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Peripheral modulation of antidepressant targets MAO-B and GABAAR by harmol induces mitohormesis and delays aging in preclinical models

May 15, 2023

Abstract Reversible and sub-lethal stresses to the mitochondria elicit a program of compensatory responses that ultimately improve mitochondrial function, a conserved anti-aging mechanism termed mitohormesis. Here, we show that harmol, a member of the beta-carbolines family with anti-depressant properties, improves mitochondrial function and metabolic parameters, and extends healthspan. Treatment with harmol induces a transient mitochondrial depolarization, a strong mitophagy response, and the AMPK compensatory pathway both in cultured C2C12 myotubes and in male mouse liver, brown adipose tissue and muscle, even though harmol crosses poorly the blood–brain barrier. Mechanistically, simultaneous modulation of the targets of harmol monoamine-oxidase B and GABA-A receptor reproduces harmol-induced mitochondrial improvements. Diet-induced pre-diabetic male mice improve their glucose tolerance, liver steatosis and insulin sensitivity after treatment with harmol. Harmol or a combination of monoamine oxidase B and GABA-A receptor modulators extend the lifespan of hermaphrodite Caenorhabditis elegans or female Drosophila melanogaster. Finally, two-year-old male and female mice treated with harmol exhibit delayed frailty onset with improved glycemia, exercise performance and strength. Our results reveal that peripheral targeting of monoamine oxidase B and GABA-A receptor, common antidepressant targets, extends healthspan through mitohormesis. Introduction Disruption of mitochondrial function has been linked to different age-associated pathologies 1 , 2 , such as diminished muscle function 3 , insulin resistance and diabetes 4 , depression or anxiety 5 , 6 . In turn, strategies aimed at improving mitochondrial function have been shown to slow aging and reduce the incidence and severity of age-related pathologies 1 . Mitohormetic compounds are defined as mild mitochondrial stressors that trigger a compensatory response coordinated between the nucleus and the mitochondria, resulting in a final improvement in mitochondrial function 1 , 2 . For this, mitohormetic compounds activate a mitochondria quality control program that relies on the elimination of dysfunctional mitochondria by the induction of a mitochondria-specific type of autophagy, termed mitophagy. Also, an increased ADP/ATP ratio upon energetic stress activates AMPK, a crucial mechanism in the mitochondrial quality control program. Mitohormesis can be induced by genetic means in animal models, such as ablation of subunits of respiratory complexes 7 or the use of conplastic mouse models 8 . Treatment with compounds that induce a reversible stress in the mitochondria have been shown to improve mitochondrial function by a mitohormetic mechanism. This results in improved diabetes and muscle function or extended lifespan in Caenorhabditis elegans, humans and/or mice, as was shown with the anti-diabetic drug metformin 9 , 10 , the NAD+ precursors nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) 11 , 12 , the green tea polyphenols epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) 13 , D-glucosamine 14 or the pomegranate-derived urolithin A 15 , 16 . Also, behavioral interventions such as exercise 7 , 17 , calorie restriction or fasting 18 have been suggested to exert mitohormetic effects. In this work, we describe the β-carboline harmol as a mitohormetic compound. Harmol simultaneously modulated monoamine oxidase B and GABAAR, two relevant regulators of psychiatric depression and anxiety, and this dual modulation was sufficient to reproduce the harmol-mediated improvement of mitochondrial function. Harmol also activated mitophagy and AMPK pathways in vitro and in vivo. Using animal models, we showed that harmol supplementation extended lifespan of Caenorhabditis elegans and Drosophila melanogaster. In mice, harmol crossed the blood-brain barrier poorly, but improved glucose homeostasis and insulin sensitivity in a model of diet-induced obesity and diabetes, and delayed neuromuscular degeneration in old mice. Our results reveal a group of compounds that target relevant psychiatric pathways in peripheral tissues and improve whole body metabolism, aging and age-related diseases. Results Screening platform to identify mitochondria-depolarizing products We first set up a screening platform for mitohormetic products. Previous reports comparing cell lines showed that the mouse myotube cell line C2C12 had good electron transport capacity and was able to efficiently perform oxidative phosphorylation from a variety of sources 19 . C2C12 muscle precursors can be easily and efficiently differentiated to mature myotubes (Fig. S1a ) and have been extensively used to perform high-throughput screenings 20 , 21 , 22 . Also, muscle tissue plays a crucial role in the pathogenesis of type 2 diabetes and it has been proposed as one of the main targets for anti-diabetic strategies 23 . For all these reasons, we chose C2C12 cells for our screening. For the mitochondrial membrane potential reporting system, previous reports had shown that several fluorescent dyes accumulate in the intermembrane space in a manner proportional to mitochondrial membrane potential 24 . From the several described options, rhodamine derivative tetramethylrhodamine methyl ester (TMRM) was the less toxic to the ETC 25 , was specially sensitive to gradual changes in mitochondrial membrane potential, and was well suited for high-throughput screenings 26 . The optimal incubation time to reach a stable TMRM fluorescent signal was determined by treating C2C12 myotubes with two intermediate concentrations of TMRM (15 nM or 25 nM, Fig. S1b and S1c ) for 10 min, 60 min or 16 h. Stability of TMRM signal was determined by repeated TMRM measures at 0 min and 40 min. As shown in Fig. S1b and S1c , TMRM incubation for short times (10 min and 60 min) resulted in variability between the first read (0 min) and the second read (40 min). Only incubation with TMRM for 16 h resulted in a stable TMRM signal with time. From this point on, we always incubated C2C12 cells with TMRM for 16 h prior to any treatment. Then, a curve with increasing concentrations of TMRM was performed to find the lowest TMRM concentration that resulted in a sensitive and robust decrease of mitochondria depolarization when treated with the mitochondria depolarizer carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). As shown in Fig. S1d , 15 nM TMRM detected FCCP-mediated depolarization with the same sensitivity as higher concentrations, but 5 nM TMRM failed to do so. Therefore, incubations with 15 nM TMRM for 16 h before the addition of compounds were the chosen conditions for the screening, resulting in very specific and sensitive mitochondria staining as shown in Fig. S1e . We also tested the maximum concentration of the solvent of our libraries (DMSO) that did not elicit significant changes in the TMRM signal. As observed in Fig. S1f , 0.1 % DMSO was the highest concentration with no significant effect on the TMRM signal. Using these conditions, we observed a dose-dependent inhibition of the mitochondrial potential by FCCP (Fig. S1g–h ). Importantly, with these conditions, we obtained a Z factor above 0.5 for FCCP concentrations as low as 250 nM (Figure  S1i ), confirming the robustness of our screening platform. Selection of mitohormetic compounds Using the platform described above, we screened 982 compounds isolated from natural sources such as plants, animals, fungi or bacteria (Supplementary Data  1 ). We tested each compound in triplicate at a final concentration of 1.3 μg/ml (the concentration used for the screening) dissolved in a final concentration of 0.1% DMSO, and using DMSO at 0.1% as negative control. First, we searched for compounds that induced a short-term mitochondrial depolarization. For this, we measured TMRM fluorescence intensity before the addition of the compounds (time point T0) to define the baseline. We then added the compounds and measured TMRM fluorescence after 30 min (T30) or 60 minutes (T60). Depolarizing compounds elicited a decrease in TMRM signal at either of these times that resulted in a strictly standardized mean difference (SSMD 27 ) of SSMD ≤ −2 compared to 0.1% DMSO controls, and were identified as hits. This way, 96 positive hits were selected (Fig. 1a ). Next, we searched for compounds that induced a transient, non-toxic mitochondrial depolarization. For this, we retested our 96 hits at T30 and T60, and included a long-term time point at 16 h after treatment. We confirmed a short-term mitochondrial depolarization (T30 and/or T60) below SSMD ≤ −2 in 50 compounds (Fig. 1b ). In addition, we selected for reversible depolarizers, one of the characteristics of mitohormetic compounds. For this, cells were kept for another 16 hours with the compounds and TMRM fluorescence was measured again. We selected the 36 hits that induced mitochondrial depolarization after a short treatment of 30 and/or 60 min, but not after a 16-hour treatment (green dots in Fig. 1c , with SSMD ≥ −2), indicating an ability of these compounds to reversibly depolarize the mitochondria. Fig. 1: Screening for mitohormetics. a Strictly standardized mean difference (SSMD) of each of the 982 compounds tested at 30 or 60 min after treatment of differentiated C2C12 myotubes (the lowest SSMD from the two timepoints in indicated). Compounds that induced a SSMD ≤ −2 are shown in orange (depolarizers). Compounds that increased mitochondrial potential or had no effect are shown in blue. b SSMD of the 96 depolarizing compounds identified in (a) were quantified 30 or 60 min after compound addition, and the lowest SSMD of both times is represented. c The same experiment as in (b), but measured 16 hours after compound addition. d–f Left panels represent the oxygen consumption rates (OCR) in differentiated C2C12 myotubes treated for 16 h with 1.3 μg/ml of harmol (d), harmine (e) or norharmane (f) following the sequential addition of oligomycin A (O), FCCP (F) and Rotenone/Antimycin A (R/A) at the indicated times (Mitostress Seahorse experiment). Panels on the right represent the spare respiratory capacity (SRC) parameter of the complete Mitostress experiment. g Chemical structure of the indicated compounds. Bars and line-connected circles represent the mean of the indicated number of replicates. Individual dots represent independent cell samples. Error bars represent the standard deviation. Isolated circles represent single replicates. Statistical significance was assessed calculating the SSMD parameter (a–c) or using the two-tailed unpaired Student t test (d–f). P values are indicated when P < 0.05. Source data are provided as a Source Data file. To select for mitohormetic compounds, we next tested the ability of our reversible depolarizers to improve mitochondrial function. For this, we performed a complete analysis of the mitochondrial function using the Mitostress protocol by Seahorse in differentiated C2C12 myotubes treated with our reversible depolarizers for 16 h. More specifically, we selected the compounds that increased the spare respiratory capacity, a parameter that reflects the ability of mitochondria to cope with stressful situations and has been associated with improved mitochondrial fitness and delayed aging 28 . As a positive control, we used the anti-diabetic drug rosiglitazone, which improves mitochondrial biogenesis and function 29 , 30 . Rosiglitazone was able to depolarize mitochondria in our C2C12-TMRM model (Fig. S1j ), and to increase the spare respiratory capacity measured by Seahorse (Fig. S1k ). We identified compounds that had been previously described to reduce mitochondrial potential, such as emodin 31 and mangostin 32 (Fig. S1l and S1m ). In addition, we found mitohormetic potential in several members of the family of β-carbolines, such as harmol (Fig. 1d ) and harmine (Fig. 1e ). Norharmane, from the same family, was identified as a reversible depolarizer (Fig. 1c ) and showed a non-significant tendency to increase the spare respiratory capacity in the Seahorse experiment (Fig. 1f ). Among all the identified compounds, (Fig. 1g ), harmol showed the most robust and reproducible increase in spare respiratory capacity. In vitro characterization of the mitochondrial effects of harmol We next studied the molecular effects of harmol on mitochondria. First, we checked by Seahorse if harmol was acting directly on mitochondrial respiration. For this, we acutely treated C2C12 cells with harmol and measured the immediate oxygen consumption rate (OCR) response using a Seahorse analyzer, reflecting changes in the mitochondrial respiration. As shown in Fig. S2a , acute treatment with harmol, in contrast to rosiglitazone, did not change mitochondrial respiration, indicating that harmol does not act directly on the electron transport chain. Next, we studied the induction of mitophagy and of AMPK, two canonical mechanisms of the mitochondrial quality control program. First, we measured the conversion of the protein LC3-I into its lipidated, autophagosome-associated form LC3-II, a well-described autophagy marker 33 . Interestingly, 1 h after harmol treatment, lipidation of the autophagy marker LC3 was increased in the harmol-treated cells, indicating that harmol treatment activated signs of autophagy (Fig. 2a ). We measured the protein levels of the mitophagy marker PINK1 34 , and again observed a clear induction in PINK1 levels (Fig. 2a ), indicating that harmol triggered autophagy specifically targeting the mitochondria. We also measured the activation of AMPK, another canonical mechanism of the mitochondrial quality control program 35 . We observed that 1 hour of treatment with harmol elicited a moderate, although not statistically significant increase in the phosphorylation of AMPK, similar to the increase observed with the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR). No change was observed in the activity of the AMPK target proteins ACC1 and ACC2 (Fig. 2a and Fig. S2b ). We then measured these same markers of the mitochondrial quality control program 3 h after treatment with harmol. At this timepoint the autophagy and mitophagy markers LC3-II/LC3-I ratio and PINK1, respectively, were not as increased as at 1 h after harmol treatment, although they were still activated when compared to baseline; whereas the AMPK target ACC1 was clearly hyperphosphorylated and activated (Fig. 2b ). ACC2, another target of AMPK, was not altered in any of these timepoints (Fig. S2b and S2c ). Fig. 2: In vitro mechanistic characterization of the mitochondrial effects of harmol. a, b Western blots of the indicated proteins in differentiated C2C12 myotubes 1 h (a) or 3 h (b) after treatment with 0.1% DMSO, 1.3 μg/ml harmol (H), 30 μM rosiglitazone (R) or 250 μM AICAR (A). Quantifications of the significantly different proteins are shown in the bar graphs to the right (n = 6 independent cell samples for each condition). c Confocal images of differentiated C2C12 cells treated with DMSO (01%), harmol (1.3 µg/ml) or FCCP (500 nM) for 45 min and stained for mitochondria with Mitotracker (Mito); for lysosomes with Lysotracker (Lyso); or for active mitochondria with TMRM. d Quantification of the colocalization of Mitotracker and Lysotracker staining, calculated as the Pearson r correlation coefficient in the same cells and treatments as in (c). e C2C12 myotubes stained with TMRM were treated with the indicated concentrations of harmol alone or combined with 20 µM chloroquine (CQ) for 30 min, and then TMRM intensity was recorded before (T0) and 60 min after (T60) treatment with harmol. f Western blots of the indicated proteins in differentiated C2C12 myotubes 16 h after treatment with 0.1% DMSO, 1.3 μg/ml harmol (H), 30 μM rosiglitazone (R) or 250 μM AICAR (A). (n = 6 independent cell samples for each condition). g Quantitative PCR experiments measuring the ratio between mitochondrial DNA and nuclear DNA (mt/nDNA) in differentiated C2C12 myotubes treated with 1.3 µg/ml harmol or 250 μM AICAR for the indicated times. h ATP content in differentiated C2C12 myotubes treated with 0.1% DMSO or with 1.3 μg/ml harmol was assessed at the indicated timepoints (a.u. arbitrary units). Bars and line-connected dots represent the average of the indicated replicates. Dots in bar graphs (a, b, d, f, g) represent independent cultured cell samples. Each lane in Western blots (a, b, f) represents independent cell samples. Error bars represent the standard deviation. Statistical significance was assessed using the one-way ANOVA (a, b, d, f) or the two-way ANOVA (e, g, h) tests with Tukey’s correction for multiple comparisons. P values are indicated when P < 0.05. Source data are provided as a Source Data file. To better understand the induction of autophagy by harmol, we stained differentiated C2C12 myotubes with a combination of Mitotracker (staining total mitochondria) and Lysotracker (staining lysosomes); or with TMRM (staining active mitochondria, with a detectable mitochondrial potential) and Lysotracker. As shown in Fig. 2c, d , after treatment with harmol we observed a strong co-localization of lysosomes and mitochondria, indicating mitochondria-specific autophagy. Of note, there was almost no co-localization of Lysotracker with TMRM up to 60 min after harmol treatment, indicating that harmol-induced mitochondria-specific autophagy only took place in inactive, TMRM-negative mitochondria (Fig. 2c ). To further prove the involvement of autophagy in the mitochondrial depolarization induced by harmol, we treated C2C12 myotubes with chloroquine, that inhibits the fusion of autophagosomes with lysosomes 36 . We observed a dramatic blockage of the harmol-induced mitochondrial depolarization after chloroquine treatment (Fig. 2e and S2d ), indicating that mitophagy was essential for harmol-mediated depolarization. Activation of AMPK and mitophagy leads to a compensatory response that finally results in improved mitochondrial amounts and/or function. To test this mechanism, we measured several markers of mitochondrial function after 16 h of treatment with harmol. We observed a significant increase in the protein expression of the mitochondrial transcription and replication factor TFAM, and of the components of the complex III (UQCRC2) and complex V ATP5a (Fig. 2f and S2e ). Regarding mitochondrial content, it remained unchanged during all the treatment, as measured by quantification of the mitochondrial-to-nuclear mt/nDNA ratio, up to 16 h (Fig. 2g ). We finally analyzed ATP production, another illustrative marker of mitochondrial fitness. We observed an initial decrease in the ATP content 3 h after treatment with harmol, in agreement with the induction of the AMPK pathway at this time; and increased ATP levels after 16 h, in agreement with the increased levels of mitochondrial function markers (Fig. 2h ). Finally, we wanted to determine if the observed increase in mitochondrial respiration and function with harmol could be associated to changes in myotube size. For this, we treated differentiated myotubes with harmol. We took photographs of the myotubes at different timepoints, and did not observe any difference in the average myotube size at any timepoint (Fig. S2f ). In vitro characterization of the mechanisms of harmol-induced mitochondrial response We next wondered how harmol elicited the mitophagy and mitohormesis responses shown above. For this, we explored the three best-known molecular targets of β-carbolines. First, the β-carboline harmine is a well-known inhibitor of DYRK1A 37 , 38 . In a recent screening searching for harmine analogs with ability to inhibit DYRK1A in neuroblastoma and glioblastoma cells, harmol was identified as an inhibitor of DYRK1A with low effectiveness against the monoamine oxidase MAO-A (but its effects on the MAO-B isoform was not tested) 39 . To check if harmol effects on mitochondrial depolarization could be caused by DYRK1A inhibition, we treated C2C12 cells with harmol for 5, 10 and 15 min, and checked DYRK1A phosphorylation levels. As shown in Fig. S3a , we did not detect any significant decrease in DYRK1A phosphorylation. These results indicate that inhibition of DYRK1A was not necessary for the mitochondrial depolarization induced by harmol in C2C12 myotubes. Next, β-carbolines have been shown to inhibit the monoamine oxidase (MAO) 40 . We validated a strong and specific inhibition of the activity of the MAO-B isoform by harmol, starting already at 60 min after treatment and becoming almost totally abrogated 16 h after treatment (Fig. 3a ), while the activity of the MAO-A isoform remained unchanged at all times (Fig. S3b ). As a positive control, we used harmine, another β-carboline previously described as a pan-MAO inhibitor 41 that we also detected in our initial screen (Fig. 1e ). When increasing concentrations of harmol were added together with the MAO-B inhibitor rasagilin, the lowest harmol concentrations tended to depolarize more efficiently than harmol alone, indicating that MAO-B inhibition participated in the depolarizing ability of harmol (Fig. 3b ). This cooperation between rasagilin and harmol was significant but not very strong, and was only observed at 3.5 h after the treatment. These results indicated that MAO-B inhibition participates in harmol mechanism of action, but it follows slower kinetics than harmol alone, and it is not sufficient to fully recapitulate harmol functions. MAO enzymes metabolize molecules presenting amine groups, including neurotransmitters as serotonin or dopamine, their best known substrates 42 . In addition, MAO enzymes can also metabolize polyamines, such as putrescine, the metabolic precursor of spermidine 43 . Based on this, we measured alterations in polyamine pools in C2C12 cells treated with harmol. Treatment with harmol significantly increased the levels of the polyamine spermidine (Fig. 3c and Supplementary Data  2 ). Fig. 3: Mechanisms of harmol functions in mitochondria. a C2C12 myotubes were treated with 0.1% DMSO, 1.3 μg/ml harmol or 1.3 μg/ml harmine, and specific MAO-B activity was measured at the indicate times. b C2C12 myotubes stained with TMRM were treated with rasagilin, and then with the indicated concentrations of harmol, and TMRM fluorescence was measured at T0 and T210. c C2C12 myotubes were treated with vehicle (0.1% DMSO, D) or with 1.3 μg/ml harmol (H) during 24 h, and spermidine levels were measured. d Frequency of spontaneous inhibitory currents (sIPSCs) events measured in mouse hippocampal slices (n = 5 from 3 different animals) before (B) and after (A) treatment with 1.3 μg/ml harmol. e C2C12 myotubes stained with TMRM were treated with the indicated concentrations of harmol and bicuculline, and TMRM fluorescence was measured at T0 and T60 after treatment. f Confocal images of differentiated C2C12 myotubes treated with DMSO (0.1%), harmol (1.3 µg/ml), bicuculline (5 µM) or the combination of harmol + bicuculline, and stained for total mitochondria with Mitotracker (green) and for lysosomes with Lysotracker (red). Size bar = 25 µm. Colocalization of Mitotracker and Lysotracker is shown in yellow. g Quantified Mitotracker and Lysotracker colocalization from (f). h, i C2C12 myotubes were treated with increasing concentrations of harmol and 1 µM FG7142 (h); or with increasing concentrations of FG7142 and 10 µM rasagilin (i), and TMRM fluorescence was measured at T0 and T60. j Spare respiratory capacity (SRC) of C2C12 myotubes treated for 16 h with 0.1% DMSO, 1 μM rasagiline (R), 2 μM FG7142 (F) or the combination of 1 µM rasagilin and 2 µM FG7142 (R + F), or with 1.3 μg/ml harmol, and then analyzed by Seahorse. Bars and line-connected dots represent the average of the indicated independent replicates. Dots in bar graphs represent independent cell samples. Error bars represent the standard deviation. Statistical significance was assessed using the two-way (a, b, e, h, i) or the one-way (g, j) ANOVA with Tukey correction for multiple comparisons; or the two-tailed unpaired Student t-test (c, d). P values are indicated when P < 0.05. Color codes of P values indicate the groups being compared. Source data are provided as a Source Data file. Since MAO-B inhibition alone did not fully recapitulate all harmol effects, we searched for other possible mechanisms of action. Another well-reported target of β-carbolines is the receptor of the neurotransmitter gamma-aminobutyric acid type A (GABAAR) 44 . To assess if harmol influences