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Adiponectin Receptor Agonist Adiporon Suppresses Adipogenesis in C3H10T1/2 Cells Through the Adenosine Monophosphate‑Activated Protein Kinase Signaling Pathway

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Adiponectin Receptor Agonist Adiporon Suppresses Adipogenesis in C3H10T1/2 Cells Through the Adenosine Monophosphate‑Activated Protein Kinase Signaling Pathway MOLECULAR MEDICINE REPORTS 16: 7163-7169, 2017 Adiponectin receptor agonist AdipoRon suppresses adipogenesis in C3H10T1/2 cells through the adenosine monophosphate‑activated protein kinase signaling pathway SHU-JUAN WANG, WEN-YI LU and KAI-YAN LIU Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Peking University People's Hospital and Institute of Hematology, Beijing 100044, P.R. China Received January 3, 2017; Accepted July 24, 2017 DOI: 10.3892/mmr.2017.7450 Abstract. The aim of the present study was to investigate Introduction the effects of AdipoRon, an adiponectin receptor agonist, on adipogenesis in C3H10T1/2 cells and to explore the underlying Obesity is a common public health problem and is a signifi- mechanisms. C3H10T1/2 cells were treated with increasing cant risk factor for a number of metabolic disorders, including doses of AdipoRon for 8 days, and Oil Red O staining was type 2 diabetes, hypertension and cardiovascular disease (1). used to assess lipid accumulation. The protein and mRNA The development of obesity is characterized by an increase in expression levels of adipogenic transcription factors and the number and size of mature adipocytes that are produced by adipocyte-specific genes were examined by western blot- differentiation and mitogenesis (2). Adipogenesis is controlled ting and reverse transcription quantitative polymerase chain by many transcription factors, including peroxisome prolif- reaction, respectively. AdipoRon treatment inhibited lipid erator-activated receptor γ (PPARγ) and CCAAT/enhancer accumulation in C3H10T1/2 cells in a dose-dependent manner binding proteins (C/EBPs) (3-5). At the initial stage of and significantly suppressed the expression of adipogenic tran- adipogenesis, C/EBPβ is activated, followed by the sequential scription factors, including peroxisome proliferator-activated activation of C/EBPα and PPARγ, two major late-adipogenic receptor γ, CAAT/enhancer binding protein (C/EBP)-β and transcription factors. Activation of C/EBPα and PPARγ subse- C/EBPα. In addition, cells treated with AdipoRon exhibited quently leads to the expression of a number of genes involved a significant decrease in the expression of adipocyte‑specific in adipocyte differentiation, including genes encoding lipid genes, including fatty acid binding protein 4, fatty acid metabolizing enzymes [such as fatty acid binding protein synthase, leptin, adiponectin, and stearoyl-CoA desaturase-1. (FABP)-4 and fatty acid synthase (FAS)] and adipokines (such Notably, AdipoRon significantly increased the phosphoryla- as adiponectin and leptin) (6). tion of adenosine monophosphate-activated protein kinase Adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). The results (AMPK) is an important cellular energy sensor, and its activa- indicated that AdipoRon exerted an inhibitory effect on adipo- tion is closely related to the balance between lipid accumulation genesis in C3H10T1/2 cells by downregulating the expression and carbohydrate metabolism (7). AMPK phosphorylation of adipogenic transcription factors and adipocyte-specific inhibits metabolic enzymes involved in fatty acid synthesis, genes and by promoting the phosphorylation of AMPK and leading to suppressed adipogenesis (8,9). ACC, which suggested that AdipoRon may be a potential drug Adiponectin is an adipokine that is mainly produced by to prevent and treat diseases caused by abnormal adipogenesis, adipocytes. Unlike other adipokines, plasma adiponectin such as obesity. levels are reduced in obese subjects (10). Adiponectin binds to the adiponectin receptors, AdipoR1 and AdipoR2, and reduces obesity-related insulin resistance (11). AdipoRon, a newly discovered, orally active small molecule, is an adiponectin receptor agonist that binds to and activates both Correspondence to: Professor Kai-Yan Liu, Beijing Key Laboratory AdipoR1 and AdipoR2, and has been previously reported to of Hematopoietic Stem Cell Transplantation, Peking University improve obesity-related insulin resistance and type 2 diabetes People's Hospital and Institute of Hematology, 11 Xi-Zhi-Men South by activating AMPK and PPARα pathways (12). However, the Street, Beijing 100044, P.R. China effects of adiponectin on obesity itself and on adipogenesis E-mail: liukaiyan@medmail.com.cn are controversial. Adiponectin may promote adipogenesis induced by weight gain (11,13). By contrast, elevated adipo- Key words: adiponectin, AdipoRon, antiadipogenesis, adenosine nectin levels were recently reported to reduce lipid content monophosphate-activated protein kinase, C3H10T1/2 