DOI 10.1515/jpem-2013-0296 J Pediatr Endocr Met 2014; 27(5-6): 479–484
Oya Sayın, Yavuz Tokgöz and Nur Arslan*
Investigation of adropin and leptin levels in pediatric obesity-related nonalcoholic fatty liver disease Abstract Aim: Nonalcoholic fatty liver disease (NAFLD) is the accumulation of excess fat in the liver in the absence of alcohol consumption, which is commonly associated with obesity and increased risk of atherosclerosis as well as insulin resistance. Adropin is a recently identified protein encoded by the gene related with energy homeostasis, which is expressed in the liver and the brain and has a role in preventing insulin resistance and obesity. The aim of this study was to investigate the serum adropin and leptin levels in obese adolescents and compare the patients with, and without, NAFLD and with healthy controls. Methods: Sixty-four obese adolescents (30 with NAFLD, 34 without NAFLD) and 36 healthy controls were enrolled in the study. Serum adropin and leptin levels were evaluated by sandwich enzyme-linked immunosorbent assay. Results: Serum adropin levels were significantly lower in obese children than healthy controls (3.2 ± 1.0 and 9.2 ± 1.2 ng/mL, respectively, p = 0.001). Serum leptin levels were significantly higher in patients than in controls (12.4 ± 1.1 and 4.1 ± 3.1 pg/mL, respectively; p = 0.000). Serum adropin levels of patients with NAFLD were significantly lower than in patients without NAFLD (2.9 ± 0.5 and 3.5 ± 1.2 ng/ mL, respectively; p = 0.023) and healthy controls (p = 0.000). Logistic regression analysis showed that a decrease in adropin levels was the only independent factor for fatty liver disease in obese adolescents (odds ratio: 3.07, 95% confidence interval 1.14–8.2, p = 0.026). Leptin, relative weight and HOMA-IR of the patients were not independent risk factors for NAFLD. Conclusions: In this study, serum adropin levels were significantly lower in obese adolescents with fatty liver disease compared to patients without fatty liver disease and healthy controls. Lower adropin level was an independent risk factor for NAFLD in obese adolescents in logistic regression analysis. Assessment of serum adropin concentrations may provide a reliable indicator of fatty liver disease in obese adolescents. Keywords: adolescent; adropin; fatty liver disease; leptin; obesity.
*Corresponding author: Nur Arslan, Division of Pediatric Gastroenterology, Hepatology and Nutrition, Dokuz Eylul University Faculty of Medicine, Department of Pediatrics, Izmir, Turkey, Phone: +90 2324126107, Fax: +90 2324126005, E-mail: [email protected]; and Dokuz Eylul University Health Science Institute, Department of Molecular Medicine, Izmir, Turkey Oya Sayın: Research Laboratory, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey Yavuz Tokgöz: Diyarbakir Children Hospital, Department of Pediatric Gastroenterology, Hepatology, Diyarbakir, Turkey
Introduction Nonalcoholic fatty liver disease (NAFLD) is characterized by the excessive accumulation of large droplets of triglycerides within hepatocytes in the absence of chronic alcohol consumption. The spectrum of NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) and cirrhosis, and, in some patients, development of hepatocellular carcinoma. It is known that obesity, insulin resistance, oxidative stress, and inflammation have an important role in the development and progression of NAFLD (1–4). Development and complications of obesity consist of complex mechanisms, including numerous adipokines, hormones, and cytokines (5). Adipokines are hormones that originated from adipose tissue. These proteins coordinate insulin homeostasis, immunity, and obesity related inflammation. Leptin is the most important adipokine produced by adipose tissue. It plays a key role in the satiety and appetite for the regulation of body weight. Serum leptin concentrations have been shown to be increased in patients with obesity, insulin resistance, metabolic syndrome, and NAFLD (1, 6, 7). Adropin (ADR, molecular weight 7.927 kDa) was recently defined by Kumar et al. in 2008 (8). Its name is derived from the Latin roots “aduro” (to combust) and “pinquis” (fats or oils). Adropin is encoded by the “energy homeostasis associated gene” (Enho) and is mainly expressed in the liver and brain (8). Adropin plays an important role in the regulation of insulin sensitivity, energy homeostasis, and probably, in obesity (9). Adropin is also present in heart, kidney (10), and in cow’s milk
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480 Sayın et al.: Adropin levels in fatty liver disease and plasma (11). To date, only a few studies have been completed assessing the serum adropin levels in human diseases such as obstructive sleep apnea (12), cardiac syndrome X (13), gestational diabetes (14), adult obesity (15), and heart failure (9). To our knowledge, adropin has not been studied in obese children and in obesity-related NAFLD in humans, and consequently, the possible role of adropin in the development of NAFLD is unknown. The aim of this study was to investigate the serum adropin and leptin concentrations in obese adolescents and compare the patients with, and without, NAFLD and with healthy controls. Another aim of this study was to evaluate the relationship between serum adropin levels and insulin resistance (IR).
