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Endocrinology Vol. 144, No. 3 754-759
Copyright © 2003 by The Endocrine Society

ARTICLE

The Role of Ghrelin and Growth Hormone Secretagogues Receptor on Rat Adipogenesis

Kichoon Choi, Sang-Gun Roh, Yeon-Hee Hong, Yogendra B. Shrestha, Daisuke Hishikawa, Chen Chen, Masayasu Kojima, Kenji Kangawa and Shin-Ichi Sasaki

Department of Food Resource Science (K.C., S.-G.R., Y.-H.H., Y.B.S., D.H., S.S.), Faculty of Agriculture, Shinshu University, Nagano-ken 399-4598, Japan; Endocrine Cell Biology (C.C.), Prince Henry’s Institute of Medical Research, Clayton 3168, Australia; Molecular Genetics (M.K.), Institute of Life Science, Kurume University, Aikawamachi 2432-3, Kurume, Fukuoka 839-0861, Japan; and Department of Biochemistry (K.K.), National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan

Address all correspondence and requests for reprints to: Dr. Sang-gun Roh, Department of Food Resource Science Faculty of Agriculture, Shinshu University Nagano-ken 399-4598, Japan. E-mail: sangroh{at}gipmc.shinshu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent research progress indicates a close link between ghrelin, a natural ligand of GH secretagogues receptor (GHS-R), and both the metabolic balance and body composition. To clarify the involvement of ghrelin and GHS-R in the process of adipogenesis, we measured the expression of GHS-R and peroxisome proliferator-activated receptor {gamma} 2 (PPAR-{gamma}2) mRNA in rat adipocytes using semiquantitative RT-PCR methods. The levels of GHS-R mRNA increased by up to 4-fold in adipose tissue from epididymal and parametrial regions as the rat aged from 4–20 wk and were significantly elevated during the differentiation of preadipocytes in vitro. Ghrelin (10-8 M for 10 d) stimulated the activity of glycerol-3-phosphate dehydrogenase and the differentiation of rat preadipocytes in vitro. Ghrelin treatment also significantly increased the levels of PPAR-{gamma}2 mRNA in primary cultured rat differentiated adipocytes. In addition, isoproterenol (10-8 M, 40 min)-stimulated lipolysis was significantly reduced by simultaneous ghrelin treatment in a dose-dependent manner in vitro. In conclusion, the expression of GHS-R in rat adipocytes increases with the age and during adipogenesis. Ghrelin in vitro stimulates the differentiation of preadipocytes and antagonizes lipolysis. Ghrelin may therefore play an important role in the process of adipogenesis in rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN, AN ENDOGENOUS ligand for the GH secretagogues receptor (GHS-R), was discovered in the stomach tissues of rat and human in 1999 (1). Functionally, ghrelin stimulates GH secretion from pituitary somatotropes (1) and increases food intake and body weight (2, 3). Based on experiments employing intracerebroventricular injection of ghrelin in rat, it is proposed that ghrelin acts directly on the hypothalamic regulatory nuclei that controls energy homeostasis as an orexigenic peptide (4, 5, 6). Furuse et al. (7) reported, however, that intracerebroventricular injection of ghrelin inhibited food intake in a dose-dependent manner in the neonatal chick indicating a clear species difference. GHS-R mRNA presents in a variety of tissues including the stomach, hypothalamic region, heart, lung, pancreas, intestine, kidney, and adipose tissues (1, 3, 8, 9). Such a wide distribution of GHS-R indicates that ghrelin produced in and secreted from the stomach may have a variety of regulatory functions in the brain and peripheral tissues through endocrine, autocrine, and/or paracrine pathways. Recently, ghrelin has been shown to have orexigenic and adipogenic effects in rodents in vivo (3, 6, 10). Published data indicate that ghrelin antagonizes leptin action in rat through the activation of the hypothalamic neuropeptide Y-Y1 receptor pathway (6). It has also been reported that ghrelin stimulates prolactin, ACTH, and cortisol secretions in human (11, 12). The ghrelin-induced increase in body weight and adipose tissue may therefore be induced indirectly through stimulation of the appetite and directly through action on adipose tissue. There is, however, no evidence whether ghrelin acts on adipose tissue directly to regulate adipogenesis.

