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Effect of diet on hepatic and intestinal lipogenesis in squirrel, Cebus, and cynomolgus monkeys
Atherosclerosis, 20 (1974) 405-416 0 Elsevier Scientific Publishing Company,
405
Amsterdam
- Printed in The Netherlands
EFFECT OF DIET ON HEPATIC AND INTESTINAL SQUIRREL, CEBUS, AND CYNOMOLGUS MONKEYS
JOYCE E. COREY Department
AND
of Nutrition,
LIPOGENESIS
IN
K. C. HAYES Harvard
School of Public Health,
Boston,
Mass. 02115 (U.S.A.)
(Received October 31st, 1973) (Accepted February
13th, 1974)
SUMMARY
Since distinct species differences were found in the serum lipid response of several species of nonhuman primates to dietary fat and cholesterol, in vitro lipogenesis by liver and intestine was studied in four of these species (Cebus albifons and apella, Macaca fascicularis, and Saimiri sciureus) in order to identify potential differences in the capacity of these tissues to synthesize cholesterol and triglyceride. Comparison of New World cebus and Old World cynomolgus monkeys demonstrated that the cebus monkey exhibited a greater potential for cholesterogenesis in the liver and for total lipogenesis in the jejunum than did the cynomolgus monkey. Proportionately, however, the cynomolgus demonstrated a higher rate of intestinal cholesterol synthesis in vitro relative to hepatic cholesterol synthesis than did the New World species. The feeding of cholesterol with butterfat to squirrel monkeys caused a 94% inhibition of cholesterol synthesis from acetate in liver slices and a 48 ‘A inhibition in the ileum. Dietary coconut oil, on the other hand, when compared to safflower oil resulted in a significant increase in the rate of triglyceride synthesis from acetate in liver from cebus monkeys and in jejunum from cebus and cynomolgus monkeys. The possible relationship of these differences in lipogenesis to species differences in the hyperlipidemic capacity of dietary fat and cholesterol is discussed.
Key words : Cholesterol - In vitro synthesis - Primates - Triglycerides
This work was supported in part by U.S. Public Health Service Grants HL-10098, GM-333 and KO4-HL-70285 and the Fund for Research and Teaching, Department of Nutrition, Harvard School of Public Health.
406
J. E. COREY, K. C. HAYES
INTRODUCTION
For reasons that are still unclear, considerable variation exists among species of nonhuman primates in their susceptibility to both spontaneous and diet-induced atherosclerosisr. Part of this variability might be attributed to differences in the serum lipid response of these primate species to dietary fat and cholesterol since hypercholesterolemia is considered a critical factor in the development of atherosclerosissJ. A previous report4 described the serum lipid changes in four primate species during manipulation of dietary fat, cholesterol, and carbohydrate. Having evaluated these serum differences, a systematic investigation of cholesterol metabolism in these species, was initiated. Maintenance of cholesterol homeostasis is controlled by a balance between absorption, synthesis, deposition, mobilization and excretion. Since the liver and gut have been shown to account for 97 % of endogenous cholesterol synthesis in the squirrel monkeys, initial efforts have focused on in vitro lipogenesis by these tissues in several species of primates studied under different dietary treatments. MATERIALS AND METHODS
Treatment of animals
The present study included two separate experiments utilizing 24 squirrel (Saimiri sciureus) (Experiment l), 7 cebus (Cebus albifrons and apella) and 12 cynomolgus (Macaca fascicularis) monkeys (Experiment 2). It is recognized that cebus albifrons and cebus apella actually represent two species of the genus cebus, but data from these two species have been treated as one since the two groups were statistically indistinguishable with respect to all parameters measured. Although the dietary treatments of the squirrel monkeys in Experiment 1 did not provide an ideal experimental design, the experiment did offer an opportunity to evaluate lipogenesis in polyunsaturated fat-fed and cholesterol-saturated fat-fed squirrel monkeys. In Experiment 1 young adult male squirrel monkeys (Tarpon Zoo, Tarpon Springs, Fla.) were fed a purified agar cake diet (Table 1) containing either 10 % cottonseed-soybean oil (Wesson Oil, Hunt-Wesson Foods, Fullerton, Calif.) (6 monkeys) or 10% butterfat plus 0.2 % cholesterol (18 monkeys) for 10 months. The juvenile male and female cebus and cynomolgus monkeys used in Experiment 2 were raised in our primate nursery6 and at the time of the study were 33 years old and had been fed a purified agar cake diet containing 8% safflower oil or coconut oil (Table 1) for 30 months. All the animals were fed on the day of sacrifice and tissues for incubation were taken 2 h postprandially under Nembutal anesthesia. Blood was collected from the posterior vena cava for the determinations of serum cholesterol7 and triglycerides, and agarose gel electrophoresis was performed according to the method of Nobleg. In vitro conditions
Incubations
were carried out in 20 ml incubation
vials containing
2.5 ml
EFFECT OF DIET ON HEPATIC
TABLE
AND INTESTINAL
407
LIPOGENESIS
1
COMPOSITION
OF EXPERIMENTAL
Zngredientsa
Casein Lactalbumin Dextrin Sucrose Safflower oil Coconut oil Butterfat Cottonseed-soybean Vitamin mixC Salt mixd Choline chloride Inositole Cellulosef Cholesterolg
MONKEY
Experiment
DIETS (g/loo
I
g) FED DURING
EXPERIMENTS
Experiment
1 AND
2
-
diet I
diet 2
diet 3
diet 4
20.0 42.5
20.0 42.5 10.0 0.5 4.0 0.34 0.1 21.6 0.2
-
-
14.6 23.7 28.9 8.0
14.6 23.7 28.9 8.0 0.4 4.0 0.24 20.0 -
oilb 10.0 0.5 4.0 0.34 0.1 21.6
0.4 4.0 0.24 20.0
2
lngredients were mixed and a hot 2.5% Bacto-agar solution added. The diet was cooled and cut into small cubes for feeding. Wesson Oil, Hunt-Wesson Foods Inc., Fullerton, Calif. The vitamin mix contained (in mg); thiamine hydrochloride, 80; riboflavin, 160; pyridoxine hydrochloride, 80; calcium pantothenate, 500; niacinamide, 800; folic acid, 20; biotin, 4; cyanocobalamin, 3; menadione, 100; dl-a-tocopherol acid succinate, 1000; retinyl acetate beadlets, 250,000 I.U.; cholecalciferol beadlets, 25,000 I.U.; ascorbic acid, 12.25 g made to 100 g with dextrin. HEGSTED, D. M., et al., J. Biol. C/tern., 138 (1941) 459. Inositol added at 0.1 g/l00 g diet. Alphacel, General Biochemicals, Chagrin Falls, Ohio. Cholesterol, U.S.P., Nutritional Biochemicals Corp., Cleveland, Ohio.
Krebs-Ringer bicarbonate buffer (pH 7.4), 3.0 pmole sodium acetate (1.2 mm), 6 ,&i [I-r*C]acetate (New England Nuclear, Boston, Mass.; specific activity 55 Ci/ mole), 5.0 mg glucose, and 0.125 mg penicillin and streptomycin, The tightly capped vials were pregassed with 95 ‘A 02-5 ‘4 CO2 for 5 min prior to incubation and for 5 min at the start of the incubation carried out with shaking at 37 “C for 2 h. At sacrifice, sections of liver and gut were quickly excised and rinsed in ice cold Krebs-Ringer bicarbonate buffer. Liver slices (0.5 mm thick) were obtained with a Stadie-Riggs hand microtome. Full thickness slices of jejunum and terminal ileum were taken approximately 3 cm below the ligament of Treitz and 5 cm from the ileocecal junction, respectively. Lipogenesis along the length of the gut was also determined in 5 cebus and 5 cynomolgus monkeys by taking sequential sections approximately 10 cm apart for incubation. Samples of tissues weighing approximately 200 mg were blotted dry and transferred to the pregassed incubation vials. After 2 h of incubation, the tissues were removed, rinsed in Krebs-Ringer bicarbonate buffer and the reaction stopped by transfer to tubes containing 10 ml of a chloroform-methanol
408
J. E. COREY, K. C. HAYES
(2:l). It had been previously determined that negligible lipid extractable radioactivity was present in the medium at the end of the incubation period. After extraction of tissues by shaking, a procedure which has been shown to remove 99 % of the lipid extractable radioactivity, the extract was filtered and washed according to the method of Folch et al.10 and the fat-free dry weight of the tissues determined to the nearest ,ug. Thin-layer chromatography of the extract was performed using 0.25 mm thick Silica Gel H plates developed in a mixture of hexane-diethyl ether-acetic acid (70:30:1). Aliquots of the extract were also used for cholesterol analysis’. Autoradiographs of the thin-layer chromatograms were developed to verify the identity of the lipid and radioactivity bands *. Thin-layer chromatograms were then scraped and the silica gel transferred directly to 10 ml of POPOP scintillation fluid (New England Nuclear, Boston, Mass.) for determination of radioactivity incorporated into free fatty acids, cholesterol, cholesterol ester and squalene, triglycerides and phospholipids. Counting efficiency was determined by the channels ratio methodlr. The results are expressed as the mean i SD. of nmoles acetate incorporated per g fat-free dry weight. Only radioactivity incorporated into total lipid, cholesterol and triglyceride is reported, since free fatty acid radioactivity was negligible. Cholesterol ester-squalene radioactivity was highly variable and phospholipid radioactivity was nonspecific, actually representing all the label remaining at the origin of the thin-layer chromatogram. Analysis of variance was performed on the data and the effects of treatment were evaluated by Duncan’s multiple range testis. mixture
TABLE PLASMA MONKEYS
2 CHOLESTEROL
AND
IN EXPERIMENTS
TRIGLYCERIDE 1 AND
Species and dietary fat
CONCENTRATIONS
OF
CEBUS,
CYNOMOLGUS
AND
SQUIRREL
2 Plasma cholesterol
Plasma
(mgldl)
(meld11
triglyceride
SquirreI
Cottonseed-soybean oil Butterfat + cholesterol
164 •t 61& 362 zt 82c
21 + 118 30 i 26a
139 f 138 254 zt 6gb
24 + 108 91 + 21b
144 + 3g8 163 i 198
35 + 17a 100 f 55b
Cebus
Safflower oil Coconut oil CynomoIgus
Safflower oil Coconut oil
Mean * SD. B*b*CValues within a column without a common superscript differ significantly (P < 0.05).
* Thin-layer chromatograms were placed in contact with Eastman Kodak Blue Brand Medical X-ray film in a tightly sealed cassette for 4 days.
EFFECT OF DIET ON HEPATIC
AND INTESTINAL
LIPOGENESIS
409
Fig. 1. Cebus and cynomolgus monkeys fed safflower oil or coconut oil (Experiment 2) demonstrated a significant positive correlation (r = 0.74) between serum triglycerides and the ‘A lipid staining in the pre-/I region after agarose gel electrophoresis.
RESULTS
Plasma lipids
The terminal plasma cholesterol and triglyceride concentrations of monkeys in Experiment 1 and 2 are shown in Table 2. Plasma cholesterol levels were low and similar in all monkeys fed cottonseed-soybean oil (Experiment 1) or safflower oil (Experiment 2). The cholesterol-butterfat fed squirrel monkeys in Experiment 1 developed a hypercholesteremia as did the coconut oil fed cebus monkeys in Experiment 2. The cholesterol levels of the cynomolgus were not significantly increased by coconut oil. Plasma triglycerides were low in safflower oil fed cebus and cynomolgus monkeys and in squirrel monkeys regardless of diet. Coconut oil induced a significant triglyceridemia in both the cebus and cynomolgus monkeys which was significantly correlated (r = 0.74) with an increase in the % lipid staining in the pre-I3 region on agarose gel electrophoresis (Fig. 1). Species diflerences in lipogenesis
The data from Experiment 2 presented in Table 3 provide a basis for interspecies comparison (cebus and cynomolgus) of rates of total lipid, cholesterol, and triglyceride synthesis from acetate in the liver and gut. Safflower oil fed cebus monkeys incorporated more acetate into total hepatic lipid than did the safflower oil fed cynomolgus monkeys. When considering individual lipid fractions, this differential was most striking for hepatic cholesterogenesis, the New World cebus demonstrating significantly more cholesterogenic activity from acetate than the Old World cynomolgus. No significant species differences were found for hepatic triglyceride synthesis due to large standard deviations and the small number of animals, but the same trend was evident-the cebus exceeding the cynomolgus. Similar species differences in acetate incorporation into total lipid were demonstrated in the jejunum, but here the enhanced lipogenesis by the cebus was confined to triglyceride synthesis. Fig. 2 illustrates the relative rates of incorporation of acetate into cholesterol by liver and gut slices for these monkeys and also includes the data from the cottonseed-soybean oil fed
410
J. E. COREY, K. C. HAYES
TABLE 3 NMOLES[~-14C]~~~~~~~~~~~~~~~~~~~INTOHEPATICANDINTESTINALLIPIDSBYCEBUSANDCYNOMOLEXPERIMENT 2
GUSMONKEYSIN
Cynomolgus
Cebus
Liver Total lipide Cholesterol Triglyceride Tissue cholesterol, mg/g
safJl0 wer oil
coconut oil
safflower oil
coconut oil
(3)
(4)
(5)
(7)
5709 f 1274& 2932 + 1713& 1174 i- 398”
4847 * 926~9~ 1147 5 417b 2504 + 346b
3323 & 1580bgC 437 + 276b 893 & 617”
1760 & 1326c 173 & 97b 990 + 8558
22.1 i 4.5a
16.9 i- 2.1”
18.1 f 3.0a
18.8 i- 7.1a
1584 & 181 & 700 &
2472 k 47d 165 +I 63& 1493 f 32c
707 k 111 i 247 *
1159 * 108 i 597 f
23.8 & 1.7b
17.5 i 0.88
Jejunum
Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g Ileutn Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g
157c 52” 85b
18.5 + 1.8& 2082 f 754” 332 & 72asb 882 i 5858 15.2 & 2.2”
1371 + 209 f 422 i-
92&sb 74& 408
78” 16& 83”
22.2 & 2.7b
1601 & 745&vb 1396 i 343 + 163%rb 322 i 515 5 357& 399 + 16.7 + l.la
15.9 i 1.38
320” 368 151b
2698*b 96apb 3578
13.9 +c 2.4a
Numbers in parentheses represent number of animals. &,b,cTdValues within a row without a common superscript differ significantly (P < 0.05). e Data are expressed as nmoles [1-WZ]acetate incorporated/g fat-free dry weight i S.D.
squirrel monkeys (Experiment 1). It is apparent that cynomolgus monkeys had a larger contribution to the total cholesterol synthesis provided by the gut than either of the New World species. Although the most accurate representation of comparative rates of cholesterogenesis for the liver and gut would clearly necessitate consideration of organ size and weighting of the rates of cholesterogenesis, the relations shown in Fig. 2 would be exaggerated due to the fact that the cynomolgus has a proportionately longer intestine than the New World species. Similarly, when acetate incorporation into cholesterol was expressed as a percentage of total lipogenesis by each organ, only 11 T/, of the total hepatic label incorporated by the cynomolgus was in cholesterol, while the corresponding figure for the cebus monkeys was 47%. Conversely, in the intestine 16% of the acetate incorporated into lipid by the cynomolgus was in cholesterol, a significantly greater percentage than the 11 ‘A incorporated by the cebus monkeys (Table 3). Comparison of the intestinal lipogenic activity at 10 cm intervals by species (Fig. 3) indicated that the ileum was the most active segment for cholesterol synthesis in both the cebus and cynomolgus species, similar to the observation made by Diet-
EFFECT OF DIET ON HEPATIC AND INTESTINAL LIPOGENESIS
411
3000
Cebus
Squire1
Cynomolgus
Fig. 2. Comparison of the rates of acetate incorporation into cholesterol by liver, ileum and jejunum of those monkeys fed unsaturated fat revealed distinct species differences in the relative rates of cholesterogenesis by these organs.
schy and Wilson in squirrel monkeys 1s. Although similar rates of cholesterogenesis were observed in the ileal and jejunal sections of the cebus and cynomolgus, the cebus synthesized more cholesterol at all intermediate points and a greater amount of triglyceride throughout the gut (Fig. 3). EfSect of diet on lipogenesis
Imposed on the differences between species were differences within species due to dietary cholesterol (Experiment 1) and to the type of dietary fat (Experiment 2). In
Fig. 3. Mapping the small intestine for acetate incorporation into cholesterol and triglyceride at 10 cm intervals revealed species and dietary differences between cebus and cynomolgus monkeys (Experiment 2).
3. E. COREY, K. C. HAYES
412 TABLE 4 NMOLES
[b-‘4c]ACETATE
EXPERIMENT
INCORPORATED
INTO HEPATIC
AND INTESTINAL
LIPIDS
BY SQUIRREL
MONKEYS
IN
1
Cottonseed-soybean oil (6)
Butterfat + cholesterol (18)
3290 & 1516 & 978 h 16.8 +
3108 f 2171& 101 f 56b 2046 f 14218 76.1 f 55.1b
Liver
Total lipide Cholesterol Triglyceride Tissue cholesterol, mg/g
1876& 7948 924& 4.0a
Jejunum
Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g
571 & 194a 46 f 1P 297 * 14P 19.9 f 2.2=
573 f 496a 42 ?? 22& 261 & 253& 23.7 f 4.4&
1504 & 476 & 458 * 17.3 f
1126 f 231 & 426 i 16.4 zt
IIeum
Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g
3638 127b 154& 2.98
347b llO& 2308 3.4”
Numbers in parentheses represent number of animals. a,b Values within a row without a common superscript differ significantly (P < 0.05). e Data are expressed as nmoles [I-W]acetate incorporated/g fat-free dry weight + S.D. Experiment
I, cholesterol-butterfat
fed squirrel
monkeys
demonstrated
a 94% re-
duction in the incorporation of acetate into hepatic cholesterol when compared with cottonseed-soybean oil fed squirrel monkeys (Table 4). This inhibition was specific for cholesterol synthesis as total lipogenesis was unaffected due to a concomitant rise in triglyceride
synthesis.
An inhibition
of approximately
48 % of cholesterol
synthesis
from acetate was seen in the ileum of butterfat-cholesterol fed squirrel monkeys compared with unsaturated fat fed animals. In contrast, no aspect of Iipogenesis in the jejunum of the squirrel monkeys was affected by the feeding of cholesterol and butterfat. In Experiment 2, although comparison of coconut oil with safflower oil fed animals demonstrated no statistical difference in total hepatic Iipogenesis from acetate in either the cebus or cynomolgus monkeys, the cebus actually exhibited a significant increase in acetate incorporation into triglyceride and a parallel decrease in cholesterol synthesis. This coconut oil-induced enhancement of triglyceride synthesis from acetate was even more marked in jejunal slices from both the cebus and cynomolgus monkeys. No changes in cholesterol synthesis were observed. Lipid synthesis by ileal slices was independent of dietary fat in both species of monkeys in which this was examined. Tables 3 and 4 also include tissue concentrations of hepatic and intestinal cholesterol for the different species of monkeys. It is apparent that, in addition to inducing hypercholesterolemia, cholesterol feeding resulted in a significant increase
EFFECT
OF DIET
ON HEPATIC
AND INTESTINAL
LIPOGENESIS
413
in hepatic cholesterol but not intestinal cholesterol in the squirrel monkey. Coconut oil, on the other hand, induced a significant increase in the cholesterol content of the jejunum, but not the liver, in both the cebus and cynomolgus monkeys (Table 3). DISCUSSION
It has been previously demonstrated that the New World cebus monkey is particularly susceptible to elevation of serum cholesterol by dietary coconut oil and much less sensitive to dietary cholesterol, while the Old World cynomolgus responds to dietary cholesterol but is relatively insensitive to coconut oil4. In this same study squirrel monkeys responded to both coconut oil and cholesterol, attaining a serum cholesterol concentration similar to the cebus. The present investigation extended these observations on serum lipids to species differences in lipogenesis by gut and liver, tissues which are important to cholesterol homeostasis. Conclusions warranted by the data relate to the effect of dietary cholesterol on lipogenesis in the squirrel monkey, to the effect of coconut oil on lipogenesis in the cebus and cynomolgus monkeys, and to species variation in hepatic and intestinal lipogenesis. Although presumably all mammalian tissues can synthesize cholesteroli3si4, it has been demonstrated in both the rat and the squirrel monkey fed low cholesterol diets that per unit weight the liver and intestine are capable of the highest rates of in vitro cholesterogenesis and that control of cholesterol synthesis may differ in these two organ@. The present study provided evidence for this high capacity for in vitro cholesterol synthesis by liver and gut in squirrel, cebus and cynomolgus monkeys. The terminal ileum had greater synthetic activity than more proximal sections of the gut in the species studied here, a finding previously reported in the squirrel monkeyls and baboonis. In Experiment 1, feeding cholesterol and butterfat to squirrel monkeys almost totally inhibited in vitro hepatic cholesterol synthesis and partially inhibited ileal cholesterogenesis. Thus, increased endogenous cholesterol synthesis was not a contributing factor in the hypercholesterolemia induced by dietary cholesterol and butterfat. Other investigators have demonstrated negative feedback inhibition of hepatic enzymes controlling cholesterol synthesis is. Control of intestinal cholesterol synthesis has been less well defined and may involve dynamics between bile acid flux and luminal cholesterolsJ7. Coconut oil was reported previously to be hypercholesterolemic and results of the present study indicate that the hypercholesterolemia in cebus monkeys, like that induced by dietary cholesterol, was not attributable to enhanced endogenous cholesterol synthesis. In fact, coconut oil feeding decreased hepatic synthesis of cholesterol in agreement with other in vitro studies 1s. In contrast to its inhibitory effect on hepatic cholesterogenesis, coconut oil significantly enhanced incorporation of acetate into triglycerides in both liver and jejunum slices from the cebus monkey and in jejunum slices from the cynomolgus. This reciprocal relation between triglyceride and cholesterol synthesis from acetate parallels reciprocal alterations in hepatic enzymes controlling fatty acid and cholesterol synthesis in rats fed different levels of dietary
414
J. E. COREY, K. C. HAYES
fatis. The greater potential for fatty acid synthesis by liver slices from animals fed saturated fat is consistent with in vitro data from mice and rats18~20-22in which dietary unsaturated fats exerted a greater suppression on fatty acid synthesis than diets containing tripalmitin, triolein, or medium chain triglycerides when compared to maximal rates exhibited by animals on a fat-free diet. In the perfused rat liver, too, triglyceride output has been shown to increase with fatty acid chain length greater than Cl2 and to decrease with the degree of unsaturation 2s. The increased triglyceride synthesis by liver and gut slices was associated with an increased concentration of serum triglycerides localized in the pre-/?-lipoproteins by electrophoresis. A triglyceridemia associated with dietary saturated fat has been reported in man24y25and in squirrel and cebus monkeys fed coconut oil without dietary cholestero14. In addition to the effect of dietary cholesterol and fat on acetate incorporation into neutral lipids, distinct species differences appeared in both the relative rates of hepatic and intestinal lipogenesis in vitro and in the total amount of acetate incorporated into the various lipid fractions. In cynomolgus, cebus and squirrel monkeys fed cholesterol-free diets, the potential for cholesterol synthesis by the small intestine relative to that by the liver was much greater in the cynomolgus than in the squirrel or cebus monkeys. The cebus, however, was capable of greater hepatic cholesterol synthesis in absolute terms than either the cynomolgus or the squirrel monkey, the difference being most apparent with the cynomolgus. Similarly, the cebus incorporated more acetate into hepatic and jejunal triglyceride than did the cynomolgus or squirrel monkeys. However, these interspecies comparisons must be tempered by the fact that the experimental designs were not strictly comparable. Although extrapolation of in vitro data on acetate incorporation to in vivo synthesis is complicated by the potential effects of altered uptake of acetate by cells and by dilution of acetate by endogenous pools, Wilson15 has demonstrated a direct relation between endogenous cholesterol production measured in viva by isotopic balance and [2-r4C]acetate incorporation into cholesterol by liver slices under conditions shown to be optimal for his assay system. The observed species differences in hepatic and intestinal lipogenesis may thus be relevant to their differing physiological responses to dietary fat and cholesterol. The observed species differences in susceptibility to diet-induced hypercholesterolemia must reflect differences in one or more of the processes maintaining cholesterol homeostasis. With respect to the hypercholesterolemia induced by dietary cholesterol, the present results suggest an association between hypercholesterolemia and the relative rates of hepatic and intestinal cholesterogenesis. Species that develop hypercholesterolemia with dietary cholesterol -the cynomolgus monkey, hamsteP, and gerbil (unpublished data) - have a proportionately high capacity for intestinal cholesterol synthesis compared to liver than do less responsive species such as the New World monkeys and the rats. Absorption of cholesterol must also be considered in this context due to the feedback regulation of hepatic cholesterol synthesis by exogenous cholesterolrs. In this respect, man, like the cholesterol-responsive species mentioned above, has an intestinal synthetic rate which equals that of the liver in the proxi-
EFFECT OF DIET ON HEPATIC
AND INTESTINAL
LIPOGENESIS
415
ma1 portion and exceeds that of the liver by four times distally27, yet man is relatively insensitive to dietary cholesterol, circulating cholesterol levels rising only 5 mg/lOO mg dietary cholesterol”*. Consideration of cholesterol absorption rates, however, reveals that unlike the Old World monkeyzg or the hamsters0 which absorb cholesterol readily, man has a limited capacity to do ~031, perhaps conferring a relative resistance to dietary cholesterol. The other dietary ingredient resulting in hypercholesterolemia was coconut oil. This fat elevated both plasma cholesterol and triglyceride levels in cebus monkeys but only triglyceride levels in cynomolgus, and enhanced incorporation of acetate into triglyceride in vitro in both species while depressing cholesterogenesis. This latter observation suggests that endogenous cholesterol synthesis was also not a factor in coconut oil-induced hypercholesterolemia. The consistent elevation of plasma triglyceride levels and of in vitro triglyceride synthesis, however, suggests that increased triglyceride synthesis by lipoprotein-forming tissues may be causally related to the enhanced levels of circulating cholesterol. Connor et al.32 have associated the hypocholesterolemic capacity of unsaturated fat with an increased excretion of cholesterol as bile acids. Since excretion is ultimately a function of lipoprotein turnover, the altered patterns of in vitro lipogenesis observed here in safflower and coconut oil fed cebus and cynomolgus monkeys may reflect alterations in synthesis and subsequent metabolism and turnover of a specific triglyceride-rich lipoprotein. Data from studies using perfused rat liver33 and infused gut34 indicate that the long chain saturated fatty acid, palmitate, is incorporated into a very low density lipoprotein containing more cholesterol per unit of triglyceride than the lipoprotein particle formed from the polyunsaturated fatty acid, linoleate. The metabolism of these particles may also vary34. Analysis of the composition and metabolism of the lipoproteins secreted by monkeys under defined dietary conditions is required in order to support any hypothesis associating lipoprotein metabolism with species variability to diet and/or atherogenesis. Since these three primate species have distinct variations in lipid metabolism and atherosclerosis, they provide good models for these comparisons.
REFERENCES
1 STRONG, J. P., EGGEN, D. A., NEWMAN, W. P. AND MARTINEZ, R. D., Naturally occurring and experimental atherosclerosis in primates, Ann. N. Y. Acad. Sci., 149 (1968) 882. 2 KANNELL, W. B., DAWBER, T. R., FRIEDMAN,G. D., GLENNON,W. E. AND MCNAMARA, P. M., Risk factors in coronary heart disease: Evaluation of several serum lipids as predictors of coronary heart disease: Framingham Study, Ann. Znt. Med., 61 (1964) 888. 3 Task Force on Arteriosclerosis, ArterioscIerosis. A report by the National Heart and Lung Institute, Department of Health, Education and Welfare Pub]. No. 72-219, Vol. 2, 1971, Washington, D.C.,
pp. 47-49. 4 COREY, J. E., HAYES, K. C., DORR, B. AND HEGSTED,D. M., Comparative
lipid response of four primate species to dietary changes in fat and carbohydrate, Atherosclerosis, 19 (1974) 119. 5 DIETSCHY,J. M. AND WILSON, J. D., Regulation of cholesterol metabolism, New Engl. J. Med., 282 (1970) 1128. 6 AUSMAN, L. M., HAYES, K. C., LAGE, A. AND HEGSTED,D. M., Nursery care and growth of Old and New World infant monkeys, Lab. Animal Care, 20 (1970) 907.
