# Cmpk2 Gene and Protein Expression in Saliva or Salivary Glands of Dyslipidemic Mice
## 4 Discussion
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 (Figures 3 and 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-\(\alpha\)[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 Immunofluorescentcence 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 pilocarppine, which approximated a parasympatheticC\(-\)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.