The effect of thyroid hormone on fibronectin messenger ribonucleic acid levels in primary cultured rat hepatocytes.

Our previous in vivo studies demonstrated that thyroid hormone promotes the expression of the fibronectin (FN) gene in the rat liver, while it inhibits the synthesis in cultured human skin fibroblasts. These results can be interpreted as either different regulation of FN synthesis or gene expression among tissues, or divergent results of experiments performed in vivo or in vitro. Here we report on the action of thyroid hormone on FN gene expression in vitro using primary cultured hepatocytes compared to that in cultured skin fibroblasts. Hepatocytes were isolated from hypothyroid rats and were cultured in medium supplemented with thyroidectomized bovine serum (TxBS) or fetal bovine serum (FBS). T3 was added 2 or 24 h after plating, and cells were harvested after 2, 6, or 24 h. Total RNA was extracted, and mRNAs for rat FN and albumin were measured. The requirement of de novo protein synthesis for thyroid hormone-mediated induction of FN mRNA was examined by the addition of cycloheximide 15 min before T3 addition. The amount of FN mRNA significantly decreased in the hepatocytes cultured with TxBS compared with those cultured with FBS. The addition of T3 to TxBS resulted in the restoration of FN mRNA to the level in hepatocytes cultured in FBS. FN mRNA increased during the course of culture in the absence of T3; however, a further increase was observed 6 h after T3 addition. The abundance of albumin RNA decreased during the course of culture, but unlike FN mRNA, it was not changed by T3 addition. The increase in FN mRNA by T3 was not influenced by cycloheximide. These results indicate that thyroid hormone enhances FN gene expression in hepatocytes by its direct action without requiring de novo protein synthesis. In contrast, T3 decreased FN mRNA in cultured skin fibroblasts. Thus, the mode of thyroid hormone action on FN gene expression is different among tissues.     

Effect of thyroxine administration on the IGF/IGF binding protein system in neonatal and adult thyroidectomized rats

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.     

Insulin-like growth factor binding protein expression in the hypothyroid rat is age dependent.

Thyroid hormone is essential for normal growth and development. For certain T4 effects, there is a critical period during ontogeny when normal T4 levels are required, and thyroid replacement after that period cannot correct the changes in hypothyroid animals. We have previously described a prolonged high expression of serum insulin-like growth factor binding protein (IGFBP)-2 during the perinatal period in congenitally hypothyroid rats. To see if this effect was confined only to a certain period during rat ontogeny, we made rats hypothyroid with methimazole treatment either prenatally, or at different postnatal ages from 1 to 14 days of life, and at adult age. Serum IGF-I levels were reduced by approximately 30% in all the 18-day-old hypothyroid animals, and did not correlate with the duration of the hypothyroid state. Serum IGF-I levels in the adult animals were 50% of control levels. At the age of 18 days, control animals had only very low levels of IGFBP-2 demonstrable by western ligand blotting, whereas the congenitally hypothyroid animals had elevated levels. Pups placed on methimazole treatment since the first day of life showed higher IGFBP-2 levels at the age of 18 days, although the change was not as prominent as in the congenitally hypothyroid animals (200% vs. 500% of control levels, respectively). Binding protein changes were approximately 2-fold at the mRNA level. Rats started on methimazole after the first 5 days of life showed normal low levels of IGFBP-2 at the age of 18 days. Abnormal IGBFP-2 expression in congenitally or neonatally hypothyroid animals could be corrected by thyroid hormone replacement, if started during the first week of the life, but not later. In adult hypothyroid animals, there was no induction of IGFBP-2 expression, but the levels of IGFBP-3 and -4 were decreased to 80% and to 30% of control levels, respectively. IGFBP-3 messenger RNA (mRNA) levels were decreased to 50% of control levels but IGFBP-4 mRNA levels were paradoxically increased in the hypothyroid animals. All these changes could be corrected by T4 replacement. In conclusion, there exists a critical period during the perinatal development of the rat, when thyroid hormone is essential for a subsequent normal IGFBP-2 ontogenic pattern. Adult animals show a completely different IGFBP response to hypothyroidism, with a decrease of IGFBP-3 and -4 levels. Thus, the effects of thyroid hormone on IGF-IGFBP axis regulation depend on the developmental stage of the animal.     

Expression of hepatic mRNAs for insulin-like growth factors-I and -II during the development of hypothyroid rats.

The effect of thyroid status on the expression of insulin-like growth factors-I and -II mRNAs in the liver of developing rats has been investigated. Northern blot analyses of the specific mRNA demonstrated the presence of four IGF-II mRNA species which were strongly expressed in fetal liver and progressively declined after birth, becoming undetectable after week 3. This decrease was markedly delayed in the liver of hypothyroid rats. In addition, expression of IGF-I mRNA, absent in fetal liver, began during week 1 after birth and progressively increased with age. This increase was markedly delayed in the liver of hypothyroid rats. The data suggest that thyroid hormones regulate rat development via the co-ordinate expression of hepatic IGF-II and IGF-I mRNAs.     

Modulation of the Insulin-Like Growth Factor System by Chronic Alcohol Feeding

Insulin-like growth factor (IGF)-I is a potent anabolic agent that plays an important role in regulating muscle protein balance. Alterations in one or more of the various components of the IGF system may be in part responsible for the muscle wasting that accompanies chronic alcohol consumption. The purpose of the present study was to characterize changes in the growth hormone-IGF axis produced by chronic alcohol consumption in rats. After 8 weeks of alcohol feeding, the IGF-I concentration was decreased in plasma (31%) as well as in the liver and skeletal muscle (40–50%), compared with pair-fed control animals. In addition, alcohol consumption decreased IGF-I mRNA abundance in liver and muscle (∼50%). IGF-I content in duodenum and kidney, however, was not altered by alcohol feeding. Concomitantly, the relative concentration of IGF binding protein (IG-FBP)-1 was increased in plasma, liver, and muscle of alcohol-fed rats, compared with control values. In contrast, no changes in the plasma concentrations of IGFBP-2, -3, or -4 were detected in alcohol-fed rats at this time point. Previous studies have indicated that elevations in glucocorticoids or decreases in insulin or growth hormone might be responsible for the decrease in IGF-I and/or the increase in IGFBP-1 in other catabolic conditions. However, there was no difference in the plasma concentrations of these hormones between alcohol-fed and control animals in this study. These data indicate that chronic alcohol feeding in rats decreases IGF-I and increases IGFBP-1 in the circulation and in skeletal muscle and that these changes appear to be independent of changes in classical hormonal regulators of the IGF system. The observed alterations in the IGF system are consistent with a reduction in the anabolic actions of IGF-I induced by chronic alcohol consumption.     

甲状腺功能低下对大鼠肝细胞核T3受体的影响

目的探讨甲低大鼠肝核Ｔ３受体特性的变化,为阐明甲状腺激素对肝细胞核Ｔ３受体的作用提供实验资料。方法用０．５％ＮａＣｌＯ４诱导大鼠甲状腺功能低下,采用该室建立起来的Ｔ３和离体肝细胞核结合实验的方法,观察甲状腺功能低下对大鼠肝核Ｔ３受体的影响。结果甲状腺功能低下大鼠肝核Ｔ３受体的亲合常数（Ｋａ）增加,最大结合容量（ＭＢＣ）明显下降,Ｔ３受体占位率与对照组无显著差别,且ＭＢＣ与血清Ｔ３、Ｔ４呈正相关。结论肝核Ｔ３受体的数目受甲状腺激素的调节与控制,甲状腺激素对肝脏的作用主要通过调节核Ｔ３受体的结合容量,而非通过改变受体占位率来实现的     

Regulation of insulin-like growth factor-binding protein messenger ribonucleic acid levels in sheep thyroid cells.

The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology.     

Regulation of a thyroid hormone-dependent protein by growth hormone: the induction of hepatic alpha 2U globulin and its messenger ribonucleic acid in adult male hypothyroid rats

