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The ontogeny of acyl coenzyme A: cholesterol acyltransferase in rat liver, intestine, adipose tissue,… Little, Marie-Térèse E. 1990

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ONTOGENY OF ACYL COENZYME A: CHOLESTEROL ACYLTRANSFERASE IN RAT LIVER/ INTESTINE, ADIPOSE TISSUE, AND AORTA by MARIE-TERESE E . LITTLE' B . S c . , The Univers i ty of V i c t o r i a A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF MEDICINE DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1990 (c) Marie-Tefese E . L i t t l e , 1990 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 of O b s t e t r i c s and G y n a e c o l o g y The University of British Columbia Vancouver, Canada Date A p r i l 10 , 1990  DE-6 (2/88) ABSTRACT Epidemiological studies have shown that cho les tero l i s a major r i s k factor for the development of a therosc leros i s . Since the a therosc lero t i c plaque develops over a long period intervent ions ear ly i n l i f e may be of some benef i t . In a d d i t i o n , i t has been shown that the enzymes involved in cho les tero l metabolism can be manipulated in ear ly l i f e . Therefore, studies of the developmental patterns of the key enzymes in cho les tero l metabolism are of great importance. Acy l coenzyme A: cho les tero l acyltransferase (ACAT) i s the primary enzyme which catalyzes the conversion of free cho les tero l to cho les tero l esters i n c e l l s . A better understanding of the ro l e and control of ACAT during development i s needed i n order to trace the poss ible causes i n ear ly l i f e that lead to atherosc leros is i n the adult . This research focused on the developmental pattern of ACAT i n the ra t l i v e r , i n t e s t i n e , brown and white adipose t i s sue (BAT and WAT, respect ively) and aorta . Age spec i f i c changes were observed i n the r a t l i v e r , in tes t ine and BAT. The r a t l i v e r and in tes t ine possess s i g n i f i c a n t amounts of ACAT a c t i v i t y throughout development and there appears to be marked v a r i a t i o n s i n a c t i v i t y during th i s time. The ra t BAT and WAT appear to be devoid of ACAT a c t i v i t y throughout i i development with the exception of adult BAT. Due to the small amount of the a o r t i c t i s sue samples and/or the i n s e n s i t i v i t y of the assay, no d e f i n i t e conclusions could be made from t h i s a o r t i c study. In searching for factors that might contro l the ACAT enzyme the immediate ef fects of short-term manipulation of d i e t on the a c t i v i t y of ACAT were s tudied. The rats were a l l weaned ear ly on day 18 to one of the fol lowing d ie t s : Purina Rat Chow, high carbohydrate (HG) , high fat (HF) , or 2% c h o l e s t e r o l . The HF was the only d i e t that cons i s tent ly increased hepatic ACAT a c t i v i t y i n a l l the age groups. The c h o l e s t e r o l d ie t s s i g n i f i c a n t l y increased the a c t i v i t y of ACAT i n the 22 and 25 day o ld r a t s . The HG d ie t increased the a c t i v i t y of ACAT i n the 22, 25, and 30 day o ld r a t s . No s i g n i f i c a n t d i f ferences were observed between the adult c o n t r o l and HG d i e t groups. Feeding rats a HF or HG d ie t p r e c i p i t a t e d a dramatic drop i n i n t e s t i n a l ACAT a c t i v i t y i n the 22 day o ld animals. These ef fects were not observed i n the o lder animals. The high cho les tero l d i e t had no s i g n i f i c a n t e f fec t on the i n t e s t i n a l enzyme's a c t i v i t y i n 22 d a y . o l d r a t s . There was no s i g n i f i c a n t change i n the BAT and WAT ACAT a c t i v i t y with the experimental d ie t s with the exception that a l l the experimental d ie t s decreased ACAT a c t i v i t y i n the adult BAT. TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES ix LIST OF FIGURES X ACKNOWLEDGEMENTS x i LIST OF ABBREVIATIONS x i i i 1. LITERATURE REVIEW 1 1.1 Introduction 1 1.1.1 Cholesterol and Cholesterol Ester 1 Metabolism 1.1.2 ACAT and i t s Role i n 13 Atherosclerosis 1.1.3 ACAT Enzyme Properties 15 1.1.4 ACAT and the Liver: Role and 17 Sp e c i f i c Properties 1.1.5 ACAT and the Intestine: Role and 19 Sp e c i f i c Properties 1.1.6 ACAT and Adipose Tissue 23 1.1.7 ACAT and the Aorta: Role and 24 Sp e c i f i c Properties i v 1.1.8 Summary 26 1.2 Contro l of ACAT 28 1.2.1 Introduction 28 1.2.2 Substrate l e v e l 29 1.2.3 Phosphorylation/Dephosphorylation 31 1.2.4 Hormonal Control 34 1.2.4.1 Progesterone and Estrogen 34 1.2.4.2 Thyroid Hormone 35 1.2.4.3 Insu l in 37 1.3 Exposure to Dietary Manipulation: 39 Introduct ion . 1.3.1 Choles tero l 40 1.3.1.1 Prenatal Period 4 0 1.3.1.2 Suckl ing Period 42 1.3.1.2.1 Dietary Intervention 45 1.3.1.3 Weaning Period 47 1.4 Object ives 53 2. EXPERIMENTAL — 2.1 Experimental Design and Rationale I 54 2.2 Experimental Design and Rationale II 56 2.3 Mater ia l s and Methods 58 2.3.1 Mater ia l s 58 v 2.3 .1 .1 Animals 58 2.3.1.2 Chemicals 58 2.3 .1 .3 Equipment 58 2.3.1.4 Diets 62 2.3.2 Methods 64 2 .3 .2 .1 Animal Care 64 2.3.2.2 Animal Breeding 64 2.3.2.3 Treatment of Animals 65 2.3.2.4 Blood C o l l e c t i o n and 65 Preparation 2 .3 .2 .5 Serum T r i g l y c e r i d e A n a l y s i s : 66 P r i n c i p l e 2 .3 .2 .6 Tota l Serum Cholesterol 66 Analys i s : P r i n c i p l e 2 .3 .2 .6 .1 Procedure Used 67 2 .3 .2 .7 Preparation of Tissues for 67 Enzyme Analys is 2 .3 .2 .8 Prote in Analys is 69 2 .3 .2 .9 Preparation of Radio labe l led 69 Coenzyme A 2.3.2.10 Enzyme Assay 69 2.3.2.10.1 P r i n c i p l e of Assay 71 2.3.2.10.2 Procedure Used 71 2.3.2.10 S t a t i s t i c a l Evaluat ion 73 v i 3. RESULTS 74 3.1 Developmental study 74 3.1.1 Development of ACAT i n the L i v e r 74 3.1.2 Development of ACAT i n the 76 Intest ine 3.1.3 Development of ACAT i n BAT 76 3.1.4 Development of ACAT i n WAT 76 3.1.5 Development of ACAT i n the Aorta 79 3.1.6 Serum Cholesterol and T r i g l y c e r i d e 79 Levels During Development 3.3 Diet study 82 3.3.1 The Ef fec t s of Diets on L i v e r ACAT 82 A c t i v i t y 3.3.2 The Ef fec t s of Diets on Intest ine 85 A c t i v i t y 3.3.3 The Ef fec t s of Diets on WAT ACAT 85 A c t i v i t y 3.3.4 The Ef fec t s of Diets on BAT ACAT 88 A c t i v i t y 3.3.5 The Ef fec t s of Diets on Aorta ACAT 88 A c t i v i t y 3.3.6 Serum Cholesterol and T r i g l y c e r i d e 91 Levels v i i 3.4 S t a t i s t i c a l Evaluat ion 91 4. DISCUSSION 94 4.1 Ontogeny of ACAT 94 4.1.1 L i v e r 94 4.1.2 Intest ine 97 4.1.3 WAT 104 4.1.4 BAT 105 4.1.5 Aorta 106 4.2 E f f e c t of Diets on ACAT a c t i v i t y 107 4.2.1 L i v e r 108 4.2.2 Intest ine 109 4.2.3 Aorta , BAT and WAT 110 5. SUMMARY AND CONCLUSIONS 112 5.1 Developmental Study I 112 5.2 Diet Study II 114 5.3 Suggestions for Future Work 116 6. REFERENCES 120 7. APPENDICES 153 7.1 ACAT i n the Placenta 153 7.2 Composition of Purina Rat Chow 155 8. BIOGRAPHICAL INFORMATION 157 v i i i LIST OF TABLES Page I . L i s t of Abbreviat ions . x i i i I I . Examples of Late E f f e c t s on E a r l y Adaptat ion. 52 I I I . Chemicals and S u p p l i e r s . 59 IV. Suppliers Names and Addresses. 60 V. Equipment Used i n Experiments. 61 V I . Composition of D ie t s . 63 V I I . Composition of High Fat and High Carbohydrate 63 Diets . V I I I . ACAT A c t i v i t y i n BAT and WAT. 78 IX. ACAT A c t i v i t y i n the A o r t a . 78 X. The Effects of Diets on ACAT A c t i v i t y i n 87 WAT. X I . The Effects of Diets on ACAT A c t i v i t y i n the 90 Aorta . XII . The Effects of Diets on Serum Choles tero l and 93 T r i g l y c e r i d e Leve l s . i x LIST OF FIGURES Page 1. Exogenous and Endogenous Fat Transport Pathways. 8 2. LDL Pathway. 12 3. Control of ACAT v i a Phosphorylation / 32 Dephosphorylation. 4. Ontogeny of ACAT i n the L i v e r . 75 5. Ontogeny of ACAT i n the Intes t ine . 77 6. Choles tero l and T r i g l y c e r i d e Levels Throughout 81 Development. 7. The Ef fec t s of Diets on ACAT A c t i v i t y i n the 84 L i v e r . 8. The Ef fec t s of Diets on ACAT A c t i v i t y i n the 86 Intes t ine . 9. The Ef fec t s of Diets on ACAT A c t i v i t y i n BAT. 89 10. The Relat ionship Between the Ontogeny of ACAT i n 102 the L i v e r and Intest ine and Serum Choles tero l and T r i g l y c e r i d e Levels . 11. ACAT a c t i v i t y i n r a t f e t a l and p lacenta l t i s sues . 154 x ACKNOWLEDGEMENTS I would l i k e to thank: Dr. Peter Hahn for h i s supervis ion, guidance, construct ive c r i t i c i s m , and support. My stay i n h i s lab was t r u l y r a p s i c h o r d i c . My supervisory committee, Dr. P. Jones, Dr. J . F r o h l i c h , and Dr. D. Seccombe for t h e i r advice and suggestions. The Chairman of my committee, Dr. P . C . K . Leung for h i s support and encouragement. Kevin G l a t i o t i s for h i s patience, understanding and unre lent ing ass istance with my work. My parents , E i l e e n and Ernie L i t t l e for t h e i r help i n put t ing everything into perspect ive . x i This Thesis Is Dedicated To My Family. My Parents, Ernie and Ei l een L i t t l e . My Brothers and S i s t e r s , P h i l , Anne-Marie, P a t r i c i a Margaret, Miriam F r a n c i s , David, John, Bernard, and Monica-Mary and to Kevin. xn |Table I | Ab b r e v i a t i o n s 3 , 5 , 3 ' - t r i i o d o t h y r o n i n e ; T3 3,5-dichloro-2-hydroxy-benzenesuulfonic a c i d ; DHBS 3-hydroxy-3-methylglutaryl coenzyme A reductase; HMG-CoA reductase AcyI-coenzyme A r c h o l e s t e r o l a c y l t r a n s f e r a s e ; ACAT Bovine serum albumin; BSA Brown adipose t i s s u e ; BAT Carbohydrate; CHO C h o l e s t e r o l e s t e r ; CE C h o l e s t e r o l esterase; CEase C h o l e s t e r o l oxidase; CO Coenzyme A; CoA C y c l i c AMP; cAMP Degree C e l c i u s ; °C D i s t i I led water; dH20 D i t h i o e r y t h r i t o l ; DTT F a m i l i a l hypercholesterolemia; FH F a t t y a c i d ; FA Free c h o l e s t e r o l ; FC Free f a t t y Acids; FFA Glucose-6-phosphate dehydrogenase; G6PDH G l y c e r o l kinase; GK High carbohydrate; HG High d e n s i t y l i p o p r o t e i n ; HDL High Fat and C h o l e s t e r o l ; HFC High f a t d i e t ; HF Hydrogen peroxide; H202 Intermediate d e n s i t y l i p o p r o t e i n ; IDL L e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e ; LCAT Low d e n s i t y l i p o p r o t e i n ; LDL Messenger RNA; mRNA Nanometer; nm P r o p y l t h i o u r a c i I ; PTU P u r i n a Rat Chow plus ZX c h o l e s t e r o l d i e t ; 2% c h o l e s t e r o l Standard Error S.E. . Strptozocin-induced d i a b e t i c ; ST2-D Thin layer chromatography; TLC Thyroxine; H T r i g l y c e r i d e ; TG Very low d e n s i t y l i p o p r o t e i n ; VLDL White Adipose t i s s u e ; WAT x i i i LITERATURE REVIEW 1.1 INTRODUCTION 1.1.1 Choles tero l and Choles tero l Es ter Metabolism The uptake of cho le s t ero l es ter in to c e l l s by receptor-mediated endocytosis and i t s subsequent d e - e s t e r i f i c a t i o n and r e - e s t e r i f i c a t i o n by i n t r a c e l l u l a r enzymes has generated much i n t e r e s t p a r t i c u l a r l y because of the l i n k between these processes and patho log ica l condit ions such as a therosc l eros i s (Brown and Golds te in , 1984). The c o n t r o l of the enzymes governing cho le s t ero l anabolism and catabolism i s i n t e g r a l to the maintenance of whole body cho le s t ero l homeostasis. Choles tero l i s an e s sent ia l component of c e l l membranes serving a v i t a l funct ion i n a l t e r i n g membrane f l u i d i t y and i t i s an important precursor for the biosynthes is of s t e r o i d hormones and b i l e ac ids . The a b i l i t y of the body to absorb and synthesize cho le s t ero l vary markedly i n d i f f e r e n t species . In comparison to the human, small mammals such as the r a t and hamster synthesize large amounts of cho les tero l (Dietschy, 1984). The human synthesizes approximately 10 mg of cho le s t ero l per day per kg body weight (while on a low c h o l e s t e r o l diet ) i n comparison to the r a t and hamster who 1 synthesize approximately 40 mg per day per kg body weight and 100 mg per day per kg body weight, re spec t ive ly (Hashimoto, et a l . , 1974; Dietschy, 1984). T r a d i t i o n a l l y , the l i v e r has been regarded as the major t i s s u e responsible for the maintenance of cho le s t ero l homeostasis. In the past the l i v e r was estimated to synthesize over 80% of the endogenous c h o l e s t e r o l . However, more recent l i t e r a t u r e suggests that the l i v e r i s responsible for 51, 40, 27, 18, and 16% of t o t a l body cho le s t ero l synthesis i n the r a t , s q u i r r e l monkey, hamster, r a b b i t , and guinea p i g , r e spec t ive ly (Dietschy et a l . , 1983). Other t i s sues such as the adrenal , ovary, small i n t e s t i n e , stomach, spleen, and heart appear to play a more subs tant ia l s t e r o l synthet ic r o l e than was prev ious ly be l ieved (Dietschy et a l . , 1983). Due to t h i s major contr ibut ion of extrahepatic t i s sues to cho le s t ero l metabolism i t i s important that some of these a d d i t i o n a l t i s sues be included i n a study about cho les tero l metabolism during development. Cho les tery l esters serve two c r u c i a l functions i n mammalian c e l l s . They provide an i n t r a c e l l u l a r storage form of cho le s t ero l and a means by which cho le s t ero l i s transported i n the blood plasma (Spector, et a l . , 1979). A storage form of cho le s t ero l provides a safeguard for c e l l s that u t i l i z e a large quantity of c h o l e s t e r o l for 2 biosynthet i c purposes. Free cho le s t ero l res ides to a large extent wi th in the phosphol ipid b i l a y e r where i t modulates the f l u i d i t y of the membrane. Any a l t e r a t i o n i n the f l u i d i t y of the membrane v i a changes i n the cho les tero l content can have detrimental e f fec ts on the permeabi l i ty and propert i e s of the membrane bound enzymes and transport systems (Spector, et a l . , 1979). Hence a s p e c i a l i z e d storage pool of cho le s t ero l i n the form of c h o l e s t e r o l ester i s present wi th in most mammalian c e l l s which abol ishes the need to extract free cho le s t ero l from the f r a g i l e membrane systems. The primary enzyme responsible for the e s t e r i f i c a t i o n of cho le s t ero l i n plasma i s L e c i t h i n : cho le s t ero l acy l transferase (LCAT) which mediates the t r a n s f e r of fa t ty acids between l e c i t h i n and cho le s t ero l (Dobiasova, 1983; F r o h l i c h and McLeod, 1987) . LCAT i s a key p layer i n c h o l e s t e r o l and t r i g l y c e r i d e (TG) homeostasis (Froh l i ch and McLeod, 1987). There are two known enzymes which cata lyze the conversion of cho le s t ero l to c h o l e s t e r o l esters (CE) i n t r a c e l l u l a r l y : cho les tero l ester hydrolase and a c y l -coenzyme A: cho le s t ero l acy l - t rans ferase (ACAT). 3 C h o l e s t e r o l e s t e r h y d r o l a s e c a t a l y z e s t h e r e a c t i o n : C h o l e s t e r o l + f a t t y a c i d <===> c h o l e s t e r o l e s t e r + H 2 0 . T h i s r e v e r s i b l e r e a c t i o n d o e s n o t r e q u i r e A T P o r c o e n z y m e A . C h o l e s t e r o l e s t e r h y d r o l a s e e s t e r i f i e s t h e f r e e f a t t y a c i d w i t h o u t n e e d f o r p r i o r c h e m i c a l a c t i v a t i o n . T h e r e a r e two f o r m s o f t h i s enzyme d i s t i n g u i s h e d f r o m e a c h o t h e r b y d i f f e r e n t pH o p t i m a ( S p e c t o r , 1 9 7 9 ) . I t i s t h e p r e v a i l i n g o p i n i o n o f m o s t r e s e a r c h e r s t h a t A C A T i s t h e m a j o r enzyme r e s p o n s i b l e f o r t h e i n t r a c e l l u l a r e s t e r i f i c a t i o n o f c h o l e s t e r o l . A C A T c a t a l y z e s t h e r e a c t i o n : C h o l e s t e r o l + A c y l - C o A > C h o l e s t e r o l E s t e r + C o A . I n c o n t r a s t t o t h e c h o l e s t e r o l e s t e r h y d r o l a s e p a t h w a y , t h e n o n - r e v e r s i b l e A C A T r e a c t i o n r e q u i r e s t h e p r i o r a c t i v a t i o n o f t h e f a t t y a c i d ; t h u s , i t r e q u i r e s b o t h c o e n z y m e A (CoA) a n d A T P ( S p e c t o r , 1 9 7 9 ) . A d d i t i o n a l p r o p e r t i e s o f t h e A C A T enzyme a n d i t s r e a c t i o n a r e d e a l t w i t h i n s e c t i o n 1 . 1 . 3 . Due t o t h e i n s o l u b i l i t y o f m o s t l i p i d s i n a q u e o u s m e d i a , t h e s e s u b s t a n c e s a r e t r a n s p o r t e d b y a g r o u p o f g l o b u l a r l i p i d - p r o t e i n c o m p l e x e s c a l l e d l i p o p r o t e i n s . T h e l i p o p r o t e i n s e a c h c o n s i s t o f a c o r e o f n e u t r a l l i p i d 4 (pr imar i ly TG and CE) surrounded by a phosphol ip id and p r o t e i n b i l a y e r . Chylomicrons are p r i m a r i l y responsible for the import of exogenous TG and c h o l e s t e r o l . Very low densi ty l i p o p r o t e i n s (VLDL) p r i n c i p a l l y transport TG o r i g i n a t i n g from the l i v e r . Low densi ty l i p o p r o t e i n (LDL) and high densi ty l ipopro te ins (HDL) are concerned with the transport of endogenous cho les tero l (Brown and Golds te in , 1977) . In humans, LDL i s the c a r r i e r of most of the c h o l e s t e r o l i n plasma and approximately three- fourths of t h i s cho le s t ero l i s i n the e s t e r i f i e d form (Brown and Golds te in , 1976). The LDL pathway regulates the synthes is , uptake, and d i sposa l of c h o l e s t e r o l . Much a t tent ion has been focused on t h i s pathway p a r t i c u l a r l y because of the we l l known and accepted c o r r e l a t i o n between elevated plasma LDL l e v e l s and the associated r i s k of coronary ar tery disease . A defect i n t h i s pathway, such as that occurr ing i n the genetic disease f a m i l i a l hypercholesterolemia (FH), causes elevated c h o l e s t e r o l l eve l s and the precocious occurrence of a therosc l eros i s (Brown and Golds te in , 1983). The normal process of a therosc leros i s begins ear ly i n l i f e with an accumulation of cho le s t ero l and cho le s t ero l esters wi th in the intima and media of the ar tery wal l (Brown and Golds te in , 1977). At b i r t h human infants possess a large 5 number of LDL receptors and they have LDL concentrations s i m i l a r to other animal species. In i n d u s t r i a l i z e d s o c i e t i e s , the human LDL l e v e l r i s e s three to f o u r - f o l d with increasing age; the increase noted as early as i n childhood (Brown and Goldstein, 1984). The l i p i d transport system can be divided into two pathways: an exogenous route for TG and cholesterol absorbed from the small i n t e s t i n e and an endogenous route for cholest e r o l and TG entering the bloodstream from the l i v e r and other non-intestinal tissues (Brown and Goldstein, 1983; Figure 1) . The exogenous pathway begins i n the small i n t e s t i n e where dietary cholesterol and other l i p i d s are passively absorbed across the brush border of the small i n t e s t i n e . Within the i n t e s t i n a l c e l l the cholesterol i s e s t e r i f i e d through the action of ACAT and packaged i n chylomicrons for entry into the lymph (Meddings et a l . , 1987). The chylomicrons eventually d e l i v e r the t r i a c y l -g l y c e r o l component to adipose tiss u e for storage and the muscle for beta-oxidation to supply energy. The ester bond of the t r i a c y l g l y c e r o l i s cleaved by the action of l i p o p r o t e i n l i p a s e , an e x t r a c e l l u l a r enzyme most active i n the cardiac and s k e l e t a l muscle, adipose t i s s u e , and the l a c t a t i n g mammary gland. The remaining cholesterol-ester r i c h chylomicron remnants are removed from the c i r c u l a t i o n 6 Figure 1. Exogenous and endogenous pathway of fat t ranspor t . Dietary cho le s t ero l i n the form of cho le s t ero l es ter i s absorbed through the wal l of the small in te s t ine and i s transported along with t r i g l y c e r i d e s i n the chylomicron p a r t i c l e . L ipoprote in l i p a s e i n the adipose t i s s u e and muscle hydrolyzes the t r i g l y c e r i d e s l i b e r a t i n g f a t t y ac ids . The c h o l e s t e r o l - r i c h remnants bind s p e c i f i c receptors on the hepatic membrane. The c h o l e s t e r o l can be secreted in to the in te s t ine as such or i n the form of b i l e a c i d or i t can be transported with t r i g l y c e r i d e i n VLDL, thus i n i t i a t i n g the endogenous pathway. The t r i g l y c e r i d e por t ion of the p a r t i c l e i s again removed i n the adipose t i s s u e and the muscle. The c h o l e s t e r o l - r i c h intermediate densi ty l i p o p r o t e i n (IDL) e i t h e r binds to the hepat ic LDL receptors or i t i s converted i n the c i r c u l a t i o n to LDL (adapted from Brown and Golds te in , 1984). 