Chronic consumption of a high-fat diet (HFD) disrupts carbohydrate metabolism and mitochondrial activity, leading to increased reactive oxygen species (ROS) production and decreased ATP production in salivary glands. Hyperglycemia exacerbates this effect by increasing mitochondrial oxygen consumption and ROS overproduction. Mitochondrial dysfunction associated with hyperglycemia contributes to insulin resistance and type 2 diabetes. N-acetylcysteine (NAC), an analog of reduced glutathione (GSH), has shown promise in enhancing mitochondrial function, particularly oxidative phosphorylation. Previous research demonstrated that simultaneous administration of NAC with HFD prevents hyperglycemia and protects against oxidative damage in the parotid glands of insulin-resistant rats. However, the effect of NAC administration *after* the onset of hyperglycemia on mitochondrial function in salivary glands remained unclear. This study aimed to determine if NAC supplementation could reverse mitochondrial dysfunction, reduce apoptosis, and decrease pro-oxidative enzyme activity in the salivary glands of rats with HFD-induced hyperglycemia.

Existing literature highlights the detrimental effects of HFD on mitochondrial function, leading to reduced ATP production and increased ROS formation in various tissues, including salivary glands. Hyperglycemia further intensifies this mitochondrial dysfunction. Studies have shown that NAC can improve mitochondrial function by increasing the activity of mitochondrial complexes and reducing ROS production in different organs like the liver, heart, and brain. However, the efficacy of NAC in reversing HFD-induced mitochondrial dysfunction in salivary glands after the development of hyperglycemia was not previously investigated. This gap in knowledge motivated the current research.

Male Wistar rats were divided into four groups (n=10 per group): control (C), control + NAC (C+NAC), high-fat diet (HFD), and high-fat diet + NAC (HFD+NAC). Rats in the HFD and HFD+NAC groups were fed a 60% fat diet for 6 weeks, after which hyperglycemia was confirmed. For the next 4 weeks, rats in the C+NAC and HFD+NAC groups received intragastric NAC (500mg/kg BW). At the end of the 10-week period, rats were sacrificed, and parotid and submandibular salivary glands were collected. Mitochondrial fractions were isolated, and various parameters were assessed, including: mitochondrial respiratory complex activities (I, II, II+III, IV), ADP/ATP ratio, H2O2 concentration, activities of ROS-generating enzymes (NOX and XO), free radical production (DCFH-DA), caspase-3 and -9 activities, Bax, Bcl-2, IL-1β, TNF-α concentrations, GSH, GSSG levels, and the GSH/GSSG ratio. The activity of enzymatic antioxidants (SOD, Px, CAT) and lipid peroxidation (MDA) were also assessed in both mitochondrial fractions and serum. Histological analysis was performed to evaluate salivary gland morphology. Statistical analysis included one-way ANOVA with Tukey's post hoc test. Correlations between parameters were evaluated using Pearson's correlation coefficient.

The HFD group showed significant increases in body weight, blood glucose, insulin levels, and HOMA-IR compared to the control group. NAC administration did not significantly affect these parameters. Mitochondrial respiratory complex activities (I, II+III) were significantly reduced in the HFD group in both salivary glands compared to the control group. NAC supplementation partially restored the activity of complex I in parotid glands but not in submandibular glands. The ADP/ATP ratio was significantly elevated in the HFD group and partially reduced by NAC in both salivary glands. H2O2 levels were significantly higher in the HFD group and were normalized by NAC in the parotid glands. The activities of ROS-generating enzymes (NOX, XO) and free radical production (DCFH-DA) were significantly increased in the HFD group and reduced by NAC, but levels remained significantly higher compared to the control group in some instances. Inflammation biomarkers (IL-1β, TNF-α) were significantly elevated in the HFD group and decreased by NAC in parotid glands, while changes in submandibular glands were less significant. Apoptosis markers (caspase-3, caspase-9, Bax/Bcl-2 ratio) were significantly increased in the HFD group and partially reduced by NAC in parotid glands. GSH/GSSG ratio was restored by NAC supplementation only in submandibular gland mitochondria and in plasma. Histological analysis revealed increased vacuolation in salivary glands of HFD rats, which was not reversed by NAC. In the HFD+NAC group, a negative correlation was observed between ADP/ATP ratio in parotid gland mitochondria and stimulated saliva secretion, and a positive correlation between complex II+III activity and stimulated saliva secretion was also found.

The findings suggest that the beneficial effects of NAC on salivary gland function are dependent on the timing of its administration. While NAC improved some aspects of mitochondrial function and reduced inflammatory and apoptotic markers, particularly in the parotid glands, it failed to completely reverse the detrimental effects of HFD-induced hyperglycemia. The partial restoration of mitochondrial function and reduced oxidative stress in some parameters suggest that NAC might have some protective effects, but these effects are limited and may not be sufficient to fully counteract the profound mitochondrial damage caused by prolonged HFD-induced hyperglycemia. The differences observed between the parotid and submandibular glands suggest potential tissue-specific responses to NAC supplementation and HFD.

This study demonstrates that NAC supplementation introduced after the onset of hyperglycemia offers limited protection against mitochondrial dysfunction, apoptosis, and inflammation in the salivary glands of HFD rats. The positive effects are more pronounced in the parotid glands than in the submandibular glands. Future studies could investigate the effects of earlier NAC administration and explore other potential therapeutic interventions for mitigating HFD-induced salivary gland damage.

The study was conducted on a rat model, which may not fully reflect human physiology. The relatively short duration of NAC supplementation (4 weeks) may not have been sufficient to observe more significant beneficial effects. Further research with longer-term NAC treatment is warranted. The study focused on mitochondrial parameters and might not fully capture the overall impact of NAC on salivary gland function. More detailed investigation into the mechanisms underlying the tissue-specific differences in response to NAC is needed.