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Administration of Acylated Ghrelin Reduces Insulin Sensitivity, Whereas the Combination of Acylated Plus Unacylated Ghrelin Strongly Improves Insulin Sensitivity

Department of Medicine (C.G., F.M.M., J.A.M.J.L.J., P.J.D.D., P.V.K., L.J.H., F.B., A.J.v.d.L.), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; Theratechnologies Inc. (T.A.), Montréal, Québec, Canada H4S 2A4; and Section of Endocrinology and Metabolism (F.B., E.G.), Department of Internal Medicine, University of Turin, 10126 Turin, Italy

Address all correspondence and requests for reprints to: C. Gauna, M.D., Section of Endocrinology, Department of Medicine, Room Ee542, Erasmus Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: c.gauna{at}erasmusmc.nl.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We investigated the metabolic actions of ghrelin in humans by examining the effects of acute administration of acylated ghrelin, unacylated ghrelin, and the combination in eight adult-onset GH-deficient patients. We followed glucose, insulin, and free fatty acid concentrations before and after lunch and with or without the presence of GH in the circulation.

We found that acylated ghrelin, which is rapidly cleared from the circulation, induced a rapid rise in glucose and insulin levels. Unacylated ghrelin, however, prevented the acylated ghrelin-induced rise in insulin and glucose when it was coadministered with acylated ghrelin. Surprisingly, the injection of acylated ghrelin induced an acute increase in unacylated ghrelin and therefore total ghrelin levels. Finally, acylated ghrelin decreased insulin sensitivity up to the end of a period of 6 h after administration. This decrease in insulin sensitivity was prevented by coinjection of unacylated ghrelin. This combined administration of acylated and unacylated ghrelin even significantly improved insulin sensitivity, compared with placebo, for at least 6 h, which warrants studies to investigate the long-term efficacy of this combination in the treatment of disorders with disturbed insulin sensitivity.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
GHRELIN IS PREDOMINANTLY produced by the stomach, but it is also detectable in many other tissues as well (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Ghrelin can stimulate GH secretion, which is mediated by the activation of the so-called GH secretagogue (GHS) type 1a receptor (GHS-R1a). However, ghrelin exhibits additional activities including, for example, stimulation of prolactin and ACTH secretion, stimulation of a positive energy balance, gastric motility, and acid secretion but also modulation of pancreatic exocrine and endocrine function as well as effects on glucose levels (2, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). Ghrelin is the first natural hormone known in which the hydroxyl group of one of its serine residues is acylated by n-octanoic acid (1). This acylation is essential for binding to the GHS-R1a receptor, for the GH-releasing capacity of ghrelin and likely also for its other endocrine actions (1, 29, 30, 31). Ghrelin is expressed by pancreatic endocrine -cells, in rat and human tissue, according to some authors (32), and by pancreatic ß-cells according to one group only (33). Moreover, ghrelin is not coexpressed with any known islet hormone, and the ghrelin cells may therefore constitute a new islet cell type (34). Ghrelin seems to exert a tonic inhibitory regulation on insulin secretion from pancreatic ß-cells, and a clear negative association between ghrelin and insulin secretion has been found in humans as well as animals by some (23, 35, 36, 37, 38, 39), although not by others (40). Also, ghrelin induces a significant increase in human plasma glucose levels, which are surprisingly followed by a reduction in insulin secretion (17). We as well as others (41, 42, 43) already reported that acute as well as chronic treatment with GHS, particularly nonpeptidyl derivatives, induces hyperglycemia and insulin resistance in a considerable number of elderly subjects and obese patients. This suggests that ghrelin exerts a significant role in the fine-tuning of insulin secretion and glucose metabolism. Also, ghrelin secretion may be suppressed, at least in part, by an increased plasma glucose level and insulin, as shown by hyperinsulinemic euglycemic clamp studies in healthy subjects (38, 44, 45). However, it has also been suggested that ghrelin could have direct stimulatory effects on glycogenolysis (17).

To further investigate the effects of ghrelin on glucose and insulin handling in humans, we studied in adult-onset GH-deficient patients the effects of a single iv administration of placebo, acylated human ghrelin (AG), unacylated human ghrelin (UAG), and a combination of AG and UAG on glucose and free fatty acid (FFA) metabolism before and after lunch and with or without the presence of GH.

