The objective of the proposal is to identify behavioral and cellular mechanisms by which inhibition of fatty acid synthase (FASi) and enhanced beta-oxidation by stimulation of carnitine-palmitoyltransferase-1 (CPT-1s) alter hypothalamic energy status and adjust food intake. The candidate will (1) gain experience with primary hypothalamic neuronal cultures and acute hypothalamic explants in static incubation, (2) learn a range of biochemical assays, and (3) build upon experience with HPLC, immunohistochemistry, and mRNA detection. The training will facilitate the candidate's transition to independent research. Experiments will be conducted at the Johns Hopkins University School of Medicine. The primary mentor is Dr. Gabriele Ronnett, a neuroscientist and biochemist who uses neuronal cultures. The co-mentor is Dr. Timothy Moran, an expert in neurophysiology of feeding. The candidate will interact with an interdisciplinary team that investigates roles of fatty acid metabolism in a range of human health problems, including obesity. C75 is a FASi/CPT-1s that reduces food intake and alters gene expression for hypothalamic feeding-related neuropeptides when given centrally to rodents. We find that icv C75 decreases feeding in rats by reducing meal frequency, not size, suggesting that FASi or CPT-1s, or both, reduce drive to initiate feeding, rather than enhance satiety. C75 affects energy status in primary cultures of hypothalamic neurons, altering intracellular signaling to modulate neuropeptide production. Novel compounds selective for potent FASi or CPT-1s are available to us to investigate individual roles of FAS and CPT-1 in feeding regulation. The overall hypothesis is that altered fatty acid metabolism in hypothalamic neurons changes their energy state and leads to homeostatic alterations in food intake. Experiments will address two Specific Aims: (1) Identify how FASi or CPT-1s in the brain alter food intake and gene expression: Experiments in mice will measure food intake, meal patterns, brain c-Fos expression, and expression of mRNA for feeding-related neuropeptides after icv C75, FASi, or CPT-1s.
The second aim i s to (2) Identify how FASi and CPT-1s change hypothalamic neuronal energy status and production of feeding-regulating neuropeptides: The compounds will be applied to (a) primary hypothalamic neuronal cultures and (b) acute hypothalamic explants. We will measure changes in intracellular AMP/ATP ratio and activity of AMP-activated protein kinase, levels of mRNAs for feeding-related neuropeptides, and neuropeptide production and release.
