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Plasma lipid variations in response to diet and exercise McKenzie, Donald Chisholm 1972

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PLASMA LIPID VARIATIONS IN RESPONSE TO DIET AND EXERCISE By DONALD CHISHOLM McKENZIE B.Sc.(P.E.), University of Guelph, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHYSICAL EDUCATION in the School of Physical Education and Recreation We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1972 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of physical Education The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The purpose of this study was to determine the plasma l i p i d variations during periods of low calorie diet and low calorie diet plus increased physical a c t i v i t y . Four male graduate students, with above normal ac t iv i ty levels , volunteered for the 1 0 week study which was divided into f ive experimental periods. The f i r s t , or control condition involved a two week period during which the subjects r e -ceived a regular diet of normal foods equivalent to approximately 3600 calories per day. During this period 'normal' a c t i v i t y was maintained. The second treatment condition involved a 1 0 day period of a low calorie diet , with continued 'normal' a c t i v i t y . The low calorie diet was equivalent to approximately 1800 calories da i ly , of natural foods, plus one multiple vitamin p i l l . The th ird experimental period was s imilar to the control period; a two week period during which the subjects received approx-imately 4 0 0 0 ca lor ies , per day, of the regular d i e t . Again, 'normal' ac t iv i ty was maintained. The fourth treatment condition was indent ical to the second experimental period with the addit ional requirement of increased d a i l y energy expenditure, per subject, of 5 0 0 ca lor ies . The f i n a l experimental period consisted of a two week period of the regular diet with 'normal' a c t i v i t y . Blood samples were taken, following an overnight fast , twice during each experimental period: once mid-way through the period and again at the end. Plasma tr ig lycer ide and free fatty acid concentrations were measured in duplicate in each sample. The results of orthogonal comparisons among treatment means showed a s t a t i s t i c a l l y s ignif icant increase i n the plasma free fat ty acid concentration during the low calorie diet and the low calorie diet plus exercise treatment conditions. Increased mobilization of free fatty acids from adipose tissue tr ig lycerides i n response to the insuff ic ient dietary supply of substrates for metabolism was cited as the mechanism responsible for the r ise i n free fatty acid concentration. Neuman-Keuls method was used to examine the effect of the increased physical a c t i v i t y during the low calorie diet periods; the results showed that the increased physical ac t iv i ty had no s ignif icant effect on the plasma free fatty acids . Similar s t a t i s t i c a l procedures applied to the plasma tr ig lycer ide values showed a s ignif icant decrease i n the plasma tr ig lycer ide concen-trat ion during the low calorie diet and the low calorie diet plus increased physical ac t iv i ty periods. The stress of the low calorie diet on the habitually active subjects was responsible for the decreased leve ls . The l i p i d and carbohydrate content of the normal and the low calorie d ie ts , as wel l as increased peripheral uptake of tr ig lycer ides , were suggested as possible explanations for the plasma tr ig lycer ide changes. The addit ional da i ly output of 500 Calories during the second stress condition was not of suff icient magnitude to e l i c i t a further decrease in plasma tr ig lycer ide concentration. ACKNOWLEDGEMENTS The author would l ike to express his sincere gratitude to the School of Home Economics, U . B . C , for the use of t h e i r research f a c i l i t i e s for the analyses of the l i p i d s . I would also l i k e to thank the members of my thesis committee; Dr. S.R. Brown, Dr. I .D . Desai, Dr. R. W. Schutz and especial ly Dr. K.D. Coutts, chairman, for their time and effort i n examining this thes i s . The author i s also indebted to Miss Jane Noble for her help i n many facets of this study. TABLE OF CONTENTS CHAPTER PAGE I . STATEMENT OF THE PROBLEM 1 Introduction . 1 Purpose of the Study 2 Delimitations 2 Limitations 2 Hypotheses 2 Significance of the Study 3 Definition of Terms 3 I I . REVIEW OF THE LITERATURE 5 Introduction 5 Related Study 5 Nutri t ional Influence on Plasma TG and FFA 7 Effects of Exercise on Plasma TG and FFA 9 Hormonal Regulation of Plasma TG and FFA 15 Growth Hormone 15 Glucagon 16 Insulin 17 Epinephrine and Norepinephrine 19 Autonomic Control of Fat Mobilization 19 Chapter Summary 20 I I I . METHODS AND PROCEDURES 25 Subjects 25 Experimental Periods 25 i i i l l CHAPTER PAGE Experimental Design . 2S S t a t i s t i c a l Analysis . , 23 IV RESULTS AND DISCUSSION 30 Results 30 S t a t i s t i c a l Analysis , 31 Discussion 37 Free Fatty Acids 37 Triglycerides 41 V SUMMARY AND CONCLUSIONS 43 Summary . . . . . . . . . . . . . . * . . . . . . . . . . . . . . . . . • « • • « * « • . < . . . 43 Conclusions ;,), REFERENCES 45 APPENDIX A RAW SCORES 55 iv LIST OF TABLES TABLE PAGE I . Experimental Design 2 ^ n . Means and Standard Deviations 30 I I I . Orthogonal Comparisons: Free Fatty Acids 34 IV. Orthogonal Comparisons: Triglycerides 34 V. Summary of Anova: Free Fatty Acids 3 5 VT. Summary of Anova: Triglycerides . . . . . . . . 36 VII . Neuman - Keuls Analyses: Plasma Lipids 37 V LIST OF FIGURES FIGURE PAGE 1. Fat Transport Cycle: At Rest 21 2. Fat Transport Cycle: Insufficient Caloric Intake ..... 22 3. Plasma Free Fatty Acid Variations 32 4. Plasma Triglyceride Variations 33 CHAPTER I STATEMENT OF THE PROBLEM Introduction This study represented a portion of a larger invest igation undertaken by the Divis ion of Human Nutri t ion, School of Home Economics, U . B . C , and the School of Physical Education and Recreation, U . B . C , during the summer of 1971. The purpose of the entire study was to i n -vestigate work performance on a semi-defined, low calorie diet during d a i l y periods of l ight and moderately heavy physical a c t i v i t y , and to measure various physiological and biochemical parameters deemed relevant. Since stress conditions such as insuff ic ient ca lor i c intake, physical ac t iv i ty , and hormonal and nervous variat ions, increase the t issue requirements for l i p i d s (Carlson and Pernow, 1961; Carlson et a l . 1965; Renold and Cahi l , 1965; Gollnick et a l . 1970); measures of l i p i d metabolism were included i n th is investigation and constitute the main effort i n th is thes is . Generally, i n response to stress, the tr ig lycer ides stored i n adipose t issue are hydrolyzed and the l iberated fa t ty acids are transported i n an albumin-free fatty acid complex to the tissues (Fredrick-son and Gordon, 1958 (b); Olson and Vester, I960). The plasma free fat ty acids have been shown to be a major fue l source for oxidative metabolism during exercise and times of insuff ic ient ca lor ic intake (Dole, 1956; Gordon and Cherkes, 1956; Laure l l , 1956; Basu et a l . I960; Rodahl, 1964; Kuel, 1970; Pruett, 1970; Horstman, 1971; Misbin et a l , 1971). On the basis of this relationship, the measurement of tr ig lycer ides and free fatty acids were made. 1 2 Purpose of the Study The purpose of this study was to determine the variations in plasma triglyceride and free fatty acid levels during a low calorie diet period and a low calorie diet plus exercise period. Delimitations 1. This study of lipids was confined to the triglycerides and free fatty acids contained in blood plasma, and their changes during the length of the experimental periods. Limitations 1. The subjects involved in this study were volunteers and highly active and therefore not representative of a random sample. Hypotheses A. Free Fatty Acids 1. The level of plasma free fatty acids increases during the low calorie diet. 2. The level of plasma free fatty acids increases during the low calorie diet plus exercise condition. B. Triglycerides 1. There is no change in the level of plasma triglycerides during the low calorie diet. 2. During the low calorie diet plus exercise period the level of plasma triglycerides is decreased. 