[show abstract][hide abstract] ABSTRACT: Transcriptional effectors of white adipocyte-selective gene expression have not been described. Here we show that TLE3 is a white-selective cofactor that acts reciprocally with the brown-selective cofactor Prdm16 to specify lipid storage and thermogenic gene programs. Occupancy of TLE3 and Prdm16 on certain promoters is mutually exclusive, due to the ability of TLE3 to disrupt the physical interaction between Prdm16 and PPARγ. When expressed at elevated levels in brown fat, TLE3 counters Prdm16, suppressing brown-selective genes and inducing white-selective genes, resulting in impaired fatty acid oxidation and thermogenesis. Conversely, mice lacking TLE3 in adipose tissue show enhanced thermogenesis in inguinal white adipose depots and are protected from age-dependent deterioration of brown adipose tissue function. Our results suggest that the establishment of distinct adipocyte phenotypes with different capacities for thermogenesis and lipid storage is accomplished in part through the cell-type-selective recruitment of TLE3 or Prdm16 to key adipocyte target genes.
[show abstract][hide abstract] ABSTRACT: Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.
PLoS ONE 01/2013; 8(8):e72511. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Methamphetamine (Meth) abuse has been shown to induce alterations in mitochondrial function in the brain as well as to induce hyperthermia, which contributes to neurotoxicity and Meth-associated mortality. Brown adipose tissue (BAT), a thermogenic site known to be important in neonates, has recently regained importance since being identified in significant amounts and in correlation with metabolic balance in human adults. Given the high mitochondrial content of BAT and its role in thermogenesis, we aimed to investigate whether BAT plays any role in the development of Meth-induced hyperthermia. By ablating or denervating BAT, we identified a partial contribution of this organ to Meth-induced hyperthermia. BAT ablation decreased temperature by 0.5°C and reduced the length of hyperthermia by 1 h, compared to sham-operated controls. BAT denervation also affected the development of hyperthermia in correlation with decreased the expression of electron transport chain molecules, and increase on PCG1a levels, but without affecting Meth-induced uncoupling protein 1 upregulation. Furthermore, in isolated BAT cells in culture, Meth, but not Norepinephrine, induced H2O2 upregulation. In addition, we found that in vivo Reactive Oxygen Species (ROS) play a role in Meth hyperthermia. Thus, sympathetically mediated mitochondrial activation in the BAT and Meth-induced ROS are key components to the development of hyperthermia in Meth abuse.
[show abstract][hide abstract] ABSTRACT: Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I-specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I- but not a class II-selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I-selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.
[show abstract][hide abstract] ABSTRACT: Gene silencing using RNA interference (RNAi) has become a prominent biological tool for gene annotation, pathway analysis, and target discovery in mammalian cells. High-throughput screens conducted using whole-genome siRNA libraries have uncovered rich sets of new genes involved in a variety of biological processes and cellular models of disease. However, high-throughput RNAi screening is not yet a mainstream tool in life science research because current screening platforms are expensive and onerous. Miniaturizing the RNAi screening platform to reduce cost and increase throughput will enable its widespread use and harness its potential for rapid genome annotation. With this aim, we have combined semi-conductor microfabrication and nanolitre dispensing techniques to develop miniaturized electroporation-ready microwell arrays loaded with siRNA molecules in which multiplexed gene knockdown can be achieved. Arrays of microwells are created using high-aspect ratio biocompatible photoresists on optically transparent and conductive Indium-Tin Oxide (ITO) substrates with integrated micro-electrodes to enable in situ electroporation. Non-contact inkjet microarraying allows precise dispensing of nanolitre volumes into the microwell structures. We have achieved parallel electroporation of multiple mammalian cells cultured in these microwell arrays and observed efficient knockdown of genes with surface-bound, printed siRNAs. Further integration of microfabrication and non-contact nanolitre dispensing techniques described here may enable single-substrate whole-genome siRNA screening in mammalian cells.
Lab on a Chip 02/2012; 12(5):939-47. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diabetic peripheral neuropathy (DPN) is characterized by myelin abnormalities; however, the molecular mechanisms underlying such deficits remain obscure. To uncover the effects of diabetes on myelin alterations, we have analyzed myelin composition. In a streptozotocin-treated rat model of diabetic neuropathy, analysis of sciatic nerve myelin lipids revealed that diabetes alters myelin's phospholipid, FA, and cholesterol content in a pattern that can modify membrane fluidity. Reduced expression of relevant genes in the FA biosynthetic pathway and decreased levels of the transcriptionally active form of the lipogenic factor sterol-regulatory element binding factor-1c (SREBF-1c) were found in diabetic sciatic nerve. Expression of myelin's major protein, myelin protein zero (P0), was also suppressed by diabetes. In addition, we confirmed that diabetes induces sciatic nerve myelin abnormalities, primarily infoldings that have previously been associated with altered membrane fluidity. In a diabetic setting, synthetic activator of the nuclear receptor liver X receptor (LXR) increased SREBF-1c function and restored myelin lipid species and P0 expression levels to normal. These LXR-modulated improvements were associated with restored myelin structure in sciatic nerve and enhanced performance in functional tests such as thermal nociceptive threshold and nerve conduction velocity. These findings demonstrate an important role for the LXR-SREBF-1c axis in protection from diabetes-induced myelin abnormalities.
The Journal of Lipid Research 12/2011; 53(2):300-10. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: PPARγ and Wnt signaling are central positive and negative regulators of adipogenesis, respectively. Here we identify the groucho family member TLE3 as a transcriptional integrator of the PPARγ and Wnt pathways. TLE3 is a direct target of PPARγ that participates in a feed-forward loop during adipocyte differentiation. TLE3 enhances PPARγ activity and functions synergistically with PPARγ on its target promoters to stimulate adipogenesis. At the same time, induction of TLE3 during differentiation provides a mechanism for termination of Wnt signaling. TLE3 antagonizes TCF4 activation by β-catenin in preadipocytes, thereby inhibiting Wnt target gene expression and reversing β-catenin-dependent repression of adipocyte gene expression. Transgenic expression of TLE3 in adipose tissue in vivo mimics the effects of PPARγ agonist and ameliorates high-fat-diet-induced insulin resistance. Our data suggest that TLE3 acts as a dual-function switch, driving the formation of both active and repressive transcriptional complexes that facilitate the adipogenic program.
[show abstract][hide abstract] ABSTRACT: Neuroactive steroids act in the peripheral nervous system as physiological regulators and as protective agents for acquired or inherited peripheral neuropathy. In recent years, modulation of neuroactive steroids levels has been studied as a potential therapeutic approach to protect peripheral nerves from damage induced by diabetes. Nuclear receptors of the liver X receptor (LXR) family regulate adrenal steroidogenesis via their ability to control cholesterol homeostasis. Here we show that rat sciatic nerve expresses both LRXα and β isoforms and that these receptors are functional. Activation of liver X receptors using a synthetic ligand results in increased levels of neurosteroids and protection of the sciatic nerve from neuropathy induced by diabetes. LXR ligand treatment of streptozotocin-treated rats increases expression in the sciatic nerve of steroidogenic acute regulatory protein (a molecule involved in the transfer of cholesterol into mitochondria), of the enzyme P450scc (responsible for conversion of cholesterol into pregnenolone), of 5α-reductase (an enzyme involved in the generation of neuroactive steroids) and of classical LXR targets involved in cholesterol efflux, such as ABCA1 and ABCG1. These effects were associated with increased levels of neuroactive steroids (e.g., pregnenolone, progesterone, dihydroprogesterone and 3α-diol) in the sciatic nerve, and with neuroprotective effects on thermal nociceptive activity, nerve conduction velocity, and Na(+), K(+)-ATPase activity. These results suggest that LXR activation may represent a new pharmacological avenue to increase local neuroactive steroid levels that exert neuroprotective effects in diabetic neuropathy.
Journal of Neuroscience 09/2010; 30(36):11896-901. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.
[show abstract][hide abstract] ABSTRACT: The Forkhead transcription factors FoxO1, FoxO3a, and FoxO4 play a prominent role in regulating cell survival and cell cycle. Whereas FOXO1 was shown to mediate insulin sensitivity and adipocyte differentiation, the role of the transcription factor FoxO4 in metabolism remains ill defined. To uncover the effects of FoxO4, we generated a cellular model of stable FoxO4 overexpression and subjected it to microarray-based gene expression profiling. While pathway analysis revealed a disruption of cholesterol biosynthesis gene expression, biochemical studies revealed an inhibition of cholesterol biosynthesis, which was coupled with decreased mRNA levels of lanosterol 14alpha demethylase (CYP51). FoxO4-mediated repression of CYP51 led to the accumulation of 24,25 dihydrolano-sterol (DHL), which independently and unlike lanosterol inhibited cholesterol biosynthesis. Furthermore, FoxO4-overexpressing cells accumulated lipid droplets and triacylglycerols and had an increase in basal glucose uptake. Recapitulation of these effects was obtained following treatment with CYP51 inhibitors, which also induce DHL buildup. Moreover, DHL but not lanosterol strongly stimulated liver X receptor alpha (LXRalpha) activity, suggesting that DHL and LXRalpha mediate the downstream effects initiated by FoxO4. Together, these studies suggest that FoxO4 acts on CYP51 to regulate the late steps of cholesterol biosynthesis.
The Journal of Lipid Research 06/2010; 51(6):1312-24. · 4.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unravelling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research.
Lab on a Chip 12/2009; 9(24):3557-66. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: The discovery, synthesis, and optimization of compound 1 from a high-throughput screening hit to highly potent and selective peroxisome proliferator-activated receptor delta (PPARdelta) agonists are reported. The synthesis and structure-activity relationship in this series are described in detail. On the basis of a general schematic PPAR pharmacophore model, scaffold 1 was divided into headgroup, linker, and tailgroup and successively optimized for PPAR activation using in vitro PPAR transactivation assays. A (2-methylphenoxy)acetic acid headgroup, a flexible linker, and a five-membered heteroaromatic center ring with two hydrophobic aryl substituents were required for efficient and selective PPARdelta activation. The fine-tuning of these aryl substituents led to an array of highly potent and selective compounds such as compound 38c, displaying an excellent pharmacokinetic profile in mouse. In an in vivo acute dosing model, selected members of this array were shown to induce the expression of pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3), genes that are known to be involved in energy homeostasis and regulated by PPARdelta in skeletal muscle.
Journal of Medicinal Chemistry 11/2009; 53(1):77-105. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Stroke is characterized by massive inflammation in areas surrounding the injury that magnifies damage to the brain. The liver X receptors (LXRs) are nuclear receptors that regulate cholesterol, lipid, and glucose metabolism. Synthetic LXR agonists have potent anti-inflammatory properties in a variety of settings, including neuroinflammation. However, the ability of LXR agonists to suppress stroke-associated inflammation has not been evaluated. Here, we have used time-lapse magnetic resonance imaging (MRI) to show that a single dose of an LXR ligand administered post-injury dramatically reduces brain damage in a model of acute brain ischemia. Neuroprotection was associated with suppression of neuroinflammation.
[show abstract][hide abstract] ABSTRACT: Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on "trans-eQTL bands", defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.
[show abstract][hide abstract] ABSTRACT: The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.
[show abstract][hide abstract] ABSTRACT: We have identified a novel liver X receptor (LXR) agonist (2) that activates the LXRbeta subtype with selectivity over LXRalpha. LXRbeta selectivity was confirmed using macrophages derived from LXR mutant mice. Despite its selectivity and modest potency, the compound can induce APO-AI-dependent cholesterol efflux from macrophages with full efficacy. Our results indicate that it is possible to achieve significant LXRbeta selectivity in a small molecule while maintaining functional LXR activity.
Journal of Medicinal Chemistry 09/2007; 50(17):4255-9. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: PPARgamma is the master regulator of adipogenesis and the molecular target of the thiazolidinedione antidiabetic drugs. By screening for compounds that promote adipogenesis, we identified a small molecule that targets the PPARgamma pathway by a distinct mechanism. This molecule, harmine, is not a ligand for the receptor; rather, it acts as a cell-type-specific regulator of PPARgamma expression. Administration of harmine to diabetic mice mimics the effects of PPARgamma ligands on adipocyte gene expression and insulin sensitivity. Unlike thiazolidinediones, however, harmine does not cause significant weight gain or hepatic lipid accumulation. Molecular studies indicate that harmine controls PPARgamma expression through inhibition of the Wnt signaling pathway. This work validates phenotypic screening of adipocytes as a promising strategy for the identification of bioactive small molecules and suggests that regulators of PPARgamma expression may represent a complementary approach to PPARgamma ligands in the treatment of insulin resistance.
[show abstract][hide abstract] ABSTRACT: The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Insight into the physiologic roles of the LXRs has been greatly facilitated by the discovery of potent synthetic agonists. Here we show that one of these compounds, T0901317, is also a high-affinity ligand for the xenobiotic receptor pregnane X receptor (PXR). T0901317 binds and activates PXR with the same nanomolar potency with which it stimulates LXR activity. T0901317 induces expression not only of LXR target genes, but also of PXR target genes in cells and animals, including the scavenger receptor CD36, a property not shared by more specific LXR ligands, such as GW3965. Activation of PXR targets may explain why T0901317 induces dramatic liver steatosis, while GW3965 has a milder effect. These results suggest that many of the biological activities heretofore associated with LXR activation may be mediated by PXR, not LXR. Since T0901317 has been widely used in animals to study LXR function, the in vivo effects of this compound ascribed to LXR activation should be re-examined.