Phenolic Compounds Reduce the Fat Content in Caenorhabditis elegans by Affecting Lipogenesis, Lipolysis, and Different Stress Responses

Abstract

Supplementation with bioactive compounds capable of regulating energy homeostasis is a promising strategy to manage obesity. Here, we have screened the ability of different phenolic compounds (myricetin, kaempferol, naringin, hesperidin, apigenin, luteolin, resveratrol, curcumin, and epicatechin) and phenolic acids (p-coumaric, ellagic, ferulic, gallic, and vanillic acids) regulating C. elegans fat accumulation. Resveratrol exhibited the strongest lipid-reducing activity, which was accompanied by the improvement of lifespan, oxidative stress, and aging, without affecting worm development. Whole-genome expression microarrays demonstrated that resveratrol affected fat mobilization, fatty acid metabolism, and unfolded protein response of the endoplasmic reticulum (UPR^ER), mimicking the response to calorie restriction. Apigenin induced the oxidative stress response and lipid mobilization, while vanillic acid affected the unfolded-protein response in ER. In summary, our data demonstrates that phenolic compounds exert a lipid-reducing activity in C. elegans through different biological processes and signaling pathways, including those related with lipid mobilization and fatty acid metabolism, oxidative stress, aging, and UPR-ER response. These findings open the door to the possibility of combining them in order to achieve complementary activity against obesity-related disorders.

Keywords: apigenin; bioactive compounds; obesity; resveratrol; vanillic acid.

Figure 1

(A) Representation of the Cohen’s d effect size for the fat-reducing activity of the different phenolic compounds in C. elegans, considering Nile Red results from all doses tested. (B) Cohen’s d effect size of all compounds considering the low (10 µM) and medium (100 µM) doses tested.

Figure 2

Resveratrol increases C. elegans healthspan. (A) Nile Red and Oil Red O staining of DMSO- and resveratrol-treated worms. (B) Nile Red and Oil Red O quantification of DMSO- and resveratrol-treated worms (100 µM). The results are expressed as the mean ± SD relative to DMSO-treated worms. Significance refers to the effect of resveratrol with respect to DMSO control worms (Student’s t-Test, *** p < 0.001). (C) Quantification of lipofuscin aging pigment of resveratrol-treated worms compared to DMSO control worms (mean ± SD relative to DMSO-treated worms). Significance refers to the effect of resveratrol with respect to DMSO control worms (ANOVA followed by LSD test, *** p < 0.001). (D) Quantification of the ROS production (measured by DHE) in resveratrol-treated worms compared to DMSO control worms (mean ± SD relative to DMSO treated worms. Significance refers to the effect of resveratrol with respect to DMSO control worms (ANOVA followed by LSD test, * p < 0.05). (E) Lifespan analysis of resveratrol-treated worms compared to DMSO control worms. (F) Size of the treated- and untreated worms on day 1 of adulthood. Significance refers to the effect of resveratrol with respect to DMSO control worms (Student’s t-Test, * p < 0.05) (G) Microscope observation of the presence of eggs (white arrows) and L1 larvae (blue arrows) in both DMSO- and resveratrol-supplemented plates.

Figure 3

Comparison of the gene expression of resveratrol (200 µM) vs. DMSO-treated worms. (A) Hierarchical clustering analysis of control (green) and resveratrol (yellow) samples. (B) Gene Ontology biological processes enriched in resveratrol-treated worms compared with DMSO. (C) qPCR analysis of genes differentially expressed in microarray analyses. Results are expressed as the fold difference expression levels of each gene in the resveratrol-treated group compared with the DMSO control group, calculated with the 2^−∆∆Ct method. Significance refers to the effect of resveratrol with respect to DMSO control worms (Wilcoxon test, * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 4

Gene expression microarray comparison of apigenin (100 µM)-treated worms vs. DMSO control worms. (A) Hierarchical clustering analysis of apigenin (yellow) and DMSO (green) samples. (B) Gene Ontology biological processes enriched in apigenin-treated worms compared with DMSO control group. (C) qPCR analysis of genes differentially expressed in microarray analyses. Results are expressed as the fold difference expression levels of each gene in the apigenin-treated group compared with the DMSO control group, calculated with the 2^−∆∆Ct method. Significance refers to the effect of resveratrol with respect to DMSO control worms (Wilcoxon test, * p < 0.05; ** p < 0.01; *** p < 0.001). (D) Interaction network obtained from GeneMANIA of the most related genes connected with cyp P450 observed after treatment with apigenin.

Figure 5

Gene expression microarray comparison of vanillic acid (100 µM)-treated worms vs. DMSO control worms. (A) Hierarchical clustering analysis of vanillic acid (yellow) and DMSO (green) samples. (B) qPCR analysis of genes differentially expressed in microarray analyses. Results are expressed as the fold-difference expression levels of each gene in the vanillic acid-treated group compared with the DMSO control group, calculated with the 2^−∆∆Ct method. Significance refers to the effect of vanillic acid with respect to DMSO control worms (Wilcoxon test, * p < 0.05; ** p < 0.01). (C) Quantification of the ROS production (measured by DHE) in vanillic acid-treated worms compared with DMSO control worms (mean ± SD relative to DMSO-treated worms). Significance refers to the effect of vanillic acid with respect to DMSO control worms (Student’s t-Test, * p < 0.05).

Figure 6

(A) C. elegans treatment with resveratrol during the L1 to L4 stages mimics the calorie restriction response, as suggested by the reduced expression of the lipid synthesis-related genes fat-7 and drd-1, together with a reduced lipid mobilization and elevated peroxisome lipid oxidation and hydrolysis, which explains the reduced fat content of the treated worms. As a result, endoplasmic reticulum-UPR and glutathione metabolism stress responses are downregulated, which might be responsible for the improvement of C. elegans health and lifespan by this phenolic compound. (B) C. elegans treatment with apigenin induced a significant upregulation of the triglyceride lipase gene lipl-5, together with changes in the expression of cytochrome P450 monooxygenase genes and gpx-3, which might explain the reduced lipid content of the worms treated with this compound. Similarly to apigenin, vanillic acid treatment also affected the expression of the antioxidant genes cyp-37B1 and gpx-3, suggesting an increased response to oxidative stress in the treated worms. Besides, vanillic acid induced the expression of the glycerophospholipid metabolism-related gene gpdh-1, which might be partially responsible of the fat-reducing activity of this compound in C. elegans and the subsequent activation of the ER-UPR.