Nitric oxide availability as a marker of oxidative stress.

Nitric oxide (NO) is widely considered one of the most important molecules produced in the human body, acting as a necessary regulator in a vast array of vital physiological functions, namely, blood pressure, immune response, and neural communication. Healthy endothelium is defined by the ability to produce adequate levels of NO. Reactive oxygen species (ROS) play a major role in NO-based cell signaling. ROS can affect NO availability both from production to post-production scavenging and lead to a myriad of vascular disorders due to compromised NO functionality. In 2004, it was identified in animal models that oxidative stress plays a significant role in the development of hypertension, in part by inactivation of NO (Ghosh et al., Br J Pharmacol 141(4):562-573, 2004). It was thus concluded that NO bioavailability was reduced in the presence of ROS. We speculated that the accurate detection of NO and quantification in biological matrices is critical as a marker of oxidative stress (Bryan et al., Proc Natl Acad Sci USA 101(12):4308-4313, 2004). The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively, and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone-based chemiluminescence with chemical derivatization to determine molecular source of NO as well as ex vivo with organ bath myography. This approach ties fundamental biochemistry to functional response.

Dietary nitrite improves insulin signaling through GLUT4 translocation.

Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes.