From the Institute of Human Nutrition, the
§ Department of Medicine, and the ¶ Institute of
Cancer Research of the College of Physicians and Surgeons, Columbia
University, New York, New York 10032 and the
Medical
Research Council, Hammersmith Hospital,
London W12 0NN, United Kingdom
Received for publication, September 5, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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Transthyretin (TTR) acts physiologically in the
transport of retinol in the circulation. We previously reported the
generation and partial characterization of TTR-deficient
(TTR The predominant retinoid in the fasting circulation is retinol (1,
2). All circulating retinol is bound to its specific plasma transport
protein, the 21-kDa retinol-binding protein
(RBP)1 (1, 2). RBP, which is
synthesized and secreted primarily by hepatocytes, is the sole specific
transport protein for retinol in the circulation (1, 2). The secretion
of RBP is strongly stimulated by its association with retinol, which
alters the conformation of the protein (1, 2). In blood, RBP is found
as a 1:1 protein-protein complex with a 55-kDa serum protein,
transthyretin (TTR) (1, 2). Association with TTR is proposed both
to facilitate RBP release from its site of synthesis in the endoplasmic
reticulum (2) and to prevent renal filtration of RBP (1). Delivery of
retinol to cells through the circulation by the RBP-TTR complex is the
major pathway through which cells and tissues acquire retinol. It is
generally accepted that cells and tissues acquire the retinoic acid
they need for regulating gene expression via intracellular oxidation of
this retinol to retinoic acid (3).
We previously reported the targeted disruption of the mouse gene for
TTR (4, 5). Although TTR-deficient (TTR Materials--
Dulbecco's modified Eagle's medium (DMEM),
Medium 199, Krebs buffer, penicillin/streptomycin, gentamicin, fetal
bovine serum, and trypsin were purchased from Life Technologies, Inc.
Collagenase type IV, collagen type I, soybean trypsin inhibitor, and
HEPES were purchased from Sigma. Fatty acyl chlorides were obtained from Nu-Chek-Prep, Inc. (Elysian, MN). [3H]Retinol,
[35S]methionine, and Na125I were purchased
from PerkinElmer Life Sciences. Hexane, methanol, chloroform, methylene
chloride, acetonitrile, and benzene, all of HPLC grade or grades of
comparable purity, were purchased from Fisher. Authentic
all-trans-retinol was a kind gift of Dr. Christian Eckhoff
of Hoffmann-La Roche. All other chemicals and supplies were purchased
from standard commercial suppliers.
Mice--
For our studies, we employed WT 129SV mice and mice
lacking immunoreactive TTR (TTR
All mice were maintained in a specific virus and pathogen free
"barrier" facility prior to use in the studies. The animals were
allowed ad libitum access to a standard rodent chow diet (Purina Products, Richmond, VA) and water. The room housing the mice
was maintained on a standard 12-h light-dark cycle.
Primary Mouse Hepatocyte Isolation and Culture--
For
hepatocyte isolations, mouse livers were perfused in situ
through the portal vein at the rate of 10 ml/min, first with Krebs
buffer (NaCl, 6.75 g/liter; NaHCO3, 2 g/liter; KCl, 0.345 g/liter; KH2PO4, 0.158 g/liter,
MgSO4, 0.287 g/liter; gentamicin, 10 mg/liter; and glucose,
1.0 g/liter) for 4 min followed by medium 199 containing 0.25 mg/ml
collagenase type IV and 10 ng/ml soybean trypsin inhibitor for 12 min.
After perfusion, the liver, with the capsule intact, was quickly
excised from the body cavity and transferred to a sterile Petri dish.
After opening the capsule, the liver digest was resuspended in medium
199 and filtered through a fine nylon mesh to remove undigested
materials. Hepatocytes were separated from nonparenchymal cells and
debris by centrifugation at 20 × g for 3 min at room
temperature. The hepatocyte-containing pellet was washed with DMEM
containing glucose (4.5 g/liter), sodium pyruvate (0.110 g/liter),
NaHCO3 (3.7 g/liter), penicillin (200 units/ml),
streptomycin (0.050 g/liter), 20 mM HEPES, pH 7.4, and 10%
fetal calf serum. Routinely, greater than 85% of the isolated cells
excluded trypan blue. The yield of isolated mouse hepatocytes averaged
~20 × 106 hepatocytes/g liver.
Isolated hepatocytes were plated at 5 × 106 cells per
100-mm plastic dish in DMEM containing glucose (4.5 g/liter), sodium pyruvate (0.110 g/liter), NaHCO3 (3.7 g/liter), penicillin
(200 units/ml), streptomycin (0.050 g/liter), 20 mM HEPES,
pH 7.4, and 10% fetal calf serum. The plastic tissue culture dishes
had been coated prior to hepatocyte plating with collagen type I, according to the supplier's instructions. This culture medium was
changed for fresh medium after incubation for 4 h at 37 °C in a
humidified atmosphere of 5% CO2, 95% air. At this time,
most hepatocytes have attached to the collagen-coated plates and have started to assume a flattened morphology. Hepatocyte cultures were
incubated at 37 °C in 5% CO2, 95% air overnight prior
to the start of an experiment. The hepatocytes isolated and maintained according to these procedures remained viable in culture for at least 3 days.
At the start of our experiments to assess the effects of TTR deficiency
on RBP synthesis and secretion, hepatocytes were washed three times
with PBS and fresh DMEM containing glucose (4.5 g/liter), sodium
pyruvate (0.110 g/liter), NaHCO3 (3.7 g/liter), penicillin (200 units/ml), streptomycin (0.050 g/liter), 20 mM HEPES,
pH 7.4, and supplemented with either 10% fetal calf serum, 5%
TTR-deficient mouse serum, or 5% TTR-deficient mouse serum
supplemented with 1 µg of human TTR per ml of medium placed over the
cells. Hepatocytes were then cultured for 12 h to assess RBP
synthesis and secretion. After removal of the medium, cultured
hepatocytes were washed 3 times with 5 ml of ice-cold PBS. The media
and cells were stored at Bilateral Nephrectomy of Mice--
Three- to four-month-old
female WT and TTR
At 0, 6, 11, 15, and 18 h after the bilateral nephrectomy, 3-5
animals of each strain were sacrificed, and blood and liver were taken
for analysis. Blood was collected through the caudal vein and allowed
to clot at 4 °C. Both serum and the livers were immediately frozen
in liquid N2 and stored at Purification of Human RBP--
Human RBP used for our studies
was purified to homogeneity from human serum by conventional column
chromatography according to procedures we have described previously
(7). The homogeneously purified human RBP migrated on
SDS-polyacrylamide gel electrophoresis as a single protein band with an
approximate mass of 21 kDa. The UV absorbance spectrum of this purified
human RBP indicated that the RBP was ~75% saturated with retinol
(A325/A280 = 0.75). Consequently, we chose to add additional unlabeled retinol to
the purified RBP to make the RBP fully saturated with retinol (7).
Unbound retinol was removed from the RBP solution through treatment of
the RBP with charcoal/dextran (8).
Clearance of Circulating Human RBP from WT and TTR Preparation of Rat Chylomicrons Labeled with
[3H]Retinyl Esters--
Rat chylomicrons containing
3H-labeled retinyl esters were prepared as described by
Goodman et al. (9). Briefly, Harlan Sprague-Dawley rats
(200-300 g) that had been fasted overnight were administered by gavage
of 0.2 ml of a solution of corn oil + olive oil (1:1 v/v) containing 4 mg/ml Tissue Uptake of Chylomicron Retinoid--
Three-month-old male
WT and TTR
To assess the levels of [3H]retinoids taken up by tissues
from the labeled chylomicrons, tissues were homogenized in 4 volumes of
10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (PBS), using a Polytron homogenizer (Brinkmann Instruments) to give
20% (w/v) homogenates. The tissue homogenates were then extracted with
6 volumes of chloroform:methanol (2:1 v/v). After centrifugation and
removal of the lower retinoid-containing chloroform phase, the upper
phase was reextracted with 6 volumes of chloroform:methanol (2:1 v/v).
The lower chloroform phase from the reextracted tissue was taken and
combined with the first chloroform extract in a 20-ml glass liquid
scintillation vial and allowed to stand overnight in an ordinary
laboratory fume hood. After the chloroform had evaporated, the
retinoid-containing lipid film was dissolved in 20 ml of Hydrofluor
liquid scintillation counting solution (National Diagnostics, Atlanta,
Georgia). 3H counts/min were assayed in a Beckman LS 1800 LSC counter using a quench-correction program for calculation of
3H disintegrations/min.
