Regulation by Dietary Fats of 3-Hydroxy-3-Methylglutaryl-Coenzyme ReducÃ-ase in Rat Liver
A
ABSTRACT The effects of various dietary fats on the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A ( HMG-CoA ) reducÃ-asein rat liver microsomes, the rate-limiting enzyme in cholesterogenesis, were examined. A series of experiments demonstrated the dependency of the HMG-CoA reducÃ-ase activity on the nature of dietary fats. When saturated fats with chain length of 12 to 18 were the dietary sources and were fed at the 10% level for 19 days, feeding fats with shorter chain fatty acids caused a lower enzyme activity compared to those with longer chain fatty acids. The activity was also regulated by the degree of unsaturation of dietary fats; when safflower oil, camellia oil or tristearin were fed at the 10% level for 18 days, the higher the unsaturation, the lower the activity. When trimyristin or tripalmitin were fed at the 10% level for 14 days, addition of essential fatty acid, at the level of minimum daily requirement (1% was replaced by safflower oil), did not affect the enzyme activity. Though the rate of incorporation of mevalonate into cholesterol in the 12,500 X g supernatant fraction of the liver was also found to be influenced by the types of dietary fats, the extent of the response appeared much smaller than that of HMG-CoA reductase. No consistent correlation between the HMG-CoA reductase activity and the content of microsomal cholesterol or cholesteryl ester and the fatty acid composition of microsomal lipids was observed. J. Nutr. 108: 601-612, 1978. INDEXING KEY WORDS hepatic cholesterogenesis •HMG-CoA reductase •dietary fats •essential fatty acid •microsomal cholesterol 3-Hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase [EC 1.1.1.34] is known to be the rate-limiting enzyme of cholesterogenesis in animal tissues. The activity of hepatic HMG-CoA reductase is controlled by the nutritional and hormonal state of the animals (1-3). One of the nutntional factors controlling the activity of this enzyme in the liver is the amount and nature of dietary fat. Craig et al. (4) observed in rats a decrease in the activity by changing the diets from a closed formula, non-purified stock diet to a fat-free diet. Goldfarb
and
PitOt
(5)
reported
that
etary corn oil stimulated the enzyme activity in proportion to the amount added to a fat-free diet. In the hamster dietary ethyl palmitate stimulated, whereas ethyl linoleate, depressed, hepatic HMG-CoA reductase (6). Further information on the effects of differences in the composition of d¡et fats Qn th e actiyify of the ,. , ., ,, u, . .Õ Ilver arf not available although there are some data showing that hepatic cholesterogenesis measured using acetate as a
di-
Received for publication
601
July 6, 1977.
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TAKASHI IDE, HIROSHI OKAMATSU AND MICHIHIRO SUGANO Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Kyushu University School of Agriculture, Fukuoka 812 Japan
602
IDE, OKAMATSU AND SUGANO TABLE
1
Fatty acid corn-position of dietary fats
Fatty acids Fats
8:0
10:0
12:0
%Coconut
14:0
16:0
18:0
18:1
18:2 Unknown
weight
substrate is regulated by the type of di etary fat (7-10). In this communication the hepatic HMGCoA reductase activity was measured in rats fed purified diets containing various fats of different compositions. Data sup porting the dependency of the activity on the nature of dietary fats, degree of unsaturation as well as chain length of constituent fatty acids, are presented. MATERIALS AND METHODS
Materials. DL-[3-14C]HMG anhydride was synthesized from DL-[3-14C]HMG1 (10,000 dpm/nmole) according to the pro cedure of Goldfarb and Pitot ( 11 ) and was then converted to DL-[3-14C]HMG-CoA by the method of Suzuki et al. (12).2 The DL-[3-14C]HMG-CoA thus synthesized was further purified through a 1 cm X 100 cm column of sephadex G-10. The quantity of DL-HMG-CoA was determined (13) and its specific radioactivity was accurately de termined. The specific radioactivity of the labeled HMG-CoA was adjusted to 5,000 dpm/nmole prior to use. DL-Mevalonolactone3 and DL-[2-14C]mevalonic acid * were dissolved together in 0.1 N NaHCOs, incubated at 37°for 90 minutes to convert the lactone to the free acid ( 14 ) and then the mixture was treated with diethyl ether to remove the salt. Prior to use for the enzymatic analysis the solu tion was neutralized with 0.1 N HC1. Animals and diets. Male Wistar rats5 were housed individually in an air condi
tioned room (22-25°) in which the light was on between 06:00 and 18:00 hours and fed a closed formula, non-purified diet6 until body weight reached 70 to 100 g. Rats were then divided into groups with equal body weights and fed purified experimental diets. The composition of the basal diet expressed as weight percent was as follows: vitamin-free casein,7 20; min eral mixture,8 (15) 4; vitamin mixture (15) 1; choline chloride, 0.15; cellulose powder, 4 and sucrose to 100. Fats (5 to 20% ) were added at the expense of su crose. Feeding periods were shown in each table. Fatty acid composition of dietary fat is shown in table 1. Preparation of liver microsomes. The rats were killed by decapitation. Livers were quickly removed, rinsed with ice cold saline, weighed and homogenized in nine volumes of 0.25 M sucrose containing 0.05 M potassium phosphate buffer (pH 7.0), 0.075 M nicotinamide, 0.02 M ß-mercaptoethanol and 0.0025 M EDTA. The homogenate was centrifuged at 12,500 X g for 20 1 New England Nuclear Corp., Boston, Mass., spe cific activity, 22.8-51.92 mCl/mmole, diluted as Indicated with unlabeled HMG, Tokyo Kasel, Co., Tokyo. 2 Coenzyme A was the product of Kyowa Hakko Kogyo, Ltd., Tokyo. » Nakaral Chemicals, Ltd., Kyoto. * DL-[2-14C]mevalonic acid dibenzylethylenedlamine salt, the Radiochemical Centre, Amersham, specific activity 36 mCl/m mole. 5 Kyudo Co.. Kumamoto. • Type NMF, Oriental Yeast Co., Tokyo. 7 Nutritional Blochemicals Co., Cleveland. 8 The mineral and vitamin mixtures according to Harper (15), were purchased from the Tanabe Amino Acid Foundation, Osaka.
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52.6— oilCorn —— — oilSafflower _ oilHydrogenated-safflower —— — —— — oilCamellia —100.0 — oilTrioctanoinTrilaurinTrimyristinTripalmitinTristearin3.5 —— — 99.4— — 2.5— —— — .————5.7————— — —20.6—————0.691.02.11.110.612.512.218.47.8——6.689.712.82.82.21.981.
DIETARY FATS AND HMG-CoA REDUCTASE
603
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minutes to sediment nuclei, cell debris and Assay of the conversion of mevalonate mitochondria. The supernatant was centri- to cholesterol. The method of Goodwin fuged at 105,000 X g for 60 minutes. The and Margolis (17) was slightly modified; microsomal pellet thus obtained was sus 15 /¿molesMgCl2 and 50 /¿molesdibasic pended in potassium phosphate buffer potassium phosphate, were added to 1 ml (pH 7.2) containing 0.02 M ß-mercapto- of the 12,500 X g supernatant solution. The ethanol and 0.02 M EDTA. An aliquot of reaction was started by the addition of a this suspension was used for the assay of solution containing 2 /¿molesNADP,6 6 /¿moles glucose-6-phosphate,6 1 enzyme HMG-CoA reducÃ-ase activity and deter mination of cholesterol (18) and protein unit glucose-6-phosphate dehydrogenase," 2 /¿molesATP2 and 0.5 /¿molesDL-[2-14C](23). Assay of HMG-CoA reducÃ-ase activity. mevalonic acid (0.2 /¿Ci),the final volume The method of Shapiro et al. (16) was was 1.2 ml, pH 7.4. Incubation was con slightly modified. The incubation mixture tinued at 37°for 60 minutes. Two ml of 95% ethanol and 0.5 ml of 60% KOH were contained 10 /¿molespotassium phosphate buffer (pH 7.2), 2 /¿moles/î-mercapto- then added and the mixture was saponified ethanol, 2 /¿moles EDTA, 2 /¿moles at 70°for 90 minutes. Unsaponifiable mat NADPH,6 0.05 /¿moles DL-[3-"C] HMG- ter was extracted twice with petroleum CoA (0.11 /¿Ci)and microsomal protein in ether (b.p. 40-60°) and the combined ex a final volume of 100 /J. Incubation was tract was washed once with water. Digicarried out in a metabolic shaker at 37°. tonin precipitable sterols (DPS) were iso Under this condition, formation of mevalo- lated and purified according to the pro nate was found to be linear at least for 45 cedure of Sperry and Webb (18). The radioactivity in DPS was counted in a minutes of incubation using 450 /¿gof pro tein per tube and also linear at least for toluene scintillation fluid (2 g of 2,5-di phenyloxazole and 50 mg of l,4-di-2-(5up to 900 /¿gof protein per tube employ per liter of tolu ing 30 minutes of incubation. Routinely, phenyloxazolyl)-benzene 200 to 700 tig of protein per tube was used ene) and corrected for quenching as pre viously. The activity was expressed as and incubation was continued for 30 min utes. The reaction was terminated by the nmoles of mevalonate incorporated into addition of 50 /¿I of 6 N HC1, and the mix DPS per hour per g of liver. ture was reincubated at 37°for 30 minutes Lipid analysis. Plasma, liver and its mi to insure the lactonization of mevalonic crosomal lipids were extracted and puri acid formed. The mixture was then cen- fied according to the procedure of Folch trifuged at 2,000 X g for 5 minutes to sedi et al. ( 19). Cholesterol was determined by ment denatured protein. An aliquot of the the method of Sperry and Webb (18). supernatant (usually 100 /¿I)was submit Separation of phosphatidylcholine was ted for thin-layer chromatography (silica carried out by thin-layer chromatography gel G),9 benzene :acetone (1:1, v/v) as a on silica gel G9 using a solvent mixture devleoping solvent. The area correspond of chloroform :methanol: water (65:25:4, ing to mevalonolactone ( Rf value, 0.6-0.7 ) v/v/v) (20, 21). Gas-liquid Chromato was directly scraped into the counting vial graphie (GLC) analyses of fatty acid and the radioactivity was counted 10 in a methyl esters were done as reported pre viously (22) using a hydrogen flame ionidioxane scintillation fluid (100 g of naph thalene and 4 g of 2,5-diphenyloxazole per zation detector " and a 2 m X 4 mm stain liter of dioxane). The radioactivity was less steel column packed with 5% dicorrected for quenching by an external ethylene glycol succinate on support standard. The HMG-CoA reducÃ-aseactivity medium,12 60-80 mesh. was expressed as pmoles of mevalonate formed per minute per mg of microsomal "E. Merck, Darmstadt. protein. The variation in the enzyme activ 10Intertechnique model SL-32 scintillation spec was used. ity from the same liver source never ex trometer 11Japan Electron Optic Laboratory JGC-750 gas ceeded ±5% by the method employed Chromatograph, Tokyo. "Chromosorb PAW DMCS, Gaschro Kogyo Co., herein. Tokyo.
604
IDE, OKAMATSU AND SUGANO
Microsomal protein determination. In order to avoid the interference of /3-mercaptoethanol with the determination of protein, the sample was initially treated 1oJ"°'-iio'S with trichloroacetic acid and then dis significanti 20Is§1IMC and 5% for ; solved in a known volume of 0.4 N NaOH. 1Co00 ICO contenti11:§•§te eroi An aliquot of this solution was assayed for oOOtÃ’-H-HO-H-H-HiC oÃOO5aO protein by the procedure of Lowry et al. (23). * a Statistical analysis. Data were analyzed CO io CN-H-H-HlOrl by the Student's t test (24). COJ-H-H1-H CN
i-«-H-HÎt^~ 1C
mPì!Õ1n.16i1ICbOQS^^ 100 ^00 O Oi-H00 Ol
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T^-H-H-HCO
1C-H-H-H"O 1C
CJsCO r~»
RESULTS
CN1-H IM
Effect of fat-free, coconut oil and corn oil diets ( experiment 1 ). Table 2 shows microsomal HMG-CoA reductase activity ¡*¡1g and cholesterol content in rats fed the diets CO COf-IN 'SOBri'H11ofree of fats or containing either coconut oil or corn oil as a fat source for 3 weeks. OSCO5îï§s§1C —iTt< 00 Dietary fat levels were 5 or 20%. On con OCO ^A,lìC CM-H-H-Hr-cNOCOCN CN -H sidering circadian rhythm of hepatic HMGCoA reductase (2, 3), rats were killed at 10:00 hours (when the enzyme activity is eC low) and at 24:00 hours (when the enzyme e CN -S'5co faßifo C! coÌ^HIM óo oó.'H0•ibd•a•88O....o1-H8o activity is high). oo88 In the experiment with low fat diets, the ^il3 growth of rats fed the fat-free diet, com 00 COCN COsoasi>ooo pared with those fed the fat containing diets, tended to be lower, though they all o ö consumed the same amount of diet. In con So3 »O^ »Oooo $Î flj> trast, food consumption of rats fed high -U-H-Hi—i -H-H-HT-ICOIOCO fat diets was lower than that in rats fed i—o-H-H-HCO cor-t-CO At the 5% dietary level the concentra ,lls'Sg tion of microsomal cholesterol was lowest 00-H-H-HCO CO in rats fed corn oil, while at the 20% level the lowest value was found in rats fed t-•* CO 00 00 o.fe IM CO'o CO X1 coconut oil, the same trend was also ob T P 0 3 < e he .05. when Time rats served in whole liver. Thus no correlation seems to exist between the enzyme activity respectively. and the content of cholesterol. Plasma cho -Sao « lesterol was significantly lower in rats fed corn oil compared with those fed a fat03 O O free diet in one instance (20% fats and IO*»C killed at midnight). oSO§1C5£
8CO
C
O5li?0»Plasma 91±7»62±5°
1Rats weighing 75 g to 85 g were fed experimental diets containing 10% of fats for 19 days and killed at midnight. 2 Mean±SEMof seven rats. Values in a column not sharing a common superscript letter are significantly different at P < 0.05.
the same but more remarkable tendency was observed in whole liver. It seemed likely that in this experiment some nega tive correlation may exist between die HMG-CoA reductase activity and the con tent of cholesterol or cholesteryl ester. No difference was found in the concentration of plasma cholesterol among groups. Effects of fats with different degree of unsaturation (experiment 4). The dietary fats examined were safflower oil (rich in linoleic acid), camellia oil (rich in oleic acid) and tristearin (rich in stearic acid). Trilaurin was also included for comparison. These fats were added to the diet at the 10% level. Rats were fed for 18 days and killed at 24:00 hours. No significant difference in the food in take due to the type of dietary fat was ob
served. Compared with rats fed trilaurin, those fed safflower oil showed a signifi cantly greater weight gain. As shown in table 5, the highest HMGCoA reductase was observed with tri stearin, followed by camellia oil and saf flower oil in decreasing order. Differences in enzyme activities among these groups were highly significant from each other. The activity observed with trilaurin was lowest and it was significantly lower than with safflower oil. Though the rate of incorporation of mevalonate into digitonin precipitable sterols (DPS) was significantly lower in rats fed trilaurin compared with the rats fed long chain fatty acid-fats, no signifi cant differences were observed among the latter three groups.
