Endocr. J./ Y. Ueta et al.: ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

REVIEW

Hypothalamic and Appetite Response in Anorexia-Cachexia Animal

YOICHI UETA, HIROFUMI HASHIMOTO, ETSURO ONUMA*, YOH TAKUWA** AND ETSURO OGATA***

Department of Physiology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan *Kamakura Research Laboratories, Chugai Pharmaceutical Co. Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan **Department of Physiology, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan ***Cancer Institute Hospital, 3-10-6 Ariake Koto-ku, Tokyo 135-855, Japan

Received June 13, 2007; Accepted June 25, 2007; Released online September 7, 2007

Correspondence to: Yoichi Ueta, M.D., Ph.D., Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

Key words: Anorexia, Cachexia, Hypothalamus, Neuropeptides, -related protein

CACHEXIA, characterized by anorexia and marked weight loss, is a complex syndrome in patients with inflammatory disease and cancer. In particular, the pathogenesis of cancer cachexia is a multifactor syndrome that includes reduced food intake, alteration in energy and substrate metabolism in the host and accelerated fat and muscle loss [1]. As anorexia-cachexia syndrome compromises the quality of life and contributes to mortality in patients, it is important to examine the essential mechanisms of the syndrome in order to develop effective therapeutics even though anorexia-cachexia syndrome may be caused by multiple factors. In this review, we focus on the hypothalamic neuropeptide gene expression in an anorexia-cachexia animal model because neuropeptides in the hypothalamus may be the key molecules that regulate the appetite and feeding behavior in cancer anorexia-cachexia syndrome [2] as well as in normal conditions [3, 4]. Quantitative changes in neuro - gene expressions in the hypothalamus and the relevance of their changes in abnormal nutritional conditions (e.g., negative energy balance) are reviewed and discussed by a comparison of three different types of anorexia and anorexia-cachexia animal models; dehydration-induced anorexia, bacterial lipopolysacchaide (LPS)-induced anorexia, and cancer-induced anorexia-cachexia syndrome.

Hypothalamic neuropeptides related to appetite and feeding behaviors It is well established that appetite and feeding behaviors are primarily controlled by a feeding center, the lateral hypothalamic area (LHA), and a satiety center, the ventromedial hypothalamic nucleus (VMH), in the hypothalamus [5]. Recently, neural networks and chemical mediators among the hypothalamic nuclei-not only the LHA and the VMH but also the arcuate nucleus (Arc) and the paraventricular nucleus (PVN) -related to feeding regulation have been studied by many investigators. Various kinds of

1 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111 neuropeptides as well as classic neurotransmitters such as dopamine and serotonin act as feeding regulatory factors in the neural networks at these sites [6]. For example, centrally administered (NPY) strongly stimulates feeding in mice and rats [7]. Many studies have demonstrated that the expression of the NPY gene was markedly increased in the neurons of the Arc after food deprivation (starvation). This type of is called an “orexigenic” neuropeptide. / hypocretins, which are produced in the neurons located in the LHA [5], are also potent orexigenic neuropeptides. On the other hand, (POMC), which encodes α melanocyte-stimulating hormone (αMSH)) in the Arc and corticotrophin-releasing hormone (CRH) in the PVN function as the inhibitory system for feeding [8]. These neuropeptides are referred to as “anorexigenic” neuropeptides. Recently, many orexigenic and anorexigenic neuropeptides that regulate appetite and body weight have been discovered in the hypothalamus [3, 4, 6]. In general, orexigenic neuropeptides in the hypothalamus such as NPY and orexins/hypocretins stimulate feeding. Starvation (negative energy balance) up-regulates the expression of the orexigenic neuropeptide genes, and anorexigenic neuropeptides in the hypothalamus such as αMSH suppress feeding; starvation down-regulates the expression of anorexigenic neuropeptide genes.

