Chronic Central Infusion of Increases Hypothalamic Y and Agouti-Related mRNA Levels and Body Weight in Rats Jun Kamegai, Hideki Tamura, Takako Shimizu, Shinya Ishii, Hitoshi Sugihara, and Ichiji Wakabayashi

Ghrelin, an endogenous for the growth (GHS-R), was originally puri- fied from the rat . Like the synthetic growth nergy intake and expenditure are tightly regu- hormone (GHSs), ghrelin specifically re- lated in mammals (1). Several neuronal popula- leases (GH) after intravenous admin- tions, particularly in the hypothalamic arcuate istration. Also consistent with the central actions of nucleus (ARC), are involved in the regulation of GHSs, ghrelin-immunoreactive cells were shown to be E energy and have been implicated as possi- located in the hypothalamic as well as the stomach. Recently, we showed that a single central ble targets of orexigenic (1). These include administration of ghrelin increased food intake and (NPY)/agouti-related protein (AGRP) co- hypothalamic agouti-related protein (AGRP) gene ex- expressing , pro-opiomelanocortin (POMC) neu- pression in rodents, and the orexigenic effect of this rons, and growth hormone–releasing hormone (GHRH) seems to be independent of its GH-releasing neurons, which are known to be stimulated (NPY and activity. However, the effect of chronic infusion of AGRP) or suppressed (POMC and GHRH) by starvation ghrelin on food consumption and body weight and their (2–10). However, central administration of NPY, AGRP, possible mechanisms have not been elucidated. In this and GHRH increased food intake in rats, whereas ␣-mela- study, we determined the effects of chronic intracere- ␣ broventricular treatment with ghrelin on metabolic fac- nocyte–stimulating hormone ( -MSH), an end product of tors and on neuropeptide genes that are expressed in POMC processing, decreased food intake when injected hypothalamic neurons that have been previously shown centrally (11–13). to express the GHS-R and to regulate food consumption. The growth hormone secretagogues (GHSs) are syn- Chronic central administration of rat ghrelin (1 ␮g/rat thetic peptide and nonpeptide compounds that stimulate every 12 h for 72 h) significantly increased food intake the pulsatile release of growth hormone (GH) after intra- and body weight. However, it did not affect plasma venous administration (14). Central administration of , glucose, , or GH concentrations. We also GHSs also stimulates feeding behavior (15). GHS receptor found that chronic central administration of ghrelin increased both neuropeptide Y (NPY) mRNA levels (GHS-R) mRNA is expressed in the and in of saline-treated controls; P < 0.05) and several areas of the brain, including the 10.1% ؎ 151.0) AGRP mRNA levels (160.0 ؎ 22.5% of saline-treated (16). In the hypothalamus, GHS-R mRNA is expressed in controls; P < 0.05) in the arcuate nucleus. Thus, the NPY/AGRP-, POMC-, and GHRH-containing neurons (17– primary hypothalamic targets of ghrelin are NPY/AGRP- 19). Ghrelin, an endogenous ligand for the GHS-R, was containing neurons, and ghrelin is a newly discovered purified from the rat stomach (20). Similar to the synthetic orexigenic peptide in the brain and stomach. Diabetes GHSs, ghrelin specifically releases GH after intravenous 50:2438–2443, 2001 administration. Recently, we showed that a single central administration of ghrelin increased food intake and hypo- thalamic AGRP gene expression in rodents, and the orexi- genic effect of this peptide seems to be independent of its GH-releasing activity (21). Thus, ghrelin has an alternative role in stimulating food intake in addition to releasing GH from the pituitary. It has been reported that previous exposure to GHS can induce desensitization of the subse- quent GH responses to GHS, indicating that homologous From the Department of Medicine, Nippon Medical School, Tokyo, Japan. desensitization is observed after continuous exposure to Address correspondence and reprint requests to Jun Kamegai, Department GHS with respect to GH release (22,23). However, the of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, Tokyo 113- 8603, Japan. E-mail: [email protected]. effect of chronic infusion of ghrelin on food consumption Received for publication 1 December 2000 and accepted in revised form 31 and body weight and their possible mechanisms has not July 2001. AGRP, agouti-related protein; ARC, hypothalamic arcuate nucleus; GH, been elucidated. In this study, we determined the effects of growth hormone; GHRH, growth hormone–releasing hormone; GHS, growth chronic intracerebroventricular treatment with ghrelin on hormone secretagogue; GHS-R, growth hormone secretagogue receptor; metabolic factors and on the hypothalamic NPY, AGRP, ␣-MSH, ␣-–stimulating hormone; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; RIA, radioimmunoassay; SS, ; SSC, saline POMC, and GHRH mRNA levels using radioactive in situ sodium citrate. hybridization histochemistry.

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RESEARCH DESIGN AND METHODS TABLE 1 Animals. Male Sprague-Dawley rats (250–280 g) were housed in air-condi- Comparisons of food intake, body weight, fat weight, plasma tioned animal quarters, with lights on between 08:00 and 20:00 h, and were glucose, insulin, leptin, and GH in rats treated with rat ghrelin or given food and water ad libitum. The animals were anesthetized with ether, saline for 3 days and a 23-gauge stainless-steel cannula was implanted into the right lateral ventricle using a stereotaxic apparatus, as previously described (24). The Control Ghrelin-treated upper incisor bar was set 3.3 mm below the interauricular line, and the bregma was taken as A-P zero. The cannula tip was placed at A Ϫ0.9, L Ϫ1.2, and V n 55 Ϫ3.6 mm and secured in place with dental acrylic. Only those rats whose Food intake (g/72 h) 97.8 Ϯ 4.5 116.0 Ϯ 6.2* cerebrospinal fluid overflowed through the cannula were used for the exper- Initial body weight (g) 373 Ϯ 12 379 Ϯ 13 iment. They were kept in individual cages and habituated by handling every Final body weight (g) 376 Ϯ 14 391 Ϯ 15 day. Injections of the peptide were performed 14 days after placement of the ⌬ Body weight (g) 2.8 Ϯ 2.5 11.8 Ϯ 2.9* cannula. Experiments were conducted according to the principles and proce- Epididymal fat weight (g) 3.6 Ϯ 0.3 3.9 Ϯ 0.3 dures outlined in the National Institutes of Health Guide for the Care and Use Retroperitoneal fat weight (g) 3.2 Ϯ 0.4 4.2 Ϯ 0.4 of Laboratory Animals. Ϯ Ϯ Experimental protocol. Rat ghrelin (1 ␮g/rat; provided by Dr. K. Kangawa, Inguinal fat weight (g) 3.8 0.3 4.0 0.2 National Cardiovascular Center Research Institute, Osaka, Japan) or saline Plasma glucose (mg/dl) 144.8 Ϯ 3.2 148.6 Ϯ 5.1 was injected every 12 h for 72 h. Food intake and body weights were measured Plasma insulin (ng/ml) 4.56 Ϯ 0.43 4.90 Ϯ 0.28 daily. Animals were anesthetized with pentobarbital (70 mg/kg i.p.) ϳ4 h after Plasma leptin (ng/ml) 5.65 Ϯ 1.39 6.99 Ϯ 0.83 the final injection of ghrelin. The blood was collected, and plasma was Plasma GH (ng/ml) 8.61 Ϯ 3.18 6.45 Ϯ 1.38 separated by centrifugation and stored at Ϫ70°C until assay. Plasma glucose was measured by the glucose oxidase method. Plasma insulin and leptin were Data are means Ϯ SD. *P Ͻ 0.05 compared with the control group. measured by radioimmunoassay (RIA) using a commercially available rat insulin RIA kit (Amersham, Tokyo) and rat leptin RIA kit (Linco Research, St. Charles, MO), respectively. GH levels were measured with a double-antibody 6.2 g for ghrelin vs. 97.8 Ϯ 4.5 g for saline; n ϭ 5 rats per RIA using materials supplied by the National Institute of Diabetes and group; P Ͻ 0.05) (Table 1) and body weight (11.8 Ϯ 2.9 g Digestive and Diseases, National Institutes of Health (Bethesda, MD), for ghrelin vs. 2.8 Ϯ 2.5 g for saline; n ϭ 5 rats per group; as previously described (21). Epididymal, retroperitoneal, and inguinal fat Ͻ pads were dissected and weighed. Anesthetized rats were perfused with 2% P 0.05 (Table 1), as compared with intracerebroventric- paraformaldehyde, and the brain was kept overnight at 4°C in the same ular injection of the vehicle. However, it did not affect the fixative containing 20% sucrose. weight of three fat deposits or of plasma insulin, glucose, In situ hybridization. Frozen coronal sections, 30 ␮m thick, were cut with leptin, and GH concentrations, although there was the a cryostat, mounted onto Silan-coated slides (Shin-Etsu Chemical, Tokyo), and air-dried. The medial basal hypothalamus from the rostral end of the third trend toward higher levels of fat pad weights and plasma ventricle to the caudal end of the ARC was processed for in situ hybridization leptin and insulin levels in the ghrelin-treated group (Table using 35S-labeled riboprobes for NPY, AGRP, POMC, and GHRH mRNA, as 1). previously described (21). The hybridization protocol has been described Hypothalamic neuropeptide gene expressions. Cen- previously (25). In brief, sections were dried, digested by proteinase K (10 tral administration of ghrelin increased the levels of NPY ␮g/ml), and acetylated with 0.25% acetic anhydride. Sections were rinsed Ϯ brieflyin2ϫ saline sodium citrate (SSC) and then air-dried. The probe was mRNA in the ARC to 151.0 10.1% of the saline-treated dissolved in hybridization buffer (10 mmol/l Tris [pH 8.0], containing 50% controls (n ϭ 5 rats per group; P Ͻ 0.05) (Fig. 1). Similarly, formamide, 10% dextran sulfate, 1ϫ Denhardt’s solution, 12 mmol/l EDTA, 30 ARC AGRP mRNA levels were increased by 60.0 Ϯ 22.5% mmol/l NaCl, 0.5 mg/ml yeast transfer RNA, and 10 mmol/l dithiothreitol). in ghrelin-treated rats, as compared with the saline-treated Thereafter, 2 ϫ 106 dpm of the probe in 100 ␮l buffer was applied to each slide ϭ Ͻ and hybridized at 55°C overnight. The slides were rinsed in 4ϫ SSC and rats (n 5 per group; P 0.05) (Fig. 2). However, the digested with ribonuclease-A (20 ␮g/ml) for 30 min, followed by two 5-min levels of POMC and GHRH mRNA in the ARC did not differ changes of 2ϫ SSC, 10 min in 1ϫ SSC, 10 min in 0.5ϫ SSC at room significantly between the ghrelin-treated and saline-treated temperature, and 30 min in 0.1ϫ SSC at 55°C. The slides were dehydrated in rats (Figs. 3 and 4). ethanol and then air-dried. These sections were dipped in Konica NR-M2 autoradiographic emulsion (Konica, Tokyo), exposed for 5 days, and devel- oped. The sections were counterstained with cresyl violet. As a control for DISCUSSION nonspecific labeling, sense NPY, AGRP, POMC, and GHRH probes were used In the present study, we demonstrated that chronic central on some adjacent sections from experimental animals, and no specific signal infusion of ghrelin significantly increases food intake and was detected. Data analysis. The hybridization signal in the brain slice was determined body weight. We also found that chronic central adminis- from the autoradiograms using an MCID image analysis system (Imaging tration of ghrelin increases hypothalamic NPY and AGRP Research, St. Catherines, Canada), as previously described (25). The ARC on mRNA levels, whereas central infusion of ghrelin does not one side was enclosed by a circle with a 1.12-mm radius as a fixed window, alter POMC and GHRH mRNA levels. However, the infu- which covered almost the whole area of the nucleus. The relative optical sion did not affect plasma insulin, glucose, leptin, or GH density within the window was measured. The background was estimated by measuring the relative optical density within a window placed over another concentrations. These results indicated that the primary area of the hypothalamus. The anatomical equivalence of hypothalamic hypothalamic targets of ghrelin are NPY/AGRP-containing sections among animals was obtained by selecting slides with the aid of a rat neurons, and ghrelin is a novel orexigenic peptide in the brain atlas (26) and verifying the anatomical identifications by staining brain and stomach. sections with cresyl violet. Six sections per animal were examined. The values reported are the mean Ϯ SE. Five rats were used for the analysis. Statistical The central regulation of pituitary GH release is medi- analysis was performed with the Student’s t test to compare the means ated by the opposing actions of GHRH and somatostatin between two groups. P Ͻ 0.05 was considered significant. (SS) (27). In this study, plasma GH levels and GHRH and SS (data not shown) mRNA levels were not altered by central infusion of ghrelin. These results are consistent RESULTS with our previous report that a single central injection of 1 Food intake, body weight, fat weight, and plasma ␮g rat ghrelin did not alter GHRH and SS mRNA levels or . Seven injections of rat ghrelin (1 ␮g) into the the episodic release of GH of freely moving adult male rats lateral ventricle of rats during a 72-h period of free feeding (21). It also has been reported that treatment with ghrelin resulted in a significant increase in food intake (116.0 Ϯ for 1 week increased the food intake of female dwarf rats,

DIABETES, VOL. 50, NOVEMBER 2001 2439 EFFECT OF GHRELIN ON THE HYPOTHALAMUS

NPY mRNA levels after a single injection of ghrelin, although there was a trend toward an increase. It remains to be determined whether this discrepancy is because 1) the single injection of 1 ␮g of ghrelin was not sufficient to increase NPY mRNA levels significantly, or 2) prolonged stimulation with ghrelin is necessary to increase NPY mRNA levels. During starvation, when circulating leptin, an –derived hormone, levels fall (31,32), NPY and AGRP mRNA levels increase in the ARC. Because the hypotha- lamic NPY/AGRP-containing neurons also express the (33), these neurons may be direct targets for leptin. Because leptin inhibits NPY and AGRP mRNA levels (5,34,35), one mechanism by which fasting leads to

FIG. 1. Effect of intracerebroventricular administration of ghrelin on NPY mRNA in the ARC of adult male rats. Rat ghrelin (1 ␮g) or its vehicle was administered into the lateral ventricle every 12 h for 72 h. NPY mRNA levels in the ARC were determined by in situ hybridization. A: Representative in situ hybridization histochemistry for NPY mRNA, as revealed by emulsion autoradiography in the ARC after central administration of ghrelin or the vehicle. Note that administration of ghrelin increased NPY mRNA levels. The scale bar indicates 500 ␮m. B: A summary of the data, which are expressed as a percentage of the ؍ vehicle-treated control group. The data represent the mean ؎ SE (n 5 animals/group). *P < 0.05 compared with the vehicle-treated control group. which are deficient in GH (28). Taken together, these data suggest that the orexigenic action of ghrelin is unlikely to be due to its ability to stimulate GH . It has been reported that central administration of a synthetic GHS induced c-fos expression, a marker of neu- ronal activity, in NPY/AGRP-containing neurons (29,30). FIG. 2. Effect of intracerebroventricular administration of ghrelin on Consistent with these reports, this study showed that AGRP mRNA in the ARC of adult male rats. Rat ghrelin (1 ␮g) or its vehicle was administered into the lateral ventricle every 12 h for 72 h. chronic central administration of ghrelin increased both AGRP mRNA levels in the ARC were determined by in situ hybridiza- NPY mRNA levels and AGRP mRNA levels, suggesting that tion. A: Representative in situ hybridization histochemistry for AGRP mRNA, as revealed by emulsion autoradiography in the ARC after ghrelin activates the NPY/AGRP neurons of the ARC. central administration of ghrelin or the vehicle. Note that administra- These findings are in agreement with our previous report tion of ghrelin increased AGRP mRNA levels. The scale bar indicates that a single central injection of ghrelin (1 ␮g/rat) in- 500 ␮m. B: A summary of the data, which are expressed as a percentage of the vehicle-treated control group. The data represent the mean ؎ SE animals/group). *P < 0.05 compared with the vehicle-treated 5 ؍ creased AGRP mRNA levels in the ARC (21). However, in (n the same report, we did not observe a significant change in control group.

