Intestinal Brush-border Membrane Vesicles Research Articles

Structure-inhibitory profiles of nucleosides for the human intestinal N1 and N2 Na+-nucleoside transporters.

To determine the structure-inhibitory profiles of nucleosides for the N1 and N2 Na+-nucleoside transporters of the human intestine. The uptake of 3H-labeled prototypic substrates of the N1 (inosine) and N2 (thymidine) transporters into human intestinal brush border membrane vesicles was measured by a rapid filtration technique in the presence and absence of various uridine and adenosine analogs and antiviral and anticancer nucleoside drugs (100 and 1000 microM). In the ribose ring, the 3'-oxygen is required for inhibition of uptake of nucleosides by both the N1 and N2 transporters. The structural requirements for such inhibition differ with respect to modifications on the 5' position of the sugar ring or on the base. The N2 transporter is more tolerant to these substitutions than is the N1 transporter. The 6 position on uracil and the 8 position on adenine are critical for inhibition of uptake of nucleosides by both the N1 and N2 nucleoside transporters. These data are the first evidence that the binding site(s) of the human N1 and N2 transporters differ in their interaction with analogs of their common substrates, uridine and adenosine. Such studies can provide insight into the critical structural determinants of the substrate necessary for recognition by the Na+-nucleoside transporters of the human intestine.

Direct inhibitory effect of rotavirus NSP4(114-135) peptide on the Na(+)-D-glucose symporter of rabbit intestinal brush border membrane.

The direct effect of a rotavirus nonstructural glycoprotein, NSP4, and certain related peptides on the sodium-coupled transport of D-glucose and of L-leucine was studied by using intestinal brush border membrane vesicles isolated from young rabbits. Kinetic analyses revealed that the NSP4(114-135) peptide, which causes diarrhea in young rodents, is a specific, fully noncompetitive inhibitor of the Na(+)-D-glucose symporter (SGLT1). This interaction involves three peptide-binding sites per carrier unit. In contrast, the Norwalk virus NV(464-483) and mNSP4(131K) peptides, neither of which causes diarrhea, both behave inertly. The NSP4(114-135) and NV(464-483) peptides inhibited Na(+)-L-leucine symport about equally and partially via a different transport mechanism, in that Na(+) behaves as a nonobligatory activator. The selective and strong inhibition caused by the NSP4(114-135) peptide on SGLT1 in vitro suggests that during rotavirus infection in vivo, NSP4 can be one effector directly causing SGLT1 inhibition. This effect, implying a concomitant inhibition of water reabsorption, is postulated to play a mechanistic role in the pathogenesis of rotavirus diarrhea.

Secretory transport of cadmium through intestinal brush border membrane via H +-antiport

The effect of pH on the secretory transport of Cd through the intestinal brush border membrane was investigated using isolated rat intestinal brush border membrane vesicles (BBMV) and the Caco-2 intestinal epithelial cell line. BBMV equilibrated at pH 5.5 or 7.5 (pH in) were mixed with an experimental buffer at pH 5.5 or 7.5 (pH out) containing CdCl 2. The initial accumulation of Cd in BBMV incubated for 1 or 3 min at pH in 5.5 and pH out 7.5 (outwardly directed H +-gradient) was significantly higher than that at pH in=pH out=7.5, but the equilibrated Cd accumulation incubated for 30 min was marginally lower. Carbonylcyanide- p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore, diminished the increasing effect of the H +-gradient on the initial Cd accumulation. Caco-2 cell monolayers cultured on permeable membranes were incubated with CdCl 2 from the basolateral medium, and the transport of Cd from the basolateral to apical medium and the accumulation of Cd in the monolayers were measured. Cd transport was increased by lowering the pH of the apical medium, and was accompanied by a decrease in the Cd accumulation. Coincubation with CdCl 2 and tetraethylammonium, a typical substrate for H +-antiport of the renal organic cation transporter, from the basolateral medium slightly but significantly decreased the basolateral-to-apical transport of Cd, with a concomitant increase in the Cd accumulation. These findings suggest the secretory transport of Cd through the intestinal brush border membrane not only via passive diffusion but also via H +-antiport of the putative organic cation transporter.

Improvement of intestinal absorption of peptides: adsorption of B1-Phe monoglucosylated insulin to rat intestinal brush-border membrane vesicles

In a previous study we glycosylated insulin to improve its intestinal absorption. When the glycosylated product, p-(succinylamido)-phenyl-α- d-glucopyranoside (SAPG)-substituted insulin (SAPG-INS), was administered intra-intestinally to rats, it showed a greater hypoglycemic effect than native bovine insulin. The enhanced hypoglycemic effect of SAPG-INS was considered to be due to an increase in membrane permeability as well as an increase in resistance to enzymatic degradation. In particular, membrane permeability may be related to an interaction with the Na +-dependent d-glucose transporter (SGLT-1) which is located in the brush-border membrane of epithelial cells. The insulin product used in the previous study, however, comprised a mixture of mono-, di- and tri-SAPG-substituted insulin. In this study SAPG-INS with a defined substitution number and position was synthesized to examine the interaction between the transporter and glycosylated insulin in more detail. The new product was mono-SAPG-substituted insulin substituted at the B1-phenylalanine position (B1-SAPG-INS) and was selectively synthesized after protection of the A1-glycine and εB29-lysine amino acids. The hypoglycemic effect of B1-SAPG-INS in rats after an intravenous dose of 71 μg/kg was almost the same as that of native bovine insulin at a dose of 1 U/kg and B1-SAPG-INS retained about 60% of the immunoreactivity of native bovine insulin. The interaction of B1-SAPG-INS with the intestinal transporter was examined by a rapid filtration technique using 125I-labeled B1-SAPG-INS and brush-border membrane vesicles (BBMVs) which were prepared from rat small intestine by the Mg-precipitation method. The amount of B1-SAPG-INS adsorbed or absorbed by BBMVs in the presence of an inward Na +-gradient into BBMVs was greater than that of native bovine insulin. This adsorption/absorption was significantly inhibited by the presence of 1 mM phloridzin. A similar inhibition was observed when Na + was replaced with K + and when B1-SAPG-INS was incubated with BBMVs at 4°C. From the effect of osmolarity on the extent of adsorption/absorption, it was considered that B1-SAPG-INS was not taken up into the intravesicular space but adsorbed onto the external membrane surface of BBMVs. These findings suggested that B1-SAPG-INS was adsorbed specifically onto the transporter. The hypoglycemic effect of insulin was enhanced by glycosylation at the B1 position in in situ experiments using normal and diabetic rats. Consequently, it is suggested that B1-SAPG-INS was adsorbed specifically onto the glucose transporter of intestinal BBM. This specific adsorption may be involved in the mechanism of the enhanced hypoglycemic effect of B1-SAPG-INS both in normal and diabetic rats.

Uptake of barbituric acid derivatives in small intestinal brush border membrane vesicles from retinyl palmitate-treated rats.

Brush border membrane was prepared from the small intestinal (jejunum) cells along the crypt-villus axis. The fluorescence spectra of 1,8-anilinonaphthalene sulfonic acid and the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene were measured in the brush border membrane vesicle suspension. The hydrophobicity of brush border membrane was found to be in the order villus tip >mid villus >lower villus. The fluidity of brush border membrane was in the order villus tip <mid villus <lower villus. The uptake of barbituric acid derivatives by brush border membrane vesicles was well correlated with their partition coefficients (isopentyl acetate/water). No significant difference was observed between the uptake of hexobarbital by brush border membrane vesicles from the villus tip and lower villus. When retinyl palmitate was administered to rats, the fluidity of brush border membrane was found to be higher in the retinyl palmitate-treated rats than in the control rats. However, no significant difference in the uptake of hexobarbital by brush border membrane vesicles was observed between the retinyl palmitate-administered rats and the control rats. Thus, the retinyl palmitate treatment seems unlikely to affect the passively transported ligands like barbituric acid derivatives in brush border membrane vesicles.

Prediction of Oral Absorption of Cephalosporins in Humans.

Orally active cephalosporin antibiotics are known to be absorbed from the small intestine via the oligopeptide transporter (PepT1). Although several methods have been proposed for the prediction of oral absorption in humans, no in vitro method has been established to predict oral absorption of the drugs that are absorbed by a carrier-mediated process. In this mini review, we propose a method to predict oral absorption of cephalosporins in humans from in vitro studies using rat intestinal brush border membrane vesicles (BBMV) or Caco-2 cells. Uptake into BBMV or Caco-2 cells via PepT1 was estimated by subtracting the uptake at 4°C from that at 25°C (BBMV) or 37°C (Caco-2 cells), which was well correlated with the extent of oral absorption according to the complete radial mixing (CRM) model reported by Amidon et al. The present method gives fairly good prediction of oral absorption of cephalosporins and may be used for the screening of well absorbed PepT1 substrates.

Modification of ceftibuten transport by the addition of non-ionic surfactants

The effects of non-ionic surfactants on the carrier-mediated transport of ceftibuten by rat intestinal brush-border membrane vesicles (BBMVs) were investigated. Ceftibuten uptake by BBMVs was measured by a rapid filtration technique. The concentration of surfactants for the uptake experiments was determined by a decrease in the turbidity of BBMV suspension and by the release of an impermeable probe, 2′,7′-bis(carboxyethyl)-4(5)-carboxyfluorescein, from the vesicle inside. In fact, the surfactant concentration of 0.03% (w/v) was selected to maintain the stability of BBMVs. The extent of ceftibuten uptake by BBMVs with various surfactants was correlated with their physicochemical properties, i.e. hydrophile–lipophile balance (HLB), critical micelle concentration (c.m.c.), average diameter of micelle colloid, and polydispersity determined by particle size distribution. The surfactants used were divided into two groups on the basis of polydispersity index ( d w/ d n), i.e. low polydispersity ( d w/ d n≅1) and high polydispersity d w/ d n>2). The ceftibuten uptake due to the addition of surfactants with low polydispersity increased with a decrease in the HLB number. These results indicate that the ceftibuten transport is modulated by the size distribution and hydrophobicity of surfactants. In addition, the effects of surfactants on the membrane lipid fluidity monitored by diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) were investigated. There was significant correlation between ceftibuten uptake and the fluorescence anisotropy of TMA-DPH-labeled membranes due to the addition of surfactants with low polydispersity ( r=−0.81, P<0.0001). These results suggest that surfactants with low polydispersity, in part, increase or decrease the outer membrane leaflet, thereby enhancing or suppressing the ceftibuten transport by BBMVs, and that ceftibuten transport caused by surfactants with low polydispersity may be strongly dependent on the hydrophobic interaction.

Antioxidant action and potential antidiabetic properties of an isoflavonoid-containing soyabean phytochemical extract (SPE).

The potential role of oestrogenic agents, antioxidants and intestinal glucose-uptake inhibitors in the treatment of diabetes is briefly reviewed. Reports in the literature suggest that oestrogen replacement therapy may favourably modulate glucose homeostasis. A soya phytochemical extract (SPE) containing the isoflavone phytoestrogens genistein and daidzein (mostly in their glycone forms as genistin and daidzin) was investigated as an antioxidant and modulator of intestinal glucose-transport. In the present study, SPE was found to protect against glucose-induced oxidation of human low density lipoproteins (LDL) in vitro. Equol (a gut bacterial metabolite of daidzein) was a more effective antioxidant than daidzein or genistein in this system and was of similar antioxidant potency to the dietary flavonols quercetin and kaempferol and to the endogenous antioxidant 17beta-oestradiol. SPE was found to be an inhibitor of glucose uptake into rabbit intestinal brush border membrane vesicles in vitro, though of weaker potency than the classical sodium dependent glucose transporter (SGLT) inhibitor, phlorizin. Thus SPE displays a range of properties which may be of benefit in diabetes, namely as an oestrogenic agent, an inhibitor of intestinal glucose-uptake and a preventive agent for glucose-induced lipid peroxidation.

