目的:在营养丰富的条件下,肝脏脂肪生成升高,将过量的碳水化合物转化为三酰甘油(TAG)。TAG的脂肪酰基部分最终被极低密度脂蛋白转运到脂肪组织中,导致TAG作为过量能量的首选储存形式的积累。TAG清除和合成之间的平衡的破坏导致脂质在肝脏中的积累,导致非酒精性脂肪性肝病(NAFLD)的进展,包括非酒精性脂肪性肝炎。蛋白质精氨酸甲基转移酶(PRMT)6已与各种代谢过程有关,包括肝糖异生,小鼠模型中的肌肉萎缩和脂肪营养不良。然而,PRMT6在肝脏脂肪生成控制中的作用至今尚未阐明.
方法:我们使用免疫共沉淀法评估了PRMT6和LXRα之间的相互作用。通过LC-MS/MS测定评估被PRMT6甲基化的LXRα的特定精氨酸残基,并且通过mSREBP-1c荧光素酶测定探索LXRα甲基化的功能后果。通过腺病毒介导的PRMT6的异位表达或通过shRNA在肝细胞中敲低PRMT6来评估PRMT6对肝脂肪生成的影响。最后,通过异位表达PRMT6介导的精氨酸甲基化缺陷的LXRα突变体或肝脏中PRMT6基因敲低,探索了PRMT6在体内肝脂质代谢中的作用.
结果:我们发现,固醇调节元件结合蛋白(SREBP)1c的启动子活性被PRMT6强烈激活。有趣的是,我们证明了PRMT6与LXRα结合,SREBP-1c的转录因子,通过其LXXLL主题,导致精氨酸残基的不对称二甲基化和该蛋白质的活化。的确,肝细胞中PRMT6的异位表达导致脂肪生成途径中LXRα靶基因的表达增强。相反,PRMT6的遗传或药理学抑制减少了脂肪生成基因的表达和原代肝细胞中的脂质积累。机械上,我们发现LXRα的不对称二甲基化导致小的异二聚体伴侣(SHP)的解离,该因子的转录共抑制剂,导致LXRα介导的转录过程的激活。最后,我们表明,肝脏中LXRα的不对称二甲基化的破坏导致脂肪生成中基因的表达减少,导致体内高脂肪饮食小鼠肝脏脂质积累减少。
结论:我们发现PRMT6通过赋予精氨酸253的不对称二甲基化来调节LXRα活性,从而阻断SHP介导的抑制并促进肝脏脂质积累。这些结果表明,PRMT6在通过调节LXRα介导的肝脏脂肪生成来控制脂质稳态方面至关重要。
OBJECTIVE: Hepatic lipogenesis is elevated in nutrient abundant conditions to convert the excess carbohydrate into triacylglycerol (TAG). Fatty acyl moiety of TAG is eventually transported into adipose tissues by very low density lipoprotein, leading to the accumulation of TAG as a preferred storage form of excess energy. Disruption of the balance between TAG clearance and synthesis leads to the accumulation of lipids in the liver, leading to the progression of non-alcoholic fatty liver disease (NAFLD) including non-alcoholic steatohepatitis. Protein arginine methyltransferase (PRMT) 6 has been linked to the various metabolic processes including hepatic gluconeogenesis, muscle atrophy and lipodystrophy in mouse models. However, the role of PRMT6 in the control of hepatic lipogenesis has not been elucidated to date.
METHODS: We assessed the interaction between PRMT6 and LXR alpha by using co-immunoprecipitation assay. The specific arginine residue of
LXR alpha that is methylated by PRMT6 was assessed by LC-MS/MS assay and the functional consequences of
LXR alpha methylation was explored by mSREBP-1c luciferase assay. The effect of PRMT6 on hepatic lipogenesis was assessed by adenovirus-mediated ectopic expression of PRMT6 or knockdown of PRMT6 via shRNA in hepatocytes. Finally, the role of PRMT6 in hepatic lipid metabolism in vivo was explored by either ectopic expression of LXR alpha mutant that is defective in PRMT6-mediated arginine methylation or knockdown of PRMT6 in liver.
RESULTS: We found that promoter activity of sterol regulatory element binding protein (SREBP) 1c is robustly activated by PRMT6. Interestingly, we demonstrated that PRMT6 binds to
LXR alpha, a transcription factor for SREBP-1c, via its LXXLL motif, leading to the asymmetric dimethylation of an arginine residue and activation of this protein. Indeed, ectopic expression of PRMT6 in hepatocytes led to the enhanced expression of
LXR alpha target genes in the lipogenic pathway. Conversely, genetic or pharmacological inhibition of PRMT6 diminished expression of lipogenic genes and the lipid accumulation in primary hepatocytes. Mechanistically, we found that asymmetric dimethylation of
LXR alpha led to the dissociation of small heterodimer partner (SHP), a transcriptional co-inhibitor of this factor, resulting in the activation of LXR alpha-mediated transcriptional process. Finally, we showed that disruption of asymmetric dimethylation of LXR alpha in the liver led to the diminished expression of genes in the lipogenesis, resulting in the reduced hepatic lipid accumulation in high fat diet-fed mice in vivo.
CONCLUSIONS: We showed that PRMT6 modulates LXR alpha activity by conferring asymmetric dimethylation of arginine 253, thus blocking SHP-mediated inhibition and promoting hepatic lipid accumulation. These results suggest that PRMT6 is critical in the control of lipid homeostasis by regulation of LXR alpha-mediated lipogenesis in the liver.