Question

You have worked on YFE1 (your favorite enzyme!) for awhile and know it is a serine/threonine kinase important in a crucial signal transduction pathway in the cell (not related to metabolism). You have developed in vitro and in vivo assays for its activity. You have the genetic tools to overexpress the protein and to produce mutants of the protein that show decreased function while still expressing the protein as well as RNAi to silence the gene. You know that eliminating the gene function leads to impaired cells that do not survive well because the pathway it is in is very important in the cell. Another researcher just published results showing that knocking out the YFE1 gene in mice leads to pups that survive less than 10 days. They found during day 1, the pups developed hyperglycemia which they believe was the cause of their early deaths. There has been no previous evidence YFE1 played a role in glucose metabolism; none of your work has ever shown a link to the pathway, but you acknowledge that you have never done experiments that directly test this. Given these new results, you want to see if you can figure out if YFE1 could be involved in glucose metabolism. Write a hypothesis. Outline the experiments you would conduct to test your hypothesis. Define the cells you will examine if that is your choice of experiments. (in particular, the tissue source). You may do any in vitro or in vivo assay – no whole animal studies. [Don't just say you are going to run a microarray. Tell me what cells you would use as control and experimental. Don't just say you are going to run a Northern blot - tell me what sample you are going to run it on and against what gene or sequence your probe will be. However, you should not write a Method section description of the experiment.]

Answer 1

We require glucose metabolism to produce energy. Glucose is generally stored in muscle and liver as glycogen and when required glycogen is broke down to glucose and relesed into blood. The glucose uptake rate in cells is controlled by insulin and glucagon.

For the glucose uptake assay , the cell line C2C12 muscle cells of mouse will be used.

C2C12 is myofibroblast which has been originated from muscle.

In this cell line YFE1 gene will be overexpressed and also knocked down using RNAi approach. Thw glucose uptake experient will be performed to see the difference between control and overexpressed or knocked down cells. To measure the glucose uptake, 2-deoxyglucose (2-DG) is used because of its structural similarity with glucose. When 2-deoxyglucose (2-DG) is added to the cells, cells will uptake it by glucose transporter. Inside the cell 2-deoxyglucose (2-DG) ts converted to 2-DG-6-phosphate (2-DG6P). However, as 2-DG6P can not further metabolized it becomes stored in cells. The stored 2-DG6P is proportional to the amount of 2-DG and can be quantified.