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. 2015 Jun;39(6):967-76.
doi: 10.1038/ijo.2015.23. Epub 2015 Mar 12.

Resveratrol induces brown-like adipocyte formation in white fat through activation of AMP-activated protein kinase (AMPK) α1

S Wang  1 , X Liang  2 , Q Yang  2 , X Fu  2 , C J Rogers  2 , M Zhu  3 , B D Rodgers  2 , Q Jiang  4 , M V Dodson  2 , M Du  2
Affiliations

Resveratrol induces brown-like adipocyte formation in white fat through activation of AMP-activated protein kinase (AMPK) α1

S Wang et al. Int J Obes (Lond). 2015 Jun.

Abstract

Objective: Development of brown-like/beige adipocytes in white adipose tissue (WAT) helps to reduce obesity. Thus we investigated the effects of resveratrol, a dietary polyphenol capable of preventing obesity and related complications in humans and animal models, on brown-like adipocyte formation in inguinal WAT (iWAT).

Methods: CD1 female mice (5-month old) were fed a high-fat diet with/without 0.1% resveratrol. In addition, primary stromal vascular cells separated from iWAT were subjected to resveratrol treatment. Markers of brown-like (beige) adipogenesis were measured and the involvement of AMP-activated protein kinase (AMPK) α1 was assessed using conditional knockout.

Results: Resveratrol significantly increased mRNA and/or protein expression of brown adipocyte markers, including uncoupling protein 1 (UCP1), PR domain-containing 16, cell death-inducing DFFA-like effector A, elongation of very long-chain fatty acids protein 3, peroxisome proliferator-activated receptor-γ coactivator 1α, cytochrome c and pyruvate dehydrogenase, in differentiated iWAT stromal vascular cells (SVCs), suggesting that resveratrol induced brown-like adipocyte formation in vitro. Concomitantly, resveratrol markedly enhanced AMPKα1 phosphorylation and differentiated SVC oxygen consumption. Such changes were absent in cells lacking AMPKα1, showing that AMPKα1 is a critical mediator of resveratrol action. Resveratrol also induced beige adipogenesis in vivo along with the appearance of multiocular adipocytes, increased UCP1 expression and enhanced fatty acid oxidation.

Conclusions: Resveratrol induces brown-like adipocyte formation in iWAT via AMPKα1 activation and suggest that its beneficial antiobesity effects may be partly due to the browning of WAT and, as a consequence, increased oxygen consumption.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Effects of resveratrol on the lipid accumulation and the expression of adipogenic marker genes in differentiated inguinal WAT (iWAT) SVC. A) Oil-Red O staining was conducted in the differentiated iWAT SVC after 7-day brown adipogenic differentiation. Microscopic pictures were taken on day 7 with × 100 magnification. B) The stained Oil-Red O was extracted with isopropanol. The absorbance of the extracted Oil-Red O was spectrophotometrically determined at 530 nm to measure triglyceride (TG) accumulation. C, D) Western blot analysis of adipogenic marker genes (PPARγ and aP2) in the differentiated iWAT SVC after 7-day brown adipogenic differentiation, and β-tubulin was used as a loading control (C). Mean ± SEM of immunoblotting bands of PPARγ and aP2 (D). The intensities of the bands were expressed as arbitrary units. ** P < 0.01 and *** P < 0.001 versus control.
Figure 2
Figure 2
Resveratrol promoted formation of brown-like adipocytes in the differentiated iWAT SVC cells from wild-type mice after 7-day adipogenic differentiation. A) Relative mRNA levels of brown adipocytes selective genes (PRDM16, UCP1, Cidea, Elovl3 and PGC-1a) and beige adipocytes selective genes (CD137, Tbx1 and TMEM26). B) UCP1 immunofluorescence staining for UCP1 in the differentiated iWAT SVC. Nuclei were stained with DAPI (scale bar, 100 μm). C, D) Western blot analysis of brown adipocytes selective genes (UCP1, PRDM16, Cyto C, and PDH) in the differentiated iWAT SVC, and β-tubulin was used as loading control (C); Mean ± SEM of immunoblotting bands of UCP1, PRDM16, Cyto C and PDH (D). The intensities of the bands were expressed as arbitrary units. E) Basal O2 consumption of differentiated iWAT SVC from control and resveratrol treated groups. *P < 0.05, **P < 0.01 and *** P < 0.001 versus control.
Figure 3
Figure 3
Effects of resveratrol on the phosphorylation of AMPKα and Sirt1 in wild type and AMPKα1 knockout iWAT SVC. A) Western blot analysis of phospho-AMPKα (p-AMPKα), t-AMPKα (t-AMPKα) and Sirt1 in the differentiated iWAT SVC of wild type (left part) and AMPKα1 deletion (right part). β-Tubulin was used as loading control. B) Mean ± SEM of immunoblotting bands of p-AMPKα, t-AMPKα, p-AMPKα/t-AMPKα and Sirt1 in wild type and AMPKα1 knockout cells. The intensities of the bands were expressed as arbitrary units. *P < 0.05 versus control.
Figure 4
Figure 4
AMPK inhibition or AMPKα1 deletion eliminated the browning effects of resveratrol on mouse differentiated iWAT SVC. A) Effects of AMPK inhibitor Compound C (CC) in the protein contents of UCP1, PRDM16, Cyto C, PDH, phospho-AMPKα (p-AMPKα), and t-AMPKα (t-AMPKα) in the differentiated iWAT SVC after 7-day brown adipogenic differentiation. β-Tubulin was used as loading control. B) Mean ± SEM of immunoblotting bands of UCP1, PRDM16, Cyto C, PDH, p-AMPKα/t-AMPKα. The intensities of the bands were expressed as arbitrary units. *P < 0.05 versus control, #P < 0.05 versus Resv 10 μM. C) Relative mRNA levels of brown adipocyte selective genes (PRDM16, UCP1, Cidea, Elovl3, and PGC1α) in the differentiated SVC after 7-day differentiation with classical brown adipogenic induction cocktails. SVC cells from iWAT of weaning Rosa26Cre/AMPKα1flox/flox mice were treated with 4-hydroxytamoxifen (4-OHT) to delete AMPKα1 before being induced to undergo brown adipogenic differentiation. D) Basal O2 consumption of differentiated AMPKα1 knockout iWAT SVC from control and resveratrol treated groups. E, F) Western blot analysis of brown adipocyte selective genes (UCP1, PRDM16, Cyto C, and PDH) in the differentiated SVC after 7-day brown adipogenic differentiation, and β-tubulin was used as loading control (E). Mean ± SEM of immunoblotting bands of UCP1, PRDM16, Cyto C and PDH (F). The intensities of the bands were expressed as arbitrary units.
Figure 5
Figure 5
Resveratrol induced brown-like adipocytes in iWAT. A) Weekly food intake were measured in control (n=6) and 0.1% Resv (n=6) groups. B) Body weight changes were compared between control and 0.1% Resv groups during 4 weeks. C) iWAT index was compared between control and 0.1% Resv groups. D) Representative images of H&E and UCP1 IHC staining in sections of iWAT of control and 0.1% Resv treated mice. All images were obtained at × 400 magnification. E) Distribution percentage of adipocyte diameters from control and 0.1% Resv treated mice. Data analysis from the H&E staining sections. F, G) Western blot analyses of p-AMPKα, t-AMPKα, UCP-1, PRDM 16, Cyto C and adipogenic marker genes (PPARγ and aP2) were performed in iWAT of control and resveratrol treated mice, and β-tubulin was used as the loading control (F). Mean ± SEM of immunoblotting bands of p-AMPKα, t-AMPKα, p-AMPKα/t-AMPKα, UCP-1, PRDM16, Cyto C, PPARγ and aP2 (G). The intensities of the bands were expressed as arbitrary units.*P < 0.05, **P < 0.01 versus control.
Figure 6
Figure 6
Resveratrol promoted the lipid oxidation of iWAT. A) O2 consumption of control and resveratrol treated mice were recorded during a 3-h period. B) CO2 production of control and resveratrol treated mice was recorded during a 3-h period. C) Respiratory exchange ratio (RER) of control and resveratrol treated mice was recorded during a 3-h period. D) Average heat production of control and resveratrol treated mice during a 3-h period. F) O2 consumption of iWAT of control and resveratrol treated mice was measured as the decrease in dissolved oxygen (DO). **P < 0.01 versus control.
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