Dibenzazepine-Loaded Nanoparticles Induce Local Browning of White Adipose Tissue to Counteract Obesity.

Inhibition of Notch signaling via systemic drug administration triggers conversion of white adipocytes into beige adipocytes (browning) and reduces adiposity. However, translation of this discovery into clinical practice is challenged by potential off-target side effects and lack of control over the location and temporal extent of beige adipocyte biogenesis. Here, we demonstrate an alternative approach to stimulate browning using nanoparticles (NPs) composed of FDA-approved poly(lactide-co-glycolide) that enable sustained local release of a Notch inhibitor (dibenzazepine, DBZ). These DBZ-loaded NPs support rapid cellular internalization and inhibit Notch signaling in adipocytes. Importantly, focal injection of these NPs into the inguinal white adipose tissue depots of diet-induced obese mice results in localized NP retention and browning of adipocytes, consequently improving the glucose homeostasis and attenuating body-weight gain of the treated mice. These findings offer new avenues to develop a potential therapeutic strategy for clinical treatment of obesity and its associated metabolic syndrome.

Halogen bonding controls the regioselectivity of the deiodination of thyroid hormones and their sulfate analogues.

The type 1 iodothyronine deiodinase (1D-1) in liver and kidney converts the L-thyroxine (T4), a prohormone, by outer-ring (5') deiodination to biologically active 3,3',5-triiodothyronine (T3) or by inner-ring (5) deiodination to inactive 3,3',5'-triiodothronine (rT3). Sulfate conjugation is an important step in the irreversible inactivation of thyroid hormones. While sulfate conjugation of the phenolic hydroxyl group stimulates the 5-deiodination of T4 and T3, it blocks the 5'-deiodination of T4. We show that thyroxine sulfate (T4S) undergoes faster deiodination as compared to the parent thyroid hormone T4 by synthetic selenium compounds. It is also shown that ID-3 mimics, which are remarkably selective to the inner-ring deiodination of T4 and T3, changes the selectivity completely when T4S is used as a substrate. From the theoretical investigations, it is observed that the strength of halogen bonding increases upon sulfate conjugation, which leads to a change in the regioselectivity of ID-3 mimics towards the deiodination of T4S. It has been shown that these mimics perform both the 5'- and 5-ring deiodinations by an identical mechanism.

A ubiquitous tire rubber additive induced serious eye injury in zebrafish (Danio rerio).

Previous studies have indicated that tire wear particles (TWPs) leachate exposure induced serious eye injury in fish through inhibiting the thyroid peroxidase (TPO) enzyme activity. However, the main TPO inhibitors in the leachate were still unknown. In this study, we identified 2-Mercaptobenzothiazole (MBT) as the potential TPO inhibitor in the TWPs leachate through references search, model prediction based on Danish QSAR and ToxCast database, molecular docking, and in vivo assay. We further explored the toxic mechanism of MBT under environmentally relevant concentrations. The decreased eye size of zebrafish larvae was mainly caused by the decreased lens diameter and cell density in the inner nuclear layer (INL) and outer nuclear layer (ONL) of the retina. Transcriptomics analysis demonstrated that the eye phototransduction function was significantly suppressed by inhibiting the photoreceptor cell proliferation process after MBT exposure. The altered opsin gene expression and decreased opsin protein levels were induced by weakening thyroid hormone signaling after MBT treatment. These results were comparable to those obtained from a known TPO inhibitor, methimazole. This study has identified MBT as the primary TPO inhibitor responsible for inducing eye impairment in zebrafish larvae exposed to TWPs leachate. It is crucial for reducing the toxicity of TWPs leachate in fish.

Simple one-step process for immobilization of biomolecules on polymer substrates based on surface-attached polymer networks.

For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.

Thyroid hormone deiodinases response in brain of spontaneausly hypertensive rats after hypotensive effects induced by mandibular extension.

PURPOSE: The deiodinases activate or inactivate the thyroid hormones (TH) in virtually all tissues in both physiological and pathological conditions. The three deiodinases, DIO1, DIO2, and DIO3, have different catalytic functions and regulate TH tissue distribution. The aim of the present study was to evaluate the modulation of gene expression of the deiodinases and TH transporters and protein levels of DIO1 in parietal and frontal areas of cerebral cortex of spontaneously hypertensive rats (SHRs), after two successive mandibular extensions (ME). METHODS: ME was performed on anesthetized rats by a dilatator appropriately designed and real-time PCR and western blotting techniques were employed for gene expression and protein level study. RESULTS: Mean blood pressure (MBP) significantly decreased in 2ME-treated rats when compared to sham-operated rats (p < 0.001) and this decrease lasted for the entire observation period. In gene expression analysis, in 2ME-treated rats we did not observe any significant variation of DIO1 and DIO3 with respect to the sham-operated rats. Differently, DIO2 gene expression significantly increased in frontal area of 2ME-treated rats, with respect to sham-operated rats (p < 0.01). Furthermore, in parietal area, protein levels of DIO1 in 2ME-treated rats were significantly higher than in sham-operated rats (p < 0.01). Moreover MCT8 and OATP1C1 both resulted significantly higher (p < 0.05 and p < 0.001) in sham frontal cortex. CONCLUSION: In summary, our data on SHRs, while confirming the hypotensive effect of two MEs, show that the treatment also solicits the three deiodinases production in the cerebral cortex.

Role for inner ring deiodination preventing transcutaneous passage of thyroxine.

Creams containing thyroid hormone are commonly employed for cosmetic purposes. To verify whether T(4) applied to the skin surface can enter the bloodstream either directly or as a metabolite, a cream containing L-T(4) [3,5,3',5'-tetraiodothyronine (T(4))] was self-applied by volunteers for 2 wk. No significant variations in urinary iodide, TSH, and serum (total and free) T(4) and T(3) concentrations were observed at any time relative to pretreatment values, whereas rT(3) concentrations increased significantly 6 and 12 h after cream application. The increased rT(3) concentration led us to investigate the presence of inner ring type III deiodinase (D3) activity in human skin. Using human surgical discard skin, we found that T(4) can be carried across human epidermis in a liposome cream. Substantial inner ring deiodination was suggested by the fact that only 10% of transferred thyroid hormone remained as T(4), and T(3) was not detected. We then measured D3 activity in a surgical skin specimen. The K(m) for T(3) was 1.74 nmol/liter, and the maximum velocity was 23.5 fmol/microg microsomal protein/h. In conclusion, our study indicates that normal human skin serves as a substantial, but incomplete, barrier to T(4) passage. D3 plays an important role in augmenting T(4) blockade by inactivating T(4) to rT(3).

Lysosomal thyroid hormone 5'-deiodinase.

Lysosomes prepared from rat liver and kidney after loading with the detergent Triton WR-1339 show membrane-bound 5'-deiodinase activity with marked specificity for 3,3',5'-triiodothyronine (reverse T3), lesser activity with respect to thyroxine (T4) and almost none towards 3,3',5'-triiodothyronine (T3). The enzyme is thiol dependent and shows maximal catalysis at pH 7.2. As many of the states known to alter thyroid hormone levels also affect lysosomal function, inhibition of the lysosomal 5'-deiodinase leading to an increase in intracellular reverse T3 may be an initiating mechanism for thyroid hormone change.

DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) increases iodide trapping, inhibits thyroperoxidase and antagonizes the TSH-induced apical iodide efflux in porcine thyroid cells.

4,4'-Di-isothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of several anionic channels and transporters including the band 3 protein of the red blood cell membrane was tested on iodide metabolism in cultured porcine thyroid cells. We used three experimental cell culture models: (i) forskolin-stimulated correctly inside-in polarized follicle-associated thyroid cells cultured onto plastic support (ii) suspensions of isolated cells derived from such cultures (iii) polarized monolayers in bicameral chambers. DIDS was observed to increase free-iodide trapping in all conditions. Organification of iodide by follicle-associated cell cultures incubated for 6 h decreased as a function of DIDS concentration with an IC50 of 5 x 10(-5) M. This block in organification is accounted for a block in thyroperoxidase activity as in vitro both purified lactoperoxidase and purified porcine thyroperoxidase were inhibited by DIDS with a similar dose-dependency the IC50 being also of 5 x 10(-5) M. Both control and DIDS-treated cells in suspension, actively trapped iodide and reached a steady concentration in about 50 min; however the plateau was 4.4-fold higher in (10(-3) M) DIDS-treated cells. Acute TSH-stimulation at this plateau of 125I-preloaded cells in suspension in the presence of 2 mM methimazole (MMI) induced a fast release of iodide from these cells as expected (first step of the TSH-biphasic effect). This TSH-induced iodide efflux was however completely inhibited by DIDS (10(-3) M). Furthermore, addition of DIDS to the apical compartment of TSH-prestimulated cell monolayers in bicameral chambers resulted in an increase in intracellular-iodide concentration and in an inhibition of iodide efflux into the apical medium. Taken together, the present results demonstrate that DIDS mainly interacts with two main components of the thyroid apical cell membrane: thyroperoxidase and a cAMP-sensitive iodide channel.

