In this study, DHEA-treated rats were used to simulate endocrine disorders and ovarian pathological changes in PCOS. The ovaries of PCOS rats showed polycystic changes. The CSA of skeletal muscle and the mean cell area of visceral fat and subcutaneous fat were significantly greater in PCOS rats than in control rats. We found that ENPP1 was expressed at higher levels in the ovaries, skeletal muscles, visceral fat, and subcutaneous fat of PCOS rats than in control rats. The serum levels of ENPP1, FSH, LH, T, FBG, FINS, MCP-1, FFAs, leptin, TC, TG, and LDL-C were significantly higher, and the levels of ADP and HDL-C were significantly lower in the PCOS group than in the control group. In addition, the concentration of ENPP1 was highly correlated with the levels of T, HOMA-IR, MCP-1, FFAs, ADP, leptin, TC, TG, and LDL-C.
Androgens are well known to increase muscle strength and mass, and muscle size is positively correlated with serum androgen levels in women with PCOS[24]. The DHEA rats in this study mimicked the increased androgens in PCOS patients, and it led to an increase in skeletal muscle fibre CSA, which represents muscle hypertrophy. In addition, excess androgens can also lead to visceral obesity, i.e., hypertrophy of visceral and subcutaneous fat cells, as verified in this study, as well as dyslipidaemia. It is believed that androgen excess leads to PCOS exhibiting IR and visceral obesity and further promotes androgen secretion from the ovaries and adrenal glands, thus fostering a vicious cycle of PCOS[25].
The abnormal expression of indicators in the serum of PCOS rats reflected the possible existence of IR and lipid metabolism disorders in PCOS. The success of the modelling was verified by a significant increase in T in the serum of PCOS rats. Serum MCP-1 was significantly higher in PCOS rats than in controls, further confirming the pathogenesis of PCOS from the viewpoint of the presence of a chronic low-grade inflammatory basis[22]. FFAs have a close relationship with IR because FFAs are involved in the production of lipid metabolites, proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1), and cellular stress, such as oxidative and endoplasmic reticulum stress[26]. The above processes may cause the development of IR. In addition, ADP and leptin were reported to be associated with IR, independent of obesity[27]. Moreover, it was reported that PCOS patients had considerably higher TG/HDL-C, TC/HDL-C, and LDL-C/HDL-C ratios than age-matched healthy women and a strong positive connection with HOMA-IR[28]. In this study, Spearman’s rank correlation analysis showed a strong correlation between ENPP1 and almost all of the detected indicators, which suggested that ENPP1 was highly correlated with IR and lipid metabolism in PCOS.
ENPP1 plays a role in the female reproductive system. A case-control study reported that the ENPP1 level in echo-guided aspirated fluids of endometriomas was considerably greater than that in the control group[29]. ENPP1 was also shown to be closely linked to ovarian cancer patient prognosis, and ENPP1 levels rose in ovarian cancer patients, suggesting that ENPP1 may promote migration[30, 31]. Some scientists have proposed a possible mechanism of action of ENPP1 in IR. A study suggested that when the missense variant K121 of the ENPP1 gene occurred, in which lysine (K) was replaced by glutamine (Q), it predisposed patients to IR[32]. ENPP1 carrying the Q121 variant had increased physical interaction with the insulin receptor at the cell membrane and became a stronger inhibitor of insulin signalling than ENPP1 carrying K121. This further affected insulin-stimulated receptor autophosphorylation and subsequent stimulation of IRS1 phosphorylation, PI3-kinase activation, and glycogen synthesis. Although there are only a few reports about ENPP1 in PCOS, it has been shown that the K121Q variant in the ENPP1 gene is significantly linked to PCOS risk[33].
ENPP1 is significantly upregulated in cancerous tissues, may be an initiator of malignant transformation of normal epithelial cells, and increases cell proliferation, migration, and invasion[7, 34]. Compared with the control group, we found that ENPP1 levels were significantly higher in both the ovaries and serum of PCOS rats. In addition, IF staining of ovaries showed that ENPP1 was mainly expressed in the cytoplasm and cell membrane. ENPP1 was more highly expressed in the granulosa cells of PCOS rats, suggesting that elevated ENPP1 was associated with higher proliferation rates and lower apoptosis rates in ovarian granulosa cells of PCOS rats.
C-Jun amino-terminal kinase (JNK)-IRS1-BAX is a signalling pathway reflecting IR, glucose homeostasis and apoptosis[35]. Since JNK requires testing for its phosphorylation and cannot be detected by qRT-PCR, we only tested two downstream indicators, namely, IRS1 and BAX. IRS1 is a major protein involved in insulin signalling that activates several signalling pathways involved in the regulation of glucose uptake, protein synthesis and gene expression[35]. BAX is a key proapoptotic protein of the BCL-2 family and a major regulator of the mitochondrial pathway[36]. In the ovaries and skeletal muscle, the mRNA levels of ENPP1, BAX, and IRS1 were higher in the PCOS group than in the control group, and the level of ENPP1 mRNA was significantly correlated with BAX mRNA, IRS1 mRNA, and the HOMA-IR index. In subcutaneous fat and visceral fat, the level of ENPP1 mRNA was higher in the PCOS rats than in the control group, and it was strongly positively correlated with the HOMA-IR index. This finding suggested that ENPP1 was highly associated with apoptosis of ovarian granulosa cells and the insulin pathway in the ovary. Meanwhile, it may also be involved in the process of IR in skeletal muscle, subcutaneous fat, and visceral fat, thus changing the general state of PCOS rats.
The strength of this study is that we systematically explored the expression of ENPP1 in different tissues of PCOS rats and its correlation with IR and lipid metabolism-related molecules, which has not been thoroughly investigated. However, one of the limitations of our study is that we have only preliminarily discussed the correlation between ENPP1, IR and lipid metabolism-related molecules. The mechanism of ENPP1 in PCOS has not yet been determined. Second, the rats modelled by DHEA cannot fully simulate all the physiological characteristics of PCOS patients. Therefore, our next research plan is to collect the blood and follicular fluid of PCOS patients, analyse the expression of ENPP1 in human specimens and regulate the expression of ENPP1 in a human granulosa-like tumour cell line to further explore the role of ENPP1 in pathology and pathological processes.