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The effect of exercise and dietary cholesterol on cholesterol synthesis in the hamster Ridgen, Julie Elizabeth 1988

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THE EFFECT OF EXERCISE AND DIETARY CHOLESTEROL ON CHOLESTEROL SYNTHESIS IN THE HAMSTER by JULIE ELIZABETHi-RIDGEN B . S c , The Un ivers i ty of Western Ontar io , 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Div is ion of Human Nutr i t ion School of Family and Nutr i t iona l Sciences We accept th i s thes i s as conforming to the required standard THE UNIVERSITY pF" BrfrTTSH COLUMBIA December,,1988 J u l i e E l izabeth Ridgen, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date I f tCTrU) \.9K\ DE-6 (2/88) ABSTRACT Physica l i n a c t i v i t y and e levated piasma c h o l e s t e r o l are w e l l character ized r i s k factors for the development of coronary heart d isease (CHD). Consequently, manipulat ion of exerc ise in tens i ty and d ie tary c h o l e s t e r o l may favourab ly a l t e r l i p i d metabolism to reduce t h i s r i s k . The present study examined both independent and i n t e r a c t i v e e f f e c t s of exerc ise and d ietary c h o l e s t e r o l on in v i v o hepatic and i n t e s t i n a l c h o l e s t e r o l synthesis as w e l l as plasma t o t a l and high density l i p o p r o t e i n (HDL) c h o l e s t e r o l l e v e l s in hamsters. Male Syrian hamsters were randomized into one of i) low d ietary c h o l e s t e r o l (0.03% w/w) sedentary (LC-S), i i ) low c h o l e s t e r o l exercise (LC-E), i i i ) high c h o l e s t e r o l (0.12% w/w) sedentary (HC-S) or iv) high c h o l e s t e r o l exerc ise (HC-E) groups. Exerc ised hamsters were t ra ined to run at increas ing speeds on a motorized t r e a d m i l l fo r 90 minutes d a i l y over a two week period. Animals were subsequently run for 1 week at 70% of V0 2 max for 90 minutes each day. C h o l e s t e r o l synthesis was determined by measuring the rate of incorporat ion of into d i g i t o n i n p r e c i p i t a b l e s t e r o l s in l i v e r and small i n t e s t i n e over 2 hours f o l l o w i n g IP i n j e c t i o n of - ^ O . Plasma t o t a l c h o l e s t e r o l was s i g n i f i c a n t l y increased by d ietary c h o l e s t e r o l in HC versus LC groups independent of an exerc i se lowering e f fect in HC-E animals. HDL c h o l e s t e r o l was a l s o e l e v a t e d in response to d ietary c h o l e s t e r o l in HC groups, however LC-E hamsters had lower plasma HDL c h o l e s t e r o l than any other group through an i n t e r a c t i o n between exerc ise and d ie t . Incorporation of into l i v e r c h o l e s t e r o l was increased in HC-S versus LC animals, whereas exerc ise lowered hepatic s t e r o l synthesis in HC-E by an exerc ise and d i e t i n t e r a c t i o n . Although exerc ise did not a f fec t i n t e s t i n a l c h o l e s t e r o l i i 1 synthes i s , d i e t a r y c h o l e s t e r o l s i g n i f i c a n t l y decreased i n t e s t i n a l c h o l e s t e r o l synthesis in HC when compared with LC groups. Thus both plasma t o t a l c h o l e s t e r o l and smal l i n t e s t i n e responded c h a r a c t e r i s t i c a l l y to changes in d ie tary c h o l e s t e r o l l e v e l s , in opposit ion to l i v e r c h o l e s t e r o l synthes is , which showed a compensatory increase to the attenuation of i n t e s t i n a l s t e r o l synthesis . The exerc ise induced decrease evident in plasma c h o l e s t e r o l l e v e l s and hepatic c h o l e s t e r o l synthesis was not, however, observed in the smal l i n t e s t i n e . Therefore the independent response of l i v e r and smal l i n t e s t i n e to exerc ise and d ietary c h o l e s t e r o l l e v e l in t h i s study ind icate important d i f fe rences in the manner through which these organs regu late whole body c h o l e s t e r o l balance. i i i TABLE OF CONTENTS Page Abstract i i Tab le of Contents iv L i s t of Tables v i i L i s t of Figures v i i i Acknowledgements x SECTION I INTRODUCTION 1 II BACKGROUND AND LITERATURE REVIEW i) E f fec ts of Exerc ise on L ip ids in Animals 3 i i ) Use of Hamster as an Animal Model 5 i i i ) E f fec t of Exerc ise on L i p i d Metabolism in Hamsters 6 iv) Energy Metabolism During Exerc ise 8 v) Substrate U t i l i z a t i o n During Post Exerc ise Recovery 11 v i ) E f fec t of Dietary Cho les tero l on Plasma -Cho lestero l in Humans 13 v i i ) E f fec t of Dietary Cho les tero l on Plasma Cho les tero l in Animals 14 v i i i ) Regulation of L i p i d Synthesis 14 ix) High Density L ipoprote in Metabolism 17 III RATIONALE 21 IV MATERIALS AND METHODS i) Methodological Considerations a) Determination of Maximal Oxygen Consumption in Hamsters 22 iv b) Measurement of Cho les tero l Synthesis 24 i i ) Experimental Design 25 i i i ) Animals - Diet and Exerc ise Procedures a) Diet 27 b) Exercise 27 c) Entry/Exit Points of Study 28 iv) Measurement of Cho lestero l Synthesis and PIasma Cho iestero l 29 v) Laboratory Ana lys i s a) HDL-Cholesterol Determination 29 b) Plasma S p e c i f i c A c t i v i t y 30 c) Cho lestero l Determination 30 d) Tissue Preparation 31 e) I s o l a t i o n of Free Cho les tero l 31 v i) C a l c u l a t i o n s a) Cho lestero l Determination 32 b) Fract iona l Synthetic Rate 33 c) Body Composition 34 v i i i ) S t a t i s t i c a l Analyses 35 V RESULTS i) Food Intake, Body Weights and Percentage Body Fat 36 i i ) Organ Weights and Cho les tero l Content 41 i i i ) Plasma Cho les te ro l L e v e l s 44 iv) Cho les te ro l Synthet ic Rate 46 v VI DISCUSSION i) Body Weights and Food Intake 52 i i ) Body Composition, Organ Weights and Organ Cho lestero l Content 54 i i i ) Tota l Plasma C h o l e s t e r o l 56 iv) Plasma High Density L ipoprote in Cholestero l L e v e l s 58 v) F ract iona l Synthet ic Rate of Cho lestero l 61 v i ) Hepatic and I n t e s t i n a l Cho les tero l Synthesis 63 v i i ) Poss ib le Mechanisms a) Intest ina l Absorption and Cho les tero l Homeostasis 64 b) Endogenous C h o l e s t e r o l Synthesis 66 c) B i l e Acid Synthesis and Cho lestero l Homeostasis 74 d) Hormonal Factors C o n t r o l l i n g Cholestero l Synthesis 76 VII GENERAL SUMMARY AND CONCLUSIONS 79 VIII REFERENCES 81 IX APPENDIX A 93 X APPENDIX B ' " 94 XI APPENDIX C 95 XII APPENDIX D 96 XIII APPENDIX E 97 vi LIST OF TABLES Table 1. Weights and Cholestero l Content of L iver and Intest ine in Hamsters Table 2. Plasma Tota l and HDL Cholesterol of Hamsters Table 3. Inf luence of Acetate plus (-)-Hydroxyc i t rate on Cholestero l and Fatty Acid Synthesis in Perfused Rat L iver v i i LIST OF FIGURES Figure 1. Major Steps in the Synthesis of Cholestero l and the Biochemical Steps Where Metabol ic Control Takes PI ace Figure 2. Equipment Used to Determine Oxygen Consumption of Hamsters During Exerc ise Figure 3. General Experimental Design of Study Figure 4. D a i l y Feeding and Exerc ise Schedule Figure 5a. Food Intake of Hamsters Fed Low Cho les te ro l Diets F igure 5b. Food Intake of Hamsters Fed High Cho les te ro l Diets Figure 6a. Body Weights of Hamsters Fed Low Cho les te ro l Diets Figure 6b. Body Weights of Hamsters Fed High Cho les te ro l Diets F igure 7. Percent Body Fat of Hamsters F igure 8. Incorporation of ^ 0 into Cho les tero l in L i v e r and Intest ine of Hamsters F igure 9. Cumulative Incorporat ion of - ^ 0 into Cho lestero l of Tota l L i v e r and Intest ine in Hamsters F igure 10. Newly Synthesized C h o l e s t e r o l Expressed as a Fract ion of Tota l Cho les te ro l Content in L i v e r and Intest ine of Hamsters vi i i Figure 11. Inf luence of (-)-Hydroxycitrate on the Rate of C h o l e s t e r o l and Fatty Acid Synthesis in Perfused Rat L i v e r ix Acknowledgements To a l l those i n d i v i d u a l s who made completion of t h i s thes is p o s s i b l e , I extend a warm and s incere thank-you. S p e c i f i c a l l y ; Dr. P e t e r J o n e s , who as my primary thes is adv isor provided expert guidance and support throughout my graduate s tud ies , and who has g iven new meaning to the term "organizat ional e d i t i n g " (!), Dr. P e t e r Hahn, who as a committee member provided not only encouragement during t h i s pro ject , but a l s o many s c i n t i l l a t i n g conversat ions that have heightened my perception of s e l f and sc ience , Drs. Mel Lee and B i l l Mi lsom, fo r serv ing on my thes i s committee and prov id ing useful ins ight into procedural problems, Dr. Ray Pederson , for g i v i n g of his t ime, lab and suggestions regarding i n s u l i n radioimmunoassays, thes is composition and baseba l l p a r t i e s , Becca Roe, for her companionship throughout my studies and for her expert l e g - h o l d i n g ass is tance (not mine), Debbie R e i d , who was my p i l l a r of wisdom during the odd emotional c r i s i s , and My P a r e n t s , for t h e i r undying l o v e and support of my asp i ra t ions in l i f e . And f i n a l l y , I would l i k e to remember my hamsters, The Nads, who who ran s e l f l e s s l y and without choice, so that I could complete t h i s thes is and make a worthy contr ibut ion to sc ience. This even inc ludes the non-ath letes , the b i t e r s , the defecators and neurot ic escapees, and of course, grandpa. x INTRODUCTION Hypercholestero lemia i s a major r i s k marker associated with the development of coronary heart disease (CHD), a disease respons ib le f o r up to 43% of a l l deaths in Canada (1). With such a large segment of the populat ion a f fected by th i s ins id ious d isease, i t is essent ia l that Canadians be informed of the r i s k s r e l a t e d to CHD which are known to be assoc iated with cer ta in l i f e s t y l e p rac t i ses . Ep idemio log ica l and experimental evidence suggest that exercise a c t i v i t y and dietary c h o l e s t e r o l intake are factors which a f fect c h o l e s t e r o l metabolism in the human (2-6) . Phys ica l i n a c t i v i t y is g e n e r a l l y considered to be a secondary r i s k f a c t o r in the development of CHD, with more a c t i v e i n d i v i d u a l s tending to experience fewer and less severe c l i n i c a l manifestat ions of the disease (7,8). Metabo l i c and p h y s i o l o g i c a l e f fec ts of long term exercise have r e c e i v e d cons iderab le attent ion in recent years due to the b e l i e f that such a c t i v i t y i s b e n e f i c i a l in lowering e levated plasma c h o l e s t e r o l , a known r i s k marker for CHD. In contrast , i t has been hypothesized that exerc i se promotes an increase in plasma high density l ipoprote ins (HDL) which may enhance the removal of c h o l e s t e r o l from per iphera l t i s sues and decrease the formation of a t h e r o s c l e r o t i c l e s i o n s . The way in which exercise protects against CHD is c o n t r o v e r s i a l , however, i t i s widely speculated that a l t e r a t i o n s in l i p i d metabolism are p a r t l y respons ib le . Dietary c h o l e s t e r o l l e v e l is an add i t iona l factor regu la t ing c h o l e s t e r o l metabolism and may therefore be important in the development of CHD. Dietary c h o l e s t e r o l has been shown to a f fec t both plasma 1 c h o l e s t e r o l l e v e l s and endogenous hepatic and i n t e s t i n a l c h o l e s t e r o l s y n t h e s i s (9,10). Thus i t was the aim of the present study to determine whether def ined l e v e l s of exerc ise and d ietary c h o l e s t e r o l independently, and/or through an i n t e r a c t i o n , i n f l u e n c e ; i) plasma t o t a l c h o l e s t e r o l l e v e l s i i ) plasma high density l i p o p r o t e i n c h o l e s t e r o l l e v e l s i i i ) the rate of c h o l e s t e r o l synthesis in l i v e r and smal l i n t e s t i n e 2 BACKGROUND AND LITERATURE REVIEW i) E f f e c t s of Exerc ise on L ip ids in Animals The e f fec ts of exerc ise durat ion, frequency and i n t e n s i t y on plasma l i p i d concentrations are d i f f i c u l t to study with human subjects and g e n e r a l l y i n v o l v e time consuming and expensive experiments (11). As w e l l , many d i r e c t and i n d i r e c t confounding factors can a l t e r plasma c h o l e s t e r o l and l i p o p r o t e i n l e v e l s in humans. In contrast , animal experimentation a l lows greater external contro l of v a r i a b l e s d i f f i c u l t to manipulate in humans. For example; exact amounts and composition of food ingested, a c t i v i t y l e v e l s , age, and l i f e - s p a n patterns of behaviora l and p h y s i o l o g i c a l change can be monitored. In a d d i t i o n , whole organ and t i s s u e samples are e a s i l y obtained for both q u a l i t a t i v e and q u a n t i t a t i v e analyses . Laboratory animals can therefore serve as more useful i n v e s t i g a t i v e models in studies where s t r ingent v a r i a b l e control and use of i n v a s i v e preparatory techniques make human experimentation undes i rab le . Papodoulos et a j^ (12) and Pels et a l . (11) found that rats exerc ised for 4 and 12 weeks, r e s p e c t i v e l y , had s i g n i f i c a n t l y lower plasma t o t a l c h o l e s t e r o l l e v e l s than t h e i r sedentary counterparts. Unfor tunate ly , these studies were designed to detect only changes in plasma c h o l e s t e r o l and measurement of the factors respons ib le fo r these changes, such as c h o l e s t e r o l synthet ic ra te , were not performed. A t h i r d study by Takashi et jfL_(13) used the conversion of [14]C-3 hydroxy 3 m e t h y l g l u t a r y l Coenzyme A (HMG CoA) to [14]C-mevalonate by HMG CoA reductase to measure hepatic c h o l e s t e r o l synthes is . Plasma c h o l e s t e r o l l e v e l s were a l s o determined. Compared to the sedentary control group, 3 exerc i s ing rats were found to have a s i g n i f i c a n t l y higher HMG CoA reductase a c t i v i t y and hepatic s t e r o l synthet ic rate. Plasma c h o l e s t e r o l was s i g n i f i c a n t l y lower in the a c t i v e group. Severa l problems were ev ident with t h i s study that may make i n t e r p r e t a t i o n of r e s u l t s d i f f i c u l t . F i r s t l y , l i m i t a t i o n s existed with the use of [14]C-mevalonate as a too l in the measurement of cho les te ro l synthes is . For example, the assumption was made that no d i l u t i o n of the l a b e l l e d mevalonate by endogenous mevalonate would occur. More important ly , i t is p o s s i b l e that the formation of mevalonic acid is l im i ted by HMG CoA reductase a v a i l a b i l i t y , in which case p a r t i a l i n h i b i t i o n of HMG CoA condensing enzyme might occur with excess mevalonate present (14). Thus, use of [14]C-mevalonate in th is study may not have been a s e n s i t i v e i n d i c a t o r of changes in enzyme a c t i v i t y occurr ing at an e a r l i e r step in the pathway. Although i n h i b i t i o n of mevalonate to squalene is an add i t iona l p o s s i b l e contro l po int , i t is secondary to the primary i n h i b i t i o n of mevalonate synthesis per se (15). Secondly, t h i s study presented data on c h o l e s t e r o l synthesis in the exerc ise group from very ear ly t imepoints 20-30 minutes after [14]C- . mevalonate i n j e c t i o n . In f a c t , from examination of the data, i t appeared as though incorporat ion decreased after 30 minutes in exercised rats versus c o n t r o l s . Since bar graphs alone were presented for HMG CoA reductase a c t i v i t y , i t is d i f f i c u l t to a s c e r t a i n whether or not s i m i l a r trends in the two groups were occurr ing. L a s t l y , i t has been observed that in ra ts fed ad l i b i t u m , hepat ic hepatic c h o l e s t e r o l synthetic a c t i v i t y e x h i b i t s a d iurna l rhythm with a maximum of a c t i v i t y at midnight and a nadir at noon (16). No reference was made in t h i s study (13) as to the exact time of s a c r i f i c e , and whether or 4 not the d iurna l c y c l e was considered. Therefore, cons istent data may not have been c o l l e c t e d . As w e l l , animals of both sedentary and exerc ise groups were maintained on an ad l ib i tum feeding regimen. The fac t that the exerc ised group was reported to have higher energy and t o t a l c h o l e s t e r o l intakes may have rendered the r e s u l t s un in terpre tab le . Very few w e l l c o n t r o l l e d experiments e x i s t descr ib ing the combined e f f e c t s of d ie tary c h o l e s t e r o l and exercise on l i p i d metabolism. C la rke et a l . (17) conducted a study designed to measure the i n t e r a c t i v e e f f e c t s of d i e t and exerc ise on blood l e v e l s of low density l i p o p r o t e i n , t r i g l y c e r i d e , high density l i p o p r o t e i n , and t o t a l plasma c h o l e s t e r o l . Although no independent exerc ise ef fects were observed on any of the b lood l i p i d measurements, an i n t e r a c t i o n was seen in high c h o l e s t e r o l - f e d exerc ised rats . This group had lower plasma c h o l e s t e r o l only i f they were exerc i sed , when compared with sedentary rats fed a low c h o l e s t e r o l d i e t . A s i m i l a r trend was observed in plasma HDL c h o l e s t e r o l l e v e l s . Better cont ro l on food intakes and energy expenditure between exerc ise and sedentary groups would have improved experimental design. Moreover, ne i ther the rate of c h o l e s t e r o l synthesis nor the a c t i v i t y of HMG CoA reductase were measured. Thus, although r e s u l t s of t h i s study are suggest ive of d i e t - and exercise- induced a l t e r a t i o n s in l i p i d metabolism, fu r ther study of the mechanism under ly ing these r e s u l t s would be in format ive . i i ) Use of the Hamster as an Animal Model There is increas ing evidence that the hamster, in comparison to other spec ies , more c l o s e l y resembles the human with regard to c h o l e s t e r o l 5 metabolism and therefore is a pre ferab le i n v e s t i g a t i v e model. Spady and Dietschy (18) found that in r a t s , whole body synthesis of c h o l e s t e r o l occurs at much higher rates (12 mg/day per 100 g body weight) than e i t h e r in the hamster (2.5 mg/day per 100 g body weight) or in man (1 mg/day per 100 g body weight). As w e l l , rats have a higher proport ion of c h o l e s t e r o l synthesized in the l i v e r versus the i n t e s t i n e compared to hamsters. I t is thought that the ra t , with higher endogenous s t e r o l synthet ic r a t e s , more r e a d i l y adapts to changes in whole body c h o l e s t e r o l f l u x than e i t h e r the hamster or man (19) by more e f f i c i e n t l y convert ing excess c h o l e s t e r o l into b i l e ac id (20). The hamster has proved to be a good model in which to study s t e r o l metabolism, since hamster plasma LDL concentrations respond to a l t e r a t i o n s in dietary l i p i d intake in a manner v i r t u a l l y i d e n t i c a l to humans (21). Exerc ise e f fects in hamsters are a l s o s i m i l a r to those observed in humans, where phys ica l a c t i v i t y reduces serum t r i g l y c e r i d e l e v e l s and body fa t content, and has v a r i a b l e e f fects on serum c h o l e s t e r o l l e v e l s . Furthermore, d iscont inuat ion of chronic exercise in hamsters causes compensatory gains of body weight and body fa t in a manner s i m i l a r to that of humans (22). Thus, the hamster would appear to be a s u i t a b l e model fo r both the dietary c h o l e s t e r o l and exerc ise components of the present s tudy . iv) E f f e c t s of Exerc ise on L i p i d Metabolism in Hamsters Tsa i et a l . (22) examined the e f f e c t s of vo luntary d isc running at about 22,000 r e v o l u t i o n s per day in female hamsters over a 35 day per iod. No s i g n i f i c a n t d i f fe rence was found in plasma c h o l e s t e r o l between sedentary (229 mg/dl) and exercised (220 mg/dl) animals. Fatty ac id 6 synthes is , as measured with the use of t r i t i a t e d water, was found to be s i g n i f i c a n t l y higher in l i v e r of the exercised group. Measurement of c h o l e s t e r o l synthet ic rates v i a incorporat ion of t r i t i a t e d water was not performed. Certa in control and design problems of the study make i n t e r p r e t a t i o n of these r e s u l t s d i f f i c u l t . F a i l u r e to pa i r - feed exercised animals to the l e v e l of food consumed by the sedentary group r e s u l t e d in higher food intakes in exercised animals. This may in turn have r e s u l t e d in higher c h o l e s t e r o l intake