Increased placental mitochondrial fusion in gestational diabetes mellitus: an adaptive mechanism to optimize feto-placental metabolic homeostasis?

Abstract

Introduction: Gestational diabetes mellitus (GDM), a common pregnancy disorder, increases the risk of fetal overgrowth and later metabolic morbidity in the offspring. The placenta likely mediates these sequelae, but the exact mechanisms remain elusive. Mitochondrial dynamics refers to the joining and division of these organelles, in attempts to maintain cellular homeostasis in stress conditions or alterations in oxygen and fuel availability. These remodeling processes are critical to optimize mitochondrial function, and their disturbances characterize diabetes and obesity.

Methods and results: Herein we show that placental mitochondrial dynamics are tilted toward fusion in GDM, as evidenced by transmission electron microscopy and changes in the expression of key mechanochemical enzymes such as OPA1 and active phosphorylated DRP1. In vitro experiments using choriocarcinoma JEG-3 cells demonstrated that increased exposure to insulin, which typifies GDM, promotes mitochondrial fusion. As placental ceramide induces mitochondrial fission in pre-eclampsia, we also examined ceramide content in GDM and control placentae and observed a reduction in placental ceramide enrichment in GDM, likely due to an insulin-dependent increase in ceramide-degrading ASAH1 expression.

Conclusions: Placental mitochondrial fusion is enhanced in GDM, possibly as a compensatory response to maternal and fetal metabolic derangements. Alterations in placental insulin exposure and sphingolipid metabolism are among potential contributing factors. Overall, our results suggest that GDM has profound impacts on placental mitochondrial dynamics and metabolism, with plausible implications for the short-term and long-term health of the offspring.

Keywords: GDM; mitochondria; placenta.

Figure 1

TEM analysis of mitochondrial dynamics in placentae from women with gestational diabetes. (A) Representative TEM images of placental cytotrophoblast cells in a normal pregnancy and in pregnancies complicated by diet-treated (D-GDM) and insulin-treated (I-GDM) gestational diabetes. (B) Methods for quantifying mitochondrial circularity, aspect ratio and Feret’s diameter on TEM images. TEM, transmission electron microscopy.

Figure 2

Placental levels of mitochondrial fusion and fission proteins in normal pregnancies compared with gestational diabetes pregnancies. (A) Representative western blots and associated densitometry analysis of fusion markers, OPA1 and MFN1, in control (n=25 and 13, respectively) versus gestational diabetes mellitus (GDM) placental tissue (n=25 and 23, respectively). (B) Representative western blots and associated densitometry analysis of fission regulator, Drp1 and its activated form, pDRP1, in control (n=19 and 12, respectively) versus GDM (n=22 and 15, respectively) placental tissue. (C) Representative western blots and associated densitometry analysis of OPA1 and pDRP1 levels in mitochondria isolated from control (n=7) versus GDM (n=16) placentae. Data are presented as median±IQR. *P<0.05 **p<0.01. D-GDM, diet-treated GDM; I-GDM, insulin-treated GDM; MFN1, mitofusin 1; OPA1, optic atrophy 1.

Figure 3

Effects of insulin and/or glucose exposure on mitochondrial fission and fusion protein expression in choriocarcinoma JEG-3 cells. Representative western blots and associated densitometry (n=3 separate experiments in duplicate) for (A) OPA1 and (B) pDRP1 in JEG3 cells treated with EMEM (VEH), glucose (GLU), insulin (INS), and glucose and insulin (GLU+INS). (C) Representative TEM images of JEG-3 cells treated with VEH, GLU, INS and GLU+INS. (D) Representative western blots and associated densitometry for OPA1 in JEG-3 cells treated with DMSO (control), 15 µM genistein (GS15), INS, and GS15+INS. Data are presented as mean±SD. *P<0.05, **p<0.01. DMSO, dimethyl sulfoxide; EMEM, Eagle’s Minimal Essential Media; OPA1, optic atrophy potein 1.

Figure 4

Immunofluorescence analysis of OPA1 and pDRP1 in choriocarcinoma JEG-3 cells exposed to glucose and/or insulin. (A) JEG-3 cells treated with glucose (GLU), insulin (INS), glucose plus insulin (GLU+INS) or EMEM vehicle (VEH) were stained for OPA1 (green), Mitotracker (red), and DAPI (blue) and imaged by confocal microscopy (n=4 separate experiments in duplicate). (B) Quantification of immunofluorescence intensity for OPA1. (C) JEG-3 cells treated with GLU, INS, GLU+INS or EMEM vehicle were stained for pDRP1 (green), Mitotracker (red) and DAPI (blue) and imaged by confocal microscopy (n=2 separate experiments in duplicate). (D) Immunofluorescence colocalization analysis (Pearson correlation coefficient) for pDRP1 and Mitotracker. Data are expressed as mean±SD. *P<0.05, **p<0.001, ****p<0.0001. EMEM, Eagle’s Minimal Essential Media; OPA1, optic atrophy potein 1.

Figure 5

Analysis of placental ceramide metabolism in gestational diabetes and control pregnancies. (A) Placental ceramide species content (left panel; n=8 control, n=8 GDM) and placental change in mitochondrial ceramide content (right panel; n=6 control, n=12 GDM) in pregnancies complicated by GDM as compared with normal control pregnancies. (B) Representative western blots and associated densitometry for ASAH1 enzyme levels in control versus GDM placentae and in JEG-3 cells (n=3 separate experiments in triplicate) treated with EMEM (VEH), glucose (GLU), insulin (INS), or glucose and insulin (GLU+INS). Data are expressed as median ±IQR. *P<0.05, **p<0.01, ****p<0.0001. CER, ceramide; EMEM, Eagle’s Minimal Essential Media; GDM, gestational diabetes mellitus.