Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Sirtuin 6 (SIRT6) is a sirtuin family member involved in a wide range of physiologic and disease processes, including cancer and glucose homeostasis. Based on the roles played by SIRT6 in different organs, including its ability to repress the expression of glucose transporters and glycolytic enzymes, inhibiting SIRT6 has been proposed as an approach for treating type 2 diabetes mellitus (T2DM). However, so far, the lack of small-molecule Sirt6 inhibitors has hampered the conduct of in vivo studies to assess the viability of this strategy. We took advantage of a recently identified SIRT6 inhibitor, compound 1, to study the effect of pharmacological Sirt6 inhibition in a mouse model of T2DM (i.e., in high-fat-diet-fed animals). The administration of the Sirt6 inhibitor for 10 d was well tolerated and improved oral glucose tolerance, it increased the expression of the glucose transporters GLUT1 and -4 in the muscle and enhanced the activity of the glycolytic pathway. Sirt6 inhibition also resulted in reduced insulin, triglycerides, and cholesterol levels in plasma. This study represents the first in vivo study of a SIRT6 inhibitor and provides the proof-of-concept that targeting SIRT6 may be a viable strategy for improving glycemic control in T2DM.-Sociali, G., Magnone, M., Ravera, S., Damonte, P., Vigliarolo, T., Von Holtey, M., Vellone, V. G., Millo, E., Caffa, I., Cea, M., Parenti, M. D., Del Rio, A., Murone, M., Mostoslavsky, R., Grozio, A., Nencioni, A., Bruzzone S. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Abscisic acid enhances cold tolerance in honeybee larvae.

The natural composition of nutrients present in food is a key factor determining the immune function and stress responses in the honeybee (Apis mellifera). We previously demonstrated that a supplement of abscisic acid (ABA), a natural component of nectar, pollen, and honey, increases honeybee colony survival overwinter. Here we further explored the role of ABA in in vitro-reared larvae exposed to low temperatures. Four-day-old larvae (L4) exposed to 25°C for 3 days showed lower survival rates and delayed development compared to individuals growing at a standard temperature (34°C). Cold-stressed larvae maintained higher levels of ABA for longer than do larvae reared at 34°C, suggesting a biological significance for ABA. Larvae fed with an ABA-supplemented diet completely prevent the low survival rate due to cold stress and accelerate adult emergence. ABA modulates the expression of genes involved in metabolic adjustments and stress responses: Hexamerin 70b, Insulin Receptor Substrate, Vitellogenin, and Heat Shock Proteins 70. AmLANCL2, the honeybee ABA receptor, is also regulated by cold stress and ABA. These results support a role for ABA increasing the tolerance of honeybee larvae to low temperatures through priming effects.

Abscisic acid enhances glucose disposal and induces brown fat activity in adipocytes in vitro and in vivo.

Abscisic acid (ABA) is a plant hormone also present in animals, where it is involved in the regulation of innate immune cell function and of glucose disposal, through its receptor LANCL2. ABA stimulates glucose uptake by myocytes and pre-adipocytes in vitro and oral ABA improves glycemic control in rats and in healthy subjects. Here we investigated the role of the ABA/LANCL2 system in the regulation of glucose uptake and metabolism in adipocytes. Silencing of LANCL2 abrogated both the ABA- and insulin-induced increase of glucose transporter-4 expression and of glucose uptake in differentiated 3T3-L1 murine adipocytes; conversely, overexpression of LANCL2 enhanced basal, ABA- and insulin-stimulated glucose uptake. As compared with insulin, ABA treatment of adipocytes induced lower triglyceride accumulation, CO2 production and glucose-derived fatty acid synthesis. ABA per se did not induce pre-adipocyte differentiation in vitro, but stimulated adipocyte remodeling in terminally differentiated cells, with a reduction in cell size, increased mitochondrial content, enhanced O2 consumption, increased transcription of adiponectin and of brown adipose tissue (BAT) genes. A single dose of oral ABA (1µg/kg body weight) increased BAT glucose uptake 2-fold in treated rats compared with untreated controls. One-month-long ABA treatment at the same daily dose significantly upregulated expression of BAT markers in the WAT and in WAT-derived preadipocytes from treated mice compared with untreated controls. These results indicate a hitherto unknown role of LANCL2 in adipocyte sensitivity to insulin-stimulated glucose uptake and suggest a role for ABA in the induction and maintenance of BAT activity.