and t o t a l plasma c h o l e s t e r o l l e v e l s . Jeske et a l . (23) have suggested that substrate a v a i l a b i l i t y may be rate l i m i t i n g for c h o l e s t e r o l synthesis . Increased f a t t y acid synthesis may a lso have been a r e s u l t of greater food intake in the exercised hamsters in comparison to c o n t r o l s . Moreover, the a p p l i c a t i o n of the t r i t i a t e d water methodology may have had l i m i t a t i o n s in the study by Tsai et a l (22). Dietschy and Spady (24) have shown that a 20 minute e q u i l i b r a t i o n period i s the minimum required for mixing of plasma and i n t r a c e l l u l a r water a f ter an i n j e c t i o n of t r i t i a t e d water. Consequently, the assumption in th i s study that e q u i l i b r a t i o n had occurred 20 minutes a f ter an i n t r a p e r i t o n e a l i n j e c t i o n was quest ionable. Thus the s p e c i f i c a c t i v i t y of plasma may have been overestimated. Nevertheless r e l a t i v e changes in fa t ty acid synthesis should have been detected. A shorter time i n t e r v a l used for the e q u i l i b r a t i o n is known to have a q u a n t i t a t i v e e f fec t that should be measured and corrected for . In a separate study, Tsai et a l . (25) measured only t o t a l serum c h o l e s t e r o l and found i t to be decreased from 221 to 198 mg/dl in sedentary versus exerc ised hamsters. This change, however was not s t a t i s t i c a l l y s i g n i f i c a n t . The experimental design was s i m i l a r to the above, except that no rad io isotopes were used. Neither experiment included measurement of 7 exerc ise i n t e n s i t y which makes comparison to other studies d i f f i c u l t , i v ) Energy Metabolism During Exerc ise The manner by which c h o l e s t e r o l synthet ic rate i s a l tered by exerc ise i s unknown, however i t may be speculated that changes in the a v a i l a b i l i t y of precursor acety l subunits p lay a r o l e in t h i s process. The t r i c a r b o x y l i c ac id (TCA) c y c l e is a f i n a l common pathway for the ox idat ion of energy substrates (26). The importance of acetyl CoA in metabolism stems from the fac t that i t is the common end product of pyruvate decarboxy la t ion , fa t ty acid ox idat ion , and amino acid catabol ism (27) and i n i t i a t e s the primary step in the TCA pathway. In the res t ing s tate , muscle der ives approximately 90% of i t s energy from f a t t y acid ox idat ion, whereas glucose uptake by muscle represents only 10% of oxygen consumed (28). During exerc ise , however, substrate u t i l i z a t i o n for energy is a l t e r e d in order to meet increased c e l l u l a r demands for g lucose, which may or ig inate from sources inc lud ing l i v e r and muscle g lycogen, l a c t i c a c i d , pyruvate, aspartate , glutamate and a l a n i n e (26). During the f i r s t 5-10 minutes of exerc ise , muscle glycogen is the major fue l u t i l i z e d (28). As exerc ise continues, blood f low to muscles increases and blood borne substrates , provided by hepatic glycogen stores and gluconeogenic pathways become i n c r e a s i n g l y important sources of energy. Blood glucose uptake increases 25-30 f o l d above basal l e v e l s in a one hour exerc ise bout with the increment being a funct ion of i n t e n s i t y and durat ion of exerc ise. This glucose may contr ibute to 30-50% of o x i d a t i v e metabolism in the muscle (28). As exerc ise continues over 1-4 hours, the a v a i l a b i l i t y of muscle 8 glycogen p r o g r e s s i v e l y dec l ines and is accompanied by a r i s e of up to 70% in uptake of f ree f a t t y acids (28). The mechanism for the s h i f t in substrate u t i l i z a t i o n is b e l i e v e d to be l inked to the ADP/ATP r a t i o . This r a t i o determines the rate of oxygen uptake, with a lower r a t i o of ATP to ADP s t i m u l a t i n g an increase in c e l l u l a r r e s p i r a t i o n (29). The e f f e c t of t h i s w i l l be to increase u t i l i z a t i o n of NADH for a l t e r n a t i v e r e d u c t i v e pathways, such as gluconeogenesis (26). In e a r l y exerc ise , before the attainment of steady s ta te , the normal rate of ATP formation by o x i d a t i v e phosphory lat ion and g l y c o l y s i s is less than ATP hydro lys i s by muscle. This r e s u l t s in an a c c e l e r a t i o n of g l y c o l y s i s due to an increase in phosphofructokinase a c t i v i t y (26). The source of c y t o s o l i c acety l CoA is of great importance in c h o l e s t e r o l formation since the i n i t i a l b iosynthet ic steps occur in the c y t o s o l . C i t r a t e is l i k e l y the primary immediate precursor of c y t o s o l i c acety l CoA and therefore suppl ies acety l units for the f i r s t step in c h o l e s t e r o l synthesis (30). Mitochondr ia l membranes are not r e a d i l y permeable to f ree acety l CoA, a bar r ie r that is normally overcome by c i t r a t e , which car r ies acety l units across the inner mitochondrial membrane (IMM) to the cytosol in the f o l l o w i n g react ion (26): (IMM) Acety l CoA + OAA > C i t r a t e > Acety l CoA + ADP + OAA ATP/CoA Acety l CoA may o r i g i n a t e i n d i r e c t l y from the oxidat ion of g lucose , f a t t y acids and p r o t e i n , or d i r e c t l y from acetate and ketone bodies (30). Acety l CoA formed during f a t t y ac id oxidat ion enters the TCA c y c l e only i f f a t and carbohydrate degradation are in balance. If there is inadequate oxaloacetate (OAA) present to condense with acety l CoA enter ing 9 the pathway, oxidat ion of pyruvate to acety l CoA w i l l not occur, and pyruvate w i l l be d i r e c t l y converted to OAA instead. Oxaloacetate needs are met by d i r e c t conversion from pyruvate, i t s e l f generated from g l y c o l y t i c and gluconeogenic-der ived glucose (27). Thus although an increase in a c t i v i t y of the TCA cyc le is seen during exerc ise , the a v a i l a b i l i t y of acety l units for b iosynthet ic react ions from glucose and f a t t y acid ox idat ion i s reduced. Excess acety l CoA units produced during f a t t y ac id ox idat ion are s imultaneously shunted to the formation of ketone bodies, acetoacetate and D-3-hydroxybutyrate in the mitochondrial matrix. Although ab le to f r e e l y permeate the mitochondrial membrane, acetoacetate has not been considered a l i k e l y precursor for cytoplasmic acety l CoA in l i v e r because of low acetoacetyl CoA synthetase a c t i v i t y . Only extrahepatic t i s s u e s such as cardiac muscle, adipose t i s s u e and brain have the a b i l i t y to metabol ize cytoplasmic acetoacetate by acetoacetyl CoA synthetase f o l l o w e d by t h i o l a s e , to y i e l d two acety l CoA units (27). It has been suggested that in the process of l i p i d biosyntheses, f a t t y acid synthesis predominates over c h o l e s t e r o l synthesis (31). Thus, whether the body was in a p o s i t i v e energy s ta te , with r e l a t i v e l y l i t t l e ketone body formation, or negative energy state with increased ketone product ion, acetoacetate would provide only a l i m i t e d amount of acety l CoA for hepatic c h o l e s t e r o l synthes is . The lack of acety l units for s t e r o l b iosynthesis is fu r ther substant iated by the fac t that high l e v e l s of c y c l i c AMP present in a low energy state cause the l i v e r to be more e f f i c i e n t in the synthesis of g lucose and oxidat ion of f a t t y ac ids , and less so in the synthesis of f a t , which a l s o requires acety l units (27). This is p a r t i c u l a r l y true in e x e r c i s i n g i n d i v i d u a l s (32). In add i t ion , c h o l e s t e r o l synthesis may be a f fected by the a v a i l a b i l i t y of NADPH which f a c i 1 i t a t e s HMG CoA convers ion 10 to mevalonate, s ince two major s u p p l i e r s of NADPH; pentose phosphate pathway and mal i c enzyme, are i n h i b i t e d under condit ions of a high ADP/ATP r a t i o as is seen in exerc ise (27). v) Substrate U t i l i z a t i o n During Post Exerc ise Recovery Metabo l i c events occurr ing in the post -exerc i se recovery (PER) or r e p l e t i o n phase would be ant i c ipated to exert a s i m i l a r e f fect on acety l CoA a v a i l a b i l i t y as the exerc ise period by a l t e r i n g cho les te ro l precursor a v a i l a b i l i t y . During the recovery phase, o x i d a t i v e and g l y c o l y t i c a c t i v i t y remains acce lerated as long as AMP and ADP are e l e v a t e d in the t issues (27). Although muscle a c t i v i t y has ceased, c a t a b o l i c processes must funct ion to regenerate the d e f i c i t of high energy compounds generated by l i v e r and muscle during previous periods of phys ica l a c t i v i t y . Increased l a c t a t e production and acce lerated oxygen uptake above res t ing l e v e l s are c h a r a c t e r i s t i c of th i s period (33). Lactate production aids in r e p l e t i o n of hepatic and muscle glycogen stores. It has been found that l ess than 18% of l a c t a t e contr ibutes to g lycogenesis during the l a t t e r stages of recovery with the majority of blood glucose for glycogen synthesis suppl ied by the gluconeogenic precursors pyruvate, a lanine and g l y c e r o l (34). Krzentowski et a l . (35) found that during PER, humans given an ora l g lucose load a f te r 3 hr at 50% VO2 max on a t r e a d m i l l , showed the f o l l o w i n g d i f fe rences in glucose metabolism as compared to non-exerc is ing c o n t r o l s . Splanchnic output of g lucose, muscle glycogen content and l i p i d oxidat ion were increased. In add i t ion , a greater proportion of g lucose was used for muscle, and not hepat ic , glycogen r e p l e t i o n . It was hypothesized 11 that glucose-6-phosphate was used p r e f e r e n t i a l l y as a precursor fo r muscle glycogen synthes is , causing i t to be shunted away from acety l CoA formation. Others have reported an increase in a c t i v i t y of glycogen synthetase during t h i s period (37). Holm et a l . (36) saw an increase in catecholamine production which p e r s i s t e d for up to 4 hours a f ter exerc ise, the ear ly phase of PER in humans exerc is ing at 70% V0 2 max for one hour. Catecholamines serve to a c t i v a t e glycogen synthase kinase in muscle and l i v e r which i n h i b i t s glycogen formation (38). The main funct ion then of catacholamines is to enhance c a t a b o l i c processes which provide precursors fo r t h i s " r e b u i l d i n g " phase. A decrease in a lan ine was a l s o observed 40 minutes into PER, which c o r r e l a t e d with an increased f r a c t i o n a l extract ion of a lan ine and an increase in gluconeogenesis (38). Moreover, plasma t r i g l y c e r i d e l e v e l s decreased, p o s s i b l y due to increased muscle uptake for resynthesis of intramuscular l i p i d poo l . B i e l i n s k i et a j^ (33) observed that postmeal, post -exerc ise fa t ox idat ion increased from 27-38% of to ta l energy expenditure over non-exerc ised c o n t r o l s . This was accompanied by a s i g n i f i c a n t increase in carbohydrate storage and a decrease in fa t storage, p o s s i b l y due to decreased a v a i l a b i l i t y of acety l CoA. F i n a l l y , i t has been postu lated that energy i s required for s t i m u l a t i o n of body prote in turnover , which is more pronounced in PER than during exercise (39). Others have observed very low plasma i n s u l i n l e v e l s and e levated blood glucagon during a 4 hour PER period in rats (38). The e f f e c t s of i n s u l i n and glucagon are discussed elsewhere. In summary, the PER phase i s necessary for replenishment of m u s c l e / l i v e r glycogen and f a t , processes which decrease the a v a i l a b i l i t y 12 of the primary c h o l e s t e r o l precursor , acety l CoA. v i ) E f f e c t of Dietary Cho les tero l on Plasma C h o l e s t e r o l in Humans Ep idemio log ica l studies and c l i n i c a l t r i a l s have shown convincing assoc iat ions between serum c h o l e s t e r o l l e v e l s greater than 220 mg/dl and increased c a r d i o v a s c u l a r disease (CVD) inc idence (9,10). It is from such studies that the r i sk of CVD from hypercho lestero lemia , which tends to increase 1% for every 1 mg/dl r i s e in plasma c h o l e s t e r o l (40), has been e s t a b l i s h e d . Current evidence suggests that lowering blood c h o l e s t e r o l w i l l in fact reduce the rate of CHD by s lowing the progression of a t h e r o s c l e r o t i c les ions or even shr ink ing them (41). It is apparent from ep idemio log ica l studies of var ious populat ions that a thresho ld l e v e l for c h o l e s t e r o l may e x i s t whereby only very low (<100 mg/day) or very high (>1000 mg/day) c h o l e s t e r o l intakes w i l l a f f e c t plasma l e v e l s (42,43) with intermediary intake having l i t t l e or no e f f e c t (44). More c a r e f u l l y c o n t r o l l e d metabol ic studies have shown, however, that, plasma c h o l e s t e r o l w i l l r i s e in humans about 10 mg/dl for every 100 mg cholestero l/1000 C a l o r i e s (40). This d i r e c t response ex is ts when c h o l e s t e r o l intakes are 500 mg/day or l e s s , although i t is not agreed upon whether the r e l a t i o n s h i p is l i n e a r or c u r v i l i n e a r (40). The area remains c o n t r o v e r s i a l in humans s ince contro l of human subjects is d i f f i c u l t , and large v a r i a t i o n occurs in response to d ie tary c h o l e s t e r o l among i n d i v i d u a l s . 13 v i i ) E f f e c t of Dietary C h o l e s t e r o l on Plasma C h o l e s t e r o l In Animals Experimental studies in animals are e a s i l y c o n t r o l l e d and r e s u l t s have shown a more consistent r e l a t i o n s h i p between plasma c h o l e s t e r o l l e v e l s and d ietary c h o l e s t e r o l . T u r l e y et a l . (20) found that c h o l e s t e r o l feeding in female hamsters (0.12% w/w) s i g n i f i c a n t l y decreased l i v e r c h o l e s t e r o l synthesis as measured by t r i t i u m incorporat ion and increased plasma c h o l e s t e r o l l e v e l s . S i m i l a r r e s u l t s were obtained by Spady et a l . (18), who in add i t ion observed a s i g n i f i c a n t decrease in HMG CoA reductase a c t i v i t y f o l l o w i n g c h o l e s t e r o l feeding. It appears from these and other studies that d ie tary c h o l e s t e r o l feeding has a d e f i n i t e e f fec t on plasma l e v e l s in animals. Thus studying the r e l a t i o n s h i p between d ie tary and plasma c h o l e s t e r o l in animals may provide v a l u a b l e information on the e f f e c t of d ie t on c h o l e s t e r o l metabolism. v i i i ) Regulat ion of L i p i d Synthesis In man, c h o l e s t e r o l is obtained e i ther by absorption from the d ie t -(300-500 mg/day) or synthes is , p r i m a r i l y in the l i v e r and small i n t e s t i n e (700-800 mg/day) (30). The rate of c h o l e s t e r o l excret ion through a l l pathways must equal the rate of accret ion through absorption and synthesis (19). The way in which t h i s balance is maintained is not e n t i r e l y c l e a r , however, r e g u l a t i o n by feedback i n h i b i t i o n , d iurna l v a r i a t i o n and substrate a v a i l a b i l i t y have been suggested (30). Regulat ion of l i v e r c h o l e s t e r o l synthesis by feedback i n h i b i t i o n i s w e l l documented. It has been found that in both rats and hamsters, 14 c h o l e s t e r o l feeding suppresses c h o l e s t e r o l synthesis and HMG CoA reductase a c t i v i t y in the l i v e r and increases serum c h o l e s t e r o l l e v e l s (45-46). The c h o l e s t e r o l e f f e c t on HMG CoA reductase may be mediated by changes in the f l u i d i t y of i t s supporting microsomal membrane. C h o l e s t e r o l from the d i e t i s thought to accumulate in microsomal membranes as c h o l e s t e r o l e s t e r s , which in turn a f f e c t normal membrane f l u i d i t y and HMG CoA reductase a c t i v i t y (30). Changes in the amount of HMG CoA reductase are p a r t l y respons ib le for changes in cho les tero l synthet ic rates (47). B i l e acids with in the enterohepatic c i r c u l a t i o n can s i m i l a r l y i n h i b i t c h o l e s t e r o l synthesis in both l i v e r and i n t e s t i n e (48). Because of s t r u c t u r a l s i m i l a r i t i e s , i t has been hypothesized that the unconjugated b i l e acid deoxychol ic ac id competes with HMG CoA for binding s i t e s and thus may c o m p e t i t i v e l y i n h i b i t c h o l e s t e r o l synthesis in i t s e a r l y stages (30). In a d d i t i o n , the detergent propert ies of b i l e acids are known to n o n - s p e c i f i c a l l y i n h i b i t many enzymes (48), perhaps inc lud ing HMG CoA reductase (48). F igure 1 i l l u s t r a t e s the primary factors i n f l u e n c i n g -c h o l e s t e r o l metabolism: FATTY ACIDS 3 HYDROXY ; ^ * BILE^ACIDS f BUTYRIC i 1 1 AC:D \ i <D ACETYL @ i Ct> T I® ' S>n FARNESYL <© <3> ! A C E T A T E - - * eOA . . . - -*ACETOACETYL C0A~*3-HMG C0A-|iMEVAl0NATE-|*PHOSPHATE-|»SQUAlENE CHOLESTEROL ! ! ! I I SECONDARY S ITES OF REDUCED ENZYMATIC | EXERCISE? ' A C T I V I T Y AFTER CHOLESTEROL FEEOING , FASTING? '-PRIHART S ITE OF FEEDBACK I K H U I T I O B AFTER CHOLESTEROL FEEOINS FIGURE 1. Major Staps i n the S y n t h e s i s o f C h o l e s t e r o l ana the 3iochenri'cal Staps Where M e t a b o l i c C o n t r o l Takes P l a c e . (Adaoted from O i e t s c h y and W i l s o n , ( 4 8 ) ) . Th is s i m p l i f i e d scheme shows on ly c a p t a i n key s teps i n the b i o c h e m i c a l sequence . The s p e c i f i c c o n v e r s i o n s i n d i c a t e d by numbers may r e p r e s e n t a s i n g l e enzymat ic s t a p ( i e . s t e p 4 ) , or a sequence o f e a r l i e r enzymat ic s teps ( i e . s tep 5 ) . The t h r e e s i t e s a t which feedback i n h i b i t i o n is thought to be mediated are 'shown by the hatched b l o c k s - Stap 4, the pr imary s i t e o f f e e d -back i n h i b i t i o n , and Steps 5 and 6 , the s i t e s o f secondary c o n t r o l . 