GPR50 is the mammalian ortholog of the avian/amphibian melatonin receptor, Mel1c, yet its ligand and physiological function remain unknown. Previous studies implicate the receptor in the control of energy homeostasis; Gpr50-/- mice demonstrate elevated basal metabolic rate, reduced fat stores, and partial resistance to diet-induced obesity. This thesis examines the physiological impact of GPR50 on a cellular level as well as in two key structures involved in metabolism and energy balance; namely white adipose and the hypothalamus.In vitro studies demonstrate GPR50's ability to activate downstream signalling pathways, specifically, inhibition of forskolin-stimulated cAMP accumulation in HEK293 cells. Co-immunoprecipitation experiments reveal association of GPR50 with Galphai1, suggesting the receptor acts through Gi/o proteins to elicit inhibition of cAMP production. This work further identifies a novel truncated variant of GPR50 protein, lacking transmembrane domains 6 and 7 and the cytoplasmic tail. This variant demonstrates altered subcellular localisation from wild-type protein, being retained in the endoplasmic reticulum instead of inserting into the plasma membrane, and does not possess the same signalling capability as the full-length receptor.This work reports the expression of GPR50 in murine white adipose tissue and explores the potential significance of the receptor in this organ. Levels of GPR50 mRNA increase during differentiation of the adipocytic 3T3-L1 cell line, and stable knockdown or overexpression of GPR50 results in decreased and increased triglyceride accumulation, respectively. Knockdown of GPR50 represses transcript levels of hormone sensitive lipase, adipose triglyceride lipase, and lipoprotein lipase, whereas overexpression of GPR50 increases levels of these adipocytic enzymes, as well increasing expression of peroxisome proliferator-activated receptor gamma. Augmentation of GPR50 expression in either direction blunts forskolin-stimulated lipolysis in differentiated 3T3-L1 cells. These data implicate GPR50 in adipocyte metabolism.Gpr50-/- mice readily enter torpor in response to fasting, and gene expression (Affymetrix) profiling of Gpr50-/- mice reveals strong induction of thioredoxin interacting protein (Txnip) in the hypothalamus during torpor, an original finding. Recent data suggests a role for Txnip as a molecular nutrient sensor important in the regulation of energy metabolism. Here, Txnip expression is shown to be strongly expressed within the ependyma lining the third ventricle, a known site of GPR50 expression. Hypothalamic Txnip expression is induced by fasting, but demonstrates exaggerated levels of expression in Gpr50-/- and also in WT mice driven into torpor. Strikingly, Txnip expression is also elevated in the hypothalamus of a model of natural seasonal torpor, the Siberian hamster. Thus, Txnip appears to have a critical role during the torpor response, potentially regulating energy expenditure and fuel utilisation during this extreme hypometabolic state.
|Date of Award
|1 Aug 2012
- The University of Manchester
|Andrew Loudon (Supervisor) & Simon Luckman (Supervisor)