Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC93

Poster Communications

Phosphatidylinositol-4,5-bisphosphate is decreased in insulin resistance provoked by multiple conditions in L6 myotubes and 3T3-L1 adipocytes.

A. Ryan1, K. A. Hinchliffe1

1. Life Sciences, University of Manchester, Manchester, United Kingdom.

  • Table 1: Conditions used to induce insulin resistance, and the effect on PIP2 levels<\#13>

In insulin resistance, a risk factor for type 2 diabetes, cells fail to respond to insulin. In striated muscle and fat, this manifests as impaired insulin-stimulated glucose uptake due to reduced plasma membrane insertion of the glucose transporter GLUT4. In 3T3-L1 adipocytes and L6 myotubes, levels of the lipid phosphatidylinositol-4,5-bisphosphate (PIP2) are decreased in insulin resistance induced by chronic insulin stimulation1,2. However, the reason for this decrease, and whether other factors that induce insulin resistance affect PIP2, is unknown. We have investigated whether PIP2 levels are perturbed in insulin resistance induced by several factors (Table 1). The ability of these pre-treatments to abolish insulin-stimulated 3H 2-deoxy-glucose uptake was verified, and PIP2 was measured in cell extracts by quantitative blotting using a PIP2 probe derived from phospholipase C (PLC) δ3. PIP2 levels were significantly reduced under all conditions tested (Mann-Whitney test). PIP2 is regulated by PIP kinases, which synthesise it, phosphatases, which degrade it, and PLCs, which hydrolyse it. To investigate whether the observed PIP2 reductions resulted from altered kinase or phosphatase activity, experiments were performed on membrane preparations, using insulin or TNFα to induce insulin resistance in 3T3-L1 cells, and insulin or Angiotensin II in L6 myotubes. For kinase assays, membrane samples were incubated with γ32P-ATP. For phosphatase assays, membrane samples were incubated with 32P- PIP2 (produced using γ32P-ATP and a recombinant PIP-kinase). Lipids were then extracted, separated by thin layer chromatography and quantified by phosphoimaging. Surprisingly, no differences in kinase or phosphatase activity were observed. Thus, although this study shows a correlation between decreased PIP2 levels and insulin resistance induced in 3T3-L1 adipocytes and L6 myotubes by several conditions, the underlying mechanism remains unknown. However, the results are consistent with a possible role for PLC, and experiments to investigate this possibility are ongoing.

Where applicable, experiments conform with Society ethical requirements