Glucose starvation increases V-ATPase assembly and activity in mammalian cells through AMP kinase and phosphatidylinositide 3-kinase/Akt signaling

Abstract

The vacuolar H⁺-ATPase (V-ATPase) is an ATP-driven proton pump involved in many cellular processes. An important mechanism by which V-ATPase activity is controlled is the reversible assembly of its two domains, namely the peripheral V₁ domain and the integral V₀ domain. Although reversible assembly is conserved across all eukaryotic organisms, the signaling pathways controlling it have not been fully characterized. Here, we identify glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. During acute glucose starvation, the V-ATPase undergoes a rapid and reversible increase in assembly and activity as measured by lysosomal acidification. Because the V-ATPase has recently been implicated in the activation of AMP kinase (AMPK), a critical cellular energy sensor that is also activated upon glucose starvation, we compared the time course of AMPK activation and V-ATPase assembly upon glucose starvation. We observe that AMPK activation precedes increased V-ATPase activity. Moreover, the starvation-induced increase in V-ATPase activity and assembly are prevented by the AMPK inhibitor dorsomorphin. These results suggest that increased assembly and activity of the V-ATPase upon glucose starvation are dependent upon AMPK. We also find that the PI3K/Akt pathway, which has previously been implicated in controlling V-ATPase assembly in mammalian cells, also plays a role in the starvation-induced increase in V-ATPase assembly and activity. These studies thus identify a novel stimulus of V-ATPase assembly and a novel signaling pathway involved in regulating this process. The possible function of starvation-induced increase in lysosomal V-ATPase activity is discussed.

Keywords: AMP-activated kinase (AMPK); Akt PKB; glucose starvation; phosphatidylinositide 3-kinase (PI 3-kinase); proton transport; regulated assembly; vacuolar ATPase.

Figure 1.

Incubation of HEK293T cells at low or high glucose increases V-ATPase assembly relative to physiological concentrations. A, HEK293T cells were maintained in serum-free DMEM containing 5 mm (physiological) glucose for ∼6 h and then treated with serum-free DMEM containing 0 mm glucose (10 min), 5 mm glucose (1 h), 25 mm glucose (1 h), or 0 mm glucose (10 min) followed by 5 mm glucose (10 min). Incubation at high glucose was performed for longer times to allow comparison with previously published results (16–18). Following treatment, cells were fractioned into membrane (M) and cytosolic (C) fractions as described under “Experimental procedures.” Samples were subjected to SDS-PAGE and Western blotting using an antibody against subunit A of the V₁ domain as a measure of assembly. Antibody staining of subunit d of the V₀ domain was used as a loading control for the membrane fraction, and staining for vinculin was used as a loading control for the cytosolic fraction. The quantity of subunit A present in the membrane fraction is an indication of the level of V-ATPase assembly. Shown is a representative Western blotting. B, Western blots performed as described in A were quantified using ImageJ software to determine the amount of V-ATPase assembly. Band intensities of subunit A in the membrane fraction were normalized to band intensities of the membrane loading control (subunit d). Results were then normalized to the baseline assembly levels observed at 5 mm glucose, which was defined as 1.0 for each individual trial. We find that the actual fraction of membrane bound versus cytosolic V₁ is 0.33 ± 0.16 (n = 6) in HEK293T cells under normal physiological glucose concentrations (5 mm). Relative to the degree of assembly at 5 mm glucose (defined as 1.0 for this comparison), the average level of assembly for cells treated with 0 mm glucose was 1.9 ± 0.5 (p < 0.02, n = 9), whereas the average level of assembly for cells treated with 25 mm glucose was 1.4 ± 0.1 (p < 0.02, n = 5). Glucose re-addition for 10 min after starvation (labeled 0/5) returned assembly levels to 0.9 ± 0.1 (n = 3). The error bars represent standard error. The asterisks indicate statistically significant differences at the indicated p values.

Figure 2.

Incubation of HEK293T cells at low or high glucose increases V-ATPase-dependent acidification of lysosomes. A, HEK293T cells were incubated with FITC-dextran to load lysosomes by fluid-phase endocytosis as described under “Experimental procedures.” Cells were then maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with serum-free DMEM containing 0 mm glucose (10 min.), 5 mm glucose (1 h), 25 mm glucose (1 h), or 0 mm glucose (10 min.) followed by 5 mm glucose (10 min.). Following treatment, lysosomes were isolated from cells as described under “Experimental procedures.” Fluorescence intensity at 520 nm (excitation at 490 nm) was measured as a function of time following addition of Mg-ATP. The rate V-ATPase-dependent proton transport activity for each condition was determined by performing a linear regression analysis on the initial rate of concanamycin A-sensitive fluorescence quenching (where present, concanamycin A was added at 1 μm). Rates of fluorescence quenching from independent trials were normalized to the baseline value observed for lysosomes isolated from cells maintained at 5 mm glucose (defined as 1.0). The average lysosomal V-ATPase activity for lysosomes from cells treated with 0 mm glucose was 1.17 ± 0.02 (p < 0.01, n = 20), and for cells treated with 25 mm glucose the average activity was 1.13 ± 0.02 (p < 0.01, n = 20). There was no significant difference in activity between lysosomes from cells treated with 5 mm glucose compared with lysosomes from cells treated with 0 mm glucose followed by 5 mm glucose (0/5). The error bars represent the mean ± S.E. The asterisks indicate statistically significant differences at the indicated p values. B, HEK293T cells were treated with glucose as described in A. Concanamycin A (ConA) at 5 μm or DMSO was added to the cells 1 h before collection. 10 min prior to cell collection, cells were incubated with LysoTracker to stain for acidic cellular compartments and then fixed as described under “Experimental procedures.” Staining was detected by confocal fluorescence microscopy (red, LysoTracker; blue, DAPI), and representative images are shown, n = 2. C, FITC-dextran loaded HEK293T cells were maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with 0 mm glucose for the indicated amounts of time. Rates of fluorescence quenching were measured as in A. n = 3.

