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A metabolic role for the VGFderived peptides ANNA MOLES Institute of Neuroscience CNR- Rome, Italy Neurotrophins are proteins required for neuronal differentiation and survival. Pioneering work by Rita Levi-Montalcini, Stanley Cohen and Viktor Hamburger in the 1950s and early 1960s identified nerve growth factor (NGF) and demonstrated that this protein was required for sympathetic neuron survival both in vivo and in vitro. The later purification and characterization of brain-derived neurotrophic factor (BDNF), a close relative of NGF, allowed the cloning of additional family members. Six members of the neurotrophin family have been identified in vertebrates, namely NGF, BDNF, NT-3, NT-4/5, NT-6 and NT-7. These polypeptides bind, with different affinities, to the Trk family of receptor tyrosine kinases (TrkA, TrkB and TrkC) and with similar affinities to the p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor receptor Vgf gene discovered in PC12 cells following NGF treatment (Levi et al., Science 1985) Possenti et al PNAS, 1992 TISSUE SPECIFIC EXPRESSION OF VGF Salton et al. Front Neuroendocrinol, 2000 VGF a possible link between neurotrophines and energy homeostasis? Nature Neuroscience 6, 736 - 742 (2003) Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptor Baoji Xu1, 5, Evan H Goulding2, Keling Zang1, David Cepoi3, Roger D Cone3, Kevin R Jones4, Laurence H Tecott2 & Louis F Reichardt1 Resistant to several types of obesity VGF-gene acts downstream of MC4R in PVN, LH or brain stem nuclei, that project via the autonomic nervous system to peripheral metabolic tissues and regulate energy homeostasis Vgf encodes a 617 amino acid protein in rodents that, upon processing by the neuroendocrine-specific prohormone convertases (PC)1/3 and PC2, yields several peptides that are stored in dense core granules and secreted through the regulated pathway Ferri & Possenti, Trends Endocrinol Metab, 1996 VGF CONSTRUCTS Trombin cleaves at : …...LVPR GSPRTLQ….. TROMBIN GST VGF pep20 pep20-10 pep10 pep10-3 pep3 NAPPEPVPPPRAAPAPTHVRSPQPPPPAPARDELPDWNEVLPPWDREEDEVFPPGPYHPFPNYIRPR TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQEEADAEERRLQEQEELENYIEHVLLHRP IDENTIFICATION OF VGF DERIVED PEPTIDE TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQE EADAEERRLQEQEELENYIEHVLLHRP Mouse TLQP-21 sequence Human TLQP-21 sequence Can TLQP-21 modulate energy homeostasis? Experimental procedure: Male CD1 mice injected icv with TLQP-21 (6nmol/day) for 14 days with Alzet microsmotic pumps. basal Chronic TLQP-21 icv treatment Body weight Food intake 0 1 3 5 days Surgery, start treatment 7 9 11 13 HYPOTHALAMUS BAT AGRP POMC b1AR NPY CRH b2AR MCH b3AR GHS-R UCP1 Body weight UCP2 Food intake UCP3 Locomotor activity Norepi Temperature Energy expenditure Triglycerides FFA/TG Leptine Ghreline BAT THYROID fT3 fT4 WAT ADRENALS UCP1 UCP3 UCP2 PPAR-d Epinephrine b1AR PGC-1a Norepinephrine b2AR Adipon Corticosterone b3AR HemeOx1 Norepi WAT Increased T, epinephrine and resting EE & adrenal weight Normal food intake and locomotor activity,and thyroid hormones Decreased TG and increased FFA/TG ratio; reduction in WAT and leptin slight No changes in the hypothalamus; b2-AR increase in BAT; UCP1, PPAR-delta and b3-AR in WAT BAT WAT WAT WAT CONCLUSION 1 Central TLQP-21 determines: Increased EE, hyperthermia, conversion of TG in FFA Proposed mechanism: Increased sympathetic stimulation to WAT and adrenomedullary release of E Increased UCP1 and catabolic mediator in the WAT Can central TLQP-21 affects development of diet induced obesity? Experimental procedure: Male CD1 mice injected icv with TLQP-21 (6nmol/day) for 14 days with Alzet microsmotic pumps. Starting the day of surgery mice received a HF diet (+20% lard). basal Chronic TLQP-21 icv treatment Body weight Food intake 0 1 3 5 days Surgery, start treatment 7 9 11 13 Body weight Leptin Ghrelin Food intake White adipose tissue Can TLQP-21 modulate energy homeostasis and somatic growth by modulating the GH/IGF-1 axis? NO EFFECTS OF TLQP-21 -Increases energy expenditure -Increase epinephrine and inhibit norepinephrine in the long term -Activate catabolic markers in the adipose tissue -Prevents diet-induced in obesity prone individuals -Increase heart rate -….. Modulation of the ANS and the sympathoadrenomodellurary pathways. GENERAL CONCLUSION 1 TLQP-21 is the first VGF-derived peptide which is involved in energy metabolism VGF -/- TLQP-21 Other VGF-derived peptides should have an anabolic role GENERAL CONCLUSION 2 TLQP-21 centrally increases energy expenditure GENERAL CONCLUSION 3 TLQP-21 blocks development of diet induced obesity by increasing energy expenditure PSYCHOSOCIAL STRESS PROCEDURE BASELINE (3 days) SOCIAL INTERACTION (twice a day x 5 days) COHABITATION (16 days) P S Y C H O S O C I A L S T R E S S FOOD INTAKE IN DOM, SUB AND CONTROL MICE DURING PS (COHABITATION) food ingested from day 5-day 21 (gr) 110 ** ** 100 90 80 70 60 50 40 30 20 10 0 ANOVA F 2,25 = 4.75 p< 0.05 ** p< 0.01 vs CONTROL MEAN DAILY METABOLIC RATE (kcal/h),AND LOCOMOTION IN DOM SUB AND CONTROL MICE BEFORE AND AFTER THE PS PROCEDURE Mean activity Mean daily Metabolic rate 600 ** 0,85 0,8 500 0,75 counts 400 0,7 0,65 300 200 0,6 100 0,55 0,5 0 dom sub con dom sub con Basale Basale Basale Post Post Post ANOVA F2,13 = 6,77 p< 0.01 **p< 0.01 vs CONTROL Dom Basal Sub Basal Con Basal Dom Stress D22 Sub Stress D22 ANOVA F2,13 = 0.00 NS Con Stress D22 Institute of Neuroscience CNR Rome FRANCESCA D’AMATO ALESSANDRO BARTOLOMUCCI FLAMINIA PAVONE LUCIANA GARBUGINO ROBERTO COCCURELLO Institute of Neurobiology and Molecular Medicine CNR Rome and Department of Neurobiology Univ. of Rome Tor Vergata ANDREA LEVI ROBERTA POSSENTI ROBERTO RIZZI CINZIA SEVERINI GIORGIO LA CORTE Univ. of Rome La Sapienza DONATELLA BARRA EUGENIA SCHININA’ PINA MIGNOGNA ALESSANDRA GIORGI Univ. of Cagliari GIAN LUCA FERRI CARLA BRANCIA S.Lucia Foundation, Rome T. PASCUCCI Sigma Tau,SPA, Pomezia R. CONTI A.CAPRIOLI F.BORSINI O.GHIRARDI Univ. Milan-Bicocca TORSELLO, V. LOCATELLI Univ. Of Milan A.E. RIGAMONTI, E.E. MULLER