Dissertations / Theses on the topic 'Regulation of food intake'

Food intake is shaped by environmental, endocrine, metabolic, and reward-related signals. A change in appetite is an outcome of integration of the relevant external and internal stimuli. While the main purpose of eating is to reverse a negative energy balance, mechanisms protecting homeostasis change appetite for other reasons. This thesis examines the role of select brain mechanisms in regulating consumption driven by aspects other than energy. In paper I, an increased percentage of c-Fos positive OT neurons was observed after mice ingested sucrose, while no change was found after Intralipid intake. Given a choice between isocaloric sugar and Intralipid solutions, mice injected with an OT receptor antagonist increase their preference for sucrose, while total calorie intake remains unchanged, suggesting that OT prevents overconsumption of sugar. Paper II addresses whether MCH, which has anxiolytic properties and mediates reward-motivated feeding, has the ability to alleviate conditioned taste aversion in rats. We found that while mRNA expression of MCH and its receptor are changed in aversive animals, central injections of MCH do not prevent the acquisition of aversion, nor do they affect the rate of extinction of the taste aversion. Paper III describes evidence that the N/OFQ system facilitates food intake by alleviating aversive responsiveness. Blocking the NOP receptor delays extinction of aversion and reduces food intake in hungry rats. Paper IV reports that leucine ingestion increases mRNA expression levels of genes known to mediate reward, as well as orexigenic gene expression in the nucleus accumbens (Nacc), a key component of the reward circuit. Adding leucine to drinking water increases activity of the reward system, which possibly contributes to the pleasure of consumption. A separate approach using Drosophila melanogaster is introduced in paper V which provides evidence that knocking down the gene for the transcription factor Ets96B during development results in a simultaneous disruption in sleep patterns and appetite, thus highlighting the interplay between these physiological parameters. We conclude that OT, MCH, N/OFQ and Ets96B belong to mechanisms regulating food intake for reasons other than energy balance. Composition of food and negative associations with diets affect neural networks controlling appetite.

The work described in this thesis investigates the actions of hypothalamic neuropeptides involved in the control of energy homeostasis and reproduction. Cerebellin 1 (Cbln1) is a 16-amino acid peptide abundantly expressed in the cerebellum and the hypothalamus. I have shown that Cbln1 increases food intake when administered centrally to rats, and that this effect may be partly mediated by neuropeptide Y. I have also demonstrated that Cbln1 mRNA expression within the ventromedial nucleus of the hypothalamus is up-regulated following a 48-hour fast, suggesting that Cbln1 may have a physiological role in the control of food intake. In addition to hypothalamic centres, the reward system has an important role in the control of feeding. The melanocortin system has a well characterised role in the homeostatic control of food intake. My work suggests that the melanocortin system may also have a previously unknown role within the ventral tegmental area (VTA) in the hedonic control of feeding. Intra- VTA administration of a melanocortin agonist reduced, whereas an antagonist increased, food intake in rats. My work suggests that the VTA melanocortin system may act upstream of dopamine signalling to influence feeding. Kisspeptin is a hypothalamic neuropeptide with a crucial role in reproduction. The hypothalamus contains two populations of kisspeptin neurones, located in the arcuate nucleus (Arc) and in the anteroventral periventricular nucleus. The physiological role of these two populations is unknown. I used recombinant adeno-associated virus (rAAV) to suppress kisspeptin expression within the Arc in female rats. Animals injected bilaterally into the Arc with rAAV-kisspeptin antisense had significantly fewer oestrous cycles, and an increase in oestrous cycle length, compared to controls. In summary, these studies have identified novel roles for Cbln1 and the melanocortin system in the control of food intake, and have demonstrated the importance of Arc kisspeptin signalling in the regulation of reproduction.

Marijuana has been used for medicinal and recreational purposes for thousands of years. Many people think of marijuana in the context of an illegal drug. Because of the antimarijuana attitude, research with cannabinoids was neglected for a long time. Although this substance is related to social problems, scientists are interested in its action and possible medicinal properties. Since the identification of the structure of Î 9--tetrahydrocannabinol, the main psychoactive ingredient of marijuana, there has been increased interest in this compound. Following the discovery of two cannabinoid receptors, CB1 and CB2 receptors, it was determined that CB1 receptors are in high density in the central nervous system while CB2 receptors are found primarily in the immune system. The endogenous cannabinoid ligands, anandamide and 2-arachidonoylglycerol, were observed in the central nervous system and peripheral tissues. Endocannabinoids differ from other "classical" neurotransmitters because they do not appear to be stored in synaptic vesicles, and they act as retrograde messengers within the brain. The endogenous cannabinoid signaling system includes cannabinoid receptors, their endogenous ligands called endocannabinoids, and the proteins for their synthesis and inactivation. The cannabinoid system appears to act as a neuromodulatory system. During the past ten years, the endogenous cannabinoid system has been implicated in a variety of physiological functions including pain reduction, motor regulation, learning, memory, and reward. Because obesity and eating disorders are prevalent, scientists are working at the molecular level to study the mechanisms controlling body weight and regulation of food intake. Several of the neuropeptides present in hypothalamic nuclei contribute to energy balance and food intake regulation. Endogenous cannabinoid and cannobinoid receptors are found in the hypothalamus and are associated with the regulation of food intake. Although the mechanisms whereby cannabinoids influence food intake remain unclear, results suggest that the cannabinoid system will be an important target in future studies in obesity. Most research on cannabinoids has focused on their role in food intake regulation in mammalian species. It is important to determine the role of endocannabinoids in other species. The effect of intracerebroventricular injection of agonists and antagonists of both CB1 and CB2 receptors in 8 to 11 week-old male Single Comb White Leghorn and 3 to 6 weeks old male broilers was investigated. It was found that agonists of both the CB1 and CB2 receptor increased food intake significantly; however, the CB2 receptor agonist had a stronger and longer lasting effect. Antagonists of both receptors decreased food intake significantly. The CB1 receptor antagonist appeared to block both cannabinoid receptors in birds, whereas the CB2 receptor antagonist did not block both receptors. Previous studies have indicated that the CB2 receptor is found only outside the brain and spinal cord, and is involved with the immune system. From the present results, it appears that both cannabinoid receptors are present in the chicken brain. Furthermore, the CB2 receptor may also be localize in the chicken brain. There are also differences in cannabinoid system between Leghorn and broilers.
Master of Science

Learning processes play a major role in controlling intake of food. Through repeated experiences an animal acquires the ability to predict the postingestive effects of a particular food (i.e., of its nutrients and energy) from its sensory characteristics. What is unclear from the literature, however, is whether an animal can anticipate the duration of subsequent food deprivation from predictive sensory qualities of a food, and hence increase the amount eaten of that cueing food. Therefore, the aim of this work was to investigate the characteristics of this under-researched type of learning, i.e., anticipatory eating, using laboratory rats trained on two lengths of fasting (short: 2-3 h, long: 8-10 h). The main findings were as follows. 1) Anticipatory eating is learnt when a choice is given between protein- and carbohydrate-rich foods as well as on a single balanced test food. 2) The learnt extra intake of food is instrumental to preventing the return of hunger, removal of which negative reinforcement extinguishes the response. 3) The resulting return of hunger induces re-learning of anticipatory eating. 4) During the training sessions, learning of anticipatory eating competes with classical conditioning of sensory preference. Conditioning of preference is likely to be stronger with the shorter than with the longer length of fasting. Therefore, the difference between intakes before the long and the short fast at each trial is the summed result of these two mechanisms of acquired increase in intake. While preference conditioning usually reaches a maximum rapidly, depletion-avoidance increases for as long as has been tested, with interruptions of rapid self-extinction and re-learning, This self-extinction contributes to the homeostatic character of this learning. 5) High-fat maintenance diet attenuates the learning of anticipatory eating. Overall, the findings provided robust evidence that eating in rats can be controlled by instrumental learning reinforced by hunger. Accordingly, the design of an experiment on such instrumental control of eating in human subjects is proposed to conclude this thesis.

Energy homeostasis is essential for survival across all vertebrate species and involves a multitude of physiological systems that are regulated by both central and peripheral neural signaling. The hypothalamus is responsible for integrating and processing these signals and thus is regarded as the regulatory center for balancing energy homeostasis. Eating disorders, such as compulsive eating behavior associated with obesity, and anorexia, are significant public health concerns worldwide. Thus, studying appetite regulation is necessary to provide novel information for the design of solutions for health concerns that stem from altered energy intake. Such information is also relevant for improving chicken health and productivity in an agricultural setting. The objective of this dissertation research was to determine the hypothalamic mechanisms underlying appetite regulation in birds. In Experiment 1, the Virginia lines of chickens were used to elucidate the mechanisms underlying stress-induced anorexia. These chickens have been selected for low (LWS) or high (HWS) body weight at 56 days of age and have different severities of anorexia and obesity, respectively. Chicks were subjected to a combination of thermal and nutritional stress after hatch and hypothalamic nuclei, including the lateral hypothalamus (LH), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and arcuate nucleus (ARC), were collected 5 days later. Real-time PCR was used to measure the mRNA abundance of appetite-associated neuropeptides and receptors in each nucleus. The results showed that the two lines displayed distinct gene expression profiles in response to stress. In particular, the PVN of the LWS was significantly affected by stress, and expression of several anorexigenic factors was up-regulated including corticotropin-releasing factor (CRF), CRF receptor sub-types 1 and 2 (CRFR1 and CRFR2, respectively), melanocortin receptor 4, and urocortin 3, suggesting that stress-induced anorexia in the LWS may result from overriding anorexigenic signaling in the PVN, primarily through CRF signaling. This CRF signaling-associated hypothesis was further supported by results showing that the original phenotypes were restored when the LWS chicks were treated with astressin (CRF receptor antagonist) before exposure to stress. In Experiments 2 and 3, we attempted to determine the mechanisms of CRF's anorexigenic effect in chickens and Japanese quail. We administered CRF by intracerebroventricular (ICV) injection and the hypothalamus was collected 1 hour later for molecular analyses. Results showed that CRF exerted a similar inhibitory effect on food intake in these two bird species, however the hypothalamic mechanisms underlying this anorexigenic effect were different. ICV injection of CRF increased c-Fos expression in the PVN, VMH, dorsomedial nucleus (DMN), and ARC in chicks while it only affected the PVN and LH in quail. Hypothalamic gene expression results suggested that CRF decreased neuropeptide Y receptor sub-type 1 (NPYR1) in chicks while it increased proopiomelanocortin (POMC), MC4R, CRF, and CRFR2 in quail. These results suggested that the anorexigenic effect of CRF may involve a dampened neuropeptide Y (NPY) system in chicks whereas it is associated with activated CRF and melanocortin systems in quail. At the nucleus level in chicks, CRF injection decreased NPY system-associated gene expression (ARC and DMN) and increased CRF (ARC and PVN) and mesotocin (MT) (VMH)-associated mRNAs, suggesting that orexigenic signaling through NPY was overridden by the heightened anorexigenic tone through CRF and MT, which led to the inhibition of food intake. In Experiments 4 and 5, we used the same experimental design as for CRF studies to determine the hypothalamic mechanisms of the anorexigenic effects of neuropeptide K (NPK) and adrenomedullin (AM) in Japanese quail. Results from Experiment 4 showed that NPK injection activated the ARC and PVN, which was associated with increased mRNAs for a group of anorexigenic factors including CRF, UCN3, cocaine and amphetamine-regulated transcript (CART), and POMC, and decreased expression of several orexigenic factors, such as NPY and agouti-related peptide (AgRP). In Experiment 5, ICV injection of AM activated the ARC, the nucleus in which POMC and CART mRNAs were increased. In conclusion, these experiments revealed novel hypothalamic mechanisms underlying stress or exogenous neuropeptide-induced anorexia in birds and may provide insights on understanding appetite regulation from evolutionary, agricultural, and biomedical perspectives.
Ph. D.
Appetite regulation is important for survival across all vertebrate species and the hypothalamus is the regulatory center for control of feeding behavior. Thus, studying the functions of the hypothalamus on appetite regulation provide novel insight into the eating disorders, such as obesity and anorexia, a worldwide health issue. Also, such information is relevant for improving productivity in the modern chicken industry. The objective of this dissertation research was to determine the hypothalamic mechanisms underlying appetite regulation in birds. In Experiment 1, the Virginia lines of chickens were used to elucidate the mechanisms underlying stress-induced anorexia. These chickens have been selected for low (LWS) or high (HWS) body weight at 56 days of age and have different severities of anorexia and obesity, respectively. Chicks were subjected to a combination of thermal and nutritional stress after hatch. The results suggested the two lines displayed distinct appetite-associated gene expression profiles in response to stress in the hypothalamus. In particular, stress-induced anorexia in the LWS may result from potent feeding-inhibitory factor corticotropin-releasing factor (CRF). Thus, in Experiments 2 and 3, we attempted to determine the mechanisms of CRF's inhibitory effect on food intake in chickens and Japanese quail. We administered CRF by intracerebroventricular (ICV) injection and the hypothalamus was collected 1 hour later for molecular analyses. Results showed that CRF exerted a similar inhibitory effect on food intake in these two bird species. However, the inhibitory effect of CRF was primarily associated with a dampened neuropeptide Y (NPY) system which is a potent stimulatory factor for feeding behavior in chickens, whereas it may involve activated CRF and melanocortin systems in quail. In Experiments 4 and 5, we used the same experimental design as for CRF studies to determine the hypothalamic mechanisms of the inhibitory effects of neuropeptide K (NPK) and adrenomedullin (AM) in Japanese quail. Results from Experiment 4 showed that the feeding-inhibitory effect of NPK was associated with a group of increased feeding-inhibitory factors such as CRF and cocaine and amphetamine-regulated transcript (CART) and decreased feeding-stimulatory factors, such as NPY and agouti-related peptide (AgRP) in the hypothalamus. In Experiment 5, AM increased gene expression of CART and proopiomelanocortin (POMC). Overall, these experiments suggested the roles of the hypothalamus in stress or exogenous neuropeptide-induced anorexia in birds and may provide insights on understanding appetite regulation from evolutionary, agricultural, and biomedical perspectives.

Four studies were performed to examine peripheral factors involved with food intake regulation in the domestic fowl. In the first study, the mechanism by which tryptophan depresses food intake was clarified. Intraperitoneal injections of tryptophan methyl ester were demonstrated to inhibit feeding in Single Comb White Leghorn (SCWL) cockerels. Intragastric intubations of tryptophan inhibited food intake and decreased body temperature of SCWL cockerels. These results, in conjunction with previous findings, indicate that tryptophan's inhibitory influence on food intake is peripherally rather than centrally based. The second study explored the role of the duodenum in food intake regulation. Intraduodenal glucose loads had no effect on food intake of SCWL or Rock Cornish (RC) commercial broiler cockerels. In addition, splanchnicectomized birds did not respond to intraduodenal glucose infusions any differently than sham-operated controls. Apparently, the duodenum does not play a significant role in food intake control in the fowl. Hepatic involvement in appetite regulation was examined in SCWL and RC cockerels in the third study. Amino acid solutions failed to influence food intake when infused intraportally in either strain of chicken. Relatively small glucose or lipid solutions depressed food intake significantly when infused intraportally in the SCWL birds but had no effect in the RC cockerels. The liver appears to be integrally involved in controlling food consumption in the SCWL chicken. In the final study, the existence of a "hunger" factor in the peripheral circulation of two lines of chickens divergently selected for body weight was explored. Intrahepatic infusions of plasma from food deprived high-weight line chickens stimulated food intake of sated low-weight line chickens. These studies indicate that peripheral mechanisms are important in regulating appetite in light-breed chickens such as the SCWL, however, such mechanisms in heavy-breed chickens such as the RC appear to be less sensitive. This desensitization in heavy-breed chickens suggests that genetic selection for increased growth has affected the food intake control systems.
Ph. D.

Enteropancreatic hormones such as pancreatic polypeptide (PP), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) are secreted post-prandially by the gut and the pancreas. They act to regulate metabolism and appetite. An understanding of the physiology of these hormones and how they can be delivered in a practical manner is required to allow their translation into clinical treatments for obesity and diabetes. Work in this thesis investigated the effect of subcutaneously injected PP in healthy human volunteers, and demonstrated a significant reduction in food intake. A novel peptidase resistant analogue of PP, PP 1420, was administered in combination with metformin to rodents. This combination reduced food intake and body weight additively, suggesting that the combination of PP 1420 and metformin may well be beneficial in patients with obesity and diabetes. A subsequent study of PP 1420 in human volunteers, in a first-in-man Phase 1 trial, confirmed that PP 1420 was safe, well tolerated and possessed an extended terminal elimination half-life compared to native PP. In this thesis, I also explored the physiological effects of gut hormone combinations. The administration of single gut hormones such as PP, PYY or GLP-1 can reduce food intake, but may cause side effects such as nausea. The combination of gut hormones offers the possibility of increased efficacy with fewer side effects, for example, PYY+GLP-1 in combination have previously been shown to possess additive effects on food intake. The effects of a PYY+GLP-1 combination on carbohydrate metabolism have not yet been investigated. Work in this thesis examined the effects of a PYY+GLP-1 combination intravenous infusion on insulin secretion and sensitivity in healthy volunteers. Administration of PYY alone did not significantly affect insulin secretion. PYY+GLP-1 in combination stimulated insulin secretion to a similar extent to GLP-1 alone. There were no significant acute effects of PYY, GLP-1 or PYY+GLP-1 on insulin sensitivity. These findings suggest that gut hormone analogues may represent safe, effective and practical treatments for obesity. Combination PYY+GLP-1 treatment may provide the metabolic benefits of bariatric surgery without the surgery itself.

Chickens from lines that have been divergently selected for either low (LWS) or high (HWS) body weight at 56 days of age for more than 57 generations serve as unique models to study eating disorders. The LWS have different severities of anorexia while all HWS become obese. Over the past decade our groups has demonstrated that these lines have differential food intake threshold responses to a range of intracerebroventricular (ICV) injected neurotransmitters. The major brain region regulating homeostatic regulation of appetite is the hypothalamus, and hence this dissertation was focused on understanding how the hypothalamus is different between LWS and HWS lines. Experiments 1 and 2 were performed as follows: whole hypothalamus as well as individual hypothalamic nuclei, respectively, were collected from 5 day-old chicks that had been fasted for 180 min or had free access to food. The hypothalamic nuclei included those primarily associated with appetite including the lateral hypothalamus, paraventricular nucleus (PVN), ventromedial hypothalamus, dorsomedial nucleus, and arcuate nucleus (ARC). Total RNA was isolated, reverse transcribed, and real time PCR performed. Hypothalamic expression of anorexigenic factors was greater in LWS than HWS, those factors including calcitonin, corticotropin-releasing factor receptor 1, leptin receptor, neuropeptide S, melanocortin receptor 3 (MC3R), and mesotocin. The gene expression data from individual hypothalamic nuclei revealed that mesotocin from the PVN may play an important role in the inhibition of appetite in the LWS. Experiment 3 was then designed to evaluate the effects of stress on food intake: besides the differences in hypothalamic gene expression between the lines, they also have different feeding responses when stressed: ICV injection of neuropeptide Y (0.2 nmol, NPY) did not increase food intake in LWS on day 5 after stress exposure. Experiment 4 was thus designed to study the molecular mechanisms underlying conditional feeding responses to exogenous NPY after stress in the LWS. The melanocortin system (AgRP and MC3R) changed in the hypothalamus after stress in the LWS, and hence may be responsible for the loss of responsiveness to exogenous NPY in stressed LWS. Experiment 5 was designed to evaluate whether hypothalamic differences exist at the protein level: label-free liquid chromatography coupled to tandem-mass spectrometry was used to measure the abundance of proteins in the hypothalamus. Hypothalamus was obtained from fed and 180 minute-fasted 5 day-old male LWS and HWS chicks. Proteins involved in energy metabolism were different between the lines. Differences were also found in proteins involved in GABA synthesis and uptake as well as protein ubiquitination. In conclusion, these results suggest that different feeding behaviors of LWS and HWS may be due to differences in gene and protein expression in the hypothalamus.
Ph. D.

This study was initiated to determine if factors exist in the blood or cerebrospinal fluid (CSF) of the domestic fowl that act upon the central nervous system to control food intake. Plasma collected from free-feeding and 24-hour fasted leghorn cockerels was lyophilized, reconstituted to 2, 4, or 5 times the original concentration, and injected, via a stereotaxically implanted 23 gauge stainless steel guide cannula, into the lateral ventricle of free-feeding leghorn cockerels. Food intake was significantly reduced following injection of 2, 4, and 5 times normal concentration of plasma from free-feeding birds. Plasma from fasted birds did not alter food intake regardless of concentration, but did significantly reduce water intake when concentrated to five times normal. A similar study was conducted with fractions of plasma of different molecular weight ranges. Plasma collected from free-feeding cockerels was partitioned by gel filtration into the following molecular weight fractions: >5000 molecular weight, <5000 molecular weight, 1500-5000 molecular weight, and <1500 molecular weight. The fractions were lyophilized and reconstituted to four times the original concentration and injected into the lateral ventricle of free-feeding leghorn cockerels. Food intake was significantly decreased by the <5000 and <1500 molecular weight fractions, whereas water intake was not affected. The 1500-5000 molecular weight fraction and the fraction above 5000 did not affect food or water intake. To determine if this food intake inhibiting factor existed in the cerebrospinal fluid (CSF) of the domestic fowl, CSF was collected from free-feeding and 24-hour fasted broilers and injected into the lateral ventricle of leghorn and broiler cockerels. Food intake was not affected by either the normal or four-times normal concentration of CSF collected from free-feeding or 24-hour fasted broilers. Water intake was significantly increased in the leghorn and broiler birds receiving the four times normal concentration of CSF collected from 24-hour fasted birds, but was not affected in the birds receiving CSF collected from the free-feeding donors. It appears, therefore, that a food intake inhibiting factor exists in the plasma of the free-feeding domestic fowl that does not exist in the CSF.
Ph. D.

While obesity is a world leading health problem, the most efficient treatment option for severely obese patients is Roux-Y gastric bypass (RYGB) surgery. However, there are large inter-individual differences in weight loss after RYGB surgery. The reasons for this are not yet elucidated and the role of genetics in weight loss-regulation is still not fully understood. The main aim for this thesis was to investigate the effects of common obesity-associated genetic variants and their effect on weight loss and food intake. We examined if the weight loss two years following RYGB surgery depends on the  FTO genotype, as well as pre-surgery vitamin D status. For FTO AA-carriers, the surgery resulted in a 3% per-allele increased excess BMI loss (EBMIL; P=0.02). When split by vitamin D baseline status, the EBMIL of vitamin D deficient patients carrying AA exceeded that of vitamin D deficient patients carrying TT by 14% (P=0.03). No such genotypic differences were found in patients without pre-surgery vitamin D deficiency. As the influence of individual single nucleotide polymorphisms may be small, we identified a novel method to combine SNPs into a genetic risk score (GRS). Using the random forest model, SNPs with high impact on weight loss after RYGB surgery were filtered out. An up to 11% lower EBMIL with higher risk score was estimated for the GRS model (p=0.026) composed of seven BMI-associated SNPs (closest genes: MC4R, TMEM160, PTBP2, NUDT3, TFAP2B, ZNF608 and MAP2K5). Pre-surgical hunger feelings were found to be associated with EBMIL and the SNP rs4846567. Before surgery, patients filled out the Three Factor Eating Questionnaire and were genotyped for known BMI and waist-hip ratio (WHR) associated SNPs. Patients with the lowest hunger scores had up to 32% greater EBMIL compared to the highest scoring patients (P=0.002). TT-allele carriers of rs4846567 showed a 58% lower hunger feelings. TT- carriers also showed a 51% decrease in disinhibition, but no significant impact on cognitive restraint was observed. Due to the association of eating behaviour and weight loss, acute effects on DNA methylation in response to a food intake intervention of a standardized meal were also investigated. After food intake, 1832 CpG sites were differentially methylated compared to the baseline after multiple testing correction. When adjusted for white blood cell fractions, 541 CpG sites remained. This may be interpreted as that the immune system is playing an active role in the response to food intake and highlights the dynamic nature of DNA-methylation. These findings will contribute to a better care for morbidly obese patients. Post-surgical treatment may be optimized so that patients with a less favourable genetic profile may receive additional support for weight loss and weight management. This may be considered as a step in the transition towards personalized medicine.