15 C h o l e s t e r o l formation in l i v e r and smal l i n t e s t i n e undergoes c i r c a d i a n v a r i a t i o n in rodents, with peak synthet ic a c t i v i t y occurr ing at the midpoint of the dark c y c l e in ad l i b i tum fed rats (16). This cont ro l i s thought to be associated with changes in rates of synthesis of HMG CoA reductase, and thus, i t s a c t i v i t y (30). Evidence from d iurna l v a r i a t i o n studies suggests that in rodents, the peak endogenous synthet ic rate occurr ing at mid-dark c y c l e (4-6 hours postprandia l ) is funct ion ing to r e p l a c e d ie tary c h o l e s t e r o l that has been converted p r i m a r i l y to b i l e acids and excreted to aid in the d igest ion and absorption of fa t (49). One would expect that i f d ietary cho les te ro l were the s o l e regu la tor of c h o l e s t e r o l synthes is , animals in the fasted state would undergo increased synthesis as descr ibed above. Tracer studies however have shown that in 24-48 hour fasted r a t s , a reduct ion in both hepatic c h o l e s t e r o l synthet i c r a t e s , and HMG CoA reductase a c t i v i t y is observed (49). It has been suggested that the formation of mevalonic ac id is l i m i t e d by the quant i ty of substrate a v a i l a b l e for HMG CoA reductase, which would r e s u l t in p a r t i a l i n h i b i t i o n of HMG CoA condensing enzyme (30). I n s u l i n and glucagon may a l s o play a r o l e in the process of r e g u l a t i n g c h o l e s t e r o l synthes is . In v i v o t racer studies have shown i n s u l i n to increase hepatic cho les te ro lgenes i s and glucagon to block t h i s e f f e c t (50). These hormones are thought to contro l both the t o t a l amount of enzyme present and the proport ion of enzyme at an a c t i v e s i t e (50). I n s u l i n and glucagon are a l s o known to have powerful e f fects on the a v a i l a b i l i t y of acety l CoA. I n s u l i n causes increased u t i l i z a t i o n of a c e t y l un i ts fo r anabo l i c pathways, p a r t i c u l a r l y l i p i d synthes is , whereas glucagon i n d i r e c t l y causes d i v e r s i o n of a c e t y l units into o x i d a t i v e , g lucogenic and ketogenic pathways (27). Since f a s t i n g causes a s h i f t in 16 these hormones s i m i l a r to that seen during and immediately f o l l o w i n g e x e r c i s e , i t i s p o s s i b l e that cho les tero l precursors are der ived from the same metabol ic pool as is used fo r energy product ion. Thus substrate a v a i l a b i l i t y as determined by energy demands may i n f l u e n c e synthesis of c h o l e s t e r o l , in addi t ion to the other discussed contro l f a c t o r s . ix) High Density L ipoprote in Metabolism High density l i p o p r o t e i n s (HDL) serve a p u t a t i v e r o l e as p r o t e c t i v e agents against premature development of a t h e r o s c l e r o s i s and coronary heart d isease. High density l i p o p r o t e i n s can o r i g i n a t e in the l i v e r or i n t e s t i n e as d i s c o i d a l nascent HDL, and both from surface remnants of t r i g l y c e r i d e r i c h l i p o p r o t e i n s or phosphol ip id-apoprote in assoc iat ions (spher ica l HDL) (51). High density l i p o p r o t e i n s ex is t in blood p r i m a r i l y as 40 A HDL2 and 60 A HDL3 subfract ions . The HDL2 component has 3-4 f o l d more c h o l e s t e r o l ester and t r i g l y c e r i d e compared with HDL3. Thus HDL2 is considered to be an e f f i c i e n t l i p i d c a r r i e r and a better p r o t e c t i v e agent against coronary heart d i s e a s e . Two mechanisms for t h i s p ro tec t ive e f f e c t have been proposed. F i r s t l y , HDL is thought to be p r o t e c t i v e against a t h e r o s c l e r o s i s through i t s funct ion in "reverse c h o l e s t e r o l t ransport" , where extra-hepat ic c h o l e s t e r o l from the periphery is e s t e r i f i e d by HDL molecules v i a l e c i t h i n c h o l e s t e r o l acyl t ransferase (LCAT), and returned to the l i v e r fo r u t i l i z a t i o n (52). During normal f a t metabolism, d i s c o i d a l HDL c i r c u l a t e s in the plasma or lymph where i t is transformed to spher i ca l HDL by the i n f l u x of c h o l e s t e r o l across a chemical gradient mainly from very low density l i p o p r o t e i n s (VLDL) and chylomicron remnants (52). Spher ica l HDL 17 t ransports newly formed c h o l e s t e r o l esters back to the l i v e r . In a d d i t i o n , some c h o l e s t e r o l ester from HDL is t ransferred to VLDL and LDL, and r e c i p r o c a l l y , t r i g l y c e r i d e s are t ransferred from these l i p o p r o t e i n s to HDL. High density l i p o p r o t e i n s r e a d i l y adapt to changes in l i p i d status in humans. For example, when endogenous l i poprote ins are exposed to a la rge i n f l u x of d ie tary fa t and c h o l e s t e r o l , HDL l e v e l s increase as part of a normal response to excess c h o l e s t e r o l in the blood. When r e g u l a t i o n of c h o l e s t e r o l balance in normal i n d i v i d u a l s is achieved through HDL reverse c h o l e s t e r o l t ransport and other l i p o p r o t e i n s , formation o f atheromas is not favoured. Converse ly , i t has been shown that i n d i v i d u a l s with absent plasma HDL show severe impairment of c h o l e s t e r o l ester and t r i g l y c e r i d e t ranspor t (51). I t has a l s o been noted that low HDL is as p r e d i c t i v e as high LDL for coronary heart disease (53). Thus important questions have been r a i s e d regarding potent ia l benef it of e levated HDL l e v e l s . F i r s t l y , do f a c t o r s other than d iet have the a b i l i t y to independently r a i s e plasma HDL l e v e l s ? And more important, would these high l e v e l s s i g n i f i c a n t l y a l t e r the rate at which c h o l e s t e r o l undergoes reverse c h o l e s t e r o l t r a n s p o r t , and thus remove c h o l e s t e r o l from the periphery? A second way in which HDL may decrease r i s k of c a r d i o v a s c u l a r d isease i s by d i r e c t l y retard ing atheromatous l e s i o n growth (51). Normally low dens i ty l i p o p r o t e i n (LDL) is taken up into the c e l l through receptor-binding and lyzosome i n t e r n a l i z a t i o n , f o l l o w e d by a r e l e a s e of c h o l e s t e r o l in to the c e l l lumen by h y d r o l y s i s of const i tuent LDL c h o l e s t e r o l es ters . The r e s u l t a n t f ree c h o l e s t e r o l in turn i n h i b i t s endogenous c h o l e s t e r o l synthesis v i a i n h i b i t i o n of HMG CoA reductase (53). As LDL concentrat ions increase , sa turat ion k i n e t i c s occur, decreasing the number of a v a i l a b l e LDL binding s i t e s and causing LDL p a r t i c l e s to accumulate in the plasma. 18 Normal ly , r e g u l a t i o n of plasma LDL is achieved in part , by enhanced HDL removal of c h o l e s t e r o l from the c e l l lumen, which in turn causes an i n f l u x of LDL to c e l l s across a favourab le concentrat ion gradient. Experimental evidence suggests that in add i t ion to i t s normal funct ion of reverse c h o l e s t e r o l t ranspor t , HDL and in p a r t i c u l a r HDL2, protects against formation of plaque and subsequent atherogenesis by reducing c e l l u l a r LDL c h o l e s t e r o l uptake. C e l l c u l t u r e studies have shown that molar r a t i o s of HDL:LDL in excess of 5:1 i n h i b i t uptake and subsequent degradation of LDL into c e l l s of cu l tu red f i b r o b l a s t s (53), v a s c u l a r e n d o t h e l i a l c e l l s (53) and smooth muscle t i ssue (54). Various fac to rs are thought to r a i s e HDL c h o l e s t e r o l l e v e l s . Chronic a l c o h o l consumption causes an increase in VLDL f l u x and is associated with increased HDL concentrat ion and a lower r i s k of c a r d i o v a s c u l a r d isease (53). S i m i l a r l y phys ica l exerc ise has been shown to e l e v a t e HDL although the consistency and mechanism of th i s response remains c o n t r o v e r s i a l . It has been hypothesized that endurance a th le tes have e l e v a t e d l e v e l s of HDL c h o l e s t e r o l and apoproteins (A l , A l l ) in part because of reduced HDL apoprotein catabol ism as w e l l as an increased a b i l i t y to c l e a r t r i g l y c e r i d e s from s k e l e t a l muscle (55). The increased t r i g l y c e r i d e c learance most l i k e l y r e s u l t s from the exercise- induced r i s e in l i p o p r o t e i n l i p a s e (LPL), c a t a l y z i n g the h y d r o l y s i s of t r i g l y c e r i d e s in VLDL and chylomicrons, the breakdown products of which enter the plasma pool as HDL precursors to form spher i ca l HDL. Mechan is t i ca l l y , HDL is be 1 ieved to compete with LDL for eel 1 ul ar receptor binding s i t e s , which in turn decreases surface binding of LDL and i t s consequent uptake, degradation and cont r ibut ion to net c e l l c h o l e s t e r o l content (51). Furthurmore, and in contrast to LDL, HDL has a 19 slower rate of i n t e r n a l i z a t i o n thereby decreasing c e l l u l a r accumulation of c h o l e s t e r o l . Thus, in smooth muscles and vascu lar e n d o t h e l i a , where high rates of LDL uptake and i n i t i a t i o n and progression of a t h e r o s c l e r o t i c l e s i o n s are known to occur (54), HDL may be of some benef i t in reducing the r i s k of a t h e r o s c l e r o s i s by decreasing substrate supply fo r plaque formation. Caution must be exerc ised in i n t e r p r e t i n g these observat ions however, as i t is unknown whether decreased c e l l u l a r uptake of LDL, p a r t i c u l a r l y in extrahepat ic t i s s u e s , can regu late whole body c h o l e s t e r o l balance. For example, endogenous synthesis may increase in response to decreased LDL uptake and counteract any net decrease in c e l l c h o l e s t e r o l due to LDL alone. In summary, the l e v e l of HDL in the blood may.be i n v e r s e l y r e l a t e d to the r i s k of CHD. The p o s i t i v e e f f e c t s of HDL might be mediated by enhanced reverse c h o l e s t e r o l t ranspor t , or i n h i b i t i o n of c e l l u l a r LDL uptake. Studying the response of HDL l e v e l s to exerc ise may be usefu l in f u r t h e r i n g our knowledge of CHD r i s k fac tors . 20 RATIONALE Plasma c h o l e s t e r o l concentrat ion is independently inf luenced by both exerc ise a c t i v i t y and the l e v e l of cho les te ro l in the d ie t . Studies suggest that exerc ise may decrease plasma c h o l e s t e r o l l e v e l whi le d ie tary c h o l e s t e r o l has the opposite e f fec t . However, the mechanism by which these e f f e c t s occcur is not understood. A poss ib le mechanism through which these e f f e c t s on plasma c h o l e s t e r o l are mediated is by a l t e r i n g the rate of endogenous c h o l e s t e r o l synthesis . Exercise may a l t e r the a v a i l a b i l i t y of acety l CoA subunits necessary fo r the i n i t i a l stages of s t e r o l synthes i s , thereby decreasing i t s rate of formation. In contrast , i t is b e l i e v e d that d ie tary c h o l e s t e r o l exerts negative feedback i n h i b i t i o n on both hepat ic and i n t e s t i n a l rates of s t e r o l synthesis . Therefore the purpose of th i s study was to determine whether exerc ise a c t i v i t y and d ie tary c h o l e s t e r o l feeding, independently, or through an i n t e r a c t i o n , i n f l u e n c e ; plasma t o t a l c h o l e s t e r o l l e v e l s , HDL c h o l e s t e r o l l e v e l s and synthesis of c h o l e s t e r o l in l i v e r and i n t e s t i n e , the organs l a r g e l y respons ib le for c h o l e s t e r o l production. F indings from t h i s study w i l l further our knowledge of contro l mechanisms of plasma c h o l e s t e r o l and c h o l e s t e r o l synthes is , v i a exerc ise a c t i v i t y and d i e t a r y c h o l e s t e r o l l e v e l . 21 MATERIALS AND METHODS i) Methodologica l Considerations a) Determination of Maximal Oxygen Consumption in Hamsters It i s w e l l known that endurance exercise enhances the r e s p i r a t o r y capacity of muscle by inducing enzymatic changes that increase the ox idat ion of pyruvate, f a t t y acids and ketones (29). This r i s e in muscle r e s p i r a t o r y capacity r e s u l t s from an increase in both the composition and t o t a l number of mitochondria present in muscle t i s s u e (29). Endurance i s a funct ion of r e l a t i v e work rate (56) which is reflected in the a b i l i t y of muscle mitochondria to u t i l i z e oxygen (32). Comparison of t ra ined and untrained i n d i v i d u a l s exerc is ing at the same r e l a t i v e work rate has shown that t ra ined i n d i v i d u a l s have metabol ic responses to exercise that are d i f f e r e n t from those of t h e i r untrained counterparts. For example, d e p l e t i o n of muscle and l i v e r glycogen stores is s lower, and a greater r e l i a n c e on fa t oxidation for generation of energy occurs in t ra ined subjects (29). Although there ex is ts some in ter - ind iY idua l v a r i a b i l i t y , uptake of oxygen reaches a reasonably constant rate given a s p e c i f i e d t r a i n i n g schedule. Thus in an exercise study i t is d e s i r a b l e to have a l l subjects working at the same capacity i f v a r i a b l e s other than oxygen consumption, but which may be r e l a t e d to the changes in o x i d a t i v e capac i ty , are to be measured. Such control would e l iminate p o t e n t i a l confounding e f fec ts of t r a i n i n g state. A p i l o t study was therefore performed to determine maximal oxygen consumption (VO2 max), to determine the idea l length of time needed for 22 the t r a i n i n g phase in order to achieve a s t a b l e V0£ max across the exerc ise group and to determine the c a l o r i c expenditure associated with exerc ise of def ined work load and durat ion. The work load and durat ion of exerc ise was manipulated, together with projected food intakes, to achieve a constant body weight in the exerc ise group. Hamsters of s i m i l a r age and weight to those in the present study were run d a i l y at the beginning of the dark c y c l e at as high a speed as p o s s i b l e u n t i l they could no longer run. The running apparatus consisted of a f r e e l y spinning exerc ise wheel pos i t ioned on a motorized t r e a d m i l l which was enclosed in an a i r - t i g h t chamber. Figure 1 i l l u s t r a t e s the equipment used to determine oxygen consumption of a hamster during exerc ise (see Appendix A for d e t a i l s ) . FLOWMETER i OXYGEN NANALYZER AIR TIGHT EXERCISE CHAMBER •• Figure 1. Samples of post-exchange a i r were analyzed with a s i n g l e channel paramagnetic oxygen analyzer (i) (model 211, Westinghouse, P i t t sburgh , PA), and oxygen concentrat ion d i f fe rences between room and exchanged a i r were recorded ( i i ) . Oxygen consumption, c a l c u l a t e d as the product of a i r f low rate (1.23 L/min) through the a i r t i g h t exerc ise chamber m u l t i p l i e d by the oxygen concentrat ion d i f f e r e n c e between room and chamber post-exchange a i r , was used to 23 b) Measurement of Cho les tero l Synthesis Regulatory mechanisms of c h o l e s t e r o l synthesis are commonly measured i n d i r e c t l y by i) determining the a c t i v i t y of microsomal HMG CoA reductase or d i r e c t l y by i i ) measuring incorporat ion of l a b e l l e d precursor such as [14]C-acetate or t r i t i u m (3H) from t r i t i a t e d water ^H20 into c h o l e s t e r o l (23-24). Since accuracy in quant i ta t ing these rates is d e s i r a b l e , the method of choice should be consistent and r e l i a b l e . Measurement of HMG CoA reductase a c t i v i t y alone is often used to assess r e l a t i v e d i f ferences of synthet ic rates in a given t i s s u e preparat ion (24). Good technique during microsome preparation is necessary to avo id underestimation of absolute rates of cho les te ro l synthes is . The l a b e l l i n g of precursor is a l s o simple but is subject to i n t r a c e l l u l a r d i l u t i o n . For example, [14]C-acetate is taken up by the c e l l and converted to [14]C-acetyl CoA in the mitochondrial matrix. I t cannot, however, be assumed that th i s w i l l be the so le source of acety l CoA for s t e r o l synthes is , since f a t t y ac id and glucose oxidation may contr ibute acety l units which would d i l u t e t h i s l a b e l l e d pool and i t s s p e c i f i c r a d i o a c t i v i t y (23,24). The magnitude of d i l u t i o n var ies both in d i f f e r e n t organs or in the same organ under d i f f e r e n t metabolic cond i t ions , and therefore represents an u n r e l i a b l e method. The use of t r i t i a t e d water in measuring cho les te ro l synthesis i s considered a better method in comparison to the above described technique. Incorporat ion of t r i t i a t e d water into c h o l e s t e r o l circumvents d i l u t i o n problems associated with carbon l a b e l l e d substrates. The method is based on the assumption that 3 H atoms from are incorporated into s t a b l e , non-exchangeable posit ions in the s t e r o l molecule. The d i l u t i o n e f f e c t i s 24 avoided by use of high s p e c i f i c r a d i o a c t i v i t y , s ince r e l a t i v e l y l i t t l e unlabel led water i s generated m e t a b o l i c a l l y w i t h i n , or between c e l l s (23). A l i m i t a t i o n with the t r i t i a t e d water technique ar i ses in the est imation of incorporated in to each carbon atom of the c h o l e s t e r o l molecu le , or ^H/C r a t i o . Eighteen acetyl CoA units containing 36 carbon atoms are needed to synthesize one c h o l e s t e r o l molecu le , having 27 carbon and 46 hydrogen atoms. Seven of these hydrogen atoms come d i r e c t l y from water and 15 from NADPH produced in reduct ive b iosynthet i c pathways (24). However, the degree of e q u i l i b r a t i o n of NADPH with t r i t i u m is quest ionable . With high enrichment l e v e l s of t r i t i u m , the assumption i s g e n e r a l l y made that the r e d u c t i v e H of NADPH i s f u l l y e q u i l i b r a t e d with - ^ 0 and that 22 ug atoms of t r i t i u m are incorporated into each umole of c h o l e s t e r o l , g i v i n g an incorporat ion r a t i o of 0.81 (24). Use of t r i t i a t e d water as a 1abel s o l v e s d i l u t i o n problems associated with carbon 1abel 1 ed substrates and was the method of choice for the present experiment. C h o l e s t e r o l synthet ic a c t i v i t y may be expressed in d i f f e r e n t ways, depending on the in tent ion of the experiment. Typ i ca l expressions i n c l u d e ; i) incorporat ion of r a d i o a c t i v e material into c h o l e s t e r o l per organ or i i ) per gram t i s s u e or i i i ) as mg of c h o l e s t r o l synthesized per mg t i s s u e c h o l e s t e r o l , or as f r a c t i o n a l synthet ic rate (FSR). i i ) Experimental Design To a l l o w simultaneous assessment of d ie tary c h o l e s t e r o l and exerc ise on parameters of l i p i d metabolism in hamsters, a two by two f a c t o r i a l experiment was designed. Because of l im i ted space on the exerc ise equipment, d i v i s i o n of the study into two separate experiments was 25 requi red . The two experimental study groups d i f f e r e d only on the basis of d i e t , and w i l l subsequently re fer red to as; Low c h o l e s t e r o l d ie t (LC), and High c h o l e s t e r o l d iet (HC). Thus for each of the two consecutive study per iods , hamsters were a l l o c a t e d into e i ther exerc ise (E) or sedentary (S) groups wi th in each of the d ie t treatment groups. Figure 3 i l l u s t r a t e s the general experimental design used in t h i s study. FIGURE 3. Experimental Design. Factors altered were exercise and level of dietary cholesterol 26 i i i ) Animals - Diet and Exerc ise Procedures a) D i e t On day one of each of two four week stud ies , Syr ian hamsters (male, 50 days o l d , 105.9 + 6 grams (+_ S.D)) obtained from Char les R iver Laborator ies Inc., Montrea l , were randomized into e i ther sedentary or exerc ise groups containing 12 animals each. Animals were i n d i v i d u a l l y housed in s t a i n l e s s s tee l cages and exposed to 12 hour l ight/dark c y c l i n g f o r two weeks p r i o r to , and during the study per iod. A l l hamsters had f ree access to water and were fed ground Purina Laboratory Rodent Diet (5001) to whi-ch 5% corn o i l had been added. Cho les te ro l concentrat ion of the d i e t was 0.03% and 0.12% (w/w) for LC and HC r e s p e c t i v e l y (see Appendix B for complete d iet composition). Exerc is ing animals were presented with an amount of food i d e n t i c a l to the averaged previous day's intake of ad l i b i t u m fed sedentary c o n t r o l s . Food cups were removed from a l l hamsters during the d a i l y exerc ise per iod. b) Exerc ise Exercised hamsters were run s imultaneously at the beginning of the dark c y c l e fo r 90 minutes d a i l y on a motorized t r e a d m i l l . Animals were subjected to a 2 phase schedule over the experimental per iod. During the i n i t i a l t r a i n i n g phase running speed was increased by 2.5 meters/minute/day for two weeks to ensure adaptation to the apparatus and attainment of a constant V 0 2 max. Phase two consisted of high i n t e n s i t y running at 70% V 0 2 max at approximately 35 meters/minute, 27 f o l l o w i n g a 5 minute warm-up. During th i s phase animals were run d a i l y at the same time f o r a minimum of one week. Both exerc ise and non-exercise groups were exposed to to i n f r a - r e d l i g h t i n g during exerc ise so as to not perturb the normal d iurna l l i g h t c y c l e . The f o l l o w i n g diagram i l l u s t r a t e s the d a i l y feeding and exerc ise schedule: start end exercise exercise LIGHT (12 hr) \\\\\\\\V .DARK (12 h r ) V \\\\\\\\\\\V / \ remove food (E & S) return food FIGURE 4. Daily feeding and exercise schedule. c) Entry/Ex i t Points of Study Although both exerc ise and sedentary groups entered the study on the same day, ex i t dates were staggered in order to test i n d i v i d u a l animals at s i m i l a r times during a defined period of the l ight/dark cyc le . S p e c i f i c a l l y , c h o l e s t e r o l synthesis in hamsters has been shown to be maximal both at the mid-point of the dark c y c l e and 4-6 hours post- feeding (49,57). Given t h i s time frame, only 4-6 animals could be k i l l e d per day. Therefore , commencing on day 22 of each study, f o l l o w i n g the two week t r a i n i n g phase and a minimum of one week intense exerc ise , 2 pa i rs of animals were s a c r i f i c e d per day as subsequently descr ibed, with one hamster from each of exerc ise and sedentary groups forming the pa i r . 