Functional Complementation of Anti-Adipogenic Phytonutrients for Obesity Prevention and Management

Figures

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  Figure 1. Suppression of PPARγ expression in differentiating preadipocytes. Expression level of PPARγ in preadipocytes on d0, d6 post-differentiation (d6), and d6 post-differentiation in the presence of (A) berberine (C1), (B) luteolin (C2), (C) resveratrol (C3), (D) fisetin (C4), (E) quercetin (C5), (F) fucoidan (C6), (G) EGCG (C7), (H) hesperidin (C8), (I) curcumin (C9), or (J) F1 composition. β-actin served as a loading control. The protein expression level was measured using multiplexed capillary Western immunoassays. Molecular markers in kilodalton (kDa) are displayed on the left of the capillary Western immunoassay data.

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  Figure 2. Enhancement of SREBP1c expression in differentiating preadipocytes. Expression level of SREBP1c in preadipocytes on d0, d6 post-differentiation, and d6 post-differentiation in the presence of (A) berberine (C1), (B) luteolin (C2), (C) resveratrol (C3), (D) fisetin (C4), (E) quercetin (C5), (F) fucoidan (C6), (G) EGCG (C7), (H) hesperidin (C8), (I) curcumin (C9), or (J) F1 composition. β-actin served as a loading control. The protein expression level was measured using multiplexed capillary Western immunoassays. Molecular markers in kilodalton (kDa) are displayed on the left of the capillary Western immunoassay data.

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  Figure 3. Suppression of FASN expression in differentiating preadipocytes. Expression level of FASN in preadipocytes on d0, d6 post-differentiation, or d6 post-differentiation in the presence of (A) berberine (C1), (B) luteolin (C2), (C) resveratrol (C3), (D) fisetin (C4), (E) quercetin (C5), (F) fucoidan (C6), (G) EGCG (C7), (H) hesperidin (C8), (I) curcumin (C9), or (J) F1 composition. β-actin served as a loading control. The protein expression level was measured using multiplexed capillary Western immunoassays. Molecular markers in kilodalton (kDa) are displayed on the left of the capillary Western immunoassay data.

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  Figure 4. Suppression of PLIN1 expression in differentiating preadipocytes. Expression level of PLIN1 in preadipocytes on d0, d6 post-differentiation, or d6 post-differentiation in the presence of (A) berberine (C1), (B) luteolin (C2), (C) resveratrol (C3), (D) fisetin (C4), (E) quercetin (C5), (F) fucoidan (C6), (G) EGCG (C7), (H) hesperidin (C8), (I) curcumin (C9), or (J) F1 composition. β-actin served as a loading control. The protein expression level was measured using multiplexed capillary Western immunoassays. Molecular markers in kilodalton (kDa) are displayed on the left of the capillary Western immunoassay data.

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  Figure 5. Suppression of FAB4 expression in differentiating preadipocytes. Expression level of FABP4 in preadipocytes on d0, d6 post-differentiation (d6), or d6 post-differentiation in the presence of (A) berberine (C1), (B) luteolin (C2), (C) resveratrol (C3), (D) fisetin (C4), (E) quercetin (C5), (F) fucoidan (C6), (G) EGCG (C7), (H) hesperidin (C8), (I) curcumin (C9), or (J) F1 composition. β-actin served as a loading control. The FABP4 protein post-translational modification profile was measured using capillary isoelectric focusing immunoassays.

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  Figure 6. Inhibition of β-catenin degradation in differentiating preadipocytes. Expression level of β-catenin in preadipocytes on d0, d6 post-differentiation, or d6 post-differentiation in the presence of (A) berberine (C1), (B) luteolin (C2), (C) resveratrol (C3), (D) fisetin (C4), (E) quercetin (C5), (F) fucoidan (C6), (G) EGCG (C7), (H) hesperidin (C8), (I) curcumin (C9), or (J) F1 composition. β-actin served as a loading control. The protein expression level was measured using multiplexed capillary Western immunoassays. Molecular markers in kilodalton (kDa) are displayed on the left of the capillary Western immunoassay data.

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  Figure 7. Potent and lasting inhibition of adipogenesis by F1 composition. First row: Undifferentiated preadipocytes in growth media. Second row: Differentiating preadipocytes or adipocytes. Third row: Differentiating preadipocytes in complete differentiation media supplemented with quercetin (C5). Fourth row: Differentiating preadipocytes in complete differentiation media supplemented with luteolin (C2) and quercetin (C5). Fifth row: Differentiating preadipocytes in complete differentiation media supplemented with berberine (C1), luteolin (C2), resveratrol (C3), and quercetin (C5). Sixth row: Differentiating preadipocytes in complete differentiation media supplemented with the F1 composition of phytonutrients. First column: d6 post-differentiation; second column: d8 post-differentiation; third column: d10 post-differentiation; fourth column: d14 post-differentiation. Cytoplasmic lipid droplets appeared as white particles under phase-contrast microscopy.

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  Figure 8. Inhibition of intracellular lipid droplet accumulation by the F1 composition of phytonutrients. (A) Brightfield images of hematoxylin-, eosin-, and Oil Red O-stained control undifferentiated preadipocytes (left panel), control differentiating adipocytes on d14 post-differentiation (middle panel), and differentiating adipocytes treated with F1 composition on d14 post-differentiation (right panel). (B) Multi-color confocal fluorescence images of control undifferentiated preadipocytes (left panel), control differentiating adipocytes on d14 post-differentiation (middle panel), and differentiating adipocytes treated with F1 composition on d14 post-differentiation (right panel). Cells were stained for nucleus (blue), actin (green), tubulin (red), and neutral lipids (pink).

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  Figure 9. F1 composition reduces weight gain in diet-induced obesity mice. (A) Average bodyweight as a function of time of LD, HFD, and HFD + F1 animal groups. (B) Average bodyweight at week seventeen as a function of LD, HFD, and HFD + F1 animal groups. (C) Average rate of weight gain in grams per week (g/w) as a function of LD, HFD, and HFD + F1 animal groups. Error bars are standard deviations of forty mice per animal group. Single and double asterisks indicate a statistical significance of p-value ≤ 0.01 versus LD and HFD animal groups, respectively.

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  Figure 10. F1 composition improves glucose tolerance in diet-induced obesity mice. (A) Blood glucose level as a function of time post-injection. (B) Fold change in blood glucose level as a function of time post-injection. Error bars are the standard deviations of forty mice per animal group. Single and double asterisks indicate a statistical significance of a p-value ≤ 0.01 versus the LD and HFD animal groups, respectively.

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  Figure 11. F1 composition reduces blood lipids. Blood (A) triglyceride, (B) cholesterol, (C) high-density lipoprotein (HDL), and (D) low-density lipoprotein (LDL) levels as functions of LD, HFD, and HFD + F1 animal groups. Error bars are the standard deviations of forty mice per animal group. Single and double asterisks indicate a statistical significance of a p-value ≤ 0.01 versus the LD and HFD animal groups, respectively.

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  Figure 12. F1 composition reduces liver steatosis and visceral adiposity. H&E histology of (A) liver tissues and (B) visceral adipose tissues collected from LD, HFD, and HFD + F1 mice. Images were acquired with brightfield microscopy. Lipid droplets are indicated as white dots. Average weight of (C) liver tissues and (D) visceral adipose tissues (VAT) as functions of animal groups on specified diets. Error bars are the standard deviations of forty mice per animal group. Single and double asterisks indicate a statistical significance of a p-value ≤ 0.01 versus LD and HFD animal groups, respectively.