I“ v u-‘uy .n‘ ‘EI )Ovfibro l . .3. gr. . r. 3.3:? . .‘ri ll'qtlifléx 2 . |. l I... 7“: . Ev! » fairway :- ‘ ‘ , ; _ cithlr ..\..}...u.o| . (a! «t. P: 15105.?) if.» 3:... 4.... . is! I. r, . {-1.}: k .. 5.... 99.45... ... 1 ”nu-"urn” -.vm “.1 ... "my“. “q. . -{l‘t'3flhv4ib . . {.7111}..va I . . . . I... , .I rll 1).! ’12.)! , .t‘ . 155.15)»: . 2 (lift?!) .qlv’ofi. v). .r tittf. .1 'plb..v .1 .1,lvl5a11..£.v...~0 kl!v!r;?v!-}Pllnv :. .13. i.k.’ll'|. t1 . .. 0.. .. ... tot-ILrvlo... man. Lfl‘ NIVERSITY LIBRARI IE IIIIIIIIIIIIIIIIIIIIIIIIIIIII I III III II 31293 This is to certify that the thesis entitled Dietary Glucose Increases Plasama Insulin and Decreases Brown Adipose Tissue Thermogenic Activity in Adrenalectomized Ob/ob Mice presented by Ye- Min Nei has been accepted towards fulfillment of the requirements for M.S. degree in Human Nutrition Major professor Date June 71 1990 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. f———.__—._——_———__—_._"_——————— DATE DUE DATE DUE DATE DUE L___ I j I I MSU Is An Affirmative Action/Equal Opportunity Institution 6mm.“ DIETARY GLUCOSE INCREASES PLASMA INSULIN AND DECREASES BROWN ADIPOSE TISSUE THERMOGENIC ACTIVITY IN ADRENALECTOMIZED OB/OB MICE By Ye-Min Nei A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1990 647* 5048 ABSTRACT DIETARY GLUCOSE INCREASES PLASMA INSULIN AND DECREASES BROWN ADIPOSE TISSUE THERMOGENIC ACTIVITY IN ADRENALECTOMIZED OB/OB MICE By Ye—Mz'n Nei Adrenalectomy reduces plasma insulin and increases brown adipose tissue metabolism in ob/ob mice fed high starch diets, but has minimal influence on these parameters in ob/ob mice fed high glucose diets. The purpose of my study was to determine if consumption of a high glucose diet would increase plasma insulin concentrations and decrease brown adi- pose tissue metabolism in adrenalectomized ob/ob mice that had previ- ously been fed a high starch diet. Adrenalectomized ob/ob mice consumed more energy and gained more weight without an increase in oxygen con- sumption when switched from a high starch diet to a high glucose diet. Within 2 days after the switch to the high glucose diet, plasma insulin concentrations increased by 70 %. Brown adipose tissue metabolism, as assessed by GDP binding to brown adipose tissue mitochondria, was decreased 4 days after the diet switch. Plasma insulin and brown adipose tissue metabolism were unaffected in adrenalectomized lean mice switched from the high starch to the high glucose diet. The high glucose diet- induced increase in plasma insulin concentrations in adrenalectomized ob/ob mice may contribute to the observed depression in brown adipose tissue metabolism. Acknowledgements I would like to express my sincere appreciation to Dr. D. R. Romsos, my advisor, for his guidance and patient. Also, I would like to thank my husband, Nai-Hsien Wang, for his encouragement and support. iii TABLE OF CONTENTS I. LITERATURE REVIEW ............................................ 1 A. Introduction ............................................................ 1 B. Energy Balance in Genetically Obese Animals 3 1. Regulation of food intake ................................. 4 2. Energy expenditure ........................................... 8 C. Therrnogenesis and Obesity ................................... 10 1. Cold-induced thermogenesis ............................ 10 2. Diet-induced thermogenesis ............................. 12 3. Brown adipose tissue ........................................ 15 3.1. Mechanism of therrnogenesis in brown adipose tissue ......................................... 15 3.2. Assessment of therrnogenesis in brown adipose tissue ......................................... 17 3.3. Brown adipose tissue thermogenesis in genetically obese animals ....................... 19 D. Relationship Between Insulin, Thermogenesis, Central Nervous System, and Obesity ................ 20 1. Regulation of plasma insulin concentration 21 1.1. Glucose stimulation of insulin secretion 21 1.2. Nervous system stimulation of insulin secretion .................................................. 22 2. Insulin and therrnogenesis ................................ 23 3. Hyperinsulinemia in ob/ob mice ...................... 24 4. Central nervous system and insulin ................. 25 E. Relationship Between Adrenalectomy, Glucocorticoid, and Diet Compositions ............... 26 l. Adrenalectomy .................................................. 26 2. Glucocorticoids ................................................. 27 3. Diet compositions and energy balance ............ 28 4. Time sequences of response to diet ................. 29 F. Objectives and Hypothesis ..................................... 31 iv II. MATERIALS AND METHODS ................................ 32 A. Animals and Diets .................................................. 32 B. Experimental Design .............................................. 33 C. Experimental Assays .............................................. 34 D. Statistical Analysis ................................................. 37 ID. RESULTS ................................................................... 38 IV. DISCUSSION ............................................................. 47 A. Thesis Discussion ................................................... 47 B. Future Research ...................................................... 50 1. Norepinephrine turnover ................................... 50 2. Peripheral insulin injection ............................... 51 3. Vagotomy or cholinergic blocker .................... 52 4. Body composition ............................................. 53 V. REFERENCES ............................................................ 54 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. LIST of FIGURES Effect of adrenalectomy on food intake, body weight gain, and oxygen consumption of mice fed a high-starch diet ........................................ 39 Food intake, body weight gain, and oxygen consumption of sham-operated mice switched from a high-starch diet to a high-glucose diet for 2 or 4 days ..................... 40 Food intake, body weight gain, and oxygen consumption of adrenalectomized mice switched from a high-starch diet to a high-glucose diet for 2 or 4 days ..................... 42 Plasma glucose and insulin concentrations of sham-operated mice switched from a high-starch diet to a high-glucose diet for 4 days .......................................................... 43 Plasma glucose and insulin concentrations of adrenalectomized mice switched from a high-starch diet to a high-glucose diet for 2 or 4 days .................................................. 44 Total protein and GDP binding in brown adipose tissue of adrenalectomized mice switched from a high-starch diet to a high-glucose diet for 2 or 4 days ..................... 45 vi 1. LITERATURE REVIEW A. INTRODUCTION Genetic factors are one of the primary factors cause of obesity. Since it is very difficult to study the metabolic causes of obesity in man, many studies use genetically obese animal models. The symptoms manifested by ob/ob mice include hyperphagia, hyperinsulinemia, hyperglycemia, high circulating levels of corticosterone and impaired therrnoregulatory thermo- genesis (13,34,64). Lowered oxygen consumption and hyperinsulinemia are observed in ob/ob mice as early as 5 days (8) and 6 days (21) after birth. Hyperglycemia and hyperphagia are observed during the third or fourth week of life (13,55,111). Adrenalectomy reduces food intake by 35-60% and body weight gain by 50-90% in several obese animal models (57,84). Adrenalectomy also reduces the high efficiency of energy retention in these animals. This reduction is not due exclusively to reduced energy intake. The reduction in efficiency of energy retention is more likely caused by reduced intake combined with increased energy expenditure per Kcal consumed, possibly resulting from increased activity of brown adipose tissue. Brown adipose tissue (BAT) has one main function, heat production. It is important in the 2 regulation of body temperature and energy balance, particularly in small mammals. Brown adipose tissue possesses mechanisms for regulating energy expenditure in nonshivering thermogenesis (N ST) or diet-induced thennogenesis (DIT). Ob/ob mice show impaired cold tolerance, reduced DIT during overfeeding, and a depressed thennogenic response to noradre- naline, which is due to decreased oxygen consumption of BAT. The pri- mary mechanism responsible for therrnogenesis in brown fat appears to be the uncoupling of the mitochondrial proton conductance pathway from ATP synthesis (64). The activity of this pathway, assessed from the bind- ing of purine nucleotides to isolated BAT mitochondria, is markedly depressed in both ob/ob and diabetic db/db mice. Effects of adrenalectomy on energy balance in ob/ob mice are diet dependent (91). Adrenalectomized ob/ob mice fed high starch diets reduce their high efficiency of energy retention to values similar to those observed in lean mice; whereas, adrenalectomized ob/ob mice fed a high glucose diet continue to retain a high proportion of dietary energy and develop obesity. The only difference between the high starch and high glucose diets is the source of carbohydrate. It would appear that dietary glucose somehow lowers the metabolic rate of adrenalectomized ob/ob mice , but direct measurements of metabolic rate have not been reported. 3 B. Energy Balance in Genetically Obese Animals Obesity results from a positive imbalance between energy intake and energy expenditure. Control of food intake is one of the most complex biological problems yet to be understood. Intake or expenditure can be manipulated to get balance, but they are not totally independent of each other. Energy balance can be achieved in normal weight subjects or in overweight subjects provided energy output equals input level. In humans, food intake and energy expenditure are not finely balanced within a day, but good balance is normally achieved over weeks or months. Smaller animals regulate better on a day to day basis than humans. Time to attain balance might be associated with the relationship of energy expenditure to body weight. Smaller animals have relatively larger surface areas per unit body weight than larger animals, so smaller animals have higher metabolic rates per gram body weight. Rats die within one week without food whereas humans can survive for several months without food. Energy input, energy expenditure and energy stores are closely related to each other. It is unclear precisely how this system is regulated even in normal weight animals or subjects. I review the components of the system below. 