3 Significance of the Study The principal value of the study was to determine the changes in plasma lipids during the experimental conditions stated previously, thus expanding the knowledge in this area. Plasma lipid variations have been extensively investigated in sedentary subjects or subjects with some form of pathological disorder. This study is unique in that the plasma triglyceride and free fatty acid concentrations are measured in subjects who are habitually active and involved in approximately two hours of daily physical activity. This study of plasma lipid variations during diet and exercise may also be of- use to researchers in the area of coronary heart disease and atherosclerosis. There is considerable evidence implicating ab-normal lipid metabolism in the development of coronary heart disease and atherosclerosis. In addition, lipid mobilization in obesity is also of interest as this condition certainly is one of the more common ailments in our society today and its presence has been correlated with many car-diovascular disorders. Definition of Terms Triglycerides. (TG) Generally defined as compounds in which each of the alcohol groups of glycerol is esterified with a fatty acid (Masoro, 1 9 6 8 ) . Fatty Acid. Any monobasic aliphatic acid containing only carbon, hydrogen, and oxygen and made up an alkyl radical attached to the carboxyl group. The saturated fatty acids have the general formula C L CL; there n <sn *L are also several series of unsaturated fatty acids having one or more double bonds. Free Fatty Acids. (FFA) Fatty acids not i n ester or amide linkage (free carboxyl group). Also termed unesterified fatty acids (UFA), or non-esteri f ied fatty acids (NEFA). Hydrolysis. Generally defined as the s p l i t t i n g of a compound by the addition of water. For the purposes of this study, i t refers to the breakdown of the tr ig lycer ide molecule to glycerol and free fatty acids. L i polys i s . The s p l i t t i n g up, or chemical decomposition of fa t . Lipase. An enzyme that catalyzes the hydrolysis of ester linkages between the fatty acids and glycerol of the t r ig lycer ides . Lipogenesis. The formation of fat; the transformation of non-fat food materials into body fa t . Es ter i f i ca t ion . For the purpose of this thesis i t may be defined as the synthesis of the tr ig lycer ide molecule, bas ica l ly the reverse of hydrolysis . Lipemia. The presence of an abnormally high concentration of fat or l i p i d i n the blood. CHAPTER II REVIEW OF THE LITERATURE Introduction The published material on l ip ids i s enormous and an effort has been made to confine the l i terature review so le ly to those areas that pertain d i r e c t l y to the problem. Reports on the effects of res tr ic ted ca lor ic intake and increased ca lor ic expenditure on plasma tr ig lycer ides and FFA are extensively reviewed, while possible neural and hormonal mechanisms involved i n these relationships are more generally recorded. Related Study There i s only one study analogous to the present invest igat ion. Carlson and Froberg (1967) observed blood l i p i d and glucose levels during a ten day period of low calorie intake and exercise. Data were obtained on 12 men, aged 20 - 50, who walked 50 km. each day, for ten days. Daily ca lor ic intake was estimated to be about 200 calories; vitamins and minerals were supplied as tablets , i n doses s l i g h t l y above minimum r e -quirements. Levels of glucose, FFA, g lycerol , cholesterol , phospholipids and TG i n serum, were measured, after an overnight fast , immediately pr ior to the study, and on the t h i r d , s ix th , and tenth day of the walk. The l eve l of blood glucose decreased s ign i f i cant ly u n t i l the s ixth day, but by the tenth day the l eve l had r isen above the i n i t i a l standard. The levels of FFA and glycerol paral le led each other. They were increased during the f i r s t period of the study but had decreased by day ten, such that the concentration of g lycerol was s t i l l s ign i f i cant ly 5 6 elevated above the i n i t i a l value, but the FFA concentration was only slightly above the i n i t i a l measurement. The serum cholesterol concentra-tion decreased progressively from the f i r s t day. The decrease was statistically significant by day three. The concentration of the phos-pholipids decreased until the third day, remained about the same until day 6, and then decreased further. The plasma triglyceride level was decreased markedly during the walk; however, this decrease was confined to the very low density lipoproteins. In their interpretation of these results, Carlson and Froberg suggest that the metabolic events changed in the middle of the study. As an example, they cite the biphasic changes in the concentrations of plasma FFA and blood TG. They also explain that the increase in plasma glycerol, accompanied by the increase in FFA, demonstrated the increased mobilization of FFA by means of increased lipolysis of adipose tissue triglycerides. The decrease in plasma TG was attributed to the effects of the walk rather than the problem of malnutrition. They believe that this decrease may be due to: 1) diminished hepatic synthesis of plasma lipo-proteins, which may have occurred as a result of low availability of fatty acids; 2) increased uptake of TG by peripheral tissues due to increased perfusion of these tissues; and/or 3) increased amounts of lipoprotein lipqse. With regards to the plasma cholesterol levels, the authors believe that exercise per se, or fasting, does not decrease the plasma 7 cholesterol leve l ; however, "when weight loss occurs i n connection with physical tra ining, there are indications that the cholesterol l eve l i n plasma f a l l s . " Carlson and Froberg. (1967:630). Nutr i t ional Influence on Plasma TG and FFA Keys and his colleagues (1950) published a comprehensive treatise on human starvation and undernutrition which has since become a c lass ic referance to studies i n this area. Thirty-two subjects v o l -unteered for the study (the Minnesota Experiment) which was divided into four experimental periods: a control period of 12 weeks with approximately 3500 Calories per day; a semi-starvation period of 24 weeks with approx-imately 1600 Calories per day; a 12 week period of restr icted r e h a b i l i t a -t i on ; and an 8 week period of unrestricted rehabi l i ta t ion . During the 168 days on the low calorie diet the subjects lost an average of 6k% of body fat ; i n spite of this change i n body fat content, there was very l i t t l e change in any of the l i p i d fractions studied. Total plasma l i p i d s , the phospholipids, and the serum cholesterol were measured and although a l l three l i p i d fractions decreased, only the results on serum cholesterol were s t a t i s t i c a l l y s igni f icant . Keys offers a p a r t i a l explanation to the absence of any lipemia on the basis that the t o t a l fat metabolism during the semi-starvation period was approximately 57£ of the value for the control period. The effect of undernutrition on the l eve l of plasma FFA- i s wel l known. The concentration of plasma FFA increased due to increased mobil-izat ion from adipose t i ssue. 8 Gordon (1957), measured the arteriovenous unesterified fatty acid (UFA) differences in adipose tissue and skeletal muscle of the forearm in normal,' fasting subjects. He reported that the fatty acids were entering the circulation from the adipose tissue and that the myocardiam and skeletal muscle tissues were capable of extracting the U.F.A. This study has been supported by other researchers who also have concluded that the adipose tissue TG represents the major source of circulating FFA (Laurell, 1956; Gordon and Cherkes, 1956; Carlson and Froberg, 1967). The effects of a low calorie diet on the plasma TG are not so well defined. Kartin et a l . (1944) studied serum lipid changes during fasting and conditions of undernutrition. His results showed only a small i n -significant alteration of the plasma TG concentration. On the other hand, Rubin and Aladjem (1954) have shown that plasma TG are significantly elevated as a result of fasting. Carlson and Wadstrom (1956) investigated glyceride levels in four healthy young individuals, two females and two males, fasted for 63 hours. Their results showed an increase in the t r i -glyceride level only, there was no change in the concentration of the di and monglycerides. This increase in the triglyceride level was a t t r i -buted to increased lipid moblilization to meet the energy requirements during caloric restrictions. R.J. Havel, in response to this paper, commented that he had found considerably reduced quantities of glycerides in fasting individuals. When glucose or sucrose was administered to previously fasted men, the plasma triglyceride level decreased (Bragdon et a l . 1957; Baker et a l . 1968). These lowered levels have been shown to persist up to 8 to 9 11 hours (Havel, 1957). It i s probable that this reduction of plasma tr ig lycer ide concentration by glucose ingestion i s mediated by the stimulation of insu l in secretion. Dole (1965) and Gordon and Cherkes (1956) suggested that one of the probable ways which glucose acutely interferes with the plasma tr ig lycer ide levels i s through the lowering of the concentration and the turnover rate of FFA. M i l l e r (1967) showed that the t o t a l amount of plasma FFA and glycerol converted into l i v e r tr ig lycer ides and the ir release into the plasma was decreased during a glucose load. Also, since the endogenous synthesis of hepatic fa t ty acids was s t i l l depressed during the f i r s t few hours after administration of glucose (Baker et a l . 1968), the formation of l i v e r and plasma tr ig lycer ide ceases simply as a result of the lack of the available precursor (FA). Effects of Exercise on Plasma TG and FFA During exercise, energy i s derived both from fats and carbo-hydrates (Astrand and Rodahl, 1970). The plasma FFA have been shown to be an important source of energy during exercise (Easu et a l , 1960j Havel et a l . 1963; M i l l e r , Issekutz and Rodahl, 1963; George and Vallyathan, 1964; Havel et a l . 1964; Keul, 1970; Horstman et a l , 1971). During the f i r s t 10 to 15 minutes of exercise the plasma concentration of FFA decreases due to increased efflux of FFA from the plasma to the peripheral tissues (Friedberg et a l . I960 (a); Havel et a l . 1963). Carlson and Pernow (1961) have suggested that the increased efflux of plasma FFA is due to the increased amount of FFA perfusing muscular tissue per unit time. I f the exercise i s continued, the FFA leve l i s increased as a result of enhanced mobilization of FFA from adipose 10 tissue (Friedberg et a l . 1963; Havel et a l . 1964.) At the cessation of exercise cardiovascular adjustments return more rapidly to basal levels than do the metabolic changes, thus the plasma concentration of free fatty acids first increases rapidly, then slowly returns to basal levels (Carlson and Pemow, 1961; Friedberg et a l . 