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Unit of Metabolic Diseases (G.M., R.M., N.M.), Endocrinology Unit (U.P., R.P.), Department of Internal Medicine and Gastroenterology, and Center for Applied Biomedical Research (U.P., R.D.I., R.P.), Alma Mater Studiorum, University of Bologna, I-40138 Bologna, Italy; and Gastroenterology Department (E.B., E.V., M.R.), University of Turin, Ospedale San Giovanni Battista, I-10126 Turin, Italy
Address all correspondence and requests for reprints to: Giulio Marchesini, M.D., Unit of Metabolic Disease, University of Bologna, Department of Internal Medicine and Gastroenterology, Via Massarenti, 9, I-40138 Bologna, Italy. E-mail: giulio.marchesini{at}unibo.it.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Nonalcoholic fatty liver disease (NAFLD) is a complex metabolic condition in which both lifestyle and genetic factors have a pathogenic role (7). It has been convincingly associated with the metabolic insulin resistance syndrome; most patients are overweight or frankly obese, with altered glucose regulation, dyslipidemia, and raised blood pressure, all contributing to the disorder (8). However, large studies have shown that approximately 10–20% of patients are lean and have normal glucose regulation, but are nonetheless insulin resistant when tested by the homeostasis model assessment (HOMA) method (9) or by the euglycemic clamp technique (10).
In the present study, we measured fasting ghrelin concentration in a large series of NAFLD patients with different phenotypes to test the relative importance of body fat, glucose regulation, hyperinsulinemia, and insulin resistance in ghrelin levels.
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Patients and Methods |
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Eighty-six NAFLD patients (79 males) (median age, 38 yr; range, 19–74 yr) and 40 control subjects (32 males) (median age, 43 yr; range, 28–77 yr) were included in the study. Their clinical and laboratory variables are presented in Tables 1
and 2. In NAFLD cases, the diagnosis was based on chronic hypertransaminasemia (alanine transaminases (ALT) of >1.5 times the upper normal values for 3 months or more), negative hepatitis B and C viral markers, absence of autoantibodies indicative of autoimmune hepatitis or celiac disease, negative or negligible alcohol consumption (<140 g/wk), and bright liver at ultrasound scanning. The diagnosis was confirmed by liver biopsy in 62 cases. According to the criteria proposed by Brunt et al. (11), 46 cases were classified as nonalcoholic steatohepatitis (NASH), and 16 were classified as pure fatty liver. Control subjects were free of hepatic and endocrine diseases. They were selected in a BMI range similar to that of NAFLD cases. Previously diagnosed diabetes mellitus [American Diabetes Association classification (12)] was an exclusion criterion for both NAFLD patients and control subjects. In 54 NAFLD patients and 28 controls, an oral glucose tolerance test (OGTT) was also performed for a complete evaluation of glucose tolerance.
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The purpose of the study was explained to all subjects, who gave their informed consent to blood sampling for ghrelin measurement. All other investigations were carried out during regular follow-up of NAFLD patients, according to specific protocols. The study was approved by the senior staff committees of the two university hospitals, institutional review boards regulating noninterventional studies.
Methods
Anthropometry. Body weight was measured in light clothing and without shoes to the nearest half-kilogram. Height was measured to the nearest half-centimeter. BMI was calculated as weight (kilograms) divided by height squared (square meters). Subjects with BMI between 25 and 30 kg/m2 and greater than or equal to 30 kg/m2 were considered overweight and obese, respectively. Waist circumference was measured at the nearest half-centimeter at the shortest point below the lower rib margin and the iliac crest, whereas hip circumference was similarly obtained at the widest point between hip and buttock. Body fat distribution was also evaluated by waist-to-hip ratio (WHR) according to World Health Organization (13).
Biochemical and hormonal measurements. Plasma immunoreactive ghrelin levels were measured in duplicate using a commercially available RIA (Phoenix Pharmaceuticals, Inc., Mountain View, CA) that uses 125I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against the C-terminal part of human ghrelin (6). Intraassay and interassay coefficients of variation were less than 5.3 and 13.6%, respectively. This assay recognizes both acylated and deacylated ghrelin (2). The antiserum does not cross-react with any relevant peptide as previously shown (2, 14).
Plasma glucose was measured in duplicate with an automated analyzer. The coefficient of variation for any single determination was ±1.5%. Insulin was measured by an immunoenzymometric assay (AIA-PACK IRI, AIA-1200 system; Tosoh Co., Tokyo, Japan) with intraassay and interassay coefficients of variation for the quality control of less than 7%. Insulin resistance was calculated on the basis of fasting values of plasma glucose and insulin, according to the HOMA method (15) as follows: insulin resistance [HOMA-R (%)] = IRI0*BG0/22.5, where fasting insulin (IRI0) is in microunits per milliliter, and glucose (BG0) is in millimoles per liter.
In patients submitted to OGTT, two more indices of insulin sensitivity were derived from basal and postload glucose and insulin concentrations. The insulin sensitivity index (ISI) was calculated according to Matsuda and DeFronzo (16) as follows:
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where average BG0–120 and average IRI0–120 represent the mean of individual values measured after glucose load. Body weight (BW) also enters the SI equation, with a correction factor for glucose space. Additional correction factors are needed to transform metric units into SI units.
Fasting serum cholesterol, high-density lipoprotein (HDL) cholesterol, uric acid, and triglyceride levels were measured by routine laboratory techniques.
Statistical analysis
Data were processed on a personal computer and analyzed using StatView 5.0 (SAS Institute, Inc., Cary, NC.). Patients were grouped according to categorical variables (sex, class of BMI, presence/absence of impaired glucose regulation, and hypertension). Ghrelin concentrations were tested for significance using unpaired t test (two-tail) or nonparametric analysis (Mann-Whitney U test or Kruskal-Wallis test). Contingency test and Fisher’s exact test were also used, whenever appropriate, to compare prevalence.
Logistic regression analysis was used to determine factors more closely associated with low ghrelin concentrations. For this purpose, a ghrelin concentration below the median of the whole group (235 pmol/liter) was considered as dependent variable, and the clinical and laboratory values of Tables 1
and 2 were tested in univariate analysis. After this, variables significantly associated with low ghrelin were tested for independency in multivariate logistic regression analysis, after correction for age and sex. Two different models were built to further adjust data for BMI and waist circumference, separately.
All data in the text and in the tables are given as means ± SD, when not otherwise indicated. Values of P < 0.05 were considered statistically significant.
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Results |
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Both controls and NAFLD patients were selected in a wide BMI range (controls, 19.2–35.2 kg/m2; NAFLD, 20.9–37.9 kg/m2), without differences in the distribution among BMI classes between groups. However, a larger waist circumference and a higher WHR, indicative of visceral adiposity, characterized NAFLD patients.
At history, diabetes, hypertension, and dyslipidemia were not more prevalent in NAFLD, but at the time of study, arterial pressure was higher in NAFLD, whereas HDL-cholesterol concentrations were lower. NAFLD patients were also characterized by a different response to OGTT. In particular, the prevalence of impaired glucose tolerance was remarkably higher in NAFLD (22.2 vs. 3.6% in controls; P = 0.030, Fisher’s exact test) without differences in the prevalence of OGTT-detected diabetes (9.3% in NAFLD vs. 7.1% in controls; P = 0.745).
Fasting glucose was not different, but both insulin concentrations and HOMA-R were increased by over 60%. The 95% confidence interval of HOMA-R in normal-weight controls ranged up to 2.74%. This cutoff was used as the upper limit of normal insulin sensitivity. According to this criterion, 13 controls were considered to be insulin resistant (32%) vs. 57 of 86 NAFLD cases (66%; P = 0.0005, Fisher’s exact test). A significant correlation was observed between BMI and HOMA-R in controls (r = 0.334; P = 0.035), but not in NAFLD patients (r = 0.202; P = 0.062).
Also, the two indices of insulin sensitivity derived by OGTT (ISI and SI) showed a significant resistance to insulin activity in the course of the glucose load in the NAFLD cohort when compared with control subjects.
Ghrelin levels
Ghrelin levels were reduced in NAFLD patients (226 ± 72 vs. 303 ± 123 pmol/liter in controls; P < 0.0001). Differences were maintained when levels were analyzed in relation to gender (males, NAFLD, 224 ± 74 pmol/liter, controls, 279 ± 100 pmol/liter, P = 0.002; females, NAFLD, 247 ± 35 pmol/liter, controls, 396 ± 164 pmol/liter, P = 0.035). Importantly, a gender effect was observed only in the control group (males vs. females, P = 0.014), whereas no statistically significant difference between males and females was observed in NAFLD.
When ghrelin levels were analyzed in relation to quartiles of BMI, in control subjects, ghrelin progressively decreased from 401 ± 130 (lower quartile; BMI range, 19.2–25.9 kg/m2) to 236 ± 75 (upper quartile; BMI range, 30.2–35.2 kg/m2; P = 0.003). In NAFLD patients, ghrelin levels were similar between subjects in the lower BMI quartile (242 ± 90 pmol/liter; BMI range, 20.9–24.6 kg/m2) compared with subjects in the upper quartile (244 ± 72 pmol/liter; P = 0.926; BMI range, 29.0–37.9 kg/m2) (Fig. 1). A significant correlation between BMI and ghrelin concentrations was present in controls (r = -0.604; P < 0.0001), but not in NAFLD (r = -0.093; P = 0.397) (Fig. 2).
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), and the correlations between HOMA-R and ghrelin were both statistically significant (controls, r = -0.591, P < 0.0001; NAFLD, r = -0.352, P = 0.0008), although the two regression lines were considerably different, both in the intercept and in the slope (Fig. 3).
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Factors associated with ghrelin concentrations were further tested by logistic regression analysis (Table 3). Ghrelin concentrations below the median of 235 pmol/liter were associated with several laboratory and anthropometric indices at univariate analysis. The association of low ghrelin with both insulin and insulin resistance was maintained after adjustment for BMI, either in the whole population or in NAFLD patients (not reported in details).
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2.74%) was the sole factor associated with low ghrelin concentrations at multivariate analysis in the whole population (Table 3), as well as separately in control subjects [odds ratio (OR), 18.90; 95% confidence interval, 3.52–101.48; P = 0.0006] and in NAFLD patients (OR, 2.96; 95% confidence interval, 1.12–7.79; P = 0.028). BMI did not enter the regression, and when data were adjusted for BMI, the effects of HOMA-R on ghrelin levels did not change (all cases, 5.58, 2.50–12.43, P < 0.0001; controls, 16.68, 2.80–99.28, P = 0.002; NAFLD, 2.83, 1.07–7.52, P = 0.037). In the model in which data were further adjusted for waist circumference, in addition to age and gender, HOMA-R was similarly the sole factor significantly associated with low ghrelin, both in the whole population (OR, 6.00; 95% confidence interval, 2.42–12.96; P < 0.0001) and in NAFLD patients (2.92; 95% confidence interval, 1.08–7.85; P = 0.034).
NAFLD patients submitted to liver biopsy were not different from subjects who had not had a liver biopsy in the parameters presented in Table 2. In particular, BMI was 27.3 ± 3.3 kg/m2 in patients who had and 26.5 ± 2.8 kg/m2 in patients who had not had a liver biopsy (P = 0.375), and HOMA-R was 3.89 ± 1.83 and 3.68 ± 2.14%, respectively (P = 0.673). Also, ALT values were similar (biopsy positive, 84 ± 38 U/liter; biopsy negative, 73 ± 39 U/liter; P = 0.294). Ghrelin levels were also similar (234 ± 78 and 224 ± 71 pmol/liter; P = 0.606), and they did not differ in relation to the severity of fat deposition, fibrosis, and necroinflammatory activity. In particular, ghrelin was not different when patients were classified according to the presence/absence of NASH (pure fatty liver, 220 ± 88 pmol/liter; NASH, 226 ± 69 pmol/liter; P = 0.781).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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NAFLD is significantly associated with the metabolic syndrome (8). Although most patients are overweight or obese, 10–20% of cases have a BMI within normal limits. This condition represents a suitable model to study the relationship of insulin sensitivity and ghrelin, dissecting the putative effects of BMI on circulating ghrelin.
Our data are derived from a large cohort of NAFLD subjects, in whom insulin resistance, measured by the HOMA technique, was nearly the rule. The control group was carefully matched to account for potential confounders. Also, gender was considered, because a recent study demonstrated that ghrelin secretion is sexually dimorphic (18). Interestingly, our data showed that the gender effect is lost in NAFLD. Sex hormones may play an important role on ghrelin circulatory pattern (6, 19, 20), and estrogen therapy is a well-known cause of secondary NAFLD (21, 22). However, relative estrogen excess, as reported in advanced liver diseases, is unlikely in these subjects with normal liver function, and the result may simply stem from the low number of female patients causing type II statistical error.
To exclude differences related to BMI, our control population also included a few overweight or obese subjects with HOMA-R values in the range of insulin resistance as well. For this reason, ghrelin levels were analyzed in relation to quartiles of BMI and of insulin resistance, respectively. Similarly to data reported in other studies (23, 24), BMI played an important role on fasting ghrelin concentrations only in normal subjects.
By contrast, when data were analyzed in relation to quartiles of HOMA-R, the correlation between HOMA-R and ghrelin observed in controls was maintained in NAFLD, and HOMA-R was the sole factor predicting low ghrelin concentration in both groups at multivariate analysis.
The HOMA method for the measurement of insulin resistance has been extensively applied to epidemiological investigations. The figure of insulin resistance obtained with this method has a relatively low reproducibility, which reflects day-to-day variability in fasting glucose and insulin, as well as analytical uncertainty. This is mainly the case for insulin levels, and a change of 1 µU/ml insulin may determine a change of up to 20% HOMA-R. Despite this, the method proved to correlate closely with quantitative, functional tests such as the glucose clamp technique (25, 26). In nondiabetic subjects, insulin concentrations account for the larger part of HOMA-R. This explains why insulin per se was also closely associated with ghrelin. However, in logistic regression, HOMA-R was mathematically preferred to insulin, suggesting that insulin resistance rather that insulin concentrations may regulate ghrelin secretion.
