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GELTRUDE MINGRONE. Ghrelin does not influence gastric emptying in obese subjects. Obes Res. 2005;13: 739–744. Objective: To evaluate the relationship ...
Ghrelin Does Not Influence Gastric Emptying in Obese Subjects Maria E. Valera Mora,* Antonino Scarfone,† Venanzio Valenza,* Menotti Calvani,* Aldo V. Greco,* Giovanni Gasbarrini,* and Geltrude Mingrone*

Abstract VALERA MORA, MARIA E., ANTONINO SCARFONE, VENANZIO VALENZA, MENOTTI CALVANI, ALDO V. GRECO, GIOVANNI GASBARRINI, AND GELTRUDE MINGRONE. Ghrelin does not influence gastric emptying in obese subjects. Obes Res. 2005;13: 739 –744. Objective: To evaluate the relationship between fasting plasma concentrations of ghrelin and gastric emptying in obese individuals compared with lean subjects. Research Methods and Procedures: We included 20 obese patients (9 men and 11 women, BMI ⬎ 30 kg/m2) and 16 nonobese control subjects (7 men and 9 women, BMI ⱕ 25 kg/m2). Gastric emptying of solids (egg sandwich labeled with radionuclide) was measured at 120 minutes with (99m)Tc-single photon emission computed tomography imaging. Ghrelin and leptin were analyzed by radioimmunoassay and ELISA methods, respectively. Results: The gastric half-emptying time was similar in obese men and women (67.8 ⫾ 14.79 vs. 66.6 ⫾ 13.56 minutes) but significantly shorter (p ⬍ 0.001) than in the control population (men: 88.09 ⫾ 11.72 minutes; women: 97.25 ⫾ 10.31 minutes). Ghrelin levels were significantly lower in obese subjects (131.37 ⫾ 47.67 vs. 306.3 ⫾ 45.52 pg/mL; p ⬍ 0.0001 in men and 162.13 ⫾ 32.95 vs. 272.8 ⫾ 47.77 pg/mL; p ⬍ 0.0001 in women). A negative correlation between gastric emptying and fasting ghrelin levels was observed only in lean subjects (y ⫽ ⫺0.2391x ⫹ 157.9; R2 ⫽ 0.95). Also, in the lean group, ghrelin was the only significant independent determinant of gastric emptying,

Received for review September 16, 2004. Accepted in final form February 10, 2005. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. *Department of Internal Medicine and †Department of Radiology, Catholic University of Rome, Rome, Italy. Address correspondence to Geltrude Mingrone, Dipartimento di Medicina Interna, Catholic University, Largo A. Gemelli, 8, 00168 Rome, Italy. E-mail: [email protected] Copyright © 2005 NAASO

explaining 98% of the variance (adjusted R2) in a multiple regression analysis. Discussion: This report shows that, in humans, gastric emptying is faster in obese subjects than in lean controls and that, whereas ghrelin is the best determinant of gastric kinetics in healthy controls, this action is lost in obesity. Key words: gastric half-emptying time, gastric scintigraphy, leptin, ghrelin

Introduction Ghrelin is a 28 amino acid peptide produced predominantly in the stomach by the enteroendocrine cellular system and, in particular, by the X/A-like cells that represent a major endocrine population in the oxyntic mucosa. Lower amounts of this hormone derive from bowel, pancreas, kidney, the immune system, placenta, pituitary, testis, ovary, and hypothalamus (1). Ghrelin strongly stimulates growth hormone (GH)1 release through the activation of the GH secretagogue receptor type 1a. Ghrelin secretion is finely regulated by several factors. During fasting, during malnutrition, and in anorexia nervosa, circulating ghrelin levels are increased, likely through stimulation of arcuate nucleus neurons by the action of two orexigenic peptides, neuropeptide Y (NPY) and agoutirelated protein (2,3). An opposite, inhibitory effect on NPY/agouti-related protein neurons is exerted by both leptin and insulin (4,5). In obesity, fasting plasma ghrelin levels are significantly lower than in lean subjects and correlate negatively with BMI, percent body fat, and/or fasting insulin and leptin concentrations (6). Ghrelin and leptin exert antagonistic activities on gastrointestinal functions; in fact, whereas ghrelin increases gastric acid secretion, motility, and emptying, leptin has an inhibitory effect.

1

Nonstandard abbreviations: GH, growth hormone; NPY, neuropeptide Y; t1/2, gastric half-emptying time; GLP-1, glucagon-like peptide-1.

