Abscisic acid, an evolutionary conserved hormone: Biosynthesis, therapeutic and diagnostic applications in mammals

Review Abscisic acid, an evolutionary conserved hormone: Biosynthesis, therapeutic and diagnostic applications in mammals Amir Gharib a,b , Carlee Marquez a , Maria Meseguer-Beltran a , Sandra Sanchez-Sarasua a,c,* , Ana M Sanchez-Perez a,* a Neurobiotecnologia Group, Institute of Advanced Materiales (INAM), Universitat Jaume I, Avda. de Vicent Sos Baynat, S/n, 12071 Castell´ o de La Plana, Spain b Department of Laboratory Sciences, Borujerd Branch, Islamic Azad University, Borujerd, Iran c CNRS UMR 5293, Institut Des Maladies Neurod´ eg´ en´ eratives, Centre Paul Broca-Nouvelle Aquitaine, University of Bordeaux, Bordeaux, France ARTICLE INFO Keywords: Mammalian hormone Therapeutic applications Biomarker tool Immune modulation Animal model Neurological disorders ABSTRACT Abscisic acid (ABA), a phytohormone traditionally recognized for its role in plant stress responses, has recently emerged as a significant player in mammalian defense mechanisms. Like plants, various mammalian cell types synthesize ABA in response to specific health challenges, although the precise pathways remain not fully elucidated. ABA is associated with the regulation of inflammation and insulin signaling, prompting extensive research into its potential as a therapeutic agent for various diseases. ABA exerts its effects through its receptors, particularly PPAR-γand LANCL-2, which serve as signaling hubs regulating numerous pathways. Through these interactions, ABA profoundly impacts mammalian health, and new ABA targets continue to be identified. Numerous studies in animal models demonstrate ABA’s benefit in Abbreviations: 6-OHDA, 6-hydroxydopamine; ABA, Abscisic acid; ACC, Anterior cingulate cortex; AChRs, Acetylcholine receptors; AD, Alzheimer’s disease; ADHD, Attention deficit and hyperactivity disorder; AE2, Anion exchanger 2; AMPK, Adenosine monophosphate-activated protein kinase; APE1, Apurinic/apyrimidinic endonuclease/redox effector-1; ApoE, Apolipoprotein E-deficient; APP, Amyloid precursor protein; ARDS, Acute respiratory distress syndrome; Arg, Arginine; ATP, Adenosine triphosphate; Aβ, Amyloid beta; Band3, Anion exchanger protein 3; BAT, Brown adipose tissue; BDNF, Brain-derived neurotrophic factor; BMP-7, Bone morphogenetic protein-7; cADRP, Cyclic adenosine diphosphate ribose; cAMP, Cyclic adenosine monophosphate; CAT, Catalase; CD34/45, Cluster of differentiation 34/45; COPD, Chronic obstructive pulmonary disease; COX2, Cyclooxygenase-2; CRABP, Cellular retinoic acid binding proteins; CRABP2, Cellular RA binding protein 2; CRH, Corticotrophin release hormone; CRS, Cardiorenal syndrome; D-Gal, D-galactose; DIDS, 4,4 ′ -Diisothiocyanatostilbene-2,2 ′ -disulfonic acid; DSM, Diagnostic and Statistical Manual of Mental Disorders; DSS, Dextran sodium sulfate; EGTA-AM, Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acidacetoxymethyl ester; eNOS, Endothelial NO synthase; ER, Endoplasmic reticulum; ERAD, ER-associated degradation; ERK, Extracellular signal-regulated kinase; ERR α , Estrogen-related receptor alpha; ET, Essential tremor; FABP5, Fatty acid binding protein 5; FAMP5, Fatty acid-binding protein 5; FRET, F¨ orster resonance energy transfer; GABA-A, Gamma-aminobutyric acid A; GLUT-4, Glucose transporter type 4; GRP78, Glucose regulated protein; hCG, Human chorionic gonadotropin; HFD, High fat diet; HGD, High glucose diet; hiPSC, Human induced pluripotent stem cells; hM-CSF, Human macrophage colony stimulating factor; HPA, Hypothalamic-pituitary-adrenal; HPCs, Hematopoietic progenitor cells; HSP70, Heat shock proteins; I-PKA, PKA inhibitor; i.c.v, Intracerebroventricular; i.p., Intraperitoneal; IBD, Inflammatory bowel disease; IL1β, Interleukin 1 beta; INS-1, Insulinoma cell line; IP3, Inositol triphosphate 3; IRS, Insulin receptor substrate; JNK, Jun Nterminal kinase; Ki67, Kiel 67; LANCL-2, Lanthionine synthetase C-like 2; LPS, Lipopolysaccharide; LSPR, Localized surface plasmon resonance; LTP, Long-term potentiation; MAPK, Mitogen-activated protein kinases; MCP-1, Monocyte chemoattractant protein-1; MDA, Malondialdehyde; MEP, 2-C-methyl-D-erythritol 4phosphate; MetS, Metabolic syndrome; MMP-9, Metalloprotease-9; mRNA, Messenger RNA; MSC, Mesenchymal stem cell; mTOR, Mammalian target of rapamycin; MVA, Mevalonic acid; NF-kB, Nuclear factor kappa B; NLRP3, NOD-like receptor family, pyrin domain containing 3; NO, Nitric oxide; NOX4, NADPH oxidase 4; Nrf2, Nuclear factor erythroid 2-related factor 2; OVA, Ovalbumin; P-53, Protein 53; p38MAPK, p38 mitogen-activated protein kinases; p70S6K, Ribosomal protein S6 kinase beta-1; PBMC, Peripheral blood mononuclear cell; PBMNC, Peripheral blood mononuclear cells; PC, Ping Chuan; PCa, Prostate cancer; PD, Parkinson’s disease; PGC-1 α , Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha; PGE2, Prostaglandin E2; PI3K, Phosphatidylinositol-3-kinase; pIC, Posterior insular cortex; pKa, Acid dissociation constant; PKA, Protein kinase A; PKC, Protein kinase C; PMA, Phorbol myristate acetate; PPAR-γ, Peroxisome proliferator activated receptor gamma; pSNL, Partial sciatic nerve ligation; PTX, Pertussis toxin; RA, Retinoic acid; RAR, Retinoic acid receptor; RIN-m, Rat insulinoma cell line; ROS, Reactive oxygen species; SIRT1, Sirtuin 1; SOD, Superoxide dismutase; STZ, Streptozotocin; TAK1, TGF-beta-activated kinase 1; TGF, Transforming growth factor; TNF α , Tumor necrosis factor alpha; UCP3, Uncoupling protein 3; UV, Ultraviolet; VCAM-1, Vascular cell adhesion molecule-1; vGAT, Vesicular GABA transporter; VSMC, Vascular smooth muscle cell; WAT, White adipose tissue. * Corresponding authors at: Neurobiotecnologia Group, Institute of Advanced Materiales (INAM), Universitat Jaume I, Avda. de Vicent Sos Baynat, s/n, 12071 Castell´ o de La Plana, Spain (S.S. Sarasua). E-mail addresses: [email protected] (S. Sanchez-Sarasua), [email protected] (A.M. Sanchez-Perez). Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm https://doi.org/10.1016/j.bcp.2024.116521 Received 9 July 2024; Received in revised form 26 August 2024; Accepted 5 September 2024 Biochemical Pharmacology 229 (2024) 116521 Available online 7 September 2024 0006-2952/© 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). managing conditions such as neurological and psychiatric disorders, cancer, and malaria infections, all of which involve significant inflammatory dysregulation. In this manuscript we review the studies covering ABA synthesis and release in cell cultures, the signaling pathways regulated by ABA, and how these impact health in preclinical models. Furthermore, we highlight recent research suggesting that measuring ABA levels in human body fluids could serve as a useful biomarker for pathological conditions, providing insights into disease progression and treatment efficacy. This comprehensive review outlines the current understanding of ABA in mammalian pathophysiology, identifying gaps in knowledge, particularly concerning ABA biosynthesis and metabolism in mammals. In addition, this study emphasizes the need for clinical trials to validate the effectiveness of ABA-based therapies and its reliability as a biomarker for various diseases. 1. Introduction In the mid-20th century, the first molecular complex regulating plant growth was identified as an acidic compound that inhibited potato plant development [1]. A few years later, acidic molecules responsible for abscission (shedding) were isolated from cotton fruits and were named abscisin I and abscisin II [2]. With time, abscisin II was renamed as Abscisic acid (ABA) [3]. Extensive research soon revealed that ABA is a key phytohormone that regulates plant growth, senescence, and abscission in response to stressors such as low temperatures, drought, nutrient deficiencies, and excessive UV irradiation (for review, see [4,5]). Over time, ABA was found present across all biological kingdoms, including fungi, cyanobacteria, and animals. Within the animal kingdom, ABA has been discovered in hydroids, sponges, and mammals (for review, see [6]). Le Page-Degivry et al. published in 1986 the presence of potentially endogenous ABA in mammal tissue. In this study, cis-ABA and ABA-conjugated glucosides and ethers (similar to those in plants) were detected in pig tissues including the brain, kidney, and heart. In a key experiment where rats were fed a synthetic ABA-deficient diet for two generations, the authors demonstrated that these subjects exhibited higher ABA levels in the brain compared to the control group [7]. This experiment provided evidence that mammalian ABA does not necessarily originate from dietary sources and can be synthesized within mammalian organisms. Now, almost forty years later, accumulating evidence confirms that ABA is synthesized in mammalian organisms, with important implications in mammalian pathophysiology. ABA is now recognized as a unique example of an evolutionary conserved hormone that triggers defense mechanisms in response to stress. Other defense systems, such as reactive oxygen species (ROS), are also well-known examples of highly conserved defense mechanisms against stressful stimuli across biological kingdoms [8]. Given ABA’s ability to activate defense signaling pathways (discussed in detail section 3 and 4), growing evidence supports its potential as a therapeutic treatment and even as a biomarker of disease. This review provides an in-depth analysis of mammalian ABA by compiling evidence on conditions that stimulate its synthesis and release, outlining the diversity of ABA-regulated signaling pathways, and reporting the interactions with mammalian receptors. This review highlights the impact of ABA on animal models of disease where its therapeutic potential has been tested. Additionally, beyond its therapeutic applications, this review underscores recent studies that suggest ABA’s potential as a diagnostic tool. 2. ABA synthesis and biosynthesis Structurally, ABA is a classical monocyclic sesquiterpene (C 15 H 2 0O 4 ), composed of a hydrophobic 4-hydroxy cyclohex-2-enone group attached to a diene chain with an olefinic E, Z-conformation, and a carboxylic acid. This carboxylic acid contributes to ABA’s weak acidity (pKa 4.8). ABA contains an asymmetric carbon atom, resulting in the formation of two enantiomers: S-(+)-ABA and R-(−)-ABA. The geometric configuration allows for cis or trans isomers, depending on the spatial arrangement of atoms around the double bonds in the molecule. Although ABA can theoretically exist in four different stereoisomeric forms, the naturally occurring form in plants is predominantly S(+)-cis, trans-ABA; however, R- (−)- ABA may be active in certain functional assays [9]. Given ABAs importance in plant development, several attempts have been made to synthesize it under controlled laboratory conditions. In 1965, the first synthesis of ABA (then known as abscisin II) was achieved from the precursor (2Z,4E)-3methyl-5-(2,6,6-trimethylcyclohexa-1,3-dienyl)-2,4, pentadienoic acid was reported [10]. In the 1980s, an alternative strategy using isophorone as a precursor was reported [11]. Subsequent synthesis methods have been attempted but none have improved yield beyond 30 %[12]. In plants, ABA biosynthesis begins with the precursor zeaxanthin, a carotenoid-derived tetraterpene, following the plastidial 2-C-methyl-Derythritol 4-phosphate (MEP) pathway. In fungi, ABA biosynthesis starts with the precursor farnesyl pyrophosphate, following the mevalonic acid (MVA) pathway (for review, see [13,14]) (Fig. 1). Early studies in the protozoan Toxoplasma gondii (T. gondii), a pathogen that causes toxoplasmosis in humans, suggested that ABA biosynthesis Fig. 1. Biosynthesis pathways of ABA in plants and fungi. The precursor in plants for ABA biosynthesis is zeaxanthine (carotenoids) and in fungi Farnesyl pyrophosphate (FPP). Interestingly, FPP is the product of FPP synthase, a wellknown enzyme of the mevalonate pathway, important for lipid biosynthesis [151]. A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 2 followed the carotene pathway, since fluridone, a beta-carotene pathway inhibitor [15], reduced ABA synthesis [16]. The genes responsible for ABA synthesis (ABA1,ABA2, and ABA3) and responsive genes were identified in the T.Gondii genome. Years later, Nagamune et al. [17] were the first to report the impact of exogenous ABA in protozoan activity. The authors showed that ABA induced a dormant cyst stage in infected cultured cells, thus preventing parasite toxicity. In animals, early studies demonstrated that ABA is synthesized in response to stressors such as heat shock