Acute Peripheral Metabolic Effects of Intraarterial Ghrelin Infusion in Healthy Young Men

This clinical trial suggests that ghrelin directly stimulates lipolysis in skeletal muscle.

Abstract

Context:

Ghrelin is the endogenous agonist for the growth hormone secretagogue receptor (GHS-R). Intravenous administration of ghrelin induces insulin resistance and hyperglycemia and increases the levels of free fatty acids (FFA).

Objective:

To investigate whether these effects are mediated directly by ghrelin in skeletal muscle tissue.

Design:

This study was single blinded, randomized, and placebo controlled. Eight healthy men (25.5 ± 3.1 years) received 240 min of intraarterial ghrelin infusion (4.2 ng × kg⁻¹ × min⁻¹) into one femoral artery and intraarterial placebo infusion into the contralateral artery. Simultaneous blood samples were drawn from both femoral veins and muscle biopsies were obtained from both legs during both a basal period and during a hyperinsulinemic and euglycemic clamp period.

Results:

Ghrelin significantly elevated venous FFA levels and venous dilution of palmitate, suggestive of increased lipolysis. Glucose metabolism was unchanged, and there were no direct effects on pertinent enzymes in the insulin signaling cascade. The metabolic clearance rate of acyl ghrelin was 12.5 ± 3.3 ml × kg⁻¹ × min⁻¹. Acyl and desacyl ghrelin levels both increased.

Conclusions:

The results of this study suggest that ghrelin may stimulate lipolysis directly in skeletal muscle.

The GH secretagogue receptor (GHS-R) was first identified in the pituitary gland and the hypothalamus (1), subsequent to which gut-derived ghrelin was identified as the endogenous ligand (2). The GHS-R is, however, also expressed in peripheral tissues such as skeletal muscle and fat (3) and in pancreatic islets (4), suggesting that ghrelin may exert peripheral effects.

Indeed, several lines of evidence demonstrate metabolic effects of ghrelin. Exogenous ghrelin suppresses insulin secretion, whereas anti-ghrelin antiserum enhances glucose-induced insulin secretion (5). Ghrelin knockout mice exhibit improved glycemic control during a glucose tolerance test (5, 6) and are insulin-sensitive as determined both by an insulin tolerance test and during a hyperinsulinemic euglycemic clamp (6). Conversely, in a ghrelin gain-of-function rodent model the opposite effects on glucose metabolism are observed (7).

The effect of ghrelin on insulin sensitivity and glucose tolerance appears also to be of importance during calorie restriction, where both ghrelin and GHS-R knockout mice exhibit low blood glucose levels (8). During an isocaloric diet, GHS-R knockout mice are also hypoglycemic (9) compared with wild-type littermates, suggesting that ghrelin is a counter-regulatory hormone or mediates counterregulatory effects.

Data in human models are sparse and largely based on short-term infusion studies, where ghrelin has been shown to lower insulin levels resulting in hyperglycemia (10) and to induce insulin resistance (11, 12). It is, however, difficult to dissect direct effects of ghrelin from those caused by its well known stimulatory effects on GH and ACTH secretion (13). Recently, we and others reported that hypopituitary subjects constitute a viable clinical model to study GH- and ACTH-independent effects of ghrelin (14, 15), and there is now evidence that ghrelin induces lipolysis and insulin resistance independent of GH and cortisol (14). It remains, however, to be determined whether these effects are attributable to direct actions in peripheral tissues such as skeletal muscle.

To investigate this, we infused ghrelin regionally into one femoral artery compared with concomitant saline infusion into the contralateral artery of the same healthy control subject. Our hypothesis was that ghrelin reduces the in situ uptake of glucose concomitantly with stimulation of lipolysis. Moreover, we hypothesized such effects would be accompanied by local alterations in pertinent insulin signaling pathways.

Subjects and Methods

Eight healthy men aged 25.5 ± 3.1 y with a body mass index of 23.7 ± 1.1 kg × m⁻² volunteered in this study. Each participant was required to complete a medical interview, receive a full physical examination, and to participate in an initial laboratory screening. The subjects were informed about the possible risks before giving oral and written consent to participate in the trial. The study protocol was approved by the local ethics committee (Central Denmark Region Ethics Committee, M-20070081), registered at www.ClinicalTrials.gov (ID NCT00771940), and performed according to the Declaration of Helsinki.

Study protocol

All subjects were examined on one occasion after an overnight fast. They reported to the laboratory at 0730 h and underwent a femoral artery infusion of acyl ghrelin (4.2 ng × kg⁻¹ × min⁻¹, GMP-grade human acylated ghrelin, Bachem, Weil am Rhein, Germany) for 240 min (Fig. 1). The subjects fasted throughout the infusion period but were allowed to drink tap water.

Fig. 1.

Study design.

A method to study local effects in leg skeletal muscle was applied (method article submitted). In short, the femoral artery and vein of both legs were visualized by ultrasound (Vivid e; GE, Milwaukee, WI) and cannulated under local anesthesia [Xylocain (lidocaine) 10 mg × ml⁻¹; AstraZeneca, Albertslund, Denmark] using the Seldinger technique. A single-lumen 5-Fr central venous catheter (CVC) (BD Careflow, Stockholm, Sweden) was inserted in a distal direction into both femoral veins. Into one of the femoral arteries, a double-lumen 4-Fr CVC, which allowed for both infusion and sampling, was inserted in proximal direction; into the contralateral femoral artery, a single-lumen 4-Fr CVC was inserted also in a proximal direction.

The study consisted of a basal period (120 min) followed by a 120-min hyperinsulinemic euglycemic clamp period (insulin (Actrapid) 0.6 mU × kg⁻¹ × min⁻¹; Novo Nordisk, Bagsværd, Denmark). Plasma glucose was clamped at ∼5 mmol × liter⁻¹ by adjusting the rate of infusion of 20% glucose according to arterial plasma glucose measurements every 10 min. Insulin sensitivity was calculated from the glucose infusion rate during the clamp.

The proximal lumina of the arterial catheters were used for infusion of ghrelin and placebo (isotonic saline), respectively, in a single-blind randomized manner from t = 0–240 min.

Indocyanine green dye (ICG ∼75 μg × min⁻¹; Akorn, Lake Forest, IL) was infused into both arterial catheters from t = 70–120 min and 190–240 min to allow for blood flow measurements.

A catheter was inserted into an arm vein for infusion of [9,10-³H]palmitate from t = 60–120 min (0.15 μCi × min⁻¹; GE, Buckinghamshire, UK), insulin and glucose from t = 120–240 min, and isotonic saline from t = 0–240 min.

Blood samples were drawn simultaneously from both femoral veins every 20 min for measurements of glucose, free fatty acids (FFAs), and ghrelin; every 10 min from t = 100–120, and from t = 220–240 min for lactate and ICG concentrations, and every 10 min from t = 100–120 for analysis of plasma palmitate concentrations and specific activity (SA). The distal lumen in the double-lumen arterial catheter was used for arterial blood sampling every 20 min. From the artery we measured glucose, palmitate, FFA, ghrelin, GH, insulin, C-peptide, and glucagon. Arterial blood was also sampled for measurement of lactate and ICG concentrations every 10 min from t = 100–120 and 220–240 min. In addition, glucose was measured every 10 min during the clamp period.

Muscle biopsies were obtained simultaneously under local anesthesia with Bergström biopsy needles from both lateral vastus muscles at t = 105 min and after 30 min of insulin stimulation (t = 150 min). Biopsies were cleaned for blood immediately, snap-frozen in liquid nitrogen, and stored at −80 C until analyzed. The protein fraction was isolated from the muscle biopsies as previously described (16), and protein phosphorylation of Akt Ser⁴⁷³ and Akt Substrate of 160 kDa (AS160) was determined by standard Western blotting methods (16). The results were normalized to total protein expression of Akt and AS160.

Preparation of synthetic ghrelin

One hundred micrograms of human acylated ghrelin (purity by HPLC 99.7%; GMP grade, Clinalfa/Bachem, Wheil am Rhein, Germany) was dissolved in 5 ml sterile water and diluted in isotonic saline to a total volume of 50 ml.

Blood samples and measurements

Plasma glucose was analyzed in duplicate using the glucose oxidase method (Beckman Instruments, Palo Alto, CA). Serum samples were frozen immediately and stored at −20 C. FFAs were analyzed by using a commercial kit (Wako Chemicals, Neuss, Germany). Lactate was analyzed in duplicate using the lactate oxidase method (YSI 2300 STAT Plus, Yellow Springs, OH). Acyl- and desacyl ghrelin were measured using two novel highly sensitive and specific two-site sandwich assays previously described by Liu et al. (17). The acyl ghrelin assay had a sensitivity of 6.7 pg × ml⁻¹ with an intraassay coefficient of variation (CV) of 9.1% at 30 pg × ml⁻¹, 12.6% at 100 pg × ml⁻¹, and 16.8% at 300 pg × ml⁻¹. The interassay CV was 17.8% at 50 pg × ml⁻¹. The desacyl ghrelin assay had a sensitivity of 4.6 pg × ml⁻¹ with an intraassay CV of 12.5% at 50 pg × ml⁻¹, 10.7% at 150 pg × ml⁻¹, and 18.0% at 500 pg × ml⁻¹. The interassay CV was 20.8% at 30 pg × ml⁻¹. GH and insulin were analyzed with a double-monoclonal immunofluorometric assay (Delfia, Wallac Oy, Turku, Finland). C-peptide was measured by ELISA (DakoCytomation, Cambridgeshire, UK). Glucagon was analyzed using a modified in-house RIA (18). ICG optical density was measured spectrophotometrically (Milton Roy Spectronic 601, Ivyland, PA) at a wavelength of 800 and 900 nm. Subsequently, blood flow was estimated by Fick's Principle. Plasma palmitate concentration and SA were determined by HPLC using [²H₃₁]-palmitate as internal standard. Palmitate was analyzed in triplicate during steady-state. Steady-state of SA was verified (t = 100–120 min) for each individual. Leg uptake and release were calculated from steady-state palmitate SA (dpm/μmol), plasma flow, and palmitate concentrations.

Statistics

Results are expressed as mean ± se or as median and range (minimum and maximum). Circulating levels of glucose, FFAs, lactate, acyl and desacyl ghrelin, GH, insulin, C-peptide, and glucagon were compared by calculating the area under the concentration-time curve (AUC) by the trapezoid method. Mean of the last two plasma ghrelin samples of the basal and the clamp period, respectively, was used as an estimate of the steady-state ghrelin concentrations. The whole body metabolic clearance rate (MCR) of acyl ghrelin (the amount of plasma cleared out of acyl ghrelin per time unit) was calculated as MCR = infusion rate/acyl ghrelin_steady-state. Plasma flow was estimated by calculating the mean flow during the last 20 min of the basal and the clamp period. All comparisons were made by a Student's two-tailed paired t test for normal distributed data and a Wilcoxon Signed Ranks test for non-normal distributed data. A P value less that 0.05 was considered significant. Statistical analysis was performed using SPSS version 17.0 for Windows (SPSS, Inc., Chicago, IL).

Results

The serum or plasma levels of individual analytes are referred to as “systemic” when measured in the femoral artery whereas they are referred to as “placebo” and “ghrelin” when measured in the femoral vein of the leg receiving saline and ghrelin infusion, respectively.

Acyl ghrelin

At baseline plasma acyl ghrelin levels (pg × ml⁻¹) (Fig. 2A) were 18.4 ± 6.8 (systemic), 17.2 ± 5.6 (ghrelin), and 15.3 ± 5.6 (placebo), (ghrelin vs. placebo: P = 0.82).

Fig. 2.

Concentrations and ratios of acyl and desacyl ghrelin during the study. A, Concentrations of acyl ghrelin in systemic arterial plasma and in plasma from both femoral veins. Acyl ghrelin was significantly increased in the ghrelin perfused leg compared with the placebo perfused leg (*). B, The level of acyl ghrelin in the femoral vein of the ghrelin perfused leg was 3.5 times higher than in the placebo perfused leg after 20 min. This increase (acyl ghrelin concentration in the femoral vein of the leg assigned to ghrelin infusion in relation to the leg assigned to placebo infusion) then decreased and stabilized around a twofold increase. C, Concentrations of desacyl ghrelin in plasma of systemic arterial plasma and in plasma from both femoral veins. Desacyl ghrelin was significantly increased in the ghrelin perfused leg compared with the placebo perfused leg (*). D, Ratio of acyl ghrelin as a proportion of desacyl ghrelin in the femoral veins on the leg assigned to ghrelin infusion and on the leg assigned to placebo infusion. Compared point-by-point there was only a significant difference at t = 180 min.

