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•The triterpenoid, α, β-Amyrin manifests anti-adipogenicity in 3T3-L1 cells.•α, β-Amyrin inhibits preadipocyte differentiation.•α,β-Amyrin suppresses adipogenic transcription factors, ...PPARγ and C/EBPα.•α,β-Amyrin stimulates the glucose transporter GLUT4 and promotes AMPK phosphorylation.
Previous studies have reported the anti-obesity effects of α, β-Amyrin in high fat-fed mice. This study aimed to evaluate whether α, β-Amyrin has an anti-adipogenic effect in 3T3-L1 murine adipocytes and to explore the possible underlying mechanisms. 3T3-L1 pre-adipocytes were differentiated in a medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Cytotoxicity of α, β-Amyrin was assessed by MTT assay. Lipid content in adipocytes was determined by Oil-Red O staining. In addition, the protein expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding proteins alpha (C/EBPα), beta (C/EBPβ), and delta (C/EBPδ) and glucose transporter 4 (GLUT4) were determined by qRT-PCR and western blot analysis. Oil-Red O staining revealed markedly reduced fat accumulation by α, β-Amyrin (6.25–50 μg/mL) without affecting cell viability. Furthermore, our results indicate that α, β-Amyrin can significantly suppress the adipocyte differentiation by downregulating the expression levels of adipogenesis-related key transcription factors such as PPARγ and C/EBPα, but not C/EBPβ or C/EPBδ. In addition, the protein expression of membrane GLUT4 in 3T3- L1 adipocytes treated with α, β-Amyrin was significantly higher than in control cells, indicating that α, β-Amyrin augments glucose uptake. These findings suggest that α, β-Amyrin exerts an anti-adipogenic effect principally via modulation of lipid and carbohydrate metabolism in 3T3-L1cells. The present in vitro findings, taken together with our earlier observation of the anti-obesity effect in vivo, suggest that α, β-Amyrin can be developed as a new therapeutic agent for treatment and prevention of obesity.
Obesity remains a global problem. In search of phytochemicals that have antiobesity potential, this study evaluated
-amyrin, a triterpenoid mixture from
, on high-fat diet-induced obesity in mice. ...Groups of mice (n = 8) were fed a normal diet or a high-fat diet, and were orally treated or not treated with either
-amyrin (10 or 20 mg/kg) or sibutramine (10 mg/kg) for 15 weeks. Variables measured at termination were body weight, visceral fat accumulation, adipocyte surface area, peroxisome proliferator-activated receptor gamma, and lipoprotein lipase expressions in adipose tissue, the levels of plasma glucose and insulin, the satiety hormones ghrelin and leptin, the digestive enzymes amylase and lipase, and the inflammatory mediators TNF-
, interleukin-6, and MCP-1. Results showed that
-amyrin treatment resulted in lower high-fat diet-induced increases in body weight, visceral fat content, adipocyte surface area, peroxisome proliferator-activated receptor gamma, and lipoprotein lipase expressions, and blood glucose and insulin levels. Additionally, the markedly elevated leptin and decreased ghrelin levels seen in the high-fat diet-fed control mice were significantly modulated by
-amyrin treatment. Furthermore,
-amyrin decreased serum TNF-
and MCP-1. These results suggest that
-amyrin could be beneficial in reducing high-fat diet-induced obesity and associated disorders via modulation of enzymatic, hormonal, and inflammatory responses.
Insulin resistance is closely related to the metabolic syndrome that is characterized by a set of diseases that include obesity, dyslipidemia and diabetes mellitus. Due to a considerable increase of ...these diseases in the last decades, several studies are being carried out worldwide aiming to find solutions to prevent and to treat insulin resistance. Therefore, we decided to investigate the effects of the mixture of triterpenes alpha, beta‐Amyrin (AMI) on insulin resistance (IR) induced by sodium palmitate in skeletal muscle cells (C2C12), since previous studies regarding AMI conducted by our laboratory showed that AMI is able to improve insulin resistance in a hypercaloric diet obesity model. For this purpose, undifferentiated C2C12 cells (myoblasts) were maintained in horse serum (2%) for 6–10 days to induce differentiation to myotubes. The cytotoxic effect of AMI (3.12–400 μg/ml) on myoblasts and myotubes cells was assessed by the 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay. The effect of AMI (12.5, 25 and 50 μg/ml) was also evaluated in a glucose uptake model with 2‐N‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl) Amino‐2‐Deoxy‐D‐glucose (2‐NBDG) in non‐resistant myotubes as well as in a model of insulin resistance induced by sodium palmitate (500 μM) complexed to 2% BSA for 24 h. After incubation, free 2‐NBDG was washed and fluorescence densities in cell monolayers were measured with a fluorescence microplate reader (Biotek Instruments, USA) set at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The protein (PT) concentration of each sample was determined by the Lowry method. Results were expressed as mean ± SEM of three independent experiments, each in triplicate. For multiple comparison of parametric data, One‐way ANOVA was used, followed by Tukey's post test. Values of p <0.05 were considered statistically significant. AMI (3.12–400 μg/mL) did not promote reduction of cell viability when compared to the control group, both in myoblasts and myotubes. In myotubes, AMI (25 ug/mL) increased glucose uptake (2.40 ± 0.60 2‐NBGD/PT) when compared to the control group (0.98 ± 0.06 2‐NBGD/PT). In the IR model, AMI (25 and 50μg/mL) also increased glucose uptake (1.3 ± 0.0 and 1.9 ± 0.1 2‐NBDG/PT) in myotubes, as well as rosiglitazone 20 μM (1.4 ± 0.0 2‐NBDG/PT) when compared to the IR control group (0.8 ± 0.1 2‐NBGD/PT). These preliminary results suggest that AMI improves glucose uptake in physiological and insulin resistance models in myotubes. These data corroborate with in vivo findings where AMI is able to improve insulin resistance in a hypercaloric diet obesity model. Studies are being conducted to elucidate the molecular mechanisms involved in these effects.
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This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Keywords alpha, beta-Amyrin; 3T3-L1 cells; Adipocyte differentiation; PPARgamma; C/EBPalpha; GLUT4; Anti-adipogenecity Highlights * The triterpenoid, alpha, beta-Amyrin manifests anti-adipogenicity ...in 3T3-L1 cells. * alpha, beta-Amyrin inhibits preadipocyte differentiation. * alpha,beta-Amyrin suppresses adipogenic transcription factors, PPARgamma and C/EBPalpha. * alpha,beta-Amyrin stimulates the glucose transporter GLUT4 and promotes AMPK phosphorylation. Previous studies have reported the anti-obesity effects of alpha, beta-Amyrin in high fat-fed mice. This study aimed to evaluate whether alpha, beta-Amyrin has an anti-adipogenic effect in 3T3-L1 murine adipocytes and to explore the possible underlying mechanisms. 3T3-L1 pre-adipocytes were differentiated in a medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Cytotoxicity of alpha, beta-Amyrin was assessed by MTT assay. Lipid content in adipocytes was determined by Oil-Red O staining. In addition, the protein expression levels of peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding proteins alpha (C/EBPalpha), beta (C/EBPbeta), and delta (C/EBPdelta) and glucose transporter 4 (GLUT4) were determined by qRT-PCR and western blot analysis. Oil-Red O staining revealed markedly reduced fat accumulation by alpha, beta-Amyrin (6.25--50 mug/mL) without affecting cell viability. Furthermore, our results indicate that alpha, beta-Amyrin can significantly suppress the adipocyte differentiation by downregulating the expression levels of adipogenesis-related key transcription factors such as PPARgamma and C/EBPalpha, but not C/EBPbeta or C/EPBdelta. In addition, the protein expression of membrane GLUT4 in 3T3- L1 adipocytes treated with alpha, beta-Amyrin was significantly higher than in control cells, indicating that alpha, beta-Amyrin augments glucose uptake. These findings suggest that alpha, beta-Amyrin exerts an anti-adipogenic effect principally via modulation of lipid and carbohydrate metabolism in 3T3-L1cells. The present in vitro findings, taken together with our earlier observation of the anti-obesity effect in vivo, suggest that alpha, beta-Amyrin can be developed as a new therapeutic agent for treatment and prevention of obesity. Author Affiliation: (a) Postgraduate Program in Pharmacology, Faculty of Medicine, Federal University of Ceara, Fortaleza, Ceara, Brazil (b) Postgraduate Program in Medical Sciences, Faculty of Medicine, Federal University of Ceara, Fortaleza, Ceara, Brazil (c) Research Center on Biodiversity and Biotechnology, Federal University of Piaui, Parnaiba, Piaui, Brazil (d) Department of Organic Chemistry, Federal University of Piaui, Teresina, Piaui, Brazil (e) Department of Physiology and Pharmacology, Natural Products Laboratory, Faculty of Medicine, Federal University of Ceara, Fortaleza, Ceara, Brazil * Corresponding author at: Department of Physiology and Pharmacology, Natural Products Laboratory, Faculty of Medicine, Federal University of Ceara, Cel Nunes de Melo, 1315, Fortaleza, Ceara, 60430-270, Brazil. Article History: Received 19 June 2018; Revised 3 November 2018; Accepted 6 November 2018 Byline: Karina Moura de Melo (a), Francisca Tuelly Bandeira de Oliveira (b), Rose Anny Costa Silva (b), Ana Luiza Gomes Quindere (b), Jose Delano Barreto Marinho Filho (c), Ana Jersia Araujo (c), Eanes Delgado Barros Pereira (b), Adonias Almeida Carvalho (d), Mariana Helena Chaves (d), Vietla Satyanarayana Rao (e), Flavia Almeida Santos fasufc@gmail.com (e,*)
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