Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase
© Bridges et al. 2016
Received: 27 May 2016
Accepted: 22 July 2016
Published: 9 August 2016
The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known.
Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments.
Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.
KeywordsNADH:ubiquinone oxidoreductase Respiratory complex I Metformin Biguanide AMP kinase
Biguanides are commonly prescribed drugs for the treatment of type II diabetes and to prevent malaria, and are under investigation for their uses in cardiovascular disease and cancer [1–3]. Although their modes of action in all of these applications are still debated, biguanides with known anti-hyperglycemic properties (metformin, phenformin, and buformin) have consistently been observed to inhibit mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase) at millimolar and high micromolar concentrations [4–6]. Although these inhibitory concentrations appear high, they are physiologically relevant because biguanides are positively charged molecules and are thus concentrated inside mitochondria by the mitochondrial membrane potential. Inhibition of complex I leads to the activation of AMP-activated protein kinase (AMPK), which is thought to contribute to biguanide anti-hyperglycemic activity [7, 8]. Furthermore, biguanides have been found to inhibit the proliferation of cancer cell lines [9, 10] and their mechanisms of action (through mTORC1 in either an AMPK-dependent or independent manner [3, 11]) are suggested to rely on the inhibition of respiratory complex I; overexpression of the yeast alternative NADH:ubiquinone oxidoreductase NDI1 was found to ablate the effect . Conversely, although the antimalarial biguanide cycloguanil and its pro-drug proguanil inhibit isolated complex I, they do not inhibit cellular or mitochondrial respiration  and so, unlike the anti-hyperglycemic biguanides, they are not associated with lactic acidosis, a side effect of complex I inhibition. Biguanides thus exhibit a wide range of physiological effects, and it is important to understand the factors that determine their anti-hyperglycemic, anti-proliferative, and anti-malarial effects, and the interplay between these desirable effects and the undesirable side effect of lactic acidosis.
For biguanides to inhibit mitochondrial respiration they must cross both the plasma and mitochondrial inner membrane to reach their binding site on complex I. Metformin transport across the plasma membrane is known to involve organic cation transporters (OCT1–3) and multidrug and toxin extrusion proteins (MATE1–2) . The human thiamine transporter (THTR-2) , the serotonin transporter (SERT), and the plasma membrane monoamine transporter (PMAT) have also been implicated in metformin transport . Biguanide uptake is tissue specific due to varying levels of transporter expression [16, 17] and proximity to the hepatic portal vein, owing to the first-pass effect . In contrast to the cellular-level understanding that has been reached about metformin uptake by the intestines and liver, and efflux by the liver and kidneys , very little is known about how, once they have entered cells, biguanides enter mitochondria.
There is no consensus yet on whether biguanides that are more hydrophobic than metformin traverse biological membranes by passive diffusion or require protein transporters. Biguanides have high pK a values, so they are positively charged at neutral pH, and low partition coefficients, suggesting they are only poorly lipophilic . Even so, many researchers have considered phenformin able to cross biological membranes without requiring active transport [6, 19, 20]. One study concluded that OCT1 mediates the uptake of metformin but not phenformin by rat hepatoma cells , because phenformin uptake was not observed to be impaired by the OCT1 inhibitor quinidine. Conversely, the organic cation/carnitine transporter 1 (OCTN1) in the mitochondrial inner membrane  was found to contribute to phenformin uptake . Irrespective of the mode of transport, the very different levels of uptake of different biguanides with comparable lipophilicity (such as phenformin and proguanil)  argues for their selective transport across the mitochondrial inner membrane.
Here, we aim to determine why some biguanides are able to enter mitochondria and inhibit respiration whereas others (of comparable or greater hydrophobicity) are not, and to determine whether uptake into the mitochondrial matrix is a prerequisite for biguanide-mediated activation of AMPK.
Phenformin, phenyl biguanide (PubChem CID: 5932) (Sigma-Aldrich Ltd.), isopropyl biguanide (PubChem CID: 9570185), chlorophenformin (PubChem CID: 67587204) (AKOS GmbH), and benzyl biguanide (PubChem CID: 9570091) (Angene International Ltd.) were added from aqueous stock solutions, and proguanil (Sigma-Aldrich Ltd.) and bis-isopropyl biguanide (PubChem CID: 23437065) (European Directorate for the Quality of Medicines and Healthcare) from stock solutions in DMSO.
Preparation of proteins, membranes, and mitochondria
Complex I and mitochondrial membranes were prepared from Bos taurus (bovine) heart . Intact mitochondria were isolated from rat liver  and mouse heart and liver . Membranes were prepared from mouse heart mitochondria by 5 s bursts of sonication at 4 °C and collected by centrifugation (75,000 × g, 1 h).
Kinetic measurements on complex I and mitochondrial membranes
Assays were performed at 32 °C in a SpectraMax 96-well plate reader. NADH:decylubiquinone oxidoreduction by complex I at 0.5 μg mL−1 was measured in 20 mM Tris-HCl (pH 7.2), 0.15 % soy bean asolectin (Avanti Polar Lipids), and 0.15 % 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate (CHAPS, Merck Chemicals Ltd), with 200 μM decylubiquinone and 200 μM NADH, and monitored using ε340–380(NADH) = 4.81 mM−1 cm−1 . Catalysis was initiated by addition of NADH and maximal rates determined by linear regression. NADH:O2 oxidoreduction by membranes was measured similarly but using 5 μg mL−1 membranes in 10 mM Tris-HCl (pH 7.4) and 250 mM sucrose using 200 μM NADH and supplemented with 0.15 mM horse heart cytochrome c (Sigma-Aldrich Ltd.). Succinate:O2 oxidoreduction was measured using 40 μg mL−1 membranes in 5 mM succinate in 10 mM Tris-HCl (pH 7.4) as described previously . Control experiments included NaCl (to match the ionic strength) or DMSO, as appropriate.
143B (CRL-8303 from ATCC), HepG2 (85011430 from The Health Protection Agency), and MDBK (CCL-22 from ATCC) cells were grown on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS, Thermo Fisher Scientific) at 37 °C in 5 % CO2. All cells were confirmed as negative for mycoplasma.
Oxygen consumption rate measurements on intact and permeabilized cells
Oxygen consumption rates (OCRs) were measured using a Seahorse XF96 extracellular flux analyzer at 37 °C. For intact cell measurements, 1.4 × 104 143B, HepG2, or MDBK cells were plated (per well) in DMEM containing 10 % FBS into Seahorse Bioscience Inc. XF96 plates and incubated for ~12 h at 37 °C in 5 % CO2. Then, the medium was exchanged for assay buffer containing DMEM, 4.5 g L−1 glucose, 1 mM pyruvate, 32 mM NaCl, 2 mM GlutaMAX, 15 mg L−1 phenol red, and 20 mM HEPES (pH 7.4 at 37 °C) and the cells placed in a CO2-free incubator at 37 °C for 30 min. Basal oxygen consumption rates were established before the addition of biguanides at one-tenth of their IC50 (IC50/10), and followed for ~ 6 h before the addition of rotenone (2 μM).
For permeabilized cell experiments, cells were seeded into XF96 plates at 0.9–1.1 × 104 per well and incubated for 48 h at 37 °C in 5 % CO2. Then, the growth medium was exchanged for assay buffer containing 3 nM plasma membrane permeabilizer ‘PMP’ (Seahorse Biosciences Inc.), 10 mM glutamate, 10 mM malate, 220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 0.2 % fatty acid-free bovine serum albumin (BSA), and 2 mM HEPES (pH 7.4 at 37 °C). The permeabilized cells were incubated with 10 mM glutamate and 10 mM malate for 25 min prior to the addition of biguanides at IC50/5 or IC50. The biguanides were allowed to accumulate for 30 min, then respiration was uncoupled by the addition of 4 mM ADP. Rotenone was added (2 μM) at the end of the experiment.
Rotenone-sensitive OCRs were calculated by subtracting the rotenone-insensitive rates (determined at the end of the experiment). OCRs were normalized by dividing by the OCR recorded immediately prior to the addition of biguanide. For rate of uptake calculations, the OCRs at time points after biguanide addition were divided by the OCRs at the same time points in control experiments. Data from wells with failed port injections were excluded from the analyses. The respiratory control ratio (RCR) values (state III versus state IV respiration) were ~11 and ~6 for permeabilized MDBK and 143B cells, respectively.
OCR measurements on isolated mitochondria
Isolated mitochondria were monitored using a Seahorse XF96 extracellular flux analyzer at 32 °C. Mouse heart mitochondria, and mouse and rat liver mitochondria were plated into XF96 plates at 4 and 16–20 μg per well, respectively, in 220 mM mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 0.2 % fatty acid-free BSA, and 2 mM HEPES (pH 7.4 at 37 °C) with 10 mM glutamate and 10 mM malate and adhered by centrifugation (2000 × g, 20 min, 4 °C). The medium was supplemented with a further 5 mM glutamate and 5 mM malate and the mitochondria incubated for 15 min. Then two baseline readings were taken before the biguanides were added at IC50/5. Biguanides were allowed to accumulate for 15–20 min before respiration was uncoupled by the addition of 4 mM ADP. Rotenone (2 μM) was added at the end of the experiment. Data from wells with failed port injections were excluded from the analyses. The RCR values (state III versus state IV respiration) were 3.4, 4.8, and 4.6 for mouse heart, rat liver, and mouse liver, respectively.
