Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK–eNOS–NO pathway

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity.

Obesity prevalence has increased over the past decades and is now a major public health problem worldwide. It is associated with an enhanced risk of developing cardiometabolic diseases such as hypertension, insulin resistance, type 2 diabetes mellitus, coronary artery disease, myocardial infarction, heart failure, and stroke 1 . Obesity involves changes in body composition as a consequence of an energetic imbalance in which caloric intake is higher than energy expenditure 1 . The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is responsible for maintaining the energy balance after depletion of energy stores, switching off ATP-consuming anabolic pathways 2,3 . Endothelial AMPK plays a key role in the regulation of vascular function through the activation of the PI3K-Akt-endothelial nitric oxide synthase (eNOS) pathway and stimulation of nitric oxide (NO) release in endothelial cells [4][5][6] . Obesity leads to AMPK dysregulation and endothelial dysfunction, which is the first step in the progression of cardiovascular disease 7 . We have shown that caloric restriction in young Zucker fa/fa rats has cardiovascular benefits by reducing endothelial dysfunction through AMPK-PI3K-Akt-eNOS activation associated to a reduction in blood pressure, plasma triglyceride levels, and cardiac hypertrophy 5 . AMPK activation might be thus an important strategy to restore vascular function in cardiometabolic alterations.
AMPK is a heterotrimeric Ser/Thr kinase of 1188 amino acids (~ 132 kDa), which is ubiquitously distributed. It is formed by three subunits: α (α1 and α2), β (β1 and β2) and γ (γ1, γ2 and γ3), which combine to give 12 different isoforms 8 . AMPKα is the catalytic subunit and contains a conventional kinase domain (αKD) located at the N-terminus of the protein, and a C-terminal domain required for interaction with the AMPKβ subunit. The C-terminal region of AMPKα subunit forms a globular domain around which the C-terminal region of the AMPKβ subunit is wrapped. The extreme terminus of the AMPKβ subunit then forms an interaction with the AMPKγ subunit, so that the AMPKβ subunit acts as the scaffold that bridges α and γ subunits. A carbohydratebinding module (CBM), located within the central region of the AMPKβ subunit, forms a binding site for allosteric activators, termed the allosteric drug and metabolite (ADaM) binding pocket. The AMPKγ subunit contains four repeats in tandem of a structural module called cystathionine β-synthase (CBS) motif. Every pair of CBS repeats provides binding sites for the regulatory adenine nucleotides AXP (X = M, D, T) 9 .
The range of AMPK modulators has been gradually expanded over the last years 10,11 , covering from AMP mimetics, such as AICAR and C2, to ADP mimetics, such as O304, which is able to protect against pThr172 or pThr174 dephosphorylation in AMPKα2 and AMPKα1 isoforms, respectively, without allosteric activation of AMPK 12  www.nature.com/scientificreports/ whereas SU6656 15 paradoxically activates AMPK signaling by directly binding at the catalytic site. All these results reveal the complex regulation of this kinase, but at the same time offer the opportunity to be exploited in the search for drugs with novel mechanisms of action.
In this work, we describe novel indolic compounds as modulators of endothelial AMPK. For this purpose, the binding mode of these compounds has been assessed by combining molecular dynamics (MD) simulations, enzymatic and Surface Plasmon Resonance (SPR) assays, together with functional activation studies targeting AMPK, eNOS and Acetyl CoA Carboxylase (ACC) phosphorylation, as well as assessing NO production in human endothelial cells (EA. Hy926) and through vascular function of rat thoracic aorta. Our findings offer new possibilities for regulating endothelial AMPK, as well as exploring the therapeutic implications of this novel mechanism of action.

Results
Identification of IND6 as AMPKα1β1γ1 modulator. Within our drug discovery program focused on the search for novel AMPK modulators 16  were screened for binding to the AMPKα1β1γ1 at a single concentration (100 µM) using the SPR technique. Abbott's product A-769662 was used as a positive binding control, and β-cyclodextrin, which binds preferentially to the AMPKβ2 isoform, as a negative binding control. Binding of IND6, IND7, or IND8 to AMPKα1β1γ1 is slightly weaker than A-769662. IND11 showed no response (0.9 RUs), suggesting the importance of the terminal aromatic ring present in the substituent at position 3 for AMPK binding, in contrast with the more polar character of the morpholine unit in IND11, which must be protonated at physiological pH (pK a ~ 8.4). on the enzymatic activity of AMPK was examined through a luminescent assay with the recombinant AMPK isoform α1β1γ1. This assay evaluates the enzymatic activity of AMPK to phosphorylate the SAMS peptide substrate, using A-769662 as positive control. All compounds in the series were found to reduce the activity of AMPK α1β1γ1 at 30 µM and therefore may be initially considered inhibitors (Table 1). At this single concentration the percentage of inhibition ranges from 39 to 62% for IND6, IND7 and IND8, and it is less pronounced for IND11 (23%). Based on these results, IND6 was selected as representative compound to carry out a detailed evaluation of the biological effect on the enzyme activity.

