University of Zurich€¦ · PD. Dr. Thomas Riediger Institute of Veterinary Physiology, University of Zurich Winterthurerstrasse 260 8057 Zurich, Switzerland Phone: +41-44-635-8815

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Year: 2008

The anti-ghrelin Spiegelmer NOX-B11-3 blocks ghrelin- but not

fasting-induced neuronal activation in the hypothalamic arcuate

nucleus

Becskei, C; Bilik, K U; Klussmann, S; Jarosch, F; Lutz, T A; Riediger, T

Becskei, C; Bilik, K U; Klussmann, S; Jarosch, F; Lutz, T A; Riediger, T (2008). The anti-ghrelin SpiegelmerNOX-B11-3 blocks ghrelin- but not fasting-induced neuronal activation in the hypothalamic arcuate nucleus.Journal of Neuroendocrinology, 20(1):85-92.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Journal of Neuroendocrinology 2008, 20(1):85-92.

Becskei, C; Bilik, K U; Klussmann, S; Jarosch, F; Lutz, T A; Riediger, T (2008). The anti-ghrelin SpiegelmerNOX-B11-3 blocks ghrelin- but not fasting-induced neuronal activation in the hypothalamic arcuate nucleus.Journal of Neuroendocrinology, 20(1):85-92.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Journal of Neuroendocrinology 2008, 20(1):85-92.

The anti-ghrelin Spiegelmer NOX-B11-3 blocks ghrelin- but not

fasting-induced neuronal activation in the hypothalamic arcuate

nucleus

Abstract

The hypothalamic arcuate nucleus (Arc) is the presumed target site for the orexigenic hormone ghrelinwhich is secreted from the stomach during fasting. Ghrelin directly activates Arc neurones. Similar toexogenous ghrelin, overnight food deprivation also induces c-Fos expression in the Arc of mice. In thisstudy we tested the role of endogenous ghrelin in the fasting-induced c-Fos expression in the Arc ofmice. We used NOX-B11-3, the latest generation of the recently developed ghrelin-binding compounds,so-called RNA Spiegelmers (SPM) to block endogenous ghrelin action during food deprivation. Thespecificity and potency of this compound was also tested in electrophysiological andimmunohistological experiments. In electrophysiological in vitro single cell recordings NOX-B11-3effectively blocked the excitatory effect of ghrelin in the medial Arc (ArcM) of rats while thebiologically inactive control SPM had no effect. Furthermore, NOX-B11-3 (15 mg/kg ip) potentlysuppressed ghrelin-induced (25 µg/kg sc, 12h after SPM injection) c-Fos expression in the Arc.However, the same dose of NOX-B11-3 when injected at the beginning of a 14h fasting period had noeffect on the c-Fos expression in the Arc of mice. These results demonstrate that NOX-B11-3 is a longacting compound, which effectively blocks the effect of exogenous ghrelin on neuronal activity in theArc under in vitro and in vivo conditions. Furthermore, increased ghrelin signalling does not appear tobe a necessary factor for the activation of Arc neurones during food deprivation or other fasting-relatedsignals might have masked or compensated the loss of the ghrelin effect.

The anti-ghrelin Spiegelmer NOX-B11-3 blocks ghrelin- but not fasting-induced

neuronal activation in the hypothalamic arcuate nucleus

Csilla Becskei1, Kerstin U. Bilik1, Sven Klussmann2, Florian Jarosch2, Thomas A. Lutz1,

Thomas Riediger1

1Institute of Veterinary Physiology and Zurich Centre of Human Integrative Physiology,

University of Zurich, 8057 Zurich, Switzerland 2NOXXON Pharma AG, 10589 Berlin, Germany

Corresponding author:

PD. Dr. Thomas Riediger

Institute of Veterinary Physiology, University of Zurich

Winterthurerstrasse 260

8057 Zurich, Switzerland

Phone: +41-44-635-8815

Fax: +41-44-635-8932

E-mail: [email protected]

Short title: NOX-B11-3 blocks ghrelin’s effects in the arcuate nucleus

Key words: immunohistochemistry, c-Fos, electrophysiology, food intake

This study was supported by the Research Committee and Young Academics Support

Committee of the University of Zurich. C.B. is a recipient of a fellowship grant from the

Zurich Centre of Integrative Human Physiology (University of Zurich).

