Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene.

Serine/arginine-rich splicing factors (SRSFs) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins in regulating mitochondrial activity has already been shown for SRSF6, but SRSF4 altered expression has never been reported as a cause of bone marrow failure. An 8-year-old patient admitted to the hematology unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance of the SRSF4 gene (p.R235W) found via whole genome sequencing analysis and inherited from the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression and altered mitochondrial function and energetic metabolism in primary lymphocytes and Epstein-Barr-virus (EBV)-immortalized lymphoblasts compared to healthy donor (HD) cells, which appeared associated with low mTOR phosphorylation and an imbalance in the proteins regulating mitochondrial biogenesis (i.e., CLUH) and dynamics (i.e., DRP1 and OPA1). Transfection with the wtSRSF4 gene restored mitochondrial function. In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to mitochondrial dysfunction. Since mitochondrial function is crucial for hematopoietic stem cell maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, the SRSF4 mutation could have substantially contributed to the clinical phenotype of our patient.

Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Cyclic fasting bolsters cholesterol biosynthesis inhibitors' anticancer activity.

Identifying oncological applications for drugs that are already approved for other medical indications is considered a possible solution for the increasing costs of cancer treatment. Under the hypothesis that nutritional stress through fasting might enhance the antitumour properties of at least some non-oncological agents, by screening drug libraries, we find that cholesterol biosynthesis inhibitors (CBIs), including simvastatin, have increased activity against cancers of different histology under fasting conditions. We show fasting's ability to increase CBIs' antitumour effects to depend on the reduction in circulating insulin, insulin-like growth factor-1 and leptin, which blunts the expression of enzymes from the cholesterol biosynthesis pathway and enhances cholesterol efflux from cancer cells. Ultimately, low cholesterol levels through combined fasting and CBIs reduce AKT and STAT3 activity, oxidative phosphorylation and energy stores in the tumour. Our results support further studies of CBIs in combination with fasting-based dietary regimens in cancer treatment and highlight the value of fasting for drug repurposing in oncology.

Terpyridine Functionalized Cyclodextrin Nanoparticles as Carriers for Doxorubicin: Analysis of In Vitro Antiproliferative Activity.

BACKGROUND/AIM: We have recently described the development of cyclodextrin-based nanoparticles (NPs) functionalized with terpyridine and decorated with biotin-terpyridine ligands via Cu(II) and Fe(II) coordination. In the present study, we report the performance of these novel NPs as a delivery system for anticancer drugs. In particular, we analyzed the feasibility of loading these new NPs with the topoisomerase II inhibitor Doxorubicin (Doxo), still administered to patients to treat different forms of cancers. We developed Doxo-encapsulated polymeric NPS to generate nanoformulations with higher efficacy than free Doxo. MATERIALS AND METHODS: We investigated the inhibition of cell proliferation in A2780, A549, SKHep1, and MDA-MB-453 cancer cell lines using the MTT assay. RESULTS: NPs loaded with Doxo displayed higher antiproliferative activity than free Doxo. CONCLUSION: The NPs generated in this study inhibited the proliferation of cancer cells and were able to entrap the classic anticancer drug Doxo. The Doxo-loaded NP showed increased cytotoxicity in comparison to free Doxo.

Crosstalk between the Rod Outer Segments and Retinal Pigmented Epithelium in the Generation of Oxidative Stress in an In Vitro Model.

Dysfunction of the retinal pigment epithelium (RPE) is associated with several diseases characterized by retinal degeneration, such as diabetic retinopathy (DR). However, it has recently been proposed that outer retinal neurons also participate in the damage triggering. Therefore, we have evaluated the possible crosstalk between RPE and photoreceptors in priming and maintaining oxidative damage of the RPE. For this purpose, we used ARPE-19 cells as a model of human RPE, grown in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) and unoxidized (UOx) or oxidized (Ox) mammalian retinal rod outer segments (OSs). ARPE-19 cells were efficient at phagocytizing rod OSs in both NG and HG settings. However, in HG, ARPE-19 cells treated with Ox-rod OSs accumulated MDA and lipofuscins and displayed altered LC3, GRP78, and caspase 8 expression compared to untreated and UOx-rod-OS-treated cells. Data suggest that early oxidative damage may originate from the photoreceptors and subsequently extend to the RPE, providing a new perspective to the idea that retinal degeneration depends solely on a redox alteration of the RPE.

Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.

Plasma-derived extracellular vesicles released after endurance exercise exert cardioprotective activity through the activation of antioxidant pathways.

Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described. The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO2max): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre_EVs) and immediately after a single bout of endurance exercise (30' treadmill, 70% heart rate (HR) -Post_EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post_EVs, compare to Pre_EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post_EVs. At functional level, Post_EVs, but not Pre_EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H2O2) treatment), resulting in a global cardioprotective effect. In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity.

Effects of Deacetylase Inhibition on the Activation of the Antioxidant Response and Aerobic Metabolism in Cellular Models of Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by a dysfunctional DNA repair and an oxidative stress accumulation due to defective mitochondrial energy metabolism, not counteracted by endogenous antioxidant defenses, which appear down-expressed compared to the control. Since the antioxidant response lack could depend on the hypoacetylation of genes coding for detoxifying enzymes, we treated lymphoblasts and fibroblasts mutated for the FANC-A gene with some histone deacetylase inhibitors (HDACi), namely, valproic acid (VPA), beta-hydroxybutyrate (OHB), and EX527 (a Sirt1 inhibitor), under basal conditions and after hydrogen peroxide addition. The results show that VPA increased catalase and glutathione reductase expression and activity, corrected the metabolic defect, lowered lipid peroxidation, restored the mitochondrial fusion and fission balance, and improved mitomycin survival. In contrast, OHB, despite a slight increase in antioxidant enzyme expressions, exacerbated the metabolic defect, increasing oxidative stress production, probably because it also acts as an oxidative phosphorylation metabolite, while EX527 showed no effect. In conclusion, the data suggest that VPA could be a promising drug to modulate the gene expression in FA cells, confirming that the antioxidant response modulation plays a pivotal in FA pathogenesis as it acts on both oxidative stress levels and the mitochondrial metabolism and dynamics quality.

Altered Mitochondrial Dynamic in Lymphoblasts and Fibroblasts Mutated for FANCA-A Gene: The Central Role of DRP1.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and aplastic anemia. So far, 23 genes are involved in this pathology, and their mutations lead to a defect in DNA repair. In recent years, it has been observed that FA cells also display mitochondrial metabolism defects, causing an accumulation of intracellular lipids and oxidative damage. However, the molecular mechanisms involved in the metabolic alterations have not yet been elucidated. In this work, by using lymphoblasts and fibroblasts mutated for the FANC-A gene, oxidative phosphorylation (OxPhos) and mitochondria dynamics markers expression was analyzed. Results show that the metabolic defect does not depend on an altered expression of the proteins involved in OxPhos. However, FA cells are characterized by increased uncoupling protein UCP2 expression. FANC-A mutation is also associated with DRP1 overexpression that causes an imbalance in the mitochondrial dynamic toward fission and lower expression of Parkin and Beclin1. Treatment with P110, a specific inhibitor of DRP1, shows a partial mitochondrial function recovery and the decrement of DRP1 and UCP2 expression, suggesting a pivotal role of the mitochondrial dynamics in the etiopathology of Fanconi anemia.

Lubeluzole Repositioning as Chemosensitizing Agent on Multidrug-Resistant Human Ovarian A2780/DX3 Cancer Cells.

In a previous paper, we demonstrated the synergistic action of the anti-ischemic lubeluzole (Lube S) on the cytotoxic activity of doxorubicin (Dox) and paclitaxel in human ovarian cancer A2780 and lung cancer A549 cells. In the present paper, we extended in vitro the study to the multi-drug-resistant A2780/DX3 cell line to verify the hypothesis that the Dox and Lube S drug association may potentiate the antitumor activity of this anticancer compound also in the context of drug resistance. We also evaluated some possible mechanisms underlying this activity. We analyzed the antiproliferative activity in different cancer cell lines. Furthermore, apoptosis, Dox accumulation, MDR1 downregulation, ROS, and NO production in A2780/DX3 cells were also evaluated. Our results confirm that Lube S improves Dox antiproliferative and apoptotic activities through different mechanisms of action, all of which may contribute to the final antitumor effect. Moderate stereoselectivity was found, with Lube S significantly more effective than its enantiomer (Lube R) and the corresponding racemate (Lube S/R). Docking simulation studies on the ABCB1 Cryo-EM structure supported the hypothesis that Lube S forms a stable MDR1-Dox-Lube S complex, which hampers the protein transmembrane domain flipping and blocks the efflux of Dox from resistant A2780/DX3 cells. In conclusion, our in vitro studies reinforce our previous hypothesis for repositioning the anti-ischemic Lube S as a potentiating agent in anticancer chemotherapy.

Preconditioned Mesenchymal Stromal Cell-Derived Extracellular Vesicles (EVs) Counteract Inflammaging.

