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Food Demand and Energy Balance

Permanent Link: http://ufdc.ufl.edu/UFE0042055/00001

Material Information

Title: Food Demand and Energy Balance a Neuroeconomic Analysis of Melanocortin System and Exercise
Physical Description: 1 online resource (115 p.)
Language: english
Creator: Atalayer, Deniz
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: demand, elasticity, energy, exercise, food, intake, mc3r, mc4r, meal, melanocortin, mice, neuroeconomics, nosepoke, operant, pvn, receptor, running
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: One of the main factors contributing to the obesity epidemic is the obesity favorable food environment of today?s modern societies that promotes excessive energy intake while minimizing the energy expenditure. The purpose of the present work is to combine the analyses of the effects of both positive and negative energy balance on food demand and meal size by exploring the some of the physiological and neuronal mechanisms of the melanocortin system and the effects of physical activity. Homozygous deletion of the melanocortin-4 receptors (MC4R-/-) is known to result in positive energy balance as MC4R-/- mice and humans develop obesity, while those with deletion of the melanocortin-3 receptors (MC3R-/-) do not become markedly obese. In the first part of this dissertation, the effects of positive energy demand on food demand were examined. MC3R-/-, MC4R-/- mice with wild type (WT) and mice lacking both of these receptors (double knockout, DKO) were compared on select feeding and neuroanatomical dimensions. In a food demand protocol, DKO and MC4R-/- were hyperphagic at low unit costs for food, due primarily to increased meal size. However, at higher costs, their intake dropped below that of WT and MC3R-/- indicating increased elasticity of food demand. To determine whether this higher elasticity was due either to the genotype or to the positive energy balance per se, the same food demand protocol was conducted in dietary obese mice. The difference between the demand functions of WT and dietary obese mice was smaller compared to that of WT and genetically obese mice. To assess a mechanism for the larger meal size in MC4R-/- and DKO, we examined the acute anorectic effect of peripherally-administered cholecystokinin (CCK) and subsequently the induction of Fos-immunoreactivity in select brain regions. The anorectic effect of CCK was comparable in MC4R-/-, DKO, and WT, but was unexpectedly absent in MC3R-/-. CCK-8 induced c-Fos was lower in PVN in MC3R-/- than the other genotypes. The second part of this dissertation examines the effects of negative energy balance on food demand. Physical activity has long been suggested as a method to better maintain weight loss and that obesity induced by a high fat diet can be treated without energy restriction by exercise. The effects of running wheel activity on food intake and meal patterns were measured under several economic conditions in CD 1 mice. Voluntary wheel running activity increased daily food intake and running animals consumed bigger but fewer meals compared to the sedentary. Although they ate more, running mice had significantly lower body fat, especially in subcutaneous depots. In the foraging protocol, when food intake was contingent on the running wheel activity, mice were able to emit more responses compared to when food is contingent upon a nose poke response. In both voluntary and foraging running protocols mice had inelastic demand functions compared to the non-running groups indicating that negative energy balance affects how much mice are willing to work for food.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Deniz Atalayer.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Rowland, Neil E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042055:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042055/00001

Material Information

Title: Food Demand and Energy Balance a Neuroeconomic Analysis of Melanocortin System and Exercise
Physical Description: 1 online resource (115 p.)
Language: english
Creator: Atalayer, Deniz
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: demand, elasticity, energy, exercise, food, intake, mc3r, mc4r, meal, melanocortin, mice, neuroeconomics, nosepoke, operant, pvn, receptor, running
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: One of the main factors contributing to the obesity epidemic is the obesity favorable food environment of today?s modern societies that promotes excessive energy intake while minimizing the energy expenditure. The purpose of the present work is to combine the analyses of the effects of both positive and negative energy balance on food demand and meal size by exploring the some of the physiological and neuronal mechanisms of the melanocortin system and the effects of physical activity. Homozygous deletion of the melanocortin-4 receptors (MC4R-/-) is known to result in positive energy balance as MC4R-/- mice and humans develop obesity, while those with deletion of the melanocortin-3 receptors (MC3R-/-) do not become markedly obese. In the first part of this dissertation, the effects of positive energy demand on food demand were examined. MC3R-/-, MC4R-/- mice with wild type (WT) and mice lacking both of these receptors (double knockout, DKO) were compared on select feeding and neuroanatomical dimensions. In a food demand protocol, DKO and MC4R-/- were hyperphagic at low unit costs for food, due primarily to increased meal size. However, at higher costs, their intake dropped below that of WT and MC3R-/- indicating increased elasticity of food demand. To determine whether this higher elasticity was due either to the genotype or to the positive energy balance per se, the same food demand protocol was conducted in dietary obese mice. The difference between the demand functions of WT and dietary obese mice was smaller compared to that of WT and genetically obese mice. To assess a mechanism for the larger meal size in MC4R-/- and DKO, we examined the acute anorectic effect of peripherally-administered cholecystokinin (CCK) and subsequently the induction of Fos-immunoreactivity in select brain regions. The anorectic effect of CCK was comparable in MC4R-/-, DKO, and WT, but was unexpectedly absent in MC3R-/-. CCK-8 induced c-Fos was lower in PVN in MC3R-/- than the other genotypes. The second part of this dissertation examines the effects of negative energy balance on food demand. Physical activity has long been suggested as a method to better maintain weight loss and that obesity induced by a high fat diet can be treated without energy restriction by exercise. The effects of running wheel activity on food intake and meal patterns were measured under several economic conditions in CD 1 mice. Voluntary wheel running activity increased daily food intake and running animals consumed bigger but fewer meals compared to the sedentary. Although they ate more, running mice had significantly lower body fat, especially in subcutaneous depots. In the foraging protocol, when food intake was contingent on the running wheel activity, mice were able to emit more responses compared to when food is contingent upon a nose poke response. In both voluntary and foraging running protocols mice had inelastic demand functions compared to the non-running groups indicating that negative energy balance affects how much mice are willing to work for food.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Deniz Atalayer.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Rowland, Neil E.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042055:00001


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1 FOOD DEMAND AND E NERGY BALANCE: A NEUROECONOMIC ANALYSIS OF MELANOCO R TIN SYSTEM AND EXERCISE By DENIZ ATALAYER A DISSERTATION PRESENTED HERE TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Deniz Atalayer

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3 To my mom, dad and my m entors : Dr. Neil E. Rowland and Dr. Philip Teitelbaum

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4 ACKNOWLEDGMENTS My dissertation is a product of ad libitu m encouragement and support by my parents, my advisor Dr. Neil E. Rowland and my mentor Dr. Phi lip Teitelbaum. Unlike the non fan crowd that I unintentionally created around myself in the lab, they believed in me at all times Today w ith this accomplish ment, I promise to make those who believed in me be proud. Today with this accomplishment, not only I got one step closer to what I would like to pursue in the future but also to whom I would like to become : Dr. Rowland; the most humble scientist I have ye t to meet, and who is my role model as a scientist, as a teacher, and as a person. I would like to end this dedication by quoting from my mentor Dr. Philip Teitelbaum, as an advice to any young scientist and for my own remembrance forever: Do not keep y our knowledge in isolation!

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS .................................................................................................. 4 page LIST OF TABLES ............................................................................................................ 7 LI ST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 12 Ec onomic Structure of the Food Environment ........................................................ 12 Obesity /Positive Energy Balance on Food Demand ............................................... 15 Melanocortin System and Obesity .................................................................... 16 Diet Induced Obesity ........................................................................................ 19 Melanocortin System and Cholecystokinin ....................................................... 20 Negative Energy Balance and Food Demand ......................................................... 21 Summary ................................................................................................................ 23 2 GENERAL METHODS ............................................................................................ 29 Animals ................................................................................................................... 29 Diets ........................................................................................................................ 30 Apparatuses ............................................................................................................ 30 3 POSITIVE ENERGY BALANCE AND FOOD DEMAND ......................................... 34 Introduction ............................................................................................................. 34 Melanocortin 3 and 4 Receptor Knock Out Mice .............................................. 34 Diet Induced Obese Mice ................................................................................. 37 Effects of CCK .................................................................................................. 38 Subjects and Housing ............................................................................................. 38 General Methods .................................................................................................... 39 Procedures ............................................................................................................. 40 Experiment 1: Food Demand Analysis in MCR Knockout Mice ........................ 40 Experiment 2: Food Demand analysis in Diet Induced Obese Mice ................. 41 Experiment 3: Behavioral Effects of Exogenous CCK 8 in MCR Knoc kout Mice .............................................................................................................. 41 Experiment 4: C Fos Inducing Effects of CCK 8 in MCR Knockout Mice ......... 43 Data Analyses ......................................................................................................... 44 Results .................................................................................................................... 44 Experiment 1: Food Demand Analysis in MCR Knockout Mice ........................ 44 Experiment 2: Food Dem and analysis in Diet Induced Obese Mice ................. 46

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6 Experiment 3: Behavioral Effects of Exogenous CCK 8 in MCR Knockout Mice .............................................................................................................. 46 Experiment 4: C Fos Inducing Effects of CCK 8 in MCR Knockout Mice ......... 48 Discussion .............................................................................................................. 48 Experiment 1: Food Demand Analysis in MCR Knockout Mi ce ........................ 48 Experiment 2: Food Demand analysis in Diet Induced Obese Mice ................. 49 Experiment 3: Behavioral Effects of Exogenous CCK 8 in MC R Knockout Mice .............................................................................................................. 50 Experiment 4: C Fos Inducing Effects of CCK 8 in MCR Knockout Mice ......... 52 Chapter Discussion .......................................................................................... 52 4 NEGATIVE ENERGY BALANCE AND FOOD DEMAND ........................................ 66 Introduction ............................................................................................................. 66 Subjects and Housing ............................................................................................. 67 General Methods .................................................................................................... 67 Procedure ............................................................................................................... 67 Experiment 5: Voluntary Running Wheel Activity and Food Demand ............... 67 Experiment 6: Running Wheel Activity as Food Foraging ................................. 6 8 Data Analysis .......................................................................................................... 70 Results .................................................................................................................... 70 Experiment 5: Voluntary Running Wheel Activity and Food Demand ............... 70 Exp eriment 6: Running Wheel Activity as Food Foraging ................................. 72 Experiment 5 and 6: Voluntary Running versus Running as Food Foraging Effort ............................................................................................................. 75 Discussion .............................................................................................................. 76 Experiment 5: Voluntary Running Wheel Activity and Food Demand ............... 76 Experiment 6: Running Wheel Activity as Fo od Foraging ................................. 79 Chapter Discussion .......................................................................................... 80 5 GENERAL DISCUSSION ....................................................................................... 99 LIS T OF REFERENCES ............................................................................................. 106 BIOGRAPHICAL SKETCH .......................................................................................... 115

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7 LIST OF TABLES Table page 1 1 Appetitiv e costs, alternative terms, and definitions ............................................. 28 3 1 Fat c ontent of mice from experiment 1 ............................................................... 62 3 2 Ef fect of CCK 8 (6 g/kg) on int ake of crunchies by undeprived mice ................ 63 3 3 Effect of CCK 8 on chow intake by deprived mice .............................................. 64 3 4 Fos positive cells in paravent ricular hypothalamus following CCK 8 (6 g/kg) ... 65 4 1 Total caloric energy intake at each FUP schedule i n experiment 5 .................... 96 4 2 Ru nning dista nce (m) of mice in experiments 5 and 6 as a function of the fixed unit price (FUP) for food ............................................................................. 97 4 3 Total caloric energy intake at each sch edule in experiment 6 ............................ 98

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8 LIST OF FIGURES Figure page 1 1 The demand function.. ........................................................................................ 25 1 2 Concurrent approach costs and unit prices imposed. ......................................... 26 1 3 The comparison between nose poke and lever press operants. ........................ 27 3 1 Daily (23 h) food intake. ...................................................................................... 56 3 2 Intake analyzed for number of meals and mean meal size. ................................ 57 3 3 Weight changes of mice during th e demand series.. .......................................... 58 3 4 Scatterplots showing fat pad amounts.. .............................................................. 59 3 5 Daily food intake, number of meals and mean meal size in DIO (HF) and LF mice ................................................................................................................... 60 3 6 Effect of CCK 8 on intakes of chow in 30 min tests after 18 h food deprivation.. ........................................................................................................ 61 4 1 Individual day to day data from a typical mouse.. ............................................... 83 4 2 Daily food intake, number of meals taken, and meal size of sedentary and runner mice ......................................................................................................... 84 4 3 Nose poke responses.. ....................................................................................... 85 4 4 The mean body weight and dissected fat pads sedentary and wheel running mice ................................................................................................................... 86 4 5 Ind ividual day to day data of a typical mouse. .................................................... 87 4 6 Bar plots for mean number of responses (SEM) for both groups ...................... 88 4 7 The numb er of responses emitted for both groups plotted against the pellets consumed. .......................................................................................................... 89 4 8 Lever press responses (FUP responses) for runGR and pokeGR. ..................... 90 4 9 Demand functions for runGR and pokeGR.. ...................................................... 91 4 10 Mean number of meals (meal frequency) taken per day for runGR and pokeGR.. ............................................................................................................ 92 4 11 Mean daily meal sizes for runGR and pokeGR.. ................................................. 93

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9 4 12 Body weight change. .......................................................................................... 94 4 13 Voluntary and forag ing running wheel activity from both experiments. ............... 95

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FOOD DEMAND AND E NERGY BALANCE: A NEUROECONOMIC ANALYSIS OF MELANOCO R TIN SYSTEM AND EXERCISE By Deniz Atalayer August 2010 Chair: Neil E. Rowland Major: Psychology One of the main factors contributing to the obesity epidemic is the obesity favorable food environment of todays modern societies that promotes excessive energy intake while minimizing the energy expenditure. The purpose of the present work is to combine the analyses of the effects of both positive and nega tive energy balance on food demand and meal size by exploring the some of the physiological and neuronal mechanisms of the melanocortin system and the effects of physical activity. H omozygous deletion of the melanocortin4 receptors (MC4R / ) is kno wn to r esult in positive energy balance as MC4R / mice and humans develop obesity while those with deletion of the melanocortin3 receptor s (MC3R / ) do not become markedly obese. I n the first part of this dissertation the effects of positive energy demand on food demand were examined. MC3R / MC4R / mice with wild type (WT) and mice lacking both of these receptors (double knockout, DKO) were compared on select feeding and neuroanatomical dimensions. In a food demand protocol, DKO and MC4R / were hyperphagic at low unit costs for food, due primarily to increased meal size. However, at higher costs, their intake dropped below that of WT and MC3R / indicating increased elasticity of food demand. To determine whether this higher elasticity was due either to

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11 the genotype or to the positive energy balance per se, the same food demand protocol was conducted in dietary obese mice. The d ifference between the demand functions of WT and dietary obese mice was smaller compared to that of WT and genetically obese mice. T o assess a mechanism for the larger meal size in MC4R / and DKO, we examined the acute anorectic effect of peripherally administered cholecystokinin (CCK) and subsequently the induction of Fos immunoreact ivity in select brain regions. The anorectic effect of CCK was comparable in MC4R / DKO, and WT, but was unexpectedly absent in MC3R / CCK 8 induced cFos was lower in PVN in MC3R / than the other genotypes. The second part of this dissertation examines the effects of negative energy balance on food demand. Physical activity has long been suggested as a method to better maintain weight loss and that obesity induced by a high fat diet can be treated without energy restriction by exercise. T he effects of running wheel activity on food intake and meal patterns were measured under several economic conditions in CD 1 mice. V oluntary wheel running activity increased daily food intake and running animals consumed bigger but fewer meals compared to the sedentary. Although they ate more, running mice had signi ficantly lower body fat, especially in subcutaneous depots. In the foraging protocol, when food intake was contingent on the running wheel activity, mice were able to emit more responses compared to when food is contingent upon a nose poke response. In bot h voluntary and foraging running protocols mice had inelastic demand functions compared to the nonrunning groups indicating that negative energy balance affects how much mic e are willing to work for food.

