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Endocrinology Vol. 143, No. 9 3341-3350
Copyright © 2002 by The Endocrine Society

ARTICLE

Delayed Short-Term Secretory Regulation of Ghrelin in Obese Animals: Evidenced by a Specific RIA for the Active Form of Ghrelin

Hiroyuki Ariyasu, Kazuhiko Takaya, Hiroshi Hosoda, Hiroshi Iwakura, Ken Ebihara, Kiyoshi Mori, Yoshihiro Ogawa, Kiminori Hosoda, Takashi Akamizu, Masayasu Kojima, Kenji Kangawa and Kazuwa Nakao

Department of Medicine and Clinical Science (H.A., H.I., E.K., K.M., Y.O., K.H., K.N.), Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; Translational Research Center (K.T., T.A., K.K.), Kyoto University Hospital, Kyoto 606-8507, Japan; Division of Molecular Genetics, Institute of Life Science (M.K.), Kurume University, Kurume, Fukuoka 839-0861, Japan; and Department of Biochemistry (H.H., K.K.), National Cardiovascular Center Research Institute, Osaka 565-8565, Japan

Address all correspondence and requests for reprints to: Kazuhiko Takaya, M.D., Ph.D., Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japan. E-mail: ktakaya{at}kuhp.kyoto-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin is an acylated peptide, whose lipid modification is essential for its biological activities. Previous studies demonstrated that it strongly stimulates GH release and has a potent orexigenic action. Meanwhile, there is enough evidence showing that feeding states influence plasma ghrelin levels. Fasting stimulates ghrelin secretion, and feeding reduces plasma ghrelin levels. In this study we examined the regulation of plasma ghrelin by fasting in genetically obese animals considering its molecular forms. Plasma levels of active form of ghrelin as well as those of total ghrelin were reduced in ob/ob and db/db mice compared with those in their control mice. Zucker fatty (fa/fa) rats also showed lower plasma ghrelin levels by fasting than the control rats. Insulin-induced hypoglycemia, however, stimulated ghrelin secretion in the fasted fatty rats. Moreover, glucose injection was revealed to reduce plasma ghrelin levels in rats. The effect of the severity of obesity on secretory regulation of ghrelin was also studied. Older fatty rats showed low plasma ghrelin levels even after 48-h fasting. These data suggest that the short-term secretory regulation of total ghrelin and the active form of ghrelin is delayed in obese animals and that blood glucose levels may be involved in the delayed regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CRUCIAL discovery of ghrelin has provided insight into the novel regulatory system of GH release (1). Artificial compounds named GH secretagogues (GHSs) release GH in vitro and in vivo through a specific receptor (2, 3, 4, 5, 6). Ghrelin was identified as an endogenous ligand for the receptor. Detailed structural analysis revealed that it is an acylated peptide of 28 amino acidic residues (1, 7, 8). It strongly stimulates GH release in a clear dose-dependent manner in animals and humans (1, 9, 10, 11, 12, 13, 14) in concert with GH-releasing hormone (15). Previous studies revealed its unique tissue distribution, that is, the stomach is the major site of production of ghrelin, and ghrelin is also expressed in the hypothalamus (1, 16, 17, 18, 19, 20), suggesting its possible involvement in energy homeostasis as well as in GH release (21). Consistent with this hypothesis, GHSs and ghrelin stimulate food intake via hypothalamic neuropeptide Y and agouti-related protein (AGRP), when it is centrally administered in rats (22, 23, 24, 25, 26, 27, 28, 29, 30). Meanwhile, GHSs and ghrelin induce adiposity in animals independently of food intake or GH release (31, 32).

Plasma ghrelin levels are regulated by acute feeding states. We and others revealed that plasma ghrelin levels are elevated by fasting and reduced by feeding in animals and humans (17, 25, 28, 33). Oral glucose intake, but not stomach expansion, reduces plasma ghrelin levels in rats (31), and ghrelin mRNA in the gastric fundi is increased by insulin injection (34). A few previous studies have studied the relationship between chronic feeding states and plasma ghrelin levels. Plasma ghrelin levels are reduced in obese human subjects (35), and ghrelin mRNA expression in the stomach is reduced in genetically obese db/db mice (34). We and others recently reported that plasma ghrelin levels are markedly elevated in patients with anorexia nervosa (17, 36). These observations raise the idea that ghrelin may serve as an indicator of energy deposit such as leptin.

However, these studies are lacking in structural information of ghrelin in the altered plasma levels. Ghrelin is a unique hormone in that it is a 28-amino acid peptide that contains an n-octanoyl modification on Ser3, and the lipid modification is essential for ghrelin-mediated stimulation of GH release. Des-acyl ghrelin, the des-n-octanoyl form of ghrelin, has almost no biological activities (7, 37, 38). Moreover, although fasting and feeding seem to be the major determinant factors of plasma ghrelin levels in subjects with normal body weight as mentioned above, little is known about the effect of short-term changes in energy balance on them in obese subjects.

The present study attempt to establish the difference between obese and lean subjects in the secretory regulation of ghrelin, considering its molecular forms. We examined plasma ghrelin levels in genetically obese ob/ob and db/db mice and Zucker fatty (fa/fa) rats using two kinds of RIAs that recognize total ghrelin and the active form of ghrelin separately. We also examined them in leptin transgenic (Lep Tg) mice. Lep Tg mice were recently generated transgenic mice on a C57BL/6J background overexpressing leptin under the control of the liver-specific human serum amyloid-P component promoter (39, 40, 41). The hyperleptinemia causes reduced food intake and disappearance of lipid from adipose tissue in these mice. Here we show that plasma levels of both total ghrelin and the active form of ghrelin after fasting are reduced in ob/ob and db/db mice and elevated in Lep Tg mice compared with those in their control mice. To study the secretory regulation of ghrelin further, we use Zucker fatty (fa/fa) rats and clearly demonstrate that insulin-induced hypoglycemia restores the reduced response of ghrelin secretion in them. In addition, we show that the secretory regulation of ghrelin by fasting is more reduced in older, i.e. more obese, fatty rats. The data in this study suggest that short-term secretory regulation of ghrelin reflects energy deposit and that blood glucose levels are involved in the altered regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All procedures in animal experiments were approved by the Kyoto University Graduate School of Medicine committee on animal research. Blood samples were collected from the inferior vena cava of the mouse or the jugular vein of the rat under anesthesia with diethyl ether unless indicated. The human study was approved by the ethical committee on human research of Kyoto University Graduate School of Medicine, and all subjects gave their written informed consent.

Obese and "skinny" mice
Twenty-week-old male genetically obese ob/ob and db/db and their control (+/?) mice were purchased from Japan CLEA (Tokyo, Japan). Generation of Lep Tg skinny mice has been reported previously (40). Twelve-week-old female Lep Tg and nontransgenic control mice were used. The transgenic mice were used as heterozygotes. These animals were housed in a temperature-, humidity-, and light-controlled room (12-h light/12-h dark cycle, lights on at 0800 h) and allowed free access to water and standard rat chow (352 kcal/100 g, CE-2, Japan CLEA) before the study. The body weight was measured, and 400 µl blood were sampled at 1100 h after 12-h fasting. Plasma ghrelin was measured using two kinds of RIAs as described below. After blood samples were drawn, the mice were killed by chloroform, and the stomach was immediately removed and frozen in liquid nitrogen. The samples were stored at -80 C until use, and then they were prepared for ghrelin RIAs as described below. To rule out the direct effect of leptin deficiency on plasma ghrelin levels in ob/ob mice, they were injected with leptin. Twenty-week-old mice were purchased and housed as described above. They were ip injected with 1.0 mg/kg leptin (PeproTech EC, London, UK) or saline after 12-h fasting, and 400 µl blood were sampled 3 h after injection for the measurement of plasma ghrelin. The effect of food restriction on plasma ghrelin levels was also studied. Eight-week-old C57/BL6 mice were purchased and housed as described above. They were fed 70% of the average daily food intake of the control mice for 14 d, and 400 µl blood were sampled after overnight fasting for the measurement of plasma ghrelin.

Plasma ghrelin levels in Zucker fatty rats and effect of insulin-induced hypoglycemia
Fifteen-week-old genetically obese Zucker fatty (fa/fa) and the control (+/?) rats were purchased from Japan CLEA. These animals were housed in a temperature-, humidity-, and light-controlled room (12-h light/12-h dark cycle, lights on at 0800 h) and allowed free access to water and standard rat chow (352 kcal/100 g, CE-2, Japan CLEA) before the study. Their body weight was 542.5 ± 27.2 g (mean ± SD). They were fasted for 24 h for the measurement of blood glucose and plasma ghrelin. The blood samples of the control rats were also subjected to reverse phase HPLC (RP-HPLC) coupled with C-RIA for the carboxyl terminal and N-RIA for the amino terminal. Then fatty rats were sc injected with 8.0 U/kg human neutral protamine Hagedorn (NPH) insulin (Humulin N, Eli Lilly Japan, Kobe, Japan) to examine the effect of hypoglycemia on plasma ghrelin levels. About 25 µl blood were obtained 30, 60, 90, 120, and 240 min after insulin injection by making a small incision on the tail for the measurement of blood glucose. For the measurement of plasma ghrelin, 600 µl blood were sampled before and after 12- and 24-h fasting and 120 and 240 min after insulin injection. Blood glucose was measured using One Touch II (Life Scan, Milpitas, CA), and plasma ghrelin was measured by two kinds of RIAs.

Effect of glucose injection on plasma ghrelin levels in fasted rats
Eight-week-old male Sprague Dawley rats were purchased, housed, and fed as described above. Their body weight was 200.0 ± 5.1 g (mean ± SD). They were fasted for 24 h and then ip injected with 2.0 ml saline or glucose solutions containing 2.0 or 5.0 g/kg glucose. For the measurement of plasma ghrelin, 600 µl blood were sampled before fasting (at 1100 h), after 24-h fasting, and 90 min after injection. Plasma ghrelin was measured by two kinds of RIAs.

Effect of severity of obesity on plasma ghrelin levels
Younger and older Zucker fatty rats were studied to determine the effect of severity of obesity on plasma ghrelin levels by fasting. Eight- and 30-wk-old Zucker fatty and the control rats were purchased and housed as described above. The body weight was measured, and they were fasted for 48 h, except for free access to water. Then the animals were given free access to food (standard rat chow) and water for 6 h. For the measurement of plasma ghrelin, 600 µl blood were sampled before and after 24- and 48-h fasting (at 1100 h) and after 6-h refeeding. Plasma ghrelin was measured using two kinds of RIAs.

Plasma ghrelin levels in obese human subjects
Seventeen obese Japanese subjects with no apparent medical illness [body mass index (BMI), >25.0 kg/m2] were recruited. They consisted of nine men and eight women. Their age and BMI were 53 ± 4 yr and 35.8 ± 1.5 kg/m2 (mean ± SD), respectively. Twenty-one sex- and age-matched control subjects were also studied. Their age and BMI were 49 ± 5 yr and 21.1 ± 0.6 kg/m2, respectively. Blood samples were drawn between 0800–1000 h after overnight fasting, and plasma ghrelin was measured by C-RIA as described below.

Preparation of stomach samples from mice
Stomach samples were prepared from mice as previously described (7, 18). Each sample was diced and boiled for 7 min in a 5-fold volume of water for the measurement of ghrelin. The solution was adjusted to 1.0 M acetic acid and 20 mM hydrogen chloride after boiling, and the tissue was homogenized. The supernatant was obtained after centrifugation at 10,000 rpm for 30 min.

Preparation of plasma samples
Plasma samples were prepared as previously described (1, 17). Blood samples were immediately transferred to chilled polypropylene tubes containing Na2EDTA (1 mg/ml) and aprotinin (Ohkura Pharmaceutical, Inc., Kyoto, Japan; 1000 kallikrein inactivator U/ml), and centrifuged at 4 C. For N-RIA, hydrogen chloride was added to the samples at final concentration of 0.1 N immediately after separation of plasma.

Measurement of ghrelin
Measurement of mouse and rat ghrelin.
Two kinds of polyclonal antibodies were raised against the amino terminal (Gly1-Lys11) and the carboxyl terminal (Gln13-Arg28) of rat ghrelin in rabbits as previously described (7, 38). Mouse ghrelin has a completely identical structure as rat ghrelin (Iwakura, H., and K. Hosoda, manuscript submitted). One milliliter of the prepared plasma sample was diluted with an equal volume of 0.9% NaCl and loaded onto a Sep-Pak C18 cartridge (Waters Corp., Milford, MA) preequilibrated with 0.9% NaCl. For the prepared stomach samples, supernatant after the centrifugation was loaded onto a Sep-Pak C18 cartridge preequilibrated with 0.9% NaCl. The cartridge was washed with 3.0 ml 5% CH3CN/0.1% trifluoroacetic acid (TFA) and eluted with 3.0 ml of 60% CH3CN/0.1% TFA. The eluate was evaporated, lyophilized, and dissolved in RIA buffer [50 mM sodium phosphate buffer (pH 7.4), 0.5% BSA, 0.5% Triton X-100, 80 mM NaCl, 25 mM EDTA-2Na, and 0.05% NaN3]. Two kinds of RIAs, C-RIA for the carboxyl terminal and N-RIA for the amino terminal of ghrelin, were carried out. Two tracer ligands were synthesized: [Tyr0]rat ghrelin for antighrelin-(1–11) antiserum and [Tyr29]rat ghrelin-(13–28) for antighrelin-(13–28). These ligands were radioiodinated by the lactoperoxidase methods. After radioiodination, monoiodinated ligands were purified by RP-HPLC on a µBondasphere C18 column (3.9 x 150 mm; Waters Corp., Milford, MA). The tracers were stored at -20 C in 0.1% BSA. Each RIA incubation mixture was composed of 100 µl standard ghrelin or unknown sample and 200 µl antiserum diluted with RIA buffer containing 0.5% normal rabbit serum. The antighrelin-(1–11) and antighrelin-(13–28) antisera were used at final dilutions of 1:6,000,000 and 1:20,000, respectively. After 12-h incubation, 100 µl 125I-labeled tracers (15,000 cpm) were added. After an additional 36-h incubation, 100 µl antirabbit IgG goat serum were added. Free and bound tracers were separated after 24-h incubation by centrifugation at 3,000 rpm for 30 min. After aspiration of the supernatant, radioactivity in the pellet was counted with a {gamma}-counter (ARC-600, Aloka, Tokyo, Japan). The minimal detectable quantities by C-RIA and N-RIA were 5.0 and 0.5 fmol/tube, respectively. The intraassay coefficients of variation of C-RIA and N-RIA were 6.0% and 3.0%, respectively, and the interassay coefficients of variation were 9.0% and 6.0%, respectively. The recoveries of ghrelin were more than 95% for both C-RIA and N-RIA.

Measurement of human ghrelin.
Plasma ghrelin was measured as reported previously (1, 17). Briefly, polyclonal antibody against the carboxyl terminal of human ghrelin, which has an identical structure as rat ghrelin, was used. The RIA was performed similarly as described above.

Characterization of plasma ghrelin in Zucker control rats
Plasma ghrelin was characterized using RP-HPLC coupled with C-RIA and N-RIA as previously described (7, 18, 34). Plasma samples of 24-h fasted Zucker control rats were prepared and loaded on the Sep-Pak C18 cartridge as described above. The eluate was subjected to RP-HPLC on a µBondasphere C18 column. The RP-HPLC was performed using a linear gradient of CH3CN from 10–60% in 0.1% TFA for 40 min. An aliquot of each fraction obtained by RP-HPLC was evaporated and lyophilized, and one fifth of each fraction was subjected to two kinds of RIAs for the measurement of ghrelin.

Data analysis
Results are expressed as the mean ± SE unless noticed. Comparisons between groups were performed with unpaired t test. The changes in body weight, blood glucose levels, and plasma ghrelin levels were compared by ANOVA using Fisher’s test. Simple linear regression analysis was used to evaluate correlation between BMIs and plasma ghrelin levels. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stomach and plasma ghrelin levels in genetically obese ob/ob and db/db mice
Body weights and stomach ghrelin levels measured by C-RIA and N-RIA of ob/ob and db/db and their control mice are summarized in Table 1Go. The ob/ob and db/db mice weighed 90.0% and 79.0% more than their control mice, respectively (P < 0.005). Stomach ghrelin levels were significantly lower in these obese mice than in their control mice (P < 0.005 by C-RIA and P < 0.05 by N-RIA, for both). Figure 1Go, A and B, shows plasma ghrelin levels in ob/ob, db/db, and their control mice. Plasma ghrelin levels by C-RIA in ob/ob and control mice were 452.5 ± 25.4 and 646.5 ± 78.0 fmol/ml, respectively (Fig. 1AGo, upper panel). Those in db/db and the control mice were 313.2 ± 23.0 and 486.2 ± 49.7 fmol/ml, respectively (Fig. 1BGo, upper panel). The differences between obese and their control mice were significant (P < 0.05 for ob/ob and P < 0.05 for db/db). Plasma ghrelin levels by N-RIA in ob/ob and control mice were 12.7 ± 2.4 and 29.3 ± 2.8 fmol/ml, respectively (Fig. 1AGo, lower panel). Those in db/db and control mice were 6.1 ± 0.6 and 20.3 ± 1.6 fmol/ml, respectively (Fig. 1BGo, lower panel). The differences between obese and their control mice were also significant (P < 0.005 for ob/ob and P < 0.0001 for db/db).


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  		Table 1. Body weights and stomach ghrelin levels measured by C-RIA and N-RIA in genetically obese ob/ob and db/db mice, Lep Tg mice, and their control mice (+/? for ob/ob and db/db mice and non-Tg for Lep Tg mice)

 


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  		Figure 1. Plasma ghrelin levels measured by C-RIA (upper panel) and N-RIA (lower panel) in ob/ob and db/db mice and Lep Tg mice. A, Plasma ghrelin levels in ob/ob and control (+/?) mice (n = 6/group). B, Plasma ghrelin levels in db/db and control (+/?) mice (n = 6/group). C, Plasma ghrelin levels in Lep Tg and control (nontransgenic, non-Tg) mice (n = 6/group). a, P < 0.05; b, P < 0.005; c, P < 0.0001 (vs. control mice).

 
Stomach and plasma ghrelin levels in Lep Tg mice
Lep Tg mice weighed 25.1% less than the control mice (Table 1Go; P < 0.005). No visible adipose tissue was found in sc, epididymal, mesenteric, or retroperitoneal fat depots from Lep Tg mice, as previously described (40). Stomach ghrelin levels in them are shown in Table 1Go. Lep Tg mice showed higher stomach ghrelin level by C-RIA than the control mice, although the difference was not significant. On the other hand, stomach ghrelin level by N-RIA was significantly lower in Lep Tg mice compared with control mice. Figure 1CGo shows plasma ghrelin levels in Lep Tg and control mice. Plasma ghrelin levels by C-RIA in Lep Tg and the control mice were 3680.5 ± 839.0 and 1283.3 ± 164.6 fmol/ml, respectively (Fig. 1CGo, upper panel). The difference was significant (P < 0.05). Plasma ghrelin levels by N-RIA in Lep Tg and the control mice were 158.1 ± 31.2 and 62.9 ± 21.2 fmol/ml, respectively (Fig. 1CGo, lower panel). The difference was also significant (P < 0.05).

Effect of leptin injection in ob/ob mice
Table 2Go shows plasma ghrelin levels in leptin-injected ob/ob mice. Plasma ghrelin levels by C-RIA or N-RIA showed no significant difference between leptin- and saline-injected ob/ob mice.


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  		Table 2. Body weights and plasma ghrelin levels by C-RIA and N-RIA in leptin-injected ob/ob mice

 
Effect of food restriction on plasma ghrelin levels
Table 3Go shows body weight changes and plasma ghrelin levels in food-restricted and ad libitum-fed mice. Body weight was increased by 12% in ad libitum-fed mice, but was decreased by 30% in food-restricted mice. Plasma ghrelin levels were elevated in food-restricted mice compared with ad libitum-fed mice. They were 7.6- and 11.2-fold higher by C-RIA and N-RIA, respectively, than those in ad libitum-fed mice (P < 0.001 for both).


