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Department of Nutrition and Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
1Correspondence: Department of Nutrition, 126-S Henderson Bldg., Pennsylvania State University, University Park, PA 16802, USA. E-mail: acr6{at}psu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and RXR-
, was not changed
after RA treatment in vivo. In a model of chronic
vitamin A ingestion during aging, CYP26 mRNA expression, determined by
Northern blot and RT-PCR analysis, increased progressively with dietary
vitamin A (P<0.0001; marginal < control <
supplemented) and age (P<0.003). The relative
expression of CYP26 mRNA was positively correlated with liver total
retinol (log10), ranging from undetectable CYP26 expression
at liver retinol concentrations below 
20 nmol/g to a three- to
fourfold elevation at concentrations >10,000 nmol/g
(r=0.90, P<0.0001). We conclude that
CYP26 expression and RA metabolism are regulated in adult liver not
only acutely by RA administration, as may be relevant to retinoid
therapy, but under chronic dietary conditions relevant to vitamin A
nutrition in humans.—Yamamoto, Y., Zolfaghari, R., Ross, A. C.
Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic
acid metabolism by retinoids and dietary vitamin A in liver of mice and
rats.
Key Words: retinoic acid oxidation • vitamin A status • aging
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
, the regulation of retinoid molecules is not well
understood.
The liver is a principal site of retinoid metabolism. Dietary vitamin
A, absorbed mainly as esterified retinol, is transported from the
intestine to liver by chylomicra and cleared rapidly into hepatocytes
(4
5
6)
. Within these cells, retinyl esters are hydrolyzed
and the retinol thus formed is subject to several potentially competing
metabolic pathways. Retinol may be re-esterified for storage in
hepatocytes and stellate cells, secreted into plasma bound to retinol
binding protein, or sequentially oxidized to form retinal and RA
(6
, 7)
. Liver cytosol contains retinol and retinoic acid
binding proteins that participate in retinoid metabolism (8
, 9)
, whereas liver microsomes and cytosol contain members of
several families of retinoid dehydrogenases and oxidases capable of
oxidizing retinol and retinal and producing RA (10
11
12
13)
.
RA either derived endogenously or administered exogenously may be
oxidized to form more polar metabolites (14)
. The majority
of exogenously administered RA was rapidly excreted (15
, 16)
and both unchanged RA and polar metabolites were recovered
in the bile of bile duct-cannulated rats and the perfusate of isolated
perfused rat liver (15
, 17)
. Less is understood of the
fate of endogenously produced RA, which is formed by the metabolism of
vitamin A precursors and is present in cells and plasma at only
nanomolar concentrations (11)
.
Catabolism appears to play a major role in limiting RA concentration
(14)
. In 1980, Roberts et al. (18)
demonstrated the sequential conversion of RA to 4-hydroxy- and 4-oxo-RA
by hamster liver microsomes. The involvement of a cytochrome
P450-dependent pathway in RA metabolism was inferred by the biochemical
properties of the oxidative process (18)
and by the
ability of known cytochrome P450 inhibitors to inhibit RA catabolism
in vitro and in vivo (19
20
21)
.
Recently, the nature of RA catabolism was clarified with the cloning of
a novel gene, CYP26, which encodes a cytochrome P450-related
hydroxylase (22
23
24
25)
. CYP26 (22
, 25)
, also
named P450RA (24)
for its RA
inducibility, was cloned from regenerating, RA-treated zebra fish fin
(25)
, murine embryonic stem cells (22
23
24)
,
and a human cDNA library (22)
. Although analysis of the
CYP26 promoter region has not been published, RAR- and RXR- were
implicated in regulating CYP26 gene expression in F9 cells
(26)
. The transfection of CYP26 cDNA into cell lines
(22
, 24
, 26)
resulted in the oxidation of exogenous
all-trans RA to products identified as 4-hydroxy- and 4-oxo
retinoids, but the metabolism of retinol, retinal, or ß-carotene was
not induced (24)
. Transfected cells exhibited reduced
sensitivity to RA-induced differentiation and reporter gene
trans-activation, implying that RA is inactivated by the
action of CYP26 (24)
.
Besides being expressed in embryonic stem cells and during development
(23
, 25
, 27)
, CYP26 mRNA was detected by reverse
transcriptase-polymerase chain reaction (RT-PCR) in adult mouse and
human liver, brain, and placenta, but not in several other visceral
organs (23)
. In mice treated with a high dose (100 mg/kg,
i.p.) of all-trans RA, CYP26 expression was induced in
liver, but not brain (23)
. However, RA metabolism was not
studied. Thus, it is still unclear whether CYP26 is involved in RA
metabolism under physiological conditions, and no studies of its
potential regulation by vitamin A nutritional status have been
reported. With this background, the present study was designed to
clarify whether the expression of CYP26 and the metabolism of RA are
regulated in liver of intact animals under conditions of acute,
nontoxic RA treatment and chronic dietary exposure to different levels
of vitamin A. We found the expression of CYP26 and RA metabolism to be
reduced during vitamin A deficiency, up-regulated by acute treatment
with RA, and modulated by chronic dietary exposure to vitamin A. In a
chronic dietary model, CYP26 expression was strongly correlated with
liver retinol concentration. These results imply a role of CYP26 in
normal retinoid metabolism, especially when dietary vitamin A intake
and hepatic retinol reserves are elevated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In
the acute model, mice fed a stock rodent diet received a single i.p.
dose of all-trans RA, in dimethyl sulfoxide that equaled
either 100 mg/kg body weight according to Ray et al. (23)
or 5 mg/kg; 24 h later mice were killed and livers were collected
and stored at -70°C. Rats fed a nutritionally complete semisynthetic
diet [AIN-93 (29)
] or the same diet lacking vitamin A
received two oral doses of 100 µg all-trans RA dissolved
in 20 µl corn oil or corn oil as placebo 2 days and 18 h before
death (30)
. In the chronic dietary model, male Lewis rats
were raised, as described previously (28)
, from the time
of weaning until they reached the ages of 2–3 months (designated
young), 8–10 months (middle-aged), or 18–20 months (old) on a
semisynthetic diet (29)
containing 0.35 mg retinol
equivalents/kg diet (denoted as vitamin A marginal), 4 mg/kg (control),
or 50 mg/kg (vitamin A-supplemented) (28)
. Body weight,
plasma retinol (28)
, liver total retinol
(31)
, and various biochemical indices of health status
(serum alanine:aspartate aminotransferase, bilirubin, protein, albumin,
triglycerides and cholesterol) have been reported for these animals
(28)
.
RNA isolation and analysis
Total RNA samples from individual livers in each treatment group
was pooled (two livers per pool) for isolation of
poly(A)+ RNA (32)
and RT-PCR
analysis. Poly(A)+ RNA samples (3 µg of each
sample) were fractionated, transferred, and hybridized with cDNA probes
labeled with -32P-[dCTP] (3000 Ci/mmol)
using the Prime-a-Gene labeling kit (Promega, Madison, Wis.)
(32)
. For CYP26, a 1.7 kbp probe was prepared from a
plasmid containing mouse P450RAI cDNA (26)
, generously
provided by Dr. Martin Petkovich, Queen’s University (Kingston, Ont.).
After hybridization and washing, blots were exposed to XAR-5 film
(Eastman Kodak Co., Rochester, N.Y.) at -80°C for up to 4 days.
After the CYP26 probe was stripped off, membranes were rehybridized
with labeled mouse RAR- or RXR- (32)
and finally
with rat skeletal muscle ß-actin as a control (32)
.
For semiquantitative RT-PCR of CYP26 mRNA, 2 µg of the pooled total
RNA samples (2 rats/pool) in diethylpyrocarbonate-treated water was
denatured with 0.2 µg of oligo dT at 70°C for 10 min, cooled on
ice, and mixed in a final volume of 20 µl with 1x Superscript II
buffer (Life Technologies, Inc., Gaithersburg, Md.), 0.5 mM each of
dNTP (Promega), and 200 U of Superscript II reverse transcriptase (Life
Technologies). The RT reaction tubes were incubated at 42°C for
1 h for first strand synthesis, followed by incubation at 70°C
for 15 min to stop the RT reaction. Based on the cDNA sequence of the
mouse and human CYP26 (26)
, PCR primers were designed to
amplify a nucleotide fragment of 213 bp; the forward and reverse
primers were 5'-TTCTGCAGATGAAGCGCAGG-3' and 5'-TTTCGCTGCTTGTGCGAGGA-3',
respectively. Five microliters of the RT reaction (diluted to 100 µl)
was used in a final volume of 25 µl for the PCR reaction, which
contained 1x PCR buffer II (PE Applied Biosystems, Foster City,
Calif.), 1.5 mM MgCl2, 0.25 mM of each dNTP, 0.3
µM of each primer, and 0.6 U Gold Taq DNA polymerase (PE
Applied Biosystems). The amplification reaction was initiated by
incubation of PCR samples at 94°C for 10 min, followed by 35 cycles
of 94°C, 30 s; 60°C, 30 s; 72°C, 1 min. The PCR
products (10 µl) were subjected to electrophoresis in 2% agarose
gel, 0.3 µg/ml ethidium bromide. Data were scanned and quantified
using the NIH Image 1.56 program and Instat 1.11 software (GraphPad
Software, San Diego, Calif.).
Preparation of liver microsomes, enzyme assay, and HPLC analysis
Liver microsomes were prepared in 5 ml of 10 mM Tris-HCl, 250 mM
sucrose, pH 7.4 (33)
, and stored at -70°C prior to
analysis. Retinoic acid metabolizing activity was measured by
incubating varying amount of microsomal protein (34)
, as
noted in figure legends, with 60 nM of
(11,12-[3H(N)]-retinoic acid (35.8 Ci/mmol, NEN
Life Science Products, Inc., Boston Mass.) in 0.5 ml buffer containing
150 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl (pH 7.4),
and an NADPH regenerating system (2.5 units of glucose 6-phosphate
dehydrogenase, 500 nmol of NADP, and 0.5 µmol of glucose-6-phosphate)
(35)
. The reaction was initiated by adding
[3H]RA; after incubation at 37°C for the
times specified, the reaction was terminated by the addition of 1 ml
ethanol/0.025 M potassium hydroxide. The retinol and retinyl ester were
first removed by extracting twice in 4 ml of hexane. The ethanolic
phase was then acidified with 70 µl 5 N HCl and the radiolabeled RA
was extracted twice into 4 ml of hexane (36)
.
Subsequently, 3H remaining in the aqueous
subnatant was measured. For high-performance liquid chromatography
(HPLC), chromatographic conditions were as described by Teerlink et al.
(37)
. The samples from the acidified hexane extracts were
concentrated under argon at 37°C and redissolved in 110 µl of 95%
methanol-5% water-40 mM ammonium acetate, pH 5.75, for injection.
Samples were resolved by reverse-phase HPLC on a 3 µm 4.6 mm x
15 cm octadecasilyl column (Supelcosil LC-18, Supelco, Bellefonte, Pa.)
using a gradient (37)
of two mobile phases: A) 40 mM
ammonium acetate buffer (pH 5.75) in methanol (50:50 v/v); and B) 100%
methanol, beginning with 85% A:15% B, 0.8 ml/min, and increasing to
100% B over 22 min, followed by 100% B for 2 min. Fractions (20 s)
were assayed for 3H content by liquid
scintillation spectrometry. 4-Oxo-RA, kindly provided by M. Klaus,
Hoffmann-LaRoche (Basel, Switzerland), was used as a standard.
Statistics
Analysis of CYP26 mRNA expression in the chronic study was
performed by a 2-factor (diet, age) analysis of variance (ANOVA),
followed by a Tukey-Kramer test whether the main effects of diet or age
were significant. Group comparisons by diet were made by
least-significant difference test (SuperANOVA, Abacus Concepts,
Sunnyvale Calif.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(100 mg/kg, 2 mg/mouse). Northern blots of
poly(A)+ RNA were hybridized with a cDNA probe
specific for CYP26 (Fig. 1
). Little CYP26 mRNA was detected in control mouse liver (Fig. 1A
), whereas CYP26 was induced slightly by RA at 5 mg/kg and
very strongly by 100 mg/kg. These treatments did not affect the
expression of ß-actin mRNA used throughout as a control.
|
Studies were also conducted in normal and vitamin A-deficient rats
treated acutely with a lower dose of RA (two 100 µg oral doses, each
0.4 mg/kg body weight given 2 days and 18 h before livers were
collected). Similar to mice treated i.p. with much higher doses of RA,
CYP26 mRNA was barely detectable in the liver of normal and vitamin
A-deficient rats but was strongly induced after treatment with RA (Fig. 1B
).
CYP26 expression was investigated in rats that were part of a study of
the long-term effects of dietary vitamin A during aging
(28)
. Rats in this chronic study had been fed from the
time of weaning one of three diets that differed in vitamin A content
but otherwise were identical and nutritionally complete, designated
vitamin A marginal, control, and vitamin A-supplemented diet. At the
time of the study rats were 2–3 months (young), 8–10 months
(middle-aged), or 20–22 months (old) of age and their livers had
accumulated different concentration of vitamin A, depending on diet and
age (31)
, as noted below. CYP26 mRNA was barely detectable
in liver of young control rats (Fig. 1C
). CYP26 expression
increased with age in rats fed the control diet. CYP26 mRNA was readily
detected in liver of vitamin A-supplemented rats and expression
increased with age (young < middle-aged <old).
Total RNA from selected treatments was subjected to RT-PCR using
primers specific for CYP26. Figure 1D
compares the CYP26 PCR
amplicon from liver of vitamin A-deficient rats before and after
treatment with RA and from old rats raised on the vitamin A marginal,
control, or vitamin A-supplemented diets. CYP26 mRNA was not detected
in liver of vitamin A-deficient rats, but was significantly induced by
18 h after treatment with RA. In the chronic dietary study, CYP26
expression increased in a progressive manner with vitamin A intake
(vitamin A marginal < control < vitamin A-supplemented).
Indeed, the expression of CYP26 in rats fed vitamin A-supplemented diet
was similar to that of vitamin A-deficient rats after treatment with
all-trans RA (lane 2 vs. lane 5).
RAR- and RXR- were previously implicated in the regulation of
CYP26 gene expression based on the level of CYP26 mRNA in RA-treated F9
cells lacking specific retinoid receptors or in wild-type cells treated
with receptor-selective retinoids (26)
. However, in normal
rat liver, RAR- mRNA is very low (32
, 38
, 39)
. To
determine whether expression of RAR- or RXR- is correlated with
that of CYP26, RAR- and RXR- mRNA were determined by Northern
blot analysis. Although transcripts for both of these receptors were
detected in liver poly(A)+ RNA, as previously
reported (32)
, neither changed noticeably in response to
RA administration or from long-term dietary treatment with vitamin A
(data not shown).
Retinoic acid metabolism
To compare RA metabolism by livers in which CYP26 expression
differed significantly, microsomes from liver of mice and rats, treated
either acutely with RA or chronically by diet, were incubated with
[3H]RA and an NADPH-generating system.
Preliminary studies using microsomes from RA-treated rat liver were
conducted to determine suitable in vitro incubation
conditions. Figure 2
shows that the production of peak 1 (denoted by an arrow in
Fig. 2C
), corresponding to the elution position of 4-oxo-RA,
was protein dependent (Fig. 2A
) and time dependent (Fig. 2B
). The metabolism of [3H]RA was
significantly greater in microsomes from RA-treated rats than controls
(Fig. 2C
), negligible in heat-inactivated microsomes (Fig. 2C
), dependent on NADPH (not shown), and competed by a
500-fold excess of unlabeled RA (not shown). Besides the main peak
1, two or three other peaks of lesser polarity than 4-oxo-RA
but greater polarity than all-trans RA were also seen after
RA treatment. Because some of these metabolites are produced
sequentially, differences in metabolite profile with time and/or extent
of induction of metabolism are to be expected. Metabolites such as
4-hydroxy-RA and RA epoxides would be expected to elute in this region,
but these peaks were not further characterized in our study.
