Cholinergic Neurotransmission Controls Orexigenic Endocannabinoid Signaling in the Gut in Diet-Induced Obesity.

The brain bidirectionally communicates with the gut to control food intake and energy balance, which becomes dysregulated in obesity. For example, endocannabinoid (eCB) signaling in the small-intestinal (SI) epithelium is upregulated in diet-induced obese (DIO) mice and promotes overeating by a mechanism that includes inhibiting gut-brain satiation signaling. Upstream neural and molecular mechanism(s) involved in overproduction of orexigenic gut eCBs in DIO, however, are unknown. We tested the hypothesis that overactive parasympathetic signaling at the muscarinic acetylcholine receptors (mAChRs) in the SI increases biosynthesis of the eCB, 2-arachidonoyl-sn-glycerol (2-AG), which drives hyperphagia via local CB1Rs in DIO. Male mice were maintained on a high-fat/high-sucrose Western-style diet for 60 d, then administered several mAChR antagonists 30 min prior to tissue harvest or a food intake test. Levels of 2-AG and the activity of its metabolic enzymes in the SI were quantitated. DIO mice, when compared to those fed a low-fat/no-sucrose diet, displayed increased expression of cFos protein in the dorsal motor nucleus of the vagus, which suggests an increased activity of efferent cholinergic neurotransmission. These mice exhibited elevated levels of 2-AG biosynthesis in the SI, that was reduced to control levels by mAChR antagonists. Moreover, the peripherally restricted mAChR antagonist, methylhomatropine bromide, and the peripherally restricted CB1R antagonist, AM6545, reduced food intake in DIO mice for up to 24 h but had no effect in mice conditionally deficient in SI CB1Rs. These results suggest that hyperactivity at mAChRs in the periphery increases formation of 2-AG in the SI and activates local CB1Rs, which drives hyperphagia in DIO.

Pseudocannabinoid H4CBD improves glucose response during advanced metabolic syndrome in OLETF rats independent of increase in insulin signaling proteins.

Cannabidiol (CBD) use has grown exponentially more popular in the last two decades, particularly among older adults (>55 yr), though very little is known about the effects of CBD use during age-associated metabolic dysfunction. In addition, synthetic analogues of CBD have generated great interest because they can offer a chemically pure product, which is free of plant-associated contaminants. To assess the effects of a synthetic analogue of CBD (H4CBD) on advanced metabolic dysfunction, a cohort of 41-wk-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were administered 200 mg H4CBD/kg by oral gavage for 4 wk. Animals were fed ad libitum and monitored alongside vehicle-treated OLETF and Long-Evans Tokushima Otsuka (LETO) rats, the lean-strain controls. An oral glucose-tolerance test (oGTT) was performed after 4 wk of treatment. When compared with vehicle-treated, OLETF rats, H4CBD decreased body mass (BM) by 15%, which was attributed to a significant loss in abdominal fat. H4CBD reduced glucose response (AUCglucose) by 29% (P < 0.001) and insulin resistance index (IRI) by 25% (P < 0.05) compared with OLETF rats. However, H4CBD did not statically reduce fasting blood glucose or plasma insulin, despite compensatory increases in skeletal muscle native insulin receptor (IR) protein expression (54%; P < 0.05). H4CBD reduced circulating adiponectin (40%; P < 0.05) and leptin (47%; P < 0.05) and increased ghrelin (75%; P < 0.01) compared with OLETF. Taken together, a chronic, high dose of H4CBD may improve glucose response, independent of static changes in insulin signaling, and these effects are likely a benefit of the profound loss of visceral adiposity.NEW & NOTEWORTHY Cannabis product use has grown in the last two decades despite the lack of research on Cannabidiol (CBD)-mediated effects on metabolism. Here, we provide seminal data on CBD effects during age-associated metabolic dysfunction. We gave 41-wk-old OLETF rats 200 mg H4CBD/kg by mouth for 4 wk and noted a high dose of H4CBD may improve glucose response, independent of static changes in insulin signaling, and these effects are likely a benefit of loss of visceral adiposity.

Diet-Induced Gut Barrier Dysfunction Is Exacerbated in Mice Lacking Cannabinoid 1 Receptors in the Intestinal Epithelium.

