Lecithin:retinol acyltransferase from mouse and rat liver. CDNA cloning and liver-specific regulation by dietary vitamin a and retinoic acid

Lecithin:retinol acyltransferase (LRAT), present in microsomes, catalyzes the transfer of the sn-1 fatty acid of phosphatidylcholine to retinol bound to a cellular retinol-binding protein. In the present study we have cloned mouse and rat liver LRAT cDNA and tested the hypothesis that LRAT mRNA, like LRAT activity, is regulated physiologically in a liver-specific manner. The nucleotide sequences of mouse and rat liver LRAT cDNA each encode a 231-amino acid protein with 94% similarity between these species, and approximately 80% similarity to a cDNA for LRAT from human retinal pigment epithelium. Expression of rat LRAT cDNA in HEK293T cells resulted in functional retinol esterification and storage. RNA from several rat tissues hybridized with liver LRAT cDNA. However, LRAT mRNA was virtually absent from the liver of vitamin A-deficient animals, while being unaffected in intestine and testis. LRAT mRNA was rapidly induced by retinoic acid (RA) in liver of vitamin A-deficient mice and rats (P < 0.01). LRAT mRNA and enzymatic activity were well correlated in the same livers of rats treated with exogenous RA (r = 0.895, P < 0.0001), and in a dietary study that encompassed a broad range of vitamin A exposure (r = 0.799, P < 0.0001). Liver total retinol of <100 nmol/g was associated with low LRAT expression (<33% of control).We propose that RA, derived exogenously or from metabolism, serves as an important signal of vitamin A status. The constitutive expression of liver LRAT during retinoid sufficiency would serve to divert retinol into storage pools, while the curtailment of LRAT expression in retinoid deficiency would maintain retinol for secretion and delivery to peripheral tissues.     

Vitamin A concentrations in serum and liver from Florida panthers

Many of the anomalies and clinical signs afflicting the Florida panther (Felis concolor coryi) are suggestive of vitamin A deficiency. Our objectives in this study were to determine if a vitamin A deficiency exists in the free-ranging panther population and to determine if there are differences in vitamin A levels among various subgroups of free-ranging panthers. Retinol concentrations were used as an index to Vitamin A concentrations and were determined in serum and liver from free-ranging (serum, n = 45; liver, n = 22) and captive (serum, n = 9; liver, n = 2) juvenile and adult Florida panthers from southern peninsular Florida (USA), and in liver from free-ranging cougars (F. concolor subspp.) from Washington (USA) and Texas (USA) between November 1984 and March 1994. Combined juvenile (6- to 24-mo-old) and adult (> 24-mo-old) free-ranging Florida panthers had mean +/- SD serum retinol concentrations of 772.5 +/- 229 pmol/ml. Adult free-ranging Florida panthers had mean liver retinol concentrations of 4794.5 +/- 3747 nmol/g. Free-ranging nursing Florida panther kittens (age < 1 mo) had mean serum retinol concentrations of 397.9 +/- 69 pmol/ml. Among subgroups of free-ranging Florida panthers, females had higher corrected mean serum retinol concentrations than males and adult free-ranging Florida panthers had higher mean liver retinol concentrations than juveniles. Retinol concentrations in free-ranging Florida panthers did not differ significantly from those in captive panthers (liver and serum) or other free-ranging cougars (liver). Based on limited published values and our controls, a vitamin A deficiency could not be demonstrated in the Florida panther population nor were any subgroups or individuals considered deficient.     

Vitamin A status regulates hepatic lecithin: retinol acyltransferase activity in rats

The activity of lecithin:retinol acyltransferase (LRAT) was determined in microsomes from the liver and small intestine of rats with differing vitamin A status. In animals depleted of retinol, as judged by undetectable liver vitamin A stores and low plasma retinol concentrations, hepatic LRAT activity was almost undetectable, whether assayed with retinol bound to cellular retinol-binding protein or solvent-dispersed retinol. In contrast, neither the activity of intestinal LRAT nor that of acyl-CoA:retinol acyltransferase in either liver or intestine differed from that of vitamin A-adequate rats. During the course of vitamin A depletion, liver LRAT activity fell progressively, nearly in parallel to the decrease in plasma retinol concentration. Oral repletion of vitamin A-depleted rats with 0.8 mg of retinol resulted in a very rapid restoration of plasma retinol concentration and full recovery of hepatic LRAT activity within 24 h, together with deposition of retinyl ester in the liver. These data strongly implicate LRAT activity in liver as responsible for the storage of hepatic retinyl esters. Retention of the intestine's capacity to esterify retinol during vitamin A deficiency provides a mechanism for capture of dietary vitamin A, while reduced hepatic LRAT activity may function to redirect retinol in liver from storage to other metabolic pathways.     

Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes

Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consistent with lecithin:retinol acyltransferase (LRAT) activity. LRAT activity exhibited the same Km (2 microM retinol) between tissues but a higher Vmax in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg microsome protein, respectively). Solvent-dispersed retinol was also esterified in a fatty acyl CoA-dependent, PMSF-resistant reaction, consistent with acyl CoA:retinol acyltransferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity displayed a similar Vmax (300 pmol/min/mg microsome protein) between tissues but Km values of 15 and 5 microM for retinol and fatty acyl CoA in mammary gland as compared to 30 and 25 microM, respectively, in the liver. Thus, when substrate was near or below Km, retinol esterification occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concentration of CRBP in the cytosol, determined by Western blotting, was approximately 2 microM in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esterification is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver.     

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin A deficiency induces prooxidant environment and inflammation in rat aorta

We evaluated whether nutritional vitamin A deficiency generates oxidative stress and inflammation in aorta. Wistar male rats (21 days old) were given free access to a control (8 mg retinol as retinyl palmitate/kg) or a vitamin A- deficient diet for three months. One group of deficient animals was fed with the control diet fifteen days before sacrifice. Thiobarbituric acid-reactive substances (TBARS) and nitrite concentration where both analyzed in serum and aorta. Aorta Copper-Zinc Superoxide dismutase (CuZnSOD), Glutathion peroxidase (GPx) and Catalase (CAT) activities were measured. In addition, binding activity of the nuclear factor- kB (NF-kB), inducible and endothelial Nitric Oxide synthase (iNOS and eNOS, respectively) and Ciclooxygenase-2 (COX-2) expressions were determinated in aorta. Rats fed the vitamin A- deficient diet were characterized by sub-clinical plasma retinol concentration and showed increased serum and aorta concentrations of TBARS compared to controls. Lower than control activities of CuZnSOD, GPx, and CAT were observed in aorta of the vitamin A- deficient group. The binding activity of NF- kB was higher in vitamin A- deficient animals than controls. In addition, NO production evaluated as nitrite concentration increased in aorta and serum, associated with a higher expression of iNOS, eNOS and COX-2 in aorta of vitamin A-deficient rats. The incorporation of vitamin A into the diet of vitamin A-deficient rats reverted the changes observed in TBARS level, CuZnSOD and GPx activities, nitrite concentration and also, iNOS, eNOS and COX-2 expression. Prooxidant environment and inflammation are induced by vitamin A deficiency in rat aorta.     

Vitamin A toxicity in wild-caught African green vervet monkeys (Chlorocebus aethiops) after 2 years in captivity

Primate lab diets typically contain high vitamin A concentrations when compared with human recommended intakes. In this study, we analyzed the vitamin A contents of liver and serum from 13 adult female African green vervet monkeys (Chlorocebus aethiops). These monkeys were wild-caught and held in captivity for 2 y, during which time they consumed a standard primate diet. Liver vitamin A concentration (mean +/- 1 standard deviation) was 14.6 +/- 2.3 micromol retinol/g liver; subtoxicity in humans is defined as at least 1 micromol/g liver. The serum retinol concentration (0.93 +/- 0.21 microM) was not elevated. Hypertrophy and hyperplasia of hepatic stellate cells were present which, in conjunction with elevated hepatic vitamin A concentrations, are evidence of toxicity. Although the ramifications of chronically toxic vitamin A status in experimental monkeys have not been defined, this state may influence nonhuman primate research outcomes and confound data interpretation. The validity of bone mineral research using nonhuman primates is of greatest concern, in light of the association between vitamin A toxicity and compromised bone health.     

