Prostaglandin and Essential Fatty Acid Deficiency

70 - 26,276

DU, Julie Yi-Fang Tsai, 1937- PROSTAGLANDIN AND ESSENTIAL FATTY ACID DEFICIENCY.

The Ohio State University, Ph.D., 1970 Biochemistry

i i University Microfilms, A XEROX Company, Ann Arbor, Michigan ]

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED PROSTAGLANDIN AND ESSENTIAL FATTY ACID DEFICIENCY

DISSERTATION esented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Julie Yi-Fang Tsai Du, B.S., M.S. # * * * # ❖

The Ohio State University 1970

Approved by

c . Adviser"*' Department of Physiological Ohemistj-y PLEASE NOTE:

Some pages have indistinct print. Filmed as received.

UNIVERSITY MICROFILMS. ACKNOWLEDGEMENTS

The author wishes to express her sincere appreci ation to Dr. Fred A. Kruger for his guidance, patience and encouragement during the course of this study and the preparation of this manuscript. I am indebted to Dr. John Allred for his kind con cern and constructive advice. I would also like to thank Dr. Ralph M. Johnson for his guidance and support in the mitochondrial studies. The encouragement and understanding of my parents and my husband are also greatly appreciated. Special thanks are also extended to the members of my research committee for their advice and help and to ell my colleagues for their kindness and cooperation. VITA

March 23, 1937 Born— Ching-Tao, China 1959 B. S*, Department of Chemistry National Taiwan University Taipei, Taiwan, China 1961-1963 M.S., Department of Chemistry Texas Technological University Lubback, Texas 1965-1966 Research Assistant Institute of Nutrition Ohio State University Columbus, Ohio 1969-1970 Teaching Assistant Department of Physiological Chemistry Ohio State University Columbus, Ohio ,

FIELD OF STUDY

Major Field: Physiological Chemistry Minor Fields: Organic Chemistry Microbiology CONTENTS

Page ACKNOWLEDGMENTS...... ii VITA ...... iii TABLES ...... vi ILLUSTRATIONS ...... viii ABBREVIATIONS ...... ix INTRODUCTION AND STATEMENT OF P R O B L E M ...... 1 CHAPTER I REVIEW OF LITERATURE ...... 4 Essential Fatty Acids ...... 4 Mitochondrial Studies ...... 7 Prostaglandin ...... 11 Role of Lipolytic Hormones in Free Fatty Acid Mobilization from AdiposeTissue. . 16 PGEn as the Regulator of Fatty Acid Mobilization ...... IS PGE-^ and Blood Glucose ...... 21 PGEn and Glucose Metabolism ...... 21 Mechanisms of the Action of Different Hor mones on the Glucose Utilization • • . • 22 CHAPTER II MITOCHONDRIAL STUDIES ...... 25 Methods and Materials • ...... 25 D i e t s ...... 25 Materials...... 27 Purification of Commercial Linoleic Acid...... 27 Liver Mitochondrial Preparation and Studies...... 32 Lipid Extraction and Fatty Acid Compo sition of Mitochondrial Phospholipids 34 Administration of Prostaglandin .... 35 Results and Discussion ...... 35 One-week PGE^ Injection...... 35 Two-week PGEi Injection...... 37 iv Page _ * Feeding Low Levels of Methyl Linoleate. • 37 Feeding Corn Oil Up to Five Percent by W e i g h t ...... Ad. CHAPTER III ADIPOSE TISSUE STUDIES ...... 55 Methods and Materials...... 55 Materials...... 55 Preparation of Isolated Fat Cells .... 55 Analytical Procedures ...... 57 Results and Discussion ...... 59 Lipogenesis...... 59 Lipolysis...... 79 SUMMARY ...... 32 CONCLUSIONS ...... 36 LITERATURE CITED ...... 37

V TABLES

Table Par:e 1 Diets ...... 25 2 Fatty Acid Composition of Dietary Oils . . . 26 3 NADH Oxidation by Liver Mitochondria from Nornal, Essential Fatty Acid-Deficient and PGEj_- Injected Hats ...... 36 4 Respiratory Control Ratio by Liver Mitochondria from Normal, Essential Fatty Acid-Deficient and PGE^-Injected R a t s ...... 36 5 Fatty Acid Composition of Rat Liver Mitochon drial Phospholipids ...... 3& 6 Summary of Two-Week PGE^ Injection Experi ment ...... 39 7 Fatty Acid Composition of Rat Liver Mitochon drial Phospholipids from PGE^ Injected R a t s ...... 40 6 Respiratory Control Ratio of Liver Mitochon dria from Rats Fed Different Levels of Methyl Linoleate or a Normal Diet • . • * 42 9 NAiDH Oxidase "Specific Activity” of Rat Liver Mitochondria fed Low Levels of Pure Methyl Linoleate or a Normal Diet •«••••• 43 10 The Fatty Acid Composition of Rat Liver Mito chondrial Phospholipids in Rats fed Low Levels of Methyl Linoleate or Control D i e t s ...... 46 11 Effect of Feeding Corn Oil or Pure Methyl Esters of Linoleate and Linolenate Upon the Fatty Acid Composition of Rat Liver Mitochondrial Phospholipids ...... 52

vi Table Page 12 Glucose Utilization in Adipocytes from Normal and EFA-Deficient Rats ••••.. 61 13 Effect of Hormones and Diets on the Utili zation of Glucose by Isolated Fat Cells From Rats ...... 63 14 The Utilization of Glucose by Fat Cells from Rats Fed Either a Fat-Free Diet, a 5% Corn Oil Diet or a 5f° Hydrogenated Coconut Oil D i e t ...... • ...... 69 15 In Vitro Metabolism of Glucose by Fat Cells From Essential Fatty Acid-Deficient and Normal Rats After PGE^ Addition...... 70

16 The Effect of Feeding Different Levels of Corn Oil or Methyl Oleate on the Metabolism of Glucose by Isolated Fat Cells from Rats • 72 17 The Effect of Feeding Pure Methyl Linoleate or Pure Methyl Oleate on the Metabolism of Glucose by Isolated Fat Cells From Rats • 73 IB Effect of Depletion of EFA on the Incorpora tion of Glucose-1--1L4c into CO2 or FA of Normal-Fed Adult Ra t s ...... 77 19 Lipolysis by Fat Cells of Normal and EFA Deficient Rats...... Bl 20 Effect of PGEn on Epinephrine Stimulated Lipolysis by Adipocytes from Normal and EFA-Deficient Rats • «••••••••• Si

vii ILLUSTRATIONS

Figure Page 1. Structures of prostaglandins and prostanoic a c i d ...... 14 2* Average body weights of rats fed various levels of dietary pure methyl linoleate for 21 days...... 45 3. The ratio of 20:3 to 20:4 in liver mitochon drial phospholipids from previously fat- depleted rats fed low levels of pure methyl linoleate for 21 days...... 47 4, The response of NADH oxidase 11 specific activity" in rat liver mitochondria to various levels of linoleate fed for 14 d a y s ...... 50

Vlll* * * ABBREVIATIONS

ADP adenosine dinucleotide c.p.m. counts per minute C.O. corn oil HCNO hydrogenated coconut oil FA fatty acid EFA essential fatty acid FFA free fatty acid NADH reduced nicotinamide adenine dinucleotide n.s. no significance PGEj^ prostaglandin E^ S.E* standard error of the mean TL total lipids GLC gas liquid chromatography Tris tris(hydroxymethyl)aminomethane EDTA ethylene diamine tetraacetic acid ra.p. melting point b.p. boiling point Omega (u) ) Nomenclature: This nomenclature is one of the methods of naming single or methylene interrupted unsaturated long chain fatty acids. The position of the terminal methyl group is designated carbon number one. Thus, linoleic acid (octadeca-9,12-dienoic acid) 4

will be called 13:2 106. The first number “IS" means the number of carbon atoms, "2 " refers to the number of double bonds, and ”006” shows the position of the double bond, C-AMP 3*»5T-cyclic adenosine monophosphate

x INTRODUCTION AND STATEMENT OF PROBLEM

The discovery that prostaglandins are biosynthesized from essential fatty acids (PGE^ from dihomo- }f -linolenic acid or 20:3 u) 6 and PGE2 from arachidonic acid) has led to the hypothesis that perhaps a part of the actual func tion of essential fatty acids is to act as precursors of prostaglandins• BergstrSm and Carlson (1) found a higher basal rate of free fatty acid and glycerol release from adipose tissue in vitro and higher concentrations of free fatty acid in the plasma and of triglycerides in the liver of essential fatty acid (EFA) deficient rats. DePury and Collins (2) also observed plasma free fatty acids ele vated above normal. Since prostaglandins have been demonstrated to be potent inhibitors of basal and hor monal stimulated lipolysis, these observations suggest that they may be lacking in EFA deficiency. Essential fatty acids are constituents of phospho lipids which in turn are important components of cellular end subcellular membranes. These unsaturated fatty acid residues are essential for maintaining the proper struc ture of the membrane (3)»

1 A deficiency in EFA results in pronounced changes in fatty acid composition (4), as well as structure (5-9), and function (10, 11) of isolated mitochondria. Altera tions in permeability to NADH have been demonstrated in mitochondria from EFA deficient rats (12). In rat adipose tissue, prostaglandin Ex(PGEx), like insulin, is one of the most potent inhibitors of basal and hormonal stimulated lipolysis (13)* also has a weak insulin-like effect on glucose metabolism in this tissue* It stimulates glucose uptake, glucose oxidation, fatty acid and triglyceride synthesis from both glucose and acetate (14-19). Alloxan diabetic rats exhibit an impaired ability to synthesize fatty acids from ^C-glucose in liver and in adipose tissue, and the administration of insulin in vivo or in vitro corrects the abnormality

(20). If PGE^l were the controlling factor for fatty acid synthesis, a lower lipogenic capacity in EFA deficient rats might be expected. Hox^ever, Allman (21,22) demon strated a ten fold increase in fatty acid synthesis from pyruvate in liver and adipose tissue of weanling mice main tained on a fat-free diet. When linoleate was added to the diet for one week, the high fatty acid synthesis returned to normal. This investigation was undertaken to determine whether the abnormalities in essential fatty acid-defi cient rats are due to a lack of prostaglandins. It involved: 1 ) mitochondrial studies to examine the in vivo effect of exogenous prostaglandin on the alterations of liver mitochondria in rats fed an essential fatty acid- deficient diet, 2 ) epididyman adipose tissue studies to determine the effect of essential fatty acid-deficiency on lipogenesis and lipolysis in epididymal fat cells from rats and to establish whether these alterations can be accounted for by a lack of prostaglandins. CHAPTER I

REVIEW OF LITERATURE

Essential Fatty Acids Two extensive reviews on essential fatty acids (EFA) are available (10, 23). The overt signs of EFA deficiency in the rat are decreased growth rate, scaly tail and feet, increased water permeability of the skin, increased calo ric consumption in relation to body weight, diminished urine output and increased basal metabolic rate (10). It is now recognized that there are at least three essential fatty acids, linoleic (9*12 -octadecadienoic acid or 1&:2 (0 6), linolenic (9,12 ,15-octadecatrienoic or 13:3(03)* and arachidonic acid (5*3,11,14-eicosatetraenoic acid or 20:4 0)6). Schlenk and Sand (24) have recently reported a new group of essential fatty acids. They observed that the 9,12 -heptadecadienoic acid (17:2 (0 5 ) and the 6,9*12 -hepta- decatrienoic acid (17*3^ 5 ) were able to alleviate exter nal symptoms of EFA deficiency as efficiently as did linoleic acid. Linoleic acid can be converted to arachidonic acid by the animal, and linolenic acid is converted to an

4 eicosapentaenoic acid (20:5CO3) in the rat. Desaturation of stearic acid vri.ll give oleic acid, and oleic acid can be further dehydrogenated and chain elongated to yield a nonessential polyunsaturated fatty acid, namely, 5,S,11- eicosatrienoic acid or 20:3 W 9 (25 ). The transformations of these acids are as follows (2 6):

1&:2 & 6 - > 16:3 i*6 - > 20:3o 6 ->-20:4o>6 (linoleic) (arachidonic) 16:31* 3 - > 16:/+ to 3 — - 20 : 4 3 — 20 :5103 — > 22 :5 u; 3 — 22:6 co 3 (linolenic)

16:1c* 9 - -16:20)9 ->-20:2609->20:3 9 (oleic)

Dehydrogenation occurs chiefly in the microsomal fraction and requires oxygen and NADPH. Chain elongation is an NADPH, ATP and biotin dependent reaction. Significant amounts of 5,6,11-eicosatrienoic acid are found in the heart, liver, adipose tissue and erythrocytes of animals fed diets low in essential fatty acids (27)• It decreases when the diet is supplemented with linoleic or linolenic acids (26). As a result of extensive studies in animal tissues of the relationship of this trienoic acid to the tetraenoic acid, arachidonic, Holman (29) has suggested that the ratio of fatty acids of 20:3 to 20:4 be used to indicate the degree of EFA deficiency in rats. A triene to tetraene ratio of 0.4 or 6 less indicates normal EFA status in rats (30). Docosa^en- taenoic acid (22:5(0 6) exhibits EFA activity (31 )> probably through a chain shortening and reduction process that finally yields arachidonic acid (i.e., 22:5 u> 6 ' 20 :5(0 6 —>20:4(0 6). Sprecher has shown that shorter chain fatty acids of the (0 6 series can also serve as precursors for the EFA (32). In EFA deficient rats, small amounts of 14:2 a>6 were converted to linoleic acid, and 16:2 a) 6 was also an efficient precursor of linoleic acid and the longer chain (0 6 fatty acids. Competition between fatty acids in the different series has been demonstrated. Holman and Mohrhauer (33) have suggested that linoleate and linolenate compete for a common system of enzymes for elongation and for con version to their more highly unsaturated derivatives, such as the 20:4 and 20:5 acids. Brenner et al. (34-39) have obtained evidence for competition in vitro by testing the effect of polyenoic acids on the conversion of linoleate (13:2 a)6) to octadecatrienoate (13:3 u)6) in a microsomal system from the livers of fat deficient rats. VThen linoleic acid-l-^C v/es the only fatty acid present, there was a 20^5 conver sion to the 13:3 acid. The addition of linolenate caused a 50^ decrease in the formation of arachidonate. Mathe matical treatment of the results indicated that linolenate acted as a competitive inhibitor. These investigators believe that the 5>S,ll-eicosatrienoic acid that arises from oleate and is increased in EFA deficiency is not decreased by direct replacement by linoleate or linolenate but rather by the more unsaturated fatty acids (namely, arachidonic, eicosapentaenoic and decosahexaenoic acids) into which linoleic and linolenic acids are converted. Dietary experiments have indicated that the accumu lation of polyunsaturated members of a particular series in animal tissues depends on the presence and the amount of the acids of the other series. Brenner and Peluffo (37)» in a very thorough study of desaturating reactions, have shown that the C-13 unsaturated acids compete for the seme oxygenase and the order of affinity is 13:3 >13:2 y 18:1. Uchiyama et al. (39) have shown that desatura tion of stearic acid to oleic acid by rat liver prepara tions was markedly inhibited by the addition of polyun saturated fatty acid such as 13:2 or 20:4. Oleic acid was less active. They concluded that fatty acid desat uration in liver is regulated at least partly by the level of unsaturated fatty acid3 in the liver.

Prostaglandins The strong smooth-muscle stimulating activity of human seminal plasma was observed independently by Gold- blatt (50,51) and von Euler (52,53)* BergstrSm (54) showed that the observed biological activity was due to prostaglandins— a new type of highly active, lipid soluble, unsaturated hydroxy acid. Several recent reviews on prostaglandins are available (13# 55-59). The isolation in pure form from sheep glands of the first two pros taglandins, PGE-l and PGF1 , v/as reported in 1957 by BergstrBm et al. (60) and Bergstr&n and Sjovall (61-63)• Ultramicroanalysis and mass spectrometry proved that they had empirical formulas and respectively (64)* The complete structure of PGE-^ (11, 15-dihydroxy- 9-oxo-13-trans-prostenoic acid) was elucidated by various degradations, and by mass spectrometry in combination with gas chromatography (65-66), All the prostaglandins contain 20 carbon atoms and have the same basic carbon skeleton, ”prostanoic acid,” The complete structure of the primary prostaglandins E, F, A and B are shown in figure 1, Prostaglandins of the E type contain charac teristic 11-hydroxy and 9-keto groups on a 5-roembered ring. This structure is easily dehydrated by weak alkali to the 10:11 unsaturated ketone (PGA) that can rearrange to the doubly conjugated ketone (PGB). These compounds have U. V, absorption maxima at 217 and 27# respec tively (69). The F prostaglandins are analogous to the E compounds but the 9-keto group is reduced to a hydroxyl. Chemical reduction of PGE-^ yields two isomeric alcohols, F ^ and F-^ (64). Only the F^* isomer occurs naturally. All the prostaglandins contain the 13, 14-trans double bond. The E^ and F^ compounds contain only this one double bond whereas Eg and Fg have two double bonds (5# 6-cis and 17» l6-ci_s additional). The actions of E, F and A prostaglandins are often dissimilar and sometimes are opposed. Thus, the PGETs Fig. 1 Structures of Prostaglandins and Prostanoic Acid. COOJI H ? COOH

11 H /a ,r t* Prostaglandin Ej PGE1 Prostanoic acid 11a, 15(S)-dihydroxy-9-oxo- 13trans-prostenolc acid

COOH .COOH

R OH

Prostaglandin E2 Prostaglandin E3 PGE2 PGE3 Ha, 15(S)-dihydroxy-9-oxo-5£is, 11a, 15(S)-dihydroxy-9-oxo-5cis, 13trans-prostadienoic acid 13trans,17cis-prostatrienoic acid

COOH COOH

H OH Prostaglandin F ^ Prostaglandin Fjp PGFla, PGF1_l PGFi r , p g f 2_i 9a, 11a, 15(S)-trihydroxy- 9p, lia, 15(S)-trihydroxy• 13trans-prostcnoic acid 13trap.s-prostenoic acid

COOH COOH

"OH

Prostaglandin Ai Prostaglandin PGAi, PGEi-217, A^FGE], PGB1, PGF.i-278 PGEi-218, PGE^-220 lh(S)-hydroxy-9 -oxo-10, 15(S)-hydroxy-9-oxo-8(12), Ladienoic acid 13trans-prostadicnoic acid 15 are vasodepressor agents, whereas the PGF*s usually have opposite effects* On the other hand, both PGEfs and PGF*s tend to increase cardiac output, and to induce contraction of isolated gastrointestinal muscle and constriction of the iris* The PGA*s are like the PGE*s with respect to their vascular actions, but are virtually inactive as gastrointestinal contracting agents* Only PGErs have significant antilipolytic activity (13)* The presence of prostaglandins has been demonstrated in a number of tissues, including lung, thymus, brain and spinal cord, kidney, iris, umbilical cord, and human decidua (13)* Prostaglandin is released from adipose tissue by nerve stimulation or catecholamines (70# 71)• Human and sheep seminal plasma exceed other tissues in both the number of different prostaglandins present, and the total concentration: over 100 /Ug/ml as compared to less than 1 /tg/g wet tissue (13)* The elucidation of the structure showed that the prostaglandins differ only in their degree of unsatura tion, the additional double bonds being located in rela tion to the carboxyl and methyl groups in the same way as in certain naturally occurring C-20 essential fatty acids. This led to the independent discovery by van Dorp et al. (72, 73) and Bergstrom et al* (74# 75) that dihomo- J- linolenic acid (all-cis-eicosa-S,ll,14-trienoic acid or 20:3 d 6, a metabolite of linoleic acid), arachidonic acid 16 (all-cis-eicosa-5>8,11,14-tetraenoic acid or 20:4^6), and all-cis-eicosa-5.6.11,14*17-pentaenoic acid (20:5 «)3> a metabolite of linolenic acid) are transformed in high yields into PGE-j_> P G ^ and PGE^ respectively*

Role of Lipolytic Hormones in Free Fatty Acid Mobilization from Adipose Tissue The mobilisation of free fatty acid (FFA) from adi pose tissue is regulated by hormonal, nutritional and nervous factors* The lipolytic hormones (epinephrine, norepinephrine, glucagon, adrenocorticotropic hormone, and thyroid stimulating hormone) activate an enzyme called hormone-sensitive triglyceride lipase (76)* This hormone- sensitive triglyceride lipase activity has been clearly distinguished from monoglyceride lipase and lipoprotein lipase. The overall rate of triglyceride breakdown is determined by the activity of the hormone sensitive lipase (77). The FFA liberated from glycerides are in part reuti lized for triglyceride synthesis. The glycerol, however, is not reutilized for triglyceride synthesis, since adipose tissue lacks glycerokinase activity (76). Thus, measure ment of the rate of release of glycerol can be taken as approximating closely the rate of lipolysis independent of changes in rates of reesterification. Experiments from several laboratories have provided evidence that cyclic AMP plays an important role in the action of the lipolytic hormones in adipose tissue (79* #0). These hormones increase the formation of cyclic AMP in intact rat epididymal fat pads (61) and in isolated fat cells (60, 62). The methyl xanthines, inhibitors of phosphodiesterase (an enzyme which breaks down cyclic AMP to 5 *-AMP) (64), were first shovm by Vaughan and Steinberg to act synergistically with epinephrine on lipolysis (65). They act to prolong the increased concentration of cyclic AMP produced by adenyl cyclase activation (62). Further, Rizack demonstrated that the addition of cyclic AMP and ATP reactivate the lipase activity in cell free prepara tions (66). Adenyl cyclase is a membrane-bound enzyme, which has not yet been isolated in pure form (60). There fore, the definitive proof of the involvement of these lipolytic hormones in the activation of adenyl cyclase has to await the preparation and purification of the enzyme. Butcher (67) as well as Birnbaumer and Rodbell (60) have reported that several lipolytic hormones at supra maximal concentrations did not act additively on adenyl cyclase activity as measured in isolated fat cells and fat cell ghost preparation respectively. In addition, Butcher (62) and Blecher et al. (66) found that £-adren ergic blocking agents (e.g., pronethalol) were very potent antagonists of catecholamine activation of adipose tissue adenyl cyclase but did not antagonize the polypeptide IS lipolytic hormones* Based on these results, Butcher has speculated recently that the adenyl cyclase system contains of a number of hormone specific regulatory subunits (S7)*

PGE^ as the Regulator of Fatty A c i d Mo b i1i z at Steinberg and Vaughan (77* #9) have shown that PGE^ added in vitro reduced basal and lipolytic hormone stimu lated lipolysis in rat adipose tissue: PGEi counteracts the enhanced glycerol and FFA release in the presence of epinephrine, norepinephrine, ACTH, TSH and glucagon. PGE^ also inhibits methyl xanthine (theophylline, for example) stimulated lipolysis in rat adipose tissue (77* 90,91)* At high concentrations of theophylline, PGEj may be relatively inactive, perhaps because in the presence of.virtually complete inhibition of phosphodiesterase, the rate of formation of cyclic AMP is no longer rate limiting (77)* At equivalent concentrations, PGE^ inhibits theophylline induced lipolysis to a greater degree than norepinephrine induced lipolysis and also the maximum inhibition is greater (90,91). The inhibition of the lipolytic action of catecholamines by PGEi is "apparent competitive": the maximum inhibition is only about 505$ (90,92). PGE^ opposes the catecholamine-induced plasma FFA and glycerol increase in a parallel fashion in intact 19 dogs (93), and in vitro PGE^ decreases basal and the epinephrine induced activation of the lipase of rat adipose tissue (39,94)* Epinephrine increases the accumulation of cyclic AMP in adipose tissue in vitro (31,32), and this increase is inhibited by PGE-^ in concentrations that also inhibit lipolysis (33*95*96)* However, PGE^ does not inhibit the stimulation of lipolysis by either cyclic AMP itself or its dibutyryl derivative. Therefore, it probably inhibits lipolysis secondary to its inhibition of adenyl cyclase (77,97,93). PGE^ is one of the most potent inhibitors of lipoly sis known: as little as 3.2 ng/ml (9x10™%) reduces sig nificantly the effect of 0.1 xtg/ral (5*5x10™%) of epine phrine and the maximal effect of PGE^ (4 inhibition) occurs at 0.1 -t^g/ral (2.3x10™%) in adipose tissue (99)* In isolated fat cells, it is perhaps more potent. As little as 4x10- % PGEi (1*4 ng/ml) antagonizes the stimu latory effect of 5.5x10“% epinephrine (l/-Lg/ral) by fifty percent (37)* The in vivo effect of PGEj is more complicated. It acts by two independent mechanisms: at low doses it stimu lates FPA mobilization by way of the sympathetic nervous system, probably as a compensatory reaction to lowered blood pressure; at high doses, it reduces FFA mobilization by inhibition of lipolysis enough to overcome the sympa thetic effect (100,101). 20 Insulin has been recognized to inhibit lipolysis and decrease cyclic AMP levels (102). Corbin (103) recently found that there is a correlation between the entilipolytic and cyclic AMP lowering effects of insulin. Blecher et al. (55) on the other hand, have demonstrated that insulin in vivo or in vitro has no effect on phospho diesterase. The effect of prostaglandins on the level of cyclic AMP, hoxvever, is more complicated than that of insulin because they interact with cyclic AMP mechanisms in so many tissues (#3 ). POE^ inhibits lipolytic hormone increased accumulation of cyclic AMP in fat pad and iso lated fat cells (33). In isolated fat cells, PGE^ alone decreases the levels of cyclic AMP (S3). In intact fat pads, however, PGE^ increases the intracellular cyclic AMP levels without affecting lipolysis. It stimulates the level of cyclic AMP in stromo-vascular cells rather than the fat cells (57). This paradoxical discovery shows that adipose tissue is far from homogeneous. In order to study the effect of PGEi in relation to lipolysis and glucose metabolism, iso lated fat cells were chosen in the present study. PGE^ has been found to increase cyclic AMP in other tissues including: lung, diaphram, spleen, brown fat, testis (53) and brain (57). Ramwell and Shaw (70,71) have demonstrated that under catecholamine or nerve stimulation, several tissues 21 ‘(spinal cord, diaphram and epididymal fat pad) release increased amounts of prostaglandins. These data suggest that the release of prostaglandins from tissues may in some way be influenced by the activation of adenyl cyclase or by cyclic AMP itself, and that the prostaglandins might then act as feedback regulators upon the adenyl cyclase in the tissue (13,56,71,63)•

PGEi and Blood Glucose PGEj., Siyen by either single injection or infusion, elevates blood glucose in dogs (100,104), rabbits (103, 106), rats, and guinea pigs (107-109), Since this hyper glycemia does not occur in rats after adrenal demedulla- tion, it is probably mediated by a reflex release of adrenal epinephrine in response to lowered blood pressure. In dogs, PGEi blocks the rise in plasma fatty acids induced by epinephrine but has no effect upon the simul taneous hyperglycemia (93,110,111),

PGEi and Glucose Metabolism

In adipose tissue, when PGEi a^ded in vitro, it increases glucose uptake (17), glucose oxidation (19), total lipid (14,16-19) and glycogen synthesis (17)* The activation of lipid synthesis by PGEi resulbs from an increase in fatty acid synthesis (17,19) as well as from an augmentation of reesterification (16,19), PGEi injected intraperitoneally into intact rats at 10-ug/kg increases 22 the incorporation of labeled glucose into glycogen in epididymal fat tissue and diaphragm (15,19)* At this dose, it also enhances adipose tissue lipid synthesis from glucose (19)*

Mechanisms of Action of Different Hormones on Glucose Utilization The rate-limiting step in glucose uptake in fat cells is transport (112 ). Rodbell (113) showed that at low concentrations, phospholipases or proteases have insulin-like activity in fat cells. He believed that the initial action of insulin on fat cells involves some com ponents of the plasma membrane. Kono (114) showed that trypsin impairs insulin responsiveness in glucose metabo lism and lipolysis, but does not seriously damage glucose transport and subsequent metabolic steps in fat cells. In the trypsin treated cells, he found that: 1) the basal rate of glucose uptake or production of labeled CO2 was not reduced; 2 ) the transport system could be accelerated many fold by raising the glucose concentration in the incubation medium; 3) glucose metabolism was still inhib ited by phloretin, a transport inhibitor, or 3-0-methyl- D-glucose; 4) epinephrine still stimulated lipolysis. Thus, he suggested that a site or sites on the fat cell • surface containing peptide components is necessary for the action of insulin on glucose transport end lipolysis. Cuatrecasas (115) demonstrated that insulin can be coval 23 ently attached to large polymers of Sepharose (agarose polymers). He further showed that such derivatives effec tively increase the utilization of glucose and suppress the hormone-stimulated lipolysis of isolated fat cells. Since the derivatives do not enter the cells, he concluded that interaction of insulin with superficial membrane structure alone may suffice to initiate transport as v/ell as other metabolic alterations. Several investigators have found that in addition to insulin, epinephrine, glucagon and ACTH also stimulate glucose uptake and utilization in adipose tissue (116-120 ), Rodbell (121) studied the effect of lipolytic hormones and theophylline on glucose utilization in fat cell ghost pre parations. He found that these lipolytic agents caused a reduction in basal glucose utilization but did not block insulin stimulated glucose oxidation. He concluded that cyclic AMP might inhibit basal glucose transport. Bray and Goodman (122) reported that epinephrine caused a very small (10^5) but statistically significant increase in the L-arabinose space in epididymal fat pads. They also found that epinephrine increased the oxidation of glucose in fat pads even when lipolytic effects were blocked by pro pranolol. Blecher et al. (123) studied the stimulatory effects of these lipolytic hormones on glucose utilization in isolated adipocytes. They found that under certain con 24 ditions in which PGE-^ and nicotinic acid were antilipolytic against epinephrine in adipocytes, these agents were with out influence on the effects of the hormone on the metabolism of glucose. They also found that while PGEj_, K8 592^- and propranolol^ all inhibited the stimulatory effects of glucagon and ACTH on lipolysis in adipocytes, neither agent influenced the action of either hormone on glucose oxidation. They concluded that the stimulatory effects of these hormones on glucose uptake and oxidation are independent from their action on lipolysis. The mechanism of the stimulation of glucose uptake by these lipolytic hormones is still obscure. The mechanism of PGE^ in stimulating glucose utili zation is also unknown.

xk8 592: l-(3-methylphenoxy)-3-isopropylaminopropan- 2 -ol hydrochloride, a p -adrenergic blocker.

^Propranolol: 1-i sopropylamino-3-(1-naphyloxy)-2- propanol hydrochloride, a ys-adrenergic blocker. CHAPTER II

MITOCHONDRIAL STUDIES

Methods and Materials Diets Male albino rats (Holtzman) were weaned at eighteen days and randomly divided into tv/o groups. The composi tion of the diets fed to these groups is given in Table 1 (45).

TABLE 1 DIETS

.J& bX. weight Ingredient Normal EFA-deficient Dextrose • • 53 56 "Vitamin-free Casein ...... 21 21 Cellulose • • • •••••«•• 16 16 Salt Mixture, U.S.P. XIV . . . 4 4 Corn Oil. • • 5 0 Vitamin Fortification Mixture*. 1 1

* (General Biochemicals, Inc.)

Food and water were presented ad libitum and the animals were maintained on these diets throughout the

25 26 experimental period unless otherwise specified. They were used from the fourth to the sixth month on the regi men, because the deficiency syndromes (decreased weight gain and scaliness of paws and tails) developed after 12 to 16 weeks.

TABLE 2 FATTY ACID COMPOSITION OF DIETARY OILS*

Percentage of total fatty acids Fatty acid Coconut oil Corn oil

8:0 5.2 10:0 5.6 12:0 43*6 14:0 20.8 16:0 11.1 12.6 16:1 0.1 13:0 10.0 2.3 13:1 3.5 28.5 13:2 54.0 13:3 2.5 20:4 0.1

* Sabine, John R,, Hope McGrath and S, Abraham J, Nutrition, v. 98, p. 312, 1969 (see ref. 125)* 27 Materials NADH and ADP were products of the Signa Chemical Company. Prostaglandin Ej was a generous gift of the Upjohn Company, Kalamazoo, Michigan, All solvents were redistilled before use. Methyl linoleate (99% plus purity) was purified by us as recorded in the following:

Purification of Commercial kinoleic Acid Commercial linoleic acid, as analyzed by gas liquid chromatography contained 55*5/5 linoleic acid, 29% oleic acid, 7*4/5 linolenic acid, 4*7/5 palmitic acid, 1*3/5 stearic acid, 1/5 palmitoleic acid, O.E>$ myristic acid and trace amounts of lauric acid. The method for purification was adapted from several published procedures and included bromination, crystallization, methylation and mild debromination with zinc in diethyl ether (126). Since dibromo- stearic acid (the dibromoderivative of oleic acid, m.p. 2^-29°), and the saturated long chain fatty acids are all soluble in petroleum ether, end the tetrabromostearic (tetrabromoderivetive of linoleic acid, m.p., 115*5°) and hexabromoster.ric acid (hexabromoderivative of linolenic acid, m.p., 1&5°) a**e not, the impurities in linoleic acid can be removed by low temperature bromination. Bromination of Commercial Iiinoleic Acid The bromination was carried out in a hood at low temperature. Linoleic acid (400 g) in 1200 ml petroleum ether was introduced to a three necked round-bottom flask immersed in a dry-ice bath, kept at -10 to 0°C. Bromine (125 ml, 375 6> the calculated amount for brominating all the unsaturated fatty acids) in 750 ml petroleum ether was added dropwise from a separatory funnel to the reaction flask which was stirred constantly by a mechanical stirrer. At the end of the addition (ca. one hour), the content appeared pasty and more petroleum ether was added to keep the stirrer moving for 30 more minutes. It was then washed with more petroleum ether and filtered through a Bflchner funnel. This filtration removed most of the dibroraoderivative, as well as the saturated fatty acids. Hexabromoderivatives were removed by either of two means: 1 ) the crude tetrabromo- derivative was dissolved in diethyl ether, fil tered, then removed by evaporation and the product washed to remove any dibromo-contami- nants; 2 ) recrystallization from hot ethylene dichloride. The solid tetrabromostearic acid obtained was washed again with petroleum ether, filtered and transferred to a glass plate and worked to a powder with a spatula. The needle shaped crystals were snow white with a silver sheen, m.p., 114-115°C (Chemical Abstract 49* 130&Lb, m.p., 114.4-115.4°C).

B• Methylation of Tetrabromo- stearic Acid The methylation was performed in methanolic sulfuric acid (10^ sulfuric acid in methanol) by refluxing. After the tetrabromostearic acid in acidic methanol (350 g tetrabromostearic acid in a total of 1500 ml acidic MeOH) was refluxed for two hours, it separated into two layers. The upper layer was acidic methanol and the lower was the methyl tetrabromostearate. Dilute aqueous NeOH [2%) was added to neutralize the reaction mixture which was immediately transferred to a separatory funnel. The lower layer was then transferred to a beaker and soon solidified. It had a m.p. 60-6l.5°C (Chemical Abstract 49* 13031b, m.p., 6l.l-6l.7°C). When cool, some solid compound appeared in the methanol layer. This was filtered and dried, and had a m.p. at 53-61°C. 30 C• Debromination of Mothvl- t etrabromost o ar ate Methyl tetrabromostearate (175 g) was dis solved in 1*75 liter of diethyl ether (3 liter flask)* The ether solution was stirred vigorously with a mechanical stirrer, and 600 g zinc power was added over a two hour period and in small portions. After all the zinc had been added, stirring was continued until the vigorous reac tion slowed down. It was then refluxed for 30 minutes. The completeness of debromination was checked by thin layer chromatography. Prelimi nary chromatographic results showed that the methyl esters of linoleic acid and tetrabromo stearic acid traveled at different rates in a solvent system of hexanejether (192:6). A few milliliters of the ether solution was taken out, washed with distilled water several times, and spotted on a silica plate. After the plate was developed for 40 min. in the solvent system, it was dried and sprayed with dichlorofluororescein. Since pure methyl linoleate traveled faster than its bromoderivative under these conditions, the incompleteness of the reaction could be shown by two distinct spots corresponding to the bromo derivative and linoleate respectively. More zinc 31 dust (200 g) was added slowly to the reaction flask and the process repeated until there was no indication of a tetrabromostearate spot on the thin layer plate. The reaction mixture was separated by decan- tation and filtration with suction through a filter coated with celite. The filtrate was washed with distilled water until there was no reaction of bromide with 5$ AgNO^. The solution was dried over Na^SO^. Solvent was removed in a rotatory evaporator at 25 ~30°C. After evaporation, the methyl linoleate was stored under nitrogen in a deep freeze.

D. Vacuum Distillation and jrHehtificTation of Methyl Linoleate The methyl linoleate obtained was vacuum dis tilled. The main fraction had a boiling point at 174-175° c/2-3mmHg*. Using the Cary 14 Recording Spectrophotometer, the near IR spectrum (0.9-3Ji) of a 4^ solution in CCl^ (w/v) indicated no peroxide. Peaks should be detected at 1.46 or

2 .OS al if peroxide were present (127). The per centage purity was estimated by GLC to be over 99$»

* b.p. 175°C/2.5 mmHg, Organic Synthesis, Collective volume 3» P* 52S. Since the debroraination was catalyzed by Zn metal there is danger of formation of trans double bonds. Reports have shown that isolated trans double bonds have a specific absorption band at about 10.36 (123,129)• Therefore, infrared spectrum was run to test this possibility. (Beck man IR-5> 0.1 mm path, 20 $ solution in CCl^). The result showed that there was about 4$ trans isomer. This amount was not considered harmful be cause of the fact that edible products such as shortenings and margarines, have been shown to contain trans isomers (mostly monoenoic) to the extent of 20 -50$ (123).

Liver Mitochondrial Prepara tion and Studies Liver mitochondria were prepared essentially accord ing to the method of Schneider (130). The tissue was minced and homogenized in a medium containing 0.3M D-mannitol, 1 mil Tris, and 0.1mI-I EDTA; pH 7*4 (131) • The cell debris was removed by centrifugation at 600xg for 3 minutes in 0 Servall RC-*2 Refrigerated Centrifuge (#40 Rotor). The supernatant fluid was then centrifuged at 3700xg for 3 minutes. The fluffy layer was removed by gentle washing with O.3M D-mannitol-1 mil Tris and the hard packed mito chondrial pellet was washed, centrifuged and then suspended 33 in this medium. It was finally diluted to 1.5 ml of mitochondrial suspension per g wet tissue. Mitochondrial protein was determined by the Folin-phenol method modified by Miller (132) with bovine serum albumin as the standard. The hypotonic reaction medium contained: 0.01M potassium phosphate buffer, pH 7*4> 0.01M triethanolamine and 0.05M magnesium chloride. The isotonic reaction medium contained 0.1M mannitol, 0.04M potassium chloride in addition to the above ingredients. Oxygen uptake was determined following the addition of 0.2 ml of suspended mitochondria to 2.6 ml of one of these media. To each reaction vessel {final volume 3 ml) was added 50 -'■'•I of a solution containing 20 mg NADH/ml in 0.1M Tris buffer, pH 7,4. NADH oxidation and respiratory control were deter mined polarographically employing the Clark oxygen elec trode (Yellow Springs Instrument Company, Yellow Springs, Ohio). A closed reaction cell was constructed to accomo date the electrode. Oxygen consumption was recorded on a Sargent Model SR Recorder. Oxygen concentration in the reaction mixture was assumed to be the same as in pure water (e.g. 236 .AM at 25 °C) Respiratory control is defined as the ratio of the respiratory rate in state 3 (in the presence of ADP) to the respiratory rate in state 4 (the added ADP having been completely depleted, 133)* When respiratory control was measured, 0.2 ml of mitochondria were added to 2.6 ml 34 reaction mixture, followed by the addition of 50 M 1 of 114 sodium succinate or 1M ^-hydroxybutyrate and 2 or 3

successive additions of 10 ju 1 each of 0.04M ADP. Respira tory control was calculated from the slopes of state 3 and state 4 respiration.

Lipid Extraction and Fatty Acid Composition of Mitochondrial h ospholi pids Mitochondrial lipids were extracted with ethanol for 1 hour followed by three extractions with ethanol-ether The extracts were combined, evaporated to dryness, taken up in a minimum volume of chloroform, and the phospho lipids were precipitated with acetone three times (134)* The phospholipids were then hydrolyzed with 0.5N methanolic KOH and the fatty acids methylated with 145^ methanol.ic BF^ (135)» find the composition of the methyl ester mixtures was determined by gas-liquid chromatography (Barber-coleman Model 5000). A six-foot glass column was employed, which contained 20% diethylene glycol succinate on Gas-Chrom P, £0-100 mesh (Applied Science Lab., Inc., State College, Pa.). The operating temperatures were 160°C for the column, 260°C for the hydrogen flame detec tor, and 260°C at the point of sample injection. Peak areas were measured as the product of peak height times the width at half the height. 35 Administration of prostaglandin Prostaglandin was administered by intraperitoneal injection, in a volume of 1 ml/injection (50 x*g/kg)« Stock solutions of 10 mg of prostaglandin per ml of ethanol were first diluted to 1 mg/ml with water and sufficient 0.01N NaOH to make the pH 7* and then further diluted with 0.9# sodium chloride. All'doses were based upon the average weight of the group of rats.

Results and Discussion

One-week Injection A group of rats fed an essential fatty acid (EFA) deficient diet were injected intraperitoneally with 50 M g/kg of prostaglandin E^PGE^), twice daily for 7 days. The rats were then killed. The NADH oxidase "specific activity" in hypotonic medium, respiratory control ratio and phospholipid composition of liver mitochondria from these rats were measured and compared with those from normal and untreated EFA deficient rats. The results are shown in Table 3 and 4* Administration of PGE^ did not change the NADH oxidase "specific activity" when compared to that of EFA deficient rats, nor did it change the respiratory control ratio. The fatty acid compositions of rat liver mitochon drial phospholipids from normal, EFA deficient and PGE^- injected rats are given in Table 5* The increased amounts TABLE) 3 NADH OXIDATION BY LIVER MITOCHONDRIA FROM NORMAL, ESSENTIAL FATTY ACID-DEFICIENT AND PGEi-INJECTED RATS^

NADH oxidase “specific activity"^ (mean + S.E.) o o Normal (7) 0.45 + • O o Cst EFA-deficient (7) 0.13 + • PGE^-injected (6) 0.12 jh 0.02

TABLE 4 RESPIRATORY CONTROL RATIO BY LIVER MITOCHONDRIA FROM NORMAL, ESSENTIAL FATTY ACID-DEFICIENT AND PGEi-INJECTED rAts1

RESPIRATORY CONTROL RATIO3 (mean +S.E.) (succinate as substrate) ~

Normal (7) 4.5 + 0.13 EFA-deficient (7) 4.05 ± 0.22 PGE^-injected (7) 3.64 + 0.16

Rats fed an essential fatty acid-deficient diet were injected intraperitoneally with 50 ^tg/kg of PGE-i, twice daily for 7 days* 2 NADH oxidase “specific activity" is defined as the ratio of NADH oxidation rate to state 3 succinate oxida tion rate in hypotonic reaction medium*

3The respiratory control ratio is defined as the ratio of rate of respiration in the presence of ADP to rate in its absence* 37 of 16:1, 16:1 and 20:3 seen in EFA deficient rats were observed to the seme extent in PGE1-injected rats, and the decreased levels of 16:2 and 20:4 in these rats were also found in rats injected with PGE^. Thus, intraperitoneal injection of 50 z^g/kg of PGE^ for 7 days did not change the fatty acid composition of liver mitochondrial phos pholipids in the EFA-deficient rats.

