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Studies on the epinephrine-sensitive lipase of adipose tissue Yamamoto, Mas 1964

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STUDIES ON THE EPINEPHRINE-SENSITIVE L I P A S E OF ADIPOSE TISSUE b y MAS YAMAMOTO A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE I n t h e D e p a r t m e n t o f P h a r m a c o l o g y We a c c e p t t h i s t h e a i a as c o n f o r m i n g t o t h e r e a u i r - e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A u g u s t 1964 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t ; c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P h a r m a c o l o g y T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r 8, C a n a d a n . 28 August 1964 - i i -ABSTRACT The study of the role of adipose tissue i n the maintenance of the caloric homeostasis of organisms i s currently the object of widespread research. In particular, the enzymes of l i p i d metabolism i n adipose tissue are being extensively investigated i n both intact f a t pads and i n broken-c e l l preparations. Special attention is being paid to factors which control the activity of these enzymes. We have examined some properties of a lipase i n epididymal f a t pads of rats. The enzyme has been assayed by measuring the free fatty acids liberated when triglycerides are incubated with crude adipose tissue extracts. Quantitative measurements of free fatty acids were performed by (a) t i t r a t i n g the liberated acid with dilute a l k a l i solution, and (b) reacting the free fatty acids with Cu' f +to form the chloroform-soluble copper soap of long chain fatty acids, then assaying the copper with diethyl-dithiocarbamate spectrophotometrically. It i s well known that lipase activity i n adipose tissue decreases during incubation i n a Krebs-Ringer bicarbonate medium at 57° 0. The de-activated enzyme can be activated by briefly exposing the intact tissue to epinephrine. The study of this epinephrine-sensitive lipase i n adipose tissue has been the main object of this thesis. When epinephrine was added to media containing intact epididymal fat pads, the dramatic mobilization of free fatty acids from the pads into the media was observed. When epinephrine was added directly to unfractionated homogenates, l i t t l e , i f any, response was el i c i t e d , indicating perhaps that some activating factor was destroyed or diluted - i i i -out during homogenization. When ATP, cyclic 5',5*-AMP and Mg^ were added to unfractionated homogenates of adipose tissue, some lipase activation was observed. Similarly, when these nucleotides and Mg^ were added to the supernatant f l u i d obtained from centrifuged homogenates, some activation of the lipase was observed, although the results obtained were not consistent. Other nucleotide 5 ' » 5 '-cyclic phosphates generally inhibited lipase activity i n the supernatant f l u i d . Our data indicates that epinephrine activates adipose tissue lipase only when added to the intact f a t pad before homogenization. L i t t l e or no activation occurred when the amine was added to homogenates. Cyclic 5',5'-AMP had some a b i l i t y to reactivate the lipase, both in unfractionated homogenates and in the supernatant f l u i d prepared by centrifugation. The effects, however, were not marked. I t is concluded that i f epinephrine-activation of adipose tissue is mediated through cyclic 5'»5,_AMP, precise conditions for showing this have not yet been achieved. Additional experiments were performed on the epinephrine-sensitive lipase. Intact adipose tissue obtained from reserpinized rats was exposed to epinephrine after a J-hour incubation period. The results indicated that epinephrine does not activate the l i p o l y t i c system in adipose tissue of reserpinized rats. Finally, some of the factors regulating the degree of inactivation of the epinephrine-sensitive lipase during incubation were investigated. Fat pads removed from rats which had been either anaesthetized or not anaesthetized prior to sacrifice were incubated for 5 hours. Data collected from a number of experiments indicated that there were virtua l l y no differences i n the extent of lipase inactivation between the two groups of rats. - ix -ACKNOWLEDGEMENT I am indebted to Dr. G. I. Drummond of the Department of Pharmacology, University of Bri t i s h Columbia, not only for providing the necessary equipment and f a c i l i t i e s required for undertaking this project, but also for his valuable guidance, helpful criticism and encouragement throughout the entire course of this work. In addition, I wish to gratefully acknowledge the Medical Research Council grants which he kindly made available to me. - i v -TABLE OF CONTENTS Page A. INTRODUCTION 1 B. EXPERIMENTAL PROCEDURE 18 Methods - Lipase Assay I 18 Lipase Assay II 20 Lipase Assay III 21 Materials » 22 0. RESULTS 25 I. Preliminary Studies 25 1. Accuracy of the Titrimetric Method 25 2. Determination of FFA by the Copper Soap Method 25 5 . Effect of Mg^ on the recovery of FFA by the Copper Soap Method 27 4. Stability of the Diethyldithiocarbamate-Copper Complex.. 27 5 . Time Dependence of the Epinephrine-sensitive Lipase 51 6. Preparation and Use of Extracted Natural Lipids as Substrate 51 7. Preparation and Use of Heated Crude Homogenate as Substrate 54 II. Experimental Results 1. Response of Epididymal Fat Pads to Epinephrine $6 2. Response of Crude Homogenates to Epinephrine 5^ 5. Effect of Epinephrine on Lipolytic Activity when Added Before, During and After Homogenization 58 4. Search for Effects of ATP, Cyclic J',5'-AMP, Mg^ on Crude Homogenate 40 (a) ATP Effects 42 (b) Cyclic 5',5'-AMP Effects 44 (0) Mg^ Effects 47 5. Effect of ATP, Cyclic 5',5'-AMP, Mg+^ on Lipolytic Activity i n Supernatant Fractions of Adipose Tissue Homogenates 49 6. Effect of Other Cyclic Nucleotides on Lipolytic Activity i n Supernatant Fractions ^2 - V -TABLE OP CONTENTS (Cont'd) Page III. Additional Experiments on Adipose Tissue Lipase 54 1. Effect of Epinephrine on Adipose Tissues Obtained from Reserpinized Rats ^4 2. Effect of Pentobarbital-induced Anaesthesia and Length of Incubation on Inactivation Rate of Adipose Tissue Lipase 56 5. Effect of Insulin on Inactivation Rate of Adipose Tissue Lipase 5^ . D. DISCUSSION 60 E. BIBLIOGRAPHY 67 - v i -LIST OP TABLES No. T i t l e Page 1. Accuracy of the Titrimetric Method 26 2. Effect of Mg^+ on the Extraction of Palmitic Acid by the Copper Soap Method 29 5. Stability of the Diethyldithiocarbamate-Oopper Complex 5° 4. Epinephrine Effects on Intact Epididymal Fat Pads 57 5. Summary of ATP, Cyclic 5',5'-AMP, and Mg+~* on Lipase Activity in the Supernatant Fraction of Adipose Tissue Homogenates... $1 6. Effect of other Cyclic Nucleotides on Lipase Activity i n Supernatant Fraction of Adipose Tissue Homogenates 55 7. Effect of Pentobarbital-induced Anaesthesia and Length of Incubation on the Inactivation Rate of the Lipolytic System 57 - v i i -L I S T OF FIGURES No. Title Page 1. Pathway of Triglyceride Synthesis i n Liver 5 2. Outline of Metabolic Pathways i n Adipose Tissue 8 5 . Activation of Phosphorylase i n Skeletal Muscle 15 5a. Palmitic Acid Standard Curve 28 4. Time Dependence of the Epinephrine-sensitive Lipase 5 . Substrate Concentration Curve (De-activated Homogenate) 55 6. Epinephrine Effect on Crude Homogenate of Adipose Tissue 59 7. Epinephrine Effects on Lipase Activity When Added Before, During, and After Homogenization 4l 8. Effect of ATP, Cyclic 5,,5t'-AMP and Mg++ at Various Concentrations on Unfractionated Adipose Tissue Homogenate 4j 9. ATP Concentration Curve with Respect to Lipase Activity (Unfrac-tionated Homogenate 1). 45 10. Cyclic 5',5'-AMP Concentration Curve with Respect to Lipase Activity (Unfractionated Homogenate) 46 11. Mg*^  Concentration Curve with Respect to Lipase Activity (Unfrac-tionated Homogenate) 48 12. Effect of ATP, Cyclic J'^'-AMP, and Mg"*""*" on Lipase Activity i n the Supernatant Fraction 5^ 15. Effect of Epinephrine on Intact Adipose Tissue Obtained from Reserpinized Rats 55 14. Effect of Insulin on the Degree of Lipase Inactivation during Incubation 59 - v i i i -LIST OP ABBREVIATIONS FPA Free Fatty Acids ATP..... Adenosine Triphosphate OoA Coenzyme A GMP Guanosine Monophosphate CMP Cytosine Monophosphate AMP Adenosine Monophosphate UMP Uridine Monophosphate TMP Thymidine Monophosphate NADH.... Nicotinamide Adenine Dinucleotide (Reduced form) NADPH... Nicotinamide Adenine Dinucleotide Phosphate (Reduced form) Tr i s . . . . Tris(hydroxymethyl)aminomethane 1. INTRODUCTION Until relatively recent times, adipose tissue was assumed to be a metabolically inert f a t depot. Its main physiological functions were considered simply to provide thermal insulation, mechanical padding, and to act as an energy storehouse which released i t s f a t reserves rather sluggishly. Perhaps the f i r s t hint that adipose tissue does, i n fact, take a very active part i n the dynamic processes of the body arose from the work of Schoenheimer and Rittenberg i n 1957 (1-5)* But i n spite of their pioneering work indicating that a rapid turnover and continuous synthesis of f a t did occur i n the animal organism, most investigators were relatively slow to accept this new concept of adipose tissue metabolism. Few textbooks on physiology and biochemistry even now mention these depots as sites of active metabolism. During the past two decades, however, and especially so i n the past few years, investigators have become keenly aware of the true role of adipose tissue i n the overall physiology of the intact organism. This awakening interest i n various aspects of adipose tissue metabolism, including the relative importance of fats as a source of energy, i s reflected i n the increasing number of fine reviews based on these topics (4-11). The mobilization of the caloric reserve of> adipose tissue occurs i n the form of free fatty acids (FFA), also frequently referred to as non-esterified fatty acids (NEFA) and unesterified fatty acids (UFA). These fatty acids are known to be readily transportable and readily metabolizable substrates. Gordon and Cherkes (12), Gordon (15), and Dole ( l 4 ) , correlated the concentra-tions of FFA i n serum with changes i n the nutritional state of organisms, and 2. concluded that fats were released from adipose tissue and transported i n blood as FFA-Albumin complexes. These acids, although representing a relatively small fraction of circulating plasma lip i d s , have a high turnover rate (15). The i n vitro release of FFA from adipose tissue was soon demonstrated i n a number of laboratories (16,17*18). I t became evident that the release of l i p i d s as FFA was the only significant means by which fats can be liberated from adipose tissue, and that the concentration of plasma FFA closely reflected the extent of body f a t u t i l i z a t i o n . Besides releasing FFA to the circulation, adipose tissue also plays a major role i n the synthesis of triglycerides. Even after the classic work of Schoenheimer and Pdttenberg (1-5)» i t was generally believed that the l i v e r , and not adipose tissue, was the main site of lipogenesis. This notion was largely dispelled, however, when Masoro et a l (19) demonstrated that hepatectomized animals could readily synthesize triglycerides from glucose. It i s now believed that lipogenesis i n the l i v e r probably represents only a fraction of body fat synthesis. Nevertheless, the mechanism of triglyceride synthesis i n adipose tissue i s similar to the pathways suggested by Weiss and Kennedy (20) and also by Tietz and Shapiro (21) for triglyceride synthesis i n l i v e r . The synthetic pathway i n the liver is outlined in Fig. 1. One important point must be emphasized concerning triglyceride synthesis i n adipose tissue. Whereas l i v e r possesses glycerokinase activity and i s therefore able to phosphorylate glycerol to oC-glycerophosphate, no such activity has been demonstrated i n adipose tissue (22). In other words, l i v e r can readily u t i l i z e free glycerol as i t s source of oC-glycerophosphate, but adipose tissue must depend on some other source for i t s supply of ^ glycerophosphate, an essential precursor for triglyceride synthesis. For this reason, l i p o l y t i c ATP Triglyceride F i g . 1. S y n t h e t i c p a t l w a y f o r t r i g l y c e r i d e s i n l i v e r (20,21). No g l y c e r o k i n a s e a c t i v i t y h a s b e e n d e m o n s t r a t e d i n a d i p o s e t i s s u e . ( © i n d i c a t e s p h o s p h a t e m o i e t y ) . 