Plasma acyl ghrelin levels increased to 695.3 ± 135.8 (ghrelin) and 441.1 ± 104.8 (placebo) pg × ml⁻¹, (P = 0.02), at the end of the basal period. At the end of the clamp period, plasma acyl ghrelin was 719.0 ± 110.9 (ghrelin) and 355.8 ± 70.5 (placebo) pg × ml⁻¹, P = 0.002.

The levels of acyl ghrelin in the femoral veins are depicted as a ratio (ghrelin/placebo) in Fig. 2B.

Desacyl ghrelin

At baseline plasma desacyl ghrelin levels were 86.8 ± 15.6 (systemic), 76.6 ± 13.6 (ghrelin), and 82.7 ± 14.8 (placebo) pg × ml⁻¹, (ghrelin vs. placebo: P = 0.74) (Fig. 2C).

Plasma desacyl ghrelin increased to 311.0 ± 26.6 (ghrelin) and 231.5 ± 24.3 (placebo) pg × ml⁻¹, (P = 0.01), at the end of the basal period. At the end of the clamp period, plasma desacyl ghrelin was 336.5 ± 52.3 (ghrelin) and 210.0 ± 16.4 (placebo) pg × ml⁻¹, P = 0.04.

Acyl-to-desacyl ghrelin ratio

At baseline, acyl/desacyl ghrelin ratios were 0.19 ± 0.06 (ghrelin) vs. 0.16 ± 0.05 (placebo), P = 0.61. The ratios increased to 2.07 ± 0.36 (ghrelin) vs. 1.84 ± 0.37 (placebo), (P = 0.43), at the end of the basal period. At the end of the clamp period, the ratios were 2.31 ± 0.41 (ghrelin) vs. 1.72 ± 0.31 (placebo), P = 0.003 (Fig. 2D).

Metabolic clearance rate of acyl ghrelin

MCR of acyl ghrelin was 12.5 ± 3.3 ml × kg⁻¹ × min⁻¹ (=18.0 liter × kg⁻¹ × day⁻¹) during the basal period and 12.9 ± 2.8 ml × kg⁻¹ × min⁻¹ (=18.6 liter × kg⁻¹ × day⁻¹) during the clamp period, P = 0.48.

Systemic serum GH levels were 5.4 ± 1.4 μg × liter⁻¹ at baseline. Ghrelin infusion stimulated endogenous GH secretion to a maximum of 19.0 ± 3.6 μg × liter⁻¹ at t = 180 min followed by a decline toward baseline levels at t = 240 min (Fig. 3A).

Fig. 3.

Hormonal response to the intraarterial infusion of acyl ghrelin, insulin sensitivity, and plasma flow. Vertical line divides basal from clamp period. A, Serum levels of GH. B, Serum levels of insulin. C, Serum levels of C-peptide. D, Serum levels of glucagon. E, Hyperinsulinemic clamp. Glucose infusion rate during the clamp (from t = 120–240 min). F, Plasma flow during the terminal 30 min of both the basal and the clamp period measured in both legs. There was no effect of ghrelin infusion on the flow rates.

Systemic serum insulin levels were 71 ± 9 pmol × liter⁻¹ at baseline and remained at a plateau during the basal period. During the clamp period, insulin achieved a steady-state level of 242 ± 18 pmol × liter⁻¹ (Fig. 3B). Systemic serum C-peptide levels were 667 ± 97 pmol × liter⁻¹ at baseline and declined modestly during the basal period (ANOVA P < 0.05) followed by a more pronounced suppression during the clamp period to a nadir of 201 ± 58 pmol × liter⁻¹ at t = 240 min (Fig. 3C).