Patients and methods Patients Sixty-four obese adolescents (37 males, 27 females, mean age 12.9 ± 2.1 years, range 11–16 years) were enrolled in the study. Patients with chronic liver disease, hypothyroidism, familial hyperlipidemia, hypertension, diabetes mellitus, Cushing syndrome, complete gonadal dysgenesis, growth hormone deficiency, and those that used corticosteroids were all excluded. Demographic data of all patients were recorded. Physical examinations and anthropometric measurements of all patients and controls were performed. Weight was obtained with a calibrated scale and recorded to the nearest 0.1 kg. Height was measured with a stadiometer and recorded to the nearest 0.1 cm. The body mass index (BMI) was calculated as weight divided by height squared (kg/m2). Obesity was defined as a BMI exceeding the 95th percentile (16). Relative weight (RW) was also calculated for each adolescent. Diagnosis of NAFLD was done based on increased echogenicity via ultrasound compatible with fatty infiltration of the liver (17, 18). Obese patients were divided into two groups: patients with NAFLD (group 1, 30 patients) and patients without NAFLD (group 2, 34 patients) according to their liver brightness at ultrasonography. The control group consisted of 36 adolescents (19 males, 17 females, mean age 13.2 ± 2.0 years, range 11–16 years) who were admitted to the well child outpatient clinic of our hospital for medical control (screening for hepatitis, thyroid functions, glucose, lipid profile or anemia, etc.). None of the adolescents had a history of drug usage and metabolic, cardiovascular, respiratory or hepatic disease. Antropometric values and abdominal ultrasound of the controls were normal. Insulin and glucose levels, lipid profiles and liver function tests were performed and found normal in the control group.
alanine amino transferase (ALT), and aspartate amino transferase (AST). Hepatotropic viruses, serum copper and ceruloplasmin levels, serum alpha 1-antitrypsin level, and autoantibodies against nuclear, smooth muscle, liver, and kidney microsomal type-1 antigens were screened to eliminate infectious, metabolic, and immunological liver pathologies if the patients’ ALT levels were above the accepted values. Homeostasis model assessment (HOMA-IR) index was used to estimate the insulin resistance and it was calculated by “fasting insulin × fasting glucose/22.5” as described by Matthews et al. (19). HOMAIR levels of > 4.0 in adolescents was defined as an insulin-resistance status. Serum adropin measurement was carried out using human adropin enzyme-linked immunosorbent assay (ELISA) kit (Catolog No. S-1438, limit determination 0–25 ng/mL; Bachem, Bubendorf, Switzerland) as recommended by the manufacturer’s protocol. Leptin serum concentration was measured using human leptin enzymelinked immunosorbent assay (ELISA) kit (Catalog No. EK0437; Boster Biological Technology Co., Ltd, Fremont, CA, USA) The samples were read on a Synergy HT, Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) plate reader by measuring the absorbance at a wavelength of 450 nm. Venous blood was collected from all patients after an overnight fasting and then centrifuged at 4°C 3000 g for 10 min. Serum samples were stored at –20°C until the day of analysis.
Ethical approval The study protocol was designed in compliance with the Declaration of Helsinki. Informed consent was obtained from both the adolescents and their parents or legal guardians on enrollment in the study. The study was started after the approval of the Ethics Committee of the Dokuz Eylul University Faculty of Medicine.
Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 15.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were given as mean ± standard deviation; categorical variables were defined as percentages. One-way analysis of variance was used for analyzing group averages among three groups. Student t-test and Mann-Whitney U-test were used for comparing two group averages as a post hoc test. Chi-square test was used for comparing group ratios. Intercorrelations between parameters were computed through the Pearson’s correlation analysis. Correlation coefficient indicated low correlation at 0.10–0.29, medium correlation at 0.30–0.49, and high correlation at ≥ 0.50. To identify the independent variables (leptin, adropin, relative weight, and HOMA-IR) that contribute to fatty liver disease, multiple logistic regression analysis was performed and values were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). All p-values are two-tailed and group differences or correlations with p
Oya Sayın, Yavuz Tokgöz and Nur Arslan*
Investigation of adropin and leptin levels in pediatric obesity-related nonalcoholic fatty liver disease Abstract Aim: Nonalcoholic fatty liver disease (NAFLD) is the accumulation of excess fat in the liver in the absence of alcohol consumption, which is commonly associated with obesity and increased risk of atherosclerosis as well as insulin resistance. Adropin is a recently identified protein encoded by the gene related with energy homeostasis, which is expressed in the liver and the brain and has a role in preventing insulin resistance and obesity. The aim of this study was to investigate the serum adropin and leptin levels in obese adolescents and compare the patients with, and without, NAFLD and with healthy controls. Methods: Sixty-four obese adolescents (30 with NAFLD, 34 without NAFLD) and 36 healthy controls were enrolled in the study. Serum adropin and leptin levels were evaluated by sandwich enzyme-linked immunosorbent assay. Results: Serum adropin levels were significantly lower in obese children than healthy controls (3.2 ± 1.0 and 9.2 ± 1.2 ng/mL, respectively, p = 0.001). Serum leptin levels were significantly higher in patients than in controls (12.4 ± 1.1 and 4.1 ± 3.1 pg/mL, respectively; p = 0.000). Serum adropin levels of patients with NAFLD were significantly lower than in patients without NAFLD (2.9 ± 0.5 and 3.5 ± 1.2 ng/ mL, respectively; p = 0.023) and healthy controls (p = 0.000). Logistic regression analysis showed that a decrease in adropin levels was the only independent factor for fatty liver disease in obese adolescents (odds ratio: 3.07, 95% confidence interval 1.14–8.2, p = 0.026). Leptin, relative weight and HOMA-IR of the patients were not independent risk factors for NAFLD. Conclusions: In this study, serum adropin levels were significantly lower in obese adolescents with fatty liver disease compared to patients without fatty liver disease and healthy controls. Lower adropin level was an independent risk factor for NAFLD in obese adolescents in logistic regression analysis. Assessment of serum adropin concentrations may provide a reliable indicator of fatty liver disease in obese adolescents. Keywords: adolescent; adropin; fatty liver disease; leptin; obesity.
*Corresponding author: Nur Arslan, Division of Pediatric Gastroenterology, Hepatology and Nutrition, Dokuz Eylul University Faculty of Medicine, Department of Pediatrics, Izmir, Turkey, Phone: +90 2324126107, Fax: +90 2324126005, E-mail: [email protected]; and Dokuz Eylul University Health Science Institute, Department of Molecular Medicine, Izmir, Turkey Oya Sayın: Research Laboratory, Dokuz Eylul University Faculty of Medicine, Izmir, Turkey Yavuz Tokgöz: Diyarbakir Children Hospital, Department of Pediatric Gastroenterology, Hepatology, Diyarbakir, Turkey
Introduction Nonalcoholic fatty liver disease (NAFLD) is characterized by the excessive accumulation of large droplets of triglycerides within hepatocytes in the absence of chronic alcohol consumption. The spectrum of NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) and cirrhosis, and, in some patients, development of hepatocellular carcinoma. It is known that obesity, insulin resistance, oxidative stress, and inflammation have an important role in the development and progression of NAFLD (1–4). Development and complications of obesity consist of complex mechanisms, including numerous adipokines, hormones, and cytokines (5). Adipokines are hormones that originated from adipose tissue. These proteins coordinate insulin homeostasis, immunity, and obesity related inflammation. Leptin is the most important adipokine produced by adipose tissue. It plays a key role in the satiety and appetite for the regulation of body weight. Serum leptin concentrations have been shown to be increased in patients with obesity, insulin resistance, metabolic syndrome, and NAFLD (1, 6, 7). Adropin (ADR, molecular weight 7.927 kDa) was recently defined by Kumar et al. in 2008 (8). Its name is derived from the Latin roots “aduro” (to combust) and “pinquis” (fats or oils). Adropin is encoded by the “energy homeostasis associated gene” (Enho) and is mainly expressed in the liver and brain (8). Adropin plays an important role in the regulation of insulin sensitivity, energy homeostasis, and probably, in obesity (9). Adropin is also present in heart, kidney (10), and in cow’s milk
Brought to you by | Universitaetsbibliothek der LMU Muenchen Authenticated | 129.187.254.47 Download Date | 7/3/14 6:33 AM
480 Sayın et al.: Adropin levels in fatty liver disease and plasma (11). To date, only a few studies have been completed assessing the serum adropin levels in human diseases such as obstructive sleep apnea (12), cardiac syndrome X (13), gestational diabetes (14), adult obesity (15), and heart failure (9). To our knowledge, adropin has not been studied in obese children and in obesity-related NAFLD in humans, and consequently, the possible role of adropin in the development of NAFLD is unknown. The aim of this study was to investigate the serum adropin and leptin concentrations in obese adolescents and compare the patients with, and without, NAFLD and with healthy controls. Another aim of this study was to evaluate the relationship between serum adropin levels and insulin resistance (IR).