It is well established that ingested protein has a greater satiating effect than other macronutrients. The mechanisms behind this effect are unknown, although it is believed that protein induces a greater increase in levels of anorectic gut hormones than carbohydrate or fat. Recent identification of a family of promiscuous L-amino acid receptors has provided a potential mechanism for the effects of protein on appetite. These amino acid receptors - the G-protein coupled receptor family C group 6 member A (GPRC6A), the Taste receptor type 1 member 1/Taste receptor type 1 member 3 (T1R1/T1R3) dimer and the calcium-sensing receptor (CaR) - are non-specific in their ligand binding but show preference for different families of amino acids. I assessed the effects of peripheral administration of a wide range of amino acids on food intake in rodents. My results demonstrate that the specific amino acids L-arginine, L-cysteine and L-lysine acutely reduce food intake following peripheral administration. The 0-1 hour food intake following both intraperitoneal (i.p.) and oral administration of amino acids in rats negatively correlated with the stimulatory efficacy of amino acids at the T1R1/T1R3. I subsequently investigated the mechanisms by which L-arginine, L-cysteine and L-lysine inhibit food intake. Oral administration, of L-arginine and L-lysine tended to increase levels of the anorectic hormones glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Intraperitoneal administration of L-cysteine reduced levels of circulating total and acyl-ghrelin. Conditioned taste aversion studies suggested that the anorectic effect of these amino acids is not secondary to visceral illness. Chronic administration of L-cysteine significantly reduces cumulative food intake. My studies suggest that specific amino acids can influence food intake, perhaps by altering circulating levels of gastrointestinal hormones. Altering dietary amino acid content may be helpful to prevent or treat obesity.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第17623号
農博第1985号
新制||農||1010(附属図書館)
学位論文||H25||N4744(農学部図書室)
30389
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 金本 龍平, 教授 河田 照雄, 教授 谷 史人
学位規則第4条第1項該当

The regulation of food-intake in Limanda limanda was investigated, including (a) the role of the stomach as a limiting factor in fish food intake, (b) the qualities of a diet dab respond to and (c) the dynamics of food intake. Fish fed on squid (4.0 U. g'', 76.2% moisture) ate similar daily rations whether fed three times daily or once per day; when fed every three days they were unable to maintain this intake. Fish fed on pellets (18.8 kJ. g'', 8% moisture) could maintain their average daily food intake for all meal intervals tested. After a satiation meal, food intake broadly increased with deprivation time for at least 96 hours with no clear indication that stomach volume was limiting. However, when the data was re-examined using a return map (where meal m is plotted against meal m-1) there was evidence that feeding was restricted by stomach fullness as the interval between meals exceeded 25 hours. Surprisingly such limitation did not occur at higher feeding frequencies. Three models of food intake were used to simulate food-intake data, in which the role of the stomach as a constraining factor was varied: 1) Food intake was assumed always to be completely limited by stomach volume. 2) Food-intake was assumed always to be driven by a systemic need 3) Food intake was assumed to be chiefly limited by a systemic need, but when this was high, stomach volume would constrain intake. Comparisons of experimental results with these models suggest that when fish are fed frequently, or on a high-energy diet, the stomach volume is probably not limiting, whereas for a low-energy diet, fed infrequently, stomach volume was limiting when systemic need was high. Dab adapted their food intake to diets of different water content (and therefore energy density). They also adapted the distensibility of their stomachs in response to the increased volume eaten; fish fed on pellets having less distensible stomachs than those fed on squid. Thus it is unlikely that stomach volume can limit food intake in the long term, unless food quality and/or meal timing is variable and the fish cannot adapt their stomachs to the diet/feeding frequency. Methodological trials proved that that observed stomach VI volume is a function of the measurement technique, as well as the diet history of the fish, and experiments examining stomach volume should take this into consideration. The question of what aspect of a diet L. limanda adapt to was examined by testing different models using path analysis, a method of inferential modelling of causal relationships, in an attempt to explain how food-intake is regulated. Dab were found to be adapting to both the energy content of the diet and to the individual nutrients. The dynamics driving food intake in groups of dab were investigated using non-linear time series analyses. These proved to be low-dimensional, significantly non-linear, deterministic systems. The data also suggests that such systems are either capable of occasional chaotic behaviour, or are on the edge of chaos i. e. complex dynamical systems. Thus food intake is under the direct control of few (two or three) variables, through which the many known factors that influence food intake must act. Comparisons were made with individuals and groups of Oncorhynchus mykiss, with a brief look at Merlangius merlangus and Dicentrarchus labrax. These results were similar to the dab, and so this dynamical behaviour may be a feature of teleost fish in general. Importantly the fact that individual trout had similar feeding behaviour to groups indicate that the findings were not a function of hierarchical dynamics. The significance of this finding should be that appetite control with these properties allows rapid adjustment of fish according to changes in diet quality.

The present set of experiments were designed to examine the role of histamine, and its interaction with corticotropin releasing factor (CRF) and bombesin (BM) in food intake regulation of chickens. The hypothesis being tested was as follows: One component of the neuroregulation of food intake involves histaminergic activity in the hypothalamus, acting on either H1 or H2 receptors, how these receptors interact with CRF neurons and if BM elicits its effects on feeding through CRF release. Single Comb White Leghorn (SCWL) and broiler cockerels were utilized for these experiments. Birds were stereotaxically implanted with a 23-gauge thin-walled stainless steel guide cannula, and were provided a mash diet and water for ad libitum consumption. All compounds were infused into the right lateral ventricle. Effects were monitored at 15-minute intervals through three hours postinjection.

Experiment 1 examined the effects of intracereboventricular (ICV) injections of histamine (HA) and two HA antagonists, the H1 receptor antagonist chloropheneramine maleate (CM) and H2 receptor antagonist cimetidine (CIM), on food and water consumption and body temperature. Histamine was infused using 0, 25, 50, and 100 µg per 10 µl of artificial cerebrospinal fluid (aCSF). Histamine significantly decreased food and water consumption (P< 0.05) over the three hour observation period in a dose-dependent manner. Histamine was then infused to observe if the decrease in water intake was dependent upon the decrease in food intake. Birds were not allowed access to feed during this experiment. Water intake was not affected by HA in either SCWL or broilers when food was not available. To observe the effects of HA on thermoregulation, HA was infused using the same dosages and body temperature recorded for three hours. Histamine produced hypothermia at a dose of 25 µg in SCWL cockerels, with a quadratic trend at 165 and 180 min. Broiler cockerels did not show hypothermia, but rather a constant hyperthermia compared to the control with a quadratic trend throughout the latter part of the experiment. The last phase of the first set of experiments, birds were pretreated with either CM or CIM (100 µg/10 µl aCSF) followed by HA. When the birds were pretreated with either CM or CIM, the hypophagic responses to HA were attenuated. The pair of experiments that utilized H1 and H2 receptors demonstrated that these receptors are involved in the neural regulation of food intake. These experiments also demonstrated that the aphagic effects of HA on food intake can be blocked with the pretreatment of antihistaminics affecting both H1 and H2 receptors.

In Experiment 2, studies were conducted to determine if neuronal CRF elicited its effects on feeding through the release of HA. Birds were infused with 0 or 20 µg CRF and either 0 or 100 µg of CM or CIM. CRF decreased food and water intake in both SCWL and broiler cockerels. When birds were pretreated with CM, the hypophagic responses to CRF were attenuated. When birds were pretreated with CIM, the hypophagic responses of CRF were attenuated in broiler cockerels; this response was not seen in SCWL cockerels. Water intake followed a similar pattern. It was concluded that, contrary to studies showing that HA causes the release of CRF in other species, CRF may cause the release of HA in chickens.

Experiment 3 was designed to investigate whether bombesin (BM) elicited its effects on feeding through the release of CRF. Birds were infused with either, 0 or 0.5 µg BM, 0 or 5 µg aCRF (9-41) (CRF antagonist), or a combination of both. These compounds were infused to test whether the effects of BM could be blocked with the pretreatment of anticorticotropics. Food and water consumption were significantly decreased (P< 0.05) with the infusion of BM in both SCWL and broiler cockerels. Food intake was not affected with the infusion of aCRF in SCWL or broilers cockerels. However, water consumption was increased when birds were given ICV injections of aCRF. When birds were pretreated with aCRF, the anorexigenic and adipsic effects of BM were attenuated. It was concluded that BM elicits its effects on feeding through the release of CRF. These results also demonstrate that the aphagic effects of BM could be blocked with the pretreatment of anticorticotropics.
Master of Science

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

L’appetito è un comportamento motivato essenziale alla sopravvivenza, crescita e riproduzione di ciascun organismo. Il controllo fisiologico dell’appetito e del senso di sazietà negli animali è regolato da un complesso sistema di segnali centrali e periferici che coinvolgono una serie di neurotrasmettitori e neuropeptidi che interagendo reciprocamente stimolano o inibiscono il comportamento alimentare. I meccanismi che stanno alla base di tale comportamento consentono di mantenere costante l’energia metabolica attraverso un circuito finemente regolato che si instaura tra sistema centrale, in particolare i nuclei arcuati dell’ipotalamo e organi periferici (tratto gastro-intestinale, tessuto adiposo, fegato..). Inoltre, considerando che il mantenimento dell’energy balance si ottiene dall’equilibrio tra energia acquisita (food intake) e consumata, risulta chiaro che la riproduzione, un processo ad elevato consumo energetico, riveste un ruolo chiave nei meccanismi fisiologici che regolano il bilancio dell’energia. E’ impossibile infatti, considerare tali meccanismi svincolati l’uno dall’altro. Tra i fattori esterni in grado di regolare l’energy balance, la luce è sicuramente il principale sincronizzatore del comportamento animale e la melatonina è il mediatore ormonale dei ritmi circadiani/circannuali secondo cui gli animali espletano le loro funzioni. Fattori invece che possono interagire negativamente su tale delicato equilibrio sono i distruttori endocrini, sostanze estrogeno-simili in grado di mimare l’azione di ormoni endogeni. Recenti ricerche affermano che l’uomo è sempre più esposto a tali inquinanti che si trovano ampiamente diffusi in molti manufatti industriali con cui veniamo a contatto giornalmente al punto che la comunità scientifica è sempre più convinta che disordini e malattie di natura endocrinologica siano proprio da imputare a tali esposizioni. La maggior parte delle conoscenze disponibili, com’è ovvio, proviene da studi su mammifero tuttavia negli ultimi anni un crescente numero di ricerche in tal senso si sta incentrando sui bassi vertebrati, in particolare sui pesci. Questo perché modelli sperimentali, come lo zebrafish Danio rerio, consentono di studiare su scala ridotta meccanismi intricati e complessi come quelli che regolano food intake ed energy balance, inoltre considerando la crescente importanza del settore dell’ acquacoltura, molti di tali studi sono proprio finalizzati ad aumentare la produttività e qualità degli allevamenti ittici. Questa tesi si è proposta di investigare i meccanismi che regolano il food intake e il metabolismo energetico nei pesci, in particolare, utilizzando due diverse specie (Danio rerio e Sparus aurata) tale studio ha lo scopo di comprendere di circuiti neuro anatomici coinvolti nel controllo dell’appetito e di mettere in luce possibili relazioni che intercorrono tra tali meccanismi e fattori esterni quali fotoperiodo e distruttori endocrini. Nella prima parte della tesi, utilizzando zebrafish come modello sperimentale, si è valutato, tramite somministrazione in vasca per 10gg, il ruolo della melatonina (dosi 100nM e 1µM) nella regolazione dell’appetito e del metabolismo a livello centrale (cervello) e periferico (fegato, tratto gastrointestinale, gonade e muscolo). Sono stati analizzati, per la prima volta tutti insieme, i principali segnali coinvolti nella regolazione dell’appetito (NPY, LPT, CB1, MC4R, Ghrelin) della crescita (IGF-1), del metabolismo lipidico (PPARs) e della riproduzione (LPT, Ghrelin, Cox2a) tramite PCR Real Time. In Western Blot è stata analizzata la proteina CB1, tramite FTIR è stata valutata la ripartizione delle risorse metaboliche (lipidi carboidrati e proteine), per quanto riguarda la gonade è stato calcolato il numero di uova ovulate per ciascun individuo. I risultati ottenuti hanno evidenziato un chiaro ruolo della melatonina nei pathways che regolano appetito, riproduzione e metabolismo. In particolare l’ormone, a livello fisiologico ha diminuito l’appetito, a livello molecolare ha stimolato positivamente i segnali anoressigenici mentre inibiti gli oressigenici nel cervello. Nel fegato e nell’intestino sono stati inibiti i segnali che stimolano l’appetito, il metabolismo lipidico e la crescita. Nel muscolo si è osservato invece una diminuzione della componente lipidica. A livello della gonade, fisiologicamente, è stato evidenziato un significativo aumento delle uova ovulate, dato confermato dall’aumento di espressione genica di Cox2a e LPT. Tali dati, nel loro insieme, dimostrano il ruolo chiave rivestito dalla melatonina nella regolazione dell’energy balance, andando ad interagire con tutte le molecole analizzate. In particolare tale ormone sembra regolare consumo e acquisizione di energia sulla base delle risorse metaboliche a disposizione, promuovendo quindi la riproduzione e inibendo l’alimentazione. Volendo, poi, analizzare pathways del food inatke anche in modello sperimentale marino siamo passati all’orata. Nonostante la grande importanza che questa specie riveste in acquacoltura, nessuno studio sui meccanismi che regolano l’appetito era stato mai effettuato prima. In questa specie è stato scelto di analizzare il ruolo del sistema endocannabinoide nella regolazione dell’appetito, a livello centrale (cervello) e periferico (fegato) ed eventuali relazioni con il neuropeptide oressigenico per eccellenza: NPY. Giovanili di orata sono state sottoposti, via acqua, a tre concentrazioni di anandamide (AEA, cannabinoide endogeno) (0,1; 1 e 10µM) per tre tempi differenti (30,60,120’). In seguito è stato utilizzato AM251 (antagonista dell’endocannabinoide) (0,1 e 1 µM) somministrato 30’ prima dell’AEA per valutare l’effetto dell’AEA (diretto o meno). Tramite Real time PCR sono stati analizzati CB1 ed NPY nel cervello, mentre tramite Western Blot CB1 è stato analizzato in cervello e fegato. I risultati acquisiti hanno evidenziato come il sistema endocannabinoide rivesta un ruolo chiave nella regolazione dell’appetito. A livello fisiologico, infatti, il food intake ha subito un forte incremento, che è stato confermato a livello molecolare sia nel cervello che nel fegato (aumento dell’espressione genica di Cb1 ed NPY). Inoltre l’effetto dell’AEA è stato specifico e diretto, poiché la somministrazione dell’antagonista ha riportato tutti valori analizzati pari al controllo, annullando l’effetto dell’AEA. Dai dati ottenuti è stata dimostrata, per la prima in orata, volta la forte connessione CB1-NPY nella regolazione dell’appetito. Nell’ultima parte della tesi, è stato valutato l’effetto che distruttori endocrini provocano al sistema che regola appetito e bilancio energetico. Adulti di zebrafish sono stati sottoposti a 0,02; 0,2 e 2 mg/L di dietylexilphthalate (DEHP) e per verificarne gli effetti estrogenici è stato somministrato etinil estradiolo. Tramite Real time PCR sono stati analizzati segnali coinvolti nella regolazione dell’appetito (ORX, LPT, CB1) nel cervello, mentre nel fegato sono stati valutati segnali chiave del metabolismo lipidico (CB1, SREBP e PPARα). I risultati ottenuti hanno dimostrato come questo tipo di composti siano in grado di modificare profondamente e a tutti i livelli gli assi che controllano appetito e metabolismo lipidico. Fisiologicamente si osserva una diminuzione del food intake, confermata, a livello centrale, dalla riduzione dell’espressione genica degli appetito stimolanti e up-regolazione di LPT. Nel fegato invece si osserva una stimolazione dei geni coinvolti nel metabolismo lipidico. Gli effetti più forti di tali modulazioni si sono osservati alle dosi più basse di DEHP, tale dato pone l’accento sull’importanza delle dosi basse nella contaminazione ambientale. Nella loro totalità, i risultai ottenuti in questa tesi di dottorato forniscono un’importante contributo alla letteratura scientifica nella comprensione del controllo dell’appetito e, più in generale, ne evidenziano le relazioni con l’ambiente esterno, ponendo l’accento sui rapporti tra bilancio energetico e metabolismo. Inoltre, i dati ottenuti su orata, sebbene preliminari, chiariscono ed ampliano la conoscenza dei meccanismi alla base del food intake in una delle specie più importanti per l’acquacultura europea.
Feeding is a motivated behavior essential to survival, growth and reproduction of each organism. The physiological control of appetite and satiety in animals is regulated by a complex system of central and peripheral signals, involving a balance of neurotransmitters and neuropeptides that interact reciprocally to stimulate or inhibit feeding behavior. Mechanisms regulating feeding processes allow to the maintenance of energy metabolism, reached through a meticulously interconnected circuitry between central nervous system, particularly hypothalamic arcuate nuclei, and peripheral organs (gastro-intestinal tract, adipose tissue, liver). Furthermore, the physiological mechanisms that control energy balance are reciprocally linked to those that control reproduction, it is difficult, in fact, to understand the physiology of energy balance without understanding its link to reproductive success. In addition to the various controls mentioned above, the mechanisms that tend to maintain energy intake (food intake) and energy expenditure in balance is highly dependent on environmental factors. Among different environmental synchronizers, the alternation of light and dark (circadian rhythm) is probably the main factor controlling animal behavior, and melatonin is the hormonal mediator of these rhythms. It is well known that a multitude of factors can influence this precise mechanism. Genetic, nutritional and environmental factors are known to impact hunger and satiety as well as basal metabolic rate and lipid/carbohydrate metabolism. In particular, endocrine disrupters are known as molecules that can interfere with endocrine signaling miming sex hormones and endogenous steroids. The scientific community is increasingly convinced that the augment of eating disorders and disease in aspects of reproductive endocrinology and health is due to xenobiotic chemicals exposure. Alongside studies obtained on mammals, a growing number of researches on appetite control are focusing now on lower vertebrates, especially fish. This is due, in one hand, to their now recognized value as experimental model for this kind of studies. Fish, such as the zebrafish Danio rerio, are useful tools because, in a small scale, they allow to understand the intricate mechanism controlling food intake. In the other hand, considering the increasing importance of aquaculture activities in the last decades, most of the studies aim to the understanding of appetite regulation to improve productivity and quality in fish farm. The present PhD thesis focuses on mechanisms regulating food intake and energy metabolism in fish, in particular, using two different species (Danio rerio and Sparus aurata) this work is aimed at the understanding of neuroanatomical circuits involved on appetite control and at the highlighting of possible relationships between these mechanisms and external cues, with particular attention to photoperiod and endocrine disrupters. In the first part of the thesis, using zebrafish as experimental model, it was assessed the role of melatonin, through 10 days via water administration (doses 100nM and 1 micron), in regulating appetite and metabolism at central (brain) and peripheral level (liver, gastrointestinal tract, gonad and muscle). The main signals involved in appetite regulation (NPY, LPT, CB1, MC4R, Ghrelin) growth (IGF-1), lipid metabolism (PPARs, SREBP) and reproduction (LPT, Ghrelin, Cox2a) were analyzed for the first time all together by Real Time PCR. Western blot technique was used to analyze CB1 protein; FTIR for the evaluation of metabolic resources distribution (lipids, carbohydrates and proteins). Finally, fecundity, in the gonads, was evaluated by counting ovulate eggs. The results here obtained showed a clear role of melatonin in the pathways that regulate appetite, metabolism and reproduction. Melatonin significantly reduces food intake and the reduction is in agreement with the changes observed at molecular level. A significant increase in genes codifying for molecules involved in feeding inhibition and a significant reduction in the major orexigenic signals including ghrelin, NPY and CB1 are here showed. In the liver and intestine melatonin inhibited the signals that stimulate appetite, lipid metabolism and growth. In the muscle it was observed a decrease in lipid component. In addition, analyses on the melatonin role on reproductive pathways showed a clear involvement of this hormone on fecundity, in terms of eggs produced and molecules analyzed. Taken together these data, demonstrate the key role played by melatonin in the regulation of the energy balance, considering its interaction with all the molecules analyzed. In particular, this hormone appears to regulate energy consumption and acquisition on the basis of the metabolic resources available, thereby promoting reproduction and inhibiting feeding. Once highlighted processes regulating food intake and homeostasis in zebrafish, we shifted to the second experimental model the seabream Sparus aurata. Despite the great importance that this species is in aquaculture, no studies on the mechanisms that regulate appetite had never been done before. This species was chosen to analyze the role of the endocannabinoid system in appetite regulation at central (brain) and peripheral (liver) and to verify any relationship of this system with the powerful enhancer of appetite: NPY. The effect of different doses of the endocannabinoid anandamide (AEA) (0,1; 1; 10 µM), administered via water, was evaluated after different exposure times (30, 60 and 120’) at both physiological and molecular levels. Moreover, in order to assess the direct effect of AEA, AM251 (AEA antagonist) was administered 30’ before AEA. By Real-time PCR CB1 and NPY were analyzed in the brain, while by Western blot, CB1 protein was analyzed in brain and liver. The results obtained showed that the endocannabinoid system plays a key role in regulating appetite. At the physiological level in fact, food intake increased significantly, this result was confirmed at molecular level by the increase of NPY and CB1in gene expression. Moreover, the effect of the AEA was specific and direct, in fact AM251 reduced all the effects induced by AEA. Data obtained demonstrated for the first time in sea bream, a strong connection between CB1 and NPY in appetite regulation. In the last part of thesis, it was evaluated the effect of endocrine disrupters on appetite and energy balance regulation. Adult zebrafish were exposed to 0.02, 0.2 and 2 mg / L dietylexilphthalate (DEHP) and to assess the estrogen effect of the compound, ethinyl estradiol was also administered. Signals involved in appetite regulation (ORX, LPT, CB1) were analyzed in the brain while in the liver key signals of lipid metabolism (CB1, SREBP and PPARα) were evaluated. The results here shown demonstrated that DEHP is able to deeply affect all signals of appetite control and fat metabolism axes. Physiologically, a significant decrease in food intake is observable, confirmed at the central level, by the reduction of gene expression of orexigenic factors and up regulation of anorexigenic ones. In the liver, there is a stimulation of genes involved in lipid metabolism. Moreover, the impressive response of all the signals analyzed to DEHP lowest dose indicates that brain and liver are very sensitive target organs for DEHP action. The findings here reported not only provide to the current scientific literature an important contribution in the understanding of appetite control mechanism but could be interesting tools in widespread diseases and disorders linked to appetite and metabolism imbalances. Moreover, these data, although preliminary, help to expand knowledge about the mechanisms that regulate food intake in one of the most important species in European aquaculture.

Bibliography: leaves 363-421. This thesis presents studies relating to the regulation of appetite, body fat stores and energy balance in the marsupial Sminthopsis crassicaudata. All of the studies presented have been published in international journals, accepted for publication, or submitted for publication. These studies have provided novel data on the regulation of food intake, body fat stores and energy balance in the marsupail Sminthopsis crassicaudata, representing fundamental advances in marsupial biology.