1963). Lipolysis during exercise is commonly thought to be controlled by the adrenergic system (Gollnick, 1970). Havel (1963:1060) suggested that: " ... mobilization of fatty acids during exercise may result from augmented activity of sympathetic nerves in adipose tissue with consequent local liberation of norepinephrine, a potent activator of triglyceride hydrolysis in this tissue". Havel and Goldfien (1959) and Vendsal (I960) have shown that the level of circulating catecholamines during exercise is elevated and the fact that both norepinephrine and epinephrine stimulate lipolysis (Horstman et al. 1971; Rudman, 1963) support the hypothesis that the adrenergic system controls lipolysis during exercise. However, numerous nonadrenergic substances have been shown to induce lipolysis and these, too, may exert some control over lipolysis during exercise. Rudman (1963) has shown that such nonadrenergic hormones as ACTH, growth hormone, glucagon and several other pituitary polypeptides possess adipokinetic activity. Of these, ACTH would seem to be the most important during exercise as i t is a rapid stimulant of lipolysis, and i t has been shown to increase in concentration during physical activity (Hunter et a l . 1965). 11 Hunter and Sukkar (1968) have shown that the plasma insulin concentration falls during exercise. This fact, combined with the knowledge that in small concentrations, insulin inhibits fat mobili-zation (Dole, 1956; Havel and Goldfien, 1959) may indicate that this f a l l in plasma insulin assists the mobilization of FFA during exercise. It is a well known principle that during heavy exercise, the blood lactate concentration increases. Miller et a l . (1963) and Issekutz (196/*.) have shown that lactate inhibits lipolysis and they suggest that the buildup of lactate during exercise may supress the plasma FFA concentration. Rodahl (1964) has demonstrated that the plasma FFA concen-tration depends upon the intensity and duration of exercise. During heavy, short work bouts the FFA concentration was shown to drop, pos-sibly due to the rapid increase of the blood lactic acid level. When the workload was such that i t could be continued for several hours, the blood lactate remained practically unchanged and the plasma FFA con-centration rose. These changes in plasma FFA are caused by the acute effects of exercise. These alterations are important to the over-all picture of FFA mobilization however, as the blood sampling period was immediately after an over-night fast, i t is the chronic effects of exercise on FFA that pertains more directly to this study. Carlson and Permow (1961) studied the plasma FFA concentration during and after exercise. Immediately after exercise the FFA level increased rapidly, then slowly decreased in concentration! 30 minutes 12 after exercise the values were only s l i g h t l y greater than the rest ing measurements. Havel et a l . (1964) investigated the change in plasma FFA during and after two hours of moderate exercise. During the exercise period the plasma FFA values rose as expected. At the cessation of exercise, again, the FFA values rose rapidly and then f e l l , u n t i l the resting levels two hours after exercise showed no difference when com-pared to the pre-exercise values. In view of the fact that FFA are the most metabolically active of the plasma l ip ids (Harper, 1971), these results are not supris ing. Thus i t is quite probable that the acute effects of exercise during the days p r i o r to the blood sampling period would not be reflected i n the rest ing values the next morning. Experiments pertaining to the acute effects of exercise on the concentration of plasma TG have indicated a decrease i n this l i p i d fract ion and although a l l the factors involved have not been c l e a r l y established, a summary of the known mechanisms contributing to the reduction i n plasma TG concentration i s presented. Since the major s i te of plasma TG pro-ducation i s the l i v e r (Carlson and Ekelund, 1963; N i k k i l a , 1969), the decreased plasma TG l eve l could be a result of reduced hepatic production of TG. FFA are important precursors to the l i v e r , and therefore, the plasma TG (Havel and Goldfien, 1961: Friedberg, 1961; Havel et a l , 1962), and although i t i s known that during physical a c t i v i t y the mobil ization of the FFA is increased, Kagenfeldt and '.telren (1971) have demonstrated a l inear increase in the uptake of FFA i f increased blood flow to the 13 hepatic region is significantly reduced. These facts would suggest that the uptake of FFA by the liver, would be decreased during exercise, which would therefore result in a decreased production of liver TG. To support this hypothesis, Havel et al (1964) have shown a reduction in the amount of labelled FA incorporated into plasma TG during exercise as compared to resting values. There is an hypothesis that during exercise the hepatic production of lipoprotein peptides (LP) may be decreased (Carlson, 1967) and thus the vehicle used to transport the TG in plasma may not be available. This could be a limiting factor in hepatic TG release (Nikkila, 1969). S t i l l another factor to be considered in the reduction of plasma TG due to ex-ercise, is the role of lipoprotein lipase activity (VLPL). Nikkila et a l . (1963) have shown that the activity of this enzyme system is increased in the myocardium as well as in skeletal muscle during exercise. As this enzyme is responsible for the hydrolysis of circulating TG i t may well be an important factor in the decrease of plasma TG. Holloszy et a l . (1964) studied the chronic effects of exercise on the serum cholesterol, phospholipid, and triglyceride levels of middle-aged men. Two groups of subjects were involved in the study. Group A consisted of 15 men, a l l of whom had led sedentary lives for three or more years. They participated in a progressively more strenuous program of endurance calisthenics and distance running (2 to 4 miles) on an average of 3.35 times per week for six months. Five nonexercising control subjects were included in this group to serve as a check on the seasonal variations in serum lipid values. Group B was made up of 12 men, also having led sed-entary lives for a number of years. They participated in a program of distance running geared to their individual capacity. 14 Three fasting blood samples were obtained on each subject over a seven day period pr ior to the exercise program to establish baseline values. Thereafter, fasting blood samples were obtained once a month u n t i l the last week of the study, when three samples were again taken over a seven day period. Total serum cholesterol levels were determined once a month, while phospholipids and tr ig lycerides were measured every other month. The serum tr ig lycerides were measured i n 14 subjects i n Group A. The mean value for this group decreased from 208+127 mg% to 125+78 mg£ during the s ix month period. At the completion of the exercise program several men with i n i t i a l l y high tr ig lycer ide levels were asked to remain sedentary for five or s ix days. Fasting levels were determined at the end of this period and then the men were instructed to run three miles . Triglyceride values were measured at 2, 3, 20, and 44 hours following this run. After the f ive or s ix days of i n ac t i v i ty the fast ing tr ig lycer ide l eve l had increased considerably over the f i n a l values determined at the end of the exercise program (an average of 150 mg$). Within two or three hours following the three mile run a reduction was evident and th is r e -duction persisted for the 44 hour period. The authors suggest that the effect of exercise may be cumulative. Certain subjects had lower fast ing serum tr ig lycer ide levels after a number of days of exercise program than they did following a single period of exercise consisting of a three mile run. Thus exercise does produce a chronic effect on the plasma t r i -glyceride concentration. 15 Hormonal Regulation of Plasma TG and FFA Hormones may have a facilitating or permissive action on lipid metabolism (Carlson et a l . 1965). In 1958, Laurell and Chris-tensson conducted an experiment to determine the effect of a single dose of various hormones on the plasma FFA, and where indicated, the plasma TG and Phospholipids. The hormones injected were: growth hormone (GH), prolactin (LH), adrenocorticotropic hormone (ACTH), noradrenaline, adrenaline and glucagon. They concluded that ACTH and GH had no sig-nificant effect on plasma FFA; LH and glucagon had a similar reducing effect on plasma FFA; noradrenaline increased the FFA concentration to roughly the same extent as a corresponding dose of adrenaline. No changes in plasma TG or phospholipids were observed in this experiment. There appears to be some conflict between these conclusions and those of more recent investigation. Growth hormone. Henneman and Henneman (I960) have shown that intravenous administration of GH results in a prompt rise of plasma FFA and that continued daily doses of GH sustain the rise in plasma FFA. In a similar study Rabinowitz et a l . (1965) demonstrated not only that GH increases the release of FFA from adipose tissue but also that GH enhances FFA uptake by muscle tissue. Recently, Felig (1971) conducted experiments which support the findings of Rabinowitz. He also noted that the increase in plasma FFA, with administration of GH, was paralleled by a rise in blood glycerol, thus supporting the theory that the increase in FFA was due to lipolysis in adipose tissue. 