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ABSTRACTDietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) and the gut microbiome affect each other. We investigated the impact of supplementation with Buglossoides arvensis oil (BO), rich in stearidonic acid (SDA), on the human gut microbiome. Employing the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we simulated the ileal and ascending colon microbiomes of four donors. Our results reveal two distinct microbiota clusters influenced by BO, exhibiting shared and contrasting shifts. Notably, Bacteroides and Clostridia abundance underwent similar changes in both clusters, accompanied by increased propionate production in the colon. However, in the ileum, cluster 2 displayed a higher metabolic activity in terms of BO-induced propionate levels. Accordingly, a triad of bacterial members involved in propionate production through the succinate pathway, namely Bacteroides, Parabacteroides, and Phascolarctobacterium, was identified particularly in this cluster, which also showed a surge of second-generation probiotics, such as Akkermansia, in the colon. Finally, we describe for the first time the capability of gut bacteria to produce N-acyl-ethanolamines, and particularly the SDA-derived N-stearidonoyl-ethanolamine, following BO supplementation, which also stimulated the production of another bioactive endocannabinoid-like molecule, commendamide, in both cases with variations across individuals. Spearman correlations enabled the identification of bacterial genera potentially involved in endocannabinoid-like molecule production, such as, in agreement with previous reports, Bacteroides in the case of commendamide. This study suggests that the potential health benefits on the human microbiome of certain dietary oils may be amenable to stratified nutrition strategies and extend beyond n-3 PUFAs to include microbiota-derived endocannabinoid-like mediators.

There is a growing evidence suggesting the association of vitamin D deficiency (VDD) and cognitive impairment. In this study we evaluated the possible involvement of gut microbiota in the cognitive impairments mediated by VDD and investigated the effects of pharmacological treatment with the oxazoline derivative of the aliamide palmitoylethanolamide, 2-Pentadecyl-2-oxazoline (PEA-OXA). Mice were submitted to behavioural, biochemical and electrophysiological analysis to assess whether their vitamin D status affected cognitive performance together with gut microbiota composition. In VDD mice we found cognitive malfunctioning associated with reduced neuroplasticity, indicated by impaired long term potentiation, and neuroinflammation at the hippocampal level. Importantly, PEA-OXA counteracted the cognitive impairments and modified the biochemical and functional changes induced by VDD. Additionally, PEA-OXA treatment enhanced gut microbiota diversity, which tended to be decreased by VDD only in female mice, elevated the relative abundance of lactic and butyric acid-producing families, i.e. Aerococcaceae and Butyricicoccaceae, and reversed the VDD-induced decrease of butyrate-producing beneficial genera, such as Blautia in female mice, and Roseburia in male mice. These data provide novel insights for a better understanding of the cognitive decline induced by VDD and related gut dysbiosis and its potential therapeutic treatment.

Abstract N-oleoylglycine (OlGly), a lipid derived from the basic component of olive oil, oleic acid, and N-oleoylalanine (OlAla) are endocannabinoid-like mediators. We report that OlGly and OlAla, by activating the peroxisome proliferator-activated receptor alpha (PPARα), reduce the rewarding properties of a highly palatable food, dopamine neuron firing in the ventral tegmental area, and the obesogenic effect of a high-fat diet rich in lard (HFD-L). An isocaloric olive oil HFD (HFD-O) reduced body weight gain compared to the HFD-L, in a manner reversed by PPARα antagonism, and enhanced brain and intestinal OlGly levels and gut microbial diversity. OlGly or OlAla treatment of HFD-L mice resulted in gut microbiota taxonomic changes partly similar to those induced by HFD-O. We suggest that OlGly and OlAla control body weight by counteracting highly palatable food overconsumption, and possibly rebalancing the gut microbiota, and provide a potential new mechanism of action for the obeso-preventive effects of olive oil-rich diets.

Abstract Background Gut microbiota are involved in the onset and development of chronic intestinal inflammation. The recently described endocannabinoidome (eCBome), a diverse and complex system of bioactive lipid mediators, has been reported to play a role in various physio-pathological processes such as inflammation, immune responses and energy metabolism. The eCBome and the gut microbiome (miBIome) are closely linked and form the eCBome - miBIome axis, which may be of special relevance to colitis. Methods Colitis was induced in conventionally raised (CR), antibiotic-treated (ABX) and germ-free (GF) mice with dinitrobenzene sulfonic acid (DNBS). Inflammation was assessed by Disease Activity Index (DAI) score, body weight change, colon weight-length ratio, myeloperoxidase (MPO) activity and cytokine gene expression. Colonic eCBome lipid mediator concentrations were measured by HPLC-MS /MS. Results GF mice showed increased levels of anti-inflammatory eCBome lipids (LEA, OEA, DHEA and 13- HODE-EA) in the healthy state and higher MPO activity. DNBS elicited reduced inflammation in GF mice, having lower colon weight/length ratios and lower expression levels of Il1b, Il6, Tnfa and neutrophil markers compared to one or both of the other DNBS-treated groups. Il10 expression was also lower and the levels of several N-acyl ethanolamines and 13-HODE-EA levels were higher in DNBS-treated GF mice than in CR and ABX mice. The levels of these eCBome lipids negatively correlated with measures of colitis and inflammation. Conclusions These results suggest that the depletion of the gut microbiota and subsequent differential development of the gut immune system in GF mice is followed by a compensatory effect on eCBome lipid mediators, which may explain, in part, the observed lower susceptibility of GF mice to develop DNBS-induced colitis.

White adipose tissue regulation is key to metabolic health, yet still perplexing. The chief endocannabinoid anandamide metabolite, prostaglandin F2α ethanolamide (PGF2αEA), inhibits adipogenesis, that is, the formation of mature adipocytes. We observed that adipocyte progenitor cells—preadipocytes—following treatment with PGF2αEA yielded larger pellet sizes. Thus, we hypothesized that PGF2αEA might augment preadipocyte proliferation. Cell viability MTT and crystal violet assays, cell counting, and 5-bromo-2′-deoxyuridine incorporation in cell proliferation ELISA analyses confirmed our prediction. Additionally, we discovered that PGF2αEA promotes cell cycle progression through suppression of the expression of cell cycle inhibitors, p21 and p27, as shown by flow cytometry and qPCR. Enticingly, concentrations of this compound that showed no visible effect on cell proliferation or basal transcriptional activity of peroxisome proliferator-activated receptor gamma could, in contrast, reverse the anti-proliferative and peroxisome proliferator-activated receptor gamma-transcription activating effects of rosiglitazone (Rosi). MTT and luciferase reporter examinations supported this finding. The PGF2αEA pharmaceutical analog, bimatoprost, was also investigated and showed very similar effects. Importantly, we suggest the implication of the mitogen-activated protein kinase pathway in these effects, as they were blocked by the selective mitogen-activated protein kinase kinase inhibitor, PD98059. We propose that PGF2αEA is a pivotal regulator of white adipose tissue plasticity, acting as a regulator of the preadipocyte pool in adipose tissue.

Abstract Nothing is known about the potential implication of gut microbiota in skeletal muscle disorders. Here, we provide evidence that fecal microbiota composition along with circulating levels of short‐chain fatty acids (SCFAs) and related metabolites are altered in the mdx mouse model of Duchenne muscular dystrophy (DMD) compared with healthy controls. Supplementation with sodium butyrate (NaB) in mdx mice rescued muscle strength and autophagy, and prevented inflammation associated with excessive endocannabinoid signaling at CB1 receptors to the same extent as deflazacort (DFZ), the standard palliative care for DMD. In LPS‐stimulated C2C12 myoblasts, NaB reduces inflammation, promotes autophagy, and prevents dysregulation of microRNAs targeting the endocannabinoid CB1 receptor gene, in a manner depending on the activation of GPR109A and PPARγ receptors. In sum, we propose a novel disease‐modifying approach in DMD that may have benefits also in other muscular dystrophies.

The health and balance of the gut microbiota are known to be linked to diet composition and source, with fermented products and dietary proteins potentially providing an exceptional advantage for the gut. The purpose of this study was to evaluate the effect of protein hydrolysis, using a probiotic beverage enriched with either cricket protein (CP) or cricket protein hydrolysates (CP.Hs), on the composition of the gut microbiota of rats. Taxonomic characterization of the gut microbiota in fecal samples was carried out after a 14-day nutritional study to identify modifications induced by a CP- and CP.H-enriched fermented probiotic product. The results showed no significant differences (p > 0.05) in the diversity and richness of the gut microbiota among the groups fed with casein (positive control), CP-enriched, and fermented CP.H-enriched probiotic beverages; however, the overall composition of the microbiota was altered, with significant modifications in the relative abundance of several bacterial families and genera. In addition, fermented CP.H-enriched probiotic beverages could be related to the decrease in the number of potential pathogens such as Enterococcaceae. The association of gut microbiota with the nutritional parameters was determined and the results showed that digestibility and the protein efficiency ratio (PER) were highly associated with the abundance of several taxa.

Consumption of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) provides multifaceted health benefits. Recent studies suggest that ω-3 PUFAs modulate the gut microbiota by enhancing health-promoting bacteria, such as the mucin specialist Akkermansia muciniphila. However, these prebiotic properties have been poorly investigated and direct effects on the gut microbiome have never been explored dynamically across gut regions and niches (lumen vs. mucus-associated microbiota). Thus, we studied the effects of 1 week EPA- and DHA-enriched ω-3 fish-oil supplementation on the composition and functionality of the human microbiome in a Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME®). Gut microbial communities derived from one individual harvested in two different seasons were tested in duplicate. Luminal and outer mucus-associated microbiota of the ileum, ascending, transverse and descending colons were cultivated over 28 d from fecal inoculates and supplemented with ω-3 PUFAs for the last 7 d. We show that ω-3 PUFA supplementation modulates the microbiota in a gut region- and niche-dependent fashion. The outer mucus-associated microbiota displayed a higher resilience than the luminal mucin habitat to ω-3 PUFAs, with a remarkable blooming of Akkermansia muciniphila in opposition to a decrease of Firmicutes-mucolytic bacteria. The ω-3 PUFAs also induced a gradual and significant depletion of non-mucolytic Clostridia members in luminal habitats. Finally, increased concentrations of the short chain fatty acids (SCFA) propionate in colon regions at the end of the supplementation was associated positively with the bloom of Akkermansia muciniphila and members of the Desulfovibrionia class.

Omega-3 fatty acids support cardiometabolic health and reduce chronic low-grade inflammation. These fatty acids may impart their health benefits partly by modulating the endocannabinoidome and the gut microbiome, both of which are key regulators of metabolism and the inflammatory response. Whole hemp seeds (Cannabis sativa) are of exceptional nutritional value, being rich in omega-3 fatty acids. We assessed the effects of dietary substitution (equivalent to about 2 tablespoons of seeds a day for humans) of whole hemp seeds in comparison with whole linseeds in a diet-induced obesity mouse model and determined their effects on obesity and the gut microbiome-endocannabinoidome axis. We show that whole hemp seed substitution did not affect weigh gain, adiposity, or food intake, whereas linseed substitution did, in association with higher fasting glucose levels, greater insulin release during an oral glucose tolerance test, and higher levels of liver triglycerides than controls. Furthermore, hemp seed substitution mitigated diet-induced obesity-associated increases in intestinal permeability and circulating PAI-1 levels, while having no effects on markers of inflammation in epididymal adipose tissue, which were, however, increased in mice fed linseeds. Both hemp seeds and linseeds were able to modify the expression of several endocannabinoidome genes and markedly increased the levels of several omega-3 fatty acid–derived endocannabinoidome bioactive lipids with previously suggested anti-inflammatory actions in a tissue specific manner, despite the relatively low level of seed substitution. While neither diet markedly modified the gut microbiome, mice on the hemp seed diet had higher abundance of Clostridiaceae 1 and Rikenellaceae than mice fed linseed or control diet, respectively. Thus, hemp seed-containing foods might represent a source of healthy fats that are not likely to exacerbate the metabolic consequences of obesogenic diets while producing intestinal permeability protective effects and some anti-inflammatory actions.

The gut microbiota is a unique ecosystem of microorganisms interacting with the host through several biochemical mechanisms. The endocannabinoidome (eCBome), a complex signaling system including the endocannabinoid system, approximately 50 receptors and metabolic enzymes, and more than 20 lipid mediators with important physiopathologic functions, modulates gastrointestinal tract function and may mediate host cell-microbe communications there. Germ-free (GF) mice, which lack an intestinal microbiome and so differ drastically from conventionally raised (CR) mice, offer a unique opportunity to explore the eCBome in a microbe-free model and in the presence of a reintroduced functional gut microbiome through fecal microbiota transplant (FMT). We aimed to gain direct evidence for a link between the microbiome and eCBome systems by investigating eCBome alterations in the gut in GF mice before and after FMT. Basal eCBome gene expression and lipid profiles were measured in various segments of the intestine of GF and CR mice at juvenile and adult ages using targeted quantitative PCR transcriptomics and LC-MS/MS lipidomics. GF mice exhibited age-dependent modifications in intestinal eCBome gene expression and lipid mediator levels. FMT from CR donor mice to age-matched GF male mice reversed several of these alterations, particularly in the ileum and jejunum, after only 1 week, demonstrating that the gut microbiome directly impacts the host eCBome and providing a cause-effect relationship between the presence or absence of intestinal microbes and eCBome signaling. These results open the way to new studies investigating the mechanisms through which intestinal microorganisms exploit eCBome signaling to exert some of their physiopathologic functions.

Obesity is associated with a cluster of metabolic disorders, chronic low-grade inflammation, altered gut microbiota, increased intestinal permeability, and alterations of the lipid mediators of the expanded endocannabinoid (eCB) signaling system, or endocannabinoidome (eCBome). In the present study, we characterized the profile of the eCBome and related oxylipins in the small and large intestines of genetically obese (ob/ob) and diabetic (db/db) mice to decipher possible correlations between these mediators and intestinal inflammation and gut microbiota composition. Basal lipid and gene expression profiles, measured by LC/MS-MS-based targeted lipidomics and qPCR transcriptomics, respectively, highlighted a differentially altered intestinal eCBome and oxylipin tone, possibly linked to increased mRNA levels of inflammatory markers in db/db mice. In particular, the duodenal levels of several 2-monoacylglycerols and N-acylethanolamines were increased and decreased, respectively, in db/db mice, which displayed more pronounced intestinal inflammation. To a little extent, these differences were explained by changes in the expression of the corresponding metabolic enzymes. Correlation analyses suggested possible interactions between eCBome/oxylipin mediators, cytokines, and bacterial components and bacterial taxa closely related to intestinal inflammation. Collectively, this study reveals that db/db mice present a higher inflammatory state in the intestine as compared to ob/ob mice, and that this difference is associated with profound and potentially adaptive or maladaptive, and partly intestinal segment-specific alterations in eCBome and oxylipin signaling. This study opens the way to future investigations on the biological role of several poorly investigated eCBome mediators and oxylipins in the context of obesity and diabetes-induced gut dysbiosis and inflammation.

Rationale: The endocannabinoidome mediators, N-Oleoylglycine (OlGly) and N-Oleoylalanine (OlAla), have been shown to reduce acute naloxone-precipitated morphine withdrawal affective and somatic responses.Objectives: To determine the role and mechanism of action of OlGly and OlAla in withdrawal responses from chronic exposure to opiates in male Sprague-Dawley rats.Methods: Opiate withdrawal was produced: 1) spontaneously 24 h following chronic exposure to escalating doses of morphine over 14 days (Experiments 1 and 2) and steady-state exposure to heroin by minipumps for 12 days (Experiment 3), 2) by naloxone injection during steady-state heroin exposure (Experiment 4), 3) by naloxone injection during operant heroin self-administration (Experiment 5).Results: In Experiment 1, spontaneous morphine withdrawal produced somatic withdrawal reactions. The behavioral withdrawal reactions were accompanied by suppressed endogenous levels of OlGly in the nucleus accumbens, amygdala, and prefrontal cortex, N-Arachidonylglycerol and OlAla in the amygdala, 2-arachidonoylglycerol in the nucleus accumbens, amygdala and interoceptive insular cortex, and by changes in colonic microbiota composition. In Experiment 2, treatment with OlAla, but not OlGly, reduced spontaneous morphine withdrawal responses. In Experiment 3, OlAla attenuated spontaneous steady-state heroin withdrawal responses at both 5 and 20 mg/kg; OlGly only reduced withdrawal responses at the higher dose of 20 mg/kg. Experiment 4 demonstrated that naloxone-precipitated heroin withdrawal from steady-state exposure to heroin (7 mg/kg/day for 12 days) is accompanied by tissue-specific changes in brain or gut endocannabinoidome mediator, including OlGly and OlAla, levels and colonic microbiota composition, and that OlAla (5 mg/kg) attenuated behavioural withdrawal reactions, while also reversing some of the changes in brain and gut endocannabinoidome and gut microbiota induced by naloxone. Experiment 5 demonstrated that although OlAla (5 mg/kg) did not interfere with operant heroin self-administration on its own, it blocked naloxone-precipitated elevation of heroin self-administration behavior.Conclusion: These results suggest that OlAla and OlGly are two endogenous mediators whose brain concentrations respond to chronic opiate treatment and withdrawal concomitantly with changes in colon microbiota composition, and that OlAla may be more effective than OlGly in suppressing chronic opiate withdrawal responses.

Obesity is a disease with high potential for fatality. It perfectly fits the disease definition, as cancer does. This is because it damages body structure and functions, both mechanically and biologically, and alters physical, mental, and social health. In addition, it shares many common morbid characteristics with the most feared disease, cancer. For example, it is influenced by a sophisticated interaction between a person’s genetics, the environment, and an increasing number of other backgrounds. Furthermore, it displays abnormal cell growth and proliferation events, only limited to white fat, resulting in adipose tissue taking up an increasing amount of space within the body. This occurs through fat “metastases” and via altered signaling that further aggravates the pathology of obesity by inducing ubiquitous dishomeostasis. These metastases can be made graver by angiogenesis, which might boost diseased tissue growth. More common features with cancer include its progressive escalation through different levels of severity and its possibility of re-onset after recovery. Despite all these similarities with cancer, obesity is substantially less agitating for most people. Thus, the ideas proposed herein could have utility to sensitize the public opinion about the hard reality of obesity. This is increasingly needed, as the obesity pandemic has waged a fierce war against our bodies and society in general, while there is still doubt about whether it is a real disease or not. Hence, raising public consciousness to properly face health issues is crucial to improving our health instead of gaining weight unhealthily. It is obviously illogical to fight cancer extremely seriously on the one hand and to consider dying with obesity as self-inflicted on the other. In fact, obesity merits a top position among the most lethal diseases besides cancer.

Inflammatory bowel disorders can be associated with alterations in gut microbiota (dysbiosis) and behavioral disturbances. In experimental colitis, administration of fish oil (FO) or cannabinoids, such as cannabidiol (CBD), reduce inflammation. We investigated the effect of combined FO/CBD administration on inflammation and dysbiosis in the dextran sulphate sodium (DSS) model of mouse colitis, which also causes behavioral disturbances. Colitis was induced in CD1 mice by 4% w/v DSS in drinking water for five consecutive days followed by normal drinking water. FO (20–75 mg/mouse) was administered once a day starting two days after DSS, whereas CBD (0.3–30 mg/kg), alone or after FO administration, was administered once a day starting 3 days after DSS, until day 8 (d8) or day 14 (d14). Inflammation was assessed at d8 and d14 (resolution phase; RP) by measuring the Disease Activity Index (DAI) score, change in body weight, colon weight/length ratio, myeloperoxidase activity and colonic interleukin (IL)-1β (IL-1β), IL-10, and IL-6 concentrations. Intestinal permeability was measured with the fluorescein isothiocyanate-dextran. Behavioral tests (novel object recognition (NOR) and light/dark box test) were performed at d8. Fecal microbiota composition was determined by ribosomal 16S DNA sequencing of faecal pellets at d8 and d14. DSS-induced inflammation was stronger at d8 and accompanied by anxiety-like behavior and impaired recognition memory. FO (35, 50, 75 mg/mouse) alone reduced inflammation at d8, whereas CBD alone produced no effect at any of the doses tested; however, when CBD (3, 10 mg/kg) was co-administered with FO (75 mg/mouse) inflammation was attenuated. FO (20 mg/mouse) and CBD (1 mg/kg) were ineffective when given alone, but when co-administered reduced all inflammatory markers and the increased intestinal permeability at both d8 and d14, but not the behavioral impairments. FO, CBD, and their combination affected gut bacteria taxa that were not affected by DSS per se. Akkermansia muciniphila, a species suggested to afford anti-inflammatory action in colitis, was increased by DSS only at d14, but its levels were significantly elevated by all treatments at d8. FO and CBD co-administered at per se ineffective doses reduce colon inflammation, in a manner potentially strengthened by their independent elevation of Akkermansia muciniphila.

ABSTRACT The intestinal microbiota and the expanded endocannabinoid (eCB) system, or endocannabinoidome (eCBome), have both been implicated in diet-induced obesity and dysmetabolism. These systems were recently suggested to interact during the development of obesity. We aimed at identifying the potential interactions between gut microbiota composition and the eCBome during the establishment of diet-induced obesity and metabolic complications. Male mice were fed a high-fat, high-sucrose (HFHS) diet for 56 days to assess jejunum, ileum, and cecum microbiomes by 16S rRNA gene metataxonomics as well as ileum and plasma eCBome by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HFHS diet induced early (3 days) and persistent glucose intolerance followed by weight gain and hyperinsulinemia. Concomitantly, it induced the elevation of the two eCBs, anandamide, in both ileum and plasma, and 2-arachidonoyl-glycerol, in plasma, as well as alterations in several other N-acylethanolamines and 2-acylglycerols. It also promoted segment-specific changes in the relative abundance of several genera in intestinal microbiota, some of which were observed as early as 3 days following HFHS diet. Weight-independent correlations were found between the relative abundances of, among others, Barnesiella, Eubacterium, Adlercreutzia, Parasutterella, Propionibacterium, Enterococcus, and Methylobacterium and the concentrations of anandamide and the anti-inflammatory eCBome mediator N-docosahexaenoyl-ethanolamine. This study highlights for the first time the existence of potential interactions between the eCBome, an endogenous system of multifunctional signaling lipids, and several intestinal genera during early and late HFHS-induced dysmetabolic events, with potential impact on the host capability of adapting to increased intake of fat and sucrose. IMPORTANCE The intestinal microbiota and the expanded endocannabinoid system, or endocannabinoidome, have both been implicated in diet-induced obesity and dysmetabolism. This study aims at identifying the potential interactions between these two fundamental systems—which form the gut microbiota-endocannabinoidome axis—and their involvement in the establishment of diet-induced obesity and related metabolic complications. We report here time- and segment-specific microbiome disturbances as well as modifications of intestinal and circulating endocannabinoidome mediators during high-fat, high-sucrose diet-induced glucose intolerance and subsequent obesity and hyperinsulinemia. This highlights the involvement of, and the interaction between, the gut microbiota and the endocannabinoidome during metabolic adaptation to high-fat and high-sucrose feeding. These results will help identifying actionable gut microbiome members and/or endocannabinoidome mediators to improve metabolic health.

The endocannabinoidome (expanded endocannabinoid system, eCBome)-gut microbiome (mBIome) axis plays a fundamental role in the control of energy intake and processing. The liver-expressed antimicrobial peptide 2 (LEAP2) is a recently identified molecule acting as an antagonist of the ghrelin receptor and hence a potential effector of energy metabolism, also at the level of the gastrointestinal system. Here we investigated the role of the eCBome-gut mBIome axis in the control of the expression of LEAP2 in the liver and, particularly, the intestine. We confirm that the small intestine is a strong contributor to the circulating levels of LEAP2 in mice, and show that: (1) intestinal Leap2 expression is profoundly altered in the liver and small intestine of 13 week-old germ-free (GF) male mice, which also exhibit strong alterations in eCBome signaling; fecal microbiota transfer (FMT) from conventionally raised to GF mice completely restored normal Leap2 expression after 7 days from this procedure; in 13 week-old female GF mice no significant change was observed; (2) Leap2 expression in organoids prepared from the mouse duodenum is elevated by the endocannabinoid noladin ether, whereas in human Caco-2/15 epithelial intestinal cells it is elevated by PPARγ activation by rosiglitazone; (3) Leap2 expression is elevated in the ileum of mice with either high-fat diet—or genetic leptin signaling deficiency—(i.e., ob/ob and db/db mice) induced obesity. Based on these results, we propose that LEAP2 originating from the small intestine may represent a player in eCBome- and/or gut mBIome-mediated effects on food intake and energy metabolism.

Vitamin D deficiency is associated with poor mental health and dysmetabolism. Several metabolic abnormalities are associated with psychotic diseases, which can be compounded by atypical antipsychotics that induce weight gain and insulin resistance. These side-effects may be affected by vitamin D levels. The gut microbiota and endocannabinoidome (eCBome) are significant regulators of both metabolism and mental health, but their role in the development of atypical antipsychotic drug metabolic side-effects and their interaction with vitamin D status is unknown. We studied the effects of different combinations of vitamin D levels and atypical antipsychotic drug (olanzapine) exposure on whole-body metabolism and the eCBome-gut microbiota axis in female C57BL/6J mice under a high fat/high sucrose (HFHS) diet in an attempt to identify a link between the latter and the different metabolic outputs induced by the treatments. Olanzapine exerted a protective effect against diet-induced obesity and insulin resistance, largely independent of dietary vitamin D status. These changes were concomitant with olanzapine-mediated decreases in Trpv1 expression and increases in the levels of its agonists, including various N-acylethanolamines and 2-monoacylglycerols, which are consistent with the observed improvement in adiposity and metabolic status. Furthermore, while global gut bacteria community architecture was not altered by olanzapine, we identified changes in the relative abundances of various commensal bacterial families. Taken together, changes of eCBome and gut microbiota families under our experimental conditions might contribute to olanzapine and vitamin D-mediated inhibition of weight gain in mice on a HFHS diet.

