Understanding the adipose tissue acetylome in obesity and insulin resistance
2022; Elsevier BV; Volume: 246; Linguagem: Inglês
10.1016/j.trsl.2022.02.008
ISSN1931-5244
AutoresMaria del Carmen Navarro-Ruiz, Jaime López‐Alcalá, Alberto Díaz‐Ruiz, Sandra Díaz del Moral, Carmen Tercero‐Alcázar, Andrea Nieto-Calonge, José López‐Miranda, Francisco J. Tinahones, Marı́a M. Malagón, Rocío Guzmán‐Ruiz,
Tópico(s)Sirtuins and Resveratrol in Medicine
ResumoObesity is a widely prevalent pathology with a high exponential growth worldwide. Altered lipid accumulation by adipose tissue is one of the main causes of obesity and exploring lipid homeostasis in this tissue may represent a source for the identification of possible therapeutic targets. The study of the proteome and the post-translational modifications of proteins, specifically acetylation due to its involvement in energy metabolism, may be of great interest to understand the molecular mechanisms involved in adipose tissue dysfunction in obesity. The objective of this study was to characterize the subcutaneous and omental adipose tissue acetylome in conditions of obesity and insulin resistance and to describe the importance of acetylation of key molecules in adipose tissue to use them as therapeutic targets. The results describe for the first time the acetylome of subcutaneous and omental adipose tissue under physiological and physiopathological conditions such as obesity and insulin resistance. New evidence showed different acetylation patterns between two main depots and highlight the molecular complexity of adipose tissue. Results showed changes in FABP4 acetylation in subcutaneous fat in relation to insulin resistance, thus unveiling a potential marker of depot-specific dysfunctional expansion in obesity-associated metabolic disease. Furthermore, it is shown that the acetylation of FABP4 affects its function, modulating the capacity of differentiation in adipocytes. In conclusion, this study demonstrates a profound, depot-specific alteration of adipose tissue acetylome, wherein the acetylation of FABP4 may play a key role in adipocyte differentiation and lipid accumulation. Obesity is a widely prevalent pathology with a high exponential growth worldwide. Altered lipid accumulation by adipose tissue is one of the main causes of obesity and exploring lipid homeostasis in this tissue may represent a source for the identification of possible therapeutic targets. The study of the proteome and the post-translational modifications of proteins, specifically acetylation due to its involvement in energy metabolism, may be of great interest to understand the molecular mechanisms involved in adipose tissue dysfunction in obesity. The objective of this study was to characterize the subcutaneous and omental adipose tissue acetylome in conditions of obesity and insulin resistance and to describe the importance of acetylation of key molecules in adipose tissue to use them as therapeutic targets. The results describe for the first time the acetylome of subcutaneous and omental adipose tissue under physiological and physiopathological conditions such as obesity and insulin resistance. New evidence showed different acetylation patterns between two main depots and highlight the molecular complexity of adipose tissue. Results showed changes in FABP4 acetylation in subcutaneous fat in relation to insulin resistance, thus unveiling a potential marker of depot-specific dysfunctional expansion in obesity-associated metabolic disease. Furthermore, it is shown that the acetylation of FABP4 affects its function, modulating the capacity of differentiation in adipocytes. In conclusion, this study demonstrates a profound, depot-specific alteration of adipose tissue acetylome, wherein the acetylation of FABP4 may play a key role in adipocyte differentiation and lipid accumulation. At A Glance CommentaryM. Carmen Navarro-Ruiz, et al.BackgroundObesity is a complex disease that requires a maximum understanding of the biology of adipose tissue, the main organ affected in this pathology, to develop effective therapies.Translational SignificanceTherefore, analysing processes that modulate the functionality of adipose tissue to store fat and