A Novel Mix of Polyphenols and Micronutrients Reduces Adipogenesis and Promotes White Adipose Tissue Browning via UCP1 Expression and AMPK Activation

Figure 1. Oil Red O staining in 3T3-L1 adipocytes. Cells were seeded at a density of 1 × 105 cells/well in a 6-well plate and differentiated with or without A5+. (a) Cells were stained with Oil Red O and lipid droplets were visualized in optical microscopy (20× magnification) and quantified by measuring absorbance. (b–d) Bar graphs illustrating the most relevant adipogenesis-related genes modulated by A5+. Results are expressed as the mean ± SEM. * p < 0.05, ** p < 0,005, *** p < 0.001. Graphs illustrate three different experiments conducted separately. FAB4: Fatty acid-binding protein 4; PPARγ (peroxisome proliferator-activated receptor gamma).

Figure 2. Cell proliferation and cell cycle analysis. (a) Cell proliferation during MCE; (b) Cell cycle analysis during MCE; (c) Cell cycle analysis at day 10. Results are expressed as the mean ± SEM. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001. Graphs illustrate three different experiments conducted separately.

Figure 3. A5+ reduced adipocytes induced inflammation. (a) Representative blot assay reporting all cytokines evaluated; (b) Bar graph illustrating the most relevant cytokines modulated by A5+ administration. Data are reported as mean ± SEM (n = 4). (* p < 0.05; ** p < 0.005; *** p < 0.001). ctr: control; BLC: B lymphocyte chemoattractant; IL-6 Interlukin-6; CXCL9: chemokine (C-X-C motif) ligand 9; RANTES: Regulated upon Activation, Normal T Cell Expressed and Secreted; TIMP1: Tissue inhibitor matrix metalloproteinase 1.

Figure 4. A5+ promotes browning related genes. Bar graph illustrating the most relevant browning-related genes modulated by A5+ administration: (a) UCP1, (b) Adrb3, (c) Cidea, (d) DIO2. Data are reported as mean ± SEM (n = 4). (* p < 0.05; **** p < 0.0001). ctr: control; UCP1: uncoupling protein 1; Adrb3: β3-adrenergic receptor; DIO2: Deiodinase, iodothyronine, type II. ns: not significant.

Figure 5. A5+-induced lipid metabolism. Bar graph illustrating the most relevant lipid metabolism-related genes modulated by A5+ administration: (a) Genes related to mitochondrial fatty acids (FA) uptake, (b) Genes involved in fatty acid oxidation (FAO), (c) Genes regulating tricarboxylic acid (TCA) cycle. Data are reported as mean ± SEM (n = 4). (* p < 0.05; ** p < 0.005; ns: not significant). ctr: control; Cpt1beta: Carnitine palmitoyltransferase I beta; Cpt2: Carnitine palmitoyltransferase II; Crat: Carnitine acetyltransferase; ACADM: acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain; ACADL: Acyl-CoA dehydrogenase, long chain; Hadha: Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, alpha subunit; Aco2: aconitase 2; Idh3a: Isocitrate dehydrogenase subunit alpha; Sdhac: Succinate dehydrogenase complex subunit C; Cs: Citrate synthase; TBP: TATA box binding protein.

Figure 6. A5+ determined activation of AMPK-ATGL pathway in 3T3-L1 adipocytes. (a) Representative immunoblots of UCP1, AMPK, and ATGL activation analysis (n = 6), (b) distribution graphs of the densitometric scanning analyses performed by ImageQuant TL software by using GAPDH as loading control. Phosphorylated forms were normalized in comparison with their total forms. * p < 0.05; *** p < 0.001.

Figure 7. Schematic representation of A5+ effects on adipogenesis and browning. A5+ administration increased the expression levels of several genes involved in FAO, FA uptake and TCA. Moreover, it also promoted the activation of the AMPK-ATGL pathway and increased the expression levels of UCP1, leading to BAT generation and reducing the pro-inflammatory state typical of obesity and white adipogenesis. Created by Bio-Render.com.