Multihormonal regulation of hepatic sinusoidal Ntcp gene expression
Francis R. Simon,
John Fortune,
Mieko Iwahashi,
Ishtiaq Qadri, and
Eileen Sutherland
Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado Health Sciences Center, Denver, Colorado 80262
Submitted 8 September 2003
; accepted in final form 10 May 2004
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ABSTRACT
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Bile acids are efficiently removed from sinusoidal blood by a number of transporters including the Na+-taurocholate-cotransporting polypeptide (Ntcp). Na+-dependent bile salt uptake, as well as Ntcp, are expressed twofold higher in male compared with female rat livers. Also, estrogen administration to male rats decreases Ntcp expression. The aims of this study were to determine the hormonal mechanism(s) responsible for this sexually dimorphic expression of Ntcp. We examined castrated and hypophysectomized rats of both sexes. Sex steroid hormones, growth hormone, thyroid, and glucocorticoids were administered, and livers were examined for changes in Ntcp messenger RNA (mRNA). Ntcp mRNA and protein content were selectively increased in males. Estradiol selectively decreased Ntcp expression in males, whereas ovariectomy increased Ntcp in females, confirming the importance of estrogens in regulating Ntcp. Hypophysectomy decreased Ntcp mRNA levels in males and prevented estrogen administration from decreasing Ntcp, indicating the importance of pituitary hormones. Although constant infusion of growth hormone to intact males reduced Ntcp, its replacement alone after hypophysectomy did not restore the sex differences. In contrast, thyroid hormone and corticosterone increased Ntcp mRNA in hypophysectomized rats. Sex differences in Ntcp mRNA levels were produced only when the female pattern of growth hormone was administered to animals also receiving thyroid and corticosterone. Thyroid and dexamethasone also increased Ntcp mRNA in isolated rat hepatocytes, whereas growth hormone decreased Ntcp. These findings demonstrate the essential role that pituitary hormones play in the sexually dimorphic control of Ntcp expression in adult rat liver and in the mediation of estrogen effects.
growth hormone; estrogen; thyroid; glucocorticoids
BILE ACIDS ARE SYNTHESIZED exclusively in the liver from cholesterol, and after cellular translocation, they are secreted into bile primarily by the ATP-dependent bile salt export pump. Bile acids are the major osmotic driving force for bile flow and also participate in the digestion and absorption of various lipophilic dietary constituents (10). After reabsorption in the distal small intestine, bile acids are very efficiently cleared from the portal circulation by hepatocytes. Efficient clearance of bile acids from the sinusoidal circulation depends in large part on both secondary active transport processes at the sinusoidal domain and primary active transport at the canalicular surface (31, 34, 48).
Hepatic uptake of taurocholate has been well characterized (33). More than 80% of conjugated bile salts undergo single-pass extraction by the liver (31). Although it is generally believed that the Na+-taurocholate-cotransporting polypeptide (Ntcp; Slc10a1) is important in bile acid uptake, recent evidence suggests that organic anion transporting polypeptide family (Oatp1) and microsomal epoxide hydrolase are also important for taurocholate uptake (23, 64).
Excretion of bile acids at the bile canaliculus is generally believed to be rate-limiting for overall hepatic transport (43). However, studies (12, 15) using steady-state kinetics and physiological bile acid concentrations have shown that uptake of taurocholate, and not bile acid secretion, was saturable at physiological levels. Few studies have examined the physiological factors important in regulation of Ntcp. Except for the unique situation in the postpartum animal in which prolactin is elevated, most studies have shown that Ntcp is downregulated (2, 19, 50). Because uptake of bile acids is the first step in hepatic clearance, agents that decrease bile acid uptake, such as steroid conjugates, bumetanide, furosemide, verapamil, rifamycin, and A-23187, also decrease secretion (6, 52, 65). In addition, endotoxin, TNF-
, IL-1
, and IL-6, as well as estrogens, decrease Ntcp (14, 57). Indeed, downregulation of sinusoidal bile acid transporters has been proposed to serve as an adaptive mechanism to prevent accumulation of potentially toxic bile acids that may lead to cellular damage and liver dysfunction (58).
We and others (7, 49) have shown that Na+-dependent uptake of taurocholate is twofold greater in males compared with female rats. Furthermore, pharmacological doses of ethinyl estradiol (a cholestatic estrogen) decreased Na+-dependent taurocholate transport, expression of hepatic messenger (m)RNA, and protein content (6, 20, 50). Previous studies (36) have shown that sexually dimorphic expression of hepatic proteins is due to the differential pattern of growth hormone secretion rather than a direct effect of sex steroid hormones. The sexually dimorphic pattern of growth hormone secretion in rats and humans has been shown to have physiological importance in the differential expression of sex-specific hepatic cytochrome P-450 drug-metabolizing enzymes (60, 62, 63). However, the mechanism(s) by which estrogens alter Ntcp gene expression is currently unknown.
Therefore, we proposed that the sexually dimorphic expression of Ntcp and the effect of estrogens on its expression in males were due to the pattern of growth hormone secretion. The present studies were undertaken to examine in vivo and in vitro the hormonal mechanisms involved in the sex-specific expression of Ntcp mRNA and particularly the role of the growth hormone secretory pattern in regulation of its dimorphic expression. Collectively, these studies demonstrate that sex differences in expression of Ntcp are determined largely by the female secretory pattern of growth hormone, which paradoxically decreases elevated Ntcp after glucocorticoids and thyroid hormone have induced its levels. Furthermore, growth hormone secretion also mediated the estrogen-induced decreased Ntcp mRNA levels. Thus there is a complex interplay of hormones, which positively and negatively regulate the dimorphic expression of Ntcp in rats.