Studies in the adult male hypothyroid rat, a known GH-deficient animal, have shown hepatic alpha 2U-globulin mRNA to be dependent on thyroid hormones. To study the effects of GH on alpha 2U-globulin synthesis in the absence of thyroid hormones, adult male rats were rendered hypothyroid before hormone treatment. The relative effects of bovine GH or T3 were studied by RIA of alpha 2U-globulin in hepatic cytosol in rats 6 weeks after thyroid ablation. alpha 2U-Globulin levels in vehicle-treated controls were 1.3 +/- 0.7 micrograms (+/- SD) alpha 2U-globulin/mg protein. After 2 days, GH (200 micrograms/100 g X day) resulted in an increase to 5.7 +/- 1.0 micrograms alpha 2U-globulin/mg (P less than or equal to 0.05), and T3 50 micrograms/100 g X day) resulted in an increase to 11.5 +/- 3.6 micrograms/mg (P less than or equal to 0.01). After 7 days, GH resulted in an increase to 12.4 +/- 4.6 micrograms/mg (P less than or equal to 0.01), and T3 resulted in an increase to 28.7 +/- 8.7 micrograms/mg (P less than or equal to 0.01). After 4 months of thyroid ablation, baseline hepatic alpha 2U-globulin levels fell to 4.8 ng alpha 2U-globulin/mg protein. Hepatic alpha 2U-globulin was determined 4 and 8 h after the injection of GH (200 micrograms/100 g). In these animals with markedly diminished hepatic alpha 2U-globulin levels, significant (P less than or equal to 0.01) increases occurred 4 h (25.4 ng/mg) and 8 h (57.2 ng/mg) after GH injection. The effects of treatment with bovine GH (200 micrograms/100 g X day) for 3 days on hepatic alpha 2U-globulin synthesis in liver slices and alpha 2U-globulin poly (A)+ RNA levels were measured in rats 10 weeks after thyroid ablation. GH significantly (P less than 0.05) increased alpha 2U-globulin synthesis as a percentage of total protein synthesis (from 0.01% to 0.035%) and alpha 2U-globulin mRNA as a percentage of total mRNA (from 0.03% to 0.24%). The results show that GH rapidly and specifically stimulates hepatic alpha 2U-globulin and its mRNA activity in thyroid hormone-deficient rats.     

The effects of thyroid hormone on insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) expression in the neonatal rat: prolonged high expression of IGFBP-2 in methimazole-induced congenital hypothyroidism.

In the rat a developmental switch in the serum insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) profile takes place during the first 3 postnatal weeks. The fetal expression pattern of high IGF-II and IGFBP-2 is replaced by the adult pattern of low levels of IGF-II and IGFBP-2 and high levels of IGF-I and IGFBP-3. The regulatory mechanisms mediating these changes are unknown, but may include perinatal changes in endocrine function. To study the effects of thyroid function and the perinatal thyroid secretory burst on IGF and IGFBP expression, we established a rat model of congenital hypothyroidism, leading to marked postnatal growth retardation during the perinatal period. The hypothyroid animals lacked the steep rise in serum IGF-I levels normally occurring during the third week of life, showing only a modest rise to approximately 50% of control levels. The pattern of serum IGF-II decline in hypothyroid animals was slightly different from that in controls, with lower IGF-II levels during the second week of life and a slower decline down to the very low final levels. The hypothyroid pups continued to express high levels of IGFBP-2 up to the age of 19 days, while the control animals, after a slow initial decline, showed an abrupt fall of IGFBP-2 serum levels during the third week of life. Liver IGFBP-2 mRNA levels reflected the serum changes, with elevated IGFBP-2 mRNA in hypothyroid animals. The expression of other IGFBPs did not differ from that in the control group. At the age of 18 days, serum GH levels in the hypothyroid animals were approximately one third of control GH levels, which suggests a role for GH as a possible mediator of thyroid hormone actions on the IGF system. The changes in growth parameters and in the IGF and IGFBP profile of hypothyroid pups could be abolished by thyroid hormone replacement from birth. We conclude that thyroid hormone is, directly or indirectly, essential for some of the neonatal changes in IGF and IGFBP profiles.     

Expression of the genes for insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding proteins-1 and -2 in fetal rat under conditions of intrauterine growth retardation caused by maternal fasting.

Evidence suggests that insulin-like growth factors-I and -II (IGF-I and II) play a role in regulating fetal growth and development. In the fetus, IGF-I and -II are complexed with two specific binding proteins (IGFBP-1 and -2), which are thought to modulate the actions of the IGFs in target tissues. We examined regulation of the genes for IGF-I, IGF-II, IGFBP-1, and IGFBP-2 in fetal rat liver in an experimental model for intrauterine growth retardation caused by maternal fasting on days 17-21 of gestation. The mean weight of fetuses from the fasted dams was 27-32% lower than the mean weight of fetuses from the fed dams. The concentration of immunoreactive IGF-I was decreased by 71% in serum of fetuses from the fasting dams. The concentration of immunoreactive IGF-II was slightly decreased (by 12%) in serum of fetuses from the fasting dams, whereas the concentration of immunoreactive pro-IGF-II E-domain peptide was decreased by 31%. The abundance of hepatic IGF-I mRNA was decreased by 55% in fetuses from the fasting dams. In contrast, the abundance of IGF-II mRNA in fetal liver was not significantly decreased by maternal fasting. Maternal fasting caused a 2-fold increase in the abundance of IGFBP-1 mRNA in fetal liver, whereas it did not change the abundance of IGFBP-2 mRNA. The induction of IGFBP-1 mRNA in liver of the growth-retarded fetuses is similar to the induction that occurs in liver of fasting adults, while the lack of regulation of IGFBP-2 mRNA differs from the strong induction of IGFBP-2 mRNA that occurs in liver of fasting adults. In summary, these results indicate that maternal fasting causes a decrease in fetal IGF-I gene expression, a decrease in fetal serum IGF-I, and a slight decrease in fetal serum IGF-II and pro-IGF-II E-domain peptide concentrations. Maternal fasting also causes an increase in fetal IGFBP-1 gene expression. Changes in fetal insulin and glucose may be related to changes in expression of the IGF-I and IGFBP-1 genes in the growth-retarded fetuses. The decreased expression of IGF-I and -II and increased expression of the IGFBP-1 gene may contribute to the fetal growth retardation observed in this model system.     

Time course of the in vivo effects of thyroid hormone on cardiac gene expression.

The rate of response to thyroid hormone on cardiac growth, heart rate, and the relative changes in messenger RNA (mRNA) coding for alpha- and beta-myosin heavy chain (MHC), slow sarcoplasmic reticulum calcium-adenosine triphosphatase, and thyroid hormone receptors in ventricular tissue of hypothyroid rats was investigated. Hypothyroid rats had significantly smaller hearts, with slower heart rates and expressed no alpha-MHC mRNA as analyzed by an S1 nuclease protection assay when compared to euthyroid animals that expressed 79% alpha-MHC. Twelve hours after treating hypothyroid rats with 20 micrograms of L-T4, detectable levels of alpha-MHC mRNA were present and the shift to alpha-MHC mRNA was complete by 72 h of treatment. Northern blot analysis showed that hypothyroidism resulted in a 60% decrease in the level of sarcoplasmic reticulum calcium-adenosine triphosphatase mRNA which increased after 12 h of T4 administration and was 2.5-fold (P less than 0.05) greater than euthyroid levels after 72 h. In contrast, thyroid hormone receptor mRNA levels measured in poly(A)+ RNA were elevated in hypothyroid rats and decreased to euthyroid levels within 24 h after thyroid hormone treatment. These changes in cardiac gene expression occurred simultaneously with changes in both cardiac size and heart rate. The current studies characterize the coordinated changes and the time course for gene expression that occur in the hypothyroid heart after acute T4 administration.     

Short-term infusion of LongR(3) insulin-like growth factor (IGF)-I decreases hepatic IGF-I mRNA but not IGF binding protein-3 mRNA expression in pigs

Infusion of pigs with an insulin-like growth factor-I (IGF-I) analogue (LongR(3)IGF-I) that does not bind to IGF-binding proteins decreases growth rate and the plasma concentration of growth hormone (GH), IGF-I, IGFBP-3, and insulin. This study was designed to determine whether the decrease is due to changes in IGF-I and IGFBP-3 gene expression. IGF-I or LongR(3)IGF-I (180 microg/kg/day) was infused into 55-kg finisher pigs for 4 days using Travenol infuser pumps. Plasma IGF-I concentration was measured by radioimmunoassay and plasma IGFBP-3 and IGFBP-2 were estimated by Western ligand blotting. Steady-state levels of IGF-I and IGFBP-3 mRNA were measured by RNase protection assay. Neither IGF-I nor LongR(3)IGF-I had a significant effect on hepatic IGF-I class 1 mRNA expression, whereas hepatic IGF-I class 2 mRNA expression was significantly reduced by both peptides. Plasma IGFBP-3 levels were unaffected by IGF-I treatment but were reduced by LongR(3)IGF-I treatment. The decrease in IGFBP-3 was not due to decreased gene expression in porcine liver or kidney, since neither IGF-I nor LongR(3)IGF-I treatment altered IGFBP-3 mRNA. This study infers a direct effect of the IGF analogue LongR(3)IGF-I on GH through its inhibition of plasma IGF-I concentration and class 2 IGF-I mRNA. The decrease in plasma IGFBP-3 was not accompanied by a decrease in hepatic or renal IGFBP-3 mRNA, suggesting that in this case, plasma IGFBP-3 protein levels are posttranslationally regulated or are derived from tissues other than liver or kidney.