or light exposure in hydroids (Eudendrium racemosum) and sponges (Axinella polypoides)[18,19] (Table 1). In hydroids, light-stimulated ABA synthesis was inhibited by fluridone, suggesting that the carotenoid pathway was involved in ABA biosynthesis. The role of ABA in mediating light-stimulated regeneration in E. racemosum was further confirmed when exogenous ABA, administered in the dark, induced regeneration in the absence of light. In mammalian cells, the specific pathways for ABA biosynthesis have not been definitively established. However, given the structural similarity between ABA and retinoic acid (RA), and the fact that RA is synthesized from carotenoid precursors [20], it has been proposed that ABA may also be synthesized from carotenoid precursors in mammals. Supporting this hypothesis, ABA secretion in monocytes was reduced in the presence of fluridone [21]. Despite the need for further research to fully elucidate ABA biosynthesis in mammals, strong evidence indicates that mammalian ABA is synthesized in response to stress stimuli to activate defense mechanisms. Thus, immune cells are a significant source of endogenous ABA. In human granulocytes ABA is detected both as a free molecule (70 %) and in alkali-hydrolysable conjugates. Free ABA levels in these cells can increase up to threefold in response to various stress stimuli (Table 1). Interestingly, high temperatures increased intracellular ABA concentrations without promoting its release [22]. While the underlying mechanism remains unexplored, this suggests that temperature stress may require intracellular ABA action rather than paracrine signaling. Compared to granulocytes, monocytes exhibit a much higher basal ABA concentration (20-fold), though the biological implications of this difference remain unclear and may relate to distinct cellular functions. However, like granulocytes, monocytes release newly synthesized ABA in response to a variety of stimuli (Table 1)[22]. Endogenous ABA has also been detected in microglial cells, which are derived from the monocyte lineage. For example, in the murine microglial N9 cell line, ABA synthesis increases in response to bacterial lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA), a chemoattractant peptide (f-MLP), and Amyloid β(Aβ) (Table 1)[23]. These findings underscore the role of ABA in regulating immune cell functions. However, a comprehensive understanding of ABA’s role in the physiology and pathology of the immune system is still lacking. Advancing this knowledge could open new avenues for improving the management of immune disorders. Beyond immune cells, ABA synthesis has also been observed in stem cells in response to specific stimuli. For instance, human mesenchymal stem cells (MSCs) produce and release ABA in response to bone morphogenetic protein-7 (BMP-7), inflammatory cytokines, and peripheral blood mononuclear cell (PBMNC)-conditioned medium, but not in response to human macrophage colony-stimulating factor (hM-CSF) [24]. These observations suggest that the ABA response is selective, occurring only in reaction to specific stressors, indicating a targeted adaptive mechanism. Supporting this specificity, certain stimuli elicit ABA synthesis only in specific cell types. For example, pancreatic βcells (including rat insulinoma cell lines such as RIN-m and INS-1) express and release ABA in response to glucose, whereas exocrine pancreatic cells and human blood-derived mononuclear cells do not [25]. Moreover, ABA can be synthesized by muscle cells. In H9c2 rat myocytes, ABA release was observed under hypoxic conditions (Table 1), where it acted on myocyte receptors in an autocrine manner [26]. This case exemplifies ABAs autocrine function in differentiated cell outside of the immune or endocrine systems (Fig. 2). While it is plausible that other differentiated cell types might also release ABA in response to stimuli, this has yet to be documented. Finally, the specific genes involved in ABA synthesis in mammals and the mechanisms by which they are regulated remain to be discovered. 3. ABA mammalian receptors Various studies have identified Lanthionine synthetase C-like 1 and 2 (LANCL-2) as the mammalian receptors for ABA. LANCL-2 is as an Nterminal myristoylated protein anchored to the inner side of plasma membrane [27]. Interestingly, the resemblance of the animal LANCL protein family to the structure of prokaryotic lanthionine synthetase C proteins may not be coincidental, since these bacterial membraneassociated proteins represent a first defense mechanism, producing antimicrobial peptides [28]. LANCL-1/2 proteins are ubiquitously expressed in mammals and are important regulators of immune and metabolic processes. LANCL2 Table 1 ABA biosynthesis detected in culture cells in response to stimulus. Tissue/cells ABA (basal) ABA (after) stimulus ref Hydroids (E. racemosum 18 pmol/g 105–30 pmol/g Light (1 h -4h) [18] Sponges (Axinella polypoides) 4.8 pmol/g (14 ◦C) 18.1; 80.0; 104.2 pmol/g 26 ◦C (1, 5, 60 min) [19] Human Granulocytes 230 pmol/g 460–390 pmol/g PMA (0,1 mg/mL) at 20 ◦C Temperature (39 ◦C) Zymosan (0.37 mg/ml) Latex beads [22] Human Monocytes and macrophages 4.11 ±0,82 (pmol/mg) 1.6-fold intracellular; 9.2-fold release 2.7-fold intracellular and release No change intracellular; 3,6-fold release Thrombin activated platelets 1 h MCP-1 (50 ng/ml) 1 h Temperature (39 ◦C) 1 h [52] Murine microglia (N9 cells) 0.31 ±0.07 pmol/mg 4-fold intracellular; 3-fold release 3.7 fold intracellular; 5-fold release 5.7fold 3.4 −fold LPS (100 ng/mL) 72 h LPS (48 h) +Aβ(24 h) f-MLP (1 h) PMA (1 h) [23] Human mesenchymal stem cells (MSCs) Released 1.34 ±0.3 Intracellular 2.1 7 ±0.4 (pmol/mg) 1.6; 6.0; 2.3fold intracellular 4.7-fold release (returned to basal 48 h) 2.8–4.1 −fold No increase 2.8–5.5 fold BMP-7 (0,2 mg/ml) 6, 24, 48 h PBMNC conditioned media TNFα (6 h; 24 h) hM-CSF (6 h; 24 h) IL-8 (6 h; 24 h) [24] Human pancreatic islet rat insulinoma cell lines (RIN-mINS-1) Intracellular and extracellular (up to 25-fold) High glucose (16,7 mM) 15 min [25] Exocrine pancreatic cells No ABA stimulation High glucose (16,7 mM) 15 min H9c2 Cardiomyocytes Released 0.3 ng/mg protein (normoxia) 3-fold increase Hypoxia (18 h) [26] A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 3 agonists have been explored for their potential therapeutic applications in inflammatory diseases [29]. Extensive computational data confirms that ABA is a LANCL-2 agonist, and several potential ABA binding sites in LANCL2 have been identified [30,31]. Site-directed mutagenesis studies (including single mutant R118I and triple mutants R118I/R22I/K362I and R118I/S41A/E46I) have pinpointed a high-affinity ABA-binding site involving the R118 residue, with a dissociation constant (KD) of 2.6 nM±1.2 nM, along with two low-affinity binding sites [32]. In addition to LANCL-2, ABA can bind and activate the peroxisome proliferator activated receptor gamma (PPAR-γ) in a ligand-site independent manner [33]. Our group compared the binding affinity of two different ABA isomers—(S)- (+)-cis, trans-abscisic acid and (S)-(+)-trans, trans-abscisic acid—to LANCL-2. We found that both isomers bind similarly, though slightly better scores were observed for cis-ABA (−8.55 kcal/mol) compared to trans-ABA (−7.63 kcal/mol). The biological implications of this difference may not be significant, as in nature, (S)-(+)-cis, trans-abscisic acid is the most prevalent form of ABA. Similarly, we have also published docking corroboration of ABA binding to amino acid motifs in PPAR-γ [34]. PPAR-γis a key transcription factor that regulates adipogenesis, insulin sensitivity, and glucose homeostasis in humans [35]. Consequently, PPAR-γagonists are considered promising therapeutic agents for a wide range of inflammation-related diseases [36]. Beyond these two well-known ABA receptors, ABA has also been shown to block the bitter taste receptor T2R4, a member of the G protein-coupled receptor superfamily. ABA competes with quinine for binding to T2R4, resulting in reduced intracellular Ca 2+ levels [37]. Molecular docking studies have identified potential T2R amino acid motif that interact with ABA [37]. This bitter taste antagonism could have significant applications in the food industry by reducing bitterness without the need for large amounts of sugar. However, T2R receptors are not unique to oral tissue, and they have been found expressed in the nervous system [38]; airway muscle cells [39], heart and gastrointestinal tract [40]. This suggests a broader mechanism of action for ABA as a T2R antagonist, potentially influencing a range of physiological processes beyond taste perception. In addition, ABA uses retinoic acid receptor (RAR) pathway to promote apoptosis, as silencing RAR silencing blocks ABAs effect [41]. However, whether ABA binds directly to RAR remains unclear, as no docking studies have been reported. Interestingly, using an ABA mimetic affinity probe, ABA was found to bind members of the heat shock protein family, such as HSP70, and the glucose regulated protein (GRP78) in insulinoma and embryonic cells. However, no functional effect or details on amino acid motif interactions were provided [42]. These findings could be therapeutically significant given the role of GRP78 as an ER stress sensor. GRP78 is known to flag misfolded proteins for endoplasmic reticulum (ER)-associated degradation (ERAD) and regulate calcium homeostasis [43], and thereby it has even been proposed as an ER protector [44]. On the other hand, a recent study showed that GRP78 and HSP70 promote insulin resistance, hyperglycemia, and non-alcoholic fatty liver in the upper gut [45]. Thus, ABA’s binding to GRP78 and HSP70 may have different effects depending on the activity of these proteins. In the gut ABA can potentially improve insulin signaling and glucose homeostasis. This suggests an unexplored mechanism by which ABA regulates GRP78. Potential ABA-binding partners have been identified by searching for amino acid motifs deduced from experimentally confirmed ABA-binding proteins [46]. This emerging field could enhance ABA pharmacology and lead to new applications. All reported ABA receptors are intracellular, leaving the mechanism of ABA’s entry into cells unaddressed. Vigliarolo et al. [47], are among the few studies to explore this aspect, proposing that ABA is transported into cells by anionic transporters of the AE2 family. In human red blood cells, ABA is transported by the Band3 transporter, which also belongs to the AE2 family. Using DIDS, an anion exchange inhibitor, the authors observed a reduction of the ABA-induced ATP. Similarly, a reduction in ABA-induced ATP was noted with LANCL-2 inhibitors, the protein kinase A-specific inhibitor (I-PKA) and the cyclic adenosine Fig. 2. ABA synthesis and release by different mammalian cells in culture. ABA synthesis and release is detected in different mammalian cells in culture in response to a variety of pathophysiological stimulus that may be specific for cell function. Immune cells respond to infection, exogenous and physiological stimulus, mesenchymal stem cells respond to cytokines and trophic factors, but not to hM-CSF. Pancreatic beta cells release ABA in response to glucose, but not pancreatic exocrine cells. Cardiomyocytes release ABA in response to hypoxia conditions. BMP, bone marrow protein; hM-CSF, human Macrophage Colony-Stimulating Factor; MCP-1, monocyte chemoattractant protein; LPS, lipopolysaccaride, PMA, phorbol myristate acetate. A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 4 monophosphate (cAMP) inhibitor (dideoxyadenosine) [48]. These comprehensive experiments demonstrated that ABA uses anion exchange transporters for cellular entry and that ATP synthesis depends on the ABA/LANCL2/PKA pathway. However, ABA metabolism, degradation, and extracellular transport remain unexplored (Fig. 3). 4. Effect of ABA on mammalian cultured cells. Mechanism of action In mammalian stem cells, ABA has been shown to increase intracellular calcium and cyclic adenosine diphosphate ribose (cADPR), analogous to the effects observed in protozoans and sponges. Table 2 and Fig. 3 provide a comprehensive summary of ABA mechanisms and their impacts on various cultured mammalian cells. Scarfi et al. [24,49] demonstrated that exogenous ABA significantly influences mesenchymal stem cells (MSCs) and hematopoietic progenitor cells (HPCs) (CD34 +)in vitro expansion, migration (chemokinesis), and proliferation, leading to increased production of prostaglandin E2 (PGE2) and cytokine release. These trophic effects were mediated by cADPR and intracellular calcium signaling. Blocking ABA effect using an anti-ABA antibody, this study demonstrated that micromolar ABA concentrations triggered nuclear factor kappa B (NF-kB) nuclear translocation in HPCs. NF-KB regulated the expression of various factors and cytokines, which, in turn, stimulated the growth and proliferation of CD34 + stromal cells and endothelial cells, thus ABA may exert both autocrine and paracrine effects, stimulating MSCs and HPCs growth and function. Intriguingly, ABA has also been identified as a differentiating factor, promoting the maturation of human induced pluripotent stem cells (hiPSCs) into CD34+/CD45 +HPCs and further differentiation into CD41 +megakaryocytes (platelet precursors). Huang et al. showed that ABA can increase significantly the population of CD41+/CD42b + megakaryocytes and platelets compared to untreated cells. Additionally, ABA supported megakaryocyte survival and platelet generation under stress conditions, such as serum deprivation. The authors confirmed that ABA’s effects were mediated through the stimulation of protein kinase A (PKA), cADPR, and mitogen-activated extracellular signal-regulated kinase (ERK) 1/2 [50]. Furthermore, the expression of both LANCL-2 and GRP78 (potential ABA receptors) changes during the differentiation of HPCs into mature megakaryocytes, underscoring their important roles in this process. Specifically, LANCL-2 and GRP78 levels were low in immature HPCs (CD34+/CD45-), peaked as HPCs matured (CD34+/ CD45 +), and returned to basal levels in fully differentiated megakaryocytes (CD41 +). These findings highlight ABA’s potential application in specific clinical settings, such as the treatment of patients with conditions like thrombocytopenia (low platelet count), where maintaining adequate platelet levels is essential [51]. In the previous section, we reviewed the literature on ABA biosynthesis in immune cells in response to stress, suggesting that ABA acts as an initial defense mechanism. Early studies on cultured immune cells suggested that ABA has a pro-inflammatory impact. Thus, exogenous ABA was shown to stimulate monocyte migration by increasing calcium levels, protein kinase C (PKC) phosphorylation, and NF-kB translocation. This, in turn, enhanced the expression of cyclooxygenase-2 (COX2), prostaglandin E2 (PGE2), monocyte chemoattractant proteinFig. 3. Potential Mechanism of ABA Action in Mammalian Cells. Abscisic acid (ABA) has been reported to enter mammalian cells via anionic transporters, though its complete life cycle within the cell remains unclear. The mechanisms of ABA secretion or export are not yet understood. Several receptors for ABA have been identified, with LANCL2 being the primary mediator, though LANCL1 can also bind ABA in certain cell types. PPAR-γis another significant mediator; ABA can bind to PPAR-γin a ligand site-independent manner and has been shown to increase PPAR-γactivity and/or expression in some models. Additionally, ABA has been reported to interact with the Retinoic Acid Receptor and the endoplasmic reticulum protein GRP78, although the precise binding sites are unknown. Only one antagonism action has been reported on the taste receptor. Further research is warranted to fully understand this action. Activation of LANCL1/2 by ABA leads to increased intracellular calcium levels, which can activate various signalling pathways depending on the cell type and environmental conditions. Reported pathways include PKA, AKT, ERK, and the p38 MAPK/JNK pathway, which subsequently leads to Sirt1 activation. ABA can also induce the activation of NF-κB, resulting in its translocation to the nucleus. Collectively, these actions allow ABA to modulate the expression of cytokines and other factors, leading to either a reduction in proinflammatory cytokines or an increase in trophic factors. It is important to note that not all pathways may be involved in every cell type; this figure aims to summarize most of the known pathways and highlight areas where knowledge is still incomplete, like degradation and/or export. Through these mechanisms, ABA can act upon different cell types, providing protection, inflammatory response and/or differentiation and apoptosis. A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 5 1 (MCP-1), and metalloprotease-9 (MMP-9). MCP-1 is crucial for promoting monocyte migration to atherosclerotic plaques and stimulating vascular smooth muscle cell (VSMC) proliferation. Since atherosclerosis is driven by the migration and proliferation of VSMCs at endothelial lesions, ABA was concluded to play a pro-inflammatory role in atherogenesis, consistent with its higher concentrations observed in atherosclerotic plaques [52]. Similarly, granulocytes exposed to ABA increased their chemotactic response in an ABAconcentration-dependent manner. However, this effect depends on ABA structure, as modifications to the ABA molecule, such as ABA-methylamide and the trans-isomer, eliminate its impact. Also, the presence of an anti-ABA antibody in the granulocyte supernatant reduced reactive oxygen species (ROS) production by 80 %, further supporting ABA’s involvement in inflammatory processes. Confirming the pro-inflammatory hypothesis, ABA administration was shown to activate N9 microglial cell migration toward amyloid-beta (Aβ), stimulating the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF α ) via intracellular calcium and cAMP pathways [23]. Contrary to these early findings, other studies, including our own, suggest that ABA also has anti-inflammatory properties. Bassaganya-Riera et al. [33] demonstrated in murine macrophages (RAW 264.7 cell line), that ABA reduced LPS-induction of PGE2 and MCP-1 expression, oxidative stress, and mitochondrial fusion/fission, primarily by increasing cAMP. Our group later showed that in the same cell line, ABA suppressed LPS-induced increment in TNF α mRNA expression; intriguingly, ABA also enhanced LPS-induced NO production, but only in the presence of LPS, not when ABA was administered alone [34]. These findings suggest that exogenous ABA plays a dual role—both Table 2 ABA effect on cultured cells and mechanisms. BAT;brown adipose tissue;CRH, corticotropin release hormone; MK; megakaryocytes (platelet precursor); NO; Nitric Oxide; RAR, retinoic acid receptor; ROS, reactive oxidative species; VSMC Vascular smooth muscle cells. Tissue/cells ABA treatment effect via Ref/ year Hydroids (E. racemosum) 5 mM Regeneration of hydroid in the dark [18] Protozoan 50 nM Facilitates parasite maturation cADPR [17] Mesenchymal stem cells 0.1, 1.0, and 10 mM 1.3-, 1.7-, 1.4-fold colony production 1.25-, 1.6-, 1.3-fold proliferation Increase intracellular Ca 2+ cADPR [24] Hematopoietic progenitors 0.5, 2, and 20 mM 10 mM (30 min) 1.6-, 2.2-, and 1.35-fold expansion 2.8-fold nuclear translocation NFkB Increase intracellular Ca 2+ cADPR [49] Human induced pluripotent cells 10 uM (19 days) Increased hiPSC differentiation to HPC and HPC to MK and Platelet formations. GRP78 and LANCL2 expression in HPC maturing: Low in CD34+/CD45- (immature); High in CD34+/CD45+(maturing); Low in CD41+ (differentiated) MKs. ERK or AKT signaling. [50] Monocytes 30 min 0.1, 1.0, 10 mM (6 h) 10 mM (6 h) 0.1 mM activation of NF-kB, 1.3-, 1.4-, and 1.6-fold COX2 2.3-fold MCP-1; 1.8-fold MMP-9 1.8-fold PGE2 Increased intracellular Ca +2 and PKC phosphorylation [52] Aorta VSMC 0.1, 10 mM VSMC proliferation Human Granulocytes 50 nM–20 mM increase phagocytosis. increase 6-fold ROS. increase NO production (1.83–9.42x) intracellular Ca 2+ cADPR [22] Murine microglia (N9 cell) 20 μ M (10 min) 20 μ M (1 min) Promote Migration Increased NO and TNFα production intracellular Ca 2 intracellular cAMP [23] Murine macrophages (RAW267.4 cells) 20 μ M (24 h) +LPS 0,1mg/mL (2 h, 24 h) Increased 1.2-fold LPS-induced NO (24 h) Decrease 74 % TNFα mRNA (2 h) Docking of ABA isomers with PPARγand LANCL2 [34] 1.25–10 μ M (20 h) Suppressed LPS-induced E2, MCP-1, NRLP3 Increased cAMP LANCL2; PPARγ [33] Rat insulinoma (RIM-n; INS-1) 10 nM (low glucose) 1 mM (no glucose) 1.6x-2x increase insulin secretion Insulin secretion Protein G (PTX-dependent) cADPR, intracellular Ca 2+ [25] Pancreatic islets (human and mice) 1 nM (low glucose) 10 nM −1 mM. 30 min Murine adipocytes (3 T3-L1) 100 nM ABA Increased Glut-4 expression, glucose uptake; fatty acid synthesis, CO2, O2 consumption, BAT genes expression LANCL-2 [53] Myoblasts (L6 cell line) 100 nM 4x glucose uptake (NBDG) 4–6 x increase Glut 4 Glut 1 activation 5 x mitochondrial respiration LANCL1 →AMPK/PGC-1 α /Sirt1 LANCL1 →uncoupling proteins sarcolipin and UCP3 NAD-synthesizing enzyme Nampt, RabGAP, TBC1D1 [54] Differentiated muscle cells from isolated human skeletal myoblasts (Lonza) 100 nM (6 h) Increased ATP production Glycogen synthesis [55] Cardiomyocytes (H9c2 cell line) 100 nM (30 min) Increased mitochondrial function. Increased NO production LANCL1/2 →ERR →AMPK/PGC-1 α /Sirt1 [26] Murine Leukaemia cells (L178Y cell line) IC50 130 µM (5 days) Reduced viability Synergistic effect with Retinoic Acid Non described [56] Glioblastoma cells (U87MG and A172 cell lines) 200 µM (24 h) Increased autophagy MAPK/JNK signalling pathway [58] 20–50 µM (24 h) Increased apoptosis and differentiation Decreased PPAR-γvia RAR α (RA receptor) [41] Prostate Cancer Human PCa cell lines (LNCaP, PC3, and DU145) in bone marrow environment 50 µM (24 h) Induced G0 phase arrest decrease Ki67 marker. Increase cell cycle inhibitors (p26, p27, p16) LANCL-2 and PPAR-γ → p38MAPK [59,60] Human neuroblastoma BE(2)-C, SHSY5Y cells Primary cortical neurons 20 µM (12–24 h) Reduced CRH expression Reduced RAR α expression in BE(2)-C RAR α (PPAR-γdoes not change levels) [61] A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 6 stimulatory and inhibitory—in immune cell cultures, indicating that ABA can modulate immune function by regulating cell migration and cytokine production. Further research is needed to fully elucidate ABA’s physiological roles, as well as the biological significance of its timing in promoting and resolving inflammatory processes. Beyond its immunomodulatory role, ABA has also been shown to enhance insulin sensitivity and stimulate insulin secretion from pancreatic cells. At low concentrations (10 nM), both enantiomers of ABA, (+)- and (−)-cis, trans-ABA, have been found to stimulate insulin secretion from rat insulinoma cell lines (RIN-m and INS-1) in a low glucose buffer. Interestingly, higher concentrations of ABA (1 mM) induced insulin secretion even in the absence of glucose. The use of ADPR cyclase inhibitors (PTX, nicotinamide), antagonists (8-Br-cADPR and ryanodine), and an intracellular calcium chelator (EGTA-AM), effectively blocked ABA effects on pancreatic cells, confirming previous findings. Similar results were observed in freshly isolated murine pancreatic islets and human pancreatic βcells [25]. Because glucose stimulates ABA release in pancreatic cells (see above), ABA can be considered as an autocrine molecule, providing a positive feedback mechanism that prolongs and amplifies the response to glucose. Moreover, ABA has been shown to modulate the response to insulin in adipose and muscle cells, both of which are highly responsive to insulin for glucose uptake. In differentiated 3 T3-L1 murine adipocytes, ABA increased the expression of glucose transporter type 4 (GLUT-4) and enhanced insulin-induced glucose uptake via LANCL-2. In addition, ABA promoted oxygen consumption and upregulated the transcription of adiponectin and brown adipose tissue (BAT) genes [53]. In rat muscle cells (L6 myoblasts), ABA activated glucose transport via the AMPK/PGC-1 α /SIRT1 pathway and increased mitochondrial respiration through the uncoupling proteins sarcolipin and UCP3. Interestingly, when LANCL-2 was knocked out in these cells, LANCL-1 was overexpressed and could mediate ABA’s effects, improving glycemic response to glucose load [54]. This suggests that LANCL-1 may also serve as a potential ABA receptor. Studies on cardiomyocytes (H9c2 cells) have further demonstrated that ABA, through either LANCL-1 or LANCL-2, activates the AMPK/ PGC-1 α pathway via ERR α , enhancing mitochondrial function and increasing NO production [26]. Moreover, in differentiated human myotubes, ABA (via LANCL-2) increased mitochondrial metabolism gene expression, mitochondrial ATP production and glycogen synthesis, thereby contributing to glucose homeostasis and significantly improving muscle performance [55]. At higher concentrations (in the micromolar range), ABA exhibits pro-apoptotic effects on cancer cells. Early studies in a murine leukemia cell line (L178Y) demonstrated that ABA reduced cell viability [56]. In glioblastoma cell lines, ABA (20–50 µM) induced