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Cholesterol homeostasis in lean and obese male Zucker rats
Cholesterol
Homeostasis
in Lean and Obese Male Zucker
Rats
Donald J. McNamara Studies were performed in male Zucker rats to determine the metabolic effect of genetic obesity on whole body cholesterol homeostasis. Lean and obese mature Zucker rats were studied during intake of either a chow diet or a semisynthetic diet containing 10% corn oil; in addition growing animals were studied during constant body weight gain on a chow diet. Under all conditions the obese Zucker rats had significantly higher levels of total plasma cholesterol and triglyceride; however. measurements of the specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and of the rate of whole body cholesterol synthesis by sterol balance techniques demonstrated that the lean and obese animals did not differ in their endogenous rates of cholesterol synthesis. When sterol balance data were calculated per kilogram body weight, lean male Zucker rats synthesized a greater amount of cholesterol per day than obese animals. These studies demonstrate that the obese male Zucker rat, in many ways a model of human obesity, does not overproduce cholesterol and thus fails to exhibit one of major characteristics of the obese human.
0
BESE HUMANS exhibit increased cholesterol turnover rates as measured by isotope kinetics’,* and sterol balance methods.*,3 This excess cholesterol production may be as much as two times normal’ and can be reversed by weight reduction.3 Compartmental analysis of plasma-cholesterol specific activity decay curves suggests a corresponding enlargement of the slowly turning over body cholesterol pool in obesity’ which presumably exists in the adipose tissue.4 The obese Zucker rat (“fatty,” fa/fa), an animal model of juvenile onset obesity,‘.’ offers the opportunity to quantitate cholesterol homeostasis in obesity in an animal model and to determine the effects of obesity on cholesterol synthesis in specific tissues, primarily liver and adipose. In this study, measurements of sterol balance and hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in growing and mature, lean and obese male Zucker rats failed to demonstrate any significant differences in whole body cholesterol synthesis. The data indicate that the obese male Zucker rat exhibits a normal rate of cholesterol and bile acid synthesis and, in this regard, does not mimic the overproduction of cholesterol that is characteristic of human obesity. MATERIALS
AND
METHODS
Materials D,L-[3-14C] HMG-CoA (26.5 mCi/mmol), [ 1,2-‘H] cholesterol (40 Ci/mmol), and [24-‘4C]cholic acid (40 mCi/mmol) were purchased from New England Nuclear, Boston. D,L-HMG-CoA was
From the Lipid Metabolism Laboratory, the Rockefeller University, New York. Supported in part by US Public Health Service Grant HL 24190, by a grant from the Weight Watchers Foundation Inc, and by a Career Scientisi Awardfrom the Irma T. Hirsch1 Trust. Address reprint requests 10 Donald J. McNamara, PhD. Associate Professor, The Rockefeller University, 1230 York Ave. New York, NY 10021. o I985 by Grune & Stratton, Inc. 0026-0495/85/3402-0007$03.0/0
130
supplied by P-L Biochemicals Piscataway. NJ: NADP. glucosc6-phosphate (sodium salt) and glucose-6-phosphate dehydrogenase were obtained from Sigma, St Louis. 3tu-hydroxysteroid dehydrogenase was obtained from Worthington. Freehold, NJ. All other reagents and solvents were analytical grade: solvents were glassdistilled prior to use. Animals Mature male lean and obese Zucker rats (6 to 8 months of age) were the gift of Dr Joel Grinker. The Rockefeller University; immature male animals (6 to 8 weeks of age) were purchased from FAB Laboratory (New Jersey). All animals were housed in 3 light-cycled room (12 hr/cycle, dark 7PM to TAM). Animals were fed ad lib either a Purina chow diet (St Louis) or a semipurified diet for a minimum of 4 weeks prior to being transferred to individual metabolic cages (Hagelton Systems, Cincinnati, OH) that allowed separate quantitative recoveries of feces and urine as well as measurements of daily food intake. Dietary intakes were measured daily, body weights determined every other day, and feces collected daily and stored at I5 o until analyzed. After a nine-day fecal collection period. rats were anesthesized with diethyl ether. exsanguinated by cardiac puncture, and livers were removed for analysis of hepatic HMG-CoA reductase activity’ and cholesterol content; the entire intestinal contents of the small bowel were removed for measurement of the bile acid pool size.* The entire body minus liver and intestinal contents was saponified in 500 mL 30R KOH in methanol for determination of tissue cholesterol content9 All animals were killed at the midpoint of the light cycle. Diets Animals were fed ad lib either Rodent Laboratory Chow 5001 (Ralston Purina: St Louis) or a semisynthetic diet (diet Bio-Mix f832. Bio Serv Inc.; Frenchtown, NJ) composed of dextrose (58.2% by weight), casein (22.00/c), corn oil (lO.O%), cellulose (3.8%). and mineral and vitamin supplements (5.96%). The chow diet had a caloric density of 3.4 kcal/g, the semisynthetic diet had a caloric density of 4.1 kcal/g. The cholesterol contents of the chow and semisynthetic diets were 0.28 and 0.17 mg/g, respectively.
Sterol Balance Measurements Three-day pools of feces were dried under vacuum at room temperature, powdered, and extracted as described by Cohen et al.“’ Neutral steroids were isolated and measured by previously described procedures”: fecal bile acids were isolated and measured by the method of Grundy et alI2 as modified by Cohen et al.“’ Total carcass cholesterol contents were measured according to the procedure of Liu et al.9 All sterol determinations were corrected for procedural losses by use of radiolabeled internal recovery standards. Metabolism,
Vol
34,
No 2 (February),
1985
CHDLESTERDL METABOLISM IN THE ZUCKER RAT
131
Whole body cholesterol synthesis rates (me/d) were calculated from the equation: Total daily fecal neutral and acidic steroids excreted + net tissue change in cholesterol content - daily dietary intake of cholesterol.“-” Net tissue change in cholesterol content calculated daily as follows: daily change in body weight (g/d) x carcass cholesterol concentration (mg/g) = tissue accumulation (mg/d), assuming steady state carcass cholesterol concentration during the balance period.‘4.‘6
Bile acid pool sizes were measured using the 3a-hydroxysteroid dehydrogenase assay described by Turley and Dietschy.18 Plasma cholesterol and triglyceride concentrations were determined by enzymatic analysis”.*’ using Bio Dynamics bmc kits. Hepatic total cholesterol concentrations were measured by enzymatic assay as described by De Hoff et al.*’
study period while exhibiting significantly different total body weights (Table 1). The relative liver weights were identical in both sets of animals and, as would be expected, the obese rats ate significantly more chow than the lean litter mates (Table 1). The obese animals had significantly higher concentrations of plasma cholesterol, plasma triglyceride, and carcass cholesterol; bile acid pool sizes were equal (Table 1). The sterol balance data presented in Table 1 demonstrate that the obese Zucker rats’ daily rate of cholesterol synthesis per kilogram of body weight is significantly less than its lean litter mates. However, the total daily synthesis rates of cholesterol per animal are the same in lean and obese animals, a finding supported by the fact that the specific activity of hepatic HMG-CoA reductase was not significantly different in lean and obese rats [56.9 + 34.4 (n = 4) v 66.7 f 18.2 (n = 4) pmoles mevalonate formed per mg microsomal protein per minute].
