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Original Studies |
Istituto di Scienze Endocrine, Università di Milano, Ospedale Maggiore IRCCS, and Istituto Auxologico Italiano IRCCS, Milan; and Istituto Clinico Humanitas, Rozzano, Italy
Address all correspondence and requests for reprints to: Luca Persani, M.D., Laboratorio Sperimentale di Ricerche Endocrinologiche, Istituto Auxologico Italiano IRCCS, Via L. Ariosto, 13–20145 Milan, Italy.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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-2,6-sialyltransferase activity within thyrotropes and
results in the secretion of high amounts of sialylated TSH isoforms
(34.6% and 26.3%). A hybrid TSH with peculiar terminal sugar residues
and enhanced bioactivity is circulating in patients with RTH (TSH B/I,
2.2). Treatment with low doses of TRIAC can initially reduce thyroid
hormone secretion in RTH, mainly through the secretion of TSH isoforms
with changed terminal sugar residues and reduced bioactivity (TSH B/I,
0.9–1.7). In conclusion, changes in the terminal sialic acid residues
modulate the biological properties of circulating TSH, play a relevant
physiopathological role in various situations, and contribute to adjust
thyroid-stimulating activity to temporary needs. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-subunit and one on the
ß-subunit) during early stages of the translational process and are
progressively modified during several maturation steps from the rough
endoplasmic reticulum through the Golgi to the secretory granules by
specific glycosyl transferases (1, 2, 6). Therefore, intrapituitary and
circulating TSH are constituted by several isoforms with heterogeneous
carbohydrate branching and variable exposed/terminal residues that
account for differences in isoelectric focusing and lectin binding (1, 2, 7). Many different regulating factors, such as thyroid hormones, TRH, dopamine, somatostatin, and glucocorticoids, influence TSH secretion (1, 2, 8, 9). A negative thyroid hormone feedback mechanism is known to affect TSH subunit transcription and the holo-TSH secretion rate (8, 9), and recent data indicate that thyroid hormone modulates the expression of several glycosyl transferases within thyrotropes (10, 11). Hypothalamic TRH exerts its main effects on thyrotrope function by regulating TSH posttranslational oligosaccharide processing and release (1, 2, 8, 9). As a final result, several physiological and pathological states are characterized by variable amounts of circulating TSH that are constituted by molecules with different carbohydrate branching, bioactivity, organ distribution, and clearance (1, 2, 3, 4, 7, 8).
The availability of huge amounts of purified TSH allowed several studies to show that variations in the terminal residues of asparagine-linked carbohydrate chains have a major impact on hormone biological properties. In particular, highly sialylated TSH, such as that circulating in primary hypothyroidism (PH) or present in the recombinant human TSH, has a clearly detectable, but reduced, in vitro bioactivity compared to intrapituitary or serum TSH from normal subjects (12, 13), probably due to a diminished ability to interact with specific regions of the TSH receptor (5). Moreover, highly sialylated TSH escapes hepatic clearance and its MCR is slow (4, 14), thus resulting in a longer circulating half-life and a prolonged in vivo biological activity once injected in the animal (12). Enzymatic removal of terminal sialic acid and exposure of the underlying galactose residues reverts all these biological properties, and asialo-recombinant human TSH shows increased MCR and intrinsic bioactivity (12, 15).
The availability of an efficient technique for the immunopurification and concentration of circulating TSH molecules along with reliable methods for both functional and structural analyses prompted the present study, which was aimed to investigate the changes in the degree of sialylation and the biological activity of circulating TSH in various physiological and clinical situations, including those characterized by normal TSH concentrations.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Twenty-six healthy volunteers (16 males and 10 females; age, 16–52 yr), whose blood was withdrawn during the morning, served as the control group. TSH bioactivity data concerning 14 of these subjects had been previously reported (13, 16). The study of the circadian oscillation of circulating TSH and free thyroid hormone (FT4 and FT3) concentrations was performed in a new homogeneous group of 7 young men (age, 16–18 yr), whose blood was withdrawn every 30 min from 0800–1300 h and from 2200–0500 h and every 60 min during the remaining hours, as described previously (16). To obtain the amount of TSH necessary to perform ricin lectin affinity chromatography as well as to measure the bioactivity at three different dilutions in triplicate, two pools of sera [daytime pool (samples withdrawn between 0800–1400 h) and nighttime pool (2200–0500 h)] were obtained from each subject.
PH
Twenty-four patients with PH of various origin (autoimmune in 19 and postthyroidectomy in 5; 20 females and 4 males, age, 32–75 yr) were included in the study. Twelve of these patients had been previously reported (13, 16). Ten patients were also studied after 1 month treatment with low doses of L-T4 (1.0–1.2 µg/kg BW·day).
Fetuses
Fetal blood was obtained by cordocentesis using a 22-gauge heparinized needle guided by ultrasonography, as previously reported (17). The reliability of fetal sampling was assessed by analysis of red cell volume with a Coulter counter (Coulter,Hialeah, FL) and was confirmed by Kleihauer smears. The normal fetuses were selected from 539 consecutive cordocentesis carried out for rapid karyotyping and early diagnosis of infection or hematological disorders in fetuses of women who gave their informed consent to the study. One hundred and thirteen fetal samples were obtained by cordocentesis, their healthy state was shown by normal oxygenation, and acid-base balance values was measured at the time of fetal sampling. Definitive confirmation of their health was achieved by clinical examination at birth and during at least 2 months of follow-up.
As the amount of plasma available for individual studies was limited, samples obtained during the third trimester (31–37 weeks) were pooled (2 pools of 10 and 12 mL, from 19 and 23 fetuses, respectively) for the evaluation of TSH bioactivity. As control, we obtained 1 pool made of samples from euthyroid pregnant women (n = 23) of the same gestational period.
Moreover, we studied one fetus with primary hypothyroidism (cordocentesis at 27th week) and one with resistance to thyroid hormones (RTH). The prenatal diagnosis of RTH was made by analyzing chorionic villi: restriction analysis demonstrated the same mutation (T337A) in exon 9 of thyroid hormone receptor ß1 (TRß1; see below) in the mother and fetus (18). In this case, cord blood samples were obtained at 28 and 33 weeks gestation while the mother was receiving triiodothyrocetic acid (TRIAC), a thyroid hormone analog able to cross the placental barrier in significant amounts (19).
RTH
RTH is a dominantly inherited genetic disease caused by
mutations located in three hot spots within the
T3-binding domain of TRß1 (20, 21, 22). The mutated
receptors have a reduced binding affinity for T3
and exert a dominant negative activity on the function of normal TR
isoforms (20, 21, 22). Clinically, some patients are asymptomatic and
present with features of compensated hypothyroidism (generalized
resistance), whereas others ]pituitary resistance (PRTH)] show
peripheral thyrotoxic features (hyperactivity/attention deficit
disorder, insomnia, tremors, and tachycardia/atrial fibrillation)
(20, 21, 22). Patients with PRTH might benefit from the treatment with
TRIAC (22), a T3 metabolite with a higher
affinity in binding wild-type and mutated TRß1 isoforms with respect
to TR, than T3 itself (23).
We studied five PRTH patients belonging to different kindreds (Table 1
). The diagnosis of RTH was based on
several clinical and laboratory findings (24, 25). Four of these
patients had been previously reported (25). Mutations in the TRß1
gene were detected in three of them, and functional properties of
mutated receptors were also previously reported (26, 27). Detected
mutations were localized to the three hot spots within the
T3-binding domain of TRß1 (22). The five
patients were studied either in the basal condition or during a short
term and low dose treatment trial with TRIAC (1–2 mg/day for 2
weeks).
