**1. Introduction**

Alzheimer's disease (AD), first described in 1907, is a neurodegenerative disorder characterized essentially by *β*-amyloid plaques and tangles of hyperphosphorylated tau proteins alongside cholinergic dysfunction [1–3]. The pathological heterogeneity characteristic of AD creates difficulties in establishing a single theory as to its cause, characterizations, and possible treatments [4]. Important risk factors include age-related biochemical and metabolic changes, vascular disease, traumatic brain injury, epigenetic factors, diet, mitochondrial malfunction, metal exposure, infections, hypertension, obesity, dyslipidemia and diabetes mellitus type 2 (T2DM) [5,6]. In diabetes mellitus, the feedback loops between insulin action and insulin secretion do not function properly. In T2DM, the pancreatic islet *β*-cell's dysfunction, together with insulin resistance in insulin-sensitive tissues, leads to increased glucose production in the liver and decreased glucose uptake in muscle, which results in an excessive amount of glucose circulating in the blood [7]. The growing occurrence of both diabetes and dementia leading to AD is becoming a social and economic challenge worldwide [8,9].

**Citation:** Kubis-Kubiak, A.; Wiatrak, B.; Piwowar, A. The Impact of High Glucose or Insulin Exposure on S100B Protein Levels, Oxidative and Nitrosative Stress and DNA Damage in Neuron-Like Cells. *Int. J. Mol. Sci.* **2021**, *22*, 5526. https://doi.org/ 10.3390/ijms22115526

Academic Editor: Anne Vejux

Received: 10 April 2021 Accepted: 21 May 2021 Published: 24 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Current research studies point out that AD and T2DM might share some underlying mechanisms and putative biochemical pathways, i.e., the desensitization of insulin signaling, most likely driven by chronic inflammation, and mitochondrial dysfunction, as a consequence of increased oxidative stress or vasculopathy [10,11]. Moreover, T2DM is a known risk factor of AD since hyperinsulinemia and insulin resistance, hallmarks of this metabolic disturbance, can lead to memory impairment [12,13]. Almost 70% of T2DM cases demonstrate diverse forms of nervous system impairment such as diabetic neuropathy, slowed digestion of food in the stomach, carpal tunnel syndrome, erectile dysfunction and other peripheral nerve problems or even central nervous system complications, including strokes and possibly cognitive impairment [14,15]. Given that numerous data points to coincidence as well as co-morbidity between those two pathological states, in 2008 prof. De la Monte [16] proposed that AD might be called "type 3 diabetes mellitus," but this term is under criticism by others [17,18]. Nowadays, despite the vast number of papers published on AD and diabetes, the underlying link between these two disorders is still unclear [19,20]. Observations from clinical trials are highly ambiguous and have been futile in deciphering clear paths responsible for the augmented risk of dementia in T2DM patients [21–23]. The origin of brain neurodegeneration connected to continuous hyperglycemia is heterogeneous and comprised of modifications in neurotransmitters' metabolism, neuronal inflammation, mitochondrial disturbances and micro- and macrovascular dysfunction [24]. In T2DM, chronic hyperglycemia, among others, is associated with an increased risk of developing memory deficits or decreased psychomotor speed [25]. The molecular and cellular pathophysiology underlying this complication is not yet well understood. Therefore, it is important to conduct research aimed at explaining the complexity of this relationship.

S100B is an extracellular alarmin, a small helix-loop-helix protein that binds up to four Ca2+ per dimer in EF-hands motifs, counteracts amyloid-*β* accumulation, and is upregulated in AD [26]. Despite its most common localization in a subtype of mature astrocytes that ensheath blood vessels and in neural/glial antigen 2-expressing cells, S100B protein is also expressed in different cell types, ranging from arterial smooth muscle to melanoma cells [27]. Uniquely for the S100 protein family, S100B location is also observed in distinct subpopulations of neurons and in adipocytes, suggesting its potential new role in the regulation of energetic metabolism [28]. The levels of S100B mRNA expressed in the cerebral cortex and adipose tissue have been shown to be very similar [29]. S100B levels are raised in the adult organism as a consequence of nervous system damage, which makes it a potential clinical marker. Moreover, it was found that before any detectable changes in intracerebral pressure, neuroimaging, and neurological examination, S100B concentrations are elevated in serum or cerebrospinal fluids, enabling fast and crucial medical treatment before permanent damage occurs [30,31]. Depending on the concentration, this Janus-faced molecule can represent both beneficial and toxic effects on neuropathological changes in cellular metabolism characteristic for AD [32]. The extracellular action of the S100B protein is primarily based on its interaction with RAGE receptors, which are highly activated during T2DM or in hyperglycemic states. S100B binds to RAGE in the extracellular space and activates a number of intracellular biochemical pathways in microglia and neurons. RAGE expression is potentiated by increased extracellular concentrations of ligands, including the S100B protein. These receptors mediate both the trophic and the toxic effects of the S100B protein on cells [33,34]. At lower concentrations, the binding of S100B protein to RAGE causes activation of the Ras/ERK pathway and stimulation of MEK/MAP kinase regulated by extracellular signals, ERK/NF-κB/Bcl-2 and Ras/Cdc42-Rac1. At higher concentrations, S100B protein leads to hyperactivation of the Ras/MEK/ERK pathway, leading to overproduction of ROS [27]. Wartchow et al. [35] exposed the existence of insulin-S100B regulation of glucose utilization in the brain tissue. Moreover, it regulates glial fibrillary acidic protein and two glycolytic enzymes: fructose-1,6-bisphosphate aldolase and phosphoglucomutase in the brain, which is why it is so crucial for the potential role of S100B protein to be revealed [36,37].

The purpose of this article is to elucidate the potential role of S100B protein in correlation with oxidative and nitrosative stress in disturbed glucose/insulin homeostasis in neuron-like cells differentiated with nerve growth factor (NGF) pheochromocytoma (PC12) cells. This cellular model is widely used in both neurobiological and neurotoxicological studies to study the mechanisms of action of neurotoxicants as well as the potential for chemicals to alter neuronal differentiation [38,39]. To our knowledge, there is no information available regarding the role of S100B protein in the pathological mechanism underlying the "dance macabre" between T2DM and AD. We hypothesized that S100B levels are increased in neurons with simultaneous conditions of metabolic disturbance and neuropathological changes, and this fact could probably be used for the detection of the first cellular disruptions connected to dementia and T2DM.
