Publications by authors named "Eric Kalkhoven"

66 Publications

Cytokine Output of Adipocyte-iNKT Cell Interplay Is Skewed by a Lipid-Rich Microenvironment.

Front Endocrinol (Lausanne) 2020 31;11:479. Epub 2020 Jul 31.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.

The complex direct and indirect interplay between adipocytes and various adipose tissue (AT)-resident immune cells plays an important role in maintaining local and whole-body insulin sensitivity. Adipocytes can directly interact with and activate AT-resident invariant natural killer T (iNKT) cells through CD1d-dependent presentation of lipid antigens, which is associated with anti-inflammatory cytokine production in lean AT (IL-4, IL-10). Whether alterations in the microenvironment, i.e., increased free fatty acids concentrations or altered cytokine/adipokine profiles as observed in obesity, directly affect adipocyte-iNKT cell communication and subsequent cytokine output is currently unknown. Here we show that the cytokine output of adipocyte-iNKT cell interplay is skewed by a lipid-rich microenvironment. Incubation of mature 3T3-L1 adipocytes with a mixture of saturated and unsaturated fatty acids specifically reduced insulin sensitivity and increased lipolysis. Reduced activation of the CD1d-invariant T-Cell Receptor (TCR) signaling axis was observed in Jurkat reporter cells expressing the invariant NKT TCR, while co-culture assays with a iNKT hybridoma cell line (DN32.D3) skewed the cytokine output toward reduced IL-4 secretion and increased IFNγ secretion. Importantly, co-culture assays of mature 3T3-L1 adipocytes with primary iNKT cells isolated from visceral AT showed a similar shift in cytokine output. Collectively, these data indicate that iNKT cells display considerable plasticity with respect to their cytokine output, which can be skewed toward a more pro-inflammatory profile by microenvironmental factors like fatty acids.
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http://dx.doi.org/10.3389/fendo.2020.00479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7412741PMC
July 2020

FXR Isoforms Control Different Metabolic Functions in Liver Cells via Binding to Specific DNA Motifs.

Gastroenterology 2020 Nov 23;159(5):1853-1865.e10. Epub 2020 Jul 23.

Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands. Electronic address:

Background & Aims: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR.

Methods: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus.

Results: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif.

Conclusions: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.
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http://dx.doi.org/10.1053/j.gastro.2020.07.036DOI Listing
November 2020

Adipocytes harbor a glucosylceramide biosynthesis pathway involved in iNKT cell activation.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 08 30;1864(8):1157-1167. Epub 2019 Apr 30.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, the Netherlands. Electronic address:

Background: Natural killer T (NKT) cells in adipose tissue (AT) contribute to whole body energy homeostasis.

Results: Inhibition of the glucosylceramide synthesis in adipocytes impairs iNKT cell activity.

Conclusion: Glucosylceramide biosynthesis pathway is important for endogenous lipid antigen activation of iNKT cells in adipocytes.

Significance: Unraveling adipocyte-iNKT cell communication may help to fight obesity-induced AT dysfunction. Overproduction and/or accumulation of ceramide and ceramide metabolites, including glucosylceramides, can lead to insulin resistance. However, glucosylceramides also fulfill important physiological functions. They are presented by antigen presenting cells (APC) as endogenous lipid antigens via CD1d to activate a unique lymphocyte subspecies, the CD1d-restricted invariant (i) natural killer T (NKT) cells. Recently, adipocytes have emerged as lipid APC that can activate adipose tissue-resident iNKT cells and thereby contribute to whole body energy homeostasis. Here we investigate the role of the glucosylceramide biosynthesis pathway in the activation of iNKT cells by adipocytes. UDP-glucose ceramide glucosyltransferase (Ugcg), the first rate limiting step in the glucosylceramide biosynthesis pathway, was inhibited via chemical compounds and shRNA knockdown in vivo and in vitro. β-1,4-Galactosyltransferase (B4Galt) 5 and 6, enzymes that convert glucosylceramides into potentially inactive lactosylceramides, were subjected to shRNA knock down. Subsequently, (pre)adipocyte cell lines were tested in co-culture experiments with iNKT cells (IFNγ and IL4 secretion). Inhibition of Ugcg activity shows that it regulates presentation of a considerable fraction of lipid self-antigens in adipocytes. Furthermore, reduced expression levels of either B4Galt5 or -6, indicate that B4Galt5 is dominant in the production of cellular lactosylceramides, but that inhibition of either enzyme results in increased iNKT cell activation. Additionally, in vivo inhibition of Ugcg by the aminosugar AMP-DNM results in decreased iNKT cell effector function in adipose tissue. Inhibition of endogenous glucosylceramide production results in decreased iNKT cells activity and cytokine production, underscoring the role of this biosynthetic pathway in lipid self-antigen presentation by adipocytes.
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http://dx.doi.org/10.1016/j.bbalip.2019.04.016DOI Listing
August 2019

Natural helix 9 mutants of PPARγ differently affect its transcriptional activity.

Mol Metab 2019 02 16;20:115-127. Epub 2018 Dec 16.

Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands; Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands. Electronic address:

Objective: The nuclear receptor PPARγ is the master regulator of adipocyte differentiation, distribution, and function. In addition, PPARγ induces terminal differentiation of several epithelial cell lineages, including colon epithelia. Loss-of-function mutations in PPARG result in familial partial lipodystrophy subtype 3 (FPDL3), a rare condition characterized by aberrant adipose tissue distribution and severe metabolic complications, including diabetes. Mutations in PPARG have also been reported in sporadic colorectal cancers, but the significance of these mutations is unclear. Studying these natural PPARG mutations provides valuable insights into structure-function relationships in the PPARγ protein. We functionally characterized a novel FPLD3-associated PPARγ L451P mutation in helix 9 of the ligand binding domain (LBD). Interestingly, substitution of the adjacent amino acid K450 was previously reported in a human colon carcinoma cell line.

Methods: We performed a detailed side-by-side functional comparison of these two PPARγ mutants.

Results: PPARγ L451P shows multiple intermolecular defects, including impaired cofactor binding and reduced RXRα heterodimerisation and subsequent DNA binding, but not in DBD-LBD interdomain communication. The K450Q mutant displays none of these functional defects. Other colon cancer-associated PPARγ mutants displayed diverse phenotypes, ranging from complete loss of activity to wildtype activity.

Conclusions: Amino acid changes in helix 9 can differently affect LBD integrity and function. In addition, FPLD3-associated PPARγ mutations consistently cause intra- and/or intermolecular defects; colon cancer-associated PPARγ mutations on the other hand may play a role in colon cancer onset and progression, but this is not due to their effects on the most well-studied functional characteristics of PPARγ.
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http://dx.doi.org/10.1016/j.molmet.2018.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358588PMC
February 2019

A Single Complex Allele in a Patient With Partial Lipodystrophy.

Front Physiol 2018 26;9:1363. Epub 2018 Sep 26.

Center for Molecular Medicine, University Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands.

Genetic lipodystrophies are a group of rare syndromes associated with major metabolic complications - including severe insulin resistance, type 2 diabetes mellitus, and hypertriglyceridemia - which are classified according to the distribution of adipose tissue. Lipodystrophies can be present at birth or develop during life and can range from local to partial and general. With at least 18 different genes implicated so far, definite diagnosis can be challenging due to clinical and genetic heterogeneity. In an adult female patient with clinical and metabolic features of partial lipodystrophy we identified via whole genome sequencing (WGS) a single complex allele [V67M;V167A], functionally equivalent to heterozygosity. encodes for an acyltransferase implicated in the biosynthesis of triacylglycerol and glycerophospholipids. So far homozygous and compound heterozygous mutations in have only been associated with generalized lipodystrophy. A SNP risk score analysis indicated that the index patient is not predisposed to lipodystrophy based on her genetic background. The partial phenotype in our patient is therefore more likely associated to the genetic variants in To test whether the resulting double-mutant AGPAT2 protein is functional we analyzed its enzymatic activity via mass spectrometry. The resulting AGPAT2 double mutant is enzymatically inactive. Our data support the view that the current classification of lipodystrophies as strictly local, partial or generalized may have to be re-evaluated and viewed more as a continuum, both in terms of clinical presentation and underlying genetic causes. Better molecular understanding of lipodystrophies may lead to new therapies to treat adipose tissue dysfunction in common and rare diseases.
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http://dx.doi.org/10.3389/fphys.2018.01363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168662PMC
September 2018

Nuclear Receptor Nur77 Limits the Macrophage Inflammatory Response through Transcriptional Reprogramming of Mitochondrial Metabolism.

Cell Rep 2018 08;24(8):2127-2140.e7

Amsterdam UMC, University of Amsterdam, Medical Biochemistry, Amsterdam Cardiovascular Sciences, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands. Electronic address:

Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.
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http://dx.doi.org/10.1016/j.celrep.2018.07.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113932PMC
August 2018

Immunometabolic Activation of Invariant Natural Killer T Cells.

Front Immunol 2018 28;9:1192. Epub 2018 May 28.

Laboratory for Translational Immunology, University Medical Center Utrecht, Utrecht, Netherlands.

Invariant natural killer T (iNKT) cells are lipid-reactive T cells with profound immunomodulatory potential. They are unique in their restriction to lipid antigens presented in CD1d molecules, which underlies their role in lipid-driven disorders such as obesity and atherosclerosis. In this review, we discuss the contribution of iNKT cell activation to immunometabolic disease, metabolic programming of lipid antigen presentation, and immunometabolic activation of iNKT cells. First, we outline the role of iNKT cells in immunometabolic disease. Second, we discuss the effects of cellular metabolism on lipid antigen processing and presentation to iNKT cells. The synthesis and processing of glycolipids and other potential endogenous lipid antigens depends on metabolic demand and may steer iNKT cells toward adopting a Th1 or Th2 signature. Third, external signals such as toll-like receptor ligands, adipokines, and cytokines modulate antigen presentation and subsequent iNKT cell responses. Finally, we will discuss the relevance of metabolic programming of iNKT cells in human disease, focusing on their role in disorders such as obesity and atherosclerosis. The critical response to metabolic changes places iNKT cells at the helm of immunometabolic disease.
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http://dx.doi.org/10.3389/fimmu.2018.01192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985373PMC
August 2019

Profiling of 3696 Nuclear Receptor-Coregulator Interactions: A Resource for Biological and Clinical Discovery.

Endocrinology 2018 06;159(6):2397-2407

Molecular Cancer Research and Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, CG Utrecht, Netherlands.

Nuclear receptors (NRs) are ligand-inducible transcription factors that play critical roles in metazoan development, reproduction, and physiology and therefore are implicated in a broad range of pathologies. The transcriptional activity of NRs critically depends on their interaction(s) with transcriptional coregulator proteins, including coactivators and corepressors. Short leucine-rich peptide motifs in these proteins (LxxLL in coactivators and LxxxIxxxL in corepressors) are essential and sufficient for NR binding. With 350 different coregulator proteins identified to date and with many coregulators containing multiple interaction motifs, an enormous combinatorial potential is present for selective NR-mediated gene regulation. However, NR-coregulator interactions have often been determined experimentally on a one-to-one basis across diverse experimental conditions. In addition, NR-coregulator interactions are difficult to predict because the molecular determinants that govern specificity are not well established. Therefore, many biologically and clinically relevant NR-coregulator interactions may remain to be discovered. Here, we present a comprehensive overview of 3696 NR-coregulator interactions by systematically characterizing the binding of 24 nuclear receptors with 154 coregulator peptides. We identified unique ligand-dependent NR-coregulator interaction profiles for each NR, confirming many well-established NR-coregulator interactions. Hierarchical clustering based on the NR-coregulator interaction profiles largely recapitulates the classification of NR subfamilies based on the primary amino acid sequences of the ligand-binding domains, indicating that amino acid sequence is an important, although not the only, molecular determinant in directing and fine-tuning NR-coregulator interactions. This NR-coregulator peptide interactome provides an open data resource for future biological and clinical discovery as well as NR-based drug design.
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http://dx.doi.org/10.1210/en.2018-00149DOI Listing
June 2018

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children.

PLoS One 2017 26;12(10):e0187068. Epub 2017 Oct 26.

Laboratory for Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children.

Methods: 13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed.

Results: Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction.

Conclusions: First, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187068PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658151PMC
November 2017

Endogenous lipid antigens for invariant natural killer T cells hold the reins in adipose tissue homeostasis.

Immunology 2018 02 26;153(2):179-189. Epub 2017 Oct 26.

Department of Molecular Cancer Research and Centre for Molecular Medicine, University Medical Centre Utrecht, Utrecht University, Utrecht, The Netherlands.

The global obesity epidemic and its associated co-morbidities, including type 2 diabetes, cardiovascular disease and certain types of cancers, have drawn attention to the pivotal role of adipocytes in health and disease. Besides their 'classical' function in energy storage and release, adipocytes interact with adipose-tissue-resident immune cells, among which are lipid-responsive invariant natural killer T (iNKT) cells. The iNKT cells are activated by lipid antigens presented by antigen-presenting cells as CD1d/lipid complexes. Upon activation, iNKT cells can rapidly secrete soluble mediators that either promote or oppose inflammation. In lean adipose tissue, iNKT cells elicit a predominantly anti-inflammatory immune response, whereas obesity is associated with declining iNKT cell numbers. Recent work showed that adipocytes act as non-professional antigen-presenting cells for lipid antigens. Here, we discuss endogenous lipid antigen processing and presentation by adipocytes, and speculate on how these lipid antigens, together with 'environmental factors' such as tissue/organ environment and co-stimulatory signals, are able to influence the fate of adipose-tissue-resident iNKT cells, and thereby the role of these cells in obesity and its associated pathologies.
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http://dx.doi.org/10.1111/imm.12839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765379PMC
February 2018

Hypoxia-Inducible Lipid Droplet-Associated Is Not a Direct Physiological Regulator of Lipolysis in Adipose Tissue.

Endocrinology 2017 05;158(5):1231-1251

Nutrition, Metabolism, and Genomics Group, Division of Human Nutrition, Wageningen University, 6708 WE Wageningen, The Netherlands.

Triglycerides are stored in specialized organelles called lipid droplets. Numerous proteins have been shown to be physically associated with lipid droplets and govern their function. Previously, the protein hypoxia-inducible lipid droplet-associated (HILPDA) was localized to lipid droplets and was suggested to inhibit triglyceride lipolysis in hepatocytes. We confirm the partial localization of HILPDA to lipid droplets and show that HILPDA is highly abundant in adipose tissue, where its expression is controlled by the peroxisome proliferator-activated receptor γ and by β-adrenergic stimulation. Levels of HILPDA markedly increased during 3T3-L1 adipocyte differentiation. Nevertheless, silencing of Hilpda using small interfering RNA or overexpression of Hilpda using adenovirus did not show a clear impact on 3T3-L1 adipogenesis. Following β-adrenergic stimulation, the silencing of Hilpda in adipocytes did not significantly alter the release of nonesterified fatty acids (NEFA) and glycerol. By contrast, adenoviral-mediated overexpression of Hilpda modestly attenuated the release of NEFA from adipocytes following β-adrenergic stimulation. In mice, adipocyte-specific inactivation of Hilpda had no effect on plasma levels of NEFA and glycerol after fasting, cold exposure, or pharmacological β-adrenergic stimulation. In addition, other relevant metabolic parameters were unchanged by adipocyte-specific inactivation of Hilpda. Taken together, we find that HILPDA is highly abundant in adipose tissue, where its levels are induced by peroxisome proliferator-activated receptor γ and β-adrenergic stimulation. In contrast to the reported inhibition of lipolysis by HILPDA in hepatocytes, our data do not support an important direct role of HILPDA in the regulation of lipolysis in adipocytes in vivo and in vitro.
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http://dx.doi.org/10.1210/en.2016-1809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5460841PMC
May 2017