In the present experiment, we studied the expression of GHS-R mRNA in adipocytes isolated from rat adipose tissue in vivo and primary cultured adipocytes in vitro. The effects of ghrelin in vitro on the differentiation of preadipocytes to adipocytes, and on isoproterenol-induced lipolysis, were also investigated. The data from the experiment indicate that ghrelin acts directly on adipocytes to stimulate adipogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male and female Wistar rats were used in this experiment. The rats were housed in a regulated environment (22 ± 2 C, 55 ± 10% humidity, 12-h light, 12-h dark cycle with light on at 0700 h) with free access to food and water. When rats reached the appropriate age (4, 7, 10, 15, and 20 wk), adipose tissues were separated aseptically and then immediately processed for the experiments. All experiments were conducted in accordance with the Shinshu University Guide for the Care and Use of Experimental Animals.

Preparation of rat adipocytes
White adipose tissues taken from the epididymal and parametrial regions were dissected free from the connective tissue and blood vessels. After being minced in Krebs Ringer Bicarbonate (KRB)-HEPES buffer with collagenase type I (Worthington Biochemical Corp., Freehold, NJ; 0.5 mg/ml), the adipose tissue was digested at 37 C for 40 min. The cell suspension was then filtered through a polypropylene mesh (177 µm) to remove undigested tissue and washed with warm KRB-HEPES buffer with 2% BSA (Sigma, St. Louis, MO). Isolated adipocytes were used for RT-PCR and for the incubation experiment on lipolysis.

Preparation of preadipocytes
Primary preadipocytes were prepared from parametrial adipose tissue obtained from 4-wk-old female rats weighing 90–110 g. The preadipocytes were prepared and cultured as described previously (13). Briefly, adipose tissue was immediately immersed in KRB buffer (pH 7.1) and carefully separated from other connective tissue and blood vessels. The adipose tissue was subsequently minced and treated with KRB buffer containing 1.1 mM glucose, 0.5 mg/ml type I collagenase, and 3.5% BSA for 80 min at 37 C with constant gentle agitation. The digested tissue was then filtered through a polypropylene mesh to separate cell suspension from undigested tissue fragments. The filtrate was subjected to centrifugation at 1400 x g for 5 min at room temperature. The supernatant was discarded, and the pellet was washed twice with DMEM (Life Technologies, Inc., Gaithersburg, MD). The cells were resuspended in DMEM complemented by 10% fetal bovine serum (FBS) (Sigma) and 1% antibiotic mixture (Nacalai Tesque, Inc., Kyoto, Japan). The cells were plated in six-well culture plates at a final density of approximately 2.5 x 104 cells/well, and cultured at 37 C in a humidified, 5% CO2 atmosphere. The culture medium was changed with FBS- containing medium every 2 d.

Differentiation of rat preadipocytes
Establishment of preadipocytes and induction of the differentiation from preadipocytes to adipocytes were carried out using the method described by Marko et al. (14) with modification. The preadipocytes were cultured in DMEM containing 10% FBS until they reached confluence (d 0). Differentiation was induced by the addition of 0.5 mM methyl-3-isobutylxanthine (Sigma), 0.25 µM dexamethasone (Sigma), 5 µg/ml porcine insulin (Sigma), and 10% FBS in DMEM. After 48 h (d 2), the medium was replaced with DMEM containing 1 µg/ml insulin and 5% FBS for 24 h. The medium was then replaced with fresh DMEM containing 5% FBS with or without ghrelin (10-8 M) every 3 d. Differentiation of cultured preadipocytes was monitored under inverted light microscope and by the activity of the marker enzyme glycerol-3-phosphate dehydrogenase (GPDH; EC1.1.1.8).

Determination of GPDH activity
GPDH activity was measured by a spectrophotometric method (15). Cells were washed twice with ice-cold PBS, and then 0.5 ml of assay buffer (100 mM triethanolamine, 2.5 mM EDTA, 0.1 mM ß-mercaptoethanol, 0.5% Nonidet P-40) was added. Cells were subsequently scraped and sonicated to generate cell lysis. The lysate was rotated at 14,000 x g for 15 min at 4 C. The supernatant was removed and GPDH activity was immediately determined in sonicated cell extracts. The protein content of the extracts was measured by the Bradford method (16).

Incubation experiment for lipolysis on isolated adipocytes
Adipocytes were prepared from 7-wk-old female rats as detailed above. The lipolysis of dissociated adipocytes was induced by incubating cells with isoproterenol for 40 min, and the effect of ghrelin was tested by including it in the incubation process. The lipolysis was determined by measuring the level of released glycerol in the incubation medium. The medium was frozen at -20 C until glycerol assay.

Semiquantitative RT-PCR analysis of GHS-R and PPAR-{gamma}2 mRNA
The total RNA was extracted from fresh dissociated adipocytes, primary cultured preadipocytes and differentiated adipocytes in six-well culture plates. Semiquantitative RT-PCR was performed as previously described (17) to measure levels of rat GHS-R and PPAR-{gamma}2 mRNA. The primers specific to GHS-R and PPAR-{gamma}2 are (pair 1): GHS-R forward primer (5'-ACCTCCTCTGCAAACTCTTCC-3'), GHS-R reverse primer (5'-CACCCGGTACTTCTTGGACAT-3); and (pair 2): PPAR-{gamma}2 forward primer (5'-TGGGTGAAACTCTGGGAGAT-3) and PPAR-{gamma}2 reverse primer (5'-CCATAGTGGAAGCCTGATGC-3). Based on preliminary experiments with the climbing phase of PCR products in relation to PCR cycle numbers, we performed PCR with 34 cycles with 61 C annealing temperature for the GHS-R (product size of 599 bp) and 30 cycles with 57 C annealing temperature for the PPAR-{gamma}2 (product size of 454 bp). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; product size of 452 bp), the housekeeping gene, was amplified as an internal control with 28 cycles and 55 C annealing temperature. The relative levels of rat GHS-R and PPAR-{gamma}2 mRNA to GAPDH mRNA were calculated and shown in the figures.

Statistical analysis
Data are presented as the percentage of control value (mean ± SEM) in at least three repeats in each experimental group. The statistical significance of the difference in mean values was assessed by Duncan’s multiple range test followed by one-way ANOVA. Statistical comparisons between control and ghrelin in data (see Fig. 3Go) were analyzed with Student’s t test.



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  		Figure 3. Effect of ghrelin on GPDH activity during differentiation of rat preadipocytes. The preadipocytes were proliferated to confluence and then were allowed to differentiate to adipocytes in differentiation medium with or without 10-8 M ghrelin for 10 d. The GPDH activity was measured at d 0, 4, 7, and 10 during the differentiation period. All data represent the means ± SEM of the three experiments. a–d, Different superscript letters on each column indicate significant difference between groups (P < 0.05). **, P < 0.01 vs. control at same day of differentiation.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Levels of GHS-R mRNA in adipocytes from rats of different ages
Between the age of 4 and 20 wk, both male and female rats grow rapidly, with dramatic increase in adipose tissue, around 0.5–3 g. We isolated adipose tissues from age groups of 4-, 7-, 10-, 15-, and 20-wk-old female and 4-, 7-, 10-, and 20-wk-old male rats. With an internal control of GAPDH, GHS-R mRNA levels were analyzed. The levels of GHS-R mRNA in adipocytes increased significantly along the age of rats (Fig. 1Go), with no difference between male and female rats. The level of GHS-R mRNA at the age of 20 wk was about 4-fold of that at the age of 4 wk (Fig. 1Go). Because the level of GHS-R mRNA is presented by a ratio between GHS-R mRNA and GAPDH mRNA, the increase in GHS-R mRNA implies an increase in GHS-R mRNA in each adipocyte, which is not a reflection of the increase in adipose tissue along the increase in age.



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  		Figure 1. Levels of GHS-R mRNA in isolated rat adipocytes. Representative ethidium bromide-stained agarose gel showing amplified GHS-R (599 bp) and GAPDH (452 bp) with molecular markers for different age of female (left panel) and male (right panel) rats. The RT-PCR results shown are representative of three separate experiments with the same protocol. Lower panel, Data were normalized using GAPDH mRNA and expressed as a percentage of the value obtained from 4-wk-old female and male rats. The data represent the means ± SEM of the three experiments. a–c, Different superscript letters on each column indicate significant difference between groups (P < 0.05).

 
Levels of GHS-R mRNA in differentiated adipocytes
To clarify whether GHS-R expression changes in the process of differentiation of preadipocytes, the levels of GHS-R mRNA in cultured preadipocytes and during differentiation at d 4, 6, 8, and 10 were analyzed (Fig. 2Go). At d 10, the differentiation rate peaked to greater than 50%. The level of GHS-R mRNA was low in confluent preadipocytes and significantly increased during the differentiation, reaching maximal level at d 8 of differentiation. Such an increasing pattern of GHS-R expression suggests an involvement of GHS-R in the process of preadipocyte differentiation.



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  		Figure 2. Levels of GHS-R mRNA in confluent preadipocytes and differentiated adipocytes. Upper panel, Representative ethidium bromide-stained agarose gel showing amplified GHS-R (599 bp) and GAPDH (452 bp) with molecular markers for different day of differentiation indicated on top. Lower panel, Data were normalized using GAPDH mRNA and expressed as percentages of the value obtained at d 0. The RT-PCR results shown are representative of three separate experiments with the same protocol. The data represent the means ± SEM of the three experiments. a–c, Different superscript letters on each column indicate significant difference between groups (P < 0.05).

 
Functional role of ghrelin on preadipocyte differentiation
To further clarify the role of GHS-R in the process of preadipocyte differentiation, an endogenous GHS-R ligand, ghrelin, was used in the differentiation medium of preadipocytes. The GPDH activity was measured in confluent preadipocytes and differentiated adipocytes at d 4, 7, and 10 of in vitro differentiation induced by insulin, methyl-3-isobutylxanthine, dexamethasone, etc. (see details in Materials and Methods). During the differentiation, GPDH activity increased significantly, and the addition of ghrelin further elevated this activity (Fig. 3Go). Likewise, the proportion of cells positive for lipid droplets was markedly greater in presence of ghrelin in culture medium than that of cells cultured in differentiated medium without ghrelin for 7 d (Fig. 4Go). Morphologically, differentiated adipocytes appeared at d 3 after cultured in differentiation medium, and the adipocytes acquired clearly lipid droplets of variable size in the cells. At d 10, more than 50% of the preadipocytes were fully differentiated into matured adipocytes in this experiment and ghrelin increased the proportion of matured adipocytes in all stages of differentiation (data not shown).



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  		Figure 4. Effect of ghrelin on rat preadipocyte differentiation. Cells were grown in DMEM containing 10% FBS for 3 d until confluent and switched to a serum-free differentiation medium with 0 M (A) or 10-8 M ghrelin (B) for the next 7 d.

 
Effect of ghrelin on the expression of PPAR-{gamma}2 mRNA
As the expression of PPAR-{gamma}2 is significantly increased in differentiated adipocytes, it has been widely used as an index of preadipocyte differentiation (18, 19, 20). In preadipocytes, the level of PPAR-{gamma}2 mRNA was not detectable (Fig. 5Go). At d 5 and 10 of preadipocyte differentiation, the levels of PPAR-{gamma}2 mRNA were significantly increased. Taking d 5 as 100% (d 0 was undetectable), the level of PPAR-{gamma}2 mRNA at d 10 was 3-fold higher. The addition of 10-8 M ghrelin during differentiation significantly increased the expression of PPAR-{gamma}2 (Fig. 5Go). This indicates also a direct facilitation of preadipocyte differentiation by ghrelin.



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  		Figure 5. Effect of ghrelin on the levels of PPAR-{gamma}2 mRNA during adipogenesis. Upper panel, Representative ethidium bromide-stained agarose gel showing amplified PPAR-{gamma}2 (454 bp) and GAPDH (452 bp) with molecular markers for different day of differentiation day indicated on top. Lower panel, Data were normalized using GAPDH mRNA and expressed as percentages of the value obtained at d 5 of control. The RT-PCR results shown are representative of three separate experiments with the same protocol. The data represent the means ± SEM of the three experiments. **, P < 0.01 vs. control at same day of differentiation.

 
Effect of ghrelin on isoproterenol-induced lipolysis in isolated adipocytes
The addition of isoproterenol (10-8 M) in freshly dissociated rat adipocytes for 40 min significantly stimulated the release of glycerol (an index of lipolysis) to a level of 6-fold of that in control group (Fig. 6Go). The addition of ghrelin alone did not modify the level of glycerol. The simultaneous addition of ghrelin and isoproterenol significantly reduced the release of glycerol induced by isoproterenol (Fig. 6Go). This result indicates that, in addition to stimulating differentiation, ghrelin also has an antilipolytic effect on adipocytes in vitro.



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  		Figure 6. Effect of ghrelin on isoproterenol-induced lipolysis in isolated rat adipocytes. Rat adipocytes were incubated with various concentrations of ghrelin and 10-8 M isoproterenol in different combinations for 40 min. Lipolysis was determined by glycerol release in the medium. All data are expressed as percentages of the basal rate of 100% in the control group without isoproterenol or ghrelin. The data represent the means ± SEM of the four experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. isoproterenol alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated first time that ghrelin acts directly on adipocytes to stimulate differentiation from preadipocytes and to antagonize lipolysis. The level of expression of GHS-R mRNA is also positively correlating to differentiation, suggesting that the ghrelin receptor in adipocytes is GHS-R already cloned (5). These results reinforce and extend the recently established concept that ghrelin regulates energy balance and adipogenesis. Ghrelin is synthesized and released primarily by the stomach and stimulates GH release in vitro and in vivo (1). GH exhibits a powerful inhibitory activity on adipocyte differentiation in primary preadipocyte culture isolated from rat (21, 22, 23). In contrast, it has been reported by others that GH promotes adipocyte differentiation in studies using preadipocyte cell lines (24, 25, 26, 27). Ghrelin has recently been considered as an orexigenic peptide (2, 5, 10, 28) and has been found to regulate gastric acid secretion and stomach movement (29). Ghrelin has also been demonstrated capable of stimulating adiposity in rodents when administered either peripherally or centrally in vivo (3, 4). This adipose-stimulating effect has been confirmed by Asakawa et al. (28), who found that ghrelin-mediated hyperphagia was a consequence of stimulating neuropeptide Y neurons. Thus, the ghrelin-inducing feeding effect is attributable to the direct hypothalamic effects of ghrelin rather than to the mediation via GH-IGF-I axis. In the present experiments, we have demonstrated a direct action of ghrelin in stimulating preadipocyte differentiation and antagonizing isoproterenol-induced lipolysis. In addition, the expression of the receptor for ghrelin (GHS-R) in adipocytes is up-regulated during the fast-growth phase of rat and during the in vitro preadipocyte differentiation. The increase in synthesis of GHS-R strongly indicates a direct adipogenesis effect of ghrelin on rat adipocytes. The positive correlation of GHS-R expression in adipose tissue and age of rats indicates that ghrelin-GHS-R system may be at least partially responsible for the clonal expansion and proliferation of adipocytes during the aging process. The current findings also suggest that ghrelin and GHS-R in adipose tissue may play a role in the development of obesity through endocrine and/or paracrine pathways.

We conducted experiments to investigate the effect of ghrelin on preadipocyte differentiation where preadipocytes were freshly isolated from rat adipose tissue. In this study, GPDH activity in differentiated adipocytes was markedly increased by the addition of ghrelin, suggesting that ghrelin facilitates adipocyte development or adipogenesis via direct action on adipocytes. This finding clearly demonstrates that GHS-R on both preadipocytes and differentiated adipocytes is fully functional and activated directly by ghrelin. Bearing in mind that the expression of GHS-R increases during differentiation, it is apparent that the action of ghrelin is strengthened during the differentiation of preadipocytes to adipocytes. In light of the in vivo physiological function, ghrelin can increase body fat accumulation or adipogenesis both directly acting on adipocytes and indirectly through acting as an orexigenic peptide. Based on available data so far, ghrelin may be a candidate to provide feedback signaling between nutrient intake, gastric motor function, and the central nervous system (30), and to induce obesity by reducing fat consumption in rodents (3). Recently, ghrelin has also been implicated in a physiological role during the initiation of meals in humans; that is, ghrelin levels increase just before mealtime (31). At present, the mechanism by which ghrelin acts on adipogenesis in any species of animal is not known. Based on the present study and data reported above from other laboratories, this new hormone, ghrelin, exhibits new roles in adipogenesis. In these new roles, ghrelin affects adipose tissues and cells at remote depots by binding to GHS-R, which is widely distributed in peripheral tissues including preadipocytes and adipocytes. This activity is distinguished from ghrelin’s frequently mentioned action as a GH secretagogue and a regulator of energy homeostasis. In addition, ghrelin has been demonstrated in this experiment to increase the expression of PPAR-{gamma}2 gene that is important for adipogenesis. PPAR-{gamma}2 is known to be a key regulator of transcriptional pathways important for adipogenesis (32). PPAR-{gamma}2 is a member of the nuclear hormone receptor superfamily of DNA-binding transcriptional activators that, in a ligand-dependent manner, activate specific target genes important to cell growth, cell differentiation, and homeostasis. Like other transcriptional activators, nuclear receptors (including PPAR-{gamma}2) act through a variety of interacting transcriptional coactivators for adipogenesis. This provides a possible mechanism for the regulation of cell-specific transcription and differentiation events through the ghrelin-GHS receptor.

Cross-talk between hormonal stimuli promoting energy storage, such as insulin, and those increasing energy expenditure, such as activation of the sympathetic nervous system, may play a crucial role in the development of obesity (33). In culture medium, ghrelin antagonizes the lipolysis induced by isoproterenol, an agonist of the adrenergic receptor. The antilipolytic effect of ghrelin is very similar to that of insulin, which inhibits lipolytic activity in fat cells (34, 35). Activation of adipocyte cAMP phosphodiesterase by insulin is believed to be the major mechanism by which insulin reduces intracellular cAMP; this reduction leads to the inactivation of not only the cAMP-dependent protein kinase but also the net dephosphorylation of hormone-sensitive lipase (34, 36, 37, 38). Ghrelin is associated with changes in energy homeostasis favoring lipogenesis in vivo. Because ghrelin in vitro partially inhibited the isoproterenol-induced lipolysis from isolated rat adipocytes, its action in adipogenesis in vivo is a combination of multiplicate activation at several sites. The imbalance between ghrelin and adrenergic signaling pathways might have pathological implications for the development of obesity, as suggested by others (3, 4).

In conclusion, data obtained in this experiment provide evidence that ghrelin directly facilitates the adipogenesis via GHS-R on adipocytes, independent of its effects on GH secretion or orexigenic action. This study also demonstrates the involvement of GHS-R in clonal expansion of adipocytes and differentiation of preadipocytes. These findings add new physiological functions to the novel gastrointestinal hormone, ghrelin, in regulating adipocytes.


   Footnotes
 
This work was partly supported by Grant-in-Aid (No. 13760194) for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to S.-G.R.) and Research Funds from Australian National Health and Medical Research Council (to C.C.).

Abbreviations: FBS, Fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHS-R, GH secretagogues receptor; GPDH, glycerol-3-phosphate dehydrogenase; KRB, Krebs Ringer Bicarbonate; PPAR-{gamma}2, peroxisome proliferator-activated receptor {gamma} 2.

Received October 25, 2002.

Accepted for publication November 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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