With this study design, we wanted to address the acute effects of human ghrelin on parameters of glucose and lipid metabolism with or without the presence of GH and determine whether UAG has any intrinsic metabolic effects as well as whether UAG can modify the effects of AG.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Eight male subjects with a pituitary insufficiency but who were otherwise healthy were asked to participate (range 21–69 yr, age 55 ± 10; mean ± SEM) and a body mass index of 29.4 ± 2.8 (mean ± SEM). All were treated by transsphenoidal surgery at least 2 yr before enrollment for nonfunctioning pituitary tumors, and all were on stable replacement therapy for their pituitary-dependent thyroidal, adrenal, and gonadal insufficiency, including GH therapy for at least more than 1 yr, and all had a serum total IGF-I concentration within the age- and sex-adjusted normal range. All subjects were admitted at the Clinical Research Unit. No alcoholic beverages were allowed from the day before admission until the end of the study. Also, all subjects were asked to skip the administration of their GH replacement every night before each of the 5 admission days. All subjects gave their written informed consent to participate in the study, which had been approved by the hospital’s ethical committee.

All subjects underwent the following five testing sessions, each after an overnight fast, in random order and at least 1 wk apart: 1) placebo (saline 3 ml iv); 2) AG (Neosystem S.A., Strasbourg, France; 1.0 µg/kg iv, using a bacterial filter system); 3) UAG (Neosystem; 1.0 µg/kg iv, using a bacterial filter system); 4) AG (1.0 µg/kg iv) but this time after the normal GH replacement dose was administered 15 min before; and 5) AG in combination with UAG (both 1.0 µg/kg iv but via separate injection sites). All tests started in the morning at 0930 h, 30 min after one or two indwelling catheters had been placed into an antecubital vein, kept patent by slow infusion of isotonic saline.

After the administration at 1000 h of AG or the combination of UAG and AG, blood samples were collected for 2 h, after which a meal was given that consisted of two slices of bread with butter and preservatives along with a glass of milk. This meal was taken by all subjects on all test days.

Assessments

Insulin was assessed with a RIA (Medgenix Diagnostics, Brussels, Belgium; intra- and interassay coefficients of variation 13.7 and 8.0%, respectively). Glucose was assessed with an automatic hexokinase method (Roche, Almere, The Netherlands). FFAs were determined with an enzymatic colorimetric method (Wako Chemicals GmbH, Neuss, Germany; intra- and interassay coefficients of variation 1.1 and 4.1%, respectively).

AG and total ghrelin concentrations were measured, using a commercially available RIA (Linco Research Inc., St. Charles, MO). This assay uses an antibody, which is specific for ghrelin with the n-octanoyl group on serine-3. The Linco ghrelin (active) assay uses ¹²⁵I-labeled ghrelin and a ghrelin antiserum to determine the level of active ghrelin in serum, plasma, or tissue culture media by the double antibody/PEG technique. The lowest level of ghrelin that can be detected by this assay is 10 pg/ml when using a 100-µl sample size. Within- and between-assay variations of the AG assay are, respectively, 7 and 13%. The Linco total ghrelin within- and between-assay variations are, respectively, 5 and 15%.

Statistical analyses

Differences between the several study days were calculated, using a Newman-Keuls multiple comparison one-way ANOVA test (GraphPad Prism 4 for Windows; GraphPad Inc., San Diego, CA). P < 0.05 was considered significant. Areas under the curve were calculated using the trapezoid rule.

The results are expressed as mean ± SEM.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Ghrelin levels

The iv administration of 1 µg/kg pure AG induced only a relatively small peak in AG levels in serum, which had already disappeared within 2 h. Apparently, most of the AG was almost immediately degraded or eliminated (Fig. 1). However, as shown in Table 1, the total ghrelin concentration after administration of AG was significantly higher than when UAG was administered (P < 0.05). Moreover, total ghrelin levels after injection of UAG + AG were significantly higher than after the injection of UAG (Table 1 and Fig. 2).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Total ghrelin () and AG () concentrations (nanograms per liter) after an iv bolus injection of 1 µg/kg AG in six GH-deficient subjects after an overnight fast.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Changes in serum total ghrelin concentrations as percent of baseline in six GH-deficient subjects after administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv); GH (normal daily replacement dose).