Patients and methods Patients Sixty-four obese adolescents (37 males, 27 females, mean age 12.9 ± 2.1 years, range 11–16 years) were enrolled in the study. Patients with chronic liver disease, hypothyroidism, familial hyperlipidemia, hypertension, diabetes mellitus, Cushing syndrome, complete gonadal dysgenesis, growth hormone deficiency, and those that used corticosteroids were all excluded. Demographic data of all patients were recorded. Physical examinations and anthropometric measurements of all patients and controls were performed. Weight was obtained with a calibrated scale and recorded to the nearest 0.1 kg. Height was measured with a stadiometer and recorded to the nearest 0.1 cm. The body mass index (BMI) was calculated as weight divided by height squared (kg/m2). Obesity was defined as a BMI exceeding the 95th percentile (16). Relative weight (RW) was also calculated for each adolescent. Diagnosis of NAFLD was done based on increased echogenicity via ultrasound compatible with fatty infiltration of the liver (17, 18). Obese patients were divided into two groups: patients with NAFLD (group 1, 30 patients) and patients without NAFLD (group 2, 34 patients) according to their liver brightness at ultrasonography. The control group consisted of 36 adolescents (19 males, 17 females, mean age 13.2 ± 2.0 years, range 11–16 years) who were admitted to the well child outpatient clinic of our hospital for medical control (screening for hepatitis, thyroid functions, glucose, lipid profile or anemia, etc.). None of the adolescents had a history of drug usage and metabolic, cardiovascular, respiratory or hepatic disease. Antropometric values and abdominal ultrasound of the controls were normal. Insulin and glucose levels, lipid profiles and liver function tests were performed and found normal in the control group.
alanine amino transferase (ALT), and aspartate amino transferase (AST). Hepatotropic viruses, serum copper and ceruloplasmin levels, serum alpha 1-antitrypsin level, and autoantibodies against nuclear, smooth muscle, liver, and kidney microsomal type-1 antigens were screened to eliminate infectious, metabolic, and immunological liver pathologies if the patients’ ALT levels were above the accepted values. Homeostasis model assessment (HOMA-IR) index was used to estimate the insulin resistance and it was calculated by “fasting insulin × fasting glucose/22.5” as described by Matthews et al. (19). HOMAIR levels of > 4.0 in adolescents was defined as an insulin-resistance status. Serum adropin measurement was carried out using human adropin enzyme-linked immunosorbent assay (ELISA) kit (Catolog No. S-1438, limit determination 0–25 ng/mL; Bachem, Bubendorf, Switzerland) as recommended by the manufacturer’s protocol. Leptin serum concentration was measured using human leptin enzymelinked immunosorbent assay (ELISA) kit (Catalog No. EK0437; Boster Biological Technology Co., Ltd, Fremont, CA, USA) The samples were read on a Synergy HT, Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) plate reader by measuring the absorbance at a wavelength of 450 nm. Venous blood was collected from all patients after an overnight fasting and then centrifuged at 4°C 3000 g for 10 min. Serum samples were stored at –20°C until the day of analysis.
Ethical approval The study protocol was designed in compliance with the Declaration of Helsinki. Informed consent was obtained from both the adolescents and their parents or legal guardians on enrollment in the study. The study was started after the approval of the Ethics Committee of the Dokuz Eylul University Faculty of Medicine.
Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 15.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were given as mean ± standard deviation; categorical variables were defined as percentages. One-way analysis of variance was used for analyzing group averages among three groups. Student t-test and Mann-Whitney U-test were used for comparing two group averages as a post hoc test. Chi-square test was used for comparing group ratios. Intercorrelations between parameters were computed through the Pearson’s correlation analysis. Correlation coefficient indicated low correlation at 0.10–0.29, medium correlation at 0.30–0.49, and high correlation at ≥ 0.50. To identify the independent variables (leptin, adropin, relative weight, and HOMA-IR) that contribute to fatty liver disease, multiple logistic regression analysis was performed and values were expressed as odds ratios (ORs) and 95% confidence intervals (CIs). All p-values are two-tailed and group differences or correlations with p
