Characterization of a ULK1 Agonist

The purpose of this project was to identify an agonist of autophagy. The agonist had to work specifically through increasing the expression level of ULK1 within the cell. ULK1 was used as the main marker of autophagy as it has an established role in the formation of the autophagosome, a step that is known to be crucial in the autophagy process, especially in the degradation of mitochondria. Autophagy is a cellular process that serves to breakdown and recycle cellular components that are malfunctioning or collecting above a useful level. Specifically, autophagy’s role in the degradation of poorly functioning mitochondria represents an interesting line of questioning into whether autophagy is involved in the presentation of the diabetic disease state; however the role of autophagy could not be fully characterized without a method to artificially increase the expression of autophagy within the cell. While it is easy enough to test in an autophagy knock-down model, it proves more difficult to create conditions where autophagy had been arbitrarily increased. Thus, it proved beneficial for an autophagy agonist to be identified and characterized. At the start of this project, forskolin was identified as a potential autophagy agonist due to literature evidence proposing it results in an increase in CREB, a potential transcription factor of ULK1. As such, an experimental protocol was developed to test forskolin’s effect on autophagy in an in vitro hepatic cell model, as measured through its effect on ULK1 levels and related autophagy factors. Following the experimentation protocol, it was determined that forskolin does reliably increase the expression levels of ULK1 in hepatic cells, however it did not affect autophagy induction. However, when the cell was exposed to insulin, the treatment of forskolin to up-regulate ULK1 expression did affect the insulin signaling pathway. This may indicate that forskolin affects autophagy through ULK1 modulation of the insulin signaling pathway. To fully characterize forskolin’s effect on autophagy, an in vivo model should also be utilized. However, for the purposes of an in vitro setting, forskolin was identified and characterized as a ULK1 agonist, however the role of ULK1 in the autophagy process lies in question. Further research using forskolin to investigate the role of ULK1 in the insulin signaling pathway can now be conducted, and in this manner this project is considered a success.