Simonetta Lisi

Human leptin tissue distribution, but not weight loss-dependent change in expression, is associated with methylation of its promoter.

Leptin is a master regulator of energy homeostasis. Its expression, prevalently localized in adipocytes, is positively related to adipose mass. Epigenetics is emerging as an important contributor to the changes in gene expression undergone by adipose tissue during obesity. We herein investigated the involvement of methylation-dependent mechanisms in leptin regulation in humans. We studied the methylation profile of a 305 bp region in the leptin promoter and analyzed the correspondent leptin expression in visceral adipocyte fraction (AF) and stromal vascular fraction (SVF) of white adipose tissue (WAT) and liver. We found an inverse relationship between methylation and leptin expression with AF displaying a lower methylation density (8%) than SVF and liver (18%, 21%). We evidenced a hot spot region, which mostly differentiates AF versus liver. This includes C15 and 21, which are within the recognition sequences for the transcription factors Sp1 and C/EBP, and C22-23/24, flanking a TATA box. In vitro studies demonstrated that demethylation (by decitabine) increase or de novo activate leptin expression in primary fibroblasts and HeLa cells, respectively. A longitudinal study carried out in patients analyzed before and after bariatric surgery-induced weight loss indicated that in this case decrease in WAT leptin expression (about 50%) does not correspond to changes in promoter methylation density. In conclusion, methylation density in the leptin promoter constitutes one control level for cell type specific leptin expression, whereas weight-loss induced changes in leptin expression does not seem to be methylation-dependent.

The obesity and inflammatory marker haptoglobin attracts monocytes via interaction with chemokine (C-C motif) receptor 2 (CCR2)

BackgroundObesity is a chronic low inflammatory state. In the obesity condition the white adipose tissue (WAT) is massively infiltrated with monocytes/macrophages, and the nature of the signals recruiting these inflammatory cells has yet to be fully elucidated. Haptoglobin (Hp) is an inflammatory marker and its expression is induced in the WAT of obese subjects. In an effort to elucidate the biological significance of Hp presence in the WAT and of its upregulation in obesity we formulated the hypothesis that Hp may serve as a macrophage chemoattractant.ResultsWe demonstrated by chemotaxis assay that Hp is able to attract chemokine (C-C motif) receptor 2 (CCR2)-transfected pre-B lymphocytes and monocytes in a dose-dependent manner. Moreover, Hp-mediated migration of monocytes is impaired by CCR2-specific inhibition or previous cell exposure to monocyte chemoattractant protein 1 (MCP1) (also known as CCR2 ligand or chemokine (C-C motif) ligand 2 (CCL2)). Downstream effects of Hp/CCR2 interaction were also investigated: flow cytometry proved that monocytes treated with Hp show reduced CCR2 expression on their surface; Hp interaction induces calcium release that is reduced upon pretreatment with CCR2 antagonist; extracellular signal-regulated kinase (ERK)1/2, a signal transducer activated by CCR2, is phosphorylated following Hp treatment and this phosphorylation is reduced when cells are pretreated with a specific CCR2 inhibitor. Consistently, blocking the ERK1/2 pathway with U0126, the selective inhibitor of the ERK upstream mitogen-activated protein (MAP)-ERK kinase (MEK), results in a dramatic reduction (by almost 100%) of the capability of Hp to induce monocyte migration.ConclusionsOur data show that Hp is a novel monocyte chemoattractant and that its chemotactic potential is mediated, at least in part. by its interaction with CCR2.

Role of thyroglobulin in the pathogenesis of Graves’ ophthalmopathy: The hypothesis of Kriss revisited

One of the hypothesis to explain the pathogenesis of Graves’ ophthalmopathy (GO) was formulated by Joseph P. Kriss in the early 1970s. He postulated that the initiating event in the pathogenesis of GO is the deposition and accumulation of thyroglobulin (Tg) in orbital tissues, followed by an autoimmune reaction against Tg. In the last 30 yr several studies have addressed this hypothesis, through various, different experimental approaches, raising results that are both in favor and against the possibility that Tg plays a role in the pathogenesis of GO. The finding that intact Tg is present in orbital tissues of GO patients supports Kriss’ hypothesis, although the role of Tg as an autoantigen seems to be unlikely, as GO is not significantly associated with serum TgAb and mice immunized with Tg do not develop GO. Whether Tg is indeed involved in the pathogenesis of GO remains to be established. Our current view is that, provided that Tg plays a role, it is unlikely the only factor involved and Tg in orbital tissues may rather reinforce or worsen a damage initiated by other mechanisms.

Thyroglobulin in orbital tissues from patients with thyroid-associated ophthalmopathy: predominant localization in fibroadipose tissue.

UNLABELLED One of the hypotheses that explains the pathogenesis of thyroid-associated ophthalmopathy (TAO) is that thyroglobulin (Tg) is transported through a retrograde lymphatic route to orbital tissues (OT), where it elicits autoimmune damage. In a previous study we demonstrated the presence of intact Tg of thyroid origin in OT from three patients with TAO. The present study was undertaken to investigate this issue further, by increasing the number of patients, by analyzing the distribution of Tg in OT, and by investigating possible relations between the presence of Tg in OT and the clinical features of patients. OT was obtained from seven patients with TAO who underwent decompressive orbitotomy via a transpalpebral approach. Patients were designated P10 to P16. Inflamed palpebral skin, retrobulbar fibroadipose tissue and extraocular muscle surgical samples were collected separately. Tissue extracts were prepared by homogenization and analyzed for the presence of Tg using two different techniques. We first performed immunoprecipitation experiments, in which a rabbit polyclonal anti-Tg antibody was used to capture Tg on protein A and a mouse monoclonal anti-Tg antibody was used to re-veal captured Tg by Western blotting. Intact 330-kd Tg was detected in retrobulbar fibroadipose tissue extracts from three patients (P10, P11, and P16), whereas no Tg was detected in retrobulbar fibroadipose tissue extracts from the remaining four patients. Tg was not detected in the extraocular muscle extracts from all patients studied. In addition, intact 330-kd Tg was found in the inflamed palpebral skin extract from one patient (P10). No Tg was detected in OT extracts from two patients without thyroid or eye disease and in abdominal adipose tissue extracts from two obese patients without thyroid or eye disease. We then searched for Tg by enzyme-linked immunosorbent assay (ELISA), using the same antibodies used for immunoprecipitation. Tg was detected in retrobulbar fibroadipose tissue extracts from four patients (P10, P11, P12, and P16) and in the inflamed palpebral skin extract from patient P10, in amounts ranging from approximately 125 to approximately 400 pg/microg of tissue protein. Again, Tg was not detected in extraocular muscle extracts. A positive gradient between Tg in OT and Tg in the serum was observed in patient P12. Using an ELISA approach, we found that Tg in OT from three TAO patients (P10, P11, and P12) contained thyroxine (T4) residues (mean T(4) CONTENT 2.42 molecules per molecule of Tg), indicating that Tg had originated in the thyroid. Combining the results obtained in our previous and present study, we found a possible relation between the presence of Tg in OT and the previous treatment with radioiodine. Thus, of the seven patients (3 in the previous and 4 in the present study) in whose OT Tg was found, six had been treated with radioiodine, whereas of the three patients with no Tg in their OT none had been treated with radioiodine. In conclusion, Tg was found in OT extracts from patients with TAO by immunoprecipitation in three of seven cases and by ELISA in four of seven cases. Tg was found in retrobulbar fibroadipose tissue, but not in extraocular muscles. There was a relation between the presence of Tg in OT and the previous treatment with radioiodine. Our results support the hypothesis that Tg may play a role as a coantigen in the pathogenesis of TAO. Further studies are needed to investigate this possibility.

Absence of primary hypothyroidism and goiter in Slc26a4 (-/-) mice fed on a low iodine diet.

Background: Mutations in the SLC26A4 gene, coding for the anion transporter pendrin, are responsible for Pendred syndrome, characterized by congenital sensorineural deafness and dyshormonogenic goiter. The physiological role of pendrin in the thyroid is still unclear and the lack of a thyroid phenotype in some patients with SLC26A4 mutations and in Slc26a4 (-/-) mice indicate the existence of environmental or individual modifiers able to compensate for pendrin inactivation in the thyroid. Since pendrin can transport iodide in vitro, variations in iodide supply have been claimed to account for the thyroid phenotype associated with pendrin defects. Aim: The Slc26a4 (-/-) mouse model was used to test the hypothesis that iodide supply may influence the penetrance and expressivity of SLC26A4 mutations. Materials and methods: Slc26a4 (-/-) and (+/+) mice were fed up to 6 months on a standard or low iodine diet and were evaluated for thyroid structural abnormalities or biochemical hypothyroidism. Results: A 27-fold iodide restriction induced similar modifications in thyroid histology, but no differences in thyroid size, T4 or TSH levels were observed between between Slc26a4 (-/-) and (+/+) mice, either in standard conditions and during iodine restriction. Conclusions: Iodide restriction is not able to induce a thyroid phenotype in Slc26a4 (-/-) mice. These experimental data, together with those coming from a review of familial Pendred cases leaving in regions either with low or sufficient iodide supply, support the idea that the expression of thyroid phenotype in Pendred syndrome is more powerfully influenced by individual factors than by dietary iodide.

Thyroid dysfunction in megalin deficient mice

Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.

Obesity-Associated Hepatosteatosis and Impairment of Glucose Homeostasis Are Attenuated by Haptoglobin Deficiency

OBJECTIVE Haptoglobin (Hp) is upregulated in both inflammation and obesity. The low chronic inflammatory state, caused by massive adipose tissue macrophage (ATM) infiltration found in obesity, and low adiponectin have been implicated in the development of insulin resistance and hepatosteatosis. The aim of this work was to investigate whether and how Hp interferes with the onset of obesity-associated complications. RESEARCH DESIGN AND METHODS Hp-null (Hp−/−) and wild-type (WT) mice were metabolically profiled under chow-food diet (CFD) and high-fat diet (HFD) feeding by assessing physical parameters, glucose tolerance, insulin sensitivity, insulin response to glucose load, liver triglyceride content, plasma levels of leptin, insulin, glucose, and adiponectin. ATM content was evaluated by using immunohistochemistry (anti-F4/80 antibody). Adiponectin expression was measured in Hp-treated, cultured 3T3-L1 and human adipocytes. RESULTS No genotype-related difference was found in CFD animals. HFD-Hp−/− mice revealed significantly higher glucose tolerance, insulin sensitivity, glucose-stimulated insulin secretion, and adiponectin expression and reduced hepatomegaly/steatosis compared with HFD-WT mice. White adipose tissue (WAT) of HFD-Hp−/− mice showed higher activation of insulin signaling cascade, lower ATM, and higher adiponectin expression. Hp was able to inhibit adiponectin expression in cultured adipocytes. CONCLUSIONS We demonstrated that in the absence of Hp, obesity-associated insulin resistance and hepatosteatosis are attenuated, which is associated with reduced ATM content, increased plasma adiponectin, and higher WAT insulin sensitivity.

Quercetin decreases proliferation of orbital fibroblasts and their release of hyaluronic acid

Background: Inhibition of fibroblast (FB) proliferation and hyaluronic acid (HA) production may be a therapeutic approach to Graves’ ophthalmopathy (GO). The flavonoid quercetin has a wide range of activities, including reduction of FB growth. Aim: To investigate the effects of quercetin in orbital FB from GO patients and control subjects. Methods: Primary cultures of orbital FB were treated with quercetin or with its glycosides rutin and quercitrin. Cell proliferation, necrosis, apoptosis, HA production, and cell cycle were measured. Results: Beginning at a 30 µM concentration, quercetin, but not rutin and quercitrin, reduced cell proliferation, with no difference between GO and control FB. The effect of quercetin on proliferation was due to necrosis and cell cycle blockade, whereas apoptosis was unaffected. Quercetin reduced HA in the cell media, with no difference between GO and control FB. Conclusions: Quercetin reduces cell proliferation and HA release in orbital FB. Whether these initial findings have any potential for the use of quercetin in the clinical practice remains to be established.

Sortilin Is a Putative Postendocytic Receptor of Thyroglobulin

The Vps10p family member sortilin is involved in various cell processes, including protein trafficking. Here we found that sortilin is expressed in thyroid epithelial cells (thyrocytes) in a TSH-dependent manner, that the hormone precursor thyroglobulin (Tg) is a high-affinity sortilin ligand, and that binding to sortilin occurs after Tg endocytosis, resulting in Tg recycling. Sortilin was found to be expressed intracellularly in thyrocytes, as observed in mouse, human, and rat thyroid as well as in FRTL-5 cells. Sortilin expression was demonstrated to be TSH dependent, both in FRTL-5 cells and in mice treated with methimazole and perchlorate. Plasmon resonance binding assays showed that Tg binds to sortilin in a concentration-dependent manner and with high affinity, with Kd values that paralleled the hormone content of Tg. In addition, we found that Tg and sortilin interact in vivo and in cultured cells, as observed by immunoprecipitation, in mouse thyroid extracts and in COS-7 cells transiently cotransfected with sortilin and Tg. After incubation of FRTL-5 cells with exogenous, labeled Tg, sortilin and Tg interacted intracellularly, presumably within the endocytic pathway, as observed by immunofluorescence and immunoelectron microscopy, the latter technique showing some degree of Tg recycling. This was confirmed in FRTL-5 cells in which Tg recycling was reduced by silencing of the sortilin gene and in CHO cells transfected with sortilin in which recycling was increased. Our findings provide a novel pathway of Tg trafficking and a novel function of sortilin in the thyroid gland, the functional impact of which remains to be established.

Glycosaminoglycans provide a binding site for thyroglobulin in orbital tissues of patients with thyroid-associated ophthalmopathy

The presence of thyroglobulin (Tg) in orbital tissues of patients with thyroid-associated ophthalmopathy (TAO) supports a role of Tg in TAO pathogenesis. To search for Tg-binding sites in orbital tissues, because Tg is a heparin-binding protein, we investigated its binding to glycosaminoglycans (GAGs) that are abundant in orbital tissues: chondroitin sulfate B (CSB) and C (CSC) and hyaluronic acid (HA). Both in solid phase and solution phase assays purified human Tg bound to GAGs. In solid-phase assays, binding was increased by coincubation with heparin or GAGs in solution, or with an antibody against a Tg heparin-binding sequence (Arg2489-Glu2503), possibly suggesting crosslinking of Tg molecules induced by GAGs or by the presumably bivalent antibody. Orbital tissue extracts from TAO patients that contained Tg were subjected to high-salt treatment, which resulted in separation of Tg from GAGs, as observed by column chromatography. After separation from GAGs, the Tg in orbital tissue extracts acquired the ability to bind to immobilized CSB, and heparin enhanced binding, resembling the findings with purified human Tg. Therefore, we conclude that GAGs provide binding sites for Tg in orbital tissues, which may explain the presence of Tg in orbital tissues of patients with TAO.