Two-week Injection The respiratory control ratio and NADH oxidase "specific activity" in hypotonic medium of rats intra- peritoneaily injected with 50 /^g/kg PGEi for 14 days are given in Table 6. Again, there is no difference between rats injected with PGEi and untreated EFA-deficient rats. The fatty acid composition of liver mitochondrial phospholipids obtained from rats which had been injected with PGEi intraperitoneally for two weeks (Table 7) was the same as that of the untreated EFA-deficient rats* The ratio of 20:3 to 20:4 was 3 in the rats injected with PGEi for 2 weeks (Table 7)> 3*01 in the rats injected with PGEi ^or one wee*c anc* 3*36 in the untreated EFA deficient rats (Table 5)* In normal rats, it was less than 0,03 (Table 5).

Feeding Low Levels of Methyl Llnbleatc The fat-free diet was supplemented with pure methyl TABLE 5 FATTY ACID COMPOSITION OF RAT LIVER MITOCHONDRIAL PHOSPHOLIPIDS Rat % Fatty Acid 20:3 Type 14:0 16:0 16:1 18:0 18:1 18:2 20:3 20:4 22:6 20:4 Nl1 trace 11.8 2 .IS 17.57 12*35 39.76 _4 16.34 N2 trace 12.49 1.77 IS. 36 11.22 33.3 0.23 22.64 - 0.01 N3 trace 20-5 5 1*42 25*35 7.34 15.95 — 23.89 — N4 0.54 22.63 1.19 28.88 9.10 14.11 0.66 22.84 - - 0.03 Normal trPce 16. 88 1*64 22*54 10.13 25.73 trace 22.68 < 0.03 Average

Dl2 trace 11.71 14*41 18.41 27.93 3.66 16.63 5.27 1.93 3.15 D2 0.72 11.87 17.70 20.38 31.46 2.86 21.16 2.86 7.40 D3 0.25 13.43 12.11 18.91 27.37 4.35 15.70 3.13 — 1.93 D4 0.26 11.44 15.12 19.31 23.61 5.08 17.79 4.94 2.46 3.60 EFA-def. Average 0.41 12.12 14.84 19.25 27.61 3.99 17.32 5.30 trace 3.36

Pl3 0.74 20.52 11.38 19.26 25.39 3.83 12.66 4.20 . 0.91 3.01 P2 0.41 11.17 16.40 18.10 28.59 6.13 14.84 4.36 — 3.40 P3 trace 11.45 12.22 18.96 27.75 4.16 16.97 6*41 2.08 2.65 PGEi-ing. Average 0.56 14.33 13.50 18.77 27.24 4.71 14.82 4.99 - 3.01 1 N denotes normal rat (5$ corn oil diet)* 3 P denotes EFA deficients rat injected intraperitoneally with 50 g PGE;j/kg 2 D denotes EFA deficient rat (fattv/ice daily for 7 days* ^ free diet)* 4 n-fl denotes no measurable fatty acid* 39 TABLE 6 SUMMARY OF TWO-WEEK PGEj_ INJECTION EXPERIMENT

A. Respiratory control ratio of liver mitochondria from normal, essential fatty acid-deficient and PGEi- injected rats^ Respiratory Control Ratio (mean + S.E.) Succinate______ft -OH butyrate o cs ro • o o Normal 4.35 + . fo (ll)2 4.59 i (7)2 EFA-deficient 3.69 + 0.09 (10) 3.16 + 0.15 (7) PGE^-injected-^ 3.79 £ 0.13 (9) 3.13 + 0.29 (7)

B. NADH oxidation by liver mitochondria of normal, essen- eial fatty acid deficient and PGE^ injected rats NADH oxidase “specific activity"*1' (mean + S.E.)

Normal 0.36 H- 0.03 (9)2 EFA-deficient 0.10 + 0.01 (9) PGE^-injected^ 0.06 + 0.01 (6)

1 Respiratory control ratio was calculated from the slopes of state 3 (in the presence of ADP) and state 4 (in the absence of ADP) respiration. Succinate or p-hydroxybutyrate was used as substrate. 2 The numbers in the parentehsis refer to the number of animals in each group. 3 Essential fatty acid deficient rats were injected intraperitoneally with 50>ug/kg of PGEj, twice daily for 7 days. 4 NADH oxidase “specific activity" is defined as the ratio of NADH oxidation rate to state 3 succinate oxi dation rate in hypotonic reaction medium. TABLE 7 FATTY ACID COMPOSITION OF HAT LIVER MITOCHONDRIAL PHOSPHOLIPIDS FROM PGEr INJECTED RATS*

Rat /3 Fatty Acid 20:3 No. 14:0 16:0 16:1 18:0 18:1 18:2 20:3 20:4 20:4

• J + 1" 0.56 13.55 12.42 21.43 25.41 4.11 16.70 5.81 2.87 2 trace 13.31 13.12 20.39 23.39 4.5 17.28 8.00 2.16 3 0.4$ 17.19 10.11 24.40 29.76 2.51 12.13 3.4! 3.55 4 0.21 10.85 13.55 19.60 26.30 4.37 17.2 7.9 2.18 5 0.25 13.25 14.9$ 24.01 28.40 3.71 11.29 4.11 2.75 6 trace 14.10 14.69 21.20 28.47 3.85 14.46 3.23 4.48

Average 0.37 13.71 13.14 21.84 26.95 3.84 14.84 5.41 3.00

* Rats were injected intraperit one ally with 50 )’■ g/kg of PGE^, twice daily for 14 days.

-o a - 41 linoleate in amounts up to 0.6# by weight (1.6# by calorie) and fed to adult EFA deficient rats for 21 days* Levels of liver mitochondrial respiratory control ratio ( /3-hydroxybutyrate as substrate), fatty acid composition of phospholipids and NADH oxidase ''specific activity" in hypotonic medium were compared with those from unsupple mented rats fed the fat-free diet. Methyl linoleate, sup- • plemented at 1.6# of the calorie or lower did not change the respiratory control ratio (Table 6) or NADH oxidase "specific activity" levels (Table 9) appreciably from those of the unsupplemented group. Nevertheless, the body weight of the intact animals (Fig. 2) and the fatty acid composition of phospholipids from liver mitochondria (Table 10) were significantly different betv/een the sup plemented and unsuppleraented rats.

Feeding Corn Oil Up to Five Percent bv~\Veight When higher levels of linoleic acid supplied from corn oil (up to 5# corn oil by weight or 7.2# linoleic acid by calorie) were fed to EFA deficient rats for 14 days, the levels of NADH oxidase "specific activity" in hypotonic medium were restored toward the normal level (Fig. 4)* When 3.2# pure methyl linoleate or 1.6# pure methyl linoleate plus 0.4# pure methyl linolenate by calorie was fed to EFA deficient rats for 2 weeks, the level of NADH oxidase "specific activity" in hypotonic medium in the 42

TABLE 6 RESPIRATORY CONTROL RATIO OF LIVER MITOCHONDRIA FROM RATS1 FED DIFFERENT LEVELS OF METHYL LINOLEATE OR A NORMAL DIET

Diet % calories Respiratory Control Ratio^ No. of from 16:2 (Mean + S. E.) rats

0.00 3.66 + 0.20 6

0.15 4.66 + 0.26 * 0*30 4.27 + 0.30 5 0.40 3.62 ± 0.22 6 0.60 4.49 + 0.19 3 0.60 4.16 + 0.22 6 1.60 4.33 + 0.17 6 normal^ 5.12 + 0.22 6

1 Rats fed an EFA deficient diet for more than three months were placed on diets containing from zero to 1*6/5 of the calories as pure methyl linoleate for 21 days* 2 Normal rats were fed a diet containing 5% corn oil by weight from weaning* 3 Betahydroxybutyrate was used as the substrate. 43

TABLE 9 NADH OXIDASE "SPECIFIC ACTIVITY" OF RAT1 LIVER MITOCHONDRIA FED LOW LEVELS OF PURE METHYL LINOLEATE OR A NORMAL DIET

Diet % calories NADH Oxidase "Specific Activity"^ from 1S:2 (mean + S.E.)

0,0 0,11 + 0.006 0.15 0.10 + 0.016 0.3 0.11 + 0.016 0.4 0.17 + 0.017 0,6 0.12 + 0.020 o.a 0.16 + 0.011 1.6 O.lS + 0.010

Normal^ 0.37 + 0.050

1 Rats fed an EFA deficient diet for more than three months were placed on diets containing from zero to 1.6$ of the calories as pure methyl linoleate for 21 days 2 Normal rats were fed a diet containing 5$ corn oil by weight from weaning. 3 NADH Oxidase "specific activity" is defined as the ratio of NADH oxidation rate to state 3 succinate oxidation rate in hypotonic reaction medium. 4 Each figure represents an average of 6. Fig. 2. Average body weights of rats fed various levels of dietary pure methyl linoleate for 21 days (mean of 6 + S.E.) % % CALORIES FROM 18:

BODY WEIGHT IN GRAMS (MEAN OF SIX ± S . E.) TABLE 10 THE FATTY ACID COMPOSITION OF RAT LIVER MITOCHONDRIAL PHOSPHOLIPIDS IN RATS FED LOW LEVELS OF METHYL LINOLEATE OR CONTROL DIETS

Diets # Fattv Acid % calorie lw9) (w6) 20:3 from IS:2 14:0 16 iO 16:1 13:0 13:1 13:2 20:3 20:3 20:4 22:5 22:6 20:4

0.0# 0.29 15.03 13.43 18.11 25.56 4.37 17.35 - 5.73 0.49 - 3.32

0.15# 0.35 10.53 14.61 16.2 24.55 8.33 11.03 2.84 10.67 trace - I.04 0.3# 0.23 11.26 12.06 16.26 22.11 11.31 10.54 3.12 11.29 1.5 - 0.94

0.4# trace 17.07 10.4 13.24 21.77 7.93 10.27 2.64 11.53, trace - 0.9

0.6# trace 13.11 9.33 16.64 21.98 13.05 7.46 2.73 12.8 1.34 - 0.59

0.3# trace 14.0 5.91 20.71 17.7 9.81 . 9.04 4.47 16.43 2.07 - 0.56 1.6# 0.23 16.5 5.64 20.4 15.17 10.54 4.96 3.07 20.64 2.76 - 0.23

Normal 0.4 14.96 3.16 19.02 11.86 24.83 — — 24.2 1.07 1.94 -

1 All figures represent the mean of five rats. 2 Rats were fed a fat-free diet for at least three months after weaning, then fed diets containing from zero to 1 . 6 # of the calories as pure methyl linoleate for 21 days. Normal (5# corn oil by weight) rats served as controls. £- 3 ,,- ,t means undetectable 47

Fig. 3* The ratio of 20:3 to 20:4 in liver mito chondrial phospholipids from previously fat-depleted rats fed low levels of pure methyl linoleate for 21 days. The dotted line represents a level of triene to tetraene ratio below vfhich the metabolism of EFA is considered to be normal. TRIENE TO TETRAENE RATIO o o o o o *-* • • • • ro 4- o cd o

o no

C o

-H m

i\j 49

Fig. 4* The response of NADH oxidase "specific activity" in rat liver mitochondria to various levels of linoleate fed for 14 days. NADH oxidase "specific activity" is defined as the ratio of NADH oxidation rate to state 3 succinate oxidation rate.

3 mean values from 5 rats fed corn oil + S.E. 1,6% 13:2 (5) mean value from 5 rats fed + S.E. 0.4^ 13:3 -

0 mean value from 5 rats fed 3*2% 13:2 + S.E.

□ mean value from 5 normal rats + S.E. NADH OXIDASE (SPECIFIC ACTIV! TY) 51 isolated liver mitochondria was 0,25 and 0.24 respec tively, These values fall between 0.28 and 0.23 obtained with liver mitochondria from rats fed 2.5/S and 1.25% corn oil (3.6 and 1,8$ linoleate by calorie) respectively (Fig. 4). Feeding methyl linoleate (3*2$ by calorie) resulted in a triene to tetraene ratio (Table 11) of 0*12 which was between 0.23 and 0.03 obtained with liver mitochondria from rats fed 1.25 and 2.5/S corn oil (1,8 and 3 ,6% by calorie from linoleate) respectively. Both methyl linoleate and methyl linolenate feeding decreased the levels of 16:1, 18:1 and 20:3* Feeding lineleate increased the levels of 18:2 and 20:4 whereas feeding linolenate increased the level of 20:5^3 and particularly the higher metabolite of linolenate, 22:6 £^>3 (Table 11). Thus, when linoleate supplied at 1,6% or more of the total calories was fed to rats previously on a fat- free diet, the fatty acid composition of liver mitochon drial phospholipids was corrected to the fatty acid compo sition of the normal rats. While the level of NADH oxidase “specific activity” increased with increased linoleate intake, it approached the normal level only when 5?* corn oil by weight (7*2^ 18:2 of the total cal ories) was fed. The hypothesis that the liver mitochondrial permea bility defect (low levels of NADH oxidase "specific activ- TABLE 11 EFFECT OF FEEDING CORN OIL OR PURE METHYL ESTERS OF LINOLEATE AND LINOLENATE UPON THE FATTY ACID COMPOSITION OF RAT2 LIVER MITOCHONDRIAL PHOSPHOLIPIDS

Diet ' ' rt --- , , /■> % Fatty Acid corn 18:2 oil from 16:0 16:1 18:0 18:1 18:2 20:3 20:3 20:4 20:5 22:5 22:6 20:3) by wt. c o m oil CJ9) (-6) (o>3) (*»6) (tc3) 20:4Go) by cal.

