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1 INVESTIGATION INTO THE EFFECTS OF ENALAPRIL ON FOOD INTAKE AND BODY COMPOSITION IN HIGHFAT FED AGED RODENTS By COLLEEN ELIZABETH BOND A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011
2 2011 Colleen Elizabeth Bond
3 To my family and friends whose humor and encouragement got me through many long nights
4 ACKNOWLEDGMENTS I would like to thank Dr. Christy Carter, Dr. Thomas Rowe, and Dr. Philip Scarpace for their advice and guidance throughout the research and writing process. I would also like to thank the members of the Carter Morgan lab for their counsel and support during my t ime in this program.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 7 ABSTRACT ..................................................................................................................... 8 CHAPTER 1 INTRODUCTION .................................................................................................... 10 Overview ................................................................................................................. 10 Rodent Model for Declining Physical Function with Age ......................................... 12 Sarcopenia and AgeRelated Increases in Adiposity .............................................. 13 The Impact of Sarcopenia and Concomitant Obesity on Physical Function ............ 15 Modulation of Body Composition and Gene Expression by Angiotensin Converting Enzyme Inhibitors .............................................................................. 16 Physiological Effects of LongTerm ACE inhibitor Treatment in Young Animals ..... 22 Future Potential for Enalapril Treatment ................................................................. 23 2 EXPERIMENTAL DESIGN AND METHODS .......................................................... 25 Animals ................................................................................................................... 25 Diets ........................................................................................................................ 25 Experimental Design ............................................................................................... 25 Food Intake ............................................................................................................. 26 Body Weights .......................................................................................................... 27 Body Composition ................................................................................................... 27 Drug Treatment ....................................................................................................... 27 High Fat Diet (HFD) ................................................................................................ 28 Statistical Analyses ................................................................................................. 28 3 RESULTS ............................................................................................................... 30 Food Intake ............................................................................................................. 30 Body Weight ........................................................................................................... 31 Body Composition ................................................................................................... 32 4 DISCUSSION ......................................................................................................... 40 Study Implications ................................................................................................... 40 Regulatory Role of Leptin in the Maintenance of Body Composition ...................... 43 Obesity and the Development of Leptin Resistance ............................................... 47 Enalapril as a Modulator of Leptin in the Context of Obesity .................................. 50 Future Directions .................................................................................................... 51
6 LIST OF REFERENCES ............................................................................................... 55 BIOGRAPHICAL SKETCH ............................................................................................ 60
7 LIST OF FIGURES Figure page 2 1 Experimental timeline. ........................................................................................ 29 3 1 Scattergram of total kcal intake over the first three days of the high fat diet. ..... 33 3 2 Food intake over the thirteenday high fat diet .................................................. 34 3 3 Food intake showing hyperphagia over the first three days of the highfat chow diet. ........................................................................................................... 35 3 4 Daily change in body weight over the thirteen day high fat diet.. ........................ 36 3 5 Percent change in body weight from baseline to the end of the high fat diet.. .... 37 3 6 Change in body weight over the first three days of high fat diet. ........................ 38 3 7 Average fat and lean mass at baseline and post highfat diet. ........................... 39
8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INVESTIGATION INTO THE EFFECTS OF ENALAPRIL ON FOOD INTAKE AND BODY COMPOSITION IN HIGHFAT FED A GED RODENTS By Colleen Elizabeth Bond May 2011 Chair: Christy Carter Major: Medical Sciences The widespread consumption of a highfat Western diet in conjunction with decreased physical activity has created an obesity epidemic with severe health ramifications in a ged individuals. Body composition and obesity may represent important mediators in the agerelated onset of physical disability. Our previous studies indicate that, with highfat feeding, aged rats experience a heightened hyperphagia and a del ayed normalization in caloric intake, resulting in an exaggerated weight gain, relative to similarly treated young rats. Angiotensin converting enzyme (ACE) inhibitors attenuate declining physical function in aged rats via modification of body composition and modulation of metabolic and inflammatory pathways. Therefore, we sought to determine if the ACE inhibitor enalapril mitigates this hyperphagic response. Twenty four month old male, F344x BN rats were administered placebo or enalapril (40 mg/kg/day; in food pellet) for four weeks. Thereafter, half of the placebo rats continued to receive regular chow. The other half of the placebo group and the entire enalapril group were given a high fat diet for thirteen days. Compared with the highfat fed placebo rat s, which reached a peak caloric intake on Day 1, enalapril highfat fed rats demonstrated a more gradual hyperphagia, peaking at Day 4. Overall, body weight in
9 the enalapril highfat fed remained lower than placebo highfat fed, which was mainly attributable to an attenuation of fat mass gain. Thus, in the context of exposure to a highfat diet, enalapril may impede the development of obesity via direct modification of body weight /composition and an initial reduction in caloric intake.
10 CHAPTER 1 INTRODUCTION Overview Developing preclinical models of latelife intervention strategies for combating declining physical function has enormous significance (de Grey, 2007; Rae et al., 2010) With the continued graying of the worldwide population, the number of individuals at r isk of developing physical disability continues to increase and the sky rocketing social, emotional and economic cost (Olshansky et al ., 2009) of caring for such individuals mandates the need for testing the effectiveness of healthpromoting interventions within this cohort. To address this need, we have used the Fischer 344 x Brown Norway (F344BN) rat as our model, since several studies have shown that this strain proceeds from 80% to 50% mortality between 24 and 30 months of age. In humans, this same pattern of survival mirrors an exponential increase in disability. In fact, in both rats and humans, assessment of functional limitations in the 50% survival range is highly predictive of future disability and ultimately mortality (Carter et al., 2002; Guralnik et al., 1994) As a result of rising economic costs and reduced overall well being of our aging populations, it is critical to determine the biological processes and lifestyle factors that contribute to decline in functi on. Reduced physical function is concurrent with aging, and often limits the ability to perform activities of daily living, or ADLS, which includes basic self care such as bathing, dressing, and feeding. The inability to perform these basic functions has a significant impact on the physical and psychological well being of elderly individuals as it often becomes necessary for these individuals to enter nursingcare facilities, which limits independence and i ncreases economic burden. In addition, a number of agerelated diseases such as cardiovascular disease and type II diabetes
11 lead to increased morbidity, mortality ( Masoro & Austad, 2006, p.45) and contribute to ADL disability. Changes in body composition are a natural part of the aging process. A principal biological process called sarcopenia, or skeletal muscle atrophy, has been identified as a principal contributor to physical disability Sarcopenia, which is derived from Greek meaning poverty of flesh (Rosenberg, 1997) is associated with agerelated changes in body composition and is highly correlated with reduced physical performance (Buford et al., 2010) Sarcopenia and the co occurrence of obesity, however, involves the invasion of adipocytes into skeletal muscle tissue and precipitates a multitude of diseases, including inflammation, insulin resistance, and cardiovascular disease. The Western lifestyle is infamous for the consumption of a highly processed, fatty diet and lack of exercise. Metabolism slows and adiposity increases as we age, therefore elderly individuals may be more susceptible to the adverse health effects of a highfat diet. Therefore, one of our future aim s is to determine the effect of a high fat diet on the progression of sarcopenia and whether pharmacological interventions, such as angiotensin converting enzyme inhibitors (ACE inhibitors), may mitigate t he effects of a high fat diet. ACE inhibitors have been shown to be highly effective in mitigating body weight and fat tissue gain, improving insulin signaling, and reducing levels of inflammatory cytokines (Santos et al., 2009; Carter et al., 2004) Physical function studies in rodents have revealed that enalapril treatment in older animals attenuates both agerelated increases in adiposity and decline in physical function (Carter et al., 2004) Thus, our present study was designed to establish preliminary data regarding the effects of ACE
12 inhibitors in the presence of a high fat diet Although exercise is a primary defense against obesity, it m ay not be recommended for older individuals who may present with conditions such as congestive heart failure (CHF), arthritis, or obesity that precludes them from exercising comfortably. Therefore, development of pharmacological interventions would be ideal for this population, and the use of enalapril as an adjuvant may increase the ability of these individuals to engage in exercise. ACE inhibitors may potentially be used as effective alternatives and provide the health benefits of diet and exercise. We hy pothesize that the use of ACE inhibitors will mitigate the adverse effects of a highfat diet, and our current study may allow for future investigations focused on determining whether ACE inhibition drugs will maintain physical function in the presence of a high fat diet. Rodent Model for Declining Physical Function with Age In aged humans, sarcopenia and concomitant adiposity result in reduced strength and physical function (Newman et al., 2003; Goodpaster et al., 2001) In order to test hypotheses regarding effective drug and lifestyle interventions, researchers need an animal model that undergoes similar changes in body composition and physical function. Thus, we use the Fischer 344 x Brown Norway (F344 x BN) rat for aging studies because the agerelated changes in body composition, strength, and longevity mimic those in human subjects (Turturro et al., 1999; Carter et al., 2004; Rice et al., 2005) F344 x BN rats, which have an average lifespan of 30 months, gain fat and lean mass from 3 to 24 months of age, lose lean mass and continue to gain fat mass from 24 to 27 months, followed by a decline in both tissue types from 24 to 30 months (Rice et al., 2005; Carter et al., 2004) The changes in body composition reflect the underlying changes in the cell signaling pathways in adipose and muscle tissue, and resultant
13 pathophysiologies, such as insulin resistance and infla mmation, are also observed in humans (Chung et al., 2009) The physiological changes that occur in F344 x BN rats are similar to those in humans, and can be used as a predictor for phy sical decline and mortality (Carter et al., 2010) Since the F344x BN model closely mimics human lifespan and agerelated changes in body composition, we used this rodent model to assess the potential of latelife pharmacological interventions in mitigating adver se changes in body composition, such as increased adiposity, in the presence of a high fat diet. Sarcopenia and AgeRelated Increases in Adiposity Sarcopenia, a natural part of the aging process, may be the principle causative factor for physical decline with age. However, a definitive criteria for the condition has not been established; present definitions are derived from epidemiological studies that base criteria on measures of upper and lower body strength and/or characterized levels of muscle mass (Baumgartner, 2000; Cruz Jentoft et al., 2010; Visser, 2009) In order to demonstrate a correlation between reduced physical function and sarcopenia, changes in both strength and muscle type must be considered. Manini and Clark coined the term dynapenia, derived from the Greek poverty of strength, which refers to the loss of strength that occurs with age (Clark & Manini, 2008) Prior studies show that reduced muscle mass is not proportional to loss of strength, and that changes in muscle quality with age lead to reduced strength (Carter et al., 2005) In addition, treatments with growth hormone or testosterone supplementation, which cause muscle hypertrophy, do not result in proportionate increases in strength (Clark & Manini, 2008; Clark & Manini, 2010) Thus, it is clear that agerelated changes in biological pathways affecting or occurring within muscle tissue are the primary causes of diminished strength.
14 Reduction in muscle quality is associated with increased adiposity that is independent of weight gain (Kershaw & Flier, 2004) and exacerbated by the invasion of adipose cells into muscle tissues (Buford et al., 2010) Humans experience a continuous increase in adiposity and reduction in lean tissue from 25 to 65 years of age, after which overall body weight declines (Schwartz, 1998) Lipotoxicity resulting from invasion of adipose tissue into skeletal muscle can lead to the dysregulation of muscle function and increased apoptosis (Kusminski et al., 2009) In addition, the increase in fat tissue leads to multiple pathologies, including chr onic inflammation, insulin resistance, and oxidative stress, all of which exacerbate reduction in the quantity and quality of skeletal muscle (Jensen & Hsiao, 2010) Adipose tissue secretes a multitude of inflammatory cytokines, such as IL6 and tumor necrosis factor related proteins. metabolism of glucose, such as IRS 1 and GLUT4, and suppresses gene activity involve in the breakdown of fatty acids that promote synthesis of fatty acids and cholesterol. IL 6 is also highly correlated with insulin resistance and obesity, and is used as a predictor for the development of type II diabetes and cardiovascular disease (Kershaw & Flier, 2004) Increased adiposity subsequently results in over expression of cytokines, promoting insulin resistance, wholebody inflammation, and reduced lifespan. Establishing an effective pharmaceutical intervention, such as ACE inhibitor treatment, that successfully mediates weight and adipose gain associated with a high fat diet, may allow for future investigation that determines if these interventions subsequently reduce inflammation and improve insulin sensit ivity in aged animals.
15 The Impact of Sarcopenia and Concomitant Obesity on Physical Function In humans, muscle mass and strength begin s to deteriorate at around age 30, with an accelerated loss occurring after age 60 (Goodpaster et al., 2001; Stenholm et al., 2008) Visceral and intramuscular adipose tissues proliferate while subcutaneous fat is diminished (Stenholm et al., 2008) A progressive i ncrease in fat mass is a normal part of the aging process, even in the absence of considerable changes in body mass index (Zamboni et al., 2008) The reduced basal metabolic rate (BMR) observed in elderly individuals has been attributed to loss of musc le mass, while decreased physical activity aggravates muscle atrophy and weight gain (Stenholm et al. 2008; Evans & Campbell, 1993) Thus, it appears that reduced physical activity accelerates BMR reduction and subsequently intensifies weight and fat tissue gain. Age related obesity is regarded as a significant risk factor for a myriad of chronic diseases, such as metabolic dysregulation (i.e. insulin and leptin resistance), increased inflammation, and dyslipidemia (Kershaw & Flier, 2004; Evans & Campbell, 1993) The conventional assumption that adipose tissue is an inert receptacle for energy storage is false; adipos e tissue secretes a variety of hormones, interacts with the central nervous system, an d responds to hormone systems. Thus, excessive or insufficient fat reserves can have adverse effects on metabolic function (Kers haw & Flier, 2004) The chronic inflammation resulting from increased adiposity may also contribute to muscle mass degeneration and accelerate agerelated muscle loss (Jensen & Hsiao, 2010) As of 2010, 30% of adults between ages 60 and 79 were obese, with a BMI kg/m2. Obesity is becoming more prevalent among older individuals, and is associated with reduced physical functi on and chronic disease (Jensen & Hsiao, 2010) Sarcopenia in conjunction with obesity is associated with adipocyte infiltration i nto lean muscle
16 tissue, or myosteatosis (Thornell, 2011) The intrusion of adipocytes into muscle tissue has been correlated with reduced lower body strength and disabili ty. Muscle strength is measured in terms of force and power; power is proportional to muscle volume, whereas force is proportional on the cross sectional area (CSA) of the muscle. Muscle power, which is responsible for everyday activities such as rising from a chair or climbing stairs, declines to a greater degree than force in aged individuals and therefore contributes to disabi lity and loss of independence. Obese aged individuals are at a much greater risk of disability; not only do they have agerelated reduction in CSA, but they also need extra power to overcome the weight of excess adipose tissue (Narici & Maffulli, 2010; Stenholm et al., 2008) In addition, obesity is strongly correlated with self reported reduction in physical function; the probability of an elderly individual experiencing reduced physical function doubles in the presence of moderate obesity, and quadruples with severe obesity (Jensen, 2005) Recent studies have indicated that a positive correlation exists between waist circumference, an indication of visceral adipos ity, and increased risk of functional decline (Jensen & Hsiao, 2010) Thus, aged obese individuals are at a much higher risk of experiencing rapid physical decline, frailty, and disability. The development of pharmacological interventions, such as ACE inhibitor treatment, may mitigate obesity in the presence of a highfat diet, possibly preventing the development of metabolic dysregulati on and inflammatory processes. Modulation of Body Composition and Gene Expression by Angiotensin Converting Enzyme Inhibitors The reninangiotensin system (RAS) is the principal regulator of blood pressure, and involves a cascade of peptide transform ations Angiotensinogen, an inactive globulin produced by the liver, is cleaved by renin to form angiotensin I (Ang I), which is