Statistical Analysis
Sterol Metabolism
HMG-CoA
Reductase Assay
Hepatic HMG-CoA reductase activity was measured in isolated microsomes prepared and assayed as previously described.‘,” Microsomal protein concentrations were measured by the procedure of Lowry et al” using bovine serum albumin as standard. Other Assays
All data are presented as mean + standard deviation. Statistical analysis was carried out using a Hewlett-Packard 97 calculator programmed for the two-tailed student t-test of unpaired means” as supplied in the Hewlett-Packard Stat Pat I (Corvallis, Ore).
In an attempt to characterize sterol metabolism in mature male lean and obese Zucker rats further and to compare the response of in vivo cholesterol homeostasis to a hypercholesterolemic diet, animals were fed a semisynthetic diet of casein, corn oil, and cellulose. Previous studies in Sprague Dawley rats have shown that semisynthetic diets significantly increase plasma cholesterol levels while decreasing endogenous cholesterol and bile acid synthesis.15
RESULTS Sterol Metabolism
in Chow Diet Fed
Mature Male Zucker Rats
Mature lean and obese male Zucker rats fed a chow diet had comparable daily weight gains during the Table 1. Sterol Homeostasis
in Mature
Zucker Rats Fed a Chow Diet
Lean*
Finalbody weight
Obese*
P
735 f 37
434 + 4
(g)
in Corn Oil Fed
Mature Male Zucker Rats
to.001
Body weight gain (g/d) (Liver W/body wt) x 100
0.67 2.81
+ 0.45 + 0.12
0.27 2.75
f 1.04 * 0.34
Feed consumption (g/d) Plasma lipids (mg/dL)
23.5
f 0.5
33.1
+ 1.9
63 + 12
137 f 44
triglyceride
35 + 18
139 f 58
to.02
1.13 j, 0.09
1.41 + 0.08
co.02
134 + 18
127 + 9
Bile acid pool (mg/kg)
NSt Obese
LWll Sterol Balance
Fecal neutral steroids* Fecal acidic sterols Tissue accumulation Total Dietary cholesterol Cholesterol synthesis
(mg/d)
kw/ke/dl
(mgld)
15.3 + 2.4
35.7
* 5.9
16.7 + 3.5
9.0 + 2.3
21.0
+ 5.2
6.9 + 0.6
0.9 f 0.6 25.2
+ 2.5
5.8 + 0.1 19.5 t 2.4
2.2 * 1.5 58.7
+ 5.8
13.5 + 0.4 45.3
t 5.9
0.6 + 1.6 24.2
+ 4.2
8.4 + 0.511 15.8 * 4.1
*Data presented as mean + SD for four animals per group. tNS. not significantly different. tExclusive of sterols ingested during fur-licking13 and of dietary plant sterols and their secondary bacterial products. <
IIP <
NSt
cholesterol Carcass cholesterol (mg/g)
§P
NSS
0.02. 0.001.
hehId)
22.8
? 4.65
9.4 f; 0.85 0.4 f 1.4 32.8
-t 2.85
11.5 * 0.411 23.4
2 2.811
132
DONALD J McNAMARA
Table 2. Sterol Homeostasis
in Mature
body weights and daily fed consumptions were significantly different between the two sets of animals. The carcass cholesterol concentrations and the bile acid pool sizes were similar. As previously demonstrated, the semisynthetic diet decreased the size of the bile acid pool as compared to chow fed animals.” Zucker rats fed the 10% corn oil diet exhibited a lower rate of daily cholesterol synthesis as compared to chow fed animals. However, the data in Table 2 support the original observation that daily cholesterol synthesis is lower (per kilogram body weight) in the obese animals as compared to their lean litter mates, while total daily synthesis per animal was the same in the two groups. The specific activity of hepatic HMGCoA reductase did not differ between the lean (14.6 of5.9 pmol/min/mg, n = 3) and obese (10.7 and 10.1 pmol/min/mg) animals. As compared to the chow fed animals, reductase activity was decreased 79% and total sterol balance 52% by feeding the 10% corn oil diet. These findings are similar to those previously reported comparing chow and semipurified diet fed rats.15
Zucker Rats
Fed a 10% Corn Oil Diet Obeset
Lean l
Final body weight (g)
501 t 47
808; 797
Body weight gain (g/d)
0.69
+ 0.54
1.08; 1.21
(Liver W/body wt) x 100
2.89
+ 0.41
2.83: 2.81
Feed consumption (g/d)
19.1 * 1.2
22.1; 26.2
cholesterol
132 i 25
197; 227
triglyceride
79 * 37
103: 206
Carcass cholesterol fmg/g)
1.18 2 0.03
1.17; 1.21
Bile acid pool tmg/kg)
25.6
16.8; 19.4
Plasma lipids (mg/dL)
+ 5.2
Lssn
Obese
(mg/dl
(mg/ko/d)
Img/d) -~
Fecal neutral steroids*
9.2 f 0.8
17.2 + 0.4
7.6
9.6
Fecal acidic sterols
3.4 + 0.4
6.7 + 1.1
4.7
6.0
StemI Balance
Tissue accumulation Total
0.6 * 1.0
1.9
2.4
e 1.4
14.2
18.0
3.3 + 0.2
6.6 * 0.4
4.3
5.5
10.0 * 0.5
18.6 + 1.6
9.9
12.5
13.3 t 0.7
Dietary cholesterol Cholesterol synthesis
1.3 i 1.2
lmg/kg/dl
26.6
*Data presented as mean -r SD for three animals. TData presented for two animals. $Exclusive of sterols ingested during fur-lickingI and of dietary plant starols and their secondary bacterial products.
Sterol Metabolism
The data presented in Table 2 present similar findings in lean and obese Zucker rats. The plasma cholesterol and triglyceride concentrations were elevated as compared to chow fed animals even though body weights, daily body weight gains, and relative liver weights are similar to the chow fed animals. Comparison of the lean and obese rats show that only the final
Table 3. Sterol Homeostasis
The data presented so far have dealt with mature lean and obese Zucker rats which have a relatively slow growth rate and a low rate of daily cholesterol synthesis. Studies were carried out in growing animals to determine if any significant differences in daily cholesterol synthesis occurred between lean and obese animals as a function of growth.
in Growing Zucker Rats Fad a Chow Diet
Lean*
Final body weight (g)
in Chow Fed Growing Rats
P
Obese*
220 i 24
282 i_ 37
I
Body weight gain (g/d)
2.15 * 1.01
2.77
(Liver wt/body wt) x 100
3.70
3.76
+ 0.1 1
Feed consumption (g/d)
16.9 5 1.9
21.4
+ 3.1
t 0.29
0.05
W NSt
+ 1.48
--0.05
Plasma lipids (mg/dL) cholesterol
44 + 3
triglyceride
39 + 12
54 f 4
0.01
122 t 46
0.02
Carcass cholesterol (mg/g)
1.88 k 0.45
1.86 f 0.19
NSt
Hepatic cholesterol (ma/g)
1.88 2 0.45
1.85 t 0.19
NSt
Bile acid pool Lmg/kg)
236 + 44
200 t 18
N6t Obese
Sterol balance
lmg/d)
h/kg/d)
Imgld)
__..-_ Iwlkgld)
19.7 + 4.0
96.8
+ 15.9
+ 2.7
91.2
* 10.3
Fecal acidic sterols
5.3 + 0.9
26.2
+ 3.4
5.6 + 1.8
22.1
t 8.2
Tissue accumulation
2.8 * 1.3
13.7 + 5.6
3.9 t 1.4
14.9 t 3.6
Fecal neutral steroidst
Total Dietary cholesterol Cholesterol synthesis
27.8
t 5.9
137.6
5.0 + 0.4
24.8
22.7
f 5.4
111.9
i 29.2 + 2.0 t 20.9
23.6
33.2
+ 3.2
126.2
6.5 t 1.1
24.8
26.9
+ 2.9
*Data presented as mean k SD for four anrmals per group. TNS = not significantly different. SExclusive of sterols ingested during fur-lrcktng’3 and of dietary plant sterols and therr secondary bacterral products.
103.9
+ 12.2 t 2.3 5 12.9
CHOLESTEROL METABOLISM IN THE ZUCKER RAT
Growing animals exhibited a much greater daily body weight gain as compared to the mature animals (Table 3). The obese animals weighed more, ate more, and exhibited hypertriglyceridemia and a mild hypercholesterolemia as compared to their lean litter mates. Hepatic and carcass cholesterol concentrations and the size of the bile acid pools were similar in the lean and obese Zucker rats. Sterol balance data in the growing animals were almost identical whether based on body weight or total synthesis (Table 3). Interestingly, the growing animals, both lean and obese, synthesized over twice as much cholesterol per kilogram per day as did the mature animals, 23the greatest increases occurring in fecal neutral steroid output and net tissue accumulation of cholesterol. DISCUSSION
Obese patients often exhibit hyperlipidemia and an overproduction of cholesterol as measured by sterol balance methods2*3 or cholesterol turnover studies.’ Calculation of whole body cholesterol synthesis rates per kilogram of body weight demonstrates that most patients, whether of normal body weight or obese, produce approximately the same amount of cholesterol per kilogram per day.24 The unresolved question has remained whether the elevated production of cholesterol found in obesity results from the high caloric intake required to maintain the obese state, sterol synthesis in the expanded mass of adipose tissue, or both. Studies by Angel and Bray2s demonstrated similar rates of cholesterol synthesis in liver and adipose tissues of obese and control patients; in both groups hepatic synthesis was 50 times that of adipose tissue when expressed in terms of total organ activity. Similar findings have been reported by Evensen et alz6 in that the rate of incorporation of 14C-acetate into sterols in liver biopsies of morbidly obese and control subjects were not significantly different. In contrast, Angelin et a12’reported an increased specific and total activity of HMG-CoA reductase in hepatic tissues obtained from obese as compared to normally weighted controls. From the available data it is difficult to account for the incremental increase of 22 mg/d in cholesterol synthesis per kilogram excess body weight in human obesity’ solely on the basis of adipose tissue sterol synthesis. Schreibman and Dell4 reported data suggesting that the adipose tissue could account for no more than 1 mg cholesterol per kilogram fat per day. Compartmental analysis of plasma and adipose tissue cholesterol kinetics support the possibility that the excess synthesis of cholesterol in obesity occurs in the liver and intestine.4 However, the use of radiolabeled small molecular precursors of cholesterol for kinetic
133
analysis suggested that adipose tissue could significantly contribute to overall cholesterol synthesis.28 In an attempt to resolve some of these conflicting reports and to examine the usefulness of an animal model system for studying sterol homeostasis in obesity, sterol balance studies were performed in lean and obese Zucker rats. The genetically obese Zucker rat inherits obesity as an autosomal recessive trait’ and is characterized by hyperphagia, hyperinsulinemia, insulin resistence, and hyperlipidemia.6 In addition, the obese Zucker rat exhibits increased hepatic lipid synthesis29 and lipoprotein secretion3’ with decreased chylomicron triacylglycerol clearance.3’ Analysis of cholesterol homeostasis in lean and obese Zucker rats demonstrate that there are no significant differences in whole body cholesterol or bile acid synthesis, whether measured in mature animals fed a chow or semipurified diet or in growing rats fed chow. When sterol balance data were calculated on a body weight basis, daily cholesterol synthesis rates were significantly lower in mature obese as compared to lean animals, irrespective of diet. In growing Zucker rats, sterol balance values were identical for both cholesterol and bile acid synthesis rates per animal and per kilogram body weight. It is interesting to note that although whole body cholesterol synthesis per kg body weight is significantly different between mature and growing animals, the absolute rate of synthesis per animal in mature and growing lean male Zucker rats is almost identical (19.5 * 2.4 v 22.7 rt 5.4 mg/d). These results are similar to those reported by Stange and Dietschy3* which demonstrated that the rate of steroi synthesis from ‘H20 per animal is not significantly different over the age range of 30 to 100 days. In contrast, there appears to be a reduction in the rate of whole body cholesterol synthesis in male obese animals with age (growing rats, 26.9 + 2.9 mg/d, n = 4 v mature rats, 15.8 f 4.1 mg/d, n = 4; P -c0.005). Why this difference between growing and mature animals exists is not clear; however, the studies of Stange and Dietschy’* suggest that in part it may arise from the hypercholesterolemia so prevalent in the mature obese animal allowing the animal to acquire its cholesterol, not from local synthesis, but rather from circulating lipoproteins as it gets older. Previous studies have demonstrated that the rate of incorporation of ‘Hz0 into hepatic cholesterol is not significantly different in lean and obese Zucker rats33*34 and in this study it was found that the specific activity of hepatic HMG-CoA reductase in lean and obese animals was not significantly different. However, the total liver reductase is greater in obese animals due to the 1.5-fold increase in liver mass and an equivalent
134
DONALD J McNAMARA
value for recovered microsomal protein per gram liver (mean value 17.8 mg/g for lean and 16.7 mg/g for obese). Why this difference in hepatic sterol synthesis is not reflected in the sterol balance measurement of whole body synthesis may in part be explained by the fact that the liver contributes less than 50% of the total production of cholesterol in vivo in the rat.34 Other possible reasons for the observed contradiction between the in vivo and in vitro results in lean and obese Zucker rats may arise from decreased hepatic clearance of chylomicrons3’ and the increased lipoprotein secretion associated3’ with obesity, which would significantly alter the rate of transfer of newly synthesized hepatic and intestinal sterols to peripheral tissues in the obese animals3* A clear delineation of the relative contributions of the various tissues of the body to whole body sterol synthesis cannot be determined by sterol balance techniques but rather requires measurement of the rate of incorporation of ‘Hz0 into sterols in vivo in the individual tissues.35
Preliminary sterol balance studies in male SpragueDawley rats, obese due to ventromedial hypothalmic lesions,‘” demonstrate that the rates of whole body cholesterol synthesis are identical in lean and obese animals (lean, 34.2 mg/d and 83.4 mg/kg . d; obese, 28.9 mg/d and 45.5 mg/kg - d, n = 2). Thus, in the rat with either hypothalmic or genetic obesity, the rate of whole body cholesterol synthesis is not increased; indeed, when calculated per kilogram body weight, obese animals synthesize less cholesterol than their lean counterparts. The data demonstrate that the obese male Zucker rat, which is hyperlipidemic, hyperphagic, and hyperinsulinemic and exhibits increased hepatic lipid synthesis and lipoprotein secretion, does not overproduce cholesterol and in this regard fails to mimic one of the characteristics of human obesity. ACKNOWLEDGMENT The excellent technical assistance of MS Lyudmila Malikin is gratefully acknowledged. I wish to express my gratitude to Dr E. H. Ahrens. Jr. for his support and encouragement of these studies.
REFERENCES 1. Nestel PJ, Whyte HM, Goodman DeWS: Distribution and turnover of cholesterol in humans, J Clin Invest 48:982-991, 1969 2. Nestel PJ, Schreibman PH, Ahrens EH Jr.: Cholesterol metabolism in human obesity. J Clin Invest 52:2389-2397, 1973 3. Miettinen TA: Cholesterol production in obesity. Circulation 44:842-850, 197 1 4. Schreibman PH. Deli RB: Human adipocyte cholesterol. Concentration, localization, synthesis and turnover. J Clin Invest 55:986-993, 1975 5. Zucker TF, Zucker LM: Heredity obesity in the rat associated with high serum fat and cholesterol. Proc Sot Exp Biol Med 1l&165-171, 1961 6. Bray GA: The Zucker-fatty rat: A review. Fed Proc 36:148153,1977 7. Shapiro DJ, Nordstrom JL, Mitschelen JJ, et al: Microassay for 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts. Biochim Biophys Acta 370:369-377. 1974 8. Uchida K, Okuno 1, Takase H, et al: Distribution of bile acids in rats. Lipids 13:42-48, 1978 9. Liu GCK, Ahrens EH Jr., Schreibman PH. et al: Measurement of squalene in human tissues and plasma: Validation and application. J Lipid Res 17:38-45, 1976 IO. Cohen Bl, Raicht RF, Salen G, et al: An improved method for the isolation, quantitation, and identification of bile acids in rat feces. Anal Biochem 64:567-577, 1975 11. McNamara DJ, Proia A. Miettinen TA: Thin-layer and gas-liquid chromatographic identification of neutral steroids in human and rat feces. J Lipid Res 22:474-484, 1981 12. Grundy SM, Ahrens EH Jr., Miettinen TA: Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J Lipid Res 6:397-410, 1965 13. Miettinen TA, Proia A, McNamara DJ: The origins of fecal neutral steroids in rats. J Lipid Res 22:485-495, 198 1 14. Proia A, McNamara DJ, Edwards KDG, et al: Cholesterol homeostasis in the rat with a portacaval anastomosis. Proc Nat1 Acad Sci USA 76:4654-4657, 1979 15. McNamara DJ, Proia A, Edwards KDG: Cholesterol ho-
meostasis in rats fed a purified diet. Biochim Biophys Acta 7 1 I:252 260, 1982 16. Young NL, McNamara DJ, Saudek CD, et al: Hyperphagia alters cholesterol dynamics in diabetic rats. Diabetes 32:8l I-819, 1983 17. Lowry OH, Rosebrough NJ, Farr AL. et al: Protein measurement with the folin reagent. J Biol Chem I93:2655275, I95 1 18. Turley SD, Dietschy JM: Re-evaluation of the 3oc-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 19:924-928. 1978 19. Allain CC. Poon LC. Chan CSG, et al: Enzymatic determination of total serum cholesterol. Clin Chem 20:47&475. 1974 20. Bucolo G, David H: Quantitation determination of serum triglyceride by the use of enzymes. Clin Chem 19:4766482. 1973 21. DeHoff JL, Davidson LM, Kritchevsky D: An enzymatic assay for determining free and total cholesterol in tissue. Clin Chem 24:433-435, 1978 22. Glantz SA: Primer of Biostatistics. McGraw Hill, New York, 1981 23. Uchida K. Nomura Y. Kadowaki M. et al: Age-related changes in cholesterol and bile acid metabolism in rats. J Lipid Res 19:532-544. 1978 24. Leijd B: Cholesterol and bile acid metabolism in obesity. Clin Sci 59:203-206, 1980 25. Angel A, Bray GA: Synthesis of fatty acids and cholesterol by liver, adipose tissue and intestinal mucosa from obese and control patients. Em J Clin Invest 9:355-362, 1979 26. Evensen S, Ritland S. Nygaard K, et al: Synthesis of sterols and proteins in liver biopsies from obese patients subjected to gastric or jejunoileal bypass operations. Stand J Gastroent 16:657-666, 1981 27. Angelin 8, Backman L, Einersson K, et al: Hepatic cholesterol metabolism in obesity: Activity of microsomal 3-hydroxy3-methylglutaryl coenzyme A reductase. J Lipid Res 23:770-773. 1982 28. Kekki M, Miettinen TA. Wahlstrom B: Measurement of cholesterol synthesis in kinetically defined pools using fecal steroid
CHOLESTEROL METABOLISM IN THE ZUCKER RAT
analysis and double labeling technique in man. J Lipid Res l&99114, 1977 29. Godbole V, York DA: Lipoprotein in situ in the genetically obese Zucker fatty rat (fa/fa): Role of hyperphagia and hyperinsulinaemia. Diabetologia 14:191-197, 1978 30. Schonfeld G, Ptleger B: Overproduction of very low-density lipoproteins by livers of genetically obese rats. Am J Physiol 220:1178-1181,197l 3 1. Redgrave TG: Catabolism of chylomicron triacyglycerol and cholesterol ester in genetically obese rats. J Lipid Res 18:604-612, 1977 32. Stange EF, Dietschy JM: Age-related decreases in tissue sterol acquisition are mediated by changes in cholesterol synthesis
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and not low density lipoprotein uptake in the rat. J Lipid Res 25:703-713,1984 33. Triscari J, Bryce GF, Sullivan AC: Metabolic consequences of fasting in old lean and obese Zucker rats. Metabolism 29:377385,198O 34. Pector JC, Winard J, Dehaye JP: Effects of portacaval shunt on the genetically obese Zucker rat. Gastroenterol 81:932-937, 1981 35. Spady DK, Dietschy JM: Sterol synthesis in vivo in 18 tissues of the squirrel monkey, guinea pig, rabbit, hamster, and rat. J Lipid Res 24:303-315, 1983 36. Goldman JK, Bernardis LL, MacKenzie RG, et al: Effects of ventromedial hypothalmic lesions on adipose tissue of weaning male rats. Diabetologia 20:357-361, 1981
Homeostasis
in Lean and Obese Male Zucker
Rats
Donald J. McNamara Studies were performed in male Zucker rats to determine the metabolic effect of genetic obesity on whole body cholesterol homeostasis. Lean and obese mature Zucker rats were studied during intake of either a chow diet or a semisynthetic diet containing 10% corn oil; in addition growing animals were studied during constant body weight gain on a chow diet. Under all conditions the obese Zucker rats had significantly higher levels of total plasma cholesterol and triglyceride; however. measurements of the specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and of the rate of whole body cholesterol synthesis by sterol balance techniques demonstrated that the lean and obese animals did not differ in their endogenous rates of cholesterol synthesis. When sterol balance data were calculated per kilogram body weight, lean male Zucker rats synthesized a greater amount of cholesterol per day than obese animals. These studies demonstrate that the obese male Zucker rat, in many ways a model of human obesity, does not overproduce cholesterol and thus fails to exhibit one of major characteristics of the obese human.
0
BESE HUMANS exhibit increased cholesterol turnover rates as measured by isotope kinetics’,* and sterol balance methods.*,3 This excess cholesterol production may be as much as two times normal’ and can be reversed by weight reduction.3 Compartmental analysis of plasma-cholesterol specific activity decay curves suggests a corresponding enlargement of the slowly turning over body cholesterol pool in obesity’ which presumably exists in the adipose tissue.4 The obese Zucker rat (“fatty,” fa/fa), an animal model of juvenile onset obesity,‘.’ offers the opportunity to quantitate cholesterol homeostasis in obesity in an animal model and to determine the effects of obesity on cholesterol synthesis in specific tissues, primarily liver and adipose. In this study, measurements of sterol balance and hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in growing and mature, lean and obese male Zucker rats failed to demonstrate any significant differences in whole body cholesterol synthesis. The data indicate that the obese male Zucker rat exhibits a normal rate of cholesterol and bile acid synthesis and, in this regard, does not mimic the overproduction of cholesterol that is characteristic of human obesity. MATERIALS
AND
METHODS
Materials D,L-[3-14C] HMG-CoA (26.5 mCi/mmol), [ 1,2-‘H] cholesterol (40 Ci/mmol), and [24-‘4C]cholic acid (40 mCi/mmol) were purchased from New England Nuclear, Boston. D,L-HMG-CoA was
From the Lipid Metabolism Laboratory, the Rockefeller University, New York. Supported in part by US Public Health Service Grant HL 24190, by a grant from the Weight Watchers Foundation Inc, and by a Career Scientisi Awardfrom the Irma T. Hirsch1 Trust. Address reprint requests 10 Donald J. McNamara, PhD. Associate Professor, The Rockefeller University, 1230 York Ave. New York, NY 10021. o I985 by Grune & Stratton, Inc. 0026-0495/85/3402-0007$03.0/0
130
supplied by P-L Biochemicals Piscataway. NJ: NADP. glucosc6-phosphate (sodium salt) and glucose-6-phosphate dehydrogenase were obtained from Sigma, St Louis. 3tu-hydroxysteroid dehydrogenase was obtained from Worthington. Freehold, NJ. All other reagents and solvents were analytical grade: solvents were glassdistilled prior to use. Animals Mature male lean and obese Zucker rats (6 to 8 months of age) were the gift of Dr Joel Grinker. The Rockefeller University; immature male animals (6 to 8 weeks of age) were purchased from FAB Laboratory (New Jersey). All animals were housed in 3 light-cycled room (12 hr/cycle, dark 7PM to TAM). Animals were fed ad lib either a Purina chow diet (St Louis) or a semipurified diet for a minimum of 4 weeks prior to being transferred to individual metabolic cages (Hagelton Systems, Cincinnati, OH) that allowed separate quantitative recoveries of feces and urine as well as measurements of daily food intake. Dietary intakes were measured daily, body weights determined every other day, and feces collected daily and stored at I5 o until analyzed. After a nine-day fecal collection period. rats were anesthesized with diethyl ether. exsanguinated by cardiac puncture, and livers were removed for analysis of hepatic HMG-CoA reductase activity’ and cholesterol content; the entire intestinal contents of the small bowel were removed for measurement of the bile acid pool size.* The entire body minus liver and intestinal contents was saponified in 500 mL 30R KOH in methanol for determination of tissue cholesterol content9 All animals were killed at the midpoint of the light cycle. Diets Animals were fed ad lib either Rodent Laboratory Chow 5001 (Ralston Purina: St Louis) or a semisynthetic diet (diet Bio-Mix f832. Bio Serv Inc.; Frenchtown, NJ) composed of dextrose (58.2% by weight), casein (22.00/c), corn oil (lO.O%), cellulose (3.8%). and mineral and vitamin supplements (5.96%). The chow diet had a caloric density of 3.4 kcal/g, the semisynthetic diet had a caloric density of 4.1 kcal/g. The cholesterol contents of the chow and semisynthetic diets were 0.28 and 0.17 mg/g, respectively.
Sterol Balance Measurements Three-day pools of feces were dried under vacuum at room temperature, powdered, and extracted as described by Cohen et al.“’ Neutral steroids were isolated and measured by previously described procedures”: fecal bile acids were isolated and measured by the method of Grundy et alI2 as modified by Cohen et al.“’ Total carcass cholesterol contents were measured according to the procedure of Liu et al.9 All sterol determinations were corrected for procedural losses by use of radiolabeled internal recovery standards. Metabolism,
Vol
34,
No 2 (February),
1985
CHDLESTERDL METABOLISM IN THE ZUCKER RAT
131
Whole body cholesterol synthesis rates (me/d) were calculated from the equation: Total daily fecal neutral and acidic steroids excreted + net tissue change in cholesterol content - daily dietary intake of cholesterol.“-” Net tissue change in cholesterol content calculated daily as follows: daily change in body weight (g/d) x carcass cholesterol concentration (mg/g) = tissue accumulation (mg/d), assuming steady state carcass cholesterol concentration during the balance period.‘4.‘6
Bile acid pool sizes were measured using the 3a-hydroxysteroid dehydrogenase assay described by Turley and Dietschy.18 Plasma cholesterol and triglyceride concentrations were determined by enzymatic analysis”.*’ using Bio Dynamics bmc kits. Hepatic total cholesterol concentrations were measured by enzymatic assay as described by De Hoff et al.*’
study period while exhibiting significantly different total body weights (Table 1). The relative liver weights were identical in both sets of animals and, as would be expected, the obese rats ate significantly more chow than the lean litter mates (Table 1). The obese animals had significantly higher concentrations of plasma cholesterol, plasma triglyceride, and carcass cholesterol; bile acid pool sizes were equal (Table 1). The sterol balance data presented in Table 1 demonstrate that the obese Zucker rats’ daily rate of cholesterol synthesis per kilogram of body weight is significantly less than its lean litter mates. However, the total daily synthesis rates of cholesterol per animal are the same in lean and obese animals, a finding supported by the fact that the specific activity of hepatic HMG-CoA reductase was not significantly different in lean and obese rats [56.9 + 34.4 (n = 4) v 66.7 f 18.2 (n = 4) pmoles mevalonate formed per mg microsomal protein per minute].
Statistical Analysis
Sterol Metabolism
HMG-CoA
Reductase Assay
Hepatic HMG-CoA reductase activity was measured in isolated microsomes prepared and assayed as previously described.‘,” Microsomal protein concentrations were measured by the procedure of Lowry et al” using bovine serum albumin as standard. Other Assays
All data are presented as mean + standard deviation. Statistical analysis was carried out using a Hewlett-Packard 97 calculator programmed for the two-tailed student t-test of unpaired means” as supplied in the Hewlett-Packard Stat Pat I (Corvallis, Ore).
In an attempt to characterize sterol metabolism in mature male lean and obese Zucker rats further and to compare the response of in vivo cholesterol homeostasis to a hypercholesterolemic diet, animals were fed a semisynthetic diet of casein, corn oil, and cellulose. Previous studies in Sprague Dawley rats have shown that semisynthetic diets significantly increase plasma cholesterol levels while decreasing endogenous cholesterol and bile acid synthesis.15
RESULTS Sterol Metabolism
in Chow Diet Fed
Mature Male Zucker Rats
Mature lean and obese male Zucker rats fed a chow diet had comparable daily weight gains during the Table 1. Sterol Homeostasis
in Mature
Zucker Rats Fed a Chow Diet
Lean*
Finalbody weight
Obese*
P
735 f 37
434 + 4
(g)
in Corn Oil Fed
Mature Male Zucker Rats
to.001
Body weight gain (g/d) (Liver W/body wt) x 100
0.67 2.81
+ 0.45 + 0.12
0.27 2.75
f 1.04 * 0.34
Feed consumption (g/d) Plasma lipids (mg/dL)
23.5
f 0.5
33.1
+ 1.9
63 + 12
137 f 44
triglyceride
35 + 18
139 f 58
to.02
1.13 j, 0.09
1.41 + 0.08
co.02
134 + 18
127 + 9
Bile acid pool (mg/kg)
NSt Obese
LWll Sterol Balance
Fecal neutral steroids* Fecal acidic sterols Tissue accumulation Total Dietary cholesterol Cholesterol synthesis
(mg/d)
kw/ke/dl
(mgld)
15.3 + 2.4
35.7
* 5.9
16.7 + 3.5
9.0 + 2.3
21.0
+ 5.2
6.9 + 0.6
0.9 f 0.6 25.2
+ 2.5
5.8 + 0.1 19.5 t 2.4
2.2 * 1.5 58.7
+ 5.8
13.5 + 0.4 45.3
t 5.9
0.6 + 1.6 24.2
+ 4.2
8.4 + 0.511 15.8 * 4.1
*Data presented as mean + SD for four animals per group. tNS. not significantly different. tExclusive of sterols ingested during fur-licking13 and of dietary plant sterols and their secondary bacterial products. <
IIP <
NSt
cholesterol Carcass cholesterol (mg/g)
§P
NSS
0.02. 0.001.
