Improvement of insulin resistance by Cyanidin 3-glucoside, anthocyanin from black beans through the up-regulation of GLUT4 gene expression
© Inaguma et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
Black beans have been suggested to have a protective effect against obesity. Cyanidin 3-glucoside (Cy-3-G) belongs to the flavonoid class of molecules and is a member of the anthocyanin family that is present in black beans. Some studies indicated that Cy-3-G from black beans may be beneficial for the improvement of obesity and type II diabetes. It has been reported that Cyanidin 3-glucoside ameliorates insulin sensitivity due by down-regulating the retinol binding protein 4 expression in diabetic mice . Accumulation of neutral lipids, triglyceride (TG) in particular, is highly related to the development of insulin resistance and its consequences, such as type II diabetes. Lipid droplet size regulation is central in the regulation of metabolism and in adipocytokines secretion such as TNF-α and adiponectin [2, 3]. However, little is currently known about how Cy-3-G influences adipocyte differentiation and insulin resistance in 3T3-L1 adipocytes. The purpose of the present study was to determine the effects of Cy-3-G on GLUT4 gene expression to improve insulin resistance in 3T3-L1 adipocytes.
Materials and methods
Cell culture and treatment
Adipocytes, 3T3-L1 cells (Riken Cell Bank, Ibaraki, Japan), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Chemical Co, St. Louis, MO, USA) supplemented with 10 % fetal bovine serum (FBS) (Biowest, Miami, FL, USA, Japan) and 1% 5,000 units/ml penicillin, 5,000 μg/mL streptomycin (PS) (Lonza Walkersville, MD,USA) and incubated in 37 °C in 5 % CO2. For adipocyte differentiation, 3T3-L1 cells were cultured at 3.0×105 cells/well in 6-well plates. Cells were maintained until full confluence, which is often around 48h, prior to treatment, . And then, cells were treated with differentiation medium containing Dexamethazone (DEX) Solution, 3-Isobuthyl-1-Methylxanthine (IBMX) Solution, Insulin Solution, and Cy-3-G. DEX Solution, IBMX Solution, and Insulin Solution were purchased from Cayman Chemicals (Ann. Arbor, MI, USA). Cy-3-G derived from black soybean was purchased from Fujicco Co., Ltd. (Kobe, Japan). After 72 h of induction, medium was changed to DMEM containing insulin and Cy-3-G was added every 2 days. After 4 days of incubation from the initiation of differentiation, bioassays were performed.
RNA extraction and real-time PCR
Total RNA was isolated from the 3T3-L1 cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. The integrity of the RNA extracted from all samples was verified and quantified using NANO DROP 2000 (Agilent Technologies, CA). Total RNA was used for the single strand cDNA synthesis with a cDNA synthesis kit, SuperScript® III Reverse Transcriptase. Gene expression levels were analyzed by quantitative real-time PCR, using the Applied Biosystems 7500 FAST INSTURMENT (Applied Biosystems, Foster City, CA, U.S.A.). The oligonucleotide primers of mouse were obtained from Applied Biosystems (Foster City, CA, U.S.A.). The cDNA was denatured at 95 °C for 10 min, followed by 40 cycles of PCR (95 °C, 15 sec; 60 °C, 60 sec).
Results and discussion
Effect of Cy-3-G on the gene expression of GLUT4 in 3T3-L1 adipocytes.
Relative GLUT4 gene expression level (% of Control)
100 ± 1.5
Cy-3-G 20 µM
208.2 ± 1.4*
Cy-3-G 100 µM
226.57 ± 15.2*
Cy-3-G has the potential to improve insulin resistance and its consequences in 3T3-L1 adipocytes through the up-regulation of GLUT4 gene expression. Additional study on insulin resistance using 3T3-L1and anti-fat effect using diabetic model mice will be needed to verify these results in vivo.
This research was partially supported by JST-JICA’s Science and Technology Research Partnership for Sustainable Development (SATREPS).
- Sasaki R, Nishimura N, Hoshino H, Isa Y, Kadowaki M, Ichi T, Tanaka A, Nishiumi S, Fukuda I, Ashida H, Horio F, Tsuda T: Cyanidin 3-glucoside ameliorates hyperglycemia and insulin sensitivity due to downregulation of retinol binding protein 4 expression in diabetic mice. Biochem Pharm. 2007, 74: 1619-1627. 10.1016/j.bcp.2007.08.008.View ArticlePubMedGoogle Scholar
- Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamoto K, Umesono K, Akanuma Y, Fujiwara T, Horikoshi H, Yazaki Y, Kadowaki T: Troglitazone increases the number of small adipocytes without the change of white adipose tissue mass in obese Zucker rats. J Clin Invest. 1998, 101: 1354-1361. 10.1172/JCI1235.PubMed CentralView ArticlePubMedGoogle Scholar
- Kadowaki T: Insights into insulin resistance and type 2 diabetes from knockout mouse models. J Clin Invest. 2000, 106: 459-465. 10.1172/JCI10830.PubMed CentralView ArticlePubMedGoogle Scholar
- Han J, Inaguma T, Isoda H: Cyanidin 3-glucoside anthocyanin from black beans has potential to protect insulin resistance on 3T3-L1 adipocytes by inhibiting TNF-α release. Br J Nutr. (in press)Google Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.