Role of the monocarboxylic acid transport system in the intestinal absorption of an orally active β-lactam prodrug: carindacillin as a model

Transport of carbenicillin (CBPC) and its orally active prodrug (carindacillin, CIPC) was studied with rat intestinal brush border membrane vesicles (BBMV). CIPC was transported uphill into BBMV in the presence of a H + gradient, indicating that CIPC absorption is carrier-mediated. Indeed, CIPC was predominantly transported by the monocarboxylic acid transport system, although it might be possible that CIPC possesses some affinity to the oligopeptide transporter. In contrast, CBPC exhibited no affinity to either the oligopeptide or the monocarboxylic acid transport system. Apparent uptake clearance of CIPC was approximately 70-fold greater than that of CBPC. It was clarified that the modification of the chemical structure of CBPC (a dicarboxylic acid) to CIPC (a monocarboxylic acid) by ester formation may have resulted in the increased affinity to the monocarboxylic acid transport system, which, in turn, led to improved absorption of the prodrug.

Osmotic equilibrium and elastic properties of eel intestinal vesicles.

Changes in volume of intestinal brush border membrane vesicles of the European eel Anguilla anguilla were measured as vesicles were exposed to media with different osmotic pressures. Preparing the vesicles in media of low osmotic pressure allowed the effects of a small hydrostatic pressure to become a significant factor in the osmotic equilibration. By applying LaPlace's law to relate pressure and volume and assuming a linear relation between membrane tension and area expansion, we estimate an initial membrane tension at 4.02 x 10(-5) N cm(-1) and an area compressibility elastic modulus at 0. 87 x 10(-3) N cm(-1). The elastic modulus estimate falls in the low range of values reported for membranes from other tissues in other species. This lower modulus quantitatively accounts for why eel intestinal vesicles show measurable changes in volume in hypotonic media while rabbit kidney vesicles do not.

Na +-dependent and Na +-independent transport of l-arginine and l-alanine across dog intestinal brush border membrane vesicles

We prepared intestinal brush border membrane vesicles (BBMVs) from beagle dogs fed a commercial diet (protein content: 24–26%), and investigated the characteristics of transport for basic and neutral amino acids across the intestinal BBMVs. To determined the kinetic parameters for l-arginine and l-alanine uptake, their total uptake was resolved into three routes: (1) Na +-dependent carrier-mediated transport; (2) Na +-independent carrier-mediated transport; and (3) simple diffusion. We could observe subtle, but clear-cut, Na +-dependent basic amino acid transport for the first time among studies with intestinal BBMVs prepared from mammals fed a normal diet. The Na +-dependent system for l-arginine transport can be best characterized as ‘low affinity, low capacity’, in contrast to that for l-alanine transport, which is ‘low affinity, high capacity’. Maximal velocities of the Na +-dependent carrier-mediated transport are estimated to be higher for both l-arginine and l-alanine in dog intestinal BBMVs than in rabbit intestinal BBMVs reported previously. These results suggest that food habit of mammals is an important factor to decide the characteristic of system B 0,+, a Na +-dependent carrier-mediated transport system common to basic and neutral amino acids across intestinal brush border membranes, as is protein content of the diet.

Characterisation of penicillin G uptake in human small intestinal brush border membrane vesicles

BACKGROUNDMany β lactams are well absorbed by the small intestine, although the reasons for this are poorly understood.AIMSTo characterise the uptake of penicillin G into human small intestinal brush border...

Localization of PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA and protein in rat kidney.

To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of approximately 90 kDa. PEPT2 antisera detected a specific broad band of approximately 85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).

Thyroid hormones modulate zinc transport activity of rat intestinal and renal brush-border membrane.

