Spheroid Culture System Confers Differentiated Transcriptome Profile and Functional Advantage to 3T3-L1 Adipocytes
This study highlights functional differences between 2-D monolayer and 3-D spheroid 3T3-L1 adipocyte culture models and explores the underlying genomic mechanisms responsible for the different phenotypes present. The spheroids showed higher triglyceride accumulation than the monolayer culture and further increase with larger spheroid size. Whole transcriptome analysis indicated significant differential expression of genes related to adipogenesis, including adipocytokine signaling, fatty acid metabolism, and PPAR-γ signaling. Spheroids also showed downregulation of matrix metalloproteinases (MMPs), integrin, actin-cytoskeleton associated genes, and Rho/GTPase3 expression relative to 2-D monolayer, indicating suppression of the Rho-ROCK pathway and thereby promoting adipogenic differentiation. When exposed to linoleic acid (500 μM) and TNF-α (125 ng/mL) to promote chronic adiposity, linoleic acid treatment resulted in increased intracellular triglycerides and subsequent TNF-α treatment resulted in significantly altered adipocytokine signaling, fatty acid metabolism, and PPAR signaling, in addition to upregulation of multiple MMPs in spheroids vs. monolayer. Overall, 3-D spheroids showed enhanced adipogenic phenotype as indicated by triglyceride synthesis and transcriptome changes while retaining sensitivity to a pro-inflammatory stimulus. The 3-D spheroid culture thus may provide a simple, convenient, and sensitive in vitro model to study adipocyte response to metabolic stresses relevant to clinical pathologies.
KeywordsGene expression Cell culture Matrix metalloproteinase (MMP) Triglyceride Adipocytes
This work was funded by the School of Dentistry and the University of Mississippi Medical Center intramural research support programs, National Science Foundation (NSF; CBET-1033525), and National Institutes of Health (NIH; R01EB020006). The work performed through the UMMC Molecular and Genomics Facility is supported, in part, by funds from NIH, including Mississippi INBRE (P20GM103476), Center for Psychiatric Neuroscience COBRE (P30GM103328) and Obesity, Cardiorenal and Metabolic Diseases COBRE (P20GM104357). Animal fat isolation work is funded by NIH (R01HL089884, R01HL107632 to SPD). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSF or NIH. This work made use of instruments in the Department of Biomedical Materials Science Shared Equipment Facility.
Conflict of interest
The authors have no conflict of interests to disclose.
- 1.Alwayn, I. P., K. Gura, V. Nosé, B. Zausche, P. Javid, J. Garza, J. Verbesey, S. Voss, M. Ollero, C. Andersson, B. Bistrian, J. Folkman, and M. Puder. Omega-3 fatty acid supplementation prevents hepatic steatosis in a murine model of nonalcoholic fatty liver disease. Pediatr. Res. 57:445–452, 2005.CrossRefPubMedGoogle Scholar
- 30.Westbrook, L. J., A. C. Johnson, K. R. Regner, J. Lee, D. L. Mattson, P. B. Kyle, J. R. Henegar, and M. R. Garrett. Genetic susceptibility and loss of Nr4a1 enhances macrophage mediated renal injury in a rodent model of chronic kidney disease. J. Am. Soc. Nephr. 25:2499–2510, 2014.CrossRefGoogle Scholar
- 31.Xu, H. E., M. H. Lambert, V. G. Montana, D. J. Parks, S. G. Blanchard, P. J. Brown, D. D. Sternbach, J. M. Lehmann, G. B. Wisely, T. M. Willson, S. A. Kliewer, and M. V. Milburn. Molecular recognition of fatty acids by peroxisome proliferator–activated receptors. Mol. Cell. 3:397–403, 1999.CrossRefPubMedGoogle Scholar