Life Sciences Vol . 18, pp . 1447-1452, 1976 . Printed in the II .S .A .

Pergamon Presa

INCREASED EXCRETION OF BILE ACIDS BY GENETICALLY HYPERLIPOPROTEINEMIC ZUCHBR, RAT M . T . R . Subbuh and P. W . Conaelly Cardiovascular Research Unit Mayo Clinic and Mayo Foundation Rochester, Minnesota 55901 (Received in final form May 17, 1976)

summary Fecal excretion oß neutral sterols and bile acids was measured in age-+matched hyperlipoproteinemic Zucker obese rata and their lean litter mates . The bile acid excretion (mg/day + SEM) in Zucker rats was sigaißicantly higher (pc0 .01) when compared to lean controls (Zucker obese rats 41 .88 + 2 .86 ; lean controls 29 .85 + 1 .50) . Neutral sterol excretion in both the groups oß rate was similar . Total ßecal steroid excretion (ag/day _+ 8E3I) in Zucker rate was signißicantly higher (pc0 .01) than in lean controls (Zucker obese rate 52 .33 + 3 .50 ; lean controls 39 .23 + 2 .18) . The Zucker rat three mimics the increased biléacid excretion noted previously in human Type IV hyperlipoproteineaia and could serve as an ideal animal model ßor studying the interrelationship between bile acid excretion and very low density lipoprotein metabolism . Human Type IV hyperlipoproteinemia [excess pre-B very low density lipoproteins (VLDL) and triglycerides in plasma] is associated with as increased excretion oß bile acids (1,2) . The rate oß bile acid excretion showed a positive correlation with tire level oß plasma triglycerides (3,4) . Obese human subjects have also been shown to excrete higher amounts oß bile acids when compared to normal weight controle (h,8) . However, no such dißßerences have been demonstrated in anq animal model . The availability oß animal models which exhibit such dißßerenoes would enable ßurther investigations into the mechanism oß this dißßereace in bile acid excretion . The availability oß genetically obese Zucker rats (7,8) provided an ideal opportunity to examine the bile acid excretion in these rats . The Zucker obese rate show typical Type IV hyperlipoproteinemia, with plasma triglyceride levels nearly thrice that Bound in lean controls (9,10) . This communication compares the Betel excretion rates oß neutral sterols and bile acids between age-matched Zucker obese rate and their lean litter mates . Methode Five 1-month-old male Zucker Batty rate and seven lean litter mates were purchased ßrom Harriet Bird Memorial Laboratory, Stow 1447

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Massachusetts . The rate were kept on a Purina rat chow diet (Ralston Purina Co ., St . Louiq, Missouri) throughout the course oß the eaperiments . The average dietary intake oß all the rats was During the determined by monitoring their ßood intake ßor 5 days . dietary restriction in the obese rats, halß oß their regular dietary intake was given . For the cholesterol balance study the rate were kept in individual metabolic cages which allowed the quantitative collection oß ßeces (separated ßrom urine) . The rate were maintained on Purina chow diet . The total intake oß ßood during the seven-day The study period was determined using pre-weighed containers . cholesterol concentration in the diet was 0 .19 mg/gm as determined The ßecal samples in the laboratory by gas-liquid chromatography . ßrom a 1-week period were pooled, weighed, and homogenized in a known volume oß water . Aliquots oß the Pecal homogenates were taken ßor the analysis oß neutral sterols and bile acids . B~itosterol which is normaily present in the chow diet (0 .28 mg/gm), as determined in the 1&boratory, was used as a aonabsorbable marker to correct ßor variations in ßecal Blow and sterol collection (11), Its concentration in the diet was measured prior to the study by gas-liquid chromatography . Total plant sterol content was not used because recent studies (12,13) have shown that campesterol is preßerentially absorbed by pigeon intestinal mucosa . The ßecal neutral sterols were eatracted and purißied as described previously (11,12,14) based on the procedure oß Miettinen, Ahrens, and Grundy (15) . Following thin-layer chromatography, the bands corresponding to cholesterol, coprostanol, and coprostanone were eluted and quantitated as trimethylsilyl ether derivatives by gas-liquid chromatography using Sac-cholestane as an internal standard (10,12) . A Packard model 409 gas chromatograph containing 3 .8~, W-98 was used ßor the sterol analysis . ßlash heater, 300° C ; detector, 270° C ; Column conditions were : column, 320 ° C; and carrier gas, helium, 50 ml/min . Losses oß sterols during the chemical procedures were corrected on the basis oß recovery oß C14-cholesterol . The ßecal bile acids were analyzed by a procedure described previously ßrom this laboratory (16) . The eatracted bile acids were chromatographed as methyl ester trißluoroacetates on 3~ QF-1 columns . A F 8r M model 402 gas chromatograph was used ßor this Column conditions were : column, 220° C ; ßlash heater, analysis . 240° C ; detector, 250° C ; and carrier gas, helium, 50 ml/min . Cholesterol and bile acid eacretioa ßor the balance study period was calculated by correcting ßor the ßecal Blow using the recovery oß ß-3ltosterol as ßollows : Total cholesterol excretion =

Total cholesterol and metabolites in feces ß-Sitosterol recovery

B-Sitosterol recovery was calculated by dividing the ß-Sitosterol eacreted in the ßeces ßor the study period by its total dietary intake . Plasma cholesterol and triglycérides were determined by the method oß Levine and Zak (17) and Blleßeoa and Caraway (18),

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respectively . The animals were Basted overnight prior to the plasma lipid determination . Plasma lipids were measured at two age periods in each animal . Results Table I shows the body weights and plasma cholesterol and triglyceride levels in the two groups oß rate . The obese rats weighed eignißicantly more when compared to their lean litter mates at both 1 and 3 months oß age . There was no dißßerence in the plasma cholesterol level between the two groups . However, the triglyceride levels were nearly 3 times higher in the obese -rats (pc0 .001) . Paper electrophoresis oß the plasma lipoproteins ßor Zucker and lean controls conßirmed the presence oß sates VLDL in the Zucker rate as reported by others (9) . The ßecal excretion of neutral sterols and bile acids in the two groups oß animals was TABL$ I Body Heights and Plasma Lipid Levels in Obese and Lean Rats

Rata

Age (months)

Obese (5)

Body wt (gm)

3

Lean (5) 3 * pc0 .001 **pc0 .01 pc0 .001 ttpc0 .05

ßor ßor for for

dißßereace dißßerence dißßerence dißßereace

Mean f 3SM P sema 3 Cholesterol

s r g ycer

261 .8* + 7 .7

73 .2 +0 .9

458 .7** + 17.5

85 .3 +1 .5

181 .8* +13 .4

73 .0 +1 .6

36 .61 +5 .6

398 .2** +16 .8

80 .1 +4 .4

85 .5tt +29 .2

between between between between

es

98 .81 +7 .8 423 .3tt +122 .8

groups . groups . groups . groups .

examined at both 1 month and 3 months oß age (Table II) . At 1 month oß age there was no dißßerence in the ßecal eacretion oß neutral sterols or bile acids between the two groups oß animals . At 3 months oß age (ßollowiag attaining maturity) the ßecal excretion oß bile acids increased nearly 3-4-Bold in both groups oß animals . i~rthermore, the bile acid excretion in obese rats was signißicantly higher (pt0 .01) than in the lean controls . The neutral sterol eacretion did not dißßer between the groups . The total ßecal steroid eacretion in the obese rate was signißicantly higher (p~0 .01) than in lean controls . At 1 month oß age the obese rate were subjected to dietary restriction (half oß their normal intake) to see what eßßect it would have on the body weight, blood lipids, and ßecal sterol excretion . As can be seen is Table III, there was no decrease in body weight ßollowing dietary restriction . The plasma cholesterol

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TABLE II Fecal Excretion oß Neutral Sterols and Bile Acide in Obese and Leaa Rats

Rate

Age (months)

Obese (5)

Lean (5)

Mean + SEM oleeterol Cholesterol excretion da intake Neutral Total ßecal mg/day sterols Bile acids steroids

1

5 .17 +0 .28

13 .09 +1 .73

11 .79 +2 .81

24 .88 +4 .01

3

5 .18 +0,28

10 .65 +0 .88

41 .88* +2 .86

52 .33** +3 .50

1

3,39 +0,08

17 .73 +1 .52

11 .05 +2 .09

28 .78 +2 .57

3

4,12 +0 .15

9 .37 +0 .88

29 .85* +1 .30

39 .23** +2,16

* pG0 .01 ßor dißßerence between the groups . ** pc0.01 ßor dißßerence between the groups .

TABLE III Eßßect oß Dietary Restriction on Fecal Excretion oß Neutral Sterols and Bile Acide in 1-Month-0ld Obese Rate Diet Determinations Food intake (gm) Body wt (gm)

Ad libitum (4)

Restricted (4)

30 .25 + 1 .87

18 .17 + 0 .38

281 .8

+ 7 .89

278 .0

+ 7 .40

Plasma cholesterol (ag/100 sl)

73 .2* + 0 .97

92 .7* + 7 .3

Plasma triglycerides (mg/100 ml)

98 .8

+ 7 .78

78 .50 + 20 .71

5 .17 + 0 .28

2 .78 + 0 .08

Neutral sterols

13 .09+ 1 .73

4 .79k*+ 0 .38

Bile acids

11 .79 + 2 .81

12 .18 + 2 .98

Total ßecal steroids

24 .88 + 4 .01

16 .97 + 3 .10

.Cholesterol intake (ng/day) Cholesterol excretion (ag/day)

* pc0,01 ßor dißßerence between groups . **pc0 .001 ßor dißßerence between groups, All ßigures are Mean + SEM.

