Glucagon-like peptide 1 (GLP-1) is a potent inhibitor of food intake. GLP-1 receptor mRNA is densely expressed in hypothalamic arcuate nucleus (ARC) and precisely overlaps the area occupied by proopiomelanocortin (POMC) neurons. Activation of POMC neurons suppresses appetite, and lack of POMC-derived peptides or inhibition of POMC neuronal firing causes obesity. Here, we identify living POMC cells in mouse ARC brain slices by targeted expression of green fluorescent protein. Using whole-cell patch-clamp recordings, we show that GLP-1 increases the spontaneous action-potential firing of POMC neurons. The stimulatory effect of GLP-1 was mimicked by GLP-1 receptor agonist exendin-4 and abolished by the receptor antagonist exendin 9-39. The effect of GLP-1 was unchanged in the presence of the synaptic blockers DAP5 (d(-)-2-amino-5-phosphonopentanoic acid)/CNQX (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt) and picrotoxin. These results suggest that GLP-1 excites POMC neurons postsynaptically, via interaction with GLP-1 receptors on POMC cells. Whole-cell Ca2+ currents increased ∼70% in the presence of GLP-1, and this effect was abolished by L-type Ca2+ channel antagonist nifedipine. Forskolin (which activates cAMP) mimicked the effects of GLP-1 and the PKA inhibitor Rp-8-Bromo-cAMPS (8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer) blocked GLP-1 action. These data indicate that GLP-1 stimulates the electrical activity of hypothalamic POMC neurons by activation of PKA and a subsequent increase in L-type Ca2+ current. This effect may contribute to the anorectic action of GLP-1, because excitation of POMC cells is well established to reduce food intake.
Glucagon-like peptide 1 (GLP-1) is a potent incretin that is produced in the L cells of the small intestine and a subset of neurons in the nucleus of the solitary tract (NTS) (Larsen et al., 1997a; Merchenthaler et al., 1999). In addition to its stimulatory action on insulin release from pancreatic β-cells, GLP-1 is a long-term inhibitor of food intake in both rodents and humans. In rats, feeding was potently inhibited by intracerebroventricular injection of GLP-1 (Tang-Christensen et al., 1996; Turton et al., 1996), and body weight was significantly reduced by repeated central injections of GLP-1 or the GLP-1 receptor agonist exendin-4 (Meeran et al., 1999). In humans, GLP-1 secretion was lower in obese men and teenage girls and rose after body weight loss (Verdich et al., 2001; Tomasik et al., 2004). Subcutaneous injection of GLP-1 for 5 d also reduced energy intake and weight in obese subjects (Naslund et al., 2004). Despite the many in vivo studies showing that GLP-1 is a potent inhibitor of food intake when infused directly into the brain, the molecular mechanism of GLP-1 action in the CNS remains essentially unknown.
GLP-1 receptor mRNA is widely expressed in the CNS, being especially high in the hypothalamic arcuate nucleus (ARC), paraventricular nucleus (PVN), and supraoptic nuclei (Campos et al., 1994; Shughrue et al., 1996). GLP-1 neurons within the NTS mainly project to the hypothalamus (Larsen et al., 1997a), including the proopiomelanocortin (POMC) neurons of the ARC (Huda et al., 2006). In rodents, GLP-1 reduced food intake concomitantly with an increase in c-fos in the ARC that was completely blocked by the GLP-1 receptor antagonist exendin 9-39 (Larsen et al., 1997b; Neary et al., 2005). Furthermore, when the ARC was destroyed by treatment with monosodium glutamate, GLP-1 was unable to inhibit food intake (Tang-Christensen et al., 1998). Thus, hypothalamic ARC neurons appear to be important for mediating the anorectic effects of GLP-1.
It is very well established that both the anorexigenic POMC neurons and the orexigenic AgRP/NPY (agouti-related protein/neuropeptide-Y-expressing) neurons of the ARC play a key role in the regulation of appetite and energy homeostasis. For example, stimulation of POMC neurons within the ARC leads to reduced appetite and body weight (Schwartz et al., 2000; Jobst et al., 2004). Conversely, ablation (Xu et al., 2005) or electrical inhibition (Plum et al., 2006) of POMC neurons causes obesity. Mice lacking POMC-derived peptides also become obese (Yaswen et al., 1999).
