**2. Results**

The MTT assay evaluated mitochondrial activity, which can be treated as a measurement of metabolic activity, and thus viability, of cells. Cell dysfunction and cytotoxicity after exposure to high concentrations of glucose or insulin were assessed by MTT assay and presented in Figure 1.

**Figure 1.** Metabolic activity measured in MTT assay of neuron-like cells after 24 h incubation with different concentrations of (**A**) glucose and (**B**) insulin. Control—untreated neuron-like cells; DMSO—PC12 cells treated with DMSO. Statistically significant differences compared to the untreated neuron-like cells: \*\* *p* < 0.01, \*\*\* *p* < 0.001.

Analysis of neuron-like cells' viability after 24 h incubation with glucose (5–500 mM) and insulin (10–750 μM) will aid the selection of optimal concentrations (40–60% viability) for further studies. The metabolic activity of neuron-like cells was decreased when they

were cultured for 24 h with solutions of both glucose and insulin as calculated with reference to untreated neuron-like cells. DMSO treatment resulted in a substantial 95.6% decrease in mitochondrial activity, and a comparable reduction (89.8%) was obtained after incubation with 500 mM glucose. Administration of 10 mM, 20 mM glucose and 0.01 μM insulin did not affect the viability of neuron-like cells, as the results are similar (90.5%, 97.5%, 99.7% and 95.9%, respectively) to those obtained with untreated cells (100%). The results obtained after incubation with glucose or insulin were clearly dependent on concentration. The optimal concentrations—50–150 mM for glucose and 50–250 μM for insulin—were chosen for planned experiments based on the obtained MTT results for tested substances.
