miR-199a Downregulation as a Driver of the NOX4/HIF-1α/VEGF-A Pathway in Thyroid and Orbital Adipose Tissues from Graves' Patients.

Graves' disease (GD) is an autoimmune thyroiditis often associated with Graves' orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.

Oxidative Stress-Induced Sirtuin1 Downregulation Correlates to HIF-1α, GLUT-1, and VEGF-A Upregulation in Th1 Autoimmune Hashimoto's Thyroiditis.

In Hashimoto's thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines' response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.

Brief O2 uploading during continuous hypothermic machine perfusion is simple yet effective oxygenation method to improve initial kidney function in a porcine autotransplant model.

With oxygenation proposed as a resuscitative measure during hypothermic models of preservation, the aim of this study was to evaluate the optimal start time of oxygenation during continuous hypothermic machine perfusion (HMP). In this porcine ischemia-reperfusion autotransplant model, the left kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia prior to 22 hours of HMP and autotransplantation. Kidneys were randomized to receive 2 hours of oxygenation during HMP either at the start (n = 6), or end of the perfusion (n = 5) and outcomes were compared to standard, nonoxygenated HMP (n = 6) and continuous oxygenated HMP (n = 8). The brief initial and continuous oxygenated HMP groups were associated with superior graft recovery compared to either standard, nonoxygenated HMP or kidneys oxygenated at the end of HMP. This correlated with significant metabolic differences in perfusate (eg, lactate, succinate, flavin mononucleotide) and tissues (eg, succinate, adenosine triphosphate, hypoxia-inducible factor-1α, nuclear factor erythroid 2-related factor 2) suggesting superior mitochondrial preservation with initial oxygenation. Brief initial O2 uploading during HMP at procurement site might be an easy and effective preservation strategy to maintain aerobic metabolism, protect mitochondria, and achieve an improved early renal graft function compared with standard HMP or oxygen supply shortly at the end of HMP preservation.

Modulation of VEGF Expression and Oxidative Stress Response by Iodine Deficiency in Irradiated Cancerous and Non-Cancerous Breast Cells.

Breast cancer remains a major concern and its physiopathology is influenced by iodine deficiency (ID) and radiation exposure. Since radiation and ID can separately induce oxidative stress (OS) and microvascular responses in breast, their combination could additively increase these responses. Therefore, ID was induced in MCF7 and MCF12A breast cell lines by medium change. Cells were then X-irradiated with doses of 0.05, 0.1, or 3 Gy. In MCF12A cells, both ID and radiation (0.1 and 3 Gy) increased OS and vascular endothelial growth factor (VEGF) expression, with an additive effect when the highest dose was combined with ID. However, in MCF7 cells no additive effect was observed. VEGF mRNA up-regulation was reactive oxygen species (ROS)-dependent, involving radiation-induced mitochondrial ROS. Results on total VEGF mRNA hold true for the pro-angiogenic isoform VEGF165 mRNA, but the treatments did not modulate the anti-angiogenic isoform VEGF165b. Radiation-induced antioxidant response was differentially regulated upon ID in both cell lines. Thus, radiation response is modulated according to iodine status and cell type and can lead to additive effects on ROS and VEGF. As these are often involved in cancer initiation and progression, we believe that iodine status should be taken into account in radiation prevention policies.

Acute iodine deficiency induces a transient VEGF-dependent microvascular response in mammary glands involving HIF-1, ROS, and mTOR.

Iodine deficiency (ID), which affects almost two billion people worldwide, is associated with breast pathologies such as fibrosis in human and induces breast atypia in animal models. Because ID induces vascular activation in the thyroid, another iodide-uptaking organ, and as breast is also sensitive to ID, we aimed to characterize ID-induced effects on the breast microvasculature in vivo and in two different breast cell lines in vitro. Virgin and lactating NMRI mice received an iodide-deficient diet and a Na+/I- symporter inhibitor for 1 to 20 days. Some virgin mice were treated with vascular endothelial growth factor A (VEGF) or VEGF receptor inhibitors. In vitro, ID was induced in MCF7 and MCF12A cells by replacing the iodide-containing medium by an iodide-deficient medium. In vivo, VEGF expression was increased following ID in mammary tissues. Consequently, ID induced a transient increase in mammary gland blood flow, measured after anesthesia, in virgin and lactating mice, which was repressed by VEGF or VEGF receptor inhibitors. In MCF7 cells, ID induced a transient increase in reactive oxygen species, followed by an increase in hypoxia-inducible factor-1α (HIF-1α) protein and VEGF mRNA expression. Antioxidant N-acetylcysteine and mammalian target of rapamycin (mTOR) inhibitor blocked ID-induced HIF-1α protein increase and VEGF transcription. However, mTOR activity was not inhibited by N-acetylcysteine. Similar responses were observed in MCF12A cells. These data indicate that ID activates the canonical VEGF pathway and mTOR in breast tissues, which provides new insights to better understand the correlation between ID, vascular activation, and breast pathologies.

Adiponectin and skeletal muscle: pathophysiological implications in metabolic stress.

Upregulation of muscular adiponectin could act as a local protective mechanism to counteract cellular damage in obesity by weakening inflammation, oxidative stress, and apoptosis. To test this hypothesis, adiponectin-knockout (KO) and wild-type (WT) mice were fed a Western diet (WD). WT mice under WD conditions displayed 63% higher adiponectin expression in myocytes than those under standard laboratory diet (SLD) conditions (P = 0.011). WD-fed KO mice exhibited approximately threefold larger myocyte degeneration than WT mice (P = 0.003). Even under SLD conditions, myotubes of KO mice displayed already moderate immunolabeling for markers of oxidative stress (peroxiredoxin-3/5) and for a lipid peroxidation product (hydroxynonenal). Expression of tumor necrosis factor-α (TNF-α) and caspase-6, a marker of apoptosis, was also present. After WD challenge, immunoreactivity for these markers was strong in muscle of KO mice, although it was detected to a lesser extent in WT mice. Activation of NF-κB and caspase-6 doubled in myocytes of WD-fed KO mice when compared to WT mice (P < 0.001). Furthermore, muscle electrotransfer of the adiponectin gene prevented these abnormalities in WD-fed KO mice. Finally, gene abrogation of the adiponectin receptor 1 (AdipoR1) by siRNA recapitulated a pro-inflammatory state in C2C12 myotubes. Thus, upregulation of muscular adiponectin may be triggered by obesity and be crucial locally to counteract oxidative stress, inflammation, and apoptosis. These effects operate in an autocrine/paracrine manner via AdipoR1 and down-regulation of NF-κB signaling.

Vitrification preserves proliferation capacity in human spermatogonia.

