The expression and activity of the apical ileal sodium-dependent bile acid transporter (asbt) was examined in the small intestine of control, pregnant, and lactating postpartum rats 2, 12, and 21 days after delivery. Western blot analysis of brush border membrane vesicles (BBMV) prepared from different regions of the small intestine demonstrated that expression of asbt was maximal in the most distal segments for all experimental groups, was not substantially affected in pregnant and 2-day postpartum rats, and was significantly increased in 12- and 21-day postpartum rats. Analysis of mRNA suggested that asbt protein was regulated at the posttranscriptional level in postpartum rats. Increased expression of asbt protein postpartum was maximal (∼2-fold) in the proximal region of the ileum, consistent with a 60% increase in taurocholate (TC) transport in BBMV from the proximal ileum in 14- to 21-day postpartum rats relative to control rats. Absorption of TC, determined from the intact proximal ileum using an intestinal loop model, demonstrated a 30% increase in TC uptake per unit weight of tissue in 14- to 21-day postpartum rats relative to control rats. Together with the marked increase in intestinal mass observed at peak lactation, these data indicate a significant increase in asbt-mediated reclamation of bile acids in the intestine of lactating rats.
- dietary fat absorption
- enterohepatic cycle
a critical function of the mammalian liver is the formation and maintenance of bile flow, accounted for by vectorial transport of active solutes, mainly bile acids, and followed by the passive movement of water (38). The efficient digestion and absorption of dietary lipids requires a high aqueous concentration of conjugated bile acids in the lumen of the small intestine, which in turn requires a high rate of hepatic secretion of bile acids, a process mediated by an ATP-dependent transporter, the bile salt export pump (bsep) (13). Once in the intestinal lumen, the bile acids are reabsorbed mainly by the apical sodium-dependent bile acid transporter (asbt), a 38-kDa glycoprotein and an integral protein of the brush border membrane of the ileal enterocyte (42). Organic anion transporter (oatp) 3 may also act as a transport protein at the apical level of proximal enterocytes (47). After internalization into the ileocyte, bile salts bind to specific cytosolic proteins (14) like the ileal bile acid-binding protein (ilbp) and subsequently exit the cell into the portal circulation by an anion-exchange protein located on the basolateral membrane (29). A truncated form of asbt may also act as an export pump of bile acids at the basolateral level (26). The absorbed bile acids return to the liver via the portal circulation and are taken up across the basolateral membrane of the hepatocyte by the Na+-taurocholate cotransporting polypeptide (ntcp; Ref.6). As a result of this highly efficient process, ∼95% of the bile salts are conserved in each enterohepatic circulation cycle, with the 5% that is excreted in the feces being replaced by hepatic synthesis (6).
Maternal milk is a complex mixture of lipids, carbohydrates, proteins, and vitamins, and its production in lactation greatly increases the energetic demands of mammals, ranging from a 25% increase in human mothers to up to a fourfold increase in rodents (15). Food intake is thus greatly increased (2- to 4-fold) in postpartum rats compared with normal females (9, 15). The large flux of nutrients that must be sent to the neonate requires extensive adaptation of the intestinal tract, such that the maternal small intestine increases in length and weight, reflecting primarily the development of the mucosal component, i.e., the villus (9-11). [3H]thymidine labeling studies combined with histological studies showed that the intestinal crypt and villous cells are increased both in size and number (4). The increased size of the small intestine results in a marked increase in the absorptive surface area that, together with the increased food consumption, greatly increases the breakdown and absorption of nutrients. Several lines of evidence indicate that enteroglucagon is trophic to the intestine and could mediate the increase in intestinal mass observed postpartum (18, 19, 22, 40).
Bile flow, the bile salt pool, basal bile acid secretion, the maximal bile acid secretory rate, and the expression of the hepatic basolateral and canalicular bile acid transporters ntcp and bsep, respectively, are all significantly increased in lactating rats (2, 23,30-32). Increases in the maximal bile acid secretory rate and ntcp and bsep expression are mediated by the anterior pituitary hormone prolactin (PRL) (30-32). In the case of ntcp, PRL acts via the long form of the PRL receptor in the hepatocyte plasma membrane to increase phosphorylation and translocation of signal transducer and activator of transcription 5 to the nucleus, where it binds to γ-interferon-activated sequence response elements in thentcp promoter to increase gene transcription (12).
