Alterations in the Lipid Metabolism of Rat Aorta: Effects of Vitamin A DeficiencyGatica L.V. · Vega V.A. · Zirulnik F. · Oliveros L.B. · Gimenez M.S.
Department of Biochemistry and Biological Sciences, Faculty of Chemistry, Biochemistry and Pharmacy, National University of San Luis, San Luis, Argentina Corresponding Author
Antioxidants are known to reduce cardiovascular disease by reducing the concentration of free radicals in the vessel wall and by preventing the oxidative modification of low-density lipoproteins. The prooxidative effect of a vitamin-A-deficient diet on the aorta has previously been demonstrated by us. In this study, the lipid metabolism in the aorta of rats fed on a vitamin-A-deficient diet was evaluated. Vitamin A deficiency induced a hypolipidemic effect (lower serum triglyceride and cholesterol levels) and a decreased serum paraoxonase 1/arylesterase activity. The concentrations of triglycerides, total cholesterol, free and esterified cholesterol, and phospholipids were increased in the aorta of vitamin-A-deficient rats. The phospholipid compositions showed an increase in phosphatidylcholine (PC), phosphatidylinositol plus phosphatidylserine and phosphatidylethanolamine, a decrease in sphingomyelin, and no change in phosphatidylglycerol. In the aorta, the increase in triglycerides was associated with an increased fatty acid synthesis and mRNA expression of diacylglycerol acyltransferase 1. The increased PC content was attributed to an increased synthesis, as measured by [methyl-14C]choline incorporation into PC and high CTP:phosphocholine cytidylyltransferase-α mRNA expression. The cholesterol synthesis, evaluated by [1-14C]acetate incorporated into cholesterol and mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, did not change. The lipoprotein lipase and lectin-like oxidized low-density lipoprotein receptor 1 mRNA expression levels increased in the aorta of vitamin-A-deficient animals. The incorporation of vitamin A into the diet of vitamin-A-deficient rats reverted all the changes observed. These results indicate that a vitamin-A-deficient diet,in addition to having a prooxidative effect, alters the aorta lipid metabolism.
Copyright © 2006 S. Karger AG, Basel
Vitamin A deficiency is recognized as a significant problem in many developing countries and has been linked to the increased risk of infant mortality [1, 2]. Vitamin A (retinol) is obtained in the diet in the form of retinyl esters or through the ingestion of β-carotene, which is converted to 2 molecules of retinol. The carboxylic acid form of vitamin A (all-trans-retinoic acid) has important effects on the development of the cardiovascular system  and modulates the growth, differentiation and morphology of the endothelial cells . In addition, the antioxidant property of vitamin A has been communicated in several works [5, 6]. We have previously found increased thiobarbituric-acid-reactive substances in serum and aorta and higher nuclear factor κB binding activity together with increased inducible nitric oxide synthase and cyclooxygenase 2 expression in the aorta of animals fed on a vitamin-A-deficient diet, suggesting that inflammation and pro-oxidant environment are associated with vitamin A deficiency in the aorta . Several studies have shown that the dietary intake of β-carotene and vitamin A inhibits atherosclerosis by protecting low-density lipoprotein (LDL), the principal carrier of β-carotene and vitamin A, from oxidation [8, 9].
In 1997, Sawamura et al.  initially identified cDNA encoding an endothelial receptor for oxidized LDL, which was designated as lectin-like oxidized LDL receptor 1 (LOX-1). LOX-1 expression in endothelial cells is relatively low in the basal condition, but it can be induced by proinflammatory cytokines and vasoconstrictive peptides in vitro , and in proatherogenic conditions in vivo . In addition, mild oxidation of LDL significantly enhances the interaction between this lipoprotein and lipoprotein lipase (LPL) [13, 14], an enzyme that converts chylomicrons to remnants and begins the cascade required for the conversion of very low-density lipoprotein (VLDL) to LDL.
The oxidation of LDL is prevented by paraoxonase 1 (PON-1). This enzyme circulates bound to plasma high-density lipoproteins (HDL), hydrolyzes phospholipid (PL) hydroperoxides and cholesterol ester hydroperoxides (esterase activity), reduces lipid hydroperoxides to the respective hydroxides, and also degrades hydrogen peroxide (peroxidase activity). Furthermore, PON-1 protects HDL from peroxidation and, thus, improves reverse cholesterol transport; it also protects plasma membranes from free radical injury .
It is known that cholesterol accumulation in the vascular wall is associated with structural and functional changes in the vessels. Experimental data suggest that the alteration in the cellular PL metabolism is an adaptive response to prevent the cellular free cholesterol:PL ratio from reaching cytotoxic levels in the macrophage foam cells of atherosclerotic lesions .
Considering that antioxidants have been hypothesized to inhibit lipid peroxidation and play a protective role against cardiovascular disease , we examined the contribution of vitamin A withdrawal to the lipid content and mRNA expression of regulatory enzymes involved in the lipid metabolism of the rat aorta. The effect of vitamin A deficiency on the PON-1 antioxidant function in serum was also determined. Finally, the effect of vitamin A restitution to vitamin-A-deficient rats on the lipid metabolism was analyzed.
Acetic acid, sodium salt [1-14C] ([14C] acetate, 2.00 mCi/mmol) and choline chloride [methyl-14C] ([14C] choline, 54.00 mCi/mmol) were purchased from Dupont, New England Company (Boston, Mass., USA). Standard lipids were acquired from Sigma Chemical Co. (St. Louis, Mo., USA.). All the other chemicals were of reagent grade and were obtained from Merck Laboratory (Buenos Aires, Argentina).
Male Wistar rats were weaned at 21 days of age and immediately assigned randomly (8 per group) to either the experimental diet, devoid of vitamin A (vitamin-A-deficient group), or the same diet with 4,000 IU of vitamin A (8 mg retinol as retinyl palmitate per kg of diet; control group) for 3 months. Also, a group of 8 vitamin-A-deficient animals was fed with the control diet 15 days before sacrifice (vitamin-A-refed group) in order to supply them with vitamin A. The rats were housed in individual cages and kept in a 21–23°C controlled environment with a 12-hour light:dark cycle. They were given free access to food and water throughout the entire 3 months of the experimental period. Diets were prepared according to AIN-93 for laboratory rodents . Both diets had the following composition (g/kg): 397.5 cornstarch, 100 sucrose, 132 dextrinized cornstarch, 200 vitamin-free casein, 70 soybean oil, 50 cellulose fiber, 35 AIN-93 mineral mix, 10 AIN-93 vitamin mix (devoid of vitamin A for the vitamin-A-deficient diet), 3 L-cystine, 2.5 choline bitartrate and 0.014 tert-butylhydroquinone. Body weight and food intake were registered daily.
Rats were killed by cervical dislocation at 9.00 h. Blood samples were collected in EDTA-coated tubes. To minimize photoisomerization of vitamin A, the plasma was taken under reduced yellow light and frozen in the dark at –70°C until the determination of retinol concentrations. Analyses were carried out within 1–3 weeks of obtaining the samples. The plasma retinol concentration was determined by high-performance liquid chromatography . Retinoids were extracted from plasma (0.5 ml) into hexane containing 5 μg of butylated hydroxytoluene/ml as antioxidant for the analysis. Retinyl acetate was used as internal standard. Chromatography was performed on a Nucleosil 125 C-18 high-performance liquid chromatography column with methanol:water (95:5, by vol.) as the mobile phase. Retinol was detected by UV absorbance at 325 nm (Model 440, Waters Associates) and peak areas were calculated by integration (Spectra Physics Analytical).
We measured serum total cholesterol (TC), HDL cholesterol (HDLc) and triglycerides (TG) by colorimetric methods (kits from Wiener, Buenos Aires, Argentina), using fresh serum. All the measurements were performed within 4 h of obtaining the samples. (LDL + VLDL) cholesterol was calculated by subtracting the HDLc value from the TC value.
PON-1 activity toward phenyl acetate (arylesterase activity) was determined by measuring the initial rate of substrate hydrolysis in the assay mixture (0.7 ml) containing 2.8 mM of the substrate, 1 mM of CaCl2 and 1 μl of serum in 20 mM Tris-HCl (pH 8.0). The absorbance was monitored for 2 min at 270 nm. A blank sample prepared as described above but without serum, representing nonenzymatic hydrolysis, was subtracted and the activity was calculated from E270 = 1,310 M–1 cm–1. The results are expressed in units per milliliter, 1 unit arylesterase hydrolyzes 1 μmol phenyl acetate per minute .
Lipids were extracted from tissue and total lipids were determined by dry weight. The lipids were resuspended in a hexane/isopropanol mixture (3:2, by vol.), containing butylated hydroxytoluene as antioxidant . Aliquots were taken to determine TC . Other parts of the extracts were used for the separation of the different lipids on thin-layer chromatography (TLC) plates coated with silica gel G (Merck, Darmstadt, Germany) using hexane/diethyl ether/acetic acid (80:20:1, by vol.) as the solvent. The lipids were detected by exposing the plates to iodine vapors. They were scraped off and used directly for the determination of PL  TG , free and esterified cholesterol .
PL were separated into the component species phosphatidylethanolamine, phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylinositol plus phosphatidylserine and phosphatidylglycerol by TLC using silica gel H plates and chloroform/methanol/water (65:25:4, by vol.) as the solvent system. Individual PL were identified, recovered and quantified for the phosphorus content as indicated above. The results were expressed as percentage of the total PL phosphorus content. The positions of neutral lipids and individual PL were determined using the respective standards.
Aorta rings were preincubated in 0.5 ml of Krebs-Ringer glucose (K-R) solution, pH 7.2, for 10 min at 37°C in a 95% air/5% CO2 atmosphere. After that, the medium was replaced by 0.5 ml of fresh K-R solution added with 1 μCi of [1-14C] acetate and the different samples were incubated for 180 min. The reaction was stopped by the addition of 0.2 ml of 6 N sulfuric acid and tissues were thoroughly washed in ice-cold K-R solution until no more radioactivity was detected in the wash solution before storing samples at –70°C until use. Lipids were saponified by treatment with 10% (w/v) KOH in ethanol:water (100:15, by vol.) for 3 h at 80°C. The free fatty acids were recovered from the lower phase after acidification with 0.3 ml of 1.2 N HCl and extracted 3 times with petroleum ether (bp 30–40), 2.5 ml each. This fraction was dried down in a stream of nitrogen before counting its radioactivity. Aliquots of the nonsaponified fraction (upper phase) were used for the separation of the cholesterol fraction by TLC before counting its radioactivity in a Wallac 1409 DSA liquid scintillation counter. The results are expressed as disintegrations per hour per milligram tissue.
Aorta rings were preincubated in 0.5 ml of K-R solution, pH 7.2, for 10 min at 37°C in a 95% air/5% CO2 atmosphere. After that, the medium was replaced by 0.5 ml of fresh K-R solution added with 1 μCi of [methyl-14C] choline for 60 min. The reaction was stopped by the addition of 0.2 ml of 6 N sulfuric acid and tissues were thoroughly washed in ice-cold K-R solution until no more radioactivity was detected in the wash solution. PL classes were separated by TLC (see Tissue Lipid Determinations) and bands were scraped off and their radioactivity quantified. The results are expressed as disintegrations per hour per milligram tissue.
Total RNA was isolated by using Trizol (Life Technologies). All RNA isolations were performed as directed by the manufacturer. Gel electrophoresis and ethidium bromide staining confirmed the purity and integrity of the samples. The quantification of RNA was based on a spectrophotometric analysis at 260/280 nm.
Ten micrograms of total RNA were reverse-transcribed with 200 units of moloney murine leukemia virus reverse transcriptase (Promega Inc.) using random hexamers as primers in a 20-μl reaction mixture, following the manufacturer’s instructions. Reverse-transcription (RT)-generated fragments coding for β-actin , LPL , diacylglycerol acyltransferase-1 (DGAT-1) , 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) , LOX-1  and CTP: phosphocholine cytidylyltransferase (CT)-α .
Polymerase chain reaction (PCR) was performed in 35 μl of a reaction solution containing 0.2 mM of dNTPs, 1.5 mM of MgCl2, 1.25 units of Taq polymerase and 50 pmol each of rat-specific oligonucleotide primers and RT products (1/10 of RT reaction). The sequences of the different primers are shown in table 1. The expected PCR product of β-actin is 243 bp, of LPL 454 bp, of LOX-1 359 bp, of CT-α 232 bp, of HMG-CoAR 462 bp and of DGAT-1 329 bp. The samples were heated to 94°C for 2 min, followed by 35 temperature cycles. Each cycle consisted of 3 periods: (1) denaturation, 94°C for 1 min; (2) annealing, 58°C for β-actin, LPL, DGAT-1 and CT-α, 57°C for HMG-CoAR and 56°C for LOX-1 during 1 min; (3) extension, 72°C for 1 min. After 35 reaction cycles, the extension reaction was continued for another 5 min.
|Table 1. Sequences of the primers used to amplify the different genes by RT-PCR and sizes of the fragments generated|
The PCR products were electrophoresed on 2% (w/v) agarose gel with 0.01% (w/v) ethidium bromide. The image was visualized and photographed under UV transillumination. The intensity of each band was measured with the NIH Image software and reported as the values of band intensity units.
Data are presented as means ± SEM. They were analyzed by one-way ANOVA. Statistical significance was accepted at p < 0.05.
The initial body weight (in grams) of the animals of the 3 dietary groups was 51 ± 3. At the time of killing, the body weight of rats fed the vitamin-A-deficient diet was significantly lower than that of the controls (326.33 ± 5.04 vs. 376 ± 6.5, p < 0.01), while that of the vitamin-A-refed group was close to that of the controls and significantly higher than that of vitamin-A-deficient rats (365.67 ± 3.33 vs. 326.33 ± 5.04, p < 0.05).
Vitamin A deficiency was determined by the content of retinol in plasma. The plasma retinol concentrations (in micromoles per liter) of rats fed the vitamin-A-deficient diet were significantly lower (0.55 ± 0.05 vs. 1.35 ± 0.04, p < 0.001) than those of the controls. Vitamin A refeeding considerably increased the plasma vitamin A concentration in relation to vitamin-A-deficient rats (1.14 ± 0.05 vs. 0.55 ± 0.05, p < 0.001).
Vitamin A deficiency affected the circulating lipid pattern. The concentrations of TG (p < 0.001), TC (p < 0.05) and HDLc (p < 0.05) decreased in the serum of vitamin-A-deficient animals in comparison with the controls, while in the vitamin-A-refed group all these lipids reached the control values. No effect of the dietary treatment was observed on the serum (LDL + VLDL) cholesterol level (table 2).
|Table 2. Lipid contents in serum and aorta|
PON-1 activity toward phenyl acetate was significantly reduced in animals receiving the vitamin-A-deficient diet, when compared to the control and vitamin-A-refed groups (fig. 1).
|Fig. 1. PON-1 activity toward phenyl acetate. Values are presented as means ± SEM (n = 8 per dietary group). There were significant differences when vitamin-A-deficient rats (D) were compared to control (C; p < 0.001) and vitamin-A-refed rats (R; p < 0.01).|
We found important variations in the lipid profile in the aorta of animals fed the vitamin-A-deficient diet (table 2). The concentrations of TG (p < 0.01), TC (p < 0.05), free cholesterol (p < 0.05), esterified cholesterol (p < 0.01) and PL (p < 0.01) increased when compared with the control group. Vitamin A refeeding restored these changes.
On the percentage basis, the PL composition was modified in the aorta of rats fed the vitamin-A-deficient diet compared with those fed the control diet. In the vitamin-A-deficient group, PC, phosphatidylinositol plus phosphatidylserine and phosphatidylethanolamine increased and SM decreased, while phosphatidylglycerol did not change in relation to the control and vitamin-A-refed animals (fig. 2).
|Fig. 2. PL pattern of the aorta. Values are presented as means ± SEM (n = 8 per dietary group); * p < 0.05,** p < 0.01: differences when vitamin-A-deficient rats (D) were compared to control (C) and vitamin-A-refed rats (R).|
As shown in table 3, the [1-14C] acetate incorporated into cholesterol (nonsaponifiable lipid fraction) by aorta rings from vitamin-A-deficient rats was not modified in relation to the control group. The [1-14C] acetate incorporated into the saponifiable lipid fraction, indicating a de novo synthesis of fatty acids, was increased in animals fed the vitamin-A-deficient diet (p < 0.05) compared with controls. This increase was significantly reversed in the aortas of vitamin-A-refed rats.
|Table 3. Incorporation of [1-14C] acetate into saponified and nonsaponified lipid fractions and [methyl-14C] choline into PC and SM|
The [methyl-14C] choline incorporated into PC by aorta rings was increased (p < 0.001) and that incorporated into SM was decreased (p < 0.05) in the vitamin-A-deficient group when compared with the control group. In the vitamin-A-refed animals, the increase in [14C] choline incorporation into PC was partially, but significantly, reversed while the decrease in its incorporation into SM was normalized (table 3).
As shown infigure 3a, the expression of LPL mRNA, an enzyme that would lead to an increase in the external contribution of fatty acids in the tissue, was significantly higher in the aorta of vitamin-A-deficient rats than the one of controls and vitamin-A-refed animals.
|Fig. 3. Effects of vitamin A deficiency on the mRNA expression of enzymes involved in the lipid metabolism in the aorta. Representative RT-PCR analysis for LPL (a), DGAT-1 (b), HMG-CoAR (c), LOX-1 (d), CT-α (e) and β-actin (f), used as internal controls. M = Molecular weight marker; D = vitamin-A-deficient rats. On the side, the quantification of the intensity of the fragment bands is shown. Identical results were obtained in 4 independent experiments; * p <0.05 different from control (C) and vitamin-A-refed rats (R); ** p <0.01 different from C, and p <0.05 from R; *** p <0.01 different from C and R.|
The expression of DGAT-1 mRNA increased in the aorta of vitamin-A-deficient animals, indicating that TG synthesis might be increased (fig. 3b).
Figure 3c shows that HMG-CoAR mRNA, the limiting enzyme of cholesterol synthesis, did not change its expression in the 3 dietary groups, suggesting that the increase in cholesterol content observed in the aorta of vitamin-A-deficient rats is not due to an increased synthesis.
The expression of LOX-1 mRNA in the aorta of vitamin-A-deficient animals was increased in relation to controls and vitamin-A-refed rats (fig. 3d) suggesting the presence of oxidized LDL in the endothelium of the aorta.
The expression of CT, the main regulator of the de novo synthesis of PC, was increased in vitamin-A-deficient rats as compared to controls, and restored by vitamin A refeeding (fig. 3e).
We have previously shown the presence of inflammation and lipoperoxidation in the serum and aorta of vitamin-A-deficient rats .Using the same experimental model, here we communicate the effects of vitamin A deprivation on lipid disturbances in the serum and aorta of rats.
We found decreased PON-1 activity in the serum of vitamin-A-deficient rats compared to that of controls, demonstrating the presence of 1 additional marker of oxidative stress in these animals. PON-1 endows HDL with its antioxidant property and is probably responsible for the principal mechanism that inhibits the oxidation of both LDL and HDL itself. This impaired activity of PON-1, together with the higher thiobarbituric acid-reactive substances and nitrite content in the serum of vitamin-A-deficient rats communicated in our previous work , suggests that vitamin A acts as an important antioxidant on the circulation level.
Our results show that vitamin A deficiency induced a hypolipidemic effect in the serum. The decrease in serum cholesterol content was associated with the low HDLc level. The possibility that vitamin A deficiency may play a role in altering the lipoprotein secretion from the liver into the circulation should be considered. It has been shown that retinoic acid, in a dose-dependent manner, increases the secretion of apo AI, B100, CIII and AII in a culture of Hep G2 cells . On the other hand, it is known that the administration of retinoids to both experimental animals  and humans  often results in an increase in serum TG levels. Oliver and Rogers  demonstrated that retinoic-acid-induced hypertriglyceridemia is associated with a suppression of LPL activity in peripheral tissues. In our case, we observed an increased expression of LPL mRNA in the aortas from vitamin-A-deficient rats. This, and the knowledge that the high reactive oxygen species levels affect the mRNA expression and activity of LPL , lead us to suggest that the impaired LPL expression contributes to the low TG levels in the serum of vitamin-A-deficient rats. It is known that LPL activity results in the generation of lipoprotein remnants and generates free fatty acids, which are taken up and used for metabolic energy or stored as TG after reesterification. According to this, we observed an increased TG content in the aorta of vitamin-A-deficient rats. In addition, the increased expression of DGAT-1 mRNA together with the increased fatty acid synthesis, as determined by the incorporation of [14C] acetate into saponifiable lipids, could directly be responsible for the increase in TG mass in the artery.
The increase in the cholesterol content in the aorta of vitamin-A-deficient rats was not a consequence of its synthesis since the incorporation of [14C] acetate into cholesterol and HMG-CoAR mRNA expression did not change. These observations suggest a contribution of the uptake of modified cholesterol-rich lipoproteins by the endothelium to the aorta cholesterol content. This hypothesis was confirmed by the increased level of LOX-1 mRNA expression in the aorta of vitamin-A-deficient rats, compared with control animals. In addition, the high degree of oxidative stress in the serum, aorta and other tissues from rats fed on the vitamin-A-deficient diet previously demonstrated by us [7, 35, 36] and the impaired PON-1 activity communicated in this work support LDL oxidation. Auerbach et al.  observed that the oxidation of LDL is a mechanism that enhances LDL retention by endothelial LPL, and that the protective effects of apoE-containing HDL are in part due to its ability to block the retention of oxidized LDL in vivo. In this context, the increased LPL mRNA level found in the vitamin-A-deficient group could contribute to the deposition of oxidized LDL on the aorta vascular wall.
Changes in the level of PL would compromise the integrity and function of cell membranes because they mainly depend on the lipid balance, especially on the cholesterol/PL ratio . In the present study, we have found that the PL content and the PL synthesis were increased in the aorta of rats fed on a vitamin-A-deficient diet. Also, the PL pattern in the aorta of this experimental group was altered. The increased relative percentage of PC in the aorta of vitamin-A-deficient rats was explained by the increased endogenous synthesis of PC, measured by the incorporation of [methyl-14C] choline into PC. This coincided with a high expression of CT-α mRNA. The increased aorta cholesterol content could lead to an enhanced expression of CT-α mRNA [39, 40]. In addition, the increased fatty acid synthesis observed in the aorta of vitamin-A-deficient rats suggests a high availability of fatty acids to form PL. On the other hand, it is known that oxidized PL formed by lipid peroxidation in membranes are biologically active in vivo, inducing a pattern of inflammatory genes in the aorta . Kaneko et al.  found that compounds which inhibit membrane PL peroxidation or the decomposition of PL hydroperoxides, are effective in preventing lipid-peroxide-induced pathological events in cultured human umbilical vein endothelial cells. According to that and to the increased lipoperoxidation previously demonstrated in the aorta of vitamin-A-deficient rats , the increased mass of PL would be expected to be a potential source of lipid peroxides.
The results communicated here provide a novel insight into the role of vitamin A as an important molecule that influences lipid disturbances in the rat aorta. The 3 months of vitamin A deficiency result in alterations of the lipid content and gene expressions of key enzymes involved in the lipid metabolism of the aorta. The prooxidant environment induced by vitamin A withdrawal could affect the lipid homeostasis of the aorta.
The authors are grateful to Mr. Rosario del Pilar Dominguez for animal care. This work was supported by a grant from CONICET (PIP 4931) and the National University of San Luis (Project 8104), Argentina.
Prof. María Sofía Gimenez
Department of Biochemistry and Biological Sciences, Chacabuco 917
National University of San Luis, 5700 San Luis (Argentina)
Tel. +54 2652 423 789, ext 152/126, Fax +54 2652 431 301
Received: November 24, 2005
Accepted after revision: August 5, 2006
Published online: October 13, 2006
Number of Print Pages : 9
Number of Figures : 3, Number of Tables : 3, Number of References : 42
Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')
Vol. 43, No. 6, Year 2006 (Cover Date: November 2006)
Journal Editor: Pohl, U. (Munich)
ISSN: 1018–1172 (print), 1423–0135 (Online)
For additional information: http://www.karger.com/JVR