Tumor cell proliferation requires sufficient metabolic flux through the pentose phosphate pathway to meet the demand for biosynthetic precursors and to increase protection against oxidative stress which in turn requires an upregulation of substrate flow through glycolysis. This metabolic poise is often coupled with a shift in ATP production from mitochondrial OXPHOS to substrate-level phosphorylation. Despite major advances that were facilitated by using tumor-derived cell lines in research areas spanning from membrane to cytoskeletal biology, this distorted metabolic profile limits their impact as a model in physiology and toxicology. Substitution of glucose with galactose in the cell culture medium has been demonstrated to shift ATP production from substrate-level phosphorylation to mitochondrial OXPHOS. This increase in oxygen utilization is coupled to a global metabolic reorganization with potential impacts on macromolecule biosynthesis and cellular redox homeostasis, but a comprehensive analysis on the effects of sugar substitution in tumor-derived cells is still missing. To address this gap in knowledge we performed transcriptomic and metabolomic analyses on human hepatocellular carcinoma (HepG2) cells adapted to either glucose or galactose as the aldohexose source. We observed a shift towards oxidative metabolism in all primary metabolic pathways at both transcriptomic and metabolomic levels. We also observed a decrease in nicotinamide dinucleotide (NAD(P)) levels and subcellular NAD+-to-NADH ratios in cells cultured with galactose compared to glucose control cells. Our results suggest that galactose reduces both glycolytic and biosynthetic flux and restores a metabolic poise in HepG2 cells that closely reflects the metabolic state observed in primary hepatocytes.
Nutrient-deprivation autophagy factor-1 (NAF-1, miner1; gene cisd2) is part of the [2Fe-2S]containing protein family which includes mitoNEET (gene cisd1) and MiNT (miner2; gene cisd3). These proteins are redox active and are thought to play an important role in cellular energy homeostasis with NAF-1 playing a critical role in calcium regulation and aging. To date, no studies have investigated potential ligand interaction with NAF-1. Here we show that the thiazolidinediones pioglitazone and rosiglitazone along with the mitoNEET ligand, NL-1, bind to NAF-1 with low micromolar affinities. Further, we show that overexpression of NAF-1 in hepatocellular carcinoma (HepG2) cells reduces inhibition of mitochondrial respiration by pioglitazone. Our findings support the need for further efforts of the rational design of selective NAF-1 ligands.
Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.
MitoNEET is a mitochondrial [2Fe‐2S] protein known for its involvement in mitochondrial bioenergetics, iron metabolism, and oxidative stress. The protein has been extensively characterized at the biochemical level since its discovery in 2004, but the mechanisms of physiological function(s) remain unresolved. Two observations seem especially relevant for understanding the physiological function of mitoNEET: (1) under anaerobic conditions in vitro thiol compounds such as glutathione (GSH) and cysteine (Cys) reduce the 2Fe‐2S cluster from the Fe+3Fe+3 to the Fe3+Fe2+ state, and (2) mitoNEET expression levels increase in cells when the glutathione redox balance is shifted towards the oxidized state. We hypothesize that mitoNEET’s in vivo function can be elucidated by bridging these two observations and postulate that mitoNEET is an enzyme responsible for the oxidation of thiol groups on either low molecular‐weight compounds or cysteine residues on target proteins involved in redox signaling. To test this hypothesis, we monitored thiol oxidation by following oxygen consumption rates and demonstrate that mitoNEET accelerates thiol oxidation of cysteine and glutathione (GSH). Four distinct experiments comparing the reactivity of Fe3+ in the form of FeCl3 to mitoNEET demonstrate that the observed thiol oxidation by mitoNEET was not due to free Fe3+contamination. These observations were: (1) rates of cysteine oxidation (8 mM) catalyzed by mitoNEET (1.5 mM) or Fe3+ (1.5 mM) show different optima in the pH range 6.4 to 9.4 and the mitoNEET‐catalyzed reaction is 137% faster at pH 8.4 than the rate observed for Fe3+ alone. (2) Oxidation rates of the nonphysiological compound, n‐acetylcysteine (NAC) by mitoNEET are 181% higher compared to Fe3+ at a pH of 9.0. (3) MitoNEET‐mediated glutathione oxidation remained unaffected by addition of cysteine while oxidation catalyzed by free Fe3+ decreased after addition of cysteine. (4) Dimedone, a probe used to inhibit thiol oxidation from sulfenic acid to sulfinic and sulfonic acid, was 25% more effective at inhibiting cysteine oxidation when free Fe3+ was the catalyst compared to mitoNEET. These results support our hypothesis that mitoNEET possesses an active site which functions to oxidize biological thiols. While the in vivo substrate is currently unknown, we have narrowed down potential candidates to small thiol containing compounds and/or cysteine residues on specific target proteins. We are currently investigating how the enzymatic activity may be regulated by endogenous compounds and through pharmacological compounds targeting mitoNEET. Support or Funding Information This work was supported by NSF CHE‐1806266 to M.A.M and M.E.K and UofL Mentored Undergraduate Research and Creative Activities Grant to R.A.S.
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