Susana B. Bravo

Regulation of visceral adipose tissue-derived serine protease inhibitor by nutritional status, metformin, gender and pituitary factors in rat white adipose tissue

Visceral adipose tissue‐derived serine protease inhibitor (vaspin) is a recently discovered adipocytokine mainly secreted from visceral adipose tissue, which plays a main role in insulin sensitivity. In this study, we have investigated the regulation of vaspin gene expression in rat white adipose tissue (WAT) in different physiological (nutritional status, pregnancy, age and gender) and pathophysiological (gonadectomy, thyroid status and growth hormone deficiency) settings known to be associated with energy homeostasis and alterations in insulin sensitivity. We have determined vaspin gene expression by real‐time PCR. Vaspin was decreased after fasting and its levels were partially recovered after leptin treatment. Chronic treatment with metformin increased vaspin gene expression. Vaspin mRNA expression reached the highest peak at 45 days in both sexes after birth and its expression was higher in females than males, but its levels did not change throughout pregnancy. Finally, decreased levels of growth hormone and thyroid hormones suppressed vaspin expression. These findings suggest that WAT vaspin mRNA expression is regulated by nutritional status, and leptin seems to be the nutrient signal responsible for those changes. Vaspin is influenced by age and gender, and its expression is increased after treatment with insulin sensitizers. Finally, alterations in pituitary functions modify vaspin levels. Understanding the molecular mechanisms regulating vaspin will provide new insights into the pathogenesis of the metabolic syndrome.

TGF-β-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-κB activation

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-β is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-β in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Graves disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-β induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-β induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-β. By contrast, in cells in which p27 was overexpressed, TGF-β had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-κB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-β induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-κB program.

Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress.

UNLABELLED Pre-slaughter stress has adverse effects on meat quality that can lead to the occurrence of Dark Firm Dry (DFD) meat in cattle. This study explores the previously uncharacterized proteome changes linked to pre-slaughter stress in the longissimus thoracis (LT) bovine muscle. Differential proteome profiles of DFD and normal (non-DFD) LT meat samples from male calves of the Rubia Gallega breed were assessed by 2-DE coupled to MS analysis (LC-MS/MS and MALDI TOF/TOF MS). A total of seven structural-contractile proteins (three different myosin light chain isoforms, two fast skeletal myosin light chain 2 isoforms, troponin C type 2 and cofilin-2) and three metabolism enzymes (triosephosphate isomerase, ATP synthase and beta-galactoside alpha-2,6-sialyltransferase) were found to have statistically significant differential abundance in sample groups. In addition, 2-DE in combination with the phosphoprotein-specific fluorescent dye Pro-Q DPS revealed that highly phosphorylated fast skeletal myosin regulatory light chain 2 isoforms underwent the most intense relative change in muscle conversion to DFD meat. Therefore, they appear to be the most sensitive biomarkers of stress just prior to slaughter in Rubia Gallega. Overall, these findings will facilitate a more integrative understanding of the biochemical processes associated with stress in cattle muscle and their effects in meat quality. BIOLOGICAL SIGNIFICANCE Pre-slaughter stress is a crucial factor in meat production. Animals destined for slaughter are stressed by a variety of endogenous and exogenous factors that negatively affect the complex post-mortem biochemical events underlying the conversion of muscle into meat. The study of the muscle proteome has a great relevance for understanding the molecular mechanisms associated with stress. However, there is no information available on the molecular changes linked to pre-slaughter stress in cattle on the proteome scale. Our study led to the identification of a number of candidate proteins associated with the response to pre-slaughter stress in the LT bovine muscle of the Rubia Gallega breed. The functions of those significantly changed proteins have a clear biological relationship with stress response. These findings contribute to a deeper insight into the molecular pathways that respond to stress in cattle.

Serum chemerin levels during normal human pregnancy

During gestation there are important changes in maternal metabolism and an increase in insulin resistance, coinciding with an increase in adiposity. Chemerin is an adipocytokine which is expressed and secreted in various tissues, including placenta, and may play an important role in metabolic regulation during pregnancy. The aim of this study was to determine serum levels of chemerin during gestation and compare them to other indicators of insulin resistance. A cross-sectional study was carried out analyzing serum chemerin levels of 20 pregnant women during three gestational periods, early, middle, and late (between the 10th and 14th, the 23rd and 26th, and the 34th and 37th week) and 20 non-pregnant women were used as a control group. An analysis of chemerin levels during the menstrual cycle was performed in an eumenorrheic group (n=16) in the early follicular (cycle day 4±1) and the midluteal phase (cycle day 22±1), demonstrating that serum chemerin levels did not fluctuate significantly. Serum levels of chemerin were significantly elevated during late gestation when compared to early (P<0.001) and middle (P=0.001) gestation and a negative correlation between serum chemerin and adiponectin levels (r=-0.1643) became more significant when the non-pregnant group was included in the calculations (r=-0.2471). There was no significant association of triglycerides, total cholesterol, LDL, HDL, insulin, and HOMA levels with chemerin. Although chemerin rose significantly and is negatively associated with adiponectin levels, it is not correlated with other markers of insulin sensitivity, suggesting that more study is needed to determine whether chemerin is useful in predicting insulin resistance during gestation.

Plitidepsin Has a Cytostatic Effect in Human Undifferentiated (Anaplastic) Thyroid Carcinoma

Undifferentiated (anaplastic) thyroid carcinoma is a highly aggressive human cancer with very poor prognosis. Although there have been a few studies of candidate treatments, the fact that it is an infrequent tumor makes it very difficult to design clinical trials. A strong association has been observed between undifferentiated thyroid carcinoma and TP53 mutations in numerous molecular genetic and expression studies. Plitidepsin (Aplidin, PharmaMar, Madrid, Spain) is a novel anticancer compound obtained from a sea tunicate. This compound has been reported to induce apoptosis independently of TP53 status. We investigated the actions of plitidepsin in human thyroid cancer cells. In initial experiments using primary cultured cells from a differentiated (papillary) carcinoma, we found that 100 nmol/L plitidepsin induced apoptosis, whereas lower doses were cytostatic. Because our aim was to study the effects of plitidepsin at clinically relevant concentrations, subsequent experiments were done with a dosage regimen reflecting plasma concentrations observed in previously reported clinical trials: 100 nmol/L for 4 hours, followed by 10 nmol/L for 20 hours (4100/2010 plitidepsin). This plitidepsin dosage regimen blocked the proliferation of a primary undifferentiated/anaplastic thyroid carcinoma culture obtained in our laboratory and of a commercial cell line (8305C) obtained from an undifferentiated thyroid carcinoma; however, it did not induce apoptosis. The proportion of cells in the G1 phase of the cell cycle was greatly increased and the proportion in the S/G2-M phases greatly reduced, suggesting that plitidepsin blocks G1-to-S transition. Levels of the cyclin D1/cyclin-dependent kinase 4/p21 complex proteins were decreased and, in line with this, the levels of unphosphorylated Rb1 increased. The decrease in cell cycle proteins correlated with hypoacetylation of histone H3. Finally, we did experiments to assess how rapidly tumor cells return to their initial pretreatment proliferative behavior after 4100/2010 plitidepsin treatment. Cells from undifferentiated tumors needed more than 3 days to recover logarithmic growth, and after 7 days, cell number was still significantly lower than in control cultures. 4100/2010 plitidepsin inhibited the growth in soft agar. Together, our data show that plitidepsin is able to block in vitro cell cycle progression at concentrations similar to serum concentrations observed in vivo, and that this effect is persistent for several days after plitidepsin removal. Whether plitidepsin will prove to be clinically useful in the treatment of undifferentiated thyroid cancers remains to be established. However, our results raise the possibility that plitidepsin might be effective alone or in combination with radiotherapy and/or other drug treatments.

Food intake regulating-neuropeptides are expressed and regulated through pregnancy and following food restriction in rat placenta

BackgroundNeuropeptide Y (NPY), agouti related peptide (AgRP), cocaine and amphetamine-regulated transcript (CART) and melanocortins, the products of the proopiomelanocortin (POMC), are hypothalamic peptides involved in feeding regulation and energy homeostasis. Recent evidence has demonstrated their expression in rat and human placenta.MethodsIn the current study, we have investigated the expression of those neuropeptides in the rat placenta by real-time PCR using a model of maternal food restriction.ResultsOur results showed that placental-derived neuropeptides were regulated through pregnancy and following food restriction.ConclusionThese data could indicate that placental-derived neuropeptides represent a local regulatory circuit that may fine-tune control of energy balance during pregnancy.

