Differential Fatty Acid Uptake in Human Trophoblast Cells as Evidence for Mitochondria-mediated Outcomes in Gestational Diabetes Mellitus

Kyle Siemers, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States; Tyler Gandy, Sanford Research/University of South Dakota, United States; Tricia Larsen, Sanford Research, United States; Michelle L. Baack, University of South Dakota, Sanford School of Medicine, Sioux Falls, SD, United States

Background: Although gestational diabetes causes placental lipid accumulation and fetal macrosomia, it also causes essential long-chain polyunsaturated fatty acid (LCPUFA) deficiency in infants which is associated with poorer neurodevelopmental outcomes and increased lifetime risk for cardiometabolic disease. Selective physiologic LCPUFA biomagnification in the fetus suggests placental fatty acid transfer varies by carbon length and that LCPUFA transport is active and ATP-dependent.

Objectives: This study aimed to determine whether differential fatty acid uptake in human trophoblasts is mitochondria-mediated.

Design/Methods: Using MitoTracker fluorescent tagging of mitochondria and 4,4-difluoro-3a,4a-diaza-s-indacene, BODIPY, a fluorophore attached to fatty acids of variable lengths, we tracked live-cell uptake in BeWo and isolated cytotrophoblasts (CTB) from consenting mothers. Experiments were repeated in the presence of oligomycin (OM), which inhibits ATP-synthase, 2-deoxyglucose (2dG) which competitively inhibits glycolysis, etomoxir (Eto) which inhibits fatty acid oxidation, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) which uncouples respiration. Statistical analysis was conducted using Student’s T-test per drug or 2- and 1-way ANOVA to determine fatty acid-specific, drug-based, and interaction effects. Significance was set at p< 0.05.

Results: Live-cell imaging of BeWo and CTB shows rapid uptake of both BODIPY-C12, which mimics a 16-carbon saturated fatty acid like palmitate, and BODIPY-C16, which mimics a 20-carbon LCPUFA. The time to reach peak uptake was longer for BODIPY-C16 than BODIPY-C12 (p < 0.0001). Interestingly, both fatty acids colocalize with mitochondria, but BODIPY-C16 stimulates visible cytoplasmic projections that facilitate peripheral uptake, followed by perinuclear accumulation. Treatment with 1uM OM, 50 mM 2dG, or 150 uM Eto significantly decreased ATP levels in BeWo while 0.3 uM FCCP did not (p=0.017, 0.002, 0.012, 0.458 respectively; n=3). All ATP-inhibitors, slowed the rate of BODIPY-C16 uptake (p < 0.0001 with 2dG and Eto; n=4). Conversely, 2dG and Eto increased the rate of BODIPY-C12 uptake (p=0.007, p=0.012 respectively; n=2). Primary cytotrophoblasts had a similar trend (n=3).

Conclusion: Differential fatty acid uptake in human trophoblasts is ATP-dependent, thus diabetes-mediated mitochondrial dysfunction may explain both placenta lipotoxicity and LCPUFA deficiency associated with poorer outcomes.
NIH/NIGMS 2P20GM103620-06.