Hyperinsulinemia restrains endometrial angiogenesis during decidualization in early pregnancy

in Journal of Endocrinology
Correspondence should be addressed to R Gao: gao_ru_fei@cqmu.edu.cn

*(W Chen and S Lu contributed equally to this work)

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Previous research on the role of insulin has focused on metabolism. This study investigated the effect of insulin on angiogenesis in endometrial decidualization. High insulin-treated mouse model was constructed by subcutaneous injection of insulin. Venous blood glucose, serum insulin, P4, E2, FSH and LH levels in the pregnant mice were detected by ELISA. Decidual markers, angiogenesis factors and decidual vascular network were detected during decidualization in the pregnant mouse model and an artificially induced decidualization mouse model. Tube formation ability and angiogenesis factors expression were also detected in high insulin-treated HUVECS cells. To confirm whether autophagy participates in hyperinsulinemia-impaired decidual angiogenesis, autophagy was detected in vivo and in vitro. During decidualization, in the condition of high insulin, serum insulin and blood glucose were significantly higher, while ovarian steroid hormones were also disordered (P < 0.05), decidual markers BMP2 and PRL were significantly lower (P < 0.05). Uterine CD34 staining showed that the size of the vascular sinus was significantly smaller than that in control. Endometrial VEGFA was significantly decreased after treatment with high insulin in vivo and in vitro (P < 0.05), whereas ANG-1 and TIE2 expression was significantly increased (P < 0.05). In addition, aberrant expression of autophagy markers revealed that autophagy participates in endometrial angiogenesis during decidualization (P < 0.05). After treatment with the autophagy inhibitor 3-MA in HUVEC, the originally damaged cell tube formation ability and VEGFA expression were repaired. This study suggests that endometrial angiogenesis during decidualization was impaired by hyperinsulinemia in early pregnant mice.

Downloadable materials

  • Supplementary Figure 1. The expression of angiogenesis-related factors in the endometrium was disturbed by high levels of insulin. The expression of ANG-1 (A), TIE2 (B), VEGFA (C) and VEGFR2 (D) in the endometrium of pregnant mice on D6, D7 and D8 was detected by real-time PCR. Real-time PCR results showed that the expression of ANG-1, TIE2, VEGFA and VEGFR2 was disordered in the endometrium of mice with artificially induced decidualization (induced side) (E). n=3. * p<0.05. CON: control group; IN: high insulin group.
  • Supplementary Table 1 Primary Antibody List

 

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    Endometrial decidualization was impaired by high maternal insulin levels. The daily intervention dose in each group is showed (A). The expression of BMP2 and PRL in the endometrium of pregnant mice on D6, D7 and D8 was analyzed by real-time PCR (B) and Western blot (C). The expression of BMP2 in the endometrium of pregnant mice on D8 was analyzed by immunohistochemistry (D). The expression of BMP2 and PRL in the endometrium of mice with artificially induced decidualization induced by oil injection was analyzed by real-time PCR (E) and Western blot (F). n = 3. *P < 0.05. AI, artificially induced decidualization model by oil injection; CON, control group; IN, high insulin group.

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    Angiogenesis in endometrium decidualization was restrained by high maternal insulin levels in vivo. The endometrial expression of CD34 in pregnant mice on D7 and D8 (A) was analyzed by immunohistochemistry. The histogram showed the size of vascular sinus area on day 7 of pregnancy. The expression of ANG-1, TIE2, and VEGFA in the endometrium of pregnant mice on D6, D7 and D8 was analyzed by Western blot (B). Immunohistochemistry analysis of the expression of ANG-1 (C) and VEGFA (D) in the endometrium of pregnant mice on D7 and D8. The endometrial expression of CD34 in decidual endometrium artificially induced by oil injection in vivo (E) was analyzed by immunohistochemistry. The histogram showed the size of vascular sinus area in decidual endometrium. The expression of ANG-1, TIE2 and VEGFA in artificially induced decidual endometrium was analyzed by Western blot (F). n = 3. AI, artificially induced decidualization model by oil-injected; CON, control group; IN, high insulin group.

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    Angiogenesis was compromised by high insulin levels in vitro. Tube formation of HUVECS was compromised under the role of high insulin levels (A). The expression of ANG-1, TIE2 and VEGFA in HUVECS was detected by Western blot (B). The histogram shows the quantification of the Western blot results. (C). Immunofluorescence staining results showed the different expression of VEGFA in HUVECS between the high insulin group and the control group (D). n = 3. *P < 0.05. CON, control group; IN, high insulin group.

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    Autophagy was triggered by high insulin levels during decidualization. The expression of autophagy-related factors LC3B, P62, Atg5 and Beclin1 in the endometrium of pregnant mice on D6, D7 and D8 was analyzed by Western blot (A). Immunofluorescence staining results showed increased expression of LC3B in HUVECS in the high insulin group (B). n = 3. CON, control group; IN, high insulin group.

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    Enhanced autophagy was responsible for the inhibition of angiogenesis resulting from high insulin levels. The expression of LC3B, P62, Atg5 and Beclin1 in HUVECS was analyzed by Western blot (A). Immunofluorescence staining with LC3B in HUVECS was compared among the CON, IN and IN + 3-MA groups (B). Tube formation of HUVECS was compared among the CON, IN and IN + 3-MA groups (C). Immunofluorescence staining with VEGFA in HUVECS was compared among the CON, IN and IN + 3-MA groups (D). n = 3. CON, control group; IN, high insulin group; IN + 3-MA, high insulin + 3-MA group.

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