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1 THE NPY SYSTEM: A NOVEL PHYSIOLOGICAL DOMAIN By MARIA DANIELA HURTADO ANDRADE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTO R OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Maria Daniela Hurtado Andrade
3 To my Mother Charlotte for all her love and support
4 ACKNOWLEDGMENTS I owe my grat efulness to all those people who have h el ped me along the long path of preparing my dissertation Thank to them my graduate caree r has been a unique experience that I will never forget I express my deepest gratitude to my advisor, Dr. Sergei Zolotukhin, who gave me the opportunity to join his l aboratory and work under his excellent guidance. I am grateful for his support, patience. Dr. Zolotukhin along with the rest of the laboratory personnel has provid ed me with an excellent atmosphere for doing research. He has always been available not only to discuss ideas related to the project but he has helped me overcome very difficult moments as well. I would like to thank Dr. Stephen Hsu, Dr. Philip Scarpace and Dr. Nicholas Muzyczka for guiding my research and for providing a continuous support for th e past years Special t hanks to Dr. Hsu, who without knowing me, was willing to participate in my committee and for this past two years, he has given me constant scientific and clinical support. I am grateful to Dr. Andres Acosta who as a good friend and a great colleague was always willing to help and give his best suggestions. He has taught me many scientific techniques and helped me to design this project. I would not be here if it was not for him. I would like to acknowledge the past and p resent membe lab oratory : George Aslanidi, Ramaz Geguchadze, Damien Marsic, Andrea Doty, D avid Duncan, Michael La Sala and Michael Spegele I am also extremely hankful to all the members of the Division of Cellular and M olecular Therapy of the Dep artment of Pediatrics at UF but especially to Dr. Arun Srivastava, our chair and, Krista Berquist, our fiscal I am also grateful to all the people that made this project possible : Amy
5 Wright, L i Zhang Dr. Martha Campbell Thomson Dr. Oleg Gorbatyuk Dr Valeriy Sergeyev Dr. Scott Herness Tamari Kholi Dr. Herbert Herzog Dr. Shawn Dotson Alicia Brown and all the Animals Care Service staff at the Cancer and Genetics Research Complex. Many friends have helped me immensely through these years not only academically but socially as well Their support and care has helped me overcome difficult moments during my graduate career I greatly value their friendship. I would finally like to thank my amazing parents, my brother and my sister. Along these years, they have always supported and encouraged me with th eir best wishes and love. A special thanks to my mother, who has always cheered me up and stood by me through the good and bad times.
6 TABLE OF CONTENTS page ACKNOWLEDG MENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATION S ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Field of Study ................................ ................................ ................................ .......... 15 Background and Hypothesis ................................ ................................ ................... 17 Significance ................................ ................................ ................................ ............ 18 2 LIT ERATURE REVIEW ................................ ................................ .......................... 20 Obesity ................................ ................................ ................................ .................... 20 Definition ................................ ................................ ................................ .......... 20 Epidemiology ................................ ................................ ................................ .... 20 Etiology and Risk Factors ................................ ................................ ................. 21 Physiopathology and Complications ................................ ................................ 23 Therapeutic Regimens ................................ ................................ ..................... 25 Ingestive Behavior ................................ ................................ ................................ .. 27 Hormonal Control of Ingestive Behavior ................................ ........................... 28 Adipose tissue signals ................................ ................................ ............... 28 Pancreatic signals ................................ ................................ ...................... 28 Gut signals ................................ ................................ ................................ 29 Central Integrating Circuits of Ingestive Behavior ................................ ............. 30 Hypothalamic neuronal pathways that regulate appetite ............................ 30 Hypothalamic R egulators of Appetite ................................ ......................... 31 The Oral Cavity and Ingestive Behavior ................................ ........................... 32 Orosensory exposure ................................ ................................ ................. 32 Saliva and taste perception ................................ ................................ ........ 33 Gastrointestinal hormones in the oral cavity ................................ .............. 33 The NPY System ................................ ................................ ................................ .... 34 NPY, PYY and PP ................................ ................................ ............................ 34 YRs ................................ ................................ ................................ .................. 36 Obesity and PYY ................................ ................................ .............................. 38 3 THE NEUROPEPTIDE Y SYSTEM IN THE ORAL CAVITY ................................ ... 39
7 Materials and Methods ................................ ................................ ............................ 40 In Vitro YR A ntibodies Validation ................................ ................................ ...... 40 Animals ................................ ................................ ................................ ............. 40 Tissues ................................ ................................ ................................ ............. 41 RT PCR ................................ ................................ ................................ ............ 41 Immunostaining ................................ ................................ ................................ 41 YRs immunostaining ................................ ................................ .................. 41 Y receptors/NCAM double immunost aining ................................ ............... 42 Cytokeratin 5 immunostaining ................................ ................................ .... 42 In Situ Hybridization ................................ ................................ ................... 43 Results ................................ ................................ ................................ .................... 43 ................................ ................................ .... 43 Expression of YRs in the Lingual Epithelia Cells ................................ .............. 44 Expression of YRs in the Taste Bud Cells ................................ ........................ 46 Expression of YR in SG ................................ ................................ .................... 47 Origin of YR2 ................................ ................................ ................................ .... 50 Expression of NPY, PYY and PP in the oral cavity ................................ ........... 50 Discussion ................................ ................................ ................................ .............. 51 The NPY System and Tongue Epithelium ................................ ........................ 51 The NPY System and Taste Tissue ................................ ................................ 53 The NPY System in SG ................................ ................................ .................... 54 4 THE ROLE OF SALIVARY PEPTIDE YY IN INGESTIVE BEHAVIOR ................... 69 Methods ................................ ................................ ................................ .................. 70 Mice ................................ ................................ ................................ .................. 70 Mouse Saliva Collection ................................ ................................ ................... 71 Plasma Collection ................................ ................................ ............................. 71 Plasma and Saliva Hormone Level s ................................ ................................ 71 Immunostaining ................................ ................................ ................................ 71 PYY3 36 Acute Augmentation Studies ................................ ............................. 73 Gen e Transfer Experiments ................................ ................................ ............. 74 Assessing Body Fat Mass In Mice ................................ ................................ .... 74 Statistical analysis ................................ ................................ ............................ 74 Results ................................ ................................ ................................ .................... 75 Dual Origin of Salivary PYY ................................ ................................ .............. 75 YR2 Is Expressed in the Basal Epithelial Cells of the Tongue .......................... 76 Oral PYY3 36 Augmentation Therapy ................................ .............................. 76 Long Term Increase in Salivary PYY3 36 Modulates FI and BW ..................... 78 Discussion ................................ ................................ ................................ .............. 80 5 SALIVARY PEPTIDE YY: PUTATIVE CIRCUIT THAT CONTROLS INGESTIVE BEHAVIOR ................................ ................................ ................................ ............. 96 Meth ods ................................ ................................ ................................ .................. 98 Animals ................................ ................................ ................................ ............. 98 Test Substances ................................ ................................ ............................... 98
8 Experimental Procedures ................................ ................................ ................. 99 Treatment with orally administered substances ................................ ......... 99 In vivo treatment of mice with 125 I PYY1 36 ................................ ............... 99 c Fos immunostaining ................................ ................................ .............. 100 Behavioral Studies ................................ ................................ ......................... 101 Liquid paradigm (Table 5 1, open cells) ................................ ................... 101 Solid food paradigm (Table 5 1, shaded cells) ................................ ......... 103 Statistics ................................ ................................ ................................ ......... 103 Res ults ................................ ................................ ................................ .................. 104 Salivary PYY3 36 Binds to Lingual YR2 Receptors ................................ ........ 104 Salivary PYY3 36 Activates Hypothalamic C Fos ................................ .......... 104 Effect of Salivary PYY3 36 on Brain Stem Neurons ................................ ....... 105 Salivary PYY3 36 Does not Induce CTA ................................ ........................ 107 Discussion ................................ ................................ ................................ ............ 109 C Fos in Fasted vs. Fed Control Animals ................................ ....................... 111 C Fos in Fasted and Fed vs. PYY i.p. Animals ................................ .............. 111 C Fos in PYY i.p. vs. PYY OS Animals ................................ .......................... 112 Conditional Taste Aversion ................................ ................................ ............. 114 6 CONCLUSIONS ................................ ................................ ................................ ... 125 The NPY System in the Oral Cavity ................................ ................................ ...... 125 Role of Salivary PYY ................................ ................................ ............................. 126 Salivary PYY: A Putative Circuit that Regulates Ingestive Behavior ..................... 127 Salivary PYY and Taste Perception ................................ ................................ ...... 128 L IST OF REFERENCES ................................ ................................ ............................. 129 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 148
9 LIST OF TABLES Table page 3 1 Gene spec ific primers used in RT PCR ................................ .............................. 56 3 2 Antibodies used for immunostaining studies ................................ ...................... 56 4 1 Antibodies used for immunostaining studies ................................ ...................... 84 5 1 Schematic timeline of the CTA trials with liquid (open cells) or solid food (shaded cells). ................................ ................................ ................................ .. 116
10 LIST OF FIGURES Figure page 3 1 Validation of YR antibodies.. ................................ ................................ ............. 57 3 2 Expression of the NPY system in the oral cavity analyzed by reverse transcriptase (RT) PCR.. ................................ ................................ .................... 58 3 3 Immunostaining of Y1, Y2, Y4 and Y5 receptors in the dorsal epithelium of a tongue.. ................................ ................................ ................................ .............. 59 3 4 Immunostaining of Y4 receptors in the d orsal epithelium of a tongue.. .............. 61 3 5 Immunostaining of YRs in TRCs.. ................................ ................................ ....... 62 3 6 Y2 receptor is synthesized in the epithelial cells of the tongue.. ......................... 63 3 7 SG immunostaining. ................................ ................................ ........................... 64 3 8 Characterization of YR2 cells in the SG and co staining with smooth muscle acti ng.. ................................ ................................ ................................ ................ 65 3 9 YR2 In situ hybridization. ................................ ................................ .................... 66 3 10 A subpopulation of epithelial progenitor cells in the tongue epithelia expresses YR2. ................................ ................................ ................................ .. 68 4 1 PYY is synthesized in TRCs.. ................................ ................................ ............. 85 4 2 gustducin (red) by co immunostaining in the same taste bud.. ................................ ................................ ........................... 86 4 3 Y2 receptor is synthesized in the epithelial cells of the tongue. .......................... 87 4 4 Neuronal filaments innervate CV papillae. ................................ .......................... 88 4 5 Oral PYY3 36 augmentation therapy. ................................ ................................ 89 4 6 Effect of PYY gene transfer to the SG in C57Bl/6J mice. ................................ ... 93 5 1 Salivary PYY binds to Y2 receptors in the tongue epithelia. ............................. 117 5 2 Effect of PYY3 36 OS on c fos expression in the arcuate nuclei (Arc, top row), paraventricular nuclei (PVN, middle row), and the lateral hypo thalamic area (LHA, bottom row) ................................ ................................ .................... 118 5 3 Effect of PYY3 36 OS on c fos expression in the rostral area of the nucleus of solitary tract (NST). ................................ ................................ ....................... 119
11 5 4 Effect of PYY3 36 OS on c fos expression in the caudal area of the nucleus of s olitary tract (NST) and the area postrema (AP).. ................................ ......... 120 5 5 Effect of PYY3 36 treatment on aversive response ................................ .......... 121 5 6 Diagram displa ying main putative anorexigenic pathways originating in the tongue epithelia and/or TRCs ................................ ................................ ........... 123
12 LIST OF ABBREVIATION S AP Area Postrema BW Body Weight CCK Cholecystokinin CTA Conditioned Taste Aversion CV Circumvallate FI Food Intake GLP 1 Glucagon Like Peptide 1 GPCR G Protein Coupled Receptor i.p. Intraperitoneal NPY Neuropeptide T OS Oral Spray PYY Peptide YY SG Salivary Gland TRC Taste Receptor Cells VIP Vasoactive Intestinal Peptide YR Y Receptor
13 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 THE NPY SYSTEM: A NOVEL PHYSIOLOGICAL DOMAIN By Maria Daniela Hurtado Andrade August 2012 Cha ir: Sergei Zolotukhin Major: Medical Sciences Physiology and Pharmacology Members of NPY family genes are represented by well characterized hormones Neuropeptide Y (NPY), Peptide YY ( PYY ), Pancreatic Polypeptide ( PP ); and their receptors YR 1, YR 2, YR 4, an d YR 5. These genes are vastly expressed in the brain and the periphery mediating multiple metabolic functions. Recently, we have shown the presence of PYY in saliva, and the expression of its preferred receptor, YR2 in the lingual epithelia. In the current report, we extend our finding to all main NPY family members and we characterize, for the first time, their expression in the lingual basal cell epithelia and in the TRC s in mice. To investigate the possible role of salivary YR signaling in energy metabol ism, we have focused on PYY PYY a hormone that induces satiety, is synthesized in L endocrine cells of the gut. It is secreted into circulation in response to food intake and induces satiation upon interaction with its cognate YR2 Herein we demonstrate that the acute augmentation of salivary PYY induces stronger satiation as demonstrated in feeding behavioral studies. The effect is mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial cells. In a long term study involving PYY deficient mice, a sustained increase in PYY was achieved using viral vector mediated
14 gene delivery targeting salivary glands. The chronic increase in salivary PYY results in a significant long term reduction in body weight gain. The anorexig enic action of salivary PYY is corroborated by an increase in neuronal activity in satiety centers. In fact, we describe a novel neural circuit that is activated in response to the acute pharmacological augmentation of salivary PYY This putative metabolic pathway is associated with YR2 (+) cells in the oral cavity and extends through brainstem nuclei into hypothalamic satiety centers. Remarkably, orally applied PYY while inducing a strong anorexic reaction, does not induce taste aversion. Thus this study p rovides evidence for a novel physiological domain for the NPY system. The discovery of the new functions of the previously characterized gut peptide PYY and the description of this alternative metabolic pathway, which regulates ingestive behavior, reinstat es the potential of PYY for the treatment of obesity.
15 CHAPTER 1 INTRODUCTION Field of Study Obesity and its related complications including dyslipidemia, insulin resistance, hypertension type 2 diabetes, and atherosclerosis is associated with high mor bidity and mortality ; it is the most important non communicable pandemic worldwide. During the last decade, its prevalence has increased dramatically, especially in large populations in the developing world where the widespread adoption of a Western diet a nd increasingly sedentary lifestyle has become the norm. The result is excessive fat accumulation in the body to such an extent that the risk of developing a medical condition increases significantly. Such medical conditions include but are not limited to the development of a variety of cardiovascular, musculoskeletal, dermatological, gastrointestinal, endocrine, respiratory, reproductive, neurologic, psychiatric and oncologic disorders. Most cases of obesity arise from a combination of a diet composed of energy dense foods (i.e. fat and sugars) and a sedentary lifestyle. Obesity is the result of an i nequality between energy intake and expenditure that leads to the storage of fat mainly in adipose tissue. In theory, obesity could be managed with adequate n utrition and a regular exercise program, nevertheless, for some reason it is hard to comply with these relatively simple measures in a long term run As a consequence obesity and its related complications represent a medical and economical burde n worldwid e. Thus, the pharmaceutical industry has significantly invested to develop drugs to treat this condition. Potential sites of therapeutic intervention include all neuroendocrine signals that regulate ingestive behavior.
16 Ingestive behavior is the most essent ial behavior since it is required for surviv al Appetite and satiation are fundamental components of the ingestive behavior; however taste plays an imp ortant role in its regulation as well. On one hand, taste is imperative for the evaluation of food comp onents quality Taste quality detection begins in taste receptor cells (TRC) which contain specific taste receptors Once food in ingested, those receptors relay a signal to the brainstem and higher centers in the central nervous system. There is evidence pointing towards the fact that taste function and perception can be modulated by several compounds, including drugs and hormones, that inter act with the ir respective receptors present in the oral cavity. Modulation of the palatability of diffe rent tastants may be a target for changing taste responsiveness and therefore this could regulate ingestive behavior as an alternative treatment for obesity. Conversely appetite and satiation are mainly regulated by the brain gut axis, which consists of the gastroin testinal system, the vagal complex, the brainstem, the hypothalamus and higher centers in the cortex. The gastrointestinal tract is the largest endocrine organ in the body. All hormones secreted by the gastrointestinal tract are essential to the regulation of body weight (BW) and energy homeostasis. During the last decade, the increased understanding of the role of gastrointestinal peptide hormones has led to the development of strategies to modulate their circulating levels as another potent ial strategy fo r treating obesity. Among these, Peptide tyrosine tyrosine ( PYY ) which belongs to the PP fold family of peptides has recently generated a lot of interest for its role in energy homeostasis. PYY is a gastrointestinal hormone secreted in response to food i ntake (FI) mainly by
17 specialized L endocrine cells of distal intestine and colon epithelia. Over the last decade, investigators have demonstrated that PYY is an important modulator of satiation. Through binding to its cognate receptor, the Y2 receptor ( YR 2 ), PYY contributes to the regulation of both short term postprandial satiation and long term BW regulation. Ther p eripheral administration of PYY results in a significant reduction of FI and BW Interestingly, PYY and other gastrointestinal hormones such as glucagon like peptide (GLP 1) cholecystokinin ( CKK ), vasoactive intestinal peptide ( VIP ), insulin, leptin and neuropeptide Y ( NPY ), have also been shown to be present in human and murine saliva. Th ese gastrointestinal hormones present in saliva appear to have another physiological effect besides the peripheral regulation of FI It has been shown that NPY GLP 1 CKK VIP and leptin, which are either produced and secreted locally by TRCs or transported into saliva from plasma, play an important role in modulating the function of the gustatory system by interacting with their respective receptors and therefore affecting ingestive behavior. Little is known about the role of salivary PYY Background and Hypothesis It has been more than a decade since it w as demonstrated that PYY has a physiological role in FI Batterham and her group showed at that time that the acute peripheral administration of PYY resulted in significant reduction of FI and BW (Batterham et al., 2002) suggesting its potential therapeu tic application for obesity treatment. To confirm these results, Acosta (2011) performed a more detailed study of the role of PYY in regulating feeding behavior, satiety an d energy homeostasis. Acosta showed that (1) PYY 3 36 is present in human and murine saliva, (2) a short term increase in the amount of this hormone in saliva by an oral spray (OS) resulted in a temporal decrease of FI in rodents, (3) the sustained expression of a PYY transgene in
18 salivary gland s (SG) cells resulted in a long term signific ant loss of BW and (4) the increase of PYY concentration in saliva did not increase its plasma concentration. From these results, we hypothesized that salivary PYY regulates ingestive behavior through an alternative pathway that has not been previously de scribed. The circuit would originate in the sensory nerves of tongue epithelium and would project via the lingual branch of the glossopharyngeal and the facial nerve s into the brain. This hypothesis was also supported by the observation that the YR2 gene, by encoding the cognate receptor for PYY 3 36, is expressed in cells of the tongue epithelium, as Acosta determined by Reverse Transcriptase Polymerase Chain Reaction (RT PCR). Significance The overall goal of the current dissertation is to conclusively d emonstrat e the existence of dual signaling pathways by clearly delineating the effects of salivary versus systemic PYY in the murine model. The proposed alternative mechanism that modulates ingestive behavior through the initiation of hormonal signaling in the oral cavity has important implications for the development of a nov el therapy for human obesity In the Chapter 2 we provide a review of the literature on obesity We also discuss the literature on the control of ingestive behavior by appetite, satiat ion and orosensory exposure to food Finally in Chapter 2, we also review the literature on the NPY system. Chapter s 3, 4 and 5, describe the s pecific aims of the project. In each o f those we briefly provide an introduction, d escribe the methods and res ults, discuss the data related to the specific aim and present tables and figures Specifically, C hapter 3 describe s the discovery of the expression of genes coding for the peptides of the NPY system in the oral cavity as well as their most studied Y rece ptors (YR) YR1 YR2
19 YR4 and YR5 C hapter 4 de scribes the role of salivary PYY on ingestive behavior and C hapter 5 describes the neural circuitry involved in the regulation of ingestive behavior by salivary PYY Finally, in C hapter 6, we discuss the res ults, conclusions and further research of the entire study. All C hapters (except C hapter 4, already published) will be submitted as stand alone manuscripts and are presented here as currently written for publication. The refore, there is some overlap in des cription s, and these will be modified/eliminated at the time of submission of each manuscript. One manuscript has been already published and has been reprinted with permission from Acosta, A ., Hurtado, M.D ., Gorbatyuk, O ., La Sala, M ., Duncan, D ., Aslanidi, G Campbell Thompson, M ., Zhang, L ., Herzog, H ., Voutetakis, A ., et al. (2011). Salivary PYY: a putative bypass to Satiety. PLoS One 6(10):e26137.
20 CHAPTER 2 LI TERATUR E REVIEW Obesity Definition Obesity and overweight are defined as the amount of body fat at which health risks to individuals begin to increase. The body mass index (BMI) is the index used to classify normal weight, overweight and obesity and it is calcula ted by dividing the weight in kilograms by the square of the height in meters (kg/m 2 ). For the adult population, according to the World Health Organization (WHO), overweight corresponds to a BMI between 25 and 30 and obesity to a BMI greater than 30 (WHO). For the pediatric population the Centers for Disease Control (CDC) has defined overweight as a BMI between the 85th 95th percentile and obesity as a BMI greater than 95th percentile for age and sex (Pavkov et al., 2006). Epidemiology Obesity and its rel ated complications including dyslipidemia, insulin resistance, hypertension type 2 diabetes, and atherosclerosis is associated with high morbidity and mortality and is the most important non communicable pandemic worldwide. During the last decade, the pr evalence of overw eight/obesity has continued to increase around the world The WHO indicated in 2005 that 1.6 billion adults were overweight and at least 400 million were obese The epidemiologic profile predicts that by 2015, approximately 2.3 billion adults will be overweight and 700 million will be obese (WHO 2004). Children and adolescents are also affected and the trend in obesity in this population is especially alarming. The annual prevalence increase rate of childhood and
21 adolescent obesity has been growing steadily. In the United States, in 2003 2006, obesity rate was 17.6% among youths 12 19 years of age (Ogden et al., 2008) and 32 % of children and adolescents were observed to be overweight (Kuczmarski et al., 2000; Ogden et al., 2008). This nu mber has more than tripled during the last forty years (Ogden et al., 2006). There is substantial evidence that data published are predictive of future rates of obesity and overweight (International Journal of Obesity 2012). T he economic costs of overweigh t and obesity are increasing just as health consequences. O besity in some developed countries, account s for 5 10 % of the total health care costs (Wolf et al., 1998). Furthermore obesity has a major impact on mortality; 300000 deaths each year are ascribe d to overweight obesity their complications and associated diseases in the United States This way behind tobacco use obesity has become the second leading cause of preventable death worldwide (Allison et al., 1999). Etiology and Risk Factors Most case s of obesity arise from a combination of a diet composed of energy dense foods (i.e. fat and sugars) and a sedentary lifestyle. Obesity is the result of an i nequality between energy intake and expenditure that leads to the storage of fat mainly in adipose tissu e The etiology of this balance shift has not being completely clarified due to the complexity of energy balance regulation. A grosso modo genetic and environment/socio cultural factors play a major role but neuroendocrine, metabolic and psychologica l factors are clearly involved as well. E vidence exists suggesting that within the same population BMI variation is largely genetically determined (60% to 80%) (Wardel et al., 2008). Genes have minimally changed over the last four decades yet the prevale nce of overweight and obesity has
22 dramatically increased The only explanation for this observation is the change in environmental factors affecting energy homeostasis. There have been identified hundreds of genetic loci that are involved in body weight (B W) regulation in humans and other species (Rankinen et al., 2006; Crino et al., 2003). Genetic mutations can affect gene function so dramatically that results in an obese phenotype without any particular environmental obesogenic condition Nevertheless, the majority of genetic factors affect BW enough to cause obesity only when specific obesogenic conditions are present. S ocietal changes during the last couple of decades are playing a major role in the development of obesity. Eating behavior and physica l activity level are largely influenced by the environment On one hand, ingestive behaviors changes that have promote d obesity over the last decades include consumption of fast food meals (Pereira et al., 2005), consumption of oversized proportions (Young et al., 2002) and intake of sweetened beverages (Ebbeling et al., 2006). On the other hand, low levels of physical activity have been promoted by an automated and automobile oriented environment that leads to a sedentary lifestyle (Ebstein et al., 2000) Increase in energy intake and decrease in energy expenditure are the two main features of the so called obesogenic environment that has a major role in determining who becomes obese. Whifley has largely described a Social Ecological Model of influences on obesity According to her, environmental influence begin with the family (e.g. breast feeding and socioeconomic status) and extend s to peers (e.g. role models) as well as neighborhoods (e.g. safe areas that encourage physical activity), schools (e.g. s ports clubs) and national factors (e.g. food policies and taxes) (Crocker et al., 2011).
23 Other determinant factors that are also known to influence BW are (1) endocrine disorders such as hypothyroidism (Ning et al., 2006), growth hormone deficiency (Hoos e t al., 2003), Cushing syndrome (Ottosson et al., 2000) and insulinoma (Bonfig et al., 2007), and structural disorders of the hypothalamus (e.g. injury or congenital malformation) (Woods et al., 2008); (2) allelic variations in genes that participate in ene rgy homeostasis such as inactivating mutations affecting leptin (Farooqi et al., 2007), its receptor (Farooqi et al., 2007), or any other molecule involved in the leptin signaling pathway (Lee et al., 2008; Farooqi et al., 2007; Feng et al., 2005; Yeo et a l., 2004); (3) common single nucleotide polymorphisms (SNP) in the FTO gene locus (Frayling et al., 2007); (4) gain of function mutations of molecules that regulate fat storage such as Peroxisome Proliferator Activated Receptors (PPAR ) (Celi et al., 2002 ); (5) multipl e genetic syndromes that in clude obesity as part of their clinical presentation such as Prader Willi (Cummings et al., 2002); (6) medications such as antidepressants, mood stabilizers, antihistamines, insulin or insulin secretagogues, antihyp ertensives, glucocorticoids, antipsychotics anticonvulsants, and chemotherapeutic agents (Aronne et al., 2003); (7) the obesity virus that belongs to the adenovirus family of viruses and has been shown to cause increased fat storage in infected animals (Psarica et al., 2008); and (8) epigenetics which refers to the differential gene expression in response to environmental conditions usually occurring during the perinatal period (Waterland et al., 2005; Wu et al., 2006). Physiopathology and Complications A dipo se tissue besides being an storage site for excessive calories is also a n active endocrine organ. A great variety of biologically active substances is synthesiz ed
24 by adypocites (Kershaw et al., 2004). Plasma free fatty acids (FFA) levels, which are a lso elevated in most obese subjects, appear to be the trigger of the proinflammatory cascade. FFA have been shown to activate the NF pathway thereby increasing other proinflammatory cytokines expression such as TNF IL6 and MCP1 (Boden et al., 1991). Interestingly, MCP1 participates in macrophage recruitment to sites of inflammation and is involved in monocyte migration to adipose tissue of obese animals where after differentiation into macrophages, they produce even more proinflammatory cytokin es (Rollins et al., 1991). Another mechanism in which FFAs are involved and contribute to the proinflammatory state is cellular stress. Adipocytes containing excess of fat express over reactive NADPH oxidase that, results in reactive oxygen species overpro duction. The net result is dysregulated production of additional proinflammatory cytokines (Furukawa et al., 2004). Cellular stress, proinflammatory cytokine production, the release of a range of other metabolic factors such leptin, resistin and adiponecti n (Kershaw et al., 2004), predispose obese individuals to insulin resistance by poorly understood mechanisms (Greenberg et al., 2006). Insulin resistance is when insulin concentration in plasma produces a less than expected biological effect and is of con siderable clinical relevance due to its association with a variety of serious medical problems that are highly prevalent among obese subjects These problems include but are not limited to dyslipidemia atherogenesis, type 2 Diabetes Mellitus, non alcoholi c fatty liver disease hypertension, and coagulation and fibrinolysis abnormalities (Bray 2004). Even though insulin resistance has many causes, obesity is by far the most common cause (British Heart Foundation 2008).
25 Furthermore, insulin resistance result s in hyperinsulinemia to overcome high blood levels of glucose. Insulin contributes to the synthesis and activity of insulin growth factor (IGF) I and II. IGFs can stimulate proinflammatory cytokines and growth factors to enhance their mitogenic effect (Sa chdev et al., 2001). Thereby, hyperinsulinemia can indirectly affect tumorigenesis. The relationship between breast (Dirat et al., 2010), endometrial (Danaei et al., 2005) and colon cancer (Mc Tiernan et al., 2005) is well documented in obese subjects. Oth er complications of obesity and overweight are: (1) respiratory problems such as decrease in thoracic distensibility resulting in collapse of the small airway (De Lucas Ramos et al., 2004), asthma (Sood A. 2010) and sleep apnea (Carter et al., 2008); (2) g astrointestinal problems such as diminished lower esophageal sphincter pressure, esophageal motor disorders and development of hiatal hernia which result in gastroesophageal reflux disease (Ayazi et al., 2009); (3) G allbladder disease (Stampfer et al., 200 9) (4) renal disease including chronic kidney disease (Sivestava T. 2006) and renal cell carcinoma (Pan et al., 2006); (5) osteoarthritis (Cicuttini et al., 1996); (6) psychopathological disorders such anxiety, depression and obsessive compulsive disorder (Rosik et al., 2005; Tunkard AJ.2002); (7) obstetric complications (Dietl 2005); (8) and gynecological disorders like infertility (Green et al., 1988) and Polycystic ovary syndrome ( The Rotterdam ESHRE/ASRM sponsored PCOS Consensus Workshop Group, 2004 ). T herapeutic Regimens The e pidemic profile of overweight and obese population along with their associated comorbidities has led to the development of a great diversity of therapies aimed at weight loss. To effectiv ely treat obesity, therapeutic regimens shou ld in clude
26 several approaches that include lifestyle modification, pharmacotherapy and in some cases surgery. L ifestyle modification i ncluding diet and other behavioral changes such as exercise, is an approach tha t consistently results in short term BW re duction. However, long term effects are not reliable because of the difficulty of maintaining such lifestyles (Sjostrom et al., 2004; Knowler et al., 2002; Togerson et al., 2004). Independently, physical activity does not produce significant body weight lo ss However, exercise i s an excellent adjuvant therapy to a weight reducing diet (Jakicik et al., 1999). Pharmacologic treatment of obesity has evolved in the past decades and may be used when non pharmacologic approaches alone. Drugs prescribed for weight loss can either suppress appetite or inhibit nutrients absorption. It has been demonstrated that adding pharmacological support to lifestyle modification can result in an extra weight loss (Li et al., 2005; Scheen et al., 2006; Pi Sunyer et al., 2006; Van Gaal et al., 2005) but it has been reported that drug cessation leads to weight regain. In contrast to pharmacological measures, studies have demonstrated that surgery for obesity results in a more sustained body weight loss (Bult et al., 2008). In spite of weel documented complications (Flum et al., 2005 ), t here is strong evidence suggesting that bariatric surgery is the most effective long term treatment for obesity. Current accepted non absolute criteria for bariatric surgery include patients with a B MI of more t 2 2 with concomitant obesity related conditions (Dixon et al., 2005). Despite the success of some of these therapies, the treatment of obesity remains a major challenge. The rising obesity figures an d associated he alth complications
27 present medical needs for effective and safe n ovel and alternative drug therapies. Currently, a remarkably wide diversity of targets and compounds is being investigated and the main targets in the central nervous system in clude serotonin and Y receptors (YR) and in the periphery, neuroendocrine peptides that modulate appetite and satiation such as glucagon like peptide (GLP 1) and PYY (Witkamp 2011). Ingestive Behavior Ingestive behavior is the most essential behavior sinc e it is required for surviv al. Understanding the mechanisms that regulate eating behavior is essential to develop alternative therapies to treat obesity, which has been denominated the epidemic of the 21st century. Ingestive behavior is regulated to mainta in energy balance by peripheral and central mechanisms. Although energy homeostasis is a vital mechanism for regulating ingestive behavior, taste and sensory exposure of f ood in the oral cavity also pla y an essential ro le in food ingestion regulation Even though obesity has a complex, multifactorial and not entirely elucidated etiology, it is well established that most cases arise from a combination of a diet composed of energy dense foods (i.e. fat and sugars) and a sedentary lifestyle. As mentioned befor e, obesity is the result of an i nequality between energy intake and energy expenditure that leads to fat storage mainly in adipose tissue. Several efforts have been focused on developing drugs that target factors that regulate ingestive behavior for treati ng obesity. we herein review what is known about some of these factors.
28 Hormonal Control of Ingestive Behavior A cardinal function of the nervous system is to coordinate a variety of processes that permit to maintain optimal levels of circulating and stor e d energy rich nutrients in response to adiposity and gastrointestinal signals. Adipose tissue signals It exists evidence that a dipose tissue is an active endocrine organ and is not sought anymore as an inert organ, solely for the storage of energ y Three hormones secreted by ad i pocytes participate in energy homeostasis: leptin (Maffei et al., 1995), adiponectin (Qi et al., 2004) and resistin (Sul 2004). Only Leptin regulates ingestive behavior. The leptin receptor is expressed widely within the hypothalamu s (Faouzi et al., 2007). After crossing the blood brain barrier (BBB), leptin inh ibits the activity of neuropeptide Y (NPY) / agouti related peptide (AgRP) neurons and reduces the expression of these two orexigenic peptides (Cowley et al., 2001) Addionally leptin stimulates proopiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART) neurons which are known to be anorexigenic Exogenous leptin administration, both centrally and peripherally, reduces food intake (FI) and when administ ered chronically results in loss of weight (Ahima et al., 1996; Halaas et al., 1995). Pancreatic signals There are two pancreatic peptides that have a role on FI : Insulin and Pancreatic polypeptide (PP) I t has been demonstrated that i nsulin acts as an an orectic signal within the brain ( Air et al., 2002). PP belongs to the NPY family of peptides which also includes PYY and NPY Circulating PP does not cross the BBB but exerts an ano rectic
29 effect via the vagal nerve to the brain stem and then to the hypothalamus which results in reduction of NPY and orexin mRNA expression (Asakawa et al., 2003). Gut signals The gastrointestinal tract is the largest endocrine organ in the body. Almost all hormones secreted by the gastrointestinal tract exert an important role in the regulation of BW and energy homeostasis. All are anorexigenic factors except for ghrelin which is the only known peripheral orexigenic compound. Ghrelin is a potent orexigen ic factor, produced and released primarily by the gastric oxyntic cells (Sakata et al., 2002). This peptide initiates hunger (Shrestha et al., 2004). A dministration of ghrelin in duces c fos expression a marker for neuronal activation, in NPY /AgRP neurons of the arcuate nucleus (Nakazato et al., 2004). PYY as PP is a member of the PP fold family of peptides and is released post prandially from L cells of the gastrointestinal tract ( Ekblad et al., 2002 ). PYY through interaction with its cognate Y2 receptor i s a potent anorectic sign al., Unlike PP PYY can cross the BBB and results in a decreased in hypothalamic NPY mRNA expression and activation of arcuate neurons expressing POMC (Challis et al., 2003). The gene encoding for preproglucagon is vastly expresse d in the pancreas, in the L endocrine cells of the small intestine, and in the brain stem (Tang et al., 2001). By the action of pro hormone convertases 1 and 2 preproglucagon is cleaved to produce glucagon in the pancreas (Holst et al., 2004). GLP 1 is se creted by the L endocrine cells of the sm all intestine following food ingestion and acts to inhibit FI via the vagus nerve (Turton et al., 1996) which further relays the signal to the hypothalamus to activate POMC neurons (Sandoval et al., 2008).
30 OXM like GLP 1 is released from the L cells (Ghatei et al., 1983) and its exogenous administration to rodents, reduce s FI BW gain and adiposity (Dakin et al., 2004). Cholecystokinin ( CKK ) is released after meal initiation and in addition to decrease the motility o f the gastrointestinal tract, it inhibits FI by interaction with central nervous system receptors (Kissileff et al., 1981) through activation of the vagus nerve which then relays the information to the hypothalamus (Schwartz et al., 2000). CKK has also bee n showed to act as a neurotransmitter that plays a role in memory, anxiety, and reward behavior (Crawley et al., 1994). Central Integrating Circuits of Ingestive Behavior The brain is a major player in th e control of energy homeostasis The central nervous system integrates hormonal and neuronal information from the periphery Hypothalamic neuronal pathways that regulate appetite Several hypothalamic regions appear to play a cardinal role in BW regulation. The arcuate and the paraventricular nuclei are the best understood. In the arcuate nucleus, two primary neu ronal populations play a crucial role in the coordination of incoming signals regulating satiety and appetite. A portion of neurons located in the medial arcuate express and produce NPY and AgRP, wel l characterized orexigenic neuropeptides (Hahn et al., 1998). Neuronal projections from this subpopulation relay the signal primarily to the ipsilateral PVN (Martin et al., 2007). A second group of neuron is located m ore laterally and their role is to prim arily inhibit FI through CART and POMC expression (Naslund et al., 2007). Neuronal projections from this second subpopulation of the arcuate relay the signal more widely within the brain not only to the PVN but also to the DMH and LHA (Mihaly et al., 2001 ). It has been proposed that the a rcuate
31 nucleus is the primary site of act ion of peripheral hormones like leptin, ghrelin, GLP 1 insulin, and PYY The arcuate acts to coordinate other nuclei in the hypothalam us to adjust energy intake and expenditure an d thus, BW The PVN receives signals from the arcuate nucleus brain stem and other CNS structures (Sawchenko et al., 1983). This nucleus also integrates endocrine functions influenced by many neuropeptides. NPY /AgRP and MSH signal from the arcuate nucleu s, regulates thyrotropin releasing hormone (TRH) and corticotropine releasing hormone (CRH) neurons in the PVN (Fekete et al., 2000). NPY /AgRP inhibits TRH and CRH gene expression (Fekete et al., MSH stimulates their expression (Sarkar et al., 2003). Hypothalamic Regulators of Appetite There are three important central nervous mediators that control energy homeostasis: NPY the melacortin system and CART. NPY is one of the most abu ndant neurotransmitters in the CNS and is the most potent orexigen known and will be described in the next section along with the PP fold family of peptides. The melanocortin system includes products of POMC cleavage and the endogenous melanocor tin antago nists AgRP and agouti (Schwartz 2006). MSH is t he endogenous ligand for the MC3R/MC4R that are highly expressed in the arcuate nucleus (see above). The melanocortin system has been shown to activate the thyroid axis (Kim et al., 2000), to induce feeding to stimulate oxygen consumption (Pierr oz et al., 2002), and to stimulate brown adipose tissue (Yasude et al., 2004) and sympathetic ne rvous activi ties Agouti and AgRP are the only known endogenous antagonists of melanocortin receptors (see above) (Ollman et al., 1997). AgRP and NPY co locali zed in the majority
32 of cells in the CNS (Hahn et al., 1998) and activation of these neurons stimulates feeding (Roseberry et al., 2004) CART is expressed in mostly with POMC in the arcuate (Simpson et al., 2009). Even though the mechanism has not been com pletely elucidated, data has demonstrated the existence of di fferent hypothalamic neuronal circuits where CART can either act as an orexigenic or as an anorexigenic signal (Simpson et al., 2009). The Oral Cavity and Ingestive Behavior The o ral cavity play s a key role on ingestive behavior. Orosensory exposure, saliva and taste perception are three components of the oral cavity that regulate FI Orosensory exposure It is well known that liquids have a weaker suppressive appetitive responses compared to soli ds (Tsuchiya et al., 2006). Even though the mechanism is not well understood, it has been suggested that the faster transit of fluids compared to solids leads to less time of sensory exposure in the oral cavity, referred as orosensory exposure. Orosensory exposure to food or liquids is indeed an important factor that regulates appetite and satiation (Zijlstra et al., 2008 ; Lavin et al., 2002). The longer the produ c t stays in the mouth, the longer the time of orosensory exposure i.e. more exposure to smell, taste, texture, and other properties of food. Ziljstra (2009) ha s shown that greater orosensory exposure leads to earlier sensory satiation a nd therefore to smaller meal size i.e. oral exposure is an important factor in FI regulation. Orosensory exposure c omprises important sensory cues such as taste and texture that initiate a cascade of pre absorptive physiological responses, known as cephalic phase responses (CPRs). CPRs function to prepare the gastrointestinal tract for optimal
33 digestion and absorption of nutrients (Nederkoom et al., 2000). Ultimately, c ephalic stimulation stimulates de vagal complex (Teff et al., 2000) and results in a great diversity of autonomic responses mostly related to an increase in gastrointestinal secretions (e.g. gastric and pancreatic fluids and saliva) (Smeets et al., 2010). Saliva and taste perception The most obvious cephalic reflex is that of increased salivation, which can be observed by anyone at the mere thought of food. Salivary flow increases strongest once food ent ers the mouth and its composition varies with the kind of stimulus (Mattes et al., 2000). Pedersen ha s shown that saliva plays an essential role in taste perception and contributes to sensory stimulation (Pedersen et al., 2002) There is strong evidence s upporting that taste perception plays a role on the development of obesity. The food supply in the Western world, in particular, is characterized by a large variety of palatable foods that are energy dense and easy to consume. This diet promotes a positive energy balance, because it s satiating effect per unit of energy provided is low. One of the principal reasons for the low satiating effect of caloric liquids, fast foods, and foods with invisible fats may be insufficient or inadequate sensory signaling fr om the mouth during consumption (Smeets et al., 2010). Gastrointestinal hormones in the oral cavity Both saliva and blood serum contain similar proteins, peptides, steroidal hormones and RNA, which is why saliva is considered a mirror to the body. (Schi pper et al., 2007). To support this metaphor, recently, PYY (Acosta A. 2009) and other gastrointestinal hormones such as GLP 1 CKK vasoactive intestinal peptide ( VIP ), insulin, leptin and NPY have also been shown to be present in saliva ( Groschl 2008 ). These peptides are all transported into saliva from plasma and some of them including
34 GLP 1 CKK and NPY are expressed in the tongue epithelium, in specific types of TRCs of the taste buds which are the specialized anatomical structures in the oral cavity that detect chemical stimuli and originate the sensation of taste (Herness et al., 2009; Shin et al., 2008). These peptides were shown to participate in the taste quality information processing: they modulate the perception of the different tastes by incr easing or decreasing their sensitivity i.e. perception The presence of gastrointestinal peptides along with their receptors in taste buds adds more evidence to the previously described similitude between the gustatory and the gastrointestinal systems. Th e evidence suggests that the taste bud may serve as an important target for positive and negative modulators of taste sensitivity, thus providing a peripheral metabolic stat e. (Shin YK et al 2008). T aken together a ll th is evidence suggests that by modulating the concentrations of gastrointestinal hormones present in saliva, and by chang ing taste responsiveness it is possible to regulate ingestive behavior. Modulating ta ste perception through this mechanism may provide a new target for the treatment of obesity. The NPY System The NPY system comprises of NPY PYY and PP that interact with the four YR s subtypes in overlapping and redundant manner NPY PYY and PP NPY PYY and PP belong to a family of peptides sharing similar hairpin like PP fold structural homology and evolutionary history (Zhang et al., 2011). These peptides mediate various complementary and often opposing metabolic functions such as appetite and satiatio n, energy intake and expenditure; cell proliferation, migration, and
35 differentiation; neuromodulation, angiogenesis, osteogenesis, and many other biological processes. NPY is a 36 amino acid peptide widely expressed in the mammalian nervous system, with h igh levels in brain regions such as the hypothalamus and the limbic system (Kask et al., 2002). NPY is an important neuromodulator in the brain and has long been implicated as being one of the body's most potent orexigenic factors (Boguszewsk et al., 2010) Other physiological functions in which NPY has been implicated are: metabolic functions, circadian rhythm, cognition, neuronal excitability and addictions and modulation of emotional responses to various stressors (Cohen et al., 2012). PYY is a 36 amino acid peptide produced mainly by the L endocrine cells of the gut (Adrian et al., 1985). Two endogenous forms, PYY 1 36 and PYY 3 36, are released in response to food ingestion into the circulation. PYY 1 36 is cleaved by dipeptidyl peptidase IV (DPP IV) in t he amino terminal to form PYY3 36 PYY 1 36 is the most abundant form during the fasted state, whilst PYY3 36 predominates in the circulation after food intake (Grandt et al., 1994). Immediately after meal initiation, PYY 3 36 levels rise within 15 minutes, peak at 90 minutes and remain high for up to 6 hours (Adrian et al., 1985). The increase in PYY 3 36 concentration is directly proportional to the amount of calories ingested (Degen et al., 2005 ; Essah et al., 2007). PYY 3 36 is an anorexigenic hormone and e xerts its effect directly in the central nervous system and also via its effects on g astrointestinal motility through the vagus nerve (Batterham et al., 2002 and 2003). Centrally, it has been shown that PYY 3 36 inhibits appetite mainly by direc t interactio n with the Y2 receptor i n the arcuate specifically ; Y2 receptor is its
36 preferred cognate receptor. This interaction increases the activity of anorexigenic MSH neurons, and inhibits orexigenic NPY neurons (Betterham et al., 2002). PYY 3 36 may also act via the vag al complex that includes brainstem hypothalamic pathway s Koda (2005 ) showed that peripheral administration of PYY 3 36 led to vagal nerve activation and that in rats, the disruption of the vagus nerve partially abolished its anorexigenic effect. PP a 36 amino acid peptide, was the first member of the family to be identifi ed. It is secreted mainly from the pancreas but a small amount is released from the distal gut. PP is released post prandially via vagal cholinergic dependent mechanisms. PP is involved in a number of physiological functions including inhibition of pancrea tic and gallbladder secretion and activity, intestinal mobility, and ileal contractions (Zac Varghese et al., 2010). YR s The diversity of functions is mediated through the extensive redundancy of PP fold NPY family of receptors. Th e NPY family of receptors comprises several receptors termed Y1, Y2, Y4 Y5 and y6 (Larsson et al., 2008), among which only Y1, Y2, Y4 and Y5 are well expressed and sufficiently studied in mammals. These receptors belong to the rhodopsin like superfamily o f metabotropic G Protein Coupled Receptors (GPCRs). All YR s act through Gi/o signaling pathway inhibiting cAMP synthesis, activating Protein Kinase C (PKC), Mitogen Activated Protein Kinase (MAPK), or Phospholipase C (PLC) thus inducing release of intracel lular Ca2+. In addition, the YR downstream signaling modulates the conductance of membrane Ca2+ and inwardly rectifying K+ (GIRK) channels. Not only PP fold peptides bind to several YR s, their pharmacological redundancy is also increased by the action of D ipeptidyl
37 Peptidase IV (DPPIV), a serine exopeptidase that truncates NPY and PYY at their N termini producing peptides NPY 3 36 and PYY 3 36 thus changing their binding specificity (Lin et al., 2004). There is a lot of literature describing the specific feat ures of each YR Kamiji (2007) ha s compiled a comprehensive review Y1 expression is widespread throughout the brain including the thalamus, the hypothalamus, the cortex, the hippocampus the amygdala, and in arterioles of peripheral tissues. The Y1 s ubtype displays preferential activation by NPY and less affinity for PYY 1 36. Y1 is a cardinal mediator of th rough which NPY induces spontaneous feeding. Y2 receptor is more predominantly expressed than Y1 in most hypothalamic nuclei of rat. PYY 3 36, is qu ite selective for the Y2 receptor and has 200 times more affinity than PYY 1 36. NPY and its truncated forms NPY 3 36 and NPY 13 36 are also full agonists. Y2 receptor subtype mediates inhibitory effects of NPY on gastric emptying. Y4 receptor, regarded as t he PP receptor, has very high affinity for PP and almos not affinity for NPY and PYY Y4 is widely distributed in the brain especially in the arcuate nucleus PVN and the area postrema and less expressed in other areas such as the r ostral forebrain, the t halamus, the nucleus of the vagus and the nucleus of the solitary tract. Peripherally, Y 4 mRNA has been found in colon, coronary artery, small intestine, pancreas, prostate, skeletal muscle, among others. The Y5 receptor is distributed in most of the rat b rain. Y5 receptor mRNA was seen less abundantly than either the Y1 or the Y2 mRNA. Pharmacologically, NPY has a greater potency than PYY1 36 and PYY3 36 for Y5 activation.
38 Obesity and PYY Interest in the function of PYY 3 36 has increased over the last de cade due to reports demonstrating potent anorectic effects when it is exogenously administered, indicating a possible therapeutic role of this peptide in appetite and body weight control and thus obesity. Batterham ha s demonstrated that peripheral administ ration of PYY 3 36 to humans and rodents results in an important decrease of FI (Batterham et al., 2002 and 2003)
39 CHAPTER 3 THE NEUROPEPTIDE Y SYSTEM IN THE ORAL C AVITY Neuropeptide Y ( NPY ), Peptide YY ( PYY ), and Pancreatic Polypeptide ( PP ) belong to a fam ily of peptides sharing similar hairpin like PP fold structural homology and evolutionary history (Zhang et al., 2011). NPY is widely expressed in the central as well as in the peripheral nervous system; PYY is mainly released by L endo crine cells of the d istal intestine and colon epithelia, while PP is produced by specialized cell in the pancreas. These peptides mediate various complementary and often opposing metabolic functions such as appetite and satiation, energy intake and expenditure; cell prolifera tion, migration, and differentiation; neuromodulation, angiogenesis, osteogenesis, and many other biological processes. The diversity of functions is mediated through the extensive redundancy of PP receptors. These rece ptors, namely Y1, Y2, Y4, Y5, and y6, belong to the rhodopsin like superfamily of metabotropic G Protein Coupled Receptors (GPCRs). All Y receptors (YR) act through Gi/o signaling pathway inhibiting cAMP synthesis, activating Protein Kinase C (PKC), Mitoge n Activated Protein Kinase (MAPK), or Phospholipase C (PLC) thus inducing release of intracellular Ca2+. In addition, the YR downstream signaling modulates the conductance of membrane Ca2+ and inwardly rectifying K+ (GIRK) channels. Not only PP fold peptid es bind to several YR s, their pharmacological redundancy is also increased by the action of Dipeptidyl Peptidase IV (DPPIV), a serine exopeptidase that truncates NPY and PYY at their N termini producing peptides NPY 3 36 and PYY 3 36 thus changing their bind ing specificity. Adding more complexity to the understanding of the physiological role of PP fold peptides, other groups have shown that NPY is present in human saliva (Dawidson et
40 al., 1997) and the expression of NPY gene in the taste receptor cells (TRC ) in rat (Zhao et al., 2005). Acosta (2009) also showed that PYY is present in saliva and by RT PCR he also demonstrated its expression in TRCs and keratinized tongue epithelium Given the widespread pattern of the expression of PP fold peptides and cognat e YR s in other tissues, and taking into account their pleiotropic functions and the redundancy of interactions, it was of interest to explore whether other members of NPY family genes are being also expressed in the oral cavity. The purpose of the current investigation, therefore, was to identify the expression of genes coding for PP fold family peptides as well as their most studied YR s : YR1 YR2 YR4 and YR5 in the tongue epithelia cells. Materials and Methods In Vitro YR Antibodies Validation HEK 293 c ells were transfected with plasmids expressing murine YR1 YR2 YR4 YR5 or GFP cDNAs under the contro actin promoter. Two days after transfection, cells were fixed on cover slips and subjected to immunostaining analysis using the respective antibodies and conditions employed for YR detection in tissue samples (see immunosta ining section). Animals The experiments for the project were approved by the respective Institutional Animal Care and Use Committees (IACUC) at the University of Florida and the NIDCR (NIH). All procedures were done in accordance with the principles of th e National e and Use of Laboratory Animals Mice were housed at 22 24 C in a 12 hours dark/light cycle and had access to water and food ad libitum unless indicated otherwise.
41 Tissues Tongues, brains and salivary glands (SG) were harvested from wild type C57Bl/6 male mice from Charles River and YR2 Knockout (KO) A colony of YR2 KO mice ( Tschenett et al., 2003 ) is maintained at the Garvan Institute of Medical Research. RT PCR Circumvallate ( CV ) papilla e enriched tissue and surrounding taste tissue were obtained via micropunch (1 mm diameter; Harris Unicore, Ted Pella, Inc., Redding, CA, USA) from C57BL/6J mice. Total RNA was extracted with Trizol, DNA was digested with RNase free DNase (Qiagen Inc, Vale ncia, CA) followed by RNA cleanup with the RNeasy Micro kit (Qiagen). RNA was reverse transcribed with Superscript III (Invitrogen, Carlsbad, CA). Products were amplified with gene specific primers ( Table 1 ). The primer set that yielded one product with th e correct predicted amplicon size as determined with molecular ruler was selected for the finished set. Intron spanning primers were designed for each gene and tested alongside of positive control tissue (brain and tongue epithelium) to confirm expression. No cDNA samples were prepared for each primer set. DNA contamination was tested with control, intron only primers for gastrin which is not expressed by cells in the tongue. Immunostaining YR s immunostaining Tissues were harvested from fasted animals and i mmediately froz en. Four thick coronal or sagitt al sections were cut using a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany), mounted onto Fisher Superfrost Plus slides and post fixed in 4% paraformaldehyde for 10 minutes. YR LI immunostaining was conducte d utilizing TSA kit (Perkin Elmer). Tissues were blocked in 0.3% H 2 O 2 in TBS
42 for 30 minutes at room temperature to eliminate endogenous peroxidase activity, followed by blocking with TNB (0.1 M Tris HCl, pH 7.5, 0.15 M NaCl and 0.5% Blocking Reagent from P erkin Elmer; for 60 minutes at room temp) to reduce nonspecific antibody binding. Sections were then incubated with primary rabbit anti YR antibody in TNT overnight at 4 C, followed by secondary goat anti rabbit IgG (Fab' ) 2 (HRP) (Abcam; 1:1000 for 60 min at room temp). Staining was detected using Fluorescein provided in the TSA kit (1:300 for 7 min at room temp). Negative controls were run concomitantly. All sections were counterstained with DAPI. Y receptors/NCAM double immunostaining Because all YR s and NCAM primary antibodies were raised in rabbits, double immune labeling were performed using modified indirect immuno staining protocol and the TSA kit allowing for the immuno localization of two proteins in the same tissue specimen when both primary antibod ies are produced in the same host. Specifically, immediately after detection with the TSA Fluorescein, sections were extensively washed in TNT and then blocked with 10% natural donkey serum in TNT for 60 min at room temperature. Tissues were subsequently i ncubated with the second primary antibody rabbit anti NCAM (Millipore; 1:250 in 10% natu ral donkey serum overnight at 4 C) and visualized, using standard methods, with AF555 donkey anti rabbit IgG ( Invitrogen 1:1000 in TNT for 60 min at room temp). All do uble labeled sections were counterstained with DAPI. Cytokeratin 5 immunostaining The same protocol as for YR s detection was used but after in cubation with the primary antibody anti cytokeratin 5 sections were blocked with Image iT FX Signal
43 Enhancer ( Invitrogen) and incubated with goat anti rabbit Cy3 (1:800, Jackson Immunoresearch) for visualization. All sections were counterstained with DAPI In Situ Hybridization YR2 RNA was visualized in 5 m paraffin embedded sections using the QuantiGene viewRNA slide based kit from Affymetrix (Cat #QV0096) according to the sets were designed by Affymetrix. Briefly, fresh ly dissected tissue was fixed in 10% neutral buffered formaline for 24 h at RT. Tissues were embedded into paraffin after alcohol dehydration. Five micrometer sections were mounted onto slides. Slides were processed strictly following the QuantiGene protoc ol. Pre hybridization conditions were found to be optimal with 10 min of boiling in pre treatment solution and 20 min of Protease QF digestion. Tissues from YR2 knockout mice were used as negative controls. Results The pur pose of this investigation was to characterize the expression of YR subtypes in the tongue epithelia. YR s belong to GPCR family of receptors and, as such, are highly homologous. It is well known in the field that significant fraction of commercially avai lable GPCR antibodies lack specificity and selectivity resulting in binding to other subtypes within the family (Saper et al., 2005; Michel et al., 2009). It was therefore essential to validate the antibodies prior to conducting an experiment For validati on we used an immunocytochemistry (ICC) protocol. The source of all antibodies, dilutions, and controls is listed in Table 3 2. To test the selectivity of YR antibodies for the target receptor vs. related subtypes when expressed in the same cell,
44 we transf ected HEK 293 cells with plasmids expressing murine YR1 YR2 YR4 YR5 or GFP cDNAs under the contro l of a strong constitutive CBV/ actin promoter. Two days after transfection, cells were fixed on cover slips and subjected to immunostaining analysis using the respective antibodies and conditions employed for YR detection in tissue samples. Fig. 3 1A clearly shows that each antibody r eagent interacts exclusively with its respective antigen, i.e. YR1 YR2 YR4 or YR5 with no detectable cross hybridization with any other subtypes. YR s expressed in mice tissues. For the positive control, we selected hippocampal dentate gyrus region in the mouse brain as this tissue robustly expresses YR s in very specific and well characterized ways. Staining of the brain sections revealed patterns of YR positive neuronal cell bodies and fibers that were similar to the previously reported (Fig. 3 1B) (Wolak et al., 2003; Stanic et al., 2006; Parker et al., 1999; Stanic et al., 2011; K opp et al., 2002). In particular, expression patterns of YR1 and YR2 appeared to be complementary, i.e. hig h levels of YR1 expression in one region corresponded to the low levels of YR2 and vice versa (Stanic et al., 2006). Expression of YR s in the Lingual Epithelia Cells The dorsal surface of the tongue is covered by a specialized mucosa consisting of keratin ized stratified epithelium (for review, please see Squier et al., 2001). In addition to its primary function protecting the underlying tissues during mastication, it also incorporates structures with gustatory functions ( CV fungiform, and foliate papillae ), mechanical structures ( filiform papillae), and mechanoreceptors (Meissner corpuscles). In addition, the glandular component of the sublingual epithelium includes specialized SG high
45 turnover rate of cells in response to mechanical and chemical contacts. Because of the known functions of YR s in cell proliferation (Mannon et al., 2000), it was of interest to explore whether epithelial cells express YR s. First, using RT PCR protoc ols, we showed the expression of all major NPY family receptors in the tongue epithelium in mice (Fig. 3 2A). To histologically characterize th e presence of these receptors, we performed immunostaining techniques. During the first set of the experiments, w e determined that morphologically different layers (Squier et al., 20001) of the lingual epithelia expressed YR s in a very distinctive yet overlapping antibodies agains t YR s were raised in rabbits), we utilized a mirror section staining method. To follow this protocol, the first section is mounted with its inner surface turned upwards on one slide, whereas the subsequent adjacent section is mounted on the next slide with its outer surface upwards. In this way, the complementary faces could be hybridized to two different antibodies without concern related to the secondary antibodies cross reactivity. Although not entirely identical, the characteristic structures of the epi thelial layers on two separate slides provide sufficient guidance to identify distinctive cell layers or even particular cells. Three separate complementary pairs of mirror sections were analyzed by the hybridization to YR antibodies: YR1 and YR2 (Fig. 3 3A and B, respectively); YR2 and YR5 (Fig. 3 3C and D, respectively); YR1 and YR5 (Fig. 3 3E and F, respectively). The monolayer of basal epithelial cell expressed both Y1 (Fig. 3 3A, E) and Y2 receptors (Fig. 3 3B, C). YR2 expression appeared to be restri cted only to this monolayer (Fig. 3 3C, right panel), while YR1 on the other hand, was present in the parabasal prickle cell
46 cell layer, and the granular layer (Fig. 3 3A, right panel). Differentiated keratinocytes displayed very low levels of YR1 protein however, they revealed a robust expression of YR5 (Fig. 3 3 F, right panel). Fig. 3 6 shows a better characterization of YR2 in the tongue epithelium. Unlike YR1 YR2 or YR5 the expression of YR4 was not detected in the basal, or keratinized epithelial cells. Instead, it was restricted to the somato sensory neuronal fibers extending within the subepithelial region close to the basal laminae of the lingual epithelia (Fig. 3 3 G and 3 4A). Very few of YR 4 (+) fibers were also positive for a neural cell adh esion molecule (NCAM) marker (Fig. 3 4B, arrow) suggesting that the majority of these sensory projections represent intraepithelial axons with free nerve endings. In addition, YR4 (+) neuronal fibers were also abundant in some areas of the lamina propria, in particular, in the fibers innervating mechanoreceptors (Meissner corpuscles) that were also positive for NCAM (Fig. 3 4B, C). Expression of YR s in the Taste Bud Cells Gustatory papillae are distinctive structures on the dorsal tongue epithelia incorpora ting several types of cells including basal epithelial cells, keratinized cells, TRC s organized in tight clusters (taste buds), and gustatory neuronal fibers innervating TRC s. Epithelial cells and TRC s derive from the lingual embryonic undifferentiated epi thelium and are constantly turned over in the adult anim al. To characterize whether TRCs similar to keratinocytes, expressed YR s, we used RT PCR for gene expression and immunostaining for protein detection. Using the former technique, we were able to iden tify all the receptors in taste tissue (Fig. 3 2C )
47 Additionally, and i n an agreement with epithelial cell expression data (Fig. 3 2A, B), epithelial cells forming CV YR1 YR2 and Y5 in a selective manner (Fig. 3 5 A, B, and D block arrows). A significant population of TRC s was positive for YR1 (Fig. 3 5A), YR2 (Fig. 3 5B), YR4 (Fig. 3 5C), or YR5 (Fig. 3 5 D). In contrast to the epithelial cells, the staining pattern did not delineate the entire contour of cells positive for Y R showing instead preferential accumulation of YR s within the microvilli of the apical part of the cells (filled arrowheads in Fig. 3 5 A C, respective zoomed images in the panels on the right). This preferential apical distribution makes YR s easily accessi ble to paracrine salivary PP fold peptides, suggesting their possible roles in modulating taste perception. On the other hand, some YR s positive TRC s accumulated YR s in the baso lateral part of TRC s (open arrowheads in Fig. 3 5 A C), which makes these cells susceptible to the PYY NPY and PP synthesized inside taste buds (Acosta 2009; Zhao et al., 2005). Within each taste bud, TRC s fall into three morphological subtypes, Types I through III, which seem to correspond to functional classes (reviewed in Yoshid a et al., 2010). To understand the putative functions of YR s, we co stained YR s with a known TRC Type III molecular marker NCAM. The majority of TRCs expressing YR s appeared to co localize with NCAM positive cells (Fig. 3 5). Expression of YR in SG SG p roduces saliva to lubricate and supply antibacterial compounds, electrolytes and various enzymes to the oral cavity in order to initiate digestion of food. In mammals, there are multiple minor SG located throughout the oral cavity within the submucosa of t he oral mucosa, and three main pairs of SG : parotid glands that produce only serous fluid (a lso k nown as secretory glands) submandibular and sublingual glands that
48 produce a mixture of serous and mucous fluid ( also known as mixed glands). In addition, in the oral cavity, within the tongue parenchyma, there are Von SG s that secret through multiple ducts into the clefts of the CV and foliate papillae. The co mposition of the secretion of v on Ebner SG is complex (e.g. amylase, lipase and acid phosphata se). I t is conceivable, therefore, that these glands have other functions in addition to providing fluid to rinse the clefts of papillae to help in taste transduction. Since we were analyzing different tissues in the oral cavity, we have collect ed submandi bular SG to analyze the expression of the NPY system family members In addition to the data described above we have demonstrated that all YR s are expressed in SG by RT PCR ( Fig.3 2E ) We have been trying to optimize our staining protocol to work with SG tissue. It is well know that the high mucous content in the SG can hinder protein immunostaining, especially due to unspecific binding and high background. Along with one of our collaborators, we have been able to overcome some of the s e limitation s and to obtain preliminary data for YR1 (Fig.3 7A), YR2 (Fig.3 7 B) and YR4 expression (Fig. 3 7 C ). A grosso modo YR1 signal is located in the apical pole of acinar cells, and has a similar distribution of Aquaporin 5 protein, which is a channel protein that regul ates the movement of water through the plasma membrane of secretory cells. YR2 signal is expressed in the basal portion of acinar cells and myoepithelial cells of SG To characterize this particular distribution, we co stained YR2 with smooth muscle actin protein, a marker of these m y oepithelial cells. Interestingly, we found that the two proteins co localize almost 100 % (Fig. 3 8). Even though the functional significance of myoepithelial cells of SG is not completely understood, they could be involve d in contract ile fashion thus helping to expel secret ed peptides from the
49 acinar cells into SG duct luminal space Finally, YR4 seem to be present in some cells in the excretory and intercalated ducts. With respect to von SG we have found that the mono layer of cell s lining YR1 (Fig. 3 5A indicated by VEG acronym) and YR2 (Fig 3 5C and 3 6C, D and F ). We have observed some unspecific staining in acinar cells ; however, these data require validation and, a s such, are not presented. To corroborate all these data, we have started to work with in situ hybridization techniques to detect m RNA in the respective lingual tissue s To demonstrate that the immunostaining for the protein is specific we have compared i t with the pattern of mRNA expression For the moment, we have only been able to work with YR2 probe In situ hybridization studies in brain, our positive control tissue for YR2 showed that the expression patterns of the protein and the mRNA are similar i f not identical (Fig. 3 1B, 3 6A and 3 9A). We showed as well that YR2 mRNA is expressed in tongue epithelium, TRCs and von Ebner SG displaying similar expression pattern identified by the immunostaining (Fig. 3 9). Interestingly, the pattern of YR2 mRNA expression in the keratinized epithelium is slightly different from what we have seen with protein immunostaining In this portion of the tongue YR2 mRNA is expressed not only in the basal layer but also in more superficial layers (Fig.3 9B). However, th e expression in these superficial layers is significantly lower. In the case of protein detection, the signal is limited to the basal layer. We speculate that since for protein detection we used a very powerful amplification system that is based on the lev el of protein expression (TSA that amplifies up to 200 times), the signal deriving from the more superficial layers may
50 be blunted by the extremely increased intensity of expression of the protein present in the basal cells. Origin of YR2 Among all the YR s, we have specifically done more studies for YR2 (Fig. 3 6). To establish the lineage identity of YR2 positive cells we used cytokeratin 5 (K5), a basal cell epithelia marker in adult (Raimondi et al., 2006) and embryonic (Knox et al., 2010) SG as well as a marker of progenitor cells of the filiform papillae (Okubo et al., 2009). Staining of sequential mirror sections with either YR2 or K5 antibodies (Fig. 3 10A, D, F, or Fig. 3 10 B, C, E, respectively) revealed that YR2 is apparently expressed in a singl e apical layer of progenitor cells in the tongue epithelium ( Fig. 3 10 A, D) as well as in von 6C, D, F, and 3 10 F), suggestin g a possible trophic role of YR2 signaling in mitotic signaling/ regeneration Expression of NPY PYY and PP in the oral cavity It has been reported that PYY and NPY are present in saliva (Acosta 2009; Dawidson et al ., 1997 respectively ). Some proteins enter saliva from SG where they are expressed and secreted in an exocri ne fashion via zymogen granules. Other peptides can enter saliva as transud at es from serum Acosta et al showed that salivary PYY is transported from plasma into saliva Using RT PCR he also showed the expression in tongue epitheliu m and taste tissue. Little is known about the origin of NPY except that it is expressed in some TRCs (Zhao et al., 2005). So far, n othing is known about the presence of PP in saliva or its expression in the oral cavity. Using RT PCR h ere we confirm that PYY and NPY are indeed synthesized in the lingual epithelia and in addition, we also describe that PP is expressed as well ( Fig. 3 2B, D and E ). Specifically, the three peptides are expre ssed in the tongue epithelium, taste tissue and
51 SG W e have been uns uccessful showing the expression pattern of PP NPY and PYY in the tongue epithelium using immunostaining techniques which contradicts RT PCR data. It has been a challenge to optimize protocols to work with taste tissue s and it took us a very long time. H o we ver, we will perform in situ hybridization as we did for YR2 mRNA. P robes have been requested from Affymetrix to be delivered within one month period of time In taste tissue, we have only performed immuno staining of PYY which is descri bed in detail in t he following C hapter 4 Discussion The NPY System and Tongue Epithelium NPY PYY and PP are widely expressed in central and peripheral nervous system and mediate various complementary and often opposing metabolic functions such as appetite and satiation, energy intake and expenditure; cell proliferation, migration, and differentiation; neuromodulation, angiogenesis, osteogenesis, and many other biological processes. The diversity of functions is mediated through the extensive redundancy of PP fold peptides previously reported that PYY is express ed in taste tissues and Zhao (2005) reported that NPY another member of this family of peptides, along with its Y1 receptor, are also expressed in TRC s. In the current report, we confirm and extend these findings. Using RT PCR protocols, we now show the expression of all major NPY family members in the tongue epithelium taste tissue and SG of the oral cavity (Fig. 3 2) The data presented suggest s that the NPY system may have an important function in the oral cavity that has not been characterized before. The pattern of expression in the keratinized epithelium of the tongue suggests that the NPY system might play a role in cell turnover. W e est ablished the lineage identity
52 of YR2 positive cells with cytokeratin 5 (K5), a basal cell epithelia marker in adult (Raimondi et al., 2006) and embryonic (Knox et al., 2010) SG as well as a marker of progenitor cells of the filiform papillae (Okubo et al., 2009). Staining revealed that YR2 and YR1 are expressed in the apical layer of progenitor cells in the tongue epithelium suggesting a possible trophic role of NPY system signaling in mitotic signaling/regeneration. The stratified epithelium is characteriz ed by the high turnover rate of cells in response to mechanical and chemical contacts. The G i signaling in K5 progenitor cells could mediate their motility, polarity and migration towards upper layer of keratinized filliform papillae (Cotton et al., 2009). More studies need to be done to study the potential role of the NPY system i n tongue epithelium; however, it would not be surprising to confirm that the NPY system regulates cell proliferation, migration and differentiation since it has been described for other tissues such as bone vessels and skin. In bone, the NPY system modulates osteoblast activity and bone formation through YR1 and YR2 signaling (Lee et al., 2010). Following the same line, there is strong evidence supporting NPY d mitogenic function on vascular smooth muscle cells and its potential role i n endothelial cells wound healing (Ghersi et al., 2001). Coincidentally, the same apical layer of YR2 / YR1 positive cell s is adjacent to fibers that are YR4 (+) which co localize w ith NCAM (+) neuron fibers as evident from the immunostaining using Neural Cell Adhesion Molecule (NCAM) neuronal marker antibodies (Fig. 3 4). Since Acosta (2009) showed that PYY is present in saliva, and the increase of this peptide induces a decrease in food intake (FI) t he anatomical location of YR2 / YR1 positive cells, combined with their somatosensory innervation implies a
53 possible func tional role for salivary PYY ligand and its preferred Y2 receptor related to the regulation of feeding behavior. This topic is extensively described in C hapters 4 and 5 The NPY System and Taste Tissue The e xpression of the NPY system in t aste tissue suggests that it may be involved in taste modulation. Recently, it has been shown that several gastrointestinal peptides s uch as glucagon, glucagon like peptide (GLP 1) cholecystokinin (CKK) NPY vasoactive intestinal peptide (VIP) ghrelin, and galanin are also expressed in TRCs in the peripheral gustatory system namely CV and foliate papillae ( Herness, 1989; Herness et al ., 2002; Zhao et al., 2005; Seta et al., 2006; Shin et al., 2008b; Elson et al., 2010; Martin et al., 2010 ). In addition, the cognate receptors for these peptide hormones are also expressed in TRCs or found in fibers of afferent taste nerves in oral mucosa ( Herness et al., 2002; Shen et al., 2005; Zhao et al., 2005; Seta et al., 2006; Shin et al., 2008b; Elson et al., 2010; Martin et al., 2010 ). It has been also demonstrated that for some of these gut hormones, the anatomical proximity of agonists and recep tors play a role in the functioning of the peripheral gustatory system, acting to modulate taste responsiveness to certain stimuli ( e.g., sweeteners; Kawai et al., 2000; Shin et al., 2008b; Elson et al., 2010; Martin et al., 2010 ). In this report we provid e evidence that PYY NPY and PP are expressed in taste tissue as well as their cognate receptors. The pattern of expression of these receptors and respective ligands in taste tissues still remains to be characterized. It is not known whether these receptor s are expressed in the same or different cells as PYY NPY or PP or if the various YR s are co expressed together. This may indeed be the case as YR s are known to form heterodimers (Gehlert et al., 2007; Parker et al., 2011). Be that
54 as it may, in addition to Zhao s data (2005), these data demonstrate that the NPY system and its cognate receptors are well positioned in the oral cavity to support both paracrine and endocrine signaling in cells of the peripheral gustatory system. Additionally, the relationship with neuronal markers supports that cells expressing these receptors may be the origin taste related neuronal circuit. Towards defining the role of the expression of the NPY system in the oral cavity as another project in our laboratory, it has been show n that disruption of PYY signaling decreases behavioral responsiveness to the bitter tasting compound denatonium benzoate and to an intralipid fat emulsion (project in progress ). Some recent discoveries in taste research have emphasized the fact that tast e perception is linked to mechanisms of appetite and satiety. The presence of gastrointestinal peptides in saliva and their expression in TRCs added to the fact that many cells in the gut express the same molecular machinery required for nutrient detection as the one found in TRCs support s this hypothesis. Data suggests that a n ponsiveness/sensitivity to diverse taste stimuli is likely an important regulator of FI It is interesting to speculate that, at least in part, salivary PYY NPY an d PP regulate ingestive behavior via changes in taste perception. More experiments will be needed to address this question. The NPY System in SG The presence of the NPY system in SG and the co express ion of Cytokeratin 5 with YR2 SG suggest that it may also have a role in cell proliferation However, our other findings have led to the hypothesis that this system may also be involved in SG production and secretion. SG are exocrine glands produce saliva to
55 lubricate and supply antibacterial com pounds, electrolytes and various enzymes to the oral cavity in order to initiate digestion of food. The presence of YR1 and YR2 SG suggests that NPY PYY and PP may have a role in the content of the fluid secreted as wel l as in the secretion per se SG is important to rinse the clefts of CV and foliate papilla e. Food particles need to be in solution in order to stimulate TRC s in the taste buds (Pedersen et al., 2002). The expression patte rn of YR s in submandibular SG is not clear and more studies need to be done in order to characterize the physiological role of the NPY system. YR1 signal, in the apic al pole of acinar cells and its similar expression pattern as Aquaporin 5 channel protein that regulates the movement of water through the pla sma membrane of secretory cells, suggests that YR1 signaling may be involved in the viscosity and amount of saliva production. YR2 co expression with myoepithelial cells of SG suggests a possible role in saliva secretion from SG We cannot speculate about the function of YR4 in duct cells due to its unspecific expression.
56 Table 3 1. Gene specific primers used in RT PCR Target Forward Primer, 5' 3' Reverse Primer, 5' 3' Annealing Temp o C Cycles Size (b p) YR1 TGGCTTTTGAAAAT GATGACTG ATAAGCGAGAGCC AAGGTGA 60 35 65 YR2 TTGGCAACTCCCT GGTAATC TTTCCACTCTCCCA TCAAGG 60 35 155 YR4 GGGCCCAGATAGG TTGGCAAGAGA CCCTTGCAGCTCA AGCCACAAAGT 65 35 128 YR5 CCGTTCCAGAAAA CCCAGGCTCG TGGAAGACGTGGA GTGGCATCCA 64 35 232 PYY GGCAC TTCATATC TCGGTGTCTCGG TGAACACACACAG CCCTCCAGTCT 62.5 35 55 NPY TCATCTCATCCCCT GAAACC CGGAGTCCAGCCT AGTGGT 61 35 66 Table 3 2. Antibodies used for immunostaining studies Antibody Host Supplier Dilution Specificity/Control Anti YR1 Rabbit Immunostar (Huds on Wiconsin, USA; cat No. 24506) 1:100 (using TSA Kit) Staining absent when primary or secondary antibodies omitted. The antibody was characterized by immunostaining and Western blot. Anti YR2 Rabbit Neuromics (Edina, MN, USA; cat. No. RA14112) 1:3000 (u sing TSA Kit) Staining absent when primary or secondary antibodies omitted, or in NPY Y2 receptor KO tissue. Use of this antibody has been reported previously. Western blot analysis on hippocampal membrane fractions revealed a single band of 44 kDa (Stanic et al., 2011) Anti YR4 Rabbit Santa Cruz Biotechnology, Inc. Cat. No. sc 98934 1:1600 (using TSA Kit) Staining absent when primary or secondary antibodies omitted. Anti YR5 Rabbit Abcam; cat. No ab43824 1:800 (using TSA Kit) Staining absent when primary or secondary antibodies omitted. Anti NCAM Rabbit Millipore (Temecula, CA, USA; cat. No. AB5032) 1:500 Staining absent when primary or secondary antibodies omitted. Anti Keratine 5 Rabbit Covance (Emerit, CA, USA; cat. No. PRB 160P) 1:1000 Staining abse nt when primary or secondary antibodies omitted.
57 Fig ure 3 1 Validation of YR antibodies. A ) Immuno staining analysis of 293HEK cells expressing murine YR cDNAs. Columns cells transfected with YR1 YR2 YR4 YR5 or GFP expressing plasmids, respective ly. Rows cells on cover slips YR1 YR2 YR4 YR5 antibodies, respectively. Please note peripheral (membrane associated) localization of YR s as oppose to diffuse, whole cell fluoresc ence of the GFP ( ) control. B) Immunostaining analysis of mouse brain (dentate gyrus) for the expression of YR s. The diffuse staining for YR1 reflects YR1 (+) neuronal fiber distribution seen in this sagittal sectioned plane.
58 A B C D E Figure 3 2 Expression of the NPY system in the oral cavity analyzed by re verse transcriptase (RT) PCR. A) YR s i n keratinized tongue epithelium B) PYY and NPY in k eratinized tongue epithelium. C) YR s in taste tissue. D) PYY and NPY in taste tis s ue. E) YR s, PYY and NPY in SG Approximately 1x2 mm section of tongue epithelium (including some fungiform papillae) directly anterior to the CV was dissected out with microscissors for control tissue for the taste receptor. Whole pancreas was extracted fo r PYY and PP positive controls. A core sample (including the hypothalaumus) of the brain was selected for positive control tissue for the YR s and NPY and a negative control for PYY RNA was extracted and purified as the CV from each all from wild type B6 m ice. Primers were designed with NCBI primer blast
59 Figure 3 3. Immunostaining of Y1, Y2, Y4 and Y5 receptors in the dorsal epithelium of a tongue. Mirror section pairs (Panels A and B, C and D, E and F) were hybridized to the respective YR antibodies (g reen), followed by DAPI counterstain (blue), as indicated in the upper left corner of each panel. For better viewing, the confocal images in B, D, and F were reflected horizontally. Randomly selected areas of the epithelium, positive for either YR (dashed rectangles in the left sided panels), are shown as close up images on the right next to the respective panel. The irregular columns structures at the epithelial surface are transversely sectioned filiform papillae. Panel G represents tongue epithelium hybr idized with YR4 Panel H is a schematic representation of YR expression in the tongue Epithelium.
