The association of angiopoietin-like peptide 4 levels with obesity and hepatosteatosis in adolescents

Cytokine 125 (2020) 154802

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The association of angiopoietin-like peptide 4 levels with obesity and hepatosteatosis in adolescents

T

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Okan Dikkera, , Nevin Çetin Dağb, Mustafa Şahinc, Emine Türkkanb, Hüseyin Dağb a

University of Health Sciences, Istanbul Okmeydani Training and Research Hospital, Department of Medical Biochemistry, Istanbul, Turkey University of Health Sciences, Istanbul Okmeydani Training and Research Hospital, Department of Pediatrics, Istanbul, Turkey c Hitit University, Erol Olçok Training and Research Hospital, Department of Medical Biochemistry, Çorum, Turkey b

A R T I C LE I N FO

A B S T R A C T

Keywords: Angiopoietin-like peptide 4 Obesity Hepatosteatosis Adolescents

Introduction: Angiopoietin-like peptide 4 (ANGPTL-4) is an adipocytokine that regulates plasma lipoprotein levels by inhibiting the lipoprotein lipase enzyme. Changes in lipid profile can be seen in obese adolescents. Nonalcoholic fatty liver disease may also be a complication of obesity. Based on this information, in this study we aimed to evaluate the relationship between serum ANGPTL-4 levels and obesity and hepatosteatosis in adolescents. Materials and methods: A total of 85 volunteer adolescents, 55 of them were obese and 30 of them were normal weight, were included in our study. The adolescents having body mass index (BMI) 95% percentile and over according to age and sex was defined as obese. Thirty patients with grade 2–3 hepatosteatosis in abdominal ultrasound (USG) were included in ‘obese adolescents with hepatosteatosis’ subgroup and 25 obese cases with no hepatosteatosis in the USG were included in the ‘obese adolescents without hepatosteatosis′ group. Thirty patients with no hepatosteatosis in the abdominal USG and having BMI in normal percentiles according to their age and sex constituted the ‘healthy control adolescents′ group. Serum ANGPTL-4 levels were measured by Enzyme Linked Immunosorbent Assay. Laboratory tests, gender, age and BMI levels were compared statistically between groups. Correlations between ANGPTL-4 and other laboratory parameters were examined statistically in obese adolescent group. Results: The BMI, ANGPTL-4, HbA1c, AST, ALT, total cholesterol, triglyceride, LDL-cholesterol, HOMA-IR and insulin levels of the obese adolescent group were found to be significantly higher than the healthy control group (p < 0.05). We found no statistically significant difference in BMI, ANGPTL-4, triglyceride, insulin and HOMAIR levels among obese adolescents with or without hepatosteatosis (p > 0.05). In all obese adolescent groups and in obese adolescent group with hepatosteatosis; there was no statistically significant relationship between ANGPTL-4 and other variables (p > 0.05). Conclusions: We found that the levels of ANGPTL-4 increases in obesity in adolescents. However, our results make it difficult to establish a relationship between hepatosteatosis and ANGPTL-4. Targeting ANGPTL-4 may be beneficial for the pathogenesis and associated complications of obesity.

1. Introduction Lipoprotein lipase (LPL) plays a major role in the clearance of triglyceride (TG)-rich plasma lipoproteins [1]. LPL is localized in capillary endothelium. LPL catalyzes the hydrolysis of endogenous very low

density lipoprotein (VLDL)-TG and exogenous chylomicron-TG to glycerol and free fatty acids (FFAs) [2]. The resulting FFAs are taken up by a plurality of tissues. FFAs are directed to oxidation or storage or they are switched to systemic circulation [3,4]. Angiopoietin-like protein 4 (ANGPTL-4) is also known as

Abbreviations: ALT, Alanine amino transferase; ANGPTL-4, Angiopoietin-like protein 4; AST, Aspartate amino transferase; BMI, Body-mass index; CRP, C-reactive protein; CV, Variation coefficient; ELISA, Enzyme linked immunosorbent assay; FFA, Free fatty acids; HDL-cholesterol, High density lipoprotein-cholesterol; HOMAIR, Homeostatic model of assessment for insulin resistance; LDL-cholesterol, Low density lipoprotein-cholesterol; LPL, Lipoprotein lipase; NAFLD, Nonalcoholic fatty liver disease; PEDF, Pigment epithelium-derived factor; PLT, Platelet; RBC, Red blood cell; SD, Standard deviation; TG, Triglyceride; TSH, Thyroid stimulating hormone; USG, Ultrasound; VLDL, Very low density lipoprotein; WBC, White blood cell ⁎ Corresponding author at: Darülaceze Street, No: 27, Sisli, Istanbul, Turkey. E-mail address: [email protected] (O. Dikker). https://doi.org/10.1016/j.cyto.2019.154802 Received 24 March 2019; Received in revised form 21 July 2019; Accepted 7 August 2019 1043-4666/ © 2019 Elsevier Ltd. All rights reserved.