Low ghrelin levels are observed in several pathological conditions characterized by insulin resistance, such as moderate to severe obesity (2), polycystic ovary syndrome (6), acromegaly (27), and primary or secondary hypogonadisms (20), but the reason(s) for such relations are unclear. Ghrelin stimulates food intake (28), and reduced levels might be teleologically aimed at preventing the increase in body mass. This conclusion holds in controls, but not in NAFLD patients, in whom ghrelin is relatively independent of BMI. There is no consensus as to the exact relationship between ghrelin and insulin. Ghrelin was reported to stimulate (29) as well as to inhibit insulin secretion (30); in turn, hyperinsulinemia, either induced by a single bolus or during a clamp, produces conflicting effects on ghrelin concentrations. In vitro animal studies (31) and human studies during insulin infusion (5, 32) support the role of insulin as a secretagogue of ghrelin, but this effect was not confirmed in similar experiments in humans (33, 34). In our study, ghrelin was inversely related to insulin, but HOMA-R values were the most significant predictor of low ghrelin concentrations. Accordingly, ghrelin secretion might be under the control of insulin or, more likely, under the control of insulin resistance, via undefined circulating factors.
Insulin resistance was also tested by means of OGTT-derived indices. Although an overall correlation was observed in controls, this relation was lost in NAFLD. The figure of insulin resistance derived from ISI and SI is largely dependent on the dynamic response of glucose and insulin to the glucose load, which is not necessarily related to fasting values. This is mainly the case of NAFLD patients, who had a larger prevalence of glucose intolerance. The assessment of ghrelin response to oral glucose in NAFLD would be needed to compare the dynamic responses of ghrelin and insulin to glucose ingestion, and to assess the role of postload insulin resistance.
In search of other potential factors responsible for low ghrelin levels, we correlated hormonal levels with liver function parameters, with negative results. Only one study has been published so far on ghrelin levels in patients with liver disease. Tacke et al. (24) reported normal ghrelin levels in noncirrhotic patients and slightly elevated concentrations in cirrhosis. In their series of patients with advanced disease evaluated for liver transplantation, ghrelin was only related to the clinical severity of disease. This correlation, however, might also be spurious, and generated by the anorexia and decreased food intake of advanced disease. In our NAFLD cases, liver function was normal, and all subjects were on a controlled dietary regimen. Their BMI was similar to controls, and also excessive food intake cannot account for hormonal changes.
In conclusion, the study of NAFLD patients, in whom insulin resistance is relatively independent of obesity, strongly supports a primary role of insulin resistance per se on fasting ghrelin concentrations. This confirms the importance of decreased insulin sensitivity on the multiple metabolic abnormalities of patients with NAFLD.
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Footnotes |
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Abbreviations: ALT, Alanine transaminase; BMI, body mass index; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; ISI, insulin sensitivity index; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OGTT, oral glucose tolerance test; OR, odds ratio; SI, sensitivity index; WHR, waist-to-hip ratio.
Received June 25, 2003.
Accepted August 29, 2003.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-cells of humans and rats and stimulates insulin secretion. Diabetes 51:124–129This article has been cited by other articles:
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  E. Bugianesi, G. Svegliati-Baroni, and G. Marchesini Authors' response Gut, March 1, 2008; 57(3): 426 - 427. [Full Text] [PDF]
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 V. Vicennati, S. Genghini, R. De Iasio, F. Pasqui, U. Pagotto, and R. Pasquali Circulating obestatin levels and the ghrelin/obestatin ratio in obese women Eur. J. Endocrinol., September 1, 2007; 157(3): 295 - 301. [Abstract] [Full Text] [PDF]
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 P. Marzullo, A. Caumo, G. Savia, B. Verti, G. E. Walker, S. Maestrini, A. Tagliaferri, A. M. Di Blasio, and A. Liuzzi Predictors of Postabsorptive Ghrelin Secretion after Intake of Different Macronutrients J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4124 - 4130. [Abstract] [Full Text] [PDF]
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G. Marchesini, G. Bianchi, P. Lucidi, N. Villanova, M. Zoli, and P. De Feo Plasma Ghrelin Concentrations, Food Intake, and Anorexia in Liver Failure J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2136 - 2141. [Abstract] [Full Text] [PDF]
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) and in NAFLD patients (
) in relation to quartiles of BMI. Significant differences are present in controls (P = 0.0043; Kruskal-Wallis test), but not in liver patients (P = 0.083).
, dotted line) and in patients with NAFLD (•, continuous line). The r coefficients of correlation and P values are separately reported for the whole group (n = 126), for control subjects (n = 40), and for fatty liver patients (n = 86).