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Department of Cardiovascular Diseases (Z.-F.G., X.Z., Y.-W.Q., J.-Q.H., S.-P.C.), Changhai Hospital, Second Military Medical University, Shanghai 200433, China; and Department of Endocrinology (Z.Z.), No. 411 Hospital of Chinese People’s Liberation Army, Shanghai 200081, China
Address all correspondence and requests for reprints to: Professor Xing Zheng, Department of Cardiovascular Diseases, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. E-mail: zhengxing57530{at}163.com.
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Abstract |
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Objective: We hypothesize that obesity might present with imbalance of circulating ghrelin and obestatin levels.
Participants and Setting: Sixteen obese (eight men, aged 58.8 ± 4.9 yr; eight women, aged 59.9 ± 9.6 yr) and 14 normal-weight individuals (seven men, aged 52.7 ± 5.9 yr; seven women, aged 56.1 ± 4.9 yr) were evaluated at the in-patient department of Changhai Hospital, Shanghai, China.
Main Outcome Measures: Total plasma ghrelin and obestatin levels, 1 h before and 2 h after breakfast, were measured by RIA.
Results: Both preprandial plasma ghrelin levels (P < 0.01) and obestatin levels (P < 0.01) were lower in the obese compared with normal-weight controls. However, unexpectedly, the ratio of preprandial ghrelin to obestatin was higher in obese compared with normal-weight controls (P < 0.01) even after adjustment for gender and age (P < 0.01). The ratio of postprandial ghrelin to obestatin was decreased both in obese (P < 0.05) and controls (P < 0.01) compared with their preprandial levels. There were no significant differences in the ratio of postprandial ghrelin to obestatin between obese and normal-weight controls. Body mass index was positively correlated with and was a significantly independent determinant of the preprandial ghrelin to obestatin ratio.
Conclusion: Circulating preprandial ghrelin to obestatin ratio is elevated in human obesity. We suggest that high preprandial ghrelin to obestatin ratio may be involved in the etiology and pathophysiology of obesity.
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Introduction |
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Contrary to the hypothesis, however, recent studies show that preprandial (4, 6, 18) circulating ghrelin levels are decreased in obese individuals, and serum ghrelin levels are inversely correlated with body mass index (BMI) both in obesity and lean subjects. Moreover, decreased postprandial circulating ghrelin levels in obese individuals are also reported by some (7) although not by all (19, 20). But ghrelin and its agonist increase food intake in obese at least as efficiently as in lean subjects (21, 22), and further reduction of the already low plasma ghrelin concentrations in obese individuals could possibly still trigger the reduction of body fat mass or at least prevent recidivism to obesity after diet-induced weight loss (23). So this negative association between ghrelin concentrations and acute feeding and chronic positive energy balance in obesity cannot be fully interpreted as an adaptive physiological response (24).
Recently, Zhang et al. (25) reported that GHRL (ghrelin/obestatin preprohormone) gene also encoded another 23-amino-acid secreted peptide, termed obestatin. The biological activity of obestatin depended on the amidation at its carboxy terminus, and it could bind to and activate the orphan receptor GPR39 (25). And what’s surprising was that obestatin, although derived from the same peptide precursor, suppressed food intake, inhibited jejunal contraction, decreased body-weight gain, and antagonized the actions of ghrelin when both peptides were coadministered (25). These facts may suggest that the intricate balance of ghrelin and obestatin is important in the regulation of energy homeostasis and body weight control.
To the best of our knowledge, plasma obestatin profiles in relation to ghrelin and food intake in human obesity has not been studied. We hypothesized that obese individuals would present with imbalance of ghrelin and obestatin levels that might contribute to the pathogenesis of obesity. Therefore, we investigated plasma obestatin and ghrelin levels before and after a meal in obese and normal-weight subjects.
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Subjects and Methods |
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According to the criteria of the guidelines for prevention and control of overweight and obesity in Chinese adults (26), 16 obese (BMI > 28 kg/m2) and 14 normal-weight healthy subjects (BMI > 18.5 kg/m2 and < 24 kg/m2) were enrolled in this study. Obese individuals were recruited from those who were admitted to our department for work-up and treatment of obesity or related diseases, and normal-weight healthy subjects were recruited from hospital staff as age- and sex-matched controls. All participants are urban inhabitants of Shanghai, China, and their clinical characteristics are detailed in Table 1
. All subjects were clinically stable at the time of evaluation and had no evidence of gastrointestinal disease, heart disease, lung disease, liver disease, kidney disease, thyroid disease, or infection. The protocol was performed according to the principles of the Declaration of Helsinki and approved by the local research and ethics committee at the Changhai Hospital. Written informed consent was obtained from all subjects before participation.
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All subjects were on a free diet after admission to our department. Weight and height as well as waist and hip circumferences were measured using standard techniques in each subject (26). BMI and waist-hip ratio (WHR) were calculated. Fasting preprandial blood samples were drawn from an antecubital vein 1 h before breakfast (
0600 h) after a 12-h overnight fast. Blood samples were also obtained from 10 of the obese and 10 of the normal-weight subjects 2 h after breakfast (0900 h) for the measurement of postprandial ghrelin and obestatin levels. The breakfast contained about 500–600 kcal with a mixture of carbohydrates, protein, and fat. Foods were among the main dietary sources in China, including steamed wheat bun, steamed rice, deep-fried wheat dough stick, Chinese sweet pastry, egg, jook, milk, soymilk, etc. The list also reflected items of intake common to urban inhabitants of Shanghai. In addition, a core group of condiments was also provided including broad-bean paste, peanut butter, fermented soybean curd, pickles, vegetables, fruits, etc. Blood samples for measurement of ghrelin, obestatin, and insulin were immediately transferred to chilled polypropylene tubes containing EDTA-2Na (1 mg/ml) and aprotinin (Phoenix Pharmaceuticals, Belmont, CA; 100 µl containing 0.6 trypsin inhibitor units per milliliter of blood), centrifuged at 4 C, 1600 x g for 15 min, and then plasma samples were stored at –80 C until assayed.
The fasting blood levels of glucose, lipids, white blood cells (WBC), and high-sensitivity C-reactive protein (hsCRP) were measured in the clinical laboratory of our hospital. The plasma glucose levels were measured by an automated glucose oxidase method (Automatic Analyzer 7600-020; Hitachi, Tokyo, Japan). Serum levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) were measured by enzymatic methods using the autoanalyzer. WBC count was measured using an automated cell counter. The hsCRP levels were measured with latex-enhanced immunonephelometry on a Behring BNProSpec Nephelometer (Dade Behring, Marburg, Germany).
Hormonal assay
Plasma obestatin levels were measured after extraction in reverse-phase C18 columns (Phoenix Pharmaceuticals) with a commercial RIA kit (Phoenix Pharmaceuticals) using 125I-labeled obestatin as a tracer and a polyclonal antibody raised in rabbits against human obestatin. According to the manufacturer’s instructions, plasma samples were acidified with an equal amount of 0.1% trifluoroacetic acid in H2O. Then they were centrifuged at 10,000 x g for 20 min at 4 C, and the supernatant was kept. A SEP-COLUMN containing 200 mg C18 was equilibrated by washing with 60% acetonitrile in 0.1% trifluoroacetic acid followed by 0.1% trifluoroacetic acid. The acidified plasma solution was loaded onto the pretreated C18 SEP-COLUMN. The column was then washed with 0.1% trifluoroacetic acid. The peptide was eluted slowly with 60% acetonitrile in 0.1% trifluoroacetic acid, and the eluent was collected in a polypropylene tube. The eluent was evaporated to dryness in a centrifugal concentrator and then reconstituted with RIA buffer when subjected to RIA analysis. No cross-reactivity was found with human ghrelin, motilin, leptin, peptide YY 3–36, cocaine- and amphetamine-regulated transcript, GHRH, neuromedin U, neuropeptide W-23, or other relevant molecules. The sensitivity of the assay was 215.5 pg/ml. Intra- and interassay coefficients of variation (CV) reported by the manufacturer were less than 5% and less than 12%, respectively. Plasma ghrelin levels were measured using a commercial RIA kit (Phoenix Pharmaceuticals) that uses 125I-labeled bioactive ghrelin as a tracer molecule and a rabbit polyclonal antibody against full-length octanoylated human ghrelin. This assay recognizes both acylated and des-acylated forms of ghrelin. The assay protocol was similar to obestatin assay kit except that extraction of plasma was not required according to the manufacturer’s instructions. No cross-reactivity was found with human obestatin, motilin, secretin, vasoactive intestinal peptide, neuromedin U, prolactin-releasing peptide-31, galanin, GHRH, neuropeptide Y, or orexin A or B. Sensitivity of the assay was 115 pg/ml. The intraassay CV was less than 5%, and the interassay CV was less than 12%. Plasma insulin levels were measured using an ELISA kit (Phoenix Biotech Co., Ltd., Beijing, China). The lower and upper detection limits were 0 µIU/ml and 20 µIU/ml, respectively. The intraassay CV was less than 4%, and the interassay CV was less than 8%. Insulin resistance was calculated by the homeostasis model of assessment for insulin resistance (HOMA-IR) approach, calculated as fasting insulin (microunits per milliliter) x fasting blood glucose (millimoles per liter)/22.5 (27).