61 Figure 3 4. Immunostaining of Y4 receptors in the do rsal epithelium of a tongue. A) YR4 positive neuronal fibers (green) are located in the subepithel ial region u nderlying the basal laminae. B) co localization of YR4 and NCAM (red) immunostaining i n some subepithelial fibers (bla ck arrow) and within mechanoreceptors Meissner corpuscles (MC), also shown in panel C.
62 Figure 3 5. Immunostaining of YR s in TRCs Mice CV s were double hybridized with YR antibodies (green) and NCAM (red) and counterstain ed with DAPI (blue). The first column is a lower magnification Randomly selected areas of the epithelium, positive for either YR (dashed rectangles in the l eft sided panels), are shown as close up images on the following columns. Column 3 shows the three channels superimposed ( Y RECEPTOR /NCAM/DAPI). Columns 2, 4 and 5 correspond to individual channels YR NCAM and DAPI respectively.
63 Figure 3 6. Y2 recepto r is synthesized in the epithelial cells of the tongue. A) Immunostaining of YR2 positive cells in the hippocampus of C57Bl/6J mouse ( wild type ), a (+) control. B) Immunostaining of YR2 in the tongue epithelia of YR2 KO mouse, a ( ) control. VEG von Ebn e C) Immunostaining of YR2 positive cells in the CV area of t he tongue of a C57Bl/6J mouse. D) Close up of C). E), and F) close ups of D), top and bottom rectangles, respectively
64 A B C Figure 3 7. SG immunostaining A) YR1 immunostai ning in green, DAPI in blue: signal located in the apical pole of acinar cells. B) YR2 immunostaining in green, DAPI in blue: signal preferentially located in the basal portion of acinar cells and epithelial cells (Fig. 3 8). C) YR4 immunostaining in green DAPI in blue: protein expressed in some cells of the excretory and intercalated ducts.
65 A B C Figure 3 8 Characterization of YR2 cells in the SG and co staining with smooth muscle acting. A ) YR2 immunostaining in green in SG B) Smooth muscle acting stainin g in red in the same section. C) Overlay of the two channels show the co expression of the two proteins (yellow).
66 Figure 3 9. YR2 In situ hybridization. All these images were taken at a 120X magnification. YR2 mRNA is visualized as red dot s and cell nuclei in blue (DAPI). A and B are (+) and ( ) controls respectively A) Positive control: visualization of YR2 mRNA in brain tissue, spe cifically in the hippocampus. B) Negative control, YR2 KO tissue: the signal is no longer visualize d in tis sue of YR2 deficient mice. A s shown in the other panels, YR2 mRNA is expressed in the basal cells of the tongue epithelium (C), TRCs of the CV papilla (D) and Von Ebner salivary lingual gland (E).
67 A B C D E
68 Figure 3 10. A s ubpopulation of epithelial progenitor cells in the tongue epithelia expresses YR2 Two sequential mirror sections of the tongue were immunostained for YR2 (A, D, and F), or Cytokeratin 5 (K5) (B, E, and C). For better viewing, K5 images were reflected hori zontally. Areas at the sulcus edge, positive for both YR2 (A) and K5 (B) (dashed rectangles), are shown as close up images in (D) and (E), respectively. Panels (C) and (F) show YR2 and K5 CV
69 CHA PTER 4 THE ROLE OF SALIVARY PEPTIDE YY I N INGESTIVE BEHAVIOR A significant portion of metabolic polypeptides has been shown to be expressed in taste receptor cells (TRC) or to be present in saliva. This long list now includes insulin, leptin, adiponectin glucagon, glucagon like peptide 1 ( GLP 1 ), cholecystokinin ( CKK ), neuropeptide Y (NPY), vasoactive intestinal peptide ( VIP ), ghrelin, and galanin (Vallejo et al., 1984; Groschl et al., 2001; Toda et al., 2007, Shin et al., 2008; Herness 1989; Herness et a l., 2002; Zhao et al., 2005; Groschl et al., 2005; Seta et al., 2006 and Elson et al., 2010). In addition, the cognate receptors for these peptide hormones are expressed in TRC s or found in fibers of afferent taste nerves in oral mucosa (Shin et al., 2008; Herness et al., 2002; Zhao et al., 2005; Seta et al., 2006; Elson et al., 2010; Kawai et al., 2000; Shen et al., 2005 and Martin et al., 2010). Anatomical proximity of agonists and receptors suggested their putative roles in taste functions. Indeed, most of these polypeptides have been implicated in modulation of different tastes such as sweet (Shin et al., 2008; Elson et al., 2010; Kawai et al., 2000 and Martin et al., 2010), salty (Shin et al., 2010), sour (Shin et al., 2008 and 2010), and umami (Martin et al., 2009). Little, however, is known whether these or other metabolic peptides that are present in Peptide YY ( PYY ), a well characterized molecular mediator of satiation, is relea sed mostly by L endocrine cells in the distal gut epithelia in response to the amount of calories ingested. The anorectic action of the truncated form PYY 3 36 is apparently mediated through the inhibitory actions of its preferred Y2 receptor ( YR2 ) Reprinted with permission from Acosta, A ., Hurtado, M.D ., Gorbatyuk, O ., La Sala, M ., Duncan, D ., Aslanidi, G ., Campbell Thompson, M ., Zhang, L ., Herzog, H ., Voutetakis, A ., et al. (2011). Salivary PYY: a putative bypass to Satiety. PLoS One 6(10):e26137.
70 highly e xpressed in orexigenic NPY neurons in the hy pothalamic arcuate nucleus Batterham et al. (2002) has shown that he acute peripheral administration of PYY 3 36 resulted in significant reduction of food intake (FI) and body weight (BW) suggesting its potential therapeutic application for obesity treatment. The latter results, however, could not be replicated by other groups (Tschop et al., 2004) highlighting the necessity of a more detailed study of the functions of PYY 3 36 in regulating feeding behavior and sa tiety Th us, more studies were done in our laboratory and in previo us experiments it has been demonstrated that PYY is present in human and murine saliva at similar levels to those found in plasma. Interestingly in humans, 30 min a fter consumption of a 450 kcal meal the concentration of PYY 3 36 increased significantly suggesting a possible association between feeding and the concentration of PYY 3 36 in saliva The purpose of this investigation was to characte rize the role of salivary PYY on ingestive behavior. Methods Mice These studies (Approval ID #02123, Gene Therapy for Obesity and Approval ID #03059, Modulation of taste sensitivity by PYY Signaling ) were approved by the respective Institutional Ani mal Care and Use Committees (IACUC) at the University of Florida and the NIDCR (NIH). All procedures were done in accordance with the Animals. Studies were conducted in male mice housed at 22 24C in a 12 hours light/dark cycle. Mice had free access to water and food unless indicated otherwise. NPY KO male mice (Erickson et al., 1996) were purchased from Jackson Labs (129
71 NPY tm1Rpa/J), and the PYY KO colony at the Univers ity of Florida was derived from the respective breeders (Boey et al., 2006). A colony of YR2 KO mice ( Tschenett et al., 2003 ) is maintained at the Garvan Institute of Medical Research Mouse Saliva Collection Salivation was stimulate d as described earlier (Nguyen et al., 2007). Whole saliva was collected for 5 min from the oral cavity into Eppendorf tubes containing 5000 U Kallikrein inhibitor (Biomedicals) and 50 mM DPP IV inhibitor (Linco Research). Saliva samples were frozen at 80 C until analyzed Plasma Collection Blood was collected from facial vein puncture into EDTA coated tubes (Capiject) containing protease inhibitors as described for saliva collection. Plasma samples were frozen at 80 C until analyz ed Plasma and Saliva Hormone Levels PYY 3 36 from saliva and plasma was measured by PYY 3 36 RIA kit (Phoenix Pharmaceuticals) The protocol provided in the kit was followed for all measurements. Immunostaining For specific information on the source of all antibodies, dilutions, and controls please see Table 4 1 PYY and gustducin. Tissues were harvested from overnight fasted animals, embedde d and section ed in a rotary microtome at 4 m thicknesses. For PYY immunostaining tissues were blocked with 3% H2O2 in methanol followed by antigen retrieval with Trypsin (DIGEST ALL 2, Invitrogen), blocking with 5% natural donkey serum in TNT (0.1 M Tris HCl, pH 7.5, 0.15 M NaCl and 0.05% Tween 20), overnight incubation with rabbit anti
72 PYY in TNT, blocking with Image iT FX Signal Enhancer (Invitrogen) and detection with donkey anti rabbit Alexa Fluor 488 in TNT (1:1000, Invitrogen) For gustducin immunostaining the same protocol was followed using as primary antibody goat anti gustducin in TNT and a secondary antibody donkey anti goat Alexa Fluor 555 in TNT (1:1000, Invitrogen). For the double labeling, the same protocol was us ed and both primary and secondary antibodies were applied at the same time. All sections were counterstained with DAPI YR2 and NCAM Tissues were harvested from fasted animals and immediately frozen. 4 mM thick sections were cut usi ng a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany) and then fixed in 4% paraformaldehyde for 10 min. YR2 immunostaining was done with the TSA kit (Perkin Elmer). Tissues were blocked in 0.9% H2O2 in TBS for 30 min followed by blocki ng with TNB (0.1 M Tris HCl, pH 7.5, 0.15 M NaCl and 0.5% Blocking Reagent from Perkin Elmer), incubation with rabbit anti YR2 in TBS, incubation with goat anti rabbit MACH 2 HRP polymer (Biocare Medical) and detection with Fluorescein provided in the TSA kit (1:300). NCAM. The same as for YR2 detection protocol was followed, but after incubation with primary antibodies, sections were blocked with Image iT FX Signal Enhancer (Invitrogen) and incubated with goat anti rabbit Cy3 (1:800, Jackson Immunoresear ch) for visualization. All sections were counterstained with DAPI Because all YR2 and NCAM primary antibodies were raised in rabbits, double immune labeling was performed using modified indirect immuno staining protocol and the TSA k it, allowing for the localization of two antigens in the same tissue specimen
73 when both primary antibodies produced in the same host. For a detailed description of the protocol pl ease see the method section in C hapter 3. PYY 3 36 Acute Augmentation Studies The concentration of PYY 3 36 in the oral cavity was acutely increased by utilizing an oral spray. One mL total volume sterile vials were obtained from Sephora. Murine PYY 3 36 (Bachem) was diluted in sterile H2O. All 8 10 weeks old mice were individually housed. Mice were conditioned to the oral spray (OS) with vehicle after 24 hours fast on three separate occasio ns. Groups were randomized by FI and BW Prior to the study day, mice were fasted for 24 hours, the OS in a form of a single puff containing PYY 3 36 (conc entration ranging from 3 to 18 g/100 g BW, as specified) or vehicle was applied without any sedation. The applied dose was estimated using calculated average volume ( ~30 L ) delivered per puff. Food was provided 10 min after the spray was applie d and the amount consumed was measured at either 1 hr after the treatment, or at several time points (1, 2, 4, 6, 12, 18, and 24 hours). Taking into account the nocturnal feeding pattern, the experiment was conducted during the night with the first measure ment taken at 2000 hrs. Each experiment was done at least 3 times in a crossover manner with 8 mice per group. For the YR2 antagonist study, BIIE0246 compound (Tocris Bioscience, Ellisville, MO) was dissolved in 100% EtOH (0.2 mM) and the stock solution wa s mixed with PYY 3 36 aqueous solution at the 50:1 molar ratio (BIIE0246/ PYY 3 36) with the final concentration of 2% EtOH in the mixture. Prior to this experiment, mice were conditioned to 2% EtOH spray on three separate occasions. Wherever practical, the b ehavioral experiments were conducted in a blind fashion with the personnel uninformed about the treatment regimen
74 Gene Transfer Experiments Long term chronic expression of PYY 3 36 was achieved by rAAV mediated PYY gene transfer tar geted to salivary glands (SG) rAAV was constructed encoding murine pre pro PYY cDNA drive n by a strong constitutive CMV/ actin promoter (Fig 4 7A ). PYY and GFP expressing casset tes were pseudotyped into rAAV2 and rAAV10 capsids as having higher transduction efficiencies in murine SG (Katano et al., 2006). The viral vector production, purification and tittering were done as described earlier ( Zolotukhin et al., 2002) A single dose of 50 L containing 1x10 10 vector genomes was administered bilaterally into each duct of the SG as described previously (Katano et al., 2006). Assessing Body Fat Mass In M ice To assess body fat in conscious live rodents we used Time Domain Nuclear Magnetic Resonance ( LF90 Minispec Time Domain Nuclear Magnetic Resonance ). This procedure involves putting a live, conscious rodent (without anesthetics) into a sample holder. Th e sample holder has a 70 mm diameter and a length of approximately 250mm with screw top that tightens to the length of mice. The sample holder is then inserted into the analyzer. Statistical analysis Fixed effects ANOVA was used to determine overall mode l adequacy of mouse weight as the response variable and treatment type as the single factor in the experiment. Protected LSD with a type we conducted using un s t
75 Results Dual Origin of Salivary PYY Previously in the laboratory it was shown that PYY is present i n human and murine saliva by RIA concentration measurement and RP HPLC/MALDI TOF (Acosta et al., 2011 ). Some proteins enter saliva from SG where they are expressed and secreted in an exocrine fashion via zymogen granules. Other peptides can enter saliva a s a transudate from serum Acosta et al already established that a fraction of salivary PYY 3 36 can be attributed to the circulating peptide (Acosta et al. 2012) To test whether PYY 3 36 is also synthes ized in the oral cavity, Acos ta analyzed RNA isolated from murine tongue epithelia and from circumvallate (CV) papillae of the tongue and showed that both sources contained PYY specific messages. To validate PYY expression data we conducted immunostaining analyses of CV W e used ce lls in pancreatic islands in C57Bl/6J mice as a positive control ( Fig. 4 1 A) and CV taste buds in PYY KO mice (Boey et al., 2006) as a negative control ( Fig. 4 1 D). W e observed PYY positive cells in the taste buds on both sides of the CV Fig. 4 1 C, F). To exclude a potential cross reactivity of PYY antibodies with NPY that had been previously shown to be expressed in TRC s (Zhao et al., 2005), we also used NPY KO mice (Erickson et al., 1996) and detected strong PYY immuno staining in TRC s in these m ice as well ( Fig. 4 1 B, E). The PYY appears to be localized in secretion granules within TRC cytoplasm ( Fig. 4 1 E) indicating its functional similarity to PYY secreted from the gut entero endocrine L cells (Bohorquez et al., ) PYY and gustducin, a G protein subunit associated with some TRCs do not appear to be co localizing in the same TRCs ( Fig. 4 2 ). Collectively these
76 data suggest that salivary PYY 3 36 originates from two independent sources: circulati ng plasma and cells in the taste buds. YR2 I s Expressed in the Basal Epithelial Cells of the Tongue To assess a possible functional role to salivary PYY we have studied the expression profile of the PYY 3 36 preferring receptor, YR2 Acosta (2011) has pre viously detected significant levels of expression of YR2 mRNA by RT PCR using mRNA isolated from murine tongue epithelia. The immunostaining analysis was conducted using brains from C57Bl/6J mice as a positive control (Stanic et al., 2006) (Fig. 4 3A) and the tongue epithelia from YR2 KO as a negative control (Tschenett et al., 2003) (Fig. 4 3B). In wild type C57Bl/6 mice, one layer of basal epithelia cells was strongly positive for YR2 (Fig. 4 3C, D, E). In addition, epithelial cells lining up ducts of the YR2 as well (Fig. 4 3C, D, F). Coincidentally, the same apical layer of YR2 positive cells appears to be innervated with neuron filaments as evident from the immunostaining using Neural Cell Adhesion Molecule (NCAM) neur onal marker antibodies ( Fig. 4 4 ). The anatomical location of YR2 positive cells, combined with their somatosensory innervation implied a possible functional role for salivary PYY 3 36 ligand and its preferred Y2 receptor related to the regulation of feedin g behavior. Oral PYY 3 36 Augmentation Therapy Intraperitoneal injection of PYY 3 36 leading to higher circulating levels of the peptide results in a dose dependent reduction in FI in rodents (Batterham et al., 2002). To test whether similar anorectic effec t could be mediated through changes in salivary PYY 3 36, Acosta (2011) developed a small spraying device that can be applied to the oral cavity in small rodent models. Using this device, we showed that after 24 hours of
77 fasting, the PYY 3 36 treated group c onsumed, on average, 12.3% less food than the control group ( Fig. 4 5 A). Additional dose response studies showed that the increased doses of PYY 3 36 led to a proportionate reduction in one hour FI, down to 81% of control values at the maximum applied dose of 12 g/100 g ( Fig. 4 5 A ). To investigate the duration of action of orally applied PYY 3 36 we conducted standard satiation studies in C57Bl/6J as well as in PYY KO mice measuring FI over 24 hours after single treatment. At the high treatment dose (12 g / 100 g BW) the anorexigenic response for both C57Bl/6J and PYY / was significant until the next treatment 24 hrs later ( Fig. 4 5B and 4 5 C respectively). However, when animals were treated with the low dose (3 g /100g BW), only PYY KO mice responded to th e treatment in significant manner observed after 18 and 24 hrs post experiment ( Fig. 4 5 C ). Because of the sustained 24 hr effect, we tested whether repeated once a day treatment would also affect the FI and BW accumulation over an extended period of time when animals were fed high fat (HF) diet ad libitum. Indeed, DIO mice treated with PYY 3 36 OS (18 g / 100 g) consumed, on a daily basis, significantly less HF food ( Fig. 4 5 D ) resulting in retarded BW accumulation which became significantly on day 17th of the treatment ( Fig. 4 5 E ). The augmentation in oral PYY 3 36 had a pronounced anorectic physiological effect similar to the previously described systemic augmentation. To verify that PYY 3 36 applied by OS did not increase the circulating concentration of the peptide, we assayed the concentration of PYY 3 36 in the plasma 10 min after OS (18 g /100 g BW) treatment or after i.p. injection (6 g /100g BW). To exclude potential interference with
78 circulating background PYY in the plasma, we used PYY KO mice (Boey et al., 2006). There was no detectable PYY 3 36 in the plasma in the vehicle and PYY 3 36 sprayed mice, while there was a significant increase in PYY 3 36 found in plasma in the i.p. injected animals implying that PYY 3 36 applied by OS acted through its r eceptors expressed in the oral mucosa ( Fig. 4 5 F ). Direct proof of PYY 3 36/ YR2 interaction was obtained by utilizing a selective Y2 receptor antagonist BIIE0246 (Dood et al., 1999). The antagonist and PYY 3 36 agonist (12 g /100 g BW) were mixed at the 50: 1 molar ratio, respectively, and the mixture was used to treat fasted mice as described above ( Fig. 4 6G ). YR2 antagonist completely ablated the anorexigenic effect of PYY 3 36 ( Fig. 4 6G ). The application of BIIE0246 alone had no effect on 2 hr FI ( data no t shown ). Long Term Increase in Salivary PYY 3 36 Modulates FI a nd BW Standard satiation studies in mice as conducted and described above, cannot be extrapolated to predict the changes in ingestive behavior in humans due to their very different feeding patt erns. Because of their intense metabolism and high caloric requirements, feeding activities become a dominant part of murine behavior manifested in frequent meals when food provided ad lib. Consequently, employing a sporadic acute elevation in salivary PYY such as obtained when using an oral spray, might not be an optimal way to modulate feeding behavior in mice. Acosta (2011) therefore, developed an alternative protocol to provide a sustained supply of exogenous PYY 3 36 in saliva using gene delivery medi ated by viral vector. The choice of targeted vector delivery was based on previous findings demonstrating efficient transduction of cells in the SG using recombinant adeno associated virus (rAAV) (Voutetakis et al., 2004) Two serotyp es were chosen taking into account that the vast majority of cells in the SG are either
79 acinar or duct al. The former cells are mainly secretory; they secrete all the fluid and 85% of salivary proteins. The latter constitute an absorptive epithelium and onl y 15% of proteins are secreted by these cells (Turner et al., 2006). Protein secretion pathways can be divided into exocrine and endocrine, and even if the mechanisms responsible for sorting secretory proteins are not well understood, it appears they could be cell type related. Using SG transgene delivery and utilizing rAAV serotype vectors characterized by selective cell type tropism we aimed to achieve either endocrine (systemic circulation) or exocrine (saliva) secretion of the transgene encoded PYY Pr e constructed rAAV encoding murine pre pro PYY cDNA driven by a strong constituti ve actin promo ter was pseudo typed into rAAV10 and rAAV2 capsids a s described earlier. These two serotypes of rAAV have different cell type tropism in SG (Baum et al., 1 999; Voutetakis et al., 2008; Baum et al., published). While AAV2 transduce s duct cells, AAV10, on the other hand, is the only serotype among tested that appears to transduce acinar cells. Therefore, using alternative rA AV vector serotypes encodi ng the same expression cassette, we achieve either systemic (rAAV10 targeting ac inar cells) or salivary (rAAV2 targeting ductal cells) reconstitution of PYY 3 36 in the PYY KO mice model PYY KO mice with the recon stituted PYY wer e used to establish the effects of chronic salivary PYY over expression on BW of obese mice. We have been breeding a colony of PYY KO mice engineered by Herbert Herzog and imported from Australia. Interestingly, only female mice show a higher BW compared w ith the wild type phenotype PYY was over expressed either in saliva of these transgenic female mice by targeting SG with serotype 2, or in plasma when targeting SG with serotype 10. To test
80 the efficiency of PYY gene transfer, levels of PYY were assayed i n the blood and in saliva ( Fig. 4 6B ) KO mice treated with serotype 2 overexpress ed PYY only in saliva and not in plasma, while mice tr eated with serotype 10, exhibited an increase of PYY in saliva and plasma ( Fig. 4 6B ). This data corroborates the notion that serotype 10 is the only one that seems to reproducibly transduce acinar cells and therefore, rAAV encoded product is secreted into saliva AND plasma Once we were able to overexpress PYY only in saliva with rAAV encoded PYY characterizing the fun ction of salivary PYY was the next step. W e asked whether a long term increase in salivary PYY would reduce FI and, perhaps, BW in KO female mice. Six weeks after transducing SG with rAAV2 PYY female mice started to gain significantly less weight compared with sham treated mice (female mice injected with rAAV2 GFP). This difference became greater when we introduced high fat diet at week 21 post injection ( Fig. 4 6C ). There was no difference in caloric ingestion (data not shown). However, body composition s tudies demonstrated that mice overexpressin g PYY in saliva have significantly less fat mass compared with controls ( F ig 4 6D ) Discussion Gut hormones play an essential role in maintaining the brain gut axis by inducing hunger or satiation in a short ter m mode. Recently, the expression of several of these peptides was detected in TRCs where they have been shown to modulate taste perception. Little, however, is known about whether these or other gut peptides could accumulate in saliva and whether they coul d play a functional role media ting satiation. In this study, we provide evidence that the spectrum of metabolically relevant peptides present in murine and human saliva includes gut hormone PYY 3 36 and that this pe ptide can be utilized to induce satiation.
