Sphingosine-1-phosphate Signaling and the Chronic Glucocorticoid Exposure Induced Glucose Homeostasis Disorder

摘要 长期和/或过量接触糖皮质激素(GC)，如长期应激和长期GC治疗， 会导致代谢紊乱，包括高血糖。细胞内GC受体(GR)被证明可直接 刺激糖异生基因的转录，如Pck 1和G6PC，以促进糖异生。 然而，慢性GC暴露可以诱导额外的机制来进一步促进肝脏的糖异生。 我们的初步研究发现，慢性GC暴露会增加肝脏鞘氨醇-1-磷酸(S1P)的水平。 S1P被输出到细胞外表面，并与膜上的S1P受体结合发挥作用。 我们发现，降低小鼠肝脏中S1PR2的表达可以减轻慢性GC暴露的促进作用 糖异生作用。在肝癌细胞中激活S1PR2可增强GC诱导的Pock1和G6pc的表达。 染色质免疫沉淀分析发现GC诱导GR向GC反应元件募集 肝脏S1PR2基因敲除可降低Pock1和G6pc的GRES。全球基因表达分析 研究发现，肝脏S1PR2基因敲除后，RAR相关孤儿受体C(RORC)的表达减少。 RORC拮抗剂抑制GC诱导的肝癌细胞糖异生基因表达及过表达 肝脏S1PR2基因敲除小鼠肝脏中的RORC促进GC诱导的GR向Pock1和G6pc的募集 格雷斯和他们的表情。有趣的是，GC抑制了RORC的表达，而RORC的表达被 S1PR2信令。基于这些结果，我们假设慢性GC暴露激活了S1PR2信号 通过抑制GC对RORC表达的抑制作用，增强GC诱导的糖异生 可与GR共同作用，强烈增强糖异生基因转录。在目标1中，我们将测试是否更改 肝细胞内S1P水平过表达对GC慢性高血糖的影响 或者击倒Sphk1和Sphk2(两者都将鞘氨醇转化为S1P)、Sgpl1(水解S1P)和Spns2 (将S1P输出到细胞外空间)。在目标2中，我们将在GC诱导的肝脏中建立RORC 降低肝脏RORC表达或用RORC拮抗剂治疗小鼠的糖异生作用。值得注意的是，4a- 胆固醇生物合成的代谢物--羧基，4b-甲基-酵素甾醇(4ACD8)已被证明是一种RORC 激动剂。我们将研究是否降低产生4ACD8的酶Sc4mol的表达， 减弱GC对肝脏糖异生的影响。在目标3中，我们将识别由 参与糖异生基因和RORC表达的GC调控的S1PR2。我们还将分析 RORC在GR调控的肝细胞基因转录中的作用最后，GC和S1PR2信号是如何调节的 将研究RORC的表达情况。总体而言，拟议的研究将建立一个新的S1PR2-RORC轴 在慢性GC暴露时特异性地诱导，以增强GC激活的糖异生。这不仅会让你 知识促进了我们对慢性GC暴露病理生理学的理解，但它也将为我们提供新的 激素性高血糖的治疗干预靶点。

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 pathophysiology of chronic GC exposure, but it also will provide novel targets for therapeutic intervention against steroid induced hyperglycemia.