0.0 0.0 12.1 14.8 19.3 27.6 4.0 17.8 5.3 3.2 0.6 0.9 16.0 9.9 17.2 15.7 12.6 8.7 2.8 16.7 — — _ 0.5 1.25 1.8 22.3 4.0 31.6 13.7 11.4 2.8 2.4 11.9 — __ 0.2 2.5 3.6 20.4 2.7 24.3 11.0 18.7 0.7 1.2 20.1 — __ 0.03 5.0 7.2 21.1 1.7 24.6 8.7 19.1 -3 1.2 20.1 - 1.9 2.2 - normal 15.0 3.2 19.1 11.9 24.8 - - 24.2 - 1.1 1.9 - Diet % Methyl esters % Fattv Acid Tyne 16:0 16:1 18:0 18:1 18:2 20:3 20:3 20:4 20:5 22:5 22:6 20:3 by wt. by cal. P9) (*>6) (<■>3) («>6) (*£) 20:4to6) 18:2 1.2 3.2 16.4 4.7 20.5 11.9 14.2 2.9 2.1 24.1 2.4 0.12 18:2 0.6 1.6 plus 16.5 5.0 20.8 13.2 10.6 6.1 3.0 16.3 1.7 0.8 5.8 0.38 18:3 0.15 0.4 1 Each number represents an average of four rats. 2 Rats were fed a fat-free diet for at least three monthsafter weaning, then fed diets containing various supplements for 14 days. Normal (5$ corn oil by weight) rats served as controls. 3 The peak area is too small to be detectable. 53 ity" in hypotonic medium) of EFA deficiency might be due to a secondary deficiency of PGEi was not substantiated*

Fifty a g PGEi/kg was injected intraperitoneally to EFA deficient rats, twice daily for 7 or 14 day3* No change could be detected in the levels of NADH oxidase "specific activity" in hypotonic medium, nor in phospholipid fatty acid composition of liver mitochondria from these rats. BiJhle and May (19) could find increased incorpora tion of glucose into lipids and glycogen in adipose tissue 2 hours after they injected 10 /i g/kg P G % intraperitoneally into rats. When they increased the level to

100 m g/kg, the effect was more pronounced. When pure methyl linoleate was fed at low levels (up to 1,6 fa of the calories) to EFA deficient rats, the fatty acid composition of liver mitochondrial phospho lipids in these rats was comparable to that of the normal rats, but the levels of NADH oxidase "specific activity" in hypotonic medium were still low. However, when the feeding level of linoleate was increased up to 7*2% of the calories (supplied from 5/S corn oil by weight), the NADH oxidase "specific activity" in hypotonic medium was brought back to values almost equal to the levels of nor mal rat liver mitochondria. These results agree with two other studies which attempted to relate EFA deficiency to a lack of prosta glandins. 54 Gottenbos, Beerthuis and van Dorp (136) found that neither oral administration or intravenous infusion of PGEj had an effect on dermal signs of EFA deficiency, nor on excessive transpiration of water through the skin of EFA deficient animals. Kupieclci, Sekhar and Weeks (137) also found that continuous intravenous infusion of PGE-^ as well as PGE2 and PGF^ in combination had no significant effect on dermal signs of EFA deficiency. Therefore, it is concluded that with liver mito chondria, the permeability defect (low levels of NADH oxidase "specific activity" in hypotonic medium) is caused by a lack of essential fatty acids per se and not a prostaglandin deficiency. CHAPTER III

ADIPOSE TISSUE STUDIES

Methods Materials Regular insulin (Iletin injection, U.S.P.) was pur chased from. Eli Lilly: Epinephrine-HC1 (injection, U.S.P.) was obtained from Wyeth Laboratories. Hydrogenated coconut oil was purchased from Durkee Pood Company and was shown by GLC to contain no essential fatty acids. Highly puri fied methyl esters of oleate and linoleate were obtained from Nutritional Biochemical Corporation (NBC) containing 99fo plus purity. Bacterial collagenase was purchased from Worthington Biochemical Corporation. Powdered bovine serum albumin (Fraction V) was obtained from Signa Chemi cal Company. The commercial methyl oleate from NBC con tained 79f° oleate, 11.6# palmitoleate, 3*6# palmitate and small amount of myristate.

Preparation of Isolated Fat Cells Isolated fat cells were prepared essentially as described by Rodbell (13#). Male Holtzman rats weighing between 300 and 400 g were used in these studies and were

55 fed, ad libitum, either the normal 5% corn oil, no fat or specified diet. Rats were killed by decapitation usually in the morning between 10 to 12 A.M. The epididymal fat pads were removed, weighed, and cut into small pieces. Three to 4 g of tissue was added to a siliconized 25 ml flask, containing 3 ml of albumin-Fain’s buffer, and 10 mg of collagenase. Incubations were carried out for 1 hour at 37°C in a Dubnoff metabolic shaking incubator. The albumin-Fain’s buffer solution, pH 7«4> contained K% Fraction V bovine albumin which had been dialyzed against the same buffer overnight at 4°C, and was filtered through a 0.3/' millipore filter. The Fain’s buffer contained: 2.7mM KC1, 137inM NaCl, 1.4mM CaCl2, 5mM MgCl2, 4mM NaH2 P04 and 12mM NaHCO^. The same buffer was used for the metab olic studies. The tissue was dispersed into small frag ments within 1 hour of incubation with collagenase. The suspension of cells was centrifuged in polyethylene centrifuge tubes for 1 min. at 400xg at room temperature in an International clinical centrifuge. The fat cells floated to the surface, and the stromalvascular cells (cappilary, endothelial, mast, macrophage, and epithelial cells) wore sedimented. The stromal vascular cells were removed by aspiration, and the fat cells were washed two times with the same buffer containing 2% albumin. Fat droplets, which may have been from the breakage of the fat cells, floated more rapidly to the surface than the fat 57 cells and were aspirated from the surface, after gentle stirring of the cell suspension. The triglyceride concentration (fat cell content) was approximately 0.9 g/ml. Twenty-five A 1 of ^C-glucose was introduced into a siliconized 25-ml Erlenmeyer flask, containing 2.9 ml of albumin-Fain’s buffer and 6 M moles of glucose per ml (1 mg/ml) with or without hormone (the volume added v/as usually less than 30 xtl). The final specific activity of glucose was 0.5 A c per xc mole. After the addition of 0.1 ml fat cells, the incubation flasks were capped with rubber serum stoppers fitted with hanging glass wells. The wells contained cylinders of VThatman No. 1 paper rolled from 2xS-cm strips. The incu bations were carried out, with shaking, at 37°C for 2 hours. The gas phase was 95$ 0g-5$ CO2* Three to four replicate flasks were incubated in each experiment.

Analytical Procedures Radioactivity was determined in a Packard model 3375 liquid scintillation counter. ^C-lipid and COg were counted in a scintillation solution consisting of 0.4$ PPO (2,5-diphenyloxazole) and 0.05$ POPOP ^p-bis-2-(phenyl- oxazolyl)-benzene7 in toluene. Bray*s scintillation solu tion (139) was used for counting ^C-glucose in the incu bation medium. It contained: naphthalene 60 g, PPO 4 g» POPOP 0.2 g, absolute methanol 100 ml, ethylene glycol 20 53 ml and p-dioxane to make the.final volume 1 liter. At the end of the incubation period, 0,2 ml of Hyamine 10X purchased from the Packard Instrument Company was injected onto the filter paper and 0,25 ml of IN sulfuric acid into the cell suspension. After the flasks were shaken for 15 min. at room temperature, the paper strips were transfered to 10 ml of scintillation fluid and counted. Two drops of methanol were added to the counting vials to increase the solubility of the Hyamine-COg in the scintillation fluid. For the determination of -^C-lipids, the cell sus pensions were transferred to centrifuge tubes with glass stoppers and extracted with 5 ml of Dole’s extraction mixture (140). This extraction mixture contained 1 part hexane, 4 parts of isopropyl alcohol and 0.1 part IN HgSO^. Redistilled hexane was used to replace heptane in the original Dole’s extraction mixture. After the mixture had stood for 15 minutes at room temperature, 3 ml of hexane were added, and the phases were allowed to separate. The lower phase was removed by aspiration and the upper phase was washed with 3 ml of water. Portions of the upper phase were analyzed for ester content (141) with tripalmitin as standard and total lipid radioactiv ity. To determine radioactivity in triglyceride fatty acids, 1 ml of the upper phase was evaporated and the lipid vras saponified by refluxing for 2 to 3 horn's with 2 ml of ethanolic ICOH (1 ml of saturated KOH per 100 ml of 95% EtOH, freshly prepared). After 2 ml of water were added, the sample was neutralized to a bromocresol green end point and the fatty acids were extracted with 3 ml of hexane. A 2 ml aliquot of the latter was evaporated to dryness, and 10 ml of scintillation fluid were added to dissolve the residue. Free fatty acid content was deter mined by titration (142) and generally agreed with the ester content of the original lipid extract. Sodium ethoxide in absolute ethanol vras used here instead of a qieous sodium hydroxide. Thus, the system vras all in one phase and the color change vras distinct (thymal blue as indicator, the original purple changed to yellow then to a dirty blue end point). Since the lipids of adipose tissue are primarily triglycerides (143)# the difference found in fatty acids and that in total lipid was assumed to represent radio activity in the glycerol moiety and is referred to as glyceride-glycerol. Results are expressed as c.p.m./mg TG.

Results and Discussion

A. Lipogenesis The utilization of glucose in adipocytes from normal and from essential fatty acid (EFA) deficient rats was studied by using uniformly labeled glucose (U-^ C- 6° glucose) to determine the incorporation of carbon-^C into the following: carbon dioxide; total lipid; fatty acids and triglyceride-glycerol (Table 12). There was a wide range in the amount of each glu cose metabolite from adipocytes of EFA deficient (fat-free diet) rats. The variability of metabolite production in the adipocytes of the normal controls was little. The adipocytes from EFA deficient rats oxidized twice as much U-^C-glucose to ^C02 in a 2-hr incubation period and synthesized 1.6 times as much total lipid as did adipocytes from normal (5% corn oil diet) rats. The most striking difference, however, was in fatty acid synthesis. The adipocytes of EFA deficient rats exhibited an eight to ten fold higher incorporation of label into fatty acids. Glyceride-glycerol synthesis in adipocytes of EFA defi cient rats did not differ from the normal. The percentage of radioactivity in fatty acids in relation to the radio activity in total lipid (FA/TL) was in the adipocytes of EFA deficient rats, whereas that of normal rats was 5.S^. The high rate of fatty acid synthesis observed in the adipocytes of EFA deficient rats was in agreement with the high fatty acid synthesis in EFA deficient mice reported by Allman (21) and Allman and Gibson (22). However, they used pyruvate rather than glucose and found a higher incorporation into fatty acids in the liver as well as TABLE 12 GLUCOSE UTILIZATION IN ADIPOCYTES FROM NORMAL AND EFA DEFICIENT RATS

Conversion of U-^KJ-glucose to: FA3 co2 TL FA Glycerol TL

I Normal (S)^ 40,4 + 3.7 123.0 + 9.5 7.4 + 1.2 121.7 + 9.4 5-3S5 II EFA-def(S) #9.1 +19.4 206.3 +33.3 79.7 ± 21.6 127.1 ± 14.0 3S.5S5 EFA-def 2.2 1.6 10.7 1.0 Normal Statistical significance: • I-II p <0.05 p <0.05 p<0.01 n.s.

1 Incorporation of uniformly labeled glucose into labeled C02* TL, FA and Glycerol in fat cells, expressed as c.p.m./mg TG (mean + S.E.) 2 The number in the parenthesis refers to the number of experiments performed with two animals in each experimental group, 3 FA/TL denotes the percent of the glucose carbon incorporated into the fatty acid portion of the total lipids. adipose tissue. Solyora et al. {144) found a greater tri glyceride synthesis from glucose in adipose tissue from EFA deficient rats, which is in agreement with these data. Since the rate of the glyceride-glycerol synthesis was essentially the same in the adipocytes of EFA deficient and normal rats, the small increase in triglyceride synthesis observed here was due to the difference in the fatty acid synthesis per se. The ratio of fatty acid to total lipid (FA/TL) as measured by radioactive incorpora tion of was in the adipocytes of EFA deficient rats and in the adipocytes of normal rats. Rodbell (13^) reported a FA/TL ratio of 30/5 in adipocytes of normal rats. The discrepancy between my results and Rod- bell’s is undecided. The difference in diet might be one of the contributing factors. These rats were fed a synthetic diet while Rodbell fed his rats commercial pellets. The effect of insulin and PGE^ on glucose utiliza tion by the adipocytes of the EFA deficient and normal rats were also studied (Table 13). Although PGE^ (lyfcg/ml) slightly stimulated glucose oxidation, fatty acids and triglyceride synthesis from glucose in both normal and EFA deficient rat adipocytes, this stimulation was only about one fifth to one fourth that of insulin

(100 a *units/ml). Insulin caused a 1.8 fold increase in CO2 production and a 2.4 fold increase in fatty acid TABLE 131 EFFECT OF HORMONES AND DIETS ON THE UTILIZATION OF GLUCOSE BY ISOLATED FAT CELLS FROM RATS

Grouo Conversion Diet No.' of U-14C- glucose to: Normal EFA deficient

I Control 42.3 ± 4*4 124.7 + 29.0

II co2 PGEq_^ 51.3 + 5.5 (1.21)3 143.9 ± 34.7 (1.19) III Insulin 76.0 ± 5.4 (1.78) 233.4 ± 45.0 (1.37) IV Epinephrine 93.6 + 16.0 (2.28) 261.0 + 37.0 (2.09)

V Control 147.3 + 12.5 259.2 + 55.3 VI TL PGEi 170.1 + 3.6 (1.15) 307.4 ± 66.2 (1.19) VII Insulin 235.4 + 6.7- (1.60) 437.0 + 94.0 (1.33) VIII Epinephrine 237.3 ± 37.0 (1.95) 476.2 ±136.0 (1.8)

IX Control 3.3 + 2.4 103.4 ± 42.0 X FA PGEX 11.4 + 2.6 (1.30) 129.0 + 50.3 (1.25) XI Insulin 21.3 ± 4*6 (2.40) 246.0 + 50.3 (2.40) TABLE 13— Continued

Group Conversion Diet No# of U-14c- glucose to: Normal EFA deficient

XII Epinephrine 14.5 + 5.8 (1.65) 168.0 + 39.0 (1.63) /

XIII Control 138.5 ± 10.7 155.8 + 17.0 XIV Glycerol POEi 158.7 ± 7.5 (1.15) 178.4 + 18.8 (1.14) XV Insulin 214.2 + 3.7 (1.55) 241.2 + 13.3 (1.55) XVI Epinephrine 273.3 ± 35.0 (2.00) 308.2 ± 99.0 (2.00)

1 Results were expressed as c.p.m./mg TG (mean + S.E.) of four experiments, two animals in each experimental group, ~ 2 Concentration of hormones added in vitro: Insulin 100 /tunits7ml PGE]_ 1 m g/ml Epinephrine 7 M g/ml 3 The number in the parenthesis denotes the ratio of the distribution of radio activity in the presence and absence of hormone# 4 Significance levels: Group No# Normal EFA deficient I-II p= 0.02 p** 0.04 r* I-III p C 0.01 p<0.01 -f TABLE 13— Continued