1. Regulation of Food Intake Precise measures of food intake in humans are very difficult to obtain except subjects are housed in metabolic wards. Food recall or diet records provide general estimates of energy intake, but lack the sensitivity to detect small treatment differences. Some subjects may for various reasons underestimate and overestimate intake. In controlled conditions, it is ques- tionable if the data represent free-living intakes. Food intake is much easier to measure in animals than in humans. We can get clues from animals studies which can apply in human studies. It has generally been assumed that food intake is a more important controlling factor than energy expenditure in energy balance. Defective appetite control (i.e, hyperphagia) is often assumed to be the primary cause of obesity in humans and experimental animals (54,55,62,70,89). Many investigators have studied the adjustment of food intake in response to variations in the caloric density of food (4,30,62,69,70). Mature rats can compensate for diluted diet within a range from 2.95 kcal/ml to 5.05 kcal/ml (69). Monkeys fed diluted diets ranging from 0.5 to 1.35 kcal/ml maintain a constant caloric intake by adjusting oral intake in response to changes in caloric density (30). In a human study (142), 5 normal weight subjects were given a 200 ml preload containing aspartame or glucose (50 gm) with equal sweetness. Then, each subject was given excess to food after one hour. A comparison was made between equi- sweet preloads of aspartame, glucose and water control. The glucose group exhibited less hunger and desire to eat than aspartame and water control groups during the test meal. Total energy intakes, preload plus meal, were the same for all three (aspartame, glucose, and water) groups. These data show that a very good compensation exists to control food intake within specific range of caloric density in normal animals and subjects. However, the ability to compensate is affected by diet composition, variety, palatabil— ity and motivation. Offering rats multichoice cafeteria diets ,which are an assortment of energy rich foods processed for human consumption, produces hyper- phagia and obesity compared to feeding a standard stock diet (62,70,73,89). Cafeteria diets selected by rats are normally high in fat content with the same amount of carbohydrate as the control diet (70). It has been suggested that a variety of flavored food items is a contributing factor to cause dietary obesity in animals. However, Naim (62) indicated that variety in diet flavor did not induce hyperphagia in rats fed a nutri- tionally controlled purified diet. Both high fat and high sucrose diets con- 6 taining a variety of flavors in a cafeteria diet, and a high fat diet without added flavors resulted in higher energy intake. Thus, the effect of a variety of food flavors on hyperphagia in rats may have a minor effect in purified diets compared to the effect of the fat in diets (62). Consequently, energy density may play an important role in the hyperphagia and obesity induced by high-fat diets. This also appears to be true in human studies (56,58). Young female subjects fed high energy and low energy lunch by changing the carbohydrate and fat ratio, did not adjust total energy intake. Males adjusted mean daily energy intake by 11% when given the high- calorie lunch to compensate for over intake (58). Lissner also found that female subjects consumed a 15.4% surfeit on the high-fat diet, resulting in significant increase in body weight (56). The situation is more complex because different type of fat fed causes different responses, at least in rats. Normally, rats consume more solid fat (shortening, lard or tallow) than liquid fat (corn oil). If corn oil is emulsified then consumption increases (71). Many manipulations used in the research of food intake cause changes in eating pattern including rate of food intake, meal size and meal fre- quency (15,73). Popplewell reported that reduction in the permitted rate of food intake contributed to a clear reduction of meal size and an increase in 7 meal frequency in rats (15). Rogers and Blundell have very detailed inves- tigations of eating patterns in rats. Offering a cafeteria diet to rats causes an immediate increase in meal size and meal frequency. Palatability has a major influence on meal size, while variety affects both meal size and meal frequency (73). They also found that the food preferred during the second meal was different from that consumed in the first meal; mixed meals were consumed more than single item meals. Overriding satiety sig- nals happened as body weight increased with a resultant decrease in meal frequency, but meal size remaining high. Obesity has an influence on feed- ing primarily through a decrease in meal frequency with elevating meal size, which are true for most models of obesity. These data from animal research in eating patterns are good references to apply for human studies. Many studies have been done which show that variation in food intake is the major cause of changes in body fat or weight. Obese animals eat more than nonobese animals (54). Also, adult obese mice and moderately diabetic rats self-selected a higher proportion of energy from fat and a lower proportion from protein and carbohydrate than did lean (4,59,75). Obese (ob/ob) mice fed a high-fat diet were 41% more efficient than ob/ob mice fed a high-carbohydrate diet, and 38 to 71% more efficient than lean mice fed high-carbohydrate or high-fat diets respectively 8 (55). After 4 weeks of age, ob/ob mice consume more food and gain more weight than lean mice (54). All these observations suggest that hyper- phagia plays an important role in the later development of obesity in ob/ob animals. 2. Energy Expenditure Studies (2,19,76,83,100,105) on energy expenditure in obese animals have focused on obese (ob/ob) mice, obese-diabetic (db/db) mice, and obese (fa/fa) rats. These obese animals exhibit high retention of dietary energy. Obese (ob/ob) mice contain more body fat than lean litterrnates as early as 7 days after birth (8). Milk intake has been measured from 7 to 21 days of age in ob/ob and lean pups, and ob/ob pups did not consume more milk than lean pups (54). Similar findings have been reported in obese (fa/fa) rat pups (10). It is clear that the early appearance of high energy content in these ob/ob mice and fa/fa rats occurs before hyper- phagia is evident and therefore must result from low energy expenditure. Oxygen consumption is lower in preobese ob/ob pups than in lean littennates; low oxygen consumption could be detected as early as 5 days of age in ob/ob mice (8). Because ob/ob pups contained 38% more fat than lean pups at 1 week of age, the reduced oxygen consumption may be 9 due to the less lean body mass. Ob/ob and lean pups suckled dams fed a high-carbohydrate diet or a high-fat diet for 2 weeks. The body weight and body fat of obese pups suckling dams fed the high-carbohydrate diet were the same as the lean pups suckling dams fed the high-fat diet. But, obese pups still consumed less oxygen than did the lean pups. Thus, the low energy expenditure in these ob/ob pups can not be explained by differences in body composition (55). The fact that obesity develops in ob/ob mice even when energy intake is limited to that Of ad lib fed lean siblings also demonstrates that energy expenditure is low in ob/ob mice (54,98). Low energy expenditure in young obese (ob/ob) mice could result from a reduced maintenance energy requirement or from an improved abil- ity to retain energy consumed above maintenance. The maintenance energy requirement is 40% less in 3- to 6-week old (ob/ob) mice than lean mice housed at 25 to 30 0C and fed either a high-fat or a high-carbohydrate diet (55). The utilization of energy above maintenance in ob/ob mice fed the high-fat or the high- carbohydrate diet was 71% and 38% more efficient than that in lean mice, respectively (55). Thus, the 40% lower maintenance energy requirement of these obese mice is a major contribu- tion to the high efficiency of energy retention. In genetically ob/ob mouse, 10 a decreased oxygen consumption and body temperature, and an increased body fat concentration appear within the first three weeks of age. This indicates that the initial development of obesity in the ob/ob mutant is entirely due to the high efficiency of energy retention caused by a low energy expenditure. C. Thermogenesis and Obesity Genetically obese animals with low energy expenditure have defects in either cold-induced thennogenesis or diet-induced therrnogenesis. I will discuss these two components of energy expenditure and compare the differences between genetically obese and lean rodents. l. Cold-Induced Thermogenesis: Since small animals have a large surface area/volume ratio, they have greater metabolic rates than large animals, particularly when small animals are housed below their lower critical temperature. Cold-acclimated rats remain lean even thought they exhibit extreme hyperphagia. They use the excess food energy for cold-induced thermogenesis to maintain body tem- perature (80). One of the earliest abnormalities observed in ob/ob mice is the failure to maintain body temperature when exposed to temperatures below the therrnoneutral zone (32-33 0 C ). Trayhum et al (103) showed 11 that 12-d-old ob/ob mice exposed to an environmental temperature of 15—20 0C had a marked fall in body temperature within 15 minutes. These researchers used this finding to identify animals bearing the ob/ob genotype before they could be distinguished visually. Db/db mice and fa/fa rats exposed to cold show the same impaired metabolic response to cold exposure as ob/ob mice (43,100). These observations indicate that the high energy efficiency in these mice and rats may be explained in part by a reduced energy expenditure for therrnoregulatory therrnogenesis (42,58,59). Some studies have examined (44,98,101) efficiency of energy retention in obese (ob/ob) mice at warm environmental temperature (33-34 0 C). When the environmental temperature is decreased the meta- bolic rate of the ob/ob mice increases less than in lean mice (101). Maintenance energy requirement of obese mice housed at 25 to 30 0 C were the same as those of obese mice housed at 33 0C (108). Similar findings were observed in db/db mice (100,105). The capacity for non- shivering therrnogenesis was reduced by 50% in the ob/ob mice compared to lean mice at 31 0 C (101). Taken together, a defect in therrnoregulatory therrnogenesis is a major factor resulting in high efficiency of dietary energy retention in young obese mice housed at normal room temperature (20—28 0C ). However, ob/ob mice housed at 33 0 C still become obese even if pair fed (74,98). Similar findings have been observed in db/db 12 mice (105). Factors other than defective thermoregulatory thermogenesis must also be involved. 