Conceived and designed the experiments: AG CP LP SS ROE LNB. Performed the experiments: URS JCV HX AG CP JPG ROE LNB. Analyzed the data: SS JPG SS LNB. Wrote the paper: SS URS AG CP JPG LP SS ROE LNB. Proofread the manuscript: JCV. Reviewed the manuscript: HX.
Current address: F. Hoffmann-La Roche, Ltd., Basel, Switzerland
Current address: Children's Hospital of Chongqing Medical University, Chongqing, China
One co-author has what might be considered a possible competing interest, in that she now works in industry: Ms. Julie Vargas, who worked on the studies reported here as a staff research associate (technician) in Dr. Bull's laboratory, now works at F. Hoffmann-La Roche, ltd. In Basel, Switerland. No other competing interest are present.
Mutations in
We investigated the effect of genetic background on phenotypes of ATP8B1-deficient and wild-type mice, using C57Bl/6 (B6), 129, and (B6-129) F1 strain backgrounds. B6 background resulted in greater abnormalities in ATP8B1-deficient mice than did 129 and/or F1 background. ATP8B1-deficient pups of B6 background gained less weight. In adult ATP8B1-deficient mice at baseline, those of B6 background had lower serum cholesterol levels, higher serum alkaline phosphatase levels, and larger livers. After challenge with cholate-supplemented diet, these mice exhibited higher serum alkaline phosphatase and bilirubin levels, greater weight loss and larger livers. ATP8B1-deficient phenotypes in mice of F1 and 129 backgrounds are usually similar, suggesting that susceptibility to manifestations of ATP8B1 deficiency may be recessive. We also detected differences in hepatobiliary phenotypes between wild-type mice of differing strains.
Our results indicate that the ATP8B1-deficient mouse in a B6 background may be a better model of human ATP8B1 deficiency and highlight the importance of informed background strain selection for mouse models of liver disease.
ATP8B1, also known as FIC1 (familial intrahepatic cholestasis 1), is an ATP-dependent membrane transport protein in the P-type ATPase family
We previously generated mice homozygous for a mutation in
Published studies have characterized phenotypes in male ATP8B1 mutant mice, and with one exception
To investigate the simultaneous effects of mutation (WT and ATP8B1 mutant), background strain (B6, 129, and F1), sex (male and female), and diet (cholate-supplemented and control), we conducted a factorial experiment. We studied ≥5 mice for each of the (2×3×2×2 = 24) factorial combinations (“full factorial experiment”)
Main Effects | Interactions | |||||||||
Phenotypes | Genotype | Strain | Diet | Sex | Genotype x Strain | Genotype x Diet | Genotype x Sex | Strain x Diet | Strain x Sex | Diet x Sex |
Pup survival | - | Y | NA | NA | ||||||
Mid-nursing weight | Y | - | NA | Y | ||||||
Weaning weight | Y | Y | NA | Y | ||||||
% weight loss/day | Y | Y | Y | Y | Y | Y | Y | |||
Baseline serum cholesterol | Y | Y | NA | Y | Y | |||||
Post-diet cholesterol | Y | Y | Y | Y | Y | Y | ||||
Baseline serum ALP | Y | Y | NA | Y | Y | |||||
Post-diet ALP | Y | Y | Y | Y | Y | Y | Y | |||
Baseline bilirubin | - | - | NA | - | ||||||
Post-diet bilirubin | Y | Y | Y | - | Y | |||||
Baseline serum bile salts | Y | Y | NA | - | ||||||
Post-diet serum bile salts | Y | - | + | - | Y | |||||
Post-cholate biliary cholesterol∧ | Y | Y | NA | - | Y | |||||
Post-cholate biliary phospholipids∧ | Y | Y | NA | - | ||||||
Post-cholate biliary bile salts∧ | Y | - | NA | - | ||||||
% liver weight relative to final body weight # | Y | Y | Y | Y | Y | Y | Y |
The first column lists the phenotypes studied. Columns 2–5 list the main effects of 4 factors- genotype, strain, diet, and sex. Columns 6–11 list the interactions between genotype, strain, diet, and sex. ‘Y’ indicates that a main effect or interaction influences the phenotype. ‘−’ indicates no main effect. ‘+’ indicates that the factor had no main effect, but influences the phenotype when interacting with one or more of the other factors. ‘NA’ indicates that the factor was not included or assessed in the experiment. ∧As more data were available for F1 and B6 mice on cholate, than control, diet, only results of analysis of cholate diet are shown here. #For percent liver weight relative to final body weight, a 3-way interaction was detected between strain, diet, and genotype.
Offspring of heterozygote couples were less likely to survive from midway through the nursing period (∼day 10) to weaning (∼day 21) if they were of B6, than of 129 or F1 background (
Previous study had suggested that mutant mice were slightly smaller at weaning than WT and heterozygote littermates
Pup weight in WT and ATP8B1 mutant mice of B6, 129, and F1 backgrounds at: A) mid-nursing period (∼day 10) and B) weaning. Weights of mutant pups were normalized to those of WT and heterozygote littermates; means and SEM are shown. Range of N's: a) B6 (n = 14−56), 129 (n = 23−57), and F1 (n = 70−136) and b) B6 (n = 12−53), 129 (n = 23−58), and F1 (n = 50−138).
Overall, among pups born to heterozygote couples, and genotyped at weaning, the mutant allele had a frequency of 46%, which is slightly lower than the expected 50% frequency (p<0.002). This indicates a mild survival benefit conferred by the wild-type allele. The genotype frequencies were in Hardy-Weinberg equilibrium (p = 0.81), and were not observed to differ with strain background (p = 0.85). Consistent with the allelic analysis, 21% of pups born to heterozygote couples, and genotyped at weaning, were mutants. This is modestly lower than the expectation of 25%, indicating a mild decrease in rate of survival to weaning for mutant pups, compared to WT and heterozygote littermates (p<0.01, chi-squared test, N = 861).
Findings were assessed at baseline, and after short-term feeding of a diet supplemented with 0.5% cholate, or a control diet.
Rate of weight change per day was affected by genotype (ATP8B1 mutant mice lost more weight than WT), strain (B6 mice lost more weight than 129 and F1 mice), diet (mice lost more weight on cholate diet), and sex (females lost more weight, or gained less, than males); there are also several interactions (
Weight change per day in WT (A) and ATP8B1 mutant (B) mice of B6, 129, and F1 backgrounds after feeding of control (grey) or cholate (black) diet for 4–8 days; means and SEM are shown. N's for cholate diet: B6 (n = 8−13), 129 (n = 18−35), and F1 (n = 13−18); and control diet: B6 (n = 5−9), 129 (n = 22−29), and F1 (n = 11−16).
Among WT mice, only B6 females lost weight on cholate, as compared to control, diet (p<0.001;
At baseline, factor analysis showed overall effects of genotype (mutants<WT), strain (B6<129 and F1), and sex (females<males) on serum cholesterol levels (
Serum cholesterol and alkaline phosphatase levels in WT (A, C) and mutant (B, D) mice of B6, 129, and F1 backgrounds at baseline (light grey) and after feeding of cholate (black) or control (medium grey) diet for 4–8 days; means and SEM are shown. N's at baseline: B6 (n = 7−15), 129 (n = 42−66), and F1 (n = 21−36); N's for cholate diet: B6 (n = 5−12), 129 (n = 15−29), and F1 (n = 6−19) and for control diet: B6 (n = 5−12), 129 (n = 18−29), and F1 (n = 11−18).
Genotype, strain, and sex have similar effects on post-diet serum cholesterol levels, as at baseline. Overall, cholate feeding lowers serum cholesterol levels in ATP8B1 mutant mice compared to control-fed mutants, and there are diet-genotype and diet-sex interactions (
In contrast, among WT mice, only B6 males had lower cholesterol after consumption of cholate, than control, diet (p<0.001). In WT mice, no strain differences were apparent fter control diet, but after cholate diet, B6 mice had lower cholesterol than 129 mice (males: p<0.05; females: p<0.01) (
At baseline, genotype (mutant>WT), strain (B6>129 and F1), and sex (females>males) affect sALP levels (
After dietary challenge, there are overall effects of genotype (mutant>WT), strain (B6>129>F1), diet (cholate>control) and sex (females>males). Cholate feeding increases sALP levels in mutant mice (diet-genotype interaction; p<0.05 to <0.001) (
At baseline, no differences in bilirubin levels between groups of mice were detected (
Proportion of WT (A) and ATP8B1 mutant (B) mice of B6, 129, and F1 backgrounds with normal (light grey), moderately elevated (medium grey), and highly elevated (black) serum bilirubin levels in after feeding cholate or control diet for 4–8 days. N's for cholate diet: B6 (n = 5−8), 129 (n = 30−31), and F1 (n = 13−25); and control diet: B6 (n = 10−13), 129 (n = 39−48), and F1 (n = 18−23).
