LY3009120, a pan-Raf kinase inhibitor, inhibits adipogenesis of 3T3-L1 cells by controlling the expression and phosphorylation of C/EBP-α, PPAR-γ, STAT‑3, FAS, ACC, perilipin A, and AMPK

Yang,Su‑Min; Park,Yu‑Kyoung; Kim,Jee In; Lee,Yun‑Han; Lee,Tae‑Yun; Jang,Byeong‑Churl

doi:10.3892/ijmm.2018.3890

LY3009120, a pan-Raf kinase inhibitor, inhibits adipogenesis of 3T3-L1 cells by controlling the expression and phosphorylation of C/EBP-α, PPAR-γ, STAT‑3, FAS, ACC, perilipin A, and AMPK

Authors:
- Su‑Min Yang
- Yu‑Kyoung Park
- Jee In Kim
- Yun‑Han Lee
- Tae‑Yun Lee
- Byeong‑Churl Jang
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Affiliations: Department of Molecular Medicine, College of Medicine, Keimyung University, Daegu 42601, Republic of Korea, Department of Microbiology, College of Medicine, Yeungnam University, Daegu 42415, Republic of Korea
Published online on: September 21, 2018 https://doi.org/10.3892/ijmm.2018.3890
Pages: 3477-3484

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Abstract

Excessive preadipocyte differentiation/adipogenesis is closely linked to the development of obesity. LY3009120 is a pan‑Raf kinase inhibitor and is known for its anticancer activities. In the present study, the effect of LY3009120 on 3T3‑L1 cell adipogenesis was investigated. The differentiation of 3T3‑L1 preadipocytes into adipocytes was measured by Oil Red O staining and AdipoRed assay. Changes of cellular protein expression and phosphorylation levels in differentiating 3T3‑L1 preadipocytes in the absence or presence of LY3009120 were determined by western blotting analysis. Cell count assay was used to assess the cytotoxicity of LY3009120 on 3T3‑L1 cells. At 0.3 µM, LY3009120 markedly inhibited lipid accumulation and decreased triglyceride content in differentiating 3T3‑L1 cells. However, it had minimal effect on the elevated expression and phosphorylation of three Raf kinase isoforms (C‑Raf, A‑Raf, and B‑Raf) observed in the cells. LY3009120 reduced not only the expression of CCAAT/enhancer‑binding protein‑α (C/EBP‑α), peroxisome proliferator‑activated receptor‑γ (PPAR‑γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A, but also reduced the phosphorylation of signal transducer and activator of transcription‑3 (STAT‑3) in differentiating 3T3‑L1 cells. LY3009120 also increased the phosphorylation of adenosine 3',5'‑cyclic monophosphate (cAMP)‑activated protein kinase (AMPK), but did not affect the phosphorylation or expression of liver kinase B1 in these cells. In summary, this is the first report, to the best of our knowledge, demonstrating that LY3009120 has an anti‑adipogenic effect on 3T3‑L1 cells, which may be mediated through control of the expression and phosphorylation of C/EBP‑α, PPAR‑γ, STAT‑3, FAS, ACC, perilipin A, and AMPK.

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1	Schwartz MW, Seeley RJ, Zeltser LM, Drewnowski A, Ravussin E, Redman LM and Leibel RL: Obesity pathogenesis: An endocrine society scientific statement. Endocr Rev. 38:267–296. 2017. View Article : Google Scholar : PubMed/NCBI
2	Karageorgi S, Alsmadi O and Behbehani K: A review of adult obesity prevalence, trends, risk factors, and epidemiologic methods in Kuwait. J Obes. 2013:3786502013. View Article : Google Scholar
3	Rubinstein M and Low MJ: Molecular and functional genetics of the proopiomelanocortin gene, food intake regulation and obesity. FEBS Lett. 591:2593–2606. 2017. View Article : Google Scholar : PubMed/NCBI
4	Cristancho AG and Lazar MA: Forming functional fat: A growing understanding of adipocyte differentiation. Nat Rev Mol Cell Biol. 28:722–734. 2011. View Article : Google Scholar
5	Farmer SR: Transcriptional control of adipocyte formation. Cell Metab. 4:263–273. 2006. View Article : Google Scholar : PubMed/NCBI
6	Mota de Sá P, Richard AJ, Hang H and Stephens JM: Transcriptional regulation of adipogenesis. Compr Physiol. 7:635–674. 2017. View Article : Google Scholar : PubMed/NCBI
7	Guo L, Li X and Tang QQ: Transcriptional regulation of adipo-cyte differentiation: A central role for CCAAT/enhancer-binding protein (C/EBP) β. J Biol Chem. 290:755–761. 2015. View Article : Google Scholar
8	Ali AT, Hochfeld WE, Myburgh R and Pepper MS: Adipocyte and adipogenesis. Eur J Cell Biol. 92:229–236. 2013. View Article : Google Scholar : PubMed/NCBI
9	Richard AJ and Stephens JM: The role of JAK-STAT signaling in adipose tissue function. Biochim Biophys Acta. 1842:431–439. 2014. View Article : Google Scholar :
10	Zhang K, Guo W, Yang Y and Wu J: JAK2/STAT3 pathway is involved in the early stage of adipogenesis through regulating C/EBPβ transcription. J Cell Biochem. 112:488–497. 2011. View Article : Google Scholar : PubMed/NCBI
11	Lenhard JM: Lipogenic enzymes as therapeutic targets for obesity and diabetes. Curr Pharm Des. 17:325–331. 2011. View Article : Google Scholar : PubMed/NCBI
12	Peng IC, Chen Z, Sun W, Li YS, Marin TL, Hsu PH, Su MI, Cui X, Pan S, Lytle CY, et al: Glucagon regulates ACC activity in adipocytes through the CAMKKβ/AMPK pathway. Am J Physiol Endocrinol Metab. 302:E1560–E1568. 2012. View Article : Google Scholar : PubMed/NCBI
13	Beller M, Bulankina AV, Hsiao HH, Urlaub H, Jäckle H and Kühnlein RP: PERILIPIN-dependent control of lipid droplet structure and fat storage in Drosophila. Cell Metab. 12:521–532. 2010. View Article : Google Scholar : PubMed/NCBI
14	Kern PA, Di Gregorio G, Lu T, Rassouli N and Ranganathan G: Perilipin expression in human adipose tissue is elevated with obesity. J Clin Endocrinol Metab. 89:1352–1358. 2004. View Article : Google Scholar : PubMed/NCBI
15	Martini CN, Plaza MV and Maria del Vila C: PKA-dependent and independent cAMP signaling in 3T3-L1 fibroblasts differentiation. Mol Cell Endocrinol. 298:42–47. 2009. View Article : Google Scholar
16	Yavari A, Stocker CJ, Ghaffari S, Wargent ET, Steeples V, Czibik G, Pinter K, Bellahcene M, Woods A, Martínez de Morentin PB, et al: Chronic activation of γ2 AMPK induces obesity and reduces β cell function. Cell Metab. 23:821–836. 2016. View Article : Google Scholar : PubMed/NCBI
17	Prusty D, Park BH, Davis KE and Farmer SR: Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxi-some proliferator-activated receptor gamma (PPARgamma) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes. J Biol Chem. 277:46226–46232. 2002. View Article : Google Scholar : PubMed/NCBI
18	Zhou Y, Wang D, Li F, Shi J and Song J: Different roles of protein kinase C-betaI and -delta in the regulation of adipocyte differentiation. Int J Biochem Cell Biol. 38:2151–2163. 2006. View Article : Google Scholar : PubMed/NCBI
19	Porras A, Muszynski K, Rapp UR and Santos E: Dissociation between activation of Raf-1 kinase and the 42-kDa mitogen-activated protein kinase/90-kDa S6 kinase (MAPK/RSK) cascade in the insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells. J Biol Chem. 269:12741–12748. 1994.PubMed/NCBI
20	Kwak DH, Lee JH, Kim DG, Kim T, Lee KJ and Ma JY: Inhibitory effects of hwangryunhaedok-tang in 3T3-L1 adipo-genesis by regulation of Raf/MEK1/ERK1/2 pathway and PDK1/Akt phosphorylation. Evid Based Complement Alternat Med. 2013:4139062013. View Article : Google Scholar
21	Choi JS, Kim JH, Ali MY, Jung HJ, Min BS, Choi RJ, Kim GD and Jung HA: Anti-adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways. Arch Pharm Res. 38:2153–2162. 2015. View Article : Google Scholar : PubMed/NCBI
22	Henry JR, Kaufman MD, Peng SB, Ahn YM, Caldwell TM, Vogeti L, Telikepalli H, Lu WP, Hood MM, Rutkoski TJ, et al: Discovery of 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-m ethyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a pan-RAF inhibitor with minimal paradoxical activation and activity against BRAF or RAS mutant tumor cells. J Med Chem. 58:4165–4179. 2015. View Article : Google Scholar : PubMed/NCBI
23	Peng SB, Henry JR, Kaufman MD, Lu WP, Smith BD, Vogeti S, Rutkoski TJ, Wise S, Chun L, Zhang Y, et al: Inhibition of RAF isoforms and active dimers by LY3009120 leads to anti-tumor activities in RAS or BRAF mutant cancers. Cancer Cell. 28:384–398. 2015. View Article : Google Scholar : PubMed/NCBI
24	Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M and Carling D: LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Cur Biol. 13:2004–2008. 2003. View Article : Google Scholar
25	Wang D, Zhou Y, Lei W, Zhang K, Shi J, Hu Y, Shu G and Song J: Signal transducer and activator of transcription 3 (STAT3) regulates adipocyte differentiation via peroxisome-proliferator-activated receptor gamma (PPARgamma). Biol Cell. 102:1–12. 2009. View Article : Google Scholar : PubMed/NCBI
26	Shang CA and Waters MJ: Constitutively active signal transducer and activator of transcription 5 can replace the requirement for growth hormone in adipogenesis of 3T3-F442A preadipocytes. Mol Endocrinol. 17:2494–2508. 2003. View Article : Google Scholar : PubMed/NCBI
27	Wolins NE, Brasaemle DL and Bickel PE: A proposed model of fat packaging by exchangeable lipid droplet proteins. FEBS Lett. 580:5484–5491. 2006. View Article : Google Scholar : PubMed/NCBI
28	Steinberg GR, Macaulay SL, Febbraio MA and Kemp BE: AMP-activated protein kinase-the fat controller of the energy railroad. Can J Physiol Pharmacol. 84:655–665. 2006. View Article : Google Scholar : PubMed/NCBI
29	Lage R, Diéguez C, Vidal-Puig A and López M: AMPK: A metabolic gauge regulating whole-body energy homeostasis. Trends Mol Med. 14:539–549. 2008. View Article : Google Scholar : PubMed/NCBI
30	Han YH, Kee JY, Park J, Kim HL, Jeong MY, Kim DS, Jeon YD, Jung Y, Youn DH, Kang J, et al: Arctigenin inhibits adipogenesis by inducing AMPK activation and reduces weight gain in high-fat diet-induced obese mice. J Cell Biochem. 117:2067–2077. 2016. View Article : Google Scholar : PubMed/NCBI
31	Vingtdeux V, Chandakkar P, Zhao H, Davies P and Marambaud P: Small-molecule activators of AMP-activated protein kinase (AMPK), RSVA314 and RSVA405, inhibit adipogenesis. Mol Med. 17:1022–1030. 2011. View Article : Google Scholar : PubMed/NCBI
32	Baek JH, Kim NJ, Song JK and Chun KH: Kahweol inhibits lipid accumulation and induces glucose-uptake through activation of AMP-activated protein kinase (AMPK). BMB Rep. 50:566–571. 2017. View Article : Google Scholar : PubMed/NCBI
33	Zordoky BN, Nagendran J, Pulinilkunnil T, Kienesberger PC, Masson G, Waller TJ, Kemp BE, Steinberg GR and Dyck JR: AMPK-dependent inhibitory phosphorylation of ACC is not essential for maintaining myocardial fatty acid oxidation. Circ Res. 115:518–524. 2014. View Article : Google Scholar : PubMed/NCBI
34	Saha AK and Ruderman NB: Malonyl-CoA and AMP-activated protein kinase: An expanding partnership. Mol Cell Biochem. 253:65–70. 2003. View Article : Google Scholar : PubMed/NCBI
35	Richter EA and Ruderman NB: AMPK and the biochemistry of exercise: Implications for human health and disease. Biochem J. 418:261–275. 2009. View Article : Google Scholar : PubMed/NCBI
36	Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA and Cantley LC: The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA. 101:3329–3335. 2004. View Article : Google Scholar : PubMed/NCBI
37	Xu Z, Liu J and Shan T: New roles of Lkb1 in regulating adipose tissue development and thermogenesis. J Cell Physiol. 232:2296–2298. 2017. View Article : Google Scholar
38	Gormand A, Henriksson E, Ström K, Jensen TE, Sakamoto K and Göransson O: Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes. J Cell Biochem. 112:1364–1375. 2011. View Article : Google Scholar : PubMed/NCBI
39	Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelmn AM, Frenguelli BG and Hardie DG: Calmodulin dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab. 2:9–19. 2005. View Article : Google Scholar : PubMed/NCBI
40	Gowans GJ and Hardie DG: AMPK: A cellular energy sensor primarily regulated by AMP. Biochem Soc Trans. 42:71–75. 2014. View Article : Google Scholar : PubMed/NCBI