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Diabetes 56:143-151, 2007
DOI: 10.2337/db05-0996
© 2007 by the American Diabetes Association
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Exendin-4 Potently Decreases Ghrelin Levels in Fasting Rats

Diego Pérez-Tilve1, Lucas González-Matías1, Mayte Alvarez-Crespo1, Roberto Leiras1, Sulay Tovar2, Carlos Diéguez2, and Federico Mallo1

1 Department of Functional Biology and Health Sciences, Faculty of Biology, Laboratory of Endocrinology, University of Vigo, Vigo, Spain
2 Department of Physiology, Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain

Address correspondence and reprint requests to Federico Mallo, PhD, Faculty of Biology, Laboratory of Endocrinology, Campus of Vigo, As Lagoas–Marcosende, University of Vigo, E-36310 Vigo, Spain. E-mail: fmallo{at}uvigo.es

Abbreviations: DPP-IV, dipeptidyl peptidase-IV; Ex, exendin; GLP, glucagon-like peptide; GLP-1r, GLP-1 receptor; PWS, Prader-Willi Syndrome; RIA, radioiummunoassay


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ghrelin is a potent orexigenic and adipogenic hormone that strongly influences fat deposition and the generation of hunger in obesity. Indeed, hyperghrelinemia appears to promote an increase in food intake as seen in Prader-Willi Syndrome (PWS). Exendin (Ex)-4 is an agonist of the glucagon-like peptide (GLP)-1 receptor (GLP-1r) that has anorexigenic and fat-reducing properties. Here, we report that Ex-4 reduces the levels of ghrelin by up to 74% in fasted rats. These effects are dose dependent and long lasting (up to 8 h), and they can be detected after both central and peripheral administration of Ex-4. Suppression of ghrelin was neither mimicked by GLP-1(7–36)-NH2 nor blocked by the GLP-1r antagonist Ex-(9–39). Moreover, it was independent of the levels of leptin and insulin. The decrease in ghrelin levels induced by Ex-4 may explain the reduced food intake in fasted rats, justifying the more potent anorexigenic effects of Ex-4 when compared with GLP-1. As well as the potential benefits of Ex-4 in type 2 diabetes, the potent effects of Ex-4 on ghrelin make it tempting to speculate that Ex-4 could offer a therapeutic option for PWS and other syndromes characterized by substantial amounts of circulating ghrelin.

Ghrelin is a potent orexigenic and adipogenic hormone predominantly produced in stomach. The circulating levels of ghrelin are elevated during fasting (1), and they are reduced after food intake or glucose infusion in rodents and humans and when mice are maintained on a high-fat diet (1–3). Ghrelin administration stimulates food intake and reduces energy expenditure, promoting obesity in rodents (1,4,5). An increase in ghrelin levels appears to be related to hyperphagia in diabetic rats (6), and it has also been reported that ghrelin is significantly elevated in patients with Prader-Willi Syndrome (PWS). PWS is the most common genetic disorder related to human obesity, and it is associated with severe adiposity and increased hunger (7). Thus, ghrelin might play a significant role in the pathogenesis of this highly pernicious, serious, life-shortening, yet still untreatable, disease (7,8).

The levels of circulating plasma ghrelin are controlled by its secretion from the stomach (9). Since the regulation of the stomach depends on both vegetative and endocrine effectors, then it is likely that ghrelin secretion will also be influenced by such factors. One of the earliest endocrine mechanisms activated by food intake is the insulinotropic incretin system (10), which involves the secretion of glucose-dependent insulinotropic peptide, followed immediately by that of glucagon-like peptide (GLP)-1. The insulinotropic effect of GLP-1 is relatively potent in both nondiabetic and type 2 diabetic subjects, increasing the first phase of insulin secretion (11). Indeed, it is accepted that the incretin insulinotropic mechanism influences the functional relationship between food intake and insulin secretion (10).

It also appears that GLP-1 and ghrelin exert certain contrasting effects on gastrointestinal function and the regulation of appetite. Whereas ghrelin increases food intake (1,2), gastric acid secretion and motility (5), and fat deposition (1), GLP-1 reduces food intake (12,13), gastric acid secretion (14), and gastric emptying (15) and displays lipolytic effects (16). An inverse relationship between GLP-1 and ghrelin levels has been demonstrated after a standard glucose tolerance test in humans (17). Furthermore, GLP-1 was shown to reduce the levels of ghrelin paradoxically elevated by vagal stimulation in vitro (18). Together, these findings suggest that GLP-1 or related peptides might be involved in regulating ghrelin.

GLP-1 acts through a membrane receptor of which only one molecular form has been identified to date (GLP-1 receptor [GLP-1r]) (19,20). However, the endogenous active form of the peptide [GLP-1(7–36)-NH2] has a very short half-life in plasma, since it is rapidly inactivated by dipeptidyl peptidase-IV (DPP-IV) (21). Exendins are also capable of binding to and activating the GLP-1r—these being peptides isolated from the venom of the lizard Heloderma Suspectum that are very resistant to enzymatic degradation in plasma. Exendin (Ex)-4 has potent insulinotropic properties in rats and humans (10,22,23), and it has recently been approved as a treatment for human type 2 diabetes (24). Indeed, like GLP-1, Ex-4 reduces food intake, weight gain, and fat deposition in Zucker obese rats (25), and it also decreases energy intake in healthy humans (26).

On the basis of the above, we have selected Ex-4 as a potent, long-lasting, DPP-IV–resistant agonist of the GLP-1r to study the in vivo variation in ghrelin levels in fasted rats and animals fed ad libitum (10,22,23,25,26). As such, we have assessed whether the anorexigenic effects of Ex-4 are at least partially due to its capacity to diminish circulating levels of ghrelin.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal housing.
Adult male Sprague-Dawley rats (250–325 g) were maintained with free access to water and standard feed (A04; Panlab, Barcelona, Spain) with a 12-h light:dark cycle (lights on 800–2000) and controlled room temperature (20–21°C). All experimental procedures were carried out in accordance with the European Union regulations regarding the protection of animals used for experimental purposes (Council Directive CEE 86/609).

Drugs and peptides.
Ex-4, Ex-3, Ex-(9–39), GLP-1(7–36)-NH2, GLP-1(1–37), and sodium pentobarbital were all provided by Sigma-Aldrich (Alcobendas, Spain). GLP-2, Ser8-GLP-1(7–36)-NH2, Ex-(3–39), and Lys-Pyrrolidide (H-Lys[4-nitro-Z]-pyrrolidide-HCl) were obtained from Bachem (Bubendorf, Switzerland).

Intraperitoneal administration.
Rats were maintained in standard conditions before fasting, and animals were handled on a daily basis to avoid stress. On the day of the experiment, rats were intraperitoneally administered the test drug dissolved in sterile 0.9% saline or vehicle alone, and they were returned to their cages. Trunk blood samples were collected after decapitation in tubes cooled on ice. The blood was centrifuged, and the serum obtained was frozen and stored at –20°C until assayed.

Intracerebroventricular administration.
A permanent polyethylene cannula (PE 20; Intramedic) was stereotaxically implanted into the lateral ventricle of rats anesthetized with sodium pentobarbital (50 mg/kg i.p.) using the coordinates established by Paxinos and Watson (27). After surgery, the rats were left to recover for at least 7 days in individual cages and were handled daily to avoid stress. On the day of the experiment, the treatments were intracerebroventricularly administered to the rats in a volume of 5 µl [Ex-4 (0.5, 1, or 5 µg); GLP-1(7–36)-NH2 (0.1, 1, or 10 µg); GLP-1(1–37) (10 µg)] or vehicle, and trunk blood was collected 1 or 2 h later. The correct placement of the cannula was confirmed by postmortem injection of 10 µl Trypan blue (0.5%) (Sigma-Aldrich), and only rats with clear staining of the third ventricle were included in the study.

Freely moving experimental protocol.
One week before the experiment, male Sprague-Dawley rats (275–325 g) were implanted with an intracerebroventricular cannula and a permanent silastic indwelling atrial cannula introduced through the right jugular vein under pentobarbital anesthesia. Rats were allowed to recover for 3 days in individual cages, and they were acclimatized to the experimental conditions in metabolic cages for a further 4 days. All animals included in the study recovered their body weight to the values before surgery, and their 24-h accumulated food intake was at least 24 g (means ± SE 25.5 ± 1.5 g). From the 8th day after implantation, the cannula rats were fasted for 48 h. The peptides assayed were administered through the intracerebroventricular cannula as follows: Ex-4 (0.5 µg/rat), GLP-1 (10 µg/rat), or saline in a volume of 5 µl. Thirty minutes later, a blood sample (300 µl) was obtained immediately after the injection of a heparin bolus (0.3 ml, 1,000 IU/ml) (Rovi, Madrid, Spain) through an extension tube attached to the free end of the venous cannula. Then, rats were given access to pulverized standard feed (time 0), and additional blood samples were obtained at 30 min and at 1, 2, 4, 8, and 24 h, when the volume extracted was replaced with saline. Partial food intake was measured at the time of blood sampling. The accumulated food intake for each interval was presented, as well as the food intake per 100 g body wt, and over each interval (minute) was considered as the weighted food intake.

Hormone measurements.
Total ghrelin was measured using a specific radioiummunoassay (RIA) kit following the manufacturer’s instructions (Phoenix Pharmaceuticals). In our hands, the intrassay coefficents of variation (CVs) were 2.5% (8 pg/tube), 2.0% (32 pg/tube), and 4.5% (128 pg/tube), whereas the interassay CVs were 7.2, 4.3, and 6.2, respectively. Leptin levels were determined using a specific rat RIA (Linco Research) with intrassay CVs of 2.4% (1.6 ng/ml) and 4.8% (11.6 ng/ml), and interassay CVs of 4.6 and 5.7%, respectively. Insulin levels were also measured with a sensitive RIA assay (DRG Systems, Marburg, Germany), with intrassay CVs of 2.3% (0.2 ng/ml) and 3.9% (1 ng/ml) and interassay CVs of 4.7 and 5.9%, respectively. C-peptide levels were determined using a specific rat RIA (DRG Systems), presenting intrassay CVs of 0.8% (200 pmol/l) and 1.9% (800 pmol/l) and interassay CVs of 1 and 2.1%, respectively.

Statistical analysis.
The data are presented as the means ± SE in all graphs. Student’s t test for independent samples was used throughout for comparison between the two groups. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between multiple independent groups.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ex-4 (5 µg/kg) significantly reduced the levels of ghrelin in unrestrained, nonanesthetized animals 30 min after a single intraperitoneal injection compared with control rats administered saline (Fig. 1). Indeed, in fasted animals, higher doses of Ex-4 (20 µg/kg i.p.) produced a 72% reduction in ghrelin levels 2 and 4 h postinjection, and since ghrelin levels were still significantly reduced after 8 h, the effect of Ex-4 appears to be potent and to have a long-lasting effect (Fig. 2A). Fasted animals displayed low levels of leptin that remained unchanged after Ex-4 administration (Fig. 2B). Ex-4 (5 µg/kg i.p.) reduced ghrelin levels in animals fasted for 12–72 h but not in rats fed ad libitum (Fig. 2C). As expected, leptin levels diminished rapidly after the onset of fasting and they fell 88% after 12 h. Similarly, leptin levels were unaffected by Ex-4 administration at any time point examined (Fig. 2D).


Figure 1
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  		FIG. 1. Ex-4 (5 µg/kg i.p.) reduces the levels of ghrelin in fasted rats (72 h). Data are means ± SE (n = 5–8 rats per group). **P < 0.01. {square}, saline; {blacksquare}, Ex-4

 

Figure 2
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  		FIG. 2. Ex-4 has a long-lasting effect on the reduction of ghrelin in fasted rats (72 h). A: Ex-4 (20 µg/kg i.p.) reduces the levels of ghrelin at 2, 4, and 8 h (n = 8 per group). B: The levels of leptin in fasted rats are not affected by administration of Ex-4. C: Ex-4 (5 µg/kg i.p.) reduces ghrelin in rats fasted for 12–72 h but not rats fed ad libitum (0 h) after 2 h (n = 6 per group). D: Ex-4 did not modify leptin accumulation after fasting. Data are means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, treatment vs. saline. ###P < 0.001, 0 h vs. 72 h. blk12, Fed saline; {square}, saline; {blacksquare}, Ex-4

 
The intraperitoneal administration of Ex-4 diminished the levels of ghrelin in fasted animals in a dose-dependent manner (Fig. 3A), with maximal inhibition at 5 µg/kg. Ex-4 reduced ghrelin levels by up to 74% with respect to saline-administered fasted rats, which represents 38% below that of the ghrelin found in rats fed ad libitum. In contrast, the inhibitory effect of Ex-4 was not reproduced by other members of the GLP-1 family, such as the endogenous insulinotropic active peptide GLP-1(7–36)-NH2, even at higher peptide doses (20 µg/kg) (Fig. 3B). Likewise, GLP-2, another active anorexigenic product of the proglucagon peptide also failed to mimic the effect of Ex-4 (28), as did the inactive GLP-1 (1–37) assayed here as a negative control (11,29) . These peptides may be rapidly inactivated by the enzyme DPP-IV, especially the native GLP-1(7–36)-NH2, which has a very short half-life in plasma (<2 min) (21). However, no effects of GLP-1(7–36)-NH2 (20 µg/kg i.p.) on ghrelin levels were observed over shorter periods of time (5, 15, 30, and 60 min) (Fig. 3C).


Figure 3
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  		FIG. 3. The effects of GLP-1 family members on ghrelin. A: Dose-response curve of ghrelin to intraperitoneal administration of Ex-4 (2 h, n = 6). B: Response of ghrelin to GLP-1(7–36)-NH2 (n = 6), GLP-1(1–37) (n = 5), GLP-2 (n = 7), and Ex-4 (n = 6) vs. saline (n = 11) (2 h i.p.). C: GLP-1(7–36)-NH2 (20 µg/kg) did not modify the levels of ghrelin at any time between 5 and 60 min (n = 6). D: The intracerebroventricular administration of Ex-4 causes a dose-dependent reduction of ghrelin (1 h, n = 6). E: The ghrelin responses 1 h after intracerebroventricular administration of GLP-1(7–36)-NH2 (n = 7), GLP-1(1–37) (n = 7), and Ex-4 (n = 13) or saline (n = 12). F: Ghrelin dose-response curve 1 h after intracerebroventricular administration of GLP-1(7–36)-NH2 (n = 8–10 per treatment; n = 18 with saline). Data are means ± SE. ***P < 0.001, treatment vs. saline.

 
Fasting levels of ghrelin were also inhibited in a dose-dependent manner by intracerebroventricularly administered Ex-4 (Fig. 3D). Interestingly, GLP-1(7–36)-NH2 at doses previously (0.1, 1, and 10 µg) (12,13) and here (10 µg) effectively reduced food intake and did not affect the ghrelin levels in fasting rats when intracerebroventricularly given (Figs. 3E and F).

Ex-(9–39) (100 µg/kg i.p.) is a specific antagonist of the GLP-1r (30) that completely blocked the increase in insulin induced by Ex-4 (Fig. 4A) but not the Ex-4–induced decrease in ghrelin (Fig. 4B). Indeed, Ex-(9–39) itself significantly reduced the fasting levels of ghrelin (>25%). In addition, the inactivation of DPP-IV by administration of the long-term blocker Lys-Pyrrolidide (31) markedly increased the circulating levels of C-peptide by preventing the enzymatic degradation of the endogenous GLP-1 (Fig. 4C). In contrast, inactivating DPP-IV did not modify ghrelin levels over the period studied (0, 30, and 60 min) (Fig. 4D). Moreover, the GLP-1 analog Ser8-GLP-1(7–36)-NH2, which has a half-life of ~1 h in body fluids (32), failed to reduce ghrelin levels when intraperitoneally administered. However, Ex-3, which differs from Ex-4 in two amino acids being one of those a serine at position two of the peptide, displayed a similar profile of ghrelin inhibition as Ex-4 (Fig. 4E). Interestingly, the Ex-4 fragment that lacks the first two amino acids [Ex-(3–39)], failed to inhibit ghrelin, indicating that those amino acids are important for Ex-4 to influence circulating ghrelin levels (Fig. 4E).