Dehydration-induced anorexia Anorexia, the loss of appetite for food, is caused by various different mechanisms. Because the pathological model of anorexia may involve multiple factors, a simple method of developing anorexia in animal models is required in order to examine the neural and molecular mechanisms responsible for anorexia. With this physiological aspect in mind, we took notice of reports on dehydration-induced anorexia animal models [9-11]. Watts and his colleagues showed that rats exhibit profound anorexia from dehydration when hypertonic saline (2.5% NaCl) instead of drinking water was given to rats for 5 days [12, 13]. This model is advantageous in the examination of the neural network and molecules associated with dehydration-induced anorexia because slowly developed anorexia after dehydration is rapidly reversed once access to drinking water is restored. Changes in the expression of the neuropeptides genes in the hypothalamus were examined in dehydration-induced anorexia animals [11-13], and in dehydrated rats, NPY mRNAs in the Arc significantly increased compared to euhydrated rats [12]. In addition, POMC and /neuromedin N (NT/N) mRNAs in the Arc and CRH mRNA in the PVN of dehydrated rats were marked decreased in comparison with those in euhydrated rats [12]. These changes are reasonable as a result of the negative energy balance because drinking hypertonic saline caused anorexia with dehydration in the rats. It has also been demonstrated that CRH and NT/N mRNAs in the LHA were increased in dehydration-induced anorectic rats and CRH mRNA in the LHA strongly correlated with the intensity of anorexia. These results suggest that the dehydration-induced elevation of CRH and NT/N production in the LHA act upon the PVN and suppress feeding in dehydrated rats [11-13]. These studies raise the hypothesis that dehydration primarily causes up-regulation of the gene expression of anorexigenic neuropeptides in the hypothalamus which induces anorexia, and then anorexia-induced starvation (negative energy balance) up-regulates the gene expression of the orexigenic neuropeptides and reduces the gene expression of the anorexigenic neuropeptides (Fig. 1). The reason why dehydration causes up-regulation of the gene expression of anorexigenic neuropeptides in a feeding center and the possible involvement of peripheral humoral factors such as and on dehydration-induced anorexia should be clarified by further study.

2 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

LPS-induced anorexia A pathological anorectic animal model as well as a physiological model such as dehydration-induced anorexia is also important for the examination of the mechanisms of anorexia. Anorexia and weight loss are common symptoms seen both in patients with inflammatory disease and in experimental animal models with inflammation. Peripheral administration of bacterial LPS (a component of the outer membrane of gram-negative bacteria) to animals is often used to develop an experimental inflammatory animal model. Sickness syndromes such as fever, activation of the hypothalamo -pituitary adrenal axis, and anorexia occurs in response to peripheral administration of LPS [14-16]. It is interesting that LPS-induced anorexia is not related to the fever induced by LPS [17, 18]. Although vagal afferents is known to be involved in the behavioral effects induced by peripheral administration of LPS, vagotomy does not attenuate the anorectic response to peripheral administration of LPS [19]. It has been postulated that the central effects of peripheral LPS may be mediated by cytokines, LPS receptors on the surface of the cerebral endothelial cells of the blood-brain barrier, and humoral mediators such as prostaglandins [20, 21]. Several studies have examined the changes in the gene expressions of orexigenic and anorexigenic neuropeptides in the hypothalamus of LPS-treated animals [22-24]. Sergeyev et al. examined the changes of neuropeptides-POMC, NPY, , MCH and cocaine- and amphetamine-regulated transcripts (CART)-in the hypothalamus after intraperitoneal (ip) injection of LPS in rats using in situ hybridization histochemistry [23]. They confirmed a reduction of food intake 4 hours after ip injection of LPS. In the hypothalamus of the LPS-treated rats POMC and CART mRNAs in the Arc showed an increase in comparison with the control. On the other hand, MCH, CART and galanin mRNAs in the LHA decreased in the LPS-treated rats. Thus, suggesting that up-regulation of anorexigenic neuropeptides (POMC/CART) in the Arc and down -regulation of orexigenic neuropepides (MCH and galanin) in the LHA may have been the cause of the reduction of food intake after ip administration of LPS [23]. In LPS-treated rats, αMSH derived from POMC has been suggested to mediate LPS-induced anorexia [25]. They postulated that NPY mRNA in the Arc did not change in either the LPS-treated or control rats because the time interval (4 hours after ip administration of LPS) may be too short for transcriptional change to be detected. In mice, NPY and agouti-related protein (AgRP) mRNAs in the hypothalamus were significantly increased at 6 hours after LPS injection [24]. These results raised the hypothesis that peripheral administration of LPS indirectly causes up-regulation of the gene expression of anorexigenic neuropeptides in the hypothalamus and down-regulation of the gene expression of orexigenic neuropeptides in the hypothalamus (Fig. 2). It is natural that these changes in hypothalamic neuropetide gene expression induce anorexia. The up-regulation of the orexigenic neuropeptide genes in the hypothalamus in mice at 6 hours after LPS injection may be a result of anorexia induced by LPS. Peripheral administration of LPS induces production and secretion of prostaglandins and various cytokines. Thus, further study is necessary to examine the crucial factors involved in the pathogenesis of LPS-induced anorexia.

Cancer anorexia-cachexia syndrome Anorexia-cachexia syndrome is found in a large number of patients in the advanced stages of cancer [26, 27]. Several metabolic and behavioral abnormalities such as appetite loss, loss of skeletal muscle and fat, general fatigue, and psychological distress are observed in this syndrome. It is difficult to find a common factor that causes cancer anorexia-cachexia syndrome because multiple factors may be involved in the etiology of this syndrome [1, 2, 28-31]. In order to reveal the etiology of cancer anorexia-cachexia syndrome, it is important

3 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111 to investigate changes in orexigenic and anorexigenic neuropeptides in the hypothalamus in cancer-associated anorexia-cachexia animal models. Although multiple factors are involved in cancer anorexia-cachexia syndrome, the changes in these neuropeptides in the hypothalamus may elucidate the mechanisms in cancer anorexia-cachexia syndrome. The involvement of cytokines and hypothalamic neuropeptides in cancer anorexia -cachexia syndrome has been well reported [1, 2, 32, 33]. It has been demonstrated that cachectic factors such as cytokines mimic the effects of leptin on the hypothalamus and induce anorexia in cancer anorexia-cachexia syndrome (Fig. 3). Leptin is the product of the ob gene. Starvation suppresses the expression of the ob gene in adipocytes and decreases plasma leptin levels [34, 35]. Leptin deficiency due to mutation of the ob gene (ob/ob mouse) and leptin resistance due to mutation of the (db/db mouse and fa/fa rat) cause severe obesity [34, 36, 37]. NPY mRNA in the Arc of ob/ob mouse, db/db mouse, and fa/fa rat were markedly increased in comparison with controls [38, 39]. Several studies have demonstrated changes in the gene expression of hypothalamic orexigenic and anorexigenic neuropeptides in cachexia animal models bearing cancer cells such as the following.

Orexigenic neuropeptides (NPY, AgRP, orexins, MCH) in anorexia-cachexia syndrome Plata-Salaman et al. reported that NPY mRNA and POMC mRNA levels in the hypothalamus were not significantly different between pair-fed normal rats and anorectic rats bearing prostate adenocarcinoma tumor cells [30]. Nara-ashizawa et al. also demonstrated that NPY mRNA in the Arc was up-regulated in cachectic nude mice bearing human tumor cells (SEKI melanoma cells) [40]. They also showed that there is no significant difference in MCH and mRNA levels between the SEKI and control mice [40]. Jensen et al. demonstrated that NPY mRNA levels in the Arc were markedly increased in anorexic rats bearing glucagonoma (MSL-G-AN) [41]. Chance et al. demonstrated that NPY mRNA levels in the medial hypothalamus were significantly elevated in anorectic tumor-bearing (methylcholanthrene-induced sarcoma) rats, and that CRH mRNA in the medial hypothalamus tend to be reduced in anorectic tumor-bearing rats and paired-fed rats [42]. Chen et al. demonstrated that hypothalamic NPY mRNA was significantly raised in cachectic MAC16 (colonic adenocarcinoma)-bearing mice and pair-fed mice [43]. Although it is difficult to explain why the orexigenic neuropeptide NPY up-regulates in the hypothalamus of cachectic tumor-bearing animals, one explanation might be that downstream of NPY mRNA, functions such as protein synthesis, transport, secretion and its receptors are disturbed by humoral factors such as cytokines and tumor-derived factors [44]. Another explanation for some cases is that hypothalamic NPY mRNA response is less sensitive in cachectic mice bearing human tumor cells (SEKI, NAGAI, and G361) in comparison with pair-fed normal rats, and that MCH and orexin mRNA in the hypothalamus follows a pattern similar to NPY [45].

