Obesity is a complex disease involving a number of

Transcription

1 ORIGINAL Endocrine ARTICLE Research Fibroblast Growth Factor 21 (FGF-21) and Its Relation to Obesity, Metabolic Syndrome, and Nonalcoholic Fatty Liver in Children: A Longitudinal Analysis Thomas Reinehr, Joachim Woelfle, Rainer Wunsch, and Christian L. Roth Departments of Pediatric Endocrinology, Diabetes, and Nutrition Medicine (T.R.) and Pediatric Radiology and Ultrasound (R.W.), Vestische Hospital for Children and Adolescents Datteln, University of Witten/ Herdecke, D Datteln, Germany; Paediatric Endocrinology Division (J.W.), Children s Hospital, University of Bonn, D Bonn, Germany; and Seattle Children s Research Institute (C.L.R.), University of Washington, Seattle, Washington Context: Fibroblast growth factor 21 (FGF-21), a potent activator of glucose uptake, has been proposed to be related to insulin resistance, metabolic syndrome (MetS), nonalcoholic fatty liver disease (NAFLD), and weight status. Objective: Our objective was to study the relationships between FGF-21, parameters of MetS, and NAFLD before and after weight loss in obese children. Design and Setting: This was a cross-sectional comparison between obese and normal-weight children and longitudinal 1-yr follow-up study in obese children participating in a lifestyle intervention in a primary care setting. Patients: Patients included 60 obese and 40 lean children of same age, gender, and pubertal stage. Intervention: The outpatient 1-yr intervention program was based on exercise, behavior, and nutrition therapy. Main Outcomes Measures: We evaluated fasting serum FGF-21, weight status [body mass index (BMI) expressed as SD score (SDS)], body fat, insulin resistance index (homeostasis model assessment), leptin, transaminases, free fatty acids (FFA), waist circumference, blood pressure, and lipids. Results: Compared with the normal-weight children, obese children demonstrated significantly (P 0.001) increased FGF-21, leptin, and homeostasis model assessment levels. FGF-21 was significantly (P 0.05) correlated to BMI, SDS-BMI, FFA, and leptin both in cross-sectional and longitudinal analyses but not to any additional analyzed parameter. Children with and without MetS or NAFLD did not differ significantly with respect to their FGF-21 concentrations. A decrease of SDS-BMI was associated with a significant (P 0.038) decrease of FGF-21 levels (mean 34%). Conclusions: FGF-21 concentrations are reversibly increased in obese children and are related to leptin and FFA. However, our data do not support a significant relationship between FGF-21, insulin resistance, and features of MetS or NAFLD in children. (J Clin Endocrinol Metab 97: , 2012) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2012 by The Endocrine Society doi: /jc Received January 26, Accepted February 23, First Published Online March 21, 2012 Obesity is a complex disease involving a number of different peptides, transmitters, and their receptors controlling energy homeostasis (1). Furthermore, these cytokines are involved in the pathogenesis of various complications of obesity, including dyslipidemia, type 2 diabetes mellitus, and arterial hypertension summarized in the definition of the metabolic syndrome (MetS) (1, 2). Recently, a novel protein, fibroblast growth factor 21 Abbreviations: ALT, Alanine transaminase; AST, aspartate transaminase; BMI, body mass index; CV, coefficient of variation; FFA, free fatty acid; FGF-21, fibroblast growth factor 21; HDL, high-density lipoprotein; IQR, interquartile range; LDL, low-density lipoprotein; MetS, metabolic syndrome; NAFLD, nonalcoholic fatty liver disease; ogtt, oral glucose tolerance test; SDS, SD score; WAT, white adipose tissue. J Clin Endocrinol Metab, June 2012, 97(6): jcem.endojournals.org 2143

2 2144 Reinehr et al. FGF-21 in Obese Children J Clin Endocrinol Metab, June 2012, 97(6): (FGF-21), has been identified that plays an important role in liver and adipose tissue metabolism (3, 4). FGF-21 is a member of the FGF family representing a group of peptides that regulate diverse biological functions, including cell differentiation, cell growth, and angiogenesis (5). FGF-21 is mainly expressed in liver, and it functions as a potent activator of glucose uptake on adipocytes (6, 7). Although FGF-21 was initially proposed as an inducer of lipolysis in adipose tissue (7), several other studies reported that FGF-21 inhibits lipolysis in adipocytes (4, 8). FGF-21 protects animals from diet-induced obesity when overexpressed in transgenic mice and lowers blood glucose and triacylglycerol levels when administered to diabetic rodents (7, 9). Comparable glucose- and triacylglycerol-lowering effects were observed in diabetic rhesus monkeys during long-term FGF-21 treatment (3). Besides its effect on glucose and lipid metabolism in hepatocytes and adipocytes via cell surface FGF receptors (10), the central nervous system has been described as a potentially important target for the beneficial effects of FGF-21 in diet-induced obesity in rats (11). In summary, FGF-21 was assumed to be a novel target with potential antidiabetic properties that might be useful in the treatment of hyperglycemia, insulin resistance, and hyperlipidemia. However, the role of FGF-21 in humans is yet unclear. An association of FGF-21 levels and BMI was found in some (12 16) but not in all human studies (17, 18). Moreover, some authors reported a relationship between FGF-21 and MetS (12, 15), nonalcoholic fatty liver disease (NAFLD) (13), insulin resistance (12, 19), and type 2 diabetes mellitus (14), but the findings are conflicting. Even if such relations exist, the direction of that relation would be unclear. For example, a circulating FGF-21 might affect body weight, but vice versa, body weight might also affect circulating FGF-21. Weight loss studies would be helpful to determine the direction of relationship. Given this partially unclear situation in humans and the lack of studies in children, we evaluated the effects of weight reduction by a lifestyle intervention on FGF-21 levels. We also compared FGF-21 concentrations between obese and normal-weight children and studied the relationships between FGF-21, leptin, free fatty acid (FFA), and parameters of NAFLD and MetS such as transaminases, insulin resistance, lipids, and blood pressure in the course of 1 yr. Longitudinal studies and studies in obese children seem preferable because 1) cross-sectional studies cannot prove causality and are prone to many confounders, 2) adverse patterns of MetS itself begin in childhood, and 3) studies in children have the advantage that there is no potential confusion with other diseases, medications, or active tobacco smoking. We hypothesized that FGF-21 concentrations are increased in obese children compared with normal-weight children and that changes of FGF-21 were associated with changes of weight status, leptin, and parameters of NAFLD and MetS including insulin resistance. The novelty of our study is the longitudinal analysis of FGF-21 concentrations in obese children participating in a lifestyle intervention. Subjects and Methods Subjects Written informed consent was obtained from all children and their parents. The study was approved by the local ethics committee of the University of Witten/Herdecke in Germany. We examined 60 obese Caucasian children and 40 normalweight children of similar age, gender, and pubertal stage. All 60 obese children participated in the lifestyle intervention Obeldicks, which has been described in detail elsewhere (20). Briefly, this outpatient intervention program for obese children is based on physical exercise, nutrition education, and behavior therapy including the individual psychological care of the child and his or her family. The nutritional course is based on a fat- and sugarreduced diet compared with the everyday nutrition of German children. None of the children in the current study suffered from endocrine disorders, premature adrenarche, or syndromal obesity. Measurements We analyzed fasting body mass index (BMI), FGF-21, leptin, and insulin resistance index omeostasis model assessment (HOMA) in 60 obese children and in 40 normal-weight children. Furthermore, we determined in the 60 obese children at baseline and 1 yr later after participating in the lifestyle intervention Obeldicks anthropometrical markers (body fat based on skinfold measurements and waist and hip circumferences), fasting serum FGF-21, leptin, transaminases, and as parameters of MetS blood pressure, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol, triglycerides, glucose, insulin, and uric acid concentrations. Height was measured to the nearest centimeter using a rigid stadiometer. Weight was measured unclothed to the nearest 0.1 kg using a calibrated balance scale. BMI was calculated as weight in kilograms divided by the square of height in meters. The degree of overweight was quantified using Cole s least mean square method, which normalized the BMI skewed distribution and expressed BMI as a SD score (SDS) (21). Reference data for German children were used (22). All children in the study were obese according to the definition of the International Obesity Task Force (23). The pubertal developmental stage was determined according to Marshall and Tanner. Triceps and subscapularis skinfold thickness was measured twice using a caliper and averaged to calculate the percentage of body fat using a skinfold thickness equation with the following formulas (24): for boys, percent body fat (subscapularis skinfold thickness triceps skinfold thickness in millimeters) 1.6; for girls, percent body fat (subscapularis skinfold thickness triceps skinfold thickness in millimeters) 9.7.

3 J Clin Endocrinol Metab, June 2012, 97(6): jcem.endojournals.org 2145 Blood pressure was measured using a validated protocol (25). Systolic and diastolic blood pressure were measured at the right arm twice after a 10-min rest in the supine position by using a calibrated sphygmomanometer and averaged. The cuff size was based on the length and circumference of the upper arm and was as large as possible without having the elbow skin crease, obstructing the stethoscope (25). The intra- and interoperator variability was less than 5% for systolic and diastolic blood pressure. Blood sampling was performed in the fasting state at 0800 h. After clotting, blood samples were centrifuged for 10 min at 8000 rpm. Serum was stored at 81 C for later determination of FGF-21, leptin, and insulin. All samples were thawed only once. FGF-21 levels were measured with a highly specific commercially available ELISA kit (BioVendor, Brno, Czech Republic). No cross-reactivity with human FGF-19 and human FGF-23 has been observed. The sensitivity of the assay was 7 pg/ml, and the intraassay coefficient of variation (CV) and the interassay CV were below 5%. Leptin concentrations were measured by a commercially available ELISA [leptin (human) ELISA Kit; BioVendor, Alexis Biochemicals, Lausen, Switzerland], and insulin concentrations were measured by microparticle enhanced immunometric assay (MEIA; Abbott, Wiesbaden, Germany). Glucose levels were determined by colorimetric test using a Vitros analyzer (Ortho Clinical Diagnostics, Neckargmuend, Germany). Transaminases [aspartate transaminase (AST), alanine transaminase (ALT)], triglycerides, uric acids, LDL-cholesterol, and HDL-cholesterol were determined by commercially available test kits (Roche Diagnostics, Mannheim, Germany). Fasting blood FFA were determined by colorimetric tests (WAKO Freie Fettsäure, Neuss, Germany) Intra- and interassay CV were below 5% in all these methods. All variables were measured in duplicate and averaged. HOMA was used to detect the degree of insulin resistance using the following formula: resistance (HOMA) [insulin (milliunits per liter) glucose (millimoles per liter)]/22.5 (26). An oral glucose tolerance test (ogtt) was performed in all obese children according to current guidelines (27). Impaired glucose tolerance was defined by 2-h serum glucose below 140 mg/dl in ogtt. We defined the MetS following the International Diabetes Foundation definition (28) (waist circumference 90th percentile plus at least two of the following criteria: impaired fasting glucose or impaired glucose tolerance, systolic blood pressure 130 mm Hg or diastolic blood pressure 85 mm Hg, triglycerides 150 mg/dl, and HDL-cholesterol 40 mg/dl). We used German percentiles for waist circumference (19). NAFLD was diagnosed by standardized criteria based on liver ultrasound and transaminases measurements as well as absence of alcohol abuse according to the American Gastroenterological Association Medical Position Statement (29). The liver ultrasound procedures were read by a single blinded radiologist and quantification of fatty liver was performed according to the criteria of Saverymuttu et al. (30). Differential diagnoses were excluded in all children with suspected NAFLD by measuring serum creatinine kinase, antinuclear antibodies, liver autoantibodies (smooth muscle antibodies, liver kidney microsal autoantibodies, soluble linear antigen), copper, ceruloplasmin, 24-h urinary copper, 1-antitrypsin, and Epstein-Barr virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus serologies according to international recommendations (29). Statistics Statistical analyses were performed using the Winstat software package (R. Fitch Software, Bad Krozingen, Germany). Normal distribution was tested by the Kolmogorov-Smirnov test. Baseline FGF-21 levels were correlated to leptin, anthropometric variables, blood pressure, lipids, uric acids, transaminases, and insulin resistance index HOMA by Spearman correlation because FGF-21 levels were not normally distributed. Changes of FGF-21 levels were correlated to changes of the above mentioned variables in the 1-yr follow-up by Spearman correlation. Furthermore, multiple linear regressions analyses with FGF-21 as dependent variable adjusted to age, gender, and BMI and the independent variables blood pressure, triglycerides, HDL-cholesterol, uric acids, and transaminases were calculated. Because FGF-21 had a nonparametric distribution, it was log transformed in the linear regression models. Furthermore, MetS (yes/no) and NAFLD (yes/no) were included as binary variables in multiple regression analyses with log-transformed FGF-21 as dependent variable and the independent variables age, gender, BMI, NAFLD, and MetS. Also, multiple regression analyses with log-transformed FGF-21 as dependent variable and the independent variables age, gender, BMI, degree of steatosis, and number of parameters of MetS were calculated. To compare variables at baseline or in the course of 1 yr, Fisher s exact test, and Student s t test for paired and unpaired observations, and Wilcoxon and Mann-Whitney U test were used as appropriate. A P value 0.05 was considered as significant. Data are presented as mean and SD or median and interquartile range (IQR). Results The FGF-21 and leptin concentrations as well as the insulin resistance index HOMA were significantly higher in the obese children compared with the normal-weight children (Table 1). Normal-weight and obese children did not differ significantly according to age, gender, or pubertal stage. FGF-21 was significantly correlated to BMI (r 0.25; P 0.012), SDS-BMI (r 0.29; P 0.003), and leptin (r 0.21; P 0.049) but not significantly to age (r 0.14; P 0.149) or insulin resistance index HOMA (r 0.20; P 0.057) in the summarized collective of normal-weight and obese children. In the obese children, FGF-21 was significantly related to FFA but not to any parameter of MetS, NAFLD, or body composition (see Table 2). In a multiple linear regression analysis adjusted for age, gender, and BMI, no parameter of the metabolic syndrome was significantly related to the dependent variable log-transformed FGF-21. Boys did not differ significantly (P 0.085) with respect to their FGF-21 levels compared with girls [for boys, median FGF-21 levels were 114 (IQR ) pg/ml; for girls, median FGF-21 levels were 173 (IQR ) pg/ ml]. The FGF-21 concentrations did not differ significantly (P 0.381) between prepubertal [median FGF-21

4 2146 Reinehr et al. FGF-21 in Obese Children J Clin Endocrinol Metab, June 2012, 97(6): TABLE 1. Baseline characteristics (anthropometrics, FGF-21, leptin, and insulin resistance index HOMA) in 60 obese and 40 normal-weight children Obese children Normal-weight children P value Age (yr) Gender 50% female 38% female Pubertal stage 37% prepubertal, 63% early pubertal a 30% prepubertal, 70% early pubertal a BMI (kg/m 2 ) SDS-BMI FGF-21 (pg/ml) b 195 ( ) 56 (33 122) Leptin (ng/ml) HOMA b 3.5 ( ) 1.2 ( ) Data are shown as percentage or mean SD; values not normally distributed are displayed as median (IQR). a All of these children were at early pubertal stage with PII PIII and BII BIII or GII-GIII. b Not normally distributed. TABLE 2. Associations between FGF-21 and parameters of fat mass, MetS, and NAFLD at baseline and associations between changes of FGF21 and changes of parameters of fat mass, MetS, and NAFLD in the course of 1 yr in 60 obese children (correlation coefficient calculated by Spearman) Changes in course of Baseline 1yr r P r P Triceps skinfold thickness Subscapularis skinfold thickness Body fat based on skinfold thickness Hip circumference Waist circumference Fasting glucose h glucose in ogtt Insulin Systolic blood pressure Diastolic blood pressure LDL-cholesterol HDL-cholesterol Triglycerides Uric acid AST ALT FFA concentrations were 139 (IQR ) pg/ml] and pubertal children [median FGF-21 concentrations were 167 (IQR ) pg/ml]. All pubertal children were at early pubertal stage (PII PIII and BII BIII or GII GIII). At baseline, all obese children hat at least one criterion of MetS, 11 children (18%) had two, 18 children (30%) had three, three children (5%) had four, and one child (2%) had all five criteria of MetS. Obese children with [n 22; median FGF-21 levels 194 (IQR ) pg/ml] and without MetS [n 38; median FGF-21 levels 195 (IQR ) pg/ml] did not differ concerning their FGF-21 concentrations (P 0.959). In a multiple regression analysis with log-transformed FGF-21 levels as dependent variable and age, gender, BMI, and the categorical variable MetS as independent variables, the occurrence of MetS was not associated with FGF-21 levels (P 0.558) at baseline. Calculation of the same model at end of follow-up also revealed no significant association between occurrence of MetS and FGF-21 levels (P 0.415). Using the number of MetS components instead of the categorical variable MetS yes/no in linear regression models also demonstrated no significant associations between log-transformed FGF-21 and number of MetS components at baseline (P 0.707) or in follow-up (P 0.517). At baseline, 48 children (80%) demonstrated no steatosis in ultrasound, three children (5%) first-degree, five children (8%) second-degree, and four children (7%) thirddegree steatosis. Furthermore, children with NAFLD [n 12; median FGF-21 levels 198 (IQR ) pg/ml] and without NAFLD [n 48; median FGF21 levels 195 (IQR ) pg/ml] did not differ with respect to FGF-21 levels (P 0.502). In a multiple regression analysis with log-transformed FGF-21 as dependent variable and age, gender, BMI, and NAFLD as independent variables, the occurrence of NAFLD was not associated with FGF-21 levels (P 0.859) at baseline as well as in follow-up (P 0.751). Using the degree of steatosis instead of the categorical variable NAFLD yes/no in linear regression models also showed no significant associations between FGF-21 and degree of steatosis components at baseline (P 0.979) or in follow-up (P 0.675). In the 1-yr follow-up period of the 60 obese children participating in the lifestyle intervention, the changes of FGF-21 correlated significantly to changes of SDS-BMI (r 0.28; P 0.020) and changes of leptin (r 0.21; P 0.021). The changes of FGF-21 were also correlated to changes of FFA but not to changes of any parameter of metabolic syndrome, NAFLD, or body composition (see Table 2). In a multiple linear regression analysis adjusted for age, gender, and change of BMI, the changes of insulin

5 J Clin Endocrinol Metab, June 2012, 97(6): jcem.endojournals.org 2147 and HOMA as well as the changes of any parameter of the metabolic syndrome were not significantly related to the dependent variable changes of log-transformed FGF-21. In the 30 obese children with reduction of SDS-BMI in the intervention period, FGF-21, leptin, and body fat based on skinfold thickness measurements decreased significantly, and most parameters of MetS improved significantly (Table 3). In the 30 obese children without a decrease of SDS-BMI in the intervention period, no significant changes could be observed except a decrease of HDL-cholesterol (Table 3). The obese children with and without SDS-BMI decrease did not differ significantly at baseline according to age, gender, pubertal stage, any anthropometric parameter, parameters of the MetS, or FGF-21, leptin, or HOMA levels at baseline (Table 3). Discussion To the best of our knowledge, this is the first study analyzing the longitudinal relationships between FGF-21, obesity, insulin resistance index HOMA, leptin, and parameters of the MetS and NAFLD in obese children participating in a lifestyle intervention. In our study, obesity in childhood was associated with an increase of FGF-21 compared with normal-weight children. Furthermore, FGF-21 correlated to BMI and leptin as a marker of white adipose tissue (WAT). These findings are in concordance with most studies in adults demonstrating an association between FGF-21 levels and BMI (12 14, 16) and with rodent studies demonstrating an increased FGF-21 expression in WAT and liver in both diet-induced obese and in genetically obese db/db and ob/ob mice (15, 31). Interestingly, FFA levels were significantly related to FGF-21 concentrations in our study both in cross-sectional and longitudinal analyses, suggesting a relevant association. Indeed, FFA were described as physiological stimulators of FGF-21 secretion in both animals and humans (32). Weight loss was associated with a significant improvement of parameters of the MetS and NAFLD, proving the clinical relevance of the achieved weight loss. Most importantly, the decrease of FGF-21 concentrations in the obese children with weight loss demonstrates the revers- TABLE 3. Changes of anthropometrics, FGF-21, leptin, FFA, transaminases, and parameters of MetS including insulin resistance HOMA in obese children in the course of 1 yr separated to changes of SDS-BMI in the intervention period Obese children with decrease of SDS-BMI Obese children with stable or increasing SDS-BMI Baseline 1 yr later P value Baseline 1 yr later P value BMI (kg/m 2 ) SDS-BMI Triceps thickness (cm) Subscapularis thickness (cm) Body fat a (%) Hip circumference (cm) Waist circumference (cm) FGF-21 (ng/ml) b 206 (98 406) 139 (66 307) ( ) 196 ( ) Leptin (ng/ml) Free fatty acids (mmol/liter) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl) Triglycerides (mg/dl) Uric acid (mg/dl) Glucose (mg/dl) Insulin (mu/liter) b 16 (11 21) 11 (8 14) (13 32) 22 (14 33) Insulin resistance index (HOMA) b 3.0 ( ) 2.2 ( ) ( ) 5.1 ( ) h glucose in ogtt (mg/dl) ALT (U/liter) AST (U/liter) Bath groups of children were 50% female and 37% prepubertal; n 30 for both groups; age yr for the obese children with decrease of SDS-BMI and yr for obese children with stable or increasing SDS-BMI. Data are shown as percentage or mean SD; values not normally distributed are displayed as median and (IQR). a Based on skinfold thickness. b Not normally distributed.

