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Division of Endocrinology (C.G., P.U., P.K., R.M.K., J.A.M.J.L.J., P.J.D.D., M.O.v.A., L.J.H., A.P.N.T., A.J.v.d.L.), Department of Internal Medicine, Erasmus Medical Centre, 3015 GE Rotterdam, The Netherlands; Division of Endocrinology and Metabolism (E.G.), Department of Internal Medicine, University of Turin, 10126 Turin, Italy; and Postgraduate School of Molecular Medicine (A.J.v.d.L.), 3000 Rotterdam CA, The Netherlands
Address all correspondence and requests for reprints to: Carlotta Gauna, M.D., Division of Endocrinology, Department of Internal Medicine, Room Ee542, Erasmus Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: c.gauna{at}erasmusmc.nl; or carlotta.gauna{at}unito.it.
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Abstract |
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Introduction |
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10%) of total ghrelin. The majority of circulating ghrelin is UAG and has been suggested to interact with a different receptor than the GHS-R1a (1). Ghrelin secretion is regulated by the nutritional state. Thus, systemic ghrelin levels are reduced in obesity and elevated in conditions of negative energy balance (5, 6, 7), and acute changes in energy disposal determine circadian ghrelin secretion, which is increased by short-term fasting (i.e. before meals) and suppressed immediately after feeding (8). The extent of ghrelin suppression after acute energy intake has been shown to be dependent on the type of macronutrient, carbohydrates and, among them, glucose, being the most potent inhibitors of peripheral ghrelin levels (9, 10, 11). Moreover, the magnitude of ghrelin suppression by glucose load was similar after oral and iv administration (10), and 3 h of glucose infusion halved ghrelin levels (9). The majority of reports in the literature about the nutritional regulation of ghrelin refer to peripheral total ghrelin levels, which may differ from the amount released by the gut source into the local circulation. In fact, because the main source of ghrelin is the gastrointestinal tract, ghrelin is secreted into the portal vein and has to pass the liver before it reaches the peripheral circulation. It is known that the liver passage is crucial for clearance/extraction of other hormones secreted into the hepatic portal vein (such as insulin and glucagon), and this process is subjected to acute nutritional regulation (12, 13, 14, 15). Moreover, it cannot be excluded that the liver also regulates the ratio between AG and UAG and thereby affects the physiological role of these hormones. To our knowledge, portal concentrations of total ghrelin, but not AG or UAG, have been reported by Mundinger et al. (16). However, these authors did not compare portal with peripheral levels (16).
Therefore, our aims were: 1) to investigate whether peripheral ghrelin levels differ from those secreted into the portal vein; 2) to investigate whether acute nutritional changes can modify ghrelin secretion and/or metabolism; and 3) to clarify whether the first pass effect by the liver on ghrelin secreted by the gut plays a role in regulating ghrelin metabolism (i.e. the ratio between AG and UAG). To address these questions, we used a fasting rat model with a catheterization of the portal and the jugular veins under anesthesia. We compared AG and UAG variations in fasted rats under basal conditions and after iv glucose load.
This study shows that: 1) the ratio of AG to UAG is very low in the portal vein and decreases further in the systemic circulation; 2) iv glucose tolerance test (IVGTT) in anesthetized fasting rats inhibits portal and systemic UAG, whereas it only blunts prehepatic, but not systemic, AG; and 3) hepatic clearance of UAG is small, whereas hepatic clearance of AG is about 40%. Thus, our results suggest that peripheral AG metabolic regulation and action are mainly confined within the gastrointestinal tract.
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Materials and Methods |
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Animals
Male Wistar rats (aged 10–12 wk; weight 350–400 g; Harlan Netherlands BV, Horst, The Netherlands) were housed in groups in a temperature-controlled room under a 12-h light, 12-h dark cycle and maintained on pelleted chow with free access to water. The animals were housed for at least 1 wk before starting the experiments to allow acclimatization. Animal protocols were in compliance with the Dutch regulations on animal welfare and approved by the institutional Animal Welfare Committee.
Surgery and experimental design
All studies were performed after a fasting period of 18 h (overnight) under anesthesia, and the rats were euthanized at the end of the experiment.
Animals were anesthetized using an ip injection of sodium pentobarbital (60 mg/kg induction, 20 mg/kg maintenance administered at the end of the surgical procedure before starting the experimental session). Deep anesthesia was confirmed by the absence of reflexes. Animals were kept on a warming mat to maintain core body temperature and were connected to a breathing apparatus (O2, 1 liter/min) to improve oxygenation for the entire duration of the experiment (including surgical procedure).
The surgical procedure was performed under aseptic conditions, as follows. First was the cannulation of the jugular vein. An incision was made just above the right clavicle, the connective and adipose tissues pushed aside, and the jugular vein exposed. After the jugular vein was mobilized, a catheter previously connected to a syringe and filled with saline solution was pushed inside the vessel until it reached the right atrium. Patency of the catheter was checked by aspirating blood and flushing the catheter with saline solution. The free end of the catheter was used for saline injection, treatment administration, and sampling. Second was the cannulation of the portal vein. A midline incision was made from the level of the symphysis pubis to the xiphoid cartilage. The intestines were lifted out and laid next to the animal on gauze moistened with warm saline solution to minimize dehydration. A purse-string (diameter 1 mm) was made in the wall of the portal vein, opposite the gastroduodenal vein, and then the center of the purse-string was cut and the cannula inserted into the portal vein and pushed in for a few millimeters, with the tip secured about 1 mm caudal to the liver. The patency of the cannula was checked by aspirating blood and injecting saline. The free end of the cannula was used for sampling procedure during the experiment.
Treatment administration and sampling
Rats (fasted overnight) were assigned to one of the following treatment groups: saline (1 ml), n = 12, or IVGTT, n = 12. IVGTT was performed by injecting D-glucose at a dose of 1 g/kg (50%, 1 ml maximal volume) through the jugular catheter. The dose of 1 g/kg was chosen, taking in account the reduction of insulin sensitivity caused by abdominal surgery (17) and the possible interference due to anesthesia (18, 19). Sodium pentobarbital was used because, compared with other anesthetics, it has been shown to interfere less with insulin secretion and glucose metabolism both in the fed and the fasted conditions (18, 19).
After baseline samples were taken from both catheters, treatments were administered through the jugular cannula at time 0, and samples were taken from both catheters at 1, 5, 10, 20, and 30 min after treatment administration to measure glucose, insulin and total and acylated ghrelin. At every time point, the blood volume withdrawn from each catheter (350 µl) was replaced by an equal volume of saline solution.
Blood samples were collected using ice-cold EDTA containing tubes, to which the aprotinin [Trasylol, 500,000 Kallikrein inhibitor units (KIU), 40 µl/ml] was added. Samples were immediately centrifuged and aliquots for total and acylated ghrelin assays were made. Aliquots for AG measurement were promptly acidified by adding 1 N HCl [1:10 (vol/vol)], whereas aliquots for total ghrelin measurement were not. All aliquots were kept at 4 C until the end of the experiment and then stored at –20 C until assay.
Multiple freeze/thaw cycles were avoided and aliquots were thawed only for the ghrelin assay. This procedure has been indicated by Hosoda et al. (20) and Groschl et al. (21) as a standard procedure for collection of blood samples to determine ghrelin concentrations.
Serum total ghrelin and AG levels (picograms per milliliter) were measured using RIA kits that uses [125I]ghrelin as a tracer. The specificity for rat ghrelin (total and AG, respectively) is 100%. Total ghrelin is detected by polyclonal rabbit antibodies that recognizes residues(14–28), thereby including AG, UAG, and ghrelin fragments(14–28) (Table 1
). The sensitivity of the assay is 93 pg/ml; the intraassay variation (average), 6.4% coefficient of variation (CV); interassay variation, 16.3% CV. AG is recognized by a guinea pig antighrelin specific for the ghrelin molecule octanoylated at its serine3 residue. This antibody recognizes octanoyl ghrelin, residues(1–10). Cross-reactivity with unacylated ghrelin is less than 0.1% and with ghrelin fragments(14–28) zero (Table 1). The sensitivity of the assay is 7.8 pg/ml; the intraassay variation: 7.4% CV, interassay is 13.5% CV.
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Ghrelin spiking experiments in rat plasma
To confirm that ghrelin was not degraded under the sampling and storing conditions used, rat blood was spiked with a known concentration of rat (acylated) ghrelin and recovery of total and acylated ghrelin was measured in the presence and absence of sample acidification. Rat blood was withdrawn by cardiac puncture from anesthetized animals. Blood was collected in ice-cold vials containing EDTA and aprotinin (Trasylol, 500,000 KIU, 40 µl/ml) and kept on ice. Before centrifugation, rat AG was added to different tubes at a concentration of 1000 pg/ml (in duplicate). Plasma samples without any addition of acylated ghrelin were kept as control samples (blank). After centrifugation, two aliquots for total ghrelin and two aliquots for acylated ghrelin measurement were made from each tube. One aliquot was acidified by adding 1 N HCl [1:10 (vol/vol)], whereas the other aliquot was not. All aliquots were kept at 4 C until the end of the experiment and then stored at –20 C until assay.
Total and acylated ghrelin levels were then measured in duplicate in both acidified and nonacidified samples (blank or nonspiked and 1000 pg/ml).
Calculations
Unacylated ghrelin.
UAG levels were calculated by subtracting AG from total ghrelin concentrations at every time point, in either the portal or jugular vein samples.
Hepatic clearance.
To estimate whether the liver may play a role in the clearance of ghrelin produced by the gut, we calculated the percentage of hepatic extraction using a method originally proposed by Kaden et al. (22). The percentage hepatic extraction of any given hormone is calculated as follows: [(hormone presented to the liver – hormone leaving the liver) x 100/(hormone presented to the liver)]. The ratio of the relative contribution of a hormone presented to the liver by the portal vein vs. the hepatic artery (concentration x flow) is assumed to be 3:1 (23). The percentage of portal hormone extraction is calculated as follows: [(hormone concentration in the portal vein – hormone concentration in hepatic vein) x 100/(hormone concentration in the portal vein)]. We adapted this calculation to: [(hormone concentration in the portal vein – hormone concentration in jugular vein) x 100/(hormone concentration in the portal vein)], being aware that jugular hormone concentrations may be affected by a greater dilution than the hepatic vein (due to the ancillary venous return) and/or by other sources of hormone production other than those tissues that drain into the portal vein.
Basal absolute levels of glucose, insulin, total ghrelin, UAG, and AG in the systemic (jugular) and the portal circulation are reported in Table 2.
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reports absolute areas under the curve (AUCs) of all parameters in the portal and the systemic circulation, for each group.
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) changes vs. baseline (mean ± SEM) and as areas under the curve (AUCs) (mean ± SEM).
Absolute baseline levels and AUC of total ghrelin and UAG are expressed in nanograms per milliliter, variations, and AUC are expressed in picograms per milliliter.
Statistical analysis
Statistical analysis was performed using SPSS for Windows 10.0 (Chicago, IL). Row data were checked for the presence of outliers that could bias the analysis. Because we could not detect any outlier value in either the saline or IVGTT groups, all cases have been included in the statistical analysis. Independent t test was run to compare different groups, whereas paired t test was performed to compare results within the same group ( changes vs. baseline and jugular vs. portal values). A difference was considered significant when P < 0.05.
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Results |
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).
In both treatment groups, mean concentrations (absolute AUCs) of insulin, total ghrelin, and AG were higher in portal than peripheral samples, whereas absolute concentrations of glucose in the portal vein were lower than in the jugular vein. UAG levels (absolute AUCs) were lower in jugular than in portal plasma in the saline group, whereas in the glucose-injected group, portal and peripheral concentrations were similar (Table 3).
Recovery of ghrelin spiked in rat plasma
Acylated ghrelin.
In blank (nonspiked) samples, acylated ghrelin levels in nonacidified aliquots were 8% of those in acidified plasma.
When a concentration of 1000 pg/ml of acylated ghrelin was added, the recovery of acylated ghrelin in acidified plasma was 94%. Conversely, in nonacidified plasma acylated ghrelin concentration was not different from nonspiked controls, indicating that the exogenous acylated ghrelin was almost entirely degraded.
Total ghrelin.
In blank, nonspiked samples, total ghrelin levels were similar in acidified and nonacidified plasma samples.
When a concentration of 1000 pg/ml of acylated ghrelin was added, the recovery of total ghrelin in acidified plasma was 99%, and this was similar to that found in nonacidified plasma.
Overall, the experiments show that recovery of acylated and total ghrelin after spiking is greater than 90% under the conditions that we have used and that almost complete deacylation of AG occurs in nonacidified plasma.
Glucose
Saline injection (1 ml) did not modify glucose levels at any time point in either the portal or peripheral samples (Figs. 1, A and B, and 2, A and B, respectively).
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1–0: 42.6 ± 2.0 mmol/liter, portal 1–0: 30.1 ± 2.3 mmol/liter, P < 0.01).
AUCs were higher in the IVGTT than the saline control group (P < 0.0005), in both the portal and jugular samples. (Figs. 1B and 2B).
Insulin
After saline injection, a slight but significant decrease of insulin levels was observed in the portal vein at 1 min (1–0: –1.0 ± 0.3 µg/liter, P < 0.01) and persisted up to 30 min, without significant changes during the time course (Fig. 1C). In the systemic samples, insulin levels were reduced by saline at 5 min (5–0: –0.5 ± 0.2 µg/liter; P < 0.05) and up to 30 min (Fig. 2C).
As expected, IVGTT induced a prompt response in insulin levels in both the portal and systemic samples. In the portal vein, insulin peaked at 1 min (1–0: 5.7 ± 1.0 µg/liter, P < 0.01) and at 5 min was back to baseline levels. However, insulin levels were higher in the IVGTT than the saline group during the whole time course (Fig. 1C), as well as the AUC (49.2 ± 19.9 µg/liter · min–1 vs. –44.61 ± 13.6 µg/liter · min–1; P < 0.0005) (Fig. 1D).
In the peripheral samples, insulin responded to glucose administration following a pattern similar to that observed in the portal vein, although the changes were less impressive (peak: 1–0: 2.2 ± 2.3 µg/liter, P < 0.01; AUC IVGTT vs. saline: 26.9 ± 7.9 µg/liter · min–1 vs. –31.9 ± 11.0 µg/liter · min–1, respectively, P < 0.0005) (Fig. 2, C and D).
UAG
UAG concentrations in the portal vein were not significantly modified by saline injection, although a slight, but not significant, transient decrease was observed at 10 min (nadir 10–0: –198 ± 95 pg/ml) (Fig. 1E). In the peripheral samples, UAG levels were significantly blunted only at 10 min (10–0: –140 ± 62 pg/ml, P < 0.05), after which they returned to baseline (Fig. 2E).
IVGTT suppressed UAG levels in the portal circulation during the whole study period (nadir 1–0: –369 ± 40 pg/ml, P < 0.0005) (Fig. 1E). AUC of UAG was lower in the IVGTT than the saline group (–8151 ± 1522 pg/mlmin–1 vs. –2421 ± 2021 pg/ml · min–1, P < 0.03) (Fig. 1F).
In the peripheral circulation, UAG was also inhibited during the whole study period (for all time points, changes vs. baseline: P < 0.01) (Fig. 2E). The inhibition of UAG was greater after IVGTT than after saline administration (AUC: –6697 ± 1195 pg/ml · min–1 vs. –2207 ± 1756 pg/ml · min–1, respectively, P < 0.05) (Fig. 2F).
AG
Portal AG levels were transiently and slightly, although not significantly, reduced by saline administration and rose above baseline levels at 30 min (P < 0.05) (Fig. 1G). variations in the peripheral samples were also slightly inhibited, being statistically significant only at 1 and 10 min (P < 0.01) (Fig. 2G).
IVGTT induced a suppression of portal AG levels up to 10 min, with a gradual return to baseline at later time points (Fig. 1G). Portal AUC was lower during IVGTT than during saline (–716 ± 404 pg/ml · min–1 vs. –16 ± 406 pg/ml · min–1), although the difference was not statistically significant (Fig. 1H). Systemic AG levels were decreased by IVGTT injection during all time courses (P < 0.01) (Fig. 2G). However, the inhibition induced by IVGTT was not different from that observed after saline (Fig. 2H).
Hepatic clearance
Insulin.
In the saline-treated group (i.e. in fasting conditions), the percentage of insulin clearance by the liver was 56.6 ± 3.7% at baseline and did not vary during the time course. (Fig. 3A).
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UAG.
In the saline group, hepatic clearance of portal UAG was 11.4 ± 3.8% at baseline, and it slightly increased (not significantly) during the time course, reaching a maximum at 30 min (19.8 ± 3.7%, P = NS vs. baseline) (Fig. 3A).
IVGTT transiently but significantly reduced the percentage of UAG cleared by the liver (from 10.2 ± 2.8% at baseline down to a nadir of –3.6 ± 3.4% at 5 min, P < 0.004), which gradually increased to rise above baseline at 30 min (23.3 ± 3.6%, P < 0.01) (Fig. 3B). However, the percentage of UAG cleared by the liver during IVGTT was not different from the clearance in the saline-treated animals (percent AUC: 7.8 ± 3.3 vs. 11.3 ± 3.0%, respectively).
AG.
In the saline-treated rats, the hepatic removal of endogenous AG was 34.0 ± 3.5% at baseline, without significant changes over the 30-min time course after saline injection (Fig. 3A).
After IVGTT, AG extraction did not change over the time course and remained similar to that observed after saline (percent AUCs: 37.8 ± 4.9 vs. 37.9 ± 6.0%, respectively) (Fig. 3B).
Ratio of AG to UAG
Portal AG at baseline was approximately 7% of total ghrelin (saline: 6.8 ± 0.8%, IVGTT: 6.9 ± 0.6%), and it slightly increased at 30 min in all animals (Fig. 4A). In the systemic circulation, the percentage of AG was also similar in the saline and IVGTT groups (baseline: 5.8 ± 0.8 and 4.7 ± 0.3%, respectively), without significant variations over the 30-min observation (Fig. 4B).
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Discussion |
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Furthermore, we found that that the liver has a preference for the clearance of AG over UAG, as shown by the observation that the prehepatic AG to UAG ratio is higher than in the peripheral circulation. In both fasting and (iv) glucose-stimulated conditions, approximately 38% of the AG was taken up by the liver. Conversely, the fraction of UAG removed after hepatic passage was relatively small (11%) and could be further reduced (down to 8%) by parenteral glucose.
Our data show for the first time that, in rats, an iv glucose load (slightly) blunts prehepatic but not systemic AG levels. In fact, in our model, glucose administration induced a decrease from baseline of portal and, to a much lesser extent and transiently, systemic AG levels. However, when compared with values obtained in the control (saline injected) group, only a slight decrease in portal AG concentrations was recorded, whereas peripheral levels were similar to those observed in the controls. Our data are, at least in part, controversial with previous reports (24, 25). Gordon and McKeever (24) found that peripheral AG levels decreased from baseline values after glucose infusion in horses, although in this study the possible effects of a saline infusion were not evaluated. Hotta et al. (25) observed a suppression of AG levels after a 2-h glucose infusion in humans. The discrepancy between our data and those presented by Hotta et al. may be due to methodological aspects and the ghrelin assay used. In fact, in this study parameters were measured at 1 and 2 h during a continuous glucose infusion, whereas we evaluated acute effects (up to 30 min) of a bolus administered at baseline. Second, in the paper by Hotta et al., glucose infusion inhibited AG (as measured by RIA and ELISA) and UAG (as measured by ELISA), whereas total ghrelin levels as measured by RIA showed a nonsignificant tendency to decrease (25) Conversely, previous studies in rodents reported an inhibitory effect of parenteral glucose administration on peripheral ghrelin levels using assays that recognized both AG and UAG (9, 10). In line with this, we observed a suppressive effect of glucose on total and UAG levels, which was more marked in the portal than in the systemic circulation. The changes in UAG and (prehepatic) AG levels were opposite to those observed in glucose and insulin concentrations, suggesting that, in accordance with the majority of data in the literature, insulin, glucose, or both suppressed ghrelin levels (5, 8, 9, 26, 27, 28, 29).
Although we cannot fully exclude that some degradation and/or desoctanoylation occurred, we took all possible precautions to protect AG stability in our samples (20, 21). Therefore, the relative changes in UAG and AG levels probably reflect a physiological regulation in these conditions.
Moreover, a disadvantage of rat model used in these studies is that we observed a slight reduction of AG, UAG, and insulin levels also in the saline-injected animals, which may reflect neuronal, neurohormonal and hemodynamic factors as a consequence of abdominal surgery and/or anesthesia. However, by comparing glucose-treated with control animals, we believe that our model is valid.
To our knowledge, this is also the first report in the literature showing that in basal and glucose-stimulated conditions, portal ghrelin levels (either AG or UAG) are higher than the systemic levels. This is in agreement with a very recent publication by Dezaki et al. (3), who reported higher concentrations of AG and UAG (of pancreatic origin) in the pancreatic vein than the pancreatic artery. However, in our rat model the higher levels in the portal vein do not only reflect an increased release of AG and UAG by the pancreas but also by the stomach and/or the intestine. Interestingly, the gradient between portal and peripheral levels of UAG (1.1-fold), although statistically significant in fasted rats (i.e. saline group), was not as striking as the difference between portal and peripheral concentrations of AG (1.6-fold) or insulin (2.5-fold). Therefore, we hypothesize that the clearance of UAG and AG by the liver differs and is responsible for the observed different concentrations in the systemic circulation. In this respect, the metabolism of ghrelin may be comparable with that of insulin in the liver. It is known that the amount of insulin released in the periphery is regulated by hepatic extraction, which varies, depending on acute nutritional changes (23, 30). We used insulin hepatic clearance as a reference to be able to compare with what extent ghrelin is taken up by the liver. We found that, in fasting conditions, the liver clears only a small fraction (11%) of UAG, which was further, but transiently, decreased by glucose, indicating that almost all of the UAG that reaches the liver is delivered to the systemic circulation. Conversely, AG was cleared by the liver by 38% and was not altered after iv glucose load. In accordance with Kaden et al. (22), insulin extraction after single liver passage was found to be 51% and, like AG, it was not significantly modified by the iv meal.
It has been shown that the liver adapts rapidly to fluctuations in portal insulin concentrations to quench the delivery of large oscillations of insulin from the portal vein to the systemic circulation (14, 31). In our study, despite the (2.5-fold) increase in portal insulin levels after a glucose load, the percentage of insulin extraction was not significantly changed, which suggests that the absolute amount of insulin cleared by the liver was largely increased in these circumstances. The fact that the decrease in portal AG levels after glucose was small, whereas the hepatic clearance of AG did not change, may account for the lack of an effect of glucose on systemic AG concentrations. Conversely, portal UAG levels were inhibited by glucose, whereas hepatic clearance of UAG was almost zero during glucose, and therefore, almost all UAG was delivered to the systemic circulation. Moreover, and in agreement with this, we found that the ratio AG accounts for only 7% of total ghrelin in the portal vein and that the ratio of AG to UAG further decreases in the systemic circulation. Although these data provide no evidence as to the fate of the AG fraction removed by the liver, it is possible that the liver is responsible, at least in part, of AG deacylation and/or degradation in inactive fragments. In this respect, De Vriese et al. (32) showed that a 2-h incubation of (acylated) ghrelin with liver homogenates leads to the formation predominantly of UAG and, to a much lesser extent, (inactive) fragments derived from cleavage of the AG molecule at its N terminus. In this study, a precise quantification N terminus fragments of AG is difficult because the assay used detects acylated peptide regions including residues(1–10), whose activity is not known. However, enzyme activity in vivo is likely to differ from that in tissue homogenates. Therefore, the hepatic mechanisms of ghrelin deacylation remain to be clarified.
Overall, these observations suggest that an acute glucose administration regulates mainly prehepatic concentrations of AG; thereby AG may play a role in glucose metabolism and/or insulin sensitivity in the liver. This is in accordance with previous studies showing a direct effect of AG on glucose output by primary porcine hepatocytes (33) and an AG-dependent modulation of insulin action in a hepatoma cell line (34). We speculate that the relative increase of UAG fraction in the peripheral circulation reflects the buffering of AG metabolic actions, perhaps to improve peripheral insulin sensitivity. Moreover, the hepatic clearance and/or deacylation of AG might be an additional physiological mechanism modulating the central effects of AG (i.e. on energy homeostasis and feeding behavior) in response to the nutritional state.
Our results show that acute nutritional changes may differentially regulate UAG and AG concentrations, at least after an acute parenteral administration. Moreover, this study suggests that liver clearance plays an important role in the regulation of the amount of AG released to the systemic circulation. Indeed, the relevance and the regulation of hepatic clearance of ghrelin need to be further elucidated. However, the assumption that systemic total ghrelin levels reflect acylated ghrelin secretion should be made with caution.
In conclusion, the present data show that: 1) The ratio of AG to UAG is already very low in the portal vein and decreases further in the systemic circulation; 2) an acute iv glucose load in anesthetized fasting rats inhibits UAG in the portal and systemic circulation, whereas it blunts only prehepatic but not systemic AG concentrations; and 3) UAG is cleared by the liver in a small amount in fasting conditions. After glucose administration, all the UAG secreted into the portal vein was delivered to the systemic circulation. Conversely, the hepatic clearance of AG was not influenced by acute changes in glucose and/or insulin levels.
Overall, our results may suggest that AG acute metabolic regulation and action are mainly confined within the gastrointestinal tract.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online August 2, 2007
Abbreviations: AG, Acylated ghrelin; , absolute 
; AUC, area under the curve; AUC, area under the curve; CV, coefficient of variation; GHS-R1a, GH secretagogue receptor type 1a; IVGTT, iv glucose tolerance test; UAG, unacylated ghrelin.
Received February 15, 2007.
Accepted for publication July 24, 2007.
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