Subchronic toxicity study with ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methylhydro cin namate) in the cynomolgus monkey: lack of stimulation of the pituitary-thyroid-liver axis.

To evaluate the toxicological profile of the phenolic antioxidant ethylene-bis-(oxyethylene)-bis-(3-tert-butyl-4-hydroxy-5-methyl- hydrocinnamate) (EOC) in a non-human primate, male cynomolgus monkeys (Macaca fascicularis) were treated for 4 weeks by oral administration of 0, 200, or 1000 mg/kg body weight/day. Special attention was directed to parameters of the pituitary-thyroid-liver axis. Moderately increased liver weights and minimal to moderate hepatocellular hypertrophy were observed in treated animals. Otherwise, no treatment-related changes were detected in hematological, clinical chemistry, or urinalysis parameters or upon histopathological examination. Except for a slight induction of microsomal testosterone 16beta-hydroxylation, liver xenobiotic-metabolising enzyme activities and peroxisomal fatty acid beta-oxidation remained unchanged. Likewise, serum levels of thyroid stimulating hormone, thyroxine, 3,3',5-triiodothyronine and 3,3',5'-triiodothyronine as well as 5'-monodeiodinase type 1 mRNA levels in the liver, heart, cerebral cortex, and thyroid were found unchanged. The results demonstrate that, in the Cynomolgus monkey, EOC is only a very weak inducer of liver xenobiotic-metabolizing enzymes and has no effect on thyroid function. In contrast, upon feeding rats at dose levels up to 1000 ppm (equivalent to between 50 and 100 mg/kg body weight/day), EOC has been identified as a strong phenobarbital- and peroxisome proliferator-type inducer of hepatic xenobiotic-metabolizing enzymes, interfering with thyroid hormone homeostasis, causing thyroid follicular hypertrophy, and, upon chronic treatment, inducing thyroid gland follicular cell tumors (Thomas et al., 1995. In Toxicology of Industrial Compounds, pp. 319-339. Taylor and Francis). Thus, the results of this study with EOC in the cynomolgus monkey show that effects of xenobiotics on the pituitary-thyroid-liver axis as frequently observed in rodents can not necessarily be extrapolated to primates including man.

Improved method of thyroid peroxidase extraction from the human thyroid gland.

This study describes a new method of solubilizing thyroid peroxidase (TPO) and partial purification of TPO from a small surgical specimen of human thyroid tissue. Graves' thyroid tissue was homogenized and centrifuged to obtain the 100 000 X g pellet. To solubilize TPO from the 100 000 X g pellet protein, the following four detergents were used: Triton X-100, digitonin, sodium deoxycholate, and 3-[(3-choramidpropyl)-dimethylammonio] 1-propanesulfate (CHAPS). For some samples, two detergents were combined and trypsin was also used. The best solubilization of TPO activity was obtained from the combination of digitonin-CHAPS-trypsin treatment or deoxycholate-CHAPS-trypsin treatment. The solubilized crude TPO was then chromatographed on a Sephacryl S 300 column. The results of chromatography indicated that detergent treatment alone did not separate TPO from other membrane proteins and the addition of trypsin was required for separation of TPO. Sephacryl chromatography of detergent-trypsin solubilized TPO was suitable as an initial step for purification of TPO from a small human thyroid tissue.

Mode of death effect on rat liver iodothyronine 5' deiodinase activity: role of adenosine 3',5' monophosphate.

Liver thyronine 5'-deiodinase activity assayed in crude homogenates in the absence of dithiothreitol (DTT) is increased in rats killed by asphyxia when compared to that of animals killed by phenobarbital injection or decapitation. The addition of cyclic adenosine monophosphate leads to a consistent decrease in observed deiodinase activity, suggesting the possible involvement of this nucleotide in the regulation of this enzyme. The addition of DTT eliminates this effect and suggest a dual regulation of the enzyme by cAMP and physiological sulfhydryl compounds.

Incorporation of bovine thyroid peroxidase in liposomes.

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropriate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (Tc) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37 degrees C, the temperature at which thyroid peroxidase works 'in vivo'. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.

[Source of enzymes of mixed human saliva]. / Proiskhozhdenie fermentov smeshannoi sliuny cheloveka.

Production of enzymes was studied in parotid, submandibular and mucosal salivary glands as well as in exfoliated epithelial cells, emigrating leukocytes and in dental bacterial plaque. alpha-Amylase and iodide peroxidase were mainly secreted into mixed saliva by parotid and submandibular glands. Alkaline phosphatase was produced mainly by mucosal salivary glands. These glands secreted also about one half of alkaline and acid proteases, acid phosphatase, both ribonucleases, lysozyme, alkaline peroxidase and lactate dehydrogenase. Role of leukocytes and epithelial cells as well as bacterial plaque was relatively negligible in formation of mixed saliva enzymes.

[Biochemical characteristics of iodperoxidase activity of human saliva].

Peroxidase-catalyzed oxidation of iodide in human saliva leads to the formation of a brown product with lambda max 287 nm and 353 nm (I3-) identified by the method of UV-spectrophotometry. I3- directly reacts with starch producing the characteristic blue complex. Salivary iodide peroxidase activity was found to be from 1.2 to 2.3 times higher then the activity of salivary peroxidases with natural substrates (SCN- and Cl-). Optimum for the iodide peroxidase activity in human saliva was found to be near pH 5.8. Salivary iodide peroxidase activity progressively lowers with the rise of pH value of the reaction mixture until total loss at the pH>7.4 was observed. Iodide peroxidase activity in human saliva at pH>7.4 is masked due to decomposition of I3- with the increase of pH along with the inhibition of peroxidases and I3- reduction by low molecular weight dialyzable salivary components possibly by Cl- and NCS-. Salivary iodide peroxidase activity was completely inhibited by peroxidase inhibitors (NaN3, 2-mercaptoethanol, thiourea), while addition of the peroxidase alternative substrates (ascorbate, quercetin, thiocyanate) resulted in partial inhibition of iodide peroxidase activity. The results of the study confirm the idea, that high activity of human saliva peroxidase with iodide as a substrate may play a crucial role in the bioavailability and metabolism of biologically active iodide.

The dodecameric vanadium-dependent haloperoxidase from the marine algae Corallina officinalis: cloning, expression, and refolding of the recombinant enzyme.

The dodecameric vanadium-dependent bromoperoxidase from Corallina officinalis has been cloned and over-expressed in Escherichia coli. However, the enzyme was found to be predominantly in the form of inclusion bodies. This protein presents a challenging target for refolding, both due to the size (768kDa) and quaternary structure (12x64kDa). Successful refolding conditions have been established which result in an increase in the final yield of active bromoperoxidase from 0.5mg to 40mg per litre of culture. The refolded protein has been characterised and compared to the native enzyme and was shown to be stable at temperatures of 80 degrees C, over a pH range 5.5-10 and in organic solvents such as ethanol, acetonitrile, methanol, and acetone. The novel refolding approach reported in this paper opens up the full potential of this versatile enzyme for use in large scale biotransformation studies.

Estradiol-17 beta silastic implants in female Rana ridibunda depress thyroid hormone concentrations in plasma and the in vitro 5'-monodeiodination activity of kidney homogenates.

Estradiol-17 beta containing silastic tubings were implanted in female Rana ridibunda. Preliminary data, concerning in vitro incubations of such tubings in saline media, revealed that high concentrations of estradiol were released out of the tubings in the incubation medium. Compared to control-implanted frogs, the frogs that had the estradiol tubings implanted for 30 days showed a significant increase of the plasma estradiol concentration, the ovarian estradiol concentration, and the weight of the oviduct. Plasma triiodothyronine (T3) levels, plasma thyroxine levels, and the in vitro T3 production in the kidney homogenates were significantly decreased. These results indicate that high estradiol levels not only influence the gonadal axis, but also cause important effects on the thyroidal axis.

Regulation of type II iodothyronine 5'-deiodinase by thyroid hormone. Inhibition of actin polymerization blocks enzyme inactivation in cAMP-stimulated glial cells.

The cellular events mediating the rapid, thyroid hormone-dependent modulation of membrane-bound, type II iodothyronine 5'-deiodinase were studied in dibutyryl cyclic AMP(bt2cAMP)-treated brain astrocytes. Unstimulated cells had undetectable type II 5'-deiodinating activity. Treating the cells with bt2cAMP and hydrocortisone induced enzyme expression by increasing transcription of the enzyme or an essential enzyme related protein(s), with steady-state levels of type II 5'-deiodinase attained after 8 h of bt2cAMP treatment. Glial cells grown in the absence of thyroid hormone had 10-15-fold higher levels of 5'-deiodinating activity than cells grown in the presence of serum. The increased type II 5'-deiodinating activity observed in serum-free cultures was due to a prolonged enzyme half-life with no change in the rate of enzyme synthesis. Addition of thyroxine or 3,3',5'-triiodothyronine to the serum-free culture medium resulted in a concentration-dependent fall in steady-state enzyme levels, with EC50 values of approximately 0.4 nM. 3,5,3'-Triiodothyronine was at least 100-fold less effective. Chloroquine, NH4Cl, tunicamycin, colchicine, and monodansylcadavarine had no effect on the t1/2 of the enzyme, while both carbonyl cynanide m-chlorophenylhydrazone and cytochalasins completely blocked the inactivation of the type II 5'-deiodinase. These data indicate that in glial cells, an intact actin-cytoskeleton is required for thyroid hormone to modulate the energy-dependent regulation of the half-life of the short-lived, membrane-bound enzyme, type II 5'-deiodinase.

