FOOD DEPRIVATION DIFFERENTIALLY MODULATES OREXIN

RECEPTOR EXPRESSION AND SIGNALLING IN THE RAT

HYPOTHALAMUS AND ADRENAL CORTEX

1Emmanouil Karteris, 1Rachel J Machado, Jing 1Chen, 1Sevasti Zervou 2Edward W

Hillhouse and 1Harpal S Randeva

1Biomedical Research Institute, Department of Biological Sciences, University of

Warwick, Coventry, CV4 7AL; 2The Dean’s Office, The Medical School, University

of Leeds, Leeds, LS2 9NL

Corresponding author: Dr E. Karteris Molecular Medicine Research Group Biomedical Research Institute University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, UK. Tele: +44 2476 524 744 Fax: +44 2476 523 701 email: e.k.karteris@warwick.ac.uk

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Articles in PresS. Am J Physiol Endocrinol Metab (January 25, 2005). doi:10.1152/ajpendo.00351.2004

Copyright © 2005 by the American Physiological Society.

ABSTRACT

Although starvation-induced biochemical and metabolic changes are perceived by the

hypothalamus, the adrenal gland plays a key role in the integration of metabolic activity

and energy balance, implicating feeding as a major synchronizer of rhythms in the

hypothalamic-pituitary-adrenal (HPA) axis. Given that orexins are involved in the

regulation of food intake and the activation of HPA axis, we hypothesized that food

deprivation, an acute challenge to the systems that regulate energy balance, should elicit

changes in orexin receptor signalling at the hypothalamic and adrenal level.

Food deprivation induced both orexin type-1 (OX1R) and type-2 (OX2R) receptors at

mRNA and protein level in the hypothalamus, in addition to a five-fold increase in

prepro-orexin mRNA. Both cleaved peptides OR-A and OR-B are also elevated at the

protein level. Interestingly, adrenal OX1R and OX2R levels were significantly

reduced in food-deprived animals, whereas there was no expression of prepro-orexin

in the adrenal gland in either state.

Food deprivation exerted a differential effect on OXR-G protein coupling. In the

hypothalamus, of food deprived rats, compared to controls, a significant increase in

coupling of orexin receptors to Gq/11, Gs and Go was demonstrated, whereas

coupling to Gi was relatively less. However, in the adrenal cortex of the food-deprived

animal there was decreased coupling of orexin receptors to Gs, Go, and Gq/11 and

increased coupling to Gi. Subsequent second messenger studies (cAMP/IP3) have

supported these findings.

Our data indicate that food deprivation has differential effects on orexin receptor

expression and their signalling characteristics at the hypothalamic and adreno-cortical

level. These novel findings would suggest orexins as potential metabolic regulators

within the HPA axis both centrally and peripherally.

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INTRODUCTION

Starvation-induced biochemical and metabolic changes are perceived by the

hypothalamus, which in turn coordinates behavioural, autonomic and neuroendocrine

responses to these stimuli (1). Studies in rodents have shown that food deprivation

induces marked ACTH and corticosterone responses, implicating feeding as a major

synchroniser of rhythms in the hypothalamic-pituitary-adrenal (HPA) axis (2, 3).

Besides nutritional states and neuropeptides, such as corticotropin-releasing hormone

(CRH) and neuropeptide Y (NPY), known to regulate both the HPA system and

feeding behaviour, several ‘signals’ are known to regulate the HPA system.

Hypoglycaemia is a potent activator of the HPA axis, reflecting the strong functional

relationship between the hypothalamic feeding centres and the HPA axis (4) and

leptin, whose concentrations are governed by nutritional status, has an inhibitory

effect on plasma corticosterone in rats (5).

More recently, orexins (orexin-A and orexin-B), produced by neurones localised in

the lateral and dorsal hypothalamic area and perifornical hypothalamus (6), have been

implicated in the central regulation of feeding and energy homeostasis (7). Both, OR-

A (a 33-residue peptide) and OR-B (a 28-residue peptide) are proteolytically cleaved

from a common precursor, prepro-orexin, and share a 46% amino acid sequence

homology. Besides playing a role in the regulation of feeding and energy homeostasis,

orexins have been reported to exert divergent physiological actions (8-12). Orexins

orchestrate their actions by binding and activating two types of G-protein coupled

receptors, orexin-1 receptor (OX1R) and orexin-2 receptor (OX2R), which display

64% homology in their amino acid sequence (6). The OX1R preferentially binds

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orexin-A, whilst OX2R binds both orexin-A and –B, apparently with similar affinity

(6).

In the hypothalamus, levels of prepro-orexin messenger RNA are regulated by several

nutritional states and signals. Fasting (6) and insulin-induced hypoglycaemia (13, 14)

increase prepro-orexin mRNA in the rat lateral hypothalamic area, whereas leptin has

the converse effect (15). Interestingly, fasting and leptin also regulate the

hypothalamic expression of orexin receptors (15, 16). These findings, and the

observation that intracerebroventricular (ICV) administration of OR-A or –B

stimulates food consumption in rats (6), suggests a physiological role for these

neuropeptides as mediators in the regulation of feeding, particularly in response to

energy deprivation.

Though the hypothalamus is considered the cornerstone for maintenance of energy

homeostasis, the adrenal gland plays a role in the integration of metabolic activity and

energy balance. For example, studies in food deprived rats have suggested that the

HPA axis is integral to a larger hypothalamic system that mediates energy flow (2),

and that in these animals catabolic activity quickly predominates, reinforced by

elevated corticosterone not driven by hypothalamic control (ACTH), implicating

adrenal activity as a metabolic regulator (3). In addition to their hypothalamic effects,

‘nutritional signals’ such as leptin and NPY have a direct action at the adrenal level,

via their receptors, including the modulation of corticosteroid secretion (17, 18). Of

interest, rat adrenals express both orexin receptors (19) and orexins stimulate

corticosterone secretion of rat adrenocortical cells, through the activation of the

adenylyl cyclase-dependent signalling cascade (20).

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The expression of orexin receptors in both rat and human adrenals is well

documented. In the rat, both receptors are expressed in the adrenal cortex.

Interestingly, the highest amount of OX2R mRNA was found in the adrenal gland of

male rats, which was 4 times higher than brain OX2R mRNA levels (19). In situ

hybridization revealed that OX2R mRNA is localized primarily in zona glomerulosa

(ZG) and zona reticularis (ZR), whereas there was no expression at the adrenal

medulla (19).

In view of these findings, and the observation that changes in orexin levels are closely

related to nutritional status rather than to the state of hunger or satiety (21), we

hypothesised that activation of orexin receptors at the adrenal level may explain the

ACTH-independent rise in corticosterone seen in ‘starved’ rats. We sought to

investigate, in both the hypothalamus and adrenal gland, a) whether levels of orexins

and its receptors, OX1R and OX2R, differ in fed and food deprived rats, by assessing

their expression at mRNA and protein level, and b) to elucidate if nutritional status

elicits functional changes of OX1R and OX2R, by studying G-protein coupling and

the subsequent activation of second messenger pathways.

