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Q I“ :. a ‘ x 3 ‘.r.;. ‘ a; v: D -\ 1- c I‘m-w: ' L _. I 1' }\ .' 3-“ :r‘ :~.‘- 'w'f Lo" , .1 [I “I? - o“ :53 ' .fl?‘;-'I'fif" -'” "T‘J‘ ‘ F" - TP.‘-“&*‘.‘?.""I'§"-‘ ‘5')” I w ' i “ 1‘0": 5 “.- ' . 1 . . gt! .Vf'v‘"g.{5¥! u ."'.' w u if.‘ ‘1‘. .' “nit-)3. , . .“ mmfimw. - ad '- r...y:c-;mmsmmmmm LIBRARY .WEW" Michigan State University This is to certify that the thesis entitled Influence of Pattern of Feeding on Weight Gain, Nitrogen Balance, and Body Composi- tion in Rats presented by Aysel Ozelci Kavas has been accepted towards fulfillment of the requirements for PhD degree in FOOd SCience 8! Human Nutrition 1/ 7 , / . flé%w¢~fl~« Major professor Date ‘7’ $.73- 0-7 639 Ill llll lllllllllllllllflllll L 3 1293 01063 8900 l FEB 2 7 2327 '| :, APR 1 o 1997 INFLUENCE OF PATTERN OF FEEDING 0N WEIGHT GAIN, NITROGEN BALANCE, AND BODY COMPOSITION IN RATS by Aysel Firdevs Ozelci Kavas A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1978 6/03 7764 ABSTRACT INFLUENCE OF PATTERN OF FEEDING ON WEIGHT GAIN, NITROGEN BALANCE, AND BODY COMPOSITION IN RATS by Aysel Firdevs Ozelci Kavas Sixteen experiments were designed to study the in- fluence of meal pattern on body weight gain, nitrogen balance, and body composition in rats. First, a series of experiments was conducted to evaluate the influence of meal frequency on weight gain, nitrogen balance, and body composition of rats. Rats were either fed two hours per 24 or 48 hours (meal- eaters), or pair-fed to meal-eaters with an automated feeding machine (nibblers). Rats weighing approximately 250 g initially were fed 10%, 20%, or 30% casein, high- carbohydrate diets or a 20% casein, high-fat diet for seven to eight weeks. Meal-eaters gained essentially the same amount of body weight as the nibblers. Meal- feeding once per 24 or 48 hours did not adversely in- fluence nitrogen balance or body composition of the rats. In one experiment, smaller rats, weighing approximately 150 g initially, were utilized. Meal-eaters, again, retained as much nitrogen as nibblers, and contained less body fat than the nibblers. Next, rats weighing 110 to 150 or 250 g initially were utilized to determine the effect of the form of the diet (dry versus liquid) and the pattern of feeding (meal-feeding, force-feeding, nibbling, or ad Zibitum) on body weight gain and body fat. A high-carbohydrate, 20% casein, or 20% lactalbumin, diet was fed for four to eight weeks. Consumption of a diet mixed with an equal weight of water increased weight gain in one of three experiments. Body fat content of the rats was not influenced by addition of water to the diet. Neither force-feeding nor meal-feeding influenced body fat gain provided the respective control rats were pairsfed dur- ing the initial adaptation period. Likewise, when rats were pair-force-fed to ad Zibitum fed rats without an initial adaptation, meal frequency did not influence body fat gain. When meal-fed rats were switched to ad libi- tum intake, their food intake increased to equal that of rats which had been continuously fed ad Zibitum; however, rats which had been switched gained more body fat than did rats continuously fed ad Zibitum. To determine the effect of initial food restriction on subsequent body weight gain and body fat accumulation, rats were restricted to 75%, 50%, or 25% of the intake of control rats for one week and were subsequently pair- fed on a food intake basis to the control rats. .As ex- pected, restricted rats gained weight at a slower rate and had less body fat at the end of the restricted period than control rats. Upon re-feeding the same amount of food as consumed by the control rats, these re-fed rats gained more_body fat than control rats. This W '_._m l”:- . cqlnPffliEF.9rx-£aggain ,9c.c_.urre_d -regardless of whether the rats were force-fed twice daily, meal-fed once daily, or allowed to consume the food throughout the day. Both a high-carbohydrate and a high-fat diet produced compensa- tory fat gain. Compensation was apparent as early as the first week of re-feeding and was_greater in ratswhich had been restricted to S0% or 25% ofad Zibitum intake than in rats less severely restricted. Restricted rats were also re-fed on a body weight basis. These gained as much weight and fat as the ad Zibitum fed controls, indi- cating that the restricted rats were more efficient in converting dietary energyto body fat when re-fed than rats fedad Zibitum continuously. The results of these experiments suggest that meal- eating does not cause a depression in nitrogen retention or an increase in body fat deposition in rats, provided the experimental animals are pair-fed to the control rats throughout the entire experiment. A shift to a higher levelof food intake may cause an increased food effi- ciency and greater rateof fat deposition than in rats icontinuously fed the higher level of intake. The initial food restriction inherent in many studies involving meal frequency may cause the subsequent increased food effi- ciency and greater accumulation of body fat often attri- buted to an alteration in meal pattern. ACKNOWLEDGEMENTS I wish to extend my sincere thanks and appreciation to: Dr. Dale R. Romsos for his invaluable guidance, counsel, and continuous encouragement throughout my program; Drs Gilbert A. Leveille, Maurice R. Bennink, Werner G. Bergen, and H. Allen Tucker for their helpful suggestions and corrections for improving this disserta- tion; Ms Kathleen L. Muiruri and Ms Mary J. Hornshuh for their technical assistance; and Ms Constance M. Prynne for typing this manuscript. I especially wish to thank my husband, Alican Kavas, for his endless encouragement, patience, and understand- ing throughout the course of this study. ii INTRODUCTION CHAPTER I CHAPTER II CHAPTER III TABLE OF CONTENTS LITERATURE REVIEW Nutritional Adaptations Frequency of Feeding and Body Metabolism Experimental Methods of Feeding Frequency Morphological and Functional Changes in Gastrointestinal Tract (GIT) Associated with Feeding Frequency Circadian Rhythms and Feeding Frequency Carbohydrate Metabolism Protein Metabolism Lipid Metabolism INFLUENCE OF DIET COMPOSITION ON NITROGEN BALANCE AND BODY COMPOSITION IN MEAL-EATING AND NIBBLING RATS Introduction Materials 8 Methods Results Discussion INFLUENCE OF A LIQUID DIET AND MEAL PATTERN ON BODY WEIGHT AND BODY FAT IN RATS iii 11 14 19 22 42 42 43 46 SS 62 Introduction Materials 8 Methods Results Discussion CHAPTER IV INFLUENCE OF INITIAL FOOD RESTRICTION ON SUBSEQUENT BODY WEIGHT GAIN AND BODY FAT ACCUMULATION IN RATS Introduction Materials 8 Methods Experimental Protocol and Results Discussion SUMMARY AND CONCLUSIONS FOOTNOTES LITERATURE CITED iv 62 64 72 81 86 86 88 92 106 111 114 115 10. LIST OF TABLES Composition of the diets. Food intake, weight gain, nitrogen balance, and carcass composition of rats fed a 30% casein, high-carbohydrate diet (Exp. 1) Food intake, weight gain, nitrogen balance, water intake, and carcass composition of rats fed different levels of dietary protein (Exp. 2) Food intake, weight gain, nitrogen balance, water intake, and carcass composition of rats fed high-fat versus high-carbohydrate diets (Exp. 3) Food intake, weight gain, nitrogen balance, and carcass composition of rats meal-fed a 20% casein, high-carbohydrate diet once every 48 hours (Exp. 4). Food intake, weight gain, nitrogen balance, and carcass composition of young meal-fed rats (Exp. 5) Composition of diets. Food intake, weight gain, carcass composi- tion, blood glucose level, and fatty acid synthesis in meal-eating (one 2-hour meal daily), nibbling, and liquid-fed rats (EXp. 1) Food intake, weight gain, and carcass fat of force-fed, pair-fed, and ad Zibitum rats (Exp. 2) Food intake, weight gain, carcass weight, and carcass fat of rats fed ad Zibitum or pair-fed a liquid diet (Exp. 3) 45 47 49 52 54 56 65 73 75 77 11. 12. 13. 14. 15. 16. Food intake, weight gain, carcass weight, and carcass energy of rats fed ad Zibitum or pair force-fed for 4 weeks (Exp. 4) Food intake, weight gain, carcass weight, and carcass fat of rats (Exp. 5) Composition of the diets. Experimental protocol. Food intake, weight gain, carcass weight, and carcass fat of rats during the first week of experiments 1 through 6. Weight gain, gain per gram food consumed, food intake per gram body weight, and fat gain in rats during a one week period immedi- ately following one week of restricted (75%) food intake (Exp. 6) vi 78 8O 89 91 93 105 LIST OF FIGURES Body weight gain and fat gain of rats during the last three weeks of experiments 1, 2, and 3. 95 Body weight gain and fat gain of rats during the second to fourth week and during the fifth to seventh weeks in experiment 4. 99 Body weight gain and fat gain of rats during the second week and during the third to fourth weeks in experiment 5. 