The role of extrahepatic retinol binding protein in the mobilization of retinoid stores
2004; Elsevier BV; Volume: 45; Issue: 11 Linguagem: Inglês
10.1194/jlr.m400137-jlr200
ISSN1539-7262
AutoresLoredana Quadro, William S. Blaner, Leora Hamberger, Phyllis M. Novikoff, Silke Vogel, Roseann Piantedosi, Max E. Gottesman, Vittorio Colantuoni,
Tópico(s)Estrogen and related hormone effects
ResumoAlthough the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP−/−) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP−/−). By comparing hRBP−/−, RBP−/−, and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical.Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed. Although the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP−/−) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP−/−). By comparing hRBP−/−, RBP−/−, and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical. Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed. Retinol is the major circulating retinoid (vitamin A and its analogs) form and is needed to maintain normal growth and development, immunity, reproduction, vision, and other important physiological processes (1Napoli J.L. 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The Retinoids: Biology, Chemistry and Medicine. Raven Press, New York1994: 257-282Google Scholar, 21van Bennekum A.M. Wei S. Gamble M.V. Vogel S. Piantedosi R. Gottesman M.E. Episkopou V. Blaner W.S. Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice.J. Biol. Chem. 2001; 276: 1107-1113Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The mechanism through which tissues acquire retinol from circulating retinol-RBP is also subject to considerable debate.Although the major site of RBP synthesis in the body is the hepatocyte, other adult organs and tissues are reported to express RBP (2Soprano D.R. Blaner W.S. Plasma retinol-binding protein.in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. Raven Press, New York1994: 257-282Google Scholar). These include kidney, adipose, lacrimal gland, retinal pigment epithelium, testes, and brain (2Soprano D.R. Blaner W.S. 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Blaner W.S. Transthyretin, thyroxine, and retinol-binding protein in human cerebrospinal fluid: effect of lead exposure.Toxicol. Sci. 2001; 61: 107-114Crossref PubMed Scopus (60) Google Scholar). However, neither of these possibilities has been tested experimentally. Moreover, it is unclear whether circulating RBP derived from different tissues performs the same basic physiological functions as the hepatic protein (i.e., mobilization of hepatic retinoid stores and delivery of retinol to tissues).To address these questions, we used two mouse strains that we had previously generated. The RBP-knockout mouse strain (RBP−/−) was obtained by targeted disruption of the genomic locus (39Quadro L. Blaner W.S. Salchow D.J. Vogel S. Piantedosi R. Gouras P. Freeman S. Cosma M.P. Colantuoni V. Gottesman M.E. Impaired retinal function and retinoid availability in mice lacking retinol-binding protein.EMBO J. 1999; 17: 4633-4644Crossref Scopus (400) Google Scholar). The mice have dramatically reduced serum retinol levels (12.5% of wild-type animals) and impaired retinal function and visual acuity during the first months of life. Fed a retinoid-sufficient diet, they recover normal retinol levels and vision by 4 months of age. In contrast, their vision deteriorates and their circulating retinol concentration decreases if they are kept on a retinoid-deficient diet from weaning. Thus, the low levels of circulating retinol in these mice arise from recent dietary intake (39Quadro L. Blaner W.S. Salchow D.J. Vogel S. Piantedosi R. Gouras P. Freeman S. Cosma M.P. Colantuoni V. Gottesman M.E. Impaired retinal function and retinoid availability in mice lacking retinol-binding protein.EMBO J. 1999; 17: 4633-4644Crossref Scopus (400) Google Scholar). Because they are dependent on a regular retinoid intake, the retinoid status of these animals is extremely tenuous (40Quadro L. Hamberger L. Colantuoni V. Gottesman M.E. Blaner W.S. Understanding the physiological role of retinol-binding protein in retinoid metabolism using transgenic and knockout mouse models.Mol. Aspects Med. 2003; 24: 421-430Crossref PubMed Scopus (108) Google Scholar, 41Quadro L. Hamberger L. Gottesman M.E. Colantuoni V. Rajashekhar R. Blaner W.S. Transplacental delivery of retinoid: the role of retinol-binding protein and lipoprotein retinyl ester.Am. J. Physiol. Endocrinol. Metab. 