The 21st century is defined as an epidemic of metabolic diseases that not only significantly reduce the quality of life of patients but are also the main cause of death worldwide. The decreased sensitivity of peripheral cells and tissues to insulin, which is called insulin resistance (IR), contributes to the metabolic disorders in the course of obesity, type 2 diabetes and metabolic syndrome [1
]. It is undisputed that the most important factors in the development of IR are a lack of physical activity and improper diet, mainly considering an increased intake of fats and sugars. Although glucose is the primary source of energy for all body cells, in the course of hyperglycemia (primarily postprandial), we can observe an intensified production of reactive oxygen species (ROS), enhanced protein glycation and the induction of polyol and hexosamine pathways [2
]. The binding of advanced glycation end products (AGEs) to a specific receptor (RAGE) not only increases ROS production but also induces the production of proinflammatory cytokines by activating the NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) and c-Jun pathways [4
]. It is believed that long-term sustained high glucose levels after meals play a key role in the development of diabetic complications [3
]. Moreover, recent studies have indicated a link between an increased carbohydrate supply as well as insulin resistance and a greater risk of stroke or increased incidence of neurodegenerative diseases such as mild cognitive impairment (MCI), Alzheimer’s disease (AD) and Parkinson’s disease (PD) [7
]. These changes are thought to be caused by disturbances in redox homeostasis and oxidative stress (OS) [10
]. The latter is defined as an oxido-reductive imbalance leading to the oxidation of cellular biomolecules and thus to disturbed cell metabolism [13
]. The excessive ROS activity causes structural and functional changes in enzymatic and regulatory proteins, damage to cell membranes as well as DNA and the induction of apoptosis [14
]. Interestingly, the brain is particularly exposed to OS. This is determined by the intensive oxygen metabolism, high content of polyunsaturated fatty acids and relatively low activity of antioxidant enzymes [15
]. Therefore, OS may be one of the mechanisms responsible for damage to brain cells under increased sugar supply.
In our previous studies, we demonstrated that a high-sugar diet (HSD) both interferes with systemic redox homeostasis (plasma/serum) and induces oxidative stress at the salivary gland level [17
]. Interestingly, redox imbalance was observed only in the submandibular glands, which indicates the occurrence of organ-dependent metabolic disorders under the influence of an HSD. However, the effect of a dietary high sugar supply on metabolic changes in the brain, particularly including the redox balance of the hypothalamus and cerebral cortex—i.e., the brain structures regulating energy metabolism/the metabolism of lipids and carbohydrates as well as cognitive functions—is still unknown. Given the increasing incidence of metabolic and neurodegenerative diseases and the key role of OS in the pathogenesis of these disorders, the aim of this study was to assess the activity of prooxidative enzymes, enzymatic and non-enzymatic antioxidative barriers, redox status and oxidative damage to proteins in the selected brain structures of rats fed a high-sugar diet. Additionally, we evaluated the effect of an HSD on the redox homeostasis of the plasma/serum as well as selected apoptosis parameters in the brains of rats.
The latest studies indicate deleterious effects of an HSD on neurotransmission, neurogenesis and synaptogenesis [46
]. However, there are no studies concerning brain redox homeostasis under high sugar intake. This study is the first to compare the effect of a high-sugar diet on redox homeostasis in the blood as well as hypothalamus and cerebral cortex of Wistar rats. We demonstrated that an 8-week HSD diet not only induces IR and systemic oxidative stress, but also disrupts redox balance at the brain level. Interestingly, the hypothalamus is much more sensitive to oxidative damage than the cerebral cortex.
It is difficult to conclude regarding the redox homeostasis solely on the basis of a single biomarker [49
]. Therefore, in our study, we examined prooxidant enzymes, enzymatic and non-enzymatic antioxidants, and redox status as well as protein glycation and oxidation.
We showed that an 8-week HSD diet induces obesity, hyperglycemia, hyperinsulinemia, insulin resistance and systemic oxidative stress in rats. In the plasma/serum of rats from the study group, we observed disturbances of the enzymatic antioxidant barrier (↑GPx and ↓SOD-1) as well as higher oxidative damage to proteins (↑Amadori products, ↑AGEs, ↑PCs, ↑AOPPs, ↑glycoxidation products and ↓total thiols) compared to in rats fed a standard diet (Table 2
). These observations are consistent with the results of other studies. Interestingly, we did not demonstrate any correlations between systemic redox homeostasis and changes at the level of the hypothalamus and cerebral cortex, which may indicate a different nature of redox imbalance.
Aerobic organisms have developed both enzymatic and non-enzymatic antioxidant systems in order to protect against ROS overproduction. However, the activity of brain antioxidant enzymes (compared to those in other tissues) is relatively low [15
]. Our study showed a decreased activity of SOD-1 and GPx as well as higher CAT activity only in the hypothalamus of HSD rats (vs. the controls) (Figure 2
). Although we did not directly evaluate the rate of ROS production (or H2
concentration), increased CAT activity may suggest an enhanced production of free radicals in the hypothalamus of the rats from the study group. Indeed, both CAT and GPx are involved in the cytotoxic decomposition of hydrogen peroxide, but CAT is active at high and GPx at low intracellular H2
Interestingly, we did not observe any changes in the total antioxidant capacity and total oxidant status or differences in glutathione metabolism between the studied brain structures. However, in the hypothalamus of HSD rats, we noted a significantly higher oxidative stress index (compared to in the controls), which indicates a shift in brain redox homeostasis towards oxidation reactions. Indeed, this parameter (OSI = TOS/TAC) predisposes the biological system to oxidative stress [52
]. Moreover, the activity of cerebral prooxidative enzymes (↑NOX and ↑XO) was significantly higher in the hypothalamus of HSD rats compared to in the control group (Figure 1
). A strong positive correlation between NOX and XO activity and CAT activity may suggest an adaptive response of the hypothalamus to increased ROS production triggered by HSD. It is well known that the strengthening of the enzymatic antioxidant barrier is the first line of defense against OS [53
], and the decreased activity of SOD-1 and GPx in the hypothalamus of HSD rats is most likely caused by the exhaustion of antioxidant reserves under free radical overproduction (↑OSI).
The consequence of the disruption of antioxidant systems as well as increased activity of prooxidant enzymes is the increased oxidation of hypothalamic proteins (↑Amadori products, ↑AGEs, ↑PCs and ↑glycoxidation products). This hypothesis is confirmed by a positive correlation between the activity of antioxidant enzymes (CAT) and the products of oxidation (PCs) and glycation of proteins (Amadori products and AGEs) in the hypothalamus of HSD rats, and a positive correlation between the activity of prooxidant enzymes and AGEs. However, such changes were not observed in the cerebral cortex of rats from the study group.
The main cell components that undergo oxidation and glycation processes are proteins [54
]. Interestingly, in a typical eukaryotic cell, over 70% of hydroxyl radicals react with proteins, and in the brain, this frequency may be even higher [55
]. The brain is characterized by a high content of prooxidant metal ions, which are known to loosely bind to protein molecules [15
]. Our study showed that an HSD increases both the oxidation (↑PC) and glycation (↑dityrosine, ↑kynurenine, ↑Amadori products and ↑AGEs) of hypothalamic proteins (Figure 5
). Although the glycation of proteins is very slow under physiological conditions, glucose binding to proteins is much faster at high glucose concentrations [56
]. However, it is not only the increased availability of carbohydrate and lipid substrates that may intensify this process; enhanced oxygen metabolism may also. This leads to glucose auto-oxidation as well as the increased formation of dicarbonyl compounds, which significantly raises the glycoxidation rate for proteins [2
]. It has been demonstrated that protein oxidation and glycation lead to damage to amino acid residues, the breaking of polypeptide chains and the formation of cross-links, thus entailing the loss of the biological activity of modified proteins and their tendency to aggregate and accumulate [54
]. This process is particularly important in neurodegenerative diseases (AD and PD) that are accompanied by an additional decrease in the activity of proteasomes responsible for damaged protein removal [60
]. Interestingly, protein advanced glycation end products (mainly AGEs) may increase ROS production by inducing NOX activity as well as activating proinflammatory signaling pathways (mainly NF-kB) [5
]. In our study, this was suggested by a positive correlation between NOX activity and AGE concentration in the hypothalamus of HSD rats. Thus, these conditions may lead to the boosted production of cytokines, chemokines and growth factors as well as increased expression of adhesion molecules (such as ICAM or VCAM). Furthermore, the accumulation of protein glycation products in the brain tissue may lead to cell death through apoptosis or necrosis [4
]. It is noteworthy that both NO concentrations and caspase-3 activity were significantly higher in the hypothalamus of HSD rats compared to the controls (Figure 6
). It is well known that, depending on the concentration, nitric oxide has different effects on the apoptosis process: an excess of NO decreases the ATP concentration as well as the potential of the internal mitochondrial membrane, which results in the inflow of calcium ions and apoptosis [65
We did not observe increased carbonyl stress or apoptosis in the cerebral cortex of HSD rats. Moreover, the efficiency of the antioxidant barrier and the activity of prooxidant enzymes in the cerebral cortex did not differ significantly between the study and control groups. Therefore, we conclude that the hypothalamus is much more sensitive to HSD-induced oxidative damage. This organ, due to the synthesis and secretion of numerous hormones, regulates lipid and carbohydrate metabolism. Indeed, the hypothalamus is characterized by a high density of insulin receptors, which are involved in the central control of the metabolism of peripheral tissues as well as regulation of appetite and satiety [67
]. However, changes in insulin transmission also lie at the root of many neurodegenerative diseases [11
]. It is believed that cerebral oxidative stress (particularly mitochondrial dysfunction) may disrupt insulin signaling within the brain [69
]. Thus, the results of our study may explain the fact that people who consume excessive amounts of carbohydrates in their diet are much more likely to develop obesity and cardiovascular complications than cognitive disorders [71
]. Unfortunately, in our experiment, we did not perform cognitive tests that could have confirmed (or excluded) the occurrence of cognitive impairment in HSD rats. However, recent studies have indicated that the increased sensitivity of the hypothalamus to ROS may be caused by the different energy metabolism of brain structures, which additionally changes with age [73
]. Nevertheless, this hypothesis requires further research and observation.
Our work, despite its undoubted advantages, also had certain limitations. We evaluated only the most commonly used redox and apoptosis biomarkers, so we cannot fully compare the effect of the HSD on redox homeostasis in the brain as well as plasma/serum. Furthermore, our study did not include the assessment of cognitive functions in rats, so we were unable to determine whether OS in the hypothalamus results from hyperglycemia or other metabolic disorders caused by the HSD. Therefore, this work is a starting point for further research, both experimental and clinical.