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12 CHAPTER 1 GENERAL INTRODUCTION Economic Struct ure of the Food Environment From an evolutionary perspective, foraging for food is essential for an animal. In order to survive it has to remember food sources, prepare for consumption, ingest sufficient amounts of food and digest them efficiently (Adan et al ., 2006). The decision to eat is controlled both by endogenous factor s (hunger and satiety), and also by environmental cues such as availability of palatable or novel foods and predator exposure when searching for food. Animals searching for food often perform a series of behaviors; traveling, catching and consuming with the inherent strategy of optimizing and allocation of scarce resources. These behaviors constitute a form of cost for the animal in the nature. The field of economics is defined as the c omputational analysis of anticipated cost benefit balance and considered a science of highly organized human behavior (Hursh, 1984). One of the main principles of the theory of economics states that if the price increases the consumption of most goods decr eases (Hursh et al ., 1988). Recently, these principles, which relate the commodity to its price, have been applied to the field of behavior analysis (Lea 1978; Hursh 1980; Collier et al ., 1986; Killeen, 1995). Earlier studies on behavioral economics found that feeding patterns vary according to this cost of access to the food (Collier, 1985; Collier et al ., 1986, Hursh, 1980). From this perspective, feeding behavior can be considered as being highly influenced by the economic structure of the current environment in which the individual lives, because whether in the natural habitat of animals or the very industrialized lifestyle of humans, the demand for food is always in an exchange for some type of

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13 cost. F or animals, at least three different types of costs related to food consumption have been defined in the literature (Morato et al ., 1995): Cost of procuring access to food procurement cost (travel effort and/or time) Cost of consuming food consummatory cost (the cost within the patch such as digging or cl imbing for food items, sucking, catching, holding etc., which are the equivalent of operant schedules in an experimental setting in most studies) Cost of processing food (physiologic consequences/digestion) Collier et al (1972) showed that number of meals eaten daily and associated meal sizes could be altered profoundly by relatively small costs imposed during the consummatory phase. They also showed that meal structure can be differentially affected by the changes in the former two cost s categorized above. I n a previous study in our lab, we analyzed the meal structure based on the systematic changes in both types of costs in mice (Atalayer and Rowland, 2009). In addition, in order to better capture the functional differenc e between the two types of cost, w e introduced a different terminology for these two costs (Table 11). We proposed that approach cost ( procurement cost as previously termed) is analogous to travel or effort that occurs while food is still spatially and/or temporally distant, and may be associated with a low er probability of success. In contrast, unit price (consummatory cost as previously termed) is the effort expended once the food item or patch is reached, such as digging in a substrate or hulling seeds; it occurs in proximity to the f ood and is usually associated with a higher (but not necessarily fixed) probability of success. In most research protocols with animal models investigating eating in terms of economics of behavior, demand functions have been most usually determined in which the price is defined as time or effort to obtain uniform morsels of food such as small, nutritionally complete pellets. Effort is usually assessed as the number of behavioral

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14 responses required per reinforcement or reward (e.g., food, water). Thus, fro m the economics of behavior point of view, the apparent question is how the animals will change the rate or amount of responding as the required effort (cost) to access to the commodity, the food, is increased (Hursh, 1980). It is recognized that the answe r is a product of a complex interaction between the availability and/or cost of the food a nd hunger state of the organism. H owever it can be defined and summarized in a demand function which relates the price and food intake (Figure 11). A demand function is a mathematical relationship that describes the effects of prices on the quantities of commodities purchased (Lea, 1978) The slope of the demand function is determined by the amount of the effort that an animal will emit to obtain a commodity as the pr ice for that commodity increases. It is also called the elasticity of the demand, defined as the ratio of the proportional changes of dependent and independent variables (Lea and Roper, 1977). It should be noted that economists u sua lly draw the curve as th e y axis because it is the dependent variable, but in the experimental designs in psychology, the pri ce (reinforcement schedule) is more or less the independent variable so that is why we draw it in the x axis and the demand quantity ( the dependent variabl e ) on the y axis The slope of the demand function, the elasticity of demand, changes in response to various factors. In eating behavior these factors could be things such as availability and substitutability of alternatives (Lea and Roper, 1977), the incentive value or amount of the food (Killeen, 1995), the deprivation level of the individual (Hursh et al ., 1988), presence of others and amount of attached cost (Johnson et al 1993) etc. Many studies using these types of designs have shown that food intake does change under different

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15 economic conditions of food availability (Sumpter et al ., 1999; RoveeCollier et al ., 1982; Hursh, 1980; Hursh et al ., 1988). Under fixed ratio ( FR ) schedules, each unit of food costs a fixed number of behavioral responses and operant behavior is used to emulate cost for food in laboratory settings FR schedules are believed to be the most direct and are the most commonly used method to set the price of a commodity (Bauman et al ., 1996; Hursh, 1984) Thus in all of our studies presented here, we will use fixed ratio reinforcement schedules. The term fixed ratio will be combined with the term unit price (the term that we presented earlier) as fixed unit price or FUP. By using these cost regimens, researchers aim to understand how animals preference for food is shaped by the different economic environments and by extrapolation to the human condition, how an individuals food preferences may be affected in these economies where different types of food are usually available at all times. In this type of continuous access protocol, parameters of meal size and frequency may also be determined as food intake is organized in meals so increased food intake could be realized by increased meal size, meal frequency, or both. Thus, different experiments designed in this project will be discussed and analyzed in terms of demand functions and meal patterns. Obesity /Positive Energy Balance on Food Demand Obesity is one of the biggest threats to contemporary human health. At least 400 million adults globally are obese and ~700 million adults are estimated to become obese by 2015 (WHO, 2005). In addition the same report reveals that, based on the NHANES (National Health and Nutrition Examination Survey), obesity increase was highest in the last decade compared to the previous time periods. This clearly indicates that the effects of changes in the food environment in terms of its availability and cost because it

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16 is such a short period of time for any mutations in the genetic pool populationw ide. Thus in this dissertation we will examine the effects of the changes in the economic settings of the food environment by using the demand functions as indirect indication of the food motivated behavior. We hypothesize that demand for food for lean ver sus obese individuals will be different as it is has been suggested that the reinforcing value of high calorie foods are higher for obese individuals than for normal weight individuals as overweight/obese individuals work harder for highcalorie snacks com pared to nor mal weight individuals (Giesen et al ., 2010; Temple et al ., 2009). Thus an animal model of these reports would be particularly necessary and beneficial for a more accurate understanding and reliable predictions for the reinforcing value of food in obese versus non obese individuals. Melanocortin S ystem and O besity Although we are interested in the effects of the environment on the rising obesity epidemic, we do not ignore the effects of genetics. Eventually we aim to combine both genetic and e nvironmental approaches to come up with better therapeutic solutions Although s tudies of animal models have identified several genes with measurable effects on body weight and composition, it has been found that polymorphisms in the melanocortin type 4 re ceptors (MC4R) of central ner vous system are implicated in ~ 6% of early onset or severe adult obesity cases (Adan et al ., 2006; LubranoB erthelier 2003, Govaerts et al ., 2005 Vaisse et al ., 2000). As the most common known monogenic cause of human obesity we chose MC4R mutant animals (MC4R / ) in this project as the genetic model of obesity. The a rcuate nucleus of hypothalamus (Arc) integrates and distributes to other parts of the brain the neural and hormonal signals from the periphery that reflect

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17 meta bolic status further into the brain. Within the Arc, neurons containing melanocortins (MCs), products of the proopiomelanocortin (POMC) prohormone, are activated by leptin, a long term satiety hormone secreted in response to increased adiposity in the body (Schwartz et al ., 1997). The biological effects of these MCs are mediated by five known melanocortin receptors ( MC1 5R ) all belong to the family of seven transmembrane G protein coupled receptors (GPCRs) (Abdel Malek et al ., 2001; Cone et al ., 1996). MC1 R is activated by melanocyte MSH and has a key role in determining skin and hair pigmentation ( Abdel Malek et al ., 2001). On the other hand unlike the other four MCRs, MC2R is an adrenocortical adrenocorticotropic hormone receptor and has a role in secretion of steroid hormone along with some ambiguous functions a ffecting adipose tissue which also differs in humans and animals (Gantz and Fong, 2003). The last MC receptor, MC5R was found to have a role in sebaceous gland secretion (Hatta et al ., 2001). However, the effects on coordinated energy homeostasis are mediated by the central MC3R and MC4R and these are expressed in several regions of the CNS. MC4Rs have a major role in the regulation of feeding behavior and body weight and are expressed predominantly in paraventricular nucleus of hypothalamus (PVN), area postrema (AP) and nucleus of the tractus solitarius (NTS). It is a 332amino acid protein encoded by a single exon gene localized on chromosome 18q22 and signals through the activation of adenyly l cyclase in response to its endogenous agonist ligand, melanocytestimulating hormone ( MSH) which is a neuropeptide produced by proconvertase 1 dependent cleavage of POMC (LubranoBerthelier et al ., 2003) There is strong evidence that the action of both leptin and insulin relies upon MSH signaling

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18 since administration of antagonists to MC3R/MC4R blocks each of their actions in the brain (Woods, 2005). Thus, studies demonstrated nonredundant roles for the MC3R and MC4R in energy homeostasis. MC3R ( o n chromosome 20q13.2) is expressed in many areas of the CNS (and in several peripheral tissues, including the adipose tissue, heart, skeletal muscle, kidney, stomach, duodenum, pancreas and placenta (Gantz and Fong, 2003; Chajlani, 1996) However, the highest concentrations of these receptors are found in Arc and they are presumed to function as autoreceptors on POMC cell bodies at this site. The dorsal part of the ventromedial hypothalamus (VMH) that is presumed to receive projections from the Arc is anoth er structure where the MC3R are abundantly located (Cowley et al ., 2001). In addition, high levels of MC3R mRNA are expressed in the ventral tegme ntal area which is a site involved in processing of the information on reward systems (Kishi and Elmquist, 200 5). Interestingly, MC3Rs are not found to be present in the NTS or AP (Blevins and Baskin, 2010) The activity of the MC system is also regulated by an endogenous antagonist agouti related protein (AgRP) which acts at MC3R and MC4R. It is produced exclus ively in the Arc in rodents, monkeys, and humans and a single dose of AgRP that is administered into the brain near the Arc increases food intake (Kishi and Elmquist 2005). An important side note is that although often described as a competitive antagonist (which binds to a receptor but does not activate it), AgRP acts in fact as an inverse agonist (which binds to receptor but has the opposite action then the agonist ) on constitutively active MC3Rs and MC4Rs (Haskell Luevano and Monck, 2001). In contrast to POMC neurons, AgRP neurons are activated during negative energy

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19 balance initiating eating and inhibited by leptin. Common literature suggests that leptin stimulates MC signaling, resulting in increased energy expenditure (oxygen consumption and diet induc ed thermogenesis) and decreased energy intake whereas opposite effects by AgRP are inhibited by leptin. An inverse agonist and an agonist acting on the same receptor indicate how tightly these systems are regulated. A l arge body of evidence of the function al roles of these receptors comes from the genetic mutation studies in mice which have elucidated distinct and complementary roles for MC3R and MC4R in food intake and body weight (Branson et al ., 2003 ). Diet Induced Obesity As we stated above w hereas ge netic factors play a key role in which individuals will develop obesity, the determinants of obesity seem to be a multifactorial phenomenon and it may be caused by environmental or genetic factors, and/or combinations of the two. The environmental factors such as a sedentary lifestyle and the overconsumption of a high fat content diet especially make critical contributions to the present high rates of excessive adiposity in our population (Chandler et al ., 2005). T hus, while not a direct cause, high fat content diet may promote obesity (Willett and Leibel 2002). Bjorntorp (1993) termed visceral obesity the civilization syndrome as the ingredients of positive energy balance, including low physical activity, stress induced hormonal changes, nicotine and alc ohol consumption are frequent features of our modern, urbanized society. Thus even when pronouncing the importance of genetics, one should also argue the influences by environmental factors leading obesity. For this reason, in paradigms in which we used genetic mutations leading an obese phenotype, we also employed diet induced obese (DIO) animal models in order to determine the

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20 contributions of either the excessive adipose tissue accumulation in the body or the metabolic changes as determinant of the behav ior of lean versus obese individual s in various economic food environments. Melanocortin System and Cholecystokinin It has been found that large meal sizes characterize many genetic animal models of overeating and obesity. This suggests that some aspect of satiation or satiety may be impaired. During meals, gut (stomach and intestine) signals such as distension of the stomach or cholecystokinin (CCK) trigger nerve impulses via glutamatergic vagal afferent fibers traveling to the hindbrain. Specifically CCK which is a gut derived satiety factor is believed to restrain meal size by stimulating the hindbrain sites, NTS and/or by direct action in the area postrema (AP) (Appleyard et al ., 2005; Berthoud, 2002; Berthoud et al ., 2006; Fan et al ., 2004; Rinaman et a l ., 1993) MSH release in the NTS from terminals of POMC neurons. CCK is generated as food is the gastrointestinal tract and acts within the time of a single meal. Exogenous administration of CCK decreases meal size in a dose dependent manner w ithout causing illness. Some of the CCK enters the blood as a hormone that stimulates the exocrine pancreas and gall bladder as discussed above; but some of the CCK also diffuses locally in a paracrine fashion within the intestinal wall to generate action potentials through the vagus nerve which transmits a message to the AP/NTS signifying the ingestion and upcoming absorption of the food. There is now strong evidence that MC4Rs modulate release of glutamate from vagal afferents in the NTS by a presynaptic mechanism (Wan et al ., 2008); in most NTS cells, the number of spontaneous excitatory postsynaptic events was enhanced by

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21 MSH action may be to augment CCK and/or other vagally mediated satiety signals. Negativ e Energy Balance and Food Demand There are many central and peripheral signals involved in energy homeostasis and regulation of food intake, and understanding these mechanisms may lead to effective treatments in the control of obesity. Along with the read ily available high calorie/highfat foods leading DIO discussed above, the lack of necessity for a more physically active lifestyle in todays industrial societies may contribute to the obesity epidemics that we suffer nationwide in U.S. and also globally is a common assumption that obesity may be related to decreased physical activity. In addition to a role for energy input (food intake) in body weight control, elective energy output (physical exercise) has been shown to reduce the risks for obesity and obesity related diseases in animals and humans (Dishman et al ., 2006; Wolfe et al ., 1993). Studies with animals models showed that exercise prevents or delays onset of obesity in young obesity prone rats, this was hypothesized to be related to the effects of exercise on the development of the neurocircuitry controlling obesity (Hansen et al ., 2008; Rhodes et al ., 2005). It has also been shown to slow the weight gain in some genetic models of obesity (Irani et al ., 2005). However, although obesity has been r elated to high fat dietary content and lack of physical activity, the methods to treat human obesity still rely predominantly on dietary restriction, which has proven difficult to sustain. One purpose our multifactorial approach reflected in this project i s to analyze the effects of unit price changes, physical activity (voluntary running), dietary obesity and the interaction of the three on demand functions, meal patterns and body weight.

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22 Running Wheel Activity as Food foraging. Free living animals must learn how to search for food in order to survive and often perform a series of behaviors including traveling and capture before consuming food. In addition to its survival value for the access of food, physical activity has been shown to have rewarding value in rodents. Thus, rats will press a press lever in order to access running wheels (Belke, 1997; Belke and Wagner, 2005; Iversen, 1993). In our studies here, running was used both as a voluntary activity and an obligatory cost to access to food. As a re minder previously we proposed the terms approach cost which is analogous to travel or effort and unit price that is the effort expended once the food item or patch is reached (Atalayer and Rowland, 2009) In the same study we showed that when concurrent approach cost and unit price were imposed, food demand again varied relatively little across the range of parameters studied. On the other hand meal number and meal size were profoundly affected by approach cost (Figure 1 2). The operant behaviors for approach cost and unit prices used were nose poke and lever press responses respectively. In that earlier study (Atalayer and Rowland, 2009) we compared the two most commonly used operants and demonstrated that when averaged across various unit price schedules (fixed, various, progressive)1 1 Fixed unit price (FUP) i n which each unit of the item costs a fixed amount (e.g., number of responses), variable unit price (VUP) in which each food item costs a mean amount but the actual cost of each item varies around that mean and progressive unit price (PUP) in which success ive food items within an episode of feeding become progressively more costly. nose poke operant response did result in a slightly higher food intake than the lever press, but both the mice in both groups changed intake comparably across the various types and increments of unit price schedules, sugges ting that nose poke and lever press may be used interchangeably as cost devices for the study of food demand (Figure 13). These findings agree with

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23 previous studies in rodents comparing nose poke and lever press operants in which acquisition of the nose poke response often occurs more rapidly and emit s slightly more responses (David et al ., 2001; Ettenberg et al ., 1981). Thus when the experimenter prefers to reduce the time of training in mouse studies, a nose poke operant might be more useful. As much as these are the two operants that have been dominantly employed in these types of studies, we recently hypothesized that when the effects of approach cost is analyzed in a laboratory setting a running wheel activity would emulate a more natural situation co mpared to other types of commonly used operants and would comprise a relatively more ecologically valid operant, thus may result in either different or more accurate analyses of such parameters of neuroeconomic analysis of food intake in animal models. Ne vertheless establishing ecological validity is beyond the scope of this project. O ne should keep in mind that, one may not be sure that the results with the running wheel activity in comparison to any other operant behavior is more accurately emulating the cost for food that the animals face in the nature. For that, observational studies in the natural habitat of these species should be referenced. However one of the aims of this project is to find out whether the various operants that are used in this type of research may be resulted in substantially different meal patterns and f ood intake. Summary Obesity and its related metabolic disorders have become an intractably large financial burden to modern societies in terms of health care c osts and loss of productivity. There has been considerable focus on predisposing genetic factors, including polymorphisms of MC4R, but it is likely that most obesity is more directly linked to the ubiquitous availability of energy dense foods. We will show that a model of

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24 genetic obesity may be highly responsive to economic cost ; this result suggests that changes to the food environment can be effective in curbing food intake and/or choice in both normal and genetically disadvantaged obese populations. It is unrealistic to believe that a purely physiological manipulation such as gene or pharmacotherapy will be effective in the face of a toxic food environment. It is more realistic to ask how the obesity epidemic might be reversed by physiological manipulations in conjunction with radical changes of approach by the health delivery and food industries. The present study is thus presented as an early and small step in the preclinical analysis of this type of approach.

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25 cost 0 2 4 6 8 10demand 5 10 15 20 25 30 35 Figure 11. T he demand function. The figure summarizes the relation between the cost and demand. The slope of the function is determined by the amount of the effort that a person or an animal will work to obtain a commodity as the price for that commodity increases (the numbers are arbitrary and the graph was purely conceptual ).

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26 Figure 12. Concurrent approach costs and unit prices imposed. A) Mea n (SEM) food intake, B) meals per day C) and meal size of 7mice in three approach cost and five fixed unit price conditions (log scale). C B A

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27 Figure 13. The comparison between nose poke and lever press operants. Mean (SEM) number of pellets taken per day as a function of uni t price under A) fixed, B) variable, and C) progressive ratio schedules. Data are for separate groups (Ns=8) required to emit either nose poke or lever press responses to obtain food, but for the same animals is left to right progression across costs and schedules. Note that the fixed price axis is scaled logarithmically. A B C

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28 Table 1 1. Appetitive costs, alternative terms, and definitions Term used in this paper Alternate term(s) Functional description or examples Approach cost (AC) Procurement cost Anticipatory activity Travel cost Distal foraging Searching for a remote food sour ce. May have low probability of success. May include moving food to a hoard. Unit price (UP) Consummatory cost Ratio requirement Proximal foraging Effort in proximity to food such as hulling seeds or nuts; picking fruit. Relatively high probability of success.

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29 CHAPTER 2 GENERAL METHODS Animals Mice (Mus musculus ) have become an important species for contemporary basic research into physiology and behavior because of their potential for genetic manipulation. The identification of several genes involved in obesity has generated considerable interest in studies of food intake in mice, yet the number of published works that have examined meal parameters is still small and the results are often inconsistent (Atalayer and Rowland, 2009). This may be in part because mice present some procedural challenges. However, the cost efficiency and genetic variety of mice species constitute advantage over other animal models esp ecially when larger sample sizes are considered. In most of the experi ments, we did not include female mice in our design to avoid the possibility that estrous cyclicity would interact with our analysis of meal patterns. In our previous studies using operant response methods in lean and genetically obese mice (Vaughan and Ro wland, 2003; Vaughan et al ., 2005; Atalayer and Rowland, 2009), we studied the effect of changes in approach cost (we and others (Collier et al ., 1972) previously termed this procurement cost) at a constant but low unit price (previously, consummatory cos t); those studies showed that meal parameters in mice are highly sensitive to approach, very similar to results reported in rats (Collier et al ., 1972). Animal use is approved by a campus wide IACUC and is compliant with the recommendation of the Guide for the Care and Use of Laboratory Animals (1996).

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30 Diets For 1 2 weeks between receipt of the animals and the start of the experiments, animal were housed individually in polycarbonate home cages with Purina 5001 Chow pellets and tap water available ad libitum. Methods, in which discrete small food items are presented sequentially, as in standard operant behavior protocols, are particularly appealing because they minimize food spoiling behavior for which laboratory mice often show a proclivity. A few such studies have appeared in the recent literature (Chi and Powley, 2003; Donovan et al ., 2007; Fox and Byerly, 2004; Tabarin et al ., 2007; Vaughan et al ., 2005), but most of these used a low and constant unit price, and did not generate demand functions. Thus for the experiments, mice obtained 20 mg nutritionally complete pellets (Purina Test Diet 5TUM, a grainbased tablet with 10.4% kcal from fat and 24.1% from protein) when they completed a price that was specified by the particular reinforcement schedule that was in effect. The diets that were used in specific experiments (e.g. diet induced obesity, acute food intake protocol for CCK action) will be described at each section for each experiment. Apparatus Operant chambers. When not in experiments, all mice were individually housed in standard polycarbonate cages containing a 23 cm layer of bedding (SaniChips, Teklad, Madison, WI) placed on standard racks and were provided with material for nest building (Nestlet, AnCare, Bellmore, NY). In the experimental procedures for analyzing demand functions, twenty four operant chambers (Med Associates: 13x13x12 cm with Plexiglas and metal walls and stainless steel grill floor plus solid nesting platform) enclosed in ventilated, noise attenuating cabinets with the s ame 12:12 light cycle as the vivarium (a 15 watt bulb in a standard light fixture run from a 24 hr timer) were used. All

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31 chambers were equipped with one lever press and one nose poke operant device, located 2cm above the floor, situated on one wall on either side of a food aperture. Depending on the design, both or only one of these operants were active during a particular protocol as stated in the method sections of each experiment. The nose poke unit includes a yellow light emitting diode stimulus light at the back of the access port, and a source and detector. According to the manual for the chambers whenever the beam is broken with an approximate of 0.64cm length of animal nose entry, it is detec ted as a response by the system Water was supplied from sipper tubes mounted on the wall opposite side to the food magazine and the two operant devices. The vivarium was temperature (23 2oC) and humidity (4070%) controlled, with a 12:12 light cycle (lights on 0700). During the experiments, mice lived in test c hambers for 23 h per day. The mice were weighed daily and kept in empty holding cages during a 1 h cleaning period without food, although water was available. The Psychology department vivaria are part of the centralized University of Florida Animal Care program with full AAALAC accreditation. For various procedures, we modified the location of some of the operants and/or cues and/or house lights based on the design which will be described at each section for each experiment. Running wheels. Eight chambe rs were additionally fitted with an external running wheel with free wheel resistance of 4 5 g and 55.8 cm outer circumference (Med Associates, St. Albans, VT: ENV3042; 1km~1800 revolutions) to which the mice had free and voluntary access. Revolutions wer e recorded by a counter on the running wheel and also by the computer program.

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32 Data analysis and software. A record of the total pellets obtained by mice and number of responses (nosepoking, leverpressing, running wheel ac tivity) were acquired by the MED PC IV computer software (MED Associates, St. Albans, VT). The computer recordings allowed an analysis of the number of meals and the amount eaten at each meal. Early studies on meal patterns in rodents have defined a meal as an eating episode initiated aft er at least 40 min of noneating period (Kissileff, 1970; LeMagnen, 1992; Clifton, 2000). In mice, spontaneous food intake occurs in a number of eating bouts separated by periods of noneating or meal to meal intervals (MMI) that typically are at least 30 min in length (Clifton, 2000). In our previous work, we compared two different MMI criteria (15 and 30 min) to define a meal and results showed that the meal distribution in mice was critically dependent on the MMI criterion, regardless of the operant empl oyed (Atalayer and Rowland, 2009). Although determining which of the MMIs is a more accurate representation for `a meal` for mice, 15min MMI seemed to better agree with some of the earlier studies with mice ( Petersen and McCarthy, 1981; Vaughan and Rowland 2003; Fox and Byerly, 2004; Vaughan et al ., 2005 ). Thus we used this criterion in all of our analyses. Note that on average an animal will have stopped in the middle of the interval preceding a zero entry and will have started eating in the middle of the interval following a zero entry; thus, this 15 min meal criterion in fact defines an average minimum inter meal interval of 30 min, but with a range from 1545 min. After the numbers of meals per day were determined for each mouse, the mean meal size was derived by dividing the number of total pellets by the number of meals for each day.

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33 The raw data from the computer recordings, showed how many responses were made and the number of pellets earned at each 15 min each day. After the numbers of meals for eac h mouse were counted by the experimenter for each day of each schedule from the raw data, the mean meal size was derived by dividing the number of total pellets by the number of meals for the particular day. In our previous study we found that mice can adj ust the changes in cost schedules typically in ~2 days (Atalayer and Rowland, 2009). The mean for number of meals, total pellets earned and meal sizes were computed for each mouse and for each reinforcement sch edule by averaging them over four days. Param eters were analyzed for significance with SPSS computer software by using repeated measures analysis of variance (ANOVA), oneway ANOVAs, t tests and post hoc Bonferroni and/or LSD and/or Tukey when it is necessary. In all cases, P < 0.05 was considered sig nificant.

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34 CHAPTER 3 POSITIVE ENERGY BALANCE AND FOOD DEMAND Introduction Previous work from our laboratory demonstrated that MC4R / mice were hyperphagic and took larger meals under specific low cost schedules (Vaughan et al ., 2006a ) However, a systema tic analysis of increasing costs and full demand functions were not determined in that study. In the first part of C hapter 3 full demand functions in wild type (WT) MC3R / MC4R / and DKO mice will be examined. Studies suggest that hyperphagia in MC4R / occurs to support that excessive body weight increase that is essentially caused by a metabolic deficiency in these mice (Marie et al ., 2000). Thus in the second part the same type of analysi s is conducted with diet induced obese (DIO) mice to compare their results with those from genetically obese mice to determine if the demand functions are the sole product of the higher level of adipose tissue in obese animals or whether the metabolic consequences of MCR mutations do play a role. In the last section of C hapter 3 the effects of CCK will be determined along with the comparison of the induction of c Fos activity as an indication of cellular activity by CCK in the brain of these various mice with deletion of MC3R and/or MC4R. Melanocortin 3 and 4 Receptor Knock Out Mice MC4R / mice. Homozygous deletion ( / ) of the MC4R results in an obese phenotype including hyperphagia, indicating that this receptor has a role in limiting food intake and sti mulation of energy expenditure. T hey also exhibit significa nt hyperinsulinaemia and alterations in metabolic rate, maintain normal levels of lean body mass and are longer than WT. (Chen et al ., 2000; Husz ar et al ., 1997 ; Haskell et al ., 2009). Heterozygous MC4R / mice present an intermediate average increase in

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35 w eight, females being more severely affected than males ; however they display a broad variability in phenotype with an adult weight ranging from that of WT to that of homozygous MC4R / mice (Vaisse et al 2000). Studies have shown the contribution of MC4R and MC3R to the regulation of metabolism. Marie et al (2000) reported increased body weight of the MC4R / female mice at 12 wk of age, and of the MC4R / male mice at 10 wk although they found no significant hyperphagia in these mice. At the end of 14 weeks, MC4R / mice of both genders were heavier than sex matched WT, and as a consequence they started to eat more. Their result indicates that MC4R / gained weight because of the changes in their metabolism and that their hyperphagia must occur to maint ain their higher weight level. They also measured the oxygen consumption which is another indication of metabolic rate and reported that MC4R / mice consume 20% less oxygen than WT (Marie et al ., 2000). Although these mice were leptinresistant, young non obese MC4R / mice are partially sensitive to the effects of leptin which suggests that leptin resistance is at least partially a consequence of the weight gain in these animals. Thus, as they are somewhat sensitive to leptin as they get older and heavier they eventually become fully leptin resistant (Marie et al ., 2000). In addition, another measure for metabolic rate is diet induced thermogenesis, the ability of leptin to induce uncoupling 1 protein (UCP1) mRNA in brown adipose tissue (BAT), a heat prod ucing tissue (Himms Hagen, 1979). Marie et al (2000) showed that partially leptin resistant young MC4R / mice were unable to produce UCP1 mRNA in BAT in response to peripheral leptin administration This suggests that the mutation has a metabolic effect because MC4R / mice show insensitivity to leptin mediated thermogenesis even before they become

