Iron Downregulates Leptin by Decreasing CREB O-GlcNAc Modification and CREB Occupancy of the Leptin Promoter

Modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) serves as a nutrient-dependent signal to regulate transcription of the leptin gene. Recently we reported that increased levels of iron, which are also a risk factor for type 2 diabetes, down regulate leptin in mice fed high iron diets (2000 mg/kg) or in 3T3L1 cells treated with iron. Iron increased cyclic AMP-dependent response element binding protein (CREB) phosphorylation and its occupancy at inhibitory sites in the leptin promoter, but without demonstrable increases in CREB kinase activities. We now demonstrate that nutrient- and iron-dependent regulation of leptin are linked through O-GlcNAc modification of CREB. CREB O-GlcNAc modification was decreased in mice fed high iron, and this was associated with 54±7% and 44±9% reductions in recruitment to its two sites in the leptin promoter as analyzed by ChIP (p<0.05). Increasing levels of O-GlcNAc through targeted deletion of the O-GlcNAcase (OGA) gene in adipocytes increased adipose leptin mRNA expression by 86±12% (p<0.05) in mice fed a low-normal iron diet (35 mg/kg) and partially rescued the effect of high iron (35±3%, p<0.05). Similar effects were seen in serum leptin levels: High iron caused a 72% decrease and OGA deletion resulted in a rescue of 34%. Treatment of 3T3L1 cells with iron decreased expression of a leptin reporter plasmid by 45±4% and glucosamine, which bypasses the rate-limiting step in synthesis of UDP-GlcNAc, the substrate for O-GlcNAcylation, increased protein O-GlcNAc and fully rescued (106±5%) the effect of high iron (p<0.001 for both). Although iron also decreased global levels of protein O-GlcNAc modification, mRNA levels of the enzymes that control the modification, namely O-GlcNAc transferase and OGA, were unchanged. We conclude that adipose tissue integrates cellular nutrient status and tissue iron levels to control the regulation of leptin, through regulation of O-GlcNAc modification of the transcription factor CREB.