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster

Abstract. Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well-established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A-supplemented and vitamin A-deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1 -9 μg/g liver, respectively, at 28 d. In contrast, in animals on the vitamin A-deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0–32 and 0–09 μg/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d; their tracheas were depleted of retinol at 28 d. No vitamin A-associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A-deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild-to-severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A-deficiency when the tissue levels of the vitamin are depleted.     

Retinoid metabolism (LRAT,REH) in the yolk-sac membrane of Japanese quail eggs and effects of mono-ortho-PCBs

Retinoids stored in the avian egg are essential for normal development, however, laboratory and field experiments suggest that they are affected by environmental contaminants. Lecithin:retinol acyltransferase (LRAT) activity was detected in the microsomal fraction of the yolk-sac membrane of the Japanese quail at day 6 of development. LRAT activity was maximal at pH 7.0 having apparent kinetic parameters of K(m)=1.35 microM and V(max)=0.21 nmol/mg protein/h and was inhibited by the sulfhydryl modifying agent N-ethyl-maleimide. Retinol ester hydrolase (REH) activity in the microsomal fraction of the yolk-sac membrane was stimulated by the bile salt analogue 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane sulfonate and was maximal at pH 9.0 with apparent K(m)=77 microM and V(max)=34.3 nmol/mg protein/h. Injection of the PCB congener 2,3,3',4,4'-pentachlorobiphenyl increased both REH and LRAT activities, whereas 2,3,3',4-tetrachlorobiphenyl stimulated LRAT. Yolk retinol concentration and the molar ratio retinol:retinyl palmitate were lower in the exposed eggs. Yolk retinol concentration decreased as LRAT increased (R(2)=0.89) suggesting that certain PCB congeners may affect vitamin A mobilization in ovo by increasing LRAT activity in the yolk-sac membrane.     

Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein

Vitamin A (all-trans-retinol) must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. Vitamin A is transported in the blood bound to retinol-binding protein (holo-RBP), and its target cells express an RBP receptor encoded by the Stra6 (stimulated by retinoic acid 6) gene. Here we show in mice that cellular uptake of vitamin A from holo-RBP depends on functional coupling of STRA6 with intracellular lecithin:retinol acyltransferase (LRAT). Thus, vitamin A uptake from recombinant holo-RBP exhibited by wild type mice was impaired in Lrat(-/-) mice. We further provide evidence that vitamin A uptake is regulated by all-trans-retinoic acid in non-ocular tissues of mice. When in excess, vitamin A was rapidly taken up and converted to its inert ester form in peripheral tissues, such as lung, whereas in vitamin A deficiency, ocular retinoid uptake was favored. Finally, we show that the drug fenretinide, used clinically to presumably lower blood RBP levels and thus decrease circulating retinol, targets the functional coupling of STRA6 and LRAT to increase cellular vitamin A uptake in peripheral tissues. These studies provide mechanistic insights into how vitamin A is distributed to peripheral tissues in a regulated manner and identify LRAT as a critical component of this process.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Rapid induction of vitamin A deficiency in rabbits

Clinical and biochemical evidence of vitamin A deficiency was produced in rabbits as early as 4-5 weeks after weaning to a vitamin A deficient diet from dams maintained during lactation on the deficient diet. Mean serum retinol levels at the time of weaning for the deficient dams were 25 +/- 6 micrograms/dl compared with 74 +/- 8 micrograms/dl for the controls. Five weeks after weaning, 25% of pups fed the vitamin A deficient diet had ocular lesions characterized by the accumulation of sloughed epithelium on the cornea. At this time, mean serum values of the pups were 10 +/- 4 micrograms/dl for the deficient group and 73 +/- 8 micrograms/dl for the controls. Evidence of critically depleted liver stores was documented in the deficient rabbits by an elevated relative dose response test (54 +/- 18%) that did not occur in the control group (6 +/- 5%). Although food consumption was similar, weight gain was lower in the deficient group when compared to the control group.     

