Insulin stimulates and diabetes inhibits O-linked N-acetylglucosamine transferase and O-glycosylation of Sp1.
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 microU/ml) and quantified the subcellular distribution of O-GlcNAc transferase (OGT) and O-GlcNAc-modified Sp1. We also examined the phosphorylation of Sp1 using both Western blot and incorporation of 32P 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 (PO4-Sp1) at 4 h. A reciprocal relationship between O-GlcNAc-Sp1 and PO4-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.
Majumdar
Gipsy
G
Research Services, VA Medical Center, Memphis, Tennessee, USA.
Wright
Jeremiah
J
Markowitz
Paul
P
Martinez-Hernandez
Antonio
A
Raghow
Rajendra
R
Solomon
Solomon S
SS
D000818:Animals; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy / Q000201:enzymology / Q000503:physiopathology*; D005934:Glucagon / Q000494:pharmacology; D006031:Glycosylation; D007328:Insulin / Q000494:pharmacology* / Q000627:therapeutic use; D007700:Kinetics; D008113:Liver Neoplasms; D008297:Male; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*
D007328:Insulin; D016329:Sp1 Transcription Factor; D005934:Glucagon; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase