Knockdown of GSK3β increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells

Abstract

High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308: , C249-C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.

Keywords: AMP-activated protein kinase (AMPK); autophagy; endothelium; forkhead box protein O1 (FOXO1); glycogen synthase kinase 3β (GSK3β).

Figure 1. Excess nutrients increase GSK3β activity and decrease lysosome acidification

(A) HAECs incubated in excess nutrient conditions have a decreased ratio of inhibitory p-GSK3β^Ser9/t-GSK3β protein and increased protein levels of p-GS^Ser641, a downstream target of GSK3β (GAPDH n=4; β-actin n=3). (B) Exposure to excess nutrients decreased lysotracker staining (n=7). (C) Excess nutrients did not decrease LAMP1 protein levels (n=5). * indicates P<0.05 by Student's t test compared with control conditions. For (B) and (C), bar=100 μm.

Figure 2. Reduction of GSK3β activity with CHIR 99021 attenuates the effects of excess nutrients on lysosome acidification

(A) Compared with excess nutrient-exposed HAECs treated with vehicle alone (DMSO), those incubated with 12.5 nM CHIR 99021 for 23 h had more lysotracker staining. Representative images are shown adjacent to graph. * indicates P<0.05 by Student's t test (n=3). (B) CHIR 99021 treatment did not affect LAMP1 staining in cells incubated in excess nutrient conditions. ns indicates no significant difference (n=3). Quantification of fluorescence or LAMP1 is relative to the number of cells in each image. Bar=100 μm.

Figure 3. CHIR 99021 treatment increases bafilomycin-induced protein levels of the autophagosome marker LC3-II in excess nutrient conditions

Treatment of excess nutrient-exposed HAECs with 12.5 nM CHIR 99021 for 23 h increased the ratio of LC3-II/GAPDH and the ratio of LC3-II/LC3-I as determined by western blot. For example, LC3-II/GAPDH in [lane 4-lane 3] > [lane 2–lane 1]. * indicates P<0.05 for an effect of CHIR 99021 under excess nutrient conditions by two-way ANOVA (n=6).

Figure 4. Reduction of GSK3β activity increases protein levels of LC3-II

Compared with HAECs-infected with control lentivirus (shc), those infected with lentivirus reducing expression of GSK3β (shGSK3β) (A) or expressing kinase-dead GSK3β (K85A GSK3β) (B) had more GFP-LC3 puncta under both control and excess nutrient conditions. Punctate structures, rather than diffuse GFP staining is indicative of LC3-II expression. Quantification is shown relative to shc for each treatment condition, adjacent to representative images. Bar=50 μm in A and 100 μm in B. * indicates P<0.05 for an effect of lentivirus infection by two-way ANOVA (A: n=3; B: n=5).

Figure 5. Knockdown of GSK3β protein increases FOXO1 activity

(A) Compared with HAECs infected with control lentivirus (shc), those infected with lentivirus reducing expression of GSK3β (shGSK3β) had less p-FOXO1^Thr24 under both control (A) and excess nutrient conditions (B). Representative western blots are shown adjacent to the graphs. (C) Knockdown of GSK3β (shGSK3β) increased mRNA levels of PIK3C3 (left) and SOD2 (right) compared with controls (shc). * indicates P<0.05 for an effect of GSK3β knockdown by two-way ANOVA (For A and B: GAPDH n=6; β-actin n=5).

Figure 6. GSK3β knockdown increases AMPK signalling and decreases Akt signalling

Compared with HAECs infected with control lentivirus (shc), those infected with lentivirus reducing the expression of GSK3β (shGSK3β) had an increased ratio of p-AMPK^Thr172/t-AMPKα1 protein (n=5), increased p-ACC^Ser79 (GAPDH n=5; β-actin n=4), a downstream target of AMPK and a decreased ratio of p-Akt^Ser473/t-Akt (n=3) under both control (A) and excess nutrient conditions (B). shGSK3β also decreased the ratio of p-Akt^Thr308/t-Akt (n=5) under control conditions (A). * indicates P<0.05 for an effect of GSK3β knockdown by ANOVA. Representative western blots are shown with densitometric analyses. Separate blots are shown for AMPK and Akt signalling due to the similarity in molecular mass of AMPK and Akt. (C) Proposed pathway by which suppression of GSK3β activity could increase autophagosome formation in HAECs exposed to excess nutrients.

Figure 7. CHIR 99021 increases LC3-II protein levels and AMPK signalling and decreases Akt signalling in the mouse aorta

Compared with aortas from control-treated mice (injected with saline/PEG400), aortas from mice treated with CHIR 99021 had higher levels of LC3-II protein (A), a marker of autophagosomes and p-ACC^Ser79 (B), a downstream target of AMPK. CHIR 99021-treated aortas also had decreased ratios of p-Akt1^Ser473/t-Akt and p-Akt^Thr308/t-Akt (C). For (A) and (B), the same eight samples were run on two different gels to optimize visualization of each protein band. Densitometry is shown adjacent to each blot. * indicates P<0.05 (n=8).

Figure 8. Potential use of GSK3β inhibitors to reduce excess nutrient-induced endothelial dysfunction

(A) In the presence of high concentrations of glucose and palmitate, AMPK activity and basal autophagy decrease whereas GSK3β activity increases. These changes predispose endothelial cells to atherogenesis by increasing risk for endothelial dysfunction. (B) Inhibition of GSK3β in the presence of excess nutrients increases AMPK activity and autophagy, thereby reducing risk for endothelial dysfunction.