臺灣博碩士論文加值系統

　　飢餓素，是一種由胃部分泌的28胜肽激素，在多種能量代謝的恆定中，例如促進攝食以及脂肪代謝等皆具有調節的功能，而在動脈硬化進程中也具有潛在調節功能的地位。在動脈硬化的發生過程中，遷徙到血管內皮中的巨噬細胞，吞噬氧化低密度脂蛋白而後形成泡沫細胞，是相當重要的環節。近年來，飢餓素被發現對於內皮細胞具有抗發炎的效果，然而其對於泡沫細胞形成之過程中，所造成的影響及其機制仍尚未釐清。因此本篇研究使用RAW264.7巨噬細胞，探討了外加飢餓素對於巨噬細胞形成泡沫細胞過程中所扮演的角色。在實驗過程中，首先利用油紅染色法，初步瞭解飢餓素對於泡沫細胞產生抑制的影響，便接著探討其對於膽固醇釋出及氧化低密度脂蛋白攝入的作用。而後，利用西方墨點法及即時聚合酶鏈鎖反應，探討巨噬細胞表面ABCA1、ABCG1、SR-B1、CD36及SRA這些與膽固醇代謝相關蛋白的蛋白質和核糖核酸表現量。最後再利用相關的激酶抑制劑進一步探討飢餓素改變蛋白表現的作用機制。實驗結果顯示，飢餓素會抑制SRA蛋白質和核糖核酸表現，但對CD36沒有影響，SRA的表現量降低進一步使氧化低密度脂蛋白 (Ox-LDL) 攝入量降低；另一方面，飢餓素對ABCG1和SR-B1蛋白質和核糖核酸表現量沒有影響，但會增加ABCA1表現量，進一步增加膽固醇的釋出。氧化低密度脂蛋白攝入量的降低，以及膽固醇釋出量的增加使細胞內脂質含量減少，共同造成了抑制泡沫細胞形成的效果。接著在前處理PI3-Kinase及p38MAP-Kinase抑制劑的實驗中顯示，飢餓素使細胞增加ABCA1表現量的效果會被P38MAP-Kinase抑制劑所消彌，而降低SRA表現量的效果則會被PI3-Kinase抑制劑所回復。另外，在前處理轉錄抑制劑的實驗中，則發現飢餓素對於膽固醇代謝相關蛋白質之穩定性無顯著影響。根據以上實驗結果，我們認為飢餓素可透過活化p38MAP-Kinase和PI3-Kinase，影響核糖核酸表現，並分別造成巨噬細胞表面ABCA1蛋白增加、SRA蛋白減少，使膽固醇釋出增加、低密度脂蛋白攝入減少，抑制泡沫細胞形成並進一步抑制動脈硬化的發生。

Ghrelin, a 28-amino-acid peptide hormone and endogenous ligand of growth hormone secretagogue receptor (GHS-R), has diversity energy-metabolism-modulatory functions, such as orexigenic activity and regulation of lipid metabolism. Clinical reports demonstrated that decreased plasma ghrelin levels are associated with pathological obesity and the severity of coronary atherosclerosis. Pathogenesis of atherosclerosis is composed of multiple cellular events occurring within vascular wells, including endothelial cell dysfunction, macrophages infiltration, and foam cell formation. Recent studies demonstrated that ghrelin inhibits proinflammatory responses in human endothelial cells. However, the roles of ghrelin on macrophages were not well-clarified. The purpose of this study was to investigate the effects of ghrelin on foam cell formation in RAW264.7 macrophages. The effect of ghrelin on changes of ox-LDL uptake, cholesterol efflux, and foam cell formation were determined, and the results showed that ghrelin can suppress the foam cell formation, while decrease ox-LDL uptake and increase cholesterol efflux. The effects of ghrelin on expression of scavenger receptor and ATP binding cassette transporter were measured by Western blotting and real-time RT-PCR. Results of Western blotting and real-time RT-PCR showed that treatment of ghrelin increased expression level of ABCA1 and decreased expression level of SRA. To investigate the signaling pathways involved in ghrelin’s function, phosphorylation of several kinases in one-hour ghrelin treatment was measured. Then, PI3K inhibitor (LY294002) and p38MAPK inhibitor (SB203580) were used to define the possible pathways of ghrelin’s effects. Results showed that ghrelin’s positive effects on ABCA1 expression were repressed by SB203580, and its negative effects on SRA expression were reversed by LY294002. In summary, we discovered that ghrelin could increase protein expression level of ABCA1 through p38MAPK signaling pathway, and also decrease protein expression level of SRA through PI3K signaling pathway. The ghrelin mediated cholesterol metabolism related protein expression level change will lead to a decrease in macrophages oil droplets accumulation and further inhibit the foam cell formation.

