Michael Schupp

Angiotensin Type 1 Receptor Blockers Induce Peroxisome Proliferator–Activated Receptor-γ Activity

Background—Angiotensin type 1 receptor (AT1R) blockers (ARB) have been shown to reduce the incidence of type 2 diabetes mellitus by an unknown molecular mechanism. The peroxisome proliferator–activated receptor-γ (PPARγ) is the central regulator of insulin and glucose metabolism improving insulin sensitivity. We investigated the regulation of PPARγ function by ARBs. Methods and Results—The ARBs irbesartan and telmisartan (10 μmol/L) potently enhanced PPARγ-dependent 3T3-L1 adipocyte differentiation associated with a significant increase in mRNA expression of the adipogenic marker gene adipose protein 2 (aP2), as measured by quantitative real-time polymerase chain reaction (irbesartan: 3.3±0.1-fold induction; telmisartan: 3.1±0.3-fold induction; both P <0.01). Telmisartan showed a more pronounced induction of aP2 expression in lower, pharmacologically relevant concentrations compared with the other ARBs. The ARB losartan enhanced aP2 expression only at high concentrations (losartan 100 μmol/L: 3.6±0.3-fold induction; P <0.01), whereas eprosartan up to 100 μmol/L had no significant effects. In transcription reporter assays, irbesartan and telmisartan (10 μmol/L) markedly induced transcriptional activity of PPARγ by 3.4±0.9-fold and 2.6±0.6-fold (P <0.05), respectively, compared with 5.2±1.1-fold stimulation by the PPARγ ligand pioglitazone (10 μmol/L). Irbesartan and telmisartan also induced PPARγ activity in an AT1R-deficient cell model (PC12W), demonstrating that these ARBs stimulate PPARγ activity independent of their AT1R blocking actions. Conclusions—The present study demonstrates that a specific subset of ARBs induces PPARγ activity, thereby promoting PPARγ-dependent differentiation in adipocytes. The activation of PPARγ demonstrates new pleiotropic actions of certain ARBs, providing a potential mechanism for their insulin-sensitizing/antidiabetic effects.

DOT1L/KMT4 Recruitment and H3K79 Methylation Are Ubiquitously Coupled with Gene Transcription in Mammalian Cells

ABSTRACT The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruitment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethylation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding transcriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation and that of H3K79 methylation in mammalian chromatin, suggesting a widespread mechanism for parallel or sequential recruitment of DOT1L and MLL to genes in their normal “on” state.

PPARγ-Activating Angiotensin Type-1 Receptor Blockers Induce Adiponectin

The adipose-specific protein adiponectin has been recently discovered to improve insulin sensitivity. Angiotensin type-1 receptor (AT1R) blockers (ARBs) reduce the incidence of type 2 diabetes mellitus by mostly unknown molecular mechanisms. To identify new antidiabetic mechanisms of ARBs, we studied the regulation of adiponectin by angiotensin II (Ang II) and different ARBs in murine 3T3-L1 adipocytes and obese Zucker rats. Adiponectin protein expression was markedly stimulated by Ang II (5 nmol/L), which was inhibited by blockade of the AT2R, and further enhanced by the ARB irbesartan. Irbesartan-mediated adiponectin upregulation started beyond the concentrations needed for AT1R blockade and was also present in the absence of Ang II, implicating an AT1R-independent mechanism of action. Recently, certain ARBs (irbesartan, telmisartan) were identified as ligands of the peroxisome proliferator-activated receptor (PPAR)&ggr;. Telmisartan also stimulated adiponectin protein expression, whereas the non-PPAR&ggr;-activating ARB eprosartan had no effect. Blockade of PPAR&ggr; activation by the PPAR&ggr; antagonist GW9662 markedly inhibited irbesartan-induced adiponectin expression. Cognate mRNA levels of adiponectin were not affected by ARBs. Kinetic studies using the protein synthesis inhibitor cycloheximide showed that irbesartan prevented the cellular depletion of adiponectin protein. Finally, administration of irbesartan to obese Zucker rats improved insulin sensitivity and attenuated adiponectin serum depletion. The present study demonstrates that AT2R activation and certain ARBs induce adiponectin in adipocytes, which was associated with an improvement of parameters of insulin sensitivity in vivo. ARB-induced adiponectin stimulation is likely to be mediated via PPAR&ggr; activation involving a post-transcriptional mechanism.

Activation of retinoic acid receptor-α favours regulatory T cell induction at the expense of IL-17-secreting T helper cell differentiation

Autoimmunity is thought to reflect an imbalance between regulatory T helper lymphocytes (Treg) and pathogenic, IL‐17‐secreting T helper (Th17) cells. Induction of both adaptive Treg and Th17 cells requires signalling from TGF‐β. We now show that, in the context of TGF‐β signalling, all‐trans retinoic acid (ATRA) leads to increased induction of CD4+ T cells expressing the Treg specification factor forkhead box protein P3 (FoxP3) and decreased frequency of cells expressing IL‐17, even in the presence of IL‐6. Using a specific agonist and antagonist, as well as retroviral over‐expression, we also provide evidence that the effects of ATRA are likely to be at least partially mediated by the nuclear retinoic acid receptor‐α (RARα). These findings indicate that signalling through a specific nuclear retinoic acid receptor can favour the decision to adopt the Treg fate at the expense of Th17 fate. Specific agonists of RARα could, therefore, be considered candidates for the treatment of autoimmunity.

Propagation of adipogenic signals through an epigenomic transition state.

The transcriptional mechanisms by which temporary exposure to developmental signals instigates adipocyte differentiation are unknown. During early adipogenesis, we find transient enrichment of the glucocorticoid receptor (GR), CCAAT/enhancer-binding protein beta (CEBPbeta), p300, mediator subunit 1, and histone H3 acetylation near genes involved in cell proliferation, development, and differentiation, including the gene encoding the master regulator of adipocyte differentiation, peroxisome proliferator-activated receptor gamma2 (PPARgamma2). Occupancy and enhancer function are triggered by adipogenic signals, and diminish upon their removal. GR, which is important for adipogenesis but need not be active in the mature adipocyte, functions transiently with other enhancer proteins to propagate a new program of gene expression that includes induction of PPARgamma2, thereby providing a memory of the earlier adipogenic signal. Thus, the conversion of preadipocyte to adipocyte involves the formation of an epigenomic transition state that is not observed in cells at the beginning or end of the differentiation process.

Endogenous Ligands for Nuclear Receptors: Digging Deeper

Nuclear receptors (NRs) are hormone-sensing transcription factors that translate dietary or endocrine signals into changes in gene expression. Therefore, the adoption of orphan NRs through the identification of their endogenous ligands is a key element for our understanding of their biology. In this minireview, we give an update on recent progress in regard to endogenous ligands for a cluster of NRs with high sequence homology, namely peroxisome proliferator-activated receptors α and γ, Rev-erbα, and related receptors. This knowledge about the nature and physiology of these ligands may create new opportunities for therapeutic drug development.

Angiotensin type 1 receptor antagonists induce human in-vitro adipogenesis through peroxisome proliferator-activated receptor-gamma activation.

Objective In clonal animal cells, certain angiotensin receptor blockers (ARB) activate the peroxisome proliferator-activated receptor-γ (PPARγ). The aim of this work was to validate that observation in human cells and humans. Methods We investigated the induction of in-vitro adipogenesis and the activation of PPARγ-target genes, adiponectin and lipoprotein lipase, by ARB in human preadipocytes. We also studied PPARγ response-element-driven luciferase reporter gene activation in human adipocytes. Finally, we treated 14 obese men for 10 days with placebo crossed over with 150 mg/day irbesartan. Subcutaneous fat was analyzed for mRNA expression of adiponectin and lipoprotein lipase. Results Telmisartan and irbesartan, and to a lesser degree losartan, induced adipogenesis and activated PPARγ-target genes. This stimulation of PPARγ-target genes was prevented by the PPARγ antagonist GW9662. Eprosartan had no effect. Paradoxically, all ARB activated the luciferase reporter gene. PPARγ activity increased approximately two-fold with pioglitazone and 1.5-fold with the ARB in all assays. In the cross-over clinical study, irbesartan lowered blood pressure but had no effect on adiponectin or lipoprotein lipase mRNA expression. Conclusions Our data are the first to show that ARB induce adipogenesis and PPARγ-target gene expression in human adipocytes. Pharmacokinetic differences may contribute to the heterogeneous effects on metabolism and preadipocyte differentiation. In humans, larger doses of ARB, longer treatments, or both may be required to activate PPARγ in adipose cells.

