Impact of Doxorubicin Treatment on the Physiological Functions of White Adipose Tissue

<div><p>White adipose tissue (WAT) plays a fundamental role in maintaining energy balance and important endocrine functions. The loss of WAT modifies adipokine secretion and disrupts homeostasis, potentially leading to severe metabolic effects and a reduced quality of life. Doxorubicin is a chemotherapeutic agent used clinically because of its good effectiveness against various types of cancer. However, doxorubicin has deleterious effects in many healthy tissues, including WAT, liver, and skeletal and cardiac muscles. Our objective was to investigate the effects of doxorubicin on white adipocytes through <i>in vivo</i> and <i>in vitro</i> experiments. Doxorubicin reduced the uptake of glucose by retroperitoneal adipocytes and 3T3-L1 cells via the inhibition of AMP-activated protein kinase Thr172 phosphorylation and glucose transporter 4 content. Doxorubicin also reduced the serum level of adiponectin and, to a greater extent, the expression of genes encoding lipogenic (<i>Fas</i> and <i>Acc</i>) and adipogenic factors (<i>Pparg</i>, <i>C/ebpa</i>, and <i>Srebp1</i>c) in retroperitoneal adipose tissue. In addition, doxorubicin inhibited both lipogenesis and lipolysis and reduced the hormone-sensitive lipase and adipose tissue triacylglycerol lipase protein levels. Therefore, our results demonstrate the impact of doxorubicin on WAT. These results are important to understand some side effects observed in patients receiving chemotherapy and should encourage new adjuvant treatments that aim to inhibit these side effects.</p></div

Doxorubicin reduces lipogenesis.

<p>Fatty acid incorporation into triacylglycerol (TAG) in isolated adipocytes of retroperitoneal adipose tissue from rats after 72 hours of doxorubicin (DX) (15 mg/kg weight) or saline (Control) injection i.p. Data are mean ± standard deviation of 4 to 6 experiments. The groups were compared using the Student t test, *p<0.05.</p

Doxorubicin reduces lipogenic enzymes.

<p>Gene expression of lipogenic enzymes <i>Fas</i> (A) and <i>Acc</i> (B) at 24 hours, 96 hours and 12 days after induction of cell differentiation in 3T3L1 cells treated with 1uM of doxorubicin. The gene expression was evaluated by real time PCR. Data are mean ± standard deviation of 4 to 6 experiments. The groups were compared using the Two way ANOVA followed by Bonferroni post test, *p <0.05, **p<0.01 and ***p<0.001.</p

Doxorubicin decreased the adiponectin levels.

<p>Adiponectin protein levels in serum (A) and retroperitoneal adipose tissue (B) of rats 72 hours after treatment with doxorubicin (15mg/kg). Concentration of adiponectin released into culture medium (C) with doxorubicin (DX) 1uM for 24 hours, after differentiation into mature adipocytes. Data are mean ± standard deviation of 6 experiments. The groups were compared using the Student t test.** p <0.01, *** p <0.001.</p

Reduction of glucose uptake by doxorubicin in adipocytes.

<p>Glucose uptake from the retroperitoneal adipose tissues of rats (A) and glucose uptake from 3T3L1 cells (B and C). Protein expression of AMPK thr 172 phosphorylated, total content of GLUT4 and FABP4 from retroperitoneal adipose tissues of rats (D) and protein expression of AMPK threonine 172 phosphorylated, total content of GLUT4 and GAPDH from 3T3L1 cells (E). Adipocytes from the retroperitoneal adipose tissues of rats treated with a single dose of doxorubicin (15 mg/kg body weight ip), euthanized 72 hours after administration. The glucose uptake was measured by uptake of 2-DG with and without insulin stimulation (100nMol) in isolated adipocytes and 3T3L1 cells. 3T3L1 cells were incubated with doxorubicin (1uM) for 30 minutes (B) and 24 hours (C and E), after differentiation into mature adipocytes. Values represent the mean ± standard deviation of 5 to 6 experiments (A, B and C). Figures represents 4 experiments per group (D and E). The groups were compared using two-way ANOVA followed by Dunn’s (A, B, and C). *p<0.05 and **p<0.01.</p

Adipogenesis is compromised by doxorubicin in 3T3L1 cells.

<p>Gene expression of adipogenics factors <i>Ppparg</i> (A), <i>C/ebpa</i> (B) and <i>Srebp1c</i> (C) at 24 hours, 96 hours and 12 days after induction of cell differentiation in 3T3L1 cells treated with 1uM of doxorubicin. The gene expression was evaluated by real time PCR. Data are mean ± standard deviation of 4 to 6 experiments. The groups were compared using the Two way ANOVA followed by Bonferroni post test, *p<0.05, **p<0.01 and ***p<0.001.</p

Doxorubicin inhibits the lipolysis process.

<p>Glycerol released in assay of lipolysis the adipocytes isolated from retroperitoneal adipose tissue of rats after 72 hours of doxorubicin administration (15mg/kg of weighti.p), with or without isoproterenol stimulation (10uM) for 30 minutes (A). Concentration of glycerol (B) and lactate dehydrogenase LDH in incubation medium of 3T3L1 (C). Lipolysis was stimulated with isoproterenol (Iso) 10uM with doxorubicin (DX) at concentrations of 0.1 and 1uM for 24 hours, the parameters are normalized by the concentration of protein extracted from the cells. Values represent the mean ± standard deviation of 4 to 8 experiments. The groups were compared using the One-Way ANOVA followed by Bonferroni post test. *p<0.05, **p<0.01, ***p<0.001 (A, B and C). Protein expression of HSL phosphorylated at residue Ser-565, Ser-660, total HSL, ATGL and GAPDH (D). Proteins extracted from retroperitoneal adipose tissue of rats 72 hours after treatment with doxorubicin (15mg/kg). Figure represents 4 experiments per group (D).</p

Doxorubicin impairs lipogenesis.

<p>3T3L1 cells stained with oil red. 3T3L1 cell were treated with doxorubicin (DX) 1uM or PBS (control) for 12 days after the differentiation cocktail. Photos were obtained by optical microscopy at a resolution of 200X.</p

Primer sequences.

<p>Primer sequences.</p

Doxorubicin promotes toxicity in 3T3L1 cell.

<p>Cell viability after 96 hours (A) of doxorubicin (DX) incubation. Concentrations range from 0.001 to 100 uM. Cell viability was assessed by MTT assay and normalized adopting the control absorbance as 100% survival. Values represent the mean ± standard deviation of 3 independent experiments. Adipocyte size of cells from the retroperitoneal adipose tissues (B) of rats treated with a single dose of doxorubicin (15 mg/kg body weight ip), euthanized 72 hours after administration. Values represent the mean ± standard deviation of 5 to 6 experiments. The groups were compared using the one-Way ANOVA followed by Dunnett's (A) and t test (B).*p <0,05, **p <0.01, *** p <0.001.</p