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The Journal of Nutrition Vol. 128 No. 10 October 1998,
pp. 1614-1619
-Carotene 15,15'-Dioxygenase Activity and Cellular Retinol-Binding Protein Type II Level Are Enhanced by Dietary Unsaturated Triacylglycerols in Rat Intestines 1,2,3
National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305-8642, Japan
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The purpose of this study was to examine effects of dietary triacylglycerols on 
-carotene 15,15'-dioxygenase (EC 1.13.11.21) activity and cellular retinol-binding protein [CRBP (II)] in rats. Six groups of eight rats (7-wk old) were fed one of the following diets: standard (STD; 2.5% soybean oil), saturated (SFA; 15% hydrogenated soybean oil), monounsaturated (MUFA; 15% olive oil), polyunsaturated (PUFA; 15% soybean oil) or clofibrate (CLF; 2.5% soybean oil + 0.2% clofibrate) for 3 wk. The dioxygenase specific activities of the intestinal homogenates in the MUFA and PUFA groups fed the high fat diets were 2.4 times that of the STD group fed a low fat diet (P < 0.01), whereas the activities of the SFA and CLF groups were not significantly different from that of the STD group. The level of CRBP (II) in the intestine of the PUFA group was 1.3-fold that of the STD group (P < 0.05), whereas there were no significant differences among the other groups. In a second experiment, the dioxygenase activity of rat intestine was followed over 3 wk of feeding the STD and PUFA diets. After the PUFA diet was consumed for 1 d, the activity was enhanced to 2.7 times the baseline level and remained thereafter at that high level, whereas the activity of the STD group remained at the low baseline level. Thus, dietary polyunsaturated triacylglycerols enhanced both -carotene 15,15'-dioxygenase activity and CRBP (II) level in rat intestine. These results suggest that the dioxygenase and CRBP (II) are regulated by the same mechanism involving long-chain fatty acids and their metabolites.
-carotene 15,15'-dioxygenase ·
cellular retinol-binding protein II ·
fatty acids ·
intestine ·
rats
Retinal, a product of the dioxygenase reaction, is thought to be bound to cellular retinol-binding protein type II [CRBP (II)],7 which has an important role as a substrate-carrier protein for the subsequent enzyme reactions leading up to retinyl esters (Viera et al. 1995 Materials.
All-trans- Animals and diets.
Male rats of the Sprague-Dawley strain (Charles River, Kanagawa, Japan), 7-wk old and 232 ± 12 g in weight were used. In Experiment 1, to determine effects of dietary fats and clofibrate on
Preparation of organ samples and tissue homogenates.
Liver and intestine were washed with an ice-cold isotonic saline (9 g/L NaCl). Liver was cut into small pieces (~2 g), frozen in liquid nitrogen and stored at
Determination of CRBP (II) level in intestinal mucosa.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to Laemmli (1970) Statistical analysis.
Values in the text were expressed as means ± SD. Data were tested for homogeneity of variances by the Bartlett test (Sokal and Rohlf 1981 There was no significant difference in food consumption among the diet groups. Body weights were 232 ± 12 and 366 ± 25 g before and after the 3-wk experiment, respectively. The body weight gain of the MUFA group was not significantly different than that of the STD group, whereas those of the SFA and CLF groups were lower by 19.7 and 25.8%, respectively, and that of the PUFA group was 17% greater (Table 2) compared with that of the STD group. The intestinal mucosa weight of the STD group was lower than those of the other groups (P < 0.05). The absolute and relative liver weights of the CLF group were significantly greater than those of the other groups (P < 0.001). They were larger by ~40% than those of the STD group.
