Chronic High-Fat Diet Downregulates M3 Muscarinic and β1-Adrenergic Receptor mRNA Expression and Alters Submandibular Gland Histology in Wistar Rats

Abstract

Introduction

A chronic high-fat diet (HFD) is associated with metabolic disturbances, inflammation, oxidative stress, and functional impairment in several organs. However, its effects on autonomic receptor expression and submandibular gland histology remain insufficiently understood. The submandibular gland contributes 65 to 75% of basal salivary secretion and is central to salivary function. This study aimed to investigate the effect of chronic HFD exposure on M3 muscarinic receptor and β1-adrenergic receptor mRNA expression, as well as histological changes, in the submandibular glands of Wistar rats.

Methods

This experimental study used a completely randomized design. Fourteen male Wistar rats were divided into two groups: a normal chow diet control group and an HFD group, with seven rats in each group. The HFD was administered for 21 weeks. Submandibular gland tissues were collected and analyzed for M3 muscarinic receptor and β1-adrenergic receptor mRNA expression using quantitative real-time polymerase chain reaction. Histological examination was performed using hematoxylin and eosin staining, and the mean number of secretory units per field of view was calculated. Data were analyzed using the Shapiro–Wilk normality test followed by an independent t-test.

Results

M3 muscarinic receptor mRNA expression was significantly lower in the HFD group than in the control group (0.189 ± 0.057 vs 0.483 ± 0.101; p < 0.001). β1-adrenergic receptor mRNA expression was also significantly reduced in the HFD group compared with the control group (0.067 ± 0.031 vs 0.169 ± 0.071; p = 0.008). Histological analysis showed that the mean number of secretory units per field of view was significantly lower in the HFD group than in the control group (9.84 ± 0.654 vs 13.52 ± 2.171; p = 0.007).

Conclusion

Chronic HFD exposure downregulated M3 muscarinic receptor and β1-adrenergic receptor mRNA expression and induced histological alterations in the submandibular glands of Wistar rats. These findings suggest that chronic HFD may affect salivary gland structure and autonomic regulation, providing a potential basis for salivary dysfunction and increased oral health risks. However, further studies incorporating direct functional assessments are needed to confirm its actual impact on saliva secretion.

Introduction

Rapid socioeconomic development has substantially altered lifestyle and dietary patterns, particularly in developing countries. These changes include a shift from a high-carbohydrate, low-fat diet toward a diet with lower carbohydrate content and a higher proportion of fat, often accompanied by reduced physical activity.^1,2 In Indonesia, data from the 2023 Indonesian Health Survey showed that 37.4% of respondents regularly consumed fatty, cholesterol-rich, or fried foods, while the prevalence of obesity among adults aged >18 years increased from 21.8% in 2018 to 23.4% in 2023.³

High dietary fat intake has been associated with obesity, insulin resistance, cardiovascular disease, and chronic low-grade systemic inflammation. The biological response to a high-fat diet is characterized by increased circulating free fatty acids, endotoxins such as lipopolysaccharide (LPS), and inflammatory mediators, which may disrupt metabolic homeostasis and impair the function of several organs.^4–7 In the oral cavity, these systemic alterations may affect salivary gland physiology and contribute to reduced salivary secretion, thereby increasing the risk of oral diseases such as dental caries. The prevalence of dental caries in Indonesia remains high, reaching 82.2% according to the 2023 Indonesian Health Survey.^8–11

In addition to its effect on dental caries, high-fat diet exposure may also contribute to periodontal tissue damage through alterations in the oral and gut microbiome. Murphy et al reported that a high-fat diet may influence the oral microbiome, including a reduction in Bacteroidetes and an increase in Firmicutes.¹⁰ In line with this, Bai et al reported that high-fat diet affected the diversity and composition of salivary and gut microbiota in an animal model, although the effect was more pronounced in the gut microbiota.¹² These microbial compositional changes indicate dysbiosis in the oral cavity, which has been associated with several oral diseases, including periodontitis. The possible mechanism involves gut dysbiosis, reduced production of short-chain fatty acids, activation of the LPS/TLR4 pathway, increased systemic and local inflammation, and reduced gingival blood flow, which may ultimately accelerate periodontal tissue damage.¹¹

Salivary glands play a critical role in maintaining oral health through saliva secretion, which supports mastication, swallowing, lubrication, buffering capacity, remineralization, antimicrobial defense, and protection against dental caries. The major salivary glands, including the parotid, submandibular, and sublingual glands, are regulated by the autonomic nervous system. Sympathetic stimulation primarily regulates the secretion of salivary proteins through beta-adrenergic receptors, including β1-adrenergic receptor (β1-AR), whereas parasympathetic stimulation regulates fluid and electrolyte secretion mainly through muscarinic cholinergic receptors, particularly muscarinic receptor M3 (M3R).^13,14 Therefore, alterations in the expression of these receptors may indicate impaired autonomic regulation of salivary gland function.

Previous studies have shown that a high-fat diet may induce oxidative stress, nitrosative stress, inflammation, and structural damage in salivary glands. These changes include mitochondrial dysfunction, increased production of reactive oxygen and nitrogen species, inflammatory cell infiltration, fibrosis, vascular changes, and intracellular lipid accumulation.^15–17 At the molecular level, high-fat diet exposure has been associated with altered expression of genes involved in inflammation, oxidative stress, and apoptosis, including TNF-α, IL-2, NF-κB, SOD2, GPx, BAX, and Bcl-2.^15,18,19 However, evidence regarding the effect of a high-fat diet on genes involved in autonomic regulation of salivary secretion remains limited.

The submandibular gland contributes approximately 65–75% of basal salivary secretion, making it an important gland for evaluating changes in salivary function.²⁰ Therefore, this study aimed to evaluate the mRNA expression of muscarinic receptor M3R and beta-adrenergic receptor β1-AR in the submandibular glands of non-diabetic rats fed a high-fat diet. The findings of this study may provide insight into the early effects of high-fat diet-induced metabolic disturbances on autonomic regulation of salivary gland function and their potential implications for oral health.

Materials and Methods

Study Design

This study was an experimental study using a completely randomized design to evaluate the effects of a high-fat diet on M3R and β1-AR mRNA expression and histological features of the submandibular glands in Wistar rats. The study used stored biological samples obtained from male Wistar rats (Rattus norvegicus) that had been subjected to high-fat diet intervention. A total of 14 rats obtained from PT Biofarma, Bandung, Indonesia, were acclimatized and randomly divided into two groups: a normal control group and a high-fat diet group.

Animals and Sample Size

The study population consisted of healthy male Wistar rats aged 6–8 weeks and weighing 200–220 g. After a 2-week acclimatization period and 21 weeks of high-fat diet intervention, the rats were estimated to be 29–31 weeks old at termination. This indicates that the animals were in the adult phase and had not yet reached reproductive senescence or post-senescence. This age range was considered relevant because age may influence salivary gland function, including salivary secretion.²¹ The inclusion criteria were healthy rats before treatment, indicated by active movement, clear eyes, and the absence of functional abnormalities. Rats that became ill, developed physical abnormalities, or died during the study were excluded.

The sample size was determined using Mead’s resource equation method:

where E represents the error degrees of freedom, N represents the total number of experimental animals minus one, B represents the blocking component, and T represents the treatment degrees of freedom minus one. Based on this calculation, the minimum required sample size was 12 rats. To anticipate unexpected losses, 14 rats were used in this study, with seven rats allocated to each group.

Experimental Groups and Dietary Intervention

After the acclimatization period, body weight was measured before treatment. The rats were randomly assigned to two groups using a lottery-based randomization method to minimize allocation bias. The control group received a normal chow diet, whereas the high-fat diet group received a high-fat diet. All animals had free access to drinking water throughout the study period.

The dietary intervention was conducted for 21 weeks as a long-term high-fat diet model. The normal chow diet and high-fat diet were provided according to the standards approved by Universitas Padjadjaran and the Indonesian Food and Drug Authority using the SNI 1992 testing method. The composition of the diets used in this study is presented in Table 1.

Table 1.

Composition of the Normal Chow Diet and High Fat Diet Used in This Study

Composition	Normal Chow Diet (g%)	High Fat Diet (g%)
Carbohydrate	48.3	26.3
Protein	24.9	26.2
Fat	9.7	37.7
Water	10,98	10,98
Ash	6.13	6.13
Calcium	0.8	0.8
NaCl	0.1	0.1
Total Energy	380 kcal	549 kcal

Abbreviations: g, gram; kcal, kilocalorie.

Animal Housing and Acclimatization

Before the intervention, the rats were acclimatized for two weeks to adapt to the diet and laboratory environment. The animals were housed in plastic cages measuring 54 × 36.5 × 28.5 cm³, with three to four rats per cage. The cages were lined with wood shavings and covered with wire mesh to allow access to food and water. Food and water were provided ad libitum. The laboratory was maintained under a 12-h light/dark cycle, with controlled temperature at 22–24°C and stable humidity. The cages were placed away from noise sources and cleaned three times per week. The animals were monitored daily, and body weight was recorded once weekly. All procedures and observations were documented in a research logbook.

Animal Termination and Tissue Collection

At the end of the study period, all rats were terminated. Anesthesia was administered intraperitoneally using ketamine at a dose of 125 mg/kg body weight until the animals showed no pain reflexes or spontaneous movement. After complete loss of consciousness was confirmed, cervical dislocation was performed as the euthanasia method. The neck region was dissected, and the submandibular glands were collected from the anterior cervical region, between the submandibular lymph nodes and the sternum. The glands were weighed using a tissue scale. The collected submandibular gland samples were immediately frozen in liquid nitrogen and stored at −80°C until further analysis. All procedures involving experimental animals were performed by trained researchers under the supervision of the Research Ethics Committee of Universitas Padjadjaran and in accordance with the Standard Operating Procedure for Ethical Animal Experimentation.

Histological Examination

Following termination, submandibular gland tissues were processed for histological examination. The tissues were fixed in 10% phosphate-buffered formaldehyde and processed using standard histological techniques. The samples were embedded in paraffin, sectioned transversely at a thickness of 5 μm, deparaffinized, rehydrated, and stained with hematoxylin and eosin to evaluate glandular structural changes. The stained sections were examined under a light microscope at 400× magnification. For each sample, five fields of view were randomly selected. The number of secretory units was counted in each field, and the mean number per field was calculated for each sample. Images were documented with a scale bar and saved in high-quality TIFF or JPEG format for further analysis. Histological observation and counting of secretory units were performed by the principal investigator.²²

RNA Isolation

RNA was isolated from submandibular gland tissue using GENEzol™ reagent according to the manufacturer’s protocol. Briefly, tissue samples were placed into microtubes, followed by the addition of 200 μL GENEzol™ reagent and incubation at room temperature for 5 min. Subsequently, 40 μL chloroform was added, and the samples were vortexed for 15 s and incubated at room temperature for 3 min. The samples were then centrifuged at 11,200 rpm for 15 min at 4°C. The supernatant was transferred into a new microtube, mixed with 100 μL isopropanol, vortexed for 15 s, and incubated at room temperature for 10 min. The samples were centrifuged at 11,200 rpm for 10 min at 4°C. The RNA pellet was washed with 200 μL of 70% ethanol and centrifuged at 11,200 rpm for 5 min at 4°C. The pellet was air-dried for 5–10 min at room temperature, resuspended in 20 μL DEPC-treated water, and incubated at 60°C for 10 min using a heat block.

Quantitative Real-Time PCR

The mRNA expression levels of M3R and β1-AR were measured using quantitative real-time polymerase chain reaction with an AriaMx Real-Time PCR system. GAPDH was used as the reference gene. The primer sequences used in this study were as follows:

M3R forward, 5′-CGGTCGCTGTCACTTCTGGTTC-3′;

M3R reverse, 5′-CGCTGCTGCTGTGGTCTTGG-3′;

β1-AR forward, 5′-CTCATCGTGCTGCCATCGTAGTG-3′;

β1-AR reverse, 5′-GATGAAGAGGTTGGTGAGCGTCTG-3′;

GAPDH forward, 5′-GTTACCAGGGCTGCCTTCTC-3′; and

GAPDH reverse, 5′-GATGGTGATGGGTTTCCCGT-3′.

