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Pathways Regulating Intramyocellular Insulin Sensitivity and Metabolism in Health and Disease

Periodic Reporting for period 2 - PRIISM-HD (Pathways Regulating Intramyocellular Insulin Sensitivity and Metabolism in Health and Disease)

Reporting period: 2018-04-01 to 2019-03-31

Currently, there is an urgent need to understand the pathogenesis of major metabolic diseases such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). Our research focuses on identifying new genes that control key metabolic pathways implicated in the pathogenesis of these diseases, with the ultimate goal of uncovering new therapeutic targets. Specifically, we are interested in how glucocorticoids regulate metabolism in the liver, and how insulin controls glucose uptake [GLUT4 trafficking] in skeletal muscle. 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 hepatocyte and myocyte 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. At the conclusion of this action, we have successfully identified 6+ genes not previously associated with regulating metabolism by glucocorticoids in the liver. Future experiments will involve further characterizing these genes and exploring their potential as new therapeutic targets for the treatment of NAFLD and T2DM.
"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 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 escaped glucocorticoid-induced apoptosis. These ""glucocorticoid resistant"" cells will likely contain mutations in genes essential for glucocorticoid action in hepatocytes. These genes were subsequently identified using a combination of next-generation sequencing and powerful bioinformatic pipelines. Using this approach, we have successfully identified 6+ genes not previously associated with regulating metabolism by glucocorticoids in the liver. We anticipate publishing our initial findings from this study in late 2021.

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. Unfortunately, due to the COVID-19 pandemic, work on this project was been delayed. But it is anticipated that this screen will be completed during 2021, and our initial findings are expected to be ready for publication in early 2022."
This highly original and innovative approach to studying metabolism has uncovered new genes that are involved in regulating key metabolic pathways implicated in the pathogenesis of NAFLD and T2DM. Ultimately, this research could lead to the development of novel therapeutic strategies for the treatment of these major metabolic diseases.
Whole-genome CRISPR screening strategy to identify novel regulators of GLUT4 trafficking in myocytes