1 Department of Medicine and Denver Veterans Affairs Medical Center, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 2 Department of Medicine and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Gender differences
in the hepatic transport of organic anions is well established.
Although uptake of many organic anions is greater in females,
sodium-dependent taurocholate uptake is greater in hepatocytes from
male rats. We examined the hypothesis that endogenous estrogens alter
the number of sinusoidal bile acid transporters and/or decrease
membrane lipid fluidity. The initial sodium-dependent uptake of
[3H]taurocholate was
75% greater in hepatocytes from males than from either intact or
oophorectomized females rats. Taurocholate maximal uptake
was increased twofold (P < 0.03)
without a significant change in the Michaelis-Menten constant.
Sinusoidal membrane fractions were isolated from male and female rat
livers with equal specific activities and enrichments of
Na+-K+-ATPase.
Males had a significant (P < 0.05)
increase in cholesterol esters and
phosphatidylethanolamine-to-phosphatidylcholine ratio. Fluorescence
polarization indicated decreased lipid fluidity in females. In females,
expression of the sodium-dependent taurocholate peptide (Ntcp) and mRNA
were selectively decreased to 46 ± 9 and 54 ± 4%
(P < 0.01), respectively, and the
organic anion transporter peptide (Oatp) and
Na+-K+-ATPase
-subunit were not significantly different. Nuclear run-on analysis
indicated a 47% (P < 0.05) decrease
in Ntcp transcription, without a significant change in Oatp. In
conclusion, these studies demonstrated that decreased sodium-dependent
bile salt uptake in female hepatocytes was due to decreased membrane
lipid fluidity and a selective decrease in Ntcp.
lipid composition; lipid fluidity; sodium-dependent taurocholate transporter; organic anion transport peptide; transcription
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INTRODUCTION |
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THE HEPATOCYTE IS PRIMARILY responsible for transport, metabolism, and excretion of many organic anions, including bile acids, bilirubin, fatty acids, and organic dyes. Sexual differences have been described for hepatic transport as well as for enzymatic activities (22), drug detoxification (13), and lipid metabolism (47, 53). Both exogenous and endogenous compounds including indocyanine green (31), bromosulfophthalein (BSP) (39, 40, 48), bilirubin (38), and fatty acids (27, 30, 49) have been reported to be transported to a greater extent by female hepatocytes. In contrast, the initial uptake of taurocholate was more rapid in males than in females (10, 11).
Bile acids undergo an enterohepatic circulation involving secondary active transport processes at the ileal brush border and the sinusoidal domain of hepatocytes (reviewed in Ref. 50). Bile acids are synthesized exclusively in the liver from cholesterol, secreted into bile where they are the major osmotic driving force for bile flow, and subsequently participate in the digestion and absorption of various lipophilic dietary constituents. In the ileum, bile acids are efficiently reabsorbed, transported in the portal circulation to the liver, and very efficiently cleared by hepatocytes (15).
The hepatic uptake of bile acids has been extensively examined (34). Taurocholate, the major bile acid, has been shown to obey Michaelis-Menton kinetics, to require energy, and to be primarily sodium dependent. Recently, two sinusoidal membrane bile acid transporters have been cloned and their functions characterized in heterologous systems (33). The results were consistent with the view that the majority of taurocholate uptake is mediated by the sodium-dependent taurocholate transporter peptide (Ntcp) and to a lesser extent by the organic anion transporter peptide (Oatp) (23).
The hepatic uptake of taurocholate in vivo is regulated by a number of factors, including development (24), diet (16), bile acids (26), hormones (18, 44), and gender (10). Although some of these factors have been shown to regulate Ntcp at the pretranslational level, the mechanism(s) involved in the gender differences is unclear. It has been suggested that differences in liver plasma membrane lipid fluidity and electrogenic driving forces may be involved (4, 49, 54). However, sexual dimorphic differences in cytochrome P-450, enzymatic activities, and receptors have generally been demonstrated to be related to pretranslational regulation of protein content (55). In addition, because estrogens have been shown to decrease the expression of Ntcp and Oatp (44), we hypothesized that the decreased taurocholate uptake was due to transcriptional differences in the expression of the sinusoidal bile acid transporters, Ntcp and/or Oatp. Therefore, the present studies were undertaken to examine whether the sexually dimorphic hepatic uptake of taurocholate was due to differences in membrane lipids, sodium driving forces mediated by Na+-K+-ATPase, or, rather, the level of bile acid transporters. The results demonstrated that Ntcp but not Oatp protein content was significantly greater in males and expression of Ntcp was transcriptionally controlled.
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MATERIALS AND METHODS |
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Experimental animals. Male and female Sprague-Dawley rats weighing 180-200 g were purchased from Harlan (Indianapolis, IN) and were allowed to acclimate in our animal facility for at least 5 days before initiation of experiments. The temperature of the animal room was maintained at 22°C. Fluorescent lighting in the room was controlled by an automatic timer so that animals were exposed to 12 h of light and darkness. Rats were fed Purina rat chow and allowed free access to food and water. Female rats were either gonadectomized or sham operated at 200 g by the breeder. Studies were performed 5-10 days after surgery.
Hepatocyte isolation and taurocholate transport. Hepatocytes were harvested from nonfasted rats under pentobarbital sodium anesthesia by the collagenase perfusion method (7). Viability was assessed by trypan blue exclusion. Hepatocytes were suspended in 3 ml of DMEM-Ham's F-12 medium (1:1) containing 8% FCS on 60-mm petri dishes and placed in a 37°C, 5% CO2 incubator. Medium was aspirated after 3 h of incubation, and attached cells were washed twice in either sodium-containing or choline substituted for sodium Hanks' balanced salt solution (HBSS). After cells were prewarmed for 5 min, buffer was aspirated and 1.5 ml of prewarmed buffer with 100 µM taurocholate (specific activity 3.47 Ci/mmol at 6.65 × 105 dpm/plate) were added for specified times in a 37°C water bath equipped with a shaking platform. Uptake was performed in duplicate at 15, 30, 45, and 70 s. Buffer was aspirated, and cells were washed rapidly four times with HBSS-HEPES at 4°C, scraped, and dissolved in 0.5 N NaOH. Taurocholate uptake was calculated from the slope of the uptake time course. Sodium-dependent taurocholate uptake was determined from the difference measured between sodium-containing and sodium-free buffers. Results are expressed as picomoles of taurocholate per microgram of DNA per minute. DNA was measured by the method of Burton (12). Kinetic parameters of [3H]taurocholate uptake were determined as previously described (6) using concentrations of 1-100 µM taurocholate. Duplicate measurements of uptake were measured at 60 s for each concentration. Radioactivity was counted in a liquid scintillation counter with 10 ml of Econo-Scint scintillation fluid from Research Products (Mt. Prospect, IL).
Isolation of sinusoidal fractions and enzyme measurements. After being fasted overnight, rats were killed under ether anesthesia, and their livers were removed rapidly to isolate sinusoidal membrane fractions as previously described (41). Briefly, 25 mM MgCl2 was added to homogenized liver tissue in 15 ml of buffer containing 1.0 mM phenylmethylsulfonyl fluoride (PMSF) (pH 7.5) to give a final concentration of 15 mM MgCl2. After 10 min on ice, the sample was centrifuged at 2,400 g for 15 min. The pellet was resuspended in the same buffer, and sinusoidal membrane fractions were separated through a discontinuous sucrose gradient, floating at 37.5% sucrose after centrifugation at 88,000 g for 2.5 h. Fractions used for immunoblotting were stored in the original homogenization buffer with the following additions (all in mM): 0.5 ZnCl2, 1 sodium orthovanadate, 100 sodium fluoride, and 100 potassium phosphate monobasic. These substances were also added to the 100 mM sodium carbonate (pH 11.0) extraction buffer used to remove nonintegral proteins before Western blotting analysis as described by Bergwerk et al. (5).
Na+-K+-ATPase activity was measured after overnight freezing atLipid composition and fluidity measurement. Total lipids were extracted from sinusoidal membrane fractions by the method of Bligh and Dyer (8). Free and esterified cholesterol was quantitated by chromatographic methods after derivatization, as previously described (14). An automatic integrator analyzed gas-liquid chromatogram peak areas after identification of individual peaks by coretention with standard compounds. Total phospholipids were quantitated by the method of Ames and Dubin (1), and individual species were measured after separation by two-dimensional TLC as previously described (17).