Conversion of
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
-Carotene is an important micronutrient both as a vitamin A source and as a natural antioxidant. A portion of the dietary -carotene absorbed in the intestine (3-6% in rats and ~20% in humans) is secreted into the lymph (Goodman et al. 1966
, Huang and Goodman 1965), whereas a substantial part of absorbed -carotene is cleaved to retinal by -carotene 15,15'-dioxygenase in the intestinal mucosa (Goodman and Huang 1965, Olson and Hayaishi 1965). Then, retinal is converted to retinol and subsequently to retinyl esters of long-chain fatty acids, which are transported with chylomicrons to the liver for storage (Viera et al. 1995). Thus, -carotene 15,15'-dioxygenase is an important enzyme whose activity controls whether provitamin A carotenoids are either cleaved to vitamin A or delivered to tissues as intact carotenoids. But few data have been reported about its regulation, especially by nutrients. It was shown that the enzyme activity was dependent on the protein content in diet (Grownowska-Senger and Wolf 1970) and on vitamin A status (Villard and Bates 1986, Vliet et al. 1996). In particular, dietary vitamin A deficiency increased significantly -carotene dioxygenase activity in rats (Villard and Bates 1986). Other studies showed that the -carotene conversion to retinol was not affected by iron or copper deficiency (Dulin et al. 1995, Swanson and Parker 1993) and copper or selenium supplementation (Seaborn et al. 1991) in rats. However, to our knowledge, no data have been reported about possible effects of dietary fats on the dioxygenase activity, although dietary fats increase the bioavailability of -carotene (Bauernfeind et al. 1981, Dimitrov et al. 1988). It is reasonable to presume that dietary fats enhance -carotene dioxygenase activity to convert -carotene efficiently to vitamin A when the absorption of -carotene into intestinal cells is promoted.
). Coupling of the dioxygenase reaction with the subsequent reactions for the synthesis of retinyl esters could be of biological relevance in terms of efficient conversion of -carotene to vitamin A. Recently, both mRNA and protein levels of CRBP (II) were found to be increased in the jejunum of rats fed a diet with a high content of unsaturated fatty acids (Suruga et al. 1995). The mechanisms underlying the enhancement of CRBP (II) expression have not been clarified. However, the promoter of the CRBP (II) gene has a specific sequence recognized by a heterodimer of nuclear hormone receptors; retinoid X receptor and peroxisome proliferator activated receptor (PPAR) (Kliewer et al. 1992). The expression of CRBP (II) gene might be regulated by dietary polyunsaturated fatty acids, their metabolites or some hypolipidemic drugs, which could serve as ligands for PPAR. Thus, these results have given rise to the hypothesis that -carotene dioxygenase activity and the closely related binding protein CRBP (II) in conversion of -carotene to retinyl ester may be regulated by a similar mechanism and that the dioxygenase activity may be modulated by dietary fatty acids and hypolipidemic drugs as suggested in the case of CRBP (II). The purpose of this work was to investigate the effects of dietary triacylglycerols and clofibrate, one of hypolipidemic drugs, on -carotene 15,15'-dioxygenase activity and its relation to the level of CRBP (II) in rat intestine.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
-carotene (type II or IV) and all-trans-retinal were purchased from Sigma Chemical (St. Louis, MO). d-
-Tocopherol was obtained from Eisai (Tokyo, Japan). HPLC-grade acetonitrile was purchased from Nacalai Tesque (Kyoto, Japan). Other chemicals and solvents were of reagent grade. All-trans--carotene and all-trans-retinal were purified as described previously (Nagao et al. 1996). Purified all-trans--carotene was stored at 
85°C in n-hexane containing d--tocopherol at a molar ratio of 0.01 to the amount of -carotene as antioxidant.
-carotene 15,15'-dioxygenase activity and CRBP (II) level, 40 rats were divided into five groups of eight rats. Each group received one of the diets (Table 1) for 3 wk. The clofibrate diet (CLF) contained 0.2% clofibrate in the standard diet (STD). The STD and CLF were low fat diets with only 2.5% soybean oil, whereas the other diets were high fat diets as follows: saturated fatty acid diet (SFA; 15% soybean oil hydrogenated), monounsaturated fatty acid diet (MUFA; 15% olive oil) and polyunsaturated fatty acid diet (PUFA; 15% soybean oil). In Experiment 2, to study the effect of feeding period of the PUFA diet on the dioxygenase activity in intestinal mucosa, 42 rats were divided into seven groups of six rats. All groups were stabilized with the STD diet for 6 d. One group served as time-zero control for the experiment. Other groups received either the STD diet or the PUFA diet (Table 1) for different periods of time up to 2 wk.
View this table:
Table 1.
Composition of the diets1
85°C until used. The upper half of the intestine was washed free of its contents. The intestinal mucosa was then gently scraped off with a microscope slide glass, and the -carotene dioxygenase activity was analyzed the same day.
-carotene dioxygenase activity. The rest of the S10 fraction was ultracentrifuged at 105,000 × g for 60 min (4°C). The resulting cytosol (S105 fraction) was quickly frozen in liquid nitrogen and kept at 85°C until used for the CRBP (II) assay.
, with bovine serum albumin as standard.
-Carotene 15,15'-dioxygenase assay.
The -carotene dioxygenase assay was carried out using the procedure described previously (During et al. 1996). Briefly, the reaction medium contained 15 µmol/L all-trans--carotene, 0.1 mol/L N-tris-(hydroxy-methyl)-methylglycine (Tricine)-KOH buffer (pH 8.0), 0.5 mmol/L dithiothreitol, 1.5 g/L Tween 40, 4 mmol/L sodium cholate, 0.1 mmol/L -tocopherol, 15 mmol/L nicotinamide and enzyme (<0.5 mg protein) in a total volume of 0.2 mL. Reaction was started by adding 40 µL of 75 µmol/L -carotene solubilized in 7.5 g/L aqueous Tween 40 and conducted at 37°C for 30 min; in the control incubation (time-zero control), the reaction was stopped immediately after the addition of the -carotene solution. The reaction was stopped by adding 50 µL of 37% (wt/wt) formaldehyde and the mixture was further incubated at 37°C for 10 min. Then, 500 µL of acetonitrile was added and proteins were precipitated by centrifugation at 10,000 × g for 10 min. The resulting supernatant (200 µL) was directly subjected to the HPLC system, which consisted of a LC-10AS pump, an SPD-10A UV-VIS absorbance detector, a 200 µL-sample loop, and a C-R3A integrator (Shimadzu, Kyoto, Japan). Retinal was separated on a TSK gel ODS-80Ts C18 reverse-phase column (Tosoh, Tokyo, Japan) with acetonitrile/water (90:10, v/v) containing 1 g/L ammonium acetate as mobile phase (flow rate of 1.0 mL/min) and monitored at 380 nm (maximum absorbance of retinal). Retinal was quantified from its peak area by using a standard curve of all-trans-retinal. The incubation and extraction procedures were carried out under dim yellow light to minimize isomerization and degradation by light irradiation.
View this table:
Table 2.
Body gain and organ weights in rats fed the different dietary fats and clofibrate1
View larger version (62K):
[in a new window]
Fig 1.
Effects of dietary fats and clofibrate on -carotene 15,15'-dioxygenase specific and total activities in (A) rat intestinal mucosa and (B) liver. Eexperiment 1 was conducted with 7-wk-old rats fed one of following diets: standard (STD; 2.5% soybean oil), saturated (SFA; 15% hydrogenated soybean oil), monounsaturated (MUFA; 15% olive oil), polyunsaturated (PUFA; 15% soybean oil), and clofibrate (CLF; 2.5% soybean oil + 0.2% clofibrate) diets for 3 wk. Values represent means ± SD, n = 8. Data were log-transformed. Values not sharing a letter are significantly different, P < 0.01, Tukey's test.
using 15% acrylamide, 1-mm-thick slab gels. The proteins in the gel were electrophoretically transferred to nitrocellulose membrane (5 V/cm, 2 h); immunoblotting was performed by the method of Tsang et al. (1983) using rabbit anti-rat CRBP (II) antiserum (Takase et al. 1993) and goat anti-rabbit immunoglobulin G (IgG)-peroxidase conjugate (Jackson Immunresearch Laboratories, West Grove, PA) as the primary and the secondary antibodies, respectively. The amount of CRBP (II) was quantified by densitometric determination using a Shimadzu CS-9000 densitometer (Shimadzu). CRBP (II) used for standard was purified from rat small intestine as described previously (Takase et al. 1993).
). When homogenous variances were confirmed, the data were tested by one-way ANOVA, followed by Tukey's test (Sokal and Rohlf 1981) to identify significantly different means. The values underwent log transformation before the tests, if necessary. When heterogeneous variances were detected, the data were analyzed nonparametrically by the Kruskal-Wallis test (Sokal and Rohlf 1981) and significant differences of means were evaluated by the Mann-Whitney U test (Sokal and Rohlf 1981). P values < 0.05 were considered significant. All analyses except for Tukey's test were performed using the StatView software (Version 4.51, Abacus Concepts, Berkeley, CA). Tukey's test was performed by using a calculation table created with Excel 97 software (Microsoft, Redmond, WA).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (65K):
[in a new window]
Fig 2.
Effects of dietary fats and clofibrate on cellular retinol-binding protein (CRBP) type II level in rat intestinal mucosa. (A) Immunoblots of CRBP (II) for rats fed the different diets. Proteins of cytosolic fraction of intestinal mucosa (8 µg) were separated by SDS-PAGE, transferred to nitrocellulose membrane and detected with anti-rat CRBP (II) antiserum. Bound antibody was stained with peroxidase coupled to anti-rabbit immunoglobulin G (IgG) as described. Lane 1, STD diet group; lane 2, CLF diet group; lane 3, PUFA diet group; lane 4, MUFA diet group; lane 5, SFA diet group; lane 6, STD diet group. (B) CRBP (II) level in intestinal homogenate of rats fed different diets. CRBP (II) levels are expressed as a ratio to cytosolic proteins and as a total amount of CRBP (II) in the mucosa of the upper intestine. Values represent means ± SD (n = 3 for STD and SFA diet groups, n = 4 for CLF diet group, and n = 5 for MUFA and PUFA diet groups).Values not sharing a letter are significantly different, P < 0.05, Mann-Whitney's U test. See legend to Figure 1 for diet abbreviations.
-carotene 15,15'-dioxygenase of the MUFA and PUFA groups fed high fat (15%) diets were 2.4 times that of the STD group fed low fat (2.5%) diet (Fig. 1, P < 0.01). The specific activities of the SFA and CLF groups were not significantly different from that of the STD group. The total activities of the intestinal mucosa in the MUFA and PUFA groups were 2.7 and 3.2 times that of the STD group, respectively (P < 0.01). In liver, the specific activities of the MUFA and PUFA groups were not significantly higher than that of the STD group, whereas the CLF group had a lower specific activity than the STD group (P < 0.01). The total activity of the PUFA group in the whole liver was 1.7 times that of the STD group (P < 0.01).
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[in a new window]
Fig 3.
Changes in -carotene 15,15'-dioxygenase activity of rat intestinal mucosa during feeding of the standard (STD) and polyunsaturated fatty acid (PUFA) diets. In Experiment 2, 7-wk-old rats were fed the standard diet (STD; 2.5% soybean oil) for 1 wk; thereafter they consumed either the STD diet or the polyunsaturated diet (PUFA; 15% soybean oil) for different time periods. Values represent means ± SD, n = 6-8. Values not sharing a letter are significantly different, P < 0.05, Mann-Whitney's U test. Data from the previous experiment corresponding to the 21-d point for both STD and PUFA diets were also plotted (see Fig. 1) but were not included in the statistical analysis.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
-carotene to retinal in intestinal cells is catalyzed in large part by -carotene 15, 15'-dioxygenase (Nagao et al. 1996), and the enzyme plays an essential role in providing vertebrates with vitamin A. However, few data have been reported about the regulation of its activity. The enhancement of -carotene bioavailability and CRBP (II) expression by dietary unsaturated fats strongly suggests a modification of dioxygenase activity by dietary lipids. In this study, we found that a high fat diet rich in polyunsaturated fatty acids enhanced both -carotene dioxygenase activity and CRBP (II) level in rat intestine.
). In addition, we found that -carotene dioxygenase activity in intestine was elevated after consumption for 3 wk of a high fat diet rich in mono- or polyunsaturated fatty acids. The elevation of the activity was also observed within 1 d of consuming the polyunsaturated diet (<6-7 h of food intake followed by overnight food deprivation). This quick response of the activity to diets was observed for CRBP (II) mRNA in the previous study. It was enhanced by 54-63% in rat jejunum within 6 h of consuming unsaturated fatty acids (Suruga et al. 1995). Thus, these observations suggest that the response of -carotene dioxygenase to the dietary fatty acids is similar to that of CRBP (II). CRBP (II) is involved not only in absorption and metabolism of preformed vitamin A in intestine, but also in the conversion of retinal to retinol. Retinal, a product of the dioxygenase reaction, is bound to CRBP (II) and is reduced to retinol by microsomal retinal reductase (Ong 1993). Therefore, the simultaneous increases of the dioxygenase activity and CRBP (II) level would have a physiologic role in the efficient conversion of -carotene to vitamin A in intestine. Indeed, the total dioxygenase activity and total CRBP (II) level in intestinal mucosa in the rats fed the PUFA diet were much more elevated relative to STD rats than those expressed per unit protein. The results suggest that high fat diets enhance the total ability of intestine to convert -carotene to vitamin A. Moreover, carotenoid absorption is promoted by dietary fat. Therefore, these responses to a high fat diet might be part of a physiologic adaptation to produce as much vitamin A as possible when retinol and provitamin A are easily available. However, it is not clear whether the increase of CRBP (II) promotes -carotene conversion to vitamin A in vivo. In fact, CRBP (II) is present in large amounts in the intestinal cytosol and is not saturated with retinol when excess amounts of -carotene are consumed (Suzuki et al. 1995).
). The CRBP (II) level was higher in the mature enterocytes than in the stem cells of chicks (Goda et al. 1993), and -carotene dioxygenase activity was also found to be present mainly in the mature enterocytes of rat jejunum (Duszka et al. 1996). These observations suggest that these enzymes and CRBP (II) work cooperatively to produce vitamin A from -carotene and that the enzyme activities and the protein level are regulated by a common mechanism.
). The enhancement of CRBP (II) expression by dietary long-chain fatty acids was thought to be due to activation of PPAR by either fatty acids or their metabolites. Indeed, some peroxisome proliferators such as ETYA, an analog of arachidonic acid, and clofibric acid enhance the CRBP (II) level in Caco-2 cells, which are derived from a human colon adenocarcinoma (Suruga et al. 1997). In this study, however, clofibrate diet did not enhance the CRBP (II) level or -carotene dioxygenase activity. Consuming the clofibrate diet for a long time induces several enzymes for lipid catabolism as well as drug metabolizing enzymes in microsomes. It might be difficult to detect the induction of CRBP (II) and the dioxygenase even if clofibrate induces them through PPAR.
). The specific activity of the dioxygenase in liver was not enhanced by the PUFA diet, whereas the total activity in the whole liver was enhanced by the PUFA diet. This may be of no physiologic importance because -carotene is not present at detectable level in liver unless rats are fed pharmacologic levels of -carotene. However, if this response occurs in humans, it might enhance the conversion of -carotene to vitamin A.
-carotene dioxygenase activity as well as the CRBP (II) level in rat intestine. These new findings help explain the regulation of -carotene conversion to vitamin A.
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FOOTNOTES |
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-Carotene 15,15'-dioxygenase activity: its tissue distribution and regulation by dietary fats and clofibrate in rats).
Manuscript received 2 December 1997. Initial reviews completed 29 January 1998. Revision accepted 8 June 1998.
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ACKNOWLEDGMENT |
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We are grateful to M. Kitagawa of School of Food and Nutritional Sciences, The University of Shizuoka for his kind analyses of CRBP (II).
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Abstract
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Methods
Results
Discussion
References
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