Ethanol impairs glucose uptake by human astrocytes and neurons: protective effects of acetyl-L-carnitine

Abstract

Alcohol consumption causes neurocognitive deficits, neuronal injury, and neurodegeneration. At the cellular level, alcohol abuse causes oxidative damage to mitochondria and cellular proteins and interlink with the progression of neuroinflammation and neurological disorders. We previously reported that alcohol inhibits glucose transport across the blood-brain barrier (BBB), leading to BBB dysfunction and neurodegeneration. In this study, we hypothesized that ethanol (EtOH)-mediated disruption in glucose uptake would deprive energy for human astrocytes and neurons inducing neurotoxicity and neuronal degeneration. EtOH may also have a direct effect on glucose uptake in neurons and astrocytes, which has not been previously described. Our results indicate that ethanol exposure decreases the uptake of D-(2-³H)-glucose by human astrocytes and neurons. Inhibition of glucose uptake correlates with a reduction in glucose transporter protein expression (GLUT1 in astrocytes and GLUT3 in neurons). Acetyl-L-carnitine (ALC), a neuroprotective agent, suppresses the effects of alcohol on glucose uptake and GLUT levels, thus reducing neurotoxicity and neuronal degeneration. These findings suggest that deprivation of glucose in brain cells contributes to neurotoxicity in alcohol abusers, and highlights ALC as a potential therapeutic agent to prevent the deleterious health conditions caused by alcohol abuse.

Keywords: Human astrocytes; acetyl-L-carnitine; glucose transporter protein; neurodegeneration.

Figure 1

EtOH inhibits glucose uptake in human astrocytes. A. Dose dependent effects of EtOH on D-(2-³H)-glucose uptake by human astrocytes over a 24 hr time period. B. Effects of the GLUT inhibitor CB (10 μM) and ALC (100 μM) on glucose uptake by astrocytes following 50 mM EtOH treatment for 24 hr. **Statistically significant, p<0.01, compared with controls.

Figure 2

EtOH down regulates the expression of GLUT1 in human astrocytes. A-F. Immunocytochemistry of GLUT1 (red) merged with GFAP (green) and DAPI (blue) in human astrocytes. The expression of GLUT1 is shown in control (A-B), 50 mM EtOH (C-D), and 50 mM EtOH+100 μM ALC (E-F). Mouse anti-GLUT1 and rabbit anti-GFAP were used as primary antibodies. An anti-mouse-IgG Alexa Fluor 594 was used to visualize GLUT1 and an anti-rabbit-IgG Alexa Fluor 488 was used to image GFAP. Scale bar = 10 μm in all panels.

Figure 3

Western blot analysis of GLUT1 in EtOH treated human astrocytes. A. Effects of EtOH on GLUT1 protein levels (45 kDa) in human astrocytes. The effects of CB and ALC for 24 hr on GLUT1 expression are also shown in the band pattern. B. Results are expressed as the ratio of GLUT1 to α-actin bands, and presented as mean values (± SEM; n = 5). (**Statistically significant, p<0.01 compared with controls).

Figure 4

Effect of EtOH on glucose uptake in human neurons. A. Dose dependent effects of EtOH (24 hr treatment) on D-(2-³H)-glucose uptake by human neurons. B. Effects of the GLUT inhibitor CB (10 μM) and ALC (100 μM) on glucose uptake by neurons following 50 mM EtOH treatment for 24 hr. **Statistically significant, p<0.01, compared with controls.

Figure 5

EtOH down regulates GLUT3 expression in human neurons. Immunocy-tochemistry of GLUT1 (red) merged with NF (green) and DAPI (blue) in control (A-B), 50 mM EtOH (C-D), and 50 mM EtOH+100 μM ALC (G-H). Rabbit anti-GLUT3 and mouse anti-NF were used as primary antibodies. An anti-rabbit-IgG Alexa Fluor 594 and an anti-mouse-IgG Alexa Fluor 488 were used to visualize GLUT 3 and NF, respectively. Scale bar = 5 μm in all panels.

Figure 6

Western blot analysis for GLUT3 expression in EtOH treated human neurons. A-B. Effects of EtOH (24 hr treatment) on GLUT3 (45 kDa) protein levels in human neurons. Bar graph shows the results, and are expressed as the ratio of GLUT3 to that of α-actin bands, and presented as mean values (± SEM; n = 5). **Statistically significant, p<0.01, compared with controls.