Leptin transiently antagonizes ghrelin and long-lastingly orexin in ...

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6350 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol November 7, 2008 Volume 14 Number 41A LY294002 50 mmol/L, 1 hGhrelin 10 -10 mol/LB LY294002 50 mmol/L, 1 hOrexin 10 -10 mol/LC Wortmannin 100 nmol/L, 1 hOrexin 10 -10 mol/LLeptin 10 -12 mol/LLeptin 10 -12 mol/LLeptin 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 /min00 20 40 60t /min00 20 40 60t /minDMilrinone at 10 mmol/L, 30 minEMilrinone at 10 mmol/L, 30 minGhrelin 10 -10 mol/LOrexin 10 -10 mol/LLeptin 10 -12 mol/LLeptin 10 -12 mol/L600600[Ca 2+ ]i (nmol/L)400200[Ca 2+ ]i (nmol/L)40020000 20 40 60t /min00 20 40 60t /minF U0126 10 mmol/L, 30 min G STAT3 inhibitor peptide 500 mmol/L, 30 minOrexin 10 -10 mol/LOrexin 10 -10 mol/LLeptin 10 -12 mol/LLeptin 10 -12 mol/L600600[Ca 2+ ]i (nmol/L)400200[Ca 2+ ]i (nmol/L)40020000 20 40 60t /min00 20 40 60t /minHNeurons inhibitedby leptin (%)6040200Ghrelin12/20 7/12 6/10 6/10Orexin809/205/10 5/104/960Control2/12LY2940021/111/10WortmanninMilrinone5/15U0126STAT3 inhibitorIAmplitudeinhibition (%)402002020Control12a12LY29400211b1010a10WortmanninMilrinoneGhrelinOrexin109 1015U0126STAT3 inhibitorFigure 2 Leptin suppresses ghrelin-induced and orexin-induced [Ca 2+ ]i increases via different signaling pathways in NPY neurons. A-G: Effects of inhibitorsfor signaling molecules on the leptin action against ghrelin and orexin. Preincubation for 1 h with 50 mmol/L LY294002, an inhibitor for PI3-kinase, interfered with leptinaction to suppress [Ca 2+ ]i responses to ghrelin (A), but not orexin (B). Preincubation for 1 h with 100 nmol/L wortmannin, another PI3-kinase inhibitor, little affected theleptin suppression of orexin-induced [Ca 2+ ]i increases (C). Preincubation for 30 min with 10 mmol/L milrinone, a PDE3 inhibitor, blocked the leptin action to suppress[Ca 2+ ]i responses to ghrelin (D), but not orexin (E). Preincubation for 30 min with 10 mmol/L U0126, an inhibitor for MAP-kinase, little affected the leptin action tosuppress orexin-induced [Ca 2+ ]i increases (F). Preincubation for 30 min with a STAT3 inhibitor peptide at 500 mmol/L, did not significantly alter the leptin suppressionof orexin-induced [Ca 2+ ]i increases (G). H: The number of neurons whose [Ca 2+ ]i responses to ghrelin or orexin were suppressed by leptin in the presence of inhibitorsis expressed by percentage. The numbers above the bars indicate the number of neurons inhibited by leptin over that responded to ghrelin or orexin-A. I: Reduction ofamplitudes of [Ca 2+ ]i responses to ghrelin or orexin-A by leptin is expressed by percentage. a P < 0.05, b P < 0.01 vs control. The data regarding ghrelin in H and I werein part taken from our previous paper [17] .www.wjgnet.com
Kohno D et al . Leptin antagonizes ghrelin and orexin in NPY neurons 6351A[Ca 2+ ]i (nmol/L)C8006004002000GhrelinGhrelin 10 -10 mol/L0 20 40t /minOrexin Orexin 10 -10 mol/LB[Ca 2+ ]i (nmol/L)D8006004002000Leptin 10 -12 mol/LGhrelin Ghrelin 10 -10 mol/L0 20 40t /minLeptin 10 -12 mol/LOrexinOrexin 10 -10 mol/LFigure 3 Prior treatment with leptins u p p r e s s e d [ C a 2 + ] i r e s p o n s e s t osubsequent administration of orexin,but not ghrelin, in NPY neurons. A, C:Repeated administrations of ghrelin ororexin-A at 10 -10 mol/L increased [Ca 2+ ]itwice in single ARC neurons; B, D: Prioradministration of leptin failed to inhibitthe effect of ghrelin added subsequently(B), but inhibited the effect of orexin-A(D); E: The number of neurons in which[Ca 2+ ]i responses to the second ghrelin ororexin addition were suppressed by prioradministration of leptin or HKRB (Control)is expressed by percentage. The numbersabove the bars indicate the numberof neurons whose second responseswere inhibited over that exhibited firstresponses to ghrelin or orexin-A.800800[Ca 2+ ]i (nmol/L)600400200[Ca 2+ ]i (nmol/L)60040020000 20 40t /min00 20 40 60t /minENeurons inhibited (%)6040201/9GhrelinOrexin1/173/148/160ControlLeptin 10 -12 mol/L4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.8 mmol/LCaCl2, 1.2 mmol/L MgSO4, and 10 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)at pH 7.4. Fura 2-acetoxymethylester was obtainedfrom Dojin Chemical (Kumamoto, Japan). Ghrelinand orexin-A were obtained from Peptide Institute,Inc. (Osaka, Japan), Leptin was from R&D Systems(Mineapolis, MN).Data presentation and 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 and values of P
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