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Original Studies |
Department of Endocrinology (M.K., A.B.G.), Academic Department of Surgery (S.A.B.), and Department of Histopathology (S.J., D.G.L.), St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, United Kingdom EC1A 7BE; Department of Biochemistry, National Cardiovascular Center Institute (M.K., K.K.), Suita, Osaka 565-8565, Japan; and Pharmaceutical Sciences Institute (E.F.A.), Aston University, Birmingham B4 7ET, United Kingdom
Address all correspondence and requests for reprints to: Ashley Grossman, M.D., Department of Endocrinology, St. Bartholomew’s Hospital, West Smithfield, London, United Kingdom EC1A 7BE. E-mail: a.b.grossmann{at}mds.qmw.ac.uk
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Abstract |
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RNA was extracted from pituitary tissue removed at autopsy and transsphenoidal surgery (n = 62), and ghrelin and GHS-R type 1a and 1b mRNA levels were investigated using real-time RT-PCR. Both ghrelin and GHS-R mRNA were detected in all samples. Corticotroph tumors showed significantly less expression of ghrelin mRNA, whereas GHS-R mRNA levels were similar to those in normal pituitary tissue. Gonadotroph tumors showed a particularly low level of expression of GHS-R mRNA. Immunohistochemistry, using a polyclonal antibody against the C-terminal end of the ghrelin molecule, revealed positive staining in the homolog of the arcuate nucleus in the human hypothalamus and in both normal and abnormal human pituitary. Pituitary tumor ghrelin peptide content was demonstrated using two separate RIA reactions for the N-terminal and C-terminal ends of the molecule. Both forms were present in normal and abnormal pituitaries, with 5 ± 2.5% octanoylated (active) ghrelin (mean ± SD) present as a percentage of the total. We suggest that the presence of ghrelin mRNA and peptide in the pituitary implies that the locally synthesized hormone may have an autocrine/paracrine modulatory effect on pituitary hormone release.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human pituitary adenomas were obtained at the time of
transsphenoidal surgery. The tumor type was determined on the basis of
clinical and biochemical findings before surgery and morphological and
immunocytochemical data. All patients with Cushing’s disease were
treated with cortisol-lowering drugs for 6–8 weeks before surgery to
produce mean serum cortisol levels within the normal range. Patients
were routinely treated with hydrocortisone (100 mg, im) with their
premedication before surgery. Normal human pituitaries (n = 7)
were also collected at autopsy (4–24 h postmortem) from patients with
no evidence of endocrine abnormality. A total of 55 pituitary adenomas
were studied for ghrelin mRNA expression: 22 somatotroph adenomas, 4
lactotroph adenomas, 12 nonfunctioning pituitary adenomas (NFPAs), 12
corticotroph tumors, and 5 FSH-secreting adenomas (Table 1A
). Several pituitaries were also
extracted for C- and N-terminal ghrelin measurement: 3 somatotroph
adenomas, 2 lactotroph adenomas, 6 NFPAs, 1 corticotroph tumor, 1
FSH-secreting adenoma, 1 TSH-secreting adenoma, and a normal pituitary
collected at autopsy. In addition, nonpituitary neuroendocrine tumors
removed at surgery were studied: 2 ACTH-secreting ectopic tumors (of
the thymus and pancreas), 3 pancreatic insulinomas, and a pancreatic
gastrinoma (Table 1B). A normal human stomach biopsy sample was removed
at gastroscopy, and a normal human hypothalamus was collected at
autopsy. All studies received institutional ethical approval.
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Total RNA was prepared using the SV isolation kit (Promega Corp., Southampton, UK), which includes a deoxyribonuclease step. RNA was quantified by spectrophotometry (Cecil CE5501 Computing Double Beam UV Spectrophotometer, Cecil Instruments Ltd., Cambridge, UK). RNA was diluted to 100 ng/µL for use in the RT-PCR assay and was stored at -50 C.
