Insulin stimulates both the biosynthesis of transcription factor Sp1 and its O-linked N-acetylglucosaminylation (O-GlcNAcylation), which promotes nuclear localization of Sp1 and its ability to transactivate calmodulin (CaM) gene transcription. To investigate this further, we incubated H-411E liver cells with insulin (10,000 U/ml) and quantified the subcellular distribution of O-GlcNAc transferase (OGT) and O-GlcNAcmodified Sp1. We also examined the phosphorylation of Sp1 using both Western blot and incorporation of 32 P into Sp1. The results demonstrate that insulin, but not glucagon, stimulates OGT synthesis and enhances cytosolic staining of OGT (histochemical). Insulin increases O-GlcNAc-Sp1, which peaks at 30 min, followed by decline at 4 h. In contrast, insulin initiates phosphorylation of Sp1 early, followed by a continued increase in phosphorylated Sp1 (PO 4 -Sp1) at 4 h. A reciprocal relationship between O-GlcNAc-Sp1 and PO 4 -Sp1 was observed. To explore the pathophysiological relevance, we localized OGT in liver sections from streptozotocin (STZ)-induced diabetic rats. We observed that staining of OGT in STZ-induced diabetic rat liver is clearly diminished, but it was substantially restored after 6 days of insulin treatment. We conclude that insulin stimulates CaM gene transcription via a dynamic interplay between O-glycosylation and phosphorylation of Sp1 that modulates stability, mobility, subcellular compartmentalization, and activity. Diabetes 53: 3184 -3192, 2004

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