Background

Diabetes has been classed as an epidemic by the World Health Organisation (WHO) with the number of people living with diabetes over four times what it was four decades ago. WHO predict that the disease will soon be the seventh largest killer across the globe.

There is currently no cure for diabetes but symptoms are largely managed through treatment with insulin and/or through diet and lifestyle. With 422 million people living with diabetes worldwide and 1.6 million deaths annually directly attributed to diabetes, a cure is still the holy grail.

Scientists at Strathclyde have developed an assay system to allow ‘hits’ for anti-diabetic and anti-obesity drugs to be identified quickly and cheaply, ready for further analysis.

Technology Overview

A major consequence of insulin action is an increase in the rate of glucose transport into fat and muscle. This is achieved through regulated trafficking of the facilitative glucose transporter GLUT4 from insulin-sensitive intracellular stores to the plasma membrane, a process terms ‘translocation’. A robust easy-to-use assay of insulin-stimulated GLUT4 translocation has been developed which is a useful read-out for insulin sensitivity and screening potential of antagonistic or synergistic small molecules or other drugs.

Researchers at Strathclyde employ a stable HeLa cell line engineered to express HA-GLUT4-GFP. This was driven by a need to establish a cell line that can be readily genetically manipulated and imaged, and by a lack of reagents that can target endogenous GLUT4 specifically at the plasma membrane. HA-GLUT4-GFP is widely used in studies of GLUT4 trafficking; it has been extensively characterised and validated. GFP reports on the distribution of GLUT4 and the HA-epitope (only accessible to anti-HA added to the media of non-permeabilised cells) allows quantification of levels of GLUT4 in the plasma membrane. GFP also reports on total levels of GLUT4.

This assay lends itself to FACS analysis and can also be multiplexed in 96-well plates.

Benefits

Rapid and robust response in easily grown cell lines
FACS assay – large numbers of cells can be quantified
Potential to multiplex into 96-well plates
Simultaneous assay of insulin-stimulated translocation and total GLUT4 levels
Confirmed ‘hits’ can be further analysed in adipocyte lines expressing the same reporter
Cardiovascular lines are in development

Applications

Anti-diabetic and anti-obesity drug screening and trials

Assay System of Insulin Action and Sensitivity

Seeking