Quantification of Low-Level Drug Effects Using Real-Time,<i>in vitro</i>Measurement of Oxygen Consumption Rate

Neal, Adam; Rountree, Austin M.; Philips, Craig W.; Kavanagh, Terrance J.; Williams, Dominic P.; Newham, Peter; Khalil, Gamal; Cook, Daniel L.; Sweet, Ian R.

doi:10.1093/toxsci/kfv208

Cited by 21 publications

(28 citation statements)

References 42 publications

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“…Inhibition of the oxygen consumption rate (19,59) or glucose (pyruvate) oxidation by high metformin are consistent with inhibition of complex 1 and mimicked by rotenone. In this study we found inhibition of glucose oxidation by 0.5 mM but not by 0.2 mM metformin, and this concurs with studies showing inhibition of oxygen consumption at Ն 0.3 mM metformin (68). Although high metformin (Ն0.5 mM) promotes a more reduced mitochondrial NADH/NAD ratio, consistent with complex 1 inhibition (31), low metformin (Յ0.2 mM) promotes a more oxidized NADH/NAD ratio and increased ␤-octanoate oxidation, implicating increased electron transport (31).…”

Section: Metformin Lowers Glucose 6-phosphate In Hepatocytessupporting

confidence: 93%

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

Moonira

Chachra

Ford

et al. 2020

Journal of Biological Chemistry

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The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the carbohydrate-response element–binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a complex 1 inhibitor (rotenone) and an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, and C-13) had opposite effects from metformin on glycolysis, gluconeogenesis, and cell G6P. The G6P lowering by metformin, which also occurs in hepatocytes from AMPK knockout mice, is best explained by allosteric regulation of phosphofructokinase-1 and/or fructose bisphosphatase-1, as supported by increased metabolism of [3-3H]glucose relative to [2-3H]glucose; by an increase in the lactate m2/m1 isotopolog ratio from [1,2-13C2]glucose; by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1; and by marked G6P elevation by selective inhibition of phosphofructokinase-1; but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMP-activated protein kinase–independent mechanism through changes in allosteric effectors of phosphofructokinase-1 and fructose bisphosphatase-1, including AMP, Pi, and glycerol 3-phosphate.

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Section: Metformin Lowers Glucose 6-phosphate In Hepatocytessupporting

confidence: 93%

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

Moonira

Chachra

Ford

et al. 2020

Journal of Biological Chemistry

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“…OCR in the two channels that were not exposed to acetaminophen remained flat, whereas the OCR was lowered 40 and 60% by 6 and 15 mM acetaminophen. These results were similar to those obtained in a previous study with our larger peristaltic pump-driven perifusion system [10].…”

Section: Resultssupporting

confidence: 92%

“…Samples of the effluent perfusate can be collected for off-line assay—we routinely measure insulin release rate, or lactate production for example. We can also simultaneously measure the tissue’s OCR using opto-electronic sensing of the fluorescence decay rate of an oxygen-sensitive dye painted either on glass beads or on the inner column surface as routinely used in our prior studies [10]. Thus, the BaroFuse can combine conventional chemical and radioimmunological assays with OCR as a powerful index of cellular health and function.…”

Section: Methodsmentioning

confidence: 99%

“…Oxygen tension in the outflow of each tissue perfusion chamber was measured as previously described [10], except that instead of painting the oxygen-sensitive dye on the inside of the tissue perifusion chamber, glass beads (710–1180 microns, Sigma-Aldrich, St. Louis, MO) coated with the dye were layered on top of the tissue in the perifusion chamber. The flow was slow enough so that the beads did not move during the experiment, and the oxygen sensors sampled a representative cross section of perfusate that after passing by the tissue was delivered by convection to the sensors.…”

Section: Methodsmentioning

confidence: 99%

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BaroFuse, a novel pressure-driven, adjustable-throughput perfusion system for tissue maintenance and assessment

Rountree

Karkamkar

Khalil

et al. 2016

Heliyon

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ObjectivesMicrofluidic perfusion systems are used for assessing cell and tissue function while assuring cellular viability. Low perfusate flow rates, desired both for conserving reagents and for extending the number of channels and duration of experiments, conventionally depend on peristaltic pumps to maintain flow yet such pumps are unwieldy and scale poorly for high-throughput applications requiring 16 or more channels. The goal of the study was to develop a scalable multichannel microfluidics system capable of maintaining and assessing kinetic responses of small amounts of tissue to drugs or changes in test conditions.MethodsHere we describe the BaroFuse, a novel, multichannel microfluidics device fabricated using 3D-printing technology that uses gas pressure to drive large numbers of parallel perfusion experiments. The system is versatile with respect to endpoints due to the translucence of the walls of the perifusion chambers, enabling optical methods for interrogating the tissue status. The system was validated by the incorporation of an oxygen detection system that enabled continuous measurement of oxygen consumption rate (OCR).ResultsStable and low flow rates (1–20 μL/min/channel) were finely controlled by a single pressure regulator (0.5–2 psi). Control of flow in 0.2 μL/min increments was achieved. Low flow rates allowed for changes in OCR in response to glucose to be well resolved with very small numbers of islets (1–10 islets/channel). Effects of acetaminophen on OCR by precision-cut liver slices of were dose dependent and similar to previously published values that used more tissue and peristaltic-pump driven flow.ConclusionsThe very low flow rates and simplicity of design and operation of the BaroFuse device allow for the efficient generation of large number of kinetic profiles in OCR and other endpoints lasting from hours to days. The use of flow enhances the ability to make measurements on primary tissue where some elements of native three-dimensional structure are preserved. We offer the BaroFuse as a powerful tool for physiological studies and for pharmaceutical assessment of drug effects as well as personalized medicine.

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“…The microfluidic flow conditions also allow for constant replenishment of oxygen and nutrients, and concomitant removal of cell products, thereby reducing potential detrimental effects of paracrine cues that can accumulate in static systems50.…”

Section: Discussionmentioning

confidence: 99%

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

Neal

Rountree

Radtke

et al. 2016

Sci Rep

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Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.

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Quantification of Low-Level Drug Effects Using Real-Time,in vitroMeasurement of Oxygen Consumption Rate

Cited by 21 publications

References 42 publications

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation

BaroFuse, a novel pressure-driven, adjustable-throughput perfusion system for tissue maintenance and assessment

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

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