The gut barrier provides protection from pathogens and its function is compromised in diet-induced obesity (DIO). The endocannabinoid system in the gut is dysregulated in DIO and participates in gut barrier function; however, whether its activity is protective or detrimental for gut barrier integrity is unclear. We used mice conditionally deficient in cannabinoid receptor subtype-1 (CB1R) in the intestinal epithelium (intCB1-/-) to test the hypothesis that CB1Rs in intestinal epithelial cells provide protection from diet-induced gut barrier dysfunction. Control and intCB1-/- mice were placed for eight weeks on a high-fat/sucrose Western-style diet (WD) or a low-fat/no-sucrose diet. Endocannabinoid levels and activity of their metabolic enzymes were measured in the large-intestinal epithelium (LI). Paracellular permeability was tested in vivo, and expression of genes for gut barrier components and inflammatory markers were analyzed. Mice fed WD had (i) reduced levels of endocannabinoids in the LI due to lower activity of their biosynthetic enzymes, and (ii) increased permeability that was exacerbated in intCB1-/- mice. Moreover, intCB1-/- mice fed WD had decreased expression of genes for tight junction proteins and increased expression of inflammatory markers in LI. These results suggest that CB1Rs in the intestinal epithelium serve a protective role in gut barrier function in DIO.

Impact of Circulating N-Acylethanolamine Levels with Clinical and Laboratory End Points in Hemodialysis Patients.

BACKGROUND: Patients with ESRD on maintenance hemodialysis (MHD) are particularly susceptible to dysregulation of energy metabolism, which may manifest as protein energy wasting and cachexia. In recent years, the endocannabinoid system has been shown to play an important role in energy metabolism with potential relevance in ESRD. N-acylethanolamines are a class of fatty acid amides which include the major endocannabinoid ligand, anandamide, and the endogenous peroxisome proliferator-activated receptor-α agonists, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). METHODS: Serum concentrations of OEA and PEA were measured in MHD patients and their correlations with various clinical/laboratory indices were examined. Secondarily, we evaluated the association of circulating PEA and OEA levels with 12-month all-cause mortality. RESULTS: Both serum OEA and PEA levels positively correlated with high-density lipoprotein-cholesterol levels and negatively correlated with body fat and body anthropometric measures. Serum OEA levels correlated positively with serum interleukin-6 (IL-6) (rho = 0.19; p = 0.004). Serum PEA and IL-6 showed a similar but nonsignificant trend (rho = 0.12; p = 0.07). Restricted cubic spline analyses showed that increasing serum OEA and PEA both trended toward higher mortality risk, and these associations were statistically significant for PEA (PEA ≥4.7 pmol/mL; reference: PEA <4.7 pmol/mL) after adjustments in a Cox model (hazard ratio 2.99; 95% confidence interval 1.04, 8.64). CONCLUSIONS: In MHD patients, OEA and PEA are significantly correlated with variables related to lipid metabolism and body mass. Additionally, higher serum levels of PEA are associated with mortality risk. Future studies are needed to examine the potential mechanisms responsible for these findings and their clinical implications.

Bile acid composition regulates GPR119-dependent intestinal lipid sensing and food intake regulation in mice.

OBJECTIVES: Lipid mediators in the GI tract regulate satiation and satiety. Bile acids (BAs) regulate the absorption and metabolism of dietary lipid in the intestine, but their effects on lipid-regulated satiation and satiety are completely unknown. Investigating this is challenging because introducing excessive BAs or eliminating BAs strongly impacts GI functions. We used a mouse model (Cyp8b1-/- mice) with normal total BA levels, but alterations in the composition of the BA pool that impact multiple aspects of intestinal lipid metabolism. We tested two hypotheses: BAs affect food intake by (1) regulating production of the bioactive lipid oleoylethanolamide (OEA), which enhances satiety; or (2) regulating the quantity and localisation of hydrolysed fat in small intestine, which controls gastric emptying and satiation. DESIGN: We evaluated OEA levels, gastric emptying and food intake in wild-type and Cyp8b1-/- mice. We assessed the role of the fat receptor GPR119 in these effects using Gpr119-/- mice. RESULTS: Cyp8b1-/- mice on a chow diet showed mild hypophagia. Jejunal OEA production was blunted in Cyp8b1-/- mice, thus these data do not support a role for this pathway in the hypophagia of Cyp8b1-/- mice. On the other hand, Cyp8b1 deficiency decreased gastric emptying, and this was dependent on dietary fat. GPR119 deficiency normalised the gastric emptying, gut hormone levels, food intake and body weight of Cyp8b1-/- mice. CONCLUSION: BAs regulate gastric emptying and satiation by determining fat-dependent GPR119 activity in distal intestine.

Circulating Endocannabinoids and Mortality in Hemodialysis Patients.

BACKGROUND: Mortality in patients with end-stage renal disease (ESRD) on maintenance hemodialysis (MHD) remains exceptionally high. While traditional risk factors such as obesity are paradoxically associated with better survival, nontraditional risk factors including cachexia increase the likelihood of poor outcomes. There is accumulating evidence that the endocannabinoid (ECB) system plays a major role in energy preservation and storage, factors which can prevent the deleterious effects of cachexia. Hence, in this study, we evaluated the association of circulating ECB levels with mortality in MHD patients. METHODS: Serum concentrations of anandamide (AEA) and 2-arachidonoyl-sn-glycerol (2-AG), major ECB ligands, were measured in MHD patients. Their correlation with various clinical/laboratory indices and association with 12-month all-cause mortality were examined. RESULTS: Serum 2-AG levels positively correlated with body mass index, serum triglycerides and body anthropometric measures. Meanwhile, serum AEA levels correlated positively with serum interleukin-6, and negatively with serum very low-density lipoprotein levels. While increased serum 2-AG levels were associated with reduced risk of all-cause mortality (hazard ratio [HR] 0.52, 95% CI 0.28-0.98), there was no clear association between serum AEA levels and mortality (HR 0.91, 95% CI 0.48-1.72). CONCLUSIONS: In MHD patients, the circulating levels of ECB ligand, 2-AG, may play an important role in determining body mass and risk of mortality. These observations were unique to 2-AG as similar findings were not obtained with serum AEA. Future studies need to investigate the mechanisms responsible for these associations and examine the modulation of the ECB system as a potential target for therapy in ESRD.

Host- and Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden.

Plasma endocannabinoid levels in lean, overweight, and obese humans: relationships to intestinal permeability markers, inflammation, and incretin secretion.

Intestinal production of endocannabinoid and oleoylethanolamide (OEA) is impaired in high-fat diet/obese rodents, leading to reduced satiety. Such diets also alter the intestinal microbiome in association with enhanced intestinal permeability and inflammation; however, little is known of these effects in humans. This study aimed to 1) evaluate effects of lipid on plasma anandamide (AEA), 2-arachidonyl- sn-glycerol (2-AG), and OEA in humans; and 2) examine relationships to intestinal permeability, inflammation markers, and incretin hormone secretion. Twenty lean, 18 overweight, and 19 obese participants underwent intraduodenal Intralipid infusion (2 kcal/min) with collection of endoscopic duodenal biopsies and blood. Plasma AEA, 2-AG, and OEA (HPLC/tandem mass spectrometry), tumor necrosis factor-α (TNFα), glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) (multiplex), and duodenal expression of occludin, zona-occludin-1 (ZO-1), intestinal-alkaline-phosphatase (IAP), and Toll-like receptor 4 (TLR4) (by RT-PCR) were assessed. Fasting plasma AEA was increased in obese compared with lean and overweight patients ( P < 0.05), with no effect of BMI group or ID lipid infusion on plasma 2-AG or OEA. Duodenal expression of IAP and ZO-1 was reduced in obese compared with lean ( P < 0.05), and these levels related negatively to plasma AEA ( P < 0.05). The iAUC for AEA was positively related to iAUC GIP ( r = 0.384, P = 0.005). Obese individuals have increased plasma AEA and decreased duodenal expression of ZO-1 and IAP compared with lean and overweight subjects. The relationships between plasma AEA with duodenal ZO-1, IAP, and GIP suggest that altered endocannabinoid signaling may contribute to changes in intestinal permeability, inflammation, and incretin release in human obesity.

Plasma fatty acid ethanolamides are associated with postprandial triglycerides, ApoCIII, and ApoE in humans consuming a high-fructose corn syrup-sweetened beverage.

Epidemiological and clinical research studies have provided ample evidence demonstrating that consumption of sugar-sweetened beverages increases risk factors involved in the development of obesity, Type 2 diabetes, and cardiovascular disease (CVD). Our previous study demonstrated that when compared with aspartame (Asp), 2 wk of high-fructose corn syrup (HFCS)-sweetened beverages provided at 25% of daily energy requirement was associated with increased body weight, postprandial (pp) triglycerides (TG), and fasting and pp CVD risk factors in young adults. The fatty acid ethanolamide, anandamide (AEA), and the monoacylglycerol, 2-arachidonoyl- sn-glycerol (2-AG), are two primary endocannabinoids (ECs) that play a role in regulating food intake, increasing adipose storage, and regulating lipid metabolism. Therefore, we measured plasma concentrations of ECs and their analogs, oleoylethanolamide (OEA), docosahexaenoyl ethanolamide (DHEA), and docosahexaenoyl glycerol (DHG), in participants from our previous study who consumed HFCS- or Asp-sweetened beverages to determine associations with weight gain and CVD risk factors. Two-week exposure to either HFCS- or Asp-sweetened beverages resulted in significant differences in the changes in fasting levels of OEA and DHEA between groups after the testing period. Subjects who consumed Asp, but not HFCS, displayed a reduction in AEA, OEA, and DHEA after the testing period. In contrast, there were significant positive relationships between AEA, OEA, and DHEA vs. ppTG, ppApoCIII, and ppApoE in those consuming HFCS, but not in those consuming Asp. Our findings reveal previously unknown associations between circulating ECs and EC-related molecules with markers of lipid metabolism and CVD risk after HFCS consumption.

Feeding-induced oleoylethanolamide mobilization is disrupted in the gut of diet-induced obese rodents.

The gastrointestinal tract plays a critical role in the regulation of energy homeostasis by initiating neural and hormonal responses to the ingestion of nutrients. In addition to peptide hormones, such as cholecystokinin (CKK) and peptide YY (PYY), the lipid-derived mediator oleoylethanolamide (OEA) has been implicated in the control of satiety. Previous studies in humans and rodent models have shown that obesity is associated with changes in CCK, PYY and other gut-derived peptide hormones, which may contribute to decreased satiety and increased energy intake. In the present study, we show that small-intestinal OEA production is disrupted in the gut of diet-induced obese (DIO) rats and mice. In lean rodents, feeding or duodenal infusion of Intralipid® or pure oleic acid stimulates jejunal OEA mobilization. This response is strikingly absent in DIO rats and mice. Confirming previous reports, we found that feeding rats or mice a high-fat diet for 7 days is sufficient to suppress jejunal OEA mobilization. Surprisingly, a similar effect is elicited by feeding rats and mice a high-sucrose low-fat diet for 7 days. Collectively, our findings suggest that high fat-induced obesity is accompanied by alterations in the post-digestive machinery responsible for OEA biosynthesis, which may contribute to reduced satiety and hyperphagia.

Modulation of sweet taste sensitivities by endogenous leptin and endocannabinoids in mice.

KEY POINTS: Potential roles of endogenous leptin and endocannabinoids in sweet taste were examined by using pharmacological antagonists and mouse models including leptin receptor deficient (db/db) and diet-induced obese (DIO) mice. Chorda tympani (CT) nerve responses of lean mice to sweet compounds were increased after administration of leptin antagonist (LA) but not affected by administration of cannabinoid receptor antagonist (AM251). db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid levels in the taste organ, and enhanced expression of a biosynthesizing enzyme of endocannabinoids in taste cells. The effect of LA was gradually decreased and that of AM251 was increased during the course of obesity in DIO mice. These findings suggest that circulating leptin, but not local endocannabinoids, is a dominant modulator for sweet taste in lean mice and endocannabinoids become more effective modulators of sweet taste under conditions of deficient leptin signalling. ABSTRACT: Leptin is an anorexigenic mediator that reduces food intake by acting on hypothalamic receptor Ob-Rb. In contrast, endocannabinoids are orexigenic mediators that act via cannabinoid CB1 receptors in hypothalamus, limbic forebrain, and brainstem. In the peripheral taste system, leptin administration selectively inhibits behavioural, taste nerve and taste cell responses to sweet compounds. Opposing the action of leptin, endocannabinoids enhance sweet taste responses. However, potential roles of endogenous leptin and endocannabinoids in sweet taste remain unclear. Here, we used pharmacological antagonists (Ob-Rb: L39A/D40A/F41A (LA), CB1 : AM251) and examined the effects of their blocking activation of endogenous leptin and endocannabinoid signalling on taste responses in lean control, leptin receptor deficient db/db, and diet-induced obese (DIO) mice. Lean mice exhibited significant increases in chorda tympani (CT) nerve responses to sweet compounds after LA administration, while they showed no significant changes in CT responses after AM251. In contrast, db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid (2-arachidonoyl-sn-glycerol (2-AG)) levels in the taste organ, and enhanced expression of a biosynthesizing enzyme (diacylglycerol lipase α (DAGLα)) of 2-AG in taste cells. In DIO mice, the LA effect was gradually decreased and the AM251 effect was increased during the course of obesity. Taken together, our results suggest that circulating leptin, but not local endocannabinoids, may be a dominant modulator for sweet taste in lean mice; however, endocannabinoids may become more effective modulators of sweet taste under conditions of deficient leptin signalling, possibly due to increased production of endocannabinoids in taste tissue.

