Previous studies report that the synergetic effect between the overproduction of androgens and insulin resistance present in many PCOS women contributes to the alteration of the function in several tissues, including the endometrium [2
]. In turn, excess androgen induces changes in the expression of proteins related to tissue homeostasis, intracellular steroid bioavailability [18
] and uterine receptivity [17
] in PCOS endometria.
Also, it has been described that hyperinsulinemia in PCOS could possibly be due to defects in the expression and/or activity of proteins downstream from the insulin receptor [21
]. Thus, understanding protein expression in insulin signaling in PCOS-IR patients could help expand knowledge about reproductive failure observed in the majority of these cases [5
Several laboratories have established that the endometrium is an insulin responsive tissue, by indicating the presence of GLUT4 mRNA and its protein, in normal and PCOS tissue [5
]. Therefore, to better understand insulin activity in this tissue, in the present investigation proteins that participate in the pathway were evaluated, specifically PI3K/PDK-1 pathway molecules that are known to have a crucial role in glucose uptake in endometrial cells tissue [5
]. Our first goal was to determine the presence and cellular distribution of some key proteins involved in the translocation of GLUT4 to the plasma membrane, in both normal and PCOS endometria. The study of these molecules in the endometrium is of special importance because glucose serves as the main energy provider, and inadequate uptake into endometrial PCOS-IR cells has been often linked with failure to conceive. In this respect, one of the proteins assayed in the present investigation was PKCζ. Previous reports have shown that PKCζ participates in the remodelling of cortical actin [7
], and the phosphorylation of various proteins in the insulin cascade [11
]. It is important to note that phosphorylated PKCζ is lower in PCOS-IR endometria (Figure ), suggesting a potential decreased translocation of activated PKCζ to the plasma membrane and thus probably a decreased glucose uptake in PCOS-IR endometrium [27
]. It is interesting that the remodelling of cortical actin for GLUT4 vesicle translocation is also orchestrated by WAVE family proteins. In fact, a previous study has shown reduced protein levels of N-WASP and WASP in endometrial tissue of women with PCOS and hyperinsulinemia [24
On the other hand, the diminished Munc18c expression in PCOS-IR endometria, as assessed by western blot (Figure ), could indicate a possible failure in the interaction of this protein with phosphorylated PKCζ [9
]. It should be mentioned that the immunodetection of Munc18c was higher in the stromal compartment on PCOS-IR endometria, we have no clear explanation for this result, but we can speculate that this discrepancy could reside in the different techniques used. Interestingly, protein levels of Syntaxin-4 were not affected by the PCOS-IR condition. Together, these data could indicate that the lowered expression of Munc18c and phosphorylated PKCζ are not directly affecting Syntaxin-4 levels, suggesting there could be other proteins responsible for the failed translocation of GLUT4 to the plasma membrane.
The results discussed up to this point, do not allow us to conclude if the condition of hyperandrogenism and/or hyperinsulinemia present in the PCOS-IR condition can affect protein levels in endometrial tissues. Therefore, in order to better understand the action of high doses of androgens and/or insulin, we employed an endometrial stromal cell line which we stimulated in vitro with exogenous hormones. The action of testosterone and insulin were through their specific receptors present in the T-HESC cells. The androgen receptor is located in the cytoplasm and nucleus of HESCs cells, whereas, the insulin receptor is observed on the cell surface (unpublished data). Therefore, we believe that testosterone and insulin could be acting through their specific receptors present in the cells and thus T-HESC cultures should respond to hyperandrogenic and hyperinsulinic environments.
Our results showed that high concentrations of insulin significantly affect the protein expression of Munc18c. This transient hyperinsulinic condition allows us to infer that the insulin resistant condition present in PCOS patients could be altering the expression of this protein without affecting the protein expression of Syntaxin-4, a protein regulated by Munc18c. On the other hand, the treatment with high concentrations of testosterone to the T-HESC cells decreased the levels of both phospho-PKCζ (Thr410) and Munc18c, suggesting that high levels of the hormone can participate in insulin resistance in the endometrium, which could result in disturbed glucose uptake [1
When T-HESC cells were stimulated with both hormones, insulin and testosterone, we observed decreased protein levels of Munc18c and phospho-PKCζ. These results are in agreement with the results obtained in this investigation in PCOS-IR endometria for the same proteins. Therefore, hormone excesses characteristic of PCOS affect the expression of key proteins involved in insulin action at endometrial level. This observation suggests a lowered GLUT4 vesicle translocation to the cell periphery, eventually leading to a deficient entrance of glucose to the cell. Therefore, the defects in the insulin signaling pathway observed at the protein level, including Munc18c, PKCζ, phospho-PKCζ, and Syntaxin-4 in PCOS patients with insulin resistance, could lead to impaired glucose uptake. Accordingly, the involvement of insulin resistance and high androgen levels in the molecular defects of the insulin cascade cannot be discarded.