LEPTIN HAS BEEN reported to regulate the levels of

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1 /01/$03.00/0 Endocrinology 142(12): Printed in U.S.A. Copyright 2001 by The Endocrine Society Direct Modification of Somatotrope Function by Long-Term Leptin Treatment of Primary Cultured Ovine Pituitary Cells SANG-GUN ROH, GUI-YING NIE, KYLIE LONERAGAN, ARIEH GERTLER, AND CHEN CHEN Prince Henry s Institute of Medical Research (S.-G.R., G.-Y.N., K.L., C.C.), Clayton, Victoria 3168, Australia; Laboratory of Animal Molecular Physiology, Faculty of Agriculture, Shinshu University (S.-G.R.), Nagano , Japan; and Institute of Biochemistry, Hebrew University of Jerusalem (A.R.), Jerusalem 76100, Israel Leptin is produced primarily in adipocytes and regulates body energy balance. A close link between leptin and pituitary hormones, including GH, has been reported. The mechanisms employed by leptin to influence somatotropes are not clear, however. Here we report a direct action of recombinant ovine leptin on primary cultured ovine somatotropes by analyzing the levels of mrna encoding for GH or the receptors for GHRH (GHRH-R) and GH-releasing peptides (GHRP). Treatment of ovine somatotropes with leptin ( M) for 1 3 d reduced the mrna levels encoding GH and GHRH-R, but increased GHRP receptor mrna levels in a time- and dose-dependent LEPTIN HAS BEEN reported to regulate the levels of several pituitary hormones in rats, humans, and sheep (1 3) in addition to its role in the regulation of food intake and energy expenditure (4). In the pituitary gland, leptin is particularly linked to GH, an anabolic hormone (3). Expression of leptin receptor mrna has been demonstrated in the anterior pituitary gland and hypothalamus by RT-PCR (5, 6). Recently, leptin receptor-like immunoreactivity has also been detected in about 70% of ovine somatotropes, but has only been observed in about 20% of gonadotropes or corticotropes in ovine pituitary gland (7). Furthermore, it has been reported that leptin receptor gene expression was increased by GH and GHRH in GHRH transgenic mice (8, 9). In individuals who are obese due to a truncated mutation of the leptin receptor, a high level of plasma leptin was proposed to cause a pituitary dysfunction (10). This observation emphasizes the important role of leptin in regulating pituitary function. Both long- and short-form leptin receptor isoforms have been identified by RT-PCR and in situ hybridization in human pituitaries, which also provides supporting evidence for a functional role of leptin in the human pituitary (11, 12). GH is released from the pituitary gland under the dual control of hypothalamic somatostatin and GHRH. The cloning of the receptor for synthetic GH-releasing peptides (GHRP) and recent identification of the endogenous GHRP, ghrelin, suggest an additional endogenous GH secretagogue system other than GHRH for the control of GH release (13, Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GHRH-R, GHRH receptor; GHRP, GH-releasing peptide; GHRP-R, GH-releasing peptide receptor; icv, intracerebroventricular; ro-leptin, recombinant ovine leptin. manner. Three-day treatment of cells with leptin decreased the GH response to GHRH stimulation, but the GH response to GHRP-2 stimulation was increased. The combined effect of GHRH and GHRP-2 on GH secretion was not altered by treatment of the cells with leptin. These results demonstrated a direct action of leptin on ovine pituitary cells, leading to a reduced sensitivity of somatotropes to GHRH. It is also suggested that GHRP may be useful to correct the decrease in GHRH-induced GH secretion by leptin. (Endocrinology 142: , 2001) 14). Clinical observation indicates that compared with normal weight men, obese patients have defects in pulsatile GH secretion resulting in hyposomatotropism (15, 16). In addition, the GH response to GHRH stimulation is decreased in obese men and women, whereas fasting or weight loss tends to restore this response (17, 18). We postulate that GH secretion can be regulated by altering the sensitivity of somatotropes to GHRH and/or GHRP. We therefore investigated the direct effect of leptin on pituitary somatotropes with a focus on the regulatory mechanism employed by GHRH and GHRP. Materials and Methods Ovine pituitary cells Ovine pituitary cells (dispersed by collagenase treatment) were subjected to Percoll gradient centrifugation to enrich the somatotrope population to 60 80% of the total cells (19). The somatotrope-enriched cells were then cultured in DMEM containing 10% sheep serum and 2% FCS in 48-well culture dishes ( cells/well) for incubation experiments testing GH secretion. Extraction of total RNA was performed on 6-well (35-mm diameter well) culture dishes ( cells/well) for RT-PCR reactions. Cell culture medium was replenished every 2 d during a culture period of less than 10 d. Leptin treatment Treatment of cells with the recombinant ovine leptin (ro-leptin) was performed on the third day of culture after replenishing the culture medium. Final concentrations of ro-leptin in culture medium were 10 9, 10 8,or10 7 m, and the cells were treated for 24, 48, or 72 h, with fresh replenishment of culture medium and ro-leptin once every 24 h. The control group cells received vehicle alone. Ovine GH secretion and RIA On the day of the experiment the culture medium in 48-well culture dishes was changed to the incubation medium (without serum; the ph 5167