Figure 4
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  		FIG. 4. The influence of different modulators of the response to Ex-4. A: Ex-(9–39) blocked the Ex-4–induced increase in insulin. B: Ex-(9–39) (100 µg/kg) administered i.p. 30 min before Ex-4 (5 µg/kg i.p.; 1 h; n = 6 per group) did not block the decrease in ghrelin levels induced by Ex-4. C: Lys-Pyrrolidide gave rise to an increase in the levels of C-peptide. D: The intraperitoneal administration of Lys-Pyrrolidide (<20 min, 40 mg/kg), did not modify ghrelin levels (n = 6–7 per group). E: Time-response curve of ghrelin levels following intraperitoneal administration of Ex-3 and Ex-4 (5 µg/kg, n = 6 per group), Ser8-GLP-1(7–36)-NH2 (20 µg/kg), and Ex-(3–39) (50 µg/kg). *P < 0.05, treatment vs. saline). Data are means ± SE, n = 6 per group; by ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, treatment vs. saline. #P < 0.05, Ex-(9–39) + Ex-4 vs. saline + Ex-4.

 
We also studied whether the decrease in ghrelin following intracerebroventricular administration of Ex-4–modified food intake in freely moving rats (Fig. 5). The elevated levels of ghrelin provoked by fasting over the previous 24 h progressively diminished on the initiation of feeding, reaching a minimum after 4 h in saline-treated rats (Fig. 5C). Administration of Ex-4 (0.5 µg i.c.v.) 30 min before allowing the rats free access to food almost completely blocked the food intake for 8 h in fasted rats, with a marked reduction still detected at 24 h (Fig. 5A). The weighted food intake values were also reduced by 95% at 30 min and >80% at 1 h, and they remained very low for up to 8 h after Ex-4 administration (Fig. 5B). It is important to emphasize that the kinetics of ghrelin circulating levels in the Ex-4–treated rats after intracerebroventricular administration were very similar to those described following intraperitoneal administration. The levels of ghrelin start to fall after 30 min, and they remain very low for at least 8 h, correlating with the marked reduction in food intake (Fig. 5C). Accordingly, insulin levels also remained low for up to 8 h after Ex-4 administration (Fig. 5D).


Figure 5
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  		FIG. 5. Effects of intracerebroventricular administration of Ex-4 and GLP-1(7–36)-NH2 on food intake, ghrelin, and insulin in freely moving rats fasted for 48 h (275–325 g). A: Ex-4 reduces food intake over the entire 24 h, whereas GLP-1(7–36)-NH2 reduces food intake over 4 h. B: The weighted food intake is shown. C: Ex-4 reduced ghrelin levels from 0 to 8 h and ghrelin reduction according to the food intake in saline–and GLP-1–treated rats. D: Insulin levels were elevated after 2 h in saline-treated rats and while they rose from 1 to 8 h in GLP-1–treated rats, they remained unchanged in Ex-4–treated rats. Data are means ± SE* = P < 0.05, treatment vs. saline; #P < 0.05, Ex-4 vs. GLP-1. {dagger}P < 0.05, treatment vs. time 0. Saline (5 µl, n = 7), Ex-4 (0.5 µg/5 µl, n = 5), GLP-1(7–36)-NH2 (10 µg/5 µl, n = 5) administered 30 min before time 0.

 
In similar experiments, GLP-1 (10 µg) was much less effective, and it reduced the accumulated food intake by 58% at 30 min, 63% at 1 h, 64% at 2 h, and 62% at 4 h. Thereafter, GLP-1–treated rats showed a catch-up phenomenon to produce a similar food intake to controls over the entire 24-h period (Fig. 5A). GLP-1(7–36)-NH2 also reduced weighted food intake values after 30 min (50%) and 1 h (60%) (Fig. 5B), and this was accompanied by a similar reduction in ghrelin levels to that found in saline-treated rats over this period (Fig. 5C). Insulin levels progressively increased from the 1st h onwards, apparently reflecting the food intake (Fig. 5D).

Finally, we found that, while still very potent, Ex-4 (5 µg/kg) was less effective at reducing food intake in fasted rats when intraperitoneally administered, although it still reduced ghrelin levels (Fig. 6). Again, GLP-1(7–36)-NH2 (50 µg/kg i.p.) did not reduce ghrelin levels at the beginning of the experiment and accordingly did not modify food intake. Furthermore, prior administration of Ex-(9–39) (100 µg/kg i.p.) neither blocked the effect of Ex-4 on ghrelin levels nor modified the reduction in food intake (Fig. 6).


Figure 6
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  		FIG. 6. Effects of Ex-4 vs. GLP-1(7–36)-NH2 on food intake and ghrelin levels after intraperitoneal administration 2 h before time 0 in rats (275–325 g) fasted for 48 h.A: Whereas Ex-4 reduced food intake in these animals, GLP-1(7–36)-NH2 did not. Morover, the effects of Ex-4 were not blocked by Ex-(9–39). B: Weighted food intake. C: In contrast to GLP-1(7–36)-NH2, Ex-4 reduced ghrelin levels at time 0, an effect that was not modified by Ex-(9–39). Data are means ± SE by ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, treatment vs. saline; #P < 0.05, Ex-4 vs. GLP-1(7–36)-NH2. Saline (n = 10), and Ex-4 (5 µg/kg, n = 6), GLP-1(7–36)-NH2 (50 µg/kg, n = 6), Ex-9–39 (100 µg/kg, n = 5).

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified the first compound that strongly diminishes circulating levels of ghrelin. Here we provide evidence that Ex-4, a peptide postulated as an agonist of the GLP-1r (19,28,36), reduces the circulating levels of ghrelin for long periods in fasted rats in a dose-dependent manner. It is noteworthy that a single intraperitoneal injection of Ex-4 reduces ghrelin levels for at least 8 h. Indeed, a reduction of 74% of the control fasting levels of ghrelin was observed, and this reduced level was 38% below the ghrelin levels in rats fed ad libitum. In this respect, no other signal or hormone has been reported to be as potent as Ex-4. However, the effects of Ex-4 on ghrelin are restricted to the fasting state, since it does not significantly reduce the concentration of ghrelin in rats feeding ad libitum.

It is thought that the reduction in food intake induced by Ex-4 may be due to its anorexigenic effects exerted over the hypothalamus (12,13,33). On the other hand, leptin appears to interfere with the generation of appetite-antagonizing ghrelin effects in the hypothalamus (34) and to reduce ghrelin levels in the rat (35). Therefore, leptin might influence the effects of Ex-4 on ghrelin inhibition. However, central administration of Ex-4 reduces food intake in both lean and obese Zucker rats, suggesting that this effect is not dependent on the circulating levels of leptin (36). Accordingly, we also found a decrease in leptin levels following fasting that was unaffected by Ex-4 administration. Since Ex-4 strongly reduced circulating ghrelin levels, it may be inferred that the effects of Ex-4 on ghrelin are independent of the changes in leptin and that the anorexigenic effects of Ex-4 could be due to its ability to reduce the levels of the orexigenic hormone ghrelin.

The effects of Ex-4 on ghrelin are evident after both peripheral (intraperitoneal) and central (intracerebroventricular) administration. Ex-4 can cross the blood-brain barrier in a manner that is not affected by fasting but that is limited by dose (37). Thus, Ex-4 activity might be explained by a single central mechanism that would also be activated by peripheral administration if sufficient Ex-4 crosses the blood-brain barrier (37). While this hypothesis seems reasonable at present, additional studies will be necessary to ascertain whether this is in fact the case.

We also analyzed the effects of other peptides of the GLP-1 family, such as GLP-1(7–36)-NH2, the endogenous GLP-1r agonist, or GLP-2. Intriguingly, none of these were able to reduce circulating ghrelin levels at any time point (minutes to hours) irrespective of the route of administration. These peptides had essentially the same effects as saline or the inactive GLP-1 analog GLP-1(1–37) used as a negative control (11,35). It could be argued that the short half-life of GLP-1(7–36)-NH2 might justify this lack of activity; however, no responses were observed even after 5 or 15 min. Moreover intracerebroventricular administration appears to augment GLP-1 anorexigenic activity precluding the degradation of GLP-1 (12,13,33). Whereas we have used doses that are effective in reducing food intake both here (Fig. 5) and in other similar studies (12,13), no significant change in ghrelin levels was produced by administration of GLP-1. The Ser8-GLP-1(7–36)-NH2 analog that has a much longer half-life than the native peptide also failed to affect ghrelin. In contrast Ex-3, which differs in two amino acids with respect to Ex-4 [being one of those the same serine as Ser8-GLP-1(7–36)-NH2, also strongly diminished ghrelin. Moreover, increasing the endogenous GLP-1 activity by blocking DPP-IV with Lys-Pyrrolidide did not produce a significant change in ghrelin levels. Furthermore, a specific antagonist of the GLP-1r, Ex-(9–39), did not block the inhibitory effect of Ex-4 on ghrelin levels when used in the pharmacologically active range; rather, it had a partial mimetic effect reducing ghrelin levels by >25%. This could not be explained by a loss of Ex-(9–39) activity, as it completely blocked the Ex-4–induced increase of insulin in the same experiment, showing that it effectively blocked GLP-1r–mediated effects (30). Together, these data suggest that ghrelin inhibition appears to correspond to an Exendin-specific mechanism, which might be independent of GLP-1r activation.

In recent years, several studies have provided evidence that the endocrine effects of Ex-4 cannot be adequately explained by activation of the GLP-1r. Indeed, Ex-4 but not GLP-1 increases insulin sensitivity and glucose transport in cultured L6 myotubules in vitro (38), and the effects of Ex-4 and Ex-(9–39) on cAMP levels in 3T3-L1 adipocytes are not mediated by the GLP-1r (39). Significantly, GLP-1 activation was unable to modify energy expenditure (measured as Vo2) or the respiratory exchange ratio in mice (40), whereas Ex-4 clearly reduced both these parameters. Moreover, other endocrine effects elicited by Ex-4 are not reproduced by GLP-1, such as the reduction of thyrotropin secretion in rats (41). Hence, we can infer that Ex-4 exerts some of its effects through a mechanism that does not involve the activation of the GLP-1r. Our data also suggest that there may be a specific pathway through which Ex-4 acts, although whether exendins may have their own specific independent receptor remains to be seen. In this sense, it would be interesting to repeat some of these studies in the GLP-1r knockout mice, although the basic phenotype of this model does not differ greatly from that of the wild-type littermates (42), and unexpectedly, these mice do not become diabetic (43). Apparently, these mice compensate for the lack of GLP-1 with an increase in the levels of the complementary incretin glucose-dependent insulinotropic peptide (44).

Although ghrelin appears to exert important biological effects in a variety of tissues, its physiological role in health and disease is still far from clear. Deletion of the ghrelin gene in mice does not impair either growth or appetite (45), although it has been shown that the constitutive absence of ghrelin prevents the obesity induced by a high-fat diet (46). Furthermore, transgenic rats expressing an antisense growth hormone secretagogue receptor mRNA that selectively attenuates growth hormone secretagogue receptor protein expression in the arcuate nucleus display a lower body weight and less adipose tissue than control rats (47). Furthermore, obestatin, a peptide generated from the ghrelin gene, inhibits food intake in normal mice, apparently by binding to the orphan receptor gpr39 (48). Therefore, the information generated by the ghrelin knockout mice might be limited by the fact that several peptides can be derived from the ghrelin-gene. Given that these may have different and even opposing biological effects, their simultaneous inactivation may make it difficult to determine the influence of ghrelin on energy balance, food intake, and body weight.

We show here that a single intracerebroventricular injection of Ex-4 strongly reduces ghrelin levels and food intake for at least 8 h without any significant changes in the levels of insulin (Fig. 5). The plasma ghrelin profile in response to intracerebroventricularly administered Ex-4 in fasted freely moving rats appears to be similar to that after Ex-4 intraperitoneal administration. Interestingly, the effects of Ex-4 on ghrelin and food intake were closely correlated following intracerebroventricular or intraperitoneal administration of Ex-4. The effects of Ex-4 on ghrelin levels seem to be exerted by a mechanism not mimicked by GLP-1 or blocked by the prior administration of Ex-(9–39). However, the influence of GLP-1r on this aspect of Ex-4 activity cannot be excluded. Although less potent, GLP-1(7–36)-NH2 (intracerebroventricular) diminished the accumulated food intake despite the fact that it does not alter ghrelin levels. These data indicate that the activation of the GLP-1r has its own role in regulating food intake, which might be independent of the variation in ghrelin expression. In fact, we believe that activation of the GLP-1r might be partially responsible for some of the effects exerted by Ex-4 on food intake.

These results could be particularly relevant to the pathophysiology of certain types of obesity, such as the PWS. Indeed, it has been established that the levels of circulating ghrelin are strongly elevated in the plasma of patients with PWS due to an increase in ghrelin-producing cells in the stomach (7,8,49). Thus, it has been proposed that ghrelin might play a significant or indeed crucial role in the pathogenesis of this still incurable disease (8). The only rodent model for PWS currently available does not survive beyond the neonatal stage due to a failure to thrive (50). We have tested the effect of Ex-4 in extensively fasted (48 and 72 h) rats with intense hyperghrelinemia, a model representative of this pathology. Ex-4 strongly inhibits ghrelin, and it may therefore be a candidate drug for the treatment of PWS or even for obesity in general. Since Ex-4 has recently been approved for the treatment of type 2 diabetes, this information is of immediate clinical relevance and utility. The assessment of ghrelin levels in human subjects treated with Ex-4 are eagerly awaited.

In conclusion, Ex-4 reduces fasting levels of ghrelin for long periods in a dose-dependent manner, both at peripheral and central sites. Ex-4 is the most potent peptide to exert this effect to date. The Ex-4–induced reduction in ghrelin levels is sufficiently potent as to open new avenues in the study of the physiological regulation of ghrelin, as well as to clarify the pathophysiological role of ghrelin in the hyperphagia of type 2 diabetes and obesity. This effect may be especially relevant for the treatment of PWS, which to date is characterized by an absence of medical treatment other than bariatric surgery (7).


   ACKNOWLEDGMENTS
 
This work was carried out with the financial support of the European Union (LSHM-CT-2003-503041 to C.D.) and (MERG-CT-2004-006383 to F.M.), and Xunta de Galicia (PGIDT-01-PXI-30118PR to F.M.).

The authors wish to thank Manuel Gil, Chus Valcarce, and Stela Alvarez for their technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication August 4, 2005 and accepted in revised form September 28, 2006


   REFERENCES
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tschop M, Smiley DL, Heiman ML: Ghrelin induces adiposity in rodents. Nature 407:908–913, 2000[Medline]
  2. Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, Purnell JQ: Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 346:1623–1630, 2002[Abstract/Free Full Text]
  3. Moesgaard SG, Ahren B, Carr RD, Gram DX, Brand CL, Sundler F: Effects of high-fat feeding and fasting on ghrelin expression in the mouse stomach. Regul Pept 120:261–267, 2004[Medline]
  4. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, Bloom SR: The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 141:4325–4328, 2000[Abstract/Free Full Text]
  5. Gualillo O, Lago F, Gomez-Reino J, Casanueva FF, Dieguez C: Ghrelin, a widespread hormone: insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 552:105–109, 2003[Medline]
  6. Ishii S, Kamegai J, Tamura H, Shimizu T, Sugihara H, Oikawa S: Role of ghrelin in streptozotocin-induced diabetic hyperphagia. Endocrinology 143:4934–4937, 2002[Abstract]
  7. Cummings DE, Clement K, Purnell JQ, Vaisse C, Foster KE, Frayo RS, Schwartz MW, Basdevant A, Weigle DS: Elevated plasma ghrelin levels in Prader Willi syndrome. Nat Med 8:643–644, 2002[Medline]
  8. DelParigi A, Tschop M, Heiman ML, Salbe AD, Vozarova B, Sell SM, Bunt JC, Tataranni PA: High circulating ghrelin: a potential cause for hyperphagia and obesity in prader-willi syndrome. J Clin Endocrinol Metab 87:5461–5464, 2002[Abstract/Free Full Text]
  9. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660, 1999[Medline]
  10. Drucker DJ: Enhancing incretin action for the treatment of type 2 diabetes. Diabetes Care 26:2929–2940, 2003[Abstract/Free Full Text]
  11. Quddusi S, Vahl TP, Hanson K, Prigeon RL, D’Alessio DA: Differential effects of acute and extended infusions of glucagon-like peptide-1 on first- and second-phase insulin secretion in diabetic and nondiabetic humans. Diabetes Care 26:791–798, 2003[Abstract/Free Full Text]
  12. Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CM, Meeran K, Choi SJ, Taylor GM, Heath MM, Lambert PD, Wilding JP, Smith DM, Ghatei MA, Herbert J, Bloom SR: A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 379:69–72, 1996[Medline]
  13. Navarro,M, Rodriquez de FF, Alvarez E, Chowen JA, Zueco JA, Gomez R, Eng J, Blazquez, E: Colocalization of glucagon-like peptide-1 (GLP-1) receptors, glucose transporter GLUT-2, and glucokinase mRNAs in rat hypothalamic cells: evidence for a role of GLP-1 receptor agonists as an inhibitory signal for food and water intake. J Neurochem 67:1982–1991, 1996[Medline]
  14. Wettergren A, Petersen H, Orskov C, Christiansen J, Sheikh SP, Holst JJ: Glucagon-like peptide-1 7–36 amide and peptide YY from the L-cell of the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man. Scand J Gastroenterol 29:501–505, 1994[Medline]
  15. ImeryuzN, Yegen BC, Bozkurt A, Coskun T, Villanueva-Penacarrillo ML, Ulusoy NB: Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms. Am J Physiol 273:G920–G927, 1997[Medline]
  16. Villanueva-Penacarrillo ML, Marquez L, Gonzalez N, az-Miguel M, Valverde I: Effect of GLP-1 on lipid metabolism in human adipocytes. Horm Metab Res 33:73–77, 2001[Medline]
  17. Djurhuus CB, Hansen TK, Gravholt C, Orskov L, Hosoda H, Kangawa K, Jorgensen JO, Holst JJ, Schmitz O: Circulating levels of ghrelin and GLP-1 are inversely related during glucose ingestion. Horm Metab Res 34:411–413, 2002[Medline]
  18. Lippl F, Kircher F, Erdmann J, Allescher HD, Schusdziarra V: Effect of GIP, GLP-1, insulin and gastrin on ghrelin release in the isolated rat stomach. Regul Pept 119:93–98, 2004[Medline]
  19. Thorens B: Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A 89:8641–8645, 1992[Abstract/Free Full Text]
  20. Bullock BP, Heller RS, Habener JF: Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor. Endocrinology 137:2968–2978, 1996[Abstract]
  21. Mentlein R: Dipeptidyl-peptidase IV (CD26)–role in the inactivation of regulatory peptides. Regul Pept 85:9–24, 1999[Medline]
  22. Parkes DG, Pittner R, Jodka C, Smith P, Young A: Insulinotropic actions of exendin-4 and glucagon-like peptide-1 in vivo and in vitro. Metabolism 50:583–589, 2001[Medline]
  23. Egan JM, Clocquet AR, Elahi D: The insulinotropic effect of acute exendin-4 administered to humans: comparison of nondiabetic state to type 2 diabetes. J Clin Endocrinol Metab 87:1282–1290, 2002[Abstract/Free Full Text]
  24. Doggrell SA: Is exenatide advancing the treatment of type 2 diabetes? Expert Opin Pharmacother 7:109–112, 2006[Medline]
  25. Szayna M, Doyle ME, Betkey JA, Holloway HW, Spencer RG, Greig NH, Egan JM: Exendin-4 decelerates food intake, weight gain, and fat deposition in Zucker rats. Endocrinology 141:1936–1941, 2000[Abstract/Free Full Text]
  26. Edwards CM, Stanley SA, Davis R, Brynes AE, Frost GS, Seal LJ, Ghatei MA, Bloom SR: Exendin-4 reduces fasting and postprandial glucose and decreases energy intake in healthy volunteers. Am J Physiol Endocrinol Metab 281:E155–E161, 2001[Abstract/Free Full Text]
  27. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates. San Diego, CA, Academic Press, 1998, p.22
  28. Tang-Christensen M, Larsen PJ, Thulesen J, Romer J, Vrang N: The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake. Nat Med 6:802–807, 2000[Medline]
  29. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC: Exon duplication and divergence in the human preproglucagon gene. Nature 304:368–371, 1983[Medline]
  30. Goke R, Fehmann HC, Linn T, Schmidt H, Krause M, Eng J, Goke B: Exendin-4 is a high potency agonist and truncated exendin-(9–39)-amide an antagonist at the glucagon-like peptide 1-(7–36)-amide receptor of insulin-secreting beta-cells. J Biol Chem 268:19650–19655, 1993[Abstract/Free Full Text]
  31. Steinbrecher A, Reinhold D, Quigley L, Gado A, Tresser N, Izikson L, Born I, Faust J, Neubert K, Martin R, Ansorge S, Brocke S: Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo. J Immunol 166:2041–2048, 2001[Abstract/Free Full Text]
  32. Hinke SA, Manhart S, Kuhn-Wache K, Nian C, Demuth HU, Pederson RA, McIntosh CH: [Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo. J Biol Chem 279:3998–4006, 2004[Abstract/Free Full Text]
  33. Goke R, Larsen PJ, Mikkelsen JD, Sheikh SP: Distribution of GLP-1 binding sites in the rat brain: evidence that exendin-4 is a ligand of brain GLP-1 binding sites. Eur J Neurosci 7:2294–2300, 1995[Medline]
  34. Ueno N, Dube MG, Inui A, Kalra PS, Kalra SP: Leptin modulates orexigenic effects of ghrelin and attenuates adiponectin and insulin levels and selectively the dark-phase feeding as revealed by central leptin gene therapy. Endocrinology 145:4176–4184, 2004[Abstract/Free Full Text]
  35. Barazzoni R, Zanetti M, Stebel M, Biolo G, Cattin L, Guarnieri G: Hyperleptinemia prevents increased plasma ghrelin concentration during short-term moderate caloric restriction in rats. Gastroenterology 124:1188–1192, 2003[Medline]
  36. Al-Barazanji KA, Arch JR, Buckingham RE, Tadayyon M: Central exendin-4 infusion reduces body weight without altering plasma leptin in (fa/fa) Zucker rats. Obes Res 8:317–323, 2000[Medline]
  37. Kastin AJ, Akerstrom V: Entry of exendin-4 into brain is rapid but may be limited at high doses. Int J Obes Relat Metab Disord 27:313–318, 2003[Medline]
  38. Yang H, Egan JM, Wang Y, Moyes CD, Roth J, Montrose MH, Montrose-Rafizadeh C: GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor. Am J Physiol 275:C675–C683, 1998[Medline]
  39. Montrose-Rafizadeh C, Yang H, Wang Y, Roth J, Montrose MH, Adams LG: Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3–L1 adipocytes. J Cell Physiol 172:275–283, 1997[Medline]
  40. Baggio LL, Huang Q, Brown TJ, Drucker DJ: Oxyntomodulin and glucagon-like peptide-1 differentially regulate murine food intake and energy expenditure. Gastroenterology 127:546–558, 2004[Medline]
  41. Malendowicz LK, Nowak KW: Preproglucagon derived peptides and thyrotropin (TSH) secretion in the rat: robust and sustained lowering of blood TSH levels in exendin-4 injected animals. Int J Mol Med 10:327–331, 2002[Medline]
  42. Scrocchi LA, Brown TJ, MaClusky N, Brubaker PL, Auerbach AB, Joyner AL, Drucker DJ: Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene. Nat Med 2:1254–1258, 1996[Medline]
  43. Preitner F, Ibberson M, Franklin I, Binnert C, Pende M, Gjinovci A, Hansotia T, Drucker DJ, Wollheim C, Burcelin R, Thorens B: Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors. J Clin Invest 113:635–645, 2004[Medline]
  44. Pederson RA, Satkunarajah M, McIntosh CH, Scrocchi LA, Flamez D, Schuit F, Drucker DJ, Wheeler MB: Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor –/– mice. Diabetes 47:1046–1052, 1998[Abstract]
  45. Sun Y, Ahmed S, Smith RG: Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 23:7973–7981, 2003[Abstract/Free Full Text]
  46. Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M Thabet K, Cox HJ, Yancopoulos GD, Wiegand SJ, Sleeman MW: Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl Acad Sci U S A 101:8227–8232, 2004[Abstract/Free Full Text]
  47. Shuto Y, Shibasaki T, Otagiri A, Kuriyama H, Ohata H, Tamura H, Kamegai J, Sugihara H, Oikawa S, Wakabayashi I: Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity. J Clin Invest 109:1429–1436, 2002[Medline]
  48. Zhang JV, Ren PG, Avsian-Kretchmer O, Luo CW, Rauch R, Klein C, Hsueh AJ: Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin’s effects on food intake. Science 310:996–999, 2005[Abstract/Free Full Text]
  49. Choe YH, Song SY, Paik KH, Oh YJ, Chu SH, Yeo SH, Kwon EK, Kim EM, Rha MY, Jin DK: Increased density of ghrelin-expressing cells in the gastric fundus and body in Prader-Willi syndrome. J Clin Endocrinol Metab 90:5441–5445, 2005[Abstract/Free Full Text]
  50. Gabriel JM, Merchant M, Ohta T, Ji Y, Caldwell RG, Ramsey MJ, Tucker JD, Longnecker R, Nicholls RD: A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes. Proc Natl Acad Sci U S A 96:9258–9263, 1999[Abstract/Free Full Text]

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