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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-08-31.

Permanent Link: http://ufdc.ufl.edu/UFE0024937/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-08-31.
Physical Description: Book
Language: english
Creator: Acosta, Andres
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Andres Acosta.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Zolotukhin, Sergei.
Electronic Access: INACCESSIBLE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024937:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024937/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-08-31.
Physical Description: Book
Language: english
Creator: Acosta, Andres
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Andres Acosta.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Zolotukhin, Sergei.
Electronic Access: INACCESSIBLE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024937:00001


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1 THE ROLE OF SALIVARY PEPTIDE YY3 36 IN FEEDING AND AGGRESSIVE BEHAVIOR By ANDRES ACOSTA CARDENAS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T HE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Andres Acosta Cardenas

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3 To my parents for all their love and support

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4 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my adviser, Dr. Sergei Zolotukhin, who gave me the opportunity to join his laboratory and work under his guidance. He was always available to discuss new ideas and gave me all the support to pursue new project s Secondly, I would like to thank the memb ers of my supervisory committee: Dr. Jeffrey K. Harrison, Dr. Philip Scarpace and Dr. N icholas Muzy c z ka. They have provided a continuous support with always great scientific advice and guidance during my P h.D studies I am grateful to Dr. George Aslanid i for teaching me many scientific techniques and helping me to do the experimental designs in this thesis; to Dr. Steve Polyak for his constant scientific and clinical support during these three years; and to Dr. Bruce Baum for collaborating with us in the project that shaped my thesis and our discoveries. I would like to thank the past and present members of the Zolotukhib lab: George Aslanidi, Ken Lamb, Vadim Kroutov, Ramaz Geguchadze, Andrea Doty, Damien Marsic, David Duncan, and Daniela Hurtado. Also all the members of the Division of Cellular and Molecular Therapy of the Dept. of Pediatrics, but specially to Arun Srivastava, our chair; Irene Zolotukhin, and Krista Berquist. I would also like to thank all the people that make my research possible: L isa R. Stow, Amy Wright, Martha Campbell Thomson and the Pathology Core ; Darragh Devine Armon Peck Oleg Gorbatyuk, Craig Meyers, Stacey Porvasnik. Annette Mach Cathryn Mah, Mark Atkinson, Clive Wasserfall, P roteomics Core and all the Animals Care Service staff at Cancer and Genetics Research Complex. In addition, I would like to thank all my friends for their support. I greatly value the enjoyable time I shared with them throughout my graduate career. Finally, I would like to thank my Mother, Father and Brothers for their constant love and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 7 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT ........................................................................................................................................ 10 CHAPTERS 1 INTRODUCTION ....................................................................................................................... 12 Obesity: 21st Century Epidemic ................................................................................................. 12 Brain Gut Axis ......................................................................................................................... 14 NPY Family / Pathway ........................................................................................................ 16 Peptide YY ........................................................................................................................... 19 Saliva ............................................................................................................................................ 21 2 THE ROLE OF SALIVARY PYY336 IN FEEDING BEHAVIOR. AN ALTERNATIVE PATHWAY FOR SAT IETY ........................................................................ 26 Saliva and Satiation Gut Hormones ........................................................................................... 26 Materials and Methods ................................................................................................................ 27 The Role of Salivary PYY336 in Feeding Behavior .................................................................. 33 Summary and Partial Conclusions ............................................................................................. 43 3 LONG TERM SALIVARY PYY3 36 TREATM ENT MODULATES AGGRESSIVE BEHAVIOR. EAT LESS, FEEL HAPPIER ............................................................................ 63 The Neuropeptide Y Pathway and Aggression Modulation ..................................................... 63 Materials and Methods ................................................................................................................ 63 The Role of Salivary PYY336 in Aggression Modulation ........................................................ 65 Summary and Partial Conclusions ............................................................................................. 66 4 LONG TERM PEPTIDE YY GENE THERAPY: ADDRESSING EXISTING CONTROVERSY. ...................................................................................................................... 74 Peptide YY Controversy: Does PYY336 Inhibits Food Intake? ............................................... 74 Materials and Methods ................................................................................................................ 75 The Effect of Long Term PYY336 Over Expression in Feeding Behavior of Diet Induced Obese Mice ................................................................................................................ 79 Summary and Partial Conclusions ............................................................................................. 84 5 CONCLUSIONS ....................................................................................................................... 107

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6 Salivary PYY3 36: Characteriza tion and Role in Food Intake ................................................. 107 Salivary PYY3 36 and Aggressive Behavior ............................................................................. 111 Long -term Over -Expression of Site -Specific PYY3 36 in a Diet Induce Obese Mice Model ..................................................................................................................................... 111 Future Directions: ...................................................................................................................... 113 New Satiation Theory: .............................................................................................................. 118 LIST OF REFERENCES ................................................................................................................. 122 BIOGRAPHICAL SKETCH ........................................................................................................... 131

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7 LIST OF TABLES Table page 2 1. Characterization of gut hormones in murine saliva. (mean SE) .......................... 48 2 2. Metabolic hormones panel in obese mice treated rAAV -PYY compared to pairfed rAAVGFP controls (means SE) ............................................................. 49

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8 LIST OF FIGURES Figure page 1 1. A schematic representation of the chief brain pathways involved in the regulation of eating beh avior. ................................................................................... 23 1 2. NPY signaling in hypothalamic feeding circuitry.. ................................................. 24 1 3. Peptide processing that occurs to produce the endog enous forms of PYY from rat preproPYY. .................................................................................................. 25 2 1. Characterization of Peptide YY in saliva. ................................................................ 50 2 2. Characterization of Peptide YY in tongue epithelium. ......................................... 51 2 3. Origin and role of Peptide YY in saliva. .................................................................. 52 2 4. Short Term increase of PYY3 36 in murin e saliva delivered by oral spray. ............ 53 2 5. Validation of recombinant adeno associated virus encoding for pre -pro PYY and regulatory secretion. ........................................................................................... 57 2 6. Effect of rAAV -PYY vs. rAAVGFP injected into submandibular salivary glands of 45 days old lean Balb/c males fed with normo -caloric chow. ................ 58 2 7. Effect of rAAV -PYY v s. rAAVGFP injected into submandibular salivary glands of 150 days old obese C57BL/6 males mice fed with high caloric high fat (60%) diet.. ........................................................................................................... 60 3 1. Diagram of rAAV-PYY and rAAVGFP vectors plasmids. ................................... 67 3 2. Weekly Food Intake for 22 weeks Food intake in lean mice treated with rAAV-PYY vs. rAAVGFP controls.. ...................................................................... 68 3 3. Body Weight Accumulation for 22 weeks. ........................................................... 69 3 4. Territorial Resident Intruder test (attack). ................................................................ 70 3 5. Territorial Re sident Intruder test (Threat). ............................................................... 71 3 6. Territorial Resident Intruder test (Chase). ............................................................... 72 3 7. Normal non aggressive behavioral analysis ............................................................ 73 4 1. Validation of recombinant adeno associated virus encoding for pre -pro PYY and regulatory secretion.. .......................................................................................... 87

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9 4 2. rAAV-PYY delivered into superior mesenteric artery (SMA) in diet-induced obese mice. ................................................................................................................. 88 4 3. rAAV-PYY delivered into submandibulary salivary glands (SG) in diet induced obese mice. ................................................................................................... 96 4 4. rAAV-PYY delivered into 3rd intra verebral ventricle (ICV) in diet -induced obese mice. ............................................................................................................... 104 5 1. Diagram for alternative pa thway of satiety induced by salivary PYY336 ............ 120 5 2. Future directions for salivary PYY336.................................................................... 121

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF SALIVARY PEPTIDE YY 3 36 IN FEEDING AND AGGRESSIVE BEHAVIOR Andres Acosta Cardenas A ugust 2009 Chair: Sergei Zol otukhin Major: Medical Sciences -Physiology and Pharmacology Peptide YY 336 is a satiation gut hormone released postprandially by the gastrointestinal neuroendocrine L cells. PYY336 induces satiation by acting on Y2 receptors in the hypothalamus and br ainstem. Here we provide evidence for the existence of a novel alternative satiation pathway mediated by PYY3 36. We found that PYY3 36 is present in saliva. In addition, we showed that both PYY336 and its respective NPY Y2 receptors are expressed in the taste cells in the circumvallate papilla of the tongue. The physiological role of salivary PYY336 was investigated in feeding behavioral studies upon acute augmentation of the peptide applied by oral spray. This short term increase of salivary PYY336 re sulted in a decrease in one hour food intake in a dose -dependent manner. c -Fos activation in the neurons in the hypothalamic arcuate nuclei suggested that PYY336 activated the neural downstream satiation pathways. To elucidate the therapeutic anti -obesity potentials of salivary PYY336, a long -term gene therapy experiment was conducted in obese mice fed high fat diet for 4 months. The chronic elevation of salivary PYY336 was achieved using a recombinant viral vector (rAAV -PYY) expressing PYY in the saliva ry glands. Eight weeks after treatment with rAAV -PYY, mice reduced their weekly food intake and displayed a 23% body weight loss compared to control. Interestingly, the chronic over expression of salivary PYY336 also reduced aggressive behavior, measured by a behavioral

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11 resident intruder test conducted in a blind manner. Furthermore, our rAAV PYY treatment delivered to salivary glands produced a decrease in body weight in diet induced obese mice compared to rAAV GFP treated mice. This diet induced obese r esistance was not observed after rAAV-PYY injected systemically by superior mesenteric artery, and weight gain was observed after intraventricular injection of rAAV -PYY in the brain. In conclusion, we have characterized a novel pathway for satiation by pep tide PYY336 in saliva acting through the respective NPY Y2 receptors and activating brain satiety centers. The possible interconnection between feeding and aggressive behavior was also established in mice with the chronic over -expression of salivary PYY336.

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12 CHAPTER 1 INTRODUCTION Obesity: 21st Century Epidemic Obesity has reached epidemic proportions in developed countries and its prevalence is increasing in developing countries (1 ). In the United States of America, 64% of adults are overweight and 30.5% are obese (2 ) In children and adolescents, the obesity prevalence increased to 17.1 % in 2004 (3 -5 ). The Worl d Health Organization indicated that globally in 2005 there were approximately 1.6 billion overweight adults and 400 million of these were obese (6 ). Obesity is defined as the amount of excess adipose tissue at which your health risks increase (7 ). Body weight can be measured and classified into normal weight, overweight and obese by the Body Mass Index (BMI). BMI is calculated by weight (kg) divided by the square of the height (m2). The BMI for a healthy weight is from 18.5 to 24.9 kg/m2, overweight is from 25 to 29.9 kg/m2 and obese is 30 kg/m2 o r above. When a persons BMI is higher than 40 kg/m2, they are considered morbid or severely obese. (8 ). In children, obesity is measured as a BMI higher than the 95th percentile related to their age and sex (4, 5). Obesity can also be measured by waist circumference (8 ). The larger waist circumference is associated with higher health risks. Understanding the etiology of overweight and obesity is multifactorial and complex because of energy balance regulation (9 ). The simplified cause for obesity is the imbalance between food intake and energy expenditure. The energy imbalance can be related to biological (10), behavioral (11) and/or environmental factors (12, 13). Obesity etiology can also be classified as neuroendocrine obesity, drug induced weight gain, smoking cessation related obesity, sedentary lifestyle, diet, psychological and social factors, socioeconomic and ethnic

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13 fact ors and congenital and genetic disorders (14). The complexity of etiology predicts the complexity of the treatment for obesity. When the energy balance is lost, the excess calories are stored in the adipose tissues. Adipose cells will become hypertrophic and hyperplasic, increasing the mass of fat in the body. The in crease in body fat will produce an excess of adipo -citokynes, inflammatory markers, vascular factors and leptin. Eventually, the excess of body fat will result in leptin resistance, chronic inflammatory state, uncontrolled fat angiogenesis, insulin resista nce, dyslipidemia, hypertension, and coronary artery disease. Leptin and insulin resistance will produce an increase in food intake by down regulating the food intake and satiety pathways, which will further contribute to the energy imbalance. In the long term, the increase of fat mass will produce musculo -skeletal injuries due to increased weight (14). Obesity is related to the top ten mortality and morbidity causes of death in the USA (2 ). Obesity is a major health problem and the mortality risk increase with an increasing BMI (15). Obesity is related to several pathologies including cardiovascular d isease (16 ), diabetes mellitus (17), sleep apnea (18-21), cancer (22), reproductive disorders (23), endocrine disorders (24), psychological disorders (25-28), bone, joint and connective tissue disorders (29, 30) and gastrointestinal disorders (31, 32). Important advance s in understanding the physiopathology of obesity have been made, but there still is no effective long term treatment. Current treatments can be divided into pharmacological and surgical (33, 34). The pharmacologi cal treatments approaches are 1) decrease food intake; 2) increase metabolism; and 3) increase energy expenditure. The surgical treatments are 1) mal absorptive (bypass Jejunoileal or Roux Y: Biliopancreatic d iversion and duodenal switch); 2) restrictive (Gastroplasty, Gastric banding or Gastric bypass). The

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14 pharmacological treatments are associated with low adherence and significant side effects. The surgical procedures decrease body weight significantly in a short time period but are associated with mala bsorptive disorders, surgery complications and mortality, and severe post -surgery side effects (35, 36). Due to the lack of a current long term effective treatment with minimal side effects, novel therapies are req uired to prevent and treat the obesity epidemic. Gene therapy can become one of these novel approaches (37). As of October 2005, 408 loci have been identified that may relate to body weight (BW) and obesity (10). Many of these genes are critical in energy intake and expenditure and several can be considered as potential targets for genetic therapy. For example, in our previous work focusing on energy expenditure, we have achieved long term beneficial weight reducing effects of transgene adiponectin expression (38), transgene Wnt10b expression (39) and inhibition of stearoyl CoA desaturase on diet induced obesity in rats (unpublished data). Our laboratory continues to search for other genes and pathways in the metabolism of energy balance to apply genetic therapy for obesity. Even though our main focus has been on energy expenditure, food intake pathways related to satiation also have a significant role in the control of energy balance. Brain Gut Axis Food intake is mainly regulated by the brain gut axis (40, 41). The brain gut axis consists of gut hormones, the vagal complex, the brainstem, the hypothalamus and higher brain centers in the cortex rela ted to appetite and satiation (40). Appetite induces secretion of ghrelin from the stomach during the fasting period. Ghrel in acts on specialized neurons in the arcuate nucleus of the hypothalamus to activate the agouti related peptide/ NPY (AgRP/NPY) pathway (42-45). The AgRP/NPY pathway is responsible for stimulating appetite centers in the cerebral cortex that prepare the gastrointestinal tract for food intake by a vagal response and stimulate food

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15 seeking behavior from brain cortical centers (46). Satiation is induced by several gu t hormones including PYY, oxyntomodulin (OXM), and glucagonlike petide 1, which are secreted after food intake. These gut hormones inhibit the agouti related peptide/ NPY (AgRP/NPY) pathway arcuate nucleus of the hypothalamus and stimulate the proopiomel -melanocyte (47, 48). The neurons in the arcuate nucleus are called first order neurons because of their direct contact with the peripheral satiety hormones. Second -order neurons can be found in the paraventricular nucleus, lateral hypothalamic area and ventromedial nucleus of the hypothalamus (49); they inhibit the vagal response to produce the sensation of gut fullness, increase hyperthermia and produce food reward (Figure 1 1) (50). The brain stem, through the nucleus of the tractus solitarium (NTS) and the vagus nerve, controls the enteric ner vous system by afferent and efferent signals from energy levels in the periphery (46, 51). The energy levels also influence nerve terminals in the circumventricular organs in the central nervous system, which have a thin blood brain barrier ( 52). In the central nervous sy stem, the limbic system plays an essential role in feeding behavior, feedinginfluence motivation and reward -mediated feeding behavior as well as motivation and reward experiences related to food consumption (53, 54). During eating and eating seeking behaviors, the cortex regulates food intake by the direct influence of smell, taste and visual factors (55-59). Gut hormones have a critical role in energy homeostasis, especia lly related to glucose metabolism and modulation of food intake by inducing satiation. Satiation gut hormones are cholecystokinin (CCK), glucagonlike peptide 1 (GLP 1), o xyntomodulin (OXM) and peptide YY (PYY). The secretion of these hormones is relat ed to food intake and caloric consumption and their effects are peripheral over the gastrointestinal system and central over the hypothalamus and the brainstem (for review see Vincent, RP. et. al, 2008) ( (60).