4. activity of adipose tissue i s frequently measured on the basis of free glycerol produced. Since re-esterification of PPA proceeds simultaneously with hydrolysis of triglycerides i n adipose tissue, the amount of glycerol produced is therefore more indicative of the actual degree of l i p o l y t i c a c t i v i t y . The source of oC-glycerophosphate i n adipose tissue appears to be dependent on the breakdown of glucose to dihydroxyacetone phosphate and ©(-glycerophosphate. Dihydroxyacetone phosphate can be readily reduced to oC-glycerophosphate by NADH. This strongly suggests that triglyceride synthesis i n adipose tissue may be dependent on the avai l a b i l i t y of glucose and i t s breakdown i n the c e l l s , and furthermore, that any factor which affects this glycolytic scheme w i l l similarly affect lipogenesis. The assimilation of triglyceride and i t s hydrolysis and subsequent breakdown to FFA and glycerol i n adipose tissue are continuous dynamic processes, and the question naturally arises as to what factor or factors oontrol these two opposing reactions. But before the metabolic regulation of adipose tissue i s discussed, a brief histological outline of adipose tissue and some emphasis on the importance of lipids as a source of energy is indicated. The epididymal f a t pads of rats have been used most extensively by investigators i n the study of adipose tissue metabolism. The mesenteric and mesometrial adipose tissues of female rats have also been frequently used. In histological sections, the cells of white adipose tissue are large, nearly spherical and usually contain a single globule of l i p i d enclosed by a very thin f i l m of cytoplasm (25). Each c e l l contains a nucleus which appears to be pressed against the inner aspect of the c e l l membrane (24). It i s most interesting that when the ratio of capillary surface to volume of active protoplasm i s calculated, the capillary bed, relative to the amount of cytoplasm, i s actually richer than i t i s i n muscle (25,25). This observation alone demands that attention be focussed on adipose tissue concerning i t s important physiological functionnin the organism. Similarly, a rich network of nervous tissue i s present i n adipose tissue (4). Triglycerides comprise more than 99% of the total li p i d s i n adipose tissue, and only traces of cholesterol and phospholipids are found (26). Gas-liquid chromatographic studies have indicated that some 22 different fatty acids occur in adipose tissue, the major acids being palmitic (20%), oleic and isomers (46$) and l i n o l e i c acid (11%). As mentioned earlier, the importance of lipids as an energy source, not only during starvation, but also during the post-absorptive state, was suggested by Fredrickson and Gordon (15,27). I t has been estimated that approximately .50% of total respiration i s derived from FFA oxidation under basal conditions (15>28), which particularly emphasizes the'important role of lipi d s i n the oxidative processes of the intact organism. Individual organs, however, vary i n their a b i l i t y to oxidize fatty acids for energy. It has been estimated that cardiac muscle of man, under basal conditions, oxidizes approximately 50 to 40 micromoles of fatty acids per minute (15,29). In fact, cardiac muscle appears to u t i l i z e l i p i d s as i t s predominant source of energy. Skeletal muscle appears to derive i t s energy from the oxidatiomof glucose during short, intensive exercise characterized by hypoxia. But during moderate, sustained work, i f the oxygen supply is adequate, li p i d s may be i t s major substrate (50,51). For example, the non-stop migration of the ruby-throated hummingbird across the 6. Gulf of Mexico causes a decrease in i t s fat content from of i t s weight to &f0 (52). In this particular instance, l i p i d s are obviously burned directly and most ef f i c i e n t l y by f l i g h t muscles for energy. Evidence has been presented which indicates that the pectoralis muscle of pigeons can also oxidize l i p i d s directly (55)• The comparative aspects of muscle metabolism, with special emphasis on the importance of l i p i d metabolism in insects, birds and fishes has been reviewed by Drummond and Black (6). The brain depends primarily on blood glucose as i t s source of energy (5^). No arteriovenous difference i n FFA concentration across the brain under basal conditions was found by Gordon and Gherkes (12). However, i t is known that the brain does possess the enzymatic machinery necessary for oxidizing lipi d s (55). The l i v e r accounts for almost one-half of the fatty acids oxidized i n man under basal conditions (15)- F r i t z (7)» however, suggests that this organ should not be considered essential for the metabolism of l i p i d s since fatty acid oxidation and other aspects of l i p i d metabolism can occur at various extrahepatic sites i n the absence of the l i v e r . Isolated adipose tissue has a relatively low rate of conversion of added fatty acids to CO2, the rate being elevated i n starved animals (56). No information is available regarding the u t i l i z a t i o n of lipid s for energy by smooth mus cle. Using washed mitochondria from various organs, Bode and Klingenberg (56) recently demonstrated that l i v e r , kidney, heart and diaphragm mitochondria could oxidize medium to long chain FFA at appreciable rates, whereas skeletal muscle mitochondria showed the lowest activity. They also observed that carnitine esters of fatty acids increased the rate of oxidation by 7-mitochondria of a l l organs. The foregoing outline on the active oxidation of l i p i d s by certain organs re-emphasizes the important role of lipid s i n providing a readily u t i l i z a b l e source of energy. It i s evident that no knowledgeable investigator can any longer hold to the belief that adipose tissue i s merely an inert accumulation of f a t . The salient features of adipose tissue metabolism already discussed are summarized schematically i n Fig. 2. Physiological control of Adipose Tissue Metabolism. The foregoing has pointed to the prominent role played by adipose tissue i n overall energy metabolism. In recent years, many investigators have sought to understand how these metabolic reactions are controlled or altered under differing physiological conditions. That the metabolic activity of adipose tissue i s markedly influenced by a number of hormones is now a firmly established fact. The effect of these various hormones are readily observed i n vivo, and i n vitro on intact f a t pads, but the inter-pretation of these effects has perplexed, and continues to perplex, investigators everywhere. Insulin. Of a l l the hormones which are known to affect adipose tissue metabolism, insulin has probably been the most widely studied. Insulin has been shown by a number of workers to increase glucose uptake by epididymal f a t pads i n vitro (57*58,59,41,42). The oxidation of glucose to 00 2 by isolated epididymal f a t pads or by isolated f a t cells is also enhanced by insulin (24,40,45). These effects are detected with as l i t t l e as 10 microunits of insulin per ml of incubation medium (24,41); hence this sensitive in vitro F i g . 2. Outline of the metabolic processes i n adipose t i s s u e c e l l , i n d i c a t i n g the r e l a t i o n s h i p between carbohydrate and l i p i d metabolism. In normal c e l l s , f a t droplet occupies almost the e n t i r e space w i t h i n c e l l . Furthermore, there i s evidence that more than one pool of FFA exi s t s i n adipose t i s s u e c e l l s (54,78). 9. responsiveness by rat epididymal f a t pads has been used as the basis for the assay of insulin or insulin-like activity i n biological fluids (44,45). Electron micrographic evidence indicating that insulin may enhance glucose uptake in adipose tissue by a pinocytotic process has been presented by Barrnett and Ball (58). Triglyceride synthesis i s enhanced by insulin (46,47,49,50). Vaughan (49) incubated adipose tissue from fasted rats with uniformly labelled glucose, and showed that insulin i n vitro increased the specific activity of adipose tissue triglycerides by approximately 400$. Raben and Hollenberg (52,55) have suggested that the action of insulin i s directly on the esterification process. Evidence has also been presented indicating that insulin may increase the availability of NADPH for synthetic processes by stimulating the pentose phosphate pathway (45,54) or that the hormone may stimulate the hexokinase reaction (57)' 0 1 1 the other hand, Jungas and Ball (51) recently suggested that insulin, i n some unknown manner, inhibits the hydrolysis of triglycerides. They indicated rather strongly that insulin may have an inhibitory action, either directly or indirectly, on tissue lipase. The more recent studies by Tarrant et al (85) with animals made diabetic with anti-insulin serum indicate that insulin may, indeed, inhibit the l i p o l y t i c system. They found that lipolysis i n adipose tissue of diabetic rats, as indicated by the amount of glycerol released, was higher than control tissue, and the addition of insulin to the medium reduced this l i p o l y t i c activity toward the control level. They speculated that the increased l i p o l y t i c activity observed i n insulin-deficiency is due to the absence of the normal restraining action of insulin on other adipokinetic hormones, thus adding support to the suggestion made earlier by Jungas and Ball (51)' Further evidence against the widely held re-esterification 10. hypothesis as an explanation of insulin's action on FFA release from adipose tissue has recently been presented. In vivo studies by Zierler and Rabinowitz (86) indicated that when insulin i s infused at concentrations which do not have any effect on glucose uptake by adipose tissue, i t s inhibitory action on FFA release is s t i l l evident. They concluded that the action of insulin on FFA release was independent of glucose translocation. The mechanism of insulin action on adipose tissue therefore remains unsettled. The d i f f i c u l t y i n interpreting the results obtained may be due to a number of factors, and at least two are apparent. F i r s t , not a l l experiments have been performed under physiological conditions. Secondly, the measurement of the release of FFA from adipose tissue does not necessarily reflect actual l i p o l y t i c activity, since much of the FFA released may be immediately re-esterified. As indicated earlier, a true indication of l i p o l y t i c activity i n adipose tissue is obtained by measuring glycerol released, as this product-of triglyceride hydrolysis i s not re-utilized i n the esterification process. Prolactin. The i n vitro effects of prolactin on adipose tissue is similar to those observed with insulin, although much higher concentrations (0.5 to 1.0 mg/ml) are necessary (59)• It should be noted that although insulin corrects the defect i n fatty acid synthesis from glucose in adipose tissue from diabetic rats, prolactin shows no such action (40). Glucagon. Glucagon has been shown to increase the rate of both FFA and glycerol release (49 ,60,61). It also stimulates glucose uptake (61,62) and increases 11. phosphorylase activity (60,61) i n adipose tissue. Serotonin. L i t t l e information i s available regarding the action of serotonin on adipose tissue. Vaughan (60) demonstrated that serotonin could increase phosphorylase activity i n adipose tissue. The hormone, at a concentration of 0.4 micromole/ml of incubation medium, increased glucose uptake but did not increase PPA release by adipose tissue (49). AOTH Adrenocorticotropic hormone (AOTH) has been demonstrated by a number of investigators to cause the release of FFA by adipose tissue i n vitro (60,65*64). Lipase activity i s stimulated (65), as well as the uptake of glucose from the medium (4l,49). Phosphorylase activity i s also "apparently inoreased by AOTH (60). The similarity in the effects of AOTH and catechol-amines on adipose tissue metabolism i n vitro is rather remarkable, considering the dissimilarity i n the structures of these hormones. Since adipose tissue which has been depleted of catecholamines no longer responds to AOTH, i t has been suggested that AOTH acts by causing the release of catecholamines, chiefly norepinephrine, i n adipose tissue. One point of difference i n the action of AGTH and catecholamines has been observed on adipose tissue (66); whereas epinephrine action i s independent of calcium ion i n the medium, this divalent cation is essential for the enhanced l i p o l y t i c effects observed with AOTH. Other Pituitary Hormones. Other pituitary hormones have been shown to have effects on adipose tissue metabolism, and these w i l l be mentioned only briefly i n passing. 