©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on the Metabolism of Retinol and Retinol-binding Protein in Transthyretin-deficient Mice Produced by Homologous Recombination (*)

(Received for publication, September 8, 1994; and in revised form, October 25, 1994)

Shuanghong Wei (1) Vasso Episkopou (2) Roseann Piantedosi (1) Shuichiro Maeda (3) Kazunori Shimada (4) Max E. Gottesman (5) William S. Blaner (1)(§)

From the  (1)Institute of Human Nutrition, Columbia University, New York, New York 10032; (2)Department of Biochemistry and Molecular Genetics, St. Mary's Hospital, Medical School, London W2 1PG, United Kingdom; (3)Department of Biochemistry, Yamanashi Medical University, 1110 Shimagato, Tamaho-machi, Nakakoma-gun, Yamanashi 409-38, Japan; (4)Department of Medical Genetics, Division of Molecular Biomedicine, Research Institute for Microbial Diseases, Osaka University, 3-1 Suita, Osaka 565, Japan; and (5)Institute of Cancer Research, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tissue needs for retinoids are believed to be satisfied through the delivery in the circulation of retinol by its specific plasma transport protein, retinol-binding protein (RBP), which circulates as a 1-to-1 protein complex with transthyretin (TTR). The binding of RBP to TTR is thought to prevent filtration of retinol-RBP in the kidney and to play a role in secretion of RBP from hepatocytes. Recently a strain of mice (TTR) that totally lacks immunoreactive TTR was produced by targeted mutagenesis. We have explored the effects of TTR deficiency on retinol and RBP metabolism in this mutant strain. In pooled plasma from the TTR mice retinol levels averaged 6% of those of wild type animals. Similarly, plasma RBP in the TTR mice was found to be 5% of wild type levels. Hepatic retinol and retinyl ester levels were similar for mutant and wild type mice, suggesting that the mutation affects neither the uptake nor storage of dietary retinol. Levels of retinol and retinyl esters in testis, kidney, spleen, and eye cups from TTR mice were normal. Plasma all-trans-retinoic acid levels for the TTR mice were 2.3-fold higher than those of wild type (425 versus 190 ng/dl). Kidney RBP levels were similar for the mutant and wild type mice and we were unable to detect intact RBP in urine from TTR mice. Hepatic RBP levels in the TTR mice were 60% higher than those of wild type mice (39.8 versus 25.0 µg of RBP/g of tissue). These data may suggest that there is a partial blockage in RBP secretion from TTR hepatocytes that leads to lessened plasma levels of retinol-RBP.


INTRODUCTION

Retinoids (vitamin A and its analogs) are essential for growth, reproduction, and maintaining the general health of the organism(1, 2) . All retinoids present in the body come from the diet either as preformed vitamin A, or as provitamin A carotenoids, and are delivered as chylomicron retinyl ester to the liver, where the majority of the body's retinoid reserves are stored(2, 3) . The liver secretes retinol bound to retinol-binding protein (RBP) (^1)into the circulation(2, 4, 5) . RBP is the sole plasma transport protein for retinol from hepatic storage depots to peripheral target tissues for retinoid action. It is generally assumed, based on the relatively high levels of retinol in tissues and plasma and the abundance in tissues of enzymes which are able to oxidize retinol to retinoic acid, that the in situ oxidation of retinol to retinoic acid is the major route through which tissue needs for retinoic acid are satisfied(6, 7, 8, 9) . However, some retinoic acid is also present in the circulation, at levels which are 0.2-0.7% of circulating retinol levels(4, 5, 10, 11, 12, 13) . It is not presently known to what extent circulating pools of retinoic acid contribute to tissue pools. The all-trans and the 9-cis isomers are the active forms of retinoic acid that regulate the expression of retinoid responsive genes and serve as the ligands for the ligand-dependent transcription factors RARalpha, -beta, and -, and RXRalpha, -beta, and -(14, 15, 16, 17, 18, 19, 20, 21) . These nuclear receptors for retinoic acid are responsible for mediating the effects of retinoids on gene expression and are thus essential for vitamin A action.

Transthyretin (TTR) plays an important functional role in the plasma transport of both thyroid hormone and retinol. In the body, the liver and the choroid plexus are the major tissue sites of synthesis and secretion of TTR(22, 23) . In the plasma, TTR exists as a 55-kDa tetramer of identical subunits(24, 25) . TTR has one high affinity binding site for thyroxine (T(4)) (24, 26, 27) and one for RBP; the two sites are independent(27, 28) . In the human and the rat, TTR-bound T(4) accounts for approximately 15 and 70%, respectively, of the T(4) present in the plasma(29, 30) . It has been postulated, based on the molecular size of RBP (21 kDa), that the formation of the RBP-TTR complex prevents glomerular filtration and renal catabolism of RBP(2, 4, 5) . Although it has been supposed that RBP and TTR do not interact intracellularly prior to their secretion (2, 3, 4, 5, 7) , recent studies employing expression constructs for RBP and TTR have shown for a model HeLa cell system that these secretory proteins can interact within the cell prior to secretion(31, 32) . Since plasma TTR concentrations are normally in a 2-3-fold molar excess over those of RBP, most RBP in the circulation is bound to TTR (2, 3, 4, 5) .

Recently, using the techniques of targeted mutagenesis, Episkopou et al.(33) have developed a mutant strain of mice (TTR) that totally lacks TTR. This null mutation at the ttr locus was generated in embryonic stem cells. Immunoblot analysis indicated that TTR is completely absent from plasma. Since TTR was thought to play an essential role in both retinoid and thyroid hormone physiology, it was anticipated that the disruption of the TTR gene would result in embryonic lethality. However, homozygous animals display no obvious phenotypic abnormalities post-natally, as determined both morphologically and by histopathological analysis. When heterozygous animals at the ttr locus were intercrossed, genotyping of the resulting progeny showed that live-born mice homozygous for the disrupted ttr gene were recovered at a frequency of approximately 25%, indicating that absence of TTR does not compromise fetal development. Plasma levels of total T(4) and T(3) in TTR mice were found to be, respectively, 35 and 64% of those of wild type mice(33) . The TTR mice are thought to be euthyroid since T(3) levels in the TTR mice are only slightly reduced from those of wild type animals and circulating levels of pituitary thyrotropin, which regulates the production of thyroid hormone, are not affected in the TTR mice(33) . The plasma levels of both retinol and RBP in the TTR mice are less than 6% of those measured in wild type mice(33) . Such low plasma levels of retinol and RBP are observed in vitamin A deficiency, in animals which show the clinical symptoms of deficiency and which are within 1-2 weeks of death(1, 34) . This would suggest that either our understanding of retinoid transport and metabolism is not complete or that the physiology of the TTR mice has in some way compensated for the steady delivery of low levels of retinol.

Our studies reported in this manuscript provide a first full characterization of retinoid transport and metabolism in the unique TTR strain of mice.


MATERIALS AND METHODS

Animal Husbandry and Genotyping

All of the mice, both TTR and wild type, used for our studies were from the MF1 outbred strain of mice. These mice were bred and maintained under clean and conventional conditions in the Columbia University Animal Facilities. The colony was maintained in boxes, housing five or six mice each, on separate racks in the same room. All animals are provided ad libitum access to a nutritionally complete purified diet containing 2.4 µg of retinol/g of diet (35) and water. For all of our studies, we employed male mice ranging in size between 25 and 35 g.

Because the wild type and TTR mice have the same phenotype, a genotype analysis for each animal used in our studies was carried out prior to its use in experiments. Genotyping was carried out by PCR or by Southern blot analysis on DNA which had been purified with phenol-chloroform and ethanol-precipitated. We have employed the exact procedure described by Episkopou et al.(33) in the original description of the generation of the TTR mice. The oligomers used for the specific amplification were: 5`-end primer 1 (5`-GAGCGAGTGTTCCGATACTCTAA-3`) which corresponds to a sequence 181 base pairs upstream from the presumed transcription initiation site of the mouse ttr gene and which is outside the targeting vector homology (36) and 3`-end primer 2 (5`-GCGCTGACAGCCGGAACACG-3`) which corresponds to a sequence 413 base pairs downstream from the beginning of the disrupting neo cassette. The annealing temperature was 64 °C. A 1.8-kilobase fragment is observed from mice carrying the disrupted ttr gene.

HPLC Analysis of Retinol and Retinyl Esters-Retinol and retinyl ester concentrations in plasma and tissues were measured by normal phase HPLC analysis. The HPLC analysis was carried out using two 5-µm silica columns (Waters Associates, Milford, MA) linked in series, exactly as described previously(37) . Retinol was separated in hexane:dioxane:diethyl ether (94.6:5.0:0.4, v/v) flowing at 0.8 ml/min and the retinyl esters were separated in hexane:diethyl ether (99.6:0.4, v/v) flowing at 0.8 ml/min. Authentic standards of retinyl palmitate, retinyl oleate, retinyl stearate, retinyl linoleate, retinyl myristate, and retinyl palmitoleate were synthesized from authentic all-trans-retinol and the corresponding fatty acyl chloride (38) . Homogenates of tissues or tissue pools were extracted with 20 volumes of chloroform:methanol (2:1 v/v) and the total lipid extract was fractionated on solid phase silica columns (Supelco Inc., Bellefonte, PA) to separate the retinol and retinyl esters from other neutral lipids. Retinyl esters were eluted from the solid phase extraction columns in 0.1% diethyl ether in hexane and retinol was eluted in 0.7% diethyl ether in hexane. For purposes of quantitation, known tracer quantities of [^3H]retinol and [^3H]retinyl palmitate were added to the chloroform:methanol extracts. The retinoids were detected by absorbance at 325 nm, and the [^3H]retinol and [^3H]retinyl palmitate levels were determined by an in-line Berthold C-1 Radiation Detector (EG& Berthold, Nashua, NH).

HPLC Analysis of Retinoic Acid

Retinoic acid was analyzed by normal phase HPLC using two silica columns (Waters Associates) linked in series and eluted with hexane:acetonitrile:acetic acid (99.5:0.4:0.1, v/v). As described above, chloroform:methanol (2:1 v/v) extracts were prepared from plasma pools each obtained from 10 mice. Retinoic acid was separated from other lipids in the extracts by solid phase extraction on aminopropyl columns (Supelco Inc.). Retinoic acid eluted from the solid phase aminopropyl columns in 2.0% acetic acid in diethyl ether. Recovery of retinoic acid was assessed by adding a known amount of tracer [^3H]retinoic acid to the extracts. Retinoic acid was detected at 350 nm, and ^3H-label counts/min were determined by in-line radiation detection. The low limit of detection for retinoic acid using this procedure is approximately 1 ng/ml of plasma.

Radioimmunoassay of RBP and CRBP

Plasma, liver, kidney, and urine levels of RBP were measured using radioimmunoassay (RIA) procedures which have been previously described(39) . This RIA procedure employs rabbit anti-rat plasma RBP and standards of purified rat plasma RBP. The anti-rat RBP fully recognizes the mouse RBP, and the RIA procedure has previously been employed for quantitation of mouse plasma, liver, and kidney RBP levels(40) . Hepatic and testes levels of cellular retinol-binding protein (CRBP) levels were also measured by RIA(39) . For the CRBP RIA, turkey anti-rat testis CRBP and purified standard rat CRBP have been used as described previously for the assay of CRBP in murine cells and tissues(39) .

RNA Isolation and Northern Analysis

Total RNA was isolated from liver, testis, and adipose tissue from both TTR and wild type mice using the method of Chomczynski and Sacchi(41) . The RNA (20 µg/lane) was electrophoresed on a 1% agarose gel in the presence of formaldehyde and transferred to nylon membranes (Stratagene, La Jolla, CA) by capillary action. Immediately after transfer, the ratios of intensities of 28 and 18 S ribosomal RNA bands in the total RNA preparations were assessed by ethidium bromide staining. This ratio was found to be approximately 2 for all RNA samples examined in our studies. The membranes were then prehybridized at 65 °C for 1 h in a buffer containing 6 times SSC (1 times SSC: 0.15 M sodium chloride and 0.015 M sodium citrate), 5 times Denhardt's solution (1 times solution: 0.02% polyvinylpyrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), 10% dextran sulfate, and 1% SDS in the presence of 100 µg/ml denatured salmon testes DNA (Sigma), and hybridized at 65 °C overnight in the above buffer with P-labeled probes (2 times 10^6 cpm/ml) specific for rat RBP and rat CRBP, and murine beta-actin mRNAs prepared by nick translation of cDNA clones(42) . The cDNA clones for rat RBP (43) and rat CRBP (44) have been previously described. The membranes were washed in 2 times SSC, 0.5% SDS for 10 min at room temperature, two times in 1 times SSC, 1% SDS for 15 min at 65 °C, and finally in 0.1 times SSC, 1% SDS for 15 min at 65 °C. The hybridized blots were then exposed to XAR-2 films at -80 °C using intensifying screens. The bands on the autoradiographs were quantitated by scanning laser densitometry (Pharmacia Biotech Inc.). To normalize for RNA load in the different lanes of the gels, beta-actin mRNA levels were determined, and RBP and CRBP expression was normalized for beta-actin expression.

Statistical Analysis

All of the studies involving comparisons of data obtained from the TTR and wild type mice were analyzed for statistical significance. For our studies, the statistical tests consisted of t tests(45) .


RESULTS

Since the binding of RBP to TTR has been hypothesized to prevent renal filtration of RBP(2, 4, 5) , we first investigated plasma retinol and RBP levels in the TTR-deficient mice. Episkopou et al.(33) reported that plasma retinol levels in outbred TTR mice were below the low limit of detection of their assay system (which was 2 µg of retinol/dl of plasma). To obtain valid measures for plasma retinol in the mutant mice, levels were determined for pools of plasma each obtained from four TTR mice and for individual plasma samples obtained from five wild type mice. Plasma retinol levels measured in pools of plasma constructed using equal volumes of plasma taken from four individual TTR mice averaged 1.8 ± 0.5 µg/dl. The mean plasma retinol level for wild type mice was found to be 30.0 ± 1.2 µg/dl. Thus, the mutant mice have plasma retinol levels which, on average, are 6% of those of the parental wild type. Plasma RBP levels were measured by RIA in plasma obtained from six individual TTR and five wild type mice. As seen in Table 1, the individual plasma RBP levels in TTR mice are less than 5% of those of wild type mice, and are commensurate with the levels of retinol measured in pooled plasma from TTR mice. The very low levels of both plasma retinol and RBP in the TTR mice appear to support the hypothesis that TTR functions to prevent loss of retinol-RBP from the circulation. Alternatively, these data could support the hypothesis that TTR plays a role in promoting the release of RBP from hepatocytes.



To investigate further these possibilities, we measured kidney levels of RBP in TTR and wild type mice (Table 1). It has been reported that rodents exposed to polychlorinated biphenyls have elevated levels of kidney RBP. This elevation arises from displacement of RBP from TTR, increased renal filtration of RBP from the circulation, and elevated kidney RBP levels(40) . As seen in Table 1, kidney levels of RBP are not significantly different for the TTR and wild type mice. Very little intact RBP could be detected by RIA in urine collected from either TTR or wild type mice.

The levels of retinol and retinyl esters present in liver, testis, kidney, and spleen of TTR and wild type mice are provided in Table 2. The livers of TTR mice possess total retinol (retinol + retinyl ester) levels similar to those of wild type mice. These hepatic total retinol levels for the TTR mice indicate that the mutant mice are able to take up retinol from the diet and that the amount of retinol (as assessed by hepatic accumulation of retinol) is not quantitatively different for the TTR and wild type mice. For both wild type and TTR mice, less than 2% of the total retinol present in liver was in the form of retinol. The remainder of the hepatic total retinol was present as retinyl ester. The relative composition of the hepatic retinyl ester showed no differences between the wild type and TTR mice. For both wild type and mutant mice, retinyl palmitate was found to be the predominant retinyl ester, accounting for approximately 75% of the total retinyl ester in liver. The remainder of the retinyl ester present in liver for both wild type and TTR mice consisted of retinyl stearate (approximately 10% of total retinyl ester), retinyl oleate (approximately 8%) and small amounts of retinyl linoleate, retinyl myristate, and retinyl palmitoleate.



As seen in Table 2, mean retinol and retinyl ester levels in testis and spleen for TTR mice appear, upon inspection, to be slightly lower than those measured for wild type mice. However, statistical comparisons of these data and for kidney retinol and retinyl ester levels indicated that the total retinol levels in the tissues were not significantly different for TTR and wild type mice. Considering the very low plasma retinol level found in TTR mice (6% of wild type), it is surprising that the mutant mice possess such high levels of tissue retinol and retinyl esters. We also measured total retinol levels in pools of eye cups, each prepared from 3 eye cups from either TTR or wild type mice. Total retinol levels for 6 pools of eye cups from TTR mice averaged 2.56 ± 0.34 µg/pool compared to 3.25 ± 1.05 µg/pool for wild type. For eye cups of both TTR and wild type, over 90% of the total retinol was present as retinyl ester. Thus, although the neural retina in the TTR mice possess slightly less total retinol than do those of wild type mice, the mutants probably possess sufficient retinol stores to support normal vision.

Although the TTR mice are fertile and reproduce normally, we asked whether specific retinol-dependent biochemical responses and functions in the testes of TTR mice were normal. Dietary retinol is known to be necessary for maintaining spermatogenesis; and dietary retinoic acid (arriving in the circulation) cannot substitute for retinol to maintain spermatogenesis(1, 46, 47) . Cellular retinol-binding protein, type I (CRBP) expression and tissue levels are influenced by retinoid availability (44, 48, 49) through the action of a retinoic acid response element which is present in the promoter region of the CRBP gene(50) . To determine if the testis of TTR mice maintain normal retinol-dependent functions, we measured CRBP mRNA levels in total RNA prepared from testis from TTR and wild type mice. As seen in Fig. 1, testis CRBP mRNA levels are not different for TTR and wild type mice. This visual observation regarding testis CRBP mRNA levels was confirmed quantitatively when these gels were scanned by laser densitometry and normalized for beta-actin expression. Similarly, hepatic CRBP expression was assessed using total RNA prepared from TTR and wild type livers. As seen visually in Fig. 1, CRBP mRNA levels in livers of TTR mice were slightly lower than those of wild type. Repeated Northern blot analyses of hepatic CRBP mRNA levels in TTR and wild type mice which were normalized for total RNA load through measure of beta-actin mRNA levels indicated that hepatic CRBP mRNA levels in the TTR mice were approximately 75% of those of wild type. Since this observation was reproducible, we also measured CRBP protein levels by RIA in cytosol preparations from six TTR and four wild type livers and testis to investigate if possible differences in CRBP mRNA levels were reflected in strain specific differences in CRBP levels. Although hepatic CRBP levels tended to be lower in TTR than in wild type mice, when these data were analyzed statistically no significant difference between TTR and wild type levels was observed. Likewise, no statistically significant difference in testis CRBP levels was observed. Thus, it would appear that the testis and liver of TTR mice are able to maintain normal retinol-dependent functions.


Figure 1: Northern blot analysis for CRBP and beta-actin mRNA levels in total RNA prepared from liver (20 µg of total RNA) and testis (20 µg of total RNA). Total RNA prepared from wild type (lanes 1, 2, 3, 7, and 8) and TTR (lanes 4, 5, 6, 9, and 10) mice were analyzed. All procedures were carried out as described under ``Materials and Methods.''



Hepatic levels of RBP present in TTR and wild type mice were also measured by RIA (Table 1). RBP levels in livers from TTR mice were significantly (p < 0.05) higher than those observed for wild type mice. This elevation in hepatic RBP levels qualitatively resembles the elevation in RBP levels observed in livers and hepatocytes of vitamin A-deficient animals(4, 5, 35, 51) . In vitamin A deficiency, both total liver and hepatocyte RBP levels are elevated by 3-10-fold, due to a blockage in the secretory pathway for RBP(4, 5, 35, 51) . The blockage in RBP secretion occurs within the endoplasmic reticulum. The biochemical mechanisms responsible for the blockage of RBP secretion in vitamin A deficiency are not understood at present(4, 5, 52) . Hepatic RBP mRNA levels remain unchanged in vitamin A deficiency(43) . Northern blot analysis of total RNA prepared from livers and adipose tissue (a tissue which expresses RBP at approximately 25% of that of liver) of TTR and wild type mice indicated that RBP mRNA levels (when normalized for beta-actin expression) are similar in these two tissues for the two mouse strains. The results of this Northern blot analysis are shown in Fig. 2.


