musls A -4 __..... “ W‘JnM ‘ r -‘- .._ h 9- _. I, LE-u—wvh- w—l I . a . I . .1, "‘ rrnflzx‘ 15-} 'JJ‘IIIt-‘Jw-n-a .r 4...)". This is to certify that the dissertation entitled IEGULATION OF ENERGY BALANCE AND EDDY COMPOSITION IN THE OBESE (OB/OB) NOISE presented by COLLEEN KAY SMI'IH has been accepted towards fulfillment ' of the requirements for PM) degreein FOOD SCI. & HUMAN NUTRITION Major professor Dale R. Ramses Date \J/IZ)! {5 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 RASKJ LIBRARIES .——_ \— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. -_ w ‘.-v "' ,‘. _ zeo'aztg ‘ not cry Exagulation of energy balance and body composition in the obese (ob/ob) mouse . by Colleen Kay Smith 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 1985 ABSTRACT REGULATION OF ENERGY BALANCE AND BODY COMPOSITION IN THE OBESE (ob/ob) NDUSE BY Colleen Kay Smith The purpose of these studies was to describe the effects of acclimation to mild cold on energy balance and body composition of ob/ob mice, and to determine the importance of the adrenal gland in the development of obesity. Cold-acclimation (4 or 8 weeks housing at 14C) normalized energy intake, body weights and efficiency of energy retention of young obese mice, but hindlimb muscle gain was suppressed to 4% that of lean mice. Plasma corticosterone concentration of control obese mice was 2-4 times that of leans and cold-acclimation resulted in a further increase in corticosterone in obese mice, with no change in leans. To determine the importance of this high corticosterone concentration in the development of obesity, obese and lean mice were adrenalectomized or sham-operated at 3 or 6 weeks of age. Adrenalectomy normalized body weights, energy intake and efficiency of energy retention of obese mice fed stock diet. Adrenalectany also improved hindlimb muscle gains 50-19035 in obese fed stock diet, and plasma insulin concentrations of these obese mice were almost normalized. These effects of adrenalectomy were probably due to a combination of the direct lack of corticosterone and the restoration of insulin sensitivity to the tissues of obese mice. Previous studies had shown that consumption of semipurified diets by obese animals after adrenalectomy prevented the effects of adrenalectany. Consistent with this, adrenalectomy of obese mice fed a high-fat diet had no effect on energy balance, muscle gains or plasma insulin concentration. The same results were observed in obese mice fed a semipurified high-carbohydrate (glucose) diet after adrenalectomy. Insulin sensitivity was not normalized by adrenalectany in obese mice fed either senipurified diet. The restoration of insulin sensitivity appears to be necessary for the effects of adrenalectomy to be expressed in obese mice. Consumption of stock diet allowed insulin sensitivity to be restored, and adrenalectany prevented the complete development of obesity. When insulin resistance was maintained in adrenalectomized obese mice by their consumption of a sanipurified high-carbohydrate or high-fat diet, adrenalectcmy did not prevent obesity. Thus, the effects of adrenal secretions on the development of obesity in ob/ob mice appear to be dependent on the insulin status of the animal. DEDICATION To my parents AC KNOWLEDGEMENTS I would first like to thank Dr. Dale Romsos, without whose patience and guidance this project would not have been possible. I could not have asked for a better advisor. I would also like to thank Dr. Jerry Vander Tuig, whose helpfulness in the lab was a model for me. Finally, I must thank my parents, and Dennis, whose moral (and financial) support was invaluable. ii Table of Contents List Of TableSOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0... V List Of FigureSOOOOOOOO00......OOOOOOOOOOOO0.0.0.0000... vi Chapter 1. Chapter 2. Chapter 3. Chapter 4. Chapter 5. General Introduction......................... 1 Cold-acclimation of obese (ob/ob) mice: Effects on energy balance.................... 13 Materials and Methods........................ 15 Results...................................... 18 Discussion................................... 31 Cold-acclimation of obese (ob/ob) mice: Effects on skeletal muscle and bone.......... 36 Materials and Methods........................ 37 Results...................................... 41 Discussion................................... 56 Effects of adrenalectomy on energy balance of obese (ob/ob) mice are diet dependent........ 60 Materials and Methods........................ 63 Results...................................... 67 Discussion....................................98 Effects of adrenalectomy on energy balance, body composition and insulin sensitivity of obese mice fed semipurified diets............107 Materials and Methods........................110 Resu1tSOOIOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0...112 DiSCUSSionOOOOOOO0....0.0.0.0....00.0.0000000123 iii Chapter 6. General summary and conclusions ..... . . . . . . . . . 136 References 0......0.0.0.0...0.00....0.0.0.00000000000000134 iv Chapter 2. Table Table Chapter 3. Table Chapter 4. Table Table Chapter 5. Table Table Table Table Table 10. List of Tables Energy intake of mice from 4-12 weeks of ageCO0.0.0.000...OOOOOOOOOOOOOOOOOOOO0.... 1-9 Energy intake of mice from 9—12 weeks of ageOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.... 28 Composition of psoas muscle............... 45 Energy balance of energy restricted mice...96 Muscle weights of restricted mice..........97 Energy consumption and body weights of obese and lean mice.......................ll3 Energy gain and efficiency of energy reten- tion of obese and lean mice...............ll6 Energy density, muscle weights and bone lengths of obese and lean mice............118 Plasma corticosterone concentrations in obese and lean mice fed high-carbohydrate diet......................................124 Plasma corticosterone concentrations in obese and lean mice fed high-fat diet.....125 Chapter 2. Figure Figure Figure Figure Figure Chapter 3. Figure Figure Figure Figure Figure Figure Chapter 4. Figure List of Figures 1. Design of Experiment 2................... l7 2. Body weights and body energy of mice from 4-12 meks Of ageOOOOCOOOOOOOOOO0.0.0.... 22 Energy efficiency of mice from 4-12 weeks Of ageOOOOOOOOOOOOOOOOOOCCOOOOOOOOOOOOOOO 24 Body weights and body energy of mice from 9-12 meks Of age0.0000000000000000000:CO 27 Energy efficiency of mice from 9-12 weeks Of ageOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 30 Experimental Design.......................38 Muscle weights of mice from 4-12 weeks of ageOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOCOOOOO 43 Bone lengths in mice from 4-12 weeks of ageOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0... 47 Total body fat and percentage body fat in mice from 4-12 WEEKS Of ageoooooooooooooo 49 10.Total hindlimb muscle weights, bone lengths and total body fat of mice from 9-12 weeks Of ageOOOOOOOOOOOOOOOOOOOOOOO. 52 ll.Plasma corticosterone and insulin concen- trations in mice at 8 weeks of age....... 55 12.P1asma corticosterone concentrations of vi mice fed stock diet from 3-6 weeks of age or 6-9 weeks of age...................... 70 Figure 13.Body weights of mice fed stock diet from 3-6 weeks of age and 6-9 weeks of age.... 72 Figure l4.Energy consumption, gain and efficiency of mice fed stock diet from 3-6 weeks of age and 6-9 weeks of age..................... 74 Figure 15.Energy density, muscle weights and bone lengths of mice fed stock diet from 3-6 weeks of age and 6-9 weeks of age........ 77 Figure 16.Plasma insulin concentrations of mice fed stock diet from 3-6 weeks of age and 6-9 weeks of age............................. 81 Figure l7.Plasma corticosterone concentrations of mice fed high-fat diet from 3-6 weeks of age and 6-9 weeks of age................. 83 Figure 18.Body weights of mice fed high-fat diet from 3-6 weeks of age and 6-9 weeks of age...................................... 86 Figure l9.Energy consumption, gain and efficiency of mice fed high-fat diet from 3-6 weeks of age and 6-9 weeks of age.............. 88 Figure 20.Energy density, muscle weights and bone lengths of mice fed high-fat diet from 3-6 weeks of age and 6-9 weeks of age.... 91 Figure 21.Plasma insulin concentrations of mice fed high-fat diet from 3-6 weeks of age vii and 6-9 weeks of age..................... 94 Chapter 5. Figure 22.Glucose challenge curves for mice fed high-carbohydrate or high-fat diet from 4-7 meks Of ageOOOOOOOOOO0.0.0.000000000122 viii Chapter 1. General Introduction Under most conditions, the ob/ob mouse is more efficient at retaining dietary energy in its carcass than its lean littermates. This is not due simply to the increased energy intake of obese mice, since energy efficiency of obese mice pair-fed to leans is still almost 2 1/2 times that of leans (79,89). Thus, the increased efficiency of energy retention in obese mice must be due to reduced energy expenditure. There is considerable evidence indicating that a reduction in thermoregulatory thermogenesis in obese mice plays a major in this reduced energy expenditure (12,78,79,80,82). At thermoneutral environmental temperature (33C), the resting metabolic rate of obese and lean mice is the same (80), although obesity still develops in the ob/ob mice at 33C. Body temperature and oxygen consumption of obese mice declines repidly upon exposure to a cold (3C) environment (12). The average duration of survival of obese at 3C was 2.2 hours, while lean mice survived for more than a week at this temperature (50). This failure to resist cold stress was attributed to a failure to increase thermogenesis rather