Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Control by the brain of vitamin A homeostasis.

Vitamin A is a micronutrient essential for vertebrate animals maintained in homeostatic balance in the body; however, little is known about the control of this balance. This study investigated whether the hypothalamus, a key integrative brain region, regulates vitamin A levels in the liver and circulation. Vitamin A in the form of retinol or retinoic acid was stereotactically injected into the 3rd ventricle of the rat brain. Alternatively, retinoids in the mouse hypothalamus were altered through retinol-binding protein 4 (Rbp4) gene knockdown. This led to rapid change in the liver proteins controlling vitamin A homeostasis as well as vitamin A itself in liver and the circulation. Prolonged disruption of Rbp4 in the region of the arcuate nucleus of the mouse hypothalamus altered retinol levels in the liver. This supports the concept that the brain may sense retinoids and influence whole-body vitamin A homeostasis with a possible "vitaminostatic" role.

Dietary Vitamin A Affects the Function of Incretin-Producing Enteroendocrine Cells in Male Mice Fed a High-Fat Diet.

BACKGROUND: Retinol-binding protein 2 (RBP2) is an intracellular carrier for vitamin A in the absorptive enterocytes. Mice lacking RBP2 (Rbp2-/-) display an unexpected phenotype of obesity, glucose intolerance, and elevated glucose-dependent insulinotropic polypeptide (GIP) levels. GIP and glucagon-like peptide 1 (GLP-1) are incretin hormones secreted by enteroendocrine cells (EECs). We recently demonstrated the presence of RBP2 and other retinoid-related proteins in EECs. OBJECTIVES: Given RBP2's role in intracellular retinoid trafficking, we aimed to evaluate whether dietary vitamin A affects incretin-secreting cell function and gene expression. METHODS: Male Rbp2-/- mice and sex- and age-matched controls (n = 6-9) were fed a high-fat diet (HFD) for 18 wk containing normal (VAN, 4000 IU/kg of diet) or low (VAL, 25% of normal) vitamin A concentrations. Body weight was recorded biweekly. Plasma GIP and GLP-1 levels were obtained fasting and 30 min after an oral fat gavage at week 16. Glucose tolerance tests were also performed. Mice were killed at week 18, and blood and tissue samples were obtained. RESULTS: Rbp2-/- mice displayed greater weight gain on the VAN compared with the VAL diet from week 7 of the intervention (P ≤ 0.01). Stimulated GIP levels were elevated in Rbp2-/- mice compared with their controls fed the VAN diet (P = 0.02), whereas their GIP response was lower when fed the VAL diet (P = 0.03). Although no differences in GLP-1 levels were observed in the VAN diet group, a lower GLP-1 response was seen in Rbp2-/- mice fed the VAL diet (P = 0.02). Changes in incretin gene expression and that of other genes associated with EEC lineage and function were consistent with these observations. Circulating and hepatic retinoid levels revealed no systemic vitamin A deficiency across dietary groups. CONCLUSIONS: Our data support a role for RBP2 and dietary vitamin A in incretin secretion and gene expression in mice fed a HFD.

Design and Rationale the SCAN-MP (Screening for Cardiac Amyloidosis With Nuclear Imaging in Minority Populations) Study.

Background Transthyretin amyloid cardiomyopathy (ATTR-CM) is an important cause of heart failure in older individuals. Misfolding and deposition of transthyretin or prealbumin protein causes ATTR-CM in the context of a normal (wild-type) or variant TTR sequence. Variant ATTR-CM is most commonly caused by the substitution of valine for isoleucine at position 122 in transthyretin (Val122Ile or pV142I, almost exclusively observed in individuals of West African ancestry), demonstrated in 3.4% of self-identified Black individuals in the United States with an estimated 1.5 million carriers. Despite the large number of known pV142I carriers, the proportion of older Black patients with heart failure attributable to ATTR-CM remains unknown. Methods To address this knowledge gap, the SCAN-MP (Screening for Cardiac Amyloidosis with Nuclear Imaging in Minority Populations) study was funded by the National Institutes of Health/National Heart, Lung, and Blood Institute (R01HL139671) to enroll a targeted population of self-identified, community-dwelling Black or Caribbean Hispanic patients (many of whom are of West African ancestry) >60 years of age with heart failure and identify ATTR-CM by noninvasive nuclear imaging. The principal objective of SCAN-MP is to determine the prevalence of ATTR-CM in this population. Secondary objectives will explore TTR genotype, demographics, progression of variant versus wild-type ATTR-CM, and biochemical mechanisms of transthyretin amyloid fibril formation. Conclusions The SCAN-MP study is the largest, prospective study of cardiac amyloidosis in Black and Hispanic individuals. Both wild-type and variant ATTR-CM are now treatable with the US Food and Drug-approved drug tafamidis. The insights gained from SCAN-MP are likely to improve those at risk for or afflicted with ATTR-CM. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03812172.