STUDY QUESTION: Does vitrification of human immature testicular tissue (ITT) have potential benefits for future fertility preservation? Does vitrification of human ITT have potential benefits in an in vivo murine xenotransplantation model? SUMMARY ANSWER: Vitrification is able to maintain proliferation capacity in spermatogonial cells after 6 months of xenografting. WHAT IS KNOWN ALREADY: Controlled slow-freezing is the procedure currently applied for ITT cryobanking in clinical practice. Vitrification has been proposed as a promising technique for long-term storage of ITT, with a view to preserving spermatogonial stem cells (SSCs) for future fertility restoration in young boys suffering from cancer. After vitrification of ITT, in vitro survival of SSCs was demonstrated, but their functionality was not evaluated. STUDY DESIGN, SIZE, DURATION: Ten ITT pieces issuing from 10 patients aged 2-12 years were used. Fragments of fresh tissue (serving as controls) and fresh, frozen-thawed and vitrified-warmed testicular pieces xenografted to the scrotum of nude mice for 6 months were compared. MATERIALS, SETTING, METHODS: Upon graft removal, histological and immunohistochemical analyses were performed to evaluate spermatogonia (SG) (MAGE-A4), intratubular proliferation (Ki67), proliferating SG and Leydig cells (3ß-HSD). The entire piece of grafted tissue was assessed in each case. MAIN RESULTS AND THE ROLE OF CHANCE: Seminiferous tubules showed good integrity after cryopreservation and xenografting for 6 months in all three groups. Survival of SG and their ability to proliferate was observed by immunohistochemistry in all grafted groups. SG were able to initiate spermatogenesis, but blockage at the pachytene stage was observed. The recovery rate of SG was 3.4 ± 3.8, 4.1 ± 7.3 and 7.3 ± 6.3%, respectively, for fresh, slow-frozen and vitrified-warmed tissue after 6 months of xenografting. LIMITATIONS, REASONS FOR CAUTION: The study is limited by the low availability of ITT samples of human origin. The mouse xenotransplantation model needs to be refined to study human spermatogenesis. WIDER IMPLICATIONS OF THE FINDINGS: The findings of the present study have potential implications for cryobanking of ITT and fertility preservation. Spermatogonial loss recorded after fresh ITT transplantation indicates that the avascular grafting technique needs to be optimized. There are so far no convincing data justifying modification of current clinical practice for ITT storage with slow-freezing, but this study demonstrates that it is worth pursuing optimization of ITT vitrification as an alternative for preservation of SSCs. STUDY FUNDING/COMPETING INTEREST(S): The present study was supported by a grant from the Fonds National de la Recherche Scientifique de Belgique (grant Télévie N° 7. 4.572.09.F). The authors declare that there is no conflict of interest.

N-acetylcysteine and 15 deoxy-{delta}12,14-prostaglandin J2 exert a protective effect against autoimmune thyroid destruction in vivo but not against interleukin-1{alpha}/interferon {gamma}-induced inhibitory effects in thyrocytes in vitro.

Reactive oxygen species (ROS) are crucial for thyroid hormonogenesis, and their production is kept under tight control. Oxidative stress (OS) is toxic for thyrocytes in an inflammatory context. In vitro, Th1 pro-inflammatory cytokines have already been shown to decrease thyroid-specific protein expression. In the present study, OS level and its impact on thyroid function were analyzed in vitro in Th1 cytokine (interleukin [IL]-1alpha/interferon [IFN] gamma)-incubated thyrocytes (rat and human), as well as in vivo in thyroids from nonobese diabetic mice, a model of spontaneous autoimmune thyroiditis. N-acetylcysteine (NAC) and prostaglandin, 15 deoxy-(Delta12,14)-prostaglandinJ2 (15dPGJ2), were used for their antioxidant and anti-inflammatory properties, respectively. ROS production and OS were increased in IL-1alpha/IFNgamma-incubated thyrocytes and in destructive thyroiditis. In vitro, NAC not only reduced ROS production below control levels, but further decreased the expression of thyroid-specific proteins in addition to IL-1alpha/IFNgamma-inhibitory effects. Thus, besides ROS, other intracellular intermediaries likely mediate Th1 cytokine effects. In vivo, NAC and 15dPGJ2 reduced OS and the immune infiltration, thereby leading to a restoration of thyroid morphology. It is therefore likely that NAC and 15dPGJ2 mainly exert their protective effects by acting on infiltrating inflammatory cells rather than directly on thyrocytes.

Downregulation of Caveolin-1 and Upregulation of Deiodinase 3, Associated with Hypoxia-Inducible Factor-1α Increase, Are Involved in the Oxidative Stress of Graves' Orbital Adipocytes.

