Response of fibroblast growth factor 19 and bile acid synthesis after a body weight-adjusted oral fat tolerance test in overweight and obese NAFLD patients: a non-randomized controlled pilot trial

Response of fibroblast growth factor 19 and bile acid synthesis after a body weight-adjusted oral... Background: Non-alcoholic fatty liver disease (NAFLD) is common both in obese and overweight patients. Fibroblast growth factor 19 (FGF19), an intestinal hormone, could play a role in the complex pathogenesis of NAFLD. The aim of our study was to investigate responses of FGF19 and bile acid (BA) synthesis after a body weight-adjusted oral fat tolerance test (OFTT) in overweight and obese NAFLD patients. Methods: For this study, we recruited 26 NAFLD patients; 14 overweight (median BMI 28.3 kg/m ), 12 obese 2 2 (35.3 kg/m ) and 16 healthy controls (24.2 kg/m ). All individuals received 1 g fat (Calogen®) per kg body weight orally. Serum concentrations of FGF19 were determined by ELISA. Concentrations of BAs and BA synthesis marker 7α-hydroxy-4-cholesten-3-one (C4) were measured by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively; all at 0 (baseline), 2, 4 and 6 h during the OFTT. Results: BMI correlated negatively with fasting FGF19 concentrations (rho = − 0.439, p = 0.004). FGF19 levels of obese NAFLD patients were significantly (p = 0.01) lower in the fasting state (median 116.0 vs. 178.5 pg/ml), whereas overweight NAFLD patients had significantly (p = 0.004) lower FGF19 concentrations 2 h after the fat load (median 163.0 vs. 244.5 pg/ ml), and lowest values at all postprandial time points as compared to controls. Baseline BA concentrations correlated positively with FGF19 values (rho = 0.306, p = 0.048). In all groups, we observed BA increases during the OFTT with a peak at 2hbutnochangeinC4levelsinoverweight/obeseNAFLD patients. Conclusions: Reduced basal gastrointestinal FGF19 secretion and decreased postprandial response to oral fat together with blunted effect on BA synthesis indicate alterations in intestinal or hepatic FXR signaling in overweight and obese NAFLD subjects. The precise mechanism of FGF19 signaling after oral fat load needs further evaluation. Trial registration: We have registered the trial retrospectively on 30 Jan 2018 at the German clinical trials register (http://www.drks.de/), and the following number has been assigned DRKS00013942. Keywords: Bile acids, FGF19, Non-alcoholic fatty liver disease, Oral fat tolerance test * Correspondence: dana.friedrich@uks.eu Department of Medicine II, Saarland University Medical Center, Saarland University, 66421 Homburg, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 2 of 10 Background Since the role of FGF19 in the pathogenesis of hu- Obesity and fatty liver disease represent increasing med- man NAFLD is unknown, we studied FGF19 and hep- ical problems in developed countries. In Germany, the atic downstream effects (C4 and BAs) in overweight prevalence of obesity increased during the years 1998 to and obese NAFLD outpatients (and healthy controls) 2011 from 18.9 to 23.3% in men and from 22.5 to 23.9% that were subjected to a body weight-adjusted oral fat in women [1]. In the United States, 37% of adults are tolerance test (OFTT). We determined serum concen- obese [2]. Obesity is an important risk factor of trations of FGF19, C4 and BAs at baseline and at 2, 4 non-alcoholic fatty liver disease (NAFLD), which has and 6 h after OFTT. We hypothized that FGF19 been reported in 30 to 40% of adults [3, 4]. levels are lower in obese compared to overweight The term NAFLD is used for a wide spectrum of fatty NAFLD patients. liver diseases that starts with simple steatosis in We aimed to answer the following questions in this non-alcoholic fatty liver (NAFL) that may progress to study: non-alcoholic steatohepatits (NASH), which is compli- cated by fibrosis, cirrhosis, and eventually hepatocellular 1. Do fasting FGF19 serum concentrations differ carcinoma [5–7]. NAFLD is often associated with the between normal-weight healthy, overweight and metabolic syndrome and requires exclusion of excessive obese NAFLD patients? alcohol consumption as well as viral and autoimmune 2. How does a body weight-adjusted oral fat tolerance liver diseases [8]. NAFLD is common in obesity but also test (OFTT) affect serum FGF19 concentrations in in overweight patients [9, 10]. The pathophysiology of these populations? NAFLD is complex and still not fully defined [11, 12]. 3. How does a postprandial FGF19 response affect Several metabolic factors have already been identified in hepatic BA biosynthesis, as assessed by C4? the development of NAFLD, including insulin resistance, diabetes mellitus and obesity. So far there have been only few studies of the im- Methods portance of gastrointestinal hormones in the patho- Study protocol genesis of NAFLD [13–15]. The gastrointestinal The study protocol was approved by the Ethics Committee hormone fibroblast growth factor 19 (FGF19) has of the Ärztekammer des Saarlandes, Saarbrücken (ID num- emerged as a novel regulator of bile acid, carbohy- ber 58/09). All subjects (≥ 18 years) were fully informed drate and lipid metabolism. In human metabolic syn- about the study objectives and methods and gave their drome associated diseases, such as type 2 diabetes written informed consent before participating in this mellitus (T2DM) and NAFLD, FGF19 signaling seems non-randomized controlled pilot trial. to be dysregulated [16]. In animals, FGF19 transgenic mice show resistance to a high-fat diet and decreased Study subjects liver triglyceride concentrations [17] while the admin- During 2009 and 2010, we recruited overweight and istration of recombinant FGF19 increases the meta- obese NAFLD outpatients in the Department of Internal bolic rate [18]. Accordingly, in humans with NAFLD, Medicine II, Saarland University Medical Center, Hom- reduced fasting FGF19 levels were found [14, 19, 20]. burg, as well as healthy controls with normal body Therefore, the present study focuses on the dietary weight. Inclusion criteria for NAFLD were ultrasound regulation of FGF19 and its potential role in the and/or biopsy findings consistent with fatty liver disease. pathogenesis of NAFLD. Exclusion criteria were increased alcohol consumption FGF19 release in the intestine is induced by bile acids in medical history and the following acute and chronic (BAs). After a meal, the entry of dietary fat in the duode- liver diseases: cirrhosis, hepatitis A virus (HAV), hepa- num causes gallbladder contraction and BA inflow into titis B virus (HBV), hepatitis C virus (HCV), hepatitis D the intestinal lumen. The reabsorption of BAs in the ter- virus (HDV), cytomegalovirus (CMV) and Epstein-Barr minal ileum activates the canonical BA sensor farnesoid Virus (EBV) infections, hemochromatosis, Wilson’s dis- X receptor (FXR), resulting in enhanced transcription ease, α -antitrypsin deficiency, and autoimmune hepa- and secretion of FGF19 [21, 22]. FGF19 binds on hepa- titis. Healthy controls included employees of the clinic tocytes to the FGF receptor 4 (FGFR4) and its cofactor and medical students with normal BMI and no diseases βKlotho [22–24], which triggers a signaling cascade that in history. In controls, no liver and laboratory diagnosis represses cholesterol 7α-hydroxylase (CYP7A1), the was performed. rate-limiting enzyme in BA synthesis from cholesterol Subjects were divided into three groups according to [25]. 