2 5168 Endocrinology, December 2001, 142(12): Roh et al. Effect of Leptin on Ovine Somatotropes value of medium buffered by 15 mm HEPES) for the GH secretion assay. Incubation medium for the first 1.5 h at 37 C was discarded (equilibration time), and then GHRH, GHRP-2, or both were added to replenished incubation medium for 60-min incubation at 37 C. The conditioned medium was then collected for ovine GH RIA using materials supplied by the National Hormone and Pituitary Program, NIDDK, NIH [ovine GH (RIA) and ovine GH antisera]. The sensitivity of the assay was 0.25 ng GH/ml, and 50% displacement on the standard curve was observed at 9.6 ng GH/ml. The intraassay coefficients of variation for pools containing 9.2 and 3.4 ng GH/ml were 5.3% and 9.4%, respectively (n 6). The interassay coefficient of variation was 14.2% (n 6). All samples from one incubation experiment were measured in the same assay. GH values were expressed as nanogram equivalents of the ovine GH standard. Semiquantitative RT-PCR The total RNA from cultured cells in 6-well culture dishes with or without leptin treatment was extracted for GH, GHRH receptor (GHRH- R), and GHRP receptor (GHRP-R) mrna assays. RT followed by PCR amplification was employed to measure levels of ovine GH, GHRH-R, and GHRP-R mrna. One microgram of total RNA extracted from each cell culture well was treated with deoxyribonuclease I to eliminate possible contamination of genomic DNA. The RNA was then reverse transcribed to cdna in a 20- l RT reaction system containing random primers and avian myeloblastosis virus reverse transcriptase. The RT reaction was carried out at 46 C. Two microliters of the RT products were used for subsequent PCR amplification for cycles, which was in the linear increasing phase of the PCR products. Primers specific for ovine GH, GHRH-R, and GHRP-R are shown in Table 1. RT-PCR was performed as previously described (20). Twenty-eight cycles of PCR amplification at a 66 C annealing temperature were used for GHRH-R, 32 cycles at a 62 C annealing temperature for GHRP-R, and 30 cycles with a 60 C annealing temperature for GH. All PCR for GH, GHRH-R, and GHRP-R were coamplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (product size, 562 bp), which is the housekeeping gene used as an internal control. Twenty microliters of PCR products were resolved in a 2% agarose gel, and the DNA was visualized by ethidium bromide staining and analyzed using NIH image software, where band intensity is expressed in pixels. Nonreverse transcribed RNA was included with the primer sets as a negative control. The relative levels of ovine GH, GHRH-R, GHRP-R mrna, and GAPDH mrna were calculated. Statistical analysis Data are presented as the mean ( sem) of three to five separate experiments with the same treatment protocol. Comparisons were made between different treatment groups using ANOVA followed by the Dunnett post-hoc test for differences from control. Data for percent changes were analyzed using the Kruskal-Wallis H test. All experiments conformed to the National Health and Medical Research Council (Australia) ethics code of practice. Results Effect of leptin treatment on GH synthesis Compared with vehicle treatment, 24-h treatment of somatotropes with ro-leptin (10 7 m) significantly (P 0.01) decreased the levels of GH mrna, although lower doses of ro-leptin did not reduce the level of GH mrna (Fig. 1). Three-day treatment of somatotropes with ro-leptin, however, significantly (P 0.01 or P 0.05) reduced the level of GH mrna at all three doses of ro-leptin (10 7,10 8, and 10 9 m) in a dose-dependent manner (Fig. 1). Two-day treatment with the ro-leptin significantly reduced the levels of GH mrna at two doses (10 7 and 10 8 m; Fig. 1). Effect of leptin treatment on GHRH-R synthesis Similar to the change in GH mrna levels after leptin treatment, levels of GHRH-R mrna were also reduced by 1-d treatment of cells with 10 7 m, 2-d treatment of cells with 10 7 and 10 8 m, and 3-d treatment of cells with m ro-leptin (Fig. 2). The reduction in GHRH-R mrna levels after 3 d of treatment with ro-leptin was also dose dependent (Fig. 2). Effect of leptin treatment on GHRP-R synthesis The effect of ro-leptin on the levels of GHRP-R mrna was different from that on GH or GHRH-R mrna levels. Treatment of cultured cells with up to 10 7 m ro-leptin for up to 2 d did not significantly