The Receptor Tyrosine Kinase c-Met Promotes Lipid Accumulation in 3T3-L1 Adipocytes
Abstract
:1. Introduction
2. Results
2.1. c-Met Expression and Phosphorylation Are Vastly Upregulated throughout 3T3-L1 Preadipocyte Differentiation
2.2. Pharmacological Inhibition of c-Met by 20 µM JNJ Suppresses Fat Deposition and Reduces TG Levels throughout 3T3-L1 Preadipocyte Differentiation in a Concentration- and Time-Dependent Manner
2.3. JNJ (20 µM) Greatly Inhibits c-Met Y1234/5 and Y1349 Phosphorylation throughout 3T3-L1 Preadipocyte Differentiation
2.4. JNJ (20 µM) Downregulates the Expression of Adipogenic Transcription Factors (C/EBP-α and PPAR-γ) throughout 3T3-L1 Preadipocyte Differentiation
2.5. JNJ (20 µM) Downregulates FAS, Perilipin A, and Leptin Expressions throughout 3T3-L1 Preadipocyte Differentiation
2.6. JNJ (20 µM) Alters the Phosphorylation and Expression Levels of AMPK, LKB-1, and ACC throughout 3T3-L1 Preadipocyte Differentiation
2.7. Knockdown (KD) of c-Met Reduces the Fat Accumulation, TG Content, and Expression and Phosphorylation Levels of C/EBP-α, PPAR-γ, FAS, ACC, and Perilipin A during 3T3-L1 Preadipocyte Differentiation
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Culture and Differentiation
4.3. Stable Transfection of Short-Hairpin RNA (shRNA) of c-Met
4.4. Oil Red O Staining
4.5. Analysis of Cell Counting
4.6. Intracellular Triglyceride (TG) Content Quantification by AdipoRed Assay
4.7. Whole-Cell Lysates Preparation
4.8. Immunoblotting Analysis
4.9. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
4.10. Intracellular ATP Levels Measurement
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Kopelman, P.G. Obesity as a medical problem. Nature 2000, 404, 635–643. [Google Scholar] [CrossRef] [PubMed]
- Hill, J.O.; Wyatt, H.R.; Peters, J.C. Energy balance and obesity. Circulation 2012, 126, 126–132. [Google Scholar] [CrossRef] [PubMed]
- Ali, A.T.; Hochfeld, W.E.; Myburgh, R.; Pepper, M.S. Adipocyte and adipogenesis. Eur. J. Cell Biol. 2013, 92, 229–236. [Google Scholar] [CrossRef] [PubMed]
- Ambele, M.A.; Dhanraj, P.; Giles, R.; Pepper, M.S. Adipogenesis: A complex interplay of multiple molecular determinants and pathways. Int. J. Mol. Sci. 2020, 21, 4283. [Google Scholar] [CrossRef]
- Cao, Z.; Umek, R.M.; McKnight, S.L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991, 5, 1538–1552. [Google Scholar] [CrossRef]
- Farmer, S.R. Transcriptional control of adipocyte formation. Cell Metab. 2006, 4, 263–273. [Google Scholar] [CrossRef]
- Zhang, K.; Guo, W.; Yang, Y.; Wu, J. JAK2/STAT3 pathway is involved in the early stage of adipogenesis through regulating C/EBPβ transcription. J. Cell. Biochem. 2011, 112, 488–497. [Google Scholar] [CrossRef]
- Lakshmanan, M.R.; Nepokroeff, C.M.; Porter, J.W. Control of the synthesis of fatty-acid synthetase in rat liver by insulin, glucagon, and adenosine 3’:5’ cyclic monophosphate. Proc. Natl. Acad. Sci. USA 1972, 69, 3516–3519. [Google Scholar] [CrossRef]
- Janovská, A.; Hatzinikolas, G.; Staikopoulos, V.; Mclnerney, J.; Mano, M.; Wittert, G.A. AMPK and ACC phosphorylation: Effect of leptin, muscle fibre type and obesity. Mol. Cell. Endocrinol. 2008, 284, 1–10. [Google Scholar] [CrossRef]
- Kern, P.A.; Gregorio, G.D.; Lu, T.; Rassouli, N.; Ranganathan, G. Perilipin expression in human adipose tissue is elevated with obesity. J. Clin. Endocrinol. Metab. 2004, 89, 1352–1358. [Google Scholar] [CrossRef]
- Wolins, N.E.; Brasaemle, D.L.; Bickel, P.E. A proposed model of fat packaging by exchangeable lipid droplet proteins. FEBS Lett. 2006, 580, 5484–5491. [Google Scholar] [CrossRef] [PubMed]
- Martini, C.N.; Plaza, M.V.; Vila, M.C. PKA-dependent and independent cAMP signaling in 3T3-L1 fibroblasts differentiation. Mol. Cell. Endocrinol. 2009, 298, 42–47. [Google Scholar] [CrossRef] [PubMed]
- Prusty, D.; Park, B.H.; Davis, K.E.; Farmer, S.R. Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes. J. Biol. Chem. 2002, 277, 46226–46232. [Google Scholar] [CrossRef] [PubMed]
- Organ, S.L.; Tsao, M.S. An overview of the c-MET signaling pathway. Ther. Adv. Med. Oncol. 2011, 3, S7–S19. [Google Scholar] [CrossRef] [PubMed]
- Peruzzi, B.; Bottaro, D.P. Targeting the c-Met signaling pathway in cancer. Clin. Cancer Res. 2006, 12, 3657–3660. [Google Scholar] [CrossRef] [PubMed]
- Shojaei, F.; Lee, J.H.; Simmons, B.H.; Wong, A.; Esparza, C.O.; Plumlee, P.A.; Feng, J.; Stewart, A.E.; Hu-Lowe, D.D.; Christensen, J.G. HGF/c-Met acts as an alternative angiogenic pathway in sunitinib-resistant tumors. Cancer Res. 2010, 70, 10090–100100. [Google Scholar] [CrossRef]
- Perera, T.; Lavrijssen, T.; Janssens, B.; Geerts, T.; King, P.; Mevellec, L.; Cummings, M.; Lu, T.; Johnson, D.; Page, M. JNJ-38877605: A selective Met kinase inhibitor inducing regression of Met-driven tumor models. Cancer Res. 2008, 68, 4837. [Google Scholar]
- Longati, P.; Bardelli, A.; Ponzetto, C.; Naldini, L.; Comoglio, P.M. Tyrosines1234–1235 are critical for activation of the tyrosine kinase encoded by the MET proto-oncogene (HGF receptor). Oncogene 1994, 9, 49–57. [Google Scholar]
- Ponzetto, C.; Bardelli, A.; Zhen, Z.; Maina, F.; dalla Zonca, P.; Giordano, S.; Graziani, A.; Panayotou, G.; Comoglio, P.M. A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 1994, 77, 261–271. [Google Scholar] [CrossRef]
- Weidner, K.M.; Di Cesare, S.; Sachs, M.; Brinkmann, V.; Behrens, J.; Birchmeier, W. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 1996, 384, 173–176. [Google Scholar] [CrossRef]
- Abdel-Magid, A.F. Treatment of obesity and related disorders with acetyl-CoA carboxylase inhibitors. ACS Med. Chem. Lett. 2013, 4, 16–17. [Google Scholar] [CrossRef]
- Woods, A.; Johnstone, S.R.; Dickerson, K.; Leiper, F.C.; Fryer, L.G.D.; Neumann, D.; Schlattner, U.; Wallimann, T.; Carlson, M.; Carling, D. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr. Biol. 2003, 13, 2004–2008. [Google Scholar] [CrossRef]
- Gowans, G.J.; Hardie, D.G. AMPK: A cellular energy sensor primarily regulated by AMP. Biochem. Soc. Trans. 2014, 42, 71–75. [Google Scholar] [CrossRef] [PubMed]
- Zhang, D.; Fei, Q.; Li, J.; Zhang, C.; Sun, Y.; Zhu, C.; Wang, F.; Sun, Y. 2-Deoxyglucose reverses the promoting effect of insulin on colorectal cancer cells in vitro. PLoS ONE 2016, 11, e0151115. [Google Scholar] [CrossRef]
- Oliveira, A.G. The role of hepatocyte growth factor (hgf) in insulin resistance and diabetes. Front. Endocrinol. 2018, 9, 503. [Google Scholar] [CrossRef]
- Sakkab, D.; Lewitzky, M.; Posern, G.; Schaeper, U.; Sachs, M.; Birchmeier, W.; Feller, S.M. Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large docking protein Gab1 and the adapter protein CRKL. J. Biol. Chem. 2000, 275, 10772–10778. [Google Scholar] [CrossRef] [PubMed]
- Gao, C.F.; Vande Woude, G.F. HGF/SF-Met signaling in tumor progression. Cell Res. 2005, 15, 49–51. [Google Scholar] [CrossRef] [PubMed]
- Comoglio, P.M. Pathway specificity for Met signalling. Nat. Cell Biol. 2001, 3, 161–162. [Google Scholar] [CrossRef] [PubMed]
- Graziani, A.; Gramaglia, D.; Cantley, L.C.; Comoglio, P.M. The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase. J. Biol. Chem. 1991, 266, 22087–22090. [Google Scholar] [CrossRef]
- Graziani, A.; Gramaglia, D.; dalla Zonca, P.; Comoglio, P.M. Hepatocyte growth factor/scatter factor stimulates the Ras-guanine nucleotide exchanger. J. Biol. Chem. 1993, 268, 9165–9168. [Google Scholar] [CrossRef]
- Nakamura, T.; Mizuno, S. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc. Jpn. Acad. Ser B. Phys. Biol. Sci. 2010, 86, 588–610. [Google Scholar] [CrossRef] [PubMed]
- Ahn, S.H.; Lee, H.J.; Pyun, D.H.; Kim, T.J.; Abd El-Aty, A.M.; Song, J.H.; Shin, Y.K.; Jeong, J.H.; Park, E.S.; Jung, T.W. Capmatinib attenuates lipogenesis in 3T3-L1 adipocytes through an adenosine monophosphate-activated protein kinase-dependent pathway. Biochem. Biophys. Res. Comm. 2021, 533, 30–36. [Google Scholar] [CrossRef] [PubMed]
- Tang, R.; Ma, F.; Li, W.; Ouyang, S.; Liu, Z.; Wu, J. MiR-206-3p inhibits 3T3-L1 cell adipogenesis via the c-Met/PI3K/Akt pathway. Int. J. Mol. Sci. 2017, 18, 1510. [Google Scholar] [CrossRef] [PubMed]
- Owusu, B.Y.; Thomas, S.; Venukadasula, P.; Han, Z.; Janetka, J.W.; Galemmo, R.A., Jr.; Klampfer, L. Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation. Oncotarget 2017, 8, 63014–63025. [Google Scholar] [CrossRef] [PubMed]
- Uehara, Y.; Minowa, O.; Mori, C.; Shiota, K.; Kuno, J.; Noda, T.; Kitamura, N. Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature 1995, 373, 702–705. [Google Scholar] [CrossRef] [PubMed]
- Kato, T. Biological roles of hepatocyte growth factor-Met signaling from genetically modified animals. Biomed. Rep. 2017, 7, 495–503. [Google Scholar] [CrossRef]
- Lehrke, M.; Lazar, M.A. The many faces of PPARgamma. Cell 2005, 123, 993–999. [Google Scholar] [CrossRef] [PubMed]
- Kershaw, E.E.; Flier, J.S. Adipose tissue as an endocrine organ. J. Clin. Endocrinol. Metab. 2004, 89, 2548–2556. [Google Scholar] [CrossRef]
- Ahima, R.S. Adipose tissue as an endocrine organ. Obesity (Silver Spring) 2006, 14, 242S–249S. [Google Scholar] [CrossRef]
- Hwang, C.S.; Loftus, T.M.; Mandrup, S.; Lane, M.D. Adipocyte differentiation and leptin expression. Annu. Rev. Cell. Dev. Biol. 1997, 13, 231–259. [Google Scholar] [CrossRef]
- Bastard, J.P.; Maachi, M.; Lagathu, C.; Kim, M.J.; Caron, M.; Vidal, H.; Capeau, J.; Feve, B. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur. Cytokine Netw. 2006, 17, 4–12. [Google Scholar] [PubMed]
- Ceddia, R.B.; Somwar, R.; Maida, A.; Fang, X.; Bikopoulos, G.; Sweeney, G. Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells. Diabetologia 2005, 48, 132–139. [Google Scholar] [CrossRef] [PubMed]
- Shehzad, A.; Iqbal, W.; Shehzad, O.; Lee, Y.S. Adiponectin: Regulation of its production and its role in human diseases. Hormones 2012, 11, 8–20. [Google Scholar] [CrossRef] [PubMed]
- Yadav, A.; Kataria, M.A.; Saini, V.; Yadav, A. Role of leptin and adiponectin in insulin resistance. Clin. Chim. Acta. 2013, 417, 80–84. [Google Scholar] [CrossRef]
- Friedman, J. Fat in all the wrong places. Nature 2002, 415, 268–269. [Google Scholar] [CrossRef]
- Garcia, D.; Shaw, R.J. AMPK: Mechanisms of cellular energy sensing and restoration of metabolic balance. Mol. Cell. 2017, 66, 789–800. [Google Scholar] [CrossRef]
- Hardie, D.G. AMPK: A key regulator of energy balance in the single cell and the whole organism. Int. J. Obes. 2005, 32, S7–S12. [Google Scholar] [CrossRef]
- Lee, H.; Kang, R.; Bae, S.; Yoon, Y. AICAR, an activator of AMPK, inhibits adipogenesis via the WNT/β-catenin pathway in 3T3-L1 adipocytes. Int. J. Mol. Med. 2011, 28, 65–71. [Google Scholar] [CrossRef]
- Poudel, B.; Lim, S.W.; Ki, H.H.; Nepali, S.; Lee, Y.M.; Kim, D.K. Dioscin inhibits adipogenesis through the AMPK/MAPK pathway in 3T3-L1 cells and modulates fat accumulation in obese mice. Int. J. Mol. Med. 2014, 34, 1401–1408. [Google Scholar] [CrossRef]
- Herzig, S.; Shaw, R.J. AMPK: Guardian of metabolism and mitochondrial homeostasis. Nat. Rev. Mol. Cell Biol. 2018, 19, 121–135. [Google Scholar] [CrossRef]
- Svensson, R.U.; Parker, S.J.; Eichner, L.J.; Kolar, M.J.; Wallace, M.; Brun, S.N.; Lombardo, P.S.; Nostrand, J.L.V.; Hutchins, A.; Vera, L.; et al. Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models. Nat. Med. 2016, 22, 1108–1119. [Google Scholar] [CrossRef] [PubMed]
- Lally, J.S.V.; Ghoshal, S.; DePeralta, D.K.; Moaven, O.; Wei, L.; Masia, R.; Erstad, D.J.; Fujiwara, N.; Leong, V.; Houde, V.P.; et al. Inhibition of Acetyl-CoA carboxylase by phosphorylation or the inhibitor ND-654 suppresses lipogenesis and hepatocellular carcinoma. Cell. Metab. 2019, 29, 174–182. [Google Scholar] [CrossRef] [PubMed]
- Saha, A.K.; Ruderman, N.B. Malonyl-CoA and AMP-activated protein kinase: An expanding partnership. Mol. Cell Biochem. 2003, 253, 65–70. [Google Scholar] [CrossRef] [PubMed]
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Park, Y.-K.; Jang, B.-C. The Receptor Tyrosine Kinase c-Met Promotes Lipid Accumulation in 3T3-L1 Adipocytes. Int. J. Mol. Sci. 2023, 24, 8086. https://doi.org/10.3390/ijms24098086
Park Y-K, Jang B-C. The Receptor Tyrosine Kinase c-Met Promotes Lipid Accumulation in 3T3-L1 Adipocytes. International Journal of Molecular Sciences. 2023; 24(9):8086. https://doi.org/10.3390/ijms24098086
Chicago/Turabian StylePark, Yu-Kyoung, and Byeong-Churl Jang. 2023. "The Receptor Tyrosine Kinase c-Met Promotes Lipid Accumulation in 3T3-L1 Adipocytes" International Journal of Molecular Sciences 24, no. 9: 8086. https://doi.org/10.3390/ijms24098086