Insulin activity on components of its signalling pathway and the effect of Ca2+ on these components

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Biol301 - Insulin activity on components of its signalling pathway and the effect of Ca2+ on these components

Abstract

Insulin is a substrate which is involved in a signalling pathway concerned with blood glucose levels along with IR, IRS1 and PI-3 kinase. The activity of PI-3 kinase is affected by the influx of Ca2+ as was demonstrated by the addition of Ca2+ channel inhibitors to hepatocytes where PI-3 kinase activity dropped. As no effect to the phosphorylation of IR and IRS1 (insulin receptors and substrates) it was determined that the influx Ca2+ has a specific role in the pathway, ie only where PI-3 kinase is involved, in the latter stages of the pathway.

Introduction

The aim of this paper is to look at the effect of insulin, insulin receptors (IR), insulin receptor substrate 1 (IRS1) and phosphatidyinositol-3 kinase (PI-3 kinase) on rat hepatocytes. Insulin is a peptide hormone which is secreted by ß-cells which are found in the islets of langerhans and is secreted as a response to rising blood glucose levels. It is part of a complex signalling pathway which involves a large number of molecule-molecule complexes and is the stimulus of many different functions such as glycogenesis, glucose transport and the transcription of genes.  IR, IRS1 and PI-3 kinase are also key components in the signalling pathway.

IR is a diametric tyrosine kinase which is composed of α chains joined by disulphide bridges on the outside of the cell, two ß transmembrane spanning chains and two tyrosine kinase domains or TDKs. If an insulin molecule binds to the receptor the TDKs autophosphorylate, each TDK phosphorylating the other in a process called transphosphorylation. IRS1 is part of the IRS family of molecules and is a key mediator in insulin signalling. It acts as a docking protein between the insulin receptor and intracellular signalling molecules with an SH2 domain and is involved with the activation of PI-3 kinase which is another key component in the signalling pathway. The PI-3 kinase enzyme phosphorylates phosphatidyinositol-4,5-bisphosphate (PIP2), a plasma membrane lipid, to phosphotidyinositol-3,4,5-triphosphate (PIP3) which acts as a secondary messenger in the pathway with the activation of protein kinase B (PKB).

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Experimental

The hepatocytes used in the experiment were extracted from rats using the procedure outlined by by Benzeroual, Pandey, Srivastava, van de Werve and Haddad {1}. The assay which was performed to measure PI-3 kinase followed the same procedure as Fukui and Hanafusa {3} but with modifications as outlined in {1}. The procedures for IR and IRS1 experimantals are as described in {1}.

Results

Figs 1-5 are graphical representations of the data collected from the experimental and can be found at the end of the paper.

Fig.1 A plot showing PI-3 kinase activity against ...

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