THE USE OF PARABIOSIS IN THE STUDY OF FOOD INTAKE REGULATION AND BODY FAT CONTENT Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY NATHAN WILLIAM SHIER 1975 This is to certify that the ‘ I thesis entitled F The use of parabiosis in the study of food intake regulation and body fat content presented by Nathan William Shier has been accepted towards fulfillment of the requirements for Doctoral degree in Food Science and Human Nutrition Major professor Date August 8. 1975 ' 0-7639 172K Ill \IAIIII ABSTRACT THE USE OF PARABIOSIS IN THE STUDY OF FOOD INTAKE REGULATION AND BODY FAT CONTENT By Nathan William Shier A technique has been developed which makes it possible to determine the individual body weights of rats joined in parabiosis. In using parabiotic animals for dietary experimentation separate weight deter- minations of each parabiont without pair separation is necessary. Flat balance pans, attached to each of two dietetic scales, were brought to close approximation. Parabiotic rats were placed dorsally on the scales with the connection between them situated between and parallel to the balance pans. The difference between balance readings or the uncorrected weight difference wasAA. The actual weight difference between the two animals was predicted by projectingAA on a standard curve. With the total weight of the parabiotic pair and the predicted weight difference of the parabionts, two equations with two unknowns were solved which gave the weights of the individual parabionts to within : 3% of their actual weights. In developing the standard Curve, giving weight differences of parabionts, forty pairs of rats with a variety of weight differences (ranging from 2 to 327 grams) and ratios were placed on the scales, as described above, andAA recorded. The animals in each pair were then separated and weighed individually with Nathan William Shier the actual differences in their weights recorded asziB. A linear function was obtained when eachIAB was plotted as a function of its respective AA (r = 0.982). Parabiosis, the union of two living organisms, has been used in studying the hormonal control of food and water intake. These studies are difficult to interpret because the investigator had no way of feeding separate diets to each parabiont. A feeder has now been developed which permits feeding each parabiotic animal a separate diet. This feeder and its use are described. Only a few days of ‘ training are required to get the parabiotic rats accustomed to proper use of the feeder. To check whether one rat can consume any of the other's ration, a chromium sesquioxide marker was placed in the food of one parabiont and the feces of the other assayed for chromium oxide by acid digestion and spectrophotometry. No chromium oxide was found in the excreta of the parabiont whose feed had not been marked. This feeder allows, without restraint, ad libitum food consumption in parabiotic rats with excellent weight gains and food consumption even when used continuously. An improved scapular supportive suture has been developed giving excellent support at the pectoral girdle with virtually no scapular separation which has been a major problem in previous parabiotic experiments. Dye dilution studies with Evans Blue indicated an improved rate of cross circulation which could have been produced by the scapular supportive suture described above. If soft tissue sutures are physically Nathan William Shier stressed, healing will be delayed and cross circulation impaired. With increased rates of cross circulation, improved hormonal responses should be evident in parabiosis especially with hormones or metabolites quickly cleared from the circulation after crossing from one parabiont to the other. The primary causes of death in parabiotic animals are rejection (32.88%), infection (13.25%) and pair separation (8.22%) out of a total sample size of 292 pairs. Graft success was greatly improved by using female-female pairs as opposed to males (42.80% vs. 30.h0% respectively). Rejection was lowest in littermates as opposed to non-littermates. No diagnosed cases of rejection were found in Osborne- Mendel female littermates. Parabiotic rats do not appear different, physiologically or anatomically, as compared to sham operated single animals except for a shorter nose to anus length, decreased body fat content and reduced body weight. Some male parabiotic animals did have reduced adrenal vitamin C content indicative of "stress" which may alter experimental variables making it advisable to index "stress" in all parabiotic experiments. When both animals in a pair are fed a grain diet and compared to single sham operated controls on the same diet, increased body weight gain (though not statistically significant) and food efficiency but decreased body fat as a percentage of body weight is evident in the parabionts. All fat depots removed from parabiotic animals fed a grain ration (high carbohydrate diet) were smaller than single grain fed controls. Nathan William Shier In parabiotic pairs where both rats were fed a high fat diet, compared to single high fat fed controls.parabionts ate fewer calories, were less efficient in converting food to body tissue, had markedly less body fat with lower fat depot weights, and had a lower rate of body weight gain. Parabionts seem more capable of "handling" increased fat in their diet without weight gain than single animals. In pairs where one animal was fed high fat while its partner was consuming grain, animals fed high fat did not become obese, whereas, the grain fed animals lost weight. The animals fed high fat diets exhibited a similar caloric hyperphagia compared to single animals fed high fat (actually, consuming significantly more kilocalories than either single or parabiotic high fat fed controls) but, in contrast, were markedly less efficient than either control group. Compared to parabiotic high fat fed controls, high fat fed animals cross circu- lating with grain fed partners had lower body fat but the difference was not significant. All fat depot weights in high fat fed parabionts cross circulating with grain fed animals were statistically lower than weights recorded for single high fat fed animals. All depot weights for the high fat fed parabionts cross circulating with grain fed partners were also lower than all depot weights for control, high fat fed parabionts but no differences were significant. Body weight gain in high fat fed parabionts cross circulating with grain fed rats was depressed significantly as compared to single high fat fed animals as well as control high fat fed parabionts. Grain fed parabionts, cross circulating with animals fed high fat, L, Nathan William Shier became thinner than parabionts cross circulating with a grain fed partner. Food intake was markedly decreased in these animals as was food efficiency compared to either single or parabiotic grain controls. Body fat, body weight gain and all fat depot weights in grain fed animals cross circulating with high fat fed animals were all markedly depressed when compared to single grain fed controls. When these same values were compared to parabiotic control grain fed rats, there were no significant differences but all values for the grain fed rats cross circulating with the high fat fed were lower. Possibly, circulatory "factors" crossing over from the high fat fed animal are depressing food intake in the grain fed animal to levels below those in parabionts where both are fed grain. Since methoxyflurane inhalation anesthetic was used in some experiments in which heart blood was taken for insulin assay, it became necessary to determine the effects of this anesthetic on serum free fatty acids, serum glucose, adrenal weight and adrenal ascorbic acid. All of the above parameters were statistically the same between animals treated with methoxyflurane and those that were not. Male parabionts were differentially fed as previously reported for female animals. Adrenal ascorbic acid concentration was similar for parabionts fed in the differential feeder as for single control animals; consequently, stress was not considered a major complicating factor in food intake responses. Food intake of grain fed male parabionts cross circulating with high fat fed partners was markedly depressed as reported for female rats. 5 Nathan William Shier These grain fed parabionts cross circulating with high fat fed animals exhibited normal post-prandial blood levels of urea, glucose, free fatty acids and insulin even with the suppressed food intake. Possibly an anorexigenic factor (glucose, free fatty acids, or a substance liberated from the gastro-intestinal tract in response to the presence of fat) was crossing over from the high fat fed animal to the grain fed rat and producing a suppression of feed intake. A certain post- prandial blood metabolic "pattern" may serve to inhibit intake. The grain fed rat in the differentially fed pair established this pattern by receiving metabolites from its high fat fed partner rather than from its own diet; consequently, when the pattern was established, in- take was suppressei. Appetite suppressive factors may be involved, decreasing the feed intake of the grain fed rats parabiosed to high fat fed rats below the intake levels for either single or parabiotic grain fed controls, that are not calorigenic. This assumption is made on the fact that if the compound "crossing over" provided calories and reduced caloric intake on a one to one basis, body weight and body fat content should have been the same as in the grain-grain fed parabionts. THE USE OF PARABIOSIS IN THE STUDY OF FOOD INTAKE REGULATION AND BODY FAT CONTENT By Nathan William Shier A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1975 (:> Copyright by NATHAN WILLIAM SHIER 19 75 ACKNOWLEDGMENTS The author wishes to acknowledge the guidance and helpful suggestions of Dr. Olaf Mickelsen throughout. his advanced graduate studies. A word of "thanks" to Dr. Dena Cederquist, who allowed me to undertake these studies; to Dr. Modesto Yang, Dr. Rachel Schemmel and Dr. Robert Schirmer for their laboratory and clinical guidance. A special acknowledgment to my wife, Patricia Jean Donahue Shier, who worked many hours in the preparation of the dissertation. The author appreciates the financial assistance from the National Aerospace Agency (NASA), the National Institutes of Health and a Human Ecology Memorial Fellowship from Michigan State University. ii TABLE OF CONTENTS Introduction Review of literature Part 1, A method for determining growth rates of individual Part Part Part Part Part rats in parabiosis Introduction Techniques Discussion An ad lib. differential feeder for parabiotic rats Introduction Techniques Results Discussion Parabiotic surgical procedures Introduction Techniques Results Discussion Dye dilution studies Introduction Technique Results Discussion Causes of death in parabiotic rats Introduction Techniques Results Discussion Various physiological and anatomical values for "normal" parabiotic rats Introduction Techniques Results Discussion iii Page 41 41 52 53 55 56 76 77 80 80 83 89 90 100 101 102 106 Part 7, Energy metabolism Introduction Techniques Results Discussion Part 8, Parabiotic rats differentially fed grain and high fat rations Introduction Techniques Results Discussion Part 9, Effects of methoxyflurane anesthetic on various biochemical parameters Introduction Techniques Results Discussion Part 10, Metabolic consequences of ad lib. differentially feeding high fat and grain rations to parabiotic rats Introduction Techniques Results Discussion Bibliography iv Page 124 124 129 130 150 150 155 173 239 240 241 241 248 254 2 56 268 299 LIST OF TABLES TABLE l 10 11 12 13 Percent error between actual and calculated weights of individual parabionts Food consumption in grams (average/day) of parabiotic rats in ad lib. differential feeder design 3 compared to an average control value for rats permitted to eat from a cup in the floor of the cage. Weight gains in grams/day for parabiotic rats in differential feeder design 3 compared to control values for rats permitted to eat from a cup in the floor of the cage. - Ad lib. feed consumption of parabiotic rats in feeder design 3 vs. control cage (total for 8 days) Pair weight gains in ad lib. differential feeder design 3 vs. control cage (total for 8 days) Cr 0 in feces of rats fed non-labeled high fat diet in feeder design 3 (all values on dry weight basis) Parabiotic M-l food intakes in differential feeder us. controls (average / animal / day for 19 days) - Sprague-Dawley female rats Parabiotic plasma exchange rate as determined by dye T-l82N injected into Osborne-Mendel female rats Causes of death for various groups of parabiotic rats Nost to anus length (cm) - 200 day old Osborne-Mendel female parabiotic rats fed either grain or high fat ration Nose to anus length (cm) Comparison of fat depot weights (grams) between single and parabiotic Osborne-Mendel female rats fed a grain ration Comparison of fat depot weights (grams/100 grams body weight) between single and parabiotic Osborne-Mendel female rats on grain (M-l) ration V PAGE 47 59 60 61 62 63 92 99 113 114 115 116 TABLE 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Comparison of organ weights (grams) between single and parabiotic Osborne-Mendel female rats on grain diet Comparison of organ weights g / 100 g body weight between single and parabiotic Osborne—Mendel female rats Adrenal weights (mg) - Osborne-Mendel female rats fed a grain (M-l) ration Thyroid weights (mg) - Osborne-Mendel female rats fed a grain (M-l) ration Blood volume (ml) in single and parabiotic Osborne-Men- del female rats fed grain ration Packed cell volume in single and parabiotic Osborne-Men- del female rats fed grain ration Energy metabolism: Parabiotic us. Control - adult Sprague-Dawley male rats Adrenal weight (mg) - Sprague-Dawley male rats Adrenal weight mg / 100 g body weight - Sprague-Dawley male rats Thyroid weights (mg) — Sprague-Dawley male rats Testicular weights (g) - Sprague-Dawley male rats Thyroid hormone iodide (conc. ug %) - Sprague-Dawley male rats Glucose in assay medium (mg %) - Sprague-Dawley male rats Insulin in plasma (micro-units / ml) — Sprague-Dawley male rats Serum free fatty acids (ueq. / ml) - Sprague-Dawley male rats Vitamin C (ug) / mg adrenal weight - Sprague-Dawley male rats Q Urea nitrogen in serum (conc. mg %) - Sprague-Dawley male rats Use of differential feeder - Sprague-Dawley female rats vi PAGE 117 118 119 120 121 122 139 140 141 142 143 144 145 146 147 148 149 181 TABLE 32 33 35 36 37 38 39 4O 41 42 43 45 46 Food intakes in grams (aberage / animal / day) - of Sprague-Dawley female rats kept in a cage containing a differential feeder; control period; M-l diet Food intakes in grams (average / animal / day) - Sprague- Dawley female rats, experimental period Food intakes in grams (average / animal / day) - Sprague- Dawley female rats, control period vs. experimental period Food intakes in grams / 100 grams body weight (average / animal / day) - Sprague-Dawley female rats, control period; M-l diet Food intakes in grams / 100 grams body weight (average / animal / day) - Sprague-Dawley female rats, experimental period Food intakes in grams / 100 grams body weight (average / animal / day) - Sprague-Dawley female rats, control period vs. experimental period Kcal. intakes (average / animal / day) - Sprague-Dawley female rats, control period: M-l diet Kcal. intakes (average / animal / day) - Sprague-Dawley female rats, experimental period Kcal. intakes (average / animal / day) - Sprague-Dawley female rats, control period vs. experimental period Kcal. intakes / 100 grams body weight (average / animal / day) - Sprague-Dawley female rats, control period: M-l diet Energy intakes Kcal / 100 grams body weight (average / animal / day) - Sprague-Dawley female rats: Experimental period Food efficiency (Kcal. / g body weight gain) — Sprague- Dawley female rats, contrdlperiod: M-l diet Food efficiency (Kcal. / g body weight gain) - Sprague- Dawley female rats: Experimental period Food efficiency (Kcal. / g body weight gain) - Sprague- Dawley female rats, control period vs. eXperimental period Tbtal body weight changes per animal (g) during experiment- al period - Sprague-Dawley female rats vii PAGE 182 183 184 185 186 187 188 189 190 191 192 193 19a 195 196 TABLE 47 Body fat as percentage of body weight - Sprague-Dawley female rats 48 49 50 Inguinal fat depot weight (g), non-operated side only Sprague-Dawley female rats Right and left combined inguinal fat depot weight (g) Sprague-Dawley female rats Inguinal fat depot weight (g) - Sprague-Dawley female rats 51 52 53 55 56 57 58 59 6O 61 62 63 65 Right and left combined renal-retroperitoneal fat depot weight (g) Sprague-Dawley female rats Right and left combined perimetrial fat depot weight (g) Sprague-Dawley female rats Total weight of fat depots removed (g) - Sprague—Dawley female rats Empty stomach weight (g) - Sprague-Dawley female rats Empty stomach weight in grams per 100 g body weight - Sprague-Dawley female rats Empty gastro-intestinal tract weight (g) - Sprague- Dawley female rats Empty gastro-intestinal tract weight in grams per 100 g body weight, Sprague-Dawley female rats Full cecum weight (g) - Sprague-Dawley female rats Full cecum weight in grams per 100 g body weight - Sprague-Dawley female rats Adrenal weight (mg) - Sprague-Dawley male rats Adrenal weight / 100 g body weight (mg) - Sprague- Dawley male rats Vitamin C (ug) / mg adrenal weight - Sprague-Dawley male rats Serum free fatty acids (ueq. / m1) - Sprague-Dawley male rats Glucose in assay medium (conc. mg %) - Sprague-Dawley male rats Thyroid weight (mg) - Sprague-Dawley male rats viii PAGE 197 198 199 200 201 202 203 204 205 206 207 208 209 243 244 245 246 247 271 TABLE 66 67 68 69 7O 71 72 73 7b, 75 76 77 78 79 80 81 82 83 Thyroid weight (mg) / 100 g body weight - Sprague- Dawley male rats Thyroid weights (mg) - Sprague-Dawley male rats Adrenal weight (mg) - Sprague-Dawley male rats Adrenal weight / 100 g body weight (mg) - Sprague- Dawley male rats Adrenal weight (mg) - Sprague-Dawley male rats Adrenal weight / 100 g body weight (mg) - Sprague- Dawley male rats Vitamin C (ug) / mg adrenal weight - Sprague-Dawley male rats Vitamin C (ug) / mg adrenal weight - Sprague-Dawley male rats Right and left combined testicular weight (g) - Sprague- Dawley male rats Right and left combined testicular weight (g) / 100 g body weight, Sprague-Dawley male rats Serum free fatty acids (ueq/ml) - Sprague-Dawley male rats Serum free fatty acids (ueq/ml) - Sprague-Dawley male rats fed a grain (M-l) ration Urea nitrogen in serum (cone mg %) - Sprague-Dawley male rats Urea nitrogen in serum (conc. mg %) - Sprague-Dawley male rats Insulin in plasma (micro-units / ml) male rats Sprague-Dawley Insulin in plasma (micro—units / ml) male rats Sprague-Dawley Glucose in assay medium (conc. mg %) male rats Sprague-Dawley Glucose in assay medium (conc. mg %) - Sprague-Dawley male rats Glucose in assay medium (conc. mg %) male rats Sprague-Dawley ix PAGE 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 LIST OF FIGURES FIGURE PAGE 1 Two parabionts being weighed on modified dietetic scales as described in text 48 2 Each point in curves (X) and (0) represents the weight difference between two attached weights or parabiotic rats plotted as a function of differences in scale readings 50 3 Cr203 in feces of rats fed high fat diet; other rat in each pair fed the same ration with 2% Cr203. 11 collection days 65 4 Photographs of ad lib. differential feeder 67 5 Drawing of the body divider of the ad lib. differential feeder. A) Side view; B) Top and C) End projections. Scale of 1:0.7. Material: Galvanized sheet metal one millimeter thick. All approximating free metal edges were secured with solder joints 68 6 Drawing of the front piece of the ad lib. differential feeder. A) Front view; B) Top and C) End projections. Scale of 1:0.7. Material: Galvanized sheet metal one millimeter thick 69 7 Drawing of a side panel of the ad lib. differential feeder. A) Side view; B) Top and C) End projections. Scale of 1:0.7. Material: Galvanized sheet metal one millimeter thick 70 8 Flat drawings of the top piece (A) and bottom platform (B) of ad lib. differential feeder. Scale of 1:0.7. Material Galvanized sheet metal one millimeter thick. The pieces are bent, at the dotted lines, in the forms shown on Figures 4A and 8A 71 8a The top piece (A) and bottom platform (B) bent into proper shape. Scale of 1:1. Material: Galvanized sheet metal one millimeter thick. Angles shown are approximate and the pieces may have to be adjusted slightly to the positions shown in Figures 4A and B. Flaps 1 and 2 are placed through the head holes and soldered to the back of the front piece as shown in Figure 4A 72 X FIGURE 10 11 12 13 14. 