Thyroid hormone status influences the Zn2+ and metallothionein levels in intestine, liver, and kidney. To evaluate the impact of thyroid hormones on Zn2+ metabolism, Zn2+ uptake studies were carried out in intestinal and renal brush-border membrane vesicles (BBMV). Steady-state Zn2+ transport in intestinal and renal cortical BBMV was increased in hyperthyroid (Hyper-T) rats and decreased in the hypothyroid (Hypo-T) rats relative to euthyroid (Eu-T) rats. In both the intestinal and renal BBMV, Hyper-T rats showed a significant increase in maximal velocity compared with Eu-T and Hypo-T rats. Apparent Michaelis constant was unaltered in intestinal and renal BBMV prepared from the three groups. Fluorescence anisotropy of diphenyl hexatriene was decreased significantly in intestinal and renal brush-border membrane (BBM) isolated from Hyper-T rats compared with Hypo-T and Eu-T rats. A significant reduction in the microviscosity and transition temperature for Zn2+ uptake in intestinal and renal BBM from Hyper-T rats is in accordance with the increased fluidity of these BBMs. These findings suggest that the increased rate of Zn2+ transport in response to thyroid hormone status could be associated with either an increase in the number of Zn2+ transporters or an increase in the active transporters due to alteration in the membrane fluidity. Thus the thyroid hormone-mediated change in membrane fluidity might play an important role in modulating Zn2+ transport activity of intestinal and renal BBM.

Uptake of Enalapril by Rabbit Small Intestinal Brush-Border Membrane Vesicles.

Uptake of the angiotensin-converting enzyme (ACE) inhibitor enalapril by rabbit small intestinal brush border membrane vesicles was examined. In the presence of an inward H+ gradient the uptake of this peptide mimetic drug was accelerated and an overshoot phenomenon was observed. The uptake was more stimulated by higher H+ gradient. Initial uptake rate was saturable in the presence of an inward H+ gradient, with apparent Km value of 4.2 mM. These findings suggest the involvement of the H+-coupled carrier-mediated transport in the uptake of enalapril. The uptake was inhibited by ACE inhibitor trandolapril but hardly or only slightly inhibited by aminocephalosporins.

PREDICTION OF ORAL ABSORPTION OF CEPHALOSPORINS IN HUMANS: STUDIES WITH RAT INTESTINAL BRUSH BORDER MEMBRANE VESICLES AND CACO-2 CELLS

It has been reported that orally active cephalosporin antibiotics are absorbed from the small intestine via the oligopeptide transporter (PepTl). In the present study, methods have been established to predict oral absorption of cephalosporins using the in vitro data obtained with rat intestinal brush border membrane vesicles (BBMV) or Caco-2 cells. Uptake into BBMV or Caco-2 cells via PepTl was estimated by subtracting the uptake at 4°C from that at 25 or 37°C, which was well correlated with the extent of oral absorption (F) according to the intestinal absorption model reported by Amidon at al. The present method gave fairly good prediction of oral absorption of cephalosporins (Tablel, Figs. 1 and 2).

Modification of ceftibuten transport by changes in lipid fluidity caused by fatty acid glycerol esters.

The effects of various fatty acid glycerol esters (FAGE) on the transport of ceftibuten in rat intestinal brush-border membrane vesicles (BBMV) were investigated. The HLB numbers of FAGE used were in the range of 6.5 to 15.7. The ceftibuten uptake by BBMV was examined by a rapid filtration method in the presence or absence of FAGE of a non-solubilizing concentration (0.03%, w/v). Tetraglycerol monostearate, hexaglycerol monostearate, hexaglycerol sesquistearate, hexaglycerol tristearate and decaglycerol tristearate were found significantly to enhance the ceftibuten uptake under an inward H+-gradient condition, while FAGE with oleate or laurate did not affect it. The extent of ceftibuten uptake with FAGE correlated with the HLB of the esters (r= -0.90, p<0.001). To elucidate the mechanism of its enhancing action, the effects of FAGE on the membrane lipid fluidity were examined using the fluorescence probes diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) which distribute in the hydrophobic inner and outer regions of the lipid layer, respectively. The increase in ceftibuten uptake induced by FAGE proved to correlate with an increase in fluidity of the outer lipid layer (r= -0.83, p<0.003) but not the inner lipid layer of BBMV. Hence, the enhancing effect of FAGE with the lower HLB on the ceftibuten transport in the presence of the H+-gradient should, at least in part, be explained by the interaction with the outer lipid regions of BBMV.