Fluorescence polarization and lifetime measurements were done on a 4800 phase-correlation nanosecond polarization spectrofluorometer (SLM Industries, Urbana, IL) with fixed emission and excitation polarization filters. The fluorescence intensity was measured perpendicularly and parallel to the polarization phase of the exciting light to eliminate incidental scattered light by using an excitation wavelength of 360 nm and KV389 emission filters (Schol, Dwyer, PA). All samples were run at 35°C with slits at 4, 4, and 8. The probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), trimethylammonium-DPH (TMA-DPH), and DL-12-(9-anthroyloxy)stearic acid (12-AS) (Molecular Probes, Junction City, OR), were dissolved in tetrahydrofuran to a final concentration of 0.6 µg/ml. Probes were added to intact membranes in a total volume of 1.2 ml (containing ~72 µg of protein) and frequently vortexed. Fluorescence lifetimes and dynamic depolarization measurements were determined at a frequency of 30 mHz with slits from lamp to sample of 16, 0.5, and 0.5 nm by using DPH in hexadecane (9.62 ns) as a lifetime reference solution. Both phase and modulation lifetimes were measured. The maximal limiting anisotropy taken for DPH and TMA-DPH was 0.365 and 0.285 for 12-AS. Fluorescence polarization measurements were also carried out on multilamellar vesicles prepared from total lipid and polar lipid fractions. The lipid extracts were dried under a stream of nitrogen, fluorescent probes were added, and vesicles were formed in PBS by vortexing and sonication, as described previously (41). The amount of total lipid extract or polar lipid fraction used was calculated to be equivalent to the amount contained in 72 µg of initial membrane protein. Measurements were performed in triplicate, and results were analyzed as described previously (41).PAGE and immunoblotting.
SDS-PAGE and immunoblotting were carried out using minigels for
enhanced chemiluminescence (ECL) or standard size gels for Ntcp using
alkaline phosphatase. Total liver proteins were prepared by
Na2CO3
extractions of liver homogenates and resuspended in PBS, pH 7.5, containing inhibitors for protease (2 µg/ml each of antipain,
pepstatin, and chymotrypsin, 5 µg/ml each of leupeptin and aprotinin,
10 µg/ml trypsin inhibitor, and 2 mM PMSF) and phosphatase (10 mM
sodium fluoride, 1 mM sodium orthovanadate, 0.5 mM zinc chloride, and
10 mM KH2PO4) as
described by Bergwerk et al. (5). Immunoblotting was also performed on
proteins from sinusoidal membrane fractions isolated in protease and
phosphatase inhibitors and suspended in 1 mM
NaHCO3 before storage at
80°C. After electrophoresis, proteins were transferred
[using the procedure of Towbin et al. (51)] at 167 V for 1 h using a high-transfer apparatus by Ideal. Gels were blocked for 1 h
using 5% Tween 20-Tris buffered saline (TBS). Blots were processed for
ECL (Amersham) detection of specific antibodies using 1% milk in TBS
for antibody diluent. For Ntcp, blots were visualized by
5-bromo-4-chloro-3-indolyphosphate p-toluidine salt-nitro blue
tetrazolium substrate system (Kirkegaard & Perry Laboratories). All
washes were done with 0.5% Tween 20-TBS for 5 min (3 times). ECL blots
were placed in plastic film and exposed to Amersham Hyperfilm for ECL
for 30-60 s. Autoradiograms were quantitated by densitometry using
a Bio-Rad laser densitometer. Immunoblots were probed with monospecific
antibodies to Ntcp, Oatp, and
Na+-K+-ATPase
-subunit [Upstate Biotechnology (UBI)]. The
characterization of Oatp and
-subunit (UBI) antibodies have been
previously described (5, 46).
RNA isolation, analysis, and transcriptional elongation assay.
Total RNA was extracted from whole liver using a RNeasy mini kit
(Qiagen). The RNA was fractionated in 1.2% agarose-formaldehyde gels
in borate buffer at 140 V for 4 h. RNA was transferred to Hybond-N+
(Amersham) with high-efficiency transfer solution (Tel-Test) by
capillary action and fixed by ultraviolet cross-linking. cDNA probes
were labeled with
[32P]dCTP (Amersham)
using the Decaprime II (Ambion) random-primed labeling system.
Unincorporated label was removed with Probequant G-50 microcolumns
(Pharmacia). Membranes were hybridized using a high-efficiency
hybridization system (Tel-Test) for 16 h at 62°C. Membranes were
washed twice in 2× sodium chloride-sodium citrate (SSC)-0.1%
SDS and then twice in 0.1× 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 30 min to
3 days. Autoradiograms were quantitated with an imaging densitometer (Bio-Rad). The following probes were used: Ntcp (provided by B. Hagenbuch), Oatp,
Na+-K+-ATPase
-subunit (provided by J. Lingrel), and 18S rRNA (Ambion). Relative
density of mRNA was normalized to 18S RNA and is expressed as a
percentage of the male control.
Data analysis. Taurocholate uptake was calculated by the least-squares regression program, and the kinetic parameters were determined by the GraphPad Instat program. One-way ANOVA and a two-tailed t-test were used to determine statistical significance. P values < 0.05 were considered significant. Results are expressed as means ± SE.
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RESULTS |
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Taurocholate transport.
The uptake of
[3H]taurocholate into
hepatocytes isolated from male and female rats was determined. Initial
uptake of taurocholate was linear for at least 70 s in both sexes (data
not shown). The results were compared with oophorectomized (ovx) rats
to determine whether endogenous estrogens inhibited taurocholate
uptake. Table 1 shows the viability of
hepatocyte preparations and the initial uptake rate of sodium-dependent
and -independent taurocholate. Viability of isolated rat hepatocytes
was similar in all three groups. The initial sodium-dependent uptake of
[3H]taurocholate in
males compared with females was greater by 71%, as previously reported
by Brock and Vore (10). Furthermore, in hepatocytes from ovx female
rats, [3H]taurocholate
uptake was not significantly different from intact females, suggesting
that the decreased transport was not related to a direct effect of
endogenous estrogens on the taurocholate transport process. On the
other hand, sodium-independent
[3H]taurocholate,
which represented 13% of the total in male hepatocytes, was not
significantly different in the three groups.
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Sinusoidal membrane lipid composition and fluidity. Previous studies have suggested that the physical state of sinusoidal membrane lipids contributes a small but significant role in the regulation of taurocholate transport (44). Although fluidity measurements of male hepatocyte plasma membranes were reported to be less fluid than those in females (4), these changes were demonstrated only in 12-wk-old rat hepatocytes using the TMA-DPH probe. Because hepatocyte plasma membrane domains showed marked polarity of their lipid composition and fluidity (43), we reexamined whether gender differences are present in lipid composition and fluidity of sinusoidal liver plasma membrane fractions. Sinusoidal membrane fractions were isolated from male and female livers by differential sucrose density gradient centrifugation, and enzyme activities, lipid composition, and fluidity were determined.
Comparison of membrane enzyme-specific activities and enrichments between male and female is shown in Table 2. Na+-K+-ATPase, a marker of the sinusoidal domain, was markedly enriched to 59- and 53-fold in male and female sinusoidal membrane fractions, respectively. Also, immunoblots of the Na+-K+-ATPase
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Measurement of sinusoidal membrane proteins. Taurocholate is transported at the sinusoidal membrane predominantly by Ntcp and to a lesser extent by Oatp (23). In previous studies, differences in the content of the hepatic sinusoidal taurocholate transport proteins Ntcp and Oatp were more important than lipid fluidity in regulating bile acid transport (44). Therefore, the content of Ntcp and Oatp was measured in liver extractions and in sinusoidal membrane fractions. As shown in Fig. 2, Ntcp protein content was selectively reduced in female liver compared with male liver. Ntcp was present as a doublet in male liver extractions as previously reported for sinusoidal membrane fractions (Fig. 2A) (44). In contrast, only a faint 56-kDa band was demonstrated in liver extractions from female liver. This band represented only 5% of male values. Because the female protein content seemed unphysiologically low, we postulated that this result might have been due to low sensitivity of the antibody (a threshold effect) or selective loss of Ntcp during extraction. Therefore, we prepared sinusoidal membrane fractions and determined the content of Ntcp. In males, density of the higher 56-kDa band was modestly greater then the lower 51-kDa band, but both bands were approximately equally distributed in female sinusoidal fractions (Fig. 2B). However, the distribution was variable from experiment to experiment, so one could not conclude the physiological relevance of this observation. To quantitate the gender differences in Ntcp, density of the two bands was combined (Fig. 2C). The protein density of Ntcp in sinusoidal membrane fractions from males was twofold (P < 0.05) greater than in those from females.
Oatp, the other major bile acid transporter in liver, was also determined in liver extractions and sinusoidal membrane fractions from male and female livers (Fig. 2). As previously reported, Oatp is present in liver as a single 80-kDa band. In contrast to Ntcp, the protein content of Oatp was not significantly reduced in female liver extractions (80 ± 8%) or sinusoidal membrane fractions (75 ± 7%) compared with those from males (Fig. 2, A and B). Furthermore, no quantitative differences in the density of the Na+-K+-ATPaseAnalysis of mRNA levels for bile acid transporters.
The gender differences in Ntcp might reflect a change in the stability
of the protein and/or in the steady-state level of Ntcp mRNA.