15 16 17 18 19 2o 21 22 23 24 25 Drawing of food cup for ad lib. differential feeder. A) Side view, B) Top and C) Front projections. Scale of 1:0.7. Material: Galvanized sheet metal one millimeter thick. A11 grain food cups were soldered at the corners but high fat cups were not Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Modified surgical procedure for parabiosis Evans Blue dye dilution curves for control and parabiotic female Sprague-Dawley rats Body weight curves for control and parabiotic female Osborne-Mendel rats. (Av. grams/Animal/Day) Food intake of control and parabiotic female Sprague- Dawley rats. (Av./Animal/Day) Food intake per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intake per 100 grams body weight of control female Sprague-Dawley rats. (Av./Anima1/Day) Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of single and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intake per 100 grams body weight of control and parabiotic female Sprague—Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) xi PAGE 73 74 82 93 123 210 211 212 213 214 215 216 217 218 219 220 221 FIGURE 26 27 28 29 3o 31 32 33 35 36 37 38 39 40 41 42 Food intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Kcal intakes of control and parabiotic female Sprague- Dawley rats. (Av./Animal/Day) Kcal intakes of control and parabiotic female Sprague- Dawley rats. (Av./Animal/Day) Kcal intakes of control female Sprague-Dawley rats. (Av./Animal/Day) Kcal intakes of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Kcal intakes of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Kcal intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Anima1/Day) Kcal intakes per 100 grams body weight of control and parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Kcal intakes of control female Sprague-Dawley rats per 100 grams body weight. (Av./Anima1/Day) Kcal intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Anima1/Day) Kcal intakes per 100 grams body weight of parabiotic female Sprague-Dawley rats. (Av./Animal/Day) Body weights of control and parabiotic female Sprague- Dawley rats. (Av. grams/Animal/Day) Body weight gains of control and parabiotic female Sprague-Dawley rats. (Av. grams/Animal/Day) xii PAGE 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 FIGURE 43 45 46 47 48 49 50 Food intakes of control and parabiotic male Sprague— Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic male Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control male Sprague-Dawley rats. (Av./Animal/Day) Food intake per 100 grams body weight of control and parabiotic male Sprague-Dawley rats. (Av./Anima1/Day) Food intakes per 100 grams body weight of control and parabiotic male Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic male Sprague-Dawley rats. (Av./Animal/Day) Food intakes per 100 grams body weight of control and parabiotic male Sprague-Dawley rats. (Av./Animal/Day) Food intake per 100 grams body weight of parabiotic male Sprague-Dawley rats. (Av./Animal/Day) xiii PAGE 291 292 293 294 295 296 297 298 INTRODUCTION Parabiosis, the union of two living organisms (302), has been used extensively for over 100 years in the study of humoral factors. Parabiotic rats can be described as a whole body tissue graft with blood exchanged between the members of a pair (304). An experimental variable is imposed on one member of a pair with adaptative responses measured in its partner. If changes do occur, it can be concluded that blood factors were important in the response. Parabiosis has not been used widely in the nutritional sciences since Obtaining separate weights of members of a pair, without separation, and feeding parabionts separate diets offered prdblems. With the development of the above procedures in addition to improved surgical techniques and with a greater knowledge as to how the parabiotic state, per se, affects physiology and anatomy of rats, it became possible to use parabiotic rats, effectively, in the study of the regulation of food intake and body fat content. There is control over the ingestion of foodstuffs and the study of food intake regulation is an extremely complex and multi-faceted problem. In order to function efficiently as a physiological control mechanism, control of food intake must have many interwoven systems. One can pose the question: Does food intake determine body energy balance or does body energy balance determine food intake? Food intake is just one system of many employed by the organism in maintaining energy homeostasis. Actually, body weight and fatness are controlled far more precisely than food intake (1). This means that the organism makes physiological adjustments to compensate for a surfeit or deficit of calorie intake as a result of the less efficient regulation of food intake. The whole system can be viewed in terms of coarse and fine adjust- ment. What is acutally regulated or the goal of regulation in the adult animal is to maintain body energy constant. Food intake brings into the body energy with a somewhat independent and coarse control. This energy is then regulated by the body with a fine adjustment in the most efficient manner considering the physiological and environ- mental conditions under which the animal is living. Food intake has an independent component of regulation apart from body weight regulation and vice versa but the two must be interrelated. When one considers any control system, a natural categorization can be made: the environment and the organism. Factors in the environ- ment can greatly affect an organism's energy balance. Availability of food, nature of the food, condition of the animal, e.g., a broken leg, air temperature (2), wind velocity (2), humidity (2) (A vapor pressure gradient is necessary for the animal to cool evaporatively 2 3 by panting or eccrine sweat gland activity.) solar radiation (2), light cycles (2), altitude (3), water availability (4,5), alterations in the food chain and levels of certain environmental elements or pollutants such as fluorides (6-8) and tannins (9) can greatly alter an animal's energy balance and food intake. The organism, whether plant or animal, compensates by a variety of mechanisms. The external morphology can be modified or the organism can move or evoke many methods of energy acquisition or dissipation. For the present discussion it will be assumed that the organism is animal and is living in a rather stable environment to which it has acclimatized. Discussion will be focused on what actually initiates or terminates the ingestion of food as one major component of energy balance. In terms of energy balance, one can break down the animal kingdom into poikilotherm or homeotherm. Poikilothermic food intake is affected by sex, age and season of the year as is homeothermic intake but, within limits, poikilothermic food intake varies directly with the environmental temperature unlike that seen in warm blooded animals (10). This is probably a result of the direct relationship between "deep core body temperature" of the poikilotherm and environmental tempera- ture. Warm blooded animals can be classified, in terms of food intake regulation, as ruminant or monogastric. Food intake regulation is considerably different between the ruminant and monogastric because of the importance of the "volatile fatty acids" and the relatively non-involvement of glucose. In lactating ewes, blood butyrate is negatively correlated and blood free fatty acid levels, positively correlated to subsequent food intake 3 by panting or eccrine sweat gland activity.) solar radiation (2), light cycles (2), altitude (3), water availability (4,5), alterations in the food chain and levels of certain environmental elements or pollutants such as fluorides (6-8) and tannins (9) can greatly alter an animal's energy balance and food intake. The organism, whether plant or animal, compensates by a variety of mechanisms. The external morphology can be modified or the organism can move or evoke many methods of energy acquisition or dissipation. For the present discussion it will be assumed that the organism is animal and is living in a rather stable environment to which it has acclimatized. Discussion will be focused on what actually initiates or terminates the ingestion of food as one major component of energy balance. In terms of energy balance, one can break down the animal kingdom into poikilotherm or homeotherm. Poikilothermic food intake is affected by sex, age and season of the year as is homeothermic intake but, within limits, poikilothermic food intake varies directly with the environmental temperature unlike that seen in warm blooded animals (10). This is probably a result of the direct relationship between "deep core body temperature" of the poikilotherm and environmental tempera- ture. Warm blooded animals can be classified, in terms of food intake regulation, as ruminant or monogastric. Food intake regulation is considerably different between the ruminant and monogastric because of the importance of the "volatile fatty acids" and the relatively non-involvement of glucose. In lactating ewes, blood butyrate is negatively correlated and blood free fatty acid levels, positively correlated to subsequent food intake 4 during the first half hour of a three hour feeding period. The lower blood butyrate levels, the higher intake and the higher free fatty acid levels, the higher intake. Blood glucose was not correlated to intake (11). Intake has also been decreased by acetic acid or acetate intravenous infusion in cows (12). Glucosensitive and acetate sensi- tive areas have not been found in the ruminant brain (13) and hypo- thalamic temperatures have not been correlated to intake (14). Cortisol acetate, given intramuscularly at 25 mg/day, increased food intake in sheep and increased blood glucose, while total glucose utilization was not reduced. There was an impairment of glucose utilization relative to blood glucose (15). There should have been a greater blood glucose utilization as a result of the absolute blood glucose level. In warm blooded, monogastric animals most work has concentrated on the human, dog, cat, monkey, rat and mouse. There obviously are enormous species variations in size, surface covering, basal metabolism, anatomy, physiology, genetics, etc., but much valuable information can be obtained on animals other than man using experimental techniques that cannot be performed on humans. Large variations in food intake also exist between male and female and the physiological state of the organism, i.e., age, pregnancy, lactation and work load. In humans, there is also the strong esthetic, psychological drive associated with food (16). Human subjects must be used in studying the psychological compo- nent to the regulation of food intake. The counterparts of these investigations in animals are classified as behavioral or motivational. Using human subjects, it would be possible to answer such questions as 5 these: Does a primary disorder in hunger cause abnormalities in food intake? or Can disorders in food intake exist with normal sensations of hunger? (19) The overweight person does not recognize a point of satiety or even displeasure in eating as readily as a lean individual. "Full” and ”not hungry" have different meanings to the overweight (17). Oralpharyngeal sensations in humans as well as animals seem to be potent regulators of behavior related to food ingestion (18). One can measure behavior or motivation in animals but it is hard to determine if this represents a change in hunger sensation. In man it may be difficult to determine if basic organic illness creates psychological distress or if psychological illness is primary to an impairment in food intake regulation. In some societies purely psychological factors such as a fear of becoming fat can cause individ- uals to greatly curtail intake when there is actually no need to do so. Social pressures as to how a society measures beauty create strong motivational stimuli to behavior (20). Psychological misper- ceptions of hunger can also develop in the youngster resulting from "misperception" by the mother. The mother tries to abate many emotional needs in the child by offering food when the emotionalism is not associated with food. The food, though, does help the situation and, therefore, the conditioned child turns to food for future emotional needs (21). A major component of the food intake regulation in any species of animal, e.g., the rat, involves the general behavior that the animal displays towards food. The total amount of food an animal consumes per 24 hours is important but equally as important are the amounts of food eaten per "meal" and the frequency of meals which establishes a 6 recognizable feeding behavioral pattern. An experimental manipulation may produce a decrease in food intake but, if another variable is also altered, it is not clear if the second alteration is a result of food intake depression or a primary effect of the experimental variable. To answer this question a second group of animals are fed the reduced intake (pair feeding) without the experi- mental variable. If the second alteration still exists then the effect is said to be a primary result of a decrease in food intake. This type of conclusion can be misleading because even though the amount of food was pair fed, the pattern of consumption may still be quite different. Correcting for the pattern of intake, as well as the amount, gave strikingly different results when rats were fed diets containing high levels of fluoride compared to correction for amount only (7). Techniques are available allowing accurate monitoring of the pattern of intake as well as offering food at preferred levels and frequencies (22, 23). Spillage is a problem in these devices. Animals may visit the food cup to play or spill the food. If this happens, the animal is usually omitted from the data. Even though food is offered at a particular frequency one can still not be sure that the animal will eat all of the food that is offered at a certain time in a distinct pattern. Food is consumed with irregular intervals, but nutrients are probably more uniformly available to the organism since the gut serves as a food reservoir. Food ingestion in rats is about 50% higher at night due to larger meals with the frequency of meals being the same for day and night. Frequency seems to be highly fixed with small variations in 24 hour intakes caused by variation in meal size. 7 Changes in intake when the palatability of the diet is altered are once again alterations in meal size and not frequency. The number of meals per day will vary greatly between species and between individuals within a species. For rats, the average is around 8.5 meals per day. Amounts eaten in a meal vary considerably from meal to meal (24). When the caloric density of the diet is altered, frequency is changed for about seven days and then the sustained increase or de- crease of intake will be achieved in meal size with frequency con- stant (24). Food intake regulation, therefore, may involve a regulation of meal size as well as meal frequency all intricately involved in the maintenance of body energy. The size of a meal isruytcorrelated to the time elapsed preceding the meal since the last intake but is correlated to the time subsequent to the meal until eating is initiated again. This type of eating behavior has been called "provisional appetite." The animal eats to satisfy its subsequent energy demand (24). It can be reasoned that if one meal size affects the size of a subsequent meal, a very precarious balance would exist. Once meal size has been established and, if meal size controls subsequent meals, then to change meal size in order to regulate calorie intake would be very difficult. Meal size correlated to subsequent time lapse is a far more labile system. Physiological and anatomical factors regulating intake have received perhaps the bulk of experimental effort. The surface area, shape and surface covering and the ability to change these parameters are all important in energy balance (2). 8 As would be expected, food intake is affected by a previous food deprivation. Cicala and Bare, (25) found that deprivation up to 24 hours failed to increase total consumption during a subsequent 24 hour feeding period although during the first hour of refeeding, intake increased as the length of deprivation increased. The lack of en- hanced intake during the 24 hour feeding period was attributed to the dominance of the day, night feeding cycle. Le Magnen and Tallon, (26) obtained similar results except that after 48 hours of deprivation an increase in consumption occurred for approximately 3 days. The influences of deprivation on food intake are greatly modified by the absence or presence of water (27). When rats are kept under constant illumination and tested with a 3 hour feeding schedule, intake increases up to 16 hours of deprivation and then is constant even up to 96 hours (28). With human, obese patients an increase in the "satiety response" was demonstrated after fasting. These patients did not feel hungry or experience an increased appetite during fasting and were satisfied with significantly smaller amounts of food when fed after the fast (29). Food intake can be altered by changing the caloric density of the diet (24). These investigations produce convincing evidence that amimals do regulate their food intake in response to calories. Since changes in intake are not produced immediately, one must con- sider changes in body energy as a component in the regulatory response. The one major labile energy source of the body is the adipose tissue. The first major study showing compensatory increase in diet intake with diet dilution was that of Adolph, (30). A major complicating factor in diluted diets is the capacity of the gastrointestinal tract. 9 This may ultimately determine the upper limit of intake or dilution (31). An additional review concerning the gastrointestinal tract will be forth coming in this review (page 18). A change in weight of food consumed in the rat may take 24 hours or longer (usually longer) in response to change in calorie density (32) and may take weeks in the dog (33). When rats are fed a high fat diet, several days are required before calorie intake approximates the previous low calorie baseline. All of these factors indicate that the oral ingestion of food is not that sensitive to changes in diet caloric density but rather a change is enacted when some physiological control mechanism within the organism is activated. Irrespective of calories, rats also respond to a dilution of only the protein fraction of the diet (34). In this manner animals can be made to consume an excess of calories. Most evidence indicates that low protein diets suppress intakes. It can be concluded that oral ingestion is controlled for meal size as well as frequency and that there are long range control mechanisms in response to a surfeit or deficit of body energy stores. This regulation involves either the nervous or endocrine systems. Several brain areas have been studied as components in the reflex are involved in the control offood intake. The dorsal dentate- hippocampal connections and the hippocampus are implicated in appeti- tive conditioning. Dentate units augment to a conditioned stimulus resulting in food but responds by inhibition if the reward.is electric shock (35). Preoptic lesions do not affect food intake even when the animal's core temperature is elevated (36). Septal lesions in rats increased not only food intake but also a non-reinforced visual discrimination 10 task which indicates an increased or enhanced behavioral motivation towards food (37). Bilateral amygdaloid lesions decreased such a response whether reinforced or not (37, 38). In cats amygdalar inhibitory areas (the basal parvocellular nucleus) and facilitatory areas (the anteromedial area) on food intake have been identified (39). It would appear that the facilitatory action is dominant since bi- lateral lesions of the amygdala results in a net inhibition of intake whereas in dogs bilateral lesions of just the dorsomedial area produced aphagia and a decreased motivation (40). In monkeys lesions in the midbrain reticular formation produce hyperphagia but caudal medullary lesions produce hypophagia (41). The entire limbic—midbrain circuit, reticular formation and hypothalamus seem to be integrally involved in the control of food intake (42-45). From the above examples one can conclude that there are several brain areas, extra-hypothalamic, when stimulated either cause an augmentation or inhibition of food intake. The brain circuitry involved in food intake regulation is very extensive reaching from the "primitive" brain stem to the cerebral cortex. The hypothalamus is only a small part of this system and the importance of the hypothalamic areas as initiators of food intake or satiety is extremely doubtful. Probably the two brain areas receivingthe mostnattention in food intake regulation are the lateral and ventromedial areas of the hypo- thalamus. These areas were the first discovered to be implicated in the control of food intake and, unfortunately, perhaps for this reason have remained in the foreground in neurological investigation. Many nerve fiber systems run through the lateral hypothalamic areas that are associated with such diverse functions as rage, fear, 11 sexual manifestation, sleep, feeding and drinking (45). When rats are lesioned in this area an aphagia and adipsia results (46). Aphagic animals, though, continued to press a lever for food which they could not eat and when the food was infected intragastrically, through chronically implanted canulas, rats fed themselves by lever pressing and regulated intake well. The rats did not eat by mouth due to a "motor" failure but the sensation or drive for food was intact. Deprivation was sensed and alleviated (47, 48). Recovery from motor failure takes far longer when laterally lesioned animals are fed intragastrically (18). Impairment of conditioned reflexes have also been reported after lateral lesions with an aversion towards foods containing large amounts of water (49). Many papers have described the voracious appetite of animals lesioned in the ventromedial nuclear area. Food intake is doubled over normal controls and gradually decreases back to normal. The syndrome is termed "dynamic" during increased food intake and weight gain and "static" when weight stabilizes and intake has returned to normal (50—52). Young, immature rats, even after starvation, do not show this syndrome after ventromedial lesions, but somatic growth may be impaired with the production of obesity (53, 54) associated with low pituitary and plasma growth hormone (55). Lactating rats also do not show hyperphagia after ventromedial lesions (53). Adult, lesioned rats will respond to nutrient dilution even faster than normal young rats during the "dynamic" phase but this type of response to caloric dilution is lost in the static phase. Older, "normal" rats with excess fat respond similarly to nutrient dilution as static, hypothalamic rats (52). Ventromedially lesioned rats also 12 display a hypokinesis and a decreased basal energy production (51). The primary cause of the obesity is usually considered to be increased food intake, although, some authors believe it to be the hypokinesis (56). Sometimes in ventromedially lesioned rats the intake is not in- creased or if hyperphagia does exist the more palatable diets are consumed more readily (57). Lesioned animals, pair fed to controls, still gain more weight but the increase is slight. Their basal oxygen consumption is lower with no change in fasting respiratory quotient, but display a respiratory quotient above controls post-absorptive. Creatinine excretion is 30% higher. There is a decreased insulin sensitivity’and, when body weight reaches 550 grams or higher, an abnormal estrus may occur. If these animals are fasted to reduce weight or are pair fed, the estrusis normal (58). Hypothalamic rats will still gain weight even when intake is decreasing (59) and display normal fasting gastric hunger contractions (60). Ad libitum food intake patterns are the same in hypothalamic animals as controls in that the meal size is related to the interval after the meal and intragastric infusion decreases oral intake and increases the post- prandial interval (61). In hypothalamic rats body calcium, phosphorus and iron are somewhat depleted (62) and these rats are more responsive to diet palatability (52, 63). In hypothalamic monkeys there is a marked hyperglycemia and glycosuria (64, 65) and an impairment in temperature regulation to moderate cold with obesity occurring, on occasion, without hyperphagia (66). Obesity has been produced in birds by ventromedial lesions but the fat accumulation was not as pronounced as in mammals (67) and aphagia has been produced by bilateral lesions in the lateral 13 hypothalamic areas (68). The ventromedial area does seem to have an inhibitory effect on the lateral areas as shown by electrophysiological studies (69) . Anatomical connections between these two brain areas were demonstrated by Arees and Mayer, (70). The ventromedial area also has functional connections with the amygdalar complex (71). Several studies have cast a doubt on the primary importance of these hypothalamic areas in controlling food intake from a motivational or behavioral approach. Rats after recovery from ventromedial or lateral lesions still are able to regulate ingestive behavior and control body weight (72). It must be remembered, though, that the body weight may be at a different level after the lesion than before. Rats lesioned in the ventromedial hypothalamus still responded normally to intragastric loads by decreasing oral intake (73) and during the "dynamic" phase rats fed themselves intragastically by lever pressing and controlled food intake and body weight very well (18). One may also question the fact that lesioned obese animals do not represent, functionally, the type of obesity that occurs naturally. Zucker genetically obese rats were compared to lesioned animals for similarity of various parameters. Both types of animals exhibit hyperphagia (74) but lesioned animals in the "static" phase do not respond to caloric dilution by increasing intake (75), whereas, Zucker rats do with a 20% cellulose dilution but compensate only partially for a 50% dilution (74). Lesioned animals do not decrease intake on a high fat diet (76) but the Zucker rats reSpond by compensating at 8% fat but not at 60% (74). Zucker rats respond differently to cold by not augmenting circulating free fatty acids, by not increasing the 14 release of radioactivity from the thyroid and by not regulating body temperature (77) as do lesioned rats and lean controls. However, ventromedially lesioned monkeys have difficulty in maintaining body temperature in response to cold (66). d—amphetamine produces a smaller depression in food intake in Zucker rats than in either lesioned rats or normal animals (74). Zucker rats consume more water than lean or lesioned rats (78). Estrus seems to be abnormal in the Zucker rats (74) as has been shown for lesioned animals (58). One can conclude that there are many similarities but also some diff- erences and it seems reasonable to say that the genetic Zucker rats may have some impairment of the ventromedial area of the hypothalamus. In interpreting lesion experiments the sex (79) as well as the age (80) of the animal are very important. Electrical recordings from the brain are also helpful in identi- fying or defining satiety or deprivation. Deprivation is character- ized, electrically, as an EEG of low voltage and high frequency whereas the fed state is characterized by low frequency, high amplitude. Electrical activity in the brain, therefore, is correlated to appetitive drives (81). When the brain is either stimulated or lesioned, various hormone titers are altered that could effect changes in appetite or food intake. Certain hypothalamic areas in fasted cats when stimulated cause a marked elevation or depression of plasma free fatty acids and, other areas, an elevated or depressed plasma glucose. Triglycer- ides and cholesterol were unaffected (82, 83). Plasma immunoreactive growth hormone levels were not affected by hypothalamic stimulation in conscious cats while cortisol levels were significantly enhanced (83). 15 Stimulation of the ventromedial hypothalamus of the rat produces a marked elevation of plasma glucose probably due to an increased hepatic glucose production, but plasma insulin levels stay at low levels even with the rise in glucose. The insulin inhibition of release was eliminated with adrenalectomy. Plasma glucagon levels rose with or without adrenalectomy. with antiglucagon serum inhibiting partially the hyperglycemic response (84). In the fasted rat lateral hypothalamic stimulation will not change free fatty acid or insulin levels but will slightly increase glucose. If the animal is fed glucose the insulin levels increase with a drop in free fatty acids. In rats recovered from lateral lesions there is a marked increase in blood glucose and insulin after a meal with fasting free fatty acid levels 50% of normal. Ventromedial lesions produced an immediate elevation in blood glucose. In hyperphagic rats, serum insulin increased with eating and remained high (85). Cholinergic and adrenergic drugs modify food intake when injected into the third ventricle of the brain (86-88). Choinergic pathways produced mainly increased water intake whereas adrenergic compounds and Cholinergic drugs enhanced food intake (88). Glucochloralose stimulates food intake but galactochloralose does not (89). Other drugs producing hyperphagia are chromic acid, bipiperidyl mustard (90) and goldthioglucose (91-105). Perhaps the most widely studied hyperphagic drug is gold thio- glucose. Mice, rats and dogs all become hyperphagic and obese after injections of goldthioglucose (91, 92). The hyperphagia, as in ventromedially lesioned animals, usually subsides with time until 16 intake is normal, but in certain instances, female animals do not return completely back to the baseline (94). After fasting, the hyperphagia begins again (93). Earlier work concludes that the primary lesion is in the ventromedial nuclear area with the oligodendroglia cells taking up gold and being destroyed (95, 96). Exceptions are diabetic animals whose oligodendroglia are not destroyed with no subsequent hyperphagia or obesity (95, 97). Animals treated with goldthioglucose usually show no change in fasting blood glucose but glucose utilization is increased. There is an increased insulin sensitivity and increased gastric secretion in the Pavlov but not the Heidenhain pouch (91). Constant light produces changes in ad libitum food intake inde- pendent of goldthioglucose (98). Other manifestations are centro- lobular fatty liver (99), estrous malfunction (94), ulcerogenesis (101), augmentation of ACTH response to stress (101), increased tumorigenesis (102, 106), increased pancreatic insulin stores and secretory capacity in vitro in response to glucose or theophylline (107), while the diaphragm and adipose tissue respond normally to insulin (108). More C-14 carboxyl-labeled acetate is retained in goldthio- glucose treated animals at weight maintenance but when fasted the C-14 retention is the same as controls (109). Daily mobilization of fat is 50 to 60% of the control level (110). Epididymal and mesen- teric adipose tissue display normal in vitro glucose metabolism in the presence of insulin and growth hormone (111), and there is no increase in lipogenesis from acetate (112). Several recent investigations have disclaimed the conclusion that the primary lesion is in the ventromedial nucleus when goldthioglucose 17 is injected (99, 103, 104). The lesion may be secondary to vascular damage and edema around the hypothalamus (99). Goldthioglucose is known to cause damage not only to the ventromedial hypothalamus but also to the fornix, premammillary nuclei, arcuate nuclei, anterior hypothalamus, preoptic nuclei, vagohypoglosal complex, dorsal hippo- campus, hypocampal commissure and septal nuclei (105). It is still extremely interesting to note that other goldthiosugar compounds, e.g., malate and galactose do not cause brain damage, hyperphagia or obesity (97). There is also a dose response to goldthioglucose for food intake as well as obesity and one can reason from this evidence that there is some quantitation of neurons influencing food intake (99). During food ingestion cardiac output, heart rate and systolic blood pressure are all elevated and blood flow is directed to the vascular bed of the superior mesenteric artery. It has been postulated that this general sympathomimetic state marks the end of eating (113). The carotid reflex is also elevated (114). Another basic theory for food intake regulation is the "thermo- static" (115-125). The theory is based on the observation that rats when exposed to cooler environmental temperatures increased their food intake but at high temperatures intake was curtailed. Food intake is used as a means of temperature regulation. If the animals are able to dissipate more heat, more food is consumed (115). Skin temperatures of the thumb increase after a high protein meal in obese as well as non-obese subjects (117). In rats, brain temperature increases have been recorded in relation to feeding (118) and the magnitude of eleva- tion was related to the length of the meal (119). In other work no correlation between brain temperature and intake was observed in 18 ruminants. This may be a major difference between ruminant and mono— gastric animals (120). Laboratory monkeys do not increase food consumption in response to cold (122). Hypothalamic hyperphagic rats have colonic temperatures higher than controls that were not caused by increased food intake, obesity, or the failure of heat dissipation mechanisms. There is also an increase of body temperature in normal animals with short and sometimes long term fasting (125) Also, thyroxin will stimulate appetite but will increase body temperature and conversely thiouracil and thyroidectomy are hypothermic but appe- tite depressing (125). The gastrointestinal tract has been widely studied in relation to food intake. It can be broken down conveniently into the oralpharyngeal surface, the stomach and the small intestine. One major variable in the buccal cavity is the teeth. When molars are extracted from rats, body weight decreases as a result of a de- crease in food intake but the decrease is more than in pair fed controls (126). This indicates that the stimulation of the teeth during eating may be important to obtain greater digestibility which in turn ‘would affect future intake. The teeth are also very important in :rendering food in a size easily swallowed (136). Animals can regulate :food intake without the influence of oralpharyngeal input but in the rusrmal animal these mechanisms are utilized for control (126-135). The oralpharyngeal surfaces are a rich source of sensory receptors (127) and combined with taste and smell are important in discriminating txetween food objects which can affect intake by conditioning or emotion especially in the human (128, 129). Sensory cues seem to be dispensable (13C0 since rats can regulate intake by feeding themselves n—‘¥Al 19 intragastrically on liquid diets (128). Under ad libitum conditions rats seem to eat for calories, ignoring taste and sensory perception but when the rat is in an energy deficit it becomes very discriminating and taste becomes more of a factor in regulation. This same effect is displayed by hypothalamic hyperphagic rats. Rats, after a period of deprivation, will decrease intake on cellulose diluted diets but increase intake on high fat diets (128, 131). In an intact animal, though, there is little doubt as to the importance of sensory perception. Satiation is felt even before finishing a meal. When food touches the mouth or tongue there is a change in certain brain EEGs from fast, low voltage to slow, high voltage, indicative of satiety (132, 133). In the hungry animal many areas of the brain and hypothalamus exhibit fast, low voltage EEGs that are completely blocked by intravenous or intraarterial glucose injections. During eating, sensation from the sensory receptors inhibit the lateral areas of the hypothalamus as well as cortical and subcortical structures. A "sensory or primary satiation" has been defined as a fast regulation coming directly from a nervous source and a "secondary or metabolic" after the food has entered the blood. Thiel et al., (134) have pointed out that in interpreting food preference one has to consider the strain, sex and prior food ixperiences of the animal. 'This would be especially important in the human. Gastric receptors, stimulated by the presence of food or gastric (distension, do seem to be involved in food intake regulation. When c one .psmfioz soon Hospoo mo owopcoonom Hovmohm c we: meonm ca Honno meow on» .mapcoswomcoo “m oocmamp so ooomam hangar we: assess HoHHmsm one .mm.fi .m :sdaoo ea Haw Mom one mm.o mm: a sesaoo me upon Has How Moshe pcoo Mom some one * 47 a." 3 a: as E .5 E 2 a: a: . S E 2 3 as a; s” .3 2 Z a: E. s. s” 3 s... as s... E as 3 3 E a: E a: 3 S a. s. s. 2: 2 5 2 .2 z E Z 2 s. :— 3 2— a... a... a = s 2 m < m < mfim. 5mm totem .5051 ..>> oo.m_:o.mo Em_o>> .m:.o< .mpcoapmuom.amzofi>ao:a mo mvnwaoz oopmasoaso one Hooves coozpop HOHHo pcoo Mom * a canoe 48 g \ a l‘ t n u o n o a]. O \I ,/' ‘u Figure 1. Two parabionts being weighed on modified dietetic scales as described in text. 49 Figure 2. Each point in curves (X) and (0) represents the weight difference between two attached weights or parabiotic rats plotted as a function of differences in scale readings (see text). (AB) equals (AA) when two attached objects weigh independently, curve (0). Curve (0) was obtained using 40 pairs of parabiotic rats with varying weight differences between the two animals of each pair. Curve (0) is described by the equation AB = -1.023 + 1.361AA; the correlation coefficient (r) of 0.982; AB intercept + 0 (°C= 0, T-test). The relationship between AA and AB is significant (analysis of variance) P<0.001; the standard error of the regression coefficient (Sb) = 0.0413; the 95% confidence limits for the true regression coefficient (6) are: 1.277Sp51.445; the standard error of the sampled mean A-B (at A-A) = 2-3549 ($533)- The 95% confidence belt for the means of the population regression and the 95% confidence intervals for a single observation were calculated but not plotted on figure 2. All points were within the 95% confidence interval for a single observation. The values given below will enable the reader to calculate and plot confidence intervals if the need arises. The mean for AA (AA) = 121.43: sum squares for AA (SSAA) = 129,969.024; the sample size (n) = 40; the mean square deviation (med) = 221.843. Appropriate values for AA are: 49.0, 69.6, 113.3. 159.7 and 233.6. The AA values in table 1 may also be used. 50 X Attached Weights O Parabiotic Rats 0 AA Equals AB tr 1 . O 100 200 AA Figure 2. See opposite page. ‘th Note: 51 Part 1 was published as Shier, N. W. and O. Mickelsen (1972) A method for determining growth rates of individual rats in parabiosis. J. Appl. Physiol., 32:425. PART 2 AN AD LIB. DIFFERENTIAL FEEDER FOR PARABIOTIC RATS IN TRODUC TION Parabiotic rats have been used for studying the regulation of body fat content and food intake for many years (283, 287). These experiments have been greatly impeded by the lack of a reliable ad lib. differential feeder for parabiotic rats. A restrictive differential feeder was designed by Wei Han et al. (284), 1963. A partition was placed lengthwise down the middle of a regular, galvanized rat cage. An adjustable opening was made in the partition through which the parabiotic union protruded. When parabionts were placed into this restrictive feeder, allowing only forward and backward motion, their food intake decreased and they lost weight. Parabionts were then placed into the feeder for approximately 15 hours, overnight, and on this schedule the rats ate enough food to just maintain body weight. Coleman and Hummel (291), 1969, tried to place parabiotic, diabetic mice into the feeder described above and reported that the animals never did.eat, struggled and even tore the skin at the site of the union. The partitioned feeder was used with some success by Fleming (285), 1969: Schmidt and Andik (295). 1969; and Schmidt (286), 1973. but only after training their rats to meal feed within a couple of hours. The lfitts seemed to associate the feeder with food and did not mind the restraint. Meal fed animals may be poor subjects for the mechanistic stnxly of the control of food intake as the urge to eat after 22 hours of 52 53 deprivation is so strong that many "normal" factors employed to suppress food intake may be overridden. It is apparent that a relatively inex- pensive, easily used and reliable ad lib. differential feeder is needed before parabiotic rats can be used routinely in laboratory investigations in which parabiotic, differential feed intakes are of importance. TECHNIQUE Female Sprague-Dawley rats, approximately one month old, were placed in parabiosis using a modification of the procedure of Bunster and Meyer (296), 1933). Three weeks to a month were required for surgical recovery and to determine whether the "union" was viable or not. The animals were, therefore, young adults when used for experimentation. Various dimensions (length from the cephalic end of the union to the tip of the nose, the width and thickness of the head, the length of the union, the nose to anus length, thickness of the scapular area and the width at the pectoral.gindle) were made on approximately 37 pairs of animals. These dimensions would determine minimal sizes for the various components of the feeder. The feeder was made universal so that as the animals grew larger the feeder could be opened for just one animal or both. ZFeeders were constructed out of sheet metal, one millimeter thick, and bolted into regular galvanized animal cages. Since the feeder occupies cxmnsiderable space, it is recommended that a double cage be used which wrnlld,give the animals sufficient room to move around. Many feeder drxaigms were tried and when one particular design seemed to work fairly vuel]., a cardboard model was made and, with blueprints, was given to a 54 metal shop for construction of five feeders for preliminary testing and, when shown to be satisfactory, an additional 35 feeders were ordered. To test whether a feeder was functioning properly or not, a chromium sesquioxide marker was incorporated at a 1% level (w/w) in the feed on just one side of the feeder. At various times during the day the pair feeding from this feeder would be placed into a partitioned cage (284) for one-half to one hour periods, so that fecal samples could be collected separately.1 The feces from the animal not fed chromium were analyzed for the marker. If only slight levels (<:.25% of dry fecal weight; See Table 6, Page 63) of chromium were found it was assumed that the animals were consuming their own diets. The feces were pre-digested with nitric acid, wet ashed and assayed for dichromate according to the method of Czarnocki et al. (297) with the following modifications. After the color changed to red during the digestion period, digestion was continued for an additional 15 minutes when 2 additional ml of 70% perchloric acid were added and digestion continued for another 10 minutes. The digestate was sus- pended in 110 ml of distilled water and immediately filtered through Whatmann qualitative paper instead of allowing it to stand overnight. l Ikater it was found not necessary to place animals in a restrictive csqge to collect feces. Fecal excreta given by rats fed the labeled dirat are easily discernable, being a bright green. Even if the cflrromium fed animal has eaten some of the non-labeled food, its feces are still easily detectable. 