Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T₃ and T₄ levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn⁺⁺ in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.     

Regulation of adiponectin gene expression in adipose tissue by thyroid hormones

Available experimental data suggest that adiponectin and thyroid hormones have biological interaction in vivo. However, the effects of thyroid hormones on adipose adiponectin gene expression in thyroid dysfunction are unclear. We induced hyper- (HYPER) and hypothyroidism (HYPO) by daily administration of a 12 mg/l of levothyroxine and 250 mg/l of methimazole in drinking water of rats, respectively, for 42 days. The white adipose tissues and serum sample were taken on days 15, 28, 42 and also 2 weeks after treatment cessation. Analysis of adiponectin gene expression was performed by real-time PCR and 2^−ΔΔct method. The levels of adipose tissue adiponectin mRNA in the HYPO rats were decreased during the 6-week treatment when compared to control rats (<0.05) and were increased significantly 2 weeks after HYPO cessation (P < 0.05). This decline in adiponectin gene expression occurred in parallel with a decrease in T3, T4, fT3 and fT4 concentrations (P < 0.05). In opposite to HYPO rats, adipose adiponectin gene expression was increased in HYPER rats during the 6-week treatment in parallel with an increase the thyroid hormones concentrations (P < 0.05), and its expression was decreased 2 weeks after HYPER cessation (P < 0.05). Adiponectin gene expression levels showed significant negative correlations with concentrations of LDL (HYPO; r = −0.806, P = 0.001 and HYPER; r = −0.749, P = 0.002), triglyceride (HYPO; r = −0.825, P = 0.001 and HYPER; r = −0.824, P = 0.001) and significant positive correlations with concentrations of glucose (HYPO; r = 0.674, P = 0.004 and HYPER; r = 0.866, P = 0.001) and HDL (HYPO; r = 0.755, P = 0.001 and HYPER; r = 0.839, P = 0.001). The current study provides evidence that adiponectin gene expression in adipose tissue is regulated by thyroid hormones at the translation level and that lipid and carbohydrate disturbances in a patient with thyroid dysfunction may be, in part, due to adiponectin gene expression changes.     

Upregulation of CHOP-10 (gadd153) expression in the mouse blastocyst as a response to stress.

CHOP-10 (also known as gadd153 or Ddit3) is one of the genes overexpressed by mammalian cells exposed to cytotoxic agents or to nutrient stress. The response of this gene to stress was studied in the mouse blastocyst and in F9 embryonal carcinoma cells. When mouse blastocysts were exposed to the alkylating agent MMS, the metabolic inhibitor sodium arsenite or an inhibitor of protein glycosylation tunicamycin, levels of the CHOP-10 mRNA were increased by two- to threefold relative to the mRNA for beta-actin. There was no increase in gene expression when blastocysts were treated with the inhibitor of nucleotide synthesis PALA. These results show that the response of CHOP-10 is dependent on the type of stress applied to the embryo. When F9 embryonal carcinoma cells were treated with MMS or sodium arsenite, CHOP-10 expression was induced by fourfold within 4 hr of treatment. The induction following tunicamycin treatment was slower requiring at least 24 hr. The response to tunicamycin was greater in cells treated with retinoic acid to induce differentiation. The results suggest that there is a link between the extent of glycoprotein synthesis and the sensitivity of CHOP-10 to tunicamycin. The inhibitor PALA did not change CHOP-10 expression in the presence or absence of retinoic acid. In F9 cells an increase in the expression of CHOP-10 was followed by cell death due to apoptosis. The overexpression of CHOP-10 may be a marker for one of the pathways that lead to apoptosis in the blastocyst. These results suggest that there is more than one control system regulating growth arrest in the blastocyst and the fetal outcome may differ depending on the type of stress encountered in culture.     

Cartilage response to thyroid hormones in the sex-linked dwarf chicken

1. Trachea cartilages were dissected from normal and dwarf chickens which had been injected with thyroxine (T4, 200 micrograms/kg) or triiodothyronine (T3, 200 micrograms/kg) for seven consecutive days, and were analysed for nucleic acids, proteins and polyamines. 2. In saline-injected control chickens, RNA, but not DNA and protein, concentration of the cartilage was higher in dwarfs than in normals. The concentration of putrescine was lower in dwarfs than in normals, while that of spermine was the reverse. 3. Thyroid hormones, especially T3, tended to increase concentrations of RNA, spermidine and spermine, and to decrease that of putrescine. However, there were no clear differences in the response to hormones between breeds.     

Microarray analysis of gene expression profiles of rat small intestine in response to heat stress

Ambient temperature is a critical factor that affects biological organisms in many ways. In this study, the authors investigated gene expression changes in rat small intestine in response to heat stress. Male Sprague-Dawley rats were randomly divided into control and heat-stressed groups. Both groups were housed at 25 °C, although the heat-stressed group was also subjected to 40 °C for 2 h each day for 10 successive days. Rats were sacrificed 1, 3, 6, and 10 days after heat treatment, and sections of their small intestine epithelial tissue were excised for morphological examination and microarray analyses. The rat rectal and body surface temperatures and serum cortisol levels were all significantly increased after heat treatment (p < 0.05). The jejuna were significantly damaged by 3 days after heat treatment began. Microarray analysis showed that 422 genes were differentially expressed, of which 290 genes were significantly upregulated and 132 genes were significantly downregulated. Subsequent bioinformatics analyses revealed that the differentially expressed genes were mainly related to stress, immune regulation, and metabolism processes. The bioinformatics analysis of the differentially expressed genes should be beneficial to further investigations on the underlying mechanisms involved in heat stress-induced damage in the small intestine.     

AdipoR1 and AdipoR2 gene expression are regulated by thyroid hormones in adipose tissue

The aim of this study was to examine whether the relative gene expression of AdipoR1 and AdipoR2 in rat adipose tissue is altered by thyroid hormones, and whether this might relate to their circulating thyroid hormones and adiponectin levels. Hyper- and hypothyroidism were induced by daily oral administration of levothyroxine and methimazole in rats, respectively, over a 42 days period. Real-time PCR analysis was performed to evaluate the changes in AdipoR1 and AdipoR2 mRNA levels in the adipose tissue on days 15, 28, 42, and also 2 weeks after the cessation of treatment. In response to treatment with methimazole, mRNA levels of AdipoR1 and AdipoR2 decreased in the white adipose tissue compared to the euthyroid rats (p < 0.05). This decline was reversible 2 weeks after treatment cessation. The mRNA levels of AdipoR1 and AdipoR2 were increased in the hyperthyroid group of animals compared to euthyroid control (p < 0.05), and its changes were reversible 2 weeks after treatment cessation (P < 0.05). Adiponectin receptors gene expression levels in the adipose tissue of treated animals have positive correlations with thyroid hormones concentrations. Our results suggest that AdipoR1 and AdipoR2 gene expression is regulated by thyroid hormones in hypo- and hyperthyroidism.     

Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.     

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.     

A mouse model of acrodermatitis enteropathica: loss of intestine zinc transporter ZIP4 (Slc39a4) disrupts the stem cell niche and intestine integrity

Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth in vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogen- or prolactin-dependent growth. Primary tumors still displayed a constitutional expression of alpha-, beta-, and gamma-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in alpha- and beta-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of alpha-, beta-, and gamma-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10-14 days. The accumulation of beta-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance beta-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Upregulation of corin gene expression in hypertrophic cardiomyocytes and failing myocardium

High levels of plasma atrial natriuretic peptides (ANP) are associated with pathological conditions such as congestive heart failure (CHF). Recently, we have identified a cardiac serine protease, corin, that is the pro-ANP convertase. In this study, we examined the regulation of corin gene expression in cultured hypertrophic cardiomyocytes and in the left ventricular (LV) myocardium of a rat model of heart failure. Quantitative RT-PCR analysis showed that both corin and ANP mRNA levels were significantly increased in phenylephrine (PE)-stimulated rat neonatal cardiomyocytes in culture. The increase in corin mRNA correlated closely with the increase in cell size and ANP mRNA expression in the PE-treated cells (r = 0.95, P < 0.01; r = 0.92, P < 0.01, respectively). The PE-treated cardiomyocytes had an increased activity in converting recombinant human pro-ANP to biologically active ANP, as determined by a pro-ANP processing assay and a cell-based cGMP assay. In a rat model of heart failure induced by ligation of the left coronary artery, corin mRNA expression in the noninfarcted LV myocardium was significantly higher than that of control heart tissues from sham-operated animals, when examined by Northern blot analysis and RT-PCR at 8 wk. These results indicate that the corin gene is upregulated in hypertrophic cardiomyocytes and failing myocardium. Increased corin expression may contribute to elevation of ANP in the setting of cardiac hypertrophy and heart failure.