Thesis
Reference
Central effects of ghrelin on glucose and lipid metabolism and possible mediators
THEANDER-CARILLO, Claudia J.
Abstract
L'obésité est une maladie reconnue dans tout le monde. Dans les pays développés, comme les Etats Unis ou l'Angleterre, cette augmentation est plus marquée. En effet, 65% des Américains adultes sont en surpoids et 30% sont obèses. De la même manière, le surpoids est en augmentation chez les enfants et les adolescents. L'homéostasie énergétique est le résultat d'une balance entre l'apport (la prise alimentaire) et la dépense énergétique (le métabolisme de base, l'activité physique et la thermogenèse). L'excès de prise alimentaire avec réduction de la dissipation d'énergie entraîne un excès calorique qui va s'accumuler.
Dans la circulation, les lipides et les hydrates de carbone sont élevée. En conséquence, ils vont s'accumuler dans les adipocytes qui forment le tissu adipeux. Cette accumulation augmentera la masse adipeuse et par conséquent le poids, de sorte que la masse corporelle sera également élevée.
THEANDER-CARILLO, Claudia J. Central effects of ghrelin on glucose and lipid
metabolism and possible mediators. Thèse de doctorat : Univ. Genève, 2008, no. Sc. 4086
URN : urn:nbn:ch:unige-21375
DOI : 10.13097/archive-ouverte/unige:2137
Available at:
http://archive-ouverte.unige.ch/unige:2137
Disclaimer: layout of this document may differ from the published version.
UNIVERSITE DE GENEVE
Département de Zoologie et FACULTE DES SCIENCES
Biologie animale Professeur J.-L.Bény
Département de Médecine Interne FACULTE DE MEDECINE Service d’Endocrinologie, Diabétologie Professeur F. Rohner-Jeanrenaud et Nutrition
Central effects of ghrelin on glucose and lipid metabolism and possible mediators
THESE
Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Claudia J. Theander-Carrillo de
Villeta (Cundinamarca) Colombie
Thèse No 4086
Genève
Centre d’impression de l’Université de Genève 2008
Contents
Abbreviations………...…...….3
Résumé………...7
English summary………13
I Introduction……….….….….…....14
1.1 Energy homeostasis………...14
1.2 Afferent signals.……….………….16
1.2.1 Adiposity signals.………....….……...16
1.2.2 Satiety signals………....…..……...17
1.3 CNS integration of adiposity and satiety signals.………...….……....17
1.4 Hypothalamic neuropetides involved in the modulation of energy balance……….20
1.4.1 Orexigenic peptides.……….………….…….…….…………..…21
Neuropeptide Y……….……….……....21
Agouti and AGRP………..26
1.4.2 Anorexigenic peptides...26
Corticotropin-releasing factor (CRF)……….…………26
Cocaine and amphetamine regulated transcript (CART)….…27 Melanocortins………27
1.5 Thyroid hormones in the regulation of energy balance..….……...…28
1.5.1 Deiodinases………...28
1.5.2 Thyroid hormones and thermoregulation……….…..30
2.0 Ghrelin..………..……….…….…...32
2.1 The discovery of ghrelin, a natural ligand of the growt hormone secretagogue receptor……….32
2.2 The ghrelin gene and its peptide products….……….………..34
2.3 The tissue distribution of ghrelin……….…..37
2.4 Regulation of ghrelin by the nutritional state………..….38
2.5 Blood ghrelin levels in physiological and pathological condition...40
2.5.1 Obesity and diabetes………..…………..……...41
2.5.2 Anorexia nervosa and cachectic state………...42
2.6 The ghrelin receptor………..……….….43
2.7 Effects of ghrelin on growth hormone………...44
2.8 Hypothalamic ghrelin effects.………...………...…45
2.9 Ghrelin signaling via the vagus nerve………....48
2.10 Metabolic effects of ghrelin………..………...49
2.10.1 Role of ghrelin in adipogenesis…………..………...49
2.10.2 Ghrelin effects on the endocrine pancreas……..………...49
2.10.3 Effects of ghrelin on hepatocytes...51
2.10.4 Ghrelin and gastrointestinal function…………..………...52
2.10.5 Ghrelin and cardiovascular function……….52
2.10.6 Ghrelin and bone repair………..…....54
2.11 Animal models of ghrelin knockout mice and GHSR-/- mice…….…..55
2.11.1 Ghrelin knockout mice………..………....…..55
2.11.2 The GHSR-/- mice………...56
2.12 Effects of des-octanoyl ghrelin and obestatin……….…...57
2.12.1 Des-acyl ghrelin……….………...…57
2.12.2 Obestatin……….….…….…...….58
II Aims of the study……….……….……....59
III Materials and Methods………..………..60
IV Results……….……….…...68
1. Ghrelin action in the brain controls adipocyte metabolism………...69
2. Effects of central ghrelin infusion of the hypothalamo-pituitary thyroid axis.……….….……70
V Discussion and perspectives…….……….…..……84
VI Bibliography……….………...….………96
VI Acknowledgments……….……….…..…..116
Abbreviations
-cells Alpha cells
-MSH Alpha-melanocyte-stimulating hormone ACC Acetyl-CoA carboxylase alpha ACTH Adrenocorticotropic hormone
ADX Adrenalectomized
AG Acylated ghrelin
AGRP Agouti related peptide
ALP Alkaline phosphatase
ALT Alanine aminotransferase
AMPK AMP activated protein kinase
ANS Autonomic Nervous System
APO-AIV Apolipoprotein AIV
ARC Arcuate nucleus
AST Aspartate aminotransferase
ATP Adenosine triphosphate
3 -AR 3-adrenoceptor
-adrenergic Beta-adrenergic receptors
-cells Beta cells
-TKO Beta triple knockout
BAT Brown adipose tissue
BBB Blood brain barrier
BMI Body mass index
Ca 2+ Calcium
cAMP Cyclic adenosine monophosphate
CART Cocaine and amphetamine-regulated transcript
CCK Cholecystokinin
CSF Cerebrospinal fluid
CNS Central nervous system
CHF Chronic heart failure
CHO Chinese Hamster Ovary
CLIP Corticotropin-like intermediate lobe peptide.
CPT-1 Carnitine - palmitoyl transferase 1alpha
CRF Corticotropin releasing factor
CRH Corticotropin-releasing hormone
D1 Type 1 iodothyronine deiodinase
D2 Type 2 iodothyronine deiodinase
D3 Type 3 iodothyronine deiodinase
DAG Diacylglycerol
DIT Diiodotyrosine
DMH Dorsomedial hypothalamus
DMN Dorsomedial nucleus
DMNV Dorsal motor nucleus of the vagus
cells Epsilon cells
EGDTA Ethylenediamine tetra-acetic acid
ELISA Enzyme-linked immunosorbent assay ERK Extracellullar signal-regulated kinase
fa/fa Zucker fatty rats
FAS Fatty acid synthase
FAO Fatty oxydation
FFA Free fatty acids
-LPH Gamma-lipotropin
GBP Gastric bypass
GDP Guanosine diphosphate
GFP Ghreen fluorescent protein
GH Growth hormone
GH Gonadotropin hormone
Ghrelin-/- Ghrelin knockout ghrl -/- Ghrelin knockout
Ghrelin-ad lib Ghrelin ad-libitum Ghrelin-pf Ghrelin pair-fed
GHRH Growth hormone releasing hormone
GHRH-R Growth hormone-releasing hormone receptor
GHS Growth hormone secretagogue
GHS-R Growth hormone secretagogue receptor GHSR-/- Growth hormone secretagogue receptor knockout GHS-R-1a Growth hormone secretagogue receptor 1a GHS-R-1b Growth hormone secretagogue receptor 1b GHSs Growth hormone secretagogues
GIR Glucose infusion rate
GLUT2 Glucose transporter 2
GLUT4 Glucose transporter 4
GLPs Glucagon like peptides
GLP-1 Glucagon like peptide 1
GLP-2 Glucagon like peptide 2
GOAT Ghrelin O-Acyltransferase
GPC-R G-protein coupled receptor
GPDH Glycerol 3 phosphate dehydrogenase GPR39 G-protein-coupled receptor39
GRB2 Growth factor receptor bound protein 2
GRP Gastrin releasing peptide
GSK-3 Glycogen synthase kinase 3
GTP Guanosinetriphosphate
GTT Glucose tolerance test HDLs High density lipoproteins
HLPC High-performance liquid chromatogrphy
HOMA-IR Homeostasis model assessment of insulin resistance HPA Hypothalamus pituitary adrenal axis
HPG Hepatic glucose production
HPT Hypothalamo-pituitary-thyroid axis
HSL Hormone sensitive lipase
HR Heart rate
I.C.V. Intracerebroventricular
ICT-EIA Immuno complex transfer-enzyme immunoassay IGF-1 Insulin like growth factor 1
IP3 1,4,5-triphosphate
IPSCs Inhibitory postsynaptic currents
IRS Insulin receptor substrate
IRS-1 Insulin receptor substrate-1 IR-AS Insulin receptor antisense
IRMA Immunoradiometric assay
ITT Insulin tolerance test
IV Intravenously
KATP Potassium adenosine triphosphate channels
LDH Lactic acid dehydrogenase
LH Lateral hypothalamus
LHA Lateral hypothalamic area
LPL Lipoprotein lipase
LV Left ventricule
LVEF Left ventricular ejection fraction MAP Mitogen-activated protein MAPK Mitogen-activated protein kinase MBOATs Membrane-bound O-acyltransferases
MCR Melanocortin receptor
MC3R Melanocortin receptor 3
MC4R Melanocortin receptor 4
MCFAs Medium chain fatty acids
MCH Melanin concentrating hormone MCH-1R Melanin concentrating hormone receptor 1 MCH-2R Melanin concentrating hormone receptor 2 MCTs Medium-chain triacylglycerol
MIT Monoiodotyrosine
mRNA Messenger ribonucleic acid
NaCl Sodium chloride
NEFA Non-esterified fatty acids NEFA
NMR Nuclear magnetic resonance
NPY Neuropeptide Y
NPY-/- NPY knockout mice
NTS Nucleus of the solitary tract
O2 Oxygen
ob/ob Leptin-deficient mice
Ob-Rb Leptin receptor subtype b
OC Osteocalcin
PC1 Prohormone convertase 1
PC2 Prohormone convertase 2
PCR Polymerase chain reaction
PEPCK Phosphoenolpyruvate carboxykinase
PFA Perifornical area
PFH Perifornical hypothalamus
PI3K Phosphoinositide 3 kinase
PIP2 Phosphatidylinositol
PKA Protein kinaseA
PKB Protein kinase B
PKC Protein kinase C
PLC Phospholipase C
POI Postoperative ileus
POMC Propiomelanocortin
PP Pancreatic polypeptide
PPAR- Peroxisome proliferative activated receptor gamma 2 PPMCH Prepro- melanin concentrating hormone
proTRH Prothyrotropin releasing hormone PSNS Parasympathetic nervous system
PTX Pertussis toxin
PVH Paraventricular hypothalamus
PVN Paraventricular nucleus
PWS Prader-Willi Syndrome
PYY(3-36) Peptide YY(3-36)
RIA Radioimunoassay
Rd Rate of glucose dissappearance
RNA Ribonucleic acid
RP-HPLC Reverse-phase HPLC
RT-PCR Reverse transcriptase polymerase chain reaction
RQ Respiratory quotient
rT3 Reverse T3 or 3,3,5’-thriiodothyronine
RT-PCR Reverse transcriptase
RV Right ventricule
SCD-1 Stearoyl -CoA desaturase-1 Ser3 Serine in position 3
SFP Sapphire fluorescent protein
SNA Autonomic nervous system
SNS Sympathetic nervous system,
STZ-DM Streptozotocin-induced diabetes mellitus
T3 Thriiodothyronine
T4 Thyroxine
TBE Tris-borate-EDTA
TEA Tetra-ethyl ammonium
Tg Transgenic
TG Triglycerides
TKO Triple knockout (1, 2 and 3)
TM Transmembrane
TRH Thyrotropin releasing hormone
TRs Thyroid hormone receptor
TSH Thyroid stimulating hormone
UAG Unacylated ghrelin
UCP-1 Uncoupling protein 1 UCP-2 Uncoupling protein 2 UCP-3 Uncoupling protein 3
UCPs Uncoupling proteins
VMN Ventromedial nucleus
WAT White adipose tissue
WATe Epididymal white adipose tissue WATi Inguinal white adipose tissue
WT Wild type
ZDF Zucker diabetic fatty rats
Résumé
L’obésité est une maladie reconnue dans tout le monde. Dans les pays développés, comme les Etats Unis ou l’Angleterre, cette augmentation est plus marquee. En effet, 65% des Américains adultes sont en surpoids et 30% sont obèses. De la même manière, le surpoids est en augmentation chez les enfants et les adolescents.
L’homéostasie énergétique est le résultat d’une balance entre l’apport (la prise alimentaire) et la dépense énergétique (le métabolisme de base, l’activité physique et la thermogenèse). L’excès de prise alimentaire avec réduction de la dissipation d’énergie entraîne un excès calorique qui va s’accumuler. Dans la circulation, les lipides et les hydrates de carbone sont élevée. En conséquence, ils vont s’accumuler dans les adipocytes qui forment le tissu adipeux. Cette accumulation augmentera la masse adipeuse et par conséquent le poids, de sorte que la masse corporelle sera également élevée.
Le corps est le régulateur de sa propre homéostasie (Bernard 1878). Au niveau central, l’hypothalamus, comprend des neuropeptides orexigéniques, comme le neuropeptide Y (NPY), l’agouti-related peptide’ (AGRP), la ‘melanin-concentrating hormone (MCH), les orexines, les opioïdes, ainsi que les endocannabinoïdes. Au contraire, il y a aussi des neuropeptides anorexigéniques qui sont synthétisés dans l’hypothalamus le ‘corticotropin releasing hormone’ (CRH), le ‘cocaine- and anphetamine-regulated transcript’ (CART), les mélanocortines, les ‘glucagon-like peptides’ (GLPs). Après avoir reçu des signaux provenant de la périphérie, comme la leptine, l’insuline ou la ghréline dont la synthèse et la sécrétion dépendent de l’état nutritionnel, les neuropeptides hypothalamiques s’activent ou s’inactivent en favorisant ou défavorisant la prise alimentaire, la production de glucose par le foie et le métabolisme du glucose et des lipides par les tissus périphériques. De cette façon, l’homéostasie métabolique est maintenue.
Notre laboratoire, s’intéresse à la régulation centrale de la balance énergétique en étudiant, entre autres, les effets de certains neuropeptides hypothalamiques sur la prise alimentaire, la thermogenèse et le métabolisme périphérique. Pour ce faire, différents neuropeptides orexigènes ou anorexigènes sont injectés dans le cerveau ou en périphérie de façon aigüe ou chronique. La prise alimentaire, la prise de poids, et la sensibilité à
l’insuline sont mesurées. L’expression génique de différents enzymes impliquées dans la thermogenèse ou le métabolisme du glucose et des lipides est quantifiée. L’effet de certains de ces neuropeptides sur le fonctionnement de la thyroïde a également été évalué.
Dans mon travail de thèse, nous nous sommes particulièrement intéressés aux effets d’une hormone récemment découverte, la ghréline sur le comportement alimentaire, la régulation du poids corporel, la dépense énergétique et le métabolisme du glucose et des lipides.
1. Effets de l’administration centrale de ghréline sur le métabolisme du glucose et des lipides.
La découverte de la ghréline date de 1999. Elle a été réalisée par Kojima et al, qui a décrit un peptide de 27 acides amines secrété par l’estomac, plus précisément le fundus, qui se lie au récepteur des sécrétagogues de l’hormone de croissance (GHS-R) et stimule la sécrétion de GH en augmentant la concentration de calcium. Ceci diffère du mécanisme d’action du GHRH sur la GH, puisque celui-ci fait intervenir une augmentation d’AMPc.
Donc, la ghréline est devenue le ligand endogène du GHS-R. Par la suite et en plus de son effet de sécrétagogue de l’hormone de croissance, il a été montré que la ghréline exerce un effet orexigène. D’autres auteurs ont observé que l’infusion centrale de ghréline augmente l’expression de NPY et AGRP dans l’hypothalamus. De tels effets de la ghréline ont été renforcés par l’observation selon laquelle les taux de ghréline circulants augmentent avant les repas et diminuent en situation postprandiale chez l’homme (Cummings, Weigle et al. 2002). D’autres résultats ont confirmé que la synthèse de ghréline et sa sécrétion sont stimulées en condition de jeûne. De cette façon, et après avoir traversé la barrière hémato-encéphalique, la ghréline circulante peut induire une stimulation de NPY dans l’hypothalamus qui, à son tour, déclenchera la prise alimentaire (Kojima, Hosoda et al. 1999; Tschop, Smiley et al. 2000; Cummings, Purnell et al. 2001; Nakazato, Murakami et al. 2001).
Notre première étude a consisté à déterminer l’impact de la ghréline centrale sur la balance énergétique et sur le métabolisme du glucose et des lipides. Les animaux ont été repartis en trois groupes: le premièr groupe était constitué d’animaux traités à la ghréline
et chez lesquels la nouriture était offerte sans restriction, ‘ghrelin ad-libitum (ghrelin-ad lib)’. Le deuxième groupe comprenait des animaux traités à la ghréline mais subissant une restriction alimentaire, pour ne consommer que la même quantité de nourriture que les animaux contrôles. Nous les avons appelés ‘ghrelin-pair fed (ghrelin-pf)’. Le troisième groupe d’animaux était un groupe contrôle qui ne recevait pas de ghréline mais le solvant de celle-ci, c'est-à-dire du NaCl. L’étude du groupe ‘ghrelin-pf’ nous a permis de distinguer les effets de la ghréline per se de ceux qui étaient occasionnés par l’augmentation de prise alimentaire induit par la ghréline.
Dans un premier temps, nous avons injecté la ghréline dans le système cérébroventriculaire de rats Wistar pendant 6 jours et nous avons observé une augmentation de la prise alimentaire, ainsi que de la prise de poids par rapport aux rats contrôles. En plus, la ghréline entraînait aussi une augmentation de la masse adipeuse et du quotient respiratoire (RQ) mesuré par calorimétrie indirecte.
Pour étudier la sensibilité à l’insuline, le test de tolérance à l’insuline (ITT), ainsi que la technique de clamps euglycémiques-hyperinsulinémiques ont été utilisés. Les niveaux de glucose plasmatique après l’injection d’insuline pendant l’ITT étaient similaires entre les trois groupes. La même observation a été réalisée en ce qui concerne l’utilisation globale de glucose pendant le clamp. La technique du 2-deoxy-glucose nous a ensuite permis de mesurer l’utilisation de glucose en réponse à l’hyper- insulinémie dans les tissus insulino- dépendants. L’utilisation de glucose par le tissu adipeux blanc epididymal (WATe) et inguinal (WATi), ainsi que par le tissu adipeux brun (BAT) était augmentée dans les deux groups de rats recevant de la ghréline. Dans les deux types de tissu adipeux, la ghréline est capable d’augmenter l’utilisation de glucose indépendamment de l’augmentation de prise alimentaire occasionnée par la ghréline.
Pour connaître l’impact de la ghréline sur le métabolisme des lipides, une deuxième série de rats a été traitée avec la ghréline de la même façon que l’expérience décrite antérieurement. Nous avons observé que dans les deux groupes d’animaux traités à la ghréline, l’expression de ARN messager de quatre enzymes impliquées dans le stockage des lipides était augmentée dans le WAT-e : la ‘lipoprotein lipase’ (LPL), ‘l’acétyl-CoA- carboxylase alpha’ (ACC), la ‘fatty acid synthase’ (FAS) et ‘la stéaroyl-CoA
desaturase-1’ (SCD1). L’expression des protéines FAS et SCD-1 était aussi augmentée.
En contrepartie, l’expression de l’ARN messager d’une des enzymes comprises dans l’oxydation des lipides, ‘la carnitine palmitoyl transferase -1 alpha’ (CPT-1α) était diminuée dans le groupe de rats ad-libitum, traité à la ghréline. Pour étudier les effets de la ghréline sur la thermogenèse, l’expression de l’ARNm des protéines découplantes 1 et 3 (UCP1 et UCP3) dans le BAT a été mesurée. Dans le deux groupes de rats traités à la ghréline, l’expression d’UCP1 et d’UCP3 était diminuée par rapport aux rats contrôles.
Une autre partie du travail a consisté à déterminer si les effets de la ghréline sur le métabolisme des lipides s’accomplissaient d’une manière aiguë ou chronique. C’est pourquoi nous avons injecté la ghréline pendant une période de 24 heures ou de 14 jours. Nous avons observé que l’expression que l’expression de LPL dans le WATe était augmentée après 24 heures dans le groupe de rats ‘ghrelin ad-lib’, alors que cette expression était diminuée au jour 14. En contrepartie, l’expression des autres enzymes ACC, FAS et SCD-1 était diminuée après 24h de traitement à la ghréline mais augmentée après 14 jours de ce traitement.
Pour contrôler si les résultats obtenus en réponse à l’administration centrale de ghréline étaiet élicités centralement ou périphériquement (après passage du système intracérébroventriculaire dans la circulation sanguine), nous avons infusé de la ghréline pour voie périphérique en utilisant la même dose que celle qui était infusée centralement.
Nous avons observé que la ghréline à cette dose n’a pas d’effet sur le poids, la balance énergétique ou sur le métabolisme du glucose ou des lipides. On peut donc conclure que les effets anabolisants observés dans cette étude sont occasionnés par action centrale de l’hormone.
Nous avons également étudié le mécanisme par lequel les rats traités à la ghréline ont eu une augmentation de la prise alimentaire. Nous avons observé que l’expression de NPY dans l’hypothalamus est augmentée dans les deux groupes d’animaux traités à la ghréline et que les effets métaboliques sont comparables aux effets provoqués par l’administration centrale de NPY. Le NPY est connu pour moduler l’activité du SNA. Pour déterminer si ceci est également le cas pour la ghréline, nous avons injecté de la ghréline par voie centrale chez des souris dépourvues de récepteur adrénergique β1, β2 et β3 (TKO) et leurs contrôles respectifs (WT). Nous avons observé que la prise alimentaire était semblable dans les deux groupes d’animaux mais que, les souris TKO traitées à la
ghréline prennent moins de poids que les WT. On peut donc suggérer que les effects centraux de la ghrelin passent en partie par la modulation du système nerveux sympatique.
Finalement, nous avons déterminé si la ghréline endogène peut influencer le métabolisme des adipocytes. Pour ce faire, nous avons mesuré l’expression des enzymes impliquées dans le métabolisme des lipides chez des souris knockout pour le gene de la ghréline.
L’expression de l’ARNm de la LPL et de la SCD-1 dans le WATe était diminuée par rapport aux souris contrôles. Cette expérience a démontré que la ghréline endogène peut jouer un rôle dans le métabolisme des lipides.
En conclusion, dans la première étude, nous avons montré que la ghréline injectée centralement peut contrôler le métabolisme des adipocytes de façon partiellement indépendante de son action orexigène. Ainsi, elle favorise le stockage des lipides au niveau du tissu adipeux, tout en inhibant l’oxydation de ces substrats. Dans le BAT, l’infusion de ghréline diminue l’expression des protéines découplantes, ce qui permet de proposer que la ghréline inhibe la thermogènese. Nous résultats permettent également de proposer que la ghréline agisse par l’intermédiaire du NPY hypothalamique et du système nerveux sympathique (SNS).
