Abstract
BACKGROUND. Low ghrelin concentration has been associated with several features of metabolic syndrome (MS), but the relationship between ghrelin concentration and MS as a cluster of metabolic aberrations has not yet been studied.
AIMS OF THE STUDY. To analyse whether ghrelin concentration is associated with MS.
RESEARCH DESIGN AND METHODS. Fasting plasma ghrelin concentrations of the population‐based cohort of 1037 middle‐aged men and women were analysed using a commercial radioimmunoassay kit (Phoenix Peptide). MS was determined using the new International Diabetes Federation criteria.
RESULTS. The prevalence of MS was 37.2%. The ghrelin concentrations were decreased in subjects with MS (635 pg/mL) compared to those without MS (687 pg/mL) (P = 0.001). Ghrelin levels decreased with an increase in the number of metabolic abnormalities. Low ghrelin was a statistically significant predictor of MS in logistic regression analysis (P = 0.005) so that the subjects in the 1st ghrelin quartile were at higher risk of having MS compared to the subjects in the 4th quartile (OR = 1.82, 95% CI: 1.27–2.60, P = 0.001). This association remained statistically significant after adjustment for age and sex (OR = 1.76, 95% CI: 1.24–2.55, P = 0.002).
CONCLUSIONS. Metabolic syndrome is associated with low ghrelin levels suggesting a relationship of ghrelin in the metabolic disturbances of MS.
| Abbreviations | ||
| ANOVA | = | analysis of variance |
| HDL | = | high‐density lipoprotein |
| IDF | = | International Diabetes Federation |
| MS | = | metabolic syndrome |
| OR | = | odds ratio |
| OPERA | = | Oulu Project Elucidating Risk of Atherosclerosis |
| RIA | = | radioimmunoassay |
| VLDL | = | very‐low‐density lipoprotein |
Introduction
Multiple metabolic aberrations, including obesity, insulin resistance and hyperinsulinaemia, dyslipidaemia, hypertension, impaired glucose tolerance, Type 2 diabetes mellitus, and other anomalies, tend to occur jointly in the same subjects more frequently than expected by chance alone Citation1. This clustering of several cardiovascular disease and diabetes risk factors has been referred to as the metabolic syndrome (MS) Citation2. The fundamental mechanism at the origin of the metabolic cluster remains unknown even though the number of putative candidates is growing Citation3. A number of new regulatory peptides have been identified, and they have been recently suggested to play a role in the pathogenesis of MS Citation4. One such molecule is ghrelin, a blood‐borne orexigenic signal from gut to brain Citation5, with diverse hormonal, metabolic, and cardiovascular activities Citation6. Low ghrelin concentrations have been associated with the features of the MS, like obesity Citation7, insulin resistance Citation8–10, and blood pressure Citation10,11. However, the relationship between ghrelin concentrations and metabolic aberrations as a cluster per se by novel criteria has not yet been studied. The aim of the present study was to analyse whether low ghrelin concentrations are associated with MS using the new International Diabetes Federation criteria.
Key messages
The plasma ghrelin concentration is significantly correlated with several components of the MS and with metabolic cluster per se.
Ghrelin levels decreased with an increase in number of metabolic abnormalities.
Plasma ghrelin may therefore become a useful biomarker for the MS.
Subjects and methods
This study is a part of the OPERA (Oulu Project Elucidating Risk of Atherosclerosis) project, which is a population‐based, epidemiological study designed to address the risk factors and disease end‐points of atherosclerotic cardiovascular diseases. The study population and selection criteria have been previously described in detail Citation12. Middle‐aged subjects of the population‐based hypertensive (n = 518) and control cohorts (n = 519) of OPERA‐study were studied. The participants visited the research laboratory of the Department of Internal Medicine for laboratory tests, physical examination, and a detailed interview. The study was approved by the Ethical Committee of the Faculty of Medicine, University of Oulu. Waist circumference was measured to the nearest 0.5 cm with a tape measure midway between the lower rib margin and the iliac crest in light expirium. Blood pressure was measured according to the recommendations of the American Society of Hypertension, in a sitting position from the right arm with an oscillometric device (Dinamap® model 18465X, Criticon Ltd, Ascot, UK) after an overnight fast and after a 10–15‐minute rest. Three measurements were made at 1‐minute intervals, and the means of the last two were used in the analyses.
All the laboratory test samples were obtained after an overnight fast. Plasma was separated from venous blood and stored at 4°C. The venous blood glucose concentration was determined with the glucose dehydrogenase method.
The concentrations of total cholesterol and triglycerides in the plasma and lipoprotein fractions were determined by enzymatic colorimetric methods (kits of Boehringer Diagnostica, Mannheim GmbH, Germany, catalogue nos. 236691 and 701912, respectively) using Kone Specific analyser (Kone Specific, Selective Chemistry Analyser, Kone Instruments, Espoo, Finland). The very‐low‐density lipoprotein (VLDL) fraction (d<1.006 g/mL) was separated from plasma by ultracentrifugation in a Kontron TFT 45.6 rotor at 105000 g and 15°C for 18 h. The VLDL fraction was removed from the ultracentrifuged preparation by tube slicing. The plasma high‐density lipoprotein (HDL)‐cholesterol concentration was determined by mixing 1 mL of the VLDL‐free fraction with 25 µL of 2.8% (w/v) heparin and 25 µL of 2M manganese chloride and by measuring the cholesterol concentration in the supernatant after centrifugation at 1000 g and 4°C for 30 minutes.
Fasting ghrelin concentrations correlate strongly with the 24‐h integrated area under the curve ghrelin values Citation13, and therefore fasting plasma ghrelin concentrations were analysed to obtain a measure of overall ghrelin concentrations Citation14. A commercial radioimmunoassay (RIA) kit recognizing both acylated and des‐acylated ghrelin was used (Phoenix Pharmaceuticals Inc., Belmont, California, USA). The sensitivity of the assay was 1.2 pg/tube (ED80), and the intra‐ and interassay coefficients of variation (CV), as given by the manufacturer, were 4.0% and 7.5%, respectively. Interassay CV in the analyses of this study was 11.2%. Due to the low intra‐assay CV of the method, only a single measurement for each sample was performed.
The 2005 International Diabetes Federation (IDF) definition of the MS was used Citation15. According to the IDF definition, for persons to be defined as having the MS, they must have: Central obesity (defined as waist circumference ⩾94 cm for Europid men and ⩾80 cm for Europid women) plus any two of the following four factors: raised serum triglyceride level (⩾1.7 mmol/L), reduced serum HDL‐cholesterol level (<1.03 mmol/L in males and <1.29 mmol/L in females) (or specific treatment for these lipid abnormalities), raised blood pressure (systolic blood pressure ⩾130 mmHg or diastolic blood pressure ⩾85 mmHg), or treatment of previously diagnosed hypertension impaired fasting glycaemia (fasting plasma glucose ⩾5.6 mmol/L), or previously diagnosed type 2 diabetes.
To compare the means of the variables measured, Student's t test and analysis of variance (ANOVA) were used. The association between ghrelin and the variables studied was assessed using linear and logistic regression analyses. The correlation between plasma ghrelin and parameters of MS was assessed by Pearson correlation coefficient. All calculations were made with the SPSS (version 9.0; SPSS, Inc.) statistical package. P‐value <0.05 was regarded as significant.
Results
The mean ghrelin concentration of the study subjects was 667 pg/mL. shows the main characteristics of the study subjects by sex. The overall prevalence of the MS was 37.2%.
Table I. Clinical characteristics of the study patients by sex.
shows the ghrelin concentrations in relation to the clinical features of the MS. Except for plasma total triglycerides, all the other features of the MS were associated with plasma ghrelin levels. The association of plasma ghrelin with HDL‐cholesterol (P = 0.015) and fasting glucose (P = 0.049) stayed after adjustment for body mass index (BMI), age and sex. The ghrelin concentrations were decreased in subjects with MS compared to those without (P = 0.001).
Figure 1. Fasting plasma ghrelin concentrations of the study subjects in relation to the independent clinical features of metabolic syndrome(MS) and MS according to International Diabetes Federation (IDF) criteria as a cluster. Subjects with blood pressure medication were classified to the hypertension group and those with Type 2 diabetes to the group fasting glucose. Data are means and standard error of means. P‐values obtained by t test. Number of subjects embedded in the columns.
Plasma ghrelin correlated negatively with waist circumference (P<0.001), plasma total triglycerides (P = 0.046), systolic (P = 0.003) and diastolic (P = 0.002) blood pressure and fasting glucose (P = 0.008). HDL‐cholesterol showed positive correlation with plasma ghrelin (P<0.001).
The ghrelin concentrations varied in relation to the number of criteria of MS IDF criteria met (P = 0.001) (). Ghrelin levels decreased with an increase in number of metabolic abnormalities.
Figure 2. The ghrelin concentrations in relation to the number of International Diabetes Federation criteria of metabolic syndrome(MS) met. Number of subjects in parentheses.
Low ghrelin was a statistically significant predictor of MS in logistic regression analysis (P = 0.005) so that the subjects in the 1st ghrelin quartile were at higher risk of having MS compared to the subjects in the 4th quartile (OR (odds ratio) = 1.82, 95% CI: 1.27–2.60, P = 0.001). This association remained statistically significant after adjustment for age and sex (OR = 1.76, 95% CI: 1.24–2.55, P = 0.002).
