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6348 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol November 7, 2008 Volume 14 Number 41s<strong>in</strong>ce this peptide is implicated <strong>in</strong> regulation of sleep/wakefulness [15] . These rhythmic changes <strong>in</strong> <strong>ghrel<strong>in</strong></strong> <strong>and</strong><strong>orex<strong>in</strong></strong> levels may alter their <strong>in</strong>puts on the feed<strong>in</strong>g center<strong>and</strong> thereby regulate feed<strong>in</strong>g. Both <strong>ghrel<strong>in</strong></strong> [9,16-17] <strong>and</strong><strong>orex<strong>in</strong></strong> [18] stimulate food <strong>in</strong>take primarily by activat<strong>in</strong>gNPY neurons <strong>in</strong> the ARC.<strong>Lept<strong>in</strong></strong>, a powerful anorectic hormone produced <strong>and</strong>released from the adipocytes, is present constantly <strong>in</strong>the plasma at the nanomolar range [14] <strong>and</strong> is consideredto enter the bra<strong>in</strong> through the blood-bra<strong>in</strong> barrier [19] .Therefore, lept<strong>in</strong> is likely to act cont<strong>in</strong>uously on thefeed<strong>in</strong>g center <strong>and</strong> thereby modulate the efficacy oforexigenic substances. The primary action of lept<strong>in</strong><strong>in</strong> the feed<strong>in</strong>g center is <strong>in</strong>hibition of NPY neuronsas well as activation of POMC neurons <strong>in</strong> the ARC.The obesity syndrome <strong>in</strong> ob/ob mice result<strong>in</strong>g fromlack of functional lept<strong>in</strong> is attenuated by the lossof neuropeptide Y [20] . Therefore, elucidation of the<strong>in</strong>teraction of lept<strong>in</strong> with <strong>ghrel<strong>in</strong></strong> <strong>and</strong> with <strong>orex<strong>in</strong></strong><strong>in</strong> the ARC NPY neurons may provide a clue tounderst<strong>and</strong> the neuronal mechanisms for physiologicalregulation of feed<strong>in</strong>g. It has recently been shown thatlept<strong>in</strong> counteracts <strong>ghrel<strong>in</strong></strong> action to <strong>in</strong>crease cytosolicfree [Ca 2+ ]i <strong>in</strong> NPY neurons <strong>in</strong> the ARC, <strong>and</strong> that thePI3-k<strong>in</strong>ase (phosphatidyl<strong>in</strong>ositol 3-k<strong>in</strong>ase)-PDE3(phosphodiesterase 3) signal<strong>in</strong>g plays a key role <strong>in</strong> thelept<strong>in</strong> action [17] . In the present study, we isolated s<strong>in</strong>gleneurons from ARC of adult rats with matured feed<strong>in</strong>gfunction <strong>and</strong> monitored their activities by measur<strong>in</strong>g[Ca 2+ ]i. We studied the time course of lept<strong>in</strong> effects on<strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced vs <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong>NPY neurons.MATERIALS AND METHODSAnimals <strong>and</strong> preparation of s<strong>in</strong>gle neurons from theARC of adult ratsAdult male Sprague-Dawley (SD) rats were ma<strong>in</strong>ta<strong>in</strong>edon a 12-h light/dark cycle <strong>and</strong> given conventionalfood <strong>and</strong> water ad libitum. The ARC was isolated fromthe bra<strong>in</strong> of 5-8-wk-old SD rats <strong>and</strong> s<strong>in</strong>gle neuronswere prepared accord<strong>in</strong>g to the procedures reportedpreviously [16,21] with slight modifications. Briefly, ratswere anaesthetized with an <strong>in</strong>traperitoneal <strong>in</strong>jection ofcarbamic acid ethyl ester (900 mg/kg) <strong>and</strong> decapitated,<strong>and</strong> their bra<strong>in</strong>s were removed. Bra<strong>in</strong> slices conta<strong>in</strong><strong>in</strong>gthe ARC were prepared <strong>and</strong> the whole ARC of theleft <strong>and</strong> right sides was cut out. The dissected tissueswere washed with 10 mmol/L HEPES-bufferedKrebs-R<strong>in</strong>ger bicarbonate buffer (HKRB) conta<strong>in</strong><strong>in</strong>g10 mmol/L glucose. Then they were <strong>in</strong>cubated <strong>in</strong> HKRBsupplemented with 20 U/mL papa<strong>in</strong> (Sigma ChemicalCo., St. Louis, MO), 0.015 mg/mL deoxyribonuclease,0.75 mg/mL bov<strong>in</strong>e serum album<strong>in</strong> <strong>and</strong> 1mmol/Lcyste<strong>in</strong>e for 15 m<strong>in</strong> at 36℃ <strong>in</strong> a shak<strong>in</strong>g water bath,followed by gentle mechanical trituration for 5-10 m<strong>in</strong>.After trituration, the cell suspension was centrifuged at100 × g for 5 m<strong>in</strong>. The pellet