Our current knowledge of the role of GLP-1 in the CNS is primarily based on evidence from in vivo studies, and little data on the effects of GLP-1 on the electrical activity of CNS neurons, and nothing for ARC neurons, has been reported. In this study, we identify ARC POMC cells by targeted expression of eGFP in brain slices of mice, and study their electrical responses to GLP-1 using the whole-cell patch-clamp technique. We show that GLP-1 stimulates the electrical activity of POMC cells by increasing whole-cell Ca2+ currents and activation of protein kinase A.
Materials and Methods
Generation of POMC–enhanced green fluorescent protein transgenic mice.
The POMC–enhanced green fluorescent protein (eGFP) mice were created using the Cre-lox system. The generation and validation of the POMC–eGFP mouse model has been described in detail previously (Balthasar et al., 2004; Plum et al., 2006).
Coronal brain slices (250–300 μm) containing the ARC were prepared from 2- to 4-week-old POMC–eGFP mice (Plum et al., 2006). After at least 30 min recovery at 35°C in artificial CSF (ACSF) gassed with 95% O2 and 5% CO2, brain slices were transferred to a recording chamber and continuously perfused at 2–4 ml/min with gassed ACSF. ACSF contained the following (in mm): 125 NaCl, 21 NaHCO3, 2.5 KCl, 1.2 NaH2PO4, 2 CaCl2, 2 MgCl2, 10 HEPES, pH 7.4, and 5 glucose.
Brain slices were viewed with a Zeiss (Welwyn Garden City, UK) Axioskop fitted with fluorescence and infrared differential interference contrast (IR-DIC) videomicroscopy. Fluorescent POMC–eGFP neurons were identified by epifluorescence and patched under IR-DIC optics. Whole-cell current-clamp and voltage-clamp recordings were made using an EPC-9 patch-clamp amplifier, as described previously (Burdakov and Ashcroft, 2002; Plum et al., 2006; Ma et al., 2007). Patch pipettes had resistances of 3–5 MΩ when filled with internal solution. Ca2+ currents were evoked by 150 ms voltage steps from a holding potential of −60 mV to between −80 and +60 mV in 10 mV increments. Currents were filtered at 3 kHz and sampled at a frequency of 10 kHz. The peak inward current and current–voltage (I–V) relationships were measured. Resting potentials of firing neurons were determined from slow time-scale recordings on which a clear basal line was evident; this baseline was taken as the resting potential (Plum et al., 2006; Ma et al., 2007). Fold increases in firing were determined individually for each neuron and then averaged.
For most experiments, patch pipettes were filled with internal solution containing the following (in mm): 128 K-gluconate, 10 KCl, 10 HEPES (pH 7.3, adjusted with KOH), 0.1 EGTA, 2 MgCl2, 0.3 Na-GTP, and 3 K2-ATP. For recording Ca2+ currents, the extracellular solution contained the following (in mm): 2.5 KCl, 21 NaHCO3, 3 CaCl2, 1 MgCl2, 1.2 NaH2PO4, 10 HEPES, pH 7.4, and 10 glucose, supplemented with 100 mm TEA-Cl and 5 mm 4-AP to block K+ currents, and 1 μm tetrodotoxin (TTX) to block voltage-gated Na+ currents. The patch pipette was filled with the following (in mm): 130 CsCl, 10 HEPES (pH 7.3 adjusted with CsOH), 5 EGTA, 2 MgCl2, 0.5 Na-GTP, and 2 Mg-ATP. Glucagon-like peptide 1 (fragment 7-36), exendin-4, exendin 9-39, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX), d(-)-2-amino-5-phosphonopentanoic acid (DAP5), picrotoxin, tetrodotoxin, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS), forskolin, and nifedipine were added to the external solution as indicated. All drugs were from Sigma (Poole, UK). Experiments were performed at 22–25°C.
Data are presented as mean ± SEM for the indicated number of experiments (n) Statistical significance was evaluated using the Student's t test or Wilcoxon matched pairs test.
GLP-1 stimulates POMC neurons by binding to GLP-1 receptors
To investigate the effects of GLP-1 on the electrical activity of identified POMC neurons, we first performed whole-cell current-clamp recordings with zero holding current (Fig. 1A). Bath application of 100 nm GLP-1 increased the rate of spontaneous action potential firing in 7 of 8 cells (Fig. 1A). This was associated with a small but significant membrane depolarization and a small decrease in membrane resistance (Table 1). The effects of GLP-1 on firing rate were fully or partially reversible in 5 of 7 POMC cells after washout of 9 min or longer (Fig. 1A).
The stimulatory effect of GLP-1 was concentration dependent. The increase in firing rate was 1.09 ± 0.04-fold (n = 3) with 30 nm, 2.22 ± 0.41-fold (n = 7) with 100 nm, and 2.59 ± 0.37-fold (n = 5) with 1 μm GLP-1. There was no significant difference between the increase in firing found for 100 nm and 1 μm GLP-1 (p = 0.53) or between the firing rate in 30 nm GLP-1 and that in its absence (p = 0.14) (supplemental Table 2, available at www.jneurosci.org as supplemental material). Thus, we used 100 nm GLP-1 in the remainder of this study.
The effects of GLP-1 are mediated via activation of GLP-1 receptors in both brain (Scrocchi et al., 1996) and pancreatic β- and α-cells (Gromada et al., 1997; Ma et al., 2005). We therefore repeated our experiments using the specific GLP-1 receptor agonist exendin-4 and the antagonist exendin 9-39. Exendin-4 (100 nm) depolarized POMC neurons and increased the rate of spontaneous firing (Fig. 1B): the mean increase in firing was 2.88 ± 0.58-fold (n = 4). By itself, the GLP-1 receptor antagonist exendin 9-39 had no effect on either the membrane potential or the firing rate of POMC neurons (Fig. 1C). However, it abolished the ability of GLP-1 to increase the firing frequency of POMC cells (Fig. 1C). Together, these results indicate that GLP-1 interacts with GLP-1 receptors on POMC neurons.
Effects of GLP-1 on membrane potential in the presence of synaptic blockers
We next examined the effect of GLP-1 in the presence of 1 μm tetrodotoxin, which blocks Na+-dependent action potentials. Under these conditions, 100 nm GLP-1 no longer had a significant effect on the membrane potential of POMC neurons (Fig. 2A). This suggests that changes in voltage-activated currents and/or in action potential-mediated presynaptic inputs may be responsible for the observed effects of GLP-1 on the membrane potential and firing rate of POMC neurons.
To distinguish between these two possibilities, we tested the effect of blocking synaptic inputs to POMC cells. Most synaptic activity in hypothalamic circuits is thought to be mediated by release of GABA or glutamate (van den Pol et al., 1998). We therefore used CNQX (10 μm) to block AMPA receptors, DAP5 (50 μm) to block NMDA receptors, and picrotoxin (100 μm) to block GABAA receptors. When all three receptor types were blocked, 100 nm GLP-1 was still capable of increasing the firing of POMC neurons (Fig. 2B, Table 2). The mean increase in firing rate was 1.54 ± 0.19-fold (n = 7), which is not significantly different from the increase in firing rate produced by GLP-1 in the absence of synaptic blockers (p = 0.19). The stimulatory effects of GLP-1 were fully reversed in all seven cells tested (Fig. 2B, Table 2). These results suggest that stimulatory effects of GLP-1 on POMC neurons are not mediated by changes in presynaptic inputs. Thus, postsynaptic voltage-activated currents are largely responsible for the effect of GLP-1 on POMC neurons.
GLP-1 increases whole-cell Ca2+ currents in POMC neurons
GLP-1 directly increases cytosolic Ca2+ concentration and thus stimulates electrical activities in rat nodose ganglion neurons (Kakei et al., 2002). Therefore, we next measured whole-cell Ca2+ currents before and after addition of 100 nm GLP-1 (Fig. 2C,D). GLP-1 increased the peak Ca2+ current by 63% from a control value of −54 ± 16 pA to −89 ± 18 pA within 7 min (Fig. 2D) (p < 0.01). GLP-1-activated Ca2+ currents were reduced to control levels by addition of the L-type Ca2+ channel blocker nifedipine (Fig. 2C). These data suggest that GLP-1 selectively activates L-type Ca2+ channels in POMC neurons.
The effect of GLP-1 is mediated by activation of PKA
The GLP-1 receptor is a G-protein-coupled receptor that acts by increasing intracellular cAMP levels (Drucker et al., 1987; Thorens, 1992). It was found that 10 nm GLP-1 produced a twofold to threefold increase in intracellular cAMP levels in cultured hippocampal neurons (Perry et al., 2002), and forskolin mimicked the effect of GLP-1 on elevation of cytosolic Ca2+ concentration in nodose ganglion neurons (Kakei et al., 2002). To determine whether GLP-1 receptor activation in POMC neurons is mediated via elevation of cytosolic cAMP and subsequent activation of PKA, we tested the effects of forskolin and Rp-8-Bromo-cAMPS. Forskolin elevates cAMP and thus should mimic the effects of GLP-1. Figure 2E shows that forskolin (25 μm) depolarized POMC neurons and significantly increased the firing rate. In contrast, GLP-1 failed to increase firing of POMC neurons in the presence of 100 μm Rp-8-Bromo-cAMPS, a membrane-permeable specific inhibitor of PKA (Fig. 2F). Together, these data suggest that the stimulatory effects of GLP-1 on POMC neurons arise from elevation of intracellular cAMP and activation of PKA.
In this study, we show that GLP-1 depolarizes ARC POMC neurons and increases the frequency of action potentials. These effects appear to be mediated by a direct postsynaptic action of GLP-1 on POMC cells. Two pieces of evidence support this idea. First, GLP-1 retained its stimulatory effect in the presence of blockers of glutamatergic and GABAergic synaptic transmission. Second, GLP-1 was capable of enhancing Ca2+ currents in the presence of TTX, which blocks presynaptic firing and reduces transmitter release. We conclude that GLP-1 mediates its effect via direct interaction with GLP-1 receptors on POMC cells.
The stimulatory effects of GLP-1 were mimicked by GLP-1 analog exendin-4 and abolished by the GLP-1 receptor antagonist exendin 9-39, indicating that GLP-1 acts via postsynaptic GLP-1 receptors. This is consistent with in situ hybridization studies that show that GLP-1 receptor mRNA is densely expressed in hypothalamic ARC neurons (Shughrue et al., 1996; Merchenthaler et al., 1999) and almost precisely overlaps the region in which hypothalamic POMC neurons reside. Furthermore, central injection of GLP-1 induced c-fos expression in ARC neurons, and this was blocked by previous injection of exendin 9-39 (Larsen et al., 1997b).
GLP-1 receptor activation leads to stimulation of adenylate cyclase and elevation of cAMP (Thorens, 1992; Holz et al., 1993; Gromada et al., 1997; Perry et al., 2002; Ma et al., 2005). Like GLP-1, the adenylate cyclase activator forskolin elevated intracellular Ca2+ concentration in nodose ganglion neurons (Kakei et al., 2002). Consistent with these findings, forskolin mimicked the effects of GLP-1 on the electrical activity of POMC neurons. Moreover, GLP-1 was unable to excite POMC neurons in the presence of a PKA inhibitor. Our data therefore suggest that GLP-1 stimulates firing of POMC neurons via binding to GLP-1 receptors, elevation of cytosolic cAMP, and activation of PKA.
GLP-1 also stimulates the electrical activity of orexin/hypocretin neurons (Acuna-Goycolea and van den Pol, 2004), in which, in contrast to POMC neurons, it reduces Ca2+ currents and activates a nonselective cationic current. This implies that different signaling pathways exist in these two cell types. The anorectic effects of GLP-1 cannot be caused by the enhanced activity of orexin/hypocretin neurons, however, because central administration of orexin/hypocretin leads to hyperphagia (Jobst et al., 2004). Thus, GLP-1 must inhibit food intake via other mechanisms. Our results suggest that this may, at least in part, involve stimulation of POMC neurons, which is well known to have an appetite-suppressing action (Schwartz et al., 2000; Jobst et al., 2004). Mice in which POMC neurons have been specifically ablated would help to further evaluate the physiological relevance of POMC neurons in mediating the anorectic effect of GLP-1.
Finally, we note that the arcuate nucleus lies close to the median eminence, one of the circumventricular organs within the brain in which the blood–brain barrier is lacking (Merchenthaler, 1991). Thus, POMC cells may respond to blood-borne GLP-1 as well as that released from presynaptic inputs. This may be of clinical relevance, given that extendin-4 is now used to treat type-2 diabetes.
This work was supported by a Wellcome Trust Integrative Physiology Initiative in Metabolic Disease. X.M. is a Wellcome Training fellow. F.M.A. is a Royal Society Research Professor. We thank Lejla Zubcevic for technical assistance and Dr. Juris Galvanovskis for discussions.
- Correspondence should be addressed to Prof. Frances Ashcroft, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.