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Clinical Studies |
Division of Endocrinology, Departments of Internal Medicine and Pharmacology (S.P), Kyunghee University School of Medicine, Seoul, Korea
Address all correspondence and requests for reprints to: Inmyung Yang, M.D., Ph.D., Division of Endocrinology, Department of Internal Medicine, Kyunghee University School of Medicine, 1 Hoiki-dong, Dongdaemoon-Ku, 130–702 Seoul, Korea. E-mail: francis{at}chollian.dacom.co.kr
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Abstract |
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Introduction |
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In theory, a stimulation test would be better to evaluate a reduction of HSA. Acute hyperglycemia is known to suppress the GH response to GHRH (4, 5), which is reversed by the administration of pyridostigmine (6). Those findings suggest that acute hyperglycemia enhances the HSA. We previously demonstrated that oral glucose administration suppresses the TRH-induced TSH response by enhancing HSA, and the combined glucose-TRH test can be a useful method to evaluate HSA (18).
Previous studies suggest that hypothalamic HSA shows wide intersubject variability (2, 3). On the other hand, the GH response to SRIH is also known to vary among acromegalic patients (7, 8, 9, 10, 11). Many investigations (12, 13, 14, 15, 16), including our earlier study (17), have reported that the GH response to octreotide, a long-acting SRIH analog, also varies within a wide range.
There are few studies that have documented the GH response of acromegalic patients to endogenous HSA. Therefore, we investigated the GH response of acromegalic patients to HSA evaluated by the combined glucose-TRH test.
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Subjects and Methods |
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Thirteen acromegalic patients (six men, aged 37–45 yr, and seven women, aged 29–63 yr) were studied. None of the patients had received treatment before the study. The diagnosis of active acromegaly was made by the lack of GH suppression under 2 µg/L during the oral glucose tolerance test (OGTT). Pituitary adenomas were identified in all patients by either computerized tomography or magnetic resonance imaging. Informed consent was obtained from each patient before the study.
Endocrine tests
Endocrine tests were performed on separate days. Before each test, subjects fasted overnight and were recumbent for 1 h. On day 1, the TRH stimulation test was performed. TRH (Relefact TRH, Hoechst, Frankfurt, Germany; 200-µg bolus) was administered iv. Blood sampling was performed every 30 min for 2 h. On day 2, the OGTT was performed by oral administration of 75 g glucose with blood sampling as described above. On day 3, blood was obtained every hour from 0900–1600 h for determination of the basal daytime GH secretory pattern. On day 4, the GnRH stimulation test was performed by iv administration of a 100-µg bolus of GnRH (Relefact LH-RH, Hoechst), followed by the same blood sampling as that described above. A responder to releasing hormones was defined as one whose GH level exceeded the respective spontaneous daytime peak and increased, as we have previously reported (17), to more than 50% above the baseline value. On day 5, the combined glucose-TRH test was undertaken by administering 75 g glucose, orally, 30 min before the injection of TRH as reported previously (18). On day 6, for determination of the octreotide response, a 100-µg bolus of octreotide (Sandostatin, Sandoz, Switzerland) was injected iv, and blood samples were taken every hour for 6 h.
Sequencing of the gsp oncogene
Genomic DNA was extracted from the frozen or formalin-fixed paraffin-embedded tumor tissue and peripheral blood leukocytes from each patient as described previously (19). Nested PCR was performed to amplify and sequence the region between codons 184 and 251. A set of primers (upper primer, 5'-GCG CTG TGA ACA CCC CAC GTG TCT-3'; lower primer, 5'-CGC AGG GGG TGG GCG GTC ACT CCA-3') was used for the first PCR. Another set of primers (upper primer, 5' GTG ATC AAG CAG GCT GAC TAT GTG-3'; lower primer, 5'-GCT GCT GGC CAC CAC CAC GAA GAT GAT-3') was used for the second PCR. The amplified fragments were purified by agarose gel electrophoresis and used in a direct PCR sequencing reaction. The cyclic sequencing kit (SequiTherm, Epicentre Technologies, Madison, WI) and a thermal cycler (Gene and PCR System 9600, Perkin-Elmer, Norwalk, CT) were used for the analysis.