During fasting, directly after administration of study drug. Fasting glucose concentrations at baseline were 100.9 ± 2.9 mg/dl (5.6 ± 0.16 mmol/liter; 1 mg/dl = 0.05551 mmol/l). Figure 3 shows the serum glucose levels after administration of placebo, AG, and UAG, alone or together with AG, the first 2 h after administration but before lunch so when these GH-deficient subjects were still fasting. The administration of AG and to a lesser extent UAG induced significant hyperglycemia. Interestingly, when GH was administered 15 min before the administration of AG, this hyperglycemia did not occur, which was also true when AG and UAG were given simultaneously.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Acute changes in serum glucose concentrations as percent of baseline in six GH-deficient subjects after administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv). Only the data from the first 2 h after administration when subjects were still fasting are shown. The inserted table indicates which parameters significantly differ in AUC.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Subacute changes in serum glucose concentrations as percent of baseline (t = 120 min) in six GH-deficient subjects after administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv); GH (normal daily replacement dose). Only the data starting 2 h after administration and after lunch are shown. The inserted table indicates which parameters significantly differ in AUC.

During fasting, directly after administration of study drug. Fasting insulin concentrations at baseline were 32.7 ± 6.2 µU/ml (196 ± 37 pmol/liter; 1 µU/ml = 6.0 pmol/liter). Figure 5 shows the serum insulin levels after administration of placebo, AG in the absence or presence of GH, and UAG, alone or together with AG, during the first 2 h after administration but before lunch, when these GH-deficient subjects were still fasting. The administration of AG + UAG induced a significant reduction in serum insulin levels (P < 0.05). All other interventions did not significantly change serum insulin levels during the first 2 h after administration.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Acute changes in serum insulin concentrations as percent of baseline in six GH-deficient subjects after administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv). Only the data from the first 2 h after administration are shown. The inserted table indicates which parameters significantly differ in AUC.

View larger version (26K): [in this window] [in a new window]   FIG. 6. Subacute changes in serum insulin concentrations as percent of baseline (t = 120 min) in six GH-deficient subjects after administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv); GH (normal daily replacement dose). Only the data starting 2 h after administration and after lunch are shown. The inserted table indicates which parameters significantly differ in AUC.

During fasting, directly after administration of study drug. Fasting concentrations of FFAs at baseline were 0.94 ± 0.09 mmol/liter. There were no significant differences in the effects of the various compounds on FFA levels during the first 2 h when subjects were still fasting. Interestingly, the administration of whatever compound, including placebo, induced an increase in FFA concentrations (data not shown).

After lunch, 2–6 h after administration of study drug. Figure 7 shows that the administration of AG and UAG impressively reduced serum FFA levels, compared with placebo, AG (with or without GH), and UAG after lunch (P < 0.001 for all comparisons). AG (with or without GH), administered 2–6 h before, significantly increased FFA levels after lunch, compared with placebo, something that could not be observed when UAG was administered.

View larger version (26K): [in this window] [in a new window]   FIG. 7. Subacute changes in serum FFA concentrations as percent of baseline in six GH-deficient subjects after administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv); GH (normal daily replacement dose). Only the data starting 2 h after administration and after lunch are shown. The inserted table indicates which parameters significantly differ in AUC.

View larger version (36K): [in this window] [in a new window]   FIG. 8. Changes in AUC of serum glucose, insulin, and FFA concentrations as percent of baseline in six GH-deficient subjects during the first 2 h after the iv administration of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv); GH (normal daily replacement dose).

View larger version (33K): [in this window] [in a new window]   FIG. 9. Changes in AUC of serum glucose, insulin, and FFA concentrations as percent of baseline in six GH-deficient subjects during the first 4 h after lunch at 1200 h after the iv administration at 1000 h of placebo, AG (with or without GH), UAG, and AG + UAG. AG (1 µg/kg iv); UAG (1 µg/kg iv); GH (normal daily replacement dose).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In concordance with this is the observation that a single iv injection of 1 µg/kg AG early in the morning after an overnight fast induces a direct increase in glucose (Figs. 3 and 4) and insulin concentrations (Fig. 6) in GH-deficient subjects. Interestingly, pretreatment of the subjects with their normal replacement dose of GH, however, prevented these hyperglycemic changes. The changes in glucose were less after a single iv injection of 1 µg/kg UAG (Fig. 3). Moreover, the coadministration of 1 µg/kg UAG together with 1 µg/kg AG (at two separate injection sites) prevented the changes that were seen after the injection of AG alone because it blunted the hyperglycemic and hyperinsulinemic effects, as was also observed by us in another study in normal individuals without GH deficiency (47). These data indicate that not only AG, but also UAG, has metabolic effects because they both can induce hyperglycemia and change insulin levels, which indicates a GHS-R1a-independent effect because UAG cannot bind to the GHS-R1a (1, 48, 49). Maybe even more important is the observation that the combination of AG and UAG can blunt the effects of AG, which therefore might give us a clue why the administration of AG in our study immediately increased the levels of UAG in an attempt of the body to temper these metabolic effects of AG.

Recently another study demonstrated a clear metabolic role for UAG; Thompson et al. (50) reported not only that AG promotes bone marrow adipogenesis in vivo by a direct peripheral action but that this effect was also observed with UAG. Moreover, this effect of UAG could not be antagonized by administration of a potent synthetic GHS-R1a agonist. They concluded that the ratio of AG and UAG production might help regulate the balance between adipogenesis and lipolysis in response to nutritional status (50).

Another important observation we made is that the administration early in the morning of AG, but not of UAG, was still able to induce a state of insulin resistance in the period after lunch (so at least 6 h after a single administration). This indicates that GHSs can influence insulin sensitivity for many hours, as we already observed in earlier work, using GH-releasing peptide-6 as a ghrelin receptor agonist (43). In that study, we found that GH-releasing peptide-6, given in the morning, was able to induce insulin resistance in the afternoon in normal subjects, provided that GH action was knocked out, using pegvisomant as a GH receptor antagonist, so again when GH action was low. This effect might be of pathophysiological relevance because we think that these changes in insulin sensitivity, induced by AG, are most prominent in those subjects with low intrinsic GH levels, e.g. GH-deficient patients and subjects with syndrome-X, as well as during physiological aging (15, 35, 36, 37, 51, 52, 53, 54, 55). In other words, when GH action is reduced, GHS and AG can apparently induce a state of insulin resistance that might explain at least in part why in these situations people become more obese. Our data also indicate that these undesired changes in metabolism and phenotype could be counteracted by an increase in GH levels again or by increasing the UAG over AG ratio, e.g. by the administration of GH.

However, there might be another way to counteract the undesired effects of AG on insulin sensitivity, especially when GH action is low, because we observed that the postprandial effects after lunch of a bolus injection of the combination of both AG and UAG in the morning not only resulted in a significant improvement of insulin sensitivity, compared with the injection of AG alone but also compared with the injection of placebo. This improvement of insulin sensitivity resulted in significant decreases in serum insulin and FFA levels. These results might indicate that administration of UAG, alone or in combination with AG, in humans might improve insulin sensitivity. This might at least be true for subjects with a relative or absolute GH deficiency but maybe also for subjects with normal GH levels because our results indicated that even in the presence of GH, we could improve insulin sensitivity by the coadministration of AG + UAG.

At least, we think that AG and UAG should be considered as separate hormones and that UAG is more than just an inactive form of ghrelin. Future studies will have to address issues such as, for example, tachyphylaxis, and which receptor system and control systems are involved. We already observed, however, that AG can induce a glucagon-independent increase in hepatic glucose output in an isolated porcine hepatocyte model, which might be one of the mechanisms by which AG can increase serum glucose levels. Again in this model, UAG was able to counteract these effects of AG (Gauna, C., P. J. D. Delhanty, L. J. Hofland, J. A. M. J. L. Janssen, F. Broglio, E. Ghigo, and A. J. van der Lely, submitted for publication).

In conclusion, we found the following:

An iv bolus injection of AG is almost immediately cleared from the circulation.

Both AG and UAG immediately increase glucose and insulin levels.

When AG and UAG are injected together, this combination can prevent the acute hyperglycemic and hyperinsulinemic effects of AG and UAG when injected alone. Moreover, this combination of AG and UAG can improve insulin sensitivity for many hours, even compared with the worsening of insulin sensitivity of AG administration and even compared with placebo administration.

AG induces an acute increase in UAG (and therefore total ghrelin) levels via either a decrease in the clearance of ghrelin or an active release of UAG or a combination of both.

AG can induce a decrease in insulin sensitivity up to at least 6 h after administration, which again can be prevented or even actively improved by coinjection of UAG.

These data clearly demonstrate that the ghrelin system, using both the acylated and unacylated molecules, is actively involved in the acute and long-term control of glucose metabolism and insulin sensitivity in humans, which might enable new treatment modalities for the many disorders in which insulin sensitivity is disturbed.

	Footnotes

 
This work was supported by The Netherlands Organization for Health Research and Development ZONMW-NWO Grant 912-03-022 and an unrestricted grant provided by Theratechnologies Inc. (Montréal, Québec, Canada).

Abbreviations: AG, Acylated ghrelin; AUC, area under the curve; FFA, free fatty acid; GHS, GH secretagogue; GHS-R1a, GHS type 1a receptor; UAG, unacylated human ghrelin.

Received February 24, 2004.

Accepted July 8, 2004.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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