manage excess energy is essential for the treatment of obesity and its comorbidities. In this line, the acetylation of proteins, that controls energy metabolism in other tissues, could be a key component in the physio(patho)logy of adipose tissue which has not yet been described.INTRODUCTIONObesity is a chronic pathology, which constantly grown worldwide during the last decades, being one of the main causes of death in the world. Moreover, it is often an independent risk factor of insulin resistance (IR) and types 2 diabetes (T2D). Therefore, it is critical to understand the mechanism involved in the triggering of insulin resistance and type 2 diabetes associated with obesity, to discover new molecularly targeted treatments. Adipose tissue (AT) plays an essential role in obesity and excess energy storage, and its subsequent release as the production of signaling molecules secreted by adipocytes that exert functions on other cells, namely adipokines.1Unamuno X Gómez-Ambrosi J Rodríguez A Becerril S Frühbeck G Catalán V. Adipokine dysregulation and adipose tissue inflammation in human obesity.Eur J Clin Invest. 2018; 48: 0-2https://doi.org/10.1111/eci.12997Crossref Scopus (225) Google Scholar Increasing our knowledge of AT biology might enable us to overcome the limitations of measuring the traditional anthropometric indices for obesity diagnosis. Obesity-related metabolic complications do not correlate with body mass index (BMI), and additional clinical parameters are necessary for risk evaluation.1Unamuno X Gómez-Ambrosi J Rodríguez A Becerril S Frühbeck G Catalán V. Adipokine dysregulation and adipose tissue inflammation in human obesity.Eur J Clin Invest. 2018; 48: 0-2https://doi.org/10.1111/eci.12997Crossref Scopus (225) Google Scholar Moreover, it is well known that adipose tissue exhibits depot-specific differences, many of which are related to their distinct capacity for expansion.2Pellegrinelli V Carobbio S Vidal-Puig A. Adipose tissue plasticity: how fat depots respond differently to pathophysiological cues.Diabetologia. 2016; 59: 1075-1088https://doi.org/10.1007/s00125-016-3933-4Crossref PubMed Scopus (233) Google Scholar,3Vishvanath L Gupta RK. Contribution of adipogenesis to healthy adipose tissue expansion in obesity.J Clin Invest. 2019; 129: 4022-4031https://doi.org/10.1172/JCI129191Crossref PubMed Scopus (173) Google Scholar Thus, omental adipose tissue accumulation is associated with insulin resistance, while subcutaneous fat expansion seems to be protective.3Vishvanath L Gupta RK. Contribution of adipogenesis to healthy adipose tissue expansion in obesity.J Clin Invest. 2019; 129: 4022-4031https://doi.org/10.1172/JCI129191Crossref PubMed Scopus (173) Google Scholar However, the molecular mechanisms underlying these differences are not yet fully understood. The identification of depot-specific lipid metabolism biomarkers that could serve as pharmaceutical targets may help counteract the onset of obesity and metabolic complications.Proteomics, which allows the identification of new protein biomarkers, has been proved useful to understand the complex biology behind obesity and its comorbidities. In contrast to genomics and transcriptomics, the analysis of the proteome allows the detection of post-translational modifications (PTM) and protein interaction.4Aleksandrova K Egea Rodrigues C Floegel A Ahrens W Omics biomarkers in obesity: novel etiological insights and targets for precision prevention.Curr Obes Rep. 2020; 9: 219-230https://doi.org/10.1007/s13679-020-00393-yCrossref PubMed Scopus (9) Google Scholar Among them, acetylation is one of the major PTM and has emerged for its role in the modulation of key enzymes that catalyze energy metabolism. Protein acetylation depends on acetyl-CoA, which is produced through several sources: glycolysis, β-oxidation of fatty acids, and acetate.5Drazic A Myklebust LM Ree R Arnesen T The world of protein acetylation.Biochim Biophys Acta - Proteins Proteomics. 2016; 1864: 1372-1401https://doi.org/10.1016/j.bbapap.2016.06.007Crossref PubMed Scopus (397) Google Scholar Thus, most of the enzymes of glycolysis, gluconeogenesis, the tricarboxylic acid cycle, or fatty acid metabolism were found to be acetylated in different tissues.6Fukushima A Lopaschuk GD. Acetylation control of cardiac fatty acid β-oxidation and energy metabolism in obesity, diabetes, and heart failure.Biochim Biophys Acta - Mol Basis Dis. 2016; 1862: 2211-2220https://doi.org/10.1016/j.bbadis.2016.07.020Crossref PubMed Scopus (55) Google Scholar, 7Zhang Y Zhou F Bai M et al.The pivotal role of protein acetylation in linking glucose and fatty acid metabolism to β-cell function.Cell Death Dis. 2019; 10https://doi.org/10.1038/s41419-019-1349-zCrossref Scopus (29) Google Scholar, 8Zhao Q Zhang Z Li J et al.Lysine acetylome study of human hepatocellular carcinoma tissues for biomarkers and therapeutic targets discovery.Front Genet. 2020; 11: 1-14https://doi.org/10.3389/fgene.2020.572663Crossref PubMed Scopus (8) Google Scholar In adipose tissue, in vivo, and in vitro studies have evidenced the importance of lysine acetylation of proteins for proper adipocyte differentiation (adipogenesis), both under physiological9Ong BX Brunmeir R Zhang Q et al.Regulation of thermogenic adipocyte differentiation and adaptive thermogenesis through histone acetylation.Front Endocrinol (Lausanne). 2020; 11: 1-21https://doi.org/10.3389/fendo.2020.00095Crossref PubMed Scopus (6) Google Scholar and pathological conditions (ie, obesity, diabetes).5Drazic A Myklebust LM Ree R Arnesen T The world of protein acetylation.Biochim Biophys Acta - Proteins Proteomics. 2016; 1864: 1372-1401https://doi.org/10.1016/j.bbapap.2016.06.007Crossref PubMed Scopus (397) Google Scholar,10Iyer A Fairlie DP Brown L. Lysine acetylation in obesity, diabetes and metabolic disease.Immunol Cell Biol. 2012; 90: 39-46https://doi.org/10.1038/icb.2011.99Crossref PubMed Scopus (89) Google ScholarA fundamental protein involved in these functions in the AT is fatty acid-binding protein 4 (FABP4), a lipid chaperone that participates in the intracellular trafficking of fatty acids. Additionally, it is considered an adipokine.11Hotamisligil GS Bernlohr DA. Metabolic functions of FABPs - Mechanisms and therapeutic implications.Nat Rev Endocrinol. 2015; 11: 592-605https://doi.org/10.1038/nrendo.2015.122Crossref PubMed Scopus (331) Google Scholar,12Furuhashi M. Fatty acid-binding protein 4 in cardiovascular and metabolic diseases.J Atheroscler Thromb. 2019; 26: 216-232https://doi.org/10.5551/JAT.48710Crossref PubMed Scopus (94) Google Scholar Moreover, different PTM such as phosphorylation, acetylation, and/or carbonylation has been reported for FABP4, which may regulate/influence its function.13Gillilan Richard E. Stephen D. Ayers and NN. Structural basis for activation of fatty acid binding protein 4.J Mol Biol. 2007; 372: 1246-1260Crossref PubMed Scopus (114) Google Scholar, 14Prentice KJ Saksi J Hotamisligil GS. Adipokine FABP4 integrates energy stores and counterregulatory metabolic responses.J Lipid Res. 2019; 60: 734-740https://doi.org/10.1194/jlr.S091793Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 15Xu Z Ande SR Mishra S. Temporal analysis of protein lysine acetylation during adipocyte differentiation.Adipocyte. 2013; 2: 33-40https://doi.org/10.4161/adip.21916Crossref PubMed Scopus (10) Google Scholar Among those, only phosphorylation has been validated by in vitro systems, and the functional relevance of such modification(s) remains to be addressed.14Prentice KJ Saksi J Hotamisligil GS. Adipokine FABP4 integrates energy stores and counterregulatory metabolic responses.J Lipid Res. 2019; 60: 734-740https://doi.org/10.1194/jlr.S091793Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar We herein describe for the first time, an exhaustive acetyl-lysine proteome profile (acetylome) of subcutaneous (SC) and omental (OM) AT during the development of obesity and insulin resistance. Thereby, the current study establishes protein acetylation in the adipose tissue as a potential link between obesity and IR, especially in subcutaneous fat, and evidences the relevance of acetylation as a key PTM of proteins, such as FABP4, to understand the complex role of protein function in obesity and IR.MATERIALS AND METHODSSubjects and sample collectionLean and morbidly obese male subjects recruited at the Endocrinology and Nutrition Unit of the Hospital Clínico Virgen de la Victoria (Málaga, Spain). The clinical characteristics of all subjects were evaluated and the biochemical and hormonal assays were carried out as described previously.16Tinahones FJ Coín-Aragüez L Mayas MD et al.Obesity-associated insulin resistance is correlated to adipose tissue vascular endothelial growth factors and metalloproteinase levels.BMC Physiol. 