was resuspended <strong>in</strong> HKRB<strong>and</strong> distributed on coverslips. The cells were kept at 20℃<strong>in</strong> moisture-saturated dishes for up to 10 h. The animalprotocols were approved by the Jichi Medical SchoolInstitute of Animal Care <strong>and</strong> Use Committee.Measurements of [Ca 2+ ]i <strong>in</strong> s<strong>in</strong>gle neurons of the ARCAt 2 to 10 h after cell preparation, [Ca 2+ ]i was measuredby ratiometric fura-2 microfluorometry <strong>in</strong> comb<strong>in</strong>ationwith digital imag<strong>in</strong>g as previously reported [16,21] . Briefly,follow<strong>in</strong>g <strong>in</strong>cubat<strong>in</strong>g with 2 mmol/L fura-2-AM for30 m<strong>in</strong> at room temperature, the cells were mounted <strong>in</strong>a chamber <strong>and</strong> superfused with HKRB at 1 mL/m<strong>in</strong> at34℃. Fluorescence images due to excitation at 340 nm<strong>and</strong> 380 nm were detected every 8.0 s with an <strong>in</strong>tensifiedcharge-coupled device (ICCD) camera, <strong>and</strong> the ratioimage was produced by an Argus-50 system (HamamatsuPhotonics Co., Hamamatsu, Japan). Ratio values wereconverted to [Ca 2+ ]i accord<strong>in</strong>g to calibration curves. Datawere taken from the cells identified as neurons by theprocedures reported previously [16,21] .Post-[Ca 2+ ]i imag<strong>in</strong>g immunocytochemistry <strong>and</strong>identification of NPY neuronsNeurochemical identification of the neurons thatexhibited [Ca 2+ ]i responses were performed accord<strong>in</strong>gto the orig<strong>in</strong>al method [22] with slight modification [16] .Briefly, the cells were fixed with 4% paraformaldehydeover night. They were blocked <strong>in</strong> 10% normal goatser um (NGS) <strong>and</strong> <strong>in</strong> 0.1 mol/L PBS for 1 h atroom temperature. Primary antiserum aga<strong>in</strong>st NPY(DiaSor<strong>in</strong>, Stillwater, MN) was diluted 1:1000 <strong>in</strong> PBSconta<strong>in</strong><strong>in</strong>g 1.5% NGS <strong>and</strong> was <strong>in</strong>cubated 24 h at 4℃.The antiserum were then r<strong>in</strong>sed <strong>and</strong> <strong>in</strong>cubated withbiot<strong>in</strong>ylated secondary antibody raised aga<strong>in</strong>st rabbitIgG (Vector Laboratories Inc., Burl<strong>in</strong>game, CA; dilutedat 1:400) for 1 h at room temperature. The secondaryantibody was r<strong>in</strong>sed, <strong>and</strong> the sections were labeled withavid<strong>in</strong>-peroxidase complex (ABC kit, Vector) for 1 h<strong>and</strong> color-developed with 3,3'-diam<strong>in</strong>obenzid<strong>in</strong>e (DAB).Control experiments were carried out by omitt<strong>in</strong>g theprimary antiserum.To correlate [Ca 2+ ]i <strong>and</strong> immunocytochemical data,photographs of all the cells <strong>in</strong> the microscopic fieldsubjected to [Ca 2+ ]i measurements were taken at the endof [Ca 2+ ]i imag<strong>in</strong>g. Based on these photographs, the cells<strong>in</strong> which [Ca 2+ ]i was recorded were correlated with theircorrespond<strong>in</strong>g immunocytochemical results.Criteria for [Ca 2+ ]i responses <strong>and</strong> determ<strong>in</strong>ation ofresponse amplitudeGhrel<strong>in</strong>, <strong>orex<strong>in</strong></strong>-A <strong>and</strong> lept<strong>in</strong> were adm<strong>in</strong>istered to thesuperfusion solution. Amplitudes of [Ca 2+ ]i <strong>in</strong>creases<strong>in</strong> response to agents were calculated by subtract<strong>in</strong>gpre-stimulatory basal [Ca 2+ ]i levels from peak [Ca 2+ ]ilevels. When <strong>in</strong>creases <strong>in</strong> [Ca 2+ ]i took place with<strong>in</strong>5 m<strong>in</strong> after addition of agents <strong>and</strong> their amplitudes were150 nmol/L or larger, they were considered responses.Suppression by lept<strong>in</strong> was judged by the follow<strong>in</strong>g criteria.In Figures 1 <strong>and</strong> 2, when the peak amplitude of <strong>ghrel<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>crease was decreased to a level of 40%www.wjgnet.com


Kohno D et al . <strong>Lept<strong>in</strong></strong> <strong>antagonizes</strong> <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong> <strong>in</strong> NPY neurons 6349AGhrel<strong>in</strong> 10 -10 mol/LBOrex<strong>in</strong> 10 -10 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L800800[Ca 2+ ]i (nmol/L)600400200←10 mm[Ca 2+ ]i (nmol/L)60040020000 20 40 60t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>COrex<strong>in</strong> 10 -10 mol/LDOrex<strong>in</strong> 10 -10 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L<strong>Lept<strong>in</strong></strong> 10 -14 mol/L800600[Ca 2+ ]i (nmol/L)600400200←10 mm[Ca 2+ ]i (nmol/L)40020000 20 40 60t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>ENeurons <strong>in</strong>hibited (%)8060402009/202-712/204/2010-15Time after treatmentwith 10 -12 mol/L lept<strong>in</strong> (m<strong>in</strong>)Ghrel<strong>in</strong>Orex<strong>in</strong>10/20FNeurons with transient or <strong>long</strong>-last<strong>in</strong>g<strong>in</strong>hibition (%)4020040200Transient4/135/201/62/122/200/8 0/61/8Long-last<strong>in</strong>g10/201/64/122/134/200/8 0/61/818 16 14 12Ghrel<strong>in</strong>Orex<strong>in</strong>-log [<strong>Lept<strong>in</strong></strong>] (mol/L)Figure 1 Ghrel<strong>in</strong>- <strong>and</strong> <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases were suppressed by lept<strong>in</strong> <strong>in</strong> a transient or <strong>long</strong>-last<strong>in</strong>g manner <strong>in</strong> NPY neurons. A-D: Ghrel<strong>in</strong> <strong>and</strong><strong>orex<strong>in</strong></strong>-A at 10 -10 mol/L <strong>in</strong>creased [Ca 2+ ]i <strong>in</strong> s<strong>in</strong>gle neurons isolated from the ARC, <strong>and</strong> these [Ca 2+ ]i <strong>in</strong>creases were suppressed by adm<strong>in</strong>istration of lept<strong>in</strong> at 10 -12 mol/L(A, C) <strong>and</strong> 10 -14 mol/L (D). Follow<strong>in</strong>g [Ca 2+ ]i measurements, the neurons <strong>in</strong> (A) <strong>and</strong> (C) were proved to conta<strong>in</strong> NPY by immunocytochemistry us<strong>in</strong>g anti-NPY antibody (A,C; right panels). The bars <strong>in</strong>dicate 10 mm. E: <strong>Lept<strong>in</strong></strong> <strong>in</strong>hibited <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> a greater number of neurons <strong>in</strong> 2-7 m<strong>in</strong> after adm<strong>in</strong>istration than <strong>in</strong>10-15 m<strong>in</strong>. In contrast, lept<strong>in</strong> <strong>in</strong>hibited <strong>orex<strong>in</strong></strong>-A-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> the majority of neurons <strong>in</strong> 2-7 m<strong>in</strong> <strong>and</strong> 10-15 m<strong>in</strong> after adm<strong>in</strong>istration. The numbers abovethe bars <strong>in</strong>dicate the number of neurons <strong>in</strong>hibited by lept<strong>in</strong> over that responded to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A. F: The number of neurons whose [Ca 2+ ]i responses to <strong>ghrel<strong>in</strong></strong>or <strong>orex<strong>in</strong></strong>-A were suppressed by lept<strong>in</strong> <strong>in</strong> either a transient or <strong>long</strong>-last<strong>in</strong>g manner is expressed by percentage. The numbers above the po<strong>in</strong>ts <strong>in</strong>dicate the number ofneurons <strong>in</strong>hibited by lept<strong>in</strong> <strong>in</strong> the specified manner over that responded to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A.or smaller for at least 5 m<strong>in</strong> <strong>and</strong> the recovery of [Ca 2+ ]i<strong>in</strong>crease was observed after wash<strong>in</strong>g out lept<strong>in</strong>, it wasconsidered <strong>in</strong>hibition. In Figure 3, repetitive additionsof <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A twice <strong>in</strong>duced repeated [Ca 2+ ]i<strong>in</strong>creases, <strong>and</strong> the second challenge to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-Awas performed <strong>in</strong> the presence of lept<strong>in</strong>. When theamplitude of the [Ca 2+ ]i response to the second additionwas less than 150 nmol/L, it was considered <strong>in</strong>hibition.Solutions <strong>and</strong> chemicalsThe measurements were carried out <strong>in</strong> HKRB solutioncomposed of 129 mmol/L NaCl, 5.0 mmol/L NaHCO3,www.wjgnet.com


6350 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol November 7, 2008 Volume 14 Number 41A LY294002 50 mmol/L, 1 hGhrel<strong>in</strong> 10 -10 mol/LB LY294002 50 mmol/L, 1 hOrex<strong>in</strong> 10 -10 mol/LC Wortmann<strong>in</strong> 100 nmol/L, 1 hOrex<strong>in</strong> 10 -10 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L[Ca 2+ ]i (nmol/L)600400200[Ca 2+ ]i (nmol/L)600400200[Ca 2+ ]i (nmol/L)60040020000 20 40 60t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>DMilr<strong>in</strong>one at 10 mmol/L, 30 m<strong>in</strong>EMilr<strong>in</strong>one at 10 mmol/L, 30 m<strong>in</strong>Ghrel<strong>in</strong> 10 -10 mol/LOrex<strong>in</strong> 10 -10 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L600600[Ca 2+ ]i (nmol/L)400200[Ca 2+ ]i (nmol/L)40020000 20 40 60t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>F U0126 10 mmol/L, 30 m<strong>in</strong> G STAT3 <strong>in</strong>hibitor peptide 500 mmol/L, 30 m<strong>in</strong>Orex<strong>in</strong> 10 -10 mol/LOrex<strong>in</strong> 10 -10 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L<strong>Lept<strong>in</strong></strong> 10 -12 mol/L600600[Ca 2+ ]i (nmol/L)400200[Ca 2+ ]i (nmol/L)40020000 20 40 60t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>HNeurons <strong>in</strong>hibitedby lept<strong>in</strong> (%)6040200Ghrel<strong>in</strong>12/20 7/12 6/10 6/10Orex<strong>in</strong>809/205/10 5/104/960Control2/12LY2940021/111/10Wortmann<strong>in</strong>Milr<strong>in</strong>one5/15U0126STAT3 <strong>in</strong>hibitorIAmplitude<strong>in</strong>hibition (%)402002020Control12a12LY29400211b1010a10Wortmann<strong>in</strong>Milr<strong>in</strong>oneGhrel<strong>in</strong>Orex<strong>in</strong>109 1015U0126STAT3 <strong>in</strong>hibitorFigure 2 <strong>Lept<strong>in</strong></strong> suppresses <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced <strong>and</strong> <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases via different signal<strong>in</strong>g pathways <strong>in</strong> NPY neurons. A-G: Effects of <strong>in</strong>hibitorsfor signal<strong>in</strong>g molecules on the lept<strong>in</strong> action aga<strong>in</strong>st <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong>. Pre<strong>in</strong>cubation for 1 h with 50 mmol/L LY294002, an <strong>in</strong>hibitor for PI3-k<strong>in</strong>ase, <strong>in</strong>terfered with lept<strong>in</strong>action to suppress [Ca 2+ ]i responses to <strong>ghrel<strong>in</strong></strong> (A), but not <strong>orex<strong>in</strong></strong> (B). Pre<strong>in</strong>cubation for 1 h with 100 nmol/L wortmann<strong>in</strong>, another PI3-k<strong>in</strong>ase <strong>in</strong>hibitor, little affected thelept<strong>in</strong> suppression of <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases (C). Pre<strong>in</strong>cubation for 30 m<strong>in</strong> with 10 mmol/L milr<strong>in</strong>one, a PDE3 <strong>in</strong>hibitor, blocked the lept<strong>in</strong> action to suppress[Ca 2+ ]i responses to <strong>ghrel<strong>in</strong></strong> (D), but not <strong>orex<strong>in</strong></strong> (E). Pre<strong>in</strong>cubation for 30 m<strong>in</strong> with 10 mmol/L U0126, an <strong>in</strong>hibitor for MAP-k<strong>in</strong>ase, little affected the lept<strong>in</strong> action tosuppress <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases (F). Pre<strong>in</strong>cubation for 30 m<strong>in</strong> with a STAT3 <strong>in</strong>hibitor peptide at 500 mmol/L, did not significantly alter the lept<strong>in</strong> suppressionof <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases (G). H: The number of neurons whose [Ca 2+ ]i responses to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong> were suppressed by lept<strong>in</strong> <strong>in</strong> the presence of <strong>in</strong>hibitorsis expressed by percentage. The numbers above the bars <strong>in</strong>dicate the number of neurons <strong>in</strong>hibited by lept<strong>in</strong> over that responded to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A. I: Reduction ofamplitudes of [Ca 2+ ]i responses to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A by lept<strong>in</strong> is expressed by percentage. a P < 0.05, b P < 0.01 vs control. The data regard<strong>in</strong>g <strong>ghrel<strong>in</strong></strong> <strong>in</strong> H <strong>and</strong> I were<strong>in</strong> part taken from our previous paper [17] .www.wjgnet.com


Kohno D et al . <strong>Lept<strong>in</strong></strong> <strong>antagonizes</strong> <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong> <strong>in</strong> NPY neurons 6351A[Ca 2+ ]i (nmol/L)C8006004002000Ghrel<strong>in</strong>Ghrel<strong>in</strong> 10 -10 mol/L0 20 40t /m<strong>in</strong>Orex<strong>in</strong> Orex<strong>in</strong> 10 -10 mol/LB[Ca 2+ ]i (nmol/L)D8006004002000<strong>Lept<strong>in</strong></strong> 10 -12 mol/LGhrel<strong>in</strong> Ghrel<strong>in</strong> 10 -10 mol/L0 20 40t /m<strong>in</strong><strong>Lept<strong>in</strong></strong> 10 -12 mol/LOrex<strong>in</strong>Orex<strong>in</strong> 10 -10 mol/LFigure 3 Prior treatment with lept<strong>in</strong>s u p p r e s s e d [ C a 2 + ] i r e s p o n s e s t osubsequent adm<strong>in</strong>istration of <strong>orex<strong>in</strong></strong>,but not <strong>ghrel<strong>in</strong></strong>, <strong>in</strong> NPY neurons. A, C:Repeated adm<strong>in</strong>istrations of <strong>ghrel<strong>in</strong></strong> or<strong>orex<strong>in</strong></strong>-A at 10 -10 mol/L <strong>in</strong>creased [Ca 2+ ]itwice <strong>in</strong> s<strong>in</strong>gle ARC neurons; B, D: Prioradm<strong>in</strong>istration of lept<strong>in</strong> failed to <strong>in</strong>hibitthe effect of <strong>ghrel<strong>in</strong></strong> added subsequently(B), but <strong>in</strong>hibited the effect of <strong>orex<strong>in</strong></strong>-A(D); E: The number of neurons <strong>in</strong> which[Ca 2+ ]i responses to the second <strong>ghrel<strong>in</strong></strong> or<strong>orex<strong>in</strong></strong> addition were suppressed by prioradm<strong>in</strong>istration of lept<strong>in</strong> or HKRB (Control)is expressed by percentage. The numbersabove the bars <strong>in</strong>dicate the numberof neurons whose second responseswere <strong>in</strong>hibited over that exhibited firstresponses to <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A.800800[Ca 2+ ]i (nmol/L)600400200[Ca 2+ ]i (nmol/L)60040020000 20 40t /m<strong>in</strong>00 20 40 60t /m<strong>in</strong>ENeurons <strong>in</strong>hibited (%)6040201/9Ghrel<strong>in</strong>Orex<strong>in</strong>1/173/148/160Control<strong>Lept<strong>in</strong></strong> 10 -12 mol/L4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.8 mmol/LCaCl2, 1.2 mmol/L MgSO4, <strong>and</strong> 10 mmol/L N-2-hydroxyethylpiperaz<strong>in</strong>e-N'-2-ethanesulfonic acid (HEPES)at pH 7.4. Fura 2-acetoxymethylester was obta<strong>in</strong>edfrom Doj<strong>in</strong> Chemical (Kumamoto, Japan). Ghrel<strong>in</strong><strong>and</strong> <strong>orex<strong>in</strong></strong>-A were obta<strong>in</strong>ed from Peptide Institute,Inc. (Osaka, Japan), <strong>Lept<strong>in</strong></strong> was from R&D Systems(M<strong>in</strong>eapolis, MN).Data presentation <strong>and</strong> statistical analysisThe data are presented as the mean ± SE (n: number ofneurons). Each study was based on at least 7 neuronsprepared from at least 3 rats. Student’s paired or unpairedt-test was used to evaluate differences <strong>and</strong> values of P


6352 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol November 7, 2008 Volume 14 Number 41<strong>Lept<strong>in</strong></strong> <strong>in</strong>hibits <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases<strong>transiently</strong> <strong>and</strong> <strong>orex<strong>in</strong></strong>-A-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>long</strong><strong>last<strong>in</strong>gly</strong><strong>in</strong> NPY neuronsTypical results of the effects of lept<strong>in</strong> on [Ca 2+ ]iresponses to <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong>-A are shown <strong>in</strong> Figure 1;lept<strong>in</strong> at 10 -12 mol/L <strong>in</strong>hibited <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i<strong>in</strong>creases <strong>in</strong> a transient manner (Figure 1A) <strong>and</strong> <strong>orex<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> a <strong>long</strong>er-last<strong>in</strong>g manner (Figure1C) dur<strong>in</strong>g the 20 m<strong>in</strong> period of lept<strong>in</strong> adm<strong>in</strong>istration.Among 20 neurons that exhibited [Ca 2+ ]i responses to<strong>ghrel<strong>in</strong></strong>, adm<strong>in</strong>istration of 10 -12 mol/L lept<strong>in</strong> <strong>in</strong>hibited[Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> 9 neurons (45%) dur<strong>in</strong>g the earlier2-7 m<strong>in</strong> of lept<strong>in</strong> treatment, but only <strong>in</strong> 4 neurons(20%) later <strong>in</strong> the 10-15 m<strong>in</strong> period of treatment(Figure 1E), show<strong>in</strong>g attenuation of the counteract<strong>in</strong>geffect of lept<strong>in</strong> for <strong>ghrel<strong>in</strong></strong> <strong>in</strong> the later period (Figure 1F).In contrast, among 20 neurons that exhibited [Ca 2+ ]iresponses to <strong>orex<strong>in</strong></strong>, adm<strong>in</strong>istration of 10 -12 mol/L lept<strong>in</strong><strong>in</strong>hibited [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> 12 neurons (60%) dur<strong>in</strong>g the2-7 m<strong>in</strong> of lept<strong>in</strong> treatment, <strong>and</strong> <strong>in</strong> 10 neurons (50%) <strong>in</strong>the 10-15 m<strong>in</strong> period of treatment (Figure 1E), show<strong>in</strong>ga <strong>long</strong>-last<strong>in</strong>g counteract<strong>in</strong>g effect