The Research Of ENPP1 In The Pathogenesis Of Polycystic Ovary Syndrome

Part 1 Comparison of ENPP1 expression in women with polycystic ovary syndrome and its correlation with glucolipid metabolismObjective: The influence of ectonucleotide pyrophosphatase phosphodiesterase 1(ENPP1)in serum and follicular fluid of healthy and polycystic ovarian syndrome(PCOS)women is the focus of this phase of the investigation.The goal was to see if the aberrant expression of this gene is linked to problems with blood glucose and lipid metabolism in patients.Methods: Forty-five female patients aged 20-35 years,of whom 20 were healthy controls and 25 were PCOS patients,attending Renmin Hospital of Wuhan University from December 2020 to December 2021 were included.According to the patient’s index of homeostasis model assessment-insulin resistance(HOMA-IR),the PCOS group was divided into 15 patients without insulin resistance(IR)(HOMA-IR<2.69)and 10 patients with IR(expressed as PCOS+IR,HOMA-IR≥2.69).Peripheral blood from day 2 to 3 of menstruation and follicular fluid on the day of egg collection were collected from the three groups.The ENPP1 levels in serum and follicular fluid,as well as serum levels of sex hormones,blood glucose and lipids were measured.The differences of ENPP1 among the three groups were assessed.Correlations between ENPP1 levels and the patients’ sex hormones,glycolipid metabolism indicators were investigated using Spearman correlation coefficient.Results: ENPP1 levels in serum and follicular fluid were significantly higher in patients with PCOS+IR than in PCOS non-IR group,and ENPP1 levels in serum and follicular fluid were significantly higher in patients with PCOS than in normal healthy women(P < 0.05).Serum ENPP1 was highly positively correlated with follicular fluid ENPP1 in PCOS patients(P < 0.05).In addition,ENPP1 in both serum and follicular fluid of patients had a significant positive correlation with AMH,LH,T,HOMA-IR,FFAs,leptin,and a significant negative correlation with HDL-C and ADP(P < 0.05).Conclusion: ENPP1 was abnormally highly expressed in serum and follicular fluid of PCOS patients,and the expression was higher in PCOS patients with combined IR.Elevated ENPP1 is associated with the disturbance of HOMA-IR and lipid molecule levels.It indicates that ENPP1 may have an important role in the pathogenesis and clinical manifestations of PCOS,and may influence the initiation of PCOS and disorders of glucolipid metabolism to some extent.Part 2 Comparison of ENPP1 expression in various tissues in a rat model of polycystic ovary syndrome and its correlation with glycolipid metabolismObjective: This research was aimed to investigate whether ENPP1 is aberrantly expressed in ovaries,skeletal muscle,subcutaneous adipose and visceral adipose tissues of PCOS rats,the localized expression of ENPP1 in ovarian tissues,and the correlation between serum ENPP1 and sex hormones and glucolipid metabolism in PCOS rats.To investigate the expression of ENPP1 in multiple tissues throughout the body in PCOS and its possible role in the pathogenesis of PCOS.Methods: A PCOS rat model was constructed by subcutaneous injection of DHEA(Dehydroepiandrosterone).PCOS rats were injected subcutaneously with DHEA dissolved in olive oil(6mg/100g)daily,while the other group of rats were injected with the same amount of olive oil daily.The modelling procedure lasted for 20 days.Vaginal smears were performed from day 11 onwards.20 days later,rats were executed and ovaries,skeletal muscle,subcutaneous fat,visceral fat and peripheral blood were collected from the rats.Pathological section plots,serum levels of ENPP1,sex hormones,glucolipid metabolism were evaluated for comparison,and the association between serum ENPP1 and glucolipid metabolism levels of rats was examined.Glucose tolerance,insulin tolerance,and estrous cycle alterations were compared.The abnormal expression of ENPP1 levels in ovaries,skeletal muscle,subcutaneous fat and visceral fat of PCOS rats were compared and the location of ENPP1 expression in ovarian tissue was explored.Results: PCOS rats showed a decrease in the number of granulosa cell layers in the ovary to 2-3 layers and exhibited increased interstitial cell hyperplasia,follicular cysts and atretic follicles,fewer follicles at different stages of development,and fewer corpus luteum.And the average cross-sectional area of skeletal muscle,the mean area of visceral fat cells and subcutaneous fat cells in PCOS rats were significantly larger than those in control rats(P<0.05).PCOS rats showed estrous cycle disorder,abnormal glucose tolerance,and insulin tolerance.Serum assays suggested that elevated ENPP1 levels in the serum of PCOS rats showed a positive correlation with FSH,LH,T,FINS,HOMA-IR,TC,TG,LDL-C,FFAs,and leptin(P<0.05)and a negative correlation with ADP levels(P<0.05).Analysis of ENPP1 expression in ovaries,skeletal muscle,subcutaneous and visceral fat in the two groups of rats suggested that ENPP1 expression was significantly higher in all tissues of rats in the PCOS group(P<0.05).Immunofluorescence experiments performed on ovarian tissues showed that ENPP1 was expressed in the cytoplasm and on the cell membrane,and it was mainly in granulosa cells and theca cells.Conclusion: DHEA-induced PCOS rats showed multiple histological changes throughout the body,and were combined with estrous cycle disorders,abnormal sex hormone levels,glucose metabolism,and lipid metabolism disorders,which were in line with the pathophysiological changes of clinical PCOS patients.The expression of ENPP1 was significantly up-regulated in ovaries,skeletal muscle,subcutaneous fat,and visceral fat of PCOS rats.The localization of ENPP1 in the ovary was mainly in ovarian granulosa cells and theca cells,suggesting that it may be involved in the functional regulation of granulosa cells and theca cells.These results may suggest that ENPP1 is not only linked to disorders of glucolipid metabolism in PCOS,but may also play a role in the pathological alterations of systemic tissues in the development of PCOS.Part 3 Regulatory role of ENPP1 in granulocyte-associated signaling pathwaysObjective: The purpose of this study was to examine that whether upregulation of ENPP1 expression KGN cells inhibits glucose transport function,promotes cellular insulin resistance,and increases apoptosis by modulating the PI3K/AKT signaling pathway.To further explore the possible mechanism of ENPP1 in KGN cells.Methods: The KGN cells were obtained,transfected with ENPP1 recombinant plasmid(OE group)and control empty vector plasmid(NC group)respectively,and two groups(NC+SC79 and OE+SC79 groups)were set up with the specific PI3K/AKT pathway activator SC79.Western blot method was used to compare the changes in the levels of PI3K/AKT signaling pathway-related proteins PI3 K,p-PI3 K,AKT,and p-AKT,and to detect the changes of apoptosis-related proteins Bax and Bcl2,as well as the changes of GLUT4 protein reflecting glucose transport function and IR.The apoptosis levels of the four groups were measured by flow cytometry.Results: The m RNA and protein levels of ENPP1 were significantly increased in the OE group compared to the NC group(P<0.05).Besides,the ratio of p-PI3K/PI3 K and p-AKT/AKT,GLUT4 protein and Bcl2 protein levels were decreased,the level of Bax protein and the level of apoptosis were increased in OE group compared to NC group,all with significant differences(P<0.05).NC+SC79 group showed increased pAKT/AKT ratio,GLUT4 protein,Bcl2 protein level,decreased Bax protein level and decreased apoptosis rate compared to NC group,with significant differences(P<0.05).Compared with the NC+SC79 group,the OE+SC79 group showed a decrease in pAKT/AKT ratio,GLUT4 protein,Bcl2 protein content,an increase in Bax protein and an increase in apoptosis rate,with significant differences(P<0.05).Conclusion: Activation of insulin-stimulated PI3K/AKT pathway can reduce KGN cell apoptosis and enhance glucose transport.Upregulation of ENPP1 expression can inhibit insulin-stimulated PI3K/AKT signaling pathway,leading to increased apoptosis of KGN cells,inhibiting the glucose transport function of granulosa cells,and promoting the process of IR.The overexpression of ENPP1 may be related to the pathogenesis of PCOS and may become a potential target for the clinical treatment of PCOS.