At baseline, factor analysis showed overall effects of mutation (mutants>WT; 129: p<0.001; F1: p<0.01; B6: p<0.05) and strain (B6 and 129>F1) (
Serum bile salt levels in WT (A) and Atp8b1 mutant (B) mice of B6, 129, and F1 backgrounds at baseline (light grey) and after feeding of cholate (black) or control (medium grey) diet for 4–8 days; means and SEM are shown. N's at baseline: B6 (n = 9−15), 129 (n = 48−43), and F1 (n = 43−45). N's for cholate diet: B6 (n = 7−13), 129 (n = 18−35), and F1 (n = 13−18); and control diet: B6 (n = 5−9), 129 (n = 22−29), and F1 (n = 11−16).
Bile was more amenable to collection by needle aspiration from mice after cholate diet, as they tended to have well-filled gallbladders, than from mice after control diet, whose gallbladders often contained very little fluid. Therefore, we analyzed bile composition after cholate feeding only.
For concentrations of cholesterol and phospholipids in bile, there were overall effects of genotype (mutant<WT) and strain (B6>129 and F1), and for cholesterol, a strain-genotype interaction (
Bile cholesterol, phospholipid, and bile salt levels in WT (A, C, & E) and ATP8B1 mutant (B, D, & F) mice of B6, 129, and F1 backgrounds after feeding of cholate diet for 4–8 days; means and SEM are shown. N's: B6 (n = 11−19), 129 (n = 39−52), and F1 (n = 19−22).
There were overall effects of genotype (mutant>WT), strain (B6>129, with F1 intermediate), diet (cholate>control), and sex (females>males) on liver weight as a proportion of final body weight. We also detected genotype-strain, genotype-diet, and strain-diet interactions, and a 3-way interaction between strain, diet, and genotype (
Liver weight as a proportion of final body weight in WT (A) and ATP8B1 mutant (B) mice of B6, 129, and F1 backgrounds after feeding of cholate (black) or control (grey) diet for 4–8 days; means and SEM are shown. N's for cholate diet: B6 (n = 7−13), 129 (n = 18−35), and F1 (n = 13−18); and control diet: B6 (n = 5−9), 129 (n = 22−29), and F1 (n = 11−16).
Triglycerides (TG), total cholesterol (TC), and free cholesterol (FC) were quantified in snap-frozen liver tissue from a representative subset (143 mice) of the study sample; amount of esterified cholesterol (EC) was calculated by subtracting FC from TC.
No overall effects of genotype, diet, strain, or sex were detected for TG and FC; however, for both, diet-genotype and diet-genotype-sex interactions were detected (
Hepatic triglycerides and cholesterol in WT (A, C, E, and G) and mutant (B, D, F, and H) mice of B6, 129, and F1 background after cholate (black) and control (grey) diet for 4–8 days; mean and SEM are shown. N's for cholate diet: B6 (n = 4−10), 129 (n = 8−20), and F1 (n = 4−9); and control diet: B6 (n = 4−11), 129 (8−19), F1 (n = 4−8).
An overall effect of diet (cholate>control) was detected for TC and EC. After consumption of cholate diet, TC was higher in 129 WT mice (p<0.05), and EC was higher in 129 mutant mice (p<0.01), than respective groups after control diet (
We have previously shown that mice lacking Atp8b1 exhibit a mild form of human ATP8B1 deficiency, but do not suffer from progressive cholestatic liver disease
ATP8B1 mutant B6 mice manifest a number of phenotypes that have correlates in human ATP8B1 deficiency, and are not detected, or less readily apparent, in the 129 strain background. In the B6 strain,
A consistent finding in our study was that serum cholesterol was decreased in mutant, as compared to WT, mice in the B6 background; after cholate feeding, this mutation effect was present in all strains. It has been well-established both in mice and humans that cholestasis leads to a decrease in HDL and its main apolipoprotein apoA1
Results for most of the evaluated phenotypes indicate greater abnormalities in mutant mice of B6, as compared to 129, strain background; however, regarding bile composition, we detect differences between WT and mutant mice of 129 background that are not apparent in the B6 strain. We have previously reported that ATP8B1 mutant mice characteristically have increased biliary secretion of cholesterol compared with WT of the same genetic background
Our findings imply the existence of modifier loci regulating the ATP8B1 mutant phenotype; the presence of similar loci in people may underlie, at least in part, the varying severity and nature of disease manifestations that can be seen, even between patients carrying the same, or similar, ATP8B1 mutations
We have also identified strain-dependent differences in WT mice apparent at baseline and/or after dietary challenge. B6 WT mice have lower serum cholesterol (both sexes), and higher sALP (females), as well as greater weight loss (both sexes), and more enlarged livers (both sexes) than do WT 129, and sometimes F1, mice. Some of these findings are magnified upon cholate feeding, suggesting a greater innate sensitivity to cholate feeding in the B6, than 129, strain. Our findings may have general implications for choice of strain when studying hepatobiliary phenotypes, either in WT mice, or in mice with targeted mutations in other genes influencing hepatobiliary phenotypes.
