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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 256-263
Copyright © 2005 by The Endocrine Society

A Polymorphism in Type I Deiodinase Is Associated with Circulating Free Insulin-Like Growth Factor I Levels and Body Composition in Humans

Robin P. Peeters, Annewieke W. van den Beld, Hans van Toor, Andre G. Uitterlinden, Joop A. M. J. L. Janssen, Steven W. J. Lamberts and Theo J. Visser

Departments of Internal Medicine (R.P.P., A.W.v.d.B., H.v.T., A.G.U., J.A.M.J.L.J., S.W.J.L., T.J.V.), Clinical Chemistry (A.G.U.), and Epidemiology and Biostatistics (A.G.U.), Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Robin P. Peeters, M.D., Department of Internal Medicine, Room Ee 502, Erasmus University Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: r.peeters{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The interaction between the GH-IGF-I axis and thyroid hormone metabolism is complex and not fully understood. T4 stimulates IGF-I activity in animals in the absence of GH. On the other hand, GH replacement therapy results in an increase in serum T3 and a decrease in T4 and rT3 levels, suggesting a stimulation of type I deiodinase (D1) activity. Recently, we demonstrated the association of two polymorphisms in D1 (D1a-C/T; T = 34%, and D1b-A/G; G = 10%) with serum iodothyronine levels. Haplotype alleles were constructed, suggesting a lower activity of the D1 haplotype 2 allele (aT-bA) and a higher activity of the haplotype allele 3 (aC-bG). In this study, we investigated whether genetic variations in D1 are associated with the IGF-I system.

In 156 blood donors and 350 elderly men, the association of the D1 haplotype alleles with circulating IGF-I and free IGF-I levels was studied. In addition, potential associations with muscle strength and body composition were investigated in the elderly population. Finally, the relation between serum iodothyronine levels and IGF-I levels was studied.

In blood donors, haplotype allele 2 was associated with higher levels of free IGF-I (302.9 ± 22.9 vs. 376.3 ± 19.1 pg/ml, P = 0.02). In elderly men, haplotype allele 2 also showed an allele dose increase in free IGF-I levels (Ptrend = 0.01) and an allele dose decrease in serum T3 levels (Ptrend = 0.01), independent of age. Carriers of the D1a-T variant also had a higher isometric grip strength (P = 0.047) and maximum leg extensor strength (P = 0.07) as well as a higher lean body mass (P = 0.03).

In blood donors, T4 and free T4 were negatively correlated with total IGF-I levels (R = –0.18, P = 0.03 and R = –0.24, P = 0.003), whereas T3 to T4 and T3 to reverse T3 ratios were positively correlated with total IGF-I (R = 0.31, P < 0.001 and R = 0.18, P = 0.03). Free IGF-I showed a negative correlation with T4 (R = –0.26, P = 0.001) and T4-binding globulin (R = –0.31, P < 0.001) and a positive correlation with T3 to T4 ratio (R = 0.21, P = 0.01).

In conclusion, a polymorphism that results in a decreased D1 activity is associated with an increase in free IGF-I levels. The pathophysiological significance of this association with IGF-I is supported by an increased muscle strength and muscle mass in carriers of the D1 haplotype 2 allele in a population of elderly men. The association of D1 haplotype allele 2 with serum T3 levels in the elderly population suggests a relative increase in its contribution to circulating T3 in old age.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE INTERACTION BETWEEN the GH-IGF-I axis and thyroid hormone metabolism is complex and not fully understood. The anabolic effects of GH are mediated by IGF-I. Free IGF-I by analogy with sex and adrenal steroids and thyroid hormones may be the major biologically active hormonal form of IGF-I (1). GH replacement therapy in GH-deficient subjects acutely increases serum T3 levels, with a decrease in serum T4 and rT3 (2, 3, 4, 5). This suggests a stimulatory effect of GH and/or IGF-I on the activity of type I deiodinase (D1), the enzyme mainly responsible for the production of serum T3 from T4 and the clearance of rT3 (6). Type II deiodinase (D2) in skeletal muscle may also contribute to serum T3 production (6).

On the other hand, euthyroidism is essential for normal growth and development of many tissues (7, 8, 9). The effects of thyroid hormone on growth have been explained by its ability to promote the secretion of GH because it is required for a normal GH expression, both in vitro (10, 11, 12) and in vivo (13). However, not all effects of thyroid hormone on the IGF-I system are mediated by GH because thyroid hormone itself also interacts with IGF-I (14, 15, 16). Treatment of hypophysectomized or thyroidectomized rats with T4 results in a stimulation of IGF-I activity in the absence of GH (14, 15), and the administration of GH to hypothyroid rats or humans fails to reverse growth impairment unless T4 is administered concurrently (16).

Previous studies reported various alterations of the GH-IGF-I axis during hyper- and hypothyroidism. Hypothyroid adults are reported to have low concentrations of IGF-I (17, 18, 19, 20), with a restoration to normal after euthyroidism is restored. Hyperthyroid patients are reported to have normal (21, 22) or high levels of IGF-I (18, 23, 24, 25). Normalization of thyroid function results in a decrease in IGF-I (18, 24, 25) or does not alter IGF-I (21). However, free IGF-I levels do not alter during hyperthyroidism (25).

Recently we observed effects of two genetic single nucleotide polymorphisms (SNPs) in the D1 gene (D1a-C/T and D1b-A/G) on plasma thyroid hormone levels, suggesting a lower D1 activity in subjects with the D1a-T variant and a higher D1 activity in subjects with the D1b-G variant (26). In this study, we investigated the relation of D1 polymorphisms with circulating IGF-I levels as well as body composition and muscle strength in elderly men. Furthermore, we investigated the correlation between serum thyroid hormone levels and both total and free IGF-I levels in healthy subjects. The ratios of the different iodothyronines were also analyzed because these ratios better reflect peripheral thyroid hormone metabolism.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study populations

Healthy blood donors. Blood was collected from 158 healthy anonymized blood donors at the Sanquin Blood Bank South West Region (Rotterdam, The Netherlands) (26). Informed consent was given by all donors. One subject was excluded because of serum free T4 (FT4) and TSH levels indicating hyperthyroidism, another because of hypothyroid FT4 and TSH levels. Gender was not documented for one subject. The mean age of the study population was 46.2 ± 12.2 yr (mean ± SD) [47.4 ± 10.9 yr in the males (n = 100) and 44.6 ± 13.9 yr in the females (n = 55)]. Donors on thyroid hormone treatment were not excluded from blood donation. Descriptive statistics of this population are shown in Table 1Go. DNA was extracted from 2 ml of blood using the PUREGENE DNA isolation kit (Gentra Systems, Minneapolis, MN) with slight modifications of the provided protocol. After isolation, DNA concentration was measured at 260 nm, and all samples were diluted to a concentration of 50 (stock) and 10 ng/µl (work solution). Purity was determined by measuring the 260:280 nm ratio.


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  		TABLE 1. Population characteristics

 
Healthy elderly men. DNA was available for 350 subjects of a cross-sectional, single-center study in 403 independently living men, 70 yr of age and higher (27). In this study, names and addresses of all male inhabitants 70 yr and older were drawn from the municipal register of Zoetermeer, a medium-sized town in the midwestern part of The Netherlands. A total of 1567 men were invited, and after exclusion of subjects who did not live independently and subjects who were not physically or mentally able to visit the study center independently, eventually 403 men participated (25.7%). Participants signed an informed consent. The study was approved by the Medical Ethics Committee of the Erasmus Medical Center Rotterdam. (See Ref.27 for a more detailed description of this population.) Twenty-six subjects in this population were not of Caucasian origin, the majority coming from Indonesia. Height and weight were measured in standing position without shoes. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. Furthermore, lean body mass and fat mass were measured by dual-energy x-ray absorptiometry (28, 29). Quality assurance for dual-energy x-ray absorptiometry, including calibration, was performed every morning, using the standard provided by the manufacturer.

