Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria methods
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Kinetic measurements on bovine mitochondrial membranes and SMPs
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- All assays were performed at 32°C in 10 mM Tris/HCl (pH 7.4) and 250 mM sucrose. NADH oxidation was measured in 100 µM NADH, and succinate oxidation in 10 mM succinate, using a coupled assay system [ ]. Complex II activity was measured in 10 mM succinate and 100 µM d...
OCR and ECAR measurements on cultured human cells and isolated mitochondria
reagent used in the protocol.
- Use
- Cells [143B (cell line 8303 from the A.T.C.C.) and Hep G2 (cell line 85011430 from The Health Protection Agency)] were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO 2...
Statistical methods
reagent used in the protocol.
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- Experimental values are reported as means±S.E.M. for small sample sizes or as means±S.D. for large sample sizes with only technical error sources. Typically three to four replicates gave S.E.M. values of less than 5% of the mean value; numbers of replicates were increased when appropriate. Comparisons betw...
Biguanides inhibit ubiquinone reduction, but not competitively
reagent used in the protocol.
- Use
- To narrow down the location at which biguanides bind to complex I, we tested their effects on different steps in the catalytic cycle: NADH oxidation by the flavin mononucleotide, intramolecular electron transfer along the chain of FeS clusters, and ubiquinone reduction [ ]. First, metformin was found to stimulate (s...
Biguanides inhibit ubiquinone reduction, but not competitively
reagent used in the protocol.
- Use
- ( A ) Effects of metformin on flavin site reactions that require nucleotide binding to the reduced flavin [NADH:HAR (blue) and NADH:paraquat (green) oxidoreduction] [ ], and nucleotide-free reduced flavin [NADH:FeCN (purple) and NADH:O 2 (red) oxidoreduction] [ ]. NADH:O 2 oxidoreduction (H 2 O 2 production) was det...
Metformin interacts with 'deactive-like' conformations of complex I
reagent used in the protocol.
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- SMPs are robust, inverted and tightly coupled vesicles of the inner membrane of bovine heart mitochondria [ ]. NADH oxidation by complex I in SMPs does not require a hydrophilic ubiquinone because ubiquinone-10 is present, and ubiquinol-10 is reoxidized by the respiratory chain. Therefore SMPs maintain steady-state...
Biguanide effects on the flavin site and H 2 O 2 production
reagent used in the protocol.
- Use
- Complex I catalyses electron transfer from NADH to hydrophilic electron acceptors by two distinct mechanisms, both localized uniquely at the flavin site. The NADH:FeCN and NADH:O 2 reactions proceed via two sequential steps in which NADH reduces the flavin and dissociates, and then the electron acceptor reacts direc...
Biguanide interactions with respiratory complexes II, III and IV
reagent used in the protocol.
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- ( A ) Rates of complex II + III + IV activity in bovine SMPs measured by a coupled enzyme assay [ ] in the presence of biguanide concentrations equivalent to IC 50 for NADH:decylubiquinone catalysis. Results are means±S.E.M. as a percentage of the biguanide-free control, n =3-4. ( B ) Rates of respiratory...
Kinetic measurements on bovine mitochondrial membranes and SMPs
All assays were performed at 32°C in 10 mM Tris/HCl (pH 7.4) and 250 mM sucrose. NADH oxidation was measured in 100 µM NADH, and succinate oxidation in 10 mM succinate, using a coupled assay system [ ]. Complex II activity was measured in 10 mM succinate and 100 µM d...
- Use
- All assays were performed at 32°C in 10 mM Tris/HCl (pH 7.4) and 250 mM sucrose. NADH oxidation was measured in 100 µM NADH, and succinate oxidation in 10 mM succinate, using a coupled assay system [ ]. Complex II activity was measured in 10 mM succinate and 100 µM d...
OCR and ECAR measurements on cultured human cells and isolated mitochondria
Cells [143B (cell line 8303 from the A.T.C.C.) and Hep G2 (cell line 85011430 from The Health Protection Agency)] were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO 2...
- Use
- Cells [143B (cell line 8303 from the A.T.C.C.) and Hep G2 (cell line 85011430 from The Health Protection Agency)] were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO 2...
