Cardiovascular Research 62 (2004) 481 – 488
www.elsevier.com/locate/cardiores
Growth hormone releasing peptide (ghrelin) is synthesized and
secreted by cardiomyocytes
Marı́a J. Iglesias a, Roberto Piñeiro a, Montserrat Blanco b, Rosalı́a Gallego b, Carlos Diéguez c,
Oreste Gualillo d, José R. González-Juanatey a, Francisca Lago a,*
a
Unidad de Investigación del Servicio de Cardiologı́a, Laboratorio de Investigación 1, Planta Baja, Area de Investigación y Docencia,
Hospital Clı́nico Universitario de Santiago de Compostela, Travesı́a Choupana s/n, 15706, Santiago de Compostela, Spain
b
Departamento de Ciencias Morfológicas, Universidad de Santiago de Compostela, Spain
c
Departamento de Fisiologı́a, Universidad de Santiago de Compostela, Spain
d
Unidad de Investigación del Servicio de Reumatologı́a, Laboratorio de Investigación 4, Hospital Clı́nico Universitario,
Santiago de Compostela, Spain
Received 12 September 2003; received in revised form 12 January 2004; accepted 20 January 2004
Time for primary review 15 days
Available online 8 March 2004
Abstract
Objective: Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), acts on the pituitary and the hypothalamus to
stimulate the release of growth hormone (GH) and promotes appetite and adiposity. It has also been reported to increase myocardial contractility,
induce vasodilation, and protect against myocardial-infarction-induced heart failure. Though principally gastric in origin, it is also produced by
other tissues. This work investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to
stress and exogenous apoptotic agents. Methods: Ghrelin and its receptor expression was studied by RT-PCR, immunohistochemistry, and
competitive binding studies in mouse adult cardiomyocyte cell line HL-1, and primary cultured human cardiomyocytes. Ghrelin accumulation
in cardiomyocyte culture medium was measured by radioimmunoassay. Viability and apoptosis assays were carried on by MTT and Hoechst dye
vital staining, respectively. Results: RT-PCR showed that HL-1 cells produce mRNAs for both ghrelin and GHS-R, and that GHS-R1a is
expressed in human cardiomyocytes; and competitive binding studies using 125I-labelled ghrelin showed efficient constitutive expression of
GHS-R at the surface of HL-1 cells. Immunohistochemistry confirmed the presence of ghrelin in the cytoplasm of HL-1 cells and of isolated
human cardiomyocytes in primary culture. Radioimmunoassay showed that ghrelin was secreted by HL-1 cells and human cardiomyocytes into
the culture medium. Ghrelin did not modify the viability of HL-1 cells subjected to 12-h starvation, but did protect against the apoptosis inducer
cytosine arabinoside (AraC). Finally, production of ghrelin mRNA in HL-1 cardiomyocytes was reduced by AraC but increased if exposure to
AraC was preceded by GH treatment. Conclusions: Ghrelin is synthesized and secreted by isolated murine and human cardiomyocytes,
probably with paracrine/autocrine effects, and may be involved in protecting these cells from apoptosis.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Apoptosis; Cell culture/isolation; Growth factors; Hormones; Cardiomyocytes; Receptors
1. Introduction
Ghrelin is a 28-amino-acid acylated peptide that was
first isolated from rat stomach endocrine cells. It is the
endogenous ligand for growth hormone secretagogues
receptor (GHS-R) [1], a G-protein-coupled receptor that
is found mainly in the pituitary and hypothalamus [2] but
* Corresponding author. Tel.: +34-981-950902; fax: +34-981-951068.
E-mail address: [email protected] (F. Lago).
also in numerous other tissues [3]. Major interest in this
peptide derives from the fact that, in addition to other
effects, it is involved in the regulation of energy balance
and body weight homeostasis [4]. In rodents, ghrelin
stimulates appetite, adiposity and the release of growth
hormone (GH) [1,5 – 8], and regulates the gonadal axis [9]
and carbohydrate metabolism [10]. In humans, ghrelin
enhances appetite [11], and plasma ghrelin levels correlate
negatively with body mass index and are subnormal in the
obese [12,13].
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.01.024
482
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
Although ghrelin is found mainly in stomach and hypothalamus [1], ghrelin and/or its mRNA have also been
detected, at lower levels, in kidney, placenta, pancreas,
testis, ovary, adrenal cortex and myocardium (in this last
without identification of what type of cell was responsible),
and in many of these tissues, including human myocardium,
GHS-R mRNA has also been found (see Ref. [14] and
references therein). This suggests that ghrelin can act as a
paracrine/autocrine hormone in these tissues.
With regard to the cardiovascular system, GHS-R
mRNA is present not only in human myocardium but also
in the aorta, left ventricle and left atrium of rat [15].
Furthermore, in both humans and experimental animals,
ghrelin can have beneficial cardiovascular effects that seem
not to be mediated by GH [15 –22]. This suggests that one
of the multiple mechanisms by which obesity favours
cardiac pathology may consist in its being associated, as
noted above, with low ghrelin levels, which may reduce
cardioprotection.
In the work described here, we investigated whether
cardiomyocytes synthesize and secrete ghrelin, and how its
production in these cells responds to exogenous apoptotic and
anti-apoptotic agents.
2. Methods
All sera and media were from Life Technologies (Poole,
UK), and all other products from Sigma Chemicals Co. (St.
Louis, MO, USA), unless otherwise stated.
2.1. Cells
HL-1 cells (a line of adult mouse atrial cardiomyocytes)
were a generous gift of Dr. W.C. Claycomb of Louisiana
State University Medical Center (New Orleans, LA, USA)
and were cultured on fibronectin-covered plates containing
ExCell 320 medium (from JRH Biosciences, Andover,
UK) supplemented with foetal bovine serum (FBS), insulin, norepinephrine, endothelial cell growth supplement
(from Upstate Biotechnology, Lake Placid, NY, USA),
and retinoic acid [23]. In experiments on apoptosis induction, HL-1 cells were pretreated for 12 h with 1 Ag/ml
growth hormone (GH) or 0.1 –5.0 AM ghrelin and were
then treated for 12 or 24 h with 100 AM AraC as
proapoptotic agent.
Primary cultures of human cardiomyocytes were
obtained from pieces of right atrial appendage excised to
allow catheterization of the right atrium during surgery
requiring cardiopulmonary bypass (this excised tissue is
normally discarded). The atrial tissue was minced in small
pieces and digested at 37 jC in three 5-min cycles with
PBS (NaCl, 0.15 M in 0.01 M phosphate buffer of pH 7.4)
containing 0.25% trypsin, 0.15% collagenase and 0.02%
glucose, and cells were then extracted from the pooled
supernatants by centrifuging for 5 min at 580 g and were
cultured in Iscove’s modified Dulbecco’s medium (Life
Technologies) supplemented with 10% FBS, 0.1 mM hmercaptoethanol, and antibiotics.
2.2. Immunocytochemistry
Tissues: Stomachs and hearts were obtained from eight
mice. Samples of normal human stomach and heart from
surgical specimens or recent autopsies (n = 7) were provided by Prof. J. Forteza of the University Clinical Hospital’s
Department of Pathology. Samples were fixed in 10%
buffered formalin for 24 h, dehydrated, and embedded in
paraffin by standard procedures. Sections 5 Am thick were
mounted on Histobond adhesive microslides (Marienfeld,
Lauda-Königshofen, Germany), dewaxed and rehydrated.
Antigens were exposed by microwaving in 0.01 M sodium
citrate buffer (pH 6.0) (three 5-min cycles at 750 W).
Sections were successively incubated in (1) goat polyclonal
anti-ghrelin antibody (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) at a dilution of 1:500 (1 h); (2) 3%
hydrogen peroxide (Merck, Darmstadt, Germany) to block
endogenous peroxidase (10 min); (3) biotinylated donkey
anti-goat Ig (Santa Cruz) diluted to 1:100 (30 min); (4)
streptavidin –biotin – peroxidase complex (Duet kit, Dakopatts, Glostrup, Denmark), prepared 30 min before use
following the manufacturer’s protocol (30 min); and (5)
3,3V-diaminobenzidine tetrahydrochloride (DAB) solution,
prepared by dissolving a DAB-buffer tablet (Merck) in 10
ml of distilled water (10 min). Between steps, sections were
washed twice for 5 min with TBS (0.05 M Tris buffer of
pH 7.6 containing 0.3 M NaCl), and after step 5 with
distilled water. All dilutions were performed with TBS
except that of the primary antibody (step 1), for which a
Dakopatts antibody diluent was used. Negative controls
were processed using anti-ghrelin antibody exposed overnight to 10 AM ghrelin (Santa Cruz) at 4 jC, or using TBS
alone (without antibody, etc.) in some other incubation
step.
