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Evidence that amiloride antagonises insulin-stimulated protein phosphorylation by inhibiting protein kinase activity
Abstract:
The diuretic drug amiloride antagonises the insulin-dependent increase in phosphorylation of ATP-citrate lyase in hepatocytes isolated from rats that had been starved and refed a fat-free diet. Studies with a range of protein kinases and protein phosphatases that have been shown to phosphorylate or dephosphorylate purified ATP-citrate lyase in vitro revealed that amiloride was a non-specific inhibitor of all protein kinases tested, but did not significantly affect any of the protein phosphatases. These results cast doubt on previous claims that growth factors stimulate phosphorylation of ribosomal protein S6 by activating an amiloride-sensitive Na+/H+ exchange system, and that insulin inhibits a protein phosphatase that is activated by amiloride.
Insights
Amiloride, a diuretic, non-specifically inhibits protein kinases involved in cell signaling. This finding questions previous research on growth factors and insulin
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Insulin stimulates ATP-citrate lyase phosphorylation in rat hepatocytes.
- Amiloride is a diuretic drug with known cellular effects.
- Previous studies suggested amiloride-sensitive mechanisms in growth factor and insulin signaling.
Purpose of the Study:
- To investigate the effect of amiloride on insulin-stimulated ATP-citrate lyase phosphorylation.
- To determine the specificity of amiloride's action on protein kinases and phosphatases involved in this pathway.
Main Methods:
- Isolated rat hepatocytes from starved and refed animals were used.
- In vitro studies were conducted with purified ATP-citrate lyase, protein kinases, and protein phosphatases.
- Amiloride's inhibitory effects were assessed on various enzymatic activities.
Main Results:
- Amiloride antagonized the insulin-dependent increase in ATP-citrate lyase phosphorylation.
- Amiloride demonstrated non-specific inhibition of all tested protein kinases.
- Amiloride did not significantly affect the activity of tested protein phosphatases.
Conclusions:
- Amiloride's non-specific inhibition of protein kinases challenges its proposed role in specific signaling pathways.
- The findings cast doubt on previous claims linking amiloride-sensitive Na+/H+ exchange to growth factor-induced ribosomal protein S6 phosphorylation.
- This study questions the proposed mechanism of insulin action involving amiloride-activated protein phosphatases.