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, 736 - 742 (2003)

Brain-derived neurotrophic factor regulates energy balance downstream of melanocortin-4 receptorBaoji 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…..

GST VGF

TROMBIN

pep20

pep20-10

pep10

pep10-3

pep3

NAPPEPVPPPRAAPAPTHVRSPQPPPPAPARDELPDWNEVLPPWDREEDEVFPPGPYHPFPNYIRPR

TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQEEADAEERRLQEQEELENYIEHVLLHRP

IDENTIFICATION OF VGF DERIVED PEPTIDE

TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQEEADAEERRLQEQEELENYIEHVLLHRP

TLQPPASSRRRHFHHALPPARHHPDLEAQARRAQEEADAEERRLQEQEELENYIEHVLLHRP

Mouse TLQP-21 sequence

Human TLQP-21 sequence

basal Chronic TLQP-21 icv treatment

0 1 3 5 7 9 11 13 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.

BATb1AR

b2AR

b3AR

UCP1

UCP2

UCP3

Norepi

WATUCP1

UCP2

b1AR

b2AR

b3AR

ADRENALSEpinephrine

Norepinephrine

Corticosterone

HYPOTHALAMUSAGRP POMC

NPY CRH

MCH

GHS-R

UCP3

PPAR-d

PGC-1a

Adipon

HemeOx1

Norepi

THYROIDfT3

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

BAT WAT

WATWAT

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?

basal Chronic TLQP-21 icv treatment

0 1 3 5 7 9 11 13 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

PSYCHOSOCIAL

STRESS

0

10

20

30

40

50

60

70

80

90

100

110

foo

d i

ng

es

ted

fro

m d

ay

5-d

ay

21

(g

r)

****

FOOD INTAKE IN DOM, SUB AND CONTROLMICE 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

dom sub con dom sub con

Basale Basale Basale Post Post Post

MEAN DAILY METABOLIC RATE (kcal/h),AND LOCOMOTION IN DOM SUB AND CONTROL MICE BEFORE

AND AFTER THE PS PROCEDURE

Mean activity

0

100

200

300

400

500

600

DomBasal

SubBasal

ConBasal

DomStress D22

SubStress D22

ConStress D22

coun

ts

**

ANOVA F2,13

= 6,77 p< 0.01

**p< 0.01 vs CONTROLANOVA 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 POSSENTIROBERTO RIZZICINZIA SEVERINIGIORGIO LA CORTE

Institute of Neuroscience CNR Rome FRANCESCA D’AMATOALESSANDRO BARTOLOMUCCIFLAMINIA PAVONELUCIANA GARBUGINOROBERTO COCCURELLO

Univ. of Cagliari

GIAN LUCA FERRI

CARLA BRANCIA

Univ. of Rome La SapienzaDONATELLA BARRAEUGENIA SCHININA’ PINA MIGNOGNAALESSANDRA GIORGI

Univ. Of MilanA.E. RIGAMONTI, E.E. MULLER

Univ. Milan-BicoccaTORSELLO, V. LOCATELLI

Sigma Tau,SPA, PomeziaR. CONTI

A.CAPRIOLIF.BORSINI

O.GHIRARDI

S.Lucia Foundation, RomeT. PASCUCCI