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16 The satiation gut hormone peptide YY is synthesized and secreted mainly from enteroendocrine L cells in the distal gastrointestinal tract. PYY active form is PYY336 and its main effect is to induce satiation by acting in the arcuate nucleus of the hypothalamus and in the brain stem. PYY336 has also been related to an increase in energy expendit ure. These effects in energy homeostasis make PYY336 a potential target for an obesity treatment (for review see Karra, E. et al. 2009) (61). The gut hormone glucagon like peptide 1 (GLP 1) comes from the pro-glucagon gene Pro -glucagon is cleaved into oxyntomodulin, GLP 1 and GLP 1 in the presence of p rohormone convertases 1/3. GLP 1 is secreted from entero endocrine L cells from the distal small intestine and ascending colon. GLP 1 is rapidly inactivated by D ipeptidyl peptidase IV (DPP IV). The main effect of GLP 1 is to stimulate insulin secretion, but it also regulates food intake by acting in the hypothalamus and in the brainstem. Several GLP 1 analogs and DPP -IV inhibitors are currently available for the t reatment of diabetes mellitus. (for review see Kim, W. et al, 2008) (62). NPY Family / Pathway The neuropeptide family consists of neuropeptide Y (NPY), polypancreatic peptide (PP) and peptide YY (PYY) (63). These neuropeptides are all 36 amino acids long when secre ted and have 70 80% sequences similarity (64). The NPY pathway modulates food intake, body weight, energy expenditure, blood pressure, cortical excitabilit y, circadian rhythms, stress response, emotions, memory, attention, learning, aggression, ethanol susceptibility and pain processing. The NPY pathway has also been related to the mechanism of epilepsy, neurogenesis, neuroprotection, analgesia, anxiety and depression (65, 66). The widespread effects of NPY are mediated by the G -protein coupled receptors Y1, Y2, Y4, Y5 and Y6 (67).

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17 NPY receptors have variety of functions and are expressed in different locations around the brain and the body. Keire et al. describe each NPY receptor su btype, the location and function (68). The subtype Y1 receptor is found in the cerebral cortex, dentate gyrus, thalamic and hypothalamic nucleus, peripheral veins and arteries. The main effects of the subtype Y1 receptor are vasoconstriction, anxi olysis / sedation, regulation of growth in human colonic epithelium, and induction of feeding (together with Y5 receptor). The subtype Y2 receptor is found in the hypothalamus and brain stem, sympathetic and parasympathetic nerve fibers, intestine, and cer tain blood vessels. The main effects of the subtype Y2 receptor are the induced suppression of transmitter release, enhancement of satiety, antisecretory effects, enhanced memory retention, suppression of carbohydrates intake, inhibition of mucocilliary ac tivity, inhibition of vasoconstriction, regulation of interdigestive motility of the small intestine, suppression of glutamate and noradrenalin release. The subtype Y4 receptor, described as Pancreatic Polypeptide receptor Y4 is found in: the gastrointesti nal tract, pancreas, heart, arteries, hypothalamus, nucleus of the solitary tract, area postrema and paraventricular nucleus and hippocampus. The effects of the subtype Y4 receptor are inhibition of pancreatic secretion, inhibition of the gall bladder cont raction, stimulation of leutinizing hormone and follicle stimulating hormone release. The subtype Y5 receptor is found in: the hippocampus and dentate gyrus with limited presence in peripheral tissue. The effects of the subtype Y5 receptor are feeding regu lation, feeding modulation after the initial response created by the NPY Y1 receptor, anti epileptic activity, attenuation of morphine withdrawal symptoms and enhancement of diuresis and natriuresis. The subtype Y6 receptor has been cloned in rabbits, rats and humans and has properties similar to NPY Y1 receptor (68).

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18 NPY is the most potent orexigenic peptide in the CNS, acting mainly in the hypothalamic feeding pathways (Figure 1 2) (69). Dur ing fasting periods, there is a higher concentration of NPY in the hypothalamus compared to the feeding period (70-72). The injection of NPY intra cerebro -ventricular or in specific hypothalamic areas (PFA PVN and VMN) produces a significant dose response increase in feeding and hyperphagic state (73-77). In the hypothalamus, the higher concentration of NPY and NPY Y2 receptors is found in the arcuate nucleus (AR C). In the ARC, there are two types of neurons: 1) NPY / AgRP (Agouti related peptide) which has an orexigenic effect; and 2) POMC /MSH (pro -opiomelanocortin / melano stimulating hormone) which has an anorexigenic effect. These two pathways inhibit each ot her and are constantly influenced by the peripheral signals of glucose homeostasis (insulin), adiposity (leptin) and the enteric nervous system (NTS -Vagus) (69). When stimulated, the NPY neuro ns release NPY, GABA and AgRP in the ARC and send projections to the VMN, DMN, PVN, PFA and LHA in the hypothalamus (78, 79). Also, the NPY release inhibits POMC neurons in the ARC which have NPY Y1 and Y2 recepto rs in their membrane (80-82). These robust feeding effects induced by NPY are not evidenced when NPY receptors are deleted in mice models (83-88). The lack of a phenotype suggests that there is probably a compensatory mechanism that normalizes food intake in these mice. (89) These phenotypic effects are more prominent in other behavioral components related to NPY. The NPY pathway is linked to aggression, anxiety and depression. For example, NPY Y1 and Y4 receptor knockout mice exhibit abnormally aggressive behavior (65). Furthermore, both pharmacological inhibition of NPY Y2 receptor and NPY Y2 receptor knockout shows an anxiolytic, antidepressant phenotype with re duced attention and increased impulsivity (90, 91), (92).

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19 Peptide YY Peptide YY (PYY) is a gastrointestinal peptide or gut hormone, that belongs to the neuropeptide Y syst em (PP family) together with Neuropeptide Y (NPY), pancreatic polypeptide (PP), and peptide YY (63). The human Peptide YY gene is located in the Chromosome 17q21.1 (93). Peptide YY2, a homolog PYY peptide, is located in chromosom e 17q11 (94). The peptide YY gene encodes for a pre -pro -Peptide YY, like most hormones. PYY pre pro -hormone undergo is posttranslational modification (68). PYY mRNA encodes a pre pro PYY sequence of 98 a mino acids (95). After translation, it is cleaved by a signal peptidase to a pro -PYY of 70 amino acids. Then, the pro PYY C terminus is cleaved by a prohormone d ibasic convertase ; this process adds Gly-Lys -Arg to the new C terminus. T he post -translation modification results in is PYY136, a biologi cally active 36 amino acid poly peptide. After secretion, dipeptidyl peptidase IV (DPP -IV) cleaves the N terminus tyros ine -proline residues forming PYY3 36. PYY136Gly can also be cleaved into PYY336Gly (Figure 1 3) (68, 96). PYY is secreted from the entero -endocrine (L) cells of the distal small intestine and colon (5)(97) and also from the pancreas and brain stem (98, 99). PYY is secreted 15 minutes after food ingestion, with a peak at 60 minutes and remains elevated up to 6 hours. The secretion is stimulated indirectly by the proximal gut through the neural and humoral pathways and directly by the luminal contents of the distal digestive tract (97). PYY secretion is related to the amount of calories ingested; fat ingestion produces a decrease in Ghrelin concentration and stimulates PYY secretion. A high pro tein diet is related to an elevated expression of PYY and PYY plasma levels and a decrease in weight gain (100).

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20 PYY136 and PYY336 bind and activate neuropeptide Y receptors. Y receptors are membrane G protein r eceptors and work by activation of cAMP and by increasing intracellular calcium (Figure 4). PYY136 binds to all NPY Y receptors but PYY3 36 binds with higher affinity to NPY Y2 receptors (101, 102). PYY336 ha s an effect in the gastrointestinal system and in the hypothalamus. In the gastrointestinal system, PYY regulates several physiological mechanisms including the inhibition of gastric pancreatic and intestinal secretion and the inhibition of gastrointesti nal motility (103). In the hypothalamus, PYY3 36 regulates food intake by stimulating the NPY Y2 receptor that stimulates the POMC/aMSH pathway and inhibits the Agrp/NPY pathway, which induces satiation (104-108). Moreover, PYY3 36 also has been related to an increase in postprandial energy expenditure (109-113). PYY has a direct link to obesity. Ma et al. reported that morbid obese Pima Indians had mutations in PYY and Y2 receptor genes. They d escribed three single nucleotide polymorphisms (SNPs) in PYY: one miss-sense substitution, two silent substitutions and eight SNPs in the PYY Y2R (114). Torekov at el. described the polymorphism Arg72 allele of PYY associated with diabetes m ellitus type 2 and o besity in the general population (115). These findings were supported by mouse models. The role of PYY in energy homeostasis was evidenced by the PYY knockout mouse that became obese, while PYY transgenic mouse is resistant to diet induced obesity (100, 116). Several studies have shown that obese subjects have a lower concentration of PYY in plasma compared to controls (117) The caloric load required to produce a PYY -satiety response in obese subjects was more than doubled the load of lean subje cts (118) and mutations on PYY and the PYY Y2R receptor were associated with severe obesity in men (114, 115). However,

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21 obese subjects do not develope resistance to PYY. On the contrary, obese subjects develope d resistance to other metabolic hormones like leptin, insulin, or adiponectin (119). The effects of acute and chronic systemic administration of PYY3 36 are controversial. Batterham et al. reported that intraperitoneal injection of PYY3 36 reduced food intake in rodents and that peripheral infusion of PYY336 had similar effects in lean and obese humans (104, 120). These results were not replicated by several groups (121) where they were replicated several times in rodents, nonhuman primates and humans (105, 106, 111, 117, 122131). On the contrary, when PYY336 is injected centrally i.e third, lateral or fourth cerebral ventricle, there is an increase in food intake (73, 132). An opposite effect is achieved when PYY336 is injected directly into the arcuate nucleus of the hypothalamus, where Y2 receptors are abundant (104). In order to understand the controversy related to PYY3 36, seve ral behavioral factors including acclimatization, stress, and administration conditions (site, frequency, dose, and time) must be considered (121). Saliva Saliva has an essential function in humans oral and genera l health. In oral health, the saliva mixture contributes to lubricate, protect, and heal the oral mucosa as well as to moisturize the mouth. Saliva also has a key function in lubrication for phonation and in the immune response containing IgA and antibacte rial peptides. Saliva is important in the initiation of the digestion of carbohydrates by amylase, the formation of the bolus and in taste by creating a hypotonic solution that allows taste buds to recognize different flavors (133). The importance of saliva in general health and disease continues to rise with the discovery of new hormones, cytokines, and peptides present in saliva. In the last decade, several peptides, proteins, and steroidal hormones have been identified in saliva (134, 135). Many of these proteins and hormones leak into saliva by passive diffusion

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22 from capillaries (136, 137); while other pro teins, like insulin are actively transported from the capillaries to the saliva, so their concentration in saliva can be similar to their concentration in plasma (135). The presence of hormones in saliva has potential clinical applications to use saliva as diagnostic tools and as biomarkers for diseases. Last year, a proteomics consortium published the identification of over a thousand proteins present in human saliva and showed that a high proportion of these proteins are present in both plasma and saliva (138). Recently, the human salivary proteome in type 2 diabetes has identified 487 unique proteins compared to normal or pre -diabetic controls. Interestingly, 42 % of these proteins are related to metabolism (139). The recent proteomics data and the previous characterization of metabolic hormones in saliva suggests that metabolic hormones must have an important role in sa liva. Metabolic hormones such as leptin and adiponectin had been characterized in saliva (140, 141). Also ghrelin, a small gut peptide which is produced in salivary glands and can be measured in saliva has been cha racterized (142) Metabolic hormones already characterized in saliva are leptin (140), insulin (135), g hrelin (142) and GIP (143). However, it is unknown if satiation gut hormones are present in saliva. Probably, these gut peptides have not been found in saliva because of the high concentration of peptidases in saliva (144) and with the appropriate peptidases inhibitors, smaller peptides could be measured in saliva.

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23 Figure 1 1 A schematic representation of the chief brain pathways involved in t he regulation of eating behavior. ARC, arcuate nucleus; NTS, nucleus of the solitary tract; CCK, cholecystokinin; GLP 1, glucagon-like peptide 1; PYY, peptide YY. PVN, paraventricular nucleus; LHA, lateral hypothalamic area; PFA, perifornical area; NPY, ne uropeptide Y; AGRP, Agouti -related peptide; POMC, pro -opiomelanocortin; CART, cocaine and amphetamine regulated transcript; CRH, corticotropin -releasing hormone; TRH, thyrotropin -releasing hormone; OX, oxytocin; MCH, melanin concentrating hormone. Published with authors authorization (50)

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24 Figure 1 2 NPY signaling in hypothalamic feeding circuitry. Schematic of hypothalamic feeding circuits illustrate the location of NPY -expressing neurons, NPY receptors, and inhibitory or excitatory synapses. Different cell types within the hypothalamus are i nnervated by inhibitory ( ) synapses. Activation of receptor systems can stimulate (+) or inhibit ( Central and circulating signals are integrated at the first -order neurons of the ARC that expre ss POMC and CART or NPY, AgRP, and GABA. The ARC NPY neurons are inhibited by activation of the MC3R, Y1R, Y2R, Y5R, ObRb, and InsR; they are stimulated by activation of the ghrelin (or GHSR) and orexin receptors. (Bottom) The secondorder neurons include those in the DMN, VMN, LHA/PFA, and PVN. The adult NS and PA cells in the PVN are modulated by the presynaptic Y1R, Y2R, Y5R, MC4R, and ghrelin receptor. NPY -expressing neurons are found in the ARC and DMN, which are negatively modulated by MC4R, CCK1R, an d CCK2R. Though other intra and extrahypothalamic connections are known, only the connections discussed in this review are included in the figure. AgRP, agouti gene related transcript; ARC, arcuate nucleus; CART, cocaine and amphetamine regulated transcr ipt; CCK1R, cholecystokinin 1 receptor; CCK2R, cholecystokinin 2 receptor; aminobutyric acid; GHSR, growth hormone secretagogue receptor; InsR, insulin receptor; LHA, lateral hypothalamic area; MC3R, melanocortin 3 recepto r; MC4R, melanocortin 4 receptor; NPY, neuropeptide Y; NS, neurosecretory; ObRb, leptin receptor; OxAR, orexin A receptor; OxBR, orexin B receptor; PA, pre autonomic; PFA, perifornical area; POMC, pro opiomelanocortin; PVN, paraventricular nucleus; VMN, ve ntromedial nucleus; Y1R, Y2R, Y5R, neuropeptide Y receptors. Published with authors authorization ( 69).

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25 Figure 1 3 Peptide processing that occurs to produce the endogenous forms of PYY fr om rat preproPYY. Rat preproPYY is transcribed as a 98 amino acid precursor that is cleaved as it passes through the endoplasmic reticulum to the 70 amino acid proPYY. ProPYY is then cleaved by a prohormone dibasic convertase (candidates include PC1, PC2 o r furin). A carboxypeptidase B like enzyme then removes the two C -terminal basic residues resulting in a carboxyl -terminal glycine -extended form. amidating monooxygenase (PAM) then transforms the glycine into a C -terminal amide. A subsequ ent step in the secretory granules or after release by DDP IV converts PYY1 36 into PYY3 36. In addition, the Glyextended peptide can be cleaved by DPP IV to form PYY336Gly. Published with authors authorization (96)

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26 CHAPTER 2 THE ROLE OF SALIVARY PYY336 IN FEEDING BEHAVIOR. AN ALTERNATIVE PATHWAY FOR SATIETY Saliva and Satiation Gut Hormones Saliva has an essential function in humans oral and general health. In ora l health, the saliva mixture contributes to lubricate, protect, and heal the oral mucosa as well as to moisturize the mouth. Saliva also has a key function in lubrication for phonation, in the immune response containing IgA and antibacterial peptides; in t he initiation of the digestion of carbohydrates by amylase and the formation of the bolus; and in taste by creating a hypotonic solution that allows taste buds to recognize different flavors (1 33). The importance of saliva in general health and disease continues to rise with the discovery of new hormones, cytokines, and peptides present in saliva. In the last decade, several peptides, proteins, and steroidal hormones have been identified in s aliva (134, 135). Many of these proteins and hormones leak into saliva by passive diffusion from capillaries (136, 137); while other proteins, like insulin are actively tr ansported from the capillaries to the saliva, so their concentration in saliva can be similar to their concentration in plasma (135). The presence of hormones in saliva has huge potential clinical applications to use saliva as diagnostic tools and as biomarkers for diseases. Last year, a proteomics consortium published the identification of o ver a thousand proteins present in human saliva and showed that a high proportion of these proteins are present in both plasma and saliva (138). Recently, the human salivary proteome in type 2 diabetes has identi fied 487 unique proteins compared to normal or pre -diabetic controls. Interestingly, 42 % of these proteins are related to metabolism (139). The recent proteomics data and the previous characterizations of metabolic hormones in saliva suggest that metabolic hormones must have an important role in saliva. Metabolic hormones such as leptin and adiponectin had been characterized in saliva (140,

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27 141). Also ghrelin, a small gut peptide which is produces in salivary glands and can be measured in saliva has been characterized (142) However, it is unknown if satiation gut hormones are present in saliva. Probably, these gut peptides have not been found in saliva due to the high concentration of peptidases in saliva (144). This suggests that with the appropriate peptidases inhibitors, smaller peptides could be mea sured in saliva. Therefore, we hypothesized that satiation gut peptides could be found in saliva that was collected under appropriate conditions. Gut hormones have a critical role in energy homeostasis, especially related to glucose metabolism and modula tion of food intake by inducing satiation. Satiation gut hormones are cholecystokinin (CCK), g lucagonlike peptide 1 (GLP 1), o xyntomodulin (OXM) and peptide YY (PYY). The secretion of these hormones is related to food intake and caloric consumption an d their effect is peripheral over the gastrointestinal system and centrally over the hypothalamus and the brainstem (for review see Vincent, RP. et. al, 2008) (60). Materials and Methods In this hypothesis based study, the answer of each hypothesis led us to formulating a new hypothesis. We started by me asuring satiation gut hormones in human and murine saliva and their origin. Then, we searched for the expression of PYY and NPY Y receptors in the tongue epithelia of mice. The presence of these hormones in saliva leads us to study their physiological/phar macological role in food intake. The effect was studied in the short term by using an oral spray and in the long term by using rAAV gene therapy approach in lean and obese mice. We also studied the mechanism by which these hormones induced satiation by mea suring c -fos activity in the hypothalamus. Mouse studies : This study was approved by the institutional Animal Care and Use committee at the University of Florida; and by the Animals Care and Use Committee of The National Institute of Dental and Craniofacial research and by the Biosafety Committee of the

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28 National Institue of Health (Bethesda, MD). All mice procedures were done in accordance with the principles of the National Research Councils guide for the Care and Use of Laboratory Animals. Studies were done in male C57Bl/6 (Charles River Laboratories) or Balb/c (Harlan Sprague Dawley) mice housed at 22 24 C in a 12 hours light/dark cycle (lights off at 1800). Mice had free access to water and food unless otherwise stated. Saliva collection : Salivation was stimulated by an i.p. injection of 100 ul of a cocktail containing isoproterenol/pilocarpine (1mg/2mg in 1ml of PBS). (145) Saliva was collected for 5 minutes from the oral cavity using a micropipette into 1.5 ml eppendorf containing 5000 U of Kalikrein inhibitor (Biomedicals) and 50 mM of 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 5000 U of Kalikrein inhibitor (Biomedicals). Tubes were incubated 30 minutes at RT for clotting, and then spined for 10 minutes at 1200 G at 4 C. Plasma was transferred into new 1.5 ml eppendorfs containing 50 mM of DPP -IV inhibitor (Linco Research). Plasma samples were frozen at 80 C until analyzed. Plasma and saliva hormone levels : The Mouse Gut Hormone Panel from plasma and saliva were measured by Lincoplex kit (Linco Research). PYY336 from saliva, plasma or cell culture supern atant were measured by PYY336 EIA kit (Phoenix Pharmaceuticals, Inc). Chromatographic characterization of saliva was performed by reverse -phase high performance liquid chromatography (RP HPLC) and the results verified by matrixassisted laser desorption i onization Time of flight (MALDI TOF) mass spectrometry. PYY3 36 synthetic peptide from Bachem was used as a positive control. Briefly, saliva was purified using a Sep -Pak C18 cartridge (Waters) and then loaded to the RP HPLC uBondapak C18 column (3.9 x 300 mm;

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29 Waters) as described (146). The extract was air dried and reconstituted in 0,1% f ormic acid in 50% acetonitrile/water prior to analysis with MALDI TOF as describe (96, 147). Tissues collection: Mice were sacrificed by CO2 and tissues were harvest for DNA, RNA and IHC studies. Tongue epithelium was collected by an injection below the epithelium of 2 mg/ml elastase (MP Biomedicals) and 2 mg/ml dispase (CellnTec) diluted in saline solution 0.9%. 5 minutes after the injection, the epithelium is removed and placed in fresh RNA later. Circumvallated p apillae were extracted from whole tongue without separating the epithelium and placed into 4% PFA for fixing and future IHC studies. Relative quantitative RT PCR analysis : RNA extraction, purification, cDNA synthesis and RT PCR amplification was done as described in Aslanadi et al (39). Briefly, tissues were isolated using Trizol reagent (invitrogen) and homogenized using Matrix A in a FP120 Homogenizer (Qbiogene). RNA integrity was verified by agarose gel (1%) ele ctrophoresis with ethidium bromide. RNA was DNAse treated by Turbo DNA -freeTM kit (Ambion). Total RNA in equals amount for each sample (6 or more tissue/group) were converted to cDNA by a SuperScriptTM III First -Strand Synthesis supermix (Invitrogen). Prim ers were designed by Primer3 algorithm available at the Whitehead Institute for Biomedical Research website (Suplemental Table S1). cDNA was amplified by PCR using SYBR GreenERTM qPCR SuperMix for iCycler. (Invitrogen) Relative expression values were det T values of the gene of interest and the housekeeping gene (bactin or S18). For each pair wise set of samples, T was calculated. The relative expression or fold change of the gene of interest was calculated as fold chan ge = 2. Immunohistochemistry : Frozen tissue sections were brought to room temperature before melting the OCT medium in 1X TBS (wash buffer). To detect peptide YY (PYY) expression,

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30 antigen retrieval was performed in Antigen Retrieval Citra Solution (Biogenex) for 30 minutes in a steamer then rinsed in wash buffer two times for five minutes each. Tissues were blocked for non -specific binding with 10% normal goat serum diluted in Antibody Diluent (Zymed) for 20 minutes. The slides were not rinsed, b ut rather had the excess diluent dabbed off the slide. Guinea pig anti -PYY (1:500; Pierce/Thermo Fisher Scientific) was diluted in Antibody Diluent and incubated on tissues overnight at 4 C, with subsequent rinses in wash buffer after equilibrating tissue s to room temperature. Incubation of control tissues in Antibody Diluent served as the negative control. Secondary antibody, Goat anti -guinea pig conjugated to Alexa Fluor 555 (1:1000, Molecular Probes/Invitrogen), was incubated on tissues at room tempe rature for one hour followed by rinses in wash buffer. Tissues were coverslipped using VectaShield HardSet Mounting Medium with DAPI (Vector Laboratories, Inc). PYY signal was viewed by Zeiss Fluorescent Axioskop microscope, Model 9850, using AxioSkop im aging software (Carl Zeiss MicroImaging, Inc). PYY3 36 replacement studies : Acute PYY336 was delivered by an oral spray. Sterile vials for the oral spray were obtained from Sephora. Mouse PYY336 was purchased from Bachem and diluted in vehicle solutio n. All mice were individually housed and fed with normal chow. Mice 8 10 weeks old were conditioned in three occasions after 24 hours fasting to the oral spray with vehicle. Groups were randomized selected by food intake and body weight. Prior to the st udy day mice were fasted for 24 hours, and then they were sprayed either 5ug / 100 g of Body weight of PYY336 or control (vehicle). Food was provided 10 minutes after the spray and measured at 1, 2, 6, and 24 hours. Each experiment was done at least 3 t imes in a cross over manner with 13 mice per group. (Figure 2 2A, 2B, 2D, 2E) The dose response study was done with 0.3, 3, 10 ug / 100 g of BW of PYY336 or control in 8 mice per group. To measure the effect on body

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31 weight, mice were conditioned for v ehicle oral spray every 6 hours (6 am, 12 pm, 6 pm, 12 am) for 4 consecutive days and food intake and body weight was measured daily. Then, mice were randomly selected by food intake and body weight into two groups (13 mice per group). Each group received 5 ug / 100 g BW of PYY336 or control every 6 hours for the following two days. Then, every three hours (6 am, 9 am, 12 pm, 3 pm, 6 pm, 9 pm, 12 am, and 3 am) for the last day. Food intake and body weight was measured daily for the following week. Chronic PYY3 36 was delivered by a vector -mediated PYY gene delivered targeted to submandibular salivary glands. We constructed a recombinant adeno associated virus (rAAV) cassette, encoding for murine pre -pro PYY (total) cDNA (ATCC) under the control of a strong constitutive CMV/B actin promoter (Figure 2 3A). rAAV-enconding pre -pro -PYY cDNA (rAAV -PYY) and the control rAAVencoding for Green Flourescence protein (rAAV -GFP) were package into rAAV serotype 5, which have higher transduction efficiency to murine submandibular salivary glands. (148) The viral production, purification and titration was done as described by Zolotukhin et al (149). In vitro studies : to test our rAAV -PYY transgene secretion we decided to use genetically engineered human intestinal NCI -H716 cells as described by Tang et al (150). Briefly, after cell differentiation, we transfected the NCI -H716 cells with rAAV-PY Y or rAAVGFP. 48 hours later, we fast the cells overnight on basal medium [DMEM (GIBCO) with 5 mM glucose and 1% fetal bovine serum]. On the study day, parallel cultures were washed with basal medium every hour two times to stabilize their basal secretion Then the cell culture was stimulated with 2% meat hydrolysate (Sigma) in basal medium for 1 hour. For the following 4 hours, the cell culture was incubated in basal medium, with consecutive replacements of the basal medium every hour. Cell culture medium was collected every hour for PYY EIA assay.

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32 In vivo studies : A single dose of 100 ul containing 1x1010 DNAI resistant particles was administered into each salivary ducts of submandibular glands. Injections were made as described in katano et al (148). For the lean mice model, 45 days old male Balb/C mice individually housed were administered rAAV PYY, rAAV-GFP or saline (controls) into each submandibular salivary gland. (5 mice per group) Mice were fed with nor mal calories diet. Food intake and body weight were measured weekly for 24 weeks with a balance. For the obese mice model, 45 days old male C57Bl/6 mice were fed with high fat (60%) high caloric diet for 16 weeks. Diet induced Obese (DIO) C57Bl/6 mice were separated from diet resistant mice. 160 days old DIO C57Bl/6 mice individually housed were administered rAAV -PYY (n=10) or rAAVGFP (n=20) into each submandibular salivary gland. Mice were kept in high fat high caloric diet and food intake and body weight were measured in a weekly basis for 8 more weeks. To measure the effect of energy expenditure in rAAV -PYY treated mice, we decided to pair fed in a caloric restrictive model, half of the rAAV -GFP treated mice (10 mice) and kept the other 10 mice in high f at diet. During the 4 weeks of caloric restrictive model, mice treated with rAAV -PYY or rAAVGFP pair fed were provided their daily caloric intake. During these 4 weeks, mice were handled only once a week to measure their body weight. Oxygen consumption studies : Oxygen consumption was measured simultaneously in 6 mice, 3 per group by an Oxyscan OXS 4 analyzer (Omni tech Electronics). Flow rate was 1.5 ml/min with a 30 -s sampling time at 5 -min intervals. Mice were placed in the chamber for 90 minutes and t he last 30 minutes of oxygen consumption were used for calculations. Results are expressed as mass adjusted consumption (ml/min/kg0.67).

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33 Human studies : Institutional review board approved informed consents and assents were obtained from 10 lean (BMI 19 2 5) males aged 18 30 with no known diseases. Participants fasted for 12 hours overnight. The next morning, participants saliva samples were collected during fasting and 30 minutes after eating 450 kcal meal. Saliva samples were collected in 50 ml conical tubes containing 5000 U of Kalikrein inhibitor (Biomedicals) and 50 mM of DPP -IV inhibitor (Linco Research). Saliva samples were frozen at 80 C until analyzed. Statistical analysis : Statistical analysis was conducted using un -paired students t test with significance at P < 0.05. The Role of Salivary PYY336 in Feeding Behavior To determine if satiation gut hormones are present in saliva, we collected saliva stimulated from 10 C57Bl/6 male mice. Samples were collected with proteinase and DPP 4 inhibitors and stored at 80 C. To measure satiation gut hormones in a standard assay, a multiplex murine ELISA assay was used. A multiplex assay allowed us to measure hormones already characterized in saliva as well as satiation gut hormones such us PYY and GLP 1 in saliva. Metabolic hormones already characterized in saliva are leptin (140), insulin (135 ), Ghrelin (142) and GIP (143). Our results suggest that satiation gut hormones can be measured in murine saliva (Table 1). However, the concentration of these hormones in saliva did not correlate with previous published data, or with the concentration of these hormones in plasma. Also, there was a high variability between the samples. This variability and lack of correlation may be attributed to the potent cholinergic/a drenergic cocktail that is required to induce satiation in mice. Even though, the saliva collection in mice causes a falsely high concentration of this hormone; we have shown that satiation gut hormones can be measured in murine saliva. Therefore, we deci ded to validate our findings by measuring satiation gut hormone PYY336 and GLP 1 in human saliva.

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34 The satiation gut hormone peptide YY is synthesized and secreted mainly from enteroendocrine L cells in the distal gastrointestinal tract. The main effect o f the PYY active form, PYY336, is to induce satiation by acting in the arcuate nucleus of the hypothalamus and in the brain stem. PYY336 also has been related to increase in energy expenditure. These effects in energy homeostasis makes PYY336 a potenti al target for the obesity treatment (for review see Karra, E. et al. 2009) (61). The gut hormones Glucagon like peptide 1 (GLP 1) comes from the pr o -glucagon gene. Pro -glucagon cleavage into OXM GLP 1 and GLP 1 in the presence of Prohormone convertases 1/3. GLP 1 is secreted from entero endocrine L cells from the distal small intestine and ascending colon. G LP 1 is rapidly inactivated by d ipeptidyl peptidase IV (DPP IV). The main effect of GLP 1 is to stimulate insulin secretion, but it also regulates food intake by acting in the hypothalamus and in the brainstem. Several GLP 1 analogs and DPP IV inhibitors are already available for diabetes treatment (for review see Kim, W. et al, 2008) (62). Saliva from 10 lean (BMI: 19 25) males ages from 18 to 30 years old was collected by dripping technique during fasting and 30 minutes after a 450 kcal meal. Saliva was handled and stored under the same conditions described previously and measured with human EIA. The concentration of PYY336 during fasting was 14.97 9.9 pg/ml (4.5 pM) and after feeding was 76 2.88 pg/ml (18.75 pM). (p=0.024) (Figure 2 1A) The concentration of PYY336 in saliva can be correlated with the concentration of PYY336 in plasma (151). To validate these results, we measured the human PYY336 in saliva by using reverse phase high performance liquid chromatography (RP HPLC) (146) and the results were verified by matrix assisted laser desorption ionization Time of flight (MALDI TOF) mass spectrometry (96, 147). PYY336 synthetic peptide from Bachem was used as a positive control. RP -HPLC

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35 showed a major peak in human saliva in the same position as human PYY336 synthetic Figure 2 1B). The extract from RP HPLC was analyzed by MOLDI TOF MS and the molecular weight for the peak extract was the same 4050 Da in human saliva an d in the positive control. These 4050 Da is the expected molecular weight for human PYY3 36 (Figure 2 1C). These results confirm that PYY336 is present in saliva and validate our previous results using EIA which characterized satiation gut hormones in s aliva. The origin of PYY336 in saliva was unclear. Salivary PYY336 could be produced endogenous in the tongue epithelium, from salivary glands, or systemically diffusing directly from plasma. Recently, Shin et al. characterized the presence of GLP 1 and GLP 1 receptors in taste buds of the circumvallate papillae of the tongue (152). Also, cholecystokinin and v asoactive intestinal peptide has also been characterized in taste cells (153, 154). To determine the origin of PYY336, mRNA and protein expression were measured to characterize PYY336. Immunohistochemistry for PYY was performed on the tongue epithelium, circumvallate papillae of the tongue, and salivary glands. W e found that PYY is expressed in the taste cells of the circumvallate papillae of the tongue (Figure 2 2A). This data was validated by RT -PCR for PYY mRNA expression levels with a 16 0.3 fold increase compared to muscle (p= 8.65853E 08), but tongue ep ithelium has a lower expression of PYY than the colon [colon/muscle = 88 1.5 fold increase (p= 1.38168E 07)] (Figure 2 2B). These findings suggest that PYY is expressed in some taste buds of the CVP. There was no expression of PYY in submandibular sal ivary glands (data not shown). Even though we identified PYY expression in taste cells, it was not clear if salivary PYY336 was produced only by taste cells or if it diffuses through the capillaries from systemic PYY336. To find the major source of PY Y336 we did a dose response study, several doses of PYY336 were