17 subsequently converted to angiotensin II (Ang II) by angiotensinconverting enzyme, or ACE. In addition, ACE induces proteolysi s of bradykinin. Ang II is a potent vasoconstrictor while BK is a potent vasodilator, so the formation of Ang II and degradation of bradykinin by ACE results in increased blood pressure. Thus, ACE inhibitors serve to lower blood pressure by blocking the c onversion of Ang I to Ang II and arresting degradation of bradykinin (Carter et al., 2005) ACE inhibitors not only lower blood pressure, but also improve physical function via direct m odulation of body composition. The Health, Aging and Body Composition Study (Health ABC Study) compared the size of the lower extremity muscle mass, or LEMM, in a cohort of elderly individuals aged 7079 who were using various antihypertensive drugs, including ACE inhibitors. The data revealed that individuals taking ACE inhibitors had a significantly larger LEMM than those taking other antihypertensive medications, indicating a dir ect effect of ACE inhibitors on body composition (Onder et al., 2002) Daily injections of the ACE inhibitor enalapril (40 or 80 mg/kg/day) over the course of six months reduced adiposity and improved physical performance in aged F344xBN rodents (Carter et al., 2004) Furthermore, upregulation of Ang II and angiotensinogen expression in rodents resulted in proliferation of adipocytes and increased triglycerides (Engeli et al., 2000) The data validate that the peptides involved in the RAS cascade directly modify body composition, and therefore, ACE inhibi tors can mitigate these effects. ACE inhibitors have been shown to attenuate agerelated physical decline and mitigate adipose tissue gain in both humans and rats in the absence of antihypertensive effects (Onder et al., 2002). A threeyear evaluation of walking speed and k nee
18 extensor muscle strength in hypertensive women over the age of 65 with moderate to severe disability was conducted to evaluate the effects of ACE inhibitors on functional decline. Of the 641 subjects, 61 had continuously used ACE inhibitors, 301 used alternate antihypertensive medications either continuously or intermittently, 133 intermittently, and 146 never. Researchers found that subjects who used ACE inhibitors continuously had a lower average decline in muscle strength and walki ng spee d (Onder et al., 2002). After adjusting for the occurrence of congestive heart failure, myocardial infarction, and stroke, the results remained unchanged, indicating that ACE inhibitors act independently of cardiovascular events (Onder et al., 2002) Apoptosis (i.e., programmed cell death) may play a key role in the development of sarcopenia. In particular, the apoptotic pathway triggered by TNFcentral role in the pathogenesis of agerelated muscle loss (Chen & Goeddel 2002). A gerelated changes in the secretion of TNFBN rats indicate that TNFses substantially between 20 and 30 months of age and that this increase occurs primarily in intra abdominal fat. We used Q PCR to evaluate TNF tibialis anterior ( TA ) muscle of aged rodents treated with either enalapril or N(G) nitro L arginine methyl ester ( L NAME) a nitric oxide blocker. We found that TNFreduced with enalapril treatment, an effect that was reversed in LNAME treated rodents. In addition, we performed gene array analyses in order to determine which cell signaling pathways are altered by enalapril. RNA was isolated from the soleus muscle of rats treated with either placebo or 40 mg/kg/day of enalapril. We found that 237 transcripts were significantly upregulated and 77 were significantly downregulated by
19 e nalapril (Carter et al., 2010). Using IPA, we generated and scored 25 functional netw orks based on transcripts differentially expressed between groups. The IPA network with the highest score consisted primarily of genes related to cellular maintenance, including various genes related to apoptosis regulation. In fact, p53 and the caspases w ere found to be at the heart of this network, indicating that enalaprils effects on aged skeletal muscle may be mainly attributed to regulation of apoptosis (Carter et al., 2010). Using western blot analysis, we next evaluated enalaprils effects on apopt osis in the gastrocnemius muscle of rats given either 20 or 40 mg/kg enalapril, 30 mg/kg losartan, or placebo between 24 and 27 months of age. We found that enalapril treated rats had significantly decreased levels of DNA fragmentation compared to their losartan and placebo treated counterparts. In addition, we observed a downregulation of cytochrome c, active caspase 9, and cleaved caspase3, all markers specific to the mitochondrial caspasedependent apoptotic pathway (Carter et al., 2010). These data s how that enalapril may exert its beneficial effects on aged skeletal muscle by influencing gene expression. A CE inhibitors also have the abilit y to alleviate the metabolic dy sregulation and inflamm ation associated with obesity. For example, ACE inhibi tors may improve insulin sensitivity by increasing cir culating levels of bradykinin. Bradykinin binds to the bradykinin B2 receptors on adipocytes, increasing tyrosine phosphorylation of insulin receptors on the adipocytes (Engeli et al., 2000) In turn, intracellular substrates, such as insulinreceptor substrate1 (IRS 1) are activated and downstream insulin signalingpathways are initiated, which may enhance insulin sign aling and glucose uptake (Olivares Reyes et al., 2009)
20 Previous studies in our lab show that enalapril treatment can mitigate agerelated gains in adi posity and functional decline. Unpublished data from our lab established that enalapril doses less than 20 mg/kg are ineffective (Carter et al., 2004) and therefore studies typically use at least 20 to 40 mg/kg dosages. A prior study assessed the effects of enalapril on physical performance and body composition; male F344 x BN rats were injected daily with either enalapril (40 mg/kg or 80 mg/kg) or saline from 24 to 30 months of age. Physical performance was evaluated via grip strength and inclined plane tests. In humans, grip strength is a predictive tool for disability, and s tamina and muscle tone are indicative of muscle quality (Rantanen et al., 1999) Forelimb grip strength was assessed with an automated grip strength meter The mean force was calculated and divided by the ani mals body weight. The inclined plane test involved placing animals in an upward posi tion on a 60 inclined screen. The time taken for the animal to fall onto the foam pads below were divided by the animals weight. The physical performance data was evaluated based on data from animals that survived for the full six month treatment period, as it was hypothesized that changes in physical performance may not be apparent until the end of the study. There was no difference between the two enalapril groups; howe ver, comparison of the combined enalapril data to the saline group revealed that the salinetreated animals had a significantly greater decline in inclined plane performance, and a moderately signifi cant decline in grip strength. As for body composition, both enalapril groups lost significant proportions of adipose tissue between 27 and 30 months, while the saline group gained fat tissue. Loss of lean mass was equivalent between the enalapril and saline groups between 24 and 27 months of age, with lean mass stabilizing thereafter (Carter et al., 2004) These results
21 demonstrate that enalapril is effective in attenuating functional dec line The significant reduction in overall adiposity in enalapril treated animals may account for this phenomenon, since there was no difference in lean mass, muscle area, or fiber loss between the enalapril and saline groups. A six month longitudinal study compared the effects of enalapril and losartan, an angiotensin receptor blocker (ARB), on physical function, body weight and body composition on aged rats. ARB and ACE inhibitor treatments were evaluated to discern if the two drugs exert similar physiolo gical effects, such as equivalent changes in body composition. In addition, comparison of the drugs effects may help elucidate the biological mechanisms of ACE inhibitors and their contribution to reduced physical performance and changing body composition. The ACE inhibitor enalapril modulates the RAS by blocking the c onversion of Ang I to Ang II. However, the ARB losartan antagonizes AT1, the Ang II receptor, and prevents Ang II physiological action without obstructing other ACE pathways. 24 month old rats were given 40 mg/kg enalapril, 30 mg/kg losartan, or placebo. Body composition and physical performance were assessed at baseline (24 months old), and at 27 and 30 months of age. Food intake, body temperature, glucose and insulin levels, and tumor devel opment were also evaluated in order to determine if outcomes were directly related to treatment or to separate mechanisms. At 27 months, differences in physical function, body weight, and body composition were significant, although by the conclusion of the study, thes e differences were negligible. Enalapril was shown to be effective in reducing adiposity and body weight, with a maximal effect occurring within the first three months of the study. Throughout the study, enalapril treated rats had a lower overall average body weight
22 than losartan or placebotreated rats. Animals in the losartan and placebo groups, however, experienced an increase in adiposity between 24 and 27 months of age. Physical performance was evaluat ed by measuring grip strength. Enalapri l and losartan were both effective in mitigating agerelated decline in grip strength In contrast to enalapril, losartan attenuated functional decline in the absence of changes in body weight or composition, indicating that both drugs exert their effects by targeting skeletal muscle. In order to test this hypothesis, gene array analysis was used to determine which pathways are acted upon by enalapril. Gene array data indicated that the direct effects of enalapril and losartan m ay be attributed to apoptosis The ELISA method was used to determine the extent of apoptosis in gastrocnemius muscle, which is known for undergoing substantial agerelated degeneration and elevation in apoptosis markers Data showed that only enalapril was effective in attenuating apoptotic activity in gastrocnemius muscle. Finally, enalapril treated animals did not undergo any changes in body temperature, food intake, or physical activity that would account for the observed effects on body com position or physical function. Relative t o the losartan and placebo groups, enalapril effectively reduced agerelated tumorigenesis. Enalapril treated animals decreased food consumption between 24 and 27 months of age, even in the presence of increasing adiposity (Carter et al., 2010) These data lends credence to t he idea that enalapril may mitigate obesity by alternate pathways, such as increased fatty acid oxidation or improved insulin signaling, instead of through lowered caloric intake or leptin signaling pathways. Physiological Effects of LongTerm ACE inhibit or Treatment in Young Animals Studies in 3 month old Wistar rats demonstrated the lifeprolonging effects of ACE inhibitor treatment during administration of a highfat diet (Santos et al., 2009) The
23 animals were divided into regular chow and hyperlipidic diet groups. Approximately half of the animals in each group served as a control group, with the other half receiving enalapril treatment for one month. Body weight, body composition, leptin transport across the blood brain barrier (BBB), and levels of antioxidants were examined in order to determine i f enalapril could reduce the detrimental effects associated with a high calorie diet The data revealed that the highfat fed animals treated with enalapril had a prolonged lifespan compared to highfat fed control animals. The increase is lifespan can lik ely be attributed to mitigation of weight gain, reduction in inflammatory cytokines, a nd improved insulin signaling. Suppressed ACE activity in enalapril treated animals level s of glucose, insulin, and triglycerides In addition, adiponectin expression was increased, contributing to decreased glucose levels, inflammation, and upregulation of fatty acid catabolism. Catalase and superoxide dismutase (SOD), which are responsible f or minimizing levels of hydrogen peroxide and superoxide radicals, were increased in enalapril treated animals, promoting longer lifespan. Lastly, enalapril did not promote leptin transport across the BBB, nor did it enhance CNS sensitivity Circulating le ptin levels were lower in enalapril treated animals, which can be attributed to their lower overall fat mass (Santos et al., 2009) However, the direct physiological effects of enalapril indicate that despite its inability to enhance leptin signaling, enalapril is an effective drug for mitigating the pat hologies associated with a hyperlipidi c diet and subsequent obesity. Future Potential for Enalapril Treatment Enalapril has the potential to prolong quality of life and lifespan in elderly and obese individuals. Enalapril has been highly effective in reducing adiposity and
24 increasing physical function, outcomes that can be attributed to reduced inflammation, improved insulin signaling, and increased antioxidant levels. With a growing elderly population and a widespread obesity epidemic, researchers hope enalapril can reduce associated disease processes in these individuals. As previously discussed, enalapril has been highly effective in mitigating obesity and pathologies in high fat fed young animals. However, it is currently unknown if enalapril will produce the same results in aged animals. Thus, the goal of the research is to investigate whether high fat fed aged animals pretreated with enalapril wil l experience the same effects. Enalapril effectively reduces adipose tissue and circulating levels of lept in. Thus, it is possible that pretreating aged animals with enalapril will reduce hyperleptinemia and moderate lept in resistance. Given that we will not have the leptin data from this experiment to support this idea, we are using this study to establish a model for future experiments involving enalaprils effect on leptin resi stance in aged animals. The present study will be used to e valuate three main hypotheses: pretreatment of highfat fed aged animals wit h enalapril will result in (1) r educed hyperphagi c response, (2) spontaneous division into the diet induced obesity (DIO) and diet resistant (DR) groups observed i n younger animals, and (3) modulate the increase in overall adiposity.
25 CHAPTER 2 EXPERIMENTAL DESIGN AND METHODS Animals 56 Male Fisc her 344 x Brown Norway (F344 x BN) rats were purchased from the National Institutes on Aging (NIA) Colony at Harlan Industries (Indianapolis, IN). Animals were received at 24 months of age and housed individually under standard temperature and lighting conditions (12 hour light:dark cycle) in a specific pathogenfree facility accredited by the American Association for Accreditation of Laboratory Anim al Care. Water was provided ad libitum at all times. The University of Floridas Animal Care and Use Com mittee approved all protocols. Health checks were performed daily in order to identify injury or disease. Diets Baconflavored food pellets were purchased from BioServ, Frenchtown, NJ. 40 mg/kg of enalapril was compounded into the bacon pellets; placebo pellets did not contain drug. High fat rodent chow was purchased from Research Diets Inc, New Brunswick, NJ and consisted of 60% fat, 20% protein, and 20% carbohydrate. Individual food pellets contained 5.24 k cal /gram of metabolizable energy. Regular Harlan Teklad rodent chow was obtained from Harlan Laboratories Inc, and was composed of 4% fat 24% protein, and 4.5% fiber. Individu al food pellets contained 3.1 k cal /gram of metabolizable energy. Experimental Design Upon arrival, animals were acclimated to the new environment (i.e. no handling by lab personnel) for one week, followed by a five day acclimation to handling. Immediately following handling acclimation, and at the start of weekly food and body
26 weights, the animals were randomly divided into a placebo (n=31) and enalapril (n=25) group; animals were randomized so that the average weight of t he two groups were equivalent. Drug acclimation began four days after dividing the animals into the placebo and enalapril groups. Three weeks after the start of the drug acclimation period, the placebo group was divided into a placebo high fat (n=17) and plac ebo regular chow (n=14) group. All enalapril a nimals were assi gned to receive high fat chow. Animals were divided so that the average weight of each group was equivalent. Approximately 5 weeks after the start of the drug acclimation phase, baseline TDNMR was performed in order to record baselin e body composition and weight. The high fat diet began three days after TDNMR. Thereafter, food intake and body weights were recorded daily in order to capture hyperphagia and subsequent weight gain. One day after the end of the high fat diet, a second TDNMR was performed in order to record changes in body composition and weight as a result of the high fat chow diet (Figure 21). Food Intake Recording of food consumption (in grams and kilocalories) began four days before the start of the drug treatment phase and continued throughout the study. Food intake was calculated by recording the quantity of chow remaining and subtracting it from the previous weeks (or days) values The difference was multiplied b y the number of metabolizable kc al/gram (Harlan Teklad Rodent Diet #8604 (Regular Chow): 3.1kc al/ gram. Research Diets #D12492 (High Fat Diet): 5.24 kc al/g). Food intake was recorded on a weekly basis during the drug treatment phase in order to establis h baseline consumption levels. Daily recording of food intake began at the start of high fat diet and continued for the duration of the study. Daily observation of intake allowed us to observe the initial hyperphagic response to the high fat diet and determine if the
27 enalapril and placebo groups responded differently to the highly palatable, energy dense chow. Body Weights Body weights were recorded (in grams) by placing the rodents in an open tupperware container on a digital scale. Values were rounded to the nearest whole number. Baseline body weights were recorded four days before the start of the drug treatment phase, and recorded on a weekly basis thereafter in order to determine the drugs effect on body weight. Upon initiation of the high fat diet, body weights were recorded on a daily basis in order to observe the effects of the hyperphagic response on body weight, and determine if enalapril was effective in mitigating weight gain. Body Composition Time domain nuclear magnetic resonance (TD NMR) was used to determine changes in body fat and lean mass during ea ch diet phase. TD NMR was performed at baseline and at the beginni ng and end of each diet phase. Live, conscious (i.e. no anesthetic) rats were restrained in sample holders (90 mm diameter and 250 mm length). Screwcap plungers were adjusted in order to r estrain the animals while adjust ing for their individual size. The sample holder was inserted into the MiniSpec (TD NMR Minispec, Bruker Optics, The Woodlands, TX, USA), which analyzed body composition via acquisition and determination of TD NMR signals from protons in the sample area. Each animal was scanned twice, with each s can averaging two minutes. Drug Treatment Animals were randomly assigned to receive 40 mg/ kg / day enalapril (n=25) or placebo (n=31) for a period of four weeks. Food and body weight s were recorded at the end of each week in order to track changes due to drug treatment.
28 High Fat Diet (HFD) After the drug treatment period, the animals were placed on a thirteen day high fat diet. All enalapril animals were placed on HFD, and approximate ly half of the placebo group (n=17) was placed on highfat chow, with the other half continuing on regular chow (n=14). Food intake and body weights were recorded daily. Statistical Analyses Statistical analyses were performed using SAS 9.1 for Windows (Cary, NC). Where appropriate, we used multivariate regression models when i nvolving time in the analyses. If the data did not meet the assumptions of the ANOVA, we transformed them using log or square root transformation. We tested for the main effects of treatment condition (placebo high fat; placebo chow; enalapril high fat), time (days) and their interaction. Multiple pairwise group comparisons were performed using the Bonferroni procedure. The level of significance was set at p <0.05 for all analyses. All data are presented as means standard error.
29 Figure 21. Experimental timeline.
30 CHAPTER 3 RESULTS Food Intake In order to determine if the enalapril HF group experienced spontaneous division into diet induced obesity (DIO) and diet resistant (DR) groups, the total kc als consumed per animal during the first three days of the highfat diet were evaluated for each group. Figure 31 demonstrates that the three groups are dis tinctly different in terms of kc al intake during the first three days of the diet. Although there is a small overlap in consumption between the enalapril HF and placebo HF groups, the enalapril HF groups intake lies primarily between that of the placebo HF a nd placebo chow groups. Thus, the enalapril HF group did not divide into DIO and DR groups, but there are distinct differences in intake among the three groups. Co nsumption was measured in both kcals and absolute gram intake over the entire 13 days of highfat feeding (Figure 32 A and B ) On average, there was no difference in either measure (ps = 0.483 and 0.500 respectively). In contrast, d uring the hyperphagic phase of the experiment (Figure 3 3 A and B ; baseline to day 0, 1, 2) t here was a main effect of treatment condition (p < 0.001) time (p < 0.001) and a condition x time interaction (p < 0.001) for both kc al and gram intake. Post hoc analyses demonstrated that across the first three days of highfat feeding gram intake was no different at baseline amongst all groups By day 1, the placebo chow and enalapril HF were not different from each other (p > 0.05); whereas both groups were different from placebo HF (both ps < 0 .001). By day 2 all groups were different from each other, such that placebo HF was greater than enalapril HF which was also greater than placebo chow (all ps < 0.001). The same trend was observed for k cal intake. These data
31 demonstrate that enalapril attenuates the hyperphagic response observed in aged rats to the presentation of highfat feeding. Body Weight Figure 34 shows that during the entire 13 days of the highfat feeding phase of the experiment, there was a main effect of condition (p = 0.005), time (p < 0.001) and a condition x time interaction (p <0.001). Post hoc analyses of the interaction revealed that by day six differences emerged between the placebo HF and placebo chow groups (p < 0.05); however no differences were observed between placebo chow and enalapril HF groups nor between the placebo HF and enalapr il HF groups (all ps > 0.05). By day nine and throughout the remainder of the experiment, all groups were significantly different from each other such that the placebo HF animals demonstrated a larger weight gain than both the enalapril HF and placebo chow groups which were also significantly different fr om each other (all ps < 0.05). Indeed, the overall percent change in body weight from base line to the end of highfat feeding followed a similar trend (all p s < 0.05) (Figur e 35 ) These data show that while all highfat fed animals gained a significant amount of body weight, enalapril mitigated the weight gain pr edicted with highfat feeding. During the hyperphagic phase of the experiment (baseline to day 0, 1 and 2) there was a main effect of treatment (p = 0.02), but no effect of time (p = 0.41), nor an interaction of the two (p = 0.96) on body weight. Overall body weight remained consistent over this time period in both the placebo chow and enalapril HF gr oups; whereas by day 2, body weight in the placebo HF was significantly higher than the other two gr oups (both ps < 0.05; Figure 36 ). These data demonstrate that body weight over both the hyperphagic and longterm phases of the experiment was significant ly
32 influenced by food intake (both grams and k cals) durin g the hyperphagic phase of the experiment. Body Composition Body composition was assessed at baseline and then after the 13 days of highfat feeding. There was a main effect of condition (p < 0.001, p = 0.003), time (both ps < 0.001) and a condition x time interaction (p < 0.001; 0.009), all respectively for fat and lean mass. At baseline, there were no differences amongst groups for either fat or lean mass grams (all ps > 0.05). However, after 13 days of highfat feeding, all groups were different from each other such that plac ebo HF animals demonstrated significantly higher fat and lean mass relative to both enalapril HF (p = 0.05 and p = 0.022) and placebo chow animals ( p < 0.001 and p = 0.007) respecti vely (Figure 37 A and B ) In addition, there was no difference between enalapril HF and placebo chow for lean mass (p = 0.11) but a significant differ ence for fat mass (p < 0.001). These data demonstrate that enalapril treatment attenuates highfat feeding induced changes in inc reases in fat mass.
33 Fig ure 31. Scattergram of total kc al (combined total of days 0,1, and 2) intake per animal per group over the first three days of the highfat diet.
34 A B Figure 32 Food intake over the thirteenday high fat diet. Days 0 through 12 indicate the first through thirteenth day of the diet. Error bars signify the SEM, or standard error of the mean, for each value. Baseline corresponds to food intake before initiation of the high fat chow diet A) Food intake in grams. B) Food intake in kcals.
35 A B Figure 33 Food intake showing hyperphagia over the first three d ays of the highfat chow diet. Baseline intake corresponds to chow consumption before t he start of the high fat diet. Days 0, 1, and 2 indicate the first, second, and third days of the diet, r espectively. Error bars signify the SEM, or standard error of the mean, for each value. A) Food intake in grams, B) Food intake in kcals.
36 Figure 34 Daily change in body weight over t he thirteen day high fat diet. Baseline values indicate the average weight of each group before the start of the diet. Error bars signify the SEM, or standard error of the mean, for each value.
37 Figure 35 Percent change in body weight from baseline to the end of the high fat diet. Error bars signify the SEM, or standard error of the mean, for each group.
38 Figure 36 Change in body weight over the firs t three days of high fat diet. The data show the degree of w eight gain due to hyperphagia. Error bars signify the SEM, or standard error of the mean, for each value.
39 A B Figure 37 Average fat and lean mass at baseline and post highfat diet. Error bars signify the SEM, or standard error of t he mean, for each value. A) Change in fat mass B) Change in lean mass.
40 CHAPTER 4 DISCUSSION Study Implications We are witnessing a rise in the number of individuals at risk for physical disability due to the aging of the global population and a growing number of obese aged individuals. As aged obese individuals are more susceptible to chronic disease and disability, it is essential that we develop latelife pharmacological interventions. Numerous studies have demonstrated that enalapril is effective in mitigating body weight and fat tissue gain in ag ed, standard chow fed rats. Thus, the goal of the present study is to determine if enalapril is efficacious in moderating food intake and subsequent weight and adipose gain in aged, highfat fed rodents. The results from our study show that the placebo HF group demonstrated a marked increase in food intake and experienced its largest weight gain on the fi rst day of the high fat diet. In contrast, the enalapril HF group steadily increased consumption during the hyperphagic period, and did not peak until day 4. Unlike the placebo HF group, which experienced rapid weight gain after the first day of the diet, the enalapril HF group demonstrated a more moderate, consistent increase in weight over the first three days of the diet, closely mirrori ng the pattern of food intake. In addition, we observed that the pretreatment effects of enalapril on food intake and body weight disappeared after the first three days of the diet. Although consumption and body weight gain were significantly dif ferent between the placebo HF and enalapril HF groups during the hyperphagic period, these groups followed a similar pattern in weight gain and consumed equivalent amounts of food after this period. We predict that continuous enalapril treatment would have maintained significant differences in food intake and
41 weight gain throughout the high fat diet. Although the placebo HF and enalapril HF groups were gaining equivalent amounts of weight after the hyperphagic period, the placebo HF group weighed 21 grams m ore on average than the enalapril HF group and exhibited a 2.1% greater increase in body fat. The rapid weight gained experienced by the placebo H F group during the hyperphagic period can likely account for the larger overall increase in body weight and adiposity. It appears that the placebo HF group could not recover from the initial surge in food intake and subsequent weight gain, in spite of the fact that the placebo HF groups food intake was equivalent to that of the enalapril HF group by the seventh day of the diet. Upon initiation of the high fat diet, the highfat fed animals discontinued eating their drug pellets, which may account for the plateau in food consu mption between days 3 and 4. The data indicate that enalapril may attenuate weight gain by mediating the hyperphagic response to highfat chow, although the precise mechanism by which enalapril exerts its effects is currently unknown. Researchers in other labs have shown that enalapril treatment may be effective i n improving insulin signaling. For example, treatment of the CohenRosenthal diabetic hypertensive rodent strain with enalapril was effective in significantly lowering blood glucose levels (Rosenthal et al., 1997) However, previous studies in our lab have shown that enalapril does not ameliorate agerelated insulin dysfunction in skeletal muscle nor whole body glucose tolerance in aged, chow fed animals. 27 month old rats treated with 20 mg/kg/day of enalapril for three months did not experience improved glucose tolerance nor an increase in GLUT4 or AKT1 levels in the extensor digitorum longus (EDL) muscle, two markers of insulin signaling. Thus, the moderate weight loss induced by enalapril treatment may not be adequate for improving insulin function.
42 Since enalapril is ineffective in improving insulin function during normal aging in standard chow fed rodents, it is unlikely that enalapril will have any insulinenhancing effects in the presence of high fat feeding. A 2009 study by Santos evaluated the effects of enalapril in highfat fed young rodents (Santos et al., 2009) As we observed in older rodents, enalapril treatment had no effect on the serum insulin levels in either the high fat or standard chow fed young animals Although it is unli kely that insulin dependent pathways are responsible for the observed effects of enalapril on highfat fed aged animals, the SOCS3 and leptin signaling pathway have the potential to elucidate enalaprils mechanism of action. It is possible that enalapril treatment may induce changes in leptin sensitivity. Leptin, a hormone secreted by adipocytes, functions in regulating energy expenditure and appetite. Circulating leptin is proportional to the quantity of adipose tissue, and exerts its effects on hypothal amic neurons in order to maintain body composition and nutritional status (Ahima & Lazar, 2008; Cohen et al. 2001) Leptin resistance is an inherent part of the aging process in both rodents and humans, and may contribute to development of agerelated obesity (Zhang & Scarpace, 2006) Since leptin signaling plays a critical role in the regulation of food intake and body composition, it is possible that enalapril ameliorates hyperphagia by acting on the leptin signaling pathway. Peripheral and central leptin resistance are thought to contribute to the pathogenesis of agerelated obesity. Human studies have shown that elevated leptin levels are ineffective in mitigating obesity, and leptin therapies are virtually futile in obese subjects (Zhang & Scarpace, 2006) Studies in rodents support the idea that agerelated lept in -
43 insensitivity may exac erbate obesity in the elderly. Therefore, what follows is a revi ew of the role of leptin in the regulation of body composit ion in the context of obesity. Regulatory Role of Leptin in the Maintenance of Body Composition Leptin, a product of the obesity gene (Ob gene) is an adipokine that communicates with the central nervous system, or CNS, regarding energy stores and nutritional state. The stomach, intestines, and skeletal muscle produce low levels of leptin, while adipose tissue produces the greatest concentration of leptin (Ahima & Lazar, 2008) Circulating leptin is proportional to the quantity of adipose tissue, adipocyte size, and triglyceride content (Ahima & Lazar, 2008) and varies depending on the abundance of certain adipose tissues. Fasting reduces circulating leptin levels, which in turn stimulates hyperphagia (increased feeding) and reduced energy expenditure. Conversely, overfed animals have a greater concentration of circulat ing leptin, resulting in reduced food consumption and increased energy expenditure (Ahima & Lazar, 2008) Leptin selectively targets adipose tissue and preserves lean muscle mass by inducing lipid oxidation and protein synthesis while reducing lipogenesis (Buettner et al., 2008) Studies in knockout mice have demonstrated leptins critical role in maintaining nutritional state and body composition. Mice with leptin mutations (ob/ob) or leptin receptor mutations (db/db) demonstrate marked hyperphagia and resultant obes it y. These mice also have concomitant metabolic and endocrine disorders, including diabetes, enlarged fatty livers, and hypercortisolemia, or the overabundance of cortisol (Cohen et al., 2001) Leptin functions by binding to receptors in the hypothalamus and inhibiting appetite while promoting energy expenditure. Leptin receptors are present in peripheral tissues and throughout the C NS, but the highest concentration of leptin receptors
44 occurs in the arcuate nucleus of the hypothalamus (Banks, 2004) The leptin receptor has five splice variants (ObRaObRe); ObRb, the long form of the receptor, appears to be the isoform responsible for the neuroendocrine effects of leptin (Cohen et al., 2001) ObRb is f ound in peripheral tissues, and is enriched in the brainstem, the arcuate, ventromedial, dorsomedial, and paraventricular hypothalamic nuclei, and cerebral regions that manage food intake, metabolism, and regulate neuroendocrine systems (Ahima & Lazar, 2008; Cohen et al., 2001) Absence of ObRb produces a phenotype that is virtually identical to that observed in leptindeficient animals, which elucidates the critical role of this receptor for body weight regulation (Cohen et al., 2001) Leptin signaling involves several pathways, but the Janus tyrosine kinase 2 (JAK2/STAT3) pathway is the most well understood. The intracellular domain of the leptin receptor contains three tyrosine residues: LepRy985, LepRy1138, and LepRy1077. Phosphoryl ation of tyrosine 985 is responsible for negative feedback regulation and reduces leptin signaling; phosphorylation of tyrosine residue 1138 is required for phosphorylation and recruitment of STAT3 (Donato, Jr. et al., 2010) Leptin binds to the OBRb receptor in the hypothalamus, initiating phosphorylation and activation of JAK2, which is constitutively attached to ObRb. Phos phorylation of Tyr1138 recruits STAT3 to the ObRbJAK2 complex. JAK2 phosphorylates STAT3 (pSTAT3), resulting in subsequent dimerization and translocation to the nucleus, where the STAT3 dimers promote gene transcription of proopiomelanocortin (POMC) and suppressor of cytokine signaling 3 (SOCS3) (Donato, Jr. et al., 2010; Zhang & Scarpace, 2006; Munzberg & Myers, Jr., 2005) STAT3 signaling is critical for the longterm regulation of food consumption, body weight, and glucose homeostasis, and does so by regulating SOCS -
45 3. SOCS3 binds to Tyr 985 of the leptin receptor and impedes JAK activity, attenuat ing leptin receptor signaling. Studies have shown that SOCS 3 expression is elevated in obese rodents, and deletion of SOCS 3 in hypothalamic neurons enhances leptin sensitivity and minimizes susceptibility to diet induced obesity (Donato, Jr. et al., 2010; Munzberg & Myers, Jr., 2005) Since SOCS3 contributes to leptin sensitivity, it is possible that enalapril may exert its effects by acting dir ectly on SOCS3 expression and signaling. Suppressors of cytokine signaling (SOCS) are essential regulators of cytokine activity and contribute to the regulation of inflammatory systems. SOCS 3 expression is induced by numerous anti inflammatory and inflammatory cytokines and acts in a negativefeedback loop with the JAK STAT pathway. SOCS proteins are normally expressed at low levels, but cytokines and hormones that activate JAK STAT signaling also induce the rapid expression of SOCS proteins, thus i nhibiting the conti nued activation of this pathway (Hanada et al., 2003; Muscogiuri et al., 2008). The increased expression of TNFand insulin in obese animals has been found to induce SOCS3 mRNA expression in the white adipose tissue of obese mice (Emanuelli et al., 2001) In addition, Ang II has been found to induce SOCS 3 expression in both cultivated cell lines and mammalian tissues (Muscogiuri et al., 2008). Thus, enalapril may exert its physiologica l effects by reducing circulating levels of Ang II subsequently reducing SOCS 3 expression and increasing leptin sensitivity. MSH), which activates melanocortin3 and 4 receptors in order to reduce food intake, body weight enhance energy expenditure, and regulate glucose metabolism
46 (Niswender et al., 2004; Munzberg & Myers, Jr., 2005; Belgardt & Bruning, 2010) Agouti rela ted protein (AgRP) neurons inhibit melanocortin 3 and 4 receptor signaling aminobut yric acid (GABA) to promote feeding and reduced energy expenditure. Neuropeptide Y (NPY), an orexigenic, or appetitestim ulating, hormone increases food intake and suppresses thermogenesis by releasing GABA and inhibiting POMC activity ObRb is located in the ARC anabolic neuron populations responsible f or production of AgRP and NPY. The interaction of leptin with its recept ors inhibits expression of both AgRP and NPY, allowing for increased metabolism and reduced appetite (Niswender et al., 2004; Stephens et al., 1995; Munzberg & Myers, Jr., 2005; Belgardt & Bruning, 2010) Although the JAK2/STAT3 pathway is the best understood and is necessary for the longterm effects of leptin, the phosphatidylinositol 3kinase, or PI3K pathway is responsible f or the leptins acute effects. PI3Ks are heterodimeric complexes that contribute to regulation of metabolism, growth, and food intake. Activated JAK2 phosphorylates IRS proteins, which subsequently recruit PI3K in hypothalamic neurons and activates the PI3K pathway in POMC neurons (Niswender et al., 2004; Donato, Jr. et al., 2010; Munzberg & Myers, Jr., 2005) The specific neuron populations responsible for the effects of the PI3K pathw ay have not been completely identified. However, we know that the leptininduced PI3K pathway has been correlated with maintaining glucose homeostasis in peripheral tissues and regulating the rapid effects of leptin signaling, such as diminished food intak e (B elgardt & Bruning, 2010)
47 Obesity and the Development of Leptin Resistance In young humans, there is a significant correlation between adiposity and plasma leptin. However, this correlation is lost in middleaged and elderly individuals, which is indicative of dysregulation of leptin signaling (Unger, 2002) Although relative fat mass between young (2040 y.o.) and older (> 40 y.o.) individuals may be comparable, leptin levels in older subjects are not correlated with fat mass. This reduced correlation is indicative of a feedback disruption between peripheral adipose stores and appetite regulation in the central nervous system of older individuals. It is hypothesized that the impaired feedback may be related to reduction of sex hormones in older individuals and contributes to agerelated obesity (Moller et al., 1 998) Studies in obese mice and humans reveal that despite elevated leptin, appetite is no longer suppressed, an indicator of leptin resistance. Although the precise mechanism of leptin resistance is currently unknown, the two main hypotheses include f ailure of circulating leptin to cross the bloodbrain barrier (BBB) and reach its receptors in the hypothalamus, and defects in leptinreceptor signaling cascade (Kershaw & Flier, 2004; Munzberg & Myers, Jr., 2005) Leptin crosses the BBB by a saturable transport system which appears to have decreased activity in overnourished, obese rodents. The transport rate of leptin across the BBB can be attributed in part to self inhibition of i ncreased serum leptin concentration in obese animals (Munzberg & Myers, Jr., 2005; Banks, 2004) Brain perfusion studies demonstrate that the BBB is most efficient w hen circulating leptin l evels are below normal levels. Lept in concentrations above 10 ng/mL, a level observed in obese animals, exceed the BBB transport threshold. As a result, as serum leptin concentration increases, the percentage of leptin transported a cross the BBB is diminished. Leptin levels of 30 ng/mL resulted in transport that was only 66% of
48 the value that was transported at 10 ng/m L Elevated triglycerides is likely a significant contributor to BBB transport defects; both starvation and obesity r esult in elevated triglyceride levels, which inhibits le ptin transport across the BBB. Inhibition of leptin transport is necessary during periods of starvation, as it prevents leptin from exerting its appetitesuppressing and metabolism enhancing effects. However, elevated triglycerides in obese animals may be misinterpreted as starvation, so obesity is exacerbated since leptin is unable to interact with its receptors in the hypothalamus (Banks, 2004) It is thought that BBB resistance occurs before central resistance; animals with only a BBB transport defect respond to central administration of leptin, but do not respond to peripheral administration. However, prolonged obesity likely results in t he development of central leptin resistance, subsequently disrupting the leptin signaling cascade (Kershaw & Flier, 2004) Studies in mice fed either a low or highfat diet show that leptin resistance initially develops with BBB transport defects, and eventually develops i nto central leptin resistance. Mice were evaluated with peripheral (ip) or central (icv) leptin injection after four weeks and fifteen weeks of a low or high fat diet. EMSA data revealed that ip injection induced equivalent STAT3 DNA binding in both diet groups after 4 weeks. However, ip injection was ineffective in inducing STAT3 binding in the high fat group after 15 weeks, indicating that BBB transport was compromised and subsequently prevented activation of STAT3 by leptin. Although icv injection induced STAT3 activation in highfat fed mice after 15 weeks, induction was 75% lower than in low fat fed mice, which indicates defects in the leptinreceptor signaling cascade (ElHaschimi et al., 2000)
49 In several rodent models of obesity, SOCS 3 expression has been elevated. When circulating leptin levels are low, baseline STAT3 activation is moderate, which in turn depresses SO CS3 expression. As leptin concentration increases incrementally, leptin receptor signaling rises i n proportion to leptin levels. H owever, when leptin concentration is chronically elevated, baseline STAT3 activity and SOCS 3 expression is augmented, which diminishes the effects of increased leptin receptor activity A dipose tissues secrete fatty acids and inflammatory cytokines such as IL6 and TNFmay enhance SOCS 3 expression (Munzberg & Myers, Jr., 2005) In addition, C reactive protein (CR P), which is secreted by the liver and elevated in inflammatory states, has been found to bind to leptin and repr ess its interaction with ObRb. Not only does CRP inhibit leptin receptor activity, but leptin itself induces hepatic CRP expression, exacerbati ng the leptinresistant state (Belgardt & Bruning, 2010) In a study conducted by Judge et al (Judge et al., 2008) osmotic mi nipumps containing murine recombinant leptin (i.e. peripheral leptin infusion) were implanted in a subcutaneous pocket in both young (3 month old) and aged (30 month old) F344xBN rodents and the animals were infused with varying concentrations of leptin. The young rats demonstrated an anorectic response and reduction in body weight, and reduction in kilocalorie consumption and weight loss was dosedependent, with the response plateauing at 0.07 mg/day. The aged rodents showed no response to leptin infusion, even at t he maximum dose of 0.5 mg/day. The aged animals did not reduce caloric intake or lose weight, indicating a resist ance to the effects of leptin. Upon initiation of the palatable, highfat diet, the young rats spontaneously divided into either a di et induced obesity (DIO) or diet resistant (DR) groups. However, the highfat fed aged rats