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MATERIALS & METHODS

Animal Preparation

The University of Warwick, UK, on use and care of animals, approved all procedures

described. Adult male (initially 250 g) Wistar rats were housed in groups of two (n=6

for each group) in environmentally controlled conditions (22 ± 2oC, humidity 40-

60%) under a 12:12-h light:dark schedule (lights on 0600 h). Rats were allowed

unrestricted access to standard laboratory pellet rodent diet (CRM, Biosure,

Cambridge, UK; 13.1 kcal/g) and access to tap water, before being subjected to the

study. After a week of habituation to these conditions, rats were randomly distributed

into two groups. The first group was allowed to eat freely/ad libitum. The second

group was food deprived for 24 h, beginning at the onset of the dark cycle (lights out

1800 h), before both groups of animals were sacrificed by CO2 inhalation or by

cervical dislocation the following day at 1800 h.

Hypothalamic and Adrenal Dissection

To isolate the hypothalamus, animals were decapitated and their brains removed

rapidly. The hypothalamus, defined by the posterior margin of the optic chiasm and

the anterior margin of the mamillary bodies to the depth of 2-3 mm, was dissected out.

Adrenal cortex fractions were freed from adipose tissue and further separated from

inner adrenomedullary tissue by pressure between glass plates. Upon removal both

tissues were immediately snap frozen in liquid nitrogen. Samples were then stored at

–70oC until further use.

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Assay of Plasma Corticosterone Concentration

Plasma corticosterone levels between fed (n=6 from each group; i.e. CO2/cervical

dislocation) and food deprived rats (n=6 from each group) were measured by

radioimmunoassay (Amersham Life Sciences, Buckinghamshire, England). The range

for this radioimmunoassay –specific for corticosterone- is between 0.78-200 ng/ml.

Total RNA Extraction & cDNA Synthesis

Total RNA was prepared from individual samples using “RNeasyTM Total RNA Kit”

(QIAGEN, Crawley, U.K.) according to the manufacturer's guidelines. First strand

cDNA synthesis was performed using RNase Reverse Transcriptase (GIBCO BRL;

Paisley, U.K.), according to manufacturer’s recommendation.

Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Quantitative PCR was performed on a Roche Light Cycler™ system (Roche Molecular

Biochemicals, Manheim, Germany). PCR reactions were carried out in a reaction

mixture consisting of 5.0 µl reaction buffer and 2.0 mM MgCl2 (Biogene, Kimbolton,

U.K.), 1.0 µl of each primer (1ng/µl), 2.5 µl of cDNA and 0.5 µl of Light Cycler

SYBR Gold (Biogene, Kimbolton, U.K.).

Protocol conditions consisted of denaturation of 95oC for 15 secs, followed by 40

cycles of 94oC for 1 sec, 58oC for 5 secs and 72oC for 12 secs, followed by melting

curve analysis. For analysis, quantitative amounts of genes of interest were

standardised against the house-keeping gene β-actin. As a negative control for all the

reactions, preparations lacking RNA or reverse transcriptase were used in place of the

cDNA. For the quantitative analysis cDNAs from 6 fed and 6 food deprived rats were

used. Serial dilutions of hypothalamic and adrenal cDNAs provided the template on

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which a line of best fit was plotted and used as a standard curve, in order to

demonstrate accuracy and reproducibility of analysis. Quantification data was analysed

using the Light Cycler analysis software. The RNA levels were expressed as a ratio,

using “Delta-delta method” for comparing relative expression results between

treatments in real-time PCR (22).

The resultant PCR products were sequenced in an automated DNA sequencer and the

sequence data was analysed using BLAST Nucleic Acid Database Searches from the

National Centre for Biotechnology Information (NCBI).

Preparation of hypothalamic and adrenocortical membranes:

Rat hypothalami and adrenal cortex were obtained from food deprived and control male

Wistar rats, as described above. Tissues were homogenized in Dulbecco's phosphate

buffered saline containing 10 mM MgCl2, 2 mM EGTA, 1.5 g/l bovine serum albumin

(w/v), 0.15 mM bacitracin, 1 mM phenylmethyl sulphonylfluoride pH 7.2 (Extraction

Buffer) at 22oC. The homogenate was centrifuged at 800 g for 30 min at 4oC. The pellet

was discarded and the supernatant spun at 45,000 g for 60 min at 4oC. The resultant

pellet was washed, resuspended in extraction buffer and spun at 45,000 g for a further

60 minutes at 4oC. The final pellet was resuspended in 5 ml of extraction buffer using a

homogenizer. The protein concentration of the membrane suspension was determined

using the bicinchoninic acid method, with BSA as a standard (23).

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Western blotting

Hypothalamic (n=6 from each group, i.e. fed and food deprived; 100 µg) and

adrenocortical membranes (n=6 from each group; 100 µg) were centrifuged at 15,000 g

for 15 min at 4o C. The supernatant was then discarded and the resultant pellets

solubilised with Laemmli Buffer (5 M Urea, 0.17 M Sodium Dodecyl Sulfate (SDS), 0.4

M Dithiothreitol, and 50 mM Tris-HCl, pH 8.0), mixed and placed in a boiling-water

bath for 5 min and allowed to cool at room temperature.

Samples were separated on a 12% SDS-polyacrylamide gel and the proteins were

electrophoretically transferred to a nitrocellulose membrane at 250 mA for 1h in a

transfer buffer containing 20 mM Tris, 150 mM Glycine, and 20% Methanol. The

filter was then blocked in PBS containing 0.1% Tween-20 and 5% milk powder (w/v),

for 2 h at room temperature. After three washes with PBS-0.1% Tween, the

nitrocellulose membranes were incubated with primary antibody for the OX1R and

OX2R (Santa Cruz Biotechnology; Santa Cruz, USA). The primary antisera were used

at a 1:1000 dilution in PBS-0.1% Tween for 1 h at room temperature. The filters were

washed thoroughly for 30 min with PBS-0.1% Tween, before incubation with the

secondary anti-rabbit HRP-conjugated immunoglobulin (1:2000) for 1 h at room

temperature and further washing for 30 mins with PBS-0.1% Tween. Antibody

complexes were visualised as previously described (24). To ensure specificity, we

also performed preabsorption of both OX1R and OX2R with their blocking peptides

(Santa Cruz Biotechnology; Santa Cruz, USA) prior to western blotting. For the

detection of OR-A and OR-B, similar procedures were followed, using specific non

cross-reactive antibodies (Phoenix Peptides, Belmont, USA) and total hypothalamic

and adrenal lysate. To ensure that the same protein amount was loaded in all of the

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samples used for western blotting, we used antibodies against the house-keeping gene

β-actin (Santa Cruz Biotechnology; Santa Cruz, USA).

Treatment of membranes with pertussis and cholera toxins

Both pertussis (50 µg/mL) and cholera (150 µg/mL) toxin were preactivated in 0.05 M

Tris buffer, pH 7.5, containing 20 mM dithiothreitol (DTT) and 50 mM glycine for 45

min at 37o C in a final volume of 50 µL and cooled on ice for 20 mins. Hypothalamic

and adrenal membranes (n=3 from each group; 100 µg) were incubated in 20 mM Tris,

pH 7.5, containing 1 mM EDTA, 1mM DTT, 1 mM ATP, 1mM GTP, 5 mM MgCl2 10

mM Thymidine, 10 µM NAD, 5 µCi [32P]NAD, together with the preactivated toxins.

