Insulin-Stimulated Calmodulin Gene Expression in Rat H-411E Cells Can Be Selectively Blocked by Antisense Oligonucleotides

S. S. Solomon, M. R. Palazzolo, J. A. Smoake, Rajendra Raghow

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Abstract

Reduced expression of calmodulin (CaM) and decreased activity of low Km cyclic AMP (cAMP) phosphodiesterase (PDE) are associated with uncontrolled diabetes. This condition can be readily mimicked in hepatocytes cultivated in insulin-depleted medium (Solomon, et al J. Lab. Clin. Med. in press, 1994). To investigate the relationship between CaM and low Km cAMP PDE gene expression in response to insulin, we specifically blocked expression of the three CaM genes by antisense oligonucleotides under insulin-deficient and -sufficient conditions in a rat hepatoma cell line, H-411E. We observed that both the low Km cAMP PDE activity and the steady state levels of CaM mRNA were increased in response to insulin by 50 and 100%, respectively. When antisense oligonucleotide to CaM I, II or III was added to the cultures, only CaM I antisense oligonucleotide blocked insulin stimulation of both CaM I mRNA and protein with concommittant marked inhibition of insulin′s expected stimulation of low Km cAMP PDE. Furthermore, in another experiment utilizing both antisense and oligonucleotide probes specific for CaM I,II, or III together, only CaM I mRNA expression was blocked. We conclude that H-411E cells respond to insulin by appropriate increases in CaM transcripts. Furthermore, the stimulatory effect of insulin on both CaM synthesis and activation of low Km cAMP PDE could be blocked by antisense to 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 cAMP PDE genes are regulated in the diabetic state.

Original languageEnglish (US)
Pages (from-to)921-930
Number of pages10
JournalBiochemical and Biophysical Research Communications
Volume210
Issue number3
DOIs
StatePublished - May 25 1995
Externally publishedYes

Fingerprint

Antisense Oligonucleotides
Calmodulin
Gene expression
Rats
Insulin
Gene Expression
Phosphoric Diester Hydrolases
Cyclic AMP
Genes
Messenger RNA
Hepatocellular Carcinoma
Insulins
Oligonucleotide Probes
Medical problems
Hepatocytes
Chemical activation

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Insulin-Stimulated Calmodulin Gene Expression in Rat H-411E Cells Can Be Selectively Blocked by Antisense Oligonucleotides. / Solomon, S. S.; Palazzolo, M. R.; Smoake, J. A.; Raghow, Rajendra.

In: Biochemical and Biophysical Research Communications, Vol. 210, No. 3, 25.05.1995, p. 921-930.

Research output: Contribution to journalArticle

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abstract = "Reduced expression of calmodulin (CaM) and decreased activity of low Km cyclic AMP (cAMP) phosphodiesterase (PDE) are associated with uncontrolled diabetes. This condition can be readily mimicked in hepatocytes cultivated in insulin-depleted medium (Solomon, et al J. Lab. Clin. Med. in press, 1994). To investigate the relationship between CaM and low Km cAMP PDE gene expression in response to insulin, we specifically blocked expression of the three CaM genes by antisense oligonucleotides under insulin-deficient and -sufficient conditions in a rat hepatoma cell line, H-411E. We observed that both the low Km cAMP PDE activity and the steady state levels of CaM mRNA were increased in response to insulin by 50 and 100{\%}, respectively. When antisense oligonucleotide to CaM I, II or III was added to the cultures, only CaM I antisense oligonucleotide blocked insulin stimulation of both CaM I mRNA and protein with concommittant marked inhibition of insulin′s expected stimulation of low Km cAMP PDE. Furthermore, in another experiment utilizing both antisense and oligonucleotide probes specific for CaM I,II, or III together, only CaM I mRNA expression was blocked. We conclude that H-411E cells respond to insulin by appropriate increases in CaM transcripts. Furthermore, the stimulatory effect of insulin on both CaM synthesis and activation of low Km cAMP PDE could be blocked by antisense to 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 cAMP PDE genes are regulated in the diabetic state.",
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