In view of the increase in nutrient absorption, intestinal mass, the bile acid pool, and hepatic bile acid secretion in postpartum rats, we postulated that intestinal absorption of bile salts must also increase to preserve recirculation of the bile salt pool and that such an increase would likely occur by increasing asbt expression. We further questioned whether PRL or enteroglucagon, the latter in the form of glucagon-like peptide (GLP)-2, could modulate asbt expression in the small intestine. The present studies demonstrate that the expression of asbt and the ileal transport of taurocholate (TC), a major bile acid, are significantly increased in maternal rat intestine during lactation.
MATERIALS AND METHODS
Leupeptin, phenylmethylsulfonyl fluoride, pepstatin A and unlabeled TC were obtained from Sigma (St. Louis, MO). 14C-labeled TC (46.40 mCi/mmol) and 3H-labeled TC (2.10 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Bromocriptine (2-Br-α-ergocriptine methane sulfate) was a gift of Sandoz Research Institute (East Hanover, NJ). Ovine prolactin (oPRL; NIDDK-oPRL-19, AFP-9221A) was kindly provided by the National Institute of Diabetes and Digestive and Kidney Diseases, the National Hormone and Peptide Program, and Dr. A. F. Parlow (Harbor University of California Los Angeles Medical Center, Torrance, CA). A specific anti-rat asbt antibody was used in Western blot studies (42). Rat GLP-2 was obtained from American Peptide (Sunnyvale, CA).
Female Sprague-Dawley rats (Harlan Industries, Indianapolis, IN and National University of Rosario) were used throughout. Pregnant and postpartum rats were timed according to the first day that sperm were detected (day 0). The rats had free access to food and water and were maintained on a 12-h automatically timed light-dark cycle. All protocols involving animals were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Kentucky or by the National University of Rosario.
Nonpregnant female control rats (C group; 180–210 g) and rats at 20 days of pregnancy (P group; 360–400 g) and at 2 (240–270 g), 12 (250–280 g), and 21 days (260–300 g) postpartum (PP2, PP12, and PP21 groups), which served as late-pregnant and early-, mid-, and late-lactating rats, respectively, were used for Western blot analysis of asbt expression. Measurements of TC transport and asbt mRNA were performed in C rats and lactating rats 14–21 days postpartum (PP14–21; 250–310 g). Litter size in postpartum animals ranged from 8 to 10 pups.
Two groups of ovariectomized (OVX) rats weighing 190–230 g were implanted with osmotic minipumps (Alzet 2001; Alza, Palo Alto, CA) attached to an intravenous catheter as described by Liu et al. (30). The minipumps were filled with solvent (0.4 M NaHCO3, 1.6% glycerol, and 0.02% sodium azide; OVX group) or with solvent plus oPRL to yield an infusion rate of 300 or 600 μg of oPRL/day for 7 days (PRL300 and PRL600 groups). The minipumps were immersed in saline at 37°C for 2 h before implantation to ensure that flow had started. To suppress endogenous PRL secretion, all rats were implanted subcutaneously with bromocriptine pellets (7.5 mg, 10-day release; Innovative Research of America, Toledo, OH) at the time of implantation of the minipumps.
Two groups of normal rats weighing 180–220 g were implanted with osmotic minipumps as described above. The pumps were filled either with phosphate-buffered saline (vehicle of GLP-2; V group) or rat GLP-2 dissolved in vehicle to yield an infusion rate of 40 μg/day for 7 days (GLP-2 group).
The whole small intestine was divided into nine equal segments, carefully rinsed with ice-cold saline, and weighed. The most proximal segment, starting from the pylorus, was given the number 1, whereas the most distal segment close to the ileocecal valve was given the number 9 (Fig. 1 A). Segments from PP12, PP14–21, and PP21 groups were longer than those from C, P, and PP2 groups (13–15 vs. 11 cm), as previously reported (36). The mass of the intestinal segments was increased by ∼115% in PP12 and 130% in PP14–21 and PP21 groups with respect to C, P, and PP2 groups. PRL treatment did not affect the intestinal mass, whereas GLP-2 produced a minor increase (∼15% over respective controls). Neither PRL nor GLP-2 induced changes in the intestinal length. The segments were carefully rinsed with saline at 4°C and snap-frozen in liquid nitrogen for RNA analysis or immediately used in mucosa tissue preparation. For Western blot analysis and TC transport in brush border membrane vesicles (BBMV), the intestinal segments were opened lengthwise, the mucus layer carefully removed, and the mucosa obtained by scraping (7). The tissue thus obtained was used for BBMV preparation.
Preparation of BBMV.
Total homogenate was prepared from mucosa samples as previously described (37), and BBMV were prepared from total homogenates by a divalent cation precipitation method (21) with some modifications (37). Aliquots of the homogenates and BBMV were used for alkaline phosphatase activity determination. The remaining BBMV were used for Western blot analysis or determination of TC transport. Protein concentration in homogenates and BBMV was measured (33) with bovine serum albumin as standard. Alkaline phosphatase activity was determined usingp-nitrophenylphosphate as substrate (kit DG1245-K; Sigma). Apical membrane enrichment was estimated by calculation of the ratio of the alkaline phosphatase activity in BBMV to the alkaline phosphatase activity in homogenates.
Western immunoblotting studies of asbt protein.
BBMV proteins prepared from each of the nine segments (Fig.1 A) were separated in 10% SDS-polyacrylamide gels using an amount of protein (15 μg) in the gels that was found to give a densitometric signal in the linear range of the response curve for the anti-asbt antibody (data not shown). After electrotransfer onto nitrocellulose membranes (Protran; Schleicher and Schuell, Keene, NH), the blots were blocked overnight at 4°C with Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk and then incubated for 1 h with the primary asbt antibody (1:2,000). The immune complex was detected by incubation with the horseradish peroxidase-linked secondary antibody (1:2,000; Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h. Immunoreactive bands were detected with a chemiluminescence kit (ECL Plus, Amersham Pharmacia Biotech), exposed to Bio-Max MR-2 film (Sigma) for 5 min, and quantified by densitometry (Shimadzu CS-9000).
asbt RNA analysis.
Ribonuclease protection assays (RPAs) were performed using an RPA II kit (Ambion, Austin, TX) and the indicated amount of RNA isolated from rat small intestine (47). The antisense32P-labeled riboprobes were transcribed with [α-32P]UTP (800 Ci/mmol; Amersham) and T7 RNA polymerase (Maxiscript Kit; Ambion) from pBluescript II KS-rat asbt (nucleotides 1–237) that was linearized with EcoRI or from the linearized pTRI-Actin-mouse template (Ambion). The sizes of the undigested probes and protected riboprobe fragments were 336/237 and 210 nucleotides for rat asbt (nucleotides 1–237) and 304/245 nucleotides for rat actin. An unlabeled sense strand transcript was synthesized from the pBluescript II KS-rat asbt construct with T3 RNA polymerase (Maxiscript Kit, Ambion) and used as a standard for quantitation. The radiolabeled riboprobes were isolated by preparative electrophoresis on a 5% acrylamide-8 M urea denaturing gel and elution into 0.5 M ammonium acetate, 1 mM EDTA, and 0.2% SDS at 37°C. The individual antisense probes were hybridized with tissue RNA at 42°C for >12 h. Single-stranded riboprobe was digested with a mixture of RNase A and T1, and the protected products were resolved by electrophoresis on a 6% acrylamide-7 M urea denaturing gel. The dried gels were exposed to Amersham Hyperfilm with an intensifying screen at −70°C.
Na+-dependent TC transport in BBMV.
