THE EFFECT OF A HIGH FAT RATiON 0N BODY WEIGHT. BODY COMPOSITIQN’ AND ADIPOSE TISSUE WEIGHTS 0F RATS AS {NFLUENCED BY AGE, STRASN ANE WElGHT REDUCTION OF OBESE RATS Thesis for the Degree of Ph. D. MiCHIGAN STATE UNEVERSITY RACHEL SCHEMMEL 19.67 ‘ -* .‘A-a‘aaxau-rp Q LIBR4 .- Y 1 Michigan S’satc University :HESIS 1111111 NUIWIUIHHII 1| 26111111111111 3 312931 This is to certify that the thesis entitled THE EFFECT OF A HIGH FAT RATION ON BODY WEIGHT! BODY COMPOSITION AND ADIPOSE TISSUE WEIGHTS OF.‘ RATS AS INFLUENCED BY AGE, STRAIN AND WEIGHT REDUCTION OF presented by OBESE RATS Rachel Schemmel has been accepted towards fulfillment of the requirements for Ph.D. degree in Nutrition ”45/” ”A 014/ [Major professor Date August 11, 1967 0-169. ABSTRACT THE EFFECT OF A HIGH FAT RATION ON BODY WEIGHT, BODY COMPOSITION AND ADIPOSE TISSUE WEIGHTS OF RATS AS INFLUENCED BY AGE, STRAIN AND WEIGHT REDUCTION OF OBESE RATS by Rachel Schemmel Rats fed a high fat ration throughout their lives became extremely obese. Up to 150 days, they had similar amounts of protein, moisture and ash in their bodies as the grain-fed "lean" controls of the same ages. At all ages,the increased weight of the high fat-fed rats consisted primarily of' fat. However, adult obese rats had approximately a 10% increase in body ’ protein, assumed to be associated largely with proliferation of adipose tissue. For all ages, adipose tissues of the obese rats were larger than those in the lean controls. In young rats, the subcutaneous adipose tissue was larger than the abdominal adipose tissue. Except for the interscapular depot subcutaneous tissue weights of male rats weighing over 450 g paralleled body weight gains. Perirenal adipose tissue weights and that of the depot surrounding the xiphoid process continued to increase more rapidly with age than body weight. Genital depots showed maximal relative weight increases at the time of sexual development and then the rate of increase declined. The mesenteric and omental depots in the young rats increased more than body weight but this was reversed in older rats. The high fat ration produced increased body weight gain, percent of body fat and adipose tissue weights in the six strains of rats to Rachel Schemmel which it was fed. The strains were: Osborne Mendel, Sprague Dawley, HOppert, Wistar-Lewis, Hooded and MSU Gray rats. Osborne Mendel rats showed a greater prOpensity to weight gain and deposition of body fat than any of the other strains tested. This was especially true between 13 and 23 weeks of age. The rats of all strains were more efficient in converting feed energy to body tissue when fed the high fat ration than when fed the grain ration. For the first 10 weeks following weaning, males fed high fat had a 22 to 32% feed efficiency whereas grain-fed males had a 11 to 14% feed efficiency. High fat-fed females had a feed efficiency of 14 to 23% whereas for grain-fed females it was 8 to 12%- Obese Osborne Mendel rats (males = 1000 g, females = 650 g) were reduced by being fed (1) the high fat ration ad libitum on 2 of 7 days, (2) the high fat ration in daily amounts which in 7 days equalled the weekly intake of (l), (3) the grain ration ad libitum and (4) a semipurified diet approximating (3) in composition. During the first week of the reducing regimen, the rats in all groups and of both sexes lost 7% of their initial body weight. After another 24 weeks, the rats were sacrificed.’ By then the rats in (l) and (2) lost 40 to 49% of their body weights; those in (3) 30 to 35% and those in (4) 20 to 25%. ‘Nearly all the weight lost by the rats fed the grain or semipurified rations was fat. A small percentage of body protein was lost besides fat by the rats reduced on the high fat ration. Rachel Schemmel A11 adipose tissues decreased in weight during weight reduction. Those showing the largest losses of weight were the subcutaneous, mesenteric and genital depots. (These latter 2 had shown some decrease in relative weights with aging). The perirenal depots and that depot surrounding the xiphoid process showed a lesser decrease in weight with weight reduction. THE EFFECT OF.A HIGH FAT RATION ON BODY WEIGHT, BODY COMPOSITION AND ADIPOSE TISSUE WEIGHTS OF RATS AS INFLUENCED BY AGE, STRAIN AND WEIGHT REDUCTION OF OBESE RATS BY A77“ 1 9' RachelASchemmel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Foods and Nutrition 1967 ACKNOWLEDGMENTS The author gratefully acknowledges the guidance and inspiration of Dr. Olaf Mickelsen throughout her graduate studies. His assurance and“' kind assistance throughout the collection of the data and the preparation of this dissertation are greatly appreciated. A special word of "thanks" goes to Dr. Dena Cederquist who made all of this possible. The continued interest of Dr. Herman Slatis and his ideas directed toward the genetic evaluation of obesity are appreciated. The author is grateful to the forementioned committee members and to Dr. Clarence Suelter and Dr. Portia Morris for the constructive criticism of this dissertation. To Dr. Stanley Garn and the Staff of Fels Research Institute, Yellow Springs, Ohio, Dr. Ulreh Mostoskey of the Veterinary Surgery and Medicine Department, M.S.U. and Miss Dixie Middleton, the author is grateful for instruction and assistance in the preparation and interpretation of the radiographs. The author appreciates the consideration shown by other graduate students in the Department of Foods and Nutrition; especially Miss Jenny Taylor for her thoughtfulness and technical.assistance. A "thank you" to Dr. Kamal Motawi and Mrs. Brenda Burroughs Watts for technical assistance and to Owen Kleinschmidt, D.V.M. for assistance with care of the rats. The aid of Dr. Frances Magrabi for programming the data for computer analysis is appreciated. Also a "thank you" to 11 Mr. Tom Johnson for the technical drawing in Part II. A "thank you" to Mrs. Olaf Mickelsen for her thoughtfulness and understanding throughout this study. The author appreciates the financial support of the National Institutes of Health, Grant No. AM osuss; also a scholarship from Alpha Chapter of Omicron Nu made possible through the efforts of Miss Faye Kinder. iii In memory of my Father Mr. Frederic A. Schemmel iv TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . 3 Feed Efficiency - its Relation to Obesity . . .. . . . 3 Interrelationships of dietary components oi - . ~- 3- Method of feeding -_ . . . . . . . . . . . 4- Breeding . . . . . . . . . . . . . . . . 5 Activity . . . . . . . . . . . . . . . 6 Characteristics of Regulatory, Genetic and Dietary Obesities 7 Regulatory obesities . . . . . - . . . . . 7 Genetic obesities . . . . . . . . 9 Dietary obesities . . . . . . . . . . . 11 Obesity and Body Composition . . . . . . . . . . 13 Effect of age and sex . . . . . . . . . . . 14-~ Body composition of the rat . . . . .. .. . ,, . 15 Use of soft tissue radiograph for in vivo measurement Of bOdy fat 0 O O O O O O O O 0 O O O 17 Characteristics of White Adipose Tissue . . . . . . 19 Formation of the fat cell . . . . . . . . . . 19 Description a o a o o o .9 o '0 o o o o 20 The lipid free component of the adipose tissue . . . 22 The lipid component of the fat cell . . . . . . . 23 Distribution . . . . . . . . . . . . . . 25. Weight Reduction . . . . . . . . . . . . . . 28 Weight loss in vital organs during weight reduction . 4. 32 ReduCing by fasting . - . . . . .. a. . . . . 35. PART I: BODY ccm‘o‘srnon AND GROWTH OF ADIPOSE TISSUE IN OBESEANDNORMALRATS . . . . . . . . 37 Introduction . . . . . - . ,, . . . . . . 37 Experimental . . . . . . - - . . . . . . 39 Results 0 O Q ‘0 O O O O O ‘0 O .. O 0 43 Body weights . . .. . . . . . . . . . . . 44 Skeletal size . . . . . . . . . . . . . . 44 Bady pro tein o o o o o c o o o o o o o o 45 Body fat . . . . . . . . . . . . . . 46 Fat depots . . . . . . . . . . . . . . 46 Subcutaneous . . . . . . . . .. . ». . . . 46 Abdominal . . . . . - . . .. - - . . . 48 Discussion . . . . . . . . . . . . . . 50 Page PART II. FEED EFFICIENCY, BODY COMPOSITION AND DEPOSITION OF BODY FAT IN SIX STRAINS OF RATS FED THE GRAIN RATION OR THE HIGH FAT RATION . . . . . . . . 74 Introduction . . . . . . . . . . . . . . . . 74 Experimental . . . . . . . . . . . . . . . . 75 Results . . . . . . . . . . . . . . . . 77 Body weights . . . . . . . . . . . . . . . 77 Feed efficienCy . . . . . . . . . . . . . . 78 Activity . . . . . . . . . . . . . . . 79 Body protein and ash . . . . . . . . . . . . 79 Fat . . . . . . . . . . . . . 1 80 Inguinal fat depots . . . . . . . . . . . . . 81 Interscapular fat depot . . . . . . . . . . . 82 Genital fat depots . . . . . . . . . . 83 Mesenteric and omental fat depots . . . . . . . 85 Perirenal fat depots . . . . . . . . . . . . 85 Discussion . . . . . . . . . . . . . . . 86 PART III. WEIGHT REDUCTION OF OSBORNE MENDEL OBESE RATS: EFFECT ON BODY COMPOSITION, WEIGHT OF ORGANS AND ADIPOSE TISSUES . . . . . . . . . . . . 107 Introduction . . . . . . . . . . . . . . . 107 Experimental . . . . . . . . . . . . . . . 109 Results . . . . . . . . . . . . . 111 Reduction in weight . . . . . . . . . . . . 111 Food intakes . . . . . . . . . . . . . 113 Mbrbidity and mortality . . . . . . . . . . . 114 Ketosis . . . . . . . . . . . . . . . 116 Body composition of reduced rats . . . . . . . . 117 Ash . . . . . . . . . . . 118 Composition of tissue lost . - . - - . - - 118 Changes in organ size with weight reduction . . . . 119 Kidneys . . . . . . . . . . . . . . . 119 Adrenals . . . . . . . . . . . . . . . 120 Heart . . . . . . . . . . . . . . . 121 Liver . . . . . . . . . . . . . . . 121 Spleen . . . . . . . . . . . . . . . 121 Summary . . . . . . . . . . . . 121 Reduction in fat depot weights . . . . . . . . 122 Inguinal depots . . . . . . . . . . . . 122 Genital depots . . . . . . . . . 123 Perirenal and retrOperitoneal depots . . . . . 123 Mesenteric and omental fat . . . . . . . . 124 Fat surrounding xiphoid process . . . . . . . 124 vi Recapitulation Depot . . . Type of reducing regimen Age . . . Sex . . . Discussion . Initial weight loss Grain vs. semipurified rations Organ weights . . . . Protein . . . Fat depots . . . SUMMARY AND CONCLUSIONS LITERATURE CITED . vii Page 124: 125: 125' 126‘ 126L 126 126 127‘ 129 131; 131* 150 154 TABLE 10 ll 12 13 14 15 16 LIST OF TABLES Body composition of rats as determined by various investigators . High fat ration (M-lS) . . . . . . Body weights, age, length and composition of Osborne Mendel rats fed either a grain or high fat fation (continued) . . . Total weight cf subcutaneous fat depots vs. abdominal fat depots . Relative weights of fat depots in rats fed either grain (M—l) or high fat (M-lS) rations . . Summary of weight changes in adipose tissues relative to body weight changes as.inf1uenced by age, diet and sex a o c o c o o o o o a Body weights of six strains of male, female and weanling rats fed either the high fat or grain ration Feed efficiency for six strains of male and female rats. Rank order of feed efficiency . Percent of calories converted to body energy Calories consumed for calories of protein gained in the body . . . . . . . . Summary of activity Rank order for activity Protein in the carcasses of six strains of male and female rats fed either a high fat or a grain ration Ash content in grams of carcasses of male and female rats of six strains - - Percent of body fat in six strains of rats viii PAGE 16 53 54 55 56 57 58 9O 91 92 93 94 95 96 97 98 99 TABLE 17 18 19 20 21 22 23 24 25 26 27 28 29 Composition of semipurified ration Proximate analysis of Grain (M-1) and semipurified (M-16) rations . Food intakes of rats during weight reduction period Cumulative average caloric intake per rat during twenty weeks of the reduction regimen Some characteristics of rats at termination of weight reduction regimen (WRR) Percent body composition of obese and lean controls and reduced Osborne Mendel male and female rats . Absolute body composition of obese and lean controls and reduced Osborne Mendel male and female rats .. Composition of weight lost by obese Osborne Mendel rats as a result of reduction of body weight . Percentage of gross body components lost during weight reduction . . . . Organ weights of obese and lean controls and reduced Osborne Mendel male and female rats Relative weights of adipose tissues . Rank order of weight reduction of individual adipose tissues . . . . . . . Influence of sex on final weights of fat depots in the reduced obese control and lean control rats. ix PAGE 133 134 135 136 137 138 139 140 141 142 143 144 145 FIGURE LIST OF FIGURES Body weights of male and female Osborne Mendel rats fed either the high fat or grain ration . . . . Increase in body ash content of male Osborne Mendel rats Increase in body ash content of female Osborne Mendel rats Increase in body protein content of male Osborne Mendel rats a o o o o a o o o o o o o o o 0 Increase in body protein content of female Osborne Mendel rate I O O O O O O O O O O O O D O 0 Increase in body fat content of male Osborne Mendel rats Increases in body fat content of female Osborne Mendel rats 0 I O O O O a . Sum of weights of right and left inguinal fat depots expressed as percent of body weight for male and female Losborne Mendel rats. . . . . 10 11 12 13 Sum of weights of right and left depots underlying forelimb eXpressed as percent or body weight for male and female Osborne Mendel rats - Weight of interscapular fat dorsal to neck expressed as percent of body weight for male and female Osborne Mendel rats . - - . . . . Sum of weights of right and left genital fat depots expressed as percent of body weight for male and female Osborne Mendel rats . . . . . . Sum of weights of right and left perirenal and retro- peritoneal fat depots expressed as percent of body weight for male and female Osborne Mendel rats. - - Weight of mesenteric and omental fat depots exPressed as percent or body weight for male and female Osborne Mendel rates 0 o o o o o o o o o o 0 PAGE 59 61 62 63 64 65 66 67 68 69 70 71 FIGURE 14 15 16 l7 18 19 20 21 22 23 Weight of fat depot surrounding xiphoid process cartilage expressed as percent of body weight for male and female Osborne Mendel rats Percent of protein in rats of various ages expressed as percent of lean body mass as determined by different investigators . . . . .. . . . . Differences in body weights and body fat contents of male rats of each strain fed the high fat and grain rations. o ‘ g o o o o o a o a o o 9 Differences in body weights and body fat contents of female rats of each strain fed the high tat and grain ratlona e s o o o o o o o o o o 0 Sum of the weights of the inguinal fat depots expressed as g per 100 g of body weight . . . . . . Weight of the interscapular depot expressed as g per 100 8 Of bady weight. 0 O O O o O O O O O 0 Sum of the weights of the genital fat depots expressed as g/100 g of body weight . . . . . . Sum of the weights of the mesenteric and omental depots expressed as g/100 g of body weight Sum of the weights of the perirenal depots expressed as g/100 g of body weight . . . . . . Decreases in male Osborne Mendel rats subjected to one of the following reducing regimens: semipurified ration, ad lib.; grain ration, ad lib.; high fat ration 2 days each week; high fat ration in restricted amounts every day and grain-fed control rats xi PAGE 72 73 100 101 102 103 104 105 106 146 FIGURE PAGE 24 Decreases in female Osborne Mendel rats subjected to 25 26 one of the following reducing regiments: semipurified ration ad lib.; grain ration, ad lib.; high fat ration 2 days each week; and high fat ration in restricted amounts every day and grain-fed control rats . . . . 147 Weight of protein in reduced Osborne Mendel rats in comparison to weight of protein in younger high fat-fed rats (Part I). . . . . . . . . . . . . . . 148 Weight of fat in reduced Osborne Mendel rats in comparison to weight of fat in younger high fat-fed rats (Part I) O O I I O O O O O O O O O O 149 xii INTRODUCTION Obesity is invariably more common in countries with continued prosperity and greater mechanization. Accompanying this trend is an increased number of deaths from degenerative diseases to which obesity appears to play a contributory role (Body Build and Blood Pressure Study, 1959; Dublin, Latka and Spiegelman, 1949; Dublin and Marks, 1953). In these studies obesity was judged exclusively on the basis of body weight. Behnke et a1. (1942) and Brdgek (1952) suggest that a more reasonable criterion for obesity is based on the percent of body fat rather than on weight alone. The importance of a more specific measure of obesity is stressed in a review by Keys and Brogek (1953). There are numerous methods for measuring of body fat content; these include densitometry, hydrometry, roentgenography, anthrOpometry, whole body counting for potassium, helium dilution, creatinine excretion (Brogek, 1963) and ultrasonic reflections (Bullen et al., 1965). All have proven useful for determination of body fat or lean body mass, and ultimately, obesity. A caloric intake beyond expenditure results in weight gain. However, many individuals, men as well as animals, without thought or concern are able to balance caloric intake and eXpenditure for long periods of time. For others, positive caloric balance is readily maintained. This condition appears to be the result of a number of disturbances, (Anand, 1960; Bruch, 1958; Chirico and Stunkard, 1960; Mayer, 1953; 1961; Young, 1964) thereby causing a complexity of obesities which may behave physiologically and metabolically quite differently and be further complicated by psychological and emotional factors. The solution of the problems in any one of these types of obesities will greatly contribute to the overall elucidation of obesity. An ultimate goal is a thorough understanding of the involvement of food in producing a healthy and attractive individual with a long life span- At the present time, socialacceptability and life insurance statistics indicate the negative role played by obesity. Due to the long life span of man, his inadaptability to the rigdrs of ' strict experimentation and the possibility of adverse effects associated with long-continued studies makes it imperative to find a substitute test animal. The laboratory rat has been chosen for many studies of different facets of the obesity problem. The insufficient criterion of weight alone in man gives way more readily in the rat to more highly controlled food efficiency data, deposition of body fat and chemical analysis for percent of body fat, so that hereditary and environmental factors contributing to obesity can be more clearly evaluated. Likewise, cheating or false insinuations of cheating, are eliminated in weight reduction studies which thereby permit a more thorough and unbiased study of the value of weight reduction than can be done in man. Data on stabilization at the reduced weight are needed since so few men or women who have been reduced maintain their reduced weights. Since overeating most frequently represents the primary physiological factor involved in human obesity, rats made obese by concentrated sources of calories (Mickelsen et al., 1955) should make a unique contribution to the further understanding of the obesities. REVIEW OF L ITERATURE FEED EFFICIENCY--ITS RELATION TO OBESITY Interrelationships of dietary components The composition and nature of the ration may have a marked influence on the rate of body weight gain of experimental animals- Unfortunately, these studies have involved so many dietary variables (e.g. prOportions of cellulose, or fiber, type of carbohydrate, type and amount of fat, etc.) that it is impossible to draw definite conclusions about what the ideal ration ought to be to support greatest weight gain. One observation does stand out, and that is: that many nutritionally adequate semipurified rations fed to rats give better weight gains than do stock or chow diets (Mickelsen et al., 1955; Reed et al., 1930; Barboriak et al., 1958). Di Giorgio et a1. (1962) reported that lipogenic activity of epididymal adipose tissue is less in rats fed stock diets than in animals fed any of the purified diets with which they worked. These rations contained either safflower, corn,olive, hydrogenated cottonseed, butter or cocoanut oil with either glucose or starch as the carbohydrate. Hajdu (1942) reported that an equal prOportion of fat and carbohydrate produced better growth and work performance in rats than did either a high or low intake of carbohydrate or fat. On the other hand, Hoagland and Snider (1940) maintained that when the basis of measurement was efficiency of utilization expressed as gain in weight per 100 calories of food consumed, rats fed the low fat diets were more efficient since the greater growth on the higher fat diets required a greater consumption of food. Data from both Forbes et al. (1946a, 1946b) and Deuel et a1. (1947) indicated that diets containing 30 to 60% fat 3 4 caused greater gains in body weight, not only because of increased feed consumption, but also because of increased utilization of calories for weight gain as determined by calories wasted for heat production. The least efficient combination of food stuff as indicated by maximum heat increments was a diet containing no fat which was composed of cellulose and beef protein (Forbes and Swift, 1944). All seem to be in agreement, however, that rats consuming a 40 to 60% hydrogenated animal or vegetable fat will grow more rapidly than controls consuming a diet lower in fat content. Glucose produced greater weight gains in rats when substituted for starch in a semi-purified ration where the percentage of protein in the diet was just adequate for growth (Howe and Gilfillan, 1963). Similarly, weight gains were not as great in baby pigs when the dietary carbohydrate was corn starch or dextrin in comparison to glucose (Sewell and Maxwell, 1966). The greater growth of the glucose-fed pigs was associated with a greater feed consumption rather than any difference in feed efficiency. Method of feeding Rats either trained (Hollifield and Parson 1962a; b) or forced to consume a daily portion of food at two feedings (Cohn and Joseph, 1959; Cohn, 1963) gained the same amount of body weight but deposited a larger percentage of it as fat if force-fed. If the rats were fed a high fat ration, this was even more pronounced (Cohn et al., 1965). After a week of adaptation to a meal eating pattern, Leveille and Hanson (1965) reported that "meal eating" rats had a greater food efficiency. Actually, the meal eaters never ate as much food in a day as the nibbler rats, but their gain in weight was greater. Increased lipogenesis (Tepperman et al., Dickerson et al., 1943) in the meal eaters has been considered a contributory factor to the increased percent of body fat. Mere specifically, Tepperman and Tepperman (1958) reported that lipogenesis from acetate and glucose was greater in liver slices from rats trained to eat their food in a short time, and Hollifield and Parson (1962a) observed increased incorporation of acetate into fatty acids in adipose tissue of meal eating rats. Breeding Slight individual differences within the same species and even the same strain allow for some differences in metabolism and utilization of feeds. By selective inbreeding of a male and female of greatest food efficiency and also inbreeding of the least efficient, it is possible to produce strains which show high or low feed efficiency. Palmer and Kennedy (1931) reported that rats of their stock colony, though inbred for several years, still maintained variations in food efficiency. By the fifth generation of selective breeding, they separated two strains with a difference in feed efficiency of 40% (Calverley et al., 1946). The difference in feed efficiency was also associated with a difference in body composition. Even at weaning time, when the rats of both strains weighed 60 g, the animals of the more efficient strain had a higher percentage of body fat and a smaller percentage of prdtein. Six weeks later both males and females of the high efficiency strain weighed around 50 g more than the low efficiency strain for both males and females and had 3.0 to 5.0% more body fat (ibid). In this study, and the one reported by Morris et al., (1933) the females were less efficient in food utilization than the males. Zucker (1960)1ikewise, after selective inbreeding of successive generations of Norway black and white hooded rats secured two strains which differed in feed efficiency. The high efficiency strain which produced better weight gains and more fat later produced an obese mutant (Zucker and Zucker, 1961) which is discussed in more detail in the section on genetic obesities.. A complicating factor has been introduced by the observation that a reduction in litter size during the suckling period produces a rat which increases its body weight very rapidly but a high prOportion of its weight is fat (Widdowson and MeCance, 1960). Activity The amount of volitional or forced exercise obviously plays an important role in the deve10pment of obesity. Increased physical activity, regardless of its nature, especially if it requires a considerable fraction of the daily energy intake, inhibits the deve10pment of obesity. At the apposite end of the scale, a decrease in activity, verging on complete inactivity, makes it difficult for the animal to adjust its caloric intake to its requirement. Consequently, obesity deve10ps (Mayer, 1955). Ingle produced obesity in rats by forced feeding (1946) and claimed that ad libitum feeding with restriction of the animals: cage size (1949) produced the same effect. Actually, Ingle's ad libitum feeding was complicated by the fact that he incorporated water into his ration. When the rats became thirsty,'they had to eat the wet ration. However, others suggest that the obesity produced by feeding normal rats a special diet (Mickelsen et al., 1955) or as a result of hypothalamic lesions in mice (Liebelt, 1963) results in curtailed activity so that additional confinement isn't really necessary. Whether the obesity results in decreased activity or whether decreased activity results in the obesity does not appear to be resolved. However, Bielschowsky and Bielschowsky (1956) claim that in N20 genetically obese mice the inactivity was the result of the obesity. CHARACTERISTICS OF REGULATORY, GENETIC AND DIETARY OBESITIES .Rggulatory obesities With Clark's modification (1939) of the Horsley-Clarke apparatus, surgical lesions could be placed specifically in the ventromedial nuclei of the hypothalamus of the rat to produce hyperphagia and obesity (Hetherington and Ranson, 1940). These first eXperiments were no doubt an outgrowth from the controversy over whether or not a faulty pituitary or hypothalamus resulted in a characteristic obesity in man described by Ffbhlich (1901; Bruch, 1939). Hetherington and Ranson (1940) described hyperphagic rats made obese with bilateral surgical lesions of the hypothalamus as showing enlargement of adipose tissues in the abdomen, in the mesenteries and around the kidneys. These rats also had large accumulations of fat in the perineal region and over the neck and upper chest. Both sexes showed enlargement of the subcutaneous fat depots as well. The weight gain in these obese rats was primarily due to the storage of fat; true growth with synthesis of protein was not apparent in hypothalamic obesity (Hetherington and Weil, l940)-_ Breaker.,. and Waxler (1949) report similar accumulations of fat in the abdominal cavity of obese mice injected intraperitoneally with gold thioglucose which caused similar lesions in the hypothalamus (Marshall et al., 1957). 8 Invariably, the nose to anus length of the obese rat with surgical or electrolytic lesions is shorter than its control (Hetherington and Ranson, 1990; Kennedy, 1951; sérélo, 1965). Tibias (sétéio, 1965) and femurs (Han et al., 1965; Han and Lin, 1966) also were shorter in the lesioned rats. Brobeck et al., (1943) indicated that the body length of lesioned rats showed no deviation from normal, but these rats were operated on as adults. Bernardis (1963) found that lesions in all sites of the hypothalamus except the ventromedial nuclei resulted in growth impairment. There is some confusion as to the effect of obesity on the mineralization of the skeleton. Brecker and Waxler (1949) on the basis of radiographic examination of gold thioglucose induced obese and contrdl mice observed no differences in size, shape or density of bones in the two groups. Furthermore, analyses of the whole carcasses indicated no differences in the weights of ash in the two groups. On the other hand, Hetherington and Weil (1940) reported some depletion of calcium, phosphorus and iron in the hypothalamic obese rats. The hypothalamic lesioned obese rats after gaining a great deal of weight developed sores and scales on their hind feet, their hair became sparse with the skin in these spots appearing scaly and irritated. Sometimes, the very obese animal would begin losing weight and die in a short period of time with necropsy revealing some slight respiratory infection (Hetherington and Ranson, 1942; Kennedy, 1951). Males usually developed shrunken scrota and the testes lay in the inguinal canal. Necropsy revealed the testes of the male to be small in most cases with atrophy of the reproductive glands (Hetherington and Ranson, 1940). Liebelt (1963) reported abnormal ovarian cycles in mice made obese by chemical lesions. These obese mice did not breed but if c0pulation occurred prior to administration of the gold thioglucose, pregnancy proceeded to term. There was some indication in these mice that lactation was impaired. In these obese mice, the adrenals were slightly enlarged while pituitaries and thyroids were smaller (Sétalo, 1965). Liebelt (1963) reported hyperfunctioning of the thyroid gland in mice. Alterations have been observed in pancreatic beta cells, (Gepts, 1963; Hausberger et al., 1964) as well as enlargement and alterations in their structure and function (Brobeck et al., 1943). Stomach weights showed no significant differences but gastric secretions did increase in hypothalamic obesity in rats (Ridley and Brooks, 1965). The increased gastric secretion may partially explain the acute gastric ulcers in the chemically lesioned mice observed by Liebelt (1963). Implantation of electrodes in the ventromedial nuclei which can be stimulated periodically will cause even greater hyperphagia in rats than the electrolytic or chemical destruction of the "satiety" center (Steinbaum and Miller, 1965) and even more excessive weight gains. Following the surgical lesions, rats became hyperactive for a couple days postOperatively, followed by a large decrease in Spontaneous running activity (Hetherington and Ranson, 1942). Liebelt (1963) calls the mice injected with gold thioglucose "active" but doesn't feel that the methods used to study activity have been entirely satisfactory. Genetic obesities Four strains of genetically obese mice have been identified, three of which have been associated with a single gene. One of these is dependent on a dominant gene associated with a yellow coat color (Danforth, 1927). Two of these are due to a single recessive gene, one 10 identified as ob (obese) (Ingalls et al., 1950) and the other as ad (adipose) (Falconer and Isaacson, 1959). The genetics of the New Zealand (NZO) strain of obese mouse has not been determined (Bielschowsky and Bielschowsky, 1956). The mice displaying the ob gene have been studied extensively by Mayer (1960). These mice with the obese hyperglycemic syndrome display many of the characteristics of the gold thioglucose induced obesity of mice. They have large deposits of body fat with decreased lean tissues, (Vlahakis and Heston, 1959) display decreased activity, enlarged liver, heart, pancreas, and small uteri and ovaries (Meyer, 1960). They are resistant to mating (Runner and Gates, 1954) and are produced from some pairs of non-obese littermates in a 1:3 ratiow They display pancreatic dysfunction with increased insulin and glucagon secretion. Weight gain is at a maximum on a high carbohydrate diet with less weight gain on a high protein or a high fat diet (Mayer, 1960). Castle (1941) indicated that the obese mouse with the yellow coat color in addition to being obese, had a slightly longer body length. Females became more obese than did males of this strain (Danforth, 1927). Adipose mice (Falconer and Isaacson, 1959) don't develop obesity for 5 to 6 weeks after birth, the main weight gain being body fat. On the other hand, Bielschowsky and Bielschowsky (1956) reported that the N20 strain of obese mice did reproduce, although reproduction was not as favorable as in other strains. Gestation and lactation, however, delayed the obesity. Estrus cycles in the obese NZO females are, more frequently than not, elongated beyond the four or five day normal cycle. The fat 11 depots of the abdominal cavity, inguinal and retrOperitoneal area were enlarged with accumulations of fat which were largely responsible for the weight increase. In conjunction with a high resistance to insulin was a very much altered pancreas with a larger pr0portion of beta cells. Until reaching extremely marked adiposity, these mice were as active as other strains of mice. An obese mutation in the rat dependent on the presence of the homozygous recessive gene, fafa,, has been described by the Zuckers (1961, 1962, 1963, 1964, 1967). The increased weight of this rat called "fatty", primarily consists of fat, with no increase in skeletal size nor muscle mass. There is a large accumulation of fat in the subcutaneous area surrounding the neck and chest. Heterozygous sibs of "fatty" rats (Genotype Fafa) became fat if fed a high fat diet with a somewhat different prOportioning, that is, more fat in the abdominal cavity (Zucker and Zucker, 1963). Fat accretion in this rat was more closely associated with weight gain than with age. Hyperlipemia was a definite characteristic of these rats (Zucker, 1964). Dietary obesities That environmental dietary factors can modify genetic prediSposition to normal weight is clearly demonstrated by Fenton and Dowling (1953) and Fenton and Chase (1951). In their studies of growth rate and efficiency of food utilization these investigators (ibid) reported that 03H and A strains of mice gained weight which was roughly prOportional to the fat content of the diet. They became obese when fed a 47.5% fat ration. Increasing the dietary fat level above 15% did not cause similar gains in ‘body weight in the C57 strain of mice. Fenton (1956) concluded that a 50% hydrogenated fat ration caused an increased percentage of carcass fat, 12 deSpite the fact that weight gains were not prOportional to percent of dietary fat (Fenton and Dowling, 1953). I strain mice do not show this similar increase in carcass fat on a 50% fat ration. Other strains which exhibited similar obesity when fed a high fat ration were DBA/ZJN, STR/N and C57L/HeN, (Sokoloff et al., 1960). By the use of rations containing 40 to 60% hydrogenated fat, Mickelsen et a1. (1955) and Barboriak et a1. (1958) reported large weight gains of rats thus fed in contrast to stock fed controls or rats fed semi- purified diets containing liquid fats. Although others (Reed et al., 1930; Hoagland and Snider, 1941; Haldi et al., 1942; Forbes et al., 1946 a, b, c, d; Lundbaek and Stevenson, 1947; Hoagland et al., 1952) reported slightly increased weight gains in rats fed high fat diets, none of these maintgiged their rats sufficiently long for true obesity to deve10p. [J'The increased weight in these rats made obese by high fat rations was composed primarily of fat (Mickelsen and Anderson, 1959). There were slight increases in skeletal length (Mickelsen et al., 1955). Those rats maintained on a 50% hydrogenated fat diet have an increased quantity of fat in the skin (Haldi et al., 1942; Page’and Babineau, 1953) in contrast to those animals fed a stock ration or even a ration containing a liquid fat. Histological work on obese 40 week old rats (Barboriak et al., 1958) indicated that these rats are resistant to arterial lesions, that the livers have slight infiltration of fat and that the kidneys show some mild swelling of the tubules. Yamamoto et al. (1959) reported increased proteinuria in these rats consuming a high fat diet. l3 Metabolism, at least in the young rats fed this high fat diet, did not differ markedly from controls on a 50% fat diet (Reynolds and Pringle, 1965). OBESITY AND BODY COMPOSITION Because of the constancy of lean body mass, and the variability in body fat, determination of any one gross component of the body as water, protein or fat can be used to calculate percent of body fat, and ultimately the degree of obesity. Brogek has comprehensively reviewed the analytical methods (1965; 1963; Brogek and HenscheL ed., 1961). The work of Reid et a1. (1955; 1956; 1957; 1963) van Niekerk et a1. (1963) and Pearson (1963) have contributed greatly to body composition data on large animals. For the most part the review here will be limited to the rat, and consideration will be given only those data obtained by direct chemical analyses for the major components of the body as fat, protein, minerals and water. Body composition as affected by weight reduction, will be considered in the section devoted to "weight reduction." Fat is the major variable in the body. However, water, in most instances, makes up the largest percentage of body mass. After reaching puberty, water tends to make up a constant portion of the lean body mass (about 72.3% of the latter) (Reid et al., 1955). Protein will make up 17 to 20% of this total body mass (21-25% of lean body mass) with the remainder being primarily minerals (ibid). In view of the large amount of work being done in this area, there is very little besides the early work of Rathbun and Pace (1945) which relates the formulae to actual results of bodies that were analyzed for fat, protein, water and ash. 14 lgffect of age The percentage of body fat in the human subject is lowest during puberty. It is high in the infant, decreases during childhood and adolescence and increases during adulthood to a peak in late maturity. Mayer (1948; 1949) states that passage from protein synthesis in the young to fat synthesis in the adult constitutes a phase of aging. During growth, lean muscle mass accounts for a large increase in intracellular water (Kerpel-Fronius, 1958; Yannet and Darrow, 1938). In senescence this intracellular compartment decreases leaving a porportionately increased extracellular compartment (Lowry et al., 1942). Associated with this is a reduction in total lean body mass with a compensatory or greater increase in fat. The adipose tissue cell engorged with fat is composed of only 6-20% water rather than 75% as is muscle tissue. Nevertheless, the percent of water in the fat-free portion of adipose tissue is comparable to other tissues. With aging, however, fat composes a considerably larger prOportion of the fat cells (Behnke, 1964). Effect of sex There are many reports that among "normal weight" human beings, adult women have a considerably higher percentage of fat in their bodies than men (Wilmer, 1940; Reynolds and Asakawa, 1950; Garn, 1957snf Edwards, 1951; Keys et al., 1950; p. 173). The values for the body fat content of men range from 10-20% (Brogek, 1952; Mitchell et al., 1945; v. Dobeln, 1956; Pascale et al., 1956; Edelman et al., 1952; Ljunggren et al., 1957) and of women from 20-30% (Brogek et al., 1953; Bro¥ek, 1962; v. Dobeln 1956; Young et al., 1961; 15 Edelman et al., 1952; Ljunggren et al., 1957). Garn (1957a)' claims that the absolute amount of fat in the bodies of normal weight men and women is the same but since women have a smaller lean body mass, the percentage of fat is greater. The period of life when this differentiation in percentage of body fat first occurs seems to be less clearly established--many claiming it first begins at puberty, while others claim it is evident at birth (Owen et al., 1962; Hanna, 1963) and perhaps even befOre birth (Forbes, 1962). Where variations in prOportion of body fat between the sexes are slight, as in fetuses and new born infants, it becomes difficult to establish whether these differences are due to sex or individual differences. Although the difference is not statistically significant, the trend indicates a greater percentage of fat is already present in the female during the gestation period. Body composition of the rat The summary review table (Table 1), indicates the composition of the four major components of the body of the rat as determined by different investigators. Procedures for preparation of the carcasses varied from grinding raw or frozen carcasses with meat grinders to partially hydrolyzing the carcasses and homogenizing. Meisture and fat were analyzed on whole animals or on aliquots while nitrogen and ash were frequently analyzed on dry, fat-free samples. 16 .oocmquMHv he noHNHDUHmo nouns mo mwmucooumm mmaaemm pmHoom co coop mommamccH cm< .uoum noumz nm< 4mm .uoum nouns .uw3 . .oz owe. Rom 44m: meom smog c0444monaoo moon Hmuoa muoumw444m>c4 4:044m9 an pocwaumump mm mumu mo co4u4momsoo hpom H manna 17 Use of the soft tissue radiograph for in vivo measurement of body fat In lieu of direct chemical analysis for body composition indirect methods have been developed for measurement of body fat or lean body compartments. One such measurement is the soft tissue radiograph from which the measurement of skin to muscle shadow can be made. Formulae can probably be developed for converting these measurements to body fat content. Through the work of Garn (1954; 1957a; 1957b; 1957c) and his associates (Garn and Harper, 1955; Garn et al., 1956) the systematic measurements of the depth of subcutaneous fat layers introduced by Stuart et al., (1940) has become recognized as a valuable tool for prediction of adiposity in man. A comprehensive review of the soft tissue radiographic technique and complications involved in its use for human subjects was presented by Garn (1961) at a symposium on the techniques for measuring body composition. Animal work is more limited. Hogreve (1938) used radiographic measurements in a study of the process of fat deposition in the hog. Wussow and Weniger (1953) measured fat thickness at four different sites on hogs and believed this method had applicability in studies of the inheritance of fatness. Stouffer (1963) used this method successfully for evaluation of fat thickness over the back of the hog. Mickelsen (1958) introduced this work in laboratory animals. Crofford and Davis (1965) in the NZO obese mouse have used the radiograph primarily to show the similarity of skeletal size of this genetically obese mouse and a lean control of another strain. Although they have done chemical analyses for body composition no correlation with radiographic measurements was attempted. 18 Since the depth of subcutaneous fat on a linear surface is being measured on the radiograph, the data obtained from this measurement are only useful insofar as they are able to be mathematically transformed into a value for body fat. Garn (1957a; b) proposed the simple straight line equation of y = ax+b wherein y (total body weight) bears a straight line relationship to the fat thickness (x) with a slope defined by the constant (a). Weight is made up of two components (Behnke 1953), the more variable fat component and the constant lean body mass (b in the above equation). If the lean body mass is subtracted from each side, y' (weight of fat) = ax. The constant (a) is determined by the equation, a =gr 3% in which r is the correlation between the fat pad thickness and total body weight, 0y and ox are the standard deviations of weight and thickness of the fat pad respectively. Garn (1957a) reported data obtained from this formula on the trochanteric fat pad in men and the iliac crest fat pad in women to be in agreement with body fat determined by densitometry and blood dilution methods of comparable groups of older men, 20-64 year old women and young men. However, Young et al., (1963) applying this formula to women found values for fat calculated from the iliac spine fat pad to be roughly two-thirds of those values determined on the same women by densitometry measurements. Thus far, application of the above formula or similar formulae to animals wherein they can be verified with actual analyses for fat, has not been attempted. In the human, they have been primarily verified with densitometry measurements. In turn, a formula for prediction of body fat from specific gravity has been derived from linear regression-equations developed from underwater weighings and chemical analyses of fat in guinea pigs (Rathbun and Pace, 1945; Pitts, 1956). l9 CHARACTERISTICS OF THE WHITE ADIPOSE TISSUE Formation of the fat cell Two theories (ledt, 1870, Flemming, 1871b) have been pr0posed for the development of the fat organ, and combinations of these theories have been held by others (Kalliker, 1886; Hammar, 1895). According to Flemming (1871a; 1871b; 1876), adipose tissues were included among the connective tissues. He concluded that they belonged to the mesenchymal cells until such time as they accumulated fat. The fact that the fat cell did not occur randomly in the connective tissue but deve10ped wherever there was a rich vascular supply tended to support the theory of Tgldt (1870) that the' adipose tissue was clearly separated from the surrounding connective tissue. Kalliker (1886) and Hammar (1895) identified small localized areas along blood vessels and their capillary networks wherein fat accumulated.‘ ledt (1870) had called these sites "primitive fat organs." Hammar (1895) - showed these "primitive organs," to be the precursors of brown fat and not white fat, so he accepted Flemming's concept that the white fat cell is a modified fibroblast originating from the connective tissue. This idea was supported even as late as 1962 by Bloom and Fawcett (1962). The development of these theories has been reviewed by Wassermann (1964; 1965), Wertheimer and Shapiro (1948) and Wells (1940). These highly organized formations which were called "primitive organs of the white adipose tissue" were demonstrated to be present in human embryos by Wassermann (1926) and in the bovine embryo by Bell (1909). Many species (man, Tedischi, 1946; rats, Hausberger, 1938; 1955; mice, Liebelt, 1956; and birds, Clara, 1923) exhibit specific anatomical sites wherein adipose tissue is present and able to proliferate and accumulate lipid. 20 "The first adipose cells appear in the mesenchymatous lobules from which hair follicles and sebaceous glands also develOp. The primitive fat cells differentiate from the reticular cells with the penetration of a capillary bud into the lobule which proliferates first along the axis of the lobule, then Spreads and sends branches into the periphery" (Wassermann, 1926). "Adipogenesis is connected to this proliferation of the capillary thus resulting in the newest cells appearing at the periphery of the lobule," (Simon, 1965). "No fat or glycogen is found in other fibroblasts of the connective tissue except in these "primitive organs"," (Wassermann, 1965). "This "primitive organ" is formed both by the growth of the endothelial and adventitial cells. These areas can first be recognized because of the relatively thick layer of adventitial cells in conjunction with the arterioles and venules of that area. By this vascular growth, the primitive organs are built into the connective tissue as separate formations from the connective tissue itself," (Wassermann, 1958). The lobules of the adipose tissue retain the original organization of the primitive body although sometimes it is difficult to identify this organization because of the accumulation of fat in the cells. Description The mature white fat cell has a spherical shape and large size. The single large fat drOplet causes the cytOplasm to be diaplaced so that it forms a thin ring around the fat dr0plet. The nucleus is flattened and pushed to the edge of the cell, so it causes a bulge of the cell membrane in that area. Around the nucleus are observed numerous elongated rod shaped mitochondria (Tedischi, 1960). 21 Electron microsc0pic studies (Napolitano and Gagne, 1963) of white adipose tissue from the inguinal or epididymal fat depots of the young white rat or mouse show a very thin rim of cytOplasm around the lipid inclusion. The cytoplasm contains mitochondria and other organelles. There appears to be no membrane separating the lipid drOplet and the cytOplasmic membrane. On the cell exterior, the cytOplasm is bound by the plasma membrane which is surrounded by an amorphous area--the basement membrane. The nucleus is diSplaced to the periphery of the cell and is flattened. The nucleus contains a distinct nucleolar region. Most of the cytOplasm is also concentrated in the area surrounding the nucleus, and the cell organelles are concentrated in this area. The mitochondria vary in shape from ovals to very fine filaments. They do not contain as many cristae nor are they as large as in other cells (Napolitano and Fawcett,l958). The cytOplasm contains tiny granules identified as ribonucleo-protein, and occasionally, endOplasmic reticulum, Golgi bodies and in immature white adipose cells, glycogen is present. After the cells attain maturity, the glycogen disappears from the cell. The basement membrane of the signet ring is surrounded by an area of collagen fibers and individual collagen fibers are seen throughout the intercellular area. This fatty area also contains an occasional non- myelinated nerve, other mesenychymal cells and blood vessels. An electron microsc0pic study by the same authors (ibid) wherein animals were maintained in good health but gradually depleted of their fat Stores, shows that the cells do undergo morphological changes. Fat depleted cells now become ovoid rather than Spherical, diminish in size and the cell surface is no longer smooth but shows many indentations. The intercellular space is enlarged and more highly concentrated with collagen fibers in 22 proximity to the outer fat cell surface. Mitochondria and nuclei appear to be the same as in the Spherical lipid-filled cells. The depleted cell contains a large nucleus. The lipid free component of adipose tissue Mature fat-free adipose tissue has a gross composition similar to other tissues and organs; approximately 79% water, and 21% residue which is chiefly protein (Allen et al., 1959). An amino acid analysis of this protein (Bowes and Kenton, 1949) shows a distribution of amino acids which [is very similar to that of collagen except lower in proline and hydroxyproline. Sugars primarily found in adipoSe tissue are the structural sugars, muc0polysaccharides and glyc0proteins. The large linear molecules of muc0polysaccharides consist of N-acetylated or sulfated hexosamines in conjunction with a hexuronic acid or hexose repeating disaccharide units (Dorfman,l959). The glyc0proteins exist in small heterOpolysaccharide units attached to peptide chains (Spiro, 1963). The nature of these sugars suggesusthey are primarily structural rather than storage depots as is glycogen. There is little or no glycogen found in the adipose tissue, with the mature cell being especially devoid in this component. The entire protOplasm of this cell occupies 2.4% of the entire volume of the fat cell (Gersh & Still, 1945). The intercellular Spaces are shown to contain a Specific nerve supply (BBecke, 1933) and an abundant vascular supply (Gersh and Still, 1945) equivalent to or greater than other tissues and organs. During periods of increased deposition of body fat, Liebelt (1956) has shown that the lipid-free component of inguinal and gonadal fat 23 depots in mice increaseiin prOportion to the increase in body weight. Pitts (1956) reported that in female guinea pigs the fat-free adipose tissue makes up a larger percentage of the lean body mass than in male guinea pigs. This comprises 7 Z of the total lean body mass in the female, while in the male guinea pig it makes up 47; of the total lean body mass. The lipid component of the fat cell Adipose tissue in the newborn rat is fat-free (Hausberger and Gujot,l937)- As early as three days after birth the inguinal fat body contained 54% fat and by the end of 2 weeks 79% in the rat (ibid). In the lean adult mammal, adipose tissue had at least 60% or more lipid substance (Behnke, 1964). The accumulation of more lipid enlarged the fat cells so that they contained a larger percentage of fat and at maximum accumulation contained 85% fat. The Hausbergers (Hausberger and Hausberger, 1957) reported that in extremely obese mice the diameters of the fat cells increased by 35% compared with cells from normal mice. This increase in diameter permitted an accumulation of about 150% more fat. However, new cells also deve10ped in the obese mouse and 75% of the increased quantity of fat was deposited in them. Actually, there were four times as many cells in the obese mouse as in the control (Hausberger, 1959). Further evidence for an increase in the number of cells was demonstrated by Bjurulf‘s (1959) measurements on cadavers which suggested that as the amount of fat under the skin increased the number of fat cells also increased. Pitts (1956) from his studies on accumulation of body fat in guinea pigs concluded that additional body fat was deposited by both mechanisms, that is, filling up the available adipose tissue cells until the total body fat reached about 25%, at which point, the lipid-free cellular component began to increase in quantity. 24 In reviews concerning adipose tissue development, Wells (1940) and Behnke (1964) stated that, when the fat cell reaches maturity, it only maintained an ability to accumulate more fat and did not divide. Hellman and Hellerstrgm (1961) using autoradiographic methods involving the uptake of the DNA precursor, thymidine, labelled with either tritium or radioactive carbon, studied mitotic division in the subcutaneous gluteal adipose tissue of the white rat from 4 to 154 days of age. They concluded that mitotic division of the fat cells continued but at a slower rate in the older rats. For this age group, renewal of fat cells was greater in the subcutaneous fat depots than in epididymal fat depots. The amount of fat deposited and the number of cells made available for this deposition is a characteristic of the tissue itself. This was demonstrated by the "boxing glove” effect of an abdominal skin graft to the back of the hand of a 12 year old girl whose hand was severely burned. Evidently Strandberg (1915) in grafting the skin from the abdomen had also grafted some of the underlying abdominal adipose tissue which with advancing age accumulated fat and resulted in the puffiness on the back of the hand (for pictures, see Strandberg, 1915; Hashim & Van Itallie, 1965 and Wells, 1940). ‘ That this prOperty is inherent in the tissue itself and that genetic factors impose Specific deve10pmental patterns on the adipose tissues is further demonstrated by the parabiotic transplants of Hausberger (1955). He tranSplanted abdominal tissue from immature genetically lean mice to immature genetically obese mice and vice versa. There was histological evidence for the original developmental pattern of the host animal with enough change to indicate that there were also hormonal and regulating 25 mechanisms involved. Liebelt's work (1963) consisting of transplants of obese tissue to the ear of the lean mouse is supporting evidence for this. Distribution The adipose tissue's capacity to accumulate or lose fat appears to be the body's defense mechanism available for periods of feast or famine in man as well as in animals (Fredrickson, 1964). How adaptive processes Operate to fill or deplete adipose tissues aren't clearly elucidated. Nevertheless, there is evidence that fat stores are sacrificed to provide energy for the deve10pment of lean body tissue and that fat depots are depleted before lean body tissue (Sarett et al., 1966; Young et al., 1963). One exception to this, however, would be the sucking pad or corpus adiposum buccae which exists deSpite emaciation (Scammon, 1918-1919; Goldzieher, 1946). In emaciated infants, the sucking pads may become extremely prominent (Neff and Billingsley, 1930). In a study to evaluate the deposition of body fat into certain adipose depots of the white rat including the intermuscular region of the forelimb, the lumbo-dorsal region, the inguinal region, the genital, perirenal and the mesenteric and omental areas, Reed et al., (1930) concluded that neither the nature of the diet nor exercise had an effect on the preportion of fat deposited in each area. The four diets which they used to study this contained 17% of the calories as casein, the other 83% of the calories coming from cornstarch, mutton tallow, "Crisco" and soybean oil reSpectively. Rats were sacrificed at 50, 150 and 250 g for each feeding regimen. The adipose tissues were approximately twice as heavy in the rats fed the three fat rations as in thaae fed the constarch ration. For rats of the same body weight, there were no differences in the prOportions of fat in each of the six adipose tissues. 26 In a study designed to investigate the effects of feeding a high fat diet for protection in the cold, Pagg’and Babineau (1953) concluded as did Reed etza141930) that the nature of the diet did not affect the distribution of body fat. However, their results showed that in rats kept at room temperature and fed the high fat diet, a higher preportion of the fat was in the adipose tissue depots with lesser amounts in the skin and skeletal area. (The skeletal area included everything except the skin, pelvian and scapular belts of fat, perirenal and retrOperitoneal fat, testes and testicular fat, G. I. tract and mesenteric and omental fat,pancreas, and adrenals.) Liebelt (1959) showed that sex and genetic factors influence the distribution of lipid between the inguinal and gonadal fat organs in mice. Although the fat-free weights of the fat organs followed a pattern similar to body weight in NH and CBA mice the lipid deposition in these two fat organs didn't necessarily do so. In the NH mice, the female deposits 25-30% more lipid in each of the two fat organs when compared to the NH male of the same body weight. The CBA female mice deposited 25-30% more gonadal fat depot but inguinal fat depots were the same size as in the CBA male. The gonadal fat depots of both male and female CBA mice were pr0portionate1y heavier (as a % of body wt.) than those in NH mice. This was true until the mice weighed 20 g when the size of the fat depots became comparable in the two strains. Insofar as the inguinal depot is concerned, CBA male and female and male NH mice had the same amount of fat deposited for comparable body weights. After reaching body weights of 12 g, female NH mice deposited considerably more fat in this area. In gold thioglucose induced obesity of CBA/Ki mice, Liebelt et a1. (1965) reported a direct relationship between 27 the amount of lipid in the inguinal or in the gonadal fat depot and body weight. The pattern was similar to that of the non-injected control mice, but both body fat and depot fat were correspondingly greater. Reed et al. (1930) studied the distribution of fat in rats as affected by' undernutrition and exercise. The rats (180 g) receiving the starch diet (83% of calories) showed a greater depletion of adipose tissue in the genital area when exercised. This was not true for the rats consuming a ration which‘ provided 83% of the calories from fat. During fasting (with no forced activity), rats previously fed the high fat diet showed a smaller relative depletion of omental fat than from other areas. This was not true for rats that had been fed the 83% cornstarch diet. No specific conclusions can be drawn on the effect of undernutrition or exercise on distribution of body ' fat. There were too few rats in this study, and the sex of the animals was ' not given. The limited data suggests that the relative distribution of fat in the rat's body is changed by exercise or undernutrition. It appears also that age affects the proportional distribution of body fat. Peckham et al. (1962) indicated that for the epididymal fat depot, the ratio of depot fat to body fat closely paralleled the age dfthe rat with a decrease in the ratio with advancing age. However, other depots were not studied in this series. The distribution of fat in human subjects shows considerable individual variation. There are, however, certain types which have been characterized by such terms as "upper extremity pattern", "girdle fat pattern" and "lower extremity pattern" (Garn, 1955). On the basis of specific gravity determinations, Young et a1. (1963) calculated that as women increased in age from 16 to 70 years, their body 28 fat increased by 55.3%. On the basis of skinfold measurements the increase in body fat content was only 46%. The lower value for body fat content secured by the skinfold technique suggested to the investigators that as women age, more fat is deposited in the areas other than the subcutaneous. WEIGHT REDUCTION Although weight reduction has been advocated for the obese subject by' both medical personnel and the lay public, there is still not too much factual evidence to indicate the lasting benefits that may accrue from such an ordeal. Most of the earlier reports were based on insurance statistics which indicated that underweight individuals live longer than those who exceed their "normal" weight (Body Build and Blood Pressure Study, 1959).‘ A few reports from the insurance companies suggest that the obese insureeS" who reduced their weight lived longer than those who didn't (Dublin et al., 1949). The inability of most overweight individuals to "stay reduced" makes the latter statistics of dubious value. It is very likely that a fair prOportion of the subjects who reduced and thereby secured a reduction in their insurance premiums may have returned to their obese state shortly after their medical examination. The psychological advantages of weight reduction have been stressed especially by the writers of articles aimed at the general public. The theory apparently is that if individuals are not likely to do something that might improve their health, they may do the same thing if it improves their physical appearance. There are undoubtedly many psychological advantages that may accrue to the obese individual once he has "slimmed down" (Stunkard and McLaren—Hume, 1959). However, weight reduction may not be an unmixed blessing for the obese individual. This has been stressed 29 by Bruch (1957) who insists that unless the proper motivation is used, more damage than good may result from attempts to get the overweight patient to lose weight. Many of the publications aimed for popular consumption suggest that the overweight individual suffers from some metabolic or physiological disturbance which can be circumvented by the use of a special diet. Credence was given to this assumption by the writings of Pennington (1953; 1954), who suggested that metabolism of pyruvic acid may be impaired in obese subjects. Since then much of the papular writing and a fair amount of professional work has been directed toward the use of a diet high in fat as a means of producing weight reduction. There is ample evidence that a high-fat diet will produce a loss of weight (Yudkin and Carey, 1960; Kekwick and Pawan, 1956, 1965). This occurs even though the subject consumes as much of the diet as he desires. However, the major share of the "lost weight" is body water (Pilkington et al., 1960; Olesen and Quaade, 1960). A diet high in fat produces a loss of body water even though the caloric intake meets or slightly exceeds the caloric requirement. Finally, the loss in body water resulting from the ingestion of a high-fat diet is undoubtedly of limited duration. Although no studies have been extended beyond 10-14 days, the loss of weight resulting from a loss of water appears to have terminated by the end of the second week. Furthermore, most diets sufficiently high in fat to produce such an effect are likely to result in nausea (Pilkington et al., 1960: Taller,l962) and a loss of appetite by the individual who consumes them for more than one day. Kekwick and Pawan (1964) showed similar results in mice but the loss of body water was not as great in mice. These authors felt the additional weight loss on the high fat diet 30 could be accounted for by an increased quantity of intermediary products containing carbon being lost. Weight changes which take place in the normal adult man primarily consist of changes in body fat (Behnke et al., 1953). These authors concluded this from determination of the tissue either-gained or lost during fluctuations in body weight. This is not true for people undergoing starvation (Keys et al., 1950) wherein protein and water as well as fat are lost. The nitrogen balance study has been used by Passmore et al. (1958) to evaluate the content of the lost tissue during weight reduction. In the first week nitrogen losses were usually large and balances-varied from a -l to -9 g N/day indicating daily losses of from 25-60 g protein- The loss of weight in the first week was always high and associated with large losses of water. On the other hand, Christian et al. (1964) found that lean body mass remained constant in 51 "clinically normal" obese subjects on a weight reduction regimen over a period of eight weeks. For determination of lean body mass they measured K40 which had a 3.5 - 4.0% in men and up to a 5.0% error in women because of the lesser quantity of lean body mass in women. This would indicate the loss of lean body mass was not greater than 3.5 to 5.0% after weight reduction. Rath and Slabochova’(1964) found that in 32 obese women, a 1200 cal. diet which consisted approximately of 90 g protein, 40 g fat, and 120 g CHO caused a weight loss of 1.2 kg per week. The lost weight consisted of 0.67 kg of fat. These values were obtained from densitometry measurements. When this was accompanied by 4 hr. of physical exercise daily, the total body weight lost was made up entirely of fat. The loss of adipose tissue in the group 31 which exercised was 30% greater than that attained by the low calorie diet alone. Young et a1. (1960) reported that 4 of 9 obese women, 18-21 yr. of age stored nitrogen or were in nitrogen equilibrium while 5 were in negative balance after 6 weeks of weight reduction. This was on a daily intake of 90 g of protein. In only one instance, however, was this loss of N greater than 5.0% of the nitrogen. The mean percentage of body fat dropped from 35.77 to 29.19%. The mean net change in body fat content was 6.58%. These latter data were determined from underwater weighings. Passmore et al. (1958) calculated the composition of tissue lost by seven patients over a 6 week period of weight reduction when the subjects received each day a 400 cal. diet (Strong et al., 1958). For the seven patients the composition of the lost tissue varied from 73 to 83% fat, from 0.4 to 7.0% protein and from 10 to 23% water. All patients lost large quantities of water during the first 5—7 days but these water losses were not maintained beyond the initial period of weight reduction. More comprehensive studies of the effect of weight reduction on body composition have been carried out with animals. Sarett et al., (1966) provided adult rats with one half their ad libitum feed intake until the animals lost 25% of their initial body weights. Under these circumstances, "normal" weight male rats lost tissue which consisted of 8 g of protein and 30 g fat. This represented a loss of 12% body protein. On the other hand, rats classified as "obese" weighing 392 g lost 47 g fat and 2 g protein representing a loss of 3% body protein. Another group of rats weighed 700 g (ibid) and had around 35% fat in their carcasses. They had been fed "Metracal" ad libitum. 32 Their weights were reduced by 234 g using one of the following regimens: (l) 60% of their previous intake, (2) 37.5% of their previous intake and (3) fasted. In the two groups receiving restricted amounts of food, the lost tissue contained only 4 to 5% protein and about 79% fat, whereas in the fasted animals the lost tissue contained 10% protein and only 64% fat. After the rats lost 234 g, the carcasses of the restricted groups had 13.0 and 15.0% fat whereas the carcasses of the fasted group had 18.7% fat. Weight loss in vital organs during weight reduction Some vital organs increase in direct proportion to body weight, others increase in weight according to allometric prOportions to body weights, while still others remain the same size after maturation, even though body weight increases. The behavior of these vital organs during the development of obesity tends to be simulated later on, if weight reduction takes place in the adult life span of the rat. During development of obesity due to hypothalamic lesions Brobeck et al., (1943) reported that heart, liver and kidneys increased in size with increases in body weight. On the other hand, the adrenals remained about the same size as those in control animals and ovaries showed a definite decrease in size. More recently, Spencer and Coulombe (1966) indicated that liver has an allometric relationship to body weight. Allometry was first advanced by Huxley (1932) and many allometric relationships of organ weight to body size were done by Brody (1945; pp. 398- 401). The formula used by Spencer and Coulombe (ibid) was W = ABx or log W = x log B + log A. W = organ weight, B = body weight and A is the. parameter calculated (that is the predicted organ weight) which increased at a constant "x". For liver the constant "x" is approximately 0.78 to 0.87 33 of the increase in body weight. When Binder et al. (1966) applied this formula to the livers of obese hyperglycemic mice, the weight of the liver was greatly underestimated. Instead, the obese mouse had a disproportionately large liver for its body weight. Marshall at al. (1957) reported that livers in both young and adult obese hyperglycemic mice weighed twice as much as those in the controls. Also mice made obese by gold thioglucose or hypothalamic lesions showed increased liver size. On the other hand, Page’ and Babineau (1953) found no increase in the absolute weights of livers in rats consuming a high fat diet. However, these "obese" rats were only 30 g heavier than the controls. On reduced food intakes, Peters and Boyd (1966) reported that livers lost weight faster than over-all body weight. In general then, the liver varies with the weight of the rat, but the nature of the diet also causes a variation in liver weight with a high protein diet producing an increase in liver weight (Eaton, 1938; Walter and Addis, 1939) as did hypothalamic or genetic obesity. When comparing rats consuming chow to rats consuming purified diets of sucrose or starch, liver weights showed no differences (Peters and Krijnen, 1966). With increase in body weight growth of kidneys, likewise, can be expressed by an allometric equation (Stahl, 1965). No doubt, this equation may be disrupted by high protein diets which cause kidney enlargement (Reid, 1963; Osborne, et al., 1923; 1927; Addis et al., 1926) as well as by hypothalamic obesity (Brobeck et al., 1943) and the obese hyperglycemic syndrome in mice (Marshall et al., 1957). On reduced food intakes loss of weight of the kidneys was equivalent to body weight lost (Peters and Boyd, 1966). 34 Beznak (1954) found that the weight of the heart followed changes in body weight. Thus, the same cardiac weight corresponds to a given body weight irrespective of whether that body weight was achieved through growth or by losses of weight as a result of starvation. Hypothalamic obesity caused an increased heart weight in rats (Brobeck, et al., 1943) and in mice (Marshall et al., 1957). On reduced food intakes Peters and Boyd (1966) and Walter and Addis (1939) likewise reported that heart weights in rats decreased in proportion to body weight. Beznak (ibid) suggested that the reduction in heart weight may be due to a decreased functional demand. In guinea pigs, however, Baton (1938) found that hearts were practically the same weight regardless of body weight. On the other hand, Peters and Krijnen (1966) reported that rats fed purified diets containing sucrose or starch had slightly heavier hearts than controls of the same body weight consuming a commercial stock ration. Weight changes in adult rats do not significantly affect the absolute size of the adrenals. Rats with hypothalamic hyperphagia did not have enlarged adrenals (Brobeck et al., 1943). Hyperglycemic obese mice only showed an enlargement of the adrenals in later adulthood (Marshall at al., 1957). Young obese mice when compared with controls did not show any increase in adrenal weights. Likewise, losses in body weight did not cause a corresponding decrease in adrenal weight (Beznak, 1952; Peters and Boyd, 1966). Consistently, the spleen and lungs of the obese mice weighed the same as those in normal weight controls (Marshall et al., 1957). With decreased food intakes, the spleen showed more rapid weight loss than did the body as a whole (Peters and Boyd, 1966). 35 Reducing by fasting Subjects undergoing semi-starvation became hyperirritable, restless, sensitive to noise, unable to concentrate and nervous (Keys et al., 1950, p. 836). On the other hand, after the human being has fasted for three days hunger pangs are mitigated, the desire for food is suppressed, and he can continue the fast without undue distress (ibid, p. 821; Bloom, 1959; Blondheim et al., 1965; Duncan et al., 1962; Drenick et al., 1964; Hashim and Van Itallie, 1965; Silverstone et al., 1966). Zucker's (1967) weight reduction studies indicate that a similar phenomenon exists in rats. In view of the above observations, clinicians and scientists interested in reducing the obese have investigated numerous parameters associated with fasting both in humans and to a lesser extent in rats. With fasting immediate weight losses occur in man. This is partially accounted for by naturesis and loss of water (Bloom and Mitchell, 1960). Upon refeeding, readjustment in body water accounts for the rapid initial weight gains observed (Rapoport et al., 1965a; Bloom, 1962). Other physiological responses to fasting include a decrease in phsma volume (Rapoport et al., 1965a) hypotension (Drenick et al., 1964; Drenick and Smith, 1964) ketonemia (Cubberley, 1965; Duncan et al., 1962; Drenick et al., 1964; Rapoport et al., 1965a) and an increase in non- esterified fatty acids in serum (Castelli et al., 1966). The obese, however, are more resistant to ketoses than lean individuals (Pawan, 1957; Azar and Bloom, 1963). Liver glycogen reserves are decreased (Haro et al., 1965; Shoemaker et al., 1959) but other aspects of liver function are apparently normal (Rapoport et al., 1965b). Serum glucose is also decreased (Haro et al., 1965; Rapoport et al., 1965b) 36 Fasting has been reported to produce an elevation of serum uric acid levels. This occurs in the human when fat is metabolized whether it be from an exogenous source such as a high fat diet (Harding et al., 1927) or from endogenous fat (Lennox, 1924; Lecocq and McPhaul, 1964; 1965; Rapoport et al., 1965b; Murphy and Shipman, 1963; Drenick and Smith, 1964; Cristofori and Duncan, 1964). According to Rapoport (1965b) the presence of increased serum urates and decreased urine urates during fasting are associated either with an increased reabsorption of filtered urate or a diminished secretion of urate by the renal tubules. In 33 patients who fasted not less than one month and not more than two months "Probenecid"l prevented hyperuricemia (Drenick and Smith, 1964). An evaluation of the composition of the weight loss during weight reduction was done previously. However, the work of Sarett et a1. (1966) indicated that slightly larger quantities of lean body mass are lost from fasting rats than from those rats restricted in food intake. l"Probenecid" is the generic name for para dipropyl sulfamyl benzoic acid. This is frequently sold under the trade name of Benamid. PART I BODY COMPOSITION AND GROWTH OF ADIPOSE TISSUES IN OBESE AND NORMAL RATS INTRODUCTION Throughout the life span, weight gains occur as the result of growth or maturation, deposition of muscle tissue and/or deposition of fat. During maturation, (Tanner, 1963; 1964) and during physical training periods (Behnke et al., 1942; Brosek et al., 1963; Pafizkovs; 1963; 1965) protein synthesis is the primary contributor to this weight gain. However, with maturation, fat synthesis normally takes preference over protein synthesis and under ordinary conditions, weight increments result in additional body fat (Brogek, 1952; Keys and Brogek, 1953; Mayer, 1948; 1949). Obesity can be produced in rats by feeding them a diet composed of 40 - 60% hydrogenated fat (Mickelsen et al., 1955; Barboriak et al., 1958; Hoagland et al., 1952; Deuel et al., 1944). These rats continue to gain weight at a fairly rapid rate throughout most of their lives. The major portion of this weight gain is due to the accumulation of fat. The deve10pment of obesity is associated with an increase in the size of the adipose tissues. As far as the adipose tissue cells are concerned, Hausberger (1959) suggests that the existing cells are filled with fat after which a growth of new cells takes place (Behnke, 1964; Hausberger, 1959; Bjurulf, 1959). There is still some controversy as to the relative growth of different fat depots. The rate at which the different fat pads increase in size as an animal matures has not been studied very completely. Reed et a1.(l930) pr0posed that in young rats the different fat depots grew at a pr0portionately equal rate unless influenced by exercise or under-nutrition. Reports contradicting this statement have appeared more recently. Liebelt (1956) stated that inguinal 37 38 and genital fat depots in mice deve10p at quite different rates. In dietary obese rats, the data of Peckham et a1. (1962) suggested that epididymal fat depots grow less rapidly with aging. Additional evidence for a differential growth of fat pads comes from studies of rats exposed to a cold environment. Rats so acclimated had epididymal fat depots that were 1/3 as large in prOportion to body weight as those in rats acclimated to warmer temperatures (Himms-Hagen, 1965). Based on differences in percent of body fat obtained from densitometry measurements and skinfold measurements in mature adult women, Skerjl (1959), Skerjl et al. (1953) and Young et al. (1963) prOposed that with aging, accumulations of fat resulted in a more rapid increase in the size of the abdominal fat depot when compared to the subcutaneous depots. Most investigators have frequently used only one or two fat depots of animals when studying physiological and biochemical phenomena. Frequently the assumption has been made that what happens in one fat depot is characteristic of what happens in all fat depots in the body. As a start in evaluating this hypothesis, it would appear desirable to determine how the different fat depots change with changes in body weight. Many individuals are presently involved in determining body fat in human beings by skinfold measurements. They depend on measurements of subcutaneous tissues only or radiographic techniques which are limited to subcutaneous depots which can be estimated on soft tissue radiographs. For these reasons, it becomes important to know whether or not fat depots all grow at the same rate or whether during certain periods of rapid weight accretion or during aging, fat is sequestered in certain depots. To this end, both normal and obese male and female OsbornetmendEI 39 rats were sacrificed at 150 g intervals to investigate the weights of individual adipose tissues. EXPERIMENTAL Weanling male and female Osborne Mendel littermates from NIH stock and bred in our laboratory were fed either (1) a grain ration (M-l), (Campbell et al., 1966) or (2) a ration containing 60% fat (Mickelsen et al., 1955) with slight modifications (M915, Table 2). The experimental design provided that 5 male and 5 female rats be sacrificed at weaning, and 5 rats of each sex when they reached weights of 150, 300, 450 and 600 g. This was true for rats fed the grain or high fat rations. Female rats consuming the grain ration never reached 450 nor 600 3, so the data reported for the female rats do not include these two groups. The work of Mickelsen et al.(l955) indicated that the rats consuming the high fat ration became obese so a group of 5 rats were included for both males and females at 750 g and for males only at 900 and 1050 g. Since there was some variation in the time required by the rats to attain a given weight, the animals were placed in their specific groups at the time of weaning. This predetermined the ultimate weight the rats should attain before being sacrificed. The rats from one litter were distributed among the different groups. Consequently, no two rats from one litter fed the same ration were sacrificed at the same weight. In addition, data from the four groups of older animals (both male and female rats that had been fed the high fat or grain ration for about 400 days) used as controls in Part III are included here since these data help in extending this .experiment time-wise. 40 At the time of sacrifice, the rats were weighed and then anesthetized until death with ether. Immediately, they were placed on their abdomens, and nose to anus lengths measured. The skin was cut at the base of the rib cage around the entire rat to permit separating the skin from the subcutaneous fat depots by gently removing the skin from the fore and hind parts of the rat. Care was taken to exPose only those portions of the rat where the individual fat depot was being removed; the skin was replaced after removing the subcutaneous depot. To further reduce the evaporation of water from the tissue, the working surface was kept moist. Nevertheless, moisture losses through evaporation could account for 0.5 to 1.1% of the body weight as determined by progressive decreases in total carcass weight during the dissecting procedure. In subsequent calculations of moisture, adjustment in the percent of moisture was made for this evaporation loss. Right and left inguinal fat depots were separated from the body wall by tearing and occasional cutting. Care was taken to exclude the femoral artery and vein. In the larger rats, the inguinal fat depot appeared to merge with subcutaneous fat of other areas. In these cases, the inguinal fat depot was arbitrarily separated from the cephalad half by an incision at the costal border and from the dorsal side by an incision parallel to the backbone and on the ventral side, by the location of the sternum. This inguinal fat depot did not include the intermuscular fat which deve10ped in the thighs of both male and female obese rats. That fat was definitely imbedded below a thin layer of muscle and did not appear to be a part of the depot. On the other hand, where the depot had expanded to incorporate the mammary gland, it was non-distinguishable and thus included in the depot weight. Subcutaneous fat cut from below both 41 the right and left forelimb extended to the upper eSOphagus and sternum which made it possible to differentiate it into a right and left side. The interscapular fat depot was separated from the skin and the back of the neck. This depot included the entire dorsal side of the rib cage and extended to the lower costal border. After the removal of the subcutaneous fat pads, an incision was made into the abdomen. The testes of the male rats were pulled up into the abdomen and the testicular fat depot separated by cutting it away from the testes and epididymis caput and testicular artery. In addition a very small fat pad was removed from the caudal end of the epididymis and called epididymal fat to differentiate it from the larger testicular fat body which frequently has been called epididymal fat. It's weight was negligible, increased in relationship to testicular fat so individual data for it are excluded. The comparable fat depot in female rats was pulled away from the perirenal fat and then cut away from the uterus and follicles and separated into right and left pads at the base by centrally cutting it in a line with the bladder. An accumulation of fat at the base of the sternum around the cartilage of the xiphoid process was observed with aging and with accretions in body weight. This was easily separated from the cartilage of the xiphoid process and weighed. The es0phagus was cut at its base, and the gastrointestinal tract and the mesenteric and omental fat depots were removed from the abdominal cavity. Any portion of the pancreas adhering to this could be distinguished from the fat by its slightly deeper brown color and thereby readily separated from the fat. The mesenteric fat depot was gently pulled from the 42 gastrointestinal tract. When resistance was felt, a slight cut facilitated its removal. The capillary plexus supplying the gastrointestinal tract remained as a part of the mesenteric fat. Lastly, the retrOperitoneal and perirenal fat depots were removed as a single mass. By cutting inward from the outer edge of the depot, then removing it from the adrenals and kidneys and, finally, separating it from the backbone and abdominal sorts the process could be completed without loss of blood. Upon removal, all of these depots were immediately weighed on a Roller-Smith or Torsion balance depending on the size of the depot; and after weighing placed into a previously tarred pint or quart wide mouth jar with a cover always intact to avoid unnecessary loss of moisture. The carcass was placed in this same jar. The gastrointestinal tract and its contents were weighed. The contents were removed from the G. I. tract which was then rinsed with water, gently blotted with absorbant paper and weighed again before being placed in the jar with the carcass. The weight of the gastrointestinal contents was subtracted from the live weight of the rat to secure the "corrected weight". Since special attention was given to prevent any loss of blood during the dissection, the "corrected weight" of the rat compensated for any evaporation losses from the carcass. By actual weighing of the jar before and after adding all the tissues, it was found that evaporative losses amounted to only a few grams for the larger rats. The jar containing the entire carcass was then autoclaved at 15 lb. pressure for 20 to 25 minutes depending upon the size and age of the rat. 43 After autoclaving and cooling, the jar and rat war weighed to verify any gain or loss of moisture during the autoclaving procedure. variations usually were no larger than 1_1 to 2 g of the original weight. The rat was then homogenized using amounts of water equivalent to body weight for the smaller rats and lesser amounts for the larger rats as described by Mickelsen and Anderson (1959). Blenders ranging in size from 1 cup to 2 gal. were used depending on the size of the rat. After homogenization, aliquots were analyzed for moisture, fat, nitrogen and ash by methods described by Mickelsen and Anderson (ibid). Since ashing was as complete at 12 hours as at the 24 hrs. which they used, samples were ashed 12 hrs. RESULTS Although every effort was made to sacrifice the rats at the weights originally chosen, this was not always possible. A variation of‘i 15 g of body weight was accepted since some rats gained this much in a week-end when it was difficult to get the animals x-rayed. All rats were sacrificed the morning of the day following the radiograph. Some of the small rats gained as much as 7 g whereas some of the obese rats lost weight during the period intervening between the radiograph and necropsy. The body weights listed in Table 3 represent the averages for each group just prior to sacrifice and the weight after removing the contents of the gastrointestinal tract. The rats fed the grain ration had about twice as much material (both on a volume and weight basis) in their gastrointestinal tracts as those fed the high fat ration. 44 Body weights Rats fed the high fat ration gained weight more rapidly than those fed the grain ration (Fig. 1). This resulted in the high fat-fed rats being younger than the grain-fed rats at the same body weights (Table 3). These differences were significant (P<0.0S) for the 450 3 male and 300 g female rats, and highly significant (P<0.0l) for the 600 g male rats. The rate of weight gain for the high fat-fed male rats was rapid and uniform until they attained a weight of 450 g. After this, weight gain occurred at a uniform but less rapid rate. A partial explanation of this decreased rate in the older animals is that some rats lost 20-30 g and then regained this weight. Female rats fed the high fat ration showed a uniform rate of body weight increase up to 855 g. The grain-fed rats showed a plateau in their body weights at about 600 g for the males and 365 g for the females. Skeletal‘gigg The skeletons of the obese rats were the same length from snout to anus as those of the lean rats for each of the weight categories (Table 3). When the obese rats began to exceed the maximum weights of the lean controls, differences in skeletal lengths began to appear. The 1050 g male rats measured 29.0 i 0.4 cm while the 600 g lean rats were 27.7 :;0.6 cm. Although this difference is significant, its interpretation is Open to some question. The large accumulation of fat in the subcutaneous area of the obese rat makes it difficult to evaluate the true skeletal length. The ash content of the obese rats was for each weight category less than that of the lean rats. A partial explanation for this difference 45 stems from the fact that the accumulation of body ash is related to the age of the animal (Fig. 2 and 3). Again up through 150 days of age, the absolute weight of ash in the bodies of both obese and lean rats is the same. However, at each age, the rats fed the high fat ration are much heavier than those fed the grain ration. Consequently, when comparisons are made on an equal weight basis, the obese rats are younger and have less ash in their bodies. Admittedly,/the total body ash provides at best only a crude index of skeletal size. For this reason, additional studies are underway to determine the rate of accretion of such specific elements as calcium, phOSphorus and magnesium in the skeleton length and weight of Specific bones and the ash content of the entire skeleton. For each weight category through 300 g the female rats have the same skeletal lengths as measured from snout to anus as the males. At each of these weights the.females were older than the males which suggests that at comparable ages, the males would have greater snout to anus lengths. Both the male and female obese rats weighing 450 g had similar body lengths (24.8 cm). However, above this weight, the females showed no appreciable changes in length whereas the males did. For the males, this apparent increase in length continued up to a weight of 1050 g. 'gggy protein The increase in body protein was related to age up through 150 days (Fig. 4 and 5). After that age, both male and female rats fed the high fat ration appeared to deposit more protein in their bodies than the grain fed rate. This may have been associated with the marked expansion of the adipose tissue in the obese rats. The cellular material within which 46 the fat was deposited contains some protein and this with an expansion in the skin may have accounted for the extra protein. was Fer both the males and females, the increase in absolute body fat is linear with time (Fig. 6 and 7) if the rats were fed the high fat ration. This was also true for rats consuming the grain ration as long as there was an increase in body weight. However, when body weights plateaued body fat behaved similarly. The rate and absolute amount of fat accumulated in the body was significantly (F0.05). Although the weights of the depots were the same, it was apparent on inapection that more fat was present in this tissue in the high fat-fed animals. For the grain-fed rats, the relative size of the depot was prOportional to the body weights of the animals whereas in the high fat-fed rats, the size of this depot increased more rapidly than body weight and showed a relative decrease in weight with aging (Fig. 13). Young rats, especially those fed the grain ration, showed very little fat around the xiphoid process (Fig. 14). The female rats fed the high fat ration showed a proportionately rapid rate of increase in the weight of this depot. This rate of increase was much greater than that for the male rats fed the same high fat ration. The latter showed an increase in the size of this depot which paralleled that of the grain-fed rats. In the obese rats, the size of this depot and other fat deposits underlying the diaphragm were so prominent as to suggest possible interference with cardiovascular activities. In the young rat, weight of the subcutaneous fat depots was greater than those in the abdominal area. However, as the animals matured, the situation was reversed. For the female rats, this occurred at a body weight of 300 g and for the males at 600 g (Table 4). 50 DISCUSSION ,The obese rat does not develOp a larger skeleton to support its additional body weight. However, since the total ash represented the minerals present in many tissues besides the skeleton, it cannot be considered a true index of size or degree of mineralization of the skeleton. The justification for the use of body ash as a crude index of skeletal size comes from the similarity in size of the lean body mass in the grain and high fat fed rats. Studies are under way to more critically evaluate skeletal size and composition in the lean and obese rats. For both male and female , the consistently lower quantity of protein in rats eating the high fat ration reflects the difference in age at sacrifice. For the same age, the development of lean body mass was similar whether the rats consumed the grain or the high fat ration. The older obese rats showed a slight increase in total protein. When adipose tissues contain a certain amount of fat, these tissues proliferate with development of more structural material (Hausberger, 1959). The increased fat-free component of the adipose tissues most likely accounts for some of this increase in protein. Future work will be directed to check this assumption. It should be pointed out that in the adult rats, protein represented 22 to 24% of the lean body mass regardless of whether the rats were fed grain or high fat ration. Other investigators have reported similar results (Fig. 15). Numerous investigators have shown that weight gains in obesity are due to increased body fat (Hetherington and Weil, 1940; Mickelsen and Anderson, 1959; Vlahakis and Heston, 1959; Zucker and Zucker, 1963). This present series of analyses would indicate that this is true for all ages. 51 In every instance, the consumption of the high fat ration caused large increases in adipose tissue weights. This difference was always highly significant (P<0.01) except in the case of the depots underlying the fore- limb where the difference was significant (P<0.05). The one exception to this was the mesenteric and omental depots in rats weighing 150;: 10 g wherein the weight of the capillary plexus obscures the difference in weight of the fat depots (Table 5). Adipose tissues displaying particularly extensive accumulations of weight with the consumption of the high fat ration were the perirenal depot, the adipose tissue surrounding the xiphoid process and the interscapular depot; the latter was cepecially true for female rats. With few exceptions, weight gains of the subcutaneous adipose tissues, the mesenteric and omental depots and that depot surrounding the xiphoid process were similar in males and females. However, accretions of weight in the interscapular area of females consuming the high fat ration were particularly pronounced. The inguinal depot and adipose tissue underlying the forelimb appear able to increase markedly in the male. Genital depots were larger in females while perirenal depots were larger in the males. The difference in the subcutaneous adipose tissue weights of the weanling male and female rats reflects the slightly heavier weight of the females (Table 3). Table 6 summarizes the effect of age upon the weight gains of adipose tissues. From weaning to 99 days, when rats were fed the high fat ration, all depots showed increases in weight which were greater than the increases in body weight. This was particularly pronounced for the genital and perirenal depots. The large percentage of increases of the 52 latter depots, however, reflect the fact that in weanling rate these depots are relatively smaller than the subcutaneous depots. This would likewise partially account for the extensive percentage increase in these two depots when rats were fed the grain ration. After the first 100 days, subcutaneous depots increased in weight similar to body weight increases. The exception to this was the interscapular fat depot when rats were fed the high fat ration. This depot continued to show increases throughout the lifespan. For this depot, the relative weight increase ranged from 120 to 199% of body weight increase}. For each 100 day interval, the increase in weight of this depot for the males was always at the lower range, while that for females was at the upper range. After the initial large increases in relative weights of the genital depots, they showed no further relative weight gains when rats were fed the high fat ration. With further aging this depot began to decrease in relative size for both males and females. When rats were grain-fed, the same trend occurred but later. At the end of 400 days, the same drOp-off in relative weight had not yet occurred in the females. The mesenteric and omental depots showed a similar drop-off in relative weight with aging for the rats fed the high fat ration. Only the perirenal depot and the depot surrounding the xiphoid process continued to gain in relative weight. However, in the case of the grain-fed female, no changes in relative weight were observed. 53 Table 2 High fat ration (M-lS) Ingredients Z Crisco 60.0 Casein 25.0 Mineral mix; 5.0 Vitamin mix 7 2.2 Non-nutrit ve fiber3 2.0 Aureomycin 0.01 Liver powder 2.00 dl-Methionine 0.25 Sucrose 3.54 Calories per g; 6.62 1 Salt mix, Wesson modified (Osborne-Mendel) General Biochemicals, Chagrin Falls, Ohio. 2 Vitamin diet fortification mixture from Nutritional Biochemicals Corp., Cleveland, Ohio. 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Dev. of 10 grain-fed female rats. rats were 400 da. and were the lean controls from the weight reduction experiment (Part III). Subsequent data (Part III) indicate the inguinal, testicular These 57 Table 5 Relative weights of fat depots in rats fed either grain (M-l) or high fat (M015) rations. The lowest and highest average values for all groups are listed. All weights expressed as g per 100 g body weight. The average weights represent 5 animals in each weight group ranging from 150 to 1162 g. Range in average depot weights. Fat depot Sex W M-l M-lS Inguinal M 1.44 2.1 - 3.94 4.8 - 9.15 F 2.00 1.8 - 3.06 4.8 - 7.17 Forelimb depots M 0.48 0.5 - 0.6 1.0 - 2.0 F 0062 003 "' 005 007 - 105 Interscapular M 0.27 0.3 - 0.8 0.7 - 3.3 F O 35 0e4" 008 104 -1003 Genital M 0.20 0.5 - 2.6 1 0 - 4.0 F 0 27 1.0 - 2 9 2 3 - 6.1 Perirenal M 0.23 0.3 - 3.7 1.4 -14.0 F 0025 005 - 1.07 1 0 "' 903 Mesenteric and M 0.62 0.9 - 1.4 1.0 - 3.0 omental F 0061 908 ' 1.04 102 - 305 Depot surrounding M tr ' 0.02- 0.15 0.02- 1.00 Xiphoid process F tr 0.02- 0.20 0.02- 0.90 1 Weanling rats. 2 Grain ration. 3 High fat ration 4 Range in rat weights; 140 to 571 g. 5 Range in rat weights; 154 to 1162 g. 6 Range in rat weights; 150 to 363 g. 7 Range in rat weights; 146 to 885 g. 58 .:owcm£o on: oHnHmmoa mommouoom no mammuocw uswwam .ANooHNv ommouoaa oaom om uo>o .ANooHHV ommouoc0 oaom OH uo>o m N .0001000 N000 on 8.3 00000009 000000 006 cu ~.o .. - .000000 0000 06 0000 60 000 00.o+ ou $0.0- uswgus o>0umfimu c0 owcmno oz . 0800 .0000000 .0000 60 0000 000000 00000 00.0 00 0.0 + .0000000 0000 60 0000 000000 00000 00.0 60 0.0 ++ .0000000 0000 60 0000 000.000 00000 00.0 60 0.0 +++ H Aommmuocw omnv umwuma moawu N.¢ ++++ + oamm + name +++ 09mm ++++ + m wmoooum.efionm0x + + + + +0. + +1. + z 3: 000000.000 000000 . oamm 08mm + + + +++ + m f. 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Body weights of male and female Osborne Mendel rats fed either the high fat or grain ration. Each point represents the mean weight of the five rats analyzed. 10 Me i ht f 4 0 d o. ' " an we 3 001113 fast r rqin fed and high fat fe__d oc nt 23.1%.. liart III) 6O 18 0. .1 w. // 12 __ 3° 2 9- // no H 52 6 0. 3 0 I l I AL 100 200 300 400 Age in days 18 r. 15 _. /// ‘,,0—1;""g”’f'. £912_ 4.) €$ o .3 9 __ ’///// 3 l l l L 300 600 900 .1200 Body weight (g) Fig. 2. Increase in body ash content of male Osborne Mendel rats. One group of rats was fed the high fat and the other, the grain ration. Five rats were analyzed for each point on the curves. :1; Mean ash of 400 da. grain-fed and high fat-fed controls (Part III). 0—0 High fat ration o—o Grain ration 61 .1 12_ /"l ________o 30 e_ U .2 DD -.-1 :2 4.. &/p 1 l J J 100 200 300 400 Age in days 01 12 __ 1 / .,,.~——o 303 _. u .c a) "-0 :2 4 __ o I l I 300 600 Body weight (g) 900 Fig. 3. Increase in body ash content of female Osborne Mendel rats. One group of rats was fed the high fat and the other, the grain ration. Five rats were analyzed for each point on the curves. :1; Mean ash of 400 daa grain-fed and high fat-fed controls (Part III)- 0—0 High fat ration o—o Grain ration 62 . 9O __ //////9’o 3° C ‘2 60 r_ // oo o ”4 Q) 3 / 30.. /9 o l l I l 100 200 300 400 Age in days 120.. ‘///jél ”/’1r’/’,/.__—1I1 90 __ 13 v H 450 60__ -.-I Q) :3 30.. ! I l l 300 600 900 1200 Body weight (g) -Fig. 4. Increase in body protein content of male Osborne Mendel rats. One group of rats was fed the‘high fat, and the other, the grain ration. Five rats were analyzed for each point on the curves. f1, Mean protein of 400 da. grain-fed and high fat-fed controls (Part III). OHigh fat ration o oGrain ration' 63 1 :25 __ / So 50 _ ./ U .c on -.-I 52 25 _, 0 I I I I 100 200 300 400 Age in days 1 775 _ .””,/‘ ol 0””” 3° / u o .5 50 _ so -H 0 3: 25 _. I l iL 300 600 900 Body weight (g) Fig. 5. Increase in body protein content of female Osborne Mendel rats. One group of rats was fed the high fat, and the other, the grain ration. Five rats were analyzed for each point on the curves. Q1, Mean protein of 400 da. grain-fed and high fat-fed controls (Phrt III). o—-o High fat ration 0..-”-..0 Grain ration 64 600 F /O 1 500 __ 400 _ 3° U ,fi) 300 _, . 0H 0) 3 200 _ / O OLO’O/I I I I 100 200 300 400 Age in days 600 _. 1 500 F o ,a 400 _ 33 U '50 300 _ -.-I 0 3 O 200 _ , O 100 _ . /&_1 / O 0::23_».»'°f“—"’ I l i 300 600 900 1200 Body weight (3) Fig. 6. Increase in body fat content of male Osborne Mendel rats. One group of rats was fed the high fat and the other, she grain ration. Five rats were analyzed for each point on the curves. ‘ ' ‘ ' " ‘ 1' ' ' 1. Mean fat of 400 dao grain-fed and high fat-fed controls (Part 121,, g—ol-Iigh fat diet o oGrain ration 65 500 _ 400 _. 3° u 300 __ .2 no .,.I Q) :3 200 L I I I I 100 200 300 400 Age in days 500 _. l 400 _. 0 3° .: so -.-I o 3 200 _. 100 _. l 0”’9 I I I 300 600 900' Body weight (g) Fig. 7. Increase in body fat content of female Osborne Mendel rats. One group of rats was fed the high fat and the other, the grain ration. Five rats were analyzed for each point on the curves. : f I ‘ '..."'f“- ‘ , ’ ,'”< 3’”77. I. Mean fat of 400 da. grain-fed and high fat-fed controls (Part III)- o——-0High fat diet c-——Mc>Grain ration ‘ 66 Bars ”.8 :3: 632mm cl o .Houucoo cwmuw .mamammol o .umau use emu: ,mamz..llu. 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Mayer has reviewed the evidence for this for man (1965) find for animals (1957). The manner of inheritance, however, continues to elude the investigators. For purely genetic studies, the choice experimental animal has been the mouse. That gene mutations have resulted in four obese types of mice is well documented (Bielschowsky and Bielschowsky, 1956; Danforth, 1927; Falconer and Isaacson, 1959; Mayer, 1960). These are the exceptional cases of obesity rather than the usual type and it would seem that in man, only the exceptional cases of obesity would parallel these. However, the individuals who are genetically obese probably account for only a small percentage of the obese pOpulation as a whole. However, a genetic predisposition to obesity frequently becomes manifested with the ingestion of a high fat diet. Fenton (Fenton and Dowling, 1953; Fenton, 1956) investigated certain strains of mice which became obese when fed a high fat ration, while other strains did not. The same strains which became obese in the foregoing experiment did not become obese when fed a high protein ration. Still other strains of mice have been known to exhibit similar phenomena (Sokoloff et al., 1960). Rats, also show a similar characteristic with certain strains becoming obese when fed a high fat ration (Mickelsen et al., 1955)- When these same strains were fed grain or chow rations, no obesity 74 _ 75 occurred. To genetically characterize this obesity, it was necessary to locate strains of rats wherein such obesity (that is, obesity with the consumption of the high fat diet) was not manifested. No previous investigation of the nature of the obesity induced by the high fat diet has been done. For this reason, information was needed on the prOpensity of several strains of rats to become obese when fed the high fat ration. This paper reports the results of such a study together with the distribution of fat in the bodies of the different strains of rats as they became obese. EXPERIMENTAL Six strains of rats have been investigated for weight gains and deposition of body fat when fed the high fat ration. These are: (1) Osborne Mendel, (2) Sprague Dawley, (3) Happert (4) Hooded, (5) MmS.Uo Gray and (6) Wistar-Lewis- The parent stock for the Osborne Mendel rats used in this study came from NIH. Weanling Sprague Dawley rats were obtained from a local producer.1 Weanling Happert rats were procured from the Department of Biochemistry, M.SoU- This strain was developed by Drs. Hunt and Happert and used extensively in their dental caries work. The Hooded and Gray rats were both descended from the Norway Black and White rats. Parent stock was obtained from the Me So U- Psychology Department. Breeding stock for the Wistar-Lewis rats was secured through the kind assistance of Mr. Samuel Poiley2 from I commercial producer.3 1Spartan Research, Williamston, Mich. 2Mr. Samuel Poiley, National Cancer Institute, NIH, Bethesda, Maryland. 3Batelle Memorial Institute, Columbus, Ohio. 76 For all strains, litters at the time of birth were cut to eight, except for the Gray rats where litters were cut to six. This was done because previous investigators indicated that the dams of this strain could only easily raise six young. For the rat strains raised in this laboratory, the rats fed the high fat and grain rations were paired according to sex, body weight and litter so that all groups were uniform. Five male and 5 female rats were sacrificed at weaning. Ten male and 10 female rats were fed the high fat ration (Table 2, Part I) and the same number were fed the grain ration (Campbell et al., 1966). Since weanling HOppert and Sprague Dawley rats were obtained from outside sources, these rats were not littermates. Individual weekly weight records and food intakes were obtained throughout the lifetime of the rats. For each strain, twenty rats or five from each group were sacrificed at 10 weeks and the remainder at 20 weeks. An estimate of activity was made by observation of the rats. The animals were observed at regular intervals throughout one day (around 9:00 A.M., 4:00 P.M. and 9:00 P.M.) each week. On each of these occasions a record was made of their activity. The following scale was devised to quantitate the observed activity. Value of "O" --rat lying down either asleep or awake, value of "1"--rat sitting either asleep or awake, value of "2" --rat eating, drinking, cleaning self or other comparable motion and value of "3"--rat climbing cage or moving rapidly. At sacrifice, fat depots were removed as described in Part I and carcass analyses carried out for moisture, fat, nitrogen and total ash (Mickelsen and Anderson, 1959). All data were analyzed by means of the computer at Michigan State University. Means and standard errors were 77 thus obtained and T tests (Dixon and Massey, 1957) between diets as well as T tests comparing each strain of rat with the Osborne Mendel strain fed the high fat ration. In 1 g of the grain ration there were 3.24 Calories and in 1 g of the high fat ration there were 6.62 Calories. These values were multiplied by the grams of the ration eaten throughout the 10 week or 20 week interval. To determine the calories in the body, the grams of fat in the body were multiplied by 9 and the grams of protein by 4. These values were added. The calories present in the body of control weanling rats were subtracted from this to correct for Calories already present before the consumption of any food. The calories consumed divided by the calories in the carcass gave a value for calories consumed to make 1 calorie of body energy. To calculate the calories consumed to make 1 calorie of body protein the calories consumed were divided by the g of protein gained in the carcass and were multiplied by 4. To calculate the Z of energy retained in the body of the ration consumed, the number of calories gained in the carcass (using 9 calories for fat and 4 for protein) was divided by the calories consumed. RESULTS Body weights In most cases, the Osborne Mendel rats were the heaviest and gained the most weight when fed the high fat ration (Table 7). In male rats of all strains this weight difference between the grain and high fat-fed animals became accentuated during the second 10 week period (Fig. 16 and 17). This was also true of female Osborne Mendel and Lewis rats. This 78 difference in the latter strain, however, was more dependent on the fact that the grain-fed Lewis rats showed little weight gain during the second ten weeks. Feed efficiency The male HOppert rats were the most efficient in converting energy into body tissue calories (Table 8). This was true for the grain-fed rats and partially so for those fed the high-fat ration. At the 20th week, the Osborne Mendel rats fed the high fat ration were the most efficient. The latter strain (Osborne Mendel) for both males and females were most efficient when fed the high fat ration (Table 9). For the other strains, no consistent pattern in feed efficiency was apparent. The percentage of calories converted to body energy was always greafer at ten weeks than at 20 weeks and greater in males than in females. The percentage of calories converted to body energy was highest in the 20 week old Osborne Mendel rats (Table 10) and the 10 week old HOppert rats. For all strains of rats, the efficiency of depositing calories in the body is about twice as great when the high fat ration is fed in contrast with the grain ration. When feed efficiency was related to body protein deposition, the differences among the strains and for the two rations became much smaller (Table 11). When feed efficiency is expressed on the basis of calories of protein gained, the only prominent difference is the greater efficiency of the males. This holds true for both rations and age groups. On the basis of increase in total body energy (Table 10), there was no difference between males and females. The efficiencies listed in Table 10 for the 10 week old male rats are essentially the same as those for the same groups in Table 11. However, for the other groups, there is a 79 pronounced increase in efficiency when based on calories per calorie of body protein gained. Activity Rats fed the grain ration consistently showed a greater degree of activity than those fed the high fat ration (Table 12). Exclusive of the Sprague Dawley and the Hoppert rats, grain fed rate increased their activity between the 10th and 20th weeks of the experiment. The activity of these two groups of rats was affected somewhat by an outbreak of murine pneumonia which very likely influenced the results. Rats consuming the high fat ration showed decreases in activity during this same period except for the Gray and Hooded rats. A rank order of activity beginning with the least active strain is given in Table 13. The Gray rats were the most active, and the Osborne Mendel rats were the least active as a whole. However, variations between all the strains were not great. Body protein and ash At the end of 10 and 20 weeks, Osborne Mendel and Sprague Dawley rats consistently had more protein (Table 14) and ash (Table 15) in their bodies than did the other strains of rats. These two strains of rats showed a more rapid formation of body protein even though there were no pronounced differences in the body protein content of the weanling rats. The Sprague Dawley rats tended to have more ash in their bodies than the other rats; this was especially true for the males and to a lesser extent for the females (Table 15). There was no consistent difference in body ash content between the rats fed the high fat or grain rations. A 1 80 plot of the body ash data indicates a rapid increase between the 10th and 20th week of the study. 135 All the male rats fed the high fat ration had at least twice as high a percentage of fat in their bodies compared with their littermates fed the grain ration (Table 16). This was also true of female rats for four strains. However, at 10 weeks, both the HOppert and Gray rats fed the high fat ration did not have twice as much fat in their carcasses as the animals fed the grain ration. For all strains, except the HOppert rats, the increased percentage of fat in the carcass of those rats fed the high fat ration was highly significant (P0.05). However, for males of this strain, the difference in carcass fat was significant (P<0.05). The percentages of fat in the male and female rats in each ration group were essentially the same. For some groups, the females had a slightly higher percentage of fat than the males but for others, the reverse was true (Table 16). The differences in the increases in body weights of the rats fed the high fat or grain ration is practically linear for the 20 weeks of the study. This is eSpecially true of the males of all but the Lewis strain (Fig. 16) and for all but the Sprague Dawley females (Fig. 17). 81 These differences in the increase in body weight are almost mirrored in the increases in body fat (Figs. 16 and 17). The increase in body fat does not quite account for the increase in body weight. For instance at the 20th week of the study, the male O.M. rats fed the high fat ration weighed 247 g more than comparable rats fed the grain ration while the difference in carcass fat content was 213 g. The increase in body fat was obviously associated with the formation of additional lean body tissue in the obese rats. At the end of the experiment, the obese rats had 97.9 g of protein in their bodies while the grain-fed rats had 95.1 (Table 14). This difference of 2.8 g of protein represents approximately 11 g of lean body tissue. Even this does not explain completely the difference between the increase in body weight and fat content. Ilggginal fat depots For all strains of rate, within each dietary group, the males had larger inguinal fat depots than the females (Fig. 18). This was true at both the 10th and 20th week of the experiment. Since the weight of the fat pads is expressed on the basis of body weight, the difference in weight of this depot in the two sexes cannot be attributed to the larger weight of the males. When weaned, female rats had inguinal depots that were as large or in some strains (Osborne Mendel, Sprague Dawley, and Lewis) larger than the male (Fig. 18). 7 T tests indicated that in four strains this depot was significantly larger in all cases 7(P<:0.0l) when rats consumed the high fat diet, than when they consumed the grain ration. For Gray and HOppert rats this significance was decreased (P<<0.05) or didn't exist (P:>0.05). 82 In comparing each of the other strains of rats to the Osborne Mendel rats fed the high fat ration only, the Hoppert rats showed no difference in depot weight (P> 0.05). The lack of significance for the inguinal fat depot in the Hoppert rats despite the fact that the average value appears to be lower (Fig. 18) is due to the great variability in this parameter. Three of the other strains (Sprague Dawley, Hooded and Lewis) had inguinal fat depots which were significantly smaller than the Osborne Mendel- (P <0.01) ranging to no significant difference (P >0.05) (these differences and degree of significance are shown in Fig. 18). When compared to the Osborne Mendel rats, only the Gray rats showed differences in inguinal fat depots based on g per 100 g body weight which were significant in all groups. For Gray males and females the difference was significant (P< 0.02) at the 10th week and highly significant at the 20th week (P< 0.01). Interscapular fat depot For all strains of rats, females had a larger percentage of their body fat just dorsal to the neck than did males. This was true whether rats consumed the grain or high fat ration (Fig. 19). However, differences between males and females were greater when the animals were fed the high fat ration. For weanling rats, this depot was relatively the same size in the two sexes, or in some strains, slightly larger in the female rats. For the Osborne Mendel and Lewis strains, the accumulation of fat in this area was significantly increased when the rats were fed the high fat ration in contrast to when fed the grain ration (P <0.01). Hooded rats behaved similarly but the level of significance for 20 week old 83 male rats was decreased (P< 0.05). The enlargement of this depot in Gray and Sprague Dawley rats fed the high fat ration varied in degree of significance when compared to the grain fed controls. The Hoppert strain was the only one in which the interscapular fat depot in the rats fed the high fat ration was not significantly larger than in the animals fed the grain ration. This was true except for the males at the 10th week where the difference was significant (P< 0.02). For all strains of rats fed the high fat ration, the size of this depot was compared to that in the comparable Osborne Mendel groUp. No significant differences existed between the relative size of these fat depots between Hoppert and Osborne Mendel rats (P> 0.05). In the Hooded, Lewis and Sprague Dawley strains, this depot varied from being significantly smaller than the Osborne Mendel (P< 0.05) to not significantly different (P >0.05). Only the high fat-fed Gray rats in all groups had a significantly smaller depot than the Osborne Mendel rats. For all groups except the twenty week high fat—fed females this depot was significantly smaller than that of the Osborne Mendel rats (P< 0.01). The level of significance decreased for 20 week high fat-fed females (P< 0.05). Genital fat depots Parametrial depots in the female rats for all strains at 10 and 20 weeks were always relatively larger than testicular depots-in males (Fig. 20). To compensate for the difference in size of the two sexes, these values are listed on the basis of percent of body weight. For male rats, the consumption of the high fat ration resulted in a significantly greater size of the-genital depot for all strains (P< 0.01). 84 This was also true of four strains of the (Lewis, Osborne Mendel, Gray, and Hooded)female rats. Female HOppert rats fed the high fat ration showed no significant increase at 10 weeks but a significant increase (P<:0.05) at twenty weeks when the relative size of this depot was compared with the grain-fed rats. Female Sprague Dawley rats showed different levels of significance at 10 weeks (P<:0.05) and at 20 weeks (P <0.01). In comparing genital depots in each strain of rats to its size in the Osborne Mendel strain two differences appeared. Male Lewis rats fed the high fat ration had a smaller testicular depot at 10 and 20 weeks. This difference is significant (P<10.02). The other difference is that at ten weeks, both male and female Gray rats have genital depots which are consistently smaller than those of the Osborne Mendel rats. This difference is highly significant (P< 0.01). After the 10th week of the experiment (13th week of life), the genital fat depot grows at almost the same rate as does the body. This is true for all strains except the Gray rats where it grows more rapidly than does the body as a whole. At 20 weeks this depot in the Gray rat is no longer significantly smaller than it was in the Osborne Mendel rat (P >0 .05) . The apparently deviant behavior of the parametrial fat in the older female gray rats may have been.associated with a pathological observation. Twenty percent of these rats had some abnormal adipose tissue in this depot. Dr. Vance Sanger of the Pathology Department indicated this to be necrotic adipose tissue apparently resulting from a restricted blood supply. 85 Mesenteric and omental fat depots The relative size of the omental and mesenteric fat depots differed in some strains of rats when the effect of diet was evaluated, but for all strains within each dietary group there was no difference between males and females (Fig. 21). As a general rule, the consumption of the high fat ration caused a significant increase in the size of the mesenteric and omental depot in comparison to grain-fed rats. For four strains this difference was always significant (P<.0-05) and frequently highly significant (P< 0.01). Only the Gray and HOppert rats did not show this significant difference. In Gray rats, the difference was not significant at 10 weeks (P)>0.05) but significant at 20 weeks (P< 0.02). At no time was the difference significant in the HOppert rats. Comparing the mesenteric depot of each strain to the Osborne Mendel rats fed the high fat ration, it was found that this depot was significantly smaller (P<:0.05) at both ten and twenty weeks in males of all strains. In contrast, the depot was significantly smaller in females of only two strains (P<:0.05). These were the Gray females at 10 weeks, and the Hooded females at 20 weeks. Perirenal fat depots With two exceptions, for all strains and both diets, the perirenal depot comprised a larger percentage of body weight in male rats than in females (Fig. 22). These two exceptions were the grain-fed Hooded and Gray rats at 10 weeks where the differences were slight. Both male and female Osborne Mendel, Lewis and Hooded rats fed the high fat ration had much larger perirenal depots than comparable rats 86 fed the grain ration. This was true for these three strains at the 10th and 20th week and for the Gray rats with the exception of the females at the 10th week. The increase was not significant in the HOppert rats. For Sprague Dawley rats, the increase was highly significant in females (P°<0.01) but significant only for the males at the 10th week (P<:0.02) but not at the 20th week (P:>0.05). Lewis and HOppert rats fed the high fat ration showed no significant differences (P)>0.05) in the weight of the perirenal depot when compared to comparable Osborne Mendel rats. This was also true of Sprague Dawley female rats. However, the perirenal depot was smaller in the male Sprague Dawley (P<:0.02) and Gray (P<:0.05) rat than the same depot in the male Osborne Mendel rat. The relative size of the perirenal depot was smaller in the Sprague Dawley (P<0.0l), Gray (P<0.01) and Hooded (P <0.02) female rats at the 20th week than in the Osborne Mendel rats. In male Hooded rats the perirenal depot weighed significantly less (P<:0.02) at ten weeks but was not significantly different in weight from the Osborne Mendel male rats at twenty weeks. DISCUSSION This study indicated that Osborne Mendel rats responded to the high fat ration with a larger gain in body weight, a greater deposition of body fat and relatively larger fat depots than did the animals of the other five strains. When fed the high fat ration, the Osborne Mendel rats were least active physically and most efficient in converting feed to body energy. There were instances when this was not the case, but in general, this was true. 87 The Gray rats were least affected by the high fat ration. They showed the smallest increase in body weight, body fat and relative size of adipose tissue. They were least efficient in converting feed to body tissue and expended more energy on physical activity than any other group of rats. Again, for certain parameters this may not be the case, but it is generally so. The other four strains were in between these two extremes as far as response to the high fat ration was concerned. For these strains, however, some parameters point to particularly important phenomena. For instance when the HOppert rats were fed the grain ration they had the largest percentage of body fat and the largest subcutaneous adipose tissues of all the strains investigated. In the Hoppert rats these subcutaneous tissues did not get significantly larger when the rats were fed the high fat ration. The fact that they were so large already is a partial explanation for this as well as the fact that greater individual differences existed in this strain of rats. This would indicate that with selective breeding it should be possible to produce a high efficiency and a low efficiency strain of HOppert rats. During the second 10 week interval, the grain-fed female Lewis rats gained very little body weight. Therefore, an accentuated response existed in those rats fed the high fat ration during this period. The relative distribution of fat in the different depots of rats varied both with strain and sex as well as with ration fed. Since the Gray rats had the smallest accumulation of body fat, it was to be expected that the relative size of their subcutaneous adipose tissue should be the smallest. The females of that strain did not show the 88 accretion of fat in the interscapular area so characteristic of female rats of other strains. When mesenteric and genital depots of Osborne Mendel, Lewis, HOppert and Sprague Dawley rats had already increased to make up a high percentage of body weight and were increasing in weight at the same rate as body weight increases, these depots in the Gray and Hooded rats were comparatively smaller and were still increasing in weight at a faster rate. This was less pronounced in the latter strain. As a whole, fat patterning in the Hooded rats was similar to that in Gray rats but in Hooded rats the depot was always composed of a slightly larger percentage of body weight. Table 10 showed that rats were twice as efficient fh converting a calorie of the high fat ration to body energy when fed the high fat ration as compared to the grain ration. This was true of both males and females of all strains. It appears that the energy of a high fat ration can be readily transferred into body tissues with a minimum of energy expended by the animal. Investigators have frequently calculated feed efficiency on the basis of grams of weight gain for grams or calories of ration consumed. Mayer and Krehl (1948) reported that 50 day old rats have a feed efficiency of 35%. This was calculated as grams of weight gain per grams of ration consumed. During periods of maximum weight gain, food efficiency for grain-fed rats (male) would be similar in this study. However, when male rats were fed the high fat ration, food efficiency increased to 50%. Forbes et al. (1946c) recognized an increase in energy retained in rats fed high fat rations. Three experimental rations used by these 89 investigators consisted of 2, 10 and 30% fat. These all contained 2% corn oil; additional fat was lard. Protein was provided daily at a level of 2-2 3 for each rat regardless of the ration fed. To provide this amount, the protein levels in the different rations had to be varied since all rations were isocaloric. The percent of calories retained in the body were 17.18, 18.43 and 19.76 respectively. Energy lost in the feces was 3.6, 4.5 and 5.3% of the calories respectively for the rats fed the 3 rations. Energy lost in the urine was 4.7, 4.8 and 4.9% respectively. The remainder of the calories were "energy output as heat". This was determined by the increased heat increment when the rats were fed an amount of the ration beyond that sufficient for maintenance. From this was subtracted the heat increment of the maintenance ration. Calories retained were determined from body composition data. The calories present in a gram of protein or fat in the body will vary. Paladines et al.(l964) reported that various parts of the bodyof sheep produced a range in calories when combusted. FFor protein, these values were 5.3 to 5.8 kcal/g; for fat 7.4 to 9.4 kcal/g. Blaxter and Roox (1953) have reported other values. In view of this, the value of 9 kcal/g fat and 4 kcal/g protein was accepted. On the other hand, if the actual caloric value for the protein in the carcass was 5.3, the percent of retained energy would be increased by 16% in the small rats and 9.5% in the larger rats in this study. 90 H .Am0.0vmv ucmOHMchHm mum umw an5 won many Hoccoz ocuonmo nuH3 mumou H m .HHo.ovav acmoHcstHm aHewHe use use emu; use was. 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H36~ $6 H86 333 2; H36: 86 H33 66; Haw: 26 H86 626 H26 632% :6 Hfii 6m; H866 26 H32 «a; H63; ~86 H36 .333 mamsam ~24 +3.2 H606 +366 H66.0 +2.2 ~66; +3.3, ~86 $66 365: 2.863 3a: 59.6 ”.3 :95 588 63 66.6: 9.2933 5856 «3003 .MM mxmoa 0H nuwu mo mcawuuu Ham cw uum avon mo uawuuwm 0H manna Differences in body weight (g) 250 200 150 100 59 100 _ 250 L- O . ‘ 3° u a I: 3 O U ‘ «U G ‘H . >5 '0 O G ' H 0) O 0 U Q N I L! d9. ”.4 / a b 1 4 L 10 20 20 weeks weeks Fig. 16 Differences in body weights and body fat contents of male rats of each strain fed the high fat and grain rations. Each point represents the ditterence in the average values for 5 rats fed each diet. ‘ DOsborne Mendel 0 Lewis ‘ Sprague Dawley o Hooded A Hopfiert I Gray Differences in body weight (g) 150 100 101 ._ E0 100.. u c m U c o O U a . ‘H o >. 'U o .D c H U) 3 50 _. c o H , ‘3 I H-a H c: J, I l l 710 720 10 20 weeks weeks Fig. 17. Differences in body weights and body fat contents of female rats of each strain fed the high fat and grain rations. Each point represents the difference in the average values for 5 rats fed each diet. 0 Osborne Mendel 0 Lewis A Sprague Dawley 0 Hooded A Happert I Gray 102 .______Q£hQInfi_M£nd£l_____1 1o _ 4——a ——o 5 ._ gj? U .C 00 '3 I J l 3 IO 10 >. B Hnnnert g r- 14.4 0 1o _ °\° U) m U o Q. Q) "U a. 5 .. 0 Q) 1! .fi w9w//,‘¢5‘::2L““-~c..:: U) Q) U > -.-4 t3 1 '1 '3 1U 10 m Hooded 10 L 22 5 _ -—-"’"2' 2 12 ———n 4n; 4 I L 10 2U’ 10 10 10 ,______Spragne_nanle¥_______ 2 b 4_; / fin we” i I I0' 20 Gray 1 41 10 20 Lewis _. Age in weeks from weaning - Fig. 18. Sum of the weights of the inguinal fat depots expressed as g per 100 g of body weight. The significance of the difference when referred to the comparable group of Osborne-Mendel rats is listed where applicable. time intervals indicate the number of weeks the rats were on the experiment. Five rats in each group were sacrificed at the designated time intervals. (1. p. .D as g 3.. 3r U :13). 2L. 2 _ "D ‘H \' o l_ m .f‘.‘ m J I- w 10 :E Hooded Lewis m '33 :z 5._ 5.. 4__ 4_. 3 _. 3 _ 2.. 1" 3 j? 3 a? 3. ’ 10 10 Age in weeks from weaning Fig. 19. Weight of the interscapular depot expressed as g per 100 g of body weight. The significance of the difference when referred to the com- parable group of Osborne Mendel rats is listed where applicable. The time intervals indicate the number of weeks the rats were on the experiment. Five rats in each group were sacrificed at the designated time intervals. (1. P<0.0l; 2. P<0.02; 3. P<0.05). |, Male, high fat; 0, Male, grain. 0, Female, high fat; 0, Female, grain. 104 Osborne Mendel Sprague Dawley 10 l. ‘10 _ \ 5 _. 5 _ _ g’ ___ ___. #__. 5 ___ e——o n__f g E! *2' I 1 l 10 ‘20 10 20 Hogpert gray 10 _. 10 _. 5 F“ S ° 9 I a 10 20 Hooded Lewis 10 _ 10 Relative size of depot as'% of body weight 5 ._. 5 ._ 2 _r. ‘9 .— / _____.——-—-—"° \0 c——e “‘° ‘_9 a ,_Age in weeks from weaning Fig. 20. Sum of the weights of the genital fat depots expressed as g/100 g of body weight. The significance of the difference when referred to the comparable group of Osborne Mendel rats is listed where applicable. The time intervals indicate the number of weeks the rats were'on the experiment. Five rats in each group were sacrificed at the weeks designated time intervals. (1. p. .8 F “3 z. .. 4.. 5\° ED 5 3 _. 31— ‘91 '8 2 2— /."‘3 ‘H / O 1 1_ _/—-d g D w _ l 1 a 10 20 > 3 Lewis m H o of. 5 _ 5 _ I 4 P 4_ 3 _ 31.. 2 2 .- / l l 1U 4U’ Age in.weaning Fig. 21. Sum of the weights of the mesenteric and omental depots expressed as g/100 g of body weight. The significance of the difference when referred to the comparable group of Osborne MEndel rats is listed where appli- cable. The time intervals indicate the number of weeks the rati were oni the experiment. Five rats in each group were sacrificed at the des gnated ti me intervals. .(1. no. 01; 2. 110.05). In other studies carried out in this laboratory, the adrenal weights of the obese rats were larger than those of the lean animals. When the adrenal weights were calculated on the basis of body weight, the females showed a range from 17 to 34 mg per 100 g while the range for the males was from 11 to 18 mg. The relatively larger size of the adrenals in the females was seen in all groups. Since there were no differences in the absolute weights of the adrenals for any of the groups, representation of these weights on a body weight basis showed relatively smaller adrenals in the obese rats with no differences for any Of the other groups. 121 Heart: Weight reduction produced a significant decrease in the size of the hearts Of all rats except those fed the semipurified ration (Table 26). The weights of the hearts in both the male and female reduced rats approached the weight of the hearts in the lean controls. Although there were no statistical differences between the heart weights of the reduced rats and the lean controls, in no reduced group did the weight equal that of the lean control. When expressed on a relative size, the hearts for all groups other than the Obese controls were essentially the same. For the obese rats, the heart weight for both males and females was 210 mg per 100 g body weight. For males in all other groups of males, the relative heart size ranged from 290 to 310 mg and in the females from 290 to 350 mg. Liver: Weight reduction produced a significant decrease in the size of the liver for both male and female rats in all groups except the ones fed the semipurified ration (Table 26). When expressed on the basis Of relative body weight, there were only minor differences in the size Of the liver for all groups. Spleen: During weight reduction, the spleens of the rat were reduced in weight to those of control rats which were never obese (Table 26). When the weight of the Spleens was expressed as percent of body weight, they were significantly smaller (P<:0.05) only in the obese control rats. Females (except obese controls) had a larger spleen than did males when the weight of the organ was related to body weight. Summary: In all rats, hearts and Spleens followed most nearly body weight losses, liver and kidney showed a lesser degree of weight loss and adrenals showed little change in weight with weight reduction. 122 Reduction in fat depot weights All fat depots were reduced in weight when rats were subjected to any one of the four reducing regimens (Table 27). However, each fat depot had a pattern of weight loss which emphasized dissimilarities in the reduction of the various depots. Inguinal depots: For all rats subjected to weight reduction, the inguinal depot lost sufficient weight to make this depot significantly different (P<0.0l) in size from that of the obese rats even when its weight was expressed as a percent of body weight (Table 26). Both male and female rats fed the semipurified ration showed lesser total body weight losses than the rats in the other groups; the inguinal depot reflected this difference -- it was significantly different (P<0.01) from that Of all other groups of reduced rats whether male or female when expressed as a percent of body weight. Male rats fed any one of the three other reducing rations had final weights of inguinal depots which were equivalent to that of the grain controls (P70.05). Obese female rats fed the grain ration ad libitum still maintained a weight about 30% greater than the lean controls fed the same ration throughout their lives. The weights of the inguinal depots in the reduced rats were almost twice as heavy as in the lean controls. However, when eXpressed as percent of body weight, the difference in weight of this depot was not significantly different (P>0.05) from that of the lean controls. Inguinal depot weights for the two groups of female rats reduced by limiting the quantity of high fat diet were likewise not significantly different from the lean controls. 123 Genital depots: Male rats reduced on the grain ration showed a larger prOportionate weight loss for the testicular depot (Table 27) than the rats reduced by any other means (P<0.05). In prOportion to body weight, the testicular depot was largest when male rats were 600 g or 148 days of age. At that time, this depot was 4% of body weight. This depot in control obese rats (about 400 days of age) represented only 2.5% of body weight. This ratio was maintained in the obese rats that were reduced by any of the regimens except the grain ration. The rats reduced by feeding the grain ration had testicular depots which represented only 1.46% of body weight; the same as the lean controls. The parametrial fat depots in the reduced female rats represented the same prOportion of body weight as in the lean controls. In all reduced groups, this depot had not only decreased in size but it represented a smaller percentage of body weight than in the obese rats (340.01 except for the group fed semipurified ration where P<0.05, Table 27). Lean control male and female rats of this age showed a slight reduction in genital depot weights when compared to younger rats (Part I, Fig. 11). Perirenal and retrOperitoneal depots: None Of the weight reduction regimens for either sex resulted in reducing the relative weights of the perirenal depots to those of the lean controls (Table 27). All remained significantly larger (£40.01) although all showed a loss of weight when compared to th% obese controls. For both males and females the semipurified ration resulted in the least weight loss of this depot. 124 This was probably related to the smaller losses in body weight for these rats. However, male and female rats fed the high fat diet ad libitum 2 days of 7 had slightly more fat in this area than those rats restricted to a limited amount of high fat ration daily or those reduced on the grain ration. * Mesenteric and omental fat: Weight reduction was highly effective in decreasing the mesenteric and omental fat depots to weights equal to or below those of the lean controls except for the female rats consuming the semipurified ration. For male rats reduced by any of the weight reduction regimens other than the purified ration, the relative weightsiofthe mesenteric and omental fat depots were significantly lower than those in the lean controls (P<0.05). Fat surrounding xiphoid process: Compared tO the lean controls, this depot remained relatively larger (£40.05) in the rats of all reduced groups except the females fed restricted amounts of the high fat ration. For those females, the relative weight of this depot was the same as that Of the lean controls. Although the differences are not significant, it is noteworthy that for both male and female rats, the high fat ration had a differential effect during the reducing program depending on how it was fed. When this ration was fed 2 days out of 7, the fat in the xiphoid process was 1.5 times larger than when the ration was fed each day in restricted amounts. Recapitulation During weight reduction of Obese rats, the loss of weight by the different depots was influenced by (l) depot, (2) type of reducing regimen, (3) age of animal and to a lesser extent by (4) sex of the animal. 125 Depot: The rank order in which the fat depots lost weight depended upon whether the obese or the lean controls were used as the reference (Table 28). In both cases, the subcutaneous fat under the forelimb appeared to lose weight most readily. The perirenal fat depots were at the end of the list in both cases. Since the interscapular fat depot of females begins to increase very rapidly in size only after the animalfs weight begins to exceed that of the lean controls, it shows a marked reduction compared to the Obese rats and much less when compared with the lean controls. The genital and mesenteric fat depots made up a relatively smaller percentage of the body weight in the older than in the younger animals (see Part I). If an adjustment were made for this, their place in the relative order might be changed. Type of reducing regimen: The ration fed during the reducing program produced some differences in the percentages of weight lost from individual fat depots. The feeding program had a pronounced effect on the final body weights of the reduced rats. This, in turn, affected the final weights of the depots and thereby, their relative weight losses. The different final weights attained by the reduced rats makes it difficult to adequately evaluate the effect of the reducing regimen on the weight lost by the different depots. The weight of the fat depots in males reduced by feeding the grain were the same weight or lighter than those rats reduced on the high fat regimens. This was true even though male rats reduced on the grain ration were 30 g heavier. For <>ther depots e.g. the interscapular and perirenal depots, there was a Eniggestion that when rats were fed restricted amounts of the high fat 126 ration, there was a difference in final depot weights depending on whether restricted amounts of the ration were fed every day or the rats were permitted to eat ad libitum two days out of seven (Table 27). Age: Weight reduction Of two depots (the genital and mesenteric) was enhanced with aging. Weight reduction of the perirenal depot and that depot surrounding the xiphoid process was curtailed by aging. Sex: When the final weights of the fat depots in the male and female rats are compared (Table 29), it becomes apparent that the male rats during the reducing program lost relatively less fat than the females from the inguinal, forelimb and perirenal depots. The females lost relatively less fat from the interscapular, the genital, mesenteric and for the rats fed the grain and semipurified rations, the xiphoid depotso= To a certain extent, the relative weight losses of each sex reflected the sex differences in the size of these depots that existed in_the obese rats (Table 29). DISCUSSION” Initial weight loss The primary factor in determining the initial rate of weight loss during a reducing program is the deficiency of calories to which the animals are exposed. The nature of the ration is of only minor consequence as evidenced by the similar weight losses experienced by all groups of obese rats during the first few weeks of the reducing programs (Fig. 23 and 24). This is in contrast to the report by Kekwick and Pawan (1964) who claimed that weight reduction in mice was greater when most of the calories in the reducing diet came from fat than when they came from carbohydrate. They maintained that the differences in the 127 composition of the reducing diets were reflected in metabolic shifts in the mice brought about by the diets. The primary difference between this work and that of KekwiCk and Pawan is the relative amount of fat in our animals. In these studies, the animals that were reduced were grossly Obese.. This condition, plus the fact that they had been fed a high fat ration for a long time prior to weight reduction, meant that they were adapted to a metabolic scheme involving primarily fat. When they were put on the reducing regimens, they still secured a fairly high percentage of their energy from the metabolism of fat part of which was of endogenous origin. The animals used by Kflodck and Pawan (1964) were "normal" mice that undoubtedly had relatively small stores of body fat--they had been fed a high carbohydrate ration throughout their lives. Although a number Of critical comments could be made about the interpretations Kekwick and Pawan made from their results, suffice it to point out that they ignored the dehydrating effect that the high fat diet has when it is first consumed. - Grain vs. semipurified rations Work of Mickelsen et al. (1955) indicated that when weanling rats were fed a grain ration from weaning, they did not become obese. When, however, they were fed a ration of purified ingredients in such amounts that the proximate analysis of the semipurified and grain rations were the same, about 80% of the rats eventually became obese. In view of this differential effect of the two rationson the initiation of obesity, they were compared as a means of producing body weight losses in obese rats. 128 The low—fat semipurified ration was not as effective as the grain ration in lowering the weight of obese rats. Actually, some of the female rats fed this semipurified ration started to regain their body weights after an initial loss; some of them weighed slightly more at the end of the reducing regimen than they did at its start. It is difficult to decide what is reSponsible for this difference in effectiveness of the two rations. There is a slight difference in moisture content of the two rations. Since the two groups of rats consumed essentially the same weight of feed, the slightly smaller moisture content of the semipurified (2.5%) compared to the grain ration (7%) resulted in a 5% difference in caloric intake. This small difference hardly appears adequate to explain a difference of 100 g of body weight which existed between the rats in these two groups. The rats more readily accepted the semipurified ration than the grain ration when they were changed to it from the high fat ration. After the 10th week these initial differences in consumption disappeared and more nearly the same quantities were consumed by the two groups. The difference in this early acceptability could easily be related to the sweetness of the sucrose ration. Sewell and Maxwell (1966) suggest this as the reason why baby pigs consumed more of a sucrose than Of a grain ration. Other work in this laboratory (Taylor, unpublished data) indicates that rats have a great avidity for sucrose solutions and when Offered this, they will drink more than half their body weight in a 24-hour period. There was an apparent lower mortality among the rats that were reduced in weight than among the Obese controls. For females, howeverbthe evidence is not nearly as clear. A number of the Obese females that were subjected 129 to weight reduction manifested lymphomas in their subcutaneous adipose tissue. Whether they deve10ped as a result of the weight reduction or became more apparent with the loss of weight could not be determined. Furthermore, a number of female rats that were being reduced on the high fat regimen died before the study was completed. The validity of these observations can be established only by further work. Organ weights Obesity, per se, is associated with enlargement Of the kidneys. The work of Sokoloff (unpublished data) has indicated that disturbances of the kidneys are one of the primary causes of death among the Obese rats. Whether the enlargement seen in these obese rats stems from any pathological alterations will have to await the report of the pathologist. The present data do, however, indicate that body weight reduction Of the obese rats is associated with a reduction in the size of the kidneys. All but one group of rats showed a decrease in kidney weights until they were, prOportionately the same size as in the lean controls. In those rats fed the semipurified ration, the weights of the kidneys remained essentially the same size as when the animals were obese. Preliminary work indicates that during the development of Obesity, the kidneys show a reduction in their ability to clear the blood of phenol- sulfonphthalein (PSP). The impairment sets in at a fairly early stage of Obesity as evidenced by a reduction in PSP in the 450 g rats fed the high fat ration from weaning. As these rats increase in weight, their ability to clear the blood of PSP decreases even more (Taylor, unpublished data). These observations indicate that another dietary factor apparently can influence the absolute size of the kidney. This is in addition to the effect of a high protein diet. For years it has been recognized that a 130 high protein diet produces hypertrOphy of the kidneys (Addis, 1926). The hypertrOphy per se according to Smith (1951, p. 473) has not been associated with any abnormality of this organ. It is likely that the hypertrOphy of the kidney in the simple dietary type of Obesity observed in this study may be similar to that occurring in the rat made Obese by hypothalamic lesions (Kennedy, 1957). Within the first week or so after the Operation while the rats were eating very large amounts of feed (25% of body weight), the kidney, heart and liver rapidly increased in size. For the kidney, the increase in size was associated with a 1.5 fold increase in deoxyribonucleic acid phOSphorus suggesting a considerable increase in the number of kidney cells. The heart showed an increase in size with the deve10pment of obesity. As in the case of the kidneys, the increase in weight of the heart of the obese rats was not prOportional to the increase in body weight. Again, Kennedy (ibid) reported a rapid increase in the size of the heart during the first few weeks after hypothalamic lesions in his rats that became obese. Unfortunately no data accompanied that statement. At this time, it cannot be stated whether the hypertrOphy of the heart is the result of an increase in the vascular bed. Although no studies of blood volume have been made in these obese rats there is some suggestion for an increase in that parameter. This is based on the volume of blood that can be removed by a syringe inserted into the renal artery. Under such circumstances more blood can be removed from the Obese than from the lean rat. The results of the present study indicated that the weight of the heart returns toward that of the lean control during a weight reduction program. Since the rat is relatively immune to any cardiovascular abnormalities associated with the obese state (Bragdon and Mickelsen, 1955), it becomes difficult to assess the role of weight reduction on that function. __'...— 131 The liver also increased in absolute weight during the deve10pment of obesity. However, this increase is closely related to changes in body weight (Table 26). The enlargement of the liver in the rats fed the high fat ration from weaning is not associated with any abnormality either in composition or histological structure (Sokoloff, unpublished data). Weight reduction is accompanied by a decrease in the size of the liver. This decrease again being prOportional to body weight changes. Protein The larger loss of total protein on the two weight reduction diets wherein fat was restricted could be associated with a lesser intake of protein on these high fat rations. Although protein made up roughly 25% of the original high fat ration, the reduction in intake of the rats fed reduced amounts of the high fat ration was such that the intake of protein ranged from 6 to 15 g per week. On the other hand, rats reduced on the semipurified and grain rations ate from 8 to 30 g of protein per week. It is doubtful whether even this difference in protein intake can eXplain the observed differences since the presence of 3.5% protein in the tissue lost by weight reduction agrees with that reported by Sarett et al. (1966). Fat depots Here, four behavioral patterns of fat depots seem worthy of discussion. Females appear to favor deposition of fat behind the neck and in the mesenteric depots. Yet, in lean female controls, the relative amount of fat deposited behind the neck is no greater than that present in males. In weight reduction, the grain ration appeared more effective in the removal of fat from this interscapular area than other diets. The rats fed restricted amounts of the high fat ration every day were more effective in 132 removal of fat from this area than the rats fed the high fat ration ad libitum two days per week. The mesenteric depots in the obese rats were reduced so that their final weights were nearly the same or less than the weights of lean controls. Could fat be more readily removed from this area because of its proximity to the gastrointestinal tract? Males showed greater evidence of weight reduction here. This appears to counterbalance the lesser reduction of the perirenal fat depot in males. Ekerjl (1959) and Young et al. (1963) suggest a reprOportioning Of fat with a larger quantity of abdominal fat appearing in women with aging. It would appear that the perirenal fat depot and fat deposits in the abdominal cavity under the ribs and around the xiphoid process are sequestering this fat while mesenteric and genital fat depots continue to reduce. Both of the high fat reducing regimens produced less reduction in testicular fat depot size than either the grain or semipurified rations. Additional work will be required to determine whether this differential effect was due to the differences in the composition of the diets or the method of feeding (i. e., ad libitum versus restricted intake). 133 Table 17 Composition of Semipurified ration (M-l6) Ingredients % Casein 22.50 Corn Oil 2.67 Minerals1 6.28 Vitamin mix2 2 .20 Liver powder 2.00 dl-Methionine 0.25 Aureomycin 0.01 Non-nutritive bulk4 3.80 Sucrose 60.29 1 Salt Mixture, Wesson MOdified (Osborne-Mendel) General Biochemicals, Chagrin Falls, Ohio. 2 Vitamin Diet Fortification Mixture, Nutritional Biochemicals, Cleveland, Ohio. 3 Generously provided by American Cyanamid Company, Princeton, New Jersey. 4 Non-nutritive Bulk, cellulose type, General Biochemicals, Chagrin Falls, Ohio. 134 Table 18 Proximate analysis of Grain (M-1) and Semipurified (M-16) Rations Grain Semipurified Component (M-l) (M-l6) % Z Protein 23.4 22.5 Fat 3.0 2.7 Fiber 3.8 3.8 ASh 603 603 The grain ration has 3.24 calories per g; the semipurified ration has 3.48 calories per g of ration. 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Decreases in male Osborne Mendel rats subjected to one of the following reducing regimens: 6—-o semipurified ration,ad lib,; o—o, grain ration,_ ad lib.; A—A, high fat ration 2 days each week; A—A , high fat ration in restricted amounts every day and n——u , grain-fed control rats. 147 700 600 ’03 v 500 .‘E 00 «4 <1) 3 >5 '0 8 400 300 Fig. 24. l l l J J 5 10 15 ~ 20 25 Weeks on weight reduction Decreases in female Osborne Mendel rats subjected to one of the following reducing regimens: o—o semipurified ration ad lib.; o—o, grain ration, ad lib.; A—A , high fat ration 2 days each week; and A—‘ , high fat ration in restricted amounts every day and 0—0 , grain-fed control rats. 148 125 100 3° c 75 -.-I Q) 4.) o H O. *4 50 O U .c on w-J :g 25 i I I J [ ! I 150 300 450 600 700 900 1050 Male; Body weight (g) 75 P o : ./n A 0 fl :3 A a. ' A .5 . .3 SO __ ‘ o H D. u.‘ o oo o.-I Q) 3: J I I I J l 150 300 450' 600 750 7900 Female; Body weight (g) Fig. 25. Weight of protein in reduced Osborne Mendel rats in comparison to weight of protein in younger high fat-fed rats (Part I). The intersect indicates the g of protein in the rats when rats were first fed reducing rations. This figure also shows comparison with lean controls of the same age. |-—|, weight of protein in g during deve10pment of obesity; a , grain- fed control; . , grain-fed,reduced; o, semipurified, reduced; A , high fat, 2 da. of 7; A , high fat, restricted. 149 600 A 500 _ 00 U 33 400 _ ’«H O E 300 _ DO -.-I :22 200 _ 100 __ / I I I i I I 150 300 450 600 750 900 1050 Male; Body weight (g) 500 _. 3% 400 _ ‘5 *4 300 _. *H O E 200 _ 00 -.-I Q) :2 / IQO - ' A . .. / ‘o I I I I I 41 J I 150 300 450 600 750 900 1050 ‘ Female; Body weight (g) Fig. 26. Weight of fat in reduced Osborne Mendel rats in comparisoh to weight of fat in younger high fat-fed rats (Part I). The intersect indicates the g of fat in the rats at the time when rats were first fed reducing rations. This figure also shows comparison with lean controls of the same age. ._. wgt. of fat in g during deve10pment of obesity; D, grain-fed control; 0 , grain-fed,reduced;o , semipurified, reduced; A , high fat, 2 da. of 7; A , high fat, restricted. SUMMARY AND CONCLUSIONS To evaluate the effect of excessive weight on body composition and deve10pment of fat depots male and female Osborne Mendel rats were fed either a high fat ration or a grain ration. Five male and five female rats fed each ration were sacrificed when they were weaned at 24 days and at weights of 150, 300, 450 and 600 g. In addition, male and female rats fed the high fat ration were sacrificed at 750 g and males at 900 and 1050 g. Analyses for total body fat, protein, moisture and ash were done on the carcasses. Individual fat depots which were dissected from the animals and weighed included the right and left inguinal tissues, right and left subcutaneous tissues underlying the forelimb,' interscapular depot, right and left genital depots, right and left perirenal depots, mesenteric and omental fat depots and a small depot surrounding the xiphoid process- Male rats fed the grain ration never exceeded 600 g in body weight and served as the lean controls. The rats fed the high fat ration gained weight more rapidly than the grain-fed animals and eventually became grossly obese as represented by weights welltover 1000 g at the end of 260 days. At all ages, the rats fed the high fat ration had more fat in their bodies both on an absolute and relative basis. Body protein'content increased in relation to age and this rate was the same for both the lean and obese rats. In other words, rats of the same age had the same amount of protein in their bodies regardless of their weight. This was true until the high fat rats became very obese. In the latter case, they had about 10% more protein in their bodies than their lean controls. 150 151 Adipose tissues doubled in weight when rats were fed the high fat ration. In the young rats so fed, the adipose tissues all increased in weight more rapidly than did body weight. In male rats fed the high fat ration and weighing 600 g or more and in comparable females weighing 300 g or more, inguinal fat depots and subcutaneous tissues underlying the forelimb increased in weight more rapidly than the body. The interscapular fat depot in high fat-fed rats showed a continual linear increase in relative weight. This was much greater for females than for males. For grain-fed rats all subcutaneous adipose tissues increased in weight relative to increases in body weight. In high fat-fed rats, the genital and mesenteric depots decreased in relative weight with greater gains in.body weight4as7age increaseda Perirenal depots showed a linear increase in relative weight when rats were fed the high fat ration. Also, the perirenal depot showed large increases in weight in the 600 g grain-fed male rats. Adipose tissue deposited around the xiphoid process showed linear increase after male high fat-fed rats reached 450 g, and females, 300 g. The susceptibility of six strains of rats (Osborne Mendel, Sprague Dawley, HOppert, Lewis, Hooded and Gray) to become obese and the pattern of changes in both body composition and weight of adipose tissues was studied. Weanling rats of each strain were fed the grain and high fat ration. At 10 and 20 weeks after the start of feeding, 5 males and 5 females from each strain and each dietary group were sacrificed. Feed consumption records indicated that the rats of all strains and both sexes were more efficient in converting the energy of the high fat ration to body tissue than that in the grain ration. The differences r 152 among the strains in this reSpect were not very great. During the first 10 weeks, the HOppert rats were most efficient as far as both rations were concerned. At the end of 20 weeks, both male and female Osborne Mendel rats fed the high fat ration were more efficient than any other strains. The Gray rats were least efficient in converting feed energy to body tissue. Final absolute values for protein and ash were similar for Sprague Dawley and Osborne Mendel rats. Other strains had less. The increase in body fat in the high fat-fed rats was greatest in the Osborne Mendel rata,iintehmedigry£in Spraguerbawley, HOppert, Lewis and Hooded rats and least in the Gray rats. In general, adipose tissues of high fat-fed rats were heaviest in Osborne Mendel rats, intermediary in the other 4 strains and least in the Gray rats. All strains of rats reaponded to the high fat ration by some increase in body fat and some increase in adipose tissue weights. In another study, 40 male and 40 female obese Osborne Mendel rats (1000 g males; 650 g females and around 260 days of age) were reduced. Ten lean and 10 obese rats of each sex and of similar age served as con- trols and were maintained on the high fat ration throughout the study. Four reducing regimens were used for weight reduction. They were (1) Grain and, (2) Semipurified rations fed ad libitum every day, (3) High fat ration fed ad libitum 2 days of 7 and, (4) Restricted quantities of high fat ration fed daily -the total amount per week to equal that in (3). Rats were reduced and maintained their reduced weights for at least 10 weeks prior to sacrifice. 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