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UBC Theses and Dissertations

Studies on the hormonal regulation of hepatic phospholipid metabolism Sommerman, Eric Frank 1982

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STUDIES ON THE HORMONAL REGULATION OF HEPATIC PHOSPHOLIPID METABOLISM by ERIC FRANK SOMMERMAN b . S . . , ' M i c h i g a n S t a t e U n i v e r s i t y , 1930 A T h e s i s S u b m i t t e d i n P a r t i a l F u l f i l m e n t f o r t h e D e g r e e o f M a s t e r o f S c i e n c e i n THE F A C U L T Y OF GRADUATE S T U D I E S DEPARTMENT OF B I O C H E M I S T R Y T H E U N I V E R S I T Y OF B R I T I S H COLUMBIA V.e a c c e p t t n i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y OF B R I T I S H COLUMBIA M a y , 1982 E r i c F . S o n m e m a n In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of l a c W r N v ^ T t A ,  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date J , ^ ]/ytl, \Cj%\ DE-6 (3/81) ABSTRACT 00 { Investigations were c a r r i e d out on the role of glucagon and calcium in the regulation of hepatic phospholipid biosynthesis. J t was found that glucagon i n h i b i t s de novo phosphatidylcholine biosynthesis in c u l t u r e d r a t hepatocytes. T h i s i n h i b i t i o n was a s s o c i a t e d w i t h an i n h i b i t i o n o f C T P : p h o s p h o c h o 1 i n e cyt i d y l y l t r a n s f e r a s e a c t i v i t y , which i s the regulatory enzyme for phosphatidylcholine biosynthesis. Calcium was shown to inhibit the uptake of choline in hepatocytes by decreasing the Vmax of the saturatable uptake system. It also s l i g h t l y inhibited the rate of phosphatidylcholine biosynthesis by the de novo pathway, but not by the N-methylation of phosphatidylethanolamine. However, these experiments were d i f f i c u l t to interpret due to the use of ionophore A23187 to vary c y t o s o l i c calcium concentrations. This ionophore has many other effects on hepatocytes which could i n d i r e c t l y alter the synthesis of phosphatidylcholine. In v i t r o studies were carried out to determine the e f f e c t of calmodulin on CTP:phosphocholine cytidylyltransferase a c t i v i t y . Although calmodulin did not effect the a c t i v i t y under the c o n d i t i o n s of the assay, an impurity of some c a l m o d u l i n p r e p a r a t i o n s was f o u n d which i n h i b i t e d the cytidylyltransferase in a calcium independent fashion. The inhibitor had some peptide l i k e p r o p e r t i e s . The e f f e c t of calcium on the i n c o r p o r a t i o n o f [ 3 - H] s e r i n e i n t o p h o s p h o l i p i d s was a l s o investigated. Calcium was found to increase the amount of label recovered in phospholipid. It was a l s o found that,the label was rapidly transfered from phosphatidyserine to phosphatidylethanolamine. On the b a s i s of these r e s u l t s , a model i s presented f o r the relationships between calcium and phosphatidylserine metabolism. GVo LIST OF ABBREVIATIONS ACS aqueous counting s c i n t i l l a n t ADP adenosine diphosphate AMP adenosine monophosphate ATP adenosine triphophate CaM calmodulin cAMP adenosine c y c l i c monophosphate CDP choline diphosphate cGMP c y c l i c guanosine monophosphate Ci Curie CL c a r d i o l i p i n CMP c y t i d i n e monophosphate CoA coenzyme A cpm - counts per minute CTP c y t i d i n e triphosphate DG d i a c y l g l y c e r o l dpm d i s i n t e g r a t i o n s per minute EDTA ethylenediaminetetracetic acid EGTA ethyleneglycol bis(B-aminoethylether) N-N-tetracetic acid g gram GTP guanosine triphosphate h hour Hepes hydroxyethyl piperazineethanesulfonic acid IPA Ionophore A23187 Ka association constant of an enzyme-activator complex Km Michaelis-Menten constant 1 l i t e r m- m i l l i -M molar MEM" Dulbeccos m o d i f i e d E a g l e s Media formula 79-5141 min minute mol moles n- nano-NAD+ oxidized n i c o t i n a m i d e - a d e n i n e dinucleotide NADH reduced nicotinamide-adenine dinucleotide PA phosphatidate PBS phosphate buffered s a l i n e , pH 7.k PC phosphatidylcholine PE phosphatidylethanolamine PG phosphatidylglycerol P i inorganic phosphate PI phosphatidylinositol PKA cAMP dependent protein kinase PKC calcium phosphatidylserine dependent protein kinase PKM calmodulin dependent protein kinase PS phosphatidylserine SAH S-adenosyl homocysteine SAM S-adenossyl methionine SD standard deviation SM sphingomyelin TG t r i a c y l g l y c e r o l TLC t h i n layer chromatography T r i s t r i s (hydroxymethyl) aminoethane u- micro-UV u l t r a v i o l e t Vmax maximal v e l o c i t y of an enzymatic reaction TABLE OF CONTENTS £ a & e INTRODUCTION 1.1 The Structure.Properties, and Function of PL 1 1.11 The Structure of Phospholipids 1 1.12 The Properties of Phospholipids 2 1.13 The Function of Phospholipids 6 1.2 The Biosynthesis of G l y c e r o p h o s p h o l i p i d s in 9 Hepatocytes 1.21 Fatty Acid Synthesis and Activation 9 1.22 The Synthesis of PA 12 1.23 The Biosynthesis of DG 13 1.24 The Synthesis of TG 14 1.25 The de novo Synthesis of PC 15 1.26 The de novo Synthesis of PE 22 1.27 The CDP-DG Pathway to Aci d i c Phospholipids 23 1.3 Intermediary Phospholipid Metabolism 24 1.31 PE-N-Methylation 24 1.3.2 The Synthesis of PS 25 1.33 Other Base Exchange A c t i v i t i e s 26 1.34 The Decarboxylation of PS 27 1.35 The Phosphorylation of DG 27 1.36 Exchange of Acyl Groups 28 1.4 The Degradation of Phospholipids 1.41 Phosphoplipases 28 29 1.5 The Hormonal Regulation of Hepatic 30 Glycerolipid Metabolism 1.51 The Effect of Fasting on Hepatocyte 31 Glycerolipid Metabolism 1.52 The Effect of Calcium on Glycerolipid 38 Metabolism MATERIALS AND METHODS 2.1 Chemicals and Isotopes 2.2 The Isolation and Culture of 2.21 Isolation of Liver Cells 2.22 The Culture of Hepatocytes 47 Rat Hepatocytes 49 49 51 2.3 Pulse-Chase Experiments 52 2.31 Choline Uptake Studies - 52 2.32 Detection of a Change in the Rate of de. 53 novo PC Biosynthesis 2.33 Detection of a Change in the Rate of PC 53 Synthesis by the Methylation Pathway 3 2.34 Incorporation of H Serine into Hepatic 54 Phospholipids 2.35 Studies on the Incorporation of Other Lipid 55 Precursors 2.36 Harvesting of Cells for Lipid Extraction 55 2.37 Thin Layer Chromotographic Analylysis of 57 Phospholipids 2.38 Scintillation Counting 58 v/n ' D 2.4 Enzyme Activation Studies 58 2.41 Incubation Conditions 58 2.42 Harvesting of Hepatocytes and C e l l 59 Fractionation 2.43 Enzyme Assays and Protein Estimation 59 2.5 Studies on CTP:Phosphochoiine 62 Cytidylyltransferase in vitro 2.51 Preparation of Calcium-Free Labeled 62 Phosphocholine 2.52 Partial Purification of Calmodulin 64 RESULTS AND DISCUSSION 3.1 The Effect of Glucagon on Phosphatidylcholine 65* Biosynthesis % 3 3.11 The Effect of Glucagon on[ H ] Choline 65 Incorporation into Hepatocyte Phospholipid 3.12 The Effect of Glucagon on the Incorporation 71 of I CJ Palmitate into Hepatocyte Glycerolipids 3 3.13 The Effect o'f Glucagon on [ H] Glycerol 75 Incorporation into Hepatocyte Glycerolipids 3.14 The Effect of Glucagon on the Enzyme -79 A c t i v i t i e s R e s p o n s i b l e f o r de_novo Phosphatidylcholine Biosynthesis 3.2 Studies on the Effect of Calcium on -Phospholipid Biosynthesis Using Ionophore AP3187 3 3.21 Pulse-Chase Studies with [ H] Serine Label 83 83 3.22 The Effect of Calcium on Choline Uptake 91 3.23 The Effect of Calcium and Ionophore A231&7 on 95 Phosphatidylcholine Biosynthesis 3-3 The Effect of Calmodulin Preparations on 99 Phosphocholine Cytidylyltransferase in vitro 3.31 The Partial Purification of Rat Liver 99 Calmodulin and i t s Effect on Phosphocholine Cytidylyltransferase 3.32 Studies with Pure Calmodulin 105 CONCLUSIONS 106 BIBLIOGRAPHY 107 LIST OF FIGURES Figure Page 1.2 Polymorphic Phases and Dynamic Molecular Shapes of 5 L i p i d s 1.3 The Pathways of G l y c e r o l i p i d Metabolism 10 1.4 A Model f o r t h e R e g u l a t i o n o f P h o s p h o c h o l i n e 21 C y t i d y l y l t r a n s f e r a s e 1.5 The E f f e c t s of I n s u l i n and Glucagon on Phospholipid 37 Biosynthesis 2.1 The Removal of Calcium from Phosphocholine Chloride 63 by Ion Exchange Chromatography on Dowex 1 Resin 3.1 Time Course o f the E f f e c t o f G l u c a g o n on the 67 3 I n c o r p o r a t i o n o f [Me- H] C h o l i n e i n t o Phosphocholine and Phosphatidycholine 3.2 Time Course o f the E f f e c t o f G l u c a g o n on the 69 3 S e c r e t i o n of [Me- H] Betaine i n t o the Media 3 3.3 A G l u c a g o n T i t r a t i o n o f [Me- H] C h o l i n e 70 Incorporation into Phosphocholine and PC > 3.4 The E f f e c t of Glucagon on the I n c o r p o r a t i o n of 72 14 [ D - C] P a l m i t a t e i n t o Hepatocyte G l y c e r o l i p i d s 3.5 The E f f e c t of Glucagon on the I n c o r p o r a t i o n of 76 3 [ ( 3 ) - H] G l y c e r o l i n t o Hepatocyte G l y c e r o l i p i d s 3 3.6 The I n c o r p o r a t i o n o f [ ( 3 ) - H] S e r i n e i n t o 84 Hepatocyte Phospholipids 3.7 The E f f e c t of Ionophore A23187 on the Incorporation 86 3 of [(3)- Hi Serine i n t o HeDatocvte Phospholipids 3.8 A Model of the PS Cycle Hypothesis 88 3.9 The E f f e c t o f I o n o p h o r e A23187 and Calcium on 93 Choline Uptake 3.10 A Lineweaver-Burke P l o t o f the E f f e c t of Ionophore 94 A231287 and Calcium on Saturatable Choline Uptake 3.11 The E f f e c t o f I o n o p h o r e A23187 on t h e 96 3 In c o r p o r a t i o n of [(Me)- H] C h o l i n e i n t o Phospho-choline and PC \ 3.12 The E f f e c t o f I o n o p h o r e A23187 on t h e 98 I n c o r p o r a t i o n o f [ ( 1 ) - ^ H ] E t h a n o l a m i n e i n t o Phosphatidylethanolamine and PC 3.13 Sepharose 6B Chromatography of Rat Liver Homogenate 101 3.14 The E f f e c t o f 'Peak 2' on the A c t i v i t y o f 104 Phosphocholine CytidylyItranferase 1.1 The Composition and Turnover of Membrane Phospholipids 1.2 R e g u l a t o r y Enzymes i n v o l v e d i n the Metabolism of Phospholipids 3 3.1 The I n c o r p o r a t i o n o f [ ( H e ) - H] Choline into Hepatocyte Metabolites 3.2 The E f f e c t of Glucagon on the Enzyme A c t i v i t i e s I n volved i n the de novo Synthesis of Phosphatidylcholine 3.3 The E f f e c t of Boiled Rat L i v e r Homogenate on the A c t i v i t y o f CTP:Phosphocholine CytidylyItransferase 3.4 The E f f e c t o f P h o s p h o l i p i d on the CTP:Phosphocholine C y t i d y l y I t r a n s f e r a s e I n h i b i t o r y P r o p e r t i e s of Dowex 1 Column Fractions 3.5 The E f f e c t o f D i a l y s i s on t h e CTPrPhosphocholine C y t i d y l y l t r a n s f e r a s e I n h i b i t o r y P r o p e r t i e s of Dowex 1 Column Fractions 3.6 T h e E f f e c t o f P u r e CaM on Cy t i d y l y l t r a n s f e r a s e A c t i v i t y ACKNOWLEDGEMENTS I am indebted to the following people without whom this work would not have been possible. Primarily, I am grateful to Dennis Vance for his unending support, guidance, and tolerance; Haydn Pritchard, for his immeasurable efforts as my chief reviewer and critic, and for / his free tutorials in the Queen's English; Steve Pelech, for providing inspiring discussion and maternal concern; Francois Audubert, for the use of his Bic lighter; Diana Crookall, for smiling in the morning; and Ronnie de Brito, for dancing in the halls. 1 INTRODUCTION Phospholipids are a major component of c e l l membranes, b i l e , lung sufactant, and l i p o p r o t e i n s . The pathways o f phospholipid metabolism have been known for many years, l a r g e l y due to the work of Kennedy and coworkers ( 1 ) . I n t e r e s t i n t h e r e g u l a t i o n o f these pathways has i n t e n s i f i e d in recent y e a r s , as i t i s becoming i n c r e a s i n g l y evident that phospholipid metabolism i s i n t i m a t e l y r e l a t e d to other c e l l u l a r p r o c e s s e s . However, our knowledge o f t h i s r e g u l a t i o n i s s t i l l fragmentary and at a s u p e r f i c i a l l e v e l . The purpose of t h i s t h e s i s i s to gain further i n s i g h t i n t o the p r o c e s s e s by which l i p i d d i v e r s i t y , as w e l l as t o t a l l i p i d s y n t h e s i s , are maintained under d i f f e r e n t p h y s i o l o g i c a l conditions. 1 . 1 . The S t r u c t u r e f P r o p e r t i e s , and Function of  P h o s p h o l i p i d s 1 .11 The S t r u c t u r e o f Phospholipids G l y c e r o p h o s p h o l i p i d s can be c o n s i d e r e d to be d e r i v a t i v e s of g l y c e r o l - 3 - p h o s p h a t e . A wide r a n g e o f compounds are p o s s i b l e by e s t e r i f y i n g d i f f e r e n t f a t t y a c i d s to the 1 and 2 p o s i t i o n s of the g l y c e r o l , a l l g i v i n g r i s e t o d i f f e r e n t t y p e s o f PA. S t e a r a t e , palmitate, oleate, and l i n o l e a t e are the predominant fa t t y acids found i n animal membranes, with a r a c h i d o n a t e being an important minor component. PA may be thought of as the parent structure of the other 2 glycerophospholipid classes, which can be generated by esterifying different organic bases or 'head groups' to the phosphate moiety (figure 1.1). This gives rise to the major phospholipids found in biological membranes, which generally contain predominantly PC, PE, PS, and PI. Glycerophospholipids which have one of their acyl groups removed are refered to as lysophospholipids (lyso-PE, lyso-PC, etc. ) . By varying the f a t t y acid and head group moieties, approximately 200 different phospholipid molecules can be generated, and most of them have been found in vivo. The term 'phospholipid' includes these compounds, as well as plasmalogens, sphingolipids, and phosphonolipids. However, with the exception of sphingomyelin, these compounds are present in only trace quantities in most animal systems. 1.12 The Properties of Glycerolipids One can make certain generalizations which apply more or less to a l l lipid classes. Most lipids are araphipathic in that they contain long chain hydrocarbons on one end and a polar group on the other end. In aqueous solution, lipids counteract the hydrophobic effect of the carbon chains with the hydrophilic nature of the polar end by forming raultimolecular aggragates, when the l i p i d concentration reachs high enough levels (the c r i t i c a l micellar concentration). One of the most prevalant structures observed in biological systems is the bilayer membrane, proposed by Gorter and Grendel (2) in 1925. Presently, the most widely accepted model of a biological membrane is the 'Fluid Mosaic Model' (see figure 1.2a), proposed by Singer and Nicolson in Fig. 1.1 The structure of glvcerophospholipids 3 o o H 2 C O C R 1 R , - C - O ^ C ^ H General Structure H 2 C O P O X 6 7 The alcohols contributing the polar X groups in the major glvcerophospholipids are shown below; Glycerophospholipid X 1,2-diacylglycerol 3-phosphate (phosphatidate) Phosphatidyl choline (lec i t h i n ) Phosphatidyl ethanolamine (cephalin) Phocphatidyl serine Phosphatidyl i n o s i t o l H H O C H 2 C H 2 N ( C H 3 ) 3 (basic) + H O C H 2 C H 2 N H 3 (basic) + N H 3 H O C H j C H - C O O -(amphoteric) OH OH H/u u \ O H H H V " (neutral) H O ^ H H/H H OH Diphocphatidyl glycerol (cardiolipin) o H O C ^ C H O H C H ^ O P - O - C H ^ C H C ^ 6- 6 6 (aci di c) 0:C C:0 R1 R2 1972 (3). This model b a s i c a l l y accepts the idea of a bilayer, but suggests that the individual l i p i d s and proteins in the structure move translationally and ro t a t i o n a l l y . Membrane proteins can be classified as to whether they are e x t r i n s i c (or e l e c t r o s t a t i c a l l y bound), or intrinsic (integrated into the hydrophobic region of the bilayer). This model has been able to assimilate the more recent concepts of: annular lipids on i n t r i n s i c proteins, phase transitions of bilayers, micro domains in bilayers d i f f e r i n g in composition and structure, and membrane turnover, to name a few. More recent physical studies (4) show that l i p i d s may a l s o form n o n - b i l a y e r s t r u c t u r e s such as hexagonal 2 phase and inverted micelle configurations ( figure 1.2 b). It i s highly possible that such structures may exist in vivo as a minor component, and i t i s l i k e l y that such stuctures are important in vivo as intermediates i n membrane fusion events. There are many good reviews on these topics (4,5). The exact structure of a l i p i d protein aggregate will depend to some degree on a l l the solutes present, but the structures of the individual l i p i d species i s c r i t i c a l l y important. Differences in the net charge of the head group are important, and l i p i d s can be classified according to whether they are ac i d i c (PI,PS,PA,PG,CL), or neutral (PC,PE,DG,TG,cholesterol) . Of course, the pH can change the charge of a l i p i d such as PE and many of i t s properties are pH dependent. Neutral l i p i d s generally outnumber the acidic species in b i o l o g i c a l membranes. A c i d i c p h o s p h o l i p i d s have the\property of forming complexes with divalent cations, p a r t i c u l a r l y calcium. The addition of calcium to an a c i d i c phospholipid mixture can cause isothermal phase transitions from bilayer to hexagonal 2 phase in some A Fig. 1.2 Multimolecular l i p i d aggregates  in aqueous solution (A) The f l u i d mosaic model. Globular proteins are inserted to varying degrees into a phospholipid b i l a y e r . The proteins and l i p i d s move r o t a t i o n a l l y and l a t e r a l l y with relative freedom. (B) The relationship between the average shape of a l i p i d species and i t s phase p r e f e r e n c e above the c r i t i c a l m i c e l l a r concentration. ( reproduced by permission (4)). Lipid Lysophosphol i pids Detergents Phase Micelar-Molecular Shape . - v - . Inverted Cone Phosphatidylcholine Sphingomyelin Phosphatidylserine Phosphat idylglycerc wm mm I Bilayer • • • • * t • • • • Q Cylindrical Phosphatidylethanol amine (unsaturated Cardidipin -Ca^+ Phosphatide acid -C a 2 + • » * * ' 'it/, -: Hexagonal (H„) '" A Cone B 6 s i t u a t i o n s (4) . The b i o l o g i c a l s i g n i f i c a n c e of these structures has not been f u l l y e l u c i d a t e d but t h e r e are some systems where they are b e l i e v e d t o be i m p o r t a n t . F o r example, the a d d i t i o n o f PA to neuroblastoma c e l l s has been shown to i n c r e a s e the p e r m e a b i l i t y to media calcium dramatically (6). L i p i d s can be considered to be c o n i c a l , c y l i n d r i c a l , or i n v e r s e c o n i c a l ( f i g u r e 1.2b), a c c o r d i n g to t h e i r average molecular shape. C y l i n d r i c a l l i p i d s , such as PC, are generally b i l a y e r s t a b i l i z i n g l i p i d s , as would be expected from t h e i r shape. The inverse conical and c o n i c a l shapes form micelles and inverted micelles r e s p e c t i v e l y , and a r e g e n e r a l l y p r e s e n t as a minor s p e c i e s i n membranes (4). The f a t t y a c i d composition can dramatically e f f e c t the shape of a molecule within a p a r t i c u l a r l i p i d c l a s s . Thus cis-enoic a c i d s i n PE can g i v e i t an i n v e r t e d cone shape, r e l a t i v e to the c y l i n d r i c a l shape of a s t r a i g h t chain PE (4). The f a t t y a c i d c o m p o s i t i o n i s a l s o important i n determining properties such as the t r a n s i t i o n temperature and the permeability of the membrane (4). O v e r a l l , i t i s e v i d e n t that large variety of l i p i d species found in a c e l l a l l o w s f o r an i n c r e d i b l e f l e x i b i l i t y i n the properties of i t s membranes. 1.13 The F u n c t i o n o f Phospholipids The most important f u n c t i o n of p h o s p h o l i p i d s i s t h e i r use in membranes, i n which l i p i d s and p r o t e i n s are the major components. PC, PE, and SM are the major s p e c i e s found i n animal membranes, with CL, PS, PI, and PG, being minor components. T r a d i t i o n a l l y , phospholipids have been thought of as b a s i c a l l y i n e r t s t r u c t u r a l components i n membranes, forming b i l a y e r s s i m i l a r to those found i n b i o l o g i c a l systems i n v i t r o . The f u n c t i o n a l a t t r i b u t e s o f membranes have usually been as s i g n e d to p r o t e i n s , with the e x c e p t i o n of the l i p i d s forming a r e l a t i v e l y n o n s e l e c t i v e p e r m e a b i l i t y b a r r i e r to i o n i c compounds and an annular sheath f o r the p r o t e i n s . I t was o r i g i n a l l y postulated that the p h o s p h o l i p i d composion of a organism was species s p e c i f i c . However, with the development o f more powerful a n a l y t i c a l techniques i t has been shown that l i p i d compositions depend more 0 on the organelle than the species ( t a b l e 1.1a). Moreover, the f a t t y a c i d composition of a p a r t i c u l a r p h o s p h o l i p i d c l a s s may be very d i f f e r e n t from t i s s u e to t i s s u e and may change s i g n i f i c a n t l y i n d i f f e r e n t b i o l o g i c a l s i t u a t i o n s . P h o s p h o l i p i d s a l s o t u r n o v e r at markedly d i f f e r e n t rates (table 1.1b), and the f a t t y acid, g l y c e r o l , and head groups tu r n o v e r i n d e p e n d e n t l y as w e l l ( 7 ) . Thus i t i s becoming i n c r e a s i n g l y e v i d e n t that the c e l l u l a r membrane i s a f i n e l y tuned, dynamic s t r u c t u r e i n which the p r o p e r t i e s of d i f f e r e n t l i p i d s are e x p l o i t e d to g i v e the o p t i m a l membrane composition to s u i t the membranes function and environment. A d d i t i o n a l f u n c t i o n s o f p h o s p h o l i p i d s i n membranes are being uncovered, p a r t i c u l a r l y i n r e g a r d to the minor components. One function of phospholipid i n a membrane i s to provide a storage form of arachidonate, which may be r e l e a s e d by the a c t i o n o f phospholipases A ?. In many systems (8), the c o n c e n t r a t i o n of arachidonate i s rate l i m i t i n g i n the s y n t h e s i s o f the v a r i o u s p r o s t a g l a n d i n s . Indeed i t appears that phospholipase A^ pla y a key r o l e i n c e r t a i n regulatory cascade systems. Another f u n c t i o n o f some g l y c e r o l i p i d t s i s t h e i r a b i l i t y to Table 1.1 V a r i a t i o n s i n l i p i d c o m p o s i t i o n and turnover (A) The l i p i d c o m p o s i t i o n h e p a t o c y t e membranes, ex p r e s s e d as a mole 8 pe r c e n t o f t o t a l p h o s p h o l i p i d . C o l l a t e d by McMurray and McGee (1 4 9 ) . (B) The h a l f - l i f e o f p h o s p h o l i p i d c l a s s e s , as measured with v a r i o u s p r e c u r s o r s . Redrawn from (150). L i p i d Plasma Nuclear Endoplasmic G o l g i M i t o c h o n d r i a l Lysosomal C l a s s membrane membranes r e c t i c u l u m membranes membranes membranes inner outer PC 34.9 61.4 60 .9 45.3 45.4 49.7 33.5 PE 18.5 22.7 18.6 17.0 25.3 23.2 17.9 PI 7.3 8.6 8.9 8.7 5.9 12.6 8.9 PS 9.0 3.6 3-3 4.2 0.9 2.2 8.9 PG 4.8 .« « * 2.1 2.5 * CL t r a c e 0 * 17.4 3.4 6.8 1-PC 3-3 1.5 4.7 5.9 * * 0 1-PE * 0 0 6.3 * * « SM 17.7 3.2 3.7 12.3 2.5 5.0 32.9 (* not determined) B T i s s u e P h o s p h o l i p i d Labeled P r e c u r s o r H a l f - L i f e C l a s s (h) Rat PC 32 [ P ] p h o s p h a t e 10.9 L i v e r PC 3 [ H j c h o l i n e 10.0 PC 14 [ C ] s e r i n e 1.2 PE 2.1 PS 7.0 Rabbit PC 3 [ H ] c h o l i n e 13-0 Lung PC L C j p a l m i t a t e 10.0 PC 3 [ H ] g l y c e r o l 8.0 PC 32 [ P ] p h o s p h a t e 88.0 Rat PS L C j s e r i n e 1.0 I n t e s t i n e PE 0.4 9 a c t i v a t e c r i t i c a l enzymes i n v i v o . F o r example, the concentration of plasma membrane PS and DG are c r u c i a l to the a c t i v i t y of protein kinase C (9) , and PS i s r e q u i r e d f o r the a c t i v a t i o n o f adenylate cyclase by glucagon (10). These and o t h e r examples o f p h o s p h o l i p i d coupling i n regulation schemes w i l l be discussed in section 1.5. L a s t l y , changes in p h o s p h o l i p i d c o n c e n t r a t i o n s in membranes have been implicated i n b a s i c c e l l u l a r p r o c e s s e s such as ion gating (6) , membrane fusion phenomena ( 1 1 ) , and nerve conduction (5). Once again membrane l i p i d metabolism responds i n a h i g h l y s p e c i f i c manner to the p a r t i c u l a r physiologic s i t u a t i o n . The uses o f p h o s p h o l i p i d s a r e not r e s t r i c t e d to membrane phenomena. P h o s p h o l i p i d i s a l s o a major component o f l i p o p r o t e i n s , b i l e , and l u n g s u r f a c t a n t . A g a i n l i p i d m e t a b o l i s m i s h i g h l y c o o r d i n a t e d i n o r d e r to p r o v i d e o p t i m a l l e v e l s o f l i p i d s f o r each process. 1.2. The Biosynthesis o f G l v c e r o p h o s p h o l i p i d s in Hepatocytes The pathways of hepatic p h o s p h o l i p i d metabolism are i n t e r r e l a t e d and complex. To f a c i l l i t a t e d i s c u s s i o n , the r e a c t i o n s have been d i v i d e d i n t o c a t a g o r i e s a c c o r d i n g t o whether they are s y n t h e t i c , intermediate, or degradative. Throughout the following, the reader i s refered to the pathway chart in figure 1.3. 1.21 Fatty A c i d S y n t h e s i s and A c t i v a t i o n Fatty acids may either be taken up from the serum or synthesized Figure 1.3: A summary c h a r t showing pathways of l i p i d metabolism. Abbreviations as i n text. 10 11 endogenously. A l l fatty acids are synthesized from ATP, NADPH, and Acetyl-CoA. Although t h i s process occurs in a l l organisms, i t i s p a r t i c u l a r l y prominent in the l i v e r and mammary glands of higher animals. The rate l i m i t i n g step in the synthesis of fatty acids is the conversion of Acetyl-CoA and CO^ to malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (EC 6.4.1.2.). The enzyme may exist as either a protomeric, inactive form, or as an active, aggregated form (12). The aggregation i s stimulated by c i t r a t e . Acyl-CoA as well as malonyl-CoA esters inhibit aggregation and activation. Thus the enzyme can be coordinated with glycolysis through citrate levels and feedback inhibited by i t s own product. The aggregate i s also depolymerized when phosphorylated by protein kinase A and i s thus inhibited when the concentration of cAMP is elevated (14). It is also phosphorylated by a cAMP independent protein kinase and th i s i n h i b i t s a c t i v i t y as well (15) . Acyl-CoA carboxylase may also be controlled transcriptionally (16) . It i s probable that the net a c t i v i t y i s determined by .a balance of a l l these factors. The malonyl-CoA produced i s then converted to fatty acid by a multi-enzyme system in the cytosol, termed the fatty acid synthetase complex . It i s a dimer with a subunit molecular weight of 230K (17). The regulation of this complex has been extensively studied in recent years and there are several excellent reviews on this subject (18,64). Fatty acids which are taken up from serum are converted to the CoA derivative by the enzyme acyl-CoA synthetase (EC 6.2.1.1). Acyl-CoA is refered to as the 'activated form' of fatty acid because i t is this derivative that i s used in subsequent reactions. Acyl-CoA may be 12. acted on by a fa t t y acid e l o n g a t i o n systems i n the mitochondria or by s p e c i f i c monooxygenase systems i n the endoplasmic recticulura; these i n t r o d u c e d o u b l e bonds i n the f a t t y a c i d . There i s p o s i t i o n a l s p e c i f i c i t y i n the placement o f double bonds (19)- Mammals can not synthesize l i n o l e i c or l i n o l e n i c a c i d s so these must be obtained from d i e t a r y s o u r c e s . These e s s e n t i a l f a t t y a c i d s a r e r e q u i r e d f o r arachidonate b i o s y n t h e s i s , the p r e c u r s o r o f prostaglandins (20). The acyl-CoAs may be used f o r g l y c e r o l i p i d b i o s y n t h e s i s or u t i l i z e d for B-oxidation. 1.22 The S y n t h e s i s o f PA The next step i n the s y n t h e s i s of g l y c e r o l i p i d s i s the synthesis of PA. PA may be s y n t h e s i z e d by two pathways: one requiring g l y c e r o l phosphate, and the other r e q u i r i n g dihydroxyacetone phosphate. These compounds are in e q u i l i b r i u m with NADH and NAD + i n the c e l l and so the r e l a t i v e amounts depend on the NADH:NAD+ r a t i o . The t o t a l amount of these t r i o s e phosphates depends on the r a t e o f g l y c o l y s i s (the major source of dihydroxyacetone phosphate) , the r a t e of l i p o l y s i s , and the rate o f g l y c e r o l uptake from the serum. The g l y c e r o l obtainedy by t h e s e l a s t two p r o c e s s e s can be p h o s p h o r y l a t e d i n v i v o by gl y c e r o l kinase (EC 2.7.1.30) (21). The s y n t h e s i s of PA from g l y c e r o l phosphate and acyl-CoA was f i r s t observed i n ginuea p i g l i v e r microsomes by Romberg and P r i c e r i n 1953 (22). Two a c y l a t i o n s are r e q u i r e d and the evidence suggests that two microsomal enzymes are r e q u i r e d (23). PA g e n e r a l l y has a saturated f a t t y acid at p o s i t i o n one and o l e i c or l i n o l e i c at position two in vivo. There i s s t i l l controversy as to whether this difference is do to substrate specificity during synthesis, or whether i t is introduced p o s t - s y n t h e t i c a l l y by a transacylase or by deacylase/reacylase a c t i v i t i e s . There is evidence that both methods could be used to some extent ( 2 3 - 2 5 ) . G l y c e r o l phosphate acyltransferase (EC 2.3.1.15) may also be controlled the level of calcium (26), and there is evidence that the enzyme can be regulated by cAMP in adipose tissue (27). The a c y l a t i o n of dihidroxyacetone phosphate to acyl-dihydroxyacetone phosphate and i t s subsequent reduction to lyso-PA has been observed in rat liver microsomes by'Hajra and Agranoff (28). The reduction requires NADPH as a cofactor. Despite numerous attempts to assess the significance of this pathway in the synthesis of PA (29) » there is no general agreement on i t s contribution to PA synthesis. A third way PA may be synthesized is by the action of DG kinase on ATP and DG. This enzyae a c t i v i t y undergoes marked fluctuations in vivo, and w i l l be discussed in more detail in section 1.3-1.23 The Biosynthesis of DG PA occupies a unique position in the synthesis of glycerolipids; it may either react with CTP to give CDP-DG, and hence be directed toward acidic phospolipid synthesis, or it may be hydrolyzed to DG and used for the synthesis of PE, PC and TG. The hydrolysis of PA to DG is catalyzed by PA phosphatase (EC 3.1.3.4). This activity was first observed in animals by Weiss et a l . in 1956 (30). PA phosphatase a c t i v i t y has been demonstrated i n mitochondria, lysosomes, microsomes, and cytosol (3D. The c y t o s o l i c enzyme i s moderately s p e c i f i c for PA containing one unsaturated f a t t y a c i d (32). B r i n d l e y et a l . have demonstrated a p o s i t i v e c o r r e l a t i o n between PA phosphohydrolase a c t i v i t y and the r a t e of h e p a t i c TG s y n t h e s i s , and have a l s o shown that the enzyme can be a c t i v a t e d by g l u c o c o r t i c o i d s i n v i v o . Thus, i t i s l i k e l y that PA phosphatase can- r e g u l a t e TG s y n t h e s i s i n some sit u a t i o n s (33). 1.24 The S y n t h e s i s of TG DG acyltranferase a c t i v i t y (EC 2.3.1.20) was f i r s t observed in a p a r t i c u l a t e chicken l i v e r f r a c t i o n i n 1956 by Weiss et a l . The enzyme shows s p e c i f i c i t y f o r 1,2-DG, and unsaturated f a t t y acids are prefered over s a t u r a t e s (35). I t has been shown that the enzyme i s in h i b i t e d by glucagon (36). However, Groener et a l . studied the ef f e c t s of fa s t i n g and refeeding on the rate of rat l i v e r TG synthesis i n v i v o (37), and found that the i n c r e a s e d r a t e of TG s y n t h e s i s a f t e r r e f e e d i n g c o u l d not be t o t a l l y e x p l a i n e d by in c r e a s e d DG acyltranferase a c t i v i t y . He concluded that the increased DG l e v e l s stimulate TG synthesis d i r e c t l y . T h i s agrees with a study by Sundler et a l . f who s t u d i e d the e f f e c t o f exogenous f a t t y a c i d on TG synthesis (38). I n c o r p o r a t i o n o f l a b e l e d g l y c e r o l increased l i n e a r l y into DG and TG with r e s p e c t to f a t t y a c i d c o n c e n t r a t i o n . The r e s u l t s imply that TG syn t h e s i s may be r e g u l a t e d by the concentration of DG, as well as glucagon l e v e l s . 15 1.25 The de novo Synthesis of PC PC is the most abundant phospholipid in animal membranes, and is also a major component of bi l e , lipoproteins, and lung surfactant. The major pathway for i t s biosynthesis was elucidated by Kennedy and coworkers in the 1950's (1). They found that the de novo synthesis of PC occurs in three steps, r e q u i r i n g three separate enzymatic ac t i v i t i e s . The f i r s t step i s the phosphorylation of choline, catalyzed by the enzyme choline kinase ( EC 2.7.1.32). ATP i s the phosphate donor in this reaction. The second step in the synthesis is catalyzed by the enzyme CTP:phosphocholine cytidylyltransferase ( EC 2.7.7.15), which makes CDP-choline froro phosphocholine and CTP. The final step is the transfer of phosphocholine from CDP-choline to DG, forming CMP and PC. This step is catalyzed by the enzyme cholinephosphotransferase (EC 2.7.8.2 ). Although this pathway was demonstrated almost 30 years ago, very l i t t l e advancement was made on the knowledge of i t s control for many years. Using the average concentrations of the PC related metabolites in rat l i v e r , as well as the p r e d i c t e d i n t r a c e l l u l a r equilibrium constants of the reactions involved, Infante made some theoretical calculations which implied that the f i r s t two steps in the synthesis are out of e q u i l i b r i u m (39). Thus e i t h e r the choline kinase or cytidylyltransferase step could be involved in the regulation of PC biosynthesis. The third step is very close to equilibrium. The phosphorylation of choline can be rate l i m i t i n g in chicken li v e r . Studies by Vigo and Vance demonstrated that the rate of liver PC biosynthesis increased two f o l d a f t e r two days of diethylstilbesterol 16 treatment (40). There was a p o s i t i v e c o r r e l a t i o n beween this stimulation and the a c t i v i t y of choline kinase (41). Phosphocholine concentrations were also increased two f o l d . The concentration of phosphocholine in chicken l i v e r was found to be about 0.15mM, and the Km of the cytidylyltransferase for phosphocholine is about 0.17mM (42). Thus the increase in choline kinase a c t i v i t y i s d i r e c t l y translated into an increase of PC synthesis in t h i s system. Subsequent studies by Paddon et a l demonstrated that the d i e t h y l s t i l b e s t e r o l treatment increased the amount of immunotitratable choline kinase, suggesting that the stimulation was a result of increased choline kinase synthesis (43) .^This agrees with the widely accepted concept that steroids act at the level of gene expression. The c y t i d y l y l t r a n s f e r a s e step can also be regulatory in this system. In the same set of experiments, the rate of PC synthesis decreased r e l a t i v e to c o n t r o l v a l u e s on the t h i r d day of diethylstilbesterol treatment (41). However, choline kinase activity as well as phosphocholine concentrations were s t i l l elevated. On the other hand the activity of the cytidylyltransferase was decreased by 2 fo l d . Thus both enzymes are i n v o l v e d i n the r e g u l a t i o n of PC biosynthesis in chicken l i v e r . The regulatory features of PC synthesis appear to be different in adult rat l i v e r and Hela c e l l s . In these c e l l s the concentration of phosphocholine is about 1-2mM, which i s well above the apparent Km of the cytidylyltransferase (0.17mM) (47). Thus, fluctuations in the concentration of this substrate would have l i t t l e effect on the rate of PC synthesis. The concentration of choline in these cells in 5-10 fold lower than the the c o n c e n t r a t i o n of. phosphocholine (45), suggesting that choline i s rapidly phosphorylated upon uptake, with the rate limiting step occuring subsequent to this step. Choline has two possible fates upon entering the c e l l : i t may be phosphorylated and hence commited to PC synthesis, or i t may be oxidized to betaine in the mitochondria. Studies by Pritchard and Vance on cultured rat hepatocytes showed that low concentrations of choline in the media decreased the amount of labeled choline that was oxidized, while the pool size of phosphocholine remained constant (45). Increasing media choline l e v e l s increased the amount of l a b e l oxidized to betaine produced. Very l i t t l e r a d i o a c t i v i t y was recovered in CDP-choline This provides good evidence that the production of CDP-choline i s rate limiting in this system. The properties of CTP: phosphocholine cytidylyltransferase have received considerable attention in the past few years. The enzyme is found predominantly in cytosol when l i v e r i s homogenized in saline, but found in the microsomes when homogenized in d i s t i l l e d water (46). This implies that the enzyme i s e x t r i n s i c l y bound to the endoplasmic rectlculum. Choy et a l . p a r t i a l l y p u r i f i e d the enzyme from rat li v e r cytosol (47) . They found that the enzyme i s markedly stimulated when rat l i v e r p h o s p h o l i p i d i s added. They also found that the cytosolic enzyme, with a molecular weight of about 2.0x10 (L-Form) , aggregates to a high molecular weight form with particle weights from 5 7 5.6x10 to about 1.3x10 (H-Form) . H-Form had an increased basal activity but was also stimulated by rat l i v e r phospholipid. Choy and Vance studied the l i p i d activation in more detail (48). They found that lyso-PE had the greatest a c t i v a t i n g properties, while PS and PI also stimulated the enzyme to a lesser extent. Some species of lyso-PC could 18 i n h i b i t the enzyme by as much as 80%. They found a positive correlation between the activation of stored cytosol and the amount of lyso-PE present. The data implies that lyso-PE and acidic phospholipids could play a role in regulating the activity of the enzyme. The aggregation of the enzyme was also studied in more detail by Choy et a l . (49). They found that DG and PG stimulated the aggregation of the enzyme, although i t was important that the DG be delivered in a proper form. Thus, phospholipase C treatment of cytosol increased the aggregation two fold. PG stimulates both the aggregation and activity of the enzyme (50). Although PG is present in only trace quantities in liver, it seems to be important in developing lung, where the production of PG and the activation of the cytidylyltransferase are closely correlated (50). Thus PG may serve to signal the production of lung surfactant in this system. The implications of these in v i t r o observations for the regulation of liver PC synthesis are quite extensive. It could be postulated that the aggregation of the enzyme in v i t r o i s a r e f l e c t i o n of an in vivo form of regulation, functioning to translocate cytosolic enzyme to local DG domains in the endoplasmic recticulum. The enzyme would then produce its product, CDP-choline, in close proximity to the DG required in the final step of PC synthesis. This would significantly increase the efficiency of PC synthesis and allow the CDP-choline pool to remain very small, as it in fact is (44). A study by Pritchard et al showed that the rate of RC synthesis by the de novo pathway is stimulated by 3-deazaadenosine (66). They demonstrated a positive c o r r e l a t i o n between the increase in PC synthesis and the amount of cytidylyltransferase found in the 19 microsomes. The t o t a l amount o f enzyme i n the c y t o s o l and microsomes remained constant. Another study showed that when embryonic chicken muscle c e l l s were grown i n the p r e s e n c e of phospholipase C, the c y t i d y l y l t r a n f e r a s e was t r a n l o c a t e d to the microsomal f r a c t i o n and activated 3 f o l d (67). T h i s was c o r r e l a t e d i t h a 3-5 f o l d increase in theincorporation of choline l a b e l . These experiments suggest that the aggregation and a c t i v a t i o n of the enzyme may be Important regulatory features of the enzyme. PC synthesis can conceivably be regulated by the concentration of CTP. Vance et a l s t u d i e d the mechanism by which p o l i o v i r u s stimulates the r a t e of PC s y n t h e s i s i n Hela c e l l s (51). They found that that the increase in PC s y n t h e s i s was due to a stimulation of the c y t i d y l y l t r a n s f e r a s e s t e p . A s u b s e q u e n t s t u d y by Choy et a l . demonstrated that the i n c r e a s e d r a t e of t h i s r e a c t i o n was due to an i n c r e a s e i n the c o n c e n t r a t i o n o f CTP i n the Hela c e l l s (52). The p o s s i b i l i t y that CTP regulates PC synthesis has not been v e r i f i e d in other systems to date. CTP i s r e q u i r e d f o r the s y n t h e s i s of a l l phospholipid classes, although i t i s u t i l i z e d i n d i f f e r e n t ways. Thus i t i s tempting to speculate that CTP may be a universal signal for the synthesis of phospholipids. I t would be i n t e r e s t i n g to do experiments w i t h s y n c h r o n i z e d m i t o t i c c e l l s and d e t e r m i n e i f CT? l e v e l s are associated with the increase i n p h o s p h o l i p i d synthesis that must occur i n order to generate new membranes. Recent s t u d i e s by P e l e c h et a l have shown that the r a t e of PC b i o s y n t h e s i s can be i n h i b i t e d by cAMP a n a l o g s i n c u l t u r e d r a t hepatocytes (53). This i n h i b i t i o n was c o r r e l a t e d with an i n h i b i t i o n of the c y t i d y l y l t r a n s f e r a s e . The i n h i b i t i o n o f the enzyme a c t i v i t y could only be demonstrated when NaF and EDTA were added in the homogenizing buffer. NaF and EDTA i n h i b i t p r o t e i n phosphatases and protein kinases, respectively, and so i t was postulated that the i n a c t i v a t i o n was due to a protein p h o s p h o r y l a t i o n . F u r t h e r support f o r t h i s hypothesis was gained with a ser i e s of i n v i t r o s t u d i e s ( 5 4 ) . I t was found that NaF i n h i b i t s c y t o s o l i c c y t i d y l y l t r a n s f e r a s e i n a time dependent fashion. The i n h i b i t i o n could be mimicked with Mg-ATP or calcium, and supressed with protein k i n a s e i n h i b i t o r p r o t e i n i s o l a t e d from rabbit s k e l e t a l muscle. However, the f i n a l p roof t h a t the enzyme i s phosphorylated must a w a i t t h e p u r i f i c a t i o n o f t h e enzyme so t h a t d i r e c t phosphorylation can be demonstrated. The presumptive phosphorylation i n h i b i t e d aggregation of c y t o s o l i c c y t i d y l y l t r a n s f e r a s e and increased the Km f o r CTP. The i n h i b i t i o n c o u l d be r e v e r s e d by a d d i t i o n of phospholipid. However, a cAMP dependence o f the i n v i t r o c o u l d not be demonstrated. This r a i s e s the q u e s t i o n of how the cAMP analogs work to i n h i b i t the c y t i d y l y l t r a n s f e r a s e in hepatocytes. In f i g u r e 1 .4 a model o f c y t i d y l y l t r a n s f e r a s e r e g u l a t i o n i s presented which combines the r e s u l t s d i s c u s s e d above i n t o a single system. I t should be emphasized t h a t much more evidence must be obtained before t h i s model can be accepted. Primarily, proof that the enzyme i s phosphorylated must be o b t a i n e d and the i d e n t i t y o f the presumptive p r o t e i n kinase must be d i s c o v e r e d . A d d i t i o n a l l y , more experiments must be performed t o demonstrate that a c t i v a t i o n and aggregation are important i n v i v o . L a s t l y , the importance of CTP in regulating the synthesis of PC must be established. Choline phosphotransferase i s an i n t r i n s i c microsomal enzyme, and Figure 1 . 4 A model for the regulation of CTP:phosphocholine  cytidylyltransferase: The model summarizes the known regulatory features of the enzyme, d e p i c t i n g the relationship between phosphorylation, aggregation, and activation. The unphosphorylated form (square form) may be translocated to the endoplasmic recticulum by interaction with aggregating lipids such as DG, increasing the particle weight, and allowing CTP-choline to be made in close proximity to DG. Al t e r n a t i v e l y , the enzyme may be inhibited by phosphorylation, (circle form). This inhibition may be reversed by acidic phospholipids, which decrease the Km for CTP.Thus the enzyme may be activated (thatched form) in either the cytosol or the microsomes. c a t a l y z e s the f i n a l r e a c t i o n i n t h e de novo s y n t h e s i s of PC. I t s enzymatic a c t i v i t y i s r e s t i c t e d t o the c y t o p l a s m i c s i d e o f the endoplasmic r e c t i c u l u m (55). The r e a c t i o n i s r e v e r s i b l e i n v i v o . Choline phosphotransferase has only a s l i g h t d i f f e r e n t i a l s p e c i f i c i t y for p a r t i c u l a r DG, i m p l y i n g that the a c y l groups i n PC are e i t h e r regulated by a v a i l a b i l i t y or a d j u s t e d post s y n t h e t i c a l l y (56). The enzyme a c t i v i t y f l u c t u a t e s s i g n i f i c a n t l y i n response to v a r i o u s stimulations but t h i s i s probably an a d a p t i v e response rather than a regulatory event. 1.26 The de novo S y n t h e s i s of PE PE i s formed from ethanolaraine and d i g l y c e r i d e by a pathway eq u i v a l e n t to the s y n t h e s i s of PC. Although the r e a c t i o n s are the same, i t i s c l e a r that the enzymes i n v o l v e d i n the two pathways are d i s t i n c t (56,57). Less i s known about the synthesis of PE than PC. It would seem that some of the r e g u l a t o r y features of the PE pathway must be d i f f e r e n t from those of the PC pathway, i n order to maintain l i p i d d i v e r s i t y . When the media c o n c e n t r a t i o n of ethanolamine i s low, i t appears that the phosphorylation o f ethanolamine can be rate l i m i t i n g to synthesis of PE ( 5 8 ) . Few experiments have been done with higher ethanolamine l e v e l s . I t seem3 probable however that i n t h i s s i t u a t i o n the s y n t h e s i s o f C D P - e t h a n o l a m i n e can be r a t e l i m i t i n g (39). Ethanolamine phosphotransferase ( EC 2.7.3.1) p r e f e r s hexenoic fa t t y acids at position 2 and t h i s may help e x p l a i n why 20$ of the PS found in rat l i v e r i n vivo c o n t a i n s hexenoic a c y l groups (56). PE may also 23 be made by the d e c a r b o x y l a t i o n of PS ( s e c t i o n 1.34) but i t i s not known how much of the c e l l s PE i s made by t h i s pathway. However, t h i s pathway does not r e s u l t i n net phospholipid synthesis. 1.27 The CDP-DG Pathway to A c i d i c Phospholipids Instead of being converted to DG, PA may r e a c t with CTP to form CDP-DG. T h i s r e a c t i o n i s c a t a l y z e d by the microsomal enzyme PA c y t i d y l y l t r a n s f e r a s e ( EC 2. 7 . 7 . 4 1 ) . L i t t l e i s known about t h i s enzyme; one study found a high percent of p o l y e n o i c CDP-DG i n vivo r e l a t i v e to PA, but a p r e f e r e n c e f o r p o l y e n o i c f a t t y acids could not be demonstrated i n v i t r o ( 5 9). I t has not be determined then whether the high a r a c h i d o n a t e c o n t e n t o f the PI produced from CDP-DG i s i n t r o d u c e d at t h i s s t e p , o r w h e t h e r i t i s i n t r o d u c e d p o s t s y n t h e t i c a l l y . The enzyme i s s t i m u l a t e d by GTP (61). This stimulation i s supressed by EDTA and NaF, and can not be seen with nonhydrolyzable GTP analogs. This suggests th a t there i s a phosphorylation involved,. T h e r e have been few s t u d i e s i n v e s t i g a t i n g the r e l a t i o n o f PA-cytidylyltransferase to PA-phosphohydrolase. One study showed that both enzymes have a high r e s e r v e c a p a c i t y and normally operate well below maximal rates (62). Once CDP-DG i s formed, i t i s r a p i d l y reacted with either i n o s i t o l phosphate or g l y c e r o l p h o s p h a t e t o p r o d u c e PI or PG phosphate, r e s p e c t i v e l y . These r e a c t i o n s have been r e p o r t e d to o c c u r i n mi c r o s o m e s , G o l g i a p p a r a t u s , p l a s m a membrane, and t h e i n n e r mitochondrial membrane (63). The PG phosphate i s hydrolyzed to PG and condenses with w i t h a n o t h e r PG t o make CL. T h i s r e a c t i o n occurs predominantly on the inner mitochondrial-membrane (64). Most of the CL formed remains i n the mitochondria, and i s enriched i n l i n o l e a t e (65 ). It i s not c l e a r how the s e l e c t i v i t y f or l i n o l e a t e i s achieved. 1.3. Intermediary PL Metabolism In t h i s s e c t i o n , r e a c t i o n s are d e a l t w i t h that i n v o l v e no net synthesis of phospholipids. Although these reactions obviously overlap with what are termed s y n t h e t i c and d e g r a d a t i v e pathways, they are separated here to emphasize t h a t t h i s i s where the ' r e t a i l o r i n g ' of phospholipids occurs, to maintain the proper f a t t y acid proportions in the phospholipid classes, and to p r o v i d e another l e v e l of control over the head groups. It i s becoming i n c r e a s i n g l y evident that the control of these r e a c t i o n s i s i n v o l v e d i n c o u p l i n g phenomena and cascade pathways. 1.31 PE-N-Methvlation In addition to the Kennedy pathway, PC may be formed from PE by three s e q u e n t i a l methy l a t i o n s . The r e a c t i o n i s c a t a l y z e d by PE m e t h y l t r a n f e r a s e ( EC 2.1.1.17). The enzyme i s i n t r i n s i c a l l y microsomal, uses SAM as a m e t h y l d o n o r , and i s i n h i b i t e d by SAH (65).The enzyme has been shown to prefer PE with polyenoic acyl groups, and t h i s may c o n t r i b u t e s i g n i f i c a n t l y to the amount of unsaturates found i n PC i n v i v o (68). I t has been r e p o r t e d that t h i s pathway accounts for up to 20% of the PC i n r a t hepatocytes (69) • The majority of PE made by the de novo pathway i s methylated to PC^  i n hepatocytes (70). The pool size's of the intermediates mono- and di-methyl-PE are extremely small (70) . The enzyme has also been reported to occur in several other c e l l types, but the s p e c i f i c a c t i v i t y of the enzyme is very low in these tissues and thus the methylation pathway i s probably of minor significance in these tissues (71). There i s evidence that the enzyme i s regulated by cAMP. Castano et a l . (72) have shown that the a c t i v i t y of PE methyltransferase increases upon addition of of glucagon to hepatocytes. Pritchard et a l . (73) have shown the enzyme i s activated by cAMP analogs, but the flux through the pathway was inhibited as measured by ethanolamine or methionine labeling experiments. It seems possible that the enzyme is activated as measured in v i t r o because there i s more endogenous PE. However, further studies w i l l be needed to accertain this. PE-N-methylation has been reported to be involved in a number of exitation-response coupling systems in the plasma membranes of some cells (74,75); however this work has recently been criticized by Vance and Kruijff (71), who point out that the smair number of PC molecules synthesized by PE-N-methylation could not account for the changes observed in membrane properties. Thus the role of PE methylation in these processes remains uncertain. 1.32 The Synthesis of PS In 1961, i t was shown that PS can be formed by a base exchange with serine and PE, forming PS and ethanolamine (76). The reaction occurs in the microsomes, i s reversible in v i t r o , and stimulated by calcium (77). The enzyme, PS synthase ( EC 2.7.8.8), is found in the 26 microsomes of some tissues, p a r t i c u l a r l y brain and li v e r . While PS may also be made by a CDP-DG pathway in 3ome bacteria and animals, no corresponding activity has been demonstrated in mammals (78). This is i n t e r e s t i n g i n that i t s h i f t s the s y n t h e s i s of PS from the mitochondria to the endoplasmic recticulum, and from the control mechanisms operating on the other a c i d i c phospholipids, which are produced by the CDP-DG pathway. Although there i s s t i l l some controversy about alte r n a t i v e pathways to PS (78-80), i t appears at present that base exchange i s the major, i f not sole, source o f PS (81). 1.33 Other Base Exchange Ac t i v i t i e s Besides the synthesis of PS, base exchange a c t i v i t i e s ha/e been reported for PC and PE in l i v e r (81) and brain (82). The enzymes are found in microsomes and are calcium stimulated, similar to the PS synthase activity. However, they are c l e a r l y distinct enzymes, having been separated by g e l f i l t r a t i o n (83). One of these enzyraesis probably the PS synthase discussed above. The other appears to be used for ethanolamine i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s (81). Rate calulations by Bjerve indicate that the biosynthesis of PC by this pathway i s probably n e g l i g i b l e i n vivo (81). His study also indicated that ethanolamine i s p r e f e r e n t i a l l y incorporated into hexenoic containing phospholipids. Such was not the case for either serine or choline. In vivo studies by Orlando et a l . indicate that the PE synthesized by t h i s pathway turns over very rapidly. This provides some basis for other reports on PE-N-methylation (84) which 27 i suggests that there are 2 pools of PE. The pool incorporated into PC from PE has more u n s a t u r a t i o n and turns over faster. Thus the combination of base exchange and methylation provides another way in which arachadonate can be incorporated selectively into PE and PC. 1.34 The Decarboxylation of PS Rat liver mitochondria contain an enzyme, PS decarboxylase (EC 4.1.1.65), which converts PS to PE and CO^ (85). Very l i t t l e is known about this enzyme, or about the contribution of t h i s pathway to the ce l l s ethanolamine, but i t is in t e r e s t i n g in that i t provides a source of ethanolamine, and hence choline, from a nonessential amino acid. Moreover, i t requires the i n t r a c e l l u l a r migration of PS from the endoplasmic recticulum to the mitochondria to make PE, and back to the endoplasmic recticulum for PC synthesis. This cyclic migration occurs at rate faster than the average turnover time of either PE or PC, as labeled from their de novo precursors ( see table 1.1b). This could also be a significant source of mitochondrial PE. 1.35 Phosphorylation of DG In the section on glycerolipid synthesis, two ways were described by which the c e l l can make PA; one using gl y c e r o l phosphate, the other employing dihydroxyacetone phosphate. A t h i r d , and potentially very important, method i s the phosphorylation of DG to PA by DG kinase ( EC 2.7-1. ). Unlike the enzymes in the other pathways, DG kinase activity undergoes marked fluctuations which have been correlated with tissue s p e c i f i c stimulation (24,86). The enzyme operates at an functional branch point, and may give the c e l l increased f l e x i b i l i t y in maintaining the r a t i o between PC&PE/acidic phospholipids. It is also important in maintaining the PI cycle, which is discussed below. 1.36 Exchange of Acvl Groups Lysolipids generated by the action of phospholipases A^  or A^ (see section 1.5) are rapidly reacylated in vivo. Lands (24) studied the specificities of the enzymes involved in rat liver and found that the A^ a c y l t r a n s f e r a s e s i n c o r p o r a t e saturated f a t t y a c i d (primarily s t e a r a t e ) . He a l s o showed that the acyltransferases specific for the 2 position incorporate unsaturated fatty acids, and are responsible for introducing arachidonate into liver phospholipids produced from oleate or l i n o l e a t e containing PA. L i t t l e else has been done toward e l u c i d a t i n g the p a r a m e t e r s d e t e r m i n i n g these speci f i c i t i e s . Thus i t i s d i f f i c u l t to say at t h i s time whether the chief factor i s enzyme s p e c i f i c i t y pr substrate availability. It seems likely that both factors are important to some degree. 1.4. The Degradation of Phospholipids The synthesis of l i p i d should obviously outweigh the breakdown of l i p i d in a growing t i s s u e . However s t u d i e s investigating l i p i d degradation have revealed that lipases are very active in many animal tissues. Considering what has already been said about the need to maintain a dynamic, f l e x i b l e membrane l i p i d composition, i t i s 29 apparent that the lipids must turn over at a significant rate in order that they be replaced with l i p i d s more suited to a changing environment. Also, in conjunction with some of the enzymes pathways mentioned above, partial degradation and resynthesis of lipid occurs to retailor fatty acid composition. Also, TG must be broken down when i t i s required for an energy source. Lastly, as was mentioned in section 1.2, phospholipases are responsible for the specific release of arachidonate. 1.41 Phospholipases There are phospholipases for every ester linkage in a phospholipid; they are signified by A^, A^, C, and D, for the acyl linkages at positions 1 and 2, the glycerol-phosphate linkage, and the base-phosphate linkage, respectively. The A type phospholipases are ubiquitous, being distributed throughout the endoplasmic recticulura, plasma membrane, lysosomes, and Golgi apparatus of most tissues (24). Phospholipase A^ activity is very important in the mitochondria as well. The A types are generally stimulated by calcium (24 ). Unsaturated fatty acids are cleaved much more rapidly in the 1 position^ while saturated fatty acids are cleaved faster at the two position (87). Combined with the reverse speci f i c i t i e s for the acyltransf erases, it is easy to understand how predominant fatty acid distributions are maintained. Phospholipase A^ type activities attack bilayers much more readily i f there are bilayerirregularities, including small radius curvature, dislocation, or any sort of defect which might be found at aboundary between g e l and l i q u i d c r y s t a l l i n e states (88 ) . I t seems possible that these c o n d i t i o n s c o u l d occur under very s p e c i f i c p h y s i o l o g i c a l c o n d i t i o n s . P h o s p h o l i p a s e A ^  a c t i v i t i e s a r e r e s p o n s i b l e f o r r e l e a s i n g a r a c h i d o n a t e from PC, PE and PS dur i n g c a l c i u m i n d u c e d p r o s t a g l a n d i n s y n t h e s i s ( 8 9 ) . M i t o c h o n d r i a l phospholipase A^ a c t i v i t y a l s o i n c r e a s e s concomraitantly with calcium release from m i t o c h o n d r i a i n hepatocytes ( 9 0 ) . Thus, phospholipase A a c t i v i t y i s very important f o r a v a r i e t y of c e l l u l a r processes. Phospholipase C and D a c t i v i t i e s have been studied far less in animal systems. Besides PA-phosphatase, the only phospholipase C that has been investigated thoroughly i s the PI s p e c i f i c a c t i v i t y found in the cytosol of some tissues ( 9 1 ) . I t i s activated at concentrations of calcium that are f a r l e s s than those r e q u i r e d f o r phospholipase A a c t i v a t i o n , and has been s t r o n g l y i m p l i c a t e d i n a variety of coupling schemes ( 9 2 ) . The r o l e of t h i s enzyme i n some of these events i s discussed i n s e c t i o n 1 . 