Glucose metabolism in neuronal tissue declines during neurodegenerative disorders in a progressive, region-specific, and disease-specific manner. Studies have shown that extracellular hyper-glycemia affects the functioning of adenosine triphosphate (ATP) sensitive potassium channels located in neurons and astrocytes. Also, hyper-insulinemia contributes to the formation and progression of Alzheimer’s disease (AD) via competition with amyloid β (Aβ) for insulin-degrading enzyme. Aβ disruption is phosphatidylinositol 3-kinase pathway dependent, and increased circulatory insulin concentrations lead to Aβ accumulation. In 2008, based on assessment of brain glucose utilization disorders and insulin signaling disruptions, it was proposed that AD could be called "type III diabetes". Proteins from the S100 family are actively secreted during metabolic and oxidative stress, but their role in neuronal cells has yet to be clarified. However, it has been demonstrated that they act in a dose-dependent manner, which may be crucial in the modulation of glucose and insulin metabolism in the brain. The goal of this paper was to elucidate the association between high glucose and insulin concentrations with extra- and intracellular S100B and S100A8 proteins levels as well as the correlation with toxic (Aβ₄₂) and physiologic (Aβ₄₀) forms of Aβ. Medium and high glucose concentrations mimicking pre-diabetic and diabetic state, caused statistically significant discharge of S100b and S100A8 protein to the extracellular compartment. Similar effect was observed after 50 nM insulin incubation. Furthermore, the correlation coefficient patterns between those proteins shows similar associations which highlights possible effective and modulating role of S100 family in the metabolic disturbances occurring in neuropathological disorders.