Isolation of Lipoprotein Fractions--
Very low density
lipoprotein (VLDL), low density lipoprotein (LDL), high density
lipoprotein (HDL), and d > 1.21 g/ml bottom fractions
were isolated by sequential ultracentrifugation of serum obtained from
wild type and TTR HPLC Analysis of Serum and Liver Retinoids--
Serum retinol
and liver total retinol (retinol + retinyl ester) concentrations were
determined by reverse phase high performance liquid chromatography
(HPLC) using a procedure we have described previously (11). Briefly, to
an aliquot of serum (or liver homogenates) an equal volume of absolute
ethanol containing a known amount of the internal standard retinyl
acetate (Sigma) was added. Endogenous retinol and retinyl esters and
the internal standard were extracted into hexane. After one backwash
with H2O, the hexane extract was evaporated to dryness
under a gentle stream of N2. Immediately upon reaching
dryness, the retinoid containing film was redissolved in 40 µl of
benzene for injection onto the HPLC. Retinol and retinyl esters were
analyzed on a 4.6 × 250-mm 5-µm Beckmann Ultrasphere C18 column (Beckmann Instruments). The mobile phase
consisted of acetonitrile/methanol/dichloromethane (70:15:15 v/v)
delivered at a flow rate of 1.8 ml/min. Retinoids were detected and
quantitated by UV absorbance at 325 nm using a Waters PDA 996 photodiode array detector.
Radioimmunoassay (RIA) of Mouse and Human RBP and of Mouse
Albumin--
Levels of mouse RBP in tissues and serum and isolated
hepatocytes were analyzed using a sensitive and specific RIA procedure (12). This procedure employs sheep anti-rat serum RBP antiserum and
standards consisting of homogeneously purified rat serum RBP. Thus, all
mouse RBP concentrations are reported as "rat equivalents." This
RIA has been used previously for the analysis of RBP in various mouse
tissues and mouse serum (4, 5, 13). Purified human RBP and human serum
give no displacement in the mouse RBP RIA. Thus, for some experiments
where both human and mouse RBP are present in the sample tissue or
serum sample, human RBP does not interfere with the accurate measure of
mouse RBP levels.
For some experiments, human RBP was injected into mice. Here, the
concentrations of human RBP in tissues and serum were assessed by a
sensitive and specific RIA for human RBP (12). This RIA procedure makes
use of polyclonal rabbit anti-human serum RBP antiserum and
homogeneously purified human serum RBP as standard. Both purified rat
serum RBP and mouse serum RBP do not give displacement in this RIA for
human RBP. Consequently, the presence of mouse RBP in tissues and serum
samples did not interfere with the accurate measure of tissue or serum
human RBP levels.
Albumin levels of mouse liver were assessed by RIA using rabbit
anti-rat serum albumin antiserum and purified standards of rat albumin
(12). Thus, mouse albumin concentrations are reported as rat equivalents.
Protein Determinations--
A modified Lowry procedure (14) was
used for determining protein levels of mouse liver homogenates. The
reagents for this assay were purchased from Bio-Rad, and the assay was
carried out according to the supplier's instructions.
Statistical Procedures--
All data are expressed as means ± 1 S.D. Statistical significance was determined by Student's
unpaired t test (two-tailed). Group differences were
rejected as not significant for p > 0.05.
These studies pursue questions raised upon the initial
characterization of the phenotype of the TTR Liver total retinol (retinol + retinyl ester) and RBP levels for
outbred and inbred WT and TTR) mice. TTR
mice have very low
circulating levels of retinol and its specific transport protein,
retinol-binding protein (RBP). We have examined the biochemical basis
for the low plasma retinol-RBP levels. Cultured primary hepatocytes
isolated from wild type (WT) and TTR
mice accumulated RBP
in their media to an identical degree, suggesting that RBP was being
secreted from the hepatocytes at the same rate. In vivo
experiments support this conclusion. For the first 11 h after
complete nephrectomy, the levels retinol and RBP rose in the
circulations of WT and TTR
mice at nearly identical
rates. However, human retinol-RBP injected intravenously was more
rapidly cleared from the circulation (t1/2 = 0.5 h for TTR
versus
t1/2 >6 h for WT) and accumulated faster in the
kidneys of TTR
compared with WT mice. The rate of
infiltration of the retinol-RBP complex from the circulation to tissue
interstitial fluids was identical in both strains. Taken together,
these data indicate that low circulating retinol-RBP levels in
TTR
mice arise from increased renal filtration of the
retinol-RBP complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) mice have no
immunoreactive TTR, they appear normal and are viable and fertile (4).
Yet, these mutant mice show marked biochemical differences when
compared with wild type (WT) mice in parameters associated with
retinoid transport and metabolism (4, 5). The plasma retinol and RBP
levels in TTR
mice are very low, ~5% of those observed
in WT mice (4, 5). These retinol levels correspond to those seen in
severely vitamin A-deficient animals that are near death (4, 5).
Nevertheless, despite these low circulating levels of retinol-RBP,
total retinol (retinol + retinyl ester) levels in tissues of
TTR-deficient mice are similar to those in tissues from age-, sex-, and
strain-matched WT mice (4, 5). These apparently contradictory
observations raise two important questions for understanding the
physiologic role(s) of TTR in living animals and the retinoid-related
physiology of the TTR-deficient mice. The first of these is the basis
of the low circulating retinol-RBP levels. Second, how do the tissues of TTR
mice acquire and maintain normal total retinol
levels? We now report data that provide an answer for the first of
these questions and some insight into the second.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mice) from the same
genetic background. These TTR
mice were created by
targeted gene disruption in ES cells, as we have described previously
(4). These ES cells were allowed to colonize blastocysts obtained from
crosses of WT 129SV male and female mice. Thus, the inbred 129SV
TTR
mice we used for our studies are from a homogeneous
genetic background. Retinoid-related parameters of TTR-deficient mice
from the 129SV strain have not been reported previously. We earlier
have characterized TTR
mice from the outbred MF-1 genetic
background (4, 5).
70 °C for up to 4 weeks prior to assay of
media and cellular levels of RBP by radioimmunoassay.
mice were bilaterally nephrectomized to
investigate the role of the kidney in maintaining retinol and RBP blood
levels. Prior to nephrectomy, the mice were anesthetized by
intraperitoneal injection of a mixture of ketaset plus zylazine (0.2 ml/100 g body weight, 10 mg of ketaset, and 2 mg of zylazine/0.2 ml).
Within 30 min after the start of surgery, the vessels leading to and
from the kidneys had been fully ligated, and the kidneys were
surgically removed, according to procedures described for rats (6).
Within 30 min after completion of this surgical procedure, mice
regained consciousness, and they remained active throughout our
studies. In trials of this surgical procedure, mice survived for
periods up to at least 24 h after completion of the surgery.
70 °C until analysis for
retinol, mouse RBP, and mouse albumin. Animals used for measures at
0 h had undergone sham operations in which a ligature had been placed around the renal veins but was left untied.
Mice--
Three-month-old male WT and TTR-deficient mice were fasted
overnight. The next day, animals were anesthetized, and 0.1 ml of
saline containing 10 µg of human holo-RBP was injected into the right
jugular vein of each mouse. At 5, 10, 30, and 60 min and 6 h after
injection, mice were bled through the caudal vein. Immediately after
removal of a 0.4-ml aliquot of blood, the carcass was subjected to a
total body perfusion with ice-cold 0.9% NaCl containing 0.5 mM EDTA flowing at 4.5 ml/min for 2-3 min. Based on the
blanching of the liver and other organs, this perfusion removed
residual blood from the organs. Following perfusion, liver, kidney,
lung, heart, epididymal fat, perirenal fat, and skeletal muscle (both
psoas and gastrocnemius muscle) were excised and immediately frozen in
liquid N2. These tissues were stored at
70 °C prior to
analysis for human and mouse RBP. Blood was allowed to clot at 4 °C,
and the serum obtained was stored at
70 °C until RIA analysis for
human and mouse RBP.
-tocopherol (Eastman Kodak Co.) and 40 µCi of
[3H]retinol ([11,12-3H]retinol, 37.3 Ci/mmol, PerkinElmer Life Sciences) to which 800 µg of unlabeled
retinol (kindly provided by Dr. Christian Eckhoff, Hoffmann-La Roche)
was added. Within 20 min after administration of the gavage, rats were
anesthetized; the mesenteric lymph duct was cannulated, and chyle was
collected on ice, under reduced light, into a sterile 15-ml tube
containing 1 ml of a solution consisting of 1 mg of EDTA/ml saline
(0.9% NaCl). Collection continued for periods up to 24 h. During
chyle production, rats were provided free access to a solution of 0.9%
NaCl, 0.05% KCl, and 5% glucose in water. To isolate chylomicrons,
the chyle was overlaid with 0.05% EDTA in saline, pH 7.4, and was spun
for 25 min at 100,000 × g and 18 °C. After
centrifugation, chylomicrons were aspirated and stored at 4 °C in
the dark for periods of up to 4 days prior to use in experiments.
Chylomicron triglyceride concentrations were determined using a
commercially available kit (Roche Molecular Biochemicals), according to
the manufacturer's instructions.
mice were fasted overnight prior to injection
with labeled chylomicrons. The next day, each animal was anesthetized,
and 100 µl of rat chylomicrons containing 3 mg of triglyceride and
trace [3H]retinyl ester (12 nmol, 0.2 µCi) was injected
into the right jugular vein. Blood samples were obtained from the
lateral tail vein at 5, 10, 30, and 60 min after chylomicron injection.
Four animals of each strain were sacrificed 10 and 60 min after
chylomicron injection. At the time of sacrifice, blood was taken, and a
total body perfusion with ice-cold PBS was performed (as described
above). The liver, spleen, kidney, lung, heart, brain, epididymal fat, and skeletal muscle (gastrocnemius) were excised and processed as
described below for assessing [3H]retinoid uptake.
mice as described by Bronzert and
Brewer (10). For isolation of these fractions, pools of mouse serum
were employed. The serum pools were constructed by mixing 300 µl of
serum obtained from 10 individual mice of the same strain, age, and
sex. The total recovery of retinyl esters that been present in the
serum pools when summed for the VLDL, LDL, and HDL fractions ranged
from 83 to 105%. Thus, essentially all of the retinyl ester present in the circulations of fasting WT and TTR
mice could be
accounted for by that present in these three lipoprotein fractions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mice.
Specifically, what is the physiological mechanism(s) responsible for
the low circulating retinol-RBP levels? For our present experiments, we
used inbred 129SV WT and TTR
mice, whereas we had earlier
characterized the TTR
phenotype in outbred MF-1 mice (4,
5). To link the two studies, we compared various biochemical
consequences of the TTR
mutation in the two genetic
backgrounds. As was the case for outbred MF-1 TTR
mice,
serum retinol and RBP levels in TTR
mice in the 129SV
genetic background were very low. For both mouse strains, serum retinol
and RBP levels in TTR deficiency were ~5% of those observed for age-
and sex-matched WT mice. Thus, in this regard, genetic background does
not influence the severity of the TTR
phenotype.
mice are shown in Table
I. Hepatic total retinol levels in WT and
TTR
male mice, whether outbred or inbred, were not
statistically different (5). Interestingly, female TTR
mice from both strains had significantly higher hepatic total retinol
concentrations than the corresponding WT controls. Since all of the
mice were maintained on the same chow diet, it is unlikely that dietary
intake accounts for this difference. Hepatic RBP levels for both male
and female 129SV TTR
mice were ~3-4-fold greater than
those of age- and sex-matched WT controls, whereas the difference in
the MF-1 background was only 1.6-fold (Table I (5)).
Nevertheless, although genetic background does quantitatively influence
the TTR
phenotype with regards to hepatic RBP levels, the
qualitative effects of TTR deficiency on serum and liver levels of
retinol and RBP are similar for outbred MF-1 and inbred 129SV
TTR-deficient mice.
Liver total retinol and RBP levels for 3-month-old inbred 129SV and
outbred MF-1 wild type and TTR mice
Liver RBP levels are elevated in vitamin A deficiency, resulting from a
blockage in the RBP secretory pathway (1, 2). We asked if TTR
deficiency likewise inhibited RBP secretion. Primary hepatocytes from
WT and TTR mice were isolated and cultured. Since
cultured hepatocytes are able to acquire TTR from culture medium (15),
we performed these experiments under three different growth conditions.
The control medium consisted of DMEM supplemented with 10% fetal calf
serum, which we estimate contains ~3-6 µg of bovine TTR per ml of
medium. Hepatocytes were also cultured in DMEM supplemented with 5%
serum obtained from the TTR
mouse or with DMEM
supplemented with 5% TTR
mouse serum and 1 µg of
purified human TTR per ml of medium. Table
II shows the rate at which these cells
secrete RBP into the medium. RBP accumulated at the same rate in media
in both WT and TTR-deficient hepatocytes in all three culture
conditions. Thus, neither endogenously synthesized nor exogenous TTR
influences the rate of RBP secretion from liver parenchymal cells.
Consistent with our observations in intact liver (5), TTR-deficient
hepatocytes had higher cellular RBP levels than WT cells.
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The suggestion that WT and TTR-deficient hepatocytes secrete RBP at
equivalent rates is supported by data obtained with nephrectomized WT
and TTR mice. Fig. 1 shows
serum RBP and retinol levels in age- and sex-matched WT and mutant mice
at various times after complete nephrectomy. RBP levels rise in the
circulation of WT and TTR
mice at nearly identical rates
for the initial 11 h (Fig. 1, panel A). Since the
kidney is thought to be the major tissue site of RBP catabolism in the
body (1), this result implies that RBP is secreted from the liver into
the blood at nearly identical rates for the two strains. Like RBP,
serum retinol levels (Fig. 1, panel B) initially rise at
similar rates for WT and TTR
mice and reach a plateau
level within 12 h. Hepatic RBP levels in both WT and
TTR
mice progressively declined in a
time-dependent manner, whereas hepatic albumin levels
remained constant (data not shown). A decline in hepatic RBP has been
reported previously for nephrectomized rats (16, 17). Overall, the data
from these in vivo studies are fully consistent with the
in vitro cell culture experiments; both indicate that TTR
does not play an essential role in RBP secretion.
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By having ruled out a defect in RBP secretion, we now asked if the low
circulating retinol-RBP levels in TTR mice were caused by
increased renal filtration of retinol-RBP complex, as proposed
previously (1). We therefore monitored the serum levels of
intravenous-injected human holo-RBP (10 µg of hRBP) in the
circulation of WT and TTR
mice. This dose of hRBP
increases the circulating retinol-RBP levels of WT mice by ~40% and
cannot, therefore, be considered a true tracer. However, since serum
TTR concentrations are usually in 2-3-fold molar excess of those of
circulating RBP (1), circulating mouse TTR should bind all the injected
retinol-hRBP in WT mice. Human RBP is known to bind rodent TTR (18).
Fig. 2, panel A, shows that
hRBP was rapidly cleared from the circulation of TTR
but
not from WT mice (t1/2 = 0.5 h and
t1/2 >6 h, respectively). Mouse RBP levels in the
serum of WT mice also declined slightly over 6 h (Fig. 2,
panel B). This decrease may reflect competition between human and mouse RBP for binding to TTR, the unbound RBP being lost from
the circulation.
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Kidney levels of hRBP rapidly rose in TTR mice, reaching
a maximum within 5-10 min after retinol-hRBP injection (Fig. 2,
panel C) and then quickly declined. Much lower levels of
hRBP were seen in the kidneys of WT mice, and these did not change over
the period measured (Fig. 2, panel C). Mouse RBP
concentrations were low in WT and TTR
mice and did not
vary over the 6 h following injection (Fig. 2, panel
D).
In contrast to kidney, hRBP concentrations in lung (Fig. 2, panel
E), skeletal muscle (Fig. 2, panel F), and liver,
heart, and fat (data not shown) of TTR mice were
consistently lower relative to wild type. These data argue against
increased infiltration of the retinol-hRBP complex into the
interstitial fluid in the absence of TTR.
Despite low serum retinol concentrations, TTR mice have
normal tissue total retinol levels. We asked if the ability to
concentrate retinol in tissues reflected a compensatory increase in the
clearance rate of chylomicron (dietary) retinoid. We injected rat
mesenteric chylomicrons labeled in vivo with
[3H]retinyl esters into the circulations of WT and
TTR
mice. Blood samples were collected 5, 10, 30, and 60 min after injection, and the levels of chylomicron-associated
[3H]retinoid were determined. No differences in
chylomicron clearance rates were observed for mutant versus
the WT mice (data not shown). At 10 and 60 min after injection, mice
from each strain were sacrificed, and tissue [3H]retinoid
levels were measured. As expected, most chylomicron retinoid was taken
up by the liver (18, 19). There was no significant difference between
TTR
and WT mice in liver [3H]retinoid
levels at either 10 or 60 min after injection. Moreover, the relative
amounts of chylomicron retinoid taken up by extrahepatic tissues were
similar in both WT and TTR
mice. Fig.
3 illustrates the tissue distribution of
[3H]retinoid 60 min after injection. We conclude that the
normal tissue retinol levels in TTR
mice cannot be
accounted for by enhanced clearance of postprandial (chylomicron)
retinoid.
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We next asked if the distribution of retinol among plasma lipoproteins
differed in WT and TTR mice. VLDL, LDL, HDL, and
d > 1.21 g/ml bottom fractions were isolated by
sequential ultracentrifugation from pools of serum obtained from age-
and sex-matched WT and TTR
mice. The concentration of
retinol and retinyl esters in each fraction was determined (Table
III). Since the mice had been fasted for
~18 h prior to sacrifice, this retinyl ester does not arise directly
from recent dietary intake. For both WT and TTR
mice, all
retinol in the serum pools was recovered solely in the
d > 1.21 g/ml bottom fraction, with an overall
recovery of 85%. This was expected since retinol is bound exclusively
to RBP in both WT and TTR
mice (5). The fasting serum for
both male and female WT and TTR
mice at three different
ages reproducibly contained low levels of retinyl esters. The level of
circulating retinyl ester in WT mice (when expressed as retinol
equivalents) was always less than 13% and usually less than 5% of the
level of circulating retinol. Because circulating retinol levels are
low in TTR
mice, a substantial percentage, 18-133%, of
the total retinol in these mice is retinyl ester. In both strains most
circulating retinyl ester is recovered in the liver-derived VLDL
fraction with lesser amounts in the LDL and HDL fractions. No retinyl
esters were detected in the d > 1.21 g/ml bottom
fraction.
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DISCUSSION |
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We reported earlier that the circulating levels of retinol and RBP
in TTR mice is ~5% that of WT (4, 5, 20).
Nevertheless, the tissue total retinol (retinol + retinyl ester) levels
of the TTR
mice were equivalent to those of WT (5). This
work addresses two issues. 1) Why are the circulating retinol and RBP
levels low in the mutant mice? 2) How do these mice maintain normal
tissue total retinol levels in the face of low serum retinol?
Early reports describing protein-protein interactions of retinol-RBP with TTR tetramer (55 kDa) speculated that association with TTR prevents renal filtration of the small (21 kDa) RBP molecule (1, 2). This hypothesis, although reasonable, had not been verified experimentally. TTR was also suggested to facilitate RBP synthesis/secretion from liver (21-23). Studies with 4-hydroxy-N-phenylretinamide, which disrupts the RBP-TTR complex and markedly lowers circulating retinol-RBP levels, suggested that unbound circulating RBP infiltrates into the interstitial space within tissues (24). In this work, we have tested these possibilities using genetically engineered TTR-deficient mice.
Although hepatic RBP levels are, indeed, significantly elevated in
TTR mice (see Table I), cultured primary hepatocytes
isolated from these mice accumulated RBP in the medium at control rates
(Table II). Furthermore, this rate was not influenced by TTR in the
culture medium. However, consistent with elevated RBP concentrations in intact liver, RBP levels in hepatocytes isolated from TTR
mice were significantly higher than WT controls. Whether TTR deficiency
inhibits RBP secretion or whether the accumulation of RBP exceeds the
secretion capacity of the hepatocyte remains to be determined. However,
the accumulation of intracellular RBP in TTR-deficient hepatocytes is
entirely consistent with observations that TTR facilitates RBP
synthesis and/or secretion from HeLa cells (21, 22) and from human
HepG2 hepatoma cells (23).
Renal failure in humans (1) and complete nephrectomy in experimental
animals (16, 17) give rise to significantly elevated circulating levels
of both retinol and RBP. Our studies of TTR and WT mice
indicate that circulating RBP levels rise at very similar rates for
both strains during the first 11 h following nephrectomy (Fig. 1).
Since liver is the major site of RBP synthesis (1, 2), this in
vivo result supports the physiological relevance of our RBP
secretion studies in isolated TTR
hepatocytes.
We showed that injected human retinol-RBP is much more rapidly cleared
from the circulation of TTR-deficient than WT mice. This result
demonstrates that TTR reduces the rate of renal filtration of
retinol-RBP and explains the low circulating levels of retinol-RBP in
TTR mice. Aside from the kidney, tissue hRBP
concentrations in the mutant mice declined at rates parallel to the
clearance rate of hRBP from the circulation. In addition, apart from
the liver where RBP is synthesized and the kidney where RBP is
filtered, mouse RBP concentrations are much lower in tissues from
TTR
than WT mice. Taken together, these observations do
not support the notion that TTR is required to keep circulating RBP
from partitioning into the interstitial fluid of tissues.
We have not yet determined how tissues of TTR mice
maintain normal total retinol concentrations in the face of low
circulating retinol-RBP concentrations, although several hypotheses are
under investigation. First, an alternative retinol delivery pathway may
compensate for the loss of RBP. Since lipoproteins transport retinyl
esters in the circulation, it is possible that either postprandial
lipoproteins (chylomicrons and their remnants) or lipoproteins of
hepatic origin (VLDL and LDL) play a more important role in retinol
delivery to tissues than RBP. Note that neither rate of clearance of
chylomicron retinyl ester nor its delivery to tissues is elevated in
TTR
mice. As a second alternative, a reduced delivery of
retinol to the tissues of TTR
mice may be compensated for
by a decrease in retinol catabolism. TTR-deficient mice rely on
circulating retinoic acid as a source of retinoid needed to maintain
normal gene expression. We reported previously that plasma
all-trans-retinoic acid levels were elevated by ~2.4-fold
in TTR
relative to WT mice (5), and studies in rats have
indicated that plasma retinoic acid can contribute substantially to
tissue retinoic acid pools (25). Enhanced acquisition of retinoic acid from circulating retinoic acid pools may reduce the need to oxidize tissue retinol to retinoic acid. This could have a sparing effect on
tissue total retinol pools and explain why tissue total retinol levels
are the same for WT and TTR
mice.
It is well established that some retinyl ester is bound to VLDL and LDL
in the fasting circulation of healthy humans (26, 27). We have extended
this observation to mice; fasting blood from both WT and
TTR mice contains low levels of retinyl ester (Table
III). Since TTR
mice have very low circulating retinol
levels, fasting lipoprotein retinyl ester concentrations represent a
sizable portion of circulating retinol concentrations. Thus,
lipoprotein transport of retinyl ester may play a relatively more
substantial role for delivering retinol to tissues of TTR
than for WT mice. As is the case with humans, most circulating retinyl
ester in WT and TTR
mice is present in the VLDL fraction
(26, 27). Since VLDL is directly secreted by hepatocytes and undergoes
metabolism in the circulation to give rise to LDL (28), we are tempted
to speculate that the retinyl ester present in VLDL may be incorporated into this lipoprotein at the time of its assembly in the hepatocyte.
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FOOTNOTES |
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* This work was supported by Grants EY12858 and DK52444 from the National Institutes of Health and grants from the United States Department of Agriculture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Medicine, Hammer Health Sciences Bldg., Rm. 502, Columbia University, 701 W. 168th St., New York, NY 10032. Tel.: 212-305-5429; Fax: 212-305-2801; E-mail: wsb2@columbia.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M008091200
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ABBREVIATIONS |
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The abbreviations used are:
RBP, retinol-binding
protein;
TTR, transthyretin;
TTR, transthyretin-deficient
strain of mice;
WT, wild type strain of mice;
DMEM, Dulbecco's
modified Eagle's medium;
HPLC, high performance liquid chromatography;
HDL, high density lipoprotein;
LDL, low density lipoprotein;
VLDL, very
low density lipoprotein;
RIA, radioimmunoassay;
hRBP, human
retinol-binding protein;
PBS, phosphate-buffered saline.
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