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Endocrinology Vol. 139, No. 12 4782-4792
Copyright © 1998 by The Endocrine Society

ARTICLES

Mechanism of Hypothyroidism Action on Insulin-Like Growth Factor-I and -II from Neonatal to Adult Rats: Insulin Mediates Thyroid Hormone Effects in the Neonatal Period1

S. Ramos2, L. Goya, C. Alvarez and A. M. Pascual-Leone

Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad Complutense de Madrid), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain

Address all correspondence and requests for reprints to: A. M. Pascual-Leone, Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: apascual{at}eucmax.sim.ucm.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of thyroid hormone deficiency on serum levels and liver messenger RNA (mRNA) expression of insulin-like growth factors (IGFs) were studied in neonatal (until 20 days of life), weaned (22–37 days), and adult (72–87 days) rats, short periods (5, 10, and 15 days) after thyroidectomy (T). Serum levels and liver mRNA expression of IGF-I, plasma and pituitary GH, plasma insulin, and glycemia were measured in all populations; and serum levels and liver mRNA expression of IGF-II were measured only in the neonatal populations. Surprisingly, plasma insulin and GH and serum and liver mRNA expression of IGF-I were found elevated in T neonatal rats, and they decreased in weaned and adult rats and in neonatal rats rendered hypothyroid by mercapto-1-methylimidazole (MMI) treatment (MMI-hypothyroid). T and MMI-treatment of neonatal rats disturbed the normal pattern of progressive decrease of IGF-II with age. A positive correlation between insulin and IGF-I and a poor correlation between GH and IGF-I were found in both hypothyroid neonates (T and MMI-hypothyroid). On the contrary, a positive correlation between GH and IGF-I and a poor correlation between insulin and IGF-I were found for control and T adult rats. Because plasma insulin and GH changed in the same direction in all groups, insulin secretion in T neonatal was suppressed by streptozotocin, and insulin was given to T adult rats. The combined results of these experiments support the idea that the effects of thyroid hormones on IGF-I secretion are age-dependent, and they are mediated mainly by insulin during the neonatal period and by GH during adulthood.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID hormones play a fundamental role in the initiation and maintenance of somatic growth in mammalian species. Thyroid hormones affect nearly all aspects of metabolism; the overall result of their action is a stimulation of catabolic and oxidative pathways. Hyperthyroidism increases both lipid and carbohydrate oxidation, usually at the level of synthesis of key regulatory enzymes or by modifying the actions of other regulatory hormones (insulin, glucagon, catecholamines) (1). The consequences of hypothyroidism on such metabolic and endocrine pathways depend on the degree of reduction of circulating thyroid hormones. T4 and GH are involved in the growth and differentiation of the skeleton (2, 3). Both thyroid hormone and GH are essential for normal growth, but the plasma levels and pituitary content of GH are regulated by thyroid hormones, as shown in vivo (4, 5) and in vitro (6), because thyroid hormones transcriptionally regulate GH gene expression (7). On the other hand, the concept of a GH/insulin-like growth factor (GH/IGF) axis, as the major mechanism mediating the effects of GH, has been well defined (8, 9); thus, the thyroid hormone actions on the IGF system have been explained to be mediated by the direct effect of thyroid hormones on GH secretion.

IGFs are peptide hormones with endocrine, paracrine, and autocrine modes of action (8); and their function is modulated by several types of IGF binding proteins (IGFBPs). Both IGFs and IGFBPs are mostly secreted by the liver, and their secretion is regulated by GH and nutritional status (10, 11). They form a complex regulatory system, which plays an important role in tissue growth and differentiation.

Hypothyroid patients show low plasma levels of IGF-I and reduced IGF bioactivity, whereas hyperthyroid patients present high plasma IGF-I levels and also low IGF bioactivity (12); and similar changes have been observed in rats (13). Besides, a decrease in hepatic IGF-I messenger RNA (mRNA) expression in experimental hypothyroid animals has been reported (14, 15). One of the most intriguing physiological events involving the IGF system occurs during the first 3 postnatal weeks. The fetal serum profile, characterized by high IGF-II and 30-Kda IGFBPs (IGFBP1 and 2), is replaced by the adult-type profile of high IGF-I and IGFBP-3, with a dramatic reduction of IGF-II and IGFBP-1 and 2 levels. These changes are retarded by lack of thyroid hormones (16, 17), and it has been suggested that the influence of thyroid hormones on the IGFs/IGFBPs complex is age dependent (18) and that all thyroid hormone effects on the IGFs system are not GH mediated (19, 20). However, the interrelationships between the thyroid function and pituitary GH/serum IGFs axis are complex and not fully understood. GH treatment does not restore serum IGF-I levels in hypothyroid rats (13). Decreased serum IGF-I levels in hypophysectomized rats increase after treatment with T4 doses in vivo, in a way significantly greater than after GH administration (21), an effect which is not observed in vitro, suggesting the presence of factors, other than GH, involved in the regulation of this axis in vivo.

The regulation of the IGF system is dependent on the nutritional status (22). Besides, recent work has strongly suggested that, during the neonatal period of the rat, the IGF/IGFBP system in the liver is regulated by an insulin/nutrients balance, rather than GH (23, 24). Thus, insulin and nutrient status might play an interesting role in the regulation of the IGF axis in a complex endocrine and metabolic situation, such as thyroid dysfunction, a role that has not been thoroughly considered in previous work in the field. The present study has two goals: 1) to explain the different effect of thyroid hormones on IGFs secretion, depending on the developmental stage of the animal; and 2) to investigate the role of other hormones likely involved, such as insulin. To achieve these goals, two different experiments were carried out. In the first experiment, use was made of three populations of rats thyroidectomized at three different stages of life: neonatal (at 5 days of life), weaned (at 22 days of life), and adult rats (at 72 days of life); and all compared with their intact sham-operated controls. Animals were killed 5, 10, and 15 days after thyroidectomy (T); and blood glucose, T3, T4, GH, insulin, IGF-I and -II, and liver IGF-I and -II mRNA expression were determined. A subgroup of neonatal rats was submitted to a milder hypothyroidism with mercapto-1-methylimidazole (MMI) (MMI-hypothyroid); and serum IGF-I and -II, insulinemia and liver IGF-I and -II mRNA expression were determined. In the second experiment of this study, neonatal rats, thyroidectomized as in the first experiment, were treated with streptozotocin (STZ) to suppress the insulin secretion; and thyroidectomized adult rats with low serum insulin were given insulin doses to assess the role of this hormone in the effect of thyroid hormones on serum levels and liver mRNA expression of IGFs. Similar parameters, as in the first experiment, were also evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IGF-I (Boehringer Mannheim, Leverkusen, Germany) was used as standard and for iodination. Ribonuclease (RNase) A and RNase T1 were also purchased from Boehringer Mannheim. STZ was a kind gift of Upjohn Co. (Kalamazoo, MI). MMI was obtained from Sigma Chemical Co. (St. Louis, MO). Na125I and Hyperfilm-MP autoradiography film were obtained from Amersham (Amersham Ibérica SA, Madrid, Spain). Polyclonal antiserum (lot no. K9147–48) raised in rabbit against human IGF-I and the C-terminal fragment (residues 57–70) was purchased from KabiGen AB (Stockholm, Sweden). (32P)Uridine 5'-triphosphate was purchased from ICN (Nuclear Ibérica SA, Madrid, Spain). Riboprobe Gemini II Core System (Promega Corp., Madison, WI) was used for the generation of RNA probes. Lente insulin was kindly supplied by Novo Nordisk A/S Pharma SA (Madrid, Spain).

Animals
Wistar rats, bred in our laboratory with controlled temperature and artificial dark-light cycle, were used throughout the study. Females were caged with males, and mating was confirmed by the presence of spermatozoa in a vaginal smear. Each dam was housed individually from the 14th day of pregnancy. After birth, the number of pups in each litter was evened out to eight, and males and females were used in equal numbers. Animals were fed a standard laboratory diet ad libitum (19 g protein, 56 g carbohydrate, 3.5 g lipid, and 4.5 g cellulose/100 g plus salt and vitamin mixtures). Water was given ad libitum. T was performed under ether anesthesia and control rats were sham operated. To prevent a possible hypocalcemia from loss of the parathyroid glands after T, 1% calcium lactate was added to the drinking water of experimental and control rats. Rats were weighed daily. Blood from rats was harvested from the trunk after decapitation. Blood samples were allowed to clot on ice for 30 min, and serum was separated and stored at -80 C until assayed. Livers were frozen in liquid N2 upon removal for RNA extraction.

All experiments were conducted in accordance with the principles and procedures outlined in the NIH (Bethesda, MD) guide for care and use of experimental animals.

Experimental models
Exp 1. Three populations of rats were analyzed: neonatal, weaned, and adult rats (N, W, and A in Fig. 1Go). Neonatal rats were divided into three groups: T5, MMI-hypothyroid, and C (control rats) (Fig. 1Go). T5 rats received MMI (0.02% wt/vol, added to the drinking water of the mother) from day 2 of life; then they were thyroidectomized on day 5 and killed at 10, 15, and 20 days of life (5, 10, and 15 days after T). MMI was given from day 2 of life, to assure that T3 and T4 were adequately reduced; MMI-hypothyroid rats were made hypothyroid from day 2 of life by MMI (0.05% wt/vol, added to the drinking water of the mother) until they were killed at days 10, 15, and 20 of life. The MMI dose was increased in MMI-hypothyroid rats to provoke a similar degree of hypothyroidism as in the thyroidectomized group.



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  		Figure 1. Exp 1: Three populations of rats were used: neonatal (N), weaned (W), and adult rats (A). N rats were divided into three groups: T5, MMI-hypothyroid, and C. T5 rats, receiving MMI (0.02% wt/vol drinking water of mother) from day 2 after birth (B), were thyroidectomized on day 5 of life. MMI-hypothyroid rats received only MMI (0.05% wt/vol to drinking water of mother) from day 2 after birth. C, control sham-operated rats. N rats were killed (S) at 10, 15, and 20 days of life. W rats were divided into two groups: T22 and C. T22 rats, receiving MMI (0.02% wt/vol through milk of mother from day 15) and thyroidectomized on day 22. W rats were killed (S) at 27, 32, and 37 days of life. A rats were divided into two groups: T72 and C. T72 rats, adults receiving 0.02% wt/vol of MMI in drinking water from 65 days of life, were thyroidectomized (T) at 72 days. C, Control rats. A rats were killed (S) at 77, 82, and 87 days of life. Exp 2: Neonatal rats were divided into two groups: T5 + STZ, and C. T5 + STZ rats, receiving MMI (0.02% wt/vol in water of mother) from day 2 after birth (B) and thyroidectomized (T) at 5 days of life, were given STZ (70 mg/kg BW) on day 15 to suppress insulin secretion. C, Control sham-operated rats. N rats were killed (S) at 20 days of life. A rats were divided into two groups: T72 + I, and C. T72 + I rats received MMI (0.02% wt/vol to the drinking water) from day 65 of life, were thyroidectomized (T) at 72 days and then treated sc with 3 IU/100 g BW daily of insulin at 82 days of life, and were killed (S) at 87 days of life. C, Control rats killed also at 87 days of life.

