Introduction
Fibroblast growth factor 19 is a member of a hormone-like subfamily of fibroblast growth factors.1 The secretory pathway of FGF-19 in the enterohepatic-biliary axis has been extensively studied in humans and murine species.2,3,4,5 In the fed state, cholecystokinin is secreted from the duodenum to promote gall-bladder contraction. Gallbladder contraction releases bile into the duodenum, and the bile solubilizes lipid-containing nutrients and inhibits additional cholecystokinin secretion.6 Absorption of bile acids in the ileum initiates a signaling cascade that acts through FXRs (enterocyte nuclear receptors).3 The FXRs bind to a response element in the FGF-19 gene to upregu-late expression of FGF-19 and promote its secretion into the portal venous circulation (Figure 1).3,6,7 Subsequently, FGF-19 interacts with a hepatocyte cell surface receptor complex (FGFR4-βKlotho) to reduce transcription of the cytochrome P450 family 7 sub-family A member 1 gene, thereby downregulating the expression of the rate-limiting enzyme in bile acid formation.8,9,10,11 Secondarily, FGF-19 binds to the gallbladder FGFR4–βKlotho receptors and induces gallbladder dilation to promote filling.2,3,6,12 Fibroblast growth factor-19 is necessary for regulation of bile acids synthesis and gallbladder filling. Mice with knockout of the FGF-19 murine ortholog, FGF-15, have increased bile acids synthesis and almost empty gallbladders.6,13 Alterations in the human FGF-19 regulatory pathway are associated with hepatobiliary and gastrointestinal diseases.14 Reduced circulating FGF-19 concentration in humans has been correlated with bile acids– related diarrhea, lower fasting gallbladder volumes, and predisposition to cholelith formation.15,16,17
Diagram to illustrate FGF-19 production and roles of FGF-19 in the enterohepatic-biliary axis. After gallbladder contraction, bile acids (BA) are taken up into ileocytes and bind to nuclear FXRs. Activation of FXRs upregulates gene expression of FGF-19, which enters the portal venous circulation. At the hepatocytes, FGF-19 activates surface receptors FGFR4 and βKlotho, which then act in the hepatocytes' nuclei to downregulate expression of the cytochrome P450 family 7 subfamily A member 1 (CYP7A1) gene, which produces the rate-limiting enzyme in bile acids formation. Concurrently, bile acids taken up by hepatocytes from portal circulation bind to FXRs in the hepatocyte nucleus. Farnesoid X receptor then activates small heterodimer partner (SHP) to further downregulate the CYP7A1 gene. Fibroblast growth factor-19 also promotes glycogen production in hepatocytes and is responsible for relaxing gallbladder smooth muscle to promote gallbladder filling.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.676
Despite extensive research on human FGF-19, no reports of studies of canine FGF-19 have been published to our knowledge. The objective of the study reported here was to examine the temporal relationship between serum FGF-19 concentration, gallbladder contraction (volume), and SBA concentrations in healthy dogs before and after feeding to determine whether serum FGF-19 concentration increases following gallbladder contraction. In humans, the postprandial gallbladder volume nadir occurs 30 to 50 minutes after eating, and serum FGF-19 concentration peaks approximately 3 hours after release of bile acids into the gastrointestinal tract.2,18,19 In dogs, maximal gallbladder contraction typically occurs within 2 hours after feeding and SBA concentration is routinely measured within this interval.20,21 We hypothesized that serum concentration of FGF-19 would increase within 180 minutes after feeding, following an increase in SBA concentration. To test this hypothesis, we measured gall-bladder volume and SBA and FGF-19 concentrations in healthy dogs after food withholding of 12 hours' duration and at 1 and 3 hours after feeding. A secondary objective of the study was to assess FGF-19 stability in serum obtained from blood samples kept under different storage conditions after collection in tubes containing no anticoagulant or in serum separator tubes. We hypothesized that prolonged refrigeration rather than prompt freezing after collection of such samples would lead to erroneous quantification of FGF-19 concentration.
Materials and Methods
Animals
Ten healthy staff- and student-owned dogs from the Louisiana State University School of Veterinary Medicine were included in the study. Inclusion in the study required that each dog undergo a physical examination and serum biochemical analysis within 30 days of study enrollment for which findings were within reference intervals. The serum biochemical screening panel included assessments of electrolyte, albumin, BUN, total bilirubin, and creatinine concentrations and alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase activities. Apart from heartworm prophylaxis, dogs were not receiving any medications. Preventative medications included moxidectin (n = 3 dogs), moxidectin and imidacloprid (4), and ivermectin and pyrantel (3). Owners provided informed consent for inclusion of their dogs in the study. The study protocol was approved by the Louisiana State University Institutional Animal Care and Use Committee.
Sample collection
For the 10 study dogs, serum FGF-19 and SBA concentrations were quantified following food withholding of 12 hours' duration (baseline) and at 1 and 3 hours after feeding. All baseline blood samples and gallbladder measurements were obtained between 8 am and 12 pm. Blood samples (3 or 6 mL; larger volume collected for FGF-19 stability testing) were collected by venipuncture of either jugular vein or either lateral saphenous vein with a 22-gauge needle and a 3- or 6-mL syringe. For serum FGF-19 analysis, blood samples were collected into tubes containing no anticoagulant.a Coagulated blood samples were centrifuged for 10 minutes at 3,000 × g. Serum for bile acids measurement was stored at 4°C until all 3 samples were collected. From samples collected at each time point, 500 μL of serum was submitted for SBA concentration measurement. No sample was refrigerated for more than 4 hours. The remaining serum was frozen at –80°C for FGF-19 analysis.
Gallbladder volume measurement
Gallbladder length, width, and height were measured ultrasonographically immediately after blood sample collection (following food withholding of 12 hours' duration [baseline] and at 1 and 3 hours after feeding). Each dog was placed in right lateral recumbency, and a small area of hair on the ventral portion of the abdomen was clipped to facilitate gallbladder imaging. Gallbladder ultrasonography was performed by a board-certified veterinary radiologist (LAG) using a 4- to 10-MHz microconvex array transducer and ultrasound scannerb at the highest frequency needed to achieve appropriate depth. Gallbladder length, width, and height measurements were used to estimate gallbladder volume with the prolate ellipsoid formula, length (cm) X width (cm) X height (cm) X 0.53.22,23,24 After the baseline measurement, each dog was fed an amount of bland canned foodc (containing 14.8% fat and 25.8% protein) equivalent to 25% of its resting energy requirements (calculated as 70 × body weight [kg]0.75) to induce gallbladder contraction.23
SBA analysis
Serum samples were stored at 4°C for 2 to 3 hours after collection before analysis. For each sample, SBA concentration was measured with an enzymatic SBA assay kit.d,e
FGF-19 ELISA
A commercially available canine-specific quantitative sandwich ELISA kit was used to quantify serum FGF-19 concentration (inter- and intracoefficient of variation, 6.4% and 7.5%, respectively; spike recovery, 92% to 101%).f The assay was validated with canine recombinant, full-length FGF-19g by the manufacturer. After thawing on ice, each serum sample was diluted 1:2 with PBS buffer (pH, 7.0). The ELISA was performed in accordance with the manufacturer's instructions. Standards, samples, and blank controls were each analyzed in duplicate. Conjugate was added, and the plate was incubated for 1 hour at 37°C. The plate was washed 3 times and blotted dry before substrates were added; the plate was then incubated for 15 minutes at 37°C. Stop solution was added, and the optical density of each well was measured at 450 nm with a spectrophotometerh to determine FGF-19 concentration (in picograms) from the standard curve established on the same plate.
Serum sample stability of FGF-19 following storage
The stability of FGF-19 in serum following storage was evaluated by analysis of serum obtained from 6 blood samples. Aliquots of serum from whole blood samples collected in tubes containing no anticoagulant were stored immediately at –80°C, for 24 hours at 4°C, or for 5 days at 4°C. Additionally, intrasample variability between whole blood samples collected in tubes containing no anticoagulant and in serum separator tubes was evaluated. Samples were analyzed in batches to decrease interassay variability; thus, samples were kept at –80°C for variable intervals prior to analysis.
Statistical analysis
A mixed-effects repeated-measures ANOVA was used to assess the fixed effect of time with animal as a random effect for serum FGF-19 concentration and gallbladder volume. Significant differences were evaluated with post hoc least significant difference comparisons. A similar analysis with a nonparametric approach (Friedman test with post hoc Wilcoxon signed rank test) was used for evaluation of SBA concentration. Two optical density readings at baseline from different types of blood collection tubes and at 24 hours and 5 days were compared via paired t test. Each pairwise comparison of serum FGF-19 concentration, SBA concentrations, and gallbladder volume was also evaluated by calculation of the Pearson correlation coefficient. The Pearson correlation coefficient assessment was also applied to the effect of age and weight on statistical outcome of serum FGF-19 concentration analysis. Assumptions of the ANOVA and t test (normality of residuals and homoscedasticity of residuals) and Pearson correlation (normality of residuals) were assessed by examining standardized residual and quantile plots, and the normality of residuals was confirmed with a Shapiro-Wilk test. All data are reported as mean ± SD except SBA concentration data, which are reported as median and range. All analyses were performed with commercially available statistical software.i A value of P < 0.05 was considered significant.
Results
Ten dogs were enrolled in the study, including 5 spayed females and 5 castrated males. The mean ± SD age was 6 ± 2 years. Types of dogs included mixed-breed dog (n = 3), Golden Retriever (2), Beagle (1), French Bulldog (1), Greyhound (1), Miniature Schnauzer (1), and pit bull–type dog (1). One dog (the Miniature Schnauzer) was censored from the final data analysis because of high postprandial SBA concentration (44 μmol/L). Although abdominal ultrasonography of that dog did not identify any abnormalities, a portosystemic vascular anomaly could not be ruled out.
Evaluation of serum FGF-19 concentration, SBA concentration, and gallbladder volume before and after feeding
Among the 10 dogs, mean ± SD serum FGF-19 concentration at baseline (after food withholding) was 84.5 ± 54.8 pg/mL (Figure 2). At 1 and 3 hours after feeding, the mean serum FGF-19 value was 113.6 ± 68.9 pg/mL and 124.5 ± 77.4 pg/mL, respectively. Serum FGF-19 concentration was significantly increased from baseline at 1 (P = 0.003) and 3 (P = 0.006) hours after feeding. The median SBA concentration at baseline was 1 μmol/L (range, 0 to 4 μmol/L; Figure 3). The median 1-hour postprandial SBA concentration was 1 μmol/L (range, 0 to 9 μmol/L). Serum bile acids concentration at baseline and 1 hour after feeding did not differ significantly (P > 0.05). However, at 3 hours after feeding, SBA concentration was significantly (P = 0.003) increased, compared with baseline SBA concentration. At baseline, the mean ± SD gallbladder volume was 22.4 ± 18.0 cm3 (Figure 4). At 1 and 3 hours after feeding, the mean ± SD gallbladder volume was 19.6 ± 18.0 cm3 and 21.2 ± 23.4 cm3, respectively. Compared with findings at baseline, gallbladder volume was significantly (P = 0.025) decreased at 1 hour after feeding but was not significantly (P > 0.05) different at 3 hours after feeding. A moderately positive correlation (r = 0.054; P = 0.004) was identified between serum FGF-19 and SBA concentrations (Figure 5). Neither weight nor age correlated with serum FGF-19 concentration (Table 1).
Serum FGF-19 concentration in 9 dogs after food had been withheld for 12 hours (baseline; 0 hours) and at 1 and 3 hours after feeding. At each time point, the outlined box and whisker represent the mean and SD, respectively. Each symbol is specific to an individual dog. Initially, 10 dogs were assessed but 1 dog was censored from the final data analysis because of high postprandial SBA concentration. The mean ± SD serum FGF-19 concentration at 0 hours was 84.5 ± 54.8 pg/mL. At 1 and 3 hours after feeding, the mean serum FGF-19 value was 113.6 ± 68.9 pg/mL and 124.5 ± 77.4 pg/mL, respectively. *At 1 hour after feeding, mean serum FGF-19 concentration is significantly (P = 0.003) greater than baseline. †At 3 hours after feeding, serum FGF-19 concentration is significantly (P = 0.006) greater than baseline.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.676
Box-and-whisker plots of SBA concentration in the 9 study dogs before (baseline; 0 hours) and at 1 and 3 hours after feeding. For each box, the horizontal line within the boxes represents the median value and the upper and lower boundaries represent the 75th and 25th percentiles, respectively. Whiskers represent the range. *At 3 hours after feeding, SBA concentration is significantly (P = 0.003) greater than baseline. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.676
Gallbladder volume (determined ultrasonographically) in the 9 study dogs before (baseline; 0 hours) and at 1 and 3 hours after feeding. The outlined box and whisker represent the mean and SD, respectively. *At 1 hour after feeding, mean gallbladder volume is significantly (P = 0.025) less than baseline. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.676
Linear regression analysis plot of SBA concentration versus serum FGF-19 concentration in the 9 study dogs. Linear regression analysis reveals a moderately positive correlation (r = 0.054; P = 0.004) between SBA concentration and serum FGF-19 concentration.
Citation: American Journal of Veterinary Research 82, 8; 10.2460/ajvr.82.8.676
Pearson correlation coefficient (r) analysis of serum FGF-19 concentration (determined after food had been withheld for 12 hours [baseline; 0 hours] and at 1 and 3 hours after feeding) with age (in years) or weight (in kg) of 9 dogs.
| Variable | Time point | Age | Weight |
|---|---|---|---|
| Serum FGF-19 concentration | 0 h | 0.5484 (0.126) | −0.4873 (0.183) |
| 1 h | 0.4341 (0.243) | −0.4183 (0.263) | |
| 3 h | 0.3396 (0.371) | −0.6461 (0.060) |
Data are reported as r (P value).
Sample stability
In serum samples obtained from blood samples collected in tubes without anticoagulant or in serum separator tubes that were exposed to various storage conditions, FGF-19 concentration was assessed by use of a canine-specific quantitative sandwich ELISA kit and spectrophotometry (at 450 nm). In aliquots of serum obtained from blood samples collected in tubes without anticoagulant and that were immediately stored at –80°C (baseline), mean ± SD optical density was 0.93 ± 0.10 absorbance units. Mean optical density of serum sample aliquots stored at 4°C for 5 days was 0.84 ± 0.13 absorbance units, which differed significantly (P = 0.029) from baseline.
In aliquots of serum obtained from blood samples collected in serum separator tubes and stored immediately at –80°C (baseline), mean ± SD optical density was 0.95 ± 0.19 nm. The mean optical density of serum sample aliquots stored at 4°C for 24 hours was 0.95 ± 0.16 nm, which was not significantly (P > 0.05) different from baseline. Mean optical density of serum sample aliquots stored at 4°C for 5 days was 0.88 ± 0.16 nm, which differed significantly (P = 0.022) from baseline. The baseline optical density of serum obtained from blood samples collected in tubes without anticoagulant did not differ (P < 0.05) from the baseline value for serum obtained from blood samples collected in serum separator tubes. The data indicated that FGF-19 stability is affected by extended storage at 4°C. Serum samples must be analyzed or stored at –80°C within 24 hours after blood sample collection to achieve consistent measurement of FGF-19 concentration by use of the ELISA.
Discussion
The objective of the study of the present report was to investigate the temporal relationships between postprandial gallbladder contraction and serum concentrations of FGF-19 and SBA in dogs. For the study dogs, results indicated that gallbladder volume decreased significantly 1 hour after feeding; in most dogs, the gallbladder had begun to refill by 3 hours after feeding. Serum FGF-19 concentration increased to a maximum value at 1 to 3 hours after feeding. A significant increase in the SBA concentration did not occur until 3 hours after feeding, slightly later than the initial mean increase in serum FGF-19 concentration. In comparison, humans typically have an increase in SBA concentration that precedes detectable concentrations of serum FGF-19 after eating.18 This disparity between species may be attributable to a relative insensitivity of the SBS assay for detection of small (nM) changes in SBA concentration or to species differences in bile acids composition. Unlike humans, in which bile acids are predominately conjugated with glycine, most bile acids in dogs are taurine conjugated. Active distal ileal transport is the major route for conjugated bile acids reuptake.25 Unconjugated or glycine-conjugated bile acids can be passively absorbed throughout the small intestine and in the colon.
Interdog variation was evident in the small study population, which may have been related to many factors including breed. There were differences in percentage gallbladder contraction, baseline serum FGF-19 concentration after food withholding, and peak serum postprandial FGF-19 concentration among dogs. In humans receiving a standardized diet, there are considerable diurnal variations in serum FGF-19 concentration, ranging from 50 to 600 pg/ mL.18 Abnormal gallbladder motility, delayed gastric emptying, or differences in long-term diet may have affected the results of the present study. Although sporadic gallbladder contractions occur in humans in a fasted state, these contractions usually only expel about 10% of the total gallbladder volume26; similar contractions were unlikely to have contributed to the variation in serum FGF-19 concentrations among dogs in the present study. The present prospective study involved a small number of dogs with variable signalment. Studies assessing a greater number of dogs at more time points before and after feeding will be necessary to determine factors that modify serum concentration of FGF-19 in dogs and to establish a reference interval for serum FGF-19 concentration in that species.
The data obtained in the present study have provided a basis for understanding the role of FGF-19 in the enterohepatic-biliary axis in dogs. Bile acids act as signaling molecules, initiating regulation of bile acids synthesis by FGF-19, gallbladder filling, and glycogen and protein synthesis.3 Fibroblast growth factor 19 is responsible for hepatic glucose metabolism, liver regeneration, bile acids production and release, and gallbladder filling. In murine species and humans, alterations in FGF-15 (mouse ortholog of FGF-19) or FGF-19 expression predispose animals to clinical disease such as metabolic dysfunction, gastrointestinal tract disease, and neoplasia.6,13,27,28 Any condition that disrupts enterohepatic circulation or bile acids outflow or uptake into enterocytes could be reflected in serum FGF-19 measurements. A clinically available test for circulating FGF-19 could be advantageous for identification of diseases that cause major disruption of the enterohepatic circulation and enterohepaticbiliary axis, such as extrahepatic bile duct obstruction, malabsorptive gastrointestinal disease, and bile acids–associated diarrhea. Pharmacologically, FXR agonists and FGF-19 analogs influence multiple facets of hepatic metabolism. When fed a high-fat diet, transgenic mice that overexpress human FGF-19 have reduced serum glucose and insulin concentrations and improved glucose tolerance and maintain a lean body condition, compared with findings among wild-type mice.29 Unfortunately, the FGF-19–FGF receptor 4 signaling pathway has been connected to the pathogenesis of several cancers.30 Although the tumorigenic potential of FGF-19 has limited pharmacological use of FGF-19 analogs in humans, the FGF 19–FGF receptor 4 signaling pathway is a treatment target for hepatocellular carcinoma.31 Further investigation of FGF-19 signaling in dogs is warranted.
Acknowledgments
This study was funded in part by the Comparative Gastroenterology Society/Idexx Veterinary Student Summer Scholar Award.
The authors declare there were no conflicts of interest.
Abbreviations
| FXR | Farnesoid X receptor |
| FGF-19 | Fibroblast growth factor 19 |
| SBA | Serum bile acids |
Footnotes
BD Vacutainer, Becton, Dickinson and Co, Franklin Lakes, NJ.
Hitachi, Tokyo, Japan.
i/d, Hill's Pet Nutrition Inc, Topeka, Kan.
Diazyme Laboratories Inc, Poway, Calif.
Performed at the Louisiana State University School of Veterinary Medicine's Clinical Pathology Service.
MyBio Source, San Diego, Calif.
UniProtKB F1PPS0, UniProt. Available at: www.uniprot.org/.
BioTek Gen5 Plate Reader, Winooski, Vt.
JMP Pro, version 14.2.0, SAS Institute Inc, Cary, NC.
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