Isometric grip strength (IGS) was measured using an adjustable handheld dynamometer (JAMAR dynamometer) at the nondominant hand (30). Each test was repeated three times, and the average was used in the analyses. Leg or knee extensor strength (LES) was measured as described previously (31), using the Hoggan MicroFET handheld dynamometer. To obtain one main outcome measurement for LES, maximum LES (maxLES) was defined as the maximum strength for the right or left leg, whichever is largest, in a position of 120-degree extension. Statistical analyses were based on the physical unit momentum (Nm), obtained by multiplying the maximum strength (in newtons) and the distance of the dynamometer to the knee joint (in meters). See Table 1Go for the descriptives of this population. Five of the 350 subjects received thyroid hormone replacement therapy; 28 of them had diabetes.

Serum and plasma analyses

Healthy blood donors. In this population, all hormone measurements were performed in EDTA plasma. T4, FT4, T3, and TSH were measured by chemoluminescence assays (Vitros ECI Immunodiagnostic System, Ortho-Clinical Diagnostics, Amersham, UK). rT3 was measured by RIA as previously described (32). T4-binding globulin (TBG) was measured using chemoluminescence assays on an Immulite 2000 (Diagnostic Products Corp., Los Angeles, CA). Plasma free IGF-I was measured with a commercially available, noncompetitive, two-site immunoradiometric assay (Diagnostic Systems Laboratories, Veghel, The Netherlands) (33). Plasma total IGF-I was measured by an IGF binding protein (IGFBP)-blocked RIA (Medgenix Diagnostics, Fleurus, Belgium), as described previously (34); the intraassay and interassay coefficients of variation were less than 11%.

Healthy elderly men. Blood samples were collected in the morning after an overnight fast. Serum was separated by centrifugation and stored at –40 C. TSH was measured using an immunometric technique (Amerlite TSH-30, Ortho-Clinical Diagnostics). Serum T4, T3, and rT3 were all measured by RIA, FT4 by Amerlite MAB FT4 assay (Ortho-Clinical Diagnostics). TBG was also measured by RIA. Intra- and interassay variability coefficients of all the assays were less than 11%. Free IGF-I and total IGF-I were measured similarly as described for the blood bank donors.

Genotyping

Healthy blood donors. PCR-restriction fragment length polymorphism (RFLP) procedures were developed for the D1a-C785T and D1b-A1814G polymorphisms (26) (Fig. 1Go). The primers used are listed in Table 2Go. Twenty nanograms of genomic DNA was amplified in a PCR with a total volume of 10 µl. The PCR mixture contained 1x PCR buffer (Invitrogen, Breda, The Netherlands), 0.2 mM of each deoxynucleotide triphosphate, 1.5 mM MgCl, 2 pmol of each primer, and 0.5 U Taq polymerase (Invitrogen). Annealing temperatures of the different PCRs are listed in Table 2Go. Five units of restriction enzyme were used for a 1-h digestion of the PCR product at the recommended temperature. Table 2Go lists the restriction enzymes used for RFLP analysis of the two polymorphisms. Digestion products were analyzed by agarose gel electrophoresis. All subjects were genotyped for the D1a-C/T and D1b-A/G polymorphisms using RFLP.



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  		FIG. 1. Location of the two different polymorphisms in the D1 gene and the four haplotype alleles that were constructed from these polymorphisms. Frequencies of the haplotype alleles in the two different populations are shown. Size of the different exons is indicated by their scale. The coding sequence is represented by {cjs2108}, whereas {cjs2113} indicates the untranslated region. The UGA codon, coding for selenocysteine, is depicted by {triangledown}. Finally, the selenocysteine insertion sequence element is indicated by {zeg001051127sr01}.

 

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  		TABLE 2. Conditions used for RFLP and SBE analysis (see Subjects and Methods)

 
The genotypes, determined by RFLP, were confirmed by single base extension (SBE) analysis (26). PCR products were generated using the same primers as used for RFLP analysis (Table 2Go) and the same conditions as described above. The SBE reactions were performed using the ABI Prism SNaPshot dideoxynucleotide primer extension kit (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands) with slight modifications of the protocol provided by the manufacturer. For SBE analysis of the D1a polymorphism, the reverse primer was used, and for the D1b polymorphism, the forward primer was used.