than to a failure to prevent heat loss; this conclusion was confirmed by succeeding studies (80,82). The increase in oxygen consumption in obese mice after norepinephrine administration was only half that observed in leans, supporting the hypothesis of impaired non-shivering thermogenesis in obese mice (88). Since brown adipose tissue (BAT) is the major organ responsible for thermoregulatory thenmogenesis in rodents (24), it seems likely that there is a defect in heat production by BAT of obese mice at environmental tenperatures l below thermoneutrality. The mechanism for heat production by BAT involves the uncoupling of oxidative phosphorylation, generating relatively large quantities of heat without the production of ATP. The sequence of events leading to heat production in BAT begins with a stimulus, such as cold stress, which activates the sympathetic nervous system. BAT is extensively innervated by sympathetic nerves. Norepinephrine, released by the sympathetic nerve endings, binds to B-adrenergic receptors on BAT cells and activates adenyl cyclase, leading to increased cAMP production. The increased cAMP activates a lipase, and accelerates lipolysis in the BAT cells. The free fatty acids which are released are used as fuel for increased mitochondrial oxidation. BAT mitochondria contain a unique proton conductance pathway which makes the inner mitochondrial membrane more permeable to the inward movement of protons. This pathway is regulated by a 32,000 D polypeptide which is on the outer surface of the inner mitochondrial membrane. The polypeptide is inhibited by purine nucleotides; removal of the inhibition allows operation of the proton conductance pathway. Fatty acids released during lipolysis stimulate this proton conductance pathway. This sequence of events in BAT provides two means of assessing the function of the pathway. Measurement of norepinephrine turnover in BAT provides information about the activity of the sympathetic nervous system in the tissue (41). The ability of the 32,000 D polypeptide to bind purine nucleotides, such as GDP, provides a means of assessing the capacity of BAT for heat production (16). At 25- 28C, both norepinephrine turnover and GDP binding to BAT mitochondria 3 are about 60% lower than in BAT of lean mice (33,41). This confirms the presence of a defect in heat production by BAT. This reduced heat production allows obese mice to expend less energy on thenmoregulation and so to be more efficient in retaining consumed energy in their carcasses. Even though obese mice die relatively rapidly during acute exposure to severe cold, they can survive exposure to less severe cold (14C) (33,41), as well as step-wise acclimation to severe cold (80). Exposure to 14C for several weeks increased both norepinephrine turnover and GDP binding in BAT of obese mice 2 - 2 1/2 times as much in obese mice as in leans, though neither norepinephrine turnover nor GDP binding reached the same level in obese mice as in leans (33,41). The effect of this mild cold exposure on overall energy balance is not known. It seems likely that since BAT of obese mice was stimulated more by cold exposure than that of lean mice, efficiency of energy retention in the obese would be reduced to be more like that of lean mice. However, calculation of efficiency of energy retention from data in the one report available on energy efficiency of obese mice housed at 17C or 23C for 10 days showed that the relative differences in energy efficiency between obese and lean mice were even greater at 17C than at 233 (79). This was a relatively short term exposure to a slightly higher temperature compared to the length of time and temperature used in the studies on BAT, so it is possible that thermogenic mechanisms were not fully activated in the mice in this 10 day study. Studies on energy balance after longer term cold exposure could help to clarify this. 4 Not only is energy retained with a higher efficiency in obese mice than in leans, it is also partitioned quite differently in the two phenotypes. This results in a markedly different body composition in obese mice as compared to leans. At 7-8 weeks of age, ad libitum fed obese mice weigh up to 1 1/2 times as much as their lean littermates (83). This difference in body weights gradually increases, so that by 37 weeks of age, obese mice weigh twice as much as leans. The percentage body fat in obese mice is almost twice that of leans at 7 weeks of age, and increases to almost 4 times that of leans by 37 weeks (83). The fat-free carcass of these obese mice averages only 17% of their body weight, compared to 32% fbr lean mice (83). Fat-free hindlimbs, representative of skeletal muscle, weigh approximately 20% less in ad libitum fed obese mice than in leans between 7 and 37 weeks of age (83). As with the elevated efficiency of energy retention, this abnormal body composition is not simply a result of excess energy intake by obese mice (8,17,35,79,86). Obese nuce pair-fed to leans still gain more body weight and body energy than lean mice, but less carcass protein (8,79). The percentage body fat of obese mice pair-gained to lean mice was still 3 1/2 times that of leans, and their percentage lean body mass was only half that of the lean mice (17). These data indicate that obese mice have a higher percentage of body fat and a smaller muscle mass than lean nuce, even when their food intake and body weight equals that of lean mice. Skeletal muscle growth may be linked to bone growth (36), so it seens likely that if muscle growth is reduced in obese mice, bone growth may also be reduced. No data are available to confirm this, 5 although there is one report in which tail length (an indicator of linear body growth) of obese mice was significantly less than that of leans (45). Further evidence supporting this link between skeletal muscle and bone growth comes from studies in the Zucker rat. In 15 week old obese rats, skeletal muscle weighed 10% less, and the tibia and femur were 10-15% shorter than in their lean counterparts (60). If the bones of obese mice are shorter than those of leans, they could exert less of a trophic effect on muscle and so contribute to the reduced muscle mass of obese mice. Envirormental temperature may influence the body composition of obese mice as well as their efficiency of energy retention. At 20 - 25C, hindlimb muscle of obese mice weighs about 20% less than that of leans; there is a preliminary observation that hindlimb muscle of obese mice housed at 14C weighs less than half as much as muscle of lean mice, suggesting that cold-acclimation may further reduce muscle growth in obese mice (46). The effects of long term cold-acclimation on body composition of obese mice have not been described, however. Several hormonal imbalances are present in obese mice which may contribute to their abnormal energy retention and body composition. The plasma insulin concentration of obese mice is 3-5 times that of lean mice from 6-60 weeks of age (26,29) . Even when obese mice were pair-gained to leans, plasma insulin of the obese mice was 2-3 times that of leans from 5-17 weeks of age (17). However, plasma glucose of obese mice is 10-40% higher than that of leans during most of this time (26), indicating impaired sensitivity to insulin in obese mice. Insulin has an important role in the regulation of both energy balance and body composition. Several studies have shown a 6 requirement for insulin in the normal functioning of brown adipose tissue. Both GDP binding and the content of the 32,000 D protein were reduced in diabetic rats compared to normo-insulinemic controls, and conversely, were increased in hyperinsulinemic rats compared to controls (71). Interscapular BAT of streptozotocin-diabetic rats was atrophied compared to controls, and basal and norepinephrine-stimulated heat production were both reduced in diabetic rats (70). Severely diabetic rats could not sustain the increased metabolic rate needed for survival in a cold environment (58). However, moderately diabetic rats retained sufficient insulin activity to survive at cold temperatures for up to 3 months (51). The hypertrophy of BAT which occurred when rats were fed a cafeteria diet was not observed in lactating rats fed the cafeteria diet (1). Plasma insulin was low in these lactating rats; insulin administration enabled the hypertrophy of their BAT stimulated by the cafeteria diet. Rothwell and Stock also found a requirement for insulin in thermogenesis induced by diet (63). These studies indicate a requirement for insulin in both non-shivering and diet-induced thermogenesis; the primary site of both of these processes is brown adipose tissue (24,62). The presence of insulin resistance in obese mice could prevent these actions of insulin on the BAT, resulting in their reduced energy expenditure and high efficiency of energy retention. The anabolic effects of insulin on skeletal muscle are blunted due to the development of insulin resistance in muscle. From 6 weeks of age on, insulin binding by skeletal muscle of obese mice is lower than in lean mice (29). Defects in glucose transport and utilization 7 occur even earlier in obese mice - by 4 weeks of age - indicating that these intracellular defects may have a more important role in the development of insulin resistance in muscle than the defects in binding, which occur when the resistance is more fully expressed (48). As mentioned earlier, plasma insulin of 3 week old obese mice is already considerably higher than that of leans, and blood glucose is also elevated in the obese mice, indicating that the insulin resistance may already be present in these 3 week old mice (18). Muscle weights of obese mice are already significantly lower than that of lean mice by 5 weeks of age and remain lower than leans through at least 37 weeks of age (83). The development of insulin resistance in the skeletal muscle of obese mice is thus consistent with their reduced muscle mass. Corticosterone is another hormone which probably has an important role in the abnormal energy balance and body composition of obese mice. Corticosterone is the primary glucocorticoid in rodents (87) and plasma corticosterone concentrations in obese mice range from 2-5 times that of lean mice (19,26,31). Functional receptors for glucocorticoids have been identified in BAT, indicating that this thermogenic tissue is a glucocorticoid target organ (21). Small amounts of glucocorticoids (below physiological concentrations) are necessary for the increase in fatty acid mobilization and thermogenesis potentiated by cold and norepinephrine (13). However, this is probably a permissive role of glucocorticoids, since chronic administration of corticosterone to intact mice, resulting in elevated plasma concentrations of the hormone, reduces GDP binding in BAT, indicating a reduction in the capacity for heat production in 8 these mice (25). Body weight gain, food intake and metabolic efficiency were all higher in the corticosterone treated mice compared to untreated mice. None of these studies could clarify whether the effects of glucocorticoids on energy balance were a direct effect of the hormone on BAT or an indirect effect, working via the sympathetic nervous system. It does seem clear, however, that glucocorticoids suppress BAT activity and reduce energy expenditure. This is consistent with the findings of elevated plasma corticosterone, reduced energy expenditure and increased efficiency of energy retention in ob/ob mice compared to their lean counterparts. The high concentration of plasma corticosterone in obese mice probably also contributes to their abnormal body composition. High doses of glucocorticoids reduce rates of muscle protein synthesis (60,61) and increase rates of muscle protein breakdown (66). Muscle protein breakdown has been shown to be higher in obese mice than in leans (83). When rats were given doses of corticosterone which tripled their nonmal plasma corticosterone concentration for 8 days, there»was a 20% reduction in the weight of their gastrocnemius muscle when expressed as a percentage of their body weight (67). Thus, the effects of corticosterone on muscle protein synthesis and breakdown appear to have a net effect in decreasing muscle weight. High concentrations of corticosterone also promote insulin resistance through decreases in insulin binding (38,55,57). This provides an indirect mechanism by which high concentrations of corticosterone can suppress muscle growth. The key role of adrenal secretions in the abnormal energy 9 balance and body composition of obese rodents can be demonstrated by adrenalectomizing these animals and following their development. Adrenalectomy reduced the food intake of several obese animals, including the Zucker obese rat, the VMH lesioned rat, the OTC-induced obese mouse and the ob/ob mouse (7,14,49,65), so that it was equal to or less than that of their lean counterparts. Adrenalectomy of obese Zucker rats also reduced their efficiency of energy retention by 50% so that it equaled that of leans (49). This reduction in efficiency could not have been due to the reduced food intake alone - pair-feeding sham—operated obese rats to leans did not result in a reduction in efficiency of energy retention (6). Thus, the decrease in efficiency of energy retention in adrenalectomized obese rats must have been due to increased energy expenditure. Consistent with this, GDP binding to mitochondria of BAT of adrenalectomized obese rats was increased more than 4 fold compared to sham-operated obese, indicating an increased capacity for heat production in BAT of adrenalectanized obese (49) . Adrenalectomy also increased norepinephrine turnover of BAT of ob/ob mice almost 3 fold, so that it almost equaled that of lean mice (84) . The effect of adrenalectomy on overall energy balance of obese mice is not known, however. The specific absence of corticosterone appears to be responsible for these effects of adrenalectomy. Administration of corticosterone, but not desoxycorticosterone, (a mineralocorticoid), prevented the effects of adrenalectomy on body weight gain and food intake in GTG-induced obese mice (14). Corticosterone administration also reversed the increase in GDP binding in adrenalectomized obese rats (34). Thus, adrenal secretions, and specifically l0 corticosterone, play an important role in controlling energy balance in obese rodents. Adrenalectomy of obese rodents also reduces their body weight so that it is equal to or only slightly greater than that of lean mice (7,14,49,65). Body composition of obese rodents must also be altered by adrenalectomy, because body energy density of adrenalectomized obese rats and ob/ob mice was 25-30% less than that of sham-operated obese, indicating a lower proportion of body fat in the adrenalectomized obese (49,84). The excess fat accumulation seen in GTG-induced obese mice was prevented by adrenalectomy (14), and in ob/ob mice, adrenalectomy reduced the weight of white adipose tissue by over 50% (65). Muscle growth of obese animals may also be improved by adrenalectomy, since the gastrocnemius muscle of adrenalectomized ob/ob mice weighed 20% more than that of Sham-operated obese (65). This increase in muscle mass was probably facilitated by the return of insulin sensitivity to skeletal muscle of adrenalectomized ob/ob mice (56). As with the effects of adrenalectomy on energy balance, these effects of adrenalectomy on body'composition also seem to be due to the absence of corticosterone. Administration of cortisone to adrenalectomized obese mice prevented the reduction in growth rate and adipose tissue weight as well as the increase in tail length and muscle weight observed in untreated adrenalectomized obese mice (65). The composition of the diet consumed by obese animals may also affect energy balance and body'composition. Obese mice fed a high-fat diet consumed more energy and gained more body weight than those fed a high-carbohydrate diet (20). Lean mice fed a high-fat ll diet from 3-6 weeks of age gained only 30% more energy than leans fed a high-carbohydrate stock diet, but obese mice fed the high-fat diet gained more than twice as much energy as high-carbohydrate fed obese (45). All of this excess body energy gain was made up of fat energy with no change in the gain in lean energy. This indicates a substantial shift in the body composition of obese mice fed high-fat diet to an even higher proportion of fat than is found in obese mice fed high-carbohydrate diet. These effects of the high-fat diet on body'composition may have been mediated by insulin - in rats fed high-fat diets, muscle and adipose tissue were resistant to the actions of insulin (43,75,91). The diet consumed by animals may also affect the outcome of adrenalectomy. When VMH-lesioned rats were ovariectomized and adrenalectomized and then fed a pelleted stock diet, thay did not demonstrate the hyperghagia and excess body weight gain normally observed in VMH-lesioned control rats (52). However, when ovariectanized-adrenalectomized rats were fed a liquid high-carbohydrate or semi-purified high-fat diet, they consumed as nmch food and gained almost as much body weight as VMH-lesioned controls. The effect of diet on the outcome of adrenalectomy in ob/ob mice is not known, since the studies reported so far have all utilized a pelleted stock diet. Thus, the overall aim of this research was to describe the effect of acclimation to mild cold on energy balance and body canposition, and to determine the importance of the adrenal gland in the development of obesity. A further aim was to determine a possible mechanism by which the changes in energy balance and body composition caused by cold-acclimation and adrenalectomy occur. The 12 following objectives were established to meet these aims: 1. To provide further data on the effect of mild cold-acclimation on energy balance, body composition and plasma corticosterone and insulin concentrations. 2. To determine the effect of adrenalectomy on the development of the obesity syndrome in ob/ob mice. 3. To examine the impact of diet on the outcome of adrenalectomy. 4. To examine the interaction of adrenalectomy and diet composition as they affect insulin responsiveness. Chapter 2. Cold—acclimation of obese (ob/ob) mice: Effects on energy balance. Obesity in the genetically obese (ob/ob) mouse is only partially a result of hyperphagia, since pair feeding these animals with their lean counterparts does not prevent the development of obesity (8,44). Thus, these obese mice must utilize dietary energy more efficiently than their lean littermates. Reduced energy expenditure for thermoregulatory thermogenesis may partially explain this increased energy efficiency (body energy gain/energy intake) of obese mice (80). When housed at a temperature near thermoneutrality (33C), body temperature is the same in lean and obese mice and obese mice are less than 1 1/2 times as efficient as leans (46,85). However, when obese mice are housed at temperatures approximating those of many vivariums (20 - 25C) their body'temperature is 1 1/2 - 2 l/2C lower than that of lean mice and they are 2 1/2 times as efficient at retaining dietary energy than lean mice (39,44,80). These differences in body'temperature and energy efficiency of obese mice at the different environmental temperatures may be caused by low heat production by brown adipose tissue (BAT) of obese mice at 23C (33,80). BAT is the major organ responsible for thermoregulatory heat production in rodents (24). The observation that obese mice die of hypothermia within only a few hours after abrupt exposure to 4C, probably because they are unable to sufficiently increase heat production in their BAT, supports this hypothesis (50). 13 l4 Obese mice can, however, survive exposure to less severe cold (14C), as well as stepwise acclimation to a lover temperature (4C) (2,33,41). Although body temperature is still lover in obese mice than in leans after 10-20 days at 14C, BAT has been shown to be activated in these mice (33,41,46). At 20-25C, both norepinephrine turnover, an estimator of sympathetic nervous system stimulation of BAT, and GDP binding, an estimator of the capacity for heat production, are about 60% lower in BAT of obese mice than in lean mice (33,41). Exposure to 14C for several weeks increased both parameters 2 - 2 1/2 times as much in obese mice as in lean mice, though neither quite reached the same level in the obese mice as in leans (33,41). Exposure to 4C for 4 weeks increased GDP binding in obese mice by almost 3 times compared to values for obese mice at 23C, resulting in no difference in GDP binding between obese and lean mice at 4C (2). Based on these observations, one might expect that differences in energy efficiency between obese and lean mice housed at 14C would be less than those observed at 20 - 25C, due to the greater activation of BAT in obese mice housed at 14C rather than at 20 - 25C relative to the responses of lean mice housed at the same two temperatures. But calculation of efficiency of energy retention from data in the one report on energy efficiency of obese mice at temperatures below normal animal roan temperature showed a greater relative decrease in energy efficiency in lean mice (from 13.0% at 23C to 8.8% at 17C) than in obese mice (from 29.6% at 23C to 24.9% at 17C) after 10 days at 17C (79). Thus, the relative differences in energy efficiency between obese and lean mice were even greater at 17C than at 23C. At 15 lWC, obese mice were 2.8 times as efficient as lean mice, whereas at 23C, obese mice were only 2.2 times as efficient as lean mice (79). It is possible, however, that thermogenic mechanisms were not sufficiently activated in obese mice during this short term study (10 days) to produce the lower efficiency of energy retention predicted from results of longer-term studies on BAT metabolism in cold-acclimated obese mice (2,33,41) , The present study was conducted to assess effects of mild cold (14C) exposure for 4 - 8 weeks on energy balance in young and near-adult obese (ob/ob) and lean mice. In addition, after 4 weeks of cold exposure, mice were returned to 23C to assess changes in energy balance during a reacclimation period of 4 weeks. Materials and Methods Animals Obese (ob/ob) and lean (ob/+ or +/+) male mice were obtained from our breeding colony of C57BL/63-ob/+ mice. The breeding colony was housed at 233 and the mother was removed from the pups at 3 weeks of age. At 3 1/2 weeks of age, obese and lean pairs were separated from their littermates and were housed individually in solid-bottom, plastic cages with wood shavings as bedding. At 4 weeks of age, mice were assigned to one of two environmental temperatures (23C or 14C). During the first week of exposure to 14C, paper was placed in each cage as extra nesting material. All mice had food (wayne Lab-Blox, Allied Mills, Chicago, IL) and water available ad libitum. Food intake and body weights were recorded weekly. Room lights were on fran man- 1999 hours daily. 16 Experimental Design Experiment 1 was designed to determine effects of chronic cold exposure on body weight and energy balance of young obese and lean mice. Both obese and lean mice at 4 weeks of age were divided into 5 groups of 9-10 mice each. Group 1 was killed at 4 weeks of age to serve as an initial group. Groups 2 and 3 were housed at 233 for 4 and 8 weeks, respectively, and groups 4 and 5 were housed at 14C for 4 and 8 weeks, respectively. In addition, one group of obese mice housed at 23C for 4 weeks was pair-fed to lean mice. The purpose of Experiment 2 was to determine effects of chronic cold exposure on near adult obese and lean mice, as well as to determine responses of mice to reacclimation to 23C after 4 weeks of cold exposure. Because Experiments 1 and 2 were conducted concurrently, mice housed at 233 from 4-12 weeks of age in Experiment 1 (group 3) served as controls in Experiment 2 (Figure l). The cold-acclimated group was housed at 23: from 4 through 8 weeks of age, and then at 14C from 9 through 12 weeks of age. The reacclimated group was housed at 14C from 4 through 8 weeks of age and then at 23C from 9 through 12 weeks of age. Analyses At the end of each experimental period, mice were killed by cervical dislocation and the intact carcasses were frozen until analysis. Body energy was measured by direct calorimetry. Carcasses were first softened by heating in an autoclave at 100C, then homogenized (Brinkmann Polytron, Brinkmann Instruments, westbury, NY) in 2-3 times their weight of water. An aliquot of homogenate was dried at 50C and used for determination of energy with an adiabatic 17 .oaau soon no one on» an on. none: 965 60.32. on: 05 93.36 v3.6: 30: coal 05 :33: us 3332.33 05 3.0.2.3 can: 73833: 25..- uuoaasz .~ ado-«would mo nausea J as»: mxooz .om< NF m V Phh -TIIIlIIIIIlIIIlIIIlIIlIIlLrIIIllllllllllllllllllllll. norm :33". com 03 AW mwphb :- uIIIIlIIIIIIIIIlIIIIIIlIIlaIIIIIIIIIIIIIIIIIIIIIIIII. _AVL:—phnvnnv —rIIIIlIlImmflmwmmIIIIIIIIILfIIIIIIIIMMflmwmmIIIIIIIII- 18 calorimeter (Parr Instrument Co., Moline, IL). The diet was also analyzed for energy content and was found to contain 3.81 kcal/g. Energy efficiency was calculated as body energy gained divided by energy consumed during a 4 week period. Body energy gain was calculated as body energy content at the end of the experimental period minus the predicted body energy content at the beginning of the experiment. Body energy at the beginning of each experimental period was predicted from a linear regression equation based on body weights and body energy contents of appropriate groups (ie,groups 1,2 and 4) fran Experiment 1. Body weights of mice at the beginning of each experimental period were then used to predict initial body energy for each rouse. Heat production was calculated as the difference between estimated metabolizable energy intake and body energy gain. Metabolizable energy intake was calculated as gross energy intake minus fecal energy with a correction for the energy associated with urinary nitrogen and was found to be 74% of gross energy intake. Data are presented as means + SEM. Data were analyzed by analysis of variance. Treatment differences were determined by Duncan's multiple-range test (P< 0.05) (74). Results Experiment 1 When housed at 23C, obese mice consumed 30% more energy per day than leans, as expected (Table 1). Lean mice at 14C consumed 50% more than leans at 23C, but cold acclimated obese mice consumed only 8% nore energy than obese controls. As a result, energy intakes of obese and lean mice housed at 14C were the same. 19 Table 1. Energy Intake of Mice from 4-12 Weeks of Age Environmental Temperature 23C 14C (keel/day) Lean 15.5 :_0.3 23.7 I 0.3+ Obese ' 21.2 I 0.5* 22.9 1 0.7+ Mean 1 SEN (n-9-11) *Significant (P< .05) difference between lean and obese at the same temperature. +Significantly (P< .05) different from the 23C group within phenotype. 20 At 23C, obese mice weighed more than leans at both 8 and 12 weeks of age (Figure 2A). Obese mice at 14C gained approximately 60% less weight than obese mice at 23C. Weight gain of lean mice at 14C, however, was depressed only slightly. As a result, obese mice at 14C weighed the same as leans. Obese mice at 23C gained over 200 kcal energy between 4 and 12 weeks of age (Figure 28) . Lean mice gained approximately 1/4 this much energy during this period. Energy gain of obese mice was markedly depressed by cold; they gained less than 100 kcal between 4 and 12 weeks of age. Thus the carcasses of obese mice housed at 14C for 8 weeks contained less than 1/2 as much energy as obese mice at 23C. Body energy in lean mice was depressed only after 8 weeks of cold exposure (Figure 28) . From 4-8 weeks of age, obese mice housed at 23C retained 22.0 + 0. 4% of the gross energy consumed; they were 3 1/2 times as efficient at retaining body energy as leans (Figure 3) . Obese mice housed at 23C fran 4-8 weeks of age and pair-fed to lean mice retained 19.8 + 0.7% of gross energy consumed. Energy efficiency of both obese and leans declined significantly with age, though efficiency of leans decreased more. Consequently, between 9 and 12 weeks of age, obese mice at 23C were 7 times as efficient as leans (Figure 3). Cold exposure, as expected, led to a decrease in energy efficiency. Energy efficiency of obese mice, however, was depressed more than that of leans so that between 4 and 8 weeks of age, obese mice at 14C were only twice as efficient as leans (Figure 3) . Between 9 and 12 weeks of age, differences between obese and lean mice at 14C were abolished; neither group retained a significant 21 Figure 2 Body weights and body energy of mice from 4-12 weeks of age. Environmental temperature noted by numbers on the right of each data line. Each point is the mean + SEM of 9-11 mice. += Significant (P<0.05) difference due to temperature within phenotype. Obese mice weighed significantly more than leans at 8 and 12 weeks of age at 23C. Body energy was significantly higher in obese mice than in leans at all ages and both temperatures. 