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MATERIALS AND METHODS
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Experimental animals and hormone therapy.
Adult male and female Sprague-Dawley rats, weighing
200 g, were purchased from Harlan Sprague Dawley (Indianapolis, IN) and obtained after hypophysectomy, thyroidectomy, adrenalectomy, ovariectomy, or castration along with size-matched controls. Control rats underwent sham surgery and were also allowed to recover for a period of at least 6 days within our animal facilities before any treatments. Control and experimental rats were maintained under standardized conditions of temperature (2224°C), humidity, light, and darkness. Animals were provided water and rat chow ad libitum until 18 h before death, when food was removed. Hypophysectomized rats that gained weight were eliminated from further study (39). Control animals were pair fed as previously described (45, 49) when receiving estrogens, because they eat poorly. Animals were killed by 9:00 AM. As indicated under specific figures, hypophysectomized, thyroidectomized, and adrenalectomized rats were given replacement doses of corticosterone 21-acetate (50 µg·kg body wt1·day1) and/or L-thyroxine (50 µg·kg body wt1·day1) for 7 days as daily injections. Recombinant bovine growth hormone was a generous gift from Protiva (Monsanto). The daily dose of bovine growth hormone was 60 µg·100 g body wt1·day1. Growth hormone was given either continuously (female-like pattern) by means of an Alzet model 2001 osmotic minipump (Alza, Palo Alto, CA) at 60 µg·100 g body wt1·day1 x 7 days or injected subcutaneously (male-like pattern) twice daily at 30 µg·100 g body wt1·day1 for 7 days. The osmotic minipumps were implanted subcutaneously on the backs of the rats under light anesthesia. The solvent for growth hormone was 0.05 M Na2PO4, pH 8.8, 1.6% glycerol, and 0.02% NaN2. 17
-Estradiol (100 µg·kg body wt1·day1), and testosterone enanthate (2 mg·kg body wt1·day1) were dissolved in corn oil and given once daily subqutaneously for 7 days, respectively, as previously described (11, 41). 17
-Ethinyl estradiol (5 mg·kg body wt1·day1) was dissolved in corn oil and administered daily subcutaneously for 5 days as previously described (50). All agents except growth hormone were purchased from Sigma and were the highest purity available. Animals received humane care in compliance with National Research Council's criteria as outlined in Guide for the Care and Use of Laboratory Animals. These studies were approved by the Institutional Animal Care and Use Committee at this institution.
RNA isolation and analysis.
Total RNA was extracted from whole liver using RNeasy Mini Kit (Qiagen). RNA was fractionated in 1.2% agarose-formaldehyde gels in borate buffer at 140 V for 4 h, transferred to hybond N+ (Amersham) with high-efficiency transfer solution (Tel-Test) by capillary action, and fixed by UV crosslinking. cDNA probes were labeled with [32P]dCTP (Amersham) using Decaprime II random primed labeling system (Ambion). Unincorporated label was removed with Probequant G-50 microcolumns (Pharmacia). Membranes were hybridized by using a high-efficiency hybridization system for 16 h at 62°C. They were washed twice in 2x SSC/0.1% SDS, followed by washing twice in 0.1x SSC/0.1% SDS all at 55°C for 20 min with each wash. Membranes were exposed to Hyperfilm MP with intensifying screen at 70°C for 3 h. Autoradiograms were quantitated with an Imaging Densitometer (Bio-Rad). The following probes were used: Ntcp (provided by B. Hagenbuch, University Hospital, Zurich, Switzerland), multidrug resistance-associated protein 3 (Mrp3; kindly provided by D. Ortiz, Tufts Medical School, Boston, MA), and IGF-1 (kindly provided by P. S. Rotwein, Washington University, St. Louis, MO). The bile salt export protein probe was a 413-bp cDNA amplified by PCR using the following primers: 1) 5'-gaggttacttaatagcctacg-3' and 2) 5'-catctatcatcacagttfcc-3'. The PCR product was purified, sequenced to verify the authenticity, and labeled with 32P. The relative density of mRNAs were normalized to 18S ribosomal (r) RNA (Ambion) on each gel and expressed as a percentage of male controls.
Immunobloting.
Total liver fractions were prepared for immunobloting by Na2CO3 extractions of liver homogenates (3), and liver plasma membrane subfractions were isolated by differential centrifugation before SDS-PAGE and immunobloting as previously reported (49). After electrophoresis, proteins were transferred to Hybond enhanced chemiluminescence (ECL) membranes (Amersham) by the procedure of Towbin et al. (56). Gels were blocked for 1 h using 5% milk in 5% Tween/TBS and processed for ECL detection (Amersham) using 1% milk in TBS for antibody diluent steps. Blots were visualized by the Streptavidin horseradish peroxidase detection system (Amersham) and were exposed to Amersham Hyperfilm for ECL for 30 to 60 s. Autoradiograms were quantitated by imaging densitometry. The following antibodies were used: polyclonal antibodies to Ntcp (49), bile salt export protein (provided by D. Ortiz) (30), and Mrp3 (amino-terminal end; Santa Cruz Biotechnology, Santa Cruz, CA). Protein loading was corrected by using either actin or aminoblack staining of gels.
Isolated rat hepatocytes.
Rat hepatocytes were isolated by in situ collagenase perfusion method (4) of Berry and Friend (5). Isolated rat hepatocytes (>95% viable by Trypan blue exclusion) were plated at a density of 3.2 x 106 on Matrigel-coated plates (100 µg/100 ml) in defined Waymouth's medium containing insulin (107 M). After 3 h, the medium was changed to fresh Waymouth's medium plus insulin (109 M) containing either T3 (108 M), dexamethasone (107 M), or growth hormone (100 ng/ml). Cells were harvested 24 h after the addition of hormones, and RNA was extracted.
Data analysis.
Results are expressed as means ± SE. For analysis of two independent groups, a Student's t-test was used to determine significance. For analysis of three or more independent groups, ANOVA with a priori comparisons was performed by using one-way ANOVA. The post hoc test was accomplished by using pairwise comparisons with Mann-Whitney's U-test and a Bonferroni adjustment, using the Graphpad statistical package program. Statistical significance was set at P < 0.05.
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RESULTS
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Protein and mRNA levels for sinusoidal and canalicular bile acid transporters.
Previous studies (49) have shown that taurocholate uptake and Ntcp are both sexually dimorphically expressed in rat liver. However, it is not clear whether taurocholate excretory transporters are also differentially expressed. Taurocholate may exit hepatocytes by at least two different transporters. The major mechanism is biliary secretion by bile salt export protein (abcc11) (21). After cholestasis, however, taurocholate may be secreted at the sinusoidal surface by the Mrp3 (abcc3) (17, 24). Because recent studies (9) have demonstrated that prolactin increases canalicular bile salt export protein as well as Ntcp gene expression, it was proposed that bile acid transporters at the sinusoidal surface, as well as the canalicular domain, are coordinately regulated. Therefore, we examined the possibility that the canalicular bile salt transporter and/or Mrp3 may also demonstrate sexually dimorphic expression. RNA was extracted from rat livers and steady-state mRNA, and protein levels of Ntcp, bile salt export protein, and Mrp3 were determined as shown in Fig. 1.

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Fig. 1. Gender differences in expression of Na+-taurocholate-cotransporting polypeptide (Ntcp), bile salt export protein (Bsep), and multidrug resistance-associated protein (Mrp3). Normal male (filled bars) and female (open bars) rats, weighing 200 g were killed at 8 wk of life. Liver samples were prepared for RNA and protein determinations. A: Northern blot of liver messenger RNA (mRNA) from male and female rats. RNA was extracted and probed with [32P]cDNAs to Ntcp, Bsep, and Mrp3 as described in MATERIALS AND METHODS. Twenty micrograms were applied in each lane. B: graph showing the selective difference in Ntcp mRNA levels (P < 0.01), whereas Bsep and Mrp3 were not significantly different. mRNA levels are reported compared with male values set at 100%. 18S was used to correct for possible loading differences. C: Western blots showing protein mass differences for Ntcp, Bsep, and Mrp3 analyzed in male and female liver samples. Protein mass was determined in liver plasma membrane subfractions for Ntcp, Bsep, and Mrp3. Five micrograms of sinusoidal or bile canalicular membrane protein were used in each lane for Ntcp and Bsep, whereas 40 µg of sinusoidal protein was applied in each lane for Mrp3 determinations. Loading differences were corrected by reprobing the samples with -actin or aminoblack protein staining. Male values are reported at 100%. Significant differences were measured only for Ntcp (P < 0.01). Both mRNA and protein blots were quantitated by densitometry. Four individual values were measured in each group, and significant differences were determined by Student's t-test. Results are expressed as the means ± SE. NS, not significant.
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As shown in Fig. 1, A and B, Ntcp mRNA was 40% greater in male than in female rat liver (P < 0.01) (49). In contrast, both bile salt export protein and Mrp3 steady-state mRNA levels were not significantly different. In contrast to previous reports (21), we identified two mRNA bands for bile salt export protein by Northern blot analysis. Both bands were quantified by densitometry and are assumed to represent bile salt export protein. Results suggested that the sex differences in Ntcp levels were specific and not coordinately expressed at the transcriptional level.
Ntcp, bile salt export protein, and Mrp3 were also quantitated by Western blot analysis using sinusoidal and bile canalicular subfractions to examine whether steady-state mRNA levels resulted in similar protein expression. Figure 1C demonstrates that Ntcp protein content in sinusoidal membrane fractions was approximately twofold greater in males compared with females, whereas bile salt export protein in bile canalicular fractions and Mrp3 in sinusoidal fractions were not differentially expressed (Fig. 1C). Thus Ntcp is selectively increased in male rat liver, whereas other bile acid transporters at the sinusoidal and canalicular domains are not coordinately regulated with Ntcp.
Sex steroid regulation of hepatic Ntcp mRNA levels.
Dimorphic expression of hepatic proteins may be due either to the direct effect of sex steroid hormones on the liver or as in most cases, to the differential patterns of growth hormone secretion that is under hormonal control (46). Because previous studies (50) have demonstrated that pharmacological doses of estrogens (such as 17
-ethinyl estradiol) downregulated Ntcp mRNA and protein levels in male rats, we wanted to determine whether physiological administration of sex steroid hormones might be responsible for the sex differences in hepatic Ntcp mRNA levels. In addition to the effect of estrogen and testosterone administration, we examined the changes in Ntcp after ovariectomy and castration on expression of Ntcp. 17
-Estradiol, a physiological estrogen, and 17
-ethinyl estradiol, a synthetic estrogen, were used as a model of high physiological levels and reversible intrahepatic cholestasis, respectively. Both estrogens were injected daily to either intact male or female rats, and hepatic mRNA levels were measured. In additional studies, male and female rats were gonadectomized and administered either testosterone or 17
-estradiol, respectively. The results are shown in Fig. 2, A and B. In intact males, 17
-estradiol significantly (P < 0.05) reduced Ntcp mRNA levels to 70% of male values, whereas 17
-ethinyl estradiol administration dramatically reduced Ntcp mRNA to 47% of pair-fed controls (P < 0.01). Pair-fed control animals did not show significant differences from controls and thus were combined in the sham controls. These results indicated a dose response to increasing levels of estrogen administration in males. In contrast as shown in Fig. 2B, administration of 17
-estradiol to intact females had no significant effect, but 17
-ethinyl estradiol modestly (P < 0.05) reduced Ntcp mRNA. Castration of male rats and testosterone administration had no significant effect on Ntcp mRNA levels (Fig. 2A). On the other hand, ovariectomy dramatically and significantly increased (P < 0.001) levels of Ntcp mRNA 2.5-fold compared with intact female controls, which were restored to control values by 17
-estradiol (Fig. 2B).
Growth hormone secretion pattern regulates hepatic gene expression.
Estrogen administration converts the male intermittent growth hormone secretory pattern to a continuous secretion (40). Also, we and others (51, 63) have reported that estrogen administration, as well as gonadectomy, in male and female rats changes the levels of CYP2C11 and CYP2C12 that are markers of male and female dominant genes, respectively. Thus sex steroid hormones might alter Ntcp mRNA levels indirectly through changing the intermittent male growth hormone secretion pattern to a continuous female-like pattern. Therefore, we hypothesized that growth hormone infusion to male rats by osmotic minipump (which mimics the female secretory pattern) should downregulate Ntcp mRNA levels. The results are shown in Fig. 3 and indicate that the steady infusion of growth hormone to intact male rats significantly (P < 0.001) decreased Ntcp mRNA levels to 74 ± 7% of sham male levels. In contrast, growth hormone infusion of female rats did not significantly reduce Ntcp mRNA levels.

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Fig. 3. Effect of growth hormone (GH) infusion [growth hormone pump (GHP)] in intact rats on Ntcp mRNA levels. Male (filled bars) and female (open bars) rats were administered GH at 60 µg·100 g body wt1·day1 by osmotic minipumps for 7 days. Male and female control rats were sham treated. A: Northern blot analysis was performed as described in MATERIALS AND METHODS. Total liver RNA (20 µg/lane) was probed with [32P]Ntcp cDNA, and densities were quantitated as described in MATERIALS AND METHODS. Loading was corrected by 18S. B: bar graph of corrected Ntcp mRNA values. Male values are depicted in the filled columns and female values in the open columns. Male values are set at 100%. Results are expressed as means ± SE, and statistical significance was determined by 2-way ANOVA. Numbers in parenthesis indicate the number of experiments.
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Do estrogens regulate Ntcp expression directly or indirectly?
It has been proposed that decreased Ntcp mRNA and protein after 17
-ethinyl estradiol administration to male rats was secondary to cholestasis (58), whereas others have suggested that the changes may be due to decreased expression of hepatic nuclear transcription factors (20). A third possibility is that estrogen administration to intact males converts the male growth hormone secretion pattern to a female one (40), resulting in downregulation of Ntcp expression. If decreased Ntcp mRNA levels are mediated by conversion of the male to a female growth hormone secretory pattern, hypophysectomy should prevent the response to estrogen administration. Therefore, male and female rats were hypophysectomized, and after stabilization, 17
-ethinyl estradiol was administered for 5 days. Figure 4 demonstrates the effect of 17
-ethinyl estradiol on Ntcp mRNA in hypophysectomized rats. In male rats (Fig. 4A), hypophysectomy selectively and significantly (P < 0.001) reduced Ntcp mRNA levels to 57% of intact male levels. Importantly, 17
-ethinyl estradiol administration to hypophysectomized male rats did not further reduce Ntcp mRNA. On the other hand, as shown in Fig. 4B, Ntcp mRNA in hypophysectomized female rats was not significantly different from basal female values, indicating the importance of pituitary hormone secretion in maintaining the sex differences in Ntcp expression. Furthermore, similar to hypophysectomized males, 17
-ethinyl estradiol did not reduce Ntcp mRNA in hypophysectomized female rats. These studies support the hypothesis that estrogen administration decreases Ntcp mRNA through mechanisms dependent on the secretion of pituitary hormones. Prompted by these results, we further examined the possible role of the sex-differentiated secretion pattern of growth hormone in the regulation of Ntcp expression.

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Fig. 4. Effect of EE administration on Ntcp mRNA levels in hypophysectomized (Hx) male and female rats. Male (filled bars) and female (open bars) rats were Hx and either untreated or administrated EE (5 mg·kg body wt1·day1) for 5 days after being allowed 1 wk to recover from surgery. Other rats were sham operated as controls. A: Northern blots measuring Ntcp mRNA. Male values were set at 100%, and values were expressed compared with males. No differences in 18S RNA levels were detected among the different groups. B: bar graph showing results in means ± SE for 46 individual animals in each group. No significant differences were measured between the EE-treated male and female Hx rats and Hx-alone animals, as determined by 1-way ANOVA. Numbers in parentheses indicate the number of animals.