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  		Table 3. Body weight changes and plasma ghrelin levels by C-RIA and N-RIA in food-restricted and ad libitum-fed mice

 
Plasma ghrelin levels in Zucker fatty rats
The Zucker fatty and control rats were studied to examine changes in plasma ghrelin levels in obese animals in detail. Plasma ghrelin levels by C-RIA after 12-h fasting in fatty and control rats were 294.9 ± 25.4 and 485.5 ± 41.4 fmol/ml, respectively (P < 0.001). Twenty-four-hour fasting produced clearer results. Plasma ghrelin levels by C-RIA and N-RIA after 24-h fasting in the control rats were 1.7- and 2.6-fold higher than those in fatty rats, respectively (Table 4Go). There was also significant difference in blood glucose levels between fatty and control rats after 24-h fasting (Table 4Go). The molecular forms of plasma ghrelin in 24-h fasted control rats were examined by RP-HPLC coupled with C-RIA and N-RIA (Fig. 2Go, A and B). The recoveries of the immunoreactivities after RP-HPLC were more than 90% by C-RIA and 95% by N-RIA, respectively. One major peak eluted at the position of des-acyl ghrelin-(1–28), and another major peak eluted at the position of acylated rat ghrelin-(1–28) by RP-HPLC coupled with C-RIA (Fig. 2AGo, a and b, respectively). The molar ratio of des-acyl ghrelin to acylated ghrelin was 4.3:1. One major peak eluted at the position of acylated rat ghrelin-(1–28) by RP-HPLC coupled with N-RIA (Fig. 2BGo, b).


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  		Table 4. Blood glucose and plasma ghrelin levels by C-RIA and N-RIA after 24-h fasting in 15-wk-old Zucker fatty (fa/fa) and the control (+/?) rats

 


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  		Figure 2. Representative RP-HPLC coupled with C-RIA and N-RIA of plasma ghrelin in 24-h fasted Zucker control (+/?) rats. A, RP-HPLC coupled with C-RIA. B, RP-HPLC coupled with N-RIA. The arrows indicate the elution position of des-acyl ghrelin (a) and acylated full-length ghrelin (b).

 
Effect of insulin-induced hypoglycemia on plasma ghrelin levels in fasted Zucker fatty rats
Plasma ghrelin levels after insulin injection in 24-h fasted Zucker fatty rats were examined to elucidate the role of blood glucose levels in the reduced plasma ghrelin levels in these animals. The blood glucose level declined to 48 ± 6 mg/dl 90 min after insulin injection, and hypoglycemia lasted until the end of the study (Fig. 3AGo). There was no significant change in blood glucose levels in saline-injected rats. Figure 3BGo shows the time course of plasma ghrelin levels in both groups. Plasma ghrelin levels by C-RIA 120 and 240 min after insulin injection were 197 and 228% of the initial values, respectively. The changes were significant (P < 0.001 vs. saline injection for both). Plasma ghrelin levels by N-RIA 120 and 240 min after insulin injection were 263% and 278% of the initial values, respectively. The changes were also significant (P < 0.005 vs. saline injection for both).



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  		Figure 3. The effect of 8.0 U/kg NPH insulin injection in 24-h fasted Zucker fatty (fa/fa) rats (n = 8/group). A, Changes in blood glucose levels after sc injection of saline ({circ}) and insulin ({bullet}). B, Changes in plasma ghrelin levels measured by C-RIA (upper panel) and N-RIA (lower panel) after sc injection of saline ({square}) and insulin ({blacksquare}). a, P < 0.005; b, P < 0.001 (vs. injection of saline).

 
Effect of glucose injection on plasma ghrelin levels
The effect of glucose injection on plasma ghrelin levels was studied in 24-h fasted Sprague Dawley rats. Plasma ghrelin levels by C-RIA and N-RIA after 24-h fasting were elevated to 149% and 289% of the values before fasting, respectively (Fig. 4Go, upper and lower panels). Plasma ghrelin levels by C-RIA after 0 (saline injection), 2.0, and 5.0 g/kg glucose injections were 98%, 84%, and 78% of the values after 24-h fasting (Fig. 4Go, upper panel). The differences between 0 and 2.0 g/kg, and 0 and 5.0 g/kg were significant (P < 0.05 for both). Plasma ghrelin levels by N-RIA were reduced in a clearer dose-dependent manner. They were 87%, 69%, and 30% of the values after 24-h fasting (Fig. 4Go, lower panel). The differences between 0 and 2.0 g/kg, 0 and 5.0 g/kg, and 2.0 and 5.0 g/kg were significant (P < 0.05, P < 0.0001, and P < 0.0001, respectively).



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  		Figure 4. Plasma ghrelin levels measured by C-RIA (upper panel) and N-RIA (lower panel) after ip injection of 0 (saline injection), 2.0, and 5.0 g/kg glucose in 24-h fasted Sprague Dawley rats (n = 8/group). a, P < 0.05; b, P < 0.001 (vs. injection of 0 g/kg glucose). c, P < 0.001 (vs. injection of 2.0 g/kg glucose).

 
Effect of severity of obesity on the secretory regulation of ghrelin
Younger and older Zucker fatty rats than 15-wk-old rats, which were used in the studies described above, were used. Eight- and 30-wk-old Zucker fatty and their control rats were studied. Body weights of 8-wk-old fatty and control rats were 282.5 ± 5.1 and 213.8 ± 1.6 g, respectively, and those of 30-wk-old fatty and control rats were 732.5 ± 22.5 and 408.7 ± 8.8 g, respectively. That is, 8-wk-old fatty rats were only 1.3-fold heavier than the control rats, whereas 30-wk-old fatty rats were 1.8-fold heavier than the control rats. Figure 5AGo shows the effect of 24- and 48-h fasting followed by 6-h refeeding in 8-wk-old rats. Plasma ghrelin levels before fasting determined by C-RIA in fatty and control rats were 295.4 ± 22.5 and 335.8 ± 36.2 fmol/ml, respectively. Plasma ghrelin levels after 24-h fasting in fatty and control rats were 96% and 187% of the values before fasting, and those after 48-h fasting were 150% and 165% of the control levels, respectively. The differences in plasma ghrelin levels between fatty and control rats were not significant before fasting, but were significant after 24- and 48-h fasting (P < 0.001 and P < 0.05, respectively). Plasma ghrelin levels by N-RIA showed similar results. Those before fasting in fatty and control rats were 45.1 ± 4.4 and 48.1 ± 9.4 fmol/ml, respectively. Plasma ghrelin levels after 24-h fasting in fatty and control rats were 95% and 222% of the values before fasting, and those after 48-h fasting were 147% and 180% of the prefasting values, respectively. The differences between fatty and control rats were not significant before fasting, but were significant after 24- and 48-h fasting (P < 0.001 and P < 0.05, respectively). Plasma ghrelin levels after 6-h refeeding in the control rats by C-RIA and N-RIA were reduced to 45% and 32% of the values after 48-h fasting, respectively. Plasma ghrelin levels after 6-h refeeding in fatty rats, however, were not reduced. They were 106% and 97% of the values after 48-h fasting by C-RIA and N-RIA, respectively. Figure 5BGo shows the effect of 24- and 48-h fasting followed by 6-h refeeding in 30-wk-old rats. Plasma ghrelin levels in older control rats were increased in the same manner as in younger ones. Plasma ghrelin levels after 24-h fasting by C-RIA and N-RIA were 200% and 269% of the values before fasting, respectively. On the contrary, plasma ghrelin levels after 24-h fasting in older fatty rats were not changed similarly to those in younger fatty rats. They remained, however, low even after 48-h fasting. They were 108.4% by C-RIA and 73.0% by N-RIA of the values before fasting. Moreover, plasma ghrelin levels after 6-h refeeding were clearly higher than those after 48-h fasting. They were 145% by C-RIA and 261% by N-RIA of the values after 48-h fasting.



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  		Figure 5. The effect of 24- and 48-h fasting followed by 6-h refeeding in older and younger Zucker fatty (fa/fa; {bullet}) and control (+/?; {circ}) rats (n = 8/group). A, Changes in plasma ghrelin levels measured by C-RIA (upper panel) and N-RIA (lower panel) in 8-wk-old rats. B, Changes in plasma ghrelin levels measured by C-RIA (upper panel) and N-RIA (lower panel) in 30-wk-old rats. a, P < 0.05; b, P < 0.005; c, P < 0.001; d, P < 0.0001 (vs. control rats).

 
Plasma ghrelin levels in obese human subjects
Plasma ghrelin levels were examined in human nondiabetic obese subjects. Figure 6Go, A and B, shows the mean of the plasma ghrelin levels and the correlation between BMIs and plasma ghrelin levels in obese and sex- and age-matched control subjects, respectively. Plasma ghrelin levels in obese and control subjects were 82.1 ± 5.4 and 145.2 ± 16.0 fmol/ml, respectively (Fig. 6AGo). The difference was significant (P < 0.005). There was a significant negative correlation between BMIs and plasma ghrelin levels (Fig. 6BGo; r = -0.51; P < 0.0001).



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  		Figure 6. Plasma ghrelin levels in 17 obese Japanese subjects (9 men and 8 women; BMI, >25.0 kg/m2) and 20 sex- and age-matched control subjects. A, Mean plasma ghrelin levels measured by C-RIA in obese and control subjects. a, P < 0.0005. B, Correlation between BMIs and plasma ghrelin levels in obese ({bullet}) and control ({circ}) subjects. r = -0.51; P < 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we reported for the first time the secretory regulation of plasma ghrelin in obese animals distinguishing the active form of ghrelin from total ghrelin. We used two kinds of RIAs for the purpose, namely C-RIA for the carboxyl terminal and N-RIA for the amino terminal of ghrelin. The levels measured by N-RIA represent those of active form of ghrelin, as n-octanoyl modification, which is essential for the biological activity of ghrelin (7, 37, 38), is located on Ser3 of ghrelin. On the other hand, the levels measured by C-RIA represent those of total ghrelin, including its inactive form (7).

Plasma ghrelin levels by C-RIA were reduced in genetically obese ob/ob and db/db mice compared with those in their control mice. Plasma ghrelin levels by N-RIA showed results similar to those by C-RIA, and they were approximately 2–5% of the latter. We thus demonstrated that plasma levels of both total ghrelin and the active form of ghrelin are reduced in obese animals. These results are compatible with previous reports on obese Caucasian subjects (35) and rats fed a high fat diet (42), in which total plasma ghrelin levels are shown to be reduced, and with a report on reduced ghrelin mRNA levels in db/db mice (34). Plasma ghrelin levels in Lep Tg mice, which are characterized by disappearance of lipid from adipose tissue (40), showed inverse results. They were highly elevated in these skinny mice and reached 2.9-fold by C-RIA and 2.5-fold by N-RIA of those in the control mice, whereas there was a discrepancy in stomach and plasma ghrelin levels in Lep Tg mice. Stomach ghrelin levels may represent only the storage of ghrelin in the stomach and may not reflect its secretion into the bloodstream. To confirm the results in Lep Tg mice in another animal model with low body weight, food-restricted mice were studied. They were fed with 70% of the average food intake of the control mice, mimicking the feeding states in Lep Tg mice, which consume approximately 70% of the food of non-Tg mice (40). We observed that plasma ghrelin levels were also highly elevated in food-restricted mice. These results of plasma ghrelin levels in Lep Tg mice and food-restricted mice are compatible with previous reports by us and others on patients with anorexia nervosa, whose plasma ghrelin levels are highly elevated (17, 36). We also observed that plasma ghrelin levels are dramatically reduced when Lep Tg mice gain weight by feeding a high fat diet (Ebihara, K., and Y. Ogawa, unpublished data). Taken together, these data indicate that plasma levels of total ghrelin and the active form of ghrelin reflect chronic feeding states. Although we observed higher plasma ghrelin levels in non-Tg mice compared with the control mice in the group of ob/ob and db/db mice, this may be accounted for by gender difference in plasma ghrelin levels. Female animals tend to show higher plasma ghrelin levels than male animals (Ariyasu, H., unpublished data).

The mechanism for the reduced plasma ghrelin levels in obese animals and humans is unknown. It is conceivable that the deficiency in leptin action could result in the reduced plasma ghrelin levels in the obese animals used in this study, as leptin is lacking in ob/ob mice (43), and leptin receptor is lacking in db/db mice and Zucker fatty rats (44, 45, 46). Previous studies, however, showed the opposite results. The characteristic negative correlation between plasma ghrelin levels and BMIs (17, 35) instead leads to the idea that ghrelin secretion may be negatively regulated by leptin, because plasma leptin levels positively correlate with BMIs (47). In addition, the present study showed that leptin replacement in ob/ob mice did not result in restored plasma ghrelin levels. It is not likely that the amount of injected leptin used in this study was not enough, because it can elevate plasma leptin levels to the extent of those in Lep Tg mice for a few hours (40, 48). Taken together, the lack of leptin action does not seem to result in the reduced plasma ghrelin levels in these obese animals. Further study, however, is needed to elucidate the role of leptin in plasma ghrelin levels in obese subjects.

Plasma ghrelin levels in genetically obese rats were also examined. Zucker fatty (fa/fa) rats showed lower plasma ghrelin levels than the control rats. The results of RP-HPLC coupled with C-RIA and N-RIA for plasma ghrelin were compatible with those for ghrelin in the stomach (7, 18, 34). These data indicate that acylated full-length ghrelin and des-acyl ghrelin are the two major forms of this hormone in rat plasma and confirm the validity of C-RIA and N-RIA. In fasted conditions, Zucker fatty rats showed higher blood glucose levels than the control rats, compatible with a previous report (49). The difference between fatty and control rats in glucose levels led us to the hypothesis that the higher glucose levels may be involved in the reduced plasma ghrelin levels in the fasted fatty rats, because sugar intake, but not stomach expansion, decreases circulating ghrelin levels in rodents (31). The effect of short-term changes in blood glucose levels on plasma ghrelin levels in Zucker fatty rats was examined. NPH insulin injection created prolonged hypoglycemia in these animals, and the nadir blood glucose values were comparable to blood glucose levels in 24-h fasted control rats. The hypoglycemia-stimulated ghrelin secretion and plasma levels of both total ghrelin and the active form of ghrelin 120 and 240 min after insulin injection reached 200–280% of the initial values, respectively. The values exceeded those in 24-h fasted control rats. It should be noted that hypoglycemia induced by rapid insulin had much less effect on plasma ghrelin levels (Ariyasu, H., unpublished data), suggesting slow secretory regulation of ghrelin by hypoglycemia. Then the effect of glucose injection on plasma ghrelin levels was examined in fasted Sprague Dawley rats. Plasma levels of both total ghrelin and the active form of ghrelin were reduced by glucose injection in a dose-dependent manner. These data suggest that reduced blood glucose results in elevated plasma ghrelin levels in fasted rats of normal weight and that high blood glucose levels may be involved in the reduced plasma ghrelin levels in obese animals. These data also indicate that short-term stimulation of ghrelin secretion, i.e. hypoglycemia, restores the reduced plasma ghrelin levels in obese animals, suggesting the exquisite secretory regulation of ghrelin in both chronic and acute phases of energy homeostasis.

Plasma ghrelin levels were further examined using younger and older Zucker fatty rats. Moreover, they were examined in various feeding states in these studies. The time course of plasma ghrelin levels by fasting followed by refeeding in 8-wk-old rats showed intriguing results. Plasma ghrelin levels showed no significant difference by C-RIA or N-RIA between fatty and control rats when they were freely fed. They showed marked elevation after 24-h fasting in the control rats, and the values reached 1.9-fold of those before fasting and remained at almost the same levels after 48-h fasting. On the contrary, plasma ghrelin levels did not show any change in fatty rats after 24-h fasting. They were elevated after 48-h fasting, but did not reach the levels in control rats. The delayed secretory regulation of ghrelin by fasting in obese animals raised the idea that short-term secretory regulation of ghrelin is modified by an excess energy deposit. The older fatty rats showed clearer results and confirmed this idea. Eight-week-old fatty rats weighed only 1.3 times as much as the control rats, whereas 30-wk-old fatty rats weighted 1.8 times as much as the control rats. Although older control rats showed almost the same pattern of plasma ghrelin levels as the younger ones, older fatty rats showed a more delayed pattern after fasting than younger fatty rats. The larger energy deposit in older fatty rats than younger ones may explain these augmented results. Refeeding experiments after 48-h fasting also showed intriguing results. Plasma ghrelin levels were reduced to basal values after refeeding in the control rats, but not in fatty rats. Plasma ghrelin levels were unchanged in 8-wk-old fatty rats and were even elevated in 30-wk-old fatty rats. Obese animals appear to be less sensitive to negative stimulation for the secretory regulation of ghrelin, and plasma ghrelin levels may be still elevated due to the preceding fasting. These results are in keeping with our observation of the feeding behavior of the refed rats in this study if we consider that ghrelin is a potent stimulator for food intake (22, 23, 24, 25, 26, 27, 28, 29, 30). The control rats almost stopped eating after 6-h refeeding with satisfaction, whereas fatty rats were still eating even after that.

Plasma ghrelin levels in obese human subjects in the present study confirmed the results in the animals mentioned above. We clearly demonstrated a negative correlation between BMIs and plasma ghrelin levels in obese Japanese subjects, expanding a previous study showing reduced plasma ghrelin levels in obese Caucasians (35). These data are also compatible with studies by us and others of patients with anorexia nervosa (17, 36). These patients show high plasma ghrelin levels, and their BMIs have a negative correlation with these levels.

We could not detect marked discrepancy between ghrelin levels measured by C-RIA and N-RIA in the present study. Measuring the acylated form of this hormone, however, may be of advantage if we consider that ghrelin is biologically active only in the acylated form (1). There may be physiological or pathological conditions under which plasma levels of total ghrelin and the active form of ghrelin are discrepant. Measuring the active form of ghrelin may be of importance in such conditions. Further study is needed for this issue.

There are several examples that support the idea that ghrelin has actions involved in energy homeostasis as well as GH release (21). GHSs have been demonstrated to have an orexigenic action, and we and others recently showed that central administration of ghrelin induces food intake in rodents (22, 23, 24, 25, 26, 27, 28, 29, 30). Ghrelin injection induces the expression of Fos protein in the hypothalamic arcuate nucleus and then stimulates the expression of NPY and AGRP (27, 28, 29). A role of NPY and AGRP as mediators of feeding effect of ghrelin is suggested by studies in which antagonists of either NPY or AGRP were shown to attenuate the orexigenic potency of ghrelin (27, 28, 29). Continuous administration of ghrelin induces food intake even in humans (50). Meanwhile, peripheral daily administration of ghrelin or ipamorelin, one of the GHSs, induces adiposity independent of food intake or GH secretion in rodents (31, 32). These data suggest that ghrelin may act in various ways, thereby increasing energy deposit. An alteration in the secretory regulation of ghrelin in obese subjects may play an important role in these actions of ghrelin.

In conclusion, the present study demonstrates that the short-term regulation of plasma levels of both total ghrelin and the active form of ghrelin is delayed in obese animals and that insulin-induced hypoglycemia restores the delayed regulation, suggesting that blood glucose levels are involved in the delayed regulation in obese animals. These observations are in keeping with the hypothesis that ghrelin is involved in acute and chronic energy homeostasis.


   Acknowledgments
 


   Footnotes
 
This work was supported by research grants from the Japanese Ministry of Education, Science, and Culture; the Japanese Ministry of Health, Labor, and Welfare; the Japanese Society for the Promotion of Science Research for the Future Program (JSPS-RFTF 98L00801); and the Foundation for Total Health Promotion.

Abbreviations: AGRP, Agouti-related protein; BMI, body mass index; C-RIA, RIA for the carboxyl terminal; GHS, GH secretagogue; Lep Tg, leptin transgenic; NPH, neutral protamine Hagedorn; N-RIA, RIA for the amino terminal; RP-HPLC, reverse phase HPLC; TFA, trifluoroacetic acid.

Received February 26, 2002.

Accepted for publication May 28, 2002.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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