Competition assays of the indolic compound IND6.
To investigate the inhibitory mechanism of IND6, dose-response assays were performed using different ATP concentrations (from 20 to 1000 µM) and two inhibitor concentrations (10 and 20 µM). The Lineweaver-Burk plot of enzyme kinetics is depicted in Fig. 1. The results suggest that IND6 acts as a mixed-type inhibitor, as noted by the increase in the K s for ATP and the decrease in V max with increasing concentration of IND6. Let us note that this modality of enzyme inhibition was also reported previously for SBI-0206965 17 . Representation of the relationship between Km/Vm and the concentration of IND6 led to inhibition constants K i and K ′ i of 6.9 and 27.1 µM, respectively. These values are 27-and 30-fold higher than the K i and K ′ i values determined for SBI-0206965, respectively (K i = 0.26 µM; K ′ i = 0.89 µM 17 ). Overall, although both IND6 and SBI-0206965 exhibit a mixed-type inhibition, which conceptually combines both competitive and uncompetitive inhibition, these results suggest that the inhibitory activity may reflect different mechanisms of action.
To further explore the IND6 binding mode, SPR studies were performed at increasing concentrations (from 10 to 100 µM) of either SBI-0206965 or IND6 (Fig. 2). The sensorgrams showed a progressive increase in the binding, which was much larger for SBI-0206965 in agreement with its stronger inhibitory potency ( Fig. 2A,B). In a separate assay, IND6 was injected in the presence of SBI-0206965, both at a 100 μM concentration. Under these conditions, the sensorgrams showed an additive effect between SBI-0206965 and IND6 (Fig. 2C), suggesting that binding to AMPK might involve distinct binding sites. When the injection of SBI-0206965 and IND6 was performed in the presence of a high ATP concentration (200 µM) (Fig. 2D), SBI-0206965 binding was notably reduced, whereas IND6 binding was less sensible to the presence of ATP.
IND6 promotes the phosphorylation and activation of AMPK and downstream targets in a concentration-independent manner. In order to examine the effect of IND6 in AMPK phosphorylation and some of its targets, such as eNOS and ACC, human endothelial cells of the EA Hy.926 line were treated with IND6 at different concentrations (0.01, 0.1, 1, 10 and 100 μM). Untreated cells were used as control (CT). AICAR (at 5 mM), a known AMPK canonic activator, and 2-deoxyglucose (2-DG, at 1 mM), a caloric restriction mimetic, were used as positive controls. AICAR, once inside the cell, is phosphorylated by adenosine kinases and is converted to ZMP, an AMP mimetic, which binds the CBS sites in γ-AMPK 18 . On the other hand, 2-DG Binding mode of IND6 to AMPK. Due to the mixed-type inhibition of IND6, we investigated the binding to the ATP-binding site using Molecular Dynamics (MD) simulations. Four independent simulations were run for the AMPKα1β1γ1 complexes with IND6 and with SBI-020695, which was used as reference system. The X-ray structure of the AMPK-SBI-020695 complex (PDB entry 6BX6) revealed that SBI-0206965 occupies a pocket located between the N-and C-lobes and the hinge region of the enzyme, overlapping with the binding site of compound C, which is a competitive inhibitor of AMPK 22 . The results obtained from the different MD simulations showed a consistent picture, where SBI-0206965 remains stably bound in the ATP-binding pocket in all simulations (Fig. 4). In particular, binding is assisted by two hydrogen bonds between SBI-0206965 and the main chain of αVal98, with distances (averaged for the four MD simulations) of 3.2 ± 0.2 Å and 2.9 ± 0.1 Å between the pyrimidine nitrogen and exocyclic nitrogen of the inhibitor and the amide NH and carbonyl oxygen of αVal98, respectively. Furthermore, the ligand is enclosed in the hydrophobic pocket shaped by residues αLeu 24, αVal32, αIle79, αMet95, αLeu148 and αAla158.
