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Article

Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice

1
Department of Geriatric Dentistry, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata 573-1121, Japan
2
Department of Oral Anatomy, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata 573-1121, Japan
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2024, 14(24), 12004; https://doi.org/10.3390/app142412004
Submission received: 18 November 2024 / Revised: 13 December 2024 / Accepted: 19 December 2024 / Published: 21 December 2024
(This article belongs to the Special Issue Recent Advancements in Biomarkers for Noncommunicable Diseases)

Abstract

:
Salivary biomarkers are promising molecules for diagnosing systemic diseases. Cytidine/uridine monophosphate kinase 2 (CMPK2) is associated with various systemic diseases. However, little is known about the role of the CMPK2 gene in saliva and dyslipidemia. This study investigated the relationship between serum lipid levels and Cmpk2 mRNA expression in the saliva of dyslipidemic mice. Additionally, immunofluorescence staining was employed to assess the localization of the CMPK2 protein in the submandibular gland. Two types of dyslipidemic mice were utilized: mice fed a high-fat and high-cholesterol (HFHC) diet and genetically dyslipidemic ApoE-deficient mice. The mice at 9 to 46 weeks were analyzed for serum lipid levels, Cmpk2 mRNA expression in saliva, and CMPK2 protein localization in the submandibular glands. Both dyslipidemic mice displayed elevated low-density lipoprotein cholesterol and total cholesterol in serum. ApoE-deficient mice apparently exhibited increased Cmpk2 expression in saliva. Immunofluorescence staining indicated that CMPK2 proteins were primarily localized in the serous acini, potentially associated with the secretion of Cmpk2 mRNA in saliva. These findings suggest that Cmpk2 mRNA increases and is detectable in the saliva of dyslipidemic mice, providing a viable experimental model to assess the potential use of CMPK2 as a biomarker for dyslipidemia.

1. Introduction

With global dietary shifts, obesity has become a widespread problem [1,2]. Obesity-induced dyslipidemia is collectively characterized by low-density lipoprotein cholesterolemia, hypertriglyceridemia, and high-density lipoprotein cholesterolemia [3]. Elevated total cholesterol (T-CHO) is also commonly associated with these dyslipidemias. Dyslipidemia increases the risk of age-related chronic inflammatory diseases, such as cardiovascular disease [4,5], diabetes [6], and stroke [7], necessitating early detection. Consequently, there is a need to develop biomarkers for early detection. To date, biomarkers have been developed using blood [8,9], urine [10,11], cerebrospinal fluid [12], and saliva [13]. Among these, saliva testing offers the advantages of being easily collected and noninvasive.
Saliva is mainly secreted by the major and minor salivary glands, containing exfoliated epithelium and gingival crevicular fluid with antibacterial and buffering properties [14]. Saliva also includes biomarkers valuable for disease identification and screening, facilitating its development for systemic and dental diseases [15]. In dental applications, saliva biomarkers are used to screen for oral cancer [16,17], dental caries [18,19], periodontal disease [20], and Sjögren’s syndrome [21,22]. Saliva has been used diagnostically for systemic diseases in Europe and the U.S., such as heart failure [23], cardiovascular disease [24], diabetes [25], Parkinson’s disease [26], and Alzheimer’s disease [27]. During the COVID-19 pandemic, saliva-based PCR testing became widely recognized [28]. Saliva consists of 99.5% water and 0.5% inorganic and organic components [29]. The proteins, lipids, mRNAs, miRNAs, and hormones that make up this 0.5% component are already being used and investigated as potential biomarkers [30]. On the other hand, mRNA levels are studied in diseases such as vascular aging [31], oral cancer [16], and asthma [32], presenting potential as a lipid abnormality biomarker.
Cytidine/uridine monophosphate kinase 2 (CMPK2), a mitochondrial DNA (mtDNA) synthase, is a molecule whose function is rapidly being elucidated [33]. mtDNA synthesis activates the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome [34]. CMPK2 activates mtDNA synthesis in atherogenic macrophages from mice with abnormal lipid metabolism, thereby exacerbating inflammation [35]. Moreover, elevated CMPK2 expression has been demonstrated to activate mitochondrial synthesis in the upstream pathway of inflammatory cytokine release [36]. CMPK2 mRNA has been implicated in diseases such as atherosclerosis [37], rheumatoid arthritis [38], systemic lupus erythematosus [39], ischemic stroke [40], sepsis therapy [41], osteoarthritis [42], and primary Sjögren’s syndrome [43]. The mechanism of CMPK2 expression activation via Toll-like receptors (TLRs) has recently been elucidated [44]. Typically, TLRs are known to accept lipopolysaccharide [45], damage-associated molecular patterns (DAMPS) [40], and lipids [46], among others. Dyslipidemia, which increases blood lipid levels, may increase lipid binding to TLRs, further upregulating CMPK2 expression in cells. The increased inflammation and mitochondrial dysfunction in the salivary glands of mice fed a high-fat diet [47] suggests that dyslipidemia may also affect the salivary glands. Given that our previous study identified CMPK2 mRNA in human saliva, we hypothesized that CMPK2 in salivary glands and saliva may be altered by dyslipidemia. However, the direct relationship between dyslipidemia and elevated CMPK2 mRNA expression in saliva has not yet been fully characterized.
This study aimed to examine the association between serum lipid levels and Cmpk2 mRNA in saliva, as well as CMPK2 protein localization in salivary glands, utilizing two types of dyslipidemic mice (high-fat and high-cholesterol [HFHC] diet mice and ApoE-deficient mice) for the prospective development of an early detection method for dyslipidemic states.

2. Materials and Methods

2.1. Animals

Eight-week-old male C57BL/6 and B6.KOR/StmSlc-Apoeshl (ApoE-deficient) mice were obtained from Shimizu Laboratory Supplies Company (Kyoto, Japan). Upon arrival, all mice were immediately transferred to microisolators, housed in horizontal laminar flow cabinets, and given ad libitum access to food and water. The mice were maintained on a 12 h light/dark cycle and divided into five groups: (C group) C57BL/6 mice at 9 weeks old (9 w) with unrestricted access to a normal diet, (A group) ApoE-deficient mice at 9 w with ad libitum access to a normal diet, (C− group) normal diet group, C57BL/6 mice with unrestricted access to a normal diet (MF, Oriental Yeast, Tokyo, Japan) from 9 w to 46 w, (C+ group) HFHC diet group, C57BL/6 mice with ad libitum access to an HFHC diet (D12108C, Research Diets, Inc., New Brunswick, NJ, USA) from 9 w to 46 w, and (A− group) ApoE-deficient mice with ad libitum access to a normal diet from 9 w to 46 w (Figure 1 and Figure 2, Table S1, n = 3). The dietary interventions were maintained for a duration of up to 37 weeks from 9 to 46 weeks of age (Figure 1). This study adhered to the animal testing guidelines established by the Osaka Dental University Institutional Animal Care and Use Committee. (Approval No. 24-01007, 21 February 2024; No. 23-01029, 6 February 2023; No. 22-07001, 31 August 2022).

2.2. Assessment of Lipid Metabolism and Obesity-Related Parameters

The experiment was started at about 10:00 a.m. under controlled conditions with a room temperature of about 23 °C. Mice at 9 w and 46 w were weighed and photographed to document body morphology. Blood samples were obtained from the tail vein under intraperitoneal anesthesia using 1.5 mL/kg of anesthetic solution comprising dexmedetomidine hydrochloride (0.075 mg/mL, Nippon Zenyaku Kogyo Co., Ltd., Koriyama, Japan), midazolam (0.4 mg/mL, SandBox, Inc., Toronto, Canada), and butorphanol tartrate (0.5 mg/mL, Meiji Seika Pharma Co., Ltd., Tokyo, Japan).
Serum T-CHO, low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) were quantified by the Oriental Yeast Co., Ltd. using the L-Type Wako CHO・M (Cat. No. 466-11894, 462-11994, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for T-CHO, the ColesteTest LDL (Cat. No. 4105-3261701, 4105-3258442, Sekisui Medical Co., Ltd., Tokyo, Japan) for LDL-C, and the L-Type Wako TG・M (Cat. No. 467-08994, 467-09094, FUJIFILM Wako Pure Chemical Corporation) for TG. Following sample collection, the mice were euthanized, and adipose tissue deposits in the submandibular and retroperitoneal regions were evaluated.

2.3. Saliva Sample Collection

Saliva sample collection began at approximately 10:00 a.m. under controlled conditions with a room temperature of approximately 23 °C. Mice were administered a unilateral intraperitoneal injection of anesthetics at a dose of 3 mL/kg. Subsequently, pilocarpine hydrochloride (Cat. No. 28008-31, Nacalai Tesque, Inc., Kyoto, Japan) was administered intraperitoneally at a dose of 0.857 mg/kg. One milliliter of saliva was collected from the anterior portion of the mouth using a pipette. Although an increased injection volume of pilocarpine has been known to enhance salivary secretion, some mice have exhibited distressing symptoms, including trembling and restlessness, following the administration of volumes exceeding 1 mg/kg. Consequently, in this experiment, the saliva of the same group of mice was pooled into a tube that had been treated to inhibit RNase activity (n = 3). The saliva was rapidly frozen to prevent degradation with liquid nitrogen immediately following each aspiration and maintained on dry ice until further analysis (Figure S1). The saliva samples were promptly analyzed.

