INSULIN-STIMULATED CALMODULIN GENE-EXPRESSION IN RAT H-411E CELLS CANBE SELECTIVELY BLOCKED BY ANTISENSE OLIGONUCLEOTIDES

Reduced expression of calmodulin (CaM) and decreased activity of low K m cyclic AMP (cAMP) phosphodiesterase (PDE) are associated with uncont rolled diabetes. This condition can be readily mimicked in hepatocytes cultivated in insulin-depleted medium (Solomon, et al J. Lab. Clin. M ed. in press, 1994). To investigate the relationship between CaM and l ow Km cAMP PDE gene expression in response to insulin, we specifically blocked expression of the three CaM genes by antisense oligonucleotid es under insulin-deficient and -sufficient conditions in a rat hepatom a cell line, H-411E. We observed that both the low Km cAMP PDE activit y and the steady state levels of CaM mRNA were increased in response t o insulin by 50 and 100%, respectively. When antisense oligonucleotide to CaM I, II or III was added to the cultures, only CaM I antisense o ligonucleotide blocked insulin stimulation of both CaM I mRNA and prot ein with concommittant marked inhibition of insulin's expected stimula tion of low Km cAMP PDE. Furthermore, in another experiment utilizing both antisense and oligonucleotide probes specific for CaM I,II, or II I together, only CaM I mRNA expression was blocked. We conclude that H -411E cells respond to insulin by appropriate increases in CaM transcr ipts. Furthermore, the stimulatory effect of insulin on both CaM synth esis and activation of low Km cAMP PDE could be blocked by antisense t o CaM I, but not II or III genes. Therefore, in addition to the above conclusions, H-411E hepatoma cells appear to be an excellent in vitro system to explore the molecular mechanisms by which CaM and low Km cAM P PDE genes are regulated in the diabetic state. (C) 1995 Academic Pre ss, Inc.