Effects of tacrolimus (FK506) on human insulin gene expression, insulin mRNA levels, and insulin secretion in HIT-T15 cells.
FK506 (tacrolimus) is an immunosuppressive drug which interrupts Ca2+-calmodulin-calcineurin signaling pathways in T lymphocytes, thereby blocking antigen activation of T cell early activation genes. Regulation of insulin gene expression in the beta cell may also involve Ca2+-signaling pathways and FK506 has been associated with insulin-requiring diabetes mellitus during clinical use. The purpose of this study was to characterize the effects of FK506 on human insulin gene transcription, insulin mRNA levels, and insulin secretion using as a model the HIT-T15 beta cell line. FK506 had no acute effect on insulin secretion in the HIT cell, but caused a reversible time- and dose-dependent (10(-9)-10(-6) M) decrease in HIT cell insulin secretion. Decreased insulin secretion in the presence of FK506 was also accompanied by a dose-dependent decrease in HIT cell insulin content, insulin mRNA levels, and expression of a human insulin promoter-chloramphenicol acetyl transferase (CAT) reporter gene. FK506 decreased HIT cell expression of the human insulin promoter-CAT reporter gene by 40% in the presence of both low (0.4 mM) at high (20 mM) glucose concentrations. Western blot analysis of HIT cell proteins gave evidence for the presence of calcineurin in the HIT cell. These findings suggest that FK506 may have direct effects to reversibly inhibit insulin gene transcription, leading to a decline in insulin mRNA levels, insulin synthesis, and ultimately insulin secretion.
Redmon
J B
JB
Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, University of Minnesota, Minneapolis 55455, USA. Redmo001@maroon.tc.umn.edu.
Olson
L K
LK
Armstrong
M B
MB
Greene
M J
MJ
Robertson
R P
RP
D015152:Blotting, Northern; D015153:Blotting, Western; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology*; D014162:Transfection / Q000235:genetics
D002148:Calmodulin-Binding Proteins; D007166:Immunosuppressive Agents; D007328:Insulin; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D016559:Tacrolimus