Livers Of Vitamin A-deficient Rats Research Articles

Daily oscillation of glutathione redox cycle is dampened in the nutritional vitamin A deficiency

Examples of hormonal phase-shifting of circadian gene expression began to emerge a few years ago. Vitamin A fulfills a hormonal function by binding of retinoic acid to its nuclear receptors, RARs and RXRs. We found retinoid- as well as clock-responsive sites on regulatory regions of Glutathione reductase (GR) and Glutathione peroxidase (GPx) genes. Interestingly, we observed retinoid receptors, as well as GSH, GR, and GPx, display daily oscillating patterns in the rat liver. We also found that feeding animals with a vitamin A-free diet, dampened daily rhythms of RARα and RXRβ mRNA, GR expression and activity, GSH, BMAL1 protein levels, and locomotor activity. Differently, day–night oscillations of RXRα, GPx mRNA levels and activity and PER1 protein levels, were phase-shifted in the liver of vitamin A-deficient rats. These observations would emphasize the importance of micronutrient vitamin A in the modulation of biological rhythms of GSH and cellular redox state in liver.

Phospho-STAT5 accumulation in nuclear fractions from vitamin A-deficient rat liver

The growth hormone (GH)-responsive cytochrome P450 (CYP) 2C11 is down-regulated in vitamin A-deficient (VAD) rat liver. This study assessed the impact of a VAD diet on the hepatic Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) system that mediates GH signalling. Nuclear tyrosine- and serine-phosphorylated STAT5 accumulated in VAD liver, whereas nuclear JAK2 tyrosine kinase and SHP-1 phosphatase were decreased. Tyrosine-phosphorylated SHP-1 was decreased to 36 ± 14% of control ( P < 0.01), indicating its impaired activation in VAD liver. Episodic GH pulses increased nuclear phospho-STAT5, especially in control liver, but nuclear phospho-JAK2 and phospho-SHP-1 were not restored. CYP2C11 protein and testosterone 16α-hydroxylation were decreased in VAD liver to 67 ± 16% and 76 ± 19% of control, and were further decreased by GH to 32 ± 8% and 30 ± 14% of control. Thus, hypo-responsiveness of JAK-STAT in VAD liver is associated with impaired nuclear phospho-STAT dephosphorylation.

Differential regulation of endobiotic-oxidizing cytochromes P450 in vitamin A-deficient male rat liver.

1. The hepatic CYP4A-dependent omega-hydroxylation of arachidonic acid and CYP2C11-dependent 2alpha-/16alpha-hydroxylations of testosterone were decreased to 74 and 60% of respective control in microsomal fractions from vitamin A-deficient rats. Decreases in the rates of arachidonic acid omega-1-hydroxylation and testosterone 6beta-, 7alpha- and 17alpha-hydroxylations were less pronounced. 2. Corresponding decreases in microsomal CYP4A and CYP2C11 immunoreactive protein expression to 64 and 68% of respective control were observed in vitamin A-deficient rat liver. Expression of CYP3A proteins was unchanged from vitamin A-adequate control. 3. Northern analysis revealed a selective decrease in CYP4A2 mRNA expression in vitamin A-deficient rat liver to approximately 5% of control; expression of the related CYP4A1/4A3 mRNAs was not decreased. CYP2C11 mRNA expression was also decreased in vitamin A-deficient male rat liver to 39% of control levels. 4. Intake of the deficient diet containing all-trans-retinoic acid (ATRA) during the final week of the experiment restored CYP4A2 mRNA and CYP4A protein. Administration of exogenous androgen or episodic growth hormone was ineffective. In contrast, CYP2C11 expression was restored by ATRA and androgen, but not by growth hormone. 5. From these studies it emerges that CYP4A2, a fatty acid omega-hydroxylase in rat liver, is highly dependent on vitamin A for optimal expression, whereas CYP2C11 is indirectly down regulated by androgen deficiency resulting from vitamin A-deficiency. Altered CYP expression in vitamin A-deficiency provides insights into the relationship between dietary constituents and the intracellular formation of vasoactive eicosanoids as well as the clearance of androgenic steroids.

Hepatic lecithin: Retinol acyltransferase activity is induced in vivo by retinoic acid, but not by triiodothyronine, in vitamin A-deficient, hypothyroid rats

The activity of the enzyme lecithin; retinol acyltransferase (LRAT) is extremely low in the liver of vitamin A-deficient rats, but is rapidly induced after administration of retinoic acid (RA). The nuclear receptors for RA are closely related to the receptors for thyroid hormone, and molecular cross-talk between these receptors has been observed in vitro and in cultured cells. Therefore we have examined whether retinoid status and thyroid hormone status interact in the regulation of hepatic LRAT activity in vivo. Vitamin A-deficient male Lewis rats, either euthyroid or made hypothyroid by treatment with propylthiouracil, were treated once or three times with 20 μg of all- trans-RA, 10 μg/100 gm body weight of triiodothyronine (T 3), or both hormones. Hepatic LRAT activity, which was negligible in all retinoid-deficient rats, was induced by RA ( P < 0.0001) regardless of the animal's thyroid hormone status. T 3 by itself had no ability to induce hepatic LRAT activity ( P = 0.42), nor did co-administration of T 3 with RA increase or decrease the response to RA ( P = 0.13). In retinoid-sufficient rats, hypothyroidism did not alter hepatic LRAT activity; however, LRAT activity was reduced by half ( P < 0.05) after three treatments with T 3. Therefore we conclude that thyroid hormone status alone does not regulate LRAT, nor does thyroid status affect the ability of RA to induce hepatic LRAT in retinoid-deficient animals. However, in retinoid-sufficiency, repeated treatment with T 3 may reduce the hepatic esterification of retinol by LRAT.

Retinol-binding protein secretion from the liver of N-(4-hydroxyphenyl) retinamide-treated rats

N-(4-Hydroxyphenyl)retinamide (HPR; Fenretinide), a synthetic retinoid possessing antitumor activity, depresses plasma retinol and retinol-binding protein (RBP) concentrations. In study 1, the ability of retinol or HPR to induce RBP secretion from the livers of vitamin A-deficient rats was compared. A large apoRBP pool accumulated in the liver rough microsomes of these rats. Following retinol repletion, 80% of the accumulated RBP was rapidly secreted into the plasma. In contrast, HPR treatment only induced two-thirds of the RBP secretion observed with retinol. Prior colchicine treatment caused a large RBP accumulation in the Golgi-enriched fraction following retinol repletion. HPR plus colchicine treatment produced significantly less accumulation of RBP in the Golgi-enriched fraction than did retinol. In study 2, HPR treatment of vitamin A-adequate rats caused RBP to accumulate in the liver rough microsomes. When vitamin A-adequate rats were treated with colchicine, the concentration of RBP in the Golgi-enriched fraction increased 2.9-fold. However, significantly less RBP accumulated in the Golgi following HPR treatment. These studies demonstrate that HPR will induce liver RBP secretion, but to a lesser degree than retinol. Further, more RBP remained in the rough microsomes of HPR treated, vitamin A-adequate rats, indicating that HPR depressed the amount of RBP secreted.

Regulation of Hepatic Lecithin: Retinol Acyltransferase Activity by Retinoic Acid

The activity of the hepatic enzyme lecithin: retinol acyltransferase (LRAT), thought to catalyze the esterification of retinol for storage, was previously shown to vary directly with the vitamin A (retinol) status of the rat [Randolph, R. K., and Ross, A. C. (1991) J. Biol. Chem. 266, 16453-16457]. The present studies were designed to determine whether liver LRAT activity is regulated in vivo by retinoic acid, a principal active metabolite of retinol. LRAT activity was negligible in the livers of vitamin A-deficient rats. Following treatment with a single 2-μg dose of retinoic acid, LRAT activity increased significantly while, following treatment with a single 20-μg dose, liver LRAT activity equalled that of vitamin A-sufficient adult rats. Retinoic acid was more effective than an equimolar quantity of retinol in restoring LRAT activity in the vitamin A-deficient rat liver. The increase in hepatic LRAT activity after administration of retinoic acid occurred rapidly, reaching a maximum within 12-16 h but declining again after 48-72 h. Studies were conducted in vivo to gain insight into the level of regulation of LRAT by retinoic acid. The increase in LRAT activity by retinoic acid in the vitamin A-deficient rat was blocked completely by both actinomycin D and cycloheximide. The ability of liver to esterify retinol in vivo was correlated with the in vitro activity of LRAT after retinoic acid induction. We conclude that retinoic acid, an important end product of retinol metabolism, regulates a key aspect of hepatic retinal metabolism through its regulatory activity on liver LRAT.

Vitamin A deficiency alters genomic expression for fibronectin in liver and hepatocytes.

Earlier work from our laboratory had shown that vitamin A-deficient rats had increased levels of fibronectin in their serum. To explain this result, we investigated the mechanism whereby vitamin A deficiency affects the production of fibronectin by liver and hepatocytes, since liver is the known source of plasma fibronectin. By use of cDNA specific for rat liver fibronectin, we showed that livers of vitamin A-deficient rats had a 2-4-fold increase in the level of fibronectin mRNA and also a higher transcription rate. Rate of synthesis and secretion of fibronectin was found to be increased 2-fold in primary cultures of hepatocytes of deficient animals. Exogenous addition of retinyl acetate or retinoic acid to the media reversed this increase to control levels. The increase was paralleled by an increase in fibronectin mRNA, also reversed by exogenous retinoic acid. At least 12 h were needed for this reversal to take place. Thus, vitamin A appears to regulate the synthesis of fibronectin through its action on fibronectin mRNA transcription. This represents the first reported observation of an action of vitamin A at the genomic level on the synthesis of a specific protein in liver.

Biosynthesis of retinoyl-β-glucuronide, a biologically active metabolite of all- trans-retinoic acid

All- trans-retinoic acid is metabolized in vitro to a biologically active metabolite, retinoyl-β-glucuronide. We have studied the synthesis of this metabolite in vitro. The identity of the product was established by cochromatography on reverse-phase high-performance liquid chromatography, β- d-glucuronidase hydrolysis, and fast atom bombardment and collisionally activated decomposition/fast atom bombardment mass spectrometry. The formation of retinoyl-β-glucuronide is catalyzed by a UDP-glucuronosyltransferase with apparent K m 's of 54.7 μ m for all- trans-retinoic acid and 2.4 m m for UDP-glucuronic acid. The reaction requires enzyme, UDP-glucuronate, and no other factor. It is strongly inhibited by millimolar concentrations of coenzyme A. The specific activity of UDP-glucuronosyltransferase is greatest in the liver and least in the kidney of those tissues examined. The specific activity of the enzyme is increased by vitamin A deficiency. The increased specific activity observed in the vitamin A-deficient rat liver is uncharacteristic of retinoic acid inactivation enzymes; therefore, retinoyl-β-glucuronide may be of functional importance.

Spatial Distribution of Retinol-Binding Protein and Retinyl Palmitate Hydrolase Activity in Normal and Vitamin A-Deficient Rat Liver

A study was conducted to explore the spatial distribution within rat liver of two proteins importantly involved in retinoid metabolism in liver, namely, retinol-binding protein (RBP) and the enzyme retinyl palmitate hydrolase (RPH). The study was conducted with both vitamin A-sufficient (control) and vitamin A-deficient rats. Livers were carefully and reproducibly dissected into 11 sections each, and RBP levels and RPH activities were measured for each section homogenate. Both RBP and RPH activity displayed highly significant spatial heterogeneity in their distributions in liver. For control rats, the mean level of RBP was 39.0 µg/g wet weight, with a section-to-section variation of 14.5. For deficient rats, the corresponding RBP mean and variation values were 283 and 56 µg/g wet weight. For RPH, the mean level was 136 pmol free fatty acids (FFA) formed/(min · mg) with a section-to-section variation of 178. Both inspection of the data and analysis of variance indicated that this significant section-to-section variation (spatial heterogeneity) did not follow a consistent anatomic pattern from rat to rat. Thus, no one specific anatomic location in the liver was consistently high or low with regard to either RBP or RPH. Since the spatial distributions of both RBP and RPH activity did not follow a consistent anatomic pattern, it is not possible to obtain an accurate measure of the total liver levels for either parameter in a homogenate made from a small section. Finally, the patterns of distribution of RBP and RPH activity observed in the liver sections from both vitamin A-sufficient and deficient rats were not significantly correlated, either directly or inversely, as determined by chi-square analysis. Thus, RBP and RPH activity levels vary independently of each other in their heterogeneous anatomic distributions in rat liver.

Metabolism of [11- 3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11- 3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient. vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11- 3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [ 3H]retinoic acid from [ 3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (R M) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite R M from [ 3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite R M could be derived from a more stable pool of vitamin A.

Decreased hepatic gluconeogenesis in vitamin A-deficient rats.

AbstractStudies were carried out to investigate the effects of vitamin A deficiency (hypovitaminosis A) on the chemical constituents and gluconeogenic activity of rat liver. Hypovitaminosis A was found not to alter the glycogen, protein, DNA, or RNA content, but to decrease significantly the level of citrate in liver. Free amino acid levels in blood and liver of vitamin A-deficient rats were markedly higher in comparison to pair fed controls. An inhibition of hepatic gluconeogenesis in vitamin A deficiency was indicated by the decreased incorporation of 14C-labeled alanine and bicarbonate into glucose and glycogen by liver slices. Vitamin A deficiency caused a significant decrease in the activities of alanine aminotransferase, ornithine aminotransferase, glucose 6-phosphatase and fructose 1,6-bisphosphatase. Activities of aspartate aminotransferase and tryptophan pyrrolase were found to be unaltered in vitamin A-deficient livers. It is suggested that the inhibition of hepatic gluconeogenesis observed in h...

Alterations in surface carbohydrates and in some functional properties of liver lysosomal membrane in vitamin A-deficient rat

A significant decrease in total carbohydrates and particularly in mannose, galactose and sialic acid has been observed in vitamin A-deficient rat liver lysosomal membrane. These alterations adversely affect the membrane permeability and structure-linked latency of the lysosomal enzymes. Significant reduction in the pH-dependent in vitro binding of the lysosomal arylsulfatase B to the highly purified membrane has been observed in vitamin A deficiency. This is attributed to the decrease in electro-negativity, mainly due to the observed reduction in negatively-charged sialic acid residues on the outer side of the membrane. Similar reduction in sialic acid content on the inner side of the membrane affects the microenvironment in the lysosomes. Intralysosomal pH, measured by computing the proteolytic activity of lysed lysosomes and of phagolysosomes, endocytosed with denatured 131I-labelled human serum albumin, is slightly but consistently higher in vitamin A-deficient groups compared to that in control one. This is reflected in the low rate of degradation of the entrapped proteins in vitamin A deficiency. The possible physiological significance of the observations is discussed with special reference to the loss of surface carbohydrates, particularly sialic acid, in vitamin A-deficient rats.

Cellular retinol-binding protein allows specific interaction of retinol with the nucleus in vitro.

Purified cellular retinol-binding protein (CRBP), a potential mediator of vitamin A action, was found to enable retinol to bind in a specific manner to isolated nuclei from livers of vitamin A deficient rats. Binding was followed after complexing [3H]retinol with CRBP. The binding was specific, saturable, and temperature dependent. CRBP charged with unlabeled retinol or CRBP without retinol diminished binding of radioactivity whereas free retinol did not. No specific binding sites could be detected for free retinol. Purified cellular retinoic acid binding protein (CRABI) complexed with retinoic acid did not diminish the amount of retinol bound to nuclei. Approximately 3 x 10(5) specific binding sites per nucleus could be detected. Fewer binding sites were found in nuclei isolated from livers of control (chow-fed) rats and also from livers of vitamin A-deficient rats 2 hr after refeeding with retinylacetate.

Enzymatic synthesis of mannosyl retinyl phosphate from retinyl phosphate and guanosine diphosphate mannose

A study was conducted to determine whether retinyl phosphate would act as substrate for the enzymatic synthesis of mannosyl retinyl phosphate. Retinyl phosphate, prepared chemically, supported the growth of vitamin A-deficient rats at the same rate as retinol. It also stimulated the uptake of [14C]mannose from GDP-[14C]mannose into total chloroform-methanol extractable lipid. This reaction occurred in the presence of ATP, Mn2+, detergent (Zonyl A), and a membrane-rich enzyme preparation from the livers of vitamin A-deficient rats, provided that a lipid extract of the membrane preparation of alpha-L-lecithin was also added. Total chloroform-methanol-extractable, labeled mannolipid was separated into two principal labeled mannolipids by thin-layer or column chromatography or by differential solvent extraction. The properties of these mannolipids identified them as glycophospholipids: one was identical with authentic synthetic dolichyl mannosyl phosphate, and the other was concluded to be mannosyl retinyl phosphate because of its incorporation of radioactivity from [3H]retinyl phosphate, its rapid hydrolysis by dilute acid, and the formation of substance that cochromatographed with retinol upon its acid hydrolysis. The presence of ATP or GTP was essential for the stimulation of mannolipid synthesis, probably because of their protective action on the substrates against phosphatases present in the crude enzyme fraction. A pH of 6.0-6.2 favored the formation of dolichyl mannosyl phosphate; a higher pH (6.7-7.0) that of mannosyl retinyl phosphate.

Biosynthesis of ubiquinone and ubichromenol in vitamin A-deficient rats

1. 1. The concentrations of ubiquinone and ubichromenol increased in the livers, but not in the intestines and kidneys, of rats maintained on a diet deficient in vitamin A. 2. 2. After short time intervals ( e.g. 2 h) following administration of the tracer, incorporation of [2- 14C]mevalonate into ubiquinone and ubichromenol in livers of vitamin A-deficient rats was lower than for normal animals; this was in contrast to later times ( e.g. 72 h) when it was higher. 3. 3. The “newly synthesized” ubiquinone in livers of vitamin A-deficient rats was distributed in all the cell fractions without specific localization. 4. 4. Absorbed exogenous [ 14C]ubiquinone and [ 14C]ubichromenol were retained in the livers of vitamin A-deficient rats to a larger extent and for a longer time than in the normal animals. 5. 5. The results suggest that the accumulation of ubiquinone and ubichromenol in the livers of vitamin A-deficient rats is due to lowered catabolism and not to increased rate of synthesis.

The influence of α-tocopherol on arylsulfatases A and B in the liver of vitamin A-deficient rats

1. 1. The effect of different levels of dietary α-tocopherol on arylsulfatases A and B (EC 3.1.6.1) was investigated in the liver of vitamin A-deficient rats and pair-fed controls. 2. 2. Arylsulfatase A was increased in the liver of vitamin A-deficient rats receiving “normal” dietary α-tocopherol, but not when the rats were fed a high level of vitamin E. 3. 3. Arylsulfatase B was increased in vitamin A deficiency regardless of the dietary α-tocopherol level. 4. 4. Incubation at 37° of the lysosome-rich liver fraction caused more rapid release of both arylsulfatases from the lysosomes of the deficient rats receiving “normal” dietary α-tocopherol. High dietary α-tocopherol reversed this phenomenon. 5. 5. The effect of retinol added in vitro appeared to be the opposite of its effect in vivo on sulfatase release from the lysosomes, whereas α-tocopherol had the same effect when added in vitro as when fed at high levels in vivo. 6. 6. The effect of vitamin A deficiency on sulfate metabolism might be mediated through the role of retinol and perhaps of α-tocopherol on the stability of biological membranes.

A biologically active metabolite of retinoic acid from rat liver

1. Four major radioactive fractions have been isolated from the livers of vitamin A-deficient rats given [6,7-(14)C(2)]retinoic acid. 2. At least one of these was more potent than retinoic acid and approximately equal to retinol in the growth assay for vitamin A activity. 3. The biologically active material was chromatographically distinct from retinoic acid, retinol and retinal. 4. Alkaline hydrolysis of this material yielded an acidic compound containing all the radioactivity. 5. The methyl ester of the acidic product was unlike the methyl ester of retinoic acid in its chromatographic behaviour. 6. It is suggested that this metabolite may represent the active form of retinol in its growth-supporting role.

Vitamin A and Lipid Metabolism

Publisher Summary The main aspects of the function of vitamin A in metabolism have been reviewed by Lowe and Morton. Since then, the problem of the functional role of vitamin A in steroid metabolism, already indicated in that review, has been developed further. Part of this problem, that concerning vitamin A and steroid hormones is discussed by Johnson and Wolf. This chapter mainly discusses the function of vitamin A in cholesterol and ubiquinone biosynthesis. The structure of ubiquinones (SA) and ubichromenol (SC) previously found to be enriched in livers of vitamin A-deficient rats has been elucidated. The ubiquinones widely distributedin natural material are dimethoxymethylbenzoquinone derivatives with long isoprenoid side chains. The ubichromenol turned out to be the corresponding chromene derivative, obviously formed by ring closure of the ubiquinone. In vitamin A deficiency, the incorporation of C 14 -mevalonic acid in both ubiquinones and ubichromenol is enhanced. A remarkable rise of the incorporation into squalene is also observed, whereas the biosynthesis of cholesterol is reduced. The sum of the total incorporation into these substances is little affected by vitamin A deficiency.