Figure 3.

Effect of glucose starvation on various signaling pathways in HEK293T cells. HEK293T cells were maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with serum-free DMEM containing 0 or 5 mm glucose for 10 min. Following treatment, whole-cell lysates were prepared as described under “Experimental procedures.” Samples were subjected to SDS-PAGE, and Western blotting was performed using antibodies directed against the phosphorylated substrates of various signaling pathways, including PI3K (P-Akt), ERK (P-ERK), PKA (P-VASP), and AMPK (P-ACC). Total levels of the substrates were also detected using the corresponding antibodies. Representative images are shown. n = 3.

Figure 4.

Time dependence of V-ATPase–dependent lysosomal acidification, AMPK activity, and ERK activity following glucose starvation of HEK293T cells. A, HEK293T cells were loaded with FITC-dextran and maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with DMEM containing 0 mm glucose for the indicated times. Lysosomes were isolated and analyzed for V-ATPase-dependent lysosomal acidification as described under “Experimental procedures.” The error bars represent the standard error (n = 3). The asterisk represents a p value less than 0.05 comparing activity at t = 0 with each of the time points indicated under the horizontal bar. B, HEK293T cells were maintained in serum-free DMEM containing 5 mm glucose for ∼6 h then treated with DMEM containing 0 mm glucose for the indicated times. Whole-cell lysates were then prepared as described under “Experimental procedures,” and samples were subjected to SDS-PAGE and Western blotting using antibodies against the indicated proteins. Representative images are shown.

Figure 5.

Effect of inhibitors of AMPK, PKA, PI3K, Akt, ERK, or Vps34 on starvation-dependent increases in V-ATPase-dependent lysosomal acidification in HEK293T cells. A, HEK293T cells were loaded with FITC-dextran and maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with serum-free DMEM containing 0 mm glucose (10 min), 5 mm glucose (1 h), or 25 mm glucose (1 h). Where present, the cells were incubated with indicated inhibitors for 1 h prior to cell homogenization. Inhibitors included dorsomorphin (5 μm, an AMPK inhibitor), H89 (50 μm, a PKA inhibitor), LY294002 (50 μm, a PI3K inhibitor), MK2206 (1 μm, an Akt inhibitor), AZD6244 (1 μm, a MEK1/2 inhibitor), and SAR405 (10 μm, a Vps34 inhibitor). After treatment, cells were homogenized, and V-ATPase-dependent lysosomal acidification was tested as described in Fig. 2A. The error bars represent standard error. The asterisks represent statistically significant differences with a p value less than 0.05 (n ≥ 3). B, HEK293T cells were maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with serum-free DMEM containing 0 mm glucose (10 min.), 5 mm glucose (1 h), or 25 mm glucose (1 h) in the presence or absence of various signaling pathway inhibitors. Inhibitors were added at the concentrations indicated in A and, where present, were incubated with cells for a total of 1 h. 10 min prior to cell collection, cells were incubated with LysoTracker to stain for acidic intracellular compartments and then fixed as described under “Experimental procedures.” Staining was detected by confocal fluorescence microscopy (red, LysoTracker; blue, DAPI). Drug-free and concanamycin A-treated controls for the experiment are shown in Fig. 2B. Representative images are shown, n = 2.

Figure 6.

Effect of inhibitors of AMPK, PI3K, and Akt on starvation-dependent increase in V-ATPase assembly in HEK293T cells. A, HEK293T cells were maintained in serum-free DMEM containing 5 mm glucose for ∼6 h and then treated with serum-free DMEM containing 0 mm glucose (10 min) or 5 mm glucose (10 min) in the presence or absence of the indicated signaling pathway inhibitors. Inhibitors were present at the concentrations indicated in Fig. 5 and, where present, were incubated with cells for 1 h prior to analysis of assembly. Following treatment, assembly was assayed as described in Fig. 1A. Representative Western blots are shown. M indicates membrane and C indicates cytosolic fractions. B, Western blotting quantitation was performed as described in Fig. 1B, with values expressed relative to assembly measured at 5 mm glucose under each set of conditions. Error bars represent standard error, n = 2.