81 PYY 3 36 enters the oral cavity at least in part from the bloodstream. It is not known whether PYY 3 36 is selectively transported from blood capillaries, or is non specifically leaking into the gingival crevicular fluid. What is clear, however, is that salivary and plasma peptide concentrations in humans increase postprandially. In addition, because PYY is also synthesized in the TRC s of the CV it is conceivable that PYY 3 36 is secreted from the se cells into saliva. Using MS analysis, we were unable to detect PYY 1 36 in human saliva, a result that is readily explained by the action of salivary DPP IV secreted from SG (Sahara et al., 1984; Ogawa et al., 2008). On the other hand, no expression of D PP IV had been detected in TRC s inside the taste buds (Shin et al., 2008), which opens a possibility that there are two distinct pools of PYY : 1) PYY 1 36 synthesized and contained within the taste buds; 2) salivary PYY 3 36 derived from plasma. Two PYY moie ties could play separate functions: for example, PYY 1 36 in TRC s modulating taste perception by interacting with YR1 and YR2 expressed in some TRCs as shown in C hapter 3 while PYY 3 36 in saliva modulating, in part, feeding behavior by interacting with YR2 in the tongue epithelial cells. Conceptually, the latter attribute of salivary PYY 3 36 appears to be redundant considering that PYY enters saliva from plasma, while at the same time inducing satiation through hypothalamic Y2 receptors in a well described fashion. In the current report, however, we have provided ample experimental evidence showing that the augmentation of salivary PYY 3 36 indeed reduced FI. The evidence was obtained in feeding behavioral studies using 1) escalating doses of PYY 3 36, 2) sel ective YR2 antagonist (BIIE0246), 3) re feeding after fasting, and 4) ad libitum feeding paradigms,
82 while utilizing both C57Bl/6J and PYY KO mice models. Moreover, we have shown that one time treatment in mice at the beginning of a dark cycle conducted ove r twenty consecutive days was sufficient to reduce the rate of BW accumulation. This surprising persistence of the anorexigenic effect can be explained, in part, by a nocturnal feeding pattern in rodents, which is in contrast to the human diurnal feeding. Rodents consume most of the food at the beginning of the dark cycle (Tschop et al., 2004) coinciding with initial effect of PYY OS treatment. To address the issue of stability of the orally applied PYY 3 36 we developed a completely different treatment par adigm, using rAAV mediated gene transfer. SG cells exhibit at least two distinct secretory pathways: a predominant regulated one leading to exocrine protein secretion into saliva via zymogen granules and a constitutive one leading to the bloodstream (Hoque et al., 2001; Castle et al., 1998). To benefit from this difference in secretion mechanisms, the transgene to be used was designed to incorporate pre pro PYY signal sequences in ductal cells which are in charge of exocrine protein sec retion. Using rAAV2 PYY transgene in female KO mice, we were able to transduce only ductal cells and thus isolate the function of salivary PYY A sustained expression of pre pro PYY transgene from the rAAV transduced cells in the SG resulted in three fold increase of PYY 3 36 in saliva with no apparent endogenous secretion into the bloodstream. Surprisingly, this modest increase over physiological levels resulted in significant less weight gain in mice fed ad libitum. After six w eeks of treatment mice treate d w ith rAAV 2 PYY gain less weight compared with controls. These data, together with YR2 selective agonist data, point to the oral mucosal epithelial YR2 positive cells as potential targets for anorexigenic actions of the salivary
83 PYY 3 36 and suggests the existence of a putative neuronal circuit initiated in the oral cavity. We observed a juxtaposition of YR2 positive cells and neuronal filaments in the basal cell layer of the murine tongue. Whether this finding observed by immunostaining methods is reflect ive of their functional synaptic connection remains to be investigated using more precise electron microscopy methods. However, if such a signaling pathway exists, it might not be inducing an aversive response that follows the peripheral administration of PYY 3 36 and activation of neurons in the circumventricular organs of the area postrema (AP) and intermediate portion of nucleus of a solitary tract (Halatchev et al., 2005). Instead, it would activate somatosensory neurons innervating the receptor field of the tongue epithelia converging with afferent gustatory neuronal pathways The neuronal circuitry involved in signal transduction to the central n ervous system will be found in Chapter 5 In summary, we have shown that the gut sati ation peptide PYY 3 36 is present in saliva where it can play a physiological role in FI The anorexigenic effect is apparently mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial cells. We have exploited this put ative metabolic circuit to control FI, in a simple and efficient way, suggesting a potential novel therapeutic application for the treatment of obesity
84 Table 4 1. Antibodies used for immunostaining studies Antibody Host Supplier Dilution Specificity and control Anti PYY Rabbit Abcam (Cambridge, MA, USA; cat. No. ab22663) 1:2000 Staining absent when primary or secondary antibodies omitted. Staining visible when PYY / tissues were used due to cross reactivity with NPY (25%). Stain ing visualized in NPY / tissues. Use of this antibody has been reported previously. Anti YR2 Rabbit Neuromics (Edina, MN, USA; cat. No. RA14112) 1:3000 (using TSA Kit) Staining absent when primary or secondary antibodies omitted, or in NPY Y2 receptor / Use of this antibody has been reported previously. Anti gustducin Goat Santa Cruz Biotechnology (sc 26890) 1:200 Staining absent when primary or secondary antibodies omitted. Anti NCAM Rabbit Millipore (Temecula, CA, USA; cat. No. AB5032) 1:500 Stai ning absent when primary or secondary antibodies omitted. Anti Keratine 5 Rabbit Covance (Emerit, CA, USA; cat. No. PRB 160P) 1:1000 Staining absent when primary or secondary antibodies omitted.
85 Figure 4 1. PYY is synthesized in TRCs A) Immunostaini ng of PYY positive cells in a cells in the m urine pancreas, a (+) control. B) Immuno staining of PYY in CV of a NPY KO mouse, a control for PYY antibodies cross reactivity. C) Immunostaining of PYY in CV of a C57Bl/6J mouse (wild type ). D) Immunostaining of PYY in CV of a PYY KO mouse, a ( ) control. E) close up of B). F) close up of C). Arrowheads point at the apical part of a taste bud.
86 Figure 4 2. Identification of PYY gustduci n (red) by co immunostaining in the same taste bud. Secreti on granules incorporating PYY predominantly gustducin.
87 Figure 4 3. Y2 receptor is synthesized in the epithelial cells of the tongue. A) Immunostaining of YR2 positive cells in the hippocampus of C57Bl/6J mouse ( wi ld type ), a (+) control. B) Immunostaining of YR2 in the tongue epithelia of YR2 KO mouse, a ( ) con trol. VEG C) Immunostaining of YR2 positive cells in the CV area of t he tongue of a C57Bl/6J mouse. D) close up of C). E), and F) close ups of D), top and bottom rectangles, respectively
88 Figure 4 4 Neuronal filaments innervate CV papillae ( CV ) as well as the basal layer of cells distant from CV Immunostaining for NCAM in CV ( A) shows subpopulation of TRCs expressing K5 (marked by arrowheads in panel D, a close up from panel A), as well as a dense mesh of filaments at the basolateral surfaces of the taste buds. Rectangles in ( B) and (C) designate the same areas in two sequential mirror sections stained for NCAM (red), or YR2 (green) The protrusions in the tongue epithelia surface (B, C, E, and F) are filiform papillae transversely sectioned. Even distant from CV the YR2 positive epithelial layer is morphologically close to neuro filament layer below (E). Some YR2 cells and NCAM fil aments appear to be juxtaposed (arrows in E and F).
89 Figure 4 5. Oral PYY 3 36 augmentation therapy. A) Dose response effect of PYY 3 36 on 2 hr FI vs. control (n=8 each group). B) Effect of PYY 3 36 OS on FI in C57Bl/6J mice measured at 1, 2, 6, 12, 18, and 24 hr post treatment (n=8/group). C) Effect of PYY 3 36 OS on FI in PYY / mice measured at 1, 2, 6, 12, 18, and 24 hr post treatment (n=8/group). D) Average 24 hr FI in DIO mice treated with daily PYY 3 36 OS (18 g/100 g). E) Effect of daily PYY 3 36 OS (1 8 g/100 g) treatment on BW change in DIO C57Bl /6J mice (n=9 per each group). F) Concentration of PYY 3 36 in plasma of PYY KO mice 10 min after PYY 3 36 (18 g/100 g BW), or control OS vs. PYY 3 36 injected i.p. (6 g/100 g BW) (n=10 per g roup). G) Effect of YR2 specific antagonist BIIE0246 on anorexigenic action of PYY 3 36 (n=8 per each group) measured as 2 hr FI after 24 hr fast.*P < 0.05, **P < 0.01.
90 A B C
91 D E F
93 Figure 4 6. Effect of PYY gene transf er to the SG in C57Bl/6J mice. A) Diagram of rAAV PYY and rAAV GFP cassettes: ITR inverted terminal repeats of rAAV serotype 2; CBA Cytomegalovirus interm ediate early enhancer sequence/ chicken b acting promoter; murine Pre pro PYY cDNA, GFP gr een fluorescence protein cDNA. B) Concentration of PYY 3 36 in plasma an d saliva during fasting. C) Effect of PYY 3 36 SG gene delivery on weekly BW in mice fed norma l regular chow until week 21 and then with HF diet; female mice were treated with rAAV GFP (control group) and rAAV PYY D) Effect of PYY 3 36 SG gene delivery on body mass composition (all groups were 8 animals/group) .*P < 0.05, **P < 0.01, ***P<0.001.
94 A B C
96 CHAPTER 5 SALIVARY PEPTIDE YY : PUTATIVE CIRCUIT T HAT CONTROLS INGESTI VE BEHAVIOR Appetite and satiation are fundament al regulators of ingestive behavior. However the relative palatability of food also strongly influences intake. Among the many mechanisms that could potentially inhibit ingestive behavior, two of the most prominent are: (1) the induction of satiation, and (2) negative modulation of palatability, i.e. conditioned taste aversion ( C TA) (Yamamoto 2008). Multiple endogenous and exogenous substances inhibit ingestive behavior and reduce food intake (FI) by inducing satiation, producing C TA or both Indeed, severa l satiation hormones were found to induce CTA if used at higher doses in rodents and human patients: glucagon like peptide 1 (GLP 1) (Thiele et al., 1997), Exendin 4 (Kolteman et al., 2003; Baraboi et al., 2011), cholecystokinin (CKK) (Deutsch et al., 1977 ), and Peptide YY (PYY) (Halatchev et al., 2005). PYY a well characterized molecular mediator of satiation, is released mostly by enteroendocrine L cells in the distal gut epithelia proportionally to the amount of calories ingested. Evidence suggests tha t circulating truncated form PYY 3 36 physiologically reduce s FI, and that its insufficient production promote s obesity under obesogenic conditions To support this notion it has been demonstrated that some obese humans have a blunted plasma PYY 3 36 in resp onse to FI (Le Roux et al., 2006), while its systemic administration inhibits FI in rodents, monkeys, and humans (Chelikani et al., 2005; Batterham et al., 2002; Degen et al., 2005; Moran et al., 2005; Talsania et al., 2005). However, in spite of an ongoin g investigation, the mechanism by which PYY controls ingestive behavior has not been fully elucidated.
97 Data from many laboratories suggest that circulating PYY 3 36 may inhibit FI through a direct action on Y2 receptors ( YR2 ) in specific brain sites known t o control FI. Reports described that (1) PYY 3 36 crosses the blood brain barrier (BBB) in mice (Nonaka et al., 2003); (2) many forebrain and hindbrain sites mediating FI express YR2 (Stanic et al., 2006; Fetissov et al., 2004); and (3) direct injections of PYY 3 36 into the arcuate nucleus inhibit FI (Batterham et al., 2002). On the other hand, there is evidence supporting the notion that circulating PYY 3 36 may also inhibit FI through direct action on YR2 expressed in abdominal sensory branches of the vagal nerve (Koda et al., 2005). However, its role is not entirely clear because vagal denervation supressed the anorexic response to peripheral administration of PYY 3 36 in rats (Koda et al., 2005) but not in mice (Halatchev et al., 2005). Adding more complex ity to the understanding of the physiological role of PYY 3 36, we have recently documented that PYY 3 36 is present in saliva (Acosta et al., 2011). Although the innate physiological functions of salivary PYY 3 36 have yet to be determined, we have shown tha t it could modulate FI and, eventually, body weight (BW) accumulation. The anorexigenic effect is apparently mediated through the activation of the specific YR2 in a subpopulation of cells in oral mucosa. These data suggest the existence of a putative neur onal circuit initiated in YR2 positive cells in the tongue epithelium and extending to hypothalamic centers via cranial nerves afferents (Acosta et al., 2011). If such a pathway existed, it would have to relay the information through the brain stem. Incide ntally, the neurons in the area postrema ( AP ) of the brain stem are known to mediate, in part, CTA in response to the PYY 3 36 administered peripherally (Halatchev et al., 2005) or intranasal (Gantz et al., 2007).
98 The purpose of this investigation, therefo re, was to identify whether salivary PYY 3 36 inhibits ingestive behavior by activating neurons in hypothalamic centers and solitary tract (NST) nucleus, areas of the brain known to control FI. We also examined whether PYY 3 36 induced reduction in feeding i nvolved aversive behavioral responses, and we evaluated the potential contribution of neurons in the AP which are known to participate in conditional CTA Methods Animals The Institutional Animal Care and Use Committee of the University of Florida approve d all experimental procedures. Male wild type C57BL/6 mice weighing 20 25 g were housed individually in hanging wire mesh cages in a room with 12:12 hr light dark cycle. Unless noted in the experimental procedures, a nimals had ad libitum access to regular mice chow and water. Test Substances The CTA experiment was performed using PYY 3 36 (canine, mouse, porcine, rat) from Bachem (Torrance, CA) and LiCl (Alfa Aesar, Ward Hill, MA; purity greater that 99.995%). The peptide was certified by the manufacturer w ith purity greater than 97%. For the i.p. injections, PYY 3 36 was dissolved in sterile distilled water at the concentration of 0.02 g / mL The solution was injected in to the peritoneal cavity at a dose of 6 g per 100 g of BW. For the oral spray (OS) trea tment, PYY 3 36 was diluted at the concentrations of 0.075, 0.15 or 0.225 g / mL The administered doses were 6, 12 or 18 g /100 g of BW. LiCl was dissolved in sterile distilled water to a final concentration of 0.15 M; mice were injected i.p. with a volume equivalent to 2% of their BW. For the CTA experiment with liquid, flavored solutions were made of diluted Kool Aid (either
99 0.15% saccharine with 0.05% cherry Kool Aid or 0.15% saccharine with 0.05% grape Kool Aid). For the CTA with solid food, we used flav ored apple and orange Crunchies (BetterPets Inc., NJ). Experimental Procedures Treatment with orally administered substances With the exception of radio labeled PYY (see section below), the oral treatment described herein refers to the substances administ ered by an OS targeting receptors in the oral cavity. The solutions were administrated into the oral cavity in a single puff, as described previously (Acosta et al., 2011), using a sterile 9/16 Dram (8x58MM) glass sampler bottle (each puff delivers about 3 0 L of solution to the oral cavity in a harmless fashion) In vivo treatment of mice with 125 I PYY 1 36 Mice were deeply anesthetized and 125 I labeled human PYY 1 36 (Phoenix Pharmaceuticals) was administered into the oral cavity by mi cropipette at the dose o f 7 Ci/100 g BW (equivalent to 18 g /100 g BW) in a total volume of 155 L or injected intra peritoneal (i.p.). BIIE0246 antagonist was applied at 50 molar excess as described previously (Acosta et al., 2011). Five min after oral administration, mice wer e sacrificed; tongue tissues were harvested and extensively washed in several changes of PBS until no above the background radioactivity was detected by Geiger counter. After systemic administration, the animal was sacrificed 15 min after injection and the tongue was treated as described above. Sagittal sections of the fresh frozen tongue tissues were exposed to a one sided X ray film (Kod ak BioMax MR) for 3 days at 20 C. Slides were then stained with hematoxylin eosin staining to visualize cell morphology
100 c Fos immunostaining Five groups of mice were fasted for 24 hours. Mice in the negative control group (n=5) were given water via OS followed by saline solution (SS) injected i.p. Mice were sacrificed 30 min after treatment. A second group of mice (n=5) w ere sprayed with water, injected with SS i.p., fed for one hour and sacrificed one hour later. The third and fourth groups (each n=5) after the 24 hour fasting were administered PYY 3 36 OS (6 g/100g of BW) and SS i.p. or water OS and PYY 3 36 i.p. (6 g/10 0g of BW), respectively; mice were sacrificed one hour after the treatment. To study the effect of PYY 3 36 OS on CTA, a fifth group of mice, positive controls (n=5), were fasted for 24 hrs and then injected with LiCl (2% of BW at a concentration of 0.15 M) LiCl was used as a positive control treatment. LiCl is a chemical compound known to cause CTA and activate regions in the central nervous system related to aversive stimuli. PYY 3 36 injected i.p. was used as another positive control previously characteri zed to induce CTA (Halatchev et al., 2005; Chelikani et al., 2006). Fifteen min post injection, mice were fed for one hour and sacrificed one hour later. To collect brains, a previously described protocol was followed (Gortbayuk et al., 2001). Briefly, mi ce were deeply anesthetized with sodium pentobarbital and perfused sequentially through the ascending aorta with: (1) 20 mL of heparinized saline; (2) 4% paraformaldehyde in 0,1M phosphate buffer, pH 7.4 (PB). The brains were postfixed in the same fixative for 4 hours and immer sed in 30% sucrose in 0.1 M PB at 4 C. A series of 40 (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany) and collected in anti freezing storage solution.