55 RESULTS Many different feeder designs were tried with varying degrees of success. Eight designs were relatively good; each was tested to determine which one would most completely prevent cross-feeding as evidenced by the chromium marker. A parabiotic pair was placed in each of the eight feeders and food intake measured. A high fat ration (298) containing a 1% chromium sesquioxide label was placed on one side of the feeder; a grain ration (317) on the other side. Fecal samples from the animal not receiving the label were collected over several days and assayed for chromium. As seen in Figure 3, feeder 3 was extremely effective in controlling the feed consumed by the rats. Food intake and weight gain in differential feeder design 3 were improved over control values secured from parabiotic animals whose feed was in a cup sunk into the floor of the cage (Tables 2 and 3). This cup was wide enough so that both parabionts could eat at the same time. From these preliminary studies it was clear that feeder design 3 insured that each member of a parabiotic pair consumed only its own ration resulting in excellent food intakes and body weight gains in each animal of a pair. Tables 4, 5 and 6 show feed consumption, ‘weight gains and the chromium results, respectively, of five pairs of':rats each placed in a differential feeder of design 3. Only negligible amounts of chromium were detected and, once again, food ijrtake and body weight gains were significantly better than that of ccnrtrols. Several photographs of the differential feeder can be seen in Ifiigure 4 (A, B, C, D, E, F) and detailed drawings are shown in U. BRI~‘ a 56 Figures 5—9. In all food intake studies, using parabiotic rats in ad lib. differential feeders, a chromium tag is placed on one side of each feeder at the beginning, middle and end of the experiment as a check on proper usage of the feeder. The label was placed in the high fat diet for studies in which one rat received high fat and its partner a grain ration because of the ease of mixing and minimal spill- age. Fecal boli under the cage from the animal not consuming chromium may be contaminated if the marker is placed in an easily spilled diet. Food intake measurements were made on 12 single, sham operated control rats and 24 pairs or 48 parabiotic rats for a 19 day period (Figure 10). Although the feed intakes of the female parabiotic rats in feeder design 3 were greater than those of the controls, this difference disappeared when the intakes were expressed on the basis of body weight (Table 7). Intakes were actually a little better in the parabiotic animals as compared to single controls. DISCUSSION The feeder described offers a reliable and easily used method .for determining ad lib. food intakes of the individual animals in parabiosis. The feeder is adjustable for different animal sizes and permits each food cup to be removed separately. When animals are first placed in the feeder their food consumption eqlnals that of the controls in 3-4 days. Another feature of the feeder is that feed spillage by the individual animals can be mea- snrred.accurately without contamination by excreta. Spillage collection ‘birus could be constructed around each food cup, or a partition could h- ‘- '< FL (x. M 57 be constructed between the food cups extending from the floor of the cage to the bottom of the excreta collection pan which would separate very accurately the feed spillage. These techniques were not necessary for work done in this laboratory since, in crucial intake measurements, one diet was high fat and, consequently, only the loose grain ration was spilled. Spillage is also quite localized around each cup making differential collection fairly simple if both animals are fed an easily spilled food. Food intakes are statistically the same for parabiotic rats fed in the differential feeder compared to single rats eating out of conventional food cups placed on the floor of the cage but compared to parabiotic rats exposed to a food cup placed in the floor of the front of the cage the differentially fed parabionts have significantly higher intakes. When using the feeder, each animal is obligated to take a proper eating position. One animal cannot keep the other from eating as is the possibility when the feeder is in the floor along the front of the cage. In such a case the dominant animal can maneuver its body so that the other animal cannot reach the food cup. Several possible errors might arise in using the differential feeder. The feeder should be placed in animal racks that allow suffi- cient room between the bottom of the cage and the excreta collection pan.to prevent animals from reaching their feces. A crucial factor as to whether the feeder is used properly or not :is the scapular supportive suture. The scapular suture must be intact zrllowing a limited mobility at the pectoral girdle; otherwise, animals can twist around and will not learn proper feeder usage. With a good scapular suture the rats are committed to use the feeder in the manner designed. 58 In chromium determinations fecal samples under the cage could be contaminated by chromium in powdered diets when the diet is spilled, as alluded to earlier. Even though the spillage does not fall directly on the fecal material, small amounts of feed containing chromium oxide may be deposited, by air currents, on the feces of the animal fed the unmarked ration. (This is suggested by the observation that the amount of spillage was correlated with the degree of chromic oxide detection in feces collected under cages where the marker was incor- porated into a loose grain ration Z; = 0.3117.) This was not a significant r value but the t-statistic was rather high (1.601) for a sample size as large as 25; consequently, some of the low levels of chromium oxide detected were probably from this source. 59 Table 2 FOOD CONSUMPTION IN GRAMS (AVERAGE/DAY) OF PARABIOTIC RATS IN AD LIB. DIFFERENTIAL FEEDER DESIGN 3 COMPARED TO AN AVERAGE CONTROL VALUE FOR RATS PERMITTED TO EAT FROM A CUP IN THE FLOOR OF THE CAGE * H Feeder Design 3 Control 15.43 i 0.77 (SE) P) 0.05 14.58 i 0.99 (SE) * Average for 7 days ** Average for 8 pairs, each on diet for 7 days; 56 values 60 Table 3 WEIGHT GAINS IN GRAMS/DAY FOR PARABIOTIC RATS IN DIFFERENTIAL FEEDER DESIGN 3 COMPARED TO CONTROL VALUES FOR RATS PERMITTED TO EAT FROM A CUP IN THE FLOOR OF THE CAGE * H Feeder Design 3 Control Combined Right Left Combined Right Left 6.0 3.0 3.0 1.9 i 0.4 1.3 i 0.3# 0.6 i 0.3 * Over a 10 day period ** Average values for 8 pairs over a 15 day period #Since this animal is gaining weight faster than its partner, it is possible that this is the dominant member of the pair. 61 Table 4 AD LIB. FEED CONSUMPTION* OF PARABIOTIC RATS IN FEEDER DESIGN 3 VS. CONTROL CAGE (TOTAL FOR 8 DAYS) Parabiotic Food consumption (g) when parabionts were in: Pair Feeder Design 3 Control Cage 1 129 102 2 135 130 3 142 120 4 123 116 5 122 116 Totals 691 584 It SE 130 t 3.3 P< 0.05 117 .t 4.0 *High fat diet 62 Table 5 PAIR WEIGHT GAINS IN AD LIB. DIFFERENTIAL FEEDER DESIGN 3 VS. CONTROL CAGE (TOTAL FOR 8 DAYS) Parabiotic Weightggains (g) whengpargbionts were in: Fair Feeder Design 3 Control Cage 1 25 10 2 30 32 3 30 20 4 48 20 5 4O _ 27 Totals 173 109 “U l+ U) m 35 i 3.7 P< 0.05 22 i 3.3 63 Table 6 Cr203 IN FECES OF RATS FED NON-LABELED* HIGH FAT DIET IN FEEDER DESIGN 3 (all values on dry weight basis) Cage** Fecal Weight Cr203 in Feces (g) The) LZEZ____. l 4.62 2.6 0.07 2 7.60 4.2 0.05 3 3.36 5.6 0.17 4 4.81 1.0 0.02 5 6.22 32.7 0.53 37;; SE 0.16 i 0.08 *Animals fed a 2% Cr203 labeled ration produced feces containing 20-23% chromium sesquioxide. This differs from above data, P< 0.001. **Rats in cages l, 2 and 5 were fed for 8 days; those in 3 and 4 were fed for 11 days. mz mmm.a mdm. .mmd. was. H_Hmm.w .Nmm. H mmN.© on ma .mH pecanmhmm .m> Honpeoo m a oocmawm> .Q .m H com: we a memo: mo confinemeoo muznv whomezoo .m> magmas aaeazmraaaea 2H maa Howaaoo m p cocoauo> .Q .m H use: me e memo: mo comaeodeoo mozov maomszoo .m> magmas aaHezmrmaaHm 2H maamo 2H mmaaeze aooa H-z oHeonamam a oases Cr203 IN FECES OF RATS FED HIGH FAT DIET; OTHER RAT IN EACH PAIR FED THE SAME RATION 11 COLLECTION DAYS q n q «2 N I- I- O eased ha a; $033394, s» E. Figure 4. 66 A. Side view of ad lib. differential feeder; B. End view; C. Top view; D and E. Parabionts feeding from differential feeder. 1) Side panels function in directing parabionts to the food cups with each animal on its proper side of the feeder; 2) Food cups. Each can be weighed separately. The end of each cup at (a, see Figure 4C) is extended slightly above the head opening 10, see Figure 4B). This acts as a head divider between the parabionts and prevents the mixing of food from one cup with that of the other; 3) Top pieces were necessary so that the animals would enter the feeder on the same plane. The top piece extends, at right angles, towards the top of the feeder to inhibit the animals from crawling over the horizontal component of part 3; 4) Body divider in which is cut a groove allowing passage of the para- biotic union (see Figure E). The width of the groove is slightly smaller than the thickness of an animal's head; so, an animal cannot stick its head through the groove to the other side. Each parabiont is therefore blocked from the other side of the feeder; 5) Bottom platform on which an animal rests its feet while eating; 6) Front piece including head holes (10) through which the animals protude their heads in order to eat; 7) Aluminum crimps for protection from sharp metal edges; 8) The solid front part of the body divider becomes part of the head divider; 9) Slip groove allowing the side panels (1) to be opened up or closed, independent of each other. 'Adjustments can be made for the body size of each individual animal. Parts 3 and 5 are soldered in place on the front piece and parts 1 and 4 are bolted to the front piece with 10-24-0.5 (3/16" diameter) machine screws. The feeder is bolted into standard galvanized rat cages with machine screws placed through holes drilled in the front piece shown on Figure 4A. Free ends of the side panels are also bolted to the galvan- ized cage. There is only one way in which the food can be reached. Both parabionts must enter the feeder so that each is on its proper side. There are then no impediments between the animals and the food. For proper usage of the feeder, a good scapular supportive suture is necessary to minimize flexibility at the pectoral girdle (see surgical procedures in this monograph). The feeder can be likened to a metabolism feeder for single animals only made with a partition for parabionts. The pictures show the feeder outside of the cage. 7 l ~6—-—-—-—-—.2 g 3* / Y c Q 3 ° ' ”$0 3* ”no '[ 4—’-—e<1~—/’-———>-4-’ I 2 2 Z \I '4———2 4—7—9-4——22’————44t . IX fi - ____J: ‘4 lé E3 Figure 5. Drawing of the body divider (number 4 in F1 A) Side View; B) Top and c) End project vanized sheet metal one millimeter thick. secured with solder joints. (R = radius; inches.) gure 4) of the ad lib. differential feeder. ions. Scale of 1:0.7. Material: 031- All approximating free metal edges were D - diameter; All dimensions are in 69 '4 i - M 3 g N 12’ 1> Y -..-----. m __ - ' CD A __ it v C a Q ‘12 "i‘e -< 6 35 - P- I -" 3 ‘3‘ ' ”72“ 3 ,2 t“ ‘2‘ P A A A 7 A _::l) 52% h~eg C“ (\J cu Y _s ‘7 Q A "R <1 25 b V ‘O as I 4 2 If - P N I I "5+ I beg-M [2 — Mzum '4 I ‘ I6 0 Q40 A a?“ 12 "W V __ A K(Io N Na; N V v V __e. A . Figure 6. Drawing of the front piece (number 6 in Figure 4) of the ad lib. differential feeder. A) Front View; B) Top and c) End projections. Scale of 1:0.7. Material; Gal- vanized sheet metal one millimeter thick. (R = radius; D = diameter; All dimen- sions are in inches.) 7O l! 5 < 3 32 [>4 3 32 1> “‘”‘”' o ‘"“"” ‘ " .A It «a ,3 v w m -+ ___i i __.,.i_i11 _ _,_ -i; E: "Q“ ON M NM‘ C ___- - _ v v <1 o i I> ° / r 5 Q F 5 A 1 7 : 45., ' v ~M ! b _- __ ‘”“‘_____i_._____-i\ ' ’ — ’5 é ‘\ i ‘ \ g \ "la; ‘ \ 3 once ‘ N 1 ~ — 7K <( I A E I I i i l ‘N Nu . 4 11> A w v y av .3... Figure 7. Drawing of a side panel (number 1 in Figure 1») of the ,ad lib. differential feeder. A) Side view; B) Top and C) End projections. Scale of 1:0.7. Material; Gal- vanized sheet metal one millimeter thick. (R = radius; D = diameter; All dimen- sions are in inches.) <1 <1 I; I i i i p_-_ F A Figure 8. . _..4-..._ _..s-—..._ —~x .N s V OJL-*° [X R? -7 8 «1+: Jr! Flat drawings of the top piece (A) (number 3 in Figure 4) and bottom plat- form (B) (number 5 in Figure 4) of ad lib. differential feeder. 1:0.7. Galvanized sheet metal one millimeter thick. Material; Scale of The pieces are bent, at the dotted lines, in the forms shown on Figures 4A and SA. (All dimensions are in inches.) Figure 8A. 72 The top piece (A) (number 3 in Figure 4) and bottom platform (B) (number 5 in Figure 4) bent into proper shape. Scale of 1:1. Material: Galvanized sheet metal one millimeter thick.- Angles shown are approximate and the pieces may have to be adjusted slightly to the positions shown in Figures 4A and B. Flaps l and 2 are placed through the head holes and soldered to the back of the front piece as shown in Figure 4A. right 4 4 a 73 A ~+ V C 4 + ,{ I> 4 2 >4 2 5 17 N I; x:- ' A “N~ °4 .-,_..._.--....-__.-. V A B Figure 9. Drawing of food cup (number 2 in Figure 4) for ad lib. differential feeder. A) Side view; B) Top and C) Front projections. Scale of 1:0.7. Material: Galvanized sheet metal one millimeter thick. All grain food cups were soldered at the corners but high fat cups were not. (All dimensions are in inches.) GRAHS 74 l‘. i i \. Ii 3 ‘ ! i i l ! l ' ‘. ! 'I \ I l 1 \ I A l . I a ) I | ' ‘\‘ a ! . i ’\ '\ l \ ’l ‘1‘ i ‘\ I \ / l / " \ .I Y i f‘\-/ \ I '1‘ I/\ ‘ ' I / I i. / " ‘. / fi IA ‘ ‘\ I\ I \ l I \\ / E ’0 \‘ // I s I n \ ’ 0 \ ' \ I . / .’ \ 'l .I' 5' I y, ’ .' .' .' ! l f .' ! .' l i (1) Single controls (in conventional cage with standard . food cups), fed grain diet from Day 1 to 23; n = 12 ._._.. _- (2) Parabiotic, fed grain ration from Day 1 to 23 in differential feeder; n B 48 10 DAYS 20 23 Food intakes per 100 ms body weight of control and parabiotic female Sprague-Dawley rats. Av./Animal/Day) Note: 75 Part 2 was presented as a full length paper at the 1972 meetings of the Federation of American Societies for Experimental Biology and published in abstract form as Shier, Nathan W., Olaf Mickelsen and Dena Cederquist (1972) An ad lib. differential feeder for parabiotic rats. Federation Proc., 31:689. PART 3 PARABIOTIC SURGICAL PROCEDURES INTRODUCTION Sauerbruck and Heyde (299). 1908, described a surgical procedure for parabiosis in which only skin and muscles were sutured together. After placing a small incision in the side of the abdominal musculature of each animal, the dorsal muscle flaps were sutured together followed by the ventral flaps producing an open cavity between the two animals (coelio-anastomosis). This procedure was used extensively until an improved method appeared in 1933 (296). In the Sauerbruck and Heyde method, animals usually became highly infected and many pairs died from infection or physical trauma. The animals would pull on the soft tissue sutures tearing them apart. Bunster and Meyer's modifications are as follows; 1) A supportive suture was placed through the scapulae and the iliac bones; 2) The cut edges cfi‘ the abdominal area were sutured together as one mass closing off the open cavity between the animals and 3) A suggestion was made to place a few sutures through the thoracic muscles closing off the thoracic pocket between the animals. This technique seemed to give adequate support and protection for the soft-tissue and muscle sutures until healing. This technique is preferred at present. One major problem still existed with the Bunster-Meyer technique. In many pairs the scapulae would become separated after a week or 10 days. Bunster and Meyer offered no reason for this but stated that the skin sutures were healed by this time and that the scapular separation 76 77 was of little significance. Williams (300), 1968, also noticed con- siderable scapular separation. In many of this author's early pairs, scapular separation was noticed before as well as after skin healing. If separation occurs after skin healing, the skin is still stretched considerably which possibly produces a "stressfl Schmidt and Andik (295), 1969, reported that half of their pairs (24 in all) separated completely after 2 to 4 weeks. The authors concluded that the separa- tions were due to immune reactions. It is possible that some of their separations could have been due to defective scapular sutures. A better supportive scapular suture was needed not only to secure better para- bionts but also to keep pairs more rigid at the pectoral girdle for proper use of the parabiotic differential feeder as described by Shier et al. (301), 1972. TECHNIQUE The surgical technique was that of Bunster and Meyer (296), 1933, except for the following modifications. The above reference contains surgical details not described below. Thoracic sutures were not used. These sutures do not seem to be necessary and may add further trauma. The major difference in the technique reported here and that of Bunster and Meyer concerns the supportive sutures. The new suturing system for the scapular area is shown in Figure 11. The caudal supportive suture is placed around the respective medial femora of each animal and tied from the dorsal aspect. The tie should be fairly loose so as not to tie off circulation in the femoral artery and vein. This type of caudal suture has been used by Hervey (283), 1959. 78 The scapular suture is made as follows: A stainless steel half- curved surgical needle (three-eighths circle, cutting edge, number 12)1 was threaded with Suprylon, gauge 3, suture.2 The left scapula of the right animal is located and elevated and the needle is pierced through the supraspinous fossa beginning from the medial side of the scapula. The right scapula of the left animal is then located and elevated and the suture is continued across to the lateral right scapular surface of the left animal and the needle pushed through the supraspinous fossa and out the medial side. The suture is then continued posteriorly and placed through the medial side of the infraspinous fossa of the right scapula of the left animal. The suture then passes through the lateral side of the infraspinous fossa of the left scapula of the right animal emerging on the medial side (Figure 11A). The scapulae are then drawn together, as in Figure 113, and A and B are tied snugly. The end of the suture at B is left long enough so that the suture can be passed through the supraspinous fossa of the left scapula of the right animal and, diagonally, through the infraspinous fossa of the right scapula of the left animal emerging on the medial side (Figure 11). The ends of A and B are once again tied across the top of the scapulae. The taxcess suture is removed and the dorsal skin suture completed over the scapular area . Other surgical techniques and materials were as follows. The 3 aJLimals were anesthetized with a mixture of methoxyflurane and l liiltex Instrument Company, Division of E. Miltenberg, Inc. New York, New York. 10010. 2 .I. Pfrimmer and Company. Erlangen, West Germany. 3 Chemically, methoxyflurane is 2,2-dichloro-l,l-difluroethylmethyl etflieru supplied by Pitman—Moore, Division of Dow Chemical Company. Fert Washington, Pa. 19034. 79 butylated hydroxytoluene, Metofane inhalation anesthetic. The hair was removed from the appropriate sides of each animal with electric clippers. The skin was prepared with Zephiran Chloride (benzalkonium chloride)“ in an aqueous solution of approximately 0.13% concentration. All incisions were made with surgical scissors. The abdominal sutures were interrupted using a (00 gauge) braided surgical silk.5 The ventral and dorsal skin closure was made using 9 mm stainless steel wound clips. 7 After surgery, each animal was given 0.2 cc Longicil S intra- muscularly. Sterile procedure was used as much as possible. All equipment and sutures were sterilized "cold" with Zephiran Chloride. If an infection developed post-surgically in the wound or if the animal experienced a general infection due to surgical stress, additional Longicil was given at a dose of 0.2 cc upwards to 0.7 cc. Treatment was usually a single injection intramuscularly but, once on occasion, treatment was continuous for two to three days. In surgery done most recently, chloromycetin8 or Mychel-S9 was given in lieu of Longicil at a dose of 0.05 cc intraperitoneally of a 10% solution as a I; Winthrop Laboratories, Division of Sterling Drug Company. New York, iNew York. 10016. 5 American Cyanamid Company, Surgical Products Division. New York, New York . 10965. 6 Autoclips supplied by Clay-Adams, Inc. New York, New York. 10010. (Made by Totco, Glendale, California) 7 Fort Dodge Laboratories, Inc. Fort Dodge, Iowa. 50501. Note: (hie cc of Longicil-S contains 150,000 units benzathine penicillin G, JJD0,000 units of procaine penicillin G, and 250 mg of dihydrostrepto- rmycin in an aqueous suspension. £3 Brand of chloramphenicol sodium succinate. Park, Davis and Company. Detroit, Michigan. 48232. 9 Brand of chloramphenicol sodium succinate. Rachelle Laboratories, Iruz. Subsidiary of International Rectifier Corporation, 700 Henry Ferd.Avenue, Long Beach, California. 90801. 80 prophylactic and a dose of 0.05 to 0.1 cc later if infection developed. In seriously infected wounds, treatment at the above dose levels was given directly into the infected area. RESULTS The new scapular suture held extremely well. Virtually no scapular sutures weakened. The animals were held together firmly for the differ- ential feeder and scapular infection was not a serious problem. Even if the animals were exhibiting immuno-incompatibility the supportive sutures still held. DISCUSSION Surgery is more easily performed on weanling animals. The central areas of the scapula are cartilaginous at this age and the needle can be easily inserted without cracking the bone. Parabiosis seems to be better tolerated. behaviorly, when performed at an early age and younger, smaller animals will physically stress the union immediately after sur- gery far less than adults. There is no need to remove the scapular suture after healing. The suture becomes encased in connective tissue and produces no subsequent problem. Non-absorbable sutures are used both for abdominal sutures and supportive sutures to avoid the suture from loosening before a firm union has been established. One other major problem developed that has not been previously reported in the literature. Many pairs of animals, after surgery, would bite at the abdominal sutures or auto clips probably because of 81 a slight irritation. The wound in many cases would be ripped open and become infected. In some instances re-suturing with a braided silk, gauge 1 suture prevented a recurrence but in many cases did not. A rather noxiously tasting substance, when placed on the area, was of little benefit. Small aluminum braces were then constructed. These braces were about 13 mm wide and extended from the tail of the animal to the pectoral girdle. The brace was sutured on the dorsal surface of the animal with four sutures (one at the caudal area, one at the mid-line and two over the pectoral region). These braces prevented the animals from "jack-knifing" and worked very well in keeping them from the wound. Within seven to ten days the brace would be sloughed off at which time the parabiotic wound would be healed. No infection ever resulted from using the braces. Sedation was never used post-surgically since, with certain experiments, it is unwise to treat with drugs that could alter experimental results unless absolutely necessary to preserve enough animals for the experiment. In all cases control animals re- ceived the same antibiotic treatments as experimental animals, but control animals were not given braces. Wound clips were removed several weeks post-surgically and any small skin areas not healed were closed with a few silk sutures. When using wound clips one must check the animal's incisors every 2 or 3 days. Several animals have had clips caught in their teeth and could not eat. The rats were fed, while convalescing, with low food cups, approx- imately %" high, wired to the bottom of the cage. All animals were fed with a standard. powdered grain ration. No liquid diets were used. Figure 11. 82 .. Animal outlines taken from Wei Han, 1963 (285) Modified surgical procedure for parabiosis. (A) and (B) in the left figure are tied together after drawing the scapulae (1) and (2) together producing the knot at (9) in the right drawing; (A) and (B) in the right figure are then tied across the top of the two scapulae after passing the suture back through the scapular area as described in the text; (C) and (D) are tied loosely around the respective femurs (7) and (8); (3) the ventral skin suture; (4) abdominal sutures; (S) and (6) the dorsal skin flaps that are shown partially sutured at (10). The arrows indicate the dir- ection of the suture and the dots indicate that the suture is under the scapula at that particular point. PART 4 DYE DILUTION STUDIES INTRODUCTION After one has perfected a surgical skill or technique, it is wise to test several pairs of parabiotic animals for the rate of cross- circulation. Many authors do not report data on rates of blood exchange but it is essential to know if parabiosis between members of a certain Species of animals or by a particular technique will produce viable cross-circulation. There are several methods available to determine rates of cross- circulation. Early workers used the compound belladonna. After in- jecting this compound into one parabiont, the pupils of the non-injected animal were observed for dilation in 20 to 30 minutes (302). Protein bound fluorescein was used by Scheff and Plagge (303), 1955. Radio- 59 iodinateiserumafllnmfin.and iron labeled erythrocytes may also be used (304) as well as chromium labeled red cells (305). Perhaps the most widely used method is that of T-l824 or Evans Blue dye1 injection. Evans Blue molecules adsorb primarily to the albumin fraction of the blood and, consequently, are mixed uniformily as it is cleared by the injected animal very slowly (306-309). Evans Blue is eventually cleared through the bile (310). Hervey (283), 1959, presented a detailed description of the tech- nique and included a mathematical formula by which one can calculate 1 Allied Chemical, National Biological Stains and Reagents Dept. (Formerly National Aniline Division), P. 0. Box 031. Morristown, New Jersey. 07960. 83 84 rate constants. Studies were not undertaken here to determine the time that cross—circulation begins in this preparation but it has been es- tablished that considerable exchange occurs at 3 days (304, 305). As the wound heals, capillaries interdigitate and grow together producing a direct blood exchange. TECHNIQUE Adult, female Osborne-Mendel rats were made parabiotic at weaning. Controls and parabionts were fed grain laboratory ration (317) until time of experiment. All optical density readings for Evans Blue were done (283) using benzalkonium chloride as solvent (311). A 17% concentration was employed instead of the original 12.8% used by Caster since the commercially available concentration is now 17% and dilution would produce consider- able frothing. The 17% solution proved to be as optically homogeneous and acceptable as the 12.8%. The absorption maximum for Evans Blue in a 17% benzalkonium chloride solution was 626 mu. All readings were, therefore, done at 626. Calculation of dye dosage: The standard curve for Evans Blue in serum was determined to be perfectly linear at least to 8 ug per ml. ZIt is linear somewhat beyond this point but further determination was lmnnecessary. As suggested by Hervey (283), a dosage of 0.2 cc of a (3.25% solution per 100 grams body weight was used. Tb determine whether that level of Evans Blue would be adequate, time dilution of the dye in a rat was determined mathematically. Assume a,j300 gram rat is injected with 0.6 cc of a 0.25% dye solution. 85 0.25 g dye X 100 ml solution — 0.6 ml dye solution injected X = 0.0015 g injected A 300 gram rat will have a blood volume approximating 8% of body weight or 24 ml blood or 12 m1 plasma. 0.0015 g dye injected = X 12 ml plasma dilution 0.1 ml plasma taken for assay X = 0.0000125 g of dye found in 0.1 m1 plasma or 12.5 ug. 0.05 ml plasma is then added to 6 ml zephiran chloride giving a final dilution of 1.03 ug dye per ml solution. This final concentration is on the linear standard curve and. therefore, the dye dosage is acceptable. Zephiran chloride was used because Evans Blue does adhere to glass surfaces and can produce an error in optical density readings. Zephiran chloride detergent frees Evans Blue from the glass and also elutes the dye from its adhesion to the albumin fraction in the blood allowing a far more accurate optical density determination (311-313). Hemoglobin was not considered to be a primary interference since its absorption maximum in zephiran chloride is absent at 540 mu and the sample reading was made at 626 mu. Hemolysis was usually slight and fairly uniform; furthermore, any major error should cancel out. A 0.2504% solution of Evans Blue was prepared in distilled water. ’The solvent fraction would be 99.7496 grams converted to volume cor- 2recting for room temperature. This solution was standardized and the Specific gravity determined. All blood samples were taken by cardiac pnnncture under sodium pentobarbital anesthesia.1 0.35 cc blood was taken frxr control serum and determination of the hematocrit. The appropriate dye injection was prepared and the syringe weighed. l Ikasage of 0.05 cc per 100 g body weight of a 6% solution given intra- peritoneally. 86 The approximate position of the femoral vein is located by palpating the femoral artery in the groin. An incision of l to 1.5 cm is made with surgical scissors after picking up the skin with tissue forceps about 4-5 mm, lateral to the point of palpation. A "mosquito" hemostat is then used to clear any skin adhesions around the femoral vein. A hemostat is clamped on each side of the wound and allowed to hang below the leg. This keeps the wound opened and exposes the vein. The entire preparation procedure takes less than a minute and there is no blood loss. The needle (27 gauge, %") is inserted, proximally, into the vein. After dye injection and needle removal, a dampened gauze is pressed lightly on the vein for a few seconds. No blood is lost from the vein during or after injection. The wound is closed with one or two small silk sutures. The syringe is then re-weighed and the exact dosage cal- culated by difference. This procedure is fast, extremely accurate (one is confident of a complete injection) and does not appear to be debilitating to the animals. Post dye injection blood samples of 0.3 ml were taken by cardiac puncture from each animal of a pair at 10 minutes, %, l and 2 hours with the ammonium salt of heparin as anticoagulant. The total amount of blood taken was always less than 1136 of the blood volume based on 8% of body weight. The blood was centrifuged at 3000 rpm for 30 minutes and 0.1 m1 ;p1asma added.to 6 ml zephiran chloride. The optical density was deter- 1nined.against zephiran chloride with 0.1 cc distilled water added. Five single controls and one member of each of 5 pairs of para- bitrtic animals were injected and plasma optical densities determined over time by the above procedures. The 2 hour sample in one of the pailxs Was lost. The total amount of blood removed from the control 87 animals was statistically the same as that removed from the parabionts (IN>0.05). Any error in disProportionate blood removal was therefore eliminated making the dilution curves comparable. The exchange rate was calculated using the % hour blood samples and expressed as the percentage of plasma volume exchanged per minute. The exchange rate best fits the curve for the hyperbolic cotangent of rate times time as described by Hervey (283), 1959. C1 Coth rt = E—» 2 r = the exchange rate expressed as a fraction of one animal's plasma volume per minute t = time in minutes after dye injection Cl and C2 = optical densities of the injected and non-injected animal, respectively It became necessary to express this equation in a more usable form which should be of benefit to future investigators. Hervey did not publish the mathematical "breakdown" of the above expression and a mathematical discussion of this particular equation was not found in the literature, although, Huff et al. (304), 1956, presented a math- ematical tretis of parabiosis with a physical model. In order to make calculations easier, El will be equal to 2. C2 Coth rt = 2 let rt = X Coth X = 2 x -x _ Cosh u e + e COth X _ Sinh u ex _ -x ex + -x _ 2 ex - e"x 88 9.!— ex x 1 = 2 e-— x e x 1 e2x + 1 e + —; = -___—;T_—' each side of the first expression was e e multiplied by ex to give the second 2x expression x 1 _ e - 1 e ._ -—; — -—-——3:——- each side of the first expression was e e multiplied by eX to give the second expression e2x + 1 X ex = e3x + ex x 2x 3x x e e - 1 e ._ e €3x+ex 3x x = 2 e '- e e3x + eX = 2e3x — 2eX ex + 2ex = 2e3x — e3x 3ex = e3x 3:21" x e 3 = e2x 1n 3 = 2x x=_1n_2 2 since x = rt r = 1%t2 = a certain fraction of plasma volume exchanged/minute let plasma volume = PV; so, (PV X 1n 2t3) = the cc of plasma volume exchanged per minute: since, 2.3026 times the common log = loge, it follows that (2.3026 x log10 3) PV 2t PV X (100) = % plasma volume exchanged/minute 89 The "real" numbers in the above expression will change as the original value for El changes. C2 Thirty-minute samples are adequate in calculating exchange rates. By 30 minutes the dye concentration in a non-parabiotic injected animal is rather stable and is not being cleared from the blood significantly per unit time; consequently, any decrease in optical density reflects a true exchange in parabionts. If one waits too long to take samples, the dye will be equilibrated between the two animals and El will become C unity and obviously flux will be 0. 2 RESULTS The dye dilution curves for single injected controls, non-injected and injected parabionts are shown in Figure 12. Dye injection was made at time 0. The parabiotic rats displayed equal optical densities at 117 minutes. In Table 8 the percent of plasma volume exchanged per minute is presented. By definition the % exchange rate will be inversely proportional to the time for each parabiont to display equal optical densities; consequently, the percentage volume exchanged should be highly but iJrversely correlated to time at equal optical density. This correlation teas determined excluding the parabiotic sample that had to be calculated ad; 1 hour instead of 30 minutes. The correlation had an r value of —O.786. This is a very good correlation but the t-statistic was 1.800 which was non-significant. The non-significance was simply a matter of a large within group variance and small sample size. The large within 90 group variance is actually an unfair hinderance in statistical inter- pretation, since, the large difference in exchange rates is a result of the effectiveness of wound healing and is totally in the realm of chance and cannot be corrected for in experimental design. Exchange rates were still highly correlated, in the correct sign, with time. As an added check on the technique the total dye removed from the ani- mals during the experiment added to the residual dye left in circulation statistically equaled the dose of dye injected. Since samples were collected shortly after injection, it was expected that these values should be very close. Theoretically, the only difference would be the dye cleared by the animal from the time of injection to the first 10 minute sample. This difference is a small percentage of actual dose. DISCUSSION The control curve in Figure 12 agrees very well with that reported by Gibson and Evans (309) in that the slope is fairly constant for 8-10 minutes after injection of the dye. Optical densities seemed to be slightly more stable after 25 minutes. At 120 minutes there is a slight discrepancy between the curves for parabionts and controls. Exactly the same procedure was used in all animals including dye dosage; consequently, theoretically all curves should terminate at approximately the same optical density. The dye dosage was given on a body weight basis and since blood volume is correlated to body weight, one would expect similar optical densities assuming that the dynamics of clearing a small percentage of the initial dose was the same in all animals. Sometimes, in parabiotic animals, 91 a small unhealed area or irritation exists within the union and that some dye is probably lost from the systemic circulation when crossing from one animal to the other. This would decrease the optical densities. The difference between all curves at 120 minutes, although, are not statistically different (P>0.05). Hervey (283) reported a range for percent exchange of one ani- mal's plasma volume to its partner per minute as 0.3 to 2.L% (1.0 i 0.2 [Tetd. error7 ) for eight pairs. Similar results were reviewed by Finerty (302), 1952. In the present study the average percentage ex- change was 2.338 : 1.802 (std. deviation) for five pairs with a range of 0.51 to 5.03. Four out of five of the present pairs had exchanges higher than Hervey's mean. The presence or absence of a coelio-anasto- mosis makes no difference on exchange rates (283). Hervey also states that his lowest rates were in pairs done by the unmodified Bunster and Meyer procedure and that the scapulae were separated. The improved exchange rates in the present study possibly are a result of the im- proved scapular supportive sutures previously described. With improved cross-circulation rates it will become easier to demonstrate the ex- change of various test substances from one animal to its partner. It is very important to reiterate the variability in cross-cir— culation data and if possible increased sample sizes should be used. Plasma volumes were calculated in parabiotic rats at the time when (xptical densities were equal in both animals. 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Hervey's parabionts grew more slowly and at any age weighed less than single controls. At 5 months the average weight of his parabiosed animals was approximately 71—85% that of control animals. Weight gained per unit of food intake, i.e., food efficiency, was the same for the above animals whether single or parabiotic. Hervey's "normal" parabionts contained one-half the body fat of the controls, the vicera appeared to be normal but smaller, and the parabionts were shorter than "normal" animals. Hervey concluded that engrgy metabolism was the same for single controls and parabionts since feed efficiencies were identical. Individual organ weights, as affected by parabiosis, for the most part, have not been reported except for a brief comment by Hervey. In his "hooded" rats the heart and kidney weights for all female and male ;parabiotic animals were slightly lower compared to controls. The liver ailso weighed less in parabiotic males when compared to single controls tnrt was almost identical in weight for females. The variability was great and no statistics were reported. 100 lOl TECHNIQUES Osborne-Mendel and Sprague-Dawley, male and female rats were used. Parabiosis was performed during the first one to two months of life. All single controls were subjected to comparable sham surgery. Surgery was performed as indicated in the surgical section of this monograph. The animals were fed either a standard grain (317) or high fat diet (298). At the beginning of experiments all animals were of the same age. Each experimental group received rats staggered over the range of body weights so that each group would have close to the same average body weight and standard deviation. Several litters were used. There- fore, each experimental group received an equal number of animals from each litter as far as possible. Nose to anus length was determined with a metric rule with the animals on their backs. Fat pads were removed following the methods of Schemmel (318), 1967. Blood volume was determined by the Evans Blue method. (See dye dilution methods in this monograph or von Porat (306), 1951). Packed cell volume was determined using heparinized, fire polished, micro- hematocrit capillary tubes purchased under the brand name capilets. All animals were pLaced on experiment immediately after surgery ill order to follow growth curves continuously from weanling on; conse- cIuently, many animals were lost during the rejection phase of parabiosis ais‘well as other complicating factors (see causes of death in parabiosis, this monograph) . l Ikude, Div. American Hospital Supply Corp. Miami, Florida. 33152. 102 RESULTS Figure 13 shows growth curves for parabiotic female Osborne-Mendel rats fed either a 60% high fat ration (298) or a ground grain diet (317) compared to controls. All rats except the high fat single controls showed a decrease in body weight for about 2 days as a consequence of surgery. The single high fat control curve deviated from the other curves almost immediately, whereas, the other 3 curves remained together until about day 70 when the parabiotic high fat animals began to gain more‘ body weight compared to controls and parabionts fed the grain ration. The grain fed parabionts did have an initial slower rate of weight gain compared to grain fed sham operated single rats, but this slight difference was made up rapidly and at the end of 181 days, the weights of the grain fed single and parabiotic rats were statistically the same. 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H 8:3 oa.mq o~.o¢ mm.¢¢ mn.oq oo.o¢ om.mq oo.m¢ m~.m¢ oo.wm om.wm oauoacauna m .n .m + one: moz AAWU nmxow=o .230: anon .9 056: mua 00.“ on.“ 8w on d 4 d J 8 ~43 mi: 9525.: E racism ~ A 3 u 5 Eéagfi Eéo IIIIII KAN 9 .. 5 89:28 :36 @8sz I IIIII. .m.\ 3ch mezosméaaefirgz........ «Q\ .n s E n 5 38:60 E :8: 5028 III . \mo\ o 00“ Gem com 8: SHYHD NI .LHDIBH M108 INTRODUCTION Food intake may be altered in parabiotic animals by a change in energy metabolism caused by the parabiotic state, per se, and not necessarily a consequence of the experimental variable. It is essential that oxygen consumption in parabiotic animals be measured to determine if the parabiotic state does influence energy metabolism. Oxygen consumption of parabiotic animals could not be found in the literature. TECHNIQUES Oxygen consumption determinations were made by a "closed circuit" technique (332) modified as follows. Room air was used in lieu of pure oxygen and was injected into a desiccator with a calibrated syringe and needle at a rate necessary to maintain constant intra-chamber pressure. Chamber pressure was monitored by a U-tube extending from the top of the desiccator and partially filled with Brodies solution. The desiccator was made air tight by coating all cracks with stopcock grease. Intra- cflramber temperature was monitored by a thermistor probe. Soda lime was used for carbon dioxide absorption. Oxygen consumption can be read directly from the calibrated syringe. Witfll this procedure the exact volume of the exposure chamber, corrected 45387. 1 Yellow Springs Instrument Company, Inc. Yellow Springs, Ohio. bkxiel.43TA; Serial 9888; Probe No. 401. 124 125 for animal size, is of no importance and saves many calibrations. All animals were tested in the same exposure chamber. Male Sprague-Dawley rats both single and parabiotic were tested. The rats were fasted for approximately 17 hours, overnight, and all measurements were made in the mid-afternoon (1:00 to 4:00). This time was chosen as the afternoon would approximate the best basal conditions for measurements. In the morning, animals are just settling down froman active night and their body temperatures Would still be rather high relative to basal. All feed prior to fasting was a standard grain ration (317). All gas volumes were converted to Kelvin before any further cal- culation or analyses. Control oxygen consumptions were done with two single animals in the desiccator to simulate the parabiotic dualty. No measurements were made until the chamber reached constant temperature and each pair of animals were tested over three or four individual periods of approx- imately 15-20 minutes each. After each "run" the chamber was opened and.flushed out with room air to avoid long exposure periods to high relative humidities as no dessicant was used. Activity was visually monitored and all determinations thrown out where activity was excessive since oxygen consumption would then be above basal levels. On each (experimental day one pair of controls and one parabiotic pair were lneasured alternating the initial determination on each day insuring a completely "staggered" design. After oxygen consumption determination the animals were sacrificed tnrzfirst lightly anesthetizing with methoxyflurane inhalation anesthetic.2 J'ZPitman-Moore, Division of Dow Chemical Co. Fort Washington, Pa. 219CX¥+. 126 This was done in order to obtain heparinized heart blood for insulin assay and to separate the parabionts. All animals, single controls and separated parabionts, were weighed and then decapitated. Immed- iately after decapitation as much blood as possible was collected into centrifuge tubes (without anticoagulent) and placed in beakers of ice water. .Proper aliquots of serum were frozen at -40°F for future assay of combined thyroxin and triiodothyronine, glucose, urea nitrogen, and free fatty acids. After blood collection, the abdomen was opened and the adrenals removed, weighed and frozen at ~400F in 5 ml of 6% trichloroacetic acid to await assay of adrenal ascorbic acid. Approximately 5 minutes were required for the entire operation. Thyroid and testicular weights were also determined. Organs were placed between layers of slightly dampened towels, after removal and before processing, to impede evap- oration. Serum thyroxin and triiodothyronine were assayed by the "Standard 11, T4 by column test."3 For that assay, thyroxin and triiodothyronine (in 0.5 ml serum) were freed from proteins by adding 5 ml of 0.15N NaOH. After making the ion exchange resin3 alkaline, the sample was placed on the column. The resin adsorbed thyroxin as well as other iodinated organic compounds, inorganic iodine, proteins and amino acids. The proteins and iodotyrosines were eluted (alcohol-acetate buffer and 15% acetic acid) from the column while thyroxin, thyronine and inorganic iodide remained adsorbed. The thyroxin and triiodothyronine were then lnoved down the column with glacialacetic acid and eluted with 50% acetic axxid.into test tubes. The hormones were then determined colorimetrically. 3 lilo-Rad Laboratories, 220 Maple Avenue, Rockville Centre, New York. 11570. 127 The colorimetric reaction is based on the fact that bromine replaces iodine on the thyroid hormone molecules and the free iodine catalyzes the reduction of cerium by arsenic as follows: 2 Ce+4 + As+3 ------------ 2 Ce+3 + As+5 Yellow Colorless As free iodine increases, the greater the catalysis of cerium reduction. As cerium is reduced, the solution turns from yellow to colorless. Higher hormone concentrations will therefore have a higher percent transmittance or lower absorbence. All samples must be read in exactly 20 minutes from the initiation of the reaction. The rate of the reaction and color disappearance is linearly related to the initial hormone concentration. Different volumes of serum were used as a standard curve, giving varying levels of thyroid hormone eluted from the column into a con- stant volume. In using the column, therefore, one can start with different volumes of equal concentration and end with equal volumes of varying concentration both lower and higher than the original con-' centration. The standard curve was used to check the linear functioning of the system and to make sure all samples were read on the linear phase of the curve. Since actual concentration values for the standard were not known, the data cannot be expressed in exact units but the relative values can be compared between experimental and control groups giving statistically valid conclusions. A value of .04 ug:I- per ml was arbitrarily ascribed to the serum used for the standard curve thus giving numerical values to the data. Before the ascorbic acid assay, the adrenals were removed from ‘the freezer and allowed to stand at room temperature for a few minutes. 128 While a few ice crystals were still present, the adrenals were cut into small pieces with operating scissors and then homogenized on a "polytron" homogenizer.4 The homogenate was transferred to disposable centrifuge tubes and centrifuged for about 10 minutes at 3000 rpm. 0.5 ml of the supernate was taken for total ascorbic acid assay by the osazone method of Schwartz and Williams (333). The three hour incubation period recommended by Schwartz was not long enough to adequately react the more concentrated samples; consequently, the in- cubation period was increased to 6 hours at 370C. Five hundred micro- liters of the supernate were used, since both adrenals were assayed together, doubling the ascorbic acid concentration. Two drops of 2,6—dichlorophenolindophenol were used instead of one. Serum glucose was measured by the glucose oxidase method of Keston (334), as modified by Teller (335), after the proteins were precipitated by the Somogyi procedure. Chromogen and enzymes were purchased under the trade-mark "Glucostat."5 The semi-micro method was used and the reaction was allowed to go to completion. Serum urea nitrogen was analyzed by the modified Berthelot reaction (336-339). Reagents were purchased under the trade-mark "UN-TEST."6 Blood for the insulin immunoassay was taken by heart puncture with heparin (ammonium salt) as anticoagulant. Heart blood was used since cerebral spinal fluid and tissue fluid proteins may bind insulin if serum is taken when the animal is decapitated. The immunoassay EPEient-Lizeng Prof. P. Willems, Luzern Kinematisches Hochbrequenz- Gerat; Kinematica Cmbh. Luzern-Schweiz. '5 Worthington Biochemical Corporation, Freehold, New Jersey. 07728. 6 Hyland Division, Travenol Laboratories, Inc. Costa Mesa, California. 92626. 129 method and calculations are according to Hales and Handle (340). Iodinated insulianS, insulin binding reagent, standard and millipore 7 filters were purchased from Amersham/Searle. Bovine albumin was used in place of horse serum, No. 2. Samples were counted by liquid scintillation. Serum free fatty acids were measured by the extraction and titra- metric method of Ko and Royer (341). 0.4 ml of serum was used with palmitate as standard. One normal NaOH was used to absorb CO2 rather than ascarite. RESULTS All oxygen consumption values when translated into energy units were the same for parabiotic and control animals for each day. The values expressed on the basis of body weights were greater but not statistically so for the parabiotic rats. Although adrenal weights on an absolute as well as body weight basis for parabiotic animals, compared to controls, were slightly larger, there was no significance to these differences (Tables 21 and 22). Within group variance was high for parabionts with the ele- vated mean and within group variance heavily biased by one reading (96.6 mg). Without this reading the means are far closer (60.0 mg compared to 57.0 mg) for parabiotic and controls respectively and would still be non-significant. Thyroid weights approached significance for the absolute weights. When these weights were expressed on a body weight basis, they were '7 Amersham/Searle Corporation, 2636 S. Clearbrook Drive, Arlington Heights, Illinois. 60005. 130 different (P <0.01); the parabiotic rats had the heavier thyroids (Table 23). Testicular weights were identical regardless of how expressed .(Table 24). The serum thyroxin and triiodothyronine iodine level for para- bionts was low but still within the normal range of 2.9 to 6.5 ug%.8 The levels for control animals were in the mid-range for normal but were significantly higher than the parabionts (P<0.05). No difference was seen for serum glucose or plasma insulin between controls and parabionts as shown in Tables 26 and 27. Serum free fatty acid levels are not elevated to the same extent in parabiotic rats as in controls in response to fasting (Table 28). In this particuLar group of parabionts the level of adrenal ascorbic acid was lower than controls (Table 29) even with no statistical diff- erence in adrenal weights; nevertheless, the adrenal weights were absolutely and per unit body weight larger for parabionts. Fasting urea levels were different between parabionts and controls only at the 0.10 level (Table 30). The variance was high for para- bionts with a couple of animals having high values. DISCUSSION An excellent measurement of metabolic rate is "specific metabolism." Cal/24 hours Body weight in Kg Specific metabolism = 0.73 Specific metabolism for control and parabiont were practically 8 Standard II, T-4 by column test, Instruction Manual, Bio-Pad labor- aixxries, 220 Maple Avenue, Rockville Centre, New York. 11570. 131 the same. The normal Kcal produced per kilogram body weight per day is 130 for the rat (229) with the values reported here agreeing quite well considering normal variability for different species of rat. All values are close to the ones normally reported for rats. There should be no effect on parabiotic food intake caused by changes in energy metabolism produced by the parabiotic state, per se. The male adrenal weights are a little heavier than those reported by Marshall et al. (323), but are still within normal species vari- ability. Weights of the adrenal per 100 grams body weight reported by Tullner and Edgcomb (342) are identical to those of the control animals reported here. The value of 0.05 grams per 100 grams body weight reported by Spector (322) seems to be incorrect. Parabiotic adrenal weights seem to be slightly heavier than controls but, as stated earlier, biased by one extremely high value. Thyroid weights for male control and parabiotic Sprague-Dawley rats lie within the general ranges reported by Braham (328) with the controls having significantly lighter thyroids than parabionts (P< 0.01). Absolute thyroid weights for both groups were not different with all weights in the normal range. The significance on a body weight basis was caused by the smaller physical size of the parabionts. Body weight for'the controls averaged 389 grams and for parabionts 334 grams. The .animals were all of the same age and in parabiosis for approximately .5% months at the time of sacrifice. Body size usually is smaller in parabionts but most internal organ weights are, on an absolute basis, tine same as single control animals. The amount of organ tissue per lurit body weight is, therefore, increased. No difference in testicular weights were found between single 132 controls and parabionts and all values were in agreement with weights reported by Tullner and Edgcomb (342), 1962, for Sprague-Dawley rats. Serum thyroid hormone iodide levels for parabiotic animals were significantly below control values. Normal ranges for serum hormonal iodine range from 3.0 to 6.0 ug% (343) and specifically for the tech- nique used here9 2.9 to 6.5 ug%. As seen from Table 25, all values fall into the normal range but the parabiotic levels are at the lower range. As shown by adrenal ascorbic acid data (to be discussed later), parabionts in this study probably were slightly stressed. A stress syndrome independent of the adrenals has been described whereby thyroid stimulating hormone secretion is inhibited. The effect is either on the pituitary or hypothalamus. This inhibition could cause a lowered serum thyroxin level (344). ACTH-corticoid activation has been shown to inhibit thyroid stim- uLating hormone secretion (345). Since these parabionts may be slightly stressed, this corticoid activation could inhibit TSH synthesis or release at the pituitary level causing a decreased thyroid activity and serum thyroid hormone. Hypervolemia in parabiotic animals (302) may slightly dilute thyroid hormone levels but other blood constituents (glucose, urea and.insulin) were normal; consequently, this does not seem to be a :factor. Blood volume in parabiotic animals reported here was slightly (alevated but not significantly over control values (see blood volume (liscussion in this thesis). Specific metabolism was the same for parabionts and controls. 9 lilo-Rad Laboratories, 220 Maple Avenue, Rockville Centre, New York. 11570. 133 This is of major concern.in food intake studies and since thyroid weights and thyroid hormone levels were normal it appears that para- biotic rats can be used in food intake experimentation without alter- ations in energy metabolism caused by the parabiotic state, per se. As stated earlier, the difference on a body weight basis between thyroflweights of parabionts and controls is probably a result of the decreased physical size of parabionts disproportionate to internal organ weights since parabionts do have normal absolute thyroid weights but a decreased body weight. Lower circulating thyroid hormone levels are not inconsistent with a statistical agreement in energy metabolism between parabionts and controls. PBI, e.g., can range from 4-8 ug% with basal metabolic rates remaining normal (346). Even hypothyroids can have normal basal metabolic rates (347). It is quite conceivable that free serum thyroxin is the same for the two groups. Only total thyroxin and T5 were mea- sured. Free hormone is only 0.10% of the total and is in equilibrium with the total bound hormone. It is the free thyroxin that enters cells and acts as a feedback regulator for TSH secretions. Usually, the concentration of free thyroxin changes proportionately with changes in bound hormone; consequently, measurement of the total hormone pool .is a good indication of thyroid function. The totalthyroid.hormone 'binding capacity can vary directly to the total bound hormone but in- ‘versely to turn-over'rate; consequently, free thyroxin (resulting from fWrnn-over) can be the same in groups of animals with different total txnrnd iodine. These animals would also have similar metabolic rates (348)- Parabiosis may affect the total circulating thyroid hormone level 134 by altering the thyroglobulin level or through some "stress" mechanism. Over a reasonable range of these kinds of effects the free serum thyroid hormone can be regulated within normal ranges. This is done by alter- ations in the "turn-over" rate if thyroglobulin levels and total bound hormone are low. Fasting serum glucose and plasma insulin levels (Tables 26 and 27) were the same for parabionts and controls. Fasting serum glucose levels agree well with those reported by Blazquez and Quijada (349, 350). .Immunoreactive insulin levels, during fasting, are reported to be around 25 micro units per ml. As shown in Table 27, fasting insulin levels for parabiotic and control animals are identical and agree with other published values (351). Chlouverakis (352) reported a small but significant decrease in blood glucose for parabiotic mice with no changes in serum insulin levels. Data reported here (Table 26) shows a slight but non-significant decrease in fasting serum glucose in parabiotic rats. There was no parabiotic effect in rats on plasma insulin levels (Table 27). This agrees with the report of Chlouverakis that parabiosis had no effect on serum insulin levels in parabiotic mice. The effect of fasting on serum free fatty acid levels is shown iJl Table 28. The difference between parabionts and controls was un- eaxpected. Fasting free fatty acid levels averaged 0.60 ueq./ml for Ixxrabionts and 1.07 ueq./ml for controls. These values (especially Ixxrabiotic values) are within normal ranges as shown by Regouw et al. (353). Takes: et al. (354). Harper (351). Gordon (355). Dole (356) arui Grossman et al. (357). Post prandial levels approximate 0.5 ueq./ml widfll fasting levels ranging from 0.6 to 0.8 ueq./ml. 135 Fasting serum free fatty acid levels for parabiotic animals were in perfect agreement with values reported in the literature for single rats. The difference between parabionts and controls was caused by an elevation, slightly over the "normaI" ranges, in the control ani- mals. Free fatty acid data determined on other single animals fed different diets were then compared to these values: (A) Animals fed a high fat diet (60% fat)measured .367 ueq./m1 serum; (B) Grain fed animals 0.252 ueq./ml; (0) Animals fasted for 17 hours, anesthetized with metofane but did not go through oxygen consumption measurement had levels of 0.685 ueq./ml; (D) The parabionts that were fasted for 17 hours, anesthetized with metofane to obtain heart blood and exposed to oxygen consumption determination displayed free fatty acid levels of 0.600 ueq./ml; and finally, (E) The controls for the latter group, 1.070 ueq./m1. It can be seen in comparing groups (C) and (D) that the oxygen consumption procedure did not effect free fatty acid levels and that parabionts agree almost exactly with controls. The second group of controls, (C) above, were housed one in a cage. The elevated free fatty acid levels in the original control group, group (E) above, could have been caused by a particular type of emotional excitement. The second control group, group (C), was killed immed- iately after the 17 hour fast. The first control group, (E), was also housed separately, but were measured in pairs for oxygen consump- tion which was done to simulate the paired parabiotic state. By pairing animals, immediately before oxygen consumption determination, that had not