Molecular characterization of insulin resistance and glycolytic metabolism in the rat uterus

Abstract

Peripheral insulin resistance and hyperandrogenism are the primary features of polycystic ovary syndrome (PCOS). However, how insulin resistance and hyperandrogenism affect uterine function and contribute to the pathogenesis of PCOS are open questions. We treated rats with insulin alone or in combination with human chorionic gonadotropin (hCG) and showed that peripheral insulin resistance and hyperandrogenism alter uterine morphology, cell phenotype, and cell function, especially in glandular epithelial cells. These defects are associated with an aberration in the PI3K/Akt signaling pathway that is used as an indicator for the onset of insulin resistance in classical metabolic tissues. Concomitantly, increased GSK3β (Ser-9) phosphorylation and decreased ERK1/2 phosphorylation in rats treated with insulin and hCG were also observed. We also profiled the expression of glucose transporter (Glut) isoform genes in the uterus under conditions of insulin resistance and/or hyperandrogenism. Finally, we determined the expression pattern of glycolytic enzymes and intermediates during insulin resistance and hyperandrogenism in the uterus. These findings suggest that the PI3K/Akt and MAPK/ERK signaling pathways play a role in the onset of uterine insulin resistance, and they also suggest that changes in specific Glut isoform expression and alterations to glycolytic metabolism contribute to the endometrial dysfunction observed in PCOS patients.

Uterine progesterone signaling is a target for metformin therapy in PCOS-like rats

“…Experiment 1: Rats were randomly divided into control (saline treatment, n = 20) and experimental (PCOS-like, n = 20) groups. The experimental group was treated with insulin plus hCG to induce a PCOS-like metabolic and reproductive phenotype, and the control rats were treated with an equal volume of saline 29 . In brief, insulin was started at 0.5 IU/day and gradually increased to 6.0 IU/day between day 1 and the day 22 to induce hyperinsulinemia and insulin resistance, and 3.0 IU/day hCG was given on all 22 days to induce hyperandrogenism.…”

“…Rats with repeated insulin injections have not shown any hypoglycemic episodes [35][36][37] . Detailed analysis of endocrine and metabolic parameters as well as the uterine morphology in these animals has been reported previously 29 . On day 23, each group of rats was divided into two subgroups of 10 rats each (Supplemental Fig.…”

“…Uterine tissues were fixed in 10% formalin, embedded in paraffin, and sectioned for hematoxylin and eosin staining according to standard procedures. Immunohistochemistry and immunofluorescence were performed according to previously described methods 28,29 . The endogenous peroxidase and nonspecific binding were removed by incubation with 3% H 2 O 2 for 10 min and with 10% normal goat serum for 1 h at room temperature.…”

“…A detailed explanation of the Western blot analysis protocol has been published 28,29 . Total protein was isolated from whole uterine tissue by homogenization in RIPA buffer (Sigma-Aldrich) supplemented with cOmplete Mini protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche Diagnostics).…”

“…To address this hypothesis, we analyzed PCOS-associated PGR isoform expression and the MAPK signaling network in human and rat uterine tissues. By combining a PCOS-like rat model 29 and in vitro tissue culture approach 30 , we aimed to determine whether metformin directly reverses aberrant PGR-targeted and implantation-related gene expression in the PCOS-like rat uterus.…”