Periodontal 5'-deiodination on forced-induced root resorption--the protective effect of thyroid hormone administration.

The present investigation was designed to study the protective effect given by thyroid hormone (TH) on root resorption: (1) whether intra-peritoneal versus oral TH administration had the same efficiency; and (2) whether this effect involved local or systemic mechanisms. For this purpose, circulating T3 levels, systemic alkaline phosphatase (APase) activity, and 5'deiodinase (5'D) activity were evaluated in the periodontal area of 80 Sprague-Dawley rats, 8 weeks of age, in which orthodontic appliances had been inserted. The results showed that TH-treated animals (intra-peritoneal or oral) had significantly less force-induced root resorptive lesions compared with a control group, without apparent changes in T3 or APase levels, and that periodontal remodelling was accompanied by a significant increase in local T3 generation as a result of T4 deiodination. This 5'D activity was higher in those animals that received exogenous TH. These results suggest that this protective TH mechanism may be achieved at a local level and that administration of low doses of TH may play a protective role on the root surface either during orthodontic treatment or in those patients that present spontaneous root resorptive lesions.

Salivary peroxidases.

Peroxidases are known to be involved in the intracellular metabolism of H2O2 coupled with various physiological functions. Apart from the thyroid gland, the enzyme has been isolated from various extrathyroidal sources of which salivary gland is one of the richest sources of the enzyme. The enzyme from bovine and goat submaxillary gland has been extensively studied in terms of their molecular, spectral, kinetic, catalytic and immunological properties and compared with the lactoperoxidase which is similar to the salivary peroxidase. The modulation of the salivary peroxidase by various factors and the probable mechanism of the modulation has been described. The enzyme has also been compared with the thyroid peroxidase as regards their physicochemical properties as well as on the immunological and functional aspects. The similarities and dissimilarities have been incorporated. The possible function of the enzyme in iodine metabolism and in bactericidal action has been discussed.

Effect of the antioxidant TK 12627 (Irganox) on monodeiodination and on the levels of messenger ribonucleic acid of 5'-deiodinase type I and spot 14.

Until now, most potent inhibitors of monodeiodination are iodinated, propylthiouracil being an exception. We report here studies on a new non-iodinated substance, triethylene glycol bis-3-(3-tert-butyl-4-hydroxy-5-methyl-phenyl) propionate (TK 12627 or Irganox), which is used as a very efficient antioxidant in the chemistry of plastics. The studies were performed with 23 hypothyroid rats that received Irganox in their daily food (8 mg.day-1 x (100 g body wt)-1) for 3 weeks. Thyroxine (T4) metabolism was studied by implanting minipumps delivering 2.3 nmol T4.day-1 x (100 g body wt)-1 for 1 week. On day 1 before sacrifice, another minipump containing [125I]-3,5,3'-triiodothyronine (T3, 2.6 microCi/day) and [131I]-3,3',5'-triiodothyronine (rT3, 2.1 microCi/day) was implanted. The results showed that with Irganox treatment serum T4 concentrations were higher (p < 0.05). Serum T3 concentrations markedly decreased (1.07 +/- 0.07 vs 0.65 +/- 0.04 nmol/l), accompanied by a decrease of free T3 concentrations (p < 0.001). Serum rT3 concentrations increased by 50% (p < 0.001). Serum thyrotropin levels were mostly unmeasurable. The plasma clearance rate decreased slightly for T4 (19%, p < 0.05) and remarkably for rT3 (46.7%, p < 0.001). The conversion rate of T4 to rT3 did not change. Deiodinase type I (5'D-I) activity decreased in both liver and kidney tissues by 54% and 52%, respectively, and correlated with T3 (r2 = 0.79 and 0.65, respectively). Brain deiodinase type III (5D-III) was unchanged and type II (5'D-II) was unmeasurable.(ABSTRACT TRUNCATED AT 250 WORDS)