The Circulating Metabolic Regulator FGF21 Is Induced by Prolonged Fasting and PPARa Activation in Man

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1 Short Article The Circulating Metabolic Regulator FGF21 Is Induced by Prolonged Fasting and PPARa Activation in Man Cecilia Gälman, 1,5 Tomas Lundåsen, 1,5 Alexei Kharitonenkov, 3 Holly A. Bina, 3 Mats Eriksson, 1 Ingiäld Hafström, 2 Maria Dahlin, 4 Per Åmark, 4 Bo Angelin, 1 and Mats Rudling 1, * 1 Department of Endocrinology, Metabolism and Diabetes, Molecular Nutrition Unit, Departments of Medicine and Biosciences and Nutrition 2 Department of Rheumatology Karolinska Institutet, Karolinska University Hospital Huddinge, S Stockholm, Sweden 3 Lilly Research Laboratories, Division of Eli Lilly and Company, Indianapolis, IN 46285, USA 4 Neuropediatric Department, Astrid Lindgren Children s Hospital, Karolinska University Hospital and Karolinska Institutet, S Stockholm, Sweden 5 These authors contributed equally to this work *Correspondence: mats.rudling@ki.se DOI /j.cmet SUMMARY FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARa in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARa activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice. INTRODUCTION The fibroblast growth factor (FGF) family comprises at least 22 members involved in development, differentiation, cell survival and growth, wound healing, and tumor formation (Itoh and Ornitz, 2004; Ornitz and Itoh, 2001). Recently, FGF19, FGF21, and FGF23 have been described as structurally and functionally diverted (Nishimura et al., 1999, 2000; Shimada et al., 2001; Xie et al., 1999). These FGFs exert hormone-like metabolic effects by interacting with FGF receptors (Fukumoto, 2008) dependent on the presence of the beta-glucuronidases Klotho or beta-klotho as coreceptors (Goetz et al., 2007; Kharitonenkov et al., 2007a; Kurosu et al., 2007; Lin et al., 2007; Ogawa et al., 2007; Urakawa et al., 2006; Wu et al., 2007). FGF23 is linked to phosphate balance (Fukumoto and Yamashita, 2007; Masuyama et al., 2006), while FGF19 is a strong metabolic regulator in mice (Fu et al., 2004; Tomlinson et al., 2002). Like its mouse homolog FGF15, FGF19 is also a negative regulator of bile acid synthesis in mice (Inagaki et al., 2005) and humans (Lundasen et al., 2006). FGF21, abundant in the liver (Nishimura et al., 2000), is structurally related to FGF19, and FGF21-transgenic animals are resistant to diet-induced weight gain and fat accumulation (Kharitonenkov et al., 2005). Diabetic rodents given FGF21 display improved blood glucose, insulin, and triglycerides (TG), (Kharitonenkov et al., 2005) as do diabetic monkeys (Kharitonenkov and Shanafelt, 2008; Kharitonenkov et al., 2007b). FGF21 has also been shown to be a direct target gene of PPARa in mouse liver (Badman et al., 2007; Inagaki et al., 2007; Lundasen et al., 2007). Hepatic FGF21 mrna is induced in mice that are fasted or exposed to a ketogenic diet (KD), partly in a PPARa-dependent manner (Badman et al., 2007; Lundasen et al., 2007), and changes in FGF21 hepatic expression correlate with its plasma levels (Badman et al., 2007). Thus, FGF21 has been described as a novel metabolic regulator (Kharitonenkov et al., 2005) and as a master hormonal switch for liver ketogenesis and overall metabolic adaptation to fasting (Reitman, 2007). Since little information is available on FGF21 functions in man, we carried out studies to analyze circulating levels of FGF21 in various conditions, such as in normal and hypertriglyceridemic (HTG) humans and during metabolic perturbations induced by fasting, a KD, and by fenofibrate treatment. Our results show that FGF21 circulates in humans at highly variable levels. While no increases in serum FGF21 levels were observed following a 48 hr fast or KD treatment despite an induction of ketosis, fasting for one week significantly increased serum FGF21. FGF21 levels were also elevated in HTG patients, while treatment with the PPARa agonist fenofibrate lowered serum TG and increased serum FGF21. RESULTS FGF21 Is Present in Human Serum and Its Levels Are Highly Variable between Individuals We first evaluated the distribution of serum FGF21 and its relation to different metabolic parameters. When FGF21 was Cell Metabolism 8, , August 6, 2008 ª2008 Elsevier Inc. 169

2 assayed by ELISA in samples from 76 healthy normal-weight volunteers with an age span of years, its levels showed considerable interindividual variation ranging from 21 to 5300 pg/ml. The skewed distribution (Figure 1A) became normal upon log transformation (not shown). There was no difference between genders (see insert in Figure 1A). In all 76 individuals, the mean and median FGF21 levels were 450 pg/ml and 156 pg/ml, respectively. FGF21 levels did not correlate to body mass index (BMI), age, blood glucose, serum total, LDL or HDL cholesterol, or TG (not shown). Neither did we find a correlation between serum FGF21 and whole body cholesterol synthesis (assessed by serum lathosterol) or bile acid synthesis (evaluated by serum 7a-hydroxy-4-cholesten-3-one, [C4]). Individual Serum FGF21 Levels Are Stable with Time We next evaluated if circulating FGF21 has a diurnal rhythm, as has FGF19 (Lundasen et al., 2006). Samples from 5 healthy volunteers drawn every 90 min over a 25.5 hr period (Galman et al., 2005) were assayed for FGF21. There were no major changes related to feeding and fasting. In one individual, subject D, FGF21 levels were stable throughout the observation period (Figure 1B). In three subjects, FGF21 levels increased somewhat from midnight to 1:30 AM. However, comparison of the means of the relative levels of all subjects did not show any significant changes during the period as compared to the start value (9 AM), indicating that FGF21 levels are stable in healthy subjects. FGF21 Is Only Induced by Prolonged Fasting Since fasting increases hepatic FGF21 mrna expression and plasma FGF21 in mice (Badman et al., 2007; Inagaki et al., 2007; Lundasen et al., 2007), we first tested if this also occurs in humans by analyzing samples from a series of experiments. First, four subjects who had been fasted overnight continued to refrain from food the next day (Galman et al., 2005). Blood was collected every hour from 9 AM to 4 PM. This short fasting did not increase serum FGF21 levels (Figure 1C). Instead, FGF21 levels were reduced the first hours of sampling. For the group, this transient reduction was significant at 11 AM and at noon (Figure 1D). FGF21 increased for the following hours so that by 4 PM the levels had returned to initial levels. To evaluate if longer fasting could alter circulating FGF21, we analyzed serum from 7 healthy subjects prior to and after 48 hr of fasting (Arner et al., 2003). During this period, subjects lost 2.5 kg ( 3.4%) in BW (Figure 2A). As expected, there was a strong 40- fold increase in serum 3-hydroxybutyrate, and plasma glucose was reduced (Figures 2B and 2C). There were, however, no changes in FGF21 levels (Figure 2D). Thus, in man, a ketogenic response induced by a 2 day fast occurred without alterations in circulating FGF21. To further explore if a more energy-deprived state would alter FGF21 levels in humans, we assayed samples from a third study (Hafstrom et al., 1988), where 5 females with rheumatoid arthritis were sampled before and at the end of a 7 day water fast. Body weight was reduced by 4.2 kg ( 6.9%), whereas serum 3-hydroxybutyrate levels increased 70-fold (Figures 3A and 3B). In this experiment, serum FGF21 increased by 74%, from 913 ng/ml to 1592 ng/ml (p < 0.05) (Figure 3C). Thus, after a 7 day fast, plasma FGF21 increased in all individuals although the levels reached were still within the normal range. We next evaluated whether the ingestion of a KD may increase plasma levels of FGF21. For this purpose, we assayed FGF21 in samples from a set of children sampled before and on a KD. As expected, 3-hydroxybutyrate increased, and plasma glucose was reduced (Figures 3D and 3E). However, plasma FGF21, although showing a similar level and broad variation as in adults (Figure 1), was not increased on the KD (Figure 3F). Fenofibrate Treatment Increases Serum FGF21 PPARa regulates FGF21 transcription necessary for the fasting response in mice, and PPARa ligands increase FGF21 transcription in human hepatocytes (Badman et al., 2007; Inagaki et al., 2007; Lundasen et al., 2007). Fibrates, a class of TG-lowering drugs, are PPARa ligands (Forman et al., 1997), and FGF21 treatment of diabetic animals reduces plasma TG (Kharitonenkov et al., 2005, 2007b). We therefore questioned if fibrate treatment in humans can increase serum FGF21. For this purpose, 19 normal-weight, nondiabetic patients with primary HTG were treated for 3 weeks with placebo or fenofibrate (200 mg/day) using a double-blind crossover design. On placebo, plasma TGs were 5-fold higher than in the 76 controls described above. Interestingly, baseline serum FGF21 was doubled as compared to normals (Figure 4A). Among HTG patients, however, there was no correlation between FGF21 and TG levels. Fenofibrate treatment reduced plasma TG 60%, whereas serum FGF21 levels increased significantly by 28%, from 964 pg/ml to 1233 pg/ml (Figure 4B). DISCUSSION The metabolic regulator FGF21 controls glucose/lipid homeostasis in rodents and nonhuman primates and is critical in mice in the physiological response to fasting, presumably acting in part as a circulating hormone (Badman et al., 2007; Inagaki et al., 2007). Since it is unclear whether these functions are present in man, we searched for evidence for a metabolic role of FGF21 in humans by measuring FGF21 levels in serum at different metabolic and pharmacological conditions. Fasting morning levels of FGF21 in sera from 76 healthy, nonobese subjects revealed a broad 250-fold interindividual variation from 21 pg/ml to 5300 pg/ml. The mean level of 450 pg/ml is similar to that reported in mice (Badman et al., 2007). In normal subjects, we found no relations between FGF21 and serum cholesterol, TG, blood glucose, BMI, age, gender. We also found no relations between FGF21 and cholesterol or bile acid synthesis, the latter two evaluated from serum markers. There were no clear diurnal changes in serum FGF21 indicating that feeding or fasting overnight does not alter FGF21 levels. The fact that subjects with a high or a low FGF21 concentration maintained their individual levels when sampled for 25 hr indicates that there is an intrinsic variation in FGF21 metabolism in healthy subjects. Since we could not identify any modifying factors related to FGF21 levels, it is tempting to speculate that variation in serum concentration is not important for metabolic regulation in healthy humans. Indeed, the manifestation of FGF21 requires either disease condition or a metabolic challenge in healthy rodents or monkeys (Kharitonenkov and Shanafelt, 2008; Kharitonenkov et al., 2005, 2007b). Furthermore, in analogy with insulin and leptin biology, high levels of FGF21 may 170 Cell Metabolism 8, , August 6, 2008 ª2008 Elsevier Inc.

3 Figure 1. FGF21 Is Highly Variable between Individuals but Stable with Time (A) Frequency distribution of serum FGF21 in healthy subjects from samples obtained after overnight fast from 76 healthy volunteers (39 women and 37 men) with an age span from yr. The insert shows distribution in males and females; bars show SD. (B) Absence of diurnal changes in serum FGF21. Blood samples were drawn from five healthy volunteers every 90 min over a 25.5 hr period (Galman et al., 2005). The first sample was taken at 9 AM after an overnight fast. All subjects received the same standardized meals at the indicated times (arrows) and slept in darkness from 12:30 AM to 7 AM. There were no significant differences when all time points (expressed as percentage of initial value) were compared to the first sampling (Dunnett s test). (C) Serum FGF21 during acute fasting (prolonged overnight fasting 9 AM 4 PM) in four subjects. Four of the subjects shown in (B) (subjects A D) were studied 2 6 weeks after the first session. Blood was collected every hour from 9 AM to 4 PM after overnight fast. During the experiment, no food was ingested, only water was consumed ad libitum. FGF21 values are expressed as percentage of the initial value taken at 9 AM. (D) Mean values and SEM (bars) from the four subjects in panel C. The * shows p < 0.05 when compared to the first sampling (Dunnett s test). suggest either a state of hormonal resistance or an adaptive individual response to combat metabolic disturbances. The lack of a clear diurnal rhythm in serum FGF21 is in contrast to FGF19, which has two peaks during daytime (Lundasen et al., 2006). Also, our data suggest that serum FGF21 is not likely influenced by bile acid metabolism since there were no correlations to markers of bile acid or cholesterol synthesis. FGF21 has been implied as an important circulating metabolic regulator in fasted mice (Badman et al., 2007; Inagaki et al., 2007). There, PPARa activation-induced FGF21 hepatic expression/secretion has been hypothesized to promote ketogenesis in situations of energy deprivation by stimulating lipolysis in adipose tissue and promoting fatty acid oxidation in the liver; thus, inducing the energy conserving state of torpor by signaling to the brain (Reitman, 2007). In normal humans, food deprivation during the day did not increase FGF21 levels; if anything, there was a transient reduction in FGF21 instead. Extending the fasting period in normal humans to 2 days resulted in a 40-fold increase of 3-hydroxybutyrate. Despite this, there was no change in serum FGF21 levels (Figure 2D). Thus, in contrast to the situation in mice (Inagaki et al., 2007; Reitman, 2007), serum FGF21 levels are not crucial for the general fasting response in humans. However, since we did not assay FGF21 directly in liver or adipose tissues (Zhang et al., 2008), it cannot be excluded that a local expression of FGF21 is induced at all stages of fasting. This possibility warrants future investigation. Fasting for 7 days was achieved as part of a study on how food deprivation may influence disease activity in patients with rheumatoid arthritis (Hafstrom et al., 1988). Conclusions from this experiment should be drawn with some caution. However, medication was withdrawn at least two weeks before the study, and initial FGF21 levels were not different from those in healthy subjects. After this long period of fasting, the levels of 3-hydroxybutyrate were doubled (6 mmol/l) as compared to those reached after a 2 day fast (3 mmol/l). Although the levels of FGF21 increased in all patients, they were however still within the normal range. Cell Metabolism 8, , August 6, 2008 ª2008 Elsevier Inc. 171

4 Figure 2. Effects of 2 Days of Fasting in Seven Healthy Volunteers (Three Women and Four Men) (A) Body Weight. (B) 3-hydroxybutyrate. (C) Plasma Glucose. (D) Serum FGF21. Animals with high metabolic rates frequently enter the energyconserving state of torpor, and it may be that the function of FGF21 as master switch of fasting is particular in species dependent on torpor for survival (Reitman, 2007). The metabolic rate is about 10-fold higher in mice than in humans, and energy stores are thus rapidly depleted in mice upon food deprivation. Interestingly, fasting of mice for 8 h did not increase plasma FGF21 (Badman et al., 2007). This time period may correspond to the present 2 day fast where we saw no FGF21 increases in man. A 24 hr fast in mice should correspond to about a week of fasting in humans, a situation where we observed increased serum FGF21 levels. As mentioned, the levels of serum ketones were about double compared to the 2 day fast, which may indicate that circulating FGF21 could be important to boost the fasting response during starvation but is not causative to its initiation in humans. Thus, although our FGF21 data following different periods of fasting in humans are in line with the results shown in rodents, they suggest that FGF21 may have somewhat different physiological roles in man and mice; and that it is not likely to be a key regulator of hepatic ketone production in humans. Ketosis is also induced during a KD, which in mice is dependent on hepatic FGF21 production and release (Badman et al., 2007). We could however not see any increases of circulating FGF21 in a set of children clearly ketotic from such a diet regimen, further supporting a different role of FGF21 in man. Figure 3. Prolonged Fasting, but not a KD, Increases Serum FGF21 (A C) Effects of 7 days of fasting in five patients with rheumatoid arthritis (Hafstrom et al., 1988). (A) indicates body weight; (B) indicates 3-hydroxybutyrate; and (C) indicates serum FGF21. (D F) Effects of a KD given to 17 children with pharmacoresistant epilepsy. (D) indicates plasma glucose; (E) indicates 3-hydroxybutyrate, and (F) indicates serum FGF21. In patients with primary HTG, serum FGF21 also varied considerably, but the mean level in those individuals was about twice that observed in healthy subjects. At this stage, though, it is difficult to make any firm conclusions about the possible pathophysiological relevance of that finding. It is important to note, however, that we did not find any correlation between TG and FGF21 levels within the HTG group, in accordance with what we observed in the healthy group. Considering the known effects of FGF21 treatments in animals, we predicted that FGF21 levels would be lower in the HTG patients. If similar findings of elevated FGF21 levels in HTG can be confirmed in other patient series, it would be more logical to ascribe them to some form of FGF21 resistance with compensatory increases in FGF21 secretion. In two recent reports on humans with varying degrees of obesity, positive correlations between FGF21 levels and TG concentrations in serum have been observed (Chen et al., 2007; Zhang et al., 2008). 172 Cell Metabolism 8, , August 6, 2008 ª2008 Elsevier Inc.

5 Treatment of HTG patients with fenofibrate increased serum FGF21 levels. This indicates that also in man, PPARa activation increases the synthesis and secretion of FGF21. The impact of the fibrate-induced elevation in FGF21 on the hypolipidemic response cannot be determined from the present work. However, the degree of stimulation was moderate, and no correlation between the percentage changes in serum TG and FGF21 was found. This lack of relation together with our finding that serum TG and FGF21 levels were not correlated among normal or HTG subjects when analyzed separately indicates that circulating serum FGF21 levels may not be causally related to serum TG. An alternative explanation could be that the individual sensitivity to FGF21 varies, a possibility supported by the wide range of serum FGF21 levels both in normal and HTG subjects. In conclusion, endogenous FGF21 is readily detectable in serum from human subjects. There is a large interindividual variation, and HTG appears to be linked to elevated levels. Prolonged fasting and fenofibrate treatment both increase FGF21, although ketogenesis can clearly be induced independent of serum FGF21 levels, indicating that FGF21 may have different physiological roles in man and mice. Further studies monitoring the responses following the administration of exogenous FGF21 in normal, hyperlipidemic, and diabetic humans will be of great interest. EXPERIMENTAL PROCEDURES Subjects and Study Design From a material of 435 healthy volunteers, 76 subjects matched for gender and age were selected to serve as healthy controls. Information on selection and other measures of samples are shown online in Table S1. For studies on diurnal changes of FGF21, samples from 5 healthy subjects were used as detailed elsewhere (Galman et al., 2005). Subjects fasted for 2 days were 7 normal volunteers from a previous study (Arner et al., 2003); some characteristics of these subjects are given online in Table S2. The subjects fasted for 7 days were 5 female patients with rheumatoid arthritis described in detail online in Table S3. The effect of the KD was evaluated in 17 children with pharmacoresistant epilepsy; details on these patients are shown online in Table S4. The effect of fenofibrate was evaluated on 20 HTG subjects, as detailed online in Table S5. All subjects or their parents gave informed consent to participation in the studies, which had been approved by the Ethics Committee of Karolinska Institutet. When not otherwise indicated, analyzed blood was drawn in the morning after overnight fast, and serum or plasma was separated and stored at 80 C. Figure 4. Serum FGF21 Is Increased in HTG Patients and Following Fenofibrate Treatment (A) Serum TG and FGF21 in 19 HTG subjects compared to 76 healthy subjects. (B) Effects of fenofibrate treatment in HTG patients on serum TG and FGF21. Bars indicate SD. Serum Analyses FGF21 levels were assayed in duplicate by sandwich ELISA developed inhouse, using affinity chromatography purified polyclonal and monoclonal antibodies generated against recombinant human FGF21 protein. The intraassay coefficient of variation (CV) was <6.3%, and interassay CV was 8.4%. Microtiter 96 well plates were coated with a monoclonal anti-fgf21 AB (clone 61-6G9) o/n at 4 C(5mg/mL, 0.1 ml/well). All assay steps were carried out in 0.1 ml/well additions with 1 hr incubations at room temperature in duplicate. After washing and blocking, standards and samples were added. Polyclonal anti-fgf-21 (1:10000) was added and detected with donkey-anti-rabbit- HRP (1:5000) from Jackson ImmunoResearch (West Grove, PA). O-phenylenediamine dihydrochloride enzyme substrate was added, and the plate was incubated at room temperature for 10 min. Color development was stopped with H 2 SO 4. Absorbance was read at 450 nm in a plate reader. The standard curve was constructed using SOFTMax Pro software, Molecular Devices (Toronto, Canada). The standard curve range was ng/ml 10 ng/ml. The sensitivity of the ELISA was ng/ml, calculated as the mean absorbance value of duplicate measurement of zero standards plus three times the SD. Human FGF19, FGF23, FGF1, and FGF2 (R&D Systems) were tested for crossreactivities, and no such reactivitiy was observed. The 7a-hydroxy-4- cholesten-3-one (C4) concentration in serum or plasma was assayed by high pressure liquid chromatography (HPLC) (Galman et al., 2003). Unesterified lathosterol was determined in 50 ml serum by isotope dilution-mass spectrometry (Galman et al., 2005). Plasma total, LDL and HDL cholesterol, TG, and glucose were determined using routine colorimetric techniques. Statistics Significances of differences between groups were tested by ANOVA according to Dunnett (Figures 1B and 1D) or by unpaired and when applicable, by paired student t test after logarithmic transformation. GraphPad Prism v4.03 was used (GraphPad Software, San Diego, CA). SUPPLEMENTAL DATA Supplemental Data include five tables and Supplemental References and can be found at ACKNOWLEDGMENTS We thank Mrs. Ingela Arvidsson for expert technical assistance and professor Gunilla Hedlin from the Astrid Lindgren Children s Hospital, Karolinska Cell Metabolism 8, , August 6, 2008 ª2008 Elsevier Inc. 173

6 University Hospital, Stockholm, Sweden for kind advice. This work was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, the Stockholm City Council (ALF), the Grönberg and the Swedish Heart-Lung Foundations, the Foundation of Old Female Servants, the Swedish Rheumatism Association, and the Karolinska Institutet. Received: February 21, 2008 Revised: May 11, 2008 Accepted: June 27, 2008 Published: August 5, 2008 REFERENCES Arner, P., Sjoberg, S., Nordin, C., and Eriksson, M. (2003). Changes in cerebrospinal fluid signalling substances and appetite scores following 48 h fast in healthy volunteers. Appetite 41, Badman, M.K., Pissios, P., Kennedy, A.R., Koukos, G., Flier, J.S., and Maratos-Flier, E. (2007). Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states. Cell Metab. 5, Chen, W.W., Li, L., Yang, G.Y., Li, K., Qi, X.Y., Zhu, W., Tang, Y., Liu, H., and Boden, G. (2007). 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