TABLE 5 Effects of trilaurin, safflower oil, camellia oil and tristearin on HMG-CoA reductase, rate of incorporation of mevalonate into digitonin precipitable sterols (DPS) and cholesterol content of microsomes and plasma (experiment 4) cholesterolTotalw/mg30.1 cholesterolTotalEstermg/100 Dietary fats'Trilaurin gaing
-»DPSpmoles/min/mg intakeg/day*14.9 reductaseMevalonate
nmoles/hr/g ml287 liver261.1 protein3 ±4«-« 62±3»' ±1.0» ± 5.5» 28.6± 5.8« ±0.9° Safflower oil 136±4» 16.0±0.4° 119.4± 6.6» 149.9±31.86 24.6±0.66 2.2 ±0.3'' 99±6°'"67±1» t Camellia oil 124±7»."15.3±0.7»177.1±13.3° 119.8± 8.3* 25.5±0.56 2.3±0.1" 103±5» 67 ±4«. 83±3° 59±2"t 2.8±0.3"Plasma 316.2±32.5° 154.3±20.56Microsomal 25.7±0.9tEsterprotein24.3±0.6» 16.4±0.6°HMG-CoA TristearinWeight108±7°Food days2113±6« / 18
1Rats weighing 70 g to 90 g were fed experimental diets containing 10% of fats for 18 days and killed at midnight. 2 Mean±SEM of 7 rats. Values in a column not sharing a common superscript letter are significantly different at P < 0.05.
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days2124 protein'92.0 protein227.3 ±2° ±0.4« •" ± 6.5" ±0.5« 120±6»'!l 13.7 ±0.4» 70.4 ± 6.6 " 29.5±1.4« Trilaurin 119±1°'6 15.1 ±0.3» 251.2±38.1° 26.5±1.3«.' Tripalmitin 284.1 ±22.8«Microsomal 25.4±0.4* TristearinWeight 111±56Food 15.0±0.7«'6HMG-CoA
cholesterolTotal
DIETARY FATS AND HMG-CoA REDUCTASE
607
TABLE 6 Effects of trimyristin, tripalmitin and supplementation of essential fatly acid (EFA) on HMG-CoA reducÃ-ase, rate of incorporation of mevalonale into digitonin precipitable sterols (DPS) and cholesterol content of microsomes and plasma (experiment 5) cholesterolTotalEsteritg/gm cholesterolTotalEstermg/100 Dietary fats'Trimyristingaing
-»DPSnmoles/hr/g intakeg/day2 reducÃ-asepmoles/min/mg
Trimyristin ±4°. " 2.3±0.6° ±6° 62 ±3». t +EFA ±3°100±3"Food 84 17.5±0.7°19.6±0.5"HMG-CoA 214.3±21.06265.4±24.7lMevalonate 170.8±15.2«177.0±18.5°Microsomal 2.0±0.5°22.8±0.6° 24.8±0.66 55±3«100 80±3" Tripalmitin Tripalmitin 2.2±0.3°Plasma ±5«69 ±4»k +EFAWeight 1Rats weighing 80 g to 100 g were fed experimental diets containing 10% of fats for 2 weeks and killed at midnight. In essential fatty acid supplemented groups, 1% of safflower oil was added at the expense of saturated fats. 2 Mean±SEMof 7 rats. Values in a column not sharing a common superscript letter are significantly different at P < 0.05.
There were no detectable differences in the concentration of microsomal cholesterol and cholesteryl ester among the groups fed C-18 fats, the values were significantly lower than those observed in rats fed trilaurin. The same tendency was observed with the liver but it was more profound than in the microsomes. Plasma cholesterol was lowest in rats fed tristearin among those fed C-18 fats. Effects of supplement of essential fatty acid to saturated fats (experiment 5). Table 6 shows the effect of trimyristin and tri palmitin on enzyme activity. In the pre ceding experiments in which saturated fats were used as a dietary source, there was some indication of essential fatty acid de ficiency. In this experiment, therefore, the effect of an addition of essential fatty acid to these saturated fats was examined. The dietary fat level was 10%. When essential fatty acid was included to the diets, 1% of safflower oil was added at the expense of each fat, the amount containing approxi mately the minimal requirement for linoleic acid in these rats. Rats were fed for 14 days and killed at 24:00 hours. Weight gains and food consumption were the largest in rats fed essential fatty acid supplemented tripalmitin, but no demonstrable alteration was found in these parameters among the other three groups. As shown in table 6, feeding tripalmitin resulted in a 2- to 2.5-fold increase in the
enzyme activity compared with trimyristin. Addition of essential fatty acid to these saturated fats had no effect on the HMGCoA reductase activity. The rate of incorporation of mevalonate into DPS tended to be higher in rats fed the diets containing tripalmitin, but the difference was not significant. Again, ad dition of essential fatty acid did not in fluence the rate of this reaction. Microsomal cholesterol content was higher in the two groups deficient in es sential fatty acid compared with the corre sponding supplemented groups. Also the value for rats fed trimyristin was higher than that of the corresponding tripalmitinfed group. No significant alteration was, however, observed in cholesteryl ester con tent. A more profound trend was observed in cholesterol content of the liver. Plasma cholesterol was lowest in rats fed tripal mitin not supplemented with essential fatty acid. Fatty acid composition of microsomal lipids. Table 7 summarizes fatty acid com position of microsomal lipids prepared from rats killed at midnight. Though the results were obtained from different lipid fractions in each experiment, fatty acid profiles were routinely reflected by the types of dietary fats. In addition, in rats fed saturated fats the composition resem bled each other irrespective of the chain length of fatty acids and there was an in-
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protein2111.6±13.7°96.2±31.0° liver2124.1±16.0«112.0±48.3° ¡2weeks2 protein2 ml2 3.5±0.6°26.3±0.96 28.9±1.0° ±8°94 16.1=tl.3017.0±0.7° 84 91 ±2° 62 ±3" •95
608
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ABSTRACT The effects of various dietary fats on the activity of 3-hydroxy-3-methylglutaryl-Coenzyme A ( HMG-CoA ) reducÃ-asein rat liver microsomes, the rate-limiting enzyme in cholesterogenesis, were examined. A series of experiments demonstrated the dependency of the HMG-CoA reducÃ-ase activity on the nature of dietary fats. When saturated fats with chain length of 12 to 18 were the dietary sources and were fed at the 10% level for 19 days, feeding fats with shorter chain fatty acids caused a lower enzyme activity compared to those with longer chain fatty acids. The activity was also regulated by the degree of unsaturation of dietary fats; when safflower oil, camellia oil or tristearin were fed at the 10% level for 18 days, the higher the unsaturation, the lower the activity. When trimyristin or tripalmitin were fed at the 10% level for 14 days, addition of essential fatty acid, at the level of minimum daily requirement (1% was replaced by safflower oil), did not affect the enzyme activity. Though the rate of incorporation of mevalonate into cholesterol in the 12,500 X g supernatant fraction of the liver was also found to be influenced by the types of dietary fats, the extent of the response appeared much smaller than that of HMG-CoA reductase. No consistent correlation between the HMG-CoA reductase activity and the content of microsomal cholesterol or cholesteryl ester and the fatty acid composition of microsomal lipids was observed. J. Nutr. 108: 601-612, 1978. INDEXING KEY WORDS hepatic cholesterogenesis •HMG-CoA reductase •dietary fats •essential fatty acid •microsomal cholesterol 3-Hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase [EC 1.1.1.34] is known to be the rate-limiting enzyme of cholesterogenesis in animal tissues. The activity of hepatic HMG-CoA reductase is controlled by the nutritional and hormonal state of the animals (1-3). One of the nutntional factors controlling the activity of this enzyme in the liver is the amount and nature of dietary fat. Craig et al. (4) observed in rats a decrease in the activity by changing the diets from a closed formula, non-purified stock diet to a fat-free diet. Goldfarb
and
PitOt
(5)
reported
that
etary corn oil stimulated the enzyme activity in proportion to the amount added to a fat-free diet. In the hamster dietary ethyl palmitate stimulated, whereas ethyl linoleate, depressed, hepatic HMG-CoA reductase (6). Further information on the effects of differences in the composition of d¡et fats Qn th e actiyify of the ,. , ., ,, u, . .Õ Ilver arf not available although there are some data showing that hepatic cholesterogenesis measured using acetate as a
di-
Received for publication
601
July 6, 1977.
Downloaded from https://academic.oup.com/jn/article-abstract/108/4/601/4770459 by guest on 19 November 2018
TAKASHI IDE, HIROSHI OKAMATSU AND MICHIHIRO SUGANO Laboratory of Nutrition Chemistry, Department of Food Science and Technology, Kyushu University School of Agriculture, Fukuoka 812 Japan
602
IDE, OKAMATSU AND SUGANO TABLE
1
Fatty acid corn-position of dietary fats
Fatty acids Fats
8:0
10:0
12:0
%Coconut
14:0
16:0
18:0
18:1
18:2 Unknown
weight
substrate is regulated by the type of di etary fat (7-10). In this communication the hepatic HMGCoA reductase activity was measured in rats fed purified diets containing various fats of different compositions. Data sup porting the dependency of the activity on the nature of dietary fats, degree of unsaturation as well as chain length of constituent fatty acids, are presented. MATERIALS AND METHODS
Materials. DL-[3-14C]HMG anhydride was synthesized from DL-[3-14C]HMG1 (10,000 dpm/nmole) according to the pro cedure of Goldfarb and Pitot ( 11 ) and was then converted to DL-[3-14C]HMG-CoA by the method of Suzuki et al. (12).2 The DL-[3-14C]HMG-CoA thus synthesized was further purified through a 1 cm X 100 cm column of sephadex G-10. The quantity of DL-HMG-CoA was determined (13) and its specific radioactivity was accurately de termined. The specific radioactivity of the labeled HMG-CoA was adjusted to 5,000 dpm/nmole prior to use. DL-Mevalonolactone3 and DL-[2-14C]mevalonic acid * were dissolved together in 0.1 N NaHCOs, incubated at 37°for 90 minutes to convert the lactone to the free acid ( 14 ) and then the mixture was treated with diethyl ether to remove the salt. Prior to use for the enzymatic analysis the solu tion was neutralized with 0.1 N HC1. Animals and diets. Male Wistar rats5 were housed individually in an air condi
tioned room (22-25°) in which the light was on between 06:00 and 18:00 hours and fed a closed formula, non-purified diet6 until body weight reached 70 to 100 g. Rats were then divided into groups with equal body weights and fed purified experimental diets. The composition of the basal diet expressed as weight percent was as follows: vitamin-free casein,7 20; min eral mixture,8 (15) 4; vitamin mixture (15) 1; choline chloride, 0.15; cellulose powder, 4 and sucrose to 100. Fats (5 to 20% ) were added at the expense of su crose. Feeding periods were shown in each table. Fatty acid composition of dietary fat is shown in table 1. Preparation of liver microsomes. The rats were killed by decapitation. Livers were quickly removed, rinsed with ice cold saline, weighed and homogenized in nine volumes of 0.25 M sucrose containing 0.05 M potassium phosphate buffer (pH 7.0), 0.075 M nicotinamide, 0.02 M ß-mercaptoethanol and 0.0025 M EDTA. The homogenate was centrifuged at 12,500 X g for 20 1 New England Nuclear Corp., Boston, Mass., spe cific activity, 22.8-51.92 mCl/mmole, diluted as Indicated with unlabeled HMG, Tokyo Kasel, Co., Tokyo. 2 Coenzyme A was the product of Kyowa Hakko Kogyo, Ltd., Tokyo. » Nakaral Chemicals, Ltd., Kyoto. * DL-[2-14C]mevalonic acid dibenzylethylenedlamine salt, the Radiochemical Centre, Amersham, specific activity 36 mCl/m mole. 5 Kyudo Co.. Kumamoto. • Type NMF, Oriental Yeast Co., Tokyo. 7 Nutritional Blochemicals Co., Cleveland. 8 The mineral and vitamin mixtures according to Harper (15), were purchased from the Tanabe Amino Acid Foundation, Osaka.
