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Endocrinology Vol. 147, No. 6 2634-2642
Copyright © 2006 by The Endocrine Society

Role of Endogenous Ghrelin in the Hyperphagia of Mice with Streptozotocin-Induced Diabetes

J. Dong, T. L. Peeters, B. De Smet, D. Moechars, C. Delporte, P. Vanden Berghe, B. Coulie, M. Tang and I. Depoortere

Center for Gastroenterological Research, Catholic University of Leuven (J.D., T.L.P., B.D.S., P.V.B., I.D.), 3000 Leuven, Belgium; Medical College of Qingdao University (J.D.), Qingdao, China; Johnson & Johnson Pharmaceutical Research and Development, Division of Janssen Pharmaceutica (D.M., B.C.), 2340 Beerse, Belgium; and Laboratory of Biological Chemistry and Nutrition, Université Libre de Bruxelles (C.D.), 1070 Brussels, Belgium

Address all correspondence and requests for reprints to: Dr. Inge Depoortere, Center for Gastroenterological Research, Gasthuisberg O&N, Bus 701, 3000 Leuven, Belgium. E-mail: inge.depoortere{at}med.kuleuven.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin is an orexigenic peptide involved in the regulation of energy homeostasis. To investigate the role of ghrelin in the hyperphagia associated with uncontrolled streptozotocin-induced diabetes, food intake was followed in diabetic ghrelin knockout (ghrelin–/–) and control wild-type (ghrelin+/+) mice and diabetic Naval Medical Research Institute noninbred Swiss mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GH-releasing peptide-6 (D-Lys3-GHRP-6) for 5 d. In diabetic ghrelin–/– mice, hyperphagia was attenuated, and the maximal increase in food intake was 50% lower in mutant than in wild-type mice. The increased food intake observed during the light period (1000–1200 h) in ghrelin+/+ mice was abolished in mutant mice. Diabetic ghrelin–/– mice lost 12.4% more body weight than ghrelin+/+ mice. In diabetic ghrelin+/+ mice, but not in ghrelin–/– mice, the number of neuropeptide Y (NPY)-immunoreactive neurons was significantly increased. Diabetic Naval Medical Research Institute noninbred Swiss mice were hyperphagic and had increased plasma ghrelin levels. Treatment with D-Lys3-GHRP-6 reduced daily food intake by 23% and reversed the increased food intake observed during the light period. The change in the number of NPY- (2.4-fold increase) and {alpha}-MSH (1.7-fold decrease)-immunoreactive hypothalamic neurons induced by diabetes was normalized by D-Lys3-GHRP-6 treatment. Our results suggest that enhanced NPY and reduced {alpha}-MSH expression are secondary to the release of ghrelin, which should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN WAS DISCOVERED 5 yr ago as the endogenous ligand of the GH secretagogue receptor (1). This receptor was first characterized, cloned, and identified as the receptor for a family of synthetic ligands [e.g. GH-releasing peptide-6 (GHRP-6)] known as GH secretagogues that stimulate the release of GH (2, 3).

It is now clear that ghrelin has other effects besides the stimulation of GH secretion, among them the regulation of food intake. This effect was not unexpected, because in 1995 it was shown that intracerebroventricular administration of a synthetic GH secretagogue increased food intake in rats (4, 5). Therefore, it was soon confirmed that exogenous ghrelin also stimulated food intake in rats and man (6, 7). The observations that endogenous plasma ghrelin levels rise shortly before meals and are rapidly suppressed by food consumption lend credence to the idea that endogenous ghrelin is a hunger hormone, dictating the timing of meals (8). In addition to short-term fluctuations in ghrelin levels over the course of 1 d, longer-term regulation of circulating ghrelin appears to occur in relation to body weight change (9, 10). Ghrelin is now considered the first systemically active orexigenic hormone that induces weight gain by stimulating an acute increase in food intake as well as by decreasing fat utilization under conditions of negative energy balance (6, 11).

The source of the consumption-related feedback that decreases ghrelin levels has not been fully elucidated, but absorption of nutrients appears to play an essential role, because intragastric infusion of glucose, proteins, or fats, but not of an equal volume of water, lowers ghrelin levels (9, 11, 12). Williams et al. (13) confirmed that gastric distension and chemosensation do not play a role in the meal-related ghrelin response. It is unclear whether ingested nutrients suppress ghrelin production directly or indirectly, e.g. through insulin, a possibility that is consistent with the reciprocal 24-h profiles of these hormones. This has been confirmed in patients with type I diabetes, in whom absolute insulin deficiency prevented prandial plasma ghrelin suppression until the insulin deficiency was corrected with an iv insulin bolus (14). Also, in rats with uncontrolled, insulin-deficient diabetes induced by the ß-cell toxin streptozotocin (STZ), ghrelin levels are increased (15). Both conditions are characterized by hyperphagia, and it has therefore been suggested that a lack of meal-induced ghrelin suppression caused by severe insulin deficiency may explain hyperphagia in uncontrolled type 1 diabetes.

Ishii et al. (15) showed that treatment with a ghrelin receptor antagonist, D-Lys3-GHRP-6, could partially reverse diabetic hyperphagia, but recent studies questioned the specificity of the ghrelin receptor antagonist (16). At the dosage used, D-Lys3-GHRP-6 contracts the fundus of the stomach via activation of 5-serotonin-2B receptors and thereby induces early satiety, leading to reduced food intake by impairing gastric accommodation. The present study therefore aimed to provide conclusive evidence for a role for endogenous ghrelin in the hyperphagia of mice with uncontrolled insulin-deficient diabetes by comparing food intake in STZ-induced diabetic ghrelin knockout (ghrelin–/–) mice and their wild-type equivalents (ghrelin+/+). For comparison, plasma ghrelin levels and food intake were also studied in STZ-induced diabetic mice treated for 5 d with either saline or the ghrelin receptor antagonist, D-Lys3-GHRP-6. In both models the changes in hypothalamic expression of neuropeptide Y (NPY), {alpha}-MSH, and orexin, neuropeptides known to be involved in ghrelin-induced feeding responses, were studied (6, 17, 18, 19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male (16 wk old) ghrelin knockout (ghrelin–/–) mice (n = 6; 129Sv/Ev x C57BL/6 mixed genetic background) and the corresponding wild-type littermates (ghrelin+/+; n = 6) were developed by Lexicon Genetics, Inc. (The Woodlands, TX). Information regarding the construction and characterization of the ghrelin knockout mice has been published previously by De Smet et al. (20). Male (10 wk old) Naval Medical Research Institute noninbred Swiss (NMRI) mice (n = 24) were purchased from Janvier (Be Uden, The Netherlands). All mice were housed in individual cages in a temperature-controlled environment (20–22 C) under a 13-h light, 11-h dark cycle. Standard commercial mouse chow (4352 Muracon G, Nutreco Belgium NV, Gent, Belgium; 4.5% fat, 4% cellulose, and 21% proteins; 1.404 kcal/g) and tap water were available ad libitum. All experiments were approved by the ethical committee for animal experiments of Catholic University (Leuven, Belgium).

Experiment 1: ghrelin–/– and ghrelin+/+ mice
After an observation period of 4 d, ghrelin+/+ (n = 6) and ghrelin–/– mice (n = 6) were fasted overnight and injected ip with either STZ (200 mg/kg; n = 3 for each genotype) or vehicle [5 mM sodium citrate (pH 4.0); n = 3 for each genotype].

On d 11, all mice were anesthetized, and transcardial perfusion was performed with 4% paraformaldehyde. Fat pads were collected, and the brain was removed for immunohistochemistry studies. Body weight, 24- and 2-h food intakes (1000–1200 h), and blood glucose were monitored daily at 1000 h.

Experiment 2: NMRI mice
After an observation period of 4 d, mice were fasted overnight, and diabetes was induced in 12 mice by ip administration of streptozotocin [140 mg/kg dissolved in 5 mM sodium citrate (pH 4.0); Sigma-Aldrich Corp., St. Louis, MO]. Another group (n = 12) served as the control group and was injected with 5 mM sodium citrate (pH 4.0). On d 5 after the induction of diabetes, half the mice in each group (n = 6) were treated during the next 5 d twice daily (1000 and 1800 h) with either saline or the ghrelin receptor antagonist, D-Lys3-GHRP-6 (100 nmol/mouse, ip; Bachem, Bubendorf, Switzerland). On d 10, mice were anesthetized, and transcardial perfusion was performed with 4% paraformaldehyde. Fat pads were collected, and brain and stomach were removed for immunohistochemistry studies.

Food intake
Mice were put in an individual cage (13-h light, 11-h dark cycle) to follow food intake and were habituated to their cages for 4 d before the start of the experiments. Mice were not transported during the experimental period. Body weight, 24- and 2-h food intake (1000–1200 h), and blood glucose were monitored daily at 1000 h.

RIA
Blood samples for ghrelin and glucagon determinations were taken by retroorbital bleeding on d 10.

Plasma glucagon.
Glucagon was measured in the plasma samples by RIA kit (Euro-Diagnostica SE, Malmo, Sweden). The sensitivity of the assay was 3 pmol/liter (10.4 pg/ml).

Plasma ghrelin.
The plasma samples (150–200 µl) were loaded onto a Sep-Pak C18 cartridge (Waters, Milford, MA), preequilibrated with 3% CH3CN/0.1% trifluoroacetic acid (TFA). After washing with 15% CH3CN/0.1% TFA, the peptides were eluted with 50% CH3CN/0.1% TFA. The eluates were lyophilized in a Speed-Vac concentrator (Savant Instruments Inc., Holbrook, NY) and processed for RIA as previously described (21). An antibody against ghrelin-(13–28)-OH was used to measure total ghrelin levels.

Immunohistochemistry
Tissue preparation.
The brain and stomach of mice transcardially perfused with 4% paraformaldehyde were removed and postfixed for 4 h at room temperature. After fixation, tissue was rinsed in 0.1 M PBS (three times, 15 min each time), placed overnight in 30% sucrose for cryoprotection, and immersed in the cryoembedding compound at –20 C. Stomach and coronal brain sections (16 µm) were cut with a cryostat and stored at –80 C until use.

Staining for ghrelin-immunoreactive (ghrelin-IR) cells in mouse stomach.
Cryostat sections were incubated for 2 h in 0.1 M PBS containing 4% goat serum, 0.5% Triton X-100, and 0.3% NaN3 at 4 C. After incubation overnight with the rabbit antighrelin antibody (directed against the N terminal 1–17 region of desacyl ghrelin; provided by Dr. Tomasetto, INSERM, Strasbourg, France; dilution, 1:500), sections were washed and incubated with fluorescein isothiocyanate-conjugated goat antirabbit IgG (dilution, 1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 h at 4 C. After three washes (5 min each) in 0.1 M PBS, the sections were mounted with Citifluor (Citifluor, London, UK).

Staining for NPY, orexin and {alpha}-MSH immunopositive neurons in mouse hypothalamus.
Brain sections were treated with 0.3% H2O2 and 0.5% Triton X-100 in 10 mM PBS, then preincubated for 1 h at room temperature in 10 mM PBS containing 0.5% Triton X-100 and 10% normal horse or rabbit serum to block nonspecific binding sites. The sections were incubated with the primary antibody, goat anti-NPY (c-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution, 1:500), goat antiorexin (c-19, Santa Cruz Biotechnology, Inc.; dilution, 1:600), or sheep anti {alpha}-MSH (C terminal; Chemicon International, Temecula, CA; dilution, 1:10,000) diluted in PBS containing 0.5% Triton X-100 for 48 h at 4 C; washed; and incubated with biotin-long spacer-conjugated AffiniPure horse antigoat IgG (H+L) (1:200) or rabbit antisheep IgG (H+L) (dilution, 1:300; Jackson ImmunoResearch Laboratories, Inc.) for 24 h at 4 C. Sections were then incubated with fluorescein (dichlorotriazinylaminofluorescein I monohydrochloride)-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.; dilution, 1:500) for 2 h at 4 C, rinsed, and mounted with Citifluor. Negative controls were determined by omission of the primary antibody.

Representative sections from each mouse section containing the arcuate (ARC) nucleus (1.58–1.82 mm posterior to bregma) and the lateral hypothalamic area (LHA; 1.78–1.94 mm) were selected for analysis using a confocal laser scanning microscope (Nikon TE 300, Noran Oz, Nikon Corp., Melville, NY). An area within the ARC nucleus or LHA was selected for counting labeled neurons using Image 4.0 software (Scion, Frederick, MD) under fixed threshold conditions. In total, 12–15 sections from three animals in each group were analyzed.

