We propose to investigate the molecular mechanisms of appetite control in the nematode Caenorhabditis elegans. Just as in mammals, C elegans appetite is promoted by hunger signals and suppressed by satiety signals. Both responses to food availability are similar behaviorally and overlap in molecular mechanism with analogous mammalian behaviors. Based on this conservation, we aim to establish in C elegans a genetic model system to study appetite control. We anticipate that molecular mechanisms will be fundamentally similar to those of mammals, but simpler and therefore easier to unravel. Furthermore, the powerful genetic tools available in the worm will, we hope, allow rapid elucidation of new pathways, whose relevance to mammalian behavior can subsequently be tested. Our broad long-term objective is to understand at a molecular level how an animal regulates its food intake. Our goal in the next five years is to investigate the cellular and molecular mechanisms that control satiety. We will test the following hypotheses: 1. ASI sensory neuron activity signals nutritional well-being and provokes a satiety response. 2. ASI's effects are mediated by the release of peptide and protein hormones, including insulin-like peptides and the TGF-2-like peptide DAF-7. 3. Expression of satiety hormone genes increases in response to nutritional well-being. 4. Satiety hormones act on downstream neurons to change behavior. 5. Cyclic GMP (cGMP) activates cGMP-dependent protein kinase (PKG) in ASI and in downstream neurons to produce satiety.
Aim 1. Identify satiety signaling mechanisms (hypotheses 1, 4, 5). Determine the time-course of satiety behavior in single worms. Determine the sites and times of action of signal and receptor genes in the insulin, TGF-2, and cGMP pathways by expression of transgenes from neuron-specific promoters in combination with laser microsurgery (hypotheses 2, 4, and 5). Determine the order of signal action by epistasis studies measuring the effects of overexpression and gain-of-function mutations in loss-of-function mutant backgrounds.
Aim 2. Find peptide and protein hormone genes whose transcription correlates with satiety (hypotheses 2, 3). Using microarrays and quantitative RT-PCR, find peptide genes whose transcription is regulated by starvation and refeeding, or by cGMP and PKG. Mutate these genes or knock down their expression and determine the effect on satiety behavior. Screen for mutants defective in satiety- induced quiescence and identify the mutated genes by whole-genome sequencing.
Aim 3. Measure synaptic activity and release of peptides from ASI (hypotheses 1, 2). Develop methods to measure synaptic activity by recovery of fluorescence after photobleaching of the synaptic vesicle pH sensor synaptopHluorin. Develop methods to measure release of fluorescent peptides from neurons. Use these methods to determine how ASI activity and peptide release respond to conditions that induce satiety.
Obesity is a serious and growing health problem, brought on by eating more than is necessary to fulfill a person's metabolic needs. A better understanding of the signals that regulate appetite may lead to better methods for controlling food intake, and therefore to better control of obesity.
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