cells and lipid metabolism in 3T3-L1 cells (14). However, the effects of AdipoRon on adipogenesis have not yet been investigated. The present study investigated the effects of AdipoRon on 7164 WANG et al: AdipoRon SUPPRESSES ADIPOGENESIS IN C3H10T1/2 CELLS adipogenesis and explored the underlying mechanism in in the cells were observed under an inverted microscope C3H10T1/2 cells. (Olympus-CKX41; Olympus Corporation, Tokyo, Japan). The ORO-stained plates were then washed and treated with Materials and methods 100% isopropanol (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and lipid accumulation was detected Reagents. AdipoRon, 5-aminoimidazole-4-carboxamide-1-β by measuring the absorbance at 490 nm using a Benchmark -D-ribofuranoside (AICAR), dexamethasone (Dex), microplate reader (Bio-Rad Laboratories, Inc.). 3-isobutyl-1-methylxanthine (IBMX), indomethacin, insulin and Oil Red O were purchased from Sigma-Aldrich (Merck Western blot analysis. Cells were cultured in 6-well plates KGaA, Darmstadt, Germany). Minimum essential Eagle's with a seeding density of 5x104 cells/ml and differentiated medium with Earle's Balanced Salts (MEM-EBSS) was as described above. Cells were lysed in radioimmunopre- purchased from HyClone (GE Healthcare Life Sciences, Logan, cipitation assay buffer (Beyotime Institute of Biotechnology, UT, USA). Fetal bovine serum (FBS) was purchased from Shanghai, China) supplemented with a protease inhibitor Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). cocktail and phosphatase inhibitors (Roche Molecular Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Diagnostics, Pleasanton, CA, USA) for 20 min and centri- Molecular Technologies, Inc. (Kumamoto, Japan). Monoclonal fuged at 12,000 x g for 20 min at 4˚C. Protein concentration antibodies, including rabbit anti-PPARγ (cat no. 2443), rabbit was determined using the Bicinchoninic Acid Protein Assay anti-FABP4 (cat no. 3544), rabbit anti-C/EBPβ (cat no. 3087), reagent kit (Beijing Solarbio Science & Technology Co., rabbit anti-C/EBPα (cat no. 2295), rabbit anti-AMPKα (cat Ltd.). A total of 30 µg of protein lysates from each sample no. 5831), rabbit anti-phosphorylated-acetyl-CoA carboxylase were separated on 10 or 12% SDS-PAGE and transferred (p-ACC; cat no. 11818), rabbit anti-adiponectin (cat no. 2789) to a polyvinylidene difluoride membrane (EMD Millipore, and rabbit anti-GAPDH (cat no. 5174) were purchased from Billerica, MA, USA). The membrane was incubated with Cell Signaling Technology (Danvers, MA, USA). Rabbit 5% bovine serum albumin in Tris-buffered saline with 0.1% polyclonal anti-p-AMPKα1/2 antibody (cat no. sc-33524) and Tween-20 (TBST) (both from Beijing Solarbio Science & goat polyclonal anti-AdipoR1 antibody (cat no. sc-46748) and Technology Co., Ltd.) for 1 h at room temperature, followed anti-AdipoR2 antibody (cat no. sc-46751) were purchased from by hybridization with primary antibodies against PPARγ Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). (1:1,000), C/EBPβ (1:1,000), C/EBPα (1:1,000), FABP4 (1:2,000), adiponectin (1:500), AMPKα (1:1,000), p-ACC Cell culture and adipocyte differentiation. The C3H10T1/2 (1:500), GAPDH (1:1,000), p-AMPKα1/2 (1:200), AdipoR1 mouse embryonic mesenchymal stem cell line was purchased (1:200) and AdipoR2 (1:200) overnight at 4˚C. Following from the Chinese Academy of Medical Sciences (Beijing, three washes with TBST, the membrane was incubated with China). Cells were cultured in MEM-EBSS supplemented with a horseradish peroxidase-conjugated goat anti-rabbit IgG 10% FBS at 37˚C in a 5% CO2 atmosphere until adipocyte secondary antibody (cat no. sc2004; 1:10,000; Santa Cruz differentiation. Two days after reaching confluence (day 0), Biotechnology, Inc.) or a horseradish peroxidase-conjugated the C3H10T1/2 cells were cultured in differentiation medium rabbit anti-goat IgG secondary antibody (cat no. ZB-2306; (DM; MEM/EBSS containing 10% FBS, 1 µM Dex, 0.5 mM 1:10,000; Beijing Zhongshan Jinqiao Biotechnology Co., IBMX, 50 mM indomethacin and 1 µg/ml insulin) for 2 days Ltd., Beijing, China) for 1 h at room temperature. Following to induce differentiation. Following incubation, MEM/EBSS three washes with TBST, the bands were visualized with an supplemented with 10% FBS and 1 µg/ml insulin was added Enhanced Chemiluminescence Substrate (Thermo Fisher for 2 days as a differentiation medium and was changed every Scientific, Inc.). Densitometry of the western blot bands was 2 days. performed using ImageJ software version 1.31 (National Institutes of Health, Bethesda, ML, USA). Cell viability assay. Cell viability was detected by using a CCK-8 kit, according to the manufacturer's instructions. RNA preparation and reverse transcription‑quantitative Briefly, C3H10T1/2 cells were seeded in 96-well plates at polymerase chain reaction (RT‑qPCR). Cells were cultured 4 4 a density of 5x10 cells/ml at 37˚C in a humidified 5% CO2 in 6-well
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