2.4. Saliva RNA Isolation and qRT-PCR

To evaluate the efficacy of total RNA isolation methods, we compared three commercially available reagents for RNA extraction: TRIzol reagent (Cat. No. 15596018, Thermo Fisher Scientific, Waltham, MA, USA), RNeasy Protect Saliva Mini Kit (Cat. No. 74004, Qiagen, Hilden, Germany), and NucleoSpin RNA (Cat. No. 740955.10, Takara Bio, Shiga, Japan) (Table S2). TRIzol reagent method (Figure S1B): The saliva samples were thawed at 4 °C and centrifuged at 12,000× g for 20 min at 4 °C. The supernatant was carefully removed, and 1 mL of TRIzol reagent (Thermo Fisher Scientific) was added to the precipitate. The mixture was subsequently thoroughly dissociated using a pipette, followed by vortexing to ensure complete homogenization. After incubation at room temperature (RT, 23 °C) for 5 min, 200 µL of cold chloroform was added and incubated for 3 min at RT (23 °C). The samples were then centrifuged at 12,000× g for 20 min at 4 °C. Approximately 500 µL of the upper aqueous phase was transferred to a new RNase-free tube, and 500 µL of cold isopropanol was added. Following incubation at −20 °C for 2 h, the samples were centrifuged at 12,000× g for 20 min at 4 °C. The supernatant was removed, and cold 75% ethanol was added. The samples were centrifuged at 10,000× g for 5 min at 4 °C, and the procedure was repeated. Finally, the RNA pellet was air-dried for 5 min and then resuspended in 20 µL of RNase-free water. The sample was incubated in a 55 °C water bath for 5 min and stored at −80 °C until further analysis. RNeasy Protect Saliva Mini Kit (Qiagen) and NucleoSpin RNA (Takara Bio) were utilized according to the manufacturer’s standard protocol (Figure S1C). NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) was employed to assess the quality of RNA.
Reverse transcription was conducted using the SuperScript IV VILO Master Mix with ezDNase Enzyme Kit (Cat. No. 11766050, Thermo Fisher Scientific) in accordance with the manufacturer’s standard protocol. In this investigation, Actb (Cat. No. 4331182, Thermo Fisher Scientific, TaqMan Gene Expression Assay, Mm02619580_g1) was selected as the endogenous control gene, while Cmpk2 (Cat. No. 4351372, Thermo Fisher Scientific, TaqMan Gene Expression Assay, Mm01287125_m1) was designated as the target transcript. TaqMan Fast Advanced Master Mix (Cat. No. 4444557, Thermo Fisher Scientific) was employed to quantitatively assess the expression levels of salivary transcripts (Table S2). The relative expression of target genes was determined using the 2−ΔΔCt method.

2.5. Histological Staining

Following euthanasia, the mice were perfused and fixed with 4% paraformaldehyde, after which the submandibular glands were isolated microscopically. The glands were treated overnight using a 12.5–25% sucrose gradient for prevention of ice crystal formation, embedded in SCEM (Cat. No. KKK-CEM001, SECTION-LAB Co., Ltd., Hiroshima, Japan), and initially stored at −80 °C before being transferred to −20 °C for sectioning. The samples were sectioned at a thickness of 6 µm and 10 µm. For hematoxylin and eosin (H-E) staining, the 6 µm sections were stained according to the manufacturer’s instructions, with the sections subsequently sealed with glycerol. Imaging was performed using the BZ-X800 all-in-one microscope (KEYENCE, Osaka, Japan), which is equipped with an integrated camera. For immunofluorescence staining, the 10 µm sections were blocked with 5% goat serum and 0.3% Triton-X in phosphate buffer saline (PBS), followed by overnight incubation at 4 °C with the primary antibodies. Subsequently, the sections were rinsed with PBS and incubated with secondary antibodies for 1 h at 37 °C, after which they were sealed with DAPI-Fluoromount-G (Cat. No. 0100-20, Southern Biotech, Birmingham, AL, USA). The primary antibodies employed were pan-cytokeratin (pan-CK, 1:100, mouse anti-wide range, Cat. No. C2562, Sigma-Aldrich, St. Louis, MO, USA) and CMPK2 (1:100, rabbit anti-human/mouse/rat, Cat. No. 25877-1-AP, Cosmo Bio Co., Ltd., Tokyo, Japan), while the secondary antibodies were Alexa Fluor 488 (1:100, goat anti-mouse, Cat. No. ab150117, Abcam, Cambridge, UK) and Alexa Fluor 555 (1:100, goat anti-rabbit, Cat. No. ab150078, Abcam). The ImageJ 1.53t software (National Institutes of Health, Bethesda, MD, USA) was utilized to quantify the intensity of immunofluorescence staining.

