IL-10 Signaling Remodels Adipose Chromatin Architecture to Limit Thermogenesis and Energy Expenditure.

Signaling pathways that promote adipose tissue thermogenesis are well characterized, but the limiters of energy expenditure are largely unknown. Here, we show that ablation of the anti-inflammatory cytokine IL-10 improves insulin sensitivity, protects against diet-induced obesity, and elicits the browning of white adipose tissue. Mechanistic studies define bone marrow cells as the source of the IL-10 signal and adipocytes as the target cell type mediating these effects. IL-10 receptor alpha is highly enriched in mature adipocytes and is induced in response to differentiation, obesity, and aging. Assay for transposase-accessible chromatin sequencing (ATAC-seq), ChIP-seq, and RNA-seq reveal that IL-10 represses the transcription of thermogenic genes in adipocytes by altering chromatin accessibility and inhibiting ATF and C/EBPß recruitment to key enhancer regions. These findings expand our understanding of the relationship between inflammatory signaling pathways and adipose tissue function and provide insight into the physiological control of thermogenesis that could inform future therapy.

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells.

The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.

Quinone-mediated hydrogen anode for non-aqueous reductive electrosynthesis.

Electrochemical synthesis can provide more sustainable routes to industrial chemicals1-3. Electrosynthetic oxidations may often be performed 'reagent-free', generating hydrogen (H2) derived from the substrate as the sole by-product at the counter electrode. Electrosynthetic reductions, however, require an external source of electrons. Sacrificial metal anodes are commonly used for small-scale applications4, but more sustainable options are needed at larger scale. Anodic water oxidation is an especially appealing option1,5,6, but many reductions require anhydrous, air-free reaction conditions. In such cases, H2 represents an ideal alternative, motivating the growing interest in the electrochemical hydrogen oxidation reaction (HOR) under non-aqueous conditions7-12. Here we report a mediated H2 anode that achieves indirect electrochemical oxidation of H2 by pairing thermal catalytic hydrogenation of an anthraquinone mediator with electrochemical oxidation of the anthrahydroquinone. This quinone-mediated H2 anode is used to support nickel-catalysed cross-electrophile coupling (XEC), a reaction class gaining widespread adoption in the pharmaceutical industry13-15. Initial validation of this method in small-scale batch reactions is followed by adaptation to a recirculating flow reactor that enables hectogram-scale synthesis of a pharmaceutical intermediate. The mediated H2 anode technology disclosed here offers a general strategy to support H2-driven electrosynthetic reductions.

The nuclear receptor LXRα controls the functional specialization of splenic macrophages.

Macrophages are professional phagocytic cells that orchestrate innate immune responses and have considerable phenotypic diversity at different anatomical locations. However, the mechanisms that control the heterogeneity of tissue macrophages are not well characterized. Here we found that the nuclear receptor LXRα was essential for the differentiation of macrophages in the marginal zone (MZ) of the spleen. LXR-deficient mice were defective in the generation of MZ and metallophilic macrophages, which resulted in abnormal responses to blood-borne antigens. Myeloid-specific expression of LXRα or adoptive transfer of wild-type monocytes restored the MZ microenvironment in LXRα-deficient mice. Our results demonstrate that signaling via LXRα in myeloid cells is crucial for the generation of splenic MZ macrophages and identify an unprecedented role for a nuclear receptor in the generation of specialized macrophage subsets.

Cholesterol Accumulation in CD11c+ Immune Cells Is a Causal and Targetable Factor in Autoimmune Disease.

Liver X receptors (LXRs) are regulators of cholesterol metabolism that also modulate immune responses. Inactivation of LXR α and ß in mice leads to autoimmunity; however, how the regulation of cholesterol metabolism contributes to autoimmunity is unclear. Here we found that cholesterol loading of CD11c+ cells triggered the development of autoimmunity, whereas preventing excess lipid accumulation by promoting cholesterol efflux was therapeutic. LXRß-deficient mice crossed to the hyperlipidemic ApoE-deficient background or challenged with a high-cholesterol diet developed autoantibodies. Cholesterol accumulation in lymphoid organs promoted T cell priming and stimulated the production of the B cell growth factors Baff and April. Conversely, B cell expansion and the development of autoantibodies in ApoE/LXR-ß-deficient mice was reversed by ApoA-I expression. These findings implicate cholesterol imbalance as a contributor to immune dysfunction and suggest that stimulating HDL-dependent reverse cholesterol transport could be beneficial in the setting of autoimmune disease.

Feedback modulation of cholesterol metabolism by the lipid-responsive non-coding RNA LeXis.

Liver X receptors (LXRs) are transcriptional regulators of cellular and systemic cholesterol homeostasis. Under conditions of excess cholesterol, LXR activation induces the expression of several genes involved in cholesterol efflux, facilitates cholesterol esterification by promoting fatty acid synthesis, and inhibits cholesterol uptake by the low-density lipoprotein receptor. The fact that sterol content is maintained in a narrow range in most cell types and in the organism as a whole suggests that extensive crosstalk between regulatory pathways must exist. However, the molecular mechanisms that integrate LXRs with other lipid metabolic pathways are incompletely understood. Here we show that ligand activation of LXRs in mouse liver not only promotes cholesterol efflux, but also simultaneously inhibits cholesterol biosynthesis. We further identify the long non-coding RNA LeXis as a mediator of this effect. Hepatic LeXis expression is robustly induced in response to a Western diet (high in fat and cholesterol) or to pharmacological LXR activation. Raising or lowering LeXis levels in the liver affects the expression of genes involved in cholesterol biosynthesis and alters the cholesterol levels in the liver and plasma. LeXis interacts with and affects the DNA interactions of RALY, a heterogeneous ribonucleoprotein that acts as a transcriptional cofactor for cholesterol biosynthetic genes in the mouse liver. These findings outline a regulatory role for a non-coding RNA in lipid metabolism and advance our understanding of the mechanisms that coordinate sterol homeostasis.

Nucleophilic Fluorination of Heteroaryl Chlorides and Aryl Triflates Enabled by Cooperative Catalysis.

Aryl and heteroaryl fluorides are growing to be dominant motifs in pharmaceuticals and agrochemicals, yet they are rare in both nature and commodity chemicals. As a consequence, there is an increasingly urgent need to develop mild, cost-effective, and scalable methods for fluorination. The most straightforward route to synthesize aryl fluorides is through the halide exchange "halex" reaction, but conditions, cost, and atom economy preclude most available methods from large-scale manufacturing processes. We report a new approach that leverages the cooperative action of 18-crown-6 ether and tetramethylammonium chloride to catalytically access the reactivity of tetramethylammonium fluoride and achieve halex fluorinations under mild conditions with operational ease. The described methodology readily converts both heteroaryl chlorides and aryl triflates to their corresponding (hetero)aryl fluorides in high yields and purities.

Self-Assembled Tetrahedral Hosts as Supramolecular Catalysts.

The field of supramolecular chemistry has its foundation in molecular recognition and selective binding of guest molecules, often with remarkably strong binding affinities. The field evolved to leverage these favorable interactions between the host and its guest to catalyze simple, often biomimetic transformations. Drawing inspiration from these early studies, self-assembled supramolecular hosts continue to capture a significant amount of interest toward their development as catalysts for increasingly complex transformations. Nature often relies on microenvironments, derived from complex tertiary structures and a well-defined active site, to promote reactions with remarkable rate acceleration, substrate specificity, and product selectivity. Similarly, supramolecular chemists have become increasingly intrigued by the prospect that self-assembly of molecular components might generate defined and spatially segregated microenvironments that can catalyze complex transformations. Among the growing palette of supramolecular catalysts, an anionic, water-soluble, tetrahedral metal-ligand coordination host has found a range of applications in catalysis and beyond. Early work focused on characterizing and understanding this host and its various host-guest phenomena, which paved the path for exploiting these features to selectively promote desirable chemistries, including cyclizations, rearrangements, and bimolecular reactions. Although this early work matured into achievements of catalysis with dramatic rate accelerations as well as enantioenrichment, the afforded products were typically identical to those produced by background reactions that occurred outside of the host microenvironment. This Account describes our recent developments in the application of these anionic tetrahedral hosts as catalysts for organic and organometallic transformation. Inspiration from natural systems and unmet synthetic challenges led to supramolecular catalysis displaying unique divergences in reactivity to give products that are inaccessible from bulk solution. Additionally, these tetrahedral assemblies have been shown to catalyze a diverse range of transformations with notable rate acceleration over the uncatalyzed background reaction. The pursuit of complexity beyond supramolecular catalysis has since led to the integration of these tetrahedral catalysts in tandem with natural enzymes, as well as their application to dual catalysis to realize challenging synthetic reactions. Variation in the structure, including size and charge, of these tetrahedral catalysts has enabled recent studies that provide insights into connections between specific structural features of these hosts and their reactivities. These mechanistic studies reveal that the solvent exclusion properties, hydrophobic effects, confinement effects and electrostatic effects play important roles in the observed catalysis. Moreover, these features may be leveraged for the design of supramolecular catalysis beyond those described in this Account. Finally, the supramolecular chemistry detailed in this Account has presented the opportunity to emulate some of the mechanisms nature engages to achieve catalysis; however, this relationship need not be entirely unidirectional, as the examples describe herein can stand as simplified model systems for unravelling more complex biological processes.

The IDOL-UBE2D complex mediates sterol-dependent degradation of the LDL receptor.

We previously identified the E3 ubiquitin ligase IDOL as a sterol-dependent regulator of the LDL receptor (LDLR). The molecular pathway underlying IDOL action, however, remains to be determined. Here we report the identification and biochemical and structural characterization of an E2-E3 ubiquitin ligase complex for LDLR degradation. We identified the UBE2D family (UBE2D1-4) as E2 partners for IDOL that support both autoubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1 Å crystal structure of the IDOL RING domain-UBE2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL-UBE2D1 interface also defined the stereochemical basis for the selectivity of IDOL for UBE2Ds over other E2 ligases. Structure-based mutations that inhibit IDOL dimerization or IDOL-UBE2D interaction block IDOL-dependent LDLR ubiquitination and degradation. Furthermore, expression of a dominant-negative UBE2D enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOL-UBE2D complex as an important determinant of LDLR activity, and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.

Deconvoluting the Role of Charge in a Supramolecular Catalyst.

We have demonstrated that the microenvironment of a highly anionic supramolecular catalyst can mimic the active sites of enzymes and impart rate accelerations of a million-fold or more. However, these microenvironments can be challenging to study, especially in the context of understanding which specific features of the catalyst are responsible for its high performance. We report here the development of an experimental mechanistic probe consisting of two isostructural catalysts. When examined in parallel transformations, the behavior of these catalysts provides insight relevant to the importance of anionic host charge on reactivity. These two catalysts exhibit similar host-substrate interactions, but feature a significant difference in overall anionic charge (12- and 8-). Within these systems, we compare the effect of constrictive binding in a net neutral aza-Cope rearrangement. We then demonstrate how the magnitude of anionic host charge has an exceptional influence on the reaction rates for a Nazarov cyclization, evidenced by an impressive 680-fold change in reaction rate as a consequence of a 33% reduction in catalyst charge.

Apoptotic cells promote their own clearance and immune tolerance through activation of the nuclear receptor LXR.

Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.

Conformational Selection as the Mechanism of Guest Binding in a Flexible Supramolecular Host.

This study offers a detailed mechanistic investigation of host-guest encapsulation behavior in a new enzyme-mimetic metal-ligand host and provides the first observation of a conformational selection mechanism (as opposed to induced fit) in a supramolecular system. The Ga4L4 host described features a C3-symmetric ligand motif with meta-substituted phenyl spacers, which enables the host to initially self-assemble into an S4-symmetric structure and then subsequently isomerize to a T-symmetric tetrahedron for better accommodation of a sufficiently large guest. Selective inversion recovery 1H NMR studies provide structural insights into the self-exchange behaviors of the host and the guest individually in this dynamic system. Kinetic analysis of the encapsulation-isomerization event revealed that increasing the concentration of guest inhibits the rate of host-guest relaxation, a key distinguishing feature of conformational selection. A comprehensive study of this simple enzyme mimic provides insight into analogous behavior in biophysics and enzymology and aims to inform the design of efficient self-assembled microenvironment catalysts.

Parameterization of Acyclic Diaminocarbene Ligands Applied to a Gold(I)-Catalyzed Enantioselective Tandem Rearrangement/Cyclization.

Computed descriptors for acyclic diaminocarbene ligands are developed in the context of a gold catalyzed enantioselective tandem [3,3]-sigmatropic rearrangement-[2+2]-cyclization. Surrogate structures enable the rapid identification of parameters that reveal mechanistic characteristics. The observed selectivity trends are validated in a robust multivariate analysis facilitating the development of a highly enantioselective process.