2. L’action centrale de la ghréline cause un hypothyroïdisme
Il est bien connu que les hormones thyroïdiennes jouent un rôle fondamental dans la thermogenèse obligatoire. Les niveaux de ghréline circulante sont modulés par les hormones thyroïdiennes: ils sont augmentés dans le cas de hypothyroïdie et sont diminués en hyperthyroïdie (Caminos, Seoane et al. 2002). Le NPY et la ghréline ont des effets similaires: ils augmentent la prise alimentaire, favorisent le gain de poids corporel, stimulent le stockage des lipides et diminuent la thermogenèse indépendamment de leurs effets orexigènes (Billington, Briggs et al. 1991; Theander-Carrillo, Wiedmer et al.
2006). En plus, l’utilisation de glucose par le tissu adipeux blanc est augmentée chez les animaux recevant centralement du NPY ou de la ghréline (Zarjevski, Cusin et al. 1994;
Theander-Carrillo, Wiedmer et al. 2006). Par ailleurs, l’infusion de NPY dans le cerveau entraîne un état d’hypothyroïdie chez le rat qui se caractérise par une diminution
TSH normale ou également diminuée (Fekete, Kelly et al. 2001). C’est pourquoi nous avons étudié l’effet d’une infusion centrale de ghréline sur les hormones thyroïdiennes.
La ghréline a été infusée centralement pendant 6 jours chez des rats Wistar et les taux plasmatiques de T3, T4 et TSH ont été mesurés. Nous avons également déterminé l’expression et l’activité des ‘iodothyronine deiodinase’ de type 1 (D1) et de type 2 (D2) puisque ces enzymes permettent la conversion de T4 à T3 au niveau tissulaire. Pour étudier si la ghréline a des effets sur l’axe hypothalamo-hypophyso-thyroïdien, nous avons mesuré l’expression de l’ARNm codant pour le TRH et le CRF dans l’hypothalamus.
Comme par la première étude, trois groupes d’animaux on été étudiés: 2 groupes étaient traités à la ghréline et la 3ème était un groupe de contrôles. Nous avons observé que le les niveaux de T3 plasmatique étaient diminués chez les rats ‘ghrelin ad-lib’, alors que les niveaux de T4 et de TSH étaient les mêmes que les chez rats contrôles. L’expression de D2 dans le BAT était diminuée dans les deux groupes de rats traités à la ghréline, et l’activité de D2 était également diminuée, ceci principalement chez les animaux ‘ghrelin ad-lib’ en comparaison des le rats contrôles. L’expression de D1 dans le foie était aussi diminuée dans les deux groupes de rats traités à la ghréline mais l’activité de D1 était similaire entre les différents groupes. Finalement, l’expression de TRH et de CRH n’était pas changée par l’administration de ghréline.
En conclusion, cette étude montre que l’infusion centrale de ghréline pendant 6 jours entraîne une réduction des niveaux plasmatiques de T3 ainsi que d’expression de D1 dans le foie et de D2 dans le BAT. Ces résultats sont comparables à ceux qui ont été obtenus en réponse à l’infusion centrale du NPY qui induit un hypothyroïdisme.
On peut donc suggérer que les rats traités centralement à la ghréline présentent un hypothyroïdisme dû à l’augmentation de NPY hypothalamique. Ont peut également hypothétiser que la réduction d’expression des protéines découplantes du BAT observée dans la première expérience soit due à une diminution des taux de T3.
English summary
Ghrelin is a hormone secreted by the stomach and acting centrally to stimulate food intake. We studied the metabolic effects of centrally administered ghrelin. We showed that chronic central ghrelin infusion resulted in an increase in food intake, body weight, food efficiency and fat mass gain. Moreover, central ghrelin infusion promoted an increase in the insulin-stimulated glucose utilization index of white and brown adipose tissue without affecting skeletal muscle. Furthermore, in white adipose tissue the expression of fat storage-promoting enzymes, such us lipoprotein lipase, acetyl CoA carboxylase fatty acid synthase and stearoyl-CoA desaturase-1 was increased, whereas that of the rate-limiting step in fatty acid oxidation, carnitine palmitoyl transferase –1, was decreased. In brown adipocytes, the expression of the thermogenesis-related mitochondrial uncoupling protein-1 and -3 was decreased. These effects were dose- dependent, occurred independently from ghrelin-induced hyperphagia and appeared to be mediated by the sympathetic nervous system since TKO mice did not show any increase in body weigh gain. Furthermore, ghrelin-deficient mice displayed a decrease in fat storage enzymes, which led us to conclude that central ghrelin controls cell metabolism in adipose tissue. Thus, the neuroendocrine network in the central nervous system (CNS) involving ghrelin, its hypothalamic target neurons, and the sympathetic nervous system directly regulates energy metabolism in adipocytes.
We studied the effects of central ghrelin infusion on the thyroid axis. We found that plasma T3 levels were significantly reduced in ghrelin-infused animals compared to controls, while no change was observed for plasma T4 and TSH levels. Moreover, the mRNA expression of type 2 deiodinase (D2) was significantly decreased in BAT in response to central ghrelin infusion compared to controls. The D2 activity in BAT was also reduced mainly in ghrelin ad libitum-fed but not in pair-fed animals. The mRNA levels of D1 in the liver were significantly reduced in ghrelin pair-fed animals. These results suggest that the reduced plasma T3 levels observed in ghrelin-infused animals largely reflect the lower D2 activity in BAT. The reduction in D2-induced T3 production in BAT could be involved in the decrease in UCP1 and UCP3 expression in this tissue, which is accompanied by impaired thermogenesis. It can be suggested that the inhibition by ghrelin of thyroid hormone activity in BAT is mediated by a decreased sympathetic nervous system activity in this tissue.
I. Introduction
1.1. Energy homeostasis
Energy homeostasis is defined as the maintenance of normal biological states during adjustments to environmental changes. It comprises energy intake (food and drink) and dissipation (expenditure), as well as storage pathways. Energy intake is regulated by several factors, including hypothalamic neuropeptides. Energy expenditure comprises thermogenesis and physical activity. In rodents, brown adipose tissue is the main site of thermogenesis which is controled by the sympathetic nervous system. A normal energy homeostasis is achieved when energy intake is balanced by an equal energy expenditure (Fig.1). On the contrary, an imbalance towards an increase in energy intake and a decrease in energy expenditure results in an excess of energy. In this condition, carbohydrates and lipids will be stored, mainly as fat in adipose tissue, resulting in an increase in fat mass and weight gain. On the other hand, an excess of energy expenditure accompanied by a decrease in energy intake leads to shortage of energy. Under such conditions, carbohydrates and lipids are consumed to supply the body’s needs, leading to weight loss.
Figure 1. Energy homeostasis. Balance between energy intake and expenditure leading to energy homeostasis.
The total body energy expenditure is given by the conversion of oxygen and food (or stored form of energy such as fat, glycogen and protein) to carbon dioxide, water, heat and work on the environment (Lowell and Spiegelman 2000).
energy intake energy expenditure
food drink
thermogenesis physical activity Energy homeostasis
Energy expenditure
Figure 2. Thermodynamic perspective of energy expenditure. Energy enters an organism as food and exits as heat and work. Energy can also be mobilized from adipose stores. Total energy expenditure can be subdivided into three principal components:
obligatory energy expenditure required for normal functioning of cells and organs;
energy expenditure resulting from physical activity; and expenditure attributed to adaptive thermogenesis, which is defined as heat production in response to environmental temperature or diet (Lowell and Spiegelman 2000).
In addition to the obligatory thermogenesis needed for normal cell function, warm- blooded animals also possess the capacity to regulate their heat production according to external or internal demands. This process, termed adaptive thermogenesis, serves to protect the organism from excessive cold and also regulates energy balance after diet changes (Lowell and Spiegelman 2000) (Fig. 2). Adaptive thermogenesis is largely controlled by the brain. Principally, this is accomplished through the activation of the sympathetic nervous system acting on thermogenic tissues, such as skeletal muscles and brown adipose tissue (BAT).