All reactions were carried out at 37o C for 30 mins, and the incubations were terminated

with 0.7 ml ice-cold 20 mM Tris buffer, pH 7.5, containing 1 mM EDTA. Control

samples were prepared by incubating membranes in the same medium, but in the

absence of any toxin. After termination, samples were centrifuged at 13,000 rpm for 20

mins, and the pellets were washed and respun three times. The resultant pellets were

resuspended in 100 µL of 2% SDS and 320 µL of buffer containing (1% (v/v) Triton X-

100, 1% Deoxycholate, 0.5% (w/v) SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1

mM DTT, 1 mM EDTA, 0.2 mM PMSF, 10 µg/mL aprotinin). Resuspended samples

were centrifuged at 11,000 rpm for 10 mins at room temperature, and the resulted

supernatants were equally aliquoted (200 µL). Into each of these aliquots 10 µL of Gi

and Gs antisera (New England Nuclear-DuPont; Boston, USA) were added and left for

continuous agitation for 2 hr, followed by addition of 60 µL of Protein Sepharose A per

tube and further agitation overnight at 4o C. The samples were then centrifuged at 12,000

rpm for 10 mins and the pellets were solubilised with Laemmli buffer, mixed and placed

in a boiling-water bath for 5 mins before cooling to room temperature. Each sample was

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loaded on an SDS-12% polyacrylamide gel and after electrophoresis, the gels were dried

and autoradiographed using Kodak x-ray film, in order to assess the extent of ADP-

ribosylation.

Synthesis of [α-32 P] GTP-AA and photoaffinity labelling of Gα-subunits

GTP-Azidoanilide (GTP-AA) was synthesised using a method previously described

(23). Hypothalamic and adrenocortical membranes (n=3 from each group; 150 µg) were

incubated for 3 min at 30oC with orexin-A (100 nM) in Buffer A (50 mM HEPES, 30

mM KCl, 10 mM MgCl2, 1 mM Benzamidine, 0.1 mM EDTA), followed by the addition

of 5 µM GDP and 6 µCi of GTP-AA. After incubation for 3 min at 30o C in a darkened

room, membranes were placed on ice and collected by centrifugation at 15,000 g for 15

min at 4oC. The supernatant was carefully removed, and the membrane pellet was

resuspended in 120 µL of modified Buffer A (1.6 mg DTT in 5 mL Buffer A). Samples

were vortexed and irradiated for 5 min at 4oC with an ultraviolet light (254 nm) from a

distance of 5 cm, to cross-link the GTP-AA to the G-proteins. Immunoprecipitation

using 10 µl of undiluted G protein antisera (New England Nuclear-DuPont; Boston,

USA) (Table 2), to the α-subunit, was then carried out as previously described (24).

Samples were subjected to gel electrophoresis using discontinuous SDS-PAGE slab gels

(10% running; 5% stacking). The gels were stained with Coomassie Blue, dried using a

slab gel dryer and exposed to Fuji X-ray film at -70oC for 2-5 days with intensifying

screens.

cAMP and IP3 Second Messenger Studies

cAMP: For cAMP studies, using a commercially available kit (New England Nuclear-

DuPont; Boston, USA), hypothalamic and adrenocortical membrane suspensions (n=3

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from each group; 100 µg) were incubated with increasing concentrations of orexin-A

and the amount of cAMP in the incubate was determined by radioimmunoassay as

previously described (25). Standard cAMP concentrations, covering the range 0.138-100

pmol/mL, were used for determination of the standard curve of the radioimmunoassay.

The interassay coefficient of variation was 8%. Cyclic AMP assay buffer (without any

membrane preparations) was used as the negative control.

IP3: For the inositol triphosphate (IP3) assay (Amersham Pharmacia Biotech; Little

Chalfont, UK), hypothalamic and adrenocortical membranes were incubated with

increasing concentrations of orexin-A, followed by the addition of 200 µl of IP3

generation buffer as previously described (24). Membranes were incubated for 3 mins

at 37o C, and the reaction was terminated by the addition of 1 M ice-cold

trichloroacetic acid, followed by extraction of inositol phosphates and neutralisation.

IP3 levels were estimated by radioimmunoassay based on the displacement of 3[H]IP3

from a specific bovine adrenocortical IP3 binding proteins. The inter-assay coefficient

of variation was 8.7%.

Statistical Analysis

Data are shown as the mean ± S.D of each measurement. For the real-time PCR

measurements, photoaffinity labelling and western immunoblotting, results were

evaluated between groups by using two-tailed Student’s t test, with significance

determined at the level of p<0.05. For western immunoblotting and photoaffinity

labelling experiments, the densities were measured using a scanning densitometer

coupled to scanning software ImageQuant; (Molecular Dynamics, Amersham

Pharmacia, Little Chalfont, UK). For the second messenger measurements, a one-way

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Analysis of Variance (ANOVA) was used, followed by Dunnett’s test, in order to

compare each treatment dose.

RESULTS

Effect of food deprivation on corticosterone level

Plasma corticosterone levels were almost two fold higher (p<0.01) in the food deprived

rats (51 ± 2.9 ng/ml) when compared to the fed ones (28±2.7 ng/ml), as they were

measured by radioimmunoassay (Fig.1 A). Given that all rats were sacrificed by CO2

inhalation, corticosterone levels of rats sacrificed by cervical dislocation were also

measured. After comparing the two subgroups, it appears that there are no apparent

differences between the two different methods of sacrificing animals, thus suggesting

that in our model, CO2 inhalation does not constitute an additional stress factor (Fig.1

A).

Effect of food deprivation on peptide and receptor expression in hypothalamus

a) Peptide expression

Serial dilutions of hypothalamic cDNA provided the template on which a line of best

fit was plotted and used as a standard curve, in order to demonstrate accuracy and

reproducibility of analysis. Melting curve analysis of the PCR products was presented

as fluorescence over time (-dF/dT) against temperature (T oC). The melting curve

analysis showed a single melting maximum of 89.20OC for the prepro-orexin gene, a

single melting maximum of 90.30OC for the β-actin gene; thus confirming product

specificity (data not shown).

There was a five-fold increase in prepro-orexin message (P<0.01), in the hypothalami

of food-deprived animals (Fig. 1B), as compared to controls. These mRNA changes of

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the prepro-orexin peptide were also confirmed by immunoblotting analysis; where

protein expression of both OR-A and OR-B was significantly (P<0.01 and P<0.05,

respectively) increased under food deprivation (Fig. 1C).

b) Receptor expression

Compared to controls, both OXR1 and OXR2 mRNA were significantly (P<0.05)

increased in the hypothalamus of the food-deprived rat (Fig. 2A). The increases were

similar for both forms of orexin receptors (2.5-fold increase for OX1R, around 2-fold

increase for OX2R). Protein expression of OXR1 and OXR2 was confirmed by

immunoblotting using specific goat polyclonal antibodies (Fig. 2B). The OXR1

antibody was raised against a peptide mapping at the carboxy terminus of the OXR1

of rat origin, whereas the OXR2 antibody was raised against a peptide mapping at the

amino terminus of the OX2R of human origin and is rat cross-reactive. The detected

protein for OX1R has an apparent molecular weight of around 50 kDa, whereas the

OX2R was detected as a 40 kDa peptide. The specificity of the response was

confirmed by pre-incubation of OX1R and OX2R antibodies with their blocking

peptides (Figure 2B). Protein expression reflected the mRNA data (Fig. 2A), with

significant increase (P<0.01) of both receptors in food deprived animals (Fig. 2C),

compared to controls.

Effect of food deprivation on orexin receptor expression in adrenal cortex

Interestingly, findings in the adrenal cortex were in marked contrast to those observed

in the hypothalamus. Quantitative analysis of the PCR products from the rat adrenal

cortex showed that both OX1R and OX2R levels were significantly (P<0.05 and

P<0.01, respectively) reduced in food deprived animals, compared to controls (Fig.

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3A). The decrease was more profound for OX2R (4-fold), whereas OX1R

demonstrated a 2-fold decrease (Fig. 3A). Similarly, both OX1R and OX2R protein

levels were significantly decreased (P<0.05; P<0.01 respectively) in the adrenal

cortex of the food-deprived animals, compared to controls. Again the decrease was

more pronounced for OXR2. In both groups studied, the protein levels for β-actin

remained unaltered (Fig. 3B).

ADP Ribosylation

Cholera Toxin Treatment: Incubating membranes with cholera toxin resulted in the

incorporation of [32P] ADP ribose, into two bands of 45 and 47 kDa for both

hypothalamic and adrenal membranes. There were no apparent differences in the

incorporation of the probe between fed and food deprived preparations from both tissues

(Fig. 4A).

Pertussis Toxin Treatment: Treatment of hypothalamic and adrenal membranes with

pertussis toxin resulted in the incorporation of [32P] ADP ribose, into a single band with

apparent molecular weights of 41 kDa (Fig. 4B). No incorporation of label was seen in

either hypothalamic or adrenal tissues in the absence of the pertussis toxin. Similarly,

there were no detectable differences between the fed and food deprived Gi subunits. This

data, not only confirms the functional integrity of G protein α-subunits, but also reveals

that the nutritional status does not interfere with the expression of these signalling

proteins.

Functional analysis of G-protein activation by OR-A: Effects of food deprivation

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To determine which G-proteins are coupled to the orexin-receptors in the food

deprived and fed state, hypothalamic and adrenocortical membranes were labelled

with GTP-AA in the presence or absence of OR-A followed by immunoprecipitation

with specific Gα subunit antibodies (Gs, Gi, Gq/11, Go).

Optimal labelling of Gα subunits with GTP-AA requires receptor activation of

heterotrimeric G-proteins with release of bound GDP. The binding of GTP-AA is

dependent upon GDP concentration, GTP affinity of the Gα subunit and agonist-

incubation time (26). Therefore, the conditions for labelling Gα subunits were

established empirically. We have demonstrated that optimum labelling was obtained

in the presence of 5 µm GDP (data not shown). OR-A induced labelling with GTP-

AA was time-dependent with an optimal incubation time of 3 min (data not shown).

Using Gsα-subunit as a paradigm, we were able to demonstrate that the coupling of

orexin receptors upon challenge with OR-A was dose dependent, with maximal

activation at a concentration of 100 nM (Fig. 4C).

The specificity of the immunoprecipitating properties of Gs α antibody were assessed

by comparing the migration positions of orexin-induced GTP-AA photoaffinity labelled

G-proteins with those ADP-ribosylated using 32P-NAD with cholera toxin to

demonstrate that the same protein band was radiolabeled and immunoprecipitated by

the specific antibody. Surprisingly our photoaffinity 32P-GTP-AA experiment indicated

that the orexin receptors in both hypothalamus and adrenal cortex preferentially activate

the 45 kDa form of the Gsα-protein. This observation requires further investigation.

Hypothalamus:

Treatment of hypothalamic membranes with OR-A (100 nM) revealed that orexin

receptors coupled to multiple G-proteins, including Go, Gi and Gs and to a lesser

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extent Gq/11 in the control group (Fig 5A). In food deprived rats, however, there was

a change in the ‘profile’ of G-protein activation. Compared to controls, there was a

significant increase in coupling of orexin receptors to Gq, Gs and Go, whereas there

was less coupling of orexin receptors to Gi (Fig 5A). Quantification of the

immunocomplexes is shown in Figure 5B.

Adrenal cortex:

Treatment of adrenocortical membranes with OR-A (100nM) revealed a similar

“promiscuity” in the G protein profile of adrenal orexin receptors. In the control group

(fed rats) OR-A increased the labelling of Gs, Gq and Go, but not of Gi (Fig 5A). As in

the hypothalamus, food deprivation modulated the G-protein activation profile.

Interestingly, in the food-deprived animal, OR-A decreased the coupling of orexin

receptors to Gs and Go, increased that of Gi, whereas there was no coupling towards

Gq (Fig 5A). Quantification of the immunocomplexes is shown in Figure 5C.

Functional analysis of intracellular second-messenger generation by orexin-A:

Effects of food deprivation

In addition to G protein data, we dissected further the signalling characteristics of

orexin receptors in the hypothalamus and adrenal cortex, by measuring the second

messengers cAMP and IP3 in both controls and food deprived animals, upon

stimulation by different concentrations of OR-A.

Hypothalamus:

To test the OR-A ability to activate hypothalamic adenylyl cyclase, we determined the

effect of OR-A on cAMP production. When hypothalamic membranes from fed rats

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were incubated with OR-A (10 pM-100 nM) for 30 mins at 25o C, there was a

significant increase in cAMP production. This increase was found to be dose-

dependent, while the maximal response (55±5 % of basal) observed at a concentration

of 100 nM (Fig. 6A). The response was amplified in the food deprived rat, where 100

nM treatment induce a cAMP response of 95±5 % of basal (Fig.6A).

Similarly, we found that OR-A treatment of hypothalamic membranes induced a rapid

inositol triphosphate (IP3) turnover, in a dose-dependent manner. This OR-A effect

has a threshold of 1nM and a maximum response at 100 nM (30±4 % of basal) (Fig.

6B). Consistent with our G-protein labelling studies, there was a significant (P<0.01)

increase in IP3 production from hypothalami of the food deprived rats, as compared to

controls (70±7 % of basal) (Fig. 6B).

Adrenal cortex:

Similarly, when rat adrenocortical membranes from fed rats were incubated with OR-A

(0.01 nM - 100 nM), there was a significant increase in cAMP and IP3 production (Fig.

6 C, D). These increases appeared to be dose-dependent, with a maximum response at

100 nM for cAMP (78±8 % of basal), and at 10nM for IP3 (50±7 % of basal).

However, in the food deprived animals, compared to controls, treatment of

adrenocortical membranes with OR-A induced a modest response towards cAMP

production that was significant only at 100nM, with no apparent effect towards IP3

turnover (Fig. 6 C, D). Again, these findings were in keeping with our photo-affinity

labelling experiments.

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DISCUSSION

In the present study, we demonstrate the effects of food deprivation (24 h) on orexins

and orexin receptor expression in the rat hypothalamus and adrenal cortex, both at

mRNA and protein level. In addition, we present the differential effects of this

‘stressful’ nutritional stimulus on the signalling characteristics of orexin receptors at

these tissues.

The hypothalamus plays a major role in the regulation of food intake and energy

balance by integrating multiple anorexigenic and orexigenic signals, including those

elicited by orexins (21, 27). A similar role for the adrenal gland is possible given that

it expresses receptors for leptin, NPY (28) and orexin (19, 29), all regulators of energy

homeostasis. In agreement with previous studies (6, 15), we were able to show that

the levels of prepro-orexin mRNA are influenced by nutritional status, being

upregulated upon fasting in the hypothalamus. Detailed analysis at protein level

confirmed that both cleaved bioactive peptides (OR-A and OR-B) are upregulated

under food deprivation. Previous studies in fasted lactating rats, demonstrated

hypothalamic OR-B levels to be raised ten-fold above those for controls (non-fasted),

whereas OR-A showed no change (30). These differences can be due to the rodent

strain used, the sex, metabolic status and duration of food deprivation.

Food deprivation leads to changes in the activity of the HPA axis within three hours,

with an increase in corticosterone (3). These findings are of interest given orexins

stimulate the HPA axis when administered centrally (31). Furthermore, orexin neurones

are sensitive to nutritional signals such as insulin and glucose, which alter with food

deprivation (3), and hypothalamic orexin neurones express leptin receptors. As with

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prepro-orexin expression, fasting and leptin also regulate hypothalamic expression of

orexin receptors (15, 16, 32).

Numerous studies have mapped in detail the expression of orexin and orexin receptors at

the hypothalamic level. Trivedi et al., has demonstrated that within the hypothalamus,

OX1R mRNA is most abundant in the ventromedial hypothalamic nucleus whereas

OX2R is predominantly expressed in the paraventricular nucleus (33). In another study,

it has been shown that after 20 h of fasting, levels of rat OX1R mRNA were significantly

increased in the ventromedial (VMH) hypothalamic nuclei and the medial division of

amygdale, whereas levels of OX2R mRNA were augmented in the arcuate nucleus, but

remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular

hypothalamic nucleus, and amygdala following fasting (16). Three different studies (34,

13, 15) indicated that prepro-orexin is upregulated in the hypothalamus upon food

deprivation in lateral hypothalamic area (LHA) of the rat. In agreement with these

studies we noted that on fasting, hypothalamic OX1R and OX2R gene expression was

induced. Given the detailed analysis these studies have provided, we shifted our interest

at the expression at protein level. Here we provide new evidence that these changes are

also mirrored at protein level, as it was assessed by semi-quantitative western blotting

analysis.

Previous studies (3, 4) have shown that food deprivation switches feeding responses to

adrenocortical responses and the time of food presentation appears to be a more potent

synchronizer of the phase of plasma corticosteroid levels than is the light-dark cycle

(35). These observations are of interest given that orexins have circadian-dependent

actions and that orexins are known to increase corticosterone production in rats (20), and

cortisol in human adrenocortical cells (36). However, in view of our data, we can

20

conclude that adrenal orexin receptors do not influence or mediate the rise in

corticosterone levels, seen under food deprivation conditions.

It is well documented that food deprivation induces multiple metabolic changes which

may directly influence orexin receptor expression in the adrenal gland. For example,

there is a negative correlation between testosterone levels and fasting (37). Studies from

our laboratory have shown that testosterone levels also decrease following 24 h of food

deprivation (unpublished observations). Interestingly, in gonadectomised rats there was a

significant down regulation of adrenal OX2R, an effect that was reversed upon

testosterone replacement (38). Although, the regulation of adrenal orexin receptors by

gonadal steroids is not directly relevant to our findings, it suggests that changes in orexin

receptor expression might be a secondary phenomenon following initial food

deprivation-induced alterations.

Future studies should concentrate elucidating the central and peripheral actions of

orexins. This can be done by systematically deleting either OX1R and/or OX2R in a

tissue-specific fashion, using the Cre-loxP system. Using this approach, a model can be

generated bearing neuronal or adrenal-specific deletions of orexin receptors.

Our data indicate that orexin signalling may be enhanced in response to energy deficit

sensed by the hypothalamus, possibly and predominantly via OX1R, but at the same

time decreased in the adrenal cortex. Orexins act through two distinct G-protein coupled

receptors, which can transduce intracellular signals by activating heterotrimeric G-

proteins. It is likely that multiple second messenger systems are involved as orexins have

been shown to increase intracellular calcium influx (6, 39, 40, 41). There is also

21

suggestion that OX2R is coupled to the inhibitory Gi protein (42). We have shown for

the first time that native orexin receptors in the hypothalamus can activate four types of

G-proteins, namely Gs, Gq, Go and Gi, in response to OR-A. Although Gi and Go are

the two major inhibitory signalling pathways in the rat brain, we have previously shown

that multiple subtypes of G-proteins couple to other G-protein coupled receptor- systems

(43). Food deprivation altered the G-protein coupling profile. Our findings are supported

by functional assays of cAMP and IP3 assays, reflecting their G-protein coupling status.

Further research is needed to investigate if the βγ-subunits are implicated in activating

second messengers and also to assess the effects of OR-B on the G protein signalling.

Although we have demonstrated functional orexin receptors in human fetal and adult

adrenal glands (23, 44), this is the first report of G-proteins coupling to orexin receptors

in the rat adrenal cortex. Interestingly, upon food deprivation we note a down regulation

of all G-protein(s) in response to OR-A, apart from Gi. Second messenger studies

confirmed these findings, with minimal response towards cAMP production and none of

IP3 in the adrenal cortex of the food deprived rat. Our findings would support the

observations that in rat adrenocortical cells orexin-A stimulates corticosterone release via

cyclic a cAMP pathway (20).

Despite demonstrating for first time that rat orexin receptors can couple differentially to

several G protein α-subunits and activate multiple second messengers, caution should be

exercised as to how to interpret these changes. Given the plethora of different cell types

that reside within the hypothalamus and the adrenal gland, no distinctions can be made

between specific sub-populations from brain regions or adrenal zones from our data.

22

Here we provide evidence about the signalling characteristics of orexin receptors under

food deprivation in total hypothalamic and adrenal preparations.

In conclusion, our study indicates that nutritional changes like food deprivation can exert

differential effects on orexin receptor expression and their signalling characteristics at

the hypothalamic and adrenocortical level. However, the significance of our novel

findings, in particular the differential expression of G-protein activation upon food

deprivation, in both the hypothalamus and the adrenal cortex, needs further elucidation.

Acknowledgements:

This work was supported by Research Grants to HSR from - The General Charities of

Coventry; The RTDF-University of Warwick; & The Clinical Endocrinology Trust,

UK. EK and RJM should be considered first co-authors by virtue of their equal

contribution to the study.