Cells: HL-1 cells and primary-cultured human cardiomyocytes were fixed by immersion for 15 min in 10%
buffered formalin, 5 min in PBS, 4 min in methanol at 20
jC and 2 min in acetone at 20 jC, and were then washed
twice for 5 min in PBS. Antigens were exposed by microwaving for 10 min at 750 W in 0.01 M sodium citrate
buffer. Anti-ghrelin antibody was used at a dilution of 1:50.
Human cells were identified as cardiomyocytes by staining
with a 1:50 dilution of an affinity-purified goat polyclonal
antibody raised against a peptide sequence near the carboxy
terminus of human myosin heavy chain (anti-MHC antibody, from Santa Cruz). Negative controls were processed
using anti-ghrelin antibody exposed for 24 h to 10 AM
ghrelin at 4 jC, or by omitting some essential step of the
reaction.
Microscopy: Sections were observed and photographed
using a Provis AX70 microscope (Olympus, Tokyo,
Japan).
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
2.3. Ghrelin binding by HL-1 cells
HL-1 cells (5 105) were deprived of serum for 4 h and
then left overnight at 4 jC in PBS containing 0.1% of
bovine serum albumin (BSA), 105 cpm/ml [125I]hGhrelin
(Amersham Biosciences, Freiburg, Germany) and various
concentrations of unlabelled ghrelin. Cells were then
washed and lysed in 0.1 N NaOH, and total associated
radioactivity was measured in a g-counter. Scatchard analysis was performed using the program Ligand [24].
2.4. Hoechst dye vital staining
HL-1 cells (104) were seeded in 24-well plates and
incubated for 45 min at 37 jC in Hoechst 33258 dye. Then
HEPES (pH 7.8) was added to a final concentration of 5
mM, and the cells were fixed with 0.4% paraformaldehyde
for 30 min and examined by fluorescence microscopy.
2.5. MTT viability assay
HL-1 cardiomyocytes (104 per treatment) were deprived
of serum for 12 h and then treated for 72 h with ghrelin
(10 12 – 10 6 M). Four hours before the expiry of this
period, 0.5 g/l MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) was added. After overnight
incubation at 37 jC, absorbance at 550 – 600 nm was
measured.
2.6. RT-PCR
RT-PCR for ghrelin and GHS-R was performed on
RNA from HL-1 cells and from tissues from the same
sources as used for immunocytochemistry (vide supra).
Total RNA was prepared using TRIzol reagent (from Life
Technologies), and 2 Ag was back-transcribed into cDNA
by incubation for 50 min at 37 jC, 10 min at 42 jC, and 5
min at 95 jC with 200 U of murine leukemia virus reverse
transcriptase (from Life Technologies) in 20 Al of a
reaction mixture containing 50 mM KCl, 20 mM Tris –
HCl (pH 8.4), 2.5 mM MgCl2, 0.1
g/l BSA, deoxyNTPs (each 1 mM) and 20 U of the ribonuclease inhibitor
RNAsin (from Promega, Madison, WI, USA). The resulting cDNA was used as a PCR template in a reaction
mixture containing 5 U of Taq DNA polymerase (from
Life Technologies), 50 mM KCl, 20 mM Tris – HCl (pH
8.4), 2.5 mM MgCl2, 0.1 g/l BSA, deoxy-NTPs (each 0.2
mM) and the specific primers for mouse preproghrelin (5VAGCATGCTCTGGATGGACATG-3V (sense) and 5VAGGCCTGTCCGTGGTTACTTGT-3V (antisense) [25],
for mouse GHS-R (5V-CTATCCAGCATGGCCTTCTC-3V
(sense) and 5V-GGAAGCAGATGGCGAAGTAG-3V (antisense); from Genebank, accession number AF332997), or
for human ghrelin (5V-TGAGCCCTGAACACCAGAGAG3V (sense) and 5V-AAAGCCAGATGAGCGCTTCTA-3V
(antisense) [26] (each 0.2 mM). GAPDH was amplified
483
as a control. Thirty-six PCR cycles were performed, each
consisting of denaturation at 98 jC for 20 s, annealing for
1 min at 63 jC (58 jC for human ghrelin), and extension
at 72 jC for 1 min. The PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and examined under UV light. RT-PCR for human
GHS-R1a (the active form [3] of the receptor) was
performed as previously described [27].
2.7. Sequence analysis
GHS-R PCR product was sequenced using a BIGDyek
Terminator kit (Amersham Biosciences) and an ABI Prism
automated DNA sequencer (Applied Biosystems, Foster
City, CA, USA).
2.8. Southern blots
To confirm their identity, electrophoresed amplimers
were transferred from the agarose gels to nylon membranes,
hybridized for 18 h at 44 jC with a 32P-labelled antisense
cDNA probe for rat ghrelin, washed to remove excess
probe, and autoradiographed. Amplimers appearing in the
autoradiographs were sized by comparison with calibration
lanes in the ethidium –bromide-stained agarose gels.
2.9. Ghrelin radioimmunoassays
Ghrelin levels in HL-1 cells and primary cultured
human cardiomyocytes culture media were determined
using specific kits from Phoenix Pharmaceuticals (Belmont, CA, USA).
Fig. 1. RT-PCR results showing expression of preproghrelin gene in HL-1
cardiomyocytes (Panel A, lanes 3 – 6) and of ghrelin gene in human
endocardium and myocardium (Panel B, lanes 3 and 4). Positive controls:
expression of the target gene in mouse or human stomach (Panel A lane 1
and Panel B lane 2, respectively), and of GAPDH (Panel A lanes 7 – 11, and
Panel B lanes 5 – 7). Negative controls: Panel A lanes 12 and 13, and Panel
B lanes 8 and 9. Molecular weight markers: Panel A lane 2 and Panel B
lane 1. (A) Bottom panel: autoradiographs of Southern blots of lanes 1 – 6
of the upper panel.
484
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
2.10. Statistical analysis
3. Results
Results shown are the means F S.E.M.s of at least three
independent experiments. The significance of differences
was estimated by ANOVA followed by Student – Newmann –Keuls multiple comparison tests; p < 0.05 was considered significant.
3.1. Preproghrelin gene expression in cardiomyocytes
RT-PCR for preproghrelin in HL-1 cells afforded a
329-bp cDNA sequence, the identity of which was
confirmed by Southern blotting with a probe for rat
Fig. 2. Ghrelin immunoreactivity in human (g) and HL-1 (e) cardiomyocytes, in human (l) and mouse (j) heart tissue, and, as positive controls, in
human (c) and mouse (a) stomach (where immunoreactivity was found in neuroendocrine cells of gastric glands). For murine and human stomach,
murine and human heart and HL-1 cells, negative controls were run with pre-saturated primary antibody (b, d, k, m and f, respectively), and for
human cardiomyocyte primary cultures by replacing primary antibody with TBS (i). Human cardiomyocyte primary cultures were also stained by antiMHC antibody (h). (b, d, i, k, m) Nomarsky differential interference contrast. Objective magnifications: c and k, 20; a, b, d, j, l and m, 40; e, f,
g, h, i, 60.
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
485
ghrelin (Fig. 1A). That reverse transcription had proceeded properly was confirmed by amplification of
GAPDH. PCR also amplified human ghrelin cDNA
obtained from RNA from human myocardium and endocardium (Fig. 1B).
of the negative controls for the cultured cells (see Methods)
(Fig. 2F and 2I).
3.2. Synthesis of ghrelin by cardiomyocytes
RT-PCR showed that, in HL-1 cells, GHS-R mRNA is
expressed at levels similar to those observed in mouse
heart (Fig. 3A.1). The identity of the PCR product was
confirmed by sequencing (data not shown), with mouse
pituitary as positive control. Statistical analysis of three
independent Scatchard analyses of ghrelin binding by HL1 cells showed efficient constitutive expression of the
receptor gene, yielding values of 3.03 F 0.8 nM for Kd
and 0.14 F 0.06 nM for Bmax (Fig. 3B), the latter figure
being equivalent to about 170,000 binding sites per cell.
The presence of GHS-R1a in human cardiomyocytes was
confirmed by RT-PCR (Fig. 3A.2).
The cytoplasm of HL-1 cells and of primary cultures
of human cardiomyocytes showed intense immunoreactivity with anti-ghrelin antibody (Fig. 2E and 2G). The
possibility that the human cells were fibroblasts rather
than cardiac muscle fibres was ruled out by their immunoreactivity with anti-MHC antibody (Fig. 2H). Similarly,
the cytoplasm of virtually all muscle fibres in sections of
murine and human heart was immunoreactive with antighrelin antibody (Fig. 2J and 2L) (the nuclei of these
cells were not stained).
In the positive controls (mouse and human stomach),
immunoreactivity with anti-ghrelin was found in the neuroendocrine cells of the gastric glands (Fig. 2A and 2C).
Anti-ghrelin antibody pre-saturated with ghrelin did not
stain either these stomach tissues (Fig. 2B and 2D) or heart
tissues (Fig. 2K and 2M), and neither was there any staining
3.3. Expression of the GHS-R gene and ghrelin binding by
cardiomyocytes
3.4. Secretion of ghrelin by cultured HL-1 cells and human
cultured cardiomyocytes
The RIA-measured concentration of ghrelin following
24-h starvation in HL-1 cell culture medium was 16.4 F 0.3
Fig. 3. (Panel A) Panel A.1: RT-PCR results showing expression of mouse GH secretagogue receptor (GHS-R) gene in HL-1 cardiomyocytes (lanes 4 and
5) and mouse heart (lane 6). Positive controls: expression of the target gene in mouse pituitary (lane 1), and of GAPDH in the aforementioned tissues and
cells (lanes 2, 7, 8 and 9, respectively). Negative controls: lanes 10 and 11. Molecular weight marker: lane 3. Panel A.2: RT-PCR results showing
expression of human GH secretagogue receptor 1a (GHS-R1a) (lanes 2 and 3) and of GAPDH (lanes 5 and 6) in human cardiomyocytes. Negative controls:
lanes 4 and 7. Molecular weight marker: lane 1. (Panel B) A representative experiment (n = 3) showing binding of 125I-labelled human ghrelin by HL-1
cells in the presence of various concentrations of unlabelled ghrelin (competitor) (Panel B.1), and Scatchard analysis of binding data from the competition
study (Panel B.2).
486
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
pg/ml (n = 3); and in primary cultured human cardiomyocytes, culture medium was 12.8 F 1.1 pg/ml (n = 4).
3.5. Ghrelin and HL-1 cardiomyocyte viability
Ghrelin did not modify the MTT-assessed proliferation or
viability of HL-1 cardiomyocytes (Fig. 4A). However, pretreatment with 0.1 AM ghrelin for 12 h prevented the
apoptosis induced by treatment with 100 AM AraC for 12
h in these cells. Hoechst dye vital staining showed that
apoptosis affected 7.6 F 1.0% of cells with ghrelin pretreatment, against 14.9 F 1.8% without ( p < 0.05, n = 3;
Fig. 4B). Similar protection was afforded when AraC
treatment was preceded by 12-h pre-treatment with GH
instead of ghrelin (results not shown). RT-PCR showed that
ghrelin mRNA levels were decreased by 33.0 F 7.1% by
12-h treatment with 100 AM AraC ( p < 0.05, n = 3), but
were increased by 48.8 F 13.9% with respect to control cells
if 12-h pre-treatment with GH preceded the AraC treatment
( p < 0.05, n = 3).
4. Discussion
Fig. 4. (Panel A) MTT assay results showing that various concentrations of
ghrelin did not affect the viability of HL-1 cells (n = 3). (Panel B) Results of
Hoechst dye vital staining assays of apoptosis in HL-1 cells treated with
100 AM cytosine arabinoside (AraC) for 12 h, with or without pre-treatment
with 0.1 AM ghrelin for 12 h. (Panel C) Changes in ghrelin mRNA levels in
HL-1 cardiomyocytes following 12-h treatment with AraC, with or without
pre-treatment with the antiapoptotic growth hormone (GH). *p < 0.05
(n = 3).
The above results show in the first place and for the first
time that ghrelin can be synthesized by cardiomyocytes of
both human and murine origin, and that it is secreted by HL1 cells (a cultured line derived from murine atrial cardiomyocytes that maintains a heart-specific phenotype [23] and
is accordingly used as an in vitro model in studies of
cardiomyocyte biology [28]) and also by human cardiomyocytes in primary culture. We also found that HL-1 cells
produce GHS-R that efficiently binds ghrelin at the cell
surface and that human myocardium expresses GHS-R1a
mRNA. Our results are in keeping with the observation of
others that GHS-R mRNAs are present in human myocardium [3] and of GHS-R mRNA in rat left ventricle and left
atrium [15], and strongly suggest that ghrelin has paracrine/
autocrine activity in cardiac muscle.
The discovery that ghrelin is the endogenous GH secretagogue immediately prompted research on its haemodynamic effects, GH being known to play a role in the
maintenance of cardiovascular health [29]. Administration
of ghrelin has been found to reduce cardiac afterload and
increase cardiac output without increasing heart rate in
healthy volunteers [15]; to induce vasodilation [19,20];
and to improve the haemodynamics of patients with chronic
heart failure (CHF) [16]. CHF-associated cachexia is attenuated by ghrelin in rats [18], and in humans is accompanied
by above-normal ghrelin levels, possibly as a compensatory
mechanism in response to catabolic – anabolic imbalance
[17]. Ghrelin also regulates cardiovascular function in rats
suffering septic shock [22]. Similar beneficial cardiovascular effects have been observed in rabbits [21].
That most of the haemodynamic and cardioprotective
effects of ghrelin may be direct, i.e. not mediated by GH, is
suggested not only by the above-noted evidence of a paracrine/autocrine mode of action, but also, in some cases, by
more direct evidence: its vasodilatory effects are not affected by GH release inhibitors [19], and the synthetic GHS-R
ligand hexarelin prevents cardiac damage after ischaemia–
reperfusion even in hypophysectomized rats [30]. Direct
action in vivo is also suggested by the facts that, in vitro,
ghrelin stimulates H9c2 cardiomyocyte cell proliferation
[31], and reduces the doxorubicin-induced mortality of
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
H9c2 cardiomyocytes and endothelial cells [32] and the
AraC-induced mortality of HL-1 cells (this work).
Ghrelin inhibits the in vitro proliferation of cells of breast
carcinoma [33], human lung carcinoma [34], and thyroid
carcinoma [35], and promotes that of hepatoma cells [36],
prostate cancer cells [27], adrenal cells [37] and, as noted
above, H9c2 cardiomyocytes [31]. In the absence of AraC,
ghrelin administration did not affect the proliferation or
viability of HL-1 cardiomyocytes in this study. This difference between HL-1 and H9c2 cells may be due to the
former being of adult and the latter of embryonic origin.
Ghrelin reduced the AraC-induced mortality of HL-1
cells, and ghrelin mRNA levels, which were decreased by
AraC, were increased by pre-treatment with GH, which
protects against AraC-induced apoptosis in these cells [38].
Exactly how GH and ghrelin interact in cardiomyocytes
remains to be elucidated. Obesity is an increasingly prevalent
condition that increases cardiovascular risk, including risk of
heart failure [39]. The fact that ghrelin has beneficial
cardiovascular effects, and the anti-apoptotic effects observed in this study, suggests that part of this increased risk
may be due to obesity-related reduction of plasma ghrelin
levels [12,13], which may reduce protection against the
cardiomyocyte apoptosis that is known to contribute to
progressive cardiomyocyte loss in heart failure [40]. Weight
reduction, which is known to be essential for reducing
cardiovascular risk in the obese [41], may therefore owe this
effect in part to its restoring normal ghrelin levels.
In conclusion, ghrelin appears to have not only GHmediated systemic effects, but also paracrine/autocrine
actions on the metabolism and viability of a variety of types
of cell and tissue. The findings of this study suggest that the
cardioprotective effects of ghrelin are due to local mechanisms of this latter kind. It may be hypothesized that failure
of these mechanisms may be determinant in the development of several cardiac pathologies.
Acknowledgements
This research was supported by the Xunta de Galicia under
projects PGIDIT02SAN91807, PGIDT01PXI90203PR, and
PGIDIT02PXIB2080PR. Dr. Francisca Lago and Dr. Oreste
Gualillo are recipients of a contract of Research from Spanish
Ministry of Health, funded by Instituto de Salud Carlos III
and Complexo Hospitalario Universitario de Santiago.
(FL:99/3040 and OG: 00/3051).
References
[1] Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K.
Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656 – 60.
[2] Howard AD, Feighner SD, Cully DF, et al. A receptor in pituitary and
hypothalamus that functions in growth hormone release. Science
1996;273:974 – 7.
487
[3] Gnanapavan S, Kola B, Buste SA, et al. The tissue distribution of the
mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans.
J Clin Endocrinol Metab 2002;87:2988 – 91.
[4] Gualillo O, Lago F, Gómez-Reino J, Casanueva FF, Diéguez C. Ghrelin, a widespread hormone:insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 2003;
552:105 – 9.
[5] Tschöp M, Smiley DL, Heiman ML. Ghrelin induces adiposity in
rodents. Nature 2000;407:908 – 13.
[6] Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the
central regulation of feeding. Nature 2001;409:194 – 8.
[7] Lawrence CB, Snape AC, Baudoin F, Luckman SM. Acute central
ghrelin and GH secretagogues induce feeding and activate brain appetite centers. Endocrinology 2002;143:155 – 62.
[8] Seoane LM, López M, Tovar S, Casanueva FF, Senaris R, Dieguez C.
Agouti-related peptide, neuropeptide Y, and somatostatin-producing
neurons are targets for ghrelin actions in the rat hypothalamus. Endocrinology 2003;144:544 – 51.
[9] Tena-Sempere M, Barreiro ML, González LC, et al. Novel expression
and functional role of ghrelin in rat testis. Endocrinology 2002;143:
717 – 25.
[10] Kvist-Reimer M, Pacini G, Ahren B. Dose-dependent inhibition by
ghrelin of insulin secretion in the mouse. Endocrinology 2003;144:
916 – 21.
[11] Wren A.M, Seal LJ, Cohen MA, et al. Ghrelin enhances appetite and
increases food intake in humans. J Clin Endocrinol Metab 2001;
86:5992.
[12] Tschöp M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML. Circulating ghrelin levels are decreased in human obesity.
Diabetes 2001;50:707 – 9.
[13] Shiiya T, Nakazato M, Mizuta M, et al. Plasma ghrelin levels in lean
and obese humans and the effect of glucose on ghrelin secretion.
J Clin Endocrinol Metab 2002;87:240 – 4.
[14] Caminos JE, Tena-Sempere M, Gaytan F, et al. Expression of ghrelin in the cyclic and pregnant rat ovary. Endocrinology 2003;144:
1594 – 602.
[15] Nagaya N, Kojima M, Uetmatsu M, et al. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol
Regul Integr Comp Physiol 2001;280:R1483 – 7.
[16] Nagaya N, Miyatake K, Uematsu M, et al. Hemodynamic, renal, and
hormonal effects of ghrelin infusión in patients with chronic heart
failure. J Clin Endocrinol Metab 2001;86:5854 – 9.
[17] Nagaya N, Uematsu M, Kojima M, et al. Elevated circulating level of
ghrelin in cachexia associated with chronic heart failure. Circulation
2001;104:2034 – 8.
[18] Nagaya N, Uematsu M, Kojima M, et al. Chronic administration of
ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation 2001;
104:1430 – 5.
[19] Okumura H, Nagaya N, Enomoto M, Nakagawa E, Oya H, Kangawa
K. Vasodilatory effect of ghrelin, an endogenous peptide from the
stomach. J Cardiovasc Pharmacol 2002;39:779 – 83.
[20] Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of
endothelin-1. Br J Pharmacol 2002;136:1146 – 52.
