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Abusive head trauma (AHT) is a leading cause of mortality and morbidity in infants. While the reported incidence is close to 40 cases per 100'000 births/year, misdiagnoses are commonly observed in cases with atypical, subacute or chronic presentation. Currently, standard clinical evaluation of inflicted intracranial hemorrhagic injury (ICH) in infants urgently requires a screening test able to identify infants who need additional investigations. Blood biomarkers characteristic of AHT may assist in detecting these infants, improving prognosis through early medical care. To date, the application of innovative omics technologies in retrospective studies of AHT in infants is rare, due also to the blood serum and cerebrospinal fluid of AHT cases being scarce and not systematically accessible. Here, we explored the circulating blood proteomes of infants with severe AHT and their atraumatic controls. We discovered 165 circulating serum proteins that display differential changes in AHT cases compared with atraumatic controls. The peripheral blood proteomes of pediatric AHT commonly reflect: i) potentially secreted proteome from injured brain, and ii) proteome dysregulated in the system's circulation by successive biological events following acute ICH. This study opens up a novel opportunity for research efforts in clinical screening of AHT cases. This article is protected by copyright. All rights reserved.

The cullin-4 paralogs CUL4A and CUL4B assemble E3 ubiquitin ligase complexes regulating multiple chromatin-associated cellular functions. Although they are structurally similar, we found that the unique N-terminal extension of CUL4B is heavily phosphorylated during mitosis, and the phosphorylation pattern is perturbed in the CUL4B-P50L mutation causing X-linked intellectual disability (XLID). Phenotypic characterization and mutational analysis revealed that CUL4B phosphorylation is required for efficient progression through mitosis, controlling spindle positioning and cortical tension. Interestingly, while CUL4B phosphorylation triggers chromatin exclusion, it critically promotes binding to actin regulators and two previously unrecognized, CUL4B-specific DCAFs, LIS1 and WDR1. Indeed, co-immunoprecipitation experiments and biochemical analysis revealed that LIS1 and WDR1 interact with DDB1, but their binding requires the phosphorylated N-terminal domain of CUL4B. Together, our study uncovers previously unrecognized DCAFs relevant for mitosis and brain development that specifically bind CUL4B, but not the CUL4B-P50L patient mutant, by a phosphorylation-dependent mechanism.

MicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Previous works suggest that up to 60% of mammalian transcripts might be subject to miRNA-mediated regulation, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrate transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. Using this integrative approach, combined with molecular validation data, we present evidence that < 10% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identify TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions., EMBO Reports, 23 (9), ISSN:1469-221X, ISSN:1469-3178

SUMMARYMicroRNA (miRNA) loaded Argonaute (AGO) complexes regulate gene expression via direct base pairing with their mRNA targets. Current prediction approaches identified that between 20 to 60% of mammalian transcriptomes are regulated by miRNAs, but it remains largely unknown which fraction of these interactions are functional in a specific cellular context. Here, we integrated transcriptome data from a set of miRNA-depleted mouse embryonic stem cell (mESC) lines with published miRNA interaction predictions and AGO-binding profiles. This integrative approach, combined with molecular validation data, identified that only 6% of expressed genes are functionally and directly regulated by miRNAs in mESCs. In addition, analyses of the stem cell-specific miR-290-295 cluster target genes identified TFAP4 as an important transcription factor for early development. The extensive datasets developed in this study will support the development of improved predictive models for miRNA-mRNA functional interactions.

Advancements in mass spectrometry‐based proteomics have enabled experiments encompassing hundreds of samples. While these large sample sets deliver much‐needed statistical power, handling them introduces technical variability known as batch effects. Here, we present a step‐by‐step protocol for the assessment, normalization, and batch correction of proteomic data. We review established methodologies from related fields and describe solutions specific to proteomic challenges, such as ion intensity drift and missing values in quantitative feature matrices. Finally, we compile a set of techniques that enable control of batch effect adjustment quality. We provide an R package, "proBatch", containing functions required for each step of the protocol. We demonstrate the utility of this methodology on five proteomic datasets each encompassing hundreds of samples and consisting of multiple experimental designs. In conclusion, we provide guidelines and tools to make the extraction of true biological signal from large proteomic studies more robust and transparent, ultimately facilitating reliable and reproducible research in clinical proteomics and systems biology., In mass spectrometry‐based proteomics, handling large sample sets introduces technical variability known as batch effects. This tutorial provides guidelines and tools for the assessment, normalization, and batch correction of proteomics data.

