| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Other Original Articles |
Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine (N.K., T.A., Y.H., H.A., K.T., K.H., M.S., K.M., K.N.), Kyoto 606-8507; Department of Biochemistry, National Cardiovascular Center Research Institute (H.H., M.K., K.K.), Osaka 565-8565; and Clinical Research Institute, Center for Endocrine and Metabolic Diseases, Kyoto National Hospital (A.S.), Kyoto 612-8555, Japan
Address all correspondence and requests for reprints to: Dr. Takashi Akamizu, Department of Medicine and Clinical Science, Kyoto University School of Medicine, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: akataka{at}kuhp.kyoto-u.ac.jp
Abstract
Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a novel acylated peptide produced in the gastrointestinal endocrine cells as well as neuroendocrine cells in the hypothalamus. The Ser3 residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. Human medullary thyroid carcinoma is known to produce a variety of gastrointestinal and neuroendocrine peptides. In the present study we investigated ghrelin production in the thyroid gland, especially in human medullary thyroid carcinoma. PCR amplification demonstrated prepro-ghrelin gene transcripts in normal human thyroid tissue and two medullary thyroid carcinoma cell lines (human TT cells and rat 6-23 cells), but not in a rat thyroid follicular cell line. TT cells showed the expression of prepro-ghrelin mRNA of about 0.6 kb by Northern blot analysis. Furthermore, production of ghrelin in TT cells was demonstrated by RIA and immunocytochemistry. Accumulation of des-n-octanoyl ghrelin in the cultured medium of the cells was confirmed. Finally, human medullary thyroid carcinoma surgical specimens showed significantly higher des-n-octanoyl ghrelin contents than normal thyroid tissues. In conclusion, we revealed that ghrelin was produced by the human thyroid parafollicular carcinoma cell line, TT cells. These findings suggest that ghrelin is produced in the thyroid C cells as well as in medullary thyroid carcinoma and may provide opportunities to investigate its physiological role in the thyroid gland.
GHRELIN HAS RECENTLY been purified and identified as an endogenous ligand for the GH secretagogue (GHS) receptor (GHS-R) (1). There are two molecular forms of ghrelin peptide: ghrelin, the Ser3 residue of which is modified by n-octanoic acid, and des-n-octanoyl ghrelin (1, 2, 3, 4). This lipid modification is essential for hormonal activity. Ghrelin immunoreactivity has been detected not only in the hypothalamus, but also more abundantly in the stomach (1, 2, 3). More recently, ghrelin was detected in the intestine, kidney, placenta, and pituitary (2, 3, 4, 5, 6). GHS-R expression has also been demonstrated in several tissues other than the hypothalamus and pituitary: heart, lung, pancreas, intestine, and adipose tissue (1, 7). These findings suggest a wide expression and action of ghrelin throughout the body. Indeed, several roles of ghrelin, including induction of GH secretion, food intake, and vagal control of gastric function, have been demonstrated (1, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18), although its physiological functions have not been fully clarified.
Motilin receptor, which is the most homologous receptor to GHS-R, is exclusively expressed in the gastrointestinal system and the thyroid (7, 19). More recently, specific binding sites for GHS in thyroid tissue have been indicated (20, 21). Human medullary thyroid carcinoma (hMTC) is known to produce several gastrointestinal hormones and neuroendocrine peptides other than calcitonin (CT): calcitonin gene-related peptide, ACTH, serotonin, chromogranin A, vasoactive intestinal peptide, etc. (22, 23). These findings led us to investigate the expression of ghrelin in thyroid tissue, especially in hMTC. In the present study we found a substantial production of ghrelin in the hMTC cell line, TT cells. In addition, normal human thyroid tissue showed prepro-ghrelin gene expression in PCR amplification, and hMTC tissues obtained at surgical operations contained significantly more des-n-octanoyl ghrelin than normal thyroid tissues.
Materials and Methods
Cell culture
The hMTC cell line, TT cells (24, 25), and the rat MTC line, 6-23 cells (26, 27), were purchased from the American Type Culture Collection (Manassas, VA). TT cells were cultured in Ham’s F-12K (Life Technologies, Inc., Grand Island, NY), 2 mM L-glutamine, and 1.5 g/liter sodium bicarbonate supplemented with 10% FBS (Sigma, St. Louis, MO) at 37 C in a humidified atmosphere containing 95% air and 5% CO2. TT cells were plated at an initial density of 1.5 x 106 cells/100-mm dish (Corning, Inc., New York, NY); 10 ml medium were used in every culture, and medium was changed twice per wk. At 48-h intervals, medium was collected from two dishes selected at random and stored at -80 C for subsequent measurements of immunoreactive ghrelin. The 6-23 cells were cultured in DMEM (Life Technologies, Inc.), 4 mM L-glutamine, and 2.1 g/liter sodium bicarbonate supplemented with 15% horse serum (ICN Biomedicals, Inc., Aurora, OH) at 37 C in a humidified atmosphere containing 95% air and 5% CO2 (27).
Rat FRTL-5 thyroid cells (American Type Culture Collection CRL 8305) are a continuous line of functioning cells derived from normal Fisher rats and were obtained from Dr. Leonard D. Kohn (NIH, Bethesda, MD). The cells were maintained in the absence (5H) or presence (6H) of TSH as described previously (28, 29). CHO cells were also obtained from Dr. Leonard D. Kohn and cultured in the same medium as TT cells.
RNA extraction, PCR, and Northern blot analysis
Polyadenylated RNA was extracted from TT cells using a FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). cDNA synthesis was performed using a cDNA synthesis module (Amersham Pharmacia Biotech, Little Chalfont, UK). The resulting cDNA was subjected to PCR amplification with 5 µM each of the sense and antisense primers and 0.5 U AmpliTaq Gold DNA polymerase (PE Applied Biosystems, Foster, CA). The PCR primers used were: human ghrelin sense, 5'-AAGGAGTCGAAGAAGCCACCA-3' [nucleotides (nt) 148–168 in accession no. AB029434, GenBank]; human ghrelin antisense, 5'-GCCAGATGAGCGCTTCTAAACTTA-3' (nt 416–439); rat ghrelin sense, 5'-TTGAGCCCAGAGCACCAGAAA-3' (nt 112–132) (1); rat ghrelin antisense, 5'-AGTTGCAGAGGAGGCAGAAGCT-3' (nt 437–458); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-ACCACAGTCCATGCCATCAC-3' [nt 586/550–605/569 (human/rat) in accession no. M33197/M17701, GenBank]; and GAPDH antisense, 5'-TCCACCACCCTGTTGCTGTA-3' (nt 1018/982-1037/1001; human/rat). All primers used in this study were located at two different exons. The PCR conditions were 40 cycles of denaturation for 1 min at 94C, annealing for 1 min at 50C, and extension for 1 min at 72C. The PCR products were electrophoresed in 2% low melting temperature agarose gels (FMC BioProducts, Rockland, ME).
For Northern blot analysis, RNA samples were electrophoresed in 2% low melting temperature agarose gels (FMC BioProducts) containing 0.66 M formaldehyde, blotted on nylon membranes (Pall BioSupport Division, Glen Cove, NY), and fixed by UV irradiation. The probes used for Northern blot analysis were a 0.6-kb cDNA fragment of the full-length human ghrelin (1) (accession no. AB029434, GenBank) and a 0.45-kb PCR product amplified using oligonucleotide primers specific for human GAPDH (described above) and extracted using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The specificity of the product was confirmed by direct sequencing using a BigDye Terminator cycle sequencing kit FS and 310 Genetic Analyzer (Perkin-Elmer Corp., PE Applied Biosystems, Wellesley, MA). The membrane was first treated for 3 h at 42 C in 5 x SSC (84 mM NaCl and 84 mM sodium citrate, pH 7.0) containing 50% formamide, 50 mM sodium phosphate buffer, 5 x Denhardt’s solution, 0.1% SDS, and 0.25 mg/ml denatured salmon sperm DNA (Sigma), then hybridized for 16 h at 42 C in an identical solution that contained a 32P-labeled ghrelin cDNA probe. The RNA blot was washed with 2 x SSC/0.1% SDS solution at 55 C and exposed to an imaging plate for a Fujix Bio-image analyzer (BAS 2500, Fuji Photo Film Co., Ltd., Tokyo, Japan) to detect ghrelin probe binding. The membrane then was washed for 30 min at 65 C in 50% formamide/6 x SSPE (900 mM NaCl, 52 mM NaH2PO4, and 7.5 mM EDTA, pH 7.4) to strip off the ghrelin probe and used for sequential hybridization with the probes for GAPDH. Hybridization signals were measured using the BAS 2500 (Fuji Photo Film Co., Ltd.).
RIA procedure
N-RIA and C-RIA (RIAs for the ghrelin N- and C-terminals, respectively) for ghrelin were performed using antihuman ghrelin-(1–11) and -(13–28) sera, respectively, as described previously (2). The antihuman ghrelin-(1–11) antiserum specifically recognized the n-octanoylated portion at Ser3 of ghrelin and did not recognize des-n-octanoylated ghrelin. The antihuman ghrelin-(13–28) antiserum equally recognized n-octanoyl-modified and des-n-octanoylated ghrelins.
Quantification and chromatographic characterization of immunoreactive (ir-) ghrelin in TT cells and human thyroid tissues
TT cells were washed three times with PBS and scraped with a
rubber policeman. The suspension was centrifuged at 2000 rpm for 10
min, and the supernatant was removed. The pellet was treated and
subjected to homogenization and subsequent application to Sep-Pak
C18 cartridges (Waters Corp.,
Milford, MA) as described previously (1, 2, 3, 4). Human thyroid
tissues obtained from surgical operation were similarly treated and
applied to Sep-Pak C18 cartridges (Waters Corp.). Some portions of the eluates were subjected to RIAs for
ghrelin, and other portions to reverse phase HPLC (Fig. 2
) (2, 4). As mentioned above, N-RIA specifically detects
n-octanoyl-modified ghrelin, but does not recognize
des-n-octanoylated ghrelin, whereas C-RIA equally recognizes
both forms of ghrelin peptide. Therefore, all HPLC fractions and the
first 20 min HPLC fractions were quantified by N-RIA and C-RIA,
respectively. Authentic human ghrelin was chromatographed using the
same HPLC system. Protein concentrations of cell homogenates were
measured by protein assay (Bio-Rad Laboratories, Inc.,
Hercules, CA), and 1 x 106 cells contained
220 ± 31 µg.
|
The TT cell culture medium collected was kept frozen at -80 C until purification by Sep-Pak C18 cartridges (Waters Corp.). The medium thawed was directly applied to Sep-Pak C18 cartridges (Waters Corp.) as described previously (2). Peripheral patient blood (10 ml) was drawn into chilled polypropylene tubes containing EDTA-2Na (1 mg/ml blood) and aprotinin (500 U/ml blood), then immediately centrifuged and kept frozen at -20 C. After the plasma had been diluted by half with 0.9% saline, it was applied to Sep-Pak C18 cartridges (Waters Corp.) preequilibrated with 0.9% saline. The cartridge was washed with saline and 5% CH3CN/0.1% trifluoroacetic acid (TFA) and then eluted with 60% CH3CN/0.1% TFA. The eluate was lyophilized, then subjected to ghrelin RIAs.
ir-ghrelin activities of synthetic ghrelin exogenously added to culture medium
Approximately 0.5 µg synthetic ghrelin lyophilized was
dissolved with 300 ml culture medium for TT cells (
500 fmol/ml) and
subjected to the cell culture conditions in the absence or presence of
cells. The cultured media were collected on various occasions as noted
in Table 1 and frozen at -80 C until
they were subjected to RIAs for ir-ghrelin measurement.
|
For immunocytochemistry TT cells were cultured on hemocytometer cover glasses (Matsunami Glass, Osaka, Japan) treated with CELL-TAK (Becton Dickinson and Co., Bedford, MA), each charged with 1 x 104 cells/cm2. After 3 d of subculturing, the medium was changed, and the culture was continued for an additional 24 h. To obtain the immunocytochemical control, CHO cells were cultured at the same time. The cells on glasses were rapidly washed in PBS and immediately fixed in 3.7% formaldehyde in PBS for 30 min at room temperature, followed by a short rinse in PBS, treatment with 0.1% Triton X-100 for 30 min, and washing in PBS three times for 5 min each time. Then they were treated with 0.3% hydrogen peroxide for 5 min to inactive endogenous peroxidases. After rinsing with PBS for 15 min, they were incubated for 20 min with antiserum against the C-fragment of ghrelin (G306 antibody, polyclonal antibodies, 1:200 dilution), the N-fragment of ghrelin (G404 antibody, polyclonal antibodies, 1:1000 dilution), or CT (polyclonal antibodies, DAKO Corp., Glostrup, Denmark; 1:200 dilution). The control study was performed using normal rabbit serum. After washing with PBS for 15 min, cells were incubated for 30 min with Histofine Simple Stain PO(R) (Nichirei Co., Ltd., Tokyo, Japan). They were stained for 10 min at room temperature with 0.5 mg/ml diaminobenzidene and 0.01% hydrogen peroxidase in 625 mM Tris-HCl buffer solution, pH 7.5 (Funakoshi Co., Ltd., Tokyo, Japan).
Tissues
All tissues were obtained from surgical operations and stored at -80 C until subsequent experiments. All hMTC cases were sporadic and harbored no RET mutations.
Results
Prepro-ghrelin gene expression in thyroid tissues and cells
At first, we examined prepro-ghrelin gene expression in the
thyroid using RT-PCR. The human prepro-ghrelin cDNA, 292 bp in size,
was amplified in the whole human thyroid tissue, but not in the human
liver tissue or lymphocytes (Fig. 1).
This finding led us to investigate prepro-ghrelin gene expression
in various thyroid cell lines of different lineages. Both MTC-derived
cells, TT cells and 6-23 cells, showed the amplification of human and
rat prepro-ghrelin cDNAs (292 and 347 bp, respectively), whereas
follicular cell-derived FRTL-5 cells cultured in the presence or
absence of TSH did not (Fig. 1). Human and rat stomach tissues were
used as positive controls. GAPDH cDNA (452 bp in size) was also
amplified to verify that RT was successfully performed for all
materials.
|
). This size was
similar to that of rat prepro-ghrelin mRNA (0.62 kb) (1).
Additionally, a 0.8-kb transcript was faintly detected in TT cells and
human stomach tissue (Fig. 2), different from the rat stomach
(1). FRTL-5 cells or 6-23 cells did not show any
significant transcript hybridized with rat ghrelin cDNA in Northern
blot analysis (data not shown). These findings indicated substantial
gene expression of prepro-ghrelin in TT cells and suggested ghrelin
gene expression in parafollicular-derived cells of the thyroid.
Considering that alternative splicing has been reported in rat ghrelin
(30), the 0.8-kb transcript suggests the presence of an
alternative form of human prepro-ghrelin mRNA. Identification of ir-ghrelin in extracts of TT cells
To identify the peptide, the homogenates of TT cells were
subjected to reverse phase HPLC, and the ghrelin immunoreactivity of
each fraction was measured. A large immunoreactive peak (a in the
upper panel of Fig. 3)
detected by N-RIA was eluted at a position identical to that of human
ghrelin. Another major peak (b in the lower panel
of Fig. 3), which was identical to the elution position of synthetic
deacyl ghrelin, was detected only using C-RIA, although the C-RIA was
performed only for the first 20 min fractions. TT cells contained 37.7
fmol/106 cells (171 fmol/mg protein, 17.1
fmol/mg wet tissue) and 8.9 fmol/106 cells (40.5
fmol/mg protein, 4.1 fmol/mg wet tissue) (27) in C- and
N-RIA, respectively. Thus, the ghrelin content in TT cells was
comparable with that in the rat lower intestine (2, 3).
|
We examined whether TT cells secreted ghrelin into the culture
medium. As shown in Fig. 4, when 1.5
x 106 TT cells were in 100-mm dishes, ir-ghrelin
in the cultured medium, determined by C-RIA, gradually increased from
the basal level (10.6 fmol/ml) to 222.4 fmol/ml on the sixth day of
culture. In contrast, ir-ghrelin, determined by N-RIA, remained low.
These findings suggested that only the des-n-octanyl form of
ghrelin was secreted or that ghrelin was des-acylated in the cultured
medium after secretion. To test the latter possibility, we exogenously
added synthetic ghrelin to the cultured medium. As shown in Table 1,
ir-ghrelin, determined by C-RIA, slowly and slightly decreased in the
culture medium. In the presence of CHO or TT cells, the degradation was
more prominent than in the absence of cells, suggesting that cells
themselves or cellular excretions augmented the proteolysis. The loss
of ir-ghrelin by C-RIA appeared to be much less in TT cells than in CHO
cells, probably because of de novo secretion of ghrelin from
TT cells. In contrast, ir-ghrelin, determined by N-RIA, markedly
decreased to 2.74 fmol/ml when lyophilized synthetic ghrelin was
reconstituted, kept frozen at -80 C for 1 wk, and subjected to RIA.
This finding suggested that des-acylation of ghrelin could occur during
the storage, handling, and/or dissolving in the medium. The ir-ghrelin
became undetectable by N-RIA after 24 h (1 d) at 37 C, pH 7.4, and
5% CO2. This phenomenon occurred regardless of
the existence of TT or CHO cells. 6-23 cells or CHO cells did not show
any increase in ir-ghrelin N- or C-terminus in the cultured medium.
|
TT cells were specifically stained with antighrelin antisera as
well as anti-CT by peroxidase (Fig. 5).
Both antighrelin-(1–11) and -(13–28) sera were stained (Fig. 5, b and
a), confirming the presence of ghrelin in the cells.
|
To clarify the ghrelin production by human medullary carcinomas,
ghrelin contents of the tissues obtained at the surgical operation were
measured using RIA after Sep-Pak C18
purification. As shown in Table 2,
ghrelin immunoreactivities detected by C-RIA of hMTC tissues were
significantly higher than those of normal thyroid tissues (10.5 ±
7.4 vs. 1.6 ± 0.58 fmol/mg). Two papillary thyroid
carcinoma tissues also showed much lower ghrelin immunoreactivities
(2.1 ± 0.36 fmol/mg) than hMTC. Levels of ir-ghrelin determined
by N-RIA in hMTC tissues were much lower than those of ir-ghrelin
determined by C-RIA and were not significantly different from those in
normal thyroid tissues, suggesting predominant production of
des-n-octanoyl ghrelin in MTC or des-acylation of ghrelin
during the storage and/or experimental process. Plasma levels of
ir-ghrelin by C-RIA or N-RIA in MTC patients were not elevated compared
with those in normal subjects, although their plasma levels of CT and
carcinoembryonic antigen were elevated (Table 2). Plasma levels of
ir-ghrelin determined by C-RIA or N-RIA 7 d after surgical removal
were also not significantly decreased, although the levels of CT and
carcinoembryonic antigen decreased to normal.
|
We identified ghrelin production in the human thyroid
parafollicular carcinoma cell line, TT cells. Expression of
prepro-ghrelin mRNA in the cell line was revealed by Northern blot
analysis. Immunoreactivity for ghrelin was determined in the cell
extract and cytoplasm by RIA and immunostaining, respectively. The
levels of ghrelin in the cells [171 fmol/mg protein (17.1 fmol/mg
wet tissue) and 40.5 fmol/mg protein (4.1 fmol/mg wet tissue)
(27) in C- and N-RIA, respectively] were comparable with
those in the lower intestine (2, 3).
Des-n-octanoylated ghrelin accumulated in the culture medium
of TT cells. Thus, substantial expression of ghrelin was clearly
demonstrated.
Two molecular forms of ghrelin peptide are identified by two ghrelin-specific RIAs; one recognizes the octanoyl-modified portion, and another the C-terminal portion of ghrelin (2, 3, 4). Using these two RIA systems, the concentrations of ghrelin in various tissues and plasmas were ascertained. Stomach and small intestine showed high concentrations of the two forms of ghrelin peptide (2, 3). TT cells demonstrated much higher production of both forms of ghrelin peptide than rat thyroid tissue (by C-RIA, 3.5 ± 3.0 fmol/mg; by N-RIA, <0.05 fmol/mg) (3) or other tissues, except stomach and small intestine. However, only des-n-octanoyl ghrelin was detected in the culture medium of TT cells. Further studies are needed to determine whether des-n-octanoyl ghrelin is a secretable form from the cells or a des-n-octanoyl-deleted form after secretion.