[21] Matsumura K, Tsuchihashi T, Fujii K, Abe I, Iida M. Central ghrelin
modulates sympathetic activity in conscious rabbits. Hypertension
2002;40:694 – 9.
[22] Chang L, Du JB, Gao LR, Pang YZ, Tang CS. Effect of ghrelin on
septic shock in rats. Acta Pharmacol Sin 2003;24:45 – 9.
[23] Claycomb WC, Lanson Jr NA, Stallworth BS, et al. HL-1 cells: a
cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A
1998;95:2979 – 84.
[24] Munson PJ, Rodbard O. Ligand: a versatile computerized approach
for characterization of ligand binding systems. Anal Biochem
1980;107:220 – 39.
488
M.J. Iglesias et al. / Cardiovascular Research 62 (2004) 481–488
[25] Mori K, Yoshimoto A, Takaya K, et al. Kidney produces a novel
acylated peptide, ghrelin. FEBS Lett 2000;486:213 – 6.
[26] Gualillo O, Caminos JE, Blanco M, et al. Ghrelin, a novel placentaderived hormone. Endocrinology 2001;142:788 – 94.
[27] Jeffery PL, Herington AC, Chopin LK. Expression and action of the
growth hormone releasing peptide ghrelin and its receptor in prostate
cancer cell lines. J Endocrinol 2002;172:R7 – 11.
[28] González-Juanatey JR, Iglesias MJ, Alcaide C, Piñeiro R, Lago F.
Doxazosin induces apoptosis in cardiomyocytes cultured in vitro by
a mechanism that is independent of alpha-adrenergic blockade. Circulation 2003;107:127 – 31.
[29] Colao A, Marzullo P, Di Somma C. Growth hormone and the heart.
Clin Endocrinol (Oxf) 2001;54:137 – 54.
[30] Locatelli V, Rossoni G, Schweiger F, et al. Growth-hormone-independent cardioprotective effects of hexarelin in the rat. Endocrinology
1999;140:4024 – 31.
[31] Pettersson I, Muccioli G, Granata R, et al. Natural (ghrelin) and
synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation. J Endocrinol 2002;175:201 – 9.
[32] Baldanzi G, Filigheddu N, Cutrupi S, et al. Ghrelin and des-acyl ghrelin
inhibit cell death in cardiomyocytes and endothelial cells through
ERK1/2 and PI3-kinase/AKT. J Cell Biol 2002;159:1029 – 37.
[33] Cassoni P, Papotti M, Ghé C, et al. Identification, characterization,
and biological activity of specific receptors for natural (ghrelin) and
synthetic growth hormone secretagogues and analogs in human
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
breast carcinomas and cell lines. J Clin Endocrinol Metab 2001;86:
1738 – 45.
Ghé C, Cassoni P, Catapano F, et al. The antiproliferative effect of
synthetic peptidyl GH secretagogues in human CALU-1 lung carcinoma cells. Endocrinology 2002;143:484 – 91.
Volante M, Allia E, Fulcheri E, et al. Ghrelin in fetal thyroid and
follicular tumors and cell lines: expression and effects on tumor
growth. Am J Pathol 2003;162:645 – 54.
Murata M, Okimura Y, Iida K, et al. Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem
2002;277:5667 – 74.
Andreis PG, Malendowicz LK, Trejter M, et al. Ghrelin and growth
hormone secretagogue receptor are expressed in the rat adrenal cortex:
evidence that ghrelin stimulates the growth but not the secretory
activity of adrenal cells. FEBS Lett 2003;536:173 – 9.
González-Juanatey JR, Piñeiro R, Iglesias MJ, Alcaide C, Lago F.
Growth hormone prevents apoptosis in cardiomyocytes cultured in
vitro. Eur Heart J 2002;23:457 [Abstract Supplement, European Society of Cardiology Congress 2002].
Kenchaiah S, Evans JC, Levy D, et al. Obesity and the risk of heart
failure. N Engl J Med 2002;347:305 – 13.
Kang PM, Izumo S. Apoptosis and heart failure: a critical review of
the literature. Circ Res 2000;86:1107 – 13.
Schunkert H. Obesity and target organ damage: the heart. Int J Obes
Relat Metab Disord 2002;26:S15 – 20.