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J. E. COREY, K. C. HAYES
7 CARPENTER,K. J., GOTSIS,A. AND HEGSTED, D. M., Estimation of total cholesterol in serum by a micro method, Clin. Chem., 3 (1957) 233. 8 MOORE, J. H., A modified method for the determination of glyceride glycerol, J. Dairy Res., 29 (1962) 141. 9 NOBLE, R. P., Electrophoretic separation of plasma lipoproteins in agarose gel, J. Lipid Res., 9 (1968) 693. 10 FOLCH, J., LEES, M. AND SLOANE-STANLEY, G. H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 11 BRUNO, G. A. AND CHRISTIAN, J. E., Correction for quenching associated with liquid scintillation counting, Anal. Chem., 33 (1961) 650. 12 STEELE, R. G. D. AND TORRIE, J. H., Principles and Procedures of Statistics with Special Reference to the Biological Sciences, McGraw-Hill, New York, N.Y., 1960, p. 72. synthesis in the squirrel monkey: Relative rates of 13 DIETSCHY, J. M. AND WILSON, J. D., Cholesterol synthesis in various tissues and mechanisms of control, J. Clin. Invest., 47 (1968) 166. 14 DIETSCHY, J. M. AND SIPERSTEIN, M. D., Effect of cholesterol feeding and fasting on sterol synthesis in 17 tissues of the rat, J. Lipid Res., 8 (1967) 97. 15 WILSON, J. D., The relation between cholesterol absorption and cholesterol synthesis in the baboon, J. C/in. Invest., 51 (1972) 1450. 16 SHAPIRO, D. J. AND RODWELL, V. W., Fine structure of the cyclic rhythm of 3-hydroxy-3-methyl 11 glutaryl CoA reductase: Differential effects of cholesterol feeding and fasting, Biochemistry, (1972) 1042. 17 SHEFER, S., HAUSER, S., LAPAR, V. AND MOSBACH, E. H., Regulatory effects of dietary sterols and J. Lipid Res., 14 (1973) 400. bile acids on rat intestinal HMG CoA reductase, 18 REISER, R., WILLIAMS, M. C., SORRELS, M. F. AND MURTY, N. L., Biosynthesis of fatty acids and cholesterol as related to diet fat, Arch. Biochem. Biophys., 102 (1963) 276. 19 CRAIG, M. C., DUGAN, R. E., MUESING, R. A., SLAKEY, L. L. AND PORTER, J. W., Comparative effects of dietary regimens on the levels of enzymes regulating synthesis of fatty acids and cholesterol in rat liver, Arch. Biochem. Biophys., 151 (1972) 128. 20 ABRAHAM, S., Effect of diet on hepatic fatty acid synthesis, Amer. J. Clin. Nutr., 23 (1970) 1120. 21 BARTLEY, J. C. AND ABRAHAM, S., Hepatic lipogenesis in fasted, refed rats and mice: Response to Biochim. Biophys. Actn, 280 (1972) 258. dietary fats of differing fatty acid composition, 22 WILEY, J. H. AND LEVEILLE, G. A., Metabolic consequences of dietary medium-chain triglycerides in the rat, J. N&r., 103 (1973) 829. 23 KOHOUT, M., KOHOUTOVA, B. AND HEIMBERG, M., The regulation of hepatic triglyceride metabolism by free fatty acids, J. Biol. Chem., 246 (1971) 5067. lowering diets. Experimental trials 24 ANDERSON, J. T., GRANDE, F. G. AND KEYS, A., Cholesterol and literature review, J. Amer. Dietet. Ass., 62 (1973) 133. 25 GRANDE, F., ANDERSON, J. T. AND KEYS, A., Diets of different fatty acid composition producing identical serum cholesterol levels in man, Amer. J. Clin. Nutr., 25 (1972) 53. 26 CHENG, C. Y. AND FELDMAN, E. B., Clofibrate, an inhibitor of intestinal cholesterogenesis, Biochem. Pharmacol., 20 (1971) 3509. 27 DIETSCHY, J. M. AND GAMEL, W. G., Cholesterol synthesis in the intestine of men: Regional influences and control mechanisms, J. Clin. Invest., 50 (1971) 872. 28 HEGSTED, D. M., MCGANDY, R. B., MYERS, M. L. AND STARE, F. J., Quantitative effects of dietary fat on serum cholesterol in man, Amer. J. Clin. N&r., 17 (1965) 281. 29 MANNING, P. J., CLARKSON, T. B. AND LOFLAND, H. B., Cholesterol absorption, turnover and excretion rates in hypercholesteremic rhesus monkeys, Exp. Molec. Puthol., 14 (1971) 75. 30 BEHER, W. T., BAKER, G. D. AND PENNEY, D. G., Comparative study of the effect of bile acids and cholesterol on cholesterol metabolism in the mouse, rat, hamster, and guinea pig, J. Nutr., 79 (1963) 523. 31 QUINTAO, E., GRUNDY, S. M. AND AHRENS, JR. E. H., Effects of dietary cholesterol on the regulation of total body cholesterol in man, J. Lipid Res., 12 (1971) 233. 32 CONNOR, W. E., WITIAK, D. T., STONE, D. B. AND ARMSTRONG, M. L., Cholesterol balance and fecal neutral steroid and bile acid excretion in normal men fed dietary fats of different fatty acid composition, J. Clin. Invest., 48 (1969) 1363. 33 HEIMBERG, M. AND WILCOX, H. G., Effect of palmitic and oleic acids on the properties and composition of the very low density lipoproteins secreted by the liver, J. Biol. Chem., 247 (1972) 875. 34 OCKNER, R. K., HUGHES, F. B. AND ISSELBACHER, K. J., Intestinal lymph: Role in triglyceride and cholesterol transport during fat transport, J. Clin. Invest., 48 (1969) 2367.
405
Amsterdam
- Printed in The Netherlands
EFFECT OF DIET ON HEPATIC AND INTESTINAL SQUIRREL, CEBUS, AND CYNOMOLGUS MONKEYS
JOYCE E. COREY Department
AND
of Nutrition,
LIPOGENESIS
IN
K. C. HAYES Harvard
School of Public Health,
Boston,
Mass. 02115 (U.S.A.)
(Received October 31st, 1973) (Accepted February
13th, 1974)
SUMMARY
Since distinct species differences were found in the serum lipid response of several species of nonhuman primates to dietary fat and cholesterol, in vitro lipogenesis by liver and intestine was studied in four of these species (Cebus albifons and apella, Macaca fascicularis, and Saimiri sciureus) in order to identify potential differences in the capacity of these tissues to synthesize cholesterol and triglyceride. Comparison of New World cebus and Old World cynomolgus monkeys demonstrated that the cebus monkey exhibited a greater potential for cholesterogenesis in the liver and for total lipogenesis in the jejunum than did the cynomolgus monkey. Proportionately, however, the cynomolgus demonstrated a higher rate of intestinal cholesterol synthesis in vitro relative to hepatic cholesterol synthesis than did the New World species. The feeding of cholesterol with butterfat to squirrel monkeys caused a 94% inhibition of cholesterol synthesis from acetate in liver slices and a 48 ‘A inhibition in the ileum. Dietary coconut oil, on the other hand, when compared to safflower oil resulted in a significant increase in the rate of triglyceride synthesis from acetate in liver from cebus monkeys and in jejunum from cebus and cynomolgus monkeys. The possible relationship of these differences in lipogenesis to species differences in the hyperlipidemic capacity of dietary fat and cholesterol is discussed.
Key words : Cholesterol - In vitro synthesis - Primates - Triglycerides
This work was supported in part by U.S. Public Health Service Grants HL-10098, GM-333 and KO4-HL-70285 and the Fund for Research and Teaching, Department of Nutrition, Harvard School of Public Health.
406
J. E. COREY, K. C. HAYES
INTRODUCTION
For reasons that are still unclear, considerable variation exists among species of nonhuman primates in their susceptibility to both spontaneous and diet-induced atherosclerosisr. Part of this variability might be attributed to differences in the serum lipid response of these primate species to dietary fat and cholesterol since hypercholesterolemia is considered a critical factor in the development of atherosclerosissJ. A previous report4 described the serum lipid changes in four primate species during manipulation of dietary fat, cholesterol, and carbohydrate. Having evaluated these serum differences, a systematic investigation of cholesterol metabolism in these species, was initiated. Maintenance of cholesterol homeostasis is controlled by a balance between absorption, synthesis, deposition, mobilization and excretion. Since the liver and gut have been shown to account for 97 % of endogenous cholesterol synthesis in the squirrel monkeys, initial efforts have focused on in vitro lipogenesis by these tissues in several species of primates studied under different dietary treatments. MATERIALS AND METHODS
Treatment of animals
The present study included two separate experiments utilizing 24 squirrel (Saimiri sciureus) (Experiment l), 7 cebus (Cebus albifrons and apella) and 12 cynomolgus (Macaca fascicularis) monkeys (Experiment 2). It is recognized that cebus albifrons and cebus apella actually represent two species of the genus cebus, but data from these two species have been treated as one since the two groups were statistically indistinguishable with respect to all parameters measured. Although the dietary treatments of the squirrel monkeys in Experiment 1 did not provide an ideal experimental design, the experiment did offer an opportunity to evaluate lipogenesis in polyunsaturated fat-fed and cholesterol-saturated fat-fed squirrel monkeys. In Experiment 1 young adult male squirrel monkeys (Tarpon Zoo, Tarpon Springs, Fla.) were fed a purified agar cake diet (Table 1) containing either 10 % cottonseed-soybean oil (Wesson Oil, Hunt-Wesson Foods, Fullerton, Calif.) (6 monkeys) or 10% butterfat plus 0.2 % cholesterol (18 monkeys) for 10 months. The juvenile male and female cebus and cynomolgus monkeys used in Experiment 2 were raised in our primate nursery6 and at the time of the study were 33 years old and had been fed a purified agar cake diet containing 8% safflower oil or coconut oil (Table 1) for 30 months. All the animals were fed on the day of sacrifice and tissues for incubation were taken 2 h postprandially under Nembutal anesthesia. Blood was collected from the posterior vena cava for the determinations of serum cholesterol7 and triglycerides, and agarose gel electrophoresis was performed according to the method of Nobleg. In vitro conditions
Incubations
were carried out in 20 ml incubation
vials containing
2.5 ml
EFFECT OF DIET ON HEPATIC
TABLE
AND INTESTINAL
407
LIPOGENESIS
1
COMPOSITION
OF EXPERIMENTAL
Zngredientsa
Casein Lactalbumin Dextrin Sucrose Safflower oil Coconut oil Butterfat Cottonseed-soybean Vitamin mixC Salt mixd Choline chloride Inositole Cellulosef Cholesterolg
MONKEY
Experiment
DIETS (g/loo
I
g) FED DURING
EXPERIMENTS
Experiment
1 AND
2
-
diet I
diet 2
diet 3
diet 4
20.0 42.5
20.0 42.5 10.0 0.5 4.0 0.34 0.1 21.6 0.2
-
-
14.6 23.7 28.9 8.0
14.6 23.7 28.9 8.0 0.4 4.0 0.24 20.0 -
oilb 10.0 0.5 4.0 0.34 0.1 21.6
0.4 4.0 0.24 20.0
2
lngredients were mixed and a hot 2.5% Bacto-agar solution added. The diet was cooled and cut into small cubes for feeding. Wesson Oil, Hunt-Wesson Foods Inc., Fullerton, Calif. The vitamin mix contained (in mg); thiamine hydrochloride, 80; riboflavin, 160; pyridoxine hydrochloride, 80; calcium pantothenate, 500; niacinamide, 800; folic acid, 20; biotin, 4; cyanocobalamin, 3; menadione, 100; dl-a-tocopherol acid succinate, 1000; retinyl acetate beadlets, 250,000 I.U.; cholecalciferol beadlets, 25,000 I.U.; ascorbic acid, 12.25 g made to 100 g with dextrin. HEGSTED, D. M., et al., J. Biol. C/tern., 138 (1941) 459. Inositol added at 0.1 g/l00 g diet. Alphacel, General Biochemicals, Chagrin Falls, Ohio. Cholesterol, U.S.P., Nutritional Biochemicals Corp., Cleveland, Ohio.
Krebs-Ringer bicarbonate buffer (pH 7.4), 3.0 pmole sodium acetate (1.2 mm), 6 ,&i [I-r*C]acetate (New England Nuclear, Boston, Mass.; specific activity 55 Ci/ mole), 5.0 mg glucose, and 0.125 mg penicillin and streptomycin, The tightly capped vials were pregassed with 95 ‘A 02-5 ‘4 CO2 for 5 min prior to incubation and for 5 min at the start of the incubation carried out with shaking at 37 “C for 2 h. At sacrifice, sections of liver and gut were quickly excised and rinsed in ice cold Krebs-Ringer bicarbonate buffer. Liver slices (0.5 mm thick) were obtained with a Stadie-Riggs hand microtome. Full thickness slices of jejunum and terminal ileum were taken approximately 3 cm below the ligament of Treitz and 5 cm from the ileocecal junction, respectively. Lipogenesis along the length of the gut was also determined in 5 cebus and 5 cynomolgus monkeys by taking sequential sections approximately 10 cm apart for incubation. Samples of tissues weighing approximately 200 mg were blotted dry and transferred to the pregassed incubation vials. After 2 h of incubation, the tissues were removed, rinsed in Krebs-Ringer bicarbonate buffer and the reaction stopped by transfer to tubes containing 10 ml of a chloroform-methanol
408
J. E. COREY, K. C. HAYES
(2:l). It had been previously determined that negligible lipid extractable radioactivity was present in the medium at the end of the incubation period. After extraction of tissues by shaking, a procedure which has been shown to remove 99 % of the lipid extractable radioactivity, the extract was filtered and washed according to the method of Folch et al.10 and the fat-free dry weight of the tissues determined to the nearest ,ug. Thin-layer chromatography of the extract was performed using 0.25 mm thick Silica Gel H plates developed in a mixture of hexane-diethyl ether-acetic acid (70:30:1). Aliquots of the extract were also used for cholesterol analysis’. Autoradiographs of the thin-layer chromatograms were developed to verify the identity of the lipid and radioactivity bands *. Thin-layer chromatograms were then scraped and the silica gel transferred directly to 10 ml of POPOP scintillation fluid (New England Nuclear, Boston, Mass.) for determination of radioactivity incorporated into free fatty acids, cholesterol, cholesterol ester and squalene, triglycerides and phospholipids. Counting efficiency was determined by the channels ratio methodlr. The results are expressed as the mean i SD. of nmoles acetate incorporated per g fat-free dry weight. Only radioactivity incorporated into total lipid, cholesterol and triglyceride is reported, since free fatty acid radioactivity was negligible. Cholesterol ester-squalene radioactivity was highly variable and phospholipid radioactivity was nonspecific, actually representing all the label remaining at the origin of the thin-layer chromatogram. Analysis of variance was performed on the data and the effects of treatment were evaluated by Duncan’s multiple range testis. mixture
TABLE PLASMA MONKEYS
2 CHOLESTEROL
AND
IN EXPERIMENTS
TRIGLYCERIDE 1 AND
Species and dietary fat
CONCENTRATIONS
OF
CEBUS,
CYNOMOLGUS
AND
SQUIRREL
2 Plasma cholesterol
Plasma
(mgldl)
(meld11
triglyceride
SquirreI
Cottonseed-soybean oil Butterfat + cholesterol
164 •t 61& 362 zt 82c
21 + 118 30 i 26a
139 f 138 254 zt 6gb
24 + 108 91 + 21b
144 + 3g8 163 i 198
35 + 17a 100 f 55b
Cebus
Safflower oil Coconut oil CynomoIgus
Safflower oil Coconut oil
Mean * SD. B*b*CValues within a column without a common superscript differ significantly (P < 0.05).
* Thin-layer chromatograms were placed in contact with Eastman Kodak Blue Brand Medical X-ray film in a tightly sealed cassette for 4 days.
EFFECT OF DIET ON HEPATIC
AND INTESTINAL
LIPOGENESIS
409
Fig. 1. Cebus and cynomolgus monkeys fed safflower oil or coconut oil (Experiment 2) demonstrated a significant positive correlation (r = 0.74) between serum triglycerides and the ‘A lipid staining in the pre-/I region after agarose gel electrophoresis.
RESULTS
Plasma lipids
The terminal plasma cholesterol and triglyceride concentrations of monkeys in Experiment 1 and 2 are shown in Table 2. Plasma cholesterol levels were low and similar in all monkeys fed cottonseed-soybean oil (Experiment 1) or safflower oil (Experiment 2). The cholesterol-butterfat fed squirrel monkeys in Experiment 1 developed a hypercholesteremia as did the coconut oil fed cebus monkeys in Experiment 2. The cholesterol levels of the cynomolgus were not significantly increased by coconut oil. Plasma triglycerides were low in safflower oil fed cebus and cynomolgus monkeys and in squirrel monkeys regardless of diet. Coconut oil induced a significant triglyceridemia in both the cebus and cynomolgus monkeys which was significantly correlated (r = 0.74) with an increase in the % lipid staining in the pre-I3 region on agarose gel electrophoresis (Fig. 1). Species diflerences in lipogenesis
The data from Experiment 2 presented in Table 3 provide a basis for interspecies comparison (cebus and cynomolgus) of rates of total lipid, cholesterol, and triglyceride synthesis from acetate in the liver and gut. Safflower oil fed cebus monkeys incorporated more acetate into total hepatic lipid than did the safflower oil fed cynomolgus monkeys. When considering individual lipid fractions, this differential was most striking for hepatic cholesterogenesis, the New World cebus demonstrating significantly more cholesterogenic activity from acetate than the Old World cynomolgus. No significant species differences were found for hepatic triglyceride synthesis due to large standard deviations and the small number of animals, but the same trend was evident-the cebus exceeding the cynomolgus. Similar species differences in acetate incorporation into total lipid were demonstrated in the jejunum, but here the enhanced lipogenesis by the cebus was confined to triglyceride synthesis. Fig. 2 illustrates the relative rates of incorporation of acetate into cholesterol by liver and gut slices for these monkeys and also includes the data from the cottonseed-soybean oil fed
410
J. E. COREY, K. C. HAYES
TABLE 3 NMOLES[~-14C]~~~~~~~~~~~~~~~~~~~INTOHEPATICANDINTESTINALLIPIDSBYCEBUSANDCYNOMOLEXPERIMENT 2
GUSMONKEYSIN
Cynomolgus
Cebus
Liver Total lipide Cholesterol Triglyceride Tissue cholesterol, mg/g
safJl0 wer oil
coconut oil
safflower oil
coconut oil
(3)
(4)
(5)
(7)
5709 f 1274& 2932 + 1713& 1174 i- 398”
4847 * 926~9~ 1147 5 417b 2504 + 346b
3323 & 1580bgC 437 + 276b 893 & 617”
1760 & 1326c 173 & 97b 990 + 8558
22.1 i 4.5a
16.9 i- 2.1”
18.1 f 3.0a
18.8 i- 7.1a
1584 & 181 & 700 &
2472 k 47d 165 +I 63& 1493 f 32c
707 k 111 i 247 *
1159 * 108 i 597 f
23.8 & 1.7b
17.5 i 0.88
Jejunum
Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g Ileutn Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g
157c 52” 85b
18.5 + 1.8& 2082 f 754” 332 & 72asb 882 i 5858 15.2 & 2.2”
1371 + 209 f 422 i-
92&sb 74& 408
78” 16& 83”
22.2 & 2.7b
1601 & 745&vb 1396 i 343 + 163%rb 322 i 515 5 357& 399 + 16.7 + l.la
15.9 i 1.38
320” 368 151b
2698*b 96apb 3578
13.9 +c 2.4a
Numbers in parentheses represent number of animals. &,b,cTdValues within a row without a common superscript differ significantly (P < 0.05). e Data are expressed as nmoles [1-WZ]acetate incorporated/g fat-free dry weight i S.D.
squirrel monkeys (Experiment 1). It is apparent that cynomolgus monkeys had a larger contribution to the total cholesterol synthesis provided by the gut than either of the New World species. Although the most accurate representation of comparative rates of cholesterogenesis for the liver and gut would clearly necessitate consideration of organ size and weighting of the rates of cholesterogenesis, the relations shown in Fig. 2 would be exaggerated due to the fact that the cynomolgus has a proportionately longer intestine than the New World species. Similarly, when acetate incorporation into cholesterol was expressed as a percentage of total lipogenesis by each organ, only 11 T/, of the total hepatic label incorporated by the cynomolgus was in cholesterol, while the corresponding figure for the cebus monkeys was 47%. Conversely, in the intestine 16% of the acetate incorporated into lipid by the cynomolgus was in cholesterol, a significantly greater percentage than the 11 ‘A incorporated by the cebus monkeys (Table 3). Comparison of the intestinal lipogenic activity at 10 cm intervals by species (Fig. 3) indicated that the ileum was the most active segment for cholesterol synthesis in both the cebus and cynomolgus species, similar to the observation made by Diet-
EFFECT OF DIET ON HEPATIC AND INTESTINAL LIPOGENESIS
411
3000
Cebus
Squire1
Cynomolgus
Fig. 2. Comparison of the rates of acetate incorporation into cholesterol by liver, ileum and jejunum of those monkeys fed unsaturated fat revealed distinct species differences in the relative rates of cholesterogenesis by these organs.
schy and Wilson in squirrel monkeys 1s. Although similar rates of cholesterogenesis were observed in the ileal and jejunal sections of the cebus and cynomolgus, the cebus synthesized more cholesterol at all intermediate points and a greater amount of triglyceride throughout the gut (Fig. 3). EfSect of diet on lipogenesis
Imposed on the differences between species were differences within species due to dietary cholesterol (Experiment 1) and to the type of dietary fat (Experiment 2). In
Fig. 3. Mapping the small intestine for acetate incorporation into cholesterol and triglyceride at 10 cm intervals revealed species and dietary differences between cebus and cynomolgus monkeys (Experiment 2).
3. E. COREY, K. C. HAYES
412 TABLE 4 NMOLES
[b-‘4c]ACETATE
EXPERIMENT
INCORPORATED
INTO HEPATIC
AND INTESTINAL
LIPIDS
BY SQUIRREL
MONKEYS
IN
1
Cottonseed-soybean oil (6)
Butterfat + cholesterol (18)
3290 & 1516 & 978 h 16.8 +
3108 f 2171& 101 f 56b 2046 f 14218 76.1 f 55.1b
Liver
Total lipide Cholesterol Triglyceride Tissue cholesterol, mg/g
1876& 7948 924& 4.0a
Jejunum
Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g
571 & 194a 46 f 1P 297 * 14P 19.9 f 2.2=
573 f 496a 42 ?? 22& 261 & 253& 23.7 f 4.4&
1504 & 476 & 458 * 17.3 f
1126 f 231 & 426 i 16.4 zt
IIeum
Total lipid Cholesterol Triglyceride Tissue cholesterol, mg/g
3638 127b 154& 2.98
347b llO& 2308 3.4”
Numbers in parentheses represent number of animals. a,b Values within a row without a common superscript differ significantly (P < 0.05). e Data are expressed as nmoles [I-W]acetate incorporated/g fat-free dry weight + S.D. Experiment
I, cholesterol-butterfat
fed squirrel
monkeys
demonstrated
a 94% re-
duction in the incorporation of acetate into hepatic cholesterol when compared with cottonseed-soybean oil fed squirrel monkeys (Table 4). This inhibition was specific for cholesterol synthesis as total lipogenesis was unaffected due to a concomitant rise in triglyceride
synthesis.
An inhibition
of approximately
48 % of cholesterol
synthesis
from acetate was seen in the ileum of butterfat-cholesterol fed squirrel monkeys compared with unsaturated fat fed animals. In contrast, no aspect of Iipogenesis in the jejunum of the squirrel monkeys was affected by the feeding of cholesterol and butterfat. In Experiment 2, although comparison of coconut oil with safflower oil fed animals demonstrated no statistical difference in total hepatic Iipogenesis from acetate in either the cebus or cynomolgus monkeys, the cebus actually exhibited a significant increase in acetate incorporation into triglyceride and a parallel decrease in cholesterol synthesis. This coconut oil-induced enhancement of triglyceride synthesis from acetate was even more marked in jejunal slices from both the cebus and cynomolgus monkeys. No changes in cholesterol synthesis were observed. Lipid synthesis by ileal slices was independent of dietary fat in both species of monkeys in which this was examined. Tables 3 and 4 also include tissue concentrations of hepatic and intestinal cholesterol for the different species of monkeys. It is apparent that, in addition to inducing hypercholesterolemia, cholesterol feeding resulted in a significant increase
EFFECT
OF DIET
ON HEPATIC
AND INTESTINAL
LIPOGENESIS
413
in hepatic cholesterol but not intestinal cholesterol in the squirrel monkey. Coconut oil, on the other hand, induced a significant increase in the cholesterol content of the jejunum, but not the liver, in both the cebus and cynomolgus monkeys (Table 3). DISCUSSION
It has been previously demonstrated that the New World cebus monkey is particularly susceptible to elevation of serum cholesterol by dietary coconut oil and much less sensitive to dietary cholesterol, while the Old World cynomolgus responds to dietary cholesterol but is relatively insensitive to coconut oil4. In this same study squirrel monkeys responded to both coconut oil and cholesterol, attaining a serum cholesterol concentration similar to the cebus. The present investigation extended these observations on serum lipids to species differences in lipogenesis by gut and liver, tissues which are important to cholesterol homeostasis. Conclusions warranted by the data relate to the effect of dietary cholesterol on lipogenesis in the squirrel monkey, to the effect of coconut oil on lipogenesis in the cebus and cynomolgus monkeys, and to species variation in hepatic and intestinal lipogenesis. Although presumably all mammalian tissues can synthesize cholesteroli3si4, it has been demonstrated in both the rat and the squirrel monkey fed low cholesterol diets that per unit weight the liver and intestine are capable of the highest rates of in vitro cholesterogenesis and that control of cholesterol synthesis may differ in these two organ@. The present study provided evidence for this high capacity for in vitro cholesterol synthesis by liver and gut in squirrel, cebus and cynomolgus monkeys. The terminal ileum had greater synthetic activity than more proximal sections of the gut in the species studied here, a finding previously reported in the squirrel monkeyls and baboonis. In Experiment 1, feeding cholesterol and butterfat to squirrel monkeys almost totally inhibited in vitro hepatic cholesterol synthesis and partially inhibited ileal cholesterogenesis. Thus, increased endogenous cholesterol synthesis was not a contributing factor in the hypercholesterolemia induced by dietary cholesterol and butterfat. Other investigators have demonstrated negative feedback inhibition of hepatic enzymes controlling cholesterol synthesis is. Control of intestinal cholesterol synthesis has been less well defined and may involve dynamics between bile acid flux and luminal cholesterolsJ7. Coconut oil was reported previously to be hypercholesterolemic and results of the present study indicate that the hypercholesterolemia in cebus monkeys, like that induced by dietary cholesterol, was not attributable to enhanced endogenous cholesterol synthesis. In fact, coconut oil feeding decreased hepatic synthesis of cholesterol in agreement with other in vitro studies 1s. In contrast to its inhibitory effect on hepatic cholesterogenesis, coconut oil significantly enhanced incorporation of acetate into triglycerides in both liver and jejunum slices from the cebus monkey and in jejunum slices from the cynomolgus. This reciprocal relation between triglyceride and cholesterol synthesis from acetate parallels reciprocal alterations in hepatic enzymes controlling fatty acid and cholesterol synthesis in rats fed different levels of dietary
414
J. E. COREY, K. C. HAYES
fatis. The greater potential for fatty acid synthesis by liver slices from animals fed saturated fat is consistent with in vitro data from mice and rats18~20-22in which dietary unsaturated fats exerted a greater suppression on fatty acid synthesis than diets containing tripalmitin, triolein, or medium chain triglycerides when compared to maximal rates exhibited by animals on a fat-free diet. In the perfused rat liver, too, triglyceride output has been shown to increase with fatty acid chain length greater than Cl2 and to decrease with the degree of unsaturation 2s. The increased triglyceride synthesis by liver and gut slices was associated with an increased concentration of serum triglycerides localized in the pre-/?-lipoproteins by electrophoresis. A triglyceridemia associated with dietary saturated fat has been reported in man24y25and in squirrel and cebus monkeys fed coconut oil without dietary cholestero14. In addition to the effect of dietary cholesterol and fat on acetate incorporation into neutral lipids, distinct species differences appeared in both the relative rates of hepatic and intestinal lipogenesis in vitro and in the total amount of acetate incorporated into the various lipid fractions. In cynomolgus, cebus and squirrel monkeys fed cholesterol-free diets, the potential for cholesterol synthesis by the small intestine relative to that by the liver was much greater in the cynomolgus than in the squirrel or cebus monkeys. The cebus, however, was capable of greater hepatic cholesterol synthesis in absolute terms than either the cynomolgus or the squirrel monkey, the difference being most apparent with the cynomolgus. Similarly, the cebus incorporated more acetate into hepatic and jejunal triglyceride than did the cynomolgus or squirrel monkeys. However, these interspecies comparisons must be tempered by the fact that the experimental designs were not strictly comparable. Although extrapolation of in vitro data on acetate incorporation to in vivo synthesis is complicated by the potential effects of altered uptake of acetate by cells and by dilution of acetate by endogenous pools, Wilson15 has demonstrated a direct relation between endogenous cholesterol production measured in viva by isotopic balance and [2-r4C]acetate incorporation into cholesterol by liver slices under conditions shown to be optimal for his assay system. The observed species differences in hepatic and intestinal lipogenesis may thus be relevant to their differing physiological responses to dietary fat and cholesterol. The observed species differences in susceptibility to diet-induced hypercholesterolemia must reflect differences in one or more of the processes maintaining cholesterol homeostasis. With respect to the hypercholesterolemia induced by dietary cholesterol, the present results suggest an association between hypercholesterolemia and the relative rates of hepatic and intestinal cholesterogenesis. Species that develop hypercholesterolemia with dietary cholesterol -the cynomolgus monkey, hamsteP, and gerbil (unpublished data) - have a proportionately high capacity for intestinal cholesterol synthesis compared to liver than do less responsive species such as the New World monkeys and the rats. Absorption of cholesterol must also be considered in this context due to the feedback regulation of hepatic cholesterol synthesis by exogenous cholesterolrs. In this respect, man, like the cholesterol-responsive species mentioned above, has an intestinal synthetic rate which equals that of the liver in the proxi-
EFFECT OF DIET ON HEPATIC
AND INTESTINAL
LIPOGENESIS
415
ma1 portion and exceeds that of the liver by four times distally27, yet man is relatively insensitive to dietary cholesterol, circulating cholesterol levels rising only 5 mg/lOO mg dietary cholesterol”*. Consideration of cholesterol absorption rates, however, reveals that unlike the Old World monkeyzg or the hamsters0 which absorb cholesterol readily, man has a limited capacity to do ~031, perhaps conferring a relative resistance to dietary cholesterol. The other dietary ingredient resulting in hypercholesterolemia was coconut oil. This fat elevated both plasma cholesterol and triglyceride levels in cebus monkeys but only triglyceride levels in cynomolgus, and enhanced incorporation of acetate into triglyceride in vitro in both species while depressing cholesterogenesis. This latter observation suggests that endogenous cholesterol synthesis was also not a factor in coconut oil-induced hypercholesterolemia. The consistent elevation of plasma triglyceride levels and of in vitro triglyceride synthesis, however, suggests that increased triglyceride synthesis by lipoprotein-forming tissues may be causally related to the enhanced levels of circulating cholesterol. Connor et al.32 have associated the hypocholesterolemic capacity of unsaturated fat with an increased excretion of cholesterol as bile acids. Since excretion is ultimately a function of lipoprotein turnover, the altered patterns of in vitro lipogenesis observed here in safflower and coconut oil fed cebus and cynomolgus monkeys may reflect alterations in synthesis and subsequent metabolism and turnover of a specific triglyceride-rich lipoprotein. Data from studies using perfused rat liver33 and infused gut34 indicate that the long chain saturated fatty acid, palmitate, is incorporated into a very low density lipoprotein containing more cholesterol per unit of triglyceride than the lipoprotein particle formed from the polyunsaturated fatty acid, linoleate. The metabolism of these particles may also vary34. Analysis of the composition and metabolism of the lipoproteins secreted by monkeys under defined dietary conditions is required in order to support any hypothesis associating lipoprotein metabolism with species variability to diet and/or atherogenesis. Since these three primate species have distinct variations in lipid metabolism and atherosclerosis, they provide good models for these comparisons.
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