*2.1. Nitrite Levels*

The levels of cellular nitrogen free radicals were measured after 1 h and 24 h incubation with glucose or insulin. The obtained results are presented in Figure 2.

**Figure 2.** NO levels in neuron-like cells after: (**A**) 1 h incubation with glucose or insulin and (**B**) 24 h incubation with glucose (50, 100 or 250 mM) or insulin (50, 100 or 250 μM). Control—untreated neuron-like cells; H2O2—cells incubated with 50 μM H2O2 (positive control). Statistically significant differences compared to the untreated neuron-like cells: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

After 1 h incubation, a statistically significant increase in the level of nitric oxide was observed both in the presence of glucose and insulin. The results obtained after 1 h incubation with glucose were clearly dependent on concentration, with a statistically substantial rise after 100 mM (x-fold—1.21) and 150 mM (x-fold—1.3) glucose. In the case of insulin, all three concentrations instigated a statistically significant upsurge in nitric oxide levels after 1 h treatment compared to untreated cells. It is interesting that the addition of 250 μM insulin for 1 h produced a similar amount (x-fold—1.44) of nitric oxide to H2O2 (x-fold—1.49).

Significantly, the impact of glucose and insulin on nitric oxide levels after 24 h incubation was less expressed; only after treatment with 100 μM and 250 μM insulin were the levels of nitric oxide considerably higher (x-fold—1.13 and 1.14, respectively) in comparison to untreated cells.