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The results of standard incubations of microsomes from RA-treated
mice and rats and from aging rats fed control and vitamin
A-supplemented diets are illustrated in Fig. 3
. Control mouse liver converted very little
[3H]RA to polar
[3H]-retinoids (Fig. 3A
). However,
microsomes from mice treated with 5 and especially 100 mg/kg of RA
produced peak 1 and other peaks migrating between 4-oxo-RA
and RA. Peak 1 was substantially induced in both vitamin
A-deficient and control rat livers after treatment with RA (Fig. 3B
).
|
RA metabolism was also studied in liver microsomes from aging rats fed
control or vitamin A-supplemented diets (Fig. 3C
).
Differences were greatest in livers of vitamin A-supplemented rats,
where there was a progressive increase in peak 1 with
increasing age (young < middle-aged < old). Overall, the
metabolism of [3H]RA was nearly fourfold
greater in old vitamin A-supplemented vs. old control rats.
The same microsomal incubations were used to determine the conversion
of [3H]RA to compounds that remained in the
aqueous phase after hexane extraction of acidic retinoids.
Aqueous-soluble 3H counts increased linearly with
microsomal protein to 150 µg and with time to 90 min (data not
shown). However, in contrast to lipid-soluble 3H
metabolites, aqueous-soluble 3H was nearly equal
in incubations from vitamin A-deficient and control rats and differed
little after acute RA or chronic dietary treatments. Thus, these
metabolites were not well correlated with CYP26 expression or
production of lipid-soluble 3H-RA metabolites.
Relationship of CYP26 mRNA to hepatic total retinol concentration
Analysis of CYP26 mRNA expression was performed by RT-PCR
for all groups of rats in the chronic diet study (three pools per
treatment with two livers per pool). The results (Fig. 4
) are expressed relative to the mean value for the young control group
(defined as 1.0); this group was selected for reference because most
studies of normal rats have used animals of approximately this age.
Values ranged from 0 (undetectable) in most vitamin A marginal rats to
three- to fourfold induced relative to the reference group. There were
significant main effects for both diet (P<0.0001, 2-factor
ANOVA) and age (P=0.0003), and a significant interaction
(P<0.05). Across all diet groups, CYP26 expression differed
by age (young<middle-aged<old, P<0.01, Tukey-Kramer
test); across all age groups, CYP26 expression differed by diet
(marginal < control < supplemented, P<0.05, Tukey-Kramer
test). Within ages, all diet groups differed (all P<0.001
by a least-squares means test, Fig. 4
).
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Because rats in the chronic dietary study were fed their respective
levels of vitamin A throughout life, age may have been a proxy for
exposure to vitamin A, which is reflected in the wide range of hepatic
vitamin A concentrations among these animals (31)
.
Therefore, the relationship between CYP26 expression and hepatic
retinol concentration was explored by regression analysis (Fig. 5
). There was a strong relationship between hepatic CYP26 mRNA and
log10 total retinol. Based on all data points
(n=27), there was a significant linear correlation
(r=0.909, P=0.0001). Since values for rats fed
the marginal diet were at or below detection limits, we reanalyzed the
data from only the control and vitamin A-supplemented groups. Again
there was a significant linear correlation (r=0.904,
P=0.0001, n=18).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 24
, 40)
or during embryonic
development (24
, 27)
or limb regeneration
(25)
. A study that investigated CYP26 expression in
tissues of adult mice (23)
examined gene induction only
after mice were treated with a very high dose of all-trans
RA (100 mg/kg) and retinoid metabolism was not examined. The principal
finding of the present investigation is that CYP26 gene expression is
barely expressed in normal adult rodent liver, but is strongly induced
not only by acute treatment with RA, including doses lower than
previously tested, but by long-term exposure to dietary vitamin A. In
rats given no exogenous RA but fed diets differing in vitamin A content
from deficient to marginal, control, and supplemented, CYP26 mRNA
increased progressively (Fig. 1D
). The level of CYP26 mRNA
in liver of old, vitamin A-supplemented rats was nearly equal to that
in liver of vitamin A-deficient rats treated with exogenous RA. Thus,
either acute retinoid treatment, as may resemble conditions when RA is
used therapeutically (see ref 19
for a review), or the
chronic exposure to dietary vitamin A at an elevated but nontoxic level
induced CYP26 gene expression similarly. In intact animals as compared
to cultured cells (26)
, the expression of RAR- and
RXR- did not correlate with CYP26 expression, suggesting their
constitutive expression was sufficient for CYP26 induction. Overall,
these data establish that a continuum of CYP26 expression exists in
liver under physiologically relevant conditions, varying from no
detectable expression in vitamin A deficiency, to minimal expression in
animals fed a vitamin A marginal diet, and progressively increasing
when dietary vitamin A was at an adequate (control) level. When vitamin
A intake is above an adequate level, as in vitamin A-supplemented rats,
CYP26 mRNA is up-regulated. The extent of up-regulation increased with
age, as did liver vitamin A contents (see below). Thus, the induction
of CYP26 was maintained as a steady-state adaptation to an elevated
intake of vitamin A; this adaptation occurred in the absence of
exogenous RA. If we consider the expression of CYP26 in young
control-fed rats to represent a normal level of CYP26 expression, the
results demonstrate down-regulation or chronic repression in rats fed
vitamin A-deficient or marginal diet, as well as chronic induction in
vitamin A-supplemented rats. It is plausible that CYP26 regulation
provides an important mechanism to conserve the body’s endogenously
formed RA when the precursor supply of dietary vitamin A is limited and
to remove RA when it builds up.
It is important to note that the vitamin A-supplemented diet used in
this study did not induce hepatic or systemic toxicity
(28)
. To test for potential hepatotoxicity, serum
alanine:aspartate transaminase, bilirubin, and albumin were analyzed,
but these did not differ with dietary treatment and all values were
within normal range (28)
. The vitamin A-supplemented rats
in this study may be a good model for human beings who are chronic
users of vitamin/mineral supplements and/or foods such as liver that
contain high levels of preformed vitamin A (see ref 28
for
a discussion).
RA metabolism
There was a generally good relationship between CYP26 mRNA and RA
metabolism when CYP26 was induced in RA-treated mice and rats and
vitamin A-supplemented aging rats. One of the retinoid peaks detected,
peak 1, comigrated with 4-oxo-RA and therefore is putatively
identified as [3H]4-oxo-RA; other peaks were
consistent with their being metabolites, such as 4- or 18-hydroxy-RA
and retinoid epoxides, which have been identified previously in
microsomal incubations (35)
or CYP26-transfected,
RA-treated cells (24
, 25)
. Previous nutritional studies
showed an increased rate of retinol metabolism and retinoid excretion
as dietary vitamin A intake increased (41)
. However,
neither the scope of RA metabolism nor the biological significance of
oxidized retinoids is well understood. RA administered to intact
animals is eliminated rapidly from plasma (16)
and is
excreted in bile in the form of unchanged RA as well as a variety of
oxidized metabolites and glucuronide conjugates (17)
. But
whether metabolites of RA are catabolic end products destined for
excretion or perhaps have functional properties in certain situations
is still uncertain. In HeLa cells, overexpression of CYP26 reduced the
ability of RA to induce the trans-activation of
retinoid-responsive promoter elements (24)
, suggesting
that a function of CYP26 is to induce RA catabolism and thus limit
RA’s biological activities. On the other hand, RA metabolites may also
be bioactive, as exemplified by the restoration of spermatogonial
division in 4-oxo-RA-treated vitamin A-deficient mice (42)
and by the differentiation of CYP26-transfected, RA-treated P19 cells
toward a neuronal phenotype, which was not induced by RA alone
(40)
. In addition, other cytochrome P450s have been
described as being regulated by retinoids (43
, 44)
or as
playing a role in retinoid 4-hydroxylation (45
46
47)
, which
may precede the formation of 4-oxo (keto)-RA (18)
. Further
studies in different cell types and intact animals are needed to
clarify the range and activities of cytochrome P450s involved in
retinoid metabolism.