Analytical methods
Octanol/PBS distribution coefficients were measured by the shake-flask method [ ] at 32°C and pH 7.5; they are similar to partition coefficients but refer to the protonated biguanide cations [ ]. Thermo-flavin experiments were carried in an ABI 7900HT real-time PCR machine, starting at 20°C for 2...
- Use
- Octanol/PBS distribution coefficients were measured by the shake-flask method [ ] at 32°C and pH 7.5; they are similar to partition coefficients but refer to the protonated biguanide cations [ ]. Thermo-flavin experiments were carried in an ABI 7900HT real-time PCR machine, starting at 20°C for 2...
Effects of biguanides on F 1 F 0 -ATPase
( A ) Inhibition of ATP hydrolysis by SMPs (colours as in B ). Data points are means±S.E.M., n =3-5, and IC 50 values are indicated in mM. ( B ) Relationship between the IC 50 values for ATP hydrolysis and the log D values. IC 50 values are in mM with 95% confidence intervals; log D values are means±...
- Use
- ( A ) Inhibition of ATP hydrolysis by SMPs (colours as in B ). Data points are means±S.E.M., n =3-5, and IC 50 values are indicated in mM. ( B ) Relationship between the IC 50 values for ATP hydrolysis and the log D values. IC 50 values are in mM with 95% confidence intervals; log D values are means±...
Selective cellular and mitochondrial uptake of biguanides
The two antimalarial biguanides, proguanil and cycloguanil, are good inhibitors of complex I, but we could find no clinical reports for either being associated with increased risk of lactic acidosis. In contrast, the anti-diabetic drugs metformin, phenformin and buformin are weaker inhibitors and they are associated...
- Use
- The two antimalarial biguanides, proguanil and cycloguanil, are good inhibitors of complex I, but we could find no clinical reports for either being associated with increased risk of lactic acidosis. In contrast, the anti-diabetic drugs metformin, phenformin and buformin are weaker inhibitors and they are associated...
Biguanides accumulate reversibly in mitochondria according to the membrane potential
( A ) The normalized rotenone-sensitive OCR 6 h after the addition of metformin or phenformin to Hep G2 (black) or 143B (grey) cells. Results are means±S.E.M., n =3-4. The IC 50 values (with 95% confidence intervals) are metformin, 240±10 µM (143B) and 330±20 µM (Hep G2)...
- Use
- ( A ) The normalized rotenone-sensitive OCR 6 h after the addition of metformin or phenformin to Hep G2 (black) or 143B (grey) cells. Results are means±S.E.M., n =3-4. The IC 50 values (with 95% confidence intervals) are metformin, 240±10 µM (143B) and 330±20 µM (Hep G2)...
Mechanisms by which biguanides may protect against ischaemia/reperfusion injury
In ischaemic tissue lack of oxygen stops respiratory chain turnover and oxidative phosphorylation, and causes the mitochondrial NADH and succinate pools to become reduced. Under these conditions, the resting complex I converts into its deactive state [ ], and (unless bound by the inhibitor protein IF 1 ) ATP hydroly...
- Use
- In ischaemic tissue lack of oxygen stops respiratory chain turnover and oxidative phosphorylation, and causes the mitochondrial NADH and succinate pools to become reduced. Under these conditions, the resting complex I converts into its deactive state [ ], and (unless bound by the inhibitor protein IF 1 ) ATP hydroly...
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Kinetic measurements on bovine mitochondrial membranes and SMPs
All assays were performed at 32°C in 10 mM Tris/HCl (pH 7.4) and 250 mM sucrose. NADH oxidation was measured in 100 µM NADH, and succinate oxidation in 10 mM succinate, using a coupled assay system [ ]. Complex II activity was measured in 10 mM succinate and 100 µM decylubiquinone using membranes solubilized in 1% dodecylmaltoside to isolate the activity. Complex II + III activity in membranes was measured by the reduction of cytochrome c (ε 550-541 =18.00 mM -1 ·cm -1 ) with 120 µM oxidized horse heart cytochrome c, 10 mM succinate and 1 mM NaCN to inhibit complex IV. Complex IV activity was measured by the oxidation of 120 µM reduced horse heart cytochrome c. ATP hydrolysis was measured using a coupled assay system [, ], using 40 µg/ml pyruvate kinase, 200 &#...