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52.6— oilCorn —— — oilSafflower _ oilHydrogenated-safflower —— — —— — oilCamellia —100.0 — oilTrioctanoinTrilaurinTrimyristinTripalmitinTristearin3.5 —— — 99.4— — 2.5— —— — .————5.7————— — —20.6—————0.691.02.11.110.612.512.218.47.8——6.689.712.82.82.21.981.
DIETARY FATS AND HMG-CoA REDUCTASE
603
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minutes to sediment nuclei, cell debris and Assay of the conversion of mevalonate mitochondria. The supernatant was centri- to cholesterol. The method of Goodwin fuged at 105,000 X g for 60 minutes. The and Margolis (17) was slightly modified; microsomal pellet thus obtained was sus 15 /¿molesMgCl2 and 50 /¿molesdibasic pended in potassium phosphate buffer potassium phosphate, were added to 1 ml (pH 7.2) containing 0.02 M ß-mercapto- of the 12,500 X g supernatant solution. The ethanol and 0.02 M EDTA. An aliquot of reaction was started by the addition of a this suspension was used for the assay of solution containing 2 /¿molesNADP,6 6 /¿moles glucose-6-phosphate,6 1 enzyme HMG-CoA reducÃ-ase activity and deter mination of cholesterol (18) and protein unit glucose-6-phosphate dehydrogenase," 2 /¿molesATP2 and 0.5 /¿molesDL-[2-14C](23). Assay of HMG-CoA reducÃ-ase activity. mevalonic acid (0.2 /¿Ci),the final volume The method of Shapiro et al. (16) was was 1.2 ml, pH 7.4. Incubation was con slightly modified. The incubation mixture tinued at 37°for 60 minutes. Two ml of 95% ethanol and 0.5 ml of 60% KOH were contained 10 /¿molespotassium phosphate buffer (pH 7.2), 2 /¿moles/î-mercapto- then added and the mixture was saponified ethanol, 2 /¿moles EDTA, 2 /¿moles at 70°for 90 minutes. Unsaponifiable mat NADPH,6 0.05 /¿moles DL-[3-"C] HMG- ter was extracted twice with petroleum CoA (0.11 /¿Ci)and microsomal protein in ether (b.p. 40-60°) and the combined ex a final volume of 100 /J. Incubation was tract was washed once with water. Digicarried out in a metabolic shaker at 37°. tonin precipitable sterols (DPS) were iso Under this condition, formation of mevalo- lated and purified according to the pro nate was found to be linear at least for 45 cedure of Sperry and Webb (18). The radioactivity in DPS was counted in a minutes of incubation using 450 /¿gof pro tein per tube and also linear at least for toluene scintillation fluid (2 g of 2,5-di phenyloxazole and 50 mg of l,4-di-2-(5up to 900 /¿gof protein per tube employ per liter of tolu ing 30 minutes of incubation. Routinely, phenyloxazolyl)-benzene 200 to 700 tig of protein per tube was used ene) and corrected for quenching as pre viously. The activity was expressed as and incubation was continued for 30 min utes. The reaction was terminated by the nmoles of mevalonate incorporated into addition of 50 /¿I of 6 N HC1, and the mix DPS per hour per g of liver. ture was reincubated at 37°for 30 minutes Lipid analysis. Plasma, liver and its mi to insure the lactonization of mevalonic crosomal lipids were extracted and puri acid formed. The mixture was then cen- fied according to the procedure of Folch trifuged at 2,000 X g for 5 minutes to sedi et al. ( 19). Cholesterol was determined by ment denatured protein. An aliquot of the the method of Sperry and Webb (18). supernatant (usually 100 /¿I)was submit Separation of phosphatidylcholine was ted for thin-layer chromatography (silica carried out by thin-layer chromatography gel G),9 benzene :acetone (1:1, v/v) as a on silica gel G9 using a solvent mixture devleoping solvent. The area correspond of chloroform :methanol: water (65:25:4, ing to mevalonolactone ( Rf value, 0.6-0.7 ) v/v/v) (20, 21). Gas-liquid Chromato was directly scraped into the counting vial graphie (GLC) analyses of fatty acid and the radioactivity was counted 10 in a methyl esters were done as reported pre viously (22) using a hydrogen flame ionidioxane scintillation fluid (100 g of naph thalene and 4 g of 2,5-diphenyloxazole per zation detector " and a 2 m X 4 mm stain liter of dioxane). The radioactivity was less steel column packed with 5% dicorrected for quenching by an external ethylene glycol succinate on support standard. The HMG-CoA reducÃ-aseactivity medium,12 60-80 mesh. was expressed as pmoles of mevalonate formed per minute per mg of microsomal "E. Merck, Darmstadt. protein. The variation in the enzyme activ 10Intertechnique model SL-32 scintillation spec was used. ity from the same liver source never ex trometer 11Japan Electron Optic Laboratory JGC-750 gas ceeded ±5% by the method employed Chromatograph, Tokyo. "Chromosorb PAW DMCS, Gaschro Kogyo Co., herein. Tokyo.