GABAAR function, we acutely treated mouse brain slices with harmol or vehicle. We measured spontaneous inhibitory postsynaptic currents (sIPSCs) in hippocampal neurons, which are predominantly GABAergic in the telencephalon and were blocked by the GABAAR antagonist SR95531 (gabazine). We observed that acute application of harmol led to a significant reduction in the frequency of GABAergic currents, and this effect was not observed in vehicle-treated slices (Fig. 3d ). Harmol treatment led to a 40% reduction in the frequency in IPSCs but did not affect the amplitude of the remaining sIPSCs observed after harmol application (Fig. S3c ). These results suggest that harmol reduced GABAergic neurotransmission but did not completely inhibit GABA binding on GABAAR. To further study the effect of harmol on the GABA receptor, we treated differentiated C2C12 myotubes with the GABAAR antagonist bicuculline or the GABA-receptor inverse agonist FG7142. Neither of these GABAAR modulators elicited any effect in mitochondrial polarization by themselves (Fig. S3d–e ). However, when combined with harmol, inhibition of the GABAAR by bicuculline significantly reduced the harmol-mediated mitochondrial depolarization in a dose-dependent manner (Fig. 3e ), and strongly blunted the mitophagy induced by harmol (Fig. 3f, g ). These results indicated that GABAAR activity is essential for harmol-mediated mitochondrial depolarization. In addition, when the GABAAR inverse agonist FG7142 was added together with very low concentrations of harmol (unable to induce mitochondrial depolarization by themselves), this combination now induced a significant mitochondrial depolarization (Fig. 3h ), indicating that harmol activity was potentiated by an inverse agonist of the GABAAR. Finally, treatment of C2C12 differentiated myotubes with a combination of the MAO-B inhibitor rasagiline and the GABAAR inverse agonist FG7142 elicited a mitochondrial membrane depolarization (Fig. 3i ), although to a lesser extent than harmol alone. Finally, we measured the effect of MAO-B and GABAAR modulators on the respiratory capacity of C2C12 myotubes. As shown in Fig. 3j and S3f–g , FG7142 and rasagiline alone did not affect the cellular spare respiratory capacity. But, when these two treatments were added together, they induced a synergistic, almost significant improvement in the spare respiratory capacity, recapitulating the effects elicited by harmol. These results indicated that the simultaneous modulation of MAO-B and GABAAR by harmol is at least partly responsible for harmol mitohormetic capacity in C2C12 differentiated myotubes. Finally, we wondered if harmol effects were dependent on functional mitochondria. To test this point, we treated C2C12 myotubes with vehicle or harmol for 16 hours and measured cellular ATP. In accordance with previous results shown in Fig. 2h , harmol treatment increased ATP production. However, this increase in ATP by harmol was completely blunted if cells were treated with the electron transport chain inhibitors rotenone and antimycin (Fig. S3h ), indicating that, indeed, harmol required functional mitochondria to exert its effects. In vivo effects of acute treatment with harmol Next, we treated young, healthy male mice with 100 mg/kg of harmol dissolved in water by oral gavage after removing their food, and kept them in fasting until sacrifice 3 and 7 h later. We took blood samples at different times during this protocol, and did not detect changes in their blood levels of glucose (Fig. S4a ). We also measured their levels of the ketone body β-hydroxybutyrate in blood before and after 7 h of fasting and treatment. Neither harmol nor rosiglitazone treatments altered the fasting-mediated increase in ketone bodies after 7 h of fasting (Fig. S4b ). We also performed basic pharmacokinetics studies, to determine the bioavailability of harmol with time. As a first in vitro approach, we performed the blood-brain barrier (BBB) specific parallel artificial membrane permeability assay (PAMPA-BBB), to determine the rate at which harmol crossed an artificial membrane resembling the BBB 45 . As shown in Supplementary Table  1 , harmol presented a low permeability to the BBB, indicated by an effective passive permeability coefficient (LogPe) of −4.012, in a scale where LogPe < −4.7 marks totally impermeable compounds, and LogPe>4.7 marks totally permeable ones. As a reference, we tested other β-carbolines as harmine and norharmane, and the neurotransmitter serotonin, that is totally impermeable to the BBB (LogPe < −4.7). While norharmane presented a similar LogPe to harmol, harmine LogPe was very low (−4.83). This was due to a very high membrane retention, measured by the R parameter: more than 92% of harmine was retained in the artificial lipidic BBB-like membrane, compared with 54.5% of harmol and only 4.8% of serotonin. After equilibrium, the percentage of harmol that had passed from the donor to the acceptor solution through the artificial BBB, measured by the Ca(t)/Cd(0), was 24.64%, similar to norharmane, but not as low as harmine or serotonin. These results indicate that harmol is quite impermeable to the BBB, with <25% crossing it, and is only partially absorbed in the lipid part of the BBB (Supplementary Table  1 ). Next, we measured by LC-MS/MS the levels of harmol in plasma, liver and brain samples (for technical details, see Supplementary Table  2 ) from 12 week-old male mice treated by oral gavage with vehicle or 100 mg/kg harmol for 0, 3, 7 or 24 h. We considered that the shortest time (3 h) would be sufficient for a proper harmol biodistribution; 7 h would be informative about the evolution of harmol with time; and 24 h would indicate how stable harmol levels are in the body. We chose these times based on our results with cultured C2C12 myotubes shown in Fig. 2 , where harmol treatment took 1–3 h to exert its full activity. As shown in Fig. 4a and Supplementary Data  3 , harmol levels increased very clearly 3 h after treatment with harmol in all the analyzed tissues. Liver reached its maximum harmol levels at 3 h and retained high levels of harmol even 24 h after treatment. Plasma also reached high levels of harmol at 3 h, that remained high after 7 h, and dropped dramatically at 24 h after treatment. In turn, we detected very low levels of harmol in the brain after 3 and 7 h of treatment, ~10 times lower than in plasma and liver (Fig. 4a ). These time-course experiments were reflected in the area under the curve, that was much lower in brain than in the other tissues (Fig. 4b ). In this regard, the brain-plasma AUC ratio through a time-course measurement is a robust biomarker of the ability of a given compound to cross the BBB 46 . According to our time-course measurements, the brain-plasma AUC ratio of harmol was 0.125 in a scale where <0.1 means BBB impermeability, values between 0.3 and 0.5 mean sufficient BBB permeability, and values > 1 mean free BBB cross ability 46 . These results indicate a poor BBB permeability of harmol, in accordance with our in vitro PAMPA-BBB assay. Fig. 4: In vivo effects of acute treatment with harmol. a Harmol levels were measured by HPLC/MS in plasma, liver and brain samples form 12 week-old C57BL/6JHsdOla male mice treated with 100 mg/kg harmol by oral gavage and sacrificed 3, 7 and 24 hours after treatment. b Area under the curve (AUC) of the harmol levels of plasma (P), liver (L) and brain (B) shown in (a). c Western blots of the indicated proteins in brain 7 h after oral gavage administration of vehicle (water, V), 100 mg/kg harmol (H) or 10 mg/kg rosiglitazone (R). Quantification of PINK1 levels is shown in the bar graph to the right. d–f Behavioral assays testing anxiety-like behavior in mice after treatment with 100 mg/kg/day (H) or vehicle (water, W) in the drinking water during 3 weeks: open field test parameters (distance, speed, time in borders and time in center, d); elevated plus maze test parameters (ratio close/open and ratio close/(open+center), e); and dark-light box test parameter (ratio dark/light, f). g–i Western blots of the indicated proteins in liver (g), brown adipose tissue (BAT, h) or gastrocnemius muscle (i) from the same mice shown in (a–c). Quantifications of the indicated proteins are shown in the bar graphs to the right. j Quantitative PCR experiment measuring the ratio between mitochondrial and nuclear DNA (mt/nDNA) in the indicated tissues from the same mice as shown in (g–i). Bars and line-connected dots represent the average of the indicated replicates. Dots in bar graphs (b–j) represent samples or measures from independent animals. Each lane in Western blots (c, g–i) represents samples from independent animals. Error bars represent the standard error of the mean. Statistical significance was assessed using the one-way ANOVA test with Tukey’s correction for multiple comparisons (b, c, g–j) or the unpaired two-tailed Student’s t test (d–f). P values are indicated when P < 0.05. Source data are provided as a Source Data file. We next wondered if the same metabolic program that we observed in the C2C12 myotubes after harmol treatment in vitro also took place in harmol-treated mice. To answer this question, we harvested brain, liver, brown adipose tissue (BAT), and skeletal muscle (gastrocnemius) from the same mice used for the LC-MS/MS analysis. In brain, 7 h after treatment with harmol we detected a tendency to increased levels of the mitophagy marker PINK1, although it did not reach statistical significance; while levels of the phosphorylation of the AMPK target ACC1 or the autophagy marker LC3-II/LC3-I were not affected by harmol treatment (Fig. 4c and S4c ). We then wondered if the small fraction of harmol crossing the BBB (Supplementary Table  1 and Fig. 4a, b ) that elicited the slight increase in PINK1 levels in the brain (Fig. 4c ) might have any behavioral effects. To test this possibility, we treated mice with 100 mg/kg/day harmol for 3 weeks. Harmol- and vehicle-treated littermates were tested in a set of paradigms aimed at assessing anxiety-related behavior in rodents. Mice first explored an open field, a novel environment in which mice are exposed to a mildly aversive open area. We did not see any alteration in the time mice spent in the aversive (center), compared with non-aversive (borders) areas, between the harmol-treated mice compared with the control mice (Fig. 4d ). We then subjected mice to an elevated plus maze test, composed of two enclosed arms and two open arms elevated over the floor and generating a higher aversive reaction than in the previous open field test. Still, no difference was observed in the harmol-treated mice compared to the vehicle group (Fig. 4e ). Lastly, we put mice in a dark-light box, where mice are free to choose between the exploration of a strongly illuminated open box and a covered dark box. In this case, we observed a significantly reduced ratio of time spent in dark/light boxes in the harmol-treated mice when compared to control (Fig. 4f ). These results indicated that harmol treatment had a mild anxiolytic effect, that only became apparent upon a strongly aversive stimulus. We also measured mitohormetic responses in peripheral tissues of mice treated with harmol, vehicle (water) or our positive mitohormetic control, rosiglitazone. Seven hours after treatment with harmol we observed significant increases in the phosphorylation of ACC2 in liver and BAT, and in the levels of the mitophagy marker PINK1 in liver, BAT and gastrocnemius, compared with water-treated mice (Fig. 4g–i ). We also observed a clear tendency, although not significant, towards increased pAMPK in liver and BAT (Fig. 4g, h ). Interestingly, at an earlier timepoint, 3 h after treatment, these changes were not yet apparent (Fig. S4d–f ). As observed in C2C12 myotubes, 7 h after harmol treatment the increase in pACC, pAMPK and PINK1 were not reflected in significant changes in the mitochondrial cellular content (Fig. 4j and S4g ). Chronic treatment with harmol improved glucose homeostasis in diet-induced obese mice We wanted to explore if chronic harmol-mediated improvement of mitochondrial function could enhance metabolic performance. To test this hypothesis, we generated diet-induced obese (DIO) mice by feeding them with HFD (45% of calories from fat) for 4 months. Next, we treated these mice with 100 mg/kg harmol in their drinking water for 3 months. We also included a positive control group treated with 10 mg/kg rosiglitazone, an anti-diabetic drug with mitohormetic effects 29 . Importantly, mice did not show any sign of toxicity, morbidity or mortality during this time, apart from the previously described body weight gain in the rosiglitazone-treated mice 47 (Fig. 5a ). We observed that harmol-treated mice showed a clear tendency to reduced body weight gain compared with rosiglitazone and water controls (Fig. 5a ). Although we detected a slight decrease in water intake in the harmol-treated mice, that reached significance at several time points (Fig. S5a ), food intake was not affected by harmol and, as previously reported, was increased with rosiglitazone during the first weeks of treatment (Fig. 5b and S5b ). We also measured the hematological profile of our mice, and we only detected a minor but significant increase in the variation coefficient and standard deviation of their red blood cell distribution width (RDW-CV and RDW-SD) and a small decrease in their hematocrit (Fig. S5c ) in the rosiglitazone-treated mice, compared with vehicle- or harmol-treated mice, always within normal rages. At the end of the treatment, we did not find any evidence of tumor appearance, or any other evident pathology in any of the mice. We measured body composition by dual energy X-ray absorptiometry (DEXA). Harmol-treated mice had a reduced fat mass compared with rosiglitazone-treated mice (Fig. 5c ), with no decrease in their lean mass (Fig. 5d ). Importantly, harmol- and rosiglitazone-treated mice showed a marked decrease in their fasting blood glucose levels (Fig. 5e ), indicating an improved glucose homeostasis. We then performed a glucose tolerance test (GTT), and again observed an improved glucose tolerance in harmol- and rosiglitazone-treated mice (Fig. 5f, g ). To determine the effect of harmol on insulin function, we first measured the fasting insulinemia, and observed a clear decrease in harmol-treated mice, although not significant (Fig. 5h ). We also measured the Homeostatic Model of Assessment for Insulin Resistance (HOMA-IR), taking into account glucose and insulin levels at fasting, and observed that the HOMA-IR of harmol- and rosiglitazone-treated mice were strongly decreased, compared with water-drinking controls, further validating an improved insulin and glucose homeostasis after treatment with harmol (Fig. 5i ). However, when an insulin tolerance test (ITT) was performed, harmol treatment did not affect insulin sensitivity (Fig. S5d–e ). Fig. 5: In vivo effects of chronic treatment with harmol. a Body weight of obese C57BL/6JHsdOla male mice (previously fed with HFD for 4 months since they were 12 weeks old) kept on HFD and treated with vehicle (water, W), 100 mg/kg harmol (H) or 10 mg/kg rosiglitazone in their drinking water for 3 months. b Average food intake in the same mice described in (a) during all the time of treatment with the indicated compounds (n = 3 cages/condition). At the end of the treatment indicated for (a), the same n = 8 mice were measured for their fat mass (c); lean mass (d); glycemia recorded after 16 h of fasting (e); glucose tolerance test (f; area under the curve is shown in (g)); insulinemia (h) and HOMA-IR (i) after 16 hours of fasting; energy expenditure (EE, j) and respiratory quotient (RQ, k) with their corresponding average values during day and night shown to the right; weight of the indicated tissues (l); liver macroscopic appearance (m); lipid and triglyceride content in the liver (n, o); blood adiponectin (AD) and transferrin (TF) measured by Western blot (p), with the band quantification shown in (q); and spermidine levels in the liver (r). Bars and line-connected dots represent the average of the indicated number of individuals. Dots in bar graphs (b–e, g–k, n, o, q, r) represent samples or measures from independent animals. Error bars represent the standard error of the mean. Statistical significance was assessed using the two-way ANOVA test with Tukey correction for