The TT cell line is the best known stabilized cell line derived from hMTC and the most reliable model system for the human parafollicular cells developed to date (24, 25). In addition to CT, TT cells were found to produce some other hormones, namely CT gene-related peptide, ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide, serotonin, synaptophysin, neuron-specific enolase, calbindin, and tyrosine hydroxylase. Some are products of gastrointestinal endocrine and/or neuroendocrine cells. In the present study we demonstrated for the first time that TT cells produce ghrelin. Although the 6-23 cell line is also a CT-producing line derived from rat MTC (26, 27) and showed amplification of rat prepro-ghrelin cDNA, it did not show any significant transcript hybridized with rat ghrelin cDNA in Northern blot analysis or any increase in ir-ghrelin by N- or C-RIA in the culture medium. The differences between these cells were also shown by others (31); the large spectrum of demonstrated hormones, functional proteins, and markers in TT cells and their absence in 6-23 cells. The ultrastructure and immunocytochemistry of TT cells indicated that the cells resembled more closely normal parafollicular cells of the thyroid and MTC cells than 6-23 cells (31, 32). Therefore, TT cells seem more suitable for studies of ghrelin.
hMTC arises from the parafollicular C cells (22, 23). The C cells, which produce CT, originate in the embryonic neural crest and enter the developing thyroid when the ultimobranchial body from the fourth pharyngeal pouch fuses with the thyroid epithelium, unlike endoderm-derived thyroid follicular epithelial cells (22, 33). Moderate amounts of CT are produced by many extrathyroidal tissues, including pulmonary neuroendocrine cells, adrenal medulla, and gastrointestinal endocrine cells (22). Ghrelin expression and immunoreactivity have also been detected in several normal tissues, including stomach, hypothalamus, intestine, kidney, placenta, and pituitary (1, 2, 3, 4, 5, 6). PCR amplification detected ghrelin transcript in the whole normal thyroid gland and C-cell derived cell lines, but not in the thyroid follicular cell line. This finding suggested that ghrelin might be expressed in normal C cells and play endocrine, paracrine, and/or autocrine roles in the thyroid gland. However, no detectable Northern blot signal in normal thyroid tissue suggested that the content of ghrelin in the C cell is not as much as that in X/A-like cells of the gastrointestinal tract. Indeed, preliminary immunohistostaining study using antighrelin antibodies has not readily identified ir-ghrelin in normal thyroid tissue (data not shown). Thus, the presence of ghrelin in C cells remains to be confirmed.
What role ghrelin may play in the thyroid gland is unclear. Recently, specific binding sites for GHS were detected in follicular-derived thyroid tissue, but not in parafollicular-derived tissue (20, 21). Therefore, ghrelin may act on thyroid follicular cells in a paracrine manner. Ghrelin binding to the receptor causes mobilization of intracellular Ca2+ (1). There have been several studies showing that activation of the phospholipase C-Ca2+ system affected thyroid functions such as hydrogen peroxide generation (34) and cAMP accumulation in FRTL-5 thyroid cells (35). Thus, ghrelin may influence thyroid functions via the phospholipase C-Ca2+ system. Furthermore, in the context of MTC, it is intriguing to examine whether ghrelin production could affect the growth of MTC or normal thyroid follicular epithelium. These physiological and neoplastic aspects should be examined in future studies on ghrelin.
Ghrelin peptide was also detected in the surgical specimens of hMTC using the C-RIA. The concentrations of des-n-octanoyl ghrelin in hMTC were higher than those in normal thyroid and papillary carcinomas. In contrast to TT cells, hMTC tissues did not show increased ir-ghrelin levels in the N-RIA. This is possibly because des-n-octanoyl ghrelin was predominantly produced in these hMTC. Alternatively, ghrelin might be des-acylated during the storage and/or handling, as suggested by the experiment in which synthetic ghrelin was added to the cell culture medium. Considering that ir-ghrelin levels in plasma and various tissues were much lower in the N-RIA than in the C-RIA (2, 3, 4), the matter of stability and degradation of ghrelin in various conditions should be further investigated.
hMTC produces several biochemical markers, including CT, CT
gene-related peptide, ACTH, serotonin, chromogranin A, vasoactive
intestinal peptide, etc. (22, 23). Some of
these hormonal products of hMTC cells may result in significant
clinical manifestations. Plasma levels of ir-ghrelin by C-RIA or N-RIA
in hMTC patients were not elevated compared with those in normal
subjects despite the increased levels of plasma CT and CEA. The plasma
levels of ir-ghrelin of the three hMTC patients tested were kept low
before and after tumor removal. These findings suggest that ir-ghrelin
is not a good candidate as a tumor marker for hMTC. Nevertheless,
considering that all of the MTC tested were rather small (
2.7 cm in
diameter), cases with larger tumors or disseminated metastasis should
be examined to explore the clinical significance in hMTC.
In summary, we identified the production of ghrelin in the hMTC cell line. The cells produced and secreted substantial amounts of ghrelin peptide and should be an excellent tool to study the regulation of ghrelin gene expression. Moreover, ghrelin production in thyroid C cells was suggested, and it may open a new path to explore the physiological role of ghrelin in the thyroid gland.
Acknowledgments
We deeply thank Dr. Akira Miyauchi (Kuma Hospital, Kobe, Japan) and Ryo Asato (Kyoto University, Kyoto, Japan) for providing thyroid tissues and patient plasma. We also thank Miss Maki Kochi for excellent secretarial work.
Footnotes
This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture and the Japanese Ministry of Health and Welfare.
Abbreviations: C-RIA, RIA for the ghrelin C-terminus; CT, calcitonin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; ir-, immunoreactive; hMTC, human medullary thyroid carcinoma; N-RIA, RIA for the ghrelin N-terminus; TFA, trifluoroacetic acid.
Received February 27, 2001.
Accepted June 19, 2001.
References
This article has been cited by other articles:
 |
|
 |
|
  T. Takahashi, T. Ida, T. Sato, Y. Nakashima, Y. Nakamura, A. Tsuji, and M. Kojima Production of n-octanoyl-modified Ghrelin in Cultured Cells Requires Prohormone Processing Protease and Ghrelin O-acyltransferase, as well as n-octanoic Acid J. Biochem., November 1, 2009; 146(5): 675 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Tong, P. T. Pfluger, and M. H. Tschop Gastric O-acyl transferase activates hunger signal to the brain PNAS, April 29, 2008; 105(17): 6213 - 6214. [Full Text] [PDF]
|
|
|
|
|
|
J. A. Gutierrez, P. J. Solenberg, D. R. Perkins, J. A. Willency, M. D. Knierman, Z. Jin, D. R. Witcher, S. Luo, J. E. Onyia, and J. E. Hale From the Cover: Ghrelin octanoylation mediated by an orphan lipid transferase PNAS, April 29, 2008; 105(17): 6320 - 6325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. D. Delhanty, P. M. van Koetsveld, C. Gauna, B. van de Zande, G. Vitale, L. J. Hofland, and A. J. van der Lely Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E302 - E309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. T. Vestergaard, T. K. Hansen, L. C. Gormsen, P. Jakobsen, N. Moller, J. S. Christiansen, and J. O. L. Jorgensen Constant intravenous ghrelin infusion in healthy young men: clinical pharmacokinetics and metabolic effects Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1829 - E1836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. De Vriese and C. Delporte Autocrine proliferative effect of ghrelin on leukemic HL-60 and THP-1 cells J. Endocrinol., January 1, 2007; 192(1): 199 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. D. Dixit, A. T. Weeraratna, H. Yang, D. Bertak, A. Cooper-Jenkins, G. J. Riggins, C. G. Eberhart, and D. D. Taub Ghrelin and the Growth Hormone Secretagogue Receptor Constitute a Novel Autocrine Pathway in Astrocytoma Motility J. Biol. Chem., June 16, 2006; 281(24): 16681 - 16690. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P L Jeffery, R E Murray, A H Yeh, J F McNamara, R P Duncan, G D Francis, A C Herington, and L K Chopin Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines Endocr. Relat. Cancer, December 1, 2005; 12(4): 839 - 850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N Govoni, R De Iasio, C Cocco, A Parmeggiani, G Galeati, U Pagotto, C Brancia, M Spinaci, C Tamanini, R Pasquali, et al. Gastric immunolocalization and plasma profiles of acyl-ghrelin in fasted and fasted-refed prepuberal gilts J. Endocrinol., September 1, 2005; 186(3): 505 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kojima and K. Kangawa Ghrelin: Structure and Function Physiol Rev, April 1, 2005; 85(2): 495 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. De Vriese, F. Gregoire, P. De Neef, P. Robberecht, and C. Delporte Ghrelin Is Produced by the Human Erythroleukemic HEL Cell Line and Involved in an Autocrine Pathway Leading to Cell Proliferation Endocrinology, March 1, 2005; 146(3): 1514 - 1522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wei, G. Wang, X. Qi, E. W. Englander, and G. H. Greeley Jr. Characterization and Regulation of the Rat and Human Ghrelin Promoters Endocrinology, March 1, 2005; 146(3): 1611 - 1625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kanamoto, T. Akamizu, T. Tagami, Y. Hataya, K. Moriyama, K. Takaya, H. Hosoda, M. Kojima, K. Kangawa, and K. Nakao Genomic Structure and Characterization of the 5'-Flanking Region of the Human Ghrelin Gene Endocrinology, September 1, 2004; 145(9): 4144 - 4153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin Endocr. Rev., June 1, 2004; 25(3): 426 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hosoda, K. Doi, N. Nagaya, H. Okumura, E. Nakagawa, M. Enomoto, F. Ono, and K. Kangawa Optimum Collection and Storage Conditions for Ghrelin Measurements: Octanoyl Modification of Ghrelin Is Rapidly Hydrolyzed to Desacyl Ghrelin in Blood Samples Clin. Chem., June 1, 2004; 50(6): 1077 - 1080. [Full Text] [PDF]
|
|
|
|
|
|
Y. Hataya, T. Akamizu, H. Hosoda, N. Kanamoto, K. Moriyama, K. Kangawa, K. Takaya, and K. Nakao Alterations of Plasma Ghrelin Levels in Rats with Lipopolysaccharide-Induced Wasting Syndrome and Effects of Ghrelin Treatment on the Syndrome Endocrinology, December 1, 2003; 144(12): 5365 - 5371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tanaka, H. Minoura, T. Isobe, H. Yonaha, H. Kawato, D. F. Wang, T. Yoshida, M. Kojima, K. Kangawa, and N. Toyoda Ghrelin Is Involved in the Decidualization of Human Endometrial Stromal Cells J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2335 - 2340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Moller, J. Nygren, T. K. Hansen, H. Orskov, J. Frystyk, and K. S. Nair Splanchnic Release of Ghrelin in Humans J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 850 - 852. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Volante, E. Allia, E. Fulcheri, P. Cassoni, E. Ghigo, G. Muccioli, and M. Papotti Ghrelin in Fetal Thyroid and Follicular Tumors and Cell Lines: Expression and Effects on Tumor Growth Am. J. Pathol., February 1, 2003; 162(2): 645 - 654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Iwakura, K. Hosoda, R. Doi, I. Komoto, H. Nishimura, C. Son, J. Fujikura, T. Tomita, K. Takaya, Y. Ogawa, et al. Ghrelin Expression in Islet Cell Tumors: Augmented Expression of Ghrelin in a Case of Glucagonoma with Multiple Endocrine Neoplasm Type I J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 4885 - 4888. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ariyasu, K. Takaya, H. Hosoda, H. Iwakura, K. Ebihara, K. Mori, Y. Ogawa, K. Hosoda, T. Akamizu, M. Kojima, et al. Delayed Short-Term Secretory Regulation of Ghrelin in Obese Animals: Evidenced by a Specific RIA for the Active Form of Ghrelin Endocrinology, September 1, 2002; 143(9): 3341 - 3350. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Findings obtained
using the C-RIA; •, measurements obtained using the N-RIA. Values
represent the mean ± SD for duplicate culture media.
The dashed line indicates the change in the number of TT
cells. Three different experiments were performed, and similar findings
were obtained in all experiments.