Hormone assays and statistical analysis
Commercial immunoradiometric assay kits were used for GH (Nichols Institute Diagnostics, San Juan Capistrano, CA) and TSH (Daiichi, Tokyo, Japan) determinations. The sensitivity of the GH assay was 0.02 µg/L. The intra- and interassay coefficients of variation were 3.3% and 5.1%, respectively. The sensitivity of the TSH assay was 0.1 mU/L, and the intra- and interassay coefficients of variation were 2.1% and 2.5%, respectively. Total T3 and T4 were measured by RIA kits [Incstar Corp. (Stillwater, MN) and Abbott Laboratories (North Chicago, IL), respectively]. The normal range was 80–200 ng/dL for T3 and 5–13 µg/dL for T4.
Data were expressed as the mean ± SE. Differences between groups were deemed significant if P < 0.05 by to unpaired t test. A significant relation was determined if P < 0.05 by Fisher’s exact test in contingency table analysis, and a correlation was determined in the linear regression analysis. All statistical analyses were performed by using a statistical software (GraphPad Prism, GraphPad Software, San Diego, CA).
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Results |
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). The nadir GH levels were 28–80% of the peak GH levels. The
paradoxical response to TRH and GnRH was found in 69% and 23% of the
patients, respectively. Most of the patients (77%) were considered
good responders to octreotide whose GH level was suppressed by >80%
of basal level as we reported previously (17), The gsp
oncogene was found in seven (54%) of the patients (Table 1). All the mutations were found to be located at codon
201, replacing C with T, resulting in cystein instead of arginine. The
sequencing data have been previously presented (19).
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). The mean TRH-stimulated TSH levels of the patients
with normal or low HSA were significantly lower than those of the
patients with high HSA (5.13 ± 0.10 vs. 11.3 ±
2.8 mU/L; P = 0.0469; Fig. 1). HSA did
not relate to sex, age, tumor size, basal GH levels, paradoxical
responses, octreotide response, or gsp oncogene (data not
shown).
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).
However, no suppression was observed during OGTT in all patients (data
not shown). The response of GH to the glucose-induced HSA did not
correlate with the HSA assessed by the glucose-induced suppression of
the TRH-stimulated TSH response. It also did not correlate with the
octreotide response (data not shown).
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, A and
B).
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Discussion |
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The suppression of TSH during the combined glucose-TRH test is likely to be attributed to the glucose-induced HSA (18), which may reflect the hypothalamic neuronal reserve of SRIH. The HSA of some patients was higher than that of normal controls, whereas some patients had lower HSA. The higher HSA is most probably a result of a positive feedback by GH. The higher HSA of the patients does not support the hypothesis that a reduction of the HSA is a primary cause of acromegaly.
The low HSA of some patients is unlikely to have been caused by
destruction of thyrotrope cells by the tumors. Patient 2 with
macroadenoma, 32 mm in diameter, showed a high HSA (53% suppression of
TSH). In contrast, patient 9 with microadenoma showed no
suppression. Indeed, a recent study reported that anterior pituitary
function is better preserved in GH-producing macroadenomas than in
nonfunctioning macroadenomas, and hypothyroidism was not found in the
patients with acromegaly (20).
The low HSA of some patients also seems not to be attributed to a primary hypothalamic defect because of at least three reasons. Firstly, the patients with low HSA had a lower TSH response to TRH stimulation. If the low HSA is a primary defect, the TSH response should be higher because SRIH suppresses basal and TRH-stimulated TSH secretion (21, 22, 23, 24). We have also previously demonstrated that pyridostigmine-induced suppression of SRIH enhanced the TSH response to TRH (25). Secondly, the paradoxical GH response to TRH was suppressed completely even in the patients with low HSA (patients 3 and 9). Thirdly, the size of tumor did not differ in the patients with higher HSA and lower HSA. As SRIH is known to negatively control growth in a variety of cell types (26, 27, 28), patients with a primary defect in HSA would be expected to have larger tumors.
Therefore, it is likely that the low HSA reflects the low somatostatinergic neuronal reserve resulting from continuous neuronal firing by increased GH levels. TSH secretion of the patients whose SRIH secretion is already maximal would not be additionally suppressed by glucose pretreatment. Seven patients (patients 1, 3, 4, 5, 8, 9, and 10) are thought to belong to this category. The patients with high HSA are believed to still have the hypothalamic SRIH reserve. A recent study (3) also demonstrated that pyridostigmine potentiated the GHRH-induced GH response in some patients, suggesting that a certain degree of hypothalamic SRIH secretion under inhibitory cholinergic control may persist in acromegaly.
HSA did not correlate with either the nadir or the peak daytime GH levels. This suggests that basal GH levels are not determined by HSA, or HSA is not determined by basal GH levels.
The previous study (2) assessed the GH response of the tumors to HSA by measuring the GH ratio during the OGTT. As we found a spontaneous fluctuation in the basal daytime GH level in the range of 28–80% of the peak levels, it did not allow us to interpret the result of OGTT as the response to HSA. In this study, we could not find any suppression of GH even in five patients whose TSH responses were significantly suppressed by glucose. Four of them even showed a paradoxical increase above the daytime peak.
We observed a significant suppression of the GH response of the tumors during the combined glucose-TRH test. As SRIH is known to suppress the paradoxical GH response to TRH (29), it is not surprising to find suppression of the response during the glucose-TRH test. The suppressive effect lasted for about 60–90 min, which is similar to the period that glucose pretreatment suppressed the TSH response to TRH in the previous study (18). It suggests that endogenous HSA can suppress the paradoxical response in some acromegalic patients.
The glucose-induced suppressive effect on the TSH response did not correlate with that on the GH response. This suggests that the response of tumor cells to SRIH differs from that of thyrotropes, and that each tumor may have different properties that affect the responsiveness to SRIH.
All patients (no. 1–4) who showed complete suppression of the GH response to HSA, had larger tumors than the patients with poor response to HSA. This suggests that SRIH is not a major determinant of tumor size even though SRIH is known to have antiproliferative effects (26, 27, 28). More cases need to be investigated to confirm the possibility that a large tumor with the paradoxical response to TRH is likely to be a good responder to endogenous HSA.
In this study, the GH response to octreotide did not correlate with the GH response to HSA. This might be attributed to different expressions of the subtypes of SSTR. Octreotide is known to bind with the highest affinity to SSTR2 (30), whereas hypothalamic SRIH, SRIH-28, has a higher affinity to SSTR5 (31).
GH-secreting adenomas with gsp oncogene show higher response to SRIH (32) or octreotide (19). In this study, the gsp oncogene was not found with a higher frequency in the tumors with good response to the endogenous HSA. This suggests that the gsp oncogene is not a major determinant for the GH response to the HSA. The frequency of the gsp oncogene also did not differ in patients with high HSA and those with low HSA. It suggests that the G protein mutation does not play a major role in determination of the HSA.
Taken together, this study supports the idea that a reduction of HSA is not a primary cause of acromegaly, and the increase in HSA seems to be caused by the autonomous secretion of GH from the pituitary adenomas. However, a reduction of HSA may be a result of the excessive firing of SRIH neurons. HSA as well as the response of tumors to HSA do not determine tumor growth. A certain property of tumor that determines HSA and the response to HSA remains to be investigated.
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Acknowledgments |
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Footnotes |
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Received February 3, 1997.
Revised April 24, 1997.
Accepted May 1, 1997.
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References |
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