hehId)
22.8
? 4.65
9.4 f; 0.85 0.4 f 1.4 32.8
-t 2.85
11.5 * 0.411 23.4
2 2.811
132
DONALD J McNAMARA
Table 2. Sterol Homeostasis
in Mature
body weights and daily fed consumptions were significantly different between the two sets of animals. The carcass cholesterol concentrations and the bile acid pool sizes were similar. As previously demonstrated, the semisynthetic diet decreased the size of the bile acid pool as compared to chow fed animals.” Zucker rats fed the 10% corn oil diet exhibited a lower rate of daily cholesterol synthesis as compared to chow fed animals. However, the data in Table 2 support the original observation that daily cholesterol synthesis is lower (per kilogram body weight) in the obese animals as compared to their lean litter mates, while total daily synthesis per animal was the same in the two groups. The specific activity of hepatic HMGCoA reductase did not differ between the lean (14.6 of5.9 pmol/min/mg, n = 3) and obese (10.7 and 10.1 pmol/min/mg) animals. As compared to the chow fed animals, reductase activity was decreased 79% and total sterol balance 52% by feeding the 10% corn oil diet. These findings are similar to those previously reported comparing chow and semipurified diet fed rats.15
Zucker Rats
Fed a 10% Corn Oil Diet Obeset
Lean l
Final body weight (g)
501 t 47
808; 797
Body weight gain (g/d)
0.69
+ 0.54
1.08; 1.21
(Liver W/body wt) x 100
2.89
+ 0.41
2.83: 2.81
Feed consumption (g/d)
19.1 * 1.2
22.1; 26.2
cholesterol
132 i 25
197; 227
triglyceride
79 * 37
103: 206
Carcass cholesterol fmg/g)
1.18 2 0.03
1.17; 1.21
Bile acid pool tmg/kg)
25.6
16.8; 19.4
Plasma lipids (mg/dL)
+ 5.2
Lssn
Obese
(mg/dl
(mg/ko/d)
Img/d) -~
Fecal neutral steroids*
9.2 f 0.8
17.2 + 0.4
7.6
9.6
Fecal acidic sterols
3.4 + 0.4
6.7 + 1.1
4.7
6.0
StemI Balance
Tissue accumulation Total
0.6 * 1.0
1.9
2.4
e 1.4
14.2
18.0
3.3 + 0.2
6.6 * 0.4
4.3
5.5
10.0 * 0.5
18.6 + 1.6
9.9
12.5
13.3 t 0.7
Dietary cholesterol Cholesterol synthesis
1.3 i 1.2
lmg/kg/dl
26.6
*Data presented as mean -r SD for three animals. TData presented for two animals. $Exclusive of sterols ingested during fur-lickingI and of dietary plant starols and their secondary bacterial products.
Sterol Metabolism
The data presented in Table 2 present similar findings in lean and obese Zucker rats. The plasma cholesterol and triglyceride concentrations were elevated as compared to chow fed animals even though body weights, daily body weight gains, and relative liver weights are similar to the chow fed animals. Comparison of the lean and obese rats show that only the final
Table 3. Sterol Homeostasis
The data presented so far have dealt with mature lean and obese Zucker rats which have a relatively slow growth rate and a low rate of daily cholesterol synthesis. Studies were carried out in growing animals to determine if any significant differences in daily cholesterol synthesis occurred between lean and obese animals as a function of growth.
in Growing Zucker Rats Fad a Chow Diet
Lean*
Final body weight (g)
in Chow Fed Growing Rats
P
Obese*
220 i 24
282 i_ 37
I
Body weight gain (g/d)
2.15 * 1.01
2.77
(Liver wt/body wt) x 100
3.70
3.76
+ 0.1 1
Feed consumption (g/d)
16.9 5 1.9
21.4
+ 3.1
t 0.29
0.05
W NSt
+ 1.48
--0.05
Plasma lipids (mg/dL) cholesterol
44 + 3
triglyceride
39 + 12
54 f 4
0.01
122 t 46
0.02
Carcass cholesterol (mg/g)
1.88 k 0.45
1.86 f 0.19
NSt
Hepatic cholesterol (ma/g)
1.88 2 0.45
1.85 t 0.19
NSt
Bile acid pool Lmg/kg)
236 + 44
200 t 18
N6t Obese
Sterol balance
lmg/d)
h/kg/d)
Imgld)
__..-_ Iwlkgld)
19.7 + 4.0
96.8
+ 15.9
+ 2.7
91.2
* 10.3
Fecal acidic sterols
5.3 + 0.9
26.2
+ 3.4
5.6 + 1.8
22.1
t 8.2
Tissue accumulation
2.8 * 1.3
13.7 + 5.6
3.9 t 1.4
14.9 t 3.6
Fecal neutral steroidst
Total Dietary cholesterol Cholesterol synthesis
27.8
t 5.9
137.6
5.0 + 0.4
24.8
22.7
f 5.4
111.9
i 29.2 + 2.0 t 20.9
23.6
33.2
+ 3.2
126.2
6.5 t 1.1
24.8
26.9
+ 2.9
*Data presented as mean k SD for four anrmals per group. TNS = not significantly different. SExclusive of sterols ingested during fur-lrcktng’3 and of dietary plant sterols and therr secondary bacterral products.
103.9
+ 12.2 t 2.3 5 12.9
CHOLESTEROL METABOLISM IN THE ZUCKER RAT
Growing animals exhibited a much greater daily body weight gain as compared to the mature animals (Table 3). The obese animals weighed more, ate more, and exhibited hypertriglyceridemia and a mild hypercholesterolemia as compared to their lean litter mates. Hepatic and carcass cholesterol concentrations and the size of the bile acid pools were similar in the lean and obese Zucker rats. Sterol balance data in the growing animals were almost identical whether based on body weight or total synthesis (Table 3). Interestingly, the growing animals, both lean and obese, synthesized over twice as much cholesterol per kilogram per day as did the mature animals, 23the greatest increases occurring in fecal neutral steroid output and net tissue accumulation of cholesterol. DISCUSSION
Obese patients often exhibit hyperlipidemia and an overproduction of cholesterol as measured by sterol balance methods2*3 or cholesterol turnover studies.’ Calculation of whole body cholesterol synthesis rates per kilogram of body weight demonstrates that most patients, whether of normal body weight or obese, produce approximately the same amount of cholesterol per kilogram per day.24 The unresolved question has remained whether the elevated production of cholesterol found in obesity results from the high caloric intake required to maintain the obese state, sterol synthesis in the expanded mass of adipose tissue, or both. Studies by Angel and Bray2s demonstrated similar rates of cholesterol synthesis in liver and adipose tissues of obese and control patients; in both groups hepatic synthesis was 50 times that of adipose tissue when expressed in terms of total organ activity. Similar findings have been reported by Evensen et alz6 in that the rate of incorporation of 14C-acetate into sterols in liver biopsies of morbidly obese and control subjects were not significantly different. In contrast, Angelin et a12’reported an increased specific and total activity of HMG-CoA reductase in hepatic tissues obtained from obese as compared to normally weighted controls. From the available data it is difficult to account for the incremental increase of 22 mg/d in cholesterol synthesis per kilogram excess body weight in human obesity’ solely on the basis of adipose tissue sterol synthesis. Schreibman and Dell4 reported data suggesting that the adipose tissue could account for no more than 1 mg cholesterol per kilogram fat per day. Compartmental analysis of plasma and adipose tissue cholesterol kinetics support the possibility that the excess synthesis of cholesterol in obesity occurs in the liver and intestine.4 However, the use of radiolabeled small molecular precursors of cholesterol for kinetic
133
analysis suggested that adipose tissue could significantly contribute to overall cholesterol synthesis.28 In an attempt to resolve some of these conflicting reports and to examine the usefulness of an animal model system for studying sterol homeostasis in obesity, sterol balance studies were performed in lean and obese Zucker rats. The genetically obese Zucker rat inherits obesity as an autosomal recessive trait’ and is characterized by hyperphagia, hyperinsulinemia, insulin resistence, and hyperlipidemia.6 In addition, the obese Zucker rat exhibits increased hepatic lipid synthesis29 and lipoprotein secretion3’ with decreased chylomicron triacylglycerol clearance.3’ Analysis of cholesterol homeostasis in lean and obese Zucker rats demonstrate that there are no significant differences in whole body cholesterol or bile acid synthesis, whether measured in mature animals fed a chow or semipurified diet or in growing rats fed chow. When sterol balance data were calculated on a body weight basis, daily cholesterol synthesis rates were significantly lower in mature obese as compared to lean animals, irrespective of diet. In growing Zucker rats, sterol balance values were identical for both cholesterol and bile acid synthesis rates per animal and per kilogram body weight. It is interesting to note that although whole body cholesterol synthesis per kg body weight is significantly different between mature and growing animals, the absolute rate of synthesis per animal in mature and growing lean male Zucker rats is almost identical (19.5 * 2.4 v 22.7 rt 5.4 mg/d). These results are similar to those reported by Stange and Dietschy3* which demonstrated that the rate of steroi synthesis from ‘H20 per animal is not significantly different over the age range of 30 to 100 days. In contrast, there appears to be a reduction in the rate of whole body cholesterol synthesis in male obese animals with age (growing rats, 26.9 + 2.9 mg/d, n = 4 v mature rats, 15.8 f 4.1 mg/d, n = 4; P -c0.005). Why this difference between growing and mature animals exists is not clear; however, the studies of Stange and Dietschy’* suggest that in part it may arise from the hypercholesterolemia so prevalent in the mature obese animal allowing the animal to acquire its cholesterol, not from local synthesis, but rather from circulating lipoproteins as it gets older. Previous studies have demonstrated that the rate of incorporation of ‘Hz0 into hepatic cholesterol is not significantly different in lean and obese Zucker rats33*34 and in this study it was found that the specific activity of hepatic HMG-CoA reductase in lean and obese animals was not significantly different. However, the total liver reductase is greater in obese animals due to the 1.5-fold increase in liver mass and an equivalent
134
DONALD J McNAMARA
value for recovered microsomal protein per gram liver (mean value 17.8 mg/g for lean and 16.7 mg/g for obese). Why this difference in hepatic sterol synthesis is not reflected in the sterol balance measurement of whole body synthesis may in part be explained by the fact that the liver contributes less than 50% of the total production of cholesterol in vivo in the rat.34 Other possible reasons for the observed contradiction between the in vivo and in vitro results in lean and obese Zucker rats may arise from decreased hepatic clearance of chylomicrons3’ and the increased lipoprotein secretion associated3’ with obesity, which would significantly alter the rate of transfer of newly synthesized hepatic and intestinal sterols to peripheral tissues in the obese animals3* A clear delineation of the relative contributions of the various tissues of the body to whole body sterol synthesis cannot be determined by sterol balance techniques but rather requires measurement of the rate of incorporation of ‘Hz0 into sterols in vivo in the individual tissues.35
Preliminary sterol balance studies in male SpragueDawley rats, obese due to ventromedial hypothalmic lesions,‘” demonstrate that the rates of whole body cholesterol synthesis are identical in lean and obese animals (lean, 34.2 mg/d and 83.4 mg/kg . d; obese, 28.9 mg/d and 45.5 mg/kg - d, n = 2). Thus, in the rat with either hypothalmic or genetic obesity, the rate of whole body cholesterol synthesis is not increased; indeed, when calculated per kilogram body weight, obese animals synthesize less cholesterol than their lean counterparts. The data demonstrate that the obese male Zucker rat, which is hyperlipidemic, hyperphagic, and hyperinsulinemic and exhibits increased hepatic lipid synthesis and lipoprotein secretion, does not overproduce cholesterol and in this regard fails to mimic one of the characteristics of human obesity. ACKNOWLEDGMENT The excellent technical assistance of MS Lyudmila Malikin is gratefully acknowledged. I wish to express my gratitude to Dr E. H. Ahrens. Jr. for his support and encouragement of these studies.
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CHOLESTEROL METABOLISM IN THE ZUCKER RAT
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