We checked cellular levels of LC3B II during the exponential growth phase, and at roughly 70 80 % confluence, and found selleck chem Dorsomorphin that LC3B II levels in the IRS 1 overexpressing cells were decreased compared to the control cells. Further, we counted the number of autop hagic vacuoles visible under an electron microscope. The number of autophagic vacuoles was greater in the control cells than in the IRS 1 overexpressing cells. These results indicate that overexpression of IRS 1 reduces the number of autophagosomes, and imply that overex pression of IRS 1 reduces autophagy. LC3B II accumulation can result from increased up stream autophagosome formation or from impaired downstream autophagosome lysosome fusion.

To distin guish between these two possible explanations for the decrease in LC3B II levels in NIH 3T3 cells that overex press IRS 1, we determined the autophagic flux using LC3 turnover assay in the presence of bafilomycin A. If the amount of LC3B II further accumulates in the pres ence of bafilomycin A, it indicates that autophagic flux is intact, however, if the LC3B II levels remain un changed, it is likely that the autophagic flux is impaired. Autophagic flux is used to denote the dynamic processes of autophagosome synthesis, delivery of autop hagic substrates to the lysosome, and degradation of autophagic substrates within the lysosome, and is a reli able indicator of autophagic activity. First, we stud ied the nutrient starvation induced autophagy in both the control cells and the IRS 1 overexpressing cells.

Both groups of cells were seeded and cultured for one day, then the culture medium was replaced with fresh DMEM containing 10 % FBS or with Earles Balanced Salt Solution, an amino acid deficient solution, for 6 h. Treatment with EBSS resulted in increased LC3B II levels in both the control cells and the IRS 1 overexpressing cells. The levels of LC3B II were greater in the presence of bafilomycin A than in the absence of bafilomycin A for both groups of cells, ei ther treated with DMEM containing 10 % FBS or with EBSS, indicating that the autophagy fluxes were intact in both groups of cells. We next investigated the effect of insulin, which inhibits autophagy, on autophagy in both the control cells and the IRS 1 overexpressing cells. Treatment with 500 nM insulin for 6 h decreased the levels Batimastat of LC3B II in both groups of cells. The levels of LC3B II were greater in the presence of bafilomycin A than in the absence of bafilomycin A for both groups of cells either without or with insulin treat ment. This finding indicates that the autophagic fluxes remain intact in both the control cells and the IRS 1 overexpressing cells. We further investigated whether overexpression of IRS 1 inhibits autophagy in this series of experiments.