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36 injected intraperitoneally and then 10 minutes later murine stimulated saliva was collected to measure salivary PYY336. We injected saline solution 0.9%, 3 ug or 10 ug /100 g murine PYY336 diluted in 100 ul saline solution 0.9% (control). We found a concentration of 0.74 0.12 ng/ml, 3.64 2.0 ng/ml, and 39.77 14.64 ng/ml of PYY336 after i.p. injection, respectively (Figure 2 3A). There was a significant difference between saline s olution injection and the higher dose of i.p. PYY336 (p=0.04). Clearly, there is a correlation between plasmatic PYY336 concentration and salivary PYY336 concentration, showed by a dose response effect after systemic increase of PYY3 36. To corroborat e the findings and to understand the role of PYY3 36 in saliva, we decided to search for NPY Y receptors. NPY Y receptors are G -protein coupled receptors, which interact with Neuropeptide Y, Poly-pancreatic Peptide and Peptide YY. There are four types, Y1, Y2, Y4 and Y5. PYY336 has higher affinity to Y2 receptor and its effect is related to food intake modulation (61). Previously, NPY and Y1 receptor has been characterized in taste cells (155). Here we characterized t he presence of Y2 receptor in tongue epithelium by RT PCR. Y2 receptor mRNA expression was 150 fold higher than in muscle (p=0.009), but the expression was lower than in the brain [brain/muscle = 1400 1.85 fold increase (p= 0.01)] (Figure 2 3B). The d irect connection between taste cells in the circumvallated papillae with the central nervous system suggested that salivary PYY336 and its receptors expressed in the tongue could have a physiological central effect. Then, we wanted to determine if the i ncrease of these hormones would have an effect in food intake. We used an oral spray to increase salivary PYY3 36. Prior to the study day, we conditioned the mice on several occasions to receive an oral spray after a 24 hour fasting. The day of the study, mice were fast ed for 24 hours and before the dark cycle started, we sprayed 0.5

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37 ug/100g PYY3 36 vs. control (vehicle). After ten minutes, food was provided and one hour later food intake was measured. PYY336 treated group consumed 12.3% less calories t han the control group (Figure 2 4A). To avoid a selection bias, we did a cross over study by switching treatment and control groups. The PYY336 treated group consumed an average of 14.6% less calories than the control (Figure 2 4B). Further, pharmac odynamics studies showed that the increase of PYY336 had a dose response in one hour food intake. (Figure 2 4C) At a lower dose, 0.3 ug/100g PYY3 36 oral spray mice caloric intake was 16% lower than controls. At a medium dose, 3 ug/100g PYY336 oral spr ay mice caloric intake was 26% lower than controls. At a high dose, 10 ug/100g PYY3 36 oral spray mice caloric intake was 42% lower than controls. These results suggest that there is a dose response effect of PYY3 36 oral spray with Y receptors expressed in tongue epithelia, which produced a significant decrease in one hour food intake. In order to better understand the mechanism of salivary PYY336, we decided to measure satiation by performing classical satiation studies. After 1 dose of PYY336 oral spray we measured food intake at 1, 2, 6 and 24 hours after 24 hours fasting. The satiation studies were done at the beginning of the dark cycle where mice are more active and consume more calories. Prior to the study day, we conditioned the mice on seve ral occasions to receive an oral spray after a 24 hour fasting and then measure d food intake at 1, 2, 6 and 24 hours. During the study day, we sprayed 5ug/100g PYY336 10 minutes before the dark cycle began. Food was provided at the beginning of the dark cycle and food intake was measure d at 1, 2, 6 and 24 hours (Figure 2 4D). There was a significant decrease in food intake during the first hour of food intake. There was no difference in food intake during the first to second hour [PYY336 4.32 0.04 kcal vs. vehicle 4.30 0.05 kcal, p=0.47]. The difference from the first hour was maintained after two

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38 hours of food intake. There was no difference in food intake in the following 4 hours (2 to 6h). Although, from hour 6 to 24 PYY336 treated group compe nsated their food intake and consumed more calories than the control group. The food intake 24 hours after the spray was similar for both groups. Previously published studies showed similar results with systemically increase of PYY336 producing an anorexi genic effect follow by a delayed orexigenic effect (156). Interestingly, the acute administration of salivary PYY3 36 induced satiation and reduced one hour food intake with a delayed orexigenic effect similar to a cute systemic administration of PYY3 36. To verify whether this satiation effect on food intake involved satiation centers in the paraventricular nucleus of the hypothalamus, we examined c Fos expression in the paraventricular nucleus. The arcuate nucleu s and the paraventricular nucleus ha ve an important role in satiation related to PYY336(104, 105). Mice treated with PYY3 36 oral spray and mice fed for 30 minutes after fasting had a similar significant increase in c -Fos expression in the paraventricular nucleus compared to mice fasting for 24 hours and sprayed with vehicle (Figure 2 4E). These observations suggest that salivary PYY3 36 induced satiation by activation of the paraventricular nucleus of the hypoth alamus. The increase of salivary PYY3 36 had a similar physiological effect to the increase of PYY336 systemically. The similarities between salivary and plasmatic PYY3 36 made us verify that PYY336 oral spray did not change the concentration of PYY336 in plasma. We sprayed with vehicle, PYY336 or i.p. injection of PYY336 to male c57bl/6 mice. Twenty minutes later we collected blood from the facial vein. There was no significant difference between mice sprayed with PYY3 36 or vehicle. There was a sig nificant increase in mice injected with PYY336 i.p compared to both groups that received oral spray (Figure 2 4F). These data suggest that the

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39 increase of salivary PYY3 36 acted only in the tongue epithelium and had a similar effect to systemic PYY336 To determine if oral PYY336 spray could modify body weight, we decided to try several doses per day. We conditioned the mice to receive the oral spray for 4 days every 6 hours (6 am, 12 pm, 6 pm and 12 am). Then we sprayed either with PYY336 or control (vehicle) every 6 hours. We measured daily food intake and body weight. There was no difference in daily food intake or body weight after 2 days of every 6 hours PYY3 36 administration (Figure 2 4G). Due to the short half life of PYY336 and pos sibly no pharmacological steady -state, we decided to increase the amount of doses to every three hours (6 am, 9 am, 12 pm, 3 pm, 6 pm, 9 pm, 12 am and 3 am). After 24 hours of this regimen, PYY3 36 oral spray every three hours produced no difference in FI and BW c ompared to controls (Figure 2 4H). The lack of decrease in FI and BW in mice is probably due to the short -half life of PYY336 and the feeding behavior of mice. Mice eat small amounts of food constantly, with a major increase in food intake during the f irst half of the dark cycle. Therefore it is difficult to induce a long term meal related satiation in a mouse model and then translate the results for a potential clinical application. In order to induce long -term satiation in a mouse model, we genetically engineered recombinant Adeno associated virus encoding for pre -pro -peptide YY (rAAV-PYY) (Figure 2 5A). Recombinant Adeno associeted virus (rAAV) has a unique life cycle, infecting both dividing and non -diving cells and producing a long -term persist ent expression (For review see Daya, S. and Bern, KI 2008) (157 ). These characteristics make rAAV a great tool to over -express proteins in a specific organ or fluid. Also, with this tool, we can avoid several factors that have been related to different results after in jection of PYY336 like mice acclimatization, conditioning, or stress. Using rAAV, we can have a constant and longterm over -expression of PYY336 after a

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40 single administration of the viral vector into submandibulary salivary glands. We used the pre pro -ho rmone of PYY3 36 under a constitutive promoter to increase the PYY stored in granules after post translational modification. Then, when the stimulus comes, PYY is secreted into saliva in higher concentrations. To verify the regulatory granule secretion pr operties of our rAAV -PYY, we infected differentiated NCI H716 cells with rAAV -PYY or rAAV-Green Fluorescence protein (rAAV GFP) as a control (150). Forty eight hours after infection, we fasted the cells overnight and changed the media at time 0. Then we incubated the infected cells in basal medium for 1 hour, then we switched to medium with 2% meat Hydrolysate (MH) for one hour; and then we switched back to basal medium for 5 hours more, collecting the medium every hour (Figure 2 5B). During the basal state, cells treated with rAAV -PYY secreted a 2 fold increase of PYY compare to rAAV -GFP cells. During stimulation with 2% MH, rAAV-PYY treated cells secreted 100 fold increase of PYY compared to rAAV GFP cells. Af ter the stimulation, both cell lines returned to their basal state. The granule secretion stimulation showed that rAAV -PYY produced PYY in a regulatory granule secretion manner, with a minor constant secretion. Despite the regulatory granule secretion, sorting the exocrine or endocrine secretion in salivary glands after vector mediated gene deliver can be complicated and is difficult to predict (158, 159). To determine the sorting of rAAV -PYY, we delivered rAAV-PY Y or rAAVGFP by ductal canulation to submandibulary salivary glands of C57Bl/6 male mice ( 148). A month after vector delivery, we collected plasma during fasting and feeding as well as stimulated saliva. Plasma a nd saliva concentration were also measured a few weeks prior to sacrificing the mice to verify long -term expression. There were no difference between rAAV -PYY treated mice and

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41 rAAVGFP controls in plasmatic PYY336 concentration during fasting and after 1 hour feeding (Figure 2 6A). There was a two -fold increase in salivary PYY3 36 concentration in rAAV PYY treated mice compared to rAAV -GFP controls after salivation stimulation (Figure 2 6B). These data suggest that rAAV PYY delivered to salivary gland s produced an exogenous secretion of PYY336 with no endogenous PYY336 secretion; increasing only salivary PYY336. To study the effect of chronic over -expression of salivary PYY3 36, we performed a satiation study as described above. We found that ther e was a significant decrease in 1 hour food intake after 24 hours fasting, with the same caloric intake during the 1st and 2nd hour. Although, different from the oral spray with rAAV PYY there was a non -significant diffe rence 24 hours after food intake. P rior to the study day, we conditioned the mice on several occasions to fast for 24 hours and then measured food intake at 1, 2, 6 and 24 hours. During the study day we fasted the mice and food was provided at the beginning of the dark cycle and then measured at 1, 2, 6, and 24 hours. There was a significant decrease in food intake during the first hour of food intake (. There was no difference in food intake during the first to second hour. The difference from the first hour was maintained after two hours of food intake. There was a minor non-significant compensation of food intake in the following 4 hours (2 to 6h). Twenty four hours after fasting, rAAv -PYY treated group at e 5% less than the control group (Figure 2 6C). Interestingly, a two fold increa se in salivary PYY3 36 by gene delivery into salivary glands induced an early satiation during the first two hours after fasting. Additionally, using the gene delivery approach the delayed orexigenic effect of PYY336 was minor. After, we validated that our long term over -expression of salivary PYY3 36 was exclusively into saliva; it was expressed in a regulatory exogenous manner d uring salivation, and induce s satiation. We wanted to determine the long term effect of delivering rAAV -PYY into salivary

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42 glan ds. In order to test the long term effect of our model, we delivered the rAAV -PYY or rAAVGFP vector to lean and o bese male mice. In the lean mous e model, we used 45 days old balb/c males and delivered rAAV -PYY or rAAV-GFP into submandibular salivary gland s. Mice were fed with normal chow in a 12 hours light/dark cycle. Food intake and body weight were measured once a week for 22 weeks. Weekly caloric intake of rAAV-PYY treated mice was significantly lower than rAAV GFP treated control mice ( Figure 2 6D) Twenty two weeks after vector delivery, the rAAV -PYY treated mice gained less weight than the control mice (Figure 2 6E). These data suggest that long -term chronic over -expression of PYY336 in saliva of lean mice decreased food intake and body weigh t. In the obese mice mod el, we used 160 days old obese C 57bl/6 male mice fed for 4 months with high caloric high fat (60%) diet. Obese mice were selected and delivered rAAV -PYY or rAAVGFP into submandibular salivary glands. Mice were kept in the same hig h fat diet and food intake and body weight was measured weekly. rAAV -PYY treated mice weekly caloric intake was significantly lower than rAAV GFP control mice (Figure 2 7A). During the first month, rAAV-PYY treated mice had a significant decrease of 6 .64% body weight compared to controls fed with high fat diet (p < 0.016) (Figure 2 7B). rAAV-PYY treated mice lost 2.91% of body weight in one month after vector delivery. rAAV -GFP control mice gained 3.73% of body weight in one month after vector deli very. During the second month, we decided to pair fe e d half of the controls to the rAAV -PYY treated mice. This new control pairfed group was under a caloric restriction model where food was provided daily (Figure 2 7C and 7D). rAAVPYY treated mice had a significant decrease of 21.77% body weight compared to controls fed with high fat diet and a significant decrease of 3.43% compare d to pairfed controls. rAAV -PYY treated mice lost 20.80% of body weight in the second month after vector delivery. rAAV GFP

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43 control mice gained 4.74% of body weight in the second month after vector delivery. rAAV GFP pairfed control mice lost 17.37% of body weight in the seco nd month after vector delivery. Our data showed that rAAV -PYY treated mice reduced 23% their body weig ht in two months after vector delivery; this body weight reduction can be compared to the body weight reduction seen after bariatric surgery (160). Furthermore, there was also a significant reduction in white adipose tissue weight in rAAV PYY treated mice compared to rAAV GFP and rAAV GFP pairfed controls (Figure 2 7E). Due to the differenc e in reduction of BW between mice treated with rAAV-PYY compared to mice treated with rAAV -GFP that were paired feed we measured energy expenditure. rAAV -PYY treated mice consumed 7.4% more VO2 than rAAV GFP pairfed mice (Figure 2 7F). This finding su ggested that rAAV -PYY increased the concentration of PYY336 in saliva and that salivary PYY3 36 over -expression in obese mice produced a 23% significant reduction in body weight two months after injection. This effect is due to a decrease in caloric intak e as well as an increase in energy expenditure. Metabolic hormones were measured with no major differences between rAAV -PYY treated mice compared to pairfed rAAV GFP controls (Table 2 2). Summary and Partial Conclusions In this study, we provide evidenc e that satiation gut hormones such as PYY3 36 and GLP 1 can be measured in human and murine saliva, and the increase of these hormones in saliva produced a significant decrease in food intake. These findings add satiation gut hormones to the already long list of metabolic hormones present in saliva and suggest that saliva must be collected in the appropriate conditions to find small peptides. In order to detect these hormones in saliva, it is essential that the sample is collected with proteinase and DPP -IV inhibitors; these conditions are similar for the collection of blood samples. The concentration of PYY336 found in human saliva during fasting and after feeding was similar to the concentration of PYY336 in

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44 plasma (151). Unfortunately, t here is inconsistency in the literature about the concentration of PYY336 in plasma; therefore, it would be interesting to correlate the concentration of PYY336 in saliva and plasma. This correlation is not possible in a mouse model, because the saliva collection in the mouse has to be under a salivation stimulation which produces a massive granule secretion and salivation. The analysis of stimulated saliva can be inaccurate due to a large variability between samples related to the effect of the cholin ergic/adrenergic cocktail in each mouse. Interesting, PYY336 and GLP 1 concentration in murine stimulated saliva is similar to the plasmatic concentration after eating which can be related to a cholinergic vagal stimuli. The finding of satiation gut hormones in saliva can contribute to develop a non invasive method to measure these hormones in saliva. This potential clinical diagnostic tool can be useful in the diabetes and obesity fields where these hormones have been measured to monitor metabolic pr ofiles as well as treatment outcomes, especially since the incorporation of gliptins (DPP 4 inhibitors) and GLP 1 analogs for the treatment of diabetes (62). Moreover, there are several clinical trials for obesity using satiation gut hormones or their analogs (61),(62). Consequently, this potential diagnos tic tool must be accurate and with high sensitivity to measure these peptides. Even though, there has been a huge improvement in saliva collection devices and saliva processing further studies are needed to achieve the sensitivity and specificity to make saliva the gold -standard to measure hormones. Although, the role of satiation gut hormones in saliva goes beyond a diagnostic tool. Interestingly, we showed that PYY and Y2 receptors are expressed in taste cells of the circumvallated papilla (CVP) in the murine tongue. These findings correlate with previous findings that showed that NPY and Y1 receptor (155), GLP 1 and GLP 1 receptor (152), and

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45 CCK (154),(153, 161) are also expressed in taste cells of the CVP in the tongue. These findings suggested that satiation gut hormones are expressed in taste cells and correlates with previous data suggesting the entero-GU ST ,a protein related to taste, and express in taste cells (162, 163). The characterization of the satiation gut hormones specific receptor in the tongue epithelium suggests a possible new pathway related to the fa cial or glosopharyngeal nerve. However, more studies are needed to understand the similarities and differences between taste cells in the tongue and entero endocrine cells in the gut and their specific relation with food intake and taste perception. In order to understand the role of salivary PYY3 36, we wanted to determine if the increase in the concentration of salivary PYY336 without altering plasmatic concentration would induce satiation and reduce food intake. To achieve an increase in salivary PYY33 6 we developed a oral spray, which increased the concentration of salivary PYY3 36 without increasing the concentration of plasmatic PYY336. After one dose of 5ug/100 g PYY336 oral spray to the murine mouth there was significant decrease in food intake c ompared to previous results after peripheral administration, with the same delayed orexigenic effect (156). In a dose response study, we showed that even almost physiological doses of PYY3 36 (0.3ug/100g) delivered to the mouth produced a decrease in one hour food intake while other investigators have not achieved any effect after lower doses of systemic delivery of PYY336 (164). At higher doses PYY336 oral spray produced a significant decrease of food intake compared to systemic delivery of PYY336 (106, 123, 156, 164-167 ). The significant effect of increasing salivary PYY3 36 and its similarity to plasmatic increase of PYY336 suggests that the modulation of salivary PYY336 can induce satiation through an alternative pathway and have a potential clinical application for the treatment of obesity.