Inflammaging is one of the evolutionarily conserved mechanisms underlying aging and is defined as the long-term consequence of the chronic stimulation of the innate immune system. As macrophages are intimately involved in initiating and regulating the inflammatory process, their dysregulation plays major roles in inflammaging. The paracrine factors, and in particular extracellular vesicles (EVs), released by mesenchymal stromal cells (MSCs) retain immunoregulatory effects on innate and adaptive immune responses. In this paper, we demonstrate that EVs derived from MSCs preconditioned with hypoxia inflammatory cytokines exerted an anti-inflammatory role in the context of inflammaging. In this study, macrophages isolated from aged mice presented elevated pro-inflammatory factor levels already in basal conditions compared to the young counterpart, and this pre-activation status increased when cells were challenged with IFN-γ. EVs were able to attenuate the age-associated inflammation, inducing a decrease in the expression of TNF-α, iNOS, and the NADase CD38. Moreover, we demonstrate that EVs counteracted the mitochondrial dysfunction that affected the macrophages, reducing lipid peroxidation and hindering the age-associated impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis. These results indicate that preconditioned MSC-derived EVs might be exploited as new anti-aging therapies in a variety of age-related diseases.

Mutated FANCA Gene Role in the Modulation of Energy Metabolism and Mitochondrial Dynamics in Head and Neck Squamous Cell Carcinoma.

Fanconi Anaemia (FA) is a rare recessive genetic disorder characterized by a defective DNA repair mechanism. Although aplastic anaemia is the principal clinical sign in FA, patients develop a head and neck squamous cell carcinoma (HNSCC) with a frequency 500-700 folds higher than the general population, which appears more aggressive, with survival of under two years. Since FA gene mutations are also associated with a defect in the aerobic metabolism and an increased oxidative stress accumulation, this work aims to evaluate the effect of FANCA mutation on the energy metabolism and the relative mitochondrial quality control pathways in an HNSCC cellular model. Energy metabolism and cellular antioxidant capacities were evaluated by oximetric, luminometric, and spectrophotometric assays. The dynamics of the mitochondrial network, the quality of mitophagy and autophagy, and DNA double-strand damage were analysed by Western blot analysis. Data show that the HNSCC cellular model carrying the FANCA gene mutation displays an altered electron transport between respiratory Complexes I and III that does not depend on the OxPhos protein expression. Moreover, FANCA HNSCC cells show an imbalance between fusion and fission processes and alterations in autophagy and mitophagy pathways. Together, all these alterations associated with the FANCA gene mutation cause cellular energy depletion and a metabolic switch to glycolysis, exacerbating the Warburg effect in HNSCC cells and increasing the growth rate. In addition, the altered DNA repair due to the FANCA mutation causes a higher accumulation of DNA damage in the HNSCC cellular model. In conclusion, changes in energy metabolism and mitochondrial dynamics could explain the strict correlation between HNSCC and FA genes, helping to identify new therapeutic targets.

Identification of NAPRT Inhibitors with Anti-Cancer Properties by In Silico Drug Discovery.

Depriving cancer cells of sufficient NAD levels, mainly through interfering with their NAD-producing capacity, has been conceived as a promising anti-cancer strategy. Numerous inhibitors of the NAD-producing enzyme, nicotinamide phosphoribosyltransferase (NAMPT), have been developed over the past two decades. However, their limited anti-cancer activity in clinical trials raised the possibility that cancer cells may also exploit alternative NAD-producing enzymes. Recent studies show the relevance of nicotinic acid phosphoribosyltransferase (NAPRT), the rate-limiting enzyme of the Preiss-Handler NAD-production pathway for a large group of human cancers. We demonstrated that the NAPRT inhibitor 2-hydroxynicotinic acid (2-HNA) cooperates with the NAMPT inhibitor FK866 in killing NAPRT-proficient cancer cells that were otherwise insensitive to FK866 alone. Despite this emerging relevance of NAPRT as a potential target in cancer therapy, very few NAPRT inhibitors exist. Starting from a high-throughput virtual screening approach, we were able to identify and annotate two additional chemical scaffolds that function as NAPRT inhibitors. These compounds show comparable anti-cancer activity to 2-HNA and improved predicted aqueous solubility, in addition to demonstrating favorable drug-like profiles.

Terpyridine functionalized cyclodextrin nanoparticles: metal coordination for tuning anticancer activity.

Multi-metal and multi-cavity systems based on the coordination properties of terpyridine functionalized cyclodextrin polymers were synthesized and characterized. Nanoparticles decorated with terpyridine derivatives via metal coordination showed high antiproliferative activity in tumor cells.

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells.

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.

Fundamental Role of Pentose Phosphate Pathway within the Endoplasmic Reticulum in Glutamine Addiction of Triple-Negative Breast Cancer Cells.

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER-mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation.

808-nm Photobiomodulation Affects the Viability of a Head and Neck Squamous Carcinoma Cellular Model, Acting on Energy Metabolism and Oxidative Stress Production.

Photobiomodulation (PBM) is a form of low-dose light therapy that acts through energy delivery from non-ionizing sources. During the recent two decades, there has been tremendous progress with PBM acceptance in medicine. However, PBM effects on potential stimulation of existing malignant or pre-malignant cells remain unknown. Thus, the primary endpoint was to assess the safety of PBM treatment parameters on head and neck squamous cell carcinoma (HNSCC) proliferation or survival. The secondary endpoint was to assess any putative anti-cancer effects of PBM treatments. Cell viability, energy metabolism, oxidative stress, and pro- and anti-apoptotic markers expression were investigated on a Human Head and Neck Squamous Cell Carcinoma cellular model (OHSU-974 FAcorr cell line). PBM therapy was administered through the 810 nm diode laser (GaAlAs) device (Garda Laser, 7024 Negrar, Verona, Italy) at the powers of 0, 0.25, 0.50, 0.75, 1.00, or 1.25 W in continuous wave (CW) mode for an exposure time of 60 s with a spot-size of 1 cm2 and with a distance of 1.86 cm from the cells. Results showed that 810-nm PBM affected oxidative phosphorylation in OHSU-971 FAcorr, causing a metabolic switch to anaerobic glycolysis. In addition, PBM reduced the catalase activity, determining an unbalance between oxidative stress production and the antioxidant defenses, which could stimulate the pro-apoptotic cellular pathways. Our data, at the parameters investigated, suggest the safeness of PBM as a supportive cancer therapy. Pre-clinical and clinical studies are necessary to confirm the in vitro evidence.

Cyclodextrin Polymers as Delivery Systems for Targeted Anti-Cancer Chemotherapy.

In the few last years, nanosystems have emerged as a potential therapeutic approach to improve the efficacy and selectivity of many drugs. Cyclodextrins (CyDs) and their nanoparticles have been widely investigated as drug delivery systems. The covalent functionalization of CyD polymer nanoparticles with targeting molecules can improve the therapeutic potential of this family of nanosystems. In this study, we investigated cross-linked γ- and ß-cyclodextrin polymers as carriers for doxorubicin (ox) and oxaliplatin (Oxa). We also functionalized γ-CyD polymer bearing COOH functionalities with arginine-glycine-aspartic or arginine moieties for targeting the integrin receptors of cancer cells. We tested the Dox and Oxa anti-proliferative activity in the presence of the precursor polymer with COOH functionalities and its derivatives in A549 (lung, carcinoma) and HepG2 (liver, carcinoma) cell lines. We found that CyD polymers can significantly improve the antiproliferative activity of Dox in HepG2 cell lines only, whereas the cytotoxic activity of Oxa resulted as enhanced in both cell lines. The peptide or amino acid functionalized CyD polymers, loaded with Dox, did not show any additional effect compared to the precursor polymer. Finally, studies of Dox uptake showed that the higher antiproliferative activity of complexes correlates with the higher accumulation of Dox inside the cells. The results show that CyD polymers could be used as carriers for repositioning classical anticancer drugs such as Dox or Oxa to increase their antitumor activity.

The Hormetic Effect of Metformin: "Less Is More"?

Metformin (MTF) is the first-line therapy for type 2 diabetes (T2DM). The euglycemic effect of MTF is due to the inhibition of hepatic glucose production. Literature reports that the principal molecular mechanism of MTF is the activation of 5'-AMP-activated protein kinase (AMPK) due to the decrement of ATP intracellular content consequent to the inhibition of Complex I, although this effect is obtained only at millimolar concentrations. Conversely, micromolar MTF seems to activate the mitochondrial electron transport chain, increasing ATP production and limiting oxidative stress. This evidence sustains the idea that MTF exerts a hormetic effect based on its concentration in the target tissue. Therefore, in this review we describe the effects of MTF on T2DM on the principal target organs, such as liver, gut, adipose tissue, endothelium, heart, and skeletal muscle. In particular, data indicate that all organs, except the gut, accumulate MTF in the micromolar range when administered in therapeutic doses, unmasking molecular mechanisms that do not depend on Complex I inhibition.