16 Possibly the most influential study on GH was conducted by Roth et al, (1963). They have shown that the rate of secretion of GH was markedly stimulated by hypoglycemia, fasting, interference with glucose utilization, and by muscular exercise. In view of the fact administration of GH results in rapid release of fatty acids from • adipose tissue (Raben and Hollenberg, 1958; Henneman and Henneman I960), they interpreted their results as follows: ... endogenous plasma growth hormone concentrations are strikingly increased in a variety of physiologic and ex-perimental conditions known to be associated with high con-centrations of unesterified fatty acids in plasma. Thus, hypoglycemia, exercise, fasting and interference with glucose utilization by means of deoxyglucose are a l l followed by secretion of growth hormone, a response that provides for increased availability of a monocarbohydrate source of oxL-dizable substrates, namely fatty acids. Roth et a l . (1963:579) Glucagon. Unger and ELsentrout (1964) have identified glucagon in the efferent plasma of the pancreas and their demonstration of alterations of its secretion, induced by changes in blood glucose concentration, supported the view that glucagon is a true hormone with a major role in blood glucose regulation. Glucagon secretion has been shown to rise during a l l forms of glucose need (Unger et a l . 1962; Unger et a l . 1963). Thus, Unger and Eisentrout (1963:1031) consider glucagon, "... a hormone of glucose need, the function of which is to maximize hepatic glucose pro-duction when food is not available, thereby serving to maintain the flow of glucose to the brain". Lipsett et al (I960) examined the effects of glucagon on plasma FFA. They injected glucagon intravenously and analyzed the blood glucose and plasma FFA concentrations at 0, 1, 2, k, and 6 hours after injection. 17 One hour after injection the plasma FFA concentration was significantly decreased; by the second hour the FFA level had returned to the i n i t i a l value. However, at the fourth and sixth hours, the levels were signi-ficantly increased above the base-line value. In view of these results the authors stated: "A reasonable hypothesis is that glucagon effects the release of unesterified fatty acids from fat depots." Lipset et a l . (1960:352). Steinberg et a l (1959) conducted studies on rat adipose tissue in vitro and demonstrated an eight fold increase in the release of unes-terified fatty acids in the presence of glucagon. They also administered glucagon to a fasting dog, which caused a i n i t i a l f a l l in plasma FFA during the hyperglycemic phase, followed by a sustained rise (2 to 10 hours) after blood glucose had returned to normal. Lipsett, Engel and Bergenstal (1959) suggest that glucagon possesses activity apart from its effects on glycogenolysis and glucose utilization. Intravenous injection of glucagon during fasting resulted i n i t i a l l y in a slight f a l l of plasma FFA. Three to six hours later, when the blood sugar concentration was normal, the plasma FFA concentration had increased two to three fold. Control injections of saline were f o l -lowed by slight increases in plasma FFA due to the continued fast. Thus, they concluded that glucagon affects FFA metabolism independently of carbohydrate metabolism. Insulin. The permeability of the ce l l membrane for glucose depends on the plasma insulin concentration. If an overnight fast is continued from 8:00 p.m. to 10:00 a.m., subjects at rest show a steady f a l l in plasma insulin concentration (Sukkar et al. 1967). 18 Insufficient caloric intake is associated with increased mobil-ization of plasma FFA from adipose tissue stores and increased utilization of fat for oxidative metabolism. Dole (1956) has shown that in very small concentrations, insulin inhibits fat mobilization and therefore the f a l l in plasma insulin during malnutrition may assist fat mobilization. Bierman, Schwartz and Dole (1957) studied the effect of insulin on the release of fatty acids from adipose tissue stores. Using labelled palmitic acid, they were able to show that insulin decreased the release of fatty acids from tissue stores but i t did not accelerate the removal of fatty acids from blood. This inhibitory effect of insulin of the release of fatty acids and glycerol from adipose tissue was investigated by Jungas and Ball (1963). They interpretated the function of insulin, with regards to FFA, in three manners: 1) Insulin may have a direct or indirect inhibitory effect on the adipose tissue lipase. 2 ) Insulin could conceivably accelerate the steps whereby monoglyceride or diglyceride recombines with free fatty acids. 3) Insulin may accelerate the conversion of glycerol to glycerophosphate by activating the latent glycerokinase enzyme. This would permit a re-synthesis of diglyceride by way of phosphatidic acid. In either of the last two cases a re-esterification of free fatty acids would be promoted. Jones and Arky (1965) reported that in normal humans a single insulin injection did not change the serum triglyceride level but a pro-longed insulin infusion produced a marked f a l l in a l l subjects. Haahti (1959) has described a nondiabetic patient with hyperlipemia, in which 19 insulin treatment effectively reduced a l l plasma lipids. Epinephrine and Norepinephrine. Studies in vivo in man (Laurel and Christensson, 1958; Havel and Goldfin, 1959), and in animals (Spitzer and Hohenleitner, 1961) have a l l shown increased levels and increased output of FFA after injection of catecholamines. Carlson and Oro (1963) have studied the effects of norepine-phrine on the release of fatty acids from adipose tissue and their results showed that the main effect of norepinephrine was on the lipolysis in adipose tissue and not on the re-esterification process. Rizack (1961) has shown a similar lipolytic response of adipose tissue when exposed to epine-phrine . White and Engel (1958) studied the effects of epinephrine and norepinephrine on rat adipose tissue in vitro. They concluded that both suprarenal hormones stimulate production of nonesterified fatty acids from rat adipose tissue, presumably by stimulating the hydrolysis of neutral fats within the tissue. Autonomic Control of Fat Mobilization The autonomic nervous system is concerned principally with internal adjustments of the organism and i t has been shown that this system is i n -volved with the responses of those endocrine glands that receive a nervous supply (Morgan, 1965). Beznak and Hasch (1937) were the fi r s t investigators to suggest that mobilization of fat from adipose tissue involved the autonomic system. This hypothesis has been confirmed by other researchers (Havel and Goldfin, 1959; Confalonieri et al . 1961). 20 Two types of adrenergic receptors have been demonstrated by Ahlquist (1948), the alpha- and the beta- adrenergic receptors. Both receptors have been shown to exist i n human adipose t issue, where the alph-adrenergic receptors inhibited l ipo ly s i s while the beta-adrenergic receptors activated l ipo lys i s i n the adipose tissue (Efendic, 1970; Fredholm and Karlsson, 1970; Ostman and Efendic, 1970). Adipose tissue contains appreciable stores of the sympathetic transmitter substance, norepinephrine, and these stores have been shown to be depleted after cutting the autonomic nerve supply (Sidman et a l , 1962; Stock and Westerman; 1963). Conversely, C o r r e l l (1963) has shown that direct stimulation of the autonomic nerve supply can result i n rapid r e -lease of fat ty acids from adipose t i ssue . Chapter Summary Figures 1 and 2 are presented as a summary of the available l i t erature on the fat transport cycle as i t pertains to plasma FFA and TG. At rest (Figure 1), there i s an equilibrium exist ing between the FFA hydro-lyzed and re-ester i f ied within the adipose t issue; the c i rcu la t ing TG hydrolyzed to FFA by the vascular l ipoprotein l ipase (VLPL); and the FFA incorporated into plasma TG i n the l i v e r . Thus the plasma concentrations of TG and FFA remain constant. Figure 2 represents the fat transport cycle during times of i n -suff ic ient caloric intake. There i s considerable evidence that the plasma concentration of FFA i s increased during stress of this nature and this increase has been shown to be a result of l ipo lys i s of the adipose tissue tr ig lycer ides . Changes i n the c ircu lat ing hormone levels of GH, glucagon and i n s u l i n mediate th is response. The fate of the plasma TG during ca lor ic 21 PLASMA To other LIVER tissues ADIPOSE TISSUE FIGURE 1. FAT TRANSPORT CYCLE : AT REST 22 FIGURE 2. FAT TRANSPORT CYCLE : CALORIC INSUFFICIENCY 23 insufficiency however, is open to controversy, and no concrete theories have been made. During exercise the plasma FFA concentration is increased due to lipolysis of the adipose tissue triglycerides. This process is controlled, to a large extent, by the adrenergic system, although numerous non adre-nergic mechanisms such as increased amounts of GH, ACTH, glucagon; decreased plasma insulin concentration, and the production of lactic acid, have been shown to exert some control over lipolysis during exercise. However, these acute effects of exercise on plasma FFA are diminished within 2 hours after the cessation of the activity and therefore these changes in plasma FFA are not reflected in the blood samples taken after an overnight fast. Thus there are no chronic effects of exercise on plasma FFA. In addition, there is no evidence present to suggest what effects habitual exercise has on the endocrine secretions that regulate the plasma FFA and TG levels. The actue effects of exercise on plasma TG are well documented; there is a decrease in the circulating levels dur to a number of factors. -The blood flow to the hepatic region is reduced during exercise, which, accompanied by the increased peripheral uptake of the FFA, accounts for reduced quantities of precursor for the endogenous TG. Also, the