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46 The induction of satiation by salivary PYY336 was validated by showing an increase in c fos neuronal activity in the paraventricular nucleus. The activation of this hypothalamic satiation center by salivary PYY336 correlates to the mechanism of satiation produced by peripheral PYY3 36 described by Batterham et. al (104). Since, the satiation mechanism is similar between salivary PYY336 and plasmatic PYY3 36; and PYY336 oral spray did not increase plasmatic concentration, the pathway for satiation inducing might be different. Also, the enhanced effect of PYY336 when increased in saliva and its correlation with plasmatic concentration suggests that the increase of plasmatic PYY3 36 can also affect this alternative pathway to induce satiation. PYY336 induced satiation by activating NPY/ AGRP neurons in the arcuate nucleus ( 104), (168) and it is still controversial if the POMC pathway is involved in the arcuate nucleus ( 169, 170). The effect of PYY336 over the arcuate nucleus in the hypothalamus is probably mediated by the permeabilit y of the blood brain barrier to PYY3 36 (171). Alternatively, salivary PYY336 activates Y1 or Y2 receptors expressed in t he CVP. The CVP is innervated by sensory fibers of the lingual branch of the glossopharyngeal nerve (cN IX) (172). The cN -IX ganglia receive the sensory fibers and send a fferent fibers to the superior part Nucleus of the solitary tract (NTS). The NTS regulates satiation by direct or indirect stimulation of satiation gut hormones including systemic PYY336 (173, 174). This alternati ve satiation pathway mediated by salivary PYY336 can be related to sensory specific satiation (57-59) which can result in short term taste aversion and postprandial malaise (164, 175). In addition, PYY expressing neurons in the Gigantocellular reticular nucleus synapse in the NTS and with fibers from the hypothalamic Orexin and Melanin concentrating hormone systems that mediate satiation in the hypothalamus ( 99). Even though, the satiation induction by acute increase of salivary PYY336 is similar to an acute increase of plasmatic PYY336, we did not see an effect on body weight after 4 days of oral

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47 spray every six hours in mice. Thi s is probably because mice were eating constantly during the night in small quantities, but humans eat large quantities a few times per day. Therefore, we decided to increase salivary PYY3 36 in a regulatory manner related to salivation and in a non invas ive longterm over -expression. We achieved this by using a vector mediated gene delivery technique. This technique also allowed us to overcome several issues that can be related to the effect of acutely deliver PYY3 36 like acclimatization, stress, time an d site of injection, and conditioning (176). Consequently the vector mediated PYY delivery to submandibular glands offered several benefits: two fold increase in salivary PYY336 without a ltering plasma concentration, one injection per life time of the experiment (22 or 8 weeks), non invasive delivery method, no need for conditioning, and no stress produced. The body weight reduction produced by rAAV -PYY can be compared to bariatric surger y, the current gold standard for obesity (160). These data suggest that the increase of PY Y3 36 in saliva can become a long term treatment for obesity. In summary, (Figure 2 6) satiation gut peptides are present in saliva where they have a physiological role in food intake. Our working hypothesis is that this effect is mediated through the activation of specific receptors in the tongue epithelium innervated by the glossopharyngeal nerve, which synapses in the NTS. From the NTS, several fibers project to vagus nerve, the hypothalamus and other satiation centers. However, additional studies are needed to understand the mechanism by which these pathways interact.

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48 Table 2 1 Characterization of g ut h ormones in m urine s aliva. (mean SE) [saliva] (pg/ml) Amylin 533.5 99.09 Ghrelin 0.0525 0.017 GIP 150.06 30.65 GLP 1 60.55 8.12 I nsulin 583.95 65.98 Leptin 54.07 7.63 PYY 44.96 4.20

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49 Table 2 2 Metabolic hormones panel in obese mice treated rAAV -PYY compared to pairfed rAAVGFP controls. ( means SE ) rAAV PYY rAAV GFP p GIP Fasting 237.9834.75 168.9417.06 0.078 Feeding 2355.33425.74 1580.6323.46 0.002 Leptin Fasting 9010.704160.15 26685.067625.18 0.043 Feeding 20516.078072.28 32948.876011.46 0.083 Insulin Fasting 1399.56226.94 1747.06409.41 0.238 Feeding 4557.82958.84 4928.69351.10 0 .350 Amylin Fasting 251.4837.86 231.6437.62 0.298 Feeding 395.2824.94 343.1536.08 0.111 Ghrelin Fasting 9.251.09 10.780.63 0.239

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50 A B C Figure 2 1 Characterization of Peptide YY in saliva. A) Concentration of PYY336 in human s aliva during fasting and after 30 minutes of feeding (n = 5). B) Validation of PYY336 in murine and human saliva by RP HPLC and C) by MALDI TOF. Values reported as mean SE.

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51 A B Figure 2 2 Characterization of Peptide YY in tongue epithelium. A) Im muno histochemistry representative picture of PYY positive taste cells in the Circumvallated papillae of murine tongue for PYY3 36 of male C57Bl/6 mice. B) RT PCR assay measured relative mRNA PYY expression of whole tongue epithelium, muscle and colon of male C57Bl/6 mice (n = 10/group).

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52 A B Figure 2 3 Origin and role of Peptide YY in saliva. A) Dose -response experiment to measure PYY in murine saliva after 10 minutes of systemic (i.p) injection of PYY336 in male C57Bl/6 mice (n = 10/group). B) R T -PCR assay measured relative mRNA Y2 receptor expression of whole tongue epithelium, muscle and brain of male C57Bl/6 mice (n = 10/group). Values are reported as mean SE. *P <0.05 vs. control.

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53 A B Figure 2 4 Short Ter m increase of PYY3 36 in murine saliva delivered by oral spray. A) Effect of PYY336 oral spray in 1 hour food intake after 24 hours fasting compared to control oral spray (n = 26). B) Effect of PYY3 36 vs. control oral spray in 1 hour food intake after 24 hours fasting n = 26). C) Dose response effect of PYY3 36 0.3, 3 and 10 ug/100g of body weight vs. controls (n=10 per group). D) Effect of PYY336 vs. control oral spray in food intake measured 1, 2, 6 and 24 hours after fasting for 24 hours (n = 10/gr oup). E) Concentration of PYY336 in plasma 10 minutes after PYY336 and control oral spray vs. PYY3 36 i.p. (n=10/group). F) Representative example of c-fos expression in the Arcuate nucleus of the hypothalamus after 5g PYY3 36 oral spray / 100 g body w eight compared to fasting and 30 minutes feeding in male C57Bl/6 mice. G)Effect in Food intake after PYY3 36 oral spray every six hours for 3 days; and H) G)Effect in Body weight after PYY3 36 oral spray every six hours for 3 days Values are reported as me an SE. *P <0.05 vs. control.

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54 C D E Figure 2 4 Continue

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55 F G Figure 2 4 Continue

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56 H Figure 2 4 Continue

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57 A B Figure 2 5 Validation of recombinant adeno associated virus encodin g for pre -pro PYY and regulatory secretion. A) Diagram of rAAV -PYY and rAAVGFP vectors plasmids. ITR: rAAV serotype 5 inverted terminal repeats; enh: Cytomegalovirus intermediate early enhancer sequence; B -Act: chicken b acting promoter; Ex1: non coding s equence; murine Pre -pro Peptide YY gene or Green Fluorescence protein; PA: bovine growth hormone polyadenylation sequence. B) Secretion study in NCI H716 cells stimulated with Meat Hydroxylate (MH) 2% to measure granule secretion. After an overnight fast, cells were incubated in basal medium (BS) for 1 hour, MH 2% for 1 hour and then BS for 5 hours. The experiment was performed on 3 different occasions with 10 wells per group. Values are reported as mean SE.

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58 A B Figure 2 6 Effect of rAAV-PYY vs. rAAVGFP injected into submandibular salivary glands of 45 days old lean Balb/c males fed with normo -caloric chow. A) Concentration of PYY336 in plasma during fasting and after feeding. B) Concentration of PYY336 in saliva. C) Effect of PYY336 vs. control oral spray in food intake measured 1, 2, 6 and 24 hours after 24 hour fasting. D) Weekly food intake. E) Body weight gain. (n = 5/group) values are reported as means SE. *P <0.05 vs. control.

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59 C D E Fig ure 2 6 Continue

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60 A B Figure 2 7 Effect of rAAV -PYY vs. rAAVGFP injected into submandibular salivary glands of 150 days old obese C57BL/6 males mice fed with high caloric high fat (60%) diet. A) Weekly measured food intake during t he first 4 weeks. B) Body weight change in mice injected with rAAV PYY vs. rAAV-GFP. 4 weeks after injection a group of rAAV -GFP mice were pair fed with rAAV-PYY group. C) Body weight on injection day (initial), 4 weeks after injection and 8 weeks after i njection. D) Body weight 8 weeks after injection. E) White adipose tissue weight 8 weeks after injection. (n = 10/group) Values are reported as mean SE. *P <0.05 vs. control.

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61 C D E Figure 2 7 Continue

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62 F Figure 2 7 Continue

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6 3 CHAPTER 3 LONG TERM SALIVARY PYY3 36 TREATMENT MODULATES AGGRESSIVE BEHAVIOR. EAT LESS, FEEL HAPPI ER The Neuropeptide Y Pathway and Aggression Modulation The NPY pathway modulates food intake, body weight, energy e xpenditure, blood pressure, cortical excitability, circadian rhythms, stress response, emotions, memory, attention, learning, aggression, ethanol susceptibility and pain processing. The NPY pathway has also been related to the mechanism of epilepsy, neuro genesis, neuroprotection, analgesia, anxiety and depression (65, 66). The widespread effects of NPY are mediated by G -protein coupled receptors Y1, Y2, Y4, Y5 and Y6 (67). Neuropeptide Y (NPY) is expressed widely in the CNS and have been linked to aggression, anxiety and depression. For example, NPY Y1 and Y4 receptor knockout mice exhibit abnormally aggressive behavior (65). Furthermore, both pharmacological inhibition of NPY Y2 receptor and NPY Y2 receptor knockout shows an anxiolytic, antidepressant phenotype with reduced attention and increased impulsivity (90-92). However, so far little is known about the role of NPY Y2 receptors in aggressive behavior. NPY Y2 receptors endogenous agonist is PYY336 (101, 102). Recently, we repor ted that augmentation of salivary PYY3 36 modifies feeding behavior in mice. The long -term increase of salivary PYY336 by using a recombinant Adenoassociated virus (rAAV-PYY), produced a significant decrease in body weight and food intake compare to cont rols in obese mice. Interestingly, besides modulating the feeding behavior, the long term over expression of salivary PYY336 also appears to modulate aggressive behavior. Materials and Methods Vector design : A recombinant adeno associated virus encoding murine pre -pro -PYY (rAAV -PYY) under the control of a strong constitutive CMV/ actin promoter (Figure 3 1A)

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64 and the control rAAV GFP were pseudotyped into rAAV serotype 5 capsids as having higher transduction in salivary glands (SG) (148). The viral production, purification and titration were performed as described previously (149). Mouse studies : This study was approved by the Animals Care and Use Committee of The Nat ional Institute of Dental and Craniofacial research and by the Biosafety Committee of the National Institue of Health (Bethesda, MD). All mice procedures were done in accordance with the principles of the National Research Councils guide for the Care and Use of Laboratory Animals. Studies were done in male Balb/c (Harlan Sprague Dawley, Walkersville, MD) mice housed at 22 24 C in a 12 hours light/dark cycle (lights off at 1800). Forty five days old male Balb/C mice (n=5) were administered a single dose o f (100 u l, 1x 1010 vector genomes) rAAV PYY, rAAV-GFP or saline control bi laterally into the duct of submandibular salivary gland as described by Katano et al (148). Metabolic profile : Mice had free access to wat er and food (normal chow). Food intake and body weight were measured weekly for 24 weeks. Behavioral studies : Aggression territorial Intruder test were performed on week 24 after the treatment. (177) Briefly, PYY -, or GFP -treated resident mice were individually housed for at least two weeks prior to testing. Bedding from cages was not changed during the testing period to avoid unnecessary stress. On the day of the experiment, a smaller size intruder was placed into t he resident cage for 10 minutes and the residents behavior was recorded with a video camera. Each experiment was repeated 3 times on three different occasions and with different intruders. The videos from the experiments were analyzed for nonaggressive and aggressive behavior by an expert in a blind manner using The Observer v5.0 software (Noldus Information Technology) (178).

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65 Statistical analysis: Statistical analysis was conducted using un paired Students t test or by a MannWhitney test with significance at P < 0.05. Data was reported in mean SEM. The Role of Salivary PYY336 in Aggression Modulation Metabolic Profile: rAAVPYY treated mice weekly caloric intake was significantly lower than rAAVGFP contr ol mice. (rAAV -PYY 95.53 2.35 kcal vs. rAAV GFP 107.44 3.22 kcal, p < 0.002) (Figure 3 2). Twenty two weeks after vector delivery, the rAAV -PYY treated mice gained significantly less weight than the controls mice (rAAV -PYY 5.33 0.63 g vs. rAAVGFP 6.28 0.68 g, p < 0.05) (Figure 3 3). These data suggest that longterm chronic increase of PYY3 36 in saliva of lean mice modulates feeding behavior by decreasing food intake and body weight. Behavior Profile : Data presented in this report indicates t hat long term expression of Peptide YY336, an agonist of NPY Y receptors with higher affinity for the Y2 receptor, abolishes aggressive behavior in mice (ref). To test these observations, we used the territorial Resident / Intruder (R/I) aggressive paradi gm (177), a standard test for evaluating rodent aggressive behavior. The test was applied on three different occasions using different intruders. Tests were recorded and analyzed in a blind manner using the Observe r v5.0 software that evaluates over 400 parameters per second (Noldus Information Technology) (178). The aggressive behavior was analyzed by the frequency, duration and latency of attacks, threats and chase from the resident to the intruder mice. PYY3 36 treated mice displayed a 44 -fold decrease in the number of attack events compare to controls [PYY336 0.07 0.067 events per 10 min, vs. Controls 3.07 1.74 events in 10 min, n =5, p <0.05) (Figure 3 4). Likewi se, PYY336 treated mice had a significant decrease in attack duration and a significant increase in attack latency. Similarly, PYY3 36 treated mice had a significant decrease in threat events and duration compare to controls (Figure 3 5) and a decrease in chase events and duration compare d to

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66 control mice (Figure 3 6). Interestingly, even though an aggressive behavior was almost completely abrogated, the normal social interactions manifested by sniffing did not change (Figure 3 7). Summary and Partial Conclusions This dramatic change in territorial aggression suggests that the long -term treatment with NPY Y2 receptors agonists such as PYY3 36 modulates both feeding (Acosta et al. submitted) and aggressive behaviors. Because PYY3 36 has recently been t ested in clinical trials for weight loss in obese adult subjects, the unintended while favorable effects shown here must be taken in consideration before such agonists are approved for the long-term treatment of obesity. This is especially important in lig ht of the Y receptors cross talk and interactions as shown in genetically modified mice models (179). These results corroborate the i mportant NPY -serotonin link in aggression and feeding behavior (180). Further studies are needed to understand the longterm effect of Y receptors agonists in feeding and aggressive behavior, as well as in depression and anxiety.

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67 Figure 3 1 Diagram of rAAV-PYY and rAAVGFP vectors plasmids. ITR: rAAV serotype 5 inverted terminal repeats; enh: Cytomegalovirus intermediate early enhancer sequence; B -Act: chicken b acting promoter; Ex1: non coding sequence; murine Pre -pro -Peptide YY gene or Green Fluoresce nce protein; PA: bovine growth hormone polyadenylation sequence.

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68 Figure 3 2 Weekly Food Intake for 22 weeks Food intake in lean mice treated with rAAV PYY vs. rAAV-GFP controls. (n=5 per group; p<0.05) Values reported as means +/ SE.

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69 Figure 3 3 Body Weight Accumulation for 22 weeks Body Weight Accumulation for 22 weeks in lean mice treated with rAAV -PYY vs. rAAV-GFP controls. (n=5 per group; p<0.05) Values reported as means +/ SE.

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70 Figure 3 4 Territorial Resident Intruder test (attack).Territorial Aggression in individually housed mice treated with rAAV PYY vs. rAAV-GFP controls after been tested in the Resident intruder paradigm, measuring attack frequency (A), duration (B), and latency (C). Values reported as means +/ SE. p<0.0 5

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71 Figure 3 5 T erritorial Resident Intruder test (Threat).Territorial Aggression in individually housed mice treated with rAAV PYY vs. rAAV-GFP controls after been tested in the Resident intruder paradigm, measuring threat frequency (A), duration (B), and latency (C). Values reported as means +/ SE. p<0.05

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72 Figure 3 6 Territorial Resident Intruder test (Chase).Territorial Aggression in individually housed mice treated with rAAV PYY vs. rAAV-GFP controls after been tested in the Resident intrud er paradigm, measuring chase frequency (A), duration (B), and latency (C). Values reported as means +/ SE. p<0.05

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73 Figure 3 7 Normal non aggressive behavioral analysis Normal non aggressive analysis in individually housed mice treated with rAAV -P YY vs. rAAVGFP controls after been tested in the Resident -intruder paradigm, measuring social sniffing frequency (A), duration (B), and latency (C). Values reported as means +/ SE.

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74 CHAPTER 4 LONG TERM PEPTIDE YY GENE THERAPY: ADDRESSING EXISTING CONT ROVERSY. Peptide YY Controversy: Does PYY336 Inhibits Food Intake? Obesity has reach ed epidemic proportions in developed countries and its prevalence is rising in developing countries (2 ). Obesity is due to a loss of the balance between food intake and energy expenditure. The food intake is mainly regulated by the brain -gut axis (40, 41). The brain gut axis consists of gut hormones, the vagal complex, the brainstem, the hypothalamus and higher brain centers in the cortex related to appetite and satiation (40). Satiation is induced by several gut hormones including PYY, oxyntomodulin (OXM), and glucagon like petide 1, which are s ecreted after food initiation. These gut hormones inhibit the agouti related peptide/ NPY (AgRP/NPY) pathway arcuate nucleus of the hypothalamus and stimulate the pro (48). The effe ct over both pathways results in satiation and food termination. Peptide YY (PYY) is a 36 amino acid hormone secreted from neuro-endocrine L cells in the distal small intestine and colon, and also from pancreas and brain stem ( 98, 99). The role of PYY in energy homeostasis was evidenced by the PYY knockout mouse became obese, while PYY transgenic mouse is resistant to dietinduced obesity (100, 116). PYY has two main form s i n circulation, PYY1 36 and PYY3 36 (181). PYY3 36 induces satiation by acting over Y2 receptors in the arcuate nucleus of the hypothalamus and in the brain stem (104), (164 ). Moreover, PYY336 also has been related to increase in energy expenditure (109-113). The effects of acu te and chronic systemic administration of PYY3 36 are controversial. Batterham et al. reported that intraperitoneal injection of PYY3 36 reduced food intake in rodents and that peripheral infusion of PYY336 had similar effects in lean and obese humans (104, 120). These results were not replicated by several groups (121); while they were replicated several

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75 times in rodents, nonhuman primates and humans (105, 106, 111, 117, 122131)(acosta et al. 2009, unpublished). On the contrary, when PYY3 36 is injected centrally i.e third, lateral or fourth cerebral ventricle, there is an increase in food intake (73, 132). An opposite effect is achieved when PYY3 36 is injected directly into the arcuate nucleus of the hypothalamus, where Y2 receptors are abundant (104). Previously, we reported the presence of salivary PY Y336 in rodents and humans. The acute and chronic / long term increase of salivary PYY336 produces a decrease in food intake and body weight in lean and obese mice (acosta et al. 2009). In order to understand the controversy related to PYY3 36 several be havioral factors including acclimatization, stress, and administration conditions (site, frequency, doses, and time) must be consider (121). To eliminate these factors and address the effect of long term over expres sion of PYY336 in diet induce d obese mice, we have developed a vector mediated recombinant adeno associated virus encoding for pre -pro PYY. A vector mediated long term over -expression of PYY will avoid behavioral factors that can influence the effect of P YY and additionally will help us understand the effect of longterm over -expression of PYY336 in a site specific manner, avoiding systemic transgenic mouse models. Therefore, we hypothesized that one time viral vector -mediated gene delivery will provide elevated levels of PYY in a long term experiment thus inducing satiation and reducing food intake and body weight. Interestingly, we found that longterm over -expression of peripherally PYY336 have no effect in body weight, contrary to over -expression of salivary PYY336 or central PYY336. The long term over expression of PYY336 peripherally, centrally and in saliva increased the expression of DPPIV suggesting a positive feedback mechanism. Materials and Methods In this hypothesis based study, we deve loped a recombinant adeno associated virus encoding for pre -pro Peptide YY to study the effect of over -expressing PYY specifically

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76 systemic, in saliva and centrally. After injection of our vector, we measured food intake and body weight for over 20 weeks a nd we performed metabolic studies. Viral Vector design, production and purification: We constructed a recombinant adeno associated virus (rAAV) cassette, encoding for murine pre -pro -PYY (total) cDNA (ATCC) under the control of a strong constitutive CMV/B actin promoter (Figure 4 1A). rAAV-enconding pre pro -PYY cDNA (rAAV-PYY) and the control rAAVencoding for Green Flourescence protein (rAAV GFP) were package into rAAV serotype 5, which have higher transduction efficiency to murine submandibulary saliv ary glands. (148) The viral production, purification and titration was done as described by Zolotukhin et al,. (149) In vitro studies: to test our rAAV -PYY transgene secre tion, we decided to use genetically engineered human intestinal NCI -H716 cells as described by Tang et. al (150). Briefly, after cells differentiation, we transfect the NCI H716 cells with rAAVPYY or rAAV-GFP. 48 h ours later, we fast the cells overnight on basal medium [DMEM (GIBCO) with 5 mM glucose and 1% fetal bovine serum]. On the study day, parallel cultures were washed with basal medium every hour for two times to stabilize their basal secretion. Then the cell culture was stimulated with 2% meat hydrolysate (Sigma) in basal medium for 1 -hour. For the following 4 hours, cell culture was incubated in basal medium, with consecutively replacements of the basal medium every hour. Cell culture medium was collected ev ery hour for PYY EIA assay. In vivo studies: This study was approved by the institutional Animal Care and Use committee at the University of Florida. All mice procedures were done in accordance with the principles of the National Research Councils guide for the Care and Use of Laboratory Animals. Studies were done in male C57Bl/6 (Charles River Laboratories) mice housed at 22 24 C in a 12 hours light/dark cycle (lights off at 1800). Mice had free access to water and food unless

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77 otherwise stated. Mice we re fed with 60% high fat high caloric diet (Research Diet s) ad libitum. Food intake and b ody weight was measured weekly for 30 weeks after virus delivery. Mice were group housed until week 20. Thereafter, mice were individually housed. Vector delivery: A single dose of rAAV-PYY or rAAV-GFP (controls) was injected to 45 days old male C57Bl/6 mice. To over -express PYY systemically from the distal intestine, we injected 100 ul containing 1x1011 DNAI resistant particles of rAAV -PYY or rAAVGFP in to the super ior mesenteric artery as described by Polyak et al (182). To over -express salivary PYY3 36 we delivered 100 ul containing 1x1010 DNAI resistant particles into each salivary ducts of submandibular glands as described in Acosta et al. To over -express PYY336 centrally we injected 5 ul containing 1x1010 DNAI resistant particles into the 3rd ventricle of the brain as described by Carty et al (183). Sa liva collection: Salivation was stimulated by an i.p. injection of 100 ul of a cocktail containing isoproterenol/pilocarpine (1mg/2mg in 1ml of PBS). (145) Saliva was collected for 5 minutes from the oral cavity u sing a micropipette into 1.5 ml eppendorf containing 5000 U of Kalikrein inhibitor (Biomedicals) and 50 mM of DPP IV inhibitor (Linco Research). Saliva samples were frozen at 80 C until analyzed. Plasma collection: Blood was collected from facial vein pu ncture into EDTA -coated tubes (Capiject) containing 5000 U of Kalikrein inhibitor (Biomedicals). Tubes were incubated 30 minutes at RT for clotting, and then spin for 10 minutes at 1200 G at 4 C. Plasma was transferred into new 1.5 ml eppendorfs containing 50 mM of DPP IV inhibitor (Linco Research). Plasma samples were frozen at 80 C until analyzed.

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78 Plasma and saliva hormone levels: The Mouse Gut Hormone Panel from plasma and saliva were measured by Lincoplex kit (Linco Research). PYY336 from saliva, pl asma or cell culture supernatant were measured by PYY336 EIA kit (Phoenix Pharmaceuticals, Inc) Tissue collection: Mice were sacrificed by CO2 and tissues were harvest for DNA, RNA and IHC studies. Relative quantitative RT PCR analysis: RNA extraction, purification, cDNA synthesis and RT PCR amplification was done as described in Aslanadi et al. (39) Briefly, tissues were isolated using T rizol reagent (invitrogen) and h omogenized by using Matrix A in a FP120 Homogenizer (Qbiogene). RNA integrity was verified by agarose gel (1%) electrop horesis with ethidium bromide. RNA was DNAse treated by Turbo DNA-freeTM kit (Ambion). Total RNA in equals amount for each sample (6 or more tissue/group) were converted to cDNA by a SuperScriptTM III First Strand Synthesis supermix (Invitrogen). Primers were designed by Primer3 algorithm, available at the Whitehead Institute f or Biomedical Research website. cDNA was amplified by PCR using SYBR GreenERTM qPCR SuperMix for iCycler. (Invitrogen) Relative expression values were determined b housekeeping gene (b The relative expression or fold change of the gene of interest was calculated as fold change = 2 CT. Immunohistochemistry: Frozen tissue sections were brought to room temperature before melting the OCT medium in 1X TBS (wash buffer). To detect peptide YY (PYY) expression, antigen retrieval was performed in Antigen Retrieval Citra Solution (Biogenex, San Ramon, CA) for 30 minutes in a steamer then rinsed in wash buffer two times for five minutes each. Tissues were blocked for non -specific binding with 10% normal goat serum diluted in Antibody Diluent

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79 (Zymed, Invitrogen, Carlsbad, CA, USA) for 20 min utes. The slides were not rinsed, but rather had the excess diluent dabbed off the slide. Guinea pig anti -PYY (1:500; Pierce/Thermo Fisher Scientific, Rockford, IL, USA) was diluted in Antibody Diluent and incubated on tissues overnight at 4 C, with subs equent rinses in wash buffer after equilibrating tissues to room temperature. Incubation of control tissues in Antibody Diluent served as the negative control. Secondary antibody, Goat anti -guinea pig conjugated to Alexa Fluor 555 (1:1000, Molecular Prob es/Invitrogen, Carlsbad, CA, USA), was incubated on tissues at room temperature for one hour followed by rinses in wash buffer. Tissues were coverslipped using VectaShield HardSet Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) PYY signal was viewed by Zeiss Fluorescent Axioskop microscope, Model 9850, using AxioSkop imaging software (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA). Statistical analysis: Statistical analysis was conducted using unpaired Students t test with significance at P < 0.05. The Effect of Long -Term PYY3 36 Over-Expression in Feeding Behavior of Diet -Induced Obese Mice The purpose of this study was to investigate the long term metabolic effect of PYY3 36 over -express in a site specific manner in a diet -induced obese mice model and to address the existing controversy about the effect of PYY336 in food intake (121). To achieve a sustained site specific over -expression of PYY336, we used recombinant adeno as sociated virus (rAAV) because it is non -pathogenic, infect undifferentiated and differentiated cells, and produce a long term persistent expression. (For review see Daya, S. and Bern, KI 2008) (157 ) In order to achieve a regulated site specific over -expression of PYY3 36 we designed our transgene cassette with a constitutive promote for long term over -expression (Figure 4 1A), but using the pre -pro PYY hormone to secrete PYY in a regulatory granule secretion manner (150).

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80 In vitro results : To verify the regulatory granule secretion properties of our rAAV -PYY, we infect ed differentiated NCI H716 cells with rAAV PYY or rAAV-Green Fluorescence protein (rAAV GFP) as a control. 48 hours after infection, we fast the cells overnight and change the media at time 0. Then we incubated the infected cells in basal medium for 1 hou r; then switch to medium with 2% meat Hydrolysate (MH) for one hour; and then we switched back to basal medium for 5 hours more, collecting the medium every hour ( Figure 4 1B). During the basal state, cells treated with rAAV -PYY secreted a 2 fold increas e of PYY compare to rAAV-GFP cells. During stimulation with 2% MH, rAAV-PYY treated cells secreted 100 fold increase of PYY compare to rAAVGFP cells. After the stimulation, both cell lines return to their basal state. The granule secretion stimulation sho ws that rAAV-PYY will produce PYY in a regulatory granule secretion manner, with a minor constant secretion. In vivo metabolic studies using rAAV -PYY in DIO mice : Peripheral over -expression of PYY336: Eight weeks after superior mesenteric artery injection we collected blood at time 0, 2, 8 and 24 hours after fasting (Figure 4 2A). We found that rAAV -PYY treated DIO mice had a two fold increase of plasmatic PYY during fasting compare to rAAV GFP controls; and a tenfold increase during feeding compare to rAAVGFP controls. These increases continue for the following 8 hours and reduce to fasting levels 24 hours after food intake. Similar plasmatic concentrations were found during fasting and feeding 30 weeks after injection. These findings verified our long term over -expression peripheral model. The satiation studies were done 20 weeks after SMA injection at the beginning of the dark cycle w h ere mice are more active and consume more calories. We found that there was a significant decrease in 1 hour food in take after 24 hours fasting (p < 0.005), with same caloric intake during the 1st and 2nd hour. There was no difference in food intake during the first to

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81 second hour. The difference from the first hour was maintained after two hours of food intake (p = 0.0 54; Figure 4 2B). Interesting, a ten -fold increase in plasmatic PYY336 by gene delivery induces an early satiation during the first two hours after fasting. Regardless, the successful over -expression efficiency of PYY336 and satiety effect seen after rAAV-PYY SMA injection, there was no effect in weekly food intake (Figure 4 2C) or body weight in diet induced obese mice(Figure 4 2D). When mice were fasted, there was no difference in body weight loss during fasting (Figure 4 2E), a parameter for s uggesting increase energy expenditure. When mice were sacrificed, white visceral adipose tissue was collected and there was no difference between groups (Figure 4 2F). rAAV-PYY SMA treated mice had no difference in glucose tolerance test, or plasmatic co ncentration of other satiation peptides (GLP 1 and OXM) compare to rAAV GFP controls (Figure 4 2 G, H, I). Unexpectedly, mice treated with rAAV -PYY SMA injection had a 12 fold increased in RNA expression of Dipeptidil peptidase IV compare to rAAV -GFP con trols (p=0.028; Figure 4 2J). Moreover, there was a 5 fold decrease in mRNA expression of endogenous PYY in the distal small intestine and colon (p=0.039 and 0.049; Figure 4 2K). There was no difference in GLP 1 mRNA expression (Figure 4 2L). To vali date the long term over -expression of our PYY transgene delivery by SMA injection we measured the transduction efficiency by vector genomic DNA copies and the transgene expression by RNA and IHC. We found that there was a efficient transduction showed by 5 00 genomic DNA copies per ug DNA in the distal intestine. The transduction was not exclusive to the gut, on the contrary was systemically showed by a higher transduction in the liver and heart (Figure 4 2M). Similar findings we found in PYY transgene expression by RT PCR compare to controls (Figure 4 2N), as well as by IHC (Figure 4 2O).

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82 Salivary over -expression of PYY3 36: Eight weeks after salivary ducts vector delivery we collected blood at time 0 and 2 hours after fasting and stimulated saliva. We found that rAAV PYY treated DIO mice had a no difference in plasmatic PYY3 36 concentrations during fasting or 2 hours after feeding (Figure 4 3A). Similar plasmatic concentrations were found during fasting and feeding 30 weeks after vector delivery. O n the contrary, we found a two fold increase in salivary PYY336 in rAAV-PYY treated mice compare to rAAV -GFP controls (Figure 4 3B). These findings verified our long term over -expression salivary PYY336 exclusive, without altering plasmatic concentrati ons. The satiation studies were done 20 weeks after salivary duct injection at the beginning of the dark cycle where mice are more active and consume more calories. We found that there was a significant decrease in 1 hour food intake after 24 hours fasti ng (p < 0.05), with same caloric intake during the 1st and 2nd hour. There was no difference in food intake during the first to second hour. The difference from the first hour was maintained after two hours of food intake (p = 0.061). (Figure 4 3C) Even though, the increase of salivary PYY3 36 induced satiation, there was no difference in weekly food intake (Figure 4 3D). Interestingly, we found a significant decrease in body weight in diet induced obese mice treated with rAAV -PYY into salivary glands compare to rAAVGFP controls (Figure 4 3E and 3F). The difference in body weight was correlated with a greater loss of body weight during fasting (Figure 4 3G), a parameter for suggesting increase energy expenditure. When mice were sacrificed, white vi sceral adipose tissue was collected and there was no significant difference between groups (Figure 4 3H). rAAV-PYY treated into salivary glands mice had no difference in glucose tolerance test (Figure 4 3I). Although, we found that rAAV PYY treated int o salivary glands had a decrease

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83 concentration of plasmatic GLP 1 compare to controls 2 hours after feeding (Figure 4 3J). There was no difference in OXM plasmatic concentr ation during fasting or feeding (Figure 4 3K). The significant decrease in GLP 1 cou ld explain the lack difference in weekly food intake. To further understand this effect, we measured DPP IV mRNA expression. We found that mice treated with rAAV-PYY had a 2.5 fold increased in RNA expression of Dipeptidil peptidase IV compare to rAAVGFP controls (p=0.036; Figure 4 3L). Furthermore, there was a 5 fold decrease in mRNA expression of GLP 1 in the small intestine compare to rAAV -GFP mice (p=0.045; Figure 4 3M). Moreover, there was a 4 fold and 13 fold increase in mRNA expression of endoge nous PYY in the distal small intestine and colon, respectively (p=0.037 and 0.041; Figure 4 3N). To validate the long term over -expression of our PYY transgene delivery by salivary ducts injection we measured the transduction efficiency by vector genomi c DNA copies and the transgene expression by RNA and IHC. We found that there was a efficient transduction showed by 500 genomic DNA copies per ug DNA in submandibular salivary glands with no systemic transduction. Similarly, there was a 23 fold increase in expression in salivary glands (p=0.008) with no PYY expression in the liver compare to controls (Figure 4 3O). The increased expression of PYY transgene was also determined by IHC (Figure 4 3P) in salivary glands. Intra -cerebral ventricular over exp ression of PYY3 36: One week after vector intra cerebral ventricular (ICV) rAAV -PYY delivery there was an increase in weekly food intake compare to rAAVGFP controls (Figure 4 4A). Consequently, there was an increase in body weight in diet induced obese mice treated with rAAV -PYY into ICV compare to rAAV-GFP controls (Figure 4 4B and 4C). There was no difference in DPP -IV or PYY mRNA expression between mice treated with rAAV -PYY ICV and rAAVGFP controls (Figure 4 4D and 4E, respectively).

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84 Interestin gly, there was a 3 fold increase in GLP 1 mRNA expression in the small intestine compare to rAAV -GFP mice (p=0.024; Figure 4 4F). Summary and Partial Conclusions Viral vector -mediated PYY delivery results in a long-term physiologically regulated elevatio n of PYY concentration either peripherally (plasma) or locally (brain, saliva). Ten -fold increase in plasmatic PYY336 has no effect on FI and BW. The over -expression of PYY336 in the 3rd ventricle of the brain produced an increase in FI and BW. The ove r -expression of PYY336 in salivary gland and 2 -fold increase in PYY in saliva resulted in a sustained reduction in FI and BW. The over -expression of PYY transgene systemically resulted in a decreased expression of endogenous PYY, while elevation of PYY i n saliva increased the expression of endogenous PYY. In addition, both systemic and salivary PYY resulted in an increased expression of hepatic DPP IV, suggesting central mechanism of regulation of the DPP IV expression. An increased DDP IV expression apparently resulted in the decreased concentration of GLP 1 effecting changes in FI and BW. Our data suggests that the long term increase of PYY336 can initiate a feedback mechanism in endogenous PYY, GLP 1 and DPP IV expression. This regulatory mechanism ha s to be taken into account in potential clinical applications using satiation gut peptides, satiation gut peptides analogs or DPP -IV inhibitors. Recently, we found that PYY3 36 is present in saliva. The acute augmentation of salivary PYY336 produced a d ecrease in food intake and the long term ectopic over -expression of PYY in salivary glands increased the concentration of salivary PYY336 and produced a decrease in FI and BW in lean and obese mice. Here, we investigated the effect of long term over -expre ssion of PYY in salivary glands in diet induced obesity mouse model. We found a significant decrease in

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85 BW with no difference in weekly FI, suggesting an increase in energy expenditure that regulates BW; and probably the decrease in GLP 1 concentration alt ers the satiation induced by FI. On the other hand, the rAAVPYY injected in the superior m esenteric artery produced a 2 fold increase PYY in plasma during fasting and 10 -fold increase 2 hours after feeding. The increased concentration of plasmatic PYY had no lasting effect on 24 hr FI after fasting and no effect in BW compared to rAAV GFP control mice. The long-term over -expression of PYY produced a down regulation of endogenous PYY and an upregulation of DPP IV. DPP IV has a major role in converting P YY136 to PYY336. In DPP IV deficient rats, acute administration of PYY136 does not decrease food intake (128). Our findings of DP P IV up regulation are probably link to a feedback mechanism when there is an excess of PYY1 36 in plasma produced by our Pre -pro PYY transgene. These effects over DPP IV have a counter effect down regulating the expression of PYY endogenous. Our PYY trans gene is not affected by this negative feedback because is regulated by a different constitutive promoter. Interestingly, the long term increase of salivary PYY3 36 also upregulate the DPP IV, endogenous PYY and GLP 1 expression. Although, the up regulati on of DPP IV bypassed the up -regulation GLP 1 mRNA; and decreased the GLP 1 concentration in plasma. The decrease in GLP 1 can be the explanation to the lack of difference in FI in mice treated with rAAV -PYY into salivary glands. The opposite effect was s een after injection of rAAV -PYY into the 3rd Ventricle of the brain, where there was a significant increase in FI and BW compared to controls. The significant increase in BW and FI produced a down regulation of DPP IV expression and an up regulation of PYY and GLP 1. This positive feedback mechanism was probably trying to overcome the long -term central stimulation of PYY336.

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86 In summary, we have found that the long term specific increase of PYY336 have site specific effects. The increase of plasmatic P YY336 has no metabolic effect in DIO mice; the increase of salivary PYY3 36 decreased BW in DIO mice; and the over -expression of centrally PYY336 increased BW and FI in DIO mice. All these site -specific effects were correlated with an opposite feedback mechanism to overcome their primary effect. The long term increase of plasmatic and salivary PYY3 36 upregulate DPP IV and down regulate PYY and GLP 1 expression. On the contrary, the over -expression of centrally PYY336 down regulate DPP IV and up -regula te PYY and GLP 1 expression. These long term effects must be taken in consideration when satiation gut hormones, their analogs or DPP IV inhibitors are use d for long term treatments like obesity and diabetes.

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87 A B Figure 4 1 Validation of recombinan t adeno associated virus encoding for pre -pro PYY and regulatory secretion. A) Diagram of rAAV -PYY and rAAVGFP vectors plasmids. ITR: rAAV serotype 5 inverted terminal repeats; enh: Cytomegalovirus intermediate early enhancer sequence; B -Act: chicken b acting promoter; Ex1: non coding sequence; murine Pre -pro Peptide YY gene or Green Fluorescence protein; PA: bovine growth hormone polyadenylation sequence. B) Secretion study in NCI H716 cells stimulated with Meat Hydroxylate (MH) 2% to measure granule secr etion. After an overnight fast, cells were incubated in basal medium (BS) for 1 hour, MH 2% for 1 hour and then BS for 5 hours. The experiment was performed on 3 different occasions with 10 wells per group. Values are reported as mean SE.

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88 A B Fig ure 4 2: rAAV-PYY delivered into superior mesenteric artery (SMA) in diet-induced obese mice. A): Secretion Studies: Blood was collected at time 0, 2, 8 and 24 hours after fasting; B) Satiation study measuring Food Intake after 24 hours fasting; C) Weekly food intake (kcal/week); D) Body weight accumulation; E) Body weight loss after 24 hours fasting;F) white visceral adipose tissue; G) Glucose Tolerance test; H) Concentration of GLP 1 during fasting and 2 hours after feeding; I) Concentration of OXM durin g fasting and 2 hours after feeding; J) Relative mRNA expression of DPP IV in rAAV-PYY vs. rAAV-GFP controls in mice liver; K) Relative mRNA expression of endogenous PYY in rAAV PYY vs. rAAV-GFP controls in mice distal small intestine and colon; L) Relativ e mRNA expression of GLP 1 in rAAV-PYY vs. rAAVGFP controls in mice distal small intestine; M) DNA vector genomic copies per ug DNA; N) Relative mRNA expression levels of transgenic PYY in liver, distal small intestine and colon; and O) IHC for PYY antibo dies in distal intestine and liver treated with rAAV -PYY compare to distal intestine rAAV -GFP control. Values are reported as mean SE (n=10 per group)*P <0.05 and **P <0.005 vs. control.

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96 A B Figure 4 3 rAAV-PYY d elivered into submandibulary salivary glands (SG) in diet induced obese mice. A) Secretion Studies: Blood was collected at time during fasting and 2 hours after feeding; B) Concentration of PYY336 in stimulated saliva; C) Satiation study measuring Food In take after 24 hours fasting; D) Weekly food intake (kcal/week); E) Body weight accumulation; E) Body weight loss after 24 hours fasting;F) Body weight at 30 weeks after rAAV -PYY vector delivery vs. rAAVGFP controls; G) Body weight loss after 24 hours fast ing ; H) white visceral adipose tissue; I) Glucose Tolerance test; J) Concentration of GLP -1 during fasting and 2 hours after feeding; K) Concentration of OXM during fasting and 2 hours after feeding; L) Relative mRNA expression of DPP IV in rAAVPYY vs. r AAV-GFP controls in mice liver; M) Relative mRNA expression of endogenous PYY in rAAV -PYY vs. rAAVGFP controls in mice distal small intestine and colon; N) Relative mRNA expression of GLP 1 in rAAV-PYY vs. rAAVGFP controls in mice distal small intestine; O) Relative mRNA expression levels of transgenic PYY in submandibulary salivary glands and liver; and P) IHC for PYY antibodies in submandibulary salivary glands treated with rAAV -PYY compare to pancreas rAAV GFP control. Values are reported as mean SE (n=10 per group)*P <0.05 and **P <0.005 vs. control.

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104 A B Figure 4 4 rAAV-PYY delivered into 3rd intra verebral ventricle (ICV) in diet -induced obese mice. A) Weekly food intake (kcal/week); B) Body weight accumulation; C) Body weight at 8 weeks after rAAV -PYY vector delivery vs. rAAV GFP controls; D) Relative mRNA expression of DPP IV in rAAVPYY vs. rAAVGFP controls in mice liver; E) Relative mRNA expression of endogenous PYY in rAAV-PYY vs. rAAVGFP controls in mice distal small intestine and colon; and F) Relative mRNA expression of GLP 1 in rAAV-PYY vs. rAAVGFP controls in mice distal small intestine. Values are reported as mean SE (n=10 per group)*P <0.05 and **P <0.005 vs. control.

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107 CHAPTER 5 CONCLUSIONS In conclusions, our results indicate that satiation gut peptides are present in saliva. Salivary PYY336 has a physiological effect in modulating food intake. This effect is mediated through the activation of specific NPY Y2 receptors in the tongue epithelium, which is innervated by the glossopharyngeal nerve (CNIX). CNIX synapses in the NTS, where several fibers project to vagus nerve, the hypothalamus and other satiation centers to stop food consumption. Moreover, we showed that the acute in crease in salivary PYY336 decreases food intake and that the chronic long -term increase in salivary PYY3 36 decreases food intake and body weight in lean and obese mice. Interestingly, besides modifying the feeding behavior, we found that the long term increase of salivary PYY336 modifies the aggressive behavior in lean mice. Furthermore, we found that the long -term increase of salivary PYY336 produce a diet induced resistant phenotype compare to an increase in plasma PYY336 or centrally PYY3 36 which are diet induce obese phenotype in C57BL/6 diet -induced obese (DIO) mice model. Interestingly, the effect of longterm increase in salivary PYY336 in DIO mice was by increas ing energy expenditure and not by decreasing food intake. The lack of decrease in food inta ke, was due to a positive vagal feedback that up regulate DPP IV, GLP 1 and endogenous PYY mRNA expression in the gastrointestinal tract. These long term effects must be taken in consideration when satiation gut hormones, their analogs or DPP IV i nhibitors are going to be use for long term treatments for obesity and diabetes (Figure 5 1). Salivary PYY3 36: Characterization and Role in Food I ntake In this study, we provide evidence that satiation gut hormones such as PYY3 36 and GLP 1 can be measur ed in human and murine saliva, and the increase of these hormones in saliva produced a significant decrease in food intake. The finding of satiation gut hormones in saliva

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108 can contribute to develop a non invasive method to measure these hormones in saliva This potential clinical diagnostic tool can be useful in the diabetes and obesity fields where these hormones have been measured to monitor metabolic profiles as well as treatment outcomes, especially since the incorporation of gliptins (DPP 4 inhibitor s) and GLP 1 analogs for the treatment of diabetes (62). Moreover, there are several clinical trials for obesity using satiation gut hormones or their analogs (61), (62). Interestingly, we showed that PYY and Y2 receptors are expressed in taste cells of the circumvallated papilla (CVP) in the murine tongue. These findings correlate with previous findings that showed that NPY and Y1 receptor (155), GLP 1 and GLP 1 receptor (152), and CCK (154), (153, 161) are also expressed in taste cells of the CVP in the tongue. These findings suggested that satiation gut hormones are expressed in taste cells and correlates with previous data suggesting the enteroexpress in taste cells (162, 163). The characteriz ation of the satiation gut hormones specific receptor in the tongue epithelium suggests a possible new pathway related to the facial or glosopharyngeal nerve. However, more studies are needed to understand the similarities and differences between taste cel ls in the tongue and entero endocrine cells in the gut and their specific relation with food intake and taste perception. In order to understand the role of salivary PYY3 36, we wanted to determine if the increase in the concentration of salivary PYY336 w ithout altering plasmatic concentration would induce satiation and reduce food intake. To achieve an increase in salivary PYY336 we developed a oral spray, which increased the concentration of salivary PYY3 36 without increasing the concentration of plasm atic PYY336. After one dose of 5ug/100 g PYY336 oral spray to the murine mouth there was significant decrease in food intake compared to previous results after

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109 peripheral administration, with the same delayed orexigenic effect (156). In a dose response study, we showed that even almost physiological doses of PYY3 36 (0.3ug/100g) delivered to the mouth produced a decrease in one hour food intake while other investigators have not achieved any effect after lower dose s of systemic delivery of PYY336 (164). At higher doses PYY336 o ral spray produced a significant decrease of food intake compared to systemic delivery of PYY336 (106, 123, 156, 164-167 ). The significant effect of increasing salivary PYY3 36 and its similarity to plasmatic incre ase of PYY336 suggests that the modulation of salivary PYY336 can induce satiation through an alternative pathway and have a potential clinical application for the treatment of obesity. The induction of satiation by salivary PYY336 was validated by show ing an increase in c fos neuronal activity in the arcuate nucleus. The activation of this hypothalamic satiation center by salivary PYY3 36 correlates to the mechanism of satiation produced by peripheral PYY336 described by Batterham et. al (104). Since, the satiation mechanism is similar between salivary PYY336 and plasmatic PYY3 36; and PYY336 oral spray did not increase plasmatic concentration, the pathway for satiation inducing might be different. Also, th e enhanced effect of PYY336 when increased in saliva and its correlation with plasmatic concentration suggests that the increase of plasmatic PYY3 36 can also affect this alternative pathway to induce satiation. PYY336 induced satiation by activating NPY / AGRP neurons in the arcuate nucleus ( 104), (168) and it is still controversial if the POMC pathway is involved in the arcuate nucleus ( 169, 170). The effect of PYY336 over the arcuate nucleus in the hypothalamus is probably mediated by the permeability of the blood brain barrier to PYY3 36 (171). Alternatively, salivary PYY336 activates Y1 or Y2 receptors expressed in the CVP. The CVP is innervated by sensory fibers of the lingual branch of the glossopharyngeal nerve (cN IX) (172). The cN -IX ganglia receive the

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110 sensory fibers and send afferent fibers to the superior part Nucleus of the solitary tract (NTS). The NTS regulates satiation by direct or indirect stimulation of satiation gut hormones including systemic PYY336 (173, 174). This alternative satiation pathway mediated by salivary PYY336 can be related to sensory specific satiation (57-59) which can result in short term taste aversion and postprandial malaise (164, 175). In addition, PYY expressing neurons in the Gigantocellular reticular nucleus synapse in the NTS and with fibers from the hypothalamic Orexin and Melanin concentrating hormone systems that mediate appetite satiation in the hypothalamus (99). Even though, the satiation induction by acute increase of salivary PYY336 is similar to an a cute increase of plasmatic PYY336, we did not see an effect on body weight after 4 days of oral spray every six hours in mice. This is probably because mice were eating constantly during the night in small quantities, but humans eat large quantities a few times per day. Therefore, we decided to increase salivary PYY3 36 in a regulatory manner related to salivation and in a non invasive longterm over -expression. We achieved this by using a vector mediated gene delivery technique. This technique also allow ed us to overcome several issues that can be related to the effect of acutely deliver PYY3 36 like acclimatization, stress, time and site of injection, and conditioning (176). Consequently the vector mediated PYY delivery to submandibular glands offered several benefits: two fold increased in salivary PYY336 without altering plasma concentration, one injection per life time of the experiment (22 or 8 weeks), non invasive delivery method, no need for conditioning, and no stress produced. The body weight reduction produced by rAAV -PYY can be compared to bariatric surgery, the current gold standard for obesity (160). These data suggest that the increase of PYY3 36 in saliva can become a long term treatment for obesity.