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36 completely leptinresistant. Thus these animals not only have an obese phenotype, but also a metabolic deficiency that was resultant of the genotypic manipulat ion. In the pilot study for this project with a small sample size and both genders, we found that the slopes of the demand functions (elasticity of demand as previously described) of MC4R / mice w ere significantly different than in WT littermates. The heterozygous deletions in MC4R (MC4R + / ) resulted in an intermediate level of variance of demand functions between that of homozygous deletion and intact receptor conditions in mice (unreported data) For this reason, to capture the main contrast between the genotypes and the effects of the receptor deletions in addition to a simplicity principle we did not include the MC4R + / mice in this project. Our hypothesis based on our preliminary data, was that the obese mice would be hyperphagic and that their demand function would be less elasti c for increasing costs for food. MC3R / mice. In contrast, homozygous deletion ( / ) of the MC3R does not result in increased body weight, but may be associated with higher adiposity although not being hyperphagic and also despite being slightly hypophagic based on some reports (Chen et al ., 2000). Fat pad extractions indicated that nutrients are preferentially stored as fat depots rather than lean mass, resulting in what is called a f eed efficiency in MC3R / They are also reported to be hypoactive compared with WT controls in terms of the ambulatory activity and this may partly explain their obesity (Chen et al ., 2000; Cummings and Schwartz, 2000) It has been shown that the growth of both female and male MC3R / mice was normal until approximately 26 weeks of age, at which time they became slightly heavier than WT although they possess normal bone mineral content, but the average length of

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37 femur bones was significantly shorter than that of agematched WT. These suggest that growth is stunted in the absence of MC3Rs (Chen et al ., 2000). Also, the absence of MC3R does not result in large changes in metabolic rate as the body temperatures of both male and female MC3R / mice were normal. The effects of reduced expression o f aforementioned MC3R in the peripheral tissue, may contribute to the phenotypes observed. We expected a similar meal p atterns and demand functions but more adiposity in MC3R / mice compared with that of WT animals. DKO (double knockout). Based on the reports that have been published to this date on both MC4R / and MC3R / mice, one may speculate that DKO mice eat excessively, due to absent MC4R signaling, and store ingested calories more efficiently, due to the absence of both receptors (Cummings and Sc hwartz, 2000). Chen et al (2000) included a description of mice with deletions of both type 3 and 4 receptors (double knockout, DKO) but to our knowledge further characterization has not been published. To examine whether the MC3R and MC4R systems may int eract, we have now bred mice with deletions of both type 3 and 4 receptors. We hypothesized that DKO mice would show a higher level of hyperphagia and the demand functions would be significantly different than that of WT mice. H owever since this was one of the very few studies employing this newly bred genotype of mice, we were not sure if these mice would be significantly different from the MC4R / mice in terms of the measures used in this project. Diet Induced Obese Mice Because of the aforementioned ef fects of homozygous deletion of MC4Rs, we also decided to conduct the same experiment with the animals that have the same obese phenotype as MC4R / but not the underlying genotypic expression of the

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38 metabolic deficiency. The question was if the meal patterns and the slope of the demand functions were shaped by the accumulated high levels of fat in these obese animals and/or metabolic deficiency has a role in it with a possible interaction effect. Effects of CCK Protocols using peripheral CCK and injection of MC4R agonists into the NTS reduce food intake. This lea ds to the general assumption that one functional aspect of MSH action may be to augment CCK and/or other vagally mediated satiety signals. In this case, absence of MC4R should diminish the satiety effect of CCK or other gut related signals, and this has been reported by some investigators (Blevins et al ., 200 9 ). Previous work in our lab has not supported this prediction (Vaughan et al ., 2006 b ). MC4R / mice showed slightly greater inhibition of liquid food intake after CCK injection compared to wild type mice, or after oral preload of t he same diet. We wanted to generalize this finding to solid diet and expected to find simil ar results to our previous works in our lab, although in the literature the general v iew was that MC4R / mice were less sensitive to the effects of exogenous CCK (A dan et al ., 2004) Subjects and Housing MC3R / mice on a mixed C57B6/129 background were generated by breeding heterozygous pairs from a colony originating at Merck Pharmaceutical Co. MC4R / mice, also on a 129/B6 background, were generated by breeding heterozygous pairs from a colony originating at Millenium Pharmaceuticals Co. DKOs were generated by crossing the MC3R / and MC4R / mice. All mice were genotyped from a tail snip taken at weaning (H uzsar et al ., 1 997). Wild type controls were the littermat es ( +/+) derived from our in house breeding colony. No consistent differences in meal patterns or intakes were found b etween males and females, so these data were not analyzed for gender

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39 differences These colonies are maintained in the Cancer Genetics viv arium at the University of Florida. At 3 6 mo of age, mice were moved to the Psychology vivaria for the test procedures. Both facilities are managed by a centralized and accredited animal care program. For the DIO protocol, sixteen male C57BL/6 (B6) mice were purchased at 3 mo of age (Jackson Laboratories; Bar Harbor, ME) and housed in Psychology Department vivarium at the University of Florida. B6 mice were used because this strain has been shown to be a particularly good model for human metabolic syndrom e and obesity when fed with highfat content diet, but remains lean and physically normal when fed a low fat diet ( Surwit et al ., 1988; Samuelsson et al ., 2008). General Methods Twenty four behavior test chambers were used for testing. All chambers were equipped with a nose poke device with a small cue light above. The nose poke device was used in these procedures as the operant behavior for the effort cost imposed on food. The devices were located approximately 2 cm above the floor and adjacent to a foo d receptacle. These data files allowed an analysis of the number of meals and the amount eaten at each meal. Data were accumulated in 15 min time bins throughout each 23 h period leaving 1 h for cleaning and weighing the mice. Each cost schedule was applied for 4 days. The experiment was run in two replications to increase the sample size, with 17 and 23 mice respectively in each phase and with each genotype represented. The data from both studies were similar and were combined for analysis

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40 Procedures Expe riment 1: Food Demand Analysis in MCR Knockout Mice A total of 40 nave mice of MC4R/ (N=12; 6 females, 6 males), MC3R / (N=8; 3 females, 5 males), DKO (N=11; 7 females, 4 males) and WT (N=9; 3 females, 6 males) littermates were used. Mice were 49 mo of age during these tests. The WT group included both MC3R +/+ and MC4R +/+, but no difference was evident so their data were combined. No consistent differences in meal patterns or intakes were found between males and females, so these data were not analyz ed for gender differences. The experiment was run in two replications to increase the sample size, with 17 and 23 mice respectively in each phase and with each genotype represented. The data from both studies were similar and were combined for analysis, as planned. Prior to the main protocol, to habituate mice to the chambers and the novel pellets, a 4day training period was applied in which pellets were delivered contingent upon FUP2 (each pellet delivery is contingent on the completion of two responses) with the same nose poke operant. Mice were deemed to have learned the contingency when they earned enough pellets over 23 h to maintain their body weight, typically within 2 days (Atalayer and Rowland, 2009). No food deprivation was imposed at any time during the experiment. During the main protocols, mice lived in test chambers for 23 h per day and obtained a 20 mg pellet when they completed n nose poke responses as specified by a programmed FUP n schedule of reinforcement. After training, mice were tested using an increasing series of unit costs (FUP 2, 5, 10, 25, 50) with 4 days at each unit price. At the end of the study in the second batch of mice (N=23), and after about 1 week of free feeding to regain any lost weight, mice were anesthetized (Sleepaway ) and the

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41 subcutaneous, visceral and pericardial (thoracic) white adipose tissue depots were dissected and weighed. Experiment 2: Food Demand analysis in Diet Induced Obese Mice A total of 16 nave B6 male mice were randomly divided in two dietary groups of 8, high fat (HF) and low fat (LF). The diets were purchased from Research Diets (New Brunswick, NJ): HF #D12492, 34.9% fat and 60% energy; LF # D01060501, 4.3% fat and 10% energy. For the next 9 weeks, mice received the designated diet in their home ca ges. Throughout this period HF fed mice gradually gained more weight than LF fed mice and displayed signif icantly higher body weight and distinct DIO. After this phase, all mice were run in the operant test chambers where they worked for 20 mg pellets (lo w fat) pellets for the same training period and main protocol as in Experiment 1. Experiment 3: Behavioral E ffects of Exogenous CCK8 in MCR Knockout Mice Three CCK studies were performed. In the first, WT, MC4R / and DKO were included. In the second, MC4R / MC3R / and corresponding WT from two different age ranges were tested. In the third, after at least one week of free chow intake, mice from the first study were used to study induction of c Fos immunoreactivity. In the first study, mice were 49 mo of age at the time of testing. Food intake was measured in 30 min test sessions using a dessert protocol in which the mice were adapted to receive a highly preferred food treat at the same time each day. The dietary treats were Fruit Crunchies (Bi o Serv; No. F05798; 52% carbohydrate, 20% protein, 6% fat by weight, with carbohydrate fraction ~75% monoand disaccharides), 190mg spherical pellets about same macronutrient composition as chow and available in grape, apple, and orange flavors (Rowland and Robertson, 2005). During training, the mice received 10 pellets of mixed flavors in a 10 ml glass beaker, hung inside the cage from

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42 a metal stirrup. On the first day, the beaker was left in place for as long as needed for robust intake to occur. Ther eafter, the time per day was tapered rapidly to 30 min. We found that most (~90%) WT mic e readily consumed Crunchies (Rowland and Robertson, 2005) whereas ~25% of MC4R / and DKO mice did not eat this novel food even af ter many days of presentation. Thus, sufficient mice were initially trained so that at least 6 reliable eaters were obtained for each genotype. Test intakes were recorded as the number of Crunchies eaten; bedding was searched for half pellets, but these or smaller crumbs were minimal: most eating of this size of food item seems to be largely all or none. After about 1 week adaptation to the dessert regimen, as well as to handling, a baseline was established by recording intake for 3 consecutive days. On test days, mice were injected subcutane ously at about 1300 h with either the vehicle (saline, .02 ml/10 g body weight) or CCK (6 g/kg). These doses were chosen on the basis of our previous work (Vaughan et al ., 2005). The procedure was repeated 57 days later, but with the alternate injection. Half the mice received saline first and half received CCK. There was no order effect, so dat a were combined for analysis. Intakes after vehicle or peptide were expressed as % of the baseline for each individual, and data were analyzed usi ng oneway ANOVA and post hoc Tukey tests ( P<0.05). CCK (as the sulfated octapeptide) was purchased from Sig ma Chemical Co (St Louis, MO). Intake after drug was also analyzed as % of the intake by each individual after vehicle injection. No food deprivation was imposed in this protocol. In the second study, MC3R / and MC4R / mice of two age ranges were used, young adults (34 mo) and middleaged (78 mo). Littermate MC3R +/+ and MC4R +/+

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43 were tested as controls as far as possible, but no middleaged MC4R +/+ were available. To address in part weight differences between groups, two doses of CCK (3 and 6 g/kg) were tested. Further, to be consistent with studies from other laboratories (Fan et al ., 2004; Blevins et al ., 2008), we used an overnight (18 h) food deprivation protocol with a standard chow pell et presented as the test food. As before, injections were made 5 min before food was presented and intake was measured after 30 min. Three tests were conducted at 1 week intervals, with each mouse receiving vehicle, 3 or 6 g /kg CCK, in random order. Data will be presented as absolute intakes and expressed as % of the individual int akes on the vehicle test day. These data were subjected to ANOVA and post hoc Tukey tests, with a significance criterion of P <0.05. Experiment 4 : C Fos Inducing Effects of CCK 8 in MCR Knockout Mice U sing the mice that had been in the first study but now 12 mo older, we examined induction of c Fos immunoreactivity by CCK. This was performed in several batches, with all genotypes represented in each batch; no differences were found as a result of batch, so data were combined, as planned. On each test day, chow pellets were removed from the home cages 12 h beforehand to prevent recent spontaneous meals. Mice were injected with CCK (6 g/kg) or saline vehicle and 1 hr later were anesthetized (Sleepaway, 1 ml/kg; Fort Dodge), and perfused transcardially with heparinized saline followed by paraformaldehyde. Brains were removed, stored in paraformaldehyde overnight, and then sliced by vibratome into coronal 75 m sections at the levels of the paraventricular nucleus (PVN) of the hypothalamus and the area postrema (AP) and adjacent nucleus of the tractus solitarius (NTS). Sections were then incubated with primary (c Fos 4 polyclonal; Santa Cruz) and s econdary (biotinylated goat anti rabbit IgG, Zymed) antibodies, and the reaction product visualized using ABC

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44 (Vector Labs), as we have described in detail elsewhere (Li and Rowland, 1993). Sections were mounted on slides and c Fos positive cells in regions of interest were examined under a microscope and counted manually by two observers; to ensure a blind procedure, animal numbers were obscured during this phase. The counts from the two observers showed <5% variation and were averaged. Data Analyses A repeated measures analysis of variance (ANOVA; SPSS) was used with genotype as a betweensubject variable and the increasing schedules as a withinsubject variable. Followup Tukey tests were employed to assess specific differences. For acute feeding test s and the fat pad analysis, oneway ANOVA (SPSS) and post hoc Tukey tests (where appropriate) were used to examine the genotype effects on the % suppression of food intake or the dissected body fat content. For the cFos studies, the numbers of Fos stained cells in each region of treated mice were c ompared across genotypes using oneway ANOVAs and Tukey post hoc contrasts. Results Experiment 1: Food Demand Analysis in MCR Knockout Mice The demand for food as a function of unit price is shown in Figure 3 1 There were differences in intake as a function of unit price ( P<0.001) with intake declining as unit price increased. There was a significant main effect of genotype ( P=0.006) and a genotype x unit price interaction ( Ps<0.001). As is evident from panel A of Figure 3 1, at the lower costs (FUP2, FUP5, FUP10) the MC4R / and the DKO mice ate more food per day than either MC3R / or WT mice ( P s< 0.001). However, as unit cost increased further, the intakes of the MC4R / and DKO declined more sharply than thos e of MC3R / or WT, so that at the highest price (FUP50) the former groups consumed the

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45 least ( P<0.05). P anel B of Figure 3 1 expresses the intake of each group as % of their intake at the lowest cost (FUP2); intakes at the highest cost (FUP50) had fallen by 4050% in MC3R / and WT, while intakes of the MC4R / and DKO had fallen by ~70%. The meal parameters are shown in Figure 3 2. The number of meals ( panel A ) taken per day did not differ as a function of genotype but declined as unit prices increased ( P s<0.001 for all 4 genotypes). Mean meal size ( panel B ) was comparable in MC3R / and WT and did not decline significantly across the unit cost series in these genotypes. In contrast, the mean meal size of the MC4R / and DKO dropped rapidly as unit cost increased ( Ps<0.001). Thus, the decline in total food intake as unit prices increased (Figure 3 1) was the result of fewer and smaller meals in MC4R / and DKO, whereas in WT and MC3/ it was a result of fewer meals only (Figure 3 2). Body weights and fat pads. The body weights of these animals during the operant protocol are shown in Figure 3 3. P anel A shows the individual weights at the beginning and end of the cost series, and panel B shows the mean group cumulative changes in weight at each cost. DKO animals were uniformly obese at the start of the study, the MC4R / were variably obese, and MC3R / and WT had comparably low weights. All animals lost some weight during the FUP series, but the greatest absolute loss was in the MC4R / and DKO animals ( F igure 33B ). One way ANOVAs on the cumulative changes were significant ( Ps<0.001) at FUP50. Interestingly, the greatest individual weight losses were not consistently associated with the highest initial weights ( Figure 33A ). These differences in body weig ht were reflected in dissected body fat (Table 31 and Figure 34 ). The mean fat from the combined subcutaneous and visceral depots

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46 rose from a mean of 3% body weight in WT to 11.6% in MC4R / and 21.7% in DKO. The thoracic cavity also contained visible fat in MC4R / and DKO: while not a large mass compared with the other pads, thoracic fat mass (<0.02 g in WT and MC3R / ) was 0.06 0 .01 g (mean SEM ) in MC4R / and 0.13 0 .02 g in DKO. Experiment 2: Food Demand analysis in Diet Induced Obese Mice Prior to the diet regimen, mice designated for HF and LF groups had similar weights (24.2 0.4 and 24.2 0.3 g respectively, mean SEM ). At the end of 9 weeks, the HF and LF groups weighed 45.6 0.7 g and 35.3 1.5 g ( P<0.001). The demand for food as a function of uni t cost is shown in Figure 3 5 A There were differences in intake as a function of unit price ( P<0.001) with intake declining as unit price increased. The only significant difference between the DIO (previously HF) and LF mice in terms of total pellets earned per day was during FUP50 ( P<0.05) as DIO mice received less pellets than LF mice. The meal parameters are also shown in Figure 3 5 The number of meals ( Figure 35 B ) taken per day did not differ as a function of phenotype but declined as the unit prices increased ( Ps<0.001 for both phenotypes). Mean meal size ( Figure 35 C ) did not change significantly across the unit cost series in HF mice. In contrast, the mean meal size of the LF mice increased as unit cost increased ( P <0.05). Experiment 3: Behavioral Effects of Exogenous CCK 8 in MCR Knockout Mice In the first study, using the dessert protocol, mean intake of Crunchies in the baseline period did not dif fer as a function of genotype. The mean ( SEM ) numbers of Crunchies eaten were: WT 8. 5 .4, MC4R / 7.1 .7, DKO 7.2 .6. After vehicle injection, mean intakes of the WT and MC4R / groups were within 5% of their baseline, but the intake of the DKO group was reduced significantly both from their baseline and

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47 from the absolute intake of the other two groups ( Ps<0.05 ) to 4.4 .5 and 5.4 .8 Crunchies (64% of their baseline). Intakes following peptide injections were thus analyzed both as % baseline and % vehicle (Table 3 2 ). The anorectic effect of CCK was at least as great in the MC4R / and DKO mice as in W T. CCK reduced food intake from vehicle and baseline in each group ( Ps<0.05). While the suppression of intake in the MC4R / and DKO groups was slightly greater than for the WT, only the DKO was significant ( P<0.05) relative to WT and only w hen expressed as % baseline. The deprivationchow protocol was used in the second study, and the results are shown in Table 33 and Figure 3 6 Table 33 shows the absolute intakes, which differed significantly across groups ( P<0.001). Post hoc tests showed that the int ake of young (34 mo) and middleaged (1011 mo) MC4R / differed si gnificantly ( Ps<0.02) from each other, but neither differed from the corresponding MC4R +/+ (for which onl y young mice were available). Two way ANOVA showed that intakes were lower in MC3R / than in MC3R +/+ groups, and were lower in y oung than middleaged cohorts. The higher dose of 6 g/kg suppressed intake relative to the vehicle condition in all groups; the lower dose of 3 g/kg suppressed in all groups except for young MC3R / and MC3R + /+ Mean intakes as % vehicle are shown in Figure 35A At the lower dose, the young MC3R / and MC3R +/+ d iffered from all other groups (oneway ANOVA; Ps<0.01) while at the higher dose there was no group difference in % suppression. Since the MC4R / gr oups weighed about 2x more than the MC 4R +/+ control (Table 3 3 ), for a given dose they received about twice the absolute amount of CCK. Thus, approximately the same absolute amounts of CCK were received by the MC4R +/+ after

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48 6 g/kg and MC4R / after 3 g/ kg, respectively; the % intakes were comparable in these conditions (Figure 3 6B ). Experiment 4: C Fos Inducing Effects of CCK 8 in MCR Knockout Mice The effect of CCK 8 on Fos ir in PVN is shown in Table 3 4. Fos was induced strongly in WT, MC4R / and D KO mice, but less so in MC3R / ( P<0.05), with this latter group not significantly different from saline treated controls. This dose of CCK induced very strong Fos in the AP and NTS of all 4 genotypes, and accurate counting was not possible. Discussion Ex periment 1: Food Demand Analysis in MCR Knockout Mice MC4R / and DKO mice were obese and hyperphagic relative to WT, but only under the specific economic conditions of low cost for food. The elastic nature of their demand functions for food was not replic ated in DIO B6 mice. The hyperphagia of MC4R / and DKO mice, when present, was due mainly to large meal size. This suggests that the disruption of the MC4R gene, and independently of obesity, is sufficient to disrupt normal physiological controls of and/or environmental effects on food intake and meal size. These findings in MC4R / mice are consistent with but expand considerably on previous reports from our laboratory. Thus, using a FUP3 schedule, we found that MC4R / ate ~33% more food per day than WT, and with larger meal size (Vaughan et al 2006b ). Hyperphagia and increased meal size was also found using progressive ratio schedules of pellet cost; under progressive schedules, MC4R / paid a higher average cost per pellet than WT (7.7 vs 5.6 lever presses under one condition, see Vaughan et al ., 2006 b Table 2). However, under a procurement consummator y dual

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49 cost foraging protocol (Vaughan et al ., 2005 ), intake of MC4R / was identical to that of WT and MC3R / across a range of procurement costs but l ow (FUP 5,10) per pellet costs. Together with the present data, these findings suggest that MC4R / are hyperphagic when proximate or unit costs per pellet are les s than ~10 responses. If unit costs increase further, including when part of the cost is di stal or approach, then the mice are no longer hyperphagic and may eat less than WT. As a result they lose weight (see Vaughan et al ., 2005 Fig ure 3) but that weight loss does not appear to influence their fooddirected operant behavior. In this regard, the first meal is smaller in MC4R / after food deprivation (Blevins et al ., 2008 ) and from Table 3 3 it can be derived that young MC4R +/+ ate 26.2 g/kg body weight/30 min compared with 11.8 g/kg in MC4R / It follows that MC4R / may be relatively insensitive to afferent information about adiposity, consistent with th e observation by Marsh et al (1999) that MC4R / mice did not show anorexia to either peripheral or c entral injection of leptin. This is to our knowledge the first detailed report of intake and feeding patterns in DKO, but o ur data agree with Chen et al (2000) that they are significantly more obese than MC4R / a difference that is due almost exclusively to more adipose tissue (Table 3 1: note that the fat pads dissected do not account for all body fat). A t 26 weeks of age, Chen et al (2000) reported that female and male DKOs were 27 and 13% heavier, respectively, relative to MC4R / counterparts. The sex difference in weight in MC4R / was no longer evident in DKO. Our body weight data cl osely confi rm those observations (Fig ure 3 3 ). We also noted considerable pericardial fat in the DKO mice. Experiment 2: Food Demand analysis in Diet Induced Obese Mice DIO and control mice in Experiment 2 showed generally similar, although flatter demand functions than the WT in Experiment 1. Interestingly, despite their obese state

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50 at the start of the FUP series, the DIO mice showed the same total intake and parameter s as the non obese ( low fat, LF) group. Note that for both groups, this involved a change to a common grainbased, low carbohydrate diet, and so it suggests that the diet and the economy determined intake more than the weight of the animal. Further, the DIO mice lost weight as FUP increased but there was no tendency to eat less than LF except at the highest cost. Clearly, the demand curves in DIO mice are inelastic or flat compared with the steep functions in MC4R / and DKO. Experiment 3: Behavioral Effects of Exogenous CCK 8 in MCR Knockout Mice Electrophysiological, biochemical, and behavior al studies suggest that activation of MC4Rs in the NTS/AP and/or dorsal motor nucleus of the vagus potentiates the effect of vagally mediated satiation signals such as CCK ( Appleyard et al ., 2005; Fan et al ., 2004; Wan et al ., 2008; Blevins and Baskin, 2010). This is consistent with our demonstration of a large meal phenotype in MC4R / and DKO at low food costs. It follows that the satiating effect of CCK 8 should be reduced in MC4R / and DKO, but probably not in MC3R / relative to WT. That result has been reported in MC4R / in two publications (Fan et al ., 2004; Blevins et al ., 2008 ) both using a food deprivation and chow protocol similar to our own. In contrast, using a 12 hr fast and a liquid diet, we found that the intake in MC4R / was suppressed by various doses of CCK 8 at l east as effectively as in WT (Vaughan et al ., 2006 a ). In the study by Fan et al (2004), mice were 9 wk of age and so obesity presumably had not fully developed. The dose they used (3 nmol/kg, close to our lower dose) reduced 3 0 min food intake in MC4R / by ~40% relative to saline, less than in WT (~70%) and not significant. Their feeding test lasted 3 hr, and at later times the suppressant effect of CCK had completely disappeared in MC4R / but not in the WT.

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51 Thus, at the s ho rt interval we used (30 min). Fans MC4R / were in fact partially responsive to CCK. Blevins et al (2008) reported a doseresponse analysis, with mice receiving 0.75, 2.5 or 7.5 nmol CCK 8/kg lean body mass, and a 30 min food intake test. They found a suppression of food intake in WT at all 3 doses, but in MC4R / only the highest dose produced a significant reduction relative to vehicle injection. However, at the highest dose, the % suppressions were not markedly different in WT and MC4R / (43 and 36%, respectively). Our doses were not based on lean body mass, but Blevins (personal communication, June 2009) graciously provided data that the lean body mass of his WT and MC4R / mice averaged 78% and 63% of body weight, respectively. Thus, the highest d ose that his mice received transform to 5.85 and 4.72 nmol/kg (~5.5 and 4.5 g/kg) in WT and MC4R / respectively, a dose that is intermediate between our two doses. In our study, there is no evidence in either young or middleaged MC4R / for reduc ed sensitivity to CCK (Fig ure 36 ). This was also evident in our nondeprived (Crunchies) protocol (Table 3 2), which showed that DKO were also responsive to CCK 8. None of the published or present protocols indicate that MC4R / are completely unresponsive to exogenous CCK, but in some protocols there may be a reduced responsiveness. Consistent with this, we were unable to find differences in Fos expression in AP/NTS of either MC 4R / or DKO compared with WT. Finally, in a study in which we stimulated endogeno us release of CCK and other meal related factors, we found that oral preloads suppressed subsequent intake equivalently in WT and MC4R / (Vaughan et al ., 200 6 a ). The relative sparseness of MC3R in the hindbrain, as well as the above results in DKO, would lead us to expect that MC3R / mice would show normal satiation

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52 responses to CCK 8. This was i n fact reported by Fan et al (2004) using mice 510 mo of age. However, our results revealed an unexpected insensitivity to CCK 8 (Figure 3 6 ). In the young cohorts, both the MC3R +/+ and / genotypes were unresponsive to the lower dose of CCK 8 and / showed a smaller effect than +/+ at the higher dose. In middle aged cohorts, the low dose was comparably effective in MC3R +/+ and / but again at the higher dose the / were less responsive. In a preliminary study, using even younger mice (~2 mo) and a higher dose of CCK 8 (9 g/kg), we found complete insensitivity in MC3R/ Experiment 4: C Fos Inducing Effects of CCK 8 in MCR Knockout Mice The Fos ir data g ive no indication for a reduced CCK activation in AP/NTS in any genotype relative to WT, but they do suggest a reduced activation in PVN of MC3R/ If peripheral CCK 8 is activating the PVN exclusively via the NTS/AP as a relay, this result would suggest that the relay has reduced effect in MC3R / either because the synaptic transmission is reduced or the target neurons are tonically inhibited, for example because inhibitory POMC arcuate afferents have increased firing rate due to loss of functional MC3R autoreceptors. If this tonic inhibitory signal were carried through MC4Rs, then DKO mice would be insensitive to any increase in arcuate firing due to the MC3R / deletion. In any event, the reduced sensitivity of MC3R / to both CCK anorexia and cFos expression in PVN, at least warrant the speculation of a functional connection. This hypothesis requires further investigation, as well as the corollary that overexpression of MC3R might increase sensitivity to CCK. Chapter D iscussion In sum, our data s how more evidence for insensitivity to CCK 8 in MC3R / than in MC4R / and that this may be agedependent. Thus, putative insensitivity to CCK is not

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53 a viable explanation for the large meal phenotype in MC4R / because MC3R / should show comparably incr eased meal size, but they do not. In addition, the elastic demand functions of the genetically obese mice indicated that a lthough MC4R signals hunger, the phenotypic expression of its deficiency was affected by the economic st ructure of the food environment. This was an unexpected finding because in human st udies it has been shown that the incentive value of food in obese humans were reported to be higher and that these individuals were more willing to pay higher prices for food (Temple et al ., 2009; Saele ns and Epstein, 1996). This was in agreement with our results here only when the price was low. As the prices increased, obese mice decreased their efforts to get the food. In an early study by Hamilton and Brobeck (1964), it has been shown that although t o a lesser extent than rats, ve ntromedial hypothalamus lesion ed monkeys decreased their efforts as the fixed unit price increases comparably more than the sham lesioned monkeys. Ela stic demand in MC4R / and DKO was also shown to be more reflective of thei r meal size s and this was not due to obesity per se as diet induced obese mice had significantly flatter demand functions compared to the genetically obese mice The literature on MC3R / mice is rather inconsistent. Chen et al (2000) had reported modestl y lower free food intake in MC3R / relative to WT and were suggested to be highly susceptible to diet induced obesity when exposed to a highfat diet. Female MC3R / mice were reported to consume no rmal amounts of food, yet gaining more body weight than either WT or MC3R / + This has not been generalized to male mice in their study. O ne study suggests that MC3R / mice are unusually susceptible to diet induced obesity when fed with high fat food (Cummings and Schwartz, 2000) This was

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54 somewhat counterintui tive to the literature on MC3R / as it has been reported by many that these mice are hypoactive and strictly maintain their weight similar to a WT mouse. Thus, we replicated this idea by feeding MC3R / with high fat content food to see if their weight increase will be any different than that of WT mice. Our data, from a small number of animals (un reported), was not consistent with this view however; there was some tendency for higher fat accumulation in MC3R / in subcutaneous depots We expected male MC3R / mice to accumulate more adipose tissue when fed with HF diet than WT mice. We used MC3R / (N=4) and WT (N=4) mice f or this protocol. Same HF diet in E xperiment 2 was used here. For the next 11 weeks, mice received the designated diet in their home c ages. Prior to the diet regimen, both genotypes had similar weights (WT; 24. 51.0 and MC3R / ; 25.81.6 g, meanSE M ). At the end of 11 weeks, there was not a significant difference between their weights (WT; 34. 62.3 and MC3R / ; 34.32.6 g, meanSE M ). Th ere was also no significant differenc e in the fat pad weight in MC3R / mice compared to the WTs. The l arge meal size phenotype in MC4R/ and DKO, appeared not to be rel ated to compromised CCK action as we found no evidence for a diminished action of CC K in MC4R / and DKO when compared to WT mice. This was shown in various doses and also with manipulating various methodological factors in order to generalize the findings and to match it with previous reports. The data from cFos studies examining the ef fects of CCK supported the behavioral finding as the number of c Fos induced cells were comparable in DKO, MC4R/ as in WT, thus, insensitivity to CCK is concluded to not to be a viable explanation for the large meal phenotype in MC4R / and DKOs. I n MC 3R / mice however, c Fos induced cells were markedly less in PVN which was an

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55 unexpected finding as MC3R have not been reported to be located in this structure. In a recent study (unreported data) we found enough evidence to believe that t he normal activa tion in PVN is presumably mediated by MC3Rs, and whereas the normal activation i n AP is thr ough MC4Rs. In this MTII study we tested the acute anorectic effect of centrally administered MTII (a MC3 and MC4R agonist), and subsequently examined the induction of Fos immunoreactivity in select brain regions. As expected, MTII had no anorectic effect in DKO mice, but was anorectic in MC3R / MC4R / and WT MTII decreasing food intake in both MC3R / and MC4R / suggests that stimulation of either receptor is sufficient enough to produce anorexia. Fos ir was induced strongly by MTII in PVN and AP in WT mice. In MC3R / the induction in PVN was minimal and the effect in AP was as strong as in WT, whereas the revers e pattern was seen in MC4R / C Fos was induced strongly in AP in all genotypes, but was reduced in PVN of MC3R / relative to other genotypes. These results from this MT II cFos study suggest a double dis sociation of region of action. Fos ir was absent in the PVN of MC3R/ but was normal in MC4R / suggesting that MC3Rs alone underlie the induction of Fos in the PVN. In contrast, in the AP, only the MC4R / mice had reduced Fos ir relative to WT, suggesting that MC4Rs alone underlie the inducti on of Fos induction in the AP.

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56 Figure 31. Daily (23 h) food intake, A) shown as number of 20mg pellets eaten, in WT, MC3R / MC4R / and DKO mice exposed to increasing fixed unit prices (nose pokes) per pellet. Each datum is the average of 4 days at each price st ep and 812 mice per genotype. B) The panel shows the intake in absolute terms; the right panel shows intake expressed relative to intake at the lowest cost. B A

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57 Figure 3 2. Intake data from Figure 3 1 analyzed for A) n umber of meals and B) mean meal size B A

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58 F igure 3 3. Weight changes of mice during the demand series. A) W eights of individual mice (different symbols) of each genotype at the beginning of the study (first 2 days of FUP2) and at the end of the study (last 2 days of FUP50). The lines thus join the start a nd end weights for each mouse. B) Weight loss as group means, the cumulative body weight changes from initial by the end of each demand schedule. B A

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59 Figure 34. Scatterplots showing fat pad amounts. A) S ubcutaneous and B) visceral fat pad weights in individual WT, MC3R / MC4R / and DKO mice as a function of body weight. Fat pad weight was strongly correlated with body weight, but note that in DKO the subcutaneous fat is more consistently elevated than visceral fat. A B

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60 Fig ure 3 5 A) Daily (23 h) food intake, as number of 20mg pellets eaten, in DIO (HF) and LF mice exposed to increasing fixed unit prices (nose pokes) per pellet. Each datum is the average of 4 days at each price step. Intake data were further analyzed for B) number of meals and C) mean meal size. B C A

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61 Figure 3 6 Effect of CCK 8 on intakes of chow in 30 min tests after 18 h food deprivation. Intakes are expressed as mean % intake after injection of vehicle ; error bars are not shown for clarity, but average about 10% of the means. A) D ata from young adult (Y: 34 mo) and middleaged (M: 78 mo) MC 3R / and MC 3R+/+ mice. B) D ata from Y and M (1011 mo) MC 4R / and Y MC 4R+/+ mice. The numbers of mice per group were 410 as shown in Table 33 B B A

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62 Table 3 1. Fat content of mice from E xperiment 1 Genotype (N) Body wt (g) Dissected fat (g) Fat (% body wt) WT (4) 25.3 + .9 a 0.8 + 0.1 a 3.0 + .4 a MC3R / (4) 20.6 + 1.9 a 1.1 + .5 a 4.9 + 1.7 a MC4R / (8) 40.4 + 9.8 b 4.9 + .9 a 11.5 + 1.7 b DKO (7) 50.4 + 2.2 c 11.1 + 1.7 b 21.5 + 2.4 c Shown are mean SEM (N in parentheses) Statistical difference ( P<0.05) between groups is indicated by different superscripts.

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63 Table 32 Effect of CCK 8 (6 g/kg) on intake of crunchies by undeprived mice Test Intake (% baseline) Test Intake (% vehicle) WT (5) 49 5 46 5 MC4R / (5) 28 9 25 8 DKO (6) 29 4* 47 7 Shown are mean SEM (N in parentheses) P<0.05, vs corresponding WT datum.

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64 Tab le 3 3 Effect of CCK 8 on chow intake by deprived mice Genotype Age (mo) N Body weight (g) Intake after vehicle (g) Intake after CCK (3 g/kg) Intake after CCK (6 g/kg) MC3R / 3 4 5 21.9 1.2 0.53 .04 0.53 .07 0.38 0 .13 MC3R / 7 8 5 26.0 0.9 0.72 .09 0.48 0 .1 0.47 .06 MC3R+/+ 3 4 4 18.9 1.2 0.69 .06 0.68+.07 0.35 0 11 MC3R+/+ 7 8 10 23.6 0 8 0.81 .04 0.52 .05 0.35 .06 MC4R / 3 4 7 42.5 1.2 0.50 .05 0.40 .05 0.24 .03 MC4R / 10 11 5 57.1 4.6 0.76 .03 0.57 .08 0.28 .03 MC4R+/+ 3 4 7 25.6 0.6 0.67 .0 4 0.56 .05 0.41 .02 Shown are mean SEM Food intake in grams. Body weights are mean of 3 weeks, predeprivation.

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65 Table 34. Fos positive cells in paraventricular hypothalamus following CCK 8 (6 g/kg) Group (N) Fos positive cells WT (6) 235 50* MC3R / (6) 122 26 MC4R / (4) 269 18* DKO (6) 218 38* Shown are mean SEM (N in parentheses). P<0.05 differs from salinetreated controls (36 5, N =4).

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66 CHAPTER 4 NEGATIVE ENERGY BALANCE AND FOOD DEMAND Introduction Although there is a great deal of evidence in the literature showing the effects of exercise as a treatment for obesity and for maintaining a healthier life style (Colbert et al ., 2006 ; Melanson et al ., 2009 ) the econom ic structure of the food environment that humans and animals live in has n ot been taken into account. Some studies suggest that obesity is to a great extent an economic issue (Drewnowski, 2004). In addition in our studies above we have shown that environme nt and genetics both play roles on the eating behavior and obesity, thus when examining the health benefits of exercise on obesity and fat accumulation one should also include the economic setting of the food environment. In the first experiment (E xperiment 5) we used an animal model of voluntary running wheel activity and compared the demand functions and body weights of running and nonrunning (sedentary) mice. The s econd experiment (E xperiment 6) included a foraging paradigm in which we employed two types of f ood costs that were defined in C hapter 1. Thus the food was c ontingent upon running wheel activity that was used as the approach cost (AC) and a concurrent fixed unit price ( FUP ) with a lever press response. We compared the effects of running wheel activity as opposed to nose poke response as the AC requirement. We also included a control group under a low cost FUP5 with a nose poke operant. The aim of this experiment was to investigate whether mice with high workloads of wheel running activity would exhibit different demand fu n ctions, as they may pay higher cos ts for food where the running is the effort

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67 compared to nose poke as a common but less effortful type of operant behavior (Atalayer and Rowland, 2009; David et al., 2001) Subjects and Housing A total of 45 nave male albino (CD1; Harlan, Indianapolis IN) were used in the protocols. CD1 mice (2 3 mo) were chosen because they are of mixed origin and have food intake near the middle of a range of strains of mice (Bachmanov et al ., 2002). Between the receipt and the experiments, mice were kept under the conditions explained in general methods in C hapter 2. During the experiments, mice lived in test chambers for 23 h per day. The mice were weighed daily and kept in empty holding cages during a 1 h cle aning period without food, although water was available. General Methods B ehavior al test c hambers were used for testing. In addition to the nose poke operants, 8 chambers were additionally fitted with an external running wheel in the test chambers as desc ribed in the general methods section of C hapter 2. M ice had free access to the wheels Data were accumulated in 15 min time bins throughout each 23 h period. Procedure Experiment 5 : Voluntary Running Wheel Activity and Food Demand A total of 15 CD1 m ice were divided randomly in two groups, one sedentary (N =8) and the other with access to running wheels (runners, N =7). Prior to the main study, in order to habituate mice to the operant chambers and to the novel pellets a 1 h training period was applied with free food available in the food receptacle at no cost and, in the runner group, access to the wheel was blocked by a removable wall panel. Then, for the next 12 days and with wheel access still blocked, a FUP2 was in effect in which

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68 one food pellet was delivered contingent upon two nose poke responses. Mice were deemed to have learned the contingency when they earned enough pellets over 23 h to maintain their body weight; this typic ally occurred within 24 days. No food deprivation was imposed at any tim e during the experimental period. After this training with the response device, access to the wheels was allowed for the runner group, and all mice were exposed an incrementing series of food cost schedules each of which was in effect for 4 consecutive days. The FUP were 2, 5, 10, 2 5 and 50 nose poke responses per pellet. At the end of the food demand study, the mice were euthanized (Sleepaway) and the subcutaneous, visceral and pericardial (thoracic) white adipose tissue depots were dissected and weighed Experiment 6 : Running Wheel Activity as Food Foraging A total of 30 mice were used in this protocol which was completed in two batches. In the first part, 24 m ic e were randomly divided in three groups (Ns= 7 8). Two experimental groups (run GR and poke G R) were tested in a concurrent schedule of AC and FUP in operant chambers. The third group was a control group representing an ad libitum food intake condition in test chambers with only a low cost (FUP5) imposed upon throughout the protocol with a nose poke operant Prior to the main study, in order to habituate mice to the operant chambers and to the novel pellets a 1 h training period was applied with free food available in the food receptacle at no cost Then, for the next 1 2 days a fixed unit price of five responses (FUP5) was in effect in which a food pellet was delivered contingent upon five responses on the designated device. For the run GR running wheel activity was designated as the approach cost (AC) response and lever press was chosen as the fi xed unit price (FUP) response. For the

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69 pokeGR, a nose poke operant was used as the AC response and lever press was also chosen as FUP response For the third group which was the ad libitum food intake simulation control (adlibGR) a nose poke operant was us ed as the FUP response. For this group no AC was applied. A nose poke operant with a low cost (FUP5) was chosen to create a minimal effort requirement for the animals. For the present purposes, this group will be representing a free food condition in an operant chamber environment. The nose poke operant was chosen for reasons that we reported previously (Atalayer and Rowland, 2009). Following separate initial trainings at FUP5 with the designated operants for each group, as the main protocol runGR was tes ted in a series of approach cost (AC 25, 50, 100, 500, 1000) with a constant FUP 5 each of which was in effect for 4 consecutive days The approach costs for the pokeGR were AC5, 10, 25, 50, 100 with the same constant FUP5. During the protocol, a fter comp letion of the designated AC on the running wheel (for runGR) or nose poke device (for pokeGR ), a cue light was illuminated above the lever operant, indicating its availability for food responding at the prevailing FUP5. In addition, a house light was illum inated concurrently at the same time and for the duration with the cue light. Subsequently whenever 15 min elapsed without a response on the lever, a meal was declared finished (as 15 min is our meal to meal interval criterion) then the lever was inactivated and the cue and the house light extinguished. Only a cue light that was lit at all times above the nose poke operant was used for the adlibGR, and the lever press operant was in active at all times for this group. Total pellets and responses were recorded every 5 min throughout 23 h sessions. A t the end of the above protocol, a second batch of running mice ( N=7 ) was added

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70 to the protocol in order to increase the sample size for runGR. Data from both batches for two runGR groups were combined in later analysis in terms of mean number of pellets, meal size, meal numbers and total daily revolutions. Data Analysis Spilled pellets found beneath the cage floor were subtracted from the total earned; in every case spillage was small (typically < 5% of the number earned). After the numbers of meals per day were determined for each mouse, the mean meal size was derived by dividing the number of total pellets by the number of meals for each day. Data were treated using a repeated measures analysis of variance (ANOVA; SPSS) with the activity level (sedentary versus runner) as a betweensubject variable and the cost schedules as a withinsu bject variable. For E xperiment 6, operant type (nose poke and running wheel activity) was used as the independent variable. Follow u p Bonferroni and/or LSD tests were employed to assess specific differences. Results Exper iment 5 : Voluntary Running Wheel Activity and Food Demand Running revolutions, m eal numbers, meal sizes and the number of pellets consumed did not differ significantl y across the 4 days that a given schedule was in force (Figure 41). Thus, to reduce the data for presentation, revolutions and meal parameters were averaged across the 4 days to give a single mean value for each mouse at each price (FUP), and group means (SEM) computed from these individual means. The numbers of pellets taken are shown as the demand functions in Figure 4 2 A and Table 41 shows the caloric energy intake in both groups. Two way ANOVA revealed that intake was significantly different between the runner and sedentary groups

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71 ( P <0.001), and the interaction between unit price and running condition was also significant ( P <0.001). The comparison between food intake of sedentary and runner mice showed significant differences at FUP10, 25 and 50 (al l P<0.05 ) and a difference in the reverse direc tion at FUP2 ( P<0.05 ). The number of pellets consumed per day by the sedentary mice declined monotonically as unit price increased while the intake of runner mice was relatively constant Thus, the f ollowing p airwise comparisons showed that at every increase in the unit price the intake of sedentary mice not running mice, decreased significantly (FUP2 compared with FUP5, 10, 25, 50; all Ps = 0.001 for all the other possible FUP comparison s; all Ps<0.05) The co rresponding meal size and frequency data are shown in Fig ure 42 B and 4 2 C R unners took consistently fewer me als than sedentary mice (Figure 4 2 B Pairwise comp arisons indicated that this was significant at each unit price condition ( P<0.05 at each FUP an d P <0.001 at FUP5) except FUP50. Thus, twoway ANOVA for meal frequency revealed a betw eensubject effect of exercise and also a main effect of unit price increases for both groups as number of meals decreased significantly as the unit price increased ( P < 0 .001 ). Meal size analysis showed r unners ate consistently larger meals than sedentary mice (Figure 42 C). Pairwise comparisons between the meal sizes of runner and sedentary mice showed that runners ate larger meals at each unit price condition ( P<0.05 at each FUP and P <0.001 at FUP10). Thus, twoway ANOVA for meal size revealed a betw eensubject effect of exercise ( P<0.0 01) and but no main effect of unit price increase as both groups consumed relatively constant meal sizes throughout the protocol.

