Glutamate-oxaloacetate transaminase activity promotes palmitate lipotoxicity in rat hepatocytes by enhancing anaplerosis and citric acid cycle flux
- Robert A. Egnatchik1,
- Alexandra K. Leamy1,
- Sarah A. Sacco1,
- Yi Ern Cheah1,
- Masakazu Shiota2 and
- Jamey D. Young1*
- ↵* Corresponding author; email: j.d.young{at}vanderbilt.edu
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Author contributions: R.A.E., A.K.L., and J.D.Y. conceptualization; R.A.E., A.K.L., S.A.S., and M.S. data curation; R.A.E. and J.D.Y. software; R.A.E., A.K.L., S.A.S., and Y.E.C. formal analysis; R.A.E., M.S., and J.D.Y. supervision; R.A.E. and J.D.Y. funding acquisition; R.A.E., A.K.L., S.A.S., Y.E.C., M.S., and J.D.Y. investigation; R.A.E., A.K.L., and Y.E.C. methodology; R.A.E. and A.K.L. writing-original draft; R.A.E. and J.D.Y. project administration; R.A.E., S.A.S., M.S., and J.D.Y. writing-review and editing; M.S. and J.D.Y. resources.
Abstract
Hepatocyte lipotoxicity is characterized by aberrant mitochondrial metabolism, which predisposes cells to oxidative stress and apoptosis. Previously, we reported that translocation of calcium from the ER to mitochondria of palmitate-treated hepatocytes activated anaplerotic flux from glutamine to alpha-ketoglutarate (αKG), which subsequently entered the citric acid cycle (CAC) for oxidation. We hypothesized that increased glutamine anaplerosis fueled elevations in CAC flux and oxidative stress following palmitate treatment. To test this hypothesis, primary rat hepatocytes or immortalized H4IIEC3 rat hepatoma cells were treated with lipotoxic levels of palmitate while modulating anaplerotic pathways leading to αKG. We found that culture media supplemented with glutamine, glutamate, or dimethyl-αKG increased palmitate lipotoxicity compared to media that lacked these anaplerotic substrates. Knockdown of glutamate-oxaloacetate transaminase (GOT) activity significantly reduced the lipotoxic effects of palmitate, while knockdown of glutamate dehydrogenase (Glud1) had no effect on palmitate lipotoxicity. 13C flux analysis of H4IIEC3 cells co-treated with palmitate and the pan-transaminase inhibitor aminooxyacetic acid (AOA) confirmed that reductions in lipotoxic markers were associated with decreases in anaplerosis, CAC flux, and oxygen consumption. Taken together, these results demonstrate that lipotoxic palmitate treatments enhance anaplerosis in cultured rat hepatocytes, causing a shift to aberrant transaminase metabolism that fuels CAC dysregulation and oxidative stress.
- metabolic flux analysis
- fatty liver disease
- fatty acid
- tricarboxylic acid cycle (TCA cycle) (Krebs cycle)
- hepatocyte
- glutamine
- lipotoxicity
- anaplerosis
- Received July 13, 2018.
- Accepted December 18, 2018.
- Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