2440 DIABETES, VOL. 50, NOVEMBER 2001 J. KAMEGAI AND ASSOCIATES

and not the hypothalamus (20). In addition, it has been reported that intraperitoneal administration of ghrelin in freely feeding rats stimulated food intake (36). These results led to the suggestion that ghrelin is the peripheral signal from the stomach that regulates food intake by acting on the hypothalamic in addition to leptin. However, additional studies are required to deter- mine how ghrelin enters the brain. We cannot, however, exclude the possibility that the ghrelin that originates in the hypothalamus acts within the brain to increase food intake, because ghrelin-immunoreactive cells were shown to be located in the hypothalamic ARC as well as the stomach (20). In the latter case, it remains to be deter- mined whether the hypothalamic levels of ghrelin are affected by nutritional status. AGRP and NPY are potent stimulators of feeding

FIG. 3. Effect of intracerebroventricular administration of ghrelin on POMC mRNA in the ARC of adult male rats. Rat ghrelin (1 ␮g) or its vehicle was administered into the lateral ventricle every 12 h for 72 h. POMC mRNA levels in the ARC were determined by in situ hybridiza- tion. A: Representative in situ hybridization histochemistry for POMC mRNA, as revealed by emulsion autoradiography in the ARC after central administration of ghrelin or the vehicle. The scale bar indicates 500 ␮m. B: A summary of the data, which are expressed as a percentage of the vehicle-treated control group. The data represent the mean ؎ SE .(animals/group 5 ؍ n) an increase in NPY and AGRP mRNA may be through the reduction of circulating leptin. However, the reduction of leptin is unlikely to be the sole mechanism by which NPY and AGRP mRNA levels are increased during fasting, because NPY and AGRP mRNA levels also are elevated by fasting in the leptin-insensitive db/db mice, which, as a result of a mutation in the leptin receptor, are resistant to the effects of leptin (8,35). It also has been reported that fasting increases plasma ghrelin levels (28), and we FIG. 4. Effect of intracerebroventricular administration of ghrelin on showed that central infusion of ghrelin increased NPY and GHRH mRNA in the ARC of adult male rats. Rat ghrelin (1 ␮g) or its AGRP mRNA levels. Therefore, it has been hypothesized vehicle was administered into the lateral ventricle every 12 h for 72 h. GHRH mRNA levels in the ARC were determined by in situ hybridiza- that part of the mechanism by which fasting increases NPY tion. A: Representative in situ hybridization histochemistry for GHRH and AGRP mRNA levels is through an elevation in plasma mRNA, as revealed by emulsion autoradiography in the ARC after ghrelin that accompanies fasting. So far, the source of the central administration of ghrelin or the vehicle. The scale bar indicates 500 ␮m. B: A summary of the data, which are expressed as a percentage ghrelin that activates the NPY/AGRP neurons is unknown, of the vehicle-treated control group. The data represent the mean ؎ SE .(animals/group 5 ؍ but the principal site of ghrelin synthesis is the stomach (n

DIABETES, VOL. 50, NOVEMBER 2001 2441 EFFECT OF GHRELIN ON THE HYPOTHALAMUS

