A Cell-based High-throughput Assay System Reveals Modulation of Oxidative and Nonoxidative Glucose Metabolism due to Commonly Used Organic Solvents

Abstract

A 96-well format screening system was generated to quantify changes in nonoxidative glucose metabolism and oxidative pyruvate metabolism. D-Glucose uptake from the supernatant media was quantified by the glucose oxidase method, and L-lactate production of cells was quantified by the lactate dehydrogenase method applied on supernatant media. Mitochondrial membrane potential was quantified using tetramethylrhodamine methyl ester (TMRM) fluorescence, and reactive oxygen species (ROS) formation was determined by quantification of dihydrodichlorofluorescein fluorescence. Adenosine triphosphate (ATP) content of myocytes was determined using the luciferin reaction, and cellular respiration was quantified using commercially available, precoated microtiter plates. These six assays were used to determine the putative influence of organic solvents, namely dimethyl sulfoxide (DMSO), ethanol, methanol, and N-methylpyrrolidone (NMP) at concentrations of 0.01, 0.1, 1.0, and 5.0% (vol/vol), respectively, on glucose and pyruvate metabolism after 4 and 24 hours. In summary, all solvents induced significant changes in regard to one or several of the parameters evaluated, affecting cellular glucose uptake, glycolysis, mitochondrial metabolism, or oxidative phosphorylation. Accordingly, this comprehensive HTS evaluation should enable researchers to choose specific organic solvents on a rational basis to avoid nonspecific effects in cultured cells and tissue culture based experimental setups.

Inhibition of Alanine Aminotransferase in Silico and in Vivo Promotes Mitochondrial Metabolism to Impair Malignant Growth

“…d -Glucose uptake from supernatant media was determined as described previously (20). l -Lactate was measured according to the method of Sweetmann et al (21) modified as described below.…”

“…Cellular ATP content was measured by using a luciferin/luciferase-based bioluminescence assay (CellTiter-Glo, Promega, Madison, WI) as described before (20). Briefly, cells were seeded in a 96-well plate (2 × 10 3 /well), washed with PBS following treatment, and lysed (CellTiter-Glo buffer), and aliquots were taken for protein determination prior to the addition of bioluminescent substrate/enzyme solution (CellTiter-Glo substrate).…”

Small‐Molecule Targeting of the Mitochondrial Compartment with an Endogenously Cleaved Reversible Tag

“…[50] In brief, HepG2 cells were loaded with 2,7-dihydrodichlorofluorescein diacetate (H2-DCF-DA; 100 mm final concentration) in RPMI medium for 30 min at 37 8C under an atmosphere containing CO 2 (5 %). H2-DCF-DA is a nonfluorescent fluorescein derivative that emits fluorescence after oxidation to DCF-DA.…”

Improvement in the Photobiological Hydrogen Production of Aggregated Chlorella by Dimethyl Sulfoxide

“…Many carbon‐containing organic substances have the ability to increase the respiration of algae; for instance, glucose and sodium acetate can be used as substrates of respiration, and fumarate, oxaloacetate, and pyruvate can take part as catalysts in cellular respiration . In addition, dimethyl sulfoxide (DMSO), a widely used organic solvent, is also reported to be a promotor of the oxygen consumption rate of fibroblasts and L6 myoblasts, but the mechanism of this promotion is still unclear . It is documented that DMSO can increase the permeability of the membrane, and DMSO is often used to help molecules go through biofilms .…”