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*Gene*UniGene
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*(D)-PENICILLAMINE
*NITRIC OXIDE
Endocrinology Vol. 143, No. 2 551-557
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

Leptin Regulates GH Gene Expression and Secretion and Nitric Oxide Production in Pig Pituitary Cells

M. Baratta, R. Saleri, G. L. Mainardi, D. Valle, A. Giustina and C. Tamanini

Department Animal Production, Veterinary Biotechnology and Food Safety, University of Parma (M.B., R.S., G.L.M., C.T.), 43100 Parma, Italy; Eli Lilly Italia (D.V.), 50019 Florence, Italy; and Internal Medicine, University of Brescia (A.G.), 25100 Brescia, Italy

Address all correspondence and requests for reprints to: Prof. Mario Baratta, Department of Animal Production, Veterinary Biotechnology and Food Safety, Via del Taglio 8, University of Parma, 43100 Parma, Italy. E-mail: mario.baratta{at}unipr.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of this study was to investigate the direct effect of leptin on GH gene expression and secretion and the role of nitric oxide as a possible mediator in pig anterior pituitary cells. Pituitary cells from adult sows were treated for 4 or 24 h with rhleptin (from 0.1 nM to 1 µM) alone or in association with GHRH (10 nM) or hexarelin (10 nM). At the end of incubation, medium was collected for GH and nitric oxide determination by ELISA and Griess test, respectively. Total RNA was collected from cells, and GH gene expression was measured by RT-PCR. Leptin significantly (P < 0.001) stimulated GH secretion in both incubation periods. The maximum response was induced by 10 nM leptin; furthermore, a significant interaction (P < 0.002) between leptin and GHRH (P < 0.03) and between leptin and hexarelin was observed when the molecules were used in association. GH gene expression was significantly increased (at least P < 0.05) by hexarelin, GHRH, and leptin (1000 and 100 nM) after 24 h of treatment. Leptin (10 nM and 1 µM) significantly (P < 0.05) increased nitric oxide production, whereas S-nitroso-N-acetyl-penicillamine (from 0.01–1000 nM) significantly (P < 0.05) stimulated GH secretion. These data demonstrate that leptin directly influences GH regulation at the pituitary level, and nitric oxide may be involved in this function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS SECRETED from adipose tissue and causes weight loss in rodents by reducing food intake and increasing energy expenditure (1). Many studies in recent years have been focused on the role of leptin in body weight regulation, aiming to clarify the pathophysiology of obesity. However, evidence suggests that leptin not only is important in the regulation of food intake and energy balance, but also functions as a metabolic and neuroendocrine hormone (2).

Leptin receptors have been found in the choroid plexus and hypothalamus (3, 4) as well as in the pituitary of different species, such as rat and mouse (5), pig ( 6), sheep (7), and human ( 8). The direct effect of leptin at the pituitary level has been reported to be carried out by either leptin secreted by adipose tissue or produced locally (5, 8).

GH has been widely investigated as an important factor of lipolysis, and the neuroregulation of GH secretion is closely related to the state of adipose tissue reserves (9). Low body weight enhances GH secretion, whereas obesity is associated with low GH levels (10, 11). GH promotes lipolysis via inhibition of lipoprotein lipase, which hydrolyzes blood triglycerides to make them available for accumulation in adipose tissue (12).

Recent studies suggest that leptin regulates GH levels (13). In fact, central administration of a leptin-neutralizing antiserum has been shown to decrease spontaneous GH secretion (14) in rats, whereas leptin administration to cultured fetal neurons from rats leads to a time-dependent decrease in basal somatostatin secretion and somatostatin mRNA levels (15, 16). These data indicate that leptin influences GH secretion by regulating hypothalamic somatostatin gene expression. As leptin receptors have been observed in pituitary cells, a relationship between leptin and GH at the pituitary level has been suggested. Some reports indicate a stimulatory effect of leptin on GH secretion from cultured pig (17) and rat ( 18) anterior pituitary cells as well as a direct effect during fetal life or after the development of pituitary tumors (19).