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Growth hormone regulation of Ntcp.
To directly test the hypothesis that the growth hormone secretory pattern is the major determinant of sex differences in Ntcp mRNA levels, we examined the effect of different patterns of growth hormone secretion on levels of Ntcp mRNA in hypophysectomized rats. The results are shown in Fig. 5. As previously shown in Fig. 4, hypophysectomy selectively decreased hepatic Ntcp mRNA levels in male but not in female rats (P < 0.001). This selective effect is most consistent with the hypothesis that loss of growth hormone secretory pattern was responsible for the dimorphic expression of Ntcp. We initially tested the possibility that growth hormone replacement alone, given either by twice-daily injection (male pattern) or infused by osmotic minipumps (female pattern), might restore Ntcp values in males and increase them in female hypophysectomized rats. Surprisingly, neither injection nor minipump infusion of growth hormone for 7 days restored male levels of Ntcp in male or female rats. Because increased levels of Ntcp mRNA were not achieved, these results suggested that additional factors, such as thyroid or glucocorticoid hormones, might be involved in the development of gender differences in Ntcp expression.

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Fig. 5. The effect of Hx and GH replacement patterns on Ntcp mRNA levels in male and female rats. Rats were hypophysectomized at 200 g and allowed to recover from surgery for 1 wk, followed by GH administration for 1 wk. One set of animals of each sex were administered GH (60 µg/day sc) by injection every 12 h (GHi), whereas the other group had GH (120 µg/day) continuously infused by osmo-minipump (GHp). Liver RNA was extracted, separated, and probed with [32P]Ntcp cDNA. Northern blots were quantitated by scanning densitometry, and male values were normalized to 100%. Numbers in parentheses indicate the numbers of animals in each group. There was no significant change in values when comparing either Hx males or Hx females animals as determined by 1-way ANOVA. Males are shown in A and female values in B. IM, intact male; IF, intact female. ***P < 0.001.
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Effect of thyroid and corticosterone on Ntcp mRNA levels.
The effect of hormonal replacement in adrenalectomized and thyroidectomized male rats was examined to determine whether these hormones individually might regulate Ntcp mRNA levels (Fig. 6). After thyroidectomy or adrenalectomy, rats were allowed to stabilize for 1 wk. These rats were then treated with physiological doses of thyroxine or corticosterone acetate, respectively, for 1 wk. Thyroidectomy significantly (P < 0.01) reduced Ntcp mRNA content to 65% of control (Fig. 6A), whereas replacement doses of T4 (20 µg·kg body wt1·d1) restored Ntcp mRNA to control levels, but not higher. This result suggested that thyroid hormone might play an important physiological role in regulating Ntcp mRNA. However, thyroid hormone also influences growth hormone secretion, and thus the changes in Ntcp mRNA might be secondary to changes in growth hormone (13). We therefore examined the ability of thyroid hormone to regulate Ntcp mRNA content in hypophysectomized rats (Fig. 6B). Similar to thyroidectomized rats, T4 administration significantly increased (P < 0.001) Ntcp mRNA, indicating that thyroxine directly regulates hepatic Ntcp expression.

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Fig. 6. Effect of thyroidectomy, adrenalectomy, and specific hormone replacements on expression of Ntcp. A: male rats were either thyroidectomized alone or treated with thyroxine (T4) and compared with sham controls. B: Hx rats alone or treated with T4. C: IM controls were compared with adrenalectomized (Adx) alone or treated with corticosterone acetate (C). D: Hx male rats compared with rats treated with Hx plus corticosterone acetate. All surgically treated rats were allowed to recover for 1 wk, before administration of hormone replacements. T4 (20 µg·kg body wt1·day1) and corticosterone acetate (10 µg·kg body wt1·day1) were given subcutaneously for 7 days. Numbers in parenthesis indicate the number of individual experimental animals in each group. Results are expressed as means ± SE relative to sham controls as analyzed by one-way ANOVA or by Student's t-test for comparisons of hormone replacements compared with Hx. *P < 0.05 and **P < 0.01 compared with sham controls or Hx.
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As shown in Fig. 6C, adrenalectomy also significantly reduced hepatic mRNA levels to 73% of control values (P < 0.05). Surprisingly, administration of corticosterone acetate (10 mg·kg body wt1·d1) significantly increased (P < 0.01) Ntcp mRNA to values above control. Glucocorticoids also modify growth hormone secretion patterns (54); therefore, we also examined the effect of corticosterone acetate administration on Ntcp mRNA levels after hypophysectomy (Fig. 6D). A similar and significant (P < 0.01) increase in Ntcp mRNA was measured, indicating that adrenal steroids also directly modify hepatic Ntcp expression. Thus thyroid hormone and glucocorticoids both directly induce Ntcp mRNA levels independent of growth hormone secretion patterns.
Effect of hormonal replacement on expression of the sexual dimorphic pattern of Ntcp expression.