Investigation of the two- and three-fragment photodissociation of the tert-butyl peroxy radical at 248 nm.

The photodissociation dynamics of the tert-butyl peroxy (t-BuOO) radical are studied by fast-radical-beam coincidence translational spectroscopy. The neutral t-BuOO radical is formed by photodetachment of the corresponding t-BuOO- anion at 700 nm (1.77 eV), followed by dissociation at 248 nm (5.00 eV). Photofragment mass and translational energy distributions are obtained. The major channel is found to be three-body fragmentation to form O, CH3, and acetone (83%), with minor two-body fragmentation channels leading to the formation of O2 + tert-butyl radical (10%) and HO2 + isobutene (7%). Experimental results show that the translational energy distribution for two-body dissociation peaks is close to zero translational energy, with an isotropic angular distribution of fragments. These results indicate that two-body fragmentation proceeds via internal conversion to the ground electronic state followed by statistical dissociation. For three-body dissociation, the translational energy distribution peaks closer to the maximal allowed translational energy and shows an anisotropic distribution of the plane of the dissociating fragments, implying rapid dissociation on an excited-state surface. A small shoulder in the three-body translational energy distribution suggests that some three-fragment dissociation events proceed by a different mechanism, involving internal conversion to the ground electronic state followed by sequential dissociation.

Scope and Mechanism of Cooperativity at the Intersection of Organometallic and Supramolecular Catalysis.

The scope and mechanism of the microenvironment-catalyzed C(sp(3))-C(sp(3)) reductive elimination from transition metal complexes [Au(III), Pt(IV)] is explored. Experiments detailing the effect of structural perturbation of neutral and anionic spectator ligands, reactive alkyl ligands, solvent, and catalyst structure are disclosed. Indirect evidence for a coordinatively unsaturated encapsulated cationic intermediate is garnered via observation of several inactive donor-arrested inclusion complexes, including a crystallographically characterized encapsulated Au(III) cation. Finally, based on stoichiometric experiments under catalytically relevant conditions, a detailed mechanism is outlined for the dual supramolecular and platinum-catalyzed C-C coupling between methyl iodide and tetramethyltin. Determination of major platinum species present under catalytic conditions and subsequent investigation of their chemistry reveals an unexpected interplay between cis-trans isomerism and the supramolecular catalyst in a Pt(II)/Pt(IV) cycle, as well as several off-cycle reactions.

Transgenic expression of dominant-active IDOL in liver causes diet-induced hypercholesterolemia and atherosclerosis in mice.

RATIONALE: The E3 ubiquitin ligase inducible degrader of the low-density lipoprotein receptor (IDOL) triggers lysosomal degradation of the low-density lipoprotein receptor. The tissue-specific effects of the IDOL pathway on plasma cholesterol and atherosclerosis have not been examined. OBJECTIVE: Given that the liver is the primary determinant of plasma cholesterol levels, we sought to examine the consequence of effect of chronic liver-specific expression of a dominant-active form of IDOL in mice. METHODS AND RESULTS: We expressed a degradation-resistant, dominant-active form of IDOL (super IDOL [sIDOL]) in C57Bl/6J mice from the liver-specific albumin promoter (L-sIDOL transgenics). L-sIDOL mice were fed a Western diet for 20 or 30 weeks and then analyzed for plasma lipid levels and atherosclerotic lesion formation. L-sIDOL mice showed dramatic reductions in hepatic low-density lipoprotein receptor protein and increased plasma low-density lipoprotein cholesterol levels on both chow and Western diets. Moreover, L-sIDOL mice developed marked atherosclerotic lesions when fed a Western diet. Lesion formation in L-sIDOL mice was more robust than in apolipoprotein E*3 Leiden mice and did not require the addition of cholate to the diet. Western diet-fed L-sIDOL mice had elevated expression of liver X receptor target genes and proinflammatory genes in their aortas. CONCLUSIONS: Liver-specific expression of dominant-active IDOL is associated with hypercholesterolemia and a marked elevation in atherosclerotic lesions. Our results show that increased activity of the IDOL pathway in the liver can override other low-density lipoprotein receptor regulatory pathways leading to cardiovascular disease. L-sIDOL mice are a robust, dominantly inherited, diet-inducible model for the study of atherosclerosis.

