Abstract
The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDHα, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.
Highlights
Obesity is a major public health problem worldwide
In our previous study[30], we demonstrated that the co-culture of adipocytes and macrophages in a 3D cell culture system results in changes in lipid and glucose metabolism, which is similar to that of the effect of GW9662 on insulin resistance in adipose tissue in diabetic mice
Our analysis identified differentially expressed proteins that are involved in glucose metabolism, fatty acid oxidation, adipogenesis, and the electron transport chain (ETC) pathway
Summary
Obesity is a major public health problem worldwide. It is one of numerous contributors to type II diabetes (T2D), hypertension, cancer, sleep-disordered breathing, insulin resistance and cardiovascular disease, which are characteristic of metabolic syndromes[1,2,3,4]. Adipose cells in a 3D culture system enable co-culturing with a diverse array of cells, including macrophages, endothelial cells, and cancer cells[17,18,28,29] Both physical and functional aspects of adipocytes that are co-cultured with the other cell types during differentiation and enlargement differed significantly in terms of cell morphology and cytokine expression compared to that of mono-cultured adipocytes[28]. Using liquid chromatography coupled with advanced tandem mass spectrometry (LC-MS/MS)[31,32,33], numerous studies aiming to identify key effectors in the metabolic regulation of adipocytes have reported the results of quantitative proteomic analyses utilising diverse labelling techniques (e.g., isobaric tags for relative and absolute quantification [iTRAQ]34–39, tandem mass tags [TMT]40,41 and stable isotope labelling by amino acids in cell culture [SILAC]42,43). We performed a global quantitative proteomic analysis of six 3T3-L1 cell types (preadipocytes, adipocytes, and co-cultured adipocytes with macrophages in 2D- and 3D-cell culture conditions) using iTRAQ-based 2D-nanoLC-ESI-MS/MS
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