed. However, all these effects were reversed via the subsequent administration of recombinant human leptin (11) in this group of subjects. In addition, leptin has recently been shown to be secreted by murine lymphocytes and has been implicated in pathogenesis of multiple sclerosis and may serve as a potential target for therapeutic intervention (12). Lymphocytes orchestrate adaptive immune responses via antigen recognition and the secretion of cytokines and growth factors (13). The expression of hormones and their receptors on lymphocytes are believed to play an important role in the maintenance of homeostasis during normal physiological processes as well as pathological states (14, 15). Lymphocytes express receptors for GH (16) and also produce GH (17, 18), which is apparently similar to its pituitary counterpart (19). GH increases the migration of fresh and activated lymphocytes and augments T cell adhesion via ␤1 and ␤2 integrins (20). GH has been shown to play an important role in the development and regulation of the immune system (21–24). Moreover, lymphocytes express GHRH and somatostatin along with their specific receptors; however, contradictory data exist on the modulation of GH secretion from lymphocytes by these neuropeptides (25, 26). We have recently demonstrated that stimulatory mechanisms involved with GH release are similar between the pituitary gland and circulating lymphocytes; however, the inhibitory control mechanisms regulating GH appear to differ (18). Based on these data, the precise mechanisms controlling GH secretion by lymphocytes remain unclear.

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Nitric oxide (NO) has emerged as an important mediator of a wide range of critical processes including neurotransmission, endocrine signal transduction, mediation of reproductive function (27), vasodilation, and immune defense (28). In biological systems, NO is produced by NO synthases (NOS) using arginine as precursor (29). NO is implicated in the neuroendocrine control of pituitary GH secretion (30). Furthermore, leptin-induced expression of LHRH and LH secretion has been reported to be mediated via nitricoxidergic mechanisms (31). Based on these data, we conducted a series of experiments to investigate the role of leptin on GH secretion and the mechanisms involved in downstream leptin receptor signaling in porcine peripheral blood mononuclear cells (PBMCs). Materials and Methods Animals Healthy German Landrace gilts (7.8 months of age, n ⫽ 25) were used in this study. Animals were slaughtered, and 500 ml of blood was collected from each gilt and placed in heparin (2500 IU/ml) at the time of slaughter. This blood was subsequently layered on lymphocyte separation medium, Lymphodex (Fresenius Diagnostik, Wiesbaden, Germany), and centrifuged at 1500 ⫻ g for 30 min at 20 C to enrich the mononuclear cells (15, 19). Upon enrichment, mononuclear cells were removed, resuspended, and washed twice with Hanks’ balanced salt solution (HBSS). Contaminating erythrocytes were lysed by hypotonic shock in double distilled water, followed by an immediate wash with HBSS. Animals were used in accordance with procedures approved by the Institute of Animal Science and Hannover Government Animal Care and Use Committee (approval no. 509C-42502-00/394).

Cell culture PBMCs (1 ⫻ 106/ml) were resuspended in a combination of 1:1 RPMI 1640 (Sigma, Munich, Germany) and HBSS solutions. The cells were subsequently seeded in four-well culture plates (Nunc Brand Product, Darmstadt, Germany) in RPMI 1640 supplemented with 1% fetal calf serum (Sigma), 1% antibiotic/antimycotic mixture containing 10,000 IU penicillin, 10 mg streptomycin, and 25 ␮g/ml amphotericin B (Sigma). These cultures were then stimulated in the presence or absence of phytohemagglutinin from Phaseolus vulgaricus (PHA-M; 10 ␮g/ml, Sigma), a plant mitogen known to stimulate hormone production from lymphocytes, for 48 h at 37 C and 5% CO2 (17). Human leptin (Sigma) was added to specific cultures at various concentrations ranging from 1–100 nm, doses previously shown to be effective on immune cells (9). To study the involvement of NO on GH secretion, PBMCs were incubated with sodium nitroprusside (SNP; Sigma), a NO donor, at nontoxic concentrations of 0.5–1 mm (32). Furthermore, PBMCs were cultured with N␻-nitro-l-arginine methyl ester (l-NAME; 0.5 mm), a NOS inhibitor, in presence or absence of the maximal dose of leptin (100 nm). The dose for l-NAME was selected for maximal inhibition of NOS (31). Calphostin C, a specific protein kinase C (PKC) inhibitor (Calbiochem, Darmstadt, Germany), was used at a concentration of 100 nm, which has been previously shown to completely inhibit PKC activity in the presence of leptin (100 nm) (33). To obtain appropriate control data, PBMCs were cultured with l-NAME (0.5 nm) or calphostin C (100 nm) or SNP (1 mm) in combination with leptin (100 nm). Cell viability assessed by trypan blue exclusion test was more than 95%. For each treatment, a total of 120 cell cultures were conducted (10 animals; three plates each per animal; four wells per each plate).

Gel chromatography At the end of the incubation period (48 h), media from the three plates (12 wells) of one treatment were pooled, and GH was eluted by chromatography using 1.5 ⫻ 30 cm Sephadex G-50 fine column. The column was equilibrated with 0.1 m PBS containing 5% BSA, and 26 aliquots of 3 ml each were collected and stored at ⫺20 C pending analysis. These

Dixit et al. • Leptin Induces GH Secretion from PBMCs

samples were subsequently lyophilized using ␤1 lyophilizer (Christ, Osterode, Germany) and then reconstituted in 300 ␮l of assay buffer [0.01 m PBS, 0.025 m EDTA, 0.25% BSA, 0.01% thimerosal (pH 7.4)].

GH assay GH was measured in duplicate by a homologous double antibody RIA according to the method described by Bauer and Parvizi (34) and adapted for GH measurements in cell culture medium. Briefly, highly purified porcine GH (Biogenesis, Dorset, UK), with a potency of 1⫻ United States Department of Agriculture B-1 standard, was used as standard and for iodination. A highly specific antiporcine GH antiserum (Biogenesis) was used as first antibody. This antiserum shows no crossreactions with other adenohypophysial hormones. All reagents were diluted in assay buffer. Half maximum displacement was achieved at 6 ng/ml. The intra- and interassay coefficients of variation were 7.2% and 12%, respectively. The coefficient of variance of cell cultures was 15%. GH was also measured in 100-␮l aliquots of culture medium (not incubated with cells). This background level (0.7 ⫾ 0.1 ng/ml) was deducted from the level measured in each sample. GH was measured in all 26 aliquots of one treatment (three plates each, four wells; see Cell culture). For statistical evaluations, GH levels measured in 7–12 fractions were pooled. To evaluate the nature of GH secreted by lymphocytes, 100 ng of highly purified (PGH, Biogenesis) I125-labeled porcine pituitary GH diluted in 1 ml of culture medium was eluted on a column similar to the one used for elution of media from cell cultures. The profiles of the elutions resembled those of media from cell cultures (18), indicating that porcine lymphocytic GH is largely similar to its pituitary counterpart.

NO measurement In aqueous solutions that contain no heme proteins, NO is oxidized to nitrite only (35), which can serve as an indirect marker for the presence of NO (36). All reagents were freshly prepared before each assay. Assay was performed as described previously (15); briefly, total nitrite in sample was assayed using equal amounts of sample and Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthylene) ethylenediamine in 5% concentrated phosphoric acid]. Amounts of nitrite were estimated from a standard curve of sodium nitrite, and the absorbance was measured at 540 nm spectrophotometrically with an assay sensitivity of 1 ␮m.

Immunofluorescence staining PBMCs were isolated from heparinized blood samples as described earlier (blood samples from three female and three castrated male adult pigs were purchased from the National Institutes of Health, Poolsville, MD). All fluorochromes were purchased from Molecular Probes (Eugene, OR). Labeling was performed as described previously (37); briefly, the PBMCs were fixed and permeabilized using 2% paraformaldehyde and 0.1% Triton X-100 for 15 min. After thorough washing of cells, nonspecific binding sites were blocked using 2% BSA in combination with 1% goat and rabbit serum and normal mouse IgG. Cells were incubated with anti-leptin receptor mouse IgG and human anti-GH goat IgG at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C. After washing, the cells were then labeled with specific secondary antibodies (chicken antimouse IgG conjugated with Alexa Fluor-594 (green; Molecular Probes, Inc., Eugene, OR) with absorbance and fluorescence emission maximum of 590 and 617 nm and donkey antigoat Alexa Fluor-488 with absorbance and fluorescence emission maximum of 495 and 519 nm). Cellular nuclei were counterstained using 4⬘,6-diaminodino-2-phenylindole dihydrochloride (1 ␮g/ml) for 10 min at room temperature. After staining, the cells were subsequently placed into cytospin funnels and spun onto glass slides using a cytospin centrifuge (Shandon, Pittsburgh, PA) at 1200 rpm for 5 min. Cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were acquired by Spot Advanced software on a Zeiss Axiovert S100 microscope under ⫻100 objective (Carl Zeiss, Thornwood, NY).

Relative quantification of GH receptor (GHR) gene expression This experiment was carried out additionally on five mature German Landrace gilts. GHR gene expression was studied in freshly separated


PBMCs as well as in cells cultured for 5 h in the presence or absence of PHA-M (10 ␮g/well) and/or leptin (100 nm).

RNA extraction and reverse transcription (RT) Total RNA was extracted from peripheral lymphocytes according to a guanidinium thiocyanate extraction method (38) with some modifications. The concentration of RNA in each sample was analyzed by absorbance at 260 nm. The RNA integrity was checked by ethidium bromide staining after formaldehyde gel electrophoresis. The RNA (1.8 ␮g) was reverse transcribed by the use of 100 U SuperScript II Rnase H⫺ (Life Technologies, Gaithersburg, MD) in reaction buffer [50 nm TrisHCl, 75 nm KCl, 3 mm MgCl2, (pH 8.3)] with 10 mm dithiothreitol, 2.5 ␮m random hexamer primers (PE, Applied Biosystems, Foster City, CA), 500 ␮m of each deoxynucleotide triphosphate, and 10 U ribonuclease inhibitor (MBI Fermentas, Vilnius, Lithuania) for 10 min at 27 C, 60 min at 42 C, and 1 min at 99 C to inactivate the reverse transcriptase in a 20-␮l total reaction volume.

PCR For amplification of the GHR cDNA by PCR, we used the forward primer 5⬘-TGA GCC CAT TTG CAT GTG AAG-3⬘ and the reverse primer 5⬘-TCT GAG CCT TCA GTC TTT TCA TC-3⬘ (0.4 ␮m of each), which correspond to the region 779-1100 bp based on the porcine GHR cDNA sequence (39). The specificity of the resulting 322-bp DNA fragment was verified by sequencing after cloning in PCR Script Amp SK (⫹) (Stratagene, La Jolla, CA).

Competitive PCR For designing of the competitor, we used the PCR MIMIC construction kit (Clontech Laboratories, Palo Alto, CA), which yielded a heterologous competitor approximately 431 bp in size. Equal efficiencies for amplification of the two fragments were checked using total RNA from adult pig liver from 21–33 cycles. The samples were screened using a series of 10-fold dilutions of competitor. In subsequent experiments, three selected concentrations of competitor (3 ␮l) at 1:2 dilution were coamplified with 3 ␮l aliquot of RT reaction product. PCR was performed using 2 U Dynazyme II DNA-Polymerase (Finnzymes OY, Espoo, Finland) in a final volume of 50 ␮l, with 25 mm Tris-HCl, 58.5 mm KCl, and 2.04 mm MgCl2 (pH 8.4) with the following cycling conditions: 33 cycles of 95 C for 60 sec; 61 C for 10 sec; 72 C for 20 sec, and final extension for 5 min at 72 C. For negative control, we used RT product without RNA. As an internal loading control, we used 18s rRNA primers (Ambion, Austin, TX), resulting in the generation of a 488-bp DNA fragment. After the appropriate number of cycles, 18 ␮l of the amplified product was visualized on a 2% agarose gel by ethidium bromide. The signals of the specific product and its competitor were quantitated via video densitometry (Image Master ID Elite, V3; Pharmacia Biotech, Uppsala, Sweden) and subsequently corrected to the calculated level of the corresponding 18s rRNA PCR fragment. The log ratio between the two fragments was expressed as arbitrary unit without correction for size differences.

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expression of leptin receptor protein. Upon immunophenotypic analysis using both polyclonal as well as monoclonal anti-leptin receptor antibodies, we observed greater than 60% of mononuclear cells to be leptin receptor positive. Interestingly, approximately 70% of leptin receptor-positive cells displayed a polarized expression pattern (Fig. 1, A–D). Also, for the first time, we demonstrated the presence of immunoreactive GH (47.3 ⫾ 6%) within the PBMCs. Dual labeling of PBMCs revealed that approximately 24.9 ⫾ 5% of these cells coexpress both leptin receptor and GH. Negative controls omitting the primary antibody and/or the use of an isotype-matched IgG failed to demonstrate any specific labeling in our cells (data not shown). Leptin induces GH secretion and GHR expression in PBMC

Leptin is currently regarded as a strong modulator of hypophyseal GH release (3). Here, we report that leptin exerts stimulatory effects on GH secretion from mononuclear cells. Unstimulated PBMCs in the culture secreted 54.2 ⫾ 2.39 ng/ml of GH by 1.2 ⫻ 107 cells (for calculation of the levels see GH assay). This secretion increased significantly (P ⱕ 0.01) in a dose-dependent manner upon incubation with leptin for 48 h (Fig. 2). Moreover, incubation with PHA-M, a plant mitogen that stimulates lymphocytes in a T cell receptor-independent fashion by cross-linking a number of cell-surface stimulatory molecules, also significantly (P ⱕ 0.05) increased GH secretion (88.9 ⫾ 4.81 ng/ml) by PBMCs. Leptin failed to induce any additive effect on GH secretion from PHA-M-activated PBMCs (Fig. 2). The production of GH by immune cells is believed to have biological significance because these cells also appear to express GHR. Therefore, we next analyzed the GHR expression in these cells using competitive PCR and demonstrated the presence of GHR mRNA in fresh, as well as activated, PBMCs. Freshly isolated PBMCs expressed 50% higher levels of GHR mRNA when compared with cells incubated alone at 37 C for 5 h; however, this difference failed to reach statistical significance. Upon leptin (100 nm) treatment, there was a 69% increase (P ⱕ 0.05) in GHR mRNA expression compared with unstimulated control cells (Fig. 3, upper panel). Interestingly, unlike leptin, activation of PBMCs with PHA-M had no effect on GH-R expression (Fig. 3, lower panel), suggesting that leptin-induced GH secretion and GHR expression are specific leptin receptor-mediated events.

Statistical analysis

Leptin regulates NO production via PKC

The results are expressed as the mean ⫾ sem. The differences between means and the effects of treatments were determined by one-way ANOVA using Tukey’s test, which protects the significance of all pair combinations (40).

NO acts as an important signaling molecule as well as a proinflammatory mediator in cells (28). Leptin is also known to exert potent proinflammatory effects in immune cells (9). To determine whether leptin exerts effects on NO production by PBMCs, we measured nitrite levels in culture supernatants as an indirect measure of NO production. Interestingly, leptin exerted a dose-dependent induction of NO production (measured as nitrite) by PBMCs that reached significant levels when 10 nm (P ⱕ 0.01) and 100 nm (P ⱕ 0.05) of leptin were applied (Fig. 4). Moreover, in the presence of the NOS inhibitor L-NAME, leptin-induced NO production was attenuated, suggesting a specific NOS-mediated pathway. We used NO donor, SNP, as a control in cultured PBMCs. SNP

Results Subcellular localization of leptin receptor and GH within porcine PBMCs

Leptin receptor mRNA has previously been demonstrated to be expressed on immune cells (9); however, the subcellular distribution of leptin receptor protein in immune cells is currently not known. As a first step to elucidate the functional role of leptin in porcine PBMCs, we demonstrated the

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FIG. 1. Subcellular localization of leptin receptor (LepR) and GH in resting porcine lymphocytes. A, Immunofluorescence double-labeling of leptin receptor and GH was performed with specific antibodies and visualized by secondary antibodies conjugated with Alexa Fluor-594 and Alexa Fluor-488. Leptin receptor displays highly polarized expression in approximately 70% of total leptin receptor-positive PBMCs. B, Close-up image of cells expressing leptin receptor and GH, merge of images demonstrate coexpression of leptin receptor and GH (yellow) in immune cells. C, Negative control using isotypematched IgG demonstrated no staining in PBMCs. D, Percentage of cells expressing leptin receptor, GH, and cells coexpressing both of the antigens. DAPI, 4⬘,6-Diamidino-2-phenylindole.




FIG. 2. Effect of leptin on GH secretion in resting and activated PBMCs. Leptin stimulates GH secretion from resting PBMCs. Activation-induced GH secretion from PBMCs is not affected by leptin. Values are expressed as mean ⫾ SEM, for calculation of values see GH assay; *, P ⱕ 0.05; **, P ⱕ 0.01 vs. control; one-way ANOVA followed by Tukey’s test.

spontaneously liberates NO when added in aqueous medium without the involvement of NOS (P ⱕ 0.001) (Fig. 4). Additions of L-NAME or calphostin C to SNP-treated cultures did not affect the NO release (data not shown). Interestingly, PKC inhibitor calphostin C was as equally effective as L-NAME in blocking (P ⱕ 0.01) leptin-induced NO production from PBMCs (Fig. 4), suggesting that activation of PKC is critical for leptin-mediated NO release. Role of NO and PKC in leptin-induced GH secretion from PBMCs

Given the dose-dependent stimulatory effect of leptin on NO production and its regulation by PKC, we further investigated the functional role of these two signaling mediators in regulating leptin-induced GH secretion from PBMCs. Treatment of cells with the NO donor, SNP, resulted in a significant increase (P ⱕ 0.01) in GH secretion (Fig. 5), further supporting a role for NO in GH production from immune cells. The GH release in response to SNP (1 mm) plus leptin (100 nm) is not significantly different from the response to SNP or leptin when given alone. Finally, through the use of NOS and PKC inhibitors, we further demonstrated the specificity of these mediators in leptin-induced GH production. Coincubation of cells with leptin (100 nm) and L-NAME blocked the leptin-induced GH secretion (P ⱕ 0.01). As seen in Fig. 5, leptin-stimulated (100 nm) GH secretion was also abolished (P ⱕ 0.01) in the presence of calphostin C, indicating that leptin binding to leptin receptor in lymphocytes and resultant signaling is dependent on PKC and NO. When applied alone, L-NAME (0.5 nm) and calphostin C (100 nm) failed to change the basal GH release significantly. Discussion

A great deal of evidence has been presented demonstrating the involvement of hormones, cytokines, and neuro-

modulators in the bidirectional communication and integration of the immune and endocrine systems (14). Our current findings demonstrate, for the first time, that an increase in GH production by PBMCs is mediated by leptin, which involves NO and PKC signals. These findings add to the growing body of evidence that metabolic signals, such as leptin, are coupled to the modulation of immune cell function. Leptin has been shown to increase GH release and pulsatility from murine, ovine, and porcine pituitaries via GHRH, somatostatin (42, 43), and GH-releasing peptide-6 (44). On the other hand, leptin can also activate lymphocytes (8) via the leptin-specific cell-surface receptors, rather than by a nonspecific mitogenic stimulus (9). As a first step in examining the biological effects of leptin on immune cells, we investigated the subcellular localization of leptin receptors and GH within porcine mononuclear cells. Leptin receptor expression on mononuclear cells revealed an interesting polarized distribution in greater than 60% of cells. Given that polarization and aggregation of receptors in immune cells is critical in facilitating the downstream signaling, highly polar expression of leptin receptors on PBMCs suggests a specific role for leptin in immunomodulation. Furthermore, double-labeling of PBMCs revealed that 25% of these cells coexpress both leptin receptors and GH, indicating a possible autocrine/paracrine relationship between these two at the immune-cell level. In support of this hypothesis, we observed a dose-dependent increase in the GH secretion upon stimulation of PBMCs with leptin. Moreover, through the use of the T cell mitogen, PHA-M, we also demonstrated activation-induced GH secretion from PBMCs. These results were further supported by the fact that PHA-M, when used at similar concentrations, also up-regulates GH secretion by human lymphocytes (44). Leptin failed to further elevate the activation-induced GH secretion from PBMCs. Interestingly, in contrast to PHA-M, leptin not

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FIG. 3. Leptin stimulates GHR mRNA expression in PBMCs. Upper panel, Percentage of GH mRNA normalized to 18s rRNA in porcine PBMCs. GH mRNA expression in nontreated lymphocytes was set as 100%. Values are expressed as mean (five animals) ⫾ SEM; *, P ⱕ 0.05. Lower panel, Representative results of competitive PCR with GHR cDNA (322 bp) and GHR-MIMIC (431 bp). For each sample, the same amount of cDNA (3 ␮l) was coamplified with 3 ␮l of recommendable 1:2 serial dilutions of the GHR-MIMIC (10 ⫻ 10⫺5 amol/␮l ⫺ 2.5 ⫻ 10⫺5 amol/␮l). M, Molecular weight marker; neg. CON, negative RT-PCR control.

only stimulated GH secretion but also up-regulated GHR gene expression in PBMCs in our studies. This up-regulation of both GH and GHR expression by PBMCs suggests that leptin may potentiate the biological effects of GH on immune cells via the induction of autocrine or paracrine regulatory loops. The finding that GH is produced locally by lymphocytes and that these cells express GHR strongly supports the functional role of this hormone within normal immune microenvironment. GH-mediated expansion of lymphocytes appears to be a specific receptor-mediated event because human T cell lines not bearing detectable GHR fail to respond to GH (21). Moreover, phorbol diester-induced down-regulation of GHR also results in a failure of immune cells to respond to GH (21). Furthermore, an antisense oligodeoxynucleotide to GH mRNA results in inhibition of lymphocyte proliferation (22). GHR has been categorized as a class 1 cytokine receptor or helix bundle peptide cytokine receptor (45), which includes leptin. In earlier studies, the presence of GH and GHR presence has been found predominantly in B lymphocytes and B

cell lines (46). Interestingly, it is reported that the predominant action of leptin on production of proinflammatory cytokines by resting PBMCs is primarily through the activation of monocytes, which have the maximum leptin receptor occupancy among leukocytes (47). Similarly, we have observed that leptin stimulates GH production by resting PBMC; however, the role of monocytes in providing costimulatory signals to lymphocytes leading to GH production requires further study. Because leptin is reported to stimulate the growth and proliferation of immune cells (9) and lymphocytederived GH is believed to be critical for cellular proliferation (22), our data suggest that leptin might regulate immune function by inducing the local GH production in lymphocytes. In addition, the signaling pathways involved in leptininduced GH secretion appear to be very specific and distinct, because mitogen-induced lymphocyte activation was associated with increased GH secretion but exhibited little to no effect on GHR expression. This seems to be a physiologically relevant event where leptin tends to potentiate actions of pituitary- and immune-derived GH. The biological signifi-



FIG. 4. Leptin stimulates NO production (measured as nitrite) from PBMCs. Nitrite levels in PBMC culture supernatants were analyzed by Griess reaction after leptin treatment. Leptininduced NO production was significantly inhibited in presence of the NOS inhibitor, L-NAME, and the PKC blocker, calphostin C. SNP was used as a positive control to asses NO production in our culture system. Values are expressed as mean ⫾ SEM. *, P ⱕ 0.05; **, P ⱕ 0.01; ***, P ⱕ 0.001 vs. control; one-way ANOVA followed by Tukey’s test.

FIG. 5. NO regulates GH secretion from PBMCs. NO donor, SNP, induces GH release from lymphocytes. There is no additive effects of leptin and SNP. Leptin-induced GH secretion is significantly attenuated by the NOS inhibitor, L-NAME, and the PKC blocker, calphostin C. **, P ⱕ 0.01 vs. control; one-way ANOVA followed by Tukey’s test.

cance might be the maintenance of immune cell homeostasis because it is quite likely that leptin and GH concentrations within the local immune microenvironment reach physiologically significant levels without undergoing the dilution typically seen upon their release into the peripheral circulation. In addition, a recent report showing leptin secretion from mouse T cells (12) suggests that regulation of GH secretion in immune cells is under complex regulatory control, possibly from peripheral as well as local sources of leptin. To further elucidate the mechanisms involved in leptininduced GH secretion by PBMCs, we studied the involvement of NO. Previous studies have revealed that NO might

be a critical mediator of the endocrine effects of leptin (31). Here, we observed a dose-dependent stimulation of NO production in response to leptin, which could be blocked by the addition of NOS inhibitor. This is the first report demonstrating a direct stimulatory effect of leptin on NO production from mononuclear cells. NO is believed to play a critical role in regulating inflammation (28), and it has recently has been shown to be up-regulated in response to proinflammatory challenge and is inhibited after stimulation of antiinflammatory pathway downstream of liver X receptors (48). Leptin also exerts proinflammatory effects and up-regulates cytokines involved in inflammation (49). Our data suggest

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that stimulation of NO production from PBMCs could be potentially involved in the proinflammatory effects of leptin. Furthermore, recent data from murine macrophage cell line J774A.1 (50) and endothelial cells (51) support our observations that leptin up-regulates NO release. Moreover, leptin has also been shown to have a dose-dependent stimulatory effect on serum NO production (52). PKC plays a crucial role in signal transduction in lymphocytes and is reported to induce long-term proliferation of B and T cells (53). Our observations indicate that PKC is also vital for leptin-induced GH production from lymphocytes. The interaction of PKC and NO pathways seems to be present in different tissues. Recently, it has been shown that, in endothelium-denuded mesenteric arteries, NO synthesis is positively stimulated by PKC (54). In addition, PKC controls induction of NO production after Staphylococcus aureus challenge from splenocytes (55). Our studies also demonstrate that PKC is a critical mediator in leptin-induced NO and GH production. Because lymphocytes are exquisitely sensitive to depletion of the cellular energy supply (12), their effector functions are particularly influenced by endocrine signals that control cellular metabolism, resulting in cell activation and proliferation. Recently, we reported that stimulation of ghrelin receptor, GH secretagogue receptor, by a synthetic analog, hexarelin, induces a substantial increase in GH production from porcine lymphocytes (18). Despite the fact that leptin and ghrelin exert opposite effects on regulation of food intake, their shared stimulatory effects on GH expression from the pituitary, as well as lymphocytes, might be mediated via a yet unknown common signaling pathway. The expression of functional leptin receptors by immune cells and the effects of leptin on production of T helper-1 cytokines and T cell activation has been well documented (9, 11, 12, 49). Additionally, our data indicate that lymphocytic GH, as a cytokine, may mediate the immune-enhancing effects of leptin. Our results impart additional support to the emerging view that leptin may be one of the peptides playing a key role in coupling the immunoendocrine system to energy balance. Acknowledgments We thank R. Wittig, M. Stu¨ nkel, and U. Beerman for technical support. We are grateful to Gary C. Collins (Laboratory of Immunology, National Institute on Aging, National Institutes of Health) for his helpful discussions. We also thank M. Pfaffl (Institute of Physiology, FML Weihenstephan, Technical University of Munich) for his help in developing the GHR primers. Received May 15, 2003. Accepted September 4, 2003. Address all correspondence and requests for reprints to: Prof. Dr. N. Parvizi, Institute of Animal Science Mariensee, (FAL), Hoelty Strasse 10, 31535, Neustadt, Germany. E-mail: [email protected]. V.D.D. carried out the work under a German Academic Exchange Service (DAAD) fellowship.

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Jensen Symposium on Nuclear Receptors and Endocrine Disorders December 5–7, 2003, Cincinnati, OH, USA In honor of Dr. Elwood Jensen, a symposium entitled “NUCLEAR RECEPTOR AND ENDOCRINE DISORDERS” will be held in Cincinnati, OH on December 5–7, 2003. The symposium will include invited talks by 30 leading scientists in the field, contributed poster presentations and short talks. For information please visit: http://cna.uc.edu/jensensymposium OR contact Ms. Katie Gouge, Department of Cell Biology ’UC College of Medicine’ Vontz Center for Molecular Studies, Cincinnati, OH 45267. E-mail: [email protected].