change the levels of GHRP-R mrna (Fig. 3). Three-day treatment of the cells with ro-leptin, however, significantly (P 0.01 or P 0.05) increased the levels of GHRP-R mrna in a dose-dependent manner (Fig. 3). Effect of leptin treatment on basal and stimulated GH secretion As the level of GH in somatotropes and the density of GHRP-R and GHRH-R on somatotrope membranes determines GHRH- and GHRP-stimulated GH release, we tested the effect of GHRP and GHRH on GH secretion. After treatment of primary cultured cells with ro-leptin (10 8 m)for3d, basal GH secretion was not changed (control cells, Fig. 4), although the levels of GH mrna were decreased by this treatment (Fig. 1). GHRH (10 8 m)-induced GH secretion was significantly decreased by 3-d treatment of cells with roleptin (Fig. 4). This treatment also significantly enhanced GHRP-2 (10 8 m)-stimulated GH secretion (Fig. 4). When both GHRH and GHRP-2 were added to the incubation medium, the stimulated GH secretion was not changed by roleptin treatment (Fig. 4). Discussion The present studies demonstrated a direct effect of leptin on pituitary somatotropes. This reinforces and extends the TABLE 1. Oligonucleotide primer sequences for PCR amplification of ovine GH, GHRH-R, and GHRP-R cdnas Name (accession no.) Sequence Position Product length (bp) GH (X15976) Forward 5 -CCTGCTCCTGGCTTTCACCCT Oligo a Reverse 5 -ATCTTCCAGCTCCCGCATCAG GHRH-R (Prof. Thorner) Forward 5 -GCCCGCCTTTCTTCTCTCAC Oligo Reverse 5 -CTGGGCAATGTGGAGGCTAAG GHRP-R (AF118636) b Forward 5 -ACCTCCTCTGCAAACTCTTCC Oligo Reverse 5 -CACCCGGTACTTCTTGGACAT a Primer sequence was designed using the Oligo program (version #6). b We submitted gene sequence information of ovine GHRP-R to GeneBank with accession no. AF Source

3 Roh et al. Effect of Leptin on Ovine Somatotropes Endocrinology, December 2001, 142(12): FIG. 1. The effect of leptin treatment on the level of GH mrna. Cells were treated with ro-leptin (10 7,10 8, and 10 9 M) for 1, 2, or 3 d before total RNA was extracted. The top band is GAPDH (562 bp), and the lower band is GH (437 bp). The levels of GH mrna were corrected by GAPDH mrna. M, Molecular size ladders (650, 500, and 400 bp). The column represents the mean SEM of three separate experiments. *, P 0.05; **, P 0.01 (vs. control group). FIG. 2. The effect of leptin treatment on the level of GHRH-R mrna. Cells were treated with ro-leptin (10 7,10 8, and 10 9 M) for 1, 2, or 3 d before total RNA was extracted. The top band is GAPDH (562 bp), and the lower band is GHRH-R (467 bp). The levels of GHRH-R mrna were corrected by GAPDH mrna. The column represents the mean SEM of three separate experiments. *, P 0.05; **, P 0.01 (vs. control group). concept that leptin regulates pituitary endocrine function. The receptors for leptin have been shown in the anterior pituitary glands of different species (6). In sheep, more than 60% of somatotropes exhibit positive staining using antibodies against the leptin receptor, whereas only 20% of gonadotropes or corticotropes had positive staining for the leptin receptor (7). The level of leptin receptor in the pituitary gland is markedly up-regulated in GHRH transgenic mice (8), which suggests a link between GHRH/GH levels and the sensitivity of pituitary cells to leptin. The effect of leptin on GH levels, however, is controversial depending on the experimental animal species, nutritional conditions, and delivery method of leptin. It has been reported that 7-d intracerebroventricular (icv) infusion of leptin increased both the nadir level and pulse amplitude of GH in male rats (21). However, a different laboratory reported that the icv injection of leptin for 3 d did not change GH levels in normally fed rats (22). Intracerebroventricular injection of antibodies against rat leptin reduced GH secretion (23), whereas icv administration of leptin protected against the inhibitory effect of restricted nutrition on GH secretion in the rat (22, 23). It is worthwhile to emphasize that restricted nutrition in sheep or humans has a stimulatory effect on circulating GH levels (24, 25). Thus, one might expect that leptin would act on the pituitary somatotropes to reduce GH secretion in sheep and humans. This appears to be true for GHRHstimulated GH secretion from primary cultured ovine somatotropes (3, 26) and may be synonymous with the reduced level of GH seen in obese individuals (25). The experiments reported in this manuscript have further clarified the mechanism for the reduction in GHRH-stimulated GH secretion by leptin. Levels of both GH and GHRH-R mrna were significantly reduced by ro-leptin, and such a decline in mrna levels was leptin dose and time dependent. After 3 d of treatment of cells with ro-leptin, all three doses of leptin employed in the experiments (1, 10, and 100 nm) significantly reduced GH and GHRH-R mrna levels in cultured somatotropes. These changes in the levels of GH and GHRH-R mrna suggest a decrease in the synthesis of GH and GHRH-R in ro-leptin-treated somatotropes. Such a decrease in the synthesis of GH and GHRH-R may lead to a decrease in GH accumulated in somatotropes and a decrease in GHRH-R density on the surface of somatotropes. These changes may therefore evoke a decrease in GHRH-stimulated GH secretion. As this has been achieved in primary cultured somatotrope-enriched (60 80% of total cells are somatotropes) cells, the action of leptin reported here is most likely a direct action on pituitary somatotropes without involvement of any hypothalamic, peripheral, or paracrine factor. Basal secretion of GH was not affected by the treatment of cells with ro-leptin, although the GH mrna level

4 5170 Endocrinology, December 2001, 142(12): Roh et al. Effect of Leptin on Ovine Somatotropes FIG. 3. The effect of leptin treatment on the levels of GHRP-R mrna. Cells were treated with ro-leptin (10 7,10 8, and 10 9 M) for 1, 2, or 3 d before total RNA was extracted. The top band is GHRP-R (659 bp), and the lower band is GAPDH (562 bp). The levels of GHRP-R mrna were corrected by GAPDH mrna. The column represents the mean SEM of three separate experiments. *, P 0.05; **, P 0.01 (vs. control group). FIG. 4. GH secretion by cultured ovine somatotropes ( cells) in response to 60-min GHRH, GHRP-2, or combined GHRH and GHRP-2 stimulation. *, P 0.05 vs. control; **, P 0.01 vs. control;, P 0.05 vs. nonleptin-treated with same stimulation of secretagogues. was decreased. This may indicate that basal GH secretion is maintained even after ro-leptin treatment. In contrast to the decline in the levels of GH and GHRH-R mrna, the level of GHRP-R mrna is increased by 3-d treatment of ovine somatotropes with ro-leptin. This indicates an increase in the synthesis of GHRP-R. In this experiment GHRP-2-induced GH secretion was increased after 3-d treatment of cells with ro-leptin. This result suggests that the increase in the synthesis of GHRP-R by ro-leptin enhances the GH response to GHRP-2, possibly through an increase in receptor density on the membrane of somatotropes. The combined effect of GHRH and GHRP-2 on GH secretion was not changed by 3-d leptin treatment of somatotropes. This indicates that the action of GHRP-2 can completely compensate for the decrease in GH response to GHRH after treatment of cells with leptin for 3 d. One recent report indicates that the level of endogenous GHRP (or ghrelin) is significantly lower in obese humans compared to normal weight counterparts (27), which suggests GHRP deficiency in overweight conditions. This report also supports our view that GHRP may be a useful therapeutic drug to treat GH deficiency in obesity patients. The level of leptin in human plasma is 5 20 ng/ml in normal weight population and increases to 600 ng/ml in obese individuals (28, 29). The dose range of leptin used in this experiment (1 100 nm or 16 ng/ml to 1.6 g/ml) is in the range of plasma leptin concentrations in obesity patients. Leptin is secreted in pulses and in a nyctohemeral rhythm (30), and the peak value of leptin may be even higher than 600 ng/ml in obese individuals. It is also possible that the local concentrations of leptin in hypothalamic-pituitary portal blood or in anterior pituitary glands may be higher that the plasma levels. Previously, the site of leptin action was not clear; in particular, whether it acted on pituitary cells has remained obscure. Here we demonstrated a direct action of leptin on pituitary somatotropes influencing the sensitivity of somatotropes to GHRH and GHRP stimulation. The cellular mechanism of leptin action on endocrine cells is another focus of this study. Previous research has mostly examined effects on hormone levels in response to leptin treatment in whole animal experiments, with little attention to changes in endocrine cell function. Here we demonstrated that leptin actually modified the cellular function of pituitary somatotropes by changing the synthesis of GH, GHRH-R, and GHRP-R. In summary, the current experiments provide evidence for the direct action of leptin on pituitary somatotropes to modify the cell function and sensitivity to GHRH and GHRP by altering the synthesis of GH and receptors for GHRH and GHRP. The effect of combined GHRH and GHRP on GH secretion is not influenced by leptin treatment, suggesting a possible use of GHRP in the treatment of GH deficiency in obese patients. Acknowledgments We thank Ms. S. Panckridge for assistance with the preparation of graphics for this manuscript. We also thank Drs. I. J. Clarke and J. W. Goding for scientific discussion, and Drs. B. D. Gaylinn and M. O. Thorner for providing the ovine GHRH-R sequence. We express special thanks to Dr. P. Stanton for his careful reading and revision of the manuscript. Received May 21, Accepted September 5, 2001.

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