Enhanced Ca2+ Entry Sustains the Activation of Akt in Glucose Deprived SH-SY5Y Cells

Abstract

The two crucial cellular insults that take place during cerebral ischemia are the loss of oxygen and loss of glucose, which can both activate a cascade of events leading to neuronal death. In addition, the toxic overactivation of neuronal excitatory receptors, leading to Ca²⁺ overload, may contribute to ischemic neuronal injury. Brain ischemia can be simulated in vitro by oxygen/glucose deprivation, which can be reversible by the re-establishment of physiological conditions. Accordingly, we examined the effects of glucose deprivation on the PI3K/Akt survival signaling pathway and its crosstalk with HIF-1α and Ca²⁺ homeostasis in SH-SY5Y human neuroblastoma cells. It was found that glucose withdrawal decreased HIF-1α protein levels even in the presence of the ischemia-mimicking CoCl_2. On the contrary, and despite neuronal death, we identified a strong activation of the master pro-survival kinase Akt, a finding that was also confirmed by the increased phosphorylation of GSK3, a direct target of p-Akt. Remarkably, the elevated Ca²⁺ influx recorded was found to promptly trigger the activation of Akt, while a re-addition of glucose resulted in rapid restoration of both Ca²⁺ entry and p-Akt levels, highlighting the plasticity of neurons to respond to ischemic challenges and the important role of glucose homeostasis for multiple neurological disorders.

Keywords: Akt kinase; Ca2+ entry; glucose deprivation; hypoxia; hypoxia-inducible factor 1; ischemia.

Figure 1

Opposite effects of glucose deprivation on p-Akt, p-GSK3α/β and HIF-1α protein levels. (A) Representative Western blot images of SH-SY5Y cells treated with increasing glucose concentrations (0–2 mg/mL in RPMI containing 10% FBS) for 24 h. (B) Quantification of p-Akt, p-GSK3α/β and HIF-1α protein levels for dose-dependent effect. Data are presented as mean ± S.E.M., n = 3, * p < 0.05, † p < 0.001, compared to control (2 mg/mL). (C) Representative Western blot images of cells incubated for 0.5–24 h in the absence of glucose (GD, glucose deprivation) in RPMI containing 10% FBS. (D) Quantification of p-Akt, p-GSK3α/β and HIF-1α protein levels for time-dependent effect. Data are presented as mean ± S.E.M., n = 3, * p < 0.05, ** p < 0.01, † p < 0.001, compared to control (0 h).

Figure 2

CoCl₂ increases p-Akt, p-GSK3α/β and HIF-1α in a dose- and time- dependent manner. (A) Representative Western blot images of SH-SY5Y cells treated with increasing CoCl₂ concentrations (50–400 μM) for 24 h. (B) Quantification of p-Akt, p-GSK3α/β and HIF-1α protein levels for dose–dependent effect. (C) Representative Western blot images of cells treated with 400 μΜ CoCl₂ for 0.5–24 h. (D) Quantification of p-Akt, p-GSK3α/β and HIF-1α protein levels for time–dependent effect. Data are presented as mean ± S.E.M., n = 3, * p < 0.05, ** p < 0.01, † p < 0.001 compared to control (0 μΜ and 0 h for (C,D) respectively).

Figure 3

Glucose deprivation decreases HIF-1α levels during hypoxia. Representative Western blot images of p-Akt and HIF-1α protein levels (from two separate experiments) in SH-SY5Y cells incubated for 2, 4, and 24 h in the absence of glucose (GD, glucose deprivation), or in the presence of 1 mg/mL glucose, either without or with CoCl₂.

Figure 4

Glucose deprivation–induced Akt phosphorylation is partially restored after the glucose re-addition. (A) Protocol of repetitive periods of glucose deprivation using a glucose-free medium and glucose re-addition using a conditioned medium (cM). Control cells were incubated in a complete growth medium. (B) Representative Western blot images and quantification of p-Akt levels in SH-SY5Y cells incubated as shown in (A), compared to control cells. Data are presented as mean ± S.E.M., n = 3, * p < 0.05, ** p < 0.01, † p < 0.001, compared to control. (C) Representative Western blot image of p-Akt in glucose-deprived SH-SY5Y cells (for 60 min) followed by glucose supplementation (1 mg/mL) for 5, 10, and 15 min.

Figure 5

Reversal of changes in Ca²⁺ homeostasis caused by short- and long-term glucose deprivation (GD) upon the re-addition of glucose. ER Ca²⁺ content was measured upon stimulation of cells with Tg in Ca²⁺ free-KRH and SOCE was measured by addition of 3 mM CaCl₂ (arrows). Glucose deprivation of SH-SY5Y cells lasted 4 and 24 h and glucose restoration for 1 and 2 h, respectively. Representative traces from 4–6 independent experiments.

Figure 6

Ca²⁺ entry activates Akt. (A) Representative Western blot images and quantification of p-Akt and HIF-1α levels in SH-SY5Y cells incubated with 20 μΜ BAPTA-AM, a Ca²⁺ chelating agent, for 0.5 up to 24 h. (B) Representative Western blot images and quantification of p-Akt, and HIF-1α levels in SH-SY5Y cells treated with thapsigargin (Tg, 0–100 nM) for 24 h. (C) Ca²⁺ entry induced by ionomycin (IONO) or miltefosine (HePC) measured in Fura-2 loaded SH-SY5Y cells in Ca²⁺ containing KRH (representative traces from 3–6 independent experiments). (D) Representative Western blot image of STIM1 levels during glucose deprivation (GD) or Tg (100 nM) treatment for 24 h. (E,F) Representative Western blot images and quantification of p-Akt levels in SH-SY5Y cells detached and re-suspended in KRH buffer supplemented with IONO (500 nM) or HePC (20 μM). In the case of CaCl₂ (3 mM), cells were pre-treated with Tg in Ca²⁺ free-KRH for 10 min. Cells were harvested after 0, 3, 6, and 10 min of incubation. All data are presented as mean ± S.E.M., n = 4, * p < 0.05, ** p < 0.01, † p < 0.001, compared to controls.