Expression of neuropeptide W in rat stomach mucosa : Regulation by nutritional status, glucocorticoids and thyroid hormones

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.

CILAIR-Based Secretome Analysis of Obese Visceral and Subcutaneous Adipose Tissues Reveals Distinctive ECM Remodeling and Inflammation Mediators

In the context of obesity, strong evidences support a distinctive pathological contribution of adipose tissue depending on its anatomical site of accumulation. Therefore, subcutaneous adipose tissue (SAT) has been lately considered metabolically benign compared to visceral fat (VAT), whose location is associated to the risk of developing cardiovascular disease, insulin resistance, and other associated comorbidities. Under the above situation, the chronic local inflammation that characterizes obese adipose tissue, has acquired a major role on the pathogenesis of obesity. In this work, we have analyzed for the first time human obese VAT and SAT secretomes using an improved quantitative proteomic approach for the study of tissue secretomes, Comparison of Isotope-Labeled Amino acid Incorporation Rates (CILAIR). The use of double isotope-labeling-CILAIR approach to analyze VAT and SAT secretomes allowed the identification of location-specific secreted proteins and its differential secretion. Additionally to the very high percentage of identified proteins previously implicated in obesity or in its comorbidities, this approach was revealed as a useful tool for the study of the obese adipose tissue microenvironment including extracellular matrix (ECM) remodeling and inflammatory status. The results herein presented reinforce the fact that VAT and SAT depots have distinct features and contribute differentially to metabolic disease.

New Insights into Thyroglobulin Pathophysiology Revealed by the Study of a Family with Congenital Goiter

CONTEXT Thyroglobulin (TG) gene mutations cause congenital hypothyroidism (CH) with goiter. A founder effect has been proposed for some frequent mutations. Mutated proteins have a defect in intracellular transport causing intracellular retention with ultrastructural changes that resemble an endoplasmic reticulum storage disease. OBJECTIVE To reveal new aspects of thyroglobulin pathophysiology through clinical, cellular, molecular, and genetic studies in a family presenting with CH due to TG mutations from Galicia, an iodine-deficient area of Spain. DESIGN The included clinical evaluation of family members, DNA sequencing for TG gene mutation and haplotyping analysis, ultrastructural analysis of thyroid tissue specimens from affected subjects, analysis of effects of mutations found on TG gene transcription, and in vitro studies of cellular production and secretion of mutated proteins. SETTING Locations included primary care and university hospitals. RESULTS Family members with CH, mental retardation, and goiter were compound heterozygous for c.886C-->T (p.R277X) and g.IVS35+1delG. For c.886C-->T, a founder effect cannot be excluded, and its transcription was hardly detectable. g.IVS35+1delG caused an in-frame deletion in exon 35 and produced a protein that, although synthesized, could not be secreted. Ultrastructural analyses showed morphological changes consistent with an endoplasmic reticulum storage disease. CONCLUSION The shorter thyroglobulin resulting from the novel g.IVS35+1delG was retained within the endoplasmic reticulum of thyrocytes, and together with p.R227X caused severe hypothyroidism with goiter. p.R277X, the most commonly described TG mutation, is caused by a TG exon-7 highly mutation-prone region, and the possibility that some cases were introduced to South America from Galicia cannot be excluded.

Functional Role of the RET Dependence Receptor, GFRa Co-Receptors and Ligands in the Pituitary

The RET receptor is a tyrosine kinase receptor implicated in kidney and neural development. In the adenopituitary RET and the co-receptor GFRa1 are expressed exclusively in the somatotrophs secreting GH. RET is implicated in a clever pathway to maintain at physiological levels the number of somatotrophs and the GH production. Thus, in absence of its ligand GDNF, RET induces apoptosis through massive expression of Pit-1 leading to p53 accumulation. In the presence of the ligand GDNF, RET activates its tyrosine kinase and promotes survival at the expense of reducing Pit-1 expression and downregulating GH. Recent data suggest that RET can also have a second role in pituitary plasticity through a second co-receptor GFRa2.