Eight-Week Consumption of High-Sucrose Diet Has a Pro-Oxidant Effect and Alters the Function of the Salivary Glands of Rats

A high-sucrose diet (HSD) is widely known for its cariogenic effects and promotion of obesity, insulin resistance, type 2 diabetes, and cancer. However, the impact of the HSD diet on the salivary gland function as well as the level of salivary oxidative stress is still unknown and requires evaluation. Our study is the first to determine both redox balance and oxidative injury in the parotid and submandibular glands of rats fed the HSD diet compared to the control group. We have demonstrated that uric acid concentration and the activity of superoxide dismutase and peroxidase varied significantly in both the submandibular and parotid glands of HSD rats vs. the control group. However, enhanced oxidative damage to proteins, lipids, and DNA (increase in advanced glycation end products, advanced oxidation protein products, 4-hydroxynonenal, and 8-hydroxy-2’-deoxyguanosine) was observed only in the parotid glands of HSD rats. Moreover, the HSD diet also reduced the total protein content and amylase activity in both types of salivary glands and decreased the stimulated salivary flow rate. To sum up, an HSD diet reduces salivary gland function and disturbs the redox balance of the parotid as well as submandibular salivary glands. However, the parotid glands are more vulnerable to both antioxidant disturbances and oxidative damage.


Introduction
Free sugars (glucose, fructose) and sucrose are the main substrates used for energy production, which facilitates a positive energy balance [1]. Sustaining the energy balance is key to maintaining a proper body weight (for adults: 25 kg/m 2 > BMI ≥ 18.5 kg/m 2 ) and ensuring optimal nutrient 70 kcal% carbohydrates (sucrose 1400 kcal, cornstarch 1260 kcal, maltodextrin 10 140 kcal), 20 kcal% protein, and 10 kcal% fat [19]. The caloricity of both diets was the same and amounted to 3.85 kcal/g.
The food consumption was measured every two days and fluid consumption was monitored weekly. The rats were weighted on the first day of the experiment and then weekly and immediately prior to sacrifice.
After eight weeks of treatment, the rats were fasted for 12 h and anesthetized with phenobarbital (80 mg/kg body weight, intraperitoneally). The rats were placed in a supine position on the heated couch (37 • C) and then had their tail blood collected to measure blood glucose concentration (Accu Check, Roche). Next, whole unstimulated saliva was collected from the oral cavity with the pre-weighted cotton ball inserted under the tongue and bilaterally medial to the teeth and oral mucosa for 15 min [20,21]. Whole stimulated saliva was being collected 5 min after the injection of pilocarpine nitrate (5 mg/kg body weight, intraperitoneally, Sigma Chemical Co, St. Louis, MO, USA) for 5 min [20,21]. The volume of unstimulated/stimulated saliva was evaluated by subtracting the initial weight of cotton balls from their final weight. One mg of the collected saliva was considered to be 1 µL [20,21].
Subsequently, whole blood was collected from the abdominal aorta into heparinized tubes and centrifuged (5 min, 4 • C, 3000× g, MPW 351, MPW Med. Instruments, Warsaw, Poland). Then the antioxidant butyl-hydroxytoluene (BHT, 10 µL 0.5 M BHT in acetonitrile per 1 mL of the plasma Sigma-Aldrich, Steinheim, Germany) was added to the collected plasma samples. The plasma was pre-cooled in liquid nitrogen and stored at −80 • C until it was analyzed [22].
After blood collection, the parotid and submandibular glands of rats were removed. Those intended for biochemical determination were weighted, freeze-clamped with aluminum tongs, frozen in liquid nitrogen, and stored at −80 • C until biochemical experiments were conducted. The right salivary glands were intended for histological analysis and were fixed with 10% buffered formalin solution.
On the day of biochemical assays, the salivary glands were thawed (4 • C), weighted, and divided into small pieces. Then they were diluted in PBS (1:10) and the fragments of salivary glands intended for the determination of carbonyl groups were diluted in 50 mM phosphate buffer (1:10). Both PBS and phosphate buffer were enriched with BHT (10 µL 0.5 M BHT/1 mL buffer) and a protease inhibitor (1 tablet/10 mL buffer, Complete Mini Roche, France). Next, salivary glands were homogenized (Omni TH, Omni International, Kennesaw, GA, USA, on ice, 5 × 20 s), which was followed by their sonification (1800 J/sample, 3 × 20 s, on ice; UP 400S, Hielscher, Teltow, Germany). The supernatants were analyzed immediately after centrifugation (for 20 min, 4 • C, 5000× g, MPW Med Instruments, Warsaw, Poland).
The insulin concentration was analyzed using the commercial ELISA kit (Shibayagi Co., Gunma, Japan; Cell Biolabs), according to the manufacturer's instructions. The insulin sensitivity was evaluated by using the HOMA-IR index (homeostasis model assessment of insulin resistance = fasting insulin (U/mL) × fasting glucose (mM)/22.5) [23].

Salivary and Plasma Antioxidants
The concentration of UA was measured colorimetrically by using the commercial kit QuantiChrom TM Uric Acid DIUA-250 (BioAssay Systems, Harward, CA, USA) in accordance with the manufacturer's instructions. In this assay, 2,4,6-tripyridyl-s-triazine (TPTZ) forms a blue complex with iron ions in the presence of UA. The intensity of the resulting complex was measured at 490 nm wavelength.
The activity of CAT was estimated in triplicate samples by measuring the decomposition rate of hydrogen peroxide (H 2 O 2 ) at 240 nm wavelength [24]. One unit of CAT activity was defined as the amount of enzyme that decomposes 1 mmol H 2 O 2 for 1 min.
The activity of SOD was assayed colorimetrically by measuring the cytosolic activity of SOD and by inhibiting the oxidation of adrenaline to adrenochrome at 480 nm [25]. It was assumed that one unit of SOD activity inhibits the oxidation of adrenaline by 50%.
The activity of Px was determined colorimetrically at a 412 nm wavelength based on the reduction of 5,5 -dithiobis-(2-nitrobenzoic acid) to thionitrobenzene acid that reacts with hypothiocyanite-the product of potassium thiocyanate oxidation by Px [26]. A decrease in the absorbance of thionitrobenzene acid was measured five times at 30-s intervals.
The activity of GPx was measured by the Paglia and Valentine colorimetric method [27]. This assay is based on the oxidation of reduced glutathione (GSH) and the reduction of organic peroxides by GPx in the presence of NADPH and glutathione reductase (GR). One unit of GPx activity was assumed to catalyze the oxidation of 1 µmol of NADPH for 1 min.
The concentration of total protein was determined via the colorimetric bicinchoninic acid (BCA) method using the commercial kit Thermo Scientific PIERCE BCA Protein Assay (Rockford, IL, USA) with bovine serum albumin (BSA) as a standard.
The concentration of AOPP was determined colorimetrically by measuring the oxidative capacity of the iodine ion at 340 nm [29]. For AOPP determination in plasma, plasma samples were diluted in PBS (pH 7.2) 1:5 (v/v) [30].
The concentration of PC was analyzed by 2,4-dinitrophenylhydrazine (2,4-DNPH) [31]. The principle of the method is based on the reaction of 2,4-DNPH with carbonyl groups in the oxidatively damaged proteins, which results in a hydrazone determined colorimetrically at a 355 nm wavelength. PC was calculated by using an absorption coefficient for 2,4-DNPH = 22,000 M −1 cm −1 .
The concentration of TOS was estimated in triplicate samples, according to the method described by Erel [32]. This assay is based on the oxidation of the ions Fe 2+ to Fe 3+ in the presence of the oxidants contained in the sample. The absorbance was measured bi-chromatically at a 560/800 nm wavelength. The oxidative stress index (OSI) was calculated from the formula: OSI = TOS/TAS × 100 [33].

Salivary and Plasma Inflammation and ROS Production
The concentration of IL-1β was estimated by the ELISA method using ready-made kits (R & D Systems, Canada, Minneapolis, MN, USA) in accordance to the manufacturer's instructions. The absorbance was analyzed at 450 nm.
The activity of NADPH oxidase was measured by a luminescence assay using lucigenin as a luminophore [34]. One unit of NADPH oxidase activity was defined as the amount of enzyme required to release 1 nmol of superoxide anion for 1 min.

Histological Examination
The left salivary glands were processed for paraffin embedding. Next, five-micron sections were prepared and stained with hematoxylin-eosin. The sections were analyzed under a light microscope (OLYMPUS BX 51, Olympus America Inc., Center Valley, PA, USA) at 40× and 60× magnification by one histologist (Wiesława Niklińska)

Statistical Analysis
The data was analyzed by using the program Statistica 10.0 (StatSoft, Krakow, Poland). The Kolmogorov-Smirnov test showed no normal distribution of the obtained results, which was the reason for using non-parametric methods. The assessment of differences in the distribution of quantitative variables between the two groups was conducted by using the U Mann Whitney test. The distribution of values of individual quantitative parameters was presented in the form of medians (minimum-maximum). The Spearman's correlation coefficient was used to study the associations between the examined parameters. The statistical significance was defined as p ≤ 0.05.

General Characteristics
Food consumption, energy intake, and the weight of submandibular glands were comparable between HSD rats and the control group (Table 1). However, the study demonstrated that the eight-week high-sucrose diet affects the final body weight and the parotid glands' weight. The medians of the final body weight and the parotid glands' weight of HSD rats were significantly raised when compared to the controls (p = 0.005 and p = 0.05, respectively). The high-sucrose diet altered glucose homeostasis because fasting glucose and insulin concentration as well as HOMA-IR were significantly increased compared to the control rats (p = 0.0008, p = 0.02, and p < 0.0001, respectively) ( Table 1). High-sucrose feeding influenced protein concentration and amylase activity in both salivary glands of HSD rats ( Figure 1). Total protein concentrations in both: the parotid (↓ 16%) and submandibular glands (↓ 36%) of HSD rats were significantly decreased when compared to the controls (p = 0.01 and p = 0.02, respectively). As presented in Figure 1, amylase activity in the tissue of parotid (↓ 87.8%) and submandibular (↓ 82%) glands of HSD rats decreased considerably in comparison to the control rats' glands (p = 0.008 and p = 0.008, respectively).
Nutrients 2018, 10, x FOR PEER REVIEW 6 of 19 submandibular glands (↓ 36%) of HSD rats were significantly decreased when compared to the controls (p = 0.01 and p = 0.02, respectively). As presented in Figure 1, amylase activity in the tissue of parotid (↓ 87.8%) and submandibular (↓ 82%) glands of HSD rats decreased considerably in comparison to the control rats' glands (p = 0.008 and p = 0.008, respectively). Figure 1 shows that high-sucrose feeding had no influence on the non-stimulated secretory function of salivary glands in HSD rats when compared to the control group. Contrary to unstimulated salivary flow, stimulated saliva secretion in HSD rats was significantly decreased (↓ 34%) when compared to the control rats (p = 0.01).

Plasma Antioxidants and Oxidative Modification Products
Plasma UA concentration and CAT activity were similar for HSD and the control group. In the plasma of HSD rats, SOD activity was decreased (p = 0.002) and GPx activity (p = 0.002) as well as TAS concentration were increased (p = 0.03) when compared to the control group ( Figure 2).  Figure 1 shows that high-sucrose feeding had no influence on the non-stimulated secretory function of salivary glands in HSD rats when compared to the control group. Contrary to unstimulated salivary flow, stimulated saliva secretion in HSD rats was significantly decreased (↓ 34%) when compared to the control rats (p = 0.01).

Plasma Antioxidants and Oxidative Modification Products
Plasma UA concentration and CAT activity were similar for HSD and the control group. In the plasma of HSD rats, SOD activity was decreased (p = 0.002) and GPx activity (p = 0.002) as well as TAS concentration were increased (p = 0.03) when compared to the control group ( Figure 2). submandibular glands (↓ 36%) of HSD rats were significantly decreased when compared to the controls (p = 0.01 and p = 0.02, respectively). As presented in Figure 1, amylase activity in the tissue of parotid (↓ 87.8%) and submandibular (↓ 82%) glands of HSD rats decreased considerably in comparison to the control rats' glands (p = 0.008 and p = 0.008, respectively). Figure 1 shows that high-sucrose feeding had no influence on the non-stimulated secretory function of salivary glands in HSD rats when compared to the control group. Contrary to unstimulated salivary flow, stimulated saliva secretion in HSD rats was significantly decreased (↓ 34%) when compared to the control rats (p = 0.01).

Plasma Antioxidants and Oxidative Modification Products
Plasma UA concentration and CAT activity were similar for HSD and the control group. In the plasma of HSD rats, SOD activity was decreased (p = 0.002) and GPx activity (p = 0.002) as well as TAS concentration were increased (p = 0.03) when compared to the control group ( Figure 2).

Plasma Inflammation and ROS Production
Plasma IL-1β level and NADPH oxidase activity were significantly higher in HSD rats when compared to the controls (p = 0.03, p = 0.0003, respectivetly). The rate of ROS production did not statistically differ between the study and the control animals ( Figure 4).

Plasma Inflammation and ROS Production
Plasma IL-1β level and NADPH oxidase activity were significantly higher in HSD rats when compared to the controls (p = 0.03, p = 0.0003, respectivetly). The rate of ROS production did not statistically differ between the study and the control animals ( Figure 4).

Plasma Inflammation and ROS Production
Plasma IL-1β level and NADPH oxidase activity were significantly higher in HSD rats when compared to the controls (p = 0.03, p = 0.0003, respectivetly). The rate of ROS production did not statistically differ between the study and the control animals ( Figure 4).

Salivary Glands Antioxidants and Oxidative Modification Products
The effect of eight-week high-sucrose diet on the parotid gland antioxidant defense is presented in Figure 5. We noted that only SOD activity in the parotid glands of HSD rats was significantly increased when compared to the control rats (↑ 35%, p = 0.02). The concentration of UA (↓ 39%, p = 0.02) and TAS (↓ 58%, p = 0.002) as well as Px activity (↓ 46%, p = 0.004) in the parotid glands of HSD rats were considerably lower than in the control rats while CAT activity in the parotid glands of the HSD group did not differ from the control group.
The effect of eight-week high-sucrose diet on the parotid gland antioxidant defense is presented in Figure 5. We noted that only SOD activity in the parotid glands of HSD rats was significantly increased when compared to the control rats (↑ 35%, p = 0.02). The concentration of UA (↓ 39%, p = 0.02) and TAS (↓ 58%, p = 0.002) as well as Px activity (↓ 46%, p = 0.004) in the parotid glands of HSD rats were considerably lower than in the control rats while CAT activity in the parotid glands of the HSD group did not differ from the control group.
We observed that the concentration of UA (↓ 22%, p = 0.03) decreased but the SOD activity (↑ 60%, p = 0.003) increased in the submandibular glands of HSD rats when compared to the control group. CAT and Px activity as well as TAS concentration were similar in the submandibular glands of the HSD and the control groups ( Figure 5). We observed that the concentration of UA (↓ 22%, p = 0.03) decreased but the SOD activity (↑ 60%, p = 0.003) increased in the submandibular glands of HSD rats when compared to the control group. CAT and Px activity as well as TAS concentration were similar in the submandibular glands of the HSD and the control groups ( Figure 5).  (Figures 6 and 7).

Salivary Glands Inflammation and ROS Production
The level of IL-1β, activity of NADPH oxidase, and the rate of ROS production were significantly enhanced in both parotid (p = 0.008, p = 0.006, p = 0.04, respectively) and submandibular glands (p = 0.002, p = 0.05, p = 0.03, respectively) of HSD rats when compared to the controls (Figure 8).

Salivary Glands Inflammation and ROS Production
The level of IL-1β, activity of NADPH oxidase, and the rate of ROS production were significantly enhanced in both parotid (p = 0.008, p = 0.006, p = 0.04, respectively) and submandibular glands (p = 0.002, p = 0.05, p = 0.03, respectively) of HSD rats when compared to the controls (Figure 8).
Total protein (p = 0.02) and UA (p = 0.01) concentrations as well as the activity of SOD (p = 0.04) and Px (p = 0.0002) were significantly increased in the submandibular glands when compared to the

Correlations
The Spearman's Correlation Coefficient showed that the level of 4-HNE protein adducts (p = 0.03, r = −0.43) in parotid glands of HSD rats was negatively correlated with a stimulated salivary flow rate. In the parotid glands of HSD rats, positive correlations were noted between PC (p = 0.03, r = 0.47) as well as AOPP (p = 0.02, r = 0.51) and plasma HOMA-IR. We also observed a positive correlation between plasma insulin concentration and PC (p = 0.03, r = 0.46) in submandibular glands of the HSD group.

Histological Analysis
In the salivary glands of both groups of rats, we observed a typical lobular structure that was similar in the acini and duct architecture. In the microscopic image, visible severe diffuse vacuolization in the cytoplasm of parotid gland acini was noted in the HSD group and vacuoles in the cytoplasm of acini of the parotid glands of control rats were very few in number and small in size (Figure 9).

Histological Analysis
In the salivary glands of both groups of rats, we observed a typical lobular structure that was similar in the acini and duct architecture. In the microscopic image, visible severe diffuse vacuolization in the cytoplasm of parotid gland acini was noted in the HSD group and vacuoles in the cytoplasm of acini of the parotid glands of control rats were very few in number and small in size (Figure 9). Similar to parotid glands of HSD rats but less numerous, vacuoles were seen in the cytoplasm of serous cells of the submandibular glands of HSD rats. Cytoplasmic vacuolization was not observed in the submandibular glands of the controls (Figure 9).

Discussion
The presented study evaluated changes in the antioxidant level, oxidative damage, and secretory function of the salivary glands of rats fed a high-sucrose diet. Generally, in the course of eight weeks Similar to parotid glands of HSD rats but less numerous, vacuoles were seen in the cytoplasm of serous cells of the submandibular glands of HSD rats. Cytoplasmic vacuolization was not observed in the submandibular glands of the controls (Figure 9).

Discussion
The presented study evaluated changes in the antioxidant level, oxidative damage, and secretory function of the salivary glands of rats fed a high-sucrose diet. Generally, in the course of eight weeks of the diet, the antioxidant barrier of salivary glands was significantly affected. We demonstrated that both the parotid and submandibular glands of HSD rats are exposed to an oxidative stress condition. The stimulated salivary flow was significantly decreased and salivary amylase activity was statistically reduced in both types of glands of HSD rats compared to the control group.
A chronic high free-sugar diet results in obesity, hyperinsulinemia, hyperglycemia, insulin resistance, and general oxidative stress [6,[36][37][38], which was confirmed by the results of our study. We noticed that an eight-week high-sucrose feeding significantly increased body weight and affected blood glucose homeostasis assessed on the basis of higher concentrations of blood insulin and fasting glucose in the HSD group rats compared to the control rats. Chronic high-sucrose feeding decreased whole-body insulin sensitivity, which was observed as a significant increase in the HOMA-IR index. According to the guidelines on diagnosing insulin-resistance [39,40], the latter result proves insulin resistance developed in the HSD group rats. Lastly, a considerable increase in the plasma concentration of almost all the examined oxidative stress markers (TOS, AGE, AOPP, PC, 8-isoP, 4-HNE protein adducts) confirmed a shift of redox balance towards the oxidative status in the HSD rats vs. the control group. The high-percentage increase in plasma 8-isoP is also noteworthy. Plasma 8-isoP promotes endothelin production, enhances platelet aggregation, and reduces the vascular effect of NO, which may consequently result in cardiovascular diseases [41].
It should be mentioned that a high-sucrose diet may contribute to the development of prooxidative milieu in several possible aspects. First of all, an excess glucose supply in the respiratory chain increases dramatically during high-sucrose feeding, which leads to the formation of excessive quantities of superoxide anions and other free radical species [5]. A high-sucrose diet has been proven to cause lipogenic effects and it has been shown that fatty acids act as mild disconnectors responsible for changes in trans-membrane potential that could result in ROS production [42]. Moreover, as confirmed by the presented results, a high-sucrose diet results in the accumulation of an advanced glycation end product (AGE). Levi and Werman [43] demonstrated that the oxidative degradation of sucrose or glucose adducts results in enhanced ROS production. It has been shown that AGE is also able to increase NADPH oxidase activity [44] and that a high-sucrose diet alters heart antioxidant enzyme activity [6] which, with the excessive production of ROS, leads to an imbalance of a redox status and accelerates oxidative stress.
The oral cavity is subjected to various external factors that have oxidizing potential and the ability to generate ROS. It is also known that salivary antioxidants and-to a lesser extent-plasma antioxidants constitute an important part of the oral antioxidant barrier. One of the key salivary antioxidants is salivary peroxidase which, together with catalase, neutralizes hydrogen peroxide (H 2 O 2 ) formed as a result of the dismutation reaction with the participation of superoxide dismutase. Blood-derived uric acid contributes to 40% of the total salivary antioxidant barrier [10].
In both salivary glands of rats fed a high-sucrose diet, we observed a significant increase in SOD specific activity, which was more likely a cellular compensatory response to intensified superoxide anion generation than an attempt to regulate oxidative stress resistance. Tsang et al. [45] proved that a raised level of superoxide anions leads to an increase in superoxide dismutase activity and its relocation to the cell nucleus. The enzyme regulates the expression of protein genes responsible for oxidative stress resistance as well as protein genes responsible for repairing oxidative damage. Excess H 2 O 2 formed through SOD-catalyzed dismutation of superoxide does not seem to be sufficiently neutralized because we did not observe an adequate response from the enzymes responsible for its neutralization (parotid glands of HSD rats: ↓ Px, CAT unchanged, submandibular glands of HSD rats: CAT and Px activities unchanged). Although it is known that H 2 O 2 is not a free radical, it is able to cross the cell membranes and diffuse into different cellular compartments. Hydrogen peroxide that is not neutralized reacts very easily with transition metals and forms the highly reactive hydroxyl radical (OH ) responsible for the majority of oxidative damage in biological compartments [46]. We cannot explicitly explain the behavior of enzymes involved in the neutralization of hydrogen peroxide solely on the basis of the performed assays. Decreased as well as unchanged enzyme activity may result from the oxidative modification of polypeptide chains or the participation of enzymes in combating free radicals during the eight weeks of the diet. The impaired antioxidant defense may be also caused by non-enzymatic glycation of these enzymes in the course of hyperglycemia, which explains the negative correlation between Px activity in the parotid glands of HSD rats and plasma glucose concentration [47]. Clearly, the efficiency of the entire system cannot be assessed based on the analysis of only three antioxidant enzymes. However, one of the evaluated parameters was also the total antioxidant capacity, which is the sum of all antioxidants tested in the biological material (TAS). A significant decrease in TAS in the parotid glands, unchanged TAS in the submandibular glands of HSD rats, and the observed enhancement of oxidative modification in both salivary glands in the course of a high-sucrose feeding confirm the inefficiency of antioxidant systems of these glands in the eradication of ROS.
The results of our study revealed a greater-percentage increase in the concentration and diversity of oxidative damage as well as the intensity of OS to the parotid glands compared to the submandibular glands of rats fed a high-sucrose diet. In the parotid glands of the HSD rats, we observed a significant increase in all the assessed parameters of oxidative stress. In the submandibular glands of these rats, only the PC and 8-isoP levels were raised. It should also be emphasized that increased concentrations of oxidation products in the submandibular glands (PC ↑ 57%, 8-isoP ↑ 139%) were less pronounced than in the case of the parotid glands (PC ↑ 82%, 8-isoP ↑ 410%) of the HSD group vs. the controls. The above results confirm that the parotid glands are subjected to more intense oxidative stress or they are more susceptible to an oxidant attack generated in the course of the high-sucrose diet than the submandibular glands. On the other hand, the lower intensity and size of oxidative damage observed in the submandibular glands may indicate the initial stage of oxidative stress development.
The systemic oxidative stress-induced plasma redox balance changed, but blood composition did not seem to reflect salivary glands composition. However, we noticed the positive correlation between PC and AOPP concentration in the parotid glands and plasma HOMA-IR of the HSD group, which suggests that only the level of parotid protein peroxidation products increases as a function of insulin resistance while parotid lipid peroxidation, 8-OHdG, the submandibular lipid, and protein peroxidation products are independent of insulin sensitivity. We also demonstrated a positive correlation between plasma insulin and PC concentrations in the submandibular glands of the HSD rats. These results could be explained by the assumption that hyperinsulinemia leads to insufficient scavenging of hydrogen peroxide that is responsible for the oxidation of protein thiol groups in the cell [48].
We observed that the weight of the parotid glands of HSD rats was increased while the submandibular gland weight did not change. In addition, the high-sucrose diet was associated with severe morphological changes in the parotid glands and a small degree of vacuolization of the submandibular glands. These morphological changes appeared to be of a lipid nature since they had been removed during fixation and processing of the sample. It was also demonstrated that the consumption of a high-sucrose diet leads to an increase in tissue triglyceride accumulation [49]. Such severity of fatty degeneration in the parotid vs. submandibular glands of the HSD group appears to be the cause of intensified oxidative modifications in the parotid glands compared to the submandibular glands of HSD rats. It has been shown that adipose tissue increases the expression of proinflammatory monocyte chemoattractant protein-1 (MCP-1), which promotes macrophage infiltration and activates the inflammatory signaling pathway in adipocytes and macrophages [50]. This condition results in an increase in the secretion of pro-inflammatory cytokines and an increase in the production of ROS [23], which was confirmed by the presented study (↑ IL-1β, ↑ NADPH oxidase, ↑ ROS). It should be emphasized that NADPH oxidase is the main source of ROS in the cell and plays a critical role in the inflammatory processes [30,51].
We observed no changes in the volume of unstimulated whole saliva secretion, but the stimulated whole saliva as well as protein concentration and amylase activity in both glands were significantly reduced. It is believed that stimulated saliva is produced mainly by the parotid glands [52]. The impairment of the secretory function of the parotid glands may be explained by severe fatty degeneration, which reduces the active surface of secretory glands and leads to the destruction of residual glandular elements [53]. A decreased response of residual acinar cells to the muscarinic agonist can also be explained by the negative correlation between 4-HNE protein adducts in the parotid glands and a stimulated salivary flow of HSD rats. Evidence showed that the level of 4-HNE protein adducts increases the expression of proinflammatory cytokines and metalloproteinase production in the course of mitochondrial ROS-mediated stimulation of Akt/NF-kappaB signaling pathways. The cytokines and metalloproteinases have been shown to disturb the stromal tissue by decreasing the number and size of fibers, focal dilatations of axons, and fiber degeneration [54] and by blocking the interaction between the neurotransmitter and its receptor in the salivary glands of diabetic rats [55]. The latter mechanisms have also been described as the cause of a sympathetic system dysfunction in the salivary glands of diabetic rats [56]. Because the activity of salivary α-amylase is considered a marker of the sympathetic nervous system activity, we can assume that HSD rats presented lower activity of this branch of the autonomic nervous system in both glands [56]. This may be the reason for the observed decrease in salivary amylase activity and protein concentration in both salivary glands. On the other hand, it has been shown that impaired amylase secretion may result from insulin resistance, i.e., reduced sensitivity of target tissues to insulins and no anabolic effect of this hormone [57].
It should be recalled that insulin resistance is a pre-diabetic metabolic state. In these conditions, the significant decrease in the salivary gland function was confirmed in many previous clinical studies [17,[58][59][60]. Moreover, the salivary gland dysfunction has been associated with higher prevalence of dental caries [17,60], oral candidiasis [61], periodontitis [58], mucous injury, and xerostomia [62].
In summary, we demonstrated redox imbalance in the salivary glands of rats fed the HSD diet and greater sensitivity of the parotid glands to oxidative cellular damage. We found that a high-sugar diet also disturbs the secretory function of the salivary glands, which-in addition to the cariogenic effect-may significantly impair the homeostasis of the oral cavity system.
Analyzing the results of our study, it ought to be noted that only the kinetic studies can unequivocally confirm whether it is a high-sucrose diet or insulin resistance that causes the observed changes in the salivary glands. The other limitations of our experiment also included: the use of an animal model, duration of the experiment, and the evaluation of only some, although the most frequently used, biomarkers of redox homeostasis and oxidative stress. On the other hand, it should be emphasized that the analysis of the redox system (antioxidant barrier, parameters describing oxidative stress) and secretory function of the rat salivary glands in the course of a high-sucrose diet is novel and has an important clinical aspect.

Conclusions
• A high-sucrose diet disturbs the redox balance of the parotid and submandibular salivary glands. However, the parotid glands are more vulnerable to both antioxidant disturbances and oxidative damage.