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111 Salivary PYY3 36 and A ggressive Behavior These dramatic chang es in territorial aggression suggest that the long term treatment with NPY Y2 receptors agonists such as PYY3 36 modulates both feeding and aggressive behaviors. Because PYY3 36 has recently been tested in clinical trials for weight loss in obese adult sub jects, the unintended while favorable effects shown here must be taken in consideration before such agonists are approved for the long -term treatment of obesity. This is especially important in light of the Y receptors cross talk and interactions as shown in genetically modified mice models (179). These results corroborate the important NPY -serotonin link in aggression and feeding behavior (180). Further studies are needed to understand the longterm effect of Y receptors agonists in feeding and aggressive behavior, as well as in depression and anxiety. Long -term Over-Expression of Site -Specific PYY336 in a D iet I nduce O bese M ice M odel Vi ral vector -mediated PYY delivery results in a long-term physiologically regulated elevation of PYY concentration either peripherally (plasma) or locally (brain, saliva). Ten -fold increase in plasmatic PYY336 has no effect on FI and BW. The over -expressio n of PYY336 in the 3rd ventricle of the brain produced an increase in FI and BW. The over -expression of PYY336 in salivary gland and 2 -fold increase in PYY in saliva resulted in a sustained reduction in FI and BW. The over -expression of PYY transgene sys temically resulted in a decreased expression of endogenous PYY, while elevation of PYY in saliva increased the expression of endogenous PYY. In addition, both systemic and salivary PYY resulted in an increased expression of hepatic DPP IV, suggesting centr al mechanism of regulation of the DPP IV expression. An increased DDP IV expression apparently resulted in the decreased concentration of GLP 1 effecting changes in FI and BW.

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112 Our data suggests that the long term increase of PYY336 can initiate a feedbac k mechanism in endogenous PYY, GLP 1 and DPP IV expression. This regulatory mechanism has to be taken into account in potential clinical applications using satiation gut peptides, satiation gut peptides analogs or DPP -IV inhibitors. Recently, we found th at PYY3 36 is present in saliva. The acute augmentation of salivary PYY336 produced a decrease in food intake and the long term ectopic over -expression of PYY in salivary glands increased the concentration of salivary PYY336 and produced a decrease in FI and BW in lean and obese mice. Here, we investigated the effect of long term over -expression of PYY in salivary glands in diet induced obesity mouse model. We found a significant decrease in BW with no difference in weekly FI, suggesting an increase in en ergy expenditure that regulates BW; and probably the decrease in GLP 1 concentration alters the satiation induced by FI. On the other hand, the rAAV PYY injected in the Superior Mesenteric artery produced a 2 -fold increase PYY in plasma during fasting and 10 -fold increase 2 hours after feeding. The increased concentration of plasmatic PYY had no lasting effect on 24 hr FI after fasting and no effect in BW compared to rAAV GFP control mice. The long-term over -expression of PYY produced a down regulation of endogenous PYY and an upregulation of DPP IV. DPP IV has a major role in converting PYY136 to PYY336. In DPP IV deficient rats, acute administration of PYY136 does not decrease food intake (128). Our findings of DPP IV up regulation are probably link to a feedback mechanism when there is an excess of PYY1 36 in plasma produced by our Pre -pro PYY transgene. These effects over DPP IV have a counter effect down regulating the expression of PYY endogenous. Our PYY transgene is not affected by this negative feedback because is regulated by a different constitutive promoter.

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113 Interestingly, the long term increase of salivary PYY3 36 als o upregulate the DPP IV, endogenous PYY and GLP 1 expression. Although, the up regulation of DPP IV bypassed the up -regulation GLP 1 mRNA; and decreased the GLP 1 concentration in plasma. The decrease in GLP 1 can be the explanation to the lack of differe nce in FI in mice treated with rAAV -PYY into salivary glands. The opposite effect was seen after injection of rAAV -PYY into the 3rd Ventricle of the brain, where there was a significant increase in FI and BW compared to controls. The significant increase in BW and FI produced a down regulation of DPP IV expression and an up regulation of PYY and GLP 1. This positive feedback mechanism was probably trying to overcome the long -term central stimulation of PYY336. In summary, we have found that the long t erm specific increase of PYY336 have site specific effects. The increase of plasmatic PYY336 has no metabolic effect in DIO mice; the increase of salivary PYY3 36 decreased BW in DIO mice; and the over -expression of centrally PYY336 increased BW and FI in DIO mice. All these site -specific effects were correlated with an opposite feedback mechanism to overcome their primary effect. The long term increase of plasmatic and salivary PYY3 36 upregulate DPP IV and down regulate PYY and GLP 1 expression. On t he contrary, the over -expression of centrally PYY336 down regulate DPP IV and up -regulate PYY and GLP 1 expression. These long term effects must be taken in consideration when satiation gut hormones, their analogs or DPP IV inhibitors are going to be use for long term treatments like obesity and diabetes. Future D irections: Our findings have open many new hypothesis related to salivary PYY3 36, its effect in food intake, reward, aggression, behaviors, negative/positive feedback and has a great potential f or a

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114 new approach to treat obesity, the new century epidemic. Here, I am going to address each new hypothesis, m ention preliminary data, if any and discuss the importance of their findings. Mouth spray with other peptides and compounds : Our findings of sat iation gut hormones in saliva, its specific receptors in the tongue epithelia and the decrease in food intake after PYY336 oral spray suggests that other compounds can have a physiological/pharmacological effect using the same delivery method. We have alr eady tested GLP 1 and Exendin4 oral spray. The results after this pilot studies are promising, due to a significant decrease in food intake. We also tested BII0246, a NPY Y2 receptor selective antagonist, showing a increase in food intake. Further studies are needed to characterize the effect of each compound as well as other compounds available in the market like Amylin, OPT (PYY second generation), OXM mutants, GCG/GLP 1 co agonists, all the gliptins, and all the exendins. Behavior studies : Our findings of longterm increase of salivary PYY3 36 in aggressive behavior suggests that long term treatment with PYY modulates the serotonin / NPY link. Our preliminary data supports a further and more complex effect of PYY in reward, emotions, memory, anxiety and depression. We should test all the behavioral profile in the short term and long term increase of PYY3 36 in saliva, plasma and centrally. These studies must be supported by analysis of metabolic pathways and the interaction with the limbic system, mood s tabilizers, anxiolytics and antidepressants. Salivary PYY336 mechanism: Our results support the theory of an alternative pathway for satiety through the glossopharingeal nerve and the NTS. Further studies are needed to characterize the pathway and verify the role of the glossopharingeal nerve in satiety, excluding the upper branches of the vagal nerve. These studies can be done using Me -MRI, pseudorabies virus, or rAAV retrograde serotypes. Also, detailed analysis of the brain stem, their nucleus and

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115 the NTS must be studied to understand the effect of the glossopharingeal nerve and the CVP related to satiety. Another interesting observation obtain while doing IHC of the tongue was the presence of exogenous PYY336 in the tongue epithelia after oral spray. My hypothesis is that PYY336 can act as a retrograde neurotrasmiter and activate the Y receptors express in neurons on the NTS and then induce satiation and a vagal response. To test this, we should radio label PYY and spray it to the oral cavity. After t he spray, collect the brainstem and the brain; and then measure for radioactive PYY. Feedback (Positive/negative): One of our most interesting findings is the upregulation of DPP IV, GLP 1 and endogenous after increase of salivary PYY336 in DIO mice. Th ese observations must be considered in when GLP 1 analogs and DPP IV inhibitors are chronically used for treatment of Diabetes Mellitus type 2. We (as our lab and the whole metabolic field) must repeat this experiments and try to understand the long term u p regulation and down regulation of this genes, understand the pathways, the feedback mechanisms and how to regulate them. We must be very careful to alter the homeostasis of gut hormones and their regulations. For now, I can advice the use of active PYY336 when DPP -IV inhibitors are been used for the treatment of Diabetes Mellitus type 2. PYY, Y2 receptor, Y1 receptor and taste cells knockouts : The best way to verify and support our findings would be to rescue the obese phenotype in the PYY KO by rAAV -PY Y deliver to the salivary glands. These findings would also show the importance of the alternative pathway and maybe show much more. The same should be done with the NPY Y2 and Y1 receptor knockouts as well with the taste cells KO. Possible it could addres s the taste aversion related to PYY and the sensory specific cortex activation during hunger and satiety.

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116 PYY taste cells (confocal microscopy): Our findings of PYY in the CVP must be correlated with double and triple staining of PYY with alpha gustducin, GLP 1, and other taste buds markers. Also, an wide search for PYY should be done in the whole tongue. I suggest would be better using In -situ hybridization to increase specificity and sensitivity. The same experiments should be done for NPY receptors in t he tongue. Food preferences and taste aversion: The biggest weakness of our findings is not to clarify the interaction of taste with salivary PYY336, especially when there are two publications that link PYY with taste aversion. These publications suggest a central effect mediated through the vagus nerve and the NTS connecting with higher brain centers, but especially with the area postrema a CVO. My theory is that PYY produce taste aversion in higher doses when stimulates the area postrema. We have to test the effect of salivary PYY in taste, taste aversion and postprandial nausea. These side effects are essential if we want to bring the PYY oral spray to clinical trials. Clinical trial saliva vs. plasma : To further support our findings of satiation gut hormones in saliva, we are going to do a clinical trial, where we collect saliva and blood at the same time during fasting and 7, 15, 30, 60 and 120 minutes after eating a 300 kcal meal. This IRB /GCRC protocol has already been submitted and is approved after revisions are corrected. We expect to find a correlation between plasma and saliva concentration of gut peptides. These findings will support our previous results and contribute to develop a non invasive devise to measure satiation gut hormones and hormones in general. Clinical Trial PYY3 36 oral spray : Our developed of a PYY3 36 oral spray and the encouraging data in rodents, motivated us to put together a big phase 1 clinical trial to study the effect of PYY336 oral spray in obese humans. Even t hough, the IRB / GCRC protocol is ready and will be submitted in the following weeks, we need to search for internal and external

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117 financing for this trial (Budget $250.000) and to get the investigational new drug (IND) approval. Initial contacts have been done with pharmaceutical companies, with a promising participation in this trial. Follow this trial; we should try a PYY336 gum, which would have a longer effect with better pharmacokinetics and pharmacodinamics. Unfortunately, this approach cannot be te sted in rodents. rAAV PYY bigger animal model trial : The impressive decrease in body weight in obese mice after treatment with rAAV -PYY and the behavioral effect suggests that we need to test our rAAV-PYY vector in a better and bigger animal model. The best animal model to study food intake and behavior are non -human primates. In them, we would be able to study the effect of rAAV-PYY in feeding behavior as well as emotional behavior including depression, anxiety and aggression. These studies are crucial to bring gene therapy for obesity using NPY Y receptor agonists. To achieve this study, we must continue our collaboration with Dr. Bruce Baum, who has extensive experience and funding to do non-human primates gene therapy to salivary glands. Mass spectometry (PYY136 vs. PYY336): One of the biggest limitations when wo rking with PYY is the lack of a specific method to differentiate PYY1 36 from PYY3 36. In our MALDI TOF MS studies, we show a method to measure PYY3 36 exclusively. We should standardize thi s procedure to be able to quantify our samples containing PYY3 36. This will also help us to safe money in expensive and inaccurate ELISAs. Gastric Bypass vs. rAAVPYY: In order to bring gene therapy for the most prevalent disease in the world, we need t o proof that gene therapy can be safer, better, cheaper and with fewer complications than the current gold standard for obesity. Many scientists including our lab are working in improving the production of rAAV, while others are studying their safety and s ide effects of viral vectors. In the mean time, we should test our rAAV -PYY against Gastric Bypass,

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118 the current goal standard for obesity. Recently, investigators from Massachusetts General Hospital lead by Dr. Kaplan have developed a gastric bypass mouse model. Their results are similar to our results using rAAV -PYY. We have discussed with them the possibility of doing a side by-side experiment comparing gastric bypass vs. rAAV -PYY in obese mice. New Satiation Theory: Our results suggest the presence of an alternative pathway for satiety. Although, these findings must be repeated by other researchers and further studies are needed to understand the mechanism with more detail, I have a new theory for satiation. It is still controversial how exactly PYY33 6 induce s satiation. Batterham et al, suggests that PYY336 acts in the ARC nucleus due to the weak B lood B rain Barrier (BBB). On the contrary, Fry et al, denies this hypothesis of a week BBB in the ARC. They also mention the existence of Cortico -V entric ular O rgan (CVO) that regulate the energy homeostasis. Although, we and many others have shown that the increase in physiological and pharmacological doses of PYY336 in the brain ventricles produces the opposite effect. Th is effect goes against the theory of CVO pathway that PYY3 36 regulates food intake centrally Cone et al, suggests a direct effect through the vagu s nerve in the NTS in the brainstem. The sensory effect of the vagus nerve has been well supported by several groups but PYY336 injected to vagotomized mice still has an effect in food intake. This effect is actually longer than in controls. This rules out the exclusively effect of the vagus nerve. Therefore, I suggest that physiological doses of PYY3 36 have a direct effect in the NPY Y2 rec eptors in the nerve terminals of the glossopharingeal nerve (CNIX) in the tongue and of the vagus nerve (CNX) in the rest of the gastrointestinal tract. New theory for satiation : Appetite induces a food seeking behavior and when food is going to be cons umed, there is a vagal parasympathetic stimulation to our gastrointestinal system to prepare to receive the food. Once the food is consumed, there is gastric distention and pyloric

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119 relaxation that induce a vagal reflex that dilates the ileocecal junction. This dilation allows the bolus to pass to the colon. The distention of the colon releases PYY136. Active PYY336 acts locally over vagal nerve terminals. Also goes to saliva and acts over glossopharingeal nerve terminals. The vagal stimulation/inhibition produces a decrease in parasympathetic stimulation, decreasing GI motility and secretions, resulting in feelings of gastric distention (postprandial malaise). The inhibition of parasympathic stimulation triggers a sympathic stimulation related with post pr andial thermo genesis. In the glossopharingeal nerve, locally change taste perception producing a sensory -specific taste perception (taste aversion in mice at higher doses), resulting in a decrease of palatability. Both nerves merge in the NTS, and inhibit the Orexin and MCH fibers to the PVN and also inhibiting NPY neurons in the ARC, which stop sending GABA signals to the PVN. From the hypothalamus, fiber project into the amygdala, limbic system and higher areas of taste, satiety, memory and reward. GLP 1 can be added to this theory because GLP 1 is released in the same manner than PYY. Also, GLP 1 has been related with enhancing sweet taste perception. Maybe that is why after a high caloric meal, we still have some space for a desert rich in sugars and s weets.

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120 Figure 5 1 Diagram for alternative pathway of satiety induced by salivary PYY336 From the periphery PYY336 leaks to the saliva and stimulate Y2 receptors in the tongue. Through the CNIX, the NTS is stimulated in the brainstem and send proj ections to the hypothalamus. In the hypothalamus satiety is induced. An acute increase of salivary PYY336 produces an acute decrease in food intake, and a chronic increase of salivary PYY336 produces a chronic decrease in food intake and body weight. Thi s chronic increase activates reward centers and decrease aggression. Also increases the mRNA expression of DPP IV, decreasing the concentration of GLP 1; and increase the energy expenditure. Arrows in red are new findings (modified from Nature Reviews).

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121 Figure 5 2 Future directions for salivary PYY336

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131 BIOGRAPHICAL SKETCH Andres Jose Acosta Cardenas was born and raised in Quito, Ecuador. Since high s chool, he had a great interest for science and medicine. He started medical school at Pontificia Unive rsidad Catolica Del Ecuador. A few years into his career, he won the Pasteur Scholarship from Universidad San Francisco de Quito because of his research in Cytochrome P450 under Dr. Cesar Paz-y -Mino. Consequently, he transferred to Universidad San Francisc o de Quito, where he graduated with higher honors and valedictorian in May 2006. His medical t hesis Cloning Peptide YY and Oxyntomodulin transgenes was under the direction of Dr. Sergei Zolotukhin from the Division of Cellular and Molecular Therapy of the Department of Pediatrics at the University of Florida. Immediately after his medical graduation, he joine d Dr. Zolotukhins lab and the interdisciplinary Ph.D program of biomedical s ciences of the College of Medicine of t he University of Florida. Durin g his Ph.D. program he continued working with satiation gut hormones and discovered the presence and the role of these hormones in murine and human saliva. He received his Ph.D. from the University of Florida in the summer of 2009. While working in his Ph.D project, Andres validated his medical diploma from Ecuador acquiring the Educational Commission for Foreign Medical Graduates Certificate by approving the United States Medical boards. In the future, he wants to pursue a physician scientist career. The refore in July 2009, he is starting the Internal Medicine Residency Program at Shands Hospital at the University of Florida.