(a) Activity-based proteome profiling (ABPP) probes combined with mass spectrometry (MS) is the tool of functional proteomics for the quantification of catalytically active enzymes. Here, we applied a depletion-dependent ABPP for serine hydrolase (SH) profiling in 30-μm thickness cryosections of optimal-cutting-temperature (OCT)-embedded tissue of advanced lung adenocarcinomas (LUAD). (b) For the proof-of-concept, we profiled SH activities in advanced LUAD disease. We retrospectively analyzed hydrolase activity differences with prognostic values related to aggressive phenotype based on clinical data collected over the course of disease progression. Interestingly, detection of proteins correlating by active-enzyme capture uncover their functional links with the metabolic SHs related to the enhanced palmitoylation-depalmitoylation cycle of lipoproteins in aggressive LUAD. In contrast, the standard DDA-MS analysis of on-bead-depleted enzymes detected 95 SH’s of which less than 60 enzymes were quantified based on spectral counting. (c) We found enhanced activities of dozen SHs in all LUAD tumors. Remarkably, the activity of 11 hydrolases emphasized by IAH1, ABHD12, LYPLA2 and MST1 discriminate aggressive tumors at the time of diagnosis. Functional analysis leveraging enzymes and associated co-depleted proteins revealed catabolic processing of lipoproteins and enrichment of enzymes displaying increased de-palmitoylase activities. This is interesting because multiple isoforms of RAS protein (K-RAS, H-RAS and N-RAS), the most frequently mutated oncogene in human cancers including LUAD (i.e. 30% of LUADs contain somatic K-Ras mutation), attain oncogenic properties only in depalmitoylated state which favor protein intracellular trafficking by detachment from hydrophobic plasma membrane. In line, mutant K-Ras protein co-localize on outer mitochondrion membrane and interact with glycolytic enzymes (hexokinase-1 and -2) only in depalmitoylated state. This interaction induce K-Ras oncogenic properties and increased glucose uptake and glycolysis even in the presence of abundant oxygen (Warburg effect). Curiously, we observed co-depletion of mitochondrial proteins including glycolytic hexokinase 2 with several lipases displaying increase depalmitoylase activity in short LUAD survivals. (d) Surprisingly, an evident disagreement in the activity of enzymes involved in lipolysis and protein depalmitoylation (i.e. IAH1, ABHD12, LYPLA2) was discovered between LUAD survival subtypes. These essential metabolic differences were uncovered by our standard proteomic analysis and to our knowledge have not been reported. Since the inhibitors of serine lipases are promising targets in other RAS-mutant carcinomas, this metabolic vulnerabilities discovered in aggressive LUAD can be explored as promising therapeutic strategies in lung cancer. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Discrimination of aggressive tumor profiles in lung adenocarcinomas using activity based protein profiling by SWATH/DIA-MS mode [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1887.

Obesity is considered as a major public health concern with strong economic and social burdens. Exposure to pollutants such as heavy metals can contribute to the development of obesity and its associated metabolic disorders, including type 2 diabetes and cardiovascular diseases. Adipose tissue is an endocrine and paracrine organ that plays a key role in the development of these diseases and is one of the main target of heavy metal accumulation. In this study, we determined by inductively coupled plasma mass spectrometry cadmium concentrations in human subcutaneous and visceral adipose tissues, ranging between 2.5 nM and 2.5 µM. We found a positive correlation between cadmium levels and age, sex and smoking status and a negative correlation between cadmium and body mass index. Based on cadmium adipose tissue concentrations found in humans, we investigated the effects of cadmium exposure, at concentrations between 1 nM and 10 µM, on adipose-derived human mesenchymal stem cells differentiated into mature adipocytes in vitro. Transcriptomic analysis highlighted that such exposure altered the expression of genes involved in trace element homeostasis and heavy metal detoxification, such as Solute Carrier Family transporters and metallothioneins. This effect correlated with zinc level alteration in cells and cellular media. Interestingly, dysregulation of zinc homeostasis and transporters has been particularly associated with the development of obesity and type 2 diabetes. Moreover, we found that cadmium exposure induces the pro-inflammatory state of the adipocytes by enhancing the expression of genes such as IL-6, IL-1B and CCL2, cytokines also induced in obesity. Finally, cadmium modulates various adipocyte functions such as the insulin response signaling pathway and lipid homeostasis. Collectively, our data identified some of the cellular mechanisms by which cadmium alters adipocyte functions, thus highlighting new facets of its potential contribution to the progression of metabolic disorders.

Summary Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitate follow-up biomarker research into early cancer detection.

Down syndrome (DS) is mostly caused by a trisomy of the entire Chromosome 21 (Trisomy 21, T21). Here, we use SWATH mass spectrometry to quantify protein abundance and protein turnover in fibroblasts from a monozygotic twin pair discordant for T21, and to profile protein expression in 11 unrelated DS individuals and matched controls. The integration of the steady-state and turnover proteomic data indicates that protein-specific degradation of members of stoichiometric complexes is a major determinant of T21 gene dosage outcome, both within and between individuals. This effect is not apparent from genomic and transcriptomic data. The data also reveal that T21 results in extensive proteome remodeling, affecting proteins encoded by all chromosomes. Finally, we find broad, organelle-specific post-transcriptional effects such as significant downregulation of the mitochondrial proteome contributing to T21 hallmarks. Overall, we provide a valuable proteomic resource to understand the origin of DS phenotypic manifestations., Trisomy 21 (T21) is a major cause of Down syndrome but little is known about its impact on the cellular proteome. Here, the authors define the proteome of T21 fibroblasts and its turnover and also map proteomic differences in monozygotic T21-discordant twins, revealing extensive, organelle-specific changes caused by T21.

(a) Proteomics relies on protein digestion into smaller fragments/peptides and converge to proteome sequence information, classification, and expression levels without a screen of the cellular protein activities. Activity-based proteome profiling (ABPP) is emerging as a tool for functional proteomics based on mass spectrometry. We established an ABPP-SWATH/DIA MS platform compatible with OCT-embedded biopsies for differential global serine hydrolase (SHs) activity profiling and determine the Relative Depletion-Detected Activity (RDDA) index that reflects the relative activity of SHs in type IIIA lung adenocarcinoma biopsies. (b) To prevent digestion on streptavidin beads and MS spectra contamination, we used SWATH/DIA-MS2 signal intensities for the assessment of the ratio between the biopsy-extract solution either treated with DMSO or which had earlier been depleted with chemical probe for active enzyme molecules. We processed 24 lung biopsies (having a solution of depleted and non-depleted proteome) of long and short survivors with type IIIA lung adenocarcinoma tumor and their normal tissue counterparts and included peptides of 65 known endogenous inhibitors of SH. In parallel experiments, we analyzed the catalytically depleted part of the molecule by conventional ABPP-MS approach integrated on bead sample digestion. (c) We quantified 117 proteins annotated to the serine hydrolase family, of which as much as 60% was depleted with a CI of 90% in the biopsy-extract solution incubated with chemical probe. Among 133 enzymes monitored over the entire clinical cohort, we found as many as 89 enzymes with significant depletion that we further used to generate the activity SH profiles of each biopsy. We found 36 enzymes exclusively depleted in tumor tissue, both in short and long survivals, and with depletion absent from normal counterparts. By quantification of the catalytically active part of the molecule on the streptavidin bead sample pull-down digest, we found only 9 proteins that contained quality data for spectral counting comparisons. Partial Least Squares Discriminant Analysis (PLS-DA) analysis revealed the activity profiles of 9 enzymes perfectly discriminating the tumor tissues of short survivals, and these enzyme signatures were enhanced by the elevated activity of 2 enzymes - ADH1C and IAH1- in short-term survivors. (d) We have demonstrated that this approach can substantially increase the differential detection of protease activity between oncogenic lung tissue and their normal tissue counterparts. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Activity based protein profiling by SWATH/DIA-MS mode (ABPP-SWATH/DIA-MS) from OCT-embedded tissues biopsies reveals lung tumor-specific protease profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6482.

To-date, most proteomic studies aimed at discovering tissue-based cancer biomarkers have compared the quantity of selected proteins between case and control groups. However, proteins generally function in association with other proteins to form modules localized in particular subcellular compartments in specialized cell types and tissues. Sub-cellular mislocalization of proteins has in fact been detected as a key feature in a variety of cancer cells. Here, we describe a strategy for tissue-biomarker detection based on a mitochondrial fold enrichment (mtFE) score, which is sensitive to protein abundance changes as well as changes in subcellular distribution between mitochondria and cytosol. The mtFE score integrates protein abundance data from total cellular lysates and mitochondria-enriched fractions, and provides novel information for the classification of cancer samples that is not necessarily apparent from conventional abundance measurements alone. We apply this new strategy to a panel of wild-type and mutant mice with a liver-specific gene deletion of Liver receptor homolog 1 (Lrh-1hep−/−), with both lines containing control individuals as well as individuals with liver cancer induced by diethylnitrosamine (DEN). Lrh-1 gene deletion attenuates cancer cell metabolism in hepatocytes through mitochondrial glutamine processing. We show that proteome changes based on mtFE scores outperform protein abundance measurements in discriminating DEN-induced liver cancer from healthy liver tissue, and are uniquely robust against genetic perturbation. We validate the capacity of selected proteins with informative mtFE scores to indicate hepatic malignant changes in two independent mouse models of hepatocellular carcinoma (HCC), thus demonstrating the robustness of this new approach to biomarker research. Overall, the method provides a novel, sensitive approach to cancer biomarker discovery that considers contextual information of tested proteins.

In medicine, there is an urgent need for protein biomarkers in a range of applications that includes diagnostics, disease stratification, and therapeutic decisions. One of the main technologies to address this need is MS, used for protein biomarker discovery and, increasingly, also for protein biomarker validation. Currently, data-dependent analysis (also referred to as shotgun proteomics) and targeted MS, exemplified by SRM, are the most frequently used mass spectrometric methods. Recently developed data-independent acquisition techniques combine the strength of shotgun and targeted proteomics, while avoiding some of the limitations of the respective methods. They provide high-throughput, accurate quantification, and reproducible measurements within a single experimental setup. Here, we describe and review data-independent acquisition strategies and their recent use in clinically oriented studies. In addition, we also provide a detailed guide for the implementation of SWATH-MS (where SWATH is sequential window acquisition of all theoretical mass spectra)-one of the data-independent strategies that have gained wide application of late.

Microsample analysis is highly beneficial in blood-based testing where cutting-edge bioanalytical technologies enable the analysis of volumes down to a few tens of microliters. Despite the availability of analytical methods, the difficulty in obtaining high-quality and standardized microsamples at the point of collection remains a major limitation of the process. Here, we detail and model a blood separation principle which exploits discrete viscosity differences caused by blood particle sedimentation in a laminar flow. Based on this phenomenon, we developed a portable capillary-driven microfluidic device that separates blood microsamples collected from finger-pricks and delivers 2 µL of metered serum for bench-top analysis. Flow cytometric analysis demonstrated the high purity of generated microsamples. Proteomic and metabolomic analyses of the microsamples of 283 proteins and 1351 metabolite features was consistent with samples generated via a conventional centrifugation method. These results were confirmed by a clinical study scrutinising 8 blood markers in obese patients., Scientific Reports, 8, ISSN:2045-2322

Summary Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP)-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events., Graphical Abstract, Highlights • Temporal proteomic profiling in primary cells using DIA/SWATH mass spectrometry • Identification of GRB2 interactors in primary T cells • Developing and mature T cells show distinct dynamics of GRB2 protein interactions • Broad applicability of our workflow for dissecting mammalian signaling systems, Caron et al. apply an advanced workflow that combines mouse genetic engineering, affinity purification-SWATH mass spectrometry, and open-source software platforms to accurately and reproducibly quantify high-confidence, time-resolved protein interactions in primary T cells isolated from mouse tissues.

Lung cancer, especially adenocarcinoma as the most common subtype, is still the most deadly human cancers. Therefore biomarker discovery for implementation of individualised therapies remains of utmost importance. Serine hydrolases (SHs), one of the largest enzyme families, have previously been shown to be implicated in the development of lung cancers. Activity-based protein profiling (ABPP) is a proteomic method that uses active site-directed chemical probes to selectively target the active form of the subsets of the enzymes in question, and then by a combination of a streptavidin-biotin enrichment step and mass spectrometry quantifies the catalytically active amount of the enzyme molecule. In this project, we monitored both forms of serine hydrolase, the catalytically active and inactive, in a patient cohort of lung adenocarcinoma biopsies. For this purpose, we combined the activity-based proteomics for serine hydrolase and SWATH mass spectrometry (MS), which ensures highly reproducible protein quantification in a large panel of clinical samples. Twenty four lung biopsies of long- and short surviving patients with stage IIIA adenocarcinomas and their normal tissue counterparts were available as OCT-embedded tissue. Although OCT represents a non-reactive compound used to preserve tissue morphology in long-term sample storage, this substance is incompatible with mass spectrometry measurements. We optimized and developed an OCT clean-up protocol compatible with enzyme-substrate binding of activity-based chemical probes and the targeted portion of the active enzyme. To prevent digestion on streptavidin beads and MS spectra contamination, we used the reproducible and accurate SWATH MS to indirectly measure the active enzyme form in the biopsy-extract solution which had earlier been depleted for all active enzyme molecules by precipitation with chemical probes attached to the beads. We identified over 4000 lung tissue proteins from a few milligrams of OCT-embedded biopsies, and confirmed good data quality for further SH enzyme quantification. In addition to the analysis of total proteome, 278 distinct proteins were identified on the parallel streptavidin bead samples, with chemical probes being used to deplete the active enzyme from the biopsy-extract. Each SWATH experiment reported the percentage of active enzyme form through the indirectly measured ratio between the inactive SHs (sample depleted for active SHs) and the total SHs (non-depleted sample). We detected around 80 enzymes with the active form comparably measured in all three independent experiments, which amount generally accounted for between 5-55% of the total enzyme concentration. We conclude that with the modified protocol compatible with OCT-embedded tissue biopsies the combination of ABPP and SWATH-MS enables highly reproducible protein quantification in biomarker discovery. Citation Format: Tatjana Sajic, Stephan Arni, Walter Weder, Rudolf Aebersold, Sven Hillinger. Modified SWATH MS analysis of serine hydrolase activity in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4934.