Figure 2: Northern blot analysis for RBP and beta-actin mRNA levels in total RNA prepared from liver (5 µg of total RNA) and epididymal adipose tissue (20 µg of total RNA). Total RNA from wild type (lanes 1 and 2), heterozygous (lane 3), and TTR (lanes 4 and 5) mice were analyzed. All procedures were carried out as described under ``Materials and Methods.''



Measurements of plasma retinoic acid levels suggest that the levels of all-trans-retinoic acid are elevated in the TTR mice. For four pools of plasma each collected from 10 TTR or 10 wild type mice, mean plasma retinoic acid levels were determined by normal phase HPLC analysis. The mean level of all-trans-retinoic acid in plasma pools from TTR mice was approximately 2.3-fold higher than that observed for plasma pools from wild type mice (425 ± 50 ng/dl versus 190 ± 40 ng/dl). Only trace amounts of 9-cis-retinoic acid were observed in any of the pools of plasma.


DISCUSSION

This report provides the first characterization of retinoid transport and metabolism in a unique animal that lacks TTR(33) . The TTR mice, created by targeted gene disruption, also represent the first animal model to show marked alterations in retinol transport. The mutant mice thus provide valuable insight into the role of retinol-RBP in total body retinoid homeostasis.

The most striking feature of the TTR mice is their low level of plasma retinol-RBP. The plasma retinol level of the TTR mice is less than 6% of that of wild type mice. This level of plasma retinol, when seen in wild type mice deprived of vitamin A, is associated with severe vitamin A deficiency. Such mice would be blind, undergoing extreme weight loss, and unless retinol or retinoic acid were restored to the diet, close to death(1, 35, 46, 49) .

The TTR mice, however, are phenotypically normal and fertile and have the same longevity as wild type mice(33) . What, then, is the vitamin A status of the TTR mice? The uptake of dietary retinoid is normal in the mutants, since total liver retinol (retinol + retinyl esters) levels are similar to those of wild type mice. If chylomicron delivery of dietary retinoid were impaired, one would expect to observe lower hepatic retinol stores. This is not the case for the TTR mice. Levels of retinol and retinyl esters present in testis, spleen, and eye cups from TTR mice are somewhat reduced (but not significantly so; see Table 2) but these tissues are not retinol deficient. Furthermore, the expression of CRBP, a gene which is regulated by nutritional retinol status(44, 48, 49) , is equivalent in the testes of TTR and wild type mice. We conclude that retinol-dependent responses and functions are not impaired in the mutant mice, and that they can both take up dietary retinoid and transport retinol to their tissues at rates sufficient to maintain normal vitamin A status.

It has been assumed that the delivery of retinol via RBP is the predominant if not the sole mechanism through which tissue retinoid needs are satisfied(2, 4, 5) . In view of their low plasma retinol-RBP levels, how do the TTR mice receive sufficient retinoid?

One possibility is that tissue uptake of plasma retinol is, in fact, more efficient when the retinol-RBP complex is not bound to TTR. Recent studies by Noy et al.(53) have demonstrated that the rate of dissociation of retinol from the RBP-TTR complex is 2.5-fold slower than its dissociation from RBP alone (uncomplexed to TTR) and suggest that TTR plays a role in modulating the release of retinol from RBP. Thus, the absence of TTR may facilitate retinol uptake by tissues and this may account for why tissues in the mutant mice are not retinol deficient. This notion is further supported by the observation that for adult male rats with low vitamin A status and reduced plasma retinol levels (averaging 2-7 µg of retinol/dl), the daily utilization rate of retinol is less than 10% of the retinol moving though the plasma (54) . In these rats, therefore, circulating retinol is still in excess of that which is actually utilized by tissues. Although, the plasma retinol levels of the mutant mice are low (1-2 µg of retinol/dl), comparable to those observed in the later stages of vitamin A deficiency, unlike vitamin A deficiency, the supply of retinol to tissues in the TTR mice is constant. Paradoxically, it is thus possible that the absence of TTR helps render tissues of the mutant mice retinol sufficient.

A second possibility is that, unlike vitamin A deficiency, the levels of plasma retinol, although low, are constant in the mutant mice, and the mutants have been able to adapt to these levels. For example, the TTR mice might have reduced tissue retinol needs by lowering the rate of tissue retinoid catabolism. We have no data, however, that directly support this hypothesis.

Finally, the survival of the TTR mice may indicate that tissue retinoid needs are or can be met by alternative retinoid delivery systems. A recent study has indicated that plasma retinoic acid contributes substantially to tissue retinoic acid pools in most rat tissues. (^2)As described under ``Results,'' we observed that plasma retinoic acid levels in the TTR mice are approximately 2.3-fold higher than in wild type. Plasma and tissue retinoic acid levels might thus be elevated in TTR mice to compensate for the relative absence of retinol in the circulation. Our data are, in fact, consistent with this possibility, since plasma retinoic acid levels in TTR mice are elevated by approximately 2.3-fold over wild type levels. This may reflect a physiological adaptation that permits the mutants to utilize efficiently this retinoid delivery pathway. Another source of tissue retinoid is dietary (chylomicron) retinyl ester, which have been shown to contribute to retinoid pools in some extrahepatic tissues, such as adipose tissue and kidney(55, 56) . Thus, the uptake of chylomicron retinyl ester by tissues in TTR mice may be a means through which some tissues acquire needed retinoid. The TTR mice have been maintained on carotenoid-free diets, obviating the possibility that tissue needs in the TTR mice were not met from in situ retinoid formation from carotenoids(7) .

Which of these hypotheses explains the absence of vitamin A deficiency in the TTR mice is the subject of active investigation in our laboratory. However, it is evident that the analysis of the mutant mice has already revealed that our understanding of retinoid delivery and utilization, based on simple assumptions as to the role of RBP, is deficient.

To understand the physiology of the TTR mice, we need also to explain the origin of the low plasma retinol and RBP levels. Our data do not unequivocally support or disprove the hypothesis that TTR prevents the filtration of retinol-RBP from the circulation. However, the urine or kidneys of the mutant mice do not contain increased levels of RBP (see Table 1). An alternative hypothesis to explain the low plasma retinol-RBP levels is that the mutant animals fail to secrete RBP into the plasma. Recall that the total liver levels of RBP are elevated in the TTR mice (see Table 2). Elevated hepatic RBP levels are observed in vitamin A-deficient animals; in the absence of retinol, newly synthesized RBP is not secreted from the hepatocyte and is retained in the endoplasmic reticulum(35, 51, 52) . It is possible that RBP is secreted by hepatocytes as the retinol-RBP-TTR complex. In fact, interaction between newly synthesized RBP and TTR within cells prior to secretion has been described(31, 32) . To test this idea, we are currently investigating RBP synthesis and secretion rates from isolated and cultured hepatocytes prepared from the livers of TTR and wild type mice.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK47389, a grant from the American Institute for Cancer Research, a grant from the United States Department of Agriculture, and a grant from the International Scientific Research Program(04044111) from the Japanese Ministry of Education, Science, and Culture. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Human Nutrition, Columbia University, College of Physicians and Surgeons, 630 W. 168th St., New York, NY 10032. Tel.: 212-305-9336; Fax: 212-305-5384.

(^1)
The abbreviations used are: RBP, retinol-binding protein; TTR, transthyretin; CRBP, cellular retinol-binding protein, type I; HPLC, high performance liquid chromatography; RIA, radioimmunoassay; T(3), triiodothyronine; T(4), thyroxine.

(^2)
S. B. Kurlandsky, M. V. Gamble, R. Ramakrishnan, and W. S. Blaner, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Joana Palha for help in genotyping and maintaining our mouse colony and Dr. Maria Joao M. Saraiva for many helpful discussions.


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