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Splanchnic Release of Ghrelin in Humans

Medical Department M (Endocrinology and Diabetes) (N.M., T.K.H., J.F.) and Institute of Experimental Clinical Research (N.M., T.K.H., H.O., J.F.), Aarhus University Hospital, DK-8000 Aarhus, Denmark; and Endocrine Research Unit (J.N., K.S.N.), Mayo Clinic and Foundation, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: K. Sreekumaran Nair, M.D., Ph.D., Mayo Clinic and Foundation, 200 First Street SW, Room 5–194 Joseph, Rochester, Minnesota 55905. E-mail: nair.sree{at}mayo.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Ghrelin is a novel polypeptide identified in rat stomach, and it increases GH release, gastric motility, and appetite and modulates many other organ functions. It has been reported that ghrelin may be released from a variety of other tissues, but the absolute contribution of the splanchnic bed remains to be defined.

We quantified the splanchnic output of ghrelin in 22 healthy people after an overnight fast, with indwelling catheters in the femoral artery and hepatic vein. Splanchnic ghrelin output was calculated by multiplying veno-arterial difference in ghrelin concentration with splanchnic plasma flow (measured by indicator dye dilution technique). Plasma ghrelin was measured using ¹²⁵I-labeled ghrelin and rabbit polyclonal antibody raised against octanoylated human ghrelin.

Ghrelin concentrations in the artery and in the hepatic veins were 960 ± 82 pg/ml and 1102 ± 90 pg/ml (P < 0.001), respectively. The veno-arterial concentration difference was 143 ± 38 pg/ml, amounting to 15% of the arterial concentration. The splanchnic output of ghrelin was 141 ± 43 ng/min (P < 0.003). Assuming that the half-life of ghrelin is less than 60 min, the splanchnic output would explain the entire amount of circulating ghrelin in the postabsorptive state.

We conclude that a substantial amount of ghrelin is being released from the splanchnic bed in the postabsorptive state in healthy human subjects and that splanchnic bed is the major source of circulating ghrelin in humans.

	Introduction

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GHRELIN IS A 28-amino-acid acylated polypeptide newly identified in rat stomach (1). The peptide binds to the GH secretagogue (GHS) receptor and stimulates GH release in healthy humans (1, 2). In addition, ghrelin increases gastric motility, gastric acid secretion, and appetite (3, 4). It has been reported that plasma ghrelin levels are decreased in obese subjects (5) and after food intake (6), whereas short-term fasting induces moderately elevated ghrelin levels (6). Finally, ghrelin may affect insulin secretion (7) and improve cardiac performance (8, 9).

The principal source of circulating ghrelin is thought to be the stomach. This concept is based on the original isolation of ghrelin from rat stomach (1) and on subsequent studies indicating a high content of ghrelin and ghrelin mRNA in rat and human stomach (10, 11, 12) and a 60–70% reduction of ghrelin levels in gastrectomized patients, together with an acute 50% reduction of ghrelin, 30 min after gastrectomy, in two patients (12). These studies do not exclude ghrelin secretion from the remainder of the splanchnic bed. Evidences suggest that ghrelin also may be secreted from a wide variety of other tissues (13, 14, 15, 16, 17, 18). The above experimental evidence indicates the potential for ghrelin production in multiple tissue beds, including gut, but the magnitude of release of ghrelin from these tissue beds to the systemic circulation remains to be determined.

The current study was, therefore undertaken to determine whether the splanchnic bed produces ghrelin in humans and to estimate its absolute contribution to the systemic ghrelin content. We thus investigated 22 healthy subjects in the fasting postabsorptive state, with indwelling catheters in the hepatic vein and femoral artery. Net splanchnic production rates of ghrelin were calculated by multiplying veno-arterial concentration differences with hepatic plasma flow.

	Subjects and Methods

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Subjects

Twenty-two healthy subjects (11 men and 11 women), 26.5 ± 1.2 yr old, with body mass indices of 23.6 ± 0.6 kg/m² and a total body weight of 72.0 ± 3.1 kg participated in the study. The total fat mass measured by dual-x-ray absorptiometry constituted 24.9 ± 1.7% of total body weight. Fasting blood glucose levels were normal (70–100 mg/100 ml) in all participants, and all had a normal physical examination, normal electrocardiograms, and normal hematological, renal, and hepatic function as assessed by biochemical screening. All subjects ingested a standardized weight-maintaining diet (20% protein, 30% fat, and 50% carbohydrate) prescribed by a research dietitian, for 3 d, before the study. Before the study, the protocol was submitted to and approved by the Mayo Foundation Institutional Review Board; the purpose and potential risks of the study were explained to all subjects; and informed, written consent was obtained from all participants.

Experimental design

The studies were conducted, after a 12-h overnight fast, in the General Clinical Research Center at Mayo Clinic. The evening before the study, an iv catheter was inserted into an antebrachial vein and kept patent with saline.

At 0900 h, cannulation of the hepatic vein and femoral artery was performed, as described (19, 20). Hepatic vein catheters were inserted under fluoroscopic guidance, and correct positioning was confirmed by contrast injection; the femoral artery line was used for infusion of indocyanine green (cardiogreen) and for blood sampling. Femoral artery and hepatic vein catheters were used to collect blood samples. Cardiogreen was infused at a rate of 30 mg/h, from 1030 h and 90 min forwards. Blood samples were collected in duplicate after 140 and 150 min.

Analysis

Plasma samples were assayed for ghrelin, using a commercial kit (Phoenix Pharmaceuticals, Inc., Belmont, CA). In brief, the assay measures immunoreactive levels of ghrelin using ¹²⁵I-labeled bioactive ghrelin as tracer and rabbit polyclonal antibodies raised against octanoylated human ghrelin. The coefficient of variation for the assay was 3.9%.

Calculations

Hepatic vein plasma flow was calculated using the equation (19) PF = F/(Conc_art – Conc_{Hep vein}), where F is the infusion rate, Conc_art is the arterial concentration, and Conc_{Hep vein} is the hepatic vein concentration of cardiogreen. Net splanchnic output of ghrelin was calculated by multiplying venoarterial differences of ghrelin with hepatic vein plasma flow.

Statistical analysis

Plasma hormone concentrations are expressed as means ± SEM. Paired t tests were used to assess whether arterial and venous concentrations differed and whether the splanchnic output of ghrelin was different from zero. A P value less than 0.05 (two tailed) was considered statistically significant.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Figure 1 shows individual ghrelin concentrations in femoral artery (960 ± 86 pg/ml), which were lower than the concentrations in the hepatic vein (1102 ± 90 pg/ml) (P < 0.001). There was a venoarterial concentration difference of 143 ± 38 pg/ml, corresponding to 15% of arterial levels. Splanchnic plasma flows, determined by indocyanine green, were 950 ± 40 ml/min, and calculated net splanchnic release of ghrelin was 141 ± 43 ng/min, a value which was significantly different from zero (P < 0.003).

View larger version (29K): [in this window] [in a new window]   Figure 1. Arterial and venous concentrations of ghrelin in each subject (n = 22), demonstrating higher hepatic venous concentrations (1102 ± 90 pg/ml) than arterial (960 ± 82) (P < 0.001).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

It has been estimated that the circulating half-life of ghrelin is below 1 h (8, 21). Given this and assuming a plasma concentration of ghrelin of 1000 ng/l and total plasma content of ghrelin of 3000 ng, a splanchnic production rate of 140 ng/min would imply that virtually all circulating ghrelin can be accounted for by the splanchnic bed. It should be underlined that the present approach measures the entire net splanchnic release of ghrelin, meaning that contributions from stomach, the remaining gastrointestinal tract, pancreas, and liver are all included and that any ghrelin cleared regionally in the liver, for example, is excluded. Furthermore, it is likely that the immunoassay used in the present study includes des-octanoylated ghrelin, which may have pharmacokinetics that differ from those of octanoylated ghrelin. Finally, it is possible that there may be bound and unbound fractions of ghrelin with distinct half-lives and different affinity for the antibodies used in various assays. Nonetheless, the present data provide strong evidence that almost all of ghrelin in the systemic circulation is derived from the splanchnic bed, and our results are in accordance with the concept of stomach being the principal site of production.

It is presently unresolved as to what extent ghrelin circulates freely or bound to carrier proteins such as albumin, but it is conceivable that the presence of an octanoyl component renders the molecule susceptible for binding to albumin. Albumin is being produced in the liver, so it is possible that ghrelin binds to recently produced albumin, generating an increased bound fraction in the hepatic vein. However, any such event will be of little significance for our results, because albumin is a slow turnover protein with low production rates (22, 23) and because alterations in bound vs. free ghrelin fractions will not affect the total ghrelin content.

Another concern could be that hemoconcentration across the splanchnic bed, caused by lymph production, for example, may spuriously elevate hepatic vein concentrations. This possibility is precluded by the observation that concentrations of albumin were equal in the hepatic vein and femoral artery [517 ± 23 (hepatic vein) vs. 532 ± 20 (artery), P = not significant], implying that no significant hemoconcentration was occurring.

Ghrelin, the missing endogenous ligand for the GHS receptor, is a newly discovered acylated peptide, attracting considerable physiological, pathophysiological, and pharmacological interest because it significantly modulates eating behavior and gastric function, cardiac performance, and endocrine pancreatic and pituitary function (1, 2, 3, 4, 7, 8, 9). Until now, it has been assumed that the gastrointestinal tract (and, more specifically, the stomach) constitutes the major site of production of ghrelin. These findings are supported by the demonstration of highly abundant ghrelin mRNA and ghrelin-like immunoreactivity in gastric tissue (6, 10, 11, 12) and by the finding of substantial decreases in circulating ghrelin after gastrectomy. On the other hand, the presence of high concentrations of mRNA and of ghrelin in certain cell types does not necessarily infer that these cells are the major origin of systemic ghrelin, and the reductions in peptide levels upon gastrectomy could be secondary to general illness, impeded gastrointestinal function, or altered food intake or body composition. These reservations are sustained by the fact that many tissues (including the pituitary, testes, the thyroid, white blood cells, the placenta, and kidney) seem to be producing ghrelin (13, 14, 15, 16, 17, 18). The current study verified the assumption that the splanchnic bed is the principal source of ghrelin in the circulation.

In summary, we find clear evidence that the splanchnic bed releases substantial amounts of ghrelin to the circulation, and it is likely that splanchnic tissue is the only significant source of ghrelin in the systemic circulation in the postabsorptive state. These findings are compatible with the notion that gastric cells are the principal sites for ghrelin production and release.

	Acknowledgments

 
We gratefully acknowledge the nursing staff of the General Clinical Research Center (Mayo Clinic); Maureen Bigelow, R.N.; and James Andrews, M.D. for support; and Kirsten Nyberg for technical support.

	Footnotes

 
This work was supported by NIH Grants RO1-DK-41973 and RR-00585 (to Mayo Foundation) and Center for Growth and Regeneration, University of Aarhus.

Abbreviations: GHS, GH secretagogue.

Received July 22, 2002.

Accepted October 22, 2002.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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