PH-dependent inhibitory effects of angiotensin-converting enzyme inhibitors on cefroxadine uptake by rabbit small intestinal brush-border membrane vesicles and their relationship with hydrophobicity and the ratio of zwitterionic species.

The inhibitory effects of five angiotensin-converting enzyme (ACE) inhibitors on the uptake of an aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush-border membrane vesicles were examined in the presence of an inward H+ gradient. Dixon plot analysis showed that all these ACE inhibitors inhibited the uptake of cefroxadine, which is transported by a H+/oligopeptide transporter in the membrane, in the order of enalapril<quinapril, benazepril<temocapril, trandolapril at extravesicular pH 6.0. These drugs, except for enalapril, which are relatively hydrophobic, exhibited a mix of competitive and noncompetitive inhibition. We also examined the inhibitory effects of quinapril and temocapril at extravesicular pH 5.5, at which the ratio of the zwitterionic species of the drugs increased. The inhibition occurred in a nearly competitive manner at this pH and the inhibitory effects were stronger than at pH 6.0. Regression analysis of the inhibitory effects suggested that the affinity of these ACE inhibitors for the transporter was regulated by the hydrophobicity of these ACE inhibitors and the ratio of the zwitterionic species of the drugs.

Transport of phenethicillin into rat intestinal brush border membrane vesicles: role of the monocarboxylic acid transport system

Transport characteristics of phenethicillin (PEPC) was examined using brush border membrane vesicles (BBMV) prepared from rat small intestine. Uptake of PEPC in the presence of an inwardly directed H + gradient (pH in 7.5 and pH out 5.5) exhibited an overshoot phenomenon, indicating that PEPC is transported uphill into BBMV. Uptake of PEPC was inhibited by various monocarboxylic acids, not by oligopeptides or the β-lactams which are the substrates of the oligopeptide transporter. Moreover, when the uptake of l-lactic acid, a model substrate of the monocarboxylic acid transporter(s), was measured in the presence of PEPC, the uptake of l-lactic acid was inhibited by PEPC in a competitive manner. It was concluded that PEPC is transported into rat small intestinal BBMV mainly via the monocarboxylic acid transporter(s), not via the oligopeptide transporter.

Increased NHE2 expression in rat intestinal epithelium during ontogeny is transcriptionally mediated.

We have previously described changes in intestinal brush-border membrane vesicle (BBMV) Na+/H+ exchange activity and characterized Na+/H+ exchanger (NHE3) expression during rat ontogeny. The current studies were designed to investigate developmental changes in NHE2 expression in rat intestine. In previous studies, pH-dependent uptake of Na+ in jejunal BBMV utilizing HOE-694 inhibition demonstrated that NHE2 functional protein levels were lowest in 2-wk-old rats, higher in 3-wk-old and adult rats, and highest in 6-wk-old rats [Collins et al. Am. J. Physiol. 273 (Cell Physiol. 42): C1937-C1946, 1997]. In the current investigation, Northern blot analyses showed that NHE2 mRNA levels in the jejunum were similar in 6-wk-old, adult, and 3-wk-old rats and three- to fivefold lower in 2-wk-old rats. In situ hybridization of 2- and 6-wk-old rat intestine with NHE2-specific probes confirmed Northern blot observations. Polyclonal antibodies were developed against an NHE2-specific peptide from amino acids 652-661. Western blots with NHE2 antiserum showed that the intensity of a specific 90-kDa band was lowest in 2-wk-old animals and four- to sixfold higher in 3- and 6-wk-old and adult animals. Immunohistochemical analysis showed specific staining of NHE2 antiserum to only the apical intestinal membrane. Furthermore, nuclear run-on analyses showed a 1.7-fold higher NHE2 transcription rate in 6-wk-old rats than in 2-wk-old rats. Overall, the current data suggest that increases in NHE2 expression upon weaning are mediated by increased gene transcription.