Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men.

CONTEXT Ghrelin, an acylated peptide hormone secreted from the gut, regulates appetite and metabolism. Elucidating its pattern of secretion in the fed and fasted states is important in the face of the obesity epidemic. OBJECTIVE Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms. DESIGN Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast. SETTING The study was conducted at the University Hospital General Clinical Research Center. PARTICIPANTS Eight male volunteers participated, mean +/- sd age 24.5 +/- 3.7 yr and body mass index 24 +/- 2.1 kg/m(2). MAIN OUTCOME MEASURES Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting. RESULTS In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged. CONCLUSIONS Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.

[1]  N. Grishin,et al.  Identification of the Acyltransferase that Octanoylates Ghrelin, an Appetite-Stimulating Peptide Hormone , 2008, Cell.

[2]  C. Delporte,et al.  Ghrelin interacts with human plasma lipoproteins. , 2007, Endocrinology.

[3]  Xiaorong Zhu,et al.  On the Processing of Proghrelin to Ghrelin* , 2006, Journal of Biological Chemistry.

[4]  E. Ghigo,et al.  Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin releasing factor 2 receptor , 2006, Molecular and Cellular Endocrinology.

[5]  A. Yamanaka,et al.  Des-acyl ghrelin induces food intake by a mechanism independent of the growth hormone secretagogue receptor. , 2006, Endocrinology.

[6]  S. Riedl,et al.  Spontaneous 24-h ghrelin secretion pattern in fasting subjects: maintenance of a meal-related pattern. , 2005, European journal of endocrinology.

[7]  K. Mori,et al.  Ingested medium-chain fatty acids are directly utilized for the acyl modification of ghrelin. , 2005, Endocrinology.

[8]  S. Bloom,et al.  Characterization of ghrelin-like immunoreactivity in human plasma. , 2005, The Journal of clinical endocrinology and metabolism.

[9]  H. Orskov,et al.  Fasting unmasks a strong inverse association between ghrelin and cortisol in serum: studies in obese and normal-weight subjects. , 2005, The Journal of clinical endocrinology and metabolism.

[10]  S. Koizumi,et al.  Lysophospholipase I identified as a ghrelin deacylation enzyme in rat stomach. , 2004, Biochemical and biophysical research communications.

[11]  C. Delporte,et al.  Ghrelin degradation by serum and tissue homogenates: identification of the cleavage sites. , 2004, Endocrinology.

[12]  H. Katakami,et al.  Plasma levels of intact and degraded ghrelin and their responses to glucose infusion in anorexia nervosa. , 2004, The Journal of clinical endocrinology and metabolism.

[13]  E. Ghigo,et al.  Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. , 2004, The Journal of clinical endocrinology and metabolism.

[14]  D. Taub,et al.  Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. , 2004, The Journal of clinical investigation.

[15]  K. Doi,et al.  Optimum collection and storage conditions for ghrelin measurements: octanoyl modification of ghrelin is rapidly hydrolyzed to desacyl ghrelin in blood samples. , 2004, Clinical chemistry.

[16]  K. Nin,et al.  Short-term secretory regulation of the active form of ghrelin and total ghrelin during an oral glucose tolerance test in patients with anorexia nervosa. , 2004, European journal of endocrinology.

[17]  D. Malan,et al.  Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln14-ghrelin. , 2003, European journal of pharmacology.

[18]  Min-Seon Kim,et al.  Changes in ghrelin and ghrelin receptor expression according to feeding status , 2003, Neuroreport.

[19]  P. Bouloux,et al.  Ghrelin Can Bind to a Species of High Density Lipoprotein Associated with Paraoxonase* , 2003, The Journal of Biological Chemistry.

[20]  Ki-Choon Choi,et al.  The role of ghrelin and growth hormone secretagogues receptor on rat adipogenesis. , 2003, Endocrinology.

[21]  K. Nakao,et al.  Plasma ghrelin and desacyl ghrelin concentrations in renal failure. , 2002, Journal of the American Society of Nephrology : JASN.

[22]  G. Frost,et al.  Ghrelin enhances appetite and increases food intake in humans. , 2001, The Journal of clinical endocrinology and metabolism.

[23]  B. Wisse,et al.  A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. , 2001, Diabetes.

[24]  M. Tschöp,et al.  Post-prandial decrease of circulating human ghrelin levels , 2001, Journal of endocrinological investigation.

[25]  M. Papotti,et al.  Identification, characterization, and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinomas and cell lines. , 2001, The Journal of clinical endocrinology and metabolism.

[26]  K. Toshinai,et al.  Upregulation of Ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration. , 2001, Biochemical and biophysical research communications.

[27]  M. Nakazato,et al.  A role for ghrelin in the central regulation of feeding , 2001, Nature.

[28]  K. Kangawa,et al.  Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. , 2000, Biochemical and biophysical research communications.

[29]  K. Nakao,et al.  Ghrelin strongly stimulates growth hormone release in humans. , 2000, The Journal of clinical endocrinology and metabolism.

[30]  M. Nakazato,et al.  Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. , 2000, Endocrinology.

[31]  S. Bloom,et al.  The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. , 2000, Endocrinology.

[32]  M. Bednarek,et al.  Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. , 2000, Journal of medicinal chemistry.

[33]  M. Tschöp,et al.  Ghrelin induces adiposity in rodents , 2000, Nature.

[34]  M. Nakazato,et al.  Ghrelin is a growth-hormone-releasing acylated peptide from stomach , 1999, Nature.

[35]  Brennan,et al.  Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia. , 1987, Clinical chemistry.

[36]  K. Courtney,et al.  A new and rapid colorimetric determination of acetylcholinesterase activity. , 1961, Biochemical pharmacology.

[37]  J. Skepper,et al.  Functional and immunocytochemical evidence for a role of ghrelin and des-octanoyl ghrelin in the regulation of vascular tone in man. , 2006, Cardiovascular research.

[38]  T. Akamizu,et al.  Separate measurement of plasma levels of acylated and desacyl ghrelin in healthy subjects using a new direct ELISA assay. , 2005, The Journal of clinical endocrinology and metabolism.

[39]  J. Chan,et al.  Ghrelin levels are not regulated by recombinant leptin administration and/or three days of fasting in healthy subjects. , 2004, The Journal of clinical endocrinology and metabolism.

[40]  I. Robinson,et al.  Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. , 2004, Endocrinology.

[41]  F. A. Neto,et al.  Evaluation of the Comparability of Commercial Ghrelin Assays , 2004 .