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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PRIISM-HD (Pathways Regulating Intramyocellular Insulin Sensitivity and Metabolism in Health and Disease)

Teaser

Summary

Currently, there is an urgent need to understand the pathogenesis underpinning major metabolic diseases, such as type 2 diabetes, due to their dramatically increasing prevalence. Our research focusses on identifying new genes controlling key metabolic pathways in response to important hormonal cues, such as insulin and glucocorticoids, with the ultimate goal of uncovering new therapeutic targets. Specifically, we are interested in delineating the regulatory features of how insulin controls glucose uptake [GLUT4 trafficking] into skeletal muscle, and how glucocorticoids regulate gene expression in the liver. Our approach harnesses whole-genome CRISPR-cas9 based technologies, which have transformed many areas of biological research since the discovery that the bacterial immune system could be re-engineered for gene editing in mammalian cells. Using both myocyte and hepatocyte cell-based models, we are employing this powerful genetic tool to simultaneously screen thousands of genes (on a genome-wide scale) for their involvement in these metabolic pathways.

In summary, our whole-genome CRISPR-cas9 based screening approach to studying metabolism has the potential to significantly advance our understanding of key pathways driving the development of major metabolic disease.

Work performed

To date, we have successfully generated a human hepatocyte reporter cell line that ties glucocorticoid receptor transcriptional activity with apoptosis and cell death. We have now successfully used this reporter to perform a whole-genome scale CRISPR-cas9 screen to identify positive regulators of glucocorticoid receptor transcriptional activity. Briefly, this involved infecting the reporter cells with a pooled lentiviral knockout library of guide RNA molecules (targeting 18,000 genes, 4 guides/gene), and harvesting the cells that survive following treatment with glucocorticoids. These surviving cells will most likely contain mutations in genes essential for glucocorticoid receptor transcriptional activity. We are currently preparing the DNA samples generated by this screen for sequencing. The genes that correspond to the guide RNAs enriched in the surviving cells will be identified and ranked using powerful computational algorithms, before top-ranking candidates are interrogated in further studies.

For the insulin-mediated GLUT4 trafficking screen: we have successfully generated a human myocyte reporter cell line that allows us to detect plasma membrane-associated GLUT4 following stimulation with insulin. We are currently optimising the final genome-wide CRISPR-cas9 screening protocol that will enable us to identify both positive and negative regulators of insulin-stimulated GLUT4 trafficking in myocytes.

Final results

A list of fully validated top hits from both screens is anticipated to be available from mid-2020. Top-ranking targets will then be interrogated in further studies, with preliminary results expected towards the end of 2020.

This highly original and innovative approach to studying metabolism has the potential to significantly advance our understanding of how important metabolic pathways are regulated by hormones. Ultimately, this research could lead to novel therapeutic strategies for the treatment of major metabolic diseases, such as type 2 diabetes.