101 vii INTRODUCTION In the evolutionary process, higher organisms de- velop adaptive mechanisms that allow them a degree of independence from their environment. Rats with the hypothalamic-hyperphagic syndrome contain more fat, less water, and less nitrogen when fed ad Zibitum than pair-fed control animals. When the animals are offered their entire daily ration at one time, the hypothalamic-hyperphagic animals quickly con- sume their food while the controls take all day to eat their diet. Under these conditions, the differences in body fat between experimental and control animals are exaggerated (Van Putten et aZ., 1955). These studies were among the first to suggest that in addition to the effect of dietary constituents offered to an animal, the timing of food ingestion may play a role also in the economy of calorie disposition (Cohn and Joseph, 1959). The significance of studying the influence of peri- odicity of eating on body metabolism may be two-fold. First, at birth man is normally a nibbler, but custom, convenience, working habits, and sociability often make him a meal eater. Changes of the living pattern in highly industrialized countries lead to the concentration of food intake in the evening hours, the calorie intake 'during the remainder of the day often being low (Fabry and Tepperman, 1970). It is quite possible that a number of diseases associated with abnormalities in fat, protein, and carbohydrate metabolism may be caused or aggravated by eating habits (Cohn and Joseph, 1960). Epidemiologic studies revealed that excessive weight and, in elderly groups, also hypercholesterolemia, impaired glucose toler- ance, and ischemic heart disease were more common among persons with an infrequent meal pattern than among those who customarily ate five or more meals per day (Fabry and Tepperman, 1970). Secondly, if the timing of protein intake influences overall nitrogen utilization in the human, alterations in the distribution of protein among meals would be expected to have more distinct effects on infants and growing chil- dren than on adults (Taylor et aZ., 1973). This will have important impact in developing countries. Large-scale efforts have been made to improve the nutritional status of school children by providing one or, occasionally, two meals a day in the school. On a smaller but growing scale, similar efforts are being made to improve the nutritional status of preschool children considered to be much more at risk. If adequate nitrogen retention and growth can be maintained with most of the protein being given at a single meal and ad Zibitum energy with lower amounts {and/or quality of protein being consumed at other meals during the day, the possibility of increasing the value .Iof fOod supplementation programs would exist (Maclean et aZ., 1975). CHAPTER I LITERATURE REVIEW Nutritional Adaptations Nutritional adaptation is an example of the ability of the body to adapt to changes in the external environ- ment. Animals and men are able to adapt within a rela- tively wide range of dietary changes, provided that the supply of essential nutrients is ensured (Fabry, 1969). A change in the amount or composition of the diet leads to a change in the activity of systems involved in the metabolism of the nutrient. This change may be induced either by the direct effect of the substrate on the en- zyme or indirectly by influencing other physiological mechanisms controlling the activity of some key enzymes. Nutrition has a marked influence on the activity of different endocrine glands and on the adaptation of target tissues and their enzyme systems (Fabry, 1969). The pancreas responds to an excess of some nutrients by the excretion of several enzymes; however, continued administration of the diet leads to an adaptively in- creased synthesis of the appropriate eynzyme. Digestive functions seem to adapt not only to the quantity of a 4 main nutrient but also to a certain type of the nutrient. A diet rich in glucose, fructose, or galactose leads to Ian increased absorption of the appropriate sugar. If the change in the dietary regimen persists for a prolonged period, morphological adaptation of the small intestine may finally occur. Changes in the composition of the diet, for instance, a high lactose or a high fat diet, can lead, in fully grown animals, to an enlargement of the small intestine (Fabry, 1969). A special situation is the adaptation of the digestive system to changes in the nutritional pattern in mammals in the early stages of postnatal life when the animal changes from suckling to the diet of a fully grown animal. The absorption rate of nutrients and the activity of some en- zymes in the intestine during lactation corresponds to that of a diet rich in fat and lactose and it changes markedly when the young animal changes to the diet of adult animals (Fabry, 1969). A changed supply of nutrients is reflected also in the tissue metabolism. An altered supply of a nutrient leads to the alteration of the systems associated with its meta- bolism and its preferential use as a source of energy. An adaptive increase or decrease in the utilization of a nutri- ent may take place in the processing of the appropriate sub- strates from endogenous as well as exogenous sources, depen- ding on the availability of the nutrient. An animal adapted to a high carbohydrate diet has increased activity of the enzymes catalyzing the stages in the glu- cose metabolism, an increased capacity to synthesize gly- cogen from glucose, a higher glucose tolerance, and is more sensitive to the administration of insulin or hypoglycemic substances (Mitchell, 1965; Bassler, 1969). Animals adapted to a high fat diet have an increased ability to metabolize fat which is manifested by an in- creased oxidation of fatty acids, increased formation of ketone bodies in the liver or their increased oxidation in peripheral tissues, and the preferential use of fat reserves as a source of energy in calorie undernutrition (Mitchell, 1965). One of the metabolic effects of feeding fat is re- duced lipogenesis from acetate and glucose in the liver and adipose tissue. In the metabolic adaptation to a high protein diet, there is a striking increase in the activity of a number of enzymes associated with catabolism of proteins and amino acids, which are broken down to an increased extent as a source of energy and needed glucose. The increased amino acid catabolism in animals adapted to a high protein diet is manifested not only by an increased formation of urea but also by an enhanced breakdown of proteins during fast- ing (Fabry, 1969). On a low protein diet, the activity of a number of amino acid catabolizing enzymes declines, which is an example of a metabolic adaptation to a reduced supply of substrate. A reduction in energy supply results in a decrease in total energy output. Depending on the length and severity of the energy deficit in man and experimental animals, the basal metabolism is reduced, the urinary nitrogen excretion declines, and the physical activity is lowered. In experi- mental animals subjected to complete or partial starvation, tissue respiration and the activity of some tissue enzymes are reduced. Similarly, an increased energy intake also leads to metabolic adaptation manifested by a higher energy output (Fabry, 1969). Frequency of Feeding and Body Metabolism Experimental Methods of FeedingFrequency. The labora- tory rat ingests food almost continuously in small amounts during the day, with a maximum intake during the night. When this nocturnal feeding pattern is changed by periodic feedings and fastings, rats "learn" to ingest all the daily food ration within a few hours. Many of Fabry's experi- ments (Fabry, 1969; Fabry and Kujalova, 1960; Fabry et aZ., 1961 and 1962) were carried out on intermittently fasting rats adapted to gradually extended periods of fasting be- tween which there were days when free access to food was permitted. These rats gradually compensated for the periods of fasting by a larger food intake on the days of feeding; however, this periodically increased intake did not cause a complete compensation of food intake or weight gain. 'Intermittently fed rats were about 30% lighter than ad Zibitum fed controls (Fabry, 1969). When the controlled feeding is necessary, feeding the animals with a stomach tube has been frequently applied. Cohn (Cohn and Joseph, 1959, 1963, 1968) fed rats twice a day by tube and indicated that this feeding pattern leads to marked metabolic changes brought about by the periodic loads of nutrients and not by forced feeding. In addition to intermittent and force-feeding, rats have also been "trained" to consume "meals," that is, only two or three hours daily. During the first few days of meal feeding, these animals consumed very little food but they gradually increased the food intake. Trained rats consumed 15-20% less food than the ad Zibitum fed rats. The weight gain of these animals is also less than the ad Zibitum fed rats (Leveille, 1970, 1975; Philippens et aZ., 1977). Morphological and Functional Changes in Gastrointes- tinal Tract (GIT) Associated with FeedingFrequency. The laboratory rat is basically a "nibbler" and, if food is readily available, eats small quantities of food more or less continuously, reaching a maximum in the nighttime. If, however, the access to food is restricted to l to 2 hours per day, or if rats are fed intermittently only 3 days a week, alternating with l to 2 day periods of fasting, the animal soon learns to ingest large amounts of food at a time (Fabry and Tepperman, 1970). The GIT displays marked functional and morphological changes including increased activity of digestive enzymes of the pancreas, increased enzyme activity in the intestinal mucosa, and an increased rate of glucose absorption from the intestine (Fabry and Tepperman, 1970). A striking consequence of intermittent hyperphagia in rats and mice is enlargement of the GIT, especially that of the stomach (Fabry and Tepperman, 1970; Philippens et aZ., 1977). The enlargement is apparent in the absolute weight of the stomach and its portions (although the total body weight of the animal is reduced), as well as in histo— logical reactions with a hypertrOpy of the mucosa and mus- culature (Fabry, 1969). Intermittent hyperphagia produces morphological and functional changes in the digestive system which allows the animal to consume and to digest a large quantity of food within a short time (Pose et aZ., 1969). The functional consequence of the enlarged stomach is its increased capacity. The organ also serves as a food reservoir during the following period of fasting. Even after a 24 hour fast, a considerable amount of food may be found in the stomach of adapted rats (Fabry, 1969). 