(Received for publication, September 8, 1994; and in revised form, October 25, 1994)
From the
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.
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) ()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 RAR
,
-
, and -
, and RXR
, -
, 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) (24, 26, 27) and one for RBP; the two sites
are independent(27, 28) . In the human and the rat,
TTR-bound T
accounts for approximately 15 and 70%,
respectively, of the T
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
and T
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
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.
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 [H]retinol and
[
H]retinyl palmitate were added to the
chloroform:methanol extracts. The retinoids were detected by absorbance
at 325 nm, and the [
H]retinol and
[
H]retinyl palmitate levels were determined by an
in-line Berthold C-1 Radiation Detector (EG& Berthold, Nashua,
NH).
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
-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
-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
-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
-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
-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.
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. (
)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.