ncomms3685 PDF

Nom original: ncomms3685.pdf

Titre: Anti-ghrelin immunoglobulins modulate ghrelin stability and its orexigenic effect in obese mice and humans

Auteur: Kuniko Takagi

Ce fichier au format PDF 1.6 a été généré par Adobe Illustrator CS2 / Acrobat Distiller 8.1.0 (Windows), et a été envoyé sur Fichier-PDF le 27/10/2013 à 19:00, depuis l'adresse IP 212.195.x.x. La présente page de téléchargement du fichier a été vue 1004 fois.
Taille du document: 1.4 Mo (11 pages).
Confidentialité: fichier public

Aperçu du document

ARTICLE
Received 8 Aug 2013 | Accepted 30 Sep 2013 | Published 25 Oct 2013

DOI: 10.1038/ncomms3685

OPEN

Anti-ghrelin immunoglobulins modulate ghrelin
stability and its orexigenic effect in obese mice
and humans
Kuniko Takagi1,2,3,*, Romain Legrand1,2,*, Akihiro Asakawa3, Haruka Amitani3, Marie Franc¸ois1,2,
Naouel Tennoune1,2, Moı¨se Coe¨fﬁer1,2,4, Sophie Claeyssens1,2,4, Jean-Claude do Rego2,5,
Pierre De´chelotte1,2,4, Akio Inui3 & Sergueı¨ O. Fetissov1,2

Obese individuals often have increased appetite despite normal plasma levels of the main
orexigenic hormone ghrelin. Here we show that ghrelin degradation in the plasma is inhibited
by ghrelin-reactive IgG immunoglobulins, which display increased binding afﬁnity to ghrelin in
obese patients and mice. Co-administration of ghrelin together with IgG from obese individuals, but not with IgG from anorectic or control patients, increases food intake in rats.
Similarly, chronic injections of ghrelin together with IgG from ob/ob mice increase food intake,
meal frequency and total lean body mass of mice. These data reveal that in both obese
humans and mice, IgG with increased afﬁnity for ghrelin enhances ghrelin’s orexigenic effect,
which may contribute to increased appetite and overeating.

1 Inserm UMR1073, Nutrition, Gut and Brain Laboratory, Rouen 76183, France. 2 Institute for Research and Innovation in Biomedicine (IRIB), Rouen University,
Normandy University, Rouen 76183, France. 3 Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental
Sciences, Kagoshima 890-8520, Japan. 4 Rouen University Hospital, CHU Charles Nicolle, Rouen 76183, France. 5 Animal Behavior Platform (SCAC), IRIB,
Rouen 76183, France. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.O.F.
(email: Serguei.Fetissov@univ-rouen.fr).

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

1

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

O

besity is commonly accompanied by increased hunger
and food intake1. However, the plasma concentrations of
ghrelin, the principal hunger hormone2, are not
elevated3,4. This indicates that in obesity ghrelin may transmit
even more powerful hunger signals to the central circuitries
regulating appetite5,6. In fact, ghrelin administration has been
shown to stimulate food intake more efﬁciently in obese than in
lean humans7 but the underlying mechanisms remain unknown.
Ghrelin is secreted by the stomach as an acylated bioactive
peptide8 that degrades to des-acyl-ghrelin9 having no orexigenic
effects10. Earlier studies assaying total ghrelin in plasma have
reported lower levels in obese subjects3, but as it was later
clariﬁed, this decrease might be due to the low levels of des-acylghrelin, whereas acyl-ghrelin remains normal4,11 suggesting
decreased degradation of acyl-ghrelin in the obese. On the
contrary, patients with anorexia nervosa (AN) display elevated
plasma levels of both total12 and acylated ghrelin13, indicating
increased ghrelin production. Recently, ghrelin-reactive
immunoglobulins (Ig), that is, autoantibodies (autoAbs), have
been identiﬁed in healthy subjects14 and in AN patients15.
Furthermore, studies in rodents showed that gastric electrical
stimulation of ghrelin secretion16 leads to simultaneous increase
of ghrelin-reactive IgG17, and that raising antibodies against
ghrelin may lead to decreased body weight in immunized
animals18. Consequently, it is possible that plasma ghrelin may
be bound to naturally present ghrelin-reactive IgG and that
changes of properties of such IgG may inﬂuence the biological
activity of circulating ghrelin, including increasing the hormone’s
orexigenic effect in obesity.
In the present work, we determine that biologically available
ghrelin in human plasma is bound to IgG, which protects it from
degradation. Using surface plasmon resonance (SPR) technology19, we characterize the afﬁnity kinetics between plasma IgG
and ghrelin in obese subjects and leptin-deﬁcient obese ob/ob
mice20 known for their hyperphagia21, revealing increased afﬁnity
of anti-ghrelin IgG in both obese mice and humans. We also
show that IgG extracted from plasma of obese humans or ob/ob
mice enhance ghrelin-induced feeding in rodents, supporting the

Anti-ghrelin IgG in patients and controls. Next, to determine
plasma levels of ghrelin-reactive IgG, we used enzyme-linked

b

80

Free ghrelin
(fmol ml–1)

Results
Ghrelin concentrations in patients and controls. Plasma ghrelin
and des-acyl ghrelin concentrations were determined in plasma
acidiﬁed for the preservation of ghrelin from venous blood
samples taken at 8:00 after overnight fast in patients with
hyperphagic obesity (n ¼ 14, body mass index (BMI)±s.e.m.,
31.08±1.56 kg m 2), restrictive AN (n ¼ 12, BMI±s.e.m. 13.33
±0.38 kg m 2) and healthy lean controls (n ¼ 14, BMI±s.e.m.,
20.92±0.48 kg m 2) (all females; age±s.e.m., 23.14±1.08 years,
21.42±1.9 years and 18.86±1.14 years, respectively).
As expected, concentrations of both ghrelin and des-acyl
ghrelin were elevated in AN patients (Fig. 1a,b), whereas ghrelin
and des-acyl ghrelin in the obese did not differ signiﬁcantly from
that in controls (Fig. 1a,b). To determine whether plasma ghrelin
was bound to circulating IgG, we measured concentrations of
both ghrelin and des-acyl ghrelin in the eluates of IgG extracted
from human plasma. We found that both ghrelin and des-acyl
ghrelin bound to IgG were well detectable in all three study
groups, although relatively more ghrelin than des acyl-ghrelin was
found in the IgG-bound form (Fig. 1c,d). Ghrelin and des-acyl
ghrelin concentrations were then assayed in the IgG-deprived
plasma efﬂuents, revealing increased levels of IgG unbound
ghrelin in obese patients versus controls (Fig. 1c), whereas IgG
unbound des-acyl ghrelin was higher only in AN patients
(Fig. 1d). The cumulative ghrelin concentrations resulting from
the sum of ghrelin measured in the IgG-bound and unbound
fractions of the same plasma samples were increased in both
obese and AN patients as compared with controls (Fig. 1c),
whereas cumulative des-acyl ghrelin was increased only in AN
patients (Fig. 1d). The ratios of ghrelin to des-acyl ghrelin did not
signiﬁcantly differ among the groups (Fig. 1e).

*

**

400

Free des-acyl
ghrelin (fmol ml–1)

a

involvement of ghrelin-reactive immunoglobulins in increased
appetite and overeating in obesity.

60
40
20

200
100

0

0

#

50

Obese

d

**

Cumulative des-acyl
ghrelin (fmol ml–1)

Cumulative ghrelin
(fmol ml–1)

60

AN

40
30
20
10

500

Contr

e

**

400
300
200
100
0

0
Contr
IgG bound

AN

Obese
IgG unbound

Contr
IgG bound

AN

Cumulative ghrelin/
des-acyl ghrelin ratios

Contr

c

***

300

AN

Obese

Obese

0.3
0.2
0.1
0.0
Contr

AN

Obese

IgG unbound

Figure 1 | Ghrelin and des-acyl ghrelin concentrations in humans. Plasma concentrations of ghrelin (a) and des-acyl ghrelin (b) before IgG extraction.
Ghrelin (c) and des-acyl ghrelin (d) bound to the plasma-extracted IgG or IgG unbound assayed in IgG-deprived efﬂuents of the same plasma samples
are shown in the same bar as cumulative ghrelin concentrations for which statistical analysis is presented. Ratios of cumulative plasma concentrations
between ghrelin and des-acyl ghrelin (e). (a) Kruskal–Wallis (K–W) test, P ¼ 0,002, Dunn’s *Po0.05, **Po0.01; (b) K–W test, P ¼ 0.0008, Dunn’s
***Po0.001; (c) K–W test, P ¼ 0.001, Dunn’s **Po0.01, Mann–Whitney test #Po0.05; (d) K–W (M-W) test, P ¼ 0,009, Dunn’s **Po0.01. (Contr. n ¼ 14,
AN n ¼ 12 and obese n ¼ 14, error bars, s.e.m.).
2

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE
–1

Plasma IgG (mg ml )

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

Ghrelin IgG
in plasma (OD)

0.4
0.3
0.2
0.1
0.0

***
1.0
0.5

Obese

10
5
0
Contr

2.5
2.0
1.5
1.0
0.5
0.0

0.0
0M
10–6 M
Ghrelin concentration

AN

Ghrelin IgG (OD)
in obese patients

Ghrelin IgG (OD)
in controls

1.5

AN

Ghrelin IgG (OD)
in AN patients

Contr

*

15

Obese

1.5

***

1.0
0.5
0.0

0M
10–6 M
Ghrelin concentration

0M
10–6 M
Ghrelin concentration

Figure 2 | Ghrelin-reactive IgG and total IgG in humans. Plasma levels of ghrelin-reactive IgG (a). Plasma concentrations of total IgG (b) measured
by the nephelometry. Plasma levels in optical density (OD) of ELISA of ghrelin-reactive IgG before and after absorptions of plasma with 10 6 M ghrelin in
control subjects (c), AN patients (d) and obese (e). Absorption was efﬁcient in controls and the obese but not in AN patients, probably due to
increased dissociation properties of some AN patients’ IgG (Fig. 4f). (b) Student’s t-test *Po0.05; (c,e) Paired t-tests, ***Po0.001. (Contr. n ¼ 14,
AN n ¼ 12 and obese n ¼ 14, error bars, s.e.m.).

immunosorbent assay (ELISA). We found that the plasma levels
of ghrelin-reactive IgG were similar in all three groups (Fig. 2a)
but total IgG concentrations assayed by immunonephelometry
were slightly lower in obese patients (Fig. 2b). As a speciﬁcity
control, we also conﬁrmed that detection of ghrelin-reactive IgG
in ELISA was inhibited by preincubation of plasma with 10 6 M
ghrelin in both controls and obese, but it was ineffective in AN
patients (Fig. 2c–e).
The ability of a protein to act as a carrier for a small molecule
depends on the speciﬁcity and reversibility of binding as
determined by their afﬁnity kinetics. In this study, we used SPR
technology to determine afﬁnity kinetics between ghrelin and IgG
puriﬁed from the plasma of obese, AN or control subjects after
plasma acidiﬁcation and peptide extraction. By comparing the
binding of each study subject’s IgG (0.5 mg ml 1 at ﬂow speed of
5 ml min 1) to ghrelin, we found different dynamics of association and dissociation, resulting in an overall reduced SPR
response in the obese as compared with controls and AN patients
(Fig. 3a). A decrease in mean SPR resonance units (RU) values in
obese was particularly evident at the end of association and was
less pronounced after dissociation (Fig. 3b,c), indicating slower
dissociation.
Next, the afﬁnity kinetics were analysed for each study subject
using ﬁve consecutive IgG dilutions (from 0.5 to 0.03 mg ml 1 at
ﬂow speed of 30 ml min 1) and the 1:1 Langmuir’s ﬁt model
(Fig. 4a–c). In all study subjects, we found that the equilibrium
dissociation constant KD values of ghrelin-reactive IgG were in
the micromolar range between 10 6 M and 10 7 M. However,
the mean KD, which is an inverse measure of afﬁnity, was
signiﬁcantly lower in obese subjects than in controls (by a factor
of 1.2) and than in AN patients (by a factor of 2.5) (Fig. 4d). This
increase in afﬁnity was mainly due to higher association rates
(small Ka values) in obese versus controls (by a factor of 3)
(Fig. 4e), as mean dissociation rates (small Kd values) in the obese
were not signiﬁcantly different from that in controls (Fig. 4f). In
contrast, increased mean small Kd values characterized IgG from
AN patients (Fig. 4f).
We also analysed plasma levels and afﬁnity kinetics of patients’
and controls’ IgG to bind des-acyl ghrelin, which showed no

signiﬁcant differences among the groups for either of their plasma
levels (Fig. 5a) or parameters of afﬁnity kinetics, with KD detected
in the micromolar range (Fig. 5b–d).
Obese patients IgG enhance ghrelin orexigenic effects. To
determine whether IgG may modulate the orexigenic effects of
ghrelin, we injected intraperitoneally in free-feeding rats ghrelin
alone or together with human IgG characterized by different
afﬁnities for ghrelin. We used six IgG with the lowest RU values
(increased afﬁnity) from obese, six IgG with the highest RU (low
afﬁnity) from AN patients and six IgG that were most similar to
the mean RU values in the control group. As we studied the
interaction of human ghrelin with human autoAbs, we also used
human ghrelin in the rat study. As it differs from rat ghrelin by
two amino acids2, we ﬁrst analysed the effect of human ghrelin on
food intake in rats. We found that 3 nmol but not 1 nmol of
human ghrelin increased 30-min food intake in rats, but it was
less potent than rat ghrelin (Fig. 6a,b). Nevertheless, we selected
the 1 nmol dose of human ghrelin for the co-administration
experiment aiming at better distinguishing potential modulatory
effects of IgG on ghrelin-induced food intake; in fact, 1 nmol of
rat ghrelin was previously identiﬁed as the smallest dose stimulating food intake in satiated rats22. After co-administration of
1 nmol of human ghrelin with 1 nmol of IgG from obese, AN
patients or controls, we found that only rats receiving the
combination with IgG from obese displayed increased food intake
as compared with the other groups (Fig. 6c). This orexigenic
effect was acute and gradually disappeared by 12 h after injection
(Fig. 6d). The enhanced feeding effect associated with obese IgG
was linked to their co-administration with ghrelin, as injection of
1 nmol of the same IgG alone had no effects on food intake in
free-feeding rats at 30 min or later (Fig. 6e,f).
Patients’ and controls’ IgG protect ghrelin from degradation.
To test whether IgG might protect ghrelin from degradation by
plasma-hydrolysing enzymes, we studied in vitro stability of
30 fmol ml 1 of ghrelin, which is in the range of physiological
plasma ghrelin levels, after its incubation for 2 h at 37 °C in the

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

3

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

5 min
assoc
0

200

400
600
800
Time (s)
Contr
AN
Obese

b

c
*

300
280
260
240
220
200
Contr

AN

**

Ghrelin/ IgG
5 min diss (RU)

5 min
diss

300
250
200
150
100
50
0
–50

Ghrelin/ lgG
5 min assoc (RU)

Ghrelin/ lgG
response (RU)

a

#

150
140
130
120
110
100

**

Obese

Contr

AN

Obese

Figure 3 | Afﬁnity binding between human ghrelin and IgG assayed by SPR. Representative SPR sensorgrams (a) of association and dissociation to
ghrelin of IgG from obese (blue), controls (green) and AN patients (red) after single injection of 0.5 mg ml 1 of IgG with low ﬂow speed of 5 ml min 1.
Mean SPR response in RU between three study groups 5 min after association (b) and 5 min after dissociation (c) as indicated by arrows in (a).
(b) ANOVA, P ¼ 0.002, Tukey’s **Po0.01, *Po0.05; (c) K–W test, P ¼ 0.009, Dunn’s **Po0.01, M-W test, #Po0.05. (Contr. n ¼ 14, AN n ¼ 12 and
obese n ¼ 14, error bars, s.e.m.).

Ghrelin/lgG
response (RU)

a

200
Control

160
120
80
40
0
150

50

250

350

450

550

Time (s)

Ghrelin/lgG
response (RU)

b

200
160

AN

120
80
40
0
50

150

250

250

450

550

Time (s)

Ghrelin/lgG
response (RU)

c

200
Obese

160
120
80
40
0
50

150

250

350

550

450

Time (s)

Ghrelin lgG
KD (M)

**

1.0×10–6
1.0×10–7
1.0×10–8

Contr

AN Obese

f
1.0×106

*

1.0×105
1.0×104
1.0×103
Contr

Ghrelin lgG
small Kd (S–1)

e
**

1.0×10–5

Ghrelin lgG
small Ka (M–1 S–1)

d

1.0×10–1

*

*

1.0×10–2
1.0×10–3
1.0×10–4

AN Obese

Contr

AN Obese

Figure 4 | Afﬁnity kinetics between human ghrelin and IgG assayed by SPR. Afﬁnity kinetics were analysed by the Langmuir’s 1:1 model ﬁt on ﬁve
serial dilutions of IgG from 0.5 mg ml 1 and ﬂow speed of 30 ml min 1. Representative examples of curve ﬁts for the afﬁnity kinetic analysis of a
control subject (a), AN (b) and obese (c) patients. The afﬁnity kinetics parameters of the given examples are the following: (a) small ka (±s.e.m.),
9.17 103±1.09 102 M 1 s 1, small Kd (±s.e.m.), 1.37 10 3±3.14 10 5 s 1, KD, 1.50 10 7 M, w2 21.1; (b) small Ka (±s.e.m.),
8.76 103±1.17 102 M 1 s 1, small Kd (±s.e.m.), 1.78 10 3±3.52 10 5 s 1, KD, 2.03 10 7 M, w2 13.7 and (c) small Ka (±s.e.m.),
1.71 104±2.53 102 M 1 s 1, small Kd (±s.e.m.), 1.77 10 3±3.98 10 5 s 1, KD, 1.04 10 7 M, w2 12.3. Afﬁnity for ghrelin of IgG in obese
patients was increased compared to controls and AN patients as represented by lower mean KD values in obese (d). Association (Ka) and dissociation
(Kd) rates are shown in (e) and (f), respectively. (d) K–W test, P ¼ 0,004, Dunn’s **Po0.01 AN versus obese, Student’s t-test **Po0.01 controls
versus obese; (e) K–W test, P ¼ 0.03, Dunn’s *Po0.05; (f) K–W test, P ¼ 0,034, Dunn’s *Po0.05 controls versus AN, M–W test, *Po0.05 AN
versus obese. (Contr. n ¼ 14, AN n ¼ 12 and obese n ¼ 14, error bars, s.e.m.).
4

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

1.0×10–6
Des-acyl ghrelin
IgG KD (M)

Des-acyl ghrelin
IgG (OD)

0.8
0.6
0.4
0.2

1.0×10–8

0.0
Control

AN

Obese

1.0×106

Control

AN

Obese

Control

AN

Obese

1.0×10–2
Des-acyl ghrelin
IgG small Kd (s–1)

Des-acyl ghrelin
IgG small Ka (M–1 s–1)

1.0×10–7

1.0×105
1.0×104
1.0×103

1.0×10–3

1.0×10–4
Contr

AN

Obese

Figure 5 | Anti-des-acyl ghrelin IgG in humans. Plasma levels of des-acyl ghrelin-reactive IgG (a). Afﬁnity kinetics between human des-acyl ghrelin
and IgG showing: dissociation equilibrium constants (KD) (b), association rates (small Ka) (c) and dissociation rates (small Kd) (d). (Contr. n ¼ 14,
AN n ¼ 12 and obese n ¼ 14, error bars, s.e.m.).

Food intake
30 min (g)

*

3
2

#

Food intake (g)

**

4

#

#

1

#

#

3

Ghrelin Rat
3 nmol

2
1
Ghrelin +
Obese IgG

Ghrelin +
AN IgG

Ghrelin +
Contr IgG

Ghrelin

Food intake (g)

3
2
1

5 7 9 11
Time (h)

4

Ghrelin+PBS
Ghrelin+Contr IgG
Ghrelin+AN IgG
Ghrelin+Obese IgG

2
0

1

3

5 7 9 11
Time (h)

12
10
8
6
4
2
0

Obese
IgG

AN
IgG

0
Contr
IgG

3

6

0

4

PBS

1

8

*

0

Food intake
30 min (g)

PBS
Ghrelin Hu 1 nmol
Ghrelin Hu 3 nmol
Ghrelin Rat 3 nmol
0

Food intake (g)

Food intake
30 min (g)

4

Ghrelin Hu
3 nmol

Ghrelin Hu
1 nmol

PBS

0

12
10
8
6
4
2
0

PBS
Contr IgG
AN IgG
Obese IgG
0

1

3

5 7 9 11
Time (h)

Figure 6 | Acute effects of human IgG on ghrelin-induced food intake in rats. Comparison of orexigenic effects of human (Hu) ghrelin and rat ghrelin in
satiated rats (a,b). Higher 30 min food intake in free-fed rats was induced by co-administration of human ghrelin (1 nmol), together with IgG puriﬁed
from plasma of obese (1 nmol) (c,d), which alone did not signiﬁcantly change food intake as a shown in (e,f). (a) ANOVA, P ¼ 0,005, Tukey’s
**Po0.01, *Po0.05, Student’s t-tests #Po0.05; (c) ANOVA, P ¼ 0,031, Tukey’s *Po0.05, Student’s t-tests #Po0.05. (n ¼ 6, error bars, s.e.m.).

IgG-deprived plasma efﬂuents alone or together with 1 nmol of
IgG extracted from the same plasma samples. We found that after
incubation of ghrelin together with IgG from either controls, AN
or obese patients, most of the initially added ghrelin could be

detected in the incubation milieu without signiﬁcant differences
between the groups (Fig. 7a). However, when ghrelin was incubated alone, its concentration was strongly decreased, indicating
its rapid degradation (Fig. 7a). Furthermore, the efﬁciency of

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

5

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

30

***

***
***

20
10
#
0

Contr
AN
Ghrelin + lgG

Obese
Ghrelin

Ghrelin protection
by lgG (folds)

Ghrelin (fmol ml–1)

40

*

30

*

20
10
0
Contr

AN

Obese

Figure 7 | Ghrelin degradation is reduced in vitro by human IgG. Ghrelin concentrations after incubation of ghrelin (30 fmol ml 1) in the IgG-deprived
plasma efﬂuents for 2 h at 37°C with and without human IgG (a). Estimation of ghrelin protection efﬁciency from degradation by the ratios of ghrelin
concentrations measured after incubations of ghrelin with IgG versus ghrelin alone (b). (a) M–W tests ***Po0.001, Student’s t-tests #Po0.05
versus controls; (b) Student’s t-tests *Po0.05. (n ¼ 6, error bars, s.e.m.).

protection of ghrelin against degradation was more pronounced
with IgG from the obese group (Fig. 7b).
Ghrelin and anti-ghrelin IgG in ob/ob and lean mice. To further
analyse the relevance of ghrelin-reactive IgG to food intake and
obesity we studied their properties in 2-month-old male obese
ob/ob mice (body weight ±s.d., 51.4±1.8 g, n ¼ 11) as compared
with male wild-type lean mice (body weight ±s.d., 25.6±1.8 g,
n ¼ 12) of the same age and the same C57Bl6 genetic background.
In vivo analysis of body composition showed increased total fat
mass in ob/ob versus lean mice (27.6±1.8 g ±s.d., versus
2.9±1.6 g ±s.d., respectively, Student’s t-test Po0.0001) but
total lean mass was not signiﬁcantly different (21.4±1.4 g ±s.d.,
versus 20.4±1.4 g ±s.d., respectively, Student’s t-test P ¼ 0.07).
Plasma concentrations of both ghrelin and des-acyl ghrelin
were slightly lower in ob/ob than in lean mice (Fig. 8a,b), but the
ratios of ghrelin to des-acyl ghrelin did not differ between the two
groups (Fig. 8c). Plasma levels of ghrelin-reactive IgG were also
slightly lower in ob/ob than in lean mice (Fig. 8d). Levels of
ghrelin-reactive IgG were also measured in tissues homogenates
of the hypothalamus and liver, showing their presence at the
detection limit with no signiﬁcant differences between the groups
(Fig. 8e,f), whereas in the stomach tissue ghrelin-reactive IgG
were undetectable.
Afﬁnity kinetics between plasma-extracted IgG and mouse
ghrelin were analysed by SPR technology as used for characterizing human IgG described above (Fig. 8g,h). We found that IgG
from ob/ob mice had increased afﬁnities for ghrelin, manifested
by lower KD values than in lean mice, which all remained in the
10 8 M range (Fig. 8i). In contrast to ghrelin-reactive IgG in
obese humans, this increase in afﬁnity was due to lower
dissociation rates (Fig. 8k), whereas association rates did not
differ signiﬁcantly between the groups (Fig. 8j).
Obese mice IgG enhances ghrelin’s orexigenic effect. To study
the potential modulatory role of IgG in ghrelin-induced food
intake in mice, mouse ghrelin was chronically injected in
1-month-old male lean C57Bl6 mice alone or in combination
with IgG extracted from plasma of ob/ob or lean mice. For this
purpose, two IgG pools were prepared from the IgG of six ob/ob
and six lean mice characterized by low and high KD values,
respectively, as determined by SPR. Mice received two daily
intraperitoneal injections of ghrelin or ghrelin together with one
of the IgG pools at the beginning of the light phase and before the
onset of the dark phase for 2 weeks. Ghrelin doses were 0.1 nmol
during the ﬁrst 4 days and 1.0 nmol thereafter. The overall
6

dynamics of body weight changes showed superior weight gain in
mice receiving ghrelin with ob/ob IgG, although after 14 days of
injections it still did not reach signiﬁcance (day 15, Student’s
t-test P ¼ 0.06, versus controls) (Fig. 9a). However, the body
composition analysis at day 15 showed that total lean mass was
increased in mice receiving ghrelin with ob/ob IgG (Fig. 9b).
Surprisingly, percentage of body fat was not increased in this
group and was even smaller than in mice receiving chronic
injections of ghrelin alone (Fig. 9c). Daily mean food intake
during the 14 days of injections was B0.5 g higher in mice
receiving ghrelin with ob/ob IgG than in controls and mice
receiving ghrelin with lean IgG (Fig. 9d). The acute (30 min)
enhancing effects of ob/ob IgG on ghrelin-induced feeding was
visible already at the 0.1 nmol dose of ghrelin, although it was
signiﬁcant only after the evening but not morning injections
(Fig. 9e,f). In contrast, with the dose of 1.0 nmol of ghrelin the
acute IgG-enhancing effects were clear after the morning injections (Fig. 9g), whereas no further increase of the stimulatory
effects of ghrelin were observed after the evening injections
(Fig. 9h). The analysis of food intake according to the light/dark
phases showed increased food intake in mice receiving 0.1 nmol
of ghrelin with ob/ob IgG in dark but not in light phase (Fig. 9i,j),
whereas the dose of 1.0 nmol increased food intake in light phase
in all ghrelin-injected groups (Fig. 9k) and in mice co-injected
with ob/ob IgG in the dark phase (Fig. 9l). The analysis of the
feeding pattern showed that increased food intake in ghrelin plus
ob/ob IgG-injected mice was due to increased meal number
(Fig. 9m) while meal size was unchanged (Fig. 9n).
Discussion
Our work brings a new putative mechanism of ghrelin-mediated
increase in appetite in obesity that may explain why ghrelin
administration stimulates food intake more potently in obese
than in normal-weight subjects, as previously reported7. We
determined that plasma ghrelin-reactive IgG autoAbs can be
responsible for this phenomenon, serving as carrier proteins that
might enhance the bioactivity of endogenous or exogenous
ghrelin. Although the existence of a ghrelin-binding protein could
be suspected in analogy to some other peptide hormones23, the
variable-afﬁnity nature of IgG molecules makes them particularly
interesting as potential modulators of ghrelin’s biological
activities. In fact, ghrelin was readily detected in the protein
G-puriﬁed IgG eluents but also in the remaining plasma efﬂuents,
indicating that in the circulation the hormone exists in both IgGbound and unbound forms. Using these two measures to estimate
the percentage of bound ghrelin might however not be accurate

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

b
*

10
5

100

Lean

0.10
0.05

Lean

ob/ob

e
Ghrelin IgG in
hypothalamus (OD)

0.8
0.6
0.4
0.2

ob/ob

f
0.03

0.04
0.03

Ghrelin IgG
in liver (OD)

*

1.0

Lean

0.02
0.01

0.02
0.01

0.00

0.00

ob/ob

Lean

ob/ob

Lean

ob/ob

300

Ghrelin IgG
SPR response (RU)

h

Ghrelin IgG
SPR response (RU)

g
Lean

250
200
150
100
50
50

150 200
Time (s)

100

250

300
ob/ob

250
200
150
100
50

300

50

j
*

6.0×10–8
4.0×10–8
2.0×10–8

100

150 200
Time (s)

300

4.0×10–3

1.5×105
1.0×105
5.0×104

ob/ob

**

3.0×10–3
2.0×10–3
1.0×10–3

0
Lean

250

k
2.0×105
Ghrelin IgG
small Kd (s–1)

8.0×10–8

Ghrelin IgG
small Ka (M–1 s–1)

Ghrelin IgG Kd (M)

0.15

0.00

ob/ob

0.0

i

0.20

0
Lean

Ghrelin IgG (OD)

*

50

0

d

c

150

Ghrelin / des-acyl
ghrelin ratios

15

Des-acyl ghrelin
–1
(fmol ml )

Ghrelin (fmol ml–1)

a

0
Lean

ob/ob

Lean

ob/ob

Figure 8 | Ghrelin and ghrelin-reactive IgG in ob/ob and lean mice. Plasma levels of ghrelin (a) and des-acyl ghrelin (b). Ghrelin to des-acyl ghrelin
ratios (c). Plasma levels of ghrelin-reactive IgG (d). Tissue levels of ghrelin-reactive IgG in the hypothalamus (e) and liver (f). Afﬁnity kinetics of
plasma-extracted IgG and ghrelin as analysed by the SPR using the Langmuir’s 1:1 model ﬁt. Representative examples of afﬁnity kinetics of IgG for
ghrelin in lean (g) and obese (h) mice with following parameters: (g) small Ka (±s.e.m.) 1.04 105±8.7 103 M 1 s 1, small Kd (±s.e.m.),
3.3 10 3±1.37 10 4 s 1, KD, 3.18 10 8 M, w2 28,5 and (h) small Ka (±s.e.m.), 8.16 104 ±2.03 103 M 1 s 1, small Kd (±s.e.m.),
1.74 10 3±4.11 10 5 s 1, KD, 2.13 10 8 M, w2 17.1. Dissociation equilibrium constants (KD) (i). Association rates (small Ka) (j). Dissociation rates
(small Kd) (k). (a,b,d) Student’s t-tests *Po0.05; (i,k) M–W tests *Po0.05, **Po0.01. (lean, n ¼ 12, ob/ob, n ¼ 11, error bars, s.e.m.).

because a part of the initially IgG-bound ghrelin could ﬂow into
the efﬂuents during the IgG linking to the column. In turn, the
cumulative (bound and unbound) ghrelin concentrations
measured in the plasma sample might be more informative.
Indeed, in contrast to ‘‘free’’ ghrelin levels measured in native
plasma, found to be similar between obese and controls in this
study, we detected increased cumulative ghrelin concentrations in
obese patients, suggesting that in the obese there is more IgGbound ghrelin than in other groups and which was not detectable
in whole plasma before IgG extraction. This might be related to
the conformational changes of the IgG molecules upon its Fc
fragment binding to protein G eventually resulting in release of
ghrelin from the antigen-binding site24. As the Fc receptors are
ubiquitously expressed in various immune cells, including brain
microglia25, ghrelin–IgG complexes binding to Fc receptors
might be one of the possible mechanisms of ghrelin release from
the IgG that occurs in vivo. The biologically available IgG-bound

fraction of circulating ghrelin, which was increased in obese
patients, may hence contribute to ghrelin-mediated increased
appetite in obesity.
We also determined that obese patients had increased afﬁnity of
their IgG for ghrelin, which may underlie their ability to more
efﬁciently bind and transport ghrelin as compared with IgG from
controls and AN patients. Plasma levels of ghrelin-reactive IgG
were not different between three study groups, further supporting
that their different afﬁnity kinetics, rather than levels, could be
responsible for the enhanced orexigenic effects of ghrelin. Given
that the increase in association rate was by factor 3 in IgG from
obese, one may consider that while IgG from controls will bind
one molecule of ghrelin, IgG from obese will bind three molecules.
Additionally, a virtual absence of dissociations of ghrelin IgG in
obese patients were visible after single injection of IgG at low ﬂow
speed, also indicating more stable ghrelin–IgG complexes. The
micromolar range of the afﬁnity of ghrelin-reactive IgG found in

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

7

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

18
17
16

0.5

Meal size (g)

12

Ghrelin+
IgG ob/ob

Ghrelin

Ghrelin+
IgG ob/ob

Ghrelin+
IgG lean

Ghrelin

0.8

Ghrelin+
IgG ob/ob

Ghrelin+
IgG lean

***

0.6
0.4
0.2
0.0

Ghrelin+
IgG ob/ob

Ghrelin+
IgG lean

0.0

*

*

0.3
0.2
0.1
0.0

10

Ghrelin+
IgG ob/ob

Ghrelin+
IgG ob/ob

Ghrelin+
IgG lean

Ghrelin

Contr

2.0

0.1

0.4

Ghrelin+
IgG ob/ob

2.5

14

Ghrelin+
IgG lean

3.0

n

**

Ghrelin

***

16

Contr

**

Meal number per day

m
3.5

Ghrelin

Contr

Ghrelin+
IgG ob/ob

Ghrelin+
IgG lean

Ghrelin

Contr

l

0.2

Ghrelin+
IgG ob/ob

1.0

0.0

0

Contr

k

1.5

***

0.3

Ghrelin

Ghrelin+
IgG lean

0.00

0.00

Ghrelin+
IgG lean

1

0.05

0.05

Ghrelin+
IgG lean

2

**

0.10

h

Light phase
food intake (g)
after ghrelin (1.0 nM)

3

#

0.15

Ghrelin

j

*

4

#

0.10

Ghrelin

Ghrelin+
IgG ob/ob

Ghrelin

0.00

Ghrelin+
IgG lean

0.02

0.15

Contr

0.04

Morning
30 min food intake (g)
after ghrelin (1.0 nM)

0.06

Light phase food
intake (g) after
ghrelin (0.1 nM)

Dark phase food
intake (g) after
ghrelin (0.1 nM)

0.08

***

0.20

0.20

Contr

0

g
0.10

Contr

Morning
30 min food intake (g)
after ghrelin (0.1 nM)

f

Ghrelin+
IgG ob/ob

1

*

0.25

Contr

6 8 10 12 14
Days

Ghrelin+
IgG ob/ob

4

Ghrelin+
IgG ob/ob

2

Contr

0

Evening
30 min food intake (g)
after ghrelin (0.1 nM)

0

2

Ghrelin+
IgG lean

1

3

e

Evening
30 min food intake (g)
after ghrelin (1.0 nM)

Control (PBS)
Ghrelin
Ghrelin+IgG lean
Ghrelin+IgG ob/ob

***

4

Ghrelin

2

Ghrelin+
IgG lean

Ghrelin

d

Ghrelin+
IgG lean

0
Contr

3

Dark phase food
intake (g) after
ghrelin (1.0 nM)

5

15

4

i

*
10

Contr

5

Food intake (g day–1)

Body weight gain (g)

6

*

19

Ghrelin

7

1.0 nM

15

Contr

0.1 nM

c

20

Total body fat (%)

b
Total lean mass (g)

a

Figure 9 | Effects of chronic ghrelin administration in lean mice with or without IgG from lean or obese mice. Body weight gain during two daily
injections of 0.1 nmol and 1.0 nmol of ghrelin alone or in combination with IgG from lean or ob/ob mice as compared to controls that received PBS (a). Body
composition analysis at day 15 showing total lean mass (b) and percentage of body fat (c). Daily food intake during 14 days (d). Thirty-minute food
intake after evening (e) and morning (f) injections of 0.1 nmol of ghrelin during 4 days. Thirty-minute food intake after morning (g) and evening
(h) injections of 1.0 nmol of ghrelin during 10 days. Dark phase (i) and light phase (j) food intake after 0.1 nmol ghrelin injections during 4 days. Light phase
(k) and dark phase (l) food intake after 1.0 nmol ghrelin injections during 10 days. Meal frequency (m) and meal size (n) during 14 days. (a) Two-way
repeated measurements ANOVA P ¼ 0.24; (b,c,e,i,m,n) Student’s t-tests *Po0.05,**Po0.01; (d,g,h,k,l) ANOVA Po0.0001, Tukey’s post tests
**Po0.01, ***Po0.001, Student’s t-tests #Po0.05. (n ¼ 6, error bars, s.e.m.).

8

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

all study subjects is well positioned to mediate the peptide
transport because it will not compete with the nano- to picomolar afﬁnity of interaction between ghrelin and its receptor26.
Instead, ghrelin-reactive IgG might protect ghrelin from
degradation by hydrolysing enzymes that deacylate ghrelin9,
which generally display millimolar afﬁnities for their substrates.
Indeed, by an in vitro assay of ghrelin preservation from
degradation by plasma enzymes we showed here that IgG
protected ghrelin from degradation in all study groups but it
was more pronounced by IgG from obese patients. Thus, a slight
increase in afﬁnity of ghrelin-reactive IgG in obesity might change
these autoAbs to better ghrelin carriers/protectors, while not
antagonizing ghrelin receptor binding. In agreement with these
data, previous studies have also reported that micromolar afﬁnity
is a common feature of natural IgG autoAbs27. This is in contrast
to high-afﬁnity ghrelin-neutralizing antibodies, as produced by
immunization, which were shown to reduce food intake after their
peripheral administration in mice28,29. We did not ﬁnd signiﬁcant
differences in the afﬁnity of des-acyl ghrelin IgG between the
controls and patients, which may indicate that ghrelin and desacyl ghrelin do not bind to the same autoAbs and that rather
ghrelin-reactive than des-acyl ghrelin-reactive IgG can modulate
ghrelin’s orexigenic effects.
As opposite to ghrelin and IgG afﬁnity kinetics found in the
obese, ghrelin-reactive IgG in AN patients displayed increased
dissociation rates for ghrelin, indicating a tendency for decreased
afﬁnity for ghrelin, meaning that they will not be able to
efﬁciently transport ghrelin and hence might diminish ghrelin’s
orexigenic effects. Indeed, co-injections of ghrelin together with
IgG from AN patients were not able to stimulate food intake in
rats more than IgG from controls.
In obese ob/ob mice, plasma IgG afﬁnity for ghrelin was also
increased mainly due to lower dissociation rates. The relevance of
the differences of IgG properties between obese and lean mice to
food intake and body weight was tested by chronic co-injections
of IgG from ob/ob and lean mice together with ghrelin. Our
results conﬁrm the ability of IgG from obese mice to enhance
ghrelin-stimulated food intake similar to the results seen in rats
for IgG from obese humans. This effect was most obvious when
measured acutely for 30 min after ghrelin injections and it was
evident already at the smallest dose of ghrelin (0.1 nmol). Higher
doses of ghrelin (1.0 nmol) strongly stimulated food intake in all
groups of mice that received ghrelin in the evening. In contrast,
the same dose of ghrelin injected in the morning produced higher
food intake in mice receiving IgG from obese as compared with
other groups. These data are in agreement with the previously
reported differences of ghrelin stimulatory effects on food intake
between light and dark phases30 and may possibly contribute to
increased overeating in obese animals in the light phase31. The
mean daily food intake was also higher in mice that received
ghrelin with ob/ob IgG, which was due to increased meal
frequency but not meal size, pointing to increased hunger but not
delayed satiation induced by this treatment, an observation in
agreement with the accepted role of ghrelin to induce hunger32.
Interestingly, while chronic injections of ghrelin with ob/ob IgG
were associated with a tendency of increased body weight gain,
the mice did not become fatter but rather gained more lean mass.
Furthermore, mice injected with ghrelin alone showed higher
total body fat than mice co-injected with ob/ob IgG. The fat
tissue-inducing effects of chronic ghrelin administration in mice,
not accompanied by increased food intake, have previously been
documented33. The absence of adipogenic effect of ghrelin in
mice co-injected with ob/ob IgG suggests that IgG induced more
selective ghrelin actions on appetite controlling pathways and
possibly also on the somatotropic axis. The mechanism of such
selectivity needs further clariﬁcation.

Our study also showed that not all obese subjects and obese
mice had increased afﬁnities of IgG for ghrelin, suggesting that
such an increase is probably not the result of the obese phenotype
or at least not its obligatory result. It means further that, although
IgG would have a protective role for ghrelin against degradation
by plasma enzymes in all subjects, it might be involved in
enhancing ghrelin orexigenic effects only in those subjects and
mice that display increased afﬁnities for ghrelin. What makes
humans and animals produce ghrelin-reactive IgG is presently
unknown but the variation in their afﬁnities in both obese and
non-obese humans and animals suggests that there could be
different antigenic stimulations possibly involving both ghrelin
and ghrelin-like antigens such as those present in the gut
microbiota for example, in proteins of Enterococcus fecalis
bacteria14. In fact, germ-free rats display increased plasma
levels of ghrelin-reactive IgG, indicating that they lack some
microbial factors that would reduce an exaggerated ghrelinreactive IgG production14. Hence, it is tempting to speculate that
targeting ghrelin-reactive IgG via immuno-nutritional approaches
may represent a new therapeutic target for appetite control in
obesity and anorexia.
In conclusion, our data reveal that plasma ghrelin is normally
bound to circulating IgG, which may have a role as carriers for
the hormone protecting it from degradation and further that IgG
from obese patients seem to bind more ghrelin than IgG from
non-obese subjects. We also show that IgG from both obese
humans and ob/ob mice display elevated micromolar afﬁnity for
ghrelin, which may be responsible for an increased ability of these
IgG to transport ghrelin and thus enhance its orexigenic and
anabolic actions. It is likely that increased appetite and overeating
observed in some obese humans and mice may be mediated at
least partly by the circulating IgG enhancing the orexigenic effect
of ghrelin, the main hunger peptide hormone.
Methods
Study subjects. Obese, AN patients and controls (all females; age±s.e.m.,
23.14±1.08 years, 21.42±1.9 years and 18.86±1.14 years, respectively) were
admitted to the Kagoshima University Hospital and gave written informed consent
for study participation. AN patients were diagnosed according to the DSM-IV
criteria34. The study was approved by the Ethical Committee of the Kagoshima
University, Japan. Venous blood samples were collected into tubes containing
ethylenediamine tetraacetic acid (EDTA, 1 mg ml 1), aprotinin (500 U ml 1) and
1 N HCl (1:10 vol). The plasma was separated by centrifugation at 4 °C and stored
at 80 °C until assayed.
Ghrelin and ghrelin-reactive IgG assays. Ghrelin and des-acyl ghrelin concentrations in all experiments including human and mouse plasma were measured
using EIA kits from Mitsubishi Chemical Med Corp, Tokyo, Japan. Plasma levels of
IgG autoAbs reacting with ghrelin and des-acyl ghrelin were measured using
ELISA according a published protocol35. For speciﬁcity controls of ghrelin-reactive
IgG, prior to ELISA, human plasma samples were preincubated overnight at 4oC
with ghrelin diluted at 10 6 M. Concentrations of plasma total IgG were assayed
by immunonephelometry using speciﬁc antibodies (Siemens, Germany) on a BNII
nephelometer (Siemens). Total IgG were puriﬁed from human and mouse plasma
samples via extraction of peptides from plasma globulins by plasma acidiﬁcation
and separation on C-18 SEP column (Phoenix Pharmaceuticals, Burlingame, CA,
USA) followed by puriﬁcation using Melon Gel Kit (Thermo Scientiﬁc, Pierce,
Rockford, IL, USA). Puriﬁed IgG were lyophilized and then reconstituted in the
HBS-EP buffer (GE Healthcare, Piscataway, NJ, USA).
Afﬁnity kinetics assay of IgG for ghrelin. Afﬁnity kinetics of human IgG
autoAbs for ghrelin was determined by SPR on a BIAcore 1000 instrument (GE
Healthcare). Ghrelin (Peptide institute, Inc.) was diluted to 0.5 mg ml 1 in 10 mM
sodium acetate buffer (pH 5.0) (GE Healthcare) and was covalently coupled on the
sensor chip CM5 (GE Healthcare), using the amine coupling kit (GE Healthcare).
All the measurements were done on the same sensor chip. To compare relative
binding response between IgG and ghrelin in obese, AN patients and controls,
puriﬁed IgG were diluted in each sample to 0.5 mg ml 1 (3360 nM) in HBS-EP
buffer (GE Healthcare) and 25 ml were injected (INJECT) into the ﬂow conduit of
BIAcore instrument with a ﬂow speed of 5 ml min 1 at 25 °C. The values of RU of
the sensorgram were recorded 5 min after association and in the end of dissociation

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

9

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

5 min after stopping the injection. All samples were analysed in a random order
and alternating injections of IgG from controls, obese and AN patients. For the
afﬁnity kinetic analysis, a multi-cycle method was run with ﬁve serial dilutions of
each patient and control IgG: 0.5, 0.25, 0.125, 0.625 and 0.03125 (mg ml 1)
including a duplicate of 0.125 mg ml 1 and a blank (buffer only). During each
cycle, 60 ml were injected (KINJECT) into the ﬂow conduit of the BIAcore
instrument with ﬂow speed of 30 ml min 1 at 25 °C followed by 5 min of dissociation. Between injections of each sample, the binding surface was regenerated
with 10 mM NaOH resulting in the return of the sensorgram to the same baseline.
The afﬁnity kinetic data were analysed using BiaEvaluation 4.1.1 programme (GE
Healthcare). For ﬁtting kinetic data, the Langmuir’s 1:1 model was used and the
sample values were corrected by subtracting the blank values resulting from the
injection of HBS-EP buffer. Afﬁnity kinetics between patients’ and controls’ IgG
and des-acyl ghrelin and between mouse IgG and ghrelin were analysed by SPR as
described above but with human des-acyl ghrelin and mouse ghrelin (both from
Peptide institute, Inc.), respectively, coupled to the CM5 chip (GE Healthcare).
Acute food intake experiments in rats. All animal care and experimentation were
in accordance with guidelines established by the National Institutes of Health, USA
and approved by the Normandy Ethical Committee for Animal Experimentation.
Two-month-old male Sprague-Dawley rats (n ¼ 24), body weight 200–250 g (Janvier
Labs, Genest-Saint-Isle, France) were kept in holding cages (three rats per cage) in a
fully equipped animal facility under regulated environmental conditions (22±1 °C,
on a 12 h light–dark cycle with lights on at 7:30) for 1 week. Standard pelleted rodent
chow (RM1 diet, SDS, UK) and drinking water were available ad libitum. Three
days before experiments, the rats were transferred to individual metabolism cages
(Tecniplast, Lyon France) where they were fed ad libitum with the same RM1 diet
but in powdered form (SDS) and drinking water was always available. Rats were
divided into four groups (n ¼ 6) to achieve similar body weight. Three food intake
experiments were performed with 4 days interval between each experiment. Intraperitoneal injections (300 ml) were done at 11:00 and immediately thereafter rats were
returned to their metabolism cages, which contained a preweighed amount of food.
Food intake was measured at 30 min, 1, 2, 4 and 12 h. In experiment 1 human ghrelin
and rat ghrelin (Peptide institute, Inc.) were injected in free-fed satiated rats. Human
ghrelin was injected with the doses of: 1 nmol or 3 nmol per rat; rat ghrelin was
injected with the dose of 3 nmol per rat; the control group received PBS. In
experiment 2, rats received injections of six different IgG puriﬁed from plasma of
either obese, or AN or control subjects and control group received PBS. In experiment 3, rats received 1 nmol of IgG as in experiment 2 but this time together with
1 nmol of human ghrelin. Each rat injected with IgG received the same IgG as in the
previous experiment. The control group that previously was injected with PBS
received 1 nmol of human ghrelin only.
In vitro ghrelin preservation assay. Human ghrelin (150 nmol; Peptide institute,
Inc.) was diluted in the PBS and added to IgG-deprived plasma efﬂuents of obese
and AN patients and controls resulting in its ﬁnal concentration of 30 fmol ml 1.
Ghrelin was incubated in each plasma efﬂuents alone or with 1 nmol of the same
subject IgG in a rotating incubator for 2 h at 37 °C. Then, the incubation milieu was
used for measuring ghrelin concentrations as described above.
Ghrelin and anti-ghrelin IgG assays in ob/ob and lean mice. Two-month-old
C57Bl6 male obese ob/ob and wild-type lean mice were purchased from Janvier
Labs and acclimated to the animal facility for 1 week. The body composition in
awake mice was analysed using EchoMRI (EchoMRI, Houston, TX, USA) and then
mice were killed by decapitation and trunk blood was collected into tubes containing EDTA (1 mg ml 1), aprotinin (500 U ml 1) and 1 N HCl (1:10 vol). The
plasma was separated by centrifugation at 4 °C and stored at 80 °C until assayed
for ghrelin, des-acyl ghrelin and ghrelin-reactive IgG. Entire hypothalamus and
fragments of the liver and of the gastric fundus were collected and homogenized in
PBS with the cocktail of protease and phosphatase inhibitors (Sigma) for ghrelin
IgG assay. Total IgG were extracted from mouse plasma and analysed by the SPR as
described above.
Chronic food intake experiments in mice. One-month-old wild-type male
C57Bl6 mice (n ¼ 32) were purchased from Janvier Labs and acclimated to the
animal facility for 1 week with 12 h light–dark cycle, lights on at 7:00. Then, the
mice were placed individually in the BioDAQ mouse cages (Research Diets, Inc.,
New Brunswick, NJ, USA), each equipped with an automatic feeding monitor.
After 3 days of acclimation to the BioDAQ cages, mice were divided into four
groups (n ¼ 8), each receiving one of four different treatments consisting of two
daily intraperitoneal injections at 10:00 and at 18:45. of either: (i) PBS, (ii) ghrelin
alone, (iii) ghrelin with IgG extracted from lean C57Bl6 mice and (iv) ghrelin with
IgG extracted from ob/ob mice. Mouse ghrelin (Peptide institute, Inc.) was injected
at the dose of 0.1 nmol during the ﬁrst 4 days and 1.0 nmol for next ten days. Two
IgG pools were prepared from IgG of six ob/ob and six lean mice characterized by
low and high KD vales, respectively, as measured by the SPR and 1 nmol of IgG was
given at each injection. Food (SERLAB, Montataire France) and drinking water
were available ad libitum and body weight was measured daily. Feeding data were
continuously monitored for 14 days and analysed using the BioDAQ data viewer
2.3.07 (Research Diets). For the meal pattern analysis, the inter-meal interval was
10

set at 300 s. After 14 days of injections, body composition in mice was analysed by
the EchoMRI.
Statistical analysis. Data were analysed and the graphs were plotted using the
GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA, USA). Normality
was evaluated by the Kolmogorov–Smirnov test. Group differences were analysed
by the analysis of variance (ANOVA) or the non-parametric Kruskal–Wallis test
with the Tukey’s or Dunn’s post tests, according to the normality results. Where
appropriate, individual groups were additionally compared using the Student’s
t-test or the Mann–Whitney test according to the normality results. Effects of
absorptions were analysed using the paired t-test. Body weight gain in mice was
analysed using the two-way repeated measurement ANOVA. Data are shown as
means±s.e.m. and for all tests, Po0.05 was considered statistically signiﬁcant.

References
1. Small, D. M. Individual differences in the neurophysiology of reward and the
obesity epidemic. Int. J. Obes. 33, S44–S48 (2009).
2. Kojima, M. et al. Ghrelin is a growth-hormone-releasing acylated peptide from
stomach. Nature 402, 656–660 (1999).
3. Tscho¨p, M. et al. Circulating ghrelin levels are decreased in human obesity.
Diabetes 50, 707–709 (2001).
4. Barazzoni, R. et al. Relationships between desacylated and acylated ghrelin and
insulin sensitivity in the metabolic syndrome. J. Clin. Endocrinol. Metab. 92,
3935–3940 (2007).
5. Wu, Q., Clark, M. S. & Palmiter, R. D. Deciphering a neuronal circuit that
mediates appetite. Nature 483, 594–597 (2012).
6. Berthoud, H.-R. Metabolic and hedonic drives in the neural control of appetite:
who is the boss? Curr. Opin. Neurobiol. 21, 888–896 (2011).
7. Druce, M. R. et al. Ghrelin increases food intake in obese as well as lean
subjects. Int. J. Obes. 29, 1130–1136 (2005).
8. Gutierrez, J. A. et al. Ghrelin octanoylation mediated by an orphan lipid
transferase. Proc. Natl Acad. Sci. USA 105, 6320–6325 (2008).
9. Satou, M., Nishi, Y., Yoh, J., Hattori, Y. & Sugimoto, H. Identiﬁcation and
characterization of acyl-protein thioesterase 1/lysophospholipase i as a ghrelin
deacylation/lysophospholipid hydrolyzing enzyme in fetal bovine serum and
conditioned medium. Endocrinology 151, 4765–4775 (2010).
10. Asakawa, A. et al. Stomach regulates energy balance via acylated ghrelin and
desacyl ghrelin. Gut 54, 18–24 (2005).
11. Paciﬁco, L. et al. Acylated and nonacylated ghrelin levels and their associations
with insulin resistance in obese and normal weight children with metabolic
syndrome. Eur. J. Endocrinol. 161, 861–870 (2009).
12. Otto, B. et al. Weight gain decreases elevated plasma ghrelin concentrations of
patients with anorexia nervosa. Eur. J. Endocrinol. 145, 669–673 (2001).
13. Germain, N. et al. Ghrelin/obestatin ratio in two populations with low
body weight: constitutional thinness and anorexia nervosa.
Psychoneuroendocrinology 34, 413–419 (2009).
14. Fetissov, S. O. et al. Autoantibodies against appetite-regulating peptide
hormones and neuropeptides: putative modulation by gut microﬂora. Nutrition
24, 348–359 (2008).
15. Terashi, M. et al. Ghrelin reactive autoantibodies in restrictive anorexia
nervosa. Nutrition 27, 407–413 (2011).
16. Gallas, S. et al. Gastric electrical stimulation increases ghrelin production and
inhibits catecholaminergic brainstem neurons in rats. Eur. J. Neurosci. 33,
276–284 (2011).
17. Gallas, S. & Fetissov, S. O. Ghrelin, appetite and gastric electrical stimulation.
Peptides 32, 2283–2289 (2011).
18. Zorrilla, E. P. et al. Vaccination against weight gain. Proc. Natl Acad. Sci. USA
103, 13226–13231 (2006).
19. Karlsson, R. SPR for molecular interaction analysis: a review of emerging
application areas. J. Mol. Recognit. 17, 151–161 (2004).
20. Zhang, Y. et al. Positional cloning of the mouse obese gene and its human
homologue. Nature 372, 425–432 (1994).
21. Halaas, J. L. et al. Weight-reducing effects of the plasma protein encoded by the
obese gene. Science 269, 543–546 (1995).
22. Wren, A. M. et al. Ghrelin causes hyperphagia and obesity in rats. Diabetes 50,
2540–2547 (2001).
23. Cortright, D. N., Nicoletti, A. & Seasholtz, A. F. Molecular and biochemical
characterization of the mouse brain corticotropin-releasing hormone-binding
protein. Mol. Cell Endocrinol. 111, 147–157 (1995).
24. Sagawa, T. et al. Conformational changes in the antibody constant domains
upon hapten-binding. Mol. Immunol. 42, 9–18 (2005).
25. Lunnon, K. et al. Systemic inﬂammation modulates Fc receptor expression on
microglia during chronic neurodegeneration. J. Immunol. 186, 7215–7224 (2011).
26. Bennett, K. A., Langmead, C. J., Wise, A. & Milligan, G. Growth hormone
secretagogues and growth hormone releasing peptides act as orthosteric superagonists but not allosteric regulators for activation of the G protein Ga01 by the
ghrelin receptor. Mol. Pharmacol. 76, 802–811 (2009).
27. Lacroix-Desmazes, S. et al. Self-reactive antibodies (natural autoantibodies) in
healthy individuals. J. Immunol. Methods 216, 117–137 (1998).

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

ARTICLE

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3685

28. Lu, S.-C. et al. An acyl-ghrelin-speciﬁc neutralizing antibody inhibits the acute
ghrelin-mediated orexigenic effects in mice. Mol. Pharmacol. 75, 901–907
(2009).
29. Zakhari, J. S., Zorrilla, E. P., Zhou, B., Mayorov, A. V. & Janda, K. D.
Oligoclonal antibody targeting ghrelin increases energy expenditure and
reduces food intake in fasted mice. Mol. Pharmaceutics 9, 281–289 (2012).
30. Gilg, S. & Lutz, T. A. The orexigenic effect of peripheral ghrelin differs between
rats of different age and with different baseline food intake, and it may in part
be mediated by the area postrema. Physiol. Behav. 87, 353–359 (2006).
31. Fetissov, S. O. & Meguid, M. M. Serotonin delivery into the ventromedial
nucleus of the hypothalamus affects differently feeding pattern and body weight
in obese and lean Zucker rats. Appetite 54, 346–353 (2010).
32. Cummings, D. E. et al. A preprandial rise in plasma ghrelin levels suggests a
role in meal initiation in humans. Diabetes 50, 1714–1719 (2001).
33. Tscho¨p, M., Smiley, D. L. & Heiman, M. L. Ghrelin induces adiposity in
rodents. Nature 407, 908–913 (2000).
34. DSM-IV. Diagnostic and Statistical Manual of Mental Disorders (Am.
Psychiatric Assoc. 1994).
35. Fetissov, S. O. Neuropeptide autoantibodies assay. Methods Mol. Biol. 789,
295–302 (2011).

Acknowledgements
This work was supported by Grants-in-Aid for Scientiﬁc Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan. It also received supports

from Fonds Franc¸ais Alimentation & Sante´, from the European Regional Development
Fund (FEDER) and from EU INTERREG IVA 2 Seas Programme (7-003-FR_TC2N). We
are grateful to Dr Emeric Gueneau, GE Healthcare, for the help with BIAcore instrumentation and afﬁnity kinetics analysis.

Author contributions
Experimental design and experiments: S.O.F., K.T. and R.L; clinical data sampling: A.A.,
H.A. and A.I; help with experiments: M.F., N.T., M.C. and SC; data analysis: K.T., R.L.
and S.O.F; manuscript writing: S.O.F; manuscript comments and laboratory support:
J.-C.d.R., P.D. and A.I.

Additional information
Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Takagi, K. et al. Anti-ghrelin immunoglobulins modulate
ghrelin stability and its orexigenic effect in obese mice and humans. Nat. Commun.
4:2685 doi: 10.1038/ncomms3685 (2013).
This work is licensed under a Creative Commons AttributionNonCommercial-ShareAlike 3.0 Unported License. To view a copy of
this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

NATURE COMMUNICATIONS | 4:2685 | DOI: 10.1038/ncomms3685 | www.nature.com/naturecommunications

& 2013 Macmillan Publishers Limited. All rights reserved.

11