Abstract
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway1, while AgRP binds competitively to block α-MSH binding2 and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor3. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R4 and the sustained effects of AgRP on food intake5.
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Acknowledgements
We thank C. Zhang and A. M. Bradshaw for advice and technical assistance in performance of experiments in the zebrafish. We thank D. M. Parichy for providing the jaguar zebrafish strain. We thank B. S. Wulff and K. W. Conde-Frieboes and Novo Nordisk A/S for the contribution of MC4-NN2-0543. This work was supported by NIH RO1DK070332 (R.D.C.), NIH 5R01 DK082884-03 (J.S.D.), and NIH R01DK064265 (G.L.M.). R.D.C. is also supported by the Vanderbilt Diabetes Research and Training Center grant DK020593.
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M.G.-L., G.J.D., J.A.S., G.L.M., R.M., H.M.C., J.S.D. and R.D.C. designed experiments, M.G.-L., G.J.D., J.A.S., R.M., B.L.P., T.G. and I.R.T. performed experiments, G.L.M. and R.P. synthesized, purified and folded the AgRP mini peptide, and M.G.-L. and R.D.C. analysed the data and wrote the manuscript. All authors reviewed and commented on the manuscript.
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Extended data figures and tables
Extended Data Figure 1 G protein signalling is not required for α-MSH-induced depolarization of PVN MC4R neurons.
a, b, A MAP kinase kinase inhibitor, 1 µM U0126, fails to block α-MSH -induced increase in firing frequency of PVN MC4R neurons recorded in whole cell configuration, (a, left panel shows a representative trace from one neuron, b, right panel shows mean ± s.e.m. of firing frequency, P < 0.05, paired t-test). c, GTPγS, a non-hydrolysable GTP analogue, fails to block the depolarization induced by α-MSH, measured in whole-cell configuration. This drug however does block the hyperpolarization induced by µ-opioid agonist, DAMGO (10 μM), (mean ± s.e.m., P < 0.001, paired t-test). d, The Gβγ inhibitor gallein fails to inhibit α-MSH-induced depolarization of PVN MC4R neurons (mean ± s.e.m., P < 0.01, paired t-test), although it blocks the effects of DAMGO (10 μM) on membrane potential. In all electrophysiological studies, each n represents an independent neuron and slice, and no more than two slices were used per animal.
Extended Data Figure 2 The charge generated by rubidium permeation through MC4R-regulated channels depolarizes PVN MC4 neurons.
a, b, Rb+ efflux through MC4R -gated Kir channels can generate greater α-MSH -induced depolarization of PVN MC4R neurons loaded with 130 mM RbCl and 4 mM K+ through the recording pipette. Data show the mean and s.e.m. (a, *P < 0.05, unpaired t-test), and a representative trace (b).
Extended Data Figure 3 Co-expression of MC4R and Kir7.1 in the PVN.
a–d, Detection of MC4R and Kir7.1 mRNA in PVN slices using fluorescent in situ hybridization (RNAscope). Images demonstrate the region of the hypothalamus under study (a; scale bar, 200 μm), colocalization of MC4R (green) and Kir7.1 (red) mRNAs (b, white open arrows, double labelled cells; yellow arrows, Kir7.1 expression only; scale bar, 10 μm), and negative controls (c, MC4R probe with tissue from MC4R knockout mice; scale bar, 10 μm; d, bacterial probe with tissue from wild-type mice; scale bar, 10 μm). Data is representative of four male mice.
Extended Data Figure 4 Quantitation of MC4R and Kir7.1 RNA in PVN cells.
Single-molecule RNA detection in sections was quantitated by counting fluorescent dots associated with individual cells (Extended Data Fig. 3). Background threshold was determined from the number of dots per cell in sections resulting from hybridization using a negative bacterial DNA control, or from hybridization of the MC4R probe to sections from the MC4R knockout mouse (top panel, columns 1 and 2). Threshold-subtracted dot numbers were then used to determine the per cent of PVN cells expressing MC4R or Kir7.1, and the per cent of MC4R cells expressing Kir7.1; cells were considered positive if the number of dots exceeded the mean of the negative controls by 3× standard deviations (bottom panel). Data from the number of cells indicated was collected from multiple PVN sections derived from four male mice.
Extended Data Figure 5 MC4R and Kir7.1 coimmunoprecipitate from transfected HEK293 cells.
a, Cells transfected with the indicated genetically flagged proteins were incubated with the reversible crosslinker dithiobismaleimidoethane (DTME) before lysis. Lysates were immunoprecipitated using the indicated antibody (F, Flag; HA, haemagglutinin; X, no antibody), crosslinking was reversed with 100 mM dl-dithiothreitol (DTT), and samples separated by SDS–PAGE. The membrane was blotted with the M2 anti-Flag antibody to detect Kir7.1. b, Relative quantitation of protein immunoprecipitation. Densitometry analysis to measure the amount of immunoreactive Kir7.1 material was performed using Adobe Photoshop. Amount of material immunoprecipitated with the Kir7.1-3X-Flag was set at 100%. Data shows relative amount of Kir7.1 immunoprecipitated using an antibody against the 3HA-MC4R protein; bars indicate range of data from 2 independent lanes. The protein molecular weight of Kir7.1 is calculated at 40 kDa, and the two larger bands represent glycosylated forms of the protein that are absent when the N-linked glycosylation site at position 93 is mutated (data not shown). Data are representative of three independent experiments.
Extended Data Figure 6 AgRP-induced increase in thallium flux does not involve Gi signalling or β-arrestin recruitment.
a, b, Subtracted Tl+ flux examining effects of 200 nM AgRP indicates that 8 h pre incubation with pertussis toxin of MC4R- and Kir7.1-expressing HEK293 cells fails to block AgRP-induced Kir7.1 regulation (mean ± s.e.m., n = 110, combined data from three independent experiments). c, Addition of α-MSH stimulates β-arrestin recruitment to the MC4R in HEK cells stably expressing MC4R and β-arrestin fused to complementary fragments of β-galactosidase (DiscoverRx PathHunter assay, black line, logEC50 = −7.69). In contrast, increasing concentrations of AgRP are without any effect using the same assay (red line). Individual points show mean ± s.e.m. n = 12, representative of 3 independent experiments.
Extended Data Figure 7 Role of PKA and cAMP in the α-MSH-induced closure of Kir7.1.
a, b, Subtracted Tl+ flux assay examining effects of 100 nM α-MSH indicate that pre-incubation with 1 µM H89, a PKA inhibitor, fails to block α-MSH -induced regulation of Kir7.1. Data (n = 16) show mean ± s.e.m. of kinetic traces (a) and maxima (b). VHC = vehicle. c, d, Subtracted Tl+ flux assay examining effects of raising intracellular cAMP by forskolin (FSK, 20 μM) and IBMX treatment, with and without 100 nM α-MSH. Data show kinetic traces (c) and maxima (d). IBMX, 100 μM 3-isobutyl-1-methylxanthine; VHC, vehicle, mean ± s.e.m., n = 64, P < 0.01, unpaired t-test. Data representative of 3 independent replicates.
Extended Data Figure 8 The α-MSH analogue MC4-NN2-0453 (NOVO) is a partial agonist of the MC4R in a murine colon mucosal assay of MC4R activity.
The activation of the MC4R inhibits vectorial ion transport across colonic epithelium, measured as reductions in the short circuit current (Isc). a, Kinetic response to a sub-maximal basolateral concentration of α-MSH or NOVO, showing more rapid achievement of maximal activity with α-MSH, P < 0.05, one-way ANOVA with Bonferroni’s post-test. b, Full concentration-response to MC4-NN2-0453 (NOVO), showing that the compound does not achieve the efficacy reached by a maximal dose of α-MSH (denoted by the dashed line. Full characterization of the MC4R mediated α-MSH response in colonic epithelium is presented elsewhere18. Each data point represents the mean of five measurements from independent colon samples, with approximately six samples per animal obtained from 15 mice.
Extended Data Figure 9 Effects of Kir7.1 and MC4R signalling in larval zebrafish.
a–c, Knock-down of the Kir7.1 gene by kcnj13 morpholino oligonucleotide (MO) suppresses the axial growth of larvae in wild-type and mc4r null zebrafish. Sibling wild-type or mc4r-null zygotes were bred and injected with antisense kcnj13 morpholino oligonucleotide at day 0. a, The axial body length was measured at 5 dpf. Each group of 30 fish was harvested for RNA extraction and cDNA synthesis. b, Relative expression of ghrh mRNA was measured and normalized to the house keeping gene ef1a with qRT–PCR. The wild type fish that were injected with MO against kcnj13 expressed significantly higher copies of ghrh mRNA than those that were injected with control MO. (control MO, n = 9, 1.056 ± 0.116 vs kcnj13 MO, n = 9, 1.935 ± 0.294, unpaired t-test, P < 0.05). MC4R-null fish that were injected with kcnj13 MO have significantly higher GHRH expression than MC4R-null fish that were injected with control MO (control MO, n = 9, 1.040 ± 0.164 vs KCNJ MO, n = 8, 2.395 ± 0.461, one-way ANOVA, P < 0.05). c, Representative WT fish injected with kcnj13 MO vs control MO. d, e, jaguar wild-type and null mutant siblings were bred and injected with 7.5 ng non-targeting standard control or 7.5 ng antisense morpholino oligonucleotide targeting agrp or kcnj13. d, Knockdown of AgRP with agrp MO in the absence of Kir7.1 also reduces larval growth (mean ± s.e.m., n = 43, P < 0.001, unpaired t-test). e, The deletion of Kir7.1 in jaguar null blocks effects of KCNJ13 MO on MC4R-mediated inhibition of growth (mean ± s.e.m., n = 58, unpaired t-test). Data are representative of three independent experiments.
Extended Data Figure 10 A model for α-MSH and AgRP signalling at PVN MC4R neurons.
Data presented here supports a model in which MC4R may couple to both Gαs signalling and regulation of Kir7.1 activity in PVN MC4R neurons. α-MSH results in elevation of intracellular cAMP through activation of Gαs, and inhibition of K+ efflux through Kir7.1, both of which are depolarizing. AgRP lowers the constitutive activity of the MC4R and blocks α-MSH binding, but data here show that AgRP also acts as an agonist to increase K+ efflux through Kir7.1, producing a strong hyperpolarizing signal. The relative distribution and composition of the MC4R signalling complex in different subcellular compartments of PVN MC4R neurons has not been directly determined. Earlier models of α-MSH and AgRP action suggested competitive binding of these peptides to individual MC4R sites (orange box). Existing neuroanatomical data characterizing POMC and AgRP neuronal projections show that α-MSH may act independently of AgRP at many sites in the central nervous system, since AgRP immunoreactive fibres are only observed in a subset of MC4R-expressing nuclei containing POMC-immunoreactive fibres (right circle, for review see ref. 25). The ability of AgRP to act independently of α-MSH as a potent hyperpolarizing agonist, via regulation of Kir7.1, suggests the likely existence of independent AgRP sites of action (left circle). Recent reconstruction of electron microscopy images of the PVN in which POMC- and AgRP-containing synaptic vesicles have been specifically labelled with a genetically encoded marker provides preliminary anatomical support for this new model26. This study demonstrates that 52% of AgRP boutons in the PVN are not found in synapses, potentially supporting volume transmission of AgRP that may lead to competition with α-MSH at synaptic and/or non-synaptic sites. Additionally, the study found the vast majority of AgRP and POMC synaptic sites localized to different subcellular compartments of PVN neurons, supporting the independent action of both peptides. Synaptic release sites on soma were almost exclusively AgRP-containing, while POMC release sites were concentrated on distal dendrites. Another MC4R signalling pathway, involving cAMP/PKA-dependent activation of KATP channels and α-MSH-induced hyperpolarization, has been demonstrated in MC4R neurons in the dorsal motor nucleus of the vagus in the brainstem (bottom right)21. Thus, while Kir7.1 signalling appears to be essential for depolarization of PVN MC4R neurons by α-MSH, Gαs signalling and elevation of cAMP may be depolarizing or hyperpolarizing, depending on the cellular context.
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Ghamari-Langroudi, M., Digby, G., Sebag, J. et al. G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons. Nature 520, 94–98 (2015). https://doi.org/10.1038/nature14051
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DOI: https://doi.org/10.1038/nature14051
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