SUMMARY Chronic and/or excess glucocorticoid (GC) exposure, such as prolonged stress and long-term GC therapy, causes metabolic disorders including hyperglycemia. Intracellular GC receptor (GR) has been shown to directly stimulate the transcription of gluconeogenic genes, such as Pck1 and G6pc, to promote gluconeogenesis. However, chronic GC exposure can induce additional mechanisms to further enhance hepatic gluconeogenesis. Our preliminary studies found that chronic GC exposure elevated hepatic sphingosine-1-phosphate (S1P) levels. S1P is exported to the extracellular surface and binds to membrane S1P receptors (S1PRs) to exert its actions. We found that reducing S1PR2 expression in mouse liver attenuated chronic GC exposure-promoted gluconeogenesis. Activating S1PR2 in hepatoma cells enhanced GC-induced Pck1 and G6pc expression. Chromatin immunoprecipitation assay found that GC-induced GR recruitment to the GC response elements (GREs) of Pck1 and G6pc was reduced by hepatic S1PR2 knockdown. Global gene expression analysis identified that RAR-related orphan receptor C (Rorc) expression was reduced by hepatic S1PR2 knockdown. Rorc antagonist attenuated GC-induced gluconeogenic gene expression in hepatoma cells and overexpressing Rorc in the liver of hepatic S1PR2 knockout mice enhanced GC-induced GR recruitment to the Pck1 and G6pc GREs as well as their expression. Intriguingly, GC suppressed Rorc expression, which was antagonized by S1PR2 signaling. Based on these results, we hypothesize that chronic GC exposure activates S1PR2 signaling to enhance GC-induced gluconeogenesis by inhibiting GC’s suppressive effect on the expression of Rorc, which can act with GR to strongly augment gluconeogenic gene transcription. In Aim 1, we will test if altering intracellular S1P levels in hepatocytes affects chronic GC treatment-induced hyperglycemia by overexpressing or knocking down Sphk1 and Sphk2 (both convert sphingosine to S1P), Sgpl1 (hydrolyzes S1P) and Spns2 (exports S1P to extracellular space) in mouse liver. In Aim 2, we will establish Rorc’s in GC-induced hepatic gluconeogenesis by reducing hepatic Rorc expression or treating mice with Rorc antagonist. Notably, 4a- carboxy, 4b-methyl-zymosterol (4ACD8), a metabolite of cholesterol biosynthesis, has been shown to be a Rorc agonist. We will investigate whether reducing the expression of Sc4mol, an enzyme generating 4ACD8, attenuates the GC effect on hepatic gluconeogenesis. In Aim 3, we will identify signaling molecules activated by S1PR2 that participate in GC regulation of gluconeogenic genes and Rorc expression. We will also analyze the role of Rorc in GR regulated global hepatic gene transcription. Finally, how GC and S1PR2 signaling regulate Rorc expression will be investigated. Overall, the proposed studies will establish a novel S1PR2-Rorc axis induced specifically upon chronic GC exposure to enhance GC activated gluconeogenesis. Not only will this knowledge advance our understand on pathoph...