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examinations, an additional 1 tube of blood will be taken for the study). After taking the informed consent form from the volunteers and their parents who wanted to participate in the study, detailed histories of the participants were obtained. During obtaining blood from the volunteers for their routine examinations, only one more biochemistry tube of blood was taken for the study. The serum was kept at room temperature for 20 min, centrifuged at 4000 rpm for 10 min and stored at −80 °C.

peroxisome proliferator-activated receptor-γ, angiopoietin-related protein, hepatic fibrinogen/angiopoietin-related protein or fasting-induced adipose factor [5,6]. Although it is found mainly in the liver, ANGPTL-4 is also expressed in a variety of tissues such as adipose tissue, placenta, small intestine and heart [7,8]. ANGPTL-4 is a molecule that inhibits the LPL enzyme, regulates plasma lipoprotein levels and thereby suppresses the clearance of TG- rich lipoproteins, which leads to an increase in plasma TG levels [9,10]. Childhood obesity is an important public health problem with an increasing prevalence in all over the world; which approximately affects 25–30% of children [11]. Approximately 50% of obese adolescents were also found to be obese in their adulthood [12]. Obesity, which is characterized by excessive accumulation of lipids and adipose tissue, and causing ectopic fat accumulation in different tissues, is a chronic proinflammatory condition with increased cytokine levels [13,14]. Increased serum TG, total cholesterol, low density lipoprotein (LDL-cholesterol) levels and decreased high density lipoprotein (HDL-cholesterol) levels may be seen in obese adolescents [15]. Nonalcoholic fatty liver disease (NAFLD), which is defined as the presence of fat, especially TGs, in the liver, for more than 5% by weight, or fulfilling of more than 5% of the hepatocytes with fat vacuoles [16,17], is seen as a complication of obesity, and affecting 22–52% of obese children [18]. Increases in the amount of fatty acid coming to the liver or fatty acid synthesis in the liver are the main causes of fatty liver disease [19]. Insulin, a hormone involved in fatty acid metabolism, activates LPL [20,21] and has been found to be high in obese adolescents [22]. Although decreased levels of ANGPTL-4, which is closely related to fatty acid metabolism, have been reported in adult fatty liver disease [23], a study investigating the levels of ANGPTL-4 in adolescents has not been reported in the literature. When we examine the pathogenesis and complications of obesity and the functions of ANGPTL-4, we thought that ANGPTL-4 may have an important role in obese adolescents. Therefore in our study; we aimed to evaluate the relationship between serum ANGPTL-4 levels and obesity and hepatosteatosis in adolescents.

2.2. Measurement of ANGPTL-4 levels On the day of analysis, sera were allowed to dissolve at room temperature. Ready-to-use Enzyme Linked Immunosorbent Assay (ELISA) kits were used for measurements of ANGPTL-4 levels in serum (Human ANGPTL-4 ELISA, Elabscience, Texas, USA. Lot No: ZE5V12UF). The analytical (linear) measurement range was 1.56–100 ng/mL for ANGPTL-4. The minimal detection limit was 0.94 ng/mL. The reported intraassay and interassay variation coefficients (CVs) were < 5.1% and < 4.8%, respectively. 2.3. Measurement of other laboratory tests Glucose, urea, creatinine, aspartate amino transferase (AST), alanine amino transferase (ALT), C-reactive protein (CRP), total cholesterol, TG, HDL-cholesterol, LDL-cholesterol and calcium tests were measured with colorimetric method; thyroid stimulating hormone (TSH), free T4 (fT4), 25-hydroxyvitamin D3 and insulin tests were measured in the autoanalyzer (Beckman Coulter Brand, AU 5800 model, USA) by chemiluminescence immunoassay method. HbA1c was measured by high performance liquid chromatography method in autoanalyser (Biorad, Variant II turbo, Japan). Hemogram parameters were measured by autoanalyser (Mindray brand, BC6800 model, China). LDL-cholesterol was calculated using the Friedewald formula [LDL-cholesterol = total cholesterol − (HDL-cholesterol) − (TG/5)]. 2.4. Statistical analysis