Dietary cholesterol promotes adipocyte hypertrophy and adipose tissue inflammation in visceral, but not in subcutaneous, fat in monkeys.

OBJECTIVE: Excessive caloric intake is associated with obesity and adipose tissue dysfunction. However, the role of dietary cholesterol in this process is unknown. The aim of this study was to determine whether increasing dietary cholesterol intake alters adipose tissue cholesterol content, adipocyte size, and endocrine function in nonhuman primates. APPROACH AND RESULTS: Age-matched, male African Green monkeys (n=5 per group) were assigned to 1 of 3 diets containing 0.002 (low [Lo]), 0.2 (medium [Med]), or 0.4 (high [Hi]) mg cholesterol/kcal. After 10 weeks of diet feeding, animals were euthanized for adipose tissue, liver, and plasma collection. With increasing dietary cholesterol, free cholesterol (FC) content and adipocyte size increased in a stepwise manner in visceral, but not in subcutaneous fat, with a significant association between visceral adipocyte size and FC content (r(2)=0.298; n=15; P=0.035). In visceral fat, dietary cholesterol intake was associated with (1) increased proinflammatory gene expression and macrophage recruitment, (2) decreased expression of genes involved in cholesterol biosynthesis and lipoprotein uptake, and (3) increased expression of proteins involved in FC efflux. CONCLUSIONS: Increasing dietary cholesterol selectively increases visceral fat adipocyte size, FC and macrophage content, and proinflammatory gene expression in nonhuman primates. Visceral fat cells seem to compensate for increased dietary cholesterol by limiting cholesterol uptake/synthesis and increasing FC efflux pathways.

The macrophage LBP gene is an LXR target that promotes macrophage survival and atherosclerosis.

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate sterol metabolism and inflammation. We sought to identify previously unknown genes regulated by LXRs in macrophages and to determine their contribution to atherogenesis. Here we characterize a novel LXR target gene, the lipopolysaccharide binding protein (LBP) gene. Surprisingly, the ability of LXRs to control LBP expression is cell-type specific, occurring in macrophages but not liver. Treatment of macrophages with oxysterols or loading with modified LDL induces LBP in an LXR-dependent manner, suggesting a potential role for LBP in the cellular response to cholesterol overload. To investigate this further, we performed bone marrow transplant studies. After 18 weeks of Western diet feeding, atherosclerotic lesion burden was assessed revealing markedly smaller lesions in the LBP(-/-) recipients. Furthermore, loss of bone marrow LBP expression increased apoptosis in atherosclerotic lesions as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Supporting in vitro studies with isolated macrophages showed that LBP expression does not affect cholesterol efflux but promotes the survival of macrophages in the setting of cholesterol loading. The LBP gene is a macrophage-specific LXR target that promotes foam cell survival and atherogenesis.

FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.

The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.

Bone marrow NR4A expression is not a dominant factor in the development of atherosclerosis or macrophage polarization in mice.

The formation of the atherosclerotic lesion is a complex process influenced by an array of inflammatory and lipid metabolism pathways. We previously demonstrated that NR4A nuclear receptors are highly induced in macrophages in response to inflammatory stimuli and modulate the expression of genes linked to inflammation in vitro. Here we used mouse genetic models to assess the impact of NR4A expression on atherosclerosis development and macrophage polarization. Transplantation of wild-type, Nur77⁻/⁻, or Nor1⁻/⁻ null hematopoetic precursors into LDL receptor (LDLR)⁻/⁻ recipient mice led to comparable development of atherosclerotic lesions after high-cholesterol diet. We also observed comparable induction of genes linked to M1 and M2 responses in wild-type and Nur77-null macrophages in response to lipopolysaccharides and interleukin (IL)-4, respectively. In contrast, activation of the nuclear receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation of signal transductor and activator of transcription 6 (STAT6) strongly suppressed M2 responses. Recent studies have suggested that alterations in levels of Ly6C(lo) monocytes may be a contributor to inflammation and atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with decreased abundance of Ly6C(lo) monocytes, but this change was not correlated with atherosclerotic lesion development. Collectively, our results suggest that alterations in the Ly6C(lo) monocyte population and bone marrow NR4A expression do not play dominant roles in macrophage polarization or the development of atherosclerosis in mice.