The β-Hydroxybutyrate-GPR109A Receptor Regulates Fasting-induced Plasticity in the Mouse Adrenal Medulla

Abstract

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is β-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of β-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.

Keywords: AgRP; GPR109A; adrenal; autonomic nervous system; fasting; ketone bodies.

Figure 1.

Fasting increases the expression of agouti-related peptide (AgRP) messenger RNA (mRNA) in the adrenal medulla. A, Examples of in situ hybridization showing expression of AgRP mRNA in the adrenal gland of fed and fasted mice. AgRP expression was present in the medulla (Med) and absent from the cortex (Ctx). B, Group data showing that fasting led to a large increase in AgRP mRNA in the adrenal medulla. Symbols show data from individual mice and lines connect data from paired animals (see “Materials and Methods”). IU; intensity units. (n = 5, mean ± SEM). P = .008, Mann-Whitney U test. Scale bar 100 µm.

Figure 2.

GPR109A agonists increase the expression of agouti-related peptide (AgRP) in chromaffin cells in vitro. A, Examples of AgRP-ir in chromaffin cells treated with 5 mM β-hydroxybutyrate for 24 hours in vitro. Right panel is a cumulative frequency distribution showing that β-hydroxybutyrate led to a significant increase in AgRP-ir (n = 3 experiments, ≥ 377 cells per distribution, P = 1.03E-10, Kolmogorov-Smirnov [K-S] test). B, 100 µM nicotinic acid led to a significant increase in the levels of AgRP-ir (n = 3 experiments, ≥ 253 cells per distribution, P = 3.4E-14, K-S test). C and D, Cumulative frequency distributions showing that 100 µM acipimox (n = 3 experiments, ≥ 246 cells per distribution, P = 6.29E-07, K-S test) and 100 µM monomethylfumarate (n = 3 experiments, ≥ 248 cells per distribution, P = 3.92E-11, K-S test) increased the levels of AgRP-ir. E, Unlike GPR109A agonists (panels A to D), which increased the levels of AgRP-ir, 1 mM propionate, a GPR41 agonist, led to a significant decrease in the levels of AgRP-ir in chromaffin cells in vitro (n = 3 experiments, ≥ 245 cells per distribution, P = 8.24E-38, K-S test). **P less than .01. Scale bar 10 µm.

Figure 3.

Effect of β-hydroxybutyrate on adrenal chromaffin cells is mediated by a pertussis-toxin sensitive G protein–coupled receptor (GPCR). A, Examples of AgRP-ir in chromaffin cells treated with 5 mM β-hydroxybutyrate, an agonist of GPR109A receptors and pertussis toxin, an inhibitor of G_i/o-coupled receptors. B, Cumulative frequency distributions confirmed that β-hydroxybutyrate incubation led to an increase in AgRP-ir and that this effect was abolished by coincubation in 100 µg/mL pertussis toxin (≥ 143 cells per distribution, n = 3 experiments). C, Reverse transcription polymerase chain reaction showing that wild-type (wt) (GPR109A^+/+) adrenal medulla contains GPR109A messenger RNA whereas amplicons were not generated using tissue from GPR109A knockout (ko) mice. Brown adipose tissue (BAT), which expresses high levels of GPR109A, was used as a positive control. **P = 5.58E-07; ns, not significant; Kolmogorov-Smirnov test. Scale bar 10 µm.

Figure 4.

Knockout (ko) of GPR109A receptors prevents the fasting-induced increase in agouti-related peptide (AgRP) expression in adrenal chromaffin cells. A, Examples of AgRP immunoreactivity (AgRP-ir) in chromaffin cells from fed and fasted GPR109A^–/– mice. B, Cumulative frequency distributions showing that fasting led to a small but significant decrease in the levels of AgRP-ir (n = 5 experiments, 263 cells per distribution; P = 1.43E-06; Kolmogorov-Smirnov [K-S] test) in ko mice. Right panel shows the change in mean AgRP-ir for each independent experiment (n = 5). C, Plasma levels of β-hydroxybutyrate show a significant increase in fasted GPR109A^–/– mice (n = 6, mean ± SEM). D, Examples of AgRP-ir in chromaffin cells from fed and fasted GPR109A^+/+ mice. E, Cumulative frequency distributions showing that fasting led to a significant increase in the levels of AgRP-ir (n = 6 experiments, 329 cells per distribution, P = 2.04E-10, K-S test). Right panel shows the mean change in AgRP-ir for each independent experiment (n = 6). F, Plasma levels of β-hydroxybutyrate show a significant increase in fasted GPR109A^+/+ mice (n = 6, mean ± SEM). G, Food deprivation leads to a significant decrease in the plasma levels of leptin both in GPR109A wild-type (wt) and ko littermates (n = 6, mean ± SEM). H, Urine levels of corticosterone show a significant increase after fasting both in GPR109A wt (GPR109A^+/+) and ko (GPR109A^–/–) littermates (n = 6 independent experiments, mean ± SEM). I, Examples of AgRP-ir in chromaffin cells from GPR109A^–/– mice treated with 100 µM nicotinic acid. J, Cumulative frequency distributions showing that nicotinic acid led to a significant decrease in the levels of AgRP-ir (n = 3 experiments, 163 cells per distribution, P = 2.84E-4, K-S test). Right panel shows the change in mean AgRP-ir for each independent experiment (black lines connect data points from individual experiments). K, Examples of AgRP-ir in chromaffin cells from GPR109A^+/+ mice treated with 100 µM nicotinic acid. L, Cumulative frequency distributions showing that nicotinic acid led to a significant increase in the levels of AgRP-ir (n = 3 experiments, 150 cells per distribution, P = 4.96E-3, K-S test). Right panel shows the change in mean AgRP-ir for each independent experiment (black lines connect data points from individual experiments). *P less than .05; **P less than .01 (C and F, t test; G and H, Mann-Whitney U test). Scale bar 10 µm.

Figure 5.

GPR109A agonists alter the expression of multiple transmitter pathways in chromaffin cells in vitro. A, Examples of neuropeptide Y immunoreactivity (NPY-ir) in chromaffin cells treated with 5 mM β-hydroxybutyrate for 24 hours in vitro. Right panel is a cumulative frequency distribution showing that β-hydroxybutyrate led to a significant increase in NPY-ir (n = 3 experiments, ≥ 52 cells per experiment; P = 2.06E-20; Kolmogorov-Smirnov [K-S] test). B, Cumulative frequency distributions showing that 100 µM nicotinic (Nic.) acid increased the levels of NPY-ir (n = 3 experiments, ≥ 52 cells per experiment, P = 2.11E-09, K-S test). C, TH-ir in chromaffin cells treated with 5 mM β-hydroxybutyrate for 24 hours in vitro. Right panel is a cumulative frequency distribution showing this led to a decrease in TH-ir (n = 3 experiments, ≥ 50 cells per experiment, P = 3.96E-42, K-S test). D, Cumulative frequency distributions showing that 100 µM nicotinic acid treatment increased the levels of NPY-ir (n = 3 experiments, ≥ 50 cells per experiment, P = 5.31E-08, K-S test). **P less than .01. Scale bar 10 µm.

Figure 6.

GPR109A agonist modulates preganglionic → chromaffin cell synaptic strength. A, Synaptic strength was monitored at the preganglionic → chromaffin cell synapse in adrenal slices. Shown are representative examples of synaptic currents evoked in response to paired presynaptic depolarizations. Food deprivation for 24 hours led to synaptic strengthening evident as a decrease in the paired pulse ratio (PPR; fed vs fasted). Incubation of adrenal slices in 100 µM nicotinic acid also led to a decrease in PPR in adrenal slices (fed vs nicotinic acid) from both male and female mice (n = 6-8 cells from 3-6 mice). B, Model pathway leading to the fasting-dependent regulation of adrenal medulla function. Fasting-induced sympathetic activity evokes epinephrine release from chromaffin cells in the adrenal medulla. Epinephrine contributes to increased lipolysis and fatty acid release from white adipose tissue during periods of negative energy balance. Hepatic ketogenesis metabolizes fatty acids to ketone bodies, including β-hydroxybutyrate, the endogenous agonist for GPR109A. Activation of this GPCR, likely in the adrenal, elevates agouti-related peptide (AgRP) and neuropeptide Y (NPY) and inhibits tyrosine hydroxylase (TH) expression in chromaffin cells. By acting at GPR109A receptors, β-hydroxybutyrate also leads to an increase in preganglionic → chromaffin cell synaptic strength. Not shown is the GPR109A-mediated negative feedback pathway that also regulates lipolysis at the level of white adipose tissue (51). Adrenal GPR109A receptors thus regulate communication between 3 tissues (adrenal-adipose-liver) that are involved in the peripheral response to food deprivation. *P less than .05; **P less than .01; ***P less than .001 (A, left and right, respectively: analysis of variance, Tukey post hoc; t test).