Healthy elderly men. All subjects were genotyped for the two polymorphisms using only SBE as described above.

Statistical analysis

Data were analyzed using SPSS 10.0.7 for Windows (SPSS Inc., Chicago, IL). Logarithmic transformations were applied to normalize variables and minimize the influence of outliers if applicable. Differences between the genotype groups were adjusted for age, sex, and TBG levels if appropriate and tested by analysis of covariance using the general linear model procedure. Pearson’s correlation coefficients were used to calculate correlations between IGF-I and thyroid hormone metabolites after correction for age and if necessary for sex and/or TBG. The effects of the polymorphisms on serum indices of thyroid function were analyzed after the exclusion of the subjects on thyroid hormone treatment (n = 5). The age-dependent changes in thyroid hormone levels are often determined or accompanied by a poor health status, and there is an increased prevalence of nonthyroidal illness in the elderly (35, 36). For this reason, the association of the polymorphisms with serum thyroid hormone levels was reanalyzed after the exclusion of all subjects with nonthyroidal illness (T3 < 88 and rT3 > 20.8 ng/dl, n = 53). Because of the interaction of diabetes and antidiabetic drugs with IGF-I metabolism, the effect of the different polymorphisms on IGF-I metabolism was analyzed after the exclusion of all subjects with diabetes (n = 28). Results are reported as mean ± SE in the figures and as mean ± SD in the tables. Deviation from Hardy-Weinberg equilibrium was analyzed using a {chi}2 test. All genotype distributions were in Hardy-Weinberg equilibrium. Frequencies of the genotypes were the same in the Caucasian and the non-Caucasian population. P values are two-sided throughout, and P < 0.05 was considered significant. Haplotype allele frequencies were estimated using the computer program 3LOCUS.pas (37).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Association of SNPs in D1 with serum and plasma levels

Healthy blood donors. Previous haplotype analysis of the D1a and D1b polymorphisms revealed only three different haplotype alleles in this population (26) (1 = aC-bA, 2 = aT-bA, 3 = aC-bG), with a frequency of 0.56, 0.34, and 0.10, respectively) (Fig. 1Go). We demonstrated that the haplotype 2 allele was associated in a dose-dependent manner with higher rT3 and lower T3/rT3 levels, suggesting a lower D1 activity in subjects with this haplotype allele (26). Haplotype allele 3 showed an opposite relation. To increase statistical power, subjects that were heterozygous and homozygous for a specific haplotype allele were combined as carriers and analyzed vs. subjects without this allele (noncarriers). The haplotype allele 2 showed a significant, positive relation with free IGF-I levels. Mean free IGF-I levels were 73.4 pg/ml lower in subjects without allele 2, compared with subjects with allele 2 (302.9 ± 22.9 vs. 376.3 ± 19.1, P = 0.02, 95% confidence interval –133.1 to –13.8) (Fig. 2Go). Adjustment for the effect of haplotype allele 2 on plasma thyroid hormone levels, or for TBG levels, did not alter the effect of this haplotype allele on circulating free IGF-I levels. No effect of allele 2 was observed on total IGF-I levels.



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  		FIG. 2. Differences in plasma-free IGF-I levels between noncarriers and carriers (n = 90) of the D1 haplotype 2 allele in a population of 156 blood donors. To convert to picograms per milliliter, multiply by 7.649.

 
Due to the lower frequency of the D1b polymorphism, no subjects homozygous for haplotype allele 3 were present in this population. No relation was observed between allele 3 and plasma-free IGF-I levels. However, plasma total IGF-I levels were 32.1 ng/ml (4.2 nmol/liter) lower in subjects without allele 3, compared with subjects with a copy of this allele (P = 0.04, 95% confidence interval –63.4 to –1.8 ng/ml). After adjustment for the effect of allele 3 on plasma thyroid hormone levels, its effect on total IGF-I levels remained significant.

Healthy elderly men. The T variant of D1a had a frequency of 33.5% in this population, whereas the G variant of D1b had a frequency of 11.4%. Again, haplotype alleles were estimated using the computer program 3LOCUS.pas, and all subjects were genotyped based on their different haplotype alleles (26, 37). In this population, only one subject with haplotype allele 4 (Fig. 1Go) was present. In line with a decreased D1 activity in subjects with haplotype allele 2, a negative effect of this allele was observed on serum T3 levels, independent of age, with evidence for linearity (Ptrend = 0.01) (Fig. 3Go). No effect of this haplotype allele was observed on other serum thyroid hormone parameters or their ratios.



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  		FIG. 3. Differences in serum T3 levels between subjects with no, one, or two copies of the D1 haplotype 2 allele in a population of 350 elderly men. To convert the values from moles per liter to nanograms per deciliter, multiply by 65.1.

 
The haplotype allele 2 showed a significant, positive allele dose response with free IGF-I levels (Ptrend = 0.01) (Fig. 4AGo) but not with total IGF-I. Haplotype allele 3 showed an opposite trend on serum-free IGF-I levels (Ptrend = 0.08) (Fig. 4BGo) but had no effect on total IGF-I or T3 levels.



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  		FIG. 4. Differences in serum-free IGF-I levels between subjects with no, one, or two copies of the of the D1 haplotype 2 allele (A) or the D1 haplotype 3 allele (B) in a population of 350 elderly men. To convert to picograms per milliliter, multiply by 7.649.

 
IGF-I levels measured in plasma are low, compared with IGF-I measured in serum (38, 39). Therefore, absolute values between this population and the population of elderly men are different and should not be compared.

After the exclusion of all subjects with nonthyroidal illness (combination of a serum T3 < 88 and rT3 > 20.8 ng/dl, n = 53), the association of haplotype allele 2 with free IGF-I and T3 levels remained significant (P = 0.048 and P = 0.003, respectively).

Association of SNPs in D1 with physical characteristics

Healthy elderly men. Carriers of the haplotype allele 2 had a higher lean body mass (50.9 ± 0.4 vs. 52.1 ± 0.4 kg, P = 0.03, adjusted for age) (Table 3Go). BMI and fat mass were not different between carriers and noncarriers of haplotype allele 2 (Table 3Go). Haplotype allele 3 showed no effect on lean body mass. Lean body mass showed a significant negative relation with serum T3 levels (R = –0.16, P < 0.01) but not with total or free IGF-I levels.


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  		TABLE 3. Physical characteristics of haplotype allele 2 noncarriers and carriers

 
Muscle strength was also different between carriers and noncarriers of haplotype allele 2. Subjects with one or two copies of allele 2 had a higher IGS (33.6 ± 0.5 vs. 35.0 ± 0.5 kP, P = 0.047) and a higher maxLES (100.8 ± 1.6 Nm vs. 104.7 ± 1.5, P = 0.07, adjusted for age). Haplotype allele 3 did not show any association with muscle strength in this group of elderly men.

Correlation between thyroid hormone parameters and plasma or serum IGF-I levels

Healthy blood donors. Total and free plasma IGF-I levels were adjusted for age. T4 and FT4 both showed a significant, negative correlation with total IGF-I levels (R = –0.18, P = 0.025, and R = –0.25, P = 0.002, respectively), whereas the T3 to T4 and T3 to rT3 ratios showed a significant, positive correlation with plasma total IGF-I levels (R = 0.302, P < 0.001, and R = 0.180, P = 0.026, respectively) (Fig. 5Go). T3, rT3, TSH, and rT3 to T4 ratio did not show any significant correlation with total IGF-I levels. Separate analysis of men and women produced similar results (data not shown).



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  		FIG. 5. Correlation of circulating total IGF-I levels with the T3 to T4 ratio in 156 healthy blood donors. To convert the IGF-I values from nanomoles per liter to nanograms per milliliter, multiply by 7.6490.

 
Free IGF-I showed a negative correlation with T4 (R = –0.26, P = 0.001) and TBG (R = –0.31, P < 0.001) and a positive correlation with T3 to T4 ratio (R = 0.21, P = 0.01). Adjusted for TBG, no correlation between thyroid hormone levels and free IGF-I was observed.

Healthy elderly men. Even in this selected, elderly population, total IGF-I levels were significantly correlated with age (R = –0.104, P = 0.037). Adjusted for age, serum T3 was positively correlated with total serum IGF-I levels (R = 0.13, P = 0.013), whereas TSH, T4, FT4, rT3, and their ratios did not show a significant correlation with total serum IGF-I levels.

Serum free IGF-I was not correlated with age or serum TBG. Free IGF-I levels were not correlated with serum thyroid parameters or their ratios.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We previously demonstrated that two SNPs in D1 are associated with differences in rT3 concentrations in healthy blood donors but not with serum T3 levels (26). Haplotype analysis suggested that the haplotype 2 allele results in decreased activity of D1, whereas the haplotype 3 allele results in an increased activity of D1 (26). Because both SNPs are located in the 3'-untranslated region of the mRNA, a change in the stability of the mRNA is an attractive explanation of their effect. An alternative explanation may be an altered folding of the mRNA, which is necessary for the incorporation of selenocysteine in the catalytic center of the protein (6). Alternatively, the SNPs could be in linkage disequilibrium with other SNPs located in the coding sequence or regulatory areas of the gene. In both populations investigated, a significant association of the haplotype allele 2 was observed with free IGF-I levels. Carriers of the haplotype allele 2, with a supposedly lower activity of D1, have higher levels of free IGF-I, whereas no effect of this allele is observed on total IGF-I levels. Because IGF-I has a stimulatory effect on D1 (5), these high levels of free IGF-I could be seen as an adaptation to normalize D1 activity. On the other hand, thyroid hormone has been reported to stimulate IGFBP-1 expression in human hepatoma cells in vitro (40). Although IGFBP-1 is far less abundant than IGFBP-2 and IGFBP-3, it can account for the greatest changes in free IGF-I levels because it is usually unsaturated and can vary widely, compared with IGFBP-2 and IGFBP-3 (41). IGFBP-1 is mainly produced in the liver (42), and a lower activation of thyroid hormone by liver D1 could result in a lower level of IGFBP-1 and thus a higher level of free IGF-I. No significant effect of haplotype 2 on circulating IGFBP-1 was observed in these subjects (data not shown), but this does not exclude an effect on intracellular hepatic IGF-I.

Free IGF-I may be the major biologically active form of IGF-I (1), and the effect of the D1 haplotype 2 allele on free IGF-I levels was further supported by its effects on several IGF-I-related end points. Carriers of this D1 haplotype allele had a significantly higher lean body mass, IGS, and a borderline significantly higher maxLES. This D1 genotype effect seems to be mediated by an effect on skeletal muscle because fat mass and BMI were not different among carriers and noncarriers of the D1 haplotype allele 2. Part of this effect could also be mediated by the effect of haplotype allele 2 on thyroid hormone metabolism because T4 and T3 showed a negative correlation with lean body mass in this population (van den Beld, A. W., R. A. Feelders, D. E. Grobbee, H. A. P. Pols, S. W. J. Lamberts, and T. J. Visser, manuscript in preparation). Both lean body mass and muscle strength are known to be influenced by physical activity. Based on the analysis of two questionnaires and a test for physical performance, we have no reason to believe that physical activity was different between the groups (data not shown).

GH replacement studies in GH-deficient adults suggest that GH stimulates D1 (2, 3, 4, 5) and that part of this stimulatory effect could be mediated by IGF-I (5). Liver D1 plays a key role in the production of plasma T3 from T4 and in the breakdown of the metabolite rT3, but D2 in skeletal muscle may also contribute to serum T3 production (6). The negative correlations we observed in healthy blood donors between IGF-I and T4 and FT4, and the positive correlation of IGF-I with T3/T4 and T3/rT3 are completely in line with a stimulatory effect of IGF-I on liver D1. In elderly men we observed a significant, positive correlation of IGF-I with serum T3, which is also in line with a stimulatory effect of IGF-I on D1 but different from the correlations observed in healthy blood donors. This might be explained by differences in the relative contribution of liver D1 and skeletal muscle D2 to serum T3 production in these different populations (see below). A negative correlation was observed between levels of free IGF-I and TBG in blood donors. This may represent a general biological effect of IGF-I on hormone binding proteins because IGF-I has been shown to inhibit corticosteroid-binding globulin and SHBG expression in HepG2 cells (43). However, free IGF-I was not correlated with TBG in elderly men.

Several changes in thyroid hormone concentrations occur during aging: TSH, T3, and FT3 levels show an age-dependent decline, T4 and FT4 levels remain unchanged, whereas rT3 levels increase with age (36). No relation of the polymorphisms in D1 with circulating rT3 levels was observed in the elderly population. In a recent study in critically ill patients, increased levels of rT3 were accompanied by an increased D3 activity (44), and perhaps variations in rT3 production by D3 in elderly subjects mask the effects of D1 on rT3 levels. However, D1 haplotype allele 2 showed an allele dose effect on serum T3 concentrations in elderly men, resulting in lower levels of T3 in carriers of the D1 haplotype 2 allele. This relation remained the same if all subjects with nonthyroidal illness were excluded from the analysis. Haplotype allele 3 showed an opposite relation, which failed to reach significance. Because D1 produces serum T3, this is in line with our hypothesis of a decreased D1 activity in carriers of the haplotype 2 allele but different from what would be expected based on animal studies. Studies on C3H mice show that these mice, which have only 10–20% D1 activity, have normal levels of circulating T3 that can be partially explained by increased levels of plasma T4 (45). Moreover, the contribution of D1 to serum T3 may be even lower in humans than in rodents, which lack D2 in skeletal muscle (6). The different associations found between the two populations may be explained by the difference in age between these populations (means 46 vs. 77 yr). In young subjects, a decreased T3 production by D1 may be masked by the production of T3 by skeletal muscle D2. Throughout adult life, skeletal muscle size and strength gradually decline (46), resulting in a decrease in D2 expressing skeletal muscle, which is believed to contribute to serum T3 production (6). Furthermore, rT3 levels increase with age and degradation of the D2 protein is accelerated when it is exposed to its own substrates T4 and rT3 (47). Because serum rT3 levels are much lower than serum T4 levels, the relative contribution of rT3 to this substrate-induced degradation should be very low, compared with T4. Although D1 activity also decreases during aging (48, 49), the relative contribution of D2 to serum T3 production may be less important in elderly than young subjects, resulting in a relatively greater contribution of D1 to serum T3 production at advanced ages.

In conclusion, we show the association of polymorphisms in D1, which result in a decreased D1 activity, with higher levels of free IGF-I in two different populations. These data are supported by the observation that carriers of this polymorphism have a higher lean body mass and muscle strength, with a similar fat mass and BMI. Furthermore, we report that this polymorphism in D1 is associated with serum T3 concentrations in elderly men. This association is in agreement with our previous data in younger blood donors but might suggest a more important role for D1 in serum T3 production in the elderly.


   Acknowledgments
 
We thank Fang Yue for help with the RFLP analysis, Wendy Hugens for help with DNA isolation, and Pascal Arp for help with the SNaPshot analysis.


   Footnotes
 
This work was supported by ZonMw Grant 920-03-146 (to R.P.P.).

First Published Online October 13, 2004

Abbreviations: BMI, Body mass index; D1, type I deiodinase; D2, type II deiodinase; FT4, free T4; IGFBP, IGF binding protein; IGS, isometric grip strength; LES, leg or knee extensor strength; maxLES, maximum LES; RFLP, restriction fragment length polymorphism; SBE, single base extension; SNP, single nucleotide polymorphism; TBG, T4-binding globulin.

Received July 9, 2004.

Accepted September 23, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Janssen JA, van der Lely AJ, Lamberts SW 2003 Circulating free insulin-like growth-factor-I (IGF-I) levels should also be measured to estimate the IGF-I bioactivity. J Endocrinol Invest 26:588–594[Medline]
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