Arjen Koppen

Natural killer T cells in adipose tissue prevent insulin resistance

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.

Peroxisome Proliferator-Activated Receptor β/δ (PPARβ/δ) but Not PPARα Serves as a Plasma Free Fatty Acid Sensor in Liver

ABSTRACT Peroxisome proliferator-activated receptor α (PPARα) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARα target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARα, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARβ/δ during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARα. Binding of PPARβ/δ to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARβ/δ by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARβ/δ. In contrast, the data did not support activation of PPARα by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARβ/δ target genes and show that upregulation of gene expression by PPARβ/δ is sensitive to plasma FFA levels. In contrast, this is not the case for PPARα, revealing a novel mechanism for functional differentiation between PPARs.

Effect of Synthetic Dietary Triglycerides: A Novel Research Paradigm for Nutrigenomics

Background The effect of dietary fats on human health and disease are likely mediated by changes in gene expression. Several transcription factors have been shown to respond to fatty acids, including SREBP-1c, NF-κB, RXRs, LXRs, FXR, HNF4α, and PPARs. However, it is unclear to what extent these transcription factors play a role in gene regulation by dietary fatty acids in vivo. Methodology/Principal Findings Here, we take advantage of a unique experimental design using synthetic triglycerides composed of one single fatty acid in combination with gene expression profiling to examine the effects of various individual dietary fatty acids on hepatic gene expression in mice. We observed that the number of significantly changed genes and the fold-induction of genes increased with increasing fatty acid chain length and degree of unsaturation. Importantly, almost every single gene regulated by dietary unsaturated fatty acids remained unaltered in mice lacking PPARα. In addition, the majority of genes regulated by unsaturated fatty acids, especially docosahexaenoic acid, were also regulated by the specific PPARα agonist WY14643. Excellent agreement was found between the effects of unsaturated fatty acids on mouse liver versus cultured rat hepatoma cells. Interestingly, using Nuclear Receptor PamChip® Arrays, fatty acid- and WY14643-induced interactions between PPARα and coregulators were found to be highly similar, although several PPARα-coactivator interactions specific for WY14643 were identified. Conclusions/Significance We conclude that the effects of dietary unsaturated fatty acids on hepatic gene expression are almost entirely mediated by PPARα and mimic those of synthetic PPARα agonists in terms of regulation of target genes and molecular mechanism. Use of synthetic dietary triglycerides may provide a novel paradigm for nutrigenomics research.

Brown vs white adipocytes: The PPARγ coregulator story

The development of adipose tissue is a process which involves the concerted cooperation of numerous transcription factors together with their coactivators and corepressors. The peroxisome proliferator‐activated receptor γ (PPARγ) is considered to be one of the master regulators of adipocyte differentiation. The presence of two functionally distinct types of adipose tissue, white and brown (WAT and BAT), requires an even more complex regulation of adipose tissue development. In this review we will focus on the role of PPARγ coregulators in adipogenesis and especially on the role of PPARγ coregulators in white and brown adipose tissue. Specificity in coregulator function in WAT and BAT may form an additional level of regulation of adipose tissue development.

Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ

ABSTRACT Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.

Nuclear Receptor-Coregulator Interaction Profiling Identifies TRIP3 as a Novel Peroxisome Proliferator-activated Receptor γ Cofactor

Nuclear receptors (NRs) are major targets for drug discovery and have key roles in development and homeostasis as well as in many diseases such as obesity, diabetes, and cancer. NRs are ligand-dependent transcription factors that need to work in concert with so-called transcriptional coregulators, including corepressors and coactivators, to regulate transcription. Upon ligand binding, NRs undergo a conformational change, which alters their binding preference for coregulators. Short α-helical sequences in the coregulator proteins, LXXLL (in coactivators) or LXXXIXXXL (in corepressors), are essential for the NR-coregulator interactions. However, little is known on how specificity is dictated. To obtain a comprehensive overview of NR-coregulator interactions, we used a microarray approach based on interactions between NRs and peptides derived from known coregulators. Using the peroxisome proliferator-activated receptor γ (PPARγ) as a model NR, we were able to generate ligand-specific interaction profiles (agonist rosiglitazone versus antagonist GW9662 versus selective PPARγ modulator telmisartan) and characterize NR mutants and isotypes (PPARα, -β/δ, and -γ). Importantly, based on the NR-coregulator interaction profile, we were able to identify TRIP3 as a novel regulator of PPARγ-mediated adipocyte differentiation. These findings indicate that NR-coregulator interaction profiling may be a useful tool for drug development and biological discovery.

Epidermis-Type Lipoxygenase 3 Regulates Adipocyte Differentiation and Peroxisome Proliferator-Activated Receptor γ Activity

ABSTRACT The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPARγ, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differentiation has remained enigmatic. Previously, we showed that lipoxygenase (LOX) activity is involved in activation of PPARγ during the early stages of adipocyte differentiation. Of the seven known murine LOXs, only the unconventional LOX epidermis-type lipoxygenase 3 (eLOX3) is expressed in 3T3-L1 preadipocytes. Here, we show that forced expression of eLOX3 or addition of eLOX3 products stimulated adipogenesis under conditions that normally require an exogenous PPARγ ligand for differentiation. Hepoxilins, a group of oxidized arachidonic acid derivatives produced by eLOX3, bound to and activated PPARγ. Production of hepoxilins was increased transiently during the initial stages of adipogenesis. Furthermore, small interfering RNA-mediated or retroviral short hairpin RNA-mediated knockdown of eLOX3 expression abolished differentiation of 3T3-L1 preadipocytes. Finally, we demonstrate that xanthine oxidoreductase (XOR) and eLOX3 synergistically enhanced PPARγ-mediated transactivation. Collectively, our results indicate that hepoxilins produced by the concerted action of XOR and eLOX3 may function as PPARγ activators capable of promoting the early PPARγ-dependent steps in the conversion of preadipocytes into adipocytes.

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

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.

The serine/threonine phosphatase PPM1B (PP2Cβ) selectively modulates PPARγ activity.

Reversible phosphorylation is a widespread molecular mechanism to regulate the function of cellular proteins, including transcription factors. Phosphorylation of the nuclear receptor PPARγ (peroxisome-proliferator-activated receptor γ) at two conserved serine residue (Ser(112) and Ser(273)) results in an altered transcriptional activity of this transcription factor. So far, only a very limited number of cellular enzymatic activities has been described which can dephosphorylate nuclear receptors. In the present study we used immunoprecipitation assays coupled to tandem MS analysis to identify novel PPARγ-regulating proteins. We identified the serine/threonine phosphatase PPM1B [PP (protein phosphatase), Mg(2+)/Mn(2+) dependent, 1B; also known as PP2Cβ] as a novel PPARγ-interacting protein. Endogenous PPM1B protein is localized in the nucleus of mature 3T3-L1 adipocytes where it can bind to PPARγ. Furthermore we show that PPM1B can directly dephosphorylate PPARγ, both in intact cells and in vitro. In addition PPM1B increases PPARγ-mediated transcription via dephosphorylation of Ser(112). Finally, we show that knockdown of PPM1B in 3T3-L1 adipocytes blunts the expression of some PPARγ target genes while leaving others unaltered. These findings qualify the phosphatase PPM1B as a novel selective modulator of PPARγ activity.

A novel RNAi lethality rescue screen to identify regulators of adipogenesis.

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an atractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting.