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CRH PVH DMH CCK LH α-msh (POMC) VMH ARC NPY AgRP Orexin MCH

Bar = 5 μm Neurosecretory protein GL (NPGL) 1. vs BBRC (214) 2. 1 3. 4. M C SP NPGL 8aa C GLGKR ( ) C Cys 2 NPGL mrna/actb mrna 8 7 6 5 4 3 2 1 GCE (217) MM IN

NPGL Gen. Comp. Endocrinol. (218 review).1 Human Rat, Mouse Chicken Western painted turtle Western clawed frog Coelacanth Spotted gar Fugu Medaka Elephant shark Lamprey Human MADPGNRGGIHRPLSFTCSLLIVGMCCVSPFFCHSQT--DLLALSQADPQCWESSSVLLL Rat MTDPGTRSRNHCPLSLTCLLLIVGMYYVSPVFCHSQT--DLLTLNQADPQCWESSSMLLL Mouse MADPGPRSRIHSSLSLACSLFIVGICYVSPVFCHSQT--DLLTLNQADPQCWESSSMLLL Chicken M-DFGNRGRIHYNMRLTYSLLVMGVFCVTPSLCHSQI--DPLALGRADPQCWESSSAVLL Western painted turtle M-DPGNRGRIHS-VRLICSLLIMGVFCVTPFFCHSQN--DPLALGRADPQCWEFSTAALL Western clawed frog M-SSRNKRREYYSMQLTCSLLFLGIFVIPAICCHGQV--DPLALGRADPQCWETSTAILL Takifugu M-APSLR------CLPAAMVLLLGCTCVSSLRDHKQVRVDPLSVPQANPHCWKSSSALLL Human EMWKPRVSNTVSGFWDFMIYLKSSENLKHGALFWDLAQLFWDIYVDCVLSRNHGLGRRQL Rat EMRKPRVSNTVSGFWDFMIYLKSSENLKHGALFWDLAQLFWDIYVDCVLSRNHGLGRREL Mouse EMRKPRVSNTVSGFWDFMIYLKSSENLKHGALFWDLAQLFWDIYVDCVLSRNHGLGRREL Chicken EMRKPRISDSVSGFWDFMIFLKSSENLKHGALFWDLAQLFWDIYVDCVLSRTHGLGKRQL Western painted turtle EMRKPRISDSVSGFWDFMIFLKSSENLKHGALFWDLAQLFWDIYVDCVLSRTHGLGRRQL Western clawed frog EMKKPRIADTVSGFWDLMIYLKSSENLKHGILFWDLAQLFWDIYVDCVLSRTHGLGRRQL Takifugu EMRVPRIADTVPAFWDLMVSLRSSDNSKHTALFWDLAQVFWDLYLDCVLSRSHGLGRRHV Human VGEEEKISAAQPQHTRSKQGTYSQLLRTSFLKKKELIEDLISMHVRRSGSSVIGKVN--L Rat AGEEQRVSKVLPRHLGIKQGAYSQLLRSIYLKKKELIGDLTSMQLQKWGPGFTGKEK--L Mouse TGEEEKVSKVLTRHSGIKQGAYSQLLRTLFLKKKELIEDLTSMQMQKRGSRFTGKKK--L Chicken AKAQQRITTLPSQFTGRNQGMFTHIQRSPVLTKKDFFEDLIKNHKHKSRSTLLGRITGEL Western painted turtle AEAEQKITALHSQLTEQKQGTFSHNQRTRVLNKKELIEDLS-IHVHKSGSALLRRVIGGL Western clawed frog NDGNKEITNLISHLTLK---TYSHGQRSILPQKKSLIEELIGIRVHKSESRLLGSIQR-- Takifugu TALHSLFTDSKSFKLPL------------------------------------------- Human EIKRK-- 181 Rat QIKRNRS 183 Mouse EVKRK-- 181 Chicken GKKRK-- 182 Western painted turtle GIKRKQ- 181 Western clawed frog GIKRK-- 177 Takifugu ------- 13

217 4 28 ScienceDaily EurekAlert! AlphaGalileo ScienceNewsline 2

NPGL ISH Endocrinology (217) Control IHC Control Bars = 1 μm

NPGL Rostral A B POMC neurons C B C Endocrinology (217) POMC NPGL DMH POMC NPGL VMH Arc D A D NPY/AgRP neurons E E Rostral Caudal NPY/AgRP NPGL Bars = 1 μm (A, D), 1 μm (B, C, E) POMC neurons NPGL neurons

NPGL Cumulative food intake (g) 4 3 2 1 P=.53 1 2 4 6 8 1 24 Time after injection (h) Endocrinology (217) n = 8,1 P<.5 P<.1 P<.5

NPGL NPGL SP NPGL 8aa GLGRR elife (217) Arcuate nucleus C C NH 2 Mediobasal hypothalamus (MBH) Relative mrna 12 1 8 6 4 2 1: NPGL 6 2: Relative mrna 4 2

NPGL Te VTM f PMD PMV ArcLP ArcMP Cell bodies elife (217) DMH LH VMH Arc Fibers ISH DMH IHC VMH Bars = 1 μm

NPGL Cumulative caloric intake (kcal) 5 4 3 2 1 AAV-CTL AAV-NPGL Body mass (g) 65 55 45 35 25 AAV-CTL AAV-NPGL elife (217) +7 +14 +21 +28 +35 +42 Days after surgery +7 +14 +21 +28 +35 +42 Days after surgery WAT mass/ Body mass (%) 6 4 2 AAV-CTL AAV-NPGL AAV-CTL AAV-NPGL Bars = 5 cm n=6-7, P<.5, P<.1, P<.5

NPGL elife (217) mrna Relative mrna 15 1 5 rwat AAV-CTL 15 AAV-NPGL 1 5 Liver AAV-CTL AAV-NPGL FAS α-tubulin AAV-CTL AAV-NPGL phsl Total HSL Relative Intensity 2 1 FAS 4 3 2 1 phsl/hsl 16:1/16:.5.4.3.2.1 18:1/18: 25 2 15 1 5 n=6-7, P<.5

NPGL elife (217) Calorie intake (kcal) 2 15 1 5 Light CTL NPGL 8 6 4 2 Dark 12 1 8 6 4 2 24 hr 1 3 n = 8, P<.5, P<.5

NPGL elife (217) Npgl mrna (STZ) 2.5 2 1.5 1.5 1.2 1.8.6.4.2 Vehicle NPGL Insulin NPGL pakt Bars = 1 μm n = 7-8, P<.5, P<.1

elife (217)

NPGL

vs

NPGL NPGM chicken NPGM MEFMWKR-R-WYLQ-LGCVLILNLVYA------NLDYLKEAPASLGQIDHQCWEVSSHGLVE 53 chicken NPGL MDFGN-RGRIHYNMRLTYSL---LVMGVFCVTPSLCHSQIDPLALGRADPQCWESSSAVLLE 58 chicken NPGM MKKLKVADPVIALWDFMLFLKESPKPKHNELFNDLAQNFWDMYVDCVLSRSHGMGRRQLVSP 115 chicken NPGL MRKPRISDSVSGFWDFMIFLKSSENLKHGALFWDLAQLFWDIYVDCVLSRTHGLGKRQLAKA 12 Sci. Rep. (218) chicken NPGM RYSSTYSHRTLQGSAFTNPF 135 chicken NPGL QQRITTLPSQFTGRNQGMFTHIQRSPVLTKKDFFEDLIKNHKHKSRSTLLGRITGELGKKRK 182

1. Shikano K, Bessho Y, Kato M, Iwakoshi-Ukena E, Taniuchi S, Furumitsu M, Tachibana T, Bentley GE, Kriegsfeld LJ, Ukena K. Localization and function of neurosecretory protein GM, a novel small secretory protein, in the chicken hypothalamus. Sci. Rep. 8:74 (218) 2. Shikano K, Taniuchi S, Iwakoshi-Ukena E, Furumitsu M, Bentley GE, Kriegsfeld LJ, Ukena K. Chronic subcutaneous infusion of neurosecretory protein GM increases body mass gain in chicks. Gen. Comp. Endocrinol. (218) in press. 3. Ukena K. Avian and murine neurosecretory protein GL participates in the regulation of feeding and energy metabolism (Review). Gen. Comp. Endocrinol. 26:164-17 (218) 4. Shikano K, Kato M, Iwakoshi-Ukena E, Furumitsu M, Matsuura D, Masuda K, Tachibana T, Bentley GE, Kriegsfeld LJ, Ukena K. Effects of intracerebroventricular infusion of neurosecretory protein GL on body mass and food and water intake in chicks. Gen. Comp. Endocrinol. 256:37-42 (218) 5. Iwakoshi-Ukena E, Shikano K, Kondo K, Taniuchi S, Furumitsu M, Ochi Y, Sasaki T, Okamoto S, Bentley GE, Kriegsfeld LJ, Minokoshi Y, Ukena K. Neurosecretory protein GL stimulates food intake, de novo lipogenesis, and onset of obesity. elife 6:e28527 (217) 6. Matsuura D, Shikano K, Saito T, Iwakoshi-Ukena E, Furumitsu M, Ochi Y, Sato M, Bentley GE, Kriegsfeld LJ, Ukena K. Neurosecretory protein GL, a hypothalamic small secretory protein, participates in energy homeostasis in male mice. Endocrinology 158:112-1129 (217) 7. Masuda K, Furumitsu M, Ooyama H, Iwakoshi-Ukena E, Ukena K. Synthesis of neurosecretory protein GM composed of 88 amino acid residues by native chemical ligation. Tetrahedron Lett. 57:84-87 (216) 8. Masuda K, Furumitsu M, Taniuchi S, Iwakoshi-Ukena E, Ukena K. Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells. FEBS Open Bio 5:844-851 (215) 9. Masuda K, Ooyama H, Shikano K, Kondo K, Furumitsu M, Iwakoshi-Ukena E, Ukena K. Microwave-assisted solid-phase peptide synthesis of neurosecretory protein GL composed of 8 amino acid residues. J. Pept. Sci. 21:454-46 (215) 1.Ukena K, Iwakoshi-Ukena E, Taniuchi S, Bessho Y, Maejima S, Masuda K, Shikano K, Kondo K, Furumitsu M, Tachibana T. Identification of a cdna encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum. Biochem. Biophys. Res. Commun. 446:298-33 (214)

H29 D3 M2 M1 B3 B3 B3 A, B,, Lance Kriegsfeld UC Berkeley George Bentley UC Berkeley