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 time which was stimuated by adding 10nM of insulin to the hepatocytes at each time interval. The activity of PI-3 kinase was measured as the increase over the base line. Each data point represents the mean of 8 repeats and +/- the standard error values.
Fig.2 A plot showing PI-3 kinase activity against the logged concentration of insulin. The PI-3 kinase activity is expressed as a fold increase above no insulin. Each data point represents the mean of 6 repeats and +/- the standard error values.
Fig.3 A bar chart showing PI-3 kinase activity in the hepatocytres under six different treatments; control; 10nM insulin added; verapamil (200µl) then 10nM insulin; nickel chloride (500µl) the 10nM insulin added; gadolinium chloride (10 µl) the 10nM insulin added and EGTA (4mM) then 10nM insulin added. The activity of PI-3 kinase is shown as fold increase over the control result. Each bar is the calculated mean of 8 repeats and +/- the standard error.
Fig4. A bar chart showing the extent of IR phosphorylation in the hepatocytes under the treatments in Fig.2. The extent of IR phosphorylation was expressed as % insulin only and each bar is the calculated mean of 8 repeats and +/- the standard error.
Fig5. A bar chart showing the extent of IRS1 phosphorylation in the hepatocytes under the treatments shown in Fig.3 IRS1 phosphorylation was measured as % of insulin only. Each bar represents the mean of 8 repeats and +/- the standard error.
The activity of PI-3 kinase in the hepatocytes was measured in the presence of 10nM of insulin at time intervals of 0,1,3,5,10 and 15 minutes. Fig.1 shows that at 0 mins the activity of PI-3 kinase was 1 (above the control of 0nM insulin). Each reading was then taken as a fold increase over 1. The activity of PI-3 kinase increases up to 3 mins where it peaks at a value of 3.9. After this the activity decreases (5 mins) then levels off over 5-15 minutes. PI-3 kinase activity was then measured at different concentrations of insulin added (0, 0.1, 1, 10 and 100nM) and as Fig.2 shows, as the concentration of insulin increases so does PI-3 kinase activity in the hepatocytes.
Fig shows PI-3 kinase activity measured under several different treatments; 10nM insulin added; verapamil (200µl) then 10nM insulin; nickel chloride (500µl) the 10nM insulin added; gadolinium chloride (10 µl) the 10nM insulin added and EGTA (4mM) then 10nM insulin added. Nickle chloride and gadolinium chloride are Ca2+ channel inhibitors, while EGTA binds to external Ca2+ and prevents it from entering Ca2+ channels thus preventing entry to the cell. The hepatocytes which were treated were all apart from the control and the insulin only cells, the remaining cells being treated for 15 minutes prior to 10nM of insulin being added. The activity of PI-3 kinase was measured as a fold increase over the control. This increase only occurs in the addition of insulin only and the verapamil treatment. Treatment with nickel chloride (500µl) the 10nM insulin added; gadolinium chloride (10 µl) the 10nM insulin added and EGTA (4mM) then 10nM insulin added all cause a decrease in the activity of PI-3 kinase.
The phosphorylation of IR and IRS1 were measured as described. Fig.4 shows that the different treatments have no significant difference on the phosphorylation of IR other than over the control. Although the agents increase phosphorylation, they do not show a difference beween the effect that they have. Fig.5 shows that the same is true for IRS1 phosphorylation.
Discussion
From the data collected and the representation in graphical format it can be determined that the activity of PI-3 kinase is affected by insulin activity. Fig.2 shows that as insulin concentrations rise, the activity of PI-3 kinase increases, therefore showing that PI-3 kinase is involved in the signalling pathway.
It can also be determined that Ca2+ directly affects the pathway. This is shown in Fig.3. When the hepatocytes were treated with Ca2+ inhibitors (nickel chloride and gadolinium chloride) PI-3 kinase activity drops. The same is shown when the cells are treated with EGTA which prevents the cells from taking up Ca2+. As there is no affect on the phosphorylation of IR and IRS1 when the inhibitors are introduced to the cells it is clear that only the earlier stages of the insulin signalling are affected by Ca2+.
References
{1}Insulin-induced Ca2+ entry in hepatocytes is important PI 3-kinase activation, but not for insulin receptor and IRS-1 tyrosine phosphorylation. Kenza Benzeroual, Sanjay K. Pandey, Ashok K. Srivastava, Gérald van de Werve and Pierre S. Haddad. Biochimica et Biophysica Acta, 1495 (2000) 14-23.
{2}Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. Sesti G, Federici M, Hribal ML, Lauro D, Sbraccia P, Lauro R. THE FASEB JOURNAL 14 (2001) 2099-111
{3} Y. Fukui, H. Hanafusa, Mol. Cell. Biol. 9 (1989) 1651-1658.
{4} Molecular Cell Biology, Lodish, Berk, Matsudaira, Kaiser, Krieger, Scott, Zipursky, Darnell. W.H. Freeman and company. 2003.
{4} Lecture notes/handouts J Owen-Lynch 2004