Figure 7.

Both IR and IGFR contribute to autophagic flux in astrocytes. A. Representative confocal images showing red and green puncta of IR/IGF1R-flox and DKO astrocytes expressing tfLC3 reporter in normal culture condition (10% FBS), overnight serum depletion, and overnight treatment of 100 nM insulin or IGF-1 following 5 h serum depletion. To induce maximal autophagy flux, all cells were incubated with 1× HBSS for 4 h before confocal live cell imaging. Scale bar: 10 μm. B–D. Quantification of RFP⁺ puncta over total cell area (B), GFP⁺ puncta over total cell area (C), and GFP⁺ to RFP⁺ ratio (D) in IR/IGF1R-flox and DKO astrocytes under different treatments indicated. Data are shown as mean ± SEM. One-way ANOVA followed by Tukey's multiple comparison test. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001. N = 50–61 individual cells. E. Illustration showing that while multiple upstream signaling cascades converge on mTOR kinase activity to regulate autophagy flux, insulin and IGF-1 trigger strong transcriptional suppression on many genes directly involved in autophagy in astrocytes. Collectively, autophagy in astrocytes is tightly regulated by metabolic states and nutrient levels.