previously been caged together established a particular state of "emotional" excitement. This type of excitement is known to primarily elaborate norepinephrine which is a potent fat mobilizer but 136 a very weak ACTH stimulator; consequently, serum free fatty acids rose but the ACTH-corticoid stress system was not stimulated as shown by adrenal ascorbic acid data (to be discussed later). This system would not be activated in parabionts as they have been living together for many weeks. The demarcation of adrenal response to different types of "stress" or emotionalism is discussed by Russell (358). As a sensitive index of "stress", adrenal ascorbic acid levels were determined. Parabiotic rats did have significantly lower adrenal ascorbic acid than controls (P<0.02) (Table 29). Adrenal weights of these parabionts were slightly but not significantly larger. The parabiotic rats, evidently, still had not completely adapted to parabiosis. The parabiotic data are biased by two vary low values but the average vitamin C levels without these values are still some- what lower than that of the controls. Adrenal ascorbic acid for control animals, fasted for 17 hours and subjected to metofane after oxygen consumption determination, was 1.96 mg/g tissue. Concentration for male, Sprague-Dawley grain fed animals was 3.05 mg/g tissue. These data demonstrate the decreased ladrenal ascorbic acid seen after fasting or hypoglycemia. Hypoglycemia :is a potent stimulator of epinephrine and ACTH secretion. ACTH stim- lrLates the adrenal cortex to secrete glucocorticoids and in the process (If stimulation ascorbic acid is decreased. Epinephrine stimulates liJner phoSphorylase activity and, therefore, to a lower degree, fat ciepxlt fatty acid mobilization. Animals, not exposed to oxygen consump- 'ticnl procedures but still fasted and subjected to metofane, had an adrenal ascorbic. acid level of 1.9 mg/g tissue. This value is almost idenrtical to the first group of controls reported above showing no 137 additional ACTH-corticoid or epinephrine activation, which gives further support for the argument that the elevated free fatty acid release in the first group of controls resulted from norepinephrine stimulation. These data also indicate that the oxygen consumption assay system did not cause an increased stress as evidenced by similar free fatty acid and ascorbic acid values as data from animals not having gone through this procedure. The lower adrenal ascorbic acid in parabionts, there- fore, was due to the parabiotic state. Parabiotic serum urea nitrogen was higher but not statistically different from controls. This possibly was due to a higher fasting rate of gluconeogenesis stimulated by a lower blood glucose. Normal serum urea nitrogen values are around 8-20 mg%.10 The values reported here fall within the normal range. In conclusion, no changes were observed in energy metabolism measurements between control and parabionts. Adrenal and testicular weights, expressed absolutely or per unit body weight, were not diff- erent between experimental groups. Thyroid weights were not different, absolutely, but were on a body weight basis because of the smaller physical size of parabionts relative to certain internal organ weights. The serum thyroid hormones were lower in parabionts but since energy Inetabolism was not affected it was concluded that the total bound hor- lnone was decreased with free thyroxin unchanged, possibly due to an increased turnover rate from the total bound hormone. Tbtal serum 'thyroid hormone could have been lower because of the "stress factor" lwith the ACTH-corticoid system inhibiting TSH at the pituitary level. :Stress was indicated by lower adrenal ascorbic acid in the parabionts. 11) Hyland, Div. Travenol Laboratories, Inc. Costa Mesa, California. 92626. 138 No change was noted in serum glucose and urea or pLasma insulin. Fasting free fatty acids were normal in parabionts but elevated in controls for reasons discussed. The only major factor that could affect food intake would be the slight degree of stress. No parabiotic adrenal weights thus far report- ed have been different from controls but ascorbic acid was lower for parabionts presented here. This difference existed even after 5% months. Usually adaptation occurs before this length of time (359). Parabiotic and control animals were fasted for the same length of time and handled in the same manner. Everything was controlled with the state of parabiosis as experimental variable. It is possible that parabiotic rats are "stressed" differently by various experimental procedures than single control rats. It is suggested that when possible some measurement of stress be made in parabiotic experiments _ to obviate the possibility that experimental results were actually a manifestation of a general "stress syndrome." 139 8m.4s4 .N8.a4 884. 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H_oo.« . +4 A .0. no mz *amm. @53o. .moem. Noam. quoom.m .oooo. “.mmfim.m +« m .w m< AI mama: mo m a. commands .Q .m + cam: mu m . :omahmmeoo moz Hmuwuauu vmuswwm3+ ummuuu mumefixouaa< *¥ Nh.m .Ho. mm.~ w sq.m .mm. m oo.m +- N .o m< . _ mammz mo moamwum> .o .m.H amoz mu m acmaumanu MUZ .Q .m + cmmz wv c com«HMQEoo mUZ .Q .m + cmmz mv : comwumano MUZ .Q .m_H cam: m6 : confinmmsoo .moz .o .m_H_:muZ mv c cemHHmano mozmd3 mm mHQNH .149 OOH. mmo.~ om.w .o©.m~ mm.~.H mm.mH .ow.Q.H em.mH HH 5 .o m< mammz mo m u wocmwum> .Q .m.H :mmz mu : cemHumanu mozmA3post prandial circulating substance in group 5, caused by high .fat.feeding, could have produced such a rapid response in group 6. Elince group 6 did regulate upwards, it also seems probable that ani- rmals in group 6 simply had their meal frequency upset by being attached to arlanimal consuming a high fat diet. It has been reported (24) 174 that altering the caloric density of the diet will change meal fre- quency in the rat but that after 6 or 7 days frequency returns to nor- mal and any alterations in grams of food intake to compensate for calorie density of the diet is accomplished by changing meal size. The major disruption of normal intake in this experiment lasted for approximately 7 days at which time it is postulated, the high fat fed animals (group 5) reverted back to a normal frequency and at this time the grain fed partner began to regulate intake upwards. Frequency of eating in the ad lib. differential feeder is extremely important since animals must both be willing to go into the feeder at the same time. The animals would now be on the same frequency but would vary in meal size and also the ease and time of actual consumption of the different diets. The grain fed animal would have to increase consumption per unit time in order to compete with the high fat fed animal's shorter meal both in grams and probably also in time. . Grain fed parabionts, cross-circulating with high fat fed partners, after regulating their intakes upwards, leveled off at an average consumption of 76.9% of their previous control values (days 1-23). It seems reasonable to conclude that the depressed intakes from day 39 to 62 (almost 3% weeks) in group 6 were caused by some factor that was cross—circulating from the high fat fed animal and would be indic- artive of a systemic component in the regulation of food intake. Perhaps Istuoies using iso-caloric diets varying in different types of food components but having the same caloric density and particulate consis- tency would be very valuable in implicating a particular dietary factor at; being appetite depressive. Caloric density problems as the one described above would thus be avoided. 2‘ M: 175 A few summarizing comments would perhaps be appropriate at this time. Three basic interactions of experimental comparisons of group means are extremely important. These interactions are group 2 vs. 4 (single grain fed controls vs. parabiotic grain fed controls), group 1 vs. 3 (single high fat fed controls vs. parabiotic high fat controls), and group 5 vs. 6 (high fat fed animals cross—circulating with a grain fed rat vs. grain fed animals cross-circulating with high fat fed ani- mals). Groups 5 and 6 constitute the parabiotic pairs that were fed different rations. The high fat fed member of a pair is in group 5 and its partner, grain fed, is in group 6. It has been shown that parabiotic animals on a "normal" diet do have less body fat as a per- centage of body weight as compared to single rats fed the same diet (comparison 2, 4; Table 47). Absolute percentages are much lower for parabionts. Statistical significance is only 0.10 but the t-value is extremely high and just misses 0.05; consequently, the probability is far less than 0.10 that the difference was due to chance. Hervey (283) reported that his parabiotic rats regulated to one-half the body fat content of the controls. Hervey's rats were in parabiosis for 8 months and the pairs reported here had been sustained for 5 months. The general trend in lowered body fat reported here does corroborate Herwey's observation with the difference in magnitude being one of length of time in parabiosis. Energy intakes were statistically the same for groups 2 and 4 with actual values slightly higher for the parabionts (group 4). Para- birnits in group 4 show a higher food efficiency for the entire exper- inmnlt. During the control period, this was not significant, but during tine experimental period it became so. Rates of body weight gain agree 176 well with efficiency data in that parabionts (group 4) did gain more body weight but the increased gain was not significant compared to grain controls (group 2). Even with an increased body weight gain and food efficiency, body fat was regulated lower in parabiotic grain fed animals. Possibly, there are also circulating factors directly responsible for regulating body fattiness. The argument proposed by Hervey (283) is that each animal in parabiosis is actually responding to approximately twice as much body fat within its own regulatory system. Responses to a surfeit of fat would tend to decrease the excess. Fur- ther support for a suppression of body fat is seen in fat organ analyses. Virtually all fat organs removed from parabiotic animals were markedly lower in actual weight (Tables 48-49; 51-52). In previous experiments practically all fat organs absolutely and per unit body weight were lower in weight compared to controls (see Part 6, this thesis). Interaction l, 3 (single high fat controls vs. parabiotic high fat controls) provides further evidence for increased suppression of body fat in parabionts. High fat fed parabionts (group 3) compared to high fat fed controls (group 1) ate fewer calories, were less effi- cient, had markedly less body fat with all fat depot weights being much smaller, and had a lower rate of body weight gain. Parabionts seem to be able to handle a high fat diet far better than single ani- Imals. The decreased food intake may be a result of a greater release of gastrointestinal tract hormones in high fat fed parabiotic rats (group 3) (see literature review, GIT hormones). In the differentially fed pairs (group 5 vs. group 6), the high fai;:fed animal did not become obese and the grain fed animal became leaxuar than the single or parabiotic control. Group 5 displayed a 177 caloric hyperphagia when fed the high fat diet compared to the high fat fed parabiotic controls (high fat - high fat) but was markedly less efficient. Body fat in group 5 was lower than in the parabiotic high fat fed controls but not statistically so. All fat depots for group 5 were statistically lower in weight compared to group 3 and body weight gain was depressed. Rats in group 6 became leaner when attached to animals fed the high fat ration compared to grain fed parabiotic controls. Food intake decreased, food efficiency was the lowest for any grain fed group, body fat was extremely low,all fat depots were lighter and the total amount of body weight gain was decreased. Group 5 at this time was depositing fat and did increase body fat higher than normally seen in grain fed parabiotic animals. The inhibition of food intake with a decreased body fat in grain fed animals attached to animals that were calorically hyperphagic and increasing body fat stores (in other words, nutritionally hyperphagic caused by high fat feeding) has not been previously reported. The reduction in weight of a lean parabiont joined to one that is overeat- ing or is obese is a phenomenon that has been observed by a number of investigators. This was true of Hervey's (283) work when he joined hypothalamically lesioned hyperphagic and Obese animals with normal :nats. Haessler and Crawford (287) noted a similar change when they joined obese, hyperglycemic mice with normal mice. The hyperphagic dietetic mouse was used by Coleman and Hummel (291); and most recently (Hnlouverakis (352) reported that lean mice, when joined to obese diabetic mice, lost weight. The data reported here, using a nutritional model, are in 178 agreement with all of the above papers. Possibly, overly fed or obese animals, in this case group 5, respond to excessive caloric intake by trying to correct the energy imbalance. This response, if humorally mediated, could cross-over to the lean animal causing a reduction of its feed intake and consequently a loss in body weight. Meal fed animals have an almost identical suppression of food intake as that seen in group 6. Even with a reduction of food con- “ I “‘3‘.‘ ~...‘_ ’3. .- .o 1 sumption equal to 25%, meal feeders have increased food efficiencies, Li stomach weights and body fat contents with body weight gains remaining constant and equal to that of the ad lib. fed controls.1 It is doubt- ful whether meal eating can explain the changes that occurred in the parabiotic rats. Although the parabiotic rats consumed only 75% of their "normal" feed intake, there was a reduction in their feed iffi- ciency (Table 44), body fat content (Table 47), and rates of body weight gain (Table 46; Figure 42). Even stomach weights were lower than in controls confirming the suggestion that "meal eating" was likely not involved in the development of the observed condition in the rats used in the present study. Animals in group 6 were probably exposed to their food with the same frequency as when their partners (animals in group 5) were also consuming grain and had, therefore, eunple opportunity to increase their consumption if desired. At the initiation of differential feeding the average body weights kar animals in group 5 and 6 were 222 and 211 grams respectively; ccwusequently, initial intake suppression in group 6 was not a result of‘Iharger, satiated animals in group 5, physically keeping the rats in ggroup 6 out of the feeder. If any animals were dominant, it should 1 Personal communication, Dr. Gilbert A. Leveille, Chairman, Dept. of FExxi Science and Human Nutrition, Michigan State University. 179 be those in group 6; they should have been able to lead the pair into the feeder if they wished to eat. Grain fed animals attached to high fat fed partners do have the ability to consume their normal daily intakes if they want to. Rela- tively normal intakes have been seen in differentially fed pairs where cross-circulation may have been extremely low which meant that the union was only a physical one. This virtually eliminates any effect of differential feeding, per se, as causing the intake depression. In other words if these animals were not cross-circulating but still attached and feeding differentially, they would have had the ability to consume their normal intakes. Evidence seems to support the contention that an anorexigenic circulating substance coming from the high fat fed animal was respon- sible for inhibiting the intake in the grain fed animal. These find— ings also indicate that the decreased food intake seen when single animals are placed on a high fat diet may partially be humorally medi- ated in addition to the long standing argument that the fat has a longer resident time in the gastro-intestinal tract increasing its satiety value. Suggestive evidence is presented for the existence of this humoral :factor in these studies. The effect would not be as marked in the ggrain fed animals (group 6) as only small amounts per unit time of line postulated "factor" could crossover from the high fat fed rat. (Irossover rates must also exceed clearance for a physiological reSponse tr) be manifested. As seen from the data (Tables 32-33) suppression of‘:intake is about 3 grams per day in the grain fed animals (group 6) tnrt may be 6-8 grams when both parabionts are fed the high fat ration. 180 If some factor is crossing from the high fat fed animal then the intake suppression in group 5 should not be as marked as in either groups 1 or 3 as some of the factor is being lost. Group 5 has the highest intake of any of Huehigh fat fed groups or, i.e., intake was not as depressed as would be eXpected, indicating that an "appetite depressive factor" was partially lost. ‘9 "'tmltiaa...‘ 'I .5 181 Fulfill-III. ;I|m.u . . . . . l . . I . muxv mz mom on mas .oo sea as o a we saw mm mm om saw as um AH . . . . . Na.m + mo.o oo.m + mm.o Awe AocoEHwoowo mo osov mz me on o om q «a w w mHmEHcm com new :wH: :uHs wcHumaooHHoImmouo mucoHnmuma pom HIE . . . . . .H.H ma.ma ao.H.H wN.mH Awe ocoeauooxo mz omq ow N mm N m m n no NO one um mucoHnmuma Houucoo pom HI: .I . . 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The "means" for the control and recovery periods are 5.00 and 5.82 grams per 100 grams body weight, respectively. This is slightly over a 16% increase but significance was lost due to the large variance. Similar food intake augmentation after deprivation has been reported in non-parabiotic rats (26-28). Food intake suppres- sion in the present study could have been due to an "anorexigenic factor" (possibly a nutrient) crossing-over from parabionts in group 3 (high fat fed) to their grain fed partners (group 4). Group 3 was then placed back on high fat and day 24 shows this first high fat intake. This time group 3 was fed high fat until the end of the experiment. During the last 8 days of the experiment, statistical analyses were done in order to compare the different levels of intake for the four groups. As with female animals, group 4 (grain fed animals cross-circulating with a high fat fed animal) showed a similar food intake depression (36.5%). All curves from day 28 through day 35 were statistically different (Figures 43-50) for all 6 compari- sons except 1, 3. A couple of days prior to scarifice, the differential feeders were checked for proper usage with a 1% dietary tag of chromium ses- quioxide (for technique description, see section on an Ad Lib. Diff- erential Feeder for Parabiotic Rats, this thesis). There was, statis- tically, no crossage of the marker from one side of the feeder to the 259 other indicating that the parabionts were consuming their correct diets (P > .05). Since intakes looked quite stable for about a week, the animals were sacrificed on day 35. After euthanasia, thyroid, adrenal and testicular weights were determined. Organ weight data are expressed absolutely and as a percentage of body weight. Absolute organ weights probably are more accurate than relative weights since there are dis- proportinate body weight to organ weight changes in parabiosis and pair feeding (Tables 65—67). For example, Table 67 shows thyroid weights, both adjusted and unadjusted, for the pair fed group and its controls. The absolute thyroid weights are statistically the same for both groups but, on a body weight percentage, the pair fed animals had significantly higher weights. The pair fed group lost an average body weight per animal of 67 grams during the paired feeding period. This loss in body weight, disproportionate to loss in organ weight, explains the signifi- cance at the adjusted levels. The subsequent discussion will focus on group 4 (grain fed parabionts cross-circulating with high fat fed part- ners) in which food intake was suppressed. In no case is any comparison of means between thyroid weights different for either unadjusted or adjusted values (Tables 65-67). On an absolute weight basis (Table 65) all mean comparisons were non- significant except comparisons l, 4 and 3, 4, because of a slight but non-significant elevation of thyroid weights in the high fat fed ani- mals and because the thyroids for group 4 were at the light end of the normal range. This assumption was verified by the observation that group 4's thyroid weights were not different from group 2 (the single grain fed controls) and are not different from either the pair fed rats 260 (group 5) or the controls for group 5 as shown on Table 67. The average absolute thyroid weight for group 4 is 16.2 mg and is almost identical to the pair fed animal's (group 5) at 16.0 mg and the pair fed controls, 16.4 mg. This, in addition to group 4's thyroid weights being in the normal range (328), indicates that the experiment produced no detri- mental effect on thyroid weights and that thyroid function was not altered severely enough to change thyroid weight. The only adrenal weight comparisons that were different, expressed absolutely or as a percentage of body weight, were 1, 3 (single high fat vs. parabiotic high fat ) and 2, 3 (single grain vs. parabiotic high fat) (Tables 68-71). The highest adrenal weights were found in the parabiotic high fat animals. Perhaps the parabiosis combined with the high fat to produce an additive effect. These adrenal weights were not hypertrophied, however, and were in the normal range (364). In the food intake suppressed rats (group 4), the adrenal weights were statistically the same as all other groups whether expressed absolutely or as a percentage of body weight. Absolute adrenal weights for the pair fed group versus their own controls (Table 70) were statis- tically the same but were different expressed on a percentage of body weight. This difference is basically the result of a loss in weight of the pair fed group. Even with the slight difference noted in adrenal weights, adrenal ascorbic acid comparisons were all non-significant (Tables 72-73): consequently, "stress" was not a component in food intake responses. Adrenal ascorbic acid values are in agreement with those previously reported for male Spargue-Dawley rats (359). Slight variances in adrenal ascorbic acid will be noted between different investigators as a result 261 of differences in analytical procedures and experimental design, e.g., in animal room environment. Tasticular weights were the same on an absolute basis with com- parisons l, 3 and 2, 3 showing differences per 100 grams body weight. Both of these comparisons were different because the testicular weights of the high fat parabionts (group 3) were slightly elevated. Group 4 exhibited testicular weights the same as controls (comparison 2, 4). As seen from Table 76, high fat fed animals had significantly higher levels of serum free fatty acids as compared to grain fed rats. This confirms other observations on the effect of lipemia and lipo- protein lipase on free fatty acid levels in blood (35?, 365). Group 4 (the food depressed rats attached to high fat fed rats or group 3) (see Table 76) had almost exactly the same levels of post—prandial serum free fatty acids as group 2 (single grain fed controls). Group 4 had one—half the free fatty acid levels as group 5 (the rats pair fed to group 4). The elevated free fatty acids, in group 5, may be parti- ally explained by the low blood insulin levels, since that hormone inhibits the release of fat depot free fatty acids. Carbohydrate in- take was also very low in group 5 and, with the decreased L- {-glycerol phosphate, free fatty acid release from depot fat should be enchanced (366). Growth hormone, glucagon, glucocorticoids, ACTH, TSH, epinephrine andnorepinephrine all stimulate free fatty acid release in hypoglycemia (366-368). Once high levels of free fatty acids are obtained, stimu- lated initially by low levels of blood glucose, free fatty acids can produce an anti-insulin effect by inhibiting the entry of glucose into muscle including the heart (366). The net effect is to supply energy 262 for muscle in the form of free fatty acids and to spare glucose for use by the nervous system. There are many other hormone interactions in the metabolic response to hypoglycemia, e.g., many of the above hormones stimulate gluconeogenesis as well as glycogenolysis. This is shown by epinephrine's inhibition of insulin secretion (366). Minute amounts of epinephrine are effective in that system since its effect is greatly amplified by the adenyl cyclase system (389). Growth hormone can be anti-insulin by decreasing glucose transport and utilization (glycolysis) in muscle (366, 370). Typical hypoglycemic responses were not seen in group 4 even with the food intake decreased by almost one-half. Free fatty acid levels for group 4 were "normal" for a grain fed post- prandial state (371) and were not elevated even with cross-circulation from a hyperlipemic rat (see comparison 2, 3; Table 76). The relatively low blood exchange rates in parabiotic animals (283) (see also Dye Dilution Studies, this thesis) and the rapid clearance rate of free fatty acids from blood (371), probably account for the failure of free fatty acids from the hyperlipemic rat to appear in the grain fed rat. Pair fed animals exhibited serum free fatty acid levels far higher than their controls2 (Table 77) but, as expected, the variability was so high that significance was lost, i.e., since animals were fed widely varying amounts of food, the serum free fatty acid levels would reflect the level of food deprivation and, consequently, the variance between different rats would be extreme. Significance was lost due to this "essential" variance. Since the statistical test used did not realistically reflect the difference 2 By necessity the pair fed group was started after the beginning of the experiment when levels of food intake for pair feeding were known; consequently, a specific control group was employed with the pair fed group. 263 between the two means which was quite large (Table 77), the data were paired and statistically analyzed by a "sign" test or corrected chi- square. The assumption was made that pair feeding or a decreased intake would increase serum free fatty acid levels, consequently, the pair fed values (Table 77) were designated "treatment 1" and the control values in Table 77, "treatment 2." Pairing was done so that pair one would constitute the largest number in each group, the second largest in each group pair 2, etc. The five pairs were then substracted (treatment 1 minus treatment 2). In every pair, the pair fed group exhibited higher free fatty acid levels, i.e., the sign values were 5 (+) and 0 (-). On a sign test, pair fed animals exhibited a marked increased tendency or trend in serum free fatty acid levels (P<.05). Table 76 shows the comparison of group 4 and group 5 (P‘<.10). Group 4 (the food depressed group) has about one-half the level of serum free fatty acids. Both of these groups of animals received the same amounts of food but since each group was fed at a different point in time in addition to other variables extremely hard to control such as, exact animal weights and ages at the beginning of the experiments, the best comparison between groups 4 and 5 is the difference for each group between experimental and control values. Group 2 values (the single M-l fed controls for group 4) were subtracted from group 4 and the specific pair fed control values were subtracted from the pair fed group. These differences were taken after pairing as previously de- scribed, followed by statistical analysis with a sign test. The serum free fatty acids were elevated to a greater extent in the pair fed group (P <.05) than in the controls. To summarize, free fatty acid levels in the food depressed group 264 (group 4) did not follow the typical trends of food deprived animals. Serum free fatty acids of group 4 were statistically as well as almost absolutely identical to the ad lib. grain fed single control animals (group 2). If group 4 had been metabolically deprived by a low food intake, serum free fatty acid levels would have been elevated. Several sources of error could not be avoided in pair feeding: 1) It was not evident that a pair fed group would be needed until after feeding experiments had begun; consequently, new animals had to be ordered. They were obtained from the same breeding farms but the original stock was still not represented in the pair fed group; 2) In order to pair body weights with group 4, younger animals were ordered; 3) With present equipment it is not possible to pair feed with the same periodicity as the intake in group 4; and 4) Control intakes of the pair fed group were a little higher than control intakes for group 2 or group 4. This was noticed only after data tabulation at the termi- nation of the eXperiment. Data were only reviewed at the end of ex- periments in order to avoid "investigator bias," while collecting data. Absolute intakes and body weights were paired quite well between the pair fed group and group 4 during the experimental phase of the feeding trials. It still seems reasonable, though, to expect the free fatty acid levels to be higher in group 4 based only on their absolute food intake. Serum urea nitrogen was normal for all groups with group 4 having statistically the same level as group 2 despite the depressed food intake (Table 78). Starvation in rats greatly increases urine urea excretion within approximately a day (372). Comparisons of groups 2 and 3 and 3 and 4 for blood urea levels 265 are statistically different because group 3 has a relatively low value compared to other groups possibly due to transfer of either urea or amino acids to the food depressed group (group 4) as indicated by group 4 having higher levels compared to group 3. As noted on Table 78, serum urea in group 4 was the same as in the pair fed group. The diff- erence between group 4 and their controls (group 2) compared with the difference between the pair fed rats and their controls was also non- significant whether tested by paired or pooled t-statistics or a sign test as described earlier. With any major involvement of amino acid gluconeogenesis, serum urea levels would have been much higher. There was no difference between the pair fed group and their controls. The decreased urea production caused by a decreased intake possibly was offset equally by an increased urea production associated with gluco- neogenesis (Table 79). Gluconeogenesis from glycerol probably was of greater importance since glycerol levels would be elevated with an in- creased fat depot mobilization of free fatty acids. The pair fed group was food deprived, consuming less than half their normal intake; nevertheless, a reasonable degree of dietary foodstuffs were still present with metabolic changes offsetting an energy or glucose deficiency not nearly as pronounced as in starvation. The length of deprivation was just ten days. It is conceivable that during this time gluconeogenesis, from glycerol, with subsequent glyco- gen synthesis and glycogenolysis would have been adequate to maintain these animals in reasonable balance without the involvement of ACTH- corticoid activation. It is evident from the adrenal vitamin C levels (Tables 72—73) that the ACTH-corticoid system was not activated with ascorbic acid levels within normal ranges. With the mobilization of 266 depot fat for added energy by growth hormone, glucagon and, to a lesser extent, catecholamines, the protein consumed would be spared. Growth hormone also increases free fatty acid movement into muscle. An increase in serum urea nitrogen would be contraindicated by the lack of the ACTH-corticoid system and by dietary protein sparing; consequently, all serum urea values were statistically the same except for the two comparisons discussed. Plasma insulin (Tables 80-81) tended to be lower in the parabiotic animals fed the high fat diet, although, the comparison between single control groups (comparison 1, 2) was non-significant. Other investi- gators have reported no statistical difference in plasma insulin between high fat fed rats compared to grain fed controls (325). Injection of a fat emulsion in humans, intravenously, caused no change in insulin levels (373). Plasma insulin levels in parabiotic high fat fed rats (group 3) were not statistically different from control high fat fed rats but were statistically lower than grain fed rats (comparison 2, 3). Insulin levels for group 4 approximated very closely the levels in group 3. There was a tendency for lower levels in high fat fed ani- mals, probably due to lower carbohydrate intakes, the lower food mass in the gastro-intestinal tract and the greater retention of the high fat diet in the stomach. Groups 3 and 4 were slightly lower, for rea- sons to be discussed later, while group 2 was higher than all other groups. The extent of this difference reached significance in compar- isons 2, 3 and 2, 4. All other comparisons were non-significant. The values in group 2 were high probably due to the ad lib. consumption of a high carbohydrate diet with the grain ration containing 53.5% carbohydrate compared to only 6.5% for the high fat ration (374). 267 Insulin levels in groups 3 and 4 approximated each other. The lower initial secretion in group 4 was expected since oral carbohydrate intake was decreased. Castro-intestinal hormone secretion would also be decreased with the lower food intake. Castro-intestinal hormones. have been shown to be stimulators of insulin secretion (161, 162, 375). There was no statistical difference in plasma insulin levels between the pair fed values with those of group 4 but group 5 did have lower absolute values. By comparing the differences between the control group for group 4 and group 4, with the difference between the control group of the pair fed animals and the pair fed group (paired as pre- viously described) a significant difference was obtained both by paired-t and sign tests (I*<.05), the pair fed group having a lower trend in plasma insulin levels. In summary, insulin levels in group 4 were higher than in pair fed animals but lower than single grain controls (group 2). Plasma insulin in group 4 approximated the levels in their high fat fed part— ners, group 3. Insulin could cross from group 3 to group 4 or vice versa since insulin can leave the pancreas by the lymph (activity is very high in the thoracic duct) and crosses to the other parabiont with- out passing through the liver. Insulin present in the interstitial fluid could also cross more readily from one parabiont to the other (366). Plasma insulin for the pair fed group was lower than their controls as a result of a lower carbohydrate or protein intake (Table 81). The insulin levels for groups 1, 3 and 4 are all normal values for moderate to low carbohydrate intakes (349). All glucose values are expressed in mg% for a constant volume of Somoygi filtrate (Tables 82-84). To approximate mg% in serum, all 268 values are multiplied by 12 but for statistical comparison of means this is not necessary. Fasted blood glucose levels were significantly lower than controls (Table 83). This comparison was made as a check on the glucose assay system. Fasting serum levels were calculated to approximately 94 mg%. Rats pair fed grain had levels of 175 mg% with single animals fed grain ad lib. averaging 201 mg%. These glucose values agree well with those previously reported (349, 350). There was a decrease in serum glucose in the pair fed group, as shown above, that approached significance (Table 84); whereas, the food depressed group (group 4) had almost identical glucose levels as both the single grain controls (group 2)and the controls for the pair fed group. By comparing the difference between the controls for group 4 and group 4 with the difference between the controls for the pair fed group and the pair fed animals, one obtains a paired-t statistic of 2.258 which misses I*<.05 by only .048 and one obtains by a sign test (I’<.05) indicating a lower trend in blood glucose in the pair fed group as opposed to group 4. It does appear that serum glucose levels are higher in group 4 (food depressed group) than what would be expected by looking at the pair fed data. DISCUSSION The ad lib. differential feeder was very efficient in permitting each animal to consume its own diet as evidenced by chromium sesqui- oxide dietary markers. Analysis of adrenal ascorbic acid indicated that changes in food intake were not a result of "stress" as resulting from parabiosis or 269 use of the differential feeder. Food intake was suppressed in grain fed animals (group 4) attached to high fat fed animals (group 3) compared to control values for group 4. This suppression occurred with blood urea, glucose, and free fatty acid levels statistically the same as single grain ad lib. fed controls. Two factors can probably be ruled out as producing this effect: 1) Insulin and 2) Castro-intestinal distension. Insulin levels were normal for group 4 but, since the absolute amount of carbohydrate consumed was about half of that consumed by group 2, insulin levels were depressed. Insulin levels in groups 1 and 3 were statistically the same as those in group 4 with groups 1 and 3 consuming more cal- ories. Lower insulin levels (compared to group 2), therefore, would not explain the depressed consumption in group 4. Castro-intestinal distension would seem unlikely as having pro- duced the depressed intake in group 4 since these animals were consuming one—half or less of their control consumptions. Several factors could explain the intake suppression in group 4. Glucose levels were the same in group 4 as group 2 indicating a possible cross-over from group 3. Free fatty acids, crossing over from group 3, could have affected the energy balance of group 4. Since free fatty acids and glucose are cleared so rapidly relative to cross-over rates, it is difficult to demonstrate elevated levels but these compounds could cross the union, be absorbed quickly, and alter energy balance. An anorexigenic substance liberated by the gut, stimulated by the presence of fat, could have crossed over from group 3 to group 4 in- hibiting intake in the grain fed animal. If this were the only mech- anism, the "blood pattern" in group 4 should have approximated a 270 semi-starved state as opposed to the well fed condition observed. As evidenced by the experiments presented, there does seem to be an independent regulation of body fat as well as food intake but this involves a very complex interrelationship. When both parabionts in a pair are eating a low fat grain ration, their food intake is statisti- cally identical to single sham operated controls but their body fat content is lower. One must conclude that body fat was acted upon independently of food intake. Grain fed animals cross-circulating with high fat fed rats, have a further reduction in body fat probably caused by the reduced food intake. It seems reasonable to conclude that the depressed food intake caused the reduction in body fat (and not the converse) since the response was so rapid. The system becomes very complex if the anorexigenic factor coming from the high fat fed animal is one that contains calories which would tend to counter the reduction in body fat already present in parabiosis. 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H om.o3 +. m . m.~ . . mcmwz MO m u mucwHuw> .n .m.H cam: 00 : cemHumano m>oo< omamHg mmog<> mmp mo mzmHZ HmoHuHuo vmustwB. 4 ammuyu wumEonuaa¢ u... o~o. «333.m .33. .Hm.N3 No. w no.5 .3m.m « o3.o3. _+- m .m 33 mammz 00 m u mocmHum> .0 .m.H cam: 00 : cemHuwano m>om< QMHmHH mm3H<> mmH mo mz .05 mHan .m asouw mm 06mm wnu mH mHmEHcm mo macaw chH H oo3. 3oo.~ 33.3 .on.m 33.3 H 33.33 .No.3 H 33.33 o m .m 33 . . . mama: 00 m u mocmHum> .0 .m.H cam: 00 c cemHummEoo m>om< DmemHH mm3H<> MIR ho mz I ‘ I 2 . / ‘ I ' . 5 '- I l | [\2 2\ ' 3 ' I, ' I ‘ ' ‘ z ‘ ’ \' ‘ I ' | ‘ II ‘ . k .- : l I 'I I! L ,' I II \/ \A ’2./\ . , I , I I ' I I I \- \‘ 1+ z - I I " I I I I I 2 . I I l I I I I I I . 1 l \ I I I 3 'I ’ ‘1' I I'- I )M. s I 2 ’5 I! \ I b/ ‘ /\ O I \ g . / A I. I I ,\ I \ o L \ \ B B é . l \ I I I ..l \ o 3 : I ‘\ II :\,I \\ o I \ I b O z ‘ \I.-I’. : I . I 2 z I : I . I : I _..—(2) SINGLE CONTROL, FED GRAIN DIET FROM DAYS 1 TO 35 . I : I ..-..-— (h) PARABIOTIC, FED GRAD! DIET FROM DAY 1 T0 35 (CRCBS CIR- 1 . .' GUIATING IIITH ANIMALS DESIGNATED (3) ABOVE) 3 0 z o o o o O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 10 20 DAYS 30 35 Figure 49. Food intakes per 100 grams body weight of control and parabiotic male SpragJe-Dawley rats. (Av./Animal/Day) 298 7 2 2 'I O 1‘ 2 I’ I II 2 . I II 6 . ? NY I \ I I o I 17 ’ I { I : l‘ I‘\‘ ‘1 ' o A I 3‘ I ‘ z [I 1.. | l 2 I ‘ ’ -' 2 f I 5 z '33.: 1",” 1.1.1, I ‘ o z :| 2: 2‘1 I I o I . J l '_ I . I. - j ,3 .‘ 3 3| 6 . I. 4 ... " 1 2| ’1 : ’3 I l' :k I 3 -_ DAY II o I} I :2 .\ I 3 3 J." I I . I" ‘ . ' \ l '. I 1 LI : I: I; \3 3 , s 1i I I 0 . I \ - I 9:30 ' ,II I.’ I 3 .1 P'f'“ 3‘ E : 5.. |’ \ 13 ‘/ 3..3..‘ ,\ u . /.' 1 v '. " \ I \ o I», .' k l ', I I \ ‘1 \ o . I - | I I 3 2 '1" 3 \\ I J I ~ \\ 2 I]. \ ' 3 1'.“ .3 \ ' ° \ I '. .' 3 0 ’1 A . .3 ~ 1 : r. “ '-. '--’ ’ o r .. O r 3 2 2 r :- r 3 I.- 2 c . . . . . . (3) PARABIO’I'IC, FED GRAIN FROM DAY 1 TO 10, HIGH FAT FROM DAY : c 11 TO 16, GRAIN FRCH DAY 17 T0 23 AND HIGH FAT FRO! DAY 24 1 ; g- TO 35 (GROSS GIRGULATING IIITR GRAIN FED ANIMAL) : _-- -- (1+) FARABIOTIG, FED GRAIN DIET FROM DAY 1 T035 (GROSS CIR- : GUIATING IIITM ANIMAIS DESIGNATED ( 3) ABOVE) 2 O.0000.0000000000000000000000000000000 10 ' 20 DAYS -30 ' ‘ 35 Figure 50. Food intake er 100 grams body weight of parabiotic male Sprague-Dawley rats. (Am/inimaI/Day) 10. 11. BIBLIOGRAPHY Mayer, J. (1967) General characteristics of the regulation of food intake. In Alimentary Canal I: Handbook of Physiology. Ed. C. F. Code, American Physiological Society, Washington, D.C.. p. 3. Brengelmann, G. and A. C. Brown (1965) Temperature regulation. In Physiology and Biophysics. Eds., T. C. Ruch and H. D. Patton, H. B. Saunders Company, Philadelphia, p. 1050. Schnakenberg, D. D.. L. F. Krabill and P. G. 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