2. Diet Induced Thermogenesis Energy expenditure is associated with the level of food intake. Energy expenditure increases as energy intake increases. Two components of diet- induced therrnogenesis are obligatory and adaptive therrnogenesis. The definition of obligatory thermogenesis, which has also been termed heat increment or SDA, is the energy costs associated with digestion, absorp- tion, and the immediate metabolism of the ingested nutrients. Adaptive thermogenesis is defined as the increase in energy expenditure associated with a meal that is over and above the obligatory component. It is techni- cally difficult to evaluate the relative contributions of obligatory and adap- tive thermogenesis to diet-induced therrnogenesis. Most researchers focus on total diet-induced therrnogenesis without attempting to separate the two components. Rothwell and Stock fed a cafeteria diet for 3 weeks to induce overeat- ing in rats (81). Cafeteria-fed rats consumed 80% more energy than did stock-fed control rats, but less than 10% of this excess intake was stored as body energy (81). The association of lowered energy efficiency with 13 increased diet-induced thermogenesis in the overfed animals was confirmed by 20-30% higher rates of resting oxygen consumption than in stock-fed rats (81). Cafeteria diet induction of diet-induced thermogenesis could occur within 2.5 days (77). The increase in diet- induced thermo- genesis is associated with an elevation of BAT wet weight, DNA, total protein and 3 times higher GDP binding in isolated BAT mitochondria of cafeteria-fed rats (36). Rats fed a low-protein diet ate considerably more than their controls fed an adequate protein diet. Nevertheless, rats fed a low—protein diet retained much less energy (92,107) indicating that protein content of the diet influences diet-induced thermogenesis. Leblanc and Brondel (53) sug- gest that palatability of the meal is another factor causing diet- induced thermogenesis which may be via sensory stimulation to activate the sym- pathetic nervous system. Eight female subjects were fed either a highly palatable meal (HPM) or a nonpalatable meal (NPM) which was presented as desiccated biscuits by mixing all the ingredients of the highly palatable meal. Resting metabolic rate (RMR) during 90 min after ingestion of HPM was 50% higher than that of NPM control group. For the 5 week post-weanling period, obese mice converted 3 to 4 times more dietary energy to body energy than did lean mice, whereas 14 obese mice consumed only 20 to 40% more energy (54). Trayhum also studied the effects of cafeteria diets compared with stock diets in 4 week old lean and obese (ob/ob) mice (104). After 3 weeks, energy intake of lean mice fed a cafeteria diet was 69% more than that of lean animals fed the stock diet. Although energy intake increased, the cafeteria-fed lean mice only showed a 19% increase in energy gain and no change in body weight gain. Energy intake of the cafeteria-fed obese (ob/ob) mice was 49% higher than in those of stock-fed obese (ob/ob) mice. In contrast to lean mice, the cafeteria-fed obese (ob/ob) mice gained 88% more energy than stock-fed obese (ob/ob) mice. Energy efficiency of the cafeteria-fed obese (ob/ob) mice was much higher than the stock-fed groups. They also found that cafeteria-fed lean mice, but not ob/ob mice, showed an increase of metabolic rate in BAT. After this study was published Triandafillou and Himms-Hagen (106) examined fa/fa rats to find out whether a cafeteria diet would activate BAT thermogenesis in these obese animals. Either a stock diet or a cafeteria diet was given to young lean and fa/fa rats housed at 28 0 C for 2 to 3 weeks. GDP-binding in BAT mitochondria was lower in fa/fa rats than in lean rats fed a stock diet, and cafeteria-fed fa/fa rats failed to activate BAT as it did in lean rats. These data suggest that DIT is reduced in obese compared with the lean rats, as previously observed in obese mice. Diet induced thermogenesis in brown adipose in response to 15 overeating leads to an attenuation of obesity in normal rodents, whereas defective diet induced thermogenesis in BAT is one of the reasons for high metabolic efficiency in the genetically obese rodents. 3. Brown Adipose Tissue Brown adipose tissue is located in small deposits throughout the body, including the interscapular, subscapular and axillary regions, at the nape of the neck, along the length of the great vessels in the thorax and abdomen and between the ribs. Brown adipocytes have several small lipid droplets and are packed with large mitochondria. In contrast white adipocytes have a single large lipid droplet and relatively few small mitochondria. Non- shivering thermogenesis in brown adipose tissue is mediated by the sym- pathetic nervous system. Brown adipose tissue has an abundant sym- pathetic innervation secreting norepinephrine by the nerve ending to regu- late BAT metabolic activity. As already indicated brown adipose may be important in regulation of energy balance although it represents only 1-3% of body weight in rats (24,33). 3.1. Mechanism of thermogenesis in brown adipose tissue Brown adipose tissue mitochondria have a unique loosely coupled respiration (32,34,37). The principal mechanism of heat production for l6 non-shivering thermogenesis in brown adipose tissue is uncoupling of oxi- dative phosphorylation; respiration proceeds without ATP synthesis with resultant heat production. The mechanism of uncoupling involves a proton conductance pathway which makes the inner mitochondria membrane more permeable to protons (32,34,37). The therrnogenic function of brown adipose tissue mitochondria is related to a specific protein (32,000 D polypeptide which is named differently as thermogenin, uncoupling protein, nucleotide binding protein, and GDP-binding protein). Thermogenin is located in the outer surface of the inner mitochondrial membrane. The main sequence of non-shivering thermogenesis in BAT involves a stimulus (i.e. cold temperature or cafeteria diet) which activates the sympathetic nervous system to release norepinephrine from sympathetic nerves ending. Thus, norepinephrine binds to B-adrenergic receptors on BAT cells and activates adenyl cyclase with a resultant increase in cAMP. The resulting increase in cAMP causes protein kinase activation of a hormone-sensitive lipase and accelerates lipolysis which serves as the intracellular signal to switch on the proton- conductance pathway, and as the fuel for oxidation in mitochondrial (32,34,37). The precise mechanisms for control of the stimulation and inhibition of non— shivering thermogenesis are still unknown. 17 3.2. Assessment of thermogenesis in brown adipose tissue The only technique to measure the quantitative contribution of BAT to whole body energy expenditure is assessment of BAT blood flow with radioactively labelled microspheres and tissue oxygen consumption (24). Foster and Frydrnan found that the 25-fold increase of blood flow to BAT that occurs in response to maximum norepinephrine-induced stimulation of metabolic rate in cold acclimated rats accounts for 60% of the overall increase in metabolic rate (24). Measurement of blood flow to BAT is a very difficult technique and not realistic to do in large numbers of animals. Investigators have thus used other methods to assess BAT function. Most studies have generally used some of the following four basic measurements: tissue weight, pro- tein content, cytochrome oxidase activity and mitochondrial GDP binding as an index of thennogenic state of BAT. The wet weight of BAT is only a rough index of the triacylglyerol content and generally this parallels the amount of lipid stored in white adipose tissue. Tissue weight is a useless measure of metabolic capacity. Measurement of total protein, DNA, or a mitochondrial marker enzyme, such as cytochrome oxidase, provides a better index of the therrnogenic capacity, but not of the activity, of the tis- sue. Binding of GDP to brown adipose tissue mitochondria is the most 18 widely used index of the activity of the proton conductance pathway. GDP binding is expressed per unit of mitochondrial protein. Chronic cold expo— sure leads to an increase in GDP binding and uncoupling protein concen- tration (1,102). When the tissue is acutely stimulated by activation of its sympathetic nerves, GDP binding is rapidly increased (94,102). This increase in binding is associated with ultrastructural changes of isolated mitochondria without an increase in uncoupling protein concentration. This acute increase in GDP binding indicates an "unmasking" of existing nucleotide binding sites. It is important that more direct experimental approaches be used to understand the molecular basis for this opponent unmasking and alteration in thennogenic activity of the tissue. The recent development of an immunoassay for direct quantitative measurement of UCP (uncoupling protein) provides the only specific means of identifying BAT. Measurement of the amount of the UCP is a good index of the potential therrnogenic capacity of BAT, but not of its actual thermogenic activity. Another useful index of the therrnogenic activity of BAT is the measurement of NE turnover which provides infor- mation about the activity of the sympathetic nervous system, the key regu- lator of the BAT thermogenesis. 19 3.3 Brown adipose tissue thermogenesis in genetically obese an- imals Himms-Hagen and Desautels provided evidenced for a defect in mito- chondria of BAT in ob/ob mice that may explain why these mice exhibit a diminished therrnogenic response to acute, severe, cold exposure (35). GDP binding to isolated BAT mitochondria of ob/ob mice was lower than in lean mice. There is considerable evidence now that the impaired ther- mogenesis in ob/ob mice is due at least in part to a defect in this pathway in the mitochondria of BAT. Acute cold-exposure in mice causes a 45% increase in GDP binding (35). A similar alteration is found in rats (94,95). GDP binding of BAT mitochondria in obese (ob/ob) mice was 50% lower than in mitochondria of lean mice at 28 0C. Additionally, an acute expo- sure at 4 0 C for 3 h increased GDP binding in BAT mitochondria from lean mice, but failed to increase binding in obese mice (35). GDP binding to BAT mitochondria was 60% lower at 14 days of ob/ob than lean mice (28). The lower GDP binding also is found in diabetic-obese (27) mice. Defective control of sympathetic nervous system in BAT is likely a major factor in the low thennogenic activity of BAT. Sympathetic nervous system activity, indicated by norepinephrine turnover, is 40% lower in BAT of preobese (2 wk) (49) and 60% lower in adult (8 20 wk) obese mice (49,50) than in same age of lean littermates housed at 25 0 C and fed a stock diet. Reduction of nonshivering thermogenesis shown by low rates of norepinephrine turnover and low GDP binding appears before the development of obesity indicating that it is not a secon- dary phenomenon. Trayhum (97) compared blood flow in BAT of obese (ob/ob) and lean mice. Lean mice showed about a 40-fold increase in blood flow to BAT in response to norepinephrine injection, whereas the increase in blood flow to BAT in obese mice was only half that of lean mice (97). Because ob/ob mice increase their metabolic rate and blood flow less after norepinephrine injection than do lean mice, it has been speculated that an alteration in BAT metabolism contributes to the increased efficiency of energy retention in ob/ob mice. D. Relationship between Insulin, Thermogenesis, Central Nervous Sys- tem, and Obesity Hyperinsulinemia in ob/ob mice is observed at a very early age (21). Insulin plays an important role in control of food intake and BAT thermo- genesis. Thus, many experiments have focused on the role of insulin in development of obesity. First, I will mention insulin secretion. Then, the 21 relationship between insulin, thermogenesis, and food intake are discussed. 1. Regulation of Plasma Insulin Concentration 1.1. Glucose stimulation of insulin secretion Many factors modulate insulin secretion. However, glucose is the pri- mary stimulus for insulin secretion. The relationship between plasma insu- lin and plasma glucose is sigmoidal. Glucose-induced insulin release has been studied in vivo within glucose ranges from 50 mg/dl to 300 mg/dl. Plasma glucose below a threshold about 50 mg/dl does not cause insan secretion, and at a level of 300 mg/dl a maximum insulin secretion is reached in humans (46). However, the exact mechanism whereby glucose stimulates insulin secretion is unclear. Lavine found that ob/ob mouse islets were more hypersensitive and hyperresponsive to glucose than were lean controls (5,52). Similar altemations have been found in humans (46). Hyperinsulinemia in ob/ob mice is partially due to this hypersecretion in response to glucose after about 30 days of age, but not at the earlier time point. Hyperinsulinemia occurred as early as 6 days (21) and hyper- glycemia is not evident until the mice are about 30 days of age (111). Since hyperinsulinemia precedes the onset of hyperglycemia in ob/ob and db/db mice (16,21,22,lll), glucose is not the only cause of insulin 22 hypersecretion. 1.2. Nervous system stimulation of insulin secretion Neural transmitters are released from nerve terminals of the autonomic nervous system, which includes sympathetic and parasympathetic nerve, near [3 cells of pancreatic islets (61). Sympathetic fibers innervating the pancreas release norepinephrine, and the adrenal medulla releases epinephrine into circulation to provide additional control of insulin secre- tion. Norepinephrine and epinephrine can react with either a (inhibitory) or B (stimulatory) receptors, but their overall effect is to play an inhibitory role in insulin secretion in rats or mice (61). However, the mechanism of norepinephrine inhibition of insulin secretion is still not clear. Acetylcholine released from parasympathetic vagus nerve terminals is responsible for cephalic phase insan secretion which can be blocked by vagotomy (93). Parasympathetic nervous system response to a meal may stimulate insan secretion over 26% (6). The mechanism of acetylcholine potentiation of meal-induced insulin secretion is still not completely under- stood. 23 2. Insulin and Thermogenesis The complex and numerous effects and interactions of insan on nutrient metabolism have important implications in energy balance regula- tion. Recently it was suggested that insulin was an important mediator of carbohydrate-induced thermogenesis and acts as a central satiety signal in the regulation of body weight (51,79,82). However, insulin can have different effects depending on central or peripheral action (14,82). Effects of insulin added in vitro with labeled glucose C 14— to slices of rat interscapular brown adipose tissue were examined. Insulin dramatically increased glucose uptake up to 60-fold and increased oxygen consumption (90). Cafeteria-fed rats require insulin to produce diet-induced thermo- genesis and to response to noradrenaline. Twelve hours after injection of P21 insulin (8 U/rat) into insulin deficient diabetic rats resting metabolic rate and noradrenaline response values increase to equal those of non- diabetic cafeteria rats (79). Rothwell also found that diabetic rats fail to maintain body temperature when exposed to 5 0C . Giving insulin to dia- betic rats causes a recovery of cold-induced thermogenesis (79). These data indicate an insulin requirement for diet-induced thermogenesis and cold-induced thermogenesis. Subjects continuously infused with insulin and glucose causing hyperinsulinemia with nonnoglycemia had an 24 increased metabolic rate of 7.6%. After propranolol administration, energy expenditure decreased by 4% indicating that one mechanism whereby insu- lin induced thermogenesis was through [3 adrenergic receptors. Cold- adapted rats exhibit hypermetabolism with lower insulin levels compared with control rats housed at 20 0C (18). This suggests a negative correla- tion between plasma insulin concentrations and thermogenesis. Cunning- ham found that cold-adapted rodents are highly sensitive to insulin with considerably improved glucose tolerance (18). The exact role plasma insu- lin plays in control of BAT thermogenesis remains unclear. 3. Hyperinsulinemia in Ob/Ob Mice Hyperinsulinemia appears in ob/ob mice as early as 6 days of age (21). The early hyperinsulinemia has been implicated as a primary cause of excess adiposity in ob/ob mice. Insulin promotes adipocyte hypertrophy and proliferation, increases activity of lipogenic enzymes, and decreases lipolysis. Hyperinsulinemia in ob/ob mice is due to either an increased secretion or a decreased clearance. Karakash (45) used streptozotocin to destroy pancreatic islets and decrease insulin secretion. In these animals with lower plasma insulin concentrations, hepatic insulin clearance increased. These data indicate that the decreased insulin clearance observed in ob/ob mice is likely a secondary consequence of 25 hyperinsulinemia. Thus, insulin secretion likely plays a major role of hyperinsulinemia in ob/ob mice. Tassava (96) also found that pancreatic islets from ob/ob mice were hypenesponsive to acetylcholine compared to islets from lean mice. Obese mice develop severe insulin resistance by 6 weeks of age (3). This may contribute to their impaired thermogenesis since increases in insulin sensitivity are seen in cafeteria-fed rats (79) and rats fed a low protein diet (78) compared with stock-fed controls. 4. Central Nervous System and Insulin Although insulin has some direct actions on metabolism of BAT, its effects on thermogenesis are probably mediated centrally. Insulin is present in most areas of the brain but concentrations are variable. Insulin receptors are present in the hypothalamus and insulin may exert its effects at periventricular sites (17). Continuous infusion of insulin into the CSF (cerebrospinal fluid) of rats decreased food intake and reduced body weight (14). CSF insulin might be a satiety signal for body weight regula- tion. An obvious site of action for insulin is within the VMH (ventrome- dial hypothalamus). Electrical stimulation of the VMH resulted in a decrease in food intake. Whereas lesions VMH caused hyperphagia and obesity in rat. Some workers (68) found that electrical stimulation of VMH showed an increased thermogenesis in BAT. These data suggest that 26 the VMH exerts an inhibitory effect on energy intake and a stimulatory effect on thermogenesis and energy output. As a result, insulin can increase sympathetically-mediated thermogenesis probably via its central actions. Obese ob/ob animals may have a defect in the brain in response to the hyperinsulinemia resulting in low thermogenesis. E. Relationship between Adrenalectomy, Glucocorticoid, and Diet Compositions Cushing’s syndrome is the clinical manifestation of the metabolic effects of hypercortisolism. Truncal obesity is commonly seen in Cushing’s syndrome patients. Also, many obese people have signs and symptoms of Cushing’s syndrome (88). Genetically obese (ob/ob) mice have increased plasma corticosterone concentrations at 3 weeks of age which is before they are visually obese. Thus, glucocorticoid must also be considered as a contributing factor. 1. Adrenalectomy Adrenalectomy completely or partially normalizes most abnormalities of the ob/ob mice and fa/fa rats. These include hyperphagia, body weight gain and obesity, hyperinsulinemia, insulin resistance, and reduced brown adipose tissue mitochondrial GDP binding (40,41,57,66,84). Thus 27 hyperadrenocorticism of the ob/ob mice has been assumed to associated with the development of obesity. 2. Glucocorticoids Many studies have shown that excess glucocorticoids cause increases in total body fat in adults (38) and in obese experimental animals (12,25). Chronic administration of glucocorticoid to adrenalectomized ob/ob mice and fa/fa rats reverses the effects of adrenalectomy. (25,99). There is evi- dence that obese animals are very sensitive to glucocorticoid administra- tion. Freedman studied fa/fa rats adrenalectomized at 4 weeks of age and given daily injections of different concentrations of glucocorticoid for 30 days. As glucocorticoid doses increased, food intake and plasma insulin markedly increased in obese fa/fa rats without altering these measures in the lean rats (25). Adrenalectomized fa/fa rats responded to glucocorticoid at low doses (25). Thus, they suggested that genetically obese animals may be hypersensitive to glucocorticoid. Hypersensitive to glucocorticoids was also demonstrated by Tokuyama et a1 after 2 weeks implantation of corticosterone-containing pellets at 8.5 weeks of adrenalectomized ob/ob mouse (99). At low physiological levels of serum corticosterone (10 ug ldl ), ob/ob mice in contrast to lean mice obviously increased body weight gain, food intake, serum insulin, and decreased BAT mitochondrial 28 GDP binding (99). Increase of food intake in lean mice only occurred at very high levels of corticosterone (30 ug/dl ). All these results indicates that the ob/ob mouse is hypersensitive and hyperresponsive to physiologi- cal levels of corticosterone resulting in hyperphagia, hyperinsulinemia, and increased weight gain. The mechanisms whereby glucocorticoids cause obesity are not clear. One hypothesis is that glucocorticoids act through the central nervous system to restore hyperphagia (20). 3. Diet Compositions and Energy Balance The effects of adrenalectomy in normalizing energy balance are diet dependent in ob/ob mice. In long term studies (3 weeks), adrenalectomy prevents the abnormalities in ob/ob mice fed a hi gh-carbohydrate stock diet, whereas adrenalectomized ob/ob mice still exhibited the obese syn- drome when they were fed a high-fat diet or a high glucose diet (29,47, 48,91,109). I only focus on a comparison of high-starch diet and high- glucose diet effects in adrenalectomized ob/ob mice in this thesis. A major difference between a high-carbohydrate stock diet and a high- glucose diet is the source of carbohydrate. Several studies found that adrenalectomized ob/ob mice reduced their high efficiency of energy reten- tion by decreased energy intake and increased energy expenditure through 29 a increase of thermogenic activity in BAT to levels comparable to lean mice when they were fed a high-starch diet. There is only a minimal effects on energy efficiency or thermogenic activity in BAT when adrenalectomized ob/ob mice fed a high-glucose. Plasma insulin concentra- tions are similar in both lean and adrenalectomized ob/ob mice fed a high-starch diet; however there is a less pronounced effect in adrenalec- tomized ob/ob mice fed a high-glucose diet (48,109). Warwick also showed that there is no additional effect due to dietary fiber when adrenalectomized ob/ob mice were fed the starch plus wheat bran diet (109). These results indicated that dietary glucose somehow lowers the metabolic rate of adrenalectomized ob/ob mice. 4. Time Sequences of Response to Diet No studies have examined how quickly dietary glucose causes these parameters to change in adrenalectomized ob/ob mice. However, some clues can be obtained from other studies to presume that dietary effects maybe happen within a few days. When young obese rats were adrenalec- tomized GDP binding of BAT mitochondria was normalized within 7 days after adrenalectomy. A initial increase of GDP binding in obese rats appeared rapidly (within 24 hr after adrenalectomy), followed by a slower rate of increase during the next 6 days (39). Acute cold (4 0C) exposure 30 of 1 to 3 hr of young rats caused a rapid change in GDP binding (67,102,106). Similar findings were showed in lean mice (35). When young fa/fa and lean rats were injected with noradrenaline, a rapid increase of GDP binding occurred within 30 min (67). These studies suggest that thermogenic actively of BAT can change within hours after a treatment is started. Glucocorticoid treatment of adrenalectomized fa/fa rats for 24 h increases plasma insan concentrations about 5 times compared to lean controls (23). An increase of plasma insan and a decrease of GDP bind- ing has also been observed within 15 h after glucocorticoid injection in both adrenalectomized ob/ob and lean mice (31). Adrenalectomized ob/ob mice are, however, more sensitive and responsive to the rapid action of corticosterone than adrenalectomized lean mice in terms of insulin secre— tion and BAT thermogenesis depression. From the above data it appears that adrenalectomy, cold temperature, and hormones (noradrenaline and glucocorticoid) can change GDP binding to BAT mitochondria and plasma insulin concentrations within one day. 31 F. Objectives and Hypothesis My objectives were 1) to determine if replacement of dietary starch with glucose affects oxygen consumption, brown adipose tissue metabol- ism and plasma insulin concentrations in adrenalectomized ob/ob mice and 2) to describe the time sequence of changes in oxygen consumption, brown adipose tissue metabolism and plasma insulin concentrations in adrenalectomized ob/ob mice. I hypothesized that thermogenic activity of brown adipose tissue (BAT) decreases and plasma insulin increases when adrenalectomized ob/ob mice are switched from a high-starch diet to a high-glucose diet. 32 II. MATERIALS AND METHODS A. Animals and Diets: Male obese (ob/ob) and lean (ob/+ or +/+) litterrnates were obtained from our breeding colony of C57Bl/6J-ob/+ mice. They were weaned at 21 days of age and housed in solid-bottom plastic cages with wood shav- ings as bedding in a room maintained at 23—25 0C with lights on from 07:00 to 19:00 h daily. Mice were offered a stock diet (Wayne Lab- Blox, Continental Grain, Chicago, IL) and water ad libitum. At 26 days of age, ob/ob and lean pairs were separated from their littermates and housed indi- vidually. At 28 days of age mice were bilaterally adrenalectomized or sham-operated through dorsal incisions while under ether anesthesia. Each adrenal gland was gently lifted and a curved scissors was used to remove each gland along with a small amount of adipose tissue. Incisions were closed with stainless steel wound clips. The total surgical procedure was completed within 6 min. Sham-operated mice underwent the same pro- cedure except the adrenal glands were left intact. Physiological saline (0.9% NaCl) was given to adrenalectomized mice after surgery. All mice had semipurified diet and water/saline ad libitum after surgery. 33 Two semipurified diets were fed. The high-glucose diet contained in g per 100 g: 65 glucose, 20 casein, 0.3 methionine, 5.0 corn oil, 3.5 mineral mixture (7), 1.0 vitamin mixture (7), 0.2 choline chloride, and 5.0 cellu- lose. The high-starch (corn starch) diet was formulated on an equal energy basis by replacing 65 g of glucose with 59.2 g starch. These diets con- tained 66, 22, and 12% of metabolizable energy as carbohydrate, protein, and fat, respectively. B. Experimental Design: Experiment 1: Two sets of mice were used. Each set contained four groups, sham and adrenalectomized ob/ob, and sham and adrenalectomized lean mice. One set of mice served as controls and was fed the high-starch diet throughout the 16 day experiment. Another set of mice was fed the starch diet for 12 days, and then the high-glucose diet for the last 4 days of the 16 day experiment. Food intake, body weight and oxygen consump- tion were measured on day 11, 12, 13, 14 and 16. Oxygen consumption was measured at 0830-1100 h daily. Mice were decapitated at 11:00 h on day 16 to obtain trunk blood in heparinized beakers for plasma corticos- terone, insulin and glucose assays. 34 Experiment 2: Only adrenalectomized mice were used. Adrenalectom- ized ob/ob and lean mice were either fed the high-starch diet throughout the day feeding trial, or fed the high-starch diet for 12 days and then switched to the high-glucose diet for 2 or 4 days. Mice were killed at 11:00 h on day 14 or 16 by decapitation to determine BAT thermogenic activity by measuring GDP binding to isolated BAT mitochondria. Blood was also collected in heparinized beakers for plasma corticosterone, insan and glucose assays. C. Experimental Assays: Oxygen consumption: Mice were placed on a wire-mesh floor in the glass bottle that contained soda lime to remove expired carbon dioxide. The bottle was closed and immersed in a water bath maintained at 25 i- 1 0C . After a 5 min adaptation period, 6 estimates of oxygen con- sumption were recorded within the next 5 to 10 min (111). Data were cal- culated as ml oxygen consumed/hI/g body weight at STP (standard tem- perature and pressure). Hormone and glucose assays: Plasma for the corticosterone assay was diluted 1:9 with borate buffer/BSA (bovine serum albumin), then incu- bated at 60 0 C for 30 min to denature corticosterone-binding proteins. 35 Corticosterone was extracted with ethanol. After evaporating 50 ul of the ethanol extract in a vacuum oven, plasma corticosterone concentrations were determined by radioirnmunoassay (Endocrine Sciences, Tarzana, CA) with modification. Only those adrenalectomized mice with plasma corticos- terone concentrations less than 1 ug ldl were included. The lowest detect- able plasma corticosterone concentration in this assay was 0.15 pg ldl plasma. Adrenalectomized ob/ob and lean mice fed the high-starch diet and then switched to the high-glucose diet had plasma corticosterone values of 0.74 i 0.04 and 0.53 i- 0.08|.tg ldl plasma, respectively. Plasma corticosterone concentrations averaged 16.5 i- 4.1 and 2.7 i 0.6 ug/dl plasma in sham- operated ob/ob and lean mice when diets were switched from the high-starch diet to the high-glucose diet, respectively. Adrenalec- tomized ob/ob and lean mice fed the high-starch diet had 0.59 :l: 0.04 and 0.51 :I: 0.06ug corticosterone/d1 plasma, respectively. Sham-operated ob/ob and lean mice fed the high-starch diet throughout the 16 day trial had plasma corticosterone concentrations of 16.8 i 2.7 and 4.6 i 0.7 ug/dl plasma, respectively. Plasma glucose concentrations were determined by the glucose oxidase-peroxidase method (Boerhinger—Mannheim, Indianapolis, IN). Plasma insan concentrations were measured by radioirnmunoassay with 36 rat insulin as the standard and antiporcine insulin serum (Nova Research Laboratories, Bagsvaerd, Denmark ) with modifications to accommodate reduced sample volumes. GDP binding to BAT mitochondria: Mice were killed and interscapu- lar and subscapular BAT depots were rapidly removed, combined, weighed, and cut in pieces, then homogenized with 5% wt/vol in ice-cold buffer containing 250 mM sucrose and 5 mM K-TES (potassium-N-tris- metlryl-Z-aminoethane sulfonic acid) pH 7.2. Mitochondria were isolated in the sucrose buffer by the procedure of Cannon et a1 (63). H 3-GDP binding to BAT mitochondria was determined by the method of Nicholls (9) with modifications. Binding of H 3-GDP to BAT mitochondria was determined by incubation for 10 min at 25 0C in a media containing 100 mM sucrose, 20 mM K-TES, 1 mM EDTA, 2 uM rotenone, 100 W potas- sium atractyloside, 2.2 x 109 dpm/ml H3—GDP (New England Nuclear, 8.2 Ci/mmole), 10 um unlabeled GDP, and 5.5 x 108 dpm/ml C 14—sucrose (New England Nuclear, 498.7 mCi/mmole). At the end of the incubation mitochondria were separated by centrifugation, and the mito- chondria pellet was dissolved in Beckman tissue solubilizer-450 and counted in a liquid scintillation counter. C 14---sucrose was included as a marker of trapped media in the final mitochondria pellet. Specific binding 37 of H 3—GDP was calculated by subtraction of H 3—GDP trapped and non- specific H3-GDP binding which was obtained from binding of H3—GDP in the presence of 200 [AM unlabeled GDP. Protein content of mitochon- drial preparations and BAT homogenates (after extraction of lipids with acetone-petroleum ether) were measured by a modified Lowry method (11,13). D. Statistical Analysis: Two—way factorial analysis of variance was used to analyze data. The Bonferroni two-tailed t-test was employed in selected groups to detect significant difference between treatment and control groups. Student t-test was applied to examine diet effects in adrenalectonrized ob/ob mice (26). Data were presented as means 1- SE, and all significant effects were at P < 0.05. 38 III. RESULTS Food intake, body weight gain and oxygen consumption of mice fed the high-starch diet during the first 12 days after surgery are shown in Fig l. Sham-operated ob/ob mice consumed more energy and gained more body weight than lean mice (Fig. l). Adrenalectomy reduced energy intake 26% and body weight gain 37% in ob/ob mice without affecting lean mice (Fig. 1). Oxygen consumption, expressed as ml/hr/gm body weight, was lower in sham-operated ob/ob mice than in lean mice, and adrenalectomy increased oxygen consumption 20% in ob/ob mice without affecting lean mice (Fig. 1). Sham-operated and ADX ob/ob mice consumed similar amount of oxygen when values were expressed per mouse. Sham-operated ob/ob mice had higher food intake and body weight gain and lower oxygen consumption than lean mice independent of diet (Fig. 2). Food intake and oxygen consumption increased in sham-operated lean mice after their diet was switched from a high-starch diet to a high- glucose diet for 4 days. Adrenalectomized ob/ob mice, but not adrenalectomized lean mice, consumed more energy and gained more weight when switched from the 39 * DSHAM Ion I: paw 3. FOOD INTAKE - kJ/day 8 3 \\\\\\\\\\ ar- perv BODY WEIGHT GAIN - par-o OXYGEN CONSUMPTION- mL h‘ d" A 03/03 I LEAN Fig. 1. Food intake, body weight gain, and oxygen consumption of sham- operated (SHAM) and adrenalectomized (ADX) ob/ob and lean mice fed a high starch diet for 12 days. Body weights at 12 days after surgery aver- aged 22.85 :t 0.54, 19.97 i 0.38, 19.01 i- 0.22, and 18.36 i 0.34 in sham- ob/ob, ADX-ob/ob, sham-lean, and ADX-lean mice, respectively. Oxygen consumption was measured between 08:30 and 11:00 on day 12. Each bar represents means :1: SE for 20-34 mice. Asterisks indicate significant differences (P<0.05) between sham-operated ob/ob mice and adrenalectom- ized ob/ob mice. P indicates significant effect (P<0.05) of phenotype; 8 indicates significant effect (P<0.05) of surgery and P - S indicates significant (P<0.05) phenotype surgery interaction. 40 SHAM 2 DAYS 4 DAYS so. DcLucose P * o _ 3° .STARCH P 501 . so . . 40 20. - . 20 o o FOOD INTAKE - kJ/day 8 . 0.2 r 0'8 - 0.8 a T P < . . 0 0.6I o 6 I- > (‘5 g 0.4 . » 0.4 E B: 3 >. o O m i if ILA i §’//////I Em” 22H o \ onion 0 OXYGEN CONSUMPTION- mL h‘ d" I: Fig. 2. Food intake, body weight gain, and oxygen consumption of sham- operated (SHAM) mice switched from a high-starch diet to a high-glucose diet for 2 or 4 days. Left panels: sham-operated (SHAM) ob/ob and lean mice were fed the high starch diet for 14 days, or were fed the high starch diet for 12 days and then switched to the high glucose diet for 2 days. Right panels: SHAM mice were fed the high-starch diet throughout the 16 day feeding trial, or switched to the high-glucose diet for the last 4 days of the 16 day experiment. Bars for food intake and oxygen consumption represent the means (ll-12 mice per treatment group) :t SE on day 14 (left panels) and 16 (right panels). Body weight gains were calculated as the average of difference between body weight on day 14 and day 12 (left panels) or day 16 and day 12 (9-12 mice per group; right panels). Aster- isks indicate a significant effect (P<0.05) of diet within phenotype. D indi- cates significant effect (P<0.05) of diet and P indicates significant pheno- type effect. 41 high- starch diet to the high-glucose diet for 4 days (Fig. 3). Adrenalec- tomized ob/ob mice fed glucose did not increase their oxygen consumption even though energy intake was elevated (Fig. 3). Diet did not influence plasma glucose or insulin concentrations in sham—operated mice (Fig. 4). As expected, plasma insulin concentrations were much higher in ob/ob mice than in lean mice. Plasma glucose concentrations in adrenalectomized mice were not influenced by diet (Fig. 5). Plasma insulin concentrations, however, were approximately 2 times higher in adrenalectomized ob/ob mice, but not in adrenalectomized lean mice, switched to a high-glucose diet for 2 or 4 days than in the respective adrenalectomized control mice fed a high- starch diet (Fig. 5). Adrenalectomy lowered the high plasma insulin con- centrations in ob/ob mice (Fig.4 & Fig.5) as has been observed before (48,91,109). Adrenalectomized ob/ob mice had a higher protein content of BAT than that of adrenalectomized lean mice (Fig. 6). BAT protein content was unaffected by diet. GDP binding to isolated BAT mitochondria, which is an indicator of thermogenic activity of BAT, was lowered 26% in adrenalectomized ob/ob mice, but not in adrenalectomized lean mice, 42 ADX 2 DAYS 4 DAYS 3°: Ectucosa P o ’ 3° . s STARCH * p 1512' 60. FL? 2' . 60 Egg .. g g .. g 20‘ g g . 20 o / , A I. , 0.6 * 0.6 g 0.5 . P 3 . 0.5 g 0.4. .0.4 c) 5 0.3« x - 03 g?» 0.2‘ § § . 0.2 >' 0.1 . \ \ . 0.1 3 \ \ m 0.0 , , 0.0 2' a g a. P P gig I s. :1: S 6 m I: \ 4 C2) .1 4‘ \ 0 E \ E 2. \ 2 g ' ‘ \ x 0 \ x 0 0 03/03 LEAN oa/oe LEAN Fig. 3. Food intake, body weight gain, and oxygen consumption of adrenalectomized mice switched from a high-starch diet to a high-glucose diet for 2 or 4 days. Left panels: adrenalectomized (ADX) ob/ob and lean mice were fed the high-starch diet for 14 days, or were fed the high starch diet for 12 days and then switched to the high glucose-diet for 2 days. Right panels: ADX mice were fed the high-starch diet throughout the 16 day feeding trial, or switched to the high-glucose diet for the last 4 days of the 16 day experiment. Bars for food intake and oxygen consumption represent the means (8-12 mice per treatment group) t SE on day 14 (left panels) and 16 (right panels). Body weight gains were calculated as the average of difference between body weight on day 14 and day 12 or day 16 and day 12 (9-21 mice per group). Asterisk indicates a significant effect (P<0.05) of diet within phenotype treatment. D indicates significant effect of diet and P (P<0.05) indicates significant effect of phenotype. 43 SHAM GLUCOSE 30° ‘ STARCH 250.. l T 200. 150 . 100 . GLUCOSE- mg/dL //////Ai 1000 L § INSULIN- uU/mL 3 .3 .3... O Fig. 4. Plasma glucose and insulin concentrations of sham-operated (SHAM) ob/ob and lean mice fed the high-starch diet for 16 days, or fed the high- starch diet for 12 days and then switched to the high-glucose diet for 4 additional days. Each bar represents means i SE for 5-10 SHAM ob/ob and lean mice. P indicates significant effect (P<0.05) of phenotype. ADX 2 DAYS 4 DAYS 300 300 IGLUCOSE 250: STARCH - 250 (“3.4 zoo. q_ "" '1' T \ . 200 O '0 \ 3 E .J 100 \ . 100 o \ 50. \ 50 0 \ \ \ o 200 ' 200 * * D . I P . 160, _T_ DP . 160 z _| d . j E 120‘ r- 120 D 5 . . U) E 1 80¢ 80 4o. . 40 o \ \ , [SS 0 08/03 OB/OB LEAN Fig. 5. Plasma glucose and insulin concentrations of adrenalectomized mice switched from a high-starch diet to a high-glucose -diet for 2 or 4 days. Left panels: adrenalectomized (ADX) ob/ob mice were fed the high-starch diet for 14 days, or were fed the high-starch diet for 12 days and then switched to the high-glucose diet for 2 additional days. Right panels: ADX mice were fed the high-starch diet for 16 days, or were fed the high-starch diet for 12 days and then switched to the high-glucose diet for 4 additional days. Each bar represents means i SE for 20-26 ADX ob/ob and lean mice. Asterisk indicates a significant effect (P<0.05) of diet within phenotype treatment. D, significant effect (P<0.05) of diet; P, significant effect (P<0.05) of phenotype; D - P, significant (P<0.05) diet phenotype interaction. 45 ADX 2 DAYS 4 DAYS GLUCOSE P STARCH . 25 ‘6’ 8 f3 20. 5- _2o TOTAL PROTEIN- m9 / m o L. a a 1c 5 400. DP.400 ('9 g 1 T . 2.: 300. \\ 300 '5 ° = I: ' 0'5 . _ gas... 200 \ 3; ms?- \ .200 n. ma- - \ . 85 1°01 \ .100 o g o \ k \ o OB/OB OBIOB LEAN Fig. 6. Total protein and GDP binding to isolated brown adipose tissue (BAT) of adrenalectomized ob/ob and lean mice fed a high starch diet for 14 (left panels) to 16 (right panels) days, or fed the high starch diet for 12 days and then switched to a high glucose diet for 2 (left panels) or 4 (right panels) additional days. BAT represents combined interscapular and sub- scapular BAT depots. Each bar represents means :1: SE for 10-16 mice. Asterisk indicates a significant effect (P<0.05) of diet within phenotype. P indicates significant effect (P<0.05) of phenotype and D - P indicates significant (P<0.05) diet phenotype interaction. 46 switched from the high-starch diet to the high-glucose diet for four days; no changes in GDP binding were noted within 2 days after switching from the high-starch to the high-glucose diet (Fig. 6). Adrenalectomized lean mice had higher GDP binding to BAT mitochondria than adrenalectomized ob/ob mice (Fig. 6). 47 IV. DISCUSSION A. Thesis Discussion Adrenalectomy reduced food intake, body weight gain and plasma insulin concentration in ob/ob mice fed a high-starch diet, as others have reported (48,91,109). Energy expenditure, as measure by oxygen consump- tion and expressed as ml/hr/gm body weight, was higher in adrenalectom- ized ob/ob mice than in sham ob/ob mice even though adrenalectomized ob/ob mice consumed less food. Thus, the decrease in efficiency of dietary energy retention observed in adrenalectomized ob/ob mice is caused by both decreased energy intake and increased energy expenditure per unit body weight. Effects of adrenalectomy are diet dependent (48,91,109). Chronic con- sumption of a high-glucose diet can partially block the effects of adrenalectomy in ob/ob mice. I investigated the mechanism of this response by determining if dietary glucose could reverse effects of adrenalectomy in ob/ob mice fed a high starch diet. Consumption of a high glucose diet for only 4 days caused an increase in food intake and body weight gain without changing oxygen consumption in adrenalectom- ized ob/ob mice. Plasma insulin also increased and GDP binding to 48 isolated BAT mitochondria decreased in these mice within 4 days after switching to the high-glucose diet. Thus, dietary glucose can within several days reverse effects of adrenalectomy in ob/ob mice fed a high- starch diet. These responses are genotype dependent as the switch from dietary starch to glucose, did not change these parameters in adrenalectom- ized lean mice. The mechanisms responsible for dietary glucose-induced increases in plasma insulin and decreases in GDP binding to BAT mitochondria in adrenalectomized ob/ob mice are not entirely clear. Plasma insulin was elevated at the earliest time point examined (2 days after the diet switch) without significant influences of the diet switch on food intake or plasma glucose concentrations. The possibility that diet-induced differences in glu- cose concentration within the gastrointestinal tract alter gastrointestinal hormones release and/or neural signals that control insulin secretion needs to be explored. The BAT response to the diet switch in adrenalectomized ob/ob mice took longer (4 days) than did the insulin response (2 days). This raises the possibility that the decrease of thermogenic activity in BAT may be caused by the earlier increase in plasma insulin. It has been demonstrated the injection of insulin into the carotid artery decreases the sympathetic 49 efferent firing rate to BAT (87). Moreover, several reports have shown that BAT thermogenesis is depressed in insulin-deficient diabetic rats (79) and in insulin-resistant obese animals (82). Thus, insulin resistance in obese animals plays an important role in thermogenesis of BAT. At 4 weeks of age ob/ob mice, before insulin resistant development, have nor- mal responses of thermogenic activity in BAT on acute exposure to cold. Whereas, the response to cold is greatly blunted when insulin resistance has developed at 5-week-old of ob/ob mice (60). Several studies (85,86) found that insulin may also act directly on the hypothalamus to regulate sympathetic activity. Sakaguchi (85,86,87) has demonstrated that injection of insulin into the ventromedial hypothalamus or paraventricular nucleus decreases sympathetic firing rate to BAT. Destruction of neurons in the VMH abolished these effects caused by insulin injection. From the above data I suggest that insulin may be one modulator for the hypothalamic control of sympathetic nervous system to BAT in ob/ob mice. Further research is needed to explore these areas. My hypothesis is that the glucose component of the diet may act through gastrointestinal receptors or hormones to cause alteration of auto- nomic neurotransmitter to the pancreatic B-cell in adrenalectomized ob/ob mice showing a two-time increase of plasma insulin. Then, insulin acts 50 through the hypothalamic control of sympathetic nervous action to BAT. This effect is mediated in part by the VMH to decrease the firing rate of sympathetic nerves to BAT resulting in a decrease in GDP binding and an increase in food intake and body weight gain. B. Future Research To further understand my data and to keep searching for the mechan- isms whereby diet composition and adrenal secretions contribute to obe- sity, I propose the following studies. 1. Norepinephrine turnover Norepinephrine turnover, which is an indicator of sympathetic nervous system activity, should be measured in adrenalectomized ob/ob mice fed a high-starch diet and then switched to a high-glucose diet for 2 to 4 days. According to my results which showed a lower thermogenic activity in brown adipose tissue of adrenalectomized ob/ob mice fed a high-glucose diet for 4 days, I predict that consumption of the glucose diet decreases stimulation of the sympathetic nervous system to BAT. Thus, measurement of norepinephrine turnover in BAT can give further information in this area. 51 2. Peripheral insulin injection I speculate that the glucose diet-induced lowering of BAT metabolism is caused by the earlier elevation in plasma insulin concentration. To test the hypothesis that hyperinsulinemia is the key regulator causing low ther- mogenic activity in BAT, I would inject long-acting insan into adrenalec- tomized ob/ob mice fed the starch diet. I would measure GDP binding to BAT mitochondria after 2 to 4 days. Insan might act directly on BAT or indirectly via the central nervous system. Therefore, I would determine if peripheral insan injection decreased the firing rate of sympathetic nervous system to BAT in adrenalectomized ob/ob mice. If this occurred I would further suspect that insulin may act at central nervous system which then modulates sympathetic nervous system to BAT. To explore this hypothesis, I would inject insulin into the hypothalamus and measure the firing rate of sympathetic nerves in BAT. If I found that hypothalamic injection of insulin reduces the efferent rate of sympathetic activity to BAT in adrenalectomized ob/ob mice but not in lean mice, this would sug- gest that insulin may be one modulator for hypothalamus to control sym- pathetic nerves efferent firing rate to BAT. A dose response curve for insulin could also be measured to provide data on sensitivity of the hypothalamus to insulin. 52 3. Vagotomy or cholinergic blocker Consumption of a high-glucose diet caused an increase in plasma insulin concentration in adrenalectomized ob/ob but not in lean mice. The mechanisms responsible are still not clear. There are at least four possibili- ties : l) nervous system regulates insulin secretion; 2) glucose directly acts on pancreas B cells; 3) glucose stimulates gut hormones secretion which acts on pancreas to modulate insulin secretion; or 4) glucose metabolites stimulate insulin secretion. I think that nervous system regulation of insu- lin secretion is the most likely pathway according to recent findings (65,85,86,87). To address the possibility that dietary glucose affects plasma insulin in adrenalectomized ob/ob mice via altered neural regula- tion of insulin secretion I would use vagotomy or cholinergic antagonist (atropine). Adrenalectomized ob/ob mice and lean mice either fed a high- starch diet or fed a high-starch diet and then switched to a high-glucose diet would be vagotomized or injected with atropine. A disadvantage of vagotomy would be that the surgery would decrease of food intake and may interfere with the results. I would therefore use atropine as the first approach. 53 4. Body composition Energy density of gain ( an indicator of relative proportions of fat and lean tissue gain) and efficiency of energy retention can give us a better idea of how quickly the dietary glucose can reverse adrenalectomy effect in energy efficiency within a short time. Generally speaking, it is difficult to detect differences in these parameters within a short time. However, my results showed that adrenalectomized ob/ob mice gained more body weight when switched from the high-starch diet to the high-glucose diet. It would be interesting to determine if detectable change in body composi- tion would be evident within 4 days after the diet switch. 54 V. REFERENCES Ashwell, M., S. Holt, G. Jennings, D. M.,Stirling, P. Trayhum, and D. A. York. Measurement by radioirnmunoassay of the mito- chondrial uncoupling protein from brown adipose tissue of obese(ob/ob) mice and Zucker(fa/fa) rats different ages. FEBS 179(2): 233-237, 1984. Bank, H. L. A quantitative enzyme-linked immunosorbent assay for rat insulin. J. of Immunoassay 9(2): 135-158.1988. Batchelor, B. R., J. S. Stern, P. R. Johnson, and R. J. Mahler. Effects of streptozotocin on glucose metabolism, insulin response, and adiposity in ob/ob mice. Metabolism 24(1):77-91, 1975. Bellush, L. L.,and N. E. Rowland. Dietary self-selection in dia- betic rats: an overview. Brain. Res. Bull. 17(5): 653-661, 1986. Beloff-Chain, A., M. E. Newman, and K. R. L. Mansford. In vitro studies on insulin secretion in the genetically obese mouse. Diabetologia 9:447-452, 1973. Berthoud, H. R. The relative contribution of the nervous system, hormones, and metabolites to the total insulin response during a meal in the rat. Metabolism 33(1):]8-25, 1984. Bieri, J. G., G. S. Stoewsand, and G. M. Briggs. Report of the American Institute of Nutrition Ad Hoc Committee on standatds for nutrition studies J. Nutr. 107: 1340-1348, 1977. Boissonneault, G. A., M. J. Homshuh, J. W. Simons, D. R. Rom- sos and G. A. Levelle. Oxygen consumption and body fat con- tent of young lean and obese (OB/OB) mice. Proc. Soc. Exp. Biol. Med. 157: 402-406, 1978. Bouillaud, F., D. Ricquier, J. Thibault, and J. Weissenbach. Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein. Proc. Nat. 10 ll 12 13 14 15 16 17 18 19 55 Acad. Sci. 82: 445-448, 1985. Boulange, A., E. Planche, and P. D. Gasquet. Onset of genetic obesity in the absence of hyperphagia during the first week of life in the Zucker rat (fa/fa). J. Lipid Res. 20:857-864, 1979. Bray, G. A. Obesity-a disease of nutrient or energy balance? Nutr. Rev. 45(2): 33-43, 1987. Bray, G. A. Endocrine factors in the control of food intake. Fed. Proc. 33(5):1140-1145, 1974. Bray, G. A., and D. A. York. Hypothalamic and genetic obesity in dxperimental animals: an autonomic and endocrine hypothesis. Physiol. Rev. 59(3): 719-809, 1979. Brief, D. J., and J. D. Davis. Reduction of food intake and body weight by chronic intraventricular insan infusion. Brain Res. Bull. 12:571-575, 1984. Clifton, P. G. Feeding rate and meal patterns in the laboratory rat. Physiol. Behav. 32(3): 369-374, 1984. Coleman, D. L., and K. P. Hummel. Hyperinsulinemia in pre- weaning diabetes (db) mice. Diabetologia 10:607-610, 1974. Corp, E. S., S. C. Woods, D. Porte, D. M. Dorsa, D. P. Figlewicz,andD.G. Baskin Localization of I(125)-insulin binding sites in the rat hypothalamus by quantitative autoradiography. Neurosci. Lett. 70:17-22, 1986. Cunningham, J. J., M. A. Gulino, P. A. Meara, and H. H. Bode. Enhanced hepatic insulin sensitivity and peripheral glusose uptake in cold acclimating rats. Endocrinology 117: 1585-1589, 1985. Deb, S., R. J. Martin, and T. V. Hershberger. Maintenance requirement and energetic efficiency of lean and obese Zucker rats. J. Nutr. 106:191-197, 1976. 20 21 22 23 24 25 26 27 28 29 56 Debons, A. F., L. D. Zurek, C. S. Tes, and S. Abrahamsen. Cen- tral nervous system control of hyperphagia in hypothalamic obe- sity: dependence on adrenal glucocorticoids. Endocrinology 118: 1678-1681, 1986. Dubuc, P. U. Non-essential role of dietary factors in the develop- ment of diabetes in ob/ob mice. J. Nutr. 111:1742-1748, 1981. Dubuc, P. U. The development of obesity, hyperinsulinemia, and hyperglycemia in ob/ob mice. Metabolism 25(12): 1567-1574, 1976. Fletcher, J. M. Effects of adrenalectomy before weming and short- or long-term glucocorticoid administration on the geneti- cally obese Zucker rat. Biochem. J. 238: 459-463, 1986 Foster, D. 0., and M. L. Frydman. Nonshivering thermogenesis in the rat. 11. measurements of blood flow with microspheres point to brown adipose tissue as the diminant site of the calorigenesis induced by noradrenaline. Can. J. Physiol. Pharmacol. 56: 110- 122, 1978. Freedman, M. R., B. A. Horwitz, and J. S. Stern. Effect of adrenalectomy and glucocorticoid replacement of obesity. Am. J. Physiol. 250: R595-R607, 1986. Gill, J. L. Design and analysis of experiments in the animal and medical sciences. Ames, Iowa State Univ. press, 1978, vol.1. Goodbody, A. E. and P. Trayhum. GDP binding to brown- adipose-tissue mitochondria of diabetic-obese (db/db)mice Biochem. J. 194: 1019-1022, 1981. Goodbody, A. E., and P. Trayhum. Studies on the activity of brown adipose tissue in suckling; pre-obese, ob/ob mice. Biochirn. Biophys. Acta. 680: 119-126, 1982. Grogan, C. K., H. K. Kim, and D. R. Romsos. Effects of adrenalectomy on energy balance in obese (ob/ob)mice fed high 3O 31 32 33 34 35 36 37 38 39 57 camohydrate or high fat diets. J. Nutr. 117: 1115-1120, 1987. Hansen, B. C., Kai-lin C. Jen, and P. Kribbs. Regulation of food intake in monkeys: response to caloric dilution. Physiol. Behav. 26(3): 479-486, 1981. Havrankova, J., J. Roth, and M. J. Brownstein. Insulin receptors in brain. Adv. Metab. Disord. 10:259-263, 1983. Hirnms-Hagen, J. Brown adipose tissue thermogenesis in obese animals. Nutr. Rev. 41(9): 261-267, 1983. Hirnms-Hagen, J. Cellular thermogenesis. Ann. Rev. Physiol. 38:315-350, 1976. Hirnms-Hagen, J. Brown adipose tissue metabolism and therrno- genesis. Ann. Rev. Nutr. 5: 69-94, 1985. Hirnms-Hagen, J. and M. Deasutels. A mitochondrial defect in brown adipose tissue of the obese (ob/ob) mouse: reduced binding of purine nucleotides and a failure to respond to cold by an increase in binding. Biochem. Biophy. Res. Commun. 83(2): 628-634, 1978. Himms-Hagen, J., J. Triandafillou, and C. Gwilliam. Brown adi- pose tissue of cafeteria-fed rats. Am. J. Physiol. 241:E116-E120, 1981. Himms-hagen, J. Thermogenesis in brown adipose tissue as an energy buffer. NEJ Med. 311: 1549-1558, 1984. Hollifield, G. Glucocorticoid-induced obesity - A model and a challenge. Am. J. Clin. Nutr. 21(12): 1471-1474, 1968. Holt, S. J., D. A. York, and J. T. R. Fitzsimons. The effects of corticosterone, cold exposure and overfeeding with sucrose on brown adipose of obese Zucker rats (fa/fa). Biochem. J. 214: 215-223, 1983. 40 41 42 43 44 45 46 47 48 49 58 Holt, S. J ., and D. A. York. The effect of adrenalectomy on GDP binding to brown-adipose-tissue mitochondria of obese rats. Biochem. J. 208: 819-822, 1982. Holt, 8. J ., and D. A. York. Effect of adrenalectomy on brown adipose tissue of obese (ob/ob) mice. Horm. Metabol. Res. 16: 378-379, 1984. James, W. P. T., and P. Trayhum. Thermogenesis and obesity. Br. Med. Bull. 37(1): 43-48, 1981. Kaplan, M. L. Consumption of 02 and early detection of fa/fa genotype in rats. Metabolism 28(11):1147-1151, 1979. Kaplan, M. L., and G. A. Leveille. Core temperature, 02 con- sumption, and early detection of ob/ob genotype in mice. Am. J. Physiol. 227(4): 912-915, 1974. Karakash, C., F. Assimacopoulos-Jeannet, and B. Jeanrenaud. An anomaly of insulin removal in perfused liver of obese- hyperglycemic (ob/ob) mice. J. Clin. Inves. 57:1117-1124, 1976. Karam, J. H., G. M. Grodsky, K. N. Ching, F. Schmid, K. Burrill, and P. H. Forsharn. "Staircase" glucose stimulation of insulin secretion in obesity: measure of beta-cell sensitivity and capacity. Diabetes 23:763-770, 1974. Kim, H. K., and D. R. Romsos. Brown adipose tissue metabolism in ob/ob mice: effects of a high-fat diet and adrenalectomy. Am. J. Physiol. 253: E149-E157, 1987. Kim, H. K., and D. R. Romsos. Adrenalectomy fails to stimulate brown adipose tissue metabolism in ob/ob mice fed glucose. Am. J. Physiol. 255:E597-E603, 1988. Knehans, A. W. and D. R. Romsos. Norepinephrine turnover in obese (ob/ob) mice: effects of age, fasting, and acute cold. Am. J. Physiol. 244: E567-E574, 1983. 50 51 52 53 54 55 56 57 58 59 Knehans, A. W.,and D. R. Romsos. Reduced norepinephrine tum- over in brown adipose tissue of ob/ob mice. Am. J. Physiol. 242: E253-E261, 1982. Landsberg, L., and J. B. Young Insulin-mediated glucose meta- bolism in the relationship between dietary intake and sympathetic nervous activity. Int. J. Obesity 9(2):63-68, 1985. Lavine, R. L., N. Voyles, P. V. Perrino, and L. Recant. Func- tional abnormalities of islets of Langerhans of obese hypergly- cemic mouse. Am. J. Physiol. 233(2):E86-E90, 1977. Leblanc, J. and L. Brondel. Role of palatability on meal-induced therrnogeneis in human subjects. Am. J. Physiol. 248:E333-E336, 1985. Lin, P. Y., D. R. Romsos and G. A. Leveille. Food intake, body weight gain, and body composition of the young obese (ob/ob) mouse. J. Nutr. 107:1715-1723, 1977. Lin, P. Y., D. R. Romsos, J. G. V. Tuig, G. A. Leveille. Mainte- nance energy requirements, energy retention and heat production of young obese (ob/ob) and lean mice fed a high-fat or a high- carbohydrate diet. J. Nutr. 109: 1143-1153, 1979. Lissner, L., D. A. Levitsky, B. J. Strupp, H. J. Kalkwarf, and D. A. Roe. Dietary fat and the regulation of energy intake in human subjects. Am. J. clin. Nutr. 46: 886-892, 1987. Marchington, D., N. J. Rothwell, M. J. Stock, and D. A. York. Energy balance, diet-incuced thermogenesis and brown adipose tissue in lean and obese (fa/fa) Zucker rats after adrenalectomy. J. Nutr. 113: 1395-1402, 1983. Mattes, R. D., C. B. Pierce, and M. I. Friedman. Daily caloric intake of norrnal-weight adults: response to changes in dietary energy density of a luncheon meal. Am. J. Clin. Nutr. 48: 214, 1988. 59 61 62 63 64 65 66 67 68 60 Mayer, J., M. M. Dickie, M. W. Bates, and J. J. Vitale. Free selection of nutrients by hereditarily obese mice. Science 113: 745-746, 1951. Mercer, S. W. and P. Trayhum. The development of insulin resis- tance in BAT may impair the acute cold-induced acrivation of thermogenesis in genetically obese (ob/ob) mice. Biosci. Rep. 4: 933-940, 1984. Miller, R. E. Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the islets of Langerhans. Endocr. Rew. 2(4):471-494, 1981. Naim, M., J. G. Brand, M. R. Kare, and R. G. Carpenter. Energy intake, weight gain and fat deposition in rats fed flavored, nutri- tionally controlled diets in a multichoice ("cafeteria") design. J. Nutr. 115: 1447-1458, 1985. Nedergaard, J. and Lindberg, O. The brown fat cell. Int. Rev. Cytol. 74: 187-286, 1982. Nicholls, D. G. Brown adipose tissue mitochondria. Biochirn. Biophys. Acta 549: 1-29, 1979. Niijirna, A., F. Rohner-Jeanemaud, and B. Jeanemaud. Role of ventromedial hypothalamus on sympathetic efferents of brown adipose tissue. Am. J. Physiol. 247:R650-R654, 1984. Ohshima, K., N. S. Shargill, T. M. Chan, and G. A. Bray. Adrenalectomy reverses insulin resistance in muscle from obese (ob/ob) mice. Am. J. Physiol. 246: E193-E197, 1984. Peachey, T., R. R. French, and D. A. York. Regulation of GDP binding and uncoupling-protein concentration brown-adipose- tissue mitochondria. Biochem. J. 249:451-457, 1988. Perkins, M. N., N. J. Rothwell, M. J. Stock, and T. W. Stone. Activation of brown adipose tissue thermogenesis by the ven- tromedial hypothalamus. Nature 289: 401-402, 1981. 69 70 71 72 73 74 75 76 77 61 Peterson, A. D., and B. R. Baumgardt. Influence of level of energy demand on the ability of rats to compensate for diet dilu- tion. J. Nutr. 101: 1069-1074, 1971. Prats, E., M. J. Castella, R. Iglwsias and M. Alemany. Energy intake of rats fed a cafeteria diet. Physiol. Behav. 45(2): 263-272, 1989. Ramirez, 1., M. G. Tordoff, and M. I. Friedman. Dietary hyper- phagia and obesity: what causes them? Physiol. Behav. 45(1):163-168, 1989. Ramirez, 1., M. I. Friedman. Dietary obesity: role of caloric den- sity vs. fat content. Fourth Ann. meeting North Am. Assoc. for the study of Obes. 12-16, Oct. 1987. Rogers, P. J ., and J. E. Blundell. Meal patterns and food selection during the development of obesity in rats fed a cafeteria diet. Neurosci. Biobehav. Rev. 8(4): 441-453, 1984. Romsos, D. R., D. Fergusion, and J. G. Vander Tuig. Effect of a warm enviroment on energy balance in obese (ob/ob) mice. Meta- bolism 34(10): 931-937, 1985. Romsos, D. R., and D. Ferguson. Self-selected intake of carbohy- drate, fat, and protein by obese (ob/ob) and lean mice. Physiol. Behav. 28(2): 301-305, 1982. Rothwell, N. J., M. E. Saville, and M. J. Stock. Acute effects of food, 2-deoxy-D-glucose and noradrenaline on metabolic rate and brown adipose tissue in normal and atropinised lean and obese (fa/fa) Zucker rat. Pflugers Arch. 392:172-177, 1981. Rothwell, N. J ., M. Elizabeth Saville, and M. J. Stock. Factors influencing the acute effect of food on oxygen consumption in the rat. Inter. J. Obes. 6:53-59, 1982. 78 79 80 81 82 83 84 85 86 87 62 Rothwell, N. J ., M. J. Stock, and R. S. Tyzbir. Energy balance and mitochondrial function in liver and brown fat of rats fed "cafeteria" diets of varing protein content. J. Nutr.112:1663-1672, 1982. Rothwell, N. J., and M. J. Stock. A role for insulin in the diet- induced thermogenesis of cafeteria-fed rats. Metabolism 30(7):673-678, 1981. Rothwell, N. J ., and M. J. Stock. Similarities between cold- and diet-induced thermogenesis in the rat. Can. J. Physiol. Pharmacol. 58:842-848, 1980. Rothwell, N. J., and M. J. Stock. A role for brown adipose tissue in diet-induced thermogenesis. Nature 281(6):31-35, 1979. Rothwell, N. J., and M. J., Stock. Insulin and thermogenesis Int. J. Obesity 12:93-102, 1988. Rothwell, N. J.,and M. J. Stock. Regulation of energy balance. Ann. Rev. Nutr. 1: 235-256, 1981. Saito, M., and G. A. Bray. Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse. Am. J. Physiol. 246: R20- R25, 1984. Sakaguchi, T. and A. Bray Sympathetic activity following paraventricular injections of glucose and insulin. Brain Res. Bull. 21(1): 25-29, 1988. Sakaguchi, T., and G. A. Bray Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves. Proc. Natl. Acad. Sci. 84: 2012-2014, 1987. Sakaguchi, T., and G. A. Bray. Ventromedial hypothalamic lesions attentuate responses of sympathetic nerves to carotid arterial infusions of glucose and insulin. Int. J. Obes. 14:127-134, 1990. 88 89 91 92 93 94 95 96 97 98 63 Schteingart, D. E. Cushing’s syndrome. Endocrinology and meta- bolism clinics of North America 18(2):311-338, 1989. Sclafani, A., and d. Springer dietary obesity in adult rats: sirnilari- ties to hypothalamic and human obesity syndromes. Physiol. Behav. 17(3): 461-471, 1976. Shackney. S. E., and C. D., Joel. Stimulation of glucose metabol- ism in brown adipose tissue by addition of insulin in vitro. J. Bio. Chem. 241(17):4004-4010, 1966. Smith, C. K., and D. R. Romsos. Effects of adrenalectomy on energy balance of obese mice are diet dependent. Am. J. Physiol. 249: R13-R22, 1985. Stirling, J. L., and M. J. Stock. Metabolic origins of thermo- genesis induced by diet. Nature(220):801-802, 1968. Storlien, L. H. The ventromedial hypothalamic area and the vagus are neural substrates for anticipatory insan release. J. Auto. Nerv. Sys. 13:303-310, 1985. Swick, A. G., and R. W. Swick. Rapid changes in number of GDP binding sites on brown adipose tissue mitochondria. Am. J. Physiol. 251: El92-El95, 1986. Swick, A. G., and R. W. Swick. Changes in GDP binding to brown adipose tissue mitochondria and the uncoupling proten. Am. J. Physiol. 255: E865-E870, 1988. Tassava, T. Effects of acetylcholine and norepinephrine on glucose-induced insulin secretion from ob/ob and lean mouse pan- creatic islets. M. S. Thesis, Mich. State Univ., 1989. Thurlby, P. L., and P. Trayhum. Regional blood flow in geneti- cally obese (ob/ob) mice. Pflugers Arch. 385: 193-201, 1980. Thurlby, P. L., and P. Trayhum. The role of therrnoregulatory thermogenesis in the development of obesity in genetically-obese 64 (ob/ob) mice pair-fed with lean siblings. Br. J. Nutr. 42: 377-384, 1979. 99 Tokuyama, K., and J. Hirnms-Hagen. Increased sensitivity of the genetically obese mouse to corticosterone. Am. J. Physiol. 252:E202-E208, 1987. 100 Trayhum, P. Thermoregulation in the Diabetic-obese (db/db) mouse: the role of non-shivering thermogenesis in energy balance. Pflugers Arch. 380:227-232, 1979. 101 Trayhum, P. and W. P. T. James. Therrnoregulation and non- shivering thermogenesis in the genetically obese (ob/ob) mouse. Pflugers Arch. 373: 189-193, 1978. 102 Trayhum, P., M. Ashwell, G. Jennings, D. Richard, and D. M. Stirling. Effect of warm or cold exposure on GDP binding and uncoupling protein in rat brown fat. Am. J. Physiol. 252: E237- E243, 1987. 103 Trayhum, P., P. L. Thurlby, and W. P. T. James. Therrnogenic defect in pre-obese ob/ob mice. Nature 266(3): 60-62, 1977. 104 Trayhum, P., P. M. Jones, M. M. Mcguckin, and A. B. Good- body. Effects of overfeeding on energy balancd and brown fat thermogenesis in obese (ob/ob) mice. Nature 295(28): 323-325, 1982. 105 Trayhum, P., and L. Fuller. The development of obesity in geneti- cally diabetic-obese (db/db) mice pair-fed with lean siblings. Dia- betologia 19:148-153, 1980. 106 Triandafillou, J., and J. Hirnms-Hagen. Brown adipose tissue in genetially obese (fa/fa) rats: response to cold and die t. Am. J. Physiol. 244:E145-E150, 1983. 107 Tulp, O. L., P. P. Krupp, E., Jr. Danforth, and E. S. Horton. Characteristics of thyroid function in experimental protein malnu- trition. J. Nutr. 109:1321-1332, 1979. 65 108 Vander Tuig, J. G., D. R. Romsos, and G. A. Leveille. Mainte- nance energy requirements and energy retention of young obese (ob/ob) and lean mice housed at 33 and fed a high-carbohydrate or a high-fat diet. J. Nutr. 110:35-40, 1980. 109 Warwick, B. P., and D. R. Romsos. Energy balance in adrenalec- tomized ob/ob mice: effects of dietary starch and glucose. Am. J. Physiol. 255:R141-R148, 1988. 110 Watts, D. T., and D. R. H. Gourley. A simple apparatus for deter- mining basal metabolism of small animals in student laboratory. Proc. Soc. Exp. Biol. Med. 84:585-586, 1953. 111 Westrnan, S. Development of the obese-hyperglycaemic syndrome in mice. Diabetologia 4:141-149, 1968. "IIIIIIILIIIIIIIIIIIIIIIIII