Short-chain fatty acids and ketones directly regulate sympathetic nervous system via G protein-coupled receptor 41 (GPR41)

Abstract

The maintenance of energy homeostasis is essential for life, and its dysregulation leads to a variety of metabolic disorders. Under a fed condition, mammals use glucose as the main metabolic fuel, and short-chain fatty acids (SCFAs) produced by the colonic bacterial fermentation of dietary fiber also contribute a significant proportion of daily energy requirement. Under ketogenic conditions such as starvation and diabetes, ketone bodies produced in the liver from fatty acids are used as the main energy sources. To balance energy intake, dietary excess and starvation trigger an increase or a decrease in energy expenditure, respectively, by regulating the activity of the sympathetic nervous system (SNS). The regulation of metabolic homeostasis by glucose is well recognized; however, the roles of SCFAs and ketone bodies in maintaining energy balance remain unclear. Here, we show that SCFAs and ketone bodies directly regulate SNS activity via GPR41, a Gi/o protein-coupled receptor for SCFAs, at the level of the sympathetic ganglion. GPR41 was most abundantly expressed in sympathetic ganglia in mouse and humans. SCFA propionate promoted sympathetic outflow via GPR41. On the other hand, a ketone body, β-hydroxybutyrate, produced during starvation or diabetes, suppressed SNS activity by antagonizing GPR41. Pharmacological and siRNA experiments indicated that GPR41-mediated activation of sympathetic neurons involves Gβγ-PLCβ-MAPK signaling. Sympathetic regulation by SCFAs and ketone bodies correlated well with their respective effects on energy consumption. These findings establish that SCFAs and ketone bodies directly regulate GPR41-mediated SNS activity and thereby control body energy expenditure in maintaining metabolic homeostasis.

Fig. 1.

Abundant Gpr41 expression in sympathetic ganglia and reduced sympathetic nerve activity in GPR41-deficient mice. (A) Gpr41 expression in postnatal mouse tissues (P49) measured by qRT-PCR (n = 3). SCG, superior cervical ganglion. Internal control: 18S rRNA expression. (B) Gpr41 mRNA localization in mouse embryos (E13.5 and E15.5) as determined by in situ hybridization using an ³⁵S-labeled antisense Gpr41 RNA probe. Red grains (arrowheads) superimposed on a hematoxylin−eosin-stained section indicate Gpr41 mRNA localization. (Scale bar: 5 mm.) (C) Gpr41 mRNA localization in postnatal day 1 (P1) mice (Left). Anti-tyrosine hydroxylase (TH) immunostaining (brown) (Right). (Scale bar: 1 mm.) (D and E) Anti-TH antibody immunostaining (brown) in SCG at P1. Total volume was measured by quantifying the TH-positive area (n = 6). (F) (Upper) Whole-mount immunostaining of the heart with anti-TH antibodies (brown). (Lower) High magnification view. (Scale bar: 1 mm.) (G) Quantitative analysis of TH+ nerve areas (n = 5). (H) TH protein expression (n = 6). β-Actin (loading control). Mice were analyzed at 12 wk of age (F–H). *P < 0.05; **P < 0.005.

Fig. 2.

Effects of SCFA on sympathetic activity via GPR41 in GPR41-deficient mice. (A) Noradrenaline (NA) concentrations in hearts (n = 10) and plasma (n = 6). (B) Effects of propranolol on heart rate in Gpr41^−/− mice. Measurement of heart rate at 10 min after propranolol administration (i.p.; n = 7–9). Both hatched bars, propranolol-treated. (C) Effects of tyramine on heart rate. Measurement of heart rate (n = 9) at 24 h following tyramine injection (i.p.). Both hatched bars, tyramine-treated. (D) Effects of propionate on heart rate in Gpr41^−/− mice (i.p.; n = 5–7). (E) Effects of octanoate on heart rate in Gpr41^−/− mice. Measurement of heart rate at 20 min after octanoate administration (i.p.; n = 4–5). (F) After pretreatment with hexamethonium (20 mg/kg) or propranolol (4 mg/kg) for 10 min, at time 0, a bolus of propionate (1 g/kg), carbachol (50 μg/kg), or isoproterenol (3 μg/kg) was administered intraperitoneally (n = 4–6). Data of was measured at 20 min (propionate) and at 3 min (carbachol and isoproterenol). Mice were analyzed at 12 wk of age. *P < 0.05; **P < 0.005.

Fig. 3.

SCFA-GPR41 signaling in sympathetic neurons. (A) Action potentials and firing frequency in Gpr41^−/− sympathetic neurons following stimulation with propionate (10 mM) (n = 3–8). (B) Change in myocyte beating rate in sympathetic neurons with and without propionate (1 mM) treatment and effects of propranolol (0.2 μM) (n = 5–6). Isoproterenol (10 μM) was used as a positive control. (C) Effects of propionate on change in myocyte beating rate in Gpr41^−/− cardiomyocytes and sympathetic neurons (n = 6–8). (D) Firing frequency in sympathetic neurons (n = 4–9) by propionate (10 mM) stimulation after pretreatment with or without PTX (100 ng/mL), Gallein (10 μM), or NF023 (10 μM) for 2 h. Carbachol (1 μM), a cholinergic agonist, was used as a positive control. (E) Effects of propionate on change in myocyte beat rate in cardiomyocytes and sympathetic neurons (n = 6–11). Cells were stimulated by propionate (1 mM) after pretreatment with or without PTX (100 ng/mL), Gallein (10 μM), or NF023 (10 μM) for 1 h. Isoproterenol (10 μM) was used as a positive control. (F) Effect of propionate (1 mM) and NGF (50 ng/mL) on the beating rate of cardiomyocytes when cocultured with Neuro2A cells either with or without transfection of Gpr41 (n = 6). (G) Effects of siRNA on propionate-induced increase in the beating rate of cardiomyocytes when cocultured with Neuro2A cells expressing GPR41 (n = 6). *P < 0.05; **P < 0.005.

Fig. 4.

Inhibitory effects of β-hydroxybutyrate on GPR41 sympathetic activity. (A) Antagonistic effects of β-hydroxybutyrate on ERK1/2 phosphorylation by propionate (1 mM) in GPR41-expressing HEK293 cells (n = 3). (B) Reduction in cAMP levels in response to propionate (0.1 mM) treatment in GPR41-expressing HEK293 cells and inhibitory effects of β-hydroxybutyrate (100 mM) (n = 3). (C) Firing frequency in sympathetic neurons (n = 5) after propionate (10 mM) stimulation with or without β-hydroxybutyrate (10 mM). (D) Inhibitory effects of β-hydroxybutyrate (500 μM) on change in myocyte beat rate in cardiomyocytes and sympathetic neurons with propionate (1 mM) treatment (n = 5–8). (E) Effects of β-hydroxybutyrate on heart rate of Gpr41^−/− mice (500 mg/kg i.p.; n = 8). Mice were analyzed at 12 wk of age. *P < 0.05; **P < 0.005.

Fig. 5.

Inhibitory effects of ketone bodies on GPR41 sympathetic activity during fasting or diabetes. (A) Heart rate following fasting in Gpr41^−/− mice (n = 7–8). (B) NA content in the heart after 48-h starvation (n = 9−10). (C) Effects of sympathetic nerve blocking on heart rate of Gpr41^−/− starved mice (n = 9–11). (D) Change in heart rate following the induction of diabetes (n = 4−10). (E) Effects of sympathetic nerve blocking on heart rate in Gpr41^−/− diabetic mice (n = 4–5). Mice were analyzed at 12–14 wk of age. *P < 0.05; **P < 0.005.

Fig. 6.

Effects of GPR41-mediated regulation of sympathetic activity on energy expenditure. (A) Effects on oxygen consumption in Gpr41^−/− mice during feeding and at 48-h starvation. Measurement of oxygen consumption at 24 h after tyramine administration (100 mg/kg i.p.) (n = 5–7). (B) Body temperature of Gpr41^−/− mice during feeding (n = 6). (C) Ucp1 expression in brown adipose tissue (BAT) in Gpr41^−/− mice during feeding (n = 6). Internal control: 18S rRNA expression. (D) Rate of oxygen consumption in propionate and PBS administration. Oxygen consumption was measured at 40 min after propionate administration (1 g/kg i.p.) (n = 4–8). (E) Rate of oxygen consumption in β-hydroxybutyrate and PBS administration. Oxygen consumption was measured at 50 min after β-hydroxybutyrate administration (500 mg/kg i.p.) (n = 5–7). Propionate and β-hydroxybutyrate were administrated after 24 h treatment of tyramine. Hatched bars, tyramine-treated (A, D, and E). (F) Inhibitory effects of β-hydroxybutyrate on oxygen consumption. After pretreatment with β-hydroxybutyrate (500 mg/kg) for 10 min, at time 0, a bolus of propionate (1 g/kg) with or without β-hydroxybutyrate (500 mg/kg) was administered intraperitoneally (n = 8). Data were measured at time 40 min. Mice were analyzed at 14–16 wk of age. *P < 0.05; **P < 0.005.