Primers and probes
RT-PCR primers and probes were designed for human ghrelin, GHS-R
types 1a and 1b, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
using Primer Express software (PE Applied Biosystems,
Warrington, UK) based on the sequence data of the genes available in
GenBank (Table 2). In
each case, the primers were designed to cross exon-intron boundaries.
The probes were labeled with a fluorescent dye (6-carboxy-fluorescein)
and a quencher dye (6-carboxy-tetramethylrodamine). Primers and probes
were purchased from PE Applied Biosystems.
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The assay relies on the nucleolytic activity of the polymerase
displacing and cleaving any probe annealed to the amplicon after target
amplification (13). After target amplification, the probe
anneals to the amplicon and is displaced and cleaved between the
reporter and the quencher dyes by the nucleolytic activity of the
polymerase. The amount of product resulting in detectable fluorescence
at any given cycle within the exponential phase of the PCR is
proportional to the initial number of the template copies. The number
of PCR cycles (threshold cycles, Ct) needed to
detect the amplicons is therefore a direct measure of the template
concentration (Fig. 1). We used a
one-enzyme/one-tube reaction protocol as previously described
(14), except that the RT step was reduced to 15 min. In
short, the RT-PCR reaction conditions were the following: 25 µL
reaction mixture including 1 µL RNA template were heated for 15 min
at 50 C in the presence of 0.01 U/µL AmpErase UNG (PE Applied Biosystems). After RT for 15 min at 60 C, the reaction mixture
was denatured at 92 C for 5 min followed by 40 cycles of PCR at 92 C
for 20 s and at 62 C for 1 min in each cycle in the presence of
the 6-carboxy-fluorescein-labeled oligonucleotide probe. The RT-PCR
reactions were performed, recorded, and analyzed by using the ABI7700
Prism Sequence Detection System (PE Applied Biosystems).
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Quantitation of mRNA samples was carried out by relating the PCR threshold cycle obtained from tissue samples to amplicon-specific standard curves (15). Serial dilutions of the single stranded sense oligodeoxynucleotide amplicons were carried out in duplicate from 1 x 108 molecules to 10 molecules and used in triplicate RT-PCR reactions. Standard curves were obtained by plotting the log [calculated copy number] against the threshold cycle. The log copy numbers (N) of unknown samples were calculated from the regression line according to the formula: logN = (CT - b)/m, where CT is the threshold cycle, b is the y-intercept, and m is the slope of the standard curve line. As normalization to the GAPDH housekeeping gene is inaccurate, mRNA expression levels are presented as the mRNA copy number per µg total RNA. Any copy number under 3000 copies/µg total RNA was assumed to be due to illegitimate transcription (16, 17).
Quality standards for RT-PCR
All serial dilutions were carried out in duplicate. The reactions to generate standard curves were repeated twice, each time in triplicate. All clinical samples were tested in duplicate. As spectrophotometric analysis alone may not accurately reflect either the quality of isolated nucleic acids or its performance in a subsequent RT-PCR analysis, amplification of GAPDH mRNA was used as a standard for the quality of the RNA samples investigated. Samples negative for GAPDH were excluded from quantification. Two no-template controls were included with every amplification run; one was prepared before opening all the tubes and dispensing the various reagents, and the other was prepared at the end of the experiment. This allowed us to monitor any contamination arising during the handling of the reagents. Contamination of ACTH- and FSH-secreting tumors and NFPAs by somato-, lacto-, or thyrotroph cells of nontumorous tissue was excluded by confirming undetectable expression of the Pit-1 gene, as described previously (7, 18).
Protein extraction
Pituitary samples were boiled in 5 mL water for 5 min. After cooling the samples on ice, AcOH and HCl were added to final concentrations of 1 N and 20 mmol/L, respectively. After homogenizing by Polytron (Brinkmann Instruments, Inc., Westbury, NY), the supernatant was collected, and the precipitate was extracted again. Both fractions were applied to a Sep-Pak Plus column (Waters Corp., Milford, MA). After lyophilization, the samples underwent two RIA reactions (see below).
Ghrelin RIA
Tissue ghrelin content was determined by two separate RIA reactions. The first one uses a polyclonal rabbit antibody (no. 3–7) against the C-terminal part of the molecule [[Cys]ghrelin-(13–28)]; it, therefore, recognizes both the octanoylated (active) form of ghrelin and the nonoctanoylated (inactive) form of both human and rat ghrelin (19). The second RIA reaction uses another polyclonal rabbit antibody (no. 4–4), which was raised against the N-terminal of the ghrelin molecule [rat ghrelin-(1–11)] with n-octanoyl modification at serine 3) and recognizes the octanoylated form of the peptide in both human and rat. Ghrelin peptide without the n-octanoyl modification (des-acyl ghrelin) is not recognized by this antibody. [125I]Tyr-ghrelin-(13–28) and [125I]Tyr29-ghrelin were used as tracers in the C- and N-terminal RIAs, respectively. The minimal detectable concentration is 10 fmol/tube for the C-terminal assay and 0.5 fmol/tube for the N-terminal RIA.
Immunohistochemistry
Paraffin sections (3 µm) of human stomach, hypothalamus, and pituitary samples were air-dried, then placed in a 60 C oven overnight. Sections were dewaxed in xylene, followed by immersion into a solution of 750 µL 30% hydrogen peroxide and 50 mL methanol for 10 min to block endogenous peroxide. Sections were rehydrated in tap water, ready for antigen retrieval. Sections required heat-mediated antigen retrieval treatment (20). Sections were superheated for 4 min in citrate buffer 0.01 mol/L, pH 6.0, then placed into tap water immediately to avoid drying of sections. The sections were then transferred to phosphate-buffered saline (PBS) before immunostaining. Immunohistochemistry was performed using a standard avidin-biotin complex (ABC) method. The primary antibody (no. 3–7, see above) was placed on sections at a 1:5000 dilution for 40 min at room temperature in a wet chamber. Sections were washed in PBS and then incubated in a biotinylated antirabbit second layer for 30 min. Sections were again washed in PBS and then incubated in the ABC (Vectastain Elite ABC peroxide kit PK6200 Vector Laboratories, Inc., Peterborough, UK) for 20 min. After three washes in PBS, sections were visualized with activated 3',3'-diaminobenzidene-tetrahydrochloride solution (Kentec DAB tablets 4170, Biostat, Stockport, UK) for 10 min; this resulted in a brown end-product. Sections were counterstained with Gill’s hematoxylin and dehydrated through graded alcohol before mounting in DPX. Specificity of the ghrelin staining was assessed initially by preabsorption of the antibody with the full-length human ghrelin peptide; this completely abolished ghrelin staining.
Immunostaining of other hormones
Pituitary hormone stains were performed to establish the histological hormone phenotype of the tumor samples. GH, ACTH, PRL, TSH, LH, FSH, gastrin, chromogranin, glucagon, insulin, and hCGß antibodies were supplied by DAKO Corp. (Oxford, UK), whereas pancreatic polypeptide and vasoactive intestinal polypeptide antibodies were supplied by Novacastra (Peterborough, UK). Sections were incubated overnight in the primary antibody at 4 C. The standard ABC protocol was followed thereafter. For pituitary staining a normal human pituitary was used as a positive control; the hormone antibody was omitted and replaced by mouse Ig as a negative control.
Statistical analysis
The data were analyzed by a nonparametric ANOVA test, the Kruskal-Wallis test, using the Arcus Quickstat Biomedical version 1.2 (Buchan I, Addison Wesley Longman Ltd., Cambridge, UK). Significance was taken at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ghrelin was expressed in normal pituitary and in adenomatous
pituitary tissue at both the mRNA and peptide level (Figs. 2 and 3).
Using real-time PCR we detected 2.5 x 103
to 7 x 106 copies of ghrelin mRNA
molecules/µg total RNA, with a significant difference among the
different pituitary tissue types (P = 0.0042).
Specifically, corticotroph tumors showed a significantly lower level of
mRNA expression compared with normal pituitary (Fig. 2), with one
sample showing a completely absent expression of ghrelin mRNA. In
patients with acromegaly we studied the relationship of circulating GH
levels before transsphenoidal surgery and the level of ghrelin mRNA
expression in the tumors, but found no significant correlation. Ectopic
ACTH-secreting tumors and insulinomas expressed ghrelin at copy numbers
of 2 x 104 to 5 x
105 and 6 x 104 to
1 x 106, respectively, whereas the
gastrinoma expressed low levels of ghrelin mRNA (3 x
103).
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, upper left panel). Ghrelin peptide
was also detected in human hypothalamus in the arcuate (infundibular)
nucleus, whereas the other hypothalamic nuclei did not stain positively
(Fig. 3, upper right and lower left panels).
Normal pituitary also stained positively for ghrelin, although only
weakly, in some, but not all, cells (Fig. 3, lower right
panel). Positive staining was also seen in a somatotroph adenoma,
an NFPA, and a prolactinoma, whereas a corticotroph adenoma was
negative. Human stomach was used as a positive control, and full-length
human ghrelin pretreatment blocked staining in both pituitary and
hypothalamus.
A subset of pituitary samples was extracted and assayed for ghrelin
content with two RIAs (Table 3). Ghrelin
peptide was present in normal and adenomatous pituitary tissue, in both
octanoylated and des-octanoylated forms. The C- terminal RIA (which
measures both octanoylated and des-octanoylated ghrelin together)
showed an average of 1.64 ± 0.93 (mean ± SD)
pmol/g tissue peptide content, whereas the N-terminal assay (which
measures octanoylated ghrelin alone) showed 0.10 ± 0.11 pmol/g
peptide content, suggesting that 5.5 ± 2.8% of the total ghrelin
content in these samples is octanoylated, i.e. biologically
active.
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GHS-R 1a expression was detected in all pituitary samples, with
significant differences between the different tumor types
(P = 0.0057; Fig. 4).
Somatotroph tumors showed the highest level of expression of GHS-R type
1a, whereas FSH-omas showed only a very low level of expression (Fig. 4). Corticotroph tumors expressed similar amounts of GHS-R type 1a as
normal pituitaries; however, the ratio of GHS-R to ghrelin expression
was about 60 times higher in this tumor type compared with other tumor
samples, significantly different from the ratio in normal pituitary and
other tumor types. All of the nonpituitary neuroendocrine tumors
expressed GHS-R type 1a. Previously, we reported GHS-R type 1a
expression in pituitary and neuroendocrine tumors using a
semiquantitative duplex RT-PCR comparing GHS-R expression to the
housekeeping gene GAPDH (7). When we compared our current
quantitative results with our previous data on a subset of samples
(n = 30), there was a close correlation between the two datasets
(Spearman correlation coefficient = 0.76; P <
0.001).
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). Two of the 12
corticotroph, 3 of the 5 gonadotroph, 6 of the 12 nonfunctioning, and 2
of the 4 lactotroph adenomas were negative for GHS-R type 1b mRNA
expression (Fig. 5). None of the neuroendocrine tumors expressed GHS-R
type 1b, other than one insulinoma.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We also showed that ghrelin mRNA is transcribed into peptide, as cytoplasmic ghrelin immunoreactivity was seen in normal and abnormal human pituitary tissue. Initially, we detected ghrelin in human stomach. We also observed ghrelin immunostaining in the human hypothalamus; specifically, many neurons in the human homolog of the arcuate nucleus showed strong cytoplasmic staining, primarily in a granular pattern suggestive of vesicular storage, whereas other hypothalamic nuclei did not contain ghrelin-positive cells. The arcuate nucleus has been identified as a site with one of the highest concentrations of the GHS-R (21, 22), and it is thus possible that locally produced ghrelin might activate these receptors (23). Ghrelin immunoreactivity was present in both normal and certain adenomatous pituitary samples. In the normal pituitary, weak cytoplasmic staining was seen in the majority of the cells; however, some pituitary cells clearly showed no ghrelin positivity. Somatotroph, lactotroph, and nonfunctioning adenomas also showed weak ghrelin staining, whereas corticotroph adenomas showed negative staining. This correlates with our findings at the mRNA level, where, again, corticotroph adenomas expressed the lowest level of ghrelin mRNA. As the antibody used for the immunohistochemistry recognizes both octanoylated and des-octanoylated ghrelin, 15 normal and adenomatous pituitary samples underwent protein extraction and RIA reactions for both octanoylated and des-octanoylated ghrelin. The data suggest that 5% of the total ghrelin content of the pituitary samples is in an active from.
Ghrelin is a 28-amino acid peptide that has been isolated from the rat stomach, but has also been shown to be present in other tissues in the rat, including the hypothalamic arcuate nucleus (24). It has been shown to specifically activate GHS-R and stimulate GH release in vitro, but has no direct effect on ACTH, PRL, FSH, LH, or TSH secretion. It has also been shown to stimulate GH release from anesthetized rats. Ghrelin has an n-octanoyl group on the third amino acid, which appears to be necessary for biological activity. Ghrelin is highly conserved between species; rat and human ghrelins differ only by two amino acids (24). Earlier in vitro and in vivo studies using synthetic GHSs suggested that they exert their effects primarily via the hypothalamus (25, 26). However, GHSs also stimulate GH release directly from isolated rat or human pituitary (3, 27). We and others have shown the expression of GHS-R mRNA in normal human pituitary and in a variety of pituitary tumors (7, 8, 9, 10, 12). The endogenous ligand, ghrelin, arising from the hypothalamus might reach these pituitary receptors via the portal system; if the ligand is synthesized elsewhere, however, it may also have access to the peripheral circulation. Locally synthesized ghrelin could also have a direct paracrine or autocrine effect via pituitary GHS-R.
Comparing our current data on GHS-R expression with our earlier data on the same samples using duplex RT-PCR (7), we found a significant correlation between the two datasets. However, although a number of samples showed no expression even at high PCR cycle numbers with the earlier method, using the real-time PCR method we were able to detect a low level of mRNA expression in the same samples. It is known that the latter method is more sensitive and especially provides a more reliable and reproducible quantitative mRNA expression data (17). The lack of an ideal housekeeping gene (with the same level of expression in all tissue types at all circumstances) renders the assessment of duplex PCR results more difficult (17). Nevertheless, the results of the two different techniques showed a good correlation in our hands.
For many years the pituitary gland has been considered to consist of different highly specialized cell types, each producing a specific hormone and each responding to specific hypothalamic hypophysiotropic hormones and peripheral hormones. However, today there is a large body of evidence supporting the hypothesis of paracrine/autocrine regulation in the pituitary (for reviews, see Refs. 28, 29, 30). Pituitary hormones themselves can have local effects, but classical hypothalamic hormones, such as GHRH, CRH, TRH, LHRH, somatostatin, urocortin, and even leptin, as well as cytokines have been shown to be synthesized in the pituitary and to modulate pituitary function (31, 32, 33, 34, 35, 36, 37, 38). It has been suggested that they may play a role in supporting basal hormone synthesis and/or hormone output from the pituitary and in maintaining the optimal responsiveness of pituitary cells to their specific pulsatile releasing hormone arising from the hypothalamus (36, 37); however, their precise functions remain unclear. It now seems likely that ghrelin may also play a role in such activity, although the specific functional importance of this peptide in the pituitary and any possible role in tumorigenesis must await functional analysis.
In summary, we have demonstrated the presence of ghrelin mRNA and peptide in the human pituitary, and we speculate that in addition to the probable hypothalamic effects of ghrelin, locally produced ghrelin in the pituitary gland may have direct paracrine and/or autocrine effects on pituitary function.
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Acknowledgments |
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Footnotes |
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Received July 27, 2000.
Revised October 4, 2000.
Accepted October 28, 2000.
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References |
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