activity of the vascular lipoprotein lipase is increased during exercise causing hydrolysis of some of the circulating TG. In addition, there is evidence that the hepatic synthesis of the lipoprotein peptides (LP) may be de-creased during exercise as well as the factor of increased peripheral uptake of the circulating TG by the working muscles. These acute effects remain for at least UU hours and there is strong evidence to suggest that habitual exercise produces a cumulative decrease in plasma TG concentration. CHAPTER i n METHODS AND PROCEDURES Subjects Four male graduate students, aged 23 to 27, from the Univer-s i t y of B r i t i s h Columbia, volunteered for the ten week study which began May 17, 1971. Prior to the f i r s t treatment condition the sub-jects were given a medical examination. This was done to ensure that a l l biochemical and physiological parameters f e l l within the normal ranges. None of the subjects was considered obese or overweight. The subjects had higher than 'normal' ac t i v i ty l eve l s . Three of the subjects were involved i n competitive athlet ics during the summer and spent approximately two hours per day i n t ra in ing; the other subject supplemented his 'normal' ac t iv i ty with swimming and jogging i n order to closely approximate the levels of ac t i v i ty for a l l subjects. Experimental Periods The subjects were housed together on campus for the entire summer, May - September, 1971. The weights of the subjects were r e -corded da i ly , upon r i s i n g , for the duration of the invest igat ion. Each meal was prepared by a qual i f ied d ie t i c ian , and the food weighed so that each subject received the same amount of food, with the same ca lor ic content. An additional serving of each meal was prepared, blended and frozen for the analyses of the constituent nutrients . 25 There were five experimental periods; these were performed consecutively. In the f i r s t , or control period, the subjects received approximately 3600 Calories per day of a regular diet which consisted of normal foods which met or surpassed those nutr i t i ona l requirements recommended i n the Dietary Standard for Canada. During th i s two week control period 'normal' a c t i v i t y was maintained; per iodica l ly , d a i l y records of ac t iv i ty were recorded to ensure that a 'normal' l e v e l was being continued. The second treatment condition involved a 10 day period of a low calorie diet with continued 'normal' a c t i v i t y . The low calorie diet amounted to approximately 1800 Calories of a semi-defined diet consisting of natural foods which, combined with one multiple vitamin p i l l per day, met or surpassed those nutr i t i ona l requirements rec -ommended i n the Dietary Standard for Canada. The entire allotment of food for the day, i n the form of eight biscuits or a loaf , plus 21 grams of margarine, was given to the subjects early i n the morning to be con-sumed, as they wished, pr ior to 12 o'clock midnight. One vitamin p i l l per day was also given to the subjects and an unlimited supply of tea or coffee (without cream or sugar) was also avai lable . There were no res tr ic t ions as to the amount of water consumed. The third experimental period was ident i ca l to the control period; a two week period during which the subjects received approx-imately 4000 Calories, per day, of the regular d i e t . Again, 'normal' ac t i v i ty was maintained. 27 The second stress situation, the fourth treatment condition, was similar to the second experimental period with the additional re-quirement of increased daily energy expenditure, per subject, of 500 Calories. In order to achieve this goal, the subjects had to jog six miles in approximately 50 minutes or swim continuously for i+0 minutes. This was generally broken into two exercise sessions. The final experimental period was identical to the third treat-ment condition; a regular diet with 'normal' activity. Blood samples were taken twice during each experimental period, once mid-way through the period and again at the end. After an over-night fast, approximately 25 milliliters of blood were drawn from the antecubital vein of each subject. Approximately three ml were stored with various reagents or used immediately for the determination of hematocrit, hemoglobin concentration, vitamin C and glucose analyses. Of the remaining whole blood, roughly one half was allowed to clot and the serum removed and frozen; the rest of the whole blood was stored using heparin as the anticoagulant. Later, upon thawing, the serum was removed from this portion also. The plasma was stored in a freezer for approximately 18 weeks prior to the analyses. The free fatty acids were analyzed in duplicate by the colorimetric, micro-method of Laurell and Tibbling, Clinica Chimica Acta. 16:57-62, 1967, using the serum stored without heparin. The heparinized serum was used in the duplicate determination of the plasma triglycerides by the enzymatic method of Schmidt and von Dahl, Z. klin. Chem. 6, 156-159, 1968, obtained in a kit form from the Boehringer Mannheim Company, Biochemical Department. 28 Experimental Design The purpose of this investigation was to determine the plasma lipid variations during diet and exercise. Thus, the lipids contained within the plasma, specifically the triglycerides and free fatty acids, represented the dependent variable, while the diet and level of physical activity, depicted the independent variables, or the variables manipulated during the study. This study is representative of a single group, repeated mea-sures design with the fi r s t treatment condition acting as a control for the remaining four experimental periods. See Table I r Statistical Analyses The data obtained on the triglyceride and free fatty acids were analyzed statistically and represented graphically. A planned, rather than post-hoc, comparison was chosen to test the hypothesis. DunnettTs method (Winer, 1962) for comparing a l l means with a control mean was used. Theoretically, as this is a planned comparison, an analysis of variance between means was not necessary, however, as the denominator of Dunnett's method require the MS error term, an analysis of variance to test the significance between means was calculated. 29 TABLE I EXPERIMENTAL DESIGN TREATMENT CONDITIONS Control Blood Sample 1 2 Day 7 14 Regular Diet + Normal 2 Stress Situation 1 3 k 6 10 Low Calorie Diet + Normal Recovery Physical Physical Act iv i ty Act iv i ty 5 6 7 14 Regular Diet + Normal Physical Act iv i ty k Stress Situation 2 7 8 5 10 Low Calorie Diet + Increased Physical Ac t iv i ty Recovery 9 10 7 14 Regular Diet + Normal Physical Act iv i ty CHAPTER IV RESULTS AND DISCUSSION Results The means and standard deviations of the plasma free fatty acid and triglyceride levels determined for each blood sampling period are presented in Table II. TABLE II MEANS AND STANDARD DEVIATIONS BLOOD SAMPLE FFA mmoles/l TRIGLYCERIDES mg/lOO ml 1 0.373 + 0.073 115.63 ± 25.97 2 0.307 + 0.121 110.27 + 18.96 3 0.592 + 0.258 87.71 + 5.40 4 0.540 + 0.168 77.33 + 7.49 5 0.325 + 0.132 94.51 + 12.37 6 0.297 + 0.119 105.97 + 19.28 7 0.688 + 0.130 70.88 + 10.22 8 0.500 + 0.187 56.56 + 10.81 9 0.267 + 0.092 98.45 ± 25.73 10 0.228 + 0.115 104.89 + 20.96 30 31 These values are also presented graphically in Figures 3 and h. Visual inspection of the data suggested a possibility of hetero-geneity of variance, which would render the analyses of variance and orthogonal comparisons invalid. However, Eartlett's test (Edwards, 1964) was applied to the data and the results concluded that the variances were homogeneous. Statistical Analyses As the recovery values of the FFA and TG did not return to the control values, Dunnett's method for comparing a l l means with a control could not be used to test the hypotheses. Orthogonal comparisons of treatment means (Edwards, 1964) were chosen as an alternate method to serve this purpose. Although there were two blood samples per experimental period, the most appropriate values to use to test for a significant difference were the final means for each condition, that i s , the values immediately prior to the experimental per-iod as compared to the final values for the treatment condition. Therefore, to test the changes in the plasma lipids due to the low calorie diet, the final lipid values of condition 1 were compared to the final values of condition 2. Si mi laxly, the final lipid values of condition 3 were com-pared to those of condition 4 in order to examine the effects of the low calorie diet plus increased physical activity on the plasma lipids. The following Tables summarize the results of these comparisons. 0.900-1 0.BD0H BLOOD SRHPLE 1 FIGURE 3. PLASMA FREE FATTY ACID VARIATIONS 150 .D-i 140.0-130.0-120.0-1)0.0-100.0-90.0-80.0-70.0-60.0-50.0-40.0-y-0 BLOOD SAMPLE FIGURE 4. PLASMA TRIGLYCERIDE VARIATIONS -1 r 3 r 2 T 4 10 TABLE III ORTHOGONAL COMPARISONS: FREE FATTY ACIDS COMPARISON df Sd CONDITION 1 VS. CONDITION 2 27 CONDITION 3 VS. CONDITION 4 27 0.083 0.083 2.81 2.45 <.01 <.05 TABLE IV ORTHOGONAL COMPARISONS: TRIGLYCERIDES COMPARISON df Sd CONDITION 1 VS. CONDITION 2 27 CONDITION 3 VS. CONDITION 4 27 10.75 10.75 3.06 4.69 < .01 <.01 35 Subsidiary issues concerning the experimental periods, such as the effect of increased physical a c t i v i t y during the low calorie diet condition, were considered relevant to the study and post-hoc analyses of these comparisons were carried out. As this was a post-hoc examination, an analysis of variance among the means for both the FFA and the TG was necessary pr ior to any further comparisons. The f a c i l i t i e s of The University of B r i t i s h Columbia Computing Centre were used and the results are summarized as follows. TABLE V SUMMARY OF ANOVA: FREE FATTY ACIDS v SOURCE df MEAN SQUARE F P SUBJECTS 3 0.096 CONDITIONS 9 0.098 7.18 < .01 ERROR 27 0.014 39 36 TABLE VI SUMMARY OF ANOVA: TRIGLYCERIDES SOURCE df MEAN SQUARE F P SUBJECTS 3 888.339 CONDITIONS 9 1438.330 6.23 < .01 ERROR _ 231.011 39 Neuman - Keuls method (Winer, 1962) of examining the nature of the differences among treatment means was used following the s ignif icant o v e r - a l l F ratios of the FFA and TG. The results of th is procedure used, i n the discussion section of th is chapter are presented below. 