Abstract 1088: Acute Hypoglycemia Induces Mitochondria-Mediated Superoxide Production and Apoptosis in Endothelial Cells: Inhibition by AMP Kinase Activation Through an eNOS Dependent Mechanism
Background: Recent studies employing intense glycemic control in type 2 diabetic and critically ill patients have highlighted the excessive cardiovascular risk associated with the development of hypoglycemic events. These data suggest acute hypoglycemia may adversely affect endothelial function. Excessive mitochondrial superoxide is an established factor in the pathogenesis of DM vascular dysfunction related to hyperglycemia, and can be blunted by metformin. However, the role of mitochondria in the development of vascular dysfunction and endothelial apoptosis during acute hypoglycemia (more frequently observed with intense control) remains unclear.
Methods: HUVECs were grown in culture medium using 90 mg/dL glucose (normal glucose; NG) and subsequently exposed to combinations of metformin (10 μM), AICAR (AMP Kinase agonist, 100 μM), L-NAME (1 mM), and Compound C (AMP Kinase antagonist, 10 μM) at 90 mg/dL glucose or 40 mg/dL (low glucose - LG;N=5 for all conditions). We measured mitochondrial superoxide production under all test conditions using the mitochondrial superoxide sensitive fluorophore MitoSOX™ and determined the percentage of apoptotic cells (Hoechst assay).
Results: 30 minutes of LG increased mitochondrial superoxide production in HUVECs (P=0.02 vs. NG control) with an associated increase in apoptosis (12% vs. 28%, P<0.001). However, concomitant treatment with metformin or AICAR blunted excess mitochondrial superoxide with similar efficacy (both conditions: P<0.05 vs. versus LG alone). The addition of Compound C to LG + metformin reversed the benefit of metformin (P=NS vs. LG alone). Further, co-incubation with L-NAME prevented the reductions in mitochondrial superoxide induced by metformin and AICAR. Metformin reversed LG-induced apoptosis (P=0.005 vs. LG alone, P=NS vs. NG alone).
Conclusions: Acute, moderate low glucose exposure induces excessive mitochondrial superoxide production and apoptosis in isolated endothelial cells. Pharmacological interventions that simulate AMP Kinase, e.g. metformin, inhibit this response in part through increased eNOS activity. These data suggest pharmacological AMP Kinase stimulation can blunt the adverse vascular effects of moderate hypoglycemia via eNOS activation.