Membrane potential measurements
143B cells were seeded at 5 × 105 cells per 10 cm2 well in DMEM containing 10 % FBS. On the following day they were treated with 34 μM phenformin (IC50/10) for 6.5 h then washed once with phosphate-buffered saline (PBS) and detached using TrypLE (Thermo Fisher Scientific). The detached cells from each well were incubated in PBS containing 25 pg/mL tetramethylrhodamine, methyl ester (TMRM) and 20 μg/mL Hoescht stain for 20 min at 37 °C then washed twice and re-suspended in 200 μL PBS. TMRM fluorescence was detected using a NucleoCounter® NC 3000 cytometer with excitation at 530 nm and emission at 575 nm, with the Hoescht stain used to check for consistent cell density between samples.
Analysis of AMPK activation
MDBK cells were seeded at 1 × 106 cells per 6 cm dish in DMEM containing 10 % FBS. Cells were then serum starved in DMEM containing 25 mM HEPES for 6 h before biguanides were added at IC50/10. After 18 h cells were harvested by a rapid lysis procedure : cells were rinsed with PBS, then lysed using ice-cold buffer containing 50 mM HEPES, 1 mM EDTA, 10 % glycerol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 % Triton-X100, 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail (Roche). The cell lysate was centrifuged at 14,000 × g for 20 min at 4 °C and the protein concentration of the supernatant determined by bicinchoninic acid assay. The lysate was analyzed using Novex 3–12 % bis-Tris gels run in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (20 μg of protein per lane) followed by transfer to low fluorescence polyvinylidene fluoride membranes by wet transfer. Primary rabbit antibodies for AMPK-β (57C12), AMPK-α (phospho-Thr172) (40H9), and acetyl-coA carboxylase (phospho-Ser79) (Cell Signaling Technology) were incubated overnight with the membrane (1:1000 dilution) in Odyssey blocking buffer at 4 °C. A fluorescent secondary antibody was used (IRDYE800CW from LI-COR Biosciences, 1:20,000 dilution) with a LI-COR Odyssey imaging system.
Octanol/PBS partition coefficients were measured by the shake-flask method  at 32 °C, pH 7.4.
Experimental values are reported as mean averages ± standard deviation (SD) of technical replicates for large sample sizes and mean averages ± standard error of the mean (SEM) of technical replicates for small sample sizes. Data were analyzed by the unpaired two-tailed Student’s t test. IC50 values were determined using the standard dose-effect relationship (activity (%) = 100 × IC50 / (IC50 + [inhibitor] m )  with the Hill Slope (m) set to unity for purified complex I) and are reported with 95 % confidence intervals.
Results and discussion
Biguanide effects on isolated complex I and mitochondrial membranes
All seven compounds described were effective inhibitors of NADH:decylubiquinone oxidoreduction by isolated bovine complex I, as well as of NADH:O2 oxidoreduction (catalysis by complexes I-III-IV) by bovine and mouse heart mitochondrial membranes (Fig. 1b and Additional file 1), and the IC50 values for each biological system were comparable. Subsequent assays on human cell lines were based on the IC50 values from bovine heart membranes, and assays on mouse mitochondria on the IC50 values from mouse heart membranes. Assays of succinate:O2 oxidoreduction (catalysis by complexes II-III-IV) by bovine membranes, using concentrations equivalent to the IC50 values for NADH:O2 oxidoreduction, confirmed the inhibition is specific to complex I (Fig. 1c and Additional file 1). Finally, partition coefficients that describe compound hydrophobicity were measured for each compound at pH 7.4 (at which pH all the biguanides are positively charged). A correlation between inhibitory efficacy and hydrophobicity was observed (Fig. 1d and Additional file 1) that extends and confirms that observed previously with a smaller number of compounds . Improved transmembrane diffusion as a result of their greater hydrophobicity has previously been suggested as a reason why phenformin and buformin are more potent inhibitors of cellular respiration than metformin , but our data explain that this correlation is observed because the more hydrophobic compounds are better complex I inhibitors.
Biguanide effects on oxygen consumption by cells and mitochondria
The molecular determinants of biguanide uptake
Our data clearly delineate compounds 1, 3, and 4 that can cross both the plasma and mitochondrial inner membrane (like phenformin) from compounds 2 and 5 that cannot (like proguanil). Thus, conjugation of a phenyl ring directly to the biguanide moiety (compound 2) and bis-substitution of the biguanide (compound 5) prevent biguanides from accessing the matrix and inhibiting respiration; both features are present in proguanil. A chloro-group on the ring (compound 1) and the iso-propyl group itself (compound 4), which are also present in proguanil, do not prevent inhibition. The inhibition observed previously from alkyl biguanides, including the anti-diabetic compounds metformin and buformin [4, 32], is fully consistent with our results.
