Establishment of an Effective Radioiodide Thyroid Ablation Protocol in Mice

Due to the high variance in available protocols on iodide-131 ((131)I) ablation in rodents, we set out to establish an effective method to generate a thyroid-ablated mouse model that allows the application of the sodium iodide symporter (NIS) as a reporter gene without interference with thyroidal NIS. We tested a range of (131)I doses with and without prestimulation of thyroidal radioiodide uptake by a low-iodine diet and thyroid-stimulating hormone (TSH) application. Efficacy of induction of hypothyroidism was tested by measurement of serum T4 concentrations, pituitary TSH\textgreekb and liver deiodinase type 1 (DIO1) mRNA expression, body weight analysis, and (99m)Tc-pertechnetate scintigraphy. While 200 µCi (7.4 MBq) (131)I alone was not sufficient to abolish thyroidal T4 production, 500 µCi (18.5 MBq) (131)I combined with 1 week of a low-iodine diet decreased serum concentrations below the detection limit. However, the high (131)I dose resulted in severe side effects. A combination of 1 week of a low-iodine diet followed by injection of bovine TSH before the application of 150 µCi (5.5 MBq) (131)I decreased serum T4 concentrations below the detection limit and significantly increased pituitary TSH\textgreekb concentrations. The systemic effects of induced hypothyroidism were shown by growth arrest and a decrease in liver DIO1 expression below the detection limit. (99m)Tc-pertechnetate scintigraphy revealed absence of thyroidal (99m)Tc-pertechnetate uptake in ablated mice. In summary, we report a revised protocol for radioiodide ablation of the thyroid gland in the mouse to generate an in vivo model that allows the study of thyroid hormone action using NIS as a reporter gene

Molekularmedizinische Grundlagen von para- und autokrinen Regulationsstoerungen

Im vorliegenden Band stehen lokal wirksame Hormonsysteme, Gewebshormone, ihre auto-, para- und intrakrinen Funktionen sowie ihre Stoerungen als Ursache von Erkrankungen im Mittelpunkt der Darstellung. Namhafte Experten haben hundert Jahre nach der Einfuehrung des Hormonbegriffes durch Starling 1905 den gegenwaertigen Wissensstand in 25 Kapiteln zusammengefuehrt, der dem interessierten Leser eine umfassende Orientierung ueber dieses interdisziplinaere Fachgebiet erlaubt. [From the Publisher

Localization of type I iodothyronine 5\u27-deiodinase to the basolateral plasma membrane in renal cortical epithelial cells

Type I iodothyronine 5\u27-deiodinase is an integral membrane protein catalyzing the phenolic ring deiodination of thyroxine. We recently showed that the substrate binding subunit of this approximately 50-kDa protein is selectively labeled with N-bromoacetyl-L-thyroxine, allowing ready identification of the type I enzyme without the need to maintain catalytic activity. In this study, we used both affinity labeling and catalytic activity to determine the regional distribution of this enzyme in rat kidney and to localize the enzyme to specific plasma membrane domain(s) of renal epithelial cells. The type I enzyme was present exclusively in tubular epithelial cells of the outer renal cortex and co-purified with basolateral plasma membranes; the renal medulla lacked activity. LLC-PK1 cells, derived from the proximal convoluted tubule, have abundant type I 5\u27-deiodinating activity. We used this homogenous cell line to verify that the type I enzyme was localized to the cytosolic surface of the basolateral membrane. Digitonin permeabilization increased affinity labeling of the enzyme 4-fold, and approximately 75% of the affinity label was incorporated into the 27-kDa substrate binding subunit. Affinity labeling of the type I enzyme in LLC-PK1 cells mimicked the affinity labeling of the substrate binding subunit of type I 5\u27-deiodinase in rat kidney (Kohrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155-6163). Subcellular fractionation of LLC-PK1 cell homogenates showed that both affinity labeled and catalytically active type I enzyme were present on the cytosolic surface of the basolateral region of the renal cell membrane

Testosterone and estradiol treatments differently affect pituitary-thyroid axis and liver deiodinase 1 activity in orchidectomized middle-aged rats

We previously reported that orchidectomy (Orx) of middle-aged rats (15-16-month-old; MA) slightly affected pituitary-thyroid axis, but decreased liver deiodinase (Dio) type I and pituitary Dio2 enzyme activities. At present, we examined the effects of subsequent testosterone-propionate treatment (5 mg/kg; Orx + T), and compared the effects of testosterone with the effects of estradiol-clipropionate (0.06 mg/kg; Orx + E) treatment. Hormones were subcutaneously administered, daily, for three weeks, while Orx and sham-operated (SO) controls received only the vehicle. The applied dose of T did not alter serum TSH, T4 and T3 concentrations in Orx- MA, though it increased TSH when administrated to Orx young adults (2.5-month-old; Orx-YA). However, pituitaries of Orx-MA + T rats had higher relative intensity of immunofluorescence (RIP) for TSHI3; in their thyroids we found increased volume and height of follicular epithelium, decreased volume of the colloid and higher RIF for T,{[}-bound to thyroglobulin (Tg-T4). Liver Diol activity was increased. E-treatment did not affect serum hormone levels, pituitary RIF for TS1-11i, or liver Diol activity in Orx-MA rats. Thyroids had decreased relative volume and height of follicular epithelium, increased relative volume of the colloid, decreased volume of sodium iodide symporter-immunopositive epithelium and lower RIF for Tg-T4. Detected changes were statistically significant. In conclusion, androgenization enhanced pituitary TSHr, RIF, thyroid activation and liver Diol enzyme activity in Orx-MA, without elevating serum TSH as in Orx-YA rats. Estrogenization induced pituitary enlargement with no effect on pituitary TSHI) RIF, serum TSH or liver Diol activity. E also induced alterations in thyroid histology that indicate mild suppression of its functioning, and contributed to thyroid blood vessel enlargement in Orx-MA rats. (C) 2015 Elsevier Inc. All rights reserved.Ministry of Education and Science of the Republic of Serbia {[}173009]; Deutsche Forschungsgemeinschaft {[}DFG-GK 1208, TP3, RE3038/1-1]; Charite Universitatsmedizin Berlin; European Society for Endocrinology (ESE

Mutation of megalin leads to urinary loss of selenoprotein P and selenium deficiency in serum, liver, kidneys and brain

Distribution of selenium (Se) within the mammalian body is mediated by SePP (selenoprotein P), an Se-rich glycoprotein secreted by hepatocytes. Genetic and biochemical evidence indicate that the endocytic receptors ApoER2 (apolipoprotein E receptor 2) and megalin mediate tissue-specific SePP uptake. In the present study megalin-mutant mice were fed on diets containing adequate (0.15 p.p.m.) or low (0.08 p.p.m.) Se content and were analysed for tissue and plasma Se levels, cellular GPx (glutathione peroxidase) activities and protein expression patterns. Megalin-mutant mice displayed increased urinary Se loss, which correlated with SePP excretion in their urine. Accordingly, serum Se and SePP levels were significantly reduced in megalin-mutant mice, reaching marginal levels on the low-Se diet. Moreover, kidney Se content and expression of renal selenoproteins were accordingly reduced, as was SePP internalization along the proximal tubule epithelium. Although GPx4 expression was not altered in testis, Se and GPx activity in liver and brain were significantly reduced. When fed on a low-Se diet, megalin-mutant mice developed impaired movement co-ordination, but no astrogliosis. These findings suggest that megalin prevents urinary SePP loss and participates in brain Se/SePP uptake

Does the aromatic L-amino acid decarboxylase contribute to thyronamine biosynthesis?

Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T3. TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the l-alanine side chain and several deiodination steps to convert e.g. l-thyroxine (T4) into the most potent 3-T1AM. Aromatic l-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T1AM in plasma of patients with AADC-deficiency at levels (46 \ub1 18 nM, n = 4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase