Endocannabinoids selectively enhance sweet taste

Abstract

Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known orexigenic mediators that act via CB(1) receptors in hypothalamus and limbic forebrain to induce appetite and stimulate food intake. Circulating endocannabinoid levels inversely correlate with plasma levels of leptin, an anorexigenic mediator that reduces food intake by acting on hypothalamic receptors. Recently, taste has been found to be a peripheral target of leptin. Leptin selectively suppresses sweet taste responses in wild-type mice but not in leptin receptor-deficient db/db mice. Here, we show that endocannabinoids oppose the action of leptin to act as enhancers of sweet taste. We found that administration of AEA or 2-AG increases gustatory nerve responses to sweeteners in a concentration-dependent manner without affecting responses to salty, sour, bitter, and umami compounds. The cannabinoids increase behavioral responses to sweet-bitter mixtures and electrophysiological responses of taste receptor cells to sweet compounds. Mice genetically lacking CB(1) receptors show no enhancement by endocannnabinoids of sweet taste responses at cellular, nerve, or behavioral levels. In addition, the effects of endocannabinoids on sweet taste responses of taste cells are diminished by AM251, a CB(1) receptor antagonist, but not by AM630, a CB(2) receptor antagonist. Immunohistochemistry shows that CB(1) receptors are expressed in type II taste cells that also express the T1r3 sweet taste receptor component. Taken together, these observations suggest that the taste organ is a peripheral target of endocannabinoids. Reciprocal regulation of peripheral sweet taste reception by endocannabinoids and leptin may contribute to their opposing actions on food intake and play an important role in regulating energy homeostasis.

Fig. 1.

Endocannabinoids enhance gustatory nerve responses to sweeteners. (A) Typical examples of CT nerve responses of WT and CB₁^−/− mice showing the effect of i.p. injection of 1 mg/kg bw of 2-AG (Lower traces) vs. vehicle-injected control (Upper traces). CT nerve responses (normalized to response to 100 mM NH₄Cl) of WT (B) and CB₁^−/− (C) mice stimulated by sweet (Suc, 500 mM sucrose; Sac, 20 mM saccharin; Glc, 500 mM glucose; SC, 1 mM SC45647), bitter (QHCl, 20 mM quinine-HCl), salty (NaCl, 100 mM NaCl), sour (HCl, 10 mM HCl), and umami (MSG, 100 mM monosodium glutamate + 10 μM amiloride) compounds 10–30 min after administration of vehicle (black bars) or 1 mg/kg bw of 2-AG (red bars) (n = 5–10). (D) Dose-dependent effect of AEA (blue symbols) or 2-AG (red symbols) treatment on normalized chorda tympani nerve responses to 500 mM sucrose (n = 5–14). (E) Concentration-dependent responses to sucrose 10–30 min after administration of vehicle (black symbols) or 1 mg/kg bw of 2-AG (red symbols) in WT (squares) (n = 7) and CB₁^−/− (circles) mice (n = 5). Asterisks indicate significant differences from control (*P < 0.05; **P < 0.01; ***P < 0.001; Fisher’s PLSD post hoc test or t test). All data are presented as the mean ± SEM.

Fig. 2.

Endocannabinoids enhance behavioral responses to sweeteners. Concentration response relationships to varying concentrations of sucrose in mixtures with 1 mM quinine 30 min after i.p. injection of vehicle (black symbols) or 1 mg/kg bw 2-AG (red symbols) (A) and AEA (blue symbols) (B) in WT (squares) and CB₁^−/− (circles) mice (n = 5). Lick responses to distilled water (DW), NaCl (300 and 1,000 mM), HCl (3 and 10 mM), quinine (QHCl; 0.3 and 1 mM), MSG + 1 mM quinine (MSG; 100 and 300 mM), and sucrose + 1 mM quinine (Suc; 500 mM) 30 min after administration of vehicle (black bars), 1 mg/kg bw 2-AG (red bars) (C), or AEA (blue bars) (D) in WT mice (n = 5). Asterisks indicate significant differences (*P < 0.05; **P < 0.01; Fisher’s PLSD post hoc test or t test). All data are presented as the mean ± SEM.

Fig. 3.

Endocannabinoids enhance sweet responses of taste bud cells. (A) The effect of 1 μg/mL 2-AG on the response of a T1r3-GFP taste cell in the isolated fungiform taste bud with the epithelium to the sweet compound saccharin. The picture shows a T1r3-GFP taste cell from which taste responses were recorded. In this cell, the response to 5 mM saccharin was increased about 2-fold by bath application of 2-AG for 2 min and returned to the control level 2 min after wash-out of 2-AG. (B) Dose-dependent effect of AEA and 2-AG on responses to saccharin of taste bud cells from WT and T1r3-GFP mice (labeled WT, n = 7–28). Responses to saccharin of taste bud cell in CB₁^−/− mice were not affected by 1 μg/mL AEA (blue open rectangles, n = 7) or 2-AG (red open circles, n = 5). (C) The CB₁ antagonist AM251 inhibited the enhancing effects of 2-AG on responses to saccharin of TCs from WT and T1r3-GFP mice (n = 10). (D) The CB₂ antagonist AM630 did not inhibit the enhancing effects of 2-AG on response to saccharin of TCs from WT and T1r3-GFP mice (n = 9). Asterisks indicate significant differences (NS: P > 0.1; *P < 0.05; **P < 0.01, t test). All data are presented as the mean ± SEM.

Fig. 4.

CB₁ and T1r3 are coexpressed in taste bud cells. (A) Expression of gustducin (40, 45, and 50 cycles), CB₁ (40, 45, and 50 cycles), CB₂ (40, 45, and 50 cycles), and β-actin (25, 30, and 35 cycles) mRNAs in fungiform taste buds (FP), circumvallate taste buds (CV), and tongue epithelium devoid of TCs (ET). M, 100-bp marker. (B) Coexpression patterns of CB₁ with: T1r3 (Left), GAD67 (Middle), and GLAST (Right) in FP and CV of T1r3-GFP or WT mice. Immunostaining for CB₁ is shown in red. T1r3-GFP expression and immunostaining for GAD and GLAST are shown in green. (Scale bar, 10 μm.) Negative control and immunostaining in CB₁^−/− mice are shown in Fig. S7.