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

Abstract

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.

Keywords: NAPE-PLD; OEA; bile acids; gastric emptying; pythocholic acid.

Figure 1.

Ad libitum food intake after single dose administration of deoxycholic acid (DCA) or python bile at 6 Pm. Food intake after treatment (A) 0 to 3 hours, (B) 3 to 6 hours, (C) 6 to 9 hours, (D) 9 to 12 hours, (E) 12 to 18 hours, (F) 18 to 24 hours, and (G) total food intake for 24 hours (n = 6-7 for each group). *P < .05, **P < .01 (1-way ANOVA).

Figure 2.

Effects of DCA and python bile on gastric emptying, blood glucose, and insulin levels after 2 days of once-daily treatment. (A) Experimental schedule, (B) gastric emptying, (C) 1-hour refed food intake after fasting, (D) plasma GLP-1 levels, (E) body weight, (F) blood glucose levels, (G) plasma insulin levels, (H) plasma AST levels, and (I) plasma ALT levels. (n = 6-7 for each group). *P < .05 (1-way ANOVA).

Figure 3.

Influence of DCA and python bile on lipid absorption. (A) Fecal free fatty acid, (B) fecal acylglycerol, and (C) fecal cholesterol levels. Feces were collected for 24 hours before the gastric emptying experiment (n = 6-7 for each group).

Figure 4.

Purification of pythocholic acid and its taurine conjugate from python bile. (A) Python bile was first fractionated by partition between organic (ethyl acetate) and aqueous phases. The ethyl acetate phase was further purified by C8 solid phase extraction to give pythocholic acid, whereas the aqueous phase was subjected to silica gel chromatography to give tauro-conjugate. (B, C) Liquid chromatography/mass spectrometry (LC/MS) analysis of the ethyl acetate extract from python bile. (B) Total ion chromatogram (TIC) in the positive mode. The major peak was observed around 4.2 to 4.3 minutes. (C) High-resolution electrospray mass spectrometry (HR ESI MS) of the major peak (4.2-4.3 minutes). The MS signal at m/z 431.2769 was consistent with the sodium adduct of pythocholic acid [M + Na]⁺ (calculated m/z 431.2765). (D-E) LC/MS analysis of the aqueous phase from python bile (after ethyl acetate extraction). (D) TIC in the positive mode. The major peak was observed around 3.5 minutes. (E) HR ESI MS of the major peak (∼3.5 minutes). The MS signal at m/z 516.2999 was consistent with the protonated form of tauro-conjugated pythocholic acid [M + H]⁺ (calculated m/z 516.2989).

Figure 5.

Ad libitum food intake after single-dose administration of DCA or pythocholic acid (PA) at 6 Pm. Food intake after treatment (A) 0 to 3 hours, (B) 3 to 6 hours, (C) 6 to 9 hours, (D) 9 to 12 hours, (E) 12 to 18 hours, (F) 18 to 24 hours, and (G) total food intake for 24 hours (vehicle; n = 12, DCA; n = 11, PA; n = 10). *P < .05, **P < .01 (1-way ANOVA).

Figure 6.

Effects of DCA and PA on gastric emptying, blood glucose, and insulin levels after 2 days of once-daily treatment. (A) Experimental schedule, (B) gastric emptying, (C) 1-hour refed food intake after fasting, (D) plasma GLP-1 levels, (E) body weight, (F) blood glucose levels, (G) plasma insulin levels, (H) BA species measured by UPLC-MS/MS from liver samples, (I) the absolute quantities of BAs, (J) plasma AST levels, and (K) plasma ALT levels. (vehicle; n = 12, DCA; n = 11, PA; n = 10). *P < .05, vehicle vs PA, **P < .01, Vehicle vs PA, ^#P < .05, DCA vs PA, ^##P < .01, DCA vs PA (1-way ANOVA). α-MCA, α-muricholic acid; β-MCA, β-muricholic acid; PA, pythocholic acid; CA, cholic aid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GDCA, glycodeoxycholic acid; GUDCA, glycoursodeoxycholic acid; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; TabMCA, tauro-α- and tauro-β-muricholic acid; TCA, tauro-conjugated cholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TLCA, taurolithocholic acid; TPA, tauro-conjugated pythocholic; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid.

Figure 7.

Influence of DCA and PA on lipid absorption. (A) Fecal free fatty acid, (B) fecal acylglycerol, and (C) fecal cholesterol levels. Feces were collected for 24 hours before the gastric emptying experiment; (vehicle; n = 12, DCA; n = 11, PA; n = 10).

Figure 8.

Effects of DCA and PA on fatty acid ethanolamides and cannabinoids in jejunal intestine after refeeding. (A) Experimental schedule, effect of DCA and PA after refeeding on the levels of (B) OEA, (C) AEA, (D) DHEA, (E) 2-AG, (F) 2-OG, (G) 2-DG, and (H) 2-LG. (vehicle; n = 12, DCA; n = 11, PA; n = 10). *P < .05 (1-way ANOVA).