 
Weaned rats were divided into two groups: T22 and C (Fig. 1Go); T22 rats received 0.02% wt/vol MMI (added to the drinking water, from day 15) and were thyroidectomized on day 22, then killed at 27, 32, and 37 days of life (5, 10, and 15 days after T, respectively), together with their controls.

Adult rats were divided into two groups: T72 and C (Fig. 1Go); T72 rats received 0.02% wt/vol MMI (added to the drinking water, from day 65) and were thyroidectomized on day 72, then killed at 77, 82, and 87 days of life (5, 10, and 15 days after T), together with their controls.

Exp 2. Neonatal rats were divided into two groups: T + STZ, and C (Fig. 1Go). T + STZ rats received 0.02% wt/vol MMI (added to the drinking water, from day 2 of life) and were thyroidectomized on day 5 of life. To suppress insulin secretion, they were ip injected with STZ (70 mg/kg BW) in 0.05 M citrate buffer/liter (pH 4.5) on day 15 of life. The suppression of insulin secretion was confirmed 3 days later by the determination of glycemia and insulinemia. Both groups were killed at 20 days of life. Adult rats were divided into two groups: T + I, and C (Fig. 1Go). T + I rats received 0.02% wt/vol MMI (added to the drinking water, from day 65 of life), were thyroidectomized at 72 days, then treated sc with 3 IU/100 g BW daily of Lente insulin (porcine-bovine) in two doses (0900 and 1800 h) at 82 days of life, and killed on day 87. Glycemia and insulinemia were measured to assess insulin function. C rats were killed on day 87 (Fig. 1Go).

Serum glucose, T3 and T4, and plasma insulin and GH determinations
Glucose was determined with a Reflolux IIM (Boehringer Mannheim) glucose analyzer (25). Serum T3 and T4 were determined at Centro de Investigaciones Biomédicas (CSIC) by highly specific RIAs, previously described by Weeke and Orskov (26), and modified for rat plasma by Obregón et al. (27). The minimal detectable doses in plasma were 2.5 pg for T4 and 0.7 pg for T3/assay tube. Plasma immunoreactive insulin was estimated with purified rat insulin as standard (NOVO, Denmark), antibody to porcine insulin, which cross-reacted similarly with pork and rat insulin standards, and monoiodinated 125I-labeled human insulin. The minimal detectable dose was 0.04 ng/ml, with a coefficient of variation, within and between assays, of 10%. Plasma and pituitary GH were determined using the reagents generously distributed by the National Hormone and Pituitary Program of the NIDDK, NIH (recombinant GH Standard RP-2). The minimal detectable dose in pituitary homogenates and serum was 0.03 ng/ml GH. To prevent circadian variations in blood, samples were obtained at the same hour (between 1000 and 1200 h) any day, from 6–8 animals. Pituitaries were also collected and were rapidly frozen in liquid nitrogen for determination of GH by RIA.

Iodination, purification, and determination of serum IGF-I AND IGF-II
Recombinant human IGF-I and IGF-II were labeled by a modified chloramine-T method (23). The specific activity achieved with this method was approximately 90–175 µCi/µg for both peptides. Before IGF-I and -II determination, serum IGFBPs were removed by standard acid gel filtration. This method has proved to be the most reliable one for use with rat serum (23). The RIA for IGF-I and rat liver membrane receptor assay for IGF-II were carried out as previously described (23). The coefficients of variation, within and between assay, were 8.0% and 12.4%, respectively.

Preparation of RNA
Total RNA was prepared by homogenization of livers in guanidinium thiocyanate, as originally described (28). Samples were electrophoresed through 1% agarose, 2.2 M formaldehide gels and were stained with ethidium bromide to visualize the 28S and 18S ribosomal RNA and thereby confirm the integrity of the RNA and normalize the quantity of RNA in the different lanes. A ß-actin probe (0.6 kb EcoRI/HindIII fragment isolated from the VC18 vector kindly provided by Dr. P. Martín-Sanz from the Instituto de Bioquímica, CSIC, Madrid, Spain) was used in a Northern blot assay to validate the ethidium bromide method for loading normalization.

Riboprobes
Rat IGF-I and IGF-II complementary DNAs (cDNAs) were kindly provided by Dr. E. Hernández (Instituto de Bioquímica, CSIC), Dr. C. T. Roberts, and Dr. D. LeRoith (NIH, Bethesda, MD). Rat IGF-I cDNA, ligated into a pGEM-3 plasmid, was linearized with HindIII, and an antisense riboprobe was produced by T7 RNA polimerase, generating two protected fragments of 224 bases (Ia) and 386 bases (Ib). Rat IGF-II cDNA, ligated into a pGEM-3 plasmid, was linearized with HindIII and incubated with T7 RNA polimerase, to generate a riboprobe that recognized a protected fragment of 500 bases (29). The above riboprobes were synthesized with (32P)uridine 5'-triphosphate.