Ileal BBMV prepared from segments 6 and 7 and from segments 8 and 9 were analyzed for TC transport activity determination (Fig. 1 B). Sodium-dependent TC uptake was assayed by a rapid filtration technique as previously described in detail (24, 41). Briefly, a 20-μl aliquot of BBMV suspension (40–60 μg protein) was preincubated for 2 min at 37°C and uptake was initiated by the addition of 80 μl of incubation solution (in mM: 125 NaCl or KCl, 50 mannitol, 10 HEPES-Tris, pH 7.5, 0.2 CaCl2, and 1 MgCl2) containing [3H]TC (10 nM). Unlabeled TC was added to the incubation medium to reach the desired final concentration (10, 25, 50, 125, and 250 μM). Uptake was stopped at varying time intervals by the addition of 3 ml of ice-cold stop solution (in mM: 100 KCl, 100 mannitol, and 10 HEPES-Tris, pH 7.5). The samples were immediately filtered through a 0.45-μm Millipore filter (Millipore, Bedford, MA) and washed with additional stop solution containing 1 mM of unlabeled TC. The filters were dissolved in 5 ml of Aquasol (New England Nuclear) and counted in a 1500 series Tri-Carb liquid scintillation analyzer (Packard Instrument, Downers Grove, IL). All determinations were performed in duplicate in freshly prepared BBMV. Na+-dependent uptake of TC was calculated as the difference between uptake in the presence and absence of the Na+gradient. The data were fit to the Michaelis-Menten equation by a nonlinear curve-fitting algorithm for Na+-dependent uptake of TC over the range of 10–250 μM TC.
Transport of TC in intestinal loops.
The in situ intestinal loop model was used to characterize the absorption of TC by the intact ileum as previously described in detail (25). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg body wt), the abdomen was opened, and one loop 15 or 25 cm (for control and postpartum rats, respectively) proximal to the ileocecal valve, corresponding approximately to segments 7–8, was selected and brought to the exterior (Fig.1 C). Small cuts were made at both ends of the intestinal loop (which was ∼10 cm long), the loop was rinsed with ice-cold saline, and the ends were ligated. Sodium heparin solution (500 U) was injected into the femoral vein. The vein draining the selected loop was cannulated with polyethylene tubing (P-10 Intramedic; Clay-Adams, Parsippany, NJ) and secured with an adhesive agent. The intestinal lumen was immediately filled by using a needle with Krebs-Ringer solution (123 mM NaCl) containing 300 μM [14C]TC (4.1 mmol [14C]TC/mol unlabeled TC) and venous blood collected at 2-min intervals for 6 min. Because of differences in the intestinal size, the volume of solution necessary to fill the intestinal loops from postpartum rats was higher than that in control rats (∼1.0 vs. 0.6 ml). Body temperature was maintained at 37°C with a warming lamp. Losses of fluid due to venous drainage and evaporation produced by externalization of the loop were compensated by infusion of saline into the femoral vein. At the end of the experiment, the intestinal segment was homogenized in Krebs-Ringer solution (25% wt/vol) and the TC concentration was determined in aliquots of blood, luminal solution, and tissue homogenate by liquid scintillation. The mass of TC absorbed by the loop was calculated as the product of TC concentration and the volume of blood collected in each time period and was expressed both per unit weight and per length of the intestinal loop.
Data are presented as means ± SD. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni test (Western blot studies and alkaline phosphatase activity determinations in C, P, and PP rats) or Student's t-test (remaining studies) (45). Values of P <0.05 were considered to be statistically significant.
Expression of asbt in pregnant and postpartum rats.