Prospective functional classification of all possible missense variants in PPARG.

Nat Genet 2016 12 17;48(12):1570-1575. Epub 2016 Oct 17.

Program in Medical & Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions. We developed a pooled functional assay in human macrophages, experimentally evaluated all protein variants, and used the experimental data to train a variant classifier by supervised machine learning. When applied to 55 new missense variants identified in population-based and clinical sequencing, the classifier annotated 6 variants as pathogenic; these were subsequently validated by single-variant assays. Saturation mutagenesis and prospective experimental characterization can support immediate diagnostic interpretation of newly discovered missense variants in disease-related genes.
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http://dx.doi.org/10.1038/ng.3700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131844PMC
December 2016

Muscle-specific inflammation induced by MCP-1 overexpression does not affect whole-body insulin sensitivity in mice.

Diabetologia 2016 Mar 12;59(3):624-33. Epub 2015 Dec 12.

Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, the Netherlands.

Aims/hypothesis: Obesity is associated with a state of chronic low-grade inflammation that is believed to contribute to the development of skeletal muscle insulin resistance. However, the extent to which local and systemic elevation of cytokines, such as monocyte chemoattractant protein 1 (MCP-1), interferes with the action of insulin and promotes insulin resistance and glucose intolerance in muscle remains unclear. Here, we aim to investigate the effect of muscle-specific overexpression of MCP-1 on insulin sensitivity and glucose tolerance in lean and obese mice.

Methods: We used Mck-Mcp-1 transgenic (Tg) mice characterised by muscle-specific overexpression of Mcp-1 (also known as Ccl2) and elevated plasma MCP-1 levels. Mice were fed either chow or high-fat diet for 10 weeks. Numerous metabolic variables were measured, including glucose and insulin tolerance tests, muscle insulin signalling and plasma NEFA, triacylglycerol, cholesterol, glucose and insulin.

Results: Despite clearly promoting skeletal muscle inflammation, muscle-specific overexpression of Mcp-1 did not influence glucose tolerance or insulin sensitivity in either lean chow-fed or diet-induced obese mice. In addition, plasma NEFA, triacylglycerol, cholesterol, glucose and insulin were not affected by MCP-1 overexpression. Finally, in vivo insulin-induced Akt phosphorylation in skeletal muscle did not differ between Mcp-1-Tg and wild-type mice.

Conclusions/interpretation: We show that increased MCP-1 production in skeletal muscle and concomitant elevated MCP-1 levels in plasma promote inflammation in skeletal muscle but do not influence insulin signalling and have no effect on insulin resistance and glucose tolerance in lean and obese mice. Overall, our data argue against MCP-1 promoting insulin resistance in skeletal muscle and raise questions about the impact of inflammation on insulin sensitivity in muscle.
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http://dx.doi.org/10.1007/s00125-015-3822-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742493PMC
March 2016

Electric Pulse Stimulation of Myotubes as an In Vitro Exercise Model: Cell-Mediated and Non-Cell-Mediated Effects.

Sci Rep 2015 Jun 19;5:10944. Epub 2015 Jun 19.

Molecular Cancer Research and Center for Molecular Medicine, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.

Regular exercise has emerged as one of the best therapeutic strategies to prevent and treat type-2-diabetes. Exercise-induced changes in the muscle secretome, consisting of myokines and metabolites, may underlie the inter-organ communication between muscle and other organs. To investigate this crosstalk, we developed an in vitro system in which mouse C2C12 myotubes underwent electric pulse stimulation (EPS) to induce contraction. Subsequently the effects of EPS-conditioned media (EPS-CM) on hepatocytes were investigated. Here, we demonstrate that EPS-CM induces Metallothionein 1/2 and Slc30a2 gene expression and reduces Cyp2a3 gene expression in rat hepatocytes. When testing EPS-CM that was generated in the absence of C2C12 myotubes (non-cell EPS-CM) no decrease in Cyp2a3 expression was detected. However, similar inductions in hepatic Mt1/2 and Slc30a2 expression were observed. Non-cell EPS-CM were also applied to C2C12 myotubes and compared to C2C12 myotubes that underwent EPS: here changes in AMPK phosphorylation and myokine secretion largely depended on EPS-induced contraction. Taken together, these findings indicate that EPS can alter C2C12 myotube function and thereby affect gene expression in cells subjected to EPS-CM (Cyp2a3). However, EPS can also generate non-cell-mediated changes in cell culture media, which can affect gene expression in cells subjected to EPS-CM too. While EPS clearly represents a valuable tool in exercise research, care should be taken in experimental design to control for non-cell-mediated effects.
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http://dx.doi.org/10.1038/srep10944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473537PMC
June 2015

Targeting FOXO1 as an option to treat obesity?

