THE GH neuroendocrine axis is exquisitely sensitive to

0013-7227/98/$03.00/0 Vol. 139, No. 9 Endocrinology Printed in U.S.A. Copyright 1998 by The Endocrine Society Leptin Is a Potent Stimulator of Spontaneous Pulsatile Growth Hormone (GH) Secretion and the GH Response to GH-Releasing Hormone* GLORIA SHAFFER TANNENBAUM, WENDY GURD, AND MARTINE LAPOINTE Departments of Pediatrics, and Neurology and Neurosurgery, McGill University; and the Neuropeptide Physiology Laboratory, McGill University-Montreal Children s Hospital Research Institute, Montreal, Québec, Canada H3H 1P3 ABSTRACT Pulsatile GH secretion is exquisitely sensitive to perturbations in nutritional status, but the underlying mechanisms are largely unknown. Leptin, a recently discovered adipose cell hormone, is thought to be a sensor of energy stores and to regulate body mass, appetite, and metabolism at the level of the brain. Receptors for leptin are abundantly expressed in hypothalamic nuclei known to be involved in GH regulation, suggesting that leptin may serve as an important hormonal signal to the GH neuroendocrine axis in normal animals. To test this hypothesis, we examined the effects of intracerebroventricular infusion of recombinant murine leptin, at a dose of 1.2 g/day for 7 days, on both spontaneous and GH-releasing hormone (GHRH)- stimulated GH secretion in free-moving adult male rats. Concomitant with suppressive effects on food intake, body weight, and basal Received March 25, 1998. Address all correspondence and requests for reprints to: Dr. Gloria S. Tannenbaum, Neuropeptide Physiology Laboratory, McGill University-Montreal Children s Hospital Research Institute, 2300 Tupper Street, Montreal, Québec, Canada H3H 1P3. E-mail: mcta@musica. mcgill.ca. * This work was supported by Grant MT-6837 (to G.S.T.) from the Medical Research Council of Canada. Chercheur de Carrière of the Fonds de la Recherche en Santé du Québec. plasma insulin-like growth factor I, insulin, and glucose concentrations, central infusion of leptin resulted in a 2- to 3-fold augmentation of GH pulse amplitude, 5-fold higher GH nadir levels, and a 2- to 3-fold increase in the integrated area under the 6-h GH response curve compared with those in vehicle-infused controls (P 0.001). The intracerebroventricular infusion of leptin also produced a 3- to 4-fold increase in GHRH-induced GH release at GH trough times (P 0.01). These studies demonstrate a potent stimulatory action of leptin on both spontaneous pulsatile GH secretion and the GH response to GHRH. The results suggest that the GH-releasing activity of leptin is mediated, at least in part, by an inhibition of hypothalamic somatostatin release. Thus, leptin may be a critical hormonal signal of nutritional status in the neuroendocrine regulation of pulsatile GH secretion. (Endocrinology 139: 3871 3875, 1998) THE GH neuroendocrine axis is exquisitely sensitive to changes in nutritional status. Spontaneous pulses of GH release are markedly suppressed in response to a whole host of metabolic perturbations, including food deprivation, insulinopenic diabetes, and intracellular glucopenia (see Ref. 1 for review). Furthermore, obesity is associated with an impairment of both spontaneous and GH-releasing hormone (GHRH)-induced GH secretion in both humans (2) and experimental animal models (3, 4). This hyposomatotropism probably results in decreased lipolysis (5) and may serve to perpetuate the underlying obese state. However, the mechanisms by which metabolic and nutritional factors contribute to the neuroendocrine regulation of GH secretion are largely unknown. One such possible regulator is leptin, the recently discovered adipose cell hormone that is the protein product of the ob gene (6). Leptin is secreted from adipocytes and is thought to be a sensor of energy stores and to regulate appetite and metabolism at the level of the brain (7, 8). Indeed, blood concentrations of leptin increase during times of caloric repletion and decrease during fasting (9). Rapidly accumulating data have implicated leptin as a humoral link between nutrition and several neuroendocrine systems (10 13). The discovery of leptin receptor expression in the brain (14) lends credence to this hypothesis. In fact, receptors for leptin are abundantly expressed in those hypothalamic nuclei known to be involved in GH regulation, including the arcuate and periventricular nuclei (15, 16), and systemic injection of leptin induces Fos protein in the arcuate nucleus (17). Recent double labeling studies have shown the presence of leptin receptor immunoreactivity in arcuate GHRHcontaining neurons (18). These observations suggest that leptin may serve as an important hormonal signal in the regulation of pulsatile GH secretion. To test this hypothesis, we examined the effects of intracerebroventricular (icv) infusion of leptin on both spontaneous and GHRH-stimulated GH secretion in normal freemoving rats. Food intake, body weight, and plasma concentrations of insulin-like growth factor I (IGF-I), insulin, and glucose were also monitored. Materials and Methods Animals and experimental procedure Adult male Sprague-Dawley rats (225 300 g) were purchased from Charles River Canada (St. Constant, Canada) and individually housed on a 12-h light, 12-h dark cycle (lights on, 0600 1800 h) in a temperature (22 1 C)- and humidity-controlled room. Purina rat chow (Ralston- Purina, St. Louis, MO) and tap water were available ad libitum. Chronic icv and intracardiac venous cannulas were implanted under sodium pentobarbitol (50 mg/kg, ip) anesthesia using previously described techniques (19, 20). The placement of the icv cannula was verified by both a positive drinking response to carbachol (100 ng/10 l) icv in- 3871

3872 LEPTIN STIMULATES SPONTANEOUS AND GHRH-INDUCED GH RELEASE Endo 1998 Vol 139 No 9 jection on the day after surgery and methylene blue dye at the time of death. After surgery, the rats were placed directly in isolation test chambers with food and H 2 O freely available until body weight returned to preoperative levels (usually within 5 7 days). They were then implanted sc on the dorsum, under local lidocaine 1% anesthesia, with osmotic minipumps (Alzet 2001, Alza Corp., Palo Alto, CA) containing either recombinant murine leptin (Amgen, Thousand Oaks, CA; 1.2 g/day over 7 days) or the phosphate-buffered saline (PBS) vehicle. The icv cannula was connected to the minipump by means of a brain infusion kit (Alzet 3 5 mm, Alza Corp.). During the 7-day period of continuous infusion, 24-h food intake and body weight were monitored daily. Food consumption was calculated by subtracting uneaten food plus spillage from the total given. On the test day, food was removed 1.5 h before the start of sampling and was returned at the end. Leptin or vehicle infusion was maintained during the sampling periods. In the first experiment, we documented the effects of icv infusion (6 7 days) of either leptin (n 7) or PBS (n 7) on spontaneous pulsatile GH release in free-moving rats. Blood samples (0.4 ml) were withdrawn every 15 min over a 6-h sampling period (1000 1600 h) from all animals. All blood samples were immediately centrifuged, and the plasma was separated and stored at 20 C for subsequent assay of GH, IGF-I, insulin and glucose. To avoid hemodynamic disturbance, the red blood cells were resuspended in normal saline and returned to the animal after removal of the next blood sample. In the second experiment, we assessed the effects of centrally infused leptin (6 7 days) on GH responsiveness to GHRH. Free-moving, chronically cannulated rats, implanted with osmotic minipumps as described above, were administered 1 g rat GRF-(1 29)NH 2 iv at two different time points during the 6-h sampling periods. The times of 1100 and 1300 h were chosen because these times reflect typical peak and trough periods of GH secretion, as previously documented (19, 21). The GHRH peptide (provided by Dr. P. Brazeau, Notre Dame Hospital, Montreal, Canada) was diluted in normal saline just before use. To document the rapidity of the GH response to GHRH, an additional blood sample was obtained 5 min after each injection of the peptide. All animal-based procedures were approved by the McGill University Animal Care Committee. Statistical analyses The plasma GH profiles of individual rats were analyzed using the Cluster Analysis Program for endocrine pulse detection (24). Briefly, a t statistic of 2.0 was selected to maintain a maximal false positive rate of 2.5% or less, using test cluster sizes of 2 in the prepeak nadir, peak, and postpeak nadir. Student s t tests for unpaired and paired data, as appropriate, were used for statistical comparisons between and within experimental groups. The integrated area under the GH response curve (AUC) was calculated by the linear trapezoidal method. The results are expressed as the mean se. P 0.05 was considered significant. Results Effects of icv infusion of leptin on daily food intake and body weight gain The adequacy of icv leptin administration was confirmed by observing the effects on daily food intake and body weight gain (Fig. 1). While PBS-infused rats gained 25.4 7.8 g after 7 days, leptin-treated animals lost 19.2 7.6 g during the same time period. Mean daily food intake in leptin-infused rats was significantly suppressed by 24 h after the onset of infusion and remained significantly reduced ( 38%) throughout the 7-day observation period compared with that in PBS-treated controls (Fig. 1). Effects of centrally infused leptin on spontaneous pulsatile GH release As shown in Fig. 2A, PBS icv-infused control rats exhibited the typical pulsatile pattern of GH secretion. Two major episodes of GH release were evident during the 6-h sampling Hormone assays Plasma GH concentrations were measured in duplicate by double antibody RIA using materials supplied by the NIDDK Hormone Distribution Program (Bethesda, MD). The averaged plasma GH values are reported in terms of the rat GH reference preparation (rgh RP-2). The standard curve was linear between 0.62 320 ng/ml; the least detectable concentration of plasma GH under the conditions used was 1.2 ng/ml. The intra- and interassay coefficients of variation were 7.7% and 10.7%, respectively, for duplicate samples of pooled plasma containing a mean GH concentration of 60.7 ng/ml. Plasma IGF-I concentrations were measured using a previously described method (22). To decrease the interference of IGF-binding proteins in the assay, the samples were prepared by acid-ethanol extraction followed by cryoprecipitation. The IGF-I/somatomedin C rabbit antiserum (UB3 189) was obtained from the NIDDK Hormone Distribution Program (gift from Drs. L. Underwood and J. Van Wyk). Recombinant human IGF-I (Eli Lilly Co., Indianapolis, IN) was iodinated by the chloramine-t method. The averaged plasma IGF-I values are reported in terms of the recombinant human IGF-I reference preparation (Eli Lilly Co.). The standard curve was linear between 0.1 25 ng/ml. The intraand interassay coefficients of variation were 4.8% and 14.8%, respectively, for duplicate samples of pooled plasma containing a mean IGF-I concentration of 0.82 ng/ml. Plasma insulin was measured by a dextran-coated charcoal method using guinea pig antiporcine insulin serum (23). Purified crystalline rat insulin (lot 615-JE 6-9, Eli Lilly Co., provided by Dr. R. Chance) served as a reference standard. The sensitivity of the assay was 0.16 ng/ml, and the intra- and interassay coefficients of variation were 8.2% and 10.3%, respectively, for duplicate samples of pooled plasma containing a mean plasma insulin concentration of 5.68 ng/ml. Plasma glucose was measured by an automated glucose oxidase method (Glucose Analyzer 2, Beckman Instruments, Palo Alto, CA). FIG. 1. Effects of central infusion of leptin on daily body weight gain and food intake. Values are the mean SE; the number of animals in each group is shown in parentheses; the arrow indicates the time of osmotic minipump implantation. *, P 0.01 or less compared with PBS-treated controls.

LEPTIN STIMULATES SPONTANEOUS AND GHRH-INDUCED GH RELEASE 3873 period, with most peak GH values greater than 125 ng/ml; in the intervening trough periods, basal plasma GH levels were mostly undetectable. Central infusion of leptin resulted in a marked stimulation of the spontaneous GH secretory episodes (Fig. 2B); in some rats, the amplitude of the GH peaks reached values as high as 700 ng/ml. Cluster analysis of GH pulse parameters showed that leptin-infused rats exhibited a 2- to 3-fold augmentation of GH peak amplitude, approximately 5-fold higher GH nadir levels, and a 2- to 3-fold increase in the 6-h GH AUC (P 0.001) compared with those parameters in vehicle-infused controls (Fig. 3). Neither the GH peak frequency (3.1 0.5 vs. 2.3 0.2), interpeak interval (105.0 17.3 vs. 139.3 19.3 min), or peak width (91.2 17.9 vs. 113.6 8.6 min) were significantly altered. Basal plasma IGF-I, insulin, and glucose concentrations were all significantly reduced in leptin-infused rats compared with those in PBS-treated controls (Table 1). Effects of centrally infused leptin on GH responsiveness to GHRH Figure 4 illustrates the mean plasma GH response to GHRH in animals infused with either PBS or leptin for 6 7 days and then challenged with GHRH. As shown in Fig. 4A, PBS-treated controls exhibited the typical intermittent male pattern of GH responsiveness to GHRH (21), with high GHRH-induced GH release observed when GHRH was injected at peak times (1100 h) and only a limited response during GH trough periods (1300 h). The magnitude of the GH response to GHRH at 1100 h was significantly greater than that at 1300 h (P 0.01). In contrast, pretreatment with leptin abolished this time-dependent difference and resulted in a marked augmentation (3- to 4-fold) of GHRH-induced GH release at 1300 h compared with that in PBS-infused controls (30 min GH AUC, 166.6 27.0 vs. 38.7 12.5 ng/ml h; P 0.01; Fig. 4B). Leptin-treated rats also exhibited a higher GH response to GHRH at 1100 h, although this difference did not reach significance (30 min GH AUC, 185.1 24.3 vs. 162.7 16.6 ng/ml h). Of interest, GH trough levels in the leptintreated animals rarely reached undetectable levels, in contrast to those in PBS-treated controls (see Fig. 4). FIG. 2. Individual representative 6-h plasma GH profiles in two PBS icv-infused control rats (A) compared with those in rats administered leptin (1.2 g/day over 7 days) icv (B). Central infusion of leptin resulted in a marked stimulation of the spontaneous GH secretory episodes. The number in parentheses is a data point off the scale of the figure. Discussion These studies clearly demonstrate that leptin exerts potent stimulatory actions on both spontaneous pulsatile GH secretion and the GH response to GHRH. The results suggest that leptin may be an important positive regulator of GH pulsatility in normal animals. The present findings are congruent with accumulating FIG. 3. Cluster analysis of the effects of centrally infused leptin on GH pulse parameters. Each bar represents the mean SE; the number of animals in each group is shown in parentheses. a, P 0.05 or less compared with PBSinfused controls.

3874 LEPTIN STIMULATES SPONTANEOUS AND GHRH-INDUCED GH RELEASE Endo 1998 Vol 139 No 9 TABLE 1. Effects of intracerebroventricular infusion of leptin on basal plasma IGF-I, insulin, and glucose levels Experimental group n IGF-I (ng/ml) Insulin (ng/ml) Glucose (mg/dl) PBS 7 614.6 34.7 0.6 0.1 133.7 6.0 Leptin 7 400.4 54.2 a 0.3 0.0 a 111.5 5.2 a Values are the mean SE. a P 0.05 or less compared to PBS-infused controls. FIG. 4. Mean plasma GH response to 1 g GHRH iv administered at 1100 and 1300 h in rats infused icv with either the PBS vehicle (A) or leptin (1.2 g/day over 7 days; B). Pretreatment with leptin resulted in a marked augmentation of GHRH-induced GH release at 1300 h compared with that in PBS-infused controls. Also note elevation of GH trough levels in leptin-treated rats. Vertical lines represent the SE; the number of animals in each group is shown in parentheses. data implicating leptin as a humoral link between nutrition and several endocrine systems, including the gonadal, adrenal, and thyroid axes (10 12). They are also in conformity with an earlier report in the rat demonstrating that icv administered leptin antiserum resulted in a decrease in spontaneous GH release (13) and with a recent study in pigs showing leptin stimulation of GH (25). Together these findings provide support for the thesis that leptin may be a critical hormonal signal of nutritional status to the GH neuroendocrine axis. The dramatic enhancement of GH pulsatility observed in the present study (i.e. 2- to 3-fold above the normal high amplitude GH pulses of control rats) was all the more remarkable given the sustained anorexia induced by centrally administered leptin, since previous studies in the rat have consistently found GH to be markedly suppressed in nutritionally deprived states (1, 22, 23). If leptin is a stimulatory signal to the GH neuroendocrine axis, a fall in circulating leptin concentrations might translate into lower GH pulses. Indeed, the observation that blood levels of leptin are significantly decreased during fasting (10) is consistent with this idea. The leptin-induced reductions in plasma concentrations of insulin, glucose, and IGF-I reported here are in agreement with the results of previous studies (26, 27). Although leptin may exert direct effects on one or more of these parameters (28), it is also possible that these responses are secondary to the reduction in food intake we observed, as plasma IGF-I, insulin, and glucose are known to be impaired in poorly nourished animals (10, 22, 23). On the other hand, as increases in GH levels are not normally seen in nutritionally deprived states in the rat, the increases in GH observed in these experiments are probably a result of the administered leptin rather than of changes in nutritional status. The mechanism(s) by which leptin influences pulsatile GH secretion is of interest. Current experimental evidence indicates that the pulses of GH secretion are due to the episodic release of hypothalamic GHRH, whereas somatostatin (SRIF) is the physiological regulator of GH trough periods (29). The present finding of a 5-fold increase in GH nadir levels in leptin-infused animals suggests that SRIF may be involved in this response. Support for this concept was obtained in our GHRH challenge experiments; pretreatment with leptin reversed the weak GH response to GHRH observed in PBSinfused controls at GH trough times, the latter known to be due to antagonization by the increased cyclical release of endogenous SRIF (21). These results, therefore, suggest that the GH-releasing activity of leptin is mediated at least in part by inhibiting hypothalamic SRIF release into hypophyseal portal blood. This interpretation would be in keeping with the recent in vitro demonstration that leptin inhibits SRIF synthesis and secretion in cultured fetal rat neurons (30). Furthermore, it is also possible that a reduction of SRIF tone within the hypothalamus contributed to the leptininduced augmentation in GH pulse amplitude, as SRIF may directly regulate GHRH release at the level of the arcuate nucleus (29). Alternatively, GHRH secretion may be a target for regulation by leptin, because GHRH neurons harbor leptin receptors (18), and hypothalamic GHRH is altered both in response to fasting (31) and in the genetically obese Zucker rat (4, 32). Finally, the GH stimulatory actions of leptin may be mediated indirectly via interactions with other neuronal pathways known to be both responsive to leptin and to influence GH, such as the neuropeptide Y and CRH neuroendocrine systems (16, 33, 34). Additional studies will be required to identify the complete neuroendocrine pathways through which leptin stimulates GH secretion. Acknowledgments We thank Drs Jeffrey Friedman and Jeffrey Halaas for stimulating discussion and the gift of leptin. We are grateful to Dr. R. Chance for the gift of rat insulin, to the NIDDK for the provision of GH and IGF-I RIA materials, and to Julie Temko for preparation of the manuscript. References 1. 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