28 i v ) Measurement of C h o l e s t e r o l Synthesis and Plasma C h o l e s t e r o l Both pa i rs of animals were fasted overnight p r io r to t h e i r f i n a l day in the study. Fo l lowing normal rout ine , exerc is ing animals were returned to t h e i r cages a f ter 90 minutes of running and given f ree access to water. One h a l f hour l a t e r , the f i r s t of four hamsters to be studied rece ived a bolus of 50% glucose s o l u t i o n (lg/kg) v i a g a s t r i c intubat ion . The remaining 3 hamsters were s i m i l a r l y intubated at 1/2 hour i n t e r v a l s . Fo l lowing a 3 1/2 hour rest the f i r s t hamster rece ived an i n t r a - p e r i t o n e a l (IP) i n j e c t i o n of approximately 30 mCi t r i t i a t e d water, f o l l o w e d again by the remaining animals at 1/2 hour i n t e r v a l s . Two hours a f ter i n j e c t i o n animals were l i g h t l y anesthet ized with ether (USP for anasthesia) and exsanguinated. It should be noted that the order of the 4 animals being s a c r i f i c e d changed d a i l y to avoid potent ia l confounding of r e s u l t s by temporal d i f fe rences from the time of exercise a c t i v i t y . During exsanguination approximately 4 ml of blood were withdrawn from the heart and centr i fuged at 1500 RPM for 20 minutes. Plasma was removed for s p e c i f i c a c t i v i t y , plasma t o t a l and HDL-cholesterol determinations. L i v e r and smal l i n t e s t i n e were immediately removed, r insed with s a l i n e , weighed, and f r o z e n . i n l i q u i d nitrogen before storage at -70°C. v) Laboratory Analyses a) HDL-Cholesterol Determination P r e c i p i t a t i o n of HDL-cholestero l was performed immediately f o l l o w i n g animal s a c r i f i c e , using a modif ied dextran sulphate method (58). HDL in 29 plasma is often expressed as the proport ion of the l i p o p r o t e i n that i s c h o l e s t e r o l , u s u a l l y around 15%. Expression of HDL concentrat ion in plasma by i t s c h o l e s t e r o l content has been es tab l i shed as a standard method for HDL determination. Thus, f o l l o w i n g c e n t r i f u g a t i o n , 200^*1 plasma samples were combined with 20^*1 reagent (1 ml heparin, 2 ml magnesium c h l o r i d e , 1 ml 0.15M sodium c h l o r i d e ) , shaken for 10 minutes and centr i fuged at 3000 rpm for 20 minutes. The r e s u l t i n g supernatant was removed and stored at -70°C u n t i l analyzed for t o t a l HDL c h o l e s t e r o l content as descr ibed below. b) Plasma S p e c i f i c A c t i v i t y Fo l lowing storage at -70°C, 100^1 samples of plasma were d i l u t e d 1000 times with d i s t i 11 ed water, and 100_^1 of the r e s u l t i n g s o l u t i o n mixed with 10 ml s c i n t i l l a t i o n f l u i d to determine d i s i n t e g r a t i o n s per minute. S p e c i f i c a c t i v i t y was used to c a l c u l a t e c h o l e s t e r o l synthet ic r a t e , and i n d i r e c t l y , percentage body fat (see page 34). c) Cho les te ro l Determination C h o l e s t e r o l concentrat ion of plasma t o t a l , HDL, and t i ssue c h o l e s t e r o l were quantitated in v i t r o using an enzymatic k i t ( B i o p a c i f i c Diagnost ics Inc.) (59). P r e v i o u s l y frozen plasma samples and f ree c h o l e s t e r o l i s o l a t e d from t i s s u e and d i s s o l v e d in i sopropanol , were assayed in d u p l i c a t e . Ten^J of sample was combined with 1 ml c h o l e s t e r o l reagent and incubated 5 minutes at 37°C. C o l o r i m e t r i c a n a l y s i s was used to determine concentrat ion of c h o l e s t e r o l r e l a t i v e to i n t e r n a l reference standards by spectrophotometery at 505 nm (Coleman Spectrophotometer, 30 model 111 -050). d) T issue Preparation L i v e r (1.0 g) and i n t e s t i n e (0.5 g) samples were saponif ied in d u p l i c a t e to l i b e r a t e f ree and e s t e r i f i e d c h o l e s t e r o l . For l i v e r , 2 ml 30% potassium hydroxide (K0H) in water were added and the mixture heated to 80-90 °C. Two ml ethanol (EtOH) were then added and the sample heated f o r 2 hours at 90 °C. For i n t e s t i n e , the procedure was i d e n t i c a l except the volumes of K0H and ETOH were ha lved . Standards containing 100^,1 [14]C-c h o l e s t e r o l and two ml 150 mg/dl unlabel led f ree c h o l e s t e r o l were run in t r i p l i c a t e with each t i ssue batch. Two ml 30% K0H were added to these standards which were then analyzed as descr ibed above. Saponi f ied t i ssue was twice extracted to remove l i p i d s and f ree c h o l e s t e r o l . L i v e r and in terna l standards were extracted after the add i t ion of 2 ml methanol (MeOH) and 12 ml hexane:chloroform (4:1 v/v) . Samples were shaken 10 minutes and f o l l o w i n g addit ion of 1 ml H 2 0, were fu r ther shaken for 10 minutes and centr i fuged at 2300 rpm for 10 minutes. The r e s u l t a n t supernatant was reta ined and s o l v e n t removed by dry ing under N 2 gas. Intest ine samples fo l lowed the same procedure except that 3 ml MeOH were added i n i t i a l l y . e) I s o l a t i o n of Free Cho les te ro l The method of Sperry (60) was used to p r e c i p i t a t e free c h o l e s t e r o l from sample extracts . The procedure was i n i t i a t e d by the addi t ion to each sample of 4 ml acetone/EtOH (1:1 v/v) , 1 ml d i g i t o n i n s o l u t i o n (2% in 80% 31 EtOH) and 1 ml d i s t i l l e d water. The samples were covered and a l lowed to s e t t l e overnight at 23°C in screw top tubes. Centr i fugat ion (15 minutes, 2300 RPM) and removal of supernatant f o l l o w e d . The remaining d i g i t o n i n complex was washed s u c c e s s i v e l y 3 times with 1.5 ml of f i r s t 80% EtOH and second 1.5 ml ether, dr ied completely under N 2 gas, and heated for 1 hour at 110°C. To remove c h o l e s t e r o l from the d i g i t o n i d e complex, 0.3 ml pyr id ine was added and the p r e c i p i t a t e d i s s o l v e d by gent le heating. Samples were again washed 3 times with ether, only t h i s time the supernatants were pooled and kept after each c e n t r i f u g a t i o n . The ether was removed v i a N 2 stream over heat as before. A l l samples were p laced in vacuum over su lphur i c acid for 24 hours. Isopropanol was subsequently added in appropriate amounts to d i l u t e the sample to with in detectab le concentrat ion range for c h o l e s t e r o l . One h a l f the sample was combined with 10 ml s c i n t i l l a t i o n c o c k t a i l and counted for ten minutes. Synthet ic rates were determined as described in " C a l c u l a t i o n s " sect ion . v i ) Cal c u l a t i o n s a) C h o l e s t e r o l Determination C h o l e s t e r o l va lues were expressed e i ther as a concentrat ion (plasma t o t a l and HDL) or absolute va lue (sample and t o t a l t i s s u e values) and were der ived from the f o l l o w i n g equation: c o n c e n t r a t i o n a b s o l u t e . o p t i c a l density ( t e s t sample X [standard] ) X (volume d i l u t i o n factor) o p t i c a l density standard = mg/dl = mg/unit t i s s u e 32 b) F r a c t i o n a l Synthetic Rate The expression of t i ssue cho les te ro l synthesis both as the r a d i o a c t i v i t y per gram of t i s sue and as a f r a c t i o n of t o t a l t i s s u e c h o l e s t e r o l were determined, to a l l o w d i f f e r e n t i a l determination of r e s u l t s and comparison to current l i t e r a t u r e . DPM/g t i s s u e per hr were c a l c u l a t e d , knowing the incubation time and t o t a l t i s s u e weight. DPM were c a l c u l a t e d using the channels ratio.method for counting e f f i c i e n c y on a Nuclear Chicago Isocap 300 Instrument and a quench curve s p e c i f i c to the s o l v e n t s and s o l u t e s used was constructed. Since the plasma s p e c i f i c a c t i v i t y (DPM/ml plasma) was known and assumed to be f u l l y e q u i l i b r a t e d with t r i t i a t e d water, the amount of t r i t i a t e d water incorporated per gram of t i s s u e per hour was c a l c u l a t e d as f o l l o w s : DPM/g tissue.hour DPM/ml plasma (or water) = ml - ^ O /gram t i ssue .hr (1) _9 D i v i d i n g t h i s number by 18 X 10 (ml water per nmol water) = nmol /gram t issue.hr (2) In order to express th i s rate as a f r a c t i o n of the t o t a l c h o l e s t e r o l p o o l , mg c h o l e s t e r o l / g t i s s u e was needed. D i v i d i n g equation (1) by (2) gave; nmol -^UO/q.hr mg c h o l e s t e r o l per hr (3) The incorporat ion of the 3H/C r a t i o as determined by the method of 33 Dietschy et a l . (24) occurred at t h i s point s ince i t was known t h e o r e t i c a l l y how much c h o l e s t e r o l a c e r t a i n amount of t r i t i a t e d water represents (22 ug atoms of 3 H f o r each umol of c h o l e s t e r o l ) . This number was d i v i d e d by two as each water molecule contains two hydrogens atoms. Thus, car ry ing equation (2); nmol H 2 0 (4) mg c h o l e s t e r o l per hr X 386 ng/nmol 11 nmol 3H nmol c h o l e s t e r o l The uni ts (g cholesterol/mg c h o l e s t e r o l per hour) were adjusted and the r e s u l t expressed as that f r a c t i o n of the t o t a l c h o l e s t e r o l pool synthesized v i a t r i t i u m incorporat ion , or; mg c h o l e s t e r o l (synthesized) X 100 mg c h o l e s t e r o l ( t o t a l ) (5) = FSR {%) c) Body Composition Body water volume of each animal was determined from the plasma water 3 H 2 0 a c t i v i t y at time of s a c r i f i c e . It was assumed that at 120 minutes the in jected t r i t i a t e d water had e q u i l i b r a t e d across the body water compartment. Lean body mass was c a l c u l a t e d as (body water volume/ 0.73). Correct ions were made for the exchange of hydrogens of body water with those of body prote ins and carbohydrates (61,62) and f o r the presence of so lu tes in plasma (62). Body fat (%) was c a l c u l a t e d as (body weight - lean body mass) assuming a two pool model. 34 v i i ) S t a t i s t i c a l Analyses A two f a c t o r , two way a n a l y s i s of var iance (ANOVA) with f i x e d e f f e c t s was used to determine the e f fect of exerc ise , d ietary c h o l e s t e r o l and t h e i r i n t e r a c t i o n on each of plasma t o t a l c h o l e s t e r o l and HDL-c h o l e s t e r o l , and c h o l e s t e r o l synthesis. A one factor ANOVA was used to check for d i f fe rences in s t a r t i n g body weights between a l l exerc ise and sedentary animals of LC and HC d iets . A l l c a l c u l a t i o n s were completed with the ass i s tance of Lotus "Symphony" (63) and CSS.STAT (64) computer packages. A l l r e s u l t s were obtained using a two factor a n a l y s i s of var iance , t e s t i n g not only for independent d iet and exercise e f f e c t s , but a l s o f o r t h e i r i n t e r a c t i o n . Therefore, the format subsequently used to descr ibe these r e s u l t s is as f o l l o w s : i) i f no i n t e r a c t i o n e x i s t e d , the grand means for combined d ie t and/or exerc ise groups w i l l be presented along with the p r o b a b i l i t y ("p") va lue i i ) i f an i n t e r a c t i o n was observed, the o v e r a l l s i g n i f i c a n c e l e v e l ("p") of both d ie t and exercise e f f e c t s , and t h e i r i n t e r a c t i o n w i l l be presented. In a d d i t i o n , i n d i v i d u a ! group means and t h e i r r e l a t i o n s h i p to one another as determined by "Tukey's" post hoc t e s t , w i l l be discussed. Anova tables and grand means for d i e t and exerc ise groups are presented in Appendices C, D, and E. 35 RESULTS: i) Food Intake, Body Weights and Percentage Body Fat Figures 5A and 5B i l l u s t r a t e average d a i l y food consumption (x +_ SD) of sedentary and exercised animals fed low and high c h o l e s t e r o l d i e t s , r e s p e c t i v e l y . A l l four study groups showed a gradual decrease in food intake over the experimental per iod. S t a t i s t i c a l comparison was made between sedentary and exercised animals both with in and between d i e t groups on days 1, 15 and 21. Resul ts showed that exerc ise s i g n i f i c a n t l y decreased food consumption (p < 0.05) on days 1, 15 and 21 regard less of the d iet treatment. Although not s i g n i f i c a n t , exerc ised animals fed the low c h o l e s t e r o l d iet consumed s l i g h t l y l ess food than exerc ised animals consuming the high c h o l e s t e r o l d ie t . These r e l a t i o n s h i p s between food consumption and exerc ise a c t i v i t y appeared to be cons is tent over the experimental period and food consumption was therefore inc luded as a c o v a r i a b l e during subsequent s t a t i s t i c a l manipulat ion of v a r i a b l e s . Body weights of sedentary and exerc ised hamsters consuming low and high c h o l e s t e r o l d iets are shown in Figures 6A and 6B r e s p e c t i v e l y . S t a r t i n g body weights of animals were not d i f f e r e n t between the four treatment groups due to systematic randomization at study onset. Sedentary animals of both d ietary treatment groups showed r e l a t i v e l y constant increases in body weight during the 21 day study per iod. In cont ras t , exerc ised hamsters i n i t i a l l y showed s i m i l a r rates of weight gain compared with sedentary groups, f o l l o w e d by a progress ive reduct ion in rate of weight gain at about day 9 as exerc ise i n t e n s i t y 36 C O FIGURE 5A. Food Intake of Hamsters Fed Low Cholesterol Diets "O c cd a> 2 15 13 11 9 7 6 • SEDENTARY • EXERCISE 3 7 9 11 13 Day of Study 15 17 19 21 Data are group means ± S.D. FIGURE 5B. Food Intake of Hamsters Fed High Cholesterol Diets - SEDENTARY • EXERCISE 7 9 11 13 Day of Study Data are group means ± S.O. 15 17 19 21 FIGURE 6A. Body Weights of Hamsters Fed Low Cholesterol Diets 140 130 120 110 ,„!.*=::: .,ET ...-Q [ i 100 p-.A=L_ SEDENTARY -• EXERCISE 7 9 11 13 Day of Study 15 17 19 21 Data are group means ± SD. o FIGURE 6B. Body Weights of Hamsters Fed High Cholesterol Diets 140 130 *-» xz "55 c CO d> 2 120 110 100 •-E3 a • SEDENTARY L O EXERCISE 7 9 11 13 Day of Study 15 17 19 21 Data are group means ± SO. increased. The pattern of weight change of i n d i v i d u a l animals with in exerc ised groups showed cons iderab le v a r i a t i o n compared to t h e i r r e s p e c t i v e sedentary c o n t r o l s such that on day 21 the var iance in body weights of the exerc ise groups was much greater than that of the sedentary groups. Low and high c h o l e s t e r o l - f e d exerc i s ing hamsters had s i g n i f i c a n t l y reduced body weights on day 21 in comparison to t h e i r sedentary counterparts (p < 0.05). The l a t t e r observat ion was due to independent e f f e c t s of both d ie t (p < 0.001) and exerc ise (p < 0.001). Greater body weight on study day 21 was observed in exerc i s ing hamsters consuming low c h o l e s t e r o l d iets compared to those exercised and fed high c h o l e s t e r o l d iets although t h e i r food consumption was s l i g h t l y lower on average than the l a t t e r group. However, weight gain r e l a t i v e to body weight on day 1 ( f i n a l body weight - i n i t i a l body weight) of exerc ised hamsters was s i m i l a r between the two groups on average, and caused by an independent d ie t (p < 0.001) and exerc ise (p < 0.001) e f f e c t in comparison to r e s p e c t i v e sedentary counterparts. Percentage body fat of hamsters upon s a c r i f i c e is shown in Figure 7. No d i f fe rences were observed between sedentary (20.8 +_ 4.0%, X +_ SEM) and exerc ised (20.6 +_ 3.8%) hamsters fed low c h o l e s t e r o l d i e t s . S i m i l a r l y , percentage body fa t of sedentary animals (25.9 + 3.9%) consuming high c h o l e s t e r o l d i e t s was not d i f f e r e n t from exerc i s ing hamsters (17.6 +_ 2.9%) consuming high c h o l e s t e r o l d i e t s . i i ) Organ Weights and C h o l e s t e r o l Content Tab le 1 contains values of l i v e r and i n t e s t i n e weights and t h e i r t o t a l c h o l e s t e r o l content. Both d ie tary c h o l e s t e r o l (p < 0.001) and 41 FIGURE 7. Percent Body Fat of Hamsters c o LL "O o GD LOW CHOLESTEROL EXERCISE SEDENTARY HIGH CHOLESTEROL Data are group means ±SEM 42 TABLE 1. Weights and Cholesterol Content of Liver and intestine in Hamsters TREATMENT GROUP LOW C H O L E S T E R O L total weight (g) liver intestine total cholesterol liver intestine (mg) sedentary 4 . 2 7 a ' ° 1.49 ± 1 . 2 3 ± 0 . 0 8 7 . 4 8 ° 5 . 2 4 d i 0 . 5 6 ± 0 . 3 6 (n=12) exercise 3 . 9 2 ^ 1.45 ± 0 . 1 1 ± 0 . 0 8 6 . 8 7 C 5 .19^ ± 0 . 4 3 ± 0 . 3 6 (n=11) HIGH C H O L E S T E R O L sedentary 5 .24* 1.51 ± 0.14 £ 0 . 0 3 . 24.62 6.57 b 2.95 t 0 . 3 7 (n=16) exercise 4.63 1.55 ± 0 . 1 5 ± 0 . 2 9 22.37 6.46 ± 1.70 ± 0 . 4 9 (n=11) a - significantly different from corresponding exercise group , p < 0.001 b - significantly different from high .cholesterol-fed groups, p < 0.002 c - significantly different ftvm high cholesterol-fed groups, p < 0.001 d - significantly different from high cholesterol-fed groups, p < 0.003 Data are group means ± SEM t o t a l c h o l e s t e r o l content. Both d ietary c h o l e s t e r o l (p < 0.001) and exerc ise (P < 0.002) exerted independent e f f e c t s on t o t a l l i v e r weights. S p e c i f i c a l l y , among high c h o l e s t e r o l - f e d animals, sedentary and exerc i se groups had s i g n i f i c a n t l y higher (p < 0.05) l i v e r weights than those fed a low c h o l e s t e r o l d iet . Independent of the l a t t e r e f f e c t , sedentary animals had o v e r a l l higher l i v e r weights (p < 0.05) than exercised animals , although t h i s e f f e c t was l a r g e l y due to exerc ise in the high c h o l e s t e r o l -fed group. Smal 1 in tes t ine weights did not d i f f e r between any of the four experimental groups. When l i v e r weight was normalized to t o t a l body weight, high c h o l e s t e r o l - f e d animals again had a higher r a t i o of l i v e r weight to body weight (0.041 + .002) (+ SEM) versus low c h o l e s t e r o l - f e d hamsters (0.032 +.002). Tota l l i v e r c h o l e s t e r o l content was not a f fected by exerc ise in e i ther d iet group, but was s i g n i f i c a n t l y higher (p < 0.001) in hamsters consuming the high c h o l e s t e r o l d ie t . S i m i l a r l y , i n t e s t i n a l c h o l e s t e r o l content was increased (p < 0.002) by the high c h o l e s t e r o l d i e t , but was not affected by exerc ise . i i i ) PIasma C h o l e s t e r o l L e v e l s Plasma t o t a l and HDL c h o l e s t e r o l l e v e l s in hamsters are presented in Tab le 2. Plasma c h o l e s t e r o l was s i g n i f i c a n t l y and independently in f luenced by d i e t a r y c h o l e s t e r o l and phys ica l a c t i v i t y . High chol e s t e r o l - f e d animals had higher plasma t o t a l c h o l e s t e r o l (207.09 + 10.10 mg/dl) l e v e l s compared with low c h o l e s t e r o l - f e d animals (108.84 + 5.17 mg/dl) (p < 0.001), r e g a r d l e s s of the exercise s tate . Exerc ise was associated with a lowering of plasma c h o l e s t e r o l (p < 0.01) independent of d iet treatment, however 44 TABLE 2. Plasma Total and HDL of Hamsters Cholesterol Plasma Total HDL- HDLTotal Cholesterol Cholesterol Ratio TREATMENT GROUP (mg/dl) (mg/dl) LOW CHOLESTEROL " sedentary 1 14.2 •f a,b 4.12 09.7 + c.d 3.72 ' 79 b exercise 103.4 + b 6.06 64.9 d 3.19 b .63 HIGH CHOLESTEROL sedentary 226.1 a 9.62 93.9 c 3.83 .42 exercise 100.2 +_ 10.42 64.5 3.39 .45 ; a - significantly different from corresponding exercise group, p < 0.01 b - significantly different from high cholesterol fed groups, p < 0.001 c - significantly different from corresponding exercise group, p < 0.001 d - significantly different fmm high cholesterol fed groups, p < 0.004 Data are group means ± SEM (see Appendix C for sample size) the l a r g e r decrease observed in high c h o l e s t e r o l - f e d animals versus those consuming the low c h o l e s t e r o l d i e t might suggest an i n t e r a c t i v e e f f e c t (p < 0.13) fo r t h i s v a r i a b l e . HDL c h o l e s t e r o l , l i k e plasma t o t a l c h o l e s t e r o l , d i d show an o v e r a l 1 i n c r e a s e (77.35 + 3.6 to 89.23 + 2.8 m g / d l ) , (p < 0.003) in response to d ietary c h o l e s t e r o l and a decrease (74.74 + 3.4 from 91.83 + 3.9 mg/dl), (p < 0.001) in response to exercise. More predominant however was the observed i n t e r a c t i o n (p < 0.05) of these two f a c t o r s on HDL c h o l e s t e r o l l e v e l s . Exerc ise appeared to lower HDL c h o l e s t e r o l s i g n i f i c a n t l y (89.74 + 3.86 to 64.95 + 3.19 mg/dl) , (p < 0.05) o n l y i f the low c h o l e s t e r o l d iet was consumed. Consequently mean HDL c h o l e s t e r o l va lues for low (89.74 + 3.86 mg/dl) or high (93.92 + 4.09 mg/dl) c h o l e s t e r o l - f e d sedentary animals were not d i f f e r e n t from animals in the high c h o l e s t e r o l exercise group (84.54 +_ 3.56 mg/dl). i v ) C h o l e s t e r o l Synthetic Rate F igure 8 shows the rate of incorporat ion of ^ 0 into newly synthesized c h o l e s t e r o l (nmol/hr.g) of l i v e r and i n t e s t i n e in hamsters. Resu l ts ind icate that although l i v e r c h o l e s t e r o l synthesis was increased by d ie tary c h o l e s t e r o l (356.64 + 41 from 240.15 + 28 nmol/hr.g), (p < 0.009) and decreased by exerc ise (242.68 +_ 45 from 354.11 +_ 41 nmol/hr.g), (p < 0.02), i t was s i g n i f i c a n t l y modif ied by the i n t e r a c t i o n of these two f a c t o r s (p < 0.013). Only when sedentary animals consumed high c h o l e s t e r o l d i e t s d id they have incorporat ion rates that were higher (466.84 +_ 58 nmol/hr.g) than high c h o l e s t e r o l exerc ise (246.43 +_ 25 nmol/hr.g), low c h o l e s t e r o l sedentary (241 .38+24 nmol/hr.g), and low c h o l e s t e r o l exerc i se (238.92 + 32 nmol/hr.g), (p < 0.05) groups. Thus exerc i se 46 FIGURE 8.' Incorporation of  3 HO Into Cholesterol in Liver and Intestine of Hamsters 1400 L O W C H O L E S T E R O L HIGH C H O L E S T E R O L Data are group means ± SEM lowering of hepatic s te ro l synthesis was observed only in animals consuming high cho les te ro l d iets (p < 0.05), even though the o v e r a l l e f f e c t of adding c h o l e s t e r o l to the d iet was to increase hepatic c h o l e s t e r o l synthesis as compared with low c h o l e s t e r o l - f e d animals. In contrast to the l i v e r , the c h o l e s t e r o l synthet ic rate in the smal l i n t e s t i n e was af fected d i f f e r e n t l y by d i e t and exerc ise. Increasing d i e t a r y c h o l e s t e r o l s i g n i f i c a n t l y decreased i n t e s t i n a l s te ro l synthesis (768.20 + 52 from 1162.47 + 85 nmol/nr.g) whi le exerc ise had no e f f e c t (944.16 + 52 vs 986.51 + 71 nmol / h r . g ) , (p < 0.05). Figure 9 i l l u s t r a t e s the cumulat ive incorporat ion of " ^ 0 into l i v e r and i n t e s t i n e of hamsters. Animals fed the low c h o l e s t e r o l d iet changed ne i ther l i v e r nor i n t e s t i n a l rates of c h o l e s t e r o l synthesis with exerc i se . Hence, the net amount of c h o l e s t e r o l synthesized in each group by l i v e r and smal l i n t e s t i n e remained unchanged. In comparison to e i ther of the low c h o l e s t e r o l - f e d groups, high c h o l e s t e r o l - f e d sedentary animals synthesized more c h o l e s t e r o l in l i v e r , and less in i n t e s t i n e , p a r a l l e l l i n g the d i r e c t i o n of change in synthet ic rate observed in Figure 7. It appears as though the net increase in l i v e r c h o l e s t e r o l production was larger in magnitude than the decrease in i n t e s t i n a l synthesis . As a r e s u l t an increase in net c h o l e s t e r o l production in t h i s group was evident . Exerc ise in the same group did not change i n t e s t i n a l s t e r o l production, but decreased l i v e r der ived c h o l e s t e r o l to l e v e l s seen in the low c h o l e s t e r o l -f e d group. F igure 10 shows c h o l e s t e r o l synthesis in l i v e r and i n t e s t i n e expressed as a f r a c t i o n of the t o t a l c h o l e s t e r o l content with in each r e s p e c t i v e t i s s u e . High c h o l e s t e r o l - f e d animals showed lower hepat ic 48 FIGURE 9. Cumulative Incorporation of HsO Into Cholesterol of Total Liver and Intestine in Hamsters 5000 i : ~ ~ I L O W C H O L E S T E R O L H I G H C H O L E S T E R O L Data are group means FIGURE 10. Newly Synthesized Cholesterol Expressed as a Fraction of Total Cholesterol Content in Liver and Intestine of Hamsters SEDENTARY EXERCISE SEDENTARY EXERCISE LOW C H O L E S T E R O L HIGH C H O L E S T E R O L Data are group means s t e r o l synthesis (0.22 + 0.37 mg/g), (p < 0.001)-compared with low c h o l e s t e r o l - f e d animals (1.36 +_ 0.15 mg/mg t i s s u e c h o l e s t e r o l ) . No e f f e c t of exerc ise in t h i s group was observed on c h o l e s t e r o l synthesis . Diet s i m i l a r l y reduced i n t e s t i n a l c h o l e s t e r o l synthesis (6.30 +_ 1.05 from 12.99 +_ 1.39 mg/mg t i s s u e c h o l e s t e r o l ) , (p < 0.001) but no e f f e c t of exerc i se was evident . Covar ia te a n a l y s i s of factors i n c l u d i n g body weight gain, food consumption and d ietary f a t content did not s i g n i f i c a n t l y i n f l u e n c e the values obtained for synthet ic rate (nmol/hr.g) between exercised and non-exerc ised groups. In a d d i t i o n , normal izat ion of synthet ic rate to lean body mass did not s i g n i f i c a n t l y a l t e r the magnitude or d i r e c t i o n of the values obtained for hepatic or i n t e s t i n a l s t e r o l synthesis in F igure 8. 51 DISCUSSION: i) Body Weights and Food Intake Body weights of sedentary animals fed high and low c h o l e s t e r o l d i e t s increased at a s i m i l a r and constant rate even though food intake p r o g r e s s i v e l y dec l ined over the study per iod. S i m i l a r l y exerc ised animals , although consuming less food than t h e i r sedentary counterparts, gained weight at a r e l a t i v e l y constant rate u n t i l the l a s t week of study where body weights piateaued. These r e s u l t s were g e n e r a l l y in agreement with previous data showing a constant increase in body weight with age, even though food intake tended to diminish (65). Although not measured in the present study, i t has been suggested that in hamsters, both somatic growth and fat storage are responsib le for t h i s weight gain over time. It was not expected however that exercised animals would consume less food than sedentary groups. F r e e l y running hamsters normally consume more food compared with sedentary c o n t r o l s of s i m i l a r age (66). Given this observat ion , i t was thought that pa i r - feed ing exercised animals to sedentary c o n t r o l s , combined with manipulation of energy expenditure, would r e s u l t in r e l a t i v e l y constant body weights of exerc i s ing hamsters over the study per iod , counteract ing the normal rate of weight gain in sedentary animals. This design was used in order to e l iminate any confounding by weight gain that might have clouded true exercise e f fec ts on c h o l e s t e r o l synthesis or other v a r i a b l e s . Constant body weights, however, were not achieved - in exercise.groups u n t i l the l a s t study week, when exerc ise i n t e n s i t y 52 highest. During the f i r s t study week increases in weight gain of<exercised groups were s i m i l a r to sedentary animals. One would have expected exerc ised hamsters, with lower food intakes and ramped l e v e l s of exerc ise i n t e n s i t y , to have immediately shown a decce le ra t ion in weight gain. It has been shown, however, that hamsters respond to disturbances in energy balance not by changing patterns of food ingest ion , but by decreasing basal metabol ic rate (BMR) (67). For example, an animal weighing 10% l e s s than i t s p a i r - f e d control would be expected to decrease metabol ic rate by 25% (67). Thus exerc i s ing hamsters may have been ab le to compensate i n i t i a l l y for increases in energy expenditure by lowering BMR. During the penult imate and f i n a l weeks of the study, BMR changes may not have been s u f f i c i e n t to overcome the energy d e f i c i t imposed by the i n t e n s i t y of t h e . exerc ise regimen. BMR was not tested throughout the study to support t h i s theory, however maintenance of consistent body weights was achieved on average, in exerc ised groups as o r i g i n a l l y intended. A p o s s i b l e improvement in the study design would have been to have inc luded an exerc ise group in which animals were a l lowed f ree access to food. Such a group would, in addit ion to prov id ing information regarding the changes in l i p i d metabolism due to weight gain as descr ibed above, be more representat ive of a f r e e - l i v i n g human subject undergoing exerc ise . It became obvious that inc lud ing an ad 1 ibitum fed exerc ise group would not have been d i f f e r e n t from the p a i r - f e d regime used. This was because the exerc ise groups, although p a i r - f e d , d id not always ingest a l l t h e i r food as ant i c ipated . In add i t ion , the exerc ise group fed the low c h o l e s t e r o l d i e t gained less weight than the exerc ise group fed the high c h o l e s t e r o l d i e t , even though the l a t t e r group consumed more food on average. It is l i k e l y that the stress imposed by running, or perhaps 53 longer s leep periods and less feeding time in th i s group, caused a decrease in food intake by exercised animals. However, neither d ietary f a t nor t o t a l food intake d i f ferences between any experimental group exerted s i g n i f i c a n t e f f e c t s on measured parameters of l i p i d metabolism as tested with a n a l y s i s of covar iance procedures. i i ) Body Composition, Organ Weights and Organ Cholestero l Content Percentage body fat was not d i f f e r e n t between sedentary and exerc ise groups, although a trend towards lower values was noted in the high c h o l e s t e r o l - f e d exercise group. These r e s u l t s were in opposit ion to studies which showed a decrease in body fa t of exerc is ing hamsters r e l a t i v e to sedentary contro ls (65,66). Subject ive v i sua l examination at s a c r i f i c e suggested less fa t was deposited in the v i scera of exerc ised hamsters compared with sedentary counterparts. The ind i rect method of water d i l u t i o n space used to determine percentage body fat is w e l l proven (61,62). Thus, procedural problems may have been a cause for the v a r i a b i l i t y in observed body fa t with in groups. It was noted on occasion that , f o l l o w i n g in t raper i tonea l i n j e c t i o n , a drop of t r i t i a t e d water would emerge from the in ject ion s i t e a f te r removal of the needle. This would have the e f fec t of increasing the apparent amount of unabsorbed l a b e l thus reducing the apparent body fa t content. I f such loss of l abe l occurred in a non-random fash ion , body composition d i f ferences between groups may have been obscured by the large v a r i a b i l i t y . L i v e r weights were increased in high c h o l e s t e r o l - f e d animals , but decreased in exercised animals of e i ther d ie t treatment. C h o l e s t e r o l 54 content of l i v e r was a l s o increased in high c h o l e s t e r o l - f e d animals, but was not a f fected by exerc ise in e i ther the high or low chol e s t e r o l - f e d groups. The increased l i v e r weights in high c h o l e s t e r o l - f e d animals were l i k e l y due to accumulation of cho les tero l in t h i s t i s s u e (71,72). Higher observed rates of hepatic c h o l e s t e r o l synthesis would a l s o have contr ibuted to the increase in t i s s u e c h o l e s t e r o l and weight. It i s not known whether greater f a t depos i t ion occurs in the l i v e r when high c h o l e s t e r o l d i e t s are consumed, but t h i s may a l s o have occurred. Prolonged exerc ise in excess of one hour causes dep le t ion o f l i v e r glycogen stores (69,70). Moreover, exogenous g lucose is taken up predominantly by muscle to rep le te i t s glycogen s tores , rather than l i v e r as in the r e s t i n g s tate (70). Perhaps the decrease in l i v e r weights of exercised hamsters was due to loss of glycogen and not to increased removal of exogenous c h o l e s t e r o l from t h i s t i s s u e . In contrast to l i v e r , i n tes t ine weight was not increased in animals consuming the high c h o l e s t e r o l d ie t . The smal l i n t e s t i n e is a medium through which c h o l e s t e r o l enters the body and although th i s organ a s s i s t s in r e g u l a t i o n of whole body s t e r o l balance, i t is not a center fo r process ing or storage of c h o l e s t e r o l . Lack of an exerc i se e f fect on i n t e s t i n e weight was consistent with the knowledge that i n t e s t i n e , u n l i k e the l i v e r , does not store glycogen and is not r e s p o n s i b l e for u t i l i z i n g glycogen as an energy source to regulate energy balance during exerc ise . Even though t o t a l weight of the in test ine did not change in high c h o l e s t e r o l - f e d animals, c h o l e s t e r o l content was s i g n i f i c a n t l y e l e v a t e d . In add i t ion to absorption of exogenous f ree c h o l e s t e r o l , 1% of c i r c u l a t i n g LDL is taken up by the smal l in test ine (19). Thus a t rans ient increase of 55 c h o l e s t e r o l content by exogenous absorpt ion, as w e l l as a net increase in uptake of e x i s t i n g e levated plasma LDL l e v e l s may have caused the t o t a l c h o l e s t e r o l content of t h i s t i s s u e to r i s e without apprec iab ly changing t o t a l t i s s u e weight. i i i ) Tota l Plasma Cho les tero l In the present study hamsters consuming a high c h o l e s t e r o l d i e t exh ib i ted t o t a l plasma c h o l e s t e r o l l e v e l s 48% higher than those of low c h o l e s t e r o l - f e d animals. While exerc ise had no e f f e c t on plasma t o t a l c h o l e s t e r o l in the l a t t e r group, a 15% decrease was seen in plasma t o t a l c h o l e s t e r o l in high chol e s t e r o l - f e d exercised hamsters. These r e s u l t s were s i m i l a r to other f ind ings of e i ther c h o l e s t e r o l induced increases (20,72,71) or exerc ise induced decreases (22,11,12) in t o t a l plasma c h o l e s t e r o l l e v e l s . P resent ly , no i n t e r a c t i v e e f fec t between d ie tary c h o l e s t e r o l and exerc ise on t h i s v a r i a b l e was observed. C h o l e s t e r o l feeding has been s p e c i f i c a l l y shown to cause an increase in net i n t e s t i n a l c h o l e s t e r o l absorpt ion, f o l l o w e d by a dose- dependent suppression in rates of receptor-mediated hepatic LDL c learance , and a r e c i p r o c a l increase in plasma LDL concentrat ion (21). In the hamster, approximately 45% of c i r c u l a t i n g c h o l e s t e r o l is car r ied in the LDL s u b f r a c t i o n , wh i le HDL c a r r i e s near ly 50% (72). In the present experiment i t was reasonable to assume that LDL was respons ib le fo r the observed increase in plasma c h o l e s t e r o l , s ince the magnitude of change of HDL would not have accounted for the large increase in t o t a l plasma c h o l e s t e r o l . HDL increased in plasma of high c h o l e s t e r o l - f e d animals by 14% when compared 56 to the low chol e s t e r o l - f e d group. This increase may have r e s u l t e d from an increased production of HDL precursors , most l i k e l y nascent HDL from l i v e r and i n t e s t i n e , and chylomicron or VLDL remnants. An increase in HDL production may r e f l e c t the body's attempt to regulate whole body c h o l e s t e r o l ba lance, since a primary funct ion of HDL is to carry excess c h o l e s t e r o l from the periphery to the l i v e r v i a "reverse c h o l e s t e r o l t ransport" , p r e v i o u s l y descr ibed. The d i r e c t e f fec t of c h o l e s t e r o l feeding on t o t a l plasma c h o l e s t e r o l l e v e l may have been exacerbated by the addi t ion of corn o i l to the d i e t . Suppression of LDL receptor synthesis and i n t r a c e l l u l a r c h o l e s t e r o l ester format ion, caused by fat feeding, have been shown to e l e v a t e plasma LDL l e v e l s (21). A l s o , carcass c h o l e s t e r o l content increases in exerc ised versus sedentary animals (25). Thus reduction in plasma c h o l e s t e r o l might have been expected in the low c h o l e s t e r o l exercise group, i f c e l l u l a r demands for c h o l e s t e r o l were increased during exerc ise. However, on ly a trend towards lower plasma c h o l e s t e r o l was observed. As with hepat ic s t e r o l synthesis in the l a t t e r group, i t is l i k e l y that plasma l e v e l s were maintained even though pools may have been depleted. It would have been usefu l to extend the durat ion of the study to eva luate the a b i l i t y of balance mechanisms to preserve plasma c h o l e s t e r o l l e v e l s . The exerc ise induced lowering of plasma c h o l e s t e r o l in high c h o l e s t e r o l - f e d hamsters p a r a l l e l e d the decrease in hepatic synthet i c rate observed wi th in the same group. Although the magnitude of change in plasma c h o l e s t e r o l was less than synthet ic rate (19% vs. 53%) the change in l e v e l might be expla ined by a reduct ion in synthesis . L imited substrate a v a i l a b i l i t y induced by exerc ise a c t i v i t y may r e s u l t in p r o p o r t i o n a t e l y l e s s c h o l e s t e r o l synthesis by the l i v e r . Even though d ietary c h o l e s t e r o l 57 would be present in high enough quant i t ies to counteract th i s e f f e c t , increased per iphera l membrane and t i ssue demand for c h o l e s t e r o l , imposed by the exerc ise paradigm, may have increased i t s i n f l u x to these compartments. As a consequence the molar r a t i o of f ree c h o l e s t e r o l to c e l l u l a r components, such as the phosphol ip id component of membranes, may have been reduced, causing an e f f l u x of c h o l e s t e r o l from the plasma into the per iphery . i v ) Plasma High Density L ipoprote in C h o l e s t e r o l L e v e l s HDL c h o l e s t e r o l (HDL-C) concentration in plasma showed a net increase in animals fed the high cho les te ro l d i e t , compared to low c h o l e s t e r o l - f e d animals, although the r a t i o of HDL:total plasma c h o l e s t e r o l decreased by 41% in the l a t t e r group. In a d d i t i o n , exerc ise caused an o v e r a l l decrease in plasma HDL l e v e l s independent of d i e t . The main e f f e c t however, was the observed i n t e r a c t i o n between d i e t and exerc i se where exerc ise lowered HDL c h o l e s t e r o l only when animals consumed the low c h o l e s t e r o l d ie t . In no instance did exerc ise independently or through an i n t e r a c t i o n with d ietary c h o l e s t e r o l , increase HDL c h o l e s t e r o l l e v e l s . Since exerc ise only s l i g h t l y increased HDL c h o l e s t e r o l l e v e l s in high c h o l e s t e r o l - f e d hamsters, the d i spropor t ionate ly large increase in plasma c h o l e s t e r o l l e v e l s was most l i k e l y respons ib le for the decreased r a t i o of HDL-C to t o t a l plasma c h o l e s t e r o l in th i s group. F a i l u r e of exercise to d i r e c t l y increase plasma HDL-C was cons is tent with r e s u l t s seen in s i m i l a r exercise studies . Tsai et a l . (22) observed no d i f f e r e n c e in e i t h e r HDL-C or HDL-C:total plasma c h o l e s t e r o l r a t i o in 58 hamsters run v o l u n t a r i l y fo r 30 days and fed low c h o l e s t e r o l d i e t s . S i m i l a r l y , exerc ise did not a l t e r HDL-C:total plasma cho les tero l r a t i o in exerc ised rats fed high or low c h o l e s t e r o l d iets (73,17). Pels et a l . found a s i g n i f i c a n t decrease in HDL-C, but no change in HDL-C:total plasma c h o l e s t e r o l r a t i o in rats fed high c h o l e s t e r o l , high fat d iets and t r a i n e d to run at 70% and 85% of V 0 2 max in comparison to sedentary c o n t r o l s (11). These and other studies in rats (73,17) have c o n s i s t e n t l y shown lower HDL-C in exerc ised animals even though the inter-study exercise condit ions have var ied considerably . In the present study, the observed i n t e r a c t i v e e f fec t of d i e t and exerc i se on HDL-C was i n t r i g u i n g , when one considers the independent e f f e c t s of exerc ise and d ietary c h o l e s t e r o l l e v e l s . If exercise were decreasing endogenous c h o l e s t e r o l synthesis by reducing substrate a v a i l a b i l i t y , then i t is p o s s i b l e that exerc is ing animals fed low c h o l e s t e r o l d ie ts might be in negative c h o l e s t e r o l balance. Increased c e l l u l a r demands for c h o l e s t e r o l would not be met by the low c h o l e s t e r o l content of the d i e t and consequently no compensation for a decrease in c h o l e s t e r o l synthesis could be achieved. The formation of HDL from VLDL and chylomicron remnants would be reduced under the pa i r - fed imposit ion of both energy and d ietary c h o l e s t e r o l s imply due to a decrease in t h e i r product ion. Thus the amount of excess c i r c u l a t i n g cho les te ro l would be l e s s and the funct iona l need for HDL to remove cho les tero l attenuated. Furthermore, i f HDL-C does slow LDL-mediated i n t e r n a l i z a t i o n of c h o l e s t e r o l into the c e l l , then i t s presence would not be d e s i r a b l e under condi t ions inducing c e l l u l a r c h o l e s t e r o l d e f i c i t . Thus, although exerc ise d id not independently increase HDL-C, the in te rac t ion between d ie tary c h o l e s t e r o l and exerc ise was s i g n i f i c a n t . When considered together with 59 the observat ion that plasma t o t a l c h o l e s t e r o l was reduced with e x e r c i s e , t h i s f i n d i n g suggests that the changes in HDL c h o l e s t e r o l may be secondary to changes in plasma c h o l e s t e r o l and may only r e f l e c t t h e i r d i r e c t f u n c t i o n a l importance in s te ro l metabolism, not an independent e f f e c t of e x e r c i s e . No d i f f e r e n c e in percentage body fa t was noted between exerc ised and sedentary hamsters. Therefore the e f f e c t of greater f a t loss and increased l i p o p r o t e i n l i p a s e a c t i v i t y often observed with exerc ise t r a i n i n g and thought to i n d i r e c t l y cause an increase in c i r c u l a t i n g HDL l e v e l s (55), cannot be eva luated in the present study. One might conclude from the present and aforementioned studies that HDL production i s not increased by phys ica l exerc ise per se. It has been shown that development of a t h e r o s c l e r o t i c l es ions was attenuated in exerc ised rats compared with sedentary c o n t r o l s even though average HDL-C concentrat ions were lower and HDL-C:total plasma c h o l e s t e r o l remained unchanged. These data suggest that although exerc ise may b e n e f i c i a l l y re tard l e s i o n development, the e f f e c t may not be d i r e c t l y mediated by changes in the l e v e l of HDL as p r e v i o u s l y suggested. It was thought that the hamster would be a usefu l model with which to study human c h o l e s t e r o l metabolism. There is increas ing evidence that the hamster is more s u i t a b l e than the ra t for studies of c h o l e s t e r o l metabolism s ince endogenous synthet ic rate (2.5 mg/day/100 g body weight) resembles that of the human (1 mg/day/100 g body weight) more c l o s e l y than the rat (12 mg/day/100 mg body weight) (18). Studies in humans have shown a dose-response r e l a t i o n s h i p between the amount of exerc i se t r a i n i n g and degree of change of plasma HDL (74). 60 Unfortunate ly many of the studies in th i s area cannot d i s t i n g u i s h between true chronic adaptive changes in l i p o p r o t e i n s from an acute or t r a n s i e n t response, due to poor control on i n t e n s i t y , duration and type of e x e r c i s e , s tate of t r a i n i n g , d ietary intakes and base l ine measurements. Consequently the evidence for (75,76,77) and against (78,79,80) a p o s i t i v e e f f e c t of exerc ise on HDL in humans is c o n t r o v e r s i a l and d i f f i c u l t to eva luate . It might have been useful in the present study to have determined b a s e l i n e HDL-C va lues for each hamster at the s t a r t of the study so as to have measured the magnitude of change in each animal. This approach might have e l iminated some of the v a r i a b i l i t y in absolute plasma HDL c h o l e s t e r o l l e v e l s measured. Human studies however have f a i l e d to show any s i g n i f i c a n t c o r r e l a t i o n s of base l ine HDL-C with degree of HDL-C change (74). In a d d i t i o n , i t is poss ib le that the study was of i n s u f f i c i e n t durat ion for adapt ive changes to have occurred. Many human studies that support an exerc ise- induced increase in HDL c h o l e s t e r o l examined exerc is ing a t h l e t e s over many years. Thus these data ind icate that exerc ise does not increase HDL-C l e v e l s independently in the hamster, an observat ion that may serve to dispute the notion that exerc ise induces changes in HDL that d i r e c t l y , or i n d i r e c t l y decrease the r i s k of c a r d i o v a s c u l a r disease. Hamsters did however appear to respond s i m i l a r l y to humans consuming high fat and c h o l e s t e r o l d i e t s , by increas ing s l i g h t l y the production of HDL (75), although the percentage of d ie tary fa t consumed in the human studies was greater than the present study. v) F r a c t i o n a l Synthetic Rate of C h o l e s t e r o l C h o l e s t e r o l synthesis expressed as that f r a c t i o n of t i s sue 61 c h o l e s t e r o l which was newly synthesized showed a s i g n i f i c a n t decrease in both l i v e r and i n t e s t i n e in response to the high c h o l e s t e r o l d i e t , wh i le exerc i se had no e f f e c t on t h i s v a r i a b l e . F r a c t i o n a l synthet ic rate provides a measure of how synthesis of c h o l e s t e r o l wi th in a def ined per iod r e l a t e s to the f r e e l y exchangeable pool of c h o l e s t e r o l within s p e c i f i c t i s s u e s . Previous studies have shown that hamsters respond to d ietary c h o l e s t e r o l by increas ing organ uptake of c h o l e s t e r o l , p r i m a r i l y as LDL, with consequent organ weight gains (81). The observed decrease in FSR of i n t e s t i n e was most l i k e l y due to both the reduction of endogenous c h o l e s t e r o l synthes is , as w e l l as increased uptake of c h o l e s t e r o l into t h i s organ. Since rates of LDL uptake were not measured, i t is imposs ib le to t e l l whether c h o l e s t e r o l uptake was larger in t h i s organ than in l i v e r . It is however reasonable to assume that the larger % increase (19 vs 2%) in organ weight of l i v e r over i n t e s t i n e of high chol e s t e r o l - f e d animals was due mainly to greater accumulation of f ree c h o l e s t e r o l in th is t i s s u e . Approximately 25-50% of l i v e r weight gained could be d i r e c t l y a t t r i b u t a b l e to an increase in c h o l e s t e r o l content. The magnitude of th is increase must have been la rger than the increase in l i v e r synthet ic ra te , s ince the trend favoured a reduct ion in FSR in high chol e s t e r o l - f e d animals. In a d d i t i o n , the observed decrease in average body weight of exerc ised animals in comparison to sedentary c o n t r o l s did not a f fec t l i v e r weight, support ing the idea that c h o l e s t e r o l was the primary contr ibutor to increased l i v e r weights in high c h o l e s t e r o l - f e d animals. In the hamster, the smal l i n t e s t i n e might be considered as more important compared with l i v e r in regu la t ing c h o l e s t e r o l balance on a low 62 c h o l e s t e r o l d ie t for severa l reasons. F i r s t l y , basal synthet ic rates in i n t e s t i n e were twice as high as those of l i v e r , regardless of d ie tary c h o l e s t e r o l content. Improvement on techniques for eva luat ing c h o l e s t e r o l synthesis have indicated that a much larger proport ion of whole body synthesis of c h o l e s t e r o l occurs in the smal l i n t e s t i n e , in both man and hamster (18), than p r e v i o u s l y b e l i e v e d . These f ind ings are supported by the notion that the in tes t ine is the s i t e at which the body f i r s t contacts c h o l e s t e r o l and thus attempts to regulate i t s balance. The l i v e r may be q u a n t i t a t i v e l y l ess important when intake of c h o l e s t e r o l is low. In t h i s s i t u a t i o n , exerc ise apparently exerts no s i g n i f i c a n t a l t e r a t i o n of s t e r o l synthesis in e i t h e r organ. v i) Hepatic and In tes t ina l Cho lestero l Synthesis Dietary c h o l e s t e r o l content and exerc ise a c t i v i t y were observed to cause an increase and decrease r e s p e c t i v e l y in the average rate of hepat ic c h o l e s t e r o l synthesis (nmol/hr.g) in hamsters. S p e c i f i c a l l y , d ie tary c h o l e s t e r o l increased hepatic synthesis in high c h o l e s t e r o l - f e d animals , wh i le exerc i se had the opposite e f fect . Exercised hamsters fed the low c h o l e s t e r o l d i e t however did not a l t e r hepatic or i n t e s t i n a l c h o l e s t e r o l synthes is . Such inconsistency between group responses to d ietary c h o l e s t e r o l and physica l a c t i v i t y was expla ined by t h e i r s i g n i f i c a n t i n t e r a c t i o n , through which the various d ie t and exercise l e v e l s exerted more d e t e c t a b l e p h y s i o l o g i c a l e f fects in combination, than when considered a lone. The i n t e r a c t i v e e f fec t of d iet and exerc ise was most obvious in l i v e r s of high c h o l e s t e r o l - f e d animals, where s t e r o l synthesis was lowered 63 in response to exerc ise . Expla ined d i f f e r e n t l y , only sedentary animals consuming a high c h o l e s t e r o l d i e t showed increased rates of t r i t i u m incorporat ion into l i v e r t i s s u e . Hamsters fed the low c h o l e s t e r o l d ie t d id not a l t e r hepatic s t e r o l synthesis in response to exerc ise . Very few studies have examined the e f f e c t of prolonged phys i ca l exerc i se on hepatic s t e r o l synthesis in animals and i t appears that an i n t e r a c t i o n between d i e t and exerc ise factors on t h i s v a r i a b l e has not p r e v i o u s l y been tested. Takashi et a l . (13) used [14]C-mevalonate to measure c h o l e s t e r o l synthesis in rats that were exerc ised at 60-75% VO2 max for two weeks and fed ad 1ibitum d ie ts with no added c h o l e s t e r o l . A s i g n i f i c a n t increase was observed in hepatic s t e r o l synthesis of e x e r c i s i n g rats in comparison to sedentary c o n t r o l s . This r e s u l t was not cons is tent with those of e x e r c i s i n g hamsters in the present study where no change in hepatic s t e r o l synthesis occurred in response to exerc ise in the low c h o l e s t e r o l group. V a l i d comparison with the l a t t e r study becomes d i f f i c u l t fo r severa l reasons as stated in the Introduct ion. Hence the a b i l i t y of a rat to adapt to disturbances in energy balance that might i n f l u e n c e c h o l e s t e r o l formation make i t an incomparable model to the hamster (20), which more c l o s e l y resembles the human (18) in t h i s regard. v i i ) P o s s i b l e Mechanisms An explanat ion of the mechanisms, of d ie t and exerc ise induced e f f e c t s , independently and i n t e r a c t i v e l y , requires examination of fac tors which are primary regu la tors of body s t e r o l balance. These inc lude a) c h o l e s t e r o l absorpt ion, b) endogenous s t e r o l synthes is , c) b i l e ac id 64 formation and d) hormonal c o n t r o l . a) I n t e s t i n a l Absorption and C h o l e s t e r o l Homeostasis I n t e s t i n a l absorption of c h o l e s t e r o l was not measured in the present study, however i t is u n l i k e l y that exerc ise caused a decrease in absorpt ion of cho les te ro l from the smal l i n t e s t i n e of low c h o l e s t e r o l - f e d animals. In genera l , the e f f e c t of intense phys ica l a c t i v i t y over the long term would be to increase membrane turnover , p a r t i c u l a r l y in the g a s t r o i n t e s t i n a l t rac t (82) and thus the requirement for c h o l e s t e r o l . This would be expected to cause e i ther an increase or no change, in absorpt ion e f f i c i e n c y of cho les tero l in the absence of any other exercise induced i n t e s t i n a l disturbance. If , however, an increase in absorption had occurred in low cho les tero l exercised hamsters, the amount would have been smal l g iven the low cho les te ro l content of the d i e t , and probably not have i n h i b i t e d hepatic or i n t e s t i n a l s t e r o l synthesis s i g n i f i c a n t l y . In c o n t r a s t , high c h o l e s t e r o l - f e d animals should have responded d i f f e r e n t l y to the la rge i n f l u x of c h o l e s t e r o l from the d i e t whether exercised or not. Dietary c h o l e s t e r o l i s known to cause an increase in net absorption of c h o l e s t e r o l from the smal l i n t e s t i n e (19). Although the rate of absorpt ion tends to plateau as d ietary concentrat ion increases, i t has been shown that up to 40 % of c i r c u l a t i n g c h o l e s t e r o l in the plasma can be from exogenous sources (83). The i n h i b i t i o n of hepatic and i n t e s t i n a l c h o l e s t e r o l synthesis in response to d ie tary c h o l e s t e r o l has been seen in the hamster (20) and other animals (48), although the magnitude of the response to d ietary cho les tero l is often l e s s in the in test ine than in the l i v e r (48,84). In any event, the expected increase in absorption of 65 c h o l e s t e r o l due in large part to excess d ietary c h o l e s t e r o l should have i n h i b i t e d both hepatic and i n t e s t i n a l s t e r o l synthesis in th i s group (20). Thus i t was i n t r i g u i n g to f i n d that only the small i n t e s t i n e decreased synthet i c rate in response to d ietary c h o l e s t e r o l , whereas increases were seen in l i v e r . I n t e s t i n a l synthesis of c h o l e s t e r o l occurs p r i m a r i l y in e n d o t h e l i a l crypt c e l l s to r e p l e n i s h c e l l membranes of r a p i d l y p r o l i f e r a t i n g g a s t r o i n t e s t i n a l t i s sue . C e l l v i l l i a l so synthesize c h o l e s t e r o l , most l i k e l y to s t a b i l i z e chylomicrons and other l i p o p r o t e i n s during t r i g l y c e r i d e absorption (85). Reduction of i n t e s t i n a l s t e r o l synthesis occurs d i r e c t l y by feedback i n h i b i t i o n of HMG CoA reductase by the presence of f ree c h o l e s t e r o l from the d iet and b i l e acids (85). The mechanism for decreased HMG-CoA reductase a c t i v i t y may inc lude both an immediate i n a c t i v a t i o n of preformed enzyme and a longer term reduct ion of enzyme synthesis (86). As w e l l , subsequent enzymes in the synthet ic pathway between mevalonate and squalene may a l s o be reduced with prolonged d i e t a r y c h o l e s t e r o l feeding (85). In the present experiment, feeding a high c h o l e s t e r o l d ie t most l i k e l y caused a net increase in c h o l e s t e r o l absorpt ion and subsequent increase in the formation of chylomicron remnants, very low density l i poprote ins (VLDL), low density l i p o p r o t e i n s (LDL) and b i l e ac ids . These components i n h i b i t HMG CoA reductase (30) and endogenous synthesis of c h o l e s t e r o l in the i n t e s t i n e . b) Endogenous C h o l e s t e r o l Synthesis It has been e s t a b l i s h e d in hamsters that increases in the hepat ic 66 c h o l e s t e r o l pool f o l l o w i n g c h o l e s t e r o l feeding r e s u l t in compensatory r e g u l a t i o n of body c h o l e s t e r o l balance in s e v e r a l ways. F i r s t l y , hepatic and i n t e s t i n a l c h o l e s t e r o l synthesis is reduced. Secondly, secret ion by l i v e r of newly synthesized and absorbed c h o l e s t e r o l into b i l e or b i l e acids is increased. As w e l l , suppression of hepatic s t e r o l synthesis is thought to be s t imulated by an increase in receptor-dependent LDL uptake (21,30). It was therefore surpr i s ing that an increase in hepatic synthesis was observed in response to d ietary c h o l e s t e r o l . Other experimental manipulat ions in hamsters have e l i c i t e d s i m i l a r increases in hepat ic s t e r o l synthes is . It was found that t r i g l y c e r i d e feeding in hamsters d is rupted the c l a s s i c a l metabol ic responses to c h o l e s t e r o l feeding in the l i v e r . LDL receptor a c t i v i t y , cho les te ro l ester formation and c h o l e s t e r o l synthes is were af fected (21). For example, with c h o l e s t e r o l feeding, e l e v a t e d saturated t r i g l y c e r i d e intakes have been shown to increase rates of hepatic s t e r o l synthesis in s i tuat ions where receptor-dependent LDL t ransport was suppressed. Furthermore, t h i s response was shown to increase as the r a t i o of c h o l e s t e r o l to fa t in the d i e t increased, and more important ly , to be reversed i f a d iet low on these l i p i d s , or with a lower r a t i o , was fed (21). Thus in the present study, moderately e levated fa t intakes may e x p l a i n why only high c h o l e s t e r o l - f e d sedentary animals , with a higher r a t i o of c h o l e s t e r o l to fa t exhib i ted e levated rates of l i v e r s t e r o l synthesis compared with low c h o l e s t e r o l groups. The mechanism for t h i s response is s p e c u l a t i v e . It has been suggested that saturated f a t , being a poor substrate fo r e s t e r i f i c a t i o n r e a c t i o n s , r e s u l t s in lower accumulation of i n t e r c e l l u l a r c h o l e s t e r o l es ters , causing a decrease in the synthesis of LDL receptors d isproport ionate to need (21). Normal ly , synthesis of the LDL receptor i t s e l f is under feedback 67 r e g u l a t i o n so that i t s a c t i v i t y , hence the amount of c h o l e s t e r o l enter ing the c e l l , is i n v e r s e l y proport iona l to c e l l u l a r cho les te ro l content (30). The a b i l i t y of c e l l s to accumulate c h o l e s t e r o l may be the e s s e n t i a l element in regu la t ing s tero l synthes is . Thus, under the present ly descr ibed cond i t ion , regulatory pools may react to decreased receptor synthesis and a c t i v i t y by compensatory increases in c e l l u l a r rates of s t e r o l synthesis in the face of e levated LDL c h o l e s t e r o l (21). In a d d i t i o n , high f a t diets have been shown to e l e v a t e plasma c h o l e s t e r o l as d ie tary c h o l e s t e r o l content increases (87). Direct a p p l i c a t i o n of t h i s mechanism to r e s u l t s of the present study however remains d i f f i c u l t s ince corn o i l , a predominantly unsaturated f a t , was consumed with two l e v e l s of d ie tary c h o l e s t e r o l . Unfortunately the e f f e c t of corn o i l feeding in conjunct ion with c h o l e s t e r o l was not examined in the study by Spady et a l . (21). As w e l l , the l e v e l of f a t in th i s study was moderately high (8%) but not as high as the former study (20%). An a l t e r n a t i v e explanat ion for the large increase in hepatic s t e r o l synthesis of high c h o l e s t e r o l - f e d animals was that the l i v e r may have responded to the large decrease observed in i n t e s t i n a l c h o l e s t e r o l output by increas ing i t s own s t e r o l synthes is , independent of normal regu la tory processes. S u r p r i s i n g l y , the t o t a l amount of cho les tero l synthesized in t h i s group was 20 % higher than any other group, even though i n t e s t i n a l synthesis was d r a m a t i c a l l y reduced. Such an overcompensation, with no obvious metabol ic c o n t r o l , has been seen in obese hamsters, or those s u f f e r i n g from essent ia l f a t t y ac id d e f i c i e n c y , however the reason for t h i s was unclear (88). Although animals in the present experiment were not obese or presumably not s u f f e r i n g from amino acid de f i c i ency , one might 68 conclude that hamsters are g e n e r a l l y s u s c e p t i b l e to a l t e r a t i o n s in l i v e r s t e r o l synthesis under a v a r i e t y of experimental condit ions. If the unexpected r e s u l t in hepatic s t e r o l synthesis was not p h y s i o l o g i c a l and instead caused by some unknown, uncontro l led v a r i a b l e , then one, would have expected to see s i m i l a r responses in the low and high c h o l e s t e r o l - f e d sedentary groups. Since t h i s was not observed, and s ince d i e t a r y c h o l e s t e r o l was the only a l t e r e d v a r i a b l e between the two sedentary groups, i t is p o s s i b l e that the r a t i o of d ietary c h o l e s t e r o l and d i e t a r y f a t , which increased on the high c h o l e s t e r o l d i e t , was a true cause f o r the d i f fe rence . In a d d i t i o n , i t is conce ivab le that some of the experimental animals were unduly exposed to s t ress . P h y s i o l o g i c a l s t ress is a condit ion known to cause an increase in hepatic c h o l e s t e r o l synthesis through the act ion of epinephrine (88) and may have been an o v e r r i d i n g factor in causing u n c h a r a c t e r i s t i c c h o l e s t e r o l synthesis in the l i v e r . Sedentary animals fed the high c h o l e s t e r o l d iet were handled a great deal on the l a s t study day, and although the l e v e l of handl ing was not d i f f e r e n t from that r e c e i v e d by the exerc ise group, sedentary animals were not handled nearly as much as those of the exerc ise group e a r l i e r during the experimental per iod. Thus excess ive handl ing or disturbance of normal d a i l y patterns may have caused the unexpected, and p o s s i b l y t rans ient response in l i v e r s of the sedentary, high c h o l e s t e r o l - f e d group. Why low c h o l e s t e r o l - f e d sedentary animals would not have s i m i l a r l y responded is not c l e a r . E l e v a t e d l i v e r s t e r o l synthesis in response to cho les te ro l feeding may w e l l be expla ined by the high r a t i o of c h o l e s t e r o l to fat in the d i e t . However, e l u c i d a t i o n of the i n t e r a c t i v e mechanism by which l i v e r lowered c h o l e s t e r o l synthesis in h igh, but not low c h o l e s t e r o l - f e d animals, 69 remains d i f f i c u l t . Understanding how a d iet and exerc ise i n t e r a c t i o n produced t h i s e f f e c t may be f a c i l i t a t e d by f i r s t examining why exerc i se , in absence of d ietary c h o l e s t e r o l e f f e c t s , caused a f a l l in l i v e r synthesis in the high chol e s t e r o l - f e d group, A p o s s i b l e mechanism by which exerc ise may reduce l i v e r c h o l e s t e r o l synthesis is through l i m i t i n g the a v a i l a b i l i t y of c h o l e s t e r o l precursor substrate during exercise. Formation of HMG CoA requires an adequate supply of precursor acety l CoA in the c e l l c y t o s o l , and therefore i n h i b i t i o n of th i s supply of acety l CoA would depress the rate of s t e r o l synthes is . Prolonged exerc ise , as described e a r l i e r , causes d i v e r s i o n of acety l uni ts into pathways of energy metabolism and f a i l s to provide an abundance of t h i s substrate for f a t t y acid or s t e r o l synthesis . It has indeed been hypothesized that HMG CoA reductase, the r a t e - l i m i t i n g enzyme for c h o l e s t e r o l synthesis , is not normally saturated with substrate in the c e l l . Consequently the a c t i v i t y of th is enzyme may be s e n s i t i v e to changes in the amolint of acety l CoA a v a i l a b l e for s t e r o l synthesis (89). When metabol ic changes occur during exerc ise which decrease acetyl CoA f l u x , the a c t i v i t y of HMG CoA reductase may a l s o decrease, r e s u l t i n g in a change of s imi1ar magnitude and d i r e c t i o n in c h o l e s t e r o l synthet ic a c t i v i t y . Furthermore, the source of acety l CoA units could be an important determinant of s t e r o l synthet ic rate. When acety l units are a v a i l a b l e in the c e l l c y t o s o l , they are most l i k e l y der ived from acetate by acety l CoA synthetase (30). The l a t t e r is important s ince i t has been shown that even t h i s supply of acety l CoA, considered secondary to c i t r a t e - d e r i v e d acety l u n i t s , i s p r e f e r e n t i a l l y used for fa t ty acid synthesis and not chol es te ro lgenes i s (90). Figure 11 i l l u s t r a t e s that c e l l preparations 70 exposed to (-)hydroxycitrate, an i n h i b i t o r of acety l CoA t rans fer from the inner mitochondria l membrane to the c e l l cytosol v i a c i t r a t e , showed decreases in both fa t ty acid and c h o l e s t e r o l synthesis . When acetate , a p o t e n t i a l acety l CoA precursor , and (-)hydroxycitrate were added s imultaneously (Table 3), both f a t t y acid and c h o l e s t e r o l synthesis increased, however the r e l a t i v e contr ibut ion of acety l units to s t e r o l synthesis was reduced (90). Converse ly , i t has been shown that the high l e v e l s of c i t r a t e that are generated during s i tuat ions of p o s i t i v e energy balance, co-ordinate g l y c o l y s i s and l ipogenes is by i n h i b i t i n g phosphofructokinase and a c t i v a t i n g a c e t y l CoA carboxylase. It may be concluded from these observat ions that the source of acety l CoA for s t e r o l synthesis is h igh ly dependent on the abundance of c i t r a t e in the c e l l . Prolonged exerc ise causes d i v e r s i o n of pyruvate, a p o t e n t i a l precursor of c y t o b s o l i c acetate, through the g l y c o l y t i c pathway for the formation of inner mitochondrial acety l CoA. This d i v e r s i o n is respons ib le , in part , for the increase in t r i c a r b o x y l i c ac id c y c l i n g , and decreased a v a i l a b i l i t y of c i t r a t e during exerc ise . Thus lack of a v a i l a b l e c i t r a t e , and acetate , may have contr ibuted to the p r e s e n t l y observed "decrease of hepatic s t e r o l synthesis . The fac t that c h o l e s t e r o l formation decreased in l i v e r , but not i n t e s t i n e is cons is tent with the l a t t e r hypothesis, s ince the smal l i n t e s t i n e , in contrast to the l i v e r , is not respons ib le for r e g u l a t i o n of energy substrate metabolism during prolonged exerc ise . In add i t ion to the immediate metabol ic changes associated with an acute bout of exerc ise , i t i s p o s s i b l e that long term enzymatic changes may a l s o have been induced by e x e r c i s e , r e s u l t i n g in depression of hepatic 71 FIGURE l i . Influence of (-)-hydroxycitrate on the rate of cholesterol and Fatty Acid Synthesis in Perfused Rat Liver ^ , 10 0 1 2 (-)-Hydroxycitrate [mM] 40 min after operation 3 H ° 0 was added to the perfusion medium. 30 min thereafter a control liver sampie was taken. Different amounts of (-)-hydroxycitrate were added and perfusion continued for another 60 min, after which 2 additional liver samples were taken. Values represent means, vertical bars: t S.E.M. TABLE S. Influence of Acetate plus (-)-hydroxycitrate on cholesterol and Fatty-Aeid Synthesis in Perfused Rat Liver Series H^^O Incorporation -Into Acetate f-VHy- Cholesterol Fatty acids droxy-citrate mM mM /jatoms 3H/g, wet wt./hour A 10 1.1 2.27 = 1.02 (5) 2S.90=1.54 (31 A — — 5.68 = 2.11 (5) 2O.«=5.20(3) B 10 — 4.02 = 1.33 (6) 18.3/=5.56 (5) Series A: Experimental design as in fig. 3; after a 30 min perfusion period without addition (line 2) (-)-hydroxy-citrate was added together with acetate. For further details see IT. Series B: In a comparable set of experiments the influence of 10 m M acetate alone on lipid synih«sit was determined. Means ± S.D. from two series of experiments are given. Number of measurements in brackets. (Decker and Barth (.9.0)). 72 s t e r o l synthesis . The mechanism of t h i s adaptation might be s i m i l a r to that seen during f a s t i n g , s ince the l a t t e r shows p a r a l l e l p h y s i o l o g i c a l responses to exercise with respect to energy metabolism. S p e c i f i c a l l y , convers ion of squalene to c h o l e s t e r o l has been shown to be reduced in 24-72 hour bouts of fas t ing in r a t s , due to decreased supply of substrate and consequent decreased a c t i v i t y of the enzymes in th is pathway (30). A d i f f i c u l t y of the substrate hypothesis ar ises when t r y i n g to e x p l a i n , in i s o l a t i o n from other f a c t o r s , why no drop in synthet ic rate was seen in exerc ise versus sedentary low c h o l e s t e r o l - f e d animals. If lack of a v a i l a b l e substrate for c h o l e s t e r o l formation alone could lower i t s synthet i c ra te , then one would have expected to see s i m i l a r r e s u l t s in exerc ised animals, regardless of d ietary cho les te ro l content. This is where the a v a i l a b i l i t y of d ietary c h o l e s t e r o l may become rate l i m i t i n g in combination with exercise^and e x p l a i n through an i n t e r a c t i o n of these two v a r i a b l e s the observed r e s u l t s . Lack of an exercise e f f e c t may have r e f l e c t e d an i n a b i l i t y of c h o l e s t e r o l regulatory mechanisms to fu r ther suppress basal rates of c h o l e s t e r o l synthesis beyond "normal" r a t e s , as they do with exposure to d ietary c h o l e s t e r o l . One reasonable explanat ion might be that exerc ise , in low chol e s t e r o l - f e d animals, did indeed have the e f fect of lowering c h o l e s t e r o l synthes is , but s ince there was no apprec iable excess of d ie tary c h o l e s t e r o l with which to compensate t h i s e f f e c t , the l i v e r and p o s s i b l y i n t e s t i n e may have responded by t r y i n g to increase endogenous synthes is to r e p l e t e body pools . It has been shown that exerc ise may a f f e c t c h o l e s t e r o l turnover ra te , and lower the t i ssues ' c h o l e s t e r o l pool (91)., Thus the net e f fect of a compensatory increase in s t e r o l synthes i s , when combined with an exerc ise lowering e f f e c t would be no change in 73 c h o l e s t e r o l synthes is , which was the current observat ion. One might a l s o speculate that f a i l u r e to decrease cho les te ro l synthesis in the t i ssues studied was e l i c i t e d as a sparing e f fec t on the e x i s t i n g low body c h o l e s t e r o l poo l . C y t o s o l i c acety l CoA, through adaptat ion of r a t e - l i m i t i n g enzyme a c t i v i t i e s , may have been shunted through pathways of s t e r o l metabolism at a higher rate than normal, in an attempt to maintain basal rates of s tero l production. In a d d i t i o n , there may have been some down r e g u l a t i o n of hepatic LDL receptors , in an attempt to reduce the s e n s i t i v i t y of cho les te ro l feedback i n h i b i t i o n and preserve the r a p i d l y d e p l e t i n g c h o l e s t e r o l pool . The exact mechanism of such a response however cannot be 'defined. c) B i l e Acid Synthesis and Homeostasis The formation of b i l e acids serve as an important excretory pathway f o r c h o l e s t e r o l . An inverse r e l a t i o n s h i p between 7-C-hydroxylase, the rate l i m i t i n g enzyme for b i l e ac id formation, and the a c t i v i t y of HMG CoA reductase in the l i v e r has been hypothesized. Hence when dietary c h o l e s t e r o l l e v e l s are high, HMG CoA reductase is i n h i b i t e d , and 7<£-hydroxylase f a c i l i t a t e s formation of b i l e acids (92). S i m i l a r l y , when b i l e ac id product ion i s low, an increase in hepatic s t e r o l synthesis occurs. I t there fore becomes d i f f i c u l t to expla in on the basis of b i l e acid response to d i e t a r y c h o l e s t e r o l a lone , why an increase in hepatic c h o l e s t e r o l synthes is was observed in animals present ly fed the high c h o l e s t e r o l d i e t . The i n t e r a c t i o n between d i e t and exercise may better exp la in t h i s occurrence. F i r s t , in low c h o l e s t e r o l - f e d animals b i l e acid production 74 would be r e l a t i v e l y low and produced from both endognous and exogenous sources i f needed for d igest ion and absorption of d ietary fa t . During e x e r c i s e , the exercised group may have had increased c e l l u l a r demands fo r c h o l e s t e r o l , a st imulatory response for hepatic s te ro l synthesis in a d d i t i o n to the d i rec t e f fects of 7<C-hydroxylase. If , however, substrate a v a i l a b i l i t y were l i m i t i n g in the formation of c h o l e s t e r o l , then no net change in synthesis would occur, as was observed. Unfortunately the converse argument for high c h o l e s t e r o l - f e d animals, where e levated b i l e ac id product ion would i n h i b i t s t e r o l synthesis along with d ie tary c h o l e s t e r o l , does not expla in the observed increase in hepatic s t e r o l synthes is of the sedentary group. Had the l i v e r responded to the high c h o l e s t e r o l load by decreasing i t s endogenous synthesis , then f u r t h e r suppression of hepatic synthesis seen in the exercise group might have been a t t r i b u t e d to physical a c t i v i t y through reduced substrate a v a i l a b i l i t y . One might speculate that in the present experiment b i l e acid feedback i n h i b i t i o n on hepatic s te ro l synthesis may have become uncoupled, however how t h i s might occur is not c l e a r . It has been observed that a l t e r a t i o n s in hepatic synthesis in hamsters are not d i r e c t l y r e l a t e d to the c h o l e s t e r o l content of b i l e under c e r t a i n experimental condit ions (93). Thus the assumption that changes in b i l e ac id c h o l e s t e r o l content preceeded any changes in s t e r o l synthes is in the present study may be incor rect . It is p o s s i b l e , that l i v e r c h o l e s t e r o l synthes is responded independently of b i l e ac id formation, as w e l l as d i e t a r y c h o l e s t e r o l l e v e l , as suggested e a r l i e r . Both plasma cho les tero l l e v e l and c h o l e s t e r o l synthesis changed in s i m i l a r d i r e c t i o n s under the present d i e t and exercise manipulat ions. P a r a l l e l responses between hepatic c h o l e s t e r o l synthesis and plasma 75 c h o l e s t e r o l are not commonly reported. Decrease in hepatic synthesis g e n e r a l l y occurs in response to high cho les te ro l feeding to maintain the balance between plasma c h o l e s t e r o l and normal body pools (94). This s i t u a t i o n u s u a l l y r e s u l t s in no change, or an increase, in plasma c h o l e s t e r o l depending on the l e v e l of c h o l e s t e r o l in the d ie t . S i m i l a r l y , an increase in hepatic synthesis u s u a l l y occurs in response to lack of d ie tary c h o l e s t e r o l , in an attempt to restore plasma c h o l e s t e r o l to normal va lues (48). Thus f a i l u r e of the hamsters under the present experimental condit ions to e l i c i t the c l a s s i c a l response of reduced l i v e r c h o l e s t e r o l synthesis in the face of e l e v a t e d plasma c h o l e s t e r o l suggests that high c h o l e s t e r o l and fa t feeding acts independently of exerc ise on these parameters of l i p i d metabolism. This was suggested for b i l e ac id contro l of s t e r o l synthes is . d) Hormonal Factors C o n t r o l l i n g Cho lestero l Synthesis Among the fac tors which may have r e s u l t e d in the observed responses of hepatic s t e r o l synthesis in the present study are short term hormonal i n f l u e n c e s . I n s u l i n is known to st imulate c h o l e s t e r o l g e n e s i s and may be p a r t l y respons ib le for maintaining the normal d iurna l rhythm of HMG-CoA reductase (95,50). In contrast , glucagon i n h i b i t s c h o l e s t e r o l synthes is , an e f f e c t mediated by c y c l i c AMP, which diminishes the a c t i v i t y of HMG CoA reductase by enhancing reductase kinase a c t i v i t y (50). I n s u l i n and glucagon are thought to contro l both the amount of enzyme present, as w e l l as the proport ion of the enzyme in the a c t i v e state (50). It is w e l l e s t a b l i s h e d that the hormonal response to exerc ise i s character ized by a 76 f a l l in plasma i n s u l i n and r i s e in plasma glucagon (28,56). Both hormones are e s s e n t i a l regu lators of g lucose metabolism and may be i n v o l v e d in the a l l o c a t i o n of acety l CoA units fo r use in energy purposes during e x e r c i s e , instead of s t e r o l synthesis . During post-exerc ise recovery , i n s u l i n r i s e s r a p i d l y to enhance precursor u t i l i z a t i o n for glycogen r e p l e t i o n (70). In c o n t r a s t , glucagon remains high af ter exerc ise and maintains hepat ic uptake of gluconeogenic precursors (35). Even though the r e l a t i v e concentrat ions of these hormones change, the net e f f e c t is to d i v e r t energy towards glycogen r e p l e t i o n v i a gluconeogenesis, which i n h i b i t the t ranspor t of acety l CoA into the cytosol for c h o l e s t e r o l synthes is . In a d d i t i o n , chronic exerc ise may suppress the response of i n s u l i n to r i s i n g blood glucose. Since i n s u l i n exerts control over the a c t i v i t y of HMG CoA reductase and c h o l e s t e r o l synthesis , long term adapt ive changes might serve to keep s t e r o l synthesis c h r o n i c a l l y lower in exerc i s ing animals. In the present study however, the secu lar trend of th i s v a r i a b l e was not measured. Growth hormone and epinephrine, two add i t iona l hormones known to be e l e v a t e d during exercise (28), have been shown to s t imulate c h o l e s t e r o l synthes is by s t imu la t ing HMG CoA reductase a c t i v i t y (95). Growth hormone may a l s o increase hepatic synthesis of c h o l e s t e r o l by augmenting thyro id funct ion (95). In hamsters, exerc ise has been shown to increase sec re t ion of growth hormone and somatic growth (96). In th i s study, however, animals were a l lowed to run v o l u n t a r i l y and were fed ad 1 ibitum. It has s ince been shown that i f exerc i s ing hamsters are p a i r - f e d to sedentary c o n t r o l s , then the increase in somatic growth is prevented (97). Thus the e f f e c t s of growth hormone on s te ro l synthesis in the present study were probably not s i g n i f i c a n t . 77 Epinephrine is known to be e levated during both the exercise and post -exerc i se recovery periods (33). Although epinephrine is thought to s t imu la te c h o l e s t e r o l synthesis by increasing HMG CoA reductase a c t i v i t y (95), t h i s e f f e c t in the present study was probably not large s ince a decrease in hepatic synthet ic rate was observed. In f a c t , i f the sedentary animals fed the high c h o l e s t e r o l d iet experienced greater stress than the exerc ise group on the day of s a c r i f i c e , as p r e v i o u s l y suggested, then e l e v a t e d epinephrine in the sedentary group might not have been d i f f e r e n t from that of the exerc ise group. If th i s were the case, then the e f f e c t s of epinephrine on the sedentary or exercise groups would be r e l a t i v e l y equa l . One could then conclude that the . increase in hepatic s t e r o l synthesis in sedentary animals fed the high c h o l e s t e r o l d iet was not due to s t r e s s . Since the hormone was not measured such a conclus ion i s only s p e c u l a t i v e . 78 GENERAL SUMMARY AND CONCLUSIONS Resu l ts of the present study have shown that in hamsters, a s i g n i f i c a n t i n t e r a c t i o n between d ie tary c h o l e s t e r o l and exerc ise a c t i v i t y i n f l u e n c e parameters of plasma t o t a l c h o l e s t e r o l , HDL c h o l e s t e r o l and c h o l e s t e r o l synthesis . Plasma t o t a l c h o l e s t e r o l was e l e v a t e d by d ietary c h o l e s t e r o l but a l s o lowered in response to exerc i se , independent of d iet . More important however, was the i n t e r a c t i v e e f f e c t between d iet and exerc ise that reduced high plasma c h o l e s t e r o l l e v e l s in exerc i s ing animals fed high c h o l e s t e r o l d i e t s . In contrast , even though HDL-C was lowered through an i n t e r a c t i o n of these two f a c t o r s , a s i g n i f i c a n t change occurred only in e x e r c i s i n g hamsters fed low c h o l e s t e r o l d i e t s . C h o l e s t e r o l synthesis in l i v e r and smal l in tes t ine did not respond s i m i l a r l y to changes in the l e v e l of d ietary cho les te ro l and exerc ise in t h i s experiment. Intest ine synthet ic rates were decreased only in response to d i e t , whi le hepatic synthet ic rates showed an opposite increase. Moreover, a d ie t and exerc ise i n t e r a c t i o n produced a decrease in hepat ic synthesis in hamsters fed high c h o l e s t e r o l d ie ts . In g e n e r a l , the l i v e r appeared to respond independently, even u n c h a r a c t e r i s t i c a l l y , to the change in body cho les te ro l balance imposed by a high c h o l e s t e r o l d iet and prolonged exerc ise . Only under the l a t t e r condi t ions did exercise lower c h o l e s t e r o l synthesis. In a d d i t i o n , the mechanism of th i s response may not have been l inked to the changes observed in HDL c h o l e s t e r o l l e v e l s . Severa l important conc lus ions can be drawn from these study r e s u l t s ; i) HDL c h o l e s t e r o l i s not increased by exercise in the hamster 79 i i ) Exerc ise may decrease precursor substrate a v a i l a b i l i t y and thus l i m i t the rate at which endogenous c h o l e s t e r o l i s synthesized i i i ) The i n t e r a c t i o n between d ietary c h o l e s t e r o l l e v e l and exerc ise a c t i v i t y that served to lower both hepatic c h o l e s t e r o l synthesis and plasma t o t a l c h o l e s t e r o l may be important only when high c h o l e s t e r o l , moderately high fa t d ie ts are consumed iv ) The l i v e r responds to a l t e r e d ra t ios o'f c h o l e s t e r o l to fa t in the d ie t independently, or as a compensation to the reduct ion in c h o l e s t e r o l synthesis in the smal l i n t e s t i n e In summary, i f the hamster serves as a representa t i ve model of human c h o l e s t e r o l metabolism with regard to diet and exerc ise condi t ions , these f i n d i n g s suggest that there may be some benef i t der ived from exerc ise i f a high c h o l e s t e r o l d ie t were h a b i t u a l l y consumed. On the contrary, a combination of phys ica l exerc ise and low d ietary c h o l e s t e r o l does not appear to have s i g n i f i c a n t e f fec ts on s tero l metabolism, that would u l t i m a t e l y reduce the r i s k of ca rd iovascu la r d isease. I n t u i t i v e l y , t h i s is what one would expect, s ince low c h o l e s t e r o l consumption and phys ica l a c t i v i t y are independent recommendations set f o r t h to lessen the r i s k of CHD in Canadians. 8 0 REFERENCES 1. N i c h o l 1 s , E. (1986) Card iovascu lar disease in Canada. S t a t i s t i c s Canada., Catalogue 82-544, Ottawa. 2. M u l t i p l e Risk Factor Intervent ion T r i a l Research Group. (1982) M u l t i p l e r i s k factor i n t e r v e n t i o n t r i a l : Risk f a c t o r changes and m o r t a l i t y r e s u l t s . J Am Med Assoc 248:1465-1477. 3. Os lo Study Research Group. (1983) Mr. F i t and the Oslo study. J Am Med Assoc 249:893 -895. 4. Kronmal, R. 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(1988) E l e v a t e d high density l i p o p r o t e i n c h o l e s t e r o l in a t h l e t e s is r e l a t e d to enhanced plasma t r i g l y c e r i d e c learance. Metab 37:569-572. 56. A h l b o r g , G., Fe 1 i g , P., H a g e n f e l d t , L., H e n d l e r , R. & Wahren, J . (1974) Substrate turnover during prolonged exerc ise in man. J C l i n Invest 53:1080-1090. 57. Ho, J . (1979) C i rcad ian rhythm of c h o l e s t e r o l b iosynthes is : Dietary r e g u l a t i o n in the l i v e r and small i n t e s t i n e of hamsters. Int J Chro B 6:39-50. 87 58. A l b e r s , J . & Warnick, R. (1974) L i p i d and l i p o p r o t e i n a n a l y s i s . In: The manual of laboratory operat ions, L i p i d research c l i n i c s program. DHEW p u b l i c a t i o n (NIH) 75:628. 59. A1 l a i n , C , Poon, L., Chen, C , Richmond, W. & Fu, P. (1974) E n z y m a t i c determination of t o t a l serum c h o l e s t e r o l . C l i n Chem 20:470-474 60. Sperry J . (1963) Quant i ta t i ve i s o l a t i o n of s t e r o l s . J L i p i d Res 4:221-224. 61. Sheng, H. & Huggins, R. (1979) A review of body ciomposition studies with emphasis on t o t a l body water and fa t . Am 0 C l i n Nutr 32:630-647. 62. S c h o e l l e r , D. & Jones, P. (1987) Measurement of t o t a l body water by isotope d i l u t i o n : A u n i f i e d approach to c a l c u l a t i o n s . In: In v i v o body composition a n a l y s i s . ( E l l i s K., Yasamura, S. & Morgan, W. Eds.) pp. 131-137, I n s t i t u t e of Physical Sciences in Medicine, London. 63. Ewing, P. & L e B l o n d , G. (1984) Us ing symphony. (Nob le , V. & Freudenberger, J . , Eds.) Que corporat ion, Indianapol is . 64. CSS. (1987) Complete s t a t i s t i c a l system with data base management and graphics. l : S t a t s o f t . 65. B o r e r , K., H a l l f r i s c h , J . , T s a i , A. , H a l l f r i s c h , C. & Kuhns, L. 1979. The e f f e c t of exerc ise and d ietary prote in l e v e l s on somatic growth, body composition and serum l i p i d l e v e l s in adult hamsters. J Nutr 109:222-228. 66. T s a i , A. & Gong, T. 1987. Modulation of the exercise and r ret i rement 88 e f f e c t s by d ietary fa t intake in hamsters. J Nutr 117:1149-1153. 57. Borer, K. (1985). Regulat ion of energy balance in the golden hamster. In: The Hamster: Reproduction and behaviour, ( S i e g e ! , H. Ed.) pp. 363-407, Plenum Press, New York. 58. Goldman, J . (1948) E f f e c t of Cho lestero l Feeding in Hamsters. Arch Path 49:169 -172. 69. S c h e e l e , K., Herzog , W., R i t t h a l e r , G., W i r t h , A. & W e i c k e r , H. (1979) Metabo l i c adaptation to prolonged exerc ise. Eur J Appl Phys io l 41:101-108. 70. Ruderman, N., B a l o n , T . , Zorzano , A., Goodman, M. & Young, J . (1986) Acute and Chronic metabolism changes f o l l o w i n g exerc ise: Mechanisms and P h y s i o l o g i c a l re levance . In: Nut r i t ion and Exerc ise . (Winick, M., Ed.), pp. 1-8, John W i l e y and Sons New York, NY. 71. L a s s e r , N., Roheim, P., E d e l s t e i n , D.& Eder , H. (1973) Serum l i p o p r o t e i n s of normal and c h o l e s t e r o l fed rats . J L i p i d Res 14:1-8 72. Sable-Amp! i s , R., S i c a r t , R. & Farre, G. (1988) Plasma c h o l e s t e r o l e s t e r i f i c a t i o n and l i p o p r o t e i n p r o f i l e in hypercholestero lemic hamsters. Nutr Res 8:219-224. 73. H a s l e r , C , Rothenbacher , D., M e ! a , D. & K r i s - E t h e r t o n , P. (1987) Exerc i se attenuates d i e t induced a t h e r o s c l e r o s i s in the adu l t ra t . J Nutr 117:986-993. 74. H icks , A., MacDougal l , J . & Muckle, T. (1987) Acute changes in high density l i p o p r o t e i n c h o l e s t e r o l with exerc ise of d i f f e r e n t 89 i n t e n s i t i e s . J Appl Physio l 63:1956-1960. 75. B r o w n e l l , K., Bachoric , P. & Ayer le R. (1982) Changes in plasma l i p i d and l i p o p r o t e i n l e v e l s in men and women a f te r a program of moderate exerc ise . C i r c u l a t i o n 65:477-484. 76. Wood, P., H a s k i l l , W., K l e i n , H., L e w i s , S., S t e r n , M. & F a r q u h a r , J . (1976) The d i s t r i b u t i o n of plasma l i p o p r o t e i n in male runners. Metabol ism 11:1249-1257. 77. Far re 1 1 , P., Maksud, M., P o l l o c k , M., F o s t e r , G. , Anholm, J . , Hare , J . & Leon, A. (1982) A comparison of plasma c h o l e s t e r o l , t r i g l y c e r i d e s , and high-density l i p o p r o t e i n c h o l e s t e r o l in speed skaters , w e i g h t - l i f t e r s and non-ath letes . Eur J Appl Physiol 48:77-82. 78. Wood, P., H a s k e l l , W., B l a i r , S., W i l l iams, P., K r a u s s , R., L i n d g r e n , F., A l b e r s , J . , Ho, P. & F a r q u h a r , J . (1983) I n c r e a s e d exerc i se l e v e l and plasma l i p o p r o t e i n concentrat ions: A one year randomized c o n t r o l l e d study in sedentary middle aged men. Metab 32:31-38. 79. A l l i s o n , T . , Iammarinop, R., Metz , K., S k r i n a r , G., K u l l e r ,L. & Robertson, R. (1981) F a i l u r e of exerc ise to increase high density l i p o p r o t e i n subfract ions and other l i p o p r o t e i n s induced by exerc i se . J Cardiac Rehab 1:257-265. 80. L u k a s k i , H., B o l o n c h u k , W., K l e v a y , L., M a h a l k o , J . , M i l n e , D. & Sandstead, H. (1984) Inf luence and type of d ietary l i p i d on plasma 90 l i p i d concentrat ions in endurance a t h l e t e s . Am J C l i n Nutr 39:35-44. 81. Q u i n t a o , E., Grundy, S; & Ahrens , E. (1971) E f f e c t s of d i e t a r y c h o l e s t e r o l on the regu lat ion of t o t a l body c h o l e s t e r o l in man. J L i p i d Res 12:233-239. 82. V a n d e r , A., Sherman, J . & L u c i a n o , D. (1980) The d i g e s t i o n and absorpt ion of food. In: Human Physiology: The mechanisms of body funct ion , pp. 402-439, Mcgraw H i l l Inc. New York. 83. A l f i n S l a t e r , R. & Aftergood, L.(1980) L i p i d s . In: Modern n u t r i t i o n h e a l t h and d i s e a s e (Goodhart . R. & S h i l s , M., Eds.) pp. 113-141, F iebeger , P h i l a d e l p h i a . 84. De La C a l l e , M. & Gibbons, G. (1988) Hepatic and i n t e s t i n a l formation of p o l a r s t e r o l s in v i v o , in animals fed on a c h o l e s t e r o l -supplemented d ie t . Biochem J 252:3959-3962. 85. Gebhard , R., S tone , B. & P r i g g e , W. (1985) 3-Hydroxy 3-methy 1 g l u t a r y 1 coenzyme-A-reductase a c t i v i t y in the human g a s t r o i n t e s t i n a l t r a c t . J L i p i d Res 26:47-53. 86. Brown, M., G o l d s t e i n , J . & Dietschy, J . (1979) A c t i v e and i n a c t i v e forms of 3-hydroxy 3-methyl gl utary 1 CoA reductase in the l i v e r of the rat . J B io l Chem 254:5144-5149. 87. Connor , W., S t o n e , D. & Hodges, R.(1964) The i n t e r r e l a t e d e f f e c t s of d ie tary c h o l e s t e r o l and fa t upon human serum l i p i d l e v e l s . J C l i n Invest 43:1691 -1696. 88. Grundy, S., Mok, H. & Bergmann, K. (1976) R e g u l a t i o n of B i l i a r y 91 C h o l e s t e r o l Secret ion in Man. In: The L i v e r : Q u a n l i t a t i v e Aspects of St ructure and Function. ( P r e i s i g , R., B i r cher , J . & Paumgartner, G. Eds.) Aulendorf .-Edit io Cantor, 393-403. 89. P u l l i n g e r , C. & Gibbons, G. (1983) The r o l e of substrate supply in the regu la t ion of c h o l e s t e r o l b iosynthes is in rat hepatocytes. Biochem J 210:625-632. 90. Decker, K. & Barth, C. (1973) Compartmentalization of the e a r l y steps of c h o l e s t e r o l b iosynthesis in mammalian l i v e r . MoT C e l l Biochem 2:179 -187. 91. P a u l , P. (1975). E f fec ts of long l a s t i n g phys ica l exercise and t r a i n i n g on l i p i d s . In: Proceedings- Internat iona l symposium on biochemistry of exerc ise, pp. 156-193, B a s e l , Birkhauser. 92. Bjorkhem, J . & Akerlund, J . (1988) Studies on the l i n k between HMG-CoA-reductase and c h o l e s t e r o l 7-alpha-hydroxy1ase in rat l i v e r . J L i p i d Res 29:136-143. 93. Anderson, J . & Cook, L. (1986) Regulation of g a l l b l a d d e r c h o l e s t e r o l concentrat ion in the hamster: Role of hepatic c h o l e s t e r o l l e v e l . Biochim Biophys Acta 875:582-592. 94. Bhattacharyya, A. & Eggen, D. (1987) R e l a t i o n s h i p between d ie tary c h o l e s t e r o l , c h o l e s t e r o l absorpt ion, c h o l e s t e r o l synthesis and plasma c h o l e s t e r o l in rhesus monkeys. A t h e r o s c l e r o s i s 67:33-39. 95. Porter , 0. & Dugan, R. (1977) Hormonal contro l of c h o l e s t e r o l synthes is . In: Biochemical actions of hormones (Litwack, G.,Ed.) 92 pp. 198-247, Academic Press, New York. 96. Borer, K. & Ke lch , R. (1978) Increased serum growth hormone and somatic growth in e x e r c i s i n g hamsters Am J Physiol 234:E611-E617. 97. B o r e r , K., H a l l f r i s c h , J . , T s a i , A., H a l l f r i s c h , C , Kuhns, L. (1979) The e f fec t of exerc ise and d ietary prote in l e v e l s on somatic growth, body compostion and serum l i p i d l e v e l s in adult hamsters. J . Nutr 109:222-228. 93 APPENDIX A V 0 2 Max Measurement Procedure: Animals were run at minimal speed for two days p r i o r to t e s t i n g and a l lowed to f a m i l i a r i z e themselves with the running apparatus. T r e a d m i l l speed on subsequent days was increased every 2 minutes a f ter 5 minutes of warm-up, u n t i l the animals could no longer run. Mean V 0 2 consumed was c a l c u l a t e d f o r the l a s t 30 seconds of each 2 minute stage and p l o t t e d d a i l y over a 2 week per iod. In add i t ion , animals were considered to have reached a constant V0 2 max when the speed/oxygen consumption curve no longer s h i f t e d to the r i g h t . Resu l t s : Average V 0 2 max of p i l o t hamsters was determined to be 10 ml 0 2 /minute, or 76 ml 0 2 /kg body weight/minute. 94 APPENDIX B Diet Composition* PROTEIN 23.40 Arg in ine 1.38 Cyst ine 0.32 G l y c i n e 1.20 H i s t i d i n e 0.55 I s o l e u c i n e 1.18 Leucine 1.70 Lysine 1.42 Methionine 0.43 Pheny la lan ine 1.03 Tyros ine 0.68 Threonine 0.91 Tryptophan 0.29 V a l i n e . 1.21 FAT 4.50 **Cholesterol 0.03 FIBER (crude) 5.80 Neutral detergent 16.00 Ac id detergent 8.20 ASH 7.30 Calc ium 1.00 Phosphorus 0.61 Magnesium 0.21 Sodium 0.40 C h l o r i n e 0.50 PPM I ron 198.0 Zinc 70.0 Manganese 64.3 Copper 18.0 Cobal t 0.6 Iodine 0.7 Chromium 1.8 Selenium 0.20 GROSS ENERGY PHYSIOLOGICAL FUEL VALUE VITAMINS Carotene Menadione Thiamine R ibof1av in Niacin Pantothenic Chol ine F o l i c Acid Pyridoxine B i o t i n Acid B12 Vitamin A Vitamin D Vitamin E Vitamin C 4.25 3.30 PPM KCal/g KCal/g 4.50 17.70 8.00 95.0 24.0 22.5 5.90 6.00 0.07 (X100) mcg/kg 22.0 IU/g 15.0 4.5 65.0 * as analyzed by Purina ** c h o l e s t e r o l content of Study "A" Powdered f ree c h o l e s t e r o l was added to ground chow to increase the concentrat ion to 0.12g/100g f o r Study "B". 95 APPENDIX C ANOVA TABLES AND GRAND MEANS TOTAL PLASMA CHOLESTEROL (mg/dl) ,MMrtMMMrttt«W««M«rttftf/CMM«M«rt«tf«MMMtfrtMMfm«tfM«M«tfMM iss/pc 1 / QUICK ANALYSIS OF VARIANCE * : ANOVA/ANCOVA . dependent variable: PLASMA : • GDDDDBDDDDDBDDDDDDDDDDBDDDDDDDDDDBDDDDDDDDDDBDDDDDDDBDD6 • : 3 3 3 3 .- E-f-fect : SS 3 tit 3 MS 3 F 3 p ; (A): diet . . ... 107367.2 3 . 1 - 3 107367.2 3 123.9502 3-. .00000 : : (B>: exercise " • 6581.1,3' 1 3 65B1.1 3 7.5975 3 .'.00847 I ft x B - -* 2061.8 3 ' 1 3 2061.8 3 2.3802 3 .' .12665 : Within ' 36380.9 3 42 3 866.2 3 3 HmmnMnnmnHnnnmnHn3nnHnn»nHnnonnnmn»nnnomnnnmmnonnnnnnm <esc> - menu IMfl««H««M«/)««fl«Hflrt«n«H««MflMK»»««f(«««««««W««K»;«»nrtfinnnnn<>nnnnn««rtH«n««««H«HMrt«; CSS/pc J QUICK .• MEANS AND STANDARD DEVIATIONS ; ANOVA/ANCOVA : dependent variable! PLASMA i 0DDDDDDDCDDBDDDDDDDDDDBVDDDDDDDDD6 ; 3 3 diet * exercise GRCAiP l mean 3 St. dev. 3 N ; L HHHMHnHHMHt1l1HHHMMnHt1HMHMnMHMHHMMhMMMHMHMHHHHMHHt1HMHHHt1XHMHM1111l1t1t1tiXt1HMMNMMMMH9 Entire sample 1 - LOU S£.06»TTAA.4 2- LOW crXeAClSG: 1 - HI6H S££)^vn-Ae.^ 2 - H l 6 H t X ^ r t C t & f e 114.2000 3 14.24746 3 103.490? 3 20.11753 3 226.0600 3 SB.48957 3 198. 1222 3 34.57538 3 14.2.57S3 3 60.e91SS 3 11 1 11 : 15 » 9 J 46 : menu . Grand Means: Low Cholesterol Diet = High Cholesterol Diet Sedentary Animals Exercise Animals 108.84(mg/dl) 202.69 170.13 H5.81 HDL CHOLESTEROL (mg/dl) 7 MMHHHMHMHHHHMMMHHMHMk : CSS/pc : QUICK • ANOVA/ANCOVA HMMMMIIHHMHMHMMMHMMHMMHMHMHHIIIIHMIIMMHMIIMHHHHHMMHMMIIIIMHtlH ANALYSIS OF VARI»4C£ dependent variable HDL_jJ' GDDDDVDVDDDBDDDDUBDDDDBDDDDDBDBl*jDDDQDtlDt>0DEVDDt>BBDDBD6 2 3 3 - " 3 E-f-fect • SS 3 tit 3 MS 3 F 3 p HMMHHMMMMHHMHHMHMI1MHNHMHMMMHMMHXMI1MMMMMMHMXMMMMMHMMHMXHMMMHMMMM (A): diet x 1598.332 3 1 3 1598.332 3 9.63370 3 .00369 : : (8): exercise ; 3305.760 3 1 3 3305.760 3 19.92496 3 .00018 . A x B ; 672. 198 3 1 3 672.198 3 4.05157 3 .04709..., ; Within .- 6968.241 3 42 3 165.911 3 3 : tHnnmunnnnmnmmnmjMnnmnftmonntittttnmmonmHnnmnnonmnnnnn <esc> - menu IHMHIIHMMMnMMHHMHHHHMHHHHHMHHMHnMHMRttHHMHHtlHHKHtlMIIMMMMKMHMHHHIIHMMMMIIHHHMIIMHHMIl! : css/pc ; '. ' QUICK , MEANS AND STANDARD DEVIATIONS l i ANOVA/ANCOVA ; dependent variable: HDL C • j « • » GDDDDUDDDDDBDDDDDDDDDDSDVDDDDDDDD6 Grand Means. t diet « exercise £>CO0P , mean 3 St. dev. 3 « J LHHH«HMHMHH><HH»HMHHHHHHM*H»-'""~""""~-1MnHMHMHHHHI1HHMHHXMKKnH>1Hnt1HxkHHl111l<>1l1HI<9 LoW Cholesterol Diet » 77.35 (mg/dl) ' . I • 1 l»u> <cO€*KPtt.H S9.74546 3 12.86619 3. 11 » ' I _ i a LPW &XfeAc\5fc ' 64 .95454 3 10.58701 3 u / High Cholesterol Diet - 89.23 ' 2. * 1 (+/6rf SeceWrXty -' '3-°2142 3 15.31521 3 14 / » 2. * i tf/Srf fetcKc-,^. •• 84.54001 3.11.26836 3 10 .- Sedentary Animals » 91.83 .- Entire sample ^" .•' B3.95654 3 16.80099 3 - 46 / HMMHnHtlHKMMHHIIMHMHIIhHHHHMHHMllHHHnHHrtHHHMMMMMJHMMMMMHtlHHOHHHrtHHIirt <esc> - menu Exercise Animals » 7A . 74 96 APPENDIX D ANOVA T A B L E S / I N T E S T I N E F R A C T I O N A L S Y N T H E T I C R A T E ( F S R ) IMHHtlHMMHMMMMMMMHHMHMKmHMMMNHMMHHNIIHHHIIMHHHHMHHHHMHIIHMHM c s s / p c : I QUICK .• ANALYSIS OF VAP?^VC£ ANOVA/ANCOVA ; dependent variab l e : . .Ant-f sr GDDDDDDVDDDBDDDDDDDDDDBDDDDDDDDDDb... .*<ji/DDVEDDDDDDDDDD6 I 3 3 3 3 E-f-fect .• SS 3 d-f J MS 3 F 3 " . p (A): diet . 489.2266 3 1 J 489.2266 3 25.36968 3 .00006 (B)t exercise A x B Within 14.3673 3 '. 1 3 14.3673 3 " .74504 3 .39739 28.9382 3 1 3 28.9382 3 1.50064 3 .22554 790.6401 3 41 3 19.2B39 3 • 3 Hnnnyinnf\mnnmnHmn»nynnnm^n^HnommnnmnnonmHm»nn^onnnmmH <esc> - menu IKHHHMMMMMIIMHIIHMMMHHMHMHMHHMMMMHMMHHMMHHMHtlMKM^^ : css/pc .- QUICK . MEANS AND STANDARD DEVIATIONS .- ANOVA/ANCOVA . dependent variable: i n t f s r GDDODDDDDDDBDDDDDDDDDDBDDDDDDDDDD* .- di e t • exercise (,(LDvp : mean 3 st . dev. 3 'N 1 * 1 (.Cu< •Sfc-DfcNT/f-A.-/ ; 14.37504 3 6.53045 3 12 1 * 2 u>p 11.60175 3 2.83504 3 10 • • 2 * 1 Hltd Sece^TrtriV 1 6.05866 3 3.33199 3 14 2 * 2. 6-%er?£J56. : 6.53923 3 3.62045 3 9 -' Entire sample . 9.60427 3 5.56820 3 45 HHHHMMHHHMHMMnMHMNHHHHHHHHHHMMHMMMHHMHHMHMMHJHMHHHHHIIMHOMHnHHMMn <esc> ' - menu L I V E R F R A C T I O N A L S Y N T H E T I C R A T E ( F S R ) irtMMN«rttf«rttftf«rttfMMrtrtMHKM««MtftfMM«tftf«K«tfMrtrtrt«rttfrtMrt«HM : css/pc : -.- QUICK . ANALYSIS OF VARIOACt' ; * ; ANOVA/ANCOVA .• dependent variable: tiv-fsr ; * ,iDDDDDDDDDDSDDDDDDDDDDBDDDDDDDDPDCSC^O^Ot/DDBDDDDDDDDDD6 : 3 3 3 ; E-f-fect I - SS 3 at 3 nS 3 F 3 p ; LMHHMMMMMHMMHHMHMMHHMNHHMMHMMHMMXHHMMHMKHMMXHMHI1HHMHHI1XM : <A>: diet 13.38519 3 1 3 13.38519 3 80.20564 3 .00000 i (B): exercise ; .12530 3 ' 1 3 .12530 3 .75079 3 .39591 .-• A x B .• .44BO0 3 1 3 .44B00 3 2.68447 3 .10586 ; ; Within ' : 6.34166 3 38 3 .16689 3 3 i HHttnnnmnmnHmHnnntinjnnnnnHnnnnoMimnHnnmonnmnttnnm <esc> - menu 'JHMMMMHKMHHMHHHHHNMHHHHHH/IHHHHHHHHHMHMI1MHMMHKHMHIIHHMHHH : QUICKC » MEANS AND STANDARD DEVIATIONS : ANOVA/ANCOVA * dependent variable: liv-fsr -CDOVDDDDXIVDBDVDDDDDDDDEDDDVDDDDDD& ! 3 3 i d i e t . » exercise CKDUP » m e a r> 3 s t . dev. 3 N iH«rtrtMrtMrtWMN*MMM«Mrt«MtfMMrtW«MrtM««MM«fl^^ t J *• 1 L6(J S&&rrA*j •• 1-20314 3 .29651 3 11 : 1 * 2 U)W £X£A2I<£ » 1.52273 3 .71567 3 . 11 • • 2 * 1 *)t&H &fcOeOT/^Vj ' - 2 f e 9 5 7 3 - i 6 8 0 9 ^ 1 2 i 2 . 2 H ( 6 H e x ^ O < T " * * .% : Ent i r e sample .• .82^53 3 . 70~>25 3. 42 H««Mrttf««rt«tfrtMKM«tftf««MMrtMMMM«rttf«tfrt0KMM«Mtf«KMHJff«M <esc> - menu 97 ' -APPENDIX E A N O V A ' T A B L E S r A N D ' G R A N D M E A N S INTESTTKE SYNTHETIC RATE (Nmol/hr.g)-immmnmmmmmmmmmmmmmmmmmmmmm} : css/pc i s : : QUICK i . •' ANALYSIS DF VARIANCE i : ANOVA/ANCOVA : dependent viriablu intniol ; : : 3 3 i 3 i : Effect ": ''/SS 3 df . K S • '. f 3 p : ••ummmmnmmnmmimmmummmumnimmummm : (A): diet : 179S767. 3 1 I .'793767. i 31.43243 3 .00002 : j (B): e w e 1 - : 20748. 3 1 3 20748. 3 -.36325 3 .55676 : . : 66. 3 ' 1 J 66. 3 .00116 3 .32333 : : 2456051. 3 43 J 57117.3 3 - J gmmutimmmimmmmmmmutmxiQttmMMi <esc> - enu immmmmmmmmmmmmmmmnnmmmumumum-, css/pc : ; 0UICK . ! JEANS AND STANDARD DEVIATIONS i ANOVA/ANCOVA : dependent YirUble: intniol ' : immmnnmmmmmmu : 3 3 : diet . « eiercise C/VUP : «ean 3 St. in. 3 N l » 1 » 2 i . 2 i Entire saiole 1142.495 3 284.8632 3 1182.441 3 291.0238 3 745.331 3 181.3373 3 790.573 3 178.3155 3 958.812 3 306.7758 3 12 : 11 : 14 10 ; 47 : mmmmmummmmmmmmmmimimoimim <esc> - ienu. Grand Means: Low C h o l e s t e r o l D i e t - 1162.46 ( n m o l / h r . g ) H i g h C h o l e s t e r o l D i e t - 768 .20 S e d e n t a r y A n i m a l s - 944.16 E x e r c i s e A n i m a l s - 986 .51 LIVER SYNTHETIC RATE (Nmol/hr.g) c s s / p c QUICK , : ANALYSIS OF VARIANCE ANOVA/ANCOVA : dependent v a r i a b l e : l i v n m o l GDDDDDDDDDD3DDDDDDDDDDBDDDDDDDDDDBDDDDDDDDDDBDDDDDDDDDD6 3 3 3 3 E f f e c t SS 3 it 3 MS 3 T 3 p (A) : d i e t (B) : e x e r c i s e A x B W i t h i n 152129.S 3 135217.3 3 133133.1 3 8 4 S 2 3 8 . 1 3 1 3 152129.5 3 1 3 1 3 9 2 1 7 . 3 3 1 3 1 3 3 1 3 3 . 1 3 42 3 2 0 2 2 0 . 0 3 7 . 5 2 3 7 3 3 . 0 0 8 7 5 6 . 8 8 515 3 .701163 6.58424 3 .01335 3 mnmHHmmHmmMmHHJHHHHHmHHMOHMmMmmtiommmHMmoHMMmHnH-. <esc> - jnenu immnmmmnmmmiuimxmmunmmmnmmnmnump, css/pc ; QUICK : MEANS AMD STANDARD DEVIATIONS ANOVA/ANCOVA : dependent virUble: livniol . mmmnimmmmnmmH " : 3 3 i diet i exercise : lean 3 st. dev. 3 H ; immimmmummmiwmmuumxmmmmumunmuuim l » 1 » 2 * 2 * Entire suplt 241.3824 3 84.4679 3 .238.320 1 3 1 06.9394 3 -466.8413 3 216.6376 3 246.4354 3. 76.0598 3 • 310.4001 3 172.6950 3 12 11 14 9 • 46 mnmmimmnnnmmmmuuunmunimnumn <esc> _- ienu Grand Means: Low C h o l e s t e r o l D i e t - 240.15 ( n m o l / h r . g ) H i g h C h o l e s t e r o l D i e t - 356.64 Sedentary A n i m a l s - 354.11 E x e r c i s e A n i m a l s - 2 4 2 . 6 8 

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