Circulating Human Metabolites Resulting from TOTUM-070 Absorption (a Plant-Based, Polyphenol-Rich Ingredient) Improve Lipid Metabolism in Human Hepatocytes: Lessons from an Original Ex Vivo Clinical Trial

Figures

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  Figure 1. Effect of Totum-070 supplementation on circulating lipid parameters in high fat-fed hamsters. (A) Experimental design. (B) Total cholesterol. (C) non-HDL cholesterol. (D) HDL cholesterol. (E) Triglycerides. Values are presented as the means ± SEM unless specified otherwise. The differences were considered statistically significant at p < 0.05 with * for p < 0.05; ** for p < 0.01; *** for p < 0.001. Total cholesterol: global p-values (time < 0.001; column factor < 0.001; time × column factor < 0.001); F values (time F = 263; column factor F = 143; time x column factor F = 57.9); HDL cholesterol: global p-values (time < 0.001; column factor < 0.001; time × column factor = 0.01); F values (time F = 14.9; column factor F = 13.3; time × column factor F = 3.43); non-HDL cholesterol: global p-values (time < 0.001; column factor < 0.001; time × column factor = 0.01); F values (time F = 212; column factor F = 89.6; time × column factor F = 48.2); Total triglycerides: global p-values (time < 0.001; column factor < 0.001; time × column factor < 0.001); F values (time F = 17.2; column factor F = 18.4; time × column factor F = 5.40).

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  Figure 2. Absorption profile. (A,B) Circulating polyphenol metabolites resulting from human absorption and metabolization of TOTUM-070. For each metabolite, 100% is the highest post-absorptive AUC value. (C) Metabolites diversity per time point following TOTUM-070 ingestion (cumulated percentage values of the 20 metabolites at each time point ± SD).

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  Figure 3. Human HepG2 hepatocytes subjected to ex vivo procedures. (A) Experimental design. (B) Phase contrast microscopy of human HepG2 hepatocytes. (C) Cell viability measured by XTT-based assay. Measures were performed in quadruplicates per condition/volunteer (n = 10 volunteers). Values are presented as the means ± SEM unless specified otherwise. The differences were considered statistically significant at p < 0.05 with * for p < 0.05; **** for p < 0.0001 and ns for p > 0.05. Cell viability 24 h (global p-value < 0.0001; F value = 10.44); 72 h (global p-value < 0.0001; F value = 69.82). Scale bar, 100 µm.

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  Figure 4. Human HepG2 hepatocytes Red Oil Staining. (A) Naïve human serum. (B) Human enriched serum with TOTUM-070 metabolites. (C) Naïve human serum + palmitate 250 µM. (D) Human enriched serum with TOTUM-070 metabolites + palmitate 250 µM. Scale bar, 100 µm.

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  Figure 5. Quantification of intracellular lipid stocks. (A) Triglyceride content. (B) Cholesterol content. Measures were performed in quadruplicates per condition/volunteer (n = 10 volunteers). Values are presented as the means ± SEM unless specified otherwise. The differences were considered statistically significant at p < 0.05 with * for p < 0.05; **** for p < 0.0001 and ns for p > 0.05. Triglycerides: (global p-value < 0.0001; Kruskal–Wallis statistic = 59.31); Total cholesterol (global p-value < 0.0001; Kruskal–Wallis statistic = 52.95).

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  Figure 6. Volcano plot from RNA sequencing analysis. Genes with significant modulation are visualized with a volcano plot, to show statistical significance (adjusted p-values) versus magnitude of change (fold-change). Genes in red have a corrected p-value less than 0.05 and a log fold-change greater than 1.

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  Figure 7. TOTUM-070 derived metabolites limit lipid synthesis. (A) Fatty acid synthase protein expression. (B) HMG coA Reductase activity. Measures were performed in quadruplicates per condition/volunteer (n = 10 volunteers). Values are presented as the means ± SEM unless specified otherwise. The differences were considered statistically significant at p < 0.05 with **** for p < 0.0001 and ns for p > 0.05. FAS: (global p-value < 0.0001; F value = 23.09); HMGCoA (global p-value < 0.0001; Kruskal–Wallis statistic = 35.25).