The Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it regulates appetite and energy expenditure. MC4R signals via the G stimulatory α-subunit (Gs) in response to its natural agonist, α-melanocyte stimulating hormone (α-MSH). Binding of agonist to MC4R induces downstream activation of adenylate cyclase resulting in increased levels of cAMP. Generation of cAMP is thought to emanate the signal to eat less and metabolize more. Here we demonstrated that MC4R agonists differ in their ability to increase intracellular cAMP over time. Our data further indicates that the extent of MC4R retention into the intracellular compartment upon prolonged agonist exposure is agonist specific. Here we hypothesize that there are unique ligand mediated MC4R receptor interactions that can result in MC4R becoming stabilized into multiple active conformations and this can influence both signaling and trafficking properties of MC4R. Many MC4R agonists have been developed to help lower food intake and promote weight loss in obese individuals. Obesity is often associated with dyslipidemia, a condition where the concentration of circulating Non-Esterified Fatty Acids (NEFAs), such as palmitic acid, is increased. We would like to determine whether high-fat feeding affects the function of MC4R. We have modeled effects of lipid stress on MC4R function by exposing neuronal Neuro2A cells and immortalized hypothalamic mHypoE-42 neurons to elevated concentrations of palmitate within the physiological range. Using confocal microscopy we found that lipid stress changes the cell shape of Neuro2A cells and inhibits internalization of both MC4R and transferrin receptors in cells. Structure-illumination fluorescence microscopy (SIM) indicates that exposure to lipid stress inhibits transit of tagged HA-MC4R-GFP to early endosomes by halting exit of the receptor from clathrin-coated pits. In addition, lipid stress blunts desensitization of MC4R in response to the synthetic agonist, Melanotan II (MTII). A single intraperitoneal injection of MTII decreases weight in obese male C57BL/6J mice exposed to a high fat diet, but not in mice fed a low-fat diet. These studies provide evidence that lipid stress may unexpectedly promote an increased response to anti-obesity treatments targeted to MC4R by changing traffic of the receptor.
|Commitee:||Hakkak, Reza, Lupashin, Vladimir, Phelan, Kevin D., Rhee, Sung|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Biochemistry|
|Keywords:||Clathrin, Endocytosis, G protein-coupled receptor, Lipid stress, Melanocortin-4 receptor, Obesity|
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