2.6. Statistical Analysis and English Editing

All data were analyzed using Student’s t-test and one-way ANOVA followed by Tukey’s or Dunnett’s post-hoc test to perform pairwise comparisons between the groups in GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The quantitative results are presented as means ± standard deviation (SD), with statistical significance defined at p < 0.05. This manuscript was edited for English language using Paperpal (Cactus Communications Pvt, Ltd., Mumbai, Maharashtra, India).

3. Results

3.1. Body Weight Changes and Visceral Fat Accumulation in Mice

HFHC diets are characterized by a high caloric density. Excessive consumption of these foods leads to an abnormal lipid metabolism, resulting in the storage of excess calories as fat tissue. An age-related increase in body size was observed in all mice (Figure 3A, n = 3). Mice that were fed the HFHC diet (C+ group) exhibited pronounced indications of obesity (Figure 3A). In comparison to the controls that were fed a normal diet (C− group), ApoE-deficient mice that were fed a normal diet (A− group) displayed a slightly leaner body composition (Figure 3A). Regarding body weight, the C+ group (49.50 ± 1.00; p < 0.0001 vs. C−, one-way ANOVA with Tukey’s post-hoc test) exhibited the highest weight gain, followed by the C− group (40.25 ± 1.71), while A− group (33.75 ± 1.26; p < 0.001 vs. C−) demonstrated the lowest weight gain (Figure 3B, n = 3). Examination of visceral fat revealed that adipose tissue primarily accumulated around the kidneys. The C+ group exhibited significant fat deposition with extensive coverage, the C− group displayed moderate adipose volume and uniform distribution, and the A− group showed relatively less fat volume around the kidneys (Figure 3C, n = 3). Additionally, a similar pattern of accumulation was observed around the submandibular gland (Figure 3C).

3.2. Serum Lipid Levels

Typical reference ranges for mouse serum T-CHO and TG are approximately 51–171 mg/dL and 29–150 mg/dL, respectively [48]. Because LDL-C levels in mice are influenced by both physiological and experimental factors, there is no relatively stable reference range. Serum from A group exhibited higher LDL-C (72.67 ± 8.14; p < 0.001, Student’s t-test) and T-CHO levels (507.00 ± 57.65; p < 0.001) than that of C group (6.00 ± 2.00; 78.67 ± 5.03) at 9 w, while there were negligible differences in TG levels (Figure 4A, n = 3). At 46 w, LDL-C levels were higher in the C+ (57.67 ± 4.93; p < 0.01 vs. C−, one-way ANOVA with Tukey’s post-hoc test, and A− groups (66.33 ± 16.26; p < 0.001 vs. C−) compared to the controls (C−, 6.67 ± 1.15). T-CHO levels exceeded the reference range in both C+ (383.00 ± 32.19; p < 0.01 vs. C−, one-way ANOVA with Tukey’s post-hoc test) and A− groups (651.33 ± 82.77; p < 0.0001 vs. C−, p < 0.01 vs. C+), while TG levels remained within the normal range in all mice (Figure 4B, n = 3). T-CHO in ApoE-deficient mice showed the most pronounced higher levels at both 9 w and 46 w (Figure 4).

3.3. Comparison of the Quality of RNA Extracted from Saliva Using Different Reagents

Based on the NanoDrop data, RNA extracted with TRIzol Reagent demonstrated a peak at absorbance at 260 nm (A260), whereas RNA samples extracted using the RNeasy Protect Saliva Mini Kit and the NucleoSpin RNA did not exhibit a distinct peak at A260 (Figure S2). From these results, the TRIzol reagent was selected as the preferred method for extracting saliva from mice and was utilized in subsequent experiments.

3.4. Cmpk2 Gene Expression in the Saliva of Dyslipidemic Mice

Cmpk2 mRNA levels in the saliva of 9 w A group (1.97 ± 0.08; p < 0.001, Student’s t-test) exhibited higher expression compared to those in the C group (1.00 ± 0.10) (Figure 5A, n = 3). Following 37 weeks of administration of a HFHC diet, although no statistically significant difference was observed (possibly due to the small sample size), the expression of Cmpk2 mRNA in the saliva of C+ group (1.68 ± 0.29; 0.05 < p ≤ 0.10 vs. C−, one-way ANOVA with Dunnett’s post-hoc test) demonstrated elevated levels relative to that of C− group (1.04 ± 0.35) (Figure 5B, n = 3). Among the three groups, the expression in the A− group (2.05 ± 0.18; p < 0.01 vs. C−) showed the highest levels (Figure 5B).