Capsaicinoids, the pungent principles of chili peppers and prototypical activators of the transient receptor potential of the vanilloid type-1 (TRPV1) channel, which is a member of the expanded endocannabinoid system known as the endocannabinoidome (eCBome), counteract food intake and obesity. In this exploratory study, we examined the blood and stools from a subset of the participants in a cohort of reproductive-aged women with overweight/obesity who underwent a 12-week caloric restriction of 500 kcal/day with the administration of capsaicinoids (two capsules containing 100 mg of a capsicum annuum extract (CAE) each for a daily dose of 4 mg of capsaicinoids) or a placebo. Samples were collected immediately before and after the intervention, and plasma eCBome mediator levels (from 23 participants in total, 13 placebo and 10 CAE) and fecal microbiota taxa (from 15 participants in total, 9 placebo and 6 CAE) were profiled using LC–MS/MS and 16S metagenomic sequencing, respectively. CAE prevented the reduced caloric-intake-induced decrease in beneficial eCBome mediators, i.e., the TRPV1, GPR119 and/or PPARα agonists, N-oleoyl-ethanolamine, N-linoleoyl-ethanolamine and 2-oleoyl-glycerol, as well as the anti-inflammatory N-acyl-ethanolamines N-docosapentaenyl-ethanolamine and N-docosahexaenoyl-ethanolamine. CAE produced few but important alterations in the fecal microbiota, such as an increased relative abundance of the genus Flavonifractor, which is known to be inversely associated with obesity. Correlations between eCBome mediators and other potentially beneficial taxa were also observed, thus reinforcing the hypothesis of the existence of a link between the eCBome and the gut microbiome in obesity.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N-acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N-acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1Z,4Z-pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N-13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G), the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis inhibition with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15–30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

Akkermansia muciniphila is considered as one of the next-generation beneficial bacteria in the context of obesity and associated metabolic disorders. Although a first proof-of-concept of its beneficial effects has been established in the context of metabolic syndrome in humans, mechanisms are not yet fully understood. This study aimed at deciphering whether the bacterium exerts its beneficial properties through the modulation of the endocannabinoidome (eCBome). Circulating levels of 25 endogenous endocannabinoid-related lipids were quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) in the plasma of overweight or obese individuals before and after a 3 months intervention consisting of the daily ingestion of either alive or pasteurized A. muciniphila. Results from multivariate analyses suggested that the beneficial effects of A. muciniphila were not linked to an overall modification of the eCBome. However, subsequent univariate analysis showed that the decrease in 1-Palmitoyl-glycerol (1-PG) and 2-Palmitoyl-glycerol (2-PG), two eCBome lipids, observed in the placebo group was significantly counteracted by the alive bacterium, and to a lower extent by the pasteurized form. We also discovered that 1- and 2-PG are endogenous activators of peroxisome proliferator-activated receptor alpha (PPARα). We hypothesize that PPARα activation by mono-palmitoyl-glycerols may underlie part of the beneficial metabolic effects induced by A. muciniphila in human metabolic syndrome.

Monoglyceride lipase (MGLL) regulates metabolism by catabolizing monoacylglycerols (MAGs), including the endocannabinoid 2-arachidonoyl glycerol (2-AG) and some of its bioactive congeners, to the corresponding free fatty acids. Mgll knockout mice (Mgll−/−) exhibit elevated tissue levels of MAGs in association with resistance to the metabolic and cardiovascular perturbations induced by a high fat diet (HFD). The gut microbiome and its metabolic function are disrupted in obesity in a manner modulated by 2-arachidonoyl glycerol (2-AG’s) main receptors, the cannabinoid CB1 receptors. We therefore hypothesized that Mgll−/− mice have an altered microbiome, that responds differently to diet-induced obesity from that of wild-type (WT) mice. We subjected mice to HFD and assessed changes in the microbiomes after 8 and 22 weeks. As expected, Mgll−/− mice showed decreased adiposity, improved insulin sensitivity, and altered circulating incretin/adipokine levels in response to HFD. Mgll−/− mice on a chow diet exhibited significantly higher levels of Hydrogenoanaerobacterium, Roseburia, and Ruminococcus than WT mice. The relative abundance of the Lactobacillaceae and Coriobacteriaceae and of the Lactobacillus, Enterorhabdus, Clostridium_XlVa, and Falsiporphyromonas genera was significantly altered by HFD in WT but not Mgll−/− mice. Differently abundant families were also associated with changes in circulating adipokine and incretin levels in HFD-fed mice. Some gut microbiota family alterations could be reproduced by supplementing 2-AG or MAGs in culturomics experiments carried out with WT mouse fecal samples. We suggest that the altered microbiome of Mgll−/− mice contributes to their obesity resistant phenotype, and results in part from increased levels of 2-AG and MAGs.

The endocannabinoids and their main receptor, cannabinoid type-1 (CB1), suppress intracellular cyclic AMP levels and have emerged as key players in the control of energy metabolism. CB1 agonists and blockers have been reported to influence the thermogenic function of white and brown adipose tissue (WAT and BAT), affecting body weight through the inhibition and stimulation of energy expenditure, respectively. The purpose of the current study was to investigate the regulation of the endocannabinoid system in WAT and BAT following exposure to either cold or specific agonism of β3-adrenoceptors using CL316,243 (CL), conditions known to cause BAT activation and WAT browning. To address this question, we performed quantitative PCR-based mRNA profiling of genes important for endocannabinoid synthesis, degradation, and signaling, and determined endocannabinoid levels by LC-MS in WAT and BAT of control, cold-exposed, and CL-treated wild-type mice as well as primary brown adipocytes. Treatment with CL and exposure to cold caused an upregulation of endocannabinoid levels and biosynthetic enzymes in WAT. Acute β3-adrenoceptor activation increased endocannabinoids and a subset of genes of biosynthesis in BAT and primary brown adipocytes. We suggest that the cold-mediated increase in endocannabinoid tone is part of autocrine negative feed-back mechanisms controlling β3-adrenoceptor-induced BAT activation and WAT browning.

Diverse metabolic disorders have been associated with an alteration of N-acylethanolamine (NAE) levels. These bioactive lipids are synthesized mainly by N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and influence host metabolism. We have previously discovered that NAPE-PLD in the intestine and adipose tissue is connected to the pathophysiology of obesity. However, the physiological function of NAPE-PLD in the liver remains to be deciphered. To study the role of liver NAPE-PLD on metabolism, we generated a new mouse model of inducible Napepld hepatocyte-specific deletion (Napepld∆Hep mice). In this study, we report that Napepld∆Hep mice develop a high-fat diet-like phenotype, characterized by an increased fat mass gain, hepatic steatosis and we show that Napepld∆Hep mice are more sensitive to liver inflammation. We also demonstrate that the role of liver NAPE-PLD goes beyond the mere synthesis of NAEs, since the deletion of NAPE-PLD is associated with a marked modification of various bioactive lipids involved in host homeostasis such as oxysterols and bile acids. Collectively these data suggest that NAPE-PLD in hepatocytes is a key regulator of liver bioactive lipid synthesis and a dysregulation of this enzyme leads to metabolic complications. Therefore, deepening our understanding of the regulation of NAPE-PLD could be crucial to tackle obesity and related comorbidities.

Lifestyle is a well-known environmental factor that plays a major role in facilitating the development of metabolic syndrome or eventually exacerbating its consequences. Various lifestyle factors, especially changes in dietary habits, extreme temperatures, unusual light−dark cycles, substance abuse, and other stressful factors, are also established modifiers of the endocannabinoid system and its extended version, the endocannabinoidome. The endocannabinoidome is a complex lipid signaling system composed of a plethora (>100) of fatty acid-derived mediators and their receptors and anabolic and catabolic enzymes (>50 proteins) which are deeply involved in the control of energy metabolism and its pathological deviations. A strong link between the endocannabinoidome and another major player in metabolism and dysmetabolism, the gut microbiome, is also emerging. Here, we review several examples of how lifestyle modifications (westernized diets, lack or presence of certain nutritional factors, physical exercise, and the use of cannabis) can modulate the propensity to develop metabolic syndrome by modifying the crosstalk between the endocannabinoidome and the gut microbiome and, hence, how lifestyle interventions can provide new therapies against cardiometabolic risk by ensuring correct functioning of both these systems.

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)—a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin—is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

The dysregulation of the endocannabinoid system is associated with cardiometabolic complications of obesity. Allelic variants in coding genes for this system components may contribute to differences in the susceptibility to obesity and related health hazards. These data have mostly been shown in Caucasian populations and in severely obese individuals. We investigated a multiethnic Brazilian population to study the relationships among the polymorphism 385C>A in an endocannabinoid degrading enzyme gene (FAAH), endocannabinoid levels and markers of cardiometabolic risk. Fasting plasma levels of endocannabinoids and congeners (anandamide, 2-arachidonoylglycerol, N-oleoylethanolamide and N-palmitoylethanolamide) were measured by liquid chromatography-mass spectrometry in 200 apparently healthy individuals of both genders with body mass indices from 22.5 ± 1.8 to 35.9 ± 5.5 kg/m2 (mean ± 1 SD) and ages between 18 and 60 years. All were evaluated for anthropometric parameters, blood pressure, metabolic variables, homeostatic model assessment of insulin resistance (HOMA-IR), adiponectin, leptin, C-reactive protein, and genotyping. The endocannabinoid levels increased as a function of obesity and insulin resistance. The homozygous genotype AA was associated with higher levels of anandamide and lower levels of adiponectin versus wild homozygous CC and heterozygotes combined. The levels of anandamide were independent and positively associated with the genotype AA position 385 of FAAH, C-reactive protein levels and body mass index. Our findings provide evidence for an endocannabinoid-related phenotype that may be identified by the combination of circulating anandamide levels with genotyping of the FAAH 385C>A; this phenotype is not exclusive to mono-ethnoracial populations nor to individuals with severe obesity.