22 BODY ENERGY, kcal/ mouse 00— amp CON OmN .oue «o .3003 -ue loom come «0 Anyone mean one Queues: zoom mxmm? .m0< a c N w o v u u d d a r . ammo. oVPw ...... .s. ..... a. L +3.0... 32.0 9.5 .30.. Tm . :25 9.6 eflfla :GOJell < L e .N enough Op 8 O O on A M B 9 On H .1 36 23 Figure 3 Energy efficiency of mice from 4-12 weeks of age. Each bar is the mean + SEM of 9-11 mice. *= Significant (P<0.05) difference between lean and obese at the same temperature and age. += Significant (P>0.05) difference due to temperature within age group and phenotype. 24 [0 0| 52 CJLean . .Obese >. 20 U E L _ 1 a: 2 5 ll. IL I“ 10 >. (5 s s - z 211' + NJ I _ 3° 14° 23° 14° 4-8 Weeks 9-12 Weeks of Age of Age Figure 3. Energy efficiency of mice from 4 - 12 weeks of age. 25 amount of the energy consumed. This represents, to our knowledge, the first demonstration of an equalized efficiency of energy retention in intact young adult obese and lean mice fed ad libitum. When housed at 233 from 4-8 weeks of age, calculated heat production was 267 and 294 kcal/mouse in obese and lean mice, respectively. In obese and lean mice housed at 14C from 4-8 weeks of age, heat production was 40% higher (364 and 422 kcal/mouse, respectively). Experiment 2 When switched from 23C to 14C at 9 weeks of age, both obese and lean mice increased their energy intake by approximately 50% (Table 2). weight gain of these mice was reduced to approximately 1/3 that of respective controls (Figure 4A and 4B). Leans in this group did not gain body energy, whereas obese mice gained 32 kcal, which is approximately 1/3 of the amount of energy gained by obese controls (Figure 4C and 4D). Both obese and lean mice retained energy with a lower efficiency than did their counterparts maintained at 23:, but obese mice still maintained a higher efficiency than leans (Figure 5). When obese mice that had been housed at 14C for 4 weeks were reacclimated to 23C, their energy intake decreased to equal that of their obese control counterparts maintained at 23C throughout the experiment (Table 2). Energy intake of lean mice during this period of reacclimation to»23C remained 12% higher than lean controls. Both obese and lean mice gained body weight and body energy at approximately the same rate as controls (Figure 4A and 4B). 26 Figure 4 Body weights and body energy of mice from 9-12 weeks of age. Mice were housed at 23C through 12 weeks of age, or at 23C through 8 weeks of age and at 14C from 9—12 weeks of age (23C to 14C) or at 14C from 4-8 weeks of age and at 23 C from 9-12 weeks of age (14C to 23C). Each point is the mean + SEM of 6-11 mice. *= Significantly (P<0.05) different from 23C group within phenotype. 27 A Lean B Obese a e—e 23° .. 4o . ..... 23°to14° .. . F:- .... 14°t023° S? m 30- -- ‘ 3 ;)//../L >.. * ... a 20 - '2... 23° 8 band 230 t01'4’ ’ o-—-o 14° 1023" f L i J L A 8 12 8 12 Q 250 . C Lean . O 3 2 h 200 " . 0 3'2 . 150 - - > 2 m 100 - ' 2 [LI __1 i5; 55C1P':*§:E: """"""" 7:7! .. - O m 8 12 8 12 AGE, Weeks Figure 6. Body weights and body energy of mice from 9-12 weeks of age. 28 Table 2. Energy Intake of Mice from 9-12 Weeks of Age Environmental Temperature 23C 23C to 14C 14C to 23C (kcal/day) Lean 15.5 1 0.2 23.6 1 0.4+ 17.4 1 0.4+ Obese 21.3 1 0.8* 31.0 1 o.s"'+ 22.4 1 0.5* Mean 1 SEM (n89-11) *Significant (P <.05) difference between lean and obese in the same treatment group. +Significantly (P (.05) different from the 23C group within phenotype. 29 Figure 5 Energy efficiency of mice from 9-12 weeks of age. Each bar is the mean + SEM of 6-11 mice. Mice were housed at 23C through 12 weeks of age (23), or at 233 through 8 weeks of age and at 14C from 9-12 weeks of age (23C to 14C), or at 14C from 4-8 weeks of age and at 233 from 9—12 weeks of age (14C to 23C). *= Significant (P 10 ’ ‘ 1° 01 , O ca : sI.’ 8 I- 5 ' ~23° ‘ 5 --2s° to 14° {1“ .................... «14° to 23° 1 Obese 9 :2 A62. WEEKS Figure 10. Total hindlimb uncle weights, bone lengths and total body fat of nice from 9-12 weeks of age. 53 10A). The tibia of reacclimated obese mice lengthened at only half the rate of obese controls so that at 12 weeks of age, it remained shorter than the tibia from obese controls (Figure 108). The tibia of reacclimated lean mice lengthened by more than two-fold that of lean controls, so that at 12 weeks of age it was the same length as the control. Lean mice reacclimated to 23C did not gain body fat, whereas reacclimated obese mice gained approximately the same amount of body fat os obese controls (Figure 10C). Reacclimated obese mice still had significantly less body fat than obese controls, however. Experiment 3 Concentration of corticosterone in plasma of obese mice housed at 23C was higher than that of lean mice, though the difference between obese and lean was only significant at 1600 hours (Figure 11A and B). This higher concentration of corticosterone in obese mice is in agreement with results of Herberg and Kley (31) and Naeser (54). Cold acclimation increased the concentration of corticosterone in plasma of obese mice compared to obese mice at 23C at both times of the day, though this was statistically significant only at 0800 hours. In lean mice, plasma corticosterone concentrations were unaffected by cold acclimation. The concentration of corticosterone in plasma of cold acclimated obese mice was thus 5-10 times that of cold-acclimated lean mice. Plasma insulin concentration of obese mice housed at 23C was 20 times that of lean mice at this temperature (Figure 11C). Cold exposure led to a dramatic reduction in insulin of obese mice, from 411 to 137 uUhits/ml. Despite this reduction in insulin 54 Figure 11 Plasma corticosterone and insulin concentrations in mice at 8 weeks of age. They had been housed at the indicated temperatures for 4 weeks. Each point is the mean + SEM for 9-10 mice. Top panel, plasma corticosterone at 0800 hours. Middle panel, plasma corticosterone at 1600 hours. Bottom panel, plasma insulin. *= Significant (P<0.05) difference between obese and lean at the same time and temperature. += Significant (P<0.05) difference due to temperature within phenotype. SS A 0800 hf 14'Lean ‘ 121 - 1o. «1 g. . 5. . 4. .. 2- D 4 23 14° PLASMA CORTICOSTERONE, ug/dl B 16001! “Luau "Obese ‘ 12p '0 3;. ‘ 10' " ‘ 8F 1D 1 6" as a 4- db 4 2.. fl 4- 1 23° 14° C Obese 420 Loan E 400 a r ‘7 r 1: 1601- 1' 1 Z 3 140- "' “ a i 120- 1' " g 100- " ‘ - 8° 1. d § ‘0 50 4s '1 a 40 0 1 20 H H 4. 4 23° 14° 23° 14° ENVIRONMENTAL TEMPERATURE Figure 11. Plasma corticosterone and insulin concentrations in nice at 8 weeks of age. 5.6 concentration in obese mice at 14C, cold exposed obese mice were still hyperinsulinemic relative to leans. The concentration of insulin in plasma of lean mice was unaffected by cold acclimation. Discussion Skeletal muscle of obese mice housed at 23C weighed only about 65% as much as much as muscle of lean mice. This effect was not seen only in hindlimb muscle - the psoas, located in the trunk, followed the same pattern of response as the other muscles examined. This is consistent with previous reports showing that skeletal muscle of obese mice, based on the weight of the fat-free carcass or hindlimb, weighed only about 80% as much as that of leans (3,83). Chronic mild cold exposure resulted in even more dramatic differences in muscle weights between obese and lean mice. After 8 weeks at 14C, skeletal muscles of obese mice weighed only 35-45% as much as muscles of lean mice, even though their body weights were the same. As mentioned earlier, skeletal growth is a stimulus for skeletal muscle growth (36). Shapira and coworkers found that the tibia and femur of obese Zucker rats were approximately 10% shorter than those of lean rats, and suggested that these shorter bones exerted less of a trophic effect on the muscle, contributing to the reduced skeletal muscle mass of obese rats (69). In our study, hindlimb bones of obese mice were significantly shorter than those of lean mice and cold acclimation reduced bone growth even more in obese mice than in lean. These changes in bone growth parallelled the changes in muscle growth. It is possible then, that the slower growth of the skeleton 57 of obese mice compared to leans, contributed to their reduced skeletal muscle mass. It is unlikely however, that the 10-15% reduction in bone length of cold acclimated obese mice was entirely responsible for their dramatic reduction in muscle accumulation. Corticosterone reduces skeletal muscle growth through several mechanisms. High doses of glucocorticoids lead to decreased rates of muscle protein synthesis (60,61) and increased rates of muscle protein breakdown (66). The 5-10 fold elevation in concentration of corticosterone observed in the cold-acclimated obese mice might contribute to their accelerated rates of muscle breakdown and reduced muscle accumulation (83). A 3-fold increase in concentration of plasma corticosterone for 8 days caused a 20% reduction in weights of gastrocnemius muscle, as a percentage of body weight, of rats (67). Elevated concentrations of plasma corticosterone also promote insulin resistance, which could reduce muscle growth (55). Further support for the involvement of glucocorticoids in reduced muscle accumulation in obese mice comes from aishima et al.; they showed that adrenalectomy increased muscle mass of obese mice (65) . Thus, there is some evidence supporting a role for elevated plasma corticosterone in the reduced muscle growth of obese mice, though the causal relationship between these must be further clarified. Although the concentration of insulin in plasma of obese mice was lowered by