Statistical analysis
Data are expressed as the mean ± SD unless otherwise indicated. Comparisons between groups were performed with unpaired Student’s t test unless otherwise indicated, with some additionally tested by ANCOVA after adjusting for age and gender. A paired t test was used to compare the differences between preprandial and postprandial measured parameters in each group. The relationships between preprandial ghrelin, obestatin, ghrelin to obestatin ratio, and various anthropometric and metabolic variables were examined by bivariate correlations (Pearson’s correlation coefficient). Multiple regression analysis was further used to assess the relationships between preprandial ghrelin, obestatin, ghrelin to obestatin ratio, and anthropometric and metabolic variables. P values < 0.05 were regarded as statistically significant. All of the analyses were performed using SPSS for Windows (version 10.0; SPSS Inc., Chicago, IL).
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Results |
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Table 1
depicts characteristics of the study population. There were no significant differences in genders, age, plasma HDL-C levels, or WBC count between the groups. Compared with normal-weight controls, the obese group had higher BMI, waist circumference, and WHR. The obese group also had higher plasma levels of TC, TG, LDL-C, fasting glucose, and fasting insulin and higher insulin resistance as calculated by the HOMA-IR approach compared with normal-weight controls, and they tended to have higher hsCRP levels.
Differences in preprandial ghrelin, obestatin, and ghrelin to obestatin ratio
No sex differences in plasma preprandial or postprandial ghrelin, obestatin, and ghrelin to obestatin ratio were found (all P > 0.1, data not shown); thus, data for men and women were pooled. Preprandial plasma ghrelin levels (Fig. 1A) were lower in the obese group compared with normal-weight controls (494.0 ± 91.8 vs. 683.9 ± 89.4 pg/ml, P < 0.01), and after adjustment for gender and age, ghrelin levels remained significantly lower in the obese group than controls (P < 0.01). Preprandial plasma obestatin levels (Fig. 1B) in the obese group were also lower compared with normal-weight controls (42.6 ± 9.8 vs. 70.5 ± 6.4 pg/ml, P < 0.01) and remained significant after adjustment for gender and age (P < 0.01). However, unexpectedly, the ratios of ghrelin to obestatin (Fig. 1C) were higher in the obese group compared with normal-weight controls (11.8 ± 1.8 vs. 9.8 ± 1.5, P < 0.01) even after adjustment for gender and age (P < 0.01).
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Postprandial ghrelin levels (Fig. 2A) were decreased significantly in the obese group (319.0 ± 83.5 vs. 467.3 ± 80.2 pg/ml, P < 0.01) and controls (408.6 ± 62.5 vs. 645.3 ± 70.8 pg/ml, P < 0.01) compared with their preprandial levels, respectively. Postprandial ghrelin levels in the obese group were lower compared with normal-weight controls (P < 0.05) and remained significantly different after adjustment for gender and age (P < 0.05). As shown in Fig. 2B, postprandial obestatin levels both in the obese group (36.7 ± 9.3 vs. 42.8 ± 9.8 pg/ml, P < 0.05) and controls (57.5 ± 10.4 vs. 71.1 ± 5.9 pg/ml, P < 0.01) were also decreased compared with their preprandial levels and were lower in the obese group compared with normal-weight controls (P < 0.01) and also remained significantly different after adjustment for gender and age (P < 0.01). The postprandial ratios of ghrelin to obestatin, as shown in Fig. 2C, were decreased both in the obese group (8.9 ± 2.3 vs. 11.1 ± 1.1, P < 0.05) and controls (7.3 ± 1.4 vs. 9.1 ± 1.2, P < 0.01) compared with their preprandial levels, but there were no significant differences in the postprandial ratios of ghrelin to obestatin between the obese group and normal-weight controls, and the differences remained nonsignificant after adjustment for gender and age.
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BMI, waist circumference, WHR, TG, LDL-C, fasting plasma insulin levels, and HOMA-IR were negatively correlated with fasting preprandial ghrelin levels (Table 2). Fasting preprandial obestatin levels were negatively correlated with BMI, waist circumference, WHR, TC, TG, LDL-C, fasting plasma insulin levels, HOMA-IR, and hsCRP but exhibited a positive correlation with HDL-C (Table 2). However, the ratios of fasting preprandial ghrelin to obestatin were positively correlated with BMI, waist circumference, TC, and LDL-C (Table 2). Based on the simple correlation coefficients, the relationships between preprandial ghrelin, obestatin, ghrelin to obestatin ratio, and related variables were further assessed in multiple regression models to adjust BMI, age, and gender. Multiple regression analysis indicated that in this state only triglyceride was an independent predictor of fasting preprandial obestatin levels (standardized coefficient = –0.325; P = 0.023). In a multiple regression model including BMI, WHR, TC, TG, LDL-C, HDL-C, fasting glucose, fasting insulin, WBC count, and hsCRP, BMI was a significantly independent determinant of fasting preprandial ghrelin to obestatin ratio (standardized coefficient = 0.816; P = 0.020), whereas other parameters did not show significant correlations with the ghrelin to obestatin ratio.
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Discussion |
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Despite no apparent changes being observed in circulating obestatin levels upon fasting or feeding in rodents by Zhang et al. (25), we found that plasma obestatin levels were significantly decreased after food intake both in obese individuals and normal-weight subjects, and moreover, obese individuals had lower preprandial as well as postprandial plasma obestatin levels than normal-weight subjects. What’s more surprising was that, although there were similar postprandial ghrelin to obestatin ratios between them, obese subjects had significantly higher preprandial ghrelin to obestatin ratios than normal-weight subjects, that is relative ghrelin levels were still higher than relative obestatin levels in obese subjects. Furthermore, the ratios of preprandial ghrelin to obestatin were positively correlated with BMI, which was also confirmed by multiple regression analysis. So, if obestatin lives up to its name (25), the findings may provide an unexpected, but logical, explanation for the puzzling discrepancy between the lower plasma ghrelin levels in obesity and the unsurpassed pharmacological effects of ghrelin on food intake as well as body weight gain and fat accumulation and may also suggest that further reduction of the already decreased plasma ghrelin concentrations in obese individuals could possibly still trigger the reduction of body fat mass.
Recently, Chanoine et al. (28) found that obestatin was present in the perinatal rat pancreas and that there were significantly positive correlations between obestatin and insulin concentrations in postnatal pancreas, suggesting that obestatin could potentially affect insulin secretion and glucose metabolism. Our study also found that fasting preprandial obestatin levels were closely correlated with some parameters of glucose and lipid metabolism in simple correlation analysis, but only triglyceride was an independent predictor of fasting preprandial obestatin levels after adjusting for BMI, age, and gender. Although considerable evidence has suggested that ghrelin directly participates in glucose and lipid metabolism (29, 30, 31), given the small sample size and cross-sectional design of the present study, more prospective and functional studies about obestatin, especially in relation to ghrelin, will help to clarify this issue in future.
Although recently, measurement of acylated and des-acylated ghrelin in human plasma has been reported (32, 33), detection methods for differentiating between circulating amidated and nonamidated obestatin are not yet available. Considering that the biological activity of ghrelin and obestatin both depend on their posttranslational modification (25), the separate measurement of the two forms of plasma ghrelin and obestatin in future studies may provide more detailed and valuable information. In addition, we didn’t evaluate the relationship between ghrelin to obestatin ratios and hunger scores and whether the postprandial suppression of ghrelin to obestatin ratios was proportional to the ingested meal calorie, so more detailed studies are merited.
The identification of obestatin has already added complexity to ghrelin physiology. But what’s more teasing was that several recent studies about obestatin reported controversial results. For example, several studies (34, 35, 36, 37) failed to observe any effect of obestatin on food intake and body weight. But the study of Bresciani et al. (38) and the study (39) in the obestatin receptor knockout mouse further supported the in vivo effects of obestatin described by Zhang et al. (25). The underlying reason is not clear, because at least in the footnote of the paper by Nogueiras et al. (35) they mentioned that "obestatin, including compound from our laboratory was efficient in decreasing food intake in mice in the laboratory of Prof. Hsueh. The identical protocol was repeated in our laboratory, but repeatedly failed to produce those results."
Nevertheless, the present study showed for the first time that circulating preprandial ghrelin to obestatin ratios were elevated in human obesity and that the ratios were positively correlated with BMI [while preparing this manuscript, we noticed that a study performed by Haider et al. was published (40). In the study, fasting serum concentrations of both ghrelin and obestatin were lower in morbidly obese subjects compared with lean controls]. These findings may support the hypothesis that obese individuals would present with an imbalance of ghrelin and obestatin levels and suggest that an elevated preprandial ghrelin to obestatin ratio may have a role in the etiology and pathophysiology of obesity. Future studies to confirm or refute our initial results are eagerly anticipated.
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Footnotes |
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First Published Online February 13, 2007
Abbreviations: BMI, Body mass index; CV, coefficient of variation; HDL-C, high-density lipoprotein cholesterol; hsCRP, high-sensitivity C-reactive protein; HOMA-IR, homeostasis model of assessment for insulin resistance; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride; WBC, white blood cell; WHR, waist-hip ratio.
Received October 23, 2006.
Accepted February 6, 2007.
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