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Laboratori de Biologia Molecular, Nutrició i Biotecnologia, Departament de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, E-07122 Palma de Mallorca, Spain
Address all correspondence and requests for reprints to: Professor Andreu Palou, Departament de Biologia Fonamental i Ciències de la Salut, Universitat de les Illes Balears, Carretera Valldemossa Km 7.5. E-07122 Palma de Mallorca, Spain. E-mail: andreu.palou{at}uib.es.
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Abstract |
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Introduction |
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Ghrelin production and secretion by the stomach is very sensitive to the nutritional state (8). In rodents, plasma ghrelin levels increase with prolonged fasting and are decreased by re-feeding or by infusion of nutrients directly into the stomach or given iv (5, 9). The decrease of ghrelin after food intake is not due to stomach distension because it is not produced by water (5) or saline (9) ingestion. In humans, there is also a preprandial rise and a postprandial fall in plasma ghrelin levels (10). Based on these findings, it has been proposed that ghrelin is a hormone that contributes to the initiation of individual meals (10). It is not clear which factors are responsible for mediating the regulation of ghrelin secretion, but blood glucose levels may be critical because oral or iv administration of glucose decreases plasma ghrelin concentration (5).
The variations of ghrelin according to the feeding state are opposite to those observed for an important anorexigenic hormone, leptin; whereas plasma ghrelin levels rise, leptin levels decrease in response to fasting (11, 12, 13). Ghrelin stimulates and leptin inhibits appetite by modulating NPY signaling in the hypothalamus; ghrelin augments NPY gene expression and blocks leptin-induced feeding reduction, implying that there is an opposite competitive interaction between ghrelin and leptin in feeding regulation (3, 4). Although adipose tissue is the main source of leptin, this hormone is also produced by the stomach (14, 15), and a role for this protein in the short-term regulation of feeding has been suggested (16, 17).
Circulating levels of ghrelin have been demonstrated to be influenced in the long term by the type of macronutrient present in the ingested diet (18, 19). Different studies show that plasma ghrelin levels are low when a high-fat diet is ingested for a long period of time, such as 30 d (18) or 14 wk (19), and are increased when the amount of carbohydrates in the diet increases (19). It has been proposed that ghrelin down-regulation by fat ingestion might serve as a counter-regulatory mechanism to limit the development of dietary-induced adiposity and may signal when a high-calorie diet is ingested (19). Besides these facts, little is known about the short-term regulation of ghrelin production and release inresponse to the intake of different macronutrients. Here we have studied ghrelin production by the stomach in response to an acute intake of two macronutrients (fat and carbohydrates given individually) after fasting and its relationship with leptin expression in the stomach and adipose tissue.
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Materials and Methods |
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After killing the animals, the stomach and the retroperitoneal white adipose tissue depot were rapidly removed. The stomach was opened and rinsed with saline containing 0.1% diethylpyrocarbonate (Sigma), and the whole epithelium was scraped off using a glass slide, obtaining approximately 0.2 g of mucosa. Gastric mucosa and adipose tissue were immediately frozen in liquid nitrogen and stored at –70 C until RNA analysis. Blood was also collected, stored at room temperature for 1 h and overnight at 4 C, and then centrifuged at 1000 x g for 10 min to collect the serum. The guidelines for the use and care of laboratory animals of the Universitat de les Illes Balears were followed.
Northern blot analysis of gastric ghrelin mRNA and of adipose tissue leptin mRNA
Gastric mucosa samples were homogenized in Tripure reagent, and total RNA was extracted following the instructions provided by Roche Diagnostics S.L. (Sant Cugat del Vallés, Spain). Thirty micrograms of total RNA, denatured with formamide/formaldehyde, were fractionated by agarose gel electrophoresis as previously described (20). The RNA was then transferred onto a Hybond Nylon membrane (Roche Diagnostics S.L.) in 20x saline sodium citrate buffer (1x saline sodium citrate is 150 mM NaCl and 15 mM sodium citrate, pH 7.0) by capillary blotting for 16 h, and fixed with UV light (20).
The mRNA for ghrelin was detected by a chemiluminescence-based procedure, using a 30-mer antisense oligonucleotide probe (5'-GTGGCTGCAGTTTAGCTGGTGGCTTCTTGG-3'), which was synthesized commercially (Genotek, Ebergberg, Germany and labeled at both ends with a single digoxigenin ligand. CDP-Star (Roche Diagnostics S.L.) was used to visualize the signals, exposing the membranes to Hyperfilm ECL (Amersham, Buckinghamshire, UK). Bands in films were analyzed by scanner photodensitometry and quantified using the Kodak 1D Image Analysis Software 3.5 (Eastman Kodak Co., Rochester, NY). Finally, blots were stripped and reprobed for 18S rRNA, as previously described (20), to check the loading and transfer of RNA during blotting.
Leptin mRNA in the retroperitoneal adipose tissue was detected essentially as described above, using a 33-mer (5'-GGTCTGAGGCAGGGAGCAGCTCTTGGAGAAGGC-3') antisense oligonucleotide probe (Genotek).
Duplicates of RNA isolation and Northern blot analysis were performed for all samples.
RT-PCR analysis of gastric leptin mRNA
For analysis of leptin mRNA in gastric samples, 0.625 µg of total RNA (in a final volume of 25 µl) was denatured at 90 C for 1 min and then reverse transcribed to cDNA using MuLV reverse transcriptase (according to the Applied Biosystems procedure; Applied Biosystems, Madrid, Spain) at 42 C for 1 h, with a final step of 5 min at 99 C in a Perkin-Elmer 2400 Thermal Cycler (PerkinElmer, Wellesley, MA). Ten microliters of the reverse transcriptase product was used for PCR amplification, following the Hot Start PCR method and using the AmpliTaq Gold DNA polymerase (Applied Biosystems). The samples were first denatured at 94 C for 10 min, and then PCR was carried out using the following parameters: 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min. The number of cycles was 36 for leptin or 24 for ß-actin. The amplification was finished by a final extension step of 10 min at 72 C. Primers for the lep gene were as follows: forward, 5'-CCA GGA TGA CAC CAA AAC CCT C-3'; and reverse, 5'-ATC CAG GCT CTC TGG CTT CTG C-3'; and primers for the ß-actin gene were as follows: forward, 5'-ACG GGC ATT GTG ATG GAC TC-3'; and reverse, 5'-GTG GTG GTG AAG CTG TAG CC-3' (21). The expected size of the products was 316 bp for the lep gene and 164 bp for the ß-actin gene, which were visualized by electrophoresis in a 1.5% agarose gel containing ethidium bromide and verified by using a DNA 100-bp ladder. The bands in the gel were quantified by scanner photodensitometry using the Kodak 1D Image Analysis Software 3.5 for Windows (Eastman Kodak). The signal for leptin mRNA was normalized to the signal of the housekeeping gene ß-actin, and the results were expressed as the leptin to ß-actin mRNA ratio.
Quantification of gastric and serum ghrelin levels
For ghrelin determination in gastric mucosa, ghrelin peptide was extracted as described by Lee et al. (18) with slight modifications. Thus, the samples of mucosa were homogenized in PBS (PBS: 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer, pH 7.4) in a Teflon/glass homogenizer (Anorsa, Barcelona, Spain). The homogenate was centrifuged at 7000 x g for 2 min at 4 C, and the supernatant was used for ghrelin quantification. The supernatant was mixed with 10 volumes of 1 M acetic acid containing 20 mM HCl. Homogenates were boiled for 20 min and centrifuged at 7000 x g for 2 min at 4 C and then lyophilized and resuspended in PBS. Ghrelin concentration in the gastric homogenates and in serum was measured with a rat ghrelin enzyme immunosorbent assay kit (Phoenix Europe GmbH, Karlsruhe, Germany).
Quantification of gastric and serum leptin levels
Gastric mucosa was homogenized at 4 C in 1:3 (wt/vol) of PBS as explained earlier. Leptin concentration in the gastric homogenates and in serum was measured with a mouse leptin ELISA kit (R&D Systems, Minneapolis, MN).
Quantification of glucose, insulin, and triacylglyceride serum levels
Serum glucose and triacylglyceride levels were measured enzymatically using commercial kits and following standard procedures (Roche and R-Biopharm, Darmstadt, Germany, for glucose and Sigma for triacylglycerides). Serum insulin was measured using an ELISA kit (DRG Instruments, Marburg, Germany).
Statistical analysis
All data are expressed as the mean ± SEM. The statistical significance was assessed by two-way ANOVA and least significant difference post hoc comparison to determine the significance of intake of the different macronutrients (standard chow diet, fat, or carbohydrates) and of re-feeding conditions (RF and PRF groups) on the measured parameters. Student’s t test was used to compare the differences of all the groups with the fasted condition and to compare the differences between the RF and the PRF groups. The threshold of significance was defined at P < 0.05.