Cell Cycle 2015 11;14(16):2558. Epub 2015 Jun 11.

a Molecular Cancer Research; Center for Molecular Medicine; University Medical Center Utrecht ; Utrecht , The Netherlands.

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http://dx.doi.org/10.1080/15384101.2015.1060779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613142PMC
October 2015

Inflammatory characteristics of distinct abdominal adipose tissue depots relate differently to metabolic risk factors for cardiovascular disease: distinct fat depots and vascular risk factors.

Atherosclerosis 2015 Apr 2;239(2):419-27. Epub 2015 Feb 2.

Department of Vascular Medicine, University Medical Center Utrecht (UMC Utrecht), Utrecht, The Netherlands. Electronic address:

Objective: Abdominal obesity is associated with insulin resistance and metabolic syndrome. However, specific contributions of distinct adipose tissue (AT) depots to metabolic complications of obesity are still unclear. In this study, the inflammatory profile of four distinct abdominal AT-depots and the relation between AT-characteristics and obesity-induced metabolic complications was evaluated.

Methods: In 28 men undergoing abdominal aortic surgery, biopsies were collected from subcutaneous fat (SAT), and 3 visceral AT-depots: mesenteric (MAT), omental (OAT) and periaortic (PAT). The AT biopsies were characterized morphologically (adipocyte size, capillary density, CD68 + macrophages and crown-like-structures (CLS)) and the ex vivo adipokine secretion profile was determined by multiplex-immunoassay. The relation between depot-specific inflammatory characteristics and clinical parameters (waist circumference, insulin resistance and metabolic syndrome) was assessed by multivariable linear regression analysis.

Results: PAT contained the smallest adipocytes, highest capillary density and secreted abundant amounts of adipokines. SAT contained the lowest amount of macrophages and adipokines, while MAT and OAT displayed a similar inflammatory profile. In contrast to the other depots, MAT inflammation was most strongly related to metabolic complications of obesity, as adipocyte size and CLS were related to insulin resistance (β2.0; 95%CI1.15-2.85 and β0.24; 95%CI0.06-0.43) and MAT adipocyte size was associated with 79% higher odds of having metabolic syndrome (OR1.79; 95% CI1.13-2.89).

Conclusions: There are significant differences in the inflammatory profile of distinct abdominal fat depots, of which MAT characteristics were mostly associated with metabolic complications of obesity. These findings suggest a differential contribution of AT-depots to systemic metabolic dysfunction which precedes type 2 diabetes and vascular diseases.
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http://dx.doi.org/10.1016/j.atherosclerosis.2015.01.035DOI Listing
April 2015

Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells.

Obesity (Silver Spring) 2014 Oct 17;22(10):2216-23. Epub 2014 Jul 17.

Department of Vascular Medicine, University Medical Center Utrecht (UMC Utrecht), Utrecht, The Netherlands; Molecular Cancer Research, Center for Molecular Medicine, UMC Utrecht, Utrecht, The Netherlands.

Objective: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined.

Methods: EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry.

Results: In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r = -0.60, P = 0.01) and OAT-EVs (r = -0.74, P = 0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction.

Conclusions: Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR.
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http://dx.doi.org/10.1002/oby.20847DOI Listing
October 2014

Paneth cell extrusion and release of antimicrobial products is directly controlled by immune cell-derived IFN-γ.

J Exp Med 2014 Jun;211(7):1393-405

Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht, 3584 CT Utrecht, Netherlands

Paneth cells (PCs) are terminally differentiated, highly specialized secretory cells located at the base of the crypts of Lieberkühn in the small intestine. Besides their antimicrobial function, PCs serve as a component of the intestinal stem cell niche. By secreting granules containing bactericidal proteins like defensins/cryptdins and lysozyme, PCs regulate the microbiome of the gut. Here we study the control of PC degranulation in primary epithelial organoids in culture. We show that PC degranulation does not directly occur upon stimulation with microbial antigens or bacteria. In contrast, the pro-inflammatory cytokine Interferon gamma (IFN-γ) induces rapid and complete loss of granules. Using live cell imaging, we show that degranulation is coupled to luminal extrusion and death of PCs. Transfer of supernatants from in vitro stimulated iNKT cells recapitulates degranulation in an IFN-γ-dependent manner. Furthermore, endogenous IFN-γ secretion induced by anti-CD3 antibody injection causes Paneth loss and release of goblet cell mucus. The identification of IFN-γ as a trigger for degranulation and extrusion of PCs establishes a novel effector mechanism by which immune responses may regulate epithelial status and the gut microbiome.
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http://dx.doi.org/10.1084/jem.20130753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076587PMC
June 2014

CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein.

J Biol Chem 2014 Aug 25;289(32):22128-39. Epub 2014 Jun 25.

From the Molecular Cancer Research, Center for Molecular Medicine and

Obesity-induced adipose tissue (AT) dysfunction results in a chronic low-grade inflammation that predisposes to the development of insulin resistance and type 2 diabetes. During the development of obesity, the AT-resident immune cell profile alters to create a pro-inflammatory state. Very recently, CD1d-restricted invariant (i) natural killer T (NKT) cells, a unique subset of lymphocytes that are reactive to so called lipid antigens, were implicated in AT homeostasis. Interestingly, recent data also suggest that human and mouse adipocytes can present such lipid antigens to iNKT cells in a CD1d-dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are up-regulated in early adipogenesis, and are transcriptionally controlled by CCAAT/enhancer-binding protein (C/EBP)-β and -δ. Moreover, adipocyte-induced Th1 and Th2 cytokine release by iNKT cells also occurred in the absence of exogenous ligands, suggesting the display of endogenous lipid antigen-D1d complexes by 3T3-L1 adipocytes. Furthermore, we identified microsomal triglyceride transfer protein, which we show is also under the transcriptional regulation of C/EBPβ and -δ, as a novel player in the presentation of endogenous lipid antigens by adipocytes. Overall, our findings indicate that adipocytes can function as non-professional lipid antigen presenting cells, which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis.
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http://dx.doi.org/10.1074/jbc.M114.551242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139226PMC
August 2014

Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

PLoS One 2014 28;9(5):e98343. Epub 2014 May 28.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Centre Utrecht, Utrecht, The Netherlands; Netherlands Metabolomics Center, Leiden, The Netherlands.

Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098343PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037199PMC
June 2015

TIPping the balance in adipogenesis: USP7-mediated stabilization of Tip60.

Adipocyte 2014 Apr 25;3(2):160-5. Epub 2014 Feb 25.

Molecular Cancer Research; Center for Molecular Medicine; University Medical Centre Utrecht; Utrecht, The Netherlands.

Adipogenesis is regulated by a complex interplay between transcription factors, in concert with-among others-transcriptional cofactors, signaling cascades and miRNAs. Several studies have implicated the transcriptional cofactor and acetyltransferase Tip60 in PPARγ signaling and adipocyte differentiation. Since Tip60 protein levels, but not mRNA levels, are upregulated during adipogenesis, and since Tip60 can be degraded by the proteasome, we hypothesized that Tip60 protein may be stabilized through deubiquitination during adipogenesis. Indeed, Tip60 is protected from proteasomal degeradation by the deubiquitinase USP7, which is particularly important for mitotic clonal expansion (MCE), an early step in adipogenesis. Besides this novel role in early differentiation, earlier studies indicated that Tip60 is also important during the later stages of differentiation, indicating a dual role for this protein in adipogenesis. Our recent study sheds new light on the role of Tip60 in cellular differentiation and provide new insights into the importance of a regulatory process that has not been studied intensively in adipogenesis: protein (de)ubiquitination.
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http://dx.doi.org/10.4161/adip.28307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979883PMC
April 2014

Fatty acid-inducible ANGPTL4 governs lipid metabolic response to exercise.

Proc Natl Acad Sci U S A 2014 Mar 3;111(11):E1043-52. Epub 2014 Mar 3.

Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6700 EV, Wageningen, The Netherlands.

Physical activity increases energy metabolism in exercising muscle. Whether acute exercise elicits metabolic changes in nonexercising muscles remains unclear. We show that one of the few genes that is more highly induced in nonexercising muscle than in exercising human muscle during acute exercise encodes angiopoietin-like 4 (ANGPTL4), an inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance. Using a combination of human, animal, and in vitro data, we show that induction of ANGPTL4 in nonexercising muscle is mediated by elevated plasma free fatty acids via peroxisome proliferator-activated receptor-δ, presumably leading to reduced local uptake of plasma triglyceride-derived fatty acids and their sparing for use by exercising muscle. In contrast, the induction of ANGPTL4 in exercising muscle likely is counteracted via AMP-activated protein kinase (AMPK)-mediated down-regulation, promoting the use of plasma triglycerides as fuel for active muscles. Our data suggest that nonexercising muscle and the local regulation of ANGPTL4 via AMPK and free fatty acids have key roles in governing lipid homeostasis during exercise.
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http://dx.doi.org/10.1073/pnas.1400889111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3964070PMC
March 2014

Identification of human exercise-induced myokines using secretome analysis.

Physiol Genomics 2014 Apr 11;46(7):256-67. Epub 2014 Feb 11.

Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands;

Endurance exercise is associated with significant improvements in cardio-metabolic risk parameters. A role for myokines has been hypothesized, yet limited information is available about myokines induced by acute endurance exercise in humans. Therefore, the aim of the study was to identify novel exercise-induced myokines in humans. To this end, we carried out a 1 h one-legged acute endurance exercise intervention in 12 male subjects and a 12 wk exercise training intervention in 18 male subjects. Muscle biopsies were taken before and after acute exercise or exercise training and were subjected to microarray-based analysis of secreted proteins (secretome). For acute exercise, secretome analysis resulted in a list of 86 putative myokines, which was reduced to 29 by applying a fold-change cut-off of 1.5. Based on that shortlist, a selection of putative myokines was measured in the plasma by ELISA or multiplex assay. From that selection, CX3CL1 (fractalkine) and CCL2 (MCP-1) increased at both mRNA and plasma levels. From the known myokines, only IL-6 and FGF 21 changed at the mRNA level, whereas none of the known myokines changed at the plasma level. Secretome analysis of exercise training intervention resulted in a list of 69 putative myokines. Comparing putative myokines altered by acute exercise and exercise training revealed a limited overlap of only 13 genes. In conclusion, this study identified CX3CL1 and CCL2 as myokines that were induced by acute exercise at the gene expression and plasma level and that may be involved in communication between skeletal muscle and other organs.
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http://dx.doi.org/10.1152/physiolgenomics.00174.2013DOI Listing
April 2014

Extracellular vesicle markers in relation to obesity and metabolic complications in patients with manifest cardiovascular disease.

Cardiovasc Diabetol 2014 Feb 5;13:37. Epub 2014 Feb 5.

Department of Vascular Medicine, University Medical Centre Utrecht (UMCU), F02,126, Heidelberglaan 100, Utrecht 3584 CX, The Netherlands.

Background: Alterations in extracellular vesicles (EVs), including exosomes and microparticles, contribute to cardiovascular disease. We hypothesized that obesity could favour enhanced release of EVs from adipose tissue, and thereby contribute to cardiovascular risk via obesity-induced metabolic complications. The objectives of this study were: 1) to investigate the relation between the quantity, distribution and (dys) function of adipose tissue and plasma concentrations of atherothrombotic EV-markers; 2) to determine the relation between these EV-markers and the prevalence of the metabolic syndrome; and 3) to assess the contribution of EV markers to the risk of incident type 2 diabetes.

Methods: In 1012 patients with clinically manifest vascular disease, subcutaneous and visceral fat thickness was measured ultrasonographically. Plasma EVs were isolated and levels of cystatin C, serpin G1, serpin F2 and CD14 were measured, as well as fasting metabolic parameters, hsCRP and adiponectin. The association between adiposity, EV-markers, and metabolic syndrome was tested by multivariable linear and logistic regression analyses. As sex influences body fat distribution, sex-stratified analyses between adipose tissue distribution and EV-markers were performed. The relation between EV-markers and type 2 diabetes was assessed with Cox regression analyses.

Results: Higher levels of hsCRP (β 5.59; 95% CI 3.00-8.18) and lower HDL-cholesterol levels (β-11.26; 95% CI -18.39 - -4.13) were related to increased EV-cystatin C levels, and EV-cystatin C levels were associated with a 57% higher odds of having the metabolic syndrome (OR 1.57; 95% CI 1.19-2.27). HDL-cholesterol levels were positively related to EV-CD14 levels (β 5.04; 95% CI 0.07-10.0), and EV-CD14 levels were associated with a relative risk reduction of 16% for development of type 2 diabetes (HR 0.84, 95% CI 0.75-0.94), during a median follow up of 6.5 years in which 42 patients developed type 2 diabetes.

Conclusions: In patients with clinically manifest vascular disease, EV-cystatin C levels were positively related, and EV-CD14 levels were negatively related to metabolic complications of obesity.
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http://dx.doi.org/10.1186/1475-2840-13-37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3918107PMC
February 2014

An oral mixed fat load is followed by a modest anti-inflammatory adipocytokine response in overweight patients with metabolic syndrome.

Lipids 2014 Mar 21;49(3):247-54. Epub 2014 Jan 21.

Department of Vascular Medicine, University Medical Center Utrecht, PO Box 85500, 3508 GA, Utrecht, The Netherlands,

We investigated the postprandial changes in plasma levels of adipocytokines in overweight patients with metabolic syndrome after an oral fat load. After an oral fat load and during a prolonged fast, blood was drawn at 0, 2, 3, 4 and 8 h for measurement of adiponectin, adipsin, cathepsin S, chemerin, hepatic growth factor, interferon-γ-inducible protein-10, leptin, macrophage chemoattractant protein-1, macrophage migration inhibitory factor, nerve growth factor, retinol binding protein-4, resistin, serum amyloid A1, tissue inhibitor of metalloproteinase-1 and thrombopoietin using a microbead-based Luminex assay. Area under the curves (AUC) were calculated and compared. Plasma adiponectin levels were higher after an oral fat load compared to fasting at t = 2 h (950 ± 513 vs. -1,881 ± 713 ng/ml) while the plasma levels for adipsin (-9 ± 5 vs. 16 ± 5 ng/ml), chemerin (-122 ± 35 vs. 13 ± 21 ng/ml), SAA-1 (-391 ± 213 vs. 522 ± 173 ng/ml) and TPO (-335 ± 144 vs. 622 ± 216 ng/ml) were lower after an oral fat load compared to fasting. The baseline corrected AUC for IP-10 was higher after fat load compared to fasting (median -116 pg h/ml; IQR -270 to 10 vs. -21 pg h/ml; IQR -136 to 418 (p = 0.047). In conclusion, in overweight male subjects with the metabolic syndrome, an oral fat load is accompanied with a modest anti-inflammatory response of adipose tissue-derived adipocytokines.
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http://dx.doi.org/10.1007/s11745-014-3877-8DOI Listing
March 2014

Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages.