2

Abstract

The hypothalamic arcuate nucleus (Arc) is the presumed target site for the orexigenic

hormone ghrelin which is secreted from the stomach during fasting. Ghrelin directly

activates Arc neurones. Similar to exogenous ghrelin, overnight food deprivation also

induces c-Fos expression in the Arc of mice. In this study we tested the role of

endogenous ghrelin in the fasting-induced c-Fos expression in the Arc of mice. We used

NOX-B11-3, the latest generation of the recently developed ghrelin-binding compounds,

so-called RNA Spiegelmers (SPM) to block endogenous ghrelin action during food

deprivation. The specificity and potency of this compound was also tested in

electrophysiological and immunohistological experiments. In electrophysiological in

vitro single cell recordings NOX-B11-3 effectively blocked the excitatory effect of

ghrelin in the medial Arc (ArcM) of rats while the biologically inactive control SPM had

no effect. Furthermore, NOX-B11-3 (15 mg/kg ip) potently suppressed ghrelin-induced

(25 µg/kg sc, 12h after SPM injection) c-Fos expression in the Arc. However, the same

dose of NOX-B11-3 when injected at the beginning of a 14h fasting period had no effect

on the c-Fos expression in the Arc of mice. These results demonstrate that NOX-B11-3 is

a long acting compound, which effectively blocks the effect of exogenous ghrelin on

neuronal activity in the Arc under in vitro and in vivo conditions. Furthermore, increased

ghrelin signalling does not appear to be a necessary factor for the activation of Arc

neurones during food deprivation or other fasting-related signals might have masked or

compensated the loss of the ghrelin effect.

3

Introduction

Ghrelin is a 28 amino acid peptide hormone, secreted predominantly from the endocrine

cells of the stomach (1). Plasma levels of ghrelin are modulated by the nutritional state.

During fasting and shortly before meals circulating ghrelin concentrations are elevated,

while feeding or glucose intake reduce circulating ghrelin levels (2,3,4,5,6). Central or

peripheral ghrelin administration stimulates food intake and body weight gain both in

rodents and humans (7,8,9,10). Neutralising circulating or central ghrelin has the opposite

effect in rats (7,11,12). Thus, ghrelin has been postulated to be an important peripheral

orexigenic signal that regulates ingestive behaviour and energy balance.

Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor type 1a

(GHS-R 1a,) which is densely expressed in the hypothalamic arcuate nucleus (Arc)

(13,14). Specific downregulation of GHS-R expression in the Arc by antisense riboprobes

reduced food intake and body weight gain in rats (15). The orexigenic effect of synthetic

growth hormone secretagogues (GHS) was also abolished in these animals, again arguing

for an Arc mediation of ghrelin’s orexigenic action.

In electrophysiological studies ghrelin (16,17) and GHS (18) activated the Arc neurones

by a direct, postsynaptic action. Furthermore, ghrelin and GHS administration induce c-

Fos expression, a marker of neuronal activation in the orexigenic neuropeptide Y (NPY)

neurones in the Arc (7,19,20). Hence, NPYergic Arc neurones are considered as a central

target site for circulating ghrelin.

Interestingly, c-Fos expression is induced in the Arc of food deprived mice (21,22), i.e.

when ghrelin levels are high. Furthermore, the fasting-induced activation of Arc neurones

seems to occur mainly in NPYergic neurones (23). Thus, it is plausible that elevated

4

ghrelin levels during fasting contribute to the induction of c-Fos expression in Arc

neurones of mice. However, due to the lack of specific ghrelin antagonists this has not

been investigated so far.

Recently a new class of L-RNA-based hormone antagonists, so-called Spiegelmers

(SPMs), has been developed. Unlike classic hormone antagonists, SPMs do not interact

with the receptor, but bind with high affinity to their target molecule and therefore block

their biological effect by preventing binding to the endogenous receptor. The anti-ghrelin

SPM, NOX-B11 is highly specific for the bioactive n-octanoylated form of ghrelin (24).