(11,12). During fasting, NPY released from NPY/AGRP hypothalamic neuropeptide mRNAs in food-restricted and food-deprived binds to and activates NPY-Y1 and -Y5 receptors in rats. 52:441–447, 1990 11. Clark JT, Kalra PS, Croeley WR, Kalra SP: Neuropeptide Y and human the hypothalamus and results in the stimulation of feeding stimulate feeding behavior in rats. behavior (1). The simultaneous release of AGRP antago- 155:427–429, 1984 nizes the -4 receptor, which suppresses the 12. Rossi M, Kim MS, Morgan DGA, Small CJ, Edwards CMB, Sunter D, effects of ␣-MSH (12). Therefore, NPY and AGRP Abusnana S, Goldstone AP, Russell SH, Stanley SA, Smith DM, Yagaloff K, increase food intake via different pathway. It has been Ghatei MA, Bloom SR: A C-terminal fragment of agouti-related protein reported that mice with a deletion in the NPY gene also increases feeding and antagonizes the effect of alpha-melanocyte stimulat- ing hormone in vivo. Endocrinology 139:4428–4431, 1998 increase food intake after central administration of ghrelin 13. Vaccarino FJ, Bloom FE, Rivier J, Vale W, Koob GF: Stimulation of food (28), raising the possibility that redundant mechanisms intake in rats by centrally administered hypothalamic growth hormone- exist to ensure that this drive is maintained, suggesting releasing factor. Nature 314:167–168, 1985 that ghrelin has a physiological relevance in the regulation 14. Smith RG, Van Der Ploeg HT, Howard AD, Feighner SD, Cheng K, Hickey of food intake. The present studies, demonstrating that GJ, Wyvratt MJ: Peptidomimetic regulation of growth hormone secretion. Endocr Rev 18:621–645, 1997 chronic central infusion of ghrelin increase body weight in 15. Okada K, Ishii S, Minami S, Sugihara H, Shibasaki T, Wakabayashi I: rats, support the hypothesis that ghrelin plays a role in the Intracerebroventricular administration of the growth hormone-releasing physiological regulation of body weight. Orexigenic factors peptide KP-102 increases food intake in free-feeding rats. Endocrinology such as NPY or AGRP may contribute to the syn- 137:5155–5158, 1996 drome and diabetes. In fact, diabetic hyperphagia induced 16. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, by streptozotocin is associated with elevated expression Chou M, Liu KK, McKee KK, Pong S-S, Chaung L-Y, Elbrecht A, Dashkevicz of the NPY and AGRP genes in the ARC (37). Additional M, Heavens R, Rigby M, Sirinathsinghji DJS, Dean DC, Melillo DG, Patchett work will be necessary to determine whether ghrelin AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith contributes to the pathophysiological mechanisms in dia- RG, Van der Ploeg LHT: A receptor in pituitary and hypothalamus that betes. We are currently investigating these possibilities. functions in growth hormone release. Science 273:974–977, 1996 17. Guan X-M, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJS, Smith RG, Van der Ploeg LHT, Howard AD: Distribution of mRNA encoding ACKNOWLEDGMENTS the growth hormone secretagogue receptor in brain and peripheral tissues. Mol Brain Res 48:23–29, 1997 This work was supported by a Grant-in-Aid for scientific 18. Tannenbaum GS, Lapointe M, Beaudet A, Howard AD: Expression of research from the Japanese Ministry of Education, Science growth hormone secretagogue-receptors by growth hormone-releasing and Culture (to J.K.) and the Foundation for Growth hormone neurons in the mediobasal hypothalamus. Endocrinology 139: Science in Japan (to I.W.). We thank Dr. K. Kangawa (Na- 4420–4423, 1998 tional Cardiovascular Center Research Institute, Osaka, 19. Willesen MG, Kristensen P, Romer J: Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Japan) for rat ghrelin and Dr. Gregory S. 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