Interplay between glucose and leptin signaling determines the strength of GABAergic synapses at POMC neurons

Regulation of GABAergic inhibitory inputs and alterations in POMC neuron activity by nutrients and adiposity signals regulate energy and glucose homeostasis. Thus, understanding how POMC neurons integrate these two signal molecules at the synaptic level is important. Here we show that leptin’s action on GABA release to POMC neurons is influenced by glucose levels. Leptin stimulates the JAK2-PI3K pathway in both presynaptic GABAergic terminals and postsynaptic POMC neurons. Inhibition of AMPK activity in presynaptic terminals decreases GABA release at 10 mM glucose. However, postsynaptic TRPC channel opening by the PI3K-PLC signaling pathway in POMC neurons enhances spontaneous GABA release via activation of presynaptic MC3/4 and mGlu receptors at 2.5 mM glucose. High-fat feeding blunts AMPK-dependent presynaptic inhibition, whereas PLC-mediated GABAergic feedback inhibition remains responsive to leptin. Our data indicate that the interplay between glucose and leptin signaling in glutamatergic POMC neurons is critical for determining the strength of inhibitory tone towards POMC neurons.


Supplementary Figure 2) Modulation of mIPSCs by glutamate and MTII
A) Representative recording samples showing mIPSCs recorded from POMC neurons after treatment with glutamate (100 M). HP = -70mV. Right panel: Summary of the effect of glutamate on mIPSC frequency in the presence of CNQX (10 M) and DL-APV (50 M) to block ligand-gated glutamate receptors. Glutamate significantly increased mIPSC frequency (139.2 ± 15.4 % of control, n = 10 neurons). Scale bar: 100 pA, 10 s. B) Representative recording samples showing mIPSCs recorded from POMC neurons after treatment with melanotan II (MTII; 100nM). HP = -70mV. Right panel: Summary of the effect of MTII on mIPSC frequency. There was a marked increase in the mean frequency of mIPSCs in POMC neurons (185.8 ± 21.8% of control, n = 8 neurons). *p < 0.05, **p < 0.05 vs. control (paired t-test). All data are shown as mean ± SEM. Scale bar: 100 pA, 10 s. C: control.

Supplementary Figure 3) Modulation by their own neurotransmitter and peptides of spontaneous GABAergic transmission onto POMC neurons
A) Images of fluorescence microscopy showing the expression of channelrhodopsin on POMC neurons (green) in the ARC of POMC-Cre::ChR2-YFP mice. Scale bar: 50 m. B) Representative recording samples showing sIPSCs recorded from POMC neurons before, during and after light stimulation. Blue light stimulation (blue line; 470 nm; 10 Hz for 1 s,10 ms pulses, 3 s interval,5 times) significantly increased sIPSC frequency. C) Pooled data of normalized sIPSC frequency from 8 POMC neurons from POMC-Cre::ChR2-YFP mice. The mean change in normalized sIPSC frequency was 148.6±11.2 % of control (n = 8 neurons). D) Summary of effect of light stimulation on sIPSC frequency. C: control, P: photostimulation. *p < 0.05, **p < 0.01 vs. control (paired t-test). All data are shown as mean ± SEM.

Supplementary Figure 4) Expression of pS6 and pSTAT3 in a subset of POMC neurons in the ARC following i.p. injection of leptin
A) Images of fluorescence microscopy showing the expression of glutamatergic POMC neurons in the ARC. Glutamatergic neurons were located adjacent to and/or within the median eminence. Scale bar: 50 m. B) Images of fluorescence microscopy showing the expression of pS6 (Red) in the hypothalamus following i.p. injection of saline or leptin (1 mg/ kg). pS6-positive cells in animals injected with leptin were observed in the ARC and the VMH of the hypothalamus, suggesting that pS6 staining would be a useful marker for leptin-mediated Jak2-PI3K signaling pathway. Scale bar: 100 m. C) Expression of pS6 (Red) in a subset of POMC-GFP neurons (Green) in the ARC. White arrows represent the neurons co-expressing GFP and pS6. Scale bar: 50 m. D) Expression of pSTAT3 (Red) in POMC neurons (Green) following i.p. injection of leptin. Leptin elevated pSTAT3 expression in POMC neurons. High-fat feeding reduced the expression of pSTAT3 in POMC neurons. White arrows represent the neurons coexpressing GFP and pSTAT3 (n = 5 animals, respectively). ***p < 0.001 (unpaired t-test). All data are shown as mean ± SEM. S: saline, L: leptin. Scale bar: 50 m.

Supplementary Figure 5) The K ATP channel blocker does not affect leptin's action on mIPSCs
A) Sample traces showing leptin's stimulatory (left) and inhibitory effects on mIPSCs. mIPSCs were recorded in the presence of tolbutamide (200 M) at 2.5 mM glucose. Scale bar: 100 pA, 10 s. B) Graph showing normalized frequency of mIPSCs from 13 POMC neurons before and after treatment with leptin in the presence of tolbutamide (n = 13 neurons). In contrast to the effect of the AMPK inhibitor that blunts glucose-sensing by POMC neurons, tolbutamide did not alter leptin's action on mIPSCs (p > 0.05, paired t-test). All data are shown as mean ± SEM. C: control, L: leptin Supplementary Figure 6) Dantrolene, an intracellular calcium release inhibitor, blocks leptin's effect A) Representative sample traces showing mIPSCs recorded from POMC neurons before and after application of leptin in the presence of dantrolene (10 M). B) Summary of the effects of leptin on mIPSCs in the presence of dantrolene at 2.5 and 5 mM glucose. Leptin failed to modulate GABA release in the presence of dantrolene (2.5 mM glucose: 96.5 ± 3.7 % of control, n = 4 neurons; 5 mM glucose: 105.9 ± 4.6 % of control, n = 7 neurons, p > 0.05, paired t-test). All data are represented as mean ± SEM. C: control.

Supplementary Table 1) Summary of leptin's effect on the membrane potential with or without the GABA A receptor inhibitor
In the absence of the GABA A receptor antagonist, a subset of POMC neurons responded to leptin with a hyperpolarization at 2.5 and 5 mM glucose. However, leptin's inhibitory effect on POMC neuron activity was completely abolished by the GABA A receptor antagonist. **P < 0.01, ***p < 0.001 vs. control (paired t-test); All data are represented as mean ± SEM.