Altered Glycolysis, Mitochondrial Biogenesis, Autophagy and Apoptosis in Peritoneal Endometriosis in Adolescents

Abstract

Energy metabolism plays a pivotal role in the pathogenesis of endometriosis. For the initial stages of the disease in adolescents, this aspect remains unexplored. The objective of this paper was to analyze the association of cellular and endosomal profiles of markers of glycolysis, mitochondrial biogenesis, apoptosis, autophagy and estrogen signaling in peritoneal endometriosis (PE) in adolescents. We included 60 girls aged 13-17 years in a case-control study: 45 with laparoscopically confirmed PE (main group) and 15 with paramesonephric cysts (comparison group). Samples of plasma and peritoneal fluid exosomes, endometrioid foci and non-affected peritoneum were tested for estrogen receptor (Erα/β), hexokinase (Hex2), pyruvate dehydrogenase kinase (PDK1), glucose transporter (Glut1), monocarboxylate transporters (MCT1 and MCT2), optic atrophy 1 (OPA1, mitochondrial fusion protein), dynamin-related protein 1 (DRP1, mitochondrial fission protein), Bax, Bcl2, Beclin1, Bnip3, P38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor 1 (Hif-1α), mitochondrial voltage-dependent anion channel (VDAC) and transforming growth factor (TGFβ) proteins as markers of estrogen signaling, glycolysis rates, mitochondrial biogenesis and damage, apoptosis and autophagy (Western-Blot and PCR). The analysis identified higher levels of molecules associated with proliferation (ERβ), glycolysis (MCT2, PDK1, Glut1, Hex2, TGFβ and Hif-1α), mitochondrial biogenesis (OPA1, DRP1) and autophagy (P38, Beclin1 and Bnip3) and decreased levels of apoptosis markers (Bcl2/Bax) in endometrioid foci compared to non-affected peritoneum and that in the comparison group (p < 0.05). Patients with PE had altered profiles of ERβ in plasma and peritoneal fluid exosomes and higher levels of Glut1, MCT2 and Bnip3 in plasma exosomes (p < 0.05). The results of the differential expression profiles indicate microenvironment modification, mitochondrial biogenesis, estrogen reception activation and glycolytic switch along with apoptosis suppression in peritoneal endometrioid foci already in adolescents.

Keywords: Bcl-2; Hif-1α; TGFβ; adolescents; apoptosis; autophagy; estrogen receptor β; exosomes; glycolysis; mitochondrial biogenesis; peritoneal endometriosis; proliferation.

Figure 1

Comparative analysis of estrogen receptor levels in exosomes and in tissues of patients in the studied groups. Levels of exosomal ERβ in peripheral blood (A) and peritoneal fluid (B) in patients with endometriosis were significantly higher than those in the comparison group. (C) ERβ and ERα protein levels (left and right panels, respectively) in the peritoneal tissues of patients with endometriosis were higher in endometrioid foci vs. intact peritoneum. Data of relative protein level quantification are presented as boxes with median, interquartile range and min–max values. Representative Western blots are presented on panels below graphs. See also corresponding Ponceau-stained images in the Figure S1A–C of Supplemental Materials, original Western Blot in Figure S7.

Figure 2

Estimation of energy metabolism markers level in peritoneal tissues. (A) Expression of Glut1 in peritoneal tissue biopsies according to RT-PCR data (see Section 4 for details). (B) Western blot analysis for glycolysis markers Glut1, Hex2, Hif1α, MCT1, MCT2, PDK1 and also VDAC1 and TGFβ. Data of relative protein level quantification and gene expression are presented as boxes with median, interquartile range and min–max values. Representative corresponding Western blots are presented below graphs on panels. See also Ponceau-stained images in Figure S2 of Supplemental Materials, original Western Blot in Figure S7.

Figure 3

Comparative analysis of glucose and lactate transporter proteins in blood exosomes of patients of studied groups. Data of relative protein level quantification are presented as boxes with median, interquartile range and min–max values. Representative Western blots are presented below corresponding graphs on panels. See also Ponceau-stained images in Figure S3 of Supplemental Materials, original Western Blot in Figure S7.

Figure 4

Mitochondrial fission and fusion markers DRP1 and OPA1 levels are changed in patients with EMS. Reliable decrease in DRP1 transcripts (A) and Drp1 protein (B) levels in the foci biopsies of patients with EMS and increase in blood exosomes (C). (D) Relative expression of OPA1 protein increased for EMS patients compared to the intact peritoneum biopsies of patients of the comparison group. Relative data of protein level quantification and gene expression are presented as boxes with median, interquartile range and min–max values. Representative Western blots are presented below graphs. See also corresponding Ponceau-stained images in Figure S4A,B of Supplemental Materials, original Western Blot in Figure S7.

Figure 5

Expression of apoptotic markers in biopsies of peritoneal tissues. (A) Increased expression of the anti-apoptotic Bcl2 and decreased expression of pro-apoptotic Bax in the foci vs. intact peritoneum of both groups. Bcl-2/Bax transcripts ratio is reliably high in EMS foci vs. intact peritoneum of the comparison group. (B) Levels of Bcl2 and Bax proteins in the peritoneal tissues. Data of relative protein level and gene expression are presented as boxes with median, interquartile range and min–max values. Representative Western blots are presented below graphs. See corresponding Ponceau-stained images in Figure S5 of Supplemental Materials, original Western Blot in Figure S7.

Figure 6

Levels of autophagy markers in peritoneal tissue biopsies and blood exosomes of patients with EMS. (A) Decreased level of p38 protein and increased level of Beclin1 protein in EMS foci. (B) Decreased exosomal level of BNIP3 in the blood of patients with EMS vs. comparison group. Data of relative proteins level quantification are presented as boxes with median, interquartile range and min–max values. Representative Western blots are presented below graphs. See corresponding Ponceau-stained images in Figure S6A,B of Supplemental Materials, original Western Blot in Figure S7.

Figure 7

Activation of aerobic glycolysis and mitochondrial biogenesis under the conditions of increased estrogen reception in the pathogenesis of peritoneal endometriosis in adolescents. Expression levels for the key effectors of glycolysis, from the uptake of glucose by membrane transporters of the GLUT family (notably GLUT1) and its phosphorylation to glucose-6-phosphate by hexokinase (Hex2), are increased. The enhanced phosphorylation of pyruvate dehydrogenase (PDH) by its kinase (PDK1) limits pyruvate conversion to acetyl-CoA, thereby disconnecting glycolysis from tricarboxylic acid cycle (TCA) in mitochondria and reinforcing pyruvate conversion to lactate by lactate dehydrogenase A (LDHA). Lactate transport out of the cell through its transporters MCT is also increased. At the same time, a decrease in the activity of oxidative phosphorylation (TCA) in mitochondria is associated with less electron leakage and reactive oxygen species (ROS) formation and, accordingly, with the control of oxidative stress. Mitochondrial biogenesis is also increased, along with stabilization of mitochondrial cristae and enhanced apoptosis resistance (fusion marker OPA1 and fission marker DRP1). Contacts with EPR (glucose-regulated protein (GRP75) facilitate mitochondria-associated ER membrane (MAM) formation) and Ca²⁺ influx (VDAC) reinforce activation of cholesterol synthesis and steroidogenesis pathways. The implementation of ER-β signals leads to changes (p38 MAPK kinase cascade) in the expression of nuclear (p53) and mitochondrial genes, particularly those responsible for protection against apoptosis (the ratio of Bcl2 to Bax increases) and autophagy activation (Beclin, Bnip). HIF-1α and TGFβ signaling has a positive feedback loop with glycolytic switch and associated cell reprogramming.