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72 All mice ran substantial amounts, usually in excess of 5 km/day, and voluntary running wheel activity did not change as a function of unit price (Table 42 ). Nose poke responses on the other hand changed as a function of the unit price increases ( P<0.001) for bo th sedentary and runner mice (Figure 43 ). However, this was more pronounced in the running group because they consumed more pellets as indicated above. Body weights and fat pads. The average body weight of runner and sedentary groups was the same at the beginning of the experiment. As soon as wheels were available, runners lost weight and conserved a significantly lower mean body weight throughout t he whole experiment (Fig ure 44 A). All body weights, except at the beginning of the experiment, were signi ficantly different between the groups ( P s< 0.05 ). The difference in body weight at the end of the experiment was reflec ted in dissected body fat (Figure 4 4 B), and especially in subcutaneous pad (individual fat pad data not shown). Runner mice had significantly less body fat compared to sedentary mice ( P<0.05). Experiment 6 : Running Wheel Activity as Food Foraging Total daily revolutions, lever press responses, meal numbers, meal sizes and the number of pellets consumed did not differ significantly across t he 4 days that a given schedule was in force (Figure 45). Thus, to reduce the data for presentation, revolutions and meal parameters were averaged across the 4 days to give a single mean value for each mouse at each foraging cost condition (ACnFUP5) and g roup means (SEM) computed from these individual means. Table 42 shows the running wheel activity. All mice ran substantial amounts, approximately 5 km/day except the lowest foraging cost condition (AC25FUP5). P anel A

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73 of F ig ure 4 6 shows the mean number of running revolutions (the approach cost response for runGR ) and p anel B of F igure 4 6 shows the number of nose pokes (the approach cost response for pokeGR ) per day. One way ANOVA analyses showed that t he total number of approach cost (AC) responses for runGR (running wheel revolutions) and pokeGR (nose pokes) increased as the AC ratio increased ( P<0.001 and P< 0.001 respectively) For runGR mice, follow up LSD indicated that at their lowest AC condition (AC25FUP 5), mice ran significantly less than at any other cost conditions, and they ran comparable amounts for all the other AC conditions (Figure 4 6A). However, for the pokeGR, follow up LSD showed that the nose poke responses were comparable at all the cost conditions except at their highest AC condition (AC100FUP5). At this highest cost, nose poke responses increase d significantly compared to any other cost condition (Figure 46B). In addition, w hen compared as number of responses emitted throughout the protocol one way ANOVA showed that AC ratio emitt ed by the running wheel operant by runGR was significantly hig h er than the nose poke operant by the pokeGR ( P<0.001) (Figure 4 7) Note that the experimenter imposed AC ratio for runGR was higher than the AC ratio for the pokeGR. One way ANOVA showed that l ever press responses on the other hand did not differ between the two groups (runGR and pokeGR) however did change as a function of the increases in the approach cost for both runGR (Figure and 48 A ) and pokeGR mice (Figure 48 B) ( Ps<0.05) Follow up LSD indicated that runGR mice increased their lever press responses significantly at the two highest cost conditions (AC500FUP5 and AC1000FUP5) ( Ps<0.05) PokeGR increased their lever press responses at the highest cost condition (AC100FUP5) ( P=0.003) Note t hat although the approach costs

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74 increased for runGR (running wheel responses) and pokeGR (nose poke responses), the fixed unit price (lever press responses) was kept constant at FUP5. The numbers of pellets taken are shown as the demand functions in Figure 4 9 One way ANOVA revealed that intake was significantly different between the three groups ( P <0.001). Follow up LSD showed that runGR mice ate more than adlibGR and pokeGR mice and also adlibGR ate more than pokeGR mice ( Ps<0.001) The number of pellets consumed per day by the pokeGR declined towards the highest AC condition (Figure 4 9 A) while the intake of runGR was relatively constant (Figure 4 9 B) The corresponding meal frequency and size data are shown in Figure 410 and 411. One way ANOVA showed a significant main effect for groups for meal frequency analysis ( P<0.001). Followup post hoc LSD revealed that adlibGR had significantly more meals compared to runGR ( P<0.05 ) and pokeGR ( P<0.001) RunGR also took consistently more number of meals than t he pokeGR mice (P<0.001) One way ANOVA also indicated that t here was als o an effect of the AC increase on the meal frequency for only runGR ( P<0.001). P anel A of F igure 410A shows that r unGR took similar number of meals throughout the protocol except at the highest cost condition (AC1000FUP5). A followup LSD showed that at this cost, number of meals decreased significantly ( P<0.001) Panel B of F igure 410 shows that number of meals for pokeGR did not change as a function of AC ratio increase. Note that in F igure 410 B, a t the highest cost condition for pokeGR (AC100FUP5) there was a high withingroup variability. The corresponding meal size data are shown in Figure 411A and 411B. Oneway ANOVA showed a significant main effect for groups for meal frequency analysis

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75 ( P<0.0 5 ). Follow up post hoc LSD revealed that adlibGR had significantly smaller meal sizes compared to runGR ( P=0.006) and pokeGR ( P<0.05 ) RunGR and pokeGR had comparable meal sizes throughout the protocol. One way ANOVA also showed that t h ere was also an effect of the approach cost increase on the meal sizes ( P<0.001). P anel A of F igure 411 shows that runGR had similar mean meal sizes per day throughout the protocol except at the two highest cost conditions (AC500FUP5 and AC1000FUP5). Follow up post hoc LSD indicated that a t these cost conditions, runGR mice increased their mean meal sizes significantly ( P<0.001) Panel B of F igure 411 shows that pokeGR had a highly significant decrease in their mean meal sizes at th e ir hig hest cost condit ion (AC100FUP5) ( P<0.001). Note that for each parameter, data were shown in separate figures for the two groups as different x axis values were needed because the cost schedule ratios are different for the runGR and pokeGR. Body weights Table 43 shows the caloric energy intake in all three groups. One way ANOVA showed that t he average weight of all three groups did not differ significantly at the beginning of the experiment However post hoc LSD showed that runGR was slightly heavier than the adlibGR initially ( P=0.38). O neway ANOVA revealed that weight was significantly different between the groups at the end of the experiment, ( P<0.001) A follow up LSD showed that runGR and adlibGR mice had comparable weights whereas pokeGR weighed significantly less than both these groups (Figure 4 12). E xperiment 5 and 6 : Voluntary Running versus Running as Food Foraging Effort Table 42 (m) and F igure 4 13 (km) show voluntary and foraging running wheel activity from both experiments. Although voluntary running mic e seemed to run somewhat more, it was not significantly different from foraging mice One way ANOVA

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76 showed that voluntary runner mice ate significantly fewer number of pellets than the foraging mice throughout the protocol ( P= 0.001 ). However, mean meal siz es and daily number of meals did not differ between the two groups. Discussion Experiment 5 : Voluntary Running Wheel Activity and Food Demand Present work reports meal patterns of mice under negative energy balance when given different "economic" environments regarding food availability. More specifically the mice were exposed to increasing degrees of difficulty in obtaining food. The degree of difficulty was in the form of the number o f perant behaviors, nosepokes that the mice had to perform in order to receive food pellets. Supporting of our previous works in our lab, the harder the animal had to work for food the less they ate (Atalayer and Rowland, 2009, 2010a, 2010b). Running mice lost weight and in this negative energy balance, runner mice cons umed more pellets per day and ate bigger but fewer meals compared to the sedentary mice. They seemed to rapidly adjust their meal patterns and food intake in order to incorporate voluntary exercise into their daily routine in the operant chambers. Although they ate more, runner mice maintained a lower body weight throughout the protocol indicating that their increase in food intake was not enough to compensate for the extra ener gy expenditure. They also had significantly less body fat than sedentary mice, e specially in subcutaneous depots. Our results agree with studies in the literature reporting that exercising mice consume significantly more energy than nonexercising mice, yet they experience weight loss and a decrease in body fat (Vaanholt et al 2007) In our experiments no food restriction was applied at any time and food was always contingent upon some level of cost (n number of nose pokes) which generated,

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77 as in previous studies (Atalayer & Rowland 2009; Bauman, 1991; Collier, 1985; Hurs h, 1980) a ch aracteristic demand function. However, the shape of these functions which define elasticity of demand function differed between running and sedentary groups. Demand functions in runner mice were less elastic than those of sedentary mice. One may argue that because of their negative energy balance, the food commodity was more crucial for the runner mice than the sedentary mice. Thus, their demand for the food commodity was insensitive to the effects of the increases in the cost. In other words, the incentive value of the reward (food) was higher for the running mice when they were in a negative energy balance state. Incentive value is one of the factors for determining the willingness of the individual for spending effort in order to get a particular reward/r einforcement (Berridge, 1996) It has been shown that the elasticity of demand functions can be affected by manipulations of such factors (Hursh, 1984; Killeen, 1995) Although running mice expended more effort in order to maintain a higher daily food intake compared to the sedentary mice, t heir energy intake still was not enough to fully compensate the energy loss resulted from voluntary running As a consequence they lost weight. H owever, they did not decrease the level of running regardless of food intak e and the associated costs. This would be consistent with the speculation that appetitive beh avior may increase running as a food foraging activit y (Vaanholt et al ., 2007). Although the running wheel activity was voluntary in this protocol, the animals ma y be increasing their running wheel activity as a foraging type of behavior each time the unit price increases. Many studies demonstrate that exercising lean mice (and rats) usually do not lose

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78 weight with wheel running since they compensate for their extr a energy expenditure by increasing their intake ( Krawczewski et al ., 2009) However, our runner mice lost weight, presumably because of a failure to increase their overall intake when the fixed unit price for food increased after a certain level This indi c ates that these mice under the experimental conditions of increasing food prices in addition to a voluntary running wheel activity may be perform ing differently than most other lean rodents given ad libitum access to food and a running wheel. Thus another control group would have been helpful to emulate an ad libitum condition for food availability With this control group, it would be possible to see the effects of voluntary running alone on the body weight change. We are currently collecting this data wi th both same weight and lighter mice. These mice are exposed to the low est cost FUP2 that is kept constant throughout the protocol to simulate an ad libitum food intake condition in the operant chambers. They are also allowed to run voluntarily as in the original protocol. This will help for the interpretation on whether the weight change in the runner and sedentary groups at the end of the experiment is the result of voluntary running activity or increase in the unit price alone or the interaction between the two. Pilot results from mice which weighed less than the voluntary running mice in the original protocol indicated that unit price increase is in fact play ing a role in the weight change in addition to the running wheel activity T hese mice in this p ilot study which were under a very low cost (FUP2) were able to maintain their body weight throughout the protocol and there was not any decrease in their weight at the end of the experiment. However, this may be initial weight depe ndent as the average ini tial weight s of the mice in the original protocol were markedly heavier than these mice in this pilot study. Thus results from the same weight

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79 group will be i mportant to resolve this issue and we are currently collecting this data. Experiment 6 : Running Wheel Activity as Food Foraging In this protocol, two different operants as the AC response were compared. Both AC responses had a concurrent lever press response requirement as the FUP in order for the animal to receive the food. One group was required to r un in order to activate a lever which, when pressed 5 times (FUP5) delivers food. T he other group was required to nose poke to activate the lever (FUP5) Results clearly indicated that with running mice were able to emit higher costs for food foraging com pared to the nose poke behavior. With the running wheel operant in order to activate a lever to receive food, mice seemed to acquire inelastic demand functions and were able to keep up with working at the higher ratios. Mice that were required to nose poke for activating the lever (pokeGR) as in our previous study (Atalayer and Rowland, 2009) had highly elastic demand functions; as the approach cost increased the demand decreased to a level where animals were not able to maintain their body weights and were in a negative energy balance. P resumably, had we gone to higher AC for pokeGR especially a further decline in demand would have occurred as it does in rats (Hursh, 1984) but we were limited in our ability to do this because of restrictions on maximal weight loss ( 15%) due to welfare considerations In our previous works (Atalayer and Rowland, 2009) we have shown that mice tested with a nose poke operant acquired slightly more pellets compared with lever press as fixed unit price responses under various reinforcement schedules in the same str ain of mice (CD1). These fi ndings agree with studies in rats comparing nose poke and lever press operants in which acquisition of the nose poke response often occurs more rapidly, but response rates in trained rats are usually comparable (David et al ., 2001; Ettenberg et al ., 1981) We previously showed

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80 that nose poke can acquire more responses than a lever press operant (Atalayer and Rowland, 2009). The p resent study shows that running wheel activity acquires even m ore number of responses than a nose poke operant so mice can bear higher cost r atio requirements compared to both a lever press and a nose poke operant. This is in agreement with the idea that running wheel activity is a more relevant activity for food for aging (Vaanholt et al ., 2007) In other words, one may argue that mice did seem to be more motivated to work for food when the foraging activity is somewhat relevant to what they would be doing in their n atural habitat. Another explanation would be that the running wheel activity may be rewarding on its own ( Belke, 1997; Belke and Wagner, 2005; Iversen, 1993) Thus, mice may be more motivated to work not only for food but also for the running activity itself. Chapter Discussion Voluntary exercise was shown to be a good stimulant of food intake in mice, even when costs are associated with food procurement. This increased intake occurs mostly t hrough the increased meal size. When food was contingent on running wheel activity, mice seemed to be able to emit higher ratios for cost for food compared to a nose poke r esponse as shown in E xperiment 6. The comparison of the running wheel activity between the E xperime nt 5 and 6 indicated that these mice ran similar distances, although voluntary mice seemed to run m ore this was not statistically significant. In contrast to the general agreement that exercise causes a negative energy balance and hence increases food intake in mice, the picture in rats is less clear. For example, Levin & DunnMeynell (2003) found that runners did not increase their chow intake or adjust their expenditure in order to compensate for their negative energy balance. They therefore lost weight but defended this lowered level of weight gain

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81 against food restriction. Exercise may reset energy homeostasis so that a lowered level of weight gain is defended (Levin & DunnMeynell, 2003; Krawczewski et al ., 2009). The commonality between rats and mice is that the adaptation in food intake does not fully offset the increased energy expenditure. Thus, in E xperiment 5 animals were motivated to nose poke more in order to eat more pellets to compensate for the energy expenditure on the running wheel (voluntary) and generate inelastic demand functions compared to the sedentary animals. This was not the cas e for the sedentary animals. Their demand functions were elastic which means that as the fixed unit price increased their food intake decreased. However, they were still heavier at the end of the protocol. In addition, when food is contingent upon the running wheel activity as the approach cost respons e, animals also show ed inelastic demand functions compared to when the nose poke was the approach cost response. For the animal, absolute approach cost may be important not only in terms of what type of a res ponse it is but also in terms of the elapsed time from the start of responding till when the food is delivered. The amount of time spent in the two different responses compared was not measured in this study, but an average of 600 nose pokes per day spent to initiate 6 meals ( Atalayer and Rowland, 2009) would very likely account for less than 2 min of total approach cost response time per meal. More detailed temporal analysis will be needed to assess further the contribution of elapsed time on meal parameters. Consistent with this analysis, time itself has been shown to be an effective cost parameter in rats (Mathis, Johnson & Collier, 1995; Collier, Johnson & Mathis, 2002) Lastly, these data give further support to the idea that foraging or appetitive cost should not be considered a unitary phenomenon (Rowland and Mathes, 2008) but

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82 instead consists of at least two phases that affect behavior quite differently. We have introduced the terms approach cost and unit price to make this distinction clear insofar as it relates to behavioral economics, but presumably there are as yet unknown underlying neural structures that support such a distinction.

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83 Figure 41 Individual data from a typical mouse to illustrate the day to day variability A) total number of pellets consumed daily, B) running revolution (runner mouse only), C) meal size, D) meal frequency. Each data point at each figure is a daily value of each parameter of a prototype mouse from each of running and sedentary groups at a FUP25 cost schedule. Data points did not differ significantly across the 4 days that a given schedule was in force for each parameter ; hence data were averaged across the 4 days for the statistical analyses.

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84 Figure 4 2 Mean ( SE M ) A) daily food intake, B) number of meals taken, and C) meal size of sedentary and runner mice from E xperiment 1plotted as a function of each FUP for 20 mg food pellets. Each data point is an average of 4 days and 8 mice. See text for statistical interpretations.

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85 Figure 43 Nose poke respons es. Mean (SEM) nose poke responses emitted. Separate lines show the running and sedentary mice. Both groups were required to emit nose poke responses to obtain food.

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86 Figure 4 4 A ) The mean ( SE M ) body weight of sedentary and wheel runni ng mice in E xperiment 5 at vari ous phases of the experiment. B) Dissected fat pads (subcutaneous + visceral + thoracic) mass, expressed relative to body weight, of sedentary and runner m ice at the end of E xperiment 5 ( P<0.01 ).

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87 Figure 45. Individual dat a from a typical mouse to illustrate the day to day variability A) t otal daily revolutions, B) lever press responses, C) total number of pellets consumed daily, D) mean meal sizes E) meal frequency. Each data point is a daily value of each parameter of a prototype mouse from running group at AC100FUP5 cost schedule. Date points did not differ significantly across the 4 days that a given schedule was in force for each parameter ; hence data were averaged across the 4 days for the statistical analyses.

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88 Figure 46. Bar plots for m ean number of responses (SEM). This figure shows A) number of running revolutions, approach cost response for runGR and B) nose pokes app roach cost response for pokeGR

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89 Fi gure 47. The number of responses emitted for both groups plotted against the pellets consumed on the y axis in a log log scale When compared as number of responses emitted throughout the protocol, oneway ANOVA showed that AC ration emitted by the running wheel operant by runGR was significantly higher than the nose poke operant by the pokeGR ( P<0.001).

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90 Figure 48 Lever press responses (FUP responses) for A) runGR and B) pokeGR.

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91 Figure 49 Mean ( SEM) number of pellets taken as the demand functions for A) runGR and B) pokeGR. The dashed line indicates the average for daily total number of pellets consumed by the adlibGR mice.

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92 Figure 410. M ean number of meals (meal frequency) taken per day ( SEM) for A) runGR and B) pokeGR. The dashed li ne indicates average daily number of meals consumed by the adlibGR mice. Each data point is averaged at every 4 days to match the timeline of the approach cost increase schedule of the two experimental groups

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93 Figure 411. Mean daily meal sizes ( SEM) fo r A) runGR and B) pokeGR. The superimposed data points are total average daily meals sizes consumed by the adlibGR mice. Each data point is averaged at every 4 days to match the timeline of the approach cost increase schedule of the two experimental groups

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94 Figure 412. Body weight change.

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95 Figure 413. Voluntary and foraging running wheel activity from both experiments

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96 Table 4 1 Total caloric energy intake at each FUP schedule i n E xperiment 5 Sedentary mice Runner mice FUP2 80.79 3.07 69.414.08 FUP5 72.14 .31 74.123.06 FUP10 64 .67 .40 74.992.05 FUP25 5 2.54 5.60 69.024.73 FUP50 40.327.74 66.715.29 Note: Shown are both mean data from 4day regimen of each schedule applied and also group meanSEM (Kcal).

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97 Table 4 2 Running distance ( m /day ) of mice in E xperiments 5 and 6 as a function of the fixed unit price (FUP) for food Experiment 5 : Voluntary running Experiment 6 : Foraging running FUP2 39101163 AC25 4359 643 FUP5 80152111 AC50 5337 721 FUP10 64191575 AC100 5443 733 FUP25 5764862 AC500 5478 637 FUP50 62551028 AC1000 6287 442 Note: Shown are group mean SEM There were no signific ant differences between FUP s (E xperiment 5) and ACs (E xperiment 6) within a stud y

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98 Table 43. Total calori c energy intake at each schedule in E xperiment 6 pokeGR runGR adlibGR AC5FUP5 65.672.6 7 AC25FUP5 82.58 2.16 AC10FUP5 66.593.61 AC50FUP5 83.04 1.68 AC25FUP5 61.393.69 AC100FUP5 81.37 1.36 FUP5 70.92.3 AC50FUP5 59.891.55 AC500FUP5 75.16 3.20 A C100FUP5 12.935.44 AC1000FUP5 79.98 0.71 Note: Shown are group means SEM (Kcal).

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99 CHAPTER 5 GENERAL DISCUSSION Some of these findings are new, some support and some contradict previous work. One of the main findings of this study was that although MC4R s signal hunger, the phenotypic expression of its deficiency can be affected by the economic structure of the food environment The high elastic ity in demand functions for food that have been sho wn in MC4R/ and DKO compared to WT and MC3R / mice was caused by the changes in meal size rather than number of meals consumed per day. The diet induced obesity protocol showed that this effect was not due to obesity per se but presumably more dominantl y due to the geneti c deletion of the MCR receptors. However even though the effect of obesity was found to be minimal compared to the effects of genetic deletion of MCRs and resultant metabolic deficiencies on the demand function differences between lean a nd obese mice, there was still a significant effect of the obesity per se when the cos t for food was too high. At this high est FUP condition, diet induced obese mice decrease d their food intake significantly compared to the lean mice even though their dema nd for food was very similar under costs lower than FUP50. Thus, although it was more pronounced in genetically obese mice, it is still true to argue that the elasticity of the demand function of diet induced obese mice is different than the lean mice. The positive energy balance state of the obese mice resulted in highly elastic demand functions as opposed to the negative energy balance state of the running mice. In the latter case, mice had inelastic demand functions that are resistant to the changes in t he cost unlike the elastic demand functions that are very sensitive to the changes in the price. Thus, negative energy balance resulted in inelastic food demand. O ne

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100 argument would be that energy balance is a n important factor determining how much animals are motivated to work for food. It would be particularly interesting to know what would be the demand functions when animals are in both conditions of negative and positive energy balance. We are currently investiga ting whether the effects of exercise on food demand vary between DIO and le an mice, and including both positive and negative energy balance in the same design. The initial data indicate that r unning did not accelerate, and may have slowed, weight loss of DIO mice upon return to a low fat diet. I n this pilot study mice were divided randomly i n four groups, as sedentary highfat (SEDHF), sedentary low fat (SEDLF), runner highfat (RUNHF) a nd runner low fat (RUNLF). HF diet groups were made fat previously by HF diet and their weight was comparable t o our genetic obesity models (Atalayer et al ., 2010b ). These mice were then replaced in the behavioral chambers for the analysis of the demand functions with or without the running wheels based on their assigned groups above. These mice were different str ain, C57BL/6 (B6) than the mice we used in C hapter 4 (CD1) experiments in the present work. B6 mice ran slightly, but not significantly more than the CD 1 mice and, as shown in this study daily voluntary running wheel activity did not c hange as unit prices increased. In addition, the running activity of initially diet induced obese (HF) and LF mice did not differ statistically at any time during the study. During the 4 consecutive FUP phases with low fat 20mg 5TUM pellets that was also used in this present work sedentary mice from the LF group maintained their body weight while those that had developed obesity from the HF diet prior to the operant protocol, lost weight almost to their original level. The runner mice from the LF group

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101 lost a small amount of w eight at the highest FUP only. Surprisingly, runner mice from the HF group lost less weight than their sedentary counterparts so that the end of each FUP they weighed more than their sedentary HF counterparts ( P < 0.05). Although not significant at the end of the experiment, RUNHF mice weighed more than SEDHF, while the opposite relationship was true in the corresponding LF groups. Thus, exercise and the attendant changes in food intake seemed to slow weight loss upon switching from a high to low fat diet, and independent of the economic conditions. Overall, runner mice ate more pellets per day than sedentary mice ( P<0.05) and pairwise comparisons were significant at each schedule except FUP5. A twoway ANOVA analysis showed a main effect for unit price and a running x unit price interaction ( P<0.001 at each FUP). Meal pattern analysis showed that r unner mice took bigger meals at each cost schedule, however this was significantly different from the sedentary group only at FUP25 and FUP50 ( P<0.05). For meal siz es, two way ANOVA analysis showed a main effect for unit price and a main effect for running ( P<0.05 for both). For the corresponding number of meals, although runner mice took consistently fewer meals at each schedule, this did not result in statistical s ignificance. In a twoway ANOVA, a main effec t for unit price was found ( P<0.001). A lthough it was not statistically different, at the end of the experiment RUNLF mice weighed less than SEDLF mice. This was the s ame trend that was reported in Experiment 5 in C hapter 4 as runner mice had less fat pad amount compared to the sedentary mice. However the opposite trend seemed to be the case for the HF mice: RUNHF mice weighed somewhat but not significantly more than SEDHF mice. It should be noted that the weight loss in the HF groups is due primarily to the diet shift to low fat

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102 5TUM chow pellets, but compounded by the unit price requirements. In addition, similar to the meal patterns of the runner mice in Experiment 5 in C hapter 4, both runner groups in in this pilot study (RUNLF and RUNHF) consumed bigger and fewer meals than their sedentary counterparts (SEDLF and SEDHF), resulting in greater total daily intake. Our results agree with studies in the literature reporting that exercising mice consume significant ly more energy than nonexercising mice, yet they experience weight loss and a decrease in body fat. Bell, Spencer & Sherriff (1995) reported that despite increased food intake and statistically similar body weights, exercising high fat fed mice had signif icantly less body fat than sedentary high fat fed controls, a result that diffe rs somewhat from the results of our pilot study in which the RUNHF group did not lo se more weight than SEDHF mice. There are several methodological differences that may account for the differences between our results and those of Bell, Spencer & Sherriff (1995). First, they used 6wk old female Swiss Albino mice as in our diet induced obesity protocol we used male 9mo old C57BL/6 (B6) mice. Older B6 mice in our study were more p rone to obesity, shown by the dramatic increase in body weight when fed with high fat diet ( 17 g) compared with the ~4 g weight gain in reported by Bell, Spencer & Sheriff (1995) It is quite possible that age and/or gender and/or strain might have played a role on the differential results in both studies (Pitts, 1984). Irani et al (2005) reported that the primary effect of exercise on body weight of the old obese MC4RKO mice was not as dramatic as that observed for these same mice when allowed to exercis e at a young age before becoming obese. In human studies a significant inverse cross sectional relationship has been shown between activity energy expenditure and percent body fat

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103 in males, whereas such relationship was not found to be apparent in females (Westerterp & Goran, 1997). In this pilot work sedentary diet induced obese mice (SEDHF) ate the lowest amount of food of any group (especially at the start of the FUP phases) and steadily lost weight, approaching their preHF diet weights. As the phenotypic expression of a condition, diet induced obesity was also shown to play a role on the effect of exercise on food intake and body weight changes. However, although, the sedentary lean mice (SEDLF) weighed more than the runner lean mice (RUNLF) at the end of the protocol, the runner HF fed mice (RUNHF) lost most of their weight end the end of the experiment at a similar level as the sedentary HF fed mice (SEDHF). This was particularly interesting because neither runner nor sedentary diet induced obese m ice defended their higher weights throughout the protocol and RUNHF mice weighed somewhat more than the SEDHF mice, albeit not significantly. Thus, exercise was not effective at promoting weight loss in already obese mice. This slightly differential effect of exercise on RUNLF and RUNHF compared to their sedentary counterparts was not caused by the differential amounts of energy expenditure on the running wheels as we found no statistical difference between the voluntary running wheel activity of RUNLF and RUNHF. Thus, although we found a preventative effect of exercise on body fat accumulation, exercise actually slowed weight loss in mice with a precondition of obesity. In this dissertation, the attempt to combine the physiological conditions with the envi ronmental economical settings is an important because of its high relevance to todays obesity epidemic. Although eating is a physiological process, environmental

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104 factors cannot be and must not be ignored when determining how much an individual eats. The s eries of studies applied here shows that the economical setting is important on the eating behavior in a way that may reverse the effects of the factors that are conventionally known to affect eating in certain ways. In the experiment where we manipulated the economical structure of the food environment of the genetically obese and m etabolically deficient mice in C hapter 3, although conventionally we were expecting that the obese mice would be hyperphagic ( LubranoBert helier, Cavazos, Dubern, 2003) or more willing to work for food ( Hamilton and Brobeck, 1964) it was clear that obese mice started to work less and eat less as a consequence when the economical setting was changed. It is not a novel idea that the food choices are economic concepts however this w ork is an original and a unique in that it reveals that not only the food intake is an economical concept but also the phenotypical expressions of genetic tendencies can be altered by these environmental changes and this work does more than just theorizing this idea. This type of research in real life settings as translational studies with human subjects would be very important and are currently being designed in various settings by some investigators (Nasser et al ., 2008) which may give insights for the hu man condition of obesity. As a conclusion, although the elasticity of their demand functions were different there was something similar between the mice in positive energy balance and mice in negative energy balance: obese mice in positive energy balance were eating more than the lean mice; however note that runner mice that are in negative energy balance were also eating more than the sedentary lean mice. One may explain the hyperphagia in both these opposite energy balance conditions with the wanting versus liking theory

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105 (Berridge, 1996). Thus while the runner mice that are in a negative energy balance were hyperphagic compared to the sedentary mice because of the appetitive/incentive motives that are considered as wanting. The obese mice that are in the positive energy balance on the other hand were hyperphagic because the pleasure/palatability motives that is considered as liking. Of course both of the motivational processes may still be effective to a certain extent at the same time in both of energy balance conditions but it is plausible to assume that one motive may be more dominant over the other in a particular energy balance condition as stated above. This understanding may give us an idea on although both groups eat more than their control gr oups; the motivational processes underneath their hyperphagia may be different. The important difference between the mice in positive energy balance and the mice in negative energy balance is that although hyperphagic, genetically obese mice became almost hypophagic and ate less when the price for food was too costly (FUP50), however the mice that are running, maintained their daily food intake at the FUP50 (E xperiment 5) and even higher to AC1000FUP5 (E xperiment 6). This is particularly promising and hopef ul in a way that although the appetitive motives of wanting are very hard to override with environmental manipulations, the pleasure/palatable related motives of liking can be overcome by such alterations. This is partly also the explanation of todays rising obesity epidemics in our obesigenic food environment. This work indicates that if an obesigenic food environment can lead metabolic deficiencies and obesity, a food environment that is promoting the healthy eating may also be very important and not useless as treatment for obesity.

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115 BIOGRAPHICAL SKETCH Deniz Atalayer graduated from Bogazici University ( Istanbul Turkiye ) in 2004 with a Bachelor of Science degree in psychology. In fall 2005, Deniz was admitted to the behavioral neuroscience program in psychology department at the University of Florida, and started to work with Dr. Neil Rowland. Her research interests include eating behavior and obesity research with genetic, neurological, physiological, and neuroeconomic approaches. She defended her masters thesis that was examining the effects of the use of different operants in the meal pattern analysis in mice in fall 2 007, and continued for her Ph.D. in the same program which she will receive in August 2010. From there, Deniz will be pursuing her postdoctoral training at St. Lukes Hospital Obesity Research Center at the Columbia University.