7α-Hydroxy-4-cholesten-3-one (C4) is an inter- their BMI (healthy controls, normal weight: 19.0– 2 2 mediate of BA synthesis, which can be measured in 25.4 kg/m , overweight NAFLD: 25.5–29.9 kg/m , obese serum [26]. NAFLD: ≥ 30.0 kg/m )[27, 28]. Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 3 of 10 Liver parameters determined by gas chromatography-mass spectrometry In overweight and obese patients (N = 26), liver status (GCMS) [32]. 7α-hydroxy-4-cholesten-3-one (C4), a valid was assessed by abdominal ultrasound and/or liver marker of bile acid biosynthesis [33], was measured by biopsy. Ultrasound was performed using the Hitachi high-performance liquid chromatography (HPLC). EUB-8500 ultrasound scanner (Hitachi Medical Systems, Wiesbaden, Germany). Hepatic steatosis results in Statistical analysis abnormal echo patterns on ultrasound scanning; the Data analysis was performed using SPSS (version 20.0, severity of steatosis was graded as mild (I), moderate IBM, Ehningen, Germany). Kruskal-Wallis test was used (II), or severe (III) [29]. Liver biopsy samples of five to analyze quantitative data for differences within the co- patients were examined by an experienced pathologist of hort. For the present study with a low number of study Saarland University Medical Center. subjects (N < 20), normal distributions were not expected [34]. Thus, data are expressed as medians and interquar- Oral fat tolerance test (OFTT) tile ranges (IQR 25–75). In addition, Mann-Whitney-U We used a body weight-adjusted OFTT to investigate test was used to test differences between two groups. The postprandial FGF19, BA and C4 responses in our study strength of associations between two parameters was esti- subjects. For the present study, a standardized test drink mated using the non-parametric Spearman correlation Calogen® (Nutricia, Erlangen, Germany) was adminis- test. Spearman’s correlation coefficient is presented as rho. tered, which is a lipid emulsion based on vegetable fat For the OFTT, FGF19 -area under the curve (AUC) (0-6h) with 50% long-chain triglycerides [30]. All individuals and, after correcting for baseline, the incremental AUC received 1 g fat per kg body weight orally. The fat load (FGF19-IAUC) were computed using GraphPad Prism was based on subjects’ body weight to adjust the OFTT (version 6.0, GraphPad Software, La Jolla, CA, USA). A to hypercaloric (especially high-fat) eating behavior of p-value < 0.05 denotes statistical significance. obese patients [31]. Results Blood samples Subject characteristics Blood samples were drawn from a peripheral vein at Table 1 summarizes the subject characteristics. A total 8:00 AM after an overnight fasting and 2, 4 and 6 h after of 42 subjects, 21 women and 21 men, were recruited the oral fat challenge. Samples were centrifuged for for our study. Study participants were between 19 and 10 min at 3000 g 30 min after blood collection 68 years old (median 47.0 years, IQR 28.8–53.8). Overall, (ROTANTA 46R, Hettich, Tuttlingen, Germany). Subse- we recruited 14 overweight and 12 obese NAFLD quently, serum was stored in aliquots at-70 °Cuntil patients as well as 16 healthy controls. Sex and age did analysis. not differ between groups. Obese patients had a median BMI of 35.3 kg/m , which corresponds to obesity grade Serum FGF19, bile acid and C4 measurements II [28]. FGF19 serum concentrations were measured in duplicate NAFLD was diagnosed by ultrasound and/or biopsy. by quantitative sandwich enzyme-linked immunosorbent In overweight and obese patients, the steatosis spectrum assay, using the FGF19 Quantikine ELISA kit (R&D Sys- ranged from grade I, II and III to NASH and fibrosis. In tems, Minneapolis, USA). Serum BA concentrations were the overweight group (N = 14), grade I liver steatosis was Table 1 Subject characteristics, basal and postprandial FGF19 serum concentrations Variables Control Overweight Obesity p-value N (men/women) 16 (7/9) 14 (8/6) 12 (6/6) n.s. Age (years) 29.5 (24.0–53.0) 49.0 (38.8–57.3) 48.0 (37.0–57.3) 0.551 2 b BMI (kg/m ) 24.2 (21.8–26.6) 28.3 (26.3–29.2) 35.3 (32.7–39.0) < 0.001 FGF19 (pg/ml) c c c t = 0 h 178.5 (101.0–257.0) 127.5 (70.0–161.3) 116.0 (51.0–134.3) 0.01 c c c t = 2 h 244.5 (161.5–377.5) 163.0 (78.5–168.3) 181.0 (85.3–393.0) 0.004 t = 4 h 332.5 (202.0–590.8) 207.0 (112.5–365.0) 220.0 (138.8–385.3) 0.445 t = 6 h 211.0 (165.3–296.3) 154.0 (124.0–254.0) 184.5 (110.5–274.3) 0.445 All data are given as median (interquartile range) Chi-square-test Kruskal-Wallis-test Mann-Whitney-U-test Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 4 of 10 found in six patients (43%), grade II in five patients lower in obese NAFLD patients as compared to controls (36%), and grade III in one patient (7%). NASH was and tended to be lower in overweight NAFLD subjects, diagnosed in one and fibrosis stage II was documented too (Table 1, Fig. 1). Basal FGF19 concentrations did not in one patient. In the obese group (N = 12), two patients differ between sexes [women, median 126.0 (IQR 80.5– (17%) displayed liver steatosis grade I, four patients 179.0) vs. men, median 138.0 (IQR 93.0–204.0) pg/ml]. (33%) showed grade II, and three participants (25%) had Interestingly, fasting FGF19 concentrations were nega- grade III. In this group, NASH was found in one and tively correlated with BMI (Fig. 2). fibrosis stage I also in one patient. In one obese study After the OFTT, FGF19 concentrations increased in participant, liver status could not be assessed by controls, overweight and obese patients (Table 1, Fig. 1). ultrasound. Additional file 1: Table S1 lists the co- Of note, overweight patients displayed lowest FGF19 morbidities in overweight and obese NAFLD patients. concentrations at all postprandial time points. Two In overweight patients, hypercholesterolemia and in hours after the OFTT, FGF19 levels ranged from 10.0 to obese patients, arterial hypertension were the domin- 697.0 pg/ml (median 178.0, IQR 116.5–255.5). At this ant concomitant diseases, respectively. Controls did time, overweight NAFLD patients showed significantly not take any drugs regularly. Three overweight and lower FGF19 levels compared with controls (Fig. 1). The eight obese patients were taking medications. These FGF19 maximum was found in all three groups after included antidiabetics, antihypertensives, thyroid hor- 4 h, with hormone levels ranging from 59.0 to 935.0 pg/ mones, analgesics, proton pump inhibitors, antidepres- ml (median 255.0, IQR 163.3–439.3). At 4 h, FGF19 sants, non-steroidal antirheumatics, corticoidsteroids and was highest in controls and twice as high as at base- allopurinol, respectively. line. After 6 h, FGF19 values ranged from 48.0 to 802.0 pg/ml (median 189.0, IQR 136.0–269.0) and Basal and postprandial FGF19 serum concentrations were still highest in controls. In the total study group (N = 42), fasting FGF19 concen- Both women and men showed the FGF19 maximum trations ranged from 17.0 to 392.0 pg/ml (median 133.5, at 4 h [women, median 270.0 (IQR 167.5–539.0) vs. IQR 82.8–190.3). Basal FGF19 values were significantly men, median 231.0 (IQR 117.5–410.5) pg/ml] but Fig. 1 Fasting and postprandial FGF19 serum concentrations measured by quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Comparison of FGF19 values between healthy controls (N = 16), overweight (N = 14) and obese (N = 12) patients with non-alcoholic fatty liver disease (NAFLD) at baseline (0 h), 2, 4 and 6 h after the oral fat tolerance test (OFTT). Significant difference between basal (0 h) FGF19 concentrationsin controls and obese NAFLD patients [controls 178.5 (101.0–257.0) vs. obese 116.0 (51.0–134.3) pg/ml, medians (IQRs), p = < 0.05, Mann-Whitney-U-test). At 2 h, lower FGF19 values in overweight NAFLD patients in comparison to controls [overweight 163.0 (78.5–168.3) vs. controls 244.5 (161.5–377.5) pg/ml, medians (IQRs), p = 0.004, Mann-Whitney-U-test), * outlier Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 5 of 10 N=42 p=0.004 rho= - 0.439 Fig. 2 Fasting FGF19 serum concentrations versus body mass index (BMI) for all study subjects. FGF19 values correlated negatively with BMI. A scattered plot is shown and the Spearman’s correlation coefficient was calculated postprandial levels were higher in women at all time to controls. FGF19-AUC and IAUC did not correlate points. Six hours after the oral fat challenge, FGF19 con- with body weight-adjusted fat load. In addition, there centrations of both sexes tended to reach a significant was no association between FGF19-AUC and BMI; difference [women, median 216.0 (IQR 145.0–390.5) vs. FGF19-IAUC tended to correlate with age (rho = 0.291, men, median 172.0 (IQR124.0–216.5) pg/ml, p = 0.051]. p = 0.062). Mean FGF19 -area and mean incremental area (0-6h) under the curve (AUC and IAUC) did not differ sig- nificantly between the groups (AUC controls: 1772.8 Basal and postprandial BA serum concentrations ± 766.6 vs. overweight: 1130.6 ± 590.0 vs. obese: Fasting and postprandial bile acid (BA) concentrations 1469.3 ± 910.0 pg/ml/6 h; IAUC controls: 699.0 ± 383.7 vs. did not differ between overweight/obese NAFLD pa- overweight: 573.3 ± 333.4 vs. obese: 921.5 ± 732.4 pg/ml). tients and controls. In all three groups, we observed a FGF19-AUC was highest in controls and lowest in BA increase after the OFTT with a peak at 2 h (Table 2). overweight patients (p = 0.053); IAUC was higher in Basal FGF19 concentrations correlated positively with obese and lower in overweight patients in comparison basal BA values (Fig. 3). Table 2 Basal and postprandial BA and C4 serum concentrations Variables Control Overweight Obesity p-value N (men/women) 16 (7/9) 14 (8/6) 12 (6/6) n.s. Bile acids (μM) t = 0 h 1.1 (0.8–1.7) 1.5 (0.8–2.2) 1.4 (0.9–1.7) 0.343 t = 2 h 1.4 (1.1–4.7) 2.1 (1.2–3.8) 2.4 (1.6–4.1) 0.311 t = 4 h 1.1 (0.7–2.0) 2.0 (1.3–2.5) 2.0 (1.0–2.3) 0.155 t = 6 h 0.7 (0.5–1.6) 1.3 (0.8–2.0) 1.2 (0.8–1.7) 0.087 C4 (nM) t = 0 h 41.4 (7.0–69.2) 58.5 (12.8–91.6) 35.1 (1.2–72.3) 0.422 t = 2 h 28.1 (7.1–49.6) 43.1 (10.0–114.0) 35.7 (1.2–118.3) 0.765 t = 4 h 13.0 (6.8–44.7) 40.8 (21.1–99.3) 32.7 (3.3–108.0) 0.343 t = 6 h 11.8 (4.1–34.4) 40.7 (18.1–80.1) 28.6 (3.5–82.4) 0.445 All data are given as median (interquartile range), p-values: Kruskal-Wallis-test, Chi-square-test Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 6 of 10 N=42 p=0.048 rho= 0.306 Fig. 3 Fasting FGF19 serum concentrations versus fasting bile acid (BA) serum concentrations for all subjects (N = 42). There was a correlation between FGF19 and BA values. A scattered plot is shown and the Spearman’s correlation coefficient was calculated Basal and postprandial C4 serum concentrations obese NAFLD patients. C4 concentrations in overweight Fasting and postprandial C4 values did not differ signifi- NAFLD patients remained unchanged for 4 h postpran- cantly between study participants (Table 2, Fig. 4). At all dially, despite increasing FGF19 values. In the total study postprandial time points, C4 concentrations were mark- group FGF19 concentrations at 2 h correlated negatively edly lower in controls in comparison to overweight/ with C4 values at 4 h after the OFTT (Fig. 5). There was Fig. 4 Fasting and postprandial C4 concentrations. C4, a valid marker of bile acid biosynthesis, was measured by high-performance liquid chromatography (HPLC). Comparison of C4 values between healthy controls (N = 16), overweight (N = 14) and obese (N = 12) patients with non-alcoholic fatty liver disease (NAFLD) at baseline (0 h), 2, 4 and 6 h after the oral fat tolerance test (OFTT). C4 concentrations did not differ between groups (Kruskal-Wallis-test),*outlier Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 7 of 10 N=42 p=0.031 rho= - 0.332 Fig. 5 FGF19 serum concentrations at 2 h versus C4 values at 4 h after the oral fat tolerance test (OFTT) for all subjects (N = 42). A scattered plot is shown and the Spearman’s correlation coefficient was calculated also an inverse correlation of FGF19 concentrations at Discussion 4 h and C4 values at 6 h after the OFTT in the study The present study investigated serum FGF19, BA, and group (Fig. 6). These correlations were confirmed for C4 profiles in overweight and obese NAFLD patients, in the control group (Additional file 2: Figure S1, comparison to normal-weight healthy controls, after a Additional file 3: Figure S2). In NAFLD patients, FGF19 body weight-adjusted oral fat load. The key findings of concentrations did not correlate with C4 values. our study are (i) fasting FGF19 concentrations were N=42 p=0.006 rho= - 0.420 Fig. 6 FGF19 serum concentrations at 4 h versus C4 values at 6 h after the oral fat tolerance test (OFTT) for all subjects (N = 42). A scattered plot is shown and the Spearman’s correlation coefficient was calculated Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 8 of 10 significantly lower in obese (grade II) NAFLD patients as contributed to differential findings at single postprandial compared to controls, (ii) overweight NAFLD patients time points and in FGF19-IAUC between controls and had significantly lower FGF19 concentrations 2 h after NAFLD patients. the fat load and lowest values at all postprandial time Before our study started, we suspected that obese points and (iii) BAs increased during the OFTT but patients would have the highest energy and fat intake. without changes in C4 levels. Therefore we decided for a body weight-based fat load Low FGF19 concentrations have been reported in to adjust the OFTT to the patient’s eating behavior. To metabolic syndrome [35], obesity [36] and type 2 dia- check our assumption, we used a 3-day nutritional betes [37, 38]. The findings by Jansen et al. [19] support protocol and calculated energy and macronutrients in- our results, i.e. the presence of fatty liver disease in take (data not shown). Since there was no significant dif- obesity is associated with lower fasting FGF19 concen- ference between groups, we suppose under-reporting of trations. Also in obese children with NAFLD [20] and food intake in our overweight and obese patients. Most young obese NASH patients [39] lower fasting FGF19 researchers agree that the reported accuracy of food in- values were detected whereas Schreuder et al. did not take decreases with increasing BMI. A systematic review observe differences in fasting FGF19 concentrations be- covering studies between 1982 and 2014 showed that a tween controls and obese NAFLD subjects [13]. In our BMI > 30 kg/m is associated with significant study, BMI correlated negatively with fasting FGF19 under-reporting of food intake. These studies were serum concentrations. Our study was too small to cor- mostly from Europe and North America [44]. For ex- relate FGF19 levels with biopsy-proven severity of ample, in morbid obese (BMI > 40 kg/m ) energy intake NAFLD. In this respect, inverse associations had been can reach more than 4000 kcal/day, with high fat intakes found in children [15, 40], but some studies did not find of about 40 to 57% of total energy intake [31]. correlations between steatosis grade and FGF19 concen- In the present study, higher fat challenge in obese trations [13, 14]. patients could explain their higher FGF19 values in To the best of our knowledge, we are the first group comparison to overweight patients, which indicates which established a body-weight adjusted oral fat toler- that obese can compensate their low fasting FGF19 ance test (OFTT) to stimulate BA secretion and subse- values by a high fat intake. Sonne and colleagues [42] quent FGF19 expression in overweight and obese found that FGF19 concentrations in patients with NAFLD patients. Only a few studies report about post- type 2 diabetes and healthy controls increased with prandial FGF19 concentrations [13, 41, 42]. Similar oral increasing fat and decreasing carbohydrate content in fat load tests have been used for determining postpran- liquid meals (500 kcal, 2.5 vs. 10.0 and 40.0 g fat). dial triglyceride concentrations, but to date no standard FGF19 values tended to be lower in type 2 diabetes method is established [43]. Our controls showed the patients compared with controls, but were not statis- highest FGF19 values during OFTT, suggesting unim- tically significant. paired intestinal FGF19 release. Remarkably, overweight Oral fat intake stimulates bile acid (BA) secretion. The NAFLD patients had significantly lower FGF19 concen- entry of dietary fat in the duodenum causes gallbladder trations 2 h after the fat load as compared to controls contraction and inflow of BA into the intestinal lumen. and lowest hormone values at all postprandial time In the ileum, BA induce secretion of FGF19 that sup- points. In another study in healthy volunteers, oral fat presses de novo BA synthesis in the liver [25]. In the load (75 g vegetable fat, mixture of Calogen®, sun flower present study fasting and postprandial BA concentra- and olive oil) also showed a stepwise increase of FGF19 tions did not differ between overweight/obese NAFLD between 2 and 4 h and a decrease at 6 h almost reaching patients and controls and C4 serum concentrations in fasting levels [41]. Schreuder et al. used whipped cream NAFLD patients did not decrease as they did in controls. for their oral fat test in NAFLD patients [13]. The fat Therefore, the hepatic BA biosynthesis was presumably challenge was applied with 30 g cream (35% w/v fat) per not repressed. One reason could be that CYP7A1 ex- m of body surface area. Single postprandial time points pression was insufficiently suppressed by FGF19 due to in plasma FGF19 concentrations did not differ between low fasting and postprandial FGF19 concentrations. controls and NAFLD patients. Interestingly, the post- Schreuder et al. [13] also reported an impaired hepatic prandial FGF19-IAUC was lower in NAFLD patients response (no decline of C4) in NAFLD patients with [13]. In our study, mean AUC and IAUC did not differ insulin resistance. The pathomechanism(s) behind the significantly, but AUC was highest in controls and low- observed blunted C4 response are unknown. One might est in overweight patients; in contrast, IAUC was highest speculate about impaired hepatic signaling after binding in obese and lower in overweight patients in comparison of FGF19 to the FGF receptor 4/βKlotho heterodimer or to controls. In comparison to Schreuder’s study [13], our other factors within the fatty liver cell that might affect fat challenge was considerably higher, which could have the feedback regulation of BA synthesis. Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 9 of 10 Our method of bile acid analysis did not yield fatty liver disease; NASH: Non-alcoholic steatohepatitis; OFTT: Oral fat tolerance test complete profiles including conjugates with glycine or taurine. Thus, distinct differences in BA profiles between Acknowledgements lean controls and NAFLD patients might have affected We thank Nutricia GmbH for providing the study test drink Calogen® for our activation of FXR. This is supported by studies in Chin- oral fat tolerance test (OFTT). ese children were levels of chenodeoxycholic acid Availability of data and materials (CDCA) were increased in the moderate/severe stage of The data sets analysed during the current study available from the NAFLD. The authors report, that decreased circulating corresponding author on reasonable request. level of deoxycholic acid (DCA) in children with mild Authors’ contributions NAFLD might have a negative effect on the activation of DF developed study design and OFTT, recruited study participants, measured FXR, which subsequently triggers an increasing produc- body fat mass, centrifuged and aliquoted blood samples, performed FGF19 tion of CDCA in patients with moderate to severe ELISA, analyzed the data and wrote the manuscript. FL developed the idea to investigate FGF19 serum levels in NAFLD. HUM measured BA and C4 NAFLD [45]. In contrast, Jiao and colleagues [39] found concentrations. All authors read, edited and approved the final manuscript. increased DCA and decreased CDCA levels in NAFLD patients. In this context, changes in BA composition Ethics approval and consent to participate could also be one reason for altered intestinal FXR sig- The study protocol was approved by the Ethics Committee of the Ärztekammer des Saarlandes, Saarbrücken (ID number 58/09). All subjects (≥ 18 years) were naling, expressed as reduced FGF19 levels, in our over- fully informed about the study objectives and methods and gave their written weight and obese NAFLD patients. informed consent before participating in this non-randomized controlled pilot trial. Conclusions Competing interests Fasting FGF19 serum concentrations were lowest in The authors declare that they have no competing interests. obese NAFLD patients and highest in normal-weight healthy controls. Our body weight-adjusted oral fat chal- Publisher’sNote lenge resulted in lowest FGF19 concentrations in over- Springer Nature remains neutral with regard to jurisdictional claims in weight NAFLD patients at all postprandial time points. published maps and institutional affiliations. Overweight and obese NAFLD patients showed impaired Author details FGF19 release in fasting and postprandial state. We 1 Department of Medicine II, Saarland University Medical Center, Saarland assume that obese NAFLD patients were able to com- University, 66421 Homburg, Germany. Department of Molecular and Clinical Medicine, Sahlgrenska Academy, Institute of Medicine, University of pensate their low fasting FGF19 values by a high (body Gothenburg, Gothenburg, Sweden. weight-adjusted) oral fat intake. Reduced FGF19 values in overweight and obese NAFLD patients might reflect Received: 7 November 2016 Accepted: 23 May 2018 altered intestinal FXR signaling. 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Br Med J (Clin Res Ed). 1986; 292:13–5. 30. Nutricia GmbH. 2012. http://produkte.nutricia.de/de_de/pim/adults/ nahrungsmodule/calogen/1300/#. Accessed 06 June 2012. 31. Harbury CM, Verbruggen EE, Callister R, Collins CE. What do individuals with morbid obesity reportas a usual dietary intake? A narrative review of available evidence. Clin Nutr ESPEN. 2016;13:e15-e22. 32. Marschall HU, Wagner M, Zollner G, Fickert P, Diczfalusy U, Gumhold J, Silbert D, Fuchsbichler A, Benthin L, Grundström R, Gustafsson U, Sahlin S, Einarsson C, Trauner M. Complementary stimulation of hepatobiliary http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Gastroenterology Springer Journals

Response of fibroblast growth factor 19 and bile acid synthesis after a body weight-adjusted oral fat tolerance test in overweight and obese NAFLD patients: a non-randomized controlled pilot trial

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Medicine & Public Health; Gastroenterology; Internal Medicine; Hepatology
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Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is common both in obese and overweight patients. Fibroblast growth factor 19 (FGF19), an intestinal hormone, could play a role in the complex pathogenesis of NAFLD. The aim of our study was to investigate responses of FGF19 and bile acid (BA) synthesis after a body weight-adjusted oral fat tolerance test (OFTT) in overweight and obese NAFLD patients. Methods: For this study, we recruited 26 NAFLD patients; 14 overweight (median BMI 28.3 kg/m ), 12 obese 2 2 (35.3 kg/m ) and 16 healthy controls (24.2 kg/m ). All individuals received 1 g fat (Calogen®) per kg body weight orally. Serum concentrations of FGF19 were determined by ELISA. Concentrations of BAs and BA synthesis marker 7α-hydroxy-4-cholesten-3-one (C4) were measured by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively; all at 0 (baseline), 2, 4 and 6 h during the OFTT. Results: BMI correlated negatively with fasting FGF19 concentrations (rho = − 0.439, p = 0.004). FGF19 levels of obese NAFLD patients were significantly (p = 0.01) lower in the fasting state (median 116.0 vs. 178.5 pg/ml), whereas overweight NAFLD patients had significantly (p = 0.004) lower FGF19 concentrations 2 h after the fat load (median 163.0 vs. 244.5 pg/ ml), and lowest values at all postprandial time points as compared to controls. Baseline BA concentrations correlated positively with FGF19 values (rho = 0.306, p = 0.048). In all groups, we observed BA increases during the OFTT with a peak at 2hbutnochangeinC4levelsinoverweight/obeseNAFLD patients. Conclusions: Reduced basal gastrointestinal FGF19 secretion and decreased postprandial response to oral fat together with blunted effect on BA synthesis indicate alterations in intestinal or hepatic FXR signaling in overweight and obese NAFLD subjects. The precise mechanism of FGF19 signaling after oral fat load needs further evaluation. Trial registration: We have registered the trial retrospectively on 30 Jan 2018 at the German clinical trials register (http://www.drks.de/), and the following number has been assigned DRKS00013942. Keywords: Bile acids, FGF19, Non-alcoholic fatty liver disease, Oral fat tolerance test * Correspondence: dana.friedrich@uks.eu Department of Medicine II, Saarland University Medical Center, Saarland University, 66421 Homburg, Germany Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 2 of 10 Background Since the role of FGF19 in the pathogenesis of hu- Obesity and fatty liver disease represent increasing med- man NAFLD is unknown, we studied FGF19 and hep- ical problems in developed countries. In Germany, the atic downstream effects (C4 and BAs) in overweight prevalence of obesity increased during the years 1998 to and obese NAFLD outpatients (and healthy controls) 2011 from 18.9 to 23.3% in men and from 22.5 to 23.9% that were subjected to a body weight-adjusted oral fat in women [1]. In the United States, 37% of adults are tolerance test (OFTT). We determined serum concen- obese [2]. Obesity is an important risk factor of trations of FGF19, C4 and BAs at baseline and at 2, 4 non-alcoholic fatty liver disease (NAFLD), which has and 6 h after OFTT. We hypothized that FGF19 been reported in 30 to 40% of adults [3, 4]. levels are lower in obese compared to overweight The term NAFLD is used for a wide spectrum of fatty NAFLD patients. liver diseases that starts with simple steatosis in We aimed to answer the following questions in this non-alcoholic fatty liver (NAFL) that may progress to study: non-alcoholic steatohepatits (NASH), which is compli- cated by fibrosis, cirrhosis, and eventually hepatocellular 1. Do fasting FGF19 serum concentrations differ carcinoma [5–7]. NAFLD is often associated with the between normal-weight healthy, overweight and metabolic syndrome and requires exclusion of excessive obese NAFLD patients? alcohol consumption as well as viral and autoimmune 2. How does a body weight-adjusted oral fat tolerance liver diseases [8]. NAFLD is common in obesity but also test (OFTT) affect serum FGF19 concentrations in in overweight patients [9, 10]. The pathophysiology of these populations? NAFLD is complex and still not fully defined [11, 12]. 3. How does a postprandial FGF19 response affect Several metabolic factors have already been identified in hepatic BA biosynthesis, as assessed by C4? the development of NAFLD, including insulin resistance, diabetes mellitus and obesity. So far there have been only few studies of the im- Methods portance of gastrointestinal hormones in the patho- Study protocol genesis of NAFLD [13–15]. The gastrointestinal The study protocol was approved by the Ethics Committee hormone fibroblast growth factor 19 (FGF19) has of the Ärztekammer des Saarlandes, Saarbrücken (ID num- emerged as a novel regulator of bile acid, carbohy- ber 58/09). All subjects (≥ 18 years) were fully informed drate and lipid metabolism. In human metabolic syn- about the study objectives and methods and gave their drome associated diseases, such as type 2 diabetes written informed consent before participating in this mellitus (T2DM) and NAFLD, FGF19 signaling seems non-randomized controlled pilot trial. to be dysregulated [16]. In animals, FGF19 transgenic mice show resistance to a high-fat diet and decreased Study subjects liver triglyceride concentrations [17] while the admin- During 2009 and 2010, we recruited overweight and istration of recombinant FGF19 increases the meta- obese NAFLD outpatients in the Department of Internal bolic rate [18]. Accordingly, in humans with NAFLD, Medicine II, Saarland University Medical Center, Hom- reduced fasting FGF19 levels were found [14, 19, 20]. burg, as well as healthy controls with normal body Therefore, the present study focuses on the dietary weight. Inclusion criteria for NAFLD were ultrasound regulation of FGF19 and its potential role in the and/or biopsy findings consistent with fatty liver disease. pathogenesis of NAFLD. Exclusion criteria were increased alcohol consumption FGF19 release in the intestine is induced by bile acids in medical history and the following acute and chronic (BAs). After a meal, the entry of dietary fat in the duode- liver diseases: cirrhosis, hepatitis A virus (HAV), hepa- num causes gallbladder contraction and BA inflow into titis B virus (HBV), hepatitis C virus (HCV), hepatitis D the intestinal lumen. The reabsorption of BAs in the ter- virus (HDV), cytomegalovirus (CMV) and Epstein-Barr minal ileum activates the canonical BA sensor farnesoid Virus (EBV) infections, hemochromatosis, Wilson’s dis- X receptor (FXR), resulting in enhanced transcription ease, α -antitrypsin deficiency, and autoimmune hepa- and secretion of FGF19 [21, 22]. FGF19 binds on hepa- titis. Healthy controls included employees of the clinic tocytes to the FGF receptor 4 (FGFR4) and its cofactor and medical students with normal BMI and no diseases βKlotho [22–24], which triggers a signaling cascade that in history. In controls, no liver and laboratory diagnosis represses cholesterol 7α-hydroxylase (CYP7A1), the was performed. rate-limiting enzyme in BA synthesis from cholesterol Subjects were divided into three groups according to [25]. 7α-Hydroxy-4-cholesten-3-one (C4) is an inter- their BMI (healthy controls, normal weight: 19.0– 2 2 mediate of BA synthesis, which can be measured in 25.4 kg/m , overweight NAFLD: 25.5–29.9 kg/m , obese serum [26]. NAFLD: ≥ 30.0 kg/m )[27, 28]. Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 3 of 10 Liver parameters determined by gas chromatography-mass spectrometry In overweight and obese patients (N = 26), liver status (GCMS) [32]. 7α-hydroxy-4-cholesten-3-one (C4), a valid was assessed by abdominal ultrasound and/or liver marker of bile acid biosynthesis [33], was measured by biopsy. Ultrasound was performed using the Hitachi high-performance liquid chromatography (HPLC). EUB-8500 ultrasound scanner (Hitachi Medical Systems, Wiesbaden, Germany). Hepatic steatosis results in Statistical analysis abnormal echo patterns on ultrasound scanning; the Data analysis was performed using SPSS (version 20.0, severity of steatosis was graded as mild (I), moderate IBM, Ehningen, Germany). Kruskal-Wallis test was used (II), or severe (III) [29]. Liver biopsy samples of five to analyze quantitative data for differences within the co- patients were examined by an experienced pathologist of hort. For the present study with a low number of study Saarland University Medical Center. subjects (N < 20), normal distributions were not expected [34]. Thus, data are expressed as medians and interquar- Oral fat tolerance test (OFTT) tile ranges (IQR 25–75). In addition, Mann-Whitney-U We used a body weight-adjusted OFTT to investigate test was used to test differences between two groups. The postprandial FGF19, BA and C4 responses in our study strength of associations between two parameters was esti- subjects. For the present study, a standardized test drink mated using the non-parametric Spearman correlation Calogen® (Nutricia, Erlangen, Germany) was adminis- test. Spearman’s correlation coefficient is presented as rho. tered, which is a lipid emulsion based on vegetable fat For the OFTT, FGF19 -area under the curve (AUC) (0-6h) with 50% long-chain triglycerides [30]. All individuals and, after correcting for baseline, the incremental AUC received 1 g fat per kg body weight orally. The fat load (FGF19-IAUC) were computed using GraphPad Prism was based on subjects’ body weight to adjust the OFTT (version 6.0, GraphPad Software, La Jolla, CA, USA). A to hypercaloric (especially high-fat) eating behavior of p-value < 0.05 denotes statistical significance. obese patients [31]. Results Blood samples Subject characteristics Blood samples were drawn from a peripheral vein at Table 1 summarizes the subject characteristics. A total 8:00 AM after an overnight fasting and 2, 4 and 6 h after of 42 subjects, 21 women and 21 men, were recruited the oral fat challenge. Samples were centrifuged for for our study. Study participants were between 19 and 10 min at 3000 g 30 min after blood collection 68 years old (median 47.0 years, IQR 28.8–53.8). Overall, (ROTANTA 46R, Hettich, Tuttlingen, Germany). Subse- we recruited 14 overweight and 12 obese NAFLD quently, serum was stored in aliquots at-70 °Cuntil patients as well as 16 healthy controls. Sex and age did analysis. not differ between groups. Obese patients had a median BMI of 35.3 kg/m , which corresponds to obesity grade Serum FGF19, bile acid and C4 measurements II [28]. FGF19 serum concentrations were measured in duplicate NAFLD was diagnosed by ultrasound and/or biopsy. by quantitative sandwich enzyme-linked immunosorbent In overweight and obese patients, the steatosis spectrum assay, using the FGF19 Quantikine ELISA kit (R&D Sys- ranged from grade I, II and III to NASH and fibrosis. In tems, Minneapolis, USA). Serum BA concentrations were the overweight group (N = 14), grade I liver steatosis was Table 1 Subject characteristics, basal and postprandial FGF19 serum concentrations Variables Control Overweight Obesity p-value N (men/women) 16 (7/9) 14 (8/6) 12 (6/6) n.s. Age (years) 29.5 (24.0–53.0) 49.0 (38.8–57.3) 48.0 (37.0–57.3) 0.551 2 b BMI (kg/m ) 24.2 (21.8–26.6) 28.3 (26.3–29.2) 35.3 (32.7–39.0) < 0.001 FGF19 (pg/ml) c c c t = 0 h 178.5 (101.0–257.0) 127.5 (70.0–161.3) 116.0 (51.0–134.3) 0.01 c c c t = 2 h 244.5 (161.5–377.5) 163.0 (78.5–168.3) 181.0 (85.3–393.0) 0.004 t = 4 h 332.5 (202.0–590.8) 207.0 (112.5–365.0) 220.0 (138.8–385.3) 0.445 t = 6 h 211.0 (165.3–296.3) 154.0 (124.0–254.0) 184.5 (110.5–274.3) 0.445 All data are given as median (interquartile range) Chi-square-test Kruskal-Wallis-test Mann-Whitney-U-test Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 4 of 10 found in six patients (43%), grade II in five patients lower in obese NAFLD patients as compared to controls (36%), and grade III in one patient (7%). NASH was and tended to be lower in overweight NAFLD subjects, diagnosed in one and fibrosis stage II was documented too (Table 1, Fig. 1). Basal FGF19 concentrations did not in one patient. In the obese group (N = 12), two patients differ between sexes [women, median 126.0 (IQR 80.5– (17%) displayed liver steatosis grade I, four patients 179.0) vs. men, median 138.0 (IQR 93.0–204.0) pg/ml]. (33%) showed grade II, and three participants (25%) had Interestingly, fasting FGF19 concentrations were nega- grade III. In this group, NASH was found in one and tively correlated with BMI (Fig. 2). fibrosis stage I also in one patient. In one obese study After the OFTT, FGF19 concentrations increased in participant, liver status could not be assessed by controls, overweight and obese patients (Table 1, Fig. 1). ultrasound. Additional file 1: Table S1 lists the co- Of note, overweight patients displayed lowest FGF19 morbidities in overweight and obese NAFLD patients. concentrations at all postprandial time points. Two In overweight patients, hypercholesterolemia and in hours after the OFTT, FGF19 levels ranged from 10.0 to obese patients, arterial hypertension were the domin- 697.0 pg/ml (median 178.0, IQR 116.5–255.5). At this ant concomitant diseases, respectively. Controls did time, overweight NAFLD patients showed significantly not take any drugs regularly. Three overweight and lower FGF19 levels compared with controls (Fig. 1). The eight obese patients were taking medications. These FGF19 maximum was found in all three groups after included antidiabetics, antihypertensives, thyroid hor- 4 h, with hormone levels ranging from 59.0 to 935.0 pg/ mones, analgesics, proton pump inhibitors, antidepres- ml (median 255.0, IQR 163.3–439.3). At 4 h, FGF19 sants, non-steroidal antirheumatics, corticoidsteroids and was highest in controls and twice as high as at base- allopurinol, respectively. line. After 6 h, FGF19 values ranged from 48.0 to 802.0 pg/ml (median 189.0, IQR 136.0–269.0) and Basal and postprandial FGF19 serum concentrations were still highest in controls. In the total study group (N = 42), fasting FGF19 concen- Both women and men showed the FGF19 maximum trations ranged from 17.0 to 392.0 pg/ml (median 133.5, at 4 h [women, median 270.0 (IQR 167.5–539.0) vs. IQR 82.8–190.3). Basal FGF19 values were significantly men, median 231.0 (IQR 117.5–410.5) pg/ml] but Fig. 1 Fasting and postprandial FGF19 serum concentrations measured by quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Comparison of FGF19 values between healthy controls (N = 16), overweight (N = 14) and obese (N = 12) patients with non-alcoholic fatty liver disease (NAFLD) at baseline (0 h), 2, 4 and 6 h after the oral fat tolerance test (OFTT). Significant difference between basal (0 h) FGF19 concentrationsin controls and obese NAFLD patients [controls 178.5 (101.0–257.0) vs. obese 116.0 (51.0–134.3) pg/ml, medians (IQRs), p = < 0.05, Mann-Whitney-U-test). At 2 h, lower FGF19 values in overweight NAFLD patients in comparison to controls [overweight 163.0 (78.5–168.3) vs. controls 244.5 (161.5–377.5) pg/ml, medians (IQRs), p = 0.004, Mann-Whitney-U-test), * outlier Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 5 of 10 N=42 p=0.004 rho= - 0.439 Fig. 2 Fasting FGF19 serum concentrations versus body mass index (BMI) for all study subjects. FGF19 values correlated negatively with BMI. A scattered plot is shown and the Spearman’s correlation coefficient was calculated postprandial levels were higher in women at all time to controls. FGF19-AUC and IAUC did not correlate points. Six hours after the oral fat challenge, FGF19 con- with body weight-adjusted fat load. In addition, there centrations of both sexes tended to reach a significant was no association between FGF19-AUC and BMI; difference [women, median 216.0 (IQR 145.0–390.5) vs. FGF19-IAUC tended to correlate with age (rho = 0.291, men, median 172.0 (IQR124.0–216.5) pg/ml, p = 0.051]. p = 0.062). Mean FGF19 -area and mean incremental area (0-6h) under the curve (AUC and IAUC) did not differ sig- nificantly between the groups (AUC controls: 1772.8 Basal and postprandial BA serum concentrations ± 766.6 vs. overweight: 1130.6 ± 590.0 vs. obese: Fasting and postprandial bile acid (BA) concentrations 1469.3 ± 910.0 pg/ml/6 h; IAUC controls: 699.0 ± 383.7 vs. did not differ between overweight/obese NAFLD pa- overweight: 573.3 ± 333.4 vs. obese: 921.5 ± 732.4 pg/ml). tients and controls. In all three groups, we observed a FGF19-AUC was highest in controls and lowest in BA increase after the OFTT with a peak at 2 h (Table 2). overweight patients (p = 0.053); IAUC was higher in Basal FGF19 concentrations correlated positively with obese and lower in overweight patients in comparison basal BA values (Fig. 3). Table 2 Basal and postprandial BA and C4 serum concentrations Variables Control Overweight Obesity p-value N (men/women) 16 (7/9) 14 (8/6) 12 (6/6) n.s. Bile acids (μM) t = 0 h 1.1 (0.8–1.7) 1.5 (0.8–2.2) 1.4 (0.9–1.7) 0.343 t = 2 h 1.4 (1.1–4.7) 2.1 (1.2–3.8) 2.4 (1.6–4.1) 0.311 t = 4 h 1.1 (0.7–2.0) 2.0 (1.3–2.5) 2.0 (1.0–2.3) 0.155 t = 6 h 0.7 (0.5–1.6) 1.3 (0.8–2.0) 1.2 (0.8–1.7) 0.087 C4 (nM) t = 0 h 41.4 (7.0–69.2) 58.5 (12.8–91.6) 35.1 (1.2–72.3) 0.422 t = 2 h 28.1 (7.1–49.6) 43.1 (10.0–114.0) 35.7 (1.2–118.3) 0.765 t = 4 h 13.0 (6.8–44.7) 40.8 (21.1–99.3) 32.7 (3.3–108.0) 0.343 t = 6 h 11.8 (4.1–34.4) 40.7 (18.1–80.1) 28.6 (3.5–82.4) 0.445 All data are given as median (interquartile range), p-values: Kruskal-Wallis-test, Chi-square-test Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 6 of 10 N=42 p=0.048 rho= 0.306 Fig. 3 Fasting FGF19 serum concentrations versus fasting bile acid (BA) serum concentrations for all subjects (N = 42). There was a correlation between FGF19 and BA values. A scattered plot is shown and the Spearman’s correlation coefficient was calculated Basal and postprandial C4 serum concentrations obese NAFLD patients. C4 concentrations in overweight Fasting and postprandial C4 values did not differ signifi- NAFLD patients remained unchanged for 4 h postpran- cantly between study participants (Table 2, Fig. 4). At all dially, despite increasing FGF19 values. In the total study postprandial time points, C4 concentrations were mark- group FGF19 concentrations at 2 h correlated negatively edly lower in controls in comparison to overweight/ with C4 values at 4 h after the OFTT (Fig. 5). There was Fig. 4 Fasting and postprandial C4 concentrations. C4, a valid marker of bile acid biosynthesis, was measured by high-performance liquid chromatography (HPLC). Comparison of C4 values between healthy controls (N = 16), overweight (N = 14) and obese (N = 12) patients with non-alcoholic fatty liver disease (NAFLD) at baseline (0 h), 2, 4 and 6 h after the oral fat tolerance test (OFTT). C4 concentrations did not differ between groups (Kruskal-Wallis-test),*outlier Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 7 of 10 N=42 p=0.031 rho= - 0.332 Fig. 5 FGF19 serum concentrations at 2 h versus C4 values at 4 h after the oral fat tolerance test (OFTT) for all subjects (N = 42). A scattered plot is shown and the Spearman’s correlation coefficient was calculated also an inverse correlation of FGF19 concentrations at Discussion 4 h and C4 values at 6 h after the OFTT in the study The present study investigated serum FGF19, BA, and group (Fig. 6). These correlations were confirmed for C4 profiles in overweight and obese NAFLD patients, in the control group (Additional file 2: Figure S1, comparison to normal-weight healthy controls, after a Additional file 3: Figure S2). In NAFLD patients, FGF19 body weight-adjusted oral fat load. The key findings of concentrations did not correlate with C4 values. our study are (i) fasting FGF19 concentrations were N=42 p=0.006 rho= - 0.420 Fig. 6 FGF19 serum concentrations at 4 h versus C4 values at 6 h after the oral fat tolerance test (OFTT) for all subjects (N = 42). A scattered plot is shown and the Spearman’s correlation coefficient was calculated Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 8 of 10 significantly lower in obese (grade II) NAFLD patients as contributed to differential findings at single postprandial compared to controls, (ii) overweight NAFLD patients time points and in FGF19-IAUC between controls and had significantly lower FGF19 concentrations 2 h after NAFLD patients. the fat load and lowest values at all postprandial time Before our study started, we suspected that obese points and (iii) BAs increased during the OFTT but patients would have the highest energy and fat intake. without changes in C4 levels. Therefore we decided for a body weight-based fat load Low FGF19 concentrations have been reported in to adjust the OFTT to the patient’s eating behavior. To metabolic syndrome [35], obesity [36] and type 2 dia- check our assumption, we used a 3-day nutritional betes [37, 38]. The findings by Jansen et al. [19] support protocol and calculated energy and macronutrients in- our results, i.e. the presence of fatty liver disease in take (data not shown). Since there was no significant dif- obesity is associated with lower fasting FGF19 concen- ference between groups, we suppose under-reporting of trations. Also in obese children with NAFLD [20] and food intake in our overweight and obese patients. Most young obese NASH patients [39] lower fasting FGF19 researchers agree that the reported accuracy of food in- values were detected whereas Schreuder et al. did not take decreases with increasing BMI. A systematic review observe differences in fasting FGF19 concentrations be- covering studies between 1982 and 2014 showed that a tween controls and obese NAFLD subjects [13]. In our BMI > 30 kg/m is associated with significant study, BMI correlated negatively with fasting FGF19 under-reporting of food intake. These studies were serum concentrations. Our study was too small to cor- mostly from Europe and North America [44]. For ex- relate FGF19 levels with biopsy-proven severity of ample, in morbid obese (BMI > 40 kg/m ) energy intake NAFLD. In this respect, inverse associations had been can reach more than 4000 kcal/day, with high fat intakes found in children [15, 40], but some studies did not find of about 40 to 57% of total energy intake [31]. correlations between steatosis grade and FGF19 concen- In the present study, higher fat challenge in obese trations [13, 14]. patients could explain their higher FGF19 values in To the best of our knowledge, we are the first group comparison to overweight patients, which indicates which established a body-weight adjusted oral fat toler- that obese can compensate their low fasting FGF19 ance test (OFTT) to stimulate BA secretion and subse- values by a high fat intake. Sonne and colleagues [42] quent FGF19 expression in overweight and obese found that FGF19 concentrations in patients with NAFLD patients. Only a few studies report about post- type 2 diabetes and healthy controls increased with prandial FGF19 concentrations [13, 41, 42]. Similar oral increasing fat and decreasing carbohydrate content in fat load tests have been used for determining postpran- liquid meals (500 kcal, 2.5 vs. 10.0 and 40.0 g fat). dial triglyceride concentrations, but to date no standard FGF19 values tended to be lower in type 2 diabetes method is established [43]. Our controls showed the patients compared with controls, but were not statis- highest FGF19 values during OFTT, suggesting unim- tically significant. paired intestinal FGF19 release. Remarkably, overweight Oral fat intake stimulates bile acid (BA) secretion. The NAFLD patients had significantly lower FGF19 concen- entry of dietary fat in the duodenum causes gallbladder trations 2 h after the fat load as compared to controls contraction and inflow of BA into the intestinal lumen. and lowest hormone values at all postprandial time In the ileum, BA induce secretion of FGF19 that sup- points. In another study in healthy volunteers, oral fat presses de novo BA synthesis in the liver [25]. In the load (75 g vegetable fat, mixture of Calogen®, sun flower present study fasting and postprandial BA concentra- and olive oil) also showed a stepwise increase of FGF19 tions did not differ between overweight/obese NAFLD between 2 and 4 h and a decrease at 6 h almost reaching patients and controls and C4 serum concentrations in fasting levels [41]. Schreuder et al. used whipped cream NAFLD patients did not decrease as they did in controls. for their oral fat test in NAFLD patients [13]. The fat Therefore, the hepatic BA biosynthesis was presumably challenge was applied with 30 g cream (35% w/v fat) per not repressed. One reason could be that CYP7A1 ex- m of body surface area. Single postprandial time points pression was insufficiently suppressed by FGF19 due to in plasma FGF19 concentrations did not differ between low fasting and postprandial FGF19 concentrations. controls and NAFLD patients. Interestingly, the post- Schreuder et al. [13] also reported an impaired hepatic prandial FGF19-IAUC was lower in NAFLD patients response (no decline of C4) in NAFLD patients with [13]. In our study, mean AUC and IAUC did not differ insulin resistance. The pathomechanism(s) behind the significantly, but AUC was highest in controls and low- observed blunted C4 response are unknown. One might est in overweight patients; in contrast, IAUC was highest speculate about impaired hepatic signaling after binding in obese and lower in overweight patients in comparison of FGF19 to the FGF receptor 4/βKlotho heterodimer or to controls. In comparison to Schreuder’s study [13], our other factors within the fatty liver cell that might affect fat challenge was considerably higher, which could have the feedback regulation of BA synthesis. Friedrich et al. BMC Gastroenterology (2018) 18:76 Page 9 of 10 Our method of bile acid analysis did not yield fatty liver disease; NASH: Non-alcoholic steatohepatitis; OFTT: Oral fat tolerance test complete profiles including conjugates with glycine or taurine. Thus, distinct differences in BA profiles between Acknowledgements lean controls and NAFLD patients might have affected We thank Nutricia GmbH for providing the study test drink Calogen® for our activation of FXR. This is supported by studies in Chin- oral fat tolerance test (OFTT). ese children were levels of chenodeoxycholic acid Availability of data and materials (CDCA) were increased in the moderate/severe stage of The data sets analysed during the current study available from the NAFLD. The authors report, that decreased circulating corresponding author on reasonable request. level of deoxycholic acid (DCA) in children with mild Authors’ contributions NAFLD might have a negative effect on the activation of DF developed study design and OFTT, recruited study participants, measured FXR, which subsequently triggers an increasing produc- body fat mass, centrifuged and aliquoted blood samples, performed FGF19 tion of CDCA in patients with moderate to severe ELISA, analyzed the data and wrote the manuscript. FL developed the idea to investigate FGF19 serum levels in NAFLD. HUM measured BA and C4 NAFLD [45]. In contrast, Jiao and colleagues [39] found concentrations. All authors read, edited and approved the final manuscript. increased DCA and decreased CDCA levels in NAFLD patients. In this context, changes in BA composition Ethics approval and consent to participate could also be one reason for altered intestinal FXR sig- The study protocol was approved by the Ethics Committee of the Ärztekammer des Saarlandes, Saarbrücken (ID number 58/09). All subjects (≥ 18 years) were naling, expressed as reduced FGF19 levels, in our over- fully informed about the study objectives and methods and gave their written weight and obese NAFLD patients. informed consent before participating in this non-randomized controlled pilot trial. Conclusions Competing interests Fasting FGF19 serum concentrations were lowest in The authors declare that they have no competing interests. obese NAFLD patients and highest in normal-weight healthy controls. Our body weight-adjusted oral fat chal- Publisher’sNote lenge resulted in lowest FGF19 concentrations in over- Springer Nature remains neutral with regard to jurisdictional claims in weight NAFLD patients at all postprandial time points. published maps and institutional affiliations. Overweight and obese NAFLD patients showed impaired Author details FGF19 release in fasting and postprandial state. We 1 Department of Medicine II, Saarland University Medical Center, Saarland assume that obese NAFLD patients were able to com- University, 66421 Homburg, Germany. Department of Molecular and Clinical Medicine, Sahlgrenska Academy, Institute of Medicine, University of pensate their low fasting FGF19 values by a high (body Gothenburg, Gothenburg, Sweden. weight-adjusted) oral fat intake. Reduced FGF19 values in overweight and obese NAFLD patients might reflect Received: 7 November 2016 Accepted: 23 May 2018 altered intestinal FXR signaling. 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