PPAR Response Element Research Articles

Metabonomic Profiling Revealed an Alteration in Purine Nucleotide Metabolism Associated with Cardiac Hypertrophy in Rats Treated with Thiazolidinediones

Thiazolidinediones (TZDs) including rosiglitazone (RSG) and pioglitazone (PIO) are synthetic agonists selective for peroxisome proliferator-activated receptor-γ (PPARγ) and have been clinically used to treat type-II diabetes as insulin sensitizers. Recent meta-analyses have shown that TZDs are associated with an increased risk for the development of heart failure. To elucidate the mechanism underlying such a cardiac adverse effect, we used a (1)H NMR-based approach to examine the metabonomic profiles in the cardiac tissues treated with RSG (15 mg/kg body weight/day) or PIO (45 mg/kg/day) for 4 weeks and found that the TZD treatments resulted in a significantly altered metabolic profile in hearts, which was associated with cardiac hypertrophy. Multivariate analysis demonstrated that TZDs led to an accumulation in adenosine monophosphate (AMP) and a depletion of inosine. Consistently, AMP kinase, a signal pathway sensitive to the change in the intracellular concentrations of AMP, was activated in the cardiac tissues from the TZDs-treated rats. Quantitative real-time reverse-transcriptase polymerase chain reaction showed a significant induction of the genes involved in the de novo synthesis of purine nucleotide but a reduction of those for the catabolism. Furthermore, the putative PPAR-responsive elements were identified in the 5'-flanking regions of the TZD-up-regulated genes such as adenylosuccinate synthase gene (Adss) and phosphoribosl pyrophosphate synthetase 1 (Prps1), and the binding of PPARγ to these motifs was confirmed by using chromatin immunoprecipitation assay. In conclusion, these results demonstrated that TZDs induced alterations in purine nucleotide metabolism in rat hearts via transcriptional regulation of the PPARγ-target genes, which may play an important role in the development of cardiac hypertrophy associated with TZDs.

Caffeic acid induces keratinocyte differentiation by activation of PPAR-α

Peroxisome proliferator-activated receptors (PPAR)-α plays an important role in epidermal differentiation and barrier recovery, and topical treatment with PPAR-α agonists restores epidermal homeostasis in essential fatty acid deficiency and permeability barrier in skin disruptions. Therefore, we performed structure-based pharmacophore screening to search for a novel PPAR-α agonist. Caffeic acid was ultimately selected and evaluated for its effects on keratinocyte differentiation and epidermal permeability barrier. The transactivation activity of PPAR-responsive element (PPRE) and cornified envelope (CE) formation were assayed. Also, immunoblot analysis and anti-oxidant activity were investigated on caffeic acid. Caffeic acid increases the transactivation activity of PPRE and CE formation in keratinocytes. In addition, caffeic acid promotes the expression of genes and proteins related to CE formation such as involucrin and transglutaminase-1. Additionally, anti-oxidant activity were improved by caffeic acid. Caffeic acid can promote keratinocyte differentiation and restore skin barrier homeostasis and is suggested to be an appropriate skin therapeutic agent for improving epidermal permeability barrier function.

5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells

Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages.

PPARγ-induced PARylation promotes local DNA demethylation by production of 5-hydroxymethylcytosine

Recent studies have shown that DNA demethylation goes through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by Tet proteins. However, it is still unclear how the target regions for demethylation are distinguished within their genomic context. Here we show that the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has the ability to direct local demethylation around its binding sites, the PPAR response elements (PPREs), during adipocyte differentiation. PPARγ is a key regulator of the differentiation process that forms a PPARγ co-activator complex on PPREs and activates the expression of adipocyte-specific genes. The complex is poly(ADP-ribosyl)ated (PARylated) on PPREs, and Tet proteins catalyse the conversion of 5mC to 5hmC locally by their ability to bind to the PAR polymer, thereby inducing region-specific demethylation. Our study demonstrates that a sequence-dependent transcription factor complex can, through its post-translational modification, serve for Tet proteins as a landmark to identify sites of DNA demethylation.