Western blot analyses of asbt carried out in BBMV from control, pregnant, and postpartum rats at three different stages of lactation demonstrated that asbt was present as two immunoreactive bands (Fig.2), with the monomer predominating in all groups. Expression of asbt protein decreased from segment 9to segment 6 in all groups and was undetectable insegments 1–5 (data not shown), so that comparative analysis of asbt content among groups was made only for segments 6–9. Pregnancy did not affect asbt expression substantially, except for a small but significant decrease in segment 9, where constitutive expression of asbt is normally maximal. Expression of asbt protein did not differ in postpartum rats 2 days after delivery but was significantly increased relative to control rats by 12 and 21 days postpartum. Upregulation of asbt was most pronounced in the most proximal segments, i.e., segments 6 and 7, where expression was increased 100%. Alkaline phosphatase activity was measured in BBMV and homogenate samples, and the corresponding ratio was calculated to ensure comparable apical membrane enrichment in the different intestinal segments and groups. Absolute alkaline phosphatase activity in BBMV decreased ∼80% from segment 1 tosegment 9 (data not shown), as previously reported (36). Table 1 shows alkaline phosphatase activity in BBMV from segments 6 and9, corresponding approximately to the most proximal and distal regions of the ileum, respectively. Enzyme activity was significantly increased only in PP21 rats, as reported previously for the most proximal segments of the small intestine (36). The ratio of alkaline phosphatase activity in BBMV vs. homogenate was similar for all segments in normal, pregnant, and lactating rats (6.0 ± 2.8; n = 135). BBMV enrichment thus estimated was comparable to that in previous studies using a similar preparation methodology (1, 36, 37).
asbt mRNA level in lactating rats.
Because asbt protein content was increased in BBMV from postpartum rats at the late stages of lactation, it was of interest to evaluate the level of the corresponding mRNA. As shown in Fig.3, asbt mRNA was highest in distal ileum. The rat asbt probe used in this study encompasses the 5′ end of the rat asbt cDNA and detects the transcripts derived from both rat asbt mRNA start sites. Whereas there was some animal-to-animal variation, neither the gradient of expression nor the absolute amount of asbt mRNA was substantially different between control rats and PP14–21 rats.
Transport of TC in BBMV.
We analyzed TC transport in BBMV prepared from PP14–21 rats because asbt expression was maximally increased at this time. Because of the low yield of BBMV protein obtained from individual segments, BBMV from segments 6 and 7 and fromsegments 8 and 9 were combined for TC transport studies. Apical membrane enrichment was similar for both regions of the ileum and did not differ between control and PP14–21 rats (data not shown). Figure 4 shows the time course (Fig. 4, A and C) and kinetic analysis (Fig. 4, B and D) of TC uptake by BBMV in control and postpartum rats. Transport activity was lower for the proximal region of the ileum (segments 6 + 7; Fig. 4,A and B), in agreement with the lower content of asbt, relative to the distal ileum (segments 8 + 9; Fig. 4, C and D). Although there were no differences between control and postpartum rats in the rates of TC transport in the most distal region of the ileum (segments 8 + 9), TC transport in segments 6 + 7 was significantly increased by ∼60% on average in postpartum rats (see Fig. 4, B and D). Calculation of the kinetic parameters indicated that the maximal rate of transport (V max) increased from 758 ± 74 pmol · min−1 · mg protein−1in control rats to 1,097 ± 197 pmol · min−1 · mg protein−1in lactating rats in segments 6 + 7, whereas it was similar in control (1,578 ± 487 pmol · min−1 · mg protein−1) and lactating (1,694 ± 383 pmol · min−1 · mg protein−1) rats for segments 8 + 9. The Michaelis-Menten constant (K m) values were similar in control (85 ± 15 μM) and lactating (74 ± 30 μM) rats for segments 6 + 7 as well as for segments 8 + 9 (80 ± 24 and 84 ± 17 μM for control and postpartum rats, respectively).
TC absorption in intestinal loop model.
Ileal absorption of TC in vivo depends not only on asbt-mediated uptake into the enterocyte but also on binding to specific cytosolic proteins and subsequent transport across the basolateral membrane. We therefore used the intestinal loop model to determine whether transport of TC was increased in the intact ileum from postpartum rats relative to control rats. TC absorption from intestinal loops was significantly increased at 4 and 6 min in postpartum rats when expressed both per unit weight and per unit length of intestine (Fig. 5,A and C). Cumulative absorption (Fig. 5,B and D) of TC was also significantly increased in postpartum rats relative to control rats. The significant 30% increase in postpartum rats seen when uptake was expressed per unit weight agrees well with the 45% increase in Vmax in BBMV observed in these animals. The greater increase in TC absorption in postpartum rats noted when data were expressed per unit length (Fig. 5,C and D) reflects the ∼80% increase in mass per unit length of intestine in lactating rats (36).
Although the length of the loop was the same for control and lactating rats, absolute blood flow was higher in postpartum animals because of the higher mass of the intestine. When corrected per unit weight, blood flow did not differ between groups and averaged 0.19 ml · min−1 · g intestinal tissue−1. After 6 min of blood collection, the content of TC remaining in the intestinal lumen in control and postpartum rats was ∼70% and 60% of the initial loading dose, respectively. TC retained in the tissue was ∼9% of the loading dose for both groups, suggesting a high efficiency for the transport process. The initial concentration of TC in the intestinal loops, 300 μM, is about fourfold greater than the calculated K m of asbt, so that the transporter would be saturated at early times. Because decreased substrate concentration in the lumen of the loop would limit its transport over time, comparison of TC absorption between control and postpartum rats was limited to the first 6 min of blood collection.
Effect of PRL on asbt expression.
To determine whether PRL is involved in the increased expression of asbt observed in the ileum in postpartum rats, we administered oPRL to ovariectomized animals. Two different doses (300 and 600 μg/day) were used, both of which increase the expression of hepatic ntcp (30) and p-nitrophenol UDP-glucuronosyltransferase (34) and the P1 subunit of glutathione S-transferase in intestine (35), thus accounting for the increased expression of these proteins in postpartum rats. Alkaline phosphatase activity in BBMV as well as apical membrane enrichment was similar in OVX and oPRL rats (data not shown). Western blot analysis of small intestinal segments 6–9 from OVX and PRL300 rats showed no significant effects of oPRL treatment on asbt expression (Fig.6). Treatment of rats with 600 μg oPRL/day also showed no effect on asbt expression (data not shown).
Effect of GLP-2 on asbt expression.
GLP-2 was administered according to a protocol found to restore intestinal weight and length and sucrase-isomaltase activity in total parenteral nutrition-fed rats (22). Alkaline phosphatase activity in BBMV as well as apical membrane enrichment were similar in control and GLP-2-treated rats (data not shown). Figure 6 shows that the hormone did not affect asbt protein content in BBMV in either of the segments analyzed.
This study clearly demonstrates that expression of asbt, TC transport activity in ileal BBMV, and TC absorption in ileal loops are all significantly increased in postpartum rats. The asbt protein expression in the proximal ileum was increased twofold and was maximal at days 12–21 of lactation, in agreement with the maximal increase in food intake and intestinal mass noted at this time (9-11,15). The increased expression of asbt was preferentially observed in the proximal region of the ileum (segments 6 and7), where constitutive expression of the transporter is low, but not in the most distal region that normally exhibits the highest constitutive expression of asbt. It is interesting to note that alkaline phosphatase activity increased in BBMV from PP21 rats not only in the most proximal segments of the small intestine (36) but also in the distal ileum, including segment 9 (see Table1), where asbt expression remained unaltered (see Fig. 2), thus indicating a differential behavior between these two apical proteins. TC uptake in BBMV prepared from the proximal region of the ileum (segments 6 + 7) from postpartum rats was significantly increased, with a 60% increase in transport activity. Transport of TC in the intact ileum was also increased ∼30% in lactating rats when expressed per unit weight. These data agree well with the increase in asbt expression in segments 6 + 7. In contrast, there was no difference in TC transport between control and postpartum rats in BBMV prepared from the most distal region of the intestine (segments 8 + 9), in agreement with the slight (segment 8) or no (segment 9) increase in asbt expression in these regions. Thus, although the main site of Na+-dependent TC transport is the most distal region of the ileum, it does not appear to participate in the adaptive increase in bile salt reclamation in postpartum rats.
The finding that asbt expression per milligram of protein was increased in the proximal ileum in lactating rats, even when intestinal mass was greatly increased, underscores the importance of increased asbt expression in lactation. Studies examining intestinal absorption of amino acids and glucose in lactating rats have shown an increase in the rate of absorption per unit intestinal length but a decrease in transport per unit weight (9, 15, 16). However, the marked increase in mass of the small intestine nevertheless results in an even greater increase in total uptake of these essential nutrients. Although the increase in TC transport in BBMV per unit mass from segments 6 + 7 and in the ileal loop observed postpartum is moderate in magnitude, the 80–100% increase in intestinal mass per unit length at peak lactation, coupled with the ∼30% increase in length, results in a two- to threefold potential increase in intestinal reabsorption of bile acids. Because bile acids undergo enterohepatic recirculation several times each day (6), increased reclamation of bile acids by the maternal ileum may constitute a key regulatory factor in the retention and/or expansion of the bile acid pool.
To establish the molecular basis for the increase in asbt expression in postpartum rats, we analyzed asbt mRNA content by RPA. The pattern of asbt mRNA expression was not affected in lactating rats in any of the segments analyzed. The data suggest that the increase in expression of the bile acid transporter is a consequence of posttranscriptional regulation. Further studies are necessary to establish whether increased synthesis and/or decreased breakdown of asbt protein is involved.
Several hypotheses have been proposed to explain regulation of intestinal bile acid uptake. In a preliminary report (27), bile acid transport in guinea pig small intestine was increased in response to depletion of bile acids and reflected proximal recruitment of ileal enterocytes. However, in an ileal resection model, asbt expression and function were only identified in those regions that normally express the transporter (8), arguing against zonal changes in asbt expression. In the present studies, increased asbt expression in postpartum rats was localized to the proximal region of the ileum, where asbt is normally expressed at low levels, so that we did not detect recruitment of new segments expressing asbt. Although our results do not support a recruitment model, they clearly indicate preferential upregulation of bile salt transport in the region where asbt expression is constitutively the lowest. An alternative hypothesis states that the expression of asbt is dependent on the substrate load, with ileal transport downregulated by increased substrate load and upregulated by decreased substrate load. Thus Lillienau et al. (28) found that in guinea pigs, ingestion of a TC-enriched diet decreased the ileal absorption rate of tauroursodeoxycholate, whereas treatment with cholestyramine increased its absorption, consistent with a negative feedback regulation. In contrast, several studies argue that an increased bile acid load increases ileal bile acid transport. Thus rat pups fed TC by gavage show a precocious development of ileal bile acid transport (43). Stravitz et al. (44) subsequently showed that expression of asbt mRNA, protein, and activity were increased two- to threefold in rats fed 1% cholic acid in the diet relative to pair-fed control rats, whereas asbt expression was decreased ∼50% in biliary-diverted rats relative to intact controls. In contrast, Arrese et al. (1) were unable to modulate expression of asbt or bile acid transport by use of a resin to sequester intestinal bile acids or by common bile duct ligation. This unresolved controversy in the role of substrate load on the regulation of intestinal bile acid uptake makes it difficult to conclude whether the increased hepatic bile acid secretion and increased intestinal reabsorption of bile acid in postpartum rats are functionally linked or reflect independent regulatory mechanisms.