604
IDE, OKAMATSU AND SUGANO
Microsomal protein determination. In order to avoid the interference of /3-mercaptoethanol with the determination of protein, the sample was initially treated 1oJ"°'-iio'S with trichloroacetic acid and then dis significanti 20Is§1IMC and 5% for ; solved in a known volume of 0.4 N NaOH. 1Co00 ICO contenti11:§•§te eroi An aliquot of this solution was assayed for oOOtÃ’-H-HO-H-H-HiC oÃOO5aO protein by the procedure of Lowry et al. (23). * a Statistical analysis. Data were analyzed CO io CN-H-H-HlOrl by the Student's t test (24). COJ-H-H1-H CN
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Effect of fat-free, coconut oil and corn oil diets ( experiment 1 ). Table 2 shows microsomal HMG-CoA reductase activity ¡*¡1g and cholesterol content in rats fed the diets CO COf-IN 'SOBri'H11ofree of fats or containing either coconut oil or corn oil as a fat source for 3 weeks. OSCO5îï§s§1C —iTt< 00 Dietary fat levels were 5 or 20%. On con OCO ^A,lìC CM-H-H-Hr-cNOCOCN CN -H sidering circadian rhythm of hepatic HMGCoA reductase (2, 3), rats were killed at 10:00 hours (when the enzyme activity is eC low) and at 24:00 hours (when the enzyme e CN -S'5co faßifo C! coÌ^HIM óo oó.'H0•ibd•a•88O....o1-H8o activity is high). oo88 In the experiment with low fat diets, the ^il3 growth of rats fed the fat-free diet, com 00 COCN COsoasi>ooo pared with those fed the fat containing diets, tended to be lower, though they all o ö consumed the same amount of diet. In con So3 »O^ »Oooo $Î flj> trast, food consumption of rats fed high -U-H-Hi—i -H-H-HT-ICOIOCO fat diets was lower than that in rats fed i—o-H-H-HCO cor-t-CO At the 5% dietary level the concentra ,lls'Sg tion of microsomal cholesterol was lowest 00-H-H-HCO CO in rats fed corn oil, while at the 20% level the lowest value was found in rats fed t-•* CO 00 00 o.fe IM CO'o CO X1 coconut oil, the same trend was also ob T P 0 3 < e he .05. when Time rats served in whole liver. Thus no correlation seems to exist between the enzyme activity respectively. and the content of cholesterol. Plasma cho -Sao « lesterol was significantly lower in rats fed corn oil compared with those fed a fat03 O O free diet in one instance (20% fats and IO*»C killed at midnight). oSO§1C5£
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1Rats weighing 75 g to 85 g were fed experimental diets containing 10% of fats for 19 days and killed at midnight. 2 Mean±SEMof seven rats. Values in a column not sharing a common superscript letter are significantly different at P < 0.05.
the same but more remarkable tendency was observed in whole liver. It seemed likely that in this experiment some nega tive correlation may exist between die HMG-CoA reductase activity and the con tent of cholesterol or cholesteryl ester. No difference was found in the concentration of plasma cholesterol among groups. Effects of fats with different degree of unsaturation (experiment 4). The dietary fats examined were safflower oil (rich in linoleic acid), camellia oil (rich in oleic acid) and tristearin (rich in stearic acid). Trilaurin was also included for comparison. These fats were added to the diet at the 10% level. Rats were fed for 18 days and killed at 24:00 hours. No significant difference in the food in take due to the type of dietary fat was ob
served. Compared with rats fed trilaurin, those fed safflower oil showed a signifi cantly greater weight gain. As shown in table 5, the highest HMGCoA reductase was observed with tri stearin, followed by camellia oil and saf flower oil in decreasing order. Differences in enzyme activities among these groups were highly significant from each other. The activity observed with trilaurin was lowest and it was significantly lower than with safflower oil. Though the rate of incorporation of mevalonate into digitonin precipitable sterols (DPS) was significantly lower in rats fed trilaurin compared with the rats fed long chain fatty acid-fats, no signifi cant differences were observed among the latter three groups.