Systemic serum glucagon levels were 62 ± 7 ng × liter⁻¹ at baseline and declined gradually to a nadir of 41 ± 6 ng × liter⁻¹ at t = 180 min, where after an increase to 55 ± 18 ng × liter⁻¹ was recorded at t = 240 min (Fig. 3D).

Leg plasma flow

Plasma flow was similar in both legs during the last 20 min of both the basal, P = 0.81, and the clamp period, P = 1.00 (Fig. 3F).

Glucose

At baseline systemic and venous plasma glucose levels were similar across both legs [(mmol × liter⁻¹) 5.6 ± 0.1 (systemic) vs. 5.6 ± 0.2 (both ghrelin and placebo), P = 0.63)]. There was a slight increase in systemic as well as venous glucose levels in both legs during the basal period (Fig. 4A), but the uptake of glucose across both legs, expressed as the area under the curve (AUC), was similar during the basal as well as the clamp period [AUC_glucose (mmol × liter⁻¹ × min), basal: 2.7 ± 2.4 (ghrelin) vs. 5.8 ± 0.7 (placebo), P = 0.22; clamp: 12.9 ± 4.0 (ghrelin) vs. 13.3 ± 6.9 (placebo), P = 0.94] (Fig. 4B).

Fig. 4.

Metabolite concentrations and arteriovenous differences in systemic arterial serum and plasma and in both femoral veins in response to intraarterial infusion of acyl ghrelin. Vertical line divides basal from clamp period. A, Plasma glucose. B, Arteriovenous differences in plasma glucose. The AV-difference increased during hyperinsulinemia, but there was no difference between the legs. C, Serum free fatty acids. D, Arteriovenous differences in serum free fatty acids. During the clamp, there was a significant difference of free fatty acids with regards to the AUC. Compared point-by-point there was significant increased free fatty acid concentration at t = 140 in the ghrelin perfused leg (*). D, Plasma lactate. E, Arteriovenous differences in plasma lactate. Plasma lactate AV-differences decreased to a similar extent in both legs during hyperinsulinemia. F, Arteriovenous difference in plasma lactate. There was no significant difference between the two legs.

FFA

Baseline FFA levels (mmol × liter⁻¹) were 0.40 ± 0.08 (systemic) vs. 0.43 ± 0.07 (ghrelin) vs. 0.39 ± 0.06 (placebo), P = 0.48. Systemic and venous FFA levels increased during the basal period (Fig. 4C), but the net release of FFA across both legs, expressed as AUC, was similar during the basal period [AUC_FFA (mmol × liter⁻¹ × min) −6.6 ± 3.3 (ghrelin) vs.−4.9 ± 2.5 (placebo), P = 0.75]. By contrast, FFA net release was significantly elevated in the ghrelin-infused leg during the clamp [AUC_FFA (mmol × liter⁻¹ × min): −4.2 ± 1.4 (ghrelin) vs. 1.7 ± 2.4 (placebo), P < 0.05] (Fig. 4D).

Palmitate

The specific activity of palmitate was reduced in the venous blood of the ghrelin perfused leg compared with the placebo perfused leg (disintegrations × min⁻¹ × μmol⁻¹): 0.81 ± 0.12 (ghrelin) vs. 0.88 ± 0.12 (placebo), P = 0.047. The levels of isotopically determined basal palmitate net uptake were insignificantly lower in the ghrelin perfused leg, whereas palmitate net release was insignificantly increased [(μmol × min⁻¹ × leg⁻¹) uptake: 15.9 ± 4.5 (ghrelin) vs. 17.3 ± 5.4 (placebo), NS; release: 25.4 ± 7.3 (ghrelin) vs. 19.5 ± 4.9 (placebo), NS]. This translated into a significant difference in flow adjusted arteriovenous palmitate differences: −9.5 ± 3.1 μmol × min⁻¹ × leg⁻¹ (ghrelin) vs. −2.3 ± 1.1 μmol × min⁻¹ × leg⁻¹ (placebo), P = 0.018. Accordingly, venous palmitate levels were increased in the ghrelin perfused leg compared with the placebo perfused leg [(μmol × liter⁻¹) 262 ± 39 (ghrelin) vs. 238 ± 35 (placebo), P = 0.04] (Fig. 5).