Nitric oxide (NO) has emerged as an important intra- and intercellular transmitter involved in the control of the hypothalamic-pituitary axis, and the inducible NO synthase (iNOS) has been identified in the pituitary cells (20). Furthermore, NO has been reported to be involved in GH secretion through a calcium- and cGMP-independent mechanism (21); in addition, leptin has been shown to enhance NO production (22) in some systems, such as rat endothelial cells.

The aims of this study were to assess the direct effect of leptin on GH gene expression and secretion [basal and induced by GHRH or hexarelin (Hex), an analog of GH- releasing peptide-6 (GHRP-6)] and to determine the possible role of NO in the modulation of leptin activity in pig pituitary cells cultured in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
DMEM/Ham’s F-12 (with L-glutamine and sodium bicarbonate) was obtained from Life Technologies, Inc. (Milan, Italy); penicillin G sodium, streptomycin, FCS, collagenase type II, hyaluronidase type V, deoxyribonuclease type I, NG-methyl-L-arginine (NMMA), and S-nitroso-N-acetyl-penicillamine (SNAP) were purchased from Sigma (St. Louis, MO); collagen S was purchased from Roche (Mannheim, Germany); recombinant human leptin (rhLep) was furnished by Eli Lilly \|[amp ]\| Co. (Indianapolis, IN); GHRH (Geref) was purchased from Serono International S.A. (Geneva, Switzerland); Hex (an analog of GHRP-6 with a 2-methyl substitution of D-tryptophan; His-D-Trp(2Me)Ala-Trp-D-Phe-Lys-NH2) was purchased from Mediolanum Farmaceutici Spa (Milan, Italy); and porcine GH (pGH; AFP10864B) and anti-pGH antiserum (AFP5672099) were provided by Dr. A. F. Parlow (National Hormone and Pituitary Program, Harbor-University of California-Los Angeles Medical Center, La Jolla, CA). All molecular biology reagents were purchased from Ambion, Inc. (Austin, TX) unless otherwise specified.

Cell cultures
Pituitaries from 7- to 8-month-old cycling sows were collected at the slaughterhouse and transported to the laboratory within 30 min in a dissociation medium (0.15 M PBS, 0.6% HEPES, 0.2% D-glucose, 0.1 mg/ml streptomycin sulfate, and 0.02 mg/ml gentamicin sulfate). Anterior pituitaries were separated from neurohypophysis and sliced (0.5 mm) with a Thomas-Stadie-Riggs (Thomas Scientific, Swedesboro, NJ) tissue slicer. Cells were dissociated as previously described (23). Briefly, the pituitaries were washed four times in the dissociation medium and dispersed with collagenase II (1.0 mg/ml), hyaluronidase V (1.0 mg/ml), and deoxyribonuclease I (0.01%) at 37 C. Cells were suspended in DMEM/Ham’s F-12 with porcine serum (30%), FCS (5%), streptomycin sulfate (0.1 mg/ml), and penicillin (1.000 IU/ml). The cell yield per pituitary was approximately 10 million, and viability was determined by trypan blue exclusion at the beginning of the culture (95%) and at the end of the experiments (>95%).

Treatments
For secretion studies, cells were plated in 24-well tissue culture plates (pretreated with 5 µg/cm2 collagen S for 1 h to improve cell adhesion) at a density of about 2 x 105 cells/well, and incubated at 37 C for 48 h. The cells were then treated for 4 or 24 h with rhLep (0.1–1000 nM) alone or in association with GHRH (10 nM) or Hex (10 nM).

NO assay
NO production was assessed by measuring the amount of nitrite (NO2-), a stable metabolic product of NO, in culture medium from treated cells (24). Briefly, 100 µl medium samples collected from cells treated for 24 h with rhLep were mixed with 100 µl Griess reagent (25), which was prepared by mixing equal volumes of stock A (1% sulfanilamide and 5% phosphoric acid) and stock B (0.1% N-[naphthyl]ethylenediamine dihydrochloride). After a 10-min incubation at room temperature the absorbance was measured at 540 nm in a Spectra Shell Microplate Reader (SLT, Tecan, Milan, Italy); when medium was processed, the absorbance at 620 nm was subtracted to eliminate the yellow interference. The standard curve was performed in culture medium by using serial dilutions of sodium nitrite (50–0.39 µM; linear regression: y = 0.0223x + 0.102; r = 0.99). The interassay variability was less than 5%.

SNAP effect on GH release
Pituitary cells were treated for 24 h with SNAP, a NO donor, at concentrations of 0.001, 0.01, 0.1, and 1 mM, then media were collected for GH determination.

NMMA effect on GH release
Pituitary cells were treated with rhLep (10–1000 nM) alone or in association with NMMA (300 µM), a NOS inhibitor; after 24 h, media were collected for GH assay.

GH assay
Culture media were assayed for GH by an ELISA previously described (26) and modified for pGH. Briefly, 40 µl unknown samples or standard (ranging from 39–5,000 pg pGH) were added to the wells and incubated with 100 µl anti-pGH antiserum (1:300,000). After incubation for 48 h at 4 C, 10 ng/well biotinyl-pGH were added and incubated for 2 h at 4 C. Then, extravidin conjugated with peroxidase (1:2,000) was added in 100 µl assay buffer and incubated for 1 h; after that, 100 µl ABTS (2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid)/H2O2 were added, and the reaction was stopped after 25 min. The absorbance was measured at 405 nm in a Spectra Shell Microplate Reader; ED90, ED50, and ED10 were 0.039, 0.166, and 5 ng/well, respectively. The intra- and interassay coefficients of variation were 6% and 8.5%, respectively. Cross-reactivities with other pituitary hormones, calculated on the basis of ED50, were: pFSH, 0.018%; pLH, 0.014%; pPRL and pTSH, less than 0.01%.

RNA isolation
For GH gene expression studies, which were performed three times with three replicates for each experiment, cells (5 x 106) were plated in petri plates and pretreated with 50 µg poly-D-lysine for 1 h. After 48 h medium was replaced, and the treatments were performed for 24 h with rhLep (0.1 nM to 1 µM) or Hex (10 nM). At the end of the incubation, medium was removed, and total RNA was extracted by RNAwiz. Briefly, 1 ml RNAwiz was added to the plate after discarding the medium, and RNA was extracted according to the manufacturer’s instructions (no. 9736, Ambion, Inc., Austin, TX).

RT-PCR
RT was carried out using a Ready-To-Go kit (Amersham Pharmacia Biotech, Uppsala, Sweden) as described by the manufacturer. One microgram of total RNA was used in the RT. Aliquots (2 µl) from the generated cDNA were used for subsequent PCR amplification in the reaction buffer containing 1.5 µl MgCl2 (50 mM), 1 µl dNTPs (12.5 mM), and 1 µl Taq DNA polymerase (1 µg/µl), to a final volume of 50 µl. Amplification was carried out for 23 cycles, when the reaction was in the middle of the linear range (before reaching the amplification plateau). Each cycle consisted of denaturation at 94 C for 1 min, annealing at 50 C for 1 min and 10 sec, and extension at 72 C for 1 min and 30 sec. The following primer sets were used for pGH (2.5 µM): forward, 5'-CTTGTCCAGCCTATTTGCCAAT-3'; and reverse, 3'-GACGAGAGGACGAAGTTCTTCC-5' (designed from cDNA encoding porcine GH, GenBank, locus E00229). The amplified product with these primers has 485 bp; QuantumRNA 18S was used (no. 1717, Ambion, Inc.) as an internal positive control for a relative quantitative RT-PCR. To amplify the 18S fragment (323 bp) without reaching the plateau phase, 18S PCR alternate primer pairs (5 µM) were coincubated with 18S PCR competimers (5 µM; ratio, 2:8) according to the instructions of the manufacturers. PCR products were visualized with ethidium bromide after electrophoresis on 2% agarose gel.

Southern blot hybridization
The PCR products were capillary transferred onto a nylon filter and cross-linked to the membrane using UV light. The blot was hybridized with psoralen-biotin-labeled oligonucleotide probes (pGH and 18S, 10 ng/ml) at 65 C overnight. The chemiluminescent detection of biotinylated DNA probes was realized with the BrightStar BioDetect Kit. An autoradiograph was produced by exposure of the membrane to x-ray film for 5 min; thereafter, the blot was analyzed on a computerized densitometry program (ImageQuant, software version 3.3, Molecular Dynamics, Inc., Sunnyvale, CA); the values are presented as the ratio of the band intensities of the GH RT-PCR product over those of the corresponding ribosomal 18S RT-PCR product [the GH/18S ratio has been expressed in relative arbitrary units (RAU)].

Statistical analysis
Each experiment was repeated four times independently, and in each experiment each treatment was performed with six replicate culture wells. Experimental data are presented as the mean ± SEM. Statistical differences between treatments and interactions were calculated with multifactorial ANOVA using the Statgraphics package (STSC, Inc., Rockville, MD). When significant differences were found, means were compared by Scheffé’s F test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH release after 4 and 24 h of rhLep treatment
GH output by 2 x 105 cells after 4 h was 65.9 ± 2.3 ng/ml. GH release was significantly (P < 0.05) enhanced by leptin in a dose-dependent manner from 0.1 to 10 nM; thereafter the stimulatory effect did not increase further (Fig. 1Go, top). GH production by 2 x 105 cells after 24 h was 298.1 ± 6.8 ng/ml. GH release was significantly (P < 0.05) enhanced by rhLep; however, a dose-related response was observed only with 0.1 and 1 nM rhLep (Fig. 1Go, bottom).



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  		Figure 1. Effect of rhLep (0.1–1000 nM) on GH secretion by 2 x 105 pig pituitary cells after 4 h (top) or 24 h (bottom) of incubation. Each value represents the mean ± SEM of six wells replicated four times in independent experiments. Statistical differences between treatments were calculated with multifactorial ANOVA. Significant differences (at least P < 0.05) among the treatments are labeled with different letters.

 
GH release after 4 and 24 h of treatment with rhLep and GHRH or Hex
When cells were stimulated with leptin in association with GHRH, a significant interaction (P < 0.001) was observed between the two substances on GH secretion. A very similar correlation was observed between leptin and Hex. Figure 2Go shows the effects of GHRH (10 nM) and Hex (10 nM) alone or in association with rhLep (0.1–1000 nM) after 4 h of incubation. rhLep also exerted a positive effect on GHRH- and Hex-induced GH release after 24 h of incubation, even though the pattern of release was not correlated with rhLep concentrations.



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  		Figure 2. Effect of rhLep (0.1–1000 nM) in association with GHRH (10 nM; top) or Hex (10 nM; bottom) on GH secretion by 2 x 105 pig pituitary cells after 4 h of incubation. The first bar (C) represents GH secretion without any treatment. Each value represents the mean ± SEM of six wells replicated four times in independent experiments. Statistical differences between treatments were calculated with multifactorial ANOVA. Significant differences (at least P < 0.05) among the treatments are labeled with different letters.

 
GH mRNA expression
After 24 h of treatment, the GH mRNA level was significantly enhanced (P < 0.05) up to 32% and 37% (0.467 and 0.485 vs. 0.354 RAU GH/18S) by Hex and GHRH, respectively, and up to 78% (0.605 RAU GH/18S) and 136% (0.836 RAU GH/18S) by 1000 and 100 nM rhLep, respectively. No differences were induced by the other concentrations (Fig. 3Go). No effects on GH mRNA level were observed after 4 h of treatment.



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  		Figure 3. Effects of Hex (10 nM), GHRH (10 nM), and rhLep (0.1–1000 nM) on GH mRNA levels after 24 h of incubation. mRNA levels were measured by semiquantitative RT-PCR. Relative amounts of mRNA were compared through an internal standard 18S ribosomal mRNA (RAU = pGH/18S). Each value represents the mean ± SEM of three samples replicated three times in independent experiments. Statistical differences between treatments were calculated with multifactorial ANOVA. Significant differences (at least P < 0.05) among the treatments are labeled with different letters.

 
NO accumulation after 24 h of rhLep treatment
Figure 4Go shows the significant (P < 0.05) effect of rhLep (10–1000 nM) on NO accumulation; the highest response was observed with 1000 nM rhLep, when NO increased up to 3-fold.