Figure 3. Odds ratios(OR) with 95% CI of ghrelin quartile for metabolic syndrome (MS) obtained by logistic regression analysis adjusted for age and sex. The fourth quartile is used as a reference category.
Discussion
In our study the MS was associated with low ghrelin levels suggesting a relationship of ghrelin deficiency in the metabolic disturbances of this state. Several pathways, such as excess body fat Citation16,17, insulin resistance Citation2, central mechanisms Citation18, hyperleptinaemia Citation19 and elevated peroxisome proliferator‐activated receptor‐gamma activities Citation20 can potentially lead to the development of the MS. Excessive adiposity is undoubtedly one of the determining factors leading to the clustering of metabolic disturbances observed in the MS Citation3. Ghrelin levels are decreased in human obesity Citation7. The negative association between ghrelin and waist circumference, a prerequisite for the MS, has also been reported Citation11. Potential candidates for low ghrelin levels in obese individuals include leptin and insulin. Because adiposity influences all other features of the MS, low ghrelin in MS could reflect only the obesity state as has been suggested by a recent study Citation21. Accordingly, the association of ghrelin with triglycerides and blood pressure observed in our study were dependent on the amount of body fat. However, the associations of HDL‐cholesterol and fasting blood glucose with plasma ghrelin levels remained significant after adjustment for BMI, which is consistent with the notion that they are not entirely dependent on the amount of body fat. A positive correlation between ghrelin and HDL cholesterol concentration had been already earlier observed and the correlations were reported to be dependent Citation11 or independent Citation21 on the amount of body fat. It is not known how ghrelin could influence HDL metabolism independently of its effect on adiposity. A significant negative association was also observed between blood pressure and ghrelin levels. Ghrelin appears to have beneficial effects on blood pressure in experimental settings Citation22. Whether ghrelin deficiency has any causal role in the development of hypertension, however, remains to be explored.
Pathways leading from obesity to the manifestations of MS involve a number of metabolic risk factors Citation23. The resistance to insulin‐stimulated glucose uptake is associated with hyperinsulinaemia as well as multiple metabolic abnormalities. Consequently, decreased insulin action, or insulin resistance, has been suggested as a primary physiological defect underlying the MS Citation2. The mechanisms behind insulin resistance are complex and have not been fully elucidated yet. Low ghrelin concentrations have been associated previously with insulin resistance in subjects with polycystic ovary Citation8 and Cushing syndrome Citation24 as well as in obese children Citation9 and adolescents Citation7. The negative correlation between fasting ghrelin and insulin has also been reported earlier Citation7. Our earlier results on the same population suggested that low ghrelin is independently associated with fasting insulin concentrations and insulin resistance Citation10. Because of cross‐sectional nature of the current study it is not possible to conclude the cause‐and‐effect relationship between low ghrelin and insulin resistance and the MS. Decreased somatotrophic effects associated with ghrelin deficiency could theoretically lead to insulin resistance. Ghrelin concentrations may also be downregulated in insulin resistance as a physiological response to hyperinsulinaemic state, and low ghrelin per se may not necessarily have any causative role in the development of insulin resistance. The best ways to examine the potential etiological role of ghrelin are prospective studies and clinical trials. Another interesting issue for future studies is the Arg51Gln mutation of ghrelin gene Citation10, which might serve as ‘a natural experiment’ of impaired ghrelin function in humans.
Several studies have provided evidence that ghrelin is involved in glucose and insulin metabolism Citation25. Most of the experimental studies concerning the effects of ghrelin on insulin metabolism have concentrated on acylated ghrelin, which has been thought to be the only endocrinologically active form of the molecule. However, recent data suggest different effects of acylated and unacylated ghrelin on insulin metabolism Citation26,27. In the present study we measured total ghrelin because it is a good surrogate of acylated ghrelin since they are well correlated Citation28, and the ratio of these two remains constant under a wide variety of conditions Citation29.
In conclusion, the plasma ghrelin concentration was significantly correlated with several components of the MS and with metabolic cluster per se. Ghrelin levels decreased with an increase in number of metabolic abnormalities. Plasma ghrelin may therefore become a useful biomarker for the MS. Ghrelin's validity as a medication tool in treating patients with the metabolic syndrome should be tested in clinical trials.
Acknowledgements
The Research Council for Health of the Academy of Finland and the Finnish Foundation for Cardiovascular Research supported this study. We acknowledge the excellent technical assistance of Ms Saija Kortetjärvi, Ms Liisa Mannermaa, and Ms Sirpa Rannikko.
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