10 The small intestine also becomes significantly en- larged (Leveille and Chakrabarty, 1968; Romsos and _Leveille, 1974a). In Fabry's experiments (Fabry and Kujalova, 1960), the absolute weight of the small intes— tine was about 35% heavier than the weight of the small intestine of ad Zibitum fed controls, even though the animals weighed about 30% less than controls. Histo- logical appearance of the intestine from meal-eaters does not differ significantly from the appearance of intestines of controls (Fabry and Kujalova, 1960). After six weeks of infrequent feeding, there is a markedly increased rate of glucose absorption from the intestines (Fabry, 1969). Leveille (1970) reported that the rate of glucose absorption increased by about 40% in the meal-fed rats. The stimulus leading to enhanced in- testinal absorption in intermittently fed rats is not the reduction of the energy intake alone, but periodic hyper- phagia on days of free access to food. Contrary to find- ings of an enhanced glucose and fat absorption, amino acid absorption does not change (Fabry, 1969). The values of glucose absorption in vitro in intermittently fed animals are more than 60% higher than in ad Zibitum fed controls (Fabry, 1969). The weights of the liver, kidneys, femur, small in- testine, and stomach were significantly greater in meal- fed rats than in continuously fed control animals (Pocknee 11 and Heaton, 1976). Circadian Rhythms and Feeding Frequency. The re- .sponse of an animal to meal-feeding may depend on the timing of the meal in relation to the animal's circadian rhythms or to other periodic factors that influence these rhythms. When food is continuously available, the timing of circadian rhythms in rats and mice is determined pri- marily by the daily alteration between light and darkness (Nelson et aZ., 1975). Mice restricted to feeding in early darkness con- sumed less food and had a lower body weight than those feeding only in early light (Nelson et aZ., 1975). Nelson et a1. (1975) also found a large increase in the overall mean for liver glycogen and serum corticosterone concentra- tion in mice restricted to 4 hours daily food accessibility as compared with animals feeding ad Zibitum. The relation among the several variables examined can be considerably different at any given elapsed time after food presentation depending on whether food is presented near the onset of light or near the onset of darkness. Philippens et a2. (1977) studied circadian rhythms in rats in relation to meal timing. Liver weight varied during the 24-hour period in the rats fed ad Zibitum and subjected to a light-dark cycle. The maximum liver weight occurred during the early part of the light period. Ref stricting the availability of food intake to certain times 12 had an effect on the waveform of the rhythm; the ampli- tude was increased in restricted feeding groups. Maxi- mum liver weight usually occurred 9 hours after the food was removed. When the overall 24-hour means of each group were compared, relative liver weight (liver weight per body weight) was significantly increased in meal- fed rats compared to ad Zibitum fed rats. However, relative weight was smaller when food was presented during the first part of the light period. Stomach weight directly reflected the restricted time of food intake, with maximum stomach weight soon after the food was made available (Philippens et aZ., 1977). The over- all 24-hour mean weights of stomach of restricted feeding groups were approximately doubled when compared with the ad Zibitum fed groups. The rats fed during mid-light had a significantly lower stomach weight than other restricted feeding groups. The restricted feeding groups gained less weight than the control groups. Those rats with feeding confined to the light phase gained less weight than the rats with feeding restricted to the dark phase. Generally, lower values of serum protein were noted when the 24-hour means for the restricted groups were compared with those of the control groups (Philippens at aZ., 1977). The glucose rhythm was synchronized to the restricted feeding schedules. The phase of the 13 oscillation always occurred just prior to the time when food was made available; peak values occurred shortly thereafter. Those rats fed during the dark exhibited higher overall 24-hour blood sugar levels when compared with controls; also, their levels were higher when compared to those rats fed during the lights. Fuller and Diller (1970) also showed that there was a high amplitude circadian rhythm in liver glycogen in ad Zibitum fed rats. Restricted mean timing altered the waveform of the rhythm as well as increasing its ampli- tude. Restricted groups demonstrated a significant increase when compared with the ad Zibitum fed rats. Rats fed during the dark had higher overall 24-hour gly- cogen levels than rats fed during the light. In those rats fed during early light or dark periods, the phasing of liver protein closely followed the ad Zibitum pattern, with the major peaks occurring the first hour of feeding. The pattern of total protein in the liver was the inverse of that for glycogen. The total protein content of re- stricted groups was lower than that of the ad Zibitum fed rats, especially in those groups fed during the dark period (Fuller and Diller, 1970). Therefore, optimal nutrition may depend not only on what is eaten but when it is eaten in relation to other schedules and demands. 14 Carbohydrate Metabolism. The tissues of the meal- fed animal utilize glucose more rapidly than do those 'of nibbling rats (Leveille, 1970). Peripheral tissues of the meal-fed rat might convert a considerable portion of this glucose to storage forms, glycogen and lipid. For up to 8 hours after the initiation of the daily meal, the respiratory quotient (RQ) is in excess of unity, in- dicating that glucose is serving as the major oxidative fuel and that lipogenesis is proceeding at a rapid rate. From 8 to 14 hours following meal initiation, the RQ decreases to a value which suggests that carbohydrate, probably glycogen, and lipid are serving as the oxidative fuel. Finally, from 14 hours after the start of the meal until the initiation of the next meal, the RQ indicates lipid is the major source of energy (Leveille, 1970). After oral or parenteral glucose administration to intermittently fed rats or in rats adapted to consuming food for 2 hours per day, the rise in blood glucose is less and the rate of its decline more rapid than in ad Zibitum fed rats (Hoffmann et aZ., 1972). The tolerance for glucose is high because of the increased capacity of liver and peripheral tissues of the adapted animal to handle the absorbed glucose, mainly by converting it to glycogen and fat (Fabry and Tepperman, 1970). This improved clearance capability of glucose in the meal-fed 15 rat is the product of higher circulating insulin levels both in fed and fasted states and of greater tissue sensitivity to insulin (Wiley and Leveille, 1970). Romsos and Leveille (1974b) conducted intraperi- toneal glucose tolerance tests in meal-fed and nibbling rats fed high carbohydrate or high fat diets with either glucose or sucrose as the source of carbohydrate. Re- gardless of diet fed, meal eaters exhibited a greater ability to clear glucose from circulation than did ad Zibitum fed controls. The increase in blood glucose after the glucose load was similar in both groups of rats; however, blood glucose values in meal-fed rats re- turned towards the basal level sooner than did the values for nibblers. Source of dietary carbohydrate was with- out effect in nibbling rats or in meal-fed rats fed the high fat diets. Replacement of dietary glucose with sucrose impaired glucose tolerance in meal-fed rats fed the high carbohydrate diet (Romsos and Leveille, 1974b). Studies in humans (Wadhwa et aZ., 1973) showed that the mean blood glucose level of the subjects was higher when they gorged than when they nibbled. Post glucose levels were significantly affected by the fre- quency of feeding, being higher in gorgers than in nibblers (Wadhwa et aZ., 1973). A delayed response of serum immune reactive insulin to glucose load in 16 gorging subjects suggests that decreased sensitivity of peripheral tissues to insulin may be a contributory .factor to greater than normal circulating blood glucose levels. This difference in the effect of meal-feeding on glucose tolerance in humans and in rats cannot be readily explained. One explanation might be the difference in diet composition (Leveille and Romsos, 1974). In studies with rats, a high carbohydrate diet has generally been used whereas, in human studies, a diet much higher in fat was used. Also, the human diets contained significant quantities of sucrose but the rat diets did not. The study of Romsos and Leveille (1974b) is in agreement with the suggestion that both fat and sucrose impaired glucose tolerance; however, this impairment was noted both in meal-fed and nibbling rats. Animals adapted to ingesting food for l to 2 hours per day or to intermittent feeding have a higher liver glycogen content than ad Zibitum fed rats (Fabry, 1969; Tepperman and Tepperman, 1958). In the heart muscle of intermittently fed animals, the glycogen concentration is increased to about double the value found in controls (Fabry, 1969). Glycogen stores in adipose tissue greatly increase above those found in nibbling animals after only five days of adaptation to meal eating and reach 17 a point in fully adapted rats several fold higher after a meal than in nibbling rats. However, prior to the .meal, glycogen concentrations in adipose tissue are similar to those found in fed or fasted nibbling rats (Leveille, 1967). The activity of liver hexokinase in rats adapted to intermittent feeding is significantly increased not only in the state of satiety but also after 24 or 48 hours fasting as compared with controls fed ad Zibitum or fasted for an equal period (Fabry, 1969). An in- creased hexokinase activity in the liver and adipose tissue is found also in rats fed for 1 hour per day (Fabry, 1969) compared with ad Zibitum fed rats. Intestinal hexokinase activity is elevated above control nibbling rats when rats were forced to consume food within a 2-hour period each day. Meal-fed animals respond to a large meal with a gradual increase in hexo- kinase activity (Romsos and Leveille, 1974a). Intestinal pyruvate kinase activity is similar in meal-fed and nibbling rats (Romsos and Leveille, 1974a), whereas pyruvate kinase activity of adipose tissue from meal-fed rats is significantly elevated above control nibbler values (Leveille, 1970). Adaptation in carbohydrate metabolism in meal-fed rats, specifically an increased capacity for glucose 18 absorption and utilization and an increased glycogenesis in muscle, adipose tissue, and liver, are thought to be mediated by insulin, in at least two ways: circulating plasma insulin levels are elevated for at least a portion of the day; and, in adipose tisSue, sensitivity to the hormone is increased. It has been suggested that some of the actions of insulin may be mediated by changes in tissue concentrations of cyclic-AMP and/or -GMP (1p et aZ., 1977). Hoffman et a2. (1972) observed that meal-feeding did not enhance the insulinogenic response to intravenous glucose. Meal-feeding elevated the concentration of in- sulin in the pancreas. In intermittently fed rats, the insulin activity is higher in the serum of fasted animals as compared with controls. It may be that insulin is released in increased amounts from the pancreas of inter- mittently fed rats; however, its reserve in the pancreas is relatively small and when the requirements and the utilization of the hormone in tissue are raised, its con- centration in the bloodstream diminishes (Fabry, 1969). In the meal-fed rats, 2 hours after administration of food, a short-term hyperinsulinemia develops and insulin concentrations exceed the maximum levels in ad Zibitum fed controls (Fabry, 1969). 19 Studies with humans showed that gorging elevated the insulin response of the subjects exhibiting relatively normal insulin levels following oral glucose. Upon nib- bling, the insulin responses of both "normal" and "abnormal" groups (subjects exhibiting delayed insulin response to oral glucose) were lowered (Pringle et aZ., 1976). The meal-eating rats exhibited a sharp increase in circulating glucagon levels that coincided with hyper— insulinemia. The ad Zibitum fed rats showed a longer sustained increase which was spread over much of the dark period. The amplitude of the diurnally changing glucagon concentrations tended to be larger in the meal-fed rats (Ip et aZ., 1977). Protein Metabolism. Frequent meals resulted in a con- siderably greater retention of absorbed nitrogen than did two mealsper day in rats (Cohn and Joseph,1963)although the digestibility and fecal nitrogen were not affected by the frequency of meals (Han, 1973). The mechanisms underlying the effect of frequency of meals on the excretion of nitro- gen are not clearly known. Since protein synthesis in vivo is a rate-limiting enzymatic reaction, it seems possible that there is a limit to the quantity of amino acids ab- sorbed which can be channelled into protein biosynthesis reaction per unit of time. Also, it seems probable that when overloading with substrates occurs, as it might under 20 the conditions of infrequent feeding of large meals, some of the amino acids would be deaminated and excreted in the 'urine. Thus, rats would have to adapt to overloading by altering the pathways of enzymatic breakdown (Cohn and Joseph, 1963; Han, 1973). Most studies with experimental animals suggest that nitrogen utilization is reduced when isocaloric and iso- nitrogenous meals are provided infrequently, but this has not been the general finding for human subjects. There was no difference in nitrogen balance of young women con- suming 30 g protein and given 3 or 6 meals during the day (Shortridge and Linkswiler, 1963). Thachange was observed in nitrogen utilization:h1young women with alterations in meal frequency (Swindells et aZ., 1968). Bortz et al. (1966) also found similar nitrogen utilization and weight loss in obese subjects given a low energy intake and either 72 or 13 g protein consumed in l, 3, or 9 meals daily. It was observed in a number of experiments that, in force-fed (Cohn and Joseph, 1959, 1963) and in intermittently or meal fed (Fabry, 1969) rats, the proportion of total body protein declines in connection with an increase in body fat. In mammals, the absorption of food protein is not con- tinuous yet, after meals, there is little accumulation of free amino acids in the tissues (Garlick et aZ., 1973). 21 The liver is usually considered to modulate the inflow of amino acids from the intestines and the concentrations of amino acids in the systemic blood fluctuate much less after a meal than the concentrations in the portal vein. Muscle tissues are exposed to more constant concentrations of plasma amino acids than the liver. The synthesis of muscle proteins may not respond to sudden dietary changes to the same extent. However, experiments in vitro have shown that muscle protein synthesis is increased by insulin. Changes in the metabolism of muscle protein could therefore occur after a feed, despite the small alterations in plasma amino acid concentrations, as a result of changes in plasma insulin concentrations (Garlick et aZ., 1973). During cyclic feeding or after feeding, dramatic changes in liver weight and pro- tein content occur. The major effect of food absorption in liver appears to be a decrease in the rate of protein break- down, with a subsequent increase in the protein content of the tissue in rats on scheduled meals (Garlick et aZ., 1973; Oblet at aZ., 1975). The rhythm in tyrosine aminotransferase activity is generated primarily by the periodicity of food intake (Cohn et aZ., 1970). The amplitude of the 24-hour rhythm in hepatic tyrosine aminotransferase activities of rats fed hourly was markedly reduced when compared with the activities of the enzyme in rats eating ad Zibitum (Cohn et aZ., 1970). 22 At any time of the day, the feeding of a protein meal re- sulted in a rise in tyrosine aminotransferase and a second .protein meal induced a second rise. This induction was not mediated by a release of either insulin or corticosteroids. The lowest enzyme activity always occurred approximately 14 hours after induction by a protein meal. There was a total absence of tyrosine aminotransferase rhythm in animals fed a protein-free diet (Girard Globa and Bouldal, 1973). The rhythm in tryptophan pyrrolase is also markedly altered by changing the pattern of food intake. The peak of the rhythm in meal-fed rats was approximately at the nadir in ad Zibitum fed rats, and vice versa. In addition, tryptophan pyrrolase activity was generally much higher in meal-fed rats (Cohn et aZ., 1970). Lipid Metabolism. Rats trained to eat their food in a single Z-hour daily meal have a tremendously increased capacity to synthesize fat from carbohydrates (Leveille, 1975; Palmquist et aZ., 1977; De Bont et aZ., 1975; Holli- field and Parson, 1962; Cohn and Joseph, 1967). These changes occurring in liver and in adipose tissue have been termed "adaptive hyperlipogenesis" by Tepperman and Tepper- man (1965). The increased lipogenic activity occurs hand in hand with an increased activity of different enzymes involved in the fat formation as well as increased syn- thesis of proteins and nucleic acids in the fat cells 23 Fabry and Tepperman, 1970). This phenomenal augmentation of fatty acid synthesis occurs after only four days of .training during which time the gorging animals eat less than ad Zibitum fed controls and always lose weight (Palmquist et aZ., 1977). Following the consumption of a meal, the rates of liver fatty acid synthesis rapidly rise such that, within two hours, the rates are five times pre-meal values (Palm- quist et aZ., 1977; Lowenstein, 1971; Sullivan et aZ., 1974). Although fatty acid synthesis rates immediately after a meal are several fold higher in livers of meal- trained rats than nibbling animals fasted 22 hours and re- fed two hours, the rate of lipogenesis in meal-fed rat liver does not reach that of the fed nibbler until 5 hours after meal initiation. The rate of hepatic fatty acid syn- thesis in meal-eating rats gradually returns to pre-meal values over the subsequent 22 hour period of food depri- vation (Lowenstein, 1971; Sullivan et aZ., 1974). Rats which were fed a high carbohydrate diet during a single 3-hour feeding period each day, developed increased rates of lipogenesis that persisted for 12 to 16 hours after the meal was terminated. To determine the cause of this prolonged lipogenesis, trained rats were fed meals ranging in size from 0.5 to 10.5 g (Wittman et aZ., 1973). Lipo- genesis, gluconeogenesis, and absorption-related parameters 24 which might affect these pathways were measured at sche- duled times after feeding. All meal sizes were equally effective in causing high rates of lipogenesis within 30 to 40 minutes; however, the duration of these rates varied directly with the size of the meal. Stomach contents, luminal glucose in the intestine, and portal blood glucose and insulin concentrations varied with the rates of lipo- genesis. Gluconeogenesis did not increase until 4 to 8 hours after lipogenesis returned to pre-feeding rates (Wittman et aZ., 1973; Miller et aZ., 1973). Adaptation to the meal-feeding regimen results in an increased rate of food ingestion and a prolonged period of intestinal absorption. The latter is associated with increased blood sugar and insulin levels which, in turn, may cause meta- bolic changes producing the increased lipogenesis (Wittman et aZ., 1973). The stimulation of adipose tissue lipogenesis which occurs after consumption of the daily meal may be related to the rapid absorption of glucose and subsequent produc- tion of a-glycerOphosphate in adipose tissue. This, in turn, provides for fatty acid esterification and reversal of potential inhibition by elevated levels of tissue-free fatty acids or CoA derivatives (Leveille, 1967). Romsos and Leveille (1972a) suggested that the enhanced lipogenic capacity observed in the adipose tissue from meal-fed rats 25 resulted from a true metabolic adaptation and not a change in cell size. The authors found that the differences in in vitrolipogenic capacity of the epididymal fat pads from meal-fed and ad Zibitum rats were of similar magnitude whether the results were expressed on a tissue weight basis or on a fat cell basis. A number of enzymes are involved in the conversion of ingested carbohydrates to storage forms of enerEY; fatty acids and glycogen (Leveille and Romsos, 1974). These en- zymes are: (a) involved in the phosphorylation of glucose and its conversion to a-glycerophosphate and