apoptosis and decreased PPAR-γexpression through the RARα pathway. Additionally, ABA increased the transcription and expression of cellular retinoic acid binding protein 2 (CRABP2), which mediates RA-induced apoptosis and growth arrest [41]. ABA exposure also decreased the transcription of fatty acid binding protein 5 (FABP5), an alternative RA-induced pathway associated with cell survival and proliferation [57]. This suggests that ABA’s pro-apoptotic effects may be RARα dependent [41]. The same research group later confirmed that this pro-apoptotic effect results from autophagic activation via the MAPK/Jun N-terminal kinase (JNK) signaling pathway [58]. Furthermore, in prostate cancer cells, ABA exposure (50 µM for 24 h) resulted in G 0 arrest due to increased cell cycle inhibitors and decreased proliferation markers [59]. The same researchers found that ABA exerted its effects via its receptors LANC-2 and PPAR-γ, activating p38MAPK signaling [60]. In undifferentiated neuroblastoma cells lines BE (2)-C and SHSY-5Y cells, 20 µM ABA has been shown to reduce corticotrophin release hormone (CRH) mRNA expression by increasing RA receptor (RARα ) expression. However, no data on cell viability was reported (Table 2) [61]. Our group investigated whether ABA could promote the differentiation of SHSY-5Y cells into neuron-like cells through the RA pathway. Contrary to the expected RA effects, 20 µM ABA had no impact on differentiation process. In addition, unlike its effects on other tumor cell lines, ABA did not affect the viability of undifferentiated SHSY-5Y [62]. The mechanism responsible for the different effects of ABA on glioblastoma and neuroblastoma cells remains unclear to date. 5. Therapeutic ABA applications. Evidence from animal models of human disease ABA has been tested in a wide range of preclinical models, and its beneficial impact is increasingly recognized. Herein, we give a comprehensive review of ABA’s impact and the mechanisms that underlie the potential therapeutic effect of this hormone (Fig. 4). 5.1. Cancer ABA’s potential role as an anti-cancerogenic molecule was first suggested in 1976, when it was shown to “neutralize”human chorionic gonadotropin (hCG) [63], a negatively charged glycoprotein that coats cancer cells and protects them from immune cell attacks [64]. More recently, ABA effect was investigated in a prostate cancer model with bone metastasis, were prostate cancer (PCa) cells were xenografted into mice by intratibial injection. ABA treatment in these mice induced PCa arrest in G 0 phase within the bone marrow microenvironment, via the PPAR-γpathway (Table 3). ABA increased the expression of cell cycle inhibitors (p27, p21, p16), decreased the proliferation marker Ki67, and suppressed the mTOR/p70S6K pathway [59] (Table 2). Similar results were observed in a different cancer model where mice were xenografted with glioblastoma cells (U87MG cells). In this model, ABA treatment (60 mg/kg ABA administered once daily for 21 days) significantly reduced tumour size and increased autophagic markers [58]. Furthermore, ABA prevented angiogenesis, both alone and in combination with prednisolone in albino rats [65] (Table 3), highlighting its potential clinical value as an adjunct in cancer therapy by inhibiting the formation of new capillary blood vessels in solid tumors [66]. The mechanisms by which ABA induces apoptosis or limit cancer cell growth are not fully understood. While ABA can activate the calcium signalling pathway, it is not yet clear whether its action resembles that of other chemotherapy drugs (e.g., staurosporine, doxorubicin, tamoxifen and etoposide), which also increase intracellular calcium leading to oxidative stress and apoptosis [67]. Given the beneficial effects of ABA in various cell types, future research should determine whether the effects of ABA are concentration-dependent or if ABA receptors in cancer cells function under different conditions. The dual role of ABA as both protective and apoptotic is of great interest and holds significant potential for therapeutic applications. 5.2. Cardiorenal syndrome Cardiorenal syndrome (→by the intricate interplay between heart and kidney dysfunction, where the impairment of one organ exacerbates the deterioration of the other. Among the various subtypes of CRS, type3 involves acute kidney dysfunction leading to acute cardiac injury [68]. Currently, the primary treatment for CRS is palliative care focusing on diuresis or ultrafiltration techniques to remove excess fluid. However, these invasive methods are often ineffective for some patient [68]. Consequently, there is significant interest in developing effective therapies with fewer side effects. To investigate the effects of ABA in a rat model of CRS-3, ABA was administered once daily for 4 weeks. Remarkably, ABA reduced cardiac hypertrophy and improved arrhythmia by shortening the ventricular potential duration. At the molecular level, ABA augmented the expression of HSP-70) and decreased levels of NADPH oxidase 4 (NOX-4) and A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 7 protein 53 (P-53). This study suggests a novel protective role of ABA in CRS, potentially mediated through its effects on P-53, NOX-4 and HSP70 [69]. 5.3. Essential tremor Essential tremor (ET) is one of the most common and progressive neurological disorders, characterized by a kinetic tremor in the upper limbs[70]. The etiology of ET is primarily genetic, with significant genetic and environmental interactions. Currently, treatment options are limited to symptomatic drug therapy [71]. Recently, the effects of ABA were tested in a rat model of ET induced by intraperitoneal (i.p.) injection of harmaline. ABA was administered via intracerebroventricular (i.c.v.) injection 30 min before harmaline. This model relies on harmaline toxicity, so no genetic alterations are involved. Remarkably, ABA reduced tremor severity, improved motor symptoms, and alleviated cognitive impairments, as assessed by the shuttle box test [72]. Notably, ABA was administered directly into the cerebroventricular system, suggesting a direct action on the brain. Although further studies are needed to elucidate ABA’s mechanism of action in ET, this study supports the idea that ABA’s anti-inflammatory effects in the brain may help alleviate ET symptom. 