Attenuated metabolism is a hallmark of obesity as revealed by comparative proteomic analysis of human omental adipose tissue.

Obesity is recognized as an epidemic health problem worldwide. In humans, the accumulation of omental rather than subcutaneous fat appears to be tightly linked to insulin resistance, type 2 diabetes and cardiovascular disease. Differences in gene expression profiles in the adipose tissue comparing non-obese and obese subjects have been well documented. However, to date, no comparative proteomic studies based on omental fat have investigated the influence of obesity in protein expression. In this work, we searched for proteins differentially expressed in the omental fat of non-obese and obese subjects using 2D-DIGE and MS. Forty-four proteins, several of which were further studied by immunoblotting and immunostaining analyses, showed significant differences in the expression levels in the two groups of subjects. Our findings reveal a clearly distinctive proteomic profile between obese and non-obese subjects which emphasizes: i) reduced metabolic activity in the obese fat, since most down-regulated proteins were engaged in metabolic pathways; and ii) morphological and structural cell changes in the obese fat, as revealed by the functions exerted by most up-regulated proteins. Interestingly, transketolase and aminoacylase-1 represent newly described molecules involved in the pathophysiology of obesity, thus opening up new possibilities in the study of obesity.

Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity.

BACKGROUND: Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals. METHODOLOGY AND FINDINGS: To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples. CONCLUSIONS: ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study.

Differential proteomics of omental and subcutaneous adipose tissue reflects their unalike biochemical and metabolic properties.

Obesity is increasing exponentially in developed countries and constitutes a public health problem by enhancing the risk for metabolic disorder and cardiovascular disease. Differences in gene expression profiles and in metabolic and biochemical properties have been well-described between omental and subcutaneous adipose tissue in humans. Because omental adipose tissue has been strongly associated with the development of insulin resistance, type 2 diabetes and cardiovascular disease, we searched for proteins differentially expressed in these two fat depots using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). In this analysis, we found 43 proteins, several of which were validated by immunoblotting and immunostaining analyses. Results demonstrated tissue-specific molecular differences in the protein makeup of the two analyzed fat depots mainly related to metabolic processes such as glucose and lipid metabolism, lipid transport, protein synthesis, protein folding, response to stress and inflammation. This suggests higher metabolic activity as well as increased cell stress in the omental compared to the subcutaneous fat. These findings provide some insights into the role of omental fat in abdominal obesity-associated co-morbidities.