The novel adrenergic agonist ATR-127 targets skeletal muscle and brown adipose tissue to tackle diabesity and steatohepatitis.

OBJECTIVE: Simultaneous activation of ß2- and ß3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of ß1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel ß2-and ß3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models. METHODS: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective ß-AR agonist isoprenaline across various rodent ß-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis. RESULTS: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis. CONCLUSIONS: Our results demonstrate that ATR-127 is a highly effective, novel ß2- and ß3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.

Impairment of Endogenous Synthesis of Omega-3 DHA Exacerbates T-Cell Inflammatory Responses.

Omega-3 (ω-3) polyunsaturated fatty acids, including docosahexaenoic acid (DHA), are involved in numerous biological processes and have a range of health benefits. DHA is obtained through the action of elongases (ELOVLs) and desaturases, among which Elovl2 is the key enzyme involved in its synthesis, and can be further metabolized into several mediators that regulate the resolution of inflammation. Our group has recently reported that ELOVL2 deficient mice (Elovl2-/-) not only display reduced DHA levels in several tissues, but they also have higher pro-inflammatory responses in the brain, including the activation of innate immune cells such as macrophages. However, whether impaired synthesis of DHA affects cells of adaptive immunity, i.e., T lymphocytes, is unexplored. Here we show that Elovl2-/- mice have significantly higher lymphocytes in peripheral blood and that both CD8+ and CD4+ T cell subsets produce greater amounts of pro-inflammatory cytokines in both blood and spleen compared to wild type mice, with a higher percentage of cytotoxic CD8+ T cells (CTLs) as well as IFN-γ-producing Th1 and IL-17-producing Th17 CD4+ cells. Furthermore, we also found that DHA deficiency impacts the cross-talk between dendritic cells (DC) and T cells, inasmuch as mature DCs of Elovl2-/- mice bear higher expression of activation markers (CD80, CD86 and MHC-II) and enhance the polarization of Th1 and Th17 cells. Reintroducing DHA back into the diets of Elovl2-/- mice reversed the exacerbated immune responses observed in T cells. Hence, impairment of endogenous synthesis of DHA exacerbates T cell inflammatory responses, accounting for an important role of DHA in regulating adaptive immunity and in potentially counteracting T-cell-mediated chronic inflammation or autoimmunity.

Elovl2-Ablation Leads to Mitochondrial Membrane Fatty Acid Remodeling and Reduced Efficiency in Mouse Liver Mitochondria.

The fatty acid elongase elongation of very long-chain fatty acids protein 2 (ELOVL2) controls the elongation of polyunsaturated fatty acids (PUFA) producing precursors for omega-3, docosahexaenoic acid (DHA), and omega-6, docosapentaenoic acid (DPAn-6) in vivo. Expectedly, Elovl2-ablation drastically reduced the DHA and DPAn-6 in liver mitochondrial membranes. Unexpectedly, however, total PUFAs levels decreased further than could be explained by Elovl2 ablation. The lipid peroxidation process was not involved in PUFAs reduction since malondialdehyde-lysine (MDAL) and other oxidative stress biomarkers were not enhanced. The content of mitochondrial respiratory chain proteins remained unchanged. Still, membrane remodeling was associated with the high voltage-dependent anion channel (VDAC) and adenine nucleotide translocase 2 (ANT2), a possible reflection of the increased demand on phospholipid transport to the mitochondria. Mitochondrial function was impaired despite preserved content of the respiratory chain proteins and the absence of oxidative damage. Oligomycin-insensitive oxygen consumption increased, and coefficients of respiratory control were reduced by 50%. The mitochondria became very sensitive to fatty acid-induced uncoupling and permeabilization, where ANT2 is involved. Mitochondrial volume and number of peroxisomes increased as revealed by transmission electron microscopy. In conclusion, the results imply that endogenous DHA production is vital for the normal function of mouse liver mitochondria and could be relevant not only for mice but also for human metabolism.

Macrophage Plasticity and Polarization Are Altered in the Experimental Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. MS is characterized by infiltrations of leukocytes such as T and B lymphocytes and macrophages. Macrophages have been identified as major effectors of inflammation and demyelination in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the activation and heterogeneity of macrophages in MS has been poorly investigated. Thus, in this study, we evaluated M1 and M2 macrophages immunophenotype from EAE and control mice by analyzing over 30 surface and intracellular markers through polychromatic flow cytometry, qRT-PCR, and ELISA assay. We showed that M1 macrophages possessed a higher proinflammatory profile in EAE compared to control mice, since they expressed higher levels of activation/co-stimulatory markers (iNOS, CD40, and CD80) and cytokines/chemokines (IL-6, IL-12, CCL2, and CXCL10), whereas M2 lost their M2-like phenotype by showing a decreased expression of their signature markers CD206 and CCL22, as well as a concomitant upregulation of several M1 makers. Furthermore, immunization of M1 and M2 macrophages with MOG35-55 led to a significant hyperactivation of M1 and a concomitant shift of anti-inflammatory M2 to pro-inflammatory M1 macrophages. Overall, we provide evidence for a phenotypic alteration of M1/M2 balance during MS, which can be of crucial importance not only for a better understanding of the immunopathology of this neurodegenerative disease but also to potentially develop new macrophage-centered therapeutic strategies.

Impairment of DHA synthesis alters the expression of neuronal plasticity markers and the brain inflammatory status in mice.

Docosahexaenoic acid (DHA) is a ω-3 fatty acid typically obtained from the diet or endogenously synthesized through the action of elongases (ELOVLs) and desaturases. DHA is a key central nervous system constituent and the precursor of several molecules that regulate the resolution of inflammation. In the present study, we questioned whether the impaired synthesis of DHA affected neural plasticity and inflammatory status in the adult brain. To address this question, we investigated neural and inflammatory markers from mice deficient for ELOVL2 (Elovl2-/- ), the key enzyme in DHA synthesis. From our findings, Elovl2-/- mice showed an altered expression of markers involved in synaptic plasticity, learning, and memory formation such as Egr-1, Arc1, and BDNF specifically in the cerebral cortex, impacting behavioral functions only marginally. In parallel, we also found that DHA-deficient mice were characterized by an increased expression of pro-inflammatory molecules, namely TNF, IL-1ß, iNOS, caspase-1 as well as the activation and morphologic changes of microglia in the absence of any brain injury or disease. Reintroducing DHA in the diet of Elovl2-/- mice reversed such alterations in brain plasticity and inflammation. Hence, impairment of systemic DHA synthesis can modify the brain inflammatory and neural plasticity status, supporting the view that DHA is an essential fatty acid with an important role in keeping inflammation within its physiologic boundary and in shaping neuronal functions in the central nervous system.

Synergistic Effects of DHA and Sucrose on Body Weight Gain in PUFA-Deficient Elovl2 -/- Mice.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) is implicated in theregulation of both lipid and carbohydrate metabolism. Thus, we questioned whether dietary DHAand low or high content of sucrose impact on metabolism in mice deficient for elongation of verylong-chain fatty acids 2 (ELOVL2), an enzyme involved in the endogenous DHA synthesis. Wefound that Elovl2 -/- mice fed a high-sucrose DHA-enriched diet followed by the high sucrose, highfat challenge significantly increased body weight. This diet affected the triglyceride rich lipoproteinfraction of plasma lipoproteins and changed the expression of several genes involved in lipidmetabolism in a white adipose tissue. Our findings suggest that lipogenesis in mammals issynergistically influenced by DHA dietary and sucrose content.

Impairment of systemic DHA synthesis affects macrophage plasticity and polarization: implications for DHA supplementation during inflammation.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid obtained from the diet or synthesized from alpha-linolenic acid through the action of fatty acid elongases (ELOVL) and desaturases. DHA plays important roles in the central nervous system as well as in peripheral organs and is the precursor of several molecules that regulate resolution of inflammation. In the present study, we questioned whether impaired synthesis of DHA affected macrophage plasticity and polarization both in vitro and in vivo models. For this we investigated the activation status and inflammatory response of bone marrow-derived M1 and M2 macrophages obtained from mice deficient of Elovl2 (Elovl2-/-), a key enzyme for DHA synthesis in mammals. Although both wild type and Elovl2-/- mice were able to generate efficient M1 and M2 macrophages, M1 cells derived from Elovl2-/- mice showed an increased expression of key markers (iNOS, CD86 and MARCO) and cytokines (IL-6, IL-12 and IL-23). However, M2 macrophages exhibited upregulated M1-like markers like CD80, CD86 and IL-6, concomitantly with a downregulation of their signature marker CD206. These effects were counteracted in cells obtained from DHA-supplemented animals. Finally, white adipose tissue of Elovl2-/- mice presented an M1-like pro-inflammatory phenotype. Hence, impairment of systemic DHA synthesis delineates an alteration of M1/M2 macrophages both in vitro and in vivo, with M1 being hyperactive and more pro-inflammatory while M2 less protective, supporting the view that DHA has a key role in controlling the balance between pro- and anti-inflammatory processes.

Both maternal and offspring Elovl2 genotypes determine systemic DHA levels in perinatal mice.

The molecular details relevant to dietary supplementation of the omega-3 fatty acid DHA in mothers as well as in their offspring are not clear. The PUFA elongase, elongation of very long-chain fatty acid (ELOVL)2, is a critical enzyme in the formation of DHA in mammals. In order to address the question regarding the origin of DHA during perinatal life, we have used DHA-deficient Elovl2-ablated mice as a model system to analyze the maternal impact on the DHA level in their offspring of various genotypes. Elovl2-/- mothers maintained on control diet had significantly lower systemic levels of DHA compared with the Elovl2+/- and Elovl2+/+ mothers. Dietary DHA administration during the pregnancy and lactation periods led to increased DHA accretion in maternal tissues and serum of all genotypes. The proportion of DHA in the liver and serum of the Elovl2-/- offspring was significantly lower than in the Elovl2+/+ offspring. Remarkably, the DHA level in the Elovl2+/- offspring nursed by DHA-free-fed Elovl2-/- mothers was almost as high as in +/+ pups delivered by +/+ mothers, suggesting that endogenous synthesis in the offspring can compensate for maternal DHA deficiency. Maternal DHA supplementation had a strong impact on offspring hepatic gene expression, especially of the fatty acid transporter, Mfsd2a, suggesting a dynamic interplay between DHA synthesis and DHA uptake in the control of systemic levels in the offspring.

Anandamide Suppresses Proinflammatory T Cell Responses In Vitro through Type-1 Cannabinoid Receptor-Mediated mTOR Inhibition in Human Keratinocytes.

The endocannabinoid system comprises cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-12 and IL-23, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1 Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-12 and IL-23 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases.

Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages.

The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases.

Endocannabinoids, related compounds and their metabolic routes.

Endocannabinoids are lipid mediators able to bind to and activate cannabinoid receptors, the primary molecular targets responsible for the pharmacological effects of the Δ9-tetrahydrocannabinol. These bioactive lipids belong mainly to two classes of compounds: N-acylethanolamines and acylesters, being N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, their main representatives. During the last twenty years, an ever growing number of fatty acid derivatives (endocannabinoids and endocannabinoid-like compounds) have been discovered and their activities biological is the subject of intense investigations. Here, the most recent advances, from a therapeutic point of view, on endocannabinoids, related compounds, and their metabolic routes will be reviewed.

The fatty acid amide hydrolase in lymphocytes from sedentary and active subjects.

PURPOSE: Endocannabinoids (eCB) and interleukin 6 (IL-6) levels change during physical activity, thus suggesting their involvement in the modulation of exercise-related processes like inflammation and energy homeostasis. To investigate whether lifestyle might affect the activity of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), active and sedentary subjects were enrolled. METHODS: Plasma IL-6 levels and lymphocyte FAAH activity of eight physically active male subjects (mean ± SEM; age = 39.3 ± 2.9 yr, body mass index = 21.1 ± 0.4 kg·m), usually practicing aerobic exercise (8.1 ± 1.2 h·wk), and eight sedentary subjects (38.8 ± 3.7 yr, body mass index = 23.1 ± 0.8 kg·m) were measured. Also, in vitro effect of IL-6 was tested on FAAH expression and activity and on FAAH promoter activity in lymphocytes from sedentary subjects. RESULTS: Under resting conditions (at least 12 h from the last exercise), the active group showed plasma IL-6 levels (2.74 ± 0.73 pg·mL) and lymphocyte FAAH activity (215.7 ± 38.5 pmol·min·mg protein) significantly higher than those measured in the sedentary group (0.20 ± 0.02 pg·mL, and 42.0 ± 4.2 pmol·min·mg protein). Increased IL-6 levels paralleled increased FAAH activity, and consistently, the in vitro treatment of lymphocytes from sedentary individuals with 10 ng·mL IL-6 for 48 h significantly increased FAAH expression and activity. Transient transfection experiments showed that IL-6 induced the expression of a reporter gene under the control of a cAMP response element-like region in the human FAAH promoter. A mutation in the same element abolished IL-6 up-regulation, demonstrating that this cytokine regulates FAAH activity at the transcriptional level. CONCLUSION: IL-6 leads to activation of the FAAH promoter, thus enhancing FAAH activity that modulates the eCB tone in physically active people.

Discovery of potent inhibitors of human and mouse fatty acid amide hydrolases.

Fatty acid amide hydrolase (FAAH, EC 3.5.1.99) is the main enzyme catabolizing endocannabinoid fatty acid amides. FAAH inactivation promotes beneficial effects upon pain and anxiety without the side effects accompanying agonists of type-1 cannabinoid receptors. Aiming at discovering new selective FAAH inhibitors, we developed a series of compounds (5a-u) characterized by a functionalized heteroaromatic scaffold. Particularly, 5c and 5d were identified as extremely potent, noncompetitive, and reversible FAAH inhibitors endowed with a remarkable selectivity profile and lacking interaction with the hERG channels. In vivo antinociceptive activity was demonstrated for 5c, 5d, and 5n at a dose much lower than that able to induce either striatal and limbic stereotypies or anxiolytic activity, thus outlining their potential to turn into optimum preclinical candidates. Aiming at improving pharmacokinetic properties and metabolic stability of 5d, we developed a subset of nanomolar dialyzable FAAH inhibitors (5v-z), functionalized by specific polyethereal lateral chains and fluorinated aromatic rings.