cold acclimation they were still hyperinsulinemic relative to lean mice (Figure 10C). Considering the high concentrations of corticosterone in plasma of cold-acclimated obese mice it is likely that their muscles remained insulin resistant (29,48). These factors together with low concentrations of growth hormone (42) and 58 testosterone (76) in plasma of obese mice probably contributed to the restriction of skeletal muscle growth in cold-acclimated obese mice even though their intake of nutrients equaled that of lean mice. Retained energy was partitioned into body fat and muscle in a pattern that enabled cold-acclimated obese mice to maintain a similar percentage fat (Figure 8) and skeletal muscle (data not shown) as age-matched obese controls. Because of the limited rate of body weight gain and concomitant increase in percentage body fat, skeletal muscle gain was severely limited in the cold-acclimated obese mice. Pair-fed obese mice (data not shown) and pair-fed obese Zucker rats (9) also maintain a body composition (percentage body fat and skeletal muscle) similar to that of ad libitum fed obese counterparts. However, their reduction in muscle gain is not as severe as that of cold-acclimated animals because the pair-fed obese animals gain more body weight than their ad libitum-fed lean counterparts. These genetically obese rodents thus tenaciously maintain an elevated percentage body fat even during periods of restricted growth. Reacclimation of the obese mice to 23C reduced their energy expenditure more than energy intake, consequently their rate of body energy gain was again accelerated (Chapter 2).Concomitantly, the rate of skeletal muscle gain increased in the obese mice (Figure 10). It is likely that muscle weights of the reacclimated obese mice would have been restored to the same level as obese controls if the study had been extended beyond 4 weeks of reacclimation. In conclusion, though body weights of obese and lean mice are equalized by chronic cold exposure, obese mice maintain their obese 59 body composition (increased percentage body fat and decreased percentage skeletal muscle) by limiting skeletal muscle accumulation. Elevated concentrations of plasma corticosterone in combination with reduced concentrations of several anabolic hormones may have a key role in maintaining this obese body composition. Finally, obese mice demonstrate the ability to accelerate their rate of muscle accumulation when reacclimated to 23C after 4 weeks of cold exposure. 60 Chapter 4. Effects of adrenalectomy on energy balance of obese (ob/ob) mice are diet-dependent. Adrenalectomy reduces food intake by 35-60% and body weight gain by 50-90% in several obese animal models, so that intake and gain are shmilar to that of their lean counterparts (7,14,49,65,9l). This nonmalization of energy intake and weight gain does not necessarily indicate that these adrenalectomized obese animals normalized their body fat content, however, since obese animals pair-fed or pair-gained to lean animals still deposit body fat at a faster rate than lean counterparts (5). Based on currently available evidence it appears that adrenalectomy may reduce body fat accumulation of obese animals to a greater extent than can be explained by the reduction in energy intake. The high efficiency of energy retention usually observed in obese Zucker rats was reduced by adrenalectomy to equal that of lean rats (49). This reduction in efficiency was probably not due to reduced energy intake, since in control obese rats, a reduction in energy intake failed to reduce their efficiency of energy retention (5). The reduction in energy efficiency in adrenalectomized obese rats was more likely caused by increased energy expenditure, possibly resulting from increased activity of brown adipose tissue. This suggestion of increased brown adipose activity is supported by the findings that mitochondrial proteincontent and GDP binding to mitochondria of brown adipose, a measure of the capacity of the tissue for heat production, were increased in adrenalectomized obese rats so that they equaled that of leans (49). Adrenalectomy also increased norepinephrine turnover in 61 brown adipose tissue of ob/ob mice to nearly the same level as in lean mice, indicating increased brown adipose tissue metabolism and perhaps a reduced efficiency of energy retention (91). Energy balance of adrenalectomized ob/ob mice has not been directly examined, however. Consistent with the reduction in efficiency of energy retention in adrenalectomized obese Zucker rats, their body composition is also altered by adrenalectomy. Adrenalectomy of obese Zucker rats prevented the 25% increase in body energy density observed in sham-operated obese rats, indicating that the adrenalectomized obese rats had a lower proportion of body fat than sham-operated obese (49). Adrenalectomy affects body composition of other obese animals as well. In GPO-treated mice, adrenalectomy prevented the accumulation of excess carcass lipid seen in sham—operated GEO-treated mice (14). In ob/ob mice, the weight of the gastrocnemius muscle was increased after adrenalectomy so that it was similar to that of lean mice (65), and presumably fat accumulation. was decreased. All of the above-mentioned studies examining the effects of adrenalectomy on obese animals were conducted with animals fed a pelleted stock diet. Several lines of evidence indicate that the diet fed to obese animals may be important in influencing the outcome of adrenalectomy. In VMH-lesioned rats, combined ovariectamy-adrenalectomy prevented the hyperphagia and excess body weight gain usually seen in VMH-lesioned rats if they were fed a pelleted stock diet. However, when a semi-purified, high-fat or a liquid, high-carbohydrate diet was fed, the 62 ovariectomized-adrenalectomized rats overate to almost the same extent as intact VMH-lesioned rats (52). Treatments other than adrenalectomy which alter the development of obesity are also affected by diet. When hypophysectomized obese rats were fed a pelleted stock diet, their body weights steadily declined and they eventually died (59). However, when the hypophysectomized obese rats were fed a softer, more palatable diet, they maintained their body weights and the obesity attained prior to hypophysectomy. Vagotomy blocked hyperphagia and the development of obesity in VMH knife-cut rats fed a pelleted stock diet, but when these rats were fed several palatable foods in addition to stock diet, they gained more veight than either non-vagotimized VMH lesioned or sham-operated rats (68) . Finally, the metabolism of brown adipose tissue, which has been shown to be altered by adrenalectomy of obese animals (34,49,91) , is also influenced by diet. Heroux et al. found that the metabolic potential of brown adipose tissue was higher in rats fed a semipurified diet than in rats fed a pelleted stock diet (32). These studies demonstrated the important role of diet in determining the outcome of several treatments of obese animals. Therefore, it was of interest to examine the influence of diet on the outcome of adrenalectomy in ob/ob mice. The present study was designed to determine the effects of adrenalectomy on energy balance and body composition of obese (ob/ob) and lean mice fed either a high-carbohydrate, stock diet or a high-fat, semipurified diet. The pelleted stock diet was chosen to facilitate comparisons of this study with those from previous studies on adrenalectomy of obese animals. The high-fat 63 diet was chosen to compare to the stock diet because ob/ob mice demonstrate a marked degree of hyperphagia and seight gain on this diet (44) so that any effects of adrenalectomy on these parameters should be obvious. Finally, the extent to which obesity has developed in animals appears to influence the effects of adrenalectomy. Adrenalectomy of GTG-treated mice which were not yet obese resulted in the maintenance of food intake and body weight at the same levels as in lean (non-GTG-treated) controls (14). But, when GIG-treated mice were adrenalectomized after obesity had developed, there was a rapiddecline in food intake and body weight to levels considerably below those of untreated lean control mice (15). Therefore, this study was designed to examine the effects of adrenalectomy before obesity in ob/ob mice was markedly obvious (3 seeks of age) and after obesity was more fully developed (6 seeks of age) . Materials and Methods Animals Obese (ob/ob) and lean (ob/+ or +/+) male mice were obtained from our breeding colony of C57BL/6J ob/+ mice. The breeding colony is housed at 23C with room lights on from 0700 to 1900 hours daily. Pups are weaned at 3 seeks of age. At 3 or 6 seeks of age, obese and lean pairs were separated from their littermates and housed individually in solid-bottom plastic cages with wood shavings as bedding. After l-2 chys of adaptation to single housing, mice sere bilaterally adrenalectomized (Adx) or sham-operated (Sh) 64 through dorsal incisions while under ether anesthesia. The adrenal glands were gently lifted to the opening of the incisions and curved scissors were used to remove the glands along with a small amount of surrounding adipose tissue, taking care not to touch the adrenals with the dissecting instruments. Shamroperation consisted of locating the adrenal glands and exposing them as for adrenalectomy, but without excising them. Incisions were closed with stainless steel wound clips. Mice sere exposed to ether for approximately 4 minutes and had recovered from the anesthesia within the following 5 minutes. After surgery, drinking water of Adx mice was replaced with physiological saline (0.9% NaCl). All mice had food (see below for diets) and water (or physiological saline) available ad libitum. Food intake and body weights were monitored 3 times weekly. Experimental Design Experiment 1 was designed to examine the effects of adrenalectomy on energy balance and body composition of obese and lean mice fed a stock diet (wayne LabABlox, wayne Pet Food Div., Continental Grain co., Chicago, IL). Obese and lean mice were adrenalectomized or shamhoperated as described above at either 3 or 6 weeks of age and were fed stock diet for 3 weeks. Initial groups of 3 or 6 week old obese and lean mice were killed for baseline data. Experiment 2 was designed to assess how consumption of a high-fat diet affected the outcome of adrenalectomy in obese and lean mice. The same procedures as followed in Experiment l.were followed here, except the mice sere fed a high-fat, semipurified diet containing (9/100g) mineral mix, 4.83 (l); vitamin mix, 1.38 (l); cellulose, 5.52; methionine, 0.41; choline chloride, 0.28; casein, 27.61; 6S cerelose 27,31; corn oil, 16.33; and tallow, 16.33 for 3 weeks after adrenalectomy or sham-operation. This diet provided 60% of metabolizable energy as fat, 20% as protein and 20% as carbohydrate. Experiment 3 was designed to examine the effects of restricted intake of the high-fat diet on the outcome of adrenalectomy. Obese and lean mice were adrenalectomized or sham-operated at 6 weeks of age. They sere fed an amount of high-fat diet to provide them with the same gross energy intake as 6-9 week old sham-operated lean mice in Experiment 1. The diet was given to the mice each day at 1000-1200 hours. Some of this food was still in the food cups at 2000 hours, indicating that the mice did not consume all of their food during the light cycle. Mice were killed at 9 seeks of age for further analyses. Analyses At the end of the experimental periods, two blood samples were taken fram the orbital sinus of each mouse at 2000 hours, the approximate time of peak corticosterone concentration in mice (64). The first sample was taken within 2 minutes of the time the mouse was removed from its cage, including 15 seconds of ether exposure, and served as a nonstressed baseline sample. The second blood sample was taken 10 minutes later and served as a stressed sample. Plasma was assayed for corticosterone concentration by radioimmunoassay (Endocrine Sciences, Tarzana, CA). Plaeta insulin concentration was also determined by radioimmunoassay (Radioassay Systems Laboratories, Inc., Carson, CA) on baseline plasma samples pooled from 2-3 mice in the same treatment group. Only those adrenalectomized mice whose baseline corticosterone was less than half that of sham-operated 66 mice and whose stressed corticosterone was not increased by more than 20% above the baseline concentration were included in the analyses. Mice were killed by cervical dislocation after the second blood sample was taken, and carcasses were frozen until analyzed. After thawing, the soleus, gastrocnemius and psoas muscles were isolated and weighed. These muscles were chosen to provide a variety of fiber types, functions and locations. The soleus, in the hindlimb, contains both red and intermediate type fibers and functions in locomotion. The gastrocnemius, also in the hindlimb, contains red and white fibers and funtions in locomotion. The psoas, located along the spinal column, contains mixed fiber types and functions in support. The remaining hindlimb muscle was also stripped from the bones and weighed. The sum of the weights of all the hindlimb muscles (soleus + gastrocnemius + remaining hindlimb muscle) is reported as total hindlimb muscle. The length of the tibia was also recorded. Averages of the weights of muscles or length of bones from both hindlimbs or sides were used to calculate means. Body energy of the carcass (including all tissues removed for weighing) was measured by direct calorimetry. Carcasses were softened in an autoclave at 100C, then homogenized (Brinkmann Polytron, Brinkmann Instruments, Westbury, NY) in 2-3 times their weight of water. An aliquot of homogenate was dried at 50C and used for the determination of energy with an adiabatic calorimeter (Parr Instrument Co., Moline, IL). Body energy density was calculated as kcal/g body weight. The diets were also analyzed for gross energy content (stock diet = 3.9 kcal/g; high-fat diet = 5.3 kcal/g). Energy efficiency was calculated as body energy gained divided by 67 gross energy consumed during the 3 week experimental period. Body energy gain was calculated as body energy content at the end of the experimental period minus the predicted body energy content at the beginning of the experiment. Body energy at the beginning of each experimental period was predicted from a linear regression equation based on body weights and body energy contents of the initial groups. Body weights of the mice at the beginning of each experimental period were used to predict initial body energy for each mouse. This linear regression method was also used to calculate gain in muscle weight and tibia length. This method of predicting initial body energy, muscle weights and bone length was validated by camparing the actual and predicted values for these parameters in a separate group of mice. By this method, actual values for initial body energy, muscle weight and bone length were within 5% of predicted values. Data are presented as means + SEM. Data were analyzed as a 2 x 2 factorial design by analysis of variance. Where interaction between treatment effects (phenotype x surgery) was significant (P<0.05), Bonferroni t-tests were used to determine specific treatment effects (80). Results Experiment 1 (Stock diet). Basal concentrations of plasma corticosterone of sham-operated obese mice were almost 3 times those of lean mice (Figure 12). Plasma corticosterone concentrations of sham—operated mice increased 68 by 3-10 ug/dl after stress. Successful adrenalectomies were confirmed in 85% of the mice that underwent adrenalectomy. These adrenalectomized mice did maintain a low concentration of plasma corticosterone, but there was little or no increase in corticosterone after stress (Figure 12). Body weights of all 4 groups of mice were similar from 3-5 weeks of age (Figure 13A). Between 5 and 6 weeks of age, sham-operated obese mice gained considerably more weight than adrenalectomized obese and both groups of lean mice. Adrenalectomy of obese mice at 3 weeks of age prevented this excess body weight gain, so that their final body weight equaled that of lean mice. Between 6 and 9 weeks of age, sham-operated obese mice gained more than 5 times as much body weight as sham-operated leans (Figure 133). Adrenalectomy of 6 week old obese mice reduced body weight gain to less than half that of sham-operated obese mice (Figure 13B). By 8 weeks of age, body weights of adrenalectomized obese were significantly less than those of sham obese, and this difference widened by 9 weeks of age. Energy consumption of shameoperated obese mice from 3-6 weeks of age was the same as that of lean mice, contrary to what is usually seen at this age (15,28) (Figure 14A). This occurred because the sham-operated obese mice consumed very little food for the first several days after surgery, and their food intake only slowly increased. Adrenalectomy reduced energy intake of obese mice by 17%, though this was not statistically significant. From 6-9 weeks of age, energy consumption of sham-operated obese mice followed a more normal pattern; sham obese mice consumed 50% more energy than sham 69 Figure 12 Plasma corticosterone concentrations of mice fed stock diet from 3—6 weeks of age (A) or 6—9 weeks of age (B). Cross-hatched portion of bars represents non-stressed concentrations. Open portion of bars represent stressed concentrations, obtained 10 minutes after initial exposure to ether. Sh = sham-operated and Adx = adrenalectomized. Vertical line on top of each bar is the SEM of 9-14 mice. Non-stressed and stressed plasma corticosterone of sham obese was significantly (P<0.05) higher than that of sham leans, except for stressed values at 6-9 weeks of age. Adrenalectomy reduced corticosterone to equally low concentrations in obese and leans at body ages (P<0.05). 70 CORTICOSTERONE-yg/dl uses: one no 0 l0 0 In N PF one we axes: can loan as: soon- oeu sous uo anode-nuances ssoueusoouuuoo else: 25... mmmmo 24m: mmmmo .22 em. «.2 cm :2 .5 .22 am rommmmmhmuzoz§. . m nmmmmmhmfl mg no meow? m6 mg no memo; one ...mE v_OO._.m .02 IO 0 IO lP/BIT-EINOHELLSOOILHOO :33 O N 71 Figure 13 Body weights of mice fed stock diet from 3-6 weeks of age (A) and 6-9 weeks of age (B). Surgical treatment noted at the end of each line; Sh = sham-operated and Adx = adrenalectomized. Different letters indicate a significant difference (P<0.05) between groups at the same age. There were no significant differences between body weights of any of the groups at 3,4, or 5 weeks of age (panel A). 72 BODY WEIGHT-g .oms uo uses: one one one no execs cum Iona as: sooua new 03-. «o 3:32.. boom .2 one»: mxmm; .m0< o O m h . 0 m V a 8 2. . . S. m ..A ON . 0 n a . 1 ON M sag mlu XU<0 O a H D 10:33.”? I.— om. a ............. a 1 , on _ xo< ........... o 5 ......w Z 400 8 5 350’ . 1330 5 (3 JL 3 C: > 300 1300 g m g 25o «250 T 11.1 r z 2001' ‘ 3°° o u: 1 r EL 3 so ‘30 rm 3 s ' 70 » 70 :1 a so » too 93 3 so» . so a, < > (3 40* ' 140 '- >- u s 1% O 30» 0 « .. ‘30 o 5 20» 1 1 s <20 (:5- z m 10’ ’ h b (10 Q, _ . 11 :5 :1, 51: Ads 50 Adx n Adx sn Adx 3 AN ossss LEAN | #2 zebrlp ) F325» 22 ). Q 20, l a 120 5'; 2 d- a) g 13» s « . 