Abstract: PO0301
Chronic AMPK Activation Reprograms Glucose Metabolism and Oxygen Respiration in Renal Tubule Epithelial Cells
Session Information
- Bioengineering
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Hunter, Kuniko, Vanderbilt University, Nashville, Tennessee, United States
- Evans, Rachel C., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Love, Harold D., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Fissell, William Henry, Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background
In vitro human renal tubule epithelial cells (HREC) exhibit a glycolytic, dedifferentiated phenotype that limits their use in bioartificial kidney development. We have identified AMP-activated protein kinase (AMPK) and Transforming Growth Factor-β (TGFβ) as critical modulators of HREC differentiation. Here we show that inhibition of TGFβ signaling enhances increased respiration induced by activation of AMPK.
Methods
Primary HREC were seeded on polystyrene tissue culture plates (100,000 cells cm-2). After one week, cells were supplemented with AMPK activator Metformin (200µM), TGFβ receptor I inhibitor SB431542 (10µM), or both. After five weeks, cell oxygen consumption (OCR) and extracellular acidification rates (ECAR) were assessed using a Seahorse XFe96 Analyzer and respiratory inhibitors oligomycin (2µM), CCCP (2µM), Rotenone (0.5µM), Antimycin A (0.5µM) and 2-deoxyglucose (50mM). Statistical differences were estimated by paired, two-tailed Student’s t-test in MatLab.
Results
Metformin and Combination treatments increased cell glycolytic capacity as shown in Fig 1A. Metformin and Combination treatments significantly decreased ATP-coupled respiration, while increasing maximal oxidative phosphorylation capacity and non-mitochondrial respiration capacity as shown by elevated OCR following injections of Oligomycin, CCCP, and Rotenone/Antimycin A, respectively, as shown in Fig 1B.
Conclusion
Concomitant increases in both glycolytic and oxidative phosphorylation capacity suggest AMPK activation and TGFβ inhibition modulate cell mitochondrial and non-mitochondrial metabolic activity.
Fig 1 Metformin and SB431542 modulate mitochondrial and non-mitochondrial respiration capacity A. Glycolytic stress test (n=7); B. Mitochondrial stress test (n=4). All data are mean ± SD.
Funding
- Private Foundation Support