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Bile Acid Szcretion

145 1

increased signißicantly (pG0 .01) and the plasma triglyceridee showed no statistically eig~tißicant decrease . The ßecal ezcretim oß neutral sterols dropped signißicaatly (pG0 .001), while the bile acid eacretion showed no signißicant changes ßollowing dietary restriction . Discussion This study has, ßor the ßiret time, identißied dißßerences in bile acid eacretion in as animal model that are eiailar to those noted between noraal and Type IV hyperlipoproteinemic buns . The Zucker Batty rat with its considerable similarities to Type IV hyperlipoproteinemia should serve as an eacelleat model ßor ~ea~ studies ezploring mechanisms responsible ßor the relationship between bile acid eacretion and triglyceride (VLDL) .metabolism . The dietary restriction studies have shown that it is rather d1ßß cult to decrease the plasma triglyceride levels by similar dietary restriction in these genetically obese rate, as ßiret noted by Barry and Bray (10) . Dietary restriction caused a strüing reduction in neutral sterols but aot.i n bile acids. A similar trend was noted previously in the spontaneously atheroecleroeis-susceptible llhite Carneau pigeon (Subbiah and Connelly, unpublished datsÛ . This indicates that the ßecal neutral sterols and bile acids are aßßected dißßerently, possibly due to their origin ßrom difßereat cholesterol pools in the body . In summary, ßurther studies oß the mechanisms involved is the role oß lipoproteins (especially VLDL) and triglyceridee in cholesterol catabolism to bile acids can be investigated using the Zucker Batty rat . AcknowledBment The authors are indebted to Dr . Bruce A . Nottke Bor hie continued support and helpßul suggestions . Thanks are due to Dr . Ralph D . Elleßeon Bor the determination oß plasma cholesterol and triglyceridee and to llr . Janes R . Penner Bor his help in animal handling and care . This work was supported in part by Research Grant HL-14198 Bor a Specialized Center oß Research (800R) Brom the National Heart and Lung Institute . Reßerences 1. 2. 3. 4. 5. 8. 7. 8. 9. 10 . 11,

B. A . B701TâE, Circulation 40 13-20 (1989) . S, EINAR880N an , Eur . J . clin . Invest . 2 _ 225- 230 (1972) . H . S . 80DHI and B . HUDCHODSAR, Lancet 1 513-519 (1973) . B . A, B90T'PgE, C, L. STA~iES, N . E . TYLBR, and M, C . CAREY, In Atherosclerosis III, edited by G . SCHETTLER and A, 11EIZEL, (P . rager- egg, Berlin (1974) . T . A. IIISiT INEN, Circulation 44 842-850 (1971) . P . J . NESTEL, P . H . 8 N, and E . H . AHRENS, JR ., _J . clin . Invest . 52 2389-2897 (1973), .~. L ~4L~ aid T . F. ZUCI~t, J . Hared. 52 275-278 (1981) . L . 1~ . ZUCBBit and H, N. ANTONIADES,~door~ology _90 1320-1330 (1972) . G . SCHONFELD, C, FELSBI, and M . A . HOIIALD, J . Lipid Rse . 15 457-484 (1974) . A . S . BARRY and G . A . BRAY, Metabolism 18 833-839 (1989) . B . A . BD!TTHE and H. T . R. SUBB~.~LaT . clin . Med . 80 530538 (1972) . - -

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Hile Acid Excretion

M . T, R, biophys . M . T . R. M, T . R, Biochem .

Vol . 18, No . 12

SUBBIAH, B, A . KOTTKE, and I, A . CARIA, Biothun . Acta 249 643-646 (1971) . BUBBIAH,Am . J . clin , Nutr . 26 219-225 (1973), 3UBBIAH, B, A, I{OTTBE, ân~ P.~ . ZOLLMAN, Comp . Ph eiol . 41B 695-704 (1972) . 1~A~ISrT~ Ski . AHRII~TS, JR . , and S . M, GRUNDY, J . Lipid Res . 6 411-424 (1965) . M . T . R . SUBBIAH, J . ~Li id Res . 14 692-694 (1973), J . B . LÉVIN$ and B . ZAG,C`1~ c~im . Acta 10 381-384 (1964) . R . D . $LLBFSON and W, C . n~WAY, In Fundamentals oß Clinical Che~, edited by N . W . Teitz, p . . B . Sann erbe ompany, Philadelphia (in press) .

Increased excretion of bile acids by genetically hyperlipoproteinemic Zucker rat.

Life Sciences Vol . 18, pp . 1447-1452, 1976 . Printed in the II .S .A . Pergamon Presa INCREASED EXCRETION OF BILE ACIDS BY GENETICALLY HYPERLIPOPR...
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