All mice were maintained in a specific-pathogen-free animal facility in San Francisco; studies were conducted under a protocol approved by the UCSF IACUC. The embryonic stem cell line used in generation of the mice was derived from the 129S4 strain, and mice of the genetically most closely related commercially available 129 substrain, 129S1, were subsequently used for breeding
At approximately 21 days after birth, pups were weaned, weighed and tagged. For many litters born to heterozygote couples, pups had also been weighed at approximately day 10, and uniquely identified at that time, so that weight gain during the 2nd half of the nursing period could be evaluated; we did not want to disturb the litters earlier than that, to avoid increasing the risk of mothers abandoning their litters. To account for effects of factors such as litter size and precise age in days on pup weight, weights of mutant pups were normalized to those of WT and heterozygous littermates.
Mice were aged a minimum of 3 months prior to study diet administration (Dyets, Inc. catalog number 101914±0.5% cholic acid, sodium salt [Calbiochem]; a small, initial pilot study was performed with a highly similar diet [K4068.02, Arie Blok Diervoeders, Woerden, The Netherlands]). Mice were anesthetized, and a ‘baseline’ blood collection was performed. Then, standard mouse chow was replaced with control or cholate-supplemented diet. To optimize the number of days mice would be on study diet, we assessed impact of diet administration for varying lengths of time, monitoring mouse body weight and condition. Mutant B6 mice could not consistently remain on the cholate diet for >6 days without demonstrating excessive weight loss. Therefore, >95% of the mice in this study underwent dietary challenge for 6–7 days. To make fullest use of data from animals studied while optimizing diet length, we assessed impact of number of days on diet on phenotypes. Regression analysis did not identify differences attributable to number of days on diet over 4–8 days, so data from all mice on the diet for 4–8 days were pooled for analysis. In total, results from 396 mice are included in this study, tallied by strain as follows: 72 B6, 108 129S1, 100 129S4, and 116 F1 (the latter including mice derived from crossing B6 with 129S1, and B6 with 129S4).
We established a standard protocol in which mice were fasted for ≥4 hours, then anesthetized, and blood, gallbladder bile, liver, and spleen (for isolation of DNA to confirm genotype) were collected at sacrifice. Serum ALP, cholesterol, and bilirubin were assayed in a clinical laboratory. Serum bile salts were assayed using the Total Bile Acid Assay kit (DZ042A-K, Diazyme Labs, USA). Cholesterol, phospholipids, and bile salts in gallbladder bile were assayed as previously described
Since 24 factorial combinations are present, there were 276 possible ways of grouping the mice into two groups based on their mutation status, genetic background, diet and sex. To reduce the number of comparisons examined, and simplify the process of determining which factors affect a trait of interest, we adopted the following procedure: For each trait of interest, we fit a full factorial model with all main effects, two-factor, three-factor and the four-factor interaction
P-values reported in the text are derived from analysis of variance (ANOVA) with Tukey's post-test (performed on the relevant sub-groups), or the chi-square test. These analyses were performed using PRISM 5.0 (Graphpad Software, Inc.) or programmed in Excel (Microsoft Corp.). Except for serum bilirubin and rate of weight loss, data were log-transformed for all analyses. (As serum bile salt levels were undetectable in a small number of mice, and therefore recorded as zero, we added 0.1 prior to log transformation.) For subtle effects, significance is occasionally obtained in the factorial analysis, but not in ANOVA, due to differences in power.
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We would like to thank R. Jaenisch for providing the 129S4 WT mouse line, M.D. Kendrick for technical assistance, A.S. Knisely and S.K. Erickson for helpful discussion, and the Pathology & Imaging Core of the UCSF Liver Center (P30 DK026743) for assistance with histopathology.