5 . There i s almost nothing i n the l i t e r a t u r e about animal phospholipase D a c t i v i t i e s i n vivo. 1.5. The Hormonal Reg u l a t i o n o f Hepatic G l y c e r o l i p i d  Metabolism It i s cl e a r that the regulation of l i p i d metabolism i s complex and operates on many - l e v e l s . C o n s i d e r i n g the enzymes d i s c u s s e d i n the previous sections, one can make some g e n e r a l i z a t i o n s about"enzymatic regulation. Regulatory enzymes are o f t e n found at the beginning of a biosynthetic pathway, or at a branch p o i n t . Such a reaction i s said to 'commit' the s u b s t r a t e t o t h e pathway. R e g u l a t o r y enzymes must catalyze reactions which are out of o f thermodynamic equilibrium, so that the net rate o f the r e a c t i o n may be s i g n i f i c a n t l y changeable by a change i n the enzymes a c t i v i t y . Also, the concentration of the reaction product involved must be l e s s than the concentration of the substrate, and the product must tur n o v e r f a s t e r than the substrate; such a reaction i s s a i d to be r a t e l i m i t i n g . In t a b l e 1.2, the enzymes i n v o l v e d i n l i p i d m e t abolism t h a t have r e g u l a t o r y f u n c t i o n s are l i s t e d . These enzymes are the substratum upon which complex regulatory schemes are superimposed . *• 1.51 The E f f e c t of F a s t i n g on H e p a t i c G l y c e r o l i p i d Metabolism I t has l o n g been known t h a t d i f f e r e n t n u t r i t i o n a l s t a t e s p r o f o u n d l y e f f e c t t h e h e p a t i c TG l e v e l s , and yet the amount of phospholipid i s much les s e f f e c t e d . F a s t i n g markedly reduces hepatic TG and DG l e v e l s , w h i l e r e f e e d i n g r a p i d l y i n c r e a s e s the amount of these compounds (93). The r a t e s o f PC and PE s y n t h e s i s are only s l i g h t l y affected by n u t r i t i o n a l s t a t e s . This would then seem to be a case where the s y n t h e s i s of v a r i o u s g l y e r o l i p i d s i s c o o r d i n a t e l y r e g u l a t e d i n response t o serum c o n d i t i o n s , and thus serves as a potential in vivo model to study h e p a t i c l i p i d regulation. In order to study this" model i n t i s s u e c u l t u r e i t i s necessary to determine what f a c t o r s i n the serum serve to c o o r d i n a t e hepatic l i p i d metabolism w i t h the n u t r i t i o n a l s t a t e o f the organism. Of a l l the serum compounds which can show dramatic changes in concentration depending on the n u t r i t i o n a l s t a t e , t h e c o n c e n t r a t i o n o f glucose remains f a i r l y constant at 4;5raM (94). This i s because the functioning Table 1.2 Regul a t o r y enzymes i n p h o s p h o l i p i d metabolism: See text for appropriate discussion and references. ENZYME INHIBITORS ACTIVATORS Acetyl-CoA Carboxylase G l y c e r o l phosphate A c y l t r a n s f e r a s e DG A c y l t r a n s f e r a s e PA-Phosphohydrolase CTP :Phosphocholine Cyt i d y l y 1 t r a n s f e r a s e CTP:phosphoethanolamine C y t i d y l y l t r a n s f e r a s e PS synthase PE-N-MethyItransferase CTP:PA: c y t i d y l y 1 t r a n s f e r a s e Acyl-CoA, cAMP cAMP, Calcium Glucagon G l u c o c o r t i c o i d s N o r a d r e n a l i n cAMP, Calcium, cAMP (?) cAMP I n s u l i n , C i t r a t e I n s u l i n I n s u l i n I n s u l i n , F a t t y a c i d s A c i d i c p h o s p h o l i p i d s Calcium GTP o f the CNS, and thus the s u s t a n e n c e o f c o n s c i o u s n e s s , c r i t i c a l l y depends on blood glucose. The l i v e r i s the t i s s u e p r i m a r i l y responsible for maintaining blood glucose d u r i n g n u t r i t i o n a l stress, by converting glycogen, amino acids, and g l y c e r o l to glucose. Two peptide hormones, i n s u l i n and glucagon, are widely accepted as the major signals which c o o r d i n a t e h e p a t i c metabolism and with the n u t r i t i o n a l state to su s t a i n o p t i m a l serum glucose. Both hormones are secreted by the pancreas, from one o f two c e l l types (95). Insuli n s e c r e t i o n i s s t i m u l a t e d when b l o o d g l u c o s e l e v e l s are s l i g h t l y elevated. I n s u l i n causes g l u c o s e uptake i n the p e r i p h e r y , glycogea synthesis i n the l i v e r and s k e l e t a l muscle, and TG synthesis in the l i v e r and adipose (96). Through these p r o c e s s e s , i n s u l i n decreases blood glucose and a c t s as a s i g n a l to s t o r e n u t r i e n t s . On the other hand, glu c a g o n i s commonly r e f e r e d to as the ' s t a r v a t i o n s i g n a l ' , and i s secreted when glucose l e v e l s are s l i g h t l y decreased. Glucagon p r i m a r i l y o perates on t h r e e types of c e l l s ; a d i p o c y t e s , s k e l e t a l muscle, and hepatoc y t e s . I t causes l y p o l y s i s in adipocytes, and g l y c o g e n o l y s i s i n muscle. I n h e p a t o c y t e s glucagon s t i m u l a t e s g l y c o g e n o l y s i s , g l u c o n e o g e n e s i s , and k e t o g e n e s i s . I t a l s o i n h i b i t s g l y c o l y s i s , lipogenesis, and glycogen synthesis (95). Glycerol and free f a t t y a c i d , which are r e l e a s e d into the serum by glucagon treated a d i p o c y t e s , are a l s o b e l i e v e d to play important r o l e s i n coordinating hepatic l i p i d metabolism to the nutrional state. G l u c o c o r t i c o i d s , s e c r e t e d by t h e a d r e n a l medula d u r i n g long term starvation and ethanol s t r e s s , are important i n regulating hepatic PA phosphatase (97), and may have other e f f e c t s as w e l l . The importance of these compounds i n r e g u l a t i n g h e p a t i c l i p i d metabolism i s discussed 34 below. Thus h e p a t i c l i p i d m e t a b o l i s m i s c o o r d i n a t e d to the n u t r i t i o n a l s t a t e o f the o r g a n i s m by f i v e major serum f a c t o r s : glucagon, i n s u l i n , g l u c o c o r t i c o i d s , f a t t y a c i d , and g l y c e r o l . Glucagon binds to s p e c i f i c r e c e p t o r s on the outside of hepatocyte plasma membranes and has a bi n d i n g constant of about 4.5nm (98). I t i s widely accepted that' the primary e f f e c t o f glucagon on hepatocytes i s to activ a t e adenylate c y c l a s e which produces cAMP from ATP (98). Many other hormones are known to a c t i v a t e a d e n y l a t e c y c l a s e , i n c l u d i n g adrenalin and v a s o p r e s s i n . T h i s a c t i v a t i o n i s a transmembrane event, i n that the glu c a g o n - r e c e p t o r complex i s on the serum side of the plasma membrane and adenylate c y c l a s e i s on the cytoplasmic side. Both the hormone r e c e p t o r and adenylate c y c l a s e are i n t r i n s i c membrane p r o t e i n s , and the r e c e p t o r - a d e n y l a t e c y c l a s e complex has been the t o p i c of many s t u d i e s . Levey (100) has s o l u b i l i s e d the adenylate cyclase complex with a nonionic detergent and found i t to be a complex with a molecular weight between 100-200K. Phospholipid added back to the system was required f o r r e s t o r a t i o n o f the basal a c t i v i t y as well as the hormone r e s p o n s i v e n e s s . Rethy et a l . (101) studied adenylate cyclase i n plasma membrane a f t e r treatment w i t h phospholipase C or e t h e r : b u t a n o l e x t r a c t i o n , a p r o c e s s which s e l e c t i v e l y removes phospholipids. They found both the b a s a l and hormonally stimulated r a t e s reduced. Recovery o f t h e b a s a l l e v e l c o u l d be achieved by a d d i t i o n of PI but t h i s had no e f f e c t on the hormonally a c t i v a t e d rate. However addition of PS could r e s t o r e adrenalin a c t i v a t i o n . These r e s u l t s imply that a c i d i c p h o s p h o l i p i d s can be important couplers i n adenylate c y c l a s e a c t i v a t i o n , but i t i s t o o e a r l y to judge the phy s i o l o g i c a l s i g n i f i c a n c e of t h i s . The adenlyate cyclase receptor has regulatory subunits which give i t GTP s e n s i t i v i t y (102). x-Adrenergic stimulation and glucagon appear to involve separate GTP subunits and this may be important in determining the differential effects of these hormones. Some prostaglandins also activate adenylate cyclase by apparently by binding directly to the complex (103). Once glucagon activates adenylate cyclase, i n t r a c e l l u l a r cAMP levels can be elevated by upto 10 fold (104). cAMP activates protein kinase A, which then phosphorylates a number of proteins and so i n i t i a t e s a cascade system (99). The consequences of th i s cascade system on heptocyte metabolism are dramatic; l i p i d biosynthetic pathways which are known to be affected by cAMP levels are shown in figure 1.5b. The effect of cAMP elevation i s reversible in hepatocytes. When adenylate cyclase acti v a t i n g stimuli are removed, cAMP lev e l s are rapidly returned to normal by the action of cAMP-phosphodiesterase, which converts cAMP to AMP (99). The proteins phosphorylated by the cAMP activated cascade system may be dephosphorylated by specific phosphoprotein phosphatases. It appears that the phosphatases are regulated as s t r i c t l y as protein kinase A, but much less i s known about these. I t i s p o s s i b l e that the p r o t e i n phosphatases can activated by insulin. In the past few years some direct evidence has been obtained for th i s hypothesis, with the discovery of a second messenger of i n s u l i n , i s o l a t e d from s k e l e t a l muscle (106) and adipocytes (107). The in s u l i n mediator appears to be a peptide with a molecular weight of about 2000 daltons, and i s released from plasma membranes after addition of i n s u l i n . It has been shown to activate the protein phosphatase responsible for the dephosphorylation of pyruvate dehydrogenase (107). The generality of t h i s i n s u l i n effect remains to be seen, but the hypothesis that i n s u l i n operates by a general scheme exactly opposing glucagon on many levels i s attractive. Figure 1.5 a&b shows the opposing e f f e c t s of i n s u l i n and glucagon on l i p i d biosynthesis. The glucagon/insulin phosphorylation system adequately explains many of the i n vivo e f f e c t s of s t a r v a t i o n on hepatic l i p i d metabolism. But there are s t i l l important e f f e c t s that remain to be resolved. For one, the i n h i b i t i o n of ?C synthesis observed in hepatocytes with cAMP analogs i s notas pronounced during starvation in vivo (93). This could be because the increased serum and glycerol and fatty acid coming from adipocytes counteracts cAMP inhibition. This i s supported by recent evidence that suggests that cAMP analog inhibition of PC biosynthesis can be counteracted by media glycerol and fatty acid in hepatocytes (54). Thus fatty acid and glucagon act together to r channel DG away fron TG in order to maintain PC synthesis. On the basis of th i s hypothesis, one would expect PC biosynthesis to be inhibited under extreme starvation conditions, when serum fatty acid and glycerol levels drop. No studies have been done to determine this. Even less i s known about the ef f e c t these serum factors on PE synthesis. Studies by Geelen et a l . on hepatocytes indicate that PE synthesis could be enhanced with the addition of glucagon (108). However, the study was inconclusive as there was not sufficient data presented to determine i f the rate of PE synthesis was enhanced, or whether the r e s u l t s were due to isotope d i l u t i o n effects..The observations could a l s o b.e exp l a i n e d by the i n h i b i t i o n of the methyltranferase pathway to PC, re s u l t i n g in an increased accumulation F i g u r e 1 .5 The e f f e c t s o f i n s u l i n and g l u c a g o n on g l y c e r o l i p i d b i o s v n t h e s i s (A) The g l u c o g e n i c , g l y c o l y t i c , l i p o g e n i c l i v e r , ( h i g h i n s u l i n ) . (B) The g l y c o g e n o l y t i c , g l u c o n e o g e n i c , ketogenic l i v e r ( h i g h g l u c a g o n ) . 7-7 ^ICardiolipin PS I CDP-d. l^^ ceri'de »lf_I PE LjSophospWatidate* p»phospha*idafe—^—»D^l^ceri'de—^HPC| I ACLJ I Co A ' r F A T T Y ACIDK " Acijl - CBrnt -tine t Mai Co A t ^ A c CoA P G P Cardioli pin PS A-CDP-d.3l4cer.de: $Pl\ [PE C+5? GP—^^-^U^ophoi^ pphospha^ ida te —'- > DT^k ceride——^ PC FATfy ACID A c ^ l - carnit ine. Mai C o A C . t B A c C o A 38 of label in PE. Such i n h i b i t i o n of the methyltransferase pathway has been shown to occur with cAMP analogs (73) .\ The effect of glucocorticoids on g l y c e r o l i p i d synthesis has not been carefully examined. One exception to th i s i s a series of studies by Brindley and coworkers (33) . They provided convincing evidence that glucocorticoids increase the a c t i v i t y of PA phosphatase, which has the effect of increasing the rate of DG synthesis. The steady state DG levels are relatively constant however ( 3 7 ) , and the fin a l effect of glucocorticoids i s to increase the production of TG and cause fatty liver (33) . This reaction prevents the fatty acid concentrations in the liver from r i s i n g to a toxic l e v e l during long term starvation. It would be interesting to see i f glucocorticoids influence the synthesis of l i p i d synthesizing enzymes. 1.52 The Effect of Calcium on Glycerolipid Metabolism The discovery of the cAMP cascade system by Sutherland and coworkers was t r u l y a breakthrough in our understanding of hormonal action (109). And while i t i s true that glucagon, ad r e n a l i n , vasopressin, and many other hormones activate adenylate cyclase, i t would be naive to postulate that these hormones a l l affect a c e l l in exactly the same way. But i t has only been within the past 5 years or so that differences in • the response to d i f f e r e n t hormones have been detected. The major d i s c o v e r y which has served a key r o l e in understanding these d i f f e r e n t responses was the r e a l i z a t i o n that calcium can serve as a messenger .in a manner equivalent to cAMP. Thus calcium dependent kinases, calcium dependent enzymes, and calcium 39 binding proteins with r e g u l a t o r y f u n c t i o n s ( CaM and Troponin C) have been p u r i f i e d , and c a l c i u m a c t i v a t e d c a s c a d e systems have been elucidated (110). Before considering s p e c i f i c c a l c i u m dependent processes operating in v i v o ? several general p o i n t s should be made regarding the ways in which c a l c i u m i s employed i n the c e l l . F i r s t , c y t o p l a s m i c calcium l e v e l s a r e g e n e r a l l y v e r y low i n c o m p a r i s o n t o serum c a l c i u m concentration ( 10uM compared to 1mM ) (111,112). T h i s gradient i s maintained by Mg-ATPases o p e r a t i n g at the plasma membrane, the endoplasmic r e c t i c u l u m , and the i n n e r m i t o c h o n d r i a l membrane, which transport calcium out of the cytoplasmic compartment (113) -Mitochondria contain about 70% of the t o t a l c e l l u l a r calcium while the endoplasmic recticulum c o n t a i n s about 20% (114), and these pools are hormonally responsive (115). Thus there are several ways i n which to increase c y t o s o l i c c a l c i u m concentra'tions, depending on the pool or pools which are gated. Concerning the r e l a t i o n of hormone a c t i v a t i o n to calcium mediated responses, c a l c i u m can be c o n s i d e r e d a second, t h i r d , or f o r t h messenger, i n c o n t r a s t to cAMP, which has always been observed as a secondary messenger. Moreover, s i t u a t i o n s have been d e s c r i b e d (see sec 3.2) where c a l c i u m can be c o n s i d e r e d a p u r e l y i n t r a c e l l u l a r messenger, i n t h a t c y t o p l a s m i c concentrations can r i s e i n response to the o x i d a t i o n s t a t e o f the mitochondria, i r r e s p e c t i v e of the hormonal state. I t i s perhaps more useful to consider calcium as a ' c o u p l i n g f a c t o r ' to p r o v i d e a common c o n c e p t u a l framework for these various p o s s i b i l i t i e s . There are currently many model systems i n which calcium mediated responses are being a c t i v e l y studied. One of the most revealing systems 40 that has been studied concerns thrombin induced platelet aggregation (116-122), shown schematically in Fig. 1.6. One of the f i r s t noticable events following thrombin i s the ac t i v a t i o n of a phospholipase C. (116). The phospholipase C i s activated by basal levels of calcium and i s s p e c i f i c for PI (92). The mode in which i t s activation i s coupled to thrombin binding i s a matter of controversy, but one intriguing possibility i s that the change in the cells shape, caused presumably by the cytoskeleton in response to thrombin binding at the plama membrane, makes the PI in the plasma membrane more available to the cytosolic phospholipase C (92). In any case, once phospholipase C i s 'activated', DG is rapidly formed in the plasma membrane which can activate protein kinase C (117). The mechanism of protein kinase C i s interesting in i t s e l f : protein kinase C requires acidic phospholipid and calcium for a c t i v i t y ; however DG reduces the Ka for calcium to basal calcium levels (118). Thus protein kinase G can be activated by PI hydrolysis or increased calcium, given that acidic phospholipids are available. The DG formed i s quickly acted on by diglyceride kinase in  vivo to form PA (119). PA has ionophore properties and physiological levels of PA can cause calcium inf l u x (120). Thus i t i s plausible that the production of PA i s responsible for the calcium influx seen in v i v o . The PA so p r o d u c e d i s a c t e d on by d i g l y c e r i d e cytidylyltransferase and i n o s i t o l phosphotransferase to complete what is known as the PI cycle. Thus the net effect of these reactions is to increase the turnover of PI, with the consequent increases in steady state DG and PA concentrations. Once cytoplasmic calcium l e v e l s increase, phospholipase A^ a c t i v i t i e s are stimulated and cause the release of arachadonate from PE, PC, and PS, and subsequently increase 41 Figure 1.6 A model for thrombin induced pl a t e l e t aggregation See text for discussion. PLC = phospholipase C, CDG = CDP-DG, Ca = calcium, PKC = protein kinase C pro = protein, pro-P = phosphoprotein, PLA = p h o s p h o l i p a s e A , PL = p h o s p h o l i p i d , LPL = lyso-phospholipid , Ar = arach? donate , PG = prostaglandin. ( denotes enzyme activation, ( •) denotes cataylsis, ( •) denotes an enzymatic reaction, and ( » denotes enzyme inhibition. 42 the production of prostaglandins (92). Protein kinase C phosphorylates membrane proteins which are- closely associated with secretion of platelet granules (121). Lastly, the prostaglandins formed activate guanylate cyclase, and the product, cGMP, feedback i n h i b i t s phospholipase C (122). Thus, in this system, calcium and phospholipid are important couplers in the stimulus-response sequence. The generality of this cascade system has not been firmly established but stimulation of PI turnover by different effectors has been noted in many tissues. Although adrenergic or vasopressin activation stimulates the PI cycle in hepatocytes, the situation is different in that it does not involve calcium influx from the serum (123). Rather, it seems that intracellular calcium pools are gated to provide transient increases in cytoplasmic" calcium. Thus, adrenergic activation causes calcium release from the mitochondria, vasopressin causes release from the mitochondria and endoplasmic recticulum, and glucagon does not significantly change cytoplasmic calcium at a l l (124). As was discussed above, a l l of these hormones activate adenylate cyclase by similar mechanisms. However, i t seems nessecary to postulate separate messengers in addition to cAM? for adrenalin and vassopressin, to produce the different effects on calcium gating. Discovery of such messengers would be an important breakthrough in understanding hormone action. One possible way in which adrenalin could cause calcium release could be through the OTP subunit. Adrenalin activation of adenylate cyclase is known to be associated with GTP hydrolysis (102). As was discussed in section 1.2, mitochondrial diglyceride cytidylyltranferase is stimulated by GTP. If 43 the adrenalin induced hydrolysis is sufficiently active to decrease mitochondrial GTP levels, i t could be that this enzyme is inhibited. If this were the case, then PA levels would increase in the inner mitochondrial membrane. This could allow calcium efflux from the mitochondria. It would be very interesting to do experiments which could test this hypothesis. Regardless of how the a d r e n a l i n message reaches the mitochondria, it is fast; maximal calcium release occurs within 5 minutes after adrenalin addition (124). Concommitantly with calcium release from the mitochondria, phospholipase A^ activities are activated on mitochondrial membranes (90). It is not clear what is causal at this step, and there is much controversy regarding the sequence of events. Changes in the oxidation state of the mitochondria alone can cause lipase activation and calcium release (125), irrespective of the hormonal state. As a consequence of the mitochondrial response, cytosolic calcium levels rise transiently, where the calcium can bind to CaM and various enzymes, most notably phosphorylase a (124). However, cytosolic calcium levels decrease to basal levels within 15 minutes, due to the rapid uptake of the calcium by the endoplasmic recticulum. As wa3 mentioned in section 1.3. PS synthase is activated by calcium, and the calcium-PS can then activate protein kinase C activity at the endoplasmic recticulum. It would also be interesting to see i f any of the lipid synthesizing enzymes can be phosphorylated by protein kinase C, particularly phosphocholine cytidylyltransferase. There is also a mystery regarding the calcium; one would intuitively expect the calcium to make it back to the mitochondria somehow, yet no mechanism 44 has been proposed. A p o s s i b l e mechanism f o r the return of calcium to the mitochondria i s presented i n the r e s u l t s section. 1.6. Experimental Approach and Rationale Clea r l y , many important q u e s t i o n s remain to be answered before the r e g u l a t i o n o f h e p a t i c l i p i d metabolism can be understood as a coordinated system. The mammalian l i v e r i s an i d e a l t i s s u e to study l i p i d metabolism f o r s e v e r a l r e a s o n s . I t i s n e a r l y homogenous i n composition, being composed o f over 80$ h e p a t o c y t e s . Tissue culture techniques have been developed that a l l o w c u l t u r e o f v i r t u a l l y 100$ hepatocytes. Thus one can e a s i l y i n t e r p r e t experiments i n terms of a s i n g l e c e l l type. In a d d i t i o n , h epatocytes have many a d d i t i o n a l functions for l i p i d s that are not found i n most other tissues in that they s e c r e t e b i l e and l i p o p r o t e i n s , and are a c t i v e i n f a t t y a c i d s y n t h e s i s . Hepatocytes a r e a l s o v e r y r e s p o n s i v e to hormones and d i f f e r e n t n u t r i t i o n a l s t a t e s and these p h y s i o l o g i c a l responses give excellent foundations for biochemical s t u d i e s on regulatory phenomena. The response o f l i v e r l i p i d metabolism to p h y s i o l o g i c a l s t i m u l i i s g e n e r a l l y c o o r d i n a t e d with the responses of other t i s s u e s such as adipose, heart, and kidney, and so l e a r n i n g about the regulation of the l i v e r w i l l undoubtedly shed l i g h t on the r e g u l a t i o n of other tissues as w e l l . L a s t l y , the r e s u l t s o b t a i n e d i n such studies are u s e f u l f o r f a r more than p u r e l y a c ademic purposes. With s t r e s s , starvation, and environmental t o x i n s i n c r e a s i n g l y a f f l i c t i n g the human populace, understanding how t h e l i v e r d e a l s w i t h these and other diseases w i l l c e r t a i n l y be o f use i n the medical community i n t h e i r search for more e f f e c t i v e c l i n i c a l treatments. Of p a r t i c u l a r i n t e r e s t i n t h i s t h e s i s was the discrepancy between the response of hepatocyte phosphocholine c y t i d y l l t r a n s f e r a s e and the c y t o s o l i c enzyme to cAMP ana l o g s . The o b s e r v a t i o n that the rate of PC biosynthesis i n hepatocytes was i n h i b i t e d by cAMP analogs prompted further studies with glucagon and c a l c i u m on t h i s pathway. Also, i n  v i t r o studies on phosphocholine c y t i d y l y l t r a n s f e r a s e were carried out in an attempt to demonstrate CaM dependent i n h i b i t i o n . 1.61 The E f f e c t o f Glucagon on PC Biosynthesis Two approaches were employed to study the e f f e c t of glucagon on PC b i o s y n t h e s i s . B o t h i n v o l v e d t h e u s e o f c u l t u r e d r a t hepatocytes. The f i r s t approach used was a s e r i e s of pulse and pulse chase s t u d i e s with r a d i o l a b e l e d c h o l i n e , g l y c e r o l and palra i t a t e r a d i o l a b e l s . These are the primary precursors for PC biosynthesis. The ef f e c t of glucagon on the i n c o r p o r a t i o n p a t t e r n of these l a b e l s as a function of time and c o n c e n t r a t i o n was s t u d i e d . The second approach was a study on the e f f e c t o f g l u c a g o n on the c h o l i n e k i n a s e , phosphocholine c y t i d y l y l t r a n s f e r a s e , and cholinephosphotransferase a c t i v i t i e s at glucagon c o n c e n t r a t i o n s - shown to have an e f f e c t on the la b e l i n g pattern. 1.62 Studies on the E f f e c t o f Calcium on Phospholipid  Metabolism The e f f e c t o f c a l c i u m on s e l e c t p h o s p h o l i p i d pathways was examined in hepatocytes using radiolabel techniques similar to above. In order to vary the cytosolic calcium levels, i t was nessecary to use the ionophore A23187 (IPA). Some hormones also raise cytosolic calcium levels transiently, but they also elevate cAMP levels, and this would make the data very d i f f i c u l t to interpret. IPA i s specific for calcium and is highly soluble in biomembranes. The major drawback in using IPA is that i t releases calcium from the mitochondria and thus decouples oxidative phosphorylation. This can cause decreases in cellular ATP levels (126), with the po s s i b i l i t y that many energy dependent pathways are inhibited. As a positive control, a serine label was used to see i f the base exchange reaction was enhanced. These conditions were then used to study the effect of calcium and IPA on PC synthesis. The limitations inherent in this approach make a l l data obtained in this way inconclusive in regard to the in vivo s i t u a t i o n but provides a good foundation for future studies nonetheless. f 1.63 In Vitro Studies on CTP:Phosphocholine  Cytidylyltransferase In order to examine the p o s s i b i l i t y that CaM or protein kinase M could effect CTP:phosphocholine cytidyltransferase activity, studies were done using the standard CTP: phosphocholine cytidylyltransferse assay described below. An attempt was made to purify liver CaM, and at a l l steps the e f f e c t of the p r e p a r a t i o n on phospocholine cytidylyltransferase could be checked. This type of study provides a good counterpart to the hepatocyte studies. MATERIALS AND METHODS 2.1. Chemicals and Isotopes Chemicals and isotopes were purchased from the following companies. Sigma Chemical Company, P.O. Box 14508, St. Louis, Missouri, 63178 U.S.A. Disodium adenosine triphosphate, disodiura cytidine triphosphate, phosphorylcholine chloride, betaine hydrochloride, insulin, glucagon, Hepes,Trisma base, collagenase type 1A, phenol red, and dithiothreitol, Dowex 1. Grand Island Biological Company, 4534 Manilla Rd. S.E., Calgary Alberta. Dulbeccos MEM formula #79-5141 (MEM-), Hanks salts without bicarbonate, Hanks s a l t s without calcium, magnesium or bicarbonate, Earles Salts without calcium, magnesium, or bicarbonate, Vitamin suppliraent 100x, essential amino acid concentrate, 100x. Amersham/Searle, 505 Iroquois Rd., Oakville, Ontario, L6H 2R3 3 3 [Me.- H ] c h o l i n e (15Ci/mM), [ 1 ( 3 ) - H] g l y c e r o l 3 ' 3 (2-5Ci/mM), [3- H] serine (19Ci/mM), [1- H] ethanolamine ( 2 5 C i / m m ) , [ 1 - ' H C ] p a l m i t i c a c i d ( 5 5 m C i / m M ) , 14 cytidine-5'-diphospho [Me- c] c h o l i n e (50raCi/mM), Aqueous Counting Scintillant (ACS). Serdary Research Laboratories, Cytidine diphosphocholine, p h o s p h a t i d y l c h o l i n e , l y s o p h o s p h a t i d y l c h o l i n e , phosphatidylethanolamine, l y s o p h o s p h a t i d y l e t h a n o l a m i n e , phosphatidylserine, di-oleoyl glycerol, sphingomyelin. Bio-Rad Laboratories, 2580 Wharton Glen Ave., Mississauga, Ontario, L4X 2A9 Protein assay dye reagent concentrate. British Drug House Chemicals, 15 West 6th Ave., Vancouver, B.C., V5Y 1K2. Choline chloride. J.T. Baker Chemical Company c/o Canadian Laboratory Supplies, 237-7080 River Rd. , Richmond, B.C. V6T 1B7. Tween 20 Brinkman Instruments, 50 Galaxy Blvd., Rexdale, Ontario, M9W 4Y5. S i l i c a Gel G-25 thin layer chromatography plates. Merckr Sharpe, and Dohme, Montreal, Canada Pla s t i c Backed S i l i c a Gel G-60 thin layer chromatography plates. Calbiochem-Behring Corporation P.O. Box 12087 San Diego, California 92112. U.S.A. Ionophore A23187, Calmodulin. Flow Laboratories, 1625 Sismet Rd. , Unit 10, Mississauga, Ontario, L4V 1V6. Fetal calf serum, Vitrogen 100. Pharmacia 2044 St. Regis Blvd., Dorval, Quebec, H8P 3C9. Sepharose 4B. 2.2. The Isolation and Culture of Rat Hepatocytes Hepatocytes were released from rat livers by collagenase perfusion, essentially by the method of Berry et a l . (127). The perfusion was carried out i n s i t u , as adapted from Davis e_t a l . (128) by Pritchard and Vance (45). The cells were purified by differential centrifugation, and cultured on collagen coated petri plates in a medium supplemented with 20$ f e t a l c a l f serum, 10ug/ral insulin, and 0.4mM ornithine, but without arginine. This media selects for hepatocytes as t h e i r urea cycle i s active and synthesizes arginine from ornithine. 2.21 Iso l a t i o n of Liver Cells A 100-125g female Wistar rat was anesthetized with Nembutal. The rat was placed under a heat lamp in a laminar flow hood and the abdomen was swabbed with ethanol. A midline incision was made through the skin from the thorax to the bladder, and the skin was torn back. The abdomen wall was cut back and the intestines were moved to the right hand side, exposing the portal vein and the inferior-vena cave proximal to the renal vein. A loose ligature was placed around the vena cava anterior to the renal vein, and two loose liga t u r e s were placed around the portal vein. The portal vein was clamped o f f , and a cut was made in i t large enough to accept a cannula needle. A cannula was inserted into the portal vein. The i n i t i a l perfusion media was Hanks sa l t s without calcium or magnesium and contained 4.5g/l glucose, 10mg/l insulin,0.5mM EGTA, and 25mM Hepes. It was kept at 40°C and was pregassed with 0^. The vena cava was cut below the renal vein and the ligatures were tightened around the cannula. The perfusion rate was adjusted to 15 ml/min for 15 seconds to flush the blood out of the l i v e r . The perfusion rate was maintained at 7.5ml/min for the rest of the p e r f u s i o n . The thorax and diaphram were removed and a loose ligature was placed around the vena cava just posterior to the atria. The a t r i a were cut with scissors and the ligatures around the vena cava were tightened. One or two small cuts were made in the t i p of each l i v e r lobe, and EGTA solution was run through the l i v e r for another 4 minutes. At that time the perfusion was changed to collagenase solution, which was Hanks salts containing 4.5 g/1 glucose, 10mg/ml i n s u l i n , 25mM Hepes, and 1g/l collagenase type 1A. The collagenase solution was pregassed with 0 and kept at 40°c. The l i v e r was perfused for 10 minutes more, at which time the l i v e r was e s s e n t i a l l y disrupted. After the perfusion was completed, the l i v e r was carefully removed and transferred to 10mls of fresh collagenase solution in a 60mm pet r i plate. The l i v e r was diced by cutting i t twelve times with scissors, and then i t was carefully poured into a 50ml centrifuge tube. The centrifuge tube was agitated at 37°C for five minutes to completely disrupt the tissue. The c e l l s were then f i l t e r e d through a coarse mesh s t e r i l e nylon f i l t e r into about MOmls of ice cold culture media and more media was added to make a suspension of 100mls. The c u l t u r e media was MEM containing 20% f e t a l c a l f serum and IOmgs/1 i n s u l i n . The suspension was transferred to two 50ml centrifuge tubes and spun at 90xg for 1 minute. The supernatant was discarded and the p e l l e t washed two times with c u l t u r e media. The c e l l s were suspended in more culture media and f i l t e r e d through a sterile 75uM mesh nylon f i l t e r and the volume was made up to lOOrals. The cells were counted in a hemocytometer and the volume was adjusted to give a c e l l d e n s i t y of 1x10 6 c e l l s / m l . The y i e l d was g generally about 4x10 c e l l s / 125g rat. 2.22 The Culture of Hepatocytes The hepatocytes were plated out by pipetting 3mls of the suspension onto each 60mm p e t r i d i s h . The pe t r i plates had previously been coated with c o l l a g e n , as follows: A s t e r i l e solution of collagen was made by adding 20rals of Vitrogen 100 ( a commercial preparation of beef akin collagen, 3mgs/ml) to I60mls of 0.01N HCl at 4°C. Two mis of t h i s solution was pipetted into each 60mm Lux Contur p e t r i plate under s t e r i l e conditions. The lids were lef t partially off for 12 hrs under a filtered air flow and UV light to allow the acid to evaporate. The plates were used the same day or stored at 4°c until use. The v i a b i l i t y of the c e l l s was accessed by their a b i l i t y to exclude 0.4$ trypan blue and was generally about 90-95$. The cells were l e f t undisturbed in a 37$ incubator under an atmosphere of air:C0^ (95:5) for 24 hours p r i o r to use. After 24 hours the media was changed to serum free MEM~ with 100nM insulin (unless otherwise mentioned) and preincubated f o r 3 hours more to diminish the hormonal e f f e c t s of the f e t a l c a l f serum. A l l experiments were started directly after this preincubation. 2.3 Pulse-Chase Experiments 2.31 Choline Uptake Studies In preliminary s t u d i e s , c o n t r o l c e l l s were incubated 3 (pulsed) for one hour with MEM c o n t a i n i n g 28uM fMe- H] choline (119mCi/mmol), 100nM i n s u l i n , and with or without CaCl^, as indicated in the f i g u r e s and tables. Other c e l l s were treated with a similar media which also contained 1uM IPA. At the end of one hour, the c e l l s were harvested as described in section 2.36. In other studies, c e l l s were incubated in MEM c o n t a i n i n g 1 OOnM i n s u l i n , 1 - 1 OOuH [M.e- H] c h o l i n e (119mCi/mraol), and with or without 200mg/l CaCl^ and 1uM IPA as indicated in the figures and tables. At the end of one hour, c e l l s were rinsed once in MEM" and harvested as described in section 2.36. 2.32 Detection of a Change i n the Rate of de novo PC  Biosynthesis 3 C e l l s were pulsed f o r one h a l f hour with 28uM [Me- H ] labeled choline (178raCi/mmol) in MEM~ containing 100nM insulin. After one half hour the media was aspirated off and the c e l l were rinsed once in MEM containing 100nM i n s u l i n . Some c e l l s were harvested at the end of the pulse period in each experiment. Labeled choline was e f f i c e n t l y taken up and metabolized as previously observed (45). The r e s t of the c e l l s were then incubated (chased) in unlabeled MEM" plus 100nM i n s u l i n , and with or without glucagon or IPA as indicated in the figures and tables. C e l l s were harvested at the times indicated in the figures and tables, as described in section 2.36. 2.33 Detection of a Change in the Rate of PC Synthesis by  the Methvlation Pathway 3 C e l l s were pulsed f o r 90 minutes with 15uCi <(1)- H] labeled ethanolamine ( 10Ci/mmol) i n MEM" containing 100nM i n s u l i n . The c e l l s were then rinsed once in MEM". Some c e l l s were harvested at the end of the pu l s e . At that time the majority of the label appeared in PE, with only about 15$ in PC and only a trace in other intermediates. The rest of the cells were then chased with MEM~ with 100nM i n s u l i n , and with or without 1uM IPA as indicated in the figures and tables. Cells were harvested at one and three hours a f t e r the pulse as described in section 2.36. During the chase period, almost a l l of the label in PE was transfered to PC with a half time of about 1.5 hours. This is consistent with previous experiments (73). The PE pool was identically labeled in a l l experiments, and so i t was assumed that any change in the rate of transfer of label from PE to PC induced by IPA during the chase period would result from a change in the rate of PC synthesis by PE N-methylation. 3 2.34 I n c o £ £ o r i t i o n _ o f _ L l £ l - j H ] S e r i n e i n t o Hepatic Phospholipids 3 Cells were pulsed with 15uCi [(2)- H] serine per dish in MEM without nonessential amino acids ( to enhance serine uptake) with 100nM i n s u l i n . For pulse studies, c e l l s were harvested at the times indicated in the tables and figures (section 2.36). Cells efficiently incorporated serine la b e l , but the majority of the l a b e l was found i n p r o t e i n r a t h e r than l i p i d . For pulse-chase studies, the labeled media was aspirated o f f the cells after one hour and the c e l l s were rinsed once with MEM". The c e l l s were then incubated in MEM" with 100nM i n s u l i n and with or without 1uM IPA. C e l l s were harvested as described in section 2.36 at the times indicated in the tables and figures. S u f f i c i e n t label was incorporated into l i p i d to observe the transfer of label from serine to PS through to. PE and PC during the chase period. A minor f r a c t i o n of the label was recovered in SM, but i t was not sufficient to allow accurate quantitation. 2.35 Studi e s on the I n c o r p o r a t i o n o f Other L i p i d  Precursors Experiments were also done by pulsing each dish with 10uCi 3 . [ ( D - H] g l y c e r o l i n MEM with 100nM i n s u l i n and with or without 100nM glucagon. C e l l s were harvested after 20, 40 and 60 minutes as described in section 2.36. Label was incorporated linearly for at least one hour into TG, PC, and PE, while the DG and other intermediates approached steady state levels within 30 minutes. 14 In a similar manner, c e l l s were pulsed with 200uM [(1)- c] palmitate (7.5mCi/mmol) per dish in MEM" with 100nM insulin, and with or without 100nM glucagon. C e l l s were harvested after 20, 40 and 60 minutes as de s c r i b e d i n s e c t i o n 2.36. The c e l l s incorporated labeled palmitate l i n e a r l y into a l l l i p i d s for at least one hour. 2.36 Harvesting of C e l l s for L i p i d Extraction In a l l of the pulse chase studies described above, cells were harvested in a manner simil a r to that described previously (45). At the time of harvesting, the media was removed, and the cells were washed with 4mls ice cold PBS (two times for pulse plates, one time for chase plates) . The c e l l s were then scraped off into 3.6mls of ice cold methanoltH^O (1:0.8,by volume) with a rubber policeman, and the suspension was pipetted into 4mls of ice cold chloroform. This is the equivalant of a Folch extraction (129). 2.37 Thin Layer Chromatography Analysis of Phospholipids Aliquots of the upper phases (0.1-1.0ml) were put directly in s c i n t i l l a t i o n v i a l s f o r counting. For c h o l i n e labeling experiments another aliquot was. saved for separation of upper phase compounds. The lower phases were washed two times with chloroform:methanol:saline (3:48 :47, by volume) to remove any contaminating non l i p i d r a d i o l a b e l . One ml aliquots of lower phase were dried down in s c i n t i l l a t i o n v i a l s for counting of label. Another 1 ml aliquot was dried dowm with 25ul of total rat l i v e r p h o s p h o l i p i d ( 20mgs/ml, prepared as previously described (130)). and taken back up in 100ul chloroform:methanol (9:1,by volume) and applied to a TLC plate. When labeled choline was used, an aliquot of the media was kept for quantitation of labeled betaine. Lipid and upper phase compounds were separated by TLC using the following systems: System #1; Methanol:0.6$ NaCl:NH 3 (10:10:1,by volume) (131). This system was to separate choline, phosphocholine, and betaine on S i l i c a Gel G-60 TLC plates. 100uL of upper phase was spotted in each lane of a G-60 s i l i c a plate and run with carrier compounds (0.6mgs c h o l i n e ; 1 .5mgs phosphocholine:0.6mgs betaine per lane) . The bands were c l e a r l y r e s o l v e d as v i s u a l i z e d by I vapors, and were scraped i n t o s c i n t i l l a t i o n v i a l s for counting. This system was also used f o r the s e p a r a t i o n of CDP-choline from phosphocholine required for CT assays (see below). System #2; CHCl^:Methanol:Acetone:H^O, (50:30 : 8 : 3 , by volume) (132). T h i s system was used with S i l i c a Gel G-25 TLC plates to separate SM, PC, PS, DPE, and MPE/PE. MPE and PE were not resolved but were scraped t o g e t h e r as one band. I t has been shown (73) that MPE has only 1$ of the l a b e l found i n PE and i t was f e l t that t h i s was not s i g n i f i c a n t f o r these studies. This system was found s u f f i c i e n t to separate a l l compounds of interest when using ethanolamine l a b e l . However, PS ran too close to PC to use for serine l a b e l i n g experiments, in which case i t was used in conjunction with system #3 described below. System #3; THF:Acetone :Methanol :H20 (50:20:40:8, by volume) (133). This system was used two improve the separation of PS from PC when using a s e r i n e l a b e l . The p l a t e was f i r s t run 15cm in system #3, and then the p l a t e was dried and run 15cm in system #2. Thi s gave a very c l e a r r e s o l u t i o n o f SM from LPC, PC, PS, DPE, and PE, as v i s u a l i z e d under I vapors. System #4; Diethyl Ether: Hexane: Acetic Acid ( 40:60:1,by volume) (134) This system was used i n conjunction with System #2 to separate the neutral l i p i d s i n p a l m i t a t e or g l y c e r o l l a b e l i n g experiments. The p l a t e was run 9cm i n System #2 to separate PE, from PC, l e a v i n g the n e u t r a l l i p i d s at the s o l v e n t f r o n t . The p l a t e was d r i e d and run f o r 20cm i n System #4 to r e s o l v e the neutral lipids into fatty acid, DG, and TG fractions. 2.38 S c i n t i l l a t i o n Counting A l l samples were counted in 1ml of water with 9mls of ACS. When counting scrapings from TLC plates, the samples were lef t in a shaker for two days to ensure that the counts would completely elute into the cocktail. A l l samples were counted for 1 minute on two channels, one with a ne a r l y open window, and the other covering only the f i r s t one t h i r d of the beta spectrum. The channels ratio was then calculated. Standards containing a known amount of the isotope of interest were counted simultaneously, and their dpm/cpm r a t i o ( the counting e f f i c i e n c y ) as well as their channels ratio was calculated. A quench curve was generated and the dpms in each sample was then calculated. 2.4 Enzyme A c t i v i t y Studies 2.41 Incubation of Hepatocytes 24 hour hepatocytes were prepared as usual,and were preincubated for 3 hours in serum free MEM with no ins u l i n . A 3 pulse-chase study with [Me- H] choline was carried out prior to t h i s to determine i f there was any difference between the glucagon response with or without i n s u l i n in the preincubation media. No s i g n i f i c a n t d i f f e r e n c e was observed. After the preincubation, c e l l s were incubated for one hour with MEM with 100nm i n s u l i n and with or without 100nm glucagon. Hepatocytes o were maintained at 37 C throughout the incubation. 2.42 Harvesting of Hepatocytes and Cell Fractionation At the end of the one hour incubation in the presence or absence of glucagon, 5 sets of t r i p l i c a t e dishs of hepatocytes were harvested and f r a c t i o n a t e d , by a m o d i f i c a t i o n of a previously described technique (12). The medium was aspirated and the c e l l s were r i n s e d once with i c e cold PBS, and then scraped off into 2.5mls of homogenizing buffer ice cold (0.145M NaCl; 10mM Tris-HCl, pH 7.4; 7mM EDTA; 10mM NaF;) with a rubber policeman. The combined suspensions from each t r i p l i c a t e were homogenized with 20 cycles of a tight f i t t i n g glass dounce, and the homogenate was transferred to a polycarbonate centrifuge tube and spun for 15 minutes at 14 ,000 rpm in a Ti75 rotor to remove nuclei, unbroken c e l l s , and mitochondria. The supernatant was spun for 60 minutes at 50,000 rpm to separate the microsomes from the cytosol. The microsomal p e l l e t was resuspended in a buffer containing .25M sucrose; 10mM Tris-HCl, pH 7.4; 1mM EDTA; and 10mM NaF. The entire process was carried out at 4^c. 2.43 Enzyme Assays and Protein Estimations Enzyme assays were performed on the microsomal and cytosolic fraction. The assays r e l y on previously established procedures which are summarized below. a) Choline Kinase (135): Assays for choline kinase were carried out on the hepatocyte c y t o s o l s . The reaction mix contained (in 100ul) 0.1M Tris-HCl,pH 8.5, 0.010MMgCl2, 0.010M ATP, and 1 .OmM [Me- 3H] l a b e l e d c h o l i n e (20mCi/mM). Assays were initiated by the addition approximately 30-50ug of protein to the other components. The mixture was incubated for 20 minutes at 37°C. The reaction was terminated by boiling for two minutes. 50ul of th i s solution was spotted onto a S i l i c a G-60 plate with 10uL of 150 mM phosphocholine carrier and run for 10cm in s o l v e n t system #1 d e s c r i b e d i n s e c t i o n 2.37. The phosphocholine band was visualized on a light box and the band was counted for r a d i o a c t i v i t y . C o n t r o l studies were carried out previously to assure that a l l assays were kineticly linear. b) Phosphocholine c y t i d y l y l t r a n s f e r a s e (53): Assays for cyti d y l y l t r a n s f e r a s e were performed on rat l i v e r cytosol or microsomes. The reaction mixture contained (in 100ul), 1.5mM [Me- H] l a b e l e d phosphocholine (40mCi/mM), 2mM CTP, 8mM MgCl^, and 0.08M Tris-HCl, pH 6.5. The reaction was started by the addition of c o c k t a i l to 40-150 ug of protein, and i t was allowed to react for 15 minutes at 37°C. In some cases the assay was c a r r i e d out i n the presence of t o t a l r at l i v e r phospholipid, which was dried down under nitrogen in the tube, and then resuspended in the enzyme preparation by vortexing. The r e a c t i o n was terminated by b o i l i n g f o r two minutes, and centrifuged for 10 minutes at 7000 rpm to remove denatured protein l i p i d aggregates. 50ul of the supernatant was spotted on a plastic backed G-60 S i l i c a TLC plate with 10ul of CDP-choline 1 carrier (50mgs/ml) and run for 10cm in solvent system #1 . The CDP-choline band was vi s u a l i z e d on a l i g h t boxand then cut out into a sc i n t i l l a t i o n v i a l for counting of radioactivity. Previous control studies were carried out to assure that a l l assays were kineticly linear. c) Choline phosphotransferase (136) : This enzyme was assayed in hepatocyte microsomes prepared as des c r i b e d above. The reaction mix contained ( i n 250ul), 50mM Tris-HCl, pH 8.0, 0.20M MgC12, 0.50mM DTT, 0.40mg/ml Tween 20, and 0.50raM [Me- 1 l*c] CDP-choline (MuCi/uM) . The reaction was started by the addition of c o c k t a i l to approximately 150-200ug of protein, and the reaction mixture was maintained at 37°C for 20 minutes. The reaction was terminated by the addition of 1.6rals CHC1 :MeOH 3 (1:1,by volume). 0.8mls of chloroform and 0.35mls H^ was added subsequently to give the equivalant of a Folch (129) extraction. 20ul of CDP-choline carrier (20mg/ml) and 20ul of total rat liver phospholipid (20mg/ml) were then added and the assay tube was spun at 7,000 rpm for 10 minutes. The upper phase was discarded and the lower phase was washed twice with t h e o r e t i c a l upper phase. An aliquot of the lower phase was dried down and counted for radioactivity. Previous control studies were carried out to assure that a l l assays were kineticly linear. d) Protein Determinations (137): Protein concentrations were estimated by a modification of the Bio-Rad protein assay system, as follows. Twenty mis of freshly f i l t e r e d Bio-Rad Protein Assay Dye Reagent Concentrate was d i l u t e to 85mls with H^O. Samples containing 10 to 125ud protein were diluted to a fina l volume of 0.5ml. To the protein sample 2.5 mis of diluted dye reagent was added and vortexed gently. A f t e r s i t t i n g for 15 minutes the absorbance was read at 595nm. The solutions were stable for at least one hour. The re s u l t s were compared to a standard curve generated with IgG protein which was l i n e a r from 0 to 125ug protein. 2.5 Studies on Phosphocholine Cytidylyltransferase in vitro 2.51 Preparation of Calcium-Free Labeled Phosphocholine 3 [Me- H] p h o s p h o c h o l i n e was p r e p a r e d as p r e v i o u s l y described (138), and unlabeled phosphocholine was added to give a specific activity of 20uCi/uM. 0.72raCi of t h i s preparation in a to t a l of 9mls was applied to a Dowex 1 (H form) ion .exchange column of bed volume 1ml and the calcium was washed through with 55 mis of d i s t i l l e d H^ O. The phosphocholine was eluted with 32mls of saturated ammonium carbonate. 3-2 ml fractions were collected and assayed for t r i t i u m . Calcium was determined by atomic a b s o r p t i o n ( s ee f i g u r e 2 .1). The c a l c i u m - f r e e phosphcholine f r a c t i o n s were pooled and concentrated by lyophilyzation. D i s t i l l e d water was added back to give a 7.5mM solution of phosphocholine. The y i e l d was 48$. The remainder of the radiolabel eluted with the calcium and was not characterized further. Atomic absorption for calcium was carried out on a l l components of the phosphocholine cytidylyltransferase assay and the total calcium in the assay was 17 micromolar. Q2. a. \ Figure 2.1 Ion exchange chromotographv of calcium phosphocholine c h l o r i d e . ( ) denotes calcium and ( ) denotes phosphocholine. See text f or d e t a i l s . 63 64 2.52 P a r t i a l P u r i f i c a t i o n of CaM CaM was partially purified from rat liver cytoplasm using the protocol of Cheung et a l . t for the is o l a t i o n of CaM (105). 40 grams of rat l i v e r were homogenized in 120 mis of cold Buffer A (20mM Tris-HCl, pH 7.5,with 20mM MgSO^). The homogenate was spun for 30 minutes at 12,000xg. It was then boiled for 4.5 minutes, and spun again to remove denatured p r o t e i n . The supernatant was dialyzed 3 times against 4 l i t e r s of the Buffer A. The d i a l y s a t e was a s s a y e d f o r p h o s p h o c h o l i n e cytidylyltransferase inhibition, then applied to a 1.5 cm x 20 cm column of DEAE Sepharill which was previously equilibrated with buffer A containing 0.15M ammonium sulfate. The sample was eluted with 200mls Buffer A containing 0.15M ammonium sulfate, then a 150mls of a linear gradient from 0.15M to 0.3M ammonium sulfate in buffer A. Subsequent to thi s another 200mls 0.3M aramounium sulfate in Buffer A was added then 30mls 0.5M ammonium sulfate in buffer A. 100 fractions of 4.5mls were collected and assayed for phosphocholine cyt i d y l y l t r a n s f e r a s e inhibition, protein, and conductance. Peak 2 (see results) was applied to an Amicon column eluate concentrator and u 1 1 r a f i 1 1 r a t e d . This destroyed the inhibitory activity. RESULTS AND DISCUSSION 3.1 .The Effect of Glucagon on Phosphatidylcholine Biosynthesis 3.11 The E f f e c t of Glucagon on T^Hl Choline Incorporation  into Hepatocyte Phospholipid These experiments were designed to test the effect of glucagon on PC biosynthesis by the de novo pathway. Choline label has proved to be the best indicator of the rate of PC biosynthesis in past •3 studies (41,45,53). The in c o r p o r a t i o n pattern of [Me- H] choline into hepatocytes af t e r a one half hour pulse i s shown in Table 3.1. These results agree with previous studies (45), and show that over half of the choline label was r a p i d l y oxidized to betaine under these conditions, while most of the rest of the label was phosphorylated. Of the label that was phosphorylated, the vast majority was found in phosphocholine, while only 2% had appeared in PC. The amount of label in CDP-choline was exceedingly small, and was not quantitated in this study. This supports the accepted view that CTP: phosphocholine cytidylyltransferase i s the rate l i m i t i n g enzyme in the synthesis of PC. The effect of adding 100nM glucagon to the chase media is shown in figure 3-1. Transfer of label from phosphocholine to PC was inhibited rapidly,amounting to 50% i n h i b i t i o n (p<.001) within 15 minutes. The inhibition was sustained to some extent throughout the 2 hour chase period (about 30% at the end of one hour). The total amount of label in 66 T a b l e 3-1 The incorporation pattern of rMe- H1 choline into cultured rat hepatocytes. Cultured rat hepatocytes were incubated for one half hour in MEM containing 100nM insulin and 28uM [Me- H] Choline (178mCi/mraol) at 37 C. The cells were harvested and analyzed as described in section 2.3. Incorporation of [Me- H] choline^ (dpm x 10~ /dish) Percent Incorporation Choline Phosphocholine Phosphatidylcholine Betaine Total 6.5±.05 18.0+.1 1 .1±.1 28 .0+.2 53.6 12$ 33% 2% 52% 100$ Figure 3.1 The e f f e c t o f g l u c a g o n on t h e d i s a p p e a r a n c e o f T Me- H 1 c h o l i n e from c e l l u l a r p h o s p h o c h o l i n e and t h e accumulation into p h o s p h a t i d y l c h o l i n e . C u l t u r e d rat hepatocytes were p u l s e l a b e l e d f o r one h a l f hour w i t h 1 0 u C i [Me- H] c h o l i n e . The c e l l s were washed and f r e s h medium with ( O) or without (0 ) 100nM glucagon was added. At v a r i o u s times, the c e l l s and media were c o l l e c t e d , and t h e r a d i o a c t i v i t y was quantitated in (A) phosphocholine; and (B) phosphatidylcholine. Each p o i n t r e p r t e s e n t s the mean o f t h r e e d i s h e s , and SE i s indicated by bars. 68 phosphocholine and PC was constant throughout the chase period, which supports the hypothesis that the phosphorylation of choline effectively commits it to the synthesis of PC (45). The halflife of phosphocholine was calculated from this experiment (1.5-2.5 h), and is similar to previous studies (53). The amount of label in choline was uneffected by this treatment. Also, there was no effect on the secretion of betaine into the media (figure 3.2). These results are similar to previous studies on the effect of cAMP analogs on the rate of PC biosynthesis (53) » and are consistent with the hypothesis that glucagon acts on hepatocytes metabolism primarily by raising the intracellular cAMP levels (102). In figure 3-3» a glucagon titration curve is presented with respect to the labeling pattern of phosphocholine and PC. The data was taken after a one hour chase period with various concentrations of glucagon. Half maximal inhibition of label transfer to PC occurs at 10nM glucagon, which is close to the Km reported for hepatic glucagon receptors (4.5nM) by Rodbell et al (98). The fact that the experimental Ki is slightly higher than the receptor binding constant can be rationalized by considering that insulin concentration in the media was 100nM, and it is well documented that the insulinrglucagon ratio may be more important to the cellular state than the absolute concentration of either hormone (95). Moreover, it has been shown that the liver degrades glucagon at a substantial rate (95 ), and thus the average glucagon concentration over the hour chase could be significantly lower than the concentration at the beginning of the chase. With these factors considered, it thus seems probable that this inhibition occurs in vivo. 69 Chase Time (h) Figure 3.2 The e f f e c t of glucagon on the s e c t e t i o n of TMe -_H2 betaine into the culture media. Cultured, rat hepatocytes were pulse labeled for 30 minutes with 10uCi [Me- H ] c h o l i n e . The c e l l s were washed and fresh medium with (v) or without (•) 100nM glucagon was added. At various times, the c e l l s and media were c o l l e c t e d , and the r a d i o a c t i v i t y was quantitiated in (A) c e l l u l a r betaine; and (B) media betaine. Each point represents the mean of three dishes, and SE i s indicated by the bars. F i g u r e 3.3 The effect o f g l u c a g o n c o n c e n t r a t i o n on t h e  incorporation of fMe- Hi c h o l i n e i n t o c e l l u l a r phosphocholine  and p h o s p h a t i d y l c h o l i n e . C u l t u r e d r a t hepatocytes were pulse l a b e l e d f o r one h a l f hour w i t h 1 0 u C i [Me- H] c h o l i n e . The c e l l s were washed and fresh medium with various concentrations of glucagon was added. A f t e r one hour, the c e l l s and media were c o l l e c t e d , and the r a d i o a c t i v i t y was q u a n t i t a t e d i n (A) p h o s p h o c h o l i n e ; and (B) p h o s p h a t i d y l c h o l i n e . Each p o i n t represents the mean of three dishes, and SE i s indicated by bars. 71 It i s known that cAMP analogs decrease choline uptake ( 53). This would act to increase the specific radioactivity of the phosphocholine pool, which means that the i n h i b i t i o n of PC synthesis by glucagon is greater than the labeling data indicates. Glucagon probably inhibits the rate of PC b i o s y n t h e s i s at a step subsequent to choline p h o s p h o r y l a t i o n . T h i s c o u l d be a c h i e v e d by decreasing the a v a i l a b i l i t y of either DG or CDP-choline. It i s unlikely that DG availability i s decreased as the amount of hepatic PC has been shown to remain be r e l a t i v e l y c o n s t a n t under c o n d i t i o n s where DG concentrations have varied markedly (37). Definitive studies have not been done on hepatocytes, however. This could be tested If a sensitive assay for DG was available. It i s more probable that the inhibition of PC s y n t h e s i s i s a r e f l e c t i o n o f an i n h i b i t i o n of the cytidylyltransferase step, caused by a decrease in CTP:phosphocholine cytidylyltransferase activity, or of CTP levels. 3.12 The Ef f e c t of Glucagon on the Incorporation of L CI Palmitate into Hepatocyte G l y c e r o l i p i d s The effect of glucagon on the incorporation of palmitate into glycerolipid was studied to see i f the inhibition of the rate of PC biosynthesis could be observed with palmitate label as well. A pulse experiment was performed as a preliminary study. The Incorporation 14 pattern of [1- c] palmitate into hepatocytes i s shown in figures 3.4a-d, with and without 100nm glucagon. The t o t a l uptake, shown in 3.4a, was linear up to one hour, and was independent of glucagon under these conditions. The incorporation into the d i f f e r e n t classes of T l F i g u r e 3-4 T h e ^ e f f e c t o f g l u c a g o n on t h e i n c o r p o r a t i o n and  metabolism o f \'\- c l p a l m i t i a t e by c u l t u r e d r a t h e p a t o c y t e s . C u l t u r e d r a t hepatocytes w e r e ^ u l s e l a b e l e d f o r v a r i o u s times in c u l t u r e media with 200uM [1- C] p a l m i t a t e and without glucagon (open symbols), or with 100nM glucagon ( c l o s e d symbols). The c e l l s were harvested and the r a d i o a c t i v i t y was determined i n (A) t o t a l l i p i d ; (B) free f a t t y acid; (C) d i g l y c e r i d e (•,•) and t r i g l y c e r i d e (O, • ); and (D) phosphatidylcholine (O and phosphatidyethanolamine ( • , • ) . Each p o i n t r e p r e s e n t s the mean of three d i s h e s , and SE i s indicated by bars. Incorporation of [%!•}- C] Palmitate (dpm x 10~ / dish) lipids, is shown in figures 3.4b&c. The pattern of incorporation was also unaffected by the addition of glucagon. The recovery of palmitate label as fatty acid plateaued at 20 minutes ( figure 3.4b). This is expected as free fatty acid is an intermediate present only in trace quantities in hepatocytes (64). Thus, the specific radioactivity of the fatty acid pool rapidly increases to a maximum because the label is rapidly oxidized or esterified. Glucagon has no significant effect on fatty acid label incorporation because fatty acid is utilized as fast as it is taken up, irrespective of the hormonal state. DG is also an intermediate, although the pool size is much larger and the label does not reach a steady state concentration within the pulse period. Glucagon has no effect on the label recovered in DG. DG levels are known to decrease during fasting, however (37). It i s possible that the effect of glucagon is masked by isotope dilution effects. Thus, the amount of label in DG is the same although the DG in the glucagon treated cells has a higher specific activity. This seems likely i f it is assumed that the amount of acyl-CoA produced from endogeneous fatty acid is decreased while that produced from exogeneous fatty acid remains the same. The linear incorporation of label into phospholipids and TG (figure 3«4c&d) is expected because the pools are much larger than their precursors and hence the label would take much longer to \ equilibrate and turnover. The finding that glucagon had no effect on the incorporation of palmitate label into these lipids can be mimicked by cAMP analogs (54). This would suggest that the decrease in glycerolipid synthesis caused by glucagon was masked by isotope dilution effects. One way to rationalize this would be to postulate that the diglyceride label was not diluted as much by endogenously synthesized fatty acid in the glucagon cells, and therefore the specific radioactivity a of the diglyceride pool is higher and masked the inhibition. This interpretation seems likely as it is well known that glucagon inhibits the rate of fatty acid biosynthesis in hepatocytes (141). Again it would be useful to measure the pool size of DG to test this possibility. These findings are to be contrasted with the findings of Geelen et al (142 ), who reported an inhibition of palmitate incorporation into TG with 85nm glucagon, although the rate of phospholipid synthesis was unaffected. However, the conditions of their experiment were different than the one described here, in that the palmitate concentration was 540uM, as opposed to the concentration of 200uM used in this study. Thus it is plausible that with the higher concentration of exogenous fatty acid the label could be channelled towards B-oxidation under a glucagon load . This would cause a decrease in the radioactivity appearing in DG, and consequently in TG, ~?S, and PC. However, recent work has shown that palmitate can increase the rate of PC synthesis when it has previously been inhibited by cAMP analogs (54). Thus under the conditions of Geelens experiment it is possible that the rate of PC synthesis was maintained by the balance of two opposing effects. This interpretation is in keeping with the in vivo situation, where serum fatty acid ( concentrations increase concommitantly with glucagon levels under starvation conditions. Thus one could propose that glucagon inhibits the rate of synthesis of both PC and TG, but as exogenous fatty acid concentrations rise, PC synthesis and B-oxidation increase, the effect being a channelling of 75 fatty acid away from TG synthesis in order to maintain the phospholipid and energy requirements of the cell. •3 3-13 The Effect of Glucagon on T Hi Glycerol  Incorporation into Hepatocyte Glycerolipids A pulse experiment was performed with glycerol radiolabel to see i f a glucagon effect on PC biosynthesis could be observed with this precursor. In figures 3-5a-d, the incorporation pattern of [1(3)- H] labeled glycerol into hepatocytes i s shown,in the presence and absence of lOOnM glucagon. The total incorporation plateaued within 30 minutes (3«5a), implying that the cells have sufficient endogeneous g l y c e r o l to maintain normal c e l l functions. The incorporation into l i p i d was approximately linear throughout the hour pulse (fig. 3.5b), while the label in the upper phase peaks and diminishes, as would be expected if the total uptake plateaus. Glucagon slightly inhibits the uptake at 15 minutes, but this is of doubtful physiological significance. The relative incorporation of glycerol label into the various l i p i d components is unaffected by glucagon. The incorporation of glycerol into phospholipids proceeds linearly throughout the pulse period (figure 3.5c). After one hour, 100nM glucagon exerted a slight stimulatory effect on the incorporation PC (15$, p<.001), but no significant effect on the incorporation into PE.This result is consistent with the hypothesis that DG is preferentially channelled into PL under- a glucagon load, although the stimulation is so small that i t lends no real support to" this theory. Geelen et al .dm) T demonstrated an increase in the incorporation of F i g u r e 3.5 The effect o f g l u c a g o n on t h e i n c o r p o r a t i o n and  metabolism of T ( 3 ) - Hi g l y c e r o l by c u l t u r e d r a t h e p a t o c y t e s . Cultured rat hepatocytes were pulse labeled for various times in culture media with 200uM [ ( 3 ) - H ] g l y c e r o l and without glucagon (open symbols), or with 100nM glucagon ( c l o s e d symbols). The c e l l s were ha r v e s t e d , and the r a d i o a c t i v i t y was quantitated in (A) t o t a l c e l l u l a r m e t a b o l i t e s (B) aqueous phase m e t a b o l i t e s ( c i r c l e s ) and t o t a l l i p i d m e t a b o l i t e s ( s q u a r e s ) ; (C) p h o s p h a t i d y l c h o l i n e ( c i r c l e s ) and p h o s p h a t i d y e t h a n o l a m i n e ( s q u a r e s ) ; and (D) t r i g l y c e r i d e s ( c i r c l e s ) and d i g l y c e r i d e s (squares). Each point represents the mean of three dishes, and SE i s indicated by bars. 1 76 glycerol label into PC and PE of about 25% after one hour. This inconsistency may be explained by considering several experimental differences. The concentration of media glycerol was 0.5mM in his study, and this could be significant as there is some indication that glycerol can stimulate PE and PC synthesis (54). Also, Geelen had no insulin in his glucagon containing media, which should act to enhance a glucagon mediated effect. Lastly, Geelen used 10uM ethanolamine in his media, and this could be neccesary to see an increase in PE synthesis . It could be that the concentration of ethanolamine is rate limiting for PE biosynthesis at very low media concentrations of ethanolamine. This is supported by the observation (73) that hepatocytes incorporate pulse ethanolamine into PE very fast when the specific activity is high enough. Thus any increase expected in the incorporation into PE in this experiment could be masked by the lack of substrate. The difference between these experiments emphasizes the need to determine the effect of various substrates on phospholipid synthesis in order to properly interpret experimental observations. In figure 3.5d the incorporation pattern of [^ H] glycerol into neutral lipids is shown. It is shown that the label in DG peaks within 30 minutes. This is to be expected as the label in the upper phase precursors peaked within 20 minutes. Glucagon had no effect on the amount of label recovered in DG. As i t is likely that glucagon decreases the pool size of DG (140) , this result implies that the pool size of glycerol phosphate available to l i p i d biosynthesis is coordinately regulated with the DG pool. In other words, the glucagon induced decrease in the rate of DG synthesis from glycerol phosphate and acyl-CoA would be coordinated with a decrease in the pool size of glycerol phosphate. Thus, in glucagon treated c e l l s , the amount of label in DG would remain constant although the specific activity would increase. Measurement of the DG and g l y c e r o l phosphate pool sizes could be done to confirm t h i s postulate. Another way to test this hypothesis would be to use higher media concentrations of glycerol, to increase experimentally the gly c e r o l phosphate pool size. A glucagon effect on DG l a b e l i n c o r p o r a t i o n should then be seen, as the inhibition of gly c e r o l phosphate acyltransf erase and PA phosphatase by glucagon could then be observed (discussed in section 1.5). The observation that the incorporation of g l y c e r o l into TG was not influenced by glucagon confirms Geelen's (142) study, as well as the palmitate study presented above. Both the palmitate and glycerol label studies are consistant with the hypothesis that most TG is produced from DG arising from the PA-phosphatase pathway. The contrast between these experiments, as well as the palmitate experiments described above, emphasize the need to do future studies that vary the concentrations of l i p i d precursors in the presence and absence of glucagon. S p e c i f i c a l l y , the effect of glucagon on the incorporation of choline into phospholipid should be examined as a function of g l y c e r o l c o n c e n t r a t i o n , to see i f g l y c e r o l has a compensating effect on the i n h i b i t i o n , as fatty acid does. Also, the effect of glucagon on the de novo pathway of PE synthesis as a function of fatty acid and glycerol should examined. This would allow a comparison of the regulation of the de novo pathways of PE and PC synthesis, and this could help elucidate the mechanisms by which these pathways respond d i f f e r e n t l y to f a s t i n g . The palmitate and glycerol studies presented above should be repeated as a function of media f a t t y a c i d and g l y c e r o l c o n c e n t r a t i o n s , i n the presence of normal serum c o n c e n t r a t i o n s o f ethanolamine and c h o l i n e . The r e s u l t s from these experiments would gr e a t l y c o n t r i b u t e to our understanding of how substrate supply and hormonal c o n t r o l mechanisms coordinately adjust the biosynthesis of hepatic g l y c e r o l i p i d s to the n u t r i t i o n a l state of the organism i n v i v o . 3-14 The E f f e c t o f Glucagon on the Enzyme A c t i v i t i e s  Responsible for de novo PC biosynthesis The enzyme a c t i v i t i e s of c h o l i n e k i n a s e , CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e , and c h o l i n e phosphotransferase from control and glucagon t r e a t e d h e p a t o c y t e s were d e t e r m i n e d i n an attempt to c o r r e l a t e the r a t e changes o b s e r v e d w i t h l a b e l e d p r e c u r s o r s with changes in enzyme a c t i v i t i e s . P r e v i o u s s t u d i e s (53) with cAMP analogs suggested that the i n h i b i t i o n o f the c y t i d y l y l t r a n s f e r a s e was caused by protein phosphorylation. In the work up of the hepatocytes, kinases and phosphatases could be a c t i v e which would preclude any attempt to o b s e r v e a g l u c a g o n dependent i n h i b i t i o n . To c i r c u m v e n t t h i s p o s s i b i l i t y , harvests were c a r r i e d out i n the presence of 10mM NaF in order to prevent the a c t i o n of phosphatases, and 10raM EDTA to i n h i b i t protein kinases. This insures that any change in the amount of protein phosphorylation w i l l be maintained throughout the work up. Glucagon i n h i b i t e d CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e by 35$ (p<0.001) a f t e r one hour, i n b o t h the c y t o s o l and the microsomes (table 3.2). T h i s c o r r e l a t e s w e l l with the i n h i b i t i o n observed with 3 [Me.- H] c h o l i n e . C h o l i n e k i n a s e a c t i v i t y i s a l s o i n h i b i t e d 80 Table 3.2 The ef f e c t of glucagon on the a c t i v i t i e s of enzymes  responsible for p h o s p h a t i d y l c h o l i n e b i o s y n t h e s i s . Cultured rat hepatocytes were treated with culture media or lOOnM glucagon for 1 hour. Values are the average of 15 dishes± SD. Enzyme Control Activity with Assayed Activity 100nM Glucagon (nmol/min/mg ) (nmol/min/mg) ±SE ,+SE Choline Kinase 0.68+.04 .56+.05 Cytosolic CTP:phosphocholine Cytidylyltransferase without Phospholipid .047±.014 .031±.004 ** Cytosolic CTP:phosphocholine Cytidylyltranferase with Phospholipid .52+. 04 .54+.03 Microsomal CTP:phosphocholine Cytidylyltranfersase .57+.2 •32+.07 Cholinephosphot ran f erase .031±.003 .024+.002 * * p<0.01 ** p<0.001 marginally by 18$ (p<0.01). It i s possible that t h i s increases the amount of choline which i s oxidized, by leaving more choline to be acted on by choline oxidase. cAMP analogs were shown to have a similar inhibitory effect on the hepatic cytidylyltransferase when cel l s were harvested in this manner (53)- The addition of phospholipid to the phosphocholine cytidylyltransferase assay supresses the inhibition and this was also found with cAMP analogs. Choline phosphotransferase was slightly inhibited but t h i s i s not s t a t i s t i c a l l y significant. Thus, this data supports the generally accepted notion of glucagon as a starvation s i g n a l , which i n h i b i t s key b i o s y n t h e t i c enzymes by elevating cAMP levels. In vitro studies with rat l i v e r cytosol (54) have shown that the a c t i v i t y of CTP:phosphocholine cytidylyltransferase can be inhibited in a time and concentration dependent manner by Mg-ATP and calcium (see section 1.25). It was also shown that the activation of cyto s o l i c c y t i d y l y l t r a n s f e r a s e at 4°c can be inhibited simply by preincubation with NaF at 4°C. The i n h i b i t i o n can be supressed by addition of protein kinase i n h i b i t o r s isolated from rabbit skeletal muscle. Moreover, studies have shown that the inhibition of hepatocyte phosphocholine cytidylyltransferase observed with cAMP analogs can not be seen i f NaF i s not present during the harvest (53). A l l of these typesof inhibition can be reversed by phospholipid, and suggest that they operate bya common mechanism involvingphosphorylation of CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e . However, a complete proof that the c y t i d y l y l t r a n s f e r a s e i s p h o s p h o r y l a t e d must await purification of the enzyme so that kinase-induced phosphorylation may be demonstrated d i r e c t l y . Moreover, as was discussed in section 1.2, the i d e n t i t y o f t h e k i n a s e r e m a i n s unknown. T h i s p r e s u m p t i v e p h o s p h o r y l a t i o n o f c y t i d y l y l t r a n s f e r a s e i n h i b i t s a c t i v i t y by increasing the Km for CTP, and also i n h i b i t s l i p i d induced aggregation ( 5 4 ) . I t seems l i k e l y t h a t glucagon i n h i b i t s the enzyme through the same m e c h a n i s m , t h e r e s u l t s a r e c o n s i s t a n t w i t h the p h o s p h o r y l a t i o n / t r a n s l o c a t i o n / a c t i v a t i o n model presented i n the introduction (see s e c t i o n 1 . 2 ) . I t would be worthwhile to repeat the glucagon experiment presented here without NaF i n the h a r v e s t i n g media and see i f the i n h i b i t i o n o f t h e c y t i d y l y l t r a n s f e r a s e i s diminished, as i t was with cAMP analogs. A model for the e f f e c t of f a s t i n g on PC b i o s y n t h e s i s can now be presented. During s t a r v a t i o n , glucagon i n h i b i t s the synthesis of PC bycausing a phosphorylation of the c y t i d y l y l t r a n s f e r a s e . This has the e f f e c t of increasing the Km f o r CTP, and tr a n s l o c a t i n g some of the microsomal enzyme to the c y t o p l a s m , away from DG micro-domains. However, when the f a t t y a c i d or a c i d i c phospholipid concentrations in the c e l l r i s e , the i n h i b i t i o n o f PC s y n t h e s i s by glucagon can be counteracted. This e f f e c t i v e l y keeps the synthesis of PC to a minuraum during f a s t i n g . However, the true i n h i b i t i o n under these conditions i s only marginal as a constant l e v e l of PC i s v i t a l to c e l l function. But i f the i n t r a c e l l u l a r CTP l e v e l s drop d u r i n g f a s t i n g , PC synthesis i s decreased much more r e a d i l y . When the animal i s fed again, glucagon l e v e l s d e c r e a s e , and the p h o s p h o r y l a t i o n l e v e l o f the enzyme decreases. S i m u l t a n e o u s l y , i n t r a c e l l u l a r f a t t y a c i d concentrations d e c r e a s e , and DG l e v e l s i n c r e a s e . The C T P : p h o s p h o c h o l i n e c y t i d y l y l t r a n s f e r a s e tends to r e a s s o c i a t e with/the microsomes, but the net a c t i v i t y remains f a i r l y constant. 83 3.2. S t u d i e s on the E f f e c t of Calcium on Phospholipid Biosynthesis Using Ionophore A23187 The effect of calcium on phospholipid biosynthesis was studied in view of the wide regulatory properties of calcium, and the failure of attempts to demonstrate a cAMP dependent i n h i b i t i o n of choline cytidylyltransferase in v i t r o (54). Ionophore A23187 was chosen as a calcium adjuvant because most other methods of varying calcium in the c e l l activate adenylate cyclase. 3 3.21 Pulse-Chase S t u d i e s with [, Hi Serine As i t has been known since the 1950* s that calcium stimulates the serine synthase enzyme in v i t r o (76) i t was f e l t that using a serine label to study the ef f e c t of IPA would serve as a positive control for a l l subsequent s t u d i e s . F i r s t , i t was neccesary to determine good pulse conditions for serine label, as there was no data in the l i t e r a t u r e on the i n c o r p o r a t i o n of serine into hepatocyte phospholipids. MEM with the nonessential amino acids deleted was used for the pulse media in order to increase the uptake of serine label. 3 Figure 3.6 shows the incorporation of 15uCi of [3- H] serine into p h o s p h o l i p i d as a f u n c t i o n of time. I t can be seen that the incorporation is f a i r l y linear into a l l l i p i d classes examined. It was fel t that the one hour pulse point was a good time to begin a chase as there are sufficient counts to detect changes in flux yet there are s t i l l 2.5 times as many counts in PS as there are in PE. 0 1 2 P u l s e Time (h) 3 F i g u r e 3.6 The i n c o r p o r a t i o n o f _ l 3 - H i s e r i n e i n t o phospholipids. C u l t u r e d r a t hepatocytes were incubated f o r various l e n g t h s o f t i m e w i t h 1 5 u C i [3- H] s e r i n e i n MEM m i n u s n o n e s s e n t i a l amino a c i d s c o n t a i n i n g 100nM i n s u l i n . The c e l l s were harvested and the r a d i o a c t i v i t y was q u a n t i t a t e d i n t o t a l l i p i d (•), phosphat i d y l s e r i n e ( • ) , p h o s pha t i d y 1 e t hano lam i ne (A) f and phosphatidylcholine (•). Each point represents three dishes and the SD of each point i s indicated by bars. In the pulse chase studies, the IPA was added in MEM with normal calcium and insulin during the chase only, as i t is known that IPA enhances serine uptake (143). IPA was added for one hour, and then the cells were chased with control media. In figure 3.7a, the effect of 3 IPA on the incorporation of 15uCi [3- H] serine into hepatocyte phospholipids is shown as a function of time. It can be seen that IPA stimulated incorporation. The lipids were separated and the results are shown in figures 3-7b-d. Virtually a l l the PS label is rapidly decarboxylated to PE. This implies that the PS formed due to the calcium stimulation is rapidly transported to the mitochondria to be decarboxylated. The further incorporation of label into PC by N-methylation is fast as well. This would require the PE synthesized in the mitochondria to be rapidly transported back to the endoplasmic recticulum. It appears that not a l l of the PE made at the mitochondria is methylated to PC. It i s known that PE made by the de novo PE pathway is committed to PC biosynthesis, at least at low ethanolamine concentrations (73). These results suggest that mitochondrial PE has another fate, as the counts lost from PE do not a l l end up in PC (fig 3.7d). This could be explained i f the PE was being converted to lyso-PE by the phospholipase A^ in the mitochondria, a reaction which is also stimulated by calcium (144). This experiment suggests that the turnover of serine in phospholipid is much faster than choline and ethanolamine. A consideration of the topology of the enzymes involved shows that the PS made in the endoplasmic recticulum must be transported to the mitochondria to be decarboxylated, and then back to the endoplasmic recticulum to be methylated to PC. The fact that this 2^ a_ Figure 3-7 The e f f e c t of ionophore A23187 on the incorporation of L3_=. Hi s e r i n e i n t o p h o s p h o l i p i d s . C u l t u r e d rat, hepatocytes were incubated f o r one h a l f hour with 15uCi [ 3 - H] serine in MEM minus n o n e s s e n t i a l amino a c i d s c o n t a i n i n g 100nM i n s u l i n . Some c e l l s were h a r v e s t e d at t h i s p o i n t . The r e s t of the c e l l s were rinsed with culture media and then incubated for one hour in MEM w i t h 100nM i n s u l i n , and w i t h (diamonds) or without (squares) 1 uM ionophore IPA. Some c e l l s were harvested at t h i s time. The rest of the c e l l s were r i n s e d with media and incubated f o r 2 or 4 hours i n MEM c o n t a i n i n g 100nm i n s u l i n , and then _ harvested. The l i p i d s were e x t r a c t e d and the r a d i o a c t i v i t y was q U a n t i t a t e d i n (A) t o t a l l i p i d , p h o s p h a t i d y l s e r i n e ( B ) , phosphatidylethanolamine (C) , and p h o s p h a t i d y l c h o l i n e (C) . Each poin t r e p r e s e n t s t h r e e d i s h e s and the SD o f each po i n t i s indicated by bars. 00 process is calcium stimulated is interesting in regard to other reports in the literature. It i s known that when hepatocytes are stimulated with adrenalin, calcium is released from the mitochondria concommitantly with the activation of mitochondrial phospholipase A^  (90). The mitochondrial phospholipase A i s known to work 2 predominantly on PE (144), generating lyso-PE and fatty acid. The calcium ends up in the endoplasmic recticulum (124), where i t presumably activates PS synthase , phospholipases, and protein kinase C. The observation made here that most of the PS made in the endoplasmic recticulum goes right to the mitochondria and is turned into PE provides a way to regenerate the PE lost in the mitochondria (figure 3-8) i and is analogous to the ?I cycle operating on the plasma membrane of many cel l s . This form of intracellular regulation could have important implications in the regulatory scheme of the hepatocyte. One possible consequence of this calcium activation is to channel DG away from TG and toward ^-oxidation, through phospholipid intermediates. Furthermore, this mechanism can channel arachadonate into prostaglandin synthesis when calcium release is stimulated in the mitochondria. Also, by the activation of protein kinase C by the calcium-PS, this mechanism provides a means of coupling protein phosphorylation to hormonal response. This process (designated as the PS cycle), can conceivably be extended to other metabolic s i t u a t i o n s . It is known that mitochondria release calcium when their cytochrome system is oxidized (125). This would activate PS synthesis from PE in the endoplasmic recticulum. The ethanolamine released from the PS base exchange would rapidly be remade into PE, assuming that the concentration of Figure 3.8 The PS cycle h y p o t h e s i s . See text f o r discussion. [ 0 = plasma membrane, |m] = inner mitochondrial membrane, H] = endoplasmic recticulum, PLC= phospholipase C, PL = phospholipid, Ca = calcium, P1A = phospholipase A , PKC = p r o t e i n kinase C, pro = protein, pro-P = phosphoprotein. ( ---•) denotes a c t i v a t i o n , (—•) denotes c a t a l y s i s , (—•) denotes a reactio n . Other abbrieviations as in text. ethanolamine i s r a t e l i m i t i n g to de novo PE s y n t h e s i s . T h i s would e f f e c t i v e l y channel DG away from TG i n t o PS. The PS synthesized would t h e n be t r a n s p o r t e d t o t h e m i t o c h o n d r i a where i t would be d e c a r b o x y l a t e d to PE, and f u r t h e r a c t e d on by phospholipase to generate free f a t t y acid at the m i t o c h o n d r i a . This could then be used for ^ - o x i d a t i o n and r e d u c t i o n o f the cytochrome system. Thus the a c t i v a t i o n of the PS c y c l e would e f f e c t i v e l y t r a n s port f a t t y acid to the m i t o c h o n d r i a r e q u i r e d f o r o x i d a t i v e p h o s p h o r y l a t i o n and prostaglandin biosynthesis v i a p h o s p h o l i p i d intermediates. This system i s novel i n that a l l m i t o c h o n d r i a l f a t t y a c i d t r a n s p o r t systems reported to date u t i l i z e c a r n i t i n e f a t t y a c i d . In t h i s regard i t i s i n t e r e s t i n g that a c y l - c a r n i t i n e i n h i b i t s p r o t e i n kinase C (152); t h i s may represent another regulatory f e a t u r e of t h i s system. A d d i t i o n a l l y , i t would provide a way to r a p i d l y channel DG away from TG synthesis without i n h i b i t i n g D G - a c y l t r a n s f e r a s e , or DG s y n t h e s i s , as the cAMP pathway does. I t i s a l s o an example o f i n t r a c e l l u l a r communication between the mitochondria and the endoplasmic recticulum, which could e f f e c t i v e l y coordinate ^ - o x i d a t i o n and TG s y n t h e s i s to the oxidation state of the c e l l . I f t h i s a u t o r e g u a t i o n model operates i n vivo, i t would i n t u i t i v e l y seem t h a t c a l c i u m r e l e a s e from the mitochondria c o u l d be a d j u s t e d c o n t i n u o u s l y to a r a t e o p t i m a l f o r both the endoplasmic recticulum and the m i t o c h o n d r i a , as opposed to the rapid release of c a l c i u m induced by a d r e n a l i n . I f t h i s were the case, i t would be u s e f u l to c o n s i d e r the r e l a t i v e amount o f c a l c i u m i n the endoplasmic r e c t i c u l u m and t h e m i t o c h o n d r i a as a measure of the oxidation state. Thus the m i t o c h o n d r i a would be 'loaded' with calcium when they are e n e r g i z e d , and d e f i c i e n t i n c a l c i u m when they require more f u e l . In l i g h t of the above d i s c u s s i o n , one would i n t u i t i v e l y expect the calcium to r e t u r n to the m i t o c h o n d r i a to complete the cycle, or when s u f f i c i e n t PS has reached the m i t o c h o n d r i a . In t h i s regard i t could be r e l e v a n t t h a t c a l c i u m and PS form c o l c l e a t e s t r u c t u r e s i n v i t r o (145), e f f e c t i v e l y sequestering both the calcium and the PS into l o c a l domains. One c o u l d p o s t u l a t e then th a t the PS and calcium r e t u r n t o the m i t o c h o n d r i a t o g e t h e r i n v i v o . To t e s t t h i s hypothesis, i t would be i n t e r e s t i n g to do dual l a b e l i n g studies with with c a l c i u m and s e r i n e to see i f t h e c a l c i u m r e t u r n s t o the m i t o c h o n d r i a , and see i f t h i s r e t u r n i s c o r r e l a t e d w i t h the PS decarboxylation. Such experiments are d i f f i c u l t to perform in p r a c t i c e , and i t i s d i f f i c u l t to say a p r i o r i whether such experiments would work. As a p r e l i m i n a r y i n v e s t i g a t i o n , i n v i t r o r e c o n s t i t u t i o n studies with r e s p i r i n g m i t o c h o n d r i a and microsomes could be c a r r i e d out, with c a l c i u m and s e r i n e l a b e l . One c o u l d then i n c r e a s e the oxidation state of the m i t o c h o n d r i a and do a timecourse on the l a b e l metabolism. With t h i s system i t might be p o s s i b l e to observe calcium r e l e a s e from the m i t o c h o n d r i a , c a l c i u m uptake by the endoplasmic recticulum, stimulated s y n t h e s i s of PS, and transport of calcium and PS to the mitochondria, i n a temporal sequence. I t i s possible that cytoplasmic factors such as p h o s p h o l i p i d exchange proteins are neccesary components of the c y c l e . One could also test for an second messenger of adrenalin with t h i s system. Plasma membranes co u l d be i s o l a t e d and adrenalin could be added to them i n v i t r o . The membranes co u l d be spun down and the supernatant could be assayed for its abi l i t y to cause calcium release from in vitro preparations of mitochondria. Alternatively, one could propose a coupling scheme involving decreased GTP levels. As was mentioned in section 1.5, adrenalin coupling with the adenylate cyclase system increases GTP hydrolysis, in contrast to glucagon (102). Adrenalin also causes calcium efflux from the mitochondria in addition to elevating cAMP levels (124). It could be that the decrease in GTP levels causes mitochondrial calcium efflux by inhibiting CDP-DG acyltransf erase, as i t inhibits it in vitro (61). If this enzyme were to 0be inhibited, the mitochondrial PA levels would almost certainly rise, as the formation of CDP-DG from PA is a crossover point in the synthesis of PI and CL. The increase in PA could increase mitochondrial calcium efflux by virtue of its calcium ionophore properties. One could test this hypothesis simply by varying 45 the GTP concentration of a [ Ca] loaded mitochondrial preparation and measuring calcium release. Measurements could also be made of. GTP levels in vivo as a functon of mitochondrial calcium efflux to see if there is a positive correlation. Such experiments could contribute greatly to our knowledge of the regulatory schemes dealt with in this thesis. 3.22 The Effect of Calcium on Choline Uptake Before studying the effect of calcium and IPA on PC biosynthesis, it was felt that it should be determined i f calcium had any effect on choline uptake, as it does with amino acid uptake (143). The effect of 3 a one hour preincubation with 1uM IPA on the uptake of 28uM [Me- H] 92 labeled choline ( 119mCi/mmol) was studied in hepatocytes, as a function of calcium concentration. A one hour pulse time was used. The concentration of IPA used in this study was taken from Kelley et al. (143), who performed a similar study on amino acid uptake. Figure 3-9 shows that IPA inhibits choline uptake in a calcium dependent manner. The maximal inhibition occurred at a calcium concentration of 200rag/l or greater, and amounted to about 40$ relative to control. Figure 3-9 also shows that the uptake of choline can be increased relative to control if the calcium is removed from the media. This inhibition is different to the effect on amino acid uptake, which is stimulated by calcium in the presence of IPA. .This difference is in keeping with what is known about hepatocyte physiology and the general effects of calcium ( see section 1 . 6 ) : hepatocytes respond to the stressful conditions mediated by calcium by increasing gluconeogenesis and hence increase uptake of amino acids. However, it is sensible that the hepatocyte would leave serum choline for other tissues which cannot make significant PC from PE and PS. In figure 3 . 1 0 , a Lineweaver-Burke Plot is presented for the effect of calcium and IPA on saturatable choline uptake. From this data it appears that the Vmax decreased from 500 to 200 pmol/rain/dish in the presence of calcium. The Km value of 14UM compares well with previously observed values (54). It has been shown that cAMP analogs inhibit choline uptake by inhibiting the Vm (146), and thus it is possible that the two effectors operate through the same final pathway. Such a mechanism is difficult to postulate though, as it is known that calcium decreases the hormonally stimulated level of cAMP 93 Figure 3.9 The effect of calcium and Ionophore A23187 on choline  uptake in cultured rat hepatocytes. Cells were pulsed for one hour with 28uM [Me- H] choline (119mCi/mmol) containing various amounts of calcium in the presence ( •) or absence ( • ) of 1uM Ionophore A23187. Cells and media were collected and the to^al uptake of choline was determined. Each point represents three dishes and the bars indicate the SE. Km=14uM 1/ [Choline] (uM"l) ° " ° ^ d ? . _ ? n d l n presence <«,*) or absence (T) of Ionophore A 2 4\ 95 in hepatocytes (143), and glucagon, a well known stimulator of cAMP, has no observable effect of calcium levels (124). One possibility is that the two effectors phosphorylate a common protein, and thus exert the same effect. Phosphorylase a is activated by both calcium and cAMP (109), and could be responsible for the inhibition of choline uptake. Further experiments would be needed to determine this. 3.23 The Effect of Calcium and IPA on PC Biosynthesis It was of interest to see i f calcium had an effect on PC biosynthesis, as it has on many other pathways. As i t had previously been determined that calcium with IPA inhibits choline uptake, IPA was •3 added during the chase only. C e l l s were pulsed with [Me- H] choline for one hour prior to treatment with IPA in the chase media. The hepatocytes took up the choline label e f f i c i e n t l y , and the incorporation pattern was similar to previous studies (45). Figure 3.11 shows the effect of adding 1uM IPA on the incorporation of label into PC and choline phosphate. There was only a marginal decrease in the rate of PC biosynthesis (20$ after a three hour chase). However, it would be premature to conclude that calcium has no significant effect on PC biosynthesis in vivo. It is known that calcium activates phospholipases (24), and hence generates fatty acid and lyso-PE. Both are known to activate phosphocholine cytidylyltransferase (54). Thus i t i s possible that the increase in fatty acid and lysolipids overcomes any inhibition caused by calcium. In view of the model proposed in section 1.3, the maintainance of PC synthesis by lyso-PE when under a calcium load would be equivalant to the effect of fatty acid under a glucagon load. However, many more studies are required to 96 0 1 2 3 . 0 1 2 . 3 Chase Time (h) Figure 3.11 Th^e e f f e c t of, J on o ph o r e A 2 318 ? _ on the disappearance of TMe- Hi choline in c e l l u l a r phosphocholine and i t s  i t s accumulation i t o phosphatidylcholine. Cultured, rat hepatocytes were pulse labeled for 30 minutes with 10uCi [Me- H] choline. The c e l l s were washed and f r e s h medium with (0) or without (^ ) 1uM Ionophore A23187 was added. At various time, the cells and media were collected, and the radioactivity was determined in (A) phosphocholine; and (B) phosphatidtylcholine. Each point represents three dishes and the SE i s indicated by bars. 97 accertain t h i s . In an experiment analogous to the c h o l i n e experiment above, an atte m p t was made t o o b s e r v e t h e e f f e c t s o f c a l c i u m on the me t h y l t r a n f e r a s e pathway, by u s i n g an ethanolamine l a b e l . C o n t r o l c e l l s r a p i d l y i n c o r p o r a t e d l a b e l i n t o PE« * i t h o n ly t r a c e amounts recovered i n the upper phase p r e c u r s o r s . During the chase period, l a b e l was transferred to PC, such t h a t only 20% remained i n PE a f t e r a three hour chase. This incorporation pattern i s s i m i l a r to observations reported previously (73 ) . As shown i n f i g u r e 3.12, addition of 1uM IPA to the chase media had no s i g n i f i c a n t e f f e c t on the incorporation o f l a b e l i n t o h e p a t o c y t e p h o s p h o l i p i d s . S u b s e q u e n t to t h e s e experiments, attempts were made to observe an e f f e c t by increasing the IPA concentration i n c r e m e n t a l l y to 25uM. No e f f e c t was seen on t h i s pathway at any IPA c o n c e n t r a t i o n . However, c o n c e n t r a t i o n s of IPA gre a t e r than about 10uM k i l l e d most of the c e l l s as observed with trypan blue. Also, the concentration of i n s u l i n was adjusted to 1, 10, 100, and 1000 nM and again no e f f e c t o f 1uM IPA was seen. L a s t l y , media c a l c i u m c h l o r i d e l e v e l s were i n c r e m e n t a l l y v a r i e d from to 500mgs/l and again 1uM IPA had no e f f e c t . At t h i s point, i t was decided that any p o s s i b l e e f f e c t t h a t c a l c i u m has on PC s y n t h e s i s might be e a s i e r to observe i n v i t r o , to a v o i d the draw backs of l a b e l i n g experiments and to avo i d compensation e f f e c t s o p e r a t i n g i n i n t a c t hepatocytes. F i g u r e 3.12 The^ e f f e c t o f Ionophore A 2 3 1 87 on the disappearance of f ( 1 ) - Hi ethanolamine in phosphatidvlethanolamine  and i t s accumulation i t o p h o s p h a t i d y l c h o l i n e . Cultured iiat hepatocytes were pulse labeled for 90 minutes with 15uCi [(1)- H] ethanolamine. Cells were washed and fresh medium with (closed symbols) or without (open symbols ) 1uM Ionophore A23187 was added. At various time, the c e l l s and media were co l l e c t e d , and the radioactivity was d e t e r m i n e d i n (•, •) p h o s p h a t i d y l e t h a n o l a m i n e ; and (•, v) phosphatidylcholine. Each point represents three dishes and the SE i s indicated by bars. 99 3.3 The Effect of CaM Preparations on CTPrPhosphocholine  Cytidylyltransferase in vitro: " Calcium and CaM are have been shown to have extensive regulatory effects on many enzymes. Previous studies have shown that phosphocholine cytidylyltransferase in cytosol can be inhibited by Mg-ATP or 2mM calcium in a time dependent fashion (54) . This raised the possibility that there may be a CaM dependent protein kinase or activator operating on the enzyme in vivo. In this set of studies, the effect of CaM on cytosolic CTP: phosphocholine cytidylyltransferase was investigated. 3.31 The Partial Purification of Rat Liver CaM and its Effect on  CTP:Phosphocholine Cvtidvlyltransferase An attempt to purify rat liver CaM was made by using a method similar to the protocol of Cheung et a l . (105), for the purification of CaM and calcium binding proteins from rat brain ( see section 2.5 ). A b o i l e d , d i a l i z e d l i v e r homogenate was assayed for cytidylyltransferase inhibition. A two fold inhibition was obtained at protein concentrations of 4ug/ml ( see table 3.4). The dialysate was applied to a DEAE Sepharill column, and the activity was eluted off with an ammonium sulfate gradient. Each 4.5 ml fraction collected was assayed for cytidylyltransferase inhibition, u D 2 g o » a n d conductance to estimate the salt concentration ( see figure 3.13) • Three peaks of cytidylyltranferase inhibition were obtained. Peak two eluted close to 100 Table 3.3 Inhibition of cvtidvlvltranferase activity by a  boiled rat l i v e r homogenate.The a b i l i t y of a boiled rat brain homogenate to inhibit cytosolic CTP: phosphocholine cytidylyltranferase was determined. Assays were done as described in the methods section. SAMPLE ENZYME ACTIVITY (nmol/min/mg) Control 0.35 With 4ug/ml dialysate 0.17 With 20ug/ml dialysate 0.25 ( \QOCK. Figure 3-13 DEAE S e p h a r i l l Chromatography of a boiled rat liver homogenate. 30mls of a boiled rat l i v e r dialysate (see section 2.52) was applied to a DEAE S e p h a r i l l column (bed volume 40ml). The major protein fraction was eluted with a buffer containing 20mM MgSO^ , 20mM Tris-HCl, pH 7.4, and .15M NH^SO^. A f t e r running 200mls of th i s buffer, the concentration of NH^SO^ was increased l i n e a r l y to .3M over a range of 150ml, and then maintained at .3M for an additional 150ml. The remaining protein was eluted with 30ml of buffer with .5M NH SO . 100 f r a c t i o n s of 4.5ml were taken, and assayed f o r 4 4 cytidylyltransferase i n h i b i t i o n ( — — ) , OD ( ), and conductance. the conductance was standardized to NH SO, (•••»•). 4 4 102 where CaM normally elutes. The three peaks were assayed for their a b i l i t y to i n h i b i t c y t i d y l y l t r a n s f e r a s e in the presence of phospholipid. In table 3.5 , it can be seen that only peaks 2 and 3 were able to do this. Because of the large amount of protein in peak 1, i t was assumed that peak 1 i n h i b i t i o n was due to nonspecific protein binding. This effect has been observed previously ( 47), and was found to be reversible by phospholipid. The inhibition from peak one was not further characterized. Peaks two and three were dialized against homogenizing buffer to remove the ammonium sulfate, and then assayed again. Peak three inhibition disappeared, while peak two inhibition-was diminished but s t i l l significant (see table 3.6). The peak 2 i n h i b i t i o n p r o p e r t i e s were studied as a function of concentration and the results are shown in figure 3.14. It can be seen only that protein concentrations of only 40ng/ml are required to elicit a maximal response. The calcium dependence of the inhibition was next examined by adding EGTA to an assay system containing only 17uM calcium. 0.2mM EGTA did not reverse the inhibition. In view of the fact that this inhibitor was not calcium dependent, no further attempt was made to purify i t . However, i t should be noted that the inhibitory activity was destroyed by ul t r a f i l t r a t i o n on an amicon concentrator, implying that the inhibitor is a protein. Moreover, the observation that the inhibitor can even reverse l i p i d activation suggests that the mechanism of i n h i b i t i o n is different from the inhibition observed by phosphorylation, LPC or salt inhibition (54). One of the major impurities found in CaM preparations obtained in this way are what are known as CaM binding proteins Q47). These proteins" have have the propertyof diminishing the activity of CaM. 103 Table 3.5 The inhibition of CTP: phosphocholine cytidylyltransferase  by DEAE sepharill fractions and the effect of phospholipid. SAMPLE ENZYME ACTIVITY (nmol/min/mg) Control 0.33 Mug/ml Peak II 0.23 Dialysate 4ug/ml Peak III 0.30 Dialysate Table 3.6 The i n h i b i t i o n of CTP: phosphocholine c v t i d v l v l t r a n f e r a s e by  DEAE s e p h a r i l l f r a c t i o n s a f t e r d i a l y s i s . SAMPLE ENZYME ACTIVITY (nmol/min/mg) Control 0.38 Control with. 0.97 Phospholipid With Peak I (4ug/ml) 0.26 With Peak I (4ug/ml) 0.89 Plus Phospholipid With Peak II (4ug/ml) 0.04 With Peak II (4ug/ml) 0.18 Plus Phospholipid With Peak III (4ug/ml) 0.08 With Peak III (4ug/ml) 0.3 Plus Phospholipid 104 Figure 3.14 I n h i b i t i o n of CTP: phosphocholine c v t i d v l v l t r a n f e r a s e  a c t i v i t y by a dialyzed DEAE s e p h a r i l l f r a c t i o n . Peak II from the DEAE s e p h a r i l l column was d i a l y z e d and i t s a b i l i t y to i n h i b i t c y t o s o l i c c y t i d y l y l t r a n s f e r a s e was examined as a function of concentration. Each point i s an avearage of two determinations. O OO ^ CM O • • • • • r H O O O O (Bui /u T U l / J OUIU) 105 3-32 Studies with Pure CaM It was possible that the CaM in the above preparation was inhibiting the cytidylyltransferase in a calcium independent manner. To test t h i s hypothesis, the a b i l i t y of pure calmodulin from C a l b i o c h e m to i n h i b i t c y t o s o l i c C T P : p h o s p h o c h o 1 i n e cyt i d y l y l t r a n s f e r a s e was i n v e s t i g a t e d . No e f f e c t was observed at •concentrations of CaM exceeding 10ug/ml ( figure 3.15). In a control study, this p r e p a r a t i o n was found to s t i m u l a t e rat brain cAMP phosphodiesterase (148), confirming that i t had normal biochemical activity. Thus i t may be concluded that CaM does not inhibit cytosolic CTP:phosphocholine cytidylyltransferase. Table 3.7 The effect of pure calmodulin on the activity of cytosolic CTP:phosphocholine cytidylyltransferase. Sample Cytidylyltransferase Activity (nmol/min/mg) Control 0.92 ith .25ug/ml 0.94 CaM With 1ug/ml 1.10 CaM With 5ug/ml 0.91 CaM With 10.25ug/ml 1.10 GENERAL CONCLUSIONS H e p a t i c p h o s p h o l i p i d m e t a b o l i s m i s c o o r d i n a t e d to the p h y s i o l o g i c a l s t a t e o f an a n i m a l t h r o u g h a v a r i e t y o f c o n t r o l mechanisms. Our u n d e r s t a n d i n g o f t h e s e p r o c e s s e s has i n c r e a s e d d r a m a t i c a l l y i n recent y e a r s , but i s at an elementary l e v e l . This t h e s i s has i n v e s t i g a t e d some o f the r e g u l a t o r y mechanisms which operate i n rat hepato c y t e s . I t was shown that glucagon i n h i b i t s the r a t e o f PC b i o s y n t h e s i s by i n h i b i t i n g t h e a c t i v i t y o f CTP:phosphocholine c y t i d y l y l t r a n s f e r a s e . T h i s f i n d i n g i s i n accord with the widely accepted view tha t glucagon i n h i b i t s key biosynthetic enzymes in order to adapt the l i v e r to s t a r v a t i o n conditions. It i s l i k e l y that t h i s hormone a c t s on the c y t i d y l y l t r a n s f e r a s e v i a a phosphorylation cascade system. Future s t u d i e s i n t h i s area should focus on the mechanism by which t h i s i n h i b i t i o n occurs, as well as the r o l e of o t h e r s t a r v a t i o n s i g n a l s ( eg. g l y c e r o l , f a t t y acid) i n the regulation of PC synthesis. The role of calcium i n the r e g u l a t i o n of phospholipid metabolism was also investigated. I t was found that calcium s l i g h t l y i n h i b i t s PC synthesis i n hepatocytes, but not through a mechanism involving CaM. I t seems p o s s i b l e t h a t p r o t e i n k i n a s e C or pho s p h o r y l a s e a are involved. Calcium a l s o i n h i b i t s the uptake of c h o l i n e , by decreasing the Vm of the uptake s y s t e m . L a s t l y , i t was shown that c a l c i u m s t i m u l a t e s the i n c o r p o r a t i o n o f s e r i n e i n t o p h o s p h o l i p i d and the t r a n f e r of l i p i d to the m i t o c h o n d r i a . In the next few years i t i s l i k e l y t h a t r e s e a r c h i n l i p i d m e t a b o l i s m w i l l e s t a b l i s h that phospholipids are of c r i t i c a l importance i n many regulatory systems. 107 BIBLIOGRAPHY 1. Kennedy, E.P., The Harvey Le c t u r e s 5JL 143 (1962) 2. Gorter, E., and Gr e n d e l , F . J . Exp. Med. 41 (1925) 3. Singer, S.J. and N i c o l s o n , G.L., S c i . JI5. 720 (1972) 4. A l o i e , R.C, ed. 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