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Results |
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). The ingestion of both carbohydrate and fat diet produced a significant decrease (P < 0.05, Student’s t test) in ghrelin mRNA expression, which, 45 min later, remained low in animals fed with carbohydrates but was recovered in animals fed with the fat diet. Ghrelin mRNA expression in stomach of fasted rats after food intake was clearly dependent on the type of macronutrients ingested (P < 0.05, two-way ANOVA), and there was an interactive effect of macronutrients ingested (chow, carbohydrates, or fat) and re-feeding conditions (RF or PRF groups; P < 0.05, two-way ANOVA).
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), and levels remained high and without significant changes after re-feeding with the different diets.
On the other hand, there was an interactive effect of macronutrients ingested and the feeding conditions (P < 0.05, two-way ANOVA) on gastric ghrelin (Fig. 1C). Ghrelin concentration in stomach decreased significantly (P < 0.05, Student’s t test) with fasting, and after re-feeding, levels remained similar in all the groups to the levels of fasted animals; a significant increase (P < 0.05, Student’s t test) was only found in the PRF vs. the RF group in the animals fed with the fat diet.
Effect of the intake of different macronutrients and different feeding conditions on gastric and adipose tissue leptin and on leptin serum levels
Twenty minutes of free access to food intake in fasted rats resulted in a decrease in the gastric content of leptin that was significant (P < 0.05, Student’s t test) 45 min after the cessation of the food stimulus in the animals eating carbohydrates and in the animals eating fat (Fig. 2A).
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Gastric leptin mRNA levels had a tendency to decrease (P = 0.080, Student’s t test) compared with fed animals after 14 hour of fasting, whereas re-feeding for 20 min with carbohydrates, but not with the other diets, induced a significant (P < 0.05, Student’s t test) increase in leptin expression (Fig. 2C).
Leptin mRNA expression in the retroperitoneal adipose tissue was significantly decreased (P < 0.05, Student’s t test) after 14 h of fasting, although re-feeding for 20 min did not significantly affect leptin expression in any of the studied groups. In the fat-fed animals, there was a significant decrease in leptin expression in the PRF group compared with the RF group (Fig. 2D).
Effect of the intake of different macronutrients and different feeding conditions on glucose, insulin, and triacylglyceride serum levels
Circulating glucose and insulin levels were significantly affected by the type of macronutrients ingested (P < 0.05, two-way ANOVA). Glucose and triacylglyceride serum levels decreased significantly (P < 0.05, Student’s t test) with fasting. Glucose levels increased significantly (P < 0.05, Student’s t test) only after re-feeding with carbohydrates; this increase was not accompanied by a significant increase in insulin levels after re-feeding either with carbohydrates or with the other diets tested. Triacylglyceride levels increased only 45 min after re-feeding with the fat diet (Table 1).
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Discussion |
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According to previous studies that showed an increase in circulating ghrelin in 24- and 48-h fasted rats (9, 13), our data show that ghrelin serum levels were significantly higher in rats after a shorter fasting period (14 h) compared with ad libitum-fed rats. In contrast, ghrelin concentration in rat stomach mucosa decreased significantly after fasting. This inverse pattern of ghrelin levels in the stomach tissue and systemic circulation has also been described previously (13) and may result from an increased secretion of ghrelin by the stomach in response to fasting.
Concerning ghrelin expression, our results show a tendency of ghrelin mRNA levels to increase in stomach in response to 14 h of fasting, whereas food intake for 20 min after fasting produced a decrease that was significant for the intake of the two macronutrients studied but not for the control diet (chow), where the decrease was significant 45 min later. This fact demonstrates that the intake of the two macronutrients, separately, can account for a sharp drop in ghrelin mRNA that is even higher than the decrease produced by the intake of a mixed meal (chow diet). Interestingly, 45 min after the ingestion, ghrelin mRNA levels increased significantly in fat-fed rats, recovering the fasted levels, whereas levels remained low when rats ate the carbohydrate diet. Serum ghrelin levels followed the same but smoother tendency. We should remark that the fat diet used contained a small percentage of protein (7.6%), so we cannot rule out that the different response of rats to both diets (carbohydrates and fat) regarding ghrelin production could be partially influenced by the presence or absence of protein in the diet.
Re-feeding with carbohydrates produces an increase in glucose serum levels that is maintained 45 min later. On the other hand, 45 min after re-feeding with fat, there is an increase in serum triacylglyceride, whereas glucose levels are not affected. So, when considering factors that could account for the difference in ghrelin expression between the RF and PRF groups in the carbohydrate- and fat-fed rats, we can hypothesize that, although the intake of both nutrients can account for a decrease in ghrelin mRNA expression, high circulating glucose levels could be important for the persistent effect of the carbohydrate diet compared with the fat diet.
Recently, a study has been published in which different macronutrients (e.g. triglycerides or dextrose) given to 2- or 3-d fasted rats were found to similarly diminish circulating ghrelin levels 90 min after ingestion (9). The different ways of the administration of nutrients, such as intragastric or iv (9) or voluntarily ingested (our study), and the difference in the fasting period can account for the velocity and the degree in which the differences in gene expression are reflected in a difference in blood levels, and this could explain why in our study, 45 min after food intake, the differences in blood levels of ghrelin were not significant.
As previously reported (22, 23, 24), we found serum leptin levels and leptin expression levels by the adipose tissue to be affected by the fed state. Both parameters were lower in 14-h fasted animals, whereas 20 min of standard chow diet intake produced an increase in serum leptin levels. Previous results have shown that gastric leptin is released into the blood in response to food intake (16, 17). Here we also found that gastric leptin levels decreased after food intake but without apparent differences after carbohydrate or fat intake, suggesting that leptin secretion from the stomach would be similarly stimulated by the intake of both macronutrients. However, carbohydrate intake, but not fat intake, stimulates gastric leptin expression and thus will probably allow the synthesis of new leptin to compensate for the emptying of the stomach stores and, later on, to reach the steady-state levels of well-fed rats.
Ghrelin and leptin circulating levels follow an inverse pattern in response to fasting. Ghrelin and leptin expression by the stomach also followed an opposite profile in response to fasting and re-feeding. These facts indicate opposite roles in the acute regulation of feeding intake of both hormones produced in the stomach, stimulation (ghrelin) and inhibition (leptin) of food ingestion, therefore transmitting fasting and satiety signals, respectively, to the hypothalamus through hypothalamic peptides, which are involved in the actions of these hormones both in the endocrine and vagal afferent pathways (25).
It is well known that different macronutrients have a different satiating capacity. Carbohydrate intake in humans has a powerful short-term satiating effect (10–15 min), which is greater than the effect of fat, although the mechanism involved has not yet been fully understood (26). Here we have demonstrated that the ingestion of a carbohydrate or fat diet can induce a sharp reduction (after 20 min of feeding) in ghrelin mRNA expression in rat stomach and, moreover, that this reduction is more persistent when the ingested macronutrient is carbohydrates; so different macronutrients would affect ghrelin expression differently, and this could be related to the satiating capacity described for these nutrients. According to these data, the inhibition of ghrelin production by the stomach in response to the different nutrients is related with the higher satiating capacity of carbohydrates compared with fat. Another study, in humans, has demonstrated that circulating ghrelin fell abruptly after a high-carbohydrate meal and an isocaloric high-fat meal and that the carbohydrate meal had a significantly greater suppressant effect on hunger feelings, with plasma ghrelin changes being associated with hunger changes (27).
On the other hand, the lack of stimulatory effect of fat-food intake on gastric leptin mRNA expression could also be related to the lower satiating capacity of this macronutrient. In addition, although the physiological implications are not clear, differences in the effect of fat intake vs. carbohydrate or mixed diet on leptin expression by the adipose tissue 45 min after food intake would agree with the differences in the satiating effects of both diets.
It has been described that nutrients may exert their inhibitory effects on ghrelin secretion luminally or systemically (9), but it is not known whether the nutrients have a direct effect on modulating ghrelin expression or whether the effect is mediated by specific hormones. It has also been proved that ghrelin release is influenced by vagal stimulation (28), which is also implicated in leptin signaling (25). More work needs to be done to clarify whether the regulation is due to a direct effect of the nutrients or is mediated indirectly by metabolic hormones and to clarify the importance of the vagal afferent transmission in signaling the hypothalamic energy homeostatic center.
In conclusion, our results agree with a role of ghrelin in the initiation of food intake and show that the inhibition of ghrelin production by the stomach in rats by feeding depends on the type of macronutrient ingested. Moreover, the different response of ghrelin production after the intake of an isocaloric amount of carbohydrates or fat can be related with the known higher satiating capacity of carbohydrates compared with fat.
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Acknowledgments |
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Footnotes |
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Abbreviations: NPY, Neuropeptide Y; PRF, post re-fed; RF, re-fed.
Received April 16, 2004.
Accepted for publication July 22, 2004.
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, P < 0.05 PRV vs. RF (Student’s t test).