2012; 12: 4https://doi.org/10.1186/1472-6793-12-4Crossref PubMed Scopus (63) Google Scholar,17Guzmán-Ruiz R Tercero-Alcázar C Rabanal-Ruiz Y et al.Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.FASEB J. 2020; 34: 7520-7539https://doi.org/10.1096/fj.201902703RCrossref PubMed Scopus (15) Google ScholarObese subjects were stratified into two groups as described18Care D Suppl SS. 2. Classification and diagnosis of diabetes: Standards of medical care in diabetesd2019.Diabetes Care. 2019; 42: S13-S28https://doi.org/10.2337/dc19-S002Crossref PubMed Scopus (1680) Google Scholar according to the criteria determined by the ADA [Obese Normoglycemic (Ob-NG): Fasting plasma glucose (FPG) < 100 mg/dL, HbA1c < 5.7%; and impaired fasting glucose (IFG) = 100–126 mg/dL, HbA1c: 5.7%–6.4%]. IFG will be referred to as Ob-IR subjects as previously was described.17Guzmán-Ruiz R Tercero-Alcázar C Rabanal-Ruiz Y et al.Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.FASEB J. 2020; 34: 7520-7539https://doi.org/10.1096/fj.201902703RCrossref PubMed Scopus (15) Google Scholar19Díaz-Ruiz A Guzmán-Ruiz R Moreno NR et al.Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity.Antioxid Redox Signal. 2015; 23: 597-612https://doi.org/10.1089/ars.2014.5939Crossref PubMed Scopus (42) Google Scholar The Ethics and Research Committee of the Hospital Clínico Virgen de la Victoria approved the experimental design. Participants gave their informed written consent.Paired plasma and biopsies of subcutaneous (SC) and omental (OM) AT were obtained from subjects and were kept at −80°C before use.Protein extractionTo extract proteins for mass spectrometry analysis, frozen tissues (100 mg) were homogenized as previously described.17Guzmán-Ruiz R Tercero-Alcázar C Rabanal-Ruiz Y et al.Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.FASEB J. 2020; 34: 7520-7539https://doi.org/10.1096/fj.201902703RCrossref PubMed Scopus (15) Google Scholar Briefly, samples were disrupted in buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton-X-100, 1 mM EDTA, 1 mM TSA (Trichostatin A, deacetylase inhibitor), and 1 µg/mL protease inhibitor cocktail. After centrifugation at 13400 rpm, the supernatant containing cytosolic proteins was kept at -80ºC. The pellet containing nuclear fraction was resuspended in buffer containing 20 mM HEPES (pH: 7.9), 1.5 mM MgCl2, 420 mM NaCl, 25% glycerol, 1 mM TSA, and 1 µg/mL protease inhibitor cocktail and homogenized to extract nuclear proteins as described.20Guzmán-Ruiz R Ortega F Rodríguez A et al.Alarmin high-mobility group B1 (HMGB1) is regulated in human adipocytes in insulin resistance and influences insulin secretion in β-cells.Int J Obes. 2014; 38: 1545-1554https://doi.org/10.1038/ijo.2014.36Crossref PubMed Scopus (67) Google ScholarAcetylated protein enrichment and MS analysisCytosolic and nuclear protein extracts (200 μg) from adipose tissue samples were pooled (pool A = 3 samples; pool B = 2 samples) to help overcome resource constraints individuals analyzed as described.21Diz AP Truebano M Skibinski DOF. The consequences of sample pooling in proteomics: an empirical study.Electrophoresis. 2009; 30: 2967-2975https://doi.org/10.1002/elps.200900210Crossref PubMed Scopus (156) Google Scholar Additionally, two different pools were processed to obtain different replicates. Recombinant acetylated bovine serum albumin (1%) was added to samples, as acetylated protein control. Samples were incubated overnight with anti-acetyl-lys antibody (100 µL) conjugated to beads (ImmnunochemTechnology, Davis, CA, U.S.A.). Beads were previously washed twice with phosphate-buffered saline (PBS). Thereafter, beads containing bound acetylated proteins were washed with immunoprecipitation (IP) buffer, IP buffer (50 mM MOPS pH 7.2, 10 mM NaHPO4, 50 mM NaCl). The supernatant was removed and beads were carefully washed twice with water. Bound proteins were eluted with 0.15% trifluoroacetic acid to obtain affinity enrichment of the acetylated proteins.LC-MS/MS analysis was performed at the Proteomics Unit of the University of Córdoba (Central Research Support Service, Córdoba, Spain). Samples were analyzed using a Dionex Ultimate 3000 nano-LC system (nano UHPLC; Thermo Fisher Scientific, Waltham, MA, U.S.A.) in tandem with a linear quadrupole ion trap-Orbitrap (LTQ Orbitrap XL) mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific, Thermo Fisher Scientific, Waltham, MA, U.S.A.). Acquired data were analyzed with Proteome Discoverer 2.1 software (Sequest HT algorithm, Thermo Fisher Scientific, Waltham, MA, U.S.A.), using a specific human database extracted from the non-redundant UNIPROT website (https://www.uniprot.org/). Peptide search was carried out with a maximum of 1 missed cleavage site. Carbamidomethylation of cysteines was set as a fixed modification, whilst methionine oxidation and lysine acetylation were set as variable modifications. Finally, the identified peptides were filtered using a false discovery rate (FDR) of 1%, calculated using a decoy database strategy.Functional annotation analysisGO enrichment analyses were performed using PANTHER classification system (http://www.pantherdb.org) against a background of Homo Sapiens proteome.22Huaiyu M Anushya M Xiaosong H et al.Protocol Update for Large-scale genome and gene function analysis with PANTHER Classification System (v.14.0).Nat Protoc. 2019; 14 (doi:10.1038/s41596-019-0128-8.Protocol): 703-721Crossref PubMed Scopus (515) Google Scholar Enrichments of GO terms (PANTHER GO-Slim Biological Process, Molecular Function, and Cellular Component) were considered statistically significant for FDR < 0.05 and were corrected for multiple testing by Bonferroni's method. The top 10 of the most significant GO terms were studied. GO Biological Process network and hierarchical clustering tree of statistically significant acetylated proteins was performed using the ShinyGO v0.61 (http://bioinformatics.sdstate.edu/go/) database.23Ge SX Jung D. ShinyGO: A graphical enrichment tool for animals and plants.bioRxiv. 2018; : 2017-2018https://doi.org/10.1101/315150Crossref Scopus (0) Google Scholar Acetylated proteins in both depots and in each experimental group were obtained using GraphPad Prism 7 (La Jolla, CA, U.S.A). Venn diagrams depicting common and specific acetylated proteins in all analyzed conditions were created using InteractiVenn web tool (http://www.interactivenn.net).24Heberle H Meirelles VG da Silva FR Telles GP Minghim R. InteractiVenn: A web-based tool for the analysis of sets through Venn diagrams.BMC Bioinformatics. 2015; 16: 1-7https://doi.org/10.1186/s12859-015-0611-3Crossref PubMed Scopus (956) Google Scholar Microsoft Excel was used to calculate the ratios and fold changes (FC) followed by log2 transformation.Site-directed mutagenesis of FABP4Based on previous bibliography,13Gillilan Richard E. Stephen D. Ayers and NN. Structural basis for activation of fatty acid binding protein 4.J Mol Biol. 2007; 372: 1246-1260Crossref PubMed Scopus (114) Google Scholar different lysine residues were selected to create a site-directed mutagenesis of FABP4. Primers were designed to introduce changes from lysine (AAA) to isoleucine (ATA) for each of the residues mutated by PCR-directed mutagenesis.25Vazquez-Martinez R Cruz-Garcia D Duran-Prado M Peinado JR Castaño JP Malagon MM. Rab18 inhibits secretory activity in neuroendocrine cells by interacting with secretory Granules.Traffic. 2007; 8: 867-882https://doi.org/10.1111/j.1600-0854.2007.00570.xCrossref PubMed Scopus (43) Google Scholar All PCRs were carried out using 100 ng of pEGFP-C2-WTFABP4 (provided by Dr Noy) as a template, and 125 ng of K22I Primers (Reverse and Forward), K32I Primers (Reverse and Forward), or K59I Primers (Reverse and Forward) (Supplementary Table 1), depending on the mutation. Both nucleic acids were diluted to a total volume of 25 μL, containing 1 unit of KAPA HIFI HOTSTART DNA polymerase (Kapabiosystem, Boston, MA, U.S.A.) and the appropriate amplification buffer. PCR was performed using the following thermocycling conditions: denaturation at 95°C for 3 minutes, followed by 25 cycles of 98°C for 30 seconds; 60°C for 15 seconds; 72°C for 1 min/kb, and a final extension at 72°C for 1 min/kb. DNA electrophoresis on 1% agarose gel was used to validate and obtain the PCR products. The appropriate fragments were gel-purified using AccuPrep Gel Purification Kit (Bioneer, Daejeon, Republic of Korea) according to the manufacturer's instructions. DNA purified was digested with 5 units of DpnI enzyme for 1 hour (ThermoFisher, Waltham, MA, U.S.A.). DNA was measured by Nanodrop 2000C instrument (Thermofisher, Wilmington, DE) and used to transform chemically DH5α competent E. coli cells. Thus, the corresponding vectors pEGFP-C2 (mock), pEGFP-C2-WTFABP4 (wild type, WT), pEGF-C2-K22IFABP4 (mutant 1, M1), pEGFP-C2-K32IFABP4 (mutant 2, M2), pEGFP-C2-K59IFABP4 (mutant 3, M3), and pEGFP-C2-TMFABP4 (triple mutant, TM) were generated. Finally, the correct position of the mutations in the different mutant plasmids was confirmed by sequencing (STABVida, Caparica, Portugal).In silico 3D structure modelingTo confirm that the substitution of Lys in the TM version of FABP4 did not alter the 3-dimensional structure (3D) of FABP4, the protein was modeled in silico. Phyre2 software (http://www.sbg.bio.ic.ac.uk/phyre2) was used to generate the alignments of GFP-FABP4-WT and GFP-FABP4-TM protein sequences, against the 3D structure of FABP4 (PDB id code:3P6D) registered in the PDB database (Protein Data Bank, NJ, U.S.A.). The 3D structures generated in silico were compared using the Swiss PDB Viewer program (Lausanne, Switzerland) and its Root Mean Squared (RMS) statistical tool, which identifies spatially related structures.FABP4 (WT or TM) and HSL protein (AlphaFold id code: AF-P54310) models were docked using HDOCK26Yan Y Zhang D Zhou P Li B Huang SY. HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy.Nucleic Acids Res. 2017; 45: W365-W373https://doi.org/10.1093/nar/gkx407Crossref PubMed Scopus (321) Google Scholar under the default setting. The binding poses of the top docking complexes were selected and compared. The interacting residues of FABP4-HSL were evaluated by Ligprot+27Laskowski RA Swindells MB. LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery.J Chem Inf Model. 2011; 51: 2778-2786https://doi.org/10.1021/ci200227uCrossref PubMed Scopus (2958) Google Scholar to generate schematic diagrams of protein-protein interactions.Culture and treatment of 3T3-L1 cell line3T3-L1 cells [American Type Culture Collection (Manassas, VA, U.S.A)] were cultured on 12-well plates (3 × 103 cells/cm2) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of newborn calf serum, 4 mM of glutamine, and 1% of antibiotic-antimycotic solution. At 100% confluence, cells were differentiated into adipocytes as previously described.17Guzmán-Ruiz R Tercero-Alcázar C Rabanal-Ruiz Y et al.Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.FASEB J. 2020; 34: 7520-7539https://doi.org/10.1096/fj.201902703RCrossref PubMed Scopus (15) Google Scholar19Díaz-Ruiz A Guzmán-Ruiz R Moreno NR et al.Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity.Antioxid Redox Signal. 2015; 23: 597-612https://doi.org/10.1089/ars.2014.5939Crossref PubMed Scopus (42) Google ScholarDifferentiated 3T3-L1 cells (D6-7) were exposed to a combination of high concentrations of glucose (4.5 g/L) and insulin (100 nM) (HGHI) for 24 hours, to induce insulin resistance. Another set of cells was exposed to lipid overload using oleate or palmitate (500 mM) for 18 hours.17Guzmán-Ruiz R Tercero-Alcázar C Rabanal-Ruiz Y et al.Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.FASEB J. 2020; 34: 7520-7539https://doi.org/10.1096/fj.201902703RCrossref PubMed Scopus (15) Google Scholar,19Díaz-Ruiz A Guzmán-Ruiz R Moreno NR et al.Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity.Antioxid Redox Signal. 2015; 23: 597-612https://doi.org/10.1089/ars.2014.5939Crossref PubMed Scopus (42) Google Scholar After treatment, cells were processed for acetylated protein analysis, lipid analysis, and immunoblotting.Oil Red-O stainingOil Red-O staining of lipid droplets was performed in 3T3-L1 cells during differentiation. Cells were washed with Dulbecco's PBS (PBS) and fixed with 4% paraformaldehyde for 10 minutes at room temperature (RT). Coverslips were washed with 60% isopropanol and cells were incubated with Oil Red-O for 30 minutes at RT in the dark and the unbound dye was rinsed with distilled water. Images were captured with an inverted light microscope coupled to a camera (MOTICAM 1080 HD, Leica, Wetzlar, Germany).Cell transfectionFor the analysis of acetylated FABP4, 3T3-L1 cells at day 3 of differentiation were transfected with Lipofectamine 2000 (Invitrogen, Barcelona, Spain) and mock, WT-, M1-, M2-, M3- or TM-FABP4 expression vectors, according to the manufacturer's instructions. At 48 hours post-transfection, the cells were processed for immunostaining, RT-PCR, and immunoblotting analysis. In a series of experiments aimed at investigating the effects of FABP4 acetylation on adipocyte differentiation at the long-term, cells were processed for microscopy and expression studies on day 10 of differentiation.Dot blot analysisFractions of crude extracts, washing phases and acetylation protein extracts from each pool of samples were collected and used for IP validation by dot blot as previously described.19Díaz-Ruiz A Guzmán-Ruiz R Moreno NR et al.Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity.Antioxid Redox Signal. 2015; 23: 597-612https://doi.org/10.1089/ars.2014.5939Crossref PubMed Scopus (42) Google Scholar Nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) were used to detect samples ( 10 μL each sample). Anti-acetyl Lys primary antibody was used overnight (4°C) and peroxidase-conjugated rabbit secondary antibody was administered for 1 hour (Supplementary Table 2). Immunoreaction was visualized using ECL (Bio-Rad Laboratories, Hercules, CA, U.S.A.). Quantification of band intensities was carried out on digital images of membrane samples provided by LAS4000 gel documentation system (GE Healthcare; Barcelona, Spain) using ImageJ software (ImageJ, https://imagej.nih.gov/ij/, NIH, Bethesda, MA, U.S.A.), and normalized with Ponceau density values as previously described.17Guzmán-Ruiz R Tercero-Alcázar C Rabanal-Ruiz Y et al.Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.FASEB J. 2020; 34: 7520-7539https://doi.org/10.1096/fj.201902703RCrossref PubMed Scopus (15) Google Scholar All comparative experiments were performed under identical conditions.Quantitative immunoblottingTwenty microgram of protein per sample were loaded onto 10% polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes (Trans-Blot Turbo Transfer System; Bio-Rad Laboratories, Hercules, CA, USA). Primary antibodies were used overnight (4°C) and peroxidase-conjugated rabbit secondary antibody was administered for 1 hour (Supplementary Table 2). Immunoreaction and posterior quantification were described above.RNA isolation and expression analysis by RT‐PCRTotal RNA from 3T3‐L1 cells was extracted using Trizol RNA isolation method (Invitrogen, Carlsbad, CA, U.S.A.) and purified with the RNeasy Lipid kit (QIAGEN, Valencia, CA, U.S.A.). Isolation and purification of RNA from 3T3-L1 cells were performed as described.2Pellegrinelli V Carobbio S Vidal-Puig A. Adipose tissue plasticity: how fat depots respond differently to pathophysiological cues.Diabetologia. 2016; 59: 1075-1088https://doi.org/10.1007/s00125-016-3933-4Crossref PubMed Scopus (233) Google Scholar Real-time PCR (RT-PCR) was carried out using the listed primers (Supplementary Table 1). Specific signals of FABP4, SREBP-1, C/EBPa, and PPARg were normalized using a normalization factor obtained from constitutively expressed Hprt and Gapdh levels (housekeeping genes, which maintain stable expression in all conditions), using the free tool BestKeeper software. All samples were run in duplicate and the average values were calculated.Table 1Anthropometric and biochemical characteristics of subjectsLean (n = 5)Ob-NG (n = 16)Ob-IR (n = 16)AnovaSex (men/women)5/09/78/8Age (years)43,4 ± 5,245,1 ± 4,643,0 ± 4,7nsWeight (kg)71,8 ± 4,3146,6 ±13,6†P < 0.001 vs lean subjects.153,6 ± 8,2†P < 0.001 vs lean subjects.<0.001BMI (Kg/m2)22,96 ± 0,751,7 ± 3,3†P < 0.001 vs lean subjects.53,1 ± 2,1†P < 0.001 vs lean subjects.<0.00
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