12. The actions of these polypeptide hormones have been reviewed by Rudman (11), Vaughan (9), Winegrad (10) and Wertheimer (5). Growth hormone possesses a l i p o l y t i c action similar to that of AOTH and epinephrine, although higher concentrations are required (64). Similarly Thyrotropin (TSH) has been demonstrated by White and Engel (64) to stimulate the release of FFA from adipose tissue i n v i t r o . However, both Vaughan (9) and Winegrad (10) stress the point that no conclusions about the part played by these pituitary hormones on adipose tissue metabolism can be drawn due to the d i f f i c u l t y of ascertaining the purity of these hormone preparations. Adrenergic Amines. The actions of adrenergic amines on adipose tissue metabolism have attracted widespread attention, and at the moment are being actively studied i n a vast number of laboratories. Since much of the experimental work on this thesis i s directly related to some of these effects observed, particularly the dramatic stimulation of l i p o l y t i c activity i n adipose tissue, the actions of these catecholamines on this tissue w i l l be discussed i n some detail. The concept that the sympathetic nervous system plays an important role i n the regulation of FFA release from adipose tissue has been supported by numerous in vivo experiments. Pharmacological blockade of norepinephrine action, using ergotamine, dibenzyline, or dibenamine has been shown to decrease plasma FFA concentration (67,68), indicating decreased FFA mobilization from adipose tissue. Unilateral denervation of symmetric" fat bodies results i n a diminished rate of l i p i d depletion from the denervated side (4). Conversely, physiological stresses of various types cause an increase i n FFA levels i n plasma (69,70). For example, fasting (12,14)" and fear (70) elevates plasma FFA levels, a l l of which indicates that the release of FFA from adipose tissue i s primarily due to an increased sympathetic discharge. I t is interesting to note that norepinephrine, i n vivo, has greater l i p o l y t i c activity than epinephrine (67,71)> although the two compounds possess similar levels of a c t i v i t i e s i n vitro (64). The l i p o l y t i c action of epinephrine on intact f a t pads iua. vitro has been adequately demonstrated by numerous investigators (16,18,46,47,71,72). This effect has been observed with very low concentrations of epinephrine (1 x 10"^ M) i n the incubation medium. The increased release of FFA i s accompanied by an increased release of glycerol into the medium (47), which would be i n accord with the increased lipolysis of tissue glycerides. Dibenamine and dibenzyline both i n h i b i t the release of FFA in vitro (75), as they have been shown to do i n vivo (67,68). In general, the observed effects of epinephrine and of insulin on adipose tissue metabolism are directly opposite. Whereas insulin appears to promote triglyceride synthesis, epinephrine enhances the breakdown of triglycerides. However, these two hormones do share at least one common feature; both increase glucose uptake by epididymal fat pads i n vitro (46,49,74), although i n this respect i t should be stated that the effect of epinephrine i s quantitatively much less than that produced by insulin (49). However, Vaughan (49) was able to demonstrate that at lower concentrations of epinephrine (0.1 microgram/ml), no measurable changes i n glucose uptake was observed, while the stimulatory effect of epinephrine on FFA release was s t i l l largely evident. Similar effects were observed 14. with AOTH and glucagon at concentrations of 0.04 units/ml and 5 microgram/ml respectively. Ball and Jungas (75) recently studied the effect of anaerobic conditions and diet on the response of adipose tissue to both insulin and epinephrine. They observed that epinephrine had no effect on glycerol and FFA production by adipose tissue from normally fed rats under anaerobic conditions. In the presence of oxygen, however, l i p o l y t i c activity was clearly indicated. Tissue from fasted-refed rats (whose glycogen content i n adipose tissue i s markedly elevated) responded to epinephrine under anaerobic conditions with a three-fold increase in glycerol production, but no increase i n tissue FFA level was observed. Under aerobic conditions, glycerol production was extremely elevated, whereas tissue FFA level remained quite low, indicating that re-esterification of the hydrolysed FFA had occurred at a very rapid rate. From observations such as these, Ball and Jungas suggested that epinephrine might increase the rate of l i p o l y s i s by increasing:.the activity of adipose tissue lipase, the enzyme responsible for hydrolysis of triglycerides. Their data also indicated that some form of high energy phosphate might be required for activation of adipose tissue lipase. They furthermore suggested that activation and inactivation of lipase may not be unlike that seen f o r skeletal muscle phosphorylase. It w i l l be recalled that epinephrine activates phosphorylase by mediating the formation of adenosine J% ^-cyclic phosphate (cyclic 5'>5'-AMP) (76), as shown schematically i n Fig. 5. Later, Rizack (77) studied lipase activity i n adipose tissue homogenates and this activity did indeed appear to be epinephrine sensitive. Rizack observed that when f a t pads were incubated i n Krebs-Ringer medium and then Adenyl C y c l a s e Epinephrine A T P , Mg ++ C y c l i c 3,5-AMP Phosphory lase b k i nase ( INACTIVE ) - ^ - P h o s p h o r y l a s e b k i n a s e ( ACTIVE ) Phosphorylase b ( INACTIVE ) V Phosphorylase a ( A C T I V E ) GLYCOGEN -^>- G lucose -1 -Phospha te F i g . 3. Activation of phosphorylase i n skeletal muscle by epinephrine, mediated by c y c l i c 3',5'-AMP. H 1 \J1 1 6 . h o m o g e n i z e d , t h e y c o n t a i n e d much l o w e r l i p a s e a c t i v i t y t h a n homogenates p r e p a r e d f r o m p a i r e d pads w h i c h h a d n o t been i n c u b a t e d . T h i s l o s s i n l i p a s e a c t i v i t y c o u l d be r e g a i n e d b y t h e a d d i t i o n o f e p i n e p h r i n e t o t h e i n c u b a t i o n medium p r i o r t o h o m o g e n i z a t i o n and c e n t r i f u g a t i o n . A c t i v i t y c o u l d be r e g a i n e d by t h e a d d i t i o n o f e p i n e p h r i n e to t h e s u c r o s e e x t r a c t a f t e r c e n t r i f l i g a t i o n o n l y i f t i s s u e s e d i m e n t f r o m a n u n i n c u b a t e d f a t p a d was a l s o a d d e d . T h i s i n d i c a t e d t h a t some f a c t o r p r o d u c e d by some s e d i m e n t a b l e p a r t i c u l a t e component o f t h e c e l l was n e c e s s a r y f o r e p i n e p h r i n e a c t i o n . F u r t h e r m o r e , he d e m o n s t r a t e d t h a t ATP c o u l d r e p l a c e t h e t i s s u e s e d i m e n t i n t h e a c t i v a t i n g s y s t e m . T h i s e v i d e n c e s t r o n g l y s u g g e s t e d t h a t e i t h e r c y c l i c J ' ^ ' - A M P o r a n u c l e o t i d e s i m i l a r to i t was i n v o l v e d i n t h e a c t i v a t i o n o f t h e l i p o l y t i c s y s t e m i n a d i p o s e t i s s u e . H o w e v e r , p r i o r t o t h i s , s e v e r a l i n v e s t i g a t o r s h a d shown t h a t c y c l i c 3 ' > 5 ' - A M P and o t h e r n u c l e o t i d e s had no e f f e c t on FFA r e l e a s e f r o m i n t a c t f a t pads ( 6 0 , 7 8 ) . D o l e ( 7 8 ) o b s e r v e d " that c y c l i c J ' , 5 ' - A M P a t c o n c e n t r a t i o n s i n t h e medium between 10~5 and 1 0 " ^ M g r e a t l y i n h i b i t e d t h e r e l e a s e o f FFA by i n t a c t f a t p a d s . S i m i l a r l y , - 5 he f o u n d t h a t A T P a t 1 x 10 ^ M e v e n more s t r o n g l y i n h i b i t e d t h e r e l e a s e o f F F A . I t i s n o t s u r p r i s i n g t h a t c y c l i c J ' , 5 ' - A M P and o t h e r n u c l e o t i d e s w o u l d f a i l t o i n c r e a s e F F A r e l e a s e f r o m i n t a c t a d i p o s e t i s s u e . T h e s e c h a r g e d m o l e c u l e s a r e known t o c r o s s c e l l b a r r i e r s w i t h g r e a t d i f f i c u l t y . I t seemed l o g i c a l t h a t i f c y c l i c J ' j ^ ' - A M P were i n v o l v e d , i t s f u n c t i o n c o u l d be more c l e a r l y a s c e r t a i n e d by t h e use o f homogenates a n d f r a c t i o n a t e d c e l l u l a r c o m p o n e n t s . When t h e p r e s e n t p r o j e c t was f o r m u l a t e d , R i z a c k ( 7 7 ) h a d a l r e a d y s u g g e s t e d ( a l t h o u g h w i t h o u t d a t a ) i n h i s f i r s t s t u d y o f t h e e p i n e p h r i n e s e n s i t i v e l i p a s e i n homogenates t h a t c y c l i c 5 ' » 5 ' - A M P may n o t 17-be the activating factor. Recently, several other J1,5*-cyclic nucleotides have become available, and i t was not i l l o g i c a l to consider that one of these or some other nucleotide may be implicated. A project was therefore formulated and undertaken i n this laboratory to examine the effects of epinephrine, ATP, cyclic 5 ' J 5 ' - A M P and other cyclic nucleotides on l i p o l y t i c activity of intact f a t pads, and on crude and fractionated homogenates prepared therefrom. It was hoped to learn something more of the factors involved i n the control of FFA production by adipose tissue. The purpose of this thesis i s to record the results to date. While this work was i n progress, Rizack presented further data on the epinephrine-sensitive lipase present i n adipose tissue homogenates (79). This recent data has indicated that cyclic 5 1 >5'-AMP may indeed mediate the activation of adipose tissue lipase. He demonstrated that when supernatant f l u i d from incubated f a t pads was pre-incubated for six minutes at 57° 0 with concentrations of ATP and cyclic 5',5'-AMP both at 2 x 10"^ M, Mg + + at 5 x 1CT 2 M, i n 0.25 M Tris buffer, pH 7 .4, the enzyme activity i n the supernate returned from Ijfo to 66% of it s original activity. The present report describes our studies regarding these problems of control of adipose tissue l i p o l y s i s . It may be pertinent to point out that this i s the f i r s t excursion into this aspect of l i p i d metabolism undertaken i n this laboratory. La some respects then, i t must be considered preliminary and does, i n fact, embody a number of preliminary and exploratory experiments. It i s hoped i t w i l l be a continuing study. 18. EXPERIMENTAL PROCEDURE Methoda. Epididymal f a t pads were obtained from normally fed Wiatar rats weighing between 275 and 525 g' Unless otherwise indicated, the rats were anaesthetized with 6 mg pentobarbital per 100 g body weight at least 50 minutes prior to sacrifice. The f a t pads were quickly removed and transferred to a freshly prepared Krebs-Ringer bicarbonate buffer, pH 7«4, at room temperature. The l e f t pad was bisected longitudinally and one of the pieces blotted, weighed on a torsion balance, and used to determine the i n i t i a l lipase activity. The other half was combined with the whole right pad, rinsed, blotted, weighed, and incubated for either 90 or 180 minutes at 57° G i n a solution of Krebs-Ringer bicarbonate buffer, pH 7-4, containing 5$ bovine albumin. At the end of the incubation period, the pads were assayed for lipase activity under different experimental conditions. Lipase Assays. Three different assay methods for the epinephrine-sensitive system were used i n the course of these studies. Assay I. This assay was performed according to Rizack (77)• Extracts of f a t pads were prepared by homogenizing the tissues i n 5 volumes of O.25 M sucrose i n a glass chamber with a Teflon pestle at 4° 0 for 50 to 60 seconds. The crude homogenate was centrifuged for 10 minutes at 12,000 x g at 0° C. The hard f a t cake which formed at the top was l i f t e d and pushed aside carefully with a metal spatula, and the supernatant f l u i d transferred to a chilled test tube using a Pasteur pipette. The pellet remaining at the bottom of the tube was discarded, along with the f a t cake. 19-Lipolytic activity was measured by placing 0.2 ml of the extract i n a glass-stoppered 20 x 1^ 0 mm tube, with 0.1 ml of a 1:4 dilution of a 50$ coconut o i l emulsion as substrate, 0.5 ml of 20$ extracted albumin solution, pH 6.8, 0.2 ml of 0.065M phosphate buffer at pH 6.8, and sufficient glass-d i s t i l l e d water to make a f i n a l volume of 2.0 ml. After 50 minutes' incubation at 57° 0» the reaction was stopped by the addition of 10.0 ml of an acidic extraction mixture of heptane and isopropanol, as described by Dole (l4) for the extraction of long chain fatty acids. Control mixtures were not incubated, but extracted immediately after the tissue extracts were added. For the extraction of FFA liberated, the tubes were placed i n a mechanical shaker and agitated vigorously f o r 10 minutes. 4 .0 ml of glass^ d i s t i l l e d water and 6.0 ml of heptane were then added, and the tubes shaken again for 10 minutes. Both blank and standard reference tubes were subjected to the same extraction procedure i n every assay. The blanks contained a total of 6 .0 ml of g l a s B - d i s t i l l e d water, 10.0 ml of the acidic organic extraction mixture, and 6 .0 ml of heptane. The standard reference tubes held 6 .0 ml of glass-distilled water, 10.0 ml of extraction mixture, and 6 .0 ml of heptane containing 156 microequivalents of palmitic acid per l i t r e . The system separated cleanly into two phases upon standing for 5 minutes, and two 5«° ™1 aliquots of the upper organic phase were transferred from each extraction tube to 15-ml conical centrifuge tubes, using 5-nO. volumetric pipettes. 1.0 ml of an ethanol solution of thymol blue was added to each J.Q wl aliquot, and titrations were performed with NaOH solutions of approximately O.O56 N concentration, using a Gilmont microburette ("Manostat Digi-pet"), capacity 0.1 ml. Nitrogen was delivered to the bottom of the tubes during t i t r a t i o n to expel C0 2 from 20. the sample and to keep the two phases well mixed. Good fluorescent lighting was essential i n order to determine the yellow-green endpoint accurately. The difference i n the fatty acid content of the non-incubated (control) mixture and the incubated mixture was taken as the l i p o l y t i c activity of the tissue extract, expressed i n milliunits, where 1.0 m i l l i u n i t : 1.0 millimicroequivalents of FFA released per minute per ml of supernatant f l u i d . Assay II. This assay was performed according to Vaughan's modification (80) of the method of Duncombe (82). Fat pads were homogenized for 50 to 60 seconds at room temperature, using 4 volumes of 0.154 KOI i n a glass chamber with a Teflon pestle. The assays were performed i n suitable glass-stoppered tubes i n a total volume of 1.0 ml containing 30 mg bovine serum albumin, pH 7«°» 20 micromoles of sodium phosphate buffer, pH 7-0, and 0.2 ml of the crude homogenate which had been f i l t e r e d through two layers of cheesecloth. The tubes were incubated at 37° 0 for either 10 or 15 minutes. The reaction was stopped at the desired time by the addition of 1.0 ml of a mixture containing 0.9 M triethanolamine, 0.1 N acetic acid, and 5$ cupric n i t r a t e • ^ 2 ° - The purpose of this treatment i s to convert the FFA formed to the chloroform-soluble copper soaps. Chloroform, 4.0 ml was added, the tubes placed horizontally i n a mechanical shaker and agitated vigorously for 30 minutes. After brief centrifugation, as much as possible of the upper aqueous phase and precipitated protein was removed by suction. Approximately % of the tubes did not yield a clear separation of the phases even after centrifugation. In such cases, a 21. glass rod was used to break up the thick, gelatinous protein precipitate i n the chloroform layer, and the tubes re-centrifuged. 2.0 ml aliquots of the chloroform layer were carefully transferred with a special long-tipped 2-ml pipette to appropriate test tubes containing 0.25 ^ 0.1% sodium diethyldithiocarbamate i n n-butanol. After mixing, the optical density was read at 440 millimicrons i n a Beckmann DU spectrophotometer, using a l i g h t path of 1.0 cm. The colour y i e l d was proportional to the various quantities of added palmitic acid tested; i.e., from 0 to J00 millimicroequivalents. In this assay, no exogenous substrate was added, since the homogenate i t s e l f provided sufficient triglycerides. Lipolytic activity, expressed as microequivalents of FFA produced per gram wet weight of tissue per 10 minutes, was taken as the difference i n the fatty acid content of the non-incubated mixture and the incubated mixture. Assay III. This assay waB both a combination and modification of the methods used i n Assays I and II. The incubation of the enzyme system was performed according to Assay I and the determination of FFA so formed was carried out using the cupric nitrate method of Assay'II. The reaction mixture contained 50 mg extracted bovine albumin, pH 6.8, 6 micromoles sodium phosphate buffer, pH 6.8, 0.1 ml of supernatant f l u i d , O.O5 ml.of a 1:4 dilution of a $0% coconut o i l emulsion (or O.I5 ml of inactivated adipose tissue homogenate), and sufficient glass-distilled water to make a f i n a l volume of 1.0 ml. Incubation of the mixture was performed at J7° 0 for 50 minutes, and the reaction stopped by the addition of 1.0 ml of the cupric nitrate solution as described for Assay II. Lipolytic activity i n 22. the supernatant f l u i d was expressed i n milliunits, as for Assay I; Materials. Bovine serum albumin (Fraction V, lot A2J B-70) was purchased from Sigma Chemical Company and purified according to the method of Goodman (81) to remove FFA. 5^ g °^ the crude albumin was dissolved i n 200 ml of glass-d i s t i l l e d water by simply placing the albumin powder over the water and allowing i t to dissolve overnight. The resultant dark amber solution was then lyophilized, powdered with mortar and pestle, covered with anhydrous 2,2,4-trimethylpentane containing 5$ acetic acid, and placed in the coldroom overnight. As much as possible of the acetic acid-trimethyl-pentane extraction solvent was then aspirated and the albumin washed twice with anhydrous trimethylpentane. Agitation of the albumin suspension during the extraction process with organic solvents was kept to a minimum to reduce the extent of protein denaturation. After aspiration of the trimethylpentane, the albumin was covered again with the anhydrous 5$ acetic acid-trimethylpentane mixture and stored i n the coldroom overnight. The removal of the acetic acid-trimethylpentane mixture and washing with anhydrous trimethylpentane was repeated. The organic solvent was removed under vacuum, and the powder obtained was taken up i n a suitable volume of glass-distilled water. To remove the last trace of acetic-acid, the albumin solution was dialyzed continuously for 5. days against a total volume of 60 l i t r e s of demineralized water, followed by 20 l i t r e s of glass-d i s t i l l e d water. The solution was then lyophilized and the extracted albumin stored i n the deep-freeze u n t i l required. The commercially prepared albumin was found to contain 0.60 eq FFA 25-per mole. After extraction by the method of Goodman (81) just described, the content of PPA was reduced to 0 . l4 eq/mole i n one of the extractions and to 0.18 eq/mole i n another. The extent of purification approached that of Goodman, who reduced the FFA concentration to 0.10 eq/mole. The recovery of albumin after extraction of FFA was approximately 90$. The 20$ albumin solutions used for the assays were prepared by sprinkling 10 g of fine l y powdered lyophilized albumin over 40 to 4^ ml of glass-distilled water i n a large Erlenmeyer flask, and allowing i t to dissolve overnight i n the coldroom. The pH of the solution, usually around 4.6, was adjusted to either pH 6.8, 7.0 or 7.4 with 5 N NaOH, and the solution made up to a f i n a l volume of 50 ml with glass-distilled water. Palmitic acid (Eastman Organic Chemicals) was re-crystallized twice before use as a reference standard. The 50$ coconut o i l emulsion was prepared by homogenizing equal parts by weight of coconut o i l and water, using B r i j 76 ® , a brand of polyoxy-ethylene fatty alcohol ether, as the emulsifying agent. Before use, the commercial coconut o i l was extracted with either chloroform : methanol (2sl) or ether and bicarbonate solution to reduce the amount of endogenous FFA. The FFA extraction mixture of Dole (l4) described for Assay I consisted of isopropanol, heptane and 1.0 N sulfuric acid i n the ratio 40:10:1 by volume respectively. Sodium diethyldithiocarbamate was re-crystallized twice before use. Its butanol solution was stored i n a refrigerator, and was kept no longer than one week due to the relative i n s t a b i l i t y of the reagent. The alkaline solution used irrthe titrimetric method of Assay I was 24. prepared daily be adding O.O5 ml of a saturated solution of NaOH to 25.0 ml of freshly boiled, gl a s s - d i s t i l l e d water. The thymol blue indicator solution was prepared as follows: 0.1 g thymol blue was dissolved i n 21.5 ml 0.01 N NaOH solution, then diluted to 25O ml with carbon dioxide-free water. 10.0 ml of this solution was further diluted to 100.0 ml with freshly d i s t i l l e d ethanol, and the pH adjusted so that 1.0 ml of the indicator required five to 10 microlitres of base for neutralization. ATP (disodium salt) was purchased from Nutritional Biochemical Corpo-ration. Cyclic J'j^'-AMP was also a commercial preparation. Cyclic 5»,5'-GMP, cyclic J'^'-CMP and cyclic 3',5'-UMP were available i n this laboratory (84). The deoxyribonucleoside-3 1 ,5 '-cyclic phosphates, namely cyclic ^ l ,^-deoxy-AMP, cyclic J1,5'-deoxy-GMP, cyclic 5* ,5'-deoxy-0MP, and cyclic 3',5'-TMP, were prepared by the method of Drummond et a l (85) and were donated by Dr. M. Smith. 25-RESULTS I. Preliminary Studies 1. Accuracy of the Titrimetric Method. The determination of FFA i n Assay I depends upon the t i t r a t i o n of FFA with a l k a l i using a microtitrator (See Experimental). Since the yellow-green endpoint was rather d i f f i c u l t to detect when t i t r a t i n g microquantities of FFA, a series of replicate tubes were titrated to determine the degree of accuracy of this method under the conditions of the assay. The subjective aspect of the method was minimized by t i t r a t i n g the f i n a l two or three microlitres with the metre-face covered. The O.O56 N NaOH used as the base was prepared under very rigi d conditions i n order to exclude CC^. Data obtained for a series of two experiments are shown i n Table 1. 2. Determination of FFA by the Copper Soap Method. The determination of FFA i n Assay II and Assay III depends upon the formation of the copper soap of FFA, extraction of these into chloroform, and subsequent reaction of the copper with diethyldithiocarbamate to form a coloured complex absorbing at 440 millimicrons. An experiment was performed to ensure that the colour yi e l d was proportional to FFA present. From 0 to $00 m i l l i -micro equivalent of palmitic acid was added to a series of reaction tubes and the extraction of the FFA performed according to Assay II. The tubes contained a l l components of Assay II except the homogenate. Optical density was read i n a Beckmann DU Spectrophotometer at 440 millimicrons, using a light path of 1.0 cm. Triplicate determinations yielded excellent agreement. Under the conditions of the assay, the colour yield was SERIES A SERIES B Tube Aliquot Microlitres of base req'd Tube Aliquot Microlitres of base req*d 1 (a) (b) 15.85 15.45 1 (a) (b) 8.85 8.92 2 (a) (b) 15.14 15.32 2 (a) (b) 8.46 8.82 3 (a) (b) 15.10 15.20 3 (a) (b) 8.50 8.43 4 (a) (b) 15.17 15.30 4 ( a ) (b) 8.57 8.63 5 (a) (b) 15.47 15.43 5 (a) (b) 8.41 8.74 Mean = 15.34 Deviation = 0.51 Standard Deviation = 0.25 Standard Error of Mean = 0.08 Mean =8.63 Deviation = 0.29 Standard Deviation = 0.20 Standard Error of Mean = 0.06 Table 1. Accuracy of the titrimetric method. In series A, five experimental tubes, each containing a l l the components (except the super-nate) used in Assay I, were extracted and titrated. In series B, five reference standard tubes, each containing 936 millimicroequivalents of palmitic acid only, were extracted and titrated. 27-directly proportional to the concentration of palmitic acid added, as indicated i n Pig. 3a. 3. Effect of ME** on the Recovery of Palmitic Acid by the Popper  Method of Assay II. The determination of PFA concentration by Assay II (and III) depends on the formation of cupric salts of long chain fatty acids and subsequent extraction with chloroform. A strong possibility existed that high concentrations of Mg*"+ i n the reaction mixture might interfere with the formation of cupric salts of long chain fatty acids. Hence a series of tubes containing 200 millimicroequivalents of palmitic acid and Mg*1- concentrations ranging from 0 to 5 x 10 M was extracted and assayed according to Assay II. The data obtained (Table 2) indicated that Mg^ had l i t t l e , i f any, effect on the extraction of palmitic acid when the copper soap method was used for the determination of long chain PPA. 4. Stability of the Diethyldithiocarbamate-copper Complex. During the course of these experiments, i t was frequently noted that when the optical density of the chloroform solutions were re-read after 15 to 50 minutes, the readings gave slightly higher values. Therefore, after one of the experiments, the solutions were re-read in the same sequence every 20 minutes and the optical densities recorded as indicated i n Table 5- The changes i n optical density with each subsequent reading showed an increase of .004 to .012 O.D. units, usually around .008 units. However, since the rate of change was constant for a l l the tubes, and furthermore, since only the difference i n O.D. between the zero time control and the experimental tubes was of any importance, it.was concluded that the observed O.D. changes F i g . P a l m i t i c a c i d s t a n d a r d c u r v e . 