The competitive binding mode of IND6 to the ATP-binding pocket was guided by the superposition with both staurosporine, SBI-020695 and compound C, taking advantage of their X-ray structures (PDB entries 4ZHX, 4CFE, 4CFF, 6BX6 and 3AQV; see Supplementary Material), which revealed the formation of hydrogen bonds between these compounds with the hinge region of the kinase, particularly involving residues αVal96 and αGlu94 (αVal98 and αGlu96 in AMPKα1).
The MD simulations performed for the AMPK-IND6 complex revealed larger fluctuations of the ligand in the binding pocket compared to SBI-020695 (Fig. 4A). This trait can be attributed to the flexibility of the benzyloxy moiety as well as to the non-planarity of the central benzene ring relative to the indole ring, enhancing also the fluctuations of the P loop (Fig. 4B). This is also reflected in the hydrogen bond distances formed between the indole NH group of IND6 with the carbonyl oxygen of αVal98 (average distance of 3.4 ± 1.1 Å), and the carboxylate oxygen of IND6 with the NH group of αGlu96 (average distance of 3.7 ± 0.9 Å), which are larger than those formed by SBI-020695. Overall, these traits agree with the 27-fold lower potency of IND6 relative to SBI-020695 (see above).  www.nature.com/scientificreports/ Additional MD simulations were also performed to examine the binding of IND6 to the ADaM site, which mediates the activation effect played by several small molecules, such as A-769662 23 . The ligand was oriented taking advantage of the close alignment exhibited by activators such as A-769662, 991 and SC4 (see Supplementary  Material). These studies showed that IND6 may adopt two distinct binding modes (Fig. 5). In one case, IND6 is deeply bound into the hydrophobic cavity of the ADaM site, and the carboxylate group forms salt bridge interactions with the protonated amino groups of αLys31 and αLys33 (average distances of 3.0 ± 0.4 and 3.5 ± 0.9 Å). It is worth noting that the top of the P-loop points to the N-terminus of the αC-helix, leaving the ATP-binding site accessible for the binding of ATP. Indeed, a significant fraction of the conformations sampled by the P-loop superpose well with the conformations adopted in the ternary complex formed by AMPK bound to A-769662 and ATP 24 (Fig. 6). This suggests that IND6 might mimic the role of A-769662 in this binding mode.
In the other binding mode, IND6 protrudes from the ADaM site toward the αC-helix, sitting on the top of the P-loop (Fig. 5B). This binding mode is assisted by electrostatic interactions between the carboxylate group and the protonated residues αLys31 and αLys53 (average distances of 4.9 ± 0.9 and 5.3 ± 1.1 Å), and a cation-π interaction between βArg83 and the benzyloxy ring of IND6 (average distance of 4.0 ± 0.5 Å). Remarkably, this binding mode imposes a structural distortion of the P-loop, which occludes the ATP-binding site (Figs. 5B and 6), making it unable to accommodate ATP. Therefore, this binding mode might explain the non-competitive mechanism of the mixed-type inhibition.   (Fig. 7A,B). To confirm that the increase in DAF-2T fluorescence is a real consequence of eNOS activation, pretreatments with the eNOS inhibitor L-NAME (100 µM), were performed in absence or in presence of IND6 (1 and 5 µM). L-NAME abolishes IND6-induced increment of DAF-2T fluorescence indicating that NO increase is a consequence of eNOS activation (Fig. 7A,C).  (Fig. 8B), which are inhibitors of AMPK and eNOS, respectively. Furthermore, concentration-response curves were also performed with A-769662 (10 -9 -10 −4 M) or AICAR (10 -5 -8 × 10 −3 M) to compare their effect with the one promoted by IND6 (Fig. 8C).
Since the three of them elicited a concentration-dependent vasodilation, their pharmacological efficacy (E max ) and potency (EC 50 ) were calculated. Although the three compounds had a similar E max , IND6 resulted as potent as A-769662 but significantly more potent than AICAR (Table 2).

