Ghrelin treatment increases receptor-bound leptin in healthy and endotoxemic obese Lewis rats

(1)

(2)

Angenommen vom Senat der Medizinischen Hochschule Hannover am 22.06.2010

Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover Präsident : Professor Dr. med. Dieter Bitter-Suermann

Betreuer : Professor Dr. med. Heike Nave Referent : Professor Dr. med. Peter Claus Korreferent : Professor Dr. med. Oliver Bock

Tag der mündlichen Prüfung: 22.06.2010

Promotionsausschussmitglieder:

Professor Dr. Ernst Ungewickel Professor Dr. Johannes Gessner Professor Dr. Roland Jacobs

(3)

Inhaltsverzeichnis Seite

Einleitung 1 - 4

Experimental and Clinical Endocrinology & Diabetes 117, 473-479 (2009) 5 - 11

Life Sciences 81, 80-87 (2007) 12 - 19

Diskussion 20 - 22

Zusammenfassung 23

Literaturverzeichnis 24 - 28

Lebenslauf 29 - 30

Erklärung nach §2 Abs. 2 Nr. 5 und 6 PromO 31

Danksagung 32

(4)

1

Einleitung

Weltweit gilt Adipositas als zunehmendes Problem, mittlerweile sind laut WHO über eine Milliarde Menschen übergewichtig und mehr als 300 Millionen Menschen adipös bei steigender Prävalenz. Als Definitionskriterium wird zumeist der Body-Mass-Index (BMI) nach den WHO-Leitlinien genutzt, wobei ein BMI von 18,5-24,9 kg/m² Körperoberfläche als normal, 25-29,5 kg/m² als übergewichtig und mehr als 30 kg/m² als adipös gilt (WHO, 2006). Die medikamentöse Therapie steckt noch in den Anfängen, ein wirklich effektives und nebenwirkungsarmes Medikament ist derzeit noch nicht erhältlich (Idelevich et al., 2009). Neben einer großen Zahl an Adipositas assoziierten Erkrankungen wie Hypertonie, Diabetes Mellitus Typ II oder koronare Herzkrankheit, zeigt sich ein Zusammenhang mit dem Hormon- und Immunsystem. So finden sich z.B. bei adipösen Patienten höhere Morbiditäts- und Mortalitätsraten während einer Sepsis als bei Normalgewichtigen (Bercault et al., 2004, Vachharajani et al., 2005).

Eine Sepsis wird durch zwei Faktoren definiert (ACCP/SCCM Consensus Conference Committee, 1992): Erstens das Vorhandensein eines systemic inflammatory response syndrom (SIRS, definiert als zwei von vier Kriterien: Körpertemperatur >38°C oder

<36°C, Herzfrequenz >90/min, Atemfrequenz >20/min oder pCO2 <32 mmHg, Leukozyten >12000/µl oder <3800/µl oder >10% unreife Leukozyten im Blutbild) und zweitens der mikrobiologische oder klinische Nachweis einer Infektion, also das Vorhandensein von Erregern oder deren Toxinen in der Blutbahn.

Eins dieser Toxine ist Lipopolysaccharid (LPS, Endotoxin), ein Bestandteil der Zellwand von gramnegativen Bakterien und wird bei deren Zerfall freigesetzt, worauf der Körper mit schwersten immunologischen Reaktionen bis hin zum Multiorganversagen reagiert. Des Weiteren induziert es eine langanhaltende Reduktion der Energieaufnahme bei

(5)

2 gleichzeitiger Steigerung des Energieverbrauchs, was zu einer schweren Katabolie und dadurch zu einer schlechten Prognose der Sepsis führt (Faggioni et al., 1997).

Die Energiebilanz wird im Körper von einem Netzwerk von Signalkaskaden und Hormonen gesteuert. Zwei wichtige Hormone dieses Systems sind Ghrelin und Leptin.

Ghrelin ist ein appetitanregendes Protein, welches hauptsächlich im Magen produziert wird (Kojima et al., 1999, Lee et al., 2002) und einer Leptin induzierten Reduktion der Nahrungsaufnahme entgegenwirkt (Nakazato et al., 2001). Es wird direkt vor Nahrungsaufnahme sezerniert und nach der Nahrungsaufnahme kontinuierlich wieder abgebaut (Beck et al., 2003). In einer Studie von Hataya und Kollegen mit LPS behandelten Ratten waren die Plasma Ghrelinwerte geringer als die der Kontrollgruppe und eine Therapie mit Ghrelin erhöhte sowohl die Nahrungsaufnahme als auch das Gewicht der Versuchstiere (Hataya et al., 2003). Im Hinblick auf einen möglichen therapeutischen Einsatz von Ghrelin im Rahmen der Sepsis war die Frage inwieweit Adipositas einen Einfluss auf exogenes Ghrelin und dessen Wirkung hat, da bekannt ist, dass bei Adipositas ein gestörtes Ghrelin-System vorliegt (Shearman et al., 2006).

Leptin ist ein 167-Aminosäuren großes Protein, welches vom Fettgewebe produziert wird und früher als reines Hormon des Fettstoffwechselzyklus galt, da es entsprechend zur Masse des Fettgewebes steigt, jedoch heutzutage mehr als Regulator von metabolischen, neuroendokrinen und immunologischen Funktionen betrachtet wird (Ahima und Flier, 2000). Um wirken zu können muss das Proteinhormon Leptin im Gegensatz zu Hormonen auf Cholesterinbasis, welche die Zellmembran ungehindert passieren können, mit einem membranständigen oder löslichen Rezeptor interagieren. Zur Zeit sind sechs verschiedene Isoformen des Leptinrezeptors (Ob-Ra-f) bekannt, der lösliche Rezeptor Ob-R (Ob-Re) geht durch Abspaltung des zellmembrangebundenen Rezeptors hervor (Ge et al., 2002).