Abscisic acid transport in human erythrocytes.

Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic ß-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [(3)H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [(3)H]ABA and [(35)S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone.

Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.

Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.

The ABA-LANCL1/2 Hormone-Receptors System Protects H9c2 Cardiomyocytes from Hypoxia-Induced Mitochondrial Injury via an AMPK- and NO-Mediated Mechanism.

Abscisic acid (ABA) regulates plant responses to stress, partly via NO. In mammals, ABA stimulates NO production by innate immune cells and keratinocytes, glucose uptake and mitochondrial respiration by skeletal myocytes and improves blood glucose homeostasis through its receptors LANCL1 and LANCL2. We hypothesized a role for the ABA-LANCL1/2 system in cardiomyocyte protection from hypoxia via NO. The effect of ABA and of the silencing or overexpression of LANCL1 and LANCL2 were investigated in H9c2 rat cardiomyoblasts under normoxia or hypoxia/reoxygenation. In H9c2, hypoxia induced ABA release, and ABA stimulated NO production. ABA increased the survival of H9c2 to hypoxia, and L-NAME, an inhibitor of NO synthase (NOS), abrogated this effect. ABA also increased glucose uptake and NADPH levels and increased phosphorylation of Akt, AMPK and eNOS. Overexpression or silencing of LANCL1/2 significantly increased or decreased, respectively, transcription, expression and phosphorylation of AMPK, Akt and eNOS; transcription of NAMPT, Sirt1 and the arginine transporter. The mitochondrial proton gradient and cell vitality increased in LANCL1/2-overexpressing vs. -silenced cells after hypoxia/reoxygenation, and L-NAME abrogated this difference. These results implicate the ABA-LANCL1/2 hormone-receptor system in NO-mediated cardiomyocyte protection against hypoxia.

Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes.

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response. Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E(2) (PGE(2)) by human granulocytes, release of PGE(2) and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.

Binding of abscisic acid to human LANCL2.

The phytohormone abscisic acid (ABA) is the central regulator of abiotic stress in plants and plays important roles during plant growth and development. In animal cells, ABA was shown to be an endogenous hormone, acting as a stress signal and stimulating cell functions involved in inflammatory responses and in insulin release. Recently, we demonstrated that Lanthionine synthetase component C-like protein 2 (LANCL2) is required for ABA binding to the plasmamembrane of granulocytes and for the activation of the signaling pathway triggered by ABA in human granulocytes and in rat insulinoma cells. In order to investigate whether ABA activates LANCL2 via direct interaction, we performed specific binding studies on human LANCL2 recombinant protein using different experimental approaches (saturation binding, scintillation proximity assays, dot blot experiments and affinity chromatography). Altogether, results indicate that human recombinant LANCL2 binds ABA directly and provide the first demonstration of ABA binding to a mammalian ABA receptor.

Identification of Biochemical and Molecular Markers of Early Aging in Childhood Cancer Survivors.

Survival rates of childhood cancer patients have improved over the past four decades, although cancer treatments increase the risk of developing chronic diseases typical of aging. Thus, we aimed to identify molecular/metabolic cellular alterations responsible for early aging in childhood cancer survivors (CCS). Biochemical, proteomic, and molecular biology analyses were conducted on mononuclear cells (MNCs) isolated from peripheral blood of 196 CCS, the results being compared with those obtained on MNCs of 154 healthy subjects. CCS-MNCs showed inefficient oxidative phosphorylation associated with low energy status, and increased lipid peroxidation and lactate fermentation compared with age-matched normal controls. According to a mathematical model based on biochemical parameters, CCS-MNCs showed significantly higher metabolic ages than their real ages. The dysfunctional metabolism of CCS-MNCs is associated with lower expression levels of genes and proteins involved in mitochondrial biogenesis and metabolism regulation, such as CLUH, PGC1-alpha, and SIRT6 in CCS, not observed in the age-matched healthy or elderly subjects. In conclusion, our study identified some biochemical and molecular alterations possibly contributing to the pathophysiology of aging and metabolic deficiencies in CCS. These results identify new targets for pharmacological interventions to restore mitochondrial function, slowing down the aging-associated pathologies in CCS.

Insulin-independent stimulation of skeletal muscle glucose uptake by low-dose abscisic acid via AMPK activation.

Abscisic acid (ABA) is a plant hormone active also in mammals where it regulates, at nanomolar concentrations, blood glucose homeostasis. Here we investigated the mechanism through which low-dose ABA controls glycemia and glucose fate. ABA stimulated uptake of the fluorescent glucose analog 2-NBDG by L6, and of [18F]-deoxy-glucose (FDG) by mouse skeletal muscle, in the absence of insulin, and both effects were abrogated by the specific AMPK inhibitor dorsomorphin. In L6, incubation with ABA increased phosphorylation of AMPK and upregulated PGC-1α expression. LANCL2 silencing reduced all these ABA-induced effects. In vivo, low-dose oral ABA stimulated glucose uptake and storage in the skeletal muscle of rats undergoing an oral glucose load, as detected by micro-PET. Chronic treatment with ABA significantly improved the AUC of glycemia and muscle glycogen content in CD1 mice exposed to a high-glucose diet. Finally, both acute and chronic ABA treatment of hypoinsulinemic TRPM2-/- mice ameliorated the glycemia profile and increased muscle glycogen storage. Altogether, these results suggest that low-dose oral ABA might be beneficial for pre-diabetic and diabetic subjects by increasing insulin-independent skeletal muscle glucose disposal through an AMPK-mediated mechanism.

Chronic Intake of Micrograms of Abscisic Acid Improves Glycemia and Lipidemia in a Human Study and in High-Glucose Fed Mice.

We tested the effect of chronic low-dose abscisic acid (ABA), a phytohormone-regulating human glucose tolerance, on the metabolic parameters that are dysregulated in prediabetes and metabolic syndrome (MS).Ten healthy subjects received 1 µg ABA/Kg body weight (BW)/day as an ABA-rich food supplement: (i) the glycemia profile after a carbohydrate-rich meal, with or without supplement, was compared; (ii) fasting blood glucose (FBG), glycated hemoglobin (HbA1c), total cholesterol (TC), and body mass index (BMI) after 75 days of daily supplementation of a habitual Mediterranean diet were compared with starting values.CD1 mice were fed a high-glucose diet with or without synthetic ABA (1 µg/Kg BW) for 4 months and the same parameters investigated in the human study were compared. The food supplement significantly reduced the area under the curve of glycemia after a carbohydrate-rich meal and FBG, HbA1c, TC, and BMI after chronic treatment. ABA-treated mice showed a significant reduction of HbA1c, TC, and body weight gain compared with untreated controls. The combined results from the human and murine studies allow us to conclude that the observed improvement of the metabolic parameters can be attributed to ABA and to advocate the use of ABA-containing food supplements in prediabetes and/or MS.

Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2.

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ±â€¯1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.

Abscisic acid influx into human nucleated cells occurs through the anion exchanger AE2.

Abscisic acid (ABA) is a hormone conserved from cyanobacteria to higher plants, where it regulates responses to environmental stimuli. ABA also plays a role in mammalian physiology, pointedly in inflammatory responses and in glycemic control. As the animal ABA receptor is on the intracellular side of the plasma membrane, a transporter is required for the hormone's action. Here we demonstrate that ABA transport in human nucleated cells occurs via the anion exchanger AE2. Together with the recent demonstration that ABA influx into human erythrocytes occurs via Band 3, this result identifies the AE family members as the mammalian ABA transporters.

G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2.

Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation.

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model.

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, ß-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.

Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.

The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 µM and 0.80 ± 0.07 µM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.