Effects of vitamin A deficiency on cell proliferation and morphology of trachea of the hamster

Regulation by vitamin A of cell proliferation and differentiation of epithelial tissues is well-established. Deficiency of vitamin A in experimental animals leads to the development of hyperplasia and squamous metaplasia. The objective of the present study was to examine, for young hamsters, the effects of variable levels of the vitamin in the liver and trachea, on cell proliferation and morphology of tracheal epithelium and on body weights. Newly born litters were maintained on vitamin A-supplemented and vitamin A-deficient diets, and various parameters were examined at different ages. Retinol and retinyl palmitate levels were determined by high performance liquid chromatography. For animals on the supplemented diet, concentrations of liver retinyl palmitate and retinol increased progressively with age, reaching highest levels of approximately 84 and 1.9 micrograms g liver, respectively, at 28 d. In contrast, in animals on the vitamin A-deficient diet, the retinyl palmitate and retinol levels decreased progressively, reaching the lowest levels of approximately 0.32 and 0.09 micrograms/g, respectively. No significant reduction in retinol was observed in the trachea of animals maintained on the deficient diet for at least 20 d: their tracheas were depleted of retinol at 28 d. No vitamin A-associated differences were, however, observed in the labelling indices, growth fraction or in the morphology of the tracheal epithelium. Both the control and vitamin A-deficient animals gained weight progressively until 36 d of age, although the weight of animals in the latter group remained below those in the former group. These results show that mild-to-severe deficiency of vitamin A had no effects on cell proliferation or tracheal morphology of the hamster. The hyperplasia and squamous metaplasia in the trachea occurs only at an extreme vitamin A-deficiency when the tissue levels of the vitamin are depleted.     

Cholate-independent retinyl ester hydrolysis. Stimulation by Apo-cellular retinol-binding protein

Apo-cellular retinol-binding protein (apoCRBP) activated the hydrolysis of endogenous retinyl esters in rat liver microsomes by a cholate independent retinyl ester hydrolase. A Michaelis-Menten relationship was observed between the apoCRBP concentration and the rate of retinol formation, with half-maximum stimulation at 2.6 +/- 0.6 microM (mean +/- S.D., n = 5). Two other retinol-binding proteins, bovine serum albumin and beta-lactoglobulin, acceptors for the rapid and spontaneous hydration of retinol from membranes, had no effect up to 90 microM. These data suggest activation of the hydrolase by apoCRBP directly, rather than by facilitating removal of retinol from membranes. The hydrolase responding was the cholate-independent/cholate-inhibited retinyl ester hydrolase as shown by: 60% inhibition of the apoCRBP effect by 3 mM cholate; apoCRBP enhancement of retinyl ester hydrolysis in liver microsomes that had no detectable cholate-enhanced activity; inhibition of cholate-dependent, but not apoCRBP-stimulated retinyl ester hydrolysis by rabbit anti-rat cholesteryl esterase. Compared to the rate (mean +/- S.D. of [n] different preparations) supported by 5 microM apoCRBP in liver microsomes of 6.7 +/- 3.7 pmol/min/mg protein [10], microsomes from rat lung, kidney, and testes had endogenous retinyl ester hydrolysis rates of 1.8 +/- 0.3 [5], 0.5 +/- 0.2 [3], and 0.3 +/- 0.2 [5] pmol/min/mg protein, respectively. N-Ethylmaleimide and N-tosyl-L-phenylalanine chloromethyl ketone were potent inhibitors of apoCRBP-stimulated hydrolysis with IC50 values of 0.25 and 0.15 mM, respectively, but phenylmethylsulfonyl fluoride and diisopropyl-fluorophosphate were less effective with IC50 values of 1 mM, indicating the importance of imidazole and sulfhydryl groups to the activity. These data provide evidence of a physiological role for the cholate-independent hydrolase in retinoid metabolism and suggest that apoCRBP is a signal for retinyl ester mobilization.     

Retinyl esters are the substrate for isomerohydrolase

Regeneration of 11-cis retinal from all-trans retinol in the retinal pigment epithelium (RPE) is a critical step in the visual cycle. The enzyme(s) involved in this isomerization process has not been identified and both all-trans retinol and all-trans retinyl esters have been proposed as the substrate. This study is to determine the substrate of the isomerase enzyme or enzymatic complex. Incubation of bovine RPE microsomes with all-trans [(3)H]-retinol generated both retinyl esters and 11-cis retinol. Inhibition of lecithin retinol acyltransferase (LRAT) with 10-N-acetamidodecyl chloromethyl ketone (AcDCMK) or cellular retinol-binding protein I (CRBP) diminished the generation of both retinyl esters and 11-cis retinol from all-trans retinol. The 11-cis retinol production correlated with the retinyl ester levels, but not with the all-trans retinol levels in the reaction mixture. When retinyl esters were allowed to form prior to the addition of the LRAT inhibitors, a significant amount of isomerization product was generated. Incubation of all-trans [(3)H]-retinyl palmitate with RPE microsomes generated 11-cis retinol without any detectable production of all-trans retinol. The RPE65 knockout (Rpe65(-/-)) mouse eyecup lacks the isomerase activity, but LRAT activity remains the same as that in the wild-type (WT) mice. Retinyl esters in WT mice plateau at 8 weeks-of-age, but Rpe65(-/-) mice continue to accumulate retinyl esters with age (e.g., at 36 weeks, the levels are 20x that of WT). Our data indicate that the retinyl esters are the substrate of the isomerization reaction.     

Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3.

Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physiological roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3, and Adh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mg.kg(-1)), metabolism of retinol to retinoic acid in liver was reduced 10-fold in Adh1 mutants and 3.8-fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD(50) value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one generation on a retinol-supplemented diet containing 10-fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild-type (92-95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol- supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity.     

Impaired retinol utilization in Adh4 alcohol dehydrogenase mutant mice

Adh4, a member of the mouse alcohol dehydrogenase (ADH) gene family, encodes an enzyme that functions in vitro as a retinol dehydrogenase in the conversion of retinol to retinoic acid, an important developmental signaling molecule. To explore the role of Adh4 in retinoid signaling in vivo, gene targeting was used to create a null mutation at the Adh4 locus. Homozygous Adh4 mutant mice were viable and fertile and demonstrated no obvious defects when maintained on a standard mouse diet. However, when subjected to vitamin A deficiency during gestation, Adh4 mutant mice demonstrated a higher number of stillbirths than did wild‐type mice. The proportion of liveborn second generation vitamin A‐deficient newborn mice was only 15% for Adh4 mutant mice but 49% for wild‐type mice. After retinol administration to vitamin A‐deficient dams in order to rescue embryonic development, Adh4 mutant mice demonstrated a higher resorption rate at stage E12.5 (69%), compared with wild‐type mice (30%). The relative ability of Adh4 mutant and wild‐type mice to metabolize retinol to retinoic acid was measured after administration of a 100‐mg/kg dose of retinol. Whereas kidney retinoic acid levels were below the level of detection in all vehicle‐treated mice (<1 pmol/g), retinol treatment resulted in very high kidney retinoic acid levels in wild‐type mice (273 pmol/g) but 8‐fold lower levels in Adh4 mutant mice (32 pmol/g), indicating a defect in metabolism of retinol to retinoic acid. These findings demonstrate that another retinol dehydrogenase can compensate for a lack of Adh4 when vitamin A is sufficient, but that Adh4 helps optimize retinol utilization under conditions of both retinol deficiency and excess. Dev. Genet. 25:1–10, 1999. © 1999 Wiley‐Liss, Inc.     

Modification of Sertoli cell functions in vitamin A-deficient rats

FSH binding and cAMP responses to FSH in Sertoli cell-enriched testes were not affected by the vitamin A (retinol) status of the animals. These results indicate that changes in Sertoli cell functions during vitamin A deficiency are independent of FSH-Sertoli cell interactions. Concentrations of serum androgen binding protein (ABP) in vitamin A-deficient rats were consistently higher than those of control animals throughout the study period. The accumulation of testicular fluid after efferent duct ligation, an indication of Sertoli cell secretory function, was normal in vitamin A-deficient rats at least until 70 days of age, but declined thereafter. ABP concentrations in seminiferous tubular fluid of vitamin A-deficient rats increased transitorily during the 70-80-day age period but returned to normal by 90 days. The increment of ABP in seminiferous tubular fluid after efferent duct ligation, and ABP concentrations in interstitial fluid were consistently lower in vitamin A-deficient rats. The higher serum ABP in vitamin A-deficient rats therefore cannot be explained by an increase in the permeability of Sertoli-cell tight junctions or basement membrane.