50 did not divide into these groups, and instead exhibited vast increases in weight. Typically, animals provided highfat chow demonstrate hyperphagia upon initiation of the HF diet. In the young rodents, leptinreceptor activity allowed for normalization of caloric intake, and these animals return to pre HF consumption levels. However, in the leptin resistant aged rats, there was a delayed normalization of caloric intake, resulting in an extended hyperphagic effect that exaggerated increases in weight and adipose tissue in addition to a peak kilocalorie intake that was much greater than the peak intake observed in the young rats (Judge et al., 2008) Enalapril as a Modulator of Leptin in the Context of Obesity Studies conducted by Santos et al (Santos et al., 2009) evaluated the effect of enalapril on both serum leptin concentration and leptin response in young rodents fed standard or high fat chow. The first study involved measuring serum leptin after young rats were fed either standard or hyperlipidic chow and simultaneously treated with 10 mg/kg/day of enalapril for six months. The data show that standard and highfat fed animals treated with enalapril had lower serum leptin concentration compared to their placebotreated counterparts. The decrease in leptin can likely be attributed to the reduced fat mass in enalapril treated animals, as leptin concentration is proportional to adiposity. The second study evaluated enalaprils effects on leptin response in the presence of a hyperlipidic diet. Intracerebroventricular (icv) leptin cannulas were implanted in thr eemonth old male Wistar rats. The rats were fed either a standard or high fat diet in conjunction with 10 mg/kg/day enalapril treatment (dissolved in the animals drinking water) for one month. 24 hours after injection, food intakes were recorded. Food intake was significantly reduced in the icv treated diet groups, although there was no
51 significant difference observed between the enalapril and control groups. Lastly, leptin transport across the BBB was evaluated in a subset of these groups by intravenous injection of radioactively labeled leptin (125I leptin). The data show that enalapril did not alter transport of 125I leptin across the BBB (Santos et al., 2009) Future Directions The studies conducted by Santos and Scarpace provide an excellent basis for investigating the potential role of enalapril on leptin signaling in highfat fed aged rodents. Judge et al. (Judge et al., 2008) demonstrated that aged rodents are leptin resistant and exhib it hyperphagia and delayed normalization of caloric intake when presented with hyperlipidic food. Santos et al. showed that icv administration of leptin effectively reduced food intake in the young highfat fed rodents, although there was no significant di fference between the control and enalapril treated animals. In addition, enalapril did not inc rease BBB transport of leptin. Although enalapril did not exert significant effects in icv treated animals or increase leptin transport, we must consider that the studies were conducted using a very low dose of enalapril (10 mg/kg/day), as opposed to the standard dose of 2040 mg/kg/day used in our studies. Thus, it is quite possible that enalapril can alter the leptin signaling pathway in highfat fed rodents, but the 10 mg/kg/day dose is too low to exert significant biological effects. As previously discussed, lept in concentrations above 10 ng/mL, a level observed in obese animals, exceed the BBB transport threshold (Unger, 2002; Banks, 2004) Therefore, enalapril may improve leptin transport by reducing overall adiposity and subsequently reducing circulating levels of leptin. Our present study has shown that enalapril alone, at a dose of 40 mg/kg/day, is effective in reducing hyperphagia in highfat fed aged animals. These findings warrant a
52 future investigation of the combined effects of leptin and enalapril in highfat fed aged animals, and the potential effects on leptin transport. Sin ce the animals ceased consumption of their drug pellets in our present study, our future study would involve administering 40 mg/kg/day of enalapril in drinking water so that the animals continue drug treatment while on the highfat diet, potentially resul ting in a more dramatic reduction in hyperphagia. ICV leptin cannulas would be implanted in half of the enalapril HF and placebo HF animals in order to determine if enalapril and leptin produce an additive effect in reducing hyperphagia. A subset of the enalapril HF and placebo HF groups would be injected with 125I leptin in order to evaluate whether enalapril improves leptin transport across the BBB in aged animals. Since our target human population includes obese aged individuals, we aim to determine if enalapril will be effective in mitigating weight gain in those who are already in an obese state. Our present study investigated enalaprils effects in normal weight rodents who were pretreated with enalapril before introduction to highfat food. Thus, we observed enalaprils effec ts before the onset of obesity. A future research aim will be to highfat feed rodents first, followed by enalapril treatment when the animals are in an obese state. Thus, pretreatment with a high fat diet may provide a more real istic rodent model of our target human population. Future experiments may also include those that focus on antagonism of various pathways. In vivo, nitric oxide synthase ( NOS ) inhibition in rats with the ubiquitous NO blocker LNAME results in reduced sk eletal muscle, cross sectional area, and subsequent disruption in walking speed (Wang et al., 2001). NO exists in three isoforms: endothelial NOS (eNOS), neuronal/mitochondrial NOS (n/mtNOS), and
53 inducible NOS (iNOS) (Dirks & Leeuwenburgh, 2005; Marzetti et al., 2010; Tracy et al., 1999). A low steady state level of NO, which is maintained by constitutively expressed eNOS and nNOS, favorably modulates cellular metabolic function. In fact, low steady state concentration reversibly inhibits cytochrome c release from mitochondria and subsequently reduces apoptosis ( Beltran et al., 2000). iNOS, however, produces large and potentially toxic levels of NO and is increased with age. Thus, we investigated enalaprils effects on NO production, based on the hypothesis that enalapril would increase production of eNOS and nNOS by reversing the proteolytic degradation of bradykinin. Subsequently, we predicted that increased production of the favorable isoforms of NO would preserve cellular metabolic function and improve s keletal muscle function. Male F344 x BN rats were treated with 20 mg/kg enalapril, 1 mg/kg LNAME, a combination of enalapril and LNAME, and placebo between 24 and 27 months of age. Evaluation of the TA muscle revealed that NOS activity was increased by enalapril treatment. However, L NAME in combination with enalapril reduced NOS activity and also reversed the decrease in body weight that is observed with enalapril treatment alone. Therefore, we may use these data as a rationale for an indepth evaluation of the bradykin pathways effects on skeletal muscle function and body weight. Bradykinin blockers such as HOE 140, a highly potent B2receptor antagonist with a long duration of action (Bao et al., 2000), may be used to address these issues. If our fut ure studies establish that enalapril significantly improves leptin signaling and subsequently reduces hyperphagia and weight gain in highfat fed aged animals, we will have a potential pharmacological intervention for effectively combating age-
54 related obes ity in humans. Consequently, we may observe a reduction in the pathologies associated with agerelated obesity, including metabolic dysregulation and inflammation. These health improvements in elderly individuals may reduce health care costs due to repeated hospitalizations, and potentially allow for increased independence and prolonged lifespan. Thus, it is imperative that we thoroughly investigate enalaprils full potenti al as a pharmacotherapy agent.
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60 BIOGRAPHICAL SKETCH Colleen Bond was born in Long Beach, CA, and lived in Negishi, Japan for seven years before relocating to Pensacola, FL in 1998. Colleen attended Pensacola High Schools International Baccalaureate Program and receiv ed the IB Diploma in May 2005. In June 2009, she graduated from the University of Florida with a Bachelor of Science degree in Integrative Biology. In May 2011, she received her Master of Science in Medical Sciences f rom the University of Florida.