REFERENCES

1) Schwartz MW, Dallman MF, and Woods SC. The hypothalamus response to

starvation: implications for the study of wasting disorders. Am J Physiol 269: 949-

957, 1995.

2) Akana SF, Strack AM, Hanson ES and Dallman MF. Regulation of activity in

the hypothalamic-pituitary-adrenal axis is integral to a larger hypothalamic system

that determines caloric flow. Endocrinology 135: 1125-1134, 1994.

23

3) Dallman MF, Akana SF, Bhatnagar S, Bell ME, Choi S, Chu A, Horsley C,

Levin N, Meijer O, Soriano LR, Strack AM and Viau V. Starvation: Early signals,

sensors, and sequelae. Endocrinology 140: 4015-4023, 1999.

4) Guillaume V, Grino M, Conte-Devlox B, Boudouresque F and Oliver C.

Corticotropin-releasing factor secretion increases in rat hypophyseal portal blood

during insulin-induced hypoglycaemia. Neuroendocrinology 49: 676-679, 1989.

5) Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E and

Flier JS. Role of leptin in the neuroendocrine response to fasting. Nature 382: 250-

253, 1996.

6) Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H,

Williams SC, Richardson JA, Kozlowski GP, Wilson S, Arch JR, Buckingham

RE, Haynes AC, Carr SA, Annan RS, McNulty DE, Liu WS, Terrett JA,

Elshourbagy NA, Bergsma DJ and Yanagisawa M. Orexins and orexin receptors: a

family of hypothalamic neuropeptides and G protein-coupled receptors that regulate

feeding behaviour. Cell 92: 573-585, 1998.

7) Kalra SP, Dube MG, Pu S, Xu B, Horvath TL and Kalra PS. Interacting

appetite-regulating pathways in the hypothalamic regulation of body weight.

Endocrine Reviews 20: 68-100, 1999.

8) Ida T, Nakahara K, Katayama T, Murakami N and Nakazato M. Effect of

lateral cerebroventricular injection of the appetite-stimulating neuropeptide, orexin

24

and neurpeptide Y, on the various behavioural aspects of rats. Brain Res. 821: 526-

529, 1999.

9) Samson WK, Gosnell B, Chang JK, Resch ZT and Murphy TC. Cardiovascular

regulatory actions of the hypocretins in the brain. Brain Res. 831: 248-253, 1999.

10) Takahashi N, Okumura T, Yamada H and Kohgo Y. Stimulation of gastric

acid secretion by centrally administered orexin-A in conscious rats. Biochem.

Biophys. Res. Commun. 254: 623-627, 1999.

11) Pu S, Jain MR, Kalra PS and Kalra SP. Orexins, a novel family of

hypothalamic neuropeptides, modulate pituitary luteinising hormone secretion in an

ovarian steroid-dependent manner. Regul. Pept. 78: 133-136, 1998.

12) Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ,

Nishino S and Mignot E. The sleep disorder canine narcolepsy is caused by a

mutation in the hypocretin (orexin) receptor 2 gene. Cell 98: 365-376, 1999.

13) Cai XJ, Widdowson PS, Harrold J, Wilson S, Buckingham RE, Arch JR,

Tadayyon M, Clapham JC, Wilding J and Williams G. Hypothalamic orexin

expression: modulation by blood glucose and feeding. Diabetes 48: 2132-2137, 1999.

14) Griffond B, Risold PY, Jacquemard C, Colard C and Fellman D. Insulin

induced hypoglycaemia increases preprohypocretin (orexin) mRNA in the rat lateral

hypothalamic area. Neuroscience Letter 262: 77-80, 1999.

25

15) Lopez M, Seoane L, Garcia MC, Lago F, Casanueva FF, Senaris R and

Dieguez C. Leptin regulation of the prepro-orexin and orexin receptor mRNA levels

in the hypothalamus. Biochem Biophys Res Commun 269: 41-45, 2000.

16) Lu XY, Bagnol D, Burke S, Akil H and Watson SJ. Differential distribution

and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the

brain upon fasting. Horm. Behav. 37: 335-344, 2000.

17) Bornstein SR, Uhlmann K, Haidan A, Ehrhart-Bornstein M and Scherbaum

W.A. Evidence for a novel peripheral action of leptin as a metabolic signal to adrenal

gland. Leptin inhibits cortisol release directly. Diabetes 46: 1235-1238, 1997.

18) Nussdorfer GG and Gottardo G. Neuropeptide-Y family of peptides in the

autocrine-regulation of adrenocortical function. Horm. Metab. Res. 30: 368-373,

1998.

19) Jöhren O, Neidert SJ, Kummer M, Dendorfer A and Dominiak P. Prepro-

orexin and orexin receptor mRNAs are differentially expressed in peripheral tissues of

male and female rats. Endocrinology 142: 3324-3331, 2001.

20) Malendowicz LK, Tortorella C and Nussdorfer GG. Orexins stimulate

corticosterone secretion of rat adrenocortical cells, through the activation of the

adenylate cyclase-dependent signaling cascade. J. Steroid Biochem. Mol. Biol. 70:

185-188, 1999.

26

21) Lubkin M and Stricker-Krongrad A. Independent feeding and metabolic

actions of orexins in mice. Biochem. Biophys. Res. Commun. 253: 241-245, 1998.

22) Pfaffl MW. A new mathematical model for relative quantification in real-time

RT-PCR. Nucleic Acids Res. 1: e45, 2000.

23) Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano

MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein

using bicinchoninic acid. Anal Biochem. 150:76-85, 1985.

24) Randeva HS, Karteris E, Grammatopoulos D and Hillhouse EW. Expression

of orexin-A and functional orexin type 2 receptors in the human adult adrenals:

implications for adrenal function and energy homeostasis. J. Clin. Endocrinol. Metab.

86: 4808-4813, 2001.

25) Karteris E, Papadopoulou N, Grammatopoulos DK, Hillhouse EW.

Expression and signalling characteristics of the corticotrophin-releasing hormone

receptors during the implantation phase in the human endometrium. J Mol

Endocrinol. 32: 21-32, 2004.

26) Offermanns S, Schultz G, Rosenthal W. Identification of receptor-activated G

proteins with photoreactive GTP analog, [alpha-32P]GTP azidoanilide. Methods

Enzymol. 195: 286-301, 1991.

27

27) Balasko M, Szelenyi Z and Szekely M. Central thermoregulatory effects of

neuropeptide Y and orexin A in rats. Acta Physiol Hung. 86: 219-222, 1999.

28) Cao GY, Considine RV and Lynn RB. Leptin receptors in the adrenal medulla

of the rat. Am J Physiol. 273: 448-452, 1997.

29) Nanmoku T, Isobe K, Sakurai T, Yamanaka A, Takekoshi K, Kawakami Y,

Ishii K, Goto K and Nakai T. Orexins suppress catecholamine synthesis and

secretion in cultured PC12 cells. Biochem. Biophys. Res. Commun. 274: 310-315,

2000.

30) Cai XJ, Denis R, Vernon RG, Clapham JC, Wilson S, Arch JR, Williams G.

Food restriction selectively increases hypothalamic orexin-B levels in lactating rats.

Regul Pept. 97:163-168, 2001.

31) Kuru M, Ueta Y, Serino R, Nakazato M, Yamamoto Y, Shibuya I and

Yamashita H. Centrally administered orexin/hypocretin activates HPA axis in rats.

NeuroReport 11: 1977-1980, 2000.

32) Yamanaka A, Beuckmann CT, Willie JT, Hara J, Tsujino N, Mieda M,

Tominaga M, Yagami K, Sugiyama F, Goto K, Yanagisawa M, Sakurai T.

Hypothalamic orexin neurons regulate arousal according to energy balance in mice.

Neuron 38:701-13, 2003.

28

33) Trivedi P, Yu H, MacNeil DJ, Van der Ploeg LH, Guan XM. Distribution of

orexin receptor mRNA in the rat brain. FEBS Lett. 438:71-5, 1998.

34) Lopez M, Seoane L, Senaris RM, Dieguez C. Prepro-orexin mRNA levels in the

rat hypothalamus, and orexin receptors mRNA levels in the rat hypothalamus and

adrenal gland are not influenced by the thyroid status. Neurosci Lett.300:171-

175,2001.

35) Krieger D.T. and Hauser H. Comparison of synchronisation of circadian

corticosteroid rhythms by photoperiod and food. Proc. Natl. Acad. Sci. USA. 75:

1577-1581, 1978.

36) Mazzocchi G, Malendowicz LK, Gottardo L, Aragona F and Nussdorfer GG.

Orexin-A stimulates cortisol secretion from human adrenocortical cells through

activation of the adenylate cyclase-dependent signaling cascade. J. Clin. Endocrinol.

Metab. 86: 778-782, 2001.

37) Bergendahl M, Perheentupa A, Huhtaniemi I. Effect of short-term starvation

on reproductive hormone gene expression, secretion and receptor levels in male rats. J

Endocrinol. 121: 409-417, 1989.

38) Jöhren O, Bruggemann N, Dendorfer A, and Dominiak P. Gonadal steroids

differentially regulate the messenger ribonucleic acid expression of pituitary orexin

type 1 receptors and adrenal orexin type 2 receptors. Endocrinology 144: 1219-1225,

2003.

29

39) Eriksson KS, Sergeeva O, Brown RE and Haas HL. Orexin/hypocretin excites

the histaminergic neurons of the tuberomammillary nucleus. J. Neurosci. 21: 9273-

9279, 2001.

40) Lund PE, Shariatmadari R, Uustare A, Detheux M, Parmentier M,

Kukkonen JP, and Akerman KE. The orexin OX1 receptor activates a novel Ca2+

influx pathway necessary for coupling to phospholipase C. J Biol Chem. 275: 30806-

30812, 2000.

41) van den Pol AN, Gao XB, Obrietan K, Kilduff TS, and Belousov AB.

Presynaptic and postsynaptic actions and modulation of neuroendocrine neurons by a

new hypothalamic peptide, hypocretin/orexin. J Neurosci. 18: 7962-7971, 1998.

42) Martin G, Fabre V, Siggins GR and de Lecea L. Interaction of the hypocretins

with neurotransmitters in the nucleus accumbens. Regul. Pept. 104: 111-117, 2002.

43) Grammatopoulos DK, Randeva HS, Levine MA, Kanellopoulou KA and

Hillhouse EW. Rat cerebral cortex corticotropin-releasing hormone receptors:

evidence for receptor coupling to multiple G-proteins. J. Neurochem. 2: 509-519,

2001.

44) Karteris E, Randeva HS, Grammatopoulos DK, Jaffe RB and Hillhouse E.W.

Expression and coupling characteristics of the CRH and orexin type 2 receptors in

human fetal adrenals. J. Clin. Endocrinol. Metab. 86: 4512-4519, 2001.

30

FIGURE LEGENDS

Figure 1. Panel (A) demonstrates that there is a significant upregulation (P<0.01) of

corticosterone production in the food deprived rats (n=6) when compared to fed ones

(n=6). The method of sacrificing, did not exert any effects on corticosterone levels of

the two groups studied.

Panel (B) demonstrates that there is a significant increase in prepro-orexin mRNA

levels in the food deprived hypothalamus of the rat (n=6). These differences in mRNA

expression can be seen from the large difference in amplification efficiency as it is

demonstrated by the delay in amplification (intercept cycle) of the fed rat sample.

Panel (C) demonstrates western blot analysis of OR-A and OR-B of rat hypothalamic

lysates. Lane 1 corresponds to positive control (OR-A or OR-B), Lane 2 corresponds

to the fed hypothalamus and Lane 3 corresponds to the food deprived hypothalamic

lysates. Quantification of the immunocomplexes, revealed that there is a significant

increase in protein expression of both OR-A and OR-B under food deprivation

conditions (n=6). *P<0.05; **P<0.01

Figure 2. Panel (A) demonstrates that there is a significant up regulation of OX1R

and OX2R mRNA levels in the food deprived hypothalamus (n=6) of the rat when

compared to the fed ones (n=6), as it was assessed by real-time PCR.

Panel (B). Western blot analysis of membrane protein extracts from rat adrenal (Lane

3) and hypothalamus (Lane 4) demonstrate that the antibody against OX1R

recognised a band with an apparent molecular weight of 50kDa. Similarly, when the

specific OX2R antibody has been used, it recognised a single band with an apparent

molecular weight of 40 kDa. Both bands appeared to be specific for orexin receptors,

since when the antibodies were preabsorbed with their respective blocking peptides

31

(Lane 1 and 2 for rat adrenal and hypothalamus respectively), there was no apparent

immunodetection.

Panel (C) demonstrates western blot analysis of OX1R and OX2R of hypothalamic

membranes from fed (n=6) and food deprived rats (n=6). Quantification of the

immunocomplexes, revealed that there is a significant increase in protein expression

of both orexin receptors under food deprivation conditions whereas the protein levels

for the house-keeping gene β-actin appeared to be unaltered. *P<0.05; **P<0.01

Figure 3. Panel (A) demonstrates that there is a significant up downregulation of

OX1R and OX2R mRNA levels in the food deprived rat adrenal cortex (n=6) when

compared to the fed adrenals, as it was assessed by real-time PCR. These changes in

mRNA expression have been shown as differences in amplification efficiency as it is

demonstrated by the delay in amplification (intercept cycle) of the food deprived

cDNAs.

Panel (B) demonstrates western blot analysis of OX1R and OX2R of adrenal

membranes from fed (n=6) and food deprived (n=6) rats. Quantification of the

immunocomplexes, revealed that there is a significant decrease in protein expression

of both orexin receptors under food deprivation conditions whereas the protein levels

for the house-keeping gene β-actin appeared to be unaltered. These changes are in

agreement with the mRNA data. *P<0.05; **P<0.01

32

Figure 4. Panel (A). ADP ribosylation by cholera toxin of hypothalamic (n=3) (H)

and adrenal (n=3) (A) membrane Gs α-subunits. Both membranes were incubated

with cholera toxin 150 µg/mL. After protein precipitation, the samples were denatured

and applied to a 12 % SDS-polyacrylamide gel. Incorporation of label resulted into the

detection of two bands of 45, and 47 kDa in hypothalamic and adrenal membranes.