To distinguish between the two regulatory mechanisms, Northern blot
analysis was performed (Fig. 3). Hepatic
Ntcp mRNA levels from female rats were 54 ± 4%
(P < 0.01) of the value obtained in
males. On the other hand, Oatp (84 ± 10%) and
Na+-K+-ATPase
-subunit mRNA (101 ± 3%) levels were similar in male and female
livers. These results suggested a selective pretranslational control of
Ntcp gene expression.
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DISCUSSION |
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Gender differences in the hepatic transport for organic anions including taurocholate (10), bilirubin (38), BSP (39), indocyanine green (31), fatty acids (49), and some steroid hormones (10) have been described. Only taurocholate has been shown to have greater transport capacity in males. The mechanism(s) responsible for increased taurocholate transport is unclear but may involve gender differences in cell size, driving forces, or membrane lipid fluidity, as well as in the density of bile acid carriers. The physiological regulation of bile acid transport is well understood. However, few studies have addressed the molecular mechanisms involved in regulation of either Ntcp or Oatp. The recent cloning of sinusoidal bile acid transporters and the development of specific antibodies have provided the tools to dissect these mechanisms (37). We have combined molecular, biochemical, and physical approaches to examine the mechanisms involved in the gender differences in hepatic taurocholate uptake. The results demonstrated that the lipid physical properties of sinusoidal membranes from female rats were less fluid than those from males. However, more importantly, males selectively expressed greater amounts of Ntcp, the major hepatic bile acid transporter. Sex differences in Ntcp mRNA were due to transcriptional regulation of the steady-state mRNA levels.
A previous study reported that initial uptake of taurocholate was reduced in hepatocytes isolated from female rats (10), although another preliminary report indicated no differences (3). Therefore, we examined the initial uptake of [3H]taurocholate in isolated hepatocytes from male, female, and female ovx rats. The initial uptake of sodium-dependent taurocholate was significantly (P < 0.01) lower in intact (42%) and ovx (46%) female hepatocytes, confirming and extending the studies of Brock and Vore (10). Moreover, because taurocholate transport in hepatocytes from ovx females was similar to that in intact females, endogenous estrogens did not contribute to the gender differences. The kinetic parameters in [3H]taurocholate transport into isolated hepatocytes indicated a significant (P < 0.03) reduction in Vmax (54%) in ovx female hepatocytes without a significant difference in Km. This suggested that differences in the number of taurocholate transporters may account for sexual dimorphic taurocholate transport.
It has been postulated that changes in organic anion uptake, for example BSP and fatty acids, might be due to differences in membrane lipid fluidity, since Vmax was unchanged but Km was greater in males (40, 49). Membrane lipid fluidity is established to be important in the regulation of liver transport processes (43). A number of studies have demonstrated gender and hormonal differences in hepatic lipid metabolism (47, 53), but only two preliminary studies have examined possible sex differences in hepatic membrane fluidity (3, 4). Because taurocholate transport has been shown, in part, to be regulated by membrane lipid fluidity (35), we determined the lipid composition and fluidity parameters in female sinusoidal membrane fractions.
Fluidity measures the bulk physical properties of membrane lipids, which are important determinants of transport properties (43). Benedetti et al. (4) reported (using TMA-DPH in isolated hepatocytes) that there is an age-related increase in female fluidity values. On the other hand, Bellentani et al. (3) (using DPH as a probe) failed to demonstrate gender differences in fluidity using liver plasma membrane vesicles. To study this question, we used both steady-state and dynamic depolarization techniques to measure membrane lipid fluidity components. Steady-state determinations were measured with three different probes to analyze the different components and domains of membrane lipid properties. DPH and TMA-DPH polarization values largely determined static components of membrane lipids in the interior and outer leaflet, respectively, whereas the 12-AS probe reflects the dynamic parameters of lipid fluidity (43). Although TMA-DPH was not significantly different, both DPH and 12-AS measurements indicated that female sinusoidal membranes were less fluid than those isolated from male rat livers. These differences were confirmed using dynamic fluorescence depolarization, in which the structural as well as the dynamic components of membrane fluidity were decreased in female sinusoidal membrane fractions. These results differed from those previously reported, which may be due, in part, to the use of multiple probes, the increased sensitivity of dynamic depolarization measurements, and the use of well-characterized highly purified sinusoidal fractions. Thus the present results suggested that differences in membrane fluidity might account, in part, for the reduced taurocholate transport in female hepatocytes.
In addition to membrane lipid fluidity, it has been suggested that
differences in organic anion transport may result from gender
differences in cell size or driving forces. However, Sorrentino et al.
(49) have shown that male and female hepatocytes are similar in size.
On the other hand, Weisiger and Fitz (54) demonstrated a modestly
greater potential difference in female hepatocytes. However, this
reported difference could only account for ~20% of the difference in
taurocholate uptake. In addition, these authors did not explore the
biochemical mechanisms, which might account for this difference.
Possibilities included activity of the sodium pump as well as membrane
potassium currents. Our studies did not uncover gender differences in
sinusoidal
Na+-K+-ATPase
activity or mRNA content of the -subunit. Because activities of the
sodium pump were similar in both genders, it is suggested that the
potential difference reported is related to changes in parameters other
than the sodium pump.
Previous studies have demonstrated sexually differentiated functions
for sinusoidal organic anion transport; however, no apparent common
mechanistic theme has emerged. Taurocholate uptake kinetics demonstrated increased
Vmax and
unaltered Km in
males, whereas both fatty acids and BSP-glutathione transport kinetics
demonstrated increased
Km with unaltered
Vmax in females.
Because decreased taurocholate
Vmax suggested,
but did not prove, a difference in the expression of Ntcp, we measured
the density of the transporter in both total liver and sinusoidal
membrane fractions. Ntcp expression was sexually dimorphic, being
present in female sinusoidal membranes at only 46% of the level found
in males. In contrast, neither Oatp nor the
Na+-K+-ATPase
-subunit was significantly different. Lack of change in Oatp was
consistent with physiological studies, which have demonstrated that
estradiol-17
-D-glucuronide,
which is a major physiological substrate for Oatp, was transported with
similar velocity in male and female hepatocytes (10, 25).
To determine if the difference in Ntcp protein content was due to pre-
or posttranslational differences, we first measured the steady-state
mRNA levels of Ntcp. The mRNA content of Ntcp in female livers was
reduced to 54% of that found in males, consistent with the difference
in protein content, and indicated that the protein differences were due
to pretranslational processes rather than altered protein turnover.
Furthermore, the difference was selective, since neither Oatp nor
Na+-K+-ATPase
-subunit was significantly different. Furthermore, nuclear run-on
assays indicated that the gender differences in steady-state mRNA
levels resulted from a selective decrease in transcription of Ntcp in
livers of females compared with that in males.
It is well established that many hepatic functions are expressed in a
sexually dimorphic fashion (36). In particular, the sexual dimorphic
expression of cytochrome P-450 enzymes
have been thoroughly characterized (22). However, a number of other
hepatic enzymes (45), transcription factors (28), and liver plasma membrane receptors (36) have also been identified to be sexually dimorphic. For the most part, these proteins are regulated by the
dimorphic secretory pattern of growth hormone rather than the direct
effect of sex steroid hormones (42). These gender differences are
regulated at the transcriptional level and may be determined by
hormonal control of transcription factors such as the signal transducer
and activator of transcription (STAT)-5b (52) and hepatic nuclear
factor-6 (28). Recently, studies have demonstrated that
prolactin-mediated postpartum upregulation of Ntcp is through the Janus
kinase (JAK)-STAT pathway of intracellular signaling (19). Growth
hormone, similar to prolactin, also utilizes the JAK-STAT signaling
pathway to induce hepatic genes containing the STAT or interferon-
activation site-like DNA binding domains (2). In
preliminary studies, we have reported that the intermittent administration ("male-like" pattern) of growth hormone to
hypophysectomized rats is an important physiological determinant of the
dimorphic expression of Ntcp (9).
In conclusion, the present studies have demonstrated that Ntcp was dimorphically expressed in rat liver due to gender differences in transcription. The lower expression of Ntcp in association with decreased sinusoidal fluidity accounts for the decreased taurocholate uptake in female hepatocytes compared with that in males. These studies suggest that sex hormones possibly working through growth hormone significantly contribute to the regulation of sinusoidal transport of taurocholate.
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ACKNOWLEDGEMENTS |
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We are indebted to Larry Zoccaro who performed the fluidity measurements and Mary Sheron and Rolf Dahl who assisted us with word processing.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-15851 and DK-34914 (F. R. Simon), DK-23026 (A. Wolkoff), and DK-41296 (Liver Center).
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. §1734 solely to indicate this fact.
Address for reprint requests: F. R. Simon, Dept. of Medicine (B-145), Univ. of Colorado Health Sciences Center, Denver, CO 80262.
Received 16 June 1998; accepted in final form 6 November 1998.
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