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. 2022 Feb 11;11(2):363.
doi: 10.3390/antiox11020363.

Bio-Waste Products of Mangifera indica L. Reduce Adipogenesis and Exert Antioxidant Effects on 3T3-L1 Cells

Giovanni Pratelli  1 , Daniela Carlisi  1 , Antonella D'Anneo  2 , Antonella Maggio  3 , Sonia Emanuele  1 , Antonio Palumbo Piccionello  3 , Michela Giuliano  2 , Anna De Blasio  2 , Giuseppe Calvaruso  2 , Marianna Lauricella  1
Affiliations

Bio-Waste Products of Mangifera indica L. Reduce Adipogenesis and Exert Antioxidant Effects on 3T3-L1 Cells

Giovanni Pratelli et al. Antioxidants (Basel). .

Abstract

Several studies highlighted the beneficial value of natural compounds in the prevention and treatment of obesity. Here, we investigated the anti-obesity effects of extracts of peel and seed of mango (Mangifera indica L.) cultivated in Sicily (Italy) in 3T3-L1 cells. Mango Peel (MPE) and Mango Seed (MSE) extracts at a 100 µg/mL concentration significantly reduced lipid accumulation and triacylglycerol contents during 3T3-L1 adipocyte differentiation without toxicity. HPLC-ESI-MS analysis showed that both the extracts contain some polyphenolic compounds that can account for the observed biological effects. The anti-adipogenic effect of MPE and MSE was the result of down-regulation of the key adipogenic transcription factor PPARγ and its downstream targets FABP4/aP2, GLUT4 and Adipsin, as well SREBP-1c, a transcription factor which promotes lipogenesis. In addition, both MPE and MSE significantly activated AMPK with the consequent inhibition of Acetyl-CoA-carboxylase (ACC) and up-regulated PPARα. The addition of compound C, a specific AMPK inhibitor, reduced the effects of MPE and MSE on AMPK and ACC phosphorylation, suggesting a role of AMPK in mediating MPE and MSE anti-lipogenic effects. Notably, MPE and MSE possess an elevated radical scavenging activity, as demonstrated by DPPH radical scavenging assay, and reduced ROS content produced during adipocyte differentiation. This last effect could be a consequence of the increase in the antioxidant factors Nrf2, MnSOD and HO-1. In conclusion, MPE and MSE possesses both anti-adipogenic and antioxidant potential, thus suggesting that the bio-waste products of mango are promising anti-obesity natural compounds.

Keywords: 3T3-L1 cells; AMPK; adipogenesis; mango peel extracts; mango seed extracts.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
MPE and MSE possess radical scavenging activity. The antioxidant activity of MPE and MSE was evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay. Different concentrations of MPE and MSE (25–200 µg/mL) were added on ethanol DPPH• solution and absorbance of each concentration was measured at 517 nm by spectrophotometer. The bar graphs represent the mean of three independent experiments ± SD. ** p < 0.01 and *** p < 0.001 with respect to the only vehicle control.
Figure 2
Figure 2
Effects of MPE and MSE on the viability of 3T3-L1 pre-adipocyte cells. 3T3-L1 cells (8 × 103/200 μL of culture medium) were exposed to different doses (25–200 µg/mL) of MPE or MSE for 8 days. Then, the percentage of viable cells was assessed by MTT assay. The values reported are the mean ± SD of three independent experiments. ** p < 0.01 with respect to control cells treated with vehicle only.
Figure 3
Figure 3
MPE and MSE reduce lipid content in 3T3-L1 adipocytes. 3T3-L1 cells were treated with pro-differentiative agents for 8 days in the presence or absence of different concentrations (25, 50 and 100 µg/mL) MPE or MSE, as reported in Methods. Representative photographs showing LDs reduction by Oil red O staining after MPE and MSE treatment (200× original magnification) (A). Lipid droplets (LDs) content was ascertained by analyzing the percentage area Red Oil stained by Image J (B) Quantitative Oil red O staining measured by spectrophotometer at 490 nm reading (C). Cellular TGs content quantified by spectrophotometer at 546 nm reading (D). The results are the mean of three independent experiments ± SD. *** p < 0.001 with respect to the undifferentiated cells (Undif.). # p < 0.05, ## p < 0.01 and ### p < 0.001 with respect to the differentiated untreated cells (Dif.).
Figure 4
Figure 4
MPE and MSE down-regulate the expression of adipogenic differentiation and lipid accumulation markers in 3T3-L1 adipocytes. 3T3-L1 cells were treated with pro-differentiative agents for 8 days in the presence or absence of 100 µg/mL MPE or MSE, as reported in Methods. Then, cell lysates were analysed by Western blotting using specific primary antibodies directed against PPARγ, FABP4/aP2, GLUT4, Adipsin and SREBP1-c. Equal loading of proteins was verified by immunoblotting for β-actin and showed values were assigned in relation to undifferentiated cells (Undif.). The bar graphs represent the mean of three independent experiments ± SD. ** p < 0.01 and *** p < 0.001 with respect to undifferentiated 3T3-L1 cells (Undif). # p < 0.05, ## p < 0.01 and ### p < 0.001 with respect to the differentiated untreated 3T3-L1 cells (Dif.).
Figure 5
Figure 5
MPE and MSE up-regulate PPARα and activate AMPK in 3T3-L1 adipocytes. 3T3-L1 cells were treated with pro-differentiative agents for 8 days in the presence or absence of 100 µg/mL MPE or MSE, as reported in Methods. Then, cell lysates were analysed by Western blotting using specific primary antibodies directed against PPARα, AMPK, phospho-AMPK and phospho-ACC. Equal loading of proteins was verified by immunoblotting for β-actin and showed values were assigned in relation to undifferentiated 3T3-L1 cells (Undif). The bar graphs represent the mean of three independent experiments ± SD. # p < 0.05, ## p < 0.01 and ### p < 0.001 with respect to differentiated untreated cells (Dif.).
Figure 6
Figure 6
Compound C counteracts the effects of MPE and MSE on AMPK activation and ACC phosphorylation. Confluent 3T3-L1 cells were pre-treated for 4 h with 10 μM AMPK inhibitor, compound C (CC), and for another 24 h in the presence or absence of 100 µg/mL MPE or MSE. Cell extracts were then prepared and immunoblotted with antibodies to AMPK, phospho-AMPK, or phospho-ACC. Equal loading of proteins was verified by immunoblotting for β-actin and showed values were assigned in relation to undifferentiated 3T3-L1 cells (Undif). The bar graphs represent the mean of three independent experiments ± SD. * p < 0.05 and *** p < 0.001 with respect to the undifferentiated untreated 3T3-L1 cells.
Figure 7
Figure 7
MPE and MSE exert anti-oxidant effects in 3T3-L1 adipocytes. 3T3-L1 cells were treated with pro-differentiative agents for 8 days in the presence or absence of 100 µg/mL MPE or MSE, as reported in Methods. (A) Intracellular ROS were detected using the redox-sensitive fluorochrome H2-DCFDA. After differentiation, the medium was replaced with 10 µM H2DCFDA solution and the incubation was protracted for 30 min at 37 °C. The oxidation of the fluorochrome generates green fluorescence, which was visualized by a Leica microscope equipped with a DC300F camera using a FITC filter. Representative micrographs of fluorescence microscopy were taken at 200× magnification. (B). Western blotting analysis of Nrf2, MnSOD and HO-1 in 3T3-L1 cells differentiated without or with 100 µg/mL MPE or MSE. Equal loading of proteins was verified by immunoblotting for β-actin and showed values were assigned in relation to undifferentiated cells (Undif.). The bar graphs represent the mean of three independent experiments ± SD. * p < 0.05, ** p < 0.01 with respect to the undifferentiated 3T3-L1 cells, # p < 0.05, ## p < 0.01 and ### p < 0.001 with respect to the differentiated untreated 3T3-L1 cells (Dif.).
Figure 8
Figure 8
The addition of MPE and MSE to the differentiation medium reduced 3T3-L1 adipocytes differentiation, as demonstrated by the loss of LDs and TGs. The anti-adipogenic and anti-lipogenic effects of MPE and MSE seem to be correlated with the ability of mango extracts to reduce PPARγ and SREBP-1c and their targets, as well as to increase AMPK and PPARα. MPE and MSE also reduce ROS content produced during adipocyte differentiation, by activating Nrf2 and its downstream targets.
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