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7 protocols using d lys3 ghrp 6

1

Rat Ghrelin and Norepinephrine Regulation

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Rat UCN1 and rat acylated ghrelin were purchased from Peptide Institute, Inc. (Osaka, Japan). Yohimbine hydrochloride, a selective α2‐AR antagonist, was purchased from Sigma‐Aldrich Chemical Co. (St. Louis, MO, USA). The GHS‐R1a antagonist, [D‐Lys3]‐GHRP‐6, was purchased from Bachem, Inc. (Torrance, CA, USA). These compounds were dissolved in saline when injected by IP and intravenous (IV) routes. Rikkunshito, a Japanese traditional Kampo medicine, was supplied from Tsumura & Co. in the form of a powdered extract obtained by spray‐drying a hot water extract mixture of the following eight crude drugs: Atractylodis lanceae rhizoma (4.0 g), Ginseng radix (4.0 g), Pinelliae tuber (4.0 g), Poria (4.0 g), Zizyphi fructus (2.0 g), Citri unshiu pericarpium (2.0 g), Glycyrrhizae radix (1.0 g), and Zingiberis rhizoma (0.5 g). Rikkunshito was dissolved in distilled water when injected by orogastric administration (PO). Other analytical reagents included commercially available highest‐purity products.
Treatments were performed on unanesthetized and lightly hand‐restrained rats using the following volumes: 10 μL/rat for ICV injection, 1 mL/kg for IP or IV injection through the tail vein, and 10 mL/kg for PO.
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2

Ghrelin and Rikkunshito Preparation

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Rat acylated ghrelin (Peptide Institute, Osaka, Japan) and ghrelin receptor antagonist, [D-Lys3]-GHRP-6 (Bachem, Bubendorf, Switzerland), were dissolved in sterilized physiological saline (Otsuka Pharmaceutical, Tokyo, Japan) before use. Rikkunshito (Tsumura & Co.), as described in the revised 16th edition of the Japanese Pharmacopoeia, was used as a powdered extract manufactured by spray drying of the hot-water extract of a mixture of eight varieties of crude drugs: Atractylodis lanceae rhizoma, Ginseng radix, Pinelliae tuber, Hoelen, Zizyphi fructus, Aurantii nobilis pericarpium, Glycyrrhizae radix, and Zingiberis rhizoma, and suspended in distilled water.
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3

Ghrelin Synthesis and Purification

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Ghrelin was synthesized by conventional solid phase synthesis and purified to at least 98% purity by HPLC by Neosystem (Strasburg, France). D-Lys3-GHRP-6 was purchased from Bachem AG (Dubendorf, Switzerland). H89 was from Sigma, Milan, Italy. Rat macrophage colony-stimulatory factor (M-CSF) and rat RANKL were from Peprotech, Rocky Hill, NJ.
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4

Cytoplasmic Calcium Regulation in Islet Cells

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Recordings of cytoplasmic calcium concentration ([Ca 21 ] c ) and measurements of ATP-sensitive K 1 (K ATP ) current and membrane potential (V m ) in the perforated-patch configuration were performed with a bath solution that contained (in mmol/L): 140 NaCl, 5 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , glucose as indicated, and 10 HEPES, at pH 7.4, adjusted with NaOH. Krebs-Ringer HEPES solution for insulin secretion was composed of (in mmol/L): 120 NaCl, 4. Islets and islet cell clusters were cultured in RPMI 1640 medium (11.1 mmol/L glucose) enriched with 10% FCS and 1% penicillin/streptomycin. MIN6 cells were cultured in DMEM (25 mmol/L glucose) containing 10% FCS and 1% penicillin/streptomycin. AG (human) was obtained from Biomol (Hamburg, Germany). UAG (human) and the ghrelin receptor inverse agonist K-(D1-NaI)-FwLL-NH 2 were purchased from Tocris Bioscience (Wiesbaden-Nordstadt, Germany); the ghrelin receptor antagonist [D-Lys 3 ]-GHRP-6 and the SSTR2-5 antagonist H6056 were from Bachem (Bubendorf, Switzerland). Fura-2 acetoxymethylester (Fura-2-AM) was ordered from Biotrend (Köln, Germany). RPMI 1640 medium, FCS, penicillin/ streptomycin, and trypsin were from Invitrogen (Karlsruhe, Germany). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany) in the purest form available.
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5

Ghrelin, DAG, and D-Lys3-GHRP-6 Synthesis

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Ghrelin and DAG were synthesized by conventional solid phase synthesis and purified to at least 98% purity by HPLC by Neosystem (Strasbourg, France). D-Lys3-GHRP-6 was purchased from Bachem AG (Budendorf, Switzerland). All compounds were dissolved in the culture medium, carefully preserving sterile conditions. E. Coli LPS (serotype 0111:B4) was purchased from Sigma (St Louis, CA, USA). The pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[Omethyl]fluoromethyl-ketone (Z-VAD-FMK, InvivoGen, Roma, Italy) was solved in dimethyl sulfoxide.
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6

Rat Ghrelin and NSC Expansion Media

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Rat ghrelin was obtained from Peptides International (Louisville, KT, USA). D-Lys-3-GHRP-6 was purchased from Bachem (Torrance, CA, USA). NSC expansion media, DMEM/F12, and B27 supplement were obtained from Gibco/Invitrogen. B-27 is an optimized serum substitute developed for low-density plating and long-term viability and growth of central nervous system (CNS) neurons (Brewer et al. 1993) . All tissue culture reagents were obtained from Gibco/Invitrogen, and all other reagents were obtained from Sigma unless otherwise indicated.
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7

Neural Stem Cell Expansion Protocol

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Rat AG, UAG and OB were obtained from Tocris (Ellisville, MO, USA). D-Lys-3-GHRP-6 was purchased from Bachem (Torrance, CA, USA). Neural stem cell expansion media, DMEM/F12 and B27 supplement were from Gibco/ Invitrogen. B-27 is an optimized serum substitute developed for low-density plating and long-term viability and growth of CNS neurons. AMPK inhibitor, compound C, was from Tocris and UCP2 inhibitor, genipin, was from Sigma-Aldrich. All tissue culture reagents were obtained from Gibco/Invitrogen, and all other reagents were obtained from Sigma-Aldrich unless otherwise indicated.
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