Rate and extent of biguanide uptake
Biguanide uptake into mitochondria depends on the mitochondrial membrane potential. However, once inside mitochondria, biguanides inhibit complex I and thus may decrease the membrane potential, resulting in a negative feedback loop that will eventually balance the two effects. To evaluate the effects of phenformin on membrane potential, 143B cells were treated with phenformin at IC50/10 to match the experiment in Fig. 2a, and the membrane potential was evaluated using the fluorescence of TMRM. After 6.5 h, the TMRM fluorescence was 22.4 ± 2.9 % (n = 3) of the control value, compared to 13.43 ± 1.7 % (n = 3) in cells treated with 1 μM of the uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (Additional file 3). Although the TMRM fluorescence is not a quantitative measure, it is clear that phenformin treatment induces a decrease in the membrane potential. We previously found that, in addition to their effects on complex I, biguanides also directly inhibit ATP hydrolysis by F1Fo ATP synthase, hindering the use of ATP hydrolysis to support the membrane potential  when the respiratory chain is inhibited.
Is biguanide transport protein mediated or determined by membrane solubility?
The transport of metformin across the plasma membrane by OCTs is widely accepted, but more hydrophobic biguanides like phenformin have been widely considered to cross biological membranes by passive transport [6, 19, 20]. The most hydrophobic biguanide tested here, proguanil, is known to cross the plasma membrane into liver cells because it is converted to cycloguanil by P450s , but it does not reach the mitochondrial matrix to inhibit complex I. Here, we have shown that selective transport across the inner membrane both prevents proguanil (and compounds 2 and 5) from accumulating in mitochondria, and facilitates the rapid uptake of phenformin (and compounds 1, 3, and 4). This selectivity strongly suggests that transport is protein mediated. By extension, the different uptake rates between cell lines can be ascribed to different expression levels of transporter proteins, particularly in the plasma membrane. Here, we observed the slowest uptake in bovine kidney cells, and it has been observed that biguanide uptake into human HEK293 kidney cells is very slow unless OCTs are overexpressed . Hepatic cells (like HepG2) are known to have high OCT1 expression levels for biguanide uptake and, conversely, kidney cells have high MATE1 expression levels for biguanide efflux . Although the simplest explanation for our data is selective biguanide uptake into mitochondria, the influence of highly active and selective efflux processes cannot be excluded.
Activation of AMPK by biguanides
Our data indicate that access to the mitochondrial matrix is necessary for biguanide-mediated AMPK activation, so it is unlikely that an extra-mitochondrial enzyme is the primary target responsible for AMPK-related downstream effects of biguanide treatments. AMP deaminase in the cytoplasm and mGPD in the intermembrane space have both been proposed as primary extra-mitochondrial targets of metformin [31, 35]; our results suggest that the inhibition of neither of them is linked to the activation of AMPK. Consistent with this picture, acute metformin treatments were observed to inhibit mGPD and cause plasma glucose to drop, but not to activate AMPK , whereas long-term metformin treatments did activate AMPK. Whether the anti-hyperglycemic effects of metformin (and phenformin) are mediated by AMPK activation [7, 36, 37] or by alternative signaling pathways [2, 38] (or both) is thus still debated. The anti-proliferative effects of biguanides have been more closely linked to inhibition of complex I, as they were ablated by overexpression of the yeast alternative NADH:ubiquinone oxidoreductase NDI1 ; they may be mediated by mTORC1 in either an AMPK-dependent or independent manner [3, 11].
All the biguanides tested inhibit mitochondrial complex I, but only some biguanides inhibit mitochondrial respiration in vivo. Here, we have identified the specific molecular features that control the uptake of substituted biguanides into mitochondria and allow them to target complex I: direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. The high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only biguanides that enter mitochondria and inhibit complex I activate AMP kinase. Our results can be applied in the design of new complex I-targeted biguanides with improved anti-proliferative activity , and to assist in deconvoluting the targets and mechanisms of action of anti-hyperglycemic biguanides like metformin.
ACC, acetyl-coA carboxylase; AMPK, AMP-activated protein kinase; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; MATE, multidrug and toxin extrusion proteins; mGPD, mitochondrial glycerol-3-phosphate dehydrogenase; OCR, oxygen consumption rate; OCT, organic cation transporter; PBS, phosphate-buffered saline; RCR, respiratory control ratio; SD, standard deviation; SEM, standard error of the mean; SERT, serotonin transporter; TMRM, tetramethylrhodamine, methyl ester
We thank David Carling and Elizabeth Hinchy for advice on AMPK experiments and Gigi Lau for preparing rat liver mitochondria.
This research was funded by the Medical Research Council (grant number U105663141 to JH). VS was supported by the Amgen Scholars program.
HRB performed all research and data analyses except preliminary assays with directly conjugated biguanides, which were performed by VS; ANAA prepared mouse mitochondria and membranes; HRB and JH designed the research and wrote the paper; JH directed the project. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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