Conditional targeting strategy to create Sur1 flox mice.

<p>(A) Illustration of the targeting construct and possible recombination event to produce founder mice carrying the neomycin resistance cassette. (B) Examples of PCR products from a total of 10 mice are shown; WT (lane 8), homozygous Sur1^loxP/loxP (lanes 1,4 and 9) and heterozygous Sur1^loxP/+ (lanes 2,3,5,6,7 and 10). The arrows show the position of 500 and 1000 base-pair markers.</p

Frequency of recombination in Cre mouse strains.

<p>epi  =  epinephrine; male mice 12-15 weeks old.</p

Analysis of Sur1^loxP/loxP;GCG-cre⁺

<p>islet cells. (A) α-Cells in 2.8 mM glucose were pulsed for five minutes with arginine (1 mM) or arginine plus diazoxide (100 μM) as indicated. In this experiment diazoxide had no effect in four cells (gray traces), while in two cells (black traces) the channel agonist blocked stimulation by arginine. All six cells were stimulated by epinephrine (5 μM). (B) In a separate experiment β-cells were stimulated with 16.7 mM glucose and pulsed for five minutes with diazoxide (100 μM). The individual traces show [Ca²⁺]_c was reduced to baseline values in all of the β-cells. The upper trace, offset by 0.3 units, shows the mean ± SEM values (n = 20).</p

Effect of diazoxide on arginine stimulation of α-cells in WT versus Sur1^−/− α-cells.

<p>The cells were in 2.8± SEM, respectively, for the indicated number of cells. The experiments were repeated four times with similar results using different islet cell preparations obtained from 1 or 2 mice. The pulse lengths are 5 minutes.</p

Stimulation of isolated pancreatic islet cells by arginine.

<p>(A) α- and β-cells were distinguished by their response to an epinephrine pulse in 2.8 mM glucose. (B) Arginine stimulation was blocked by nifedipine (10 μM). The solid traces and shaded areas are the means ± SEM, respectively, for the indicated number of cells. These experiments were repeated four times with similar results using different islet preparations derived from 1 or 2 mice. (C and D) Responses of WT and Sur1^−/− α- and β-cells to increasing concentrations of arginine at three concentrations of glucose. Each trace is an average of Ca²⁺ values from 4–10 cells; the experiments were repeated 4 times with similar results using different islet preparations. Islet preparations were from 1 or 2 mice. The pulse lengths are 5 minutes. The 5.6 and 16.7 mM glucose α-cell traces are offset 0.1 and 0.2 units respectively, for clarity.</p

Mouse body weight and blood glucose values.

<p>Mean ± SEM, n = 6∼10.</p

Direct versus indirect actions of ghrelin on hypothalamic NPY neurons

<div><p>Objectives</p><p>Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons.</p><p>Materials and methods</p><p>Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca²⁺ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus.</p><p>Results</p><p>Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca²⁺]_c, with an EC₅₀ ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP₃) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na⁺]_o suppressed activation, suggesting a role for Na⁺-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons <i>in situ</i>. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca²⁺]_c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca²⁺ channels, respectively, while Ni²⁺, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca²⁺ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca²⁺ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action <i>in vivo</i>.</p><p>Conclusions</p><p>NPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons <i>in situ</i>. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na⁺ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca²⁺ channels, which could contribute to NPY neuronal activity <i>in situ</i>.</p></div

NYP-expressing neurons of the arcuate nucleus express subunits of both SUR1-Kir6.2 (K_ATP) and SUR1-Trpm4 channels.

<p>Coronal sections of the hypothalamus from an NPY-GFP mouse, showing NPY-GFP neurons (green) immunolabeled for SUR1(A), or Kir6.2 (B) or Trpm4 (C), as indicated (red); merged images show co-expression of GFP and all three channel subunits by NPY neurons (yellow) DAPI nuclear labeling shown in blue. The images (low power view, <i>upper panel</i>s, and high power view, <i>lower panels</i>) shown are representative of findings in 2 NPY-GFP mice.</p

Ghrelin directly activates NPY-GFP neurons.

<p>(A) Representative field of isolated neurons from NPY-GFP mice. (B) Same field after addition of ghrelin. (C) Merged image of fields (A) and (B). Arrows point to non-NPY cells. (D) Average values (n = 4) of Relative [Ca²⁺]_c after sequential addition of indicated concentrations of acylated ghrelin. (E) Ghrelin dose-response curve. (F) Ghrelin induced [Ca²⁺]_c increases are blocked by the GHS-R antagonist [D-lys3]-GHRP-6 (50 μM). (G) CNQX (2 μM), an AMPA receptor antagonist, did not affect ghrelin action. Ghrelin (200 pM; red bars) was applied in F and G. Traces are mean values from 6–12 neurons.</p

Ghrelin stimulation of GHS-R in isolated NPY-GFP neurons activates both adenylate cyclase (AC)-PKA and PLC-IP₃ signaling pathways.

<p>(A) The adenylate cyclase agonist, Forskolin (10 μM), mimics the effect of ghrelin. (B) The PKA inhibitor, H89 (10 μM) inhibits the effect of ghrelin. (C) U73122 (1 μM), an irreversible PLC inhibitor, reduced ghrelin action. (D) Thapsigargin (0.5 μM), an inhibitor of SERCA, the ER Ca²⁺ pump, did not inhibit ghrelin induced [Ca²⁺]_c increases. Ghrelin was applied at 100 pM (red bars). Traces are mean values from 5–20 cells.</p