Majumdar Gipsy, Harmon Ashley, Candelaria Rosalind, Martinez-Hernandez Antonio, Raghow Rajendra, Solomon Solomon S
Research Services, Veterans Affairs Medical Center, Memphis, TN 38104, USA.
Am J Physiol Endocrinol Metab. 2003 Sep;285(3):E584-91. doi: 10.1152/ajpendo.00140.2003.
Both insulin and glucagon stimulate steady-state levels of Sp1 transcription factor, but only insulin stimulates transcription of the calmodulin (CaM) gene in liver. Because O-glycosylation of Sp1 by O-linked N-acetylglucosamine (O-GlcNAc) is thought to regulate its ability to activate transcription, we assayed the levels of Sp1 with anti-Sp1 and anti-O-GlcNAc antibodies in Western blots by use of extracts of H-411E liver cells treated with insulin (10,000 microU/ml) or glucagon (1.5 x 10(-5) M). We also assessed subcellular localization of the native and glycosylated Sp1 in H411E cells treated with either hormone in the presence of deoxynorleucine (DON, an indirect inhibitor of O-glycosylation) or streptozotocin (STZ, an indirect stimulator of O-glycosylation). Insulin stimulated both total and O-GlcNAc-modified Sp1 primarily in the nucleus and induced CaM gene transcription (P < 0.0001). In contrast, glucagon promoted accumulation of Sp1 in the cytoplasm but not the nucleus, without significantly stimulating (P = not significant) either its O-glycosylation or transcription of the CaM gene. DON inhibited O-glycosylation of Sp1 and its ability to migrate to the nucleus and transactivate CaM gene transcription. In contrast, cotreatment of cells with STZ and glucagon enhanced O-glycosylation of Sp1, promoting its migration to the nucleus and resulting in increased CaM gene transcription. Thus O-glycosylation of Sp1 by insulin, but not glucagon, apparently enhances its (Sp1) nuclear recruitment and results in activation of CaM gene transcription.
胰岛素和胰高血糖素均可刺激Sp1转录因子达到稳态水平，但只有胰岛素能刺激肝脏中钙调蛋白（CaM）基因的转录。由于O-连接的N-乙酰葡糖胺（O-GlcNAc）对Sp1进行O-糖基化被认为可调节其激活转录的能力，因此我们使用抗Sp1抗体和抗O-GlcNAc抗体，通过对用胰岛素（10,000微单位/毫升）或胰高血糖素（1.5×10⁻⁵ M）处理的H-411E肝细胞提取物进行蛋白质免疫印迹分析，来检测Sp1的水平。我们还评估了在脱氧正亮氨酸（DON，一种O-糖基化的间接抑制剂）或链脲佐菌素（STZ，一种O-糖基化的间接刺激剂）存在的情况下，用这两种激素处理的H411E细胞中天然和糖基化Sp1的亚细胞定位。胰岛素主要在细胞核中刺激总Sp1和O-GlcNAc修饰的Sp1，并诱导CaM基因转录（P < 0.0001）。相比之下，胰高血糖素促进Sp1在细胞质中积累，但不在细胞核中积累，对其O-糖基化或CaM基因转录均无显著刺激作用（P = 无显著性差异）。DON抑制Sp1的O-糖基化及其迁移至细胞核并反式激活CaM基因转录的能力。相反，用STZ和胰高血糖素共同处理细胞可增强Sp1的O-糖基化，促进其向细胞核迁移，并导致CaM基因转录增加。因此，胰岛素而非胰高血糖素对Sp1的O-糖基化显然增强了其（Sp1）向细胞核的募集，并导致CaM基因转录激活。
