The present investigation has identified 3 novel findings. First, acute exposure of endothelial cells to low glucose concentrations in a clinically relevant range (40–80 mg/dL) induces a rapid reduction in NO bioavailability at least in part through dephosphorylation of at the serine 1177 site. Second, the loss of NO bioavailability is mechanistically linked to concomitant increases in mitochondria specific superoxide production likely through mitochondrial hyperpolarization. Third, while native AMPK activation is not sufficient to maintain levels of activated eNOS and inhibit excessive mitochondrial superoxide production, pharmacological AMPK activation inhibits excessive mitochondrial oxidative stress through improved NO bioavailability that may involve phosphorylation at eNOS’s Ser633 activation site. Importantly, we demonstrate our findings have relevance to human vascular endothelial function by demonstrating that acute low glucose exposure profoundly impairs endothelial function in intact human adipose arterioles, while treatment with metformin or a superoxide scavenger designed to pass through membranes inhibits the adverse effects of low glucose. Taken together, these data suggest a new mechanism of inducing vascular mitochondrial oxidative stress and endothelial dysfunction; namely, brief hypoglycemia, typical of the clinical situation involving tight glycemic regulation.
The consequences of tight glycemic control include intermittent hypoglycemia in the range that we identify as capable of increasing mitochondrial oxidative stress and endothelial dysfunction. There have been few prior investigations of the effect of low glucose on endothelial function, and prior work in this area has been conflicting. Exposure of porcine aortic endothelial cells to 2-deoxyglucose in a glucose-free environment inhibits bradykinin stimulated production of prostacyclin and possibly nitric oxide as well.16
In a study of rabbit aortas, inhibition of glycolysis with either 2-deoxyglucose or iodoacetate in buffer containing 16.6 mM glucose failed to alter endothelium-dependent vasodilation to acetylcholine.17
A third study assessed the effect of moderate and severe insulin-induced hypoglycemia on cerebral blood flow in goats, finding an NO-dependent increase in cerebral blood flow under hypoglycemic conditions.18
. These studies are significantly heterogeneous with respect to their study designs, including significant differences in the species studied, vascular beds tested, and methodologies employed to induce low glucose states. The porcine and rabbit studies employed inhibitors of glycolysis enzymes to mimic the effects of glucose deprivation limiting their generalizability to physiologically relevant low glucose states. Our findings significantly expand our knowledge base on the effects of low glucose on human vascular endothelial function through the use of human tissues and clinically relevant concentrations of glucose.
Our data demonstrate low glucose exposure rapidly inhibits NO bioavailablity at least in part through reducing the level of Ser1177 phosphorylated eNOS. Mechanistically, the loss of NO bioavailability may also relate to our observed increase total cellular oxidative stress observed in this study by an increase in H2
under LG conditions. We were unable to demonstrate evidence of excessive total superoxide levels under low glucose conditions. While we cannot exclude an effect on total superoxide concentrations for longer exposure periods, our data suggest that a 60 minute period of LG exposure does not induce excessive total superoxide levels. Our data are also consistent with prior work demonstrating that glucose deprivation inhibits the phosphatydylinositol 3-kinase/Akt signaling pathway intrinsic to the activation of eNOS.19
Our data significantly expand on this finding by 1) extending the prior study’s findings to clearly demonstrate an effect on eNOS and 2) demonstrating the potential clinical relevance of inhibition of this pathway at physiological levels of low glucose. Further work to delineate the mechanisms of eNOS inactivation by low glucose is necessary to determine potential targets for interventions.
NO is a key regulator of the mitochondrial electron transport chain (ETC), the major source of mitochondrial superoxide.20
NO modulates ETC activity through inhibition of complexes I and IV.21, 22
Under normal homeostatic conditions, relatively high levels of NO in endothelial cells significantly inhibit ETC activity.23
Our data suggest that low glucose induced losses of bioavailable NO remove homeostatic restrictions on mitochondrial electron transport chain flux, leading to mitochondrial hyperpolarization and superoxide production. Mitochondrial hyperpolarization, as induced in our study by LG, is well-known to be mechanistically related to increased mitochondrial ROS production. 10, 24, 25, 25, 26
Our novel findings in the setting of LG are consistent with prior work demonstrating that the amount of glucose oxidation via the Krebs cycle, near dormant under normal glucose conditions in endothelial cells, is significantly increased with low glucose exposure.27
Data from the neurological literature demonstrate hypoglycemia shifts mitochondria from state 3 to state 4 respiration in central neurons, an alteration well-known for increasing mitochondrial superoxide production.28
Subsequent work also shows neuronal hypoglycemia induces marked increases in both mitochondrial superoxide and hydrogen peroxide production.29, 30
Further work using inhibitors of the mitochondrial ETC and measurements of specific activity levels of components of the ETC will be necessary to determine the exact ETC sources of mitochondrial ROS under low glucose conditions.
We found that LG exposure enhances endothelial cell H2
content with a corresponding increase in mitochondrial superoxide content. Increases in ROS production are linked to increased oxidative damage, reduced NO bioavailability, and endothelial dysfunction.31
. In the context of our findings, the biological role of increased mitochondrial superoxide under LG conditions likely includes both the adverse sequelae of increased oxidative damage as well as modulation of important pathological cell signaling cascades. Mitochondrial superoxide can reduce NO bioavailability by rapidly reacting locally with NO to form peroxynitrite. Peroxynitrite formation reduces NO bioavailability, and itself can damage the electron transport chain, antioxidant defenses, and mitochondrial proteins and nucleic acids.32
Our data are also consistent with the emerging concept of that mitochondrial ROS, particularly hydrogen peroxide (H2
), are central regulators of homeostatic and pathological cell signaling in endothelial cells.33, 34
While superoxide can escape from mitochondria in modest quantities, the concentrations of more stable H2
(produced by rapid dismutation of superoxide to H2
in both the mitochondrial matrix and intermembrane space) far exceed that of superoxide in mitochondria.35
can easily pass through mitochondrial membranes.36
has previously been reported to activate AMPK via calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ).37
Prior work also establishes the ability of NO to reduce mitochondrial H2
production via suppression superoxide production from the mitochondrial ETC.38
Our data are entirely consistent with this previously established regulatory nexus of H2
, AMPK, and NO. We have importantly extended these findings by showing exposure to low glucose at clinically relevant concentrations and exposure times triggers this regulatory pathway, beginning with a loss of NO bioavailability, a concomitant increase in mitochondrial H2
production, and an increase in AMPK activity.
Interestingly, we found treatment with either metformin or AICAR, known pharmacological activators of AMPK through activation of tumor suppressor product LKB1, reversed the loss of NO bioavailability and inhibited excessive mitochondrial oxidative stress. Further, metformin reversed endothelial dysfunction in intact human arterioles acutely exposed to moderate low glucose concentrations. These data are consistent with prior work demonstrating AMPK activation of eNOS and NO suppression of mitochondrial ETC activity.12, 23
Inhibition of eNOS using L-NAME prevented the effects of metformin and AICAR, further suggesting that the ameliorative effects of pharmacological AMPK activation are mechanistically linked to increased NO bioavailability.
One potential paradox in our findings is that increased pAMPK with LG exposure alone failed to increase NO levels even out to 2 hours of LG exposure. Based on prior work and our data in , we suspect this initially puzzling finding relates to differences in the isoform of AMPK activated by metformin and AICAR (AMPKα2 via activation of LKB1)39
versus the isoform activated by H2
(AMPKα1 via activation of CaMKKβ)37
and the relative importance of each isoform in regulating eNOS activity. While AMPKα1 is more abundant in endothelial cells, AMPKα2 activity appears to be of significantly greater importance in eNOS regulation. In endothelial cells, pAMPKα2 plays a key role in activating eNOS and increasing NO bioavailability, while there is minimal to no role for AMPKα1 in eNOS regulation.12
Further, these data would suggest that pharmacological AMPKα2 activation may have a protective effect against LG-induced endothelial dysfunction. Future work is clearly indicated to confirm this attractive hypothesis. provides a graphical synthesis of our findings with previously reported work in this area.
LG-Induced Endothelial Dysfunction: Role of Mitochondrial Superoxide Production and Prevention by AMPK Activation
Our findings with respect to fatty acid and L-carnitine suggest mechanistically that free fatty acid utilization in vivo
would not be sufficient to suppress LG induced NO suppression and excessive mitochondrial superoxide production, particularly in diabetic patients who have significant impairment in free fatty acid utilization and reduced basal plasma and tissue carnitine levels. 40, 41
Interestingly, our data does suggest that pharmacological supplementation with L-carnitine may be able to inhibit the adverse effects of LG on mitochondrial superoxide production and perhaps improve endothelial dysfunction. L-carnitine plays a key role in both transporting and intracellular handling of fatty acids,42
and L-carnitine supplementation has been shown to improve NO bioavailability and improve free-fatty acid induced endothelial dysfunction.43
The mechanism of the vascular benefits of L-carnitine remains unclear, and further work is necessary to delineate the efficacy and mechanism of action of L-carnitine in the setting of low glucose.
Our data have limitations. First, HUVECs may not be representative of other vascular endothelial cells. However, we tested arteriolar endothelial function of intact human vessels and verified that LG induces a state of endothelial dysfunction reversible with metformin and anti-oxidant therapy, suggesting our findings have physiological relevance to human arteriolar endothelial function. Second, the mechanism of eNOS inactivation beyond dephosphorylation at Ser1177 under LG condintions remains unknown and will need to be elucidated in future work. While our data demonstrate eNOS phosphorylation at the 1177 serine site occurs upon exposure to low glucose conditions in a time-dependent manner, experiments with eNOS 1177 mutants would reinforce our findings. Third, we did not specifically measure the contributions of AMPKα1 and AMPKα2. Future work will be necessary to verify the AMPK isoform specific effects we suspect explain the differential effects of physiological H2
based AMPK activation and pharmacological AMPK activation. Finally, metformin’s ameloriative effects may also include augmentation of endothelial cell glucose uptake through activated AMPK directed increases in GLUT1 activity and/or blockade of mitochondrial ETC complex I.44, 45
Both mechanisms could also blunt mitochondrial superoxide production. Further investigation will be necessary to determine the contribution of this metformin effect to our findings. Balanced against these limitations is the novelty of our findings, our evidence of physiological relevance in human arterioles, and the potential clinical implications of these data regarding the cardiovascular dangers of hypoglycemia.