PhD thesis

Clinical aspects of ghrelin: Pharmacokinetics and

metabolic effects

PhD dissertation

Esben Thyssen Vestergaard, MD

Faculty of Health Sciences University of Aarhus 2008

Esben Thyssen Vestergaard Clinical aspects of ghrelin

Clinical aspects of ghrelin: Pharmacokinetics and

metabolic effects

PhD dissertation

Esben Thyssen Vestergaard, MD

Faculty of Health Sciences University of Aarhus Medical Department M and Medical Research Laboratories Aarhus University Hospital DK-8000 Aarhus C Denmark

1


Publications This dissertation is based on the following articles

i. ET Vestergaard, TK Hansen, LC Gormsen, P Jakobsen, N Møller, JS Christiansen,

JOL Jørgensen. Constant intravenous ghrelin infusion in healthy young men: Clini-

cal pharmacokinetics and metabolic effects. 2007. American Journal of Physiology

– Endocrinology and Metabolism 292(6): E1829-1836.

ii. ET Vestergaard, CB Djurhuus, J Gjedsted, S Nielsen, N Møller, JJ Holst, JOL Jør-

gensen, O Schmitz. Acute effects of ghrelin administration on glucose and lipid

metabolism. 2007. Journal of Clinical Endocrinology & Metabolism. Epub.

iii. ET Vestergaard, LC Gormsen, N Jessen, S Lund, TK Hansen, N Møller, JOL Jør-

gensen. Ghrelin infusion in humans induces acute insulin resistance and lipolysis

independent of GH-signaling. Article submitted for publication.

2


Contents Publications ............................................................................................................... 2 Contents .................................................................................................................... 3 List of abbreviations ................................................................................................... 6 Preface ...................................................................................................................... 8 Background...............................................................................................................10 Methods ...................................................................................................................12

Subjects ................................................................................................................12

Pharmacokinetic study ........................................................................................12

Somatostatin study .............................................................................................13

Hypopituitary study.............................................................................................13

Study design..........................................................................................................14




Pharmacokinetic modeling ......................................................................................17

Tracers..................................................................................................................19

Indirect calorimetry................................................................................................20

Microdialysis ..........................................................................................................21

Western blotting and PI3K assay.............................................................................21

Analyses................................................................................................................22

Statistics................................................................................................................23

Summary of results ...................................................................................................24 Pharmacokinetic study............................................................................................24

Ghrelin pharmacokinetics ....................................................................................24

Correlations........................................................................................................24

Hormones and metabolites..................................................................................24

Appetite .............................................................................................................25

Somatostatin study ................................................................................................26


Indirect calorimetry ............................................................................................26

Glucose and palmitate metabolism.......................................................................27

Correlations........................................................................................................28

Interstitial glycerol concentrations .......................................................................28

Hypopituitary study................................................................................................29

3



Resting energy expenditure.................................................................................30

Glucose metabolism and substrate oxidation ........................................................30

Lipid metabolism ................................................................................................30

Regional substrate metabolism (microdialysis)......................................................31

GH and insulin signaling......................................................................................31

Discussion.................................................................................................................31 Methodological considerations ................................................................................31




Considerations of possible mechanisms and explana- ...............................................33

tions - implications and review of literature..............................................................33

Endogenous ghrelin excursions and previous pharmacokinetic studies ...................33

Pharmacokinetics in a clinical context...................................................................33

Predictors of ghrelin levels ..................................................................................34

Effects on appetite..............................................................................................35

Hormonal and metabolite effects .........................................................................35

Effects on substrate metabolism ..........................................................................36

Effects on resting energy expenditure ..................................................................38

Methodological limitations and pitfalls .....................................................................38




Conclusions & Perspectives.....................................................................................40

Conclusions ........................................................................................................40

Perspectives .......................................................................................................41

Summary ..................................................................................................................43 Summary in Danish ...................................................................................................44 Acknowledgements....................................................................................................45 References................................................................................................................46

Appendix 1

Original manuscript “Constant intravenous ghrelin infusion in healthy young men: Clinical

pharmacokinetics and metabolic effects”

Appendix 2

4


Original manuscript “Acute effects of ghrelin administration on glucose and lipid metabolism”

Appendix 3

Original manuscript “Ghrelin infusion in humans induces acute insulin resistance and lipolysis

independent of GH-signaling”

Appendix 4

Author declarations

5


List of abbreviations A Coefficient with unit of concentration ACC Acetyl-CoA carboxylase ACTH Adrenocorticotropic hormone AMPK adenosine 5′ monophosphate-activated protein kinase ANOVA Analysis of variance AS160 Akt substrate AUC and AUC∞ Area under concentration-time curve AUMC Area under the first moment curve B Coefficient with unit of concentration BMI Body mass index C Concentration Cl Clearance Cmax Maximal serum or plasma concentration Co Corticotropin Css Steady state concentration dpm Disintegrations per minute e Mathematical constant, base e EE Energy expenditure ELISA Enzyme-Linked ImmunoSorbent Assay EGP Endogenous glucose production ERK Extracellular signal-regulated kinase F Tracer infusion rate f1 Fraction eliminated associated with initial exponential term f2 Fraction eliminated associated with terminal exponential term FFA Free fatty acids GCP Good Clinical practice GH Growth hormone GHD Growth hormone deficiency GHRH Growth hormone releasing hormone GHS Growth hormone secretagogue GHS-R Growth hormone secretagogue receptor GIR Glucose infusion rate GOX Oxidative glucose disposal rate Gn Gonadotropin HDL High-density lipoprotein HPLC High-performance liquid chromatography IGF-I Insulin-like growth factor I IMP Investigational medicinal product IRS Insulin receptor substrate ITT Insulin tolerance test JAK Janus kinase K01 Elimination rate constant λ1 Slope of the initial elimination concentration-time curve λ2 Slope of the initial terminal concentration-time curve MRT Mean residence time M value Glucose metabolized NOGD Non-oxidative glucose disposal NS Non-significant

6


p Denotes phosphorylation of an enzyme p- Plasma levels of a specific compound PI3K Phosphatidylinositol 3-kinase PKB Protein kinase B Q Pool size QUICKI Quantitative insulin sensitivity check index R0 Rate of infusion Rd Rate of disappearance RIA Radio immuno assay RQ Respiratory quotient s- Serum levels of a specific compound SA Specific activity s.c. Subcutaneous SE Standard error of the mean STAT Signal transducer of activation and transcription T Infusion period Th Thyrotropin t Time t

½ Half-life V Vasopressin VAS Visual analogue scale Vc Volume of distribution, central compartment VD Volume of distributionV (λ2) Volume of distribution, terminal phase Vs. Versus Vss Volume of distribution, at steady state concentrations

7


Preface Ghrelin is a pleiotropic gut-derived peptide-hormone and more than 2,500 papers on

ghrelin have already been published since the first and famous Nature paper appeared on

December 9 1999. For more than three years I have with much joy and thrill had the chance

to participate in the ever evolving field of ghrelin research. This PhD dissertation is based on

clinical trials performed at Medical Department M and the Medical Research Laboratories at

Aarhus University Hospital, which have offered unique scientific surroundings.

Many skilled and dedicated people contributed to these studies. First of all, I would like to

mention and express my extensive acknowledgments to my principal supervisor Professor

Jens Otto Lunde Jørgensen for inviting and introducing me to metabolic research and for

always keeping focus, being optimistic ye realistic and for scientific guiding as well as for

introducing me to correct scientific writing. I am grateful to Professor Jens Sandahl

Christiansen for providing a stimulating scientific environment with many PhD-students as

colleagues and for providing funding. I wish to thank my supervisors Professor Niels Møller

and Dr. Troels Krarup Hansen for scientific tuition and guidance as well as stimulating scien-

tific discussions. I have enjoyed collaborating with Professor Ole Schmitz and would like to

acknowledge his assistance and continuous interest in my research projects and thank for

the scientific discussions we have had. I am indebted to Preben Jakobsen for pharmacoki-

netic tuition and discussions.

t

rt

I have enjoyed working with and appreciate all the experienced and highly skilled tech-

nicians at the Medical Research Laboratories and I am especially indebted to Susanne Søren-

sen, Elsebeth Hornemann, Merete Møller, Joan Hansen, and Anette Mengel for excellent

technical assistance and for sharing their invaluable experience on clinical research with me.

I would also like to thank Bente Mo ensen and Elin Carstensen for excellent technical assis-

tance.

I have enjoyed working with Dr. Sten Lund, Dr. Jan Frystyk, Dr. Søren Nielsen, Dr. Claus

Gravholt, Dr. Keld Sørensen, Dr. Erik Sloth, Dr. Rolf Dall, and Professor Lars Melholt Rasmus-

sen, and they are all thanked for their continuous help and supervision. I appreciate the col-

laboration with Dr. Kai Lange, Professor Michael Kjær, Dr. Steen Nielsen, and Professor Jens

Juul Holst.

I have had the great joy of working together with a number of enthusiastic present and

former PhD-students who have all contributed to a unique scientific environment by contin-

ued guidance, taking part in fruitful discussions and providing support in all imaginable ways.

8


I am especially indebted to Dr. Lars Christian Gormsen, Dr. Niels Jessen, Dr. Niels Holmark

Andersen, Dr. Christian Born Djurhuus, Dr. Jakob Gjedsted, Dr. Mads Buhl, Dr. Ande s Boje-

sen, Dr. Signe Gjedde, Dr. Britta Hjerrild, Dr. Charlot e Nielsen, Dr. Tina Parkner and Dr.

Jian-Wen Chen.

r

t

Finally, I avail myself of expressing my sincere gratitude to my family for their extraordi-

nary support and patience.

9


Background The history of ghrelin is already fascinating. It began and evolved years before the identi-

fication of ghrelin by reverse pharmacology and before the ghrelin receptor was discovered.

Bowers and colleagues demonstrated that met enkephalins were weakly active growth hor-

mone (GH) releasing factors and

they designed more potent ana-

logues in a series of experiments

(1). These molecules are now

denoted GH secretagogues

(GHS). In 1996, Howard and

colleagues characterized and

cloned the GHS receptor (GHS-R

or ghrelin receptor) (2). After

more than a year of systematic

search for an endogenous ligand for the GHS-R, ghrelin was successfully discovered in 1999

by Masayasu Kojima and colleagues (3). Unexpectedly, ghrelin was primarily produced by

gastric cells and the structure turned out to be a unique n-octanoyl acylated peptide (4),

Figure 1.

Figure 1

Administration of ghrelin was shown to potently induce GH secretion (5). Traditionally,

the pulsatile secretion of GH from the pituitary gland is considered to be under both positive

and negative regulation by hypothalamic factors. GH releasing hormone (GHRH) stimulates

GH release, whereas somatostatin exerts an inhibitory influence (6). Circulating ghrelin is

mainly derived from the gut (7;8), and although the ghrelin receptor is abundantly present in

the pituitary gland (9;10), it remains to be convincingly demonstrated that stomach-derived

ghrelin is a physiological regulator of GH secretion. Conditions characterized by high GH lev-

els such as exercise (11), fasting (12), and insulin-induced hypoglycemia (13), are not pre-

ceded by consistent elevations in serum ghrelin levels.

The GHS-R is present not only in the hypothalamus and the pituitary gland but also in

many peripheral organs and tissues (9;10) for which reason pleiotropic effects of the en-

dogenous ligand could be expected. Subsequently, ghrelin research has included a variety of

different research fields such as endocrinology, metabolism, gastroenterology, cardiology,

reproduction, bone growth, and immunology. Additional effects of exogenous ghrelin espe-

cially of interest in relation to this PhD project include stimulation of adrenocorticotropic

10


hormone (ACTH) and prolactin secretion (5;14). Moreover, there is accumulating evidence to

support that ghrelin plays an important role in the regulation of food intake (15-19), energy

metabolism (20-22), and gastric motility (23;24). Administration of ghrelin stimulates appe-

tite and food intake in rodents as well as in humans (16-19), and circulating ghrelin levels

increase prior to food intake and are suppressed postprandially, which has led to the hy-

pothesis that gastric ghrelin acts as a meal-initiator (15). In addition, ghrelin accelerates

gastric emptying (23;24) and increases gastric acid secretion (25;26). In rodents, admini-

stration of ghrelin induces adiposity (20).

There does not seem to be a simple causal link between ghrelin secretion and conditions

such as simple obesity and anorexia nervosa. By contrast, ghrelin levels are suppressed in

obesity and elevated in anorexia nervosa (27;28), which is more compatible with a compen-

satory response to increased and reduced fat stores, respectively. The endocrine response to

ghrelin (i.e. GH-release) is, however, attenuated in obesity (17). It remains to be studied

whether this decrease in ghrelin levels in obesity is caused by increased ghrelin degradation

or decreased secretion and whether any biochemical or anthropometrical variables predict

ghrelin turnover. These issues point to the study object of the first paper, namely the phar-

macokinetics of ghrelin. Until now, only two articles have reported pharmacokinetic data

(29;30) and both describe first-order elimination in a one-compartment model with a half-life

between 9-31 minutes. Several trials involving ghrelin, ghrelin agonists as well as antagonists

are presently ongoing. Optimal administration calls for knowledge about distribution and

elimination as well as the kinetics of these processes.

In clinical trials, administration of ghrelin increases plasma glucose and free fatty acids

(FFA) (31;32) and reduces glucose disposal rates (33) compatible with an impairment of

insulin sensitivity, but it has been difficult to dissect direct peripheral effects from indirect

GH- and cortisol-mediated effects. Several lines of evidence suggest that ghrelin exerts direct

metabolic effects: (i) in rodent pancreatic islets both ghrelin and GHS-R are present, and

ghrelin inhibits glucose-induced insulin secretion via a paracrine mechanism (34). In a clinical

setting ghrelin also inhibits insulin secretion (22). (ii) a GHS-R antagonist enhanced glucose-

induced insulin secretion from perfused rodent pancreas, whereas exogenous ghrelin sup-

pressed insulin secretion (35). (iii) ghrelin knockout mice display enhanced glucose-induced

insulin release from isolated islets (35;36) and exhibit increased peripheral insulin sensitivity

(36). Ghrelin/GHS-R double knockout mice show lower glucose levels after a glucose toler-

ance test and a more rapid drop in plasma glucose levels after an insulin tolerance test (37).

11


The objective of the second study was therefore to determine the GH- and cortisol-

independent effects of ghrelin on glucose and lipid metabolism in a clinical setting. Ghrelin

has previously been shown to cause GH-release despite concomitant somatostatin infusion

(38). In order to suppress GH secretion, the somatostatin dose therefore was doubled as

compared to the previous study by De Vito et al. (38) and a constant infusion of ghrelin was

used instead of bolus administration.

The second study revealed that somatostatin does not fully suppress ghrelin-induced GH

and cortisol secretion. Hypopituitary patients replaced with GH and hydrocortisone constitute

an in vivo model for studying putative GH- and ACTH-independent effects of ghrelin. Hypopi-

tuitary patients have participated in one study reporting the effect of ghrelin on metabolites

only (31). In the third study, the objective was to further determine potential direct effects

of ghrelin on whole body and regional substrate metabolism.

Methods Subjects The studies were conducted in accordance to the Helsinki Declaration and all subjects

gave their oral and written informed consent to participate in the respective trials. Study

protocols were approved by the local Ethics Committee of Aarhus County. According to the

International Committee of Medical Journal Editors the protocols were registered in a public

database (www.clinicaltrials.gov). All subjects fasted for at least 9 hrs before study com-

mencement and remained fasting throughout the study day.

Pharmacokinetic study

Seventeen healthy men participated in this study. The quite large number of participants

for a cross-over pharmacokinetic study was chosen to obtain sufficient statistical power (39)

for a cardiovascular study performed in parallel in the same subjects (40). The baseline

characteristics are provided in Table 1.

12


All participants

had a normal

physical examina-

tion, normal he-

matological indi-

ces, and normal

renal and liver

function tests. All

were non-smokers

and none were abusing alcohol or taking any medication. Subjects were recruited among

university students.

Ta

F

ble 1. Baseline characteristics of the subjects in the pharmacokinetic studyMean ± SE Range(n = 17)

Age (years) 23.1 ± 0.4 21 - 26Height (m) 1.84 ± 0.01 1.70 - 1.96Weight (kg) 77.6 ± 1.5 68.8 - 88.8BMI (kg/m2) 23.0 ± 0.3 20.7 - 25.6Fasting plasma glucose (mmol/l) 4.9 ± 0.1 4.2 - 6.0asting serum ghrelin (µg/l) 0.74 ± 0.06 0.41 - 1.23

Total cholesterol (mmol/l) 3.98 ± 0.16 2.90 - 5.70HDL cholesterol (mmol/l) 1.58 ± 0.09 1.1 - 2.7Plasma albumin (µmol/l) 676.0 ± 5.6 624 - 713

The study protocol was approved by the Danish Medicines Agency and the Good Clinical

Practice (GCP) Unit of Aarhus University Hospital.

Somatostatin study

Eight healthy men aged 27.2 ± 0.9 years with a body mass index (BMI) of 23.4 ± 0.5

kg/m2 volunteered in this study. All had a normal physical examination.

Hypopituitary study

Eight hypopituitary men on stable replacement therapy with GH and hydrocortisone (for

> 3 months) participated. None of the patients had diabetes or any other concomitant

chronic disease. Their baseline characteristics are provided in Table 2.

Table 2. Characterization of the hypopituitary subjectsPatient Age BMI Diagnosis IGF-I Insufficient pituitary axes

yr kg/m2 µg/l1 60 28.8 Pituitary apoplexy 143 GH, Th, Co, Gn2 51 27.5 Prolactinoma 188 GH, Th, Co, Gn3 56 36.0 Cushing disease 193 GH, Th, Co4 60 33.8 Pituitary apoplexy 245 GH, Th, Co, Gn5 62 31.7 Pituitary apoplexy 196 GH, Th, Co, Gn6 46 29.9 Clinically nonfunctioning pituitary adenoma 199 GH, Th, Co, Gn7 31 30.6 Craniopharyngeoma 222 GH, Th, Co, Gn, V8 60 34.4 Cushing disease 270 GH, Th, Co, Gn

BMI, body mass index; IGF-I levels at baseline during GH substitionGH, growth hormone; Th, thyrotropin; Co, corticotropin; Gn, gonadotropin; V, vasopressin

13


Study design


This study was initiated after May 1 2004 and accordingly assigned to EMEAs guidelines

for clinical trials of Investigational Medicinal Products (IMPs). Thus, the IMP (ghrelin) was

required to fulfill both the legislation for Good Manufacturing Practice and Good Distribution

Practice and the clinical trial itself should follow the GCP directive. After completing a com-

prehensive application process we obtained the first national permission from the Danish

Medicines Agency to conduct clinical trials using ghrelin as a trial medication.

Synthetic human acylated ghrelin (NeoMPS, Strasbourg, France) was dissolved in isotonic

saline and sterilized by double passage through a 0.8 and a 0.2 µm pore size filter (Super

Acrodisc, Gelman Sciences, Ann Arbor, MI, USA) by the local University Hospital Pharmacy

Service.

Two volunteers participated in a pilot study, whereafter tolerability, safety, and serum

ghrelin levels were evaluated. The pilot study revealed no safety concerns and subsequently

15 additional subjects were screened, enrolled, randomized, and investigated.

The University Hospital Pharmacy generated a randomization scheme using the Web

hosted generator at http://www.randomization.com. The study design was a randomized,

placebo-controlled double-blind cross-over study.

Two intravenous cannulaes were placed in cubital veins for blood sampling and infusion,

respectively. At t = 0 min, a constant 3-h infusion of ghrelin (5 pmol/kg body weight per

min) or an isovolumetric saline infusion commenced. Blood samples were drawn every 20

min for the 3-h infusion period whereafter blood was drawn at 5-min intervals for a 1-h pe-

riod and subsequently blood was drawn at 10-min intervals for the last two hours.

A muscle biopsy was obtained at t = 90 min from the lateral vastus muscle with a

Bergström biopsy needle under local anesthesia (1% lidocaine). A total amount of ~ 200 mg

muscle was aspirated; biopsies were cleaned for blood within 15 sec and snap-frozen in liq-

uid nitrogen. Muscle biopsies were stored at -80°C until analyzed.

To determine the effects of ghrelin infusion on appetite related scores, a visual analogue

scale (VAS) was used. The subjects reported VAS ratings of (VAS1) hunger, (VAS2) satiety,

(VAS3) desire to eat, and (VAS4) prospective food consumption. Subjects were instructed to

make a single vertical mark on a horizontal line (possible scores 0 to 100 mm) to indicate

their current feelings, Figure 2:

14


Ghrelins farmakokinetik hos raske kontroller Appetitregulation

Appetitvurdering ved infusionsstart og -slut

Initialer: __ __ __ Projekt id: __ __ __ Dato: __/ __ - __

Sult

Hvor sulten er du?

Slet ikke sulten _______________________________________________ Maximalt sulten

Mæthed

Hvor mæt er du?

Slet ikke mæt _______________________________________________ Helt mæt

Hvor stor er din trang til at spise?

Slet ikke trang til at spise Størst mulig trang til at spise

_______________________________________________

Hvor meget kan du spise?

Ingenting _______________________________________________ Så meget som muligt

Figure 2

Hunger was defined as "the subjective driving force for the search for, choice of, and in-

gestion of food.” Satiety was defined as "the sensation after eating so that a person does not

feel the need to eat for some time afterwards.” The desire to eat was defined as "the per-

son's subjective feeling of the need to consume a meal.” The prospec ive food consumption

was defined "the quantity of food the subject imagined himself able to eat at the given

time.” Baseline evaluations were collected prior to the infusion period (t = 0) and at termina-

tion of the infusion period (t = 180 min). A blinded observer measured the scores.

t

Somatostatin study

All participants were examined on two occasions separated by a minimum of five weeks.

The subjects were studied at 07.00 am in a quiet, thermo neutral in-door environment.

One intra-venous cannula was inserted in the antecubital region for infusion, and one in-

tra-venous cannula was inserted in a dorsal hand vein for blood sampling. The latter was

placed in a heated box at 65°C allowing for arterialized blood samples to be drawn. Plasma

glucose levels were determined every 10 min and blood samples were drawn every 30 min

and analyzed for ghrelin, GH, cortisol, glucagon, insulin, C-peptide, and FFA.

The study design is illustrated in Figure 3. In a randomized, single-blind, placebo-

controlled cross-over design a constant infusion of human acylated ghrelin lasting 300 min-

utes at 5 pmol/kg body weight per min or saline (as placebo) was used. The period from t =

15


0 to t = 180 min is referred to as the basal period and the period from t = 180 to t = 300

min as the clamp period.

At t = 0 min infusion of somatostatin (Ferring Pharmaceuticals, Copenhagen, Denmark)

330 µg/h, insulin (Actrapid, Novo Nordisk, Bagsværd, Denmark) 0.1 mU/kg/min, GH

(Norditropin, Novo Nordisk)

2 ng/kg/min was com-

menced. Infusion of gluca-

gon (Glucagen, Novo Nord-

isk) 0.5 ng/kg/min was

added at t = 30 min. Eu-

glycemia was intended and

if necessary isotonic glu-

cose was infused to main-

tain a plasma glucose level

of ~ 5.0 mmol/l in order to

prevent somatostatin-

induced hypoglycemia.

From t = 180 min and on-

wards a hyperinsulinemic

euglycemic clamp (insulin

0.6 mU/kg/min) was per-

formed. Plasma glucose was clamped at 5.0 mmol/l by adjusting the rate of infusion of 20%

glucose according to plasma glucose measurements every 10 min. Insulin sensitivity was

estimated by the level of glucose infusion rate (GIR) during the hyperinsulinemic euglycemic

clamp.

Figure 3

Hypopituitary study

All patients were studied on two occasions (ghrelin 5 pmol/kg body weight per min and

saline for 300 min) in a randomized, double-blind, placebo-controlled cross-over design. In-

tra-venous cannulaes were inserted as in the Somatostatin study.

16


The study design is il-

lustrated in Figure 4. At t

= 0 saline or a primed-

continuous ghrelin infu-

sion was commenced.

The bolus dose was esti-

mated from the elimina-

tion rate constant of

ghrelin k01 calculated in

the pharmacokinetic

study and infused over a

20-min interval to avoid

an overshoot of steady

state levels. Subse-

quently, ghrelin was infused at a rate of 5 pmol/kg body weight per min.

Figure 4

Muscle and fat biopsies were obtained at t = 120 min. A hyperinsulinemic euglycemic

clamp was performed from t = 120 to 300 min. Blood samples were obtained as indicated in

Figure 4. Indirect calorimetry was performed for the terminal 30 min of both the basal and

the clamp period.

Pharmacokinetic modeling

A review of PubMed indexed journals1 showed that ghrelin has been administered more

than 500 times in patients and healthy volunteers. Ghrelin has mostly been administered as

intravenous bolus injections, but also intravenous infusions as well as subcutaneous (s.c.)

administrations have been tested. Surprisingly, the pharmacokinetics of ghrelin is reported

by two papers only (29;30). Both reported first-order elimination in a one-compartment

model with a half-life between 9-31 minutes but the pharmacokinetic approach was not de-

scribed in detail. We decided to extend the current pharmacokinetic knowledge and used a

constant intravenous ghrelin infusion to investigate (i) if steady state levels were obtained

after ~ 6 times the reported half-life i.e. ~ 180 min as expected, (ii) if systemic ghrelin levels

could be predicted based on experimentally derived pharmacokinetic variables and (iii) the

1Review of papers retrieved by search term “ghrelin” on the Web-hosted database

http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed by end of September 2007. The sum of ghrelin administrations does not take non-identifiable double publications into account.

17


mean residence time (MRT) of ghrelin in the human body. MRT is a summary pharmacoki-

netic term, which refers to the average time (in min) a molecule spends in the human body.

The postinfusion concentration-time curve was analyzed from each of the 17 subjects us-

ing a nonlinear least-squares regression analysis based on both a mono- and a biexponential

decay model. By visual inspection of the concentration-time curve on a linear and a log-

transformed scale the more complicated biexponential model:

tt eBeAC ⋅−⋅− ⋅+⋅= 21 '' λλ

provided the best fit and, subsequently by statistical testing, had significantly lower sum-of-

squares, 0.92 vs. 0.04. The statistical extra sum-of-squares F-test was applied to compare

the two nes ed models and favored the 2-phase model, P < 0.0001. λt 1 and λ2 denote the

initial and terminal slopes. Pharmacokinetic analyzes were performed by using the computer

software GraphPad Prism v4.00 for Windows (GraphPad Software, San Diego, USA). To cor-

rect the serum ghrelin levels for endogenous ghrelin secretion, serum ghrelin levels on the

placebo day were subtracted from the levels measured during active treatment. Because

pharmacokinetic parameters were estimated based on a post-infusion curve, intercepts

should be corrected according to Loo and Riegelman (41):

,1

',

1

'21

21TT e

TBB

eTA

A⋅−⋅− −

⋅⋅=

−

⋅⋅=

λλ

λλ

where T is the infusion period.

The distribution half-life t½(λ1) and the elimination (terminal) half-life t½(λ2) were calculated

as

11½

)2ln()(

λλ =t and

22½

)2ln()(

λλ =t ,

respectively. The area under the serum concentration-time curve AUC∞ was calculated as

21 λλBAAUC +=∞ .

The so-called area under the first moment curve AUMC – a prerequisite to estimate MRT –

was calculated as

22

21 λλ

BAAUMC += .

Total clearance Cl was calculated as follows:

18


∞

=AUCDoseCl ,

MRT was calculated as

∞

=AUCAUMCMRT .

Simulation of the serum concentration during the infusion was done according to the follow-

ing equation (42)

CleRf

CleRf

Ctt )1()1( 21

0201⋅−⋅− −⋅⋅

+−⋅⋅

=λλ

,

where R0 is the rate of infusion and f1 and f2 denominate the fraction of elimination associ-

ated with the first and last exponential term, respectively:

∞

=AUC

Af 1

1λ

and∞

=AUC

Bf 2

2λ

.

Serum concentration at steady state CSS was calculated as

ClR

C ss0= .

An elaborate list of pharmacokinetics including the volume of distribution of the central com-

partment Vc, the volume of distribution during the terminal phase V (λ2) and the volume of

distribution at steady state Vss is reported in the first paper.

Tracers

A primed-continuous infusion of [3-3H]-glucose was used (Somatostatin study: 17 µCi bo-

lus followed by 0.17 µCi/min; Hypopituitary study: 12 µCi bolus followed by 0.17 µCi/min,

NEN Life Science Products, Boston, MA, USA). Glucose flux rates were calculated at 10-min

intervals during the last 30-min intervals of the basal and clamp periods, respectively, using

Steele’s nonsteady-state equations (43) and, based on empirical observations, a pool fraction

of 0.65 and a volume of distribution of 200 ml/kg body weight are used (43;44):

.SA

dtdSACVQF

RglcD

a

×××−=

19


During the clamp period, endogenous glucose production (EGP) was calculated by sub-

tracting the rate of exogenous glucose infusion from the rate of appearance of [3-3H]-

glucose:

.GIRREGP a −=

In the Hypopituitary study, oxidative glucose disposal rate (GOX) was calculated from

energy expenditure (EE) and respiratory quotient (RQ) after correction for protein oxidation

rates, which was estimated from the urinary excretion of urea

.29.0

71.0 EEnonproteinRQnonproteinGOX ×−

=

Nonoxidative glucose disposal (NOGD) was calculated as whole body glucose disposal Rd

minus the rate of GOX adjusted for body weight and calculated per minute (45)

,GOXRNOGD d −= where

.dt

dCVQRR glc

Dad ××−=

[9,10-3H]-palmitate (Danish Medicines Agency, Copenhagen, Denmark and Department

of Nuclear Medicine, Aarhus University Hospital, Denmark) was infused continuously at 0.3

µCi/min. Blood samples were drawn for analysis of palmitate concentration and specific ac-

tivity (SA). Steady state of SA was verified (last 30-min interval of a palmitate-infusion pe-

riod). Plasma palmitate concentration and SA were determined by HPLC using [2H31]-

palmitate as internal standard. Systemic palmitate flux (Ra, µmol/min) was calculated using

the [9,10-3H]-palmitate infusion rate (dpm/min) divided by the steady state palmitate SA

(dpm/µmol)

.SAFRa =

Indirect calorimetry

RQ and EE were assessed by indirect calorimetry (45) (Deltatrac; Datex Instruments, Hel-

sinki, Finland), which was performed for the terminal 30 min of the basal and clamp periods

in the Somatostatin and the Hypopituitary studies as indicated in Figures 3 and 4. An addi-

tional calorimetry period was included at baseline in the Somatostatin study before infusion

of saline and ghrelin. In the Hypopituitary study, lipid oxidation was calculated from EE and

RQ after correction for protein oxidation rates,

20


.29.0

1 RQnonproteinEEnonproteintionLipidoxida −×=

Microdialysis

Muscle microdialysis catheters (CMA-60, CMA, Stockholm, Sweden) were inserted after

applying local analgesic of 1 ml 1% lidocaine superficial to the fascia. Correct placement of

the microdialysis catheters was confirmed by the presence of muscle twitches during inser-

tion. In the Somatostatin study, two catheters were used in order to ensure viability of at

least one (gastrocnemius and the lateral vastus muscle approximately 10 cm below and 14

cm above the patella, respectively). In the Hypopituitary study, only one muscle microdialy-

sis catheter was used because no membranes were disrupted in the earlier study. Subse-

quently, two additional microdialysis catheters were positioned in the s.c. adipose tissue ap-

proximately 5 cm lateral of the umbilicus and in the femoral s.c. adipose tissue after 0.25 ml

of lidocaine s.c. pre-administration.

The microdialysis catheters have a molecular cut-off of 20 kDa and a membrane length

of 30 mm. Prior to insertion the catheters were manually flushed with perfusion fluid (Ringer

Chloride, T1, CMA, Na+ 147 mmol/l; K+ 1.4 mmol/l; Ca2+ 2.3 mmol/l; Cl- 156 mmol/l, pH 6;

osmolality 290 mosmol/kg) to allow for clearance of air bubbles from the microdialysis mem-

branes. The microdialysis systems were perfused at a flow rate of 1 µl/min using CMA-107

perfusion pumps (CMA).

The relative recovery of interstitial glycerol was assessed by the internal reference

method using 3H-glycerol (46). 3H-glycerol was added to the perfusate in order to obtain

approximately 1000 cpm/µl. Perfusate and dialysate were counted using a Wallac 1450 liquid

scintillation counter applying the Optiphase supermix scintillation fluid (Wallac, Turku,

Finland) for muscle and Ultima Gold scintillation fluid (Packard Biosciences, Meriden, CT,

USA) for adipose tissue. Changes in interstitial glycerol concentration correspond to regional

lipolysis (47).

60 min of equilibration was allowed in order to minimize the influence of local edema and

hemorrhage. The sampling was performed every 30 min and continued until t = 300 min.

Western blotting and PI3K assay

Muscle biopsies were homogenized as previously described (48). Aliquots of protein were

resolved by SDS-PAGE, and proteins were electro-blotted onto nitrocellulose membranes.

Immunoblotting was performed using primary antibodies as follows: pSTAT5a and -b,

21


STAT5, pSTAT3, STAT3, pERK1 and -2, ERK1 and -2, pAMPKα, AMPKα-pan, pACC, pAkt, Akt,

PAS, and AS160. Membranes were incubated with HRP-coupled secondary antibodies, visu-

alized by BioWest enhanced chemiluminescence (UVP LabWorks, Upland, CA, USA) and

quantified by UVP BioImaging System. Densitometric measurements were adjusted to an

internal control.

PI3K activity was assessed as previously described (48).

Analyses

Biochemical methods were similar in all three studies unless indicated and specified. Se-

rum total ghrelin levels were measured in duplicate by an in-house assay as described previ-

ously (12). The assay measures immunoreactive levels of ghrelin using 125I-labelled bioactive

ghrelin tracer and rabbit polyclonal antibodies raised against octanoylated human ghrelin.

The assay recognizes the COOH-terminal of ghrelin and as such determines acylated as well

as des-acylated ghrelin. The intraassay coefficient of variation is usually low in this assay

(2.8% in a random sample). Samples from each individual were analyzed in the same assay

run. A double commercial monoclonal immunofluorometric assay (DELFIA, Perkin Elmer, Wal-

lac, Turku, Finland) was used to measure serum levels of GH, cortisol and insulin. Plasma

glucose levels were measured in duplicate on a glucose analyzer (Beckman Instruments,

Palo Alto, CA, USA). Serum FFA was determined using a commercial kit (Wako Chemicals,

Neuss, Germany). Plasma ACTH was determined using a commercial method (Immulite Di-

agnostic Product Corporation Scandinavia). Plasma albumin, total cholesterol and high-

density lipoprotein (HDL) cholesterol levels were determined by commercial methods (Cobas

Integra 800) using an immunoturbidimetric measurement, an enzymatic colorimetric and a

homogeneous enzymatic colorimetric method, respectively. In the Somatostatin study, corti-

sol was measured with a solid-phase, time-resolved fluoroimmunoassay (Delfia), insulin and

C-peptide were measured with an immunoassay (DAKO, Denmark), and plasma glucagon

was measured by an in-house radioimmuno assay modified from (49).

Glucose, glycerol, lactate, and urea in the microdialysis dialysate were measured in dupli-

cate by an automated spectrophotometric kinetic enzymatic analyzer (CMA 600; CMA).

22


Statistics

Results are expressed as mean ± SE.

Systemic levels of ghrelin, GH, ACTH, prolactin, cortisol, glucagon, insulin, C-peptide,

epinephrine, norepinephrine, glucose (and skeletal muscle interstitial glucose), FFA, GIR, and

the time series of revised QUICKI estimates were analyzed by two-way analysis of variance

(ANOVA). The interaction between time and treatment ("time x treatment") was considered

the term of interest. The Bonferroni correction was used to account for multiple comparisons

when appropriate.

Comparisons of baseline values were carried out by Student’s two-tailed paired t test.

Glucose kinetics, M value, palmitate fluxes, RQ, EE, lipid oxidation, time averaged intersti-

tial concentrations, changes in VAS, densitometric quantitative measures of Western blots,

and PI3K activity were examined by Student’s two-tailed paired t test when appropriate.

In the Pharmacokinetic study, backward regressions analyses were performed including

BMI, total and HDL cholesterol levels as independent variables, and the following variables

for ghrelin: MRT, AUC, half-lives, k01, or Cl were included as dependent variables.

In the Somatostatin study, effects of GH, cortisol, glucagon, insulin levels, and GIR (in-

cluded as independent variables) on lipolysis (palmitate flux and FFA increase) (included as

dependent variables) were analyzed by comparing the changes in rates (GIR and palmitate

flux) and areas under concentration-time curves (∆AUC, GH, cortisol, glucagon, insulin, and

NEFA) by backward regression analyses.

A P value less than 0.05 was considered significant. Statistical analysis was performed us-

ing SPSS versions 13.0 and 14.0 for Windows.

23


Summary of results Pharmacokinetic study

Ghrelin pharmacokinetics

Mean levels of circulating ghrelin (total ghrelin) vs. time are shown in Figure 5. Serum

ghrelin increased to 4.41 ± 0.29 µg/l above placebo levels after 180 min infusion. This level

was comparable to predicted Cmax (4.35 ± 0.28 µg/l, P = 0.45), whereas it was significantly

smaller than predicted CSS (5.00 ± 0.35 µg/l, P = 0.03).

CSS

Cmax

3.32e-0.033t

1.35e-0.007t

Figure 5. Levels of total serum ghrelin above baseline vs. time and key pharmacokinetic

parameters.

The initial and terminal ghrelin half-lives, t½(λ1) and t½(λ2) were 24.2 ± 2.5 and 146.0 ±

35.6 min, respectively and MRT was 92.7 ± 16.3 min.

Correlations

MRT was included as a dependent parameter in a backward stepwise multiple regression

analysis. Both BMI (r = 0.51, P < 0.001) and HDL cholesterol (r = 0.75, P < 0.001) were

positively associated with MRT.

Hormones and metabolites

The hormonal and metabolite responses to ghrelin and saline infusions are shown in Fig-

ure 6. Ghrelin caused an increase in systemic levels of GH, prolactin, ACTH, cortisol, FFA,

and glucose, whereas insulin remained constant until termination of the infusion period. Ele-

vations in levels of both FFA and glucose concomitantly with constant and elevated insulin

levels suggested a significant decrease in insulin sensitivity. Insulin sensitivity was not meas-

24


ured directly, as a hyperinsulinemic euglycemic clamp would have hampered or interfered

with the measurements of

appetite and cardiovascular

indexes. However, to obtain

a measure for insulin sensi-

tivity revised QUICKI (50)

for the time series was used

although this model is vali-

dated on fasting subjects

only. Revised QUICKI indi-

cated that insulin sensitivity

decreased after 40 min of

ghrelin infusion and did not

reach pre-infusion levels

throughout the study day.

Appetite

scores were

sim

0

All VAS

ilar at baseline on both

study days (placebo vs.

ghrelin, in cm: VAS1: 4.2 ±

.5 vs. 3.9 ± 0.5, P = 0.52;

VAS2: 3.6 ± 0.5 vs. 4.1 ±

0.5, P = 0.32; VAS3: 4.2 ±

0.5 vs. 4.7 ± 0.5, P = 0.43; and VAS4: 5.6 ± 0.4 vs. 5.3 ± 0.5, P = 0.40, respectively) and

after 180 min of infusion (placebo vs. ghrelin, in cm: VAS1: 5.9 ± 0.5 vs. 5.4 ± 0.6, P =

0.45; VAS2: 2.9 ± 0.4 vs. 2.5 ± 0.4, P = 0.38; VAS3: 6.3 ± 0.3 vs. 6.2 ± 0.4, P = 0.83; and

VAS4: 6.4 ± 0.4 vs. 6.8 ± 0.3, P = 0.28, respectively). Comparing the changes in VAS scores

during the 3-h infusion period revealed that ghrelin affected the delta score of “satiety” only,

P = 0.03. However, it was obvious that some of the subjects experienced a transient in-

crease of hunger after 40 - 90 min of ghrelin infusion, but these observations were not for-

mally recorded.

Time (minutes)

0 100 200 300

Rev

ised

QU

ICK

I

0.0

0.2

0.4

0.6

*

G

**

* ** * *

Seru

m in

sulin

(pm

ol/l)

0

10

20

30

40

50

*

E

* *

*

*

Pla

sma

gluc

ose

(mm

ol/l)

0.0

4.8

5.0

5.2

5.4

5.6

**C

Ser

um F

FA (m

mol

/l)0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6GhrelinPlacebo

A

*

* *

*

* *

Time (minutes)

0 100 200 300

Ser

um c

ortis

ol (n

mol

/l)

0

100

200

300

400

500

*

H

*

* *

*

Seru

m A

CTH

(ng/

ml)

0

5

10

15

20

25

30 *F

Ser

um p

rola

ctin

(µg/

l)

0

2

4

6

8

10

12

14

*

D

** *

Seru

m G

H (n

g/m

l)

0

10

20

30

40

50

*

B

*

*

*

** * * *

Infusion period Infusion period

Figure 6

25


Somatostatin study

Hormones and metabolites

Serum ghrelin levels were similar at baseline on both study days, P = 0.30. Infusion of

ghrelin resulted in a 4.5–fold increase in circulating ghrelin levels during both the basal and

the clamp period. In the saline study serum ghrelin levels decreased moderately and gradu-

ally with time, ANOVA P =

0.001. The hormonal and

metabolite responses are

shown in Figure 7.

Serum GH increased

moderately in response to

ghrelin infusion, ANOVA P

< 0.001, and reached a

maximum of 4.6 ± 1.3

ng/ml (i.e. somatostatin

infusion blunted the GH

response by a factor 10 as

compared with the phar-

macokinetic study) after

60 min of infusion where-

after a gradual decline

towards placebo levels

was observed. Serum cor-

tisol increased approxi-

mately 1.9–fold after 90

min of ghrelin exposure as

compared to placebo fol-

lowed by a decline to-

wards placebo levels after

270 minutes.

Indirect calorimetry

The rates of EE and RQ were

1552 ± 39 kcal/24-h, P = 0.30

Figure 7

Time (minutes)

-100 0 100 200 300

Plas

ma

gluc

agon

(pg/

ml)

0

20

40

60

80

100

120

Ser

um g

hrel

in (µ

g/l)

0

1

2

3

4

5

6

7

GhrelinSaline

P < 0.001

Ser

um g

row

th h

orm

one

(ng/

ml)

0

2

4

6

8

10

*

***

*

P < 0.001

Seru

m c

ortis

ol (n

mol

/l)

0

200

400

600

800

*

P < 0.001

****

**

P < 0.01

Ser

um in

sulin

(pm

ol/l)

0

50

100

150

200

250

300

P = 0.41

P = 0.61

Seru

m C

-pep

tide

(pm

ol/l)

0

200

400

600P = 0.93

Plas

ma

gluc

ose

(mm

ol/l)

0

4

6

8

P = 0.63 P = 0.28

Time (minutes)

-100 0 100 200 300

Ser

um F

FA (µ

mol

/l)

0

200

400

600

800

1000

P = 0.10 P < 0.001

**

Basal Clamp

A

B

C

D

E

F

G

H

*

*

Pancreatic clampBasal ClampPancreatic clamp

comparable in the basal period [EE: 1521 ± 43 (ghrelin) vs.

and RQ: 0.89 ± 0.01 (ghrelin) vs. 0.88 ± 0.01, P = 0.53, re-

26


spectively], whereas insulin-stimulated EE was significantly lower during ghrelin infusion as

compared with placebo [EE: 1539 ± 28 (ghrelin) vs. 1608 ± 32 kcal/24-h, P = 0.048]. RQ

was similar during the clamp period [0.92 ± 0.03 (ghrelin) vs. 0.93 ± 0.01, P = 0.65].