7 D i e t a r y Fat I Figure 1. Exogenous and Endogenous pathway C h o l e s t e r o l E s t e r TG ADIPOSE TISSUE, MUSCLE CHYLOMICRON REMNANT C h o l e s t e r o l r i c h remnant endogenous exogenous 7 alpha h y d r o x y l a s e by s p e c i f i c hepatic receptors . Once i n the l i v e r c e l l , the c h o l e s t e r o l i s secreted into the i n t e s t i n e as such or as b i l e ac ids or i t i s packaged along with t r i a c y l g l y c e r o l i n VLDL i n i t i a t i n g the endogenous pathway. Once again the t r i a c y l g l y c e r o l component i s removed r e s u l t i n g i n a c h o l e s t e r o l - r i c h intermediate density l i p o p r o t e i n (IDL) . A por t ion of the IDL binds to LDL receptors and i s r a p i d l y engulfed by the l i v e r c e l l . The remaining IDL p a r t i c l e stays wi th in the c i r c u l a t i o n u n t i l i t s apoprotein E d i s soc ia tes and the p a r t i c l e i s converted to LDL. HDL, o r i g i n a t i n g from the l i v e r , i s secreted into the plasma as a nascent discoid-shaped p a r t i c l e almost completely devoid of cho le s t ero l e s ter . These nascent p a r t i c l e s are converted to spher i ca l l i p o p r o t e i n s by the accumulation of cho le s t ero l e s ters . LCAT catalyzes t h i s reac t ion i n the plasma. The cho le s t ero l esters are then t rans ferred from HDL to VLDL or LDL (Brown and Golds te in , 1984) . The c i r c u l a t i n g plasma LDL i s removed from the plasma by two d i s t i n c t mechanisms. Approximately 2 0% to 40% of LDL clearance involves non-spec i f i c endocytosis and i s re ferred to as receptor-independent LDL uptake (Meddings, et a l . , 1987) . The uptake of 60% to 80% of the l ipoprote in-bound c h o l e s t e r o l and cho le s t ero l esters occurs through the 9 process of receptor-mediated endocytosis (Brown and Go lds t e in , 1983; Meddings, et a l . , 1987; Figure 2) . LDL i s bound by a receptor on the surface of the c e l l i n a coated c l a t h r i n p i t . This membrane involutes and pinches o f f thus engulf ing the LDL and the receptor . Once ins ide the c e l l the receptor and the LDL d i s s o c i a t e . The receptor i s recyc led back to the c e l l surface and the LDL i s de l ivered to a lysosome. Apoprotein B-100 i s broken down into amino acids and the cho le s t ero l es ter bond i s cleaved to y i e l d u n e s t e r i f i e d cho le s t ero l and fa t ty ac ids . When an adequate amount of cho le s t ero l has accumulated through t h i s pathway to meet the b iosynthet ic requirements of the c e l l three metabolic processes r e s u l t . F i r s t , the oversupply of c h o l e s t e r o l i n h i b i t s 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) , the rate l i m i t i n g enzyme i n c h o l e s t e r o l synthes is . Second, the t r a n s c r i p t i o n of the receptor gene into messenger RNA (mRNA) i s suppressed thus i n h i b i t i n g the synthesis of a d d i t i o n a l LDL receptors . T h i r d , and most s i g n i f i c a n t for t h i s t h e s i s , the ACAT enzyme i s ac t iva ted promoting the r e - e s t e r i f i c a t i o n of cho le s t ero l to cho le s t ero l es ter for storage purposes (Brown and Golds te in , 1984). 10 Figure 2 The LDL pathway. The cho le s t ero l containing LDL p a r t i c l e i s bound to s p e c i f i c receptors located i n coated c l a t h r i n p i t s and i n t e r n a l i z e d v i a receptor-mediated endocytosis . As a d i r e c t r e s u l t of c h o l e s t e r o l uptake HMG-CoA reductase, the rate l i m i t i n g enzyme i n cho le s t ero l synthesis i s i n h i b i t e d , the receptors are down-regulated and the ACAT enzyme i s ac t ivated promoting e s t e r i f i c a t i o n of c h o l e s t e r o l (adapted from Brown and Golds te in , 1984). 11 Figure 2. LDL PATHWAY 1.1.2 ACAT and i t s Role i n Atherosc l eros i s Atherosc l eros i s i s the primary cause of death i n the western c i v i l i z a t i o n (Ross, 1988). There were approximately 12,000 a therosc leros i s re la ted deaths i n Canada i n 1986. ( S t a t i s t i c s Canada, 1988). The development of athero-s c l e r o s i s i s progress ive , beginning i n e a r l y chi ldhood with the accumulation of fa t ty streaks and manifest ing the sequelae of t h i s disease i n adulthood with advanced a o r t i c f ibrous plaques (Ross, 1988). The development of the a t h e r o s c l e r o t i c plaque i s character ized by an accumulation of free cho le s t ero l and cho le s t ero l esters i n the intima and media of the a r t e r i a l w a l l . The scavenger c e l l s wi th in the plaque become so dense with c h o l e s t e r y l esters that they take on a foamy appearance, hence the term foam c e l l . The c h o l e s t e r y l ester- ladened LDL's are de l i vered to the macrophages of the damaged smooth muscle c e l l and are subsequently hydrolyzed by ac id c h o l e s t e r y l esterase (ACE) (Brown and Golds te in , 1983a; Spr inkle et a l . , 1987). In add i t i on to the exogenous LDL c h o l e s t e r o l , the a r t e r i a l t i s s u e i s capable of synthes iz ing c h o l e s t e r o l de novo. When the macrophage takes up more cho le s t ero l than i t can use or export the excess i s e s t e r i f i e d as i s the case i n other t i s s u e s . Although, CEase and ACAT are both capable of performing t h i s funct ion , i t i s now bel ieved that ACAT plays 13 the predominant r o l e . Neutral c h o l e s t e r y l esterase (NCE) wi th in the c e l l can hydrolyze these c h o l e s t e r y l esters (Sprinkle et a l . , 1987). There i s some dispute as to whether or not LCAT i s present i n a r t e r i a l t i s s u e s . Abdul la et a l . (1968) noted an increase i n LCAT i n the human a t h e r o s c l e r o t i c a r t e r y . Even though the LCAT may be present i n a r t e r i a l t i s s u e i t appears that ACAT plays a more subs tant ia l r o l e i n atheroma formation because the increase i n a c t i v i t y of ACAT i n the atheroma l e s i o n was greater than the increase i n a c t i v i t y of LCAT (Abdulla et a l . ' , 1968; Hashimoto et a l . , 1974). The a t h e r o s c l e r o t i c plague i s a complicated s t ruc ture . The int ima les ions cons i s t , of p r o l i f e r a t e d smooth muscle c e l l s , macrophages, lymphocytes and a large amount of e x t r a c e l l u l a r mater ia l inc lud ing co l lagen , f i b r i n , glycosaminoglycans, and c h o l e s t e r o l . In the advanced a t h e r o s c l e r o t i c s tate the l e s i o n may include large areas of c a l c i f i c a t i o n , thrombosis, and e x t r a c e l l u l a r depots of connective t i s sue and c h o l e s t e r o l . These deposits protrude in to the lumen compromising the normal flow of blood (Brown and Golds te in , 1983a; Ross, 1988). I t i s wel l documented that a r t e r i e s undergoing atherogenic change c h a r a c t e r i s t i c a l l y show increased e s t e r i f i c a t i o n of cho le s t ero l and an accumulation of 14 c h o l e s t e r o l esters (St. C l a i r , 1976). Hashimoto et a l . and others have reported that the a c t i v i t y of ACAT i s increased up to 50- fo ld i n rabb i t a t h e r o s c l e r o t i c a r t e r i e s compared with non-diseased a r t e r i e s (Hashimoto et a l . , 1974; St C l a i r et a l . , 1970; Gal lo et a l . , 1977; Helgerud et a l . , 1982). Choles tero l oleate i s the major l i p i d associated with diseased a r t e r i e s . Spr inkle and co-workers (1987) conducted an enzymatic developmental study on rabbi t s and they found convincing r e s u l t s which suggest that ACAT may play a s i g n i f i c a n t r o l e i n an accumulation of c h o l e s t e r y l esters during the ear ly phases of l e s i o n development. These same inves t igators suggest that the noted increase i n ACAT a c t i v i t y i n the rabbi t s fed cho le s t ero l i s due to enzyme induct ion , membrane f l u i d i t y changes, or increased substrate concentrat ion at the enzyme s i t e . 1.1.3 ACAT Enzyme Propert ies Acyl-coenzyme A: cho le s t ero l acy l transferase i s a membrane bound microsomal enzyme that i s respons ible for the i n t r a c e l l u l a r e s t e r i f i c a t i o n of cho le s t ero l (Erickson and Cooper, 1980; Helgerud et a l . , 1982) to long-chain f a t t y -a c y l cho le s t ero l esters (Gavey et a l . , 1983). There are a number of t i s sues from d i f f e r e n t species possessing ACAT a c t i v i t y inc lud ing human, r a t , guinea p i g , and char l i v e r 15 (Erickson and Cooper, 1980; Gavey et a l . , 1983; Mukherjee, et a l . , 1958; Drevon, 1978; Dannevig and Norum, 1983) and i n t e s t i n e (Norum et a l . , 1979; Suckl ing et a l . , 1983; Dannevig and Norum, 1983), r a b b i t , pigeon and monkey a r t e r i e s (Hashimoto et , a l . , 1974; Brecher and Chobian, 1974) and mouse E h r l i c h asc i tes tumor c e l l s (Brenneman, 1977) . ACAT has a lso been i s o l a t e d i n the adrenals , ovar ies , r a t sk in (Norum, 1974) , and human sk in f i b r o b l a s t s (Spector et a l . , 1979). I t has been found that oleoyl-CoA i s the best substrate for the r a t l i v e r ACAT enzyme, with the rates of synthesis from a c y l CoA decreasing from c h o l e s t e r y l o leate > palmitate > s tearate > l i n o l e a t e (Goodman et a l . , 1964). This appears to be the case i n several other t i s sues and i t i s the consensus of most inves t igators that c h o l e s t e r y l oleate i s the predominant ester formed v i a 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 synthesis (Spector et a l . , 1979). Goodman and col leagues a lso observed that the highest ACAT a c t i v i t i e s are found i n the mitochondrial and microsomal f r a c t i o n s (1964). Hepatic ACAT i n v i t r o i s i n h i b i t e d by f a t t y acids i n concentrations above 1 x 10~ 5M while t h i s i n h i b i t i o n i s reversed by the add i t ion of serum albumin. The hepatic ACAT enzyme i s s ens i t i ve to detergents; taurocholate and glycocholate i n h i b i t ACAT (Goodman et a l . , 16 1964; Spector et a l . , 1979). The a c t i v i t y of ACAT i s dependent on acy l CoA concentrat ions . The pH optimum of ACAT from d i f f e r e n t species range from 7.2 to 7.8 (Spector et a l . , 1979; Norum et a l . , 1981; Helgerud, 1981). 1.1.4 ACAT and the L i v e r : Role and S p e c i f i c Propert ies The l i v e r i s the major s i t e of c h o l e s t e r o l synthesis and the predominant route for cho le s t ero l catabol ism. I t i s genera l ly accepted that hepatic ACAT plays an i n t e g r a l ro l e i n i n t r a c e l l u l a r cho le s t ero l homeostasis. I t s p r i n c i p a l r o l e i s the i n t r a c e l l u l a r e s t e r i f i c a t i o n of c h o l e s t e r o l . By doing so ACAT may also mediate cho le s t ero l homeostasis i n severa l other c a p a c i t i e s . I t i s speculated that i t might p a r t i c i p a t e i n d i r e c t i n g the pool of free c h o l e s t e r o l into the d i f f e r e n t excretory pathways: b i l i a r y c h o l e s t e r o l , b i l e a c i d synthes is , and VLDL (Nervi et a l . , 1984). ACAT might maintain membrane i n t e g r i t y and funct ion by e s t e r i f y i n g the excess c h o l e s t e r o l . An a l t e r a t i o n i n the c h o l e s t e r o l : phosphol ip id r a t i o invokes a change i n membrane permeabi l i ty and membrane enzyme a c t i v i t i e s (Erickson et a l . , 1984). In the r a t , a great percentage of VLDL i s of hepat ic o r i g i n and i t i s speculated that the cho les tero l es ter por t ion of t h i s p a r t i c l e i s assembled by the ACAT reac t ion (Erickson and Cooper, 1980). The contr ibut ion of VLDL to the t o t a l plasma 17 c h o l e s t e r o l pool i s small i n humans. Thus, i t i s u n l i k e l y that ACAT p a r t i c i p a t e s to any great extent i n the contro l of plasma cho le s t ero l (Erickson and Cooper, 1980), s ince LCAT i s the major enzyme responsible for the synthesis of plasma c h o l e s t e r o l esters (Glomset, 1968). ACAT a c t i v i t y has been found i n the l i v e r of many species . In decreasing order the r e l a t i v e a c t i v i t i e s are r a t , p i g , dog, monkey, r a b b i t , c a l f , and guinea p i g (Spector et a l . , 1979; Stokke, 1974). Although o r i g i n a l l y i t was thought that ACAT was absent i n the human l i v e r (Stokke, 1974), more recent l i t e r a t u r e states that i t i s indeed present (Erickson and Cooper, 1980; Balasubramaniam et a l . , 1978; Angel in et a l . , 1987). The human enzyme appears to e x h i b i t a c t i v i t i e s s i m i l a r to those found i n the rabb i t (Erickson and Cooper, 1980). A c i r c a d i a n rhythm of low amplitude has been detected for r a t hepat ic ACAT a c t i v i t y and i t p a r a l l e l s the a c t i v i t y of HMG-CoA reductase and thus cho le s t ero l b iosynthes is (Erickson et a l . , 1984). ACAT a c t i v i t y i n ra t l i v e r microsomes i s associated with the RNA-rich v e s i c l e s ; ACAT i s l o c a l i z e d i n membranes o r i g i n a t i n g from the rough endoplasmic ret iculum (Balasubramaniam et a l . , 1978b). In contras t , both HMG-CoA reductase and cho le s t ero l 7-alpha hydroxylase are confined to endoplasmic ret iculum membranes with low ribosomal 18 coat ing (Balasubramaniam et a l . , 1978b). Balasubramaniam and co-workers have shown that when l a b e l l e d cho le s t ero l i s added to hepatic c e l l s with the add i t i on of cofactors for both ACAT and cho le s t ero l 7-alpha hydroxylase, the s p e c i f i c a c t i v i t i e s of the two enzymes were found to be d i f f e r e n t (1978b). This suggested that there might be at l eas t two d i s t i n c t cho le s t ero l substrate pools i n the endoplasmic re t i cu lum; one for the ACAT react ion and one pool that acts as a substrate for cho les tero l 7-alpha hydroxylase (Balasubramaniam et a l . , 1978b; Spector et a l . , 1979). 1.1.5 ACAT and the Intes t ine: Role and S p e c i f i c Propert ies Numerous i n v i t r o studies have demonstrated that the propert i e s of the i n t e s t i n a l ACAT enzyme are i n many ways very s i m i l a r to those of the hepatic enzyme. Only those c h a r a c t e r i s t i c s which are unique to the i n t e s t i n e w i l l be discussed i n t h i s s ec t ion . The small i n t e s t i n a l mucosa i s considered a key player i n c h o l e s t e r o l metabolism because i t i s the s i t e for uptake of the s t e r o l s present i n the i n t e s t i n a l contents and because i t i s one of the most ac t ive t i s sues responsible for endogenous cho le s t ero l production (Stange and Dietschy, 1985; Turley and Dietschy, 1981). In the i n t e s t i n a l 19 e p i t h e l i a l c e l l i n t r a c e l l u l a r e s t e r i f i c a t i o n i s considered a regulatory step i n the absorption of c h o l e s t e r o l . Eighty to ninety percent of the cho le s t ero l t rans ferred into the lymph i s e s t e r i f i e d with fa t ty acids from both d ie tary and endogenous sources (Treadwell and Vahouny, 1968). In the past i t has been suggested that regu la t ion of cho le s t ero l absorption i n the gut and the re tent ion of cho le s t ero l i n i n t e s t i n a l c e l l s as cho le s t ero l esters i s under the contro l of cho l e s t ero l ester hydrolase of pancreat ic o r i g i n (Angel and Farkas, 1974; Ga l lo et a l . , 1977). However, more recent l i t e r a t u r e suggests that t h i s regu la t ion i s under the c o n t r o l of ACAT (Helgerud et a l . , 1982; Norum et a l . , 1983). I f t h i s i s the case, ACAT i s indeed an important regulatory enzyme given the unequivocal evidence that d i e tary c h o l e s t e r o l plays a causal r o l e i n the pathogenesis of coronary ar tery disease i n man (Kannel, 1988). What ro l e ACAT plays i n the i n t e s t i n e during development i s l a r g e l y unknown. Norum and co-workers (1983) suggest that the funct ion of ACAT i n cho le s t ero l absorption i n the adult ra t i n t e s t i n e i s i n e s t e r i f y i n g cho le s t ero l so that cho le s t ero l es ters can be exported by the enterocyte in to the lymph. Norum and co-workers have found that the a c t i v i t y of ACAT i s greater i n the v i l l o u s c e l l s than i n the crypt c e l l s of the i n t e s t i n a l epithel ium (1983). These f indings seem 20 l o g i c a l given the s p e c i f i c functions of each of these c e l l s . Absorptive v i l l o u s c e l l s evolve from d i v i s i o n , d i f f e r e n t i a t i o n , and migration of the stem c e l l s at the base of the i n t e s t i n a l v i l l o u s . The mature v i l l o u s c e l l s , i n contrast to the immature crypt c e l l s , house an assortment of enzymes and transport prote ins associated with d iges t ion and uptake of n u t r i e n t s . Hence, the ACAT enzyme would serve a usefu l purpose here i n e s t e r i f y i n g c h o l e s t e r o l to ready i t for transport into the lymph. On the other hand, the crypt c e l l s c o n t i n u a l l y u t i l i z e cho les tero l for membrane synthesis associated with c e l l d i f f e r e n t i a t i o n and p r o l i f e r a t i o n , thus 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 i s an unwanted process (Norum et a l . , 1983). Rat i n t e s t i n a l ACAT a c t i v i t y d i sp lays a d i u r n a l v a r i a t i o n r e l a t e d to the animal's feeding schedule: increase i n ACAT a c t i v i t y with fa s t ing and a decrease with feeding (Helgerud, 1982). ACAT a c t i v i t y i s the highest i n the proximal in te s t ine and lowest i n the d i s t a l port ion i n r a t (Haugen and Norum, 1976) and humans (Helgerud, 1981). In r a b b i t s , the mid-gut exh ib i t s the highest ACAT a c t i v i t y . The suggestion that ACAT plays a r o l e i n the absorption of c h o l e s t e r o l i s given further credence by the fact that the c l u s t e r of c h o l e s t e r o l absorptive s i t e s are predominantly located i n 21 the proximal in te s t ine (Sylen and Nordstrom, 1970); Suckl ing and Stange, 1985). I t should a lso be noted that compounds such as Sandoz 58-035 [3 - (decy ld imethy l - s i l y l )N[2 - (4 -methylphenyl)1-phenylethyl propanamide], when administered i n v i v o , provoke a dramatic decrease i n the a c t i v i t y of ACAT which i s accompanied by a decrease i n absorption of c h o l e s t e r o l and a decrease i n c h o l e s t e r y l esters entering the lymph (Suckling and Stange, 1985). Choles tero l esterase (CEase) seems to predominantly work to hydrolyze and take up c h o l e s t e r o l ; r e - e s t e r i f i c a t i o n i s thought to occur v i a the ACAT r e a c t i o n . Therefore, i t appears that CEase and ACAT work i n tandem: CEase aids i n the uptake and hydro lys i s of c h o l e s t e r o l esters and ACAT r e - e s t e r i f i e s the cho le s t ero l for transport in to the lymphatic system. Norum et a l . , (1981) summarize the a d d i t i o n a l ro les of ACAT i n the in te s t ine i n three po int s . F i r s t , ACAT may act as a pro tec t ive enzyme, protec t ing the c e l l from excessive accumulation of cho le s t ero l by e s t e r i f y i n g i t . Second, ACAT promotes storage of cho le s t ero l for future use i n membrane or l i p o p r o t e i n synthes is . L a s t l y , ACAT may a f fec t l i p o -p r o t e i n metabolism. 22 1.1.6 ACAT and Adipose Tissue Adipose t i s sue contains one of the larges t pools of exchangeable cho les tero l and i t i s a major s i t e for c h o l e s t e r o l storage (Angel and Farkas, 1974). Fat t i s sue contains more cho le s t ero l than most other organs or membranes when i t i s expressed per un i t of pro te in (Farkas et a l . , 1973; Pittman et a l . , 1975). A high l e v e l of c h o l e s t e r o l esterase (CEase) has been reported i n ra t s and humans (Pittman et a l . , 1975). ACAT a c t i v i t y has been reported to be low or absent i n adul t r a t adipose t i s sue concomitantly with l i t t l e c h o l e s t e r y l ester stores (Angel and Farkas, 1974). This seems p e c u l i a r s ince i n other t i s sues a considerable part of c h o l e s t e r o l i s stored as acy l e s t er s . In a d d i t i o n , c h o l e s t e r o l i s reported to be released from the adipose t i s sue when catecholamines are administered (Farkas et a l . , 1973) and during s tarvat ion of adult ra t s (Angel and Farkas, 1974) . No data are a v a i l a b l e for in fant or f e t a l animals. White adipose t i s sue may play a s p e c i a l r o l e i n the maintenance of t o t a l plasma c h o l e s t e r o l , s ince HMG-CoA reductase a c t i v i t y i n WAT of obese mice was found to be h igh , even though the hepatic enzyme showed low a c t i v i t y (Hahn, 1980). Whether or not ACAT i s present i n adipose t i s s u e during development i s unknown. Brown adipose t i s sue 23 d i f f e r s i n metabolic development from WAT. The primary ro l e of BAT i s to produce heat by o x i d i z i n g f a t t y a c i d s . Brown fat i s found i n a l l h ibernators and newborn babies , r a t s , and mice (Johansson, 1959). I t contr ibutes about 25% of the t o t a l l i p i d s to the body i n the suck l ing r a t ; the only t i s s u e that produces more i s the sk in (Hahn, 1986) . Although the developmental pattern of HMG-CoA reductase i n BAT has been determined, (HMG-CoA reductase i s high i n the f e t a l BAT and decreases to the 14th postnata l day; Hahn and Smale, 1982) , there has been no developmental studies p e r t a i n i n g to the ro l e of ACAT i n adipose t i s s u e . 1.1.7 ACAT and the Aorta : Role and S p e c i f i c Propert ies ACAT a c t i v i t y has been found i n the a r t e r i e s of the r a t , r a b b i t , pigeon, s q u i r r e l monkey, rhesus monkey, dog, swine, and humans (Suckling and Stange, 1985). The d i s t r i b u t i o n of ACAT i n a o r t i c t i s sue i s s i m i l a r to that i n the l i v e r (Goodman et a l . , 1964; Suckl ing and Stange, 1985). However the concentrat ion of ACAT i n t h i s t i s sue i s lower than i s found i n the hepatic t i s s u e . In many ways the a r t e r i a l enzyme i s s i m i l a r to the i n t e s t i n a l and hepatic enzymes. The enzyme prefers fa t ty acy l CoA as a cofactor , i t i s i n h i b i t e d by detergents, and i t has a pH optimum of 7.4 (Hashimoto et a l . , 1974; Suckl ing and Stange, 1985). 24 O l e i c a c i d i s synthesized i n the a t h e r o s c l e r o t i c a r t e r y . Due to the a o r t i c ACAT's preference for o l eoy l CoA i t seems l i k e l y that t h i s l o c a l l y synthesized fa t ty a c i d i s used by ACAT. The i n t r a c e l l u l a r cho le s t ero l esters are pre -dominantly c h o l e s t e r y l oleate whereas the LDL derived c h o l e s t e r y l esters that f i l t e r into the int ima and media i s mostly c h o l e s t e r y l l i n o l e a t e (Suckling and Stange, 1985). I t has been suggested that ACAT operates wi th in the c e l l to protect the c e l l from excess cho le s t ero l accumulation. This i s a c o n t r o v e r s i a l issue s ince i t i s not known whether the increase i n ACAT a c t i v i t y i n the a t h e r o s c l e r o t i c ar tery represents an e t i o l o g i c a l event, an exacerbating fac tor , or a passive consequence of an increased a v a i l a b i l i t y of free cho le s t ero l ( G i l l i e s , et a l . , 1986). Spector et a l . (1979) suggests that the accumulation of c h o l e s t e r y l esters i s a r e s u l t of an increased a v a i l a b i l i t y of cho le s t ero l and that the ACAT react ion provides a mechanism for the removal of a p o t e n t i a l l y harmful ,excess of c h o l e s t e r o l . Other inves t iga tors have shown that ACAT i n h i b i t o r s reduce the extent of the atheromatous l e s i o n suggesting that t h i s increased e s t e r i f i c a t i o n by ACAT serves as a biochemical trapping mechanism for cho le s t ero l entering the a o r t i c wal l (Be l l and Schaub, 1986; G i l l i e s et a l . , 1986). Both of these 25 inves t iga tors have used adult rabb i t aortae and a r t e r i e s and again no studies have been completed looking at these parameters during development. Since the a t h e r o s c l e r o t i c plaque develops over a long per iod of time i t i s important that the ontogeny of ACAT, one of the key enzymes i n c h o l e s t e r o l metabolism, be c l e a r l y e s tab l i shed . In a d d i t i o n , i t has been shown that some enzymes i n cho le s t ero l metabolism can be manipulated i n e a r l y l i f e by ear ly in tervent ion therefore knowledge of the developmental pat tern of the ACAT enzyme i s of i n t e r e s t (Rymaszewski et a l . , 1985; Subbiah and Hassan, 1982). 1.1.8 Summary The LDL pathway regulates the synthes is , uptake, and d i sposa l of c h o l e s t e r o l . The uptake of c h o l e s t e r y l esters v i a receptor mediated endocytosis and an accumulation of excess cho le s t ero l mediates three important processes. The excess cho le s t ero l i n h i b i t s HMG-CoA reductase and the synthesis of a d d i t i o n a l LDL receptors , and i t ac t ivates the ACAT enzyme, promoting e s t e r i f i c a t i o n of cho le s t ero l for storage. As mentioned, the ACAT enzyme has a number of important propert ies that d i s t i n g u i s h i t from the other c h o l e s t e r o l e s t e r i f y i n g enzymes. The propert ies of the ACAT 26 enzyme have been wel l character ized however the developmental p r o f i l e of t h i s enzyme has not been e luc idated . 27 1.2 CONTROL OF ACAT 1.2.1 Introduct ion The u n e s t e r i f i e d cho le s t ero l wi th in the c e l l ex i s t s p r i m a r i l y wi th in the membrane where i t functions to modulate the f l u i d i t y of the phosphol ipid b i l a y e r thus a l lowing the proper funct ioning of the membrane bound enzymes and transport systems (Spector et a l . , 1979). I t i s general ly accepted that the maintenance of the concentrat ion of the u n e s t e r i f i e d cho le s t ero l pool i s c r u c i a l . By e s t e r i f y i n g c h o l e s t e r o l and prov id ing a storage pool of c h o l e s t e r o l , ACAT p a r t i c i p a t e s i n the contro l of free c h o l e s t e r o l wi th in the c e l l . The contro l of the ACAT enzyme has often been l inked to the c o n t r o l of cho le s t ero l 7-alpha hydroxylase and HMG-CoA reductase. Usual ly when the a c t i v i t y of HMG-CoA reductase i s high ACAT a c t i v i t y i s low and v i c e versa . The mechanisms d i r e c t i n g the a c t i v i t y of these enzymes are not wel l understood. I t i s thought that long term regula t ion i s governed by enzyme pro te in synthesis and degradation (Beirne et a l . , 1977; Mitropoulos et a l , 1978). These three enzymes appear to be r e c i p r o c a l l y regulated i n the short term by phosphorylat ion/dephosphorylat ion and substrate supply. 28 There are a number of factors which appear to s p e c i f i c a l l y regulate ACAT a c t i v i t y i n the short term. Those that w i l l be dea l t with i n t h i s sect ion include 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 , phosphorylation and dephosphorylation, and hormones inc lud ing progesterone, estrogen, t h y r o i d hormone, and i n s u l i n . The l a s t segment deals with the e f fec t s of d i e tary manipulation on cho le s t ero l metabolism. 1.2.2 Substrate Level The most obvious fac tor that contro l s ACAT a c t i v i t y i s the substrate supply or 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 . Many inves t igators have shown that a l t e r i n g the l e v e l of c h o l e s t e r o l i n the l i v e r i n v ivo causes a change i n the a c t i v i t y of ACAT. In r a t s , i f the hepat ic cho le s t ero l concentrat ion i s increased by c h o l e s t e r o l or cholate feeding, an atherogenic d i e t , f a s t i n g , or the adminis trat ion of mevalonic a c i d , ACAT a c t i v i t y increases (Erickson et a l . , 1980) . S i m i l a r l y , the ACAT a c t i v i t y i n the r a t i n t e s t i n a l mucosa appears to be suscept ib le to the change i n c h o l e s t e r o l content because an increase i n ACAT a c t i v i t y was noted when rats were fasted and a decrease i n a c t i v i t y occurred when rats were fed a contro l HG d i e t (Helgerud et a l . , 1982). This might be due to the a l t e r a t i o n i n the unes ter i fed cho les tero l content i n the microsomes which was 29 high a f t er fa s t ing and low a f ter feeding (Helgerud et a l . , 1982). Others have suggested that as opposed to ACAT being regulated by the substrate supply, cho le s t ero l or one of i t s substrates may regulate ACAT a c t i v i t y by in t erac t ions with one or more of the enzyme's regulatory s i t e s or by changing the f l u i d i t y of the endoplasmic r e t i c u l a r membrane (Helgerud et a l . , 1982; Suckl ing et a l . , 1982). On the other hand i f the substrate supply of c h o l e s t e r o l i s the major regulator of ACAT a c t i v i t y then i t would be expected that reducing the absorption of c h o l e s t e r o l by adminis trat ing cholestyramine would subsequently i n h i b i t the ACAT enzyme. This appears to be the case i n the r a b b i t i n t e s t i n e ( F i e l d and Salome, 1982) but not so i n the ra t (Stange et a l . , 1983). Although cholestyramine appeared to have no e f f ec t on r a t i n t e s t i n a l ACAT, i t st imulated cho les tero l synthesis and increased the c h o l e s t e r o l content of the enterocyte. Stange and co-workers (1983) propose an explanation for t h i s i n t h e i r substrate pool theory which suggests that there are several d i f f e r e n t funct ional pools of cho le s t ero l that meet the d i f f e r e n t metabolic needs of the i n t e s t i n a l c e l l . The pool that suppl ies the substrate for the ACAT react ion i s d i f f e r e n t from the newly synthesized (or HMG-CoA reductase supplied) p o o l . Thus, i n t h i s case HMG-CoA reductase and 30 ACAT appear to be regulated by d i f f e r e n t mechanisms a phenomena which does not support e a r l i e r reports of r e c i p r o c a l regulat ion of these two enzymes. 1.2.3 Phosphorylation/Dephosphorylation In add i t ion to substrate-suppl ied regu la t ion of the ACAT enzyme discussed i n the previous sect ion i t appears that ACAT may also be regulated i n the short term by phosphorylat ion and dephosphorylation. I t i s thought that a c t i v a t i o n of r a t hepatic and i n t e s t i n a l ACAT by phosphorylat ion occurs through a ATP-dependent prote in kinase pathway and that pro te in phosphatase inac t iva te s t h i s enzyme v i a dephosphorylation (Gavey et a l . , 1983; Suckl ing et a l . , 1983b; Sca l l en and Sanghvi, 1983; Figure 3) . The second messenger for t h i s system i s unknown, but i t might be cAMP-mediated (Basheeruddin et a l . , 1982; Suckl ing and Stange, 1985). I t should be noted that others have found no evidence for t h i s system of contro l i n human l i v e r microsomal preparations (Einarsson et a l . , 1989). This same group of inves t igators reported that HMG-CoA reductase of human l i v e r o r i g i n was i n a c t i v a t e d / a c t i v a t e d by dephosphorylat ion/ phosphorylat ion. S c a l l e n and Sanghvi (1983) propose that the three enzymes i n hepatic cho le s t ero l metabolism, HMG-CoA 31 Figure 3. Schematic diagram of the proposed short term control of ACAT by phosphorylation and dephosphorylation. (Adapted from Gavey et a l . , 1983). ACAT (Phosphorylated / Active) Protein phosphatase Protein kinase ACAT (Dephosphorylated / Inactive) 32 reductase, 7 alpha-hydroxylase, and ACAT are r e c i p r o c a l l y regulated by phosphorylat ion/ dephosphorylation. The enzyme involved i n synthesis of cho le s t ero l i . e . HMG-CoA reductase i s inac t iva ted by phosphorylation whereas the enzymes involved i n u t i l i z a t i o n of the c h o l e s t e r o l , ACAT and 7 alpha-hydroxylase are ac t ivated by phosphorylat ion. Therefore, t h i s coordinated regu la t ion of the three key enzymes i n cho les tero l metabolism provides a short term mechanism for the contro l of i n t r a c e l l u l a r u n e s t e r i f i e d c h o l e s t e r o l homeostasis. Although there i s subs tant ia l i n v i t r o evidence that ACAT and HMG-CoA reductase are coordinate ly c o n t r o l l e d t h i s way there are condit ions and t i s sues i n which these two enzymes appear to be regulated independently. For instance, ACAT a c t i v i t y i s increased i n cu l ture hepatocytes by the a d d i t i o n of 25-hydroxycholesterol and mevalonic ac id but t h i s e f f ec t i s absent a f t er an 18 hour incubat ion per iod . In contras t , the i n h i b i t i o n by these treatments on HMG-CoA reductase pers i s t ed for at l eas t 22 hours, thus i t i s poss ib le that the mechanism of contro l i s d i f f e r e n t for these two enzymes (Suckling and Stange, 1985). Other treatments such as feeding cholestyramine or a f a t - f r e e d i e t to r a t s increases HMG-CoA reductase but has no apparent impact on ACAT (Suckling and Stange, 1985; Innis , 1986). 33 This suggests that other mechanisms apart from or i n a d d i t i o n to phosphorylat ion/dephosphorylat ion might be involved i n the contro l of the HMG-CoA reductase and ACAT enzymes. However, a d e f i n i t e answer to t h i s phosphorylat ion/ dephosphorylation debate w i l l have to await fur ther experiments using p u r i f i e d enzyme. 1.2.4 Hormonal Control Hormonal regulat ion of ACAT a c t i v i t y has not been wel l e s tabl i shed except for the e f fec t s of estrogen and progesterone. However, one can speculate that hormones such as the t h y r o i d hormone and i n s u l i n p a r t i c i p a t e i n c h o l e s t e r o l metabolism during development. 1 .2.4.1 Progesterone and Estrogen A number of i n v i t r o experiments have demonstrated that e t h i n y l e s t r a d i o l and progesterone a f fec t the hepatic a c t i v i t y of ACAT. E t h i n y l e s t r a d i o l increases ACAT a c t i v i t y i n the r a t poss ib ly by increas ing the substrate supply to the l i v e r v i a increas ing the number of l i p o p r o t e i n receptors (Del Pozo et a l . , 1983; Chao et a l . , 1979; Suckl ing and Stange, 1985). Progesterone i n h i b i t s hepatic ACAT a c t i v i t y i n add i t i on to increas ing the b i l i a r y cho le s t ero l output i n male ra t s (Erickson and Cooper 1980; Del Pozo et a l . , 1983). 34 This i n h i b i t i o n was reversed by feeding the ra t s a c h o l e s t e r o l d i e t or by i n j e c t i o n of e t h i n y l e s t r a d i o l . I f t h i s progesterone-related decrease i n enzyme a c t i v i t y occurs i n v ivo one could postulate that t h i s may be a contr ibut ing fac tor to the sex-re lated di f ferences i n c h o l e s t e r o l and l i p o p r o t e i n metabolism (Erickson and Cooper, 1980). 1.2.4.2 Thyroid Hormone I t i s wel l known that t h y r o i d hormones are e s sent ia l for normal growth and t i s sue d i f f e r e n t i a t i o n (Nathanielsz, 1976). A l t e r a t i o n s i n t h y r o i d status causes changes i n plasma l i p i d s and l i p o p r o t e i n s . There i s an inverse r e l a t i o n s h i p between t h y r o i d a c t i v i t y and plasma cho le s t ero l l e v e l s . Thus, hyperthyroidism as a pa tho log ica l condi t ion i n a pat i ent or i n the hyperthyroid- induced experimental animal i s associated with hypocholesterolemia and the reverse i s true for hypothyroidism (Heimberg et a l . , 1985). The e t io logy of hypercholesterolemia associated with decreased l eve l s of t h y r o i d hormones may r e s u l t from increased hepatic output of the VLDL or decreased uptake of LDL (Heimberg et a l . , 1985). Choles tero l synthesis i n ra t l i v e r i s reduced to o n e - f i f t h the l e v e l of contro l animals a f t er thyroidectomy. This reduct ion of cholesterolgenes is and the i n h i b i t i o n on HMG-CoA reductase can be counteracted 35 by the adminis trat ion of t h y r o i d extract or t r i i odothyron ine (Goldfarb, 1980). The contro l of the t h y r o i d gland on c h o l e s t e r o l metabolism i s l i k e l y through the e f fec t s these hormones have on cho le s t ero l 7 alpha-hydroxylase rather than a d i r e c t e f fec t on HMG-CoA reductase (Hahn and Innis , 1984; Heimberg et a l . , 1985). I t i s poss ib le that maternal t h y r o i d hormones do cross the placenta s ince T4 given to pregnant ra t s was shown to have a d i r e c t e f fec t on the enzyme phosphoenolpyruvate carboxykinase i n r a t fetuses (Hahn and Hassanal i , 1982). Hahn and others (1977) have shown that plasma cho le s t ero l l e v e l s are reduced i n suckl ing animals when T4 i s given. Given the apparent e f fec ts of t h y r o i d hormones on HMG-CoA reductase i t i s probable that these hormones a lso mediate, to some extent, the ACAT react ion during development. The only experiments l i n k i n g t h y r o i d hormones and ACAT a c t i v i t y are those by Hahn (1986b) . I t was found that in fant rat i n t e s t i n a l ACAT a c t i v i t y increased almost 5 - fo ld when PTU was added to the mother's dr ink ing water. HMG-CoA reductase a c t i v i t y was only s l i g h t l y decreased i n t h i s case. However, i t has been shown by others that the adminis trat ion of thyroxine causes a decrease i n exogenous and endogenous c h o l e s t e r o l absorption and thyroidectomy enhances the 36 absorption and transport of cho le s t ero l (Ponz de Leon et a l . , 1984). Given the proposed r o l e ACAT plays i n the absorption of cho le s t ero l one could speculate that the t h y r o i d hormones may p a r t i c i p a t e i n the contro l of the ACAT enzyme during development. 1.2.4.3 I n s u l i n I n s u l i n i s regarded as a prime candidate for the c o n t r o l of cho le s t ero l metabolism for a number of reasons. Diabetes m e l l i t u s i s often accompanied by high l e v e l s of LDL and VLDL and low l eve l s of HDL which predisposes the i n d i v i d u a l to precocious a therosc l eros i s . Diabetes as wel l as the s treptozocin- induced diabetes are frequently associated with hypercholesterolemia. The r i s e i n plasma c h o l e s t e r o l l eve l s noted i n the s treptozocin- induced d i a b e t i c (STZ-D) ra t s i s accentuated when the ra t s are fed an atherogenic d i e t (Jiao et a l . , 1988; Goldfarb, 1980). In a d d i t i o n , cho le s t ero l synthesis i s reported to be increased two to three times normal rates i n the i n t e s t i n e of d iabe t i c animals and humans (Jiao et a l . , 1989). Diabetes a l so causes changes i n the key enzymes i n c h o l e s t e r o l metabolism. HMG-CoA reductase and cho le s t ero l 7 alpha-hydroxylase are both increased s u b s t a n t i a l l y i n the STZ-D r a t i n t e s t i n e (Goldfarb, 1980). J i a o et a l . (1988) 37 reported that microsomal ACAT a c t i v i t y i n i n t e s t i n a l e p i t h e l i a l c e l l s i s higher i n STZ-D ra t s than i n normal r a t s . This would i n f e r that cho le s t ero l e s t e r i f i c a t i o n i s increased by the d i a b e t i c s tate and that i n s u l i n might i n h i b i t cho le s t ero l e s t e r i f i c a t i o n v i a the ACAT reac t ion . I t i s i n t e r e s t i n g to note that J i a o and co-workers (1988) reported that the i n s u l i n ac t ion on the regu la t ion of ACAT i s opposite i n the l i v e r and the i n t e s t i n e ; ACAT a c t i v i t y i s decreased i n the l i v e r whereas i t i s increased i n the i n t e s t i n e . These inves t igators suggest that the i n s u l i n de f i c i ency i n diabetes could be a major fac tor i n the enhanced ACAT a c t i v i t y i n the i n t e s t i n e . The exact mechanism whereby i n s u l i n def ic iency regulates ACAT a c t i v i t y i s d i f f i c u l t to assess without the complete p u r i f i c a t i o n of the enzyme p r o t e i n , however i t i s proposed that t h i s regu la t ion occurs through pro te in synthes is , phosphory-l a t i o n / dephosphorylation or by the a l t e r a t i o n i n membrane f l u i d i t y (Jiao et a l . , 1988). 38 1.3 Exposure to Dietary Manipulat ion: Introduct ion I t has been shown that environmental factors ac t ing on the i n d i v i d u a l ear ly i n l i f e may a l t e r the l a t e r development of that i n d i v i d u a l (Hahn, 1989; Hahn, 1987; Hahn and Koldovsky, 1966; Hassan and Subbiah, 1989; Reiser et a l . , 1977) . Thus any adaptive response i n adul t l i f e may be condit ioned by the f i r s t adaptation to an external stimulus ear ly i n l i f e . This sec t ion s h a l l deal with the contro l exerted by n u t r i t i o n a l factors during ear ly l i f e on the development of cho le s t ero l metabolism. Mammalian development has a prenata l , a suck l ing and a weaning stage, d i s t inguished by the mode of food intake and the composition of t h i s food. Obviously, the prenata l d i e t i s suppl ied v i a the placenta , and any change i n the composition of the d i e t must be through the mother. Immediately a f t er b i r t h , the d i e t i s mostly or exc lus ive ly mi lk , depending on the degree of maturity of the species at b i r t h . For instance, the r a t , mouse, and hamster are born very immature and are thus completely dependent on maternal mi lk , whereas the more mature human neonate and the guinea p i g neonate can be p a r t l y dependent on and completely independent of maternal mi lk , r e s p e c t i v e l y . The d i e t composition of the neonate can be manipulated e a s i l y i n the case of animals capable of eat ing independently of the 39 mother, and less e a s i l y i n those dependent on mother's mi lk . F i n a l l y , the weaning per iod s t a r t s when the in fant commences to eat other food instead of or i n add i t i on to mi lk . In some species , t h i s co incides with the suck l ing per iod , e .g . i n guinea p i g s . 1.3.1 Choles tero l 1.3.1.1 Prenatal Period The mammalian fetus receives i t s nutr ients v i a p lacenta l t r a n s f e r . Glucose i s the primary component of the f e t a l "diet ." There appears to be materna l - f e ta l t rans fer of c h o l e s t e r o l i n some species ( P i t k i n et a l . , 1972), but i t i s a l so synthesized de novo i n a number of f e t a l t i s sues , inc lud ing the human adrenal , l i v e r , t e s tes , b r a i n , and ovary (Carr and Simpson, 1982). The rate of cho le s t ero l synthesis i n the fetus as judged by the a c t i v i t y of the rate l i m i t i n g enzyme i n cho le s t ero l synthes is , 3-hydroxy-3-methyl g l u t a r y l coenzyme A (HMG-CoA) reductase, increases i n the l i v e r jus t p r i o r to b i r t h to rates two to three times higher than those i n the adul t (McNamara et a l . , 1972). Concomitantly, the f e t a l plasma l e v e l of cho le s t ero l i s low during t h i s i n t e r v a l and does not depend on the maternal l e v e l (Shafr ir and Khass is , 1982). The hepatic production of b i l e acids i s the major pathway of cho le s t ero l catabol ism. Choles tero l 7 40 alpha-hydroxylase, the major regulatory enzyme i n b i l e ac id synthes is , i s low i n the fetus (Hahn and Inn i s , 1984) , pos s ib ly r e f l e c t i n g the immaturity of the systems of b i l e a c i d synthes is , hepatic uptake, and secre t ion and i l e a l reabsorpt ion (Innis , 1985). I t has been suggested that b i l e ac ids of maternal o r i g i n i n d i r e c t l y a f fec t f e t a l cho les tero l metabolism by cross ing the placenta and i n h i b i t i n g f e t a l hepatic HMG-CoA reductase (Hahn, 1987) . Much a t tent ion has been focused on the p o t e n t i a l e f fec ts of ear ly n u t r i t i o n on the development of cho le s t ero l metabolism i n the adul t . However, l i t t l e information has been c o l l e c t e d on the f e t a l and neonatal outcome of manipulation of the maternal d i e t . I t appears that hepatic HMG-CoA reductase a c t i v i t y i n f e t a l ra t s i s a l t e r e d by the quanti ty of fa t and the f a t t y ac id composition of the maternal d i e t (Haave et a l . , 1989). Feeding cholestyramine, a non-absorbable r e s i n that binds b i l e ac ids , to pregnant ra t s r e s u l t s i n a 50% increase i n f e t a l hepatic HMG-CoA reductase a c t i v i t y with no apparent change i n the maternal or f e t a l plasma l i p i d l e v e l s (Innis , 1988) . In contrast , pregnant ra t s fed a high fat and c h o l e s t e r o l (HFC) d i e t had s i g n i f i c a n t l y increased plasma cho le s t ero l l e v e l s , but the HMG-CoA reductase a c t i v i t y i n the fetus was not affected (Innis , 1988). I t should be noted that others have found that feeding cholestyramine or 41 cho le s t ero l to pregnant ra t s has no e f f ec t on f e t a l c h o l e s t e r o l synthesis (Miguel and Abraham, 1976). Hassan and co-workers have shown that cholestyramine fed to pregnant ra t s r e s u l t s i n a 2 . 6 - f o l d increase i n f e t a l cho le s t ero l 7 alpha-hydroxylase a c t i v i t y compared to the c o n t r o l values (1985). 1.3.1.2 Suckl ing Period The suckl ing per iod , which const i tutes the second phase of development, begins at b i r t h with a dramatic change i n supply, ra te , and composition of food intake . The t r a n s i t i o n from a prenatal high carbohydrate "diet" to a neonatal high fa t milk d i e t i s preceded by a per iod of s tarva t ion which may l a s t from 10 minutes to several hours. As mentioned, the maturity of the species at b i r t h d ic ta te s the dependency on the mother for mi lk . The cho le s t ero l content of the milk a lso d i sp lays i n t e r - s p e c i e s v a r i a t i o n . Rat mi lk contains approximately 120 mg/dl ( C a r r o l l , 1964), while human breast milk contains 20-24 mg/dl (Hahn, 1982). Blood c h o l e s t e r o l l eve l s dramat ica l ly increase from low f e t a l l e v e l s to high postnata l values i n most mammals ( C a r r o l l and Hamilton, 1973), while HMG-CoA reductase a c t i v i t y i n the l i v e r , i n t e s t i n a l mucosa and brown adipose t i s s u e (BAT) decrease 42 af t er b i r t h (McNamara et a l . , 1972; Hahn and Smale, 1982; Kroeger and Hahn, 1983; Hahn and Walker, 1979). This has a lso been confirmed by more d i r e c t methods, i . e . the incorporat ion of t r i t i a t e d water in to c h o l e s t e r o l (Belknap and Dietschy, 1988; Stange and Dietschy, 1984), the technique considered to be the most r e l i a b l e way to study l i p i d metabolism (Jeske and Dietschy, 1980; Dietschy and Spady, 1984). Using the same method, i t was a lso shown that c h o l e s t e r o l synthesis i s low i n a l l t i s sues of in fant ra t s except i n the bra in (Belknap and Dietschy, 1988; Hahn, 1986) . I t has been suggested that the postnata l r i s e i n t o t a l c h o l e s t e r o l i s l inked to the c h o l e s t e r o l content of the mi lk (Friedman and Byers, 1961) and of the d i e t (Mott et a l . , 1978; Whatley et a l . , 1981). However, i t was found that the postnatal r i s e i n cho le s t ero l occurs i n two steps. The f i r s t (immediately a f t er b ir th) occurs independent of d i e t . The second increase seems to depend on d i e t , s ince the r i s e d i d not occur i n infants fed only tea with sugar. Thus, even though plasma cho le s t ero l l e v e l s seem to depend to some degree on the consumption of mi lk , t h i s i s not the so le regulator (Hahn and Koldovsky, 1966). Undoubtedly, other factors are involved. There appears to be l i t t l e d i f f erence between the rate of cho le s t ero l synthesis i n 43 various t i s sues during t h i s time, with the exception of the high rate of synthesis noted i n the b r a i n (Hahn, 1986). Previous inves t igators have proposed that the decrease i n HMG-CoA reductase during the suck l ing per iod i s due to a "factor" i n the milk (McNamara et a l , 1972; Boguslawski and Wrobel, 1974) . To date no such fac tor has been i s o l a t e d . However, i t i s wel l es tabl i shed that there i s an inverse r e l a t i o n s h i p between blood l eve l s of t o t a l cho le s t ero l and hepat ic HMG-CoA reductase, thus i t i s more probable that c h o l e s t e r o l or one of i t s metabol i tes , or VLDL, i s responsible (Park and Subbiah, 1988). The low a c t i v i t y of HMG-CoA reductase during the suck l ing phase could be a t t r i b u t e d to a decrease i n the synthesis of the enzyme and an increase i n phosphorylation ( inact ivat ion) of the enzyme p r o t e i n , p a r t i c u l a r l y i n the in fant r a t i n t e s t i n a l mucosa (Hahn, 1987). Choles tero l 7 alpha-hydroxylase a c t i v i t y i s very low during the suckl ing per iod (Hahn and Innis , 1984). This low rate of turnover of b i l e acids (Hahn, 1986) i s i n d i c a t i v e of the considerable demand for cho le s t ero l for growth and development during the suckl ing per iod , even though one might th ink that b i l e ac id synthesis should be high given the increased consumption of c h o l e s t e r o l . To date there are no studies that have looked at the ontogeny of ACAT i n the r a t . 44 1.3.1 .2 .1 Dietary Intervention In the past few years , there has been a renewed i n t e r e s t i n the e f fec ts of d i e t a r y manipulation on c h o l e s t e r o l metabolism during the suck l ing p e r i o d . Just as the placenta regulates the passage of nutr ients from the mother to the fetus , the mammary glands maintain that funct ion p o s t n a t a l l y . Given that s tudies of breast milk have shown that approximately two t h i r d s of mi lk cho les tero l i s derived from the maternal blood ( P i t k i n et a l , 1972), i t i s not s u r p r i s i n g that the maternal d i e t inf luences c h o l e s t e r o l metabolism i n the suckl ing animal. I t i s , however, not always easy to come to the r i g h t conclus ion concerning the r o l e of d i e t i n development. An example i s the often c i t e d and refuted paper by Reiser and Sidelman (1972), which concluded that an ear ly n u t r i t i o n a l change i n the cho le s t ero l content of milk has an e f fec t on the plasma cho le s t ero l l eve l s of the o f f s p r i n g that p e r s i s t s in to adulthood. This conclusion was based on the c h o l e s t e r o l determination i n the milk of only one mother r a t . Nevertheless, mi lk undoubtedly contains substances that a f f ec t the immediate and probably a lso the l a t e r metabolic pat tern of the i n d i v i d u a l . Thus, the o f f spr ing of mother 45 ra t s d r i n k i n g milk or water from the 14th day of pregnancy up to the 30th day a f t er d e l i v e r y showed the e f fec t of the mother's d i e t (Kritchevsky et a l . , 1983). That i s , the hepat ic cho le s t ero l 7 alpha-hydroxylase was three times more ac t ive i n the o f f spr ing of mi lk - f ed mothers than i n those whose mothers received water throughout. The main conclus ion to be drawn from t h i s experiment i s that i t i s the i n t r a - u t e r i n e per iod that i s more important for future development (Kritchevsky et a l . , 1983). T h i s , however, i s not borne out by other data, which ind ica te that postnatal changes i n d i e t composition play an equal ly important r o l e . Overfeeding neonatal ra t s by reducing the l i t t e r s i ze to 3 from 14 r e s u l t s i n an e levat ion of t o t a l blood c h o l e s t e r o l , thought to be due to the concomitant r i s e i n i n s u l i n l e v e l s . HMG-CoA reductase a c t i v i t y i s decreased i n t h i s circumstance, and the rate of growth i s increased (Hahn and Walker, 1979) . Feeding a HFC d i e t to pregnant rats decreased the a c t i v i t y of HMG-CoA reductase i n suckl ing rats up to the 14th postnatal day, a f t er which no e f fec t was found (Innis , 1985). The decrease i n enzyme a c t i v i t y i s i n agreement with other studies of 21-day-old ra t s born to dams fed a HFC d i e t and suckled by dams fed a contro l d i e t (Naseem et a l . , 1980a; Naseem et a l . , 1980b). 46 E a r l y d i e tary intervent ion with cholestyramine appears to a f f ec t the key enzymes i n cho le s t ero l metabolism. HMG-CoA reductase a c t i v i t y was increased on postnata l days 8 and 14 i n pups born to dams fed _ cholestyramine and cross fostered to dams fed a contro l d i e t (Innis , 1988). Cholestyramine given to nursing dams had a t rans i en t e f fect on the suckl ing pups' hepatic HMG-CoA reductase a c t i v i t y , s ince an increase was seen only to the 14th postnata l day (Innis , 1988; Hassan and Subbiah, 1989). HMG-CoA reductase a c t i v i t y was found to be enhanced three to f i v e times i n the l i v e r s of 17-day-old rat s a r t i f i c i a l l y reared on a low c h o l e s t e r o l d i e t compared to pups i n normal cho le s t ero l and mother-reared groups (Auestad et a l . , 1988). 1.3.1.3 Weaning Period The suckl ing phase gradual ly merges in to the weaning per iod , when s o l i d food increas ing ly replaces the milk d i e t . In the r a t , the d ie tary t r a n s i t i o n per iod commences around the 16th or 17th postnatal day, with the completion of weaning occurr ing around postnatal day 3 0 (Krecek and Kreckova, 1957; Henning, 1981). The rate of cho le s t ero l synthesis increases i n most t i s sues at weaning (except the b r a i n ) , with the l i v e r and the i n t e s t i n a l mucosa becoming the main cho le s t ero l producers (Hahn, 1986). The a c t i v i t y 47 of hepat ic HMG-CoA reductase increases r a p i d l y at weaning i f a high carbohydrate (HG) d i e t i s fed, but remains low i f the r a t i s weaned to a d i e t high i n fat (HF) (McNamara et a l . , 1972; Hahn et a l , 1978). This phase of development i s a lso marked by a decrease i n blood cho le s t ero l (McNamara et a l , 1972; Hahn, 1989). As r a t milk i s thought to contain an i n h i b i t o r of HMG-CoA reductase a c t i v i t y (Hahn, 1987) , the dramatic r i s e i n HMG-CoA reductase i s the r e s u l t of a d i e t -r e l a t e d removal of the i n h i b i t i o n of the reductase a c t i v i t y and a net synthesis i n the reductase p r o t e i n . Rat hepatic 7 alpha-hydroxylase a c t i v i t y i s low i n the suck l ing per iod and r i s e s at the time of weaning (Hahn and Innis , 1984) . The fac tors c o n t r o l l i n g t h i s r i s e are unknown. The high reductase a c t i v i t y associated with lower 7 alpha-hydroxylase a c t i v i t y might be a manifestat ion of the great demand for c h o l e s t e r o l during t h i s ac t ive per iod of growth and development (Naseem et a l , 1980b). The ontogeny of ACAT reveals a dramatic and sudden increase i n i n t e s t i n a l a c t i v i t y at weaning. Hepatic a c t i v i t y decreases at t h i s time ( L i t t l e and Hahn, 1989) . The sudden r i s e i n ACAT a c t i v i t y i n the i n t e s t i n a l mucosa may be a r e f l e c t i o n of the increased rate of cho le s t ero l synthesis occurr ing at weaning. I t i s probable that the developmental changes 48 noted i n the key enzymes i n cho le s t ero l metabolism are a l t e r e d to some extent by the change i n d i e t . A considerable amount of research has been completed i n the area of weaning and n u t r i t i o n . The e f fec t s of premature weaning are l o n g - l a s t i n g and often p e r s i s t into adult l i f e . Premature weaning of female ra t s r e s u l t s i n a 50% increase i n serum cho le s t ero l compared to ra t s weaned at the usual time and subsequently exposed to a high c h o l e s t e r o l d i e t i n adul t l i f e (Hahn et a l , 1978) . Subbiah and others have shown that premature weaning of guinea pigs causes a s l i g h t decrease i n hepatic cho les tero l 7 alpha-hydroxylase that p e r s i s t s to 6 weeks of age and i s not r a i s e d by adding c h o l e s t e r o l to the d i e t (1985). A decrease i n plasma c h o l e s t e r o l l eve l s coupled with a decrease i n glucagon and an increase i n i n s u l i n l eve l s occurs when rats are weaned to a HG d i e t (Hahn et a l . , 1980). Intrauter ine or postnatal exposure to a HFC d i e t r e s u l t s i n a 75% decrease i n HMG-CoA reductase and a 164% increase i n 7 alpha-hydroxylase a c t i v i t y . Weanlings nursed by dams fed a HFC d i e t had a 66% decrease i n HMG-CoA reductase and a 150% increase i n c h o l e s t e r o l 7 a lpha-hydroxylase a c t i v i t y compared to those nursed by mothers on a normal lab d i e t (Naseem et a l . , 1980b). 49 Cholestyramine included i n the d i e t s of post-weanling male rabb i t s immediately reduced a r t e r i a l ACAT a c t i v i t y . This decrease pers i s t ed for an a d d i t i o n a l 9 weeks while the animals were fed a normal HG d i e t (Rymaszewski et a l . , 1986). Feeding cholestyramine-enriched d i e t s to male pups and t h e i r mothers from postnatal day 14 to 21 had no e f fect on the plasma cho le s t ero l l eve l s but s i g n i f i c a n t l y increased reductase a c t i v i t y temporari ly (Innis , 1988). The fact that cholestyramine lacks the a b i l i t y to have any l a s t i n g e f fects on HMG-CoA reductase, supported by the r e s u l t s of other studies that demonstrate the pers i s t en t e f fec t s of ear ly d i e t a r y manipulation on HMG- CoA reductase, suggests that there might be a " c r i t i c a l period" i n development when the adjustment of cho les tero l metabolism can have permanent e f fec t s on metabolism l a t e r i n l i f e (Innis , 1988; Naseem, 1980a; Subbiah et a l . , 1985). To my knowledge the e f fects of a d i e t a r y manipulation (high c h o l e s t e r o l , HF, or HG diet) on the ACAT enzyme has not been looked at prev ious ly . In the past , inves t igators have been concerned with the a b i l i t y of environmental factors to c o n t r o l and a l t e r g e n e t i c a l l y regulated development. This sec t ion has reviewed a number of animal studies which suggest that d i e t a r y manipulation of cho les tero l metabolism during the three phases of development can have pers i s t ent and 50 p e r m a n e n t e f f e c t s o n m e t a b o l i s m . S i n c e d e v e l o p m e n t i s a n o n - r e v e r s i b l e p r o c e s s , t h e r e a p p e a r s t o b e a c r i t i c a l p e r i o d d u r i n g w h i c h c h a n g e s i n t h e d i e t c a n h a v e l a s t i n g c o n s e q u e n c e s . 51 |Table II | Examples of late Effects of Early Adaptation EFFECTOR RESULT IN ADULT PW to HG Diet More prone to hypercholesterolemia PW TO HF Diet Prevents hypercholesterolemia Overnutrit ion * Elevated plasma cholesterol and insul in Undernutrition * Obesi ty Insulin * Obesity and hypercholesterolemia * Between days 3 and 10 after birth 52 1.4 Object ives Very l i t t l e information i s a v a i l a b l e concerning the developmental p r o f i l e of the ACAT enzyme. A bet ter understanding of the r o l e and c o n t r o l of ACAT during development i s needed i n order to trace the poss ib le causes ear ly i n l i f e that lead to a therosc leros i s i n the adul t . The s p e c i f i c object ive of t h i s study was to determine changes i n ACAT a c t i v i t y throughout development i n r a t l i v e r , i n t e s t i n e , aor ta , and adipose t i s sue (BAT and WAT). To further the understanding of the contro l of ACAT the second study aimed to determine the e f fec t s of d ie tary manipulation on the a c t i v i t y of the ACAT enzyme. The t h i r d object ive was to demonstrate whether or not there i s any r e l a t i o n s h i p between serum cho le s t ero l l e v e l s and hepatic and i n t e s t i n a l ACAT a c t i v i t y . 53 2. EXPERIMENTAL 2.1 EXPERIMENTAL RATIONALE AND DESIGN I ACAT i s responsible for the i n t r a c e l l u l a r e s t e r i f i c a t i o n of free cho les tero l to long-chain f a t t y - a c y l c h o l e s t e r o l e s ters . Very l i t t l e information i s ava i l ab l e concerning ACAT a c t i v i t y during development. Of the three enzymes thought to be rate l i m i t i n g i n cho le s t ero l metabolism, HMG-CoA reductase and 7 alpha-hydroxylase show very low a c t i v i t i e s i n the l i v e r of suckl ing ra t s (Hahn and Walker, 1979) and HMG-CoA reductase i s low i n the gut of in fant ra t s compared to prenatal a c t i v i t y l e v e l s (Hahn and Smale, 1981). There i s no information regarding the developmental aspects of ACAT i n the l i v e r and i n t e s t i n e . Due to the major contr ibut ion of extrahepatic t i s sues to c h o l e s t e r o l metabolism i t i s important that some of these a d d i t i o n a l t i s sues be included i n a study about cho les tero l metabolism during development. According ly , the f i r s t study was designed to e s t a b l i s h the ontogeny of ACAT a c t i v i t y i n r a t l i v e r , i n t e s t i n e , aor ta , and adipose t i s sue (BAT and WAT). The serum c h o l e s t e r o l l e v e l s were monitored throughout development i n order to determine i f a r e l a t i o n s h i p ex i s t s between the ACAT enzyme a c t i v i t y and serum cho le s t ero l l e v e l s . 54 To determine the ontogeny of ACAT (study I) the ra t s were k i l l e d on postnatal days 10, 14, 18, 21, 22, 30, and 60. F e t a l t i s sues were obtained on day 21 of gestat ion by caesarean sec t ion . Newborn rats were taken immediately upon d e l i v e r y (no suckling) and 24 hours pos tna ta l l y (suckl ing ad  l ib i tum) before t i s sues were removed fo l lowing decap i ta t ion . The proximal j e j u n a l sect ion of the small i n t e s t i n e was used i n t h i s study because Haugen and Norum have shown that ACAT a c t i v i t y i s the highest i n t h i s por t ion of the gut (1976) . A l l ra t s were weaned on postnatal day 21 to Purina Rat Chow. ACAT a c t i v i t y was measured by the rate of incorporat ion of [ 1 - 1 4 C ] o l eoy l coenzyme A into cho le s t ero l esters according to a method developed by Helgerud et . a l (1981). 55 2.2 EXPERIMENTAL RATIONALE AND DESIGN II I t has been shown that environmental factors ac t ing on the i n d i v i d u a l ear ly i n l i f e may a l t e r the l a t e r development of that i n d i v i d u a l (Hahn, 1989; Hahn, 1987; Hahn and Koldovsky, 1966; Hassan and Subbiah, 1989; Re i ser , 1977). Animal studies have shown that d i e tary manipulation of c h o l e s t e r o l metabolism during an animal's ear ly development can have pers i s t en t and permanent e f fec ts on that organism l a t e r i n l i f e . High plasma cho le s t ero l i s a known r i s k fac tor for a t h e r o s c l e r o s i s . This disease i s the major cause of death and d i s a b i l i t y i n the i n d u s t r i a l i z e d nat ions . Due to the unequivocal l i n k between d i e tary c h o l e s t e r o l and coronary ar tery disease and the fact that ACAT plays a major r o l e i n o v e r a l l cho le s t ero l homeostasis, the c o r o l l a r y of d ie tary manipulation on ACAT i s i n t e g r a l to the complete understanding of the regulat ion of cho le s t ero l metabolism throughout development. The e f fec t of exposure to high d i e t a r y c h o l e s t e r o l , f a t , and carbohydrate upon ACAT a c t i v i t y throughout development i s unknown. Therefore, the second study was designed to determine whether or not a change i n the r a t ' s d i e t has any e f fec t on the a c t i v i t y of the ACAT enzyme. 56 Invest igated i n the second part of t h i s study were the changes i n the a c t i v i t y of ACAT i n the l i v e r , i n t e s t i n e , adipose t i s s u e , and aorta associated with s p e c i f i c modif icat ions i n the d i e t . The d i e tary change included weaning ra t s ear ly on day 18 to one of the fo l lowing d i e t s : 2% c h o l e s t e r o l , high f a t , and high carbohydrate. The rats used i n t h i s experiment were 22, 25, 30 and 60 days o l d . The experimental protoco l was the same as that used i n study I . 57 2.3 MATERIALS AND METHODS 2.3.1 Mater ia l s 2 .3 .1 .1 Animals Wistar s t r a i n (our own breed and from the U n i v e r s i t y of B r i t i s h Columbia, Animal Care) were used i n a l l experiments. A t o t a l of 306 and 111 Wistar ra t s were used i n the developmental (study I) and d i e t (study II) experiments, r e s p e c t i v e l y . 2 .3.1.2 Chemicals A complete l i s t of the chemicals used i n t h i s study and the respect ive suppl iers i s given i n Table I I I AND IV. 2 .3 .1 .3 Equipment A complete l i s t of the equipment used i n the experiments i s out l ined i n Table V. 58 |Table III | Chemicals and Suppliers Suppli es Supplier Chemical;. Chloroform BDH Cupric Sulfate Fisher Diethyl Ether BOH Dithioerythr i tol Sigma Ethanol Fisher Glacial Acetic Acid BDH Methanol Fisher Petroleum Ether BDH Phenol Reagent BDH Potassium Chloride BDH Potassium Phospahte JT Baker Potassium Sodium Tartrate Crystal USBC Sodium Carbonate Fisher Sodium Chloride Fisher Sucrose BDH Proteins and Enzymes Albumin Bovine Sigma Oleoyl Coenzyme A Sigma Radiochemicals Oleoyl Coenzyme A NEN [Oleoyl-1-KC] (Specif ic Act iv i ty = 53.5mCi/mmoI) Diagnostic Kit? Cholesterol and Tr iglyceride B i opac i f i c-D i agnos t i c ChromstO'irE'ehic and Sc in t i l l a t ion Supplies Iodine Fisher L ip id Standards S i gma S i l i c a Gel TLC glass plates BDH Diet Supplies Agar ICN Casein ICN Cholesterol S i gma Choline bi tartrate ICN Corn oiI USBC Dextrose USBC dl-methionine ICN Mineral Mix (AIN) ICN Vitamin Mix (AIN) ICN 59 [Table IV | Suppliers names and addresses NAME ADDRESS Amersham Amersham Canada Ltd. Oakvi l le, Ont. BDH Br i t ish Drug House, Vancouver, B.C. Biopaci f ic Sc ien t i f i c Biopacif ic S c i e n t i f i c Diagnostic Inc., W. Vancouver, B.C. Chemonics Sc ien t i f i c Chemonics S c i e n t i f i c Co. , Richmond, B.C. E.M. Science E.M. Science, Gibbstown, NJ, USA Fisher Fisher S c i e n t i f i c Co. , Vancouver, B.C. J . T . Baker J . T . Baker Chemical , Phi 11ipsburg, NJ, USA • Medigas Medigas Pac i f ic Limited, Vanouver, B.C. NEN New England Nuclear (Canada), Lachine ,Que. S i gma Sigma Chemical Co. , St . Louis Missouri , USA USBC United States Biochemical Corporation, Cleveland, Ohio, USA 1 60 Equipment used in the experiments Equipment TYPE Balance Mettler AE100 Balance Mettler PE300 Centrifuge Beckman model J6B Hand held homogenizer Wheaton 2ml I EC centrifuge IEC N- Analyt ical Evaporator Meyer N-Evap III Polytron homogenizer Brinkman Instruments S c i n t i l l a t i o n counter Beckman LS9000 Spectrophotometer Gi l ford Stasar II Ultracentrifuge Beckman model L7-55 UItracentrifuge Beckman model L8-55 Vert is homogenizer Vertis-23 Vortex-type mixer Fisher Vortex-Genie Waterbath Dubnoff Metabolic Shaking Incubator I 61 2.3.1.4 Diets Male and female white Wistar ra t s used i n the developmental experiments (experiment I) were a l l weaned on postnata l day 21 and fed Purina Rat Chow and water ad  l i b i t u m . Newborn rats were taken immediately at b i r t h and thus they were not allowed access to t h e i r mother's mi lk . The pups taken 24 hours pos tnata l ly were allowed to suckle ad l i b i t u m . Suckl ing rat s were kept i n the same cage as t h e i r mothers; therefore , they had free access to t h e i r mother's mi lk , food, and water. In experiment II (effect of d i e tary manipulation on ACAT a c t i v i t y ) male and female rats aged 22, 25, 30, and 60 days were fed d ie t s of HF, HG, cho le s t ero l - enr iched Purina Rat Chow (2% choles terol ) , or normal Purina Rat Chow. Rats were prematurely weaned on postnatal day 18. The adult rats (day 60) were weaned on postnatal day 18 to Purina Rat Chow and subsequently fed the experimental d i e t for at l eas t 5 days p r i o r to the removal of the t i s s u e s . The composition of the i n d i v i d u a l d i e t s are given i n Tables V I , V I I , and Appendix B. 62 |Table VI | Approximate composition of diets given as a percentage of protein, fa t , carbohydrate (CHO), and other nutrients (minerals, vitamins, elements, f ibre) DIETS (Per cent) Protein Fat CHO Other Purina Rat Chow 23.0 4.5 65.0 7.5 High Carbohydrate 23.0 2.0 69.0 6.0 High Fat 34.0 46.0 11.0 9.0 Cholesterol (2%) 22.0 6.5 64.0 7.0 I |Table VII \ Composition of high fat and high carbohydrate d ie ts . NUTRIENTS High Fat High Carbohydrate (9) (9) Casein 200.0 200.0 D1-methionine 3.0 3.0 Dextrose 66.7 600.0 Corn o i l 268.0 20.0 Mineral Mix (AIN) 35.0 35.0 Vitamin Mix (AIN) 10.0 10.0 Choline bi tartrate 2.0 2.0 Water and Agar 650ml of 1.0% Agar 285ml of 1.0% Agar I 2.3.2 Methods 2 .3 .2 .1 Animal Care The animals were housed i n a temperature c o n t r o l l e d ( 2 3 - 2 5 ° C ) animal un i t with food (Purina Rat Chow or experimental diets) and water a v a i l a b l e ad l i b i t u m . L i g h t i n g was automatical ly regulated prov id ing a 1600-0400 l i g h t and 0400-1600 dark c y c l e . 2 .3 .2 .2 Animal Breeding The ra t s used for experiments performed on postnatal days 10 to 60 were harem-bred by p l a c i n g one male and two females together i n an animal cage for a per iod of approximately 10 days. A f t e r the 10-day breeding per iod the females were removed and housed together u n t i l a few days before the estimated time of d e l i v e r y when they were separated into cages of t h e i r own. On postnata l day 2, the l i t t e r s were sexed and cross - fos tered with l i t t e r s born on the same day; the l i t t e r r a t i o was ten pups per dam. The prenata l , neonatal , and d i e t s tudies required dated pregnancies, thus two female ra t s were placed i n a cage with one male and day 1 of pregnancy was determined by the presence of spermatozoa i n vag ina l smears. The females were 64 then placed alone i n a cage for the remainder of ges tat ion . A d d i t i o n a l dated pregnant ra t s were obtained from the U n i v e r s i t y of B r i t i s h Columbia animal care u n i t . Male and female ra t s were used for a l l experiments. 2 .3 .2 .3 Treatment of Animals Experiments were performed between 0730 and 1000 hours. A l l ra t s (except the f e t a l groups) were k i l l e d by a sharp blow to the head followed by decap i ta t ion . The dams were anesthetized (sodium pentothal 40 mg/kg) and the fetuses were removed by rap id hysterectomy and k i l l e d by decap i ta t ion . 2 .3.2.4 Blood C o l l e c t i o n and Preparat ion Blood samples were c o l l e c t e d immediately fo l lowing decapi ta t ion and then placed on i c e . The blood from f e t a l , neonatal , and the infant ra t s were pooled i n order to c o l l e c t 30 u l of serum for subsequent assays. The samples were centr i fuged at 4000 rpm (Beckman model J6B centrifuge) for 15 minutes i n order to separate the serum from the c e l l s . The serum port ion was then removed and stored i n Eppendorf tubes at - 2 0 ° C u n t i l serum c h o l e s t e r o l and t r i g l y c e r i d e analys i s was performed. 65 2.3 .2 .5 Serum T r i g l y c e r i d e Ana lys i s : P r i n c i p l e Serum t r i g l y c e r i d e s are hydrolyzed to g l y c e r o l and free f a t t y acids (FFA). The g l y c e r o l i s converted to g l y c e r o l - 1 -phosphate when ATP and g l y c e r o l kinase (GK) are added i n v i t r o . The g lycero l - l -phosphate i s then ox id ized to hydrogen peroxide(H 2 0 2 ) by g l y c e r o l phosphate oxidase. In the presence of peroxidase the H 2 0 2 condenses with 3,5-dichloro-2-hydroxy-benzenesulfonic ac id (DHBS) to y i e l d a red co lored quinoneimine dye. The i n t e n s i t y of the c o l o r i s d i r e c t l y proport iona l to the concentrat ion of t r i g l y c e r i d e s i n the sample. 2 .3 .2 .6 Serum Choles tero l A n a l y s i s : P r i n c i p l e Choles tero l esters are hydrolyzed to free cho le s t ero l by cho le s t ero l esterase (CE). In the presence of c h o l e s t e r o l oxidase (CO) free cho le s t ero l i s ox id ized to cholesten-3-one and H 2 0 2 . When peroxidase i s added the H 2 0 2 couples with 4-aminoantipyrine and phenol to y i e l d quinoneimine and water. The i n t e n s i t y of the c o l o r i n the sample i s d i r e c t l y proport iona l to the concentrat ion of t o t a l cho le s t ero l (Diagnostic Chemicals Manual, 1987). 66 2 .3 .2 .6 .1 Procedure Used Blood for serum cho le s t ero l and t r i g l y c e r i d e analys i s was obtained immediately fo l lowing decap i ta t ion . T o t a l serum cho le s t ero l and t r i g l y c e r i d e assay k i t s (B iopac i f i c Diagnostic) were used for i n v i t r o quant i ta t ive determination of t o t a l serum cho le s t ero l and t r i g l y c e r i d e s . 2 .3 .2 .7 Preparat ion of Tissue for Enzyme Assay The r a t t i s sues ( l i v e r , BAT, WAT, and aorta) were r a p i d l y excised and c h i l l e d on i c e . The duodenal segment of the bowel was removed and d iscarded. The next 20-cm was removed and washed with 0.9% ice co ld s a l i n e containing ImM of D i t h i o e r y t h r i t o l (DTT). This segment was opened l o n g i t u d i n a l l y and the mucosa was scraped with a g lass s l i d e to a consis tent depth and placed i n 1ml of 0.2M sucrose s o l u t i o n . The i n t a c t segment d i s t a l to the duodenum i n the f e t a l and neonatal ra t s was taken and homogenized without p r i o r scraping . Mucosal samples from the in fant and young adul t ra t s were pooled together. The mucosa/sucrose s o l u t i o n was gently mixed and centr i fuged at 2500 x g for 10 minutes at 4°C (Beckman model J6B centrifuge) to remove the unbroken c e l l s and debr i s . The mucosal p e l l e t was then suspended i n 1 ml of the sucrose buffer and homogenized with a motor-driven v e r t i s homogenizer with 4 continuous passes 67 with the pes t l e . The glass tubes were placed on i ce during homogenization. The homogenate was centr i fuged at 6800 x g for 15 minutes at 4 °C i n an IEC centr i fuge with an Internat iona l IEC ro tor (cat.# 874) i n order to sediment the c e l l u l a r debr i s , mitochondria, n u c l e i , and most of the lysosomes (Norum et a l . , 1979). The 'microsomal f r a c t i o n ' was obtained by centr i fug ing the supernatant at 140,000 x g for 30 min at 4 °C i n a Beckman L7-55 or a L8-55 u l t r a c e n t r i f u g e using a Type 50.3 or a Type 25 T i r o t o r . The p e l l e t was suspended i n 0.2M potassium phosphate (KH 2 P0 4 ) buffer (pH 7.4) and manually homogenized using a Wheaton hand-held t i g h t - f i t t i n g homogenizer. The homogenate was then stored at -80 ° C . I t has been found that the ACAT enzyme i s s table at t h i s temperature for long periods of a time (up to 1 1/2 years) (Helgerud et a l . , 1981). Procedure for the l i v e r , adipose t i s s u e , and aorta were as stated for the i n t e s t i n a l mucosa excluding suspension of the t i s s u e i n the sucrose buffer and the i n i t i a l c en tr i fuga t ion step (2500 x g for 10 minutes); t h i s step was omitted. The l i v e r t i s sues were homogenized as explained for the i n t e s t i n a l mucosa. The adipose t i s s u e and a o r t i c samples were homogenized i n .02M sucrose buf fer using a Brinkman Polytron homogenizer. The aorta and BAT samples 68 were always pooled together (2-9 t i s sues per sample). Subsequent steps were as stated for the i n t e s t i n a l mucosa. 2 .3 .2 .8 Prote in Analys i s The pro te in content of the samples was analyzed according to Lowry et a l . (1951) using bovine serum albumin (BSA) as a standard ( G i l f o r d Stasar II spectrophotometer at 660 nm). 