Acute Hypoglycemia Induces Mitochondrial Dysfunction in the Isolated Brain Microvessels: Possible Role of Endothelial Nitric Oxide Synthase

Background Insulin therapy for patients with diabetes mellitus (DM) carries the risk for acute hypoglycemia (HG). Recurrent HG increases the ischemic brain injury in experimental stroke. Furthermore, HG has been shown to affect mitochondrial function in the cultured endothelial cells. Previously, we have shown that vascular mitochondria regulate endothelial nitric oxide synthase activity (eNOS). Therefore, we hypothesized that exposure to acute insulin‐induced HG in vivo negatively affects the ex vivo cerebral microvascular respiratory function. Here, we investigated for the first time, the effect of acute HG on mitochondrial respiratory function ex vivo in the isolated brain microvessels from wild‐type and endothelial nitric oxide synthase (eNOS) knock‐out mice. Methods Three month‐old C57bl6 mice (n=6) were injected intraperitoneally (i.p) with insulin (3 U/ Kg body weight) to induce and maintain hypoglycemic state (blood glucose level < 70mg/dL) for 90 minutes. In order to evaluate the HG‐independent effects of hyperinsulinemia, a group of mice (n=6) were maintained at normoglycemia after the insulin injection by i.p injection of glucose. Control mice (n=6) were injected with saline. Brain parenchymal microvessles (40μ to 300μ diameter) were isolated and oxygen consumption rate (OCR) and mitochondrial respiratory parameters were measured using Agilent Seahorse XFe24 analyzer. Basal and maximal respiration were measured along with the spare respiratory capacity, proton leak and the non‐mitochondrial respiration. Parameters were reported as OCR normalized to the protein concentration of cell lysates in each well in picomoles/L of O2/μg protein. Similar studies were also conducted in eNOS KO mice. Results The maximal respiration was decreased by 47.3% (3.0±0.4 vs 5.7±0.7, p=0.01, n=6) in the isolated mouse brain microvessels from mice exposed to HG compared to saline exposed animals. Furthermore, spare respiratory capacity decreased by 76% (0.3±0.1 vs 1.3±0.1, p=0.0004, n=6) along with the non‐mitochondrial respiration (1.0±0.2 vs 2.5±0.3, p=0.01, n=6). Basal respiration and proton leak showed a strong trend towards decrease by 32.5% (2.9±0.5 vs 4.3±0.5, p=0.06, n=6) and 28% (1.8±0.3 vs 2.5±0.3, p=0.12, n=6) respectively along with the ATP production (1.4±0.2 vs 1.8±0.2, p=0.24, n=6) in microvessels from mice exposed to HG. Interestingly, similar mitochondrial respiratory changes were not observed in brain microvessels from the mice that were maintained under normal glucose levels after the insulin injection, indicating that the observed changes were solely due to the HG. Interestingly, brain microvessels from eNOS knock‐out mice exposed to HG failed to exhibit the impairments of mitochondrial respiratory function observed in wild type mice. Conclusion Acute HG induces mitochondrial respiratory dysfunction in the cerebral microvasculature. eNOS might play a key role in mediating the observed effects. Support or Funding Information Support: National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .