Changes in the expression of insulin signaling pathway molecules in endometria from polycystic ovary syndrome women with or without hyperinsulinemia

Abstract

Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder associated with insulin resistance and compensatory hyperinsulinemia. Scarce information is available on the expression of molecules involved in the insulin pathway in endometria from women with PCOS. Therefore, we examined the protein levels of insulin-signaling molecules, like insulin receptor, insulin-receptor substrate (IRS)-1, pIRS-1Y612, Akt, AS160, pAS160T642 and GLUT4 in endometria from PCOS women with or without hyperinsulinemia. Protein levels were assessed by Western blot and immunohistochemistry in 21 proliferative-phase endometria from control women (CE = 7), normoinssulinemic PCOS women (PCOSE-NI = 7) and hyperinsulinemic PCOS women (PCOSE-HI = 7). The data show no differences in the expression of insulin receptor between all groups as assessed by Western blot; however, IRS-1 and pIRS-1Y612 were lower in PCOSE-HI than controls and PCOSE-NI (P < 0.05). AS160 was detected in all analyzed tissues with similar expression levels between groups. Importantly, PCOSE-HI exhibited lower levels of pAS160T642 (P < 0.05) and of GLUT4 (P < 0.05) compared with CE. The immunohistochemistry for insulin receptor, IRS-1, Akt, AS160 and GLUT4 showed epithelial and stromal localization; IRS-1 staining was lower in PCOSE-HI (P < 0.05). In conclusion, human endometrium has the machinery for glucose uptake mediated by insulin. The diminished expression of GLUT4, as well as the lower level of pIRS-1Y612 and pAS160T642 exhibited by PCOSE-HI, suggests a disruption in the translocation of vesicles with GLUT4 to the cell surface in these patients.

Figure 1

Immunohistochemistry for proteins of the insulin-signaling pathway. Representative image from immunohistochemical detection of insulin receptor, IRS-1, Akt, AS160 and GLUT4 in paraffin wax sections of proliferative endometria obtained from CE women (n = 7) (left panel), women with PCOS without (PCOSE-NI, n = 7) (middle panel) and with hyperinsulinemia (PCOSE-HI, n = 7) (right panel). The hematoxylin/eosin staining showed comparable architecture in the three analyzed groups (A, B, C). Positive staining was detected in epithelial and stromal cells of all studied endometria for all antigens. A similar positive staining for insulin receptor was observed in all groups (D, E, F). (G, H, I) Immunostaining for IRS-1, which is less intense in the PCOSE-HI group (I). For Akt-1 (J, K, L), the immunostaining was more intense in the epithelia of PCOSE-HI compared with CE. The three analyzed groups showed positive staining for AS160 in a comparable manner (M, N, O). GLUT4 (P, Q, R) was immunodetected in all groups with less staining intensity in PCOSE-NI and PCOSE-HI compared with CE. Magnification 400× in all panels. Inserts show negative controls for all immunohistochemical assays. Scale bars represent 50 μm.

Figure 2

Ratio between pIRS-1Y612 and IRS-1 in proliferative endometria from CE and PCOSE-HI women, assessed by Western blotting. Equal amounts of protein were loaded in each lane, and pIRS-1Y612 and IRS-1 band intensities were quantified by scanning densitometry and normalized to intensities observed for β-actin as internal control. A representative image of the media of bands obtained from 7 CE and 7 PCOSE-HI endometrial is shown. The results are expressed as AU and the values shown are mean ± SEM in CE and PCOSE-HI. a = P < 0.05 in PCOSE-HI compared with CE.

Figure 3

Ratio between pAS160T642 and AS160 in proliferative endometria from CE and PCOSE-HI women, assessed by Western blotting. Equal amounts of protein were loaded in each lane, and pAS160T642 and AS160 band intensities were quantified by scanning densitometry and normalized to intensities observed for β-actin as internal control. A representative image of the media of bands obtained from 7 CE and 7 PCOSE-HI is shown. The results are expressed as AU and the values shown are mean ± SEM in CE and PCOSE-HI. a = P < 0.05 in PCOSE-HI compared with CE.

Figure 4

Western blot analysis of protein levels of GLUT4 in proliferative endometria from CE and PCOSE-HI women. Equal amounts of protein were loaded in each lane. GLUT4 was detected as a band with a molecular mass of 46 kDa. Band intensities were quantified by scanning densitometry and normalized to intensities observed for β-actin as internal control. A representative image of the media of bands obtained from 7 CE and 7 PCOSE-HI is shown. The results are expressed as AU and the values shown are means ± SEM in CE and PCOSE-HI. a = P < 0.05 in PCOSE-HI compared with CE.