3T3-L1 cells were obtained from the American Type Culture Collection and cultured at 37°C in 5% CO₂ in Dulbecco's modified Eagle's medium supplemented with 25 mmol/l glucose, 1.0 mmol/l pyruvate, 4.02 mmol/l L-alanyl-glutamine and 10% fetal bovine serum (Gibco, New York, NY, USA). Cell differentiation began 24 h after confluency and occurred over 4 days in a medium containing 0.25 µM dexamethasone, 0.5 mmol/l 3-isobutyl-1-methylxanthine and 5 µg/ml insulin (Sigma, St. Louis, MO, USA). Following differentiation, the cells were cultured for 10 days in growth medium containing 5 µg/ml of insulin. Each parameter was evaluated using a 6-well aliquot from this culture. On day 10 after differentiation, the cells were incubated for 24 h in a medium containing 0.5% fetal bovine serum. Cells were treated with 10 µl of TPM (50 µM) or isoproterenol (20 µM) for 30 min. At the end of the incubation, the glycerol and non-esterified fatty acid (NEFA) contents (Wako Chemical, Richmond, Inc., VA, USA) of the incubation medium were used as an index of lipolysis and were measured using enzymatic-colorimetric kits. In addition, the release of lactate dehydrogenase (LDH) induced by plasma membrane disruption in culture medium was used as a cellular viability index.

Cells were incubated in Krebs/Ringer/phosphate buffer (pH 7.4), supplemented with 1% bovine serum albumin and 1 mmol/l palmitate, at 37°C under a CO₂ (5%)/O₂ (95%) gas mixture. Aliquots (450 µl) were transferred to polypropylene test tubes containing 5 µl of [1-¹⁴C]-palmitate, in the presence or absence of insulin (10 nmol/l), and in the presence or absence of TPM (50 µM). These samples were subsequently incubated for 1 h at 37°C in a water bath. Following incubation, the mixture was acidified with 0.2 ml H₂SO₄ (8 N) and incubated for an additional 30 min. At the end of the incubation, the reaction mixture was treated with 2.5 ml of Dole's reagent (isopropanol:n-heptane:H₂SO₄, 4:1:0.25, v/v/v) for lipid extraction.

The whole cell extracts were prepared by lysis of the cells in extraction buffer [1% Triton X-100, 100 mmol/l Tris (pH 7.4), containing 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium vanadate, 2 mmol/l phenylmethylsulphonyl fluoride and 0.1 mg of aprotinin/ml] at 4°C. The extracts were centrifuged at 9,000 × g at 4°C (5804R; Eppendorf AG, Hamburg, Germany) for 40 min to remove insoluble material, and the supernatants of these tissues were used for protein quantification, according to the Bradford method. Proteins were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol, run on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked, probed and blotted with primary antibodies. Antibodies used for immunoblotting were phospho HSL^ser660, phospho HSL^ser563, HSL total (Cell Signaling Technology, Beverly, MA, USA), and phospho PKA^Thr198, PKA total, CGI-58, perilipin A (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phospho ATGL^Ser406, ATGL total, β-actin (Abcam, Eugene, OR, USA), and phospho perilipin A (Vala Sciences, Inc., San Diego, CA, USA). Chemiluminescent detection was performed with horseradish peroxidase-conjugated secondary antibodies (Thermo Scientific, Rockford, IL, USA). Autoradiographs of membranes were obtained for visualization of protein bands. The results of the blots are presented as direct comparisons of the area of the apparent bands in autoradiographs and quantified by densitometry using the Scion Image software (Scion Image software; Scion Corp., Frederick, MD, USA).

Bars represent 6 different experiments. Differences between the groups were evaluated using one-way analysis of variance followed by the Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference. The software used for data analysis was the Statistical Package for the Social Sciences (SPSS) version 17.0 for Windows (SPSS, Inc., Chicago, IL, USA).

To evaluate TPM-induced physiological alterations in lipolysis, the glycerol and NEFA contents were assayed in the incubation medium. The contents of glycerol and NEFA in isoproterenol-treated cells were higher compared with the control group. Similar results were observed for the isoproterenol group (Fig. 1A and B). To analyze lipogenesis, insulin-induced [1-¹⁴C]-palmitate incorporation into lipids was assayed. These results show that the use of TPM decreased palmitate incorporation into lipids compared to the control group regardless of insulin stimulus (Fig. 1C). To evaluate whether TPM exposure effects cellular viability, the cytosolic enzyme LDH release was quantified. The analyzed groups did not demonstrate significant differences, suggesting that TPM was not cytotoxic for the times and dosages used (Fig. 1D).

Based on the current findings, we hypothesize that TPM treatment could increase the phosphorylation of the main lipolytic enzymes. Subsequently, the phosphorylation of PKA^Thr198, HSL^Ser660, HSL^Ser563, ATGL^Ser406 and perilipin A, as well as the protein levels of CGI-58, PKA^Thr198, HSL^Ser660, HSL^Ser563, ATGL^Ser406 and perilipin A were analyzed (Fig. 1E–J). TPM treatment led to a higher enzymatic phosphorylation compared to the control group. Therefore, this result may explain, at least in part, why the glycerol and NEFA levels were increased. Notably, the analyzed protein phosphorylation and the CGI-58 protein levels were similar for cells treated with TPM or isoproterenol (Fig. 1E–J).