Fig. 5.

Palmitate. The figure depicts the mean levels of palmitate net uptake, palmitate net release, and venous concentrations of palmitate in the placebo and the ghrelin perfused leg, respectively, from t = 100–120 min.

Lactate

The absolute levels and arteriovenous differences of plasma lactate during the terminal 20 min of the basal and the clamp periods are shown in Fig. 4, E and F, respectively. No significant effects of ghrelin were recorded.

M-value

A plateau of glucose infusion rate was obtained from t = 190 to t = 240 min (ANOVA P = 0.16). Insulin sensitivity, as assessed by the M-value, reached a maximum of 2.0 ± 0.3 mg × kg⁻¹ × min⁻¹ from t = 210 min and onwards (Fig. 3E).

Insulin activation of Akt and AS160

Densitometric quantitative bar graphs and representative Western blots from the skeletal muscle biopsies are provided in Fig. 6. Insulin stimulated phosphorylation of Akt and AS160 by ∼2 fold compared with basal, and this increase was similar in both the ghrelin and the placebo-perfused leg. There was no difference in total expression of Akt and AS160 when comparing the ghrelin-perfused leg with the placebo-perfused leg in the basal or in the clamp period.

Fig. 6.

The figure shows quantitative bar graphs and representative Western blots regarding effects of ghrelin and placebo infusion on insulin stimulated Akt Ser⁴⁷³ and AS160 PAS phosphorylation in skeletal muscle in study subjects. The bar graphs depict the percentage increase of phosphorylation from the basal to the clamp period on the leg assigned to ghrelin infusion and on the contralateral leg assigned to placebo infusion. There was no difference in basal phosphorylation levels.

Discussion

Several studies in rodent models and human subjects indicate that ghrelin in the systemic circulation impacts on peripheral glucose and FFA metabolism, and evidence also suggests that at least some of these actions are independent of the well-known ghrelin-induced stimulation of GH and ACTH (5, 6, 9, 11, 12, 15, 19). The aim of the present study was to investigate whether ghrelin exerts any such effects directly in human skeletal muscle in vivo. The basis of our model is regional perfusion with ghrelin in one femoral artery compared with simultaneous perfusion with saline in the contralateral femoral artery of the same individual. The outcome measures were uptake or release of pertinent metabolites across the same vascular beds as assessed by arteriovenous differences and concomitant measurements of arterial blood flow in both legs. In addition, we also used a palmitate tracer as a more specific means to assess the regional uptake and release of FFA. Activation of basal and stimulated insulin signaling pathways was assessed in muscle biopsies from both legs. Finally, our model also included measurements of acyl and desacyl ghrelin concentrations in different vascular beds.

Both legs received the exact same amount of GH, insulin, cortisol, glucose, FFAs, and other hormones and metabolites except ghrelin. Based on the measurements of acyl ghrelin in the veins, there was a clear and significant difference in acyl ghrelin levels (although acyl ghrelin also increased many fold in the placebo leg). Thus, the differences in FFAs and palmitate in the veins of the legs cannot be attributable to different exposures of GH, insulin, cortisol, etc. The only difference from the ghrelin perfused leg to the placebo perfused leg was the different ghrelin exposure. Furthermore, there was no day-to-day variability because each subject was examined only once—hence, the usual problem with crossover trials was eliminated. Therefore, our results cannot be solely explained by the increased GH levels but must be directly attributable to the ghrelin infusion.

We also observed that systemic levels of desacyl ghrelin increased suggesting that acyl ghrelin is metabolized to desacyl ghrelin in the peripheral circulation.

The present study is the first to report the metabolic clearance rate of acyl ghrelin during a constant intraarterial infusion. The metabolic clearance rate of constant intraarterial acyl ghrelin infusion was similar to the clearance rate after an intravenous bolus injection of 3 μg × kg⁻¹ acyl ghrelin (20). Paolo et al. (20) showed that the clearance of acyl ghrelin correlates inversely with dose and concentration. Thus, in future studies using a lower infusion rate of intraarterial acyl ghrelin would allow for increased clearance and subsequently for a lower proportion of ghrelin spillover to the contralateral placebo perfused leg.

