Peptide YY(3-36) inhibits both anorexigenic proopiomelanocortin and orexigenic neuropeptide Y neurons: implications for hypothalamic regulation of energy homeostasis

Abstract

Peptide YY(3-36) (PYY(3-36)) is released by endocrine cells of the gut and may serve as an important long-distance neuropeptide signal relating energy balance information to the brain to depress food intake. The postulated mechanism is the activation of anorexigenic proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. In striking contrast, using voltage and current-clamp recording, we found that PYY(3-36) consistently, dose dependently, and reversibly inhibited POMC cells by reducing action potentials, hyperpolarizing the membrane potential, decreasing input resistance and inward calcium currents, increasing G-protein-gated inwardly rectifying K+ channel currents, and presynaptically inhibiting release of excitatory glutamate. Importantly, we found PYY(3-36) had similar inhibitory effects on identified orexigenic neuropeptide Y (NPY) neurons. In both cell types, these effects were blocked by BIIE0246, a Y2 receptor antagonist. Together, these data argue that anorexigenic actions of PYY(3-36) are mediated more likely by inhibition of NPY neurons. Dual PYY(3-36) inhibition of both NPY and POMC cells may temporarily reduce the contribution of arcuate cells to feeding circuits, enhancing the role of other CNS loci.

Figure 1.

PYY_3-36 inhibits GFP-expressing POMC neurons in the hypothalamus. A, Identification of hypothalamic POMC neurons in a slice from GFP-POMC transgenic mouse. Fluorescent (left, white) and IR-DIC (right) images of a POMC cell body (arrow) being approached by the recording pipette (arrowhead). B, When negative current steps (-50 to -100 pA for 50-200 ms; blue trace, bottom) were delivered to POMC neurons, low-threshold spikes (LTS) became evident (blue trace, top). Positive current steps (+20 to +30 pA; red trace, bottom) generated a burst of spikes. Spike-frequency adaptation was typically observed in POMC cells during extended current injection. C, Under current clamp, PYY_3-36 blocked spike frequency and hyperpolarized POMC-expressing neurons. D, Flow pipe application of the vehicle (ACSF) did not change the spike frequency or membrane potential in POMC neurons. E, In voltage clamp, PYY_3-36 induced a consistent outward current in these hypothalamic cells.

Figure 2.

The depressing actions of PYY_3-36 are consistent with a postsynaptic activation of Y₂ receptors in POMC neurons. A, PYY_3-36 reduced the spike frequency in a dose-dependent manner. B, The graph shows the time course of PYY_3-36 (open circles) and NPY_13-36 (filled circles) inhibitory actions on the frequency of action potentials in identified arcuate POMC cells. C, The actions of PYY_3-36 and NPY_13-36, two agonists of Y₂ receptors, on POMC neuron membrane potential were dose dependent. D, Both Y₁ (Pro₃₄NPY or d-Arg25NPY) and Y₂ (PYY_3-36 or NPY_13-36) receptor agonists hyperpolarized POMC neurons. BIBP3226, a selective Y₁ receptor antagonist, eliminated the Y₁ receptor-mediated hyperpolarization but did not affect Y₂ receptor-dependent changes in membrane potential.

Figure 3.

PYY_3-36 activates GIRK-like currents and depresses whole-cell calcium currents in POMC neurons. A, Voltage-ramp protocols (from -140 to -10 mV for 800 ms; top) revealed that PYY_3-36 activates an inwardly rectifying current reversing at approximately -60 mV in 15 mm external potassium. B, The PYY_3-36-induced current (gray trace) was attenuated by 800 μm barium (black) in the ACSF, consistent with an inwardly rectifying subtype. C, Voltage steps (see protocol at bottom) showing the effect of PYY_3-36 on the inward current response in a typical arcuate GFP-expressing neuron in the absence (left) and the presence (right) of 200 nm Tertiapin-Q. Without pretreatment with Tertiapin-Q, PYY_3-36 activated an inward current that, in addition, was blocked by 100 μm barium in the external solution. In the presence of Tertiapin-Q, PYY_3-36 failed to activate the inward current. These data suggest that PYY3-36 activates a GIRK-like current in GFP-containing cells. D, Bar graph summarizing the effect of high (800 μm) and low (100 μm) doses of external barium and Tertiapin-Q (200 nm) on the current induced by PYY_3-36 after voltage steps from -60 to -120 mV during 200 ms. E, PYY_3-36 also reduced POMC current responses to voltage ramps from -60 to +40 mV. F, Whole-cell calcium current evoked by voltage steps from -80 to 0 mV (top) before (left) and during (right) PYY_3-36 application to POMC cells. PYY_3-36 depressed voltage-dependent calcium currents in these hypothalamic neurons. G, Bar graph showing the mean depressing effect of PYY_3-36 on calcium current.

Figure 4.

PYY_3-36 and NPY_13-36, Y₂ receptor agonists, attenuated POMC glutamate-mediated synaptic activity by presynaptic mechanisms. A, In voltage clamp (-60 mV holding potentials), EPSCs were detected as fast inward currents in POMC neurons. EPSCs were completely abolished after NMDA and AMPA receptor antagonist application (using 50 μm AP-5 and 10 μm CNQX, respectively), confirming their glutamatergic nature (left). PYY_3-36 reduced EPSC frequency in POMC cells (right). B, The graph shows the time course of the inhibitory PYY_3-36 (open circles) and NPY_13-36 (filled circles) actions on the frequency of EPSCs in hypothalamic POMC neurons. Recovery was achieved after 10-20 min of peptide washout. C, Bar graph summarizing the depressing actions of PYY_3-36 (left), NPY_13-36 (middle), and NPY (right) on mEPSC frequency. D, Little change in mean amplitude of mEPSCs was observed after the application of PYY_3-36 (top trace), NPY_13-36 (middle traces), and NPY (bottom traces) as shown in these three typical hypothalamic POMC cells.

Figure 5.

Inhibitory actions of PYY_3-36 on identified arcuate NPY neurons. A, This representative recording shows the suppression in firing rate and membrane hyperpolarization of a GFP-expressing NPY neuron evoked by application of 100 nm PYY_3-36 to the slice. B, Time course graph presenting the mean inhibitory actions of PYY_3-36 (open circles) and NPY (filled circles) on NPY neurons. C, The bar graph compares the dose-dependent effects of PYY_3-36 in POMC neurons (open bars) and NPY neurons (filled bars). D, This graph shows the direct hyperpolarizing action of PYY_3-36 and NPY on GFP-expressing NPY neurons. memb. pot., Membrane potential. E, PYY_3-36 reduced excitatory synaptic transmission in NPY neurons. F, Bar graph presenting the mean reduction in the frequency of both EPSCs and mEPSCs evoked by PYY_3-36. G, H, Neither the cumulative distribution of mEPSC amplitude (G) nor the mean amplitude of miniature events (H) was altered by PYY_3-36 consistent with a presynaptic action on glutamate release.