Kinetic measurements on isolated complex I
All assays were performed at 32°C in 20 mM Tris/HCl (pH 7.2). NADH:decylubiquinone oxidoreduction was measured using 200 µM NADH and 200 µM decylubiquinone, in 0.075% soya bean asolectin (Avanti Polar Lipids) and 0.075% CHAPS (Merck Chemicals) and quantified by the absorbance of NADH (ε 340-380 =4.81 mM -1 ·cm -1 ) [ ]. Catalysis was initiated by the addition of NADH, following a 2 min pre-incubation, and rates measured as the linear regression of the maximal rate (discarding any initial lag phases). Biguanides were added immediately before NADH, unless otherwise stated, and the level of inhibition did not depend on the length of pre-incubation. Initial rates for the NADH:FeCN (ferricyanide), NADH:HAR [hexaammineruthenium(III)] and NADH:paraquat reactions were measured in 100 µM NADH with 1 mM FeCN (...
EPR spectroscopy
EPR samples of ~11 mg/ml bovine complex I were prepared anaerobically. The samples were incubated for 15 min at 4°C with 100 mM NaCl, 100 mM metformin, 2.5 mM phenformin, or 0.35 mM proguanil, then 5 mM NADH was added and the samples frozen immediately. Spectra were recorded with 1 mW microwave power, microwave frequency 9.36-9.38 GHz, modulation frequency 100 kHz, modulation amplitude 1 mT, time constant 81.92 ms and conversion time 20.48 ms at 12 K, using a Bruker EMX X-band spectrometer with an ER 4119HS high-sensitivity cavity, maintained at low temperature by an Oxford Instruments ESR900 continuous-flow liquid helium cryostat.
OCR and ECAR measurements on cultured human cells and isolated mitochondria
Cells [143B (cell line 8303 from the A.T.C.C.) and Hep G2 (cell line 85011430 from The Health Protection Agency)] were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO 2. Per well, 3 × 10 4 or 1.5 × 10 4 cells were plated into XF24 or XF96 (Seahorse Bioscience) plates respectively, and incubated for 12 h at 37°C in 5% CO 2. The medium was exchanged for assay buffer containing DMEM, 4.5 g/l glucose, 1 mM pyruvate, 32 mM NaCl, 2 mM GlutaMAX, 15 mg/l Phenol Red and 20 mM Hepes (pH 7.4) and the cells placed in a CO 2 -free incubator at 37°C for 60 min. OCRs (oxygen consumption rates) and ECARs (extracellular acidification rates) were measured in a Seahorse extracellular flux analyse...
Statistical methods
Experimental values are reported as means±S.E.M. for small sample sizes or as means±S.D. for large sample sizes with only technical error sources. Typically three to four replicates gave S.E.M. values of less than 5% of the mean value; numbers of replicates were increased when appropriate. Comparisons between samples are marked * P <0.05, ** P <0.01 and **** P <0.0001 as determined using an unpaired two-tailed Students t test. Differences between measurements with P >0.05 were considered not significant. IC 50 values were determined using the standard dose-effect relationship {activity (%)=100 × IC 50 /(IC 50 +[inhibitor] m ); [ ] with the Hill Slope (m) set to unity for complex I} and are reported with 95% confidence intervals.
Biguanides inhibit ubiquinone reduction, but not competitively
To narrow down the location at which biguanides bind to complex I, we tested their effects on different steps in the catalytic cycle: NADH oxidation by the flavin mononucleotide, intramolecular electron transfer along the chain of FeS clusters, and ubiquinone reduction [ ]. First, metformin was found to stimulate (see below and ), not inhibit, the NADH:FeCN oxidoreduction reaction. The NADH:FeCN reaction is widely used to test the rate of NADH oxidation by complex I, because the whole reaction is localized exclusively at the flavin site [ ]. Furthermore, even extremely high concentrations of metformin only affected the thermal stability of the flavin site slightly, showing that the flavin environment is not significantly altered (the thermal stability is measured as a melting temperature that varies from 56.3±0.4 in 200 mM NaCl to 55.3±0.1 in 200 mM metformin). We...
Biguanide effects on the flavin site and H 2 O 2 production
Complex I catalyses electron transfer from NADH to hydrophilic electron acceptors by two distinct mechanisms, both localized uniquely at the flavin site. The NADH:FeCN and NADH:O 2 reactions proceed via two sequential steps in which NADH reduces the flavin and dissociates, and then the electron acceptor reacts directly (see E, left-hand reaction) [ ]; for the NADH:HAR and NADH:paraquat reactions NADH reduces the flavin, but the electron acceptor reacts only if a nucleotide is bound in the reduced flavin site (see E, right-hand reaction) [ ]. Note that although NADH:O 2 oxidoreduction produces superoxide, we detect the H 2 O 2 formed following superoxide dismutation, and thus refer to 'H 2 O 2 production'. Data showing the effects of biguanides on these different reactions are shown in. (A) shows that metformin stimulates the rates of the two reactions (the NADH:FeCN and N...
Effects of biguanides on F 1 F 0 -ATPase
( A ) Inhibition of ATP hydrolysis by SMPs (colours as in B ). Data points are means±S.E.M., n =3-5, and IC 50 values are indicated in mM. ( B ) Relationship between the IC 50 values for ATP hydrolysis and the log D values. IC 50 values are in mM with 95% confidence intervals; log D values are means±S.E.M., n =3. Linear fit with R 2 =0.948. ( C ) Relative inhibition of ATP production in SMPs in the presence of piericidin A (○) or metformin (●). ATP synthesis was driven using 200 µM NADH; NADH oxidation was measured spectroscopically, and the concentration of ATP determined after 3.5 min (during this time a linear rate is observed). NADH oxidation rates were adjusted using 0-50 nM piericidin A or 0-250 mM metformin, with the ionic strength kept constant at 250 mM using NaCl. ( D - F ) Dose-dependent effects of...
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Octanol/PBS distribution coefficients were measured by the shake-flask method [ ] at 32°C and pH 7.5; they are similar to partition coefficients but refer to the proto...
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Experimental values are reported as means±S.E.M.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Octanol/PBS distribution coefficients were measured by the shake-flask method [ ] at 32°C and pH 7.5; they are similar to partition coefficients but refer to the proto....
from paperStatistical comparison
Experimental values are reported as means±S.E.M. for small sample sizes or as means±S.D. for large sample sizes with only technical error sources. Typically three to f...; Complex I has been proposed as the major respiratory-chain target of metformin [, ], but inhibition of complexes II and IV have also been reported in rat liver mitochondria [ ]...; ( A ) Rates of complex II + III + IV activity in bovine SMPs measured by a coupled enzyme assay [ ] in the presence of biguanide concentrations equivalent to IC 50 for NADH:decy...; The effects of biguanides on ATP hydrolysis by F 1 F O -ATPase were tested in SMPs by using a coupled assay to detect the formation of ADP [, ]. (A) shows that all five biguani...
from paperReporting output
Report representative outputs alongside summary comparisons for Octanol/PBS distribution coefficients were measured by the shake-flask method [ ] at 32°C and pH 7.5; they are similar to partition coefficients but refer to the proto....
inferred from protocolStructured statistical methods
Experimental values are reported as means±S.E.M. for small sample sizes or as means±S.D. for large sample sizes with only technical error sources. Typically three to f...; Complex I has been proposed as the major respiratory-chain target of metformin [, ], but inhibition of complexes II and IV have also been reported in rat liver mitochondria [ ]...; ( A ) Rates of complex II + III + IV activity in bovine SMPs measured by a coupled enzyme assay [ ] in the presence of biguanide concentrations equivalent to IC 50 for NADH:decy...; The effects of biguanides on ATP hydrolysis by F 1 F O -ATPase were tested in SMPs by using a coupled assay to detect the formation of ADP [, ]. (A) shows that all five biguani...
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All assays were performed at 32°C in 10 mM Tris/HCl (pH 7.4) and 250 mM sucrose. NADH oxidation was measured in 100 µM NADH, and succinate oxidation in 10 mM succinate, using a coupled assay system [ ]. Complex II activity was measured in 10 mM succinate and 100 µM decylubiquinone using membranes solubilized in 1% dodecylmaltoside to isolate the activity. Complex II + III activity in membranes was measured by the reduction of cytochrome c (ε 550-541 =18.00 mM -1 ·cm -1 ) with 120 µM oxidized horse heart cytochrome c, 10 mM succinate and 1 mM NaCN to inhibit complex IV. Complex IV activity was measured by the oxidation of 120 µM reduced horse heart cytochrome c. ATP hydrolysis was measured using a coupled assay system [, ], using 40 µg/ml pyruvate kinase, 200 µM phosphoenol pyruvate and 50 µg/ml lactate dehydrogenase, with 4 mM MgSO 4, 2 mM K 2 SO 4, 4 µM rotenone and 5 µg/ml gramicidin, by monitoring the oxidation of 200 µM NADH (rotenone and gramicidin were omitted for purified enzyme measurements)...
All assays were performed at 32°C in 20 mM Tris/HCl (pH 7.2). NADH:decylubiquinone oxidoreduction was measured using 200 µM NADH and 200 µM decylubiquinone, in 0.075% soya bean asolectin (Avanti Polar Lipids) and 0.075% CHAPS (Merck Chemicals) and quantified by the absorbance of NADH (ε 340-380 =4.81 mM -1 ·cm -1 ) [ ]. Catalysis was initiated by the addition of NADH, following a 2 min pre-incubation, and rates measured as the linear regression of the maximal rate (discarding any initial lag phases). Biguanides were added immediately before NADH, unless otherwise stated, and the level of inhibition did not depend on the length of pre-incubation. Initial rates for the NADH:FeCN (ferricyanide), NADH:HAR [hexaammineruthenium(III)] and NADH:paraquat reactions were measured in 100 µM NADH with 1 mM FeCN (ε 420-500 =1 mM -1 ·cm -1 ), 3.5 mM HAR or 200 µM paraquat (ε 340-380 =4.81 mM -1 ·cm -1 ) [, ]. H 2 O 2 formation was followed in 30 µM NADH as the catalase-sensitive horseradish peroxidase-dependent oxidation of 10...
EPR samples of ~11 mg/ml bovine complex I were prepared anaerobically. The samples were incubated for 15 min at 4°C with 100 mM NaCl, 100 mM metformin, 2.5 mM phenformin, or 0.35 mM proguanil, then 5 mM NADH was added and the samples frozen immediately. Spectra were recorded with 1 mW microwave power, microwave frequency 9.36-9.38 GHz, modulation frequency 100 kHz, modulation amplitude 1 mT, time constant 81.92 ms and conversion time 20.48 ms at 12 K, using a Bruker EMX X-band spectrometer with an ER 4119HS high-sensitivity cavity, maintained at low temperature by an Oxford Instruments ESR900 continuous-flow liquid helium cryostat.
Cells [143B (cell line 8303 from the A.T.C.C.) and Hep G2 (cell line 85011430 from The Health Protection Agency)] were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO 2. Per well, 3 × 10 4 or 1.5 × 10 4 cells were plated into XF24 or XF96 (Seahorse Bioscience) plates respectively, and incubated for 12 h at 37°C in 5% CO 2. The medium was exchanged for assay buffer containing DMEM, 4.5 g/l glucose, 1 mM pyruvate, 32 mM NaCl, 2 mM GlutaMAX, 15 mg/l Phenol Red and 20 mM Hepes (pH 7.4) and the cells placed in a CO 2 -free incubator at 37°C for 60 min. OCRs (oxygen consumption rates) and ECARs (extracellular acidification rates) were measured in a Seahorse extracellular flux analyser; 200 nM rotenone was used to inhibit complex I where stated. To calculate the normalized rotenone-sensitive OCR rates, the rotenone-insensitive rates (determined at the end of the experiment) were subtracted and the traces normalized to 100% before biguanide addition. ECAR data were normalize...
Experimental values are reported as means±S.E.M. for small sample sizes or as means±S.D. for large sample sizes with only technical error sources. Typically three to four replicates gave S.E.M. values of less than 5% of the mean value; numbers of replicates were increased when appropriate. Comparisons between samples are marked * P <0.05, ** P <0.01 and **** P <0.0001 as determined using an unpaired two-tailed Students t test. Differences between measurements with P >0.05 were considered not significant. IC 50 values were determined using the standard dose-effect relationship {activity (%)=100 × IC 50 /(IC 50 +[inhibitor] m ); [ ] with the Hill Slope (m) set to unity for complex I} and are reported with 95% confidence intervals.