Discussion
The results presented in this study point out that the novel indolic derivatives appear to act as paradoxical activators of the endothelial AMPK α1β1γ1, although they exhibit a mixed-type inhibition in the enzymatic assays. Dysregulation in the AMPK signaling pathway in over-nutrition and obesity contributes to the development of metabolic disorders and endothelial dysfunction 1 , which is considered the first step in the progression of cardiovascular disease 2 . Reduced endothelial AMPK phosphorylation leads to down-regulation of the PI3K-Akt-eNOS pathway together with low rates of NO synthesis 5,7 . Contrarily, activation of endothelial AMPK restores impaired endothelial function and normalizes systolic blood pressure through the stimulation of the PI3K-Akt-eNOS pathway 5,25 . In this context, identification of new chemical entities that can activate endothelial AMPK could be of significant interest for the treatment of obesity-related disorders. Starting with the use of SPR techniques, we selected IND6, which exhibits one of the best affinity values against recombinant AMPKα1β1γ1 (RU 13.2, 100 µM). In parallel, the AMPK α1β1γ1 enzymatic activity was assessed by means of a luminescent assay, which revealed inhibitory activity values in the micromolar range against all tested compounds (Table 1). Hence, IND6 was subjected to enzymatic kinetic analysis to examine its competition with ATP. We varied both ATP and IND6 concentrations with a constant concentration of the peptide substrate used in the enzymatic reaction. The double reciprocal plot of data ( Fig. 1) indicated that IND6 behaves as a mixed-type AMPK inhibitor. A similar inhibition mode has been recently reported for SBI-0206965, which was described as type IIb AMPK inhibitor 17 . Furthermore, SPR sensorgrams showed that IND6 and SBI-0206965 presented an additive effect on the binding to AMPK, suggesting that they may bind at different binding sites. Moreover, when we performed the same experiments in the presence of a high ATP concentration (200 µM), there was a significant reduction in the ability of SBI-020696 to bind to AMPK, while the affinity of IND6 for AMPK remained at large extent  www.nature.com/scientificreports/ unchanged. All these results suggest that IND6 and SBI-020696 may bind the ATP-binding site, thus leading to competitive inhibition of the enzyme, but also suggest that IND6 may regulate the AMPK activity through binding to an additional pocket. The biological effect of IND6 may be explained from the distinct binding modes observed for IND6 in the ADaM site, and the drastic influence exerted on the structural conformation of the P-loop. Thus, the similar arrangements observed for the P-loop when IND6 is deeply inserted into the ADaM site and in the X-ray structure of the AMPK bound to A-769662 suggest that IND6 may mimic the activating role attributed to this latter compound. Nevertheless, the structural distortion of the P-loop caused by the alternative binding mode, where IND6 protrudes from the ADaM site, might explain the non-competitive component of the mixed-type inhibition, in conjunction with the direct competition exerted by IND6 upon binding to the ATP-binding site. At this point, let us remark that the adoption of two partially overlapping binding modes at the ADaM site may be facilitated by the lack of direct interactions between IND6 and α-Asp90, in contrast to A-769662, which was found to form a hydrogen bond interaction with this residue in our previous studies of the AMPKα2β1-A769662 complex 24 . In fact, previous experimental studies demonstrated that the interaction between βArg83, αAsp90 and A-769662 is crucial for the enzyme activation 10 .
Despite the mixed-type inhibition, cellular assays showed that IND6 promotes AMPK phosphorylation (% p-AMPK/tubulin vs. CT) in the human endothelial cell line, EA Hy.926, which expresses the α1β1 isoform. This suggests a paradoxical activation of AMPK similar to the one described for the indolic compound SU6656, which seems to promote AMPK's LKB1 dependent phosphorylation 15,26 . In any case, the activation of AMPK by IND6 is functional since it translates to ACC, an ubiquitous AMPK target 20 , as well as to eNOS and NO release. The inhibition elicited by SBI-0206965 on the concentration-dependent relaxation induced by IND6 in arterial rings confirms specific activation of vascular AMPK, whereas its inhibition by L-NAME confirms activation of endothelial eNOS. This is in accordance with the AMPK-dependent eNOS activation described in endothelial cells 5,21 , as well as in arteries associated to a reduction in blood pressure, plasma triglyceride levels, and cardiac hypertrophy 5 . Activation of eNOS by AMPK is a well-described pathway, although an attenuated NO production in response to AMPK activation has also been reported 27 .