100, 200, a n d J00 m i l l i m i c r o e q u i v a l e n t s o f t w i c e r e - c r y s t a l l i z e d p a l m i t i c a c i d , d i s s o l v e d i n c h l o r o f o r m , were p i p e t t e d i n t o g l a s s - s t o p p e r e d t u b e s . The c h l o r o f o r m was e v a p o r a t e d t o d r y n e s s , a l b u m i n s o l u t i o n was a d d e d , and t h e t u b e s h e a t e d t o 5O 0 0 a n d s w i r l e d u n t i l no t r a c e o f t h e p a l m i t i c a c i d r e s i d u e was v i s i b l e . P h o s p h a t e b u f f e r and w a t e r were a d d e d a s d e s c r i b e d i n A s s a y I I b e f o r e e x t r a c t i o n was p e r f o r m e d . Mg++ Concentration in Reaction Mixture Optical Density at 440 millimicrons (d = 1.0 cm) None .570 - .010 2.0 x 10"5 M .529 - .001 5.0 x 10"3 M .574 - .001 _2 + 5.0 x 10 M .553 - .007 Table 2. Effect of Mg++ on the extraction of added palmitic acid, using Assay II. Each tube contained 200 millimicroequivalents of palmitic acid, 30 mg bovine serum albumin, phosphate buffer, 6.0 x 10 M in a final volume of 1.0 ml. The experiment was performed in duplicate, and excellent agreements of values were obtained. Tube No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 O.D. at 10 min. 266 381 255 383 280 341 308 355 249 293 260 297 251 285 252 293 258 O.D. at 30 min. 276 390 262 392 288 352 318 364 258 300 265 306 258 294 260 302 266 A O.D. 010 009 007 009 008 Oil 010 009 . 009 007 005 009 007 009 008 009 008 O.D. at 50 min. 285 402 269 400 297 357 325 371 265 306 274 310 266 302 267 312 270 A O.D. 009 012 007 008 009 006 007 007 007 006 009 004 008 008 007 010 004 O.D. at 70 min. 292 412 276 408 303 365 233 380 272 313 280 320 272 308 273 318 277 A O.D. 007 010 007 008 006 008 008 009 007 007 006 010 006 006 006 006 007 O.D. at 90 min. 300 A O.D. 008 Table 3. Observed changes in optical density on subsequent readings when EPA concentration was determined by the copper technique of Assay II. Optical densities were read 10 minutes after mixing 2.0 ml chloroform extract with 0.25 ml diethydithiocarbamate, then re-read in the same sequence at 20 minute intervals. 51-were of l i t t l e consequence insofar as the accuracy in the assay for FFA was concerned. A most l i k e l y explanation f o r these observed changes i n optical density is that during and after the process of transferring the coloured chloroform solutions between the tubes and cuvette, sufficient chloroform evaporates to cause a slight increase i n the concentration of the diethyldithiocarbamate-copper complex. 5« Time Dependence of Epinephrine-sensitive Lipase. At the outset i t was necessary to determine the effect of time of incubation on l i p o l y t i c activity i n homogenates in order to ensure a valid assay. Fat pads were homogenized i n 5 volumes of O.25 M sucrose at 4° 0 and centrifuged at 12,000 x g for 10 minutes at 0° 0. 0.2 ml of the supernate was incubated at 57° 0 with coconut o i l emulsion as substrate, and albumin and phosphate buffer as described for Assay I. Reactions were stopped every 10 minutes by the addition of 10.0 ml of acidified organic extraction mixture. The results of two such experiments are shown i n Fig. 4. In contrast to Rizack (77), who i n i t i a l l y incubated his. reaction mixture for 60 minutes, i t was immediately apparent that the reaction approached the upper limits of linearity at 60 minutes' incubation only when l i p o l y t i c activity was low. At elevated levels of activity, the reaction proceeded i n a linear manner for about 40 minutes only. Therefore, our reaction mixtures were incubated i n Assays I and III for only JO minutes. I t was noted that Rizack, i n his later studies (79), also reduced his incubation time from 60 to JO minutes, thus adding support to our observations. 6. Lipids Extracted from Adipose Tissue as Substrate for the Lipolytic  Enzyme System. Reference has already been made to the wide variety of 52. 0 /S  0 20 40 60 80 I N C U B A T I O N T I M E ( M i n . ) F i g . 4. T i m e c o u r s e c u r v e s f o r t h e e p i n e p h r i n e - s e n s i t i v e l i p a s e . Two e x p e r i m e n t s were p e r f o r m e d , u s i n g t h e s u p e r n a t a n t f r a c t i o n o f a d i p o s e t i s s u e homogenates o b t a i n e d f r o m two d i f f e r e n t r a t s . A s s a y I was u s e d t o d e t e r m i n e FFA p r o d u c e d . 55-methods, tissue preparations, and substrates used by various investigators i n this f i e l d . Selecting the most suitable substrate i n particular has been a major problem. Most investigators use a commercial coconut o i l emulsion, Ediol® . Rubenstein et a l (88) used lipids extracted from fat pads as substrate. We also attempted to employ this more "physiological" material i n our work. A total of 15«57 g of epididymal adipose tissue was obtained from 4 rats weighing between 460 and 490 grams and homogenized i n a Servall Omnimixer using 17 volumes of chloroform t methanol (2:1) mixture as described for the extraction of l i p i d s by Folch et al (87). An additional 5 volumes of the chloroform t methanol mixture was used to rinse out the cup and flask. After f i l t r a t i o n through fat-free paper into a separatory funnel, the organic solvent was shaken with 0.2 volumes of glass-distilled water. The lower organic phase was collected and the solvent removed under vacuum i n a rotary evaporator. The clear, yellow, viscous f l u i d obtained was subjected to further treatment under a mechanical vacuum pump to remove the last traces of organic solvent i n the l i p i d extract. The yield of o i l obtained was 75$. A 20$ emulsion was then prepared by homogenizing 1.0 ml of the o i l with 4.0 ml of glass-distilled water, using 0.1 g of Bri j 76® as the emulsifying agent. The emulsion was diluted with an equal volume of water before being used as substrate. Preliminary experiments indicated that v i r t u a l l y no l i p o l y t i c activity was evident with this preparation. Despite its great potential as a substrate f o r adipose tissue lipase, no further attempt was made to improve the quality of this substrate, i n view of the limited time available. 5*. 1- Heated Crude Homogenate of Adipose Tissue as Substrate. Since our preliminary attempt to use lipid s extracted from f a t pads as substrate f a i l e d to give adequate reaction rates, an attempt was made to u t i l i z e the triglycerides available i n a homogenate of f a t pads without extraction with organic solvents. In this case, enzymatic activity in the homogenate was destroyed by heat. Epididymal f a t pads were homogenized i n 5 volumes of 0.25 M sucrose at room temperature, then immediately f i l t e r e d through two layers of cheesecloth into a test tube immersed i n a boiling water bath. After the heated extract was re-homogenized i n a Potter-Elvehjem homogenizer, a rather smooth, creamy preparation resulted. Preliminary experiments using this simple, natural substrate yielded highly encouraging results. Although l i p o l y t i c activities observed were about equal to that obtained with coconut o i l emulsion, the major advantage over the latter a r t i f i c i a l substrate was that the preparation, i f Immediately deactivated by heat, gave much lower blanks. In fact, the level of endogenous FFA found in this substrate was negligible. An experiment was performed to determine the minimum volume of this-substrate required for the l i p o l y t i c system under study, and the results are shown i n Fig. 5. The substrate concentration curve indicated that O.15 ml of the heated crude homogenate was required i n order to saturate the enzyme. Although this material shows considerable promise as a substrate, i t was not widely used i n the present studies. ~!00 .02 .04 .06 .08 .10 -12 .14 .16 .18 S U B S T R A T E ( m l ) F i g . 5- S u b s t r a t e c o n c e n t r a t i o n c u r v e , u s i n g t r i g l y c e r i d e s p r e s e n t i n a b o i l e d e x t r a c t as s u b s t r a t e . (See t e x t f o r p r e p a r a t i v e p r o c e d u r e ) . I n c u b a t i o n was p e r f o r m e d a t 37° 0 f o r J O m i n u t e s , u s i n g A s s a y I I I . 56. II. Experimental Results 1. Response of Epididymal Fat Pads to Epinephrine. The dramatic response of adipose tissue to epinephrine Is most readily demonstrated when fa t pads are incubated for a few minutes with this drug. In a series of experiments with adipose tissues obtained from normal fed 200 g rats, the addition of epinephrine to the incubation medium caused a marked increase i n the release of FFA into the medium, presumably by stimulating the hormone-sensitive l i p o l y t i c system (Table 4). When epinephrine was injected into rats before removal of f a t pads, the response was approximately 1/6 that obtained when epinephrine was added directly to the intact pad. Untreated control pads showed a high degree of variation in their a b i l i t y to release or remove FFA from the medium. For instance, adipose tissues from rats 1 and 2 (Table 4) indicated levels of l i p o l y t i c activity equivalent to those from epinephrine-injected rats, whereas adipose tissues from the remaining controls suggested an uptake of FFA from the medium. The extreme variation found i n these control tissues might be explained on the basis of later experiences; that i s , the handling of rats just prior to sacrifice probably causes a high degree of sympathetic stimulation with the result that endogenous catecholamine levels are increased. This, i n turn, w i l l cause a marked increase i n l i p o l y t i c activity, as probably was the c a B e of adipose tissues obtained from rats 1 and 2. These results are typical of many available i n the literature and serve here to emphasize the marked a b i l i t y of epinephrine to mediate the release of FFA from intact adipose tissue. Clearly the nature of such action can only be completely understood by a study of events at a subcellular level. 57. Rat Untreated Epinephrine Injected Epinephrine Added to Medium 1 +1040 2 + 630 3 - 760 4 - 20 5 - 730 6 - 140 7 +490 8 +870 9 +4270 10 +4490 Table 4. Effect of epinephrine on net release or uptake of EPA by terminal sections of rat epididymal fat pads. Epinephrine, 25 micrograms/kg, was injected into rats No. 7 and 8 approximately 5 minutes prior to removal of fat pads. Pads from rats No. 9 and 10 were incubated in a medium containing 2 micrograms epinephrine per ml. Incubations were performed under 95$ 02/5?6 C0 2 in a Dubnoff metabolic shaker for 1 hour at 37° C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 5$ extracted bovine serum albumin. 2.0 ml aliquot of the medium were removed for the assay of EPA by the extraction and titrimetric methods of Dole (14) as described in Assay I. The activities are expressed as millimicroequivalents PPA/g wet weight/hr. Either the l e f t or the right pad served as zero time control for the other side in each experiment. (-) indicates EPA uptake by tissue and (+) indicates EPA released. 38. 2. Epinephrine Effects on Crude Homogenate8 of Adipose Tissues. As indicated i n the foregoing section, the l i p o l y t i c effect of epinephrine on intact f a t pads is a particularly outstanding feature of this hormone's action. This is presumably due to increased activity of adipose tissue lipase. It might be reasonable to assume that epinephrine could have a similar effect i n homogenates assayed directly for lipase activity. Thus f a t pads were removed and incubated as previously described to reduce the activity of the l i p o l y t i c system. Thereafter they were homogenized and assayed for lipase activity with and without epinephrine added to the homogenate. Another portion of the f a t pad was homogenized without incubation to serve as the pre-incubation control. In the presence of epinephrine, only slight activation of the l i p o l y t i c system was observed (Fig. 6 ) . At higher, and admittedly even less physiological concentrations of epinephrine, the a c t i v i t i e s increased only sl i g h t l y . This relatively small increase i n lipase activity observed with epinephrine on crude homogenates has also been documented elsewhere (88). I t i s pertinent to note that the substrate used i n this experiment was the natural triglycerides present i n an unfraction-ated homogenate. The results indicate that although epinephrine causes a marked release of FFA from intact f a t pads, i t does not cause activation of lipase present i n an unfractionated homogenate. 3 ' Effect of Epinephrine on Lipolytic Activities of Adipose Tissues  When Added Before, During and After Homogenization. The relatively small effect of epinephrine on l i p a B e a c t i v i t y observed when i t is added to crude homogenates contrasted so sharply with the large effects observed when epinephrine was added i o a medium containing Intact fat pads that further probing into the matter was clearly necessary. Hence fat pads which had been 59-P r e -incub. P o s t -incub. 5/ig/ml IS^g/ml 30ug/m| E P I N E P H R I N E A D D E D F i g . 6. E f f e c t of epinephrine on l i p o l y t i c a c t i v i t y when added to crude homogenate of adipose t i s s u e . Epinephrine was added (5 to JO microgram/ml reaction mixture) and l i p o l y t i c a c t i v i t y determined according to Assay II. Results were averaged from duplicate assays using homogenate obtained from one r a t . One determination was made f o r the post-incubation a c t i v i t y . A c t i v i t y i s expressed as microequivalents FFA produced/g/lO minutes. 40. incubated for 3 hours were assayed f o r lipase activity by (a) pre-treating the pads with epinephrine 10 minutes before homogenizing, (b) homogenizing the pads with epinephrine, and (c) adding epinephrine after homogenizing was complete. The averaged results of experiments with 4 rats are shown i n Fig. 7-The average l i p o l y t i c activity after the 3-hour incubation was approxi-mately JOfo of the original activity indicating the degree of loss of lipase activity. When epinephrine was added directly to the intact pad prior to homogenizing, a large increase i n lipase activity was observed i n the assay. Epinephrine added during homogenization caused only a slight increase i n activity, and when added after homogenization, no effect on lipase was obtained. Again, i n this experiment, the lipase assay employed the triglycerides present i n the unfractionated homogenate. The data indicate that epinephrine can activate the lipase only when the tissue i s intact. I t might be reasoned that during homogenization or assay by the technique used, some necessary factor i s either destroyed or diluted out, or that epinephrine i t s e l f may be destroyed. 4. Search for Effect of Cyclic 5',5'-AMP, ATP and Mg+* on Lipase  Activity of TJnfractionated Homogenates. The data previously recorded indicated that epinephrine, when added to crude homogenates, was unable to increase lipase activity. If cyclic 5',5'-AMP were involved in epinephrine-induced l i p o l y s i s , homogenization may have destroyed some factor or disrupted some cellular component necessary for i t s formation. It is less l i k e l y that epinephrine i t s e l f would have been destroyed, but i f this occurred, the conversion of endogenous ATP to cyclic 5,»5,-AMP by adenyl cyclase would D B F i g . 7- E f f e c t of epinephrine on l i p o l y t i c a c t i v i t y i n 5-hour incubated pads when added before, during and a f t e r homogenization. Bars "A" and "B" represent i n i t i a l and poat -5"h°ur inoubation l i p o l y t i o a c t i v i t i e s r e s p e c t i v e l y . Epinephrine (medium concentration 17 microgram/ml) was added to the medium containing pad " 0 " 10 minutes before homogen-i z i n g . The 0 . 2 5 M sucrose homogenizing s o l u t i o n used f o r pad "D" contained epinephrine ( 1 7 micrograms/ml). "E" represents l i p o l y t i c a c t i v i t y when epinephrine (17 micro-graas/ml) was added to the reaction mixture. L i p o l y t i c a c t i v i t y , expressed as microequivalents FFA produced/g/10 minutes, was determined according to Assay I I . Data shown were averaged from experiments on 4 animals. 42. not occur. The addition of cyclic 3',5'-AMP and ATP directly to the tissue homogenate was clearly indicated. A series of experiments was^ therefore f undertaken to determine whether these nucleotides had any effect on lipase activity i n crude homogenates, and.. i f so, to determine the concentrations of each which gave maximal activation. A preliminary experiment was performed in which the concentrations of both ATP and cyclic 5',5'-AMP were increased from J . 2 x 10"? M to 1.0 x 10"5 M in the reaction mixture, while Mgt+ concentrations were varied from 1.6.x 10~5 to 5.0 x 10~2 M as indicated i n Fig. 8. Lipolytic activity was again determined according to Assay II and i s expressed as microequivalents of FFA produced per gram wet weight of tissue per 10 minutes. The results indicate that supplementation of the extract with ATP and cyclic J'^'-AMP in the range of 1.0 x 10"^ M and 1.0 x lO""^ " M may have some a b i l i t y to increase lipase activity. At these concentrations, the l i p o l y t i c activity was increased from 2.6 to J.2 microequivalents/g/lO minutes. The degree of a c t i -vation nevertheless fa i l e d to attain the i n i t i a l lipase activity of 3.9 microequivalent/g/lO minutes. At higher concentrations of nucleotides and Mg*"% definite inhibition of lipase activity occurred. (a) Effect of Varying the Concentration of ATP. The concentrations of cyclic 3' ,5'-AMP, ATP and Mg*;1" used i n the preceding preliminary study indicated that cyclic 5 'J 5 , -AMP might indeed mediate a lipase-activating system at least to a small degree. Since the concentrations of the components used i n the preliminary study were chosen arb i t r a r i l y , a systematic search for the optimal concentrations of each component was considered worthwhile i n order to settle the matter. Thus, in a series of assays, the concentration 3-2x«0~7|-6xlO"6 8-OxlO"6 i-6x!0"5 8-0xl0"5 4-0xl0~4 4-OxiO-5 2-0xf0"4 1-OxlO"3 2-OxlO"3 l-OxlO'2 5-OxlO"2 (Upper L i ne=ATP, C y c l i c 3'5 -AMP concent ra t ions) (Lower Line = Mg*"'" concentrat ion) Pig.. 8. E f f e c t of various concentrations of ATP, c y c l i c 5',5'-AMP, and Mg++ on l i p o l y t i c a c t i v i t y , determined according to Assay I I . Concentrations of nucleotides and Mg++ were varied as indicated, and represent the f i n a l concentration i n the 1.0 ml re a c t i o n mixture. The i n i t i a l and post-2-hour incubation a c t i v i t i e s were 5.9 and 2.6 microequivalents/g/10 minutes r e s p e c t i v e l y . 44. of one of the three components was varied i n turn, while the concentrations of the other two components were held constant. F i r s t , ATP concentration I —8 —4 was varied from 6.4 x 10 M to 2.0 x 10 M in the reaction.mixture, while the concentration of cyclic 51,5'-AMP and Mg++ were held constant at 8.0 x 10"^ M and 4.0 x 10""^  M respectively. The result of an experiment under these conditions i s shown i n Fig. 9> and indicates that the optimal concentration of ATP was around $.2 x 10~7 M. At these concentrations of ATP, cyclic J'^'-AMP and Mg++ , lipase activity increased from 2.9 to 5.4 microequivalent/g/lO minutes, but again failed to reach the i n i t i a l lipase activity of 6.4 microequivalent/g/10 minutes. Again, i t i s pertinent to emphasize that the natural triglyceride present i n the homogenate was the substrate used (Assay II). (b) Effect of Varying the Ooncentration of Cyclic 5' >5'-AMP. When the concentration of cyclic 5'>5'~AMP was varied from 12.8 x 10"^ M to 1.0 x 10~5 M, while the concentrations of ATP and Mg"1^were held constant at 5.2 x lO"^ M and 4.0 x 10"*^  M respectively, the optimum activation occurred at cyclic Jl,^-AMP concentration of %2 x 10 -? M (Fig. 10). It was interesting to note that equimolar concentrations of both ATP and cyclic 5',5'-AMP were apparently required. The experiment was repeated with adipose tissue homogenates obtained from another rat, and the resulting concentration curve was vi r t u a l l y identical i n form to the f i r s t , except that the level of lipase a c t i v i t i e s i n the two homogenates differed (Fig. 10). In one of the experiments, lipase activity was increased from 5»7 to 4.4 microequivalents/ g/10 minutes, but again f a i l e d to attain the i n i t i a l activity of 5.6 mioro-equivalents/g/10 minutes. In the other experiment, l i p o l y t i c activity was 6-4xlCf 8 3'2xl0" 7 )-6x I0" 6 8-OxlO""6 4-0x !0~5 2-0 x 10"" A T P \> F i g . 9- E f f e c t of various concentrations of ATP on l i p o l y t i c a c t i v i t y . The f i n a l ATP concentrations i n the 1 . 0 ml incubation mixture were varied from 6.4 x 10 M to 2 . 0 x 10 M. C y c l i c 3',51-AMP and .Mg44- concentrations were held constant a t 8 . 0 x 1 0 " ° M and 4 . 0 x 1 0 ~ 4 M re s p e c t i v e l y . The i n i t i a l and post - 9 0-minute incubation a c t i v i t i e s were 6.4 and 2 - 9 microequivalents/g/lO minutes r e s p e c t i v e l y . Lipase a c t i v i t y was determined according to Assay I I . 46. - 5 - 0 -3'0i 1 1 1 1 I : I I I-I28xl0~9 6-4XICT8 3-2 xlO"7 l-6xl0"6 8-OxlO"6 4-0xl0"5 20xl0" 4 1-0 xlO' CYCLIC 3 ' 5 - A M P F i g . 1 0 . E f f e c t of various concentrations of c y c l i c 5',5'-AMP on l i p a s e a c t i v i t y . The f i n a l concentrations of c y c l i c J'jJ'-AMP were v a r i e d from 1 2 . 8 x l O - ? M to 1 . 0 x 10~5 M i n the 1 . 0 ml incubation mixture. The concentrations of ATP and Kg"*-1" were held constant at J.2 x 1 0 ~ 7 M and 4 . 0 x 10 M r e s p e c t i v e l y . The i n i t i a l and post -90-minute incubation a c t i v i t i e s were ^.6 and p.7 microequivalents/g/ 1 0 minutes f o r experiment 1 (lower curve, c i r c l e s ) , and 5 - 7 and 4 . J mi.croequivalents/g/lO minutes f o r experiment 2 (upper curve, t r i a n g l e s ) r e s p e c t i v e l y . Lipase a c t i v i t y was determined according to Assay I I . 1 47. increased from 4.5 to 4.9 microequivalents/g/lO minutes when the i n i t i a l lipase activity had been 5«7 microequivalents/g/10 minutes. (c) Effect of Mgr-*" at Various Concentrations. The results obtained so far indicated that the optimum concentrations of ATP and cyclic 5',5'-AMP were 5«7 x: 10"^  M; Therefore, i n another experiment, this particular concentration of the nucleotides was used, while the Mg + + concentration was varied from 9.6 x 1QT7 M to 7«5 x 1°~ 2 M (Fig. 11). A marked inhibition of lipase activity in the homogenate was evident when the Mg"*"*" concentration exceeded J.O x 10~5 M. On the other hand, l i t t l e activation of the l i p o l y t i c system was observed at lower Mg"M' concentrations, despite the presence of what were considered optimal concentrations of ATP and cyclic 5',5'-AMP. The experiment was repeated with a homogenate obtained from another rat, and an identical concentration curve was obtained (Fig. 11). The possibility that Mg++ at high concentrations may interfere with the copper method of Assay II was eliminated (see Preliminary Results), hence the inhibition of lipase activity i n adipose tissue homogenates by high Mg"r"t" concentrations must be considered real. The limited time available prevented a more thorough investigation into the effects of ATP, cyclic 5',5'-AMP and Mg + + on li p o l y t i c activity i n crude homogenates. These preliminary experiments appear to indicate that ATP and cyclic 5'>5,-AMP c a n activate the l i p o l y t i c system i n homogenates to a limited extent, but we were unable to raise the levels of activity in any experiment to that of the unincubated controls. If cyclic 5',5'-AMP is involved, optimal conditions have not yet been achieved. It might be mentioned, however, that increase i n activity obtained was greater than that achieved by adding epinephrine to the homogenates. 48. £ g CT a o o-o 9 - 6 x l 0 " 7 4 - 8 x l 0 ~ 6 2 4 x l 0 ~ 5 l - 2 x ! 0 " 4 6 0 x l 0 " 4 3 0 x l 0 " 3 l-5x I0" 2 7 5 x I 0 " 2 Mg F i g . 1 1 . E f f e c t s of various concentrations of Mg"*"* on l i p a s e a c t i v i t y i n unfractionated adipose tissue homogenates. The concentration of Mg44" was va r i e d from 9 - 6 x 1 0 " ' M to 7 . 5 x 1 0 ~ 2 M i n the r e a c t i o n mixture. C y c l i c 5',5'-AMP and ATP concentrations were held constant a t J . 2 x 10""7 M. The i n i t i a l and p03t - 9 0-minute incubation a c t i v i t i e s were 4 . 6 and 2 - 5 microequivalents/g/lO minutes f o r experiment 1 (upper curve, c i r c l e s ) and 4 . 6 and 2 . 7 microequvalents/g/lO minutes f o r experiment 2 (lower curve, t r i a n g l e s ) r e s p e c t i v e l y . Lipase a c t i v i t y was measured according to Assay I I . 49-5' Study of Lipolytic Activity i n Supernatant Fluid of Adipose Tissue  Homogenates. A l l the experiments described so far u t i l i z e d unfractionated homogenates in which the endogenous triglycerides served as the substrate (Assay II). I t was during the course of these experiments that Rizack 1s paper (79) appeared, i n which he showed that cyclic 5'>5'-AMP caused an activation of the lipase present in the supernatant obtained by centrifu-gation of an adipose tissue homogenate at low velocity. In his studies, commercial coconut o i l emulsion, Ediol® , served as the substrate. The lipase system described by Rizack (79) was then studied to determine whether i t responded i n our hands to cyclic 5 'J5 , ~AMP better than the unfractionated homogenates. Fat pads were pre-incubated as necessary, homogenized and centrifuged at 12,000 x g for 10 minutes at 0° 0. The supernatant solution was removed and served as the source of enzyme (see Experimental). Our preliminary studies involved the titrimetric method (Assay I) with coconut o i l emulsion as substrate, and the spectrophotometric method (Assay III) i n which coconut o i l emulsion again served as the substrate. The results of two such experiments are shown in Fig. 12. They appear to indicate that f u l l l i p o l y t i c activity was restored when the supernatant f l u i d was pre-incubated with ATP, cyclic 5 ' ,5 1 -AMP and Mg + +. However, i t must be emphasized at this point that the activation of the enzyme system under these conditions was not always reproducible. A summary of the effects of these components on the activation of the l i p o l y t i c enzyme system i n the supernatant fraction i s presented i n Table 5* It is evident that the degree of activation varies considerably from one experiment to another. Significant activation was obtained i n 6 of 11 experiments, but l i t t l e or no activation was obtained i n the remaining 5 experiments. Experiment "a" A B Exper iment "b" F i g . 1 2 . A c t i v a t i o n of the epinephrine-sensitive l i p a s e i n the supernatant f r a c t i o n of adipose tissue with c y c l i c 3',5'-AMP. Bars "A" and "B" represent i n i t i a l and post-incubation l i p a s e a c t i v i t i e s r e s p ectively. Bar " 0 " represents a c t i v i t y observed when the supemate obtained from the incubated f a t pad was pre-incubated at 3 7 ° 0 f o r 6 minutes with f i n a l concentrations of c y c l i c J 1 ,5 '-AMP and ATP, both a t 2 . 0 x 10~5 M, and Mg++ a t 5 . 0 x 1 0 ~ 2 M. In add i t i o n , T r i s b u f f e r O .25 M f i n a l concentration, pH l . k , was used i n experiment "a" (Assay I) and phosphate buffer, 6 . 0 x 10~5 M, pH 7 . 4 , was used i n experiment "b" (Assay I I I ) . The data represents r e s u l t s from experiments on adipose t i s s u e s from two d i f f e r e n t r a t s , both performed i n duplicate. 51-Experiment No. Lipolytic Activity (milliunits) Supernatant A Supernatant B Supernatant B + Cyclic 31,5*-AMP Activation 1 36 26 36 + 2 62 36 47 + 3 51 28 28 0 4 60 18 42 + 5 48 20 15 0 6 31 22 31 + 7 46 35 38 0 8 50 34 40 + 9 41 11 13 0 10 31 5 11 + 11 59 20 18 0 Table 5. Summary of the effects of ATP and cyclic 3',5'-AMP at 2.0 x —5 ++ —2 10 M, and Mg at 5.0 x 10 M. Experiments 1 to 5 were performed according to Assay I, and experiments 6 to 11 according to Assay III. Refer to legend, Fig. 12, for details of activation method. Supernatants "A" and "B" were obtained from non-incubated and incubated pads respectively. 52. Although our procedure used i n these experiments is closely similar to that of Rizack (79)> our results are considerably less impressive than his. Our data indicate that cyclic 5',5,~AMP can activate the lipase present i n the supernatant f l u i d obtained from centrifuged homogenates of adipose tissue. It does so i n an inconsistent mannerj some extracts were restored to f u l l activity, some part i a l l y restored, and some were unaffected by the nucleotides. Rizack, however, makes no mention as to how consistent his results were or whether his data represent the mean of a series of experiments. We feel that more work i s warranted before definite conclusions can be drawn regarding the role of cyclic 5 ,»5I-AMP. Perhaps we have fai l e d to achieve the precise conditions necessary for activation. 6. Possible Effect of Other Cyclic Nucleotides on Lipase from the  Supernatant Fluid., A number of other cyclic nucleotides were tested at concentrations of 2.0 x 10"^ M and 2.0 x i 0 ~ ^ M. ATP and Mg^ concentrations —5 -2 were held at 2.0 x 10 ' M and 5«0 x 10 M respectively. The results are presented i n Table 6. Cyclic 5',5,-G-MP and cyclic 5'^'-deoxy-CMP at both concentrations, and cyclic 5'>5'-TMP at 2.0 x 10"^ M appeared to cause some inhibition of l i p o l y t i c activity. Very slight increases i n l i p o l y t i c a c t i v i t i e s may have occurred with cyclic 5'»5,~^eoxy-AMP at both concentra-tions, with cyclic 5 1>5 * - & e o x y - ( M P at 2.0 x 10"^ M, and also with cyclic 5 ,,5,-UMP at 2.0 x 10~5 M. Generally, most of the nucleotides tested caused decreased lipase a c t i v i t i e s at the higher of the two concentrations used, namely 2.0 x 10 M. Activities Supernatant A Activities Supernatant B Cyclic Nucleotides Final Concentrations of Nucleotides Activities 39 33 3' ,5'-GMP 2 x 10"^ M 2 x 10 M 23 24 50 34 3",5'-d-AMP 2 x lO"5^ M 2 x 10~4 M 39 38 3',5»-d-GMP ! 2 x 10~Jj M 2 x 10 M 14 11 41 11 3',5«-d-CMP 2 x 10~Ji 2 x 10~11 6 8 3',5»-AMP 2 x 10"5M 11 31 5 3',5,-QSP 2 x 10~JM 2 x lOTH 7 4 3',5'-UMP 2 x 10~jj M 2 x 10 M 9 6 59 20 3',5'-TMP 2 x 10~5 M 17 Table 6. Effect of various cyclic nucleotides on lipolytic activity in the supernatant f l u i d obtained from incubated fat pads. The supernatant flu i d was pre-incubated for 6 minutes with the nucleotides indicated, plus ATP 2.0 x 10"5 M and Mg*"4" 5.0 x 10 - 2 M in phosphate buffer 6.0 x 10~3 M at pH 7.4. Lipase activity was determined by Assay III and is expressed in milliunits. 54. A d d i t i o n a l E x p e r i m e n t s on A d i p o s e T i s s u e L i p a s e 1. E f f e c t o f E p i n e p h r i n e on A d i p o s e T i s s u e O b t a i n e d f r o m R e s e r p i n i z e d  R a t s . T h r o u g h o u t o u r w o r k , we have o b s e r v e d v a r y i n g d e g r e e s o f i n a c t i v a t i o n o f l i p a s e u p o n i n c u b a t i o n o f f a t pads i n K r e b s - R i n g e r medium. O t h e r i n v e s t i g a t o r s h a v e o b s e r v e d t h i s a l s o . We have c o n s i d e r e d t h i s may be due t o t r a u m a o r e x c i t e m e n t i n d u c e d u p o n t h e a n i m a l p r i o r t o s a c r i f i c e , r e s u l t i n g i n t h e endogenous r e l e a s e o f c a t e c h o l a m i n e s . I t i s common k n o w l e d g e t h a t r e s e r p i n e d e p l e t e s t h e s t o r a g e s i t e s o f endogenous c a t e c h o l a m i n e s i n v a r i o u s t i s s u e s . I t was c o n s i d e r e d o f i n t e r e s t t o examine t h e r a t e o f i n a c t i v a t i o n o f a d i p o s e t i s s u e l i p a s e f r o m f a t pads o f r e s e r p i n i z e d a n i m a l s , and t o d e t e r m i n e t h e e f f e c t o f e p i n e p h r i n e when added t o t h e medium c o n t a i n i n g i n t a c t f a t pads : w h i c h h a d b e e n so d e p l e t e d o f endogenous c a t e c h o l a m i n e s . T h r e e r a t s w e i g h i n g J00 g each g i v e n s u b c u t a n e o u s i n j e c t i o n s o f 0.5 mg r e s e r p i n e ( R e s e r p i n e I n j e c t i o n , U . S . P . O i b a ) d a i l y f o r 4 days and s a c r i f i c e d on t h e f i f t h d a y . The f a t pads were b i s e c t e d l o n g i d u d i n a l l y and one o f t h e p i e c e s a s s a y e d i m m e d i a t e l y f o r i n i t i a l l i p a s e a c t i v i t y by A s s a y I I I . The r e m a i n i n g p i e c e s were i n c u b a t e d s e p a r a t e l y i n t h e u s u a l manner f o r 5 h o u r s i n K r e b s - R i n g e r b i c a r b o n a t e - a l b u m i n medium. T o one o f t h e p i e c e s , e p i n e p h r i n e (17 m i c r o g r a m s / m l medium) was a d d e d 10 m i n u t e s b e f o r e t h e end o f t h e i n c u b a t i o n p e r i o d . T h e i n c u b a t e d t i s s u e s " w e r e t h e n h o m o g e n i z e d , o e n t r i f u g e d a n d t h e s u p e r n a t a n t f l u i d a s s a y e d f o r l i p a s e a c t i v i t y . The r e s u l t s o f two o f t h e e x p e r i m e n t s a r e shown i n F i g . 15. Two n o t e -w o r t h y o b s e r v a t i o n s were made. S u r p r i s i n g l y , t h e e x t e n t o f l i p a s e A B C F i g . I p . E f f e c t of epinephrine on f a t pads obtained from re s e r p i n i s e d r a t s . Bars "A" and "B" represent i n i t i a l and p o 3 t-p-hour incubation l i p o l y t i c a c t i v i t i e s r e s p e c t i v e l y . Bar "0" represents l i p a s e a c t i v i t y of pads t o which epinephrine (17 micrograms/ml f i n a l concentration) was added 10 minutes p r i o r to homogenizing and assaying f o r l i p a s e a c t i v i t y by Assay i l l . Experimental r e s u l t s from 2 r a t s are shovm. Lipase a c t i v i t y i s expressed i n m i l l i u n i t s . 56. inactivation after 5 hours' incubation was very small. Furthermore, epinephrine appeared to have l i t t l e effect on activating the l i p o l y t i c system even when added to the intact pad. The average i n i t i a l level of lipase activity i n adipose tissues from normal rats, as determined during the course of these studies, i s approximately 47 milliunits. Hence the i n i t i a l lipase a c t i v i t i e s i n adipose tissue from these reserpine-treated rats were probably elevated to some extent. The results are interesting but, at the moment, d i f f i c u l t to interpret. 2. Effect of Pentobarbital-induced Anaesthesia and Length of Incubation  on the Inactivation Rate of the Lipolytic System. It has been mentioned that throughout this project we noted that the extent of inactivation of the l i p o l y t i c system varied widely from experiment to experiment. In our experiences, the degree of inactivation varied from 17% to 86% of the i n i t i a l pre-incubation le v e l . Furthermore, i t became apparent that pre-treatment of rats with pentobarbital did not always cause a large decline i n post-incubation lipase activity. Even more discerning was the observation that when l i p o l y t i c a c t i v i t i e s were determined by Assay II, i n which incubation periods were in many cases for only 90 minutes, many tissues showed a high degree of lipase inactivation. Therefore, data was collected from a large proportion of the experiments performed during the course of this study, and tabulated under three categoriesj: namely, adipose tissue from rats which were (a) anaesthet-ized and incubated for 5 hours, (b) not anaesthetized and incubated for 5 hours, and (c) anaesthetized and incubated for 90 minutes, (Table 7). I t i s most interesting to note from the averages computed that there is very l i t t l e difference i n the ^degree of inactivation of the J-hour 3-hour Incubation 1-1/2 hour Incubation Anaesthetized with Pentobarbital Not Anaesthetized Anaesthetized with Pentobarbital Pre-incubati on Post-incubation Pre-incubation Po st-incubation Pre-incubation Post-incubation Activity* Activity* Activity** Activity** Activity*** Activity*** (milliunits) (milliunits) (milliunits) (milliunits) (ueq/g/ 1 0 min) (ueq/g/10 min) 62 43 54 46 10.0 3.5 59 51 54 29 3.3 0.7 31 22 37 24 4.1 0.7 46 35 47 38 4.4 1.1 70 58 36 26 6.5 2.9 50 34 115 52 5.6 3.7 41 11 62 36 5.7 4.3 31 5 51 28 4.6 2.5 22 10 60 18 4.6 2.7 59 20 39 33 5.4 3.3 49 34 Mean 47 29 Mean 55 33 Mean 5.4 2.5 % Initi a l Activ Lty 62% jSlnitial Activi ty 60% % I n i t i a l Activity 47% *Assay III **Assay I ***Assay II ' Table 7. Effect of pretreatment of rats with pentobarbital and the length of incubation time of fat pads on the degree of lipase inactivation. Lipolytic activities of the 3-hour incubated pads are expressed as milliunits of activity, and were determined according to Assay I or Assay III as indicated. The lipase activities of the l-j-hour incubated pads are expressed as inicroequivalents EPA produced/g wet weight of tissue/10 minutes, and were assayed according to Assay II. 5 8 . incubated pads whether or not the rats had been anaesthetized prior to sacrifice. Even more surprising is the fact that the extent of deactivation was greater i n pads which had been incubated for only 9 0 minutes, as compared with the 5-hour incubated pads. It would seem that some unknown factors determine the rate or degree of inactivation of the enzymes during incubation, and perhaps the handling of the animal, or the degree of trauma produced by the removal of the pad, is not as important as believed by several investigators. 5' Effect of Insulin on the Rate of Inactivation of the Epinephrine-sensitive Lipase System. The suggestion that insulin may inhibit a l i p o l y t i c system i n adipose tissue prompted a brief investigation into the effect of insulin on the rate of inactivation of the l i p o l y t i c system when fat pads are incubated for given periods of time. Epididymal fat pads were obtained from normal fed rats, and assayed for i n i t i a l and post-incubation a c t i v i t i e s by Assay II. Insulin was added to both mediums containing the experimental pads. Glucose was added to one of the insulin-treated pads. The lipase activities observed under these conditions are shown in Fig. l 4. Incubation of the pads for 90 minutes caused the l i p o l y t i c activity to f a l l to approximately 60% of i t s i n i t i a l l e v e l . However, when insulin was present i n the medium (with or without glucose), the rate of depression of l i p o l y t i c activities was delayed. If insulin does have an inhibitory effect on an adipose tissue lipase, the presence of insulin would have been expected to accelerate the rate of lipase inactivation, rather than decrease the rate, as suggested by this experiment. c ' £ O \ 5-0-cn •v. \ CT <D 3. 4-0->-f— 30-> h-O < 20-O 10-o _J B D F i g . l 4 . E f f e c t of i n s u l i n on the d e a c t i v a t i o n rate of the epinephrine-sensitive l i p a s e . Bars "A" and "B" represent l i p a s e a c t i v i t i e s before and a f t e r 9 0 minutes' incubation respectively. Both .pads represented by "C" and "D" were incubated i n a medium containing 2 units/ml i n s u l i n . "D" also contained glucose, 1 . 5 mg/ml. Experiment was performed i n duplicate from adipose tissue obtained from one r a t . L i p o l y t i c a c t i v i t i e s were determined by Assay II and are expressed as microequivalents FFA produced/g/10 minutes. 60. DISCUSSION In the past, most of the studies on adipose tissue metabolism have been performed either i n vivo or i n vitro on isolated, intact f a t pads. The i n vivo studies consisted largely of measuring the metabolic products of l i p o l y s i s i n the circulation, and although this method has yielded, and continues to yield, much information on. l i p i d metabolism, particularly with respect to the u t i l i z a t i o n of l i p i d s for energy, i t has contributed l i t t l e towards elucidating the nature of the intracellular metabolic regulation of li p i d s i n adipose tissues. In v i t r o studies of isolated, intact adipose tissue have contributed considerably more to our knowledge of the metabolic processes i n these tissues. In this respect, epididymal f a t pads of rats are particularly suitable for incubation studies, not only because they are easily isolated as discrete organs, but also because of their extremely thin, le a f - l i k e structure, requiring no further manipulation before incubating i n a suitable medium. However, in order to seek answers to the problem of metabolic regulation i n adipose tissue, (particularly to study the enzymes of this tissue), investigators have recently turned to crude homogenates and supernatant fluids of adipose tissue. Presently, only a relatively small number of workers are using broken-cell preparations of this tissue, and the reasons for this w i l l become apparent when the problems of working with adipose tissue extracts i s discussed. In our studies, we have examined the activity of the hormone- or epinephrine-sensitive lipase system, mainly i n crude homogenates and i n the supernatant fractions of rat epididymal fat pads. The problems of working with enzyme systems in adipose tissue 61. homogenates w i l l be discussed to some extent, since the interpretation of some of the results obtained may be better understood on the basis of the techniques used. Unlike other tissues such as skeletal or cardiac muscle, adipose tissue yields greasy homogenates (at temperatures near 0° 0) which are vi r t u a l l y impossible to pipette. Only by centrifugation at low temperatures and removal of the f a t cake accumulated at the top is i t possible to work with this type of preparation. On the other hand, homogenization at room temperature yields a creamy suspension which, upon f i l t e r i n g through two layers of cheesecloth to remove pieces of the tough outer membrane, allows the preparation to be pipetted. However, unless this suspension is constantly agitated, a difference i n composition between different aliquots of the same homogenate may result. The assay of lipase activity naturally involves the use of a suitable substrate, and the hormone-sensitive lipase i s known to be specific for triglycerides of long chain fatty acids. The question then arises as to what substrate to use. A brief examination of some recent work by various investigators on enzyme systems of adipose tissue indicates that there are almost as many substrates being used as -there are investigators, working i n this particular f i e l d . For instance, Rizack (77) has used Ediol®, a commercial preparation of a 50$ coconut o i l emulsion. Vaughan (80) used no exogenous substrate since i n her assay system the crude homogenate i t s e l f supplied sufficient substrate. Rubinstein et a l (88) obtained l i p i d extracts from adipose tissues and prepared a 20$ emulsion of l i p i d s i n Krebs-Ringer bicarbonate medium, florin and Shafrir (89) used commercial tripalmitin i n a 5$ solution of gum acacia. In our laboratory 6 2 . we have used our own preparation of a 50$ coconut o i l emulsion, Vaughan1 s method (80), and homogenates which were heated to.100° 0, as substrates.. We found that the deactivated homogenate was most suitable for our assays. Other workers have occasionally used Tween 20 as an emulsifying agent, despite the fact that this agent i t s e l f serves as a substrate for the lipase i n adipose tissue. Vaughan (80) has indicated that commercially prepared Ediol ® contains a considerable amount of monoglyceride (about 10 micro-moles of monostearin per ml). Since monoglycerides are rapidly - hydrolysed by adipose tissue extracts, results obtained with Ediol® as substrate must therefore be interpreted with this fact i n mind. The extent of decrease i n lipase activity during incubation was extremely variable from f a t pads of one animal to another. Our data indicate that i n every case inactivation did occur, but the degree of inactivation varied widely. Vaughan (80) has indicated that i n her studies, the activity remaining after a 5-hour incubation was Usually about 20 to 50$ of the original activity, although in some cases l i t t l e or no detectable decrease was observed. In our experience, the average activity remaining after a 5-hour incubation was about 60$ of the original activity. On the other hand, when fa t pads were incubated for only 1-1/2 hours rather than 5 hours, the average post-incubation activity was approximately 47$ of the original activity. Rubenstein et al (88) have indicated that i n their work no incubation was necessary to inactivate the enzyme system, and suggest that this may be due to the fact that they prepared their homogenates i n a bicarbonate-containing medium. They indicated that this particular medium may have inactivated the endogenous-activated lipase, or perhaps oxidized any endogenous epinephrine present. 6J. A number of factors could be involved to account for the variable degree of inactivation. For example, the nutritional and/or emotional state of the animal, or the age of the animal at the time of sacrifice, would most certainly have an effect on the rate of inactivation. Further-more, i t i s even possible that injury to the central nervous system during decapitation or cervical dislocation may cause widespread sympathetic discharge, resulting i n the release of catecholamines in a l l tissues, including adipose tissue. The fact that the degree of inactivation i n f a t pads from pentobarbital-anaesthetized rats was no different from that of unanaesthetized animals might suggest that other factors are involved i n determining the degree of inactivation. The marked release of FFA from adipose tissue mediated by epinephrine i s consistent with the idea of activation of tissue lipase. Our results obtained with unfractionated homogenates would indicate that epinephrine can cause activation of the enzyme only i n intact fat pads. When epinephrine was added during or after homogenization, l i t t l e or no activation of lipase occurred. These results agree with those of Rubinstein et a l (88) who used triglycerides extracted from adipose tissue as substrate. They are not i n accord with the findings of Rizack (77 )» who used commercial coconut o i l , Ediol as substrate and a supernatant f l u i d obtained from a centrifuged homogenate as the enzyme source. The present experiments suggest that cyclic 5,>5'~AMP has some a b i l i t y to activate the lipase present in unf ractionated homogenates of adipose tissue. I t must be emphasized that the effects of the nucleotides are not marked, and i n no case did the activation approach that of the control level present i n the homogenate prepared from unincubated tissue. 64. Cyclic 5'»5'-AMP seemed to have a better a b i l i t y to activate the lipase present i n centrifuged supernatant f l u i d prepared from adipose tissue homogenates when coconut o i l was used as substrate. However, the degree of activation was variablej some extracts responded well, some not at a l l . Our data is not as positive as that of Rizack, although the system we have used seems vi r t u a l l y identical to his. Clearly more work is necessary to settle this matter. Results of one investigator are exceedingly d i f f i c u l t to compare with that of another, since each seems to use a different system and different conditions to study lipase activity. This is i n part due to the technical d i f f i c u l t i e s inherent i n working with adipose tissue homogenates, and i n obtaining preparations of highly l i p o p h i l i c materials such as triglycerides i n a form suitable for enzyme studies. I t i s clear from the published material at hand that the study of factors governing l i p i d metabolism i n broken ce l l preparations is s t i l l i n an embryonic state. The problem, however, because of i t s physiological importance, i s now attracting widespread attention. It i s hoped that some effort w i l l be made to unify the techniques and preparations used i n different laboratories so as to c l a r i f y the situation and f a c i l i t a t e a solution to the problem. The re-activation of an enzyme system i n intact fat pads by epinephrine indicates that the triglyceride-splitting lipase exists i n two forms; that i s , an active and an inactive form. Our data strongly suggests that a lipase system i n crude homogenates of adipose tissue was activated by ATP and cyclic ^'^'-AMP at 5.2 x 10~7 M, and Mg^ at 4 x 10"^ M, despite the fact that epinephrine used under similar conditions f a i l e d to produce any 65. significant degree of activation. We have also demonstrated that ATP and cyclic J1,5'-AMP at 2 x 10~5 M and Mg"*""1" at 5 x 10""2 M could also activate a lipase i n the supernatant fraction of adipose tissue homogenates, thus supporting Rizack's latest findings (79). The sharp inhibition of l i p o l y t i c activity i n crude homogenates at Mg"*"1" concentrations exceeding 3 x 10"^ M is d i f f i c u l t to interpret, especially i n view of the optimum concentration of 5 x 10~2 M required for the activation of the enzyme system i n the supernatant fraction. The synthetic analogues of cyclic 5',5'-AMP, with the exception of cyclic 3',5'-deoxy-AMP, showed no particular indication of activating the l i p o l y t i c system i n the supernatant f l u i d under the conditions of the assay system. The almost total lack of lipase activation with epinephrine, observed i n our studies on adipose tissues from reserpinized rats, i s d i f f i c u l t to understand. Since reserpine causes the depletion of catecholamines i n tissues, i t might be expected that the extent of decrease i n lipase activity upon incubation would be minimized and, i n fact, this was observed. On the other hand, i f the tissues were depleted of catecholamines, their response to added epinephrine might be expected to be highly exaggerated. However, even a normal response to epinephrine was not elicit e d i n our studies. It was observed that the rats had lost an average of 15% of their body weight, their epididymal fat pads weighed about 1/3 that of untreated rats and, i n addition, the i n i t i a l levels of lipase activities were elevated above the normal lev e l . Therefore, i n view of these observations, we suggest that either the dose of reserpine given (1.5 mg/kg/daily for 4 days) was inadequate to cause a marked depletion of catecholamines or, alternatively, 66. that the catecholamines were,, in fact, depleted and that the high level of lipase activity observed was mediated through some mechanism other than that involving catecholamines. Whether working in vivo with intact organisms or i n vitro with isolated tissues and broken c e l l preparations, the ultimate aim of most scientists has been to seek the answer to the question of metabolic control i n the normal physiological processes of the intact organism. Since adipose tissue i s practically the only source of FFA (which are subsequently oxidized by the heart and other organs for energy) the answer to the question of metabolic regulation of lipid s must be sought i n this tissue. 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