2004; 286: E844-E851Crossref PubMed Scopus (67) Google Scholar). Finally, because retinol mobilization from hepatic stores is compromised, RBP−/− animals accumulate retinol and retinyl ester in the liver at a higher rate than do wild-type mice (39Quadro L. Blaner W.S. Salchow D.J. Vogel S. Piantedosi R. Gouras P. Freeman S. Cosma M.P. Colantuoni V. Gottesman M.E. Impaired retinal function and retinoid availability in mice lacking retinol-binding protein.EMBO J. 1999; 17: 4633-4644Crossref Scopus (400) Google Scholar, 42Paik J. Vogel S. Quadro L. Piantedosi R. Gottesman M.E. Lai K. Hamberger L. De Morais Vieira M. Blaner W.S. Retinoid: overlapping delivery pathways to tissues from the circulation.J. Nutr. 2004; 134: 276-280Crossref Google Scholar).We also generated a transgenic mouse that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in the rbp-null background (hRBP−/−) (43Quadro L. Blaner W.S. Hamberger L. Van Gelder R.N. Vogel S. Piantedosi R. Gouras P. Colantuoni V. Gottesman M.E. Muscle expression of human retinol-binding protein (RBP). Suppression of the visual defect of RBP knockout mice.J. Biol. Chem. 2002; 277: 30191-30197Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The transgenic hRBP is produced at high levels, and, like endogenous murine RBP (mRBP), binds transthyretin and delivers retinol to tissues. Moreover, the hRBP suppresses the visual defect of the rbp-null mice and allows peripheral tissues to acquire normal levels of retinol (43Quadro L. Blaner W.S. Hamberger L. Van Gelder R.N. Vogel S. Piantedosi R. Gouras P. Colantuoni V. Gottesman M.E. Muscle expression of human retinol-binding protein (RBP). Suppression of the visual defect of RBP knockout mice.J. Biol. Chem. 2002; 277: 30191-30197Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).The experiments reported here using mutant and wild-type mice address two important issues related to RBP and retinoid metabolism. First, we demonstrate that extrahepatically synthesized hRBP cannot mobilize hepatic stores. Only RBP synthesized in the liver can mobilize such stores. Second, we ask if RBP plays a role in mediating the distribution of retinoids between liver hepatocytes and stellate cells. RBP does not mediate cellular retinoid trafficking within the liver.METHODSAnimalshRBP−/− mice were generated and characterized as described by Quadro et al. (43Quadro L. Blaner W.S. Hamberger L. Van Gelder R.N. Vogel S. Piantedosi R. Gouras P. Colantuoni V. Gottesman M.E. Muscle expression of human retinol-binding protein (RBP). Suppression of the visual defect of RBP knockout mice.J. Biol. Chem. 2002; 277: 30191-30197Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).Plasma clearance and liver uptake of [3H]retinolThree month old female hRBP−/−, RBP−/−, and wild-type mice received an oral bolus of [3H]retinol (106 cpm/100 μl) in peanut oil via gavage. Plasma samples were obtained after centrifugation of the blood that had been collected into a tube containing EDTA at 14,000 g. Dissected tissues were immediately placed in liquid nitrogen and stored at −70°C until analysis. To assess [3H]retinoid concentrations in total plasma, 20 μl of each plasma sample was transferred to a scintillation vial and dissolved in 20 ml of Hydroflor liquid scintillation counting solution. To analyze liver levels of [3H]retinoids, tissues were weighed, homogenized in 3 volumes of PBS using a Polytron homogenizer (Brinkman Instruments, Westbury, NY), and extracted with chloroform-methanol (2:1, v/v). After centrifugation at 500 g for 10 min, the lower chloroform phase was transferred to scintillation vials and evaporated in a fume hood. The retinoid-containing lipid film remaining after evaporation of the chloroform was dissolved in 20 ml of Hydroflor liquid scintillation counting solution. 3H-cpm present in plasma and tissues samples was measured in a Beckman LS 1800 liquid scintillation counter.Preparation of protein extracts from mouse tissues and Western blot analysisTo survey RBP protein expression in mouse tissues, organs were removed after PBS perfusion and homogenized with a Waring blender in a buffer containing 20 mM potassium phosphate buffer, pH 7.0, 0.25 M sucrose, 50 mM NaCl, 5 mM EDTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 10 mM benzamidine-HCl (5 ml/g wet weight). Nuclei were pelleted by centrifugation at 700 g for 5 min. All operations were performed at 4°C. Protein concentrations were determined by the method of Lowry et al. (44Lowery O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 263-275Google Scholar) using BSA as a standard. Western blot analysis was performed as described (39Quadro L. Blaner W.S. Salchow D.J. Vogel S. Piantedosi R. Gouras P. Freeman S. Cosma M.P. Colantuoni V. Gottesman M.E. Impaired retinal function and retinoid availability in mice lacking retinol-binding protein.EMBO J. 1999; 17: 4633-4644Crossref Scopus (400) Google Scholar) by using a rabbit polyclonal anti-rat serum RBP (45Muto Y. Smith J.E. Milch P.O. Goodman D.S. Regulation of retinol-binding protein metabolism by retinoid status in the rat.J. Biol. Chem. 1972; 247: 2542-2550Abstract Full Text PDF PubMed Google Scholar).Antibody productionA peptide corresponding to amino acids 185–201 of the primary sequence of mouse RBP (46Jessen K.A. Satre M.A. Induction of mouse retinol binding protein gene expression by cyclic AMP in Hepa 1-6 cells.Arch. Biochem. Biophys. 1998; 357: 126-130Crossref PubMed Scopus (13) Google Scholar) was synthesized by the Columbia University Howard Hughes Protein Core Facility. The peptide was linked to an eight-armed matrix (47Mertz J.R. Banda P.W. Kierszenbaum A.L. Rat sperm galactosyl receptor: purification and identification by polyclonal antibodies raised against multiple antigen peptides.Mol. Reprod. Dev. 1995; 41: 374-383Crossref PubMed Scopus (16) Google Scholar), and this multiple antigen peptide (MAP) served as the immunogen. The MAP was sent to Pocono Rabbit Farm and Laboratory, Inc. (Canadensis, PA) for antibody production. For this purpose, a New Zealand White rabbit was injected intradermally with 1 mg of MAP in Complete Freund's Adjuvant. One booster intradermal injection of 100 μg of MAP in Complete Freund's Adjuvant was given on day 14. On day 28, the rabbit was given a subcutaneous injection of 100 μg of the MAP in Incomplete Freund's Adjuvant. Test bleeds began 42 days after the initial injection, followed by injection of 50 μg of MAP in Incomplete Freund's Adjuvant on day 56 and every 4 weeks thereafter. Test bleeds were taken 2 weeks after each injection (days 42, 70, and 98). On day 126 after the initial immunization, the rabbit was exsanguinated.HPLC analysisRetinol and retinyl ester concentrations in plasma and tissues were measured by reverse-phase HPLC as described (48Blaner W.S. Das S.R. Gouras P. Flood M.T. Hydrolysis of 11-cis- and all-trans-retinyl palmitate by homogenates of human retinal epithelial cells.J. Biol. Chem. 1987; 262: 53-58Abstract Full Text PDF PubMed Google Scholar, 49Wei S. Episkopou V. Piantedosi R. Maeda S. Shimada K. Gottesman M.E. Blaner W.S. Studies on the metabolism of retinol and retinol-binding protein in transthyretin-deficient mice produced by homologous recombination.J. Biol. Chem. 1995; 270: 866-870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).Diet and animal husbandryA purified nutritionally complete control retinoid-sufficient diet (Purified Test Diet 5755; W. F. Fisher and Son, Inc.) containing 22 IU retinol/g diet and a retinoid-deficient but otherwise nutritionally complete diet (Purified Test Diet 5822; W. F. Fisher and Son, Inc.) containing by actual lot analysis <0.22 IU retinol/g diet were obtained from standard sources. All nutrients other than retinol were present in these two purified diets at the same concentrations. Mice used for these studies were bred and maintained continuously in a specific virus- and pathogen-free (barrier) facility operating on a 12 h dark/light cycle.Light microscopy analysisFor light microscopic studies, the liver was removed from wild-type, RBP−/−, and hRBP−/− mice under ether anesthesia. Approximately 1 mm slices of the liver were cut by hand and placed in a fixative containing 4% paraformaldehyde/2.5% glutaraldehyde/0.1 M cacodylate buffer for 5 h at 0°C with shaking. Frozen sections (∼20 μm) were prepared from the liver slices using a freezing microtome and treated as follows: 1) immersion in 0.5% oil red O/60% triethyl phosphate solution for 15 min at room temperature to detect neutral lipids in stellate cells and hepatocytes; 2) staining in methyl-green pyronin for histology; and 3) preparation of toluidine blue-stained 1 μm sections after embedding into Epon, according to protocols established for electron microscopy, to confirm lipid distribution.RESULTSCirculating hRBP of extrahepatic origin cannot mobilize liver retinoidTo determine if RBP of extrahepatic origin can mobilize hepatic retinoid stores, we measured plasma and hepatic concentrations of [3H]retinol at 2, 4, and 24 h after oral administration of a bolus dose of [3H]retinol in peanut oil. Figure 1Ashows the total radioactivity present in plasma at these time points. These values represent both the rate of clearance and secretion into plasma of the ingested [3H]retinoid. Note that 2 h after administration, plasma [3H]retinoid levels in RBP−/− and hRBP−/− mice were significantly lower than in