Proteomics analysis of human obesity reveals the epigenetic factor HDAC4 as a potential target for obesity

Abstract

Sedentary lifestyle and excessive energy intake are prominent contributors to obesity; a major risk factors for the development of insulin resistance, type 2 diabetes and cardiovascular diseases. Elucidating the molecular mechanisms underlying these chronic conditions is of relevant importance as it might lead to the identification of novel anti-obesity targets. The purpose of the current study is to investigate differentially expressed proteins between lean and obese subjects through a shot-gun quantitative proteomics approach using peripheral blood mononuclear cells (PBMCs) extracts as well as potential modulation of those proteins by physical exercise. Using this approach, a total of 47 proteins showed at least 1.5 fold change between lean and obese subjects. In obese, the proteomic profiling before and after 3 months of physical exercise showed differential expression of 38 proteins. Thrombospondin 1 (TSP1) was among the proteins that were upregulated in obese subjects and then decreased by physical exercise. Conversely, the histone deacetylase 4 (HDAC4) was downregulated in obese subjects and then induced by physical exercise. The proteomic data was further validated by qRT-PCR, Western blot and immunohistochemistry in both PBMCs and adipose tissue. We also showed that HDAC4 levels correlated positively with maximum oxygen consumption (VO2 Max) but negatively with body mass index, percent body fat, and the inflammatory chemokine RANTES. In functional assays, our data indicated that ectopic expression of HDAC4 significantly impaired TNF-α-dependent activation of NF-κB, establishing thus a link between HDAC4 and regulation of the immune system. Together, the expression pattern of HDAC4 in obese subjects before and after physical exercise, its correlation with various physical, clinical and metabolic parameters along with its inhibitory effect on NF-κB are suggestive of a protective role of HDAC4 against obesity. HDAC4 could therefore represent a potential therapeutic target for the control and management of obesity and presumably insulin resistance.

Figure 1. Effect of obesity on protein expression in PBMCs.

Venn diagram showing the number of unique and overlapping proteins identified from lean (LN) and obese (OB) subjects (A). Distribution of all the identified proteins according to their biological processes (B). Both panels were generated using ProteinCenter software (Thermo Scientific, Germany). qRT-PCR data carried out on 10 genes to validate the observed differential protein expression at the mRNA levels using total RNA isolated from PBMCs of lean and obese male subjects (n=5 each) and the data are presented as fold changes in obese compared to lean subjects (C). The gene names and primer sequences are shown in Table S6. * P < 0.05 as determined using student’s t-test.

Figure 2. Effect of physical exercise on protein expression in PBMCs of obese subjects.

Venn diagram showing the number of unique and overlapping proteins identified from obese before and after 3 months of physical exercise (A). qRT-PCR data carried out on 9 genes to validate the observed differential protein expression at the mRNA levels. Total RNA were isolated from PBMCs of male obese before and after exercise obese (n=5 each) and the data are presented as fold changes in obese compared to lean subjects (B). * P < 0.05 as determined using student’s t-test.

Figure 3. Representative Western blot confirming differential expression of HDAC4 and TSP1 in obese subjects.

(A and B) Total proteins were extracted from PBMC of lean (n=7) and obese (n=7) non-diabetic participants and subjected to western blot using the indicated antibodies. The bands were quantified as described in materials and methods and the relative intensity was determined after correction with GAPDH that was used as internal control to monitor loading efficiency (A). The data are presented in the form of graphs as fold changes compared to lean group (B). (C and D) Nuclear (N) and cytoplasmic (C) extracts were prepared from PBMCs isolated from lean (LN) and obese (OB) male subjects and subjected to Western blot using the indicated antibodies. The bands representing the cytoplasmic fractions were quantified as described in materials and methods and the relative intensity was determined after correction with Tubulin from the cytoplasmic fraction that was used as internal control to monitor loading efficiency. Lamin B was used as control to monitor nuclear localization (C). The data are presented as fold changes in obese compared to lean subjects (D). The blots shown are representatives of at least two independent experiments with consistent results.

Figure 4. HDAC4 and TSP1 are also differentially expressed in the adipose tissue of obese humans.

Representative immunohistochemical (IHC) staining using fat adipose biopsies from lean (n=4) and obese (n=8) male subjects illustrating the expression pattern of HDAC4 (A) and TSP1 (B). Aperio software was used to quantify positive staining (indicated by arrows) and the values are illustrated at the bottom as fold changes compared to lean. As negative control (NC) for the experiment, the primary antibodies were omitted (A, B). qRT-PCR analysis confirming differential expression in of HDAC4 and TSP1 at the mRNA levels in the adipose tissue. Total RNA was extracted from subcutaneous adipose tissue from lean and obese subjects (n=10 each) and analyzed by qRT-PCR. The data are presented as fold changes in obese compared to lean subjects (C). Graphic illustration of IHC quantification of HDAC4 and TSP1 proteins in subcutaneous adipose tissue collected from obese before and after exercise (n=8 each). Aperio software was used to quantify positive staining (D). qRT-PCR analysis showing the effect of physical exercise on the expression of HDAC4 and TSP1 mRNA in the adipose tissue. Total RNA was extracted from subcutaneous adipose tissue from obese subjects before and physical exercise (n=10 each) and analyzed by qRT-PCR. The data are presented as fold changes in obese before exercise compared to obese after exercise (E). * p< 0.05 and ** p< 0.01 as determined using student’s t-test.

Figure 5. Ectopic expression of HDAC4 impaired NF-κB activation by TNF-α in luciferase assays.

Reporter and expression vectors were used in transient transfection assays in HEK293 cells as indicated in materials and methods. Reporter vector in which the firefly luciferase gene is under the control of 3 copies of wild type (3xwt-κB) or mutant (3xmut-κB) NF-κB binding was cotransfected with HDAC4 expression vector. pCMV was used a control empty vector. 24 hours post-transfection, cells were treated with 25 ng/ml of TNF-α for overnight and then, the luciferase activity was carried out as described in materials and methods. Protein concentration was used for normalization.