6 2148 Reinehr et al. FGF-21 in Obese Children J Clin Endocrinol Metab, June 2012, 97(6): ibility of the rise of FGF-21 in obesity. This finding suggests that the increase of FGF-21 is a consequence and not a cause of childhood obesity. One study in obese adults also reported a decrease of FGF-21 levels in weight loss (14), whereas two other adult studies did not find a significant change of FGF-21 due to weight loss (33, 34). However, the weight loss of one of these latter studies was only very moderate (34), and in the other study, the weight loss was achieved by bariatric surgery and not by lifestyle intervention (33), probably explaining the difference to our study. Because FGF-21 has been reported to contribute to regulating lipolysis in WAT, increase insulin-independent glucose uptake in 3T3-L1 adipocytes, and improve insulin resistance (4, 7, 35), in other words, improving the underlying pathomechanisms of the MetS and NAFLD, it is a surprising finding that FGF-21 was increased in obesity, which is frequently associated with MetS and NAFLD. Furthermore, all parameters of the MetS and NAFLD as well as insulin resistance HOMA did not correlate with FGF-21 in our study both in cross-sectional and longitudinal analyses, questioning a direct link between FGF-21 and parameters of MetS and NAFLD in children. However, these findings fit with the hypothesis that obesity is a FGF-21-resistant state (35). In this state, FGF-21 fails to exert its expected effects on glucose homeostasis and lipid oxidation. Consistent with this, one recent study reported that acute continuous infusion of FGF-21 to control mice leads to reduced hepatic glucose output and increased insulin sensitivity although having no effect on obese ob/ob mice (36). However, a lack of leptin signaling in the ob/ob mice may also explain this finding because FGF-21 concentrations were related to leptin levels in our study. Interestingly, despite high endogenous levels in obese mice, exogenous FGF-21 administered at pharmacological doses appears to exert actions to improve metabolic parameters and induces weight loss (37, 38). A state in which high endogenous levels of a physiological regulator appear to be ineffective but in which high pharmacological doses induce the expected results suggests a state of hormone resistance. A down-regulation of FGF receptors has been discussed as a cause of FGF-21 resistance in obesity (10, 35). However, a recent study reported a lack of overt FGF-21 resistance in two mouse models of obesity and insulin resistance (39). Our study has a few potential limitations. First, BMI percentiles were used to classify overweight. Although BMI is a good measure for overweight, one needs to be aware of its limitation as an indirect measure of fat mass. Thus, we have also analyzed skinfold measurements to determine body fat. Because we measured skinfold thickness only in obese children, this may explain the lack of association between FGF-21 and body fat based on skinfold thickness because the degree of obesity was relatively homogeneous in our obese children. Second, we studied only prepubertal children and early pubertal children, probably missing an effect of later pubertal stages on FGF-21 levels. Third, the HOMA model is only an assessment of insulin resistance. Clamp studies are actually the gold standard for analyzing insulin resistance. However, validation studies in youth demonstrated a good correlation between HOMA and clamp techniques (40). Fourth, we are not able to differentiate the effect of diet, increased physical exercise, and weight status on FGF-21 concentrations due to our study protocol. Fifth, we found no correlation between transaminases and FGF-21 concentrations. However, the levels of transaminases have been demonstrated to be a poor marker of the severity of NAFLD. Fifth, diagnosis of NAFLD was not confirmed by liver biopsy. Therefore all conclusions concerning FGF-21 and NAFLD have to be interpreted very cautiously. Liver biopsies in children are difficult to perform, also for ethical reasons, because no specific therapy follows histological diagnosis of NAFLD apart from recommending reduction of overweight, which is generally advised to all obese children. Finally, because we studied children, and age is a well-recognized parameter that clearly contributes to insulin resistance and the MetS severity, all statements can be applied only to children and not to adults. In summary, this is the first study in childhood demonstrating that obese children had increased FGF-21 levels compared with normal-weight children. Overweight reduction due to lifestyle intervention was associated with a decrease of FGF-21 levels, suggesting that the increase of FGF-21 in childhood obesity is the consequence and not the cause of obesity. Because our cross-sectional and longitudinal data do not support a significant relationship between FGF-21, insulin resistance, and parameters of MetS and NAFLD, these findings support the hypothesis of a FGF-21-resistant state in obese children. Acknowledgments We thank Ms. K. Schark-Zimmer, Children s Hospital University of Bonn, for her kind support and great expertise in the laboratory. Address all correspondence and requests for reprints to: Prof. Dr. Thomas Reinehr, Department of Pediatric Nutrition Medicine, Vestische Hospital for Children and Adolescents Datteln, University of Witten/Herdecke, Dr. F. Steiner Strasse 5, D Datteln, Germany. T.Reinehr@ kinderklinik-datteln.de. Thisstudyisregisteredatwww.clinicaltrials.gov(NCT ). T.R. received grant support ( ) from of the German Ministry of Education and Research (Bundesministerium

7 J Clin Endocrinol Metab, June 2012, 97(6): jcem.endojournals.org 2149 für Bildung und Forschung Obesity network, LARGE Grant 01 GI0839), the National Genome Research Network (NGFNplus Grant 01GS0820), and by Hexal AG; however, the hypothesis development, analysis, interpretation, and conclusions contained in this study are those of the author s alone. T.R. and C.L.R. developed the study design, T.R. performed the anthropometrical measurements, R.W. performed the ultrasound measurements, and C.L.R. performed the laboratory measurements. T.R., J.W., R.W., and C.L.R. discussed the findings. T.R. wrote the first draft of the paper. Disclosure Summary: The authors have nothing to disclose. References 1. Roth CL, Reinehr T 2010 Roles of gastrointestinal and adipose tissue peptides in childhood obesity and changes after weight loss due to lifestyle intervention. Arch Pediatr Adolesc Med 164: Reaven GM 1991 Insulin resistance and compensatory hyperinsulinemia: role in hypertension, dyslipidemia, and coronary heart disease. Am Heart J 121: Kharitonenkov A, Wroblewski VJ, Koester A, Chen YF, Clutinger CK, Tigno XT, Hansen BC, Shanafelt AB, Etgen GJ 2007 The metabolic state of diabetic monkeys is regulated by fibroblast growth factor-21. Endocrinology 148: Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos- Flier E 2007 Hepatic fibroblast growth factor 21 is regulated by PPAR and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab 5: Böttcher RT, Niehrs C 2005 Fibroblast growth factor signaling during early vertebrate development. Endocr Rev 26: Cuevas-Ramos D, Almeda-Valdes P, Aguilar-Salinas CA, Cuevas- Ramos G, Cuevas-Sosa AA, Gomez-Perez FJ 2009 The role of fibroblast growth factor 21 (FGF21) on energy balance, glucose and lipid metabolism. Curr Diabetes Rev 5: Inagaki T, Dutchak P, Zhao G, Ding X, Gautron L, Parameswara V, Li Y, Goetz R, Mohammadi M, Esser V, Elmquist JK, Gerard RD, Burgess SC, Hammer RE, Mangelsdorf DJ, Kliewer SA 2007 Endocrine regulation of the fasting response by PPARalpha-mediated induction of fibroblast growth factor 21. Cell Metab 5: Arner P, Pettersson A, Mitchell PJ, Dunbar JD, Kharitonenkov A, Rydén M 2008 FGF21 attenuates lipolysis in human adipocytes: a possible link to improved insulin sensitivity. FEBS Lett 582: Kharitonenkov A, Shiyanova TL, Koester A, Ford AM, Micanovic R, Galbreath EJ, Sandusky GE, Hammond LJ, Moyers JS, Owens RA, Gromada J, Brozinick JT, Hawkins ED, Wroblewski VJ, Li DS, Mehrbod F, Jaskunas SR, Shanafelt AB 2005 FGF-21 as a novel metabolic regulator. J Clin Invest 115: Murata Y, Konishi M, Itoh N 2011 FGF21 as an Endocrine Regulator in Lipid Metabolism: From Molecular Evolution to Physiology and Pathophysiology. J Nutr Metab 2011: Sarruf DA, Thaler JP, Morton GJ, German J, Fischer JD, Ogimoto K, Schwartz MW 2010 Fibroblast growth factor 21 action in the brain increases energy expenditure and insulin sensitivity in obese rats. Diabetes 59: Tyynismaa H, Raivio T, Hakkarainen A, Ortega-Alonso A, Lundbom N, Kaprio J, Rissanen A, Suomalainen A, Pietiläinen KH 2011 Liver fat but not other adiposity measures influence circulating FGF21 levels in healthy young adult twins. J Clin Endocrinol Metab 96:E351 E Dushay J, Chui PC, Gopalakrishnan GS, Varela-Rey M, Crawley M, Fisher FM, Badman MK, Martinez-Chantar ML, Maratos-Flier E 2010 Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology 139: Mraz M, Bartlova M, Lacinova Z, Michalsky D, Kasalicky M, Haluzikova D, Matoulek M, Dostalova I, Humenanska V, Haluzik M 2009 Serum concentrations and tissue expression of a novel endocrine regulator fibroblast growth factor-21 in patients with type 2 diabetes and obesity. Clin Endocrinol (Oxf) 71: Zhang X, Yeung DC, Karpisek M, Stejskal D, Zhou ZG, Liu F, Wong RL, Chow WS, Tso AW, Lam KS, Xu A 2008 Serum FGF21 levels are increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes 57: Dostálová I, Kaválková P, Haluzíková D, Lacinová Z, Mráz M, Papezová H, Haluzík M 2008 Plasma concentrations of fibroblast growth factors 19 and 21 in patients with anorexia nervosa. J Clin Endocrinol Metab 93: Li L, Yang G, Ning H, Yang M, Liu H, Chen W 2008 Plasma FGF-21 levels in type 2 diabetic patients with ketosis. Diabetes Res Clin Pract 82: Gälman C, Lundåsen T, Kharitonenkov A, Bina HA, Eriksson M, Hafström I, Dahlin M, Amark P, Angelin B, Rudling M 2008 The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPAR activation in man. Cell Metab 8: Kromeyer-Hausschild K, Glässer N, Zellner K 2008 Waist Circumference percentile in Jena children (Germany) 6 to 18 years of age. Aktuel Ernaehr Med 33: Reinehr T, de Sousa G, Toschke AM, Andler W 2006 Long-term follow-up of cardiovascular disease risk factors in children after an obesity intervention. Am J Clin Nutr 84: Cole TJ 1990 The LMS method for constructing normalized growth standards. Eur J Clin Nutr 44: Ziegler A, Geiss HC, Hesse V, von Hippel V, Jäger U, Johnson D, Korte W, Kunze D, Menner K, Müller G, Müller M, Niemann- Pilatus A, Remer T, Wittchen H, Zabransky S, Zelner K, Hebebrand J 2001 Percentiles of body mass index in children and adolescents evaluated from different regional German studies. Monatsschr Kinderheilkd 149: Cole TJ, Bellizzi MC, Flegal KM, Dietz WH 2000 Establishing a standard definition for child overweight and obesity worldwide: international survey. BMJ 320: Slaughter MH, Lohman TG, Boileau RA, Horswill CA, Stillman RJ, Van Loan MD, Bemben DA 1988 Skinfold equations for estimation of body fatness in children and youth. Hum Biol 60: National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents 2004 The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics 114(2 Suppl 4th Report): Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC 1985 Homeostasis model assessment: insulin resistance and -cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28: American Diabetes Association 2004 Diagnosis and classification of diabetes mellitus. Diabetes Care 54(Suppl 1):S5 S Zimmet P, Alberti KG, Kaufman F, Tajima N, Silink M, Arslanian S, Wong G, Bennett P, Shaw J, Caprio S 2007 The metabolic syndrome in children and adolescents: an IDF consensus report. Pediatr Diabetes 8: American Gastroenterological Association 2002 American Gastroenterological Association medical position statement: nonalcoholic fatty liver disease. Gastroenterology 123: Saverymuttu SH, Joseph AE, Maxwell JD 1986 Ultrasound scanning in the detection of hepatic fibrosis and steatosis. Br Med J (Clin Res Ed) 292: Muise ES, Azzolina B, Kuo DW, El-Sherbeini M, Tan Y, Yuan X, Mu J, Thompson JR, Berger JP, Wong KK 2008 Adipose fibroblast growth factor 21 is up-regulated by peroxisome proliferator-activated receptor and altered metabolic states. Mol Pharmacol 74: Mai K, Andres J, Biedasek K, Weicht J, Bobbert T, Sabath M, Meinus S, Reinecke F, Möhlig M, Weickert MO, Clemenz M, Pfeiffer

8 2150 Reinehr et al. FGF-21 in Obese Children J Clin Endocrinol Metab, June 2012, 97(6): AF, Kintscher U, Spuler S, Spranger J 2009 Free fatty acids link metabolism and regulation of the insulin-sensitizing fibroblast growth factor-21. Diabetes 58: Woelnerhanssen B, Peterli R, Steinert RE, Peters T, BorbélyY,Beglinger C 2011 Effects of postbariatric surgery weight loss on adipokines and metabolic parameters: comparison of laparoscopic Roux-en-Y gastric bypass and laparoscopic sleeve gastrectomy-a prospective randomized trial. Surg Obes Relat Dis 7: Mai K, Schwarz F, Bobbert T, Andres J, Assmann A, Pfeiffer AF, Spranger J 2011 Relation between fibroblast growth factor-21, adiposity, metabolism, and weight reduction. Metabolism 60: Fisher FM, Chui PC, Antonellis PJ, Bina HA, Kharitonenkov A, Flier JS, Maratos-Flier E 2010 Obesity is a fibroblast growth factor 21 (FGF21)-resistant state. Diabetes 59: Berglund ED, Kang L, Lee-Young RS, Hasenour CM, Lustig DG, Lynes SE, Donahue EP, Swift LL, Charron MJ, Wasserman DH 2010 Glucagon and lipid interactions in the regulation of hepatic AMPK signaling and expression of PPAR and FGF21 transcripts in vivo. Am J Physiol Endocrinol Metab 299:E607 E Coskun T, Bina HA, Schneider MA, Dunbar JD, Hu CC, Chen Y, Moller DE, Kharitonenkov A 2008 Fibroblast growth factor 21 corrects obesity in mice. Endocrinology 149: Xu J, Lloyd DJ, Hale C, Stanislaus S, Chen M, Sivits G, Vonderfecht S, Hecht R, Li YS, Lindberg RA, Chen JL, Jung DY, Zhang Z, Ko HJ, Kim JK, Véniant MM 2009 Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58: Hale C, Chen MM, Stanislaus S, Chinookoswong N, Hager T, Wang M, Véniant MM, Xu J 2012 Lack of overt FGF21 resistance in two mouse models of obesity and insulin resistance. Endocrinology 153: Henderson M, Rabasa-Lhoret R, Bastard JP, Chiasson JL, Baillargeon JP, Hanley JA, Lambert M 2011 Measuring insulin sensitivity in youth: How do the different indices compare with the gold-standard method? Diabetes Metab 37:72 78 Members receive free electronic delivery of FDA drug safety alerts from the PDR Network.