The relative mRNA expression levels were calculated and expressed as fold change.

Statistical Analysis

The data were analyzed using IBM SPSS Statistics version 24. Data normality was assessed using the Shapiro Wilk test. Normally distributed data were analyzed using the independent samples t-test. If the data were not normally distributed, the Mann Whitney U-test was used. A p-value of <0.05 was considered statistically significant.

Results

M3R mRNA Expression in the Submandibular Gland

M3R mRNA expression in the submandibular glands was significantly lower in the HFD group than in the NCD group. The comparison of M3R mRNA expression between the two groups is presented in Table 2, and the graphical representation is shown in Figure 1. The mean M3R mRNA expression was 0.483 ± 0.101 in the NCD group and 0.189 ± 0.057 in the HFD group. Statistical analysis using the independent samples t-test showed a significant difference between the two groups (p < 0.001), as shown in Table 2. These findings indicate that long-term HFD exposure reduced M3R mRNA expression in the submandibular glands of Wistar rats.

Table 2.

Comparison of M3R mRNA Expression in the Submandibular Glands Between the NCD and HFD Groups

Variable	NCD Group (Mean ± SD)	HFD Group (Mean ± SD)	P Value
M3R mRNA expression	0.483 ± 0.101	0.189 ± 0.057	<0.001

Abbreviations: HFD, high fat diet; M3R, muscarinic receptor 3; NCD, normal chow diet; SD: standard deviation.

Figure 1.

M3R mRNA expression in the submandibular glands of rats in the NCD and HFD groups.

Notes: Data are presented as mean ± SD. ***p < 0.001 compared with the NCD group.

β1-AR mRNA Expression in the Submandibular Gland

β1-AR mRNA expression in the submandibular glands was significantly lower in the HFD group than in the NCD group. The comparison of β1-AR mRNA expression between the two groups is presented in Table 3, and the graphical representation is shown in Figure 2. The mean β1-AR mRNA expression was 0.169 ± 0.071 in the NCD group and 0.067 ± 0.031 in the HFD group. Statistical analysis using the independent samples t-test showed a significant difference between the two groups (p = 0.008), as shown in Table 3. These findings indicate that long-term HFD exposure reduced β1-AR mRNA expression in the submandibular glands of Wistar rats.

Table 3.

Comparison of β1-AR mRNA Expression in the Submandibular Glands Between the NCD and HFD Groups

Variable	NCD Group (Mean ± SD)	HFD Group (Mean ± SD)	P Value
β1-AR mRNA expression	0.169 ± 0.071	0.067 ± 0.031	0.008

Abbreviations: β1-AR, beta-1 adrenergic receptor; HFD, high fat diet; NCD, normal chow diet; SD, standard deviation.

Figure 2.

β1-AR mRNA expression in the submandibular glands of rats in the NCD and HFD groups.

Notes: Data are presented as mean ± SD. **p < 0.01 compared with the NCD group.

Histological Findings of the Submandibular Gland

Representative histological images of the submandibular glands from the NCD and HFD groups are shown in Figure 3a and b, respectively. In the NCD group, the secretory units of the submandibular gland tissue appeared as well-organized clustered structures,²³ indicating relatively preserved glandular architecture (Figure 3a). In contrast, the HFD group showed fewer secretory units per field of view (Figure 3b).

Figure 3.

Histological appearance of the submandibular glands in the NCD (a) and HFD (b) groups (H&E staining, 400× magnification).

Notes: Arrows indicate the submandibular gland. Scale bar = 100 µm.

The comparison of the mean number of secretory units per field of view between the NCD and HFD groups is presented in Table 4, and the graphical representation is shown in Figure 4. The mean number of secretory units per field of view was significantly lower in the HFD group than in the NCD group. The NCD group showed a mean value of 13.52 ± 2.171 secretory units per field of view, whereas the HFD group showed a mean value of 9.84 ± 0.654 secretory units per field of view. Statistical analysis using the independent samples t-test showed a significant difference between the two groups (p = 0.007), as presented in Table 4. These results indicate that long-term HFD exposure reduced the number of secretory units in the submandibular glands of Wistar rats.

Table 4.

Comparison of the Mean Number of Secretory Units per Field of View in the Submandibular Glands Between the NCD and HFD Groups

Variable	NCD Group (Mean ± SD)	HFD Group (Mean ± SD)	P Value
Secretory units per field of view	13,52 ± 2171	9.84 ± 0.654	0.007

Abbreviations: HFD, high-fat diet; NCD, normal chow diet; SD, standard deviation.

Figure 4.

Mean number of secretory units per field of view in the submandibular glands of rats in the NCD and HFD groups.

Notes: Data are presented as mean ± SD. **p < 0.01 compared with the NCD group.

Discussion

In this study, long-term high-fat diet exposure was associated with reduced M3R and β1-AR mRNA expression and a decreased number of secretory units in the submandibular glands of Wistar rats. These findings suggest that HFD may impair submandibular gland function through alterations in autonomic receptor expression and histological structure.

Based on a previously published study, 12 weeks of HFD exposure induced lipid profile alterations consistent with dyslipidemia. The HFD group showed higher levels of total cholesterol, triglycerides, and LDL-C than the control group, whereas HDL-C levels were lower. These changes were statistically significant.²⁴ Although lipid profile was not analyzed as a primary variable in the present study, these previously published lipid profile data²⁴ indicate that the stored biological samples used in this study were obtained from animals with HFD-induced metabolic disturbance. Therefore, this metabolic background may support the interpretation of the observed changes in M3R and β1-AR mRNA expression and submandibular gland histology.

A previous study by Chen et al reported that HFD administration for eight weeks reduced the expression of M3R and β1-AR in the salivary glands of rats with diabetes mellitus.²⁵ However, because this study used a diabetes-induced model, it remains unclear whether the observed changes were caused directly by HFD exposure or by diabetes-related complications such as hyperglycemia and vascular damage. Therefore, studies evaluating the direct effects of a HFD on salivary glands in non-diabetic conditions are needed. This is particularly relevant because individuals with obesity or high-fat dietary patterns may develop subclinical salivary gland dysfunction even in the absence of diabetes. In the present study, reduced M3R and β1-AR mRNA expression occurred without experimental diabetes induction, suggesting that chronic HFD exposure accompanied by metabolic disturbance may also impair receptors involved in salivary secretion regulation.

The significant reduction in M3R mRNA expression observed in the HFD group indicates that HFD exposure may affect parasympathetic regulation of salivary secretion. M3R is the major muscarinic receptor in salivary glands and plays an essential role in regulating salivary volume and composition through parasympathetic stimulation.²⁶ Salivary secretion begins when acetylcholine binds to M3R on the surface of salivary acinar cells. This activation stimulates the formation of inositol 1,4.5-trisphosphate (IP3), which induces Ca²⁺ release from the endoplasmic reticulum into the cytoplasm. Increased intracellular Ca²⁺ then activates Ca²⁺-sensitive Cl⁻ and K⁺ channels, creating an osmotic gradient that drives water movement into the glandular lumen. In addition, increased intracellular Ca²⁺ promotes aquaporin-5 (AQP5) translocation to the apical membrane of acinar cells, facilitating water transport and saliva formation.^27,28 Therefore, reduced M3R expression may disrupt parasympathetic-mediated fluid secretion and contribute to impaired salivary gland function.

The reduction in M3R mRNA expression may be related to oxidative and nitrosative stress induced by HFD. Previous studies have shown that HFD exposure increases oxidative stress biomarkers, such as malondialdehyde (MDA) and total oxidant status, while reducing total antioxidant capacity in salivary gland tissue.^19,29,30 Increased oxidative stress may cause lipid peroxidation and membrane damage, which can impair receptor integrity and signaling. Lipid peroxidation products, including isoprostanes, have been reported to disrupt membrane fluidity and receptor function and may also reduce neurotransmitter release.^31–33 These mechanisms may weaken parasympathetic stimulation and contribute to the downregulation of M3R expression.

Inflammation may also contribute to reduced M3R expression in the HFD group. Accumulation of reactive oxygen species can activate inflammatory signaling pathways, including NF-κB, leading to increased production of pro-inflammatory cytokines and cellular damage.³⁴ Previous studies have reported increased expression of p-NF-κB, TNF-α, IL-1β, IL-6, IL-10, IL-22, and IFN-γ in the submandibular glands of HFD-fed animals, indicating activation of inflammatory pathways.^19,35 Pro-inflammatory cytokines may interfere with parasympathetic neurotransmission by reducing acetylcholine release from nerve endings, thereby decreasing M3R stimulation.³⁶ In the long term, this reduced stimulation may contribute to decreased M3R expression, as observed in the present study. HFD has also been reported to reduce AQP5 expression, which may further impair water transport and salivary secretion.¹⁹

In addition to reduced M3R expression, this study demonstrated a significant reduction in β1-AR mRNA expression in the submandibular glands of HFD-fed rats. This finding suggests that HFD affects not only parasympathetic regulation but also sympathetic regulation involved in salivary protein secretion. β1-AR is expressed in salivary gland acinar cells and mediates the response to sympathetic stimulation through norepinephrine release. Norepinephrine binds to β-adrenergic receptors and activates the cyclic adenosine monophosphate (cAMP) pathway, which increases protein kinase A activity, amylase secretion, and transient salivary flow. This pathway is closely related to salivary protein secretion, whereas M3R-mediated parasympathetic stimulation primarily regulates fluid secretion through intracellular Ca²⁺ signaling.^14,27,37 Therefore, decreased β1-AR expression may reduce acinar cell sensitivity to sympathetic stimulation and impair salivary protein secretion.

The decrease in β1-AR mRNA expression observed in this study is consistent with previous findings showing that HFD can affect sympathetic regulation in salivary glands. De Carvalho et al reported that three months of HFD exposure reduced adiponectin and tyrosine hydroxylase (TH) levels, suggesting impaired sympathetic innervation and reduced adrenergic-mediated protein secretion.³⁸ Yamamoto et al also showed that HFD reduced TH levels in the submandibular glands of rats.³⁹ Because TH is the rate-limiting enzyme in catecholamine synthesis and an important marker of sympathetic nerve activity, reduced TH may decrease catecholamine synthesis and release, thereby weakening adrenergic receptor stimulation. Chronic oxidative stress, nitrosative stress, and inflammation induced by HFD may further contribute to impaired sympathetic activity and β1-AR downregulation.⁴⁰ Thus, reduced β1-AR mRNA expression may reflect impaired sympathetic regulation, potentially leading to reduced salivary protein secretion and decreased protective salivary function.

The present study also showed that HFD exposure significantly reduced the mean number of secretory units per field of view in the submandibular glands. This finding indicates that HFD may affect not only molecular regulation of salivary secretion but also the histological structure of the submandibular gland. Salivary secretory units, encompassing both acini and ductal structures like GCTs are vital components that produce saliva and essential proteins involved in oral lubrication, mucosal protection, and maintenance of oral homeostasis.^27,41 Therefore, a reduction in secretory units may indicate structural impairment of the gland and may contribute to altered secretory function.

These histological findings are consistent with previous studies reporting degenerative changes in salivary glands after HFD exposure, including reduced acinar cell number, vacuolization, reduced serous granules, and acinar atrophy.⁴² The structural changes observed in this study may be related to chronic oxidative stress, nitrosative stress, and inflammation. Increased production of reactive oxygen and nitrogen species, along with activation of inflammatory pathways such as NF-κB, may damage secretory cells and reduce the ability of glandular tissue to maintain its normal structure. These processes may contribute to the reduced number of secretory units observed in the HFD group.

It is important to note that rodent submandibular glands contain abundant granular convoluted tubules (GCTs), which can appear as distinct eosinophilic structures on H&E staining. Previous studies have indicated that a HFD and obesity trigger excessive lipid accumulation (fatty degeneration) in salivary gland cells. This condition leads to ductal atrophy, a reduction in the size and number of granules within GCTs, and disruption of cellular architecture due to oxidative stress and apoptosis.^15,42 In our study, the prominent eosinophilic structures observed could potentially represent these GCTs or altered secretory units undergoing structural atrophy. However, because our current histological assessment utilized only H&E staining, we were unable to definitively distinguish between these possibilities. Nevertheless, it is highly plausible that chronic HFD exposure concurrently induces structural damage across different glandular compartments.

These combined structural deteriorations also support the qRT-PCR results, which showed reduced M3R and β1-AR mRNA expression in the HFD group. Reduced M3R expression suggests impaired parasympathetic regulation of fluid secretion, whereas reduced β1-AR expression suggests impaired sympathetic regulation of protein secretion. Therefore, the decreased secretory units count strengthens the interpretation that HFD exposure may impair submandibular gland function through both molecular and structural alterations.

Overall, the findings of this study suggest that long-term HFD exposure may disrupt autonomic regulation and histological integrity of the submandibular glands. These changes may represent early mechanisms underlying HFD-related salivary gland dysfunction and may contribute to increased susceptibility to oral health problems associated with reduced salivary secretion. Thus, chronic HFD exposure may indirectly increase the risk of dental caries by impairing salivary gland function and reducing the protective role of saliva.

This study has several limitations. First, salivary function was not directly evaluated; therefore, the association between reduced M3R and β1-AR mRNA expression, decreased secretory units count, and impaired salivary secretion should be interpreted cautiously. Future studies should include functional parameters such as salivary flow rate, salivary volume, salivary protein concentration, amylase activity, and salivary IgA levels to further clarify the effects of HFD exposure on salivary gland function. Second, our molecular investigation focused primarily on transcriptional levels without protein-level validation techniques (eg, immunohistochemistry or Western blotting), and lacked the measurement of specific oxidative stress and inflammatory biomarkers to comprehensively support the proposed mechanisms. Finally, the histological observation was performed by a single, unblinded observer (the principal investigator), which may introduce potential observational bias. Furthermore, this study relied solely on hematoxylin and eosin staining and did not utilize specific staining, such as Alcian blue. Consequently, it was challenging to definitively differentiate between mucous acini, potentially altered acini, and other eosinophilic structures like GCTs, which is a limitation of our histological assessment. Future studies should utilize blinded observations, involve multiple observers to assess intra- and inter-observer reliability, and implement a validated histomorphometric scoring system to provide a more objective quantitative evaluation.

Conclusion

This study demonstrated that HFD exposure reduced M3R and β1-AR mRNA expression in the submandibular glands of Wistar rats compared with the control group. In addition, HFD exposure was associated with histological alterations in the submandibular glands, as indicated by a decreased mean number of secretory units per field of view. These findings suggest that HFD exposure may affect salivary gland structure and autonomic regulation, providing a potential basis for salivary dysfunction. However, because salivary secretion was not directly evaluated, this functional implication should be interpreted cautiously. Future studies incorporating direct functional assessments, protein-level validation, and complementary oxidative stress and inflammatory biomarkers are necessary to fully substantiate these mechanistic conclusions.

Data Sharing Statement

The corresponding authors can provide the data supporting the findings of this study upon reasonable request.

Ethics Approval and Consent to Participate

All animal procedures were approved by the Research Ethics Committee of Universitas Padjadjaran (Approval number: 926/UN6.KEP/EC/2025). All experiments were conducted in accordance with ARRIVE guidelines and the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Author Contributions

Conceptualization: DF, HG, FV; Methodology: DF, HG, FV; Investigation: DF; Formal Analysis: DF, PTR, KIS; Data Curation: DF, PTR, KIS; Writing – Original Draft: DF; Writing – Review & Editing: DF, HG, FV, PTR, KIS; Supervision: HG, FV. All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The author(s) declare (s) no conflict of interest.