115 -< (J rm - 1° 4 ‘ ’ ‘10 "- 11‘ l 5L D 1 5 - I“ J b D D Q (’5 sn Ads 51: Adx' 371' Ad: so: Adx. g a: 03255 LEAN ossss LEAN o g “f u: 8° Figure 14. Energy consumption, gain and efficiency of nice fed stock diet fro. 3-6 weeks of age and 6-9 weeks of age. 75 leans (Figure 148) . Recovery from sham-surgery was more rapid in these older obese mice. Adrenalectomy of 6 week old obese mice reduced their energy intake to equal that of leans. Sham-operated obese mice gained almost 2 1/2 times as much body energy between 3 and 6 seeks of age as sham-operated leans (Figure 14C). Adrenalectomy of 3 seek old obese mice fed stock diet prevented this excess energy gain. The difference in energy gain betseen sham obese and lean mice was even more pronounced from 6-9 weeks of age, when sham obese mice gained 15 times as much body energy as sham leans (Figure 14D) . Adrenalectomy of 6 week old obese mice prevented the large energy gain observed in sham obese mice. The high body energy gain of 3—6 seek old sham obese mice coupled with their only slightly elevated energy intake resulted in an efficiency of energy retention more than twice that of sham leans (Figure 14E) . Adrenalectomy of 3 seek old obese mice normalized their energy efficiency to equal that of lean mice. Betseen 6 and 9 seeks of age, sham-operated obese mice continued to retain body energy with a higher efficiency than sham leans (Figure 14F) . As in the younger mice, adrenalectomy of 6 week old obese mice normalized their efficiency of energy retention to the level of lean mice. Adrenalectomy of lean mice did not affect any of these parameters of energy balance as compared with sham leans. Energy density of sham-operated obese mice increased by 40% between 3 and 6 seeks of age, compared to a 20% increase in sham-operated leans (Figure 15A) . The higher energy density of sham-operated obese mice indicates that they had a higher proportion of body fat than lean mice. Adrenalectomy of obese mice fed stock 76 Figure 15 Energy density, muscle weights and bone lengths of mice fed stock diet from 3-6 weeks of age (A,C,E,G) and 6-9 weeks of age (B,D,F,H). Cross-hatched portion of bars represents means of predicted values for mice at the beginning of the experiment. All predicted values for obese mice at the beginning of the experiment were significantly different (P<0.05) than initial leans. Open portion of bars represents gain during the experimental period. Sh = sham-operated, Adx = adrenalectomized. Vertical lines on each bar are the SEM of 9.14 muce. Different letters above the bars indicate significant (P<0.05) differences between groups at the end of the experimental period. m2m20< Om2w_._.<1_.an.\¢ 404>F 33:30 awn-Inigo mmwmmmwmm 1 a 7 a C 3 2 two>m130 ammo 0-9 WEEKS OF AGE 435103 STOCK DIET T—fi 34 WEEKS OF AGE ”2232. 3.834523 552m 05.33:: 3.32.: 4:2 3.309. E0150: Energy density, mscle weights and bone lengths Figure 15 . of nice fed stock diet fron 3-6 weeks of age and 6-9 weeks of age. 78 diet prevented the increase in body energy density of 3-6 week old mice, though it remained significantly higher than that of lean mice. Energy density of 6-9 week old sham—operated obese mice increased by 25%, and, as in the 3-6 week old mice, this increase was prevented by adrenalectomy (Figure 158) The lower energy density of adrenalectomized obese mice compared to sham-operated obese indicates that adrenalectomy increased the proportion of lean body mass in obese mice. This was confirmed by the muscle weight data. The soleus and gastrocnemius muscles showed the same pattern of changes as the total hindlimb muscle, so they are not presented. Hindlimb muscles of 3 week old obese mice already weighed 17% less than muscles of lean mice, and sham-operated obese mice gained only 48% as much hindlimb muscle as lean mice from 3-6 weeks of age (Figure 15C). Adrenalectomy improved muscle gain of obese mice by almost 50%, so that their final hindlimb muscle weight was 80% of leans. Hindlimb muscle of unoperated 6 week old obese mice weighed 30% less than that of lean mice (Figure 150). Sham-operated obese mice gained only 80 mg hindlimb muscle during the 3 week experimental period, so that muscle weight at 9 weeks of age was only 65% of leans. Adrenalectany of 6 week old obese mice significantly increased their hindlimb muscle gain so that by 9 weeks of age, the muscle of adrenalectomized obese mice weighed 87% as much as leans. Results for the psoas muscle indicate that these effects on muscle were not restricted to the (Figure 1515:). Adrenalectomy improved gain in psoas weights of 3 and 6 week old obese mice by 40%, but psoas weights of adrenalectomized obese were still less than those of lean controls because muscles of obese mice weighed less 79 than muscles of lean mice at the time of surgery. The tibia of 3 week old obese mice was 95% as long as the tibia of lean mice, and between 3 and 6 weeks of age the tibia of sham-operated obese mice lengthened by less than that of leans (Figure 15G). Adrenalectomy of obese mice improved gain in tibia length by 30%, but final tibia length was still only 96% that of leans. Adrenalectomy of 6 week old obese mice did not significantly improve gain in tibia length; the tibia of 9 week old adrenalectomized obese mice was 98% as long as that of lean mice (Figure 15H). Plasma insulin concentration of 6 week old sham-operated obese mice was 4 l/2 times that of sham leans (Figure 16A). Adrenalectomy of 3 week old obese mice fed stock diet prevented this hyperinsulinemia fran developing, and at 6 weeks of age plasma insulin concentration of adrenalectomized obese equaled that of leans. By 9 weeks of age, plasma insulin concentration of sham-operated obese mice was 20 times that of sham leans (Figure 16B) . Adrenalectomy of 6 week old obese mice reduced plasma insulin concentration so that plasma insulin of 9 week old adrenalectomized obese mice was only 4 times that of lean mice. Experiment 2 (High-fat diet) Basal concentrations of plasma corticosterone of 3-6 week old and 6-9 week old sham—operated obese mice were 2 - 2 1/2 times that of sham leans (Figure 17A and 178). Plasma corticosterone was doubled or tripled after stress in all groups of sham—operated mice. Successful adrenalectomies were confirmed in 65% of the mice that underwent adrenalectany. As in Experiment 1, the adrenalectomized 80 Figure 16 Plasma insulin concentrations of mice fed stock diet from 3-6 weeks of age (A) and 6-9 weeks of age (B). Sh = sham-operated and Adx = adrenalectomized. vertical line on top of each bar is the SEM of 3—5 samples of plasma pooled from 2-3 mice each. Different letters above the bars indicate a significant (P<0,GS) difference between groups of the same age. 81 INSULIN-pU/ ml .0». «a 3.00? «no can one no .3003 cum scum no.2. sue: vow 03-. we uguusuuaogu c.3903 was; .3 one»; Z0¢mzm .noxnamzzw >0¢mzm $I>Uzm_o_n_u_m >O¢mzm hergy consumption, gain and efficiency of nice fed high fat diet fron.3-6 weeks of age and 6-9 weeks of age. Figure 19. 89 gain significantly more energy than sham leans from 6-9 weeks of age (Figure 19D). Adrenalectomized obese mice gained 30% less energy than sham obese from 6-9 weeks of age, but this was still 4 1/2 times the energy gain of lean mice. Thus, the normalization in efficiency of energy retention seen in adrenalectomized obese mice fed stock diet was not seen when adrenalectomized obese mice were fed the high-fat diet (Figure 19E and 19F). Efficiency of energy retention of 3-6 week old adrenalectomized obese mice was only 10% lower than that of sham-operated obese mice and was 3 1/2 times the efficiency of lean mice. Fran 6-9 weeks of age, sham-operated obese mice retained energy with 6 times the efficiency of sham leans (Figure 19F). Efficiency of energy retention of adrenalectomized obese mice was slightly (25%) less than that of sham-operated obese, but was still 4 1/2 times the efficiency of lean mice. Consumption of the high-fat diet also blocked the effects of adrenalectomy on changes in body composition in obese mice. Energy density of sham—operated obese mice fed high-fat diet increased by 67% between 3 and 6 weeks of age, so that at 6 weeks of age, energy density of obese mice was more than twice that of leans (Figure 20A). Adrenalectany of 3 week old obese mice did not prevent this increase in energy density as it had in obese mice fed stock diet. Energy density of sham-operated obese mice increased by 38% between 6 and 9 weeks of age (Figure 208). Adrenalectomy of 6 week old obese nuce did not prevent the increase in energy density from 6-9 weeks of age, and body energy density of adrenalectomized obese mice remained Kore than twice that of lean mice. These results indicate that consumption of the high-fat diet prevented the improvement 90 Figure 20 Energy density, muscle weights and bone lengths of mice fed high-fat diet from 3-6 weeks of age (A,C,E,G) and 6-9 weeks of age. Striped portion of bars represents means of predicted values for mice at the beginning of the experiment. All predicted values for obese mice at the beginning of the experiment were significantly different (P<0.05) than initial leans. Open portion of bars represents gain during the experimental period. Sh = sham—operated and Adx = adrenalectomized. Vertical lines on each bar are the SEM of 9-14 mice. Different letters above the bars indicate a significant (P<0.05) difference between groups at the end of the experimental period. 91 m2mao< Om2m3miso in; rmzoqzuaa o o o o o a m s . E x . m mu " m ...“. s K u as m w is D 4 RB " t H “A a . x I . b l A b A e! b a n e m. w . .. H... A .m H S 5 SH. 3 a b “L K E a m. a M6 mm a ms 3 mm H .- flmC .- a“ a 986 a a“ 5 4 3 2 1 i mmmmmmmmm 3mm. “33 O\_aoxu>b.m2wa >G¢wzm aElmJOmDS 02-402.: 4 _ 1111111111111111111111 11 1111