TABLE 5 Effects of trilaurin, safflower oil, camellia oil and tristearin on HMG-CoA reductase, rate of incorporation of mevalonate into digitonin precipitable sterols (DPS) and cholesterol content of microsomes and plasma (experiment 4) cholesterolTotalw/mg30.1 cholesterolTotalEstermg/100 Dietary fats'Trilaurin gaing
-»DPSpmoles/min/mg intakeg/day*14.9 reductaseMevalonate
nmoles/hr/g ml287 liver261.1 protein3 ±4«-« 62±3»' ±1.0» ± 5.5» 28.6± 5.8« ±0.9° Safflower oil 136±4» 16.0±0.4° 119.4± 6.6» 149.9±31.86 24.6±0.66 2.2 ±0.3'' 99±6°'"67±1» t Camellia oil 124±7»."15.3±0.7»177.1±13.3° 119.8± 8.3* 25.5±0.56 2.3±0.1" 103±5» 67 ±4«. 83±3° 59±2"t 2.8±0.3"Plasma 316.2±32.5° 154.3±20.56Microsomal 25.7±0.9tEsterprotein24.3±0.6» 16.4±0.6°HMG-CoA TristearinWeight108±7°Food days2113±6« / 18
1Rats weighing 70 g to 90 g were fed experimental diets containing 10% of fats for 18 days and killed at midnight. 2 Mean±SEM of 7 rats. Values in a column not sharing a common superscript letter are significantly different at P < 0.05.
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days2124 protein'92.0 protein227.3 ±2° ±0.4« •" ± 6.5" ±0.5« 120±6»'!l 13.7 ±0.4» 70.4 ± 6.6 " 29.5±1.4« Trilaurin 119±1°'6 15.1 ±0.3» 251.2±38.1° 26.5±1.3«.' Tripalmitin 284.1 ±22.8«Microsomal 25.4±0.4* TristearinWeight 111±56Food 15.0±0.7«'6HMG-CoA
cholesterolTotal
DIETARY FATS AND HMG-CoA REDUCTASE
607
TABLE 6 Effects of trimyristin, tripalmitin and supplementation of essential fatly acid (EFA) on HMG-CoA reducÃ-ase, rate of incorporation of mevalonale into digitonin precipitable sterols (DPS) and cholesterol content of microsomes and plasma (experiment 5) cholesterolTotalEsteritg/gm cholesterolTotalEstermg/100 Dietary fats'Trimyristingaing
-»DPSnmoles/hr/g intakeg/day2 reducÃ-asepmoles/min/mg
Trimyristin ±4°. " 2.3±0.6° ±6° 62 ±3». t +EFA ±3°100±3"Food 84 17.5±0.7°19.6±0.5"HMG-CoA 214.3±21.06265.4±24.7lMevalonate 170.8±15.2«177.0±18.5°Microsomal 2.0±0.5°22.8±0.6° 24.8±0.66 55±3«100 80±3" Tripalmitin Tripalmitin 2.2±0.3°Plasma ±5«69 ±4»k +EFAWeight 1Rats weighing 80 g to 100 g were fed experimental diets containing 10% of fats for 2 weeks and killed at midnight. In essential fatty acid supplemented groups, 1% of safflower oil was added at the expense of saturated fats. 2 Mean±SEMof 7 rats. Values in a column not sharing a common superscript letter are significantly different at P < 0.05.
There were no detectable differences in the concentration of microsomal cholesterol and cholesteryl ester among the groups fed C-18 fats, the values were significantly lower than those observed in rats fed trilaurin. The same tendency was observed with the liver but it was more profound than in the microsomes. Plasma cholesterol was lowest in rats fed tristearin among those fed C-18 fats. Effects of supplement of essential fatty acid to saturated fats (experiment 5). Table 6 shows the effect of trimyristin and tri palmitin on enzyme activity. In the pre ceding experiments in which saturated fats were used as a dietary source, there was some indication of essential fatty acid de ficiency. In this experiment, therefore, the effect of an addition of essential fatty acid to these saturated fats was examined. The dietary fat level was 10%. When essential fatty acid was included to the diets, 1% of safflower oil was added at the expense of each fat, the amount containing approxi mately the minimal requirement for linoleic acid in these rats. Rats were fed for 14 days and killed at 24:00 hours. Weight gains and food consumption were the largest in rats fed essential fatty acid supplemented tripalmitin, but no demonstrable alteration was found in these parameters among the other three groups. As shown in table 6, feeding tripalmitin resulted in a 2- to 2.5-fold increase in the
enzyme activity compared with trimyristin. Addition of essential fatty acid to these saturated fats had no effect on the HMGCoA reductase activity. The rate of incorporation of mevalonate into DPS tended to be higher in rats fed the diets containing tripalmitin, but the difference was not significant. Again, ad dition of essential fatty acid did not in fluence the rate of this reaction. Microsomal cholesterol content was higher in the two groups deficient in es sential fatty acid compared with the corre sponding supplemented groups. Also the value for rats fed trimyristin was higher than that of the corresponding tripalmitinfed group. No significant alteration was, however, observed in cholesteryl ester con tent. A more profound trend was observed in cholesterol content of the liver. Plasma cholesterol was lowest in rats fed tripal mitin not supplemented with essential fatty acid. Fatty acid composition of microsomal lipids. Table 7 summarizes fatty acid com position of microsomal lipids prepared from rats killed at midnight. Though the results were obtained from different lipid fractions in each experiment, fatty acid profiles were routinely reflected by the types of dietary fats. In addition, in rats fed saturated fats the composition resem bled each other irrespective of the chain length of fatty acids and there was an in-
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protein2111.6±13.7°96.2±31.0° liver2124.1±16.0«112.0±48.3° ¡2weeks2 protein2 ml2 3.5±0.6°26.3±0.96 28.9±1.0° ±8°94 16.1=tl.3017.0±0.7° 84 91 ±2° 62 ±3" •95
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