Anorexigenic neuropeptides (αMSH, CART, CRF) in anorexia-cachexia syndrome Plata-Salaman et al. reported that POMC mRNA levels in the hypothalamus were not significantly different between pair-fed normal rats and anorectic rats bearing prostate adenocarcinoma tumor cells [30]. Chance et al. demonstrated that POMC mRNA was not changed in cachectic tumor-bearing rats [46]. Nara-ashizawa et al. demonstrated that CRH mRNA in the PVN was up-regulated in cachectic tumor-bearing mice [40]. Jensen et al. demonstrated that hypothalamic CART is highly expressed in anorectic rat glucagonoma [47]. As we mentioned above, it may be difficult to come to a conclusion on the relationship between the hypothalamic neuropeptides and appetite loss in cachectic animal

4 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111 models because previous reports are not concordant. One of reasons may be based on the different tumor cells used for tumor-bearing models and the different methods of evaluation such as Northern blot analysis, RNA protection assay, and in situ hybridization histochemistry. In addition, the studies have not examined all of the orexigenic/ anorexigenic neuropeptides in the hypothalamus. Another weak point is that the cachexia animal models are not recovered by therapeutic treatment, thus it is difficult to compare the changes of the neuropeptides in the hypothalamus at all stages- pre-cachectic, cachectic, and recovery from the cachexia. To resolve these issues we selected what we suggest is an ideal cachexica animal model as described below.

Anorexia-cachexia syndrome in parathyroid hormone-related protein (PTHrP)-secreting tumor bearing animals PTHrP is frequently produced and secreted by cancers and is a special type of leukemia/lymphoma (adult T cell leukemia/lymphoma). PTHrP is known to be a principal factor responsible for humoral hypercalcemia of malignancy (HHM) [48, 49]. Both polyclonal and monoclonal antibodies that neutralize PTHrP exerted anti-hypercalcemic and anti-cachectic effects in animal models [50-52]. Onuma et al. generated a humanized anti-PTHrP monoclonal antibody [53]. We demonstrated that peripheral administration of this antibody improved anorexia-cachexia syndrome in PTHrP-secreting tumor bearing animals [54, 55]. In this rat model, the mRNA levels of orexigenic neuropeptides such as NPY were significantly higher than those found in nontumor-bearing rats, whereas mRNA levels of anorexigenic peptides such as POMC and CRH were lower than in nontumor-bearing rats [56]. Bolus intravenous administration of anti-PTHrP antibody returned these mRNA levels to control levels [56]. The results suggest that starvation caused these changes in the hypothalamus and improved feeding was a consequence of the restoration from the administration of anti-PTHrP antibody. In this model, the leptin level in plasma was markedly decreased and anti-PTHrP restored it. Many appetite-regulating peptides are under the control of leptin [3, 34, 57-59]. Thus, it seems that adipose tissues and the hypothalamus are not the primary target sites of PTHrP to induce anorexia and cachexia. On the other hand, the mRNA levels of orexins and MCH that are expressed in the neurons of the LHA and enhance feeding [60, 61] were not markedly altered in this rat model and an anti-PTHrP antibody had little effect. PTHrP may affect factors and mechanisms that function between feeding-regulating neuropeptides in the hypothalamus and pathways in the CNS downstream of these neuropeptides (Fig. 4). We cannot disregard the possibility that hypercalcemia in plasma may inhibit appetite and feeding through the CNS because severe hypercalcemia was observed in this model. However, there is a potent possibility that PTHrP may act as a neurotransmitter/neuromodulator in the CNS to inhibit feeding because PTHrP and its receptor, PTH1R mRNAs, were widely expressed in the brain of the rat [62].

Conclusions The etiology of anorexia and appetite loss in cancer anorexia-cachexia syndrome is not explained by modification of the two centers theory in the hypothalamus and possible involvement of orexigenic and/or anorexigenic neuropeptides in the hypothalamus. Dehydration-induced anorexia and LPS-induced anorexia seem to be the result of the up-regulation of anorexigenic neuropeptides in the hypothalamus and starvation signals that stimulate the up-regulation of orexigenic neuropeptides. However, it is very difficult to understand the etiology of feeding-regulated hypothalamic neuropeptides of cancer anorexia-cachexia syndrome. Unknown factors/pathways cause anorexia/cachexia, and anorexia-induced up-regulation of orxigenic neuropeptides and down-regulation of anorexigenic neuropeptides in the hypothalamus does not stimulate feeding. Why do

5 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111 hypothalamic neuropeptide signals that stimulate eating not cause feeding? Does a common chemical mediator such as PTHrP exist in anorexia-cachexia syndrome? A PTHrP-secreting tumor may give us new insight into cancer anorexia-cachexia syndrome. Further study is required to find a common factor responsible for cancer anorexia-cachexia syndrome and the action site of the anorexic signal in the CNS.

Acknowledgements We thank F. Ford for editing the manuscript.

References 1. Muscaritoli M, Bossola M, Aversa Z, Bellantone R, Fanelli FR (2006) Prevention and treatment of cancer cachexia: New insights into an old problem. Eur J Cancer 42:31-41. 2. Inui A (1999) Cancer anorexia-cachexia syndrome: are neuropeptides the key? Cancer Res 59:4493-501. 3. Cummings DE, Schwartz MW (2003) Genetics and pathophysiology of human obesity. Annu Rev Med 54:453-471. 4. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, Kalra PS (1999) Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20:68-100. 5. Oomura Y (1980) Input-output organization in the hypothalamus relating to food intake behavior. Handbook of the Hypothalamus (Vol. 2). New York: Marcel Dekker, p557-620. 6. Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW (2006) Central nervous system control of food intake and body weight. Nature Rev 443:289-295. 7. Stanley BG, Leibowitz SF (1985) Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior. Proc Natl Acad Sci USA 82:3940-3943. 8. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD (1997) Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385:165-168. 9. Schoorlemmer GHM, Evered MD (2002) Reduced feeding during water deprivation depends on hydration of the gut. Am J Physiol Regulatory Integrative Comp Physiol 283:1061-1069. 10. Watts AG (1999) Dehydration-associated anorexia: development and rapid reversal. Physiol Behav 65: 871-878. 11. Watts AG (2001) Neuropeptides and the integration of motor responses to dehydration. Annu Rev Neurosci 24: 357-384. 12. Watts AG, Kelly AB, Sanchez-Watts G (1995) Neuropeptides and thirst: the temporal response of corticotropin-releasing hormone and neurotensin/neuromedin N gene expression in rat limbic forebrain neurons to drink hypertonic saline. Behav Neurosci 109: 1146-1157. 13. Watts AG, Sanchez-Watts G, Kelly AB (1999) Distinct patterns of neuropeptide gene expression in the lateral hypothalamic area and arcuate nucleus are associated with dehydration-induced anorexia. J Neurosci 19 (14): 6111-6121. 14. O’Reilly B, Vander AJ, Kluger MJ (1988) Effects of chronic infusion of lipoplysaccharide on food intake and body temperature of the rat. Physiol Behav 42: 287-291. 15. Lugarini F, Hrupka BJ, Schwartz GJ, Plata-Salaman CR, Langhans W (2002) A role for cyclooxygenase-2 in lipopolysaccharide-induced anorexia in rats. Am J Physiol Regul Integr Comp Physiol 283: R862-868. 16. Von Meyenburg C, Hrupka BH, Arsenijevic D, Schwartz GJ, Landmann R, Langhans W (2004) Role for CD14, TLR2, and TLR4 in bacterial product-induced anorexia. Am J Physiol Regul Integr Comp Physiol 287: R298-305.

6 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

17. Larson SJ, Collins SM, Weingarten HP (1996) Dissociation of temperature changes and anorexia after experimental colitis and LPS administration in rats. Am J Physiol Regul Integr Comp Physiol 271: R967-972. 18. McCarthy DO, Kluger MJ, Vander AJ (1984) The role of fever in appetite suppression after endotoxin administration. Am J Clin Nutr 40: 310-316. 19. Porter MH, Hrupka BJ, Lannghans W, Schwartz GJ (1998) Vagal and splanchnic afferents are not necessary for the anorexia produced by peripheral IL-1, LPS and MDP. Am J Physiol Regul Integr Comp Physiol 275: R384-389. 20. Cao C, Matsumura K, Yamagata K, Watanabe Y (1995) Induction by lipopolysaccharide of cyclooxygenase-2 mRNA in rat brain: its possible role in the febrile response. Brain Res 697: 187-196. 21. Cao CY, Matsumura K, Yamagata K, Watanabe Y (1997) Involvement of cyclooxygenase-2 in LPS-induced fever and regulation of its mRNA by LPS in the rat brain. Am J Physiol Regul Integr Comp Physiol 272: R1712-1725. 22. Gayle D, Ilyin SE, Plata-Salaman CR (1999) Feeding status and bacterial LPS-induced cytokine and neuropeptide gene expression in hypothalamus. Am J Physiol Regul Integr Comp Physiol 46: R1188-1195. 23. Sergeyev V, Broberger C, Hokfelt T (2001) Effect of LPS administration on the expression of POMC, NPY, galanin, CART and MCH mRNAs in the rat hypothalamus. Brain Res Mol Brain Res 90(2): 93-100. 24. Ogimoto K, Harris MK, Wisse BE (2006) MyD88 is a key mediator of anorexia, but not weight loss, induced by lipopolysaccharide and interleukin-1b. Endocrinology 147: 4445-4453. 25. Huang QH, Hruby VJ, Tatro JB (1999) Role of central in endotoxin-induced anorexia. Am J Physiol Regul Integr Comp Physiol 276: R864-871. 26. Tisdale MJ (2001) Cancer anorexia and cachexia. Nutrition 17: 438-442. 27. Bruera E (1997) ABC of palliative care. Anorexia, cachexia, and nutrition. BMJ 315:1219-1222 28. Tisdale MJ (1997) Biology of cachexia. J Natl Cancer Inst 89:1763-1773. 29. Plata-Salaman CR, Borkoski JP (1994) Chemokines/intercrines and central regulation of feeding. Am J Physiol 266:R1711-1715. 30. Plata-Salaman CR, Ilyin SE, Gayle D (1998) Brain cytokine mRNAs in anorectic rats bearing prostate adenocarcinoma tumor cells. Am J Physiol 275:R566-573. 31. Schwartz MW, Dallman MF, Woods SC (1995) Hypothalamic response to starvation: implications for the study of wasting disorders. Am J Physiol 269:R949-957. 32. Ramos EJ, Suzuki S, Marks D, Inui A, Asakawa A, Meguid MM (2004) Cancer anorexia-cachexia syndrome: cytokines and neuropeptides. Curr Opin Clin Nutr Metab Care 7:427-434. 33. Laviano A, Meguid MM, Inui A, Muscaritoli M, Rossi-Fanelli F (2005) Therapy insight: Cancer anorexia-cachexia syndrome—when all you can eat is yourself. Nat Clin Pract Oncol 2:158-165. 34. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372:425-432. 35. Frederich RC, Lollmann B, Hamann A, Napolitano-Rosen A, Kahn BB, Lowell BB, Flier JS. (1995) Expression of ob mRNA and its encoded protein in rodents. Impact of nutrition and obesity. J Clin Invest 96:1658-1663. 36. Chua SC Jr, Chung WK, Wu-Peng XS, Zhang Y, Liu SM, Tartaglia L, Leibel RL. (1996) Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science 271:994-996. 37. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM. (1996) Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632-635. 38. Yamamoto Y, Ueta Y, Serino R, Nomura M, Shibuya I, Yamashita H (2000) Effects of food restriction on the hypothalamic prepro-orexin gene expression in genetically obese mice. Brain Res Bull 51:515-521. 39. Yamamoto Y, Ueta Y, Yamashita H, Asayama K, Shirahata A (2002) Expressions of the prepro-orexin and orexin type 2 receptor genes in obese rat. Peptides 23:1689-1696.

7 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

40. Nara-ashizawa N, Tsukada T, Maruyama K, Akiyama Y, Kajimura N, Nagasaki K, Iwanaga T, Yamaguchi K (2001) Hypothalamic appetite-regulating neuropeptide mRNA levels in cachectic nude mice bearing human tumor cells. Metabolism 50(10):1213-1219. 41. Jensen PB, Blume N, Mikkelsen JD, Larsen PJ, Jensen HI, Holst JJ, Madsen OD (1998) Transplantable rat glucagonomas cause acute onset of severe anorexia and adipsia despite highly elevated NPY mRNA levels in the hypothalamic arcuate nucleus. J Clin Invest 101:503-510. 42. Chance WT, Sheriff S, Kasckow JW, Regmi A, Balasubramaniam A (1998) NPY messenger RNA is increased in medial hypothalamus of anorectic tumor-bearing rats. Regul Pept 75-76:347-353. 43. Chen B, Taylor S, Tisdale MJ, Williams G (2001) Cachexia in MAC16 adenocarcinoma: suppression of hunger despite normal regulation of leptin, and hypothalamic neuropeptide Y. J Neurochem 79:1004-1012. 44. McCarthy HD, McKibbin PE, Perkins AV, Linton EA, Williams G (1993) Alterations in hypothalamic NPY and CRF in anorexic tumor-bearing rats. Am J Physiol 264:E638-643. 45. Nara-ashizawa N, Tsukada T, Maruyama K, Akiyama Y, Kajimura N, Yamaguchi K (2001) Response of hypothalamic NPY mRNAs to a negative energy balance is less sensitive in cachectic mice bearing human tumor cells. Nutr Cancer 41:111-118. 46. Chance WT, Sheriff S, Dayal R, Balasubramaniam A (2003) Refractory hypothalamic alpha-MSH satiety and AGRP feeding systems in rats bearing MCA sarcomas. Peptides 24:1909-1919. 47. Jensen PB, Kristensen P, Clausen JT, Judge ME, Hastrup S, Thim L, Wulff BS, Fogeg C, Jensen J, Holst JJ, Madsen OD (1999) The hypothalamic satiety peptide CART is expressed in anorectic and non-anorectic pancreatic islet tumors and in the normal islet of Langerhans. FEBS Lett 447:139-143. 48. Grill V, Rankin W, Martin TJ (1998) Parathyroid hormone-related protein (PTHrP) and hypercalcaemia. Eur J Cancer 34: 222-229. 49. Suva LJ, Winslow GA, Wettenhall RE, Hammonds RG, Moseley JM, Diefenbach Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI (1987) A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science 237: 893-896. 50. Kukreja SC, Shevrin DH, Wimbiscus SA, Ebeling PR, Danks JA, Rodda CP, Wood WI, Martin TJ (1988) Antibodies to parathyroid hormone-related protein lower serum calcium in athymic mouse models of malignancy-associated hypercalcemia due to human tumors. J Clin Invest 82: 1798-1802. 51. Sato K, Yamakawa Y, Shizume K, Satoh T, Nohtomi K, Demura H, Akatsu T, Nagata N, Kasahara T, Ohkawa H, Ohsumi K (1993) Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors. J Bone Miner Res 8: 849-860. 52. Iguchi H, Onuma E, Sato K, Sato K, Ogata E (2001) Involvement of parathyroid hormone-related protein in experimental cachexia induced by a human lung cancer-derived cell line established from a bone metastasis specimen. Int J Cancer 94: 24-27. 53. Onuma E, Sato K, Sato H, Tsunenari T, Ishii K, Esaki K, Yabuta N, Wakahara Y, Yamada-Okabe H, Ogata E (2004) Generation of a humanized monoclonal antibody against human parathyroid hormone-related protein and its efficacy against humoral hypercalcemia of malignancy. Anticancer Res 24: 2665-2674. 54. Iguchi H, Onuma E, Sato K, Ogata E (2001) Involvement of parathyroid hormone-related protein in experimental cachexia induced by a human lung cancer-derived cell line established from a bone metastasis specimen. Int J Cancer 94:24-27. 55. Onuma E, Sato K, Saito H, Ysunenari T, Ishii K, Esaki K, Yabuta N, Wakahara Y, Yamada-Okabe H, Ogata E. (2004) Generation of a humanized monoclonal antibody against human parathyroid hormone-related protein and its efficacy against humoral hypercalcemia of malignancy. Anticancer Res 24:2665-2673.

8 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

56. Hshimoto H, Azuma Y, Kawasaki M, Fujihara H, Onuma E, Yamada-Okabe H, Takuwa Y, Ogata E, Ueta Y (2007) Parathyroid hormone-related protein induces cachectic syndromes without directly modulating the expression of hypothalamic feeding-regulating peptides. Clin Cancer Res 13:292-298. 57. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG (1996) Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101-1106. 58. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG (1997) Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119-2123. 59. Mizuno TM, Mobbs CV (1999) Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology 140:814-817. 60. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA, Elshourbagy NA, Bergsma DJ, Yanagisawa M (1998) Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92:573-585. 61. Qu D, Ludwig DS, Gammeltoft S, Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Przypek R, Kanarek R, Maratos-Flier E (1996) A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380:243-247. 62. Weaver DR, Deeds JD, Lee K, Segre GV. (1995) Localization of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor mRNAs in rat brain. Mol Brain Res 28:296-310.

9 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

Fig. 1. Neuropeptides in dehydration-induced anorexia

Fig. 2. Neuropeptides in lipopolysaccharide (LPS)-induced anorexia

10 Endocr. J./ Y. Ueta et al.: NEUROPEPTIDE ROLE IN ANOREXIA-CACHEXIA doi: 10.1507/endocrj.KR-111

Fig. 3. Neuropeptides in cancer-induced anorexia-cachexia

Fig. 4. Neuropeptides in parathyroid hormone-related protein (PTHrP) tumor-induced anorexia-cachexia

11