OBJECTIVE:NADPH oxidase 4 (NOX4) is a reactive oxygen species (ROS) producing NADPH oxidase that regulates redox homeostasis in diverse insulin sensitive cell types. In particular NOX4 derived ROS is a key modulator of adipocyte differentiation and mediates insulin receptor signaling in mature adipocytes in vitro. Our study was aimed at investigating the role of NOX4 in adipose tissue differentiation whole body metabolic homeostasis and insulin sensitivity in vivo.DESIGN:Mice with genetic ablation of NOX4 (NOX4 deficient mice) were subjected to chow or high fat containing diet for 12 weeks. Body weight gain adiposity insulin sensitivity and adipose tissue and liver gene and protein expression were analyzed and compared with similarly treated wild type mice.RESULTS:Here we report that NOX4 deficient mice display latent adipose tissue accumulation and are susceptible to diet induced obesity and early onset insulin resistance. Obesity results from accelerated adipocyte differentiation and hypertrophy and an increase in whole body energy efficiency. Insulin resistance is associated with increased adipose tissue hypoxia inflammation and adipocyte apoptosis. In the liver more severe diet induced steatosis was observed due to the lack of proper upregulation of mitochondrial fatty acid ß oxidation.CONCLUSION:These findings identify NOX4 as a regulator of metabolic homeostasis. Moreover they indicate an anti adipogenic role for NOX4 in vivo and reveal its function as a protector against the development of diet induced obesity insulin resistance and hepatosteatosis.International Journal of Obesity advance online publication 20 March 2012; doi:10.1038/ijo.2011.279.

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) analyses. First, the salt gradient (using K(+) as displacing agent) was evaluated from 25 to 500mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC-MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.

A comparative study of three detergent-free protein extraction protocols--a differential centrifugal fractionation, a delipidation protocol based on the Bligh and Dyer method, and the trifluoroethanol addition as cosolvent to an aqueous buffer--was performed on white adipose tissue. The performance of the protocols directly compatible with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was evaluated based on the total protein extraction yield and the protein recovery from different functional and cellular compartments. The most suitable method for the extraction of white adipose tissue proteins from a wide range of cellular and structural compartments was the delipidation protocol based on the Bligh and Dyer method.

STAT6 interacts with PPARγ to elicit macrophage polarization towards an anti-inflammatory, insulin-sensitizing phenotype. Mice deficient in STAT6 display liver lipid accumulation (hepatosteatosis). Rosiglitazone (RSG), a PPARγ agonist, ameliorates hepatosteatosis and enhances insulin sensitivity. To elucidate the role of STAT6 in PPARγ action on hepatosteatosis we compared liver proteomes of RSG-treated wild type and STAT6-deficient mice and we identified pyruvate kinase M2 (PKM2), a glycolysis and proliferation-regulating enzyme that displayed STAT6-dependent expression. RSG induced PKM2 within inflammatory cells in liver but suppressed its expression in adipose tissue. RSG diminished hepatosteatosis and oxidative stress, enhanced fat accumulation and improved insulin sensitivity in STAT6-deficient mice. Our data reveal a complex interaction between STAT6 and PPARγ in the regulation of liver and adipose tissue lipid depot distribution and design STAT6 as a novel link between inflammatory cell metabolism and adipocyte and hepatocyte function.

Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis.