LXR␣ is a nuclear receptor that has previously been shown to regulate the metabolic conversion of cholesterol to bile acids. Here we define a role for this transcription factor in the control of cellular cholesterol efflux. We demonstrate that retroviral expression of LXR␣ in NIH 3T3 fibroblasts or RAW264.7 macrophages and͞or treatment of these cells with oxysterol ligands of LXR results in 7-to 30-fold induction of the mRNA encoding the putative cholesterol͞phospholipid transporter ATP-binding cassette (ABC)A1. In contrast, induction of ABCA1 mRNA in response to oxysterols is attenuated in cells that constitutively express dominant-negative forms of LXR␣ or LXR␤ that lack the AF2 transcriptional activation domain. We further demonstrate that expression of LXR␣ in NIH 3T3 fibroblasts and͞or treatment of these cells with oxysterols is sufficient to stimulate cholesterol efflux to extracellular apolipoprotein AI. The ability of oxysterol ligands of LXR to stimulate efflux is dramatically reduced in Tangier fibroblasts, which carry a loss of function mutation in the ABCA1 gene. Taken together, these results indicate that cellular cholesterol efflux is controlled, at least in part, at the level of transcription by a nuclear receptor-signaling pathway. They suggest a model in which activation of LXRs by oxysterols in response to cellular sterol loading leads to induction of the ABCA1 transporter and the stimulation of lipid efflux to extracellular acceptors. These findings have important implications for our understanding of mammalian cholesterol homeostasis and suggest new opportunities for pharmacological regulation of cellular lipid metabolism.H uman and murine macrophages express cell surface receptors that bind and then rapidly internalize oxidized or modified (e.g., acetylated) low density lipoprotein (LDL) (1). This rapid and unregulated uptake of the cholesterol-rich LDL results in the formation of intracellular cholesteryl ester inclusions that give the macrophages a foamy appearance (2). Such cholesteryl ester-rich foam cells have been identified in both early and advanced atherosclerotic lesions of humans and animals and are thought to play a critical role in the development of the disease (3, 4). Thus, the differential expression of specific genes in lipid loaded macrophages is likely to play an important role in the pathology of atherosclerosis.Recent studies have identified two genes, ATP-binding cassette (ABC)A1 (5) and ABCG1 (6, 7), whose mRNAs are induced 7-to Ͼ100-fold in lipid-loaded macrophages. In earlier publications, ABCA1 was referred to as ABC1, whereas ABCG1 was referred to as either ABC8 (murine) or white (human). ABCA1 and ABCG1 are members of the ATP binding cassette (ABC) superfamily of transporter proteins that bind and hydrolyze ATP to provide energy for transmembrane transport (8-10). The ABCA1 gene encodes a full transporter protein containing 12 putative transmembrane domains and two ABCs (11), whereas the ABCG1 gene encodes a half transporter protein containing six putative transme...

The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

The liver has a central role in glucose homeostasis, as it has the distinctive ability to produce and consume glucose. On feeding, glucose influx triggers gene expression changes in hepatocytes to suppress endogenous glucose production and convert excess glucose into glycogen or fatty acids to be stored in adipose tissue. This process is controlled by insulin, although debate exists as to whether insulin acts directly or indirectly on the liver. In addition to stimulating pancreatic insulin release, glucose also regulates the activity of ChREBP, a transcription factor that modulates lipogenesis. Here we describe another mechanism whereby glucose determines its own fate: we show that glucose binds and stimulates the transcriptional activity of the liver X receptor (LXR), a nuclear receptor that coordinates hepatic lipid metabolism. d-Glucose and d-glucose-6-phosphate are direct agonists of both LXR-alpha and LXR-beta. Glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands. Cholesterol homeostasis genes that require LXR for expression are upregulated in liver and intestine of fasted mice re-fed with a glucose diet, indicating that glucose is an endogenous LXR ligand. Our results identify LXR as a transcriptional switch that integrates hepatic glucose metabolism and fatty acid synthesis.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine–imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Macrophages play a central role in the development of atherosclerosis through the accumulation of oxidized LDL (oxLDL). AIM (Spalpha/Api6) has previously been shown to promote macrophage survival; however, its function in atherogenesis is unknown. Here we identify AIM as a critical factor that protects macrophages from the apoptotic effects of oxidized lipids. AIM protein is induced in response to oxLDL loading and is highly expressed in foam cells within atherosclerotic lesions. Interestingly, both expression of AIM in lesions and its induction by oxidized lipids require the action of LXR/RXR heterodimers. AIM-/- macrophages are highly susceptible to oxLDL-induced apoptosis in vitro and undergo accelerated apoptosis in atherosclerotic lesions in vivo. Moreover, early atherosclerotic lesions in AIM-/-LDLR-/- double knockout mice are dramatically reduced when compared to AIM+/+LDLR-/- controls. We conclude that AIM production facilitates macrophage survival within atherosclerotic lesions and that loss of AIM decreases early lesion development by increasing macrophage apoptosis.

Lipid-loaded macrophage "foam cells" accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2-14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXR␣/␤ null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells.

Available drugs are unable to effectively rescue the folding defects in vitro and ameliorate the clinical-phenotype of cystic fibrosis (CF), caused by deletion of F508 (ΔF508 or F508del) and some point mutations in the CF transmembrane conductance regulator (CFTR), a plasma membrane (PM) anion channel. To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutants expression and function at the PM. High throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at the nucleotide binding domain (NBD1), NBD2 and their membrane spanning domains (MSDs) interfaces. While individually these compounds marginally improve ΔF508-CFTR folding efficiency, function, and stability, their combinations lead to ~50–100% of wild type-level correction in immortalized and primary human airway epithelia, and in mouse nasal epithelia. Likewise, corrector combinations were effective for rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.