101 For the bri ght field photomicrographs, sections were pre incubated first with 0.5% H 2 O 2 10% methanol for 15 min and then with 5% normal goat serum for 1 h. Sections were incubated for 36 hours at 4C with anti c Fos primary antibody (Santa Cruz Biotech., 1:2000 dilut ion). Incubation with secondary goat anti rabbit biotinylated antibody (Dilution 1:400 for 4 hours) was followed by incubation with avidin biotin peroxidase complex (ABC; Vector Laboratories, Burlingame, CA, USA). Reactions were visualized using 3,3 diamin obenzidine (DAB) as a chromagen. Behavioral Studies Two complementing paradigms were used to study the induction of CTA by PYY 3 36: one with liquid and one with solid food. Both protocols were performed as previously described by Halatchev et al., (2005) a nd Chelikani et al., (2006). with the following modifications (Table 5 1). Mice were habituated to individual housing two weeks before the experiments cages between 1900 and 2100 hrs (dark peri od from 1900 and 700 hrs). Animals had free access to regular rodent chow at all times. Water was withdrawn 23 hours before the start of the first day of training. Mice had access to water or to the flavored solutions every day for 1 hour. Liquid paradigm (Table 5 1, open cells) Habituation procedure. T o habituate to timing of liquid presentation, mice were conditioned to consume water during a 5 day training period. After 23 hours of liquid deprivation, water was offered for one hour (1900 to 2000 hrs) in two different bottles that were situated equidistant from the food hopper. To determine t he amount consumed bottles were weighed before and after each training session. To acclimate mice to i.p. injections and to the OS, after each training session, anima ls were injected
102 i.p. with a volume of sterile isotonic saline solution equal to 2% of BW and, at the same time, water was administered to the oral cavity via an OS. Conditioning procedure. Immediately following training, animals were subjected to a 12 day conditioning procedure consisting of the following three, four day sequences (Table 1). The animals were assigned at random to either of two flavors conditions and were subjected to a regimen of OS and i.p. injections. During the same 1 hour period (1900 to 2000 hrs) on Day 1, mice were offered a novel flavored liquid in both bottles (that could be either grape or cherry Kool Aid prepared as described previously (Halatchev et al., 2005)), followed by one of the following treatment regimens: 1) PYY 3 36 OS a t doses indicated, accompanied by i.p. injection of saline solution; 2) water OS and PYY 3 36 i.p.; 3) water OS and LiCl i.p.; or 4) water OS and sterile saline (ss) solution i.p. On Day 2, all the mice received water for 1 hour to allow for recovery from t he treatment regimen. On Day 3, each mouse received the alternative novel flavor (i.e., if an animal received grape during the first day, it received cherry in the third and vice versa), and after 1 hour, all mice received saline solution i.p. injection an d water OS. On Day 4, they received again water during the 1 hour period. We repeated t his four day sequence three times At the end, over twelve consecutive days mice were exposed to three conditioning trials. Preference trials. On the two days following the conditioning period, we gave each mouse simultaneously access to the two flavored solutions and we measure the amount consumed of each stimulus after an hour For each mouse, the left right position of the bottles containing the two flavored solutions was reversed during the second day. Water was presented as flavored solution in two separate bottles
103 equidistant from the food. Treatment consisted either of the following regimens: (1) PYY 3 36 OS at different doses and saline solution (ss) i.p., (2) wate r OS and PYY 3 36 i.p., (3) water OS and LiCl i.p., or (4) water OS and ss i.p. Solid food paradigm (Table 5 1, shaded cells) For the CTA experiment with solid food, procedures were the same as those described above, but flavored Crunchies were used instea d flavored solutions. Mice were fasted for 23 hours instead of being water deprived for 23 hours. Regular chow or flavored crunchies were presented in two separate trays equidistant from water. Treatment consisted either of the following regimens: (1) PYY 3 36 OS at different doses and saline solution (ss) i.p., (2) water OS and PYY 3 36 i.p., (3) water OS and LiCl i.p., or (4) water OS and ss i.p. Statistics Statistical analyses were performed using IBM SPSS Statistics Version 17 software. Data are expressed as group means +/ SE. For the CTA experiments, significance across individual treatments was determined using one way ANOVA with (two tailed) t test was used to determine the significance when two groups were compared. For c Fos activation experiments, one posthoc was used comparing different treatments to the fasting control group, followed by one osthoc tests to determine rate resulted from multiple pairwise comparisons. The statistical rejection criterion was
104 Results Salivary PYY 3 36 Binds to L ingual YR2 Receptors In C hapters 3 and 4, we showed that there is a subpopulation of YR2 (+) cells in taste receptor cells (TRC) we have also shown that in mice, salivary PYY 3 36 mediates anorexigenic re sponses in YR2 dependent fashion. To determine whether salivary PYY binds to YR2 expressed on tongue epithelia cells, we have utilized 125 I labeled PYY 1 36 administered into the oral cavity of PYY KO mice. Five minutes after treatment, radio labeled PYY wa s bound to both dorsal and ventral tongue surface epithelia (Fig. 5 1A). When labeled PYY was mixed with YR2 specific antagonist BIIE0246 (Doods et al., 1999), the binding was abrogated providing additional support for the specificity of the interaction (F ig. 5 1B). Moreover, when radio labeled PYY was administered i.p., the binding of 125 I PYY to the that systemic PYY is efficiently transported into saliva (Acosta 200 9). The exact localization of radiolabeled PYY was visualized by staining the same slide with hematoxylin eosin. The experiments described below focus on identifying putative neural pathways downstream of PYY / YR2 interaction. Salivary PYY 3 36 Activates Hyp othalamic C Fos The mechanism of the anorexigenic action of peripherally applied PYY 3 36 could be related to its action on hypothalamic neurons (Batterham et al., 2002). Alternatively, peripheral PYY 3 36 may inhibit FI by signaling through YR2 expressed i n the vagus nerve (Koda et al., 2005). Both pathways have been shown to activate c fos in the hypothalamus. To test whether salivary PYY augmentation activates hypothalamic centers, four groups of mice were conditioned for repeated cycles of fasting for 24 hrs
105 followed by refeeding. A treatment combination of i.p. injections and OS administration was incorporated into the conditioning protocol. Three control groups were sprayed with vehicle and either not treated (Group 1, Fig. 5 2, column Fast ), fed for 1 hr (Group 2, Fig. 5 2, column Fed ), or injected with PYY 3 36 i.p. (Group 3, Fig. 5 2, column PYY i.p. ). Mice in group 4 (Fig. 5 2, column PYY OS ) were treated with PYY 3 36 OS (6 g /100 g BW). Mice in Groups 1, 2, and 4 were also sham injected so th at all mice in all groups were subjected to the same combination of spray/i.p. injections. Mice in groups 1, 3, and 4 were fasted over the duration of the experiment. All mice were sacrificed at one hour after the treatment. Brains were harvested and neuro nal activity was evaluated by probing the induction of c fos expression. Similar to the mice from the fed control group, orally treated and i.p. injected PYY 3 36 groups showed activation of neurons in hypothalamic arcuate nucleus (Arc, Fig. 5 2, top row), paraventricular nucleus (PVN, Fig. 5 2, middle row), and lateral hypothalamic area (LHA, Fig. 5 2, bottom row). Although PYY 3 36 i.p. injected mice displayed an increase in a number of c fos positive PVN neurons, the trend, however, did not reach statisti cal significance. Effect of Salivary PYY 3 36 on Brain Stem Neurons To investigate the afferent neuronal pathways further, we studied the patterns of c fos activation in the nucleus of the solitary tract (NST) in the rostral and caudal brainstem; the cauda l portion known to relay satiation signals from the alimentary tract to the hypothalamus (Hamilton et al., 1984). Both OS and i.p. groups were treated with the ide ntical doses of the PYY 3 36 (6 g/100g BW) that were previously identified to reliably reduce FI (Batterham et al., 2002; Acosta et al., 2011). Two prominent areas of the NTS were analyzed separately: rostral and caudal, as well as area postrema (Fig. 5 3A ) shaded areas unilaterally shown on the right aspect of the solitary tract). To study
106 these areas, we introduced an additional control group of mice injected i.p. with LiCl to induce visceral malaise. Rostral NST (rNST). In the rostral subdivision, we combined c Fos positive neurons in several sub nuclei constituting medial part of NTS: rostral medial (Rm), rostral intermedial (Ri), and rostral ventrolateral (Rvl). All three areas showed similar responses trends and, thus, were combined in one morphological entity (the respective shaded areas in Fig. 5 3B; and the dashed ovals in the brain secti ons, Fig. 5 3D). Surprisingly, both PYY 3 36 i.p. and OS treated groups showed a significant reduction in the numbers of c Fos positive neurons as compared with either fasting or PYY i.p. group (Fig. 5 3C). Animals in the fed group responded by activating neurons, while there was no significant effect in the rostral NST neurons in LiCl group. Caudal NS T (cNST). In the caudal aspect, we studied the intermediate NST (also known as NST at the level of AP shaded area, Fig. 5 3A). Within this region, we studi ed the medial NST (mNST, areas outlined in Fig. 4A). There were few c Fos positive cells in fasted animals. Unlike rostral part, the caudal NST responded to LiCl treatment in a very robust fashion. In addition, both fed and PYY 3 36 i.p. control groups show ed significant increase, while there was no response in the OS group (Fig. 5 4B). Area Postrema ( AP ). In the AP all four groups showed significant activation of c Fos neurons when pair wise compared to the fasted group (Fig. 5 4C). Similar to the caudal a rea, the neurons in the PYY 3 36 OS group showed the least activation that was significantly lower than in PYY 3 36 i.p. group, and not different from the fed group. Overall, neurons in both rostral and caudal brainstem clearly responded in a distinctive wa ys to the PYY treatment, dependent on the administration route. For
107 example, rostral neurons in the OS group showed significantly higher degree of inhibition as compared to the i.p. group. At the same time, caudal mNST neurons were either not activated in the OS group or showed significantly lower degree of activation in the AP Such differential pattern could reflect the distinctive mechanisms of PYY 3 36 action: humoral via circumventricular organs when administered systemically vs neuronal if applied by o ral spray. Salivary PYY 3 36 Does not Induce CTA PYY 3 36 administered systemically had been shown to reduce FI (Batterham et al., 2002) while at the same time inducing CTA (Halatchev et al., 2005). The latter manifestation is apparently related to the acti vation of neurons in AP brainstem area mediating, in part, aversive reactions (Halatchev et al., 2005). Because we observed a distinct pattern of brainstem neurons activation after OS or i.p. administered PYY 3 36,Ithen asked whether these differences m behavior as well. Inducing CTA with flavored liquid. PYY 3 36 OS at doses that reliably and reproducibly inhibit FI (6 g /100g of BW) (Acosta el at. 2011) did not produce CTA in mice while PYY 3 36 i.p. at the same do se did (Fig. 5 5 A). Negative controls that received saline i.p. and water OS paired to both flavors, did not show any preference for either of the flavors and drank equally from both stimuli. Positive controls that were treated with LiCl, on the contrary, showed the largest reduction of treatment paired flavor: the difference of consumption between the two flavors was 75%. Mice that received PYY 3 36 i.p. (6 g /100g of BW) drank 65% less of the PYY 3 36 i.p. paired flavor vs. the saline paired flavor. PYY 3 36 OS treated mice drank equally from the two flavors.
108 To compare the effect of the treatment (saline i.p. and water OS versus PYY 3 36 OS, PYY 3 36 i.p. or LiCl) on the relative consumption of the treatment paired flavor, we also expressed the results in rati os in which the amount of treatment paired flavor was divided by the total volume consumed by an animal (Figure 5 5 B). Both the PYY 3 36 i.p. group and LiCl injected controls had reduced ratios compared with the saline control group. PYY 3 36 i.p. injected m ice had a ratio of 0.24 +/ The drastic reduction of treatment paired flavor consumed by LiCl treated mice translated to a ratio of 0.19 +/ PYY 3 36 OS showed a ratio close to the negative controls ratio (p=0.5). Previously, we had shown that higher doses of PYY 3 36 applied orally for 21 consecutive days resulted in a significant reduction of BW accumulation in mice (Acosta et al., 2011). Therefore, to exclude the possibility of mounting CTA at higher doses, the ab ove experiment was repeated using PYY 3 36 OS at 12 and 18 g /100 g of BW. Likewise, neither of these doses resulted in preference or aversion for any of the flavors (Fig. 5 5 C, D). LiCl control group consistently showed a reduction of the paired flavor con sumption. Inducing CTA with flavored solid food. To corroborate these data and to reproduce potential therapeutic application scenario, we repeated the behavioral experiment using flavored solid food. Using just two PYY 3 36 OS treatment doses (6 g /100g, or 18 g /100g BW),we have observed similar results (Fig. 5 5 E, F). PYY 3 36 OS paired flavors had no effect on amount of food consumed, while there was significant difference documented for PYY 3 36 i.p. treated group, and even more so for LiCl control group
109 Discussion Throughout the gastrointestinal system, mechanical and chemical stimuli induce endocrine cells respon se They release satiety signals in response to FI thereby inducing cellular responses along the entire gastrointestinal tract. Released signa ls are transmitted neurally and reach the brain through vagal afferents or humorally as circulating ligands targeting specific receptors in the brain. These signals are interpreted by the CNS and result in ingestive behavior modifications. PYY 3 36 plays a major role as a satiety signaling hormone. It is released from intestinal L endocrine cells into the bloodstream primarily in response to the amount of calories ingested. Circulating PYY 3 36 freely crosses the blood brain barrier gaining access to brain po steriorly (Nonaka et al., 2003) and activating arcuate neurons directly (Batteham et al., 2002; Halatcheve t al., 2004), and/or through the intermediate NST and area postrema in the caudal brainstem (Halatchev et al., 2005). Adding to the growing complexit y of the PYY 3 36 targeted neuronal network, we have recently described a putative signaling pathway originating in the oral cavity and responsive to salivary PYY 3 36 (Acosta et al., 2011). Here, we also show that circulating PYY 3 36 rapidly binds to YR2 re ceptors in the tongue epithelia (Fig. 5 1C). Moreover, orally applied PYY binds to the lingual YR2 receptors (Fig. 1A, B) without increasing the circulating concentration (Acosta et al., 2011). Although the PYY was administered as the full length form PYY 1 converted into the truncated form PYY 3 36 by the peptidase DPPIV present in saliva (Ogawa et al., 2008; Sahara et al., 1984). My previous results suggested the existence of an alternative anorexig enic circuitry mediated by salivary PYY 3 36 and its cognate receptors in the oral cavity.
110 Moreover, because systemic PYY 3 36 had been implicated in mounting CTA by activating area postrema neurons (Halatchev et al., 2005; Chelikani et al., 2006), it was of interest to test whether orally administered PYY 3 36 induced aversive responses as well. The data presented in this report have to be interpreted with the following notions in mind. On one hand, we have previously shown that PYY 3 36 administered periphera lly will be transported, or will leak into the oral cavity from the bloodstream (Fig. 5 1C). Thus, in the i.p. injected positive controls utilized in this study, PYY 3 36 will activate target neurons as characterized previously (Halatchev et al., 2006; Mora n et al., 2005), and, upon diffusion into the oral cavity, it will also affect the putative pathway that originates in the lingual epithelia cells. On the other hand, PYY 3 36, applied by OS, will not leak retrogradely i nto the bloodstream (Acosta et al. 20 11 ). As a result, it would not positive cells and putative afferent pathways. The immediate structure responding to the afferent information from the oral cavity and the lingual receptors is the nucleus of the solitary tract. An important point to consider is that the rostral and caudal aspects of the NST are innervated by overlapping but distinct neuronal projections. rNST contains, predominantly, overlapping terminals of the two cranial nerves: branches of the facial nerve (VII) the chorda tympani and the greater superficial petrosal innervating, respectively, the anterior 2/3 of the tongue and the palate; and the linguotonsilar branch of the glossopharyngeal nerve (IX) origina ted in the posterior part of the tongue. cNST and AP on the other hand, are innervated mostly by the superior laryngeal branch of the vagus nerve (X). Due to the anatomical differences and taking into account functional studies, it was suggested that the NST
111 consists of two major divisions: rostral, and caudal, mediating and integrating gustatory and visceral information, respectively (Hamilton et al., 1984). Moreover, this partition is further manifested in distinctive afferent projections to the higher b rains areas (Fig. 5 6). C Fos in Fasted v s. Fed Control Animals Data in this report are consistent with previously published findings describing activation of neurons in hypothalamic areas in anticipatory response to feeding in habituated animals (Johnston e et al., 2006). There were few c Fos positive cells in fasted animals in Arc, PVN, and LHA and their numbers were markedly induced in all areas after feeding. All these activated areas are known to mediate both satiety and hunger, and, therefore, without additional morphological studies involving immuno staining it is not possible to determine the precise nature of activated hypothalamic c Fos expressing neurons. In the hindbrain, rNST, cNST, and area postrema reacted similarly by increasing the numbers o f activated neurons. This increase is explained by the induction of the afferent signaling from gustatory neural fibers innervating lingual and mucosal TRCs (rNST), as well as from the stimulated chemo and mechanoreceptors in the gut (cNST, AP ). C Fos i n Fasted and Fed vs. PYY i.p. Animals Rostral and caudal NST in PYY i.p. treated animals reacted in distinctively different ways. There was significant reduction of c Fos (+) neurons in rNST as compared with both fasting and feeding conditions. It appeared that systemic PYY was inhibiting the activation of the gustatory neurons in this area that might have resulted from a) either blocking afferent signaling after passing of PYY from blood into saliva or b) activating brain structures mediating aversive resp onses. The latter option is
112 reinforced by the fact that there is a significant induction of area postrema neurons after PYY i.p. administration as shown in this report and by other groups (Halatchev et al., 2005). The response of the cNST and area postrem a to the systemic PYY administration is determined by their close anatomical association. AP a circumventricular organ directly affected by the plasma hormones, projects neuronal afferents into the medial NST (Cunningham et al., 1994). Both cNST and area postrema groups responded by significantly increasing the numbers of c Fos (+) cells compared to the fasted, but not to the fed group. Both nuclei responded to LiCl in a very dramatic way consistent with the view that the area postrema projects into the cN TS (Date et al., 2006) and that it is a chemoreceptor trigger zone mediating nausea (Bernstein et al., 1992). C Fos in PYY i.p. vs. PYY OS Animals Administration of an anorexigenic dose of PYY 3 36, whether it is i.p. or by an OS, increased the number of c Fos positive neurons in the forebrain Arc, PVN, and LHA nuclei. These findings do not directly confirm or contradict other studies suggesting that peripherally administered supraphysiological PYY 3 36 inhibits FI through direct activation of Y2 receptors in the arcuate nucleus (Halatchev et al., 2005; Batterham et al., 2202; Halatchev et al., 2004) This is because circulating PYY 3 36 in i.p. injected control animals can enter the oral cavity (Fig. 5 1C) and induce an anorexigenic response through the putati ve pathway initiated in the oral mucosa. What is clear, however, is that supraphysiological salivary PYY 3 36 activates Arc, PVN, and LHA nuclei in a very robust fashion (Fig. 5 2) and, thus, can modulate satiety/feeding centers by circumventing humoral pha se. This notion refers to the hypothetical therapeutic
113 application whereby PYY 3 36 could be administered into the oral cavity thus inducing satiety and reducing the size of the meal that follows. This data support the notion of the existence of a separate anorexigenic signaling pathway initiated in the oral cavity. Interestingly, the activation of the neurons in LHA nucleus feeding center in fasted animals that were treated with PYY OS was significantly higher (p=0.002) than in the fed mice. Whether such a putative pathway plays a meaningful regulatory role under physiological salivary PYY 3 36 concentrations remains to be determined. However, in favor of such a possibility, is the fact that a significant postprandial increase of salivary PYY 3 36 (Acosta e t al., 2011) mirrors the similar postprandial increase in plasma PYY concentration. The pattern of neuronal activation in hindbrain areas by salivary PYY 3 36 also lends credence to the notion of a separate dedicated pathway. There was a marked difference i n responses to PYY treatment depending on the route of administration: in the OS treated mice, rNST neurons were inhibited in a more pronounced way, while the activation was either minimal ( AP ) or not significant (rNST). We have in Chapters 3 and 4 shown the extensive expression of the PYY 3 36 preferred receptor YR2 in the basal cell epithelia of the tongue, as well as in TRCs of the circumvallate (CV) papillae. These PYY 3 36 responsive cells could be candidates to transduce the information from salivary P YY 3 36. Othe r members of the Neuropeptide Y (NPY) family NPY and its preferred receptor, YR1 have been previously shown to be expressed in TRCs regulating inwardly rectifying K+ currents (Zhao et al., 2005; Herness et al., 2009). Although their direct roles in modulating taste perception remains to be determined, it is possible that both salivary PYY and NPY modulate signaling
114 manifested as c Fos positive neurons in the arcuate and PVN nuclei. Therefore, while peripheral PYY 3 36 may exerts its effects t hrough the vagal nerve, salivary PYY 3 36 could affect the facial and glossopharyngeal nerves which carry afferent gustatory and somatosensory signals. At least one ascending noradrenergic pathway links the NST to the arcuate (Date et al., 2006), and there exists strong evidence of ascending NST PVN projections that are involved in leptin and CKK satiation effects (Blevins et al., 2010). Oral inputs could also reach the area postrema directly via mandibular trigeminal afferents (Jacquin et al., 1982), from t he cervical vagus nerves (Kaia et al., 1982), and \ or indirectly from NST, which receives trigeminal afferents input (Hamilton et al., 1984) and projects to the area postrema (Shapiro et al., 1985). Taken together these data provide support for the existenc e of anatomical substrates connecting oral mucosa and satiety centers. Conditional Taste Aversion To corroborate brain mapping data, we have conducted feeding behavioral studies with flavored liquid and solid food. Although PYY 3 36 i.p. injected mice indee d developed aversive reaction to an associated flavor, no such response was documented in mice treated with OS PYY 3 36, even at the highest dose of 18 g /100 g BW. This fact confirms my previous observation showing that 1) orally applied PYY 3 36 does not l eak into the bloodstream; and that 2) there apparently exists a metabolic circuit associated with YR2 positive cells in the oral cavity and extending through brainstem nuclei into hypothalamic satiety centers (Fig. 5 6). This putative alternative pathway o riginates in sensory nerves of the tongue epithelium and/or taste buds and projects, via the facial and glossopharyngeal nerves, into the brainstem. From the brainstem, specifically in the NST, the signal could be relayed into the forebrain
115 activating the arcuate and paraventricular nuclei. The precise phenotype/s of the neurons and connections involved remain to be identified at this time. However, due to the activation patterns of the NST, we can infer that PYY 3 36 could be inducing an anorectic effect th portion of the NTS). To the best of our knowledge, this is the first report demonstrating that PYY 3 36 administered into the oral cavity does not induce the adverse effect that is obser ved when PYY 3 36 is administered systemically. Degen et al., (2005) demonstrated in their clinical trial that exogenous administered PYY 3 36 can suppress FI in humans only when used at the supraphysiological doses. Importantly, inhibition of feeding induce d with such doses was accompanied by subjective dose dependent side effects associated with gastrointestinal malaise (apparently related to the CTA reported in animal models). As a result, the potential of PYY to emerge as a powerful drug to treat obesity was challenged by its narrow therapeutic index. The discovery of an alternative pathway mediated by salivary PYY 3 36 and its receptors in the oral cavity that regulates ingestive behavior without inducing CTA reveals the existence of a novel, albeit yet to be fully characterized domain for the NPY system and reinstates the potential of PYY 3 36 for the treatment of obesity.
1 16 Table 5 1. Schematic timeline of the CTA trials with liquid ( bottle content ) or solid food ( rack content ). Habituation Conditioning Trials Days 1 2 3 4 5 6, 10, 14 7, 11, 15 8, 12, 16 9, 13, 17 18 bottle content water flavor 1 in both bottles water flavor 2 in both bottles water flavors 1 or 2 in separate bottles regimen water OS + ss i.p. 1 of 4 treatment regimens water OS + ss i. p. water OS + ss i.p. water OS + ss i.p. none rack content regular chow flavor 1 in both trays regular chow flavor 2 in both trays Regular chow flavors 1 or 2 in each tray regimen water OS + ss i.p. 1 of 4 treatment regimens water OS + ss i.p. water OS + ss i.p. water OS + ss i.p. none
117 A 125 I PYY OS B 125 I PYY OS + BIIE0246 C 125 I PYY i.p. D E F Fig ure 5 1 Salivary PYY binds to Y2 recept ors in the tongue epithelia. A) Represen tative image of a sagittal section of the murine tongue subjected to 125 I PYY bin ding applied orally in vivo; B) image of the tongue from the animal where radio labeled PYY was co administered with YR2 specific antagonist BIIE0246 (please note a shade out l ine of the tongue); C) image of the tongue from the animal where 125 I PYY was injected i.p. Images D, E and F are from the same tissues, but after H /E staining and visualized in the bright field Silver grains associated with the cells in the lingual epith elia could be distinctively identified.
118 Figure 5 2 Effect of PYY 3 36 OS on c fos expression in the arcuate nuclei (Arc, top row), paraventricular nuclei (PVN, middle row), and the lateral hypothalamic area (LHA, bottom row). Shown are repr esentative photomicrographs of the c fos activity in mice fasted for 24 hrs and either not treated (Column Fast ), fed for 1 hr (Column Fed ); injected with PYY 3 36 i.p., 6 g /100 g BW (Column PYY i.p. ), or treated with PYY 3 36 using oral spray, 6 g /1 00 g BW (Column PYY OS ). Panels in the rightmost column show tabulated values expressed as average number of c Fos positive cells per section (n=4 mice per group). Data are expressed as mean SEM. Statistics calculated by one st post hoc (overall p=0.01), pairwise treatment comparisons
119 Figure 5 3 Effect of PYY 3 36 OS on c fos expression i n the rostral area of the nucl eus of solitary tract (NST). A) Diagram for clarity, only one side is shown. The course of the solitary tract i s also shown unilaterally. Filled irregular shaped ovals indicate the overlapping termination patterns of the facial nerve (VII), the linguotonsilar branch of the glossopharyngeal nerve (IX), and the superior laryngeal branch of the vagus nerve (X). Shaded areas on the right aspect indicate the sectioned areas in the rostral NST and the AP ; sections were collected bilaterally; B) Diagram of the coronal representation of the medial rostral area of the solitary tract: sol solitary tract, Rm rostral medial ; Ri rostral intermedial; Rvl rostral ventrolateral, area postrema AP ; 4V fourth ventricle. Filled ova l indicates tabulated areas; C) tabulated values expressed as average number of c Fos positive cells per s ection (n=4 mice per group). D) Shown ar e representative photomicrographs of the c fos activity in the ovals indicate areas included in the tabulations (see panel B, filled oval). Data are expressed as mean SEM. Statistics was calculated by one hoc (overall p=0.000), pairwise treatment 0.001. The numerical p value above the bar graph indicates the significance calculated by less stri ngent LSD test.
120 Figure 5 4. Effect of PYY 3 36 OS on c fos expression in the caudal area of the nucleus of solitary tract (NST) and the area postrema ( A P ). A) Diagram of the coronal representation of the intermediate area of the solitary tract. area postrema AP ; mNTS medial nucleus of the solitary tract; C central canal; X dorsal motor nucleus of the vagus; XII hypoglossal nucleus; dashed rectan gle designates the areas shown as photomicrographs; dashed ovals designate areas included in the c Fos stai Tabulated values expressed as average number of c Fos positive cells per section in the mNST (n=4). The treatment gro ups are as follows: Fast animals fasted for 24 hrs; Fed after 24 hrs fast, animals fed for 1 hr; LiCl after 24 hrs fast animals injected with LiCl i.p.; PYY 3 36 i.p. after 24 hrs fast, the hormone was injected i.p.; PYY 3 36 OS after 24 hrs fast, the hormone was administered by the oral spray. All animals were sac rificed 1 hr post treatment. C) Tabulated values expressed as average number of c Fos positive cells per section in the AP Statistics was calculated by one way ost hoc (overall p=0.000), pairwise treatment the bar graph indicates the significance calculated by less stringent LSD test.
121 Figure 5 5. Effect of PYY 3 36 treatment on aversiv e response. Liquid paradigm. A) Individual flavor consumption: saline paired flavor (black bar) vs treatment paired flavor (grey bar). The treatment groups are as follows: Saline sal ine injected i.p. + water OS; PYY OS PYY 3 36 administered orally (6 g/100g BW) + saline injected i.p.; PYY i.p. PYY 3 36 injected i.p. (6 g/100g BW) + Water OS; LiCl Li Cl injected i.p. + Water OS; B) Ratios of volume of treatment paired flavor consu med vs total volume consumed across treatment groups. Treatment gr oups are same as in Panel A; C) Individual flavor consumption: saline paired flavor (black bar) vs treatment paired flavor (grey bar). The treatment groups are as follows: Saline saline in jected i.p. + water OS; 6 g, 12 g, 18 g PYY 3 36 administered orally at 6, 12, or 18 g/100g BW respectively + saline injected i.p.; LiCl Li Cl injected i.p. + Water OS; D) Ratios of volume of treatment paired flavor consumed vs total volume consumed across treatment groups. Treatment groups are same as in Panel tailed) t test for A and C; or one hoc for 0.001. Solid food paradigm. E) Individual flavor consumption: saline paired flavor (black bar) vs treatment paired flavor (grey bar). The treatment groups are as follows: Saline saline injected i.p. + water OS; PYY OS 6 g, and 18 g PYY 3 36 administer ed orally (6, or 18 g/100g BW, respectively) + saline injected i.p.; PYY i.p. 6 g PYY 3 36 injected i.p. (6 g/100g BW) + Water OS; LiCl Li Cl injected i.p. + Water OS; F) Ratios of grams of treatment paired flavor consumed vs total grams consumed acro ss treatment groups. Treatment groups are same as in (two tailed) t test for A, or one hoc for B,
123 Figure 5 6 Dia gram displaying main putative anorexigenic pathways originating in the tongue epithelia and/or TRCs innervated with afferent projections of neurons from cranial nerve VII (chorda tympani branch), glossopharyngeal nerve IX, or superior laryngeal branch of t he cranial nerve X. For clarity, only ascending projection are shown, although the majority of these pathways include reciprocal descending fibers. The rostral (gustatory) and caudal (visceral) subdivisions of the NTS are shown by white and shaded areas, r espectively. The distinctive shading of PBN is used to show the existence of functionally segregated nuclei. Anatomically and functionally related nuclei of the forebrain areas are designated by similar shaped and shaded ovals, their functional roles are d isplayed in italics. Abbreviations are as following: rNST rostral nucleus of the solitary tract; cNST caudal nucleus of the solitary tract; area postrema Area postrema ; PBN parabrachial nucleus; VPMpc parvicellular part of the posteromedial ventr al thalamic nucleus; IC insular cortex; PFC prefrontal complex; Amy amygdala; VTA ventral tegmental area; NAac nucleus accumbens; VP ventral pallidum; LHA lateral hypothalamic area; PVN paraventricular nucleus.
125 CHAPTER 6 CONCLUSIONS As shown in Chapters 3, 4 and 5 of this dissertation, w e have extensively characterized the expression of the neuropeptide Y (NPY) system family members in the oral cavity and described several novel function s of the previously well characterized family membe r satiation gut peptide PYY The NPY System in the Oral Cavity Members of NPY family genes are represented by well characterized hormones NPY PYY Pancreatic Polypeptide (PP) ; and their cognate Y receptors (YR) YR 1, YR 2, YR 4, and YR 5. These genes are w idely expressed in the brain as well as on the periphery mediating multiple and diverse metabolic functions. Recently, we have shown the presence of PYY in the saliva, and the expression of its preferred receptor, YR2 in the lingual epithelia. In the curre nt report, we extended our finding to all main NPY family members and characterized their expression in the lingual basal cell epithelia and in the taste receptor cells (TRC) in mice. Using immuno staining and RT PCR protocols, we showed the expression of t he genes coding for all three hormones, NPY PYY and PP in the tongue epithelia and TRCs In the stratified keratinized lingual epithelial cells in the dorsum of the tongue, YR s are expressed in the cascade fashion following (and, possibly, mediating) ep ithelial cells differentiation. The cascade manifested in switching from YR1 / YR2 (+) progenitor cells in the basal layer, to Y1Y (+) cells in the prickle cell layer, to YR1 / YR5 (+) cells in the granular layer, to YR5 (+) in the keratinocytes. In addition, YR4 was shown to be expressed in somatosensory neurons innervating basal layer.
126 In the taste buds of the circumvallate (CV) papillae, YR4 was shown to be expressed in nerve fibers innervating TCRs. Moreover, significant population of TCRs was positive for YR1 YR2 YR4 or YR5 showing preferential accumulation of YR s within the microvilli of the apical part of the cells. TCRs expressing YR s also expressed Neural Cell Adhesion Molecule NCAM suggesting their possible role in the gustatory signal transduction Due to the characteristic pattern expression of YR1 and YR2 in the basal layer cells of the tongue epithelium, we established the lineage identity and showed that these cells are dividing progenitor cells. The dorsal stratified epithelium of the tongue i s characterized by a high turnover rate of cells in response to mechanical and chemical insults Because of the known functions of YR s in cell proliferation we speculate that the NPY system in the oral cavity plays a role i n cell s turnover. The role of the NPY system in taste tissue is currently under investigation in our laboratory. For the moment, by assessing all taste qualities in mice, it has been found that PYY apparently, mediates lipid sensing and, perhaps, bitter taste perception. Role of Salivar y PYY To investigate the possible role of salivary YR signaling in energy metabolism, we focused our research on PYY PYY a hormone that induces satiety, is synthesized in L endocrine cells of the gut. It is secreted into circulation in response to food i ntake (FI) and induces satiation upon interaction with its cognate YR2 Herein, along with Dr. PYY enters the oral cavity at least in part from the bloodstream. In addition, because PYY is also synthesized in the TRC s of the CV it is conceivable that PYY is secreted from these cells into saliva. Two PYY moieties could play separate functions: for example, PYY in
127 TRC s modulating taste perception by interacting with YR1 and YR2 expressed in some TRCs while PYY in s aliva modulating, in part, feeding behavior by interacting with YR2 in the tongue epithelial cells. With respect to the latter, we provided evidence that the acute augmentation of salivary PYY induces stronger satiation as demonstrated in feeding behaviora l studies. The effect is mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial cells. In a long term study involving PYY deficient mice, a sustained increase in PYY was achieved using viral vector mediated gene del ivery targeting salivary glands (SG) The chronic increase in salivary PYY resulted in a significant long term reduction in body weight (BW) gain. Collectively, the data point to oral mucosal epithelial YR2 positive cells as potential targets for anorexige nic actions of the salivary PYY and suggests the existence of a putative neuronal circuit initiated in the oral cavity. Salivary PYY : A Putative Circuit that Regulates Ingestive Behavior Circulating PYY freely crosses the blood brain barrier gaining acce ss to brain posteriorly and activating arcuate nucleus neurons, and/or through the intermediate nucleus of the solitary tract and area postrema (AP) in the caudal brainstem. The data presented in this report have to be interpreted with the following notion in mind: PYY applied in the oral cavity, does not leak retrogradely into the bloodstream. As a result, it positive cells and putative afferent pathways. In this manus cript we showed that salivary PYY rapidly binds to YR2 receptors in the tongue epithelia to initiate a metabolic response which is inhibition of ingestive behavior. Brain activation studies suggest that the signal from the oral cavity is relayed to the cen tral nervous system where it extends through brainstem nuclei into
128 hypothalamic satiety centers. The precise phenotype/s of the neurons and connections involved remain to be identified at this time. Whether such a putative pathway plays a meaningful regula tory role under physiological salivary PYY concentrations remains to be determined as well. However, in favor of such a possibility, is the fact that a significant postprandial increase of salivary PYY mirrors the similar postprandial increase in plasma PY Y concentration. The neural connection s between the oral cavity and the brainstem which are responsible for the afferent signaling remain to be fully characterized. However we speculate that this putative alternative pathway originates in sensory nerves of the tongue epithelium and projects, via the facial and glossopharyngeal nerves, into the brainstem. Salivary PYY and Taste Perception Because systemic PYY had been implicated in mounting CTA by activating area postrema neurons, it was of interest to test whether orally administered PYY induced aversive responses as well. From our results, we can infer that PYY does not induce an anorectic effect through CTA adverse effect that is observed when PYY is administered systemically. The potential of PYY to eme rge as a powerful antiobesity drug was challenged by its narrow therapeutic index. The discovery of an alternative pathway mediated by salivary PYY and its receptors in the oral cavity that regulates ingestive behavior without inducing CTA reveals the exis tence of a novel, albeit yet to be fully characterized domain for the NPY system and reinstates the potential of PYY for the treatment of obesity.
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148 BIOGRAPHICAL SKETCH Maria Daniela Hurtado Andrade was born and raised in Quito, Ecuador. Since high school, she had great interest for science and medicine. Thus, she pursued her medical education at the Pontificia Universidad Catlica Del Ecuador, from where she graduated with honors and salutat orian in November 2008. laboratory at the University of Florida as a research scholar and months later she was accepted into the Interdisciplinary Program of Biomedical Scien ces of the College of Medicine at the same institution to start her doctoral training While working on her Doctoral project, Daniela validated her medical diploma from Ecuador. After taking the United States M edical Licensing Boards, she obtained the Educ ational Commission for Foreign Medical Graduates Certificate. During her doctoral training, she published a co authored article and presented her research at several national and international meetings. Due to academic excellence, Daniela has received sev eral awards and certificates of outstanding achievements. In the future, she wants to pursue a physician scientist career. Therefore in 2013, she will start her Internal Medicine Residency training in the United States.