ELENA TIMOFEEVA

LE SYSTÈME À CORTICOLIBÉRINE (CRF) DANS

Mémoire

présen té

à la Faculté des étude supérieures

de l'université Laval

pour l'obtention

du grade de maître ès sciences (M.Sc.)

Département de Physiologie

FACULTÉ DE MÉDECINE

UNIVERSITÉ LAVAL

MARS 1997

O Elena Timofeeva, 1997

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AVANT-PROPOS

Je tiens à exprimer ma reconnaissance à mon superviseur, le docteur Denis Richard, pour

sa constante collaboration au cours de la réalisation de ce travail, sa grande disponibilité et ses

conseils efficaces.

Je tiens également à remercier les docteurs Serge Rivest et Nicholas Barden pour avoir

bien voulu accepter de faire l'évaluation finale de ce mémoire.

Je remercie aussi les docteurs Robert Rivest et Naceur Naïmi qui ont contribué à cette

recherche, de même que Chantale Roberge, Josée Lalonde, Frédéric Picard, Qingling Huang et

Antoine Labrie pour leur aide technique.

Enfin, je tiens à exprimer ma gratitude à mon époux et ma fille pour leur complicité

morale.

TABLE DES MATIÈRES

PAGE TITRE

AVANT-PROPOS

LISTE DES FIGURES

LISTE DES TABLEAUX

CHAPITRE 1 In traduction générale

1.1 The corticotropin-re1e;ising factor (CRF) system

1.1.1 CRF

1.1.2 CRFi and C m receptors.

1.1.3 The hypothalamo-pituitary-adrenal a i s (HPA)

1.1.4 CRF in the stress reaction

1.2 CRF and obesity

1.2.1 Role of the CRF in food intake and energy expenditure

1.2.2 The Zucker fatty rats as a mode1 of obesity

1.2.3 The HPA axis and the CRF in the Zucker rats

CHAPITRE II Première étude

Expression of corticotropin-releasing factor and its receptors in the brain

of lean and obese Zucker rats 2.1 Résumé

2.2 Abstract

2.3 Introduction

Page

2.4 Materials and methods

2.4. Z Animals and diet

2.4.2 Treadmill running

2.4.3 B rain preparation

2.4.4 In situ hybridization histochemistry

2.4.5 Antisense 3%-labeled cRNA and hnRNA probe

2.4.6 Quantitative analysis of the hybridization signals

2.4.7 Plasma determinations

2.4.8 Statistics

2.5 Results

2.5.1 Plasma glucose, insulin, corticosterone and ACTH

2-52 CRF mRNA and hnRNA

2.5.3 mRNAs encoding CRFl and CW2 receptors

2.6 Discussion

2.7 References

CHAPITRE ID Deuxième étude

Activiîy of CRF nerrrons and expression of the genes encoding CRF and its receptors in food-deprived lean and obese Zucker rats

3.1 Résumé

3.2 Abstract

3.3 Introduction

3.4 Methods

3.4.1 Anirnals and treatrnents

3.4.2 Plasma determinations

3.4.3 Brain and pituitary preparation

3 -4.4 In situ hybridization histochemistry

3.4.5 Combination of immunocytochemistry within situ hybridization

3 A.6 Antisense 3%-labeled cRNA probe

3.4.7 Quantitative analysis of the hybridization signals

3.4.8 S tatistical analysis

3.5 Results 3.5.1 Body weight, plasma levels of glucose and corticosterone 3.5.2 Fos immunoreactivity (ir)/CRF mRNA

3.5.3 CRF mRNA expression 3.5.4 CRFI receptor rnRNA 3 5 5 C E 2 receptor mRNA

3.6 Discussion 3 -7 References

CHAPITRE IV Conclusion générale

RÉFÉRENCES DE L'INTRODUCTION ET DE LA CONCLUSION

GÉNÉRALE

LISTE DES FIGURES

CHAPITRE II

Figure 1 Darkfield photomicrographs of coronal sections

h m the hypothalarnic PVN, depicting CRF hnRNA

Figure 2 DarkfieId photomicrographs of coronal sections

from the MPOA depicting CRF h n W A

Figure 3 ODs of the hybridization signais for the CRF rnRNA and

hnRNA in the hypothalamic PVN and two regions of the MPOA 46

Figure 4 Darkfield photomicrographs of coronal sections from

the hypothalarnic PVN, depicting CRFi receptor mRNA

Figure 5 Darkfield photomicrognphs of coronal sections from

the hypothalamic VMH, depicting C m receptor mRNA

Figure 6 ODs of the hybridization signals for the CRFI receptor mRNA

in the hypothalamic PVN and the hybridization signals for

the CW2 receptor mRNA in the hypothalamic VMH

CHAPITRE III

Figure 1 Time course of the action of food deprivation on the number of

cells colocaiizing Fos ir and CRF mRNA 79

Figure 2 Photomicrographs of the PVN displaying cells colocalizing

Fos ir and CRF rnRNA 80

Figure 3 Time course of the action of food depnvation on the ODs

of the hybridization signal of the CRF mRNA 8 1

Figure 4 Dark-field photomicrographs of the PVN depicting CRF mRNA 82

Figure 5 Time course of the action of food deprivation on the ODs of the

hybridization signal of the CRFl receptor mRNA in the PVN

and pituitary 83

Figure 6 Dark-field photomicrographs of the PVN depicting

CRFl receptor rnRNA

Figure 7 Dark-field photomicrographs of the pituitary

depicting CRF receptor mRNA

Figure 8 Time course of the action of food deprivation on the ODs of the

hybridization signal of the C R b receptor rnRNA in the VMH 86

Figure 9 Film autoradiograms of the hybridization signal

of the CRF2 receptor mRNA in the VMH

LISTE DES TABLEAUX

CHAPITRE 11

Table 1 Body and epididymal fat weights of lean (Fa/?) and

obese (fafia) Zucker rats

Table 2 Plasma levels of glucose, insulin, conicosterone, and

ACTH of lean (Fd?) and obese ( M a ) Zucker rats

CHAPITRE III

Table 1 Time course of food deprivation on body weight, glucose

and corticosterone plasma levels

Table 2 Brain structures containing cells coexpressing Fos

and CRF mRNA

CHAPITRE 1

INTRODUCTION GÉNÉRALE

1.1 THE

1.1.1

CORTICOTROPIN-RELEASING

CRF

Corticotropin-releasing factor (CRF) was

FACTOR (CRF) SYSTEM

first demonstrated in the hypothalamic

extracts in 1955 by Guillemin and Rosenberg (1955) and Shaffran and Schally (1955). The

fractions that contained ACTH-releasing activity from 490,000 ovine hypothalami was findly

extracted in 198 1 by Vale and CO-workers (Vale W et al., 198 1). The active fraction tumed out

to be a 4 1-amino-acid straight-chain peptide (ovine CRF-41) with a molecular weight of 4670.

The full carboxy-terminal region of the peptide is required for biological activity, while some

variability in the amino terminus is apparently tolerated without loss of a potency (Vale W et

al., 1981).

Recent studies employing molecular biological techniques indicate that CRF, like many

other hypothalarnic releasing and inhibiting factors, is synthesized from a larger precursor

rnolecule (Fumtani Y et ni., 1983; Shibahara S et al., 1983). The precursor contains 190 amino

acids and the sequence of CRF-41 is found near its C-terminal region. Cleavage of the peptide

from the prohormone is mediated by the action of the processing enzymes, and occurs at

dibasic residues (lysine, arginine) which Rank the carboxy and amino terminus of the peptide

(Steiner DF et al., 1980). CRF sequence is preceded by the paired basic residues arginine-

arginine, and followed by the sequence glycine-lysine (Furutani Y et al., 1983). The oCRF-41

sequence itself contains a pair of basic amino acid residues (arpinine-lysine) at positions 35

and 36. There is some evidence that in vivo cieavage may occur at this site as weil, to yield

smaller, biologically inactive peptide fragments. Such cleavage may be glucocorticoid sensitive

(Smith AI et al., 1987). The physiological significance of these posttranscriptional

modifications remains to be elucidated.

Chxacterization of the CRF-41 of the different species indicates that its structure is

highly conserved; the human and rat peptides are identical and differ from oCRF-41 only by

seven amino acid residues (Rivier J et al., 1983) and by two residues from porcine CRF

(Patîhy M et al., 1986).

The nucleotide sequences CRF-41 cDNA of the ovine (Furutani Y et aL, 1983), and rat

(Jinpami H et OZ., 1985) and CRF-41 gene sequences of the human (Shibahara S et al., 1983)

and rat (Thompson RT et ai., 1987) have been determined. It appears that the CRF-41 gene is

also highly conserved through evolution. The gene sequences encoding human and rat CRF-

41 show very high (94%) homology and, for the rest of the precursor, the homology is about

80%. The CRF gene contains two exons separated by an intervening sequence. The first exon

of the rat C E gene contains approximately 160 bp of the 5' untranslated region. Exon II

contains 15 bp of the 5' untranslated region, the protein coding region, and the complete 3'

untranslated region (Thompson RT et nL, 1987).

CRF-41 is expressed in many regions of the CNS. These regions can be divided into

three major classes: (1) the anatomic sites clearly involved in the control of ACTH release

from the pituitary, (2) the CRF neurons of the cerebral cortex, and (3) the subcortical areas

associated with the regdation of autonomie function (Petnisz P, Merchenthaler 1, 1992).

The highest level of CRF in the central nervous system was rneasured in the median

eminence (Palkovits M et al ., 1985). Irnmunohistochemical studies indicate that the lesions of

paraventricular nucleus of hypothalamus (PVN) virtually eliminate CRF-immunoreative

terminais from the median eminence (Antoni FA et al., 1983). CRF neurons have been

demonstrated in d l eight major parts of the PVN, although a large proportion is concentrated

in the dorsal medial parvocellular part (PVNmpd) (Swanson LW et ni., 1983). The CW

motoneuron pool is defined as those neurons which express C W and project to the median

eminence (Swanson LW et al., 1987). This population of the PVN cells is extremely

heterogeneous with respect to different expressed peptides (CRF, vasopressin,

cholecystokinin, angiotensin, enkephalin, neurotensin, oxytocin) (Sawchenko PE et al., 1984;

Mezey E et al., 1986; Hokfelt T et al., 1983). There is evidence that some neurons may

contain CRF plus other peptides. The physiological significance of this colocalization of neuropeptides are not well understood. The synergistic action of C W and vasopressin on

ACTH secretion, however, is well established (Abou-Samra AB et al., 1987; Aguilera G ,

1991). C W is also expressed in a small group of PVN neurons that project to the lower brain

stem and to the spinal cord (Swanson LW et al., 1982) as well as in a subset of oxytocin-

containing neurons of the magnocellular subdivision of the PVN (Burlet A et al., 1983) that

send their axons to the posterior lobe of the pituitary, in which the CRF-ir fibers were

demonstrated (Bloom FE et al., 1982). The cerebral cortex contains CRF perikarya (Palkovits M et al., 1985). The

physiological significance of CRF in neocortex remains to be elucidated.

CRF-ir cells have been found in different nuclei of the telencephalon, the diencephalon

and the brain stem. The largest densities of CRF-ir cells are located in the media1 preoptic area

(MPOA), the bed nucleus of stria terminalis, the periventricular nucleus, the central nucleus of

amygdala, the substantia innominata, the periaqueductal gray, the media1 and dorsal nuclei of

the raphe, the Barrington's nucleus, the dorsal vagai complex (Bissett G, 1990).

1.1.2 CRFl AND CRFl RECEPTORS

Radioligand binding (De Souza EB, Insel TR, 1990; W y n n PC et al., 1983) and molecular cloning studies (Chang CP et al., 1993; Chen R et al., 1993; Pemn MH et aL, 1993)

indicate that the CRF actions are mediated by distinct receptors that exhibit specific

pharmacological and anatomical characteristics.

Molecular cloning studies revealed the existence of at least two major classes of

mammalian CRF receptors. They belong to the calcitonidvasoactive intestinal peptide/growth

hormone-releasing hormone family of receptors. These receptors are built of one polypeptide

chain. They have an extracellular domain at the N terminus with a glycosylation sites. The

CRF receptors have five potential glycosylation sites. The receptors of this group have seven

hydrophobic regions that form membrane-spanning domains. These receptors have been

called seven transmembrane (7TM) or heptahelical receptors (Hucho F, Tsetlin V, 1996).

These receptors have also the intracellular C-terminus.

There are potential phosphorylation sites in the three intracellular loops, as well as in

the C terminus that are phosphorilated by protein kinase C, casein kinase II, and protein kinase

A. The phosphorylation of receptors may be involved in the modulation of the receptor activity

and for the G protein-coupling. The third intracellular loop of CRF recepton contains a

sequence for binding and activating G proteins (Chen R et al., 1993).

Two major groups of CRF receptors have been cloned. The first type of receptors referred now as CRFl was obtained from the pituitary and the brain (Chen R et al.. 1993:

Perrin MH et al., 1993; Chang CP et al., 1993; Vita N et al., 1993). The splice variant of the CRFl receptor, CRF-RA2 was found in a human Cushing disease corticotropic adenorna, in

which 29 amino acids are inserted into the first intracellular loop (Chen R et al., 1993). Recently the other type of C W receptors that was referred to as CRF;! (CWzcr and CRI?@)

were described (Lovenberg TW et al., 1995; Perrin MH et al., 1995). The amino acid

sequences of the receptors are different in the extracellular signaling N-terminus.

Activation of CRF receptors by their ligands leads to CAMP accumulation. A dose-

dependent increase in intracellular CAMP following CRF binding has been shown (Vita N et

al., 1993; Chen R et ni., 1993). Moreover, the CAMP accumulation induced by forskolin, a

known stimulator of adenylate cyclase, was similar to that induced by hCRF and oCRF (Vita N et al., 1993). CAMP accumulation following the stimulation of CRF2 receptors has also

been shown (Lovenberg W et al., 1995; Perrin MH et al., 1995). The maximal CAMP accumulation induced through the CRFl in the ceils expressing this type of receptors was

greater than that induced through CRF2 in cells transfected with this receptor (Lovenberg W

et al., 1995).

CRF receptors are coupled to G-proteins. The modulation of ion channels by G-

protein have been shown (Gilman AG, 1987). Indeed, CRF produces inhibition of the neurons

in the central nucleus of amygdala and increases a number of evoked spikes in the basolaterd

amygdala (Rainnie DG et al., 1992). It has been shown that the CRF-induced membrane

depolarization of rat corticoprophs is due to P-type, and possibly L-type of high-threshold

~ a 2 + channels (Kuryshev YA et al., 1996).

CRF can modulate gene expression by changing intracellular CAMP. CAMP-

responsive elements (CRE) that have core motif of nucleotide sequence TGACGTCA were

identified in the promoter region of different genes, for example in the promoter region of the

phosphoenolpyruvate carboxykinase gene and in those of proopiomelanocortin (POMC) gene

(Lu G et ai., 1992; Kraus J, Hollt V, 1995; Drust DS et al., 199 1). The CRE - binding protein

(CREB)/activating transcription factor is a constitutive protein that regulates transcription of

genes that contain the CRE sequence (Drust DS et al., 199 1; Cole et al., 1992). The CREB is

active in a phosphorylated state. CREB is activated by CAMP-dependent protein kinase

(PKA), that was demonstrated by genetic inactivation of PKA that completely abolishes the

CAMP effect on the POMC gene transcription (Boutillier AL et ai., 1994).

The CRE sequence is found in the CRF promoter, so that CRF could influence its own

expression via CRF receptors. Mutation of the CRE sequence of the CRF gene prornoter

completely abolishcs the stimulatory effect by CAMP (Spengler D et al., 1992). Moreover, a

CAMP-dependent PKA pathway of the CRF gene expression in the rat hypothalamus has been

reported (Itoi K et al., 1996).

The different types of CRF receptors have been described in the brain. The CRFl is

widely distributed in the brain and expressed in the intermediate and anterior lobe of pituitary

(Potter E et al., 1994). In the forebrain CRFI rnRNA is expressed in the neocortical areas, the

hippocampal formation, the entorinal cortex, and the subicular cortex. Receptor transcripts

have been found in functionally associated ce11 groups of the forebrain (substantia innominata,

magnocellular preoptic nucleus, subthalamic nucleus) and the midbrain (substantia nigra,

ventral tegmental area). In the limbic region CRFl mRNA was located in the media1 septa1

nucleus, the nucleus of the diagonal band, in the lateral division of the bed nucleus of the stria

terminalis, in the amygdaloid cornplex. In the hypothalamus CRFl rnRNA was described in the dorsomediai and supramammilary nuclei and in the posterior hypothalamic area. Within

the neurosecretory structures, CRFi mRNA was found at low levels in the paraventricular and

arcuate nuclei of the hypothalamus under basal conditions whereas stress enhanced expression

of CRFl rnRNA in the PVN (Rivest S et al., 1995).

CRFi mRNA expression has been shown in the brainstem cell groups involved in the

processing of somatic sensori information (dorsal colurnn, laterodorsal and pedunculopontine

tegrnental nuclei), the lateral parabrachial nucleus, the pontine gray, the lateral reticular, and the

red nucIei. Strong mRNA signals were found in the nuclei of cerebelhm (Potter E et al..

1994). CRFI and CRF2a distribute differently in the brain (Chalmers DT et nl., 1995).

CRF2cr mRNA expression was found in the lateral septum (CRFI more abundant in the

medial septum), in the cortical and media1 amygdaloid nuclei (CRFI receptor expression was

very high in the basolateral area but low in the cortical amygdala). Within hypothalamus, the highest levels of CRFza mRNA expression was found in the ventromedial hypothalamic

nucleus (VMH) but undetectable within DMH, while CRFl was abundant in the DMH but

low within the VMH (Chalmers DT et al., 1995). CRF2B mRNA expression was found predominantly in the non-neuronal cells (e.g.

choroid plexus, and arterioles) (Lovenberg TW et al., 1995).

1.1.3 THE HYPOTHALAMO-PITUITARY-ADRENAL AXIS (HPA)

Baseline levels of glucocorticoids are necessary for the normal function of most tissues

(Baxter JD, Rousseau GG, 1979). Even srnall deviations from normal levels of circulating

glucocorticoids result in significant changes in a wide variety of physiological and biochernical

variables in the body (Dallman MF et al., 1987).

Corticosterone, the main glucocorticoid in the rat is secreted by the adrenals, which

together with the pituitary and the hypothalamus form the hypothalamic-pituitary-adrenal

(HPA) a i s . The Ievels of plasma corticosterone are regulated by the HPA axis (WhitnaIl MH,

1993). The HPA axis contains the neurons in the hypothalamic paraventricular nucleus that

express the CRF (Vale W et al., 198 1). As mentioned above, the CRF neurosecretory cells are

located within the dorsal media1 parvocellular subdivision of the paraventricular nucleus

(Bloom FE et al., 1982; Swanson LW, Kuypers HGJM, 1980; Antonini FA et al-, 1983;

Burlet A et al., 1983; Hokfelt T et al., 1983). These cells send axons to the portal capillary

plexus in the external zone of the median eminence (Zimmerman EA et al., 1977; Swanson

LW, Kuypers HGJM, 1980). About half of the parvocellular CRF neurosecretory cells

express aIso another ACTH releasing factor, vasopressin (VP) (Whitnall MH et al., 1987).

CRF and VP are secreted from axon terminals in the median eminence, then the portal

capillaries bring them to the anterior pituitary, where the CRF and VP synergistically stimulate

the synthesis and release of ACTH from the pituitary neurohormones cells (Vale W et al.,

1983; Abou-Samra AB et al.. 1987). ACTH, in mm, stimulates the synthesis and secretion of

glucocorticoids from the adrenal cortex (Rarnachandran J et al., 1987). Circulating

glucocorticoids (corticosterone in rats and mice. cortisol in humans) modulate energy

utilization throughout the organism, affect cardiovascular tone, and also are potent

immunosuppressors (Baxter ID, Rousseau GG, 1979) and exert negative feedback on the

hypothalamic neurosecretory cells and the pituitary corticotrophs (Dallman MF et al., 1994).

This feedback regulation by circulating glucocorticoids is an important mechanisrn for

controlling the activity of the HPA mis.

1.1.4 CRF IN THE STRESS REACTION

It has been shown that a single exposure to an acute stressful stimulus is followed by a

rapid increase in plasma ACTH and corticosterone, which reach maximal levels in 5 to 30 min

and retum to basal levels within the following 6 hours, depending on the nature and intensity

of the stimulus (Jia LG et aL., 1992). When the stressful stimulus becomes persistent or

repeated, plasma glucocorticoid levels usually remain above basal values, while plasma ACTH levels Vary depending on the stress paradigm (Aguilera G, 1994).

It has been also dernonstrated that several stressors, including hypertonic saline

injection (Lightman SL, Young WS, 1988), insulin-induced hypoglycemia (Suda T et ai.,

l988), streptococcal cell-wall-induced arthritis (S tenberg EM et ni.. l989), electroconvulsive

shock (Herman JP et al., 1989b), and restraint as well as swim stresses (Harbuz MS,

Lightman SL 1989), are capable of increasing hypothalamic CRF mRNA. It was shown that

chronic, but not acute, exposure to footshock stress increases CRF mRNA levels in the PVN

(Imaki T et al., 1991). Probably a large basal pool of CRF mRNA renders difficult the

detection of changes in mRNA levels after some acute stressful stimuli. To study the changes

in the CRF levels after acute stress, the probes hybridizing to sequences inherent to

heteronuclear RNA (CRF hnRNA) have been utilized. These probes detect RNA before the

processing to mRNA and, therefore, provide good index of the transcriptional activation

(Herman JP et al., 199 1). It has been shown that acute ether exposure stress induced a marked

rapid expression of CRF hnRNA that peaked only 5 min after stress, that is the time of the

maximal ACTH secretory response, without any significant changes in the CRI? mRNA levels

during the 4 hours of examination (Kovàcs EU, Sawchenko PE, 1996). Nonetheless, several

other acute stress paradigms produced a significant increase in hypothalamic CRF mRNA

levels after several hours (Lightman SL, Young WS, 1988; Harbuz MS, Lightman SL 1989:

Stenberg EM et a l , 1989). These discrepancies probably could be explained by different

neuronal circuits that are jnvolved in the different stress paradigrns. Thus neurogenic and

systemk stresses by different pathways differently activate the parvocellular and magnocellular

C W cells in the PVN (Ericsson LHY, Sawchenko PE, 1996; Sawchenko PE et al., 1996). The influence of the corticosterone levels on the stress-induced activation of the CRF

system has been challenged intensively. It is well established that CRF mRNA and peptide

expressions are negatively regulated by glucocorticoids in the PVN (Jingami H et al., 1985;

Young WS et al., 1986; Kovàcs KJ, Mezey E, 1987). This negative influence of elevated

corticosterone levels by acute stresses on the CRF mRNA and POMC mRNA as well as on

the secretion of ACTH have been revealed. In the case of chronic stresses, the lowering of the

negative corticosterone feedback on the HPA axis has been demonstrated. Plasma

corticosterone levels and CRF mRNA levels in the PVN are still elevated 24h after repeated

irnmobilization, and show a further rise after a subsequent irnmobilization (Mamalaki E et ai.,

1992). It has been shown that corticosterone (30 mg/100g body weight. SC) reliably decreased

ACTH responses to swim stress. This feedback inhibition does not occur in rats that are either

exposed to chronic footshock or in rats that are chronically treated with corticosterone (Young

EA et ai., 1 990).

Makino and CO-workers (Makino S et al.. 1995) have studied the responses to acute

and chronic irnmobilization stresses in the rats with adrenalectomy and corticosterone

replacement (ADX+CORT). The dose of CORT administered kept basal CRF levels in the

PVN and POMC levels in anterior pituitary in ADX rats in the sarne range as those in sham-

opented rats. Acute stress significantly increased plasma CORT in sham-operated rats, but not

in ADX+CORT treated animals. Acute stress increased the CRF mRNA levels in the

parvocellular division of the PVN in both the sham and ADX+CORT groups of animals. The

magnitude of the increase in CRF mmA levels during acute stress was significantly higher in

the ADX+CORT group. Repeated stresses also increased the levels of CRI mRNA in the

parvocellular division of the PVN in both sham and ADX+CORT rats. However, the C W

mRNA levels reached a similar magnitude in sham and ADX+CORT rats despite the

significantly higher corticosterone levels in the sharn groups (Makino S et al., 1995).

The reduction of the corticosterone negative feedback regulation in the case of the

chronic stress is due, probably, to the reduction of the GR mRNA levels in al[ subfields of the

hippocampus and the PVN after repeated stresses in the sham group, with robust elevated

corticosterone plasma levels, but not in the ADX+CORT group with basal corticosterone

plasma levels (Makino S et al., 1995).

It has been demonstrated also that chronic stresses significantl y down-regulate MR

mRNA expression in subfields CAL, CA3 and the dentate gyrus (DG) of the hippocampus,

and GR mRNA expression in subfields CA1, the DG and in the frontoparietal cortex. At the

level of parvocellular PVN of chronically stressed rats, GR mRNA transcription was

decreased to 60% of control values (Herman JP et al., 1995). GR mRNA expression was

negatively correlated with PVN CRF mRNA expression, suggesting a relationship between

elevated CRF gene expression and down-regulation of GR at the level of the PVN in

chronically stressed animals.

1.2 CRF AND OBESITY

1.2.1 ROLE OF THE CRF IN FOOD INTAKE AND ENERGY EXPENDITURE

In recent years, the pituitary-unrelated action of CRF on the energy balance has been

studied profoundly (Richard D, 1993; York DA, 1992). The CRF treatment blunts energy

storage by concornitantly reducing energy intake and augmenting energy expenditure (Richard

D, 1993).

The centrally administered CRF has an anorectic effects in the rat (Morley JE, Levine

AS, 1982; Britton DR et al., 1982). Also the C W injection significantly inhibited starvation-

induced feeding (Levine AS et al., 1983). The antagonist of CRF, alpha-helical CRF (9-41)

attenuated anorectic effects of the central CW injection (Krahn DD et ni., 1986). There are

convinced evidences that stress-induced anorexia is mediated by the CRF, because the alpha-

helical CRF (9-41) prevented the anorectic effects of an acute exercise (Rivest S, Richard D,

1990) and restraint stress (Krahn DD et nl., 1986).

The site for CRF anorectic actions in the central nervous system remains to be

determined. For studing this aspect, Krahn DD et al. ( 1988 ) administrated CRF by

microinjection into five different sites within the central nervous system. These sites included

the PVN, the ventromedial hypothalamus, striatum. the lateral hypothalamus, and the globus

pallidus. Only after injection into PVN, feeding was decreased by CRF. The PVN represents

an area in the hypothalamus that appears to influence the feeding behavior. Within the PVN

the effects of different putative neurotransmitter modulaton of feeding (such as CRF, NPY,

galanin, bombesine) have been demonstrated (Krahn DD et a l , 1988; Dryden S et al., 1994;

Akabayashi A et al., 1994; Plamondon H, Merali 2, 1994). The rnechanisrn of the CRF

anorectic effect is unclear, nevertheless it seems to be distinct from HPA axis. Indeed, the CRF still decreases feeding in hypophysectornized rats (Morley JE, Levine AS, 1990). This

suggests that the anorectic effect of the CRF is independent of its ability to release ACTH andor beta-endorp hin.

The thermogenic effects of CRF are also well established. The central administration of

CRF produces dose-dependent thermogenic response in conscious or anaesthetized rats

(LeFeuvre RA et al.. 1987; Rothwell NJ, 1990) and increases the firing rate of the sympathetic

nerves supplying the brown adipose tissue (Holt SJ, York DA, 1989; Egawa M et al., 1990

[a]). These sympathetic stimulation by CRF was dose-dependent and appeared at a very low

dose of C W , such as 125 pmol (Holt SJ, York DA, 1989). The areas in the central nervous

system involved in the thermogenic CRF effects are not clear. The fact that an injection of CRF

in the media1 preoptic area (MPOA) led to a 50% increase in the firing rate of the sympathetic

fibers enervating brown adipose tissue (Egawa MH et al., 1990 [b]) suggests the implication

of MPOA in the thermogenic CRF effects.

1.2.2 THE ZUCKER FATTY RATS AS A MODEL OF OBESITY

Genetically obese (fdfa) Zucker rats and their lean (Fa/?) controls rats were first

described by Zucker LM and Zucker TF in 1961. The homozygotes fa/fa develop early

hyperphagia and extreme obesity through the genenl growth of al1 fat deposits during L month

(> 40% of the body weight). The fernates are completel y infertile and the males also exhibit

low fertility. Lean heterozygous Zucker rats are used for breeding and the incidence of obesity

in the offspring is about 25%. The obesity and hyperlipoproteinemia are accompanied by mild

hyperglycemia, hypercorticosteronemia. striking hyperinsulinemia, detectable as early at 2-3

weeks of age, and profound insulin resistance (Shafrir E, 1992). The pancreatic islets of fa/fa

rats are hypertrophie and hyperplastic and sustain the mode of hypersecretion throughout life,

showing a high rate of exocytosis and microtubule formation (Shino A et al., 1973). Insulin

oversecretion has been demonstrated as the earliest detectable abnormality in young genetically

pre-obese fa /h rats. The pre-obese fdfa rat pups oversecreted insulin relative to normal

controls in response to glucose (Rohner-leanrenaud F, Jeanrenaud B, 1985). This abnormality

was normalized by an acute atropine bolus given just pior to the glucose bolus. This suggests

that the hyperinsulinemia of preobesity was of early occurrence and a vagally-mediated defect.

Moreover, the sympathetic tone reaching periphery is additionally decreased in the obesity

syndrome; its inhibitory effect at the level of the islets of Langerhans presumably is smaller,

which would further favor insulin oversecretion (Niijirna A et al., 1984). The turnover of catecholamines at the level of the pancreas of adult fa/fa rats was found to be markedly

decreased compared to the lean controls (Levin BE et al., 198 1) . Nevertheless, the observation

of an hypersecretion of insulin by isolated p-cells from the pancreas of fdfa rats for as long

as 2 1 days suggests that oversecretion probably is a metabolic feature of the pancreas of fatty

rats (Hayek A, 1980). The hyperinsulinemia appears to be one of the important factors in the

development of the obese phenotype. However, the dissociation between hyperinsulinemia and

hyperphagia suggests that this is not the only factor. The destruction of the p-cells by

streptozotocin and the supplementation with exogenous insulin at a different concentrations

does not prevent weight gain and increased food consürnption in the Zucker rats (Stolz DJ,

Martin RJ, 1982). Recently, the adipocyte-derived hormone leptin (the ob gene product) was discovered

(Zhang Yet nL, 1994). The injection of Ieptin to the mutant obese mouse (ob/ob) Iowered their

body weight, body fat, food intake, and semm concentrations of glucose and insulin. None of

these effects was found in diabetic (db/db) mice (Pelleymounter MA et al.. 1995; Halaas JL et

al., 1995).

Tartaglia and CO-authors (Tartaglia LA et ni., 1995) have identified the leptin receptor

(OB-R), which closely relates to other members of the class 1 cytokine receptor family and

transfers signals through a JAK-STAT transduction pathway. Chua and CO-authors (Chua

SCJ et al., 1996) developed genetic and physical mapping of the chromosome 4 regions

containing the db (diabetic) and fa (fatty) loci. They have concluded that db, fa and Obr

(gene of the OB-R) lie in the same locus of chromosome 4. The investigation of the db

mutation led to the conclusion that only one point mutation G - T results in the abnormal

splicing of the RNA . The mutation db form has an insertion of 106 bp and, as a result, the

mutant OB-R protein lacks of most of the cytoplasmic region including the Box 2 - JAK

binding motif (Chen H et cd., 1996; Lee GH et al., 1996). In the case of the fa mutation in the

Zucker rats, the only point missense mutation A - C results in a change of Glu to Pro in a

highly conserved domain of OB-R ( Phillips MS et ni., 1996). The OB-R mRNA is expressed

in the hypothalamic nuclei that are implicated in the regulation of food intake and energy

balance, such as the arcuate nucleus, the ventromedial, the paraventricular nucleus (Mercer JG

et ni., 1996).

The absence of the leptin signaling pathway in the Zucker rats could influence to

developing of obese phenotype. Indeed, leptin deficiency led to an increase in neuropeptide Y

expression in the arcuate nucleus (Stephens TW et al., 1995; Schwartz MW et al. , 1996), and

to an increase in CRF expression in the PVN of food-deprived obese mice (Richard Dy Huang

Q, in press).

1.2.3 THE HPA-AXIS AND THE CRF IN THE ZUCKER RATS

The adrenal glands of fdfa Zucker rats have a higher weight, a thicker cortex, and an

hypertrophie zona fasciculata compared with lean littermates (Bestetti GE et al., 1990). The

obese Zucker rats have enhanced corticosterone turnover (White JD, Kershaw M, 1989), and

the absence of the normal diumal variation of corticosterone levels in obese Zucker rats also

have been shown (Martin RJ et nL, 1978). The treatment of 5-week old obese fdfa rats with

antiglucocorticoid RU 486 (mifepnstone), that binds to the GR but not to the MR, effectively

reversed the development of obesity (Langley SC, York DA, 1990).

The role of corticosterone in the development of obesity in the fatty Zucker rats was

demonstrated by the effects of adrenalectomy and corticosterone replacement. It has been

shown that obese Zucker rats decrease both caloric intake and body weight gain following

adrenalectomy and daily injections of corticosterone attenuate the effect of adrenalectomy in a

dose-dependent fashion (Yukimura Y, Bray GA, 1978; Yukimura Y er 01.. 1978). Moreover,

Castonguay TW et al. ( 1986) have reported that several rnetabolic parameters that are affected

following adrenalectomy in the obese Zucker rats are prevented with corticosterone

replacement. For example, the drop in circulating levels of glucose and triglycerides after

adrenalectomy was prevented w i th a corticosterone replacement. The decrease in adipose tissue

ce11 size following adrenalectomy was also prevented by a treatment with steroids. The obese

rat are hyperinsulinemic. However, after adrenalectomy, insulin levels faIl within lean levels.

With steroid replacement, the insulin levels increased to preoperative levels (Castonguay TW

et 01. , 1986).

The increase of the levels of the GR and MR receptors in hippocarnpus and GR in

hypothalamus of obese rats was reported by Langley and York ( 199 1). The elevation of the

corticosterone receptor numbers in the brain of the obese rats is a tissue-specific defect,

because for example there are no phenotypic differences in hepatic corticosterone receptor

number or affinity (Langley SC, York DA , 1991). The increase in both the number of

corticosterone receptors and in the levels of plasma corticosterone in the obese falfa rats

suggests a defect in the feedback down-regulation of the corticosterone receptors by

corticosterone in these animais, compared with those reported in lean rats (Spencer RL et al.,

199 1 ; Herman JP, 1993). Moreover the binding affinity for GR in the lean rats increased rapidly after

adrenalectomy, whereas there was no regulation of the binding affinity for GR in the obese rats

in response to changes in circulating corticosterone by adrenalectomy (Langley SC, York DA,

1 99 1).

The putative abnormalities of the HPA a i s of the faffa rat became more evident when

the animals were placed into stressful conditions. It has been shown that immobilization, ether,

or cold stresses result in a higher increasing of the ACTH and corticosterone responses in

obese rats compared to lean control (Guillaume-Gentil C et al., 1990; Bestetti GE et al., 1990).

In contrast, Plotsky PM and CO-worken (1992) found that C W contents in the stalk

median eminence and CRF mRNA levels in the parvocelIuIar subdivision of the PVN had no

significant differences between phenotypes. Moreover, the response of hypophysial-portal

CRF to nitroprusside-induced hypotension, was no greater in Zucker fdfa rats. However, the

norepinephrine-evoked CRF secretion from hypothalamic fragments of obese rats was

significantly greater compared with lem control and the retum of ACTH and corticosterone to

initial values were delayed after nitroprusside or exogenous CRF administration in obese

Zucker rats (Plotsky PM et a[., 1992).

The elevated basai adrenocortical activity and the decrease of sensitivity of ACTH to

corticosterone feedback led Walker and CO-workers to the suggestion that the tonically elevated

drive of the HPA a i s reflects a state of chronic stress in rats of the obese phenotype (Walker

CD et al., 1992).

Because of the CRF anorectic and thermogenic effect and, on the other hand, the

hyperphagia, hypercorticosteronemia, decreased sympathetic tone in the fatty Zucker rats, a

hypothesis has been put forth suggesting that the development of obesity may be due to a

reduction in the central CRF activity. Indeed, CRF blocks weight gain in obese rats with less

effect in lean anirnals (Rohner-Jeanrenaud F et nL, 1989).

The present study was conducted to explore the contributions of the CRF and CW

receptors in the response to stress or rnetabolic rate change in genetically obese Zucker rats to

elucidate the function of the CRF system in obese animals.

CHAPITRE 11

PREMIÈRE ÉTUDE

EXPRESSION OF CORTICOTROPIN-RIELEASING FACTOR AND ITS

RECEPTORS IN THE BRAIN OF LEAN AND OBESE ZUCKER RATS

Endocrinology 1996; 137: 4786-4795.

EXPRESSION OF CRF AND ITS RECEPTORS IN THE BRAIN OF LEAN AND OBESE ZUCKER RATS

Denis RICHARDI, Robert RIVESTI, Naceur NAÏMII, Elena TIMOFEEVA~, and Serge

RIVEST~

l~éparternent de physiologie, Faculté de médecine, université Laval, Québec (Qué), G 1 K 7P4, CANADA and * Laboratoire d'Endocrinologie moléculaire, Centre hospitalier de l'université

Laval, 2705 boulevard Laurier, Québec (Qué), GIV 4G2

Running Title: CRF and its receptors in obesity.

Mailing address: Dr Denis Richard, département de physiologie. Faculté de médecine,

université Laval, Québec (Qué), CANADA, G I K 7P4

Tel., 4 18-656-3348; FAX, 4 18-656-7898; E-mail, denis.richard8phs.ulaval.ca

L'expression de I'ARNm et 1'ARN hétéronucIéaire du CRF, de même que I'ARNm

codant les récepteurs du CRF de type 1 ( C E r - R ) et de type 2a (CRF2-R) a été étudié dans le

cerveau des rats Zucker maigres (Fa/?) et obèses (falfa). La technique d'hybridation in sitrr a été

employée pour mesurer le niveau de 1'ARNm et 1'ARNhn dans les rats sacrifiés avant (au

repos), pendant, et 120 minutes après une course sur tapis roulant. L'expression I'ARNhn du

CRF dans le noyau paraventriculaire de I'hypothaIamus (PVN) à l'état du repos était minimal

chez les rats obèses et comparable à celui des rats maigres. Pendant la course sur le tapis

roulant, cette expression a été plus élevée chez les rats obèses, que chez les rats maigres. Chez

les rats obèses, la transcription de I'ARNm du CRFi-R dans le PVN était élevée au repos, a

diminué considérablement pendant la course, et a augmenté encore 120 minutes après la course.

Chez les rats maigres, I'ARNm du CRFI-R dans le PVN était minimal avant et après la course,

mais a augmenté jusqu'au niveau des rats obèses 120 minutes après la course. Dans le PVN des

rats obèses, l'expression du gène du Cmi -R mesurée au repos était comparable à selle

observée pendant de la course, et dépendante de l'état nutritionnel. L'expression du CRF2-R a été

réduite dans le noyau ventromédian de l'hypothalamus (VMH) des rats obèses. La

concentration de l'hormone adrenocorticotripe (ACTH) dans le plasma pendant la course était

moins élevée chez les rats obèses que chez les animaux maigres. Les niveaux de base et d'aprtts

le course de corticostérone étaient plus élevés chez les rats fdfa que chez les rats Fa/?. Pourtant,

les niveaux de corticostérone chez les rats maigres et obèses étaient similaires durent la course.

Ces résultats fournissent l'évidence de modulations différentes de l'expression du CRF et ses

récepteurs chez les rats maigres et obèses dans les certains noyaux hypothalamique. Compte

tenu des effets anorexian: et thermogéne du CRF, de même que le rôle du PVN et du VMM

dans la régulation de la balance énergétique, les altérations observées dans le PVN et VMH aux

niveaux de la biosynthèse du CRF et de ses récepteurs pourraient être impliquées dans le

développement de l'obésité.

ABSTRACT

Expression of CRF mRNA and heteronuclear RNA (hnRNA) as well as the mRNAs

encoding the CRF receptors of type 1 (CRFI R) and 2a (CRF:! R) in the brain, has been

investigated in lean (Fa/?) and obese (falfa) Zucker rats. Exonic and intronic in situ

hybridization histochemistry was employed to measure the mRNA and hnRNA levels in rats

sacrificed prior to (resting state), during and, 120 minutes after a treadmill running session.

The resting expression of CRF hnRNA in the hypothalamic paraventricular nucleus (PVN) of

obese rats, was minimal and comparable to that of lean rats. However, during treadmilt

running, this expression was higher in obese than in lean rats. In obese rats, the transcription of

the CRFl R mRNA in the PVN was high in the resting conditions, dropped considerably

during running and raised again to elevated levels 120 minutes following the treadmill session.

In iean rats, CRFi R mRNA in the PVN was minimal before and during running, but rose to a

value similar to that of obese rats 120 minutes after running. In the PVN of obese rats, the

expression of the CRFI R gene measured during resting conditions was comparable to the

level seen after running and proved to be dependent upon the feeding state of the rats. The

expression of the CRF2 R transcript was reduced in the ventrornedial nucleus of the

hypothalamus (VMH) of the obese rat. Plasma adrenocorticotropic hormone (ACTH)

concentrations during treadmill running were lower in obese than in lean animals. Basal and

post running levels of circulating corticosterone were higher in falfa than in Fa/? rats.

However, there was no difference in corticosterone levels between lean and obese animals

during running. The present results provide evidence for differences between lean and obese

rats in the expression of CRF and its receptor within selective hypothalamjc nuclei. Given the

anorectic and thermogenic properties of CRF and the role of PVN and VMH in the regulation

of energy balance, it can be argued that the observed alterations in the biosynthesis of CRF and

its receptors within the PVN and VMH might be related to the deveiopment of obesity.

Kev words: Adrenocorticotropic hormone, brain, corticosterone, corticotropin-releasing factor,

corticotropin-releasing factor type 1 receptor, corticotropin-releasing factor type 2 receptor,

glucose, heteronuclear RNA, hypothalamus, in situ hybridization histochemistry, insulin,

media1 preoptic area, obesity, paraventricular nucleus of the hypothalamus, ventromedial

nucleus of the hypothalamus.

INTRODUCTION

Corticotropin-releasing factor is a 41-residue peptide widely distributed throughout the

brain and particularly concentrated in the medial parvocellular division of the hypothalamic

paraventricular nucleus (PVN) (1). The CRF-containing cells of the PVN are predorninantly

involved in the control of the pituitary-adrenal axis (2), representing the most recognized,

though not the sole, action of CRF. Among the best documented effects of CRF which are

unrelated to the pituitary-adrenal axis, are the thermogenic and anorectic actions of this peptide

(3,4). CRF has been reported to be involved in the anorectic effects of treadmill running (3,

restraint stress (6) , estradiol (7) and caffeine (8), and in the thermogenic actions of

fenflurarnine (9) and the serotonin 2A/2C receptor agonist, (+)-1-(2,5-dirnethoxy-4-

iodopheny1)-2-aminopropane (DOI) (10).

Because of its anorectic and thermogenic attributes, CRF has emerged throughout

years as a potentid neuroreguIator of energy balance. In this regard, a hypothesis has been put

forth supgesting that the developrnent of obesity may be due to a reduction in the central CRF

activity (3). However, although this inference has gained support from studies emphasizing the

inescapabie action of adrenalectomy in preventing, attenuating or, reversing obesity ( 1 1 - 17 j

and from CRF's ability to block weight gain in obese rats with littIe effect in lean animais (18),

any clear demonstration that the activity of the CRF system is reduced in obese rats has yet to

be provided. Apart from the suggestion of a deficit in hypophyseal portal plasma CRF (19),

there has been no proposed mechanisms nor brain regions which could be involved in a

potentiai reduction of the CRF central activity in obese rats. Furthemore, the characteristic

increase in the hypothalamic-pituitary-adrenal axis seen in obese Iaboratory animds is stiIl

interpreted as a demonstration of the hyperactivity of the CRF neuroendocrine system in

obesity (20-22). As underlined recently (22), the situation of the C E system in animal obesity

remains compIex due to the concomitant increased activity of the HPA axis and the accelerated

rate of energy deposition, which predict hyper- and hypoactivity of the CRF system,

respec tively .

This study was designed to investigate the CRF neurosystem in obesity by assessing

the expression of CRF and its receptors within the rat brain. In an initial series of experiments,

resting, running and post running expression of CRF mRNA and heteronuclear RNA

(hnRNA) was investigated in lean (Fa/?) and obese (faifa) rats using exonic and intronic in sit~r

hybridization histochemistry. In a second series of experiments, expression of the mRNAs

encoding the CRF type 1 (CRFI R) and type 2a (CRF2 R) receptors was examined in the

brains of Fa/? and fdfa rats. The role of CW receptors in obesity is not known but the

distribution of these receptors in the brain (23,24) suggests that CRFI R and CRF;? R may

have specific functional roles in mediating the effects of CRF in the control of food intake and

energy expenditure. Indeed, CRF receptors have been Iocated in two brain regions particularly

known to be involved in the regulation of energy balance; the biosynthesis of CRFi R, which

has been reported to be induced following irnmobilization (25), immune challenge (25) or

injection of dexfenfluramine (26), has been found in the PVN whereas the biosynthesis of

CRF2 R mRNA is particularly concentrated in the ventromedial nucleus of the hypothalamus

(VMH) (24,27).

MATERIALS AND METHODS

Animals and diet

Fa/? and fdfa rats, aged 6 weeks, were purchased from the Canadian Breeding

Laboratories (St-Constant, Canada). Al1 rats were cared for and handled according to the

Canadian Guide for the Care and Use of Laboratory Anirnals and the present protocol was

approved by our institutional animal care committee. Table 1 emphasizes differences between

Fal? and fdfa rats in body and epididymal fat weights. Rats were killed in the resting state,

during, and 120 minutes after a treadmill running session. The animaIs were housed

individuatly in wire-bottom cages suspended above absorbent paper. They were subjected to a

12:lZh light-dark cycle (lights on between 06:OO and 18:00) and kept under an ambient

temperature of 23 f 1°C. The rats had free access to a stock diet (Agway Prolab,

Rat/Mouse/Hamster 1000 Formula) except on the night preceding their sacrifice, when they

were not alIowed to eat folIowing 23:OO so that dl groups wouid be overnight fasted at the

time of kiliing. Additional groups of lean and obese rats were fed ad libitum and killed in the

resting state.

TreadmilI running

On the last morning of the experiment, both Fa/? and falfa rats were either killed at rest

or subjected to a treadmill mnning session of 60 minutes. During the running session, the

speed of the motor-driven treadmill was raised by increments of five meters/rninute every 15

minutes, from 10 to 25 meterslminute. The rats were forced to run continuously for 60

minutes and were, in appearance, exhausted at the end of the running session. The running

session took place between 08:OO and 10:OO. During the five days preceding the experimental

session, al1 rats included in the study had been accustomed to treadmill running by being

subjected to daily running sessions of fifteen minutes a& speeds not exceeding 15

metedminute.

Brain preparation

Brains from Fa/? and fdfa rats assigned to resting or running conditions were prepared

for histochemical deteminations as previously described (26). Brains of the treadmill-run rats

were removed either immediately after or 120 minutes after the running session. Immediately

prior to the removal of the brains, rats were anesthetized with 1.5 ml of a mixture containing

20 mglml of ketarnine and 2.5 mglml of xylazine, injected intraperitoneally. Without delay,

they were perfused intracardially with 30 ml of ice-cold isotonic saline followed by 200 ml of

a paraformaldehyde (4%) solution. The brains were rernoved at the end of perfusion and kept

in paraformaldehyde for an additional period of 2 days. They were then transferred to a

solution containing paraformaldehyde and sucrose (10%) before being cut 24 hours later using

a sliding microtome (Histoslide 2000, Reichert-Jung). Brain sections were taken from the

olfactory bulb to the brain stem. Thirty-mm-thick sections were collected and stored at -30°C

in a cold sterile cryoprotecting solution containing sodium phosphate buffer (50 mm, ethylene

glycol (30%), and glycerol (20%).

In situ hyb ridization histochemistry

In situ hybridization histochemistry was used to localize CW mRNA, CRF hnRNA,

CRFl R and CRF;! R rnRNAs on brain sections taken from the entire brain. The protocol used

was largely adapted from the technique described by Simrnons et al. (28). Briefly, 1 out of

every 5 brain sections was mounted ont0 poly-L-lysine coated slides and allowed to desiccate

ovemight under vacuum . The sections were then successively fixed for 20 minutes in

parafomaldehyde (4%), digested for 30 minutes at 37" C with proteinase K ( I O mg/mI in 100

mM Tris-HC1 containing 50 rnM EDTA, pH &O), acetylated with acetic anhydride (0.25% in

0.1 M trietholamine, pH 8.0) and dehydrated through graded concentrations (50, 70, 95, and

100%) of alcohol. After vacuum drying for at least 2 hours, 90 ml of the hybridization

mixture, which contains an antisense 3% labeled cRNA probe (107cprn/ml) was spotted on

each slide. The slides were sealed under a coverslip and incubated ovemight at 60°C in a slide

warmer. The next day, the coverslips were removed and the slides rinsed 4 times with SCC

(0.6 M NaCl, 60 m M trisodium citrate buffer, pH 7.0), digested for 30 minutes at 37' C with

RNAse-A (20 mg/ml in H20), washed for 30 minutes at 65" C in descending concentrations

of SSC (2 x SCC, 10 minutes, 1 x SCC 10 minutes, 0.5 x SCC 10 minutes and 0.1 x SCC)

and dehydrated through graded concentrations of alcohol. After a 2 hour-period of vacuum

drying, the slides were exposed on X Ray film (Kodak) for periods varying between 12 to 48

h, depending upon the nature of the probe used. Once removed from the autoradiography

cassettes, the slides were defatted in xylene and dipped in NTB2 nuclear emulsion (Eastman

Kodak, Rochester, NY). Depending on the probe used, the slides were exposed from 7 to 2 1

days, before being developed in D 19 developer (Kodak) for 3.5 minutes at 14- 15°C and fixed

in rapid fixer (Kodak) for 5 minutes. Finally, tissues were rinsed in mnning distilled water for

1 to 2 h, counterstained with thionin (0.258), dehydrated through graded concentrations of

alcohol, cleared in xylene, and coverslipped with DPX.

Antisense 3%-labeled cRNA and hnRNA probes

The CRF cRNA probe was generated from the EcoR1 fragment of a rat CRF cDNA

(Dr. K. Mayo, Northwestern University, Evanston, IL) subcloned into pBluescript SK-1

(Stratagene, La Jolla, CA), and linearized with Hind III (Pharmacia). The cRNA probe for the

CRF intron was produced from a 530 bp fragment of the CRF intron 1 (Dr. S. Watson, The

University of Michigan, Ann Arbor) contained in a pGem3 plasmid and linearized with Hind

III. The CRFl R cRNA probe was generated from a 1.3 kb PstI-Pst1 fragment of the cDNA

of the rat CRF[ R (Dr. W. Vale, Peptide Biology Laboratory, The Salk Institute) subcloned

into pBluescript II SK (Stratagene, La Jolla, CA) and linearized with BamH 1 and HindIII

(Phamacia) for antisense and sense probes, respectively. The CRF2 R cRNA probe was

prepared from a 275 bp cDNA fragment of the CRF2 R (Dr. T.W. Lovenberg, Neurocrine

Biosciences Inc.) subcloned into a pBluescript SK+ (Stratagene, La Jolla) and Iinearized with

Xbal. Radioactive sense CRNA copies were aIso prepared to verify the specificity of each

probe.

Quantitative analysis of the hybridization signals

The various hybridization signals revealed on NTB2 dipped nuclear emulsion slides

were analyzed and quantified under a Iight microscope (Olympus, BX50) equipped with a

black and white video camera (Sony, XC-77) coupled to a Macintosh cornputer (Power PC

7 100/66) using Image software (version 1.55 non-FPU, Wayne Rasband, NIH, Bethesda,

24

MD). The optical density (O.D.) for the hybridization signal was measured under dark-field

illumination at a magnification of 25x. Brain sections from the different groups of rats were

rnatched for rostrocaudal levels as closely as possible. When no hybridization signal was

visible under dark-field illumination, the brain structures of interest were digitized under bright

field illumination and then subjected to densitometric analysis under dark field illumination.

The O.D. for each specific region was corrected for the average background signal which was

detemined by sampling unlabeled areas outside of the areas of interest. Brain sections of three

to six rats were analysed for semi-quantification of O.D.

Plasma determinations

An intracardial blood sample was taken in anesthetized rats immediately before the

beginning of the intracardial perfusion with paraformaldehyde. Plasma glucose was

determined (glucose oxydase method) using a glucose analyzer (Beckman, Pa10 Alto, CA).

Insulin levels were determined by radioimmunoassay (sensitivity, 0.035nrnol/L; inter-assay

coefficient of variation, 9.2%) using reagent kits from Incstar (Stillwater, MN). Serum

corticosterone was determined by a cornpetitive protein-binding assay (sensitivity,

0.058nmoIL; inter-assay coefficient of variation, 9.0%) using plasma from a dexamethasone-

treated fernale rhesus monkey as the source of transcortin (29). The plasma levels of ACTH

were measured by irnmunoassay (sensitivity, 0.22pmollml; inter-assay coefficient of variation,

7.3%) (Allegro ACTH irnmunoassay kit, Nichols Institute, San Juan Capistrano, CA).

Statistics

A 2 x 3 analysis of variance (ANOVA) was used to examine the main and interaction

effects of phenotype [lean (Fa/?), obese (fdfa)] and activity [resting, running, post mnning] on

the various dependent variables rneasured in this study. Where relevant, a posteriori

25

cornparisons were performed using the BonferoniDunn multiple-cornparison-procedure.

One-way ANOVA was used to compare obese and lean rats fed ad-libitrcm.

RESULTS

Plasma glucose, insulin, corticosterone and adrenocorticotropic hormone

Table 2 presents the mean concentrations of plasma glucose, insulin, corticosterone and

ACTH of Fa/? and fdfa rats measured prior to (resting), during (running 60 min) or,

following (120 min post mnning) the treadmill running session. Glucose and insulin levels

were significantly higher in obese rats than in lean animals. In obese rats, insulin levels during

running were higher than the levels measured at rest or 120 minutes following running. In lean

rats, there was no significant difference between the resting, ninning and post running levels of

insulin. ANOVA revealed a significant phenotype by activity interaction effect on both

corticosterone and ACTH plasma levels. In resting and post running conditions, corticosterone

levels were markedly higher in obese rats than in lean animals whereas there was no difference

between lean and obese rats in their levels of ACTH. During running, there was no difference

between phenotypes in the levels of corticosterone. The effect of mnninp on corticosterone

levels was significant in both obese and iean rats. Similarly, ACTH levels measured

immediately after running were higher in lean than in obese rats; in the latter, running did not

significantly elevate ACTH levels above resting values.

CRF mRNA and hnRNA

The effects of the various treatments used in this study on the expression of the C W

gene were evaluated by measuring expression of CRF mRNA and CW hnRNA using exonic

and intronic in situ hybridization histochemistry . Figures 1 and 2 presents darkfield

photomicrographs of brain sections taken from lean and obese rats. The sections were

hybridized with an antisense riboprobe complementary to the rat CRF intron 1. In both the

26

PVN (figure 1) and MPOA (figure 2), the hybridization signal was weak under resting

conditions and 120 minutes following running. In fa/fa rats. treadmill mnning induced a rapid

and transient expression of CRF hnRNA that was clearly apparent in the MPOA and the PVN.

Within the MPOA, CRF hnRNA transcnpt was expressed in the dorsal part of the nucleus and

in the medial preoptic nucleus (MPN). Figure 3 surnrnarizes the O.D. of the hybridization

signals for CRF mRNA and CRF hnRNA in the PVN, the dorsal MPOA and the MPN.

Analysis of optical density (O.D.) confirmed that running expression of CRF hnRNA was

higher in obese than in lem rats (figure 3). Analysis of O.D. also revealed a significant effect

of phenotype on the CRF mRNA hybridization signal in the PVN; faNa rats displayed higher

PVN levels of CRF mRNA compared to Fa/? rats.

mRNAs encoding CRFI and CRF2 receptors

The CRFl R mRNA was found in abundance in neocortical, amygdaloid (basdateral

and media1 nuclei). rnesencephalic (interpeduncular and red nuclei) and medullary (cerebellar

cortex, pontine gray, and lateral donal tegmental and rnotor trigemmal nuclei) stmctures. The

highest concentrations of CRF2 R mRNA were found in the lateral septum and the VMH.

There was no clear expression of C m R mRNA in the PVN. Two main differences were

found between lean and obese animals in the pattern of expression of the CRFi and CRI?;?

receptor genes. The intensities of the hybridization signals for the CRFl R transcript in the

PVN and the CRF;! R mRNA in the VMH were found to be respectively stronger and less

intense in fdfa rats compared to the Fa/? rats. The hybridization signal of the CRFI R

transcript in resting obese rats was comparable to that measured in rats killed 120 minutes after

running (figure 4 and 6A). It is interesting to note that, the expression of the CRFl R gene in

fdfa rats appeared to depend largely on the feeding state of the rats as there was no phenotypic

difference in ad-libitum fed rats in the levels of PVN CRFl R (figure 6, panel C). During or

120 minutes post mnning, there was no difference between lean and obese rats in the level of

CRFI R rnRNA in the PVN (fig 6A). Treadmill mnning suppressed the expression of the

27

CRFl R transcript in obese rats as no hybridization signal was detected in these rats

immediately after running. The hybridization signal of the CRF2 R transcript in the W H

(figure 5 and 6) was weaker in faKa rats than in Fa/? animals. The difference between lean and

obese rats in the expression of the CRF;! R mRNA was also detectable in ad-libitum fed rats

under basal conditions (fig 6D).

DISCUSSION

The results of this study provide clear evidence that CRF gene expression in obese rats

can be triggered by stressful conditions such as treadmill ninning. Indeed, the levels of CRF

hnRNA in the MPOA and PVN of fa/fa rats were found to be higher during running than

under resting conditions. Within the PVN, the expression was enhanced in the parvocellular

division of the structure, in which there is a concentration of neuroendocrine CRF neurons

involved in the control of the pituitary-adrenal axis. In the MPOA, the expression was

increased in the dorsal aspect of the structure as well as in the MPN. The running levels of

CRF hnRNA in both the MPOA and PVN were higher in obese rats than in lean animals,

suggesting that obese rats are hyperesponsive to stress. Although consistent with previous

investigations (30), this suggestion has nonetheless to be cautiously interpreted inasmuch as

the factors responsible for the enhanced expression of CW displayed by the obese animals

after treadmill running are still unclear. Because of its larger body weight, and possibly other

factors inherent to its phenotype, the obese rat might have experienced a more laborious

running session than its lean counterpart and could therefore have responded with a higher

CRF biosynthesis. Notwithstanding the factors involved, it seems clear that CRF expression

can be effectively induced in obese Zucker rats.

In contrast to the PVN, where the induction of CRF gene expression can be linked to

an increase in the HPA activity, enhanced biosynthesis of CRF in the MPOA cannot be readily

28

related to a specific function of the CRF system. The role of the CRF-containing cells of the

MPOA has yet to be clearly defined. Although there appears to be some hypophysiotrophic

CRF neurons in the MPOA (3 l), it is not yet docurnented whether these cells significantly

contribute to the activation of pituitary corticotropic celIs. It is also not clear whether the CRF

neurons contained within the MPOA can act directly on the CRF cells of the P V N to enhance

activity of the CRF neuroendocrine system. A possible impact of the stimulation of the CRF

neurons in the MPOA during running could be the impairment of the reproductive function

that is concomitantly induced by treadmili running in the rat (32). CRF neurons are involved in

the stress-induced inhibition of gonadotropin-releasing hormone (GnRH) and this inhibition

has been reported to occur in the MPOA (33). AIthough the site of the CRF: neuron perikarya

involved in this action has not been determined, there is no a priori indication that it could not

be the MPOA.

The levels of ACTH measured immediately after running were higher in lean than in

obese rats, in which they were not even raised significantly above resting values. The reason

for this blunted ACTH response to running in obese rats remains unclear in the light of

elevated PVN CRF mRNA levels which predict increases in both CRF and ACTH secretions.

One hypothetical explanation for this blunted response would be that a negative feedback of

high levels corticosterone on CRF secretion occurred as a short-terrn mechanism to prevent

excessive activation of the HPA s i s . Such an inhibitory effect of corticosterone on CRF and

ACTH secretion could be detected prior to or without being reflected by a change in CRF

biosynthesis. Plotsky et al. (19) reported rhat the portal secretion of CRF in anesthetized rats

with high levels of corticosterone was lower in obese rats than in lean animals with no

alteration in the levels of PVN mRNA. In addition, Plotsky et al. (19) reported that the portal

secretion of CRF was enhanced to a greater extent in fdfa than in Fa/? rats in response to

pharmacological adrenalectomy (191, suggesting that, when the levels of circulating

corticosterone are high, the PVN CRF neurosecretory system of fdfa rats might be more

sensitive to the negative feedback action of corticosteroids than that of Fd? rats. However, this

increased inhibitory response would likely not represent a chronic condition in the obese

Zucker rat. From the hypertrophy of the zona fasciculata of the adrenal cortex (34) and the

atrophy of the thymus gland that have been reported in the fdfa rat (20), it can be confidently

presumed that there is a net increase in the activity of the pituitary-adrenal a i s in €''/fa

compared to Fa,? rats. The possibility that this increase relates to augmented sensitivity to

ACTE! and CRF, respectively, cannot also be mled out.

Resting as well as post running levels of corticosterone were higher in fdfa rats than in

Fa/? animals. Such a phenotypic difference has previously been reported (20,30,34) in lean and

obese rais from which blood was sampled in the first part (rnorning) of the Iight phase of the

lighvdark cycle. The higher levels of corticosterone in falfa compared to Fa/? rats have been

ascribed to a hypersecretion of corticosterone from the adrenal gland (34) since, the metabolic

clearance rate of corticosterone in fdfa rats is known to be higher than that observed in Fa/?

animals (35). Walker et nL(20) observed rnoming IC5o values for corticosterone on ACTH

secretion that tended to be higher in fdfa rats than in Fa/? animals, suggesting that there might

be some resistance to steroid feedback in obese rats. However, this resistance would not seem

to be present in the evening (20) or during stress (19). indicating that it could be dependent

upon the levels of circulating corticosterone. The phenotypic difference in sensitivity to steroid

feedback could be dictated by the type of glucocorticoid receptors that are activated. Less

sensitivity to the negative feedback action of corticosteroids, in the presence of low

corticosterone levels, could indicate an impairment of the functioning of glucocorticoid type I

receptors in fdfa compared to Fa/?. Glucocorticoid type 1 receptors, which have a high affinity

for corticosteroids, are known to mediate the negative feedback action of corticosterone in the

rnoming, when its levels are low (36). Conversely, more sensitivity to the negative feedback

action of corticosteroids, in the presence of high corticosterone levels, would be consistent with

the occupancy of more glucocorticoid type II receptors in faffa compared to Fa/?(37)

(Timofeeva and Richard, unpublished resul ts). Glucocorticoid type II receptors, which are

widely distributed throughout the brain and found in the pituitary as wel1 as within the PVN

CRF neurons (38), are occupied and activated when corticosterone levels are high (36).

The results on CRF mRNA and CRF hnRNA obtained in this study tend to refute the

hypothesis that the activity of the C W system is reduced in obese rats. No reduction in the

expression of the CRF gene was observed in fa/fa rats as compared to the Fa/? rats.

Furthemore, obese rats displayed even higher levels of PVN CRF rnRNA than Fa/? rats. This

finding, which is consistent with the view of a higher HPA activity in obese laboratory rodents

(20-22,39), is not dissonant with the antiobesity effect of CRF. Because of the presence of

many CRF groups of neurons in the brain, likely with different functions, i t is clear that the

stimulation of the CRF neurons controlling the HPA activity does not invariably predict a

concurrent increase in the activity of another CRF system potentially involved in the control of

food intake and energy expenditure. The existence of a CRF system, including CRF cells and

receptors, specifically involved in the regulation of energy balance has yet to be fully

demonstrated. However, this remains a likely possibility, in light of the potent effects of CW

in inhibiting food intake and promoting energy dissipation (3,4), and the convincing

demonstration of the ability of a brain infusion of a non-hypophysiotropic dose or CRF to

reduce the weight gain of obese rats (18).

The phenotypic difference in the resting induction of CRFl R gene in the PVN

emerged as an interesting finding from this study in light of its dependence on the feeding

state. In fact, whereas there was no apparent difference between lean and obese rats in the

resting expression of CRFl R transcript in the PVN when rats had unrestricted access to food.

the relatively short food deprivation inherent to the experimental protocol used in this snidy

brought about a phenotypic difference in the expression of the CRFi R gene. CRFi R mRNA

levels assessed in the PVN of partially food deprived fdfa rats were as high as those seen in

Fa/? and fdfa animals 120 minutes after running. Given that stressful conditions has been

reported to trigger the expression of CRFl R gene in the PVN (25). it can be argued that the

short deprivation of food to which rats were subjected in this study resulted in a stressful

condition in obese rats. The possibility that obese rats are more sensitive than lean rats to a

food deprivation stress has been suggested before (40).

The impact of the increase in the PVN CRFI R expression in ovemight fasted obese

rats rats cannot yet be readily delineated. We recentiy observed the presence of CRFI R within

CRF-containing neurons (26) of the PVN, indicating that CRF may directly modulate its own

secretion. However, it remains unclear whether the occupation of this receptor type is coupled

to an inhibitory or a stimulatory response of the CRF cells. Recent studies provided evidence

that CRF injection in the cerebral ventricles enhanced expression of CRF and its type 1

receptor within the parvocellular portion of the hypothalamic PVN (41.42). The potential

enhancement of CRF neuronal activity by CRF itself could represent a mechanism whereby

CRF neuroendocrine activity wouid be increased in obese rats. However. the possibility that

CRF exerts a negative feedback o n its own secretion cannot be ruled out (43). Autoinhibition

by CRF of the CRF neuroendocrine function would represent a mechanism by which CRF can

prevent the excessive activation of the pituitary-adrenal axis during stress. The mechanisms

responsible for the stimulation of CRFi R gene expression in the PVN by food deprivation

also needs to be further investigated. The presence of high levels of corticosterone in obese rats

is unlikely to be part of such mechanisms, as recent results tended to dismiss the role of

corticosterone in the increased stimulation of CRFI R gene expression (44). Increased activity

of the noradrenergic system has recently been associated with the expression of CRFi R (44).

However, the activity of the noradrenergic system has been reported to be reduced in obese

rats (45), thus calling this into question as a mechanism for stimulating the expression of

CRFl R in the endocrine hypothalamus of fdfa rats.

32

The reduction in the VMH levels of CRF;? R mRNA could represent an important

feature of the obese phenotype. The involvement of the VMH in the regulation of energy

balance, because it is associated more with the control of energy expenditure than on the

control of food intake (22), is concordant with the action of CRF, which is capable of

preventing weight gain in obese rats by essentially augmenting energy expenditure through

brown adipose tissue (BAT) thermogenesis (18). BAT thermopnesis is strongly dependent

on SNS activity (46), which increases following injection of CRF into the VMH (47). In

addition, VMH lesion (48) and VMH stimulation (49) have been associated with a reduction

and a stimulation of BAT thermogenesis, respectively. Moreover, the reduction in VMH levels

of CRF2 R mRNA could also be involved in the hyperinsulinemic state of Zucker rats. The

infusion of CRF reverses this state of hyperinsulinemia (18) which has been ascribed to the

increased activity of vagal efferents to the pancreas (50). It has been suggested that the VMH

could be involved in producing an inhibitory tone on vagal efferents reaching the pancreas

(22). There is also evidence that CRF can reduce the parasympathetic nervous systern activity

to the gut (51). Although the mechanisms underlying the reduction in the VMH levels of

CRF2 R mRNA remain to be delineated, the neuromolecular phenomenon might be

determinant in the ontogeny of obesity.

The results of this study provide clear evidence that the transcription of neurocnne C W

can be triggered in obese rats in response to stressful conditions and, also emphasize a

phenotypic difference in the resting expression of the PVN CRFi R transcript. This difference

appeared to be closely dependent on the feeding state which somehow suggests that obese rats

could be more sensitive to a food deprivation stress than lean animals. In addition, the results

of this study demonstrate that the levels of expression of CRF2-R mRNA in the VMH were

lower in fa/fa than in Fa/? rats. Finally, the present results also confirmed the States of

hyperglycemia, hyperinsulinernia and hypercorticosteronemia commonly observed in obese

33

laboratory rodents. Alterations in the function of the hypothalarnic CRF system might be part

of the mechanisms leading to the development of obesity.

ACKNOWLEDGMENTS

This work was supported by the Medical Research Council of Canada. Dr. Serge Rivest holds

a Scholarship from the Medical Research Council of Canada.

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FIGURE LEGENDS

Figure 1. Dark-field photomicrographs of coronal sections from the hypothalamic

paraventricular nucleus (PVN) depicting CRF heteronuclear (hn) RNA. The brain sections (30

mm thick) were obtained from rats killed before (resting) during (mnning 60 min) or

following (post running 120 min) a session of treadmill mnning. Magnification, x40.

Figure 2. Dark-field photomicrographs of coronal sections from the medial preoptic area

(MPOA) depicting CRF heteronuclear (hn) RNA. The brain sections (30 mm thick) were

obtained from rats killed before (resting) during (running 60 min) or foilowing (post mnning

120 min) a session of treadmill mnning. Magnification, x40.

Figure 3. Optical densities (O.D.) of the hybridization signals for the CRF mRNA and

heteronuclear RNA (hnRNA) in the hypothalamic paraventricuiar nucleus (PVN) and two

regions of the medial preoptic area (MPOA). The brain sections (30 mm thick) were obtained

from rats killed before (resting) during (running 60 min) or following (post running 120 min)

a session of treadmill running. A 2 x 3 analysis of variance (ANOVA) was used to examine

the main and interaction effects of phenotype (P) [Iean, obese] and activity (A) [resting.

mnning, post running] on the various dependent variables measured in this study. In the cases

of significant P x A interactions, n posteriori comparisons were performed using the

BonferoniIDunn multiple-cornparison-procedure. * different from Lean (Fd?) within the same

level of activity, p < 0.005; t different from Resting within the same phenotype, p < 0.005.

Figure 4. Dark-field photomicrographs of coronal sections from the hypothalamic

paraventricular nucleus (PVN) depicting CRFI receptor mRNA. The brain sections (30 mm

thick) were obtained from rats killed before (resting) during (running 60 min) or following

(post mnning 120 min) a session of treadmill running. Magnification, x40.

Figure 5. Dark-field photomicrographs of coronal sections from the hypothalamic

ventromedial nucleus (VMH) depicting CRF;! receptor mRNA. The brain sections (30 mm

thick) were obtained from rats killed before a session of treadmill ninning. Magnification, x25.

Figure 6. The upper panels (A, B) dernonstrate the optical densities (O.D.) of the

hybridization signals for the CRFl receptor mRNA in the hypothalamic paraventricular

nudeus (PVN). and the CRF2 receptor mRNA in the hypothalamic ventromedial nucieus

(VMH). The brain sections (30 m m thick) were obtained from rats killed before (resting)

during (running 60 min) or following (post mnning 120 min) a 60-minute session of treadmill

running. The lower panels (C, D) illustrate optical densities (O.D.) of the hybridization signals

for the PVN CRFi receptor mRNA and the VMH CRF2 receptor mRNA of rats fed ad-

libirrtrn and killed at rest (see 'Materials and Methods' for more details). A 2 x 3 analysis of

variance (ANOVA) was used to examine the main and interaction effects of phenotype (P)

[lean, obese] and activity (A) [resting, running, post running] on the various dependent

variables measured in this study. In the cases of significant P x A interactions, a posteriori

cornparisons were performed using the BonferoniDunn multiple-comparison-procedure (*

different from Lean (Fa/?) within the same level of activity. p c 0.005; t different from

Resting within the same phenotype, p .= 0.005). One-way ANOVA was used to compare

obese and lean rats fed ad-libitum (* different from Lean (Fa/?) < 0.05).

Table 1. Body and epididymal fat weights of lean (Fa/?) and obese (falfa) Zucker rats

1 Lean Fa/?) Obese (fa/fa) 1

Body weight (g)

1 Epididyrnal fat weight (g)

* Different from Lean (Fa/?), p < 0.00 1

Table 2, Plasma levels of glucose, insulin, corticosterone and adrenocorticotropic hormone (ACTH) of lean (Fa/?) and obese (falfa) Zucker rats before (resting) during (running 60 min) or following (post running 120 min) a session of treadmill running.

Lean (Fa/?) Obese (fdfa)

Resting Running Post tunning Resting Running Post tunning Significant (60 min) ( 1 20 min) (60 min) (120 min) effects

(P < 0.05)

Glucose 8.40 4 0.46 8.3M 0.56 7.99 f0.54 10.18 f 0.54 13.53 4 1 -06 10.57 f 0.93 P (mmolll) n = 6 n = 8 n = 6 n = 6 n = 8 n = 6

Insulin (nmolll) 0.10 f 0.02 0.16 f 0.04 0.07 f 0.02 1.06 f 0.28 * 1.86 f 0.22 *t 0.69 f 0.27 P, A, P x A n = 6 n = 8 n = 5 n = 6 n = 8 n = 5

Corticosterone n. d. 1.60f0.06t 0.015f0.03 1.13f0.18* 1.67f0.06f- 1.06f0.16* P ,A ,PxA (mmolll) n = 6 n = 8 n = 6 n = 6 n = 8 n = 6

Values represent means * s.e.m and the number (n) of observations. See RESULTS for more details on the statistical analyses. ACTH, adrenocorticotropic hormone; P, phenotype main effect; A, activity main effect; P x A, phenotype x activity interaction. In the cases of significant P x A interactions, a posteriori comparisons were performed using the Bonferoni/Dunn multiple-comparison-procedure. * different from 'Lean (Fd?)' within the same level of activity , p < 0.005; t different from 'Resting' within the same phenotype, p c 0.005. n. d., non detectable.

PVN CRF hnRNA Resting

Running (60 min)

Post r u h g (120 min)

Lean (Fa/?) Obese (faRa)

Figure 1

MPOA CRF hnRNA

Runaing (60 min)

Lean (Fa/?) Obese (fa/fa)

Figure 2

Lean Obese PVN CRF mRNG

ANOVA P Phenotype (P) 0 . 2 Activity (A) 0.271 P x A 0,509

Resting Running Post running (60 min) ( 1 20 min)

MPOA CRF mRNA

ANOVA - P Phenotype (P) 0.056

0.D. Activity (A) 0.292

50 1 P x A 0.823

Resting Running Post running (60min) (120min)

MPN CRF mRNA

ANOVA P Phenotype (P) 0.227

Resting Running Post running (60 min) ( 1 20 min)

ANOVA P Phenotype (P) o . O ~

0.D. Activity (A) 0.001 P x A 0.003

1 *+

Resting Running Post runninl (60 min) ( 1 20 min)

MPOA CRI? hnRNA ANOVA P Phenotype (P) 0 .20

O.D. Activity (A) 0.004 - P x A 0.097

Resting Running Post runninj (60 min) ( 1 20 min)

MPN CRF hnRNA ANOVA P Phenotype (P) 0.01 8

0.D. Activity (A) 0.006 0.028

*t 10

Resting Running Post mnnin; (60 min) ( 1 20 min)

Figure 3

PVN CRF, receptor mRNA Resting

Running (60 min)

Post ninning (120 min)

Lean i Fa/?) Obese (fa/fa)

Figure 4

VMH CR& receptor mRNA

--

Lean (Fa/?) Obese (falfa)

Figure 5

A - PVN CRFl receptor mRNA

ANOVA P Phenotype(P) 0.004 Lean

0.D. Activity (A) 0.001 Y

P x A 0.023 Obe~e

Resting Running Post running (60 min) (1 20 min)

C - PVN CRFl receptor mRNA

O.D. 0 Lean 20 4 ai Obese

B - VMH C q receptor mRNA ANOVA P Phenotype(P) 0.002

0.D. Activity (A) 0.057 Lean

50 1 P X A 0.339 Obese

Resting During Following (60 min) ( 120 min)

VMH CRF2 receptor mRNA

0 Lean

4 a Obese 401 T

Resting Resting

Figure 6

CHAPITRE III

FUNCTlONAL ACTIVATION OF CRF' NEURONS AND EXPRESSION OF THE

GENES ENCODING CRF AND ITS RIECEPTORS IN FOOD-DEPRIVED LEAN

(Fa/?) AND OBESE (faRa) ZUCKER RATS.

Neuroendocrinology 1997; 66: 327-340.

FUNCTIONAL ACTIVATION OF CRI? NEURONS AND EXPRESSION OF THE GENES ENCODING CRF AND ITS RlECEPTORS IN FOOD-DEPRIWD LEAN

(Fa/?) AND OBESE (fdfa) ZUCKER RATS.

Elena TIMOFEEVA and Denis RICHARD

Département de Physiologie, Faculté de Médecine, Université Laval, Québec (Qué), G1K 7P4,

CANADA

Runnine TitIe: Food deprivation, obesity and the CRF system.

Kev words: Brain, corticotropin-releasing factor, corticotropin-releasing factor type 1 receptor.

corticotropin-releasing factor type 2 receptor, glucose, hypothalamic-pituitary-adrenal axis,

obesity, paraventricuIar nucleus of the hypothalamus, ventromedial nucleus of the

hypothalamus.

Mailing address: Dr Denis Richard, Département de Physiologie, Faculté de Médecine,

Université Laval, Québec (Qué), CANADA, G 1 K 7P4

Tel., 4 18-656-3348; FAX, 418-656-7898; E-mail, denis.richard@phs.ulaval.ca

L'effet de la privation de nourriture de différentes durées sur l'activation fonctionnelle

des neurones de la corticolibérine (CRF), de même que sur I'expression des gènes codant le

CRF et ses récepteurs de type 1 (CRFI-R) et de type 2 ( C e - R ) a été évalué dans le cerveau

des rats Zucker maigres (Fa/?) et obèses (falfa). Les rats Fa/? et fdfa étaient soumis à une

privation de noumture pour les périodes de 0 ,3 ,6 , 12, et 24 heures. Des mesures de Fos et de

I'ARNm du CRF ont été effectuées sur la même tranche de cerveau pour confirmer un état

d'activation des neurones à CRF. La technique d'hybridation in situ a été utilisée pour mesurer

I'ARNrn codant pour le CRF et ses récepteurs. La privation de nourriture a provoqué

I'expression rapide de Fos dans les cellules à CRF de certaines régions du cerveau des rats

obèses (fdfa), telles que le noyau paraventriculaire de l'hypothalamus (PVN), le noyau du lit de

la stria terminale (BNST), le noyau préoptique antérodorsal (ADP), le noyau préoptique médian

(MPN), la substantia innominata (SI) et le noyau de Barrington (BN). La colocalisation

Fosll'AEWm du CRF a été remarquable particulièrement dans le PVN des rats fdfa, où presque

toutes les cellules à CRF de la division parvoceIlu1aire du noyau ont manifesté la présence du

Fos dans les noyaux, après 12 heures de privation de nourriture. Chez les rats obèses, la

privation de nourriture a aussi entraîné l'augmentation des nivaux d'ARNm du CW dans le

PVN et le BNST de même que des hausses et baisses de I'expression de 1'ARNm des CRFI-R

dans le PVN et l'hypophyse antérieure. L'expression de CRFI-R dans le PVN des rats obèses

s'est produite après 12 heures de privation de nourriture. Chez les rats maigres (Fa/?), la

privation de noumture n'a pas causé une activation prononcée des cellules à CRF, et a produit

une diminution légère du niveau de I'ARNm du CRF dans le PVN, le BNST et le noyau central

de l'amygdale (CeA), et une diminution graduelle de l'expression du gène de C w - R dans le

noyau ventromédian de Ifhypothalamus (VMH). Ces résultats démontrent que la privation de

nourriture des rats Zucker obèses est capable de produire la réponse semblable au stress, qui se

reflète par l'activation particulière de l'axe corticotrope V A ) . Cette réponse est différente de

53

celle qui a été observée chez les rats Fa/?, laquelle dans ce cas-là était compatible avec des effets

du CRF sur la régulation de la balance énergétique.

The time course of the action of food deprivation on the functiond activation of corticotropin-

releasing hormone (Cm) neurons and on the expression of the genes encoding CRF and its

receptors of type 1 (CRFI-R) and 2a (CRF2-R) in the brain were assessed in lean (Fa/?) and

obese (fdfa) Zucker rats. Fa/? and fdfa rats were assigned to food deprivation periods of 0, 3,

6, 12, and 24 hours. Measurements of Fos immunoreactivity (ir) and CRF mRNA were carried

out on the same brain sections to assess the state of activation of CRF neurons. In sitri

hybridization histochemistry was ernployed to measure the rnRNAs encoding CRF and its

receptors. In fdfa rats, food deprivation induced a rapid expression of Fos in CRF cells of

several brain regions that include the panventricular nucleus of the hypothaiamus (PVN), the

bed nucleus of the stria terminalis (BNST). the anterodorsd preoptic nucleus (ADP), the media1

preoptic nucleus (MPN), the substantia innominata (SI) and the Barrington's nucleus (BN). The

colocalization of Fos and ir/CW mRNA was particularly noticeable in the PVN of falfa rats,

where most CRF cells of the parvocellular division of the nucleus displayed Fos-positive nuclei,

12 hours after the onset of fasting. In obese rats, food deprivation also produced an increase in

the CRF mRNA levels in the PVN and BNST as wei1 as high and low expressions of the

CRFI-R in, respectively, the PVN and the anterior lobe of the pituitary. The expression of

CRFI-R in the PVN of obese rats occurred 12 hours after the onset of the deprivation. In Fa/?

rats, food deprivation induce no marked activation of the CRF cells, a slow decrease in the CRF

mRNA Ievels in the PVN, the BNST and the central nucleus of the amygdala (CeA), and a

uradual decrease in the expression of CRF2-R gene in the ventromedial nucleus of the b

hypothalamus (VMH). These results demonstnte that food deprivation is capable of generating

in obese Zucker rats a stress-iike response that translates into a particularly striking activation of

the hypothalamic-pituitary-adrenal (HPA) a i s . This response contrasts with rhat observed in

Fa/? rats, in which the action of food deprivation on the CRF system seems more compatible

with the known effects of CRF in the regdation of energy balance.

INTRODUCTION

Corticotropin-releasing factor (CRF) is a 4 1 -residue peptide widely distri buted in the

brain and that plays a pivotal role in orchestrating the overall response of the body to stress

[1,2]. The most recognized of the numerous CRF actions is the control of the pituitary-adrenal

a i s , which is insured by CRF-containing neurons located in the media1 parvocellular division

of the hypothalamic paraventricular nucleus (PVN). Among the best documented effects of

CRF which are unrelated to the control of the pituitary-adrenal axis. are the anorectic and

thermogenic actions of this peptide [3,4]. CRF has been reported to be involved in the anorectic

effects of treadrnill running [5 ] , restraint stress [6] , estradiol [7] and caffeine [8], as well as in

the thermogenic actions of fenflurarnine [9] and the serotonin 2N2C receptor agonist, (f)- 1-

(2,s-dimethoxy-4-iodopheny1)-2-aminopropane (DOI) [IO]. In line with the anorectic and

thermogenic attributes of CRF, the hypothesis has been put forth that the development of

obesity can be due to a reduction in the central CW activity [3]. This inference has gained

strong support from studies emphasizing the inescapable action of adrenalectomy in preventing,

attenuating or, reversing obesity [ 1 1 - 171 and from the ability of CRF to block weight gain in

obese rats with little effect in lean animals [18].

The proposition of a deficient CRF system in animal obesity, though concordant with

the effects of CRF on food intake and energy expenditure, has yet to be ascertained. So far,

there has been no identification of either clear mechanisms whereby CRF could be involved in

the reguiation of energy balance or precise neuronal circuitries through which the anorectic and

thermogenic messages of CRF could be channeled. Furthemore, the characteristic increase in

the activity of the hypothdarnic-pituitary-adrenal (IIPA) axis seen in obese laboratory animals

clearly speaks in favor of the existence of an hyperactive CRF system in obesity [19-2 11.

The present study was conducted to investigate the effects of food deprivation on major

functional determinants of the CRF systern. Because it induces fat store variations leading to

marked adjustments in the control of food intake and energy expenditure, the food deprivation

paradigm proves very useful to investigate the regulation of energy balance. The effect of food

deprivation on the CRF system has only been partly investigated. A reduction in the CRF

mRNA Ievels has been observed following deprivation [22-241. However, this reduction has

been only observed in the medial parvocellular division of the PVN, where the function of the

CRF neurons seems oriented more towards the control of the HPA axis than towards the

regulation of energy balance. So far, there has been no report demonstrating effects of food

deprivation on the CRF mRNA levels outside the PVN. There is also no report documenting

the effects of food deprivation on the expression of the CRF receptor genes within the brain. In

this study, the functional activation of CRF neurons and the brain expression of the genes

encoding CRF and its receptors of type 1 (CRFI-R) and 2 a (CRF2-R) were detemined in

food-deprived lean (fafia) and obese (Fa/?) Zucker rats.

METHODS

Animais and treatments

Fa,? and fdfa Zucker rats, aged 6 weeks, were purchased from the Canadian

Breeding Laboratones (St-Constant, Canada). AI1 rats were cared for and handled according

to the Canadian Guide for the Care and Use of Laboratory Animals and the present protocol

was approved by our institutional animal care committee. The animals were housed

individuaily in wire-bottom cages suspended above absorbent paper and, otherwise specified,

fed ad libitum with a stock diet (Agway Prolab, RatlMouseIHamster 1000 Formula). They

were subjected to a 14:OO: 10:UO h dark-light cycle (lights on between 12:OO and 22:OO) and

kept under an ambient temperature of 23 f 1°C. Rats were killed following food deprivation

penods of 0, 3, 6, 12 and 24 hours. Because we intended to keep the time of sacrifice

constant, the food-removal times were varied according to the Iength of the food deprivation

periods; the time of sacrifice was at the beginning of the light period, the deprivation started 3,

6, 12, or 24 hours earlier.

Plasma determination

An intracardial blood sarnple was taken in anesthetized rats immediately before the

beginning of the intracardial perfusion with paraformaldehyde. Plasma glucose was determined

(glucose oxydase method) using a glucose analyzer (Beckman, Palo Alto, CA). Serum

corticosterone was determined by a cornpetitive protein-binding assay (sensitivity, 0.05 nmol/L;

inter-assay coefficient of variation, 9.0%) using plasma from a dexamethasone-treated fernale

rhesus monkey as the source of transcortin [25].

Brain and pituitary preparation

The brains and pituitaries were prepared as previously described [26]. Briefly, rats were

anesthetized with 1.5 ml of a mixture containing 20 mg/ml of ketamine and 2.5 mg/ml of

xylazine. Without delay, they were perfused intracardially with 30 ml of ice-cold isotonic saline

followed by 200 ml of a paraformaldehyde (4%) solution. The brains and pituitaries were

removed at the end of perfusion and kept in paraformaidehyde for an additionai period of 7

days. They were then transfened to a solution containing paraformaldehyde and sucrose (10%)

before being cut 12 hours later using a sliding microtome (Histoslide 2000, Reichert-Jung).

Brain sections were taken from the olfactory bulb to the brain stem. Thirty-mm-thick sections

were collected and stored at -30°C in a cold sterile cryoprotecting solution containing sodium

phosphate buffer (50 mM), ethylene glycol (30%), and glycero1(20%).

In situ hybridization histochemistry

In situ hybndization histochemistry was used to Iocalize CRF mRNA, CRFl-R and

CRF2-R -As on tissue sections taken from the entire brain and the pituitary. The protocol

58

used was largely adapted from the technique described by Simrnons et al. (1989) 1261.

Briefly, I out of every 5 brain sections as well as al1 the pituitary specimens were mounted

onto poly-L-lysine coated slides and ailowed to desiccate overnight under vacuum . The

sections were then successively fixed for 20 minutes in paraformaldehyde (4%), digested for

30 minutes at 37" C with proteinase K (10 pg/ml in 100 rnM Tris-HC1 containing 50 mM

EDTA, pH 8.0), acetylated with acetic anhydride (0.25% in O. 1 M trietholamine, pH 8.0) and

dehydrated through graded concentrations (50,70,95, and 100%) of alcohol. After vacuum

drying for at l eu t 2 hours, 90 ml of the hybridization mixture, which contains an antisense

3% labeled cRNA probe (107cpm/ml), were spotted on each slide. The slides were sealed

under a coverslip and incubaced overnight at 60°C in a slide warmer. The next day, the

coverslips were removed and the slides rinsed 4 tirnes with 4x SSC (0.6 M NaCI, 60 mM

trisodium citrate buffer, pH 7.0), digested for 30 minutes at 37' C with RNAse-A (20 pglml

in 10 mM Tris-500 rnM NaCl containing 1 mM EDTA)), washed in descending

concentrations of SSC (2x, 10 min; lx, 5 min; OSx, 5 min; O.lx, 30 min at 60' C) and

dehydrated through graded concentrations of alcohol. After a 2 hour-period of vacuum

drying, the slides were exposed on a X Ray film (Eastman Kodak, Rochester, NY) for

periods varying between 24 to 72 h, depending upon the nature of the probes used. Once

removed from the autoradiography cassettes, the slides were defatted in xylene and dipped in

NTB2 nuclear emulsion (Kodak). Again depending on the probe used, the slides were

exposed from 7 to 21 days, before being developed in Dl9 developer (Kodak) for 3.5

minutes at 14-lS°C and fixed in rapid fixer (Kodak) for 5 minutes. Finally, tissues were

rinsed in running distilled water for 1 to 2 h, counterstained with thionin (0.25%). dehydrated

through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX.

Combination of imrnunocytochemistry with in situ hybridization

Immunocytochemical detection of Fos, the protein encoded by the oncogene c-fos,

was combined with detection of CRF mRNA to determine whether CRF cells were activated

during food deprivation. Brain sections were first processed for irnmunochernical detection

of Fos using a conventional avidin-biotin-immunoperoxidase method. Briefly, brain slices

were washed in sterile 0.05M potassium phosphate-buffered saline (KPBS) that was treated

with DEPC water. They were then incubated for 24 hours at 4 OC with a Fos antibody (rabbit

polyclonal IgG, Oncogene Science, NY). The Fos antibody was used at a 150,000 dilution in

KPBS (50 rnM) with heparin (0.25%), triton X-100 (0.4%) and bovine semm albumin (28).

Twenty-four hours following incubation at 4°C with the first antibody, the brain slices were

rinsed in sterile KPBS and incubated with a mixture of KPBS, triton X-100, heparin, and

biotinylated goat antirabbit IgG (1: 1,500 dilution; Vector Laboratories, CA) for 90 min.

Sections were then rinsed with KPBS and incubated at room temperature for 60 min with an

avidin-biotin-peroxidase complex (Vectastain ABC Elite Kit, Vector Laboratories, CA),

followed by a second incubation with a mixture of KPBS, triton-X-100, heparin and

biotinylated goat antirabbit IgG with the ABC Elite solution. After several rinses in sterile

KPBS, the brain slices were allowed to react in a mixture containing sterile KPBS, the

chromagen 3,3'-diarninobenzidine tetrahydrochloride (Dm, O.OS'%), and 1% hydrogen

peroxide. Thereafter, tissues were rinsed in sterile KPBS, mounted ont0 poly-L-lysine-coated

slides, desiccated overnight under vacuum, fixed in paraformaldehyde (4%) for 30 min, and

digested for 30 min at 3 7 O C with proteinase K (10 pg/ml in 100 m M Tris-HC1 containing 50

mlM EDTA, pH 8.0). Prehybridization, hybridization, and post-hybridization steps were

performed as described above except for the dehydration step, which was shortened to avoid

decolorization of Fos-immunoreactive (Fos-ir) cells. After vacuum drying for 2 hours,

sections were exposed ont0 X-ray film, defatted in xylene, and dipped in the NTB2 nuclear

emulsion. Slides were exposed for 7 days, developed in D l 9 developer for 3.5 min at 15' C,

and fixed in rapid fixer for 5 min. Thereafter, tissues were rinsed in running distilled water

for 1 to 2 h, rapidly dehydrated through graded concentrations of alcohol, cleared in xylene,

and coverslipped with DPX.

60

Antisense 3%-labeled cRNA probes

The CRF cRNA probe was generated from the 1.2 kb EcoR 1 fragment of rat CRF

cDNA (Dr. K. Mayo, Northwestem University, Evanston, IL, USA) subcloned into pGEM4

plasmid (Stratagene, La Jolla, CA), and linearized with Hind III and EcoR I (Pharmacia

Biotech Inc., Canada) for antisense and sense probes, respectively. The CRFI-R cRNA probe

was generated from a 1.3 kb Pst 1-Pst 1 fragment of the rat prCRF PP 1.343s cDNA (Dr. W.

Vale, Peptide Biology Laboratory, The Salk Institute) subcloned into pBIuescript II SK

(Stratagene, La Jolla, CA) and linearized with BamH 1 and Hind III to generate antisense

and sense probes, respectively. The specific rat CRF2-R CRNA probe was prepared from a

275 bp fragment of the 5' region cDNA of the CRFza receptor (Dr. T.W. Lovenberg,

Neurocrine Biosciences Inc.) subcloned into a pBluescript SK+ (Stratagene, La Jolla, CA)

and linearized with EcoR I and BamH I for antisense and sense probes, respectively. The

specificity of each probe was confirmed by the absence of positive signal in sections

hybridized with sense probe. Radioactive riboprobes were synthesized by incubation of 250

np linearized plasmid in 10 rnM NaCl, 10 mM dithiothreitol, 6 rnM MgCl, 40 rnM Tris (pH

7.9), 0.2 mM ATP/GTP/CTP, a-%-UTP, 40 U RNasin (Promega, Madison, WI), and 20 U

SP 6 , T7 or T3 RNA polimerase for CRF, CRFI-R and CRF2-R antisense probes,

respectively, for 60 min at 37OC. The DNA templates were treated with LOO ml of DNAse

solution (1 ml DNAse, 5 ml of 5 rng/ml RNA, 94 ml of 10 rnM TrisllO rnM MgC12). The

preparation of the riboprobes was accomplished the phenol-chloroform extraction and

ammonium acetate precipitation.

Quantitative analysis of the hybridization signals

The hybridization signals revealed on NTBZ dipped nuclear emulsion slides were

analyzed and quantified under a light microscope (Olympus, BX50) equipped with a black and

white video camera (Sony, XC-77) coupled to a Macintosh cornputer (Power PC 7100/66)

using Image software (version 1.55 non-FPU, Wayne Rasband, NIH, Bethesda, MD). The

61

optical density (OD) of the hybridization signal was measured under dark-field illumination at

a magnification of 25x. The brain sections from the different groups of rats were matched for

rostrocaudal levels as closely as possible. The OD determination was performed on each side

of the brain and the tow OD values obtained were averaged. This average was included in the

statistical analyses as the individual score of a rat. When no hybridization signal was visible

under darkfield illumination, the brain structures of interest were outlined under brigthfield

illumination and then subjected to densitometric analysis under darkfield illumination. The OD

for each specific region was corrected for the average background signal, which was

determined by sampling unlabeled areas outside of the areas of interest.

Statistical analysis

A 2 x 5 analysis of variance (ANOVA) was used to detect significant (Pc0.05) main

and interaction effects of phenotype [lean (Fa/?), obese (fdfa)] and time [O (fed ad libitum), 3,

6, 12, 24 hours] on the various dependent variables measured in this ctudy. Each of the ten

groups used included 4 animals. When a significant main effect of time or a significant

phenotype x time interaction occurred, a posteriori comparisons were performed using the

BonferoniDunn multiple-cornparison-procedure. In the case of a significant main effect of

time, 4 cornparisons were performed, the level of significance (P value) being accordingly

adjusted to 0.0125 (0.05/4); the mean of each of the food-deprived groups (3, 6 , 12, 24 hours)

was compared to the mean of the group fed ad libitum (time O). In the case of a significant

phenotype x time interaction, 13 comparisons were performed with a level of significance (P

value) adjusted to 0.0038 (0.05/13); within each level of phenotype. the mean of each of the

food-deprived groups (3, 6 , 12, 24 hours) was compared to the mean of the group fed ad

libitum (time O), and within each level of tirne, obese rats were compared to lean animals.

RESULTS

Body weight, plasma levels of glucose and corticosterone

Table I presents the time course of the action of food deprivation on epididymal fat

weight, plasma levels of glucose and plasma levels of corticosterone in Fa/? and falfa rats.

Values for fat weight, plasma glucose and plasma corticosterone were al1 higher in obese rats

than in lean anirnals. In Fa/?, plasma glucose levels tended to decrease throughout the food

restriction period but this effect was not significant. Regardless of the phenotype of the rats,

corticosterone levels were significantly above ad libifrrm levels following 12 and 24 hours of

deprivation.

Fos immunoreactivity (ir)/CRF mRNA

CRF cells of many structures displayed Fos-positive nuclei in food-deprived obese

rats. This is emphasized in table 2, which presents the list of the brain structures containing

Fos ir/CRF mRNA neurons with the number of these cells for each structure, 12 hours

following the onset of food deprivation. In al1 the regions examined, the number of double

stained cells were unequivocaIly higher in fdfa rats than in Fa/? rats.

Figure 1 illustrates the time course of the action of food deprivation on the number of

Fos-positive CRF cells in the PVN of Fa/? and fdfa rats. The amount of cells colocaiizing

Fos ir and CRF mRNA was very low in the PVN of Fa/? rats. In falfa animals, 3 hours of

food deprivation were sufficient to sharpIy increase the number of double-labeled cells in the

PVN. The number of these cells remained etevated even after 24 hours of food deprivation.

In obese anirnals, the majority the PVN CRF cells were found to be Fos-positive, 12 hours

following the removal of food. This is illustrated in figure 2, which demonstrates coronal

PVN sections labeled for Fos (dark brown nuclei) and CRF mRNA (silver grains).

CRF mRNA expression

Figure 3 ilhstrates the time course of the action of food deprivation on the CRF

rnRNA in different brain regions of Fa/? and fdfa rats. ANOVA revealed a significant effect

of phenotype on CRF mRNA in the PVN, the levels being higher in obese than in Iean rats.

Although the phenotype x time interaction was not statistically significant, the bar graph

(figure 3, upper right panel) suggests that the effect of phenotype on PVN CRF mRNA was

largely dependent on the presence of food-restricted groups. In the BNST, ANOVA revealed

a significant phenotype by time interaction; 24 hours of food deprivation led to reduction in

the BNST IeveIs of CRF mRNA in lean rats and an increase in these levels in obese animals.

In the centrai nucleus of the amygdala (CeA), CRF mRNA leveIs decreased following

deprivation in Fa/? whereas they remain stable in faifa rats. The CRF mRNA expression in

the MPN and the BN was not significantly affected by the various experimental treatments of

this study. Figure 4 illustrates the effects of 12 hours of food deprivation on the CRF mRNA

levels in the PVN of Fa/? and fdfa rats.

CRFl receptor mRNA

The CRFI-R message was found in abundance in neocortical, amygdaloid

(basolateral and media1 nuciei), mesencephalic (interpeduncular and red nuclei) and rnedullary

(cerebellar cortex, pontine gray, and lateral dorsal tegmental and motor trigemmal nuclei)

structures (data not shown). Figure 5 illustrates the time course of the action of food

deprivation on CRFl-R mRNA in the PVN and the pituitary of Fa/? and fdfa rats. CRFi-R

mRNA levels were very low (barely detectable) in the PVN of fed lean and obese animais

(figure 6 ) and remained low during food deprivation in Fa/? rats. In contrast, food deprivation

led to a marked increase in the CRFI-R mRNA levels in the PVN of falfa rats. The increase

in the C m l - R message was significant from 6 to 24 hours after the beginning of the

deprivation and manifested itself within the parvocelluIar division of the PVN. In the

pituitary, the effects of food deprivation on the expression of the CRFI-R gene were

phenotype- and site-dependent (figure 5 and 7). In the anterior lobe (AL) of the pituitary, food

deprivation Ied to a graduai increase in the expression of CRFI-R mRNA in Fal? rats,

whereas it generates a slow decrease in this expression in falfa rats. In the intermediate lobe

(IL) of the pituitary, food deprivation induced a progressive increase in the expression of

CRFI-R m W A in both Fa/? and fdfa rats. The levels after 12 and 24 hours of deprivation

were higher than the levels measured in animals fed ad libitrim. The levels of mRNA were

higher in fdfa rats than in Fa/? animals.

CRF2 receptor mRNA

The CRF2-R rnRNA is concentrated in the lateral septum and in the ventromedial

nucleus of the hypothalamus (VMH). Whereas our treatments did not affect the expression

of CRF2-R mRNA in the septum, they markedly affected the expression of the gene in the

VMH (Figures 8 and 9). Figure 8 illustrates the time course of the action of food deprivation

on the expression of CRF2-R mRNA in the VMH of Fal? and fdfa rats. In Fa/? rats, food

deprivation led to a reduction in the VMH levels of CRF2-R M A . This reduction became

significant 6 hours after the onset of the deprivation and remained significant until the end of

the fasting period. In fdfa rats, food deprivation did not induce any significant changes in the

VMH levels of CRF2-R mR!\JA. However, the VMH levels of CRF2-R mRNA in fed W h

rats were lower than those of fed Fa/?rats and already at the Ievel seen in Fa/? rats after 24

hours of deprivation. Figure 9 depicts the C R b - R mRNA hybridization signal in the VMH

of fed and food-deprived lean and obese rats.

DISCUSSION

The results of this study demonstrate that food deprivation can induce a quickly

occumng and long lasting stimulation of the CRF system in fdfa rats. In fact, less than three

hours of deprivation turned out ta be sufficient to induce expression of Fos in CRF ceIIs of

many brain regions, and this enhanced expression was still manifest 24 hours following the

beginning of the deprivation. Fos is a phosphoprotein encoded by the immediate-early gene c-

fos, and this protein is quickly and transiently expressed in activûted cells of various brain

regions in response to a panoply of external stimuli [27,28]. The mapping of Fos (or c-fos

mRNA) has been extensively and advantageousty used as a general approach to identify and

characterize functionally activated neurons. The presence of Fos in CRF neurons has been

positively coupled to the activation of CRF neurons in numerous experimental paradigms. Fos

expression in CRF neurons has been associated with an increased activity of the CRF system in

experimental conditions that inciude immobilization [29], treadmill running [30], hernorrhage

1311, dehydration 1321, salt loading [33], immune challenge 1341 and treatment with

serotoninergic anorectic agents [35,36]. Despite the link between the expression of Fos and the

activity of the CRF neurons, i t seems now clear that there is no direct functional relationship

between Fos and the CRF gene. The promoter of the CRF gene Iacks the DNA sequence,

TGACTCA [3 11, known as the consensus site of the aminopyridine- 1 (AP- 1) binding site,

upon which Fos acts to regulate the transcription of target genes [27,28]. There is aiso evidence

that the gene transcription of CRF precedes that of c-fos, in response to a stressor 137,383.

The excitation of CRF neurons that was induced by fasting in faffa rats was particularly

striking in the parvocellular division of the PVN, where the majority of the CRF celis displayed

Fos-positive nuclei after 12 hours of food deprivation. Given that the CRF cells of the

parvocelluIar division of the PVN fulfiIl the endocrine function of CRF, it can be argued that

food deprivation stimulates the activity of the pituitary-adrenal axis via a centrai mechanism.

The observation that food deprivation can be associated to a stimulation of the HPA a i s in fdfa

rats is consistent with the generai view that obese laboratory rodents exhibit overactive HPA

axes [19-21,391. At variance with the PVN, the activity of CE2F neurons in other regions such

as the substantia innominata (SI), the MPN, the BNST and the BN cannot be readily assigned

to a specific CRFergic function. Indeed, a very substantial number of CE-containing celIs

outside the PVN displayed Fos immunoreactivity after deprivation. The possibility that sorne

CRF-positive cells of the BNST and the MPOA can assist the PVN in the control of the HPA

axis cannot be excluded [40]. Similarly, the possibility that the stimulation of CRF cells in the

MPOA and the B N can be implicated in the changes induced by food deprivation on,

respectively, reproduction [30] and miction [41] cannot be precluded. The factors responsible

for the activation of CRF system following food deprivation has yet to be described. The fat9

Cfa) phenotype is due to a nuII mutation of the leptin receptor and the possibility of a functional

relationship between the lack of ieptin responsiveness and the stress-like response to food

deprivation in fa/fa rats deserves attention. Leptin has been reported to attenude the activation of

the HPA axis in food-restricted [42] and in ob/ob (Huang, Q. and Richard, D., unpublished

results) mice. In addition, leptin has been shown to improve the functionality of the

reproductive system in fasted [42] and ob/ob [43] mice, which is consonant with an inhibitory

effect of leprin on sorne CRF neurons, given CRF represents as an effective inhibitor of the

reproductive system [30].

The present resuIts indicate that food deprivation is capable of inducing a transient

expression of CRFI-R mRl\tA in the PVN of fdfa rats. The expression of the CRFI-R gene

occurs in the parvoceIluIar division of the PVN, and the peak of this expression takes place

twelve hours after the onset of the deprivation. This finding is consistent with the results of a

previous study [44], in which we observed an increased expression of the CRFI-R gene in

obese rats subjected to an ovemight fast. The mechanisms responsible for the stimulation of the

CRFl-R gene expression in the PVN of fdfa rats are not clear. The presence of high Ievels of

corticosterone in obese mutants is unlikely to be part of such rnechanisms, as recent results

tended to dismiss the role of corticosterone in the increased stimulation of CRFI-R gene

expression [45]. The effects of food deprivation on the expression of PVN CRFI-R mRNA

were similar to the effects of treadmill running and immobilization, which also induce transient

and delayed expression of the CRFI-R gene [34.44]. In contrast to what was observed in the

PVN, the expression of the CRFI-R gene in the AL of the pituitary of obese rats decreased

during food deprivation. Given the acknowledged down-regulating effect of CRF on the

expression of the CRFI-R gene in cells of the AL of the pituitary 1461, the present results may

be seen as supportive of an increased activity of the CRF neurons in the PVN of obese rats.

In Fa/? rats, food deprivation induced no marked expression of Fos within CRF

neurons. Furthemore, it reduced CRF mRNA levels in the BNST and the CeA, suggesting that

the action of food deprivation on the CRF system in lean rats is rather inhibitory than

stimulatory. The factors responsible for the reduction of CRF mRNA levels in the BNST and

the CeA of Fa/? rats are not known. The possibility that this reduction is caused by the high

circulating levels of corticosterone induced by food deprivation cannot be excluded. In fact,

CRF biosynthesis in namely the BNST and the CeA has been reported to be negatively

regulated by corticosterone 1471. The observation that the activity of the CRF system is

decreased in food-deprived lean rats is consistent with the demonstration that the high plasma

level of corticosterone measured in these rats probably emeges from a reduced turnover rate

[48] than from an increased central activation of the secretion.. In contrast to what is seen in

obese rats. there indeed appears to be no central activation of the CRF system in lean rats up to

24 hours of deprivation. Another factor that could contributed to the reduction of the CRF

expression in fasted lean rats is the reduction of Ieptin levels that occurred following food

deprivation. Acute intracerebroventricuiar administration of Ieptin has been reported to restore

the CRF rn RNA expression to normal levels in the PVN of food-restricted lean rats [49].

The present results provide evidence that food deprivation c m reduce the expression of

the CRF2-R gene in the VMH. The mechanisrns underlying this reduction remain to be

delineated. Because of the role played by VMH in the regulation of energy balance, the

reduction in the VMH levels of CRF2-R mRNA could represent an important adaptation

contributing to the increased energetic efficiency led to by food deprivation. The destruction of

the VMH has been known for years to promote energy deposition. The hypothesis associating

VMH lesion to the attenuation of the CW action in this structure warrants verification. Indeed,

the reduction in the CRF2-R message, being seen in both fdfa [44] and food-depnved rats,

represents the only finding reported so far, which is congrnous with the view of a reduced CRF

activity tone in conditions of enhanced energetic efficiency.

Given the anorectic and thermogenic propenies of CRF [3,4], one would not a priori

predict, in obese rats, a general excitation of the CRF system following a condition leading to a

negative energy balance. However, although the strong CRFegic response of falfa rats to food

deprivation emerges from this study as unexpected, it does not invariably invalidate the role of

C W in the regulation of energy balance. The convincing demonstration of the ability of a brain

infusion of a non-hypophysiotropic dose of CRF to reduce weight gain in obese rats [18]

strongly supports the hypothesis that the activity of the CRF system is reduced in obese rats.

The fact that food deprivation induced a prompt and widespread stimulation of CRF neurons as

well as high and Iow expression of the CWl-R transcript in the PVN and the AL of the

pituitary demonstrates that obese rats respond to food deprivation in a similar manner as they

react to stress [44]. The possibility that the marked stress-like response induced by food

removal in fa/fa rats drives fdfa rats to eat cannot be precluded, especially since that the

generalized stimulation the CRF system causes an important secretion of corticosteroids that

can overcome the potential anorecticlthermogenic effects of CRF and enhance fat deposition

[50]. In addition, a generalized activation of the CRF neurons does not exclude the possibility of

adjustments that could reduce the activity of the CRF action in brain regions pnmarily devoted

to the regulation of energy balance. The befow-control CRF2-R message in the VMH of fdfa

rats [44] could represent such an adjustment to reduce the CRF tone and to enhance energetic

efficiency. Another adaptation that could contribute to reduce CRF activity in obese animals is

an increase in the production of the CRF-binding protein (CRF-BP) in nuclei involved in the

regulation of energy balance. CRF-BP is regarded as a CRF inactivator protein [5 11. The use of

CRF-molecule fra,sments blocking the access to CRF-BP but not that to the CRF receptors [53]

proved recently capable of blunting the excessive weight gain of fdfa rats [53].

The present results demonstrate that food deprivation exerts site- time- and genotype-

dependent effects on major functional determinants of the CRF neurosystem. In fa/fa rats, food

deprivation induced i) a prompt and widespread stimulation of CRF neurons, ii) high and low

expressions of the CRF type 1 transcript in the PVN and the AL of the pituitary. In Fa/? rats,

food deprivation led to i) no marked stimulation of the CRF neurons, i i ) a gradua1 decrease in

the CRF mRNA levels in the PVN, the BNST and the CeA, ii i) a slow decrease in the

expression of CRF2-R mRNA in the VMH. AI1 together the results of this study provide

evidence that food deprivation induces a stress-like response in fdfa rats that translates in a

particularly striking activation of the HPA axis. This response differs from that exerted in Fa/?

rats, in which food deprivation alter the activity of the CRF system in a way more compatible

with the known effects of CRF in the regulation of energy balance.

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LEGENDS TO FIGURES

Figure 1. Time course of the action of food deprivation on the number of cells colocalizing

Fos immunoractivity (ir) and corticotropin-releasing hormone (CRF) mRNA on the sarne

coronal brain sections taken from the paraventricular nucleus of the hypothalamus (PVN) of

lem (Fa/?) and obese (fa/fa) Zucker rats. A 2 x 5 analysis of variance (ANOVA) was used to

examine the main and interaction effects of phenotype [Iean (Fa/?), obese (fdfa)] and food

deprivation [O, 3, 6 , 12, 24 hours] on the number of ceI1s colocalizing Fos ir and CRF

mRNA, followed by a posteriori Bonferroni-Dunn multiple-comparison procedure. * Significantly different from ad libitum group within the same phenotype, p < 0.05.

Figure 2. Photomicrographs of coronal brain sections taken from the paraventricular nucleus of

the hypothalamus (PVN) and displaying cells colocalizing Fos immunoractivity (ir) (brown

nuclei) and corticotropin-releasing hormone (CRF) mRNA (silver grains within cytoplasm).

Brain sections (30 mm thick) were obtained from lean (Fa/?) and obese (fdfa) Zucker rats

killed when fed ad libitum or following 12 houn of deprivation. Some of the colocalizations are

indicated by arrows. The scale bars correspond to 100 pm and 20 pm for, respectively, the four

upper and two lower panels.

Figure 3. Time course of the action of food deprivation on the optical densities (ODs) of the

hybridization signal of corticotropin-releasing hormone (CRF) mRNA for different brain

regions. The ODs were detennined under microscope on coronal brain sections taken from the

paraventricular nucleus of the hypothalamus (PVN), the media1 preoptic nucleus (MPN), the

bed nucleus of the stria terminalis (BNST), the central nucleus of the amygdala (CeA), and the

Barrington's nucleus (BN) lean (Fa/?) and obese (fdfa) Zucker rats. A 2 x 5 analysis of

variance (ANOVA) was used to examine the main and interaction effects of phenotype [lean

(Fa/?), obese (fdfa)] and food deprivation [O, 3, 6, 12, 24 hours], followed by a posteriori

Bonferroni-Dunn multiple-cornparison procedure. * Significantly different from ad libittm

group within the same phenotype, # Significantly different frorn lean group of the same time

point, p < 0.05.

Figure 4. Dark-field photornicrographs of coronal brain sections taken from the paraventricular

nucleus of the hypothalamus (PVN) depicting corticotropin-releasing hormone (CRF) mRNA.

The brain sections (30 mm thick) were obtained from lean (Fa/?) and obese (fa/fa) Zucker rats

killed when fed ad libitum or following 12 hours of deprivation. The scale bar corresponds to

200 Pm.

Figure 5. Time course of the action of food deprivation on the optical densities (ODs) of the

hybridization signal of corticotropin-releasing hormone (CRF) receptor of type 1 (CRFI

receptor) in the paraventricular nucleus of the hypothalamus (PVN) and in the pituitary. The

ODs were determined under microscope on coronal PVN sections taken from lean (Fa.?) and

obese (fdfa) Zucker rats. A 2 x 5 analysis of variance (ANOVA) was used to examine the

main and interaction effects of phenotype [lean (Fal?), obese (fdfa)] and food deprivation [O,

3, 6 , 12, 24 hours]. followed by a posteriori Bonferroni-Dunn multiple-cornparison

procedure. * Significantly different €rom ad libitrcrn group within the same phenotype. ** -

Significantly different from al1 the other groups of rats. i Significantly different from Lean

(Fa/?), p < 0.05.

Figure 6. Dark-field photomicrographs of coronal brain sections taken from the paraventricular

nucleus of the hypothalamus (PVN) depicting mRNA signals for corticotropin-releasing

hormone (0 receptor of type 1 (CRFI receptor). The brain sections (30 mm thick) were

obtained from lem (Fa/?) and obese (fa/fa) Zucker rats killed when fed ad libitum or following

12 hours of deprivation. The scale bar corresponds to 200 Pm.

Figure 7. Dark-field photomicrographs of coronal brain sections illustrating hybridization

signal for corticotropin-releasing hormone (CRF) receptor of type 1 (CRFI receptor) mRNA in

the anterior (Ai,) and intermediate (IL) lobes of the pituitary. The brain sections (30 mm thick)

were obtained from lean (Fa/?) and obese (idfa) Zucker rats killed when fed ad libitum or

followinp 12 hours of deprivation. The scale bar corresponds to 200 Fm.

Figure 8. Time course of the action of food deprivation on the optical densities (ODs) of the

hybridization signal of corticotropin-releasing hormone (CRF) receptor of type 2 (CRF;!

receptor) in the ventromedial nucleus of the hypothalamus (VMH). The ODs were

determined under microscope on coronal PVN sections taken from lean (Fat?) and obese

(falfa) Zucker rats. A 2 x 5 analysis of variance (ANOVA) was used to examine the main

and interaction effects of phenotype [lean (Fa'?), obese (fatfa)] and food deprivation [O, 3,6,

12, 24 hours], followed by a posteriori Bonferroni-Dunn multiple-cornpanson procedure. * L

Significantly different from ad libitum group within the same phenotype. 1 Significantly

different from Lean (Fat?), p < 0.05.

Figure 9. Film autoradiograms of coronal brain sections illustrating hybridization signal for

corticotropin-releasing hormone (CRF) receptor of type 2 (CRF;! receptor) mRNA in the

ventromedial nucleus of the hypothalamus (VMH). Coronal brain sections (30 mm thick) were

obtained from lean (Fa/?) and obese ( W h ) rats that were killed either in fed state or following a

food deprivation period of 48 hours.

Fos irICRF mRNA in the PVN

Number of cells

ANOVA

T h e (T) Phenotype (Ph) T x Ph

1 Ad libitum

Lean Obese

Figure 1

PVN Fos-ir 1 CRF mRNA

Lean (Fa/?) Obese (fa/fa) Figure 2

CRF mRNA

Ad libitum

Fooddep. 3h

E l Il 6h

la II 12h II 24h

l ANOVA

Time (T) 0.052 1 Phenotype (Ph) O. 1 28

0.297

Lean Obese

Time (T) < 0.00 1 OD Phenotype (Ph) 0.172

1 ANOVA E I O ,

Time 0.79 1 l Phenotype (Ph) < 0.00 1

OD 1 T x P h 0.101 1

Lean

T T ,

Obese ANOVA

Time (T)

Lean Obese

Lean Obese

BN

Lean

ANOVA - P Time (T) 0.222

Obese

Phenotype (Ph) 0.483 T x Ph 0.914

Figure 3

PVN CRF mRNA

Ad libitum

Lean (Fa/?) Obese (falfa)

Figure 4

CRFl receptor mRNA

1 Ad libitum

Food dep. 3h

la 11 6h I I 12h 1 I 24h

- -

ANOVA - P 0.268

ph) il189 < 0.001

Lean Obese

1 ANOVA

Time (T) < 0.00 1

OD Phenotype (Ph) c 0.00 1 0.067

120 7 i T i p h 1

Lean Obese

Figure 5

Lean Obese

PVN CRF 1 receptor mRNA

Ad libitum

Lean (Fa/?) Obese (falfa)

Figure 6

Pituitary CRF 1 receptor mRNA

Ad libitum

Lean (Fa/?) Obese (falfa)

Figure 7

CRI?, receptor mRNA in the VMH

ANOVA - P

Tirne (T) O .O02 Phenotype (Ph) c 0.001 T x Ph 0.048

/ Ad libitum

Food dep. 3h

ta II 6h 11 12h

rn II 24h

Lean Obese

Figure 8

VMH CRFWY receptor mRNA

Ad libitum

Lean (Fa/?) Obese (falfa)

Figure 9

Table 1. Time course of the action of food restriction on body weight, plasma levels of glucose and plasma levels of corticosterone in lean (Fa/?) and obese (falfa) Zucker rats.

Epididymal fat Glucose Corticosterone

(g) (m0W (mmoV1)

Lean (Fa/?)

Ad libitum

Food deprived 3h II 6h II 12h 1 t 24h

Obese (fa+fa) --

Ad libitum

Food deprived 3h

Significant effects

(P .c 0.05)

Values represent means f SEM of four anirnals per experimentd conditions. T, tirne; Ph - p heno type.

Table 2. Brain structures containing cells coexpressing Fos and CRF rnRNA and number

of doubly-labeled cells for lean (Fa/) and obese (falfa) Zucker rats 12 hours after the onset

of food-deprivation

BRAIN STRUCTURES Fat? fdfa

Bed nucleus of stria terminalis

Piriform cortex

Substantia innominata (rostral)

Substantia innominata (caudal)

Anterodorsal preoptic nucleus

Mediai preoptic area

Medial preoptic nucleus

Central arnygdaloid nucleus

Periventricular hypothalamic nucleus

Paraventricular hypothalarnic nucleus

Arcuate hypothalarnic nucleus

Lateral mamrnillary nucleus

Dorsal raphe nucleus

Barrington's nucleus

Pontine central gray (including dorsal and -

laterodorsal tegmentai nuclei)

These values represent the meankSEM of four animals per group. The double-labeled cells were calculated in each side of nuclei for one in five series of brain sections.

CHAPITRE IV

CONCLUSION GÉNÉRALE

91

The understanding.of the central mechanisms of the regulation of energy balance is an

important goal for the treatment of human obesity and obesity-reIated health problerns, such as

cardio-vascular diseases and non-insulin-dependent diabetes mellitus. CRF plays a key role in

the metabolic balance because of its hypophisiotropic (Vale W et al.. 198 1). anorectic and

thermogenic effects (Rothwell W, 1990; Richard D, 1993). We performed these studies in

genetically obese Zucker rats to explore the contributions of CRF and its receptors in the

regulation of energy balance.

Treadrnill mnning and food deprivation were used to investigate the responsiveness of

the CRF system in genetically obese Zucker rats. The measurement of the plasma

corticosterone and ACTH was performed to monitor the pituitary-adrenal activity. The efiects

of the treatments on the expression of CRF hnRNA, CRF mRNA, CW receptors transcripts

were investigated using the method of in situ hybridization histochemistry. The combination

of immunocytochemistry with in situ hybridization histochemistry was performed to study

the expression of the Fos protein in the CRF synthesizing neurons.

The mnning levels of CRF hnRNA in both the PVN and MPOA were higher in obese

rats than in lean animals. Also, we recently showed an increase in the CRF mRNA expression

after 48 hours of food deprivation in the PVN of obese rats and its decrease in lean rats in the

same experimental conditions (Timofeeva E et al., 1996). The observation of an increase in the

PVN CRF transcripts in obese rats in spite of higher corticosterone levels in post mnning and

food deprived animals, suggests a lower negative feedback regulation by the corticosterone of

the CRF synthesis in fdfa rats. This dysfunction has the similarity with the described

weakening of the negative corticosterone feedback regulation of HPA axis that occurred in

chronically stressed anirnals (Young EA et ai., 1990; Makino S et ai., 1995; Herman JP et al..

1995). Notwithstanding the involved rnechanisms, Our results strongly suggest an enhanced

sensitivity of the CRF neurocrine system in obese fdfa rats to stressful conditions. The Fos

protein synthesis was observed in the parvocellular PVN of obese but not in that of lem rats as

soon as after 3 hours of food deprivation. CRF synthesizing cells that expressed Fos protein in

obese food-deprived rats were a i s 0 found in other hypothalamic nuclei as well as in the nuclei

of limbic system and in the pons. The parvocellular PVN CRFl receptors levels were highly

increased in obese but not in lean rats after 12 houn of food deprivation. This increase in the

C W i receptors levels was similar to those seen in the PVN of both lean and obese rats after

treadmill running. Taken together the enhanced the CRFi receptor expression in the

parvocellular PVN as well as the synthesis of Fos protein in the CRFergic neurones of the

PVN and other brain nuclei in obese rats suggest that food deprivation is a stressful condition

for fdfa rats. The obtained patterns of brain nuclei stimulation in falfa rats after treadmill

running as well as after food deprivation is similar to that seen in neurogenic stresses

(Sawchenko PE et al., 1996). The neuronal circuits, involved in stimulation of the CRF

system in response to food deprivation remain to be elucidated.

Because of the anorectic and thermogenic effects of CRF (Rothwell NJ, 1990; Richard

D, 1993), and the convincing demonstrations of the ability of a brain infusion of a

nonhypophysiotropic dose of CRF to reduce the weight gain of obese rats (Rohner-Jeanrenaud

F et al., 1989), a reduced activity of the CRF system in obese rats could be suggested. The

low levels of the VMH CRF2a receptors in both the obese and food-deprived rats could

represent a mechanism leading to a reduction of CRF tone, affecting energy balance. The

source of CRF secreted in the VMH remains to be investigated.

These studies emphasized the hyperresponsiveness of the HPA axis in obese (fa/fa)

Zucker rats in a stressful conditions. The food deprivation induces the widespread stimulation

of the CRF neurons in obese but not in lean rats. The stress-like response to food deprivation

in obese fdfa rats resembles that seen in neurogenic (Sawchenko PE et al., 1996) or

processive (Herman JI?, Cullinan WE, 1997) stresses. Probably this hypersensitivity of HPA

axis of obese rats to different stimuli influences the development of the obese phenotype.

Indeed, hyperactivity of the HPA axis produces hypercorticosteronemja. The role of

corticosterone in the development of obesity is well established by the demonstration the

inescapable effect of adrenaiectomy or antiglucocorticoids in preventing, attenuating, or

revening obesity (LngIey SC, York DA, 1990; Castonguay TW et aL. 1986; Romsos DR et

ai., 1987).

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