Abstract
Purpose :
RPE cells can oxidize both glucose and fatty acids to produce acetyl-CoA. They can use acetyl-CoA from fatty acid oxidation to produce citrate in the Krebs cycle or ketone bodies such as β-hydroxybutyrate (β-HB) in ketogenesis. In biochemistry textbooks, it’s taught that ketogenesis in liver cells occurs when glucose is scarce due to insufficient levels of oxaloacetate, which would limit utilization of acetyl-CoA by the Krebs cycle. We have observed formation of β-HB in RPE cells supplied with palmitate in the presence of abundant glucose. We therefore hypothesize that glucose supplied to RPE cells is poorly oxidized by RPE mitochondria relative to the fatty acid palmitate.
Methods :
We cultured primary human fetal RPE cells in serum free Miller media either with 5.5 mM 13C-glucose and 300 μM unlabeled palmitate, or with 5.5 mM unlabeled glucose and 300 μM 13C-palmitate. Medium was collected at 0, 2, 4, 7, 10, and 13 hours after the medium change. Glucose and palmitate remaining in the medium were quantified. 13C labeling of glycolytic and Krebs cycle intermediates was determined by GC-MS.
Results :
Labeling of intermediates from 13C glucose increased steadily over time. Labeling of intermediates from 13C palmitate increased and then decreased as the RPE cells depleted palmitate rapidly from the medium. We found that citrate can be labeled with 13C from glucose or from palmitate demonstrating that the RPE cells use carbons from both glucose and from palmitate to make acetyl-CoA, which is a substrate for synthesis of citrate and for β-HB. Remarkably, we found that β-HB incorporates 13C only from palmitate, but not from glucose.
Conclusions :
The design of this experiment and our findings from it show that RPE cells can sort acetyl-CoA derived from fatty acids away from acetyl-CoA derived from glucose, using acetyl-CoA produced only from fatty acids for ketogenesis. This sorting mechanism may contribute to the ability of RPE cells to synthesize β-HB even when glucose is abundant.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.