When investigating the underlying mechanisms of glucose uptake promoting effects, the GLUT4 levels of plasma membrane (PM) and cell lysate (CL) are determined in order to assess the GLUT4 translocation into the plasma membrane. Here, the insulin receptor beta is used as an internal standard to determine the GLUT4 levels in the PM and CL, and I want to know the reason behind this logic.
Insulin promotes massive entry of the glucose into the cell, which induces the internalization of the signal and the activation of glucose transporter GLUT4 following the concentration gradian.
Have seen papers using actin and GAPDH for glut4 for total lysates. If they use IR-beta sub unit for plasma membrane glut4 content, it makes sense. isnt it?
I wouldn't use Insulin receptor as an internal standard. As Barnabe mentioned the Insulin Receptor is internalised after insulin binds to it, therefore it is not a good loading control. I would use actin or GAPDH. You can use insulin receptor as a PM marker to make sure you have purified the PMs.
Following insulin binding to its receptors at plasma membrane and internalization of the beta-subunit, succession of signals follow, ending with translocation of of GLUT-4 from cytoplasm to plasma membrane. Therefore, there is a sort of coupling between inernalisation of beta-subunits and GLUT-4 onplasma membrane .
Because the GLUT4 transporter is the only the twelve glucose transporters that is sensitive to insulin.
Hence, a significant reduction in Glut4 mRNA levels, IR-β and IRS-1 be involved in the decrease in insulin sensitivity.
Moreover, Desrois reported the association between decreased IR-β, IRS-1, and Glut4 protein levels and insulin resistance in type 2 diabetic rats. (Desrois et al., DOI: http://dx.doi.org/10.1016/j.cardiores.2003.11.021 288-296).
Glucose uptake mediated by insulin involves activated PDK1 to phosphorylate some isoforms of protein kinase C (PKC) which phosphorylates intracellular vesicles containing the glucose transporter GLUT4, which results in their migration and fusion with the plasma membrane, GRB2 activation results in signal transduction through G protein monomer, RAS. These transcriptional effects regulate increasing glucokinase, pyruvate kinase liver (L-PK), lipoprotein lipase (LPL), fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC) gene expression, and decreased glucose 6-phosphatase, fructose 1,6 bisphosphatase and phosphoenolpyruvate carboxykinase-(PEPCK) gene expression.
Thank you very much for all for your informative answers. I found an article which describes the underlying mechanism of theasinensin-induced glucose uptake into skeletal muscle cells. Here, the level of GLUT4 in the Plasma Membrane (PM) has been calculated as a ratio of the levels of GLUT4 to IR-beta, which has been expressed as a percentage to total GLUT4 level in the cell lysate (CL). The authors observed a significant increase in the GLUT4 level in the PM, while there was no sig. difference in the level of IR-beta in the PM and CL. This observation has led them to conclude that theasinensin promoted GLUT4 translocation from inter-cellular vesicles to the PM. I am thinking the logic behind this.
http://www.sciencedirect.com/science/article/pii/S0006295213007132
- Badges
- Science topic