Because growth hormone frequently regulates hepatic genes in concert with other pituitary-mediated hormones (55), and its administration alone to hypophysectomized rats did not reproduce the sexual dimorphic pattern of Ntcp expression, we then explored the possibility that glucocorticoids and thyroid hormone were required to achieve the dimorphic expression of Ntcp. The effect of different growth hormone secretory patterns was examined on Ntcp mRNA levels in combination with glucocorticoids and thyroid hormone replacement in hypophysectomized male and female rats. The results are shown in Fig. 7. Male and female rats were hypophysectomized and after stabilization were given the combination of corticosterone acetate and thyroid hormone replacements for 7 days. This hormone combination alone significantly (P < 0.001) increased Ntcp mRNA levels in both male and female hypophysectomized rats. Because the combination of thyroid and corticosterone induced Ntcp mRNA in both sexes to similarly high levels, the results suggested that another hormone, possibly growth hormone, might be involved in the lower values of Ntcp mRNA measured in female rats.

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Fig. 7. The effect of multihormonal replacements on Ntcp mRNA levels in rats after Hx. Rats were hypophysectomized at 200 g body wt. After 1 wk, the second set of animals was given a combination of thyroid hormone (T) (50 µg·kg body wt1·day1) and corticosterone acetate (C) (50 mg·kg body wt1·day1) for 7 days (T/C). The third and fourth sets of animals were given in addition to thyroid and corticosterone acetate either growth hormone (60 µg/day) by injection every 12 h (GHi) or growth hormone (120 µg/day) by continuous pump infusion (GHp) along with T/C for 7 days. Liver RNA was extracted, separated, and probed with [32P]Ntcp cDNA. Northern blots were quantitated, and all values were compared with intact males and set at 100%. A: Northern blot and quantitation of liver Ntcp in male rats (closed bars). B: Northern blots and quantitation of Ntcp mRNA levels in females (open bars). Four animals were used in each group as indicated in the parentheses. 18S probing was used to correct for possible differential loading. Significant values are determined compared with sex-specific Hx male and females. **P < 0.01 ***P < 0.001 analyzed by ANOVA.
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We then tested the possible role of different patterns of growth hormone secretion on Ntcp expression. Hypophysectomized rats were treated for 1 wk with thyroxine and corticosterone acetate plus similar daily doses of growth hormone administered either twice daily by injections (male-like pattern) or by steady infusion with minipumps (female-like pattern). As shown in Fig. 7, twice daily injection of growth hormone did not reduce Ntcp mRNA levels in either male or female livers to those measured in hypophysectomized animals. In contrast, infusion of growth hormone in combination with thyroid and corticosterone acetate reduced Ntcp mRNA to values comparable with hypophysectomy (or intact female values) in both males and females. Thus the differential effect of growth hormone infusion vs. injection recapitulated the sexually dimorphic expression of Ntcp.
In vitro effects of hormones on expression of Ntcp mRNA.
The previous in vivo studies demonstrated induction of Ntcp by both thyroid hormone and glucocorticoids, whereas the steady infusion of growth hormone decreased Ntcp mRNA. To determine whether these responses in intact animals are due to direct action on the liver, we examined their effect on the endogenous induction of Ntcp mRNA in isolated rat hepatocytes. Hepatocytes were isolated from male rats, plated on Matrigel-coated dishes in medium with low concentrations of insulin (109 M). In preliminary studies, we found that hepatocytes plated on Matrigel with hormone-free medium plus insulin gave stable levels of Ntcp mRNA expression (data not shown). Ntcp mRNA levels were found to stabilize after 18 h in culture (at values
18% of control). Therefore, hormones were added to hepatocyte cultures after overnight stabilization (18 h). Thyroxine (108 M), dexamethasone (107 M), and growth hormone (100 ng/ml) were added 24 h before lyses of cells and preparation of RNA. Figure 8A shows the effect of these hormones on Ntcp mRNA levels, whereas Fig. 8B demonstrates the effect of growth hormone on expression of IGF-1 mRNA levels. Thyroxine and dexamethasone significantly (P < 0.01) increased Ntcp mRNA levels 2.3- and 2.9-fold, respectively. Thus as previously demonstrated in in vivo studies, these hormones directly induce hepatic Ntcp. On the other hand, as shown in Fig. 8A, addition of growth hormone (100 ng/ml) significantly decreased Ntcp mRNA levels to 33% of control (P < 0.05). This effect was not due to a toxic effect of growth hormone, because in the same experiment, IGF-1 mRNA was increased 2.3-fold (Fig. 8B) as shown by others (55). Furthermore, the addition of growth hormone in addition to thyroxine and dexamethasone restored Ntcp values to basal level. Because growth hormone was maintained at a relatively constant level during 24 h of the experiment, these results are consistent with the in vivo infusion of growth hormone and indicate that growth hormone downregulates Ntcp at the hepatocyte level. Thus these in vitro studies recapitulate the in vivo effects of growth hormone on Ntcp mRNA.

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Fig. 8. A: in vitro effects of hormones on expression of Ntcp mRNA in isolated rat hepatocytes. Hepatocytes were isolated from nonfasted male rats weighing 225 g by the 2-step collagenase perfusion method (3). The hepatocyte suspension was added to 60-mm dishes precoated with Matrigel and incubated in a humidified atmosphere of 5% CO2 in air at 37°C overnight in Waymouth's medium. After 24 h, the medium was changed, T3 (108 M), dexamethasone (Dex) (107 M), and GH (100 ng/ml) were added, and cells were isolated another 24 h later. RNA was extracted and quantitated by densitometry. [32P]Ntcp, IGF-1, and 18S cDNAs were used as probes. A: effect of hormones on Ntcp mRNA levels. B: effect of GH on IGF-1 mRNA. Data are corrected for loading with 18S. Results are expressed as means ± SE and analyzed by 1-way ANOVA and Student's t-test. Number of determinations in each group is indicated in parentheses.
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DISCUSSION
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Sex differences in transport of drugs and endogenous substrates exist in humans as well as animals (37). Transport of bile acids as well as many xenobiotics is known to be greater in male than female rats (8, 43). Our previous study (49) has shown that reduced uptake of taurocholate in female rats, as well as after estrogen administration to males, is due to decreased transcription of Ntcp mRNA. The present studies were therefore undertaken to examine the possible hormonal mechanism(s) responsible for the differential expression of Ntcp mRNA and to determine whether the mechanism(s) for estrogen-induced cholestasis involves direct effects on the liver expression of Ntcp. The current results demonstrate that the sexually dimorphic expression of Ntcp mRNA involves a complex interaction of hormones including thyroid, glucocorticoids, and growth hormone leading to sex differences in Ntcp expression. Furthermore, the effect of estrogens to reduce Ntcp mRNA is not due to a direct action on the liver but rather requires pituitary hormones, particularly the steady secretion of growth hormone.
Hepatic bile salt excretion is very efficient with little evidence of intrahepatic accumulation of substrate during its enterohepatic circulation (42). Because previous studies (49) have shown that taurocholate uptake was two times greater in males compared with females, we proposed that bile salt export protein levels might be coordinately regulated with Ntcp to prevent intrahepatic accumulation of bile acids. However, in contrast to Ntcp, the steady-state level of bile salt export protein mRNA and protein were not significantly different. In addition, the sex differences in sinusoidal Ntcp expression were selective, because Mrp3, which is also capable of bile salt transport, was not different (24). Thus expression of transporters involved in bile salt secretion was not coordinately regulated with uptake.
Sex steroid hormones, especially estrogens, are known to modify hepatic function, including biliary secretion (44). However, the physiological role of estrogen is unclear, because 17
-ethinyl estradiol is usually administered at pharmacological doses. Therefore, to investigate the possible physiological role of estrogens in decreasing taurocholate uptake, 17
-estradiol was administered to male and female rats at physiological levels (41). In males, 17
-estradiol administration selectively reduced Ntcp mRNA to 70% of control values (P < 0.05), whereas it did not significantly alter Ntcp in female rats. These results suggested that physiological levels of estrogen might be responsible for the sex differences in Ntcp expression. This postulate was supported by increased Ntcp mRNA after ovariectomy and its restoration to normal levels with 17
-estradiol replacement.
Estrogens may modify hepatic gene expression either directly or secondary to altering the growth hormone secretory pattern. The rat and human Ntcp promoters have been characterized (29, 47). In addition to the critical role of hepatic nuclear factor-1
, potential steroid nuclear protein elements and functional STAT binding sites have been identified in the rat promoter (19). The direct effect of estrogens on liver gene expression may involve the classical ligand-dependent pathway or alternative pathways including ligand-independent activation of estrogen receptors or nonnuclear actions through cell-surface receptors (22, 38). Previous studies (20) demonstrated that 17
-ethinyl estradiol administration to male rats reduced critical hepatic nuclear binding proteins, including HNF1 and C/EBP. However, it was unclear whether these changes were directly involved in the dimorphic expression of Ntcp. On the other hand, the selective effect of 17
-estradiol administration in males and ovariectomy in females suggested that estrogens might modify other hormones rather than having a direct effect on hepatic genes (38). Sex steroids regulate the pattern of growth hormone secretion by mediating the balance of secretion between growth hormone-releasing hormone and somatostatin (Fig. 9) (27, 28). Estrogen administration has been demonstrated to convert the male growth hormone secretory pattern to a female pattern (40). The specific molecular mechanism(s) controlling the sex-specific expression of hepatic genes is unclear but may involve nuclear content of STAT 5b (53, 59).

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Fig. 9. Model of the hypothalamic-pituitary-liver axis and hormonal regulation of Ntcp by the dimorphic pattern of GH secretion. The intermittent (male) and steady (female) pattern of GH secretion is regulated by the balance between the hypothalamic release of GH releasing hormone (GHRH) and somatostatin, which in turn is determined in part by sex steroid hormones. The differential pattern of GH secretion is responsible for the direct effects on the sexually dimorphic expression of specific hepatic genes including Ntcp. On the other hand, corticosterone from the adrenal gland (A) and T4 from the thyroid glands directly increases Ntcp expression equally in both males and females. 17 -E administration in normal males converts the male GH secretory pattern to a steady secretion and thus decreases Ntcp mRNA levels.
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It is well established that the sexually dimorphic secretion of growth hormone is responsible in many instances of sex differences in hepatic gene expression (Fig. 9). A number of hepatic genes including surface receptors and cytochrome P-450s are regulated in large part by the growth hormone secretory pattern (1). In adult rats, the pattern of growth hormone secretion differs markedly between the sexes (Fig. 9) (18). Males produce episodes of high growth hormone secretion at regular intervals of 34 h separated by periods of
60 min in which plasma levels fall below detection. Secretion of growth hormone in females is more continuous, with many smaller peaks and a baseline plasma level that rarely falls to undetectable levels (61). Sex-specific secretory patterns can be mimicked in vivo in which it has been shown that differences in gene expression can be reproduced in the hypophysectomized animal by either growth hormone injections (male pattern) or constant infusions with a minipump (female pattern) (16).
Several lines of evidence in this study suggest that regulation of Ntcp may involve pituitary hormones, especially growth hormone. First, the infusion of growth hormone to intact male rats decreased Ntcp mRNA (Fig. 3), consistent with its reduction after estrogen administration. We (51) previously demonstrated that estrogen administration decreased CYP2C11 and increased CYP2C12 and 5
-reductase mRNA content in male livers, consistent with converting the male growth hormone pattern to one of continuous secretion. Second, ovariectomy converts the female growth hormone secretory pattern to a male-like pattern (40), consistent with the demonstration that ovariectomy increased Ntcp mRNA in female rats. Thus the inductive effects of thyroid hormone and glucocorticoids are unopposed by the male growth hormone secretory pattern. Third, 17
-ethinyl estradiol administration to hypophysectomized male and female rats did not decrease Ntcp expression, indicating that estrogens require pituitary hormones to alter the liver Ntcp mRNA levels. Fourth, loss of pituitary hormones (Fig. 4) including thyroid, corticosterone, and growth hormone, selectively decreased Ntcp mRNA.
The previous studies indicated that pituitary hormones, in particular growth hormone, may be involved in the mediation of sex differences as well as the effect of estrogens on Ntcp mRNA levels. However, it was unclear whether the effect of growth hormone was a positive effect of the male pattern of growth hormone secretion or, rather, a negative effect of the female pattern. To examine this question, we tested the direct effect of different patterns of growth hormone secretion on Ntcp expression in the hypophysectomized model. Surprisingly, the results indicated that although hypophysectomy reduced Ntcp selectively in male rats, suggesting a positive role of growth hormone, the addition of growth hormone by injection, as well as by constant infusion, did not significantly increase Ntcp expression.
In contrast to growth hormone, the effect of thyroid and corticosterone administered either alone or together to male and female rats significantly increased Ntcp mRNA levels to basal male values (Figs. 7 and 8). This result indicated the importance of one or both of these hormones in the maintenance of high levels of Ntcp expression. However, Ntcp values did not demonstrate dimorphic expression until different patterns of growth hormone were administered. As anticipated from previous results in intact animals, growth hormone infusion to hypophysectomized rats supplemented with both thyroid hormone and corticosterone reduced Ntcp values to those measured in hypophysectomized or intact female rats. On the other hand, after injection of growth hormone, Ntcp mRNA values were still at male levels and significantly (P < 0.01) greater than hypophysectomy. Thus sex differences in expression of Ntcp are due to downregulation of elevated Ntcp levels by infusion of growth hormone in the female pattern.
Both glucocorticoid and thyroid hormones have been shown to regulate bile flow and bile acid secretion (32, 35), although the cellular mechanism is unclear. The present results indicate that both thyroid and corticosterone may be important physiologically in regulating Ntcp mRNA. Putative DNA recognition sites have been identified for these hormones in the 5'-flanking region of rat Ntcp, although the functional significance has not been established (29). Previous studies have indicated that thyroid hormone and glucocorticoids increased bile salt excretion, but their effect on Ntcp expression in isolated rat hepatocytes has not been reported. These studies demonstrate that both thyroxine and dexamethasone significantly increased Ntcp mRNA. In contrast, growth hormone alone reduced Ntcp mRNA, whereas together with thyroxine and glucocorticoids, it reduced the elevated Ntcp to basal levels. Although these in vitro results are similar to our animal studies, they differ from those reported by Cao, et al. (8) who have shown that growth hormone, as well as prolactin, increased Ntcp mRNA in isolated rat hepatocytes. However, a number of experimental differences are present in our studies. First, we used male hepatocytes instead of female cells (53). Second, isolated rat hepatocytes were cultured on Matrigel in contrast to collagen. Finally, hormones were added after 18 h when Ntcp values were stable, and mRNA was isolated 24 h later. In contrast, Cao et al. (8) examined the effect of growth hormone shortly after isolation when basal levels were unstable.
Although the physiological significance for differences in Ntcp expression are unclear, reports indicate functionally significant polymorphisms in human Ntcp (25). In addition, disorders associated with estrogen administration such as intrahepatic cholestasis of pregnancy may have abnormalities in hepatic uptake (44). Because recent studies (26, 62) have established that the growth hormone secretory pattern in humans is an important determinant of CYP3A4 expression, it is possible that estrogen administration may also contribute to disorders of bile acid transport.
In conclusion, sex differences in Ntcp mRNA are transcriptionally regulated, and its expression is under multihormonal control involving thyroid hormone, corticosterone, and growth hormone. Both thyroid and glucocorticoids increase Ntcp in vivo in both sexes, whereas growth hormone on the other hand negatively regulates Ntcp mRNA expression when infused in the female secretion pattern. Thus the current results strongly suggest that the sexually dimorphic differences in Ntcp expression, as well as the effect of estrogens in downregulating Ntcp, results from the female pattern of growth hormone secretion.
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GRANTS
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This work was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15851.
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ACKNOWLEDGMENTS
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We are grateful to Drs. I. M. Arias, A. Mode, and P. Meier for sharing antibodies and cDNA probes with us.
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FOOTNOTES
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Address for reprint requests and other correspondence: F. R. Simon, Univ. of Colorado Health Sciences Center, Dept. of Medicine (B-145), 4200 E. 9th Ave., Denver, CO 80262 (E-mail: franz.simon{at}uchsc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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