Proteomic Analysis of 3T3-L1 Adipocyte Mitochondria during Differentiation and Enlargement

Abstract

The increase in adipose tissue mass arises in part from progressive lipid loading and triglyceride accumulation in adipocytes. Enlarged adipocytes produce the highest levels of pro-inflammatory molecules and reactive oxygen species (ROS). Since mitochondria are the site for major metabolic processes (e.g., TCA cycle) that govern the extent of triglyceride accumulation as well as the primary site of ROS generation, we quantitatively investigated changes in the adipocyte mitochondrial proteome during different stages of differentiation and enlargement. Mitochondrial proteins from 3T3-L1 adipocytes at different stages of lipid accumulation (days 0-18) were digested and labeled using the iTRAQ 8-plex kit. The labeled peptides were fractionated using a liquid phase isoelectric fractionation system (MSWIFT) to increase the depth of proteome coverage and analyzed using LC-MS/MS. A total of 631 proteins in the mitochondrial fraction, including endoplasmic reticulum-associated and golgi-related mitochondrial proteins, were identified and classified into 12 functional categories. A total of 123 proteins demonstrated a statistically significant change in expression in at least one of the time points over the course of the experiment. The identified proteins included enzymes and transporters involved in the TCA cycle, fatty acid oxidation, and ATP synthesis. Our results indicate that cultured adipocytes enter a state of metabolic-overdrive where increased flux through the TCA cycle and increased fatty acid oxidation occur simultaneously. The proteomic data also suggest that accumulation of reduced electron carriers and the resultant oxidative stress may be attractive targets for modulating adipocyte function in metabolic disorders.

Autophagy inhibition in early but not in later stages prevents 3T3-L1 differentiation: Effect on mitochondrial remodeling

“…It has been previously demonstrated that adipocyte differentiation is accompanied by profound changes in the mitochondrion, at the level of gene expression, proteome composition and even mitochondrial network organization (Newton et al, 2011;WilsonFritch et al, 2003;Ducluzeau et al, 2011). In our experiments, we also found fundamental changes in mitochondrial network organization between predipocytes and adipocytes subjected to an 8-day differentiation protocol.…”

“…3T3-L1 fibroblasts, have been the most frequently used tool for the characterization of the differentiation program (Tang and Lane, 2012;Gregoire et al, 1998;Poulos et al, 2010). Both proteomic and transcriptomic analyses revealed that differentiating adipocytes undergo profound changes resulting in cytoplasm remodeling (Farmer, 2006;Newton et al, 2011). Adipocyte mitochondria are both the major site of fatty acid oxidation as well as the site for synthesis of key intermediates in de novo lipogenesis.…”

Redox modulation of adipocyte differentiation: hypothesis of “Redox Chain” and novel insights into intervention of adipogenesis and obesity

“…Data regarding to the changes of mitochondrial DNA (mtDNA) copy number during adipocyte differentiation are inconsistent [137,145,148,149]. Skop et al [146] reported that in the first two days, mtDNA copy number declined by 50%, which was followed by a rapid increase and culminated on day 5.After that, mtDNA copy number was moderately reduced to the level observed in preadipocytes [146].…”

Characterisation of the relationship between Heme-oxygenase-1 and adiponectin

“…As a consequence of chronic positive energy balance, adipose tissue expands. This occurs either by hypertrophy or by hyperplasia [14]. Hypertrophy involves increased accretion of lipids within existing adipocytes.…”

“…For decades it was believed that the number of adipocytes is set during childhood and maintained through adulthood [39], which indicated that adipose tissue expansion is due to increases in the size of adipocytes rather than increases in the number of adipocytes. However, recent studies show that adipocytes do not have an unlimited capacity for hypertrophy [14,40] [48]. Hypertrophic adipocyte-derived free fatty acids (FFAs) can also lead to an increase in TNFα production by binding to macrophage toll-like receptor-4 (TLR-4) and activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [49,50].…”