Syncytialization and prolonged exposure to palmitate impacts BeWo respiration

Abstract

Placental villous trophoblast mitochondrial respiratory function is critical for a successful pregnancy and environmental influences such as maternal obesity have been associated with respiratory impairment at term. More recently, a gestational high fat diet independent of maternal body composition, has been highlighted as a potential independent regulator of placental mitochondrial metabolism. The current study aimed to characterize the direct impact of a prolonged and isolated exposure to the dietary fatty acids Palmitate (PA) and Oleate (OA) upon placental cell mitochondrial respiratory function. BeWo cytotrophoblast (CT) and syncytiotrophoblast (SCT) cells were treated for 72 hours with 100 µM PA, OA or PA+OA (P/O). Live-cell metabolic function was analyzed via the Seahorse XF Mito and Glycolysis Stress tests. Immunoblots and spectrophotometric activity assays were utilized to examine the protein expression and function of electron transport chain (ETC) complexes and key mitochondrial regulatory enzymes. Syncytialization of BeWo cells resulted reduced respiratory activity in conjunction with altered complex I and II activity and decreased pyruvate dehydrogenase (PDH) protein expression and activity. PA and P/O treatments were associated with increased basal and maximal respiratory activities in BeWo CT cells without alterations in protein expression or activity of individual ETC complexes and mitochondrial substrate regulators. The metabolic suppression in BeWo SCTs was consistent with that previously observed in primary human trophoblast cell cultures, while the observed increases in respiratory activity in PA-treated BeWo CTs may be indicative of an early timepoint of specific dietary saturated fat-mediated placental cell mitochondrial dysfunction.

The impact of hyperglycemia upon BeWo trophoblast cell metabolic function: A multi-OMICS and functional metabolic analysis

“…In brief, BeWo cells were plated at 1.4×10 5 cells/well in 6-well plates containing coverslips coated with laminin (2 µg/cm 2 ) and grown under HG conditions as described. Cellular expression of ZO-1 was then examined via immunofluorescent microscopy as previously detailed [37].…”

“…BeWo trophoblasts were plated at 9.5x10 5 cells/plate in 100 mm cell culture dishes and cultured under LG and HG conditions as described. At T72h cells were washed once in ice-cold PBS and subsequently lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors as previously described [37]. Protein concentrations were then adjusted to 2 µg/µL in Laemmli loading buffer (62.5 mM Tris-Cl (pH 6.8); 2% SDS 10% glycerol; 0.002% bromophenol blue; 4% β-mercaptoethanol).…”

“…Cells were then pelleted (400g, 5 mins), snap frozen in liquid nitrogen and stored at -80℃ until analyzed. Cell pellets were subsequently lysed and Complex I activity was assessed as the rate of rotenone-sensitive NADH oxidation, and Complex II activity was assessed as the rate of DCPIP oxidation as previously detailed [37]. ETC complex activity assays were normalized to cell lysate protein content via Bicinchoninic Acid (BCA) assay (Pierce, ThermoFisher Scientific) as per manufacturer's instructions.…”

“…BeWo cells were plated at 7.5x10 3 cells/well in Seahorse XF24 V7PS plates and cultured under LG and HG conditions as described above. At T72h mitochondrial activity was assessed using the Seahorse XF Mito Stress Test, as previously optimized for BeWo trophoblast cells [37].…”

“…LDH activity was assessed as the rate of NADH oxidation, and CS activity was assessed as the rate of Ellman's reagent consumption as previously detailed [37]. The enzyme activity of LDH and CS were normalized to cell lysate protein content via BCA assay.…”