Group No. Normal EFA deficient I-IV p<0.01 p= 0.05

V-VI p ^ 0.02 p== 0.02 V-VII p < 0.01 p < 0.05 V-VIII ■ p < 0.02 P < 0.05

IX-X p< 0.05 p <. 0.10 IX-XI p< 0.05 p= 0.05 IX-XII p= 0.05 p < 0.10 x i i i -x i v p ^ 0.02 p= 0.05 XIII-XV p<0.01 p= 0.05 XIII-XVI p <0.01 p < 0.01 66 {Synthesis in both groups but an increase in total lipid synthesis of 1.6 times in the normal adipocytes and a 1.9 times in EFA deficient adipocytes when compared to untreated. The in vitro effect of epinephrine on glucose oxida tion and synthesis of total lipid, glycerol and fatty acids in adipocytes from normal and EFA deficient rats was also studied (Table 13)* Epinephrine (7>^g/ml) caused a marked stimulation in glucose oxidation and in glycerol synthesis in both normal and EFA deficient adipocytes. It also caused an increase in total lipid synthesis (1.95 fold increase in normal and 1.6 fold increase in EFA deficient adipocytes). Total fatty acid synthesis (free fatty acids and glyceride-fatty acids) was increased 1.6 fold in both the normal and EFA deficient adipocytes. The stimulatory effect of these three hormones on glucose metabolism in adipocytes from normal rats was in agreement with the findings of other workers (19,136) although the degree of stimulation was lower. The hormonal responses in the adipocytes from my normal control rats were lower than that reported by Rodbell (133) probably because of the age difference of the rats. The rats used by Rodbell weighed between 150- 200 g. In these experiments, however, rats had to be kept on a fat-free diet for at least three months in order to develop EFA deficiency, so the average weights 67 of normal and EFA deficient rats were 400 g and 300 g respectively (4 ar*d one half to 6 months old)* Cries and Steinke (145) studied the in vitro metabolism of l-^fC- glucose by adipose tissue segments and isolated fat cells* They found that the basal glucose metabolism as well as the response to insulin decreased with increasing body weight of the rats. These results showed that EFA defi ciency did not block the effect of insulin, PGEj_ and epinephrine on glucose metabolism in rat adipocytes. The percentage of hormonal response in the adipocytes from both the normal and EFA deficient rats was similar although the absolute values were quite different. Feeding 5% hydrogenated coconut oil for 7 days to rats previously fed a fat-free diet had no significant effect on lipogenesis nor on glucose oxidation in their adipocytes (Table 14)* These rats were still hyper- lipogenic and synthesized S.l times more fatty acids (26 c.p.m./mg TG) than adipocytes from normal rats (3*2 c.p.m./mg TG). In a subsequent experiment, 5% hydroge nated coconut oil was fed to EFA deficient rats for 26 days (Table 15). Even then, the high rate of fatty acid synthesis was not lowered significantly (24.2 c.p.m./mg TG, i.e., 7 times higher than the normal levels). The effect of PGE^ on glucose metabolism (measured as incorporation of 1-^C-glucose into ^C02, total lipid end fatty acids) in adipocytes from the normal, fat-free 66 find 5% hydrogenated coconut oil fed rats was also compared (Table 14)- Adipocytes from both of the EFA deficient rats (fat- free diet and 5$ hydrogenated coconut oil diet fed for 7 days) were slightly more responsive to PGE^ (1 xtg/ml) addition than were normal cells. Production of ^C02 from l„14c-glucose was increased 1.3 times in the EFA deficient adipocytes and 1.1 times in the normal adipocytes. Fatty acid synthesis increased 1.2 to 1.4 times in the EFA deficient adipocytes and 1.1 times in the normal adipocytes after PGE^ addition. The PGE-^ effect was more pronounced in the adipocytes from EFA deficient rats receiving the 5fo hydrogenated coconut oil diet for 26 days (Table 15)* PGE^ produced a 1.6 fold increase in fatty acid synthesis, a 1.6 fold increase in glucose oxidation and a 1.5 fold increase in total lipid synthesis by adipocytes in the group fed hydrogenated coconut oil for 26 days, whereas in the normal group, it produced 1.1, 1.2 and 1.4 fold increases in fatty acid, CO2 and total lipid synthesis respectively. The greater response of adipocytes from hydrogenated coconut oil fed EFA deficient rats (fed for 26 days) to exogenous PGE-^ could mean that these rats are more defi cient in EFA and hence perhaps more deficient in prosta glandin than cells from rats on a fat-free diet. This is in agreement with results reported in Aaes-J^gensen’s TABLE 14 THE UTILIZATION OF GLUCOSE BY FAT CELLS FROM RATS FED EITHER A FAT-FREE DIET, A 5# CORN OIL DIET OR A % HYDROGENATED COCONUT OIL DIET

Conversion EFA-deficient^ of Glucose- Normal l-14c to: {5% Corn Oil) % HCNO1 (7 days) Fat-free

Control 19.6 + 2.4 35-9 + 4.4 36.6 + 2.20 C02 PGEi5 22.2 + 2.4 (1.13)4 45.6 + 11.7 (1.27) 49.7 ± 5.23 (1.35) Control 99.5 + 6.6 141.7 + 27.0 137.1 + 23.0 TL PGE]_ 117.3 + 4.1 (1.1$) 1*4.7 + 35.3 (1.30) 159.0 + 15.0 (1.16) Control 3.2 + 0.12 26.0 + 2.0 3 0.7 + 3.5 FA PGEi 3.55+ .16(1.12 37.3 + 3.6 (1.45) 36.2 + 3.7 (1.25)

1 Hydrogenated coconut oil (HCNO, 5$ by wt.) was fed for 7 days to rats previously on a fat-free diet from weaning. 2 Results were expressed as c.p.m./mg TG (mean + S.E.). 3 RGEi added in vitro at a concentration of 1 a g/ml. 4 The number in the parenthesis denotes the ratio of the results in the presence and absence of PGEi* 5 No significant difference was found in the results from the rats fed 5$ hydrogenated coconut oil for 7 days and fat-free diet in all the measurements including the incorporation of glucose-l-^c into labeled COg, total lipids and fatty acids. 6 Each number represents samples from 4 animals. TABLE 15

IN VITRO METABOLISM OF GLUCOSE BY FAT CELLS FROM ESSENTIAL FATTY ACID DEFICIENT Aim NORMAL RATS1 AFTER PGE-l ADDITION (c.p.m./mg TG)

Conversion of Glucose- 5% HCNoS i-!4c to: Normal^ 26 days HCNO/Nomal

control 21.53 35.2 1.6 co2 PGE1 25.66 (1.2)^ 56.1 (1.65)

control 61.76 115.9 TL 1.4 PG% 112.44 (1.37) 176.4 (1.53) control 3.46 24.2 FA 7.0 PGEi 3.96 (1.14) 46.2 (1.6)

1 Two animals were used in each group, with triplicate determinations for each animal. 2 A diet containing % corn oil was fed to rats after weaning, 3 A diet containing 5% hydrogenated coconut oil (HCNO) was fed to EFA deficient rats for 26 days. 4 The number in the parenthesis refers to the ratio of the level of metabolite with PGE-l to the control level. 71 review (10) stating that feeding saturated fats enhances EFA deficiency syndromes in rats* The feeding of corn oil at 1.4$ (approximately 2$ of the total calories) and 2,8% (4$ of the total calories). decreased the lipogenesis in adipocytes to 10.9 c*p*m./mg TG and 7 c.p.m./mg TG respectively after 7 days (Table 16). The rate of lipogenesis in adipocytes from rats fed a fat-free diet was 30.7 c.p.m./mg TG. Feeding unpurified methyl ole ate-*- (2.3 $^ oleate plus 2.7$ hydrogenated coconut oil) for 7 days decreased- the hyperlipogenesis from the fat-free level dovm to 19.6 c.p.m./mg TG (Table 16). That linoleate was responsible for inhibiting lipo genesis was confirmed by feeding pure methyl esters of oleate or linoleate for 10 days (2 .3$ oleate or linoleate plus 2.7$ hydrogenated coconut oil, Table 17). The latter was far more efficient in inhibiting lipogenesis than the former. The rates of fatty acid synthesis in the adi pocytes from the linoleate fed rats and the oleate fed rats were 5 c.p.m./mg TG and 24 c.p.m./mg TG respectively. Carbon dioxide production in adipocytes from linoleate fed rats was also lower than that from oleate fed.rats (17*4

The unpurified methyl oleate contains 79*5/2 oleate, 11.6$ palmitoleate, 3.3$ palmitate, 2 .6$ myristate, and some stearate.

^In 2 .6$ corn oil, the total amount of linoleate plus oleate is approximately 2 .3$. 72 TABLE 16 THE EFFECT OF FEEDING DIFFERENT LEVELS OF CORN OIL OR METHYL OLEATE ON THE METABOLISM OF GLUCOSE BY ISOLATED FAT CELLS FROM RATS1

Conversion of Glucose-•l-^C to Diets FA/TL co2 TL FA

Normal 19.13 67.56 3.63 5.4 % 2.8% C.O. 2 22.50+1.29 63.02+ 3.13 6.97+0.52 10,05% 1.4/5 C.O. 31.76+3.50 111.32+ 7.30 10,66+0.57 9.75% 2.3/5 Oleate 29.96+2.57 95.90+ 9.00 19.60+1.50 20 . 40^ 2.7% HCNO2 EFA-def 36.60+2.15 137.10+23.0 30.70+3.50 22.1(0%,

1 Corn oil or methyl oleate plus hydrogenated coconut oil were fed to EFA deficient (fat free diet) for 7 days. 2 C.O. and HCNO refer to corn oil and hydrogenated coconut oil respectively. 3 Results from normal (5% corn oil diet), corn oil and oleate plus hydrogenated coconut fed rats were obtained from 2 animals, with 3 determina tions for each. 4 The results of the EFA deficient (fat free diet) were from the previous experiment as shown in Table 14. 5 Results were expressed as c.p.m./mg TG (mean + S.E.). 73

TABLE 17 THE EFFECT OF FEEDING PURE METHYL LINOLEATE OR PURE METHYL OLEATE ON THE METABOLISM OF GLUCOSE BY ISOLATED FAT CELLS FROM RATS

Conversion of Glucose-l-*^fC to Diets o o TL FA FA/TL

2.3 fo linoleate 17.40+2.9 109.3+23.4 5.0+1.20 4*6^ 2.7% HCNO (3) 2*3% Oleate 29.15+1.3 129.2+30.3 23.9+6.29 2.7fo HCNO (3)

1 All results were expressed as c.p.m./mg TG (mean + S.E.) 2 Different diets were fed to EFA deficient rats for 10 days. 3 The number in the parenthesis refers to the number of animals in each group, three determi nations for each animal. 74 c.p.m./mg TG compared to 29 c.p.m./mg TG). These results showed that hydrogenated coconut oil fed to rats at the 5fa level had little or no effect on the rate of lipogenesis in cells from rats on a fat-free diet. Oleate had only a slight inhibitory effect while corn oil or methyl linoleate markedly supressed the lipogenesis. This is in agreement with the work of Sabine et al. (125)> Allman and Gibson et al. (32,146). Sabine et al. fed diets containing corn oil, safflower oil, coconut oil, tripalmitin, triolein and oleic acid at the 10^ level to mice for 3 days and studied acetate incopora- tion into liver fatty acids. They found that only corn oil or safflower oil (both rich in linoleic acid) were effec tive in reducing fatty acid synthesis. Allman and Gibson et al. (22,146) showed that with rats and mice on a fat-free diet, both liver and adipose tissue lipogenesis (acetyl CoA or malonyl CoA incorpora tion into fatty acids) were supressed by feeding 2 fo linoleate. They also reported that the high rate of hepatic fatty acid synthesis was correlated with a decrease in the linoleate content in liver lipids. It has been reported that hepatic and adipose tissue lipogenesis are profoundly influenced by nutritional status (147-149). Fasting or the feeding of diets rich in fat and low in carbohydrate leads to decreased lipo genesis. Leveille (150) found that increasing the level 75 of dietary fat up to or beyond 20 % decreased the ability of adipose tissue to incorporate glucose into fatty acids and concurrently, the activities of several enzymes involved in lipogenesis including glucose-6-phosphate dehy drogenase, 6-phosphogluconate dehydrogenase and malic enzyme in adipose tissue were all reduced. Reiser et al. (151) using.rats, found that all tri glycerides containing short, medium and long chain fatty acids, fed at the 30^ level, depressed hepatic fatty acid synthesis from acetate in vivo, but trilinolein and safflower oil were the most effective. The alteration in lipogenesis subsequent to the inclusion of fat in the diet will depend not only on the nature of the fatty acids, but also on their level in the diet. It has been generally believed that fatty acid syn thesis is regulated by long chain acyl CoA. Bortz et al. (152) reported that acetyl CoA carboxylase activity was blocked by fatty acyl CoA in vitro. In addition, he has reported an increase in the hepatic level of acyl CoA (after feeding corn) with a concomitant reduction in fatty acid synthesis (153)• The inhibition observed in these studies may not be entirely due to acyl CoA feedback inhibition alone, other authors have reported that fatty acid synthetase and other lipogenic enzymes are also decreased (154-156). 76 Inkpen et al. (154) have shown that linoleate exerts control upon the enzymatic activity for 9-desaturation of stearate to oleate. They have speculated that linoleate plays a regulatory role in desaturation and lipogenesis in the liver. Chu et al. (155) have shown that the regulatory effect of linoleate on hepatic fatty acid synthetase and glucose-6-phosphate dehydrogenase is also exerted by arechidonate and linolenate. Recently, Gibson et al. (156) found that several lipogenic enzymes in liver, including acetyl CoA carboxy lase, fatty acid synthetase, malic enzyme, glucose-6- phosphate dehydrogenase and citrate cleavage enzyme are all suppressed by feeding polyunsaturated fatty acids (linoleate, cf- and ^-linolenate and arachidonate). Other studies in their laboratory indicated that there is a net loss of enzyme protein during the adaptive fall in enzyme activity in vivo. Therefore, it is reasonable to speculate that polyunsaturated fatty acids (or a component containing or derived from them) may function as repressors in the synthesis of the lipogenic enzyme set (156). There appears to be no ready explanation as to why the various dietary fats differed in their ability to suppress fatty acid synthesis. The effect of depletion of essential fatty acids on adipocyte glucose oxidation and fatty acid synthesis was 77 studied (Table 13). Normal (5$ corn oil diet) adult rats (400 g) were placed on a fat-free or a 5?° hydrogenated coconut oil diet for either 7 or 32 days. Feeding the fat- free diet to normal adult rats for one week seemed to increase lipogenesis (from 3 .4 c.p.m./mg TG to 5.9 c.p.m./ rag TG) more than feeding hydrogenated coconut oil (from 3 .4 c.p.in./mg TG to 3.9 c.p.m./mg TG). However, after feeding fat-free or 5# hydrogenated coconut oil diets for 32 days, the rate of fatty acid synthesis was still very low. The increase was less than two fold compared to that of normal (from 3.4 c.p.m./mg TG to 5.5 c.p.m./mg TG).

TABLE 13 EFFECT OF DEPLETION OF EFA ON THE INCORPORATION OF GLUCOSE-l-^C INTO C02 OR FA OF NORMAL-FED ADULT RATS (c.p.m./rag TG)

Days of Depletion Type co2 FA

0 Normal 17.5(16.3-13.1) 3.4(3.2 -3 .6 ) 7 N->HCNO* 21.3(19.9-23.3) 3.9(3.3-4.0) 7 N no fat 22.7(22.0-23.4) 5.9(5.5-6.3) 32 N-HCNO 25.3(23.9-29.2) 5.4(4.3-6.2 ) 32 N->no fat 24.2(24.0-24.4) 5.5(3.7 -7 .3 )

* HCNO: 5$ hydrogenated coconut oil Allman and Gibson (22) have noted that mouse adipose tissue reflected the same changes in fatty acid composi tion as liver under conditions of linoleic acid deficiency but the degree was not as severe. They found that after placing young mice from laboratory chow to a fat-free diet for 21 days, the rate of hepatic fatty acid synthetase was increased 5 to 10 times that of the control animals. In these studies, when normal 4 month-old rats (400 g) were switched to a fat-free or a 5/5 hydrogenated coconut oil diet for 7 or 32 days, the rate of fatty acid synthesis from glucose in adipocytes was increased to 2 times that of the normal control but not increased up to the same level as that of the deficient (which had risen & to 10 times as high as the normal control). This per haps shows that it is very difficult to deplete linoleic acid from the adipocytes of an adult normal rat. This agrees with the finding of Stein and Stein (157) that the fatty acids of the adipose tissue of rats have a long half life (e.g., linoleic acid has a half life estimated to be 167 days). Oswald et al. (156) have shown that during development of linoleic acid deficiency in rats, the liver linoleic acid content (mg/liver) maintained roughly the same level during a 63-day period of depletion. The relative linoleic acid composition decreased because of the gain in the other fatty acids as the liver grew in sine. 79 B. Lipolysis The adipocytes from EFA deficient rats had a two fold higher basal and hormonal stimulated FFA release* Insulin (100 /Aunits/ml) and PGEi (l,Ag/ral) added in vitro had a tendency to lower the free fatty acid release in adipocytes from both normal and EFA deficient rats, but the decreases were not statistically significant. Epine phrine (0.1>ug/ml) caused a 1.36 fold increase in free fatty acid release in cells from normal rats and a 1.6 fold increase in cells from deficient rats. Epinephrine at a supramaximal concentration (7 Mg/ml) increased the free fatty acid release significantly (7 fold increase in cells from both normal and EFA deficient rats) (Table 19)* When epinephrine (7/Ug/ml) and PGEi (1/Hg/ml) were added together in vitro to adipocytes, the degree of inhibition by PGEi of epinephrine stimulated FFA release was about the same in both normal and EFA deficient adi pocytes, 50% inhibition (Table 20). The reason that insulin and PGEi not inhibit lipolysis significantly in adipoytes is perhaps also due to the age of the animals used. These results of lipolysis agree with the work of Paoletti's group (159) who found that essential fatty acid deficient rats were hypersensitive to lipid mobilizing agents including norepinephrine, theophylline, and dibutyryl cyclic AMP in epididymal adipose tissue or fat cells. so

?, They suggested that PGEjl may be lacking in the deficient animals. TABLE 19 LIPOLYSIS BY FAT CELLS OF NORMAL AND EFA DEFICIENT RATS

Basal Insulin PGE EoineDhrine 100/a units/ml 1/ig/ml O.l^g/ml 7/*g/ml (6) (4) (4) (3) (6) Normal 1.00+0.24 0.78 + 0.52 0.S9 + 0.28 1.86 + 0.40 7.06 + 1.3 Deficient 2.23 + 0.34 1.77 + 0.40 1.97 + 0.25 3.50 + 0.95 15.80 + 3.9

1 All results expressed as " m eq. EFA/g TG/2 hr" (mean + S.E.) 2 The number in the parenthesis equals to the number of animals used.

TABLE 20 EFFECT OF PGE, ON EPINEPHRINE STIMULATED LIPOLYSIS BY ADIPOCYTES FROM NORMAL AND EFA-DEFICIENT RATS

* Basal Epinephrine Ep inephrine +P GE^ Normal 0.77 6.5 3.3 Deficient 1.60 10.9 5.5

1 Three animals were used in each group. 2 All results are expressed as "/i eq. EFA/g TG/2 hr". 3 Concentrations of hormones added in vitro epinephrine = 7 >*g/ml PGB^ ® lyug/ml SUMMARY

1. Intraporitoneal injection of prostaglandin into rats fed an essential fatty acid-deficient diet did not enhance the lo\tf NADH oxidase ’’specific activity” of liver mitochondria, nor did it alter the abnormal fatty acid composition of the mitochondrial lipids. Feeding corn oil (57^) for two weeks increased NADH oxidase specific activity" to values approaching nor mal, restored the fatty acid composition of mitochon drial phospholipids to normal, and brought the body weight into the normal range.

2. Epididymal fat cells from rats fed a fat-free diet incorporated ten times more -^C from uniformly labeled glucose into fatty acids than did adipocytes from normal rats {5% corn oil diet). Adipocytes from rats fed a fat-free diet also had a two fold higher •^C-carbon dioxide production than those from normal rats. The rate of ^C-glycerol synthesis in adipocytes was not different in the two groups of rats although the rate of total lipid synthesis in adipocytes from the essential fatty acid deficient group was 1.6 times higher than that from normal. The percent of 62

o o the total lipid radioactivity in the fatty acid frac tion was 32 .5$ in adipocytes from rats fed a fat-free diet and 5*3$ in adipocytes from rats fed a 5$ corn oil diet. The high rate of lipogenesis observed in the adipocytes from rats 011 a fat-free diet was related to the absence of essential fatty acids in the diet, because when 5$ hydrogenated coconut oil was fed to these rats for 7 days, the rate of fatty acid synthesis in adipocytes was not significantly lowered. Even when the feeding period was extended to 26 days on the 5$ hydrogenated coconut oil diet, the level of lipogenic activity in ‘jhe adipocytes from these rats was still 7 fold higher compared to that of normal controls, When corn oil was fed at only 2 ,?% of the diet to rats for 7 days, the level of lipogenic activity in the adi pocytes dropped to 2 fold of normal. The inhibitory effect on lipogenesis was specifically related to the linoleic acid content of the diet. When 2.3/S pure methyl linoleate plus 2.7$ hydrogenated coconut oil were fed for ID days to rats previously on a fat-free diet, the rate of incorporation of from l-^C-glucose into fatty acids in adipocytes was 5 c.p.m./rag TG, whereas that in adipocytes from rats fed 2 .3$ pure methyl oleate plus 2 .7$ hydrogenated coconut oil diet for 10 days was almost 5 times higher When corn oil in the normal diet v/as either omitted or replaced by 5/5 hydrogenated coconut oil for 7 or 32 days in adult rats, the rate of fatty acid synthesis by the adipocytes was only doubled. The responses of the adipocytes from rats fed a normal or EFA deficient diet toward insulin, FOE-^, and epine phrine in glucose metabolism were compared. PGEi (1 yUg/ml) had a weak stimulatory effect on glucose oxidation, total lipid synthesis and fatty acid syn thesis. Epinephrine (7 /tg/ml) and insulin (100 ju units/ml) had a strong stimulatory effect on glucose oxidation and total lipid synthesis. The former enhanced glycerol synthesis whereas the latter increased fatty acid synthesis. Although the abso lute values were different in adipocytes from the two groups of rats, their percent stimulation toward these hormones were similar. Lipolysis (measured as free fatty acid release into the incubation medium) by epididymal fat cells from rats fed a normal or essential fatty acid deficient diet was aiso studied. As expected, cells from EFA deficient rats exhibited 2 times higher free fatty acid release both in the basal state and when stimu lated by epinephrine. PGE^ added in vitro at 1/tg/ml inhibited epinephrine stimulated free fatty acid release by 50% in adipocytes from both normal and EFA deficient rats. CONCLUSIONS

Studies in liver mitochondria show that intraperitoneal injection of prostaglandin E^ to rats can not correct the alterations in permeability to NADH in liver mitochondria isolated from rats fed an essential fatty acid deficient diet. Studies on lipogenesis in epididymal fat cells demonstrate that the effects of prostaglandin E;l and that of linoleic acid are completely opposite in direction under these experimental conditions. Prostaglandin E-i > added in vitro enhances lipogenesis slightly, but feeding methyl linoleate decreases it considerably. The high rate of lipogenesis observed in adipocytes from rats fed an essential fatty acid-deficient diet is due to a defi ciency in linoleic acid rather than a deficiency in prostaglandin. Studies on lipolysis in epididymal fat cells show that adipocytes from rats fed an essential fatty acid- deficient diet are hyperlipolytic, possibly related to a lack of prostaglandins.

66 LITERATURE CITED

.• 1. Bergstrom, S. and Carlson, L. A*, Acta Physiol. Scand., 64, 479 (1965). 2. De Pury, G. G. and Collins, F. D., Biochim. Biophys. Acta, 106. 213 (1965). 3. Das, M. L, Hiratsuka, H., Machinist, J. M. and Crane, F. L., Biochim. Biophys. Acta, 60. 433? (1962). 4. Rahm, Joseph J. and Holman, R. T., J. Lipid Res., £, 169, (1964). 5. Smith, J. A. and DeLuca, H. F., J. Cell Biol., 21, 15, (1964). 6. Smithson, Janet E., Lab. Invest. 20, 207, (1969). 7. Hayashida, T. and Portman, 0. V7., Proc. Soc. Exp. Biol. Med., 102., 656 (I960). 6. Johnson, R. M., Exptl. Cell Res., $ 2 , 11$, (1963). 9. Houtsmuller, U. M. T., Van Dor Beek, A., and Zaalberg, J., J. Lipids, 4» 571 (1969). 10. Aaes-J^rgensen,E., Physiol. Rev., 1* (1961). 11. MacMillan, A. L. and Sinclair, H. M., in H. M. Sinclair (Editor), Fourth International Con ference on Biochemical Problems of Lipids: Essential Fatty Acids, Butterv/orths Scientific Publishers, London, 1956, p. 206. 12. Ito, T. and Johnson, R. M., J. Nutr., %6, 215 (1966). 13. Berstrom, S., Carlson, Lars A. and Weeks, J. R., Pharmacol. Rev., 20, 1, (1966). 14. B8hle, E., Distschuneit H., Ammon, J. and Dobert, R., Deut. Ges. Inn. Med. 572, 465 (1966).

67 S3 15* Bohle, E., Rettberg, H., Dischunneit, H. H., Dobert, R. and Dischuncit, II., Verhandl. Deut. Ges. Inn. Med. 21* 797, (196?). 16. Haessier, II. A. and Crawford, J. D., J. Clin. Invest. 46, 1065 (1967). 17. Vaughan, M. In S. Bergstrom and B. Samuelsson (Editors), Prostaglandins, Proc. 2nd Nobel Sym posium, Stockholm, Juno, 1966. Almqvist and Wiksell, Stockholm; Interscience Publishers, New York, 1967, p. 139. 13. Crawford, J. D. and Haessier, II. A. in P. VI. Ramwell and J. E. Shaw (Editors), Prostaglandin Sympo sium of the Worcester Foundation for Experimental Biology, 1967, Interscience Publishers, New York, 1963, p. 103. 19. B8hle, E. and May, B., in P. V/. Rnmwell and J. E. Shaw (Editors"), Prostaglandin Sympossium of the V/ocestor Foundation for Experimental Biology, 1967, Interscience Publishers, Nev/ York, 19o8, p. 115* 20. Burton, D. N., Collins, J. M., Kennan, A. L. and Porter, J. W., J. Biol. Chem., 244* 4510, (1969). 21. Allman, D. V/., Fed. Proc., 21, 233 (1962). 22. Allman, D. W. and Gibson, D. M., J. Lipid Res • * 6, 51 (1965). 23. Alfin-Slater, R. B. and Aftergood L., Physiol • Rev. 43, 753 (1963). 24. Schlenk, H. and Sand, D. M., Biochim. Biophys. Acta, 1/tit, 305 (1967). 25. Fulco, A. J. and Mead, J. F., J. Biol. Chem., m * 1411 (1959). 26. Mead, J. F., Fed. Proc., 20, 952 (1961). 27. Mohrhauer, H. and Holman, R. T., J. Lipid Res * * lk» 346 (1963). 23. Rieckehoff, I. G., Ho3nan, R. T. and Burr, G. 0., Arch. Biochem., 20, 331 (1949). 69 29. Holman, R. T. J. Nutr., JO, 405 (I960). 30. Mohrhauei’, H. and Holman, R. T., J. Lipid Res., 4, 151 (1963). 31. Verdino, B. Blank, M. L., Privett, 0. S. and Lundberg, V/. 0., J. Nutr., 63., 234 (1964). 32. Sprecher, H., Lipids 2.* 14 (1966). 33. Holman, R. T., and Mohrhauer, H., Acta Chcm. Scand., 12 , S84 (1963). 34. Brenner, R. R., De Tomas, M. E., and Peluffo, R. 0., Biochim. Biophys. Acta, 106, 64O (1965). 35. Brenner, R. R. and Nervi, A. M., J. Lipid Res., 6, 363 (1965). 36. Brenner, R. R. and Jose, P., J. Nutr., 65, 196 (1965). 37* Brenner, R. R. and Peluffo, R. 0., J. Biol. Chem., 241, 5213 (1966). 38. Pefullo, R. 0., Brenner, R. R. and Hercuri, 0., J. Nutr., 61, 110 (1963). 39* Uchiyama, M., Nakagawa, M. 14. and Okui, S., J. Biochem., 62, 1 (19o7). 40. Stein, 0. and Stein, Y., Biochim. Biophys. Acta, 84, 621 (1964). 41. Johnson, R. M., and Ito, T., J. Lipid Res., 6, 75 (1965). 42. Hayashida, T. and Portman, 0. W., Arch. Biochem. Biophys., Jl, 206 (I960). 43. Tulpule, P. G. and Williams, J. N., Jr., J. Biol. Chem., 212, 229 (1955). 44. Haya.shida, T. and Portman, 0. W., J. Nutr., 81, 103 (1963). 45. Levin, E., Johnson, R. M. and Albert, S., J. Biol. Chem., 228, 15 (1957). 46. StancliiT, R. C., Williams, M. A., Utsumi, K. and Packer, L., Arch. Biochem. Biophys., ljl, 629(1969) 90 47* Ito, T. and Johnson, R. M., J, Biol* Chem., 239. 3201 (1964). 4$. Trojan, L. E., Ph.D. Thesis, Ohio State Univ., 1966 49. Chance, B. and Williams, G. R., J. Biol. Chem., 217. 409 (1955). 50. Goldblatt, M. W., J. Soc. Chem. Ind. (London) 52, 1056 (1933). 51. Goldblatt, M. V7., J. Physiol. (London) S4, 20S, (1935). 52. Euler, U. S. von, Arch. Exp. Pathol. Pharmakol., 76, (1934). 53* Euler, U. S. von, J. Physiol. (London) C4, 21P, (1935). 54* Bergstrom S., Nord. Med., 42 , 1465 (1949). 55. Horton, E. V7., Physiol. Rev., 7^2, 122 (1969). 56. Bergstrom, S., Science, 157. 3&2 (1967). 57* Berstrom. S., Recent Prog. Hormone Res., 22, 153 (1966). 53. Pickles, V. R., Bio. Rev., 42 , 614 (1967). 59* Samuelsson, B., Angev/andte Chmie 4., 410 (1965). 60. Bergstrom, S., Krabisch, L. and Sjovall, J., Acta Chem. Scand. 14. 1706 (I960). 61. Bergstrom, S. and Sjovall, J., Acta Chem. Scand. 11, 1066 (1957). 62. Bergstrom, S. and Sjovall, J., Acta Chem. Scand. 14. 1693 (I960). 63. Bergstrom, S.. and Sjovall, J., Acta Chem. Scand. 14, 1701 (I960). 64* Bergstrom, S., Krabisch, L., Samuelsson, B. and SjUvall, J., Acta Chem. Scand. 16, 969 (1962). 65. BergstrHm, S., Ryhage, R., Samuelsson, B. and Sjovall, J., Acta. Chem. Scand. 12271 (1963). 91 66. Bergstrom, S., Ryhnge, It.., Samuelsson and Sjovall, J., J. Biol. Chem., 2.33. 3555 (1963). 67* Bergstrom, S. and Samuelsson, B., J. Biol. Chem., 237. PC 3005 (1962). 66. Kugteren, D. H., Dorp, D. A. van, Bergstrom, S., Hemberg, M. and Samuelsson, B., Nature (London) 212. 38 (1966). 69* Hamberg, M. and Samuelsson, B. in S. Bergstrom and B. Samuelsson (Editors), Prostaglandins, Proc. 2nd Nobel Symposium, Stockholm, June, 1966, Almqvist and Wiksell, Stockholm: Interscience Publishers, New York, 1967* p. o3* 70. Remwell, P. W. and Shav;, J. E. in S. Bergstrom and B. Samuelsson (Editors), Prostaglandins, Proc. 2nd Nobel Symposium, Stockholm, June, 1966, Almqvist and Widsell, Stockholm; Interscience Publishers, New York, 1967* P* 283. 71. Shaw, J. E. and Remwell, P. V/., J. Biol. Chem., 243. 1498 (1968). 72. Dorp. D. A. van, Beerthuis, R. K., Nugteren, D. H. and Vonkeman, H., Biochim. Biophys. Acta, £0, 204 (1964). 73. Dorp. D. A. van, Beerthuis, R. K., Nugteren, D. H. and Vonkeman, H., Nature (London) 203, 839 (1964). 74* Bergstrom, S., Danielsson, H., Klenberg, D. and Samuelsson, B., J. Biol. Chem., 239. PC 4006 (1964). 75* Bergstrom, S., Danielsson, H. and Samuelsson, B., Biochim. Biophys. Acta, 90. 207 (1964). 76. Vaughan, M. and Steinberg, D. in A. E. Renold and G. F. Cahill, Jr. (Editors), Adipose Tissue, Handbook oT Physiology, Sec. 5* Am. Physiol. Soc., Washington D. C., 1965, p. 239. 77. Steinberg, D. and Varghan. in S. Bergstrom and B. Samuelsson (Editors), Prostaglandins, Proc. 2nd Nobel Symposium. Stockholm, June, 1966, Almqvist and Wiksell, Stockholm; Interscience Publishers, Hex-; York, 1967* p. 109. 92 76. Steinberg, D* end Varghan, M. in A. E. Renold and G. F. Cahill, Jr. (Editors), Adipose Tissue, Handbook of Physiology, Sec. 5, Am. Physiol. Soc., Washington D.C., 1965, p. 335* 79. Robinson, G. A., Butcher, R. V7. and Sutherland, E. V/., Ann. Rev. Biochem., 2Z> 149 (1966). 60. Birnbaumer, L. and Rodbell, M., J. Biol. Chem., 244. 3477 (1969). 61. Butcher, R. V7., Ho, R. J., Meng, H. C., Sutherland, E. W., J. Biol. Chem., 2^0 4515 (1965). 62. Butcher, R. V7., Baird, C. E., Sutherland, E. W., J. Biol. Chem., 24?. 1705 (1966). 63. Butcher, R. W., Baird, C. E., Sutherland, E. W., J. Biol. Chem., 2M , 1713 (1966). 64. Butcher, R. W. and Sutherland, E. V/., Jr., J. Biol. Chem., 222, 1244 (1962). 65. Vaughan, M. and Steinberg, D., J. Lipid Res., 4, 123 (1963). 66. Rizack, II., J. Biol. Chem., 239* 392 (1964). 67. Butcher, R. W. and Baird, C. E. in W. L. Holmes, L. A. Carlson and R. Paoletti (Editors), Drugs Affecting Lipid Metabolism, Plenum Press, New York, 1969, p. 5. 66. Blecher, M., Merlino, N. S. and Ro*Ane, J. T., J. Biol. Chera., 24,3. 3973 (1966). 69. Steinberg, D., Vaughan, M., Nestel, P. J,, Strand, 0. and Bergstrom, S., J. Clin. Invest., 43. 1533 (1964). 90. Stock, K., Aulich, A. and Y7esterinann, E., Life Sciences, 2» 113 (1966). 91. Paoletti, R., Puglisi, L. and Usaidi, 11. M., Pharma cology of Hormonal Polypeptides and Proteins, Plenum Press, New York, 1966, p. 425. 92. Fain, J. II., Ann. N. Y. Acad. Sci. 139. 679 (1967). 93. Bergstrom, S., Carlson, S. A. and Oro, L., Acta Physiol. Scand. 60, 1?0 (1964). 93 94. ICupiecki, F. P., J. Lipid Res., 3, 577 (1967). 95. Butcher, R. W. end Sutherland, E. V/., Ann. N. Y. Acad. Sci., 1£2, 349 (1967). 96. Butcher, R. V/., Pike, J. E. and Sutherland, E. V/. in S. Bergstr&i and B. Samuelsson (Editors), Prostaglandins, Proc. 2nd Nobel Symposium, Stockholm, June, 1966, Almqvist and Wiksell, Stockholm; Intorscience Publishers, New York, 1967, p. 133. 97. Humes, J. L., Mandel, L. R., and Kuehl, F. A. Jr., in P. V/. Ramwe.li and J. E. Shaw (Editors), Prostaglandin Symposium of the Worcester Founda tion for Experimental Biology, 1967, Interscience Publishers, New York, 1963, p. 79. 93. Fain, J. E. in P. W. Ram well and J. E. Shaw (Edi tors), Prostaglandin Symposium of the Worcester Foundation for Experimental. Biology, 1967, Interscience Publishers, Hew York, 1963, p. 67. 99. Weeks, J. R. and Walk, H. A., Pharmacologist, 2* 254 (1967). 100. Bergstrom, S., Carlson, L. A. and Oro, L., Acta Physiol. Scand., 6£, 135 (1966). 101. Carlson, L. A., Progr. Biochem. Pharmcol., 2* 94 (1967). 102. Butcher, R. W., Sneyd, J. G. T., Park, C. R. and Sutherland, E. W., Jr., J. Biol. Chem., 241. 1651 (1966). 103. Corbin, J. D., Sneyd, J. G. T. and Park, C. R., in preparation. 104. Sanbar, S. S., Mitchell, S. A., Lockell, S. A. Lockey, R. F., Vleck, E. A. and Greene, Jr., J. Am. Clin. Res., 1£, 410, (1967). 105. BcJhle, E., Dobert, R., Ammon, J. and Beyer, J. in N. Henning and G. Berg (Editors), Fette in der Medizin, Series 7, Pallas Verlag, Munich, 1966, p. 20. 94 106. Paoletti, R., Lentati, R. L. and Korolkiewievri.cz, Z. in S. Bergstrom, and B. Samuelsson (Editors), Prostaglandins, Proc. 2nd Nobel Symp., Stockholm, June, 1966, Almqvist and l/iksell, Stockholm, Interscience, New York, 1967, p. 147* 10?. Berti, F., Kabir Naimzada, M., Lentati, R., Mantegazsa, P. and Paoletti, R., Progr., Biochem. Pharmcol. 3., 110 (1967). 106. Berti, F., Lentati, II. E. Grafnetter, D., Boll. Soc. Itnl. Biol. Sperim., l£_, 515 (1967). 109. Berti, F. Lentati, R. Usardi, M. II., Boll. Soc. Xtal. Biol. Sperim., 41, 1327 (1965). 110. Bergstrom, S., Carlson, L. A. and 0r&, L., Acta Physiol. Scand., 67, 14-1 (1966). 111. Steinberg, D. and Pittman, R., Proc. Soc. Exp, Biol. Med., 122, 192 (1966). 112. Crofford, 0. B. and Remold, A. E., J. Biol, Chem. 240, 14 (1965). 113. Rodbell, M., Recent Prog, in Hormone Res., 24. 215 (1965). 114. Kono, T., J. Biol. Chem., 244. 1772 (1969). 115. Cuatrecasas, P., Proc. Natl. Acad. Sci., 63, 450 (1969) 116. Lynn, W. S., Mac Leod, R. M. and Brown, R. H., J. Biol. Chem., 221, 1904 (I960). 117. Hagen, J. H., J. Biol. Chem., 236, 1023 (1961). 116. Vaughan, M., J. Biol. Chem., 236. 2196 (1961). 119. Rodbell, M. in A. E. Renold and G. F. Cahill, Jr. (Editors), Handbook of Physiology, Sec. 5, Adipose Tissue, Am. Physiol. Soc., V7ash. D.C., 1965, P. 47. 120. Butcher, R. V7. and Sutherland, E. W., Ann. N. Y. Acad. Sci. 12.9, 471 (1967). 121. Rodbell, M v J. Biol. Chem., 242 , 5751 (1967). 95 122. Bray, G. A. and Goodman, H. M., J. Lipid Res., 2» 714 (196$). . 123. Blecher, M., Merlino, N. S., RofAne, J. T, and Flynn, P. D., J. Biol. Chem., 244, 3423 (1969). 124. Favarger, P in A. E. Renold and G. F. Cahill, Jr. (Editors), Handbook of Physiology, Sec. 5, Adipose Tissue, Ara. Physiol. Soc., Washington D.C. 1965, p. 19. 125. Sabine, J. R., McGrath, Grath, H. and Abrahem, S., J. Nutr., 2$, 312 (1969). 126. Baer, E. and Buchnea, D., J. Biol. Chem., 230. 447 (195$). 127. Holman, R. T., J. Am. Oil Chem. Soc., 422 (1956) 12$. Sreenivassan, B. and Holla,K. S., J. Am. Oil Chem. Soc., 44, 313 (1967). 129. AOCS Spectroscopy Committee, 1959-1960, J. Am. Oil Chem. Soc., 2$, 1$0 (1961). 130. Schneider, W. C., J. Biol. Chem., 176. 259 (1946). 131. Hayahara, B., Symposia on Enzyme Chem. (Japan), 12, 312 (1961). 132. Miller, G. L., Anal. Chem., £L, 964 (1959). 133. Chance, B. and Williams, G. R,, J. Biol. Chem., 217. 363 (1955). 134. Johnson, R. M. and Kerur, L., Biochim. Biophys. Acta, 20, 152 (1963). 135. Metcalf, L. D, and Schmitz, A. A., Anal. Chem. 33. 363 (1961). 136. Gottenbos, J. J., Beerthuis. R. K. and Dorp, D. A. van, in S. Bergstrom, Proc. 2nd Nobel Symposium, Stockholm, June, 1966, Almqvist and Wiksell, Stockholm, Interscience Publishers, New York, 1967, p. 57. 137. ICupiecki, F. P., Sekhnr, M. C. and Weeks, J. R., J. Lipid Res., % 602 (196$). 13$. Rodbell, M., J. Biol. Chem., 239, 375 (1964). 96 139. Bray, G. A., Anal. Biochem., 1, 279 (I960). 140. Dole, V. P., J. Clin. Invest. 21> 150 (1956). 141. Stein, I. and Shapiro, H. R., Brit. J. Clin, Path., 6, 15iJ (1953). 142. Dole, V, P. and Ileinertz, H., J. Biol. Chem., 235. 1965 (I960). 143. Hirsch, J., Farguhar, J. V/., Ahren, E, H., Peterson, M. L. and Stofl’el, W., Am. J. Clin. Nutr., 6, 499 (I960). 144. Solyom, A., I-luhlbachova E. and. Puglisi, L., Biochim. Biophys. Acta, 137. 427 (1967). 145. Gries, F. A. and Sleinke, J., J. Clin. Invest,, 46, 1413 (1967). 146. Allman, D. 17. Hubbard, D. D. and Gibson, D. M., J. Lipid Res., 6, 63 (1965). 147. Masoro, E. J., Chnikoff, I. L., Chernick, S. S. and Felts, J. M., J. Biol. Chem., 13£, 4^3 (1950). 14&. Felts, J. M., J. Biol. Chem., 1S£, 4S3 (1950). 149. Hausberger, F. X. and Milstein, S. V/., J. Biol. Chem. 2JJb, 463 (1955). 150. Leveille, G. A., J. Nutr., £1, 267 (1967). 151. Reiser, R., Williams, M. C., Sorrels, M, F. and Murty, M. L., Arch. Biochem. Biophys., 102. 276 (1963). 152. Bortz, V/. M., and Lynen, F., Biochem. Z., 337. 505 (1963). 153. Bortz, 17. II., Biochim. Biophys. Acta, 137* 533 (1967). 154. Inkpen, C. A., Harris, R. A. and Quackenbush, F. V/., J. Lipid Res., 10, 277 (1969). 155. Chu, L. C., Me Intosh, D. J., Hincenbergs, I. and Williams, M. A., Biochim. Biophys. Acta, 1^2, 573 (1969). 97 156. Kuto,' Yasutoshi find. Gibson. I). Me, Biochem. Biophys. Res. Comm., 1 (197GJ.

157. Stein. Y. end Stein, 0., Biochim. Biophys. Acta 60, 53 (1962).

156'. Oswald, R., Bouchard, P., ililjanich, P. and Lyman, R. L., Biochem. J., 4S5 (1965).