3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions

Keisuke Endo; Tatsuya Sato; Araya Umetsu; Megumi Watanabe; Fumihito Hikage; Yosuke Ida; Hiroshi Ohguro; Masato Furuhashi

doi:10.1016/j.heliyon.2023.e20713

3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions

Heliyon. 2023 Oct 5;9(10):e20713. doi: 10.1016/j.heliyon.2023.e20713. eCollection 2023 Oct.

Authors

Keisuke Endo¹, Tatsuya Sato^{1

2}, Araya Umetsu³, Megumi Watanabe³, Fumihito Hikage³, Yosuke Ida³, Hiroshi Ohguro³, Masato Furuhashi¹

Affiliations

¹ Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
² Department of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, Sapporo, Japan.
³ Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.

Abstract

Adipose tissues are closely related to physiological functions and pathological conditions in most organs. Although differentiated 3T3-L1 preadipocytes have been used for in vitro adipose studies, the difference in cellular characteristics of adipogenic differentiation in two-dimensional (2D) culture and three-dimensional (3D) culture remain unclear. In this study, we evaluated gene expression patterns using RNA sequencing and metabolic functions using an extracellular flux analyzer in 3T3-L1 preadipocytes with and without adipogenic induction in 2D culture and 3D culture. In 2D culture, 565 up-regulated genes and 391 down-regulated genes were identified as differentially expressed genes (DEGs) by adipogenic induction of 3T3-L1 preadipocytes, whereas only 69 up-regulated genes and 59 down-regulated genes were identified as DEGs in 3D culture. Ingenuity Pathway Analysis (IPA) revealed that genes associated with lipid metabolism were identified as 2 out of the top 3 causal networks related to diseases and function in 3D spheroids, whereas only one network related to lipid metabolism was identified within the top 9 of these causal networks in the 2D planar cells, suggesting that adipogenic induction in the 3D culture condition exhibits a more adipocyte-specific gene expression pattern in 3T3-L1 preadipocytes. Real-time metabolic analysis revealed that the metabolic capacity shifted from glycolysis to mitochondrial respiration in differentiated 3T3-L1 cells in the 3D culture condition but not in those in the 2D cultured condition, suggesting that adipogenic differentiation in 3D culture induces a metabolic phenotype of well-differentiated adipocytes. Consistently, expression levels of mitochondria-encoded genes including mt-Nd6, mt-Cytb, and mt-Co1 were significantly increased by adipogenic induction of 3T3-L1 preadipocytes in 3D culture compared with those in 2D culture. Taken together, the findings suggest that induction of adipogenesis in 3D culture provides a more adipocyte-specific gene expression pattern and enhances mitochondrial respiration, resulting in more adipocyte-like cellular properties.

Keywords: 2D culture; 3D culture; 3T3-L1 preadipocytes; Enrichment analysis; Gene ontology (GO); Ingenuity pathway analysis (IPA); RNA sequencing analysis; Seahorse Bioanalyzer.