Because PRL is a major hormone secreted postpartum and has been shown to increase expression of hepatic bile acid transporters (5), we investigated its role in asbt upregulation. Western blot analysis did not demonstrate any effect of PRL on asbt protein expression in BBMV. Because upregulation of asbt expression coexisted with intestinal hypertrophy, it is likely that other hormonal factors, such as pancreatic or intestinal glucagon (enteroglucagon) or, alternatively, the large increase in food intake, which have been shown to be partially responsible for the intestinal hypertrophy postpartum, are also involved. Elias and Dowling (10) examined the relative contribution of luminal nutrition in intestine hypertrophy postpartum and found that, unlike other models of small intestinal adaptation, the increase in luminal nutrition observed in lactating rats only partially explained intestinal hypertrophy. Several lines of evidence suggest that enteroglucagon is also trophic to the intestine and probably acts as a complement to hyperphagia. Preliminary reports indicate that in the rat, plasma enteroglucagon levels increase markedly during lactation (19) and in response to some models of intestinal adaptation such as partial starvation (40), hypothermic hyperphagia (18), and jejunal resection (19). More recently, it was demonstrated that enteroglucagon, in the form of GLP-2, restored the decrease in intestinal length and weight induced by total parenteral nutrition in rats (22). Moreover, GLP-2 treatment increased expression of sucrase-isomaltase in small intestine from rats and mice (3,22). We therefore tested whether GLP-2 could be responsible for the increased expression of asbt in lactating rats. However, GLP-2 did not affect asbt protein expression in any of the segments analyzed. GLP-2 administration increased the expression of multidrug resistance-associated protein 2 in rat duodenum and proximal jejunum by ∼50% (unpublished results), clearly suggesting that the hormone differentially affects apical transporters and/or enzymes in small intestine. It remains unclear whether other factors such as the significant increase in food intake alone or in combination with increases in the level of systemic and/or local hormones are responsible for asbt protein upregulation postpartum. More complex experimental models such as simultaneous administration of PRL and GLP-2 together with a forced feeding strategy might help to clarify these issues.
Although food intake increases only slightly by late pregnancy and immediately after delivery (∼50% over normal females), it dramatically increases by middle and late lactation (100–300% increase), agreeing well with the pattern of induction of asbt (15). Thus increased bile salt output and intestinal reclamation postpartum may represent an adaptive response to improve digestion and absorption of dietary fats, particularly of the long-chain unsaturated fatty acids, which are essential components of breast milk (46). The total increase in intestinal mass in late lactation, which is ∼130% of controls (36), represents an adaptive response to the marked increase in energy demands imposed by lactation (15). A very strong correlation exists between increased energy demand that is determined by the litter mass, increased food intake, the result of increased energy demand, and hypertrophy of the small intestine (15). At the peak of lactation in rodents, the energetic demands necessitate up to a fourfold increase in energy intake. This marked increase in energy demand requires adaptation of the intestinal mass to permit the digestion and absorption of needed nutrients. It seems reasonable to postulate that the increased bile acid secretion, pool size, and intestinal reclamation observed at the peak of lactation are necessary to increase absorption of the increased dietary lipids, both to provide energy for the dam and for incorporation into milk. Lipoprotein lipase activity is significantly increased in mammary tissue but is decreased in peripheral adipose tissue in lactation, thus ensuring incorporation of dietary and stored lipids into milk fat (20).
In summary, the present studies have demonstrated an increased expression of asbt protein and an increased capacity for bile acid transport in small intestine from postpartum rats during the latter stages of lactation. This effect is likely mediated by the preferential increase in asbt expression in the proximal region of the ileum coupled with the marked increase in intestinal mass during peak lactation. Neither intravenous administration of PRL to ovariectomized rats nor administration of GLP-2 to normal rats increased expression of asbt, indicating that other factors must be involved. These data support a role for increased asbt expression in enhancing bile acid reclamation in maternal intestine, resulting in an increased bile acid pool that would facilitate absorption of dietary lipids for incorporation into milk.
We express gratitude to José M. Pellegrino for technical assistance.
This work was supported by National Institutes of Health Grants GM-55343 and DK-46923 to M. Vore and DK-47987 to P. A. Dawson and by grants from Subsecretarı́a de Investigación y Tecnologı́a, Ministerio de Salud de la Nación, Consejo Nacional de Investigaciones Cientı́ficas y Técnicas and Universidad Nacional de Rosario, Argentina to A. D. Mottino.
Present address of A. D. Mottino: Instituto de Fisiologı́a Experimental, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Suipacha 570, 2000 Rosario, Argentina.
Address for reprint requests and other correspondence: M. Vore, Graduate Center for Toxicology, 306 Health Sciences Research Bldg., Univ. of Kentucky, Lexington, KY 40536-0305 (E-mail:).
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.
- Copyright © 2002 the American Physiological Society