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 (TR proportional to) 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 Tukey's test was used at 5% significance level. Results: T3 increased TR proportional to 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 TRa is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). The presence of CHX completely abrogated levels TRa 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 TRa gene expression in 3T3-L1 adipocytes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Cardiac dysfunction induced by obesity es not related to beta-adrenergic system impairment at the receptor-signalling pathway

Obesity has been shown to impair myocardial performance. Some factors have been suggested as responsible for possible cardiac abnormalities in models of obesity, among them beta-adrenergic (βA) system, an important mechanism of regulation of myocardial contraction and relaxation. The objective of present study was to evaluate the involvement of βA system components in myocardial dysfunction induced by obesity. Thirty-day-old male Wistar rats were distributed in control (C, n = 25) and obese (Ob, n = 25) groups. The C group was fed a standard diet and Ob group was fed four unsaturated high-fat diets for 15 weeks. Cardiac function was evaluated by isolated papillary muscle preparation and βA system evaluated by using cumulative concentrations of isoproterenol and Western blot. After 15 weeks, the Ob rats developed higher adiposity index than C rats and several comorbidities; however, were not associated with changes in systolic blood pressure. Obesity caused structural changes and the myocardial responsiveness to post-rest contraction stimulus and increased extracellular calcium (Ca2+) was compromised. There were no changes in cardiac function between groups after βA stimulation. The obesity was not accompanied by changes in protein expression of G protein subunit alpha (Gsα) and βA receptors (β1AR and β2AR). In conclusion, the myocardial dysfunction caused by unsaturated high-fat diet-induced obesity, after 15 weeks, is not related to βAR system impairment at the receptor-signalling pathway.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

(A) Glucose tolerance profile, (B and D) hormone serum levels and (C) homeostatic model assessment index (HOMA-IR).

<p>Control (C; n = 20) and obese (Ob; n = 17) groups. n = number of animals. (A) Data presented as the means ± standard deviation; Two way ANOVA for independent samples and Bonferroni’s <i>post-hoc</i> analysis. (B, C and D) Values shown are median ± interquartile range; Mann-Whitney test. *p<0.05 <i>versus</i> C.</p

Changes in body weight (BW) during 15 weeks of experimental protocol after the initiation of obesity at week 3.

<p>Data presented as the mean ± SD. * p<0.05 <i>versus</i> C. Two way ANOVA for independent samples and Bonferroni’s post-hoc analysis.</p

Baseline data from isolated muscle preparation.

<p>Values expressed as mean ± SD. n = number of animals. C: control; Ob: obese; Baseline condition: 2.5 mM [Ca²⁺]. DT: maximum developed tension normalized per cross-sectional area of the papillary muscle; RT: resting tension normalized per cross-sectional area of the papillary muscle; peak of the positive (+dT/dt) and negative (-dT/dt) tension derivatives normalized per cross-sectional area of the papillary muscle; CSA: cross-sectional area. Student’s t-test for independent samples.</p><p>Baseline data from isolated muscle preparation.</p

(A, B and C) Post-rest contraction (PRC) and (D, E and F) effects of increasing extracellular Ca²⁺ concentration in papillary muscles from control (white bars) and obese rats (cross-hatched bars).

<p>Baseline calcium concentration (0.5 mM) is presented as 100%. Maximum developed tension normalised per cross-sectional area [DT, g/mm²] and positive [+dT/dt, g/mm²/s] and negative [-dT/dt, g/mm²/s] tension derivative normalized per cross-sectional area of the papillary muscle. Data presented as the mean percent of baseline ± standard deviation.*p < 0.05 <i>versus</i> C. Repeated-measures two-way ANOVA and Student-Newman-Keuls <i>post-hoc t</i>est.</p

(A, B and C) Effects of increasing isoproterenol concentration in papillary muscles (10^<i>−7</i> to 10⁻⁵ M) and (D, E and F) protein expression of β-adrenergic receptors (β-AR) and stimulatory G-protein (G_sα) from control (white bars) and obese rats (cross-hatched bars).

<p>Baseline calcium concentration (1.0 mM) is presented as 100%. Maximum developed tension normalised per cross-sectional area [DT, g/mm²] and positive [+dT/dt, g/mm²/s] and negative [-dT/dt, g/mm²/s] tension derivative normalized per cross-sectional area of the papillary muscle. D: β₁AR, E: β₂AR and F: G_sα. (A, B and C) Data presented as the mean percent of baseline ± standard deviation; Repeated-measures two-way ANOVA and Student-Newman-Keuls <i>post-hoc</i> test. (D, E and F) Values shown are mean ± standard deviation; Student’s t-test for independent samples (D, E and F).*p < 0.05 <i>versus</i> C.</p

Systolic blood pressure, lipid profile and cardiac morphology

<p>Data presented as means ± SD. control (C) and obese (Ob) groups; n: animals numbers; Systolic blood pressure and lipid profile (<i>n</i> = 7 animals); SBP: systolic blood pressure; TG: triglycerides; T-Chol: total cholesterol; HDL: high-density lipoprotein; LDL: Low-density lipoprotein. Cardiac parameters <i>(C</i>, <i>n = 20; Ob</i>, <i>n = 17)</i>; HW: heart weight; LVW: left ventricle weight;</p><p>*p<0.05 versus C; Student’s t-test for independent samples.</p><p>Systolic blood pressure, lipid profile and cardiac morphology</p