Upregulated expression of neuronal nitric oxide synthase by insulin in both neurons and astrocytes

Both insulin and nitric oxide (NO) play important roles in the brain. However, there are no unequivocal evidences pointing to a direct effect of insulin on nitric oxide pathway in the brain. In the present study, the effects of insulin on the expression and activity of neuronal nitric oxide synthase (nNOS) were investigated in the cultured cerebellum cell line R2, cerebral cortical astrocytes, and neurons of rats by using flow cytometry, in situ hybridization, RT-PCR, and electron spin resonance (ESR) techniques. In astrocytes, the expression of nNOS was significantly stimulated by insulin in a concentration-dependent manner, with a maximal increase of about 47.6% compared with the control values (p<0.05, t test, n=5). Furthermore, in situ hybridization analysis showed that the expression of nNOS was also significantly increased by insulin (0.64 ng/ml, 6 h), reaching 134.2+/-9.6% of the control values (p<0.05, t test, n=3). In addition, by using nNOS specific primers, RT-PCR analysis also demonstrated the same effect of insulin (0.64 ng/ml, 6 h) on nNOS mRNA expression. Similarly, significant increase of the expression of nNOS protein and mRNA were also observed in both R2 cells and neurons of rats after incubation with insulin. In addition, significant increase of the activity of nNOS in R2 cells and astrocytes were also detected after incubation with insulin (0.64 ng/ml, 9 h) by using ESR technique. Overall, our results suggested that exogenous insulin could upregulate the expression and activity of nNOS in R2 cells, cerebral cortical astrocytes, and neurons of rats. The phenomena opened new insights for further investigation of the physical and pathological significances of insulin in the brain.