Statistical analysis
Results are represented as the mean ± SEM. The effects on blood glucose levels, food intake, and body weight were analyzed by repeated measures ANOVA with time as continuous factor and type (experiment 1, ghrelin–/– or ghrelin+/+; experiment 2, diabetic or vehicle) and treatment (experiment 1, diabetic or vehicle; experiment 2, antagonist or saline) as random factors. In case of significant factor effects, tests with contrasts were performed to locate pairs of factor levels with significant differences in the examined variables. The effects on plasma glucagon levels, plasma ghrelin levels, fat pad mass, and neuropeptide expression were analyzed by two-way ANOVA, followed by Tukey post hoc testing. Data were analyzed with Statistica 6.0 (StatSoft, Inc., Tulsa, OK), and significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: studies in ghrelin–/– and ghrelin+/+ mice
Effects on blood glucose and plasma glucagon levels.
In ghrelin+/+ mice, blood glucose levels were already significantly (P < 0.001) increased 24 h after the injection of STZ; in ghrelin–/– mice, the increase was only apparent after 32 h and was steeper than that in wild-type littermates (Fig. 1Go). Blood glucose levels obtained at the end of the study (d 11) were comparably elevated in both genotypes. Also, glucagon levels did not differ between ghrelin+/+ (127 ± 24 pg/ml) and ghrelin–/– mice (161 ± 18 pg/ml).


Figure 1
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  		FIG. 1. Changes in blood glucose levels in vehicle- or STZ-treated, diabetic ghrelin+/+ and ghrelin–/– mice as a function of time. Results are represented as the mean ± SEM (n = 3 animals/group). *, P < 0.05; **, P < 0.001 (STZ-diabetic ghrelin+/+ vs. ghrelin–/– mice).

 
Effects on food intake, body weight, and fat pad mass.
Daily food intake was significantly (P < 0.05) increased in ghrelin+/+ mice 5 d after the induction of diabetes compared with that of vehicle-treated wild-type mice, with a maximal increase of 141% on d 8 (Fig. 2AGo). By contrast, in ghrelin–/– mice, the induction of hyperphagia was delayed. Twenty-four-hour food intake was first decreased between d 2 and 4, normalized between d 5 and 7, and increased from d 8 on, with a maximal increase of 70% on d 9. From d 10 on, daily food intake was comparably increased in both genotypes.


Figure 2
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  		FIG. 2. Daily (24 h) food intake (A), 2-h food intake (from 1000–1200 h; B), and changes in body weight (C) in control (vehicle treated) and STZ-induced, diabetic ghrelin+/+ and ghrelin–/– mice as a function of time. Results are represented as the mean ± SEM (n = 3 animals/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (STZ-treated diabetic ghrelin+/+ vs. ghrelin–/– mice).

 
Two-hour food intake was significantly increased in diabetic wild-type mice from d 3 on, with maximal effects on d 7 compared with vehicle-treated mice (Fig. 2BGo). In mutant mice, a significant increase was only observed from d 8 on. At the end of the observation period, no significant differences in 2-h food intake were observed between the genotypes.

In both genotypes, body weight loss was apparent from d 2 after the induction of diabetes, but from d 4 on, ghrelin–/– mice started to lose more weight than ghrelin+/+ mice (Fig. 2CGo). At the end of the observation period, mutant mice had lost 12.4% more weight than wild-type mice.

Body fat pad mass, which was the sum of inguinal, epididymal, and retroperitoneal fat, disappeared after the induction of diabetes in both ghrelin+/+ and ghrelin–/– mice.

Effects on neuropeptide expression in hypothalamus
NPY expression in ARC nucleus.
In ghrelin+/+ mice, the number of NPY-immunopositive neurons in the ARC nucleus was increased (P < 0.001) from 187 ± 22 in the vehicle-treated group to 936 ± 40 neurons/mm2 in the diabetic group (Fig. 3Go). In diabetic ghrelin–/– mice, the number of NPY-immunopositive neurons also tended to increase from 257 ± 38 to 473 ± 92 neurons/mm2, but this did not reach statistical significance (P = 0.09). However, in diabetic mutant mice, the number of NPY-immunopositive neurons was significantly (P < 0.01) lower than that in diabetic wild-type littermates.


Figure 3
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  		FIG. 3. Effects of STZ-induced diabetes on NPY (A and B) and {alpha}-MSH (C and D) expression in ARC nucleus and on orexin expression (E and F) in LHA of ghrelin+/+ and ghrelin–/– mice. Microphotographs (left) and bar figures (right) show the number of IR neurons per square millimeter on d 11 in vehicle-treated and diabetic, wild-type, and mutant mice. Results are represented as the mean ± SEM (n = 3 animals/group; n = 12–15 sections/animal).

 
{alpha}-MSH expression in ARC nucleus.
In control ghrelin–/– mice the number of {alpha}-MSH-immunopositive neurons per square millimeter (1128 ± 92) was higher (P < 0.05) than that in ghrelin+/+ mice (745 ± 52; Fig. 3Go, C and D). Induction of diabetes decreased {alpha}-MSH expression in both genotypes by 57.3% (ghrelin+/+) and 49.7% (ghrelin–/–).

Orexin expression in LHA.
No significant differences were observed in orexin expression after the induction of diabetes in both genotypes (Fig. 3Go, E and F).

Experiment 2: studies of NMRI mice; effect of D-Lys3-GHRP-6 treatment
Effects on blood glucose and plasma glucagon levels.
NMRI mice were more sensitive to STZ than mice (129Sv/EV x C57BL/6 mixed genetic background) with targeted knockout of the ghrelin gene (ghrelin–/–) and wild-type littermate controls (ghrelin+/+). In NMRI mice, a dose of 140 mg/kg STZ induced comparable increases in blood glucose levels as those in ghrelin+/+ mice. Within 1 d after STZ administration, blood glucose levels were significantly increased compared with those in citric acid (vehicle)-treated control mice (Fig. 4AGo). Treatment with the ghrelin receptor antagonist reduced (P < 0.05) blood glucose levels in diabetic mice, but not in the vehicle-treated control group (Fig. 4AGo).


Figure 4
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  		FIG. 4. Changes in blood glucose levels (A) and plasma glucagon levels (B) in control (vehicle treated) and STZ-induced diabetic mice treated for 5 d with either saline or D-Lys3-GHRP-6 5 d after the induction of diabetes. Plasma glucagon levels were measured on d 10. Results are represented as the mean ± SEM (glucose, n = 6 animals/group; glucagon, n = 3 animals/group). *, P < 0.05; **, P < 0.005 (compared with saline-treated diabetic mice). #, P < 0.01 (compared with saline-treated control mice).

 
At the end of the experiment, plasma glucagon levels were lower (P < 0.05) in the diabetic saline-treated group (199 ± 30 pg/ml) than in the vehicle-treated control group (401 ± 24 pg/ml; Fig. 4BGo). Treatment of diabetic mice with D-Lys3-GHRP-6 normalized plasma glucagon levels. In control mice, treatment with the antagonist did not affect plasma glucagon levels.

Effects on plasma ghrelin levels and ghrelin protein expression
Plasma.
Plasma ghrelin levels were higher in STZ-induced diabetic mice (1266 ± 200 pg/ml) than in vehicle-treated control mice (576 ± 108 pg/ml; P < 0.05; Fig. 5AGo). Treatment with the ghrelin receptor antagonist further increased plasma ghrelin levels to 1240 ± 246 pg/ml in control mice and to 1570 ± 373 pg/ml in diabetic mice.


Figure 5
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  		FIG. 5. Plasma ghrelin levels (A) and ghrelin-IR cells in sections from mouse stomach (B) on d 10 in control (vehicle treated) and STZ-induced diabetic mice treated for 5 d with either saline or D-Lys3-GHRP-6 5 d after the induction of diabetes. Plasma ghrelin levels are represented as the mean ± SEM (n = 6 animals/group). *, P < 0.05 (compared with saline-treated control mice).

 
Stomach.
Immunohistochemical staining for ghrelin was performed on sections of gastric mucosa from control and diabetic mice treated with saline or the ghrelin receptor antagonist (Fig. 5BGo). The number of ghrelin-IR cells in the oxyntic mucosa of diabetic mice was increased (P < 0.05) compared with that in vehicle-treated control mice. Treatment with D-Lys3-GHRP-6 also increased (P < 0.05) the number of ghrelin-IR cells in the gastric mucosa of both control and diabetic mice.

Effects on food intake, body weight, and fat pad mass.
Daily (24 h) food intake and 2-h food intake (from 1000–1200 h) were significantly increased 4 d (P < 0.001) and 2 d (P < 0.01) after the induction of diabetes, respectively (Fig. 6Go). Treatment with the ghrelin receptor antagonist decreased 24-h food intake by 23% after 5 d of treatment (P < 0.05) in diabetic mice, but not in control mice (Fig. 6AGo). However, after the first injection of D-Lys3-GHRP-6, 2-h food intake in diabetic mice was normalized (Fig. 6BGo). Although the antagonist tended to decrease 2-h food intake in the control group, this did not reach significance.


Figure 6
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  		FIG. 6. Daily (24 h) food intake (A), 2-h food intake (from 1000–1200 h; B), and changes in body weight (C) in control (vehicle treated) and STZ-induced diabetic mice treated for 5 d with either saline or D-Lys3-GHRP-6 5 d after the induction of diabetes. Results are represented as the mean ± SEM (n = 6 animals/group). *, P < 0.05; **, P < 0.001; ***, P < 0.0001 (compared with saline-treated diabetic mice).

 
Despite hyperphagia, diabetic mice continued to lose weight. Treatment with the antagonist could not reverse this process (Fig. 6CGo).

Body fat pad mass was significantly (P < 0.0001) decreased from 1.73 ± 0.23 g in vehicle-treated mice to 0.04 ± 0.02 g in diabetic mice. Treatment with D-Lys3-GHRP-6 significantly decreased (P < 0.05) body fat pad mass in control mice to 1.13 ± 0.23 g, but had no effect in diabetic mice (0.06 ± 0.03 g).

Effects on neuropeptide expression in hypothalamus
NPY expression in ARC nucleus.
In diabetic mice, the number of NPY-immunopositive neurons per square millimeter in the ARC nucleus of the hypothalamus was significantly (P < 0.005) increased from 249 ± 7 (vehicle-treated control mice) to 587 ± 9 neurons/mm2 (Fig. 7Go, A and B). After treatment of diabetic mice with D-Lys3-GHRP-6, the number of NPY-positive neurons was normalized to 324 ± 36/mm2. In the vehicle-treated control group, NPY expression was decreased (155 ± 16/mm2) by treatment with the ghrelin receptor antagonist.


Figure 7
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  		FIG. 7. Effects of STZ-induced diabetes and D-Lys3-GHRP-6 treatment on NPY (A and B) and {alpha}-MSH (C and D) expression in the ARC nucleus and on orexin expression (E and F) in the LHA. Microphotographs (left) and bar figures (right) show the number of IR neurons per square millimeter on d 10 in control and diabetic mice treated for 5 d with either saline or D-Lys3-GHRP-6. Results are represented as the mean ± SEM (n = 3 animals/group; n = 12–15 sections/animal). *, P < 0.05; **, P < 0.001 (compared with saline-treated vehicle control mice). #, P < 0.0005 (compared with saline-treated diabetic mice).

 
{alpha}-MSH expression in ARC nucleus.
The number of {alpha}-MSH-immunopositive neurons in the ARC nucleus was decreased 1.7-fold in diabetic mice. This effect was reversed by treatment for 5 d with the ghrelin receptor antagonist (Fig. 7Go, C and D). The number of {alpha}-MSH-immunopositive neurons in the vehicle-treated control group was not significantly increased after treatment with D-Lys3-GHRP-6.

Orexin expression in LHA.
The number of orexin-immunopositive neurons in the LHA of the hypothalamus was not significantly affected by induction of diabetes with STZ or treatment with D-Lys3-GHRP-6 (Fig. 7Go, E and F).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin deficiency induced by the ß-cell toxin STZ provides a highly reproducible rodent model of uncontrolled insulin-deficient diabetes mellitus, characterized by weight loss, hyperglycemia, and sustained hyperphagia (22). Although the pathogenesis of diabetic hyperphagia is incompletely understood, several observations suggest a key role for reduced signaling by insulin and leptin, hormones involved in the regulation of energy balance (23). Indeed, in several models of STZ-induced diabetes, it has been shown that decreased action of leptin/insulin in the arcuate nucleus stimulates the release of the orexigenic peptides NPY and agouti-related peptide and inhibits the release of the anorectic peptide {alpha}-MSH, resulting in increased food intake (24, 25, 26, 27). In the present study, indirect evidence for decreases in plasma insulin and leptin levels was provided by the dramatic changes in fat pad mass.

