Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation

Abstract

A deficit in glucose uptake and a deposition of amyloid beta-peptide (A beta) each occur in vulnerable brain regions in Alzheimer's disease (AD). It is not known whether mechanistic links exist between A beta deposition and impaired glucose transport. We now report that A beta impairs glucose transport in cultured rat hippocampal and cortical neurons by a mechanism involving membrane lipid peroxidation. A beta impaired 3H-deoxy-glucose transport in a concentration-dependent manner and with a time course preceding neurodegeneration. The decrease in glucose transport was followed by a decrease in cellular ATP levels. Impairment of glucose transport, ATP depletion, and cell death were each prevented in cultures pretreated with antioxidants. Exposure to FeSO4, an established inducer of lipid peroxidation, also impaired glucose transport. Immunoprecipitation and Western blot analyses showed that exposure of cultures to A beta induced conjugation of 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, to the neuronal glucose transport protein GLUT3. HNE induced a concentration-dependent impairment of glucose transport and subsequent ATP depletion. Impaired glucose transport was not caused by a decreased energy demand in the neurons, because ouabain, which inhibits Na+/K(+)-ATPase activity and thereby reduces neuronal ATP hydrolysis rate, had little or no effect on glucose transport. Collectively, the data demonstrate that lipid peroxidation mediates A beta-induced impairment of glucose transport in neurons and suggest that this action of A beta may contribute to decreased glucose uptake and neuronal degeneration in AD.

Fig. 1.

Aβ induces a concentration-dependent decrease in glucose uptake in hippocampal and cortical cell cultures with a time course that precedes cell death. A, Neocortical and hippocampal cell cultures were exposed for 2 hr to vehicle or the indicated concentrations of Aβ25-35, and cellular uptake of [³H]-glucose uptake was quantified. Values are the mean and SD of determinations made in six separate cultures. *p < 0.01, **p < 0.001 for hippocampal cultures; #p < 0.01 for cortical cultures, compared to cultures exposed to vehicle (0 [Aβ]).B, Cortical and hippocampal cultures were exposed to 10 μm Aβ25-35 for the indicated time periods, and [³H]-glucose uptake was quantified. Values represent the mean and SD of determinations made in four to seven separate cultures. The decrease in glucose uptake was significant for both cortical and hippocampal cultures at the 2 hr (p < 0.05), and 4, 6, and 10 hr (p < 0.01) time points. ANOVA with Scheffe’s post hoc tests.C, Hippocampal cultures were exposed to vehicle or 10 μm Aβ25-35 for the indicated time periods, and neuronal survival was quantified (see Materials and Methods). Values represent the mean and SD of five separate cultures. *p < 0.005 (ANOVA with Scheffe’s post hoc test).D, Hippocampal cultures were exposed for 6 hr to Aβ1-40 at the indicated concentrations, and [³H]-glucose uptake was quantified. Values represent the mean and SD of determinations made in four separate cultures. *p < 0.05, **p < 0.001 compared to control (0 Aβ) value. ANOVA with Scheffe’s post hoc analysis.

Fig. 2.

Evidence for the involvement of reactive oxygen species in Aβ-induced impairment of glucose uptake. Hippocampal and cortical cultures were pretreated for 16 hr with vehicle or 50 μg/ml vitamin E (Vit E), or for 2 hr with 10 μmn-propyl gallate (nPG). Cultures were then exposed for 2 hr to water (Control), 50 μm Aβ25-35, 10 μmouabain, or 50 μmFeSO₄. The levels of [³H]-glucose uptake were quantified, and values (expressed as percentage of control) represent the mean and SD of determinations made in at least eight separate cultures. *p < 0.01 compared to control cultures; **p < 0.001 compared to Aβ-treated cultures. ANOVA with Scheffe’s post hoc analysis.

Fig. 3.

A, Aβ induces a decrease in cellular ATP levels with a time course that follows impairment of glucose uptake. Neocortical cultures were exposed to 10 μm Aβ25-35 for the indicated time periods and intracellular ATP levels were quantified (see Materials and Methods). Additional cultures were exposed to 5 μm phloretin for 2 hr. Values are expressed as percentage of the value in untreated control cultures and represent the mean and SD of determinations made in 5–15 separate cultures. *p < 0.05 compared to control cultures; ANOVA with Fischer’s post hocanalysis. The level of ATP in untreated control cultures was 29.2 ± 0.98 nmol ATP/mg protein. B, Phloretin induces a time- and concentration-dependent decrease in neuronal survival. Hippocampal cultures were exposed to the indicated concentrations of phloretin, and neuronal survival was determined 3 and 20 hr later. Values represent the mean and SD from five separate cultures. *p < 0.01, **p < 0.001 as compared to control cultures. ANOVA with Scheffe’s post hoc analysis.

Fig. 4.

HNE impairs glucose uptake in hippocampal cell cultures. A, Cultures were exposed for 3 hr to the indicated concentrations of HNE, and [³H]-glucose uptake was quantified. Values represent the mean and SD of determinations made in five separate cultures. *p < 0.005.B, Other aldehydic products of lipid peroxidation have no effect on the rate of glucose uptake. Cortical cultures were treated with 10 μm HNE or 250 μm of the indicated aldehydes for 3 hr, and [³H]-glucose uptake was quantified. Values are the mean and SD of determinations made in six separate cultures. *p < 0.05, **p < 0.001 compared to control cultures. ANOVA with Scheffe’s post hoc analysis.

Fig. 5.

GLUT3 is expressed at high levels in cultured hippocampal neurons. Shown are phase-contrast (left) and bright-field (right) micrographs of cultured hippocampal neurons (top) and cortical astrocytes (bottom) immunostained with an antibody to GLUT3. Note that neurons exhibit considerable GLUT3 immunoreactivity, whereas astrocytes do not. Arrowheads point to neuron cell bodies (top panels).

Fig. 6.

Aβ induces HNE production and conjugation to the glucose transporter. Neocortical cultures were exposed for 4 hr to 10 μm Aβ25-35 (A), 10 μm HNE (H), or vehicle (V). Solubilized total cell protein was immunoprecipitated with an antibody against the glucose transporter, and the antibody-bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoreacted with an HNE antibody (see Materials and Methods).