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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.
MitoNEET (gene cisd1) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow guidelines of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols to the nomenclature of classical bioenergetics. We endeavour to provide a balanced view on mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of databases of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.
MitoNEET is a CDGSH iron-sulfur protein which has been a target for drug development for diseases such as type-2 diabetes, cancer, and Parkinson's Disease. Functions proposed for mitoNEET are as a redox sensor and regulator of free iron in the mitochondria. We have investigated the reactivity of mitoNEET towards the reactive electrophiles 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) that are produced from the oxidation of poly-unsaturated fatty acid during oxidative stress. Proteomic, electrophoretic and spectroscopic analysis has shown that HNE and ONE react in a sequence selective manner that was unexpected considering the structure similarity of these two reactive electrophiles.
Nutrient-deprived 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.
As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow IUPAC guidelines on terminology in physical chemistry, extended by considerations on open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols to the nomenclature of classical bioenergetics. We endeavour to provide a balanced view on mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately support the development of databases of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.
The dimeric protein, CISD2, belongs to a unique family of [2Fe-2S] cluster containing proteins (CISD proteins), which are implicated in a variety of disorders including type-2 diabetes, Wolfram syndrome 2, and neurodegeneration. Localizing to membranes of the mitochondrion, endoplasmic reticulum, and mitochondrial-associated ER membranes, CISD2 has been linked to autophagy and mitochondrial bioenergetics, but the specific mechanism(s) of action remain ill-defined. Further, CISD2 has been demonstrated to bind the anti-diabetes drug pioglitazone, but the therapeutic impact of binding remains uncharacterized. To investigate the potential function(s) of CISD2 in human hepatocellular carcinoma cells (HepG2) the protein was overexpressed and respirometry was utilized to assess mitochondrial performance. Surprisingly, increased CISD2 levels enhanced cellular oxygen consumption by 86% above control, however no differences were detected during chemically uncoupled respiration. To shift metabolic flux from glycolysis to oxidative phosphorylation, glucose was replaced by galactose as the primary carbon source. Under these conditions, we observed that both control and CISD2 overexpressing cells displayed increased routine respiration, but cells overexpressing CISD2 had 64.5% higher chemically uncoupled respiration than non-transfected controls. Permeablized cells also demonstrated significantly higher uncoupling rates compared to control regardless of the substrate used. Finally, molecular docking studies were used to investigate the pioglitazone-CISD2 interaction and 30 uM pioglitazone was introduced during respirometric studies and did not negate the enhanced respiratory capacity. Together our results suggest that CISD2 is a critical, yet often overlooked regulator of mitochondrial bioenergetics and may impact the supply of reduced redox equivalents to the electron transport system.
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.
The dimeric proteins CISD1 and CISD2 belong to a unique family of iron-sulfur cluster containing proteins and are implicated in a variety of disorders including type-2 diabetes, Wolfram syndrome 2, and neurodegeneration. Localized at the mitochondrion, endoplasmic reticulum, and mitochondria-associated ER membranes, CISD proteins are directly or indirectly involved in cellular energy homeostasis, but the specific mechanism(s) of regulation remain still ill defined. Our lack of a mechanistic understanding how CISD proteins integrate into cellular bioenergetics is in part due to the fact that interacting proteins and ligands are still mostly uncharacterized. Recent studies have suggested that both proteins, CISD1 and CISD2, are involved in redox sensing via binding to NADPH, but a more comprehensive study of interaction with other cellular metabolites is missing. We demonstrate that the soluble portion of both proteins have one tyrosine and tryptophan residue per monomer which undergo fluorescent emission quenching upon ligand binding. This feature allows for the development of a simple fluorescence-based assay to characterize binding of physiologically relevant organic ligands to CISD1 and CISD2. Excitation at l = 280 nm results in fluorescent emission of both tyrosine and tryptophan, while exaction at l = 295 nm generates only tryptophan emission. By monitoring the fluorescent emission of CISD1 and CISD2 excited at l = 280 nm or l = 295 nm, we could demonstrate that reduced nicotinamide adenine dinucleotide (NADH) binds with significant affinity to two different binding sites, while the oxidized form and adenine nucleotides such as ATP and ADP only interact with one type of site. The physiological concentration of NAD + ranges between 0.3-0.4 mM, only approximately 2-3-fold lower than the apparent K D280 for both CISD1 and CISD2 (K D280 = 1.1 mM and 0.7 mM, respectively). Furthermore, binding affinities of CISD1 to ATP and ADP are within the physiological range of the phosphorylated nucleotides (K D280 = 3.4 ± 0.2 mM and 1.5 ± 0.1 mM). Our approach offers a versatile system to characterize physiological relevant binding partners and to rapidly screen for potential therapeutics that interact with CISD proteins. Importantly, we demonstrate that CISD1 and CISD2 could be governing or responding to the cellular redox state by binding with physiologically significant affinities to a variety of compounds that are directly or indirectly related to the cellular energy state (Supported by NSF CHE-160944).
The Warburg effect is ameliorated by culturing transformed cells in the presence of galactose instead of glucose as the primary carbon source. However, metabolic consequences may occur in addition to sensitizing the cells to mitochondrial toxins. The screening of pharmaceutical agents against transformed cells while using galactose must therefore be carefully evaluated. Pioglitazone is employed in clinical applications to treat type-2 diabetes but clearly has other off-target effects. Human hepatocellular carcinoma cells (HepG2) were cultured in glucose or galactose-containing medium to investigate the role of pioglitazone on cellular bioenergetics by calorimetry and respirometry. Compared with cells cultured in 10 mM glucose, HepG2 cells cultured in the presence of 10 mM galactose showed decreased metabolic activity as measured by cellular heat flow. Interestingly, cellular heat flow increased after the addition of pioglitazone for cells cultured in glucose, but not for cells cultured in galactose. Our calorimetric data indicated that a reduction in cellular capacity for glycolysis was the mechanism responsible for the increase in sensitivity to pioglitazone, and possibly to mitochondrial toxins in general, for cells cultured in galactose. Furthermore, oxygen consumption rates were decreased after the addition of pioglitazone to cells grown in glucose but remained unchanged for cells grown in the presence of galactose. We have demonstrated that pioglitazone induces a reduction in mitochondrial activity that is partially compensated via an increase in glycolysis in the presence of glucose.
MitoNEET is a protein that was identified as a drug target for diabetes, but its cellular function as well as its role in diabetes remains elusive. Protein pull-down experiments identified glutamate dehydrogenase 1 (GDH1) as a potential binding partner. GDH1 is a key metabolic enzyme with emerging roles in insulin regulation. MitoNEET forms a covalent complex with GDH1 through disulfide bond formation and acts as an activator. Proteomic analysis identified the specific cysteine residues that participate in the disulfide bond. This is the first report that effectively links mitoNEET to activation of the insulin regulator GDH1.