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Immunoreactive TSH (TSH-I) was measured by a third generation immunofluorometric assay (Delfia, Pharmacia, Milan, Italy), using a two-step procedure and TSH International Reference Preparation 80/558 as reference (normal values, 0.24–4.0 mU/L). Serum FT3 and FT4 levels were measured by direct back-titration methods, using Delfia technology (Pharmacia; normal FT3 and FT4 range, 4–8 pmol/L and 9–18 pmol/L, respectively). cAMP in incubation medium was measured using a commercial RIA (RIANEN, DuPont, Billerica, MA).
Chronobiological analysis
Chronobiological analysis was performed by means of single and population mean-cosinor methods, as previously reported (16). These procedures consist of fitting raw data for a given variable to a cosine function, with a period equal to 24 h, by the method of least squares to detect a rhythm and to estimate the following rhythmometric parameters: mesor, i.e. rhythm-determined average; amplitude, i.e. difference between the mesor and the peak of the best fitting cosine function; acrophase, i.e. the time of the crest of the best fitting function; and finally, the percent rhythm (PR), i.e. the percent variability in the data that is accounted for by the best fitting function. The rhythm is statistically validated when the amplitude zero hypothesis is rejected at the P < 0.05 level.
TSH bioassays
Immunoconcentration of circulating TSH. As human serum contains several factors interfering in the TSH bioassay response, circulating TSH was immunopurified using polystyrene tubes coated with a monoclonal antibody directed against a conformational epitope, as previously described (13). The total volume of serum samples varied from 0.75–52 mL depending on the relative circulating levels of immunoreactive TSH. The recovery before the concentration step was 92–98%, whereas final recovery ranged between 52–68%; the discrepancy was due to nonspecific losses and not to selection of particular molecular forms of TSH (13). Furthermore, the possible presence in the purified samples of non-TSH substances able to alter cAMP production in the bioassays was ruled out by the observation of unmodified basal cAMP accumulation after incubation of the cells with the immunopurified material from a Graves’ patient with suppressed TSH secretion.
CHO cells expressing human TSH receptor (CHO-R). The CHO-R strain JP-26 (supplied by Dr. G. Vassart, Brussels, Belgium) was used (28). Three different dilutions of immunoconcentrated TSH were bioassayed in triplicate, as was TSH standard. Extracellular cAMP was measured in the medium collected at the end of incubation. The sensitivity of this system was 1.6 ± 0.25 mU/L, and the other characteristics of the assay were similar to those previously reported (13). The accumulation of cAMP was exclusively due to the stimulation of transfected TSH receptor in strain JP-26, as no stimulation was observed when immunopurified samples were tested in the control cells not expressing the TSH receptor (strain JP-02) (28).
FRTL-5 cells. Immunopurified TSH from fetuses and mothers were also tested in the bioassay based on the use of FRTL-5 cells, as previously described (16). The TSH bioassay was performed under conditions similar to those used for CHO-R cells, using hypotonic buffer and the same TSH standard. The sensitivity of the assay in different experiments was 2.2 ± 0.3 mU/L, and the other characteristics of the assay were similar to those previously reported (13, 16).
As our experiments fulfilled the criteria illustrated by Chappel (29), i.e. the same reference preparation was always employed, and parallelism between immunoconcentrated sample dilutions and the TSH standard curve was observed in both immuno- and biological assays, the results of the TSH bioassay are expressed as the biological (B) to immunological (I) ratio (B/I) of immunoconcentrated TSH samples (mean ± SD; n = 9), thus giving an estimation of the biological potency of immunopurified materials.
Ricin lectin affinity chromatography
This investigation was performed as previously described (7). Briefly, columns containing 1 mL Ricinus communis unsolubilized on beaded agarose (RCA 120, Sigma Chemical Co., St. Louis, MO) were equilibrated with phosphate buffer (PB; pH 7.4) and 0.05% BSA. Specimens (25 µL of immunopurified samples and 100 µL PB, pH 6.6) with or without neuraminidase treatment (NAM; 10 mU; 4 h at 37 C) were loaded onto the column and allowed to interact for 1 h at room temperature. Unbound TSH was collected by repeated centrifugation (20 times with 1 mL PB-BSA). Bound fractions were eluted using the same procedure with PB-BSA containing 200 mmol/L galactose (Sigma). Unbound and bound fractions were pooled and dried using a Speed-Vac (JOVAN, Milan, Italy); dried samples were solubilized in 1 mL PB-BSA, and TSH was measured by immunoassay. The final recovery was always above 83%. Ricin lectin specifically binds to exposed galactose or N-acetylglucosamine residues, whereas NAM treatment specifically removes terminal sialic acid, thus exposing the underlying galactose residue. The difference between the percentages of TSH bound to ricin before and after NAM treatment represents the amount of sialylated molecules (7, 12).
Statistical analysis
Data were analyzed by Student’s t test and ANOVA, as appropriate. Differences were considered statistically significant if P < 0.05. Results are expressed as the mean ± SD.
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Results |
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TSH-I and FT4 and FT3 concentrations in the serum samples withdrawn from 26 normal subjects during the morning hours ranged between 0.6–2.4 mU/L (mean ± SD, 1.3 ± 0.5), 10.7–16.0 pmol/L (13.7 ± 1.6), and 4.8–8.0 pmol/L (5.7 ± 0.7), respectively. In the control group, circulating TSH B/I measurements ranged from 0.6–2.2 (1.5 ± 0.5).
Cosinor analysis revealed the presence of a significant TSH circadian
rhythm in all seven cases studied (P < 0.01), with
acrophases ranging between 23.33–3.35 in the night (TSH-I zenith,
1.3–3.7 mU/L), and amplitudes between 19–48% of mesor values (TSH-I
mesor, 0.85–2.7 mU/L). The analysis did not show any significant
increment in free thyroid hormone concentrations in the samples
collected after the nocturnal TSH zenith (data not shown). TSH
molecules purified from daytime serum pools had B/I values (range,
1.6–2.2; mean, 2.0 ± 0.2) significantly higher than those for
nighttime TSH from the same subjects (0.7–2.0; 1.3 ± 0.4,
P < 0.007; Fig. 1), in
all cases but one (TSH B/I: 2.1 ± 0.5 vs. 2.0 ±
0.4. Ricin analysis revealed that 0.1–8.8% (5.9 ± 2.8%) of
daytime TSH bound to the lectin, whereas after NAM treatment,
22.0–36.9% (29.7 ± 6.2%) was bound; the sialylation degree of
TSH molecules circulating in seven euthyroid subjects during the day
thus ranged between 15.0–30.5% (23.8 ± 5.8%; Fig. 1). The
nighttime TSH in the same euthyroid subjects was less retained on the
lectin column (0.1–3.3%; 1.7 ± 1.3%; P < 0.02
vs. day-time), but NAM treatment caused a marked increment
in TSH binding to ricin lectin (24.7–54.3%; 37.5 ± 9.7%); the
degree of sialylation of nighttime TSH was significantly higher
than that in daytime samples (24.7–52.5%; 35.8 ± 9.7%;
P < 0.03; Figs. 1 and 2).
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TSH-I, FT4, and FT3
concentrations in the serum samples from 24 patients with PH ranged
between 12.2–836.0 mU/L (118 ± 164), 0.9–7.5 pmol/L (3.1
± 1.6), and 1.0–4.3 pmol/L (2.0 ± 0.9), respectively. In this
group, circulating TSH B/I measurements were significantly lower than
those in controls (0.35–1.5; 0.7 ± 0.3; P <
0.001; Fig. 1
) despite a 50% overlap with the reference range. In 10
of these patients, 1-month treatment with
L-T4 caused the expected decrement in
TSH-I concentrations that was accompanied by a trend toward
normalization of both parameters: TSH B/I was higher (before:
0.35–1.1; 0.7 ± 0.3; after: 0.6–1.6; 1.1 ± 0.3;
P < 0.01), and the degree of sialylation,
i.e. the difference in hormone binding to ricin lectin
before and after NAM treatment, was lower (before: 33.8–59.1;
45.4 ± 7.6; after: 28.6–50.0; 36.8 ± 7.0%;
P < 0.02; Fig. 1). Taken together, the results
obtained in normal subjects (day- and nighttime samples) and in PH
patients (before and during L-T4
treatment), there was a highly significant inverse correlation between
TSH B/I values and the degree of sialylation of the glycoprotein
hormone (Fig. 2).
Fetuses
TSH-I and FT4 concentrations in the serum
samples from 42 normal fetuses at 31–37 weeks gestation were 2.1–8.8
mU/L (4.1 ± 1.5 mU/L) and 10.5–36.2 pmol/L (16.7 ± 5.2
pmol/L), respectively. In both FRTL-5 and CHO-R bioassays,
immunopurified TSH from the fetuses in the third trimester displayed an
extremely high B/I value (Fig. 1). In contrast, the control pool made
with samples from pregnant women of the same gestational period had a
TSH B/I ratio similar to that observed in nonpregnant adult controls
(1.4 ± 0.4). Ricin analysis revealed the absence of sialylated
TSH isoforms in third trimester fetuses, as the amounts of TSH bound to
ricin before and after NAM treatment were 7.6–8.9% (8.2 ±
0.5%) and 8.3–10.2% (9.2 ± 0.8%), respectively (Fig. 1).
Fetuses with PH or RTH had very high TSH-I levels (PH, 94 mU/L; RTH:
28th week, 277 mU/L; 33rd week, 135 mU/L). FT4
concentrations were low in hypothyroid and RTH fetuses (PH, 1.1 pmol/L;
RTH: 28th week, 8.4 pmol/L; 33rd week, 6.8 pmol/L). TSH B/I ratios in
these fetuses were similar to those in adults (PH, 0.4 ± 0.1;
RTH: 28th week, 1.1 ± 0.4; 33rd week, 1.7 ± 0.3; Fig. 1).
Ricin analysis revealed the presence of several terminal sialic
residues in these immunopurified TSH samples, as NAM treatment caused
increases in TSH bound to the lectin of 28.6–40.8% (34.6 ±
6.1%) and 22.5–31.5% (26.3 ± 3.9%) in PH and RTH,
respectively (Fig. 1).
RTH
Table 1 illustrates the biochemical data observed at baseline as
well as the results of TRß gene analysis in the five RTH patients.
After 15 days of TRIAC treatment, normalization of
FT4 levels and a clear improvement in clinical
manifestations were seen in all cases but one (no. 5 in Table 1,
patient with TRß mutation V264D; Fig. 3). The decrease in TSH-I concentrations
was absent (case 5) or very poor (-26 ± 9%; Fig. 3), whereas
the circulating TSH B/I normalized in all subjects (0.9–1.7; normal,
0.6–2.2), except in patient 5 (2.2 ± 0.1). Bioactive levels of
circulating TSH dramatically fell after 15 days of TRIAC administration
(-51 ± 11%; Fig. 3). Ricin binding analysis showed that TSH
from RTH patients had a higher number of exposed
galactose/N-acetylglucosamine residues (TSH bound before
NAM, 9.3–27.2%; 18.2 ± 6.8) and was more sialylated (TSH bound
after NAM, 42.3–59.2%; 55.2 ± 6.1) than that in controls [TSH
bound before NAM, 5.4 ± 1.5% (P < 0.04
vs. RTH); TSH bound after NAM, 29.6 ± 5.5%
(P < 0.02 vs. RTH)]. TRIAC treatment
resulted in the normalization of the exposed residues before NAM
treatment (<8.8%) in all cases except patient 5 (13.2 ± 1.0%),
whereas the degree of sialylation was unchanged. Percent variations in
bioactive TSH concentrations significantly correlated
(P < 0.05) with those in FT4
levels and with percent modifications of TSH binding to ricin lectin
before NAM treatment (Fig. 4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In normal subjects, we confirm and expand our previous data (16), obtaining evidence that the nocturnal TSH surge is constituted by molecules with a lower bioactivity and a higher sialylation degree than those of TSH molecules circulating in the same subject during the day. As highly sialylated TSH has a prolonged half-life in serum and diminished intrinsic bioactivity (5, 12, 14), the higher sialylation degree of nighttime TSH partially accounts for the lower bioactivity and contributes to the generation and the shape of the nocturnal TSH surge, which is characterized by a steep evening rise followed by a prolonged decline from a nighttime plateau lasting until the late morning hours without any evident stimulation of thyroid hormone secretion (16, 30). Indeed, TSH is secreted in a pulsatile fashion, and increased pulse frequency and amplitude are the main underlying mechanisms believed to give rise to the nocturnal TSH surge (31). The nocturnal secretion of long lasting, highly sialylated TSH isoforms contributes to generation of the nocturnal TSH zenith.
The results obtained in a large group of patients with PH confirm that
this condition is associated with the secretion of highly sialylated
TSH with reduced intrinsic bioactivity (1, 2, 3, 7, 16). Moreover,
L-T4 treatment can partially reverse
such alterations, confirming the important role played, directly at the
pituitary level or indirectly through modifications of TRH secretion,
by thyroid hormone feedback in the regulation of specific enzymatic
activities within thyrotropes (2, 8, 10, 11). The secretion of highly
sialylated, long lasting TSH molecules in primary hypothyroidism may
represent an economical advantage for hypothyroid thyrotrope cells,
perhaps contributing to the prevention of thyrotrope hyperplasia of the
so-called feedback tumor, which is a rare event occurring in severe and
long standing PH (24). We previously reported (16) the lack of a
significant circadian oscillation of serum TSH-I and TSH-B levels in
severe PH; the restoration of a normal circadian rhythm of TSH
secretion and of a significant daynight difference in TSH bioactivity
during L-T4 therapy (16) appears to
be associated with the normalization of -2,6-sialyltransferase
activity within thyrotropes.
The great impact that terminal sialylation has on the biological properties of TSH circulating in normal subjects, during the circadian oscillation of the hormone, or in PH is clearly demonstrated by the inverse correlation between TSH B/I values and the amount of sialylated TSH in each sample.
Fetal TSH, like that circulating during the third trimester of
pregnancy, was known to be poorly sialylated (3, 7), and here we
demonstrate, by means of two different bioassays, that maturation of
the hypothalamic-pituitary-thyroid axis during gestation is accompanied
by the secretion of TSH with extremely elevated bioactivity. This
finding is in agreement with the data obtained several years ago using
intrapituitary TSH from aborted fetuses (32) and might contribute to
the progressive maturation of fetal thyroid and to the marked rise of
fetal FT4 concentrations during the third
trimester despite minor changes in TSH-I concentrations (33). The
secretion of TSH molecules with such unique biological potency might
result from incomplete maturation of the complex enzymatic machinery
within fetal thyrotropes due to the perinatal activation of
hypothalamic TRH neurons (34, 35). Alternatively, these findings might
reveal the existence of a distinct subpopulation of thyrotropes in
human fetal pituitaries similar to that described in the rostral part
of the adenohypophysis in rodents (8). As previously reported in rats
during fetal development (35), the impaired thyroid hormone action
occurring in hypothyroid or RTH fetuses can induce an early expression
of -2,6-sialyltransferase activity and result in the secretion of
high amounts of sialylated TSH isoforms such as adult thyrotropes,
probably through an anticipated activation of hypothalamic TRH neurons.
Despite interference by the concomitant administration of TRIAC to the
mother, a thyroid hormone analog able to cross the placental barrier in
significant amounts (19), the data collected in the RTH fetus strongly
suggest a transient biochemical hypothyroid phenotype of RTH subjects
during intrauterine development. Indeed, the secretion of high amounts
of hyperactive TSH in RTH fetuses would result in the generation of
congenital goiter, which is a rare finding in neonates with RTH.
The results obtained in adult RTH patients extend previous data (25) and confirm that this syndrome is associated with the secretion of TSH isoforms with altered carbohydrate chains and increased bioactivity, thus explaining the presence of goiter and thyroid hypersecretion despite normal TSH-I levels (3, 25). In addition, in the present work we studied the terminal residues of carbohydrate branching of TSH circulating in five patients with PRTH before and after TRIAC treatment. TRIAC has a higher potential than T3 in overcoming the genetic defect of RTH patients (23), i.e. the dominant negative activity exerted by mutated TRß1 on wild-type receptor functions (20, 21, 22). We used low doses of TRIAC, and after 15 days, although the reduction in TSH-I concentrations was negligible, we observed normalization of FT4 circulating levels accompanied by the regression of clinical manifestations in four of five cases. Serum TSH bioactivity decreased significantly during TRIAC administration (51 ± 11%; P < 0.01), and TSH B/I normalization was observed in all cases except patient 5, in whom TRIAC administration did not produce any significant biochemical or clinical effect. These data indicate that during the first weeks of treatment, low doses of TRIAC can reduce thyroid hormone secretion in RTH patients mainly through the secretion of TSH isoforms with changed terminal sugar residues and reduced bioactivity. The patient refractory to TRIAC therapy is bearing a mutation located in the hinge region of TRß1, V264D. The molecular basis of the therapeutic potential of TRIAC in PRTH patients was tested in vitro using TRß isoforms mutated in the two hot spots within the hormone-binding domain, but not in the hinge region of the receptor (23). The observed refractoriness of V264D mutation might indicate a lower efficacy of TRIAC therapy in RTH patients bearing mutations localized in the third hot spot of the receptor. Ricin lectin analysis showed that TSH from patients with RTH has an increased percentage of exposed galactose/N-acetylglucosamine residues and an elevated sialylation degree compared to that in controls. TRIAC treatment caused the normalization of exposed galactose/N-acetylglucosa-mine residues only in the patients in whom the clinical and biochemical improvements were observed (only V264D showing a discrepant pattern), without any variation in hormone sialylation. Although the number of patients was limited, the calculated variations in bioactive TSH induced by TRIAC treatment correlated significantly with the observed changes in TSH terminal residues as well as with those in FT4 concentrations. Thus, ricin analysis produced an important contribution for elucidation of the carbohydrate structure of TSH molecules from patients with RTH: the increased sialylation is similar to that observed in primary hypothyroidism, whereas the high percentage of exposed galactose/N-acetylglucosamine residues makes it similar to the hyperactive molecules circulating during fetal life. Interestingly, we previously showed a relatively high percentage of isoforms that are firmly bound to concanavalin A in both RTH patients and normal fetuses (i.e. the less mature isoforms with high mannose or hybrid carbohydrate structures) (7, 25, 36). Similar to that of TRIAC, T3 administration normalized the circulating TSH bioactivity in RTH patients and was accompanied by a significantly increased percentage of isoforms that are not bound to concanavalin A (i.e. the secreted isoforms bearing mature complex carbohydrate chains) (25); this is probably produced by a resetting of the biosynthetic process within resistant thyrotropes.
In conclusion, our results indicate that changes in the terminal sugar residues have a great impact on the biological properties of circulating TSH and occur in various situations in vivo. They may represent an additional mechanism contributing to adjustment of thyroid-stimulating activity to temporary needs. In fact, in some physiological conditions, such as the nocturnal TSH surge or fetal life, pituitary thyrotropes preferentially secrete particular TSH isoforms. These findings imply the existence of definite mechanisms modulating the biosynthetic process within thyrotropes by acting at either the transcriptional or posttranscriptional level, leading to the regulation of terminal sugar residues. In conditions of increased TSH secretion, the release of highly sialylated isoforms is preferred, as in adult or fetal primary hypothyroidism, but this also occurs during the nocturnal TSH surge, within the normal circadian rhythm of the hormone. The expression and activity of sialyltransferase as well as those of other enzymes involved in earlier steps of the glycosylation process are known to be regulated by thyroid hormone negative feedback and hypothalamic TRH (1, 2, 8, 9, 10, 11, 35), but the data obtained in the study of circadian TSH rhythm indicate that terminal glycosylation can also be modulated by sleep-related mechanisms. Finally, the secretion of TSH molecules with altered terminal sugar residues contributes to the typical phenotype of some diseases, such as RTH. In these patients, all of the hypothalamic-pituitary-thyroid axis is activated to overcome the tissue refractoriness to thyroid hormone action; as 80% of untreated RTH patients have immunoreactive TSH within the normal range (20), the increased stimulation of the thyroid gland is achieved in most cases mainly through the secretion of hyperactive TSH.
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Acknowledgments |
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Footnotes |
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Received January 12, 1998.
Revised March 6, 1998.
Accepted April 7, 1998.
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 R. Frenzel, K. Krohn, M. Eszlinger, A. Tonjes, and R. Paschke Sialylation of Human Thyrotropin Receptor Improves and Prolongs Its Cell-Surface Expression Mol. Pharmacol., October 1, 2005; 68(4): 1106 - 1113. [Abstract] [Full Text] [PDF]
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L. C. Moeller, S. Kimura, T. Kusakabe, X.-H. Liao, J. Van Sande, and S. Refetoff Hypothyroidism in Thyroid Transcription Factor 1 Haploinsufficiency Is Caused by Reduced Expression of the Thyroid-Stimulating Hormone Receptor Mol. Endocrinol., November 1, 2003; 17(11): 2295 - 2302. [Abstract] [Full Text] [PDF]
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R. M. Calvo, E. Jauniaux, B. Gulbis, M. Asuncion, C. Gervy, B. Contempre, and G. Morreale de Escobar Fetal Tissues Are Exposed to Biologically Relevant Free Thyroxine Concentrations during Early Phases of Development J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1768 - 1777. [Abstract] [Full Text] [PDF]
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J. H. A. Oliveira, L. Persani, P. Beck-Peccoz, and J. Abucham Investigating the Paradox of Hypothyroidism and Increased Serum Thyrotropin (TSH) Levels in Sheehan's Syndrome: Characterization of TSH Carbohydrate Content and Bioactivity J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1694 - 1699. [Abstract] [Full Text]
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 D. N. Orth, R. C. Shelton, W. E. Nicholson, P. Beck-Peccoz, A. J. Tomarken, L. Persani, and P. T. Loosen Serum Thyrotropin Concentrations and Bioactivity During Sleep Deprivation in Depression Arch Gen Psychiatry, January 1, 2001; 58(1): 77 - 83. [Abstract] [Full Text] [PDF]
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L. Persani, E. Ferretti, S. Borgato, G. Faglia, and P. Beck-Peccoz Circulating Thyrotropin Bioactivity in Sporadic Central Hypothyroidism J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3631 - 3635. [Abstract] [Full Text]
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C. Asteria, O. Rajanayagam, T. N. Collingwood, L. Persani, R. Romoli, D. Mannavola, P. Zamperini, F. Buzi, F. Ciralli, V. K. K. Chatterjee, et al. Prenatal Diagnosis of Thyroid Hormone Resistance J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 405 - 410. [Abstract] [Full Text]
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, P < 0.02 vs.
baseline in primary hypothyroidism; 
, P < 0.001
vs. normal pool in fetuses. A high degree of sialylation
is always accompanied by a significantly lower TSH B/I.