NOX-B11 potently and specifically blocked the biological effects of exogenous ghrelin,

e.g. the stimulation of growth hormone secretion, food intake and body weight gain

(24,25,26). In the current study we used the latest generation of the anti-ghrelin SPMs

(NOX-B11-3) which shows a 5-fold higher ghrelin binding potency than the original

NOX-B11 compound (27). In our studies we first tested the potency and specificity of

this new compound under our experimental conditions. Using in vitro extracellular single

unit recordings and in vivo c-Fos immunohistochemistry, we investigated whether NOX-

B11-3 blocks the excitatory effect of ghrelin on Arc neurones. Second, we explored in

immunohistological c-Fos studies whether endogenously released ghrelin contributes to

the activation of Arc neurones during fasting in mice.

Material and Methods

Spiegelmers

NOX-B11-3 is a 44 nucleotide L-RNA, containing a hexaethylene glycol linker (L) with

the sequence 5’-GCGUAAGACCGAAGGUAACCAAUCCUACCG---L---

5

CGGUGAGGCAGCAC-3’. It is a derivative of Spiegelmer NOX-B11 and its selection

has been described previously (27). The biologically inactive control Spiegelmer (cSPM)

has the sequence

5’UAAGGAAACUCGGUCUGAUGCGGUAGCGCUGUGCAGAGCU-3’. Spiegelmers

used in the in vivo studies were modified with a 40 kD polyethylene glycol (PEG) moiety

to delay renal excretion. All Spiegelmers were synthesised at NOXXON Pharma AG.

All animal procedures were approved by the Veterinary Office of the Canton Zurich.

Electrophysiological studies

In contrast to mice, NPY and POMC neurons are regionally clustered in rats in the medial

and lateral subregions of the Arc respectively, which makes it easier to specifically target

these cells in our electrophysiological approach. Furthermore, in our previous

electrophysiological studies characterizing the neuronal effects of ghrelin have all been

conducted in rats, thus in order to ensure compatibility between our studies we used rats

in the present experiments as well. Male adult Wistar rats (200-250g) were used, which

had ad libitum access to standard laboratory rat chow (#3430, Kliba Nafag AG,

Switzerland) and water. All animals were maintained in a temperature-controlled room

on an artificial 12 h dark-light cycle (21 ± 1 °C, lights on at 6.00 am). The experimental

procedures for the extracellular recordings were the same as previously described (17,28).

Rats were decapitated, their brains were quickly removed and superfused with modified

ice-cold artificial cerebrospinal fluid (aCSF) of the following composition (in mM): NaCl

124; KCl 5; NaH2PO4 1.2; MgSO4 1.3; CaCl2 1.2; NaHCO3 26; glucose 10, equilibrated

with 95% O2 and 5% CO2, pH 7.4; 290 mosm/kg. 400 µm thick coronal whole brain

6

slices at the mid rostro-caudal level of the Arc were cut using a custom made vibratome.

A rectangular (2x2 mm) piece of tissue containing the Arc was dissected by hand and

transferred to a temperature controlled (37.0°C) recording chamber constantly superfused

with pre-warmed aCSF at a rate of 1.6 ml/min.

Extracellular recordings were obtained from neurones of the ArcM using glass-coated

platinum-iridium electrodes. According to the neuroanatomical brain map of Paxinos and

Watson (29), the recorded neurones were located within a distance of 300 µm lateral to

the third ventrical wall and were thus defined as “ventromedial” Arc neurones (17).

Ghrelin (Bachem, Switzerland) was applied at a concentration of 10-8 M, which has been

shown to represent an effective dose without causing receptor desensitization (16,17).

After a stable recording from a single neurone had been established ghrelin was

superfused in combination with cSPM, with NOX-B11-3 (both 25 x 10-8 M) or without

any SPM compound. All cells were tested with all three stimuli. To facilitate the binding

between ghrelin and SPMs, ghrelin was co-incubated with the SPMs 15 min at room

temperature prior to superfusion. When superfused alone, ghrelin was kept under the

same conditions without SPM. In order to exclude that the SPMs cause any changes in

the firing rate, control superfusions of these substances (25 x 10-8 M) were carried out

without ghrelin. Only ghrelin responsive neurones were included in the analysis. From all

continuously recorded rate meter counts, the average discharge rate of each neurone was

evaluated for 60 seconds prior each stimulus. This value (spontaneous discharge rate) was

used to normalise changes in the firing rate, expressed as % change of the spontaneous

discharge rate. If the averaged change of discharge rate during the response was larger

than 20%, the neurone was considered sensitive to the applied stimulus. In addition to the

7

mean change of the discharge rate during the entire responses, the peak values of the

responses were calculated on a basis of a 30 s interval during which the firing rate was

maximal. Finally, the duration between the application of the drugs and the onset of the

effects (latency) and the duration of the entire responses were determined.

c-Fos studies

Animals

c-Fos expression is only upregulated in the Arc of mice during fasting, but not in rats, at

least during a moderate food deprivation, thus we used mice in the immunohistological

studies. Adult male C57Bl/6N (Charles Rivers, Germany) mice, housed in individual

cages in a temperature-controlled room (21 ± 1 °C) were used. The mice were kept under

a 12 h light/dark cycle (lights off 9 am). They had free access to rodent chow and water,

except in the food deprivation study, when animals were fasted for 14h. All animals were

handled daily for at least three weeks before the experiments, according to the procedure

recommended by Ryabinin et al. (30) which has been shown to reduce handling- and

injection-induced c-Fos expression.

Experimental procedure

Twenty mice which had ad libitum access to food during the studies were randomly

assigned to one of four experimental groups (n=5). On the day of the experiment two

groups received an intraperitoneal injection of the biologically inactive cSPM at the

beginning of the light phase (9pm). The other two groups received NOX-B11-3

(15mg/kg; 10ml/kg in saline). Twelve hours after Spiegelmer application (i.e. at the

8

beginning of the dark phase) one cSPM and one NOX-B11-3 treated group received a

ghrelin injection (25 µg/kg sc), while the other two groups received a saline (10ml/kg sc).

Mice were sacrificed 2h after ghrelin or saline injection (11am, i.e. 2h into the dark

phase). Two additional groups of mice (n=5) were food deprived at the beginning of the

light phase (9pm) and received a cSPM or NOX-B11-3 injection at this time point. These

mice remained fasted without any further treatment until sacrifice 14h later (11am).

Immunohistochemistry

Detection of c-Fos immunoreactive neurones in the Arc has been described previously

(28). Briefly, animals were deeply anaesthetised with pentobarbital (Nembutal, Abbott

Laboratories USA 0.1 ml/mouse ip) and perfused transcardially with phosphate buffer

(PB 0.1M), followed by 4% paraformaldehyde (in 0.1M PB). The brains were removed,

postfixed for 2h in the same fixative, cryoprotected in 20 % sucrose solution (in 0.1M

PB) for 48h and snap frozen in CO2. Coronal sections (20 µm) were cut in a cryostat (CM

3050 Leica, Nussloch, Germany) and every second hypothalamic section was thaw

mounted on microscopic glass slides (SuperFrost Plus, Faust, Schaffhausen Switzerland).

After air-drying at room temperature and rehydrating in phosphate buffered saline (PBS),

sections were incubated in blocking solution for 2h (1.5% rabbit normal serum + avidin,

Vector Laboratories). The primary antibody (polyclonal goat anti-c-Fos, Santa Cruz;

1:10’000 + biotin, Vector Laboratories) was applied for 48 hours at 4° C. The unbound

antibody was removed by washing in PBS before the sections were incubated with the

secondary antibody (biotinylated rabbit-anti-goat, Vectastain®-Elite ABC Kit, Vector

Laboratories; 1:200) for 2 hours at room temperature. After incubation in ABC solution

9

(Vectastain®-Elite ABC Kit, Vector Laboratories) DAB was used as a chromogen [0.04%

in PBS with 0,02% H2O2 and for colour enhancement 0.08% NiCl2 (x 6 H2O), 0.01%

CoCl2 (x 6 H2O)]. Finally, the sections were dehydrated in graded alcohols, cleared in

xylenes and coverslipped with Entellan (Merck, Darmstadt, Germany).

The localization of c-Fos expressing neurones was identified according to the mouse

brain atlas by Hof et al. (31). For each animal c-Fos positive cells were counted manually

and in a blinded fashion in eleven corresponding sections of the Arc bilaterally, in an area

within 150 µm from the wall of the third ventricle. The mean value of the cell

counts/section of an individual animal was used for statistical analyses. Representative

photomicrographs were taken by a digital camera (AxioCam, Carl Zeiss AG).