There are some limitations to the present study. Ghrelin dose-dependently increases GH secretion (21), and to minimize GH excursions we selected to use only a fifth of the ghrelin dose used in the majority of previous ghrelin infusion studies. Besides, by using intraarterial infusion, ghrelin would circulate for a longer period before reaching the hypothalamus and the pituitary gland. Nevertheless, a pronounced GH response was observed, which may have obscured potential peripheral effects of the infused ghrelin.

Acyl ghrelin also increased in the opposite, saline-perfused leg due to spillover. While some studies report a dose–response relationship between ghrelin dose and hormonal responses (21), none exists on the dose–response of metabolic effects of ghrelin. One in vitro study in human heart muscle tissue using a radioactive labeled ghrelin isotope showed that binding of ghrelin agonist to the GHS-R was saturable (22). Thus, it is possible that a maximal or near maximal metabolic effects of ghrelin were induced in both legs.

In our study, acyl ghrelin concentration doubled in the ghrelin-perfused leg compared with the saline-perfused leg. However, desacyl ghrelin also increased in both legs although to a lesser extent. Acylation of ghrelin is essential for its binding to the GHS-R and for its hormonal and endocrine properties (2), but desacyl ghrelin may possess effects opposite to intact ghrelin via unknown mechanisms (15, 23, 24). Such opposing metabolic effects of desacyl ghrelin may partly explain why the present study did not reveal overt hyperglycemic effects or more pronounced lipolytic effects in the ghrelin perfused leg.

Lyophilized acyl ghrelin with a purity of approximately 100% was dissolved in sterile water and isotonic saline immediately before infusion. Ghrelin was not measured in the infusate, and instability may have contributed to the significant elevation of desacyl ghrelin in the peripheral circulation. Desacyl ghrelin levels increased during the initial 80 min where after it reached a plateau indicating that desacylation of the infusate was not of major significance.

Recently, it has become clear that hormones and metabolites affect the central nervous system (CNS), which in turn regulates glucose homeostasis (25). Ghrelin directly affects the CNS by crossing the blood–brain barrier and stimulating GHS-R on the NPY and AgRP neurons in the arcuate nucleus of the hypothalamus (26) and indirectly affects the CNS by activating neural mechanisms (i.e. the vagal nerve) (27). Whether peripheral administration of ghrelin and physiological surges of ghrelin activate NPY neurons and subsequently cause insulin resistance remains to be elucidated. Future studies should address whether ghrelin directly stimulates the arcuate nucleus in the hypothalamus and thereby regulates metabolism.

Previously, ghrelin-induced FFA excursions (11, 12, 15, 19) have been difficult to determine due to interference from GH and cortisol, but a study in hypopituitary adult patients showed that acute ghrelin infusion induced whole body lipolysis (14). To explore further whether these metabolic effects of ghrelin are caused by direct peripheral effects, we performed the present study. Our study suggests that ghrelin exerts direct lipolysis in peripheral tissues, and it adds to understanding the metabolic role of ghrelin. In earlier experiments (13, 14) it was not possible to determine whether ghrelin-induced lipolysis was attributable to insulin resistance or vice versa. This study shows that lipolysis is a primary effect of ghrelin.

We did not record any regional effects of acyl ghrelin infusion on glucose uptake. A number of reasons may apply, but the massive GH burst is most likely of major importance. In this regard, it is noteworthy that the observed GIR rate was low compared with previously published studies from our group in healthy subjects after overnight fasting conditions (16); this most likely reflects the insulin antagonistic effects of GH. We did not detect direct effects on pertinent enzymes (Akt and AS160) of the insulin signaling cascade, which is in agreement with recent data obtained after intravenous ghrelin infusion (14) and may be due to the concomitant increase in systemic GH levels.

In conclusion, the results of this study suggest that ghrelin directly stimulates lipolysis in skeletal muscle. We also show evidence to suggest that intact ghrelin may be desacylated in peripheral tissues, which theoretically may counterbalance the metabolic effects of intact ghrelin. It would be of future interest to investigate whether systemic ghrelin levels impact cerebral glucose metabolism in human subjects in vivo.