New role for the anandamide metabolite prostaglandin F2α ethanolamide: Rolling preadipocyte proliferation.

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.

Obesity: The Fat Tissue Disease Version of Cancer.

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.

Spontaneous and Naloxone-Precipitated Withdrawal Behaviors From Chronic Opiates are Accompanied by Changes in N-Oleoylglycine and N-Oleoylalanine Levels in the Brain and Ameliorated by Treatment With These Mediators.

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.

Alterations of brain endocannabinoidome signaling in germ-free mice.

We investigated the hypothesis that the endocannabinoidome (eCBome), an extension of the endocannabinoid (eCB) signaling system with important functions in the CNS, may play a role in the microbiota-gut-brain axis. Using LC-MS/MS and qPCR arrays we profiled the brain eCBome of juvenile (4 weeks) and adult (13 weeks) male and female germ-free (GF) mice, which are raised in sterile conditions and virtually devoid of microbiota, present neurophysiological deficits, and were found recently to exhibit a strongly altered gut eCBome in comparison to conventionally raised age/sex-matched controls. The causal effect of the gut microbiome on the eCBome was investigated through the re-introduction into adult male GF mice of a functional gut microbiota by fecal microbiota transfer (FMT). The concentrations of the eCB, 2-arachidonoylglycerol (2-AG), and its 2-monoacylglycerol congeners, were significantly reduced in the brain, but not in the hypothalamus, of both juvenile and adult male and adult female GF mice. FMT rendered these decreases non-statistically significant. The eCB, anandamide (AEA), and its congener N-acylethanolamines (NAEs), were instead increased in the brain of adult female GF mice. Saturated fatty acid-containing NAEs were decreased in adult male GF mouse hypothalamus in a manner not reversed by FMT. Only few changes were observed in the expression of eCBome enzymes and receptors. Our data open the possibility that altered eCBome signaling may underlie some of the brain dysfunctions typical of GF mice.

Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.

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.

Rapid and Concomitant Gut Microbiota and Endocannabinoidome Response to Diet-Induced Obesity in Mice.

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.

Highly efficient sample preparation and quantification of constituents from traditional Chinese herbal medicines using matrix solid-phase dispersion extraction and UPLC-MS/MS.

In this work, a rapid and simple method based on matrix solid-phase dispersion (MSPD) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed. Guge Fengtong preparation (GGFT), a traditional Chinese herbal medicine, was investigated for validation, and eight major constituents were determined including four saponins (protodioscin, protogracillin, pseudoprotodioscin and dioscin) and four gingerols (6-gingerol, 8-gingerol, 10-gingerol and 6-shogaol). Response surface methodology and desirability function were employed to optimize the extraction conditions, such as dispersant, dispersant/sample ratio, solvent concentration, and elution volume, of MSPD. Results showed that MSPD using C18 (1.75 g) as the dispersant material and methanol (89%, v/v) as the eluting solvent (12.00 mL) resulted in a high extraction efficiency. MSPD extraction had the advantages of combining extraction and clean-up in a single step, was less time consuming and required lower solvent volumes compared with conventional methods. Quantification of chemical compounds from GGFT preparations were performed using UPLC-MS/MS in multiple-reaction monitoring mode. The proposed method afforded a low limit of detection ranging from 0.02 to 0.40 ng for saponins and gingerols. For all the analytes, recoveries ranged from 80.9% to 103% and repeatabilities were acceptable with relative standard deviations of less than 6.81%. The proposed MSPD-UPLC-MS/MS method was successfully utilized to analyze five batches of GGFTs, and the results demonstrated that this method is simple, efficient and has potential to be applied for the quality control of herbal preparations.