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Gastric leptin and ghrelin are involved in the hypothalamic regulation of food intake, gastrointestinal function, metabolic control, and growth regulation, although it is not yet clear how they interact in the stomach. Ghrelin is a potent gastro-prokinetic factor that accelerates the normal emptying mechanism in rats and mice with doses compatible with those required to stimulate appetite and GH release (4). An enhanced gastrointestinal transit might reduce the satiating effect of food and, thereby, promote obesity. Gastric emptying rate has previously been compared between obese and lean subjects with conflicting outcomes (7,8). Percentage of gastric emptying during the initial 30 minutes after a solid meal was found to be increased in obese men compared with lean individuals and was normalized after a major weight reduction (9). To our knowledge, no data have been reported in the literature concerning the relationship between ghrelin and leptin circulating levels and gastric emptying in obesity. The aim of this study was to evaluate the relationship between fasting plasma concentrations of ghrelin and leptin and gastric emptying in obese individuals compared with lean subjects using a radionuclide technique, which is currently considered the gold standard for the assessment of gastric motility in humans.

Table 1. Anthropometric and biochemical characteristics of the study population

Male Female Age (years) Men Women BMI (kg/m2) Men Women Body weight (kg) Men Women Plasma leptin (ng/mL) Men Women Plasma ghrelin (pg/mL) Men Women t1/2 (min) Men Women

Obese

Lean

9 11

7 9

45.88 ⫾ 7.37 43.33 ⫾ 13.94

38.97 ⫾ 15.29 41 ⫾ 4.84

38.10 ⫾ 3.60 35.90 ⫾ 1.71

23.44 ⫾ 1.4 21.69 ⫾ 1.99

111.22 ⫾ 14.11 97.44 ⫾ 5.45

57.09 ⫾ 24.18 58.11 ⫾ 5.22

61.06 ⫾ 15.86 81.17 ⫾ 9.53

4.84 ⫾ 3.91 17.91 ⫾ 12.16

131.37 ⫾ 47.67 306.33 ⫾ 45.5 162.13 ⫾ 32.95 272.88 ⫾ 47.7 67.88 ⫾ 14.79 66.63 ⫾ 13.56

97.25 ⫾ 10.3 88.09 ⫾ 11.7

Research Methods and Procedures Study Subjects The study group included 20 obese patients (9 men and 11 women, BMI ⬎ 30 kg/m2) and 16 nonobese control subjects (7 men and 9 women, BMI ⱕ 25 kg/m2). Obesity was defined as BMI ⬎30 kg/m2, according to the criteria of both the World Health Organization and the International Obesity Task Force. The BMI mean values of obese subjects were 38 ⫾ 3.6 kg/m2 for men and 35.9 ⫾ 1.7 kg/m2 for women, whereas in control subjects, BMI mean values were 21.7 ⫾ 1.9 kg/m2 for men and 23.4 ⫾ 1.4 kg/m2 for women (Table 1). All participants were between 35 and 50 years of age. None of the study participants had impaired glucose tolerance or diabetes according to an oral glucose tolerance test. They were not taking any medications. Medical histories, physical examinations, routine laboratory tests, and electrocardiograms showed that all patients were in good health. All were normotensive, with diastolic blood pressure ⬍85 mm Hg. This study was approved by the Ethical Committee of the Catholic University, and all subjects signed an informed consent document before participation. Gastric Scintigraphy After an overnight fast, subjects ate a test meal that consisted of two pieces of toasted white bread with 10 grams of margarine, two eggs to which 10 MBq of 99mTc 740

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colloid had been added before cooking in a microwave oven, and 150 mL water. The test meal was consumed within 10 minutes. Immediately after meal ingestion (usually considered as time 0), the subjects were asked to sit in front of a single head ␥-camera system capable of small and large field of view imaging and equipped with a low-energy collimator (General Electric 3200 StarCam i, Fairfield, CT), which was positioned on the entire abdominal region. Image acquisition began immediately, with the abdomen close to the collimator, at a rate of one frame every 60 seconds for 120 minutes. After the examination, regions of interest were manually drawn around the stomach in each image, and the counts were recorded and corrected for physical decay. Data were processed, and curves of gastric activity versus time were generated. A correction for radioactive decay was performed. The following parameters were calculated using established methods, as described by Nusynowitz and Benedetto (10): gastric half-emptying time (t1/2), defined as the time at which 50% of the radiolabeled material had left the stomach); lag-phase, defined as the time period from the end of the meal until 90% of radioactivity remained still in the stomach; and residual activity at 120 minutes, defined as the percentage of radioactivity remaining in the stomach at 120 minutes.

