The normal mechanisms of pregnancy-induced liver growth are not maintained in mice lacking the bile acid sensor Fxr

Abstract

Rodents undergo gestational hepatomegaly to meet the increased metabolic demands on the maternal liver during pregnancy. This is an important physiological process, but the mechanisms and signals driving pregnancy-induced liver growth are not known. Here, we show that liver growth during pregnancy precedes maternal body weight gain, is proportional to fetal number, and is a result of hepatocyte hypertrophy associated with cell-cycle progression, polyploidy, and altered expression of cell-cycle regulators p53, Cyclin-D1, and p27. Because circulating reproductive hormones and bile acids are raised in normal pregnant women and can cause liver growth in rodents, these compounds are candidates for the signal driving gestational liver enlargement in rodents. Administration of pregnancy levels of reproductive hormones was not sufficient to cause liver growth, but mouse pregnancy was associated with increased serum bile acid levels. It is known that the bile acid sensor Fxr is required for normal recovery from partial hepatectomy, and we demonstrate that Fxr(-/-) mice undergo gestational liver growth by adaptive hepatocyte hyperplasia. This is the first identification of any component that is required to maintain the normal mechanisms of gestational hepatomegaly and also implicates Fxr in a physiologically normal process that involves control of the hepatocyte cell cycle. Understanding pregnancy-induced hepatocyte hypertrophy in mice could suggest mechanisms for safely increasing functional liver capacity in women during increased metabolic demand.

Fig. 1.

Gestational liver growth precedes changes in maternal body weight and is proportional to fetal number. A: liver weight in nonpregnant and pregnant mice. B: liver weight as a percentage of body weight at 12 days (12D) and 18 days (18D) postconception. C: liver weight plotted against number of fetuses in utero. Results are shown as means ± SE (n = 6). Studentapos;s t-test, *P < 0.05 compared with nonpregnant animals.

Fig. 2.

Pregnancy causes liver growth by hepatocyte hypertrophy. A: representative 5-μm liver sections immunostained for β-catenin. B: number of cells per microscope field. Proportion of Ki-67+ (C) and mitotic (phospho-histone-H3 positive) (D) cells is shown. E: hepatocyte nuclear DNA content from 6 nonpregnant and 6 pregnant mice; 1,500 nuclei were considered from each group. F: protein levels of cell-cycle regulators. Results are shown as means ± SE (n = 6). Studentapos;s t-test, *P < 0.05 compared with nonpregnant animals.

Fig. 3.

Pregnancy and cholate feeding cause liver growth and increase serum bile acids. A: liver weight. B: total serum bile acid concentration. Results are shown as means ± SE [n = 12 pregnancy and cholate-fed animals and n = 5 17β-estradiol (E) + progesterone (P4)-treated animals]. One-way ANOVA, *P < 0.05 compared with controls.

Fig. 4.

Cholate feeding causes liver growth by hepatocyte proliferation. A: Liver weight as a proportion of body weight. B: hepatocytes per microscope field. Proportion of Ki-67+ (C) and mitotic (phospho-histone-H3 positive) (D) cells is shown. E: hepatocyte nuclear DNA content of 6 control-fed and 6 30-day cholic acid (CA)-fed mice; 2,000 nuclei were considered from each group. F: protein levels of cell-cycle regulators. Results are shown as means ± SE (n = 6). One-way ANOVA, *P < 0.05 compared with control-fed animals.

Fig. 5.

Pregnancy induced liver growth and serum bile acid concentrations in long-term cholate-fed, Fxr^−/− and Pxr^−/− mice. A: total serum bile acids. B: liver weight. Results are shown as means ± SE (n = 6 or more). Two-way ANOVA, *P < 0.05 compared with wild-type (WT), #P < 0.05 compared with nonpregnant animals.

Fig. 6.

Fxr is required for the normal mechanisms of gestational liver growth. A: number of cells per microscope field. Proportion of Ki-67+ (B) and mitotic (phospho-histone-H3 positive) (C) cells is shown. D: hepatocyte nuclear DNA content. The sum of the DNA content of 100 nuclei per animal was determined as described in materials and methods. E: proportion of apoptotic hepatocytes. F: protein levels of cell-cycle regulators. Results are shown as means ± SE (n = 6). Two-way ANOVA, *P < 0.05 compared with wild-type, #P < 0.05 compared with nonpregnant animals.

Fig. 7.

Schematic representation of the cellular effects of pregnancy in normal and Fxr^−/− mice. During pregnancy, the hepatocytes of the liver enter the cell cycle and progress through S-phase. The majority of cells in normal pregnant mice do not progress past G2-phase, resulting in enlarged hepatocytes with increased nuclear DNA content. By contrast, in pregnant Fxr^−/− mice, a significant proportion of hepatocytes progress to M-phase, resulting in cells of similar size and nuclear DNA content as those in nonpregnant animals.