Leptin and insulin have also been shown to be peripheral regulators of ghrelin secretion, and a reciprocal relationship exists between plasma ghrelin and leptin or insulin levels (8, 10, 14, 28, 29, 30). Vice versa, ghrelin is able to inhibit insulin secretion at the level of pancreatic islets (31, 32). Electrophysiological studies demonstrated that ghrelin activates a subpopulation of neurons in the arcuate nucleus that is inhibited by leptin (33). These findings together with recent reports of elevated plasma ghrelin levels in STZ-induced diabetic rats suggest that increased ghrelin signaling may also contribute to diabetic hyperphagia (15, 34, 35).

In agreement with these studies in diabetic rats, plasma ghrelin levels were significantly increased in the present study in mice with STZ-induced diabetes. The increase in plasma ghrelin levels was associated with an increase in the number of ghrelin-IR cells in oxyntic mucosa. This is in contrast with the observations in rats (34). In this respect it should be noted that the immunohistochemistry study by Masoka et al. (34) was performed 4 wk after the induction of diabetes compared with 10 d after diabetes induction in our study. In the former case, the longer disease process may have depleted ghrelin cells more profoundly. In the present study, plasma ghrelin levels were also increased in vehicle-treated control mice after treatment with the ghrelin receptor antagonist. Because Dass et al. (36) reported very strong GH secretagogue receptor immunoreactivity in cells associated with gastric glands, blockade of ghrelin receptor at the ghrelin cell by ghrelin antagonist may stimulate the number and release of ghrelin from ghrelin-synthesizing cells via an autocrine effect.

To provide conclusive evidence for a role for ghrelin in diabetic hyperphagia, food intake was followed in ghrelin–/– and ghrelin+/+ mice and in NMRI mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GHRP-6, for 5 d. In both ghrelin+/+ mice and NMRI mice, daily food intake was increased within 5 d after the onset of diabetes. In diabetic ghrelin–/– mice, hyperphagia was attenuated until d 8, and maximal increases in food intake were much lower in mutant (70%) than in wild-type (141%) mice. In NMRI mice, hyperphagia was partially reversed by treatment with ghrelin receptor antagonist. The effect of D-Lys3-GHRP-6 on 24-h food intake is consistent with the findings of a previous report in which D-Lys3-GHRP-6 was administered intraventricularly to rats (15). It is still a matter of debate whether ghrelin signals from the periphery to the brain via vagal afferents that contain ghrelin receptors in the nodose ganglion (37) or via direct activation of ghrelin receptors in the ARC nucleus, a region where the blood-brain barrier is incomplete. In the present study we could not discriminate which ghrelin receptors are blocked by peripheral administration of ghrelin antagonist.

Two-hour food intake measured during periods of reduced food intake (between 1000 and 1200 h) was already markedly elevated in diabetic mice within 2 d after the onset of diabetes. This suggests that during diabetic hyperphagia, the active feeding period is no longer limited to the dark period. Our results suggest an important role for ghrelin in the increased food intake observed during the light period, because the hyperphagia between 1000 and 1200 h was drastically reduced in ghrelin–/– mice and by treatment of diabetic NMRI mice with D-Lys3-GHRP-6. A previous study performed in ghrelin–/– mice suggested a role for ghrelin as an endogenous factor dictating the timing of meals (20).

Despite the attenuation of food intake in diabetic ghrelin–/– mice compared with ghrelin+/+ mice, blood glucose levels and glucagon levels remained similar in the two diabetic genotypes, but body weight loss was more pronounced in ghrelin–/– mice (31%) than in ghrelin+/+ mice (19%). This is in contrast to studies in NPY-deficient mice where induction of STZ-induced diabetes further decreased food intake in NPY–/– compared with NPY+/+ mice without enhancing body weight loss (26).

Reduced food intake during D-Lys3-GHRP-6 treatment of NMRI mice also resulted in lower glucose levels, and this was reflected by normalization of plasma glucagon levels, which were initially reduced due to the hyperglycemic state of the animals. These effects were insufficient to reduce body weight loss, which probably requires a more profound normalization of changes in energy balance, including normalization of fat pad mass. Treatment with D-Lys3-GHRP-6 did not restore body fat mass in diabetic mice and even reduced fat pad mass in control mice. This is in line with previous reports on the adipogenic effects of ghrelin (11).

To stimulate food intake, ghrelin activates arcuate NPY/agouti-related peptide neurons by binding to presynaptic terminals of NPY neurons to increase the secretion of NPY, agouti-related peptide, and {gamma}-aminobutyric acid. This altered neuropeptide secretion then modulates the activity of postsynaptic, secondary order neurons in the paraventricular nucleus, dorsomedial nucleus, and LHA (e.g. orexin neurons) to stimulate food intake, whereas activation of proopiomelanocortin neurons by{gamma}-aminobutyric acid inhibits the anorectic melanocortin signaling pathway (6, 18, 19, 38, 39, 40, 41). Therefore, we studied how increased ghrelin signaling in STZ-induced diabetes alters the expression of NPY, {alpha}-MSH, and orexin-IR neurons. The induction of diabetes increased the number of NPY-IR neurons, consistent with previous reports that increased hypothalamic NPY signaling is required for the development of diabetic hyperphagia (26, 42). In ghrelin–/– mice, the induction of diabetes did not increase NPY expression. In addition, treatment of diabetic and vehicle-treated control mice with ghrelin receptor antagonist decreased NPY expression. These results, therefore, indicate that NPY is the downstream mediator of ghrelin in the hyperphagic responses of mice with insulin-deficient diabetes.

Induction of diabetes decreased the expression of the anorectic peptide, {alpha}-MSH, in agreement with the study by Havel et al. (25). Evidence for a role for ghrelin in the regulation of {alpha}-MSH expression was provided by the observation that {alpha}-MSH expression was significantly increased in ghrelin-deficient control mice and that D-Lys3-GHRP-6 treatment of diabetic mice normalized {alpha}-MSH expression. However, in diabetic ghrelin–/– mice, {alpha}-MSH expression was again decreased compared with that in control ghrelin–/– mice, independently of ghrelin. It should be pointed out that changes in the expression of hypothalamic neuropeptides were measured at a point when ghrelin–/– mice again became hyperphagic. It is also possible that due to the redundancy in orexigenic peptides, a compensatory mechanism might have taken over the role of ghrelin in the regulation of {alpha}-MSH expression, but not of NPY, in ghrelin–/– mice. In none of the models studied were changes in orexin expression observed.

In general, changes in diabetic ghrelin–/– mice paralleled changes in diabetic NMRI mice treated with the ghrelin receptor antagonist, D-Lys 3-GHRP-6; this suggests that D-Lys3-GHRP-6 interferes with ghrelin signaling pathways to reduce food intake and does not solely reduce food intake by contraction of the stomach via activation of 5-serotonin-2B receptors.

In conclusion, the attenuation of hyperphagia in ghrelin–/– mice and the reduced food intake during treatment with ghrelin receptor antagonist suggest that ghrelin should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes. Enhanced release of NPY and reduced release of {alpha}-MSH, therefore, are secondary to the release of ghrelin.


   Footnotes
 
This work was supported by grants from the Bilateral Scientific Cooperation Flanders-China (Project 01/13), the Flemish Foundation for Scientific Research (Contract FWO.0144.04), the Belgian Ministry of Science (Contracts GOA 03/11 and IUAP P5/20), and the Fund for Medical Scientific Research Belgium (Grant 3.4510.03). Pieter Vanden Berghe is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek.

J.D., T.L.P., B.D., C.D., P.V., M.T., and I.D. have nothing to declare. D.M. and B.C. are employed by Johnson & Johnson Pharmaceutical Research and Development (Beerse, Belgium).

First Published Online February 16, 2006

Abbreviations: ARC, Arcuate; GHRP-6, GH-releasing peptide-6; IR, immunoreactive; LHA, lateral hypothalamic area; NMRI, Naval Medical Research Institute noninbred Swiss; NPY, neuropeptide Y; STZ, streptozotocin; TFA, trifluoroacetic acid.

Received October 20, 2005.

Accepted for publication February 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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