Ghrelin and Gastric Emptying in Obese Subjects, Valera Mora et al.

Analytical Assays Fasting plasma samples were collected in tubes in an ice bath and frozen immediately at ⫺80 °C. Plasma glucose was measured by the glucose oxidase method (Beckman, Fullerton, CA). Leptin was measured using an ELISA human kit (Diagnostic Systems Laboratories, Webster, TX). Ghrelin was measured using a ghrelin (Active) radioimmunoassay kit (Linco Research, St. Charles, MO). The intraassay and interassay coefficients of variation for the high control were 2.65% and 16.2%, respectively; the intraassay and interassay coefficients of variation for the low control were 1.15% and 15.1%, respectively. The sensitivity of the ghrelin assay was 2.98 pM. Statistical Analysis Data are given as means ⫾ SD. Comparison between groups was made by the Bonferroni adjusted t test. Multiple linear regression analysis was used to identify predictors of gastric emptying; the coefficient of determination (R2) was used as a measure of goodness of fit of the generated equation. A two-tailed p value ⬍0.05 was considered statistically significant.

Results The circulating ghrelin level in the group of obese men was 131.37 ⫾ 47.67 pg/mL, corresponding to ⬃50% of the average values found in lean male controls (306.3 ⫾ 45.52 pg/mL; p ⬍ 0.0001). Obese women had a mean ghrelin plasma concentration of 162.13 ⫾ 32.95 pg/mL, which was 59% lower than in the control group (272.8 ⫾ 47.77 pg/mL; p ⬍ 0.0001). Because no sexual dimorphism in the circulating levels of ghrelin was found in the population studied, data from men and women were pooled together in each group. Mean circulating levels of leptin in obese men and women were 61.06 ⫾ 15.86 and 81.17 ⫾ 9.53 ng/mL, respectively, whereas a highly statistically significant difference was found in lean control subjects (4.84 ⫾ 3.91 ng/mL in men vs. 17.91 ⫾ 12.16 ng/mL in women; p ⬍ 0.0001). t1/2 was similar in obese men and women (67.8 ⫾ 14.79 vs. 66.6 ⫾ 13.56 minutes, respectively) but was significantly shorter (p ⬍ 0.001) than in the control population (men: 88.09 ⫾ 11.72 minutes; women: 97.25 ⫾ 10.31 minutes). The lag phase was similar in obese men and women (11.4 ⫾ 5.7 vs. 12.3 ⫾ 3.4 minutes, respectively) but shorter (p ⬍ 0.001) than in the control group (men: 28 ⫾ 5.4 minutes; women: 25.2 ⫾ 5.9 minutes). Furthermore, the percentage of residual activity at 120 minutes was similar in obese men and women (20.31 ⫾ 10.84% and 21.38 ⫾ 11.47%, respectively), but it was reduced (p ⬍ 0.001) compared with control subjects (men: 41.75 ⫾ 5.68%; women: 35.36 ⫾ 7.56%).

Figure 1: Correlation between BMI and ghrelin in the obese group.

Only in the obese group did BMI inversely (p ⬍ 0.001) correlate with the level of plasma ghrelin (y ⫽ ⫺9.0212x ⫹ 482.63; R2 ⫽ 0.37; Figure 1). A negative correlation (Figure 2) between gastric emptying and fasting ghrelin levels was observed in lean subjects (y ⫽ 0.2391x ⫹ 157.9; R2 ⫽ 0.95), whereas no significant correlation was found in the obese group (y ⫽ 1 ⫻ 10⫺5x ⫹ 0.00,138; R2 ⫽ 0.03). A typical scintigraphic curve of gastric emptying in an obese subject is shown in Figure 3. In a multiple regression analysis, including sex, age, BMI, body weight, leptin, and ghrelin as determinants, ghrelin was the only significant independent determinant of t1/2, explaining 98% of the variance (adjusted R2), but this was only in the control group. Table 2 reports the univariate regressions between ghrelin and age, BMI, body weight, and leptin in controls.

Discussion The principal findings of this study are that obese subjects experience more rapid gastric emptying than lean controls; however, whereas in controls the gastric kinetics seem to be regulated by ghrelin, in obese individuals, this mechanism seems to be lost. The average reduction of t1/2 in obese subjects of both sexes was ⬃27% of the value found in controls, possibly inducing a reduced satiating sensation and, thus, increasing food consumption.

