Circumventing the Crabtree Effect: Replacing Media Glucose with Galactose Increases Susceptibility of HepG2 Cells to Mitochondrial Toxicants

Abstract

Many highly proliferative cells generate almost all ATP via glycolysis despite abundant O(2) and a normal complement of fully functional mitochondria, a circumstance known as the Crabtree effect. Such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, such as the inhibitors rotenone, antimycin, oligomycin, and compounds like carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that uncouple the respiratory electron transfer system from phosphorylation. These cells are also resistant to the toxicity of many drugs whose deleterious side effect profiles are either caused, or exacerbated, by impairment of mitochondrial function. Drug-induced mitochondrial toxicity is shown by members of important drug classes, including the thiazolidinediones, statins, fibrates, antivirals, antibiotics, and anticancer agents. To increase detection of drug-induced mitochondrial effects in a preclinical cell-based assay, HepG2 cells were forced to rely on mitochondrial oxidative phosphorylation rather than glycolysis by substituting galactose for glucose in the growth media. Oxygen consumption doubles in galactose-grown HepG2 cells and their susceptibility to canonical mitochondrial toxicants correspondingly increases. Similarly, toxicity of several drugs with known mitochondrial liabilities is more readily apparent in aerobically poised HepG2 cells compared to glucose-grown cells. Some drugs were equally toxic to both glucose- and galactose-grown cells, suggesting that mitochondrial impairment is likely secondary to other cytotoxic mechanisms.

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…The viability of cells loaded with probe was tested (after trypsinization) on a PCA-96 flow cytometer using ViaCount TM kit and Cytosoft 2.5.5 software (all Guava Technologies), as per the manufacturer's instructions. To assess the contribution of glycolysis to ATP production, d PC12 cells were preincubated for 3 h in serum-free RPMI containing 10 mM galactose and 1 mM pyruvate (32). To reduce neurotransmission through cleavage of synaptobrevin 2, d PC12 were treated with 20 nM tetanus neurotoxin (TeNT) for 6 h after transfection.…”

Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells

“…To do this, we measured the levels of intracellular ATP in cells cultured in glucosecontaining media (which produce ATP by both respiration and glycolysis) and in galactose-containing media (which inhibit glycolytic production of ATP resulting in reliance on respiration (OxPhos) (Marroquin et al, 2007)). No significant difference was observed between PNC1-deficient cells and control cells cultured in glucose (Figure 2a).…”

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

“…We therefore measured Strap in cells grown in galactose-supplemented (to promote oxidative phosphorylation) and glucose-supplemented (to promote glycolysis) medium. 25,26 Strap protein levels increased under galactose compared with glucose-supplemented conditions in HCT116 cells (Figure 2c), with a similar pattern apparent in mitochondrial fractions ( Figure 2d); moreover, this increase in Strap levels required p53 activity (Supplementary Figure S1g). We then reasoned that the increased protein level might influence Strap binding to the ATP synthase b-subunit and tested this possibility by immunoprecipitating the Strap-ATP synthase complex.…”

“…Cells were seeded in DMEM containing 5% foetal calf serum in the presence of antibiotics and were allowed to grow for 24-48 h. Cells were then washed twice with PBS and received glucose-or galactose-supplemented media for 24 h. Glucose-supplemented media consisted of 25 mM glucose and 2 mM glutamine supplemented with antibiotics, and the galactose-supplemented media consisted of glucose-free media with 10 mM galactose, 5 mM HEPES and 2 mM glutamine supplemented with antibiotics. 26 The following plasmids have previously been described: pcDNA3 HA-WT Strap, 9,10 pCMV-L-p53 was a generous gift from Prof Ute Moll 22 and p3xFlag-CMV-7.1 expression vector (Sigma, Dorset, UK). L-Strap was prepared by fusing the Bcl2 mitochondrial targeting signal (DFSWLSLKTLLSLALV-GACITLGAYGHK) onto the C-terminal region of Strap in HA-WT Strap.…”