Effects of Genistein on Differentiation and Viability of Human Visceral Adipocytes

Abstract

Obesity can lead to pathological growth of adipocytes by inducing inflammation and oxidative stress. Genistein could be a potential candidate for the treatment of obesity due to its antioxidant properties. Specific kits were used to examine the effects of genistein vs adiponectin on human visceral pre-adipocytes differentiation, cell viability, mitochondrial membrane potential, and oxidative stress in pre-adipocytes and in white/brown adipocytes. Western Blot was performed to examine changes in protein activation/expression. Genistein increased human visceral pre-adipocytes differentiation and browning, and caused a dose-related improvement of cell viability and mitochondrial membrane potential. Similar effects were observed in brown adipocytes and in white adipocytes, although in white cells the increase of cell viability was inversely related to the dose. Moreover, genistein potentiated AMP-activated protein kinase (AMPK)/mitofusin2 activation/expression in pre-adipocytes and white/brown adipocytes and protected them from the effects of hydrogen peroxide. The effects caused by genistein were similar to those of adiponectin. The results obtained showed that genistein increases human visceral pre-adipocytes differentiation and browning, protected against oxidative stress in pre-adipocytes and white/brown adipocytes through mechanisms related to AMPK-signalling and the keeping of mitochondrial function.

Keywords: adiponectin; browning; cell survival; differentiation; genistein; mitochondria; peroxidation.

Figure 1

Quantitative (A) and qualitative (B) analysis of the effects of genistein on pre-adipocytes differentiation by using Oil Red O Staining: In A, effects of genistein (G) 10 pM, 1 µM, 50 µM and 200 µM, adiponectin (Adipo; 100 µM) for all the differentiating period. C = control (pre-adipocytes treated only with differentiation medium). Reported data are means ± SD of five independent experiments for each experimental protocol executed in pre-adipocytes taken from each patient. In A, ^# p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM. Short square brackets indicate significance between groups (p < 0.05). In (B), in each panel one example for each treatment is reported. The images were taken at 10 × magnification.

Figure 2

Effects of genistein in pre-adipocytes, cultured in physiological condition, on cell viability (A) and mitochondrial membrane potential (B): In A and B, the effects of genistein (G) 10 pM, 1 µM, 50 µM and 200 µM, adiponectin (Adipo; 100 µM), wortmannin (W; 200 µM), dorsomorphin (D; 200 µM). C = control. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 3

Effects of genistein in white adipocytes, cultured in physiological condition, on cell viability (A) and mitochondrial membrane potential (B), and in brown adipocytes, on cell viability (C) and mitochondrial membrane potential (D). Abbreviations are as in Figure 1 and Figure 2. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 4

Effects of genistein in pre-adipocytes, cultured in peroxidative condition, on cell viability (A) and mitochondrial membrane potential (B). N acetyl-cysteine (NAC, 200 µM). Other abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; ^## p < 0.05 vs. H₂O₂ 200 µM; ** p < 0.05 vs. Adipo 100 µM + H₂O₂ 200 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 5

Effects of genistein in white adipocytes, cultured in peroxidative condition, on cell viability (A) and mitochondrial membrane potential (B), and in brown adipocytes, on cell viability (C) and mitochondrial membrane potential (D). Abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; ^## p < 0.05 vs. H₂O₂ 200 µM; ** p < 0.05 vs. Adipo 100 µM + H₂O₂ 200 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 6

Effects of genistein on ROS production in pre-adipocytes, cultured in peroxidative (A) and physiological (B) conditions, and in white (C) and brown (D) adipocytes. Abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM; ^## p < 0.05 vs. H₂O₂ 200 µM; ** p < 0.05 vs. Adipo 100 µM + H₂O₂ 200 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 7

Effects of genistein on UCP1 activation in pre-adipocytes (A), and brown adipocytes (B), cultured in physiological and peroxidative conditions: Abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM; ^## p < 0.05 vs. H₂O₂ 200 µM; ** p < 0.05 vs. Adipo100 µM + H₂O₂ 200 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 8

Effects of genistein on Akt activation in pre-adipocytes (A), white adipocytes (B) and brown adipocytes (C), cultured in physiological and peroxidative conditions: Abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: ^# p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM; ^## p < 0.05 vs. H₂O₂ 200 µM; ** p < 0.05 vs. Adipo 100 µM + H₂O₂ 200 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 9

Effects of genistein on Mfn2 expression in pre-adipocytes (A), white adipocytes (B) and brown adipocytes (C), cultured in physiological and peroxidative conditions: Abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: # p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM; ## p < 0.05 vs. H2O2 200 µM; ** p < 0.05 vs. Adipo 100 µM + H2O2 200 µM. Short square brackets indicate significance between groups (p < 0.05).

Figure 10

Effects of genistein on AMPKα/β activation in pre-adipocytes (A), white adipocytes (B) and brown adipocytes (C), cultured in physiological and peroxidative conditions: Abbreviations are as in previous figures. Reported data are means ± SD of five independent experiments for each experimental protocol executed in adipocytes taken from each patient. Significance between groups: # p < 0.05 vs. control; * p < 0.05 vs. Adipo 100 µM; ## p < 0.05 vs. H2O2 200 µM; ** p < 0.05 vs. Adipo 100 µM + H2O2 200 µM. Short square brackets indicate significance between groups (p < 0.05).