3.5. CMPK2 Immunofluorescence Staining in the Submandibular Gland of Dyslipidemic Mice

The macroscopic view and histological characteristics of the submandibular gland were observed and documented (Figure 6A). The presence of serous acini and mucous acini could be observed in the H-E staining of the submandibular gland (Figure 6B, n = 4). Immunofluorescence staining of the submandibular gland revealed that the intensity of CMPK2 protein in the A group (31.92 ± 0.82; p < 0.0001, Student’s t-test) at 9 w was higher than that of the C group (27.87 ± 0.15) (Figure 7A,B, n = 4). In 46 w mice, the CMPK2 staining intensity demonstrated a gradual increase in the order of C− (28.57 ± 0.61), C+ (31.60 ± 1.19; p < 0.05 vs. C−, one-way ANOVA with Tukey‘s post-hoc test), and A− group (35.11 ± 1.85; p < 0.05 vs. C+, p < 0.001 vs. C−) (Figure 7A,B, n = 4). Moreover, the expression of CMPK2 protein was predominantly localized in the serous acini. CMPK2 exhibited a more pronounced aggregation restriction in the serous demilune (Figure 7C).

4. Discussion

To date, limited information exists regarding the expression of Cmpk2 mRNA in saliva. In this investigation, we demonstrate that Cmpk2 mRNA secretion into saliva is elevated in the presence of lipid abnormalities. Immunofluorescence analysis of salivary glands confirmed the localization of CMPK2 protein in serous cells rather than mucous cells within the submandibular gland.
The relationship between increased body weight and abnormal lipid levels has been demonstrated [49,50]; however, it is not observed universally [51,52]. In this study, two dyslipidemic animal models were employed, an HFHC diet group and a genetic model (ApoE-deficient mice), to elucidate the effects of varying body weights and fat accumulation while exhibiting similar states. In the HFHC diet group, there was an increase in body weight and accumulation of fat (Figure 3). In ApoE-deficient mice, an increase in body weight with increasing weekly age was noted, yet without significant lipid accumulation (Figure 3). Conversely, serum LDL-C and T-CHO significantly increased in both groups at 46 w (Figure 4). These findings are consistent with previous studies [53,54], indicating that the two animal models can be reliably established in this experiment.
mRNA has been extensively investigated in human saliva [32]. Particularly after the onset of the COVID-19 pandemic, the relationship between saliva studies and the mRNA transcripts of COVID-19 has increased significantly [55]. The salivary glands are located in close proximity to blood vessels, allowing the exchange of metabolites between the oral cavity and the circulatory system [56]. As a result, certain proteins in saliva are similar to those found in blood. Research has shown that a large number of biomarkers found in saliva correlate with their levels in the blood [57]. In addition, studies have shown that salivary biomarkers have excellent accuracy, sensitivity, and specificity in distinguishing the early stages of oral squamous cell carcinoma [58]. This suggests that saliva is a potential alternative to blood-based tests. Although blood tests are essential for diagnosing dyslipidemia, the non-invasive nature of saliva offers significant advantages. It is our contention that early prevention of dyslipidemia could be achieved if screening could be conducted by saliva testing during physical examinations. However, salivary mRNA studies in rodents remain extremely limited. In this experiment, we initially utilized two commercially available total RNA collection methods. However, no distinct waveform was observed at A260 using the NanoDrop (Figure S2). Conversely, the waveform was confirmed by the TRIzol-based method, leading to the implementation of RNA extraction using the TRIzol method in the subsequent experiment. Even using the TRIzol methods, the existence of phenol seems to be identified at the peak at A270 [59]. The persistent peak indicated that additional purification techniques would need to be necessary to purify RNA in mouse saliva.
In both 9 w and 46 w ApoE-deficient groups, serum LDL-C and T-CHO levels were elevated compared with the controls (C and C−), accompanied by increased Cmpk2 expression. At 46 weeks, the HFHC diet group (C+) also showed elevated LDL-C and T-CHO levels compared to the controls (C−), with a trend toward increased Cmpk2 expression, although the difference was not statistically significant. On the other hand, there were no differences in TG levels among groups, which was not consistent with the observed patterns of Cmpk2 expression. There is evidence that lipoproteins can activate TLRs, and it has been demonstrated that the TLR signaling pathway plays a pivotal role in regulating the expression of CMPK2 [60,61]. A line of these results suggests that at least the increase of LDL-C levels in serum may have contributed to Cmpk2 expression via TLR activation.
