Humulus japonicus stimulates thermogenesis and ameliorates oxidative stress in mouse adipocytes

in Journal of Molecular Endocrinology
Correspondence should be addressed to J H Jeong: jhjeong3@cau.ac.kr
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An aqueous extract of Humulus japonicus (AH) has been documented to ameliorate hypertension and non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effects of an aqueous extract of AH on thermogenesis and palmitate-induced oxidative stress in adipocytes. To verify the effect of AH on browning, we measured the expression levels of specific markers in 3T3-L1 adipocytes using qPCR and Western blotting, respectively. To assess the role of oxidative stress, cells were stained with DCFDA and observed by fluorescence microscopy. AH increased the expression of brown adipose tissue-specific markers. Additionally, it induced fatty acid oxidation and lipolysis and suppressed both lipogenic markers and lipid accumulation. Furthermore, AH ameliorated hydrogen peroxide-induced oxidative stress. Enhanced expression of these markers contributed to fat browning, fatty acid oxidation and lipolysis of 3T3-L1 adipocytes via the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor delta (PPARδ) signaling pathways. Moreover, AMPK and PPARδ resulting in protective effects of AH against oxidative stress. In sum, AH could promote the browning, lipolysis and thermogenesis in 3T3-L1 adipocytes and would suppress the hydrogen peroxide-induced oxidative stress and lipogenesis during differentiation. We therefore suggest that AH could be used as a potential candidate for treating obesity and related metabolic disorders.

 

      Society for Endocrinology

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    AH stimulates adipocyte browning. (A) Viability of 3T3-L1 adipocytes treated with AH at various concentrations (0, 20 and 100 μg/mL) for 24 h measured by MTT assay. (B) Quantitative real-time-PCR assay of Ucp1, Pgc1α and Prdm16 in 3T3-L1 adipocytes treated with various concentrations of AH for 24 h. (C) Western blotting analysis of UCP-1, PGC1α and PRDM16 in 3T3-L1 adipocytes treated with various concentrations of AH for 24 h. Mean ± s.e.m. was obtained from three separate experiments. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to the controls.

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    AH suppresses lipogenesis during differentiation and increases fatty acid oxidation and lipolysis in 3T3-L1 adipocytes. (A) Oil red O staining in preadipocytes and differentiated 3T3-L1 cells treated with AH (0, 20 and 100 μg/mL) for 24 h. Quantitative analysis of lipid accumulation was performed using an isopropanol extraction method. (B) Western blotting analysis of processed SREBP1, FAS and SCD1 in 3T3-L1 adipocytes treated with AH (0, 20 and 100 μg/mL) for 24 h. (C) Quantitative real-time-PCR assay of Cpt1, Aco and Fabp3 in 3T3-L1 adipocytes treated with AH (0, 20 and 100 μg/mL) for 24 h. (D) Intracellular acetyl Co-A and intracellular ATP levels were measured in 3T3-L1 adipocytes treated with AH (0, 20 and 100 μg/mL) for 24 h. Western blotting analysis of CPT1, ACO and FABP3 (E) and lipolysis (F) in 3T3-L1 adipocytes treated with AH (0, 20 and 100 μg/mL) for 24 h. Mean ± s.e.m. was calculated from data obtained from three independent experiments. ***P < 0.001, **P < 0.01 and *P < 0.05 compared to controls. !!! P < 0.001, !! P < 0.01 and ! P < 0.05 compared to 3T3-L1 adipocytes. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0010.

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    AH increases browning, fatty acid oxidation and lipolysis and suppresses lipogenesis during differentiation in 3T3-L1 adipocytes through AMPK or PPARδ pathway. (A) Western blotting analysis of AMPK phosphorylation in 3T3-L1 adipocytes treated with AH (0, 20 and 100 μg/mL) for 24 h. (B) Western blotting analysis of PPARδ expression in scrambled or AMPKsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. (C) Western blotting analysis of AMPK phosphorylation in scrambled or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. Quantitative real-time-PCR assay of Ucp1, Pgc1α and Prdm16 (D) and Western blotting analysis of UCP1, PGC1α and PRDM16 expression (E) in scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. Quantitative real-time-PCR assay of Cpt1, Aco and Fabp3 (F) and intracellular acetyl Co-A and intracellular ATP levels (G) were measured in scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. (H) Western blotting analysis of CPT1, ACO and FABP3 expression in scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. (I) Oil red O staining in preadipocytes and differentiated 3T3-L1 cells scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. Quantitative analysis of lipid accumulation was performed using an isopropanol extraction method. (J) Western blotting analysis of processed SREBP1, FAS and SCD1 in preadipocytes and differentiated 3T3-L1 cells scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. Mean ± s.e.m. was calculated from data obtained from three independent experiments. ***P < 0.001 and **P < 0.01 compared to controls. !!! P < 0.001, !! P < 0.01, and ! P < 0.05 compared to AH treatment in 3T3-L1 adipocytes or control in 3T3-L1 adipocytes. ## P < 0.01 and # P < 0.05 compared to controls in 3T3-L1 adipocytes. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0010.

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    AH ameliorates oxidative stress through AMPK or PPARδ-mediated pathway. (A) ROS staining of preadipocytes and differentiated 3T3-L1 cells scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h. Western blotting analysis of SOD1, catalase and GPx-1 expression in preadipocytes and differentiated 3T3-L1 cells in the presence of AH (0, 20 and 100 μg/mL) (B) and in preadipocytes and differentiated 3T3-L1 cells scrambled, AMPKsiRNA or PPARδsiRNA-transfected 3T3-L1 adipocytes treated with AH for 24 h (C and D). Mean ± s.e.m. was calculated from data obtained from three independent experiments. ***P < 0.001 and *P < 0.05 compared to controls. !!! P < 0.001, !! P < 0.01 and ! P < 0.05 compared to AH treatment or H2O2 treatment. ## P < 0.01 compared to H2O2 and AH treatment in 3T3-L1 adipocytes. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0010.

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    Schematic diagram of the effects of AH on adipocytes. A full colour version of this figure is available at https://doi.org/10.1530/JME-19-0010.

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