Fasting stimulates 2-AG biosynthesis in the small intestine: role of cholinergic pathways.

The endocannabinoids are lipid-derived signaling molecules that control feeding and energy balance by activating CB1-type cannabinoid receptors in the brain and peripheral tissues. Previous studies have shown that oral exposure to dietary fat stimulates endocannabinoid signaling in the rat small intestine, which provides positive feedback that drives further food intake and preference for fat-rich foods. We now describe an unexpectedly broader role for cholinergic signaling of the vagus nerve in the production of the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG), in the small intestine. We show that food deprivation increases levels of 2-AG and its lipid precursor, 1,2-diacylglycerol, in rat jejunum mucosa in a time-dependent manner. This response is abrogated by surgical resection of the vagus nerve or pharmacological blockade of small intestinal subtype-3 muscarinic acetylcholine (m3 mAch) receptors, but not inhibition of subtype-1 muscarinic acetylcholine (m1 mAch). We further show that blockade of peripheral CB1 receptors or intestinal m3 mAch receptors inhibits refeeding in fasted rats. The results suggest that food deprivation stimulates 2-AG-dependent CB1 receptor activation through a mechanism that requires efferent vagal activation of m3 mAch receptors in the jejunum, which, in turn, may promote feeding after a fast.

Endocannabinoid signaling in the gut mediates preference for dietary unsaturated fats.

Dietary fat exerts a potent stimulatory effect on feeding. This effect is mediated, at least in part, by a cephalic mechanism that involves recruitment of the vagus nerve and subsequent activation of endocannabinoid signaling in the gut. Here, we used a sham-feeding protocol in rats to identify fatty-acid constituents of dietary fat that might be responsible for triggering small-intestinal endocannabinoid signaling. Sham feeding rats with a corn oil emulsion increased endocannabinoid levels in jejunum, relative to animals that received either mineral oil (which contains no fatty acids) or no oil. Sham-feeding emulsions containing oleic acid (18:1) or linoleic acid (18:2) caused, on average, a nearly 2-fold accumulation of jejunal endocannabinoids, whereas emulsions containing stearic acid (18:0) or linolenic acid (18:3) had no such effect. In a 2-bottle-choice sham-feeding test, rats displayed strong preference for emulsions containing 18:2, which was blocked by pretreatment with the peripherally restricted CB1 cannabinoid receptor antagonists, AM6546 and URB447. Our results suggest that oral exposure to the monoenoic and dienoic fatty acid component of dietary fat selectively initiates endocannabinoid mobilization in the gut, and that this local signaling event is essential for fat preference.

Endocannabinoid signal in the gut controls dietary fat intake.

Oral sensory signals drive dietary fat intake, but the neural mechanisms underlying this process are largely unknown. The endocannabinoid system has gained recent attention for its central and peripheral roles in regulating food intake, energy balance, and reward. Here, we used a sham-feeding paradigm, which isolates orosensory from postingestive influences of foods, to examine whether endocannabinoid signaling participates in the positive feedback control of fat intake. Sham feeding a lipid-based meal stimulated endocannabinoid mobilization in the rat proximal small intestine by altering enzymatic activities that control endocannabinoid metabolism. This effect was abolished by surgical transection of the vagus nerve and was not observed in other peripheral organs or in brain regions that control feeding. Sham feeding of a nutritionally complete liquid meal produced a similar response to that of fat, whereas protein or carbohydrate alone had no such effect. Local infusion of the CB(1)-cannabinoid receptor antagonist, rimonabant, into the duodenum markedly reduced fat sham feeding. Similarly to rimonabant, systemic administration of the peripherally restricted CB(1)-receptor antagonist, URB 447, attenuated sham feeding of lipid. Collectively, the results suggest that the endocannabinoid system in the gut exerts a powerful regulatory control over fat intake and might be a target for antiobesity drugs.

A Sexually Dimorphic Role for Intestinal Cannabinoid Receptor Subtype-1 in the Behavioral Expression of Anxiety.

Background: Increasing evidence suggests that the endocannabinoid system (ECS) in the brain controls anxiety and may be a therapeutic target for the treatment of anxiety disorders. For example, both pharmacological and genetic disruption of cannabinoid receptor subtype-1 (CB1R) signaling in the central nervous system is associated with increased anxiety-like behaviors in rodents, while activating the system is anxiolytic. Sex is also a critical factor that controls the behavioral expression of anxiety; however, roles for the ECS in the gut in these processes and possible differences between sexes are largely unknown. Objective: In this study, we aimed to determine if CB1Rs in the intestinal epithelium exert control over anxiety-like behaviors in a sex-dependent manner. Methods: We subjected male and female mice with conditional deletion of CB1Rs in the intestinal epithelium (intCB1-/-) and controls (intCB1+/+) to the elevated plus maze (EPM), light/dark box, and open field test. Corticosterone (CORT) levels in plasma were measured at baseline and immediately after EPM exposure. Results: When compared with intCB1+/+ male mice, intCB1-/- male mice exhibited reduced levels of anxiety-like behaviors in the EPM and light/dark box. In contrast to male mice, no differences were found between female intCB1+/+ and intCB1-/- mice. Circulating CORT was higher in female versus male mice for both genotype groups at baseline and after EPM exposure; however, there was no effect of genotype on CORT levels. Conclusions: Collectively, these results indicate that genetic deletion of CB1Rs in the intestinal epithelium is associated with an anxiolytic phenotype in a sex-dependent manner.

The 16α-hydroxylated Bile Acid, Pythocholic Acid Decreases Food Intake and Increases Oleoylethanolamide in Male Mice.

Modulation of bile acid (BA) structure is a potential strategy for obesity and metabolic disease treatment. BAs act not only as signaling molecules involved in energy expenditure and glucose homeostasis, but also as regulators of food intake. The structure of BAs, particularly the position of the hydroxyl groups of BAs, impacts food intake partly by intestinal effects: (1) modulating the activity of N-acyl phosphatidylethanolamine phospholipase D, which produces the anorexigenic bioactive lipid oleoylethanolamide (OEA) or (2) regulating lipid absorption and the gastric emptying-satiation pathway. We hypothesized that 16α-hydroxylated BAs uniquely regulate food intake because of the long intermeal intervals in snake species in which these BAs are abundant. However, the effects of 16α-hydroxylated BAs in mammals are completely unknown because they are not naturally found in mammals. To test the effect of 16α-hydroxylated BAs on food intake, we isolated the 16α-hydroxylated BA pythocholic acid from ball pythons (Python regius). Pythocholic acid or deoxycholic acid (DCA) was given by oral gavage in mice. DCA is known to increase N-acyl phosphatidylethanolamine phospholipase D activity better than other mammalian BAs. We evaluated food intake, OEA levels, and gastric emptying in mice. We successfully isolated pythocholic acid from ball pythons for experimental use. Pythocholic acid treatment significantly decreased food intake in comparison to DCA treatment, and this was associated with increased jejunal OEA, but resulted in no change in gastric emptying or lipid absorption. The exogenous BA pythocholic acid is a novel regulator of food intake and the satiety signal for OEA in the mouse intestine.

Increased body weight in mice with fragile X messenger ribonucleoprotein 1 (Fmr1) gene mutation is associated with hypothalamic dysfunction.

Mutations in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene are linked to Fragile X Syndrome, the most common monogenic cause of intellectual disability and autism. People affected with mutations in FMR1 have higher incidence of obesity, but the mechanisms are largely unknown. In the current study, we determined that male Fmr1 knockout mice (KO, Fmr1-/y), but not female Fmr1-/-, exhibit increased weight when compared to wild-type controls, similarly to humans with FMR1 mutations. No differences in food or water intake were found between groups; however, male Fmr1-/y display lower locomotor activity, especially during their active phase. Moreover, Fmr1-/y have olfactory dysfunction determined by buried food test, although they exhibit increased compulsive behavior, determined by marble burying test. Since olfactory brain regions communicate with hypothalamic regions that regulate food intake, including POMC neurons that also regulate locomotion, we examined POMC neuron innervation and numbers in Fmr1-/y mice. POMC neurons express Fmrp, and POMC neurons in Fmr1-/y have higher inhibitory GABAergic synaptic inputs. Consistent with increased inhibitory innervation, POMC neurons in the Fmr1-/y mice exhibit lower activity, based on cFOS expression. Notably, Fmr1-/y mice have fewer POMC neurons than controls, specifically in the rostral arcuate nucleus, which could contribute to decreased locomotion and increased body weight. These results suggest a role for Fmr1 in the regulation of POMC neuron function and the etiology of Fmr1-linked obesity.

Glucoregulatory disruption in male mice offspring induced by maternal transfer of endocrine disrupting brominated flame retardants in DE-71.

Introduction: Polybrominated diphenyl ethers (PBDEs) are commercially used flame retardants that bioaccumulate in human tissues, including breast milk. PBDEs produce endocrine and metabolic disruption in experimental animals and have been associated with diabetes and metabolic syndrome (MetS) in humans, however, their sex-specific diabetogenic effects are not completely understood. Our past works show glucolipid dysregulation resulting from perinatal exposure to the commercial penta-mixture of PBDEs, DE-71, in C57BL/6 female mice. Methods: As a comparison, in the current study, the effects of DE-71 on glucose homeostasis in male offspring was examined. C57BL/6N dams were exposed to DE-71 at 0.1 mg/kg/d (L-DE-71), 0.4 mg/kg/d (H-DE-71), or received corn oil vehicle (VEH/CON) for a total of 10 wks, including gestation and lactation and their male offspring were examined in adulthood. Results: Compared to VEH/CON, DE-71 exposure produced hypoglycemia after a 11 h fast (H-DE-71). An increased fast duration from 9 to 11 h resulted in lower blood glucose in both DE-71 exposure groups. In vivo glucose challenge showed marked glucose intolerance (H-DE-71) and incomplete clearance (L- and H-DE-71). Moreover, L-DE-71-exposed mice showed altered glucose responses to exogenous insulin, including incomplete glucose clearance and/or utilization. In addition, L-DE-71 produced elevated levels of plasma glucagon and the incretin, active glucagon-like peptide-1 (7-36) amide (GLP-1) but no changes were detected in insulin. These alterations, which represent criteria used clinically to diagnose diabetes in humans, were accompanied with reduced hepatic glutamate dehydrogenase enzymatic activity, elevated adrenal epinephrine and decreased thermogenic brown adipose tissue (BAT) mass, indicating involvement of several organ system targets of PBDEs. Liver levels of several endocannabinoid species were not altered. Discussion: Our findings demonstrate that chronic, low-level exposure to PBDEs in dams can dysregulate glucose homeostasis and glucoregulatory hormones in their male offspring. Previous findings using female siblings show altered glucose homeostasis that aligned with a contrasting diabetogenic phenotype, while their mothers displayed more subtle glucoregulatory alterations, suggesting that developing organisms are more susceptible to DE-71. We summarize the results of the current work, generated in males, considering previous findings in females. Collectively, these findings offer a comprehensive account of differential effects of environmentally relevant PBDEs on glucose homeostasis and glucoregulatory endocrine dysregulation of developmentally exposed male and female mice.

Sympathetic activity controls fat-induced oleoylethanolamide signaling in small intestine.

Ingestion of dietary fat stimulates production of the small-intestinal satiety factors oleoylethanolamide (OEA) and N-palmitoyl-phosphatidylethanolamine (NPPE), which reduce food intake through a combination of local (OEA) and systemic (NPPE) actions. Previous studies have shown that sympathetic innervation of the gut is necessary for duodenal infusions of fat to induce satiety, suggesting that sympathetic activity may engage small-intestinal satiety signals such as OEA and NPPE. In the present study, we show that surgical resection of the sympathetic celiac-superior mesenteric ganglion complex, which sends projections to the upper gut, abolishes feeding-induced OEA production in rat small-intestinal cells. These effects are accounted for by suppression of OEA biosynthesis, and are mimicked by administration of the selective ß2-adrenergic receptor antagonist ICI-118,551. We further show that sympathetic ganglionectomy or pharmacological blockade of ß2-adrenergic receptors prevents NPPE release into the circulation. In addition, sympathetic ganglionectomy increases meal frequency and lowers satiety ratio, and these effects are corrected by pharmacological administration of OEA. The results suggest that sympathetic activity controls fat-induced satiety by enabling the coordinated production of local (OEA) and systemic (NPPE) satiety signals in the small intestine.