Comparative analysis of ontogenic changes in renal andintestinal biotin transpo - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Svetlana M.Nabokina, Veedamali S.Subramanian,  Hamid M.Said,作者单位：2 Veterans Affairs Medical Center, Long Beach 9082 and University of California, Irvine, California 92697 & m1 P" V- S* ]5 X. x8 Z4 Z

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      【摘要】0 R6 k$ D* W; k$ T: }# H
   Biotin, an essential water-solublemicronutrient, cannot be synthesized by mammals; rather, it is obtainedfrom exogenous sources via uptake by intestinal epithelia. Renalepithelia reclaim the vitamin that is filtered in the glomeruli. Bothepithelia take up biotin via the sodium-dependent multivitamintransporter (SMVT). Little is known about ontogenic regulation of therenal and intestinal biotin transport processes and about themechanism(s) involved in any such regulation. In this study, we soughtto examine and compare ontogenic aspects of the renal and intestinalbiotin uptake processes using purified brush-border membrane vesicles (BBMV) isolated from the kidney cortex and jejunum of suckling andadult rats. Clear ontogenic changes were observed in the intestinal biotin uptake process, which were mediated via changes in V max and apparent K m.Parallel changes were also seen in protein, mRNA, and transcriptionrate of SMVT as indicated by results of Western blotting, RT-PCR, andnuclear run-on assays, respectively. In contrast, biotin uptake byrenal BBMV did not show ontogenic changes; i.e., it was similar insuckling and adult rats. Also, the levels of SMVT protein and mRNA weresimilar in the kidneys of both age groups. These data show that biotinuptake by renal and intestinal epithelial cells responds differently toontogenic regulation. In addition, the ontogenic changes observed inthe intestinal biotin uptake process involve the entry step of thevitamin at the BBM and appear to be mediated via a transcriptional mechanism(s). * k: ?. j" v) |9 o
      【关键词】 ontogenic regulation renal biotin uptake intestinal biotin uptake
   INTRODUCTION( B4 k4 C, s* Q( C8 D) u

BIOTIN (VITAMIN H) IS AN ESSENTIAL micronutrient that acts as a coenzyme for fivemammalian carboxylases that catalyze essential steps in four pathwaysinvolving fatty acid biosynthesis, gluconeogenesis, and catabolism ofcertain amino acids and fatty acids ( 5, 29 ). Theimportance of biotin for human normal health and well-being isunderscored by the serious clinical abnormalities that result from itsdeficiency, which include neurological disorders, growth retardation,and dermal abnormalities ( 5, 10, 29, 36 ). Biotindeficiency and suboptimal levels are being reported with increasedfrequency in recent years and occur in a variety of conditions,including inborn errors of biotin metabolism/transport, after chronicuse of anticonvulsant drugs, after long-term use of parenteralnutrition, and during pregnancy ( 10, 13, 29, 32, 36 ).
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Humans and other mammals cannot synthesize biotin and thus must obtainthe vitamin from exogenous sources. Intestinal epithelial cells absorbdietary biotin as well as the biotin that is synthesized by the normalmicroflora of the large intestine ( 22, 27 ). Renalepithelial cells reabsorb biotin that is filtered into the urine. Thusintestinal and renal epithelial cells play a central role inmaintaining and regulating normal biotin body homeostasis. Previousstudies have shown the transport process of biotin in both renal andintestinal epithelia to be via a specialized,Na -dependent, carrier-mediated mechanism ( 3, 4, 7, 14, 23-25 ). The Na - dependent uptake was foundto be located at the brush-border membrane (BBM) of these epithelialcells ( 14, 25, 26 ). The molecular identity of thetransport system has been recently determined after cloning of the cDNAfrom human, rat, and rabbit tissues ( 8, 15, 16 ). Thisbiotin uptake system was also found to be able to transport two othernutrients, namely, the unrelated vitamin pantothenic acid and themetabolically important substrate lipoate ( 8, 15, 16, 17, 22 ). For this reason, the uptake system has been named thesodium-dependent multivitamin transport (SMVT). So far, SMVT appears tobe the only significant biotin carrier identified in intestinal andrenal epithelia.
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Previous studies have shown that a variety of renal and intestinaltransport events undergo ontogenic changes during early stages of life( 2, 11, 28, 34, 35 ). These changes, however, do not followa unified pattern and in certain cases are tissue specific in nature( 9, 11, 19, 34 ). To date, however, there is nothing knownabout the effect of ontogeny on renal biotin transport. As to theintestinal biotin uptake process, previous studies from our laboratoryhave shown that transport of the vitamin across intact intestinalepithelia undergoes clear ontogenic changes during the early stages oflife ( 21 ). However, little is known about the molecularmechanism(s) that mediate these changes in intestinal biotin transport.Whether these changes involve the transport step of biotin at the entrylevel of BBM of the polarized enterocyte is unclear. In this study, weexamined the effect of ontogeny on biotin transport across the BBM ofrenal and intestinal epithelia using isolated purified BBM vesicle(BBMV) preparations. We also investigated the molecular mechanism(s) that mediates the effect(s). Our results showed that, while biotin transport across the intestinal BBM undergoes ontogenic changes, nosuch changes occur in the biotin uptake process across renal BBM. Inaddition, our results suggest that the ontogenic changes observed inintestinal biotin transport involve transcriptional mechanism(s).( X; r: F3 i6 r' j9 |7 ^+ f

MATERIALS AND METHODS1 h  P3 \7 a; b& u% s1 z

Chemicals and animals. D -[8,9- 3 97%) was obtained from PerkinElmerLife Science (Boston, MA). All other chemicals, reagents, and kits usedin this study were of analytical/molecular biology grade and wereobtained from commercial sources. Cellulose nitrate filters (0.45-µmpore size) for use in uptake studies with renal and intestinal BBMVwere purchased from Sartorius Filters (Hayward, CA).4 `6 S, v( l! B
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Suckling (13-14 days old) and adult (65-70 days old)Sprague-Dawley rats were used in the study (Harlan Sprague Dawley,Indianapolis, IN). Mothers and adult rats were fed Purina Chow diet adlibitum and had free access to water. The National Council'sguidelines for the care and use of laboratory animals were followed,and all studies were approved by our Animal Studies Subcommittee." \* v: C  L& V4 r: |9 w
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Isolation of renal and intestinal BBMV and transportinvestigations. Rats were euthanized with CO 2, and their kidneys and smallintestine were removed. BBMV were then isolated from the renal cortexand jejunum using validated procedures previously established in ourlaboratory ( 25, 26, 33 ). The suitability of renal BBMV forstudying ontogeny of a transport process has been previously established ( 11, 35 ). This has been confirmed in ourlaboratory by demonstrating a similar enrichment in the activity of theBBM marker enzyme alkaline phosphatase in the final BBMV preparations compared with initial cortical homogenates of the rat groups (8-10 fold for both ages). Similarly, intestinal BBMV have been validated andused by us and others to study ontogenic regulation ( 1, 2, 6, 20 ). Isolated BBMV were preloaded with a buffer of (in mM) 280 mannitol and 20 Tris-HEPES, pH 7.4, and were incubated in a buffer of(in mM) 140 mannitol, 170 NaCl, 17 Tris-HEPES, and 17 Tris-Mes, pH 7.4, in the presence of [ 3 H]biotin. Uptake studies wereperformed at 10 s (initial rate) ( 24, 25 ) at 37°Cusing a rapid-filtration technique described previously ( 12, 24, 25 ).
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Western blot analysis. BBMV were isolated in the presence of 2 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 0.5 µg/ml leupeptin as reported previously by us ( 18 ). BBMV proteins (150 µg) wereresolved on 7.5% SDS-PAGE and electroblotted on Hybond enhancedchemiluminescence nitrocellulose membranes (Amersham PharmaciaBiotech). For Western blot analysis, the membrane was blocked with 5%nonfat dry milk in PBS containing 0.1% Tween 20 and then incubatedwith specific rabbit polyclonal anti-peptide antibodies raised againstthe LYHACRGWGRHTVGELLMADRK peptide, which corresponds to amino acids43-64 of the rat SMVT sequence (Alpha Diagnostic, San Antonio,TX). Immunodetection was performed using goat anti-rabbit IgG secondaryantibodies conjugated to horseradish peroxidase and an enhancedchemiluminescence kit (Amersham, Arlington Heights, IL). Specific bandswere quantitated using the Eagle Eye II System (Stratagene, La Jolla, CA).
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Semiquantitative RT-PCR. A gene-specific primer and 1 µg of poly(A) RNA isolatedfrom rat renal cortex and jejunum and were used with a SuperScript RT-PCR kit (Life Technologies) to synthesize first-strand cDNA asdescribed by the manufacturer. To amplify the open reading frame (ORF)of rat SMVT, two gene-specific primers (5'-GAGGATGACTGTGGCGAGCAC-3' and5'-CAGCTCACCAACAGTATGGC-3'), corresponding to the sequence in the ORFof rat SMVT, were used in a PCR, yielding a 180-bp product. The PCR wasperformed within the linear range of amplification. The conditions forPCR were denaturation at 95°C for 30 s, annealing at 58°C for30 s, and extension at 72°C for 2 min (20 and 30 cycles forkidney and jejunum, respectively). To amplify the variant II of SMVTmRNA, two gene-specific primers (5'-CAGGGCAAAATCGGAGTT-3' and5'-GTATAGGAGGGAGCCGCA-3'), spanning the sequence in the variant IIof rat SMVT mRNA, were used in a PCR, yielding a 463-bp product. Theconditions for PCR were denaturation at 95°C for 1 min, annealing at48°C for 1.5 min, and extension at 76°C for 1.5 min (35 cycles). The products were analyzed on 2% agarose gels, the images were captured using the Eagle Eye II system (Stratagene), and the amplified RT-PCR products were normalized to amplified -actin controls.

Nuclear run-on assay. Nuclei were isolated from jejunum of 10 suckling and 3 adult rats usinga modification of the method of Traber et al. ( 30 ). Themucosa was suspended in 10 vol of homogenization buffer [(in mM) 250 sucrose, 10 MgCl 2, 2 DTT, 1 PMSF, and 10 HEPES, pH 8.0, aswell as 2.5% Nonidet P-40], allowed to stand on ice for 1 h, andthen homogenized using a Potter-Elvehjem homogenizer at 700 rpm for 10 strokes (adult rats) and 5 strokes (suckling rats). The suspension wasfiltered through two layers of gauze and centrifuged at 700 g for 5 min, and the pellet was resuspended in 2.5 ml of homogenization buffer. To purify the nuclei, the suspended pellet waslayered on the top of a sucrose cushion (2.5 M sucrose, 1 mMMgCl 2, 1 mM HEPES, pH 6.8) and centrifuged at 50,000 g for 80 min at 4°C. The nuclear pellet was suspended in astorage buffer [(in mM) 5 MgCl 2, 2 DTT, 0.1 EDTA, and 20 HEPES, pH 8.0, as well as 40% glycerol] and stored at 80°C until use.

Nuclear transcription reactions were carried out using equal amounts ofnuclei from suckling and adult rats as described by Traber et al.( 30 ). RNA transcripts were isolated by sequential treatment with deoxyribonuclease and proteinase K, followed by phenol-chloroform extraction, ethanol precipitation, and purification on a Sephadex G-50 spin column (Boehringer Mannheim,Indianapolis, IN). Denatured cDNAs were immobilized on nylonfilters and hybridized with the nascent RNA transcripts for 72 hat 42°C with gentle mixing. cDNAs tested for hybridization were thefull-length ORF of rat SMVT, cloned in pcDNA3.1 (250 ng), and rat -actin (positive control) (50 ng). After stringent washings,membranes were exposed to film for 3-5 days at 80°C withintensifying screens. The intensity of the hybridization signal wasquantitated using the Eagle Eye II System (Stratagene). Data werenormalized relative to rat -actin.

Statistical analysis. Transport studies were the results of multiple separate determinationsusing different BBMV preparations isolated from various rats ondifferent occasions. Data are as means ± SE expressed aspicomoles per milligram protein per unit time. Data were analyzed usingStudent's t -test. Kinetic parameters of the saturablecomponent of the renal and intestinal biotin uptake processes [i.e.,maximal velocity ( V max ) and apparent Michaelisconstant ( K m )] were determined using acomputerized model of the Michaelis-Menten equation as described byWilkinson ( 31 ). RT-PCR, Western blotting, and nuclear run-on assays were performed on at least three separate occasions usingsamples from different rats; representative data are presented in this report.9 ^$ _+ T+ b3 w7 |* `! V
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RESULTS
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The effect of ontogeny on carrier-mediated biotin transport acrossthe BBM of rat renal cortex and jejunum was examined using purifiedBBMV preparations isolated from suckling and adult rats. Biotintransport was measured by determining the initial rate of[ 3 H]biotin uptake as a function of concentration. Kineticparameters of the biotin carrier-mediated uptake process were thencalculated as described in MATERIALS AND METHODS. Theresults (Fig. 1, A and B ) show a significantly( P V max in adultcompared with suckling rat jejunum (4.7 ± 0.33 and 1.9 ± 0.10 pmol · mg protein 1 · 10 s 1,respectively). A change in the apparent K m wasalso observed, with values being significantly ( P with suckling rat jejunum (11 ± 1.6 and 5.5 ± 0.84 µM, respectively). In contrast, the resultswith renal BBMV (Fig. 1, C and D ) showed similar V max values for the biotin uptake process insuckling and adult rats (2.2 ± 0.12 and 2.1 ± 0.08 pmol · mgprotein 1 · 10 s 1,respectively). In addition, the apparent K m wasfound to be similar in suckling and adult rat renal BBMV (30 ± 3.8 and 27 ± 2.5 µM, respectively).
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Fig. 1. Transport of biotin in jejunal and renal brush-border membranevesicles (BBMV) from suckling and adult rats as a function ofconcentration. Uptake studies were performed using jejunal [suckling( A ); adult ( B )] and renal [suckling( C ); adult ( D )] BBMV as described in MATERIALS AND METHODS in the presence of the specificconcentrations of unlabeled biotin shown. Each data point representsthe mean ± SE of 3-6 separate uptake determinations fromdifferent BBMV preparations isolated from 6-18 rats.' X9 R7 J/ L) U7 [) C

In another study, we used Western blot analysis to investigate thelevel of expression of the biotin transporter SMVT in purified jejunaland renal BBMV preparations isolated from suckling and adults rats (see MATERIALS AND METHODS ). Specific polyclonal anti-SMVT antibodies were used in the study. The results (Fig. 2 ) showed the polyclonal antibodies torecognize two major protein bands (~120 and ~140 kDa) in jejunalBBM of adult rats, whereas only one band (~120 kDa) was found injejunal BBM of suckling rats. Immunodetection of both proteins wasblocked by pretreatment of the antibody with the synthetic antigenicpeptide. With densitometry it was determined that the intensity of thesignal of the 120-kDa band increased with maturation (51.5 ± 4.9 and 67.4 ± 3.7 for suckling and adult rats, respectively). Theintensity of the signal of the 140-kDa band of adult rat jejunal BBMwas estimated at 22.6 ± 1.4. With regard to renal BBM, theresults of Western blot analysis (Fig. 3 )showed that the anti-SMVT antibodies recognize two protein bands withapparent molecular masses of ~105 and 69 kDa (the latter band has thepredicted molecular mass of rat SMVT) ( 16 ) in both agegroups. No protein bands were detected when the antibody waspreincubated with the antigenic peptide. Direct estimation of theintensity of these two specific bands showed similar levels of SMVTprotein.% w! z+ ?/ a! S1 y! v' B' y5 g, K) p2 r

Fig. 2. Western blot analysis of jejunal BBM proteins fromsuckling and adult rats. BBMV proteins (150 µg) were resolved on7.5% SDS-PAGE and electroblotted on Hybond enhanced chemiluminescence(ECL) nitrocellulose membrane. Blots were incubated with either rabbitpolyclonal anti-peptide sodium-dependent multivitamin transporter(SMVT) antibodies (Ab; left ) or anti-SMVT antibodiespretreated with the antigenic peptide ( right ).Immunodetection was performed as described in MATERIALS AND METHODS.. w( |9 p) I! L) Z

Fig. 3. Western blot analysis of renal BBM proteins from sucklingand adult rats. BBMV proteins (150 µg) were resolved on 7.5%SDS-PAGE and electroblotted on Hybond ECL nitrocellulose membrane.Blots were incubated with either rabbit polyclonal anti-peptide SMVTantibodies ( left ) or anti-SMVT antibodies pretreated withthe antigenic peptide ( right ). Immunodetection was performedas described in MATERIALS AND METHODS.5 V9 Y5 ~& N: B5 G' X
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We also determined the level of mRNA expression of SMVT in the jejunumand kidney cortex of suckling and adult rats. We used semiquantitativeRT-PCR and gene-specific primers within the ORF of SMVT in the study(see MATERIALS AND METHODS ). Data were normalized relativeto -actin. The results showed a 2.61 ± 0.15-fold higher SMVTmRNA level in the jejunum of adult compared with suckling rats (Fig. 4 A ). In contrast, the level ofSMVT mRNA expression was found to be the same in renal cortex of ratsin the two age groups (Fig. 4 B ).

Fig. 4. Semiquantitative RT-PCR analysis of mRNA from jejunum andkidney of suckling and adult rats. RT-PCR products obtained from thejejunum ( A ) and kidney ( B ) of suckling and adultrats were analyzed on a 2% agarose gel. Primers and PCR conditionsused are described in MATERIALS AND METHODS. ORF, openreading frame.7 U8 w2 L4 ]. J8 K* [3 ~
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Previous studies in our laboratory have shown the existence of fourvariants (I, II, III, and IV) for rat SMVT, which have significantheterogeneity at the 5'-untranslated region ( 8 ). We havealso shown variant II to be the predominant form expressed in adult ratsmall intestine ( 8 ). In this study, we examined the effectof developmental maturation on the level of variant II expression inthe small intestine using semiquantitative RT-PCR and variantII-specific primers. The results (Fig. 5 A ) show a 1.92 ± 0.13-fold higher level of variant II expression in adult compared withsuckling rat jejunum. To examine the effect of ontogeny on level ofexpression of the predominant variant in rat kidney cortex, we firstdetermined the identity of that variant and then examined its level insuckling and adult rat kidney cortex. The results (Fig. 5 B )showed variant II to be the only SMVT variant expressed in rat kidneycortex. In addition, kidney cortexes of suckling and adult rats werefound to express similar levels of this variant.9 @2 W" F* ?1 b3 M
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Fig. 5. Expression of 5'-untranslated region SMVT variant II injejunum and kidney of suckling and adult rats. RT-PCR products obtainedusing primers specific for the SMVT variant II and RNA from the jejunum( A ) and kidney ( B ) of suckling and adult rats(see MATERIALS AND METHODS ) were analyzed on a 2% agarosegel., V$ ~4 W0 C: r( f& P* N- @, q* b

The results described above suggest possible involvement oftranscriptional mechanism(s) in ontogenic regulation of the intestinal biotin uptake process. To further examine this possibility, we performed a nuclear run-on assay using nuclei isolated from suckling and adult rat jejunum and the full-length ORF of rat SMVT in the study.The results (Fig. 6 ) showed a 1.86 ± 0.07-fold higher nascent transcription rate for SMVT in adultcompared with suckling rats.

Fig. 6. Nuclear run-on assay of SMVT transcriptional rate injejunum of suckling and adult rats. Nuclear run-on assays wereperformed on equal amounts of nuclei isolated from the jejunum ofsuckling and adult rats (see MATERIALS AND METHODS ). Blotswere exposed to film for 3-5 days at 80°C with intensifyingscreens. SMVT, full-length ORF for rat SMVT cloned in pcDNA3.1; -actin, rat -actin cDNA.
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The purpose of the present study was to examine and compareontogenic aspects of the biotin uptake processes in renal and intestinal epithelia and to determine the mechanism involved in anyobserved changes. Purified renal and intestinal BBMV isolated fromsuckling and adult rats were used in the studies. The results showedthat while the renal biotin uptake process is not ontogenically regulated, clear evidence was obtained to indicate that the intestinal biotin uptake process is under such ontogenic regulation.

The results of biotin uptake studies performed on renal cortex BBMVshowed a similar V max and apparent K m for the vitamin uptake process in sucklingand adult rats. In contrast, a significant increase in the V max and apparent K m ofthe vitamin uptake process was observed in jejunal BBMV of adultcompared with suckling rats. The latter observations suggest thatdevelopmental maturation is associated with an increase in the number(and/or activity) of the intestinal biotin uptake carriers and adecrease in their affinity, respectively. The physiological importanceof such changes could be related to the need for efficient biotinabsorption in suckling compared with adult rats. The findings describedabove also demonstrate that the previously reported developmentalchanges in the transepithelial transport of biotin in intact ratintestinal tissue preparations ( 21 ) involve the entry stepof the vitamin at the BBM level of the polarized enterocytes.

The lack of changes in biotin uptake kinetics with ontogeny in the ratrenal cortex was associated with a lack of change in the level of SMVTprotein in suckling and adult rats. It is interesting to mention here,however, that two specific protein bands were observed on a Westernblot of renal cortical BBM. One band appeared at ~69 kDa, which isthe estimated molecular mass of the SMVT protein calculated from itspredicted amino acid sequence ( 16 ), whereas the otherappeared at 105 kDa. The latter band suggests that some of the SMVTprotein in renal BBM undergoes posttranslational modification (e.g.,glycosylation). As to the changes observed in rat intestinal biotinuptake kinetics with maturation, these changes were associated with anincrease in the level of the SMVT protein from suckling to adult ratsas shown by Western blot analysis. In addition, ontogeny was associatedwith the appearance on Western blots of an additional band for SMVT inadult rats of ~140 kDa in addition to the 120-kDa band that alsoexisted in suckling rat jejunal BBM. Because the predicted molecularmass of the SMVT protein is 69 kDa, the above findings suggest that theSMVT protein also undergoes posttranslational modifications in the ratintestine. It may also be possible that the 140-kDa band represents adimer of SMVT. Further studies are required to determine the nature ofthe posttranslational modifications (and dimerization) of SMVT in renaland intestinal tissues. We should mention here that the observedchanges in molecular mass of SMVT with maturation are not unique to thebiotin transporter but have been observed with other carriers (e.g.,the intestinal type IIb Na-P i cotransporter) ( 2 ).

The pattern of expression of the SMVT ORF was found to parallel theobserved changes in biotin uptake and SMVT protein levels in renal andintestinal BBM. Similar SMVT mRNA levels were found in suckling andadult rat renal cortex, whereas the level was 2.61 ± 0.15-foldhigher in the jejunum of adult compared with suckling rats. In previousstudies from our laboratory, we have shown that rat SMVT exists in theform of four different variants (I, II, III, and IV) ( 8 ).These variants arise from significant heterogeneity in the5'-untranslated region of SMVT ( 8 ). In addition, variantII was found to be the predominant variant expressed in the smallintestine of adult rats ( 8 ). In the present study, we alsofound variant II to be the predominant form expressed in the renalcortex of adult rats. In addition, the variant II level was found toparallel the level of the SMVT ORF in suckling and adult rats renal andjejunal tissues. Thus while similar levels of variant II expressionwere found in suckling and adult rat renal cortex, a 1.92 ± 0.13-fold higher level of this variant was found in the jejunum ofadult compared with suckling rats.

The above-described changes in the intestinal biotin uptake process andSMVT mRNA levels with maturation suggest the involvement of regulationby transcriptional mechanism(s). To further test this possibility, weperformed a nuclear run-on assay to determine the transcription rate ofSMVT in suckling and adult rat jejunum. The results showed a highertranscription rate for SMVT in adult compared with suckling rats,clearly supporting the suggestion stated above.' @$ o) X2 d  X0 @5 y4 j+ M/ |

The findings that the intestinal, but not the renal, biotin uptakeprocess is subjected to ontogenic changes suggest that the twoprocesses are under different regulation during maturation. Thereason(s) behind this difference between intestinal and renal biotinuptake processes in response to ontogenic regulation is unclear.However, it could reflect the physiological importance of renal biotinreabsorption in both suckling and adult animals. It is interesting tomention here that the tissue-specific ontogenic regulation observed inthis study for biotin uptake in renal and intestinal epithelia is notunique to biotin but has also been reported for transport of othersubstrates (e.g., transport of bile acid in renal and intestinaltissues) ( 9 ). In summary, the results of this study showthat biotin uptake by intestinal and renal epithelial cells respondsdifferently to ontogenic regulation. In addition, the results suggestthat the changes in intestinal biotin uptake with maturation appear toinvolve regulation by transcriptional mechanism(s).7 J2 _# m/ ~" L% S& s
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ACKNOWLEDGEMENTS
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This study was supported by grants from the Department of VeteransAffairs and the National Institute of Diabetes and Digestive and KidneyDiseases (DK-56061 and DK-58057).& |/ R( Z1 W& i/ W& w* n! ]7 ^# `
      【参考文献】
1. Al-Mahroos, F,Abumrad N,Said HM,andGhishan FK. Maturational changes in glutamine transport by rat jejunal brush border membrane vesicles. Pediatr Res 27:516-524,1990.$ n  o, s$ p$ n4 I- q
9 X3 u+ {4 ~% S, a/ ^1 g. L
" Q  \' v. Y3 k; C. Y; d

2. Arima, K,Hines ER,Kiela PR,Drees JB,Collins JF,andGhishan FK. Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-P i contransporter during ontogeny. Am J Physiol Gastrointest Liver Physiol 283:G426-G434,2002 .0 S: ?$ m0 f( l1 x

% v5 P5 F) D- I; N# L

3. Baur, B,andBaumgarther ER. Na -dependent biotin transport into brush-border membrane vesicles from human kidney cortex. Pflügers Arch 422:499-505,1993  .

4. Baur, B,Wick H,andBaumgarther ER. Na -dependent biotin transport into brush-border membrane vesicles from rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 258:F840-F847,1990 .$ `' x& s5 z1 o" a, q5 z
* r/ y, T/ M5 Y! i4 p4 b! D
0 `' q  L5 B( ~' x7 n6 ^
% p  s8 C2 R7 F9 i3 x1 S4 _
5. Bonjour, JP. Biotin.In: Handbook of Vitamins; Nutritional Biochemical and Clinical Aspects, edited by Machlin LJ.. New York: Dekker, 1984, p. 403-435., u% T7 M! N( n7 f+ M# s' x: E

1 r: Z! k( o& Y  [. s
6. Borowitz, SM,andGhishan FK. Maturation of jejunal phosphate transport by rat brush border membrane vesicles. Pediatr Res 19:1308-1312,1985  .0 g- K0 B* e% h% x- D5 y0 q6 @

  K. T6 O/ `8 V( ]

7. Brown, B,Selhumb J,andRosenberg IH. Intestinal absorption of biotin in the rat. J Nutr 116:1266-1271,1986 .+ L. i% z1 R/ j  G5 }3 l

" T7 P2 w+ a& ^# O# c8 J* Z
* H5 u1 A9 |+ B% r0 J" [
8. Chatterjee, NS,Kumar CK,Ortiz A,Rubin SA,andSaid HM. Molecular mechanism of the intestinal biotin transport process. Am J Physiol Cell Physiol 277:C605-C613,1999 ., u9 \0 D2 O3 A1 ~
  d( Q/ @% J" P5 f7 |

9. Christie, DM,Dawson PA,Thevananther S,andShneider BL. Comparative analysis of the ontogeny of a sodium-dependent bile acid transporter in rat kidney and ileum. Am J Physiol Gastrointest Liver Physiol 271:G377-G385,1996 .0 B7 A, ?) K& Z" c' x9 e

5 R- [: Y' w$ \- w* @% }# D2 @
10. Dakshinamurti, K,andChauhan J. Regulation of biotin enzymes. Annu Rev Nutr 8:211-233,1988  .
5 x; {' G& h) X' g0 [% L
3 t, x4 x6 s# g3 D$ N3 z

11. Guner, YS,Kiela PR,Xu H,Collins JF,andGhishan FK. Differential regulation of renal sodium-phosphate transporter by glucocorticoids during rat ontogeny. Am J Physiol Cell Physiol 277:C884-C890,1999 .; U( K6 W! _" @* P7 G% o

' Y2 t% }2 s( N: _& J* ?2 o

12. Hopfer, U,Nelson K,Prerotto J,andIsselbacher KJ. Glucose transport in isolated brush border membrane from rat intestine. J Biol Chem 248:25-32,1973 .

$ |4 n/ ^0 K2 W; _/ i( I9 G% R) I& @

13. Krause, KH,Berlit P,andBonjour JP. Impaired biotin status in anticonvulsant therapy. Ann Neurol 12:485-486,1982  .

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14. Podevin, RA,andBarbarat B. Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. Biochim Biophys Acta 856:471-481,1986 .0 }1 @# {/ u" \2 ^) c9 n% C% R

$ z8 D& d$ U; a# o$ L. N

15. Prasad, PD,Wang H,Huang W,Fei YJ,Leibach FH,Devoe LD,andGanapathy V. Molecular and functional characterization of the intestinal Na -dependent multivitamin transporter. Arch Biochem Biophys 366:95-106,1999  .
$ h% @% ^- F- N" v' j9 u

16. Prasad, PD,Wang H,Kekuda R,Fujita T,Feis YJ,Devoe LD,Leibach FH,andGanapathy V. Cloning and functional expression of a cDNA encoding a mammalian-sodium dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate. J Biol Chem 273:7501-7506,1998 .
. I4 [' D# R* ~9 U  h* A
4 o6 w# ?; T/ V# x* M& A8 L
3 A' k- u% ?# d
17. Said, HM. Cellular uptake of biotin: mechanisms and regulation. J Nutr 129:490S-493S,1999  .! Y6 p' J' J, ^, f* V3 K
  t+ m4 `$ J$ ^0 L7 ~

18. Said, HM,Chatterjee NC,Riaz H,Subramanian VS,Ortiz A,Matherly LH,Sirotnak FM,Halsted C,andRubin SA. Adaptive regulation of intestinal folate uptake: effect of dietary folate deficiency. Am J Physiol Cell Physiol 279:C1889-C1895,2000 .

' m/ K- D4 u& ~  b
19. Said, HM,Ghishan FK,andMurrell JE. Ontogenesis of intestinal transport of 5-methyltetrahydrofolate in the rat. Am J Physiol Gastrointest Liver Physiol 249:G567-G571,1985 .
& s3 C3 c+ w7 A0 |9 s5 ^0 x

: @, z% I6 y3 {: s
20. Said, HM,Ghishan FK,andRedha R. Transport of glycyl- L -proline in intestinal brush border membrane vesicles of the suckling rat: characteristics and maturation. Biochim Biophys Acta 941:232-240,1988 .7 g4 v! n1 c3 n" W& O' I
; {+ m; y3 v  b9 e
; w- ~7 Q/ P& j+ T7 q

21. Said, HM,andMohammedkhani R. Ontogenesis of the intestinal transport of biotin in the rat. Gastroenterology 94:68-72,1988  .
/ B3 \0 L2 U: i2 p+ l. C
2 _: `, b) r- _5 f
1 H  M) Z  [# k% a- p8 |
22. Said, HM,Ortiz A,McCloud E,Dyer D,Moyer MP,andRubin S. Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process shared with pantothenic acid. Am J Physiol Cell Physiol 275:C1365-C1371,1998 .1 o# N; l1 `- m& |6 @
' w) H2 V9 K' N: J% N/ |
- \# s2 C. [! M$ _) w

23. Said, HM,andRedha R. A carrier-mediated system for transport of biotin in rat intestine in vitro. Am J Physiol Gastrointest Liver Physiol 252:G52-G55,1987 .5 Q: z! d& y5 g! A8 g% p
3 c4 X; B2 c4 J/ E
$ ^; u; Q) V7 T& u

24. Said, HM,andRedha R. Biotin transport in brush border membrane vesicles of rat small intestine. Biochim Biophys Acta 945:195-201,1988 .

; l& M- `* x% }

25. Said, HM,Redha R,andNylander W. A carrier-mediated, Na gradient-dependent transport system for biotin in human intestinal brush border membrane vesicles. Am J Physiol Gastrointest Liver Physiol 253:G631-G636,1987 .5 z, w2 t6 m# q9 k9 o5 ?) l
6 s" p0 N. n9 C. I5 K

$ _- p3 m7 e& g4 J, h0 g
26. Said, HM,Redha R,andNylander W. Biotin transport in basolateral membrane vesicles of human intestine. Gastroenterology 94:1157-1163,1988  .

" Y/ F/ f9 p. ^, m

27. Said, HM,Redha R,andNylander W. Biotin transport in human intestine: site of maximum transport and effect of pH. Gastroenterology 95:1312-1317,1988  .9 j6 J# z" F' H' e

$ g' `5 g5 h" G0 V. b

28. Sweet, DH,Bush KT,andNigam SK. The organic anion transporter family: from physiology to ontogeny and the clinic. Am J Physiol Renal Physiol 281:F197-F205,2001 .  h8 A' ?& w, m0 [( k
4 A. u5 e, q8 h0 U; k0 T

: _: r8 M- z/ H+ {
29. Sweetman, L,andNyhan WL. Inheritable biotin-treatable disorders and associated phenomena. Annu Rev Nutr 6:314-343,1986.

8 U' Q2 ~9 H% q+ U
30. Traber, PG,Wang W,McDonnel M,andGumucio JJ. P450IIB expression in rat small intestine: cloning of intestinal P450IIB1 mRNA using the polymerase chain reaction and transcriptional regulation of induction. Mol Pharmacol 37:810-819,1990 ." c- o3 w! S8 g. O$ n+ H
" T+ Y  R* I0 a3 K0 h
8 g. Y/ e3 K3 e! R% z
: ~7 [/ G$ u2 k3 x  j2 k7 R
31. Wilkinson, GN. Statistical estimation in enzyme kinetics. Biochem J 80:324-332,1961  .: @3 ?0 G4 t/ X" Z/ {! n0 y+ h
' u/ Z. \% G! R) h/ _, S2 E
: d4 u* B( h; c
/ \& h0 ]+ G5 t: j6 ~1 R( C  b
32. Wolf, B,Heard GS,Jefferson LG,Proud VK,Nance WI,andWeissbecker KA. Clinical findings in four children with biotinidase deficiency detected through a state-wide neonatal screening program. N Engl J Med 313:16-19,1985 .
/ ^4 L( i+ @0 a, p
' s3 t: D0 s; L6 [+ \7 J
" Y0 M; U' F* |1 a
33. Yanagawa, N,Jo OD,andSaid HM. Riboflavin transport by rabbit renal brush border membrane vesicles. Biochim Biophys Acta 1330:172-178,1997 .* M3 r2 e$ V. W3 h2 F( z4 y

8 v' J1 ?' G% _# P% G
34. Zelikovic, I,andChesney RW. Development of renal amino acid transport systems. Semin Nephrol 9:49-55,1989  ." f! p4 Y" n4 ^* V! K5 {- b

2 k0 P( k2 s( o4 }" t! V
" N, M6 ]1 N! b2 X2 i; D2 O
35. Zelikovic, I,Stejskal E,Lohstroh P,Budreau A,andChesney RW. Developmental maturation of Na -H exchange in rat renal tubular brush-border membrane. Am J Physiol Renal Fluid Electrolyte Physiol 261:F1017-F1025,1991 ." I' w! C  W7 {% A$ G2 [* y9 N/ a( n
* G* w( ~7 a' W4 T- Z0 d, b

% `. u7 j( ]% ^% p, q, X! G
36. Zempleni, J,andMock DM. Marginal biotin deficiency is teratogenic. Proc Soc Expt Biol Med 223:14-21,2000 .

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