Investigation of Phosphatidylinositol 5-Phosphate’s Role in Insulin-Stimulated Glucose Uptake in a Skeletal Muscle Cell Line

Access to files

•   FULL-TEXT.PDF (pdf)

Abstract

Phosphatidylinositol 5-phosphate (PtdIns5P) is the least well-characterised member of the phosphoinositide family of essential regulatory phospholipids. PtdIns5P levels are altered within cells in response to a number of stimuli and evidence is accumulating to suggest that it possesses important functions in cellular signalling. However, the physiological role of this lipid remains imperfectly understood. Previous studies have shown that PtdIns5P is elevated in adipocytes in response to insulin, and microinjection of PtdIns5P into these cells promotes plasma membrane insertion of the insulin-regulated glucose transporter GLUT4 (Sbrissa et al., 2004). This finding suggests a potential role of PtdIns5P as a mediator in insulin-stimulated glucose uptake, a process essential for efficient glucose homeostasis. As approximately 75% of postprandial glucose disposal is carried out by skeletal muscle, it is important to investigate the role of PtdIns5P in the response of this tissue to insulin. Therefore, this work has used differentiated myotubes of the rat muscle cell line, L6, to explore the effects of altered PtdIns5P levels on insulin-stimulated glucose uptake. This cell model had not been previously used in the laboratory so it first required characterisation. Here insulin is shown to stimulate a transient increase of PtdIns5P in L6 myotubes, indicative of a signalling role in response to insulin. This project developed several tools to further investigate this potential role for PtdIns5P in the insulin response of myotubes. One such development was the successful overexpression of the PtdIns5P 4-kinase PIP4KIIα in these cells, which was able to abolish the insulin-stimulated PtdIns5P rise. This correlated with a loss of insulin-stimulated glucose uptake (upon PIP4KIIα expression). Interestingly, artificial elevation of PtdIns5P in L6 myotubes increases glucose uptake in the absence of stimulation. This phenomenon appears to result from the activation of PI3-kinase signalling, as it is abolished by the PI3-kinase inhibitor wortmannin, and involves activation of the PI3-kinase effector Akt. These results are consistent with the idea that insulin-stimulated PtdIns5P production contributes to the robust PI3-kinase/Akt activation necessary for insulin-stimulated glucose uptake in muscle.

Layman's Abstract

Blood glucose levels need to be tightly regulated as loss of this regulation can cause diseases such as type 2 diabetes. The high levels of glucose seen in badly managed type 2 diabetes are damaging to the body’s tissues and cause further (secondary) complications. Some examples of these are nerve and kidney damage and loss of sight. Type 2 diabetes is a major health problem in the UK today. It affects over 2 million people and its care costs the NHS around £1 million per hour to provide. This expense is largely due to the irreversible nature of diabetes; once it has developed, there is no real cure. Furthermore, most cases of diabetes are identified long after its development, by presentation of secondary complications. To reduce the burden of type 2 diabetes on both patients and healthcare organisations, better treatments and earlier diagnostic tools are required. To achieve this, more research into the finer detail of how the body’s tissues regulate blood glucose is needed. Insulin is a hormone released by the pancreas following a meal. Its principal role is to normalise blood sugar levels, which are elevated by the digestion of dietary starch and the release of the resulting glucose into the bloodstream. Muscle and fat tissues play a part in this by responding to insulin and taking up the excess glucose. Muscle is especially important in maintaining healthy blood glucose regulation; it accounts for the majority of glucose uptake in response to insulin. For this reason it has been chosen for further study in this project. Not everything is known about how muscle (and fat) achieves glucose uptake, so any work which may further this knowledge is important. Previously, a molecule commonly known as PI5P has been implicated in glucose uptake in fat tissue. Its levels rise in fat cells in response to insulin. The work here concerns the role of PI5P, in the process of glucose uptake by muscle in response to insulin. This project found a similar rise in PI5P in a muscle cell model and set about investigating this in more detail.

Keyword(s)

GLUT4; Glucose uptake; Insulin; L6; PtdIns5P; phosphatidylinositol 5-phosphate; skeletal muscle cell line

Bibliographic metadata

Institutional metadata

Record metadata