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• W2104030002 abstract "Article15 September 1999free access Cellular retinol-binding protein I is essential for vitamin A homeostasis Norbert B. Ghyselinck Norbert B. Ghyselinck Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Claes Båvik Claes Båvik Present address: Department of Human Ecology, University of Texas, Austin, TX, 78812 USA Search for more papers by this author Vincent Sapin Vincent Sapin Present address: INSERM U384 UFR de Médecine et de Pharmacie, BP 38, 63001 Clermont-Ferrand, Cedex, France Search for more papers by this author Manuel Mark Manuel Mark Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Dominique Bonnier Dominique Bonnier Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Colette Hindelang Colette Hindelang Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Andrée Dierich Andrée Dierich Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Charlotte B. Nilsson Charlotte B. Nilsson Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Helen Håkansson Helen Håkansson Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Patrick Sauvant Patrick Sauvant INRA, Centre de Recherches en Nutrition Humaine, Equipe Vitamines, Clermont-Ferrand, France Search for more papers by this author Véronique Azaïs-Braesco Véronique Azaïs-Braesco INRA, Centre de Recherches en Nutrition Humaine, Equipe Vitamines, Clermont-Ferrand, France Search for more papers by this author Maria Frasson Maria Frasson Laboratoire de Physiopathologie Rétinienne, INSERM/ULP, Hôpital Civil de Strasbourg, France Search for more papers by this author Serge Picaud Serge Picaud Laboratoire de Physiopathologie Rétinienne, INSERM/ULP, Hôpital Civil de Strasbourg, France Search for more papers by this author Pierre Chambon Corresponding Author Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Norbert B. Ghyselinck Norbert B. Ghyselinck Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Claes Båvik Claes Båvik Present address: Department of Human Ecology, University of Texas, Austin, TX, 78812 USA Search for more papers by this author Vincent Sapin Vincent Sapin Present address: INSERM U384 UFR de Médecine et de Pharmacie, BP 38, 63001 Clermont-Ferrand, Cedex, France Search for more papers by this author Manuel Mark Manuel Mark Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Dominique Bonnier Dominique Bonnier Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Colette Hindelang Colette Hindelang Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Andrée Dierich Andrée Dierich Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Charlotte B. Nilsson Charlotte B. Nilsson Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Helen Håkansson Helen Håkansson Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Patrick Sauvant Patrick Sauvant INRA, Centre de Recherches en Nutrition Humaine, Equipe Vitamines, Clermont-Ferrand, France Search for more papers by this author Véronique Azaïs-Braesco Véronique Azaïs-Braesco INRA, Centre de Recherches en Nutrition Humaine, Equipe Vitamines, Clermont-Ferrand, France Search for more papers by this author Maria Frasson Maria Frasson Laboratoire de Physiopathologie Rétinienne, INSERM/ULP, Hôpital Civil de Strasbourg, France Search for more papers by this author Serge Picaud Serge Picaud Laboratoire de Physiopathologie Rétinienne, INSERM/ULP, Hôpital Civil de Strasbourg, France Search for more papers by this author Pierre Chambon Corresponding Author Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France Search for more papers by this author Author Information Norbert B. Ghyselinck1, Claes Båvik2, Vincent Sapin3, Manuel Mark1, Dominique Bonnier1, Colette Hindelang1, Andrée Dierich1, Charlotte B. Nilsson4, Helen Håkansson4, Patrick Sauvant5, Véronique Azaïs-Braesco5, Maria Frasson6, Serge Picaud6 and Pierre Chambon 1 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, 67404 Illkirch, Cedex, France 2Present address: Department of Human Ecology, University of Texas, Austin, TX, 78812 USA 3Present address: INSERM U384 UFR de Médecine et de Pharmacie, BP 38, 63001 Clermont-Ferrand, Cedex, France 4Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 5INRA, Centre de Recherches en Nutrition Humaine, Equipe Vitamines, Clermont-Ferrand, France 6Laboratoire de Physiopathologie Rétinienne, INSERM/ULP, Hôpital Civil de Strasbourg, France ‡C.Båvik and V.Sapin contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4903-4914https://doi.org/10.1093/emboj/18.18.4903 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The gene encoding cellular retinol (ROL, vitA)-binding protein type I (CRBPI) has been inactivated. Mutant mice fed a vitA-enriched diet are healthy and fertile. They do not present any of the congenital abnormalities related to retinoic acid (RA) deficiency, indicating that CRBPI is not indispensable for RA synthesis. However, CRBPI deficiency results in an ∼50% reduction of retinyl ester (RE) accumulation in hepatic stellate cells. This reduction is due to a decreased synthesis and a 6-fold faster turnover, which are not related to changes in the levels of RE metabolizing enzymes, but probably reflect an impaired delivery of ROL to lecithin:retinol acyltransferase. CRBPI-null mice fed a vitA-deficient diet for 5 months fully exhaust their RE stores. Thus, CRBPI is indispensable for efficient RE synthesis and storage, and its absence results in a waste of ROL that is asymptomatic in vitA-sufficient animals, but leads to a severe syndrome of vitA deficiency in animals fed a vitA-deficient diet. Introduction Retinol (ROL), or vitamin A (vitA), is indispensable for embryonic development, growth, vision and survival of vertebrates (Blomhoff et al., 1991). With the exception of vision, retinoic acid (RA) has been shown to be the active vitA derivative, whose pleiotropic effects are transduced by two families of nuclear receptors, the RARs and the RXRs (Chambon, 1996). In the cytoplasm, ROL and RA are bound to cellular retinol-binding proteins (CRBP type I and II) and to cellular retinoic acid-binding proteins (CRABP type I and II), that are highly conserved in mammals (Ong, 1994) and belong to a family of cytosolic proteins binding small hydrophobic ligands (Newcomer, 1995). During development, CRBPI is specifically expressed in several tissues including motor neurons, spinal cord, lung, liver (Dollé et al., 1990; Maden et al., 1990; Ruberte et al., 1991; Gustafson et al., 1993) and placenta (Johansson et al., 1997; Sapin et al., 1997). In adults, CRBPI is highly expressed in liver, kidney, lung (Eriksson et al., 1984), brain (Zetterstrüm et al., 1994), retinal pigment epithelium (de Leeuw et al., 1990) and genital tract (Kato et al., 1985; Porter et al., 1985; Wardlaw et al., 1997). In contrast, CRBPII expression is restricted to the yolk sac between embryonic day post-coitum (E)10.5 and E15.5 (Sapin et al., 1997; our unpublished data), the liver at the end of gestation and the small intestine throughout life (Levin et al., 1987; Schaefer et al., 1989). Numerous studies in vitro using purified proteins and/or cell extracts have suggested that CRBPI and CRBPII could play important roles in ROL metabolism, being involved in esterification of ROL with long-chain fatty acids (Ong et al., 1988; Yost et al., 1988; Herr and Ong, 1992), oxidation of ROL to retinaldehyde (RAL; Ottonello et al., 1993; Boerman and Napoli, 1996) and hydrolysis of retinyl esters (RE) into ROL (Boerman and Napoli, 1991). However, the physiological functions of CRBPs are unclear (Troen et al., 1996). To investigate the actual role of CRBPI during mammalian development and post-natal life, we have knocked out the CRBPI gene. Mutant mice are healthy and fertile when fed a vitA-enriched diet. However, CRBPI deficiency results in a marked reduction of liver retinyl palmitate (RP, the main ester of vitA) levels, due to a decreased synthesis from ROL and a faster turnover rate. Accordingly, CRBPI-null mice reared on a vitA-deficient (VAD) diet fully exhaust their RE stores within 5 months, and develop abnormalities characteristic of post-natal hypovitaminosis A (HVA). Results Disruption of the CRBPI gene Mapping and sequencing of genomic clones revealed that the mouse CRBPI gene organization is very similar to that of its human homologue (data not shown; Nilsson et al., 1988). The structure of the targeting vector in which a neomycin (NEO) cassette was inserted into exon E2 is depicted in Figure 1A. One out of 157 G418-resistant clones (QK10) was shown to exhibit the Southern blot pattern expected for a single homologous recombination event (Figure 1B). It was injected into C57BL/6 blastocysts to create chimeric mice, of which two males transmitted the mutation to their offspring (see Figure 1C). Figure 1.Disruption of the CRBPI gene. (A) Schematic representation of the mouse CRBPI locus. Exons are represented as solid boxes and the promoter as a broken arrow. Structures of the targeting vector and recombinant allele are shown. Primers for PCR genotyping are indicated by arrowheads. Genomic fragments obtained after BamHI digest are indicated for WT and recombinant (HR) alleles. Restriction sites: B, BamHI; C, ClaI; E, EcoRI; X, XbaI; Xh, XhoI. (B) Genomic DNA from D3 and targeted ES cells (QK10) were analyzed with 5′ external and neomycin probes (A). Size is indicated in kb. (C) Southern blot (BamHI digest, upper panel) and PCR analyses (lower panel) of DNA from offspring of a CRBPI+/− intercross. (D) Western blot analysis of 40 μg of cytosolic extracts from E13.5 WT (+/+), heterozygous (+/−;) and homozygous (−/−) fetuses using a CRBPI-specific antiserum. Note the absence of CRBPI in mutant embryos. After stripping, the blot was reprobed with CRABPI and CRABPII antisera. Download figure Download PowerPoint To check that CRBPI gene disruption was efficient, mRNA (data not shown) and protein expression were analyzed. Antibodies directed against CRBPI detected the protein (∼16 kDa) in extracts from E13.5 wild-type (WT) and heterozygous fetuses, but not in homozygous mutants (Figure 1D), whereas CRABPI and CRABPII expression were not modified. We conclude that the present CRBPI gene disruption is a null mutation. CRBPI-null mice appear essentially normal Heterozygous matings (n = 35) yielded 24.8% (n = 72) of WT, 50% (n = 145) of heterozygous and 25.2% (n = 73) of homozygous mice, which corresponds to the Mendelian ratio. Male and female mutant mice fed a vitA-enriched diet grew normally, were fertile, healthy up to 20 months of age and indistinguishable from WT littermates. Serial sections of E14.5, E16.5 and E18.5 mutant fetuses (n = 3), placentas (n = 3) and adult eyes (n = 5) did not reveal any histological abnormality, and whole-mount skeletal analysis of CRBPI-null newborns (n = 20) did not reveal any malformation. Thus, despite the specific expression pattern of CRBPI in embryonic and adult tissues (see Introduction), mutant mice fed a vitA-enriched diet did not show any abnormality, either during development or after birth. As this lack of obvious defects might reflect a functional compensation of CRBPI by CRBPII, the expression of the latter was investigated. From E8.5 to E18.5, the expression pattern of CRBPII transcripts analyzed using in situ hybridization was identical in WT and CRBPI-null embryos, fetuses and placentas (data not shown; see Dollé et al., 1990; Ruberte et al., 1991; Sapin et al., 1997). At E18.5 and at birth, CRBPII mRNA was co-expressed with CRBPI in liver (Figure 2D), in which the CRBPII protein was confined to hepatocytes (Figure 2C), while the CRBPI protein was detected mainly in cells lining liver blood vessels (Figure 2A). The level of CRBPII mRNA was 2-fold higher in CRBPI-null liver than in WT liver (Figure 2D). The reason for this increase is unclear. It might be due to a higher local production of RA, as (i) CRBPII expression is known to be inducible by RA (Nakshatri and Chambon, 1994), and (ii) the expression level of the RA-inducible RARβ2 gene (Sucov et al., 1990) was also increased in the liver of newborn and 2-week-old CRBPI-null mice (Figure 2D). CRBPII mRNA was not detected in liver at any other fetal or post-natal stage (Figure 2D), nor in any other tissue known to express CRBPI (see Introduction; data not shown). Thus, with the possible exceptions of the yolk sac between E10.5 and E15.5 and of the liver during the neonatal period, it is unlikely that the apparent dispensability of CRBPI during development could correspond to a functional redundancy with CRBPII. Figure 2.CRBPII may compensate for the lack of CRBPI in the liver during the neonatal period. (A-C) Immunohistochemical localization of CRBPI and CRBPII in WT (+/+) and CRBPI-null (−/−) liver at 1 day of age. (A) CRBPI is expressed in sinusoid lining cells (arrowheads) and hepatocytes, albeit at very different levels. (B) Lack of immunostaining with anti-CRBPI antibody in the CRBPI-null liver. (C) CRBPII expression is restricted to hepatocytes. The immunostaining pattern is identical in WT liver (not illustrated). C, capillaries (liver sinusoids); H, hepatocytes; HE, hematopoietic cells; V, venules. Arrowheads point to endothelial cells. Magnifications ×400. (D) RNase protection assays using 50 μg of total RNA extracted from livers. A tRNA sample was used as background control. Note that signals obtained with the CRBPI probe in CRBPI-null samples corresponded to background. (E) Example of an HPLC analysis of retinoids extracted from adult mouse liver. Peaks 1-5 have retention times corresponding to ROL (1), RAL (2), retinyl acetate (3, internal standard), retinyl palmitate (RP) + retinyl oleate (4) and retinyl stearate (RS; 5), respectively. Note that RP and retinyl oleate have identical retention times, but the latter is a minor RE component in liver. RP and RS represented 90 and 10% of mouse liver RE, respectively. The RP/RS ratio was always the same in CRBPI-null and WT livers. (F) RP concentrations in livers of +/+ (white bars) and −/− (black bars) E16.5 and E18.5 fetuses, newborn (NB) and 2-week-old mice. An asterisk indicates a significant difference with WT values (P <0.05). NS, not statistically significant. Download figure Download PowerPoint Retinyl ester stores are decreased in liver of CRBPI-null mice We analyzed ROL homeostasis in liver, which expresses high levels of CRBPI and metabolizes and stores ROL. Endogenous retinoids were quantified by HPLC (Figure 2E). Oxidation of ROL did not appear to be modified in adult CRBPI-null mice, as their hepatic RAL concentration was not significantly different from that of WT mice (Table I). Retinyl palmitate (RP), which represented 90% of RE (Figure 2E), was detected in liver of E16.5 WT fetuses and its level increased in E18.5 fetuses and suckling newborns (Figure 2F). RP was also found in the liver of CRBPI-null fetuses (E16.5 and E18.5), indicating that mutants can take up and store vitA. However, RP levels were 3-fold lower than in WT fetuses (P <0.05; Figure 2F). In contrast, at post-natal day 1 and at 2 weeks of age, the content of RE in liver of suckling mutants was not different from that of their WT littermates. At these stages, similarly low levels of ROL were detected in liver of WT and CRBPI-null mutants (data not shown). From 4 weeks of age, the amount of liver RP (Figure 3A) and ROL (not shown) were ∼50% lower in CRBPI-null mice than in WT animals. After weaning, a slight decrease of RP accumulation was observed (Figure 3A, inset). This probably reflects the nutritional modification from maternal milk to diet pellets. Figure 3.Hepatic accumulation of retinyl palmitate and histology of stellate cells. (A) Mean RP concentrations in liver of WT (+/+, filled squares) and CRBPI-null (−/−, open circles) mice from 2 to 30 weeks of age. Each point represent an average of 8-20 determinations, and vertical bars indicate SEM. Asterisks indicate significant differences from +/+ values (P <0.01). The inset represents an enlargement of the early post-natal period. Note that RP and RS amounts were always 1.5-fold higher in females than in males (not shown). (B) Semi-thin sections of 8-week-old male livers stained with toluidine blue. Arrows indicate lipid droplets which are more abundant and bigger in WT than in CRBPI-null hepatic stellate cells (HSC). The bar represents 10 μm. (C) Electron microscopy showing that CRBPI-null HSC contained only one or a few small lipid droplets in their cytoplasm. The bar represents 1 μm. D, Disse's space; E, endothelium; L, lipid droplet; N, nucleus of the stellate cell; P, parenchymal cell; RC, red blood cell. Download figure Download PowerPoint Table 1. Accumulation of retinaldehyde (μg/g of tissue) in liver, kidney and lung of WT (+/+) and CRBPI-null (−/−) mice CRBPI genotype Tissue Age (weeks) +/+ −/− Liver 6 0.24 ± 0.05 0.19 ± 0.04 Lung 2 1.44 ± 0.15 1.08 ± 0.25 6 1.42 ± 0.21 1.07 ± 0.20a Kidney 2 0.98 ± 0.13 0.71 ± 0.07 6 0.46 ± 0.04 0.26 ± 0.02a Data (μg/g of tissue) shown represent mean ± SEM values for 10-15 samples per data point. a Significantly different from the WT value (P <0.05). In mammals, ROL is stored as large cytoplasmic lipid droplets composed of RE in hepatic stellate cells (HSC) located within the interstitial space between hepatocytes and endothelial cells (Wake, 1980; Blomhoff et al., 1991). HSC contain high levels of CRBPI and enzymes esterifying ROL (Blaner et al., 1985; Blomhoff et al., 1985). Light microscopy showed that HSC of CRBPI-null mice contained less abundant and smaller lipid droplets than their WT counterparts (Figure 3B). However, the CRBPI-null HSC were ultrastructurally normal (Figure 3C), and immunostaining for vimentin indicated that HSC were as numerous in mutant as in WT liver (data not shown). Retinol homeostasis in liver of adult CRBPI-null mice Retinoid homeostasis in liver results from a dynamic balance between storage (re-esterification of ROL originating from blood chylomicrons) and mobilization of stores (hydrolysis of RE in HSC). Esterification of ROL is catalyzed by two enzymes, lecithin:retinol acyltransferase (LRAT) and acyl-CoA:retinol acyltransferase (ARAT). Mobilization of ROL requires retinyl ester hydrolase (REH) activities classified into two classes, according to their dependency upon bile salts: (i) bile-salt-dependent neutral REH (nREH; Cooper and Olson, 1986; Harrison, 1993); and (ii) bile-salt-independent acidic activity (aREH; Mercier et al., 1994). LRAT, ARAT, nREH and aREH whole-liver activities were similar in WT and CRBPI-null mutants (Table II and data not shown). Table 2. Comparison of enzymatic activities in WT (+/+) and CRBPI-null (−/−) adult livers CRBPI genotype Enzyme activity (pmol/min/mg) +/+ −/− LRAT 23 ± 2 25 ± 2 ARAT 1.1 ± 0.1 1.2 ± 0.1 aREH 276 ± 20 255 ± 15 nREH 121 ± 6 105 ± 6 EROD 90 ± 2 114 ± 8a PROD 65 ± 3 71 ± 3a Each value (pmol/min/mg) is the average ± SEM of at least eight individual liver homogenates. Note that ARAT and LRAT activities were 1.5-fold higher in females than in males (not shown). a Significantly different from the WT value (P <0.05). We next analyzed vitA turnover. A single dose of tritiated ROL was given orally to WT and CRBPI-null mice. Passage of [3H]ROL through the gastrointestinal tract seemed normal in mutants, as the recovery of radioactivity in small intestine 6 h after dosing was similar in WT and CRBPI-null mice (data not shown). The amount of tritium present in liver after 6 h reflects the uptake of radiolabeled chylomicron remnants from blood, but also, and principally the esterification of [3H]ROL originating from them (Blomhoff et al., 1991). In WT liver, 10% of the radioactive dose was taken up after 6 h (Figure 4A), out of which 80% co-eluted with RP, while the remaining 20% co-eluted with ROL (HPLC data not shown). In contrast in CRBPI-null liver, only 5% of the dose was taken up after 6 h, of which 65 and 35% co-eluted with RP and ROL, respectively. Two days later, the [3H]ROL present in the liver no longer reflects uptake and esterification of ROL, but rather the turnover of RE stores (Blomhoff et al., 1991). Their estimated half-life (t1/2) was 60 days for WT and 10 days for CRBPI-null mice (Figure 4A), thus indicating a 6-fold shorter turnover time of RE in mutant liver. Figure 4.Turnover of [3H]retinol. WT (+/+) and CRBPI-null (−/−) mice are represented by filled squares and open circles, respectively. Each point represents an average of eight observations, and vertical bars indicate SEM. Asterisks indicate a significant difference from +/+ values (P <0.01). (A) The amount of tritium (d.p.m.) in whole liver is plotted on a logarithmic scale against time. Regression lines, calculated between days 2 and 20 after [3H]retinol administration, gave estimated tritium half-lifes (t1/2) of 60 days in +/+ and 10 days in −/−. (B) The amounts of tritium in blood and in whole kidney (inset) are plotted on a logarithmic scale against time. Download figure Download PowerPoint We also investigated whether ROL might be degraded faster in the liver of CRBPI-null mutants. Enzymes of the cytochrome P450 (CYP) system play an active role in the oxidative degradation of retinoids (reviewed in Duester, 1996). The mRNA level for CYP26 (Abu-Abed et al., 1998) was identical in liver of CRBPI-null and WT mice (data not shown). However, CYP1A- and CYP2B-mediated catabolism (estimated by analyzing EROD and PROD activities in whole-liver homogenates) were slightly, but significantly, increased in the liver of CRBPI-null mutants (Table II). We therefore conclude that in the liver of CRBPI-null mice (i) a lower amount of dietary ROL could be taken up, (ii) a lower proportion of newly incoming ROL is esterified as RP, (iii) RE stores have a faster turnover time, and (iv) the degradation of ROL may be slightly increased. Retinol homeostasis in lung and kidney of CRBPI-null mice Although 90% of total body vitA is stored in liver, lungs, which express high levels of CRBPI, also store vitA (Shenai and Chytil, 1990). At E16.5, the lung RP content was significantly lower (P <0.05) in CRBPI-null fetuses (14.3 ± 0.7 μg/g of tissue) than in WT (27.6 ± 1.4 μg/g of tissue), but the RP/RS ratio was unchanged (75% RP and 25% RS, see legend to Figure 2E). Only low levels of ROL were detected in fetal lungs, with a ∼35% decrease in CRBPI-null mutants when compared with WT (9.4 ± 0.9 μg/g of tissue in mutants versus 14.6 ± 1.4 μg/g of tissue in WT; P <0.05). At later stages, ROL and RP contents were similar in WT and CRBPI-null lungs (data not shown), whereas the RAL level was significantly (P <0.05) decreased by ∼25% in adult mutants (Table I). The kidney is also rich in CRBPI (Eriksson et al., 1984) and is known to play a role in ROL homeostasis (Peterson et al., 1973). At all stages after birth, ROL and RP contents were decreased by ∼30% in kidney of CRBPI-null mutants (data not shown). However, whole-kidney esterifying activities were similar (LRAT: 2.87 ± 0.41 versus 2.13 ± 0.53 pmol/min/mg; ARAT: 1.02 ± 0.83 versus 1.88 ± 0.82 pmol/min/mg) in WT and CRBPI-null mutant mice, respectively. The level of RAL was significantly (P <0.05) decreased by ∼25% in adult mutants compared with WT (Table I). Finally, both plasma (0.35 μg/ml) and urinary (0.1 μg/ml) ROL concentrations were normal. We also measured ROL turnover in blood and kidney (Figure 4B). In both cases, following a rapid decrease of [3H]ROL during the first 24 h, a more gradual decline was observed over a period of 20 days. The decay curves for CRBPI-null mutants were similar to those of WT. The observation that [3H]ROL was more abundant in blood and kidney of mutants might reflect a higher rate of depletion from liver stores (see above). It therefore appears that blood clearance and turnover of ROL in kidney are similar in WT and CRBPI-null mice. Dietary hypovitaminosis A results in a vitA deficiency syndrome in CRBPI-null mice As the turnover of ROL was faster in mutants, CRBPI deficiency may result in a depletion of ROL stores under conditions of dietary HVA. To investigate this possibility, CRBPI-null and WT mice initially weighing ∼16 g were reared from weaning under a VAD diet. Food consumption was not different between WT and mutants (data not shown), and growth was initially used to monitor their retinoid status. WT mice grew steadily during 23 weeks on VAD diet, while CRBPI-null mice grew at a slower rate from the 5th to the 12th week, and then stopped growing during the next 11 weeks (Figure 5A). Every 3 weeks, three to five mice were killed and their liver RP and serum ROL levels were determined. RP levels rapidly decreased in CRBPI-null mutants during the first 14 weeks to become undetectable (Figure 5B, open red circles). In contrast, WT mice still had important RP liver stores, even at 23 weeks (∼120 μg/g of liver; Figure 5B, filled red squares). RP half-life was estimated to be 14 days in CRBPI-null mutants versus 84 days in WT. Thus in agreement with RE turnover (Figure 4A), mutant mice exhausted their liver RP stores six times faster than WT littermates. For all mice, ROL serum levels remained stable for 12 weeks (∼0.35 μg/ml of serum). When CRBPI-null mutant RP stores dropped below 2 μg/g of liver (14 weeks), ROL serum levels decreased to reach ∼0.05 μg/ml at 23 weeks (Figure 5B, open blue circles). In contrast, WT mice maintained a normal serum ROL level during the same period (Figure 5B, filled blue squares). Thus, CRBPI appears to be indispensable for maintaining homeostasis of ROL under conditions of dietary vitA deprivation. Figure 5.CRBPI-null mice fed a VAD diet exhaust their RP stores and present symptoms of HVA. (A) Schematic representation of the nutritional protocol. Mice were fed a vitA-enriched diet from birth to 4 weeks (w), and then a VAD diet for 23 weeks. The weight of CRBPI-null mice (−/−; open circles) was significantly below that of WT mice (+/+; filled squares) from VAD week 5 onwards (P <0.05). (B) Liver RP (red lines) and blood ROL (blue lines) concentrations in WT (+/+; filled squares) and mutant (−/−; open circles) mice, as a function of time. Each point represents the average of three to five observations, and vertical bars indicate SEM. Regression lines calculated between VAD weeks 1 and 14 gave estimated half-lifes (t1/2) of 84 days in WT and 14 days in CRBPI-null mutants. ND, not detectable. (C) Typical example of dark-adaptated ERG responses from WT (+/+; black line) and mutant (−/−; green line) mice fed the vitA-enriched diet (left panel) or fed the VAD diet for 23 weeks (right panel). a and b denote a- and b-waves, respectively. (D-K) Histological sections through testes (D-G), cranial prostate (H and I) and urinary bladder (J and K) of WT (+/+; D, E, J and H) and CRBPI-null (−/−; F, G, K and I) males maintained on a VAD diet during 23 weeks. All WT tissues were unaffected. In contrast, testes of VAD mutants were degenerated; the glandular epithelium (G) of the cranial prostate, which normally secretes part of the seminal fluid (F), was completely keratinized and the lumen of the gland was filled with both desquamated keratinized cells (K) and leucocytes (LE); the bladder showed foci of squamous metaplasia (SQ) which were often adjacent to hyperplastic areas of the urinary epithelium (HY). BM, basement membrane of the seminiferous tubules; E, elongated spermatids; F, seminal fluid; G, normal (pseudostratified, columnar) prostate glandular epithelium; HY, hyperplastic urinary epithelium; K, desquamated keratinocytes; L, Leydig cells; LE, leucocytes; LP, lamina propria of the urinary bladder; LU, lumen of the bladder; M, smooth muscle cell layers of the bladder; R, round" @default.
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• W2104030002 title "Cellular retinol-binding protein I is essential for vitamin A homeostasis" @default.
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