5.4. Inflammatory diseases ABAS anti-inflammatory properties have been evaluated in several models of inflammatory diseases affecting arteries, intestine, and lungs (Table 3). In an atherosclerosis model, ABA was tested in Apolipoprotein E-deficient (ApoE −/-) mice fed a high-fat diet (HFD), replicating human atherosclerotic traits such as plaque accumulation in the arteries, which increases cardiovascular risk [73]. Systemic ABA administration over 3 months improved systolic blood pressure, reduced CD11b (+) macrophages, and decreased CD4(+) T-cell accumulation in the aortic root walls. Notably, ABA enhanced endothelial NO synthase (eNOS) expression while reducing aortic VCAM-1 and MCP-1 levels [73]. Given that atherosclerosis in humans is linked to high LDL cholesterol levels, diabetes, and smoking, and typically treated with LDL-lowering drugs [74,75]. ABA’s anti-inflammatory effects present a novel approach for managing this condition. In Inflammatory bowel diseases (IBD), where the colon mucosal lining becomes acutely or chronically inflamed [76,77], ABA was tested in a mouse model of dextran sodium sulfate (DSS)-induced colitis. ABA was administered in the diet for 35 days before DSS treatment. This preventive approach improved disease symptoms and reduced colonic leukocyte infiltration and inflammation. Similar to the atherosclerosis model, ABA downregulated VCAM-1 and E-selectin expression but paradoxically increased CD4(+) and CD8(+) T-lymphocytes in the blood. The authors suggested that ABA’s effects might be mediated Fig. 4. The beneficial impact of ABA treatment on a wide range of animal model of human disease. In a wide range of animal model of human disease, ABA treatment, mostly chronic and mostly systemic, has demonstrated a great beneficial impact preventing disease progression, reducing symptoms, and improving health status. It is remarkable the diversity of disease etiology than ABA can impact on. This fact maybe because ABA is produced and release by various cell types in response to stress/toxic stimulus to promote defense. It is tempting to speculate that when this defense is not sufficient, symptoms appear and therefore exogenous ABA improves disease. This hypothesis is supported by human studies where high ABA levels correlate with better prognosis, or in cases where ABA circulating levels can be used a biomarker of disease status. A. Gharib et al. Biochemical Pharmacology 229 (2024) 116521 8 Table 3 Exogenous ABA administration in vivo. Potential therapeutic effect demonstrated in animal model of disease. 3x Tg, triple transgenic;AD, Alzheimers disease; ADHD, Attention deficit and hyperactivity disorder; FAD, Familiar AD; DIO; Diet-induced obesity; HFD, High fat diet; HGD, High Glucose diet; PD; Parkinsons disease; STZ, Streptozotocin; RA, Retinoic Acid. (i.p, intraperitoneal; i.c.v intracerebro-ventricular). ABA treatment Disease Animal model Dose Duration Effect Via ref Cancer PCa xenograft in mice 20 mg/kg i.p. Twice daily for 8 weeks •Induction of dormancy state of PCa cells in the bone marrow microenvironment [59] UG87MG xenograft mice 60 mg/kg i.p. Once daily for 21 days •Reduce tumour size; increase autophagy markers MAPK/JNK signalling [58] Albino male rats 100 μ g/ml 1 time (ex vivo)•Prevention of angiogenesis alone and in combination with prednisolone [65] Cardiorenal syndrome Surgically induced CRS type 3 rat model 1 mg/kg orally Once daily, for 4 weeks •Shortening of the ventricular action potential duration •Decrease of heart weight. Reduced P-53, NOX-4 Increased HSP-70 levels [69] Essential tremor (ET) Harmaline-induced ET rat model 10 μ g i.c.v 30 min before harmaline administration •Improvement of tremor and motor symptomatology, and of cognitive alterations associated to the disease [72] Atherosclerosis ApoE(−/-) HFD-fed mice (atherosclerosis mouse model) 100 mg/kg (dietary administration) 84 days •Improvement of systolic blood pressure •Reduced aortic eNOS, VCAM-1 and MCP-1 expression. •Reduction of CD11b (+) macrophages and CD4(+) T-cells accumulation in aortic root walls [73] Intestinal diseases DSS-induced colitis/IBD mouse model 100 mg/kg (dietary administration) 35 days (pre DSS administration) •Improvement of the disease progress and decrease of colonic leukocyte infiltration and inflammation. •Reduced VCAM-1 and E-selectin, expression. •Increased CD4(+) and CD8(+) Tlymphocytes in blood [78] Transgenic mice with defective PPAR-γ expression in colonic epithelial cells +DSS 100 mg/kg (dietary administration) 35 days (pre DSS administration) •Reduction of disease severity and prevention of colitis •Improvement of colonic cells histopathology •Increased levels of antiinflammatory T-cells phenotypes. (Cd8+; IL10 +) •Effects independents of PPAR-γ expression in intestinal cells LANCL-2 upregulation PPARγ independent [79] Pulmonary diseases OVA-induced asthma mouse model 60 mg/kg i.p. 7 days •Prevention of the development of airway inflammation •Downregulation of NLRP3 inflammasome activation, oxidative stress and mitochondrial fusion/ fission markers PPARγ[82] LPS-induced ARDS mouse model 100 mg/kg i.p. 7 days (pre LPS administration) •Reduction of pulmonary injury •Decreased ROS production and ER stress •Increase PPAR-γlevels and activation of Nrf2 PPARγ Nrf2 [85] Influenza-associated pulmonary inflammation 100 mg/kg (dietary administration) 1 time (day of influenza infection) or 10 days (post influenza infection) •Improvement of the disease •Inhibition of leukocyte infiltration and MCP-1 expression. •Increase of IL-10 expression and regulation of genes involved in resolution PPARγ[88] Malaria Plasmodium falciparuminduced malaria mosquito model 100 nM in the blood containing Plasmodium falciparum 1 time •Decrease of infection prevalence •Increase of NOS activity and TAK1 expression [91] Plasmodium yoelii-induced malaria mouse model 2.56 mM in drinking water 3 days (before infection) •Reduction of parasitemia and spleen liver pathology •Increase of PPAR-γexpression in spleen and liver •Decrease of eNOS in spleen and liver PPAR-γ[90] Metabolic syndrome Adult male rats and mice 1 μ g/kg Single oral dose or 1 month •Increment of glucose uptake in BAT (single oral dose) •Upregulation of BAT genes markers in WAT (1 month treatment) [94] (continued on next page) A. 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