UCP2 mediates ghrelin's action on NPY/AgRP neurons by lowering free radicals

Abstract

The gut-derived hormone ghrelin exerts its effect on the brain by regulating neuronal activity. Ghrelin-induced feeding behaviour is controlled by arcuate nucleus neurons that co-express neuropeptide Y and agouti-related protein (NPY/AgRP neurons). However, the intracellular mechanisms triggered by ghrelin to alter NPY/AgRP neuronal activity are poorly understood. Here we show that ghrelin initiates robust changes in hypothalamic mitochondrial respiration in mice that are dependent on uncoupling protein 2 (UCP2). Activation of this mitochondrial mechanism is critical for ghrelin-induced mitochondrial proliferation and electric activation of NPY/AgRP neurons, for ghrelin-triggered synaptic plasticity of pro-opiomelanocortin-expressing neurons, and for ghrelin-induced food intake. The UCP2-dependent action of ghrelin on NPY/AgRP neurons is driven by a hypothalamic fatty acid oxidation pathway involving AMPK, CPT1 and free radicals that are scavenged by UCP2. These results reveal a signalling modality connecting mitochondria-mediated effects of G-protein-coupled receptors on neuronal function and associated behaviour.

Figure 1. Mitochondrial and transcriptional effects of ghrelin are UCP2-mediated

a–c, Ghrelin alters hypothalamic mitochondrial respiration. a, Oligomycin inhibits ATP synthase and uncouples substrate oxidation from ATP phosphorylation. The resulting increase in uncoupled oxygen consumption in ghrelin-treated mice is due to UCP2, as Ucp2^–/– mice exhibit no response to ghrelin. b, Ghrelin enhances respiration after palmitate addition in wild-type but not Ucp2^–/– mice. [Author: Revised sentence ok?Yes.] c, An increase in respiration after FCCP shows that ghrelin enhances the maximal respiratory capacity in wild-type but not in Ucp2^–/– mice. d, Ghrelin increases (10 nmol) hypothalamic Ucp2 mRNA expression as measured by real-time PCR (n = 6). The data are expressed as a fold increase relative to saline levels. e, Arcuate Npy gene expression is increased in response to ghrelin in wild-type but not Ucp2^–/– mice (n = 6). f, Ghrelin promotes mitochondrial proliferation in a UCP2-dependent manner in arcuate NPY/AgRP cells. [Author: Should this be NPY/AgRP cells, as per panel label?Yes.] g, Ghrelin increases Nrf1 mRNA expression in the hypothalamus of wild-type but not Ucp2^–/– mice. h, Representative traces showing that ghrelin decreases the mitochondrial membrane potential after addition of energy substrates (state 2) and after addition of oligomycin (state 4). Asterisk indicates statistically significant differences (P < 0.05) with respect to saline controls. All error bars indicate s.e.m. [Author: Correct definition of error bars?Yes.] Olig, oligomycin; Palm, palmitate; Pyr/Mal, pyruvate and malate. [Author: Correct definitions? ΔΨ_m correct for y axis label?Yes.]

Figure 2. Effects of ghrelin on neuronal firing, synaptic plasticity and feeding are UCP2-mediated

a, UCP2 potentiates NPY c-fos activation in the arcuate nucleus in response to ghrelin (10 nmol). Stereological analysis of total NPY–GFP/c-fos cells in the arcuate nucleus of wild-type (n = 6) and Ucp2^–/– mice (n = 6) is shown. b, Ghrelin increases mean action potential frequency of NPY–GFP neuronal perikarya in whole-cell recordings from wild-type mice (n = 7); this effect is diminished in Ucp2^–/– mice (n = 7). [Author: Revised sentence ok?] c, Ghrelin increases the frequency of mIPSCs on POMC–GFP perikarya in wild type but not in Ucp2^–/– mice (n = 5 for both groups). d, Ghrelin increases the number of symmetric, putatively inhibitory perikaryal synapses on POMC neurons in wild-type mice but not in Ucp2^–/– mice. [Author: ‘Number per 100 μm [micrometres] perikarya’ correct for y axis of d?Yes.] e, Peripheral ghrelin administration (intraperitoneal) significantly (P < 0.05) enhances food intake relative to saline controls in both wild-type (n = 6) and Ucp2^–/– (n = 6) mice at 1, 2 and 3 h after injection. However, food intake in Ucp2^–/– mice is significantly attenuated compared to wild-type mice 1, 2 and 3 h after injection. f, Mediobasal hypothalamus ghrelin injection (500 pmol μl⁻¹) significantly increases food intake of both genotypes at 1, 2 and 3 h after injection. However, Ucp2^–/– mice display severely attenuated food intake at all time points compared to wild-type controls. Asterisk indicates statistically significant differences (P < 0.05) with respect to saline controls; dagger indicates statistically significant (P < 0.05) reduction with respect to ghrelin-treated wild-type mice. All error bars indicate s.e.m. [Author: Correct definition of error bars?Yes.]

Figure 3. Ghrelin activates an intracellular pathway of fatty acid metabolsim

a, Ghrelin increases the pAMPK/AMPK ratio in wild-type but not Ghsr^–/– mice (n = 5). Ghr, ghrelin; Sal, saline. b, Ghrelin increases the pAMKP/AMPK ratio and phosphorylates downstream ACC in both wild-type and Ucp2^–/– mice, indicating that activation of AMPK is upstream to increased UCP2 activity (n = 6). c–e, AMPK regulation affects food intake. c, AICAR significantly increases food intake relative to saline controls in wild-type and Ghsr^–/– mice. d, AICAR-induced food intake is significantly suppressed in Ucp2^–/– mice compared to controls, indicating that UCP2 is required to translate AMPK activity into a complete food intake response (n = 5). e, AICAR significantly increases cumulative 3-h food intake relative to saline controls; however, this effect is completely blocked with a concurrent intracerebroventricular injection of compound C (CC, 6 μg μl⁻¹), an inhibitor of AMPK activity. AICAR did not affect the cumulative 3-h food intake in Ucp2^–/– mice and this was not affected by concurrent intracerebroventricular injection of compound C. f, Intraperitoneal ghrelin administration increases hypothalamic Ucp2 mRNA; however, this increase is blocked by concurrent injection of etomoxir or CC, indicating that activation of the AMPK–CPT1 pathway is important in the regulation of Ucp2 mRNA expression. g, Intraperitoneal ghrelin administration increases hypothalamic Cpt1 mRNA expression; [Author: On the figure, f is labelled Ucp3 mRNA and g is labelled Cpt1 mRNA. Please correct as necessary.] however, the induction of Cpt1 mRNA with ghrelin (Ghr) is blocked when the CPT1 inhibitor etomoxir (Eto), or compound C (CC), is injected intracerebroventricularly. h, Peripheral ghrelin injection increases hypothalamic expression of Npy mRNA, which is completely suppressed by intracerebroventricular injection of etomoxir and CC. Note that in f–h mRNA expression was not affected by intracerebroventricular injection of etomoxir or compound C with saline injected intraperitoneally. [Author: Revised sentence ok?Yes.] i, Inhibition of CPT1 activity with an intracerebroventricular injection of etomoxir suppresses ghrelin-induced (IP 10 nmol) food intake compared to mice receiving intraperitoneal ghrelin and intracerebroventricular saline (Ghr/Sal). Neither intracerebroventricular etomoxir nor compound C affected food intake in mice treated intraperitoneally with saline (n = 5–7). j, Ghrelin increased Cpt1 mRNA expression in wild-type but not in Ucp2^–/– mice (n = 6, P < 0.05). Asterisk, statistically significant (P < 0.05) with respect to saline controls; dagger, statistically significant (P < 0.05) with respect to Ghr/Sal. [Author: Please clarify ‘c’ label in panel g; statistically significant (P<0.05) with respect to Ghr/Eto.] All error bars indicate s.e.m.

Figure 4. ROS is a critical regulator of cellular and behavioural responses to ghrelina

Ghrelin increases hypothalamic synaptosomal ROS production in Ucp2^–/– mice but not wild-type mice (n = 4–5). b, Quantification of ROS production shows that ghrelin markedly increases in situ ROS in NPY neurons in Ucp2^–/– but not wild-type mice. c, Suppressed Cpt1 mRNA expression in ghrelin-treated Ucp2^–/– mice after 3 h is completely reversed and elevated above wild-type levels by concurrent intracerebroventricular injection of free radical scavenging (FRS) cocktail (n = 5, P < 0.05). i.c.v., intracerebroventricular; i.p., intraperitoneal. d, FRS cocktail reverses suppressed Npy mRNA in ghrelin-treated Ucp2^–/– mice. e, Incubating arcuate slices with FRS restores suppressed NPY neuronal action potential firing in Ucp2^–/– mice. Neurons were from NPY–GFP/Ucp2^–/– mice. Asterisk, significant to 100% saline controls. f, The FRS cocktail reverses the suppressed ghrelin-induced food intake after 1 h in Ucp2^–/– mice (n = 9, P < 0.05). Except for e: asterisk, significant to saline controls; dagger, significant decrease compared to wild-type mice administered ghrelin intraperitoneally and saline intracerebroventricularly; double dagger, significant with respect to Ucp2^–/– mice administered ghrelin intraperitoneally and saline intracerebroventricularly.