Experimental strategy
To delineate the effects that determine whether biguanides are able to cross the mitochondrial inner membrane we focused on phenformin and proguanil. They have similar partition coefficients [4], so their passive transport across the membrane should be comparable; however, they are taken up into the mitochondrial matrix with very different rates and to very different extents. Both compounds are effective inhibitors of isolated complex I, but only phenformin inhibits complex I in mitochondria [4]. The molecular structure of proguanil differs from that of phenformin in four ways (Fig. 1a): its phenyl ring is chlorinated; it has no linker between the phenyl ring and the biguanide moiety (phenformin has a two-carbon linker); its biguanide moiety is bis-substituted (phenformin is mono-substituted); and it contains an isopropyl substitution. Five biguanides were designed to test each difference (Fig. 1a): 2-[2-(4-chlorophenyl)ethyl]-1-(diaminomethylidene)guanidine) (chlorophenformin, compound 1) tests the effect of adding a chlorine to the phenyl ring; phenyl biguanide (compound 2) and 2-benzyl-1-(diaminomethylidene)guanidine (benzyl biguanide, compound 3) test the effect of the linker between the phenyl ring and the biguanide moiety; 1-(diaminomethylidene)-2-(propan-2-yl)guanidine (isopropyl biguanide, compound 4) tests the effect of the isopropyl derivative; and N-(propan-2-yl)-1-3-(propan-2-yl)carbamimidamidomethanimidamide (bis-isopropyl biguanide, compound 5) tests the effect of bis-substituting the biguanide functionality.
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 [4]. 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 [30], 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
All the compounds studied inhibit isolated complex I, so inhibition of oxygen consumption by the respiratory chain can be exploited as a marker for their presence and concentration in the mitochondrial matrix, the compartment containing the complex I biguanide-binding site. Notably, this approach to evaluating the matrix biguanide concentration avoids the need for radioactive biguanides, estimation of matrix volume, or lengthy isolation protocols. To account for the wide range of complex I IC50 values observed across the compound series, concentrations relative to the individual IC50 values were used. Biguanides were added to cells at one-tenth of their IC50 concentrations, and the rotenone-sensitive OCRs monitored for 6 h. At IC50/10 a biguanide that does not enter mitochondria does not inhibit respiration, whereas a biguanide that can enter the matrix accumulates based on its intrinsic positive charge and the membrane potential and so progressively inhibits respiration. Experiments with human osteosarcoma 143B (Fig. 2a) and hepatocarcinoma HepG2 cells (Fig. 2b) revealed that compounds 1, 3, and 4 inhibit respiration (like phenformin they enter mitochondria), but compounds 2 and 5 do not (like proguanil they do not enter mitochondria). The same pattern was observed in experiments using a kidney cell line from Bos taurus (MDBK) (Fig. 2c) although the drugs were much slower to take effect. Thus, after 6 h of exposure, 143B and HepG2 cells were inhibited to a similar level, and MDBK cells were less inhibited (Fig. 2d and Additional file 1).
The data on intact cells suggest the presence of a selectivity barrier at either the plasma membrane and/or the mitochondrial inner membrane. Therefore, the compounds were tested at concentrations of IC50/5 on MDBK cells in which the plasma membrane had been permeabilized (Fig. 3a, b), and on isolated mitochondria from mouse heart, mouse liver, and rat liver (Fig. 3c, d). The same pattern of inhibition was observed in all cases, showing that transport across the mitochondrial inner membrane is selective. Even much higher (IC50) concentrations of compounds 2, 5, and proguanil elicited only limited inhibition of the OCR in permeabilized 143B cells (Fig. 3b and Additional file 1). Furthermore, much less inhibition was observed in an equivalent experiment using mouse heart mitochondria in which ADP was added at the same time as phenformin [oxygen consumption was 85 ± 10 % of the control value (n = 8 with phenformin and n = 6 for control, see Additional file 2), compared to 30 % in the absence of ADP (see Fig. 3d and Additional file 1)], confirming that biguanide accumulation into mitochondria depends on the membrane potential [12]. Finally, the inhibition of the OCR observed in intact mitochondria respiring on glutamate and malate confirms that it is due to inhibition of complex I, not mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) [31]. mGPD has also been proposed as a target for biguanides [31] and could contribute to the inhibition of the OCR in intact cells because it couples oxidation of the NADH produced by glycolysis in the cytoplasm to reduction of O2 by complex IV. However, the malate/aspartate shuttle also performs this function, and mGPD does not contribute to oxygen consumption by isolated mitochondria because they lack any connection with cytoplasmic processes.
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 [4] when the respiratory chain is inhibited.
In order to evaluate how the matrix biguanide concentration varies in intact cells, inhibition of the OCR was exploited as a real-time marker by comparison with the IC50 data from complex I in membranes (Fig. 4a). Note that we are unable to include the effects of the decreased membrane potential (which acts to increase the OCR, opposing the inhibition) in our calculation, so the calculated values are underestimates of the true matrix concentration. Figure 4a shows how the estimated matrix phenformin concentration evolves over time, with maximal rates of accumulation of 136, 85, and 3.2 μM min−1 for HepG2, 143B, and MDBK cells, respectively. Figure 4b compares rates of uptake of each compound into each cell line, confirming that the weak inhibition in MDBK cells (Fig. 2c) is due to slow biguanide uptake. For the four biguanides (phenformin and compounds 1, 3, and 4) that are taken up into mitochondria, the estimated rates range from >1.5 mM min−1 for compound 4 into 143B and HepG2 cells (3 mM in the extracellular medium) to 1 μM min−1 for compound 1 into MDBK cells (37 μM in the extracellular medium); because the biguanides are present in the medium at different absolute concentrations, the rates are not directly comparable. Figure 4c compares the extent of accumulation after 30 min of biguanide exposure for intact and permeabilized MDBK cells, calculated in the same way (see also Additional file 1). On this timescale the intramitochondrial concentrations in intact cells hardly reach the concentration in the extracellular medium, whereas in permeabilized cells the compounds accumulate significantly into mitochondria, with compound 3 accumulating more than 200-fold. Therefore, the slow uptake of the mitochondria-permeant biguanides observed in MDBK cells is due to slow transport across the plasma membrane.
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 [33], 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 [34]. 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 [16]. 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
The anti-hyperglycemic and anti-proliferative activities of biguanides have been linked to the activation of AMPK [7, 8] and so the ability of each biguanide to activate AMPK was investigated in MDBK cells. Figure 5 shows the levels of activation of AMPK (and its downstream effector acetyl-coA carboxylase, ACC) achieved following 18-h treatments with IC50/10 biguanide concentrations. The antibodies to AMPK-α and ACC detect the phosphorylated forms (with the detection of AMPK-β as a control) and AMPK activation was observed only for those biguanides that inhibit mitochondrial respiration. Indeed, the slightly lower activation observed for phenformin and compound 1 is consistent with their less efficient inhibition in this cell line (Fig. 2c).
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 [31], 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 [12]; they may be mediated by mTORC1 in either an AMPK-dependent or independent manner [3, 11].