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  		Figure 4. Effect of rhLep (0.1–1000 nM) on nitrite release after 24 h of incubation. Each value represents the mean ± SEM of six wells replicated four times in independent experiments. Statistical differences between treatments were calculated with multifactorial ANOVA. Significant differences (at least P < 0.05) among the treatments are labeled with different letters.

 
Effect of SNAP on GH release
SNAP (0.001–1 mM) significantly (P < 0.05) increased GH secretion after 24 h of incubation, even if a dose-response effect was not observed (Fig. 5Go).



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  		Figure 5. Top, Effect of SNAP (0.001–1 mM) on GH secretion after 24 h of incubation. Each value represents the mean ± SEM of six wells replicated four times in independent experiments. Different letters mean at least P < 0.05. Bottom, The final nitrate concentrations measured for each SNAP treatment.

 
Effect of NMMA on leptin-induced GH release
The association between NMMA (300 µM) and leptin (10, 100, and 1000 nM) significantly (P < 0.001) reduced leptin- induced GH secretion after 24 h of incubation. Figure 6Go shows the effect of NMMA associated with 10 nM rhLep.



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  		Figure 6. Effect of NMMA (300 µM) alone or in association with 10 nM rhLep on GH secretion after 24 h of incubation. Each value represents the mean ± SEM of six wells replicated twice in independent experiments. Statistical differences between treatments were calculated with multifactorial ANOVA. Significant differences (at least P < 0.05) among the treatments are labeled with different letters.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GH not only is involved in development and growth, but also plays a role in body composition and fat distribution through its influence on energy metabolism as well as its lipolytic and nitrogen-sparing effect. Furthermore, abnormalities in the GH-IGF-I axis have been shown to be associated with obesity. Serum levels of GH and IGF-binding proteins are often reduced in human obese subjects (27), resulting in high free IGF-I levels, whereas starvation induces an increase in GH secretion (28). Administration of GH stimulates energy expenditure (29) and decreases the amount of body fat.

The effect of leptin on GH regulation has not been extensively investigated. Leptin may be involved in GH secretion, because this protein is produced and secreted mainly by adipose tissue, and a specific relationship between leptin and GH in weight regulation and metabolic functions might exist. Many studies indicate that leptin activity could take place through specific receptors, which have been demonstrated in those hypothalamic nuclei known to be involved in GH regulation (30). In addition, leptin has been shown to be effective in inhibiting somatostatin gene expression and secretion (15), and its central infusion stimulates basal GH secretion in rats and pigs (16, 17, 31).

The effect of peripheral leptin on GH secretion has been indirectly evidenced in vivo. In fact, the administration of an antileptin antiserum has been demonstrated to reduce GH release in normal fed rats (14). High concentrations of porcine leptin (10-6 and 10-7 M), generally considered supraphysiological, have been reported to significantly stimulate GH secretion from porcine pituitary cells (17). Our study documents a direct effect of leptin on GH secretion even at lower doses and after both short and prolonged stimulations. The different responsiveness may be due to the different reproductive status of the animals or the different leptin preparations. We also report the effectiveness of leptin in stimulating GH mRNA levels, even if at high concentrations and after a prolonged exposure (24 h). The physiological relevance of this effect needs to be further investigated. The present study also shows that leptin potentiates GHRH- and Hex-induced GH release. The current literature reports contrasting data concerning the effect of leptin on GHRH activity at the pituitary level. Both positive and negative effects have been observed (17, 18). To our knowledge, there are no data available on the in vitro effects of the association between Hex and leptin on GH secretion. The positive effect of the association between leptin and GHRH or Hex in food-deprived rats has been recently reported (32), and the authors hypothesize that this effect may be present at the hypothalamic and pituitary levels. Interestingly, they conclude that the primary alteration for the blunted GH responses to GHRH induced by food deprivation could be mediated by decreased leptin activation of the hypothalamic-GH axis. We report a significant correlation between the effect of leptin and that of both GH secretagogues at the pituitary level. Both molecules are positively influenced by leptin in stimulating GH, even if there are differences in the dose-dependent manner. It should be taken into account that Hex, an analog of GHRP-6, exhibits a different intracellular pathway from that of GHRH to enhance GH output, and that the natural ligand for GHRP-6 receptor has been discovered only recently (33, 34, 35). From the present data we hypothesize that leptin plays an important role in GH secretion directly at the pituitary level, possibly extended to gene expression and independent from that of the GH secretagogue. It would be important to identify the physiological source of leptin that induces this effect; adipose tissue or the pituitary itself has been recently proposed in rodents and humans (5, 8). Furthermore, many human pituitary cell types have been demonstrated to be effective in producing leptin, including ACTH, GH, and FSH cells in both normal and tumoral tissues (8). We may therefore hypothesize an autocrine/paracrine modulation exerted at the pituitary level. However, adipose tissue is the main source of peripheral leptin, and pituitary cells may receive direct information on energy balance through the secreting activity of fat cells. The inhibitory effect of the GH-IGF-I axis on leptin secretion is not yet clear, and it is still uncertain whether this effect is direct or indirect through stimulation of lipolysis in adipocytes (36, 37). In fact, several in vitro studies have demonstrated opposite effects of GH in isolated adipocytes (38, 39).

NO appears to be involved in the control of pituitary function, and its role in LH basal secretion as well as in the modulation of LHRH-induced LH production has been demonstrated (40). The expression of brain constitutive NOS in the anterior pituitary has been reported in rats (41, 42) and humans ( 43), and more recently, iNOS has also been detected in rats (20). Brain constitutive NOS seems to be expressed in all secretory cell types, whereas iNOS seems to be confined to a subpopulation of folliculostellate cells as well as an unidentified population of nonhormone-secreting cells, proposed as progenitor cells (20). Our data indicate that SNAP, an NO donor, stimulates GH secretion. These results agree well with those of a previous study (21) indicating that another NO donor, sodium nitroprusside, positively influences GH secretion. The researchers suggested that this modulation occurs through a specific calcium-cGMP-independent mechanism. In our study nitrite accumulation in medium after SNAP treatments showed that NO is effective in stimulating GH secretion at low concentrations even if the physiological levels are difficult to determine; however, the concentrations used in our experiments agree well with those previously reported in different systems (21, 44). NO has been also shown to be effective in stimulating basal GH in acromegalic humans (44) and to potentiate the strong GH-releasing activity of Hex in both young and old beagle dogs. This effect seems to be exerted through PGE, which is hypothesized to be the downstream effector of the chain of events triggered by the activation of the NO-ergic system (45).

Leptin significantly stimulates NO release in pig pituitary cells. Leptin receptors have been identified in different types of pituitary cells, such as corticotropes, somatotropes, and gonadotropes (7), and leptin receptor mRNA has also been recently detected in rat folliculostellate cells (46). This last observation leads us to hypothesize that folliculostellate cells may exert a paracrine effect, through NO, on GH-producing pituitary cells even if we do not have direct evidence to support this model. However, it is interesting to point out that the association of NO with NMMA (47), a competitive inhibitor of all three NOS isoforms, impairs the positive effect of leptin on GH secretion.

In conclusion, our study confirms a positive direct effect of leptin on GH mRNA levels and secretion in primary pig pituitary cells. Furthermore, leptin potentiates Hex-induced GH release and is effective in stimulating NO production that, in turn, enhances GH secretion. These results, taken together, strongly suggest that leptin exerts direct control on GH gene expression and secretion and, therefore, on metabolic resources.


   Acknowledgments
 
We thank Dr. M. Heiman (Eli Lilly \|[amp ]\| Co., Indianapolis, IN) for having provided leptin.


   Footnotes
 
This work was supported in part by a MURST COFIN grant and an FIL grant.

Abbreviations: GHRP-6, GH-releasing peptide-6; Hex, hexarelin; iNOS, inducible nitric oxide synthase; NMMA, NG-methyl-L-arginine; NO, nitric oxide; pGH, porcine GH; RAU, relative arbitrary units; rhLep, recombinant human leptin; SNAP, S-nitroso-N-acetyl-penicillamine.

Received August 7, 2001.

Accepted for publication October 25, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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