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Insulin-stimulated calmodulin gene expression in rat H-411E cells can be selectively blocked by antisense
S S Solomon1, M R Palazzolo, J A Smoake
1Research Service, Veterans Affairs Medical Center, Memphis, TN 38104, USA.
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.
Insights
Insulin stimulates calmodulin (CaM) and cyclic AMP phosphodiesterase (PDE) in liver cells. Blocking CaM I gene expression with antisense oligonucleotides prevented this stimulation, suggesting CaM I
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Uncontrolled diabetes is linked to reduced calmodulin (CaM) expression and decreased low Km cyclic AMP (cAMP) phosphodiesterase (PDE) activity.
- Hepatocytes in insulin-depleted media mimic conditions associated with diabetes.
- Insulin's role in regulating CaM and low Km cAMP PDE gene expression requires further investigation.
Purpose of the Study:
- To investigate the relationship between CaM and low Km cAMP PDE gene expression in response to insulin.
- To determine the specific CaM gene isoform involved in insulin signaling.
- To explore the utility of H-411E hepatoma cells as an in vitro model for studying diabetes-related molecular mechanisms.
Main Methods:
- H-411E rat hepatoma cells were cultured in insulin-deficient and insulin-sufficient conditions.
- Specific antisense oligonucleotides were used to block the expression of CaM I, II, and III genes.
- CaM mRNA and protein levels, as well as low Km cAMP PDE activity, were measured.
Main Results:
- Insulin significantly increased both low Km cAMP PDE activity (50%) and CaM mRNA levels (100%).
- Antisense oligonucleotides targeting CaM I, but not CaM II or III, blocked insulin's stimulatory effects on CaM I mRNA and protein.
- CaM I antisense treatment also inhibited insulin's stimulation of low Km cAMP PDE activity.
Conclusions:
- H-411E cells demonstrate insulin-induced increases in CaM transcripts.
- Insulin's stimulatory effects on CaM synthesis and low Km cAMP PDE activation are mediated specifically through CaM I.
- H-411E hepatoma cells provide a valuable in vitro model for studying the molecular regulation of CaM and low Km cAMP PDE in diabetes.