To narrow down the location at which biguanides bind to complex I, we tested their effects on different steps in the catalytic cycle: NADH oxidation by the flavin mononucleotide, intramolecular electron transfer along the chain of FeS clusters, and ubiquinone reduction [ ]. First, metformin was found to stimulate (see below and ), not inhibit, the NADH:FeCN oxidoreduction reaction. The NADH:FeCN reaction is widely used to test the rate of NADH oxidation by complex I, because the whole reaction is localized exclusively at the flavin site [ ]. Furthermore, even extremely high concentrations of metformin only affected the thermal stability of the flavin site slightly, showing that the flavin environment is not significantly altered (the thermal stability is measured as a melting temperature that varies from 56.3±0.4 in 200 mM NaCl to 55.3±0.1 in 200 mM metformin). We conclude that the biguanides do not inhibit complex I by inhibiting NADH oxidation. Secondly, (C) shows that biguanides do not affect the 'fingerprint' EPR spectra of the FeS clusters of NADH-reduced complex I [ ], suggesting they do not inhibit intramolecular electron transfer. Thirdly,...
Complex I catalyses electron transfer from NADH to hydrophilic electron acceptors by two distinct mechanisms, both localized uniquely at the flavin site. The NADH:FeCN and NADH:O 2 reactions proceed via two sequential steps in which NADH reduces the flavin and dissociates, and then the electron acceptor reacts directly (see E, left-hand reaction) [ ]; for the NADH:HAR and NADH:paraquat reactions NADH reduces the flavin, but the electron acceptor reacts only if a nucleotide is bound in the reduced flavin site (see E, right-hand reaction) [ ]. Note that although NADH:O 2 oxidoreduction produces superoxide, we detect the H 2 O 2 formed following superoxide dismutation, and thus refer to 'H 2 O 2 production'. Data showing the effects of biguanides on these different reactions are shown in. (A) shows that metformin stimulates the rates of the two reactions (the NADH:FeCN and NADH:O 2 reactions) that rely on oxidation of the nucleotide-free reduced flavin [, ], but, conversely, it inhibits the rates of two reactions [the NADH:HAR and NADH:paraquat reactions] that rely on oxidation of the nucleotide-bound reduced flavin [ ]. Dose-response stimulatory effects were al...
( A ) Inhibition of ATP hydrolysis by SMPs (colours as in B ). Data points are means±S.E.M., n =3-5, and IC 50 values are indicated in mM. ( B ) Relationship between the IC 50 values for ATP hydrolysis and the log D values. IC 50 values are in mM with 95% confidence intervals; log D values are means±S.E.M., n =3. Linear fit with R 2 =0.948. ( C ) Relative inhibition of ATP production in SMPs in the presence of piericidin A (○) or metformin (●). ATP synthesis was driven using 200 µM NADH; NADH oxidation was measured spectroscopically, and the concentration of ATP determined after 3.5 min (during this time a linear rate is observed). NADH oxidation rates were adjusted using 0-50 nM piericidin A or 0-250 mM metformin, with the ionic strength kept constant at 250 mM using NaCl. ( D - F ) Dose-dependent effects of biguanides on succinate-driven ATP production. Data points are means±S.E.M., n =3, as a percentage of ATP production in the absence of biguanide. ATP concentrations were determined after 3.5 min (○) then corrected for the rate of succinate oxidation, detected spectroscopically using...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria methods",
"description": "Evidence-backed execution summary for Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria methods from Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria.",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Kinetic measurements on bovine mitochondrial membranes and SMPs",
"text": "All assays were performed at 32°C in 10 mM Tris/HCl (pH 7.4) and 250 mM sucrose. NADH oxidation was measured in 100 µM NADH, and succinate oxidation in 10 mM succinate, using a coupled assay system [ ]. Complex II activity was measured in 10 mM succinate and 100 µM decylubiquinone using membranes solubilized in 1% dodecylmaltoside to isolate the activity. Complex II + III activity in membranes was measured by the reduction of cytochrome c (ε 550-541 =18.00 mM -1 ·cm -1 ) with 120 µM oxidized horse heart cytochrome c, 10 mM succinate and 1 mM NaCN to inhibit complex IV. Complex IV activity was measured by the oxidation of 120 µM reduced horse heart cytochrome c. ATP hydrolysis was measured using a coupled assay system [, ], using 40 µg/ml pyruvate kinase, 200 &#..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Kinetic measurements on isolated complex I",
