Guanosine monophosphate reductase regulates uncoupling protein 1 activity

Abstract

Non-shivering thermogenesis is mediated by brown adipose tissue (BAT) specific uncoupling protein 1 (UCP1). Activity of UCP1 is regulated by free fatty acids and by the di- and triphosphate forms of purine nucleotides. GMP reductase (GMPR) interconverts guanosine into adenosine nucleotides and is upregulated in response to cold. We engineered human embryonic kidney (HEK) cells to stably express UCP1 (HEK-UCP1). In HEK and HEK-UCP1 cells we transiently overexpressed GMPR and measured the concentration of all purine nucleotides. In HEK cells GMPR expression did not lead to altered concentrations of nucleotides, while in HEK-UCP1 cells GMPR decreased the ratio of triphosphate to mono- and diphosphate purine nucleotides, indicating a lowered capacity for oxidative phosphorylation. Upon GMPR expression in HEK-UCP1 both the basal proton leak (in the presence of substrate plus oligomycin) and the palmitate-inducible, UCP1-specific proton leak was greater than in the absence of either UCP1, GMPR or both. This implies an augmentation of UCP1 activity by GMPR. Surprisingly, we were able to several-fold enrich GMPR by isolation of mitochondrial protein from BAT. Possibly, GMPR is associated with mitochondria and is implicated in the maintenance of a microdomain of altered purine concentrations to modify UCP1 activity, as reflected in our respiration and nucleotide concentration measurements.