As body weight increased, the HFHC diet group exhibited an accumulation of fat tissue around the kidneys, while the ApoE-deficient mice demonstrated lower body weight and fat accumulation compared to the control group (C−) (Figure 3). Conversely, both the HFHC diet and ApoE-deficient mice displayed higher salivary Cmpk2 expression than the control mice on a normal diet (C−). Based on these findings, we posit that increased body weight due to fat accumulation does not necessarily influence the expression of the Cmpk2 mRNA in saliva (Figure 3 and Figure 5). Adipocytes secrete various adipokines, known to exert positive and negative effects on surrounding tissues through their release into the bloodstream [62]. These adipokines, such as TNF-α [63], are cytotoxic and latently induce apoptosis, potentially leading to the formation of DAMPS and activation of CMPK2 through TLRs [40]. Given that the Cmpk2 mRNA expression was elevated even in ApoE-deficient mice with low-fat accumulation, lipid abnormalities had a more pronounced direct effect than the indirect enhancement of the Cmpk2 expression via adipokines resulting from weight fluctuations.
To estimate the cells secreting the Cmpk2 gene in saliva, we performed Immunofluorescence staining of CMPK2 in the submandibular gland (Figure 7). Intense staining was observed in the serous acini of the HFHC diet group and ApoE-deficient group with lipid abnormalities. The mechanism underlying the strong localization of CMPK2 protein to serous cells remains unknown. However, serous saliva is secreted when the parasympathetic nervous system is dominant, while viscous saliva is secreted when the sympathetic nervous system is dominant [64]. In the present study, mice were stimulated with pilocarpine, which approximated a parasympathetic-dominant state, thereby possibly increasing the secretion of serous saliva and the detection of Cmpk2 in the saliva. Moreover, mitochondria are generally known to be involved in a variety of cellular functions, including cell differentiation, calcium homeostasis, and apoptosis [65,66]. In salivary gland cells, serous acini are particularly active in protein secretion; mitochondrial activity is closely related to protein secretion. Considering the high secretion in serous acini and the possibility that CMPK2 is involved in the same pathway, it is suggested that CMPK2 localizes specifically to serous acini. In addition, environmental temperature and diurnal variation also affect salivary secretion [67,68]. Given these results, caution is warranted when diagnosing using the salivary CMPK2 gene, as the conditions under which saliva is collected may significantly influence the results.
In this study, we found that the Cmpk2 gene in saliva shows a similar tendency to lipid abnormalities. However, there are several limitations to this study. First, although dyslipidemia has been reported to be related to age, this study used young mice up to 46 w. The effect of aging on Cmpk2 expression under dyslipidemic conditions remains unknown. Additionally, the differences between humans and mice need to be scrutinized. Compared to clinical studies, research on saliva in animal experiments is extremely limited. To date, few studies have investigated the relationship between lipid metabolism disorders and salivary secretory factors, and this study represents the first report on the expression of CMPK2. Consequently, quantitative discussions based on the obtained data are challenging at this stage, highlighting the need for further data accumulation and comprehensive analysis in future studies. The largest of the major salivary glands in humans is the parotid gland, while the largest in mice is the submandibular gland [69]. There are also some differences in the protein composition and digestive enzyme content of human and mouse saliva. Human saliva contains a more diverse range of proteins and has stronger immune defense functions, while mouse saliva has relatively fewer digestive enzymes and weaker digestive activity [70,71]. Differences between CMPK2 in salivary secretion in humans and rodents need to be further carefully investigated. Furthermore, the secretory mechanisms of Cmpk2 mRNA in serous cells of the submandibular gland or other salivary glands are still unclear. However, our results suggest that the detection of CMPK2 mRNA in saliva may be used to postulate lipid abnormalities.

5. Conclusions

In the present study, dyslipidemic mice (both HFHC mice and ApoE-deficient mice) exhibited elevated levels of serum LDL-C and T-CHO. Consistent with these trends, Cmpk2 mRNA expression in the saliva of dyslipidemic mice was higher compared to that of the control mice with a normal diet (C and C−). Furthermore, we observed that the CMPK2 protein was locally expressed within serous acini, at least in the submandibular gland. Although the precise mechanisms associated with dyslipidemia and salivary Cmpk2 mRNA expression remain unclear, these results may provide valuable insight into the relationship between Cmpk2 and dyslipidemia and could potentially facilitate the development of early diagnostic tools and therapeutic strategies for dyslipidemia and related lifestyle diseases.

