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.

Nutritional and metabolic regulation of the metabolite dimethylguanidino valeric acid: an early marker of cardiometabolic disease.

Dimethylguanidino valeric acid (DMGV) is a marker of fatty liver disease, incident coronary artery disease, cardiovascular mortality, and incident diabetes. Recently, it was reported that circulating DMGV levels correlated positively with consumption of sugary beverages and negatively with intake of fruits and vegetables in three Swedish community-based cohorts. Here, we validate these results in the Framingham Heart Study Third Generation Cohort. Furthermore, in mice, diets rich in sucrose or fat significantly increased plasma DMGV concentrations. DMGV is the product of metabolism of asymmetric dimethylarginine (ADMA) by the hepatic enzyme AGXT2. ADMA can also be metabolized to citrulline by the cytoplasmic enzyme DDAH1. We report that a high-sucrose diet induced conversion of ADMA exclusively into DMGV (supporting the relationship with sugary beverage intake in humans), while a high-fat diet promoted conversion of ADMA to both DMGV and citrulline. On the contrary, replacing dietary native starch with high-fiber-resistant starch increased ADMA concentrations and induced its conversion to citrulline, without altering DMGV concentrations. In a cohort of obese nondiabetic adults, circulating DMGV concentrations increased and ADMA levels decreased in those with either liver or muscle insulin resistance. This was similar to changes in DMGV and ADMA concentrations found in mice fed a high-sucrose diet. Sucrose is a disaccharide of glucose and fructose. Compared with glucose, incubation of hepatocytes with fructose significantly increased DMGV production. Overall, we provide a comprehensive picture of the dietary determinants of DMGV levels and association with insulin resistance.

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.

Residual effects of intra-articular betamethasone and triamcinolone acetonide in an equine acute synovitis model.

BACKGROUND: Intra-articular (IA) corticosteroids are regularly used in equine athletes for the control of joint inflammation. OBJECTIVES: The goal of this study was to use an acute synovitis inflammation model to determine the residual effects of IA betamethasone and triamcinolone acetonide on various inflammatory parameters and lameness. STUDY DESIGN: Crossover randomised trial. METHODS: Five mixed-breed, 2-year-old horses were randomly allocated to an IA treatment of the radiocarpal joint with 9 mg of either betamethasone or triamcinolone acetonide. Two weeks following treatment, horses were injected with 1 µg of lipopolysaccharide (LPS) diluted in 1 ml of saline. Following LPS injection, horses were crossed-over and both sets of injections were repeated after a washout period. Blood samples were collected at multiple time points for mRNA analysis, as well as serum amyloid A (SAA) and cortisol determination. At each time point, lameness was also subjectively scored. Additional injections with saline-only or LPS-only (twice) were conducted as negative and positive controls, respectively. Two-way repeated measures analysis of variance was used to analyse all data. RESULTS: Corticosteroid-only treatments result in significant mRNA expression differences, as well as significant and prolonged cortisol suppression. Following LPS injection, there was a residual treatment effect with triamcinolone evidenced by a significant treatment effect on IL-6 and PTGS1 (cyclooxygenase-1), lameness, SAA and cortisol concentrations, while only IL-6 expression was affected by betamethasone. MAIN LIMITATIONS: The acute synovitis model used here results in significant inflammation and is not representative of the low-grade inflammation seen with typical joint disease and residual anti-inflammatory effects may be more profound in naturally occurring joint disease. CONCLUSIONS: Current regulatory guidelines may be insufficient if the concern is residual anti-inflammatory effects. Additionally, intra-articular corticosteroid administration is not without risk, as evidenced by a significant suppression of serum cortisol concentration and, as such, the benefits of their administration should be weighed against those risks.

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.

Adolescent exposure to low-dose THC disrupts energy balance and adipose organ homeostasis in adulthood.

One of cannabis' most iconic effects is the stimulation of hedonic high-calorie eating-the "munchies"-yet habitual cannabis users are, on average, leaner than non-users. We asked whether this phenotype might result from lasting changes in energy balance established during adolescence, when use of the drug often begins. We found that daily low-dose administration of cannabis' intoxicating constituent, Δ9-tetrahydrocannabinol (THC), to adolescent male mice causes an adult metabolic phenotype characterized by reduced fat mass, increased lean mass and utilization of fat as fuel, partial resistance to diet-induced obesity and dyslipidemia, enhanced thermogenesis, and impaired cold- and ß-adrenergic receptor-stimulated lipolysis. Further analyses revealed that this phenotype is associated with molecular anomalies in the adipose organ, including ectopic overexpression of muscle-associated proteins and heightened anabolic processing. Thus, adolescent exposure to THC may promote an enduring "pseudo-lean" state that superficially resembles healthy leanness but might in fact be rooted in adipose organ dysfunction.

Equine Peripheral Gene Expression Changes in Response to Dose-Dependent Lipopolysaccharide-Induced Synovitis.

The use of lipopolysaccharide to induce a localized source of inflammation (acute synovitis) and allow for monitoring of changes in systemic mRNA expression has been recently reported. Here, the goal was to maintain a significant systemic mRNA response while limiting the severity of lameness such that this model can be used to examine the effects of various anti-inflammatory treatment modalities on mRNA expression. Three mixed breeds, four-year-old geldings were utilized for this study. One milliliter of phosphate-buffered saline containing 1,000 ng or less of lipopolysaccharide from E. coli O111:B4 was aseptically injected into alternating radiocarpal joints following washout periods. Blood for complete blood cell count, serum amyloid A concentration, and mRNA analysis via RT-qPCR for 23 different genes were collected before each injection, as well as at multiple times post-injection. Lameness severity was also graded at each time point. Two-way, repeated measures analysis of variance was used for statistical analysis (P < .05). Results largely replicated those previously reported, with multiple genes exhibiting significant expression changes during the acute inflammatory period (including increases in CD14, TLR4, IL-1ß, IL1RN, MMP1, and MMP9 expression) while some demonstrated dose-dependent changes; significant increases in complete blood cell count parameters and serum amyloid A concentrations were also noted. Attempts to temper the severity of lameness were not successful as nonweight bearing lameness was noted at doses of 10ng or higher, while a dose of 1ng elicited neither a detectable lameness nor a significant change in mRNA expression.

UPLC-MS/MS Method for Analysis of Endocannabinoid and Related Lipid Metabolism in Mouse Mucosal Tissue.

The endocannabinoid system is expressed in cells throughout the body and controls a variety of physiological and pathophysiological functions. We describe robust and reproducible UPLC-MS/MS-based methods for analyzing metabolism of the endocannabinoids, 2-arachidonoyl-sn-glycerol and arachidonoyl ethanolamide, and related monoacylglycerols (MAGs) and fatty acid ethanolamides (FAEs), respectively, in mouse mucosal tissues (i.e., intestine and lung). These methods are optimized for analysis of activity of the MAG biosynthetic enzyme, diacylglycerol lipase (DGL), and MAG degradative enzymes, monoacylglycerol lipase (MGL) and alpha/beta hydrolase domain containing-6 (ABHD6). Moreover, we describe a novel UPLC-MS/MS-based method for analyzing activity of the FAE degradative enzyme, fatty acid amide hydrolase (FAAH), that does not require use of radioactive substrates. In addition, we describe in vivo pharmacological methods to inhibit MAG biosynthesis selectively in the mouse small-intestinal epithelium. These methods will be useful for profiling endocannabinoid metabolism in rodent mucosal tissues in health and disease.

Telephonic description of sepsis among callers to an emergency dispatch centre in South Africa.

INTRODUCTION: Sepsis is an acute, life-threatening condition caused by a dysregulated systemic response to infection. Early medical intervention such as antibiotics and fluid resuscitation can be life-saving. Diagnosis or suspicion of sepsis by an emergency call-taker could potentially improve patient outcome. Therefore, the aim was to determine the keywords used by callers to describe septic patients in South Africa when calling a national private emergency dispatch centre. METHODS: A retrospective review of prehospital patient records was completed to identify patients with sepsis in the prehospital environment. A mixed-methods design was employed in two-sequential phases. The first phase was qualitative. Thirty cases of sepsis were randomly selected, and the original call recording was extracted. These recordings were transcribed verbatim and subjected to content analysis to determine keywords of signs and symptoms telephonically. Once keywords were identified, an additional sample of sepsis cases that met inclusion and exclusion criteria were extracted and listened to. The frequency of each of the keywords was quantified. RESULTS: Eleven distinct categories were identified. The most prevalent categories that were used to describe sepsis telephonically were: gastrointestinal symptoms (40%), acute altered mental status (35%), weakness of the legs (33%) and malaise (31%). At least one of these four categories of keywords appeared in 86% of all call recordings. CONCLUSION: It was found that certain categories appeared in higher frequencies than others so that a pattern could be recognised. Utilising these categories, telephonic recognition algorithms for sepsis could be developed to aid in predicting sepsis over the phone. This would allow for dispatching of the correct level of care immediately and could subsequently have positive effects on patient outcome.

Cannabinoid CB1 Receptors in the Intestinal Epithelium Are Required for Acute Western-Diet Preferences in Mice.

The endocannabinoid system plays an important role in the intake of palatable food. For example, endocannabinoid signaling in the upper small-intestinal epithelium is increased (i) in rats after tasting dietary fats, which promotes intake of fats, and (ii) in a mouse model of diet-induced obesity, which promotes overeating via impaired nutrient-induced gut-brain satiation signaling. We now utilized a combination of genetic, pharmacological, and behavioral approaches to identify roles for cannabinoid CB1Rs in upper small-intestinal epithelium in preferences for a western-style diet (WD, high-fat/sucrose) versus a standard rodent diet (SD, low-fat/no sucrose). Mice were maintained on SD in automated feeding chambers. During testing, mice were given simultaneous access to SD and WD, and intakes were recorded. Mice displayed large preferences for the WD, which were inhibited by systemic pretreatment with the cannabinoid CB1R antagonist/inverse agonist, AM251, for up to 3 h. We next used our novel intestinal epithelium-specific conditional cannabinoid CB1R-deficient mice (IntCB1-/-) to investigate if intestinal CB1Rs are necessary for WD preferences. Similar to AM251 treatment, preferences for WD were largely absent in IntCB1-/- mice when compared to control mice for up to 6 h. Together, these data suggest that CB1Rs in the murine intestinal epithelium are required for acute WD preferences.