Background: Even though the clinical features of Graves' orbitopathy (GO) are well known, its exact pathogenesis remains controversial. The imbalance of redox homeostasis in the connective tissue could play a crucial role leading to an inflammatory state and edema of soft orbital tissues, thus contributing to orbital hypoxia and increase in hypoxia-inducible factor (HIF)-1α. This oxidative stress appears to target the orbital cells such as fibroblasts and also adipocytes. This study aims to explore which pathways can lead to the aforementioned oxidative stress in GO adipose cells and therefore offers new plausible therapeutic targets. Methods: Orbital fat samples were obtained from patients with GO (Western blot [WB]: n = 8, immunohistochemistry [IHC]: n = 8) and from control patients (WB: n = 5, IHC: n = 3-5). They were processed for WB analysis and IHC of the antioxidants (catalase, superoxide dismutase 1) and for HIF-1α. The expression of caveolin-1 (Cav-1) and deiodinase 3 (DIO3), known to be regulated by HIF-1α, was also analyzed by WB and IHC, as well as the targets of Cav-1: glucose transporter type 4 (Glut-4), NADPH oxidase (NOX)-2, and endothelial nitric oxide synthase (eNOS). Triiodothyronine (T3) expression was also analyzed by IHC. Results: In GO adipocytes, the expression of catalase was reduced, whereas that of HIF-1α was strongly increased. A decreased local T3 supply was associated with DIO3 upregulation. The low expression of Cav-1 in GO adipocytes was associated not only with low expression of Glut-4 but also with an increased expression of NOX-2 and active eNOS phosphorylated on serine 1177. Conclusions: Cav-1 and DIO3, both sensitive to hypoxia and to the increase of HIF-1α, play a pivotal role in the oxidative stress in GO adipocytes. DIO3 regulates the cellular supply of T3, which is essential for the cell homeostasis. Cav-1 determines the cellular glucose supply through Glut-4 and regulates the activity of NOX-2 generating superoxide anions and that of eNOS generating nitric oxide (NO).

Influence of Different Partial Pressures of Oxygen During Continuous Hypothermic Machine Perfusion in a Pig Kidney Ischemia-reperfusion Autotransplant Model.

BACKGROUND: The optimal perfusate partial pressure of oxygen (PO2) during hypothermic machine perfusion (HMP) is unknown. The aims of the study were to determine the functional, metabolic, structural, and flow dynamic effects of low and high perfusate PO2 during continuous HMP in a pig kidney ischemia-reperfusion autotransplant model. METHODS: The left kidneys of a ±40 kg pigs were exposed to 30 minutes of warm ischemia and randomized to receive 22-hour HMP with either low perfusate PO2 (30% oxygen, low oxygenated HMP [HMPO2]) (n = 8) or high perfusate PO2 (90% oxygen, HMPO2high) (n = 8), before autotransplantation. Kidneys stored in 22-hour standard HMP (n = 6) and 22-hour static cold storage (n = 6) conditions served as controls. The follow-up after autotransplantation was 13 days. RESULTS: High PO2 resulted in a 3- and 10-fold increase in perfusate PO2 compared with low HMPO2 and standard HMP, respectively. Both HMPO2 groups were associated with superior graft recovery compared with the control groups. Oxygenation was associated with a more rapid and sustained decrease in renal resistance. While there was no difference in functional outcomes between both HMPO2 groups, there were clear metabolic differences with an inverse correlation between oxygen provision and the concentration of major central metabolites in the perfusion fluid but no differences were observed by oxidative stress and metabolic evaluation on preimplantation biopsies. CONCLUSIONS: While this animal study does not demonstrate any advantages for early graft function for high perfusate PO2, compared with low perfusate PO2, perfusate metabolic profile analysis suggests that aerobic mechanism is better supported under high perfusate PO2 conditions.

Role of caveolin-1 in thyroid phenotype, cell homeostasis, and hormone synthesis: in vivo study of caveolin-1 knockout mice.