Ghrelin levels

Ghrelin levels were measured at 11 am in ad libitum fed (n=3) and 14h fasted mice (n=5)

in a separate experiment. Mice were anesthetised with pentobarbital as described in the c-

Fos studies and blood was collected into EDTA coated tubes by cardiac puncture. Plasma

total ghrelin levels were analyzed by RIA (Linco Research). Unfortunately, direct

analytical assessment of the inactivation of ghrelin in the plasma is limited, because

immunoassays do not distinguish SPM-bound from unbound ghrelin. For this reason

SPM treated groups could not be included in these studies.

Statistics

The electrophysiological data were analyzed by one way ANOVA on ranks for repeated

measurements, followed by Student-Newman-Keuls post-hoc test. In the

10

immunohistological studies group means were compared by Student’s t-test or by two

way ANOVA, followed by Student-Newman-Keuls post-hoc test. P<0.05 was considered

significant. Results are presented as means ± SEM.

Results

Electrophysiological studies

Nine single unit recordings from ghrelin-excited neurones, located in the ArcM and

presumably containing NPY, were obtained from 9 different slice preparations.

Superfusion of ghrelin alone or in combination with the cSPM resulted in reversible

excitatory responses (Figure 1A; Table 1). The mean excitatory effect of ghrelin in

combination with cSPM was not different from the mean excitatory action induced by

ghrelin alone (Table 1). However, co-application of NOX-B11-3 effectively blocked the

ghrelin-induced activation in the ArcM (p<0.05) (Figure 1A; Table1). When applying the

general criteria of an excitatory effect as outline above, 1 of the 9 neurones was excited

by ghrelin in the presence of NOX-B11-3. However, the mean increase in the discharge

rate in this neurone was 73% smaller when compared to the co-application of ghrelin and

cSPM (1.7 vs. 6.3 Hz). All mean effect parameters for the different stimuli are

summarised in Table 1. Notably, all effect parameters were significantly lower following

the combined superfusion with NOX-B11-3 and ghrelin compared to the other two

stimuli. Neither cSPM (n=4) nor NOX-B11-3 (n=3) affected neuronal activity when

superfused alone (Figure 1B).

c-Fos studies

11

Influence of SPMs on ghrelin-induced c-Fos expression in the Arc

Ad libitum fed, saline injected animals showed hardly any c-Fos positive cells in the Arc,

irrespective of NOX-B11-3 or cSPM pretreatment. Compared to saline injected animals,

ghrelin significantly increased the number of c-Fos immunoreactive cells in the Arc of

cSPM treated mice (p<0.001), but not in the NOX-B11-3 pretreated animals (Figure 2).

Two way ANOVA revealed a significant main effect of NOX-B11-3 (p<0.01) and

ghrelin (p<0.001) treatment and a significant interaction between NOX-B11-3 and

ghrelin injection (p<0.05) in the ad libitum fed animals.

Influence of SPMs on fasting-induced c-Fos expression in the Arc

Fasted mice had 5 fold higher blood ghrelin levels (8.3 ±0.8 ng/ml) compared to ad

libitum fed mice (1.4 ± 0.3 ng/ml; p=0.001).

Fasting produced strong c-Fos expression in the Arc of mice. The number of c-Fos

positive cells was similar in animals that were pretreated with NOX-B11-3 or cSPM

(Figure 3).

Discussion

The orexigenic effect of ghrelin, the only peripheral orexigenic peptide known to date, is

presumably mediated via Arc neurones. We investigated whether the increase in plasma

ghrelin concentrations and the parallel increase in c-Fos expression in the Arc of fasted

mice might be causally linked. Although ghrelin concentrations rose 5 fold in the fasted

mice, the anti-ghrelin Spiegelmer NOX-B11-3, had no effect on the fasting-induced c-Fos

expression in the Arc. This suggests that an increase in circulating ghrelin does not seem

12

to be a decisive factor for the fasting-induced neuronal activation in the Arc. As shown in

our electrophysiological and immunohistological studies NOX-B11-3 potently and

specifically blocked the excitatory effect of exogenous ghrelin on Arc neurones.

The SPMs directly bind to their target molecules and thus neutralise their effects. Such an

interaction minimises the risk of undesired side effects, e.g. partial agonistic action that is

a common phenomenon for numerous receptor antagonists, particularly when used at

higher doses. In our studies we did not find any indication for agonistic effects of the

SPMs. When applied alone neither the inactive cSPM nor NOX-B11-3 caused any

changes in firing rate and c-Fos expression was minimal in mice treated with cSPM and

NOX-11B-3 alone. Furthermore, there was no detrimental impact of NOX-B11-3 on

neuronal function as shown by the undisturbed responsiveness to ghrelin after washout of

the drug.

In previous electrophysiological studies we characterised the effect of the GHS-R

antagonist (D-Lys3)-GHRP-6 under the same experimental conditions (16). Even in

1000-fold excess this compound only weakly antagonised the ghrelin-induced responses.

Compared to this very low potency, NOX-B11-3 was clearly superior because it almost

completely blocked the ghrelin-induced excitation when used in only 25 fold molar

excess.

Spiegelmers are L-isomer oligonucleotides which are not degraded by endogenous

nucleases. Hence, SPMs are unusually stable in biological fluids (over 60 hrs) (32),

allowing a long-lasting peptide neutralisation after single application. This makes these

13

compounds very useful for experimental purposes and possibly as therapeutic agents. Our

c-Fos studies are consistent with a long-lasting effect, because NOX-B11-3 abolished the

neuronal effects of exogenous ghrelin in the Arc when applied 12h before a ghrelin

stimulus. These results provide evidence that the lack of NOX-B11-3 effect on the

fasting-induced activation of Arc neurones was not due to the loss of effect during the

time between injection and sacrifice. Rather, our data argue against a dominant

contribution of endogenous ghrelin to the fasting-induced activation in the Arc.

Nevertheless, based on our results a stimulatory effect of circulating ghrelin on Arc

neurons cannot be completely excluded. It is plausible that other fasting-related signals

could have compensated for the loss of ghrelin’s effect in the NOX-B11-3 treated fasted

mice. However, the identity of these factors, which might be involved in the activation of

the Arc during food deprivation, remains unknown at this point.

The Arc is a receptive site for various peripheral signals, the blood levels of which

change according to the feeding status, including glucose, leptin, insulin and

corticosterone. These factors are likely candidates to modulate Arc activity during

fasting. The Arc contains glucosensitive neurones, (for reviews see 33,34) and the

orexigenic NPY neurones are excited (35,36) and increase c-Fos expression (37,38) when

glucose level decrease. Lean fasted mice have a blood glucose level about 30% below

that of ad libitum fed mice under our experimental conditions (unpublished observation).

Thus, it is plausible that fasting-induced hypoglycaemia might have contributed to the c-

Fos expression in the Arc. This would be consistent with our observations that a

peripheral injection of glucose effectively reverses the fasting-induced activation in Arc

neurones (39).

14

Insulin and leptin are considered two of the most important peripheral signals involved in

the long-term regulation of energy balance and body weight. Both peptides have well-

established inhibitory effects on orexigenic Arc neurones and the blood level of both

decreases in fasted animals (40). Therefore it is plausible that during fasting a

disinhibition of these neurones occurs, which then either per se may induce c-Fos

expression or may facilitate the action of other fasting-related signals in the Arc.

In addition to humoral signals, the Arc receives neuronal inputs that convey information

reflecting the energy status. The NPY neurones receive excitatory projections form the

orexin containing neurones (41,42) located in the lateral hypothalamic area (43, 44).

Orexins stimulate food intake and their expression is upregulated during fasting (45).

Hence, it is conceivable that Arc-intrinsic orexin release might contribute to the neuronal

activation in the Arc during food deprivation. Furthermore, recent studies suggest a role

for hindbrain projections to the hypothalamus in the control of food intake and in the

mediation in ghrelin’s neuronal and behavioural effects (46).

The physiological impact of neuronal activation in the Arc during fasting is not yet

known. Many of the NPY containing neurones have been shown to express c-Fos in the

fasted state (23) and it is commonly accepted that an activation of these cells stimulates

food intake (40). Arc neurones, especially NPY neurones, are however involved in the

regulation of several neuroendocrine functions as well, which induce adaptive responses

to starvation via controlling growth, gonadal, adrenal and thyroid function. In fasted

animals non-vital functions are inhibited: growth hormone secretion is reduced, in female

15

mice oestrus cycle is delayed, in male mice testosterone levels are severely reduced (47).

