Grape seed proanthocyanidin extract inhibits glutamate-induced cell death through inhibition of calcium signals and nitric oxide formation in cultured rat hippocampal neurons

<p>Abstract</p> <p>Background</p> <p>Proanthocyanidin is a polyphenolic bioflavonoid with known antioxidant activity. Some flavonoids have a modulatory effect on [Ca²⁺]_i. Although proanthocyanidin extract from blueberries reportedly affects Ca²⁺buffering capacity, there are no reports on the effects of proanthocyanidin on glutamate-induced [Ca²⁺]_ior cell death. In the present study, the effects of grape seed proanthocyanidin extract (GSPE) on glutamate-induced excitotoxicity was investigated through calcium signals and nitric oxide (NO) in cultured rat hippocampal neurons.</p> <p>Results</p> <p>Pretreatment with GSPE (0.3-10 μg/ml) for 5 min inhibited the [Ca²⁺]_iincrease normally induced by treatment with glutamate (100 μM) for 1 min, in a concentration-dependent manner. Pretreatment with GSPE (6 μg/ml) for 5 min significantly decreased the [Ca²⁺]_iincrease normally induced by two ionotropic glutamate receptor agonists, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). GSPE further decreased AMPA-induced response in the presence of 1 μM nimodipine. However, GSPE did not affect the 50 mM K⁺-induced increase in [Ca²⁺]_i. GSPE significantly decreased the metabotropic glutamate receptor agonist (<it>RS</it>)-3,5-Dihydroxyphenylglycine-induced increase in [Ca²⁺]_i, but it did not affect caffeine-induced response. GSPE (0.3-6 μg/ml) significantly inhibited synaptically induced [Ca²⁺]_ispikes by 0.1 mM [Mg²⁺]_o. In addition, pretreatment with GSPE (6 μg/ml) for 5 min inhibited 0.1 mM [Mg²⁺]_o- and glutamate-induced formation of NO. Treatment with GSPE (6 μg/ml) significantly inhibited 0.1 mM [Mg²⁺]_o- and oxygen glucose deprivation-induced neuronal cell death.</p> <p>Conclusions</p> <p>All these data suggest that GSPE inhibits 0.1 mM [Mg²⁺]_o- and oxygen glucose deprivation-induced neurotoxicity through inhibition of calcium signals and NO formation in cultured rat hippocampal neurons.</p

ATM and GLUT1-S490 phosphorylation regulate GLUT1 mediated transport in skeletal muscle.

The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1.L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity.In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL.These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction

Glucose transport in skeletal muscle.

<p>Soleus and extensor digitorum (EDL) longus muscles were incubated in the absence or presence of indinavir (IND) and then in the absence or presence of the ATM inhibitor CP466722 (CP). Values are means and standard errors (n = 3).</p>*<p>indicates a CP effect (p<0.5), and.</p>†<p>indicates an indinavir effect (P<0.05).</p

ATM inhibition reduces cell surface GLUT1, GLUT1-mediated transport, ATM phosphorylation of GLUT1 and GLUT1-GIPC1 association.

<p><b>A)</b> L6 myoblasts were subjected to a glucose transport assay in the presence of GLUT4 inhibitor, indinavir, and in the presence and absence (0.1% DMSO vehicle) of ATM inhibitor, KU55933 (KU) at 10 µM or CP466722 (CP) at 6 µM, for one hour (n = 9/group); Or <b>B)</b> DHA transport assay in the presence or absence (0.1% DMSO) of ATM inhibitors, KU and CP at 10 µM and 6 µM, respectively (n = 9/group). <b>C)</b> L6 myoblasts transiently transfected with FLAG-GLUT1 constructs were incubated in the presence or absence of ATM inhibitors, KU and CP, 10 µM and 6 µM, respectively, for one hour, then put through a GLUT1 cell surface assay. (n = 18/group) <b>D)</b> FLAG-GLUT1 L6 myoblasts were incubated in the presence or absence of ATM inhibitor, CP, at 6 µM for one hour, and subjected to a GLUT1 internalization assay (n = 12/group), <b>E)</b> subjected to a GLUT1 externalization assay. (n = 6/group), or <b>F)</b> subjected to Western blot analysis probing for FLAG or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (n = 4). <b>G)</b> Transiently transfected FLAG-GLUT1 L6 myoblasts exposed to 6 µM CP or DMSO (vehicle) for one hour were subjected to immunoprecipitation with antibodies against FLAG. Immunoprecipitates were probed with antibodies against FLAG, phosphorylated substrates of ATM (P-ATM Sub, n = 3), G_α-interacting protein-interacting protein, C-terminus (GIPC1, n = 6), and stomatin (STOM, n = 6). H) Non-transfected NT and FLAG-GLUT1 transfected L6 myoblasts incubated plus or minus 6 µM CP for one hour were subjected to a glucose transport assay (n = 6/group), Or I) a DHA transport assay (n = 6/group), both in the presence of indinavir. J) GLUT1 and GAPDH levels in non-transfected or FLAG-GLUT1-transfected myoblasts (n = 6/group). For panels H and I: †p<0.05 relative to FLAG-GLUT1. For all panels *p<0.05 relative to NT.</p

ATM activation enhances GLUT1 cell surface localization, GLUT1-mediated transport, and the GLUT1/GIPC1 interaction.

<p>L6 myoblasts were incubated with 1 µM doxorubicin (DXR), 6 mM CP, CP and DXR in tandem, or vehicle for 1 h before western analysis of A) phosphorylated ATM S1981 (P-ATM) and total ATM (n = 3/group) or B) phosphorylated ATM substrates (P-ATM Sub) and tubulin (n = 3–6/group) (*p<0.05 compared to Control; †p<0.05 compared to DXR). C) L6 myoblasts were subjected to a DHA transport assay in the presence or absence (0.1% DMSO vehicle) of 1 µM DXR or 1 µM DXR in tandem with 10 µM KU or 6 µM CP for one hour (n = 9/group). <b>D)</b> FLAG-GLUT1 L6 myoblasts were incubated in the presence or absence of 1 µM DXR or 1 µM DXR in tandem with 10 µM KU or 6 µM CP for one hour, then subjected to an assay for cell surface GLUT1 (n = 6/group). <b>E)</b> FLAG-GLUT1 L6 myoblasts were incubated in the presence or absence of ATM activator, DXR, at 1 µM for one hour and subjected to Western blot analysis probing for FLAG and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (n = 4). <b>F)</b> Transiently transfected FLAG-GLUT1 L6 myoblasts incubated with 1 µM DXR or vehicle for one hour were lysed and immunoprecipitated with FLAG antibodies. Immunoprecipitates were probed for, phosphorylated substrates of ATM (P-ATM Sub, n = 3), GIPC1 (n = 6), stomatin (STOM, n = 6), and FLAG. For all panels *p<0.05.</p

The effects of chronic ATM deficiency and acute ATM inhibition on GLUT1-mediated glucose transport in skeletal muscle.

<p>Wild type (WT) and ATM mutant (ATM −/−) mouse muscles were excised and subjected to a glucose transport assay in the presence of indinavir, a GLUT4 inhibitor. <b>A)</b> WT versus mutant soleus muscles (n = 8/group). <b>B)</b> WT versus mutant EDL muscles (n = 6/group) <b>C)</b> Wild type soleus muscles plus/minus 6 µM CP (n = 5/group). <b>D)</b> WT EDL muscles plus/minus 6 µM CP (n = 5/group). <b>E)</b> WT soleus muscles plus/minus 10 µM KU (n = 6/group). <b>F)</b> WT soleus muscles plus/minus DXR (n = 7/group). G) ATM−/− soleus muscles plus/minus DXR relative to WT (n = 3/group). H) WT and ATM−/− EDL muscles in the presence or absence of KU (n = 3/group). I) WT soleus muscles plus/minus 1 µM DXR and in the presence or absence of indinavir (IND) (n = 3/group). J) WT EDL muscles plus/minus 10 µM KU and in the presence or absence of IND (n = 3/group). For panels I and J: †p<0.05 relative to IND. For all panels, *p<0.05.</p

The effects of chronic ATM loss and ATM inhibition on GLUT1 protein levels.

<p>Mouse muscles were excised and subjected to western analysis for GLUT1 A) Wild type (WT) and ATM mutant (ATM −/−) EDL muscles (n = 3/group). B) WT EDL muscles plus/minus 10 µM KU (n = 6/group). C) WT and ATM −/− soleus muscles (n = 4/group). <b>D)</b> Wild type soleus muscles plus/minus 6 µM CP (n = 5/group). *p<0.05.</p

Effects of chronic ATM deficiency or ATM inhibition on GLUT1-mediated DHA transport in skeletal muscle.

<p>Wild type (WT) and ATM mutant (ATM −/−) mouse muscles were excised and subjected to a DHA transport assay in the presence of indinavir, a GLUT4 inhibitor. <b>A)</b> WT versus mutant soleus muscles (n = 10–12/group). <b>B)</b> WT versus mutant extensor digitorum longus (EDL) (n = 8–10/group) <b>C)</b> Wild type soleus muscles plus/minus 6 µM CP, <b>D)</b> 10 µM KU (n = 5–10/group), or <b>E)</b> 1 µM DXR (n = 5–6/group). For all panels, *p<0.05.</p