Comparative Proteomic Profiling of 3T3-L1 Adipocyte Differentiation Using SILAC Quantification

Abstract

Adipocyte differentiation is a general physiological process that is also critical for metabolic syndrome. In spite of extensive study in the past two decades, adipogenesis is a still complex cellular process that is accompanied by complicated molecular mechanisms. Here, we performed SILAC-based quantitative global proteomic profiling of 3T3-L1 adipocyte differentiation. We report protein changes to the proteome profiles, with 354 proteins exhibiting significant increase and 56 proteins showing decrease in our statistical analysis. Our results show that adipocyte differentiation is involved not only in metabolic processes by increasing TCA cycle, fatty acid synthesis, lipolysis, acetyl-CoA production, antioxidants, and electron transport, but also in nicotinamide metabolism, cristae formation, mitochondrial protein import, and Ca2+ transport into mitochondria and ER. A search for Chromosome-Centric Human Proteome Project (C-HPP) using neXtprot highlighted one protein with a protein existence uncertain (PE5) and 17 proteins as functionally uncharacterized protein existence 1 (uPE1). This study provides quantitative information on proteome changes in adipogenic differentiation, which is helpful in improving our understanding of the processes of adipogenesis.

Proteomic profiling of metformin effects in 3T3‐L1 adipocytes by SILAC‐based quantification

“…The cells were cultured for 10 days. In our previous work, we described the protein expression profiles of adipogenesis‐related markers in 3T3‐L1 adipocyte differentiation using SILAC quantification [19]. Intracellular lipids in adipocytes were visualized by Oil Red O staining as shown in Supplementary Figure S2.…”

“…Mouse 3T3‐L1preadipocytes were grown in SILAC DMEM (#88425, Thermo Scientific) containing light lysine (Sigma–Aldrich) and arginine (Sigma–Aldrich) or heavy lysine (Lys8, 13 C 6 15 N 2 – 13 C 6 , 99%; 15 N 2 , 99%, #CNLM‐291‐H‐PK, Cambridge Isotope laboratories Inc.) and arginine (Arg10, 13 C 6 15 N 4 – 13 C 6 , 99%; 15 N 4 , 99%, #CNLM‐539‐H‐PK, Cambridge Isotope laboratories Inc.), supplemented with 10% dialyzed fetal bovine serum (FBS) in 5% CO 2 humidified atmosphere at 37°C. 3T3‐L1 preadipocytes were cultured for six passages for complete labeling, and then induced to differentiate into adipocytes by addition of 0.5 mM 3‐isobutyl‐1‐methyxanthine (IBMX), 0.25 M dexamethasone, and 1 M insulin as described in our previous study [19]. Adipocytes were cultured for 48 h in the absence (control) or presence of 5 mM metformin (Sigma–Aldrich) [20].…”

“…The following search parameters were employed: carbamidometylation of cysteine as a fixed modification, N ‐terminal acetylation and oxidation of methionine as variable modifications, and heavy arginine (Arg10) and lysine (Lys8) for SILAC quantification. The tolerance parameters were applied as follows: 20 ppm for first search mass accuracy tolerance, 4.5 ppm for main search mass accuracy tolerance, 20 ppm for FTMS MS/MS tolerance, minimum peptide length of seven amino acids, peptide spectrum match false discovery rate (FDR), and protein FDR both set to 0.01 as calculated by the revert database approach [19, 23].…”

“…Mouse 3T3-L1preadipocytes were grown in SILAC DMEM (#88425, Thermo Scientific) containing light lysine (Sigma-Aldrich) and arginine (Sigma-Aldrich) or heavy lysine (Lys8, 13 3T3-L1 preadipocytes were cultured for six passages for complete labeling, and then induced to differentiate into adipocytes by addition of 0.5 mM 3-isobutyl-1-methyxanthine (IBMX), 0.25 M dexamethasone, and 1 M insulin as described in our previous study [19].…”

Quantitative proteomics characterization of cancer biomarkers and treatment

“…Stable isotope labeling by amino acids in cell culture (SILAC) technology, isobaric tags for relative and absolute quantification (iTRAQ) technology, and tandem mass tags (TMTs) technology are the main methods used for the labeling quantitative proteomics. 23 , 24 , 25 , 26 SILAC technology is suitable for analyzing living cells in culture with accurate quantification and good repeatability. 27 SILAC removes the false positives in protein-interaction studies, reveals the large-scale kinetics of proteomes, and directly uncovers the important points in the cellular signaling pathways as a quantitative phosphoproteomics technology.…”