Furthermore, thyroid hormone concentrations are decreased while glucocorticoid levels

are increased (47). Similar responses were reported in human subjects (48). Thus the

activation in the Arc neurones, besides enhancing energy repletion when food is

available, might also be involved in the re-partitioning of endogenous energy sources

during fasting and in the reduction energy expenditure.

In summary our study provides in vitro electrophysiological and in vivo

immunohistochemical evidence that the recently developed L-RNA Spiegelmer, NOX-

B11-3 is a potent and specific ghrelin antagonist, which exerts a long-lasting action after

a single peripheral administration. Based on these results the persistence of fasting-

induced c-Fos expression in the Arc of NOX-B11-3 treated mice argues against a

predominant contribution of endogenous ghrelin to the stimulation of Arc neurones

during food deprivation. Hence, the sum of other fasting-related signals, possibly a drop

in glucose, insulin and leptin concentrations, may be sufficient to activate the Arc. Which

of these factors plays the predominant role in this process awaits further clarification.

Acknowledgements

This study was supported by the Research Committee and Young Academics Support

Committee of the University of Zurich. C.B. is a recipient of a fellowship grant from the

Zurich Centre of Integrative Human Physiology (University of Zurich). We are grateful

to Stefan Vonhoff for the synthesis of NOX-B11-3.

16

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Figure legends

Figure 1. Electrophysiological single unit recordings of ghrelin-sensitive neurones

located in the medial arcuate nucleus (rat). A: In contrast to the inactive control

24

Spiegelmer (cSPM) the ghrelin antagonist NOX-B11-3 effectively blocked the ghrelin-

induced excitation. Stimulation with ghrelin alone induced a similar excitatory effect as

ghrelin in combination with cSPM. B: Neither the inactive control Spiegelmer nor the

ghrelin antagonist NOX-B11-3 affected the neuronal activity when superfused alone.

25

Figure 2. Anti-ghrelin Spiegelmer NOX B11-3, but not biologically inactive control

Spiegelmer (cSPM) completely blocked ghrelin-induced c-Fos expression in the arcuate

nucleus (Arc) of ad libitum fed mice. Representative Arc sections immunostained for c-

Fos of mice pretreated with NOX-B11-3 (15 mg/kg, ip) or cSPM. 12h after SPM

injection mice received a ghrelin (25 µg/kg sc) or saline injection (n=5/group). Bar charts

show the number of c-Fos positive neurons in the Arc of the different treatment groups.

Different letters indicate significant differences between the groups (ANOVA; p<0.05).

3V: 3rd ventricle, Scale bar: 100 µm.

26

Figure 3. The anti-ghrelin Spiegelmer NOX B11-3 (15 mg/kg, ip) had no effect on c-Fos

expression in the Arc of 14h fasted mice. Representative Arc sections immunostained for

c-Fos of mice pretreated with NOX-B11-3 (15 mg/kg, ip) or biologically inactive control

Spiegelmer (cSPM). At the time of injections mice were food deprived and remained

fasted until sacrifice 14h later. Bar charts show the number of c-Fos positive neurons in

the Arc of the different treatment groups. (n=5/group; Student’s t-test). 3V:3rd ventricle,

Scale bar: 100 µm.

27

Table 1:

Table 1: Effect parameters of the electrophysiological responses induced by ghrelin (10-8 M) in the medial arctue nucleus (rat) when superfused alone and in combination with inactive control Spiegelmer (cSPM) or the anti-ghrelin Spiegelmer NOX-B11-3 (both 25 x 10-8 M). NOX-B11-3 significantly blocked the ghrelin-induced excitatory response compared to superfusion of ghrelin alone or in combination with the cSPM. Different letters indicate significant differences (one-way ANOVA; p<0.05). Values are means ± SEM.

Effect parameter ghrelin + cSPM (n=9)

ghrelin + NOX-B11-3 (n=9)

ghrelin (n=9)

Mean latency [s] 106 ± 16 a 43 ± 10 b 102 ± 22 a

Mean response [%] 85 ± 16 a 8 ± 4 b 91 ± 20 a Mean response [Hz] 2.6 ± 0.6 a 0.4 ± 0.2 b 2.5 ± 0.5 a Mean peak response [Hz] 4.6 ± 1.2 a 1.0 ± 0.4 b 4.5 ± 1.1 a Mean response duration [s] 894 ± 122 a 409 ± 69 b 921 ± 141 a