Dieser Rezeptor spielt eine wichtige Rolle bei der hormonellen Wirkung des Leptins, da

(6)

3 wohl primär das rezeptorgebundene Leptin (bl) physiologische Wirkung hat (Brabant et al., 2000). Bei Adipositas kommt es zu einer zentralen und peripheren Leptinresistenz des Organismus und eine mögliche Erklärung hierfür ist ein Missverhältnis des rezeptorgebundenen Leptins zum ungebundenen / freien Leptin (fl), insofern als dass bei Adipositas hohe fl bei gleichzeitig prozentual erniedrigten bl Serumwerten vorherrschen (Brabant et al., 2002).

Ein weiteres wichtiges Organ in der Leptinkaskade scheint neben dem Fettgewebe die Leber zu sein, denn es konnte in verschiedenen Studien gezeigt werden, dass bei Adipositas die hepatische Ob-R-Expression nahezu aufgehoben ist (Brabant et al., 2005, Huang et al., 2004).

Nun führen hohe Leptinwerte bei einer Sepsis zu Reduktion der Energieaufnahme (Sarraf et al., 1997, Schwartz et al., 1997) und man könnte folgern, dass dadurch eine schlechtere Prognose entstände. Jedoch zeigten verschiedene Studien einen Anstieg der Überlebenswahrscheinlichkeit in normalgewichtigen Tieren während einer Sepsis wenn vorher Leptin durch eine Stimulation des Leptingens erhöht wurde (Arnalich et al., 1999, Bornstein et al., 1998). Allerdings wurde hier nicht zwischen bl und fl unterschieden.

Die Fragen sind nun ob eine Therapie mit Ghrelin während einer Sepsis zur Verhinderung der Reduktion der Energieaufnahme sinnvoll ist, ob exogen zugeführtes Ghrelin die Serum Leptinwerte insgesamt oder relativ zueinander beeinflusst oder ob andernfalls erhöhte Leptinwerte bei Adipositas eine Therapie mit Ghrelin beeinflussen.

Das Ziel der hier vorliegenden Doktorarbeit war zu klären, welchen Effekt eine singuläre intravenöse (i.v.) Injektion von Ghrelin auf die absoluten und relativen bl und fl Serumwerte und die hepatische Expression von Leptin, Ob-R und Ghrelin selbst hat. Zu diesem Zweck wurden nach Ghrelin- oder Kochsalzinjektion die eben genannten Werte in gesunden oder mittels LPS-Injektion endotoxinämischen normalgewichtigen und adipösen

(7)

4 männlichen Lewis-Ratten über 24 Stunden gemessen. Zu diesem Zweck erfolgte eine dauerhafte i.v. Katheterisierung der Tiere um eine größtmögliche Stressfreiheit zu gewährleisten (Nave et al., 1998).

(8)

received 05.01.2009

ﬁ rst decision 11.03.2009

accepted 20.04.2009

Bibliography

DOI 10.1055/s-0029-1220769 Exp Clin Endocrinol Diabetes 2009; 117: 473 – 479 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349

Correspondence

Prof. Dr. H. Nave Institute for Functional and Applied Anatomy Hannover Medical School Carl-Neuberg-Stra ß e 1 30625 Hannover Germany

Tel.: + 49 / 511 / 532 36 90 Fax: + 49 / 511 / 29 48 nave.heike@mh-hannover.de Key words

● ^▶ ghrelin ● ^▶ leptin

● ^▶ receptor-bound leptin ● ^▶ diet-induced obesity ● ^▶ endotoxemia

Ghrelin Treatment Increases Receptor-bound Leptin in Healthy and Endotoxemic Obese Lewis Rats

a meal. After meal ingestion, it progressively decreases ( Beck et al., 2003 ). Hataya and colleagues (2003) showed that plasma ghrelin levels were suppressed in LPS-injected rats and ghrelin treatment induced an increased food intake associated with a signiﬁ cant increase in body weight.

Gilg and Lutz (2006) showed that the eff ect of an intraperitoneal ghrelin treatment depends on the age of the rat, the baseline food intake and is altered when the treatment is repeated over several days. In a recent study we could show a benefi cial eff ect of ghrelin on the food consumption of normal weight rats in endotoxemia whereas this benefi cial eff ect was abrogated in obese animals ( Prenzler et al., 2007 ). Since the ghrelin system is altered in diet-induced obesity ( Shearman et al., 2006 ), we hypothesize that in addition to the above mentioned parameters the body weight of the animal infl uences the modulating capaci- ties of exogenous ghrelin.

Leptin is a 167 amino-acid peptide mainly produced by the adipose tissue. It was ﬁ rst thought to be an anti-obesity hormone, but now a more complex description has emerged of a regulator of metabolism, neuroendocrine and immune Introduction

&

Obese patients with sepsis have higher morbidity and mortality rates than normal weight subjects, but the underlying mechanisms are largely unknown ( Bercault et al., 2004 ; Vachharajani et al., 2005 ). Lipopolysaccharide (LPS; endotoxin), a major cell wall component of gram-negative bacteria, is the central constituent of endotoxemia. It induces long-lasting reduced energy intake and augmented energy expenditure and the resulting malnutrition is a crucial factor for the prognosis of septic subjects ( Faggioni et al., 1997 ). Energy homeostasis both in a physiological and pathophysiological context such as endotoxemia is maintained by a complex multifactorial regulation network and ghrelin and leptin are central components of this signaling system.

Ghrelin is an octanoylated orexigenic peptide, mainly produced in the stomach ( Kojima et al., 1999 ; Lee et al., 2002 ). Ghrelin blocks leptin- induced feeding reduction, implying that there is a competitive interaction between ghrelin and leptin in body weight regulation ( Nakazato et al., 2001 ). Ghrelin is released before the beginning of Authors C. Macke ¹ , N. K. Prenzler ¹ , R. Horn ² , G. Brabant ³ , H. Nave ¹

Aﬃ liations ¹ Institute for Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany

² Clinic for Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany ³ Department of Endocrinology, Christie ’ s Hospital, Manchester, U.K.

Abstract &

Obese patients with sepsis have higher morbidity and mortality rates than normal weight subjects. One crucial factor is the disease-associated disturbed energy balance. Ghrelin is an orexigenic peptide, mainly produced in the stomach.

Leptin is an adipose-tissue derived peptide, cir- culating as free (fl ) and receptor-bound protein (bl) acting antagonistically to ghrelin ’ s eff ects on food intake. In the present study we tested the weight dependent infl uence of an intravenous (i.v.) ghrelin injection on leptin levels as well as hepatic protein expression in healthy and endotoxemic rats. Male Lewis rats were

randomly divided into four diet-induced obese and four normal weight groups. Application of either ghrelin or NaCl was followed by a bolus injection of LPS or NaCl. Blood was collected at fi ve time points (up to 24 h) to measure fl and bl by radioimmunoassay. Furthermore, hepatic leptin, leptin receptor and ghrelin expression were investigated immunohistochemically. Results revealed a late shift from high elevated fl to signifi cantly enhanced levels of bl in ghrelin treated obese animals. Both fl and bl levels remained unaff ected in lean rats. The fi ndings suggest that an increased body weight of the treated animals is associated with altered hormone levels after therapeutic interventions with ghrelin.

(9)

functions ( Ahima and Flier, 2000 ). To date at least six diff erent leptin receptor isoforms (Ob-Ra-f) are known. The soluble Ob-R (Ob-Re) is generated by ectodomain shedding of membrane- spanning receptors ( Ge et al., 2002 ). Obesity is accompanied by a central and peripheral leptin resistance of unknown origin and one possible explanation is the misbalance between the amounts of free (fl ) and receptor-bound (bl) leptin in diet-induced obesity ( Brabant et al., 2002 ). Obese individuals have high elevated serum levels of fl , whereas the relative proportion of bl is severely decreased. The liver seems to play a major role in the leptin signaling cascade, since hepatic Ob-R expression and consecutive post-receptor signaling is abolished in diet-induced obesity ( Brabant et al., 2005 , Huang et al., 2004 ).

Sarraf and colleagues (1997) and Schwartz and Seeley (1997) reported that the increase of leptin in normal weight subjects as a result of the elevation of cytokine levels may also contribute to anorexia and weight loss in infl ammatory conditions. However, several studies demonstrate that the stimulation of the leptin gene expression and leptin release following an endotoxin challenge positively correlates with survival ( Arnalich et al., 1999 , Bornstein et al., 1998 ). Thus, on the one hand ghrelin may be a useful agent for the therapy of infl ammation-induced weight loss. On the other hand it is not clear whether exogenous ghrelin aff ects serum leptin levels or results in a shift of fl to bl or vice versa.

The aim of the present study was to evaluate the eﬀ ects of a single intravenous (i.v.) injection of the leptin antagonist ghrelin on serum ﬂ and bl amounts and hepatic leptin, Ob-R and ghrelin expression. Investigations were performed in male normal weight and diet-induced obese healthy and LPS-challenged rats over 24 h.

Materials and Methods &

Animals

Male Lewis rats (7 weeks old, n = 32) were obtained from the Central Animal Facility, Hannover Medical School, Hannover, Germany and individually housed in plastic-based cages (40 × 26 × 15 cm), in sound-proofed, air-conditioned and artifi - cially lighted rooms (lights on at 0700 h – 1900 h) at an ambient temperature of 24.0 ± 0.5 ° C. The animals were kept under spe- cifi c pathogen free conditions. Animals were randomized into two groups. One group was fed a high-calorie diet (34 % carbohydrate, 17 % protein, 4 % water, 35 % fat and 5.2 kcal / g; Altromin C1057, Altromin GmbH & Co KG, Lage, Germany) for fi ve weeks, after which the experiments started with the normal weight animals (mean body weight 302 g ± 15 g, n = 16) and the diet- induced obese littermates (mean body weight 350 g ± 15 g, n = 16). Standard rat chow (50 % carbohydrate, 19 % protein, 12 % water, 4 % fat and 2.1 kcal / g; Altromin 1234, Altromin GmbH &

Co KG) and tap water were available ad libitum throughout the whole experiment for all experimental animals. All research and animal care procedures had been approved by the Lower Saxony district government in Hannover, Germany (AZ 04 / 753).