2. Materials and methods

Laboratory tests, gender, age and BMI levels were compared statistically between the groups. Correlations between ANGPTL-4 and other laboratory parameters were examined statistically in obese adolescent group. For this, IBM SPSS Statistics 22 (SPSS Inc, Chicago Illinois) programs were used. The conformity of the parameters to the normal distribution was evaluated by Shapiro-Wilks test. As well as the descriptive statistical methods (mean, standard deviation, frequency), One-way ANOVA test was used to compare the normally distributed quantitative data, and Tukey HSD test and Tamhane′s T2 test were used to determine the group that caused the difference. The Kruskal Wallis test was performed to compare the non-normally distributed data between groups and Mann Whitney U test was used for the determination of the groups causing difference. The Student′s t test was used for the comparison of the parameters with normal distribution between two groups, and the Mann Whitney U test was used for the comparison of the parameters that did not show normal distribution. Chi-Square test and Continuity (Yates) Correction were used to compare qualitative data. Pearson correlation analysis was performed to examine the relationships between the parameters that are compatible with normal distribution. Significance was evaluated as p < 0.05.

2.1. Patients and design A total of 85 volunteer adolescents were included in our study, aged between 10 and 16 years, who were admitted to the pediatrics outpatient clinic between 1 February and 8 March 2019. Among study participants, 55 were obese and 30 were normal weight, healthy, control cases. The body mass index (BMI) was obtained by division of weight of the patient in kg to the square of the height in meters. Adolescents having a BMI of 95% percentile and over according to their age and sex were defined as obese [24]. Thirty patients with grade 2–3 hepatosteatosis in abdominal ultrasound (USG) were defined as obese adolescents with hepatosteatosis; 25 obese cases with no hepatosteatosis in the USG were defined as obese adolescents without hepatosteatosis. Thirty patients with no hepatosteatosis in the abdominal USG and having BMI in normal percentiles according to their age and sex constituted the healthy control adolescents group. Homeostatic model of assessment for insulin resistance (HOMA-IR) was calculated with the formula; (insulin IU/L × glucose mg/dL)/405 [25]. Obese adolescents with HOMA-IR values higher than 2.5 were evaluated as having insulin resistance. Smokers, patients with chronic diseases, infections, malignancy and those with metabolic and endocrinological diseases were not included in obese or control groups. Any of the adolescents in the control and obese groups were not taking any medications, including the statin group. In addition, adolescents with insulin resistance were not included in the healthy control group. The patients who were observed to be in compliance with the inclusion criteria were informed about the study (in addition to routine

2.5. Ethical approval The study was approved by the Ministry of Health Okmeydanı Training and Research Hospital Ethics Committee (date: 09.10.2018 and number: 1003). 3. Results The mean age of adolescents was 12.80 ± 1.86 years. The mean 2

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Table 1 The data of obese and control groups.

Age (year) Gender n (%) Female Male BMI (kg/m2) ANGPTL-4 (ng/mL) HbA1c (%) Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) AST (U/L) (median) ALT (U/L) (median) CRP (mg/L) Total cholesterol (mg/dL) Triglyceride (mg/dL) (median) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Calcium (mg/dL) TSH (mU/L) Free T4 (ng/L) 25-Hydroksivitamin D3 (ug/L) HOMA-IR (median) Insulin (IU/L) (median) WBC (×103/μL) RBC (median) (x106/μL) PLT (x103/μL)

Obese adolescent group (n:55)

Healthy control adolescent group (n:30)

p

Mean ± SD

Mean ± SD

12.76 ± 2.06

12.88 ± 1.83

1

24 (43%) 31 (57%) 31.19 ± 3.75 53.48 ± 12.31 5.49 ± 0.37 89.9 ± 9.66 24.71 ± 6.25 0.54 ± 0.11 25.02 ± 9.14 (23) 24.85 ± 11.55 (21) 6.39 ± 4.09 167.78 ± 38.93 126.46 ± 59.47 (1 1 2) 44 ± 9.43 105.85 ± 32.76 10.03 ± 0.3 2.76 ± 1.12 8.71 ± 1.53 19.3 ± 6.17 4.09 ± 2.08 (3.6) 18.35 ± 8.75 (16.8) 8.24 ± 1.58 4.92 ± 0.33 (4.8) 307.1 ± 76.87

14 (46%) 16 (54%) 23.36 ± 3.17 35.87 ± 12.03 5.23 ± 0.31 88.46 ± 9.05 23.92 ± 6.55 0.53 ± 0.14 21.25 ± 5.7 (19.5) 15.96 ± 7.95 (13) 5.01 ± 3.68 148.25 ± 29.2 81.75 ± 27.16 (80) 50.29 ± 11.53 85.96 ± 23.3 9.93 ± 0.38 2.3 ± 1.13 8.4 ± 1.28 17.26 ± 6.92 1.92 ± 0.75 (1.7) 8.7 ± 3.06 (8.3) 7.79 ± 2.02 5 ± 0.46 (4.9) 296.25 ± 90.47

2

0.816 0.231

1

< 0.001* < 0.001* 0.006* 1 0.554 1 0.630 1 0.825 3 0.032* 3 < 0.001* 1 0.181 1 0.037* 3 0.002* 1 0.020* 1 0.011* 1 0.219 1 0.116 1 0.398 1 0.223 3 < 0.001* 3 < 0.001* 1 0.325 3 0.749 1 0.609 1 1

ALT: Alanine amino transferase, AST: Aspartate amino transferase, ANGPTL-4: Angiopoietin-like protein 4, BMI: Body-Mass Index, CRP: C-reactive protein, HOMAIR: Homeostatic model of assessment for insulin resistance, HDL-cholesterol: High density lipoprotein- cholesterol, LDL-cholesterol: Low density lipoprotein- cholesterol, PLT: Platelet, RBC: Red blood cell, SD: Standard deviation, TSH: Thyroid stimulating hormone, WBC: White blood cell. 1 Student t test. 2 Continuity (Yates) correction. 3 Mann Whitney U test. * p < 0.05.

BMI values were 28.30 ± 5.19 kg/m2. There was no statistically significant difference regarding age, gender distribution, glucose, urea, creatinine, CRP, calcium, TSH, fT4, 25-hydroxyvitamin D3, white blood cell (WBC), red blood cell (RBC) and platelet (PLT) levels between obese adolescents and healthy control groups (p > 0.05) (Table 1). The BMI, ANGPTL-4, HbA1c, AST, ALT, total cholesterol, TG, LDLcholesterol, HOMA-IR and insulin levels of the obese adolescent group were significantly higher than the healthy control group (p < 0.05). HDL-cholesterol levels of obese adolescents were found to be significantly lower than healthy controls (p < 0.05) (Table 1). No statistically significant difference was found between the groups in terms of age, gender, glucose, urea, creatinine, AST, calcium, TSH, fT4, 25-hydroxyvitamin D3 levels, WBC, RBC and PLT levels (p > 0.05) (Table 2). The BMI, ANGPTL-4, TG, insulin and HOMA-IR levels of the healthy control group were significantly lower than those without hepatosteatosis (p < 0.05). There was no statistically significant difference in BMI, ANGPTL-4, TG, insulin and HOMA-IR levels between obese adolescents with or without hepatosteatosis (p > 0.05) (Table 2). CRP and HbA1c levels of the healthy control group were significantly lower than the obese adolescents with hepatosteatosis (p < 0.05). There was no statistically significant difference between the other groups in terms of CRP and HbA1c levels (p > 0.05) (Table 2). ALT levels of the healthy control group were found to be significantly lower than the obese adolescents with and without hepatosteatosis (p < 0.05). ALT levels of obese adolescents without hepatosteatosis were significantly lower than that of obese adolescents with hepatosteatosis (p < 0.05) (Table 2).

