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Keywords:

  • bile acids;
  • cholesterol;
  • diurnal rhythm;
  • enterohepatic circulation;
  • FXR;
  • lipid metabolism

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conflict of interest statement
  7. Acknowledgements
  8. References

Bile acids (BAs) traversing the enterohepatic circulation exert several important metabolic effects. Their hepatic synthesis, controlled by the enzyme cholesterol 7α-hydroxylase (CYP7A1), has a unique diurnal variation in man. Here we provide evidence that the transintestinal flux of BAs regulates serum levels of intestinal fibroblast growth factor 19 (FGF19) that in turn modulate BA production in human liver. Basal FGF19 levels varied by 10-fold in normal subjects, and were reduced following treatment with a BA-binding resin and increased upon feeding the BA chenodeoxycholic acid. Serum FGF19 levels exhibited a pronounced diurnal rhythm with peaks occurring 90–120 min after the postprandial rise in serum BAs. The FGF19 peaks in turn preceded the declining phase of BA synthesis. The diurnal rhythm of serum FGF19 was abolished upon fasting. We conclude that, in humans, circulating FGF19 has a diurnal rhythm controlled by the transintestinal BA flux, and that FGF19 modulates hepatic BA synthesis. Through its systemic effects, circulating FGF19 may also mediate other known BA-dependent effects on lipid and carbohydrate metabolism.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conflict of interest statement
  7. Acknowledgements
  8. References

The maintenance of adequate concentrations of the lipophilic bile acids (BAs) within the enterohepatic circulation is of critical importance for several aspects of metabolic homeostasis, and disturbances of BA metabolism are associated with clinically important disease entities such as liver disease, cholesterol gallstones and malabsorption. BAs exert several important regulatory effects on lipid and carbohydrate metabolism by binding to the nuclear farnesoid X receptor (FXR) [1–4]. In the liver, BAs can inhibit their own synthesis from cholesterol by feedback inhibition of the rate-limiting hepatic enzyme cholesterol 7α-hydroxylase [5] (CYP7A1). However, the regulation of BA synthesis differs in fundamental aspects between humans and other species. Of particular relevance is the difference in the diurnal regulation of BA production where BA synthesis in humans shows two distinct peaks during the day [6] whereas in rodents one peak occurs at night [7].

The mechanisms for feedback regulation of CYP7A1 in the liver are multiple but all seem to involve mutual BA response elements [8]. The BA-activated nuclear receptor FXR suppresses CYP7A1 transcription not only through the short heterodimer partner (SHP)-liver receptor homologue 1 (LRH1) cascade [9, 10] but also by mechanisms independent of SHP gene induction [11–13]. As the liver harbours all components of this regulatory machinery, it has generally been assumed that the predominant events of feedback regulation of BA synthesis occur in the liver. Recent experiments in mice indicate that intestinal fibroblast growth factor 15 (FGF15) may function as a secretory signal to the liver where Cyp7a1 expression is repressed [14]. The transcription of FGF15 and its human ortologue FGF19 is stimulated by FXR [11, 14, 15]. FGF19 can suppress CYP7A1 expression by a mechanism independent of SHP-gene induction [11, 16] and is expressed in the small intestine [17] but not in human liver [18]. FGF19 and FGF15 interact with FGF receptor 4 (FGFR4) which is abundant in the liver [14, 17, 19]. Mice lacking FGFR4 have induced BA synthesis and an increased BA pool size [20]. The physiological relevance of a mechanism involving FGF15/19 signalling from the intestine to the liver in the regulation of BA synthesis is however still unclear, and there is no published information as regards the presence of circulating FGF15/19 in mouse or human blood serum.

We hypothesized that endogenous FGF19 may be important for the regulation of BA synthesis in man. In the present study we therefore determined circulating FGF19 and its relation to BA metabolism. Our results indicate that the transintestinal flux of BAs regulates circulating FGF19 which in turn modulates hepatic BA synthesis, both in response to pharmacological perturbations and during the physiological diurnal changes in BA synthesis.

Materials and methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conflict of interest statement
  7. Acknowledgements
  8. References

Subjects and study design

Blood serum was obtained after overnight fast from 15 normal volunteers (eight men, seven women). For the studies on the effects of manipulations of BA enterohepatic circulation serum samples from gallstone patients previously reported on [21] were analysed. They had been treated for 2–3 weeks with either cholestyramine (Questran®; Bristol-Myers, New York, NY, USA), 16 g day−1 (n = 4), or chenodeoxycholic acid (Chenofalk; Dr Falk Pharma GmbH, Freiburg, Germany), 15 mg kg day−1 (n = 6) as described previously [21]. For the studies on diurnal variation, previously obtained samples from five healthy volunteers [6] were analysed. All subjects gave informed consent for participation in the studies, which had been approved by the Ethics Committee of the Karolinska Institute.

Serum analyses

A sandwich enzyme-linked immunosorbent assay (ELISA) kit was used for colorimetric detection of FGF19 in serum (FGF19 Quantikine ELISA kit, Cat. No. DF1900; R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions. All serum samples were assayed in duplicate, the coefficient of variation was 2%. Serum levels of 7α-hydroxy-4-cholesten-3-one (C4) were used to monitor CYP7A1 enzymatic activity (BA synthesis). They were analysed from individual serum samples after sample work up followed by high-pressure liquid chromatography as described previously [7]. C4 values were corrected for total serum cholesterol; the use of uncorrected values did not give significantly different results. Total serum BAs were assayed by gas chromatography/mass spectrornetry [6]. Total cholesterol and triglyceride levels were determined with a Monarch automated analyser using commercially available kits (Instrumentation Laboratory Company, Lexington, MA, USA).

Quantitative real-time PCR

cDNA was synthesized from total RNA pools obtained from three to four individuals (FirstChoice Human Total RNA Survey Panel, Cat. No. 600, Lot No. 016K10; Ambion, Austin, TX, USA) using SuperscriptIII according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR was performed employing the ABI 7500 Fast Real-Time PCR System and relative mRNA expression was calculated according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA) using the deltaCt method. Human RPLPO was used as endogenous control (Applied Biosystems, Part No. 4333761T). FGF19 primers/probe was an Assay on Demand (Applied Biosystems, AssayID No. Hs00192780_m1).

Statistical analysis

Data are presented as mean ± SEM. The significance of differences (Fig. 1) was tested by two-tailed paired Student's t-test. In order to stabilize the variances, data were log-transformed when a correlation between means and variances was found. Comparisons of temporal changes (Figs 2–4) were performed by one-way anova with Dunnett's test, using 09:00 hours’ data of day 1 as control.

image

Figure 1.  Effects of treatment with cholestyramine and chenodeoxycholic acid (CDCA) on serum FGF19 and BA synthesis (C4/chol.). Blood samples (overnight fasting morning samples) were drawn prior to (basal) and after 2 weeks of treatment with cholestyramine (16 g day−1, n = 4) (a, c), or 3–4 weeks of treatment with the BA chenodexycholic acid (CDCA) (15 mg kg day−1, n = 6) (b, d), as described previously [21].

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image

Figure 2.  Diurnal changes of serum FGF19 and BA synthesis (C4/chol.) in five healthy volunteers. Blood samples were collected (from an indwelling venous catheter in the forearm) every 90 min over a 25.5 h period as described previously [6]. The first sample was drawn at 9 am after an overnight fast. The subjects remained at the metabolic ward and received the same standardized meals of natural type: breakfast at 9 am, lunch at noon and dinner at 6 pm respectively; the meals are indicated with arrows. The subjects slept in total darkness from 0:30 to 0:7 am. (a–e) Diurnal variation of FGF19 (black circles) and C4/chol (red triangles) in five normal volunteers. (f) All diurnal FGF19 data from (a–e) presented as percentage change from start value of each individual (mean ± SEM). Significant differences compared with first sampling (Dunnett's test) are indicated (*P < 0.05; **P < 0.01). Data on C4/chol. have been published previously [6].

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image

Figure 3.  Diurnal changes of total BAs, FGF19, and BA synthesis (C4/chol.) in five healthy volunteers. Samples were collected every 90th minute for 25.5 h as described in legend to Fig. 2. Data are expressed as percentage change from the initial morning value 9 am, day 1. Each symbol represents the mean value from five subjects. Significant differences compared with first sampling (Dunnett's test) are indicated (*P < 0.05; **P < 0.01). Data on C4/chol. and BAs have been published previously [6].

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image

Figure 4.  Changes in serum total BAs, FGF19, BA synthesis (C4/chol.) and serum triglycerides (TG) during acute fasting (9 am–4 pm) in four subjects. (a) Temporal data (day 1) from the five subjects having regular meals presented in Fig. 2. Arrows indicate when meals were ingested. (b) Four of the subjects shown in Fig. 2(a–d) were restudied 2–6 weeks after the first session. Subjects were on a fast overnight and the following day no food was ingested during the experiment, only tap water was consumed ad libitum [6]. All data are mean values presented as percentage change from the individual start value (initial morning value at 9 am). Significant differences compared with first sampling (Dunnett's test) are indicated (*P < 0.05; **P < 0.01). Data on C4/chol. TGs, and BAs have been published previously [6].

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Results and discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conflict of interest statement
  7. Acknowledgements
  8. References

When assayed with ELISA, serum FGF19 levels were readily measurable in serum from fasting healthy individuals. The concentration in 15 normal subjects (eight men, seven women) averaged 193 ± 36 (SEM) pg mL−1 (range, 49–590 pg mL−1).

To determine whether fasting serum levels of FGF19 were altered in response to perturbations of BA enterohepatic circulation, we next assayed FGF19 levels in serum samples from two previously published experiments: subjects treated with the resin cholestyramine [21] which prevents BAs from being absorbed, and subjects treated with the BA chenodeoxycholic acid (CDCA) [21] which increases the amount of BAs undergoing enterohepatic circulation. We monitored BA synthesis in these individuals by determining the serum levels of C4, a marker for CYP7A1 enzymatic activity [7]. Treatment with cholestyramine reduced serum FGF19 levels by 87% whilst serum C4 levels increased 18-fold (Fig. 1a). In contrast, CDCA treatment increased FGF19 levels by 250% whereas serum C4 levels were reduced by 26%. Thus, following interference with BA enterohepatic circulation leading to reduced (cholestyramine) and increased (CDCA) intestinal exposure to BAs, circulatory FGF19 levels were altered as predicted from our hypothesis.

It is of interest to note that, in contrast to the pronounced changes in FGF19 levels, the fasting portal venous (or peripheral venous) BA levels are not markedly affected by cholestyramine or CDCA treatment [21]. Given that FGF19 reduces CYP7A1 expression [11, 14], these data indicate that during these experimental perturbations the hepatic synthesis of BAs is under hormonal control from the intestine via FGF19. We screened human tissue RNA using quantitative real-time PCR and found that FGF19 mRNA was readily detected in the small intestine, cervix, testes and thymus (data not shown). The expression in the small intestine and cervix was 5- and 8-fold higher, respectively, than that of testes whereas in the thymus the mRNA expression was 40% of that in the testes. No signals were obtained in the liver, skeletal muscle, adipose tissue or kidney. These results are in agreement with those reported by Nishimura et al. who did not detect any FGF19 mRNA in human liver or kidney [18]. We have not yet been able to obtain intestinal specimens for assay of FGF19 mRNA from patients treated with cholestyramine or CDCA. However, in response to cholestyramine treatment, the intestinal expression of the rat ortologue FGF15 is reduced by >95% whilst serum C4 increases by 3.5-fold and hepatic Cyp7a1 mRNA by 4.5-fold (L. Persson, unpublished data).

As the enterohepatic circulation undergoes dynamic changes during a 24-h day with periods of fasting and feeding, we investigated whether serum levels of FGF19 showed any diurnal variation that could be related to the known temporal changes in BA synthesis [6]. For this purpose, we used serum samples previously collected over 25.5 h and assayed for C4 and BAs [6]. Serum levels of FGF19 in this experiment are shown in Fig. 2(a–e) along with previously published C4 data. The FGF19 levels showed marked temporal changes in striking synchronicity with the changes in C4. When all individual FGF19 data were combined (Fig. 2f), it was evident that FGF19 showed a diurnal rhythm with two major peaks occurring around 3 and 9 pm. Analysis of serum BA levels, reflecting portal venous inflow of BAs from the intestine [22], showed that FGF19 peak in serum with a delay of about 1.5–3 h following the peak of serum BAs (Fig. 3). These results are compatible with the concept that FGF19 is secreted from the intestine in response to the postprandial increase in transintestinal BA flux. The temporal relationship between FGF19 and C4 levels with a delay of roughly 2 h for the suppressive phase of BA synthesis measured as C4 would be in agreement with the concept that circulating FGF19 in turn suppresses BA synthesis.

The onset of BA synthesis in the morning is not influenced by food intake, as we have demonstrated an increase in serum C4 peaking at about 1 pmwhen subjects are fasting [6]. If FGF19 is secreted from the intestine in response to the transintestinal flux of BA, we would expect its normal diurnal rhythm to be abolished in this situation. To test this, we determined FGF19 levels in four of the subjects shown in Fig. 2 on a second occasion when food was omitted (Fig. 4b) [6]. In this experiment, serum BAs and triglycerides gradually declined with time, whereas the synthesis of BAs was induced in the morning regardless of food intake as shown previously [6] (compare Fig. 4a and b). In agreement with our prediction, serum FGF19 levels did not change in the fasting subjects. As discussed previously [6], the subsequent reduction of BA synthesis in the afternoon (after 1:30 pm) when food was regularly ingested (Fig. 4a) was less evident during fasting (Fig. 4b). This is compatible with the concept that circulating FGF19 levels are important for the suppression of BA synthesis in the physiological situation of feeding – fasting during the day.

Whether circulating FGF19 could be actively involved as a mediator of other metabolic regulatory effects exerted by BAs should be considered. In addition to the suppression of Cyp7a1 in mice overexpressing or treated with FGF19, such animals also respond with a reduction in body weight together with reduced plasma levels of triglycerides and glucose and induced energy expenditure [16, 23]. This is of interest as hepatic triglyceride synthesis in humans is reduced following CDCA feeding and increased during resin treatment [24]. Thus, altered FGF19 levels may be a plausible explanation for the effects on plasma lipids seen during interference with BA circulation in humans.

Considering the known effects of FGF19 on glucose and energy metabolism in animals [16, 23], it is also of interest to note that BA treatment of mice has recently been reported to elicit similar responses, viz. reduction in plasma glucose and triglycerides, decreased body weight and increased energy expenditure [25]. These effects have been ascribed to the interaction of BAs with the G protein coupled receptor TGR5 [25]. The exact role of the FGF19-FGFR4 pathway in this situation needs to be studied in humans. Our finding in normal humans of a pronounced diurnal variation in circulating FGF19 levels related to BA uptake in the intestine also raises interesting questions on how interindividual differences in endogenous FGF19 secretion in relation to BA enterohepatic circulation may influence important aspects not only of lipid but also of carbohydrate and intermediary metabolism.

In conclusion, our results indicate that the transintestinal flux of BAs in humans regulates the release of FGF19 from the intestine, and that circulating FGF19 suppresses BA synthesis in the liver. This mechanism seems to operate not only during extreme pharmacological perturbations of BA circulation but also during the physiological diurnal changes of BA synthesis in humans. The relative contribution of intestinal FGF19 in the complex regulation of hepatic BA synthesis, as well as the role of FGF19 as a mediator of BA-induced effects on triglyceride and carbohydrate metabolism, are important questions to be addressed in future studies.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conflict of interest statement
  7. Acknowledgements
  8. References

We thank Ingela Arvidsson for expert technical assistance, and Ingemar Björkhem for critical review of the manuscript. This work was supported by grants from the Swedish Research Council, the Swedish Foundation for Strategic Research, the Grönberg and the Swedish Heart-Lung Foundations, the Foundation of Old Female Servants, Novo Nordisk Fonden and the Karolinska Institute.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conflict of interest statement
  7. Acknowledgements
  8. References
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Mats Rudling MD, PhD, M 63, CME, Karolinska University Hospital Huddinge, S 141 86 Stockholm, Sweden. (fax: +46 8 711 07 10; e-mail: mats.rudling@cnt.ki.se).