Figure 2: Correlation between ghrelin and t1/2 in lean subjects.

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Figure 3: (Top) Selection of the regions of interest over the stomach. (Bottom left) A typical curve of gastric activity versus time in an obese subject. (Bottom right) Functional parameters (lag-phase, t1/2, residual activity at 120 minutes).

Ghrelin showed a significant negative correlation with t1/2, suggesting a direct influence of ghrelin on gastric emptying; this result was also supported by a multivariate analysis showing that the best determinant of gastric emptying was circulating ghrelin levels. However, this relationship was not found in the obese group, indicating that different mechanisms might intervene in the control of gastric kinetics in obesity.

It has been previously shown (11) that accelerated gastric emptying in obese subjects is an important determinant of the reduction of the satiating effect of food, leading to an increased food consumption. However, no data are reported in the literature concerning the possible mechanisms inducing the accelerated emptying of the stomach in humans. Several peptides synthesized and secreted by the gastrointestinal tract are known to regulate food intake. Ghrelin,

Table 2. Univariate regressions between ghrelin and age, body weight, BMI, and leptin

Ghrelin Ghrelin Ghrelin Ghrelin

vs. vs. vs. vs.

BMI body weight leptin age

B



p

t

R2

⫺9.967 ⫾ 5.456 0.082 ⫾ 2.522 0.184 ⫾ 1.043 0.067 ⫾ 2.528

⫺0.520 0.011 0.059 0.009

NS NS NS NS

⫺1.827 0.033 0.177 0.026

0.27 0.00 0.00 0.00

NS, not significant.

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the first identified peripherally active orexigenic factor, acts by increasing hunger, whereas peptide YY, pancreatic polypeptide, and NPY, all belonging to a peptide family known as the PP-fold peptides, induce satiety. Glucagonlike peptide-1 (GLP-1) in humans inhibits gastrointestinal motility and decreases hunger and energy intake (12). Cummings et al. (13) showed that the circulating levels of ghrelin rise preprandially and fall postprandially in healthy subjects, suggesting a role of this hormone in meal initiation in humans. Twenty-four-hour profiles of circulating ghrelin in patients with simple obesity and anorexia nervosa consistently showed lower and higher levels, respectively, compared with healthy, normal-weight subjects (14). On the basis of the above reported data, Shiiya et al. (14) advanced the hypothesis that nutritional state is a determinant of plasma ghrelin in humans and that ghrelin secretion is up-regulated under conditions of negative energy balance and down-regulated in the setting of positive energy balance. Weight loss promotes ghrelin secretion in obese individuals (15,16). The decrease of ghrelin secretion in obesity has been considered a physiological adaptation to long-term positive energy balance. Furthermore, obese subjects do not exhibit the decline in plasma ghrelin observed after a test meal in lean subjects. The lack of suppression after a meal in obese subjects could lead to increased food consumption and suggests that ghrelin may be involved in the pathophysiology of obesity (17). A correlation between food intake and gastrointestinal fed and fasted motor activities has been recently shown in rodents (18). This action is mediated by NPY, a hormone that is able to change gastroduodenal motor activity from fed to fasted conditions through the action of Y2 receptors (19) and that is involved in the inhibition of gastric emptying of solid and liquid meals (20). NPY seems to be the link between ghrelin and gastric kinetics. In obesity, ghrelin levels are significantly lower than in lean subjects and correlate negatively with BMI, percent body fat, and/or fasting insulin and leptin concentrations (6). Accordingly, we have shown a negative relationship between ghrelin and BMI in our series (Figure 1). Leptin has opposite effects from ghrelin (21). However, in our study, leptin did not significantly affect gastric emptying in obese and lean subjects. Among the hormones that are involved in the regulation of gastric motility, the gastric inhibitory peptide results in a decrease of acid gastric secretion, whereas GLP-1 allows a decrease of both gastric acid secretion and emptying through the inhibitory action of vagal nerve activity (22). Because gastric inhibitory peptide and GLP-1 responses to a mixed meal are impaired in obesity (23,24), the augmented gastric emptying observed in our series might be ascribed to a reduced inhibition of gastrointestinal motility. In conclusion, this report in humans shows that gastric emptying—assessed by the gold standard technique of gas-

tric scintigraphy—in obese subjects is significantly accelerated compared with lean controls and that, whereas ghrelin is the best determinant of gastric kinetics in healthy controls, this action is lost in obesity.