multiple comparisons (a, f); and the one-way ANOVA test with Tukey correction for multiple comparisons in panels b–e, g–I, l, and n–r. P values are indicated when P < 0.05. Source data are provided as a Source Data file. We also performed an indirect calorimetry experiment with the harmol- and rosiglitazone-treated mice. We observed a remarkable decrease in their energy expenditure (Fig. 5j ) and in their VO2 (Fig. S5f ), compared with the vehicle-treated control mice, whereas rosiglitazone-treated mice did not show any alteration in these parameters. We assessed the metabolic flexibility of these mice by calculating the Respiratory Quotient (RQ). As shown in Fig. 5k and previously reported 48 , rosiglitazone-treated mice showed a significant decrease in their RQ, indicating a stronger usage of lipids than control mice. In turn, harmol-treated mice showed no difference in their RQ values compared with control mice. Of note, neither harmol- nor rosiglitazone-treated mice showed alterations in their locomotor activity (Fig. S5g ). After sacrifice, we measured the weight of several tissues at the time of sacrifice. As shown in Fig. 5l , rosiglitazone-treated mice showed a significant increase in their subcutaneous (sWAT) and perigonadal (pWAT) white adipose depots compared to harmol-treated and/or control mice, as expected from their increased body weight and fat mass shown in Fig. 5a, c . Since DIO mice accumulate lipids in their liver, we wondered if harmol treatment could have a beneficial effect in liver lipid content. Indeed, harmol-treated mice displayed less steatotic livers (Fig. 5m ), and accumulated less lipids (Fig. 5n ) and triglycerides (Fig. 5o ) than control mice. In accordance, liver weight tended to be lighter in harmol-treated mice compared with water-treated mice (Fig. 5l ), although this difference did not reach significance. We also measured the plasma levels of adiponectin, an adipokine consistently found downregulated in diabetic individuals in mice 49 and humans 50 . As shown in Fig. 5p , and quantified in Fig. 5q , harmol-treated mice showed a significant increase in their serum adiponectin levels, indicating improved metabolic fitness. To rule out possible toxicity effects of harmol, we performed a histological evaluation of kidney sections (Fig. S5h ), and did not observe any difference between harmol-treated and control mice (Fig. S5i ). In addition, blood urea nitrogen (BUN) was not affected by harmol treatment (Fig. S5j ). These results indicate that chronic treatment with harmol did not affect renal function. To test if increased skeletal muscle mass and/or energy expenditure could be involved in the metabolic effects of harmol, we measured the levels of all isoforms of the muscle protein myosin heavy chain (MHC) in the soleus muscle from mice of the three treatment groups. As shown in Fig. S5k , soleus MHC levels were not changed by the treatment with harmol nor rosiglitazone. Furthermore, we analyzed the muscle fiber type in these soleus skeletal muscles, and we did not detect any change between the treatment groups (Fig. S5l ). Finally, we also measured the accumulation of polyamines in tissues, as the polyamine spermidine was increased in C2C12 myotubes treated with harmol (Fig. 3c ), and spermidine has been associated to autophagy-dependent metabolic fitness in the liver 51 . Mice treated with harmol for 3 months accumulated significantly more spermidine in their livers (Fig. 5r and Supplementary Data  2 ). Treatment with harmol extended lifespan in invertebrates Mitohormetic compounds have also been shown to delay aging and extend lifespan, a conserved process often reported in invertebrate models such as Caenorhabditis elegans and Drosophila melanogaster 52 . These organisms have a much shorter lifespan compared to mammalians, so they are ideal to perform this type of experiment. To test if harmol had any effect in the aging process, we treated Caenorhabditis elegans worms with harmol since birth and quantified their total lifespan. As shown in Fig. 6a , S6a and Supplementary Data  4a , harmol-treated worms lived significantly longer than vehicle (DMSO)-treated controls, showing a median lifespan extension of 37.5%. In this regard, treatment of C. elegans worms with harmol from birth to the end of development slightly decreased phosphorylation of AMPK, although not significantly (Fig. S6b ); did not affect the expression of the telomerase catalytic subunit trt-1 mRNA (Fig. S6c ); nor the mitochondrial/nuclear DNA ratio (Fig. S6d ). We then tested the lifespan in harmol-treated Drosophila melanogaster flies. Again, as shown in Fig. 6b , S6e and Supplementary Data  4b , harmol-treated flies showed a significant lifespan extension of 11.3% compared with control-treated flies. As observed before with worms and mouse cells and tissues, treatment with harmol did not alter the mitochondrial/nuclear DNA ratio in flies (Fig. S6f ). To explore the mechanisms of this harmol-mediated lifespan extension, we measured lifespan in mutant C. elegans worms for the AMPK homologue AAK-2. Ablation of aak-2 in worms completely prevented harmol-mediated lifespan extension (Fig. 6c , S6g and Supplementary Data  4c ). Next, we wanted to explore the relevance of harmol target proteins in harmol-mediated lifespan extension. For this, we treated the worm strain with the deletion of the GABAAR gene homologue unc-49 or with the triple deletion of all MAO homologues amx-1/2/3. Ablation of the GABAAR homologue unc-49 completely abrogated harmol-mediated lifespan (Fig. 6d , S6h and Supplementary Data  4d ). In contrast, harmol treatment maintained its ability to extend lifespan in the absence of the three MAO homologues amx-1/2/3, while treatment with the AMPK inhibitor AICAR actually slightly shortened lifespan (Fig. 6e , S6i and Supplementary Data  4e ). These results support our previous mechanistic insights in cultured cells, indicating that harmol inhibits MAO (and, thus, MAO ablation does not affect harmol effects in lifespan); and that a functional GABAAR is necessary for the mitohormetic properties of harmol. Finally, we wanted to determine if this lifespan extension ability of harmol could be reproduced by modulating harmol target proteins pharmacologically, as partly observed in vitro in Fig. 3 . For this, we treated C. elegans and D. melanogaster with the MAO-B inhibitor selegiline, that has been shown to increase lifespan in rats 53 , and with the GABAAR inverse agonists FG7142. C. elegans worms did not have increased lifespan after treatment with a combination of selegiline and FG7142 (Fig. S6j and Supplementary Data  4f ). However, D. melanogaster flies treated with this combination lived longer than vehicle-treated flies (Fig. 6f and S6k , and Supplementary Data  4g ). We next tested this compound combination in invertebrate models of neurological stress, where MAO and GABAAR modulation may result in more pronounced effects. We first tried an Alzheimer’s model, the GMC101 mutant C. elegans model 54 , 55 . We determined viability of these mutant worms by measuring their crawling speed, and worms moving below a threshold were considered non-viable. As shown in Fig. S6l , treatment with harmol resulted in the highest increase in viability in all the days of measurement, although it only reached significance at day 1. In contrast, treatment with selegiline, FG7142 or a combination of these two compounds only increased viability at day 1, but not at the following days. Finally, we also tested flies carrying the bang senseless (parabss1) mutation, that induces epilepsy 56 . When we treated these flies with the combination of selegiline and FG7142, they lived significantly longer than the vehicle-treated flies (Fig. 6g and S6m and Supplementary Data  4h ). Fig. 6: Treatment with harmol extends lifespan in worms and flies. a–d Kaplan–Meier survival curves including data from at least two independent replicates of the indicated genotypes of hermaphrodite C. elegans worms of the N2 strain treated from birth with vehicle (DMSO, D) or with 15 μg/ml harmol (H) (a, c–e); of the indicated genotypes of Canton-S x w1118 female D. melanogaster flies (b, f, g) treated with vehicle (DMSO) or 25 μg/ml harmol (b); or with vehicle (ethanol, EtOH) or a combination of 0.75 μg/ml selegiline and 10 μg/ml FG7142 (f, g) since birth. The number of individual flies or worms is indicated in each panel. Statistical significance was assessed using the Log-rank (Mantel-Cox) test. Source data are provided as a Source Data file. Treatment with harmol delayed frailty onset in aged mice We next wondered if these age-delaying effects of harmol would also take place in mammals. To test this, we performed the Valencia Score for frailty 57 , 58 on 2-year-old mice before and after treatment with 100 mg/kg harmol in their drinking water for 2 months. Mice treated with harmol did not show any weight loss, indicating that harmol treatment was safe at this age (Fig. S7a ). Recapitulating what we observed in young obese mice (Fig. 5 ), old mice treated with harmol significantly decreased their fasting glycemia, again pointing to an improved glucose tolerance after harmol treatment (Fig. 7a ). Also of interest, mice treated for 2 months with harmol had lower total blood cholesterol (Fig. 7b ). Mice treated with harmol showed a clear tendency to maintain their motor coordination for a longer time in the rotarod test (Fig. 7c ) and to maintain their grip strength with time (Fig. 7d and S7b ), compared with vehicle-treated mice. When muscular strength was tested, harmol-treated mice showed a remarkable improvement compared to vehicle-treated animals, as shown in Fig. 7e, f . When they ran at 75% of their maximal aerobic capacity, harmol-treated mice ran for a longer distance than control-treated mice (Fig. 7g ). Cardiorespiratory fitness, as measured by maximal oxygen uptake (VO2max) was significantly higher in the harmol-treated animals when compared with the vehicle-treated ones (Fig. 7h ). VO2max is a strong and independent predictor of all-cause and disease-specific mortality in humans 59 . Moreover, at maximum speed, harmol-treated mice produced far less lactate than control-treated mice (Fig. 7i ), indicating a later onset of anaerobic glycolysis and better muscular function. We also measured the amount of total myosin heavy chain (MHC) in the soleus muscle from these mice, and we observed increased total MHC levels in harmol-treated mice, more similar to MHC levels in muscle from younger mice (Fig. 7j and S7c ), associated with improved muscle function 60 . In addition, we compared the fiber area and fiber type switch in soleus muscle between water- and harmol-treated old mice. We observed that harmol treatment did not affect the muscle fiber size (Fig. S7d–f ). However, soleus muscles of mice treated with harmol showed a higher percentage of type II fibers and a lower percentage of type I fibers compared to the control group, a fiber pattern associated to improved muscle function (Fig. S7g–h ). When all these parameters were put together and scored according to the Valencia Score, harmol-treated mice scored much lower than control-treated mice (Fig. 7k ), indicating that harmol treatment in aged mice protected from frailty. Fig. 7: Treatment with harmol delayed aging and reduced frailty in old mice. (a–f) Longitudinal assessment of healthspan in 2 year-old male and female C57BL/6JHsdOla mice before (Pre) and after (Post) 2 months of treatment with 100 mg/kg harmol in the drinking water: (a) fasting (5 h) glucose levels (n = 5–7); (b) total blood cholesterol levels at the end of the intervention (n = 6–7); (c) rotarod test (n = 6–7); (d) maximal grip strength in grams relativized by animal body weight (BW) (n = 5–7); (e) maximal carrying load in the ladder climbing test represented as a percentage of the animal BW (n = 6–7); (f) hanging endurance in the grid hanging test (n = 6–7); (g) endurance capacity in a running treadmill test (n = 5–7); (h) maximal oxygen consumption at the end of the intervention (n = 6–7); (i) lactate increment in the VO2max test (n = 6–7). (j) Western blot against total myosin heavy chain (MHC) in protein extracts from soleus muscle of aged male mice treated with the indicated treatments and normalized to actin. (k) Percentage of frail mice (from a total of n = 5–7 at the end of the intervention. Horizontal lines in the dot-plots represent the average of the indicated number of individuals. Dots represent samples or measures from independent animals. Error bars represent the standard error of the mean. Statistical significance was assessed using the two-way ANOVA test with Sidak’s correction for multiple comparisons (a, c–g); the unpaired two-tailed Student t-test (b, h–j); or the Chi square test (k). P values are indicated when P < 0.05. Source data are provided as a Source Data file. Mouse maintenance Animal experiments were conducted at the CNIO (Madrid) and at the University of Valencia, and performed according to protocols approved by the CNIO‐ISCIII Ethics Committee for Research and Animal Welfare (CEIyBA) (PROEX 161/18); and by the University of Valencia Ethics Committee for Research and Animal Welfare (License reference: A1444079171882). All the mice used in this work were of C57BL/6JOlaHsd or C57BL/6J backgrounds and, unless otherwise indicated, were fed with the following chow diets: Teklad Global 18% Protein Rodent Diet (Teklad, 2018S) at the CNIO; 2014 Teklad Global 14% Protein Rodent Maintenance Diet (Teklad, 2914) at the Valencia University. All mouse experiments were performed with male mice, except for the 2- year-old experiment where males and females were used. This sex selection was due to availability of mice and cohort homogeneity. Mice were housed at 22 °C, with 12-h light/dark cycles (7:00–19:00) and 55 +/− 15% humidity, and kept in pathogen-free conditions. In vivo metabolic assays 12-week-old male C57BL/6JOlaHsd mice were rendered pre-diabetic by feeding them with 45% high-fat diet (Research Diets D12451i) for 4 months. For both acute and chronic treatments, harmol and rosiglitazone were dissolved in water. Harmol was purchased from Alfa Aesar (Catalog number: J63827) and rosiglitazone from MedChem Express (Catalog number: HY-17386). For measurement of glucose, ketone bodies, fasting insulin and adiponectin protein quantification experiments, blood was collected from the tail. Glucose and ketone bodies concentrations were determined using Stat Strip Xpress GLU/KET system (Menarini). For fasting insulin and adiponectin protein quantification experiments, plasma was obtained by centrifuging blood at 1500 × g for 5 min. For GTT, mice were fasted for 16 hours (from 17:00 to 09:00). Dextrose was dissolved in PBS and injected intraperitoneally at the concentration of 2 g/kg. For ITT, insulin was injected intraperitoneally at the concentration of 0.75 U/kg. In both assays, blood was collected from the tail vein and glucose levels were measured at the indicated times. Fasting insulin quantification was performed using Ultra Sensitive Mouse Insulin ELISA kit (Crystal Chem Inc - 90080). HOMA–IR was calculated using the following equation (HOMA-IR = [(fasting insulin, µU/mL) × (fasting glucose, mg/dl] / 405) 101 . Body composition (fat and lean content) was determined by dual energy X-ray Absorptiometry (DEXA) using a Lunar PIXImus Densitometer (GE Medical Systems). Image acquisition lasted 5 min with mice under anesthesia by inhalation of 2% isofluorane. Image analysis was performed using the PIXImus II Series Densitometers software version 1.46.007 (GEHC). Mouse behavioral analysis Adult C57BL/6J male mice (3–4 month old) were used for behavioral tests. Mice were maintained under standard housing conditions in 12 h dark/light cycles with food and water ad libitum. Harmol-treated mice were given 1.17 mg/ml harmol in their drinking water ad libitum for 3 weeks (a final dose of 100 mg/kg/day harmol), while control mice were given plain water. All tests were implemented during the light phase with experimenter blind to condition. Mice were handled and habituated to the experimenter for 3 days before the behavioral assays. All behavioral tests were separated at least by 24 h. The order of tests was as follows: open field, elevated plus maze (EPM) and dark-light box. Testing apparatuses were cleaned with a solution of 70% ethanol in water after each trial to avoid olfactory cues. Behavioral tasks were recorded and analyzed as previously described 102 . Open field. Spontaneous locomotor activity was measured in an open field apparatus. It consisted of a rectangular chamber of 48 × 48 × 48 cm that was made of plastic under uniform light conditions (70 lux). Mice were allowed to explore the arena for 10 minutes. The arena was delimited for analysis into two different regions: center (square area of 30 × 30 cm equidistant from the walls) and the remaining borders. The time spent, velocity, distance as well as transitions between the zones was calculated as described in 102 . Elevated plus maze. The elevated plus maze consisted of four arms (50 × 10 cm) elevated 50 cm above the floor. The plus maze had two closed arms with black acrylic glass walls (30 cm high) and two open (wall-free) arms connected by a central platform. Indirect illumination provided 70 lux to the open arms and 15 lux to the closed arms. Mice were gently placed in the center of the maze and their behavior was recorded for 5 minutes. Time spent and the number of entries in each arm was quantified post hoc 102 . Dark-light box. The test apparatus consisted of two boxes (25 × 25 cm each) connected by a small aperture: the light box (open box with direct illumination of 450 lux) and the dark box (opaque). Mice were placed facing the dark box and their behavior was recorded for 5 min. Mice were video-tracked and ratio of time in dark box over time in light box was calculated post hoc 102 . Longitudinal assessment of healthspan in old mice Thirteen 23-month-old C57BL/6J mice (9 males, 4 females) were randomly divided in two groups: control (n = 6; 4 males, 2 females) and harmol-treated (n = 7; 5 males, 2 females). A dose of 100 mg/kg of harmol was dissolved in water and administered to the treated group for 8 weeks. All mice were evaluated before and after the intervention for different functional parameters to assess frailty. Animals were acclimated to all the performed tests with 2 sessions of habituation before the evaluation day. Body weight, food and drink intake. Animals body weight and their food and drink intake were recorded every 7 days using a PB3002 Delta Range balance (Mettler Scales, Toledo, OH). Fasting glycemia and total cholesterol. For the measurement of glycemia, mice were fasted for 5 h (from 7:00 to 12:00); for the measurement of total cholesterol, mice were fasted for 12 h (from 21:00 to 09:00). We collected blood from the saphenous vein and measured both parameters by using a Multicare Lux System (Model 3820001A). Motor coordination. We used the Rotarod (Panlab, Harvard Apparatus) to evaluate motor coordination. The protocol consisted in increasing the speed of the Rotarod from 4 rpm to 40 rpm in 5 min, and the time that the mouse was able to stay on the rod until falling was recorded. Each animal performed three attempts with 5 min of rest between each attempt and the final data recorded was the maximal time achieved in any of the tests. Grip strength. Maximal grip strength was evaluated by using the Grip Strength Meter (Panlab, Harvard Apparatus). The mice were held by the base of the tail by allowing them to grasp the “T” drawbar of the apparatus with their front paws for a few seconds. The peak amount of force applied was automatically registered in grams by the apparatus. Each mouse performed 3 sets of 10 consecutive attempts with 5 min of rest between each set, and the best performance was recorded as maximal grip strength. Since the body weight of mouse influences its force, the grip strength was relativized by dividing the animal weight. Ladder climbing test. We followed a modification of the protocol of Hornberger and Farrar 103 to evaluate maximal strength. Ladder climbing (1 × 0.18 m, 1 cm grid, 80° incline) was used to assess maximal carrying load with weights secured with adhesive strip to the mouse proximal portion of the tail. The initial climb consisted of carrying a load that was 75% of the animal body weight. Each animal needed ~40 dynamic movements for climbing the entire length of the ladder. When mice reached the top of the ladder, they rested for 5 min. The load was increased by 25% of the mouse body weight in each repetition until the load prevented the animal from climbing the entire ladder. Failure was established after three non-successful attempts to reach at least two-thirds of the ladder. The highest load successfully carried over was considered the maximal carrying load and was represented as a percentage of the mice body weight. Grid hanging test. This is a widely used test that can be considered a more robust assay than grip strength in aging studies because its improvements as a result of geroprotective interventions has been previously demonstrated 104 . A horizontal rectangular grid was used to measure the muscular strength and hanging endurance against the gravitational force with the four limbs of the mouse. The animal was placed on the grid and it was quickly turned upside down. The grid was at a distance of 1 m from the floor with a soft pad underneath to absorb the impact of the fall. We recorded the latency time to fall (in seconds) three times, allowing the mice to rest for 5 min in between. The best time was considered for the statistical analysis. Maximal oxygen consumption. A treadmill for mice with an indirect calorimetry analyzer (Panlab, Harvard Apparatus, Columbus Instruments, Oxylet Pro LE405 gas analyzer) was used for the measurement of their maximal oxygen consumption (VO2max, ml/min/kg0.75) during an incremental treadmill test. After a warm-up period of 4 min at 6 m/min, the treadmill band velocity was increased by 2.4 m/min every 2 min until exhaustion. Total running time (min) and maximal running speed (m/min) were recorded. The maximal running speed was considered the maximal aerobic workload capacity of the animal 105 . The increase in blood lactate concentration was studied by obtaining blood from the saphenous vein 5 min after the end of the test and comparing with the value at rest. Endurance capacity. From the maximal running speed achieved in the VO2max test, mice performed a continuous running treadmill test to evaluate their endurance. After an incremental warm-up period of 7 min, each mouse ran at 75% of its maximal running speed. Total running distance to exhaustion (km) was registered. Frailty assessment (Valencia Score). The “Valencia Score” was performed to evaluate frailty in the old animals. This score is based on the previous one for frailty developed for humans by Linda Fried and co-workers 106 . The score includes the measurement of the following five components: unintentional weight loss (change in body weight), low activity level (motor coordination), weakness (grip strength), poor endurance (total running time in the incremental treadmill test), and slowness (maximal running speed in the incremental treadmill test). The frailty score for each age group of animals is calculated as follows: total number of tests failed by the animals at each age group, divided by the total number of tests performed by these animals, expressed in percentage. For more details see refs. 57 , 58 . Caenorhabditis elegans experiments No ethical approval was required for experiments using invertebrate animals, which have been performed according to EU and national legislation as well as local biosafety regulations. C. elegans strains N2 Bristol, GMC101 (dvIs100 [unc-54p::A-beta-1-42::unc-54 3’-UTR + mtl-2p::GFP]), aak-2(ok524), RB834 amx-1(ok659), RB1190 amx-2(ok1235), RB1533 amx-3(ok1838), unc-49(e407), and E. coli strain OP50 were obtained from the Caenorhabditis Genetics Center (CGC, University of Minnesota). amx-1, amx-2, and amx-3 mutants were gene

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Senseonics (NYSE: SENS) operates as a medical device startup company. It develops and commercializes transformative glucose monitoring products that enable people with diabetes. It utilizes fluorescence sensing technology for diabetes management. It was formerly known as Sensors for Medicine and Science. The company was founded in 2011 and is based in Germantown, Maryland.

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