The brain also controls energy expenditure via the hypothalamo-pituitary-thyroid axis
by mechanisms that are not fully understood, but which involve changes in ion cycling and mitochondrial proton leaks (Lowell and Spiegelman 2000).
It has been demonstrated that a period of fasting, with its associated weight loss, is followed by a transient period of increased food intake (hyperphagia), such that the body weight is returned to its value before fasting. This suggests that body weight is first sensed or measured by the brain and then regulated via changes in feeding behaviour.
Changes in body weight essentially reflect changes in fat mass. In 1953, Kennedy proposed that the brain receives signals which are proportional to the size of body fat stores and that these signals act in the brain to reduce food intake. In case of weight loss, such adiposity signals would be reduced, thereby leading to increased food intake.
Conversely, weight gain would promote increased levels of adiposity signals, causing a reduction in food intake. This model would account for the brain’s capacity to sense or measure body weight, but does not explain the control of food intake during and between individual meals. To explain this phenomenon, Gibbs and Smith introduced the term of satiety factors to imply that after a meal, peptides secreted from the gastrointestinal tract would lead to decreased food intake (Gibbs and Smith 1978; Lowell and Spiegelman 2000; Schwartz, Woods et al. 2000).
1.2 Afferent Signals 1.2.1 Adiposity signals
Presently, only two molecules, insulin and leptin, are classified as being genuine adiposity signals. Both hormones are circulating in proportion to body fat stores. They are also both able to cross the blood-brain barrier and receptors for these two hormones are found in neurons regulating food intake. Furthermore, when exogenous insulin or leptin is infused in the brain, food intake decreases, whereas blocking the signal of leptin and insulin in the brain causes an increase in food intake and body weight gain (Baskin, Figlewicz Lattemann et al. 1999).
Apart from their central role in regulating energy intake, insulin and leptin also have important peripheral actions contributing to glucose homeostasis and the control of body weight regulation.
1.2.2 Satiety signals
Satiety signals were introduced as a concept to explain termination of food intake during an individual meal. The following characteristics have been suggested for a compound (endogenous or exogenous) acting as a satiety signal: The signal should be generated as soon as the food is ingested, resulting in a decreased meal size. Exogenous administration should be effective and be without side effects. They should, furthermore, act in a dose- dependent manner and should not prevent a meal from occuring. Satiety signals are released by special enteroendocrine cells along the wall of the gastro-intestinal tract and are activated when reached by components of digested food. Generally, satiety signals are reaching the hindbrain via the circulation. However, satiety signals can also convey their message by modulating the activity of afferent fibres and, in particular, those carried by the vagus nerve of the autonomic nervous system (Woods 2005). Some of the known satiety signals are: cholecystokinin (CCK), glucagon like peptide-1 (GLP-1), apolipoprotein AIV (APO-AIV), enterostatin, bombesin/gastrin releasing peptide (Bombesin/GRP), oxyntomodulin, gastric leptin, amylin, peptide YY (3-36) (PYY3-36) and pancreatic polypeptide (PP) (Woods 2005).
1.3 CNS integration of adiposity and satiety signals
Adiposity and satiety signals reach the brain by different pathways (Fig. 3). The adiposity signals insulin and leptin are freely circulating in the blood. They reach the brain after having crossed the blood-brain-barrier and act at the level of the arcuate nucleus in the hypothalamus. This area of the brain is rich in receptors for insulin and leptin. It contains neurons which coexpress NPY and AGRP, as well as other neurons which coexpress the melanocortin precursor (prohormone), proopiomelanocortin and the cocaine and amphetamine-regulated transcript (CART). The adiposity signals modulate the activity of both of these neuron types. This step is called “first-order signalling” (Fig 4). These two groups of neurons then project to other areas of the hypothalamus, such as the paraventricular nucleus (PVN) and the lateral hypothalamic area (LHA). Projections from the ARC to the PVN and LHA constitute the so-called “second-order signalling” (Fig 4).
Animal experiments in which either the PVN or LHA has been lesioned show that the activation of the PVN activates catabolic pathways, whereas activation of the LHA leads
to activation of anabolic pathways. Projections from both these nuclei terminate on the nucleus of the solitary tract (NTS).
The NTS is the focal point where adiposity and satiety signals meet and are integrated into a behavioural signal (eat or stop eating). Indeed, this nucleus receives the message of adiposity signals from the PVN and LHA, as well as that of satiety signals arising from the gastro-intestinal tract and the liver via the vagus nerve and cervical spine afferents.
Adiposity and Satiety Signals
Figure 3. Model showing how a change in body adiposity is coupled to compensatory changes of food intake. Leptin and insulin are adiposity signals, secreted in proportion to body fat content, which act in the hypothalamus to stimulate catabolic, while inhibiting anabolic effector pathways. These pathways have opposing effects on energy balance (the difference between calories consumed and energy expended) that in turn determines the amount of body fuel stored as fat. Satiety signals, released from the stomach and liver are transmitted to the NTS, mainly through afferent signals such as the vagus nerve and sympathetic afferents. The efferent signals from the NTS, accompanied by the activation of catabolic pathways will determine meal size. Adiposity and satiety signals both converge in the NTS to determine energy intake and energy expenditure (Schwartz, Woods et al. 2000).
Arcuate and Paraventricular nucleus in the hypothalamus
Figure 4. NPY/AGRP and POMC/CART (propiomelanocortin, cocaine and amphetamine-regulated transcript) neurons in the arcuate nucleus. Populations of first-order NPY/AGRP (green) and POMC/CART (red) neurons in the arcuate nucleus (ARC) are regulated by leptin and project to the PVN, to the lateral hypothalamic area (LHA) and to the perifornical area (PFA), which are locations of second-order hypothalamic neuropeptidergic neurons involved in the regulation of food intake and energy homeostass. Modified from (Schwartz, Woods et al. 2000).
To explain the regulation of energy balance, alternative theories to those involving adiposity and satiety signals have been proposed. These include the glucostatic and the lipostatic or adipostatic theory. In the first theory, glucose is considered the main signal for reporting the body’s nutritional status, whereas in the second, this role is attributed to lipids. As a whole, these theories are based on the fact that circulating levels of glucose, insulin, leptin, fatty acids and glycerol are proportional to the amount of food consumed and of stored lipids. These signals reach the hypothalamus and activate efferent pathways to regulate food intake, hepatic glucose output, glucose and lipid metabolism to maintain energy balance (Fig. 5) (Lam, Schwartz et al. 2005).
Oxy CRH TRH
orexin MCH
second-order neuronal
first-order neuronal
Afferent and efferent signals to regulate energy balance
Figure 5. Afferent and efferent signals to regulate energy balance. Messages from peripheral tissues to the brain are conveyed by afferent signals, such as leptin, insulin, ghrelin, glucose and lipids. They converge in the hypothalamus and efferent signals via the autonomic nervous system, thyroid and hypothalamio-pituitary-adrenal axis will modulate the activity of target organs to regulate food intake, hepatic glucose output, glucose and lipid metabolism and energy dissipation.
1.4 Hypothalamic neuropeptides involved in the modulation of energy balance
Neuropeptides are peptides that are released by neurons. In the hypothalamus, they can be divided into two groups, according to their action on food intake. Thus, the so-called
“orexigenic” peptides stimulate food intake, whereas the so-called “anorexigenic”
peptides decrease food intake.
Neuropeptide Y (NPY), agouti related peptide (AGRP), melanin concentrating hormone (MCH), orexin A and B and opioids represent the main orexigenic neuropeptides, while corticotropin releasing factor (CRH), cocaine and amphetamine-regulated transcript (CART) and the melanocortins are the principal anorexigenic peptides.
