ANNA MOLES Institute of Neuroscience CNR- Rome, Italy A metabolic role for the VGF- derived peptides

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

Nature Neuroscience 6, (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 VGF a possible link between neurotrophines and energy homeostasis?

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….. NAPPEPVPPPRAAPAPTHVRSPQPPPPAPARDELPDWNEVLPPWDREEDEVFPPGPYHPFPNYIRPR TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQEEADAEERRLQEQEELENYIEHVLLHRP

IDENTIFICATION OF VGF DERIVED PEPTIDE TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQE EADAEERRLQEQEELENYIEHVLLHRP

Mouse TLQP-21 sequence Human TLQP-21 sequence

basalChronic TLQP-21 icv treatment Body weight Food intake days Surgery, start treatment 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.

BAT b1AR b2AR b3AR UCP1 UCP2 UCP3 Norepi WAT UCP1 UCP2 b1AR b2AR b3AR ADRENALS Epinephrine Norepinephrine Corticosterone HYPOTHALAMUS AGRP POMC NPY CRH MCH GHS-R UCP3 PPAR-d PGC-1a Adipon HemeOx1 Norepi THYROID fT3 fT4 Body weight Food intake Locomotor activity Temperature Energy expenditure Triglycerides FFA/TG Leptine Ghreline BAT 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; slight reduction in WAT and leptin

No changes in the hypothalamus;  2-AR increase in BAT; UCP1, PPAR-delta and  3-AR in WAT BATWAT

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? basalChronic TLQP-21 icv treatment Body weight Food intake days Surgery, start treatment 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).

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 Other VGF-derived peptides should have an anabolic role VGF -/- TLQP-21

TLQP-21 centrally increases energy expenditure GENERAL CONCLUSION 2

GENERAL CONCLUSION 3 TLQP-21 blocks development of diet induced obesity by increasing energy expenditure

BASELINE (3 days) SOCIAL INTERACTION (twice a day x 5 days) COHABITATION (16 days) PSYCHOSOCIAL STRESS PROCEDURE PSYCHOSOCIALPSYCHOSOCIAL STRESSSTRESS

food ingested from day 5-day 21 (gr) ** FOOD INTAKE IN DOM, SUB AND CONTROL MICE DURING PS (COHABITATION ) ANOVA F 2,25 = 4.75 p< 0.05 ** p< 0.01 vs CONTROL

Mean daily Metabolic rate 0,5 0,55 0,6 0,65 0,7 0,75 0,8 0,85 domsubcondomsubcon Basale Post MEAN DAILY METABOLIC RATE (kcal/h),AND LOCOMOTION IN DOM SUB AND CONTROL MICE BEFORE AND AFTER THE PS PROCEDURE ** ANOVA F 2,13 = 6,77 p< 0.01 **p< 0.01 vs CONTROL ANOVA F 2,13 = 0.00 NS

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 Institute of Neuroscience CNR Rome FRANCESCA D’AMATO ALESSANDRO BARTOLOMUCCI FLAMINIA PAVONE LUCIANA GARBUGINO ROBERTO COCCURELLO Univ. of Cagliari GIAN LUCA FERRI CARLA BRANCIA Univ. of Rome La Sapienza DONATELLA BARRA EUGENIA SCHININA’ PINA MIGNOGNA ALESSANDRA GIORGI Univ. Of Milan A.E. RIGAMONTI, E.E. MULLER Univ. Milan-Bicocca TORSELLO, V. LOCATELLI Sigma Tau,SPA, Pomezia R. CONTI A.CAPRIOLI F.BORSINI O.GHIRARDI S.Lucia Foundation, Rome T. PASCUCCI