of lept<strong>in</strong> (Figure 1F).The <strong>long</strong>-last<strong>in</strong>g effect was also evoked by lept<strong>in</strong> at alower concentration of 10 -14 mol/L (Figure 1D). <strong>Lept<strong>in</strong></strong>at both 10 -14 mol/L <strong>and</strong> 10 -12 mol/L counteracted <strong>ghrel<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases predom<strong>in</strong>antly <strong>in</strong> a transientmanner <strong>and</strong> <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases ma<strong>in</strong>ly <strong>in</strong> a<strong>long</strong>-last<strong>in</strong>g manner (Figure 1F).Pretreatment with lept<strong>in</strong> <strong>in</strong>hibited <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced, butnot <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced, [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> NPY neuronsThe data that lept<strong>in</strong> <strong>in</strong>hibited <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i<strong>in</strong>creases <strong>transiently</strong> prompted us to hypothesize thatefficacy of lept<strong>in</strong> is attenuated by time. Therefore, weexam<strong>in</strong>ed whether prior adm<strong>in</strong>istration of lept<strong>in</strong> is lesseffective <strong>in</strong> counteract<strong>in</strong>g <strong>ghrel<strong>in</strong></strong> action. Repetitiveadditions of <strong>ghrel<strong>in</strong></strong> or <strong>orex<strong>in</strong></strong>-A twice <strong>in</strong>ducedrepeated [Ca 2+ ]i <strong>in</strong>creases twice <strong>in</strong> a similar manner(Figure 3A <strong>and</strong> C). Follow<strong>in</strong>g <strong>in</strong>fusion of lept<strong>in</strong> thathad started 8 m<strong>in</strong> <strong>in</strong> advance, the addition of <strong>ghrel<strong>in</strong></strong><strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases with amplitudes comparable tothose <strong>in</strong> the control without lept<strong>in</strong> (Figure 3B), <strong>and</strong> thesimilar result was observed <strong>in</strong> the majority of neurons(Figure 3E). This result <strong>in</strong>dicates a marked attenuationof <strong>in</strong>hibitory ability of lept<strong>in</strong> by time. In contrast,<strong>in</strong>fusion of lept<strong>in</strong> that started 8 m<strong>in</strong> <strong>in</strong> advance <strong>in</strong>hibited[Ca 2+ ]i responses to the addition of <strong>orex<strong>in</strong></strong>-A <strong>in</strong> 8 of 16<strong>orex<strong>in</strong></strong>-responsive neurons (50%) (Figure 3D <strong>and</strong> E).This <strong>in</strong>cidence of the <strong>in</strong>hibition by lept<strong>in</strong> adm<strong>in</strong>istered<strong>in</strong> prior to <strong>orex<strong>in</strong></strong> was comparable to the <strong>in</strong>hibition bylept<strong>in</strong> adm<strong>in</strong>istered after <strong>orex<strong>in</strong></strong> (12 of 20 neurons, 60%)(Figure 1E). These data <strong>in</strong>dicate that the ability of lept<strong>in</strong>to counteract <strong>orex<strong>in</strong></strong> action is well preserved withoutappreciable attenuation.We next exam<strong>in</strong>ed whether the difference <strong>in</strong> thetime dependence of lept<strong>in</strong> action on <strong>ghrel<strong>in</strong></strong>-vs <strong>orex<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases could <strong>in</strong>volve different lept<strong>in</strong>signal<strong>in</strong>g mechanisms <strong>in</strong> NPY neurons. <strong>Lept<strong>in</strong></strong> isl<strong>in</strong>ked to several signal<strong>in</strong>g pathways, which <strong>in</strong>cludephosphatidyl<strong>in</strong>ositol 3 (PI3)-k<strong>in</strong>ase <strong>and</strong>, its downstreameffecter phosphodiesterase 3 (PDE3) [23] , signal transducer<strong>and</strong> activator of transcription 3 (STAT3) [24] , <strong>and</strong> mitogenactivatedprote<strong>in</strong> (MAP)-k<strong>in</strong>ase [25] . We have previouslyshown that lept<strong>in</strong> suppresses <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i<strong>in</strong>creases via PI3-k<strong>in</strong>ase- <strong>and</strong> PDE3-, but not MAP-k<strong>in</strong>ase<strong>and</strong>STAT3-, mediated pathway [17] . Therefore, whetherthese signal<strong>in</strong>g mechanisms could be <strong>in</strong>volved <strong>in</strong> the lept<strong>in</strong>action to counteract <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases wasexam<strong>in</strong>ed. Pretreatment with <strong>in</strong>hibitors for PI3-k<strong>in</strong>ase,LY294002 (Figure 2A) or wortmann<strong>in</strong> (data not shown),blocked the lept<strong>in</strong> action to suppress [Ca 2+ ]i responses to<strong>ghrel<strong>in</strong></strong> <strong>in</strong> both the response <strong>in</strong>cidence (Figure 2H) <strong>and</strong>response amplitude (Figure 2I). Likewise, pretreatmentwith an <strong>in</strong>hibitor for PDE3, milr<strong>in</strong>one, blocked the lept<strong>in</strong>action aga<strong>in</strong>st <strong>ghrel<strong>in</strong></strong> (Figure 2D, H <strong>and</strong> I). These resultsconfirm previous report [17] . In contrast, LY294002 (Figure2B), wortmann<strong>in</strong> (Figure 2C) <strong>and</strong> milr<strong>in</strong>one (Figure 2E)failed to significantly affect the lept<strong>in</strong> suppression of<strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> both the response<strong>in</strong>cidence <strong>and</strong> amplitude (Figure 2H <strong>and</strong> I). Furthermore,pretreatment with a MAP k<strong>in</strong>ase <strong>in</strong>hibitor U0126 (Figure2F) or a STAT3 <strong>in</strong>hibitor peptide (Figure 2G) little alteredthe lept<strong>in</strong> ability to <strong>in</strong>hibit [Ca 2+ ]i responses to <strong>orex<strong>in</strong></strong> <strong>in</strong>both the response <strong>in</strong>cidence <strong>and</strong> amplitude (Figure 2H<strong>and</strong> I), the results similar to those reported for <strong>ghrel<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases [17] .DISCUSSIONThe present data <strong>in</strong>dicate that lept<strong>in</strong> <strong>in</strong>hibits <strong>ghrel<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> a transient manner <strong>and</strong> <strong>orex<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> a <strong>long</strong>-last<strong>in</strong>g manner. Thetransient action of lept<strong>in</strong> to <strong>in</strong>hibit <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i<strong>in</strong>creases is not due to <strong>in</strong>sufficient concentration of lept<strong>in</strong>,s<strong>in</strong>ce lept<strong>in</strong> at a lower concentration of 10 -14 mol/L isalready maximal <strong>in</strong> counteract<strong>in</strong>g the <strong>ghrel<strong>in</strong></strong> effect [17] <strong>and</strong>more specifically <strong>in</strong> exhibit<strong>in</strong>g the transient <strong>in</strong>hibitoryproperty (Figure 1F). Furthermore, the transient propertyfor lept<strong>in</strong> <strong>in</strong>hibition of <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creasesis neither due to excessive concentration of <strong>ghrel<strong>in</strong></strong>,s<strong>in</strong>ce the <strong>ghrel<strong>in</strong></strong> concentration of 10 -10 mol/L used <strong>in</strong>the present study is close to a maximal, but never supermaximalconcentration <strong>in</strong> activat<strong>in</strong>g NPY neurons [16] .Therefore, the transient manner with which lept<strong>in</strong>counteracts <strong>ghrel<strong>in</strong></strong> action reflects the <strong>in</strong>tr<strong>in</strong>sic propertyof <strong>in</strong>teraction between lept<strong>in</strong> <strong>and</strong> <strong>ghrel<strong>in</strong></strong>.The present study clearly <strong>in</strong>dicated that the lept<strong>in</strong>signal<strong>in</strong>g underly<strong>in</strong>g the <strong>in</strong>hibition of [Ca 2+ ]i responsesto <strong>orex<strong>in</strong></strong>-A <strong>in</strong> NPY neurons is dist<strong>in</strong>ct from thatto <strong>ghrel<strong>in</strong></strong>. The transient action of lept<strong>in</strong> to <strong>in</strong>hibit<strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> NPY neurons maybe mediated by the lept<strong>in</strong> signal<strong>in</strong>g via PI3-k<strong>in</strong>ase <strong>and</strong>PDE3, s<strong>in</strong>ce the <strong>in</strong>hibitors for these enzymes block thelept<strong>in</strong> action (Figure 2A <strong>and</strong> D) [17] . The <strong>ghrel<strong>in</strong></strong> signal<strong>in</strong>gvia the cAMP system could be the target for this lept<strong>in</strong>signal<strong>in</strong>g [17] . On the other h<strong>and</strong>, the <strong>long</strong>-last<strong>in</strong>g actionof lept<strong>in</strong> to counteract <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creaseswww.wjgnet.com


Kohno D et al . <strong>Lept<strong>in</strong></strong> <strong>antagonizes</strong> <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong> <strong>in</strong> NPY neurons 6353was not affected by <strong>in</strong>hibitors for PI3-k<strong>in</strong>ase, PDE3,MAP-k<strong>in</strong>ase <strong>and</strong> STAT3, well known lept<strong>in</strong> signal<strong>in</strong>gmolecules. This result suggests that the <strong>long</strong>-last<strong>in</strong>gcounteract<strong>in</strong>g action of lept<strong>in</strong> for <strong>orex<strong>in</strong></strong> is mediatedby a yet unidentified lept<strong>in</strong> signal<strong>in</strong>g, which may <strong>long</strong><strong>last<strong>in</strong>gly</strong><strong>in</strong>hibit the <strong>orex<strong>in</strong></strong>-stimulated PLC-PKC-IP3pathway reported previously [18] , though further study isdef<strong>in</strong>itely needed to elucidate the signal<strong>in</strong>g <strong>in</strong>teractionbetween lept<strong>in</strong> <strong>and</strong> <strong>orex<strong>in</strong></strong>.The transient nature with which lept<strong>in</strong> counteractsthe <strong>ghrel<strong>in</strong></strong> action on NPY neurons may serve as theneuronal mechanism that allows fast<strong>in</strong>g-associated<strong>in</strong>creases <strong>in</strong> <strong>ghrel<strong>in</strong></strong> levels to activate the ARC NPYneurons <strong>in</strong> the cont<strong>in</strong>uous presence of lept<strong>in</strong> [14] <strong>and</strong>thereby stimulate feed<strong>in</strong>g.Both <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong> have been suggested to beconcerned with obesity <strong>and</strong> type 2 diabetes [26,27] . Basedon the present results, lept<strong>in</strong> resistance could alter thesensitivity to <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong> <strong>in</strong> the ARC NPY neurons,which may alter the energy metabolism <strong>and</strong> thereby<strong>in</strong>fluence the pathogenesis of obesity <strong>and</strong> type 2 diabetes.COMMENTSBackgroundGhrel<strong>in</strong> <strong>and</strong> <strong>orex<strong>in</strong></strong>s are potent orexigenic peptides work<strong>in</strong>g primarily via activat<strong>in</strong>gneuropeptide Y (NPY) neurons <strong>in</strong> the arcuate nucleus (ARC). NPY neurons<strong>in</strong> the ARC also serve as a major target for the anorexigenic lept<strong>in</strong>. Therefore,<strong>in</strong>teractions of lept<strong>in</strong> with <strong>ghrel<strong>in</strong></strong> <strong>and</strong> <strong>orex<strong>in</strong></strong> <strong>in</strong> the ARC NPY neurons may playa key role <strong>in</strong> physiological regulation of feed<strong>in</strong>g.Research frontiersThe authors study the time course of lept<strong>in</strong> effects on <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced vs <strong>orex<strong>in</strong></strong><strong>in</strong>duced[Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> NPY neurons.Innovations <strong>and</strong> breakthroughsThe study clearly <strong>in</strong>dicated that the lept<strong>in</strong> signal<strong>in</strong>g underly<strong>in</strong>g the <strong>in</strong>hibition of[Ca 2+ ]i responses to <strong>orex<strong>in</strong></strong>-A <strong>in</strong> NPY neurons is dist<strong>in</strong>ct from that to <strong>ghrel<strong>in</strong></strong>.The transient action of lept<strong>in</strong> to <strong>in</strong>hibit <strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> NPYneurons may be mediated by the lept<strong>in</strong> signal<strong>in</strong>g via PI3-k<strong>in</strong>ase <strong>and</strong> PDE3, The<strong>ghrel<strong>in</strong></strong> signal<strong>in</strong>g via the cAMP system could be the target for this lept<strong>in</strong> signal<strong>in</strong>g.ApplicationsThe transient nature with which lept<strong>in</strong> counteracts the <strong>ghrel<strong>in</strong></strong> action on NPYneurons may serve as the neuronal mechanism that allows fast<strong>in</strong>g-associated<strong>in</strong>creases <strong>in</strong> <strong>ghrel<strong>in</strong></strong> levels to activate the ARC NPY neurons <strong>in</strong> the cont<strong>in</strong>uouspresence of lept<strong>in</strong> [14] <strong>and</strong> thereby stimulate feed<strong>in</strong>g.Peer reviewA well designed, <strong>in</strong>-depth paper about the time course of lept<strong>in</strong> effects on<strong>ghrel<strong>in</strong></strong>-<strong>in</strong>duced vs <strong>orex<strong>in</strong></strong>-<strong>in</strong>duced [Ca 2+ ]i <strong>in</strong>creases <strong>in</strong> NPY neurons.REFERENCES1 Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Bask<strong>in</strong> DG.Central nervous system control of food <strong>in</strong>take. 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