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Third Division (Y.D., M.S.M., S.M., M.N.), Department of Internal Medicine, and Second Department of Anatomy (A.S., T.S.), Miyazaki Medical College, Miyazaki 889-1692; and National Cardiovascular Center Research Institute (M.K., H.H., K.K.), Osaka 565-8565, Japan
Address all correspondence and requests for reprints to: Masamitsu Nakazato, M.D., Ph.D., Third Department of Internal Medicine, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan. E-mail: nakazato{at}post.miyazaki-med.ac.jp
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Introduction |
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We have shown that ghrelin is likely to be present in gastric endocrine cells by in situ hybridization and immunohistochemistry (4). Gastrointestinal peptides, part of a complex biological signaling system, act as substrates for intracellular communication both in the digestive system and between the digestive system and body organs. More than 18 endocrine cell types have been identified in the gastrointestinal tract, and the number continues to grow. We used in situ hybridization histochemistry combined with immunohistochemistry, immunohistochemical double staining, and electron microscopy immunostaining to investigate the cellular origin of ghrelin in the digestive systems of rats and humans. We developed two RIAs specific for the N- and C-terminal regions of ghrelin. We also studied the distribution of ghrelin in rat gastrointestinal tract by the RIAs and RT-PCR, and its receptor by RT-PCR.
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Materials and Methods |
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Peptide synthesis
C-Terminally Cys-extended rat ghrelin (position 1–11) with
octanoylated Ser3, C-terminally Tyr-extended rat
ghrelin (position 1–28) with octanoylated Ser3,
N-terminally Cys- or Tyr-extended rat ghrelin (position 13–28), and
des-acyl ghrelin (position 1–28) were synthesized using solid phase
techniques. Cys-extended peptides were used for immunization and
Tyr-extended peptides for radioiodination as described below. The
validity of the synthesis was confirmed by amino acid analysis,
sequencing, and spectrometric analysis.
Preparation and characterization of antisera
To generate antighrelin antisera, synthetic
[Cys12]ghrelin-(1–11) (4 mg) and
[Cys0]ghrelin-(13–28) (10 mg) were separately
conjugated with maleimide-activated mariculture keyhole limpet
hemocyanin (Pierce Chemical Co., Rockford, IL; 6 mg).
Amino acid analysis of the conjugate showed that one hemocyanin
molecule was coupled with an average 280
[Cys12]ghrelin-(1–11) or 275
[Cys0]ghrelin-(13–28) molecules. The antigenic
conjugate solution (1.5–3 ml) was administered to three New Zealand
White rabbits. The antirat ghrelin-(1–11) antiserum (G606)
specifically recognized ghrelin with n-octanoylated
Ser3 and did not recognize des-acyl ghrelin. The
antirat ghrelin-(13–28) antiserum (G107) equally recognized
n-octanoyl-modified and des-acyl ghrelin. Both antisera had
100% cross-reactivity with human ghrelin-(1–28).
RIA procedure
Synthetic rat [Tyr29]ghrelin-(1–28) and
[Tyr0]ghrelin-(13–28) were radioiodinated by
the lactoperoxidase method. The 125I-labeled
peptides were purified on a TSK ODS SIL 120A column (Tosoh Co. Ltd.,
Tokyo, Japan) by reverse phase HPLC (RP-HPLC). The RIA incubation
buffer was 50 mM sodium phosphate (pH 7.4) that contained
0.5% BSA treated with N-ethylmaleimide, 80
mM NaCl, 25 mM EDTA·2Na,
0.05% NaN3, and 0.5% Triton X-100. A diluted
sample or a standard peptide solution (100 µl) was incubated for
24 h with 100 µl of the antiserum diluent [final dilution of
antighrelin-(1–11) antiserum, 1:620,000; that of antighrelin-(13–28)
antiserum, 1:20,000]. The tracer solution (16,000 cpm in 100 µl) was
added, and the mixture again incubated for 24 h. The bound and
free ligands were separated by second antibody (200 µl). All
procedures were performed at 4 C. Samples were assayed in duplicate.
Half- maximum inhibition by rat ghrelin-(1–28) on the standard RIA
curve with antighrelin-(1–11) antiserum was 3.8 fmol/tube, and that
with antighrelin-(13–28) antiserum was 80 fmol. The dilution curves
for the extracts of rat stomach and jejunum paralleled both standard
curves. The respective intra- and interassay coefficients of variation
in the RIA for ghrelin N-terminus were 3.5% and 3.2% at 50% binding,
and those for ghrelin C-terminus were 3.7% and 3.3% at 50% binding.
The recoveries of rat ghrelin-(1–28) (1 ng) and
[125I]rat ghrelin-(1–28) (5,000 cpm) added to
the tissue homogenates in the extraction done with a Sep-Pak
C18 cartridge (Waters Corp.,
Milford, MA), respectively, were 92.2 ± 0.4%
(±SEM) and 88.9 ± 0.6%.
Quantification and chromatographic characterization of
immunoreactive (ir-) ghrelin in stomach and intestine
The stomach and intestine were resected immediately after
decapitation of three 12-week-old male Wistar rats fed ad
libitum. The glandular stomach was divided into fundus and
pylorus. The jejunum was resected 10–20 cm from the pyloric ring, the
ileum 20–30 cm above the terminal ileum, and the colon 5–15 cm below
the terminal ileum. The tissues were heated at 95–100 C for 10 min in
a 10-fold volume of water to inactivate intrinsic proteases. After
cooling to 4 C, CH3COOH and HCl were added to the
respective final concentrations of 1 M and 20
mM, after which the tissue was homogenized in a
Polytron (Brinkmann Instruments, Inc., Westbury, NY) for
10 min. The homogenate was centrifuged at 11,500 x g
for 30 min. The supernatants were applied to Sep-Pak
C18 cartridges, then the peptides were eluted
with 60% acetonitrile (CH3CN) solution
containing 0.1% trifluoroacetic acid (TFA). Some portions of the
eluates were subjected to two RIAs for ghrelin, and other portions to
RP-HPLC (Fig. 1
, A and B). All HPLC
fractions were quantified by the RIAs for ghrelin. Authentic rat
ghrelin-(1–28) was chromatographed with the same HPLC system.
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In situ hybridization histochemistry
Three Wistar rats were anesthetized by an ip injection of sodium
pentobarbital (75 mg/kg BW) and perfused transcardially for 10 min with
100 ml 0.1 M phosphate buffer (pH 7.4) containing heparin
(100 U/100 ml), then for 15 min with 150 ml fixative containing 4%
paraformaldehyde and 0.2% picric acid in 0.1 M phosphate
buffer. Their stomachs were removed, postfixed with the same fixative
for 24 h at 4 C, then incubated for 24 h in 0.1 M
PBS (pH 7.4) containing 30% sucrose. The glandular stomachs were
quickly frozen on a dry ice and stored at -80 C until use for the
in situ hybridization analysis. They were cut at -20 C with
a cryostat in slices 12 µm thick, then thaw-mounted on silane-coated
slides and kept at -80 C. The stored slides were allowed to dry for 10
min at room temperature, then fixed in 4% formaldehyde in PBS (pH 7.4)
for 5 min and washed twice in PBS (pH 7.4). Next, they were incubated
for 10 min in 0.9% saline containing 0.1 M
triethanolamine and 0.25% acetic anhydride, dehydrated in a graded
ethanol series, and delipidated in 100% chloroform for 5 min, after
which they were immersed in 100% ethanol, then in 95% ethanol, and
allowed to dry briefly in air. Hybridization was performed at 37 C
overnight in 45 µl hybridization buffer containing 50% formamide and
4 x SSC (1 x SSC = 150 mM NaCl
and 15 mM sodium citrate) with 500 µg/ml
sheared salmon sperm DNA (Sigma, St. Louis, MO), 250 µl
yeast total RNA (Sigma), 1 x Denhardt’s solution
(0.02% Ficoll, 0.02% polyvinylpyrolidone, and 0.02% BSA), and 10%
dextran sulfate (500,000 mol wt) under a Nescofilm (BDH/Merck,
Dagenham, UK) coverslip. Three 45-mer antisense oligonucleotide probes
against rat ghrelin cDNA (nucleotides 90–134, 213–257, and 421–465
in Ref. 4) were used. These probes are not closely
homologous to other known cDNA sequences. A 100-fold molar excess of
each unlabeled probe served as the controls. A total of 5 x
105 cpm/slide of 3'-end
35S-labeled probes were used. After
hybridization, sections were washed for 1 h in four changes of
1 x SSC at 55 C, then for 1 h in two changes of 1 x
SSC at room temperature. After washing, they were dehydrated in a
graded alcohol series and air-dried. The sections were coated with
Kodak NTB3 emulsion (Eastman Kodak Co.,
Rochester, NY) for autoradiography and exposed for 24 h in
light-tight boxes at 4 C. After development in Kodak D-19
and fixing in Fujifix (Fuji Photo Film Co., Ltd., Tokyo,
Japan), they were rinsed with distilled water and coverslipped.
In the second protocol, after washing as described above some sections were subjected to immunohistochemical procedures. They were treated with 0.3% hydrogen peroxide for 30 min to inactive endogenous peroxidases, then incubated with normal goat serum for 1 h to block nonspecific binding. These sections were incubated overnight at 4 C with antisomatostatin (anti-SRIF) antiserum (DAKO Corp., Glostrup, Denmark; dilution, 1:200), antihistidine decarboxylase (anti-HDC) antiserum (EURO-DIAGNOSTICA, Malmö, Sweden; dilution, 1:2,000), antichromogranin A antiserum (DAKO Corp.; dilution, 1:500), or antiserotonin antiserum (DAKO Corp.; dilution, 1:5). After washing with PBS, the slides were incubated overnight at 4 C with goat biotinylated antirabbit IgG (Vectastain, Vector Laboratories, Inc., Burlingame, CA). They were stained for 10 min at room temperature using the avidin-biotin-peroxidase complex method (Vectastain Elite ABC kit, Vector Laboratories, Inc.) with 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.006% hydrogen peroxide in 50 mM Tris-HCl buffer solution (pH 7.2). After dehydration in a graded alcohol series, they were covered with emulsion for autoradiography as described above and counterstained with hematoxylin. The number of cells that have ghrelin messenger RNA (mRNA) signal and the immunoreactivity of one of the above substances were quantified by counting two randomly selected visual fields in two sections from each of three rats under a light microscope (x20 objective lens; magnification, x200). In this study we defined the selected visual fields as the areas that contain both ghrelin mRNA signal and the immunoreactivity of each of the above substances in the rat oxyntic gland. We also considered that cells with grain densities at least 5 times higher than the background densities are positively labeled for ghrelin mRNA, and a distinctive brown chromogen in the cell cytoplasm indicates the immunoreactivity of SRIF, HDC, chromogranin A, or serotonin.
Light microscopy immunostaining
Frozen 12-µm-thick sections of the glandular stomachs were
prepared from the same three rats as those used in in situ
hybridization histochemistry. Rat small intestine was fixed with 4%
paraformaldehyde and 0.2% picric acid and embedded in paraffin. Human
gastric fundi and small and large intestines obtained at autopsy from
three patients who had died of cardiovascular disease were fixed and
embedded in paraffin as described above, and the tissues were cut in
3-µm-thick slices. After pretreatment with 0.3% hydrogen peroxide
and incubation with normal goat serum, all slices were incubated
overnight at 4 C with antighrelin-(1–11) antiserum (final dilution,
1:10,000) or antighrelin-(13–28) antiserum (final dilution, 1:10,000).
All of the sections were stained by the avidin-biotin complex method as
described above. Control studies were performed with normal rabbit
serum or antighrelin-(1–11) and antighrelin-(13–28) antisera that had
been absorbed with 10 µg rat ghrelin.
Immunohistochemical double staining
The human gastric fundi sections were incubated with
antighrelin-(1–11) antiserum, then with Alexa Fluor 488 goat
antirabbit IgG (Molecular Probes, Inc., Eugene, OR). After
being washed with PBS, they were incubated first with mouse
antichromogranin A antiserum (DAKO Corp.; dilution,
1:500), then with Alexa Fluor 568 goat antimouse IgG (Molecular Probes, Inc.), after which they were observed under a BH2-RFC
microscope (Olympus Corp., Tokyo, Japan). In the double
staining for ghrelin vs. SRIF, HDC, and serotonin in human
gastric fundi, ghrelin first was stained using the avidin-biotin
complex method. The specimens then were washed with 0.1
M glycine-HCl buffer (pH 2.2) and incubated
overnight at 4 C with anti-SRIF, -HDC, or -serotonin antiserum. They
were stained by the streptavidin-alkaline phosphatase method using a
labeled streptavidin biotin kit (DAKO Corp.) and AP
Substrate Kit III (Vector Laboratories, Inc.). The number
of cells in which two peptides were colocalized was quantified by
counting two randomly selected visual fields in two sections from each
of three human subjects under a fluorescence or light microscope (x20
objective lens; magnification, x200).
Electron microscopy immunostaining
Three Wistar rats were perfused with 2% paraformaldehyde and
2.5% glutaraldehyde in 0.1 M PBS. Their stomachs were
excised and fixed at 4 C overnight with the above fixative, then
postfixed at 4 C for 90 min with 1% osmium tetroxide in 0.1
M PBS. They were dehydrated in a graded ethanol series and
embedded in Epon. Ultrathin sections of the specimens were cut and
treated for 30 min with 5% sodium meta-periodate (5),
after which they were immersed for 10 min in 5% normal goat serum and
PBS containing 1% BSA, then incubated overnight at 4 C with
antighrelin-(13–28) antiserum (dilution 1:1000). Next they were
incubated with 8 nm colloidal gold conjugated antirabbit donkey IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA; dilution, 1:50), after which the sections were counterstained with
uranyl acetate and lead citrate. For the controls, antighrelin
antiserum was omitted or replaced by normal rabbit serum. The sections
were examined in a JEOL 1200EX electron microscope (JEOL USA, Inc., Tokyo, Japan). The sizes of 200 ghrelin-containing
granules were measured.
RT-PCR for ghrelin and GHS-R
Total RNAs were extracted from the stomachs and small and large
intestines of three Wistar rats by the acid guanidinium
thiocyanate-phenol-chloroform method (6). First strand
cDNA was synthesized from 2.5 µg of a RNA sample and 7
µM oligo(deoxythymidine)18 primer
with ReverTra Ace-
(Toyobo Co., Ltd., Osaka, Japan). The resulting
cDNA was subjected to PCR amplification with 2 µM each of
the sense and antisense primers and 2.5 U Pyrobest DNA polymerase
(Takara Shuzo Co., Ltd., Shiga, Japan). The PCR primers specific for
ghrelin were 5'-TTGAGCCCAGAGCACCAGAAA-3' for sense and
5'-AGTTGCAGAGGAGGCAGAAGCT-3' for antisense (nucleotides 112–132 and
437–458 in Ref. 4), and the primers specific for GHS-R
were 5'-GAGATCGCTCAGATCAGCCAGTAC-3' for sense and
5'-TAATCCCCAAACTGAG-GTTCTGC-3' for antisense
(nucleotides 880–903 and 1170–1192 in accession no. AB001982,
GenBank). The reaction volume was 25 µl, and the PCR conditions were
35 cycles of denaturation for 5 sec at 94 C, annealing for 10 sec at 65
C, and extension for 1 min at 72 C. The PCR products were
electrophoresed on a 2% agarose gel (FMC BioProducts, Rockland,
ME).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
detected by two RIAs was eluted at the position identical to that of
n-octanoylated ghrelin-(1–28). Another major peak (peak a
in Fig. 1) in both stomach and jejunum was detected only by the RIA for
ghrelin C-terminus. This molecule was eluted at a position identical to
that of des-acyl ghrelin-(1–28), indicating that it was des-acyl
ghrelin-(1–28). Ghrelin immunoreactivity was present from the stomach
to the colon, with the highest content in the gastric fundus (Table 1). The plasma concentration of
ir-ghrelin measured by RIA for C-terminus was 556.2 ± 43.8
fmol/ml (mean ± SE), and that measured by
RIA for N-terminus was 94.6 ± 14.0 fmol/ml.
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) when the oligonucleotide probe
90–134 was used, but were infrequent in the pyloric gland (Fig. 2B).
The signals disappeared when a 100-fold molar excess of the unlabeled
probe was present (Fig. 2C). The same ghrelin mRNA localization
patterns were detected when the other two probes were used (data not
shown). Colocalization of ghrelin, chromogranin A, SRIF, HDC, and
serotonin was investigated by in situ hybridization
histochemistry combined with immunohistochemistry. Ghrelin mRNA
hybridization signals were present in 21% (93 of 443) of chromogranin
A-immunoreactive endocrine cells (Fig. 2D), but none was found in
SRIF-, HDC-, or serotonin- immunoreactive cells (Fig. 2, E-G).
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, A and B). Small numbers of ghrelin
cells were present in the upper small intestine (Fig. 3, C and D), and
very small numbers were present in the lower small intestine and large
intestine (not shown). Cells immunostained with antisera for
ghrelin-(1–11) and -(13–28) were colocalized extensively in the
gastrointestinal tract (Fig. 3, E and F). In humans, too, ghrelin cells
were most abundant in the oxyntic gland and were infrequent in the
pyloric gland and upper small intestine (Fig. 3, G-J).
Ghrelin-immunoreactive materials were concentrated in the basal
cytoplasm of ghrelin cells of both rats and humans (Fig. 3, D, H, and
J). In the human gastric fundus, ghrelin cells accounted for 23% (30
of 128) of chromogranin A-immunoreactive endocrine cells in
immunofluorescence double staining (Fig. 3, K-M). Human ghrelin cells
did not have SRIF (Fig. 3N), HDC (not shown), or serotonin (not shown)
immunoreactivity in double staining. No immunoreactivities for
ghrelin-(1–11) and -(13–28) were detected in the tissues when normal
rabbit serum or antiserum absorbed with excessive ghrelin was used (not
shown).
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). Ghrelin cells were round to ovoid and
of a closed type that had no contact with the lumen. They were
positioned close to the capillary. The ghrelin-containing granules were
120 ± 30 nm (mean ± SEM) in diameter, smaller
than the granules of D cells and enterochromaffin-like (ECL) cells.
Neither D nor ECL cells reacted with ghrelin antiserum (not shown).
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, upper panel). A
GHS-R transcript product corresponding to the predicted 313 bp size
also was present in all of these organs (Fig. 5, lower
panel).
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Discussion |
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We here identified ghrelin-producing endocrine cells in the digestive tracts of rats and humans. They were most abundant in the oxyntic mucosae of both species. To date, four types of endocrine cells, ECL, D, enterochromaffin (EC), and X/A-like cells, have been identified in the oxyntic mucosa by means of ultrastructural and immunohistochemical criteria (7, 8). The relative percentages of these four cells in rat oxyntic gland are 60–70% for ECL cells, 20% for X/A-like cells, 2–5% for D cells, and 0–2% EC cells; those in human oxyntic gland are 30% for ECL cells, 20% for X/A-like cells, 22% for D cells, and 7% for EC cells (9, 10). Major products in the granules of the first three cell types have been shown to be histamine and uroguanylin (11), SRIF, and serotonin, respectively, whereas no products have been reported in the granules of X/A-like cells. X/A-like cells represent a major endocrine cell population in the oxyntic mucosae of both rats and humans. X/A-like cells are round to ovoid, with round, compact, electron-dense granules (8, 12, 13). They are found mainly in the oxyntic gland and infrequently in the pyloric gland and small intestine. The localization, population, and ultrastructural features of ghrelin-immunoreactive cells in the gastrointestinal tract indicate that X/A-like cells, whose hormonal product has not previously been clarified, are ghrelin cells. Ghrelin cells can be abbreviated as Gr cells according to the precedented nomenclature of other enteroendocrine cells. Ghrelin cells in the oxyntic mucosa are closed-type cells that have no continuity with the lumen, suggesting that they respond to physical stimuli from the lumen, chemical stimuli from the basolateral site, or both. Ghrelin cells are closely associated with the capillary network running through the lamina propria in electron microscopy. Ghrelin circulates in the rat plasma. These findings suggest that ghrelin cells function in an endocrine fashion, a mechanism feasible for delivering ghrelin to remote tissues that express GHS-R.
Although Northern blot analysis of rat tissues showed that prepro-ghrelin mRNA occurs only in the stomach (4), RT-PCR analysis and RIAs detected it in the intestine as well. GHS-R mRNA also was present in the stomach and intestine. Ghrelin’s possible function in the digestive tract, such as regulation of motility of the gut wall, gastric acid secretion, and renewal of gut epithelium, is a fascinating area that requires further investigation.
Future determination of ghrelin content and the amount of its mRNA in the gastrointestinal tract under various physiological and pathophysiological conditions should provide information on what mechanisms govern the biosynthesis and secretion of this peptide. The findings presented here will help establish new ways to clarify the additional, as yet undefined, physiological functions of this novel gastrointestinal hormone, ghrelin.
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Footnotes |
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Received June 1, 2000.
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F. Piccoli, L. Degen, C. MacLean, S. Peter, L. Baselgia, F. Larsen, C. Beglinger, and J. Drewe Pharmacokinetics and Pharmacodynamic Effects of an Oral Ghrelin Agonist in Healthy Subjects J. Clin. Endocrinol. Metab., May 1, 2007; 92(5): 1814 - 1820. [Abstract] [Full Text] [PDF]
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D. Kohno, M. Nakata, F. Maekawa, K. Fujiwara, Y. Maejima, M. Kuramochi, T. Shimazaki, H. Okano, T. Onaka, and T. Yada Leptin Suppresses Ghrelin-Induced Activation of Neuropeptide Y Neurons in the Arcuate Nucleus via Phosphatidylinositol 3-Kinase- and Phosphodiesterase 3-Mediated Pathway Endocrinology, May 1, 2007; 148(5): 2251 - 2263. [Abstract] [Full Text] [PDF]
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A. J. Page, J. A. Slattery, C. Milte, R. Laker, T. O'Donnell, C. Dorian, S. M. Brierley, and L. A. Blackshaw Ghrelin selectively reduces mechanosensitivity of upper gastrointestinal vagal afferents Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1376 - G1384. [Abstract] [Full Text] [PDF]
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 M. Mottershead, E. Karteris, J. Y Barclay, S. Suortamo, M. Newbold, H. Randeva, and C. U Nwokolo Immunohistochemical and quantitative mRNA assessment of ghrelin expression in gastric and oesophageal adenocarcinoma J. Clin. Pathol., April 1, 2007; 60(4): 405 - 409. [Abstract] [Full Text] [PDF]
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A. S Bang, S. G Soule, T. G Yandle, A M. Richards, and C. J Pemberton Characterisation of proghrelin peptides in mammalian tissue and plasma J. Endocrinol., February 1, 2007; 192(2): 313 - 323. [Abstract] [Full Text] [PDF]
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F. Fak, L. Friis-Hansen, B. Westrom, and N. Wierup Gastric ghrelin cell development is hampered and plasma ghrelin is reduced by delayed weaning in rats J. Endocrinol., February 1, 2007; 192(2): 345 - 352. [Abstract] [Full Text] [PDF]
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 R. R. Kraemer and V. D. Castracane Exercise and Humoral Mediators of Peripheral Energy Balance: Ghrelin and Adiponectin Experimental Biology and Medicine, February 1, 2007; 232(2): 184 - 194. [Abstract] [Full Text] [PDF]
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H. Chung, E. Kim, D. H. Lee, S. Seo, S. Ju, D. Lee, H. Kim, and S. Park Ghrelin Inhibits Apoptosis in Hypothalamic Neuronal Cells during Oxygen-Glucose Deprivation Endocrinology, January 1, 2007; 148(1): 148 - 159. [Abstract] [Full Text] [PDF]
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 E. Naslund and J. G. Kral Impact of Gastric Bypass Surgery on Gut Hormones and Glucose Homeostasis in Type 2 Diabetes Diabetes, December 1, 2006; 55(Supplement_2): S92 - S97. [Abstract] [Full Text] [PDF]
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S. L Dun, G C |
, Data obtained using a
RIA for ghrelin N-terminus; •, data obtained using a RIA for ghrelin
C-terminus. The fundus (A; 5 mg) and jejunum (B; 125 mg) were
chromatographed on a TSK ODS SIL 120A column (4.6 x 150 mm). A
linear gradient of 10–60% CH3CN containing 0.1% TFA was
run for 40 min at 1.0 ml/min. The fraction volume was 0.5 ml.
Arrows indicate the elution positions of des-acyl rat
ghrelin-(1–28) (a) and n-octanoylated rat
ghrelin-(1–28) (b).