Obesity (Silver Spring) 2014 May 9;22(5):1296-308. Epub 2014 Jan 9.

Department of Vascular Medicine, University Medical Center Utrecht (UMC Utrecht), Utrecht, The Netherlands; Section Metabolic Diseases, Molecular Cancer Research, UMC Utrecht, Utrecht, The Netherlands.

Objective: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined.

Methods: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes.

Results: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR.

Conclusions: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated.
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http://dx.doi.org/10.1002/oby.20679DOI Listing
May 2014

The relation between body iron stores and adipose tissue function in patients with manifest vascular disease.

Eur J Clin Invest 2013 Dec 23;43(12):1240-9. Epub 2013 Sep 23.

Department of Vascular Medicine, University Medical Center Utrecht, Utrecht, the Netherlands.

Background: We investigated whether plasma ferritin levels through the pro-inflammatory effects of free iron are associated with adipose tissue dysfunction in a relevant population of patients with manifest vascular disease who would potentially benefit the most from further aetiological insights.

Materials And Methods: In a cohort of 355 patients with vascular diseases, the association between plasma ferritin and adiponectin levels was quantified using linear regression analysis. Interleukin-6 and adiponectin levels were measured in medium from pre-adipocytes and adipocytes after incubation with increasing concentrations of Fe(III)-citrate and after co-incubation with iron chelators or radical scavengers.

Results: Increasing ferritin plasma concentrations were not related to plasma adiponectin levels in patients without (β -0·13; 95% CI -0·30 to 0·04) or with the metabolic syndrome (β -0·04; 95% CI -0·17 to 0·10). Similar results were found in patients who developed a new cardiovascular event in the follow-up period. In vitro, incubation with increasing concentrations of Fe(III)-citrate-induced inflammation in pre-adipocyte cultures as witnessed by increased IL-6 secretion at 30 μm Fe(III)-citrate vs. control (500 ± 98 pg/mL vs. 194 ± 31 pg/mL, P = 0·03). Co-incubation of pre-adipocytes with iron chelators or radical scavengers prevented this inflammatory response. Incubation of adipocytes with 30 μm Fe(III)-citrate did not influence adiponectin secretion compared with control.

Conclusions: In patients with vascular disease, there is no association between plasma ferritin and adiponectin levels. In vitro, free iron induces an inflammatory response in pre-adipocytes, but not in adipocytes. This response was blocked by co-incubation with iron chelators or radical scavengers. Adiponectin secretion by adipocytes was not influenced by free iron.
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http://dx.doi.org/10.1111/eci.12165DOI Listing
December 2013

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60.

Nat Commun 2013 ;4:2656

Section Metabolic Diseases, Molecular Cancer Research, and Netherlands Metabolomics Centre, University Medical Centre Utrecht, Utrecht 3584 CG, The Netherlands.

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.
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http://dx.doi.org/10.1038/ncomms3656DOI Listing
May 2014

Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity.

Immunity 2013 Aug;39(2):259-71

Department of Immunology, University Medical Center Utrecht, Utrecht 3584EA, The Netherlands.

Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.
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http://dx.doi.org/10.1016/j.immuni.2013.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133784PMC
August 2013

PPARγ regulates expression of carbohydrate sulfotransferase 11 (CHST11/C4ST1), a regulator of LPL cell surface binding.

PLoS One 2013 16;8(5):e64284. Epub 2013 May 16.

Department of Metabolic Diseases, University Medical Centre Utrecht, Utrecht, The Netherlands.

The transcription factor PPARγ is the key regulator of adipocyte differentiation, function and maintenance, and the cellular target of the insulin-sensitizing thiazolidinediones. Identification and functional characterization of genes regulated by PPARγ will therefore lead to a better understanding of adipocyte biology and may also contribute to the development of new anti-diabetic drugs. Here, we report carbohydrate sulfotransferase 11 (Chst11/C4st1) as a novel PPARγ target gene. Chst11 can sulphate chondroitin, a major glycosaminoglycan involved in development and disease. The Chst11 gene contains two functional intronic PPARγ binding sites, and is up-regulated at the mRNA and protein level during 3T3-L1 adipogenesis. Chst11 knockdown reduced intracellular lipid accumulation in mature adipocytes, which is due to a lowered activity of lipoprotein lipase, which may associate with the adipocyte cell surface through Chst11-mediated sulfation of chondroitin, rather than impaired adipogenesis. Besides directly inducing Lpl expression, PPARγ may therefore control lipid accumulation by elevating the levels of Chst11-mediated proteoglycan sulfation and thereby increasing the binding capacity for Lpl on the adipocyte cell surface.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064284PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3655946PMC
December 2013