In human thyroid, caveolin-1 is localized at the apex of thyrocytes, but its role there remains unknown. Using immunohistochemistry, (127)I imaging, transmission electron microscopy, immunogold electron microscopy, and quantification of H(2)O(2), we found that in caveolin-1 knockout mice thyroid cell homeostasis was disrupted, with evidence of oxidative stress, cell damage, and apoptosis. An even more striking phenotype was the absence of thyroglobulin and iodine in one-half of the follicular lumina and their presence in the cytosol, suggesting that the iodide organification and binding to thyroglobulin were intracellular rather than at the apical membrane/extracellular colloid interface. The latter abnormality may be secondary to the observed mislocalization of the thyroid hormone synthesis machinery (dual oxidases, thyroperoxidase) in the cytosol. Nevertheless, the overall uptake of radioiodide, its organification, and secretion as thyroid hormones were comparable to those of wild-type mice, suggesting adequate compensation by the normal TSH retrocontrol. Accordingly, the levels of free thyroxine and TSH were normal. Only the levels of free triiodothyronine showed a slight decrease in caveolin-1 knockout mice. However, when TSH levels were increased through low-iodine chow and sodium perchlorate, the induced goiter was more prominent in caveolin-1 knockout mice. We conclude that caveolin-1 plays a role in proper thyroid hormone synthesis as well as in cell number homeostasis. Our study demonstrates for the first time a physiological function of caveolin-1 in the thyroid gland. Because the expression and subcellular localization of caveolin-1 were similar between normal human and murine thyroids, our findings in caveolin-1 knockout mice may have direct relevance to the human counterpart.

Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content.

In basal conditions, thyroid epithelial cells produce moderate amounts of reactive oxygen species (ROS) that are physiologically required for thyroid hormone synthesis. They are not necessarily toxic because they are continuously detoxified either in the process of hormone synthesis or by endogenous antioxidant systems. Using a rat model of goiter formation and iodine-induced involution, we found that compared with control thyroids, the oxidative stress, assessed by the detection of 4-hydroxynonenal, was strongly enhanced both in hyperplastic and involuting glands. The level of antioxidant defenses (glutathione peroxidases and peroxiredoxins) was also up-regulated in both groups, although somewhat less in the latter. Of note, increased oxidative stress came along with an inflammatory reaction, but only in involuting glands, suggesting that although antioxidant systems can adequately buffer a heavy load of ROS in goiter, it is not necessarily the case in involuting glands. The effects of 15-deoxy-Delta(12,14)-prostaglandin J2 (15dPGJ2), an endogenous ligand of peroxisome proliferated-activated receptor gamma (PPARgamma) with antiinflammatory properties, were then investigated in involuting glands. This drug strongly reduced both 4-hydroxynonenal staining and the inflammatory reaction, indicating that it can block iodine-induced cytotoxicity. When experiments were carried out with the PPARgamma antagonist, bisphenol A diglycidyl ether, 15dPGJ2-induced effects remained unchanged, suggesting that these effects were not mediated by PPARgamma. In conclusion, thyroid epithelial cells are well adapted to endogenously produced ROS in basal and goitrous conditions. In iodine-induced goiter involution, the increased oxidative stress is accompanied by inflammation that can be blocked by 15dPGJ2 through PPARgamma-independent protective effects.

Reciprocal negative regulation between thyrotropin/3',5'-cyclic adenosine monophosphate-mediated proliferation and caveolin-1 expression in human and murine thyrocytes.

The expression of caveolins is down-regulated in tissue samples of human thyroid autonomous adenomas and in the animal model of this disease. Because several cell types present in thyroid express caveolins, it remained unclear if this down-regulation occurs in thyrocytes and which are the mechanism and role of this down-regulation in the tumor context. Here we show that prolonged stimulation of isolated human thyrocytes by TSH/cAMP/cAMP-dependent protein kinase inhibits caveolins' expression. The expression of caveolins is not down-regulated by activators of other signaling pathways relevant to thyroid growth/function. Therefore, the down-regulation of caveolins' expression in autonomous adenomas is a direct consequence of the chronic activation of the TSH/cAMP pathway in thyrocytes. The down-regulation of caveolin-1 occurs at the mRNA level, with a consequent protein decrease. TSH/cAMP induces a transcription-dependent, translation-independent destabilization of the caveolin-1 mRNA. This effect is correlated to the known proliferative role of that cascade in thyrocytes. In vivo, thyrocytes of caveolin-1 knockout mice display enhanced proliferation. This demonstrates, for the first time, the in vivo significance of the specific caveolin-1 down-regulation by one mitogenic cascade and its relation to a human disease.

