Finding Links between Obesity and Diabetes: Using Diacylglycerol Kinase to Regulate Insulin Signaling

In the U.S., two-thirds of adults are overweight or obese, with 35.7% of them - nearly 78 million people – obese. What used to be a disease that only afflicted adults is now also increasingly being diagnosed in children, contributing to ever-growing obesity rates. In particular, obesity and diabetes are closely linked, with obese people at higher risk for developing type 2 diabetes. All this only further intensifies the urgent need to address obesity and type 2 diabetes. In particular, we need to understand how the two conditions are linked and what metabolic processes are involved, in order to develop treatment and prevention strategies that will ultimately reduce the burden of type 2 diabetes on healthcare. My project looks at how the triacylglycerol synthesis and insulin signaling pathways are linked. In particular, we are studying how lipid intermediates cause insulin resistance in hepatocytes. We are interested in whether phosphatidic acid (PA) and diacylglycerol (DAG) affect Akt phosphorylation, and the mechanism by which PA and DAG might inhibit insulin signaling. Diacylglycerol kinase (DGK) is the enzyme that catalyzes conversion of DAG to PA, and was used as a tool to manipulate the cellular content of PA and DAG. The content and activity of DGK was adjusted by DGK overexpression in mouse hepatocytes, thus altering the balance of PA and DAG. The physiological consequences were then used to determine how insulin signaling is affected. Our hypothesis is that DGK regulates insulin signaling by changing intracellular PA and DAG levels. The isoform used was the DGKθ isoform. Our results showed that PA is associated with impaired insulin action in mouse hepatocytes, but DAG is not.Bachelor of Science in Public Healt

Inhibited Insulin Signaling in Mouse Hepatocytes Is Associated with Increased Phosphatidic Acid but Not Diacylglycerol

Although an elevated triacylglycerol content in non-adipose tissues is often associated with insulin resistance, the mechanistic relationship remains unclear. The data support roles for intermediates in the glycerol-3-phosphate pathway of triacylglycerol synthesis: diacylglycerol (DAG), which may cause insulin resistance in liver by activating PKCϵ, and phosphatidic acid (PA), which inhibits insulin action in hepatocytes by disrupting the assembly of mTOR and rictor. To determine whether increases in DAG and PA impair insulin signaling when produced by pathways other than that of de novo synthesis, we examined primary mouse hepatocytes after enzymatically manipulating the cellular content of DAG or PA. Overexpressing phospholipase D1 or phospholipase D2 inhibited insulin signaling and was accompanied by an elevated cellular content of total PA, without a change in total DAG. Overexpression of diacylglycerol kinase-θ inhibited insulin signaling and was accompanied by an elevated cellular content of total PA and a decreased cellular content of total DAG. Overexpressing glycerol-3-phosphate acyltransferase-1 or -4 inhibited insulin signaling and increased the cellular content of both PA and DAG. Insulin signaling impairment caused by overexpression of phospholipase D1/D2 or diacylglycerol kinase-θ was always accompanied by disassociation of mTOR/rictor and reduction of mTORC2 kinase activity. However, although the protein ratio of membrane to cytosolic PKCϵ increased, PKC activity itself was unaltered. These data suggest that PA, but not DAG, is associated with impaired insulin action in mouse hepatocytes