Peripheral endocannabinoid signaling controls hyperphagia in western diet-induced obesity

Abstract

The endocannabinoid system in the brain and periphery plays a major role in controlling food intake and energy balance. We reported that tasting dietary fats was met with increased levels of the endocannabinoids, 2-arachidonoyl-sn-glycerol (2-AG) and anandamide, in the rat upper small intestine, and pharmacological inhibition of this local signaling event dose-dependently blocked sham feeding of fats. We now investigated the contribution of peripheral endocannabinoid signaling in hyperphagia associated with chronic consumption of a western-style diet in mice ([WD] i.e., high fat and sucrose). Feeding patterns were assessed in male C57BL/6Tac mice maintained for 60days on WD or a standard rodent chow (SD), and the role for peripheral endocannabinoid signaling at CB₁Rs in controlling food intake was investigated via pharmacological interventions. In addition, levels of the endocannabinoids, 2-AG and anandamide, in the upper small intestine and circulation of mice were analyzed via liquid chromatography coupled to tandem mass spectrometry to evaluate diet-related changes in endocannabinoid signaling and the potential impact on food intake. Mice fed WD for 60days exhibited large increases in body weight, daily caloric intake, average meal size, and rate of feeding when compared to control mice fed SD. Inhibiting peripheral CB₁Rs with the peripherally-restricted neutral cannabinoid CB₁ receptor antagonist, AM6545 (10mg/kg), significantly reduced intake of WD during a 6h test, but failed to modify intake of SD in mice. AM6545 normalized intake of WD, average meal size, and rate of feeding to levels found in SD control mice. These results suggest that endogenous activity at peripheral CB₁Rs in WD mice is critical for driving hyperphagia. In support of this hypothesis, levels of 2-AG and anandamide in both, jejunum mucosa and plasma, of ad-libitum fed WD mice increased when compared to SC mice. Furthermore, expression of genes for primary components of the endocannabinoid system (i.e., cannabinoid receptors, and endocannabinoid biosynthetic and degradative enzymes) was dysregulated in WD mice when compared to SC mice. Our results suggest that hyperphagia associated with WD-induced obesity is driven by enhanced endocannabinoid signaling at peripheral CB₁Rs.

Keywords: 2-AG; Anandamide; Endocannabinoid; Obesity; Peripheral; Western diet.

Figure 1

Chronic consumption of a western diet is associated with hyperphagia. Male mice maintained for 60 days on a western diet (Western) become obese (a, cumulative change in body weight; b, gross body weight) and display increases in 24 h caloric intake, meal size, and rate of intake (c–e) when compared to mice maintained on a standard chow diet (Stand). Meal duration, frequency, and post meal interval do not significantly differ between diets (f–h). Repeated measures two-way ANOVA, with Sidak’s multiple comparison post hoc test, *** = p<0.001 (a); unpaired Student’s t-test (two-tailed), *** = p<0.001 between Stand and Wetern. Results are expressed as means ± SEM; n=16/condition (a, b), n=8/condition (c–h).

Figure 2

Mice fed a western diet display increases in levels of 2-AG and anandamide in jejunum mucosa and plasma. Mice maintained on a standard diet (Stand) that were fasted for 24 h (FD) display increases in 2-AG levels in jejunum mucosa, when compared to free feeding controls [FF (a)], and FF male mice maintained on western diet (Western) display increases in levels of 2-AG in jejunum mucosa to levels found in fasted Stand mice (a). Western mice display elevated anandamide in jejunum mucosa when compared to Stand mice, irrespective of feeding condition (b). Western mice display increases in plasma levels of 2-AG (c) and anandamide (d) when compared to Stand mice. Two-way ANOVA with Student-Newman-Keuls multiple comparison post hoc test. * = p<0.05, *** = p<0.001, ns = not significant. Results are expressed as means ± SEM; n=7/condition for jejunum, n=9–10/condition for plasma.

Figure 3

Inhibiting peripheral CB₁Rs reduces food intake in mice fed a western diet, but not a standard diet, and normalizes intake and meal patterns in western diet fed mice. Pharmacological blockade of peripheral CB₁Rs with AM6545, when compared to vehicle treatment (Veh), inhibits the caloric intake of male mice maintained for 60 days on western diet [WD (a)] during a 6 h test, but has no effect on caloric intake in mice maintained on standard chow diet [b (SD)]. Vehicle-treated WD mice display an increase in caloric intake (c), average meal size (d), and rate of feeding [e (kcal per minute of feeding)], when compared to Veh-treated SD mice during a 6 h test. Pharmacological blockade of peripheral CB₁Rs with AM6545 reduces total caloric intake (c), meal size (d), and rate of intake (e) in WD mice to levels indistinguishable from SD control mice, and has no effect on meal parameters in SD mice (c–e). Repeated measures (a, b) or regular (c–e) two-way ANOVA, with Sidak’s or Student-Newman-Keuls, respectively, multiple comparison post hoc test, *= p<0.05, ** = p<0.01, *** = p<0.001. Results are expressed as means ± SEM; n=7–8/condition.

Figure 4

Expression of genes for components of the endocannabinoid system is modified in mice fed a western diet. Expression of mRNA encoding CB₁R [a (CNR1)], CB₂R [b (CNR2)], DAGL-α [c (DAGLA)], and FAAH (f) are elevated in standard chow-fed (Stand) 24 h fasted (FD) male mice, an effect absent in mice fed a western diet (Western). No changes are found under all conditions for DAGL-β [d (DAGLB)]. Expression of MAGL and FAAH genes are elevated in Western mice when compared to Stand (e, f). Two-Way ANOVA with Student-Newman-Keuls multiple comparison post hoc test. ** = p<0.01, * = p<0.05, ns = not significant. Results are expressed as means ± SEM; n=3–4/condition in triplicate.