Intravenous cannulation

Animals were anesthetized intramuscularly with ketamine hydrochloride (10 % , 0.35 ml, Graeub, Aulendorf, Germany) and medetomidine hydrochloride (0.001 % , 0.05 ml, Pﬁ zer GmbH, Karlsruhe, Germany) and provided with a central venous catheter in the right external jugular vein as reported before ( Nave

et al., 1998 ). To avoid perioperative hypothermia and dehydra- tion, animals were operated on a warming blanket (surface temperature of 30 ° C) and were subcutanously injected with 2 ml of 5 % dextrose (dissolved in 0.9 % saline) in each fl ank before the operation ( Felies et al., 2005 ). For the fi rst two postoperative days, rats were given water containing 0.4 mg metamizole / ml for an appropriate analgesia. Animals were allowed to recover over fi ve days to avoid surgical stress-induced impairment of the experiments.

Endotoxin and ghrelin

One batch of Escherichia coli endotoxin (#L3880, Sigma-Aldrich, Taufkirchen, Germany) was dissolved in 0.9 % saline and a dose of 100 μ g / kg in 0.2 ml saline was i.v. applied. The dosage was determined by pilot studies (data not shown). Octanoylated rat- ghrelin (H-4862, Bachem, Weil am Rhein, Germany) was dissolved in 0.9 % saline and a dose of 10 nmol / kg in 0.2 ml saline was i.v. applied. The dosage was determined by previous studies ( Chang et al., 2003 ; Tschop et al., 2000 ).

Experimental protocol

Animals were randomly divided into two groups: 1) normal weight and 2) diet-induced obese; and each of these into four groups a) controls (NaCl + NaCl, n = 3), b) ghrelin-treated controls (Ghrelin + NaCl, n = 5), c) endotoxemic rats (NaCl + LPS, n = 3) and d) ghrelin-treated endotoxemic rats (Ghrelin + LPS, n = 5). The baseline blood sample (500 μ l) was taken 24 h before the investi- gation. The experiments started with an i.v. bolus injection of either 0.2 ml saline or ghrelin, followed 10 min later by another i.v. bolus injection of either 0.2 ml saline or LPS. In addition to the baseline blood sample, further samples were taken at three diﬀ erent time points after the second i.v. injection (1 h, 3 h and 6 h). After each blood withdrawal through the central catheter, the volume was replaced by an i.v. application of an isotonic Ringer solution (Ringer-Laktat, B.Braun Melsungen AG, Melsun- gen, Germany). All animals survived until the end of experiments. Animals were i.v. anesthetized 24 h after the second injection with the same anesthesia used for the catheterization.

Blood was collected after puncture of the heart for further endo- crinological analyses.

Leptin measurements

All assays have been previously described in detail ( Brabant et al., 2000 ). In short, antibodies to the N-terminal portion of the protein (leptin amino acid number 26 – 38) were generated by coupling to hemocyanine by the carbodiimide method. N-terminal antibodies selectively detect protein bound leptin immuno- reactivity. Receptor-bound leptin was measured at fi ve diff erent time points: baseline, 1 h, 3 h, 6 h and 24 h. In addition free leptin was measured using a commercially available polyclonal antibody based assay (Linco Research, St. Charles, USA) at fi ve diff erent time points: baseline, 1 h, 3 h, 6 h and 24 h. The maximal cross-reactivity between the diff erent leptin assays was below 0.001. It has to be mentioned that although commercially available leptin assays show high correlation, they can provide diff erent measures for leptin levels ( Chan et al., 2007 ). In the present study the Linco assay was used, measuring predominantly free leptin.

Immunohistochemistry of ghrelin, leptin and Ob-R After collecting the 24 h blood sample, animals were transfused transcardially through the left ventricle with 100 ml of phos-

(10)

phate-buﬀ ered saline (PBS). Parts of the intermediate lobe of the liver were removed and immediately frozen in liquid nitrogen.

The organ samples were stored at − 70 ° C until used for immunohistochemistry. The detections of leptin (alkaline phophatase- antialkaline phosphatase complex and Fast Red as substrate) and ghrelin (ABC complex and visualization of peroxidase by diaminobenzidine) were performed as described previously ( Brabant et al., 2004 ; Prenzler et al., 2007 ). For detection of the leptin receptor, liver sections were incubated with the primary monoclonal antibody (Ob-R B3; Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a dilution of 1:100 overnight at 4 ° C, followed by incubation with mouse IgG (Dako, Hamburg, Germany). To visualize the binding an alkaline phosphatase-anti alkaline phosphatase complex (1:50) was used, followed by the repeti- tion of the last two steps. The sections were stained with Fast Blue for 25 min and counterstained with hematoxylin. Method speciﬁ city was checked by isotype controls (not shown). For each animal ﬁ ve sections were examined. Semiquantitative assessment of protein expression was evaluated by ImageJ image analyzer.

Data analysis

Data are expressed as means ± SEM (standard error of the mean).

To analyze the eff ect of the LPS application on the diff erent parameters over time data were analyzed by using two-factor analyses of variance (ANOVA) for repeated measurements (factors: intervention / treatment and time) and were followed by one-factor (treatment) ANOVA and Fisher-PLSD post hoc analysis, where appropriate. Treatment and / or group diff erences were considered signifi cant if p < 0.05. For the calculation of the relative proportion of bl vs. total leptin the mean values were used.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Time [h] after second i.v. injection normal weight NaCl+NaCl

normal weight ghrelin+NaCl obese NaCl+NaCl obese ghrelin+NaCl

Baseline

* *

*

#

# #

#

free serum leptin (ng/ml)

1 3 6 24

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Time [h] after second i.v. injection normal weight NaCl+LPS

normal weight ghrelin+LPS obese NaCl+LPS obese ghrelin+LPS

Baseline

*

#

# #

#

free serum leptin (ng/ml)

1 3 6 24

Fig. 1 Free leptin levels (mean + SEM; baseline [ − 24 h] and at four diff erent time points [1 h, 3 h, 6 h, 24 h] after second i.v. application) in the sera of normal weight and obese rats (n = 3 – 5). A ) free leptin, non-endotoxemic rats, and B ) free leptin, endotoxemic rats. Signifi cant post hoc eff ects vs. baseline value within one group are indicated by an asterisk * (p < 0.05). Signifi cant post hoc eff ects of the obese vs. the normal weight animals within one group at the same time point are indicated by a rhomb #(p < 0.05).

Table. 1 Relative proportion ( % ) of bound leptin levels vs. total leptin levels (baseline and 24 h).

baseline 24 h

normal weight, NaCl + NaCl 75.2 71.7 normal weight, ghrelin + NaCl 69.0 70.8 normal weight, NaCl + LPS 72.6 73.4 normal weight, ghrelin + LPS 73.6 80.6

obese, NaCl + NaCl 54.4 54.8

obese, ghrelin + NaCl 59.0 66.6

obese, NaCl + LPS 53.0 56.9

obese, ghrelin + LPS 50.5 75.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Time [h] after second i.v. injection normal weight NaCl+NaCl

normal weight ghrelin+NaCl

obese NaCl+NaCl obese ghrelin+NaCl

Baseline

*#

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5

Time [h] after second i.v. injection normal weight NaCl+LPS

normal weight ghrelin+LPS obese ghrelin+LPS obese NaCl+LPS

Baseline

*#

bound serum leptin (ng/mL)bound serum leptin (ng/mL)

1 3 6 24

Fig. 2 Bound leptin levels (mean + SEM; baseline [ − 24 h] and at four diff erent time points [1 h, 3 h, 6 h, 24 h] after second i.v. application) in the sera of normal weight and obese animals (n = 3 – 5). A ) non- endotoxemic rats, B ) endotoxemic rats. Signifi cant post hoc eff ects vs.

baseline value within one group are indicated by an asterisk * (p < 0.05).

Signiﬁ cant post hoc eﬀ ects of the obese vs. the normal weight animals within one group at the same time point are indicated by a rhomb

#(p < 0.05).

(11)

Results &

Serum levels of free leptin

Obese animals showed signifi cantly higher baseline serum levels of fl than normal weight littermates ( ● ^▶ Fig. 1A,B ). Surprisingly, the non-endotoxemic obese rats had a decrease in fl levels at the 3 h and 6 h time points but returned to baseline values at 24 h ( ● ^▶ Fig. 1A ). However, there were signifi cant diff erences between the ghrelin-treated endotoxemic rats ( ●^▶ Fig. 1B ). Fl levels of the endotoxemic obese decreased dramatically and reached control levels of normal weight animals with a short increase at the 3 h time point whereas the ghrelin application had no eff ect on the lean littermates ( ● ^▶ Fig. 1B ).

Serum levels of bound-leptin

Since bl and ﬂ are diﬀ erentially regulated and serve independent functions in health and various pathophysiological conditions it was of major interest whether ghrelin and / or LPS alter these components of the leptin system in a distinct manner. The relative proportion of bl vs. total leptin in the sera of obese control animals was decreased compared to lean littermates ( Table. 1 ).

In normal weight animals bl levels were not signiﬁ cantly altered during endotoxemia or after ghrelin injection. Ghrelin treatment

modulated serum levels of bl in obese rats in a diﬀ erent manner.

Both, in healthy and endotoxemic obese animals, ghrelin application signiﬁ cantly increased bl levels with a late maximum 24 h after treatment ( ●^▶ Fig. 2A,B ). This resulted in an almost equally high relative proportion of bl in ghrelin treated obese rats (66.6 % in healthy and 75.0 % in endotoxemic) compared to the corresponding normal weight animals (70.8 % and 80.6 % ) ( Table. 1 ). In view of baseline levels it was an increase from 59.0 % to 66.6 % in healthy and even from 50.5 % to 75.0 % in endotoxemic rats 24 h after ghrelin injection. This corresponded approximately to the baseline bl levels of the normal weight animals.

Immunohistochemistry of hepatic leptin expression Since the liver plays a central role in the formation of bl and the expression of leptin and Ob-R ghrelin- and LPS-induced hepatic protein expression was determined immunohistochemically.

The leptin expression was comparable in all normal weight groups. The amount of leptin in the liver was slightly enhanced in obese littermates ( ● ^▶ Fig. 3A ). Opposite eﬀ ects were seen in obese vs. lean endotoxemic (NaCl + LPS) animals as compared with their corresponding healthy controls: a tendency to decreased staining density was observed in both normal weight endotoxemic groups, whereas the leptin expression was

0 100 200 300 400 500 600

ghrelin+LPS ghrelin+LPS

NaCl+LPS NaCl+LPS

ghrelin+NaCl ghrelin+NaCl

NaCl+NaCl NaCl+NaCl

#

nw ob

hepatic leptin expression [mean density]

*#

A B

C

0 100 200 300

NaCl+LPS NaCl+LPS

NaCl+NaCl NaCl+NaCl

nw ob

hepatic Ob-R expression [mean density]

D

Fig. 3 Mean immunohistological density of hepatic ( A ) leptin and ( D ) leptin receptor (Ob-R) staining 24 h after second i.v. injection (mean ± SEM). Signifi cant post hoc eff ects of the obese ghrelin + LPS vs. the NaCl + LPS group are indicated by an asterisk * (p < 0.05). Signifi cant post hoc eff ects of the obese (ob) vs.

the normal weight (nw) animals within one group are indicated by a rhomb #(p < 0.05). Representative immunohistological detection of leptin in ( B ) normal weight and ( C ) obese ghrelin-treated endotoxemic (ghrelin + LPS) rats.

(12)

increased in obese endotoxemic rats. The treatment with ghrelin resulted in a signiﬁ cant enhancement of the hepatic leptin expression in obese endotoxemic (ghrelin + LPS) animals compared to the corresponding normal weight animals ( ●^▶ Fig. 3B,C ) and the healthy ghrelin-treated (ghrelin + NaCl) obese littermates.

Immunohistochemistry of the hepatic Ob-R expression The expression of Ob-R detected with a monoclonal antibody recognizing the long (Ob-Rb) and several short (Ob-Ra, Ob-Rc) isoforms was nearly identical in all investigated normal weight and diet-induced obese groups. However, the amount was slightly decreased in obese animals ( ● ^▶ Fig. 3D ).

Immunohistochemistry of the hepatic ghrelin expression

No signifi cant diff erences between the normal weight groups could be detected ( ● ^▶ Fig. 4A ). Obese control animals (NaCl + NaCl) had a signifi cantly decreased ghrelin expression compared to normal weight littermates. Interestingly, after an i.v. ghrelin application in healthy obese rats the protein expression of ghrelin signifi cantly increased ( ●^▶ Fig. 4B,C ). An endotoxin challenge slightly enhanced the hepatic ghrelin expression of obese animals compared to their controls, but the treatment with ghrelin was without eff ect.

Discussion &

Ghrelin, a potent orexigen mainly produced by enteroendocrine cells in the stomach, exerts antagonistic eﬀ ects on the leptin- induced decrease in food intake through activation of the hypothalamic neuropeptide Y-Y1 pathway ( Wren et al., 2000 ).

Some reports have shown a role of ghrelin in other physiological functions such as cardiovascular regulation, infl ammation pro- cess or septic shock ( Chang et al., 2003 ; Nagaya et al., 2001 ). Sys- temic infl ammations are characterized by reduced energy intake and augmented energy expenditure. The resulting lack of food is a crucial factor for the prognosis of septic subjects ( Faggioni et al., 1997 ). Several experimental and clinical studies report that LPS or infections with Helicobacter pylori result in a reduc- tion of plasma ghrelin levels in rodents and humans ( Basa et al., 2003 ; Osawa et al., 2005 ). In a recent study of our group the eff ect of a single ghrelin application in normal weight and diet- induced obese rats signifi cantly enhanced ghrelin serum levels by ~ 300 % in all investigated groups. However, this eff ect solely persisted for a short time period of two hours ( Prenzler et al., 2007 ). Thus, only with repeated applications might ghrelin be an interesting orexigenic tool to increase feeding (or decrease energy expenditure) in septic individuals.

To date only few data exist showing the impact of ghrelin as a therapeutic agent in severe infl ammations and little information is available concerning the infl uence of body weight on septic or endotoxemic subjects ( Chang et al., 2003 ; Wu et al., 2005 ). As ghrelin and leptin are functional antagonists in regard to food intake it can be assumed that the application of ghrelin as a therapeutic agent in severe infl ammations has profound eff ects on serum leptin levels. Tsubone and colleagues (2005) observed a downregulation of ghrelin levels in diabetic mice after a subcutaneous leptin injection, whereas Schmid and colleagues (2005) could not detect an impact of an i.v. ghrelin injection on plasma leptin levels i n healthy normal weight humans. As it is known

0 100 200 300 400 500 600

NaCl+LPS NaCl+LPS

NaCl+NaCl NaCl+NaCl

§

*#

*

nw ob

hepatic ghrelin expression [mean density]

A

B

C

Fig. 4 Mean immunohistological density of hepatic ghrelin staining ( A ) 24 h after second i.v. injection (mean ± SEM). Signifi cant post hoc eff ects vs. the NaCl + NaCl control group are indicated by an asterisk * (p < 0.05). Signifi cant post hoc eff ects vs. the Ghrelin + NaCl group are indicated by a rhomb #(p < 0.05). Signifi cant post hoc eff ects of the obese (ob) NaCl + NaCl vs. the corresponding normal weight (nw) animals group are indicated by a paragraph sign § (p < 0.05). Representative immunohistological detection of ghrelin in obese ( B ) control (NaCl + NaCl) and ( C ) ghrelin treated healthy rats. Ghrelin positive cells / areas are indicated by black arrows.

(13)

that obese patients have an increased incidence of systemic infections, higher morbidity and mortality rates than normal weight subjects and an altered leptin and ghrelin system, it is of special interest to examine the diff erences in terms of bl and fl levels between lean and obese animals after a ghrelin application in health and disease ( Loff reda et al., 1998 ; Matarese et al., 2002 ). The vast majority of studies do not diff erentiate between the both forms of leptin. It can be hypothesized that bl is the physiologically active leptin component ( Brabant et al., 2000 ).

The present study demonstrates a late shift of ﬂ , the prevalent component of leptin in diet-induced obesity, to bl after ghrelin treatment both in healthy and endotoxemic obese rats. One day after the single ghrelin injection the obese ghrelin-treated rats had an almost equally high percentage of bl as compared to their corresponding normal weight littermates. In contrast, serum leptin amounts and composition remained nearly unaﬀ ected in normal weight littermates. Regard to ghrelin as a potential drug target to regulate energy homeostasis in diet-induced obese animals, the elevation of bl in healthy obese animals by means of a single ghrelin administration has to be investigated in future projects.

Brabant and colleagues (2005) as well as Ikejima and colleagues (2004) showed that the liver plays a central role in the formation of bl and the expression of leptin and Ob-R. In this study no eﬀ ect was seen on the hepatic leptin or Ob-R expression after ghrelin treatment, only an LPS application elevated the hepatic leptin expression in obese animals. In a recent study we showed that ghrelin does not elevate the energy uptake in endotoxemic ghrelin-treated obese rats as much as it does in the normal weight littermates ( Prenzler et al., 2007 ). Thus, a stronger impact of an LPS challenge in obese as compared to lean animals on the metabolic system can be assumed.

There are indications that plasma leptin levels are elevated in endotoxemia and sepsis and this fact is associated with benefi ts such as positive immunomodulatory eff ects of leptin and enhanced survival times ( Bornstein et al., 1998 ; Lord et al., 1998 ). In the present study no eff ect of a sublethal endotoxin challenge on serum fl or bl levels was observed. Although the present model of a sublethal endotoxemia is accompanied by distinct immunological and physiological changes ( Felies et al., 2004 ; Nave et al., 2004 ), the relatively low concentration of LPS may be insuffi cient to trigger a signifi cant increase of serum leptin levels.

The present study documents the positive eﬀ ect of ghrelin on bl levels in the serum of acute endotoxemic obese rodents. Consid- ering that bl appears to serve an independent physiological role compared to the unbound ﬂ and that the balance between both leptin forms is disrupted in obesity it is interesting that exogenous ghrelin can alter bl levels in obese rats.

Acknowledgments &

The correction of the English by S. Fryk is gratefully acknowl- edged. The study was supported by the Eli Lilly International Foundation, Bad Homburg, Germany and the Danone Founda- tion, Haar, Germany.

Conﬂ ict of interest : None.

References

1 Ahima R , Flier J . Leptin . Annu Rev Physiol 2000 ; 62 : 413 – 437 2 Arnalich F , Lopez J , Codoceo R et al . Relationship of plasma leptin to

plasma cytokines and human survival in sepsis and septic shock . J Infect Dis 1999 ; 180 : 908 – 911

3 Basa NR , Wang L , Arteaga JR et al . Bacterial lipopolysaccharide shifts fasted plasma ghrelin to postprandial levels in rats . Neurosci Lett 2003 ; 343 : 25 – 28

4 Beck B , Richy S , Stricker-Krongrad A . Ghrelin and body weight regula- tion in the obese Zucker rat in relation to feeding state and dark/light cycle . Exp Biol Med 2003 ; 228 : 1124 – 1131

5 Bercault N , Boulain T , Kuteifan K et al . Obesity-related excess mortality rate in an adult intensive care unit: A risk-adjusted matched cohort study . Crit Care Med 2004 ; 32 : 998 – 1003

6 Bornstein SR , Licinio J , Tauchnitz R et al . Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm, in cortisol and leptin secretion . J Clin Endocrinol Metab 1998 ; 83 : 180 – 183

7 Brabant G , Horn R , von zur M ü hlen A et al . Free and protein bound leptin are distinct and independently controlled factors in energy regulation . Diabetologia 2000 ; 43 : 438 – 442

8 Brabant G , M ü ller G , Horn R et al . Hepatic leptin signaling in obesity . FASEB J 2005 ; 19 : 1048 – 1050

9 Brabant G , Nave H , Horn R et al . In vivo and in vitro evidence for a hepatic modulation of the leptin signal in rats . Eur J Clin Invest 2004 ; 34 : 831 – 837

10 Brabant G , Nave H , Mayr B et al . Secretion of free and protein-bound leptin from subcutaneous adipose tissue of lean and obese women . J Clin Endocrinol Metab 2002 ; 87 : 3966 – 3970

11 Chan JL , Wong SL , Orlova C et al . Pharmacokinetics of recombinant methionyl human leptin after subcutaneous administration: variation of concentration-dependent parameters according to assay . J Clin Endocrinol Metab 2007 ; 92 : 2307 – 2311

12 Chang L , Zhao J , Yang J et al . Therapeutic eﬀ ects of ghrelin on endotoxic shock in rats . Eur J Pharmacol 2003 ; 473 : 171 – 176

13 Faggioni R , Fuller J , Moser A et al . LPS-induced anorexia in leptin-deﬁ - cient (ob/ob) and leptin receptor-deﬁ cient (db/db) mice . Am J Physiol 1997 ; 273 : R181 – R186

14 Felies M , von H ö rsten S , Pabst R et al . Neuropeptide Y stabilizes body temperature and prevents hypotension in endotoxemic rats . J Physiol 2004 ; 561 : 245 – 252

15 Felies M , Poppendieck S , Nave H . Perioperative normothermia depends on intraoperative warming procedure, extent of surgical intervention and age of the experimental animal . Life Sci 2005 ; 77 : 3133 – 3140 16 Ge H , Huang L , Pourbahrami et al . Generation of soluble leptin recep-

tor by ectodomain shedding of membrane-spanning receptors in vitro and in vivo . J Biol Chem 2002 ; 48 : 45898 – 45903

17 Gilg S , Lutz TA . The orexigenic eff ect of peripheral ghrelin diff ers between rats of diff erent age and with diff erent baseline food intake, and it may in part be mediated by the area postrema . Physiol Behav 2006 ; 87 : 353 – 359

18 Hataya Y , Akamizu T , Hosoda H et al . Alterations of plasma ghrelin levels in rats with lipopolysaccharide-induced wasting syndrome and eﬀ ects of ghrelin treatment on the syndrome . Endocrinology 2003 ; 144 : 5365 – 5371

19 Huang W , Dedousis N , Bhatt BA et al . Impaired activation of phos- phatidylinositol 3-kinase by leptin is a novel mechanism of hepatic leptin resistance in diet-induced obesity . J Biol Chem 2004 ; 279 : 21695 – 21700

20 Ikejima K , Lang T , Zhang YJ et al . Expression of leptin receptors in hepatic sinusoidal cells . Comp Hepatol 2004 ; 14 : 1 – 12

21 Kojima M , Hosoda H , Date Y et al . Ghrelin is a growth-hormone-releas- ing acylated peptide from stomach . Nature 1999 ; 402 : 656 – 660 22 Lee H-M , Wang G , Englander EW et al . Ghrelin, a new gastrointestinal

endocrine peptide that stimulates insulin secretion: enteric distribu- tion, ontogeny, inﬂ uence of endocrine, and dietary manipulations . Endocrinology 2002 ; 143 : 185 – 190

23 Loﬀ reda S , Yang SQ , Lin HZ et al . Leptin regulates proinﬂ ammatory immune responses . FASEB J 1998 ; 12 : 57 – 65

24 Lord GM , Matarese G , Howard JK et al . Leptin modulates the T-cell

response and reverses starvation-induced immunosuppression .

Nature 1998 ; 394 : 897 – 901

25 Matarese G , La Cava A , Sanna V et al . Balancing susceptibility to infec- tion and autoimmunity: a role for leptin? Trends Immunol 2002 ; 23 : 182 – 187

26 Nagaya N , Uematsu M , Kojima M et al . Chronic administration of ghre- lin improves left ventricular dysfunction and attenuates development

(14)

of cardiac cachexia in rats with heart failure . Circulation 2001 ; 104 : 1430 – 1435

27 Nakazato M , Murakami N , Date Y et al . A role for ghrelin in the central regulation of feeding . Nature 2001 ; 409 : 194 – 198

28 Nave H , Bedoui S , Moenter F et al . Reduced tissue immigration of monocytes by neuropeptide Y during endotoxemia is associated with Y2 receptor activation . J Neuroimmunol 2004 ; 155 : 1 – 12

29 Nave H , von Hoersten S , Helfritz F et al . Intravenous cannulation of the freely behaving rat: frequent blood sampling and volume-dependent eﬀ ect on blood leukocyte counts . J Exp Anim Sci 1998 ; 39 : 67 – 77 30 Osawa H , Nakazato M , Date Y et al . Impaired production of gastric

ghrelin in chronic gastritis associated with Helicobacter pylori . J Clin Endocrinol Metab 2005 ; 90 : 10 – 16

31 Prenzler NK , Macke C , Horn R et al . Obesity inﬂ uences the food consumption and cytokine pattern in ghrelin-treated endotoxemic rats . Life Sci 2007 ; 81 : 80 – 87

32 Schmid DA , Held K , Ising M et al . Ghrelin stimulates appetite, imagina- tion of food, GH, ACTH, and cortisol, but does not aﬀ ect leptin in normal controls . Neuropsychopharmacology 2005 ; 30 : 1187 – 1192 33 Schwartz MW , Seeley RJ . Neuroendocrine responses to starvation and

weight loss . N Engl J Med 1997 ; 336 : 1802 – 1811

34 Sarraf P , Frederich RC , Turner EM et al . Multiple cytokines and acute inﬂ ammation raise mouse leptin levels: potential role in inﬂ ammatory anorexia . J Exp Med 1997 ; 185 : 171 – 175

35 Shearman LP , Wang SP , Helmling S et al . Ghrelin neutralization by a ribonucleic acid-SPM ameliorates obesity in diet-induced obese mice . Endocrinology 2006 ; 147 : 1517 – 1526

36 Tschop M , Smiley DL , Heiman ML . Ghrelin induces adiposity in rodents . Nature 2000 ; 407 : 908 – 913

37 Tsubone T , Masaki T , Katsuragi I et al . Leptin downregulates ghrelin levels in streptozotocin-induced diabetic mice . Am J Physiol Regul Integr Comp Physiol 2005 ; 289 : R1703 – R1706

38 Vachharajani V , Russell JM , Scott KL et al . Obesity exacerbates sepsis- induced inﬂ ammation and microvascular dysfunction in mouse brain . Microcirculation 2005 ; 12 : 183 – 194

39 Wren AM , Small CJ , Ward HL et al . The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion . Endo- crinology 2000 ; 141 : 4325 – 4328

40 Wu R , Dong W , Zhou M et al . Ghrelin improves tissue perfusion in severe sepsis via downregulation of endothelin-1 . Cardiovasc Res 2005 ; 68 : 318 – 326