Glucose and palmitate metabolism

Glucose metabolism is depicted in Figure 8. The rate of isotopically determined total glu-

cose turnover (Rd) during the basal [1.9 ± 0.1 (ghrelin) vs. 2.3 ± 0.1 mg/kg/min, P = 0.03]

as well as the clamp period [3.9 ± 0.6 (ghrelin) vs. 6.1 ± 0.5 mg/kg/min, P = 0.02] was sig-

nificantly lower during ghrelin administration as compared with placebo infusion. By contrast,

endogenous glucose production was similar during ghrelin and placebo administration in the

basal period and was suppressed to a similar degree during the hyperinsulinemic clamp (P

NS). Significantly lower GIR during the ghrelin study resulted in similar plasma glucose lev-

els.

Rate of glucose disappearance

Saline Ghrelin Saline Ghrelin

Rd

(mg/

kg/m

in)

0

1

2

3

4

5

6

7

8

Glucose infusion rate

Time (minutes)-100 0 100 200 300

GIR

(mg/

kg/m

in)

0

2

4

6

8

GhrelinSaline

P < 0.001

* **

*

*

**** *

**

A

C

M-value

Saline Ghrelin

M-v

alue

(mg/

kg/m

in)

0

2

4

6

8

10

P = 0.007

Basal Clamp

P = 0.02

P = 0.03

Endogenous glucose production

Saline Ghrelin Saline Ghrelin

EGP

(mg/

kg/m

in)

0

1

2

3

4

P = 0.48

P = 0.44

Basal Clamp

Figure 8

27


Insulin sensitivity, as assessed by GIR, was reduced during ghrelin infusion as compared

with placebo infusion [4.0 ± 0.7 (ghrelin) vs. 6.9 ± 0.9 mg/kg/min, P = 0.007].

Basal systemic palmitate flux was significantly increased during ghrelin administration

(131 ± 26 (ghrelin) vs. 69 ± 5 µmol/min, P = 0.048).

Correlations

Backward regressions analyses excluded ∆AUC of glucagon, insulin, and GIR and re-

vealed that ∆AUC of GH, P = 0.005, and cortisol, P = 0.002 were both significant predictors

of ∆AUC of FFA. However, similar analyses where ∆AUC of FFA was replaced by palmitate

flux (as a marker for lipolysis) as the dependent variable excluded all independent variables

of the model as predictors of lipolysis.

Interstitial glycerol concentrations

In general, the hyperinsulinemic clamp suppressed interstitial glycerol levels in muscle

and adipose tissue during both ghrelin and placebo treatment. Glycerol levels were elevated

in femoral s.c. fat during ghrelin and clamp conditions, P = 0.01, and overall, the regional

glycerol release was elevated during ghrelin infusion, although it failed to reach statistical

significance. Interstitial glycerol levels decreased, as expected, during the hyperinsulinemic

clamp period, but the suppression of interstitial glycerol levels was attenuated on the ghrelin

day as compared to the saline day. In abdominal s.c. fat tissue hyperinsulinemia caused a

reduction of 56.3 ± 10.5 µmol/l during ghrelin infusion as compared to a reduction of 131.6

± 19.4 µmol/l during saline infusion, P = 0.002.

28


Hypopituitary study

Hormones and metabolites Hormonal and metabolite responses are shown in Figure 9. Serum ghrelin was similar at

baseline (t = 0) on the two study days [0.49 ± 0.06 (ghrelin) vs. 0.51 ± 0.06 µg/l, P = 0.38]

and correlated negatively with BMI on both study days [r = -0.73, P = 0.04 (ghrelin) and r =

-0.83, P = 0.01 (saline)]. By means of the bolus infusion, steady state ghrelin levels were

achieved already at t = 20

min. Infusion of ghrelin in-

creased systemic ghrelin

levels to a plateau of 5.33 ±

0.45 µg/l in the basal state

and 5.86 ± 0.50 µg/l during

the clamp period [mean

ghrelin levels basal period

s. clamp period P =

0.001]. Serum levels of

prolactin, GH, cortisol, insu-

lin, and plasma levels of

norepinephrine and epi-

nephrine were comparable

on both study days.

v

Glucose. Basal plasma

glucose levels were similar

on both study days [5.1 ±

0.3 mmol/l (ghrelin) vs. 5.1

± 0.2 mmol/l, P = 0.94].

Ghrelin caused an increase

in plasma glucose levels to

6.1 ± 0.2 mmol/l at t = 120

min, P = 0.009. During the clamp, plasma glucose levels were maintained at ~ 5.0 mmol/l

on the saline day and were gradually lowered towards postabsorptive levels on the ghrelin

day resulting in comparable glucose levels during the final 30 min of the clamp, P = 0.88.

Ser

um g

hrel

in ( µ

g/l)

0

2

4

6

8

10

Ser

um G

H ( µ

g/l)

0

1

2

3

4

5

GhrelinSaline

CP = 0.12

Ser

um p

rola

ctin

(ng/

ml)

0

2

4

6

8

10

12P = 0.21

B

Ser

um c

ortis

ol (n

mol

/l)

0

200

400

600

800

1000

1200 DP = 0.08

A

Time (minutes)0 100 200 300

Pla

sma

gluc

ose

(mm

ol/l)

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Basal period Clamp periodP < 0.01 P = 0.88

Time (minutes)

0-30

30-60

60-90

90-12

0

120-1

50

150-1

80

180-2

10

210-2

40

240-2

70

270-3

00

Inte

rstit

ial g

luco

se (m

mol

/l)

1

2

3

4

5

6

7

8

9

Basal period Clamp period

P = 0.01 P = 0.64

F

H

Time (minutes)

0 100 200 300

Seru

m F

FA (m

mol

/l)

0.0

0.2

0.4

0.6

0.8

1.0 G P < 0.01

Ser

um in

sulin

(pm

ol/l)

0

200

400

600

Basal period Clamp periodP = 0.21 P = 0.21

E

Figure 9

29


FFA. The basal levels were comparable [0.38 ± 0.05 (ghrelin) vs. 0.45 ± 0.08 mmol/l, P

= 0.47]. Ghrelin infusion induced an 80% increase in FFA to 0.62 ± 0.03 mmol/l at t = 120

min, P < 0.05, followed by a decline and a return to placebo levels during the clamp period.

Resting energy expenditure

Resting energy expenditure (EE) was not significantly affected by ghrelin neither in the

basal nor in the clamp period.

Glucose metabolism and substrate oxidation

Glucose metabolism is

shown in Figure 10. GIR

was significantly decreased

during ghrelin administra-

tion (P < 0.01, Figure 10A)

and the M value was re-

duced by ~ 60% (P =

0.0001, Figure 10A insert).

Ghrelin reduced the

rates of oxidative, non-

oxidative and total glucose

disposal during the clamp

period (P = 0.009, P =

0.033, and P = 0.012, re-

spectively; Figure 10B),

whereas ghrelin did not

significantly have an impact

on glucose metabolism in

the basal state.

Lipid metabolism

Ghrelin did not signifi-

cantly affect RQ in the basal

state, but showed a trend

towards increased lipid oxidation during the clamp period. Overall, ghrelin caused metabolic

inflexibility, in the sense that RQ increased 0.07 ± 0.01 in the saline study (RQclamp – RQbasal)

Time (minutes)

50 100 150 200 250 300

Glu

cose

infu

sion

rate

(mg

x kg

-1 x

min

-1)

0

1

2

3

4

5

GhrelinSaline

P < 0.01

Saline Ghrelin

Glu

cose

infu

sion

rate

(mg

x kg

-1 x

min

-1)

0

1

2

3

4

5P < 0.001

Basal period Clamp periodSaline Ghrelin Saline Ghrelin

Glu

cose

Rd

(mg

x kg

-1 x

min

-1)

0

1

2

3

4

5

6

NOGD

GOX

*

*

P = 0.012

GOX clamp P = 0.009NOGD clamp P = 0.033

A

B

Figure 10

30


as compared to 0.03 ± 0.01 in the ghrelin study (P = 0.03). Infusion of ghrelin had no sig-

nificant effects on neither basal nor insulin-stimulated rates of lipid oxidation (P = 0.37 and P

= 0.19) although ghrelin caused a trend towards metabolic inflexibility (percentage decrease

in lipid oxidation rates: saline 55 ± 5% vs. ghrelin 22 ± 17%, P = 0.11) implicating that

ghrelin caused a tendency to increase rate of lipidoxidation.

Regional substrate metabolism (microdialysis)

Interstitial skeletal muscle glucose levels mirrored the fluctuations in the plasma levels of

glucose during ghrelin infusion (Figure 9H). By contrast, interstitial levels of glucose in ab-

dominal and femoral fat remained at placebo levels. Ghrelin did not significantly influence

the levels of interstitial glycerol, lactate, or urea in neither skeletal muscle nor s.c. tissues.

GH and insulin signaling

AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation

were similar during saline and ghrelin administration, P = 0.57 and P = 0.87, respectively.

Moreover, ghrelin did not evoke Janus kinase/signal transducer of activation and transcrip-

tion (JAK/STAT) signaling as assessed by STAT5, P = 0.20, or STAT3 phosphorylation, P =

0.51, MAPK-stimulation as assessed by extracellular signal-regulated kinase (ERK) 1, P =

0.15, and ERK2 activation, P = 0.94, or suppression of insulin signaling pathways as as-

sessed by protein kinase B (PKB/Akt), P = 0.58, and Akt substrate (AS160) phosphorylation,

P = 0.43. IRS-associated phosphatidylinositol 3-kinase (PI3K) activity was not modified by

ghrelin infusion either [IRS-1 (arbitrary units): 84.7 ± 11.4 (ghrelin) vs. 115.3 ± 19.3, P =

0.36; IRS-2 (arbitrary units): 77.6 ± 22.1 (ghrelin) vs. 122.4 ± 29.6, P = 0.29].

Discussion Methodological considerations


Serum ghrelin was measured by our in-house ghrelin assay in all three studies. Methods

for measurement of ghrelin are much debated and no gold standard exists. Several commer-

cially available assays based on RIA and ELISA techniques recognize different epitopes of the

ghrelin amino acid sequence with or without the acyl side chain, which implies that results

obtained by different assays are not comparable. For example, Hotta et al. applied several

different ghrelin assays on the same plasma samples and depending on the assay, acylated

31


ghrelin levels were either reported to be increased, similar and decreased in women with

anorexia nervosa as compared with age-matched healthy controls (51). This fundamental

problem is even more complicated because the two most popular kits (from Phoenix Phar-

maceuticals and Linco Research) yield a 10-fold difference in measured ghrelin levels (52)!

This variation may be reduced by time-consuming RIA-HPLC methods or a new liquid chro-

matography tandem mass spectrometry technique (53). The latter method has also revealed

that ghrelin degrades rapidly in serum (53), but only recently systematic in vitro studies has

resulted in detailed sample preparation protocols that describe optimal acidification and es-

terase-inhibition procedures.2

When the pharmacokinetic study was designed, the pharmacokinetics (half-life) of ghrelin

was reported by one other study only (30). We aimed to estimate the pharmacokinetics in

detail and to investigate if the previously reported half-life could predict time to steady state.

Therefore a constant intravenous ghrelin infusion was applied.

To investigate if 180-min of ghrelin infusion induced hunger in the post-absorptive pe-

riod, similar visual analogue scales as previously used during ghrelin infusion and ad libitum

meals (19) and in patients with anorexia nervosa (54) were applied.

Muscle biopsies were included to study ghrelins direct putative effects of on JAK/STAT,

insulin, ghrelin and AMPK signaling.

Somatostatin study

From the pharmacokinetic study it was clear that ghrelin infusion caused metabolic

changes, but it was not elucidated if elevations in plasma glucose and serum FFA were

caused by ghrelin itself or by the concomitant release of GH and cortisol. A rough measure of

insulin sensitivity (revised QUICKI) revealed that ghrelin infusion caused insulin resistance

from t = 40 min and during the rest of the study day. The pharmacokinetic study also dem-

onstrated that GH and cortisol peaked after 60 and 120 min, respectively, whereafter a

gradual decline towards baseline levels was observed. In a previous study, concomitant

somatostatin infusion clearly impaired the ghrelin-induced GH release. Therefore, we used a

higher somatostatin infusion rate together with a constant ghrelin infusion and applied gold

standard technique for measurement of insulin sensitivity (hyperinsulinemic euglycemic

2 Bruce D Gaylinn et al. 2007 Long Term Fasting Inhibits Ghrelin Acylation but Not Ghrelin Secre-

tion in Normal Young Men; Evidence from New Assays for Full Length Acyl- and Des-Acyl Ghrelin. Proc 89th Ann Meeting Endocrine Soc P1-62 Abstract

32


clamp) in addition to tracer methodology and microdialysis to determine peripheral, hepatic,

and regional s.c. effects of ghrelin.

Hypopituitary study

The Somatostatin study revealed a moderate but detectable GH and cortisol break-

through. Although the GH and cortisol elevations were minute and comparable to – or lower

than – levels measured in the postabsorptive state (GH) (55) and during nighttime (cortisol)

(56) both GH and cortisol elevations reached statistical significance and may in part have

had metabolic effects. Therefore, a similar study comprising hypopituitary patients (GH re-

sponse to a dynamic GH stimulation test 0.3 ± 0.1 µg/l) on stable replacement therapy with

GH and hydrocortisone was undertaken. In addition to the methods applied to the Soma-

tostatin study, muscle and fat biopsies were performed and the Hypopituitary study included

more regional s.c. measurements to elaborate regional effects of ghrelin. Muscle biopsies

also enabled comparisons with biopsies from the pharmacokinetic study thereby delineating

JAK/STAT signaling by endogenous GH.

Considerations of possible mechanisms and explana-

tions - implications and review of literature

Endogenous ghrelin excursions and previous pharmacokinetic

studies

Endogenous ghrelin exhibits approximately 22 surges per 24 hours (57). Nagaya et al.

reported that total ghrelin levels disappeared from plasma with a half-life of 10 minutes after

a bolus injection (30). Subsequently, Akamizu et al. reported half-lives of both total (t½ 27 to

31 minutes) and acylated plasma ghrelin (t½ 9 to 13 minutes) in a one-compartment model

calculated from ghrelin concentrations in blood samples taken every 15 minutes (29). These

previously reported half-lives of total ghrelin are in line with the initial half-life (t½ 24.2 min-

utes) in our pharmacokinetic study.

Pharmacokinetics in a clinical context

Our study revealed that a bi-exponential equation gave the best fit as compared to a

mono-exponential approach. One explanation can be attributed to the frequency of blood

sampling since the concentration-time curve appears monophasic when the fraction of a

substance eliminated by the last exponential term (f2) is relatively large (in this study 33%)

33


and blood is drawn less frequently. To circumvent this problem, blood samples were col-

lected every five minutes for the first hour after termination of the ghrelin infusion in order

to detect the two distinct slopes of the decay curve. Another plausible explanation is attrib-

utable to our assay that recognizes ghrelin as well as desacyl ghrelin. Recently, ghrelin has

been demonstrated to degrade to desacyl ghrelin (53) and because desacyl ghrelin has a

larger half-life than ghrelin (29), ghrelin elimination appears to follow a bi-exponential decay

curve. Of note, Gauna et al. detected higher total serum ghrelin levels after administration of

acylated ghrelin than after an equal dose of desacyl ghrelin (31). Systematic in vitro experi-

ments demonstrated that this surprising observation was not caused by an assay problem.

Rather, they suggested that the apparent increase in desacyl ghrelin following administration

of acylated ghrelin stemmed from endogenous release of desacyl ghrelin or was caused by

an acute change in the capacity to degrade ghrelin (31).

However, pharmacokinetic parameters from our bi-exponential approach predicted the

experimentally obtained ghrelin levels during the infusion period. A small tendency to overes-

timate the experimental ghrelin levels by the two-compartment analysis indicates that simpli-

fying distribution kinetics of ghrelin to a one-compartment model is inadequate.

The peak ghrelin concentration observed was very similar to the mathematically pre-

dicted level, but a steady state level was not reached again in accordance with a two-

compartment model. Our observations contrast previous ghrelin infusion studies reporting

steady state within 60 to 90 minutes (18;19;33;58). However, none of the previous reports

provides the pharmacokinetic approach, making it difficult to draw direct comparisons to our

results. The clinical implications of the two-compartment characteristics of ghrelin are that

longer infusion periods are required to obtain steady state levels of ghrelin in clinical ghrelin

infusion studies.

Predictors of ghrelin levels

Systemic ghrelin levels change dynamically to feeding state (59;60). The present studies

revealed a positive correlation between BMI and MRT of ghrelin. Thus, the counter-

regulatory decline in ghrelin levels that presumably serves to compensate for a positive en-

ergy expenditure in obese individuals (61-63) is not attributable to increased clear-

ance/decreased MRT of ghrelin. Therefore, decreased ghrelin levels in obesity appear to be

caused by decreased secretion rates. Our observation (MRT of ghrelin correlates positively

with BMI) is substantiated by a previous report, where a constant ghrelin infusion increased

circulating ghrelin levels more in obese than in lean subjects (17), and it is compatible with

34


previous studies reporting that obese individuals have a reduced postprandial decrease in

ghrelin levels as compared to lean subjects (61-63).

A positive correlation between MRT and HDL cholesterol levels was also revealed. Al-

though correlation does not imply causality, it has previously been shown that the majority

of circulating acylated ghrelin is bound to larger molecules (64), and the high-density lipo-

protein-fraction has been demonstrated to bind acylated ghrelin in vi (65). HDL choles-

terol levels could thus be an independent biological determinant of ghrelin bioavailability in

humans and may account for the 2

tro

nd compartment.

Effects on appetite

The appetite related results were moderate and mostly non-significant as compared to

previous publications (17-19;66). Temporal and concentration differences between the ear-

lier reports and the present results may have caused these discrepancies. Wren et al. (19)

studied orexigenic and appetite effects of more physiological increments (approximately 2-

fold elevations from baseline), significantly less than the 6.5-fold increase we obtained. High

ghrelin levels may entail compensatory mechanisms such as internalization of the GHS-R

from the cell surface in order to desensitize the cell responsiveness (67) and the effect of

ghrelin on food intake in rodents is indeed bell-shaped (68). The studies by Druce et al. (17),

Schmid et al. (66), and Neary et al. (18) reported the orexigenic effects of ghrelin after 45,

60, and 90 minutes respectively.

Hormonal and metabolite effects

In the pharmacokinetic study, significant elevations were observed in circulating levels of

GH, ACTH, cortisol, prolactin, glucose and FFAs in accordance with most previous reports

(4;5;14;17-19;29-33;66;69;70). A small but significant increase in plasma glucose levels was

also observed, but in contrast to one former study (22), this was associated with an increase

rather than a decrease in insulin levels. From this study it is not possible to determine any

direct metabolic effect of ghrelin, but our results appear to corroborate earlier observations

reporting that ghrelin induces insulin resistance in humans (31)

At first glance, the increase in cortisol levels contrasts the previous established inverse

correlation between ghrelin and cortisol observed during fasting (12). However, the ACTH

concentration-time curve reveals a transient increase in ACTH levels after 60 minutes of

ghrelin infusion only, whereafter ACTH levels return to baseline levels. The present ghrelin

infusion period is not sufficient to disclose any effect of ghrelin on cortisol levels after nor-

35


malization of ACTH levels, but the decrease in cortisol levels at t = 360 minutes indicates

that ghrelin may actually inhibit cortisol levels on the long term.

Pancreatic clamp technique and adjustable glucose infusion in the Somatostatin study

ensured comparable levels of glucose, FFA, insulin, C-peptide, and glucagon. Somatostatin

attenuated the ghrelin-induced GH response to levels comparable to physiological GH bursts

in the postabsorptive state (55), but cortisol excursions recorded during ghrelin infusion

were comparable to the levels reported in the pharmacokinetic study.

In the Hypopituitary study, similar hormonal levels during ghrelin and saline infusion

were successfully achieved. The increases in glucose and FFA levels therefore seemed di-

rectly attributable to ghrelin exposure.

Effects on substrate metabolism

In the Somatostatin study, ghrelin infusion combined with a pancreatic clamp caused a

significant reduction in glucose disposal rates and GIR, and lipolysis in terms of increased

palmitate rate of appearance and FFA levels. Although the GH response was clearly attenu-

ated as compared with previous ghrelin infusion studies (22;32;33;71;72), small and physio-

logical GH excursions may affect substrate metabolism, although its significance is disputable

(73-76). In the present study cortisol levels during ghrelin infusion surpassed placebo levels

from time 60 to 240 min and reached ~ 2-fold the corresponding placebo level after 90 min.

The metabolic impact of such increments in cortisol levels on glucose and lipid metabolism is

also uncertain (77-80). In our study, a correlation between GH, cortisol and FFA indicate that

GH and cortisol actually caused lipolysis, which, in turn can induce insulin resistance. How-

ever, neither GH nor cortisol was predictor, when palmitate turn-over rate was included as

the dependent variable.

In the Hypopituitary study, we accomplished to factor out GH and cortisol increments.

Ghrelin infusion did indeed cause a decrease in glucose disposal rates, GIR, and lipolysis. We

recorded significantly increased interstitial glucose levels in skeletal muscle substantiating

that insulin sensitivity was significantly attenuated in this tissue. The molecular mechanisms

by which, ghrelin influences glucose and lipid metabolism remain however to be determined,

because no detectable aberrations in AMPK, PI3K, PKB/Akt, or AS160 were retrieved in

skeletal muscle. In particular, it is still not evident whether ghrelin causes insulin resistance

directly and, as a secondary phenomenon, lipolysis or vice versa. It is possible to suppress

lipolysis pharmacologically and the role of the concomitant ghrelin-induced FFA increase

could be explored by using an inhibitor of lipolysis (e.g. acipimox). It is, however, important

36


to note that biopsies were only obtained in the basal state, and it remains to be demon-

strated whether ghrelin may influence the same signaling pathways during concomitant insu-

lin stimulation.

Although our results demonstrate an insulin antagonistic effect rather than a stimulation

of insulin signaling, which is evident in in vitro experiments (81-85), our results are in line

with the in vivo evidence that ghrelin induces insulin resistance (31;36).

FFA levels increased in response to ghrelin administration, but this increase did not trans-

late into an increase in whole body lipid oxidation or energy expenditure contrasting obser-

vations in rodent models (20;86-88). The mechanism by which lipid oxidation remained unaf-

fected in the present study is undetermined. However in rodents, it has been demonstrated

that ghrelin induces a hepatic lipogenic gene expression together with a reduction in AMPK

levels, which is a stimulator of FFA oxidation (89). It remains to be elucidated if ghrelin also

affects expressions of genes involved in lipid oxidation in humans as indicated by our clinical

observations.

It remains speculative what the exact physiological role of ghrelin is in humans. The role

of ghrelin in appetite regulation and food intake has been studied extensively. Numerous

rodent models comprising ghrelin knockouts, ghrelin receptor knockouts, measurements of

neuropeptide Y and agouti-related peptide expressions following ghrelin administration,

blocking antibodies, ghrelin antagonists, and blocking RNA Spiegelmers indicate that ghrelin

stimulates food intake. In humans, ghrelin increases before food intake and decreases

shortly after feeding even without time and food-related cues and ghrelin has therefore been

suggested to initiate food intake (90). This study reported total levels of ghrelin only, so the

relationship between appetite and acylated ghrelin (i.e. the orexigenic subtype) is still not

clarified and one study by Avram et al. found no effects of either fasting or feeding on acy-

lated ghrelin levels (91).

The role of ghrelin in regulation of GH secretion has been addressed in a number of stud-

ies. The preprandial and nocturnal increments of ghrelin may hypothetically cause subse-

quent GH secretion, but analyses of the ghrelin-GH interrelationship have reported inconsis-

tent results: 1) positive association between ghrelin and GH pulsatility (92), 2) no relation

between GH and ghrelin pulses (91), and 3) a correlation depending on certain conditions

(93). Moreover, physiological conditions characterized by a GH increase are not preceded by

elevated peripheral ghrelin levels (11-13). Thus, it remains to be convincingly demonstrated

that gut-derived ghrelin is a significant GH releasing factor.

37


The role of ghrelin in substrate metabolism is currently a matter of debate. Numerous ro-

dent models demonstrate that ghrelin administration increases adiposity, whereas data from

ghrelin and ghrelin receptor knockout models have not convincingly supported the adipo-

genic properties of ghrelin (86;88;94;95). There seems to be more evidence that ghrelin

affects glucose homeostasis and decreases insulin sensitivity (22;34-37). All of our three

studies in this dissertation support that ghrelin acutely causes insulin resistance directly as

well as indirectly by elevation of GH and cortisol levels. It therefore seems possible that a

natural metabolic domain for ghrelin action is in conditions characterized by short term en-

ergy shortage, where ghrelin gradually increases during the time interval until the next meal,

whereafter food intake causes the next ghrelin decrease (15;96). Ghrelin could be specu-

lated to counteract fasting-induced hypoglycemia and thereby secure fuel supply for glucose

dependent tissues such as the central nervous system (97). Ghrelin does not increase consis-

tently after prolonged fasting (12), and it therefore remains uncertain whether ghrelin in this

condition regulates GH secretion or substrate metabolism.

Effects on resting energy expenditure

Resting energy expenditure decreased in response to ghrelin and the pancreatic clamp

during hyperinsulinemia, whereas no change was evident in the Hypopituitary study. In GHD

rodents, 24-h EE was not affected, but transient and small negative deflections in EE were

recorded immediately following ghrelin injections (20) indicating that ghrelin exerted some

effects on EE. Numerous rodent studies report either increased or decreased spontaneous

activity following ghrelin administration, whereas the effect of ghrelin on resting EE in hu-

mans, to our knowledge, has not been reported previously. Our present studies suggest that

ghrelin is not a major determinant of EE, but more powerful studies need to be undertaken

to further investigate this matter.

Methodological limitations and pitfalls


Only total levels of serum ghrelin were measured. Total ghrelin comprises of the acylated

as well as the desacyl 28 amino-acid peptide, probably along with other shorter fragments

and elongated ghrelin forms. Of note, the endocrine effects of ghrelin are mainly attributed

to the octanoylation on serine number 3 (4;98). By the time the pharmacokinetic study was

designed, this was not an obvious limitation, because we planned to infuse the genuine acy-

lated human ghrelin form and the assay would therefore presumably mainly measure acy-

38


lated ghrelin levels. Recently, it became evident that ghrelin is indeed degraded by cleaving

off of the acyl side chain, because a sensitive, quantitative mass spectrometry method

documented that serum levels of ghrelin decreased, whereas desacyl ghrelins levels in-

creased in a stoichiometric ratio (53). Thus, the assay used in this study is a limitation, and

this should be kept in mind when interpreting the results and conclusions with regards to the

pharmacokinetics. However, the equation used to predict steady state concentrations ap-

pears to be a useful tool, since it was successfully used in the Hypopituitary study, where a

primed continuous ghrelin infusion accurately predicted steady state concentrations (5.12 ±

0.45 µg/l vs. predicted 5.00 ± 0.35 µg/l).

The hunger data are conflicting and therefore difficult to interpret. The lack of compelling

appetite effects in this study may in part be attributed to the infrequent measurements on

the visual analogue scale. Instead, study participants could have been offered an ad libitum

buffet to measure energy intake rather than relying on a hypothetical response.

Somatostatin study

The somatostatin clamp did not abolish ghrelin-induced GH release. A small but signifi-

cant GH peak occurred and at least part of the effects on glucose and lipids are presumably

explained by GH. The study question: whether ghrelin per se has direct effects on glucose

and lipid metabolism can therefore not be fully answered by the study.

The fact that glucose levels fell during the basal period of the saline/ghrelin period indi-

cates that the low-dose insulin infusion had an effect, and at the end of the basal period the

glucose levels rose and the glucose infusion rate dropped. This indicates that lipolysis was

ongoing and glucose uptake reduced, most likely due to the small dose of GH infusion and

on the ghrelin day also due to the GH peak induced. In, addition, regression analysis re-

vealed that ∆AUC of GH, P = 0.005, and cortisol, P = 0.002, were both significant predictors

of ∆AUC of FFA, but the correlation was not supported when palmitate flux was included

instead of ∆AUC of FFA as dependent variable. The discrepancy between the effects of ghre-

lin on ∆AUC of FFA and palmitate flux may be attributable to a smaller method sensitivity of

the tracer technique as compared to the measurements of FFA concentrations. However, the

exact cause of the discrepancy has not been elucidated.

Plasma glucagon was stable during initial 30-min, whereafter an increasing trend was

evident. The reason for this increase is not clear and contrasts similar somatostatin gluca-

gon-replacement studies, where plasma glucagon remains at steady state. However, plasma

39


glucagon levels were similar during both saline and ghrelin administration. Therefore, it

seems unlikely that glucagon caused the effects on GIR and substrate metabolism.

Hypopituitary study

Due to the extensive and complex study design the number of biopsies was limited. Per-

forming a biopsy in the basal period favored the study of GH signaling, whereas a biopsy

during hyperinsulinemia would have been optimal for studying insulin signaling. The basal

period was chosen in order to make it comparable to the biopsy timing of the pharmacoki-

netic study. In the basal state, insulin signaling activity is low a priori, and it would have

been relevant and informative also to have biopsies during the hyperinsulinemic clamp pe-

riod.

In the basal period plasma glucose increased in response to ghrelin, although glucose

disposal rate, endogenous glucose production and glucose infusion were constant. This

seems to be attributable to methodological insensitivity unless, unlikely so, changes in vol-

ume of distribution for glucose could explain the apparent discrepancy.

Conclusions & Perspectives

Conclusions

In conclusion, the kinetics of infused ghrelin follows a two-compartment model and the

mean residence time of infused ghrelin correlates positively with both HDL cholesterol and

BMI. The pharmacokinetic study provides useful data when tailoring ghrelin infusion rates in

clinical experiments. Ghrelin infusion induces a robust release of both GH and cortisol ac-

companied by an increase in systemic glucose and FFA levels.

Concomitant somatostatin infusion blunts the ghrelin-induced GH release, but the effects

on systemic glucose and FFA levels prevail in addition to a reduction of insulin sensitivity.

Hypopituitary patients with GH and ACTH deficiency constitute an attractive in vivo model

for studying putative GH- and cortisol-independent effects of ghrelin. In the Hypopituitary

study, we demonstrate that ghrelin infusion per se causes insulin resistance and increments

of systemic glucose and FFA levels. These effects were not attributed to direct detectable

effects on AMPK, insulin or JAK/STAT signaling.

40


Perspectives

Potential therapeutic applications of ghrelin in conditions characterized by

impaired ghrelin secretion

Conditions characterized by low GH levels such as obesity (99) and aging (100) and con-

ditions characterized by “ghrelin resistance”, e.g. cachexia (101) and anorexia nervosa (102),

may benefit from prolonged treatment with ghrelin or GHS. These conditions have been the

objective of a few controlled trials, but the potential therapeutic role of ghrelin and GHS still

needs to be elucidated in detail. Ghrelin administration has, indeed, proved successful in

short term treatment of cardiac and pulmonary cachexia; three weeks of ghrelin administra-

tion to 10 patients suffering from chronic heart failure improved lean body mass, muscle

strength, peak workload and oxygen consumption during exercise (103). A similar trial com-

prising a three week period and equal ghrelin doses improved body composition, muscle

wasting, and functional capacity in patients suffering from cachexia due to chronic pulmo-

nary obstructive disease (104).

Certain subgroups of patients with classic GHD such as patients suffering from pituitary

stalk lesions (105) may benefit from chronic treatment with GHS or ghrelin. In a randomized,

placebo-controlled, double-blind study 11% (n = 83) of adult GHD patients responded with a

peak GH response ≥ 5 µg/L after the first or the last dose of the oral GHS NN703 (106). Ad-

ministration of another oral GHS, MK-0677, during a 7 – 8 day period in 18 prepubertal chil-

dren with idiopathic GHD also induced an increase in GH in the subjects receiving the highest

dose (n = 12) (107). Patients with baseline serum IGF-I levels greater than 50 µg/L showed

a more pronounced GH response to MK-0677 in comparison with patients whose baseline

serum IGF-I levels were below 50 µg/L (107). A third randomized, double-blind, cross-over

study confirmed that the effect of GHS correlates inversely with the degree of GHD (108).

A potential advantage of GHS or ghrelin treatment in the subgroups above includes pres-

ervation of the negative IGF-I feedback on GH secretion and for GHS treatment the possibil-

ity of oral administration is appealing. Future studies may reveal whether GHS or ghrelin

actually have therapeutic potential in the above-mentioned conditions.

Limitations for ghrelin treatment in a clinical setting

Ghrelin is one of the most potent GHS and in comparison to hexarelin, a synthetic GHS

for intravenous administration, ghrelin has a stronger GH-releasing effect (14). The major

limitation of ghrelin treatment in a clinical setting is attributable to a very short half-life when

injected intravenously and oral bioavailability is probably negligible although it has never

41


been tested. S.c. ghrelin administration elicits a bell-shaped GH response after 30 minutes,

but the response only lasts between 60 and 90 minutes (69).

Diagnostic potential of ghrelin

Ghrelin levels are not altered in patients with GHD (11;109;110), and ghrelin measure-

ments therefore do not seem helpful for the diagnosis and follow-up of these patients. The

diagnosis of GHD is established by provocative tests and the test of choice is the insulin tol-

erance test (ITT) (111). Certain contraindications apply to this test, e.g. patient history of

ischemic heart disease or seizure disorder, in the event of which the GHRH plus arginine test

is considered as a second choice. GHS binds to hypothalamic and pituitary cells (112) and

augments the effect of GHRH on GH-release (113). The GHS GHRP-6 in combination with

GHRH has also proved useful as a diagnostic tool in adult GHD (114;115). If appropriate cut-

off levels are taken into account, these tests may prove an alternative to the ITT.

It is a potential concern that patients with GHD secondary to pituitary stalk lesions may

be misdiagnosed by exhibiting a normal response to exogenous ghrelin or a GHS. Whether

this is a clinically significant problem is, however, uncertain; at least it has been reported

that GHRH in combination with pyridostigmine correctly diagnoses patients with severe GHD,

irrespective of etiology, provided that an appropriate cut-off is available (116).

Other aspects of ghrelin administration to hypopituitary patients

The vast majority of clinical and many animal studies reporting effects of exogenous

ghrelin are hampered by the concomitant increase of GH secretion in addition to cortisol in-

crements. Many scientific articles should be interpreted with care, because they report the

combined effects of ghrelin, GH, IGF-I and cortisol rather than effects of ghrelin per se.

Therefore, hypopituitary patients constitute an interesting model for studying GH- and ACTH-

independent effects of ghrelin and future studies comprising this group of patients may re-

sult in more valid conclusions regarding direct effects of ghrelin.

Potential therapeutic applications of ghrelin antagonists

Obesity, the metabolic syndrome, type 2 diabetes, and the associated complications are

major and growing problems in the health care services and the lay public. It is evident that

preventing or treating obesity, hyperlipidemia, and diabetes by life style interventions and

medical therapy improves quality of life and health. Among numerous factors leading to a

positive energy balance, ghrelin has attracted attention because it induces appetite

(17;19;66), increases food intake (17-19), decreases insulin secretion (22;34;68;117-119),

and causes insulin resistance (33). In turn, antagonists of ghrelin and animal ghre-

42


lin(receptor) knockout models have demonstrated beneficial effects on appetite and food

intake (86;88;119), insulin secretion (35;36;119) and insulin sensitivity (36;37;94). Future

clinical trials will reveal whether results from rodent studies apply to human physiology; i.e. if

antagonism of ghrelin enhances insulin secretion to match increased insulin requirements in

diet-induced obesity and improves glycemic control, and if ghrelin antagonists thereby have

therapeutic potential in the treatment of the metabolic syndrome and type 2 diabetes in ad-

dition to body weight control.

Summary Ghrelin is a gastrointestinal peptide hormone originally discovered as the endogenous

ligand for the GHS-R. The GHS-R is widely distributed and ongoing research suggests plei-

otropic effects of ghrelin. In view of the potential therapeutic applications of ghrelin the

overall aim of this PhD study was to investigate clinical pharmacokinetics and acute meta-

bolic effects of ghrelin in detail.

The PhD dissertation consists of three original papers based on three randomized, pla-

cebo-controlled cross-over clinical protocols comprising both healthy subjects and hypopitui-

tary patients: 1) Pharmacokinetic study, 2) Somatostatin study, and 3) Hypopituitary study.

Two articles have been published in international peer-reviewed journals and a third manu-

script has been submitted for review.

In the first protocol, acylated ghrelin was infused into 17 healthy subjects. By measure-

ments of ghrelin levels and pharmacokinetic modeling an extensive list of pharmacokinetic

parameters was calculated and correlated with central parameters of individual characteris-

tics. In addition, muscle biopsies were sampled to study pertinent ghrelin signaling path-

ways.

In the second protocol comprising eight healthy subjects receiving ghrelin and placebo

infusion on two different occasions, the aim was to suppress the ghrelin-induced GH- and

cortisol-secretion by concomitant somatostatin infusion in order to measure the direct effects

of ghrelin on glucose and lipid metabolism. Insulin sensitivity was measured by a hyperinsu-

linemic euglycemic clamp and substrate metabolism was measured by tracer and microdialy-

sis technique. The Somatostatin study revealed a moderate but detectable GH and cortisol

breakthrough.

To circumvent the effects of endogenous GH and cortisol on glucose and lipid metabo-

lism, a third study comprising eight hypopituitary adults on stable replacement therapy with

43


GH and hydrocortisone was performed. In this protocol, we also used the pharmacokinetic

parameters to calculate a ghrelin bolus dose to rapidly achieve steady state concentrations.

The pharmacokinetic results showed that ghrelin infusion followed a two-compartment

model. The MRT of exogenous ghrelin correlated positively with both HDL cholesterol levels

and BMI. The pharmacokinetic parameters are useful for designing future clinical ghrelin

infusion studies. Concomitant somatostatin infusion attenuated ghrelin induced GH secretion,

but GH levels were significantly increased as compared to GH levels during placebo admini-

stration. In the Hypopituitary study, it was demonstrated that ghrelin infusion per se caused

insulin resistance and increments in levels of systemic glucose and FFA levels. These effects

were not attributed to direct detectable effects on AMPK, insulin or JAK/STAT signaling. No

aberrations in putative ghrelin signaling pathways were detected. In addition, our studies

demonstrated that ghrelin-induced endogenous GH release translated into GH receptor sig-

naling in skeletal muscle.

Summary in Danish Ghrelin er et gastrointestinalt peptid-hormon bestående af 28 aminosyrer med en unik

acyl-sidekæde. Ghrelin blev opdaget i 1999 af en forskergruppe, som ledte efter en endogen

ligand for den såkaldte GHS receptor. GHS receptoren var forinden karakteriseret og klonet i

1996. GHS receptoren forefindes i størstedelen af kroppens væv. Man kunne derfor forvente,

at dette relativt nyopdagede peptid-hormon ville frembyde en række forskelligartede effek-

ter. Talrige kliniske, dyreeksperimentelle og laboratoriebaserede forsøg indenfor dette områ-

de har efterfølgende vist, at ghrelin i overensstemmelse med receptorens lokalisation, har

adskillige effekter. Heriblandt er det muligt, at ghrelin kan have et terapeutisk potentiale og

dette PhD-studium havde til formål at undersøge og tilvejebringe ny viden om ghrelins klini-

ske farmakokinetiske og akutte metaboliske effekter.

PhD-afhandlingen består af tre originalartikler, som er baseret på tre kliniske, randomise-

rede, placebo-kontrollerede overkrydsningsstudier, hvor både raske og hypofyseinsufficiente

patienter blev undersøgt. Studierne er 1) Farmakokinetikstudiet, 2) Somatostatinstudiet og

3) Hypofyseinsufficiensstudiet. Artiklerne fra det første og andet studium er allerede publice-

ret i internationale peer-reviewed tidsskrifter, mens artiklen vedr. det sidste studium er ind-

sendt til bedømmelse ligeledes ved et internationalt tidsskrift.

I det første studium deltog 17 raske, unge mænd. Vi infunderede henholdsvis ghrelin og

saltvand på to forskellige undersøgelsesdage og målte efterfølgende ghrelinniveauerne i se-

rum. Ved hjælp af farmakokinetisk modellering og beregninger estimerede vi en række væ-

44


sentlige farmakokinetiske variabler og udførte korrelationsstudier, hvor centrale individaf-

hængige karakteristika indgik. Målinger af GH, kortisol, glukose og frie fedtsyrer viste, at

ghrelin medførte sekretion af begge hormoner og øgede de systemiske niveauer af begge

substrater.

I det andet studium deltog 8 raske, unge mænd. Vi infunderede henholdsvis ghrelin og

saltvand på to forskellige undersøgelsesdage samtidig med somatostatin for at undertrykke

GH og kortisol sekretionen og for dermed at undersøge ghrelins direkte effekter på glukose-

og lipidmetabolisme. Insulinfølsomhed blev målt med hyperinsulinæmisk euglykæmisk

clampteknik og substratmetabolisme blev målt med både tracerteknik og mikrodialyse. I det-

te studium var der på trods af somatostatininfusionen en moderat GH og kortisol sekretion.

For at undgå den endogene GH og kortisolsekretion og disses effekter på glukose- og li-

pidmetabolisme, udførte vi et tredje studium med hypofyseinsufficiente voksne patienter

behandlet med GH- og hydrokortisonsubstitution. I dette forsøg anvendte vi også den far-

makokinetiske viden fra det første forsøg til at beregne en ghrelin bolus-dosis, således at

ghrelin ligevægtsniveauet blev opnået hurtigere end ved en konstant ghrelininfusion.

Det farmakokinetiske studium viste, at infunderet ghrelin følger to-kompartment-

karakteristik. Det farmakokinetiske begreb mean residence ime (et udtryk for den gennem-

snitlige tid et givent molekyle opholder sig i kroppen) udviste positiv korrelation med både

højdensitet kolesterol niveauer og body mass index. De beregnede farmakokinetiske para-

metre kan med fordel benyttes i designet af fremtidige ghrelininfusionsstudier og har allere-

de fundet anvendelse i Hypofyse-insufficiensstudiet. Somatostatinstudiet viste, at somatosta-

tin nedsatte den endogene GH sekretion betydeligt, men GH niveauerne var signifikant høje-

re end de tilsvarende niveauer under placebo- og somatostatinindgift. Hypofyseinsufficiens-

studiet viste, at ghrelininfusion uafhængigt af GH og kortisol medførte insulinresistens og

øgede plasmaniveauerne af glukose og serumniveauerne af frie fedtsyrer. Effekterne kunne

ikke tilskrives målbare ændringer af AMPK, insulin eller JAK/STAT signalvejs-molekyler og vi

fandt ingen ændring af formodede ghrelinsignalvejs enzymer. Derudover viste vores studier,

at ghrelininduceret GH sekretion medførte aktivering af GH signalvejen i skeletmuskulatur.

t

Acknowledgements I am indebted to Aarhus University for providing a research fellowship and to Novo Nord-

isk for an unrestricted educational grant. I wish to thank the following foundations for pro-

viding me financial support: The World Anti-Doping Agency (WADA), The Research Initiative

of Aarhus University Hospital, The John and Birthe Meyer Foundation, The FOOD Study

45


Group/Ministry of Food, Agriculture and Fisheries & Ministry of Family and Consumer Affairs,

The Maersk McKinney Møller and wife Chastine McKinney Møller Foundation, The Danish

Diabetes Association, and Roche Diagnostics for kindly providing microdialysis catheters.

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