Below, the main neuropeptides on which circulating hormones, such as insulin, leptin ghrelin and others act are described. These neuropeptides are all synthetized in the ARC nucleus.
liver pancreas
pc
stomach Leptin Insulin
Glucose
muscle adipose
tissue
Ghrelin
brain
ANS
afferent signals
efferent signals
thyroid pituitary adrenal glands Lipids
Thyroid
HPA
BAT
1.4.1 Orexigenic peptides Neuropeptide Y
Neuropeptide Y (NPY) is a 36 aminoacid peptide belonging to the same family as pancreatic polypeptide. It is the most abundant peptide found in the hypothalamus. NPY is mainly synthetized in the ARC nucleus, but it is also found in the PVN, dorsomedial nucleus (DMN) and perifornical hypothalamus (PFH). NPY receptors are G-protein coupled receptors and are known as Y1, Y2, Y4, Y5, Y6 receptors.
In 1984, Clark JT et al showed for the first time that NPY infused centrally increases appetite. This effect was blocked by the opiate antagonist, naloxone and the dopamine antagonist, haloperidol. NPY-infused animals preferred high carbohydrate over high fat or high protein-containing diets (Stanley, Daniel et al. 1985; Morley, Levine et al. 1987).
It was then showed that semi-chronic (6 days) intracerebroventricular NPY infusion in normal rats leads to a marked increase in food intake and body weight gain (Sainsbury, Rohner-Jeanrenaud et al. 1997).
NPY plays a role not only in energy intake, but also in modulating the activity of the sympathetic nervous system since central administration of NPY suppresses the activity of sympathetic nerves innervating BAT in a dose-dependent manner (Egawa, Yoshimatsu et al. 1990; Egawa, Yoshimatsu et al. 1991). Similar results were found by Billintong et al who demonstrated that centrally injected NPY leads to a decrease in guanosine diphosphate (GDP)-binding, used as a marker of brown fat thermogenic activity (Billington, Briggs et al. 1994). NPY infusion into the PVN decreases uncoupling protein 1 (UCP1) mRNA expression in BAT. No effect was observed on uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3) mRNA expression in white adipose tissue (WAT) and muscle, respectively (Kotz, Wang et al. 2000).
The ob mRNA expression in inguinal white adipose tissue (WATi) is higher in NPY- infused rats compared to controls (Sainsbury, Cusin et al. 1996). Moreover, intracerebroventricular injection of NPY leads to an increase in the activity and mRNA expression of lipoprotein lipase (LPL) in WAT (Billington, Briggs et al. 1994).
Rats intracerebroventricularly infused with NPY for 7 days exhibit a series of metabolic alterations that are similar to those encountered during the development of obesity. Thus, centrally infused NPY leads to basal hyperinsulinemia, to an enhanced insulin response to meal feeding and to an increase in insulin-stimulated glucose uptake by adipose tissue.
Such increase in glucose uptake may be due to an increase in glucose transporter 4 (GLUT-4) mRNA and protein levels in adipocytes (Zarjevski, Cusin et al. 1994).
Furthermore, the NPY-induced hyperinsulinemia is accompanied by muscle insulin resistance (Zarjevski, Cusin et al. 1994). These effects are prevented by adrenalectomy (ADX). Adrenal glands and corticosterone are thus necessary for the establishment of the anabolic defects induced by NPY (Sainsbury, Cusin et al. 1997). Interestingly, in ADX rats, central glucocorticoid supplementation is sufficient to allow for the obesity-inducing effects of NPY to occur. This was observed for most of the NPY effects, except for the expression of UCP1 and UCP3 in BAT which is decreased in NPY-infused rats, an effect that is not observed in ADX rats supplemented with central dexametasone infusion. It can be concluded that centrally infused NPY has obesity-promoting effects which require the presence of glucocorticoids, while the NPY effect on thermogenesis appears to be independent from these hormones (Zakrzewska, Sainsbury et al. 1999).
Other actions of NPY are to stimulate the hypothalamo-pituitary-adrenal-axis (HPA) and to inhibit the gonadotropic axis. Central NPY also leads to decreases in plasma levels of thriiodothyronine (T3) and thyroxine (T4), accompanied by abnormal or low thyroid stimulating hormone (TSH) levels and reduced prothyrotropin releasing hormone (proTRH) mRNA in the PVN of the hypothalamus. Thus, centrally infused NPY results in a state of central hypothyroidism (Fekete, Sarkar et al. 2002).
Fasting or diet induces weight loss and decreases fat stores (low leptin) in type 1 diabetes (low insulin). Both low leptin and insulin lead to an increase in NPY synthesis and secretion. This effect is reversible, since a decreased expression is seen after insulin treatment of type 1 diabetic animals. Similarly, an increase in leptin leads to a decrease in NPY expression. In animal models of energy deficiency, NPY synthesis and secretion are up-regulated. Thus, the adiposity signals insulin and leptin control the expression of hypothalamic NPY (Ramos, Meguid et al. 2005; Woods 2005).
To study the in vivo physiological effects of NPY, rodent models of NPY overexpression or deletion were constructed and their metabolic profile was analyzed. Inui et al demonstrated that transgenic mice overexpressing NPY by 18% in the arcuate nucleus have no increase in food intake and body weight, but show more signs of anxiety that controls (Inui, Okita et al. 1998). Later it was observed that these animals, when given a 50% sucrose-rich diet, have a significant increase in body weight compared to controls, accompanied by hyperinsulinemia and hyperglycemia. NPY-overexpressing animals also increase their food intake, an effect that lasts several weeks after termination of the 50%
sucrose diet. The effects on food intake could be significantly inhibited by antagonists of NPY Y-1 but not of Y-5 receptors (Kaga, Inui et al. 2001).
Initial studies on NPY knockout mice did not show any effect on food intake and body weight, whether the animals were fed a standard or a high fat diet (Erickson, Clegg et al.
1996). However, peripheral treatment with leptin leads to a more pronounced reduction of food intake in NPY-/- animals compared to controls, demonstrating that leptin action is enhanced in NPY knockout animals and therefore suggesting that NPY may antagonize leptin (Hollopeter, Erickson et al. 1998). Further investigations on the relationship between leptin and NPY were published by Erickson et al, demonstrating that NPY-/- mice crossed with the leptin knockout mice (NPY-/-/ob/ob) present a reduction in food intake and body weight accompanied by an increase in energy expenditure. Thus, leptin- deficient mice require the presence of NPY to develop an obese phenotype (Erickson, Hollopeter et al. 1996). Bannon et al also demonstrated that mice lacking the NPY gene (NPY-/-) have a reduction in food intake after 1, 2 and 4h after re-feeding following 24h and 48h of food deprivation (Bannon, Seda et al. 2000).
A very convincing demonstration of the importance of NPY/AGPR pathway for food intake was given by Luquet et al who ablated the NPY/AGRP neurons in adult mice by targeting the human diphtheria toxin receptor to the locus of AGRP. Injection of diphtheria toxin led to a more than 80% destruction of NPY neurons accompanied by a sharp drop in food intake and body weight, the latter decreasing by 20% in two days in all animals (Luquet, Perez et al. 2005). Similar results were published simultaneously by Gropp et al (Gropp, Shanabrough et al. 2005). In performing the same ablation on neonate mice, however, only a minor reduction of body weight and no effect on food intake were observed. Furthermore, these mice were near normal with respect to body
demonstrate that compensatory mechanisms can restore a normal feeding behaviour and, importantly, they offer a possible explanation for the lack of effect of NPY gene deletion on food intake obtained by Erickson et al.
Models of NPY receptor (Y1, Y2, Y4 and Y5) knockout mice have been constructed, most of them on a mixed 129Sv/C57BL/6 or 129Sv/Balb/c background. Y1-/- mice display only a slight reduction in food intake, while fasting-induced re-feeding is more diminished (Pedrazzini 2004). Kushi et al, however, found that these mice are slightly hyperinsulinemic and, quite contrary to expectations, develop obesity in later life. They also display increased levels of UCP1 in BAT, suggesting increased energy expenditure (Kushi, Sasai et al. 1998). In the ARC, CART as well as POMC mRNA were strongly decreased, whereas no effects were observed on NPY or AGRP mRNA levels (Herzog 2003). A more distinct phenotype with respect to body weight was produced by crossing Y1-/- and ob/ob mice. This double knockout has a significantly lower body weight compared to ob/ob mice (Pralong, Gonzales et al. 2002).
Sainsbury et al generated both a conventional germ-line Y2 knockout and a conditional hypothalamic Y2 knockout and compared their phenotypes. For the germ-line knockout, a reduction in body weight gain was observed despite the fact that food intake was increased (females) or unchanged (males). Re-feeding after starvation was, furthermore, substantially increased in Y2-/- mice. These mice also displayed significant changes in neuropeptides related to energy balance. Thus, POMC and CART in the ARC and CRF in the PVN were decreased, whereas hypothalamic NPY and AGRP were increased at the mRNA level (Sainsbury, Cooney et al. 2002). In the conditional hypothalamic Y2
knockout model, significant decreases in body weight associated with significant increases in food intake were observed. Interestingly, the effects on food intake and body weight in this model were transient and fully overcome after ~12 and ~24 days after gene deletion, respectively, emphasising the capacity of the organism to compensate for a missing gene. Furthermore, mRNA levels of NPY, AGRP, POMC and CART were all increased in knockout animals in this model (Sainsbury, Schwarzer et al. 2002).
Y2 receptors are highly expressed on NPY neurons in the ARC (Batterham, Cowley et al.
2002). A specific (Dumont, Cadieux et al. 2000) and endogenous ligand of Y2 is the gut- derived anorexigenic peptide, PYY(3-36). Peripheral administration of PYY(3-36) decreases hypothalamic NPY mRNA levels, electrical activity of ARC NPY neurons and inhibits food intake in wild-type but not in Y2-/- mice, suggesting that Y2 receptors
mediate the autoinhibition of NPY synthesis (Batterham, Cowley et al. 2002; Acuna- Goycolea and van den Pol 2005).
Y4-/--mice display a small but significant reduction in body weight gain and a reduction in food intake. These mice did not display any change in neuropeptides in the ARC.
However, such changes appeared when crossed with ob/ob mice. Thus Y4-/-, ob/ob mice displayed significant reductions in both NPY and AGRP mRNA levels compared to ob/ob mice (Sainsbury, Schwarzer et al. 2002). Furthermore, a double knockout of Y2
and Y4 receptors has recently been shown to provide a substantial protection against diet- induced obesity (Sainsbury, Bergen et al. 2006).Y5 receptor knockout mice, finally, display a late onset obesity with increases in food intake and body weight. These mice are normal with respect to fasting-induced re-feeding (Marsh, Hollopeter et al. 1998;
Kanatani, Mashiko et al. 2000).
In conclusion, centrally infused NPY can control food intake and body weight. NPY also exerts a negative control on the sympathetic nervous system, decreasing thermogenesis.
NPY infusion furthermore leads to an increase in insulin-stimulated glucose uptake in WAT accompanied by an increase in ob and LPL mRNA expression and in LPL activity.
NPY-infused animals develop an obesity syndrome in which glucocorticoid contribution is essential. NPY also induces hypothyroidism.
Leptin and insulin can regulate the hypothalamic NPY expression. Overexpression of NPY does not have any effect on food intake and body weight. The lack of a strong phenotype of NPY knockout mice has been given a satisfactory explanation due to the works of Luquet et al and Gropp et al. Studies of double knockout (NPY-/-/ob/ob-/-) animals show a decrease in the obese phenotype, demonstrating that NPY acts downstream of leptin and, moreover, that the presence of NPY is essential for the development of obesity in leptin-deficient mice. The results from knockouts of the NPY receptors are in part contrary to expected results and age-related compensatory mechanisms, as demonstrated by Luquet et al, may have occured. Another possibility is that hypothalamic and non-hypothalamic NPY receptors affect energy homeostasis differently (Baldock, Allison et al. 2007).
Agouti and AGRP
The agouti protein was identified in agouti (Ay/a) mice which is an autosomal dominant model of genetic obesity. The protein is constitutively expressed throughout the body of the yellow agouti Ay mice. These mice have a characteristic yellow coat colour, an increase in body length and an obese phenotype, accompanied by insulin resistance and hyperglycemia. The agouti molecule is expressed in the skin of these mice and is an antagonist of alpha-melanocyte stimulating hormone (MSH) on MC-1 receptors.
Agouti-related peptide (AGRP), a 132 amino acid peptide has sequence similarity to agouti. It is synthetized mainly in the ARC nucleus in neurons which co-express NPY.
AGRP is an antagonist of MSH on the melanocortin receptors 3 and 4 (MC3-R and MC4-R) which are expressed in the brain (Arora and Anubhuti 2006).
Inhibition of these receptors by AGRP leads to an increase in food intake and caloric efficiency and to an impaired thermogenesis, leading to an obese phenotype. Central AGRP administration has a surprisingly long-lasting effect. Thus, acute intracerebroventricular injection of a single bolus of AGRP causes an increase in food intake that lasts for at least one week. Furthermore, AGRP expression changes with diet and the nutritional state. Thus, rats fed on a high fat diet for 22 weeks exhibit reduced hypothalamic AGRP mRNA expression with a concomitant increase in MC4-R mRNA expression (Ramos, Meguid et al. 2005).
1.4.2 Anorexigenic peptides
Corticotropin-releasing factor (CRF)
Corticotropin-releasing factor (CRF) is a 41 amino acid peptide, which stimulates ACTH secretion. CRF also decreases food intake. The effect is mainly mediated by corticotropin releasing factor 1 and corticotropin releasing factor 2 (CRF-1 and CRF-2) receptors.
Intracerebroventricular injection of CRF in the PVN decreases food intake in fasted rats and chronic administration of CRF decreases food intake, resulting in concomitant weight loss. The infusion of urocortin, a potent activator of CRF-2 decreases food intake and body weight gain. Thus, CRF is an anorexigenic peptide and promotes weight loss trough the CRF-2 receptor (Arora and Anubhuti 2006).
Cocaine- and amphetamine-regulated transcript (CART)
Cocaine- and amphetamine-regulated transcript (CART) is synthetized in the periventricular nucleus, the dorsomedial nucleus, the perifornical region and the lateral nucleus. In the paraventricular nucleus, it is colocalized with vasopressin and corticotropin-releasing factor-containing neurons. In the arcuate nucleus, it is colocalized with POMC. CART is a potent anorexigenic peptide. Intracerebroventricular CART administration produces a decrease in food intake, not only in ad libitum fed, but also in fasted rats. Moreover, the CART peptide is synthetized by neurons which express Ob-Rb within the hypothalamus. In both fa/fa and ob/ob mice, a decrease in CART mRNA expression in the arcuate nucleus was observed. In ob/ob mice, this decrease could be reversed by exogenous leptin administration. Similarly, diabetes and fasting decrease CART mRNA expression. Thus, CART mRNA expression in the hypothalamus is modulated by the nutritional state and also by peripheral hormones (Arora and Anubhuti 2006).
Melanocortins
In the arcuate nucleus, in addition to the NPY/AGRP neurons, there is another group of neurons which express a prohomone called proopiomelanocortin, POMC, from which the melanocortins are derived.
The gene is expressed in the skin, immune system, pituitary (anterior and intermediate) and hypothalamic neurons. ACTH, -, -, and -MSH (melanocyte-stimulating hormone), - and gamma-lipotropin-LPH), corticotropin-like intermediate lobe peptide (CLIP), and -endorphin are all derived from POMC. ACTH is derived by cleavage due to a prohormone convertase 1 (PC1) expressed in the anterior pituitary. Moreover, prohormone convertase 2 (PC2) cleaves POMC to yield alpha melanocyte-stimulating hormone, MSH. AlphaMSH is a tridecapeptide which is expressed mainly in the ARC. The MSH containing neurons project to the DMN, the medial preoptic area, the anterior, posterior and lateral hypothalamus, the PVN, and also to the central nucleus of the amygdala. MSH acts on the melanocortin 3 and 4 (MC3 and MC4) receptors to inhibit food intake. Thus, central administration of MSH is anorexigenic and promotes
a concomitant body weight loss. Additionally, MC3 and MC4 receptors agonists, such as MTII decrease food intake, whereas the antagonist SHU-9119 increases food intake.
Obesity and hyperphagia are observed in MC4-R knockout mice, as well as in humans and in mice with mutations of the MC4-R gene. MC3-R knockout mice are only overweight with an increased fat mass without any increase in food intake. The MC3 and MC4 receptors are expressed in the ARC, ventromedial nucleus (VMN) and LHA nuclei.
The MC4-R is also expressed in the PVN and DMN, the medial preoptic area and in the anterior hypothalamus. The MC3-R is expressed in the preoptic nucleus, and posterior hypothalamic area (Ramos, Meguid et al. 2005; Woods 2005; Arora and Anubhuti 2006).
1.5 Thyroid hormones in the regulation of energy balance
Thyroid hormones thyroxine (T4) and triiodothyronine (T3) are produced in the thyroid gland. However, of the circulating T3, only about 20% originates from the thyroid gland, the remaining 80% being produced by deiodination of T4 in humans. In rat, 50%
originates from the thyroid gland and 50% from deiodination. T3 is the most potent of the two thyroid hormones and produces its effects within hours, whereas the action of T4 takes several days. For these reasons, T4 may be considered a storage form or even a prohormone of T3 (Bianco, Salvatore et al. 2002). Thyroid hormones have effects on cellular differentiation and growth, but most importantly on metabolism in a wide variety of ways.
1.5.1 Deiodinases
The tissue availability of the various forms of thyroid hormones is detemined by the action of tissue specific enzymes called deiodinases. As the name suggests, these enzymes catalyze the removal of iodine atoms from either the outer ring or the inner ring or both from the thyroxine (T4) molecule (Fig 6). There are three iodothyronine deiodinases: D1, D2 and D3. Of the three types, only the type 1 iodothyronine deiodinase (D1) can function both as an outer (5’) or inner (5) ring iodothyronine deiodinase. In rat, D1 is expressed in the liver, kidney, CNS, pituitary, thyroid gland, intestine and placenta.
A similar expression is found in humans with the exception of the CNS, which does not express D1. In the liver, D1 catalyzes the T4 to T3 conversion, thereby supplying a large
part of T3 in plasma of euthyroid humans and even more in thyrotoxic patients (Bianco, Salvatore et al. 2002).
Type 2 iodothyronine deiodinase (D2) is an outer ring deiodinase, which catalyzes the conversion of T4 to T3 and of rT3 to 3,3’-T2. D2 is particularly important in the brain where it produces more than 75% of the nuclear T3 in the rat cerebral cortex (Crantz, Silva et al. 1982). In the rat, D2 is mainly expressed in the brain (pituitary gland, pineal) and BAT. In humans, D2 is expressed in skeletal muscles and in the heart, thereby contributing to a higher fraction of T3 than in rats. (Bianco, Salvatore et al. 2002) D2 activity has furthermore been shown to be the principal source of circulating T3 in humans (Maia, Kim et al. 2005).
D3 is the major T3 and T4 inactivating enzyme. It catalyzes the conversion of T4 to rT3 and the conversion of T3 to 3,3’-T2 This enzyme contributes to the thyroid hormone homeostasis by protecting tissues from an excess of thyroid hormones. D3 in the rat is highly expressed in the CNS, skin, placenta and uterus (Kaplan and Yaskoski 1980;
Kaplan and Yaskoski 1981). It is also found in the human embryonic liver and human hemangiomas (Richard, Hume et al. 1998; Huang, Tu et al. 2000).
Reverse T3 and 3,3’-T2 were previously considered as being biologically inactive (Bianco, Salvatore et al. 2002). This view has, however, recently been criticized by Moreno et al who points out that rT3 appears to play a role in actin polymerization and microfilament organization (Farwell, Dubord-Tomasetti et al. 2006). Moreover, production of 3,3’-T2 has been observed in various states of nonthyroidal illness (Pinna, Hiedra et al. 1998; Moreno, de Lange et al. 2008). Another metabolite of thyroid hormones is 3,5’-T2 (T2). T2 appears to be biologically active since in a preparation of isolated and perfused liver of hypothyroid rats, it stimulates oxygen consumption (Horst, Rokos et al. 1989) and its circulating levels have also been shown to vary in disease (Pinna, Hiedra et al. 1998).
Structures and interrelationships between the principal iodothyronines
Figure 6. Structures and interrelationships between the principal iodothyronines formed by catalysis by the selenodeiodinases (Bianco, Salvatore et al. 2002).
1.5.2 Thyroid hormones and thermoregulation
Thermoregulation in the newborn human, hibernating mammals and rodents is largely controlled by BAT (Bianco, Maia et al. 2005; Sprague, Yang et al. 2007) and thermogenesis in BAT is mediated by UCP1. In BAT, which is heavily innervated by the sympathetic nervous system (SNS), the expression levels of UCP1 are tightly controlled by synergistic actions between thyroid hormones and pathways activated by norepinephrine (Bianco, Sheng et al. 1988; Bianco, Maia et al. 2005). In addition, it is also known that D2 in BAT of mammals and newborn humans is able to control energy expenditure. Thus, D2 knockout mice develop hypothermia when they are exposed to cold.
In adult humans, however, skeletal muscle is responsible for most of the energy expenditure and D2 plays an important role in this tissue (Bianco, Maia et al. 2005). In
contrast to the effect of thyroid hormone on D1 in BAT, the mechanisms involved in the thyroid hormone-induced thermogenesis in skeletal muscle are less known. They appear to include a stimulation of the Ca2+-ATPase responsible for pumping Ca2+ from the sarcoplasm into the sarcoplasmic reticulum (Bianco, Maia et al. 2005). A role of UCP3 in thyroid hormone-induced thermogenesis in skeletal muscle has recently been proposed (Sprague, Yang et al. 2007).
In view of the central role played by thyroid hormones in metabolism, it is not surprising that their levels are sensitive to the nutritional state. Thus, in conditions of fasting or starvation, thyroid hormone levels are known to fall, both in rats (Rondeel, Heide et al.
1992) and humans (Spencer, Lum et al. 1983). These changes in thyroid hormone levels may be seen as an adaptation to conditions of starvation, leading to a reduced metabolism (Boelen, Wiersinga et al. 2008). Further information on this role of thyroid hormones has been obtained by studying their influence on leptin and it has been found that thyroid hormones are able to regulate leptin secretion, both in vitro and in vivo. Thus, it has been show that thyroid homones have an inhibitory action on leptin secretion from rat adipose tissue in vitro (Medina-Gomez, Calvo et al. 2004). This is in keeping with in vivo data showing that thyroidectomized rats have higher leptin levels than controls and infusion of T3 or T4 to these rats lead to lower plasma leptin levels (Escobar-Morreale, Escobar del Rey et al. 1997). On the other hand, leptin is known to stimulate the hypothalamo- pituitary-thyroid axis. Thus, central injection of leptin resulted in increased levels of T3 and decreased levels of T4, effects that were likely caused by the observed increases in BAT D2 mRNA expression and activity (Cettour-Rose, Burger et al. 2002). Furthermore, subcutaneous leptin injection in euthyroid rats leads to an increase in D1 activity in the liver, pituitary and thyroid gland (Cabanelas, Lisboa et al. 2006). In contrast to the results of central leptin injections by Cettour-Rose et al, subcutaneous leptin administration was reported to downregulate BAT D2 activity in euthyroid (Cabanelas, Lisboa et al. 2006) as well as in hypo- and hyperthyroid rats (Cabanelas, Lisboa et al. 2007). This discrepancy may possibly indicate differences between central and peripheral effects of leptin.