2 .3 .2 .9 Preparation of Radio labe l l ed Oleoyl Coenzyme A 0 1 e o y l - l - 1 4 C (53.5 mCi/mmol) was purchased from NEN and stored at -20 ° C . P r i o r to i t s use i n the ACAT assay, the radio isotope was reconst i tuted i n 3.3 ml of 0.01 M sodium acetate and ethanol buffer (pH 6.0) and 1.7 ml of unlabe l l ed o l eoy l CoA containing 5ml of the sodium acetate and ethanol buffer and 5mg of o l eoy l CoA. The o l e o y l - l - 1 4 C preparat ion was kept i n approximately 1ml a l iquot s and the unused port ions of the l a b e l l e d substrate and buf fer was stored at -20 °C for use at a l a t e r date. 2 .3.2.10 Enzyme Assay 2 .3 .2 .10.1 P r i n c i p l e of Assay The ACAT enzyme has been extensively s tudied s ince i t s discovery by Mukherjee and co-workers i n 1958. The ear ly 69 experiments inves t iga t ing the e s t e r i f i c a t i o n of cho les tero l by s u b c e l l u l a r f rac t ions used exogenous l a b e l l e d cho les tero l as a substrate . However, t h i s method evoked questions concerning the heterogeneity of the l a b e l l e d substrate . (Spector, 1979). The most popular ACAT assay employed today uses 1 4 C - l a b e l l e d fa t ty acyl-CoA as a substrate . I t has been found that oleoyl-CoA i s the best substrate for the ACAT assay, with the rates of synthesis from a c y l CoA being c h o l e s t e r y l oleate > palmitate > stearate > l i n o l e a t e (Goodman et a l . , 1964). In a d d i t i o n , c h o l e s t e r o l esters formed within the c e l l are synthesized p r i m a r i l y from c h o l e s t e r y l oleate (Spector et a l . , 1979). There appears to be no value i n adding any exogenous c h o l e s t e r o l to the system when an a c y l CoA i s used (Spector et a l . , 1979). The highest ACAT a c t i v i t y i n r a t and guinea p i g l i v e r homogenates i s found i n the microsomal f r a c t i o n (Helgerud et a l . , 1981; Stokke and Norum, 1970; Beck and Drevon, 1978). Although many v a r i a t i o n s of the ACAT assay ex i s t the procedures a l l have a number of un iversa l c h a r a c t e r i s t i c s . Customari ly , about 1 mg of microsomal p r o t e i n i s incubated with a f a t t y acyl -CoA, a t h i o l , and fa t ty a c i d free BSA. The BSA serves as a protec t ive mechanism thus preventing the s o l u b i l i z a t i o n of the membrane by fa t ty acyl-CoA when high concentrations of the acyl-CoA are used. The rate of the 70 cho le s t ero l ester formation i s dependent upon the concentrat ion of the substrate added to the system. Moderate but increas ing concentrations of f a t t y acyl-CoA w i l l s t imulate the formation of cho le s t ero l e s ters , however, a f t er a plateau region i s achieved a d d i t i o n a l substrate causes i n h i b i t i o n . This i s probably due to the detergent propert ies of the fa t ty acyl -CoA. I t has been es tabl i shed that a 2 minute react ion time i s pre ferred when using o leoy l CoA as a substrate and 10 minutes must be allowed for when using palmitoyl -CoA. Upon completion of the reac t ion period the reac t ion i s general ly hal ted by adding chloroform-methanol. The extracted l i p i d s , t r i g l y c e r i d e (TG), free c h o l e s t e r o l (FC), cho le s t ero l es ter (CE), f a t t y acids (FA) are then separated by t h i n layer chromatography (TLC). A s o l u t i o n of petroleum ether, d i e t h y l ether, and a c e t i c ac id i s genera l ly used as an e luent . 2 .3 .2 .10.2 Procedure Used The assay of ACAT a c t i v i t y was performed as described by Helgerud, Saarem, and Norum (1981) with a few modi f i ca t ions . The a c t i v i t y of t h i s enzyme was determined by the formation of l a b e l l e d cho le s t ero l esters from [ 1 - 1 4 C ] o l eoy l CoA and endogenous c h o l e s t e r o l . The f i n a l incubation mixture contained 120-150 ug microsomal p r o t e i n , 100 u l of 71 5% BSA i n 0.2 M potassium phosphate (KH 2 P0 4 ) buffer(pH 7.4) , and 200 -300 u l of KH 2 P0 4 buffer to make the f i n a l volume to 450 u l . Incubations were c a r r i e d out i n g lass tubes shaken continuously i n a shaking water bath for 5 minutes at 37 ° C . The reac t ion was s tar ted by the add i t ion of [ 1 - 1 4 C ] o leoy l CoA. A standard react ion time of 2 minutes was used. The reac t ion was stopped by adding 10 ml of chloroform-methanol (2:1 v/v) and 0.5 ml of dH 2 0. The reac t ion mixture was then spun at 2000 rpm for 5 minutes 4 °C (Beckman model J6B centrifuge) to separate the phases. The top water phase was then removed and 2ml of 0.88% potassium c h l o r i d e (KCl) was added to wash the extract before spinning at 2000 rpm for 10 minutes. The chloroform phase was then t rans ferred to glass v i a l s and d r i e d under n i trogen. The remaining l i p i d extract was resuspended i n lOOul of chloroform and appl ied to s i l i c a ge l G t h i n layer chromatography (TLC) p l a t e s . The p lates were developed with petroleum ether: d i e t h y l ether: ace t i c a c i d (83:17:3 v/v) and the band of c h o l e s t e r o l ester was located by v i s u a l i z i n g with iodine vapor. The cho les tero l es ter zone was scraped o f f the p la te with a small putty kn i fe and placed i n s c i n t i l l a t i o n v i a l s . The r a d i o a c t i v i t y was measured by l i q u i d s c i n t i l l a t i o n with 2 ml s c i n t i l l a t i o n f l u i d i n a Beckman LS9000 L i q u i d S c i n t i l l a t i o n System. 72 E s t e r i f i c a t i o n rates were ca lcu la ted as pmol of cho l e s t ery l [ 1 - 1 4 C ] oleate formed per mg microsomal pro te in per minute. 2 .3 .2 .11 S t a t i s t i c a l Analys i s Data are given as means + standard e r r o r of 6 or more determinations. A one way analys i s of variance (ANOVA) model was used to t e s t the hypothesis of equa l i ty of ACAT a c t i v i t y throughout development (developmental study) and the equa l i ty of responses to d i f f e r e n t d i e t a r y regimes (diet s tudy) . Once the n u l l hypothesis was re jec ted the Tukey-Kramer mul t ip le comparisons t e s t was performed to e s t a b l i s h which means were s i g n i f i c a n t l y d i f f e r e n t from each other. This was done for both s tudies . The computerized s t a t i s t i c a l package SYSTAT was used. 73 3. RESULTS 3 .1 . Developmental Study Developmental changes i n the a c t i v i t y of the ACAT enzyme as measured by the rate of e s t e r i f i c a t i o n of ( 1 - 1 4 C ) o l e o y l CoA to cho les tero l i n r a t l i v e r and i n t e s t i n e are shown i n Figure 4 and 5 and the r e s u l t s for the adipose t i s sue (BAT and WAT) and aorta appear i n Tables VIII and IX. ACAT a c t i v i t y i s expressed as pmol'mg p r o t e i n - 1 ' m i n - 1 , mean + the standard error ( S . E . ) . 3 .1.1 Development of ACAT i n L i v e r A c t i v i t y was low i n f e t a l t i s sue (age = -1) and decreased to a nadir at b i r t h (age = 0) . A c t i v i t y progress ive ly increased during the suck l ing per iod to postnata l day 14 (161.30 + 19.438 pmol'mg p r o t e i n " 1 ' m i n - 1 ) and then dec l ined to less than h a l f of t h i s value on postnata l days 18, 21, 22, 25, and 30. The ra t s were weaned on day 21. The highest values were found i n the adult animal (166.68 + 15.30 pmol.mg p r o t e i n - 1 .min" 1 ) (Figure 4). 74 c ' E \ c '<L> ~o C l > I— U < I— < < 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 AGE (DAYS) F i g u r e 4 . O n t o g e n y o f A C A T in L i v e r . A C A T a c t i v i t y ( m e a n + S . E . o f 6 o r m o r e r a t s ) e x p r e s s e d a s p m o l / m g P r / m i n a s a f u n c t i o n o f a g e . T h e f e t a l a n d n e o n a t a l v a l u e s w e r e o b t a i n e d o n d a y 2 1 g e s t a t i o n ( - 1 ) , a t b i r t h ( 0 ) , a n d 2 4 h o u r s p o s t n a t a l ( 1 ) , r e s p e c t i v e l y . N u m b e r s n e x t t o t h e d a t a p o i n t s d e n o t e a g e in d a y s . P < . 0 0 1 v s . - 1 ( F e t u s ) f o r a l l g r o u p s e x c e p t d a y s 0 , 1 , 2 2 , 2 5 ; P < . 0 0 2 v s . 1 4 d a y f o r a l l a g e g r o u p s e x c e p t d a y 6 0 ; P < 0 . 0 5 v s 2 5 d a y f o r d a y s 1 0 , 1 4 , 1 8 , a n d 6 0 ; • P < . 0 0 1 v s . 6 0 d a y f o r a l l a g e g r o u p s e x c e p t d a y 1 4 . 75 3.1.2 Development of ACAT i n I n t e s t i n a l Mucosa ACAT a c t i v i t y decreased s i g n i f i c a n t l y from a moderately high f e t a l value ( 70.93 + 5.326 pmol.mg p r o t e i n - 1 . m i n - 1 ) to v i r t u a l l y no a c t i v i t y on day 14 (Figure 2) . I t then increased reaching maximal values on day 22 (151.65 + 12.298 pmol.mg pro te in .min •L) before decreasing again to day 60 (9.43 + 3.677 pmol* mg p r o t e i n - 1 'min""1 ) . 3.1.3 Development of ACAT i n BAT ACAT a c t i v i t y was low i n BAT throughout development (Table V I I I ) . During the post-weaning per iod the ACAT a c t i v i t y increased from 6.47 + 1.663 pmol.mg p r o t e i n - 1 . m i n - 1 (postnatal day 30) to 20.28 + 4.093 pmol.mg p r o t e i n - 1 . m i n - 1 (postnatal day 60). 3.1.4 Development of ACAT i n WAT WAT contained i n s i g n i f i c a n t amounts c h o l e s t e r o l -e s t e r i f y i n g a c t i v i t y throughout development compared to the a c t i v i t y i n the l i v e r and i n t e s t i n a l mucosa during the same per iod (Table V I I I ) . The r e s u l t s for WAT ACAT a c t i v i t y from postnata l day 10 to 60 were a l l l e s s than 3.50 pmol'mg p r o t e i n - 1 ' m i n - 1 . 76 180-1 c E \ 1 6 0 -c 1 4 0 -o ct en 1 2 0 -E \ 1 0 0 -o c b CL 8 0 -VITY 6 0 -i — o < 4 0 -i — < < 2 0 -0 -^ W E A N I N G (21) T -+- -+- -+-I N T E S T I N E -+- •+-F i g u r e 5 . - 5 0 5 10 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 AGE (DAYS) O n t o g e n y o f A C A T a c t i v i t y in I n t e s t i n a l M u c o s a . A C A T a c t i v i t y ( m e a n + S . E . o f 6 o r m o r e r a t s ) e x p r e s s e d a s p m o l / m g P r / m i n u t e a s a f u n c t i o n o f a g e . The f e t a l a n d n e o n a t a l v a l u e s w e r e o b t a i n e d o n d a y 21 g e s t a t i o n ( - 1 ) - ° t b i r t h ( 0 ) , a n d 2 4 h o u r s p o s t n a t a l ( 1 ) , r e s p e c t i v e l y . P < .001 v s . 2 2 d a y f o r a l l g r o u p s ; P < . 0 5 v s . - 1 ( F e t u s ) f o r a l l g r o u p s e x c e p t d a y s 0 a n d 2 5 ; P < . 0 5 v s . 1 4 d a y o l d f o r d a y s - 1 , 0 , 2 2 , 2 5 . 77 liable VI i T ACAT ac t i v i t y (mean ± S .E . of 5 or more rats ) expressed as a pmol/mgPr/min throughout development in rat adipose t issue. ADIPOSE TISSUE Age B A T WA T (Days) ACAT Act iv i ty Standard Error ACAT Act iv i ty Standard Error Fetus 3.59 (a) 1.20 n/a n/a 0 0.15 (a) 0.06 n/a n/a 1 1.48 (a) 0.86 n/a n/a 10 1.30 (a) 0.53 3.32 (b) 1.30 14 0.12 (a) 0.07 0.21 0.07 21 0.40 (a) 0.15 2.24 (b) 1.20 22 3.89 (a) 1.90 2.15 0.66 25 0.32 (a) 0.23 0.15 0.10 30 6.48 (a) 1.70 1.05 0.28 60 20.28 4.10 1.22 0.32 (a) P < .001 vs . 60 day old (b) P < .05 vs . 14 day old |Table IX ' ACAT ac t i v i t y (mean ± S .E . of 3 or more samples ) expressed as pmol/mgPr/min throughout development in rat aorta . . Five or more rat aortae' were pooled together to make up 1 sample. Age (Days) A O R T A ACAT Act iv i ty Standard Error Fetus n/a n/a 0 n/a n/a 1 n/a n/a 10 0.74 0.32 14 7.65 4.67 21 2.75 0.31 25 10.69 10.62 30 7.80 4.25 60 9.63 5.12 78 3.1.5 Development of ACAT i n Aorta A c t i v i t y of ACAT i n the r a t aorta was found to be n e g l i g i b l e i n comparison to the other t i s s u e s . However, r e l a t i v e l y speaking, the a c t i v i t y increased from 0.743 + — i • — l 0.32 pmol.mg prote in .min at day 10 to reach a peak on days 21 and 25 (10.87 + 10.617 pmol.mg p r o t e i n - 1 . m i n - 1 ) (Table IX) . The cho les tero l e s t e r i f y i n g a c t i v i t y i n the aorta demonstrated large v a r i a t i o n s wi th in the age groups. 3.1.6 Serum Choles tero l and T r i g l y c e r i d e During Development Further studies were done to evaluate the r e l a t i o n s h i p of ACAT a c t i v i t y to serum cho le s t ero l concentrat ions . Serum t o t a l cho le s t ero l l eve l s (Figure 6) increased from low l e v e l s i n the fetus (58.65 + 4.85 mg/dl) to high l e v e l s on the 10th postnatal day (171.90 + 2.89 mg/dl ) . I t r a p i d l y decreased with increas ing age during the mid-suckl ing period reaching a low the day immediately fo l lowing weaning (day 2 2 = 85.72 + 6.42 mg/dl ) . Choles tero l l e v e l s then increased on days 25 and 30 (second peak on day 30 = 135.53 mg/dl) and then i t plummeted on day 60 down to 72.75 mg/dl . Serum t r i g l y c e r i d e (TG) concentrations (Figure 6) revealed a s i m i l a r two-peak developmental curve. F e t a l TG 79 l e v e l s were high (152.75 mg/dl) but values decreased to 118.40 mg/dl at b i r t h . The serum TG l e v e l s were s l i g h t l y increased i n animals who were allowed to suckle for up to 24 hours. Peak concentrations were obtained on day 10 (172.45 mg/dl) . The serum TG l eve l s then decreased on days 14 (141.50 mg/dl) and 21 (119.50 mg/dl ) . The l e v e l s increased again on day 22 (149.59 mg/dl) and stayed v i r t u a l l y the same through to postnatal day 30 before decreasing to 117.94 mg/dl on day 60. 80 2 2 0 2 0 0 1 8 0 ^ 1 6 0 1 4 0 -1 2 0 -1 0 0 -8 0 -6 0 -4 0 -2 0 -+-• C H O L E S T E R O L • T R I G L Y C E R I D E + u r e 6 . - 5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 AGE (DAYS) P l a s m a c h o l e s t e r o l a n d t r i g l y c e r i d e l e v e l s ( m g / d l ) t h r o u g h o u t d e v e l o p m e n t . T h e a g e s - 1 , 0 , a n d 1 r e f e r t o t h e f e t u s ( d a y 2 1 g e s t a t i o n ) , a n d n e o n a t e s ( a t b i r t h a n d 2 4 h o u r s o l d ) . M e a n + S . E . o f 2 o r m o r e d e t e r m i n a t i o n s . ( P < . 0 5 ) . 81 3.2 Diet Study In study I I , the ra t s were weaned e a r l y at postnatal day 18 to one of the fol lowing experimental d i e t s : HG, HF, or 2% c h o l e s t e r o l . With the exception of adul t r a t s , the experimental d i e tary regime was maintained u n t i l the rats were k i l l e d . The adult ra t s were fed the experimental d ie t s for at l eas t 5 days before they were k i l l e d . 3.2.1 L i v e r The high fat d i e t was the only d i e t that cons i s t ent ly increased hepatic ACAT a c t i v i t y throughout development (P < .001; Figure 7) . In general a l l three d ie t s caused an increase i n the enzyme's a c t i v i t y . The exception was i n the adult animal where i t was found that the 2% c h o l e s t e r o l d i e t s i g n i f i c a n t l y decreased ACAT a c t i v i t y and the HG d i e t had no e f f e c t . There was a s i g n i f i c a n t increase i n the 22 and 25 day o ld hepatic ACAT a c t i v i t y i n a l l the experimental groups (P < 0.001). The HF and HG d ie t s s i g n i f i c a n t l y increased ACAT a c t i v i t y i n the 30 day o ld r a t s . Both the HF and HG d ie t s caused marked v a r i a t i o n s i n ACAT a c t i v i t y throughout the per iod of development that was s tudied (across age groups). The 2% cho le s tero l d i e t caused a s i g n i f i c a n t change i n a c t i v i t y of ACAT i n the 30 and 60 day o ld animals compared to the 22 day o ld animals fed the contro l d i e t . 82 However, there was no d i f ference between the 22 day o ld contro l a c t i v i t y and the 25 day o l d animals fed 2% c h o l e s t e r o l . 83 c 3 2 0 n E 3 0 0 : \ c 2 8 0 : <D 2 6 0 -O _^ Q_ 2 4 0 : o> 2 2 0 : E 2 0 0 -\ o 1 8 0 -E 1 6 0 -CL 1 4 0 : > 1 2 0 : > 1 0 0 -o < 8 0 . -1— 6 0 -< 4 0 : < 2 0 : 0 J C O N T R O L ( P u r i n a R a t C h o w ) HIGH C A R B O H Y D R A T E L S HIGH FAT E S 2% C H O L E S T E R O L L I V E R F i g u r e 7 . 2 5 A G E ( D A Y S ) T h e e f f e c t s o f d i e t s o n l i ve r A C A T a c t v i t y ( m e a n + S . E . o f 4 o r m o r e r a t s ) e x p r e s s e d a s p m o l / m g P r / m i n a s - a f u n c t i o n of a g e . * Day 2 2 P < .001 v s . C o n t r o l ; Day 2 5 P < . 001 v s . C o n t r o l : D a y 3 0 P < . 0 0 2 v s . C o n t r o l D a y 6 0 P < . 0 2 v s . C o n t r o l 84 3.2.2 Intest ine The most s t r i k i n g feature of the r e s u l t s of d i e tary manipulation i s the dramatic decrease i n a c t i v i t y i n the HF and HG d i e t groups at day 22 (Figure 8) . The 2% cho les tero l d i e t s i g n i f i c a n t l y increased the ACAT a c t i v i t y i n r a t s aged 25 and 30 but no e f fec t was noted on days 22 and 60. The HG d i e t decreased the enzyme a c t i v i t y i n a l l the age groups except the 30 day o ld r a t s . 3.2.3 WAT As mentioned before i t was found that the WAT contained i n s i g n i f i c a n t amounts of ACAT a c t i v i t y throughout development. The e f fec ts of the d i e t s during the post-weaning per iod were marginal i n t h i s t i s sue with one exception: HG d i e t increased ACAT a c t i v i t y more than 4 - fo ld (Table X ) . 85 c "E c IV o o_ cn E \ o E CL < I— < < 1 6 0 1 4 0 1 2 0 1 0 0 8 0 6 0 4 0 2 0 -0 C O N T R O L ( R o t C h o w ) H I G H C A R B O H Y D R A T E H I G H F A T K S 3 2% C H O L E S T E R O L E i rh —, 6 0 2 5 A G E ( D A Y S ) F i g u r e 8 . T h e e f f e c t s o f d i e t s o n I n t e s t i n a l M u c o s a . A C A T a c t i v i t y ( m e a n + S . E . o f 4 o r m o r e s a m p l e s ) e x p r e s s e d a s p m o l / m g P r / m i n a s a f u n c t i o n o f a g e . * D a y 2 2 P < . 0 0 1 v s . C o n t r o l D a y 2 5 P < . 0 0 5 v s . C o n t r o l D a y 3 0 P < . 0 0 1 v s . C o n t r o l . 86 |Tabte X ~| The ef fects of diets on ACAT ac t iv i ty (pmol/mgPr/min) in UAT + standard error for sample size (n) 01 ETS Age Control High Carbohydrate «fgh Fat 2% C hotesteral (Days) ACAT Act iv i ty Standard Error (n) ACAT Act iv i ty Standard Error (n) ACAT Act iv i ty Standard Error (n) ACAT c t i v i t y Standard Error (n) 22 25 30 60 2.153 0.153 1.049 1.217 0.659 1.000 0.281 0.319 12 8 32 16 1.755 1.080 4.850 1.160 0.562 0.389 0.205 0.690 11 6 6 5 0.645 0.448 0..643 1.128 0.345 0.176 0.245 0.446 10 6 7 4 0.800 2.450 1.478 0.328 1.644 0.755 7 8 4 |»--X-M-:v.-K-:-:.: 87 3.2.4 BAT The most s a l i e n t feature i n Figure 9 i s the reduced ACAT a c t i v i t y i n adult BAT for a l l the d i e t groups, p a r t i c u l a r l y the dramatic decrease noted i n the adult 2% c h o l e s t e r o l group (P < 0.05 for a l l 3 d i e t s versus the c o n t r o l ) . No s i g n i f i c a n t changes were found i n the 22 and 30 day o ld animals that were given the experimental d i e t s . The HG and HF d ie t s appear to cause a s l i g h t increase i n the ACAT a c t i v i t y i n the 25 day-old animals, however t h i s increase was not s i g n i f i c a n t . No change was noted with the high cho le s t ero l d i e t . 3.2.5 Aorta A l l three d i e t s r e l a t i v e l y decreased the ACAT a c t i v i t y i n the 30 and 60 day o ld a o r t i c samples (Table XI) . There was not enough t i s sue and data from postnatal day 22 for a n a l y s i s . 88 c "E \ c '<u o l_ CL E \ o E CL > o < I— < < CONTROL HIGH CARBOHYDRATE HIGH FAT ^ 2% CHOLESTEROL 25 i 20 J 10 5 0 Figure 9. BAT i 22 I X * 60 25 AGE (DAYS) The ef fects of diets on BAT ACAT activity (mean + S.E. of 4 or more samp les ) expressed as p m o l / m g p ro te in /m inu te as a funct ion of age. (* P < .05 vs. Control) 89 |Table XI The ef fects of diets on ACAT act iv i ty (pmol/mgPr/min) in Aorta ± standard error for sample size (n) DIETS Age CGH1R0. High- Carbohvcrate High Fat :;:;<? s;12%: C idtes.terol (Days) ACAT Standard ACAT Standard ACAT Standard ACAT Standard Act iv i ty Error (n) Act iv i ty Error (n) Act iv i ty Error (n) Ac t i v i t y Error (n) 21 2.748 0.313 4 . . 0 _ 0.000 0 . -25 10.687 10.617 3 0.000 0.000 3 0.000 0.000 0 0.000 - 4 30 7.801 4.251 9 1.500 0.000 4 0.335 0.215 4 6.925 6.055 3 60 9.633 5.118 6 5.960 0.000 4 4.690 0.351 5 2.357 0.807 4 90 3.2.6 Serum Cholesterol and T r i g l y c e r i d e Levels The resul t s from the analys is of the serum cho les tero l and t r i g l y c e r i d e l eve l s appear i n Table XII . From t h i s data i t i s apparent that the HF and HG d ie t s increased the serum cho les tero l l eve ls i n the rats aged 21, 22, 25, and 30. The 2% choles tero l d ie t increased the serum c h o l e s t e r o l l eve l s i n the 30 and 60 day o ld animals. 3.4 S t a t i s t i c a l Analys i s A one way analys is of variance (ANOVA) model was used to t e s t the hypothesis of equa l i ty of ACAT a c t i v i t y throughout development (developmental study I ) . The one way ANOVA suggests that hypothesis i s re jec ted at the s ign i f i cance l e v e l of: L iver P < .001 (F = 25.756) Intestine P < .001 (F = 21.462) WAT P < .006 (F = 3.216) BAT P < .000 (F = 11.194) Aorta P < .784 (F = 0.484). In the d ie t study (II) s ign i f i cances of d i f f erence were tested using the one-way ANOVA t e s t . The one-way ANOVA 91 suggest that the ACAT a c t i v i t y means (for contro l and experimental d i e t s wi th in one t issue) are s i g n i f i c a n t l y d i f f e r e n t at a s i gn i f i cance l e v e l of: L i v e r P < .001 Intest ine P < .003. 92 |Tabte XII | Plasma Cholesterol and Triglyceride levels in animals fed Control, High Carbohydrate, High Fat and 2% Cholesterol Diets. Age (Days) Cholesterol and Triglyceride levels (mg / d l ) C0MTR.0L High CSFI aOKydrate High Fat 2% Cholesterol TG Cholesterol TG Cholesterol TG Cholesterol TG Cholesterol 21 136 74 103 91 142 136 - -22 155 72 104 86 250 143 - • 25 133 108 110 115 198 140 - -30 94 93 61 124 88 109 110 92 60 95 62 162 69 93 4. DISCUSSION 4.1 Ontogeny of ACAT 4.1.1 L i v e r This study examines for the f i r s t time the developmental patterns of ACAT a c t i v i t y i n the r a t l i v e r , i n t e s t i n e , aorta and adipose t i s sue (BAT and WAT) from the l a t e f e t a l stage to the adu l t . The major features of the ontogeny of r a t hepatic ACAT a c t i v i t y are the very low a c t i v i t i e s noted i n the fetus and newborn, the sharp r i s e i n a c t i v i t y during the suckl ing per iod followed by an equal ly sharp dec l ine fo l lowing spontaneous weaning, and the highest hepatic a c t i v i t y throughout development noted i n the adult animal. The fact that the developmental pat tern of hepatic ACAT reveals that there i s l i t t l e i f any cho les tero l e s t e r i f i c a t i o n mediated by the ACAT reac t ion prenata l ly ( late term) and at b i r t h and that t h i s a c t i v i t y increases s i g n i f i c a n t l y 24 hours pos tnata l ly indicates that ACAT may belong to "neonatal" c l u s t e r of enzymes described by Greengard (1970). Greengard suggests that the severe neonatal hypoglycemia associated with b i r t h t r i g g e r s the re lease of glucagon which i n turn st imulates the synthesis of t h i s c l u s t e r of enzymes. The fact that J i a o and co-94 workers (1988) have reported that i n s u l i n i n h i b i t s ACAT a c t i v i t y i n adult ra t s further supports the idea that ACAT may be ac t ivated by glucagon i n the neonatal p e r i o d . The peak a c t i v i t y observed on postnatal day 14 i s most l i k e l y r e l a t e d to the cho les tero l i n the d i e t . More c h o l e s t e r o l i s c i r c u l a t i n g i n the body at the mid-suckl ing stage as i s evident from the high serum c h o l e s t e r o l values . One could speculate that the ACAT enzyme i s induced during the suckl ing per iod by the high fat milk d i e t although the e f fec ts are not apparent for some time as i s the case i n several other enzymes. As mentioned i n the in t roduc t ion , several authors have proposed that there i s an inverse r e l a t i o n s h i p between HMG-CoA reductase and ACAT a c t i v i t y . I f t h i s i s the case then one would expect the a c t i v i t y of ACAT to be high during the suckl ing per iod because hepatic HMG-CoA reductase a c t i v i t y i s the lowest during t h i s time (McNamara et a l . 1972). Furthermore, plasma cho le s t ero l l e v e l s reach a peak during t h i s time (McNamara et a l . 1972). In the present study the a c t i v i t y of ACAT reached a peak at mid-suckl ing on postnatal day 14. Thus cho les tero l metabolism during the suckl ing per iod i n the r a t l i v e r appears to be fo l lowing the same sequence as the LDL pathway proposed by Brown and Goldste in (1984; see Figure 2) . The 95 dramatic increase noted at the mid-suckl ing stage could be due to d i e t - r e l a t e d induct ion of the enzyme. S i m i l a r l y , the decrease i n hepat ic ACAT a c t i v i t y observed between days 18 and 25 seems to be d i e t - r e l a t e d . This r a p i d decrease may be a t t r i b u t e d to the in fant rats n i b b l i n g on t h e i r mother's high carbohydrate chow. I t has been shown that the d i e tary t r a n s i t i o n per iod commences around the 16th or 17th postnatal day i n the r a t (Krecek and Kreckova, 1957; Henning, 1981). The decrease i n serum c h o l e s t e r o l l eve l s i n t h i s study r e f l e c t s the change i n the r a t ' s d i e t . The a c t i v i t y of ACAT increased almost four-f o l d from postnatal day 25 to 60. Why t h i s enzyme would be so ac t ive i n the adult male ra t s i s d i f f i c u l t to assess. However, one can speculate that the endogenous cho le s t ero l synthesized i s not been u t i l i z e d for b iosynthet i c purposes and thus the excess i s stored as a c y l e s ters . I t i s poss ib le that t h i s high rate of cho le s t ero l e s t e r i f i c a t i o n might be sex -re la ted . Hahn (unpublished data) has observed that the ACAT a c t i v i t y i n adult female r a t s were much lower than that of t h e i r male l i t t e r - m a t e s . 96 4.1.2 Intest ine I t i s apparent from the data presented that the r a t i n t e s t i n a l mucosa possesses s i g n i f i c a n t amounts of ACAT a c t i v i t y at various stages of development. The key observations i n the developmental pat tern of ACAT i n the i n t e s t i n e are as fol lows: r e l a t i v e l y high prenata l values which decrease with age up to the 14th postnata l day; low values throughout the suckl ing phase; a dramatic r i s e i n ACAT a c t i v i t y fo l lowing complete weaning; and an equal ly dramatic decrease i n ACAT a c t i v i t y to the 60th postnatal day. One of the most i n t r i g u i n g observations i n t h i s study i s the high ACAT a c t i v i t y i n the f e t a l i n t e s t i n a l mucosa. Why t h i s enzyme i s so ac t ive at a time when the fetus i s r e c e i v i n g i t s nutr ients from the maternal blood v i a the u m b i l i c a l veins i s unknown. Hahn and Smale have a lso shown that the a c t i v i t y of HMG-CoA reductase i n the fetus i s high i n the proximal por t ion of the gut but low i n the d i s t a l por t ion (1982). I t i s poss ib le that the increased e s t e r i f i c a t i o n observed i n t h i s study i s a r e f l e c t i o n of the increased endogenous cho le s t ero l synthes is . In searching for factors that might c o n t r o l ACAT a c t i v i t y during prenatal l i f e , Rymaszewski et a l . found that the add i t ion of amniotic f l u i d to r a b b i t a o r t i c homogenates 97 causes a l i n e a r increase i n ACAT a c t i v i t y (1988). Given that the fetus does swallow amniotic f l u i d (approximately 500 ml per day i n the sheep and human; Wintour, 1986) i n the l a t t e r parts of gestat ion i t i s poss ib le that the high ACAT a c t i v i t y i n the r a t f e t a l i n t e s t i n a l mucosa i s due to a factor(s) i n the amniotic f l u i d . Choles tero l i s present i n human amniotic f l u i d (19.6 mg/dl; Natelson, 1974). Park and Subbiah have confirmed that LDL and VLDLs are present i n human amniotic f l u i d (1987). Hepatic and i n t e s t i n a l ACAT a c t i v i t i e s have been shown to be increased when the a v a i l a b i l i t y of cho le s t ero l i s increased (Mitropoulos et a l . , 1978; Helgerud et a l . , 1982; Suckl ing et a l . , 1982). High plasma c h o l e s t e r o l l e v e l s are i n d i c a t i v e of t h i s increased substrate supply. However, the serum cho le s t ero l l e v e l i n the fetus (58.7 mg/dl) was lower when compared to the 10 day o ld values (171.9 mg/dl) , despite the 6 - fo ld d i f ference between the high f e t a l and low 10 day o ld ACAT a c t i v i t i e s . This would ind ica te that the substrate supply of cho le s t ero l does not play an exclus ive r o l e i n regu la t ing ACAT a c t i v i t y i n the fe tus . Rymaszewski and co-workers (1985) suggest that the high ACAT a c t i v i t i e s observed i n the f e t a l aorta are due e i t h e r to s t imulat ing factor(s) i n the amniotic f l u i d or to the development of an endogenous i n h i b i t o r a f t er b i r t h . This might a lso be an 98 explanation for the high f e t a l i n t e s t i n a l ACAT values observed i n the present study. The i n t e s t i n e i s an important t i s sue i n the regulat ion of whole body cho le s t ero l metabolism. I t i s the organ which i s i n d i r e c t contact with exogenous d i e tary c h o l e s t e r o l , i t i s the s i t e of cho le s t ero l absorption and one of the major c h o l e s t e r o l synthet ic t i s sues i n the body. The re su l t s ind ica ted that the r a t i n t e s t i n a l ACAT a c t i v i t y i s low throughout the suckl ing phase. The developmental pattern suggests that the in troduct ion of the high fat milk d i e t i s a f f e c t i n g the enzyme's a c t i v i t y . Rat mi lk contains 47% f a t , p r i m a r i l y as saturated fa t ty acids (McNamara et a l . , 1972; Luckey, et a l . , 1954) and approximately 120 mg/dl of c h o l e s t e r o l ( C a r r o l l , 1964). Diets r i c h i n unsaturated fats s i g n i f i c a n t l y increase the i n t e s t i n a l ACAT a c t i v i t y i n r a b b i t s ; however, saturated-enriched fat d i e t s appear to have no e f f ec t on t h i s enzyme ( F i e l d and Salome, 1982). ACAT a c t i v i t y has been shown to be dramat ica l ly increased by unsaturated fa t ty acids more so than by saturated fats ( F i e l d and Salome, 1982) . I t has been observed that the status of the membrane i s an important modulator of ACAT a c t i v i t y . Perhaps the in f lux of saturated fats from the ra t milk to the small i n t e s t i n e causes more r i g i d i t y wi th in the 99 membrane thus a l t e r i n g i t s f l u i d i t y and the propert ies of the ACAT enzyme. Berberian and others (1977) have shown that prostaglandin E2 i n h i b i t s the ACAT enzyme and thus 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 i n the r a b b i t aor ta . The idea that prostaglandins have an e f fec t on i n t e s t i n a l ACAT i s pure speculat ion . However, i t i s conceivable that they e x h i b i t some e f fec t because prostaglandins are present i n the maternal milk and there i s very l i t t l e synthesis i n the small i n t e s t i n e of suckl ing rat s (Koldovsky, 1990). Thyroid hormone may a lso play a r o l e i n the enzyme a c t i v i t y during the suckl ing phase. Ind irec t evidence by Hahn (1986b) suggests that t h y r o i d hormones i n h i b i t i n t e s t i n a l ACAT a c t i v i t y during development i n r a t s . At b i r t h t h y r o i d hormone l eve l s are very low. Rat T4 and T3 concentrations increase progress ive ly from b i r t h achieving adul t l e v e l s around postnatal days 14-16 for T4 and around postnata l day 24-26 for T3 (Sohal, 1988; Ar tan-Sp ire et a l . , 1983). Thus the low ACAT a c t i v i t y i n the suckl ing r a t i n t e s t i n e could be due to t h y r o i d hormone i n h i b i t i o n . However, t h i s does not expla in the dramatic r i s e i n enzyme a c t i v i t y 24 hours post-weaning. The dramatic r i s e i n the a c t i v i t y of the ACAT enzyme at weaning suggests that the d ie tary change from a high fat to 100 a high carbohydrate has some impact on t h i s enzyme i n the i n t e s t i n e . The funct ion of t h i s increased cho les tero l e s t e r i f i c a t i o n i s not c l e a r . However i t i s known that i n t e s t i n a l cho le s t ero l synthesis i s increased at t h i s time (Hahn and Smale, 1982; Kroeger and Hahn, 1983). Perhaps the increased e s t e r i f i c a t i o n i s a r e f l e c t i o n of t h i s and a means of handling the rap id increase i n c h o l e s t e r o l b iosynthes i s . Nevertheless , the high ACAT a c t i v i t y noted i n the immediate post-weaning per iod suggests that t h i s enzyme plays an important r o l e i n the gut. What exact ly i s t h i s r o l e i s d i f f i c u l t to p r e d i c t from these r e s u l t s however Norum and other inves t iga tors (1981) propose several functions for ACAT i n the i n t e s t i n a l mucosa. They suggest that the high ACAT a c t i v i t y may be a protec t ive mechanism i n the membrane preventing excess accumulation c h o l e s t e r o l . As i n other t i s s u e s , ACAT might be e s t e r i f y i n g c h o l e s t e r o l for future hydro lys i s and use i n membrane and l i p o p r o t e i n synthes is . ACAT may a lso be p lay ing an i n t e g r a l r o l e i n true absorption of c h o l e s t e r o l from the gut. I t should be noted that the ACAT enzyme appears to have d i f f e r e n t developmental patterns and perhaps d i s t i n c t functions i n the l i v e r and the i n t e s t i n e (Figure 10). I t i s poss ib le that the i n t e s t i n a l ACAT enzyme i s responsible for the endogenously produced cho le s t ero l and the hepatic enzyme 101 THE RELATIONSHIP BETWEEN THE ONTOGENY OF ACAT IN THE RAT LIVER AND INTESTINAL MUCOSA AND PLASMA CHOLESTEROL AND TRIGLYCERIDE LEVELS. ^WEANING (21) T INTESTINE - 5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 AGE (DAYS) 220-] X) \ 200-• 180-160-140-120-O 100-80-O 60-tj 40-a E 20 o CL 0' • • C H O L E S T E R O L • - • TRIGLYCERIDE 10 15 20 25 30 35 40 45 50 55 60 65 AGE (DAYS) Figure 1 0 . Ontogeny of ACAT octvity ( p m o l / m g pro te in /min) in the rat liver and intestinal mucosa compared to changes in p lasma cholesterol and triglyceride levels ( m g / d l ) during the same period. 102 i s accountable for the exogenous c h o l e s t e r o l . However an exc lus ive r o l e for each i s d i f f i c u l t to fathom since both t i s sues seem to respond at c e r t a i n times i n development to a change i n the d i e t . I t would seem more reasonable that the i n t e s t i n a l enzyme would be responsible for the exogenous 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 given the proposed r o l e of ACAT i n the e s t e r i f i c a t i o n of d i e tary c h o l e s t e r o l p r i o r to packaging i n the chylomicron. I f t h i s i s the case, the low a c t i v i t y during the suckl ing phase may be a t t r i b u t e d to the suppression of t h i s enzyme by some factor(s ) d i e tary or hormonal. Drevon and co-workers (1980) s tate that un l ike adul t r a t hepatic ACAT the i n t e s t i n a l ACAT appears to predominantly e s t e r i f y exogenous cho le s t ero l from d ie tary or b i l i a r y sources as opposed to l o c a l l y synthesized c h o l e s t e r o l . Whether or not t h i s i s so during development remains to be e luc idated . There appears to be no d i r e c t r e l a t i o n s h i p between serum cho le s t ero l l e v e l s and ACAT a c t i v i t y i n the l i v e r and i n t e s t i n e throughout development (Figure 10) . This i s not s u r p r i s i n g because other inves t igators have not found any c o r r e l a t i o n between plasma cho le s t ero l and ACAT a c t i v i t y i n other t i s sues (Rymaszewski et a l . , 1985). The developmental 103 pattern of serum cho le s t ero l reported here i s compatible with e a r l i e r reports (McNamara et a l . , 1972; C a r r o l l and Hamilton, 1973; Hahn, 1982; Hahn, 1978). 4.1.3 WAT White adipose t i s sue contains more c h o l e s t e r o l per mg of p r o t e i n than a l l other organs except s k e l e t a l muscle when expressed on a whole organ bas is (Krause and Hartman, 1984). I t i s general ly assumed that cho le s t ero l i n t i s sues ex i s t s as a s t r u c t u r a l component of the b i o l o g i c a l membrane, however t h i s does not appear to be the case i n adipose t i s sue (Angel and Farkas, 1974). In the adul t most of the c h o l e s t e r o l i s stored i n the c e n t r a l o i l drople t rather than i n the membrane. I t ex i s t s p r i m a r i l y i n the u n e s t e r i f i e d form which i n i t s e l f d i s t inguishes WAT as unique amongst the t i s sues i n the body (Krause and Hartman, 1984). Choles tero l i s reported to be released from the adipose t i s sue when catecholamines are administered (Farkas et a l . , 1973) and during s tarvat ion of adul t ra t s (Angel and Farkas, 1974). I t i s thought that c h o l e s t e r o l i s released i n these s i tua t ions through the l i p o l y t i c ac t ion of c y c l i c -AMP on cho le s t ero l esterase. Neutral c h o l e s t e r o l esterase has been reported to be present i n WAT and functions to hydrolyze the incoming cho le s t ero l esters (Arnaud and Boyer, 104 1974; Krause and Hartman, 1984). ACAT a c t i v i t y has been reported to be low or absent i n adult r a t adipose t i s sue concomitantly with l i t t l e c h o l e s t e r y l es ter stores (Angel and Farkas, 1974). This argument was strengthened by the r e s u l t s presented here which ind ica te that the a c t i v i t y of ACAT i s undetectable not only i n the adul t animals but throughout development. As suggested by others i t i s poss ib le that cho le s t ero l i s stored as free cho le s t ero l i n a c e n t r a l o i l droplet of the adipocyte and i s re leased as such upon demand (Angel and Farkas, 1974). 4.1.4 BAT Results indicated that there i s l i t t l e i f any ACAT a c t i v i t y i n BAT throughout development. I t was thought, perhaps na ive ly , that because HMG-CoA reductase i s ac t ive even i n the f e t a l t i s sue there might be s i m i l a r or inverse a c t i v i t i e s of the ACAT enzyme. This does not appear to be the case. In contrast to what was found i n WAT, ACAT a c t i v i t y appeared to be elevated i n the adult animal. S u r p r i s i n g l y , t h i s post-weaning r i s e occurs at a time when the amount of brown fat r e l a t i v e to body weight i s decreasing with postnatal age, thus there i s l i t t l e i f any c e l l growth and l i t t l e c e l l membrane requirement (Aherne and H u l l , 1966; Tarkkonen and Ju lku 1968). In a d d i t i o n , 105 c h o l e s t e r o l i s not a precursor for any known hormone synthesis i n t h i s t i s sue (Nedergaard 1986). I t i s poss ib le that the ACAT enzyme of BAT o r i g i n i s p a r t i c i p a t i n g i n the regu la t ion of plasma cho le s t ero l l e v e l s i n the adul t animal. 4.1.5 Aorta ACAT and i t s r e l a t i o n to cho le s t ero l es ter metabolism i s of p a r t i c u l a r in teres t because of i t s r o l e i n a t h e r o s c l e r o s i s . I t i s wel l documented that a r t e r i e s undergoing atherogenic change c h a r a c t e r i s t i c a l l y show an increase i n cho le s t ero l e s t e r i f y i n g a c t i v i t y by ACAT and a progress ive increase i n cho le s t ero l es ter content (St. C l a i r , 1976). The a c t i v i t y of ACAT has been reported to be increased up to 50- fo ld i n a t h e r o s c l e r o t i c a r t e r i e s (Hashimoto, 1974; St . C l a i r , 1976). I t has been suggested that ACAT operates i n t r a c e l l u l a r l y to protect the c e l l from excess cho le s t ero l accumulation. This i s a c o n t r o v e r s i a l issue s ince i t i s not known whether the increase i n ACAT a c t i v i t y i n the a t h e r o s c l e r o t i c ar tery represents an exacerbating fac tor or a passive consequence of an increased a v a i l a b i l i t y of free cho le s t ero l ( G i l l i e s , et a l . , 1986). A c t i v i t y of ACAT i n the r a t aorta was found to be n e g l i g i b l e i n comparison to the other t i s s u e s . The v a r i a b i l i t y of ACAT a c t i v i t y i n the in fant ra t s may be due 106 i n part to the small amount of a r t e r i a l t i s s u e . A small sample s i ze i n a l l of the age groups may have contr ibuted to wide v a r i a t i o n s i n a c t i v i t y . I t i s l i k e l y that the ACAT assay used i s not s ens i t i ve enough for t h i s small amount of t i s s u e . Further studies with l a r g e r pools of t i s sue or with t i s s u e from a d i f f e r e n t animal model are needed. Needless to say the r e s u l t s presented here do not support Spr inkle and co-worker's f indings i n which ACAT a c t i v i t y from rabb i t aortae decreased as a quadratic funct ion with age (Sprinkle et a l . , 1987) . 4.2 Ef f ec t s of Diets on ACAT A c t i v i t y In the previous study r e s u l t s ind icated that there i s a marked v a r i a t i o n i n the a c t i v i t y of ACAT i n the l i v e r , i n t e s t i n e , and BAT throughout development. An extension of t h i s work was a d ie tary study which focused on the immediate e f fec t s of short-term manipulation of d i e t on the a c t i v i t y of ACAT. The rat s i n t h i s study were a l l weaned ear ly on day 18 to one of the fo l lowing d i e t s : Purina Rat Chow, high carbohydrate, high f a t , or 2% c h o l e s t e r o l . 107 4.2.1 L i v e r The argument that d i e t s high i n fa t increase the a c t i v i t y of ACAT was strengthened by the present r e s u l t s . In fac t the high fat d i e t was the only d i e t that cons i s t en t ly increased hepatic ACAT a c t i v i t y throughout development (Figure 7) . The response of ACAT to a fat d i e t seems to be dependent on the type of fa t used. Spector, Kaduce, and Dane (1980) fed ra t s d i e t s enriched i n e i ther saturated or unsaturated fats and observed that the hepatic ACAT a c t i v i t y was 70-90% higher i n the unsaturated f a t - f e d animals than the a c t i v i t y of those ra t s fed saturated f a t s . Presumably, the fa t ty ac id composition of the membrane has some inf luence on the enzyme a c t i v i t y ( F i e l d and Salome, 1982) poss ib ly by causing a more f l u i d endoplasmic r e t i c u l a r membrane. Unfortunately only corn o i l (unsaturated fat) was used i n t h i s study thus a comparison between d i f f e r e n t fat d i e t s cannot be made. Seccombe and col leagues (1987) a lso used corn o i l i n t h e i r rabb i t s tudies . They too found an increase i n ACAT a c t i v i t y (1.5 times) with the high fat d i e t . S u r p r i s i n g l y , an even greater increase i n ACAT a c t i v i t y (4.5 times) was observed when c a r n i t i n e was added to the fat d i e t . The cho le s t ero l d i e t s i g n i f i c a n t l y increased the ACAT a c t i v i t y i n the 22 and 25 day old animals. This confirms 108 e a r l i e r reports which state that 0.1 - 5% c h o l e s t e r o l - r i c h d i e t s increase ACAT a c t i v i t y 2-3 f o l d (Erickson et a l . , 1978; Er ickson et a l . , 1980; Mitropoulos et a l . , 1978). The obvious mechanism would be that t h i s increase provides a d d i t i o n a l substrate for the r e a c t i o n . Suckl ing et a l , (1982) suggest that the normal supply to the microsomal enzyme i s probably suboptimal which allows the enzyme to respond to changes i n cho le s t ero l supply. 4.2.2 Intes t ine The most s t r i k i n g feature of the r e s u l t s of d i e tary manipulation i s the dramatic decrease i n a c t i v i t y i n the HF and HG d i e t groups at day 22 (Figure 8) . The e f fec ts of d i e t a r y fa t on i n t e s t i n a l ACAT var i e s with the type of fa t used and the amount of fa t administered (Suckling and Stange, 1985) . In the present study a 45% corn o i l d i e t was given to the r a t s . This p r e c i p i t a t e d a r a p i d drop i n enzyme a c t i v i t y i n the 22 day o ld r a t s . Others have shown that a intraduodenal perfus ion of c h o l e s t e r o l - f r e e l i p i d causes s i m i l a r changes i n ACAT a c t i v i t y plus a decrease i n mucosal free c h o l e s t e r o l content and decreased secre t ion of c h o l e s t e r y l ester into the mesenteric lymph (Bennett C l a r k , 109 1979; Suckl ing and Stange, 1985). Stange and co-workers (1983) observed no s i g n i f i c a n t e f fec t s on ACAT a c t i v i t y when r a t s were fed a 10% corn o i l d i e t for three days. The 2% cho le s tero l d i e t s i g n i f i c a n t l y increased the ACAT a c t i v i t y i n 30 day o ld ra t s as seen i n the l i v e r but no e f fec t was noted on days 22, 25, and 60. This lack of s t imulat ion i n these ra t s was unexpected because many inves t iga tors have reported an increase i n ACAT a c t i v i t y with cho le s t ero l feeding i n ra t s (Stange et a l . , 1983a; Helgerud et a l , 1982; Norum et a l . , 1983), guinea pigs (Drevon, 1978), and rabbi t s ( F i e l d and Salome, 1982). The HG d i e t decreased the enzyme a c t i v i t y i n a l l the age groups except the 30 day o ld r a t s . 4.2.3 Aor ta , BAT, and WAT Examination of the e f fec ts of d i e tary manipulation on the a o r t i c ACAT a c t i v i t y provided no conclus ive r e s u l t s . This was l i k e l y due to a small sample s i z e , small amounts of t i s s u e , and an assay that was not s ens i t i ve enough for t h i s small amount of t i s s u e . From the developmental study i t was apparent that there was l i t t l e i f any ACAT a c t i v i t y i n BAT throughout development except i n the adult t i s s u e . I t was proposed / that t h i s dramatic increase i n ACAT a c t i v i t y might be 110 affected by a change i n d i e t . A l l three d i e t s s i g n i f i c a n t l y decreased ACAT a c t i v i t y i n the adult BAT (postnatal day 60). However such was not the case for the other age groups. There was no s i g n i f i c a n t d i f ferences between the BAT i n rats fed an experimental d i e t and the BAT of ra t s fed the Purina Rat Chow. The developmental r e s u l t s ind icated that the r a t white adipose t i s sue i s devoid of ACAT a c t i v i t y . S i m i l a r l y , there appeared to be no d i e t - i n d u c t i o n of ACAT a c t i v i t y . I t was thought that there might be a change i n a c t i v i t y with the 2% c h o l e s t e r o l d i e t because a d i r e c t c o r r e l a t i o n between d i e t a r y cho le s t ero l l e v e l and adipocyte c h o l e s t e r o l content has been demonstrated by Angel and Farkas (1974). However, the r e s u l t s presented here ne i ther confirm nor deny t h i s c o r r e l a t i o n they merely suggest that the cho le s t ero l accumulated i s not stored i n the ester form v i a the ACAT reac t ion ( L i t t l e and Hahn, 1989; L i t t l e and Hahn, 1990). I l l 5. SUMMARY AND CONCLUSIONS 5.1 Developmental Study I The uptake of cho le s t ero l ester in to c e l l s by receptor-mediated endocytosis and i t s subsequent d e - e s t e r i f i c a t i o n and r e - e s t e r i f i c a t i o n by i n t r a c e l l u l a r enzymes has generated much i n t e r e s t p a r t i c u l a r l y because of the l i n k between these processes and patho log ica l condit ions such as a therosc l eros i s (Brown and Golds te in , 1984). I t has been shown that the pathogenesis of the a t h e r o s c l e r o t i c plaque begins ear ly i n l i f e although the c l i n i c a l symptoms are not apparent u n t i l mid or l a t e adulthood. Animal s tudies have shown that d i e tary manipulation of c h o l e s t e r o l metabolism during an animal's ear ly development can have p e r s i s t e n t and permanent e f f e c t s . Therefore i t i s important that the ontogeny of ACAT, one of the key enzymes i n cho le s t ero l metabolism, be c l e a r l y es tab l i shed . The primary focus of the research presented here was i n e s t a b l i s h i n g the ontogenic pattern of the ACAT enzyme. Using an ACAT assay developed by Helgerud, Saarem, and Norum (1981) the developmental p r o f i l e of ACAT a c t i v i t y i n the ra t l i v e r , i n t e s t i n e , brown and white adipose t i s s u e , and aorta was def ined. A t o t a l of 306 Wistar ra t s were used. The ACAT a c t i v i t y was measured i n the la te - term fetus , newborn, 112 and on postnatal days 1, 10, 14, 18, 21, 22, 25, 30, and 60. The f indings of these studies can be summarized as fol lows: i . The r a t l i v e r and i n t e s t i n e possess s i g n i f i c a n t amounts of ACAT a c t i v i t y throughout development and there appears to be marked v a r i a t i o n s i n a c t i v i t y during t h i s time. These r e s u l t s suggest that the ACAT enzymes o r i g i n a t i n g from these two t i s sues play major ro l e s i n cho le s t ero l metabolism throughout development i n the r a t ; i i . The r a t brown and white adipose t i s sues appear to be devoid of ACAT a c t i v i t y throughout development with the exception of adult BAT. The high ACAT a c t i v i t y i n the adul t BAT suggests that t h i s t i s s u e may p a r t i c i p a t e i n the regulat ion of cho le s t ero l metabolism; i i i . Due to the small amount of the a o r t i c t i s sue samples and/or the i n s e n s i t i v i t y of the assay, no d e f i n i t e conclusions could be made from t h i s a o r t i c study. 113 5.2 Diet Study II In searching for factors that might contro l the ACAT enzyme the immediate e f fec ts of short-term manipulation of d i e t on the a c t i v i t y of ACAT was s tudied . A t o t a l of 111 Wistar r a t was used i n t h i s study. The ra t s were a l l weaned ear ly on day 18 to one of the fo l lowing d i e t s : Purina Rat Chow, high carbohydrate, high f a t , or 2% c h o l e s t e r o l . The r e s u l t s can be summarized as fol lows: i . The HF was the only d i e t that cons i s t ent ly increased hepatic ACAT a c t i v i t y . These r e s u l t s suggest that fa t ty acids play an important r o l e i n the contro l of t h i s enzyme poss ib ly by a l t e r i n g the f l u i d i t y of the r e t i c u l a r membrane. The cho le s t ero l d i e t s s i g n i f i c a n t l y increased the a c t i v i t y of ACAT i n the 22 and 25 day o ld r a t s . This suggests that the substrate supply to the ACAT enzyme i s a lso an important regulator of a c t i v i t y i n these animals. The HG d i e t increased the a c t i v i t y of ACAT i n the 22, 25, and 30 day o ld r a t s . No s i g n i f i c a n t d i f ferences were observed between the adult contro l and HG d i e t groups; i i . Feeding the ra t s a HF or HG p r e c i p i t a t e d a dramatic drop i n i n t e s t i n a l ACAT a c t i v i t y i n the 22 114 day o ld animals. These e f fec ts were not observed i n the o lder animals suggesting that the enzyme of the 22 day o l d i n t e s t i n e i s p a r t i c u l a r l y s e n s i t i v e to changes i n the d i e t . The high cho le s t ero l d i e t had no s i g n i f i c a n t e f fec t on the enzyme's a c t i v i t y ; i i i . There was no s i g n i f i c a n t change i n the BAT and WAT ACAT a c t i v i t y with the experimental d i e t s . Examination of the e f fec ts of d i e tary manipulation on a o r t i c ACAT a c t i v i t y provided no conclus ive r e s u l t s . Acyl-coenzyme A: cho le s t ero l acy l - t rans ferase undoubtedly plays an important r o l e i n cho le s t ero l e s t e r i f i c a t i o n and hence t h i s enzyme may be involved i n the pathogenesis of a therosc l eros i s . 115 5.3 Suggestions for Future Work Although the present study es tabl i shed the ontogenic pat tern of ACAT i n the i n t e s t i n e and l i v e r the r o l e the enzyme plays i n these two t i s sues during development i s s t i l l unknown. I t has been suggested that ACAT aids i n the absorption of c h o l e s t e r o l . ACAT a c t i v i t y has been found to be decreased by the i n h i b i t o r Sandoz compound 58-035 (Suckling and Stange, 1985; Sampson et a l . , 1987). This compound a lso causes pronounced malabsorption of cho le s t ero l when administered i n v ivo (Suckling and Stange, 1985). This compound could be used i n an experiment designed to t e s t whether or not i n t e s t i n a l ACAT plays a r o l e i n the absorption of exogenous cho les tero l and at what per iod of development does t h i s enzyme d i s p l a y t h i s funct ion . Labe l l ed cho le s t ero l could be given to the ra t s i n add i t ion to the i n h i b i t o r and the changes i n the cho le s t ero l ester content i n the i n t e s t i n a l mucosa and the lymph could be monitored. I t i s l i k e l y that there i s some hormonal contro l of the ACAT enzyme during development. I t seems su i tab le that further studies should incorporate the e f fec t s of such hormones as T3, T4, i n s u l i n , and glucagon on t h i s enzyme. These hormones probably play an important r o l e i n 116 c o n t r o l l i n g t h e a c t i v i t y o f t h e A C A T enzyme e s p e c i a l l y d u r i n g d e v e l o p m e n t . I t was f o u n d i n t h e p r e s e n t s t u d y t h a t t h e H F a n d HG d i e t s h a d p r o f o u n d e f f e c t s o n t h e A C A T a c t i v i t y i n t h e 22 d a y o l d r a t i n t e s t i n e . H o w e v e r , t h e q u e s t i o n o f w h e t h e r o r n o t t h e s e d i e t s h a d a n y l a s t i n g e f f e c t s o n t h e enzyme was n o t a d d r e s s e d i n t h i s s t u d y . F u r t h e r s t u d i e s a r e n e e d e d i n o r d e r t o d e t e r m i n e w h e t h e r o r n o t t h e s e c h a n g e s i n A C A T p e r s i s t i n t o a d u l t h o o d e v e n a f t e r t h e a n i m a l i s g i v e n a r e c o v e r y p e r i o d t o n o r m a l i z e o n a P u r i n a Chow d i e t . T o u n d e r s t a n d c o m p l e t e l y t h e r e g u l a t i o n o f c h o l e s t e r o l s t o r a g e o f a d i p o s e t i s s u e , one m u s t e s t a b l i s h w h e t h e r o r n o t t h e i n c r e a s e i n c h o l e s t e r o l c o n t e n t i n t h e a d i p o s e t i s s u e o f c h o l e s t e r o l - f e d r a t s i s i n t h e e s t e r o r f r e e f o r m . I f t h e r e i s a n i n c r e a s e i n c h o l e s t e r o l e s t e r c o n t e n t i t i s p r o b a b l y d u e t o t h e a c t i o n o f C E a s e . T h e d a t a p r e s e n t e d h e r e s u g g e s t t h a t A C A T a c t i v i t y o f a d i p o s e t i s s u e o r i g i n i s n o t a f f e c t e d b y a n i n c r e a s e i n d i e t a r y c h o l e s t e r o l o r f a t . I t h a s b e e n f o u n d t h a t A C A T i s a l s o p r e s e n t i n t h e s t e r o i d - p r o d u c i n g t i s s u e s . T h e c o n c e n t r a t i o n o f c h o l e s t e r y l e s t e r s v a r i e s among t h e s e t i s s u e s . F o r i n s t a n c e t h e r a t a d r e n a l c o r t e x c o n t a i n s l a r g e a m o u n t s o f c h o l e s t e r y l e s t e r w h e r e a s t h e L e y d i g c e l l f r o m t h e t e s t i s a p p e a r s t o c o n t a i n 117 very l i t t l e cho le s t ero l es ter (Suckling and Stange, 1985). I was curious as to whether or not ACAT plays the same ro l e i n the placenta as i t does i n the adrenal , that i s to provide a storage pool for s t e r o i d hormone synthes is . I t was found i n t h i s study that there i s some ACAT a c t i v i t y i n the r a t placenta a l b e i t i n low concentrations (Appendix A) . Based on the Brown and Golds te in LDL uptake model one might propose that the uptake of l i p o p r o t e i n s by the p lacenta l c e l l s would st imulate the formation of c h o l e s t e r y l e s ters . However, i t i s we l l known that c h o l e s t e r y l esters are not present i n the placenta to any great degree, a f ind ing which i s incons i s tent with the Brown and Goldste in model (Simpson and Burkhart, 1980). There are at l e a s t two poss ib le explanations for t h i s . The f i r s t i s that the placenta cont inua l ly u t i l i z e s c h o l e s t e r o l for s t e r o i d hormone synthesis thus the ACAT reac t ion i s an unwanted process . The second p l a u s i b l e explanation i s that the s t imulat ion of ACAT (or CEase) by the LDL uptake i s overridden by an i n h i b i t o r y pathway. I t has been found by Simpson and Burkhart (1980) that the ACAT enzyme i s i n h i b i t e d by progesterone and pregnenolone i n humans. I t appears that cho le s t ero l ester synthesis i s i n h i b i t e d when progesterone i s synthesized r e s u l t i n g i n a decrease i n free 118 c h o l e s t e r o l and an i n h i b i t i o n of ACAT (Simpson and Burkhart, 1980) . 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E f f e c t of d i e t a r y fat and cho le s t ero l on mi lk composition, milk intake , and cho le s t ero l metabolism i n the r a b b i t . J . Nutr. 111. 424-441 (1981). Wintour, E . M . Amniotic f l u i d - Our f i r s t environment. NIPS. 1, 95-97 (1986). 152 7.1. APPENDIX A. ACAT ACTIVITY IN RAT FETAL AND PLACENTAL TISSUES 153 A p p e n d i x A A C A T ACTIVITY IN RAT FETAL A N D P L A C E N T A L T I S S U E S £ \ c '<d O CL o> E \ o £ C L < I— < < 8 0 n 6 0 4 0 2 0 -F i g u r e 1 1 I LIVER INTESTINE BAT PLACENTA T I S S U E T Y P E A C A T a c t i v i t y ( p m o l / m g P r / m i n ) in t h e l i ve r i n t e s t i n a l m u c o s a , BAT, a n d p l a c e n t a in t h e ra t o n d a y 21 g e s t a t i o n . 154 7.2 APPENDIX B. COMPOSITION OF PURINA RAT CHOW AND PURINA PLUS 2% CHOLESTEROL DIET 155 |Appendix B. Composition of Pu r i n a Rat Chow and Pu r i n a Rat Chow plus 2% d i e t . NUTRIENTS Ground extruded corn Choline c h l o r i d e Sybean pulp F o l i c a c i d F i s h meal R i b o f l a v i n Gound oats Thiamin Brewers' d r i e d yeast N i a c i n A l f a l f a meal Py r i d o x i n e h y d r o c h l o r i d e Cane molasses Ferrous s u l f a t Wheat germ meal Vitamin A supplement D r i e d whey D-activated animal s t e r o l Meat meal Vitamin E supplement Animal f a t with BHA Calcium iodate D i c a l c i u m phosphate Ferrous carbonate S a l t Manganous oxide Wheat midd l i n g s Cobalt carbonate Calcium carbonate Copper s u l f a t e V itamin B-12 supplement Zinc s u l f a t e OL-methionine Zinc oxide Calcium pantothenate I 156 

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