Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms

Abstract

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. However, the underlying mechanisms for these effects are unclear. It was suggested that SCFAs may regulate gut hormones via their endogenous receptors Free fatty acid receptors 2 (FFAR2) and 3 (FFAR3), but direct evidence is lacking. We examined the effects of SCFA administration in mice, and show that butyrate, propionate, and acetate all protected against diet-induced obesity and insulin resistance. Butyrate and propionate, but not acetate, induce gut hormones and reduce food intake. As FFAR3 is the common receptor activated by butyrate and propionate, we examined these effects in FFAR3-deficient mice. The effects of butyrate and propionate on body weight and food intake are independent of FFAR3. In addition, FFAR3 plays a minor role in butyrate stimulation of Glucagon-like peptide-1, and is not required for butyrate- and propionate-dependent induction of Glucose-dependent insulinotropic peptide. Finally, FFAR3-deficient mice show normal body weight and glucose homeostasis. Stimulation of gut hormones and food intake inhibition by butyrate and propionate may represent a novel mechanism by which gut microbiota regulates host metabolism. These effects are largely intact in FFAR3-deficient mice, indicating additional mediators are required for these beneficial effects.

Figure 1. Effects of dietary SCFAs on body weight and glucose homeostasis.

Three-month-old lean C57BL/6N mice were switched to HFD containing molarity-matched sodium salts of butyrate (5% w/w), propionate (4.3%), and acetate (3.7%) for four weeks. (A) Body weight was measured weekly, and four-week cumulative weight gain is expressed as a percentage of initial body weight. (B) Oral glucose tolerance test was performed in overnight fasted mice four weeks after diet switch. Blood glucose levels and total glucose area-under-the-curve (AUC) are shown. (C, D) Plasma levels of insulin and leptin were determined in overnight fasted mice four weeks after diet switch. Data are mean ± SEM. N=8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control HFD.

Figure 2. Effects of dietary SCFAs on food intake and locomotor activity.

(A-C) Three-month-old lean C57BL/6N mice were switched to HFD containing sodium salts of butyrate (5%), propionate (4.3%), and acetate (3.7%) for nine days. Daily food intake, cumulative food intake, and cumulative locomotor activity are shown. L: light phase. D: dark phase. (D, E) Dose titration of sodium butyrate and sodium propionate in HFD was performed in three-month-old lean C57BL/6N mice. Eight-day cumulative body weight change and food intake are shown. Data are mean ± SEM. N=8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control diet.

Figure 3. Effects of orally administered fatty acids on incretins and other hormones.

(A-F) Three-month-old lean C57BL/6N mice were fasted overnight and orally dosed with saline, sodium butyrate, sodium propionate, sodium acetate, an SCFA admixture (65% sodium acetate, 20% sodium propionate, 15% sodium butyrate), octanoic acid (OA), or α-linolenic acid (LA), all at 400mg/kg body weight. Plasma levels of total GLP-1, active GLP-1, GIP, PYY, insulin, and amylin were measured 10 minutes after dosing. Intra-assay CV% was below 8.9% for all immunoassays. Data are mean ± SEM. N=8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. saline. NS: not significant.

Figure 4. Effects of butyrate and propionate on energy homeostasis in Ffar3 knockout mice.

(A) Body weight of three-month-old Ffar3 knockouts and wild-type littermates on standard chow diet and one week after switching to HFD. N=34–41. (B-D) After one week of HFD feeding, Ffar3 knockouts and wild-type littermates were switched to HFD containing sodium butyrate (5%) or sodium propionate (4.3%) for eight days. Cumulative body weight change and daily food intake are shown. Data are mean ± SEM. N=8–13. *P<0.05, **P<0.01, ***P<0.001 vs. control diet. #P<0.05 vs. wild-type mice on control diet. NS: not significant.

Figure 5. Effects of butyrate and propionate on gut hormones in Ffar3 knockout mice.

(A) Ffar3 mRNA expression determined by quantitative RT-PCR in the mucosal and smooth muscle (SM) layers of different intestinal segments from lean C57BL/6N mice and in GLUTag cells. Data are normalized against Rplp0 mRNA. N=3. (B-H) After four weeks of HFD feeding, five-month-old Ffar3 knockout mice and wild-type littermates were fasted overnight and dosed with saline, sodium butyrate, or sodium propionate (400mg/kg). Plasma levels of total GLP-1, active GLP-1, GIP, PYY, insulin, and ghrelin were measured 10 minutes after dosing. Intra-assay CV% was below 7.6% for all immunoassays. N=8. Data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, NS: not significant.

Figure 6. Normal body composition and glucose homeostasis in Ffar3 knockout mice.

(A) Body composition was determined by quantitative NMR in Ffar3 knockouts and wild-type littermates after five months of HFD feeding. (B, C) Plasma leptin levels were determined in overnight fasted Ffar3 knockouts and wild-type littermates maintained on standard chow diet or HFD. (D) Blood glucose was measured in ad libitum fed mice maintained on HFD three hours after the start of the light phase. (E) Oral glucose tolerance test after overnight fasting and (F) intraperitoneal insulin tolerance test after five-hour daytime fasting in Ffar3 knockouts and wild-type littermates maintained on HFD. Data are mean ± SEM. N=8–14.