Uptake and turnover of mono-iodinated thyroid hormone metabolites by PCCL3 thyrocytes

Tyrosine and phenolic ring de-iodination of thyroid hormones (TH) is crucial for regulating their physiological activity. Furthermore, reactions such as de-carboxylation to thyronamines (TAM) and de-amination to thyroacetic acids (TAc) produce TH metabolites (THM) with distinct biological properties. This needs to be considered when studying effects of TH and THM. The accurate and precise quantitative analysis of TH and THM in cell culture supernatants and cell lysates are key procedures required for studying the in vitro metabolism of TH. We report here the development of a liquid-liquid extraction/isotope dilution-liquid chromatography-electrospray tandem mass spectrometry (LC- MS/MS) method for the quantification of 9 thyronines (TN) and 6 TAM in human hepatocellular carcinoma Hep G2 cell lysate extracts. In addition, we adapted the method to quantify TH, TAM and TAc, in cell lysates of FBS-depleted rat thyroid epithelium PCCL3 cells. The methods for both cell lines were validated by rigorous assessment of linearity, limits of quantification and detection (LLOQ and LLOD respectively), intra- and inter-day accuracy, precision, process efficiency (PE), matrix effect (ME) and relative recovery (RE). Calibration curves covering 11 concentrations (based on 400 μl of lysate) were linear in the range 0.016–50 nM and 0.010–50 nM for Hep G2 and PCCL3 cells respectively. The lower limits of quantification were in the range 0.031 to 1 nM. We applied the PCCL3 version of the LC-MS/MS method to the analysis of lysed cell extracts from PCCL3 cells that had been incubated with 3-iodo-L-thyronine (T1), 3-iodothyronamine (3-T1AM) and 3-iodothyroacetic acid (3-T1Ac). Over the course of 30 minutes incubation 3-T1AM was de-iodinated to 4-[4-(2-aminoethylphenoxy)]phenol (thyronamine, T0AM) and de-aminated to 3-T1Ac respectively, whilst T1 underwent de-iodination to T0. This data indicates avid metabolism of these mono-iodinated compounds and the utility of LC-MS/MS to quantify such cellular metabolism

Serotonin transporter-deficient mice display enhanced adipose tissue inflammation after chronic high-fat diet feeding.

INTRODUCTION Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and mice. A possible link and interaction between the inflammatory effects of serotonin and metabolic derangements in SERT-deficient mice has not been investigated so far. METHODS SERT-deficient (Sert -/-) and wild type (WT) mice were fed a high-fat diet, starting at 8 weeks of age. Metabolic phenotyping (metabolic caging, glucose and insulin tolerance testing, body and organ weight measurements, qPCR, histology) and assessment of adipose tissue inflammation (flow cytometry, histology, qPCR) were carried out at the end of the 19-week high-fat diet feeding period. In parallel, Sert -/- and WT mice received a control diet and were analyzed either at the time point equivalent to high-fat diet feeding or as early as 8-11 weeks of age for baseline characterization. RESULTS After 19 weeks of high-fat diet, Sert -/- and WT mice displayed similar whole-body and fat pad weights despite increased relative weight gain due to lower starting body weight in Sert -/-. In obese Sert -/- animals insulin resistance and liver steatosis were enhanced as compared to WT animals. Leukocyte accumulation and mRNA expression of cytokine signaling mediators were increased in epididymal adipose tissue of obese Sert -/- mice. These effects were associated with higher adipose tissue mRNA expression of the chemokine monocyte chemoattractant protein 1 and presence of monocytosis in blood with an increased proportion of pro-inflammatory Ly6C+ monocytes. By contrast, Sert -/- mice fed a control diet did not display adipose tissue inflammation. DISCUSSION Our observations suggest that SERT deficiency in mice is associated with inflammatory processes that manifest as increased adipose tissue inflammation upon chronic high-fat diet feeding due to enhanced leukocyte recruitment

Role of the thyroid hormone metabolite 3-iodothyronamine in the regulation of the thyroid hormone homeostasis