There was no apparent difference between the fed and food deprived samples.

Panel (B). ADP ribosylation by pertussis toxin of hypothalamic (n=3) (H) and adrenal

(n=3) (A) membrane Gi α-subunits. After protein precipitation, the samples were

denatured and applied to a 12 % SDS-polyacrylamide gel. Incorporation of label

resulted into the detection of a single band of 41 kDa in hypothalamic and adrenal

membranes. There was no apparent difference between the fed and food deprived

samples.

Panel (C). Autoradiograph of OR-A induced photolabelling (with 32P-GTP-AA) of Gs

α-subunits from rat hypothalamic (H) and adrenal (A) membranes. Membranes were

incubated with 32P-GTP-AA and different concentrations of OR-A (10-11-10-7M),

followed by UV crosslinking and immunoprecipitation of the Gs α-subunit using a

specific antibody. Proteins were resolved on SDS-PAGE gels, followed by

autoradiography. Immunodetected bands were quantified by scanning densitometric

analysis. Identical results were obtained from three independent experiments (mean ±

S.D). *p<0.05 compared to basal activity, +p<0.05 compared to OR-A in the adrenal

membranes.

33

Figure 5. Panel (A): Autoradiograph of agonist-induced photolabeling G-protein α-

subunits with GTP-AA. Hypothalamic and adrenal cortex membranes from fed rats

(n=3 from each group) were incubated with GTP-AA and Orexin A (100 nM). The

experiment has been repeated in food deprived rats (n=3 from each group). Following

UV crosslinking, G-protein a-subunits were immunoprecipitated with specific antisera

for Gs, Gq/11, Gi1/2, and Go, and resolved on 12 % SDS polyacrylamide gels.

Immunodetected bands were quantified by scanning densitometric analysis. Data are

expressed as the mean ± S.D OD units were expressed as the ratio of treated over

untreated membranes for the rat hypothalamus (Panel B) and the rat adrenal cortex

(Panel C). *P<0.05; **P<0.01

Figure 6.

cAMP (Panel A) and IP3 (Panel B) accumulation from rat hypothalamic membranes

(100 µg protein) in the presence of different concentrations of OR-A. Results are

expressed as the mean ± S.D. of three independent experiments.

cAMP (Panel C) and IP3 (Panel D) accumulation from rat adrenal cortex membranes

(100 µg protein) in the presence of different concentrations of Orexin-A. Results are

expressed as the mean ± S.D. of three independent experiments. *P<0.05 **P<0.01

compared to basal activity, +p<0.05 comparing the food deprived and control

treatments for both the hypothalamus and adrenal cortex.

34

A.

CO2 Inhalation Cervical Dislocation

Fed 28 ± 2.7 ng/ml 27 ± 3.2 ng/ml

Food Deprived 51 ± 2.9 ng/ml 52 ± 2.1 ng/ml

Method of Sacrifice

Nutritional Status

0

1

2

3

4

5

6

7

Fed Food Deprived

mR

NA

(fol

d ab

ove

fed

rats

)

B.

Food Deprived

Fed

C.1 2 31 2 3

0

5

10

15

20

25

30

35

40

Fed Food Deprived

OD

Uni

ts

OrexinOrexin--AA

OrexinOrexin--A A (3.5 kDa)(3.5 kDa)

0

5

10

15

20

25

30

35

Fed Food Deprived

OD

Uni

ts

OrexinOrexin--BB1 2 31 2 3

OrexinOrexin--BB

(3.5 kDa)(3.5 kDa)

Figure-1

0

0.5

1

1.5

2

2.5

3

3.5

Fed Food Deprived

mR

NA

(fol

d ab

ove

fed

rats

) OX1R

0

0.5

1

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2

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mR

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(fol

d ab

ove

fed

rats

) OX2R

A.

B.

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 kDakDa250250150150

10010075755050

3737

25251515

OX1ROX2R

0

10

20

30

Fed Food Deprived

OD

Uni

ts

C.Food

DeprivedFed

OX1R

0

10

20

30

40

Fed Food Deprived

OD

Uni

ts

OX2R

0

5

10

15

Fed Food Deprived

OD

Uni

ts

β-actin

Figure-2

0

0.5

1

1.5

2

2.5

Fed Food Deprived

mR

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(fol

d in

crea

se a

bove

food

de

priv

ed ra

ts)

OX1R

FedFed

FoodFoodDeprivedDeprived

0

1

2

3

4

5

6

Fed Food DeprivedmR

NA

(fol

d in

crea

se a

bove

food

de

priv

ed ra

ts)

OX2R

FedFed

Food DeprivedFood Deprived

B.Fed Food

Deprived

0

10

20

30

Fed Food Deprived

OD

Uni

ts

0

10

20

30

40

Fed Food Deprived

OD

Uni

ts

0

5

10

15

Fed Food Deprived

OD

Units

OX1R

OX2R

β-actin

Figure-3

Gs(A) (B)Gi

Fed Food Deprived Fed Food Deprived-- + + -- ++CTxCTx -- + + -- ++PTx PTx

47 kDa45 kDaH H 41 kDa

A 47 kDa 45kDa A 41 kDa

(C)

1

1.5

2

2.5

3

basal 0.01 0.1 1 10 100concentration (nM)

Gs-

α: F

old

incr

ease

abo

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asal

(O

D u

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)

AdrenalHypothalamus* +

* +

**

*

*

Basal 10-11 10-10 10-9 10-8 10-7 M (OR-A)

Hypothalamus

GsGsαα

Adrenal Cortex

Figure 4

A.

H

- + - +Fed Food Deprived

- No Supplement+ OR- A (100nM)

H= HypothalamusA= Adrenal Cortex

GsA

H

A

Gi

H

A

Gq

HGo

A

Hypothalamus Adrenal CortexB. C.

1

2

3

4

5

6

7

8

9

Fed Food Deprived

Rat

io o

f OD

Uni

ts (+

/-)

1

1.2

1.4

1.6

1.8

2

2.2

2.4

Fed Food Deprived

Rat

io o

f OD

Uni

ts (+

/-)

1

1.5

2

2.5

3

3.5

Fed Food Deprived

Ratio

of O

D Un

its (+

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11.21.4

1.61.8

22.22.4

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3

Fed Food Deprived

Ratio

of O

D Un

its (+

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1

1.1

1.2

1.3

1.4

1.5

1.6

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Fed Food Deprived

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1234567891011

Fed Food Deprived

Rat

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1.8

2

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2.6

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Fed Food DeprivedR

atio

of O

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Figure-5

Hypothalamus Adrenal Cortex

A

0

20

40

60

80

100

††††

††

cAM

P (%

incr

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abo

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)

C

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120

†††† ††

cAM

P (%

incr

ease

abo

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asal

)

OR-A 0.01nM 0.1nM 1nM 10nM 100nM OR-A 0.01nM 0.1nM 1nM 10nM 100nM

BD

0

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IP3

(% in

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IP3

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†† †† ††

OR-A 0.01nM 0.1nM 1nM 10nM 100nM OR-A 0.01nM 0.1nM 1nM 10nM 100nM

Food DeprivedFed