Liver-Specific Peroxisome Proliferator-Activated Receptor α Target Gene Regulation by the Angiotensin Type 1 Receptor Blocker Telmisartan

OBJECTIVE—The angiotensin type 1 receptor blocker (ARB) and peroxisome proliferator–activated receptor (PPAR) γ modulator telmisartan has been recently demonstrated to reduce plasma triglycerides in nondiabetic and diabetic hypertensive patients. The present study investigates the molecular mechanisms of telmisartans hypolipidemic actions, in particular its effect on the PPARα pathway. RESEARCH DESIGN AND METHODS—Regulation of PPARα target genes by telmisartan was studied by real-time PCR and Western immunoblotting in vitro and in vivo in liver/skeletal muscle of mice with diet-induced obesity. Activation of the PPARα ligand binding domain (LBD) was investigated using transactivation assays. RESULTS—Telmisartan significantly induced the PPARα target genes carnitine palmitoyl transferase 1A (CPT1A) in human HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in murine AML12 cells in the micromolar range. Telmisartan-induced CPT1A stimulation was markedly reduced after small interfering RNA–mediated knockdown of PPARα. Telmisartan consistently activated the PPARα-LBD as a partial PPARα agonist. Despite high in vitro concentrations required for PPARα activation, telmisartan (3 mg · kg−1 · day−1) potently increased ACSL1 and CPT1A expression in liver from diet-induced obese mice associated with a marked decrease of hepatic and serum triglycerides. Muscular CPT1B expression was not affected. Tissue specificity of telmisartan-induced PPARα target gene induction may be the result of previously reported high hepatic concentrations of telmisartan. CONCLUSIONS—The present study identifies the ARB/PPARγ modulator telmisartan as a partial PPARα agonist. As a result of its particular pharmacokinetic profile, PPARα activation by telmisartan seems to be restricted to the liver. Hepatic PPARα activation may provide an explanation for telmisartans antidyslipidemic actions observed in recent clinical trials.

Adipocyte-specific Expression of Murine Resistin Is Mediated by Synergism between Peroxisome Proliferator-activated Receptor γ and CCAAT/Enhancer-binding Proteins

Resistin antagonizes insulin action in mouse, making it a potential therapeutic target for treating metabolic diseases such as diabetes. To better understand how mouse resistin gene (Retn) expression is restricted to fat tissue, we identified an adipocyte-specific enhancer located ∼8.8-kb upstream of the transcription start site. This region contains a binding site for the master adipogenic regulator peroxisome proliferator-activated receptor γ (PPARγ), and binds endogenous PPARγ together with its partner retinoid-X receptor α (RXRα). It also contains three binding sites for CCAAT/enhancer-binding protein (C/EBP), and is bound by endogenous C/EBPα and C/EBPβ in adipocytes. Exogenous expression of PPARγ/RXRα and C/EBPα in non-adipocyte cells synergistically drives robust expression from the enhancer. Although PPARγ ligands repress Retn transcription in adipocytes, rosiglitazone paradoxically stimulates the enhancer activity, suggesting that the enhancer is not directly involved in negative regulation. Unlike expression of Retn in mouse, human resistin (RETN) is expressed primarily in macrophages. Interestingly, the region homologous to the mouse Retn enhancer in the human gene contains all three C/EBP elements, but is not conserved for the sequence bound by PPARγ. Furthermore, it displays little or no binding by PPARγ in vitro. Taken together, the data suggest that a composite enhancer binding both PPARγ and C/EBP factors confers adipocyte-specific expression to Retn in mouse, and its absence from the human gene may explain the lack of adipocyte expression in humans.

Retinol saturase promotes adipogenesis and is downregulated in obesity

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor γ (PPARγ). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity. On the other hand, ectopic RetSat with an intact, but not a mutated, FAD/NAD dinucleotide-binding motif increased endogenous PPARγ transcriptional activity and promoted adipogenesis. Indeed, RetSat was not required for adipogenesis when cells were provided with exogenous PPARγ ligands. In adipose tissue, RetSat is expressed in adipocytes but is unexpectedly downregulated in obesity, most likely owing to infiltration of macrophages that we demonstrate to repress RetSat expression. Thiazolidinedione treatment reversed low RetSat expression in adipose tissue of obese mice. Thus, RetSat plays an important role in the biology of adipocytes, where it favors normal differentiation, yet is reduced in the obese state. RetSat is thus a novel target for therapeutic intervention in metabolic disease.