Caffeic and Chlorogenic Acids Synergistically Activate Browning Program in Human Adipocytes: Implications of AMPK- and PPAR-Mediated Pathways

Abstract

Caffeic acid (CA) and chlorogenic acid (CGA) are phenolic compounds claimed to be responsible for the metabolic effects of coffee and tea consumption. Along with their structural similarities, they share common mechanisms such as activation of the AMP-activated protein kinase (AMPK) signaling. The present study aimed to investigate the anti-obesity potential of CA and CGA as co-treatment in human adipocytes. The molecular interactions of CA and CGA with key adipogenic transcription factors were simulated through an in silico molecular docking approach. The expression levels of white and brown adipocyte markers, as well as genes related to lipid metabolism, were analyzed by real-time quantitative PCR and Western blot analyses. Mechanistically, the CA/CGA combination induced lipolysis, upregulated AMPK and browning gene expression and downregulated peroxisome proliferator-activated receptor γ (PPARγ) at both transcriptional and protein levels. The gene expression profiles of the CA/CGA-co-treated adipocytes strongly resembled brown-like signatures. Major pathways identified included the AMPK- and PPAR-related signaling pathways. Collectively, these findings indicated that CA/CGA co-stimulation exerted a browning-inducing potential superior to that of either compound used alone which merits implementation in obesity management. Further, the obtained data provide additional insights on how CA and CGA modify adipocyte function, differentiation and lipid metabolism.

Keywords: adipocytes; anti-obesity effect; browning; caffeic acid; chlorogenic acid; molecular docking; obesity.

Figure 1

Chemical structures of caffeic and chlorogenic acids and molecular docking models of putative interactions with target proteins. Caffeic acid chemical name: (E)-3-(3,4-dihydroxyphenyl)prop-2-enoic acid; Mw 180.16 g/M (A). Chlorogenic acid chemical name: (1S,3R,4R,5R)-3-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy-1,4,5-trihydroxycyclohexane-1-carboxylic acid; Mw 354.31 g/M (B). Putative interaction between caffeic acid and AMP-activated protein kinase (AMPK; PDB: 4CFF); Free energy of binding (ΔG) and affinity (Ki) are −6.4 kcal/M and 20.6 µM, respectively (C). Chlorogenic acid and AMPK (PDB: 4CFF); ΔG −8.1 kcal/M; Ki 1.2 µM (D). Caffeic acid and CAAT/enhancer-binding protein alpha (C/EBPα; PDB: 1NWQ); ΔG −4.7 kcal/M; Ki 362.5 µM (E). Chlorogenic acid and C/EBPα (PDB: 1NWQ); ΔG −6.1 kcal/M; Ki 34.2 µM (F). Caffeic acid and peroxisome proliferator-activated receptor α (PPARα; PDB: 2P54); ΔG −6.0 kcal/M; Ki 40.5 µM (G). Chlorogenic acid and PPARα (PDB: 2P54); ΔG −7.6 kcal/M; Ki 2.7 µM (H). Caffeic acid and peroxisome proliferator-activated receptor γ (PPARγ) (PDB: 2P4Y); ΔG −5.8 kcal/M; Ki 56.8 µM (I). Chlorogenic acid and PPARγ (PDB: 2P4Y); ΔG −7.4 kcal/M; Ki 3.8 µM (J).

Figure 2

Caffeic and chlorogenic acids decreased lipid accumulation and enhanced glycerol release in human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes. Representative pictures captured with 20× magnification (scale bar 50 μm) after the Oil red O staining (A). Absorbance of the Oil red O solution at 495 nm (B). Free glycerol concentration (µM) in the cell culture media (C). Data are expressed as mean ± SEM; each experimental group consisted of at least six technical replicates from three independent biological experiments. * p < 0.05 compared to vehicle control group.

Figure 3

Combined treatment with caffeic and chlorogenic acids altered adipogenic gene expression and induced browning markers’ transcriptional activation in human SGBS adipocytes. Relative mRNA expression (∆∆Cq) normalized to vehicle control group for the following genes: (A) acetyl-coA-carboxylase (ACC), (B) adiponectin (ADIPOQ), (C) AMP-activated protein kinase (AMPK), (D) tumor necrosis factor receptor superfamily member 9 (CD137), (E) CAAT/enhancer-binding protein alpha (CEBPA), (F) cell death activator CIDE-A (CIDEA), (G) fatty acid binding protein 4 (FABP4), (H) fatty acid synthase (FASN), (I) pyruvate dehydrogenase kinase isoform 4 (PDK4), (J) peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1A), (K) peroxisome proliferator-activated receptor alpha (PPARA), (L) PPARG, (M) sterol regulatory element-binding protein 1 (SREBP1) and (N) uncoupling protein 1 (UCP1) from the RT-qPCR. RPL13A and TUBB were applied as reference genes. Each sample was analyzed in triplicate from three independent experiments. Data are presented as mean ± SEM. * p < 0.05 compared to the vehicle control group.

Figure 4

Caffeic and chlorogenic acids diminished adiponectin, CAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) protein production in human SGBS adipocytes. Western blot analysis was performed to examine the protein level of adiponectin (A), C/EBPα (B) and PPARγ (C). Data are expressed as mean ± SEM and are representative of three independent experiments. * p < 0.05 compared to the vehicle control group.