Proteomic profiling of cold thyroid nodules.

Cold thyroid nodules (CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs (10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs.

Overexpression of adiponectin targeted to adipose tissue in transgenic mice: impaired adipocyte differentiation.

Adiponectin (ApN) is an adipokine whose expression and plasma levels are inversely related to obesity and insulin-resistant states. Chronic repercussions of ApN treatment or overexpression on adiposity and body weight are still controversial. Here, we generated a transgenic (Tg) mouse model allowing persistent and moderate overexpression of native full-length ApN targeted to white adipose tissue. Adipose mass and adipocyte size of Tg mice were reduced despite preserved calorie intake. This reduction resulted from increased energy expenditure and up-regulation of uncoupling proteins, and from abrogation of the adipocyte differentiation program, as shown by the loss of a key lipogenic enzyme and of adipocyte markers. Adipose mass remodeling favors enhanced insulin sensitivity and improved lipid profile of Tg mice. Alteration of the adipocyte phenotype was likely to result from increased expression of the preadipocyte factor-1 and from down-regulation of the transcription factor, CCAAT/enhancer binding protein-alpha, which orchestrates adipocyte differentiation. We further found that recombinant ApN directly stimulated pre- adipocyte factor-1 mRNA and attenuated CCAAT/enhancer binding protein-alpha expression in cultured 3T3-F442A cells. Conversely, opposite changes in the expression of these genes were observed in white fat of ApN-deficient mice. Thus, besides enhanced energy expenditure, our work shows that impairment of adipocyte differentiation contributes to the anti-adiposity effect of ApN.

Iodine deficiency induces a VEGF-dependent microvascular response in salivary glands and in the stomach.

Despite efforts to optimize iodine supply in iodine deficient countries, iodine deficiency (ID) remains a global problem worldwide. Activation of the local microvasculature by ID in the thyroid gland aims at improving the local supply of iodide. For this purpose, the thyrocytes secrete vascular endothelial growth factor (VEGF) that acts on adjacent capillaries, via a reactive oxygen species (ROS)/Hypoxia Inducible factor (HIF)-dependent pathway. Beside the thyroid, other organs including salivary glands and the stomach do express the sodium/iodide symporter (NIS) and are able to take iodide up, potentially rendering them sensitive to ID. To verify this hypothesis, ID-induced effects on the local microvasculature were studied in salivary glands and in the stomach. ID was induced by feeding young mice with an iodide-deficient diet and NIS inhibitor perchlorate in the drinking water. In salivary glands, ID induced a transient increase in HIF-1α protein expression accompanied by a transient, VEGF-dependent increase in blood flow. In the gastric mucosa, ID transiently increased VEGF expression in the mucin-secreting epithelium and in ghrelin-secreting endocrine cells. These observations suggest that microvascular changes in response to ID occur in NIS-expressing tissues other than the thyroid. NIS expressing cells could be viewed as iodide sensors that respond to ID by inducing vascular changes, probably to optimize iodide bioavailability at regional or systemic levels.

Extrapancreatic effects of incretin hormones: evidence for weight-independent changes in morphological aspects and oxidative status in insulin-sensitive organs of the obese nondiabetic Zucker rat (ZFR).

Incretin-based therapies are widely used to treat type 2 diabetes. Although hypoglycemic actions of incretins are mostly due to their insulinotropic/glucagonostatic effects, they may also influence extrapancreatic metabolism. We administered exendin-4 (Ex-4), a long-acting glucagon-like peptide receptor agonist, at low dose (0.1 nmol/kg/day) for a short period (10 days), in obese nondiabetic fa/fa Zucker rats (ZFRs). Ex-4-treated ZFRs were compared to vehicle (saline)-treated ZFRs and vehicle- and Ex-4-treated lean rats (LRs). Blood glucose levels were measured at days 0, 9, and 10. Ingested food and animal weight were recorded daily. On the day of sacrifice (d10), blood was sampled along with liver, epididymal, subcutaneous, brown adipose, and skeletal muscle tissues from animals fasted for 24 h. Plasma insulin and blood glucose levels, food intake, and body and epididymal fat weight were unchanged, but gross morphological changes were observed in insulin-sensitive tissues. The average size of hepatocytes was significantly lower in Ex-4-treated ZFRs, associated with decreased number and size of lipid droplets and 4-hydroxy-2-nonenal (HNE) staining, a marker of oxidative stress (OS). Myocytes, which were smaller in ZFRs than in LRs, were significantly enlarged and depleted of lipid droplets in Ex-4-treated ZFRs. Weak HNE staining was increased by Ex-4. A similar observation was made in brown adipose tissue, whereas the elevated HNE staining observed in epididymal adipocytes of ZFRs, suggestive of strong OS, was decreased by Ex-4. These results suggest that incretins by acting on OS in insulin-sensitive tissues may contribute to weight-independent improvement in insulin sensitivity.

Pioglitazone, a PPARγ Agonist, Upregulates the Expression of Caveolin-1 and Catalase, Essential for Thyroid Cell Homeostasis: A Clue to the Pathogenesis of Hashimoto's Thyroiditis.

BACKGROUND: Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates the expression of multiple target genes involved in several metabolic pathways as well as in inflammation. The expression and cell localization of caveolin-1 (Cav-1), thyroperoxidase (TPO), and dual oxidase (DUOX), involved in extracellular iodination, is modulated by Th1 cytokines in human normal thyroid cells and in Hashimoto's thyroiditis (HT). OBJECTIVES: The objectives of this study were (i) to analyze the PPARγ protein and mRNA expression at the follicular level in HT versus controls in correlation with the one of Cav-1; (ii) to study the effects of Th1 cytokines on PPARγ and catalase expression in human thyrocyte primary cultures; and (iii) to study the effects of pioglitazone, a PPARγ agonist, on thyroxisome components (Cav-1, TPO, DUOX) and on catalase, involved in antioxidant defense. RESULTS: Although the global expression of PPARγ in the whole gland of patients with HT was not modified compared with controls, there was great heterogeneity among glands and among follicles within the same thyroid. Besides normal (type 1) follicles, there were around inflammatory zones, hyperactive (type 2) follicles with high PPARγ and Cav-1 expression, and inactive (type 3) follicles which were unable to form thyroxine and did not express PPARγ or Cav-1. In human thyrocytes in primary culture, Th1 cytokines decreased PPARγ and catalase expression; pioglitazone increased Cav-1, TPO, and catalase expression. CONCLUSION: PPARγ may play a central role in normal thyroid physiology by upregulating Cav-1, essential for the organization of the thyroxisome and extracellular iodination. By upregulating catalase, PPARγ may also contribute to cell homeostasis. The inhibitory effect of Th1 cytokines on PPARγ expression may be considered as a new pathogenetic mechanism for HT, and the use of PPARγ agonists could open a new therapeutic approach.

Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin.

BACKGROUND: The dual oxidases (Duox) are involved in hydrogen peroxide generation, which is essential for thyroid hormone synthesis, and therefore they are markers of thyroid function. During inflammation, cytokines upregulate DUOX gene expression in the airway and the intestine, suggesting a role for these proteins in innate immunity. It was previously demonstrated that interleukin-4 (IL-4) upregulates DUOX gene expression in thyrocytes. Although the role of IL-4 in autoimmune thyroid diseases has been studied extensively, the effects of IL-4 on thyroid physiology remain largely unknown. Therefore, a new animal model was generated to study the impact of IL-4 on thyroid function. METHODS: Transgenic (Thyr-IL-4) mice with thyroid-targeted expression of murine IL-4 were generated. Transgene expression was verified at the mRNA and protein level in thyroid tissues and primary cultures. The phenotype of the Thyr-IL-4 animals was characterized by measuring serum thyroxine (T4) and thyrotropin levels and performing thyroid morphometric analysis, immunohistochemistry, whole transcriptome sequencing, quantitative reverse transcription polymerase chain reaction, and ex vivo thyroid function assays. RESULTS: Thyrocytes from two Thyr-IL-4 mouse lines (#30 and #52) expressed IL-4, which was secreted into the extracellular space. Although 10-month-old transgenic animals had T4 and thyrotropin serum levels in the normal range, they had altered thyroid follicular structure with enlarged follicles composed of elongated thyrocytes containing numerous endocytic vesicles. These follicles were positive for T4 staining the colloid, indicating their capacity to produce thyroid hormones. RNA profiling of Thyr-IL-4 thyroid samples revealed modulation of multiple genes involved in inflammation, while no major leukocyte infiltration could be detected. Upregulated expression of Duox1, Duoxa1, and the pendrin anion exchanger gene (Slc26a4) was detected. In contrast, the iodide symporter gene Slc5a5 was markedly downregulated resulting in impaired iodide uptake and reduced thyroid hormone levels in transgenic thyroid tissue. Hydrogen peroxide production was increased in Thyr-IL-4 thyroid tissue compared with wild-type animals, but no significant oxidative stress could be detected. CONCLUSIONS: This is the first study to show that ectopic expression of IL-4 in thyroid tissue upregulates Duox1/Duoxa1 and Slc26a4 expression in the thyroid. The present data demonstrate that IL-4 could affect thyroid morphology and function, mainly by downregulating Slc5a5 expression, while maintaining a normal euthyroid phenotype.

Oxidative Stress and Upregulation of Antioxidant Proteins, Including Adiponectin, in Extraocular Muscular Cells, Orbital Adipocytes, and Thyrocytes in Graves' Disease Associated with Orbitopathy.

BACKGROUND: Graves' orbitopathy (GO) is the main extrathyroidal manifestation associated with Graves' disease (GD). It is characterized by reduced eye motility due to an increased volume of orbital fat and/or of extraocular muscles (EOMs) infiltrated by fibrosis and adipose tissue. The pathogenetic mechanisms leading to fibrosis and adipogenesis are mainly based on the interaction between orbital fibroblasts and immune cells (lymphocytes and mast cells) infiltrating the GO EOMs. METHODS: Analysis of the morphological status, oxidative stress (OS), and antioxidant defenses in the orbital muscular cells and adipocytes in GO patients compared with controls was conducted. RESULTS: Both cell types are affected by OS, as shown by the increased expression of 4-hydroxynonenal, which leads to apoptosis in muscular cells. However, the EOMs and the adipocytes possess antioxidant defenses (peroxiredoxin 5 and catalase) against the OS, which are also upregulated in thyrocytes in GD. The expression of adiponectin (ApN) and proliferator-activated receptor gamma (PPARγ) is also increased in GO muscular cells and adipocytes. OS and antioxidant proteins expression are correlated to the level of blood antithyrotropin receptor antibodies (TSHR-Ab). CONCLUSION: Even when TSHR-Ab level is normalized, OS and antioxidant protein expression is high in EOM muscular cells and adipocytes in GO compared with controls. This justifies a supplementation with antioxidants in active as well as chronic GO patients. Orbital muscular cells are also the sources of PPARγ and ApN, which have direct or indirect local protective effects against OS. Modulation of these proteins could be considered as a future therapeutic approach for GO.