目次
致謝........................................................i
目次......................................................iii
圖目次......................................................v
重要中英文名詞與縮寫.......................................vi
中文摘要..................................................vii
Abstract...................................................ix
第一章、前言................................................1
第二章、文獻回顧............................................2
一、動脈硬化................................................2
1. 動脈硬化的過程...........................................2
2. 泡沫細胞形成過程.........................................3
二、飢餓素..................................................3
1. 飢餓素的結構.............................................3
2. 飢餓素的生理作用.........................................4
3. 飢餓素與能量恆定.........................................5
4. 飢餓素與心血管疾病.......................................6
三、研究動機................................................7
第三章、實驗設計............................................8
實驗目的 (一)：Ghrelin對RAW264.7巨噬細胞形成泡沫細胞的影響..8
實驗目的 (二)：Ghrelin對RAW264.7巨噬細胞吞噬DiI-ox-LDL能力
的影響......................................................8
實驗目的 (三)：Ghrelin對RAW264.7巨噬細胞釋出膽固醇能力的影
響..........................................................8
實驗目的 (四)：不同劑量Ghrelin對RAW264.7巨噬細胞表面與膽固
醇代謝相關蛋白的影響........................................9
實驗目的 (五)：不同處理時間Ghrelin對RAW264.7巨噬細胞表面與
膽固醇代謝相關蛋白的影響....................................9
實驗目的 (六)：Ghrelin對RAW264.7巨噬細胞表面與膽固醇代謝相
關蛋白之mRNA表現的影響......................................9
實驗目的 (七)：可能參與Ghrelin作用之訊息傳遞路徑............9
實驗目的 (八)：Ghrelin影響RAW264.7巨噬細胞表面與膽固醇代謝
相關蛋白之訊息傳遞路徑.....................................10
實驗目的 (九)：Ghrelin對於RAW264.7巨噬細胞表面與膽固醇代謝
相關蛋白穩定性之影響.......................................10
第四章、材料與方法.........................................11
一、藥品及抗體來源.........................................11
二、實驗方法...............................................12
1. 細胞繼代 (Subculture)...................................12
2. 冷凍細胞 (Frozen cells).................................12
3. 解凍細胞 (Cells recovery)...............................12
4. 蛋白質樣本製備分析......................................13
5. 核糖核酸樣本製備分析....................................15
6. 細胞膽固醇釋出分析 (Cholesterol efflux).................16
7. 螢光標定氧化低密度脂蛋白汲取分析 (DiI-ox-LDL uptake)....16
8. 油紅染色法 (Oil-red O stain)............................17
9. 統計方法................................................17
第五章、實驗結果...........................................18
一、Ghrelin對RAW264.7巨噬細胞形成泡沫細胞的影響............18
二、Ghrelin對RAW264.7巨噬細胞吞噬DiI-ox-LDL能力的影響......18
三、Ghrelin對RAW264.7巨噬細胞釋出膽固醇能力的影響..........18
四、不同劑量Ghrelin對RAW264.7巨噬細胞表面與膽固醇代謝相關
蛋白的影響.................................................19
五、不同處理時間Ghrelin對RAW264.7巨噬細胞表面與膽固醇代謝
相關蛋白的影響.............................................19
六、Ghrelin對RAW264.7巨噬細胞表面與膽固醇代謝相關蛋白之RNA
表現的影響.................................................19
七、可能參與Ghrelin作用之訊息傳遞路徑......................20
八、Ghrelin影響RAW264.7巨噬細胞表面與膽固醇代謝相關蛋白之
訊息傳遞路徑...............................................20
九、Ghrelin對於RAW264.7巨噬細胞表面與膽固醇代謝相關蛋白穩
定性之影響.................................................21
第六章、討論...............................................23
第七章、結論...............................................26
參考文獻...................................................27
圖表與說明.................................................33

圖目次
圖一、Ghrelin對RAW264.7巨噬細胞形成泡沫細胞的影響..........33
圖二、Ghrelin對RAW264.7巨噬細胞吞噬DiI-ox-LDL能力的影響....35
圖三、Ghrelin對RAW264.7巨噬細胞釋出膽固醇能力的影響........36
圖四、不同劑量Ghrelin對RAW264.7巨噬細胞表面與釋出膽固醇相
關蛋白的影響...............................................37
圖五、不同劑量Ghrelin對RAW264.7巨噬細胞表面與吞噬脂蛋白相
關蛋白的影響...............................................38
圖六、不同處理時間Ghrelin對RAW264.7巨噬細胞表面釋出膽固醇
相關蛋白的影響.............................................39
圖七、不同處理時間Ghrelin對RAW264.7巨噬細胞表面吞噬脂蛋白
相關蛋白的影響.............................................40
圖八、Ghrelin對RAW264.7巨噬細胞表面與膽固醇代謝相關蛋白之
RNA表現的影響..............................................41
圖九、可能參與Ghrelin作用中的訊息傳遞路徑..................42
圖十、Ghrelin影響RAW264.7巨噬細胞表面與膽固醇代謝相關蛋白
之訊息傳遞路徑.............................................43
圖十一、Ghrelin對於RAW264.7巨噬細胞表面與膽固醇代謝相關蛋
白穩定性之影響.............................................45
圖十二、飢餓素影響RAW264.7泡沫細胞形成之示意圖.............46
附圖一、國人十大死因死亡率 (衛生福利部統計處2018-6-15).....47
附圖二、十年國人十大死因比較圖 (衛生福利部統計處2018-6-15).47
附表一、抗體列表...........................................48

1. Baldanzi G, Filigheddu N, Cutrupi S, Catapano F, Bonissoni S, Fubini A, Malan D, Baj G, Granata R, Broglio F, Papotti M, Surico N, Bussolino F, Isgaard J, Deghenghi R, Sinigaglia F, Prat M, Muccioli G, Ghigo E, and Graziani A. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. J Cell Biol 159: 1029-37, 2002.
2. Bednarek MA, Feighner SD, Pong SS, McKee KK, Hreniuk DL, Silva MV, Warren VA, Howard AD, Van Der Ploeg LH, and Heck JV. Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem 43: 4370-6, 2000.
3. Bewick GA, Kent A, Campbell D, Patterson M, Ghatei MA, Bloom SR, and Gardiner JV. Mice with hyperghrelinemia are hyperphagic and glucose intolerant and have reduced leptin sensitivity. Diabetes 58: 840-6, 2009.
4. Chen C-Y, Fujimiya M, Asakawa A, Lee S-D, and Inui A. Regulation of Ingestive Behavior, the Upper Gastrointestinal Motility and Gastric Acid Secretion by Ghrelin in Mammals. Current Nutrition & Food Science 4: 32-40, 2008.
5. Chen CY, Asakawa A, Fujimiya M, Lee SD, and Inui A. Ghrelin gene products and the regulation of food intake and gut motility. Pharmacol Rev 61: 430-81, 2009.
6. Cheng B, Wan J, Wang Y, Mei C, Liu W, Ke L, and He P. Ghrelin inhibits foam cell formation via simultaneously down-regulating the expression of acyl-coenzyme A:cholesterol acyltransferase 1 and up-regulating adenosine triphosphate-binding cassette transporter A1. Cardiovasc Pathol 19: e159-66, 2010.
7. Choi K, Roh SG, Hong YH, Shrestha YB, Hishikawa D, Chen C, Kojima M, Kangawa K, and Sasaki S. The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis. Endocrinology 144: 754-9, 2003.
8. Cui C, Ohnuma H, Daimon M, Susa S, Yamaguchi H, Kameda W, Jimbu Y, Oizumi T, and Kato T. Ghrelin infused into the portal vein inhibits glucose-stimulated insulin secretion in Wistar rats. Peptides 29: 1241-6, 2008.
9. Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura S, Kangawa K, and Nakazato M. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141: 4255-61, 2000.
10. De Smet B, Depoortere I, Moechars D, Swennen Q, Moreaux B, Cryns K, Tack J, Buyse J, Coulie B, and Peeters TL. Energy homeostasis and gastric emptying in ghrelin knockout mice. J Pharmacol Exp Ther 316: 431-9, 2006.
11. Demers A, Caron V, Rodrigue-Way A, Wahli W, Ong H, and Tremblay A. A concerted kinase interplay identifies PPARgamma as a molecular target of ghrelin signaling in macrophages. PLoS One 4: e7728, 2009.
12. Endemann G, Stanton LW, Madden KS, Bryant CM, White RT, and Protter AA. CD36 is a receptor for oxidized low density lipoprotein. J Biol Chem 268: 11811-6, 1993.
13. Fujino K, Inui A, Asakawa A, Kihara N, Fujimura M, and Fujimiya M. Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats. J Physiol 550: 227-40, 2003.
14. Fukuda H, Mizuta Y, Isomoto H, Takeshima F, Ohnita K, Ohba K, Omagari K, Taniyama K, and Kohno S. Ghrelin enhances gastric motility through direct stimulation of intrinsic neural pathways and capsaicin-sensitive afferent neurones in rats. Scand J Gastroenterol 39: 1209-14, 2004.
15. Glass CK and Witztum JL. Atherosclerosis. the road ahead. Cell 104: 503-16, 2001.
16. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, and Korbonits M. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 87: 2988, 2002.
17. Gutierrez JA, Solenberg PJ, Perkins DR, Willency JA, Knierman MD, Jin Z, Witcher DR, Luo S, Onyia JE, and Hale JE. Ghrelin octanoylation mediated by an orphan lipid transferase. Proceedings of the National Academy of Sciences 105: 6320-6325, 2008.
18. Hansson GK and Hermansson A. The immune system in atherosclerosis. Nat Immunol 12: 204-12, 2011.
19. Hashimoto H, Fujihara H, Kawasaki M, Saito T, Shibata M, Otsubo H, Takei Y, and Ueta Y. Centrally and peripherally administered ghrelin potently inhibits water intake in rats. Endocrinology 148: 1638-47, 2007.
20. Hattori N. Expression, regulation and biological actions of growth hormone (GH) and ghrelin in the immune system. Growth Horm IGF Res 19: 187-97, 2009.
21. Ji A, Meyer JM, Cai L, Akinmusire A, de Beer MC, Webb NR, and van der Westhuyzen DR. Scavenger receptor SR-BI in macrophage lipid metabolism. Atherosclerosis 217: 106-12, 2011.
22. Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, and Wakabayashi I. Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression. Endocrinology 141: 4797-800, 2000.
23. Kaplan M, Aviram M, and Hayek T. Oxidative stress and macrophage foam cell formation during diabetes mellitus-induced atherogenesis: role of insulin therapy. Pharmacol Ther 136: 175-85, 2012.
24. Kawczynska-Drozdz A, Olszanecki R, Jawien J, Brzozowski T, Pawlik WW, Korbut R, and Guzik TJ. Ghrelin inhibits vascular superoxide production in spontaneously hypertensive rats. Am J Hypertens 19: 764-7, 2006.
25. Kleinz MJ, Maguire JJ, Skepper JN, and Davenport AP. Functional and immunocytochemical evidence for a role of ghrelin and des-octanoyl ghrelin in the regulation of vascular tone in man. Cardiovasc Res 69: 227-35, 2006.
26. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, and Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656-60, 1999.
27. Konturek PC, Konturek JW, Czesnikiewicz-Guzik M, Brzozowski T, Sito E, and Konturek SJ. Neuro-hormonal control of food intake: basic mechanisms and clinical implications. J Physiol Pharmacol 56 Suppl 6: 5-25, 2005.
28. Korbonits M, Bustin SA, Kojima M, Jordan S, Adams EF, Lowe DG, Kangawa K, and Grossman AB. The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors. J Clin Endocrinol Metab 86: 881-7, 2001.
29. Levin F, Edholm T, Schmidt PT, Gryback P, Jacobsson H, Degerblad M, Hoybye C, Holst JJ, Rehfeld JF, Hellstrom PM, and Naslund E. Ghrelin stimulates gastric emptying and hunger in normal-weight humans. J Clin Endocrinol Metab 91: 3296-302, 2006.
30. Li AC and Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med 8: 1235-42, 2002.
31. Li B, Zeng M, He W, Huang X, Luo L, Zhang H, and Deng DY. Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/beta-catenin signaling and suppresses lung inflammation. Endocrinology 156: 203-17, 2015.
32. Li WG, Gavrila D, Liu X, Wang L, Gunnlaugsson S, Stoll LL, McCormick ML, Sigmund CD, Tang C, and Weintraub NL. Ghrelin inhibits proinflammatory responses and nuclear factor-kappaB activation in human endothelial cells. Circulation 109: 2221-6, 2004.
33. Libby P. Inflammation in atherosclerosis. Nature 420: 868-74, 2002.
34. Lusis AJ. Atherosclerosis. Nature 407: 233-41, 2000.
35. Min KJ, Cho KH, and Kwon TK. The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells. Cell Signal 24: 1215-21, 2012.
36. Moore KJ, Sheedy FJ, and Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nat Rev Immunol 13: 709-21, 2013.
37. Muccioli G, Pons N, Ghe C, Catapano F, Granata R, and Ghigo E. Ghrelin and des-acyl ghrelin both inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a growth hormone secretagogue receptor. Eur J Pharmacol 498: 27-35, 2004.
38. Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, and Chihara K. Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem 277: 5667-74, 2002.
39. Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, Hayashi Y, and Kangawa K. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol 280: R1483-7, 2001.
40. Okumura H, Nagaya N, Enomoto M, Nakagawa E, Oya H, and Kangawa K. Vasodilatory effect of ghrelin, an endogenous peptide from the stomach. J Cardiovasc Pharmacol 39: 779-83, 2002.
41. Oner-Iyidogan Y, Kocak H, Gurdol F, Oner P, Issever H, and Esin D. Circulating ghrelin levels in obese women: a possible association with hypertension. Scand J Clin Lab Invest 67: 568-76, 2007.
42. Park YM, S RK, J AM, and Silverstein RL. Insulin promotes macrophage foam cell formation: potential implications in diabetes-related atherosclerosis. Lab Invest 92: 1171-80, 2012.
43. Podrez EA, Febbraio M, Sheibani N, Schmitt D, Silverstein RL, Hajjar DP, Cohen PA, Frazier WA, Hoff HF, and Hazen SL. Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species. J Clin Invest 105: 1095-108, 2000.
44. Poykko SM, Kellokoski E, Ukkola O, Kauma H, Paivansalo M, Kesaniemi YA, and Horkko S. Plasma ghrelin concentrations are positively associated with carotid artery atherosclerosis in males. J Intern Med 260: 43-52, 2006.
45. Rak-Mardyla A and Gregoraszczuk EL. ERK 1/2 and PI-3 kinase pathways as a potential mechanism of ghrelin action on cell proliferation and apoptosis in the porcine ovarian follicular cells. J Physiol Pharmacol 61: 451-8, 2010.
46. Rodriguez A, Gomez-Ambrosi J, Catalan V, Gil MJ, Becerril S, Sainz N, Silva C, Salvador J, Colina I, and Fruhbeck G. Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes. Int J Obes (Lond) 33: 541-52, 2009.
47. Rosenson RS, Brewer HB, Jr., Davidson WS, Fayad ZA, Fuster V, Goldstein J, Hellerstein M, Jiang XC, Phillips MC, Rader DJ, Remaley AT, Rothblat GH, Tall AR, and Yvan-Charvet L. Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport. Circulation 125: 1905-19, 2012.
48. St-Pierre DH, Bastard JP, Coderre L, Brochu M, Karelis AD, Lavoie ME, Malita F, Fontaine J, Mignault D, Cianflone K, Imbeault P, Doucet E, and Rabasa-Lhoret R. Association of acylated ghrelin profiles with chronic inflammatory markers in overweight and obese postmenopausal women: a MONET study. Eur J Endocrinol 157: 419-26, 2007.
49. Sun N, Wang H, and Wang L. Ghrelin inhibits oxLDL-induced inflammation in RAW264.7 mouse macrophages through down-regulation of LOX-1 expression via NF-kappaB signaling pathway. Cell Mol Biol (Noisy-le-grand) 62: 57-61, 2016.
50. Sun Y, Asnicar M, Saha PK, Chan L, and Smith RG. Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice. Cell Metab 3: 379-86, 2006.
51. Tall AR. Cholesterol efflux pathways and other potential mechanisms involved in the athero-protective effect of high density lipoproteins. J Intern Med 263: 256-73, 2008.
52. Thompson NM, Gill DA, Davies R, Loveridge N, Houston PA, Robinson IC, and Wells T. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 145: 234-42, 2004.
53. Tian L, Luo N, Klein RL, Chung BH, Garvey WT, and Fu Y. Adiponectin reduces lipid accumulation in macrophage foam cells. Atherosclerosis 202: 152-61, 2009.
54. Tschop M, Smiley DL, and Heiman ML. Ghrelin induces adiposity in rodents. Nature 407: 908-13, 2000.
55. Wang L, Chen Q, Ke D, and Li G. Ghrelin inhibits atherosclerotic plaque angiogenesis and promotes plaque stability in a rabbit atherosclerotic model. Peptides 90: 17-26, 2017.
56. Waseem T, Duxbury M, Ito H, Ashley SW, and Robinson MK. Exogenous ghrelin modulates release of pro-inflammatory and anti-inflammatory cytokines in LPS-stimulated macrophages through distinct signaling pathways. Surgery 143: 334-42, 2008.
57. Weber C, Zernecke A, and Libby P. The multifaceted contributions of leukocyte subsets to atherosclerosis: lessons from mouse models. Nat Rev Immunol 8: 802-15, 2008.
58. Willesen MG, Kristensen P, and Romer J. Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology 70: 306-16, 1999.
59. Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, and Bloom SR. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86: 5992, 2001.
60. Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, and Bloom SR. Ghrelin Causes Hyperphagia and Obesity in Rats. Diabetes 50: 2540-2547, 2001.
61. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, and Bloom SR. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 141: 4325-8, 2000.
62. Xiang Y, Li Q, Li M, Wang W, Cui C, and Zhang J. Ghrelin inhibits AGEs-induced apoptosis in human endothelial cells involving ERK1/2 and PI3K/Akt pathways. Cell Biochem Funct 29: 149-55, 2011.
63. Yang J, Brown MS, Liang G, Grishin NV, and Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132: 387-96, 2008.
64. Yasuda T, Masaki T, Kakuma T, and Yoshimatsu H. Centrally administered ghrelin suppresses sympathetic nerve activity in brown adipose tissue of rats. Neurosci Lett 349: 75-8, 2003.
65. Zhang M, Fang W, Yuan F, Qu X, Liu H, Chen H, Yu Y, Zheng Z, and Shen Y. Plasma ghrelin levels are closely associated with stenosis severity and morphology of angiographically-detected coronary atherosclerosis in patients with coronary artery disease. Int J Cardiol 151: 122-3, 2011.
66. Zhang M, Fang WY, Yuan F, Qu XK, Liu H, Xu YJ, Chen H, Yu YF, Shen Y, and Zheng ZC. Plasma ghrelin levels are closely associated with severity and morphology of angiographically-detected coronary atherosclerosis in Chineses patients with diabetes mellitus. Acta Pharmacol Sin 33: 452-8, 2012.