Miriane de Oliveira

Tributyltin chloride leads to adiposity and impairs metabolic functions in the rat liver and pancreas

Tributyltin chloride (TBT) is an environmental contaminant used in antifouling paints of boats. Endocrine disruptor effects of TBT are well established in animal models. However, the adverse effects on metabolism are less well understood. The toxicity of TBT in the white adipose tissue (WAT), liver and pancreas of female rats were assessed. Animals were divided into control and TBT (0.1 μg/kg/day) groups. TBT induced an increase in the body weight of the rats by the 15th day of oral exposure. The weight gain was associated with high parametrial (PR) and retroperitoneal (RP) WAT weights. TBT-treatment increased the adiposity, inflammation and expression of ERα and PPARγ proteins in both RP and PR WAT. In 3T3-L1 cells, estrogen treatment reduced lipid droplets accumulation, however increased the ERα protein expression. In contrast, TBT-treatment increased the lipid accumulation and reduced the ERα expression. WAT metabolic changes led to hepatic inflammation, lipid accumulation, increase of PPARγ and reduction of ERα protein expression. Accordingly, there were increases in the glucose tolerance and insulin sensitivity tests with increases in the number of pancreatic islets and insulin levels. These findings suggest that TBT leads to adiposity in WAT specifically, impairing the metabolic functions of the liver and pancreas.

A Comparative Genotoxicity Study of a Supraphysiological Dose of Triiodothyronine (T3) in Obese Rats Subjected to Either Calorie-Restricted Diet or Hyperthyroidism

This study was designed to determine the genotoxicity of a supraphysiological dose of triiodothyronine (T3) in both obese and calorie-restricted obese animals. Fifty male Wistar rats were randomly assigned to one of the two following groups: control (C; n = 10) and obese (OB; n = 40). The C group received standard food, whereas the OB group was fed a hypercaloric diet for 20 weeks. After this period, half of the OB animals (n = 20) were subjected to a 25%-calorie restriction of standard diet for 8 weeks forming thus a new group (OR), whereas the remaining OB animals were kept on the initial hypercaloric diet. During the following two weeks, 10 OR animals continued on the calorie restriction diet, whereas the remaining 10 rats of this group formed a new group (ORS) given a supraphysiological dose of T3 (25 µg/100 g body weight) along with the calorie restriction diet. Similarly, the remaining OB animals were divided into two groups, one that continued on the hypercaloric diet (OB, n = 10), and one that received the supraphysiological dose of T3 (25 µg/100 g body weight) along with the hypercaloric diet (OS, n = 10) for two weeks. The OB group showed weight gain, increased adiposity, insulin resistance, increased leptin levels and genotoxicity; T3 administration in OS animals led to an increase in genotoxicity and oxidative stress when compared with the OB group. The OR group showed weight loss and normalized levels of adiposity, insulin resistance, serum leptin and genotoxicity, thus having features similar to those of the C group. On the other hand, the ORS group, compared to OR animals, showed higher genotoxicity. Our results indicate that regardless of diet, a supraphysiological dose of T3 causes genotoxicity and potentiates oxidative stress.

Triiodothyronine Increases mRNA and Protein Leptin Levels in Short Time in 3T3-L1 Adipocytes by PI3K Pathway Activation

The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of leptin mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. We examined the involvement of this pathway in mediating TH effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI=100 nM) T3 dose during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey’s test was used at 5% significance. T3 increased leptin mRNA expression in P (2.26 ± 0.36, p< 0.001), SI (1.99 ±0.22, p< 0.01) compared to C group (1± 0.18). This increase was completely abrogated by LY294002 in P (1.31±0.05, p< 0.001) and SI (1.33±0.31, p< 0.05). Western blotting confirmed these results at protein level, indicating the PI3K pathway dependency. To examine whether leptin is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). In P, the presence of CHX maintained the levels mRNA leptin, but was completely abrogated in SI (1.14±0.09, p> 0.001). These results demonstrate that the activation of the PI3K signaling pathway has a role in TH-mediated direct and indirect leptin gene expression in 3T3-L1 adipocytes.

Triiodothyronine and breast cancer.

The thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), are essential for survival; they are involved in the processes of development, growth, and metabolism. In addition to hyperthyroidism or hypothyroidism, THs are involved in other diseases. The role of THs in the development and differentiation of mammary epithelium is well established; however, their specific role in the pathogenesis of breast cancer (BC) is controversial. Steroid hormones affect many human cancers and the abnormal responsiveness of the mammary epithelial cells to estradiol (E2) in particular is known to be an important cause for the development and progression of BC. The proliferative effect of T3 has been demonstrated in various types of cancer. In BC cell lines, T3 may foster the conditions for tumor proliferation and increase the effect of cell proliferation by E2; thus, T3 may play a role in the development and progression of BC. Studies show that T3 has effects similar to E2 in BC cell lines. Despite controversy regarding the relationship between thyroid disturbances and the incidence of BC, studies show that thyroid status may influence the development of tumor, proliferation and metastasis.

Tributyltin impacts in metabolic syndrome development through disruption of angiotensin II receptor signaling pathways in white adipose tissue from adult female rats

White adipose tissue (WAT) dysfunction and obesity are a consequence of a low-grade inflammation state. These WAT irregularities could result from abnormal metabolic renin-angiotensin system (RAS) control. Recently, tributyltin (TBT) has been found to play a critical role in these metabolic irregularities. However, TBT actions on the WAT-RAS functions are not currently well understood. In this study, we assessed whether TBT exposure resulted in metabolic syndrome (MetS) development and other metabolic complications as a result of abnormal modulation of WAT-RAS pathways. TBT (100 ng/kg/day) was administered to adult female Wistar rats, and their WAT morphophysiology and adipokine profiles were assessed. We further assessed the expression of Angiotensin-II receptor proteins (AT1R and AT2R) and proteins involved in downstream pathways mediating inflammation and adipogenesis modulation. TBT-exposed rats exhibited increases in body weight and adiposity. TBT rats present dyslipidemia and insulin resistance, suggesting MetS development. TBT promoted WAT inflammatory infiltration, AT1R protein overexpression and reduced Angiotensin-(1-7) expression. These TBT WAT abnormalities are reflected by NFκB activation, with higher adipokine levels (leptin, TNF-α and IL-6) and overexpression of AKT, ERK, P38, FAS and PPARγ protein. In vitro, TBT exposure stimulates lipid accumulation, reduces AT2R protein expression, and increases leptin, AKT and ERK protein expression in 3T3L1 cells. These findings suggest that TBT exposure participates in MetS development via the improper function of WAT-RAS metabolic control.

Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells

Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.

Short-term effects of triiodothyronine on thyroid hormone receptor alpha by PI3K pathway in adipocytes, 3T3-L1

OBJECTIVE The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of TH receptor alpha (TRα) mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. MATERIALS AND METHODS It was examined the involvement of PI3K pathway in mediating T3 effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI =100 nM) T3 doses during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukeys test was used at 5% significance level. RESULTS T3 increased TRα mRNA expression in P (1.91±0.13, p<0.001), SI (2.14±0.44, p<0.001) compared to C group (1±0.08). This increase was completely abrogated by LY294002 in P (0.53±0.03, p<0.001) and SI (0.31±0.03, p<0.001). To examine whether TRα is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). The presence of CHX completely abrogated levels TRα mRNA in P (1.15±0.05, p>0.001) and SI (0.99±0.15, p>0.001), induced by T3. CONCLUSION These results demonstrate that the activation of the PI3K signaling pathway has a role in T3-mediated indirect TRα gene expression in 3T3-L1 adipocytes.

Obesity and Weight Loss: The Influence of Thyroid Hormone on Adipokines

The primary function of adipose tissue is storing energy in triacylglycerol (TG) form, neutralizing the excess of circulating lipids and saving non-adipose tissues of a fat overload. Under normal conditions, in the postprandial state, there is lipogenic endocrine system stimulation, allowing that positive energy balance can be stored as TG in adipose tissue, a process called lipogenesis. In contrast, the mobilization of fat in adipocytes occurs through the hydrolysis of TG by hormone sensitive lipase (HSL), a phenomenon called lipolysis. At the center of this interface lipolysis and lipogenesis is the insulin hormone, which exerts a potent inhibitory role on the HSL, allowing lower rates of lipolysis and hence, highest fat mass [1]. However, adipose tissue is not only a passive stock organ of triacylglycerol, being currently recognized as an endocrine organ with multiple functions [1, 2]. Produces several biologically active substances called adipokines, among them tumor necrosis factor- (TNF-), monocyte chemoattractant protein 1 (MCP-1), interleukin-6 (IL-6), leptin, resistin and adiponectin. These substances actively participate in, among others, body energy regulation, mainly, by endocrine, paracrine and autocrine signals, which allow the adipocyte play a metabolic role in other tissues [3-5].