37 TABLE VII NEUMAN - KEULS ANALYSES: PLASMA LIPIDS COMPARISON PLASMA LIPID q.95 (r;27) cal .q.95 RESULT FFA .169 .040 N.S. CONDITION 2 VS. CONDITION 4 TG 26.68 20.77 N.S. Discussion The subjects had a higher a c t i v i t y l eve l than "normal" and although the d a i l y recommended allowance for the subjects (3600 C a l -ories) was observed for the f i r s t treatment condition, the subjects showed a gradual loss of weight, indicat ing that a d a i l y intake of 3600 Calories was not suff ic ient to maintain ca lor ic balance. Therefore, you would ex-pect the low calorie d ie t , which contained f i f t y percent of the recommended calorie allowance, to be a considerable stress on the ca lor ic equil ibrium of the body and th is stress to be further compounded by the effect of the increased physical a c t i v i t y . Free Fatty Acids. The results of the orthogonal comparisons have shown that the plasma FFA leve l was s ign i f i cant ly elevated (F£05) during both the 38 low calorie diet and the low calorie diet plus exercise conditions. This i s not surprising as i t i s wel l known that the plasma FFA are related to the nutr i t iona l state of the subject (Dole, 1956; Gordon and Cherkes, 1956). The low calorie diet in i t i a t ed a negative ca lor ie balance of approximately 1800 Calories per day per subject. In addition, during the low calor ie diet plus exercise period, da i l y increased physical a c t i v i t y accounted for an addit ional 500 Calories creating a t o t a l d e f i c i t of approximately 2300 Calories per day for this period. Therefore, as the diet was not providing suff ic ient substrates to supply the metabolic processes of the body, the source of these substrates must have been of an endogenous nature. It i s an accepted principle that the plasma FFA serve d i r -ect ly , along with glucose, as the major substrates for metabolism (Andres et a l , 1956; Carlson and Pernow, 1959; Friedberg et a l , I960 a; Friedberg et a l , I960 b ) . In addition, Gordon and Cherkes (1956), Carlson and Oro (1963), Baker et a l (1968) and others have shown that there i s a def ini te relationship that exists between carbohydrates and l i p i d s i n substrate u t i l i z a t i o n . I f the nutr i t iona l requirements are sat i s f ied by carbo-hydrates, the plasma FFA concentration drops to a low l e v e l , probably due to the secretion of i n s u l i n , which i s a known inh ib i tor of FFA release (Dole, 1956 a ) . Conversely, i f glucose i s not available i n suff ic ient amounts to sat is fy the ce l lu lar requirements, the l eve l of FFA i n plasma r i ses . S t a t i s t i c a l analysis of the serum glucose l eve l ^ showed a s i g -nif icant decrease (p<.05) i n this variable during the diet and the diet plus exercise conditions, indicat ing that the glucose stores were being depleated (Issekutz et a l , 1966). Therefore, the r ise i n plasma FFA (1) Variables, other than l ip ids taken during this study are available from the Divis ion of Human Nutri t ion, School of Home Economics, UBC. 39 concentration, during the low calorie diet and the low calorie diet plus exercise periods, followed a logical and previously demonstrated pattern. The rise in plasma FFA was a result of increased mobil-ization of FFA from the triglycerides stored as adipose tissue (Gordon and Cherkes, 1956; Fredrickson and Gordon, 1958 a; Fredrickson and Gordon, 1958 b; Basu, I960; Carlson and Oro, 1963). The contribution of the short chain fatty acids (C<10), absorbed directly from the gastrointestinal tract, can be considered very minor (Fredrickson and Gordon, 1958 a), especially considering the small amount of fat in the low calorie diet. The only other source of FFA is the circulating plasma TG; however, the activity of the vascular lipoprotein lipase, which acts on the TG-lipoprotein com-plex to liberate the FFA, would not contribute significantly to the plasma FFA level at rest (Honkhouse et al, 1961; Persson et al, 1970). Considering the data obtained from the anthropometrical measurements, which showed a decrease in various skin fold thicknesses during the low calorie diet and the low calorie diet plus exercise periods, and the accompanying decrease in the percentage of body fat, as calculated from the body density mea-sures, a decrease in the size of the adipose tissue during these periods was indicated and supports the fact that hydrolysis of adipose tissue t r i -glyceride was responsible for the elevated plasma FFA level. Neuman - Keuls method was used to examine the differences between the lipid values observed during the low calorie diet and the low calorie diet plus increased physical activity periods. The results showed no significant difference (P>,05) between the final FFA values observed during each period, nor was there statistical significance be-tween the samples taken at the mid-points of these experimental conditions. Therefore the increased caloric expenditure, during the low calorie d ie t , had no s t a t i s t i c a l l y s ignif icant effect on the plasma FFA leve l as mea-sured during this study. Certainly a l l the l i terature concerning plasma FFA levels and exercise has indicated that these levels are increased s ign i f i cant ly during exercise; however, as the FFA are known to be the most metabolically active of the plasma l i p i d s (Harper, 1971), i t i s probable that the acute effect of exercise was diminished pr ior to the blood sampling. The graph of the plasma FFA variations (Figure 3) shows that the highest values of the FFA were attained at the mid-point of each stress s i tuat ion, rather than at the termination of each condition as would be expected. Similar findings have been reported by Carlson and Froberg (1967) who studied 12 men during a 10 day period of very low calorie diet and exercise. The plasma FFA l eve l increased during the f i r s t s ix days but had decreased by the tenth day to a concentration only s l i g h t l y above the i n i t i a l value. . - The authors did not attempt to explain this phenomenon and merely proposed that the metabolic events changed during the middle of the study. However, the changes i n the plasma FFA were paral le led by the plasma glycerol levels which indicates that the FFA increase was due to l ipo lys i s of the adipose tissue TG (Carlson and Oro, 1963). This would suggest that at the end of the 10 day period the adipose tissue was mobilizing less FFA than at the mid-point; therefore, i t i s possible that other substrates were being used to sat isfy the ce l lu lar requirements, thus decreasing the demand for FFA. Triglycerides. The plasma triglyceride level was significantly decreased (P <.01) during the low calorie diet and the low calorie diet plus i n -creased physical activity periods; a finding which contradicts the hypo-thesis that the plasma TG concentration would not change during the low calorie diet period. There was no significant change in the plasma TG concen-tration during the control period (blood samples 1 and 2) , indicating that any effects of habitual exercise on the plasma TG levels had plateaued. Also, Neuman - Keuls method of comparing treatment means showed no statis-tically significant difference (P >.01) between the TG values obtained during the low calorie diet and the low calorie diet plus increased physical activity treatment conditions. Therefore the decrease in plasma TG con-centration during the two stress situations is attributed solely to the effects of the low calorie diet, quite a unique finding. In addition, i t is evident that an additional daily energy output of 500 Calories does not affect the plasma TG concentration of these subjects. This additional output represents an increase of 12.5% above the daily caloric requirement (approximately 4000 Calories), and apparently this increase is not of sufficient magnitude to e l i c i t a further decrease in TG concentration. There are two possible explanations for the decreased plasma triglyceride concentration. Ballard et a l . (I960) and Gousios et a l . (1963) have demonstrated that peripheral tissues can utilize esterified fatty acids as a source of energy. In agreement with these findings, Hollenberg (I960) has also reported a significant increase in the lipoprotein lipase activity in the myocardium and diaphragm during fasting, a factor which would account for the hydrolysis of some circulating triglycerides. Therefore, i f the low calorie diet represents a stress, such that the increase in plasma FFA can not supply suff ic ient substrates, i t i s possible that the plasma TG could be u t i l i z e d . The amount of carbohydrate and l i p i d in the diets could also be responsible for the change i n the c ircu lat ing TG. With the essential fat ty acids providing the only source of fat i n the low calorie d i e t , the l eve l of c irculat ing chylomicrons would be ins ign i f i cant . How-ever, the normal diet contained approximately 35 to 40 per cent fat ; i t i s possible that the change i n fat content of the diets alone might be res-ponsible for a reduction i n c ircu lat ing TG. Dietary carbohydrates supply much of the "raw materials" needed i n the synthesis of long-chain fatty acids (Wakil and Barnes, 1 9 7 1 ) . Glucose i s broken down v i a the glycolyt ic pathway to pyruvate, which i s then oxidized to acetyl - CoA. Acetyl - CoA is condensed with oxaloacetate to form c i tra te , which diffuses out of the mitochondria. Citrate i s then cleaved by an extramitochondrial enzyme to oxaloacetic acid and acetyl -CoA, which can be used for fatty acid synthesis and as the source of C2 units for elongation after conversion to malonyl - CoA. The low calorie diet did not provide suff ic ient substrates for metabolism and i t i s probable that a l l the carbohydrate i n the diet was oxidized rather than converted to glycogen or long-chain fatty acids. As these fatty acids are the precursors of plasma TG, i t i s apparent that the l eve l of carbohydrate i n the diet plays an important role i n a l ter ing the concentration of plasma TG. CHAPTER V SUMMARY AND CONCLUSIONS Summary The purpose of th is study was to determine the plasma l i p i d variations during periods of low calorie diet and low calorie diet plus increased physical a c t i v i t y . The study was divided into five con-secutive experimental periods; the f i r s t , or control period, was s imilar to the recovery periods, which followed each stress s i tuat ion. During these three periods the subjects maintained 'normal' physical ac t i v i t y and consumed a regular diet containing 3600 Calories per day (4000 C a l -ories for the recovery periods). The f i r s t stress s ituation involved a ten day period on a semi-defined, low calorie diet (1800 Calories per day) with continued 'normal' physical a c t i v i t y . Following the fourteen day recovery period, the second stress s i tuation began; i t was ident i ca l to the f i r s t stress condition with addit ional d a i l y energy expenditure of 500 Calories above the established 'normal' a c t i v i t y . This was approximately equivalent to a s ix mile run in 50 minutes. Blood samples were taken, following an overnight fast , twice during each period, once mid-way through the period and again at the end. Plasma tr ig lycerides and free fatty acids were measured i n the samples taken from four male graduate students with a high i n i t i a l l eve l of a c t i v i t y . The results of orthogonal comparisons among treatment means showed a s t a t i s t i c a l l y s ignif icant r i se i n plasma free fat ty acids during the low calorie diet and the low calorie diet plus increased physical 43 activity periods. This was attributed to increased mobilization of free fatty acids from adipose tissue triglycerides in response to the insufficient dietary supply of substrates for metabolism. Neuman - Keuls method was used to examine the effect of the increased physical activity during the low calorie diet periods; the results showed that the increased caloric ex-penditure had no significant effect on the plasma free fatty acids. Similar statistical procedures applied to the plasma t r i -glyceride values showed a significant decrease in the plasma triglyceride concentration during the low calorie diet and the low calorie diet plus increased physical activity periods. The stress of the low calorie diet on the habitually active subjects was responsible for the decreased levels. The lipid and carbohydrate content of the normal and the low calorie diets, as well as increased peripheral uptake of triglycerides were suggested as possible explanations for the plasma triglyceride changes. The additional daily output of 500 Calories during the second stress condition was not of sufficient magnitude to e l i c i t a further decrease in plasma triglyceride concentration. Conclusions 1. The plasma free fatty acids were significantly increased in active male subjects during a low calorie diet and a low calorie diet plus increased physical activity. 2. During the same treatment conditions mentioned above, the plasma triglyceride concentration was significantly reduced. 3. The increase in physical activity during the second stress condition had no significant effect on the plasma lipids. REFERENCES Ahlquist, R.P. "A study of the adrenotropic receptors." Am. J. of Physiol., 1 5 3 : 5 8 6 - 6 0 0 , 1 9 4 8 . Andres, R., G. Cader, and K.L. Zierler. "The quantitatively minor role of carbohydrate in oxidative metabolism by skeletal muscle in intact man in the basal state. Measurements of oxygen and glucose uptake and carbon dioxide and lactate pro-duction in the forearm." J. Clin. Invest., 3 5 : 6 7 1 - 6 8 2 , 1 9 5 6 . Astrand, P.O., and K. Rodahl. Textbook of Work Physiology. . Toronto: McGraw - H i l l Book Company, 1970 . Baker, N., A.S. Garfinkel, and M.C. Schotz. "Hepatic triglyceride secretion in relation to lipogenesis and free fatty acid mobilization in fasted and glucose - refed rats." J. Lipid Research., 9 : 1 - 7 , 1968 . Ballard, E.B., W.H. Danforth, S. Haegle, and R.J. Bing. "Myocardial metabolism of fatty acids." J. Clin. Invest., 3 9 : 7 1 7 - 7 2 3 , I 9 6 0 . Basu, A., R. Passmore, and J.A. Strong. "The effect of exercise on the level of non-esterified fatty acids in the blood." Quart. J.  Exp. Physiol., 4 5 : 3 1 2 - 3 1 7 , I 9 6 0 . Beznak, A.B.L., and Z. Hasch. "The effect of sympathectomy on the fatty deposit in connective tissue." Quart. J. Exp. Physiol. , 27 1-15, 1 9 3 7 . Bierman, E.L., I.L. Schwartz, and V.P. Dole. "Action of insulin on the release of fatty acids from tissue stores." Am. J. Phvsiol., 1 9 1 : 3 5 9 - 3 6 2 , 1 9 5 7 . Eragdon, J.H., R.J. Havel, and R.3. Gordon Jr., "Effects of carbohydrate feeding on serum lipids and lipoproteins in the rat." Am. J. Physiol., 1 8 9 : 6 3 - 6 7 , 1 9 5 7 . Canadian Bulletin on Nutrition. Dietary Standard for Canada. Vol. 6 , Mo. 1 , March 1 9 6 4 . 45 46 Carlson, L.A., "Plasma lipids and lipoproteins and tissue lipids during exercise", in Nutrition and Physical Activity, ed. by G.Blix, Sweden: Almquist and Wikseus, 1 9 6 7 . and L.B. Wadstrom. "On the occurence of t r i , di, and mono-glycerides in human serum." Third International Conference, Biochemical Problems of Lipids, Brussels, 1 2 3 - 1 3 5 , 1 9 5 6 . and B. Pernow. "Studies on blood lipids during exercise. Arterial and venous plasma concentrations of unesterified fatty acids." J. Lab. Clin. Med., 53:833-841, 1959. and B. Pernow. "Studies on blood lipids during exercise. 2. The arterial plasma free fatty acid concentration during and after exercise, and its regulation." J. Lab. Clin. Med., 58:673-681, 1961. and L. 6ro. "Studies on the relationship between the con-centration of plasma free fatty acids and glycerol in vivo." Metabolism, 12:132-142, 1963. and L.G. Ekelund. "Splanchnic production and uptake of en-dogenous triglycerides in the fasting state in man." J. Clin. Invest42:714-718, 1963. and F. Mossfeldt. "Acute effects of prolonged, heavy ex-ercise on the concentration of plasma lipids and lipoproteins in man." Acta Physiol. Scand., 62:51-59, 1964. J. Boberg, and B. Hogstedt. "Some physiological and clinical implications of lipid metabolism from adipose tissue." in Handbook of Physiology, Section 5: Adipose Tissue, Ch. 63 og. 625-644, American Physiological Society, Washington D.C, 1965. and S.O. Froberg. "Blood lipid and glucose levels during a ten day period of low calorie intake and exercise in man." Metabolism, 1 6 : 6 2 4 - 6 3 4 , 1 9 6 7 . Confalonieri, C, M.V. Mazzucchelli, and P. Schlechter. "The nervous system and lipid metabolism of adipose tissue. 1. Influence of denervation on lipid mobilization processes, lipid synthesis, and lipopexia in the interscapular Metabolism. 1 0 : 3 2 4 - 3 2 9 , 1 9 6 1 . 47 Correll, J.W. "Adipose tissue: ability to respond to nerve stimulation in vitro." Science, 140:387-388, 1963. Dole, V.P. "A relation between non-esterified fatty acids in human plasma and the metabolism of glucose." J.Clin. Invest., 35:150-154, 1956 (a). "Fractionation of plasma non-esterified fatty acids." Proc. Soc. Sxp. Biol. • Med., 93:532-533, 1956 (b). Edwards, A.L. Experimental Design in Psychological Research, New York: Holt, Rinehart, and Winston, 1964. Efendic, S. "Catecholamines and metabolism of human adipose tissue." Acta Med. Scanaa., 187:477-481, 1970. Felig, P. "Metabolic response to human growth hormone during prolonged starvation." J. Clin. Invest., 50:411-414, 1971. Fredholm, B.B., and J. Karlsson. "Metabolic effects of prolonged sympathetic nerve stimulation in canine subcutaneous tissue." Acta  Physiol. Scand., 80:567-577, 1970. Fredrickson, D.S. and R.S. Gordon. "Transport of fatty acids." Physiol. Rev. 38:585-630, 1958 (a). . "The metabolism of albumin - bound C^ - - labelled unesterified fatty acids in normal human subjects." J. Clin. Invest., 37: 1504-1515, 1958 (b). Friedberg, S.J., D.B. Sher, M.D. Bogdonoff, and E.H. Estes Jr. "The dynamics of plasma free fatty acid metabolism during exercise ", J. Lipid Research, 4:34-38, 1963. . W.R. Harlan, E.H. Estes Jr., and D.L. Trout. "The effect of exercise on the concentration and turnover of plasma non-esterified fatty acids." J. Clin. Invest., 39:215-220, I960 (a) . R.F. Klein, D.L. Trout, M.D. Bogdonoff, and E.H. Estes Jr. "The characteristics of the peripheral transport of Cl4 -labelled palmitic acid." J. Clin. Invest., 39:1511-1515, I960 (b). 48 George, J.C and N.V. Vallyathan. "Effect of exercise on free fatty acid levels in the pigeon." J. Applied Physiol., 19:619-622, 1964. Gollnick, P.D., R.G. Soule, A.W. Taylor, C. Williams, and CD. Ianuzzo. "Exercise - induced glycogenolyses and lipolysis in the rat: hormonal influence." Am. J. Phvsiol., 219:729-733, 1970. Gordon, R.S. "Unesterified fatty acids in human blood plasma. II: the transport function of unesterified fatty acids." J. Clin. Invest., 36:810-815, 1957. and A. Cherke3. "Unesterified fatty acids in human blood plasma." J. Clin. Invest., 3 5 : 2 0 6 - 2 0 9 , 1956 . Gousios, A., J.M. Felts, and R.J. Havel. "The metabolism of serum t r i -glycerides and free fatty acids by the myocardium." Metabolism, 12:75-80, 1963. Haahti, E. "Effect of insulin in a case of essential hyperlipemia." Scand. J. Clin. Lab. Invest., 1 1 : 3 0 5 - 3 1 2 , 1959 . Hagenfeldt, L. and J. Wahren. "Metabolism of free fatty acids and ketone bodies." in Muscle Metabolism During Exercise, edited by B. Pernow and B. Saltin, New York: Plenum Press, 1 9 7 1 . Harper, H.A. Review of Physiological Chemistry. California: Lange Med-ica l Publications, 1971. Havel, R.J. "Early effects of fat ingestion on lipids and lipoproteins of serum in man." J. Clin. Invest., 36:848-854, 1957i and A. Goldfien. "The role of the sympathetic nervous system in the metabolism of free fatty acids." J. Lipid  Research, 1:102-108, 1959. A. Naimark, and CF. Borchgrevink. "Turnover rate and oxidation of free fatty acids in the blood plasma in man during exercise. Studies during continuous infusion of palmitate -l-C 1^." J. Clin. Invest., 42 :1054-1063, 1963. 49 . L.A. Carlson, L.G. Ekelund, and A. Holmgren. "Turnover rate and oxidation of different free fatty acids in man during exercise." J. Applied Physiol., 19:613-618, 1964. Henneman, D.H. and P.H. Henneman. "Effects of HGH on levels of blood and urinarv carbohydrates and fat metabolites in man." J. Clin. Invest"., 3 9 : 1 2 3 9 - 1 2 4 5 , I 9 6 0 . Hollenberg, C.H. "The effect of fasting on the lipoprotein lipase activity of rat heart and diaphragm." J. Clin. Invest., 39:1282-1287, I960. Holloszy, J.O., J.S. Skinner, G. Toro, and T. Cureton. "Effect of a six month program of endurance exercise on the serum lipids of middle aged men." Am. J. Cardiology, 14:753-760, 1964. Horstman, D., J. Mendez, E.R. Buskirk, R. Boileau, and W.C. Nicholas. "Lipid metabolism during heavy and moderate exercise." Medicine and Science in Sports, Vol. 3 , Ko. 1, p 18, Spring, 1971. Hunter, W.M., C.C. Fonseka, R. Passmore. "Growth hormone: important role in muscular exercise in adults." Science, 150:1051-1052, 1965. and M.Y. Sukkar. "Changes in plasma insulin levels during muscular exercise." J. Physiol., 196:110P, 1968. Issekutz, B. Jr. "The effect of exercise on the metabolism of free fatty acids." in Fat as a Tissue, edited by K. Rodahl and B. Issekutz Jr., Toronto: McGraw-Hill Book Co., p. 228-238, 1964. . H.I. Miller, and K. Rodahl. "Lipid and carbohydrate meta-bolism during exercise." Fed. Proc, 25:1415-1420, 1966. Jones, D.P. and R.A. Arky. "Effects of insulin on triglyceride and free fatty acid metabolism in man." Metabolism, 14:1287-1293, 1965. Jungas, R.L. and E.G. Ball. "Studies on metabolism of adipose tissue:XII. The effects of insulin and epinephrine on the free fatty acids and glycerol production in the presence and absence of glucose." Biochemistry, 2 : 3 8 3 - 3 8 8 , 1963:. 5 0 Kartin, E.L., E.E. Kan, A.W. Winkler, J.B. Peters. "Elood ketones and serum lipids in starvation and water deprivation." J. Clin. Invest., 23:824-835, 1944. Keul, J., E. Doll, and G. Haralambie. "Free fatty acids, glycerol, and triglycerides in arterial and femoral venous blood before and after a training period of 4 weeks." Pflugers Arch t > 316: 194-204, 1970. Keys, A. The Biology of Human Starvation, Minneapolis: The University of Minnesota Press. Vol. 1 and 2, 1950. Laurell, S., "Plasma free fatty acids in diabetic acidosis and starvation." Scand. J. Clin. Lab. Invest. 8:81-86, 1956. and B. Christensson. "Effect of a single dose of some hor-mones on plasma unesterified fatty acids." Acta Physiol. Scand., 44:248-255, 1958. and G. Tibbling. "Colorimetric micro-determination of free fatty acids in plasma." Clinica Chimica Acta, 1 6 : 5 7 - 6 3 , 1 9 6 7 . Lipsett, M.B., H.R. Engel, and D.M. Bergenstal. "Evidence for an action of glucagon unrelated to carbohydrate metabolism." Clinical  Research, 7:251-252, 1959. H.R. Engel, and D.M. Bergenstal. "The effects of glucagon on the plasma unesterified fatty acids and in nitrogen met-abolism." J. Lab. Clin. Med., 5 6 : 3 4 2 - 3 5 4 , I 9 6 0 . Masoro, E.J. Physiological Chemistry of Lipids in Mammals. Philadelphia: W.B. Saunders Co., 1968. Miller, H.I. "Plasma free fatty acid appearance in plasma triglycerides." Metabolism, 16:1096-1105, 1967. B. Issekutz Jr., and K. Rodahl. "Effect of exercise on the metabolism of fatty acids in the dog." Am. J. Physiol., 205:167-172, 1963. 51 Misbin, R.I., P.J. Edgar, and H. Lockwood. "Influence of adrenergic receptor stimulation on glucose metabolism during star-vation in man: Effects on circulating levels of insulin, growth hormone, and free fatty acids." Metabolism, 20: 544, 1971. Monkhouse, R.C., J. Strachan, and F. McClain. "Studies on clearing factor lipase of post-heparin plasma." Can. J. Biochem. Physiol., 39:1027-1036, 1961. Morgan, C.T. Physiological Psychology. Toronto: McGraw-Hill Book Co., 1965. Nikkila, E.A. "Control of plasma and liver triglyceride kinetics by carbohydrate metabolism and insulin." Advances in Lipid Research, 7:63-81, 1969. A. Torstl, and 0. Penttila. "The effect of exercise on lipoprotein lipase activity of rat heart, adipose tissue, and skeletal muscle." Metabolism, 12:863-865, 1963. Olson, R.E. and J.W. Vester. "Nutrition - endocrine interrelationships in the control of fat transport in man." Physiol. Rev., 40: 677-733, I960. Ostman, J. and S.Efendic. "Catecholamines and metabolism of human adipose tissue. II Effect of isopropylnoradrenaline and adrenergic blocking agents on lipolysis in human omental tissue in vitro." Acta Medica Scand., 187:471-482, 1970. Persson, B. "Effect of a prolonged fast on lipoprotein lipase activity elated from human adipose tissue." Acta Medica Scand., 188:225-228, 1970. Pruett, E.D.R*. "Free fatty acid mobilization during and after graded exercise." Acta Physiol. Scand., 78-79: 39A, 1970. Raben, M.S. and C.H. Hollenberg. "Effect of growth hormone on plasma free fatty acids." J. Clin. Invest., 37:922-923, 1958. Rabinowitz, D., G.A. Klassen, and K.L. Zierler. "Effect on human growth hormone on muscle and adipose tissue metabolism in the forearm of man." J. Clin. Invest., 44:51-61, 1965. 52 Renold, A.E. and G.F. Cahill. "Metabolism of adipose tissue: a summary." in Handbook of Physiology, Section 5- Adipose Tissue, p . 483-490, American Physiological Society, Washington D.C., 1965. Rizack, M.A. "An epinephrine - sensitive lipolytic activity in adipose tissue" J. Eiol. Chem., 236-657-662, 1961. Rodahl, K., H.I. Miller, and B. Issekutz, Jr. "Plasma free fatty acids in exercise." J. Applied Physiol., 19:489-492, 1964. Roth, J., S.M. Glick, R.S. Yalow, and S.A. Berson "Secretion of human growth hormone: physiologic and experimental modifications." Metabolism, 12:577-579, 1963. Rubin, L. and F. Aladjem. "Serum lipoprotein changes during fasting in man." Am. J. Physiol. 178:263-266, 1954. Rudman, D. "The adipokinetic action of polypeptides and amine hormones upon the adipose tissue of various animal species." J. Lipid  Research 4:119-129, 1963. Schmidt, V.F.H. and K. von Dahl. "Zur Methode der enzymatischen Neutralfett-bestiramung in biologischem material." Z. Klin. Chem., 6, 156-159, 1968. Sidman, R.L., M. Perkins, and N. Weiner. "Noradrenaline and adrenaline content of adipose tissue." Nature, 193:36-37, 1962. Spitzer, J.J. and F.J. Hohenleiter. "Release of free fatty acids by adipose tissue in vivo." J. Lipid Research, 2:396-399, 1961. Steinberg, D., E. Shafrir, and M. Vaughan. "Direct effect of glucagon on release of unesterified fatty acids (UFA) from adipose tissue." Clinical Research, 7:250, 1959. Stock, K. and E.O. Westerman. "Concentration of norepinephrine, serotonin and histamine, and amine-metabolizing enzymes in mammalian adipose tissue." J. Lipid Research, 4:297-304, 1963. Sukkar, M.Y., W.M. Hunter, and R. Passmore. "Changes in plasma levels of insulin and growth-hormone levels after a protein meal." Lancet , 2:1020-1022, 1967. 53 Unger, R.H., A.M. Eisentraut, M.S. McCall, and L.L. Madison, "Measure-ments of endogenous glucagon in plasma and the influence of blood glucose concentration upon its secretion." J. Clin. Invest., 41:682-689, 1962. . A.M. Eisentraut, and L.L. Madison. "The effects of total staroation upon the levels of circulating glucagon and insulin in man." J. Clin. Invest., 42:1031-1039, 1963 . and A.M. Eisentraut. "Studies on the physiologic role of glucagon." Diabetes, 13:563-568, 1964. Vendsalu, A. "Studies on adrenaline and noradrenaline in human plasma." Acta Physiol. Scand. Suppl., Vol. 49. No. 1 7 3 : 8-123, I960. Wakil, S.J. and E.M. Barnes, "Fatty acid metabolism." in Comprehensive  Biochemistry 18s: Pyruvate and Fatty Acid Metabolism, edited by M. Florkin and E. Stotz. New York: Elsevier Publishing Company, 1971. White, J.E. and F.L. Engel, "A lipolytic action of epinephrine and nore-pinephrine on rat adipose tissue in vitro." Proc. Soc. Exp. Biol. Med., 99:375-378, 1958. Winer, B.J. Statistical Principles in Experimental Design. Toronto: McGraw-Hill Book Co., 1962. J A P P E N D I X RAW SCORES Free Fatty Acids ( m moles / 1) Subject Blood Sample 1 2 3 4 1 .333 .299 .395 .464 2 .280 .405 .395 .549 3 .549 .537 .949 .333 4 .449 .687 .675 .348 5 .200 .349 .500 .253 6 .240 .309 .457 .181 7 .836 .739 .532 .647 8 .405 .692 .616 .287 9 .174 .244 .393 .257 10 .127 .233 .387 .164 Triglycerides (mg / 1 0 0 ml ) Subject Blood Sample 1 2 1 H8.93 103 .10 2 1 2 4 . 5 8 85.92 3 80.19 88.78 4 83.06 67.30 5 100.24 77.33 6 131.61 94.51 7 63.01 6 7 . 3 0 8 41.53 67.30 9 110.26 87.35 10 128.88 85.92 3 4 121.72 88.78 121.02 104.54 88.78 93.08 75.90 83.06 94.51 105.97 94.51 100.24 85.92 67.30 58.71 58.71 127.45 68.74 115.99 88.78 

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