3-Iodthyronamin (3-T1AM) ist ein in Menschen und Nagern endogener Schilddrüsenhormon (SDH)-Metabolit, der in murinem Darmgewebe durch Decarboxylierung und Deiodierung aus T4 gebildet werden kann. Die einmalige Injektion von 50 mg/kg 3-T1AM führt unter anderem zu Bradykardie und Hypothermie in Mäusen und Hamstern. Diese Effekte sind den Wirkungen eines Überschusses an klassischem SDH T3 entgegengesetzt. Es wurde außerdem gezeigt, dass dieselbe 3-T1AM-Dosis in Ratten Plasma T4 und TSH supprimiert, also mit der Hypothalamus-Hypophysen-Schilddrüsen (HPT)-Achse interferiert. Die Hypothese dieser Doktorarbeit ist, dass 3-T1AM durch direkte Wirkung auf die Schilddrüse (SD) an der Feinregulation der SDH-Homöostase beteiligt ist. Männlichen C57BL/6 Mäusen wurden 7 d lang täglich 5 mg/kg 3-T1AM oder die entsprechende Lösungsmittelmenge intraperitoneal appliziert. Die mRNA- Expression der SDH-Synthesegene Natrium-Iodid-Symporter (Nis), Thyreoglobulin und Pendrin waren nach 3-T1AM-Behandlung vermindert, was potenziell zu eingeschränkter SDH-Biosynthese führen kann. Eine Beteiligung der HPT-Achse konnte jedoch nicht nachgewiesen werden. Dies wurde an unveränderter Expression T3-responsiver Gene, die in die Regulation der HPT-Achse involviert sind, und unveränderten SDH-Serumkonzentrationen festgemacht. Eine funktionelle Auswirkung der 3-T1AM-Behandlung auf die SD trotz unveränderter zirkulierender SDH im Serum wurde in der Vergrößerung der SD-Follikellumina sichtbar. Versuche mit dem Iod-freien 3-T1AM-Metaboliten T0AM zeigen, dass dieser unter gleichen Behandlungsbedingungen ein eigenes Wirkprofil in Mäusen besitzt, jedoch auch thyroidale Genexpression und Morphologie beeinflusst. In vitro Studien in PCCL3 Rattenthyreozyten ergänzen die Befunde der Mausstudien. 3-T1AM wird von PCCL3 Zellen aufgenommen und verstoffwechselt. Die entstandenen Metaboliten T0AM und 3-Iodthyroessigsäure (3-TA1) wurden von den Zellen in das Zellkulturmedium sezerniert. Sowohl Nis-Expression als auch Funktion in Form von Iodidaufnahme wurden durch 3-T1AM in PCCL3 Zellen vermindert. Auch eine partielle Inhibition der Dio1-Aktivität, sowie der Glucoseverwertung wurden beobachtet. Mechanistisch konnte 3-T1AM-Signalgebung über adrenerge oder Taar1-vermittelte cAMP-Modulation in diesem Modell weitgehend ausgeschlossen werden, während ein starker 3-T1AM-abhängiger Anstieg intrazellulären Calciums beobachtet wurde. T0AM könnte den inhibitorischen Effekt von 3-T1AM auf Nis in PCCL3 Zellen (teilweise) vermitteln. Zusammenfassend hat 3-T1AM das Potenzial auf mehreren Ebenen mit der SDH-Biosynthese zu interferieren und so die SDH-Homöostase durch direkte Wirkung auf die SD zu regulieren. Ein in vitro Modell funktioneller SD- Follikel aus murinen embryonalen Stammzellen, das 2012 entwickelt wurde, wurde im Rahmen der Doktorarbeit erlernt und im Labor etabliert. In diesem Modell soll in zukünftigen Studien die direkte Wirkung von 3-T1AM auf die SDH- Biosynthese untersucht werden.3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone (TH) metabolite in humans and rodents, which can be formed in murine intestinal tissue by decarboxylation and deiodination from T4. A single injection of 50 mg/kg 3-T1AM leads to bradycardia and hypothermia in mice and Djungarian hamsters. These effects are opposite to the effects of an excess of the classic TH T3. It has also been demonstrated that the same 3-T1AM dose suppresses plasma T4 and TSH in rats, indicating interference with the hypothalamus pituitary thyroid (HPT) axis. The hypothesis of this doctoral thesis is that 3-T1AM is involved in the fine tuning of the TH homeostasis by direct action on the thyroid gland. Male C57BL/6 mice were intraperitoneally injected with 5 mg/kg 3-T1AM or the corresponding amount of solvent for 7 d daily. The mRNA expression of the TH synthesis genes sodium iodide symporter (Nis), thyroglobulin, and pendrin were reduced after 3-T1AM treatment, potentially leading to reduced TH biosynthesis. However, no involvement of the HPT axis was observed. This was attributed to unchanged expression of T3 responsive genes involved in the regulation of the HPT axis and unaltered TH serum concentrations. A functional effect of 3-T1AM treatment on the thyroid despite unchanged circulating TH was observed in the enlargement of thyroid follicular lumina. Experiments with the iodine-free 3-T1AM metabolite T0AM show that the latter exerts its own activity profile in mice under the same treatment conditions, but also affects thyroid gene expression and morphology. The findings of the mouse studies were complemented with in vitro studies in PCCL3 rat thyrocytes. 3-T1AM is taken up by PCCL3 cells and is intracellularly metabolized. The resulting metabolites T0AM and 3-iodothyroacetic acid (3-TA1) were secreted from the cells into the cell culture medium. Both, Nis expression and function in the form of iodide uptake were reduced by 3-T1AM in PCCL3 cells. A partial inhibition of Dio1 activity as well as glucose utilization were also observed. Mechanistically, 3-T1AM signaling via adrenergic or Taar1-mediated cAMP modulation could be largely excluded in this model, whereas a strong 3-T1AM-dependent increase in intracellular calcium was observed, which needs to be further characterized. T0AM could (partially) mediate the inhibitory effect of 3-T1AM on Nis in PCCL3 cells. In summary, 3-T1AM has the potential to interfere with the TH biosynthesis machinery at several levels and thus regulate the TH homeostasis by direct action on the thyroid gland. An in vitro model of functional thyroid follicles from murine embryonic stem cells, which was published in 2012, was implemented within the framework of the doctoral thesis and established in the laboratory. In this model, the direct effect of 3-T1AM on TH biosynthesis will be investigated in future studies

Reticulated Platelets—Which Functions Have Been Established by In Vivo and In Vitro Data?