pyruvate; (b) involved in fatty acid synthesis; and (c) involved in the generation of NADPH for the support of fatty acid syn- thesis. The activities of a-glycerOphOSphate dehydrogenase and pyruvate kinase were increased as a consequence of meal- feeding, whereas there was no change in phosphofructokinase activity (Leveille, 1970, 1975; Leveille and Romsos, 1974). Citrate cleavage enzyme, acetyl coenzyme A carboxy- lase and fatty acid synthetase were all over 100% higher in adipose tissue of meal-fed as compared with nibbling rats (Fabry, 1969; Leveille, 1970; Leveille and Romsos, 1974; Armstrong et aZ., 1976). The activity of the fatty acid synthetase complex was not in excess of that of acetyl CoA carboxylase. Although acetyl CoA carboxylase may be the 26 rate-limiting enzyme for fatty acid biosynthesis, this regulatory role would result from the allosteric nature .and not the total activity of this enzyme. High acti- vity of citrate cleavage enzyme in adipose tissue of meal-fed rats implies that the generation of acetyl CoA via this enzymatic step is not likely to limit fatty acid synthesis (Leveille, 1970). During enhanced lipogenesis , the pentose pathway supplies more than half of the reduced NADP needed for the synthesis of fatty acids in rodent adipose tissue (Fabry, 1969). The activities of two pentose pathway enzymes, glucose-6- phosphate and 6-phospho-g1uconate dehydrogenase, were in- creased as a result of meal-feeding (Leveille, 1970; Tepper- man and Tepperman, 1958, 1961, 1964; Leveille, 1967; Hollifield and Parson, 1962; Muiruri and Leveille, 1970). Of the enzymes involved in the transhydrogenation cycle, the activities of pyruvate carboxylase and malic enzyme in rats (Leveille, 1970; Muiruri and Leveille, 1970; Tepper- man and Tepperman, 1964; Leveille and Hanson, 1966) and mice (Romsos and Leveille, 1972b) were markedly increased by meal-feeding. The activity of malic dehydrogenase was not significantly increased but was high in contrast to that of the other two enzymes of the transhydrogenation cycle and not likely to be limiting (Leveille, 1970). The activity of isocitrate dehydrogenase was not increased 27 in rat epididymal adipose tissue as a result of meal feeding. There is a marked difference between rats and mice with respect to adaptations to a gorging pattern of food intake. Thus far, there is no evidence from in viva tracer experiments that rates of fatty acid synthesis from carbohydrate are any greater in gorging mice than in nibblers (Palmquist et aZ., 1977). Despite the ap- parent species differences in lipogenic activation of rats and mice, both species respond similarly with respect to accumulation of depot fat. In both Species, total food consumption is drastically reduced and in neither species does body fat accumulate during the early stages of food restriction to a single large meal per day. Mice that are on an intermittent fasting (24 hours) and refeeding (48 hours) schedule show no change in body composition relative to ad Zibitum fed mice. However, relative rates of fatty acid synthesis in adipose tissue and liver vary considerably (Romsos and Leveille, 1972b). In the guinea pig, there was a reduction in average daily weight gain and gross feed efficiency after meal feeding. In vivo lipogenesis indicated a definite diurnal pattern on both meal feeding and ad Zibitum feeding, with lower glucose incorporation into lipid by the meal-fed animals (Kuhl and Reid, 1973). 28 DiGirolamo and Rudman (1966) compared the rate of in vitra conversion of glucose to fatty acid by slices of ‘ epididymaladipose tissue from rat, guinea pig, rabbit, and hamster. The relative rates were 157 : 83 : 12 : 1, respectively. Adding insulin to the culture medium re- sulted in 188-290% increase in the rate for rat tissue, but it had no effect on hamster tissue. Hamster adipose tissue may also convert glucose to fatty acids at a low and rather inflexible rate in viva (Silverman and Zucker, 1976). Noncompensation may result from intake of low fat diet being held to current metabolic needs plus the small amount which can be converted to fat. Presumably, intake in excess of this amount inhibits further consumption through negative feedback (Silverman and Zucker, 1976). In contrast to results obtained in adipose tissue of inter- mittent fasting hamster, synthesis of liver lipids and gly- cogen increase after the meal, as has been established in the rat (Simek, 1975). In the livers of chickens adapted-to a single daily meal, the rate of fatty acid synthesis is increased (Leveille, 1967; Leveille and Hanson, 1965a). Ingestion of the daily 2-hour meal increases hepatic lipogenesis to a level which sis more than twice that observed for livers of ad Zibitum ’7fed chicks. Hepatic lipogenic enzyme activity in the chick does not appear to be increased by meal feeding (Leveille, 1967; Leveille and Hanson, 1965a). The 22 hour 29 period of fast between daily meals depresses hepatic lipogenesis much less in adapted chicks than in a similar -period of food restriction in ad Zibitum fed chicks (Leveille et aZ., 1975). The capacity for fatty acid synthesis was signifi- cantly reduced in both meal-fed and nibbling rats as a consequence of increasing the percentage of energy derived from fat. The response to meal feeding decreased as the level of dietary fat increased, although the rate of lipo- genesis in adipose tissue of meal-fed animals was still higher than that of tissue from nibbling rats. The ac- tivities of malic enzyme (ME) and glucose-6-phosphate de- hydrogenase were higher in tissues of meal-fed animals and decreased as the level of dietary fat was increased (Leveille, 1970, 1975). As the level of protein in the diet increased and, hence, the level of carbohydrate decreased, the rate of fatty acid synthesis was diminished. The difference be- tween meal-fed and nibbling rats was apparent at all levels of dietary protein. As in the studies with dietary fat, the activity of ME paralleled the observed rates of fatty acid synthesis. The observed effects could be due to a reduction in dietary carbohydrate or to the increased amount of protein or fat ingested (Leveille, 1975). Hepatic lipogenesis and the activities of ME and 3O citrate cleavage enzyme are reduced by feeding chicks high fat or high protein diets (Yeh and Leveille, 1969). Increased dietary fat, at the expense of carbohydrate but with a constant calorie/protein ratio, resulted in a significant impairment of lipogenesis as did increasing the level of dietary protein (Leveille et aZ., 1975). This suggests that dietary protein and fat exert a direct effect on the capacity for fatty acid synthesis which cannot be totally accounted for by reduction in dietary carbohydrate. Intermittent feeding leads, in addition to enhanced ability of the liver to synthesize fatty acids, to an increased ability to oxidize fatty acids (Fabry,'l969). A new equilibrium between anabolic and catabolic processes means that the enhanced lipid formation during the period of free access to food is balanced by more intense cata- bolic processes during the subsequent period of fasting. In the course of subsequent fasting, anabolic changes de- cline to an even lower level, while catabolic changes become markedly enhanced (Petrasek at aZ., 1969). Cohn at al. (1959, 1960, 1963, 1968) reported, in a series of experiments, that rats force-fed twice daily gained the same amount of weight but significantly more fat than ad Zibitum fed controls on an equal food intake. The increase in fat was at the expense of nitrogenous 31 constituents. In the experiments of Fabry et al. (1969), the ratio of body fat in relation to body weight or total body pro- tein was significantly higher in intermittently fed rats or in rats fed for 2 hours a day, than in control groups. On the other hand, Stevenson et a1. (1964) confirmed the enhanced lipogenesis in adipose tissue but found a slightly reduced ratio of fat as compared with heavier controls fed ad Zibitum. Leveille and Romsos (1974) found that approximately 70% of ingested energy is stored during the absorptive period following the initiation of a meal in the rat: 20% in the form of glycogen, and 50% in the form of fat. The remaining 30% of ingested energy serves to support metabolic functions during the absorptive period. Muscle is the major site of glycogen storage in both meal-fed and ad Zibitum fed animals, accounting for over 90%. Most of the remainder is stored in the liver in the case of nibbling animals, and in adipose tissue in the case of meal-fed rats. Fifty percent of ingested energy stored in the form of lipid is synthesized and stored in adipose tissue (Leveille and Romsos, 1974). Protein in- gested during the meal period in excess of that needed for immediate protein synthesis would be converted to lipid for storage. 