Despite the known health risks associated with smoking, up to 20% of the US population persist in this behavior. Many of these individuals smoke to control their body weight or appetite and fear of post-cessation weight gain motivates continued smoking. In clinical populations, nicotine and tobacco use is associated with lower body weight, and cessation yields an average weight gain of about 10 lbs, mostly within the first year. While the health benefits of quitting outweigh the costs of moderate weight gain, excessive weight gain can occur, and is more likely in already obese smokers. Nicotine replacement therapies can delay this weight gain, but do not prevent it altogether, and the underlying mechanism for how nicotine is able to reduce weight is not fully understood.
In rodent models, nicotine reduces weight gain, reduces food consumption, and alters energy expenditure, but these effects vary with duration and route of nicotine administration. Nicotine, acting through nicotinic acetylcholine receptors (nAChRs), increases the firing rate of both orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC), and viral-mediated knockdown of the β4 nAChR subunit in all neuronal cell types in the ARC prevents another nicotinic agonist, cytisine, from decreasing food intake, however, it is unknown exactly how this yields reduced food intake. This thesis tackles several unanswered questions related to cholinergic signaling in the ARC and how nicotine acts within this circuit.
First, I explore the anatomical source of the endogenous nAChR ligand, acetylcholine (ACh) in the ARC. I probe the connectivity of both distal and local ACh sources using a combination of retrograde and anterograde tracing techniques, and examine the mRNA and protein expression of cholinergic cell markers within the ARC itself. These experiments show that cholinergic neurons within the hypothalamus are likely to provide cholinergic input to the ARC, and more distal sources of cholinergic input are discussed.
The subunit composition of nAChRs directly controls their regulation of neuronal excitability, and while it has been shown that the β4 subunit is expressed on POMC neurons in the ARC, it was not known whether this expression is selectively expressed in anorexogenic neurons or how other nAChR subtypes are distributed in this nucleus. Using translating ribosome affinity purification (TRAP) on ARC tissue from mice with ribosomes tagged in either AgRP or POMC cells, I examined nAChR subunit mRNA levels using real-time PCR. Further, I used immunoprecipitation to probe the subunit composition of functionally assembled nAChR. Numerous common and rare nAChR subtypes are expressed in the ARC, and there are fewer differences in expression between AgRP and POMC neurons than previously hypothesized. Further, the functional nAChR in this region are unique with respect to other brain areas that express the β4 subunit.
AgRP and POMC neurons have opposing functions with respect to controlling food intake, yet these cells are anatomically intermingled within the ARC. To determine the role of distinct nAChR subunits in either cell population, cell type selective manipulations are necessary. I next show validation for cell type-selective, virally-delivered small hairpin RNAs targeting either the β4 or α7 subunit. I then used these viruses to examine the contribution of each subunit in either AgRP or POMC cells to the behavioral response to nicotine. While β4 and α7 subunits have different roles in the ARC with respect to responding to the nicotinic drugs nicotine and cytisine, their roles in AgRP and POMC cells are not dramatically different, despite the different functions of these cell populations.
Finally, I examined the sex-specific effects of long term self-titrated nicotine exposure combined with high fat diet (HFD) on body weight and metabolism-regulating signals. Male and female animals respond differently to coadministered nicotine and high fat diet, with respect to changes in body weight. Nicotine reduced weight gain in male mice with access to HFD, but did not impact the weight gain of other groups. Further, the regulation of both neuropeptides involved in energy balance and uncoupling proteins varied with diet and nicotine in a sex-dependent manner.
Taken together these experiments further inform our understanding of nAChR signaling in the ARC and provide the basis for a new hypothesis regarding the regulation of this circuit by nicotinic drugs.
|Advisor:||Picciotto, Marina R.|
|School Location:||United States -- Connecticut|
|Source:||DAI-B 79/12(E), Dissertation Abstracts International|
|Keywords:||AgRP, Food Intake, Hypothalamus, Nicotine, POMC, nAChR|
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