The efficacy of IND6 to promote AMPK stimulation is comparable to AICAR (5 mM), 2-DG (1 mM) or A-769662 (0.1 mM). However, the potency of IND6 is in the micromolar range, one and three orders of magnitude higher than A-769662 and AICAR, respectively, as clearly observed in the relaxation curves. To note, the apparent discrepancy between the concentrations of IND6 tested in the in vitro assays with recombinant proteins (SPR or inhibition studies) and the active concentrations observed in cell culture, which are much lower than in the former case. This could be explained by the synergic effect of the intracellular machinery, which leads to signal amplification 28 .
In summary, IND6 binding profile provides a basis to rationalize the activating behavior of IND6 in EA Hy.926 cells and arteries by increasing AMPK activity in a functional manner, as demonstrated by the increment in both ACC and eNOS phosphorylation, two well-known targets of p-Thr172/174AMPK 20,21 . Moreover, this study shows that IND6 increases NO levels in both endothelial cells and arteries, demonstrating again a functional activation of AMPK. Our findings provide evidence that IND6 holds potential as treatment of vascular complications associated with obesity, where intracellular ATP levels are high due to the energy surplus and AMPK activity is reduced 5 . AMPK activation by IND6 might be thus an important strategy to restore vascular function in cardiometabolic alterations.

Methods
Chemistry. All reagents were of commercial quality. Solvents were dried and purified by standard methods.
Analytical TLC was performed on aluminum sheets coated with a 0.2 mm layer of silica gel 60 F254. Silica gel 60 (230-400 mesh) was used for flash chromatography. Analytical HPLC-MS was performed on Waters equipment coupled to a single quadrupole ESI-MS (Waters Micromass ZQ 2000) using a reverse-phase SunFire C18 4.6 × 50 mm column (3.5 μm) at a flow rate of 1 mL/min and by using a diode array UV detector. Mixtures of CH 3  www.nature.com/scientificreports/ gun and directed analysis: Parallel Reaction Monitoring, PRM), obtaining an m/z MS-MS spectrum that was used for the subsequent database search and identification based on the results. After comparing the information obtained with the data dumped in the databases, it was concluded that the most abundant isoform of AMPK in these cells is AMPKα1β1 with 99.99% accuracy. Although the identification of the subunit γ was not conclusive, we chose the recombinant isoform AMPKα1β1γ1 for the affinity and kinase assay studies.

Binding studies by surface plasmon resonance (SPR). SPR experiments were performed at 25 °C
with a Biacore X-100 apparatus (Biacore, GE) in HBS-EP (10 mM Hepes, 150 mM NaCl, 3 mM EDTA), with 2% de DMSO, 0.05% Tween 20 and 200 µM ATP when was required, at 25 °C. The protein AMPk was inmobilized on a CM5 sensor chip (Biacore, GE) following standard amine coupling method 29  Molecular modelling simulations. Molecular Dynamics (MD) simulations were used to examine the binding mode of SBI-020695 and IND6 to AMPK, considering the binding to both the ATP-binding site and the ADaM site, which is implicated in the enzyme activation by small molecules, such as A-769662. The X-ray structure of AMPK in the PDB entry 6C9J 30 , which consists of chains α 1 , β 1 and γ 1 , was utilized to build up the protein, and the position of SBI-020695 in the ATP-binding pocket was defined after superposition with the X-ray structure 6BX6 31 , which contains SBI-020695 bound to AMPK (isoform α2). To generate the IND6-bound complexes, SBI-020695 and A-769662 (PDB entry 4CFF) were replaced by IND6 in order to build up the simulated systems with IND6 in the ATP-binding and ADaM sites, respectively. Following our previous studies 23 , the γ-subunit was not considered in MD simulations because it does not participate in the inhibition process by SBI and IND6. Moreover, due to the lack of information of C-terminal tails of α and β subunits in the X-ray structure, these parts were not treated in our simulated systems. Finally, Thr174 was simulated in the phosphorylated form (pThr174).
Simulations were performed using the AMBER18 package 32 and the Amber ff99SB-ildn force field 33 for the protein, whereas the ligand (SBI, IND6) was parameterized using the GAFF force field in conjunction with restrained electrostatic potential-fitted (RESP) partial atomic charges derived from B3LYP/6-31G(d) 34 calculations. The two simulated systems were immersed in an octahedral box of TIP3P water molecules 35 . The final systems contained around 370 residues, the ligand, around 26,000 water molecules, and one/two Na + atom for the complexes with SBI/IND6, which were added to maintain the neutrality of the simulated systems.
Simulations were done in the NPT ensemble for equilibration and NVT for MD productions. The simulations for SBI and IND6 were performed for 4 independent replicas. The minimization of the two systems was performed refining the position of hydrogen atoms in the protein (2000 cycles of steepest descent algorithm followed by 8000 cycles of conjugate gradient), and subsequently of the whole system (4000 cycles for steepest descent and 1000 cycles of conjugate gradient). Then, the temperature of the system was gradually increased from 100 to 300 K in 5 steps (50 ps each) using the NVT ensemble, followed by an additional 5 ns step performed in the NPT ensemble to equilibrate the density of the system. In this process, restraints were imposed to avoid artefactual changes in the hydrogen bonds between the ligand with Val98, as well as between pThr174 and Arg140. Production MD simulations were run for 250 ns per replica, leading to a total simulation time of 1.0 μs per ligand. Restraints were gradually eliminated during the first 100 ns in order to avoid changes in the ligand binding mode due to structural fluctuations in the ATP-binding pocket, and the analysis of the trajectories was performed on the snapshots sampled in the last 150 ns unrestrained MD simulation. Determination of the phospho-proteins by WB. The expression of the phosphorylated forms of: AMPKα in the residue Thr174 (pThr 174 -AMPKα; 62 kDa), ACC in the residue Ser79 (pSer 79 -ACC; 265 kDa), eNOS in the residue Ser 1177 (p-Ser 1177 eNOS; 133 kDa) and tubulin (55 kDa) were determined by WB 36 (as described in Supplementary Material) in EA.Hy926 cells lysates. Cells were seeded in 6-well plates (Sarstedt) with a density of 120,000 cells/well. After 48 h, once subconfluent, they were treated with modulating compounds (AICAR at 5 mM, 2-deoxyglucose at 1 mM, and IND6 at 0.01, 0.1, 1, 10 and 100 μM) and in control untreated cells (CT). All the treatments were kept at 37 °C in a humid atmosphere and 5% CO 2 for 1 h, except of the 2-deoxyglucose, which were maintained only during 10 min.
For the detection of all the proteins, acrylamide gels at 7% [H2O (5.1 mL); 1.5 M Tris-HCl pH = 8.8 (2.5 mL); SDS at 20% (50 μL) were used, acrylamide/bisacrylamide 30% (2.3 mL); ammonium persulphate 10% (50 μL), TEMED (5 μL)]. Polyclonal rabbit antibodies against the pSer 79 -ACC, pThr 174 -AMPK (1:500, Cell Signaling Technology) and pSer 1177 -eNOS (1:500, EMD Millipore Corporation) were used as primary antibodies and an anti-rabbit antibody (IgG) marked with peroxidase (1:2000, Santa Cruz Biotechnology) was used as secondary antibody. Tubulin was detected with a monoclonal mouse antibody (1:5000, Abcam) as primary antibody and an anti-mouse antibody (IgG) marked with peroxidase (1:10,000, GE Healthcare) as secondary. Quantification was carried out by establishing the relationship between the phosphorylated form of the different proteins and tubulin (p-protein/tubulin) based on the concentration of the modulating compounds administered, in addition to the negative control, on which no treatment was performed. It is worth to be mentioned that in order to obtain the maximum efficiency of the technique, WB gels were cut before the transference following the molecular weight markers depending on where the proteins were expected to appear.
Detection of nitric oxide (NO) by fluorescence microscopy. EA. Hy926 cells were seeded on 8-well plates (Sarstedt) at a density of 6000 cells per well and allowed to grow in DMEM until a 60-70% confluence was reached. The medium was then aspirated and the cells for NO detection were incubated for 1 h with no treatment (control, CT), with DMSO 0.01% (maximum solvent concentration achieved with IND6 5 µM), AICAR 5 mM as a control of activation of NO production, IND6 1 µM and IND6 5 µM. Once the incubation time had elapsed, the medium was aspirated and the cells were incubated with 4.5-diaminofluorescein diacetate (Molecular Probes) (DAF-2DA 10 -5 M) for 30 min in the dark, at 37 °C in a humid atmosphere and 5% CO 2 , in DMEM. The DAF-2DA is a non-fluorescent permeable probe capable of diffusing through the cell membrane. Inside the cell it is degraded by esterases to 4.5-diaminofluorescein-2 (2-DAF), which when reacting with intracellular NO gives rise to thiazolofluorescein (DAF-2T), capable of emitting green fluorescence (excitation wavelength 488 nm and emission wavelength 530 nm), so that the intensity of fluorescence emitted will be proportional to the production of NO in the cells. To assess eNOS involvement in DAF-2T fluorescence increase, endothelial cells were also incubated 30 min with the eNOS inhibitor L-NAME (100 µM) before stimulation with 1 and 5 µM IND6. The medium was then removed, the wells were washed twice with PBS for 15 min and then incubated with 4′,6-diamino-2-phenylindol (DAPI, 1 μg/mL; Molecular Probes) for 15 min at room temperature and in the dark. The DAPI (excitation wavelength 405 nm and emission wavelength 430 nm) stains the cell nuclei that are visualized by fluorescence microscopy. The cells were fixed in paraformaldehyde (PFA) for 1 h and washed 4 times with PBS 5 min. They were kept in PBS in darkness and at 4 °C until they were used for fluorescence microscopy (LeicaDM 2 000 Led). The quantification of the fluorescence intensity was performed with the ImageJ software and the fluorescence intensity at 530 nm is represented with respect to the control, which are the untreated cells in percentage form.
Animals. Eight-week-old male Wistar rats were housed under controlled dark-light cycles (12 h:12 h from 8:00 to 20:00) and temperature (25 °C) conditions with standard food and water ad libitum. Animals were housed individually for two weeks. Then, they were anesthetized with ketamine (Rompun, Bayer; 0.8 mg/100 g) and xylazine (Imalgene, Merial; 0.4 mg/100 g) and sacrificed by exsanguination. Thoracic aorta was dissected and used for vascular function assays. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH publication No. 85-23, revised in 2011) and was approved by the Ethics Committee of Universidad Complutense de Madrid (PROEX 206/18). All efforts were made to avoid animal suffering in accordance with the ARRIVE guidelines for reporting experiments involving animals. Vascular reactivity in the thoracic aorta artery. Vascular function studies were performed on the thoracic aorta, as previously described 5,37 . Once isolated, it was placed in a Petri dish containing Krebs Henseleit www.nature.com/scientificreports/ were separated. The artery was then cut into segments of 2 mm in length. The experiments were carried out in intact arteries with endothelium. Each of these segments was inserted between two horizontal rigid steel wires (300 μm in diameter) according to the method described previously 5,37 . Arterial segments were placed in an organ bath containing a KH solution at 37 °C continuously bubbled with a mixture of 95% O 2 and 5% CO 2 , maintaining a physiological pH between 7.3 and 7.4 constantly. These were subjected to an initial tension of 1.5 g; which was periodically readjusted for 45 min until stabilization. Once the preparations were stabilized, their functional integrity was checked with KCl (75 mM). This value represented 100% of vascular contraction for each of the arterial segments. Next, concentration-response relaxation curves were performed after contraction to a single dose of NA (10 −7 M), both with acetylcholine to test the integrity of endothelial function and with the different AMPK activators (AICAR 10 −5 M-8 × 10 −3 M; A-769662 10 −9 M-10 −4 M or IND6 10 −9 M-10 −4 M) to assess vasodilatation. To test the possible involvement of both AMPK and eNOS in the IND6-dependent vasodilation, arteries were preincubated for 30 min with the AMPK and eNOS inhibitors, SBI-0206965 (10 −4 M) and L-NAME (10 −4 M), respectively. After each curve, the preparations were washed 3 times with KH solution, and a 20-min rest period was left between each curve to ensure that the effects observed in each curve were not due to the agents used previously. The relaxation results were expressed as percentage relaxation with respect to the previous contraction obtained with NA (10 −7 M). The analysis of the recordings obtained was performed with the aid of ACQ Knowledge 3.9 software (BioPac Systems INC).
Preparation of drugs. AICAR (Toronto Chemical Research) was prepared at a concentration of 1.3 × 10 −1 M in distilled water and kept at − 20 °C until use. 2-deoxyglucose (Sigma) was prepared at a concentration of 10 −1 M in distilled water and used immediately. SBI-0206965 (Sigma-Aldrich), A-769662 (Tocris Bioscience) and the different modulator candidates were prepared in DMSO (Sigma). NA (Sigma Aldrich) was prepared in a saline-ascorbic solution (0.9% NaCl/0.01% ascorbic acid), Ach and L-NAME (Sigma Aldrich) were prepared in 0.9% NaCl solution. A stock solution (10 −2 M) was prepared for each of them and stored at − 20 °C until use (maximum 3 months). www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.