Obesity is a physiological consequence of dysregulated energy homeostasis. Energy homeostasis depends on energy intake and energy expenditure. Factors controlling the development of different adipose tissue deposits in the body and their distinct metabolic phenotypes are of considerable interest from both an agricultural and biomedical perspective. Following the literature review, the first chapter was devoted to studies designed to bridge the neural-adipose interface in understanding the relationship between appetite regulation and adipose tissue deposition in chickens, using chickens selected for low or high juvenile body weight as a model. Appetite regulation in the brain, particularly the hypothalamus, is the main factor governing food intake. Neuropeptide Y (NPY), known as a potent orexigenic factor, also promotes energy storage in fat in mammals and thus has a dual role in promoting energy intake via appetite regulation in the brain and energy storage/expenditure via direct effects on adipose tissue function. There have been no reports of the effects of NPY on adipose tissue function in any avian species. By exposing chicken preadipocytes to different concentration of NPY, we found that NPY enhances both proliferation and differentiation and thus appears to play a major role in chicken adipogenesis, an effect that has not yet been reported, to our knowledge. In the body weight selected chicken lines, we found that NPY and receptor sub-type expression was elevated in the abdominal fat of chickens from the high body weight chicken line and expression of these genes displayed heterosis in the reciprocal crosses of the parental lines as compared to both the high and low body weight selected lines. Intriguingly, expression of those same genes was greater in the low weight than high weight chickens in the hypothalamus. Hypothalamic transcriptomic profiling revealed that genes involved in serotonergic and dopaminergic systems may also play an important role in both appetite regulation and insulin-regulated energy homeostasis in the body weight chicken lines. Intracerebroventricular injection of serotonin in broiler chicks was associated with a dose and time dependent reduction in food intake that was coupled with the activation of the ventromedial hypothalamus and arcuate nucleus, as determined by c-fos immunoreactivity. The remainder of this dissertation project describes the effects of knocking down expression of a recently discovered transcription factor, ZBED6, on mouse preadipocyte proliferation and differentiation. The dissertation ends with a study using diet-induced porcine prepubertal obesity as a model to examine differences in adipokine gene expression between different fat depots from pigs that consumed diets that differed in carbohydrate composition. Overall, we conclude that both NPY and monoamines such as serotonin and dopamine are of importance in the regulation of energy balance in chickens. Moreover, we propose that NPY is a factor that mediates hypothalamus and adipose tissue crosstalk in chickens. An understanding of this system may provide a new avenue for the treatment of obesity and associated disease complications by re-orchestrating the neuronal outputs or adiposity inputs. This information may also be of value in developing strategies to improve feed conversion and meat yield in commercial broilers.
Ph. D.

This study was to determine (1) if genetic selection for high (HWS) or low (LWS) body weight in chickens has altered the hypothalamic AMP-activated protein kinase (AMPK) system and (2) if this alteration contributes to the dissimilar feeding response to various appetite modulators between HWS and LWS lines. Compared to HWS, LWS chickens had higher levels of AMPK α and acetyl-CoA carboxylase (ACC) phosphorylation, which was caused by upregulation of the upstream factor calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK β). There was greater mRNA expression of carnitine palmitoyltransferase I (CPT1), leptin receptor (LEPR) and neuropeptide Y (NPY) and less mRNA expression of ACC α, fatty acid synthase (FAS), fat mass and obesity associated gene (FTO), pro-opiomelanocortin (POMC) and orexin in LWS than HWS chickens. At 5 days of age, intracerebroventricular (ICV) injection of AICAR, 5-amino- 4-imidazolecarboxamide riboside, caused a quadratic dose-dependent decrease in food intake in LWS but not HWS chicks. Compound C, (6-(4-(2-piperidin-1-yl-ethoxy)- phenyl))-3-pyridin-4-yl-pyrazolo(1,5-a)-pyrimidine, caused a quadratic dose-dependent increase in food intake in HWS but not LWS chicks. The anorexigenic effect of AICAR in LWS chicks and orexigenic effect of Compound C in HWS chicks resulted from either activation or inhibition of other kinase pathways separate from AMPK. There is a lower threshold for the anorexigenic effect of ghrelin in LWS than HWS chicks, which was associated with differential hypothalamic AMPK signaling. ICV injection of ghrelin iii inhibited corticotrophin-releasing hormone (CRH), 20-hydroxysteroid dehydrogenase (20HSD), glucocorticoid receptor (GR), CPT1 and FTO expression in LWS but not HWS chicks. Additionally, the hypothalamic mRNA level of ghrelin was significantly higher in LWS than HWS chicks, which may also contribute to the differential threshold response to ghrelin in these two lines. Obestatin caused a linear dose-dependent increase in food intake in HWS but not LWS chicks. The orexigenic effect of obestatin in HWS chicks was not associated with altered AMPK. Obestatin inhibited LEPR and FTO expression in HWS but not LWS chicks. Thus, selection for body weight may alter the hypothalamic response to ghrelin by the AMPK pathway, CRH pathway, CPT1 and FTO, and to obestatin by LEPR and FTO.
Ph. D.

Novel secreted peptides represent an important field of research because delineation of their function affords new insights into the pathophysiological processes causing disease, and provides opportunities for the development of pharmacological therapies. Augurin is a recently identified secreted peptide of unknown function expressed in the central nervous system (CNS), pituitary, and several endocrine tissues. Because of its expression pattern I hypothesised that augurin may be involved in the neuroendocrine system. I therefore investigated the effects of central injection of augurin on the hypothalamo-pituitary axes and food intake in male Wistar rats. Intracerebroventricular (ICV) or intraparaventricular nucleus (iPVN) injection of augurin increased plasma adrenocorticotrophic hormone (ACTH) and corticosterone levels compared with vehicle-injected controls. Augurin also increased the release of corticotrophin releasing hormone (CRH) from hypothalamic explants, and pre-treatment with a CRH receptor antagonist in vivo prevented the rise in ACTH and corticosterone caused by ICV augurin, suggesting activation the hypothalamo-pituitary-adrenal (HPA) axis via by the release of CRH from neurons in the PVN. In addition to stimulating the HPA axis, iPVN injection of augurin at the start of either the light or dark phase caused a robust increase in food intake. To assess whether augurin might have a physiological role in the regulation of the HPA axis or food intake, I investigated its endogenous distribution in the rat CNS using immunohistochemistry and in situ hybridisation histochemistry. Augurin positive neurons were present in several regions of the CNS including the hypothalamus and the dorsal vagal complex of the brainstem, a distribution consistent with a possible role in neuroendocrine function. Augurin is therefore anatomically positioned to play a role in the hypothalamic regulation of the HPA axis and food intake. While the data presented suggest a novel role for augurin, these are preliminary studies. Currently, little is known about the physiology of the augurin system. Further characterisation of the neuroanatomy, identification of receptors, and the development of receptor agonists and antagonists would provide further data on the physiological role of augurin, and on the potential therapeutic benefits of manipulating the augurin system.

This research aimed to characterize the physiological role of bombesin-like peptides (BN-LP) in the control of ingestion. The first experiment assessed the developmental profile of BN response in rats, and demonstrated that BN effectively suppressed feeding from postnatal day (PD) 1 through PD 15. Pretreatment with BN receptor antagonist blocked this suppression, suggesting that BN receptors are functional and may participate in feeding regulation from the first hours following birth. We then examined whether endogenous levels of BN-LP in the brain changed in a meal-dependent manner. Of the 15 distinct nuclei examined, meal-related alterations in BN-LP were observed at the hypothalamic paraventricular (PVN), arcuate and dorsomedial nuclei and at the nucleus accumbens. These alterations appeared site and peptide specific since changes in CRF levels were restricted to the hypothalamic lateral and ventromedial nuclei and the central nucleus of the amygdala. To determine what these changes meant in terms of peptide utilization, we then monitored the in vivo release of BN-LP as compared to the preprandial and/or postprandial conditions, where the interstitial levels of BN-LP were relatively high. The next study examined whether sustained central exposure to a BN agonist affected spontaneous feeding, ingestive response to acute BN, or the density of BN receptors within the CNS. Feeding was suppressed over the initial 48 h of BN infusion, however, tolerance to this effect was apparent by 72 h and was associated with receptor down-regulation at the PVN and dentate gyrus. Acute BN administration suppressed feeding in both the chronic BN exposed and control groups indicating lack of tolerance to the acute fluctuations of BN. These findings imply the existence of different neural mechanism(s) mediating the acute versus long-term effects of BN. Finally, the potential interactions of BN with other satiety peptides were investigated. These studies revealed that BN partly mediates its satiety effects through interactions with CRF. The specificity of this interaction was supported by the lack of interaction between BN and/or CRF with oxytocin. This series of experiments provide novel data supporting the view that BN-LP play an important role in the regulation of food intake, and provide some new insights into their possible mechanism(s) of action.

It is generally accepted that human appetite is under multifactorial control, and that investigation of each facet implicated in appetite regulation is required to develop understanding in this area. The current research aimed to investigate subjective appetite ratings and ad libitum food intake in response to manipulated preloads, while simultaneously measuring the hormone and metabolite response and gastric emptying rate of those preloads. This tripartite approach was then used to explore the relationship between the gastrointestinal hormones GLP-1 and CCK, and appetite. Manipulated preloads differing in their carbohydrate and fat content were used to investigate the post-prandial response. Increasing the carbohydrate and energy content of a preload was shown to decrease ad libitum food intake, although there were no obvious effects on subjective appetite ratings. In addition the secretion of GLP-1, GIP, insulin and glucose were significantly increased as preload carbohydrate and energy content increased. The gastric emptying of the preload was slower as carbohydrate and energy content increased. The manipulation of preload fat and energy content showed a similar pattern of response, with decreased ad libitum energy intake and delayed gastric emptying as preload fat and energy content increased. Hormone and metabolite responses showed significant increases in CCK and GLP-1 with increasing preload fat and energy content. To investigate further the role of GLP-1 in the regulation of human appetite, appetite responses during a GLP-1 infusion were assessed against a saline control. The infusion of GLP-1 did not have a significant effect on self rated appetite or ad libitum energy intake, and was not supportive of a major role for GLP-1 as a satiety hormone in man. However GLP-1 infusion significantly delayed gastric emptying of a water load, supporting the role of GLP-1 as a regulator of gastric emptying. The role of CCK in human appetite was similarly investigated, using an infusion of the CCKA receptor antagonist loxiglumide against a saline control. Loxiglumide infusion significantly increased ad libitum energy intake, and significantly increased the rate of gastric emptying of a high energy liquid preload. The infusion was also found to significantly increase GLP-1 and insulin responses, and the increased ad libitum energy intake was observed in spite of high GLP-1 levels with loxiglumide infusion. The ad libitum test meal intake data from the manipulated preload studies showed that a small number of subjects did not adjust their intake according to preload energy intake. Thus the role of habitual exercise levels on food intake response was assessed to determine if this poor response was related to lifestyle differences. Individuals who participated in regular recreational exercise were found to better adjust ad libitum energy intake in response to manipulated preloads than those who undertook no regular recreational exercise. These data provide support a role for exercise in the regulation of food intake. Taken together these data provide evidence that the differing hormonal responses to manipulated preloads are involved in the regulation of appetite response. Investigations of the roles of GLP-1 and CCK in appetite suggest that CCK is a more potent regulator of appetite than GLP-1. The role of exercise in determining the appetite response to manipulated preloads suggests that the investigation of lifestyle differences is also of importance in the understanding of human appetite.

Réguler la prise alimentaire et la dépense énergétique permet en général de limiter d'importants changements de poids corporel. Hormones (leptine, ghréline, insuline) et nutriments sont impliqués dans ces régulations. La résistance à la leptine, souvent associée à l'obésité, limite la régulation de la prise alimentaire. La modélisation mathématique de la dynamique du poids contribue en particulier à une meilleure compréhension des mécanismes de régulation (notamment chez l’humain). Or les régulations hormonales sont largement ignorées dans les modèles existants.Dans cette thèse, nous considérons un modèle de régulation hormonale du poids appliqué aux rats, composé d'équations différentielles non-linéaires. Il décrit la dynamique de la prise alimentaire, du poids et de la dépense énergétique, régulés par la leptine, la ghréline et le glucose. Il reproduit et prédit l'évolution du poids et de la prise alimentaire chez des rats soumis à différents régimes hypocaloriques, et met en évidence l'adaptation de la dépense énergétique. Nous introduisons ensuite le premier modèle décrivant le développement de la résistance à la leptine, prenant en compte la régulation de la prise alimentaire par la leptine et ses récepteurs. Nous montrons que des perturbations de la prise alimentaire, ou de la concentration en leptine, peuvent rendre un individu sain résistant à la leptine et obèse. Enfin, nous présentons une simplification réaliste de la dynamique du poids dans ces modèles, permettant de construire un nouveau modèle combinant les deux modèles précédents
The regulation of food intake and energy expenditure usually limits important loss or gain of body weight. Hormones (leptin, ghrelin, insulin) and nutrients (glucose, triglycerides) are among the main regulators of food intake. Leptin is also involved in leptin resistance, often associated with obesity and characterized by a reduced efficacy to regulate food intake. Mathematical models describing the dynamics of body weight have been used to assist clinical weight loss interventions or to study an experimentally inaccessible phenomenon, such as starvation experiments in humans. Modeling of the effect of hormones on body weight has however been largely ignored.In this thesis, we first consider a model of body weight regulation by hormones in rats, made of nonlinear differential equations. It describes the dynamics of food intake, body weight and energy expenditure, regulated by leptin, ghrelin and glucose. It is able to reproduce and predict the evolution of body weight and food intake in rats submitted to different patterns of caloric restriction, showing the importance of the adaptation of energy expenditure. Second, we introduce the first model of leptin resistance development, based on the regulation of food intake by leptin and leptin receptors. We show that healthy individuals may become leptin resistant and obese due to perturbations in food intake or leptin concentration. Finally, modifications of these models are presented, characterized by simplified yet realistic body weight dynamics. The models prove able to fit the previous, as well as new sets of experimental data and allow to build a complete model combining both previous models regulatory mechanisms

Xenin, a gastrointestinal peptide, is structurally similar to neurotensin which functions as a satiety factor via neurotensin receptor 1 (Ntsr1). Metabolic effect of the adipocyte hormone leptin is partially mediated through the Ntsr1 and interleukin 1 type I receptor (IL-1RI) in the central nervous system (CNS). Xenin reduces food intake when administered centrally and peripherally. Apart from its acute feeding-suppressing effect, the distinct metabolic action of xenin and the mechanism of xenin-induced anorexia remain to be elucidated. We hypothesized that prolonged xenin treatment reduces food intake and body weight and increases energy expenditure. We also hypothesized that xenin reduces food intake by activating CNS signalling pathways including Ntsr1 and IL-1RI and by interacting with leptin. To address these hypotheses, we examined (1) the effect of xenin treatment on food intake, energy expenditure and body weight in wild-type, Ntsr1-deficient and IL-1RI-deficient mice, (2) the effect of xenin on hypothalamic Fos and interleukin 1β (IL-1β) expression in wild-type mice, and (3) the effect of co-injection of xenin and leptin on food intake and body weight in wild-type mice. Daily intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) xenin treatment (6-10 days) significantly reduced body weight gain and adiposity with a transient reduction in food intake in wild-type mice. Xenin treatment (i.p.) caused a significant reduction in respiratory quotient without changes in energy expenditure. Xenin treatment increased hormone sensitive lipase (HSL) mRNA levels and reduced acyl-coenzyme A: diacylglycerol acyltransferase 2 (DGAT2) mRNA levels in white adipose tissue. Xenin (i.p.) increased the number of Fos-immunoreactive cells in the hypothalamus and the brainstem and increased hypothalamic IL-1β mRNA levels. The anorectic effects of xenin and leptin were abolished or attenuated in mice lacking Ntsr1 or IL-1RI. I.p. co-administration of xenin and leptin caused greater reductions in food intake and body weight compared to leptin alone and xenin alone. These data suggest that long-term xenin treatment reduces body weight by reducing food intake and increasing fat oxidization. Xenin reduces food intake by activating CNS signalling pathways involving Ntsr1 and IL-1 possibly through the interaction with leptin. These findings implicate xenin and its downstream mediators as potential targets for anti-obesity drugs.