The total cholesterol and LDL-cholesterol levels of the healthy control group were significantly lower than the obese adolescents without hepatosteatosis (p < 0.05). There were no statistically significant differences between the other groups in terms of total cholesterol and LDL-cholesterol levels (p > 0.05) (Table 2). HDL-cholesterol levels of the healthy control group were significantly higher than the obese adolescents with hepatosteatosis (p < 0.05). There was no statistically significant difference between the other groups in terms of HDL-cholesterol levels (p > 0.05) (Table 2). In addition, when the correlation of ANGPTL-4 levels with other parameters were analyzed with Pearson correlation analysis; in the obese adolescent group (n: 55) and in the obese adolescent group with hepatosteatosis (n: 30); there was no statistically significant correlation between age, BMI, HbA1c, glucose, urea, creatinine, AST, ALT, CRP, cholesterol, TG, HDL-cholesterol, LDL-cholesterol, calcium, TSH, fT4, 25-hydroxyvitamin D3, HOMA-IR, insulin WBC, RBC and PLT levels with ANGPTL-4 (p > 0.05).

4. Discussion Lipoproteins, such as chylomicrons created by VLDL produced by the liver or dietary TG, mediate the transfer of TG for oxidation to produce energy or storage in various tissues [26,27]. ANGPTL-4 is a molecule which reduces the hydrolysis of TGs in TG-rich lipoproteins by inhibiting LPL [9,10]. ANGPTL-4 also functions as an early pro-angiogenic cytokine that induces adipocyte differentiation and endothelial cell growth necessary for adipose tissue expansion [28,29]. In our study, we found increased ANGPTL-4 levels of obese adolescents compared to healthy control adolescents. However, in our study, we did not 3

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Table 2 Data of healthy control adolescents and obese adolescents with and without hepatosteatosis.

Age (years) Gender n (%) Female Male BMI (kg/m2) ANGPTL-4 (ng/mL) HbA1c (%) Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) AST (U/L) (median) ALT (U/L) (median) CRP (mg/L) Total cholesterol (mg/dL) Triglyceride (mg/dL) (median) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Calcium (mg/dL) TSH (mU/L) Free T4 (ng/L) 25-Hydroxivitamin D3(ug/L) HOMA-IR (median) Insulin (IU/L) (median) WBC (x103/μL) RBC (median) (x106/μL) PLT (x103/μL)

Obese adolescent group without hepatosteatosis (n:25)

Obese adolescent group with hepatosteatosis (n:30)

Healthy control Adolescent group (n:30)

p

Mean ± SD

Mean ± SD

Mean ± SD

12.55 ± 2.13

13 ± 2

12.88 ± 1.83

1

11 (44%) 14 (56%) 30.04 ± 2.88 51.62 ± 6.84 5.4 ± 0.34 87.91 ± 9.46 25.18 ± 6.48 0.54 ± 0.1 24.05 ± 7.56 (22) 21.14 ± 8.06 (18) 5.15 ± 3 177.91 ± 38.51 133.91 ± 63.72 (1 2 2) 46.05 ± 9.76 113 ± 31.42 10.05 ± 0.32 2.98 ± 1.13 8.85 ± 1.65 19.73 ± 6.66 3.61 ± 1.27 (3.3) 16.71 ± 5.76 (15.7) 8.06 ± 1.7 4.92 ± 0.37 (4.8) 299.18 ± 60.67

11 (36%) 19 (64%) 32.52 ± 4.25 55.63 ± 16.53 5.58 ± 0.39 92.21 ± 9.61 24.16 ± 6.11 0.54 ± 0.13 26.16 ± 10.79 (26) 29.16 ± 13.58 (26) 7.81 ± 4.77 156.05 ± 36.99 117.84 ± 54.56 (99) 41.63 ± 8.7 97.58 ± 33.13 10.01 ± 0.28 2.51 ± 1.08 8.55 ± 1.4 18.81 ± 5.7 4.64 ± 2.68 (4.1) 20.24 ± 11.16 (17.4) 8.45 ± 1.45 4.92 ± 0.29 (4.8) 316.26 ± 93.12

14 (46%) 16 (54%) 23.36 ± 3.17 35.87 ± 12.03 5.23 ± 0.31 88.46 ± 9.05 23.92 ± 6.55 0.53 ± 0.14 21.25 ± 5.7 (19.5) 15.96 ± 7.95 (13) 5.01 ± 3.68 148.25 ± 29.2 81.75 ± 27.16 (80) 50.29 ± 11.53 85.96 ± 23.3 9.93 ± 0.38 2.3 ± 1.13 8.4 ± 1.28 17.26 ± 6.92 1.92 ± 0.75 (1.7) 8.7 ± 3.06 (8.3) 7.79 ± 2.02 5 ± 0.46 (4.9) 296.25 ± 90.47

2

0.746 0.276

1

< 0.001* < 0.001* 0.006* 1 0.291 1 0.783 1 0.970 3 0.077 3 < 0.001* 1 0.039* 1 0.017* 3 0.008* 1 0.026* 1 0.010* 1 0.441 1 0.118 1 0.559 1 0.433 3 < 0.001* 3 < 0.001* 1 0.479 3 0.950 1 0.706 1 1

ALT: Alanine amino transferase, AST: Aspartate amino transferase, ANGPTL-4: Angiopoietin-like protein 4, BMI: Body-Mass Index, CRP: C-reactive protein, HOMAIR: Homeostatic model of assessment for insulin resistance, HDL-cholesterol: High density lipoprotein-cholesterol, LDL-cholesterol: Low density lipoprotein-cholesterol, PLT: Platelet, RBC: Red blood cell, SD: Standard deviation, TSH: Thyroid stimulating hormone, WBC: White blood cell. 1 Oneway ANOVA test. 2 Chi-square test. 3 Kruskal wallis test. * p < 0.05.

find any correlation between ANGPTL-4 levels and BMI, HbA1c HOMAIR, TG levels and other parameters. Cinkajzlová et al. found high ANGPTL-4 levels in a study of obese adults [30]. Barja-Fernandez et al. found high ANGPTL-4 levels in obese subjects with impaired glucose tolerance. They reported that increased ANGPTL-4 levels with weight gain decreased with weight loss. Also, ANGPTL-4 levels showed positive correlation with BMI, HbA1c, HOMAIR, TG, CRP and leukocyte levels, and negatively correlated with HDLcholesterol [31]. Ortega-Senovilla et al. reported that in overweight and obese pregnant women, ANGPTL-4 concentrations increased continuously during pregnancy, and increase in plasma ANGPTL-4 levels were reported to be positively associated with gestational weight gain [32]. In accordance with these studies in the literature, in our study we showed that there is a relationship between obesity and increased ANGPTL-4 levels in adolescents. In contrast to all these studies, Donma et al. reported no changes in ANGPTL-4 levels in morbidly obese children less than 10 years of age in their study [33]. Adipose tissue increases in obese individuals. Abnormal secretion of adipokines (or adipocytokines) from adipose tissue triggering a significant increase in intracellular lipid and excessive accumulation of adipose cells play a causal role in obesity [34]. Reduced adiponectin levels [35] and increased levels of interleukin-6 have been reported in obese patients [36]. Makoveichuk et al. showed that the effect of a cytokine which decreases fat tissue such as tumor necrosis factor-alpha was by increasing the synthesis of ANGPTL-4 and its inhibition on LPL [37]. The role of subclinical inflammatory process in the pathogenesis of obesity has been shown [38]. In addition, pigment epithelium-derived factor (PEDF) which is secreted by adipocytes (an adipocyte-secreted factor) and increased in obesity is effective on adipose triglyceride lipase, a regulator of triacylglycerol metabolism in the liver.

PEDF increases adipocyte lipolysis, promotes lipid accumulation associated with insulin resistance in muscle and liver and induces proinflammatory signaling [39]. In this regard, in our study inflammatory cytokines and molecules increased in adolescent obesity may have led to an increase in ANGPTL-4 levels. An increase in the amount of fatty acids in the liver, in some conditions such as obesity and hunger; excessive carbohydrate intake by diet or total parenteral nutrition and increased fatty acid synthesis in the liver are among the causes of fatty liver disease [19]. In our study, we divided the obese adolescents into two groups as with or without fatty liver and found no significant difference in ANGPTL-4 levels between the groups. In contrast to obesity in adolescents, this data made it difficult to establish a relationship between hepatosteatosis and ANGPTL-4 levels. In their study, Altun et al. found a significant decrease in ANGPTL-4 levels in adult hepatosteatosis patients compared to healthy control group. In addition, they found negative correlation between TG, total cholesterol, LDL-cholesterol and AST with ANGPTL-4; but did not report any correlation between glucose, ALT, HOMA-IR and HDL-cholesterol. Insulin and HOMA-IR levels were significantly higher in patients with hepatic steatosis [23]. However, insulin and HOMA-IR levels were not significantly different between our obese adolescents with or without hepatosteatosis. This data suggests that insulin may be effective on ANGPTL-4 serum levels. Insulin increases the synthesis of TG and FFA in liver and fat tissue. The resulting fatty acids are taken up by adipocytes and muscle cells. Insulin causes the fatty acids to be used according to the energy needs, and the unused ones are converted into TGs in the adipose tissue to accumulate through lipogenesis. After meals, TGs are stored by the action of insulin [20]. Insulin increases LPL activity [20,21]. LPL 4

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difficult to establish a relationship between hepatosteatosis and ANGPTL-4. Targeting ANGPTL-4 may be beneficial for the pathogenesis and associated complications of obesity.

hyperactivity is associated with decreased plasma TG levels, whereas loss of function results in severe hypertriglyceridemia [20]. In fasting, insulin levels are low and LPL activity decreases in adipose tissue to promote the use of TGs in peripheral tissues [40,41]. Indeed, we also found high levels of insulin in the adolescent obese group where we found high levels of ANGPTL-4 levels. We believe that ANGPTL-4 levels have increased for the purpose of compensation for suppressing activation in LPL, which increases with the effect of increased insulin levels in obese patients. In addition, the ANGPTL-4 levels were similar in groups having similar insulin and HOMA-IR levels (in obese patients with or without hepatosteatosis), and there was no difference in TG levels in these two groups, which suggested that ANGPTL-4 levels increased secondary to insulin increase. ANGPTL-4 expression is stimulated in vitro with FFA [6]. Accordingly, diet modulations that increase plasma FFA levels, including longterm fasting, very low-calorie, and high-fat, high-energy diets, increase plasma ANGPTL-4 levels [5,42]. Wang et al. showed that overexpression of ANGPTL-4 decreased body weight, increased total cholesterol, TG levels, increased intracellular hydrolysis of TGs, and increased levels of FFA in plasma by adipolysis. They also reported that ANGPTL-4 overexpression promotes liver steatosis in mice, enhancing insulin sensitivity and glucose tolerance [43]. Xu et al. reported that the expression of adenovirus-mediated ANGPTL-4 induces hyperlipidemia, hepatomegaly and hepatosteatosis in rats [10]. These studies show the relationship between high ANGPTL-4 levels and hepatosteatosis in contrast to the study of Altun et al. [23] and our study. Therefore, more clinical studies are warranted to explain the alterations of ANGPTL-4 levels in fatty liver disease. Koster et al. demonstrated that hepatic overexpression of ANGPTL-4 in transgenic mice resulted in hypertriglyceridemia and decreased LPL activity, whereas ANGPTL-4 deficiency resulted in hypotriglyceridemia [44]. In ANGPTL-4 null mice or in mice injected with ANGPTL-4 antibody, circulating TG levels were shown to decrease [44–46]. Increased levels of ANGPTL-4 may be responsible for increased plasma TG levels in adolescent obesity. Specific approaches targeting ANGPTL-4 can be used to treat plasma lipid content. Lichenstein et al. reported that ANGPTL-4 protects against severe pro-inflammatory effects of dietary saturated fat by inhibiting lipoprotein lipase-dependent uptake of fatty acids in mesenteric lymph node macrophages [47]. Georgiadi et al. reported that ANGPTL-4 suppresses foam cell formation and reduces atherosclerosis. They have also reported that stimulation of ANGPTL-4 with oxidized low-density lipoprotein in macrophages may provide protection against lipid overload [48]. The protective role of ANGPTL-4 has been shown in these studies. In addition, in our study, CRP and HbA1c levels were significantly lower in control group than those of obese adolescents with hepatosteatosis. Elevated CRP levels suggests a low-grade inflammation in obesity [38], elevated HbA1c suggests insulin resistance and predisposition to type 2 diabetes mellitus [49]. Also the ALT levels of the healthy control group were significantly lower than the obese adolescents. ALT levels of obese adolescents without hepatosteatosis were significantly lower than that of obese adolescents with hepatosteatosis. Slight increases in ALT levels are among the laboratory findings seen in obesity and slightly higher ALT levels are commonly seen in fatty liver disease [50]. There are some limitations of our study. First, the sample size could have been larger. Secondly, we could not measure inflammatory cytokine levels and LPL activity in the sera of volunteer adolescents. It should also be noted that investigating PEDF and/or other adipocytesecreted factors may strengthen our results.

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5. Conclusions We believe that ANGPTL-4 levels increase in obesity in adolescents for compensation with protective effects. However, our results make it 5

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