"text": "All assays were performed at 32°C in 20 mM Tris/HCl (pH 7.2). NADH:decylubiquinone oxidoreduction was measured using 200 µM NADH and 200 µM decylubiquinone, in 0.075% soya bean asolectin (Avanti Polar Lipids) and 0.075% CHAPS (Merck Chemicals) and quantified by the absorbance of NADH (ε 340-380 =4.81 mM -1 ·cm -1 ) [ ]. Catalysis was initiated by the addition of NADH, following a 2 min pre-incubation, and rates measured as the linear regression of the maximal rate (discarding any initial lag phases). Biguanides were added immediately before NADH, unless otherwise stated, and the level of inhibition did not depend on the length of pre-incubation. Initial rates for the NADH:FeCN (ferricyanide), NADH:HAR [hexaammineruthenium(III)] and NADH:paraquat reactions were measured in 100 µM NADH with 1 mM FeCN (..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "EPR spectroscopy",
"text": "EPR samples of ~11 mg/ml bovine complex I were prepared anaerobically. The samples were incubated for 15 min at 4°C with 100 mM NaCl, 100 mM metformin, 2.5 mM phenformin, or 0.35 mM proguanil, then 5 mM NADH was added and the samples frozen immediately. Spectra were recorded with 1 mW microwave power, microwave frequency 9.36-9.38 GHz, modulation frequency 100 kHz, modulation amplitude 1 mT, time constant 81.92 ms and conversion time 20.48 ms at 12 K, using a Bruker EMX X-band spectrometer with an ER 4119HS high-sensitivity cavity, maintained at low temperature by an Oxford Instruments ESR900 continuous-flow liquid helium cryostat."
},
{
"@type": "HowToStep",
"position": 4,
"name": "OCR and ECAR measurements on cultured human cells and isolated mitochondria",
"text": "Cells [143B (cell line 8303 from the A.T.C.C.) and Hep G2 (cell line 85011430 from The Health Protection Agency)] were grown on DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Thermo Fisher Scientific) and 100 units/ml penicillin and 100 µg/ml streptomycin at 37°C in 5% CO 2. Per well, 3 × 10 4 or 1.5 × 10 4 cells were plated into XF24 or XF96 (Seahorse Bioscience) plates respectively, and incubated for 12 h at 37°C in 5% CO 2. The medium was exchanged for assay buffer containing DMEM, 4.5 g/l glucose, 1 mM pyruvate, 32 mM NaCl, 2 mM GlutaMAX, 15 mg/l Phenol Red and 20 mM Hepes (pH 7.4) and the cells placed in a CO 2 -free incubator at 37°C for 60 min. OCRs (oxygen consumption rates) and ECARs (extracellular acidification rates) were measured in a Seahorse extracellular flux analyse..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Statistical methods",
"text": "Experimental values are reported as means±S.E.M. for small sample sizes or as means±S.D. for large sample sizes with only technical error sources. Typically three to four replicates gave S.E.M. values of less than 5% of the mean value; numbers of replicates were increased when appropriate. Comparisons between samples are marked * P <0.05, ** P <0.01 and **** P <0.0001 as determined using an unpaired two-tailed Students t test. Differences between measurements with P >0.05 were considered not significant. IC 50 values were determined using the standard dose-effect relationship {activity (%)=100 × IC 50 /(IC 50 +[inhibitor] m ); [ ] with the Hill Slope (m) set to unity for complex I} and are reported with 95% confidence intervals."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Biguanides inhibit ubiquinone reduction, but not competitively",
"text": "To narrow down the location at which biguanides bind to complex I, we tested their effects on different steps in the catalytic cycle: NADH oxidation by the flavin mononucleotide, intramolecular electron transfer along the chain of FeS clusters, and ubiquinone reduction [ ]. First, metformin was found to stimulate (see below and ), not inhibit, the NADH:FeCN oxidoreduction reaction. The NADH:FeCN reaction is widely used to test the rate of NADH oxidation by complex I, because the whole reaction is localized exclusively at the flavin site [ ]. Furthermore, even extremely high concentrations of metformin only affected the thermal stability of the flavin site slightly, showing that the flavin environment is not significantly altered (the thermal stability is measured as a melting temperature that varies from 56.3±0.4 in 200 mM NaCl to 55.3±0.1 in 200 mM metformin). We..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Biguanide effects on the flavin site and H 2 O 2 production",
"text": "Complex I catalyses electron transfer from NADH to hydrophilic electron acceptors by two distinct mechanisms, both localized uniquely at the flavin site. The NADH:FeCN and NADH:O 2 reactions proceed via two sequential steps in which NADH reduces the flavin and dissociates, and then the electron acceptor reacts directly (see E, left-hand reaction) [ ]; for the NADH:HAR and NADH:paraquat reactions NADH reduces the flavin, but the electron acceptor reacts only if a nucleotide is bound in the reduced flavin site (see E, right-hand reaction) [ ]. Note that although NADH:O 2 oxidoreduction produces superoxide, we detect the H 2 O 2 formed following superoxide dismutation, and thus refer to 'H 2 O 2 production'. Data showing the effects of biguanides on these different reactions are shown in. (A) shows that metformin stimulates the rates of the two reactions (the NADH:FeCN and N..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Effects of biguanides on F 1 F 0 -ATPase",
"text": "( A ) Inhibition of ATP hydrolysis by SMPs (colours as in B ). Data points are means±S.E.M., n =3-5, and IC 50 values are indicated in mM. ( B ) Relationship between the IC 50 values for ATP hydrolysis and the log D values. IC 50 values are in mM with 95% confidence intervals; log D values are means±S.E.M., n =3. Linear fit with R 2 =0.948. ( C ) Relative inhibition of ATP production in SMPs in the presence of piericidin A (○) or metformin (●). ATP synthesis was driven using 200 µM NADH; NADH oxidation was measured spectroscopically, and the concentration of ATP determined after 3.5 min (during this time a linear rate is observed). NADH oxidation rates were adjusted using 0-50 nM piericidin A or 0-250 mM metformin, with the ionic strength kept constant at 250 mM using NaCl. ( D - F ) Dose-dependent effects of..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Kinetic measurements on bovine mitochondrial membranes and SMPs"
},
{
"@type": "HowToTool",
"name": "OCR and ECAR measurements on cultured human cells and isolated mitochondria"
},
{
"@type": "HowToTool",
"name": "Analytical methods"
},
{
"@type": "HowToTool",
"name": "Effects of biguanides on F 1 F 0 -ATPase"
},
{
"@type": "HowToTool",
"name": "Selective cellular and mitochondrial uptake of biguanides"
},
{
"@type": "HowToTool",
"name": "Biguanides accumulate reversibly in mitochondria according to the membrane potential"
},
{
"@type": "HowToTool",
"name": "Mechanisms by which biguanides may protect against ischaemia/reperfusion injury"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Kinetic measurements on bovine mitochondrial membranes and SMPs"
},
{
"@type": "HowToSupply",
"name": "OCR and ECAR measurements on cultured human cells and isolated mitochondria"
},
{
"@type": "HowToSupply",
"name": "Statistical methods"
},
{
"@type": "HowToSupply",
"name": "Biguanides inhibit ubiquinone reduction, but not competitively"
},
{
"@type": "HowToSupply",
"name": "Biguanides inhibit ubiquinone reduction, but not competitively"
},
{
"@type": "HowToSupply",
"name": "Metformin interacts with 'deactive-like' conformations of complex I"
},
{
"@type": "HowToSupply",
"name": "Biguanide effects on the flavin site and H 2 O 2 production"
},
{
"@type": "HowToSupply",
"name": "Biguanide interactions with respiratory complexes II, III and IV"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria",
"datePublished": "2014",
"author": [
{
"@type": "Person",
"name": "Hannah R. Bridges"
},
{
"@type": "Person",
"name": "Andrew J. Y. Jones"
},
{
"@type": "Person",
"name": "Michael N. Pollak"
},
{
"@type": "Person",
"name": "Judy Hirst"
}
],
"identifier": "10.1042/bj20140620"
}
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"name": "Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria methods",
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