PPARγ agonists upregulate sphingosine 1-phosphate (S1P) receptor 1 expression, which in turn reduces S1P-induced [Ca2 +]i increases in renal mesangial cells

We previously identified peroxisome proliferator-activated receptor gamma (PPARγ) agonists (thiazolidinediones, TZDs) as modulators of the sphingolipid metabolism in renal mesangial cells. TZDs upregulated sphingosine kinase 1 (SK-1) and increased the formation of intracellular sphingosine 1-phosphate (S1P), which in turn reduced the expression of pro-fibrotic connective tissue growth factor. Since S1P also acts as extracellular ligand at specific S1P receptors (S1PR, S1P1–5), we investigated here the effect of TZDs on S1PR expression in mesangial cells and evaluated the functional consequences by measuring S1P-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). Treatment with two different TZDs, troglitazone and rosiglitazone, enhanced S1P1 mRNA and protein expression in rat mesangial cells, whereas S1P2–5 expression levels were not altered. Upregulation of S1P1 mRNA upon TZD treatment was also detected in human mesangial cells and mouse glomeruli. PPARγ antagonism and promoter studies revealed that the TZD-dependent S1P1 mRNA induction involved a functional PPAR response element in the S1P1 promoter. Pharmacological approaches disclosed that S1P-induced [Ca2+]i increases in rat mesangial cells were predominantly mediated by S1P2 and S1P3. Interestingly, the transcriptional upregulation of S1P1 by TZDs resulted in a reduction of S1P-induced [Ca2+]i increases, which was reversed by the S1P1/3 antagonist VPC-23019, the protein kinase C (PKC) inhibitor PKC-412, and by S1P1 siRNA. These data suggest that PPARγ-dependent upregulation of S1P1 leads to an inhibition of S1P-induced Ca2+ signaling in a PKC-dependent manner. Overall, these results reveal that TZDs not only modulate intracellular S1P levels but also regulate S1PR signaling by increasing S1P1 expression in mesangial cells.

HIV-1 Vpr Enhances PPARβ/δ-Mediated Transcription, Increases PDK4 Expression, and Reduces PDC Activity

HIV infection and its therapy are associated with disorders of lipid metabolism and bioenergetics. Previous work has suggested that viral protein R (Vpr) may contribute to the development of lipodystrophy and insulin resistance observed in HIV-1-infected patients. In adipocytes, Vpr suppresses mRNA expression of peroxisomal proliferator-activating receptor-γ (PPARγ)-responsive genes and inhibits differentiation. We investigated whether Vpr might interact with PPARβ/δ and influence its transcriptional activity. In the presence of PPARβ/δ, Vpr induced a 3.3-fold increase in PPAR response element-driven transcriptional activity, a 1.9-fold increase in pyruvate dehydrogenase kinase 4 (PDK4) protein expression, and a 1.6-fold increase in the phosphorylated pyruvate dehydrogenase subunit E1α leading to a 47% decrease in the activity of the pyruvate dehydrogenase complex in HepG2 cells. PPARβ/δ knockdown attenuated Vpr-induced enhancement of endogenous PPARβ/δ-responsive PDK4 mRNA expression. Vpr induced a 1.3-fold increase in mRNA expression of both carnitine palmitoyltransferase I (CPT1) and acetyl-coenzyme A acyltransferase 2 (ACAA2) and doubled the activity of β-hydroxylacyl coenzyme A dehydrogenase (HADH). Vpr physically interacted with the ligand-binding domain of PPARβ/δ in vitro and in vivo. Consistent with a role in energy expenditure, Vpr increased state-3 respiration in isolated mitochondria (1.16-fold) and basal oxygen consumption rate in intact HepG2 cells (1.2-fold) in an etomoxir-sensitive manner, indicating that the oxygen consumption rate increase is β-oxidation-dependent. The effects of Vpr on PPAR response element activation, pyruvate dehydrogenase complex activity, and β-oxidation were reversed by specific PPARβ/δ antagonists. These results support the hypothesis that Vpr contributes to impaired energy metabolism and increased energy expenditure in HIV patients.

ANGPTL4 expression induced by butyrate and rosiglitazone in human intestinal epithelial cells utilizes independent pathways

Short-chain fatty acids (SCFAs), such as butyrate and propionate, are metabolic products of carbohydrate fermentation by the microbiota and constitute the main source of energy for host colonocytes. SCFAs are also important for gastrointestinal health, immunity, and host metabolism. Intestinally produced angiopoietin-like protein 4 (ANGPTL4) is a secreted protein with metabolism-altering properties and may offer a route by which microbiota can regulate host metabolism. Peroxisome proliferator-activated receptor (PPAR)-γ has previously been shown to be involved in microbiota-induced expression of intestinal ANGPTL4, but the role of bacterial metabolites in this process has remained elusive. Here, we show that the SCFA butyrate regulates intestinal ANGPTL4 expression in a PPAR-γ-independent manner. Although PPAR-γ is not required for butyrate-driven intestinal ANGPTL4 expression, costimulating with PPAR-γ ligands and SCFAs leads to additive increases in ANGPTL4 levels. We suggest that PPAR-γ and butyrate rely on two separate regulatory sites, a PPAR-responsive element downstream the transcription start site and a butyrate-responsive element(s) within the promoter region, 0.5 kb upstream of the transcription start site. Furthermore, butyrate gavage and colonization with Clostridium tyrobutyricum, a SCFA producer, can independently induce expression of intestinal ANGPTL4 in germ-free mice. Thus, oral administration of SCFA or use of SCFA-producing bacteria may be additional routes to maintain intestinal ANGPTL4 levels for preventive nutrition or therapeutic purposes.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

Peroxisome Proliferator-activated Receptor α Positively Regulates Complement C3 Expression but Inhibits Tumor Necrosis Factor α-mediated Activation of C3 Gene in Mammalian Hepatic-derived Cells

Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor α (PPARα) in human hepatoma HepG2 cells and mouse liver. Using PPARα siRNA and synthetic PPARα agonist WY-14643 and antagonist MK886 we showed that activation of PPARα results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPARα in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNFα increases C3 gene via NF-κB and, to a lesser extent, MEK1/2 signaling pathways, whereas TNFα-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPARα abolishes TNFα-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-κB via PPRE-dependent mechanism in HepG2 cells. TNFα decreases PPARα protein content via NF-κB and MEK1/2 signaling pathways and inhibits PPARα binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPARα and TNFα in the regulation of complement system.

Clofibrate Induces Heme Oxygenase 1 Expression through a PPARα-Independent Mechanism in Human Cancer Cells

Background and Aims: Clofibrate, an established PPARα ligand, has recently been shown to have anticancer activity yet its mechanisms of action remain to be characterized. This study examined the effect of clofibrate on heme oxygenase-1 (HO-1) gene expression in A2780 (human ovarian cancer) and DU145 (human prostate cancer) cells. Methods and Results: We demonstrate that clofibrate induces HO-1 expression in a concentration- and time-dependent manner. The induction of HO-1 by clofibrate was detected at both mRNA and protein levels and the HO-1 gene promoter activity was also dramatically induced by clofibrate, indicating that clofibrate up-regulates HO-1 gene transcription. Surprisingly, the induction of HO-1 by clofibrate was mediated by the Nrf2 signaling pathway, not by the PPARα pathway. This was primarily demonstrated by siRNA knockdown of Nrf2 expression that significantly attenuated clofibrate-induced HO-1 gene transcription, and siRNA knockdown of PPARα that had no effect on clofibrate-induced HO-1 promoter activity. Furthermore, deletion of the antioxidant response elements (AREs) in the HO-1 gene promoter diminished clofibrate-induced HO-1 transcription and deletion of the PPAR response elements (PPREs) had no such effect. Likewise, application of PPARα antagonists had no effect on clofibrate-induced HO-1 expression. Conclusion: Clofibrate induces HO-1 gene expression in cancer cells through a PPARα-independent mechanism and the Nrf2 signaling pathway is indispensible for this induction.

Abstract 18845: PPARalpha/Sirt1 Complex is Involved in the Deterioration of Fatty Acid Metabolism During the Pathological Development of Heart Failure

The major substrate shifts from fatty acid to glucose during the pathological development of heart failure, which may promote left ventricular (LV) dysfunction through energetic shortage. PPARα transcribes fatty acid oxidation (FAO) genes through the PPAR response element /direct repeat 1 (DR1), which is composed of two units of AGGTCA-like sequences that directionally align, separated by a single spacer. In addition to activating transcription through DR1, PPARα, in complex with Sirt1, suppresses transcription through an isolated single hexad element which is not configured into a DR1. Since many PPREs have imperfect DR1s in which one AGGTCA sequence is conserved but the other is not, we hypothesized that the PPARα/Sirt1 complex suppresses a subset of PPAR target genes harboring the imperfect DR1 under pressure overload (PO) conditions. Both PPARα and Sirt1 are upregulated in the heart during PO, and PO-induced cardiac systolic dysfunctions are attenuated in PPARα+/- (LV ejection fraction (EF) (%), WT 51, PPARα+/- 68*, p<0.05 vs WT), and Sirt1+/- (LV EF (%), WT 40, Sirt1+/- 62*, p<0.05 vs WT) mice. PO-induced downregulation of a subset of PPAR target genes harboring imperfect DR1s, such as Cpt2 and Mcad, was partly normalized in PPARα+/- (relative mRNA vs Wild type (WT) sham, Cpt2: WT TAC 0.7, PPARα+/- sham 1.1, PPARα+/- TAC 1.05*, Mcad: WT TAC 0.49, PPARα+/- sham 1.1, PPARα+/- TAC 0.77* p<0.05 vs WT TAC) and Sirt1+/- (relative mRNA vs WT sham, Cpt2: WT TAC 0.37, Sirt1+/- sham 1.1, Sirt1+/- TAC 0.78*, Mcad: WT TAC 0.49, Sirt1+/- sham 1.2, Sirt1+/- TAC 0.73* p<0.05 vs WT TAC) mice. Forced expression of PPARα resulted in increased occupancies of PPARα and Sirt1 on the proximal regions of the imperfect DR1s of the Cpt2 and Mcad promoters in the heart, indicating that PPARα recruits Sirt1 to the imperfect DR1. FAO activity was impaired by PO, but was partly normalized in PPARα+/- mice (relative FAO activity vs WT sham, WT TAC 0.50, PPARα+/- Sham 0.88, PPARα+/- TAC 0.89*, * p<0.05 vs WT TAC). PPARα and Sirt1 cooperatively suppressed reporter gene activity driven by the imperfect DR1 in the Mcad promoter. These results suggest that PPARα and Sirt1 play a role in the impaired FAO in the failing heart though transcriptional suppression of imperfect DR1s.

Peroxisome proliferator-activated receptor α mediates enhancement of gene expression of cerebroside sulfotransferase in several murine organs

Sulfatides, 3-O-sulfogalactosylceramides, are known to have multifunctional properties. These molecules are distributed in various tissues of mammals, where they are synthesized from galactosylceramides by sulfation at C3 of the galactosyl residue. Although this reaction is specifically catalyzed by cerebroside sulfotransferase (CST), the mechanisms underlying the transcriptional regulation of this enzyme are not understood. With respect to this issue, we previously found potential sequences of peroxisome proliferator-activated receptor (PPAR) response element on upstream regions of the mouse CST gene and presumed the possible regulation by the nuclear receptor PPARα. To confirm this hypothesis, we treated wild-type and Ppara-null mice with the specific PPARα agonist fenofibrate and examined the amounts of sulfatides and CST gene expression in various tissues. Fenofibrate treatment increased sulfatides and CST mRNA levels in the kidney, heart, liver, and small intestine in a PPARα-dependent manner. However, these effects of fenofibrate were absent in the brain or colon. Fenofibrate treatment did not affect the mRNA level of arylsulfatase A, which is the key enzyme for catalyzing desulfation of sulfatides, in any of these six tissues. Analyses of the DNA-binding activity and conventional gene expression targets of PPARα has demonstrated that fenofibrate treatment activated PPARα in the kidney, heart, liver, and small intestine but did not affect the brain or colon. These findings suggest that PPARα activation induces CST gene expression and enhances sulfatide synthesis in mice, which suggests that PPARα is a possible transcriptional regulator for the mouse CST gene.

PPARγ Regulates Resistance Vessel Tone Through a Mechanism Involving RGS5-Mediated Control of Protein Kinase C and BKCa Channel Activity

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) by thiazolidinediones lowers blood pressure, whereas PPARγ mutations cause hypertension. Previous studies suggest these effects may be mediated through the vasculature, but the underlying mechanisms remain unclear. To identify PPARγ mechanisms and transcriptional targets in vascular smooth muscle and their role in regulating resistance artery tone. We studied mesenteric artery (MA) from transgenic mice expressing dominant-negative (DN) mutant PPARγ driven by a smooth muscle cell-specific promoter. MA from transgenic mice exhibited a robust increase in myogenic tone. Patch clamp analysis revealed a reduced large conductance Ca(2+)-activated K(+) (BKCa) current in freshly dissociated smooth muscle cell from transgenic MA. Inhibition of protein kinase C corrected both enhanced myogenic constriction and impaired the large conductance Ca(2+)-activated K(+) channel function. Gene expression profiling revealed a marked loss of the regulator of G protein signaling 5 (RGS5) mRNA in transgenic MA, which was accompanied by a substantial increase in angiotensin II-induced constriction in MA. Small interfering RNA targeting RGS5 caused augmented myogenic tone in intact mesenteric arteries and increased activation of protein kinase C in smooth muscle cell cultures. PPARγ and PPARδ each bind to a PPAR response element close to the RGS5 promoter. RGS5 expression in nontransgenic MA was induced after activation of either PPARγ or PPARδ, an effect that was markedly blunted by DN PPARγ. We conclude that RGS5 in smooth muscle is a PPARγ and PPARδ target, which when activated blunts angiotensin II-mediated activation of protein kinase C, and preserves the large conductance Ca(2+)-activated K(+) channel activity, thus providing tight control of myogenic tone in the microcirculation.

Abstract 251: Regulator of G Protein Signaling 5 is a Novel Peroxisome Proliferator-Activated Receptor (PPAR)-gamma and PPAR-delta Target Gene and Regulator of Resistance Vessel Tone

PPARγ is a ligand-activated transcription factor expressed in the vasculature. Targeted expression of dominant negative (DN) PPARγ to vascular muscle of transgenic mice (S-P467L) caused hypertension and aortic dysfunction. Recently, we demonstrated a robust increase in myogenic tone in S-P467L resistance artery and a reduced large conductance Ca 2+ -activated K + (BKCa) current in S-P467L smooth muscle cells (SMC). Inhibition of protein kinase C (PKC) corrected enhanced myogenic constriction and impaired BKCa channel function. Here, we tested the hypothesis that altered expression of PPARγ target genes in S-P467L resistance artery contributes to increased vessel tone. Gene expression profiling in mesenteric artery revealed a marked loss of the regulator of G protein signaling 5 (RGS5) in S-P467L compared to non-transgenic (NT) (fold change compared to NT; 0.18±0.03, p<0.05). Knock down of RGS5 by siRNA in SMC cultures enhanced PKC activity. Consistent with the function of RGS5 as a negative regulator of angiotensin (Ang) II signaling, S-P467L mesenteric artery exhibited an increased Ang II-induced constriction (at 3 nM: 31.4±4% in S-P467L vs 12.9±2% in NT, p<0.05). Pre-incubation of the artery for 30 minutes with the AT1 receptor (AT1R) antagonist losartan completely abolished Ang II-induced vasoconstriction. Neither AT1aR nor AT1bR mRNA expression was different between the groups, implicating a post-receptor mechanism in response to Ang II. Using a sequence-based model, we identified potential PPAR response elements (PPREs) close to the RGS5 promoter. Electrophoretic mobility shift assays showed that both PPARγ and PPARδ can bind at one of the identified PPREs, suggesting that RGS5 might be a direct target of both PPARs. Whereas a significant induction of RGS5 expression was observed in normal mesenteric artery following PPARγ (3.3±0.4 fold) or PPARδ (3.5±0.3 fold) agonist treatment, these inductions were completely lost in S-P467L. We conclude that PPARγ is required for normal resistance vessel tone and have identified RGS5 as a functionally important PPAR target gene.

Clinical and molecular characterization of a severe form of partial lipodystrophy expanding the phenotype of PPARγ deficiency

Familial partial lipodystrophy (FPLD) is characterized by abnormal fat distribution and a metabolic syndrome with hypertriglyceridemia. We identified a family with a severe form of FPLD3 with never-reported clinical features and a novel mutation affecting the DNA binding domain of PPARγ (E157D). Apart from the lipodystrophy and severe metabolic syndrome, individuals presented musculoskeletal and hematological issues. E157D heterozygotes had a muscular habitus yet displayed muscle weakness and myopathy. Also, E157D heterozygotes presented multiple cytopenias and a susceptibility to autoimmune disease. In vitro studies showed that the E157D mutation does not decrease the receptor's affinity to classical PPAR response elements or its responsiveness to a PPARγ agonist, yet it severely reduces its target gene transcription. Microarray experiments demonstrated a decreased activation of a wide array of genes, including genes involved in the PPAR response, the immune response, hematopoiesis, and metabolism in muscle. In addition, a subset of genes with cryptic PPAR response elements was activated. In summary, we describe a large family with a novel PPARγ mutation, which extends the clinical phenotype of FPLD3 to include muscular, immune, and hematological features. Together, our results support the role of PPARγ in controlling homeostasis of multiple systems beyond lipid metabolism.

A Novel Mechanism for Hepatic Lipid Accumulation: A Physiological Role for Hepatic PPARγ-fsp27 Signal

Fat-specific protein 27 (fsp27) was originally isolated by screening for genes specifically expressed in fully differentiated mouse adipocytes. Fsp27 and cell death-inducing DFF45-like effector (CIDE) C, the human homologue of fsp27, belong to the CIDE family. Fsp27, which is highly expressed in mouse white and brown adipose tissues, was recently reported to be a lipid droplet (LD)-binding protein that promotes lipid accumulation in adipocytes. In contrast, we showed that fsp27 was also expressed in the fatty liver of the ob/ob type II diabetes model mouse. The expression of fsp27 was markedly decreased in livers lacking the nuclear receptor peroxisome proliferator-activated receptor γ(PPARγ). A functional PPAR response element site was identified in the fsp27 promoter region. Forced expression of fsp27 in hepatocytes in vitro or in vivo led to increased LD through increased triglyceride levels. The current status of the physiological roles of the PPARγ-fsp27 signal in fatty liver are discussed along with its significance as a factor involved in the development of metabolic disorders.

Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPARγ, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPARγ agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPARγ, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPARγ through the modulation of MAP kinase family members.

Editorial: Molecular Endocrinology Articles in the Spotlight for May 2012

This month’s issue of Molecular Endocrinology features many excellent articles. We have picked some of the most compelling new research to spotlight: In “Mutations in MCT8 in Patients With AllanHerndon-Dudley-Syndrome Affecting Its Cellular Distribution,” Kersseboom et al (freely available at http://mend.endojournals.org/) observes cell-type dependent effects of mutations within the monocarboxylate transporter 8 (MCT8) thyroid hormone transporter, suggesting tissue-specific effects on thyroid hormone transport in patients with MCT8 mutations. Patients with Allan-Herndon-Dudley-syndrome exhibit severe psychomotor retardation illustrating the importance of MCT8 in maintaining thyroid hormone homeostasis in the brain, particularly during development. The authors identify 2 distinct cellular pathogenic mechanisms of MCT8 mutations: some mutations result in partial or complete loss of thyroid hormone transport activity without affecting cellular MCT8 protein distribution, whereas other mutations mainly disturb protein expression and trafficking. The severity of the phenotype of patients with MCT8 mutations is independent of the underlying pathogenic mechanism, suggesting that even minor perturbations in thyroid hormone transport via MCT8 could have severe consequences on brain development. The report by Katano-Toki et al, “THRAP3 Interacts With HELZ2 and Plays a Novel Role in Adipocyte Differentiation,” used coimmunoprecipitation coupled with liquid chromatography and tandem mass spectrometry to identify Thrap3 (thyroid hormone receptor-associated protein 3), a putative component of the Mediator/tartrate-resistant acid phosphatase complex, as a Helz2 associating protein. The authors also showed that endogenous Helz2, Thrap3, and peroxisome proliferator-activating receptor(PPAR) associate with each other in mature 3T3-L1 adipocytes. Furthermore, Thrap3 and Helz2 are corecruited to PPAR-response elements within the Fabp4/aP2 and Adipoqenhancers in 3T3-L1 cells in response to PPARligands andcooperativelyenhancePPAR-mediatedgenetranscription in vitro. Thus, Thrap3 is a novel PPAR cofactor that plays an important role in the terminal differentiation of adipocytes. “An Essential Role for Insulin and IGF1 Receptors in Regulating Sertoli Cell Proliferation, Testis Size, and FSH Action in Mice” by Pitetti et al provides in vivo evidence that IGFs are the major regulators of Sertoli cell number and testis size in mammals. Both the insulin receptor and the type I IGF1 receptor are required to obtain an optimal number of adult Sertoli cells and testis size. It was also found that the pituitary hormone FSH requires the insulin/IGF1 signaling pathway to mediate its proliferative effects on immature Sertoli cells and thus testis size. These resultssuggestthatgermcellsthemselvesmayregulatethe number of Sertoli cells and thus testis size and daily sperm outputthroughthesecretionofgrowthfactorspromoting Sertoli cell survival and maturation during postnatal testicular development. Congratulations to these researchers as well as those who produced all of the outstanding science in the May issue.

Transcriptional regulation of methionine adenosyltransferase 2A by peroxisome proliferator-activated receptors in rat hepatic stellate cells

Methionine adenosyltransferases (MATs) are critical enzymes that catalyze the formation of the methyl donor S-adenosyl methionine (SAM). The MAT2A gene, which encodes the catalytic subunit α2, is induced in dedifferentiated liver. We previously demonstrated that MAT2A expression is enhanced in activated hepatic stellate cells (HSCs) and that silencing this gene reduces HSC activation. In this study, we examined the molecular mechanisms responsible for the transcriptional regulation of the MAT2A gene in HSCs. We identified peroxisome proliferator-activated receptor (PPAR) response elements (PPREs) in the rat MAT2A promoter. The PPARγ agonist rosiglitazone (RSG) promoted quiescence in the activated rat HSC cell line (BSC) or culture-activated primary rat HSCs, decreased MAT2A expression and promoter activity, and enhanced PPARγ binding to MAT2A PPREs. In vivo HSC activation in bile duct-ligated rats lowered PPARγ interaction with MAT2A PPREs. Silencing PPARγ increased MAT2A transcription, whereas overexpressing it had the opposite effect, demonstrating that PPARγ negatively controls this gene. Site-directed mutagenesis of PPREs abolished PPARγ recruitment to the MAT2A promoter and its inhibitory effect on MAT2A transcription in quiescent HSCs. PPRE mutations decreased the basal promoter activity of MAT2A in activated HSCs independent of PPARγ, indicating that other factors might be involved in PPRE interaction. We identified PPARβ binding to wild-type but not to mutated PPREs in activated cells. Furthermore, silencing PPARβ inhibited MAT2A expression and promoter activity. Forced expression of MAT2A in RSG-treated HSCs lowered PPARγ and enhanced PPARβ expression, thereby promoting an activated phenotype. We identified PPARγ as a negative regulator of MAT2A in quiescent HSCs. A switch from quiescence to activation abolishes this control and allows PPARβ to up-regulate MAT2A transcription.

A PPARγ-FGF1 axis is required for adaptive adipose remodelling and metabolic homeostasis.

Although feast and famine cycles illustrate that remodelling of adipose tissue in response to fluctuations in nutrient availability is essential for maintaining metabolic homeostasis, the underlying mechanisms remain poorly understood. Here we identify fibroblast growth factor 1 (FGF1) as a critical transducer in this process in mice, and link its regulation to the nuclear receptor PPARγ (peroxisome proliferator activated receptor γ), which is the adipocyte master regulator and the target of the thiazolidinedione class of insulin sensitizing drugs. FGF1 is the prototype of the 22-member FGF family of proteins and has been implicated in a range of physiological processes, including development, wound healing and cardiovascular changes. Surprisingly, FGF1 knockout mice display no significant phenotype under standard laboratory conditions. We show that FGF1 is highly induced in adipose tissue in response to a high-fat diet and that mice lacking FGF1 develop an aggressive diabetic phenotype coupled to aberrant adipose expansion when challenged with a high-fat diet. Further analysis of adipose depots in FGF1-deficient mice revealed multiple histopathologies in the vasculature network, an accentuated inflammatory response, aberrant adipocyte size distribution and ectopic expression of pancreatic lipases. On withdrawal of the high-fat diet, this inflamed adipose tissue fails to properly resolve, resulting in extensive fat necrosis. In terms of mechanisms, we show that adipose induction of FGF1 in the fed state is regulated by PPARγ acting through an evolutionarily conserved promoter proximal PPAR response element within the FGF1 gene. The discovery of a phenotype for the FGF1 knockout mouse establishes the PPARγ–FGF1 axis as critical for maintaining metabolic homeostasis and insulin sensitization.