Acknowledgment This study was supported by Miur, Rome, Italy. References 1. Broglio F, Gottero C, Arvat E, et al. Endocrine and nonendocrine actions of ghrelin. Horm Res. 2003;59:109 –17. 2. Kamegai J, Tamura H, Shimizu T, et al. Chronic central infusion of ghrelin increases hypothalamic neuropeptide Y and Agouti-related protein mRNA levels and body weight in rats. Diabetes. 2001;50:2438 – 43. 3. Williams G, Bing C, Cai XJ, et al. The hypothalamus and the control of energy homeostasis: different circuits, different purposes. Physiol Behav. 2001;74:683–701. 4. Inui A, Asakawa A, Bowers CY, et al. Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ. FASEB J. 2004;18:439 –56. 5. Wren AM, Seal LJ, Cohen MA, et al. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab. 2001;86:5992–5. 6. Tschop M, Weyer C, Tataranni PA, et al. Circulating ghrelin levels are decreased in human obesity. Diabetes. 2001; 50:707–9. 7. Wright RA, Krinsky S, Fleeman C, et al. Gastric emptying and obesity. Gastroenterology. 1983;84:747–51. 8. Jackson SJ, Leahy FE, McGowan AA, et al. Delayed gastric emptying in the obese: an assessment using the non-invasive 13 C-octanoic acid breath test. Diabetes Obes Metab. 2004;6: 264 –70. 9. Horowitz M, Collins PJ, Cook DJ, et al. Abnormalities of gastric emptying in obese patients. Int J Obes Relat Metab Disord. 1983;7:415–21. 10. Nusynowitz ML, Benedetto AR. The lag phase of gastric emptying: clinical, mathematical and in vitro studies. J Nucl Med. 1994;35:1023–7. 11. Sasaki H, Nagulesparan M, Dubois A, et al. Gastric function and obesity: gastric emptying, gastric acid secretion, and plasma pepsinogen. Int J Obes Relat Metab Disord. 1984;8: 183–90. 12. Flint A, Raben A, Ersboll AK, et al. The effect of physiological levels of glucagon-like peptide-1 on appetite, gastric emptying, energy and substrate metabolism in obesity. Int J Obes Relat Metab Disord. 2001;25:781–92. 13. Cummings DE, Purnell JQ, Frayo RS, et al. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes. 2001;50:1714 –9. 14. Shiiya T, Nakazato M, Mizuta M, et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab. 2002;87: 240 – 4. 15. Hansen TK, Dall R, Hosoda H, et al. Weight loss increases circulating levels of ghrelin in human obesity. Clin Endocrinol. 2002;56:203– 6. OBESITY RESEARCH Vol. 13 No. 4 April 2005

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16. Cummings DE, Weigle DS, Frayo RS, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med. 2002;346:1623–30. 17. English PJ, Ghatei MA, Malik IA, et al. Food fails to suppress ghrelin levels in obese humans. J Clin Endocrinol Metab. 2002;87:2984 –7. 18. Fujimiya M, Inui A. Peptidergic regulation of gastrointestinal motility in rodents. Peptides. 2000;21:1565– 82. 19. Fujimiya M, Itoh E, Kilhara N, et al. Neuropeptide Y induces fasted pattern of duodenal motility via Y2 receptors in conscious fed rats. Am J Physiol. 2000;278:G32– 8. 20. Matsuda M, Aono M, Moriga M, et al. Centrally administered neuropeptide Y (NPY) inhibits gastric emptying and intestinal transit in the rat. Dig Dis Sci 1993;38:845–50.

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21. Asakawa A, Inui A, Kaga T, et al. Ghrelin is an appetite stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology. 2001;120:337– 45. 22. Wettergren A, Schojoldager B, Mortensen PE, et al. Truncated GLP-1 (proglucagon 78 –107-amide) inhibits gastric and pancreatic functions in man. Dig Dis Sci. 1993; 38:665–73. 23. Lugari R, Dei Cas A, Ugolotti D, et al. Glucagon-like peptide 1 (GLP-1) secretion and plasma dipeptidyl peptidase IV (DPP-IV) activity in morbidly obese patients undergoing biliopancreatic diversion. Horm Metab Res. 2004;36:111–5. 24. Meier JJ, Nauck MA, Schmidt WE, et al. Gastric inhibitory polypeptide: the neglected incretin revisited. Regul Pept. 2002;107:1–13.