Mitoregulin and its Effect on Pancreatic Beta Cells Under Glucolipotoxic Conditions​
I will be working to understand the role of mitoregulin (MTLN) within pancreatic beta cells, specifically within an INS-1 cell line. The goal of my research is to understand if MTLN will be able to help INS-1 cells under glucolipotoxicity as exhibited in type 2 diabetes. MTLN has been shown in cardiac, muscle, plasma and fat cells to affect fatty acid oxidation (FAO), radicalized oxygen species (ROS), lipolysis, and has been shown to associate with multiple proteins and complexes within the electron transport chain (ETC). Glucolipotoxicity has been shown to cause pancreatic beta cell dysfunction through the creation of ceramides from chronic exposure to lipids creating ceramides, inhibiting transcription factors for insulin. It has also been shown to cause increased ER stress, ROS which can damage proteins and other structures within the cell, and an increase in cytokines. These culminating factors ultimately leading to apoptosis.
Methodology
To investigate the indirect and direct effects of MTLN within pancreatic beta cells under glucolipotoxicity, various methods and materials will be used.
Cell Culturing
INS-1 cells have to be grown as to have controls for experiments and be able to test for necessary conditions.
Metabolism
If possible, I would like to conduct a sea horse assay to see how FAO is upregulated or to see if glycolysis is altered.
Transfection
Transfections will be used to input over/under expression plasmids in order to knock out or overexpress MTLN.
Plasmids
Plasmids will be created to overexpress and knock out MTLN. The over expression plasmid will also be used to see where MTLN localizes as it will have GFP attached. Silencing will occur through the use of a siRNA specific to MTLN.
Survivability
Survivability will be assessed through cells incubate with glucolipotoxic media (GLM) (0.25 mM Palmitate, and 30 mM glucose). This will be followed up by an MTS assay to assess metabolic activity and thus survivability.
ELISAs
ELISAs will be conducted to see how cells insulin secretion and cytokine production is affected by glucolipotoxicty.
Western Blotting
Western blotting will be used to see if proteins are present within cells
ROS
ROS quantification within cell lysate will be used to see if ROS production is higher in cells under glucolipotoxicity.
Localization
MTLN will be visualized with a confocal microscope to show where MTLN is located within the cell