32 Cohn and Joseph (1968) suggested that, during a weight reduction regimen for obese rats, the magnitude of the .load of ingested nutrients and the frequency of its con- sumption play a role in the body weight loss and in the alterations in body composition. After rats had been made obese by force-feeding excess calories, they were allowed to ingest their food ad Zibitum or were pair force-fed against a control. The rats with free access to food ate sparingly until a body weight consistent with their age and sex was achieved; at this time, the food intake in- creased to approximately normal with consequent slow gain in body weight. The force-fed animals not only tended to lose less and regain more weight than their partners eating ad Zibitum but, in addition, these rats fed by a tube ended the experiment containing more body fat and less protein and water. When the amount of food eaten was re- stricted, there was no influence of feeding frequency on the rate of weight loss and on body composition. Han (1973) demonstrated that rats force-fed two meals a day contained more fat and energy but less water than did ad Zibitum fed rats. The proportion of tissue gained as fat was much higher in rats fed two meals per day than in those fed ad Zibitum. Han (1973) suggested that higher fat deposition in the rats fed two meals per day indicated that overloaded carbohydrate may stimulate the lipogenesis 33 of the adipose tissues. In mice, witholding food two or three separate 24- hour periods per week produced increased percentages of body fat but had no effect upon food intake or weight gains during experimental periods ranging from 14 to 44 days (Welch, 1968). Han (1967) reported that sheep ingesting 8 meals per day gained body protein, fat, and energy at a more rapid and efficient rate than sheep fed one meal per day. Lepkovski at al. (1960) showed that chickens trained to eat their daily feed in two hours were no different in body fat content than chickens fed the same amount of feed ad Zibitum. Han et a2. (1967) also concluded that frequency of meals caused no effect on the body composition of chick- ens. However, Feigenbaum at al. (1962) obtained results with chickens which were contrary to those obtained by Cohn with rats. The fat content of chicks fed two meals per day was considerably lower than that of the control group fed ad Zibitum meals. These observations suggest that body fat accumulation varies from species to species, and even within the same species different results may be obtained under different experimental conditions. When restricted diets with a predominance of carbo- hydrates are used, the ratio of body fat in relation to the body weight or total body protein was significantly 34 higher in intermittently fasting rats or in rats fed for two hours per day, than in control ad Zibitum groups (Fabry, 1969). The differences in the fat content were, 'in most instances, smaller than in Cohn's experiments. With the exception of Cohn's experiments on tube-fed rats where an equal caloric intake is ensured, the incre- ment of body fat is quantitatively not so marked and readily reproducible in intermittent fasting animals. One might have expected a large increment of body fat in intermittently fasting animals in view of the high lipo- genic activity in these animals (Fabry, 1969). Rats fed a high fat diet had a higher concentration of body fat and deposited more fat daily than those fed a low fat diet (Wood and Reid, 1975). Fatty acid synthesis was also greatly increased as a result of meal feeding with the low fat diet but not with the high fat diet. The for- mation of body fat from dietary fat resulted in a greater deposition of fat in epididymal and perirenal fat pads, as well as in the carcasses of the rats given the high fat diet. Rats meal-fed the low fat diet, as a result of their greatly increased capacity for lipogenesis, had larger fat pads than nibbling rats fed the low fat diet. Conversely, rats meal-fed the high fat diet had smaller fat pads than nibbling rats fed the high fat diet. These results suggest that meal-fed and nibbling rats respond differently to changes in diet composition. 35 Wardlaw et a1 (1969) observed that decreased feeding frequency did not affect fat deposition in mature rats, 'whereas, in the younger animals, infrequent feeding in- creased body fat content. Heggeness (1965) used a feeding procedure for rats which involved alternation between three days of ad Zibitum food intake and three days of food re- striction. He found an increase in body fat as a result of the alternation in feeding pattern in animals which were placed on the experiment at 25 days but not in those placed on experiment at 55 days of age. Friend (1967) was unable to show a significant difference in fat deposition of rats between single feeders and multiple feeders except when young rats with an initial weight of 117 g were used. With these young rats, there was an increased deposition of fat when only one feeding was given. In the studies of Van Putten et al (1955) and Cohn (1959, 1960, 1963, 1968) in which increased deposition of fat with decreasing feeding frequency was demonstrated, rats which were relatively im- mature at the beginning of the experiment were utilized. The fat that is deposited in adipose tissue and other organs such as the liver may be derived from either dietary fat or de nava synthesis. Both synthesis and mobilization L are processes that are self-regulating to some extent but many hormones influence the rates of lipid metabolism and, thus, the balance between lipogenesis and lipolysis (Meier 36 and Burns, 1976). Much of the lipogenic influence of insulin is attri- buted to its role in carbohydrate metabolism. It promotes the utilization of glucose with the formation of a-glycerol phosphate and accelerates the conversion of pyruvate to acetyl CoA. Insulin also promotes lipogenesis in adipose tissue by making glucose available through facilitated transport. Insulin promotes the production of NADPH by directing carbohydrate metabolism through the hexose mono- phosphate shunt. In addition to its rapid lipogenic in- fluence, insulin also has a delayed effect by increasing the synthesis of enzymes involved in lipogenesis. Insulin is a potent inhibitor of fat mobilization. It facilitates the movement of glucose into the adipocytes with subsequent production of a-glycerol phosphate. Thus, when glucose is available, most of the fatty acids mobilized from the tri- glycerides are re-esterified and little fatty acid is re- leased to the blood. Insulin also has an inhibitory in- fluence on the activity of cyclic-AMP, an effect that could reduce mobilization of fat (Ip et aZ., 1977; Meier and Burns, 1976). The lipogenic effect of insulin on fat cells from fasted/re-fed rats was found to be enhanced (Braun and Fabry, 1969) as indicated by an increased incorporation of ‘“C-l-glucose into total lipids of fat cells isolated from 37 adipose tissue of fasted/re-fed rats as compared to fat cells from ad Zibitum fed rats. Similarly, an increased lipogenic effect of exogenous insulin on adipose tissue from periodically fed rats has been demonstrated in vitra and in viva (Braun at aZ., 1967). Meal-fed rats show a greater insulin sensitivity than do ad Zibitum fed rats (Reiser and Hallfrisch, 1977; Wiley and Leveille, 1970). Glucagon has an inhibitory effect on lipogenesis. The Opposing activities of insulin and glucagon may be related to their inhibitory and stimulatory effects, re- spectively, on cyclic-AMP which inhibits hepatic lipogene- sis (Meier and Burns, 1976). Although growth hormone has an immediate inhibitory effect on fatty acid synthesis in vitra, administration of the hormone for several days can repair lipogenesis in hypophysectomized rats and pigeons. Obesity resulting from chronic growth hormone administration is also accompanied by the development of progressive pancreatic islet hyper- tropy and hyperplasia indicative of increased insulin pro- duction (Meier and Burns, 1976). Prolactin has been shown to have marked stimulatory influence on body fat stores. In vitra studies of fish indicate that prolactin stimulates the rapid formation of hepatic lipids. Prolactin given in viva also stimulates the activities of several enzymes involved in lipogenesis 38 in the pigeon (Meier and Burns, 1976). Studies of rat adipose tissue indicate that prolactin has several lipo- 'genic activities that are similar to those of insulin. Provided that glucose is present, it stimulates the syn- thesis of fatty acids in vitra from acetate and pyruvate, and directs carbohydrate metabolism through the hexose monophosphate shunt (Meier and Burns, 1976). On the other hand, prolactin does not repair fatty acid synthesis in alloxan-diabetic rats whereas insulin does. Prolactin delays the degradation of insulin thereby increasing its activity (Meier and Burns, 1976). Prolactin stimulates increases or decreases in fat storage depending on whether it is present in larger quantities during daily intervals of lipogenic or lipolytic sensitivities (Meier and Burns, 1976). Hormonal regulation of fat mobilization centers on the hormone-sensitive lipase which catalyzes the conversion of triglycerides to fatty acids. Epinephrine, norephinephrine, and glucagon have strong stimulatory influences on this reaction. ACTH has similar effects in vitra but it is not clear whether it has a physiological role. Cyclic-AMP also promotes the conversion of triglycerides to fatty acids and it is generally accepted that many of the lipolytic activities of hormones are consequences of their effects on adenyl cyclase which catalyzes the conversion of ATP to c-AMP. 39 Thyroxin has a supportive effect on lipolysis, perhaps by way of augmenting catecholamine activity. Glucocorticoids also have supportive influences on the mobilization of fat (Meier and Burns, 1976). Results of several experiments in different species suggest that infrequent feeding might cause a rise of serum cholesterol levels and worsening of experimental atherosclerosis. Okey et al. (1960) investigated the serum cholesterol levels of ad Zibitum fed rats and rats fed for one hour per day. On the high cholesterol diet, female rats fed one hour had higher blood but lower liver choles- terol content than rats fed ad Zibitum. Cohn at al. (1961) fed chicks an atherogenic diet either ad Zibitum or for only two hours per day. After five weeks of the experiment, the chicks fed for two hours had doubled their serum cholesterol levels and had an in- creased incidence of atherosclerotic vascular lesions rela- tive to birds fed ad Zibitum, despite the fact that the ad Zibitum fed chicks consumed more food and ingested more cholesterol. When the chicks were switched to a cholesterol- free diet, the return of the cholesterol levels to normal values and the regression of vascular plaques were more rapid in ad Zibitum fed animals. Leveille and Hanson (19653, 1965b) also reported an increased cholesterol synthesis in liver in rats and chicks fed for two hours per day. 40 On a high-fat diet, monkeys fed twice for 30 minutes daily had significantly greater serum cholesterol levels 'than monkeys with free access to food (Gopalan et aZ., 1962). In humans, a dietary pattern of large infrequent feedings is associated with higher serum cholesterol levels than a pattern of frequent smaller meals (Fabry and Tepperman, 1970). In some hyperlipidemic patients, the level of serum lipids declined if the diet, otherwise unchanged, was divided into a greater number of small portions (Cohn, 1961). Enhanced biosynthesis of lipids and triglycerides can play a significant part in the development of obesity if the energy balance is favorable for energy storage (Fabry and Tepperman, 1970). A study of several years' duration evaluated the feeding patterns in a group of subjects with "resistant" obesity and a group of lean but otherwise healthy individuals (Pawan, 1972). In the obese group, 60% of the women and 54% of the men were found to have a feeding pattern of two meals daily, the larger meal being consumed in the evening, whereas only 16% of the women and 32% of the men consumed more than three meals daily. The relationship between frequency of eating and adi- posity was also studied in a cross-sectional population of men and women ages 35 to 69 (Metzner, 1977). Frequency of eating was related inversely to the adiposity index. The 41 proportion of overweight people tended to decrease as fre- quency of meals increased from three or fewer to five or more per day. The proportion of men with normal.weight increased with meal frequency. Studies by Young at al. (1971a, 1971b) showed that there was a slightly greater weight loss among subjects when taking more frequent meals. The increased cholesterol synthesis in liver can par- ticipate in the hypercholesterolemic effects of infrequent feeding observed in many species (Fabry and Tepperman, 1970). Serum phospholipid concentrations were higher in the subjects having one meal per day than in those on more fre- quent regimens. A similar effect was observed in the values for serum triglycerides (Young et aZ., 1971a). The percentage of subjects in which the ischemic heart disease was diagnosed decreased significantly with the in- creased meal frequency (Fabry, 1969). In conclusion, meal frequency might play a significant role in regulation of metabolism and may even cause patho- logical changes under certain conditions. Force-fed rats were utilized in previous studies to demonstrate that feeding frequency increased nitrogen ex- cretion and body fat accumulation. It is possible thatfbrce feeding per ae caused the metabolic alterations. Consequently, these experiments were undertaken to determine the influence of meal frequency on body weight gain, body composition, and nitrogen balance in rats. CHAPTER II INFLUENCE OF DIET COMPOSITION ON NITROGEN BALANCE AND BODY COMPOSITION IN MEAL-EATING AND NIBBLING RATS Introduction The time distribution of food intake has been shown to influence metabolism in several species. When the laboratory rat, which is by nature a nibbler, is forced to become a meal-eater, the alterations in the ingestion and absorption of food may produce changes in various enzymatic activities and in body composition (Leveille, 1970, 1975). Pair-fed rats given food by a stomach tube twice daily gained essentially the same amount of body weight as did rats with free access to food; however, the force-fed rats had a marked increase in total fat content in comparison to rats eating ad Zibitum (Cohn and Joseph, 1963; Han, 1973). Cohn et a1. (1963) also showed in rats that decreasing the number of meals was accompanied by increased urinary nitrogen excretion, nitrogen intake being constant. They concluded that the capacity of rate- limiting enzymatic reactions concerned with protein ana- bolism may have been exceeded by the load of nutrients presented to the animal. Thus, an increased quantity of absorbed amino acids was catabolized; the nitrogen was 42 43 excreted as urea, and the residual carbon chains utilized for fat synthesis. In addition to alteration of meal pattern by force- feeding, rats have also been trained to eat meals. Rats trained to consume their food in a short period of time each day usually ingest only 60% to 80% of the amount of nutrients eaten by the control rats with free access to food. However, the trained rats gain as much body weight as the ad Zibitum fed rats (Leveille, 1970; Cohn and Joseph, 1970). Thus, these trained rats are more effi- cient in converting food to body weight than ad Zibitum fed rats. These results suggest that the pattern of food intake may alter protein and energy metabolism. The ob- jectives of this study were to investigate the influence of diet composition on nitrogen balance and body composi- tion in rats fed one 2-hour meal per 24 or 48 hours com- pared with pair-fed rats continuously fed with a feeding apparatus. Materials 6 Methods Male Sprague-Dawley rats1 weighing approximately 150 g or 250 g initially, were individually housed in metal cages having raised wire floors. The room was lighted from 0700 hours to 1900 hours. Water was avail- able ad Zibitum. Five experiments were conducted. The composition of the four semipurified diets used in the 44 various experiments is presented in Table 1. The high- fat diet was prepared by substituting tallow on an equal 'energy basis for all the glucose in the 20% casein diet. Food intake was recorded daily and body weights were re- corded weekly. Meal-fed rats were given access to food for only 2 hours per 24 or 48 hours (0900-1100 hours). Nibblers were pair-fed to their meal-eating pair-mates by means of an automated feeding apparatus (Romsos and Leveille, 1974b). The feeding apparatus continuously delivered food to the nibblers. The rats consumed the diet as it was delivered to the food cup, without allowing the diet to accumulate in the food cup. During the fourth or fifth week of the experiment, the rats were placed in metabolic cages to collect urine and feces separately. About 1 ml of 4 N HCl was added to each urine collection flask. The cages were rinsed daily to quantitatively recover urine. Daily specimens of urine and feces were pooled at the end of 7 days. At the termination of the experiments, rats were decapitated at 1200 hours and the carcasses were frozen until analyzed. Carcass weight represented the weight ' of the rat minus blood loss and minus weight of the stomach contents. Carcasses were dried to constant weight, and ground for the body composition analyses. 45 TABLE 1 COMPOSITION OF THE DIETS Diet 1 2 3 4 Casein, g 10.0 20.0 30.0 20.0 Basal,‘ g 13.9 13.9 13.9 13.9 Glucose,2 g 76.1 66.1 56.1 - ranow,2 g - - - 28.0 Total 100.0 100.0 100 0 61.9 1The basal mix contained (in g/13.9 g): methionine, 0.3; vitamin mix, 0.4 (see Yeh and Leveille, 1969); mineral oil mix, 4.0 (see Leveille and O'Hea, 1967); choline chloride, 0.2; cellulose, 4.0; and corn oil, 5.0. 2Energy values used were: glucose 8 3.64 kcal/g, tallow = 8.59 kcal/g (Brambila and Hill, 1966). 46 Urinary, fecal, feed, and carcass nitrogen were determined by the semi-micro or micro Kjeldhal procedure (Horwitz, -l960) and body protein was calculated as nitrogen x 6.25. Body fat was determined gravimetrically after chloroform: methanol (3:2 v/v) extraction. In experiments 3 and 4, the body fat and protein were calculated from the follow- ing equations which were derived from direct carcass analysis of 48 rats: % fat - 60.62 - 0.77 x % H20 (r 8 0.76; p < 0.01) % protein = -19.48 + 0.559 x % H20 (r = 0.80; p < 0.01) The data were analyzed statistically by means of the Student's t-test or analysis of variance. When the F value was significant, the means were compared by Scheffé test (Gi11, 1977). Results Experiment 1. The food intake and body weight of the rats fed the 30% casein, high-carbohydrate diet are shown in Table 2. There was no difference in weight gain between the meal-eaters and the pair-fed nibblers. Average increases in body weights were parallel during the 8 week study. Urine and feces were collected for 7 days during the sixth week of the experiment. Meal-eaters excreted slightly more urinary, but less fecal nitrogen. There was 47 TABLE 2 FOOD INTAKE, WEIGHT GAIN, NITROGEN BALANCE, AND CARCASS COMPOSITION OF RATS FED A 30% CASEIN, HIGH CARBOHYDRATE DIET’(EXP. 1)1 Parameter Meal-Eater Nibbler Food intake (g/day) 17.4 1 0.3 17.4 1 0.3 Weight gain (g) 145.0 1 3.0 155.0 1 4.0 Urinary nitrogen (mg/day)2 446.0 1 24.0 383.0 1 26.0 Fecal nitrogen (mg/day)2 44.0 1 2.0 54.0 1 4.03 Nitrogen balance (mg/day) 334.0 1 18.0 347.0 1 24.0 Carcass weight (g) 371.0 1 8.0 395.0 1 7.0 Carcass protein (%) 19.2 1 0.8 18.9 1 0.6 Carcass fat (%) 12.8 1 1.2 14.6 1 1.3 1Values represent mean 1 SEM fOr 10 rats weighing 251 1 3 g initially and fed fOr 8 weeks. Meal-eaters = one meal per 24 hours. 2Urine and feces were collected during the Sixth week. ’Significant change (p < 0.05), meal-eaters va nibblers. 48 no difference in overall nitrogen balance between the two groups (Table 2). Carcass weight, and carcass protein and fat are presented in Table 2. Carcass protein values agree with our nitrogen balance data in that there were no differences in either carcass protein or nitrogen balance between the two groups. Carcass weights and fat contents of meal-fed and nibbling groups also were simi- lar. Experiment 2. Diets containing 10%, 20%, or 30% casein were fed to investigate whether the level of pro- tein would affect the nitrogen balance and the body composition of meal-fed rats. Meal-eaters and nibblers fed the same amount of protein gained practically the same amount Of body weight. Weight gain increased as the level of casein in the diet increased. Urinary and fecal nitrogen excretions increased as the level of casein in the diet increased (p < 0.01) (Table 3). There was no significant difference in urin- ary nitrogen excretion between the meal-eaters and nibblers. Fecal nitrogen excretion tended to be less in meal-eaters than in nibblers and was significantly lower (p < 0.05) in meal-eaters than in nibblers fed 30% casein. 7 Again, meal frequency did not influence nitrogen balance when the three levels of casein were fed (Table 3). 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Changes in carcass weight were essentially the same for both groups. Force-fed rats gained slightly, but not significantly, more total carcass energy during the last 3 weeks than did the ad Zibitum fed rats. The energy gain contributed by fat was not greater in the force-fed rats than in the ad Zibitum fed rats. Thus, we conclude that force-feeding has only a minimal influence on body fat when the force-fed rats are pair-fed throughout the entire experiment. Experiment 5. During the first 2-week period of meal—feeding (two l-hour meals per day), rats consumed about 12 g of food, in contrast to consumption of about 17 g food by the ad Zibitum fed group (Table 12). The ad Zibitum fed group gained nearly twice as much weight com— pared to the meal-fed rats during this period. During the last 3 weeks, ad Zibitum fed rats con- sumed about 25% more food than did the meal-fed rats or the pair-fed nibblers. 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Experiment 3. A carbohydrate-free, high-fat diet 'was fed ad Zibitum for 4 weeks or fed at 75% of ad Zibitum intake during the first week and pair-fed for the last 3 weeks (Table 14). Rats were killed after 1 and 4 weeks and carcass fat was analyzed. Ad Zibitum fed rats con- sumed about 14 g of food daily during the first week of the experiment; the restricted group consumed 9 g food daily (Table 15). The ad Zibitum fed rats gained more body weight and had heavier carcasses which contained more fat than the restricted rats at the end of the first week. During the last 3 weeks, both groups consumed 13.6 g food daily. The weight gain from weeks 2 to 4 was significantly greater in the group which had been re- stricted during the first week (Figure 1). Also, the rats restricted during the first week accumulated more body fat in the last 3 weeks than did the ad Zibitum fed rats. Thus, compensatory fat gain was evident in rats fed the high-fat diet as well as in rats fed the high- carbohydrate diet (experiments 1 and 2). Total body fat content at the end of 4 weeks did not differ between the two groups: 52.5 t 3.3 g in the ad Zibitum fed rats; and 98 53.7 1 3.1 g in rats restricted during the first week. Experiment 4. Rats were killed after 1, 4, and 7 weeks to determine the duration of increased food ef- ficiency in the restricted/re-fed rats. One group of rats was allowed to eat ad Zibitum for 7 weeks, the other group was restricted to 75% of ad Zibitum intake during the first week and pair-fed for the remaining 6 weeks (Table 14). The ad Zibitum fed group consumed 19.8 g food per day, and the restricted rats consumed 14 g food per day during the first week (Table 15). Again, ad Zibitum fed rats gained weight at a faster rate, their carcasses were heavier, and had significantly more fat at the end of 1 week than the restricted rats. Be- tween weeks 2 and 4, both ad Zibitum and pair-fed rats consumed about 23.3 g food daily. The body weight gain during this period was greater in the rats which had been restricted during the first week (Figure 2). Also, as in experiments 1 to 3, the fat gain was greater in the re- stricted, pair-fed rats from weeks 2 to 4 (Figure 2) than in ad Zibitum fed rats. During the last 3 weeks of the experiment (weeks 5-7), ad Zibitum and pair-fed rats consumed similar quantities of food per day: 26.6 g and 25.3 g, respectively. The increased food efficiency observed during the previous 3 weeks of pair-feeding was no longer apparent; both groups 99 a b '60 '- . , u-i Pi DWeighi Gain I40 - ‘ ‘ , .. a. ///l Fat Gain a a 1’ IOOF .1 . {1 8 .2 a 80 - - 8 8 3 60 - - n B a 40 " a b '1 C '3 o 20 "' % a O c! o M M” add A Ad- Restricted Ad- Restricted lib pair-fed lib pair- fed Weeks 2 to 4 5 to 7 Fig. 2. Body weight gain and fat gain of rats during Weeks 2-4 and Weeks 5-7 in Exp. 4. Rats were fed ad Zibitum for 7 weeks or restricted to 75% of ad Zibitum intake during the first week and were pair-fed to the ad'Zibitum fed rats for the last 6 weeks (Table 14). Each bar represents the mean and SEM for 10 rats. Treatment means not sharing a common superscript letter differ significantly (p < 0.05). 100 gained similar amounts of weight and body fat (Figure 2). Experiment 5. This experiment was designed to .evaluate the effect of the severity of the initial food restriction on subsequent body weight gain and body fat accumulation. Rats were fed ad Zibitum for 4 weeks or were restricted to 75%, 50%, or 25% of ad Zibitum intake during the first week, and pair-fed on a food-intake basis to ad Zibitum eating rats for the last 3 weeks (Table 14). In this experiment, rats were killed after 1, 2, and 4 weeks to obtain data on the time sequence of the compensatory gain. During the first week, as in the other experiments of this study, ad Zibitum fed rats consumed about 19 g food daily and the rats restricted to 75%, 50%, and 25% of ad Zibitum intake were fed 14.3 g, 9.6 g, and 4.8 g food daily, respectively (Table 15). The weight gain, carcass weight, and carcass fat content of the ad Zibitum fed group were greater than for the other groups during this week; values for the restricted groups related to the degree of food restriction during the first week. After the first week, all the restricted rats were pair- fed to the ad Zibitum fed rats. All rats consumed about 22 g of food daily during the second week. Figure 3 shows the weight and fat gain during the second week and during the third to fourth weeks. Weight gain and fat gain were 101 RS C) P DWeiqht Gain " FE F11 '8“ O I Imu- . . .0- _. .3 % Fat Gain 5 neo— .. - x a a a.b i N 60" "' -1 8 " i z 40- 4- - 2 i a db b b 8 20!- d. ~£ 7/i- j? .- 32}; V1. ,‘ ,;,f,', , .’ O 7%, K” a. “4 ,’ Ad! 25 °/. so °/. 75% A d- 25 °/. so °/. 757. lib restricted restricted restricted lib restricted restricted restr:cted paw-ted panefed paw-ted paw-fed paw-fed pan-ted W e a it 2 We 9 ks 3 to 4 Fig. 3. Body weight gain and fat gain of rats during the 2nd week and during Weeks 3-4 in Exp. 5. Rats were fed ad Zibitum for 4 weeks or restricted to 75%, 50%, or 25% of ad Zibitum intake during the first week and were pair-fed to the ad Zibitum fed rats for the last 3 weeks (Table 14). Each bar represents the mean and SEM for 10 rats. Treat- ment means not sharing a common superscript letter differ significantly (p < 0.05). 102 greatest in rats that had been most severely restricted during the first week. The effects of the previous food _restriction were evident during both the second and the third and fourth weeks of the study. At the end of the 4 week experiment, the rats which had been restricted to 75% and 50% of ad Zibitum food intake during the first week weighed as much as the ad Zibitum fed rats; the rats restricted to 25% of ad Zibitum intake weighed less than the ad Zibitum fed rats but not less than the other restricted pair-fed rats. Ad Zibitum fed rats contained 31.6 t 1.3 g fat; rats which were re- stricted to 75%, 50%, and 25% of ad Zibitum intake during the first week contained 39.2 i 3.0 g, 43.1 i 3.1 g, and 42.6 t 3.5 g body fat, respectively. Body fat content of the rats restricted to 50% and 25% of ad Zibitum intake during the first week was greater than observed in rats fed ad Zibitum. These observations demonstrate that the catch-up growth was evident as early as the first week after refeeding and that the increased efficiency of con- verting the food energy to body fat gain was more pro- nounced in the more severely restricted rats. Experiment 6. Rats refed ad Zibitum after a period of food restriction consume more food than rats fed ad Zibitum continuously (Wilson and Osbourn, 1960; Meyer and Clawson, 1964; Drew and Reid, 1975a, 1975b, 1975c; 103 Robinson et aZ., 1975; Levitski et aZ., 1976; Szepesi and Vojnik, 1975; Szepesi et aZ., 1975; Szepesi and Epstein, 1976; Ashworth, 1968; Miller and Wise, 1976). In experi- ments 1 through 5, rats were pair-fed on a food intake basis to the control group. However, the restricted rats weighed less than the control rats after the initial week of food restriction; consequently, the restricted pair-fed rats consumed more food per gram body weight than did the control rats. In this experiment, one group of restricted rats was pair-fed on a food intake basis and the second group was pair-fed on a body weight basis (Table 14). During the first week of the experiment, ad Zibitum fed rats consumed 21 g food per day and the restricted rats consumed an average of 5.2 g food per day (Table 15). As in experiment 5, ad Zibitum fed rats gained about 50 g body weight and the restricted group last body weight during this period. The carcasses of the restricted rats weighed less and contained less fat at the end of the first week than did carcasses of the ad Zibitum controls (Table 15). Rats pair-fed on a food intake basis grew faster than the ad Zibitum fed rats during each of the last three weeks. 0n the other hand, rats which were pair-fed on a body weight basis gained weight at a slower rate during the first and second weeks of refeeding, but gained weight faster during 104 the third week than the ad Zibitum fed rats. The total weight gain of the ad Zibitum fed rats and of the rats fed on the weight basis during the last 3 weeks was simi- lar, whereas the rats which were pair-fed the same amount of food as the ad Zibitum groups gained more body weight than either of the other groups (Table 16). The total food intake per average body weight (weeks 2 to 4) was 18% greater in rats pair-fed on a food intake basis than in either ad Zibitum fed rats or in rats pair-fed on a body weight basis. Weight gain per gram of food intake during the last 3 weeks was greater in rats which had been re- stricted than in ad Zibitum fed rats (Table 16). Body fat gain showed a similar trend to the weight gain; rats pair-fed on a food intake basis deposited more fat during the last three weeks than ad Zibitum fed rats. There was no difference in fat accumulation between rats pair-fed on a body weight basis and the ad Zibitum fed rats (Table 16). Total 4 week food intake of the ad Zibitum fed rats was greater than either of the groups of rats that were restricted during the first week; food intake averaged 25 g daily in ad Zibitum fed rats, 20 g in rats pair-fed on a food intake basis, and 14 g in rats pair-fed on a body weight basis. 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