Reticulated platelets (RP) are the youngest platelet fraction released into the circulation. These immature platelets have increased RNA content, a larger cell volume, more dense granules, higher levels of surface activation markers and are thought to be more reactive compared to their mature counterparts. RP have been associated with cardiovascular disease, diabetes and increased mortality. Yet only a few animal studies investigating RP have been conducted so far and further investigations are warranted. Established methods to count RP are flow cytometry (staining with thiazole orange or SYTO13) or fully automated hematology analyzers (immature platelet fraction, IPF). IPF has been established as a diagnostic parameter in thrombocytopenia, cardiovascular disease and, in particular, the response to antiplatelet therapy. This review seeks to provide an overview of the key features of RP as well as preanalytical and analytical aspects that need to be considered when working with this platelet population

Oncostatin M produced in Kupffer cells in response to PGE2: possible contributor to hepatic insulin resistance and steatosis

Hepatic insulin resistance is a major contributor to hyperglycemia in metabolic syndrome and type II diabetes. It is caused in part by the low-grade inflammation that accompanies both diseases, leading to elevated local and circulating levels of cytokines and cyclooxygenase (COX) products such as prostaglandin E2 (PGE2). In a recent study, PGE2 produced in Kupffer cells attenuated insulin-dependent glucose utilization by interrupting the intracellular signal chain downstream of the insulin receptor in hepatocytes. In addition to directly affecting insulin signaling in hepatocytes, PGE2 in the liver might affect insulin resistance by modulating cytokine production in non-parenchymal cells. In accordance with this hypothesis, PGE2 stimulated oncostatin M (OSM) production by Kupffer cells. OSM in turn attenuated insulin-dependent Akt activation and, as a downstream target, glucokinase induction in hepatocytes, most likely by inducing suppressor of cytokine signaling 3 (SOCS3). In addition, it inhibited the expression of key enzymes of hepatic lipid metabolism. COX-2 and OSM mRNA were induced early in the course of the development of non-alcoholic steatohepatitis (NASH) in mice. Thus, induction of OSM production in Kupffer cells by an autocrine PGE2-dependent feed-forward loop may be an additional, thus far unrecognized, mechanism contributing to hepatic insulin resistance and the development of NASH

Photophysics of Platinum Tetrayne Oligomers: Delocalization of Triplet Exciton

A series of platinum tetrayne oligomers, <i>all-trans</i>-Cl–Pt­(P₂)–[(CC)₄–Pt­(P₂)]_<i>n</i>–Cl, where P = tri­(<i>p</i>-tolyl)­phosphine and <i>n</i> = 1–3, was subjected to a detailed photophysical investigation. The photoluminescence of each oligomer at low temperature (<i>T < </i>140 K) in a 2-methyltetrahydrofuran (Me-THF) glass features an intense and narrow 0–0 phosphorescence band accompanied by a vibronic progression of sub-bands separated by ca. 2100 cm^–1. The emission arises from a ³π,π* triplet state concentrated on the (CC)₄ carbon chain and the vibronic progression originates from coupling of the excitation to the ν­(CC) stretch. All of the experimental data including ambient temperature absorption, low-temperature photoluminescence, and ambient temperature transient absorption spectroscopy provide clear evidence that the triplet state is localized on a chromophore consisting of approximately two −[(CC)₄–Pt­(P₂)]– repeat units. Density functional theory calculations support the hypothesis that the triplet–triplet absorption arises from transitions that are delocalized over two repeat units

Inter-day (n = 8) accuracy and precision for TH and TAM spiked lysed Hep G2 cell extracts in 24-well plates.

<p>Inter-day (n = 8) accuracy and precision for TH and TAM spiked lysed Hep G2 cell extracts in 24-well plates.</p

Negative ion electrospray tandem mass spectrometry and retention time (R_t) parameters for thyroacetic acids and internal standards.

<p>Negative ion electrospray tandem mass spectrometry and retention time (R_t) parameters for thyroacetic acids and internal standards.</p

Application of the LC-MS/MS method to extracts of PCCL3 rat thyrocytes.

<p>THM in extracts of PCCL3 (A, B) cell and supernatants (C, D) after incubation with 500 nM 3-T₁AM (A, C) or 3-T₁ (B, D) in Krebs-Ringer buffer as measured by LC-MS/MS. Bars represent means of 3 independent experiments carried out on different days +/- SEM.</p