Correlation of CYP26 mRNA with liver retinol
Significant differences in CYP26 expression due to diet and age
were apparent from the analysis of data shown in Fig. 4
. Hepatic
vitamin A is considered one of the best indicators of vitamin A status
(48)
. Rats in the chronic dietary study were known to have
a very wide range of liver total retinol concentration
(31)
; therefore, it was of interest to determine the range
of liver total retinol concentration over which CYP26 expression varied
(Fig. 5)
. While there was little CYP26 mRNA in any of the vitamin A
marginal groups regardless of age or liver vitamin A, CYP26 mRNA
increased nearly linearly with the log of liver retinol concentration
in control and vitamin A-supplemented rats. It has been inferred from a
variety of data that human beings with liver total retinol
concentrations <20 µg/g (70 nmol/g) are at risk of vitamin A
deficiency (see ref 48
). Indeed, plasma retinol also tends
to fall when liver retinol is <20 µg/g (48)
. Based on
the relationship between CYP26 expression and liver retinol shown in
Fig. 5
, CYP26 mRNA expression equal to half that of the mean young
control value would correspond to a liver vitamin A concentration of
50 nmol total retinol/g, or 14 µg/g. Thus, CYP26 expression is
regulated in a range where other biochemical processes indicative of
vitamin A status, such as regulation of plasma retinol, are also
regulated. The regulation of hepatic CYP26 expression may be an
important mechanism for tightly controlling the concentration of RA, a
potent hormone, in the face of wide, but still physiological,
differences in vitamin A intake and hepatic vitamin A accumulation. In
addition, it has been reported that a major portion or even the
preponderance of liver RA is derived by uptake from plasma
(16)
; this RA presumably represents RA produced in
peripheral tissues. Thus the liver, through the regulation of CYP26
expression, may play a central role in regulating RA concentrations
throughout the body. If so, then the hepatic regulation of CYP26
expression could, in turn, have far-reaching effects on peripheral
retinoid concentrations and on retinoid-responsive gene transcription
in target tissues as well as within liver.
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ACKNOWLEDGMENTS |
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and RXR-, and M. Klaus for
4-oxo-retinoic acid. This work was supported by National Institutes of
Health grants DK-46869 and AG-09839 and by funds from the Howard Heinz
Endowment.
Received for publication February 7, 2000.
Revision received April 11, 2000.
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REFERENCES |
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 L. Liu and L. J. Gudas Disruption of the Lecithin:Retinol Acyltransferase Gene Makes Mice More Susceptible to Vitamin A Deficiency J. Biol. Chem., December 2, 2005; 280(48): 40226 - 40234. [Abstract] [Full Text] [PDF]
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 M. D. Gulkac, G. Akpinar, H. Ustun, and A. Ozon Kanli Effects of vitamin A on doxorubicin-induced chromosomal aberrations in bone marrow cells of rats Mutagenesis, May 1, 2004; 19(3): 231 - 236. [Abstract] [Full Text] [PDF]
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A. C. Ross and R. Zolfaghari Regulation of Hepatic Retinol Metabolism: Perspectives from Studies on Vitamin A Status J. Nutr., January 1, 2004; 134(1): 269S - 275. [Abstract] [Full Text] [PDF]
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A. C. Ross Retinoid Production and Catabolism: Role of Diet in Regulating Retinol Esterification and Retinoic Acid Oxidation J. Nutr., January 1, 2003; 133(1): 291S - 296. [Abstract] [Full Text] [PDF]
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 R. Zolfaghari and A. C. Ross Lecithin:retinol acyltransferase from mouse and rat liver: cDNA cloning and liver-specific regulation by dietary vitamin A and retinoic acid J. Lipid Res., December 1, 2000; 41(12): 2024 - 2034. [Abstract] [Full Text]
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