Supplementary Materials

The following supporting information can be downloaded at: ttps://www.mdpi.com/article/10.3390/app142412004/s1, Figure S1: Salivary sample collection and RNA extraction; Figure S2: Assessment of extracted RNA from the saliva samples using NanoDrop spectrophotometry; Table S1: Composition of feed ingredients; Table S2: Reagents used for RNA extraction, reverse transcription, and qRT-PCR.

Author Contributions

Conceptualization, Y.H. and A.K.; formal analysis, B.Z. and M.N.; funding acquisition, A.K.; investigation, B.Z.; methodology, B.Z. and M.N.; project administration, A.K. and Y.H.; supervision, A.K., Y.H. and K.T.; validation, Y.H., A.K. and M.N.; writing—original draft, B.Z., Y.H. and A.K.; writing—review and editing, M.N. and K.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Scientific Research (C) (24K13060).

Institutional Review Board Statement

The animal study protocol was approved by the Osaka Dental University Institutional Animal Care and Use Committee. (Approval No. 24-01007, 21 February 2024; No. 23-01029, 6 February 2023; No. 22-07001, 31 August 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We thank Shanshan Zheng, Zi Deng, Yutian Wang from Osaka Dental University for sharing insights and experiences. A schematic view was created in BioRender. tyou, b. (2025) https://BioRender.com/j46x112 (accessed on 18 November 2024); https://BioRender.com/a36x486 (accessed on 18 November 2024); https://BioRender.com/c68t451 (accessed on 18 November 2024); https://BioRender.com/n51o643 (accessed on 18 November 2024).

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic illustration of mice husbandry process: Diet and rearing duration. C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a high-fat and high-cholesterol (HFHC) diet, A−: ApoE-deficient mice fed a normal diet. (n = 3).
Figure 1. Schematic illustration of mice husbandry process: Diet and rearing duration. C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a high-fat and high-cholesterol (HFHC) diet, A−: ApoE-deficient mice fed a normal diet. (n = 3).
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Figure 2. Representative images of diets. (A) Normal diet. (B) HFHC diet.
Figure 2. Representative images of diets. (A) Normal diet. (B) HFHC diet.
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Figure 3. Body size and fat accumulation. (A) Body size of 9 w and 46 w mice. Body size of 9 w C mice (white dotted line). (B) Body weight changes. *** p < 0.001, **** p < 0.0001 (comparison to the C−): one-way ANOVA with Tukey’s post-hoc test. (C) Visceral fat of 9 w and 46 w mice (submandibular space and retroperitoneal space). Adipose tissue (red arrow). C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet (n = 3).
Figure 3. Body size and fat accumulation. (A) Body size of 9 w and 46 w mice. Body size of 9 w C mice (white dotted line). (B) Body weight changes. *** p < 0.001, **** p < 0.0001 (comparison to the C−): one-way ANOVA with Tukey’s post-hoc test. (C) Visceral fat of 9 w and 46 w mice (submandibular space and retroperitoneal space). Adipose tissue (red arrow). C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet (n = 3).
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Figure 4. Quantitative biochemical analysis of serum lipid levels. (A) Low-density lipoprotein cholesterol (LDL-C), total cholesterol (T-CHO), and triglyceride (TG) levels in 9 w mice. (B) LDL-C, T-CHO, and TG in 46 w mice. Reference ranges: T-CHO (51–171 mg/dL), TG (29–150 mg/dL). Data are mean ± SD (n = 3). ns: no significant difference, ** p < 0.01, *** p < 0.001, **** p < 0.0001: Student’s t-test for 9 w mice, one-way ANOVA with Tukey’s post-hoc test for 46 w mice. C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
Figure 4. Quantitative biochemical analysis of serum lipid levels. (A) Low-density lipoprotein cholesterol (LDL-C), total cholesterol (T-CHO), and triglyceride (TG) levels in 9 w mice. (B) LDL-C, T-CHO, and TG in 46 w mice. Reference ranges: T-CHO (51–171 mg/dL), TG (29–150 mg/dL). Data are mean ± SD (n = 3). ns: no significant difference, ** p < 0.01, *** p < 0.001, **** p < 0.0001: Student’s t-test for 9 w mice, one-way ANOVA with Tukey’s post-hoc test for 46 w mice. C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
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Figure 5. Cytidine/uridine monophosphate kinase 2 (Cmpk2) mRNA expression in saliva. (A) The expression of Cmpk2 mRNA in the saliva of 9 w mice was quantified using qRT-PCR and normalized to Actb. (B) The expression of Cmpk2 mRNA in the saliva of 46 w mice was detected by qRT-PCR, and then the relative expression of Cmpk2 in the saliva of the C+ group and A− group was compared with C−. Data are mean ± SD (n = 3). ns: no significant difference, ** p < 0.01, *** p < 0.001: Student’s t-test for 9 w mice, one-way ANOVA with Dunnett’s post-hoc test for 46 w mice. C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
Figure 5. Cytidine/uridine monophosphate kinase 2 (Cmpk2) mRNA expression in saliva. (A) The expression of Cmpk2 mRNA in the saliva of 9 w mice was quantified using qRT-PCR and normalized to Actb. (B) The expression of Cmpk2 mRNA in the saliva of 46 w mice was detected by qRT-PCR, and then the relative expression of Cmpk2 in the saliva of the C+ group and A− group was compared with C−. Data are mean ± SD (n = 3). ns: no significant difference, ** p < 0.01, *** p < 0.001: Student’s t-test for 9 w mice, one-way ANOVA with Dunnett’s post-hoc test for 46 w mice. C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
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Figure 6. Macroscopic view of the submandibular gland and its histology. (A) Schematic view of the submandibular gland (position and morphology). (B) Hematoxylin-Eosin (H-E) staining of the submandibular gland (n = 4). D: duct (black arrow), SA: serous acini (white arrowhead), MA: mucous acini (black arrowhead). C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
Figure 6. Macroscopic view of the submandibular gland and its histology. (A) Schematic view of the submandibular gland (position and morphology). (B) Hematoxylin-Eosin (H-E) staining of the submandibular gland (n = 4). D: duct (black arrow), SA: serous acini (white arrowhead), MA: mucous acini (black arrowhead). C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
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Figure 7. Immunofluorescence staining of the submandibular gland and its quantitative data. (A) The submandibular gland was stained with pan-cytokeratin (pan-CK), DAPI (DAPI-Fluoromount-G), and CMPK2. Pan-CK binds to salivary gland epithelial cells, while DAPI binds to cell nuclei. (B) The quantification of CMPK2 fluorescence intensity was quantified using ImageJ software (National Institutes of Health). Data are mean ± SD (n = 4). * p < 0.05, *** p < 0.001, **** p < 0.0001: Student’s t-test for 9 w mice, one-way ANOVA with Tukey’s post-hoc test for 46 w mice. (C) Focus on serous demilune in H-E and immunofluorescence staining images of the submandibular gland (merge image of pan-CK, CMPK2, and DAPI); CMPK2 is localized in the serous acini. SA: serous acini, MA: mucous acini. SeD: serous demilune (white dotted line and arrow). C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
Figure 7. Immunofluorescence staining of the submandibular gland and its quantitative data. (A) The submandibular gland was stained with pan-cytokeratin (pan-CK), DAPI (DAPI-Fluoromount-G), and CMPK2. Pan-CK binds to salivary gland epithelial cells, while DAPI binds to cell nuclei. (B) The quantification of CMPK2 fluorescence intensity was quantified using ImageJ software (National Institutes of Health). Data are mean ± SD (n = 4). * p < 0.05, *** p < 0.001, **** p < 0.0001: Student’s t-test for 9 w mice, one-way ANOVA with Tukey’s post-hoc test for 46 w mice. (C) Focus on serous demilune in H-E and immunofluorescence staining images of the submandibular gland (merge image of pan-CK, CMPK2, and DAPI); CMPK2 is localized in the serous acini. SA: serous acini, MA: mucous acini. SeD: serous demilune (white dotted line and arrow). C: C57BL/6 mice, A: ApoE-deficient mice, C−: C57BL/6 mice fed a normal diet, C+: C57BL/6 mice fed a HFHC diet, A−: ApoE-deficient mice fed a normal diet.
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Zhang, B.; Kawamoto, A.; Nakagawa, M.; Honda, Y.; Takahashi, K. Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice. Appl. Sci. 2024, 14, 12004. https://doi.org/10.3390/app142412004

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Zhang B, Kawamoto A, Nakagawa M, Honda Y, Takahashi K. Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice. Applied Sciences. 2024; 14(24):12004. https://doi.org/10.3390/app142412004

Chicago/Turabian Style

Zhang, Baiyan, Akiyo Kawamoto, Masato Nakagawa, Yoshitomo Honda, and Kazuya Takahashi. 2024. "Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice" Applied Sciences 14, no. 24: 12004. https://doi.org/10.3390/app142412004

APA Style

Zhang, B., Kawamoto, A., Nakagawa, M., Honda, Y., & Takahashi, K. (2024). Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice. Applied Sciences, 14(24), 12004. https://doi.org/10.3390/app142412004

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