wild-type animals. Triglyceride and cholesterol concentrations in plasma pools of all three strains were equivalent (data not shown), suggesting that their chylomicron retinoid clearance rates were similar. We also analyzed by HPLC [3H]retinol levels in the circulation of all animals at the same time point (2 h). Table 1 shows that ∼70% of the 3H label in the circulation of wild-type female animals was [3H]retinol, compared with only ∼25% of the 3H label in female RBP−/− and hRBP−/−mice. An identical study was carried out using male mice from the three strains and gave the same results. These data support the hypothesis that wild-type mice secrete newly absorbed [3H]retinol back into the circulation. In contrast, RBP−/− and hRBP−/−mice retain this retinol because they cannot mobilize hepatic retinoid stores. The levels of plasma radioactivity at 4 and 24 h were similar in the three strains. The distribution of this radioactivity between [3H]retinol and derivatives was not determined.TABLE 1Total plasma retinol 2 h after oral administration of [3H]retinol to wild-type, RBP−/−, and hRBP−/− miceGenotype[3H]RetinolMice%nWild type725RBP−/−285hRBP−/−234hRBP, human retinol binding protein; RBP, retinol binding protein. Values represent the percentage of total [3H]retinol (retinol and retinyl ester) present in the circulation as [3H]retinol. Plasma samples from female mice of each genotype were pooled. An identical duplicate experiment was carried out using age-matched male mice from each of the three strains. The data obtained from the male mice were the same as for the females. Open table in a new tab Liver uptake curves of the three strains (Fig. 1B) support the above conclusion. At 2 h, liver [3H]retinoid content was similar in all strains. Note that this observation does not conflict with our finding that wild-type animals secrete [3H]retinol at this time point, because the amount of circulating [3H]retinol is very small compared with the large amount of retinoid stored in liver (3Blaner W.S. Olson J.A. Retinol and retinoic acid metabolism.in: Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids: Biology, Chemistry and Medicine. Raven Press, New York1994: 229-256Google Scholar). Failure to find a statistically significant decrease in hepatic retinoid levels does not, therefore, indicate the presence or absence of secretion. At 4 h, hepatic [3H]retinol levels in RBP−/− and hRBP−/− mice were significantly higher than in wild-type animals. At 24 h, wild-type hepatic [3H]retinol concentrations started to increase, likely reflecting reuptake of circulating retinol. Nevertheless, wild-type [3H]retinol levels did not reach the levels of RBP-null mutants. Taken together, these results are consistent with the notion that RBP synthesized in the liver can mobilize liver stores but that RBP of extrahepatic origin cannot. In muscle and in other tissues analyzed, [3H]retinol uptake curves were similar in the three mouse strains (data not shown).Circulating hRBP is not taken up by liverThe data provided in Fig. 1 indicate that liver retinoid stores cannot be mobilized by hRBP secreted from muscle and suggest that RBP must be synthesized in liver to mobilize hepatic retinoid. Our data imply that circulating hRBP is not internalized by hepatocytes. To test this hypothesis, protein extracts were prepared from liver and muscle of hRBP−/− mice and from livers of wild-type and RBP−/− animals that were extensively perfused before dissection to remove blood from tissues. Western blot analysis was performed using a rabbit polyclonal anti-rat serum RBP (45Muto Y. Smith J.E. Milch P.O. Goodman D.S. Regulation of retinol-binding protein metabolism by retinoid status in the rat.J. Biol. Chem. 1972; 247: 2542-2550Abstract Full Text PDF PubMed Google Scholar) that cross-reacts with both endogenous mRBP and exogenous hRBP (Fig. 2). RBP was detected only in muscle extracts from hRBP−/− mice. As expected, no RBP was detected in liver extracts from RBP−/− mice, whereas a strong immunoreactive band was observed in extracts from wild-type mice. Critically, although the serum concentration of hRBP in serum from hRBP−/− mice (2.27 ± 0.43 mg/dl) is similar to the concentration of mRBP present in the circulations of wild-type mice (21van Bennekum A.M. Wei S. Gamble M.V. Vogel S. Piantedosi R. Gottesman M.E. Episkopou V. Blaner W.S. Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice.J. Biol. Chem. 2001; 276: 1107-1113Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 43Quadro L. Blaner W.S. Hamberger L. Van Gelder R.N. Vogel S. Piantedosi R. Gou
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