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  • Lactate preconditioning promotes a HIF-1α-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts.

Lactate preconditioning promotes a HIF-1α-mediated metabolic shift from OXPHOS to glycolysis in normal human diploid fibroblasts.

Scientific reports (2020-05-21)
Alexandra M Kozlov, Asad Lone, Dean H Betts, Robert C Cumming
ABSTRACT

Recent evidence has emerged that cancer cells can use various metabolites as fuel sources. Restricting cultured cancer cells to sole metabolite fuel sources can promote metabolic changes leading to enhanced glycolysis or mitochondrial OXPHOS. However, the effect of metabolite-restriction on non-transformed cells remains largely unexplored. Here we examined the effect of restricting media fuel sources, including glucose, pyruvate or lactate, on the metabolic state of cultured human dermal fibroblasts. Fibroblasts cultured in lactate-only medium exhibited reduced PDH phosphorylation, indicative of OXPHOS, and a concurrent elevation of ROS. Lactate exposure primed fibroblasts to switch to glycolysis by increasing transcript abundance of genes encoding glycolytic enzymes and, upon exposure to glucose, increasing glycolytic enzyme levels. Furthermore, lactate treatment stabilized HIF-1α, a master regulator of glycolysis, in a manner attenuated by antioxidant exposure. Our findings indicate that lactate preconditioning primes fibroblasts to switch from OXPHOS to glycolysis metabolism, in part, through ROS-mediated HIF-1α stabilization. Interestingly, we found that lactate preconditioning results in increased transcript abundance of MYC and SNAI1, key facilitators of early somatic cell reprogramming. Defined metabolite treatment may represent a novel approach to increasing somatic cell reprogramming efficiency by amplifying a critical metabolic switch that occurs during iPSC generation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Acetyl-L-cysteine, Sigma Grade, ≥99% (TLC), powder
Sigma-Aldrich
D-(+)-Glucose, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
Sigma-Aldrich
Phenylmethanesulfonyl fluoride, ≥98.5% (GC)
Sigma-Aldrich
Sodium pyruvate, ReagentPlus®, ≥99%
Sigma-Aldrich
Sodium orthovanadate, ≥90% (titration)
Sigma-Aldrich
Sodium L-lactate, ≥99.0% (NT)
Supelco
Sodium bicarbonate, Meets ACS Specifications, Meets Reagent Specifications for testing USP/NF monographs GR ACS
Sigma-Aldrich
KC7F2, ≥98% (HPLC)
Sigma-Aldrich
PhosphoDetect Anti-PDH-E1α (pSer²³²) Rabbit pAb, liquid, Calbiochem®
Sigma-Aldrich
Amyloid Protein Non-Aβ Component, ≥80% (HPLC)