Solution hybridization/RNase protection assay
Solution hybridization/RNase protection assays were performed as previously described (23). Briefly, 20 µg liver total RNA were hybridized with 500,000 cpm of the 32P-labeled riboprobes, described above, for 18 h at 45 C in 75% formamide and 400 mM NaCl. After RNase digestion with a buffer containing 40 µg RNase A/ml and 2 µg RNase T1/ml for 1 h at 37 C, protected RNA-RNA hybrids were resolved on denaturing 8% polyacrylamide and 8 M urea gels. Autoradiography was performed at -70 C against a Hyperfilm MP film between intensifying screens. Bands representing protected probe fragments were quantified using a Molecular Dynamics, Inc. scanning densitometer and accompanying software.

Statistical analysis
All data are presented as means ± SD. Statistical comparisons were performed by one-way ANOVA, followed by the protected least-significant-difference test (30). Linear regression analysis was used to correlate serum IGF-I values with serum insulin and GH. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
Neonates. Table 1Go shows the expected increase of body weight with age in control and hypothyroid (thyroidectomized and MMI-hypothyroid) populations. Body weight of hypothyroid rats was decreased vs. control rats, starting 5 days after T (10 days of life) and also at 10 days of life in MMI-hypothyroid rats. In all stages, body weight reduction was greater in thyroidectomized, than in MMI-hypothyroid rats, when both were compared with control rats. Similarly, serum T3 and T4 were diminished in both hypothyroid groups vs. controls; no significant differences were observed when both hypothyroid groups were compared. MMI-hypothyroid rats showed reduced levels of plasma and pituitary GH vs. controls in all stages except at 20 days of life, when plasma GH was similar to control values. At 10 and 20 days, plasma and pituitary GH in MMI-hypothyroid were lower than in thyroidectomized animals (Table 1Go). Surprisingly, an increase in plasma GH was observed in thyroidectomized rats, 5 and 15 days after T; besides, an increase in pituitary GH was found at 5 and 15 days after T vs. control values. Plasma insulin was increased in thyroidectomized rats and decreased in MMI-hypothyroid rats vs. controls (Fig. 2Go), a similar pattern of changes to the one found for serum IGF-I, which was elevated in thyroidectomized and reduced in MMI-hypothyroid rats vs. controls (Fig. 2Go). Increased plasma insulin was followed by a low serum glucose in thyroidectomized neonates (Table 1Go), whereas serum glucose in MMI-hypothyroid was unchanged, compared with controls, but was higher than in the thyroidectomized population at all stages studied.


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  		Table 1. Body weight, serum T3 and T4, plasma and pituitary GH content, and glycemia of three groups of neonatal rats

 


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  		Figure 2. Serum levels of IGF-I and plasma insulin in neonatal rats. Populations were as follows: thyroidectomized at 5 days of life, T5 (see Fig. 1Go); C control rats; MMI, treated from 2 days with MMI 0.05% wt/vol (see MMI-hypothyroid in Fig. 1Go). All three groups studied were killed at 10, 15, and 20 days of life (D). These ages for T rats represented 5, 10, and 15 days after T (aT). A and B, Means ± SD. Number of animals: 8–10. *, P < 0.05, relative to control rats; {blacktriangleright}, P < 0.05, relative to thyroidectomized rats.

 
Figures 2Go and 3Go show a positive correlation between serum IGF-I and liver IGF-I mRNA expression in both groups of hypothyroid animals (r = 0.92, P < 0.05). Statistical analysis of densitometric results shows an increase of liver IGF-I mRNA in thyroidectomized rats at days 5, 10, and 15 after T (10, 15, and 20 days of life), whereas a decrease of IGF-I mRNA was found in liver of MMI-hypothyroid rats vs. control rats, only on day 15 of life.



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  		Figure 3. Liver mRNA expression of IGF-I in neonatal rats, by RNase protection assay, and optical density of bands, shown as arbitrary units (5–6 assays each point of different neonatal rats (T, C, and MMI). Means ± SD. T, Rats thyroidectomized at 5 days of life (see T5 in Fig. 1Go); C, controls; MMI, rats treated with 0.05% wt/vol (see MMI-hypothyroid in Fig. 1Go). Days when killed are shown at the bottom of plots. For T rats, these days represent 5, 10, and 15 days after T (aT). *, P < 0.05, relative to control rats; {blacktriangleright}, P < 0.05, relative to thyroidectomized rats.

 
Because IGF-II is the most abundant IGF in mammalian prenatal stages, serum levels and liver mRNA expression of IGF-II were evaluated in neonatal populations at all stages, and the results are shown in Fig. 4Go, A and B. Reduced serum levels and IGF-II mRNA expression in liver of thyroidectomized rats were observed 5 and 10 days after T, compared with controls. On the contrary, increases in liver IGF-II mRNA expression were found in MMI-hypothyroid (MMI) rats on day 15, and increases in serum levels and liver mRNA expression of IGF-II were found at 20 days of life in both groups, thyroidectomized (T) and MMI-hypothyroid (MMI). These results show a parallel variation between serum levels and liver mRNA expression of IGF-II in both neonatal populations.



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  		Figure 4. A, Serum levels of IGF-II in neonatal rats. Number of animals: 8–10. *, P < 0.05, relative to control rats; {blacktriangleright}, P < 0.05, relative to thyroidectomized rats. B, Liver mRNA expression of IGF-II in neonatal rats, by RNase protection assay, and optical density of bands, shown as arbitrary units (6–7 assays each point of different neonatal rats T, C, and MMI); T, thyroidectomized at 5 days of life (see T5 in Fig. 1Go); C, controls); MMI, treated with 0.05% wt/vol (see MMI-hypothyroid in Fig. 1Go). Days when killed for A and B are shown at the bottom of plots. For T rats, these days represent 5, 10, and 15 days after T (aT). Mean ± SD. *, P < 0.05, relative to control rats; {blacktriangleright}, P < 0.05, relative to thyroidectomized rats in A and B.

 
Weaned and adult rats. Thyroidectomized rats, both weaned and adult, showed a reduction of body weight vs. controls at all stages studied and a parallel decrease of plasma T3 and T4 (Tables 2Go and 3Go). In weaned rats, pituitary GH was decreased at 5 and 15 days after T, and plasma GH was decreased at 15 days after T (Table 2Go). Plasma and pituitary GH were very reduced in thyroidectomized adult rats vs. controls, starting from day 5 after T (Table 3Go). The percent decrease of pituitary GH, 5 days after T, was 35% in weaned and 54% in adults, and this difference was over 65% in both groups 15 days after T (Tables 2Go and 3Go). Plasma insulin was decreased in weaned rats 5 days after T but not 10 and 15 days after T. In adult rats, a decrease in plasma insulin vs. control values was observed at 10 and 15 days after T. Serum glucose was inversely correlated to plasma insulin in all cases (data not shown).


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  		Table 2. Body weight, serum T3 and T4, plasma insulin and GH, and pituitary GH content of two groups of weaning rats

 

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  		Table 3. Body weight, serum T3 and T4, plasma insulin, and GH and pituitary GH content of two groups of adult rats

 
Figure 5AGo shows reduced serum IGF-I in hypothyroid weaned and adult rats. The decreased IGF-I mRNA expression in liver of the same rats, at all stages after T, is shown in Fig. 5BGo. Figure 6Go shows regression lines and correlation coefficients between serum IGF-I and plasma insulin and GH in thyroidectomized (T) and control neonatal and adult rats. A strong positive correlation is observed between serum IGF-I and plasma insulin in T neonatal rats (r = 0.83, P < 0.05) and controls (r = 0.96, P < 0.05). Both thyroidectomized and control neonatal rats present a negative (r = -0.58) or a slight correlation (r = 0.75), respectively, between plasma GH and IGF-I, but a high correlation is found between plasma GH and IGF-I in both thyroidectomized (r = 0.91, P < 0.05) and control (r = 0.99, P < 0.05) adult rats. A higher correlation between serum IGF-I and plasma insulin (y = 8.72 + 5.09x, r = 0.85, P < 0.05) than between serum IGF-I and plasma GH (y = 44.39 + 45.58x, r = 0.46) was also observed in MMI-hypothyroid rats. However, a poor or negative correlation was found between insulin and serum IGF-I in thyroidectomized and control adult rats (r = 0.47 and r = -0.36, respectively) (Fig. 6Go). Results similar to those found in adults were obtained between IGF-I and insulin in thyroidectomized (r = -51, P < 0.05) and control (r = 0.40, P < 0.05) weanling rats. On the contrary, a high correlation between serum IGF-I and GH levels was found for adults and for thyroidectomized (0.94, P < 0.05) and control (0.96, P < 0.05) weanling rats. It is worth noting that correlations between serum IGF-I and serum insulin or GH found for adult rats were very similar to those found for weanling rats.



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  		Figure 5. Serum concentration (A) and liver mRNA expression levels (B) of IGF-I in weaned and adult rats thyroidectomized (T) and controls (C). Weaned and adult rats were thyroidectomized at 22 days and 72 days of life, respectively, and killed at 5, 10, and 15 days after T (aT), corresponding to 27, 32, and 37 days of life in weaned and 77, 82, and 87 days of life (D) in adults (see Fig. 1Go). A, Mean ± SD serum IGF-I. Number of animals: 6–8. B, RNase protection assays and optical density of bands shown as arbitrary units (7–8 assays each point of different animals). *, P < 0.05, relative to control rats for (A and B); D, days.

 


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  		Figure 6. Relationships among serum IGF-I and plasma insulin and GH in thyroidectomized (T) and control (C) neonatal and adult rats. Data were pooled in neonatal rats, from five litters each, at 10, 15, and 20 days of life. Data in adult rats were pooled from each 8–12 animals from 77, 82, and 87 days of life. Regression lines and correlation coefficient are shown. Statistical differences (P < 0.05) were found in both control and T rats between insulin and IGF-I in the neonatal period and between GH and IGF-I in adults.

 
In conclusion, thyroidectomized neonates (T5) showed high plasma insulin, elevated plasma and pituitary GH, and high serum levels and liver mRNA expression of IGF-I vs. controls. On the contrary, MMI-hypothyroid neonates presented low plasma insulin and GH and decreased serum levels and liver mRNA expression of IGF-I. However, a positive correlation between circulating IGF-I and plasma insulin was found in both neonatal hypothyroid populations. Thyroidectomized adult rats (T72) showed low plasma insulin, low plasma and pituitary GH, and decreased serum levels and liver mRNA expression of IGF-I vs. controls on day 87 of life (Table 3Go and Fig. 5Go). Interestingly, in adult hypothyroid rats, a positive correlation was found between serum IGF-I and plasma GH (Fig. 6Go). Similar changes were obtained in weanling rats, although the results for this group were less conclusive (Table 2Go). Nonetheless, in the three hypothyroid populations (neonatal, weaned, and adult), plasma insulin and plasma GH changed in the same direction as serum IGF-I. Thus, to further investigate the role of insulin mediating IGF-I secretion in hypothyroid conditions, a second experiment (in which insulin secretion in neonatal rats is suppressed by administration of STZ, and insulin is given to adult rats) was carried out, and the results are shown in Table 4Go and Figs. 7Go and 8Go.


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  		Table 4. Serum T3, T4, and pituitary GH content of neonatal and adult rats thyroidectomized at 5 (T5) and 72 (T72) days of life and treated with STZ (T5 + STZ) or insulin (T72 + I) at 15 and 82 days of life, respectively

 


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  		Figure 7. A, Serum IGF-I and -II of neonatal rats thyroidectomized on day 5 (T), treated with STZ on day 15 (T + STZ), and serum IGF-I serum levels in adult thyroidectomized on day 72 (T), treated with insulin on day 82 (T + I) (see Fig. 1Go, T5 + STZ, and T72 + I, Exp 2). Rats were killed on day 20 (neonates) or 87 (adults). Values are mean of 8–10 animals ± SD. B, Serum insulin and GH in same populations as in panel A killed at same days. Values are mean of 8–10 animals ± SD. *, P < 0.05, relative to controls; {blacktriangleright}, P < 0.05, relative to thyroidectomized rats for A and B.

 


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  		Figure 8. Liver mRNA expression of IGF-I (A) and IGF-II (B) in neonatal rats and liver mRNA expression of IGF-I in adult rats (C). mRNA expression levels were assayed by RNase protection assay, followed by densitometric measurement of bands, shown as arbitrary units (5–6 assays each point of different neonatal animals). Populations were: Panels A and B: T rats, neonatal rats thyroidectomized on day 5 of life (see T5, in Fig. 1Go); C, control rats; T + STZ, same as T rats (see above) but given STZ from day 15 of life (see Fig. 1Go, Exp 2). All three neonatal groups were killed at 20 days of life. Panel C: T rats, adult rats thyroidectomized at 72 days of life (see T72 in Fig. 1Go); C, control rats; and T + I rats, same as T (see above) but treated from day 82 on with 3 IU/100 g BW daily of Lente insulin in two doses (see T72 + I, Fig. 1Go). All three groups of adult animals were killed at 87 days of life. Means ± SD. *, P < 0.05, relative to control rats; {blacktriangleright}, P < 0.05, relative to only thyroidectomized adult rats for panels A–C.

 
Exp 2
Treatment of thyroidectomized neonates (T5) with STZ on day 15 (T5 + STZ) maintained the plasma GH level above that of controls, decreased pituitary GH and plasma insulin, and decreased serum IGF-I vs. controls at 20 days of life (Table 4Go, Fig. 7Go). Although plasma GH and pituitary GH content decreased in T5 + STZ vs. T5 (P < 0.05, Fig. 7Go, Table 1Go), a bigger reduction was found in plasma insulin, which was above control values in T5 at 20 days (Fig. 7Go) and decreased well below control levels after STZ treatment (Fig. 7Go). But plasma T3 and T4 values remained below those of control after STZ (Table 4Go). Insulin treatment of thyroidectomized adult rats (T72), starting on day 82 (T72 + I), increased plasma and pituitary GH above values observed in T72, P < 0.05 (Table 3Go), but the values were still lower than those of controls (Fig. 7Go, Table 4Go). Plasma insulin in T72 + I was higher than both T72 (P < 0.05, Table 3Go) and controls (Fig. 7Go), and serum IGF-I was lower than in T72, P < 0.05, (Table 3Go) and controls (Fig. 7Go). Plasma T3 and T4 values remained below those of controls (Table 4Go). In agreement with these results a positive correlation between serum IGF-I and plasma insulin (y = -90.95 + 12.85x, r = 0.97, P < 0.05) and a negative correlation between serum IGF-I and plasma GH were obtained for T5 + STZ (y = 76.38 - 0.41x, r = -0.83). In the T72 + I group, a positive correlation between serum IGF-I and plasma GH was observed (y = 49.02 + 16.78x, r = 0.98, P < 0.05). These results support the role of insulin, rather than GH, mediating the effect of thyroid hormones on IGF-I secretion in neonatal stages of development, contrary to what happens in the adult period, when such an effect is mostly dependent on circulating GH.

Figure 8Go, A and B, and Fig. 7AGo show the parallel variation between liver IGF-I and -II mRNA expression and serum values of IGF-I and -II, both in T5 (T) and T5 + STZ (T + STZ) neonatal rats. Elevated liver IGF-I mRNA expression and serum IGF-I in thyroidectomized neonates vs. controls were both decreased below control values after STZ treatment. However, STZ treatment increased liver mRNA expression and serum levels of IGF-II. On the contrary, Fig. 8CGo shows that diminished liver mRNA expression of IGF-I transcripts in adult thyroidectomized rats increased after insulin treatment but still remained below control values. The parallel variation between circulating levels (Fig. 7AGo) and liver mRNA expression of IGF-I in adults (Fig. 8CGo), and of IGF-I and -II in neonates, strongly suggest transcriptional regulation of the system in these populations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is increasing evidence to suggest that thyroid hormones are intimately involved in the regulation of the GH/IGF axis at a number of levels. Although thyroid hormones have direct actions on the pituitary, both by altering the number of somatotroph cells (31) and influencing expression of the GH gene (7), all thyroid hormone effects on IGFs/IGFBPs do not seem to be GH-mediated (19, 20, 21). Indeed, GH (and not insulin) has been the target of most studies as the putative factor mediating the effects of thyroid hormones on the IGF/IGFBP system (13, 19, 32), despite the facts that: 1) thyroid hormones affect most aspects of metabolism by modifying hormones, such as insulin and catecholamines (33); and 2) IGF secretion is regulated by the metabolic and nutritional status (10, 11). In the present study, rats were thyroidectomized to achieve a maximal reduction of thyroid hormone levels and, moreover, to prevent differential effects of goitrogen drugs on plasma insulin in rats (34, 35). The results from this study, obtained a few days after T, show the short-term effects of highly reduced levels of thyroid hormones on different parameters, analyzed from neonatal to adult stages, and point out the fact that the effects of thyroid function on the IGF/IGFBP system depend on the developmental life stage of the animals, as previously reported in other experimental models (17, 18).

Plasma T3 and T4 and body weight severely decreased in all three hypothyroid populations (neonatal, weanling, and adult), and barely detectable values of thyroid hormones were found 5 days after T; these results are in agreement with those of Coiro et al. (36) and Walker and Dussault (37). Both in weanling, and more clearly in adult rats, body weight reduction takes place along with a decrease of thyroid hormones and serum and pituitary GH. On the contrary, the body weight decrease in neonatal thyroidectomized rats was not accompanied with a reduction of plasma GH or serum IGF-I; these results support the crucial role of thyroid hormones in the regulation of growth during immature stages of development, as previously described in infant hypo-physectomized rats, and in contrast with the limited effectiveness of T4 in promoting growth in older hypophysectomized rats (38). In weanling rats, we found a decrease of plasma and pituitary GH, 15 days after T, in agreement with other authors who have previously reported such a decrease 10 days after surgery (36); but in adult rats, we report a reduction of plasma and pituitary GH, 5 days after T. A possible explanation for this difference is the fact that the hypothalamic regulatory mechanism for pituitary GH release develops during the neonatal period in rats (39) and, probably, responsiveness of GH synthesis to thyroid hormone matures throughout the neonatal period and becomes fully established at adulthood. Glydon (40) demonstrated that the primary plexus of the median eminence is not formed in rats until the 5th postnatal day and that SRIF concentration rises markedly during the neonatal period, to attain peak values at 28 days of life (39). This immaturity of the endocrine system could explain the fact that neonatal rats on day 10 of life (5 days after T) showed an unexpected increase of plasma and pituitary GH and plasma insulin vs. controls, which remained until day 20 of life (15 days after surgery), contrary to what was observed in adults. A parallel study of neonatal rats treated with MMI, discussed later, was necessary to confirm the differences found between neonates and adults.

A direct correlation between plasma insulin and GH is found in all populations, but both parameters increased in thyroidectomized neonatal rats, whereas they decreased in weanling rats 15 days after T, and in T adult rats at all stages. In agreement with the fact reported in this study (that both GH and insulin change in all hypothyroid groups in the same direction), thyroid hormone deprivation in adult animals results in a decrease of circulating levels of plasma insulin and GH (41, 42), and diabetes in adults is characterized by an inhibitory effect on GH secretion, whereas insulin treatment restored normal plasma and pituitary GH (43). Furthermore, the parallel changes in plasma insulin and GH, as well as the concentrations of pituitary GH observed in this study, support the value of plasma variations of GH obtained at a single time point, because it is well known that GH secretion is episodic and follows a circadian rhythm (44). Notwithstanding, the mechanism by which thyroid hormones contribute to the regulation of glucose and insulin homeostasis is a complex subject (45). Experimental hypothyroidism may be accompanied by normal, increased, or decreased basal plasma insulin (45), and variations can be explained by the different metabolic adaptation to thyroid hormone deprivation, depending on the stage of development, because SRIF rises during the neonatal period (39) and exerts an inhibitory influence on insulin secretion (46). Lenzen et al. (47) have found increased insulin secretion from the isolated pancreas in rats after T, as we report for neonatal rats. To investigate whether the increase of GH and insulin in neonatal rats can simply be explained by the immaturity of the animal or whether adaptation to a situation of absence of thyroid gland is also involved, a population of MMI-hypothyroid rats was assayed at the same stages as thyroidectomized rats. MMI-hypothyroid rats showed very low levels of thyroid hormones, together with decreased plasma and pituitary GH and plasma insulin. This result suggests that, in immature stages of the hypothalamic system, the imbalance provoked by thyroid withdrawal increases plasma insulin and GH; ß adrenergic stimulation increases plasma insulin concentration in the euthyroid organism (48), and treatment of rats with glucocorticoids increases basal insulinemia (49, 50), but the study of the consequences of adaptations to surgical stress under a situation of absence of thyroid hormones remains a subject for further investigation. Besides, the possible effect per se of goitrogens on plasma insulin (34, 35) could play a role in the reduced insulin observed in MMI-hypothyroid rats. This different adaptive process of GH and insulin, short periods after T, between neonates and adults is currently being studied in our laboratory. However, the comparative study of two neonatal populations, rendered hypothyroid by different means, has made it possible to find clear differences between both groups: thyroidectomized neonates show increased serum levels and liver mRNA expression of IGF-I in the presence of high plasma insulin, contrary to the reduction found in all three parameters in MMI-hypothyroid rats. Although IGF-I reduction in MMI-hypothyroid neonatal animals has been reported previously (16, 17, 19), insulin values were never determined and, therefore, correlation with IGF-I was never calculated in such conditions. The high positive correlation between circulating insulin and IGF-I in neonatal stages was confirmed by the linear regression analysis, where a poor correlation between plasma GH and IGF-I was also found (Fig. 6Go). Similar correlations between insulin, IGF-I, and GH were observed in MMI-hypothyroid rats, which supports previous results showing that a balance of insulin and nutrients, rather than GH, regulates secretion of IGF-I in immature stages of development (23, 24).

Both T and MMI treatment disturb the normal pattern of progressive decrease of IGF-II with age, shown by control rats (51), but differences between the two groups were found that can be explained by the effect of blood glucose on IGF-II regulation previously suggested in immature rats (23), because blood glucose was reduced in thyroidectomized rats but not in MMI-hypothyroid rats.

Weanling and adult hypothyroid rats in this study show decreased serum values and liver mRNA expression of IGF-I at all time points, together with a reduction of insulin and GH, in agreement with results previously reported (52). Linear regression analysis in these stages (weanling and adult) shows a high positive correlation between plasma GH and serum IGF-I and a very poor correlation for insulin. However, the decrease of plasma and pituitary GH and plasma insulin after T was faster in adult than in weanling rats, suggesting weanling as an intermediate stage in the development of this regulatory system between immaturity (neonatal period) and adulthood. All results, taken together, lead to the conclusion of an age-dependency of the effect of thyroid hormones on IGF secretion, as previously suggested for IGFBPs (17, 18). This time dependency seems to be caused not only by the immaturity of endocrine systems to adaptation but also by a different regulatory pathway between both stages. Nonetheless, to confirm our hypothesis and to rule out the putative regulatory role of GH (which changes, together with insulin) in Exp 2, insulin secretion in thyroidectomized neonatal rats was prevented by administration of STZ, and thyroidectomized adults were treated with insulin.

As expected, thyroidectomized neonates treated with STZ (T55 + STZ) showed reduced plasma insulin, 5 days after starting STZ treatment (day 20 of life); but the plasma GH level remained above that of controls (Fig. 7Go), and pituitary GH started to decrease (Table 4Go), as reported in diabetic situations (41). In these conditions (reduced thyroid hormones, elevated plasma GH, and reduced plasma insulin vs. controls), a decrease of serum IGF-I was observed, and a positive correlation between insulin and IGF-I in T5 + STZ was obtained (r = 0.98, P < 0.05). However, STZ treatment evoked an increase of serum levels and mRNA expression of IGF-II in thyroidectomized neonates, suggesting, as previously mentioned for MMI-hypothyroid neonates (see above), the regulatory role of glycemia on IGF-II. On the contrary, administration of insulin to adult thyroidectomized rats increased plasma insulin over control values and reduced serum IGF-I and pituitary GH content, compared with untreated hypothyroid rats. The data presented strongly suggest that insulin, not GH, mediates thyroid hormone effects on IGF-I secretion in neonatal stages of development; however, as the neuroendocrine system matures and connections are established, GH gradually takes over, to mediate thyroid effects on IGF-I in adulthood. The complex regulation of the thyroid hormone/IGF system will benefit from the study of IGFBPs in these populations and experiments of hypothyroidism recovery with T4 administration, both currently in progress in our laboratory.

We firmly believe that the role of insulin, which had been neglected so far, should be considered when studying the relationship between thyroid function and IGF-I in immature stages of development. The precise role of insulin in a mature organism when IGF-I regulation by GH is fully established remains a subject of further investigation.


   Acknowledgments
 
We are grateful to the National Hormone and Pituitary Program for the supply of immunoreactants for the determination of rat GH, as well as the Upjohn Co. for supplying STZ, and NOVO Nordisk Pharma SA for supplying Lente Insulin. The authors especially thank Dr. Morreale de Escobar and the member of her laboratory Socorro Durán and M. Jesús Presas for plasma T3 and T4 determination.


   Footnotes
 
1 This work was supported by Grant PB940030-A from DGICYT (Ministerio de Educación y Ciencia) Spain and Grant 08.50009/97 from CAM (Comunidad Autónoma de Madrid). Back

2 Supported by a fellowship from Conserjería de Educación y Cultura from CAM. Back

Received February 13, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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