Vitamin A regulates tissue-specific organ remodeling in diet-induced obesity independent of mitochondrial function.

Background: Perturbed mitochondrial energetics and vitamin A (VitA) metabolism are associated with the pathogenesis of diet-induced obesity (DIO) and type 2 diabetes (T2D). Methods: To test the hypothesis that VitA regulates tissue-specific mitochondrial energetics and adverse organ remodeling in DIO, we utilized a murine model of impaired VitA availability and high fat diet (HFD) feeding. Mitochondrial respiratory capacity and organ remodeling were assessed in liver, skeletal muscle, and kidney tissue, which are organs affected by T2D-associated complications and are critical for the pathogenesis of T2D. Results: In liver, VitA had no impact on maximal ADP-stimulated mitochondrial respiratory capacity (VADP) following HFD feeding with palmitoyl-carnitine and pyruvate each combined with malate as substrates. Interestingly, histopathological and gene expression analyses revealed that VitA mediates steatosis and adverse remodeling in DIO. In skeletal muscle, VitA did not affect VADP following HFD feeding. No morphological differences were detected between groups. In kidney, VADP was not different between groups with both combinations of substrates and VitA transduced the pro-fibrotic transcriptional response following HFD feeding. Conclusion: The present study identifies an unexpected and tissue-specific role for VitA in DIO that regulates the pro-fibrotic transcriptional response and that results in organ damage independent of changes in mitochondrial energetics.

Retinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice.

Retinoids are potent transcriptional regulators that act in regulating cell proliferation, differentiation, and other cellular processes. We carried out studies in male mice to establish the importance of local cellular retinoid stores within the lung alveolus for maintaining its health in the face of an acute inflammatory challenge induced by intranasal instillation of lipopolysaccharide. We also undertook single cell RNA sequencing and bioinformatic analyses to identify roles for different alveolar cell populations involved in mediating these retinoid-dependent responses. Here we show that local retinoid stores and uncompromised metabolism and signaling within the lung are required to lessen the severity of an acute inflammatory challenge. Unexpectedly, our data also establish that alveolar cells other than lipofibroblasts, specifically microvascular endothelial and alveolar epithelial cells, are able to take up lipoprotein-transported retinoid and to accumulate cellular retinoid stores that are directly used to respond to an acute inflammatory challenge.

Products of the visual cycle are detected in mice lacking retinol binding protein 4, the only known vitamin A carrier in plasma.

Efficient delivery of vitamin A to the retinal pigment epithelium is vital to the production of the light-sensitive visual chromophore 11-cis-retinal. Nevertheless, retinol binding protein 4 (RBP4) is the only known carrier of vitamin A in plasma. Here, we present new findings that further characterize the visual cycle in the presence of Rbp4 deficiency. In the face of impaired delivery of retinol in Rbp4-/- mice, we determined that 11-cis-retinaldehyde reached levels that were ∼60% of WT at 4 months of age and all-trans-retinyl ester was 18% of normal yet photoreceptor cell loss was apparent by 8 months of age. The lack of Rbp4 appeared to have a greater impact on scotopic rod-mediated responses than on cone function at early ages. Also, despite severely impaired delivery of retinol, bisretinoid lipofuscin that forms as a byproduct of the visual cycle was measurable by HPLC and by quantitative fundus autofluorescence. In mice carrying an Rpe65 amino acid variant that slows visual cycle kinetics, Rbp4 deficiency had a less pronounced effect on 11-cis-retinal levels. Finally, we found that ocular retinoids were not altered in mice expressing elevated adipose-derived total Rbp4 protein (hRBP4+/+AdiCre+/-). In conclusion, our findings are consistent with a model in which vitamin A can be delivered to the retina by Rbp4-independent pathways.

Vitamin A preserves cardiac energetic gene expression in a murine model of diet-induced obesity.

Perturbed vitamin-A metabolism is associated with type 2 diabetes and mitochondrial dysfunction that are pathophysiologically linked to the development of diabetic cardiomyopathy (DCM). However, the mechanism, by which vitamin A might regulate mitochondrial energetics in DCM has previously not been explored. To test the hypothesis that vitamin-A deficiency accelerates the onset of cardiomyopathy in diet-induced obesity (DIO), we subjected mice with lecithin retinol acyltransferase (Lrat) germline deletion, which exhibit impaired vitamin-A stores, to vitamin A-deficient high-fat diet (HFD) feeding. Wild-type mice fed with a vitamin A-sufficient HFD served as controls. Cardiac structure, contractile function, and mitochondrial respiratory capacity were preserved despite vitamin-A deficiency following 20 wk of HFD feeding. Gene profiling by RNA sequencing revealed that vitamin A is required for the expression of genes involved in cardiac fatty acid oxidation, glycolysis, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation in DIO as expression of these genes was relatively preserved under vitamin A-sufficient HFD conditions. Together, these data identify a transcriptional program, by which vitamin A preserves cardiac energetic gene expression in DIO that might attenuate subsequent onset of mitochondrial and contractile dysfunction.NEW & NOTEWORTHY The relationship between vitamin-A status and the pathogenesis of diabetic cardiomyopathy has not been studied in detail. We assessed cardiac mitochondrial respiratory capacity, contractile function, and gene expression by RNA sequencing in a murine model of combined vitamin-A deficiency and diet-induced obesity. Our study identifies a role for vitamin A in preserving cardiac energetic gene expression that might attenuate subsequent development of mitochondrial and contractile dysfunction in diet-induced obesity.

Whole Genome Association Study of the Plasma Metabolome Identifies Metabolites Linked to Cardiometabolic Disease in Black Individuals.

Integrating genetic information with metabolomics has provided new insights into genes affecting human metabolism. However, gene-metabolite integration has been primarily studied in individuals of European Ancestry, limiting the opportunity to leverage genomic diversity for discovery. In addition, these analyses have principally involved known metabolites, with the majority of the profiled peaks left unannotated. Here, we perform a whole genome association study of 2,291 metabolite peaks (known and unknown features) in 2,466 Black individuals from the Jackson Heart Study. We identify 519 locus-metabolite associations for 427 metabolite peaks and validate our findings in two multi-ethnic cohorts. A significant proportion of these associations are in ancestry specific alleles including findings in APOE, TTR and CD36. We leverage tandem mass spectrometry to annotate unknown metabolites, providing new insight into hereditary diseases including transthyretin amyloidosis and sickle cell disease. Our integrative omics approach leverages genomic diversity to provide novel insights into diverse cardiometabolic diseases.

Analysis of lumbar spine stenosis specimens for identification of amyloid.

BACKGROUND: Lumbar spinal stenosis (LSS) is a common reason for spine surgery in which ligamentum flavum is resected. Transthyretin (TTR) amyloid is an often unrecognized and potentially modifiable mechanism for LSS that can also cause TTR cardiac amyloidosis. Accordingly, older adult patients undergoing lumbar spine (LS) surgery were evaluated for amyloid and if present, the precursor protein, as well as comprehensive characterization of the clinical phenotype. METHODS: A prospective, cohort study in 2 academic medical centers enrolled 47 subjects (age 69 ± 7 years, 53% male) undergoing clinically indicated LS decompression. The presence of amyloid was evaluated by Congo Red staining and in those with amyloid, precursor protein was determined by laser capture microdissection coupled to mass spectrometry (LCM-MS). The phenotype was assessed by disease-specific questionnaires (Swiss Spinal Stenosis Questionnaire and Kansas City Cardiomyopathy Questionnaire) and the 36-question short-form health survey, as well as biochemical measures (TTR, retinol-binding protein, and TTR stability). Cardiac testing included technetium-99m-pyrophosphate scintigraphy, electrocardiograms, echocardiograms, and cardiac biomarkers as well as measures of functional capacity. RESULTS: Amyloid was detected in 16 samples (34% of participants) and was more common in those aged ≥ 75 years of age (66.7%) compared with those <75 years (22.3%, p < 0.05). LCM-MS demonstrated TTR as the precursor protein in 62.5% of participants with amyloid while 37.5% had an indeterminant type of amyloid. Demographic, clinical, quality-of-life measures, electrocardiographic, echocardiographic, and biochemical measures did not differ between those with and without amyloid. Among those with TTR amyloid (n = 10), one subject had cardiac involvement by scintigraphy. CONCLUSIONS: Amyloid is detected in more than a third of older adults undergoing LSS. Amyloid is more common with advancing age and is particularly common in those >75 years old. No demographic, clinical, biochemical, or cardiac parameter distinguished those with and without amyloid. In more than half of subjects with LS amyloid, the precursor protein was TTR indicating the importance of pathological assessment.

Intestinal Enteroendocrine Cell Signaling: Retinol-binding Protein 2 and Retinoid Actions.

Retinol-binding protein 2-deficient (Rbp2-/-) mice are more prone to obesity, glucose intolerance, and hepatic steatosis than matched controls. Glucose-dependent insulinotropic polypeptide (GIP) blood levels are dysregulated in these mice. The present studies provide new insights into these observations. Single cell transcriptomic and immunohistochemical studies establish that RBP2 is highly expressed in enteroendocrine cells (EECs) that produce incretins, either GIP or glucagon-like peptide-1. EECs also express an enzyme needed for all-trans-retinoic acid (ATRA) synthesis, aldehyde dehydrogenase 1 family member A1, and retinoic acid receptor-alpha, which mediates ATRA-dependent transcription. Total and GIP-positive EECs are significantly lower in Rbp2-/- mice. The plasma transport protein for retinol, retinol-binding protein 4 (RBP4) is also expressed in EECs and is cosecreted with GIP upon stimulation. Collectively, our data support direct roles for RBP2 and ATRA in cellular processes that give rise to GIP-producing EECs and roles for RBP2 and RBP4 within EECs that facilitate hormone storage and secretion.

Retinol-binding protein 2 (RBP2): More than just dietary retinoid uptake.

Retinol-binding protein 2 (RBP2, also known as cellular retinol-binding protein 2 (CRBP2)) is a member of the fatty acid-binding protein family and has been extensively studied for its role in facilitating dietary vitamin A (retinol) uptake and metabolism within enterocytes of the small intestine. RBP2 is present in highest concentrations in the proximal small intestine where it constitutes approximately 0.1-0.5% of soluble protein. Recent reports have established that RBP2 binds monoacylglycerols (MAGs) with high affinity, including the canonical endocannabinoid 2-arachidonoylglycerol (2-AG). Crystallographic studies reveal that retinol, 2-AG, or other long-chain MAGs alternatively can bind in the retinol-binding pocket of RBP2. It also has been demonstrated recently that Rbp2-deficient mice are more susceptible to developing obesity and associated metabolic phenotypes when exposed to a high fat diet, or as they age when fed a conventional chow diet. When subjected to an oral fat challenge, the Rbp2-deficient mice release into the circulation significantly more, compared to littermate controls, of the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP). These new findings regarding RBP2 structure and actions within the intestine are the focus of this review.

Alcohol induced hepatic retinoid depletion is associated with the induction of multiple retinoid catabolizing cytochrome P450 enzymes.

Chronic alcohol consumption leads to a spectrum of liver disease that is associated with significant global mortality and morbidity. Alcohol is known to deplete hepatic vitamin A content, which has been linked to the pathogenesis of alcoholic liver disease. It has been suggested that induction of Cytochrome P450 2E1 (CYP2E1) contributes to alcohol-induced hepatic vitamin A depletion, but the possible contributions of other retinoid-catabolizing CYPs have not been well studied. The main objective of this study was to better understand alcohol-induced hepatic vitamin A depletion and test the hypothesis that alcohol-induced depletion of hepatic vitamin A is due to CYP-mediated oxidative catabolism. This hypothesis was tested in a mouse model of chronic alcohol consumption, including wild type and Cyp2e1 -/- mice. Our results show that chronic alcohol consumption is associated with decreased levels of hepatic retinol, retinyl esters, and retinoic acid. Moreover, the depletion of hepatic retinoid is associated with the induction of multiple retinoid catabolizing CYPs, including CYP26A1, and CYP26B1 in alcohol fed wild type mice. In Cyp2e1 -/- mice, alcohol-induced retinol decline is blunted but retinyl esters undergo a change in their acyl composition and decline upon alcohol exposure like WT mice. In conclusion, the alcohol induced decline in hepatic vitamin A content is associated with increased expression of multiple retinoid-catabolizing CYPs, including the retinoic acid specific hydroxylases CYP26A1 and CYP26B1.

Vitamin A and Vitamin E: Will the Real Antioxidant Please Stand Up?

Vitamin A, acting through its metabolite, all-trans-retinoic acid, is a potent transcriptional regulator affecting expression levels of hundreds of genes through retinoic acid response elements present within these genes. However, the literature is replete with claims that consider vitamin A to be an antioxidant vitamin, like vitamins C and E. This apparent contradiction in the understanding of how vitamin A acts mechanistically within the body is a major focus of this review. Vitamin E, which is generally understood to act as a lipophilic antioxidant protecting polyunsaturated fatty acids present in membranes, is often proposed to be a transcriptional regulator. The evaluation of this claim is another focus of the review. We conclude that vitamin A is an indirect antioxidant, whose indirect function is to transcriptionally regulate a number of genes involved in mediating the body's canonical antioxidant responses. Vitamin E, in addition to being a direct antioxidant, prevents the increase of peroxidized lipids that alter both metabolic pathways and gene expression profiles within tissues and cells. However, there is little compelling evidence that vitamin E has a direct transcriptional mechanism like that of vitamin A. Thus, we propose that the term antioxidant not be applied to vitamin A, and we discourage the use of the term transcriptional mediator when discussing vitamin E.

Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands.

Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format. By examining chemical libraries of bioactive lipids, we provided evidence for the selective interaction of CRBP2 with a subset of nonretinoid ligands with the highest affinity for sn-1 and sn-2 MAGs that contain polyunsaturated C18-C20 acyl chains. We also elucidated the structure-affinity relationship for nonretinoid ligands of this protein. We further dissect the molecular basis for this ligand's specificity by analyzing high-resolution crystal structures of CRBP2 in complex with selected derivatives of MAGs. Finally, we identify T51 and V62 as key amino acids that enable the broadening of ligand selectivity to MAGs in CRBP2 as compared with retinoid-specific CRBP1. Thus, our study provides the molecular framework for understanding the lipid selectivity and diverse functions of CRBPs in controlling lipid homeostasis.

Retinol-binding protein 2 (RBP2): biology and pathobiology.

Retinol-binding protein 2 (RBP2; originally cellular retinol-binding protein, type II (CRBPII)) is a 16 kDa cytosolic protein that in the adult is localized predominantly to absorptive cells of the proximal small intestine. It is well established that RBP2 plays a central role in facilitating uptake of dietary retinoid, retinoid metabolism in enterocytes, and retinoid actions locally within the intestine. Studies of mice lacking Rbp2 establish that Rbp2 is not required in times of dietary retinoid-sufficiency. However, in times of dietary retinoid-insufficiency, the complete lack of Rbp2 gives rise to perinatal lethality owing to RBP2 absence in both placental (maternal) and neonatal tissues. Moreover, when maintained on a high-fat diet, Rbp2-knockout mice develop obesity, glucose intolerance and a fatty liver. Unexpectedly, recent investigations have demonstrated that RBP2 binds long-chain 2-monoacylglycerols (2-MAGs), including the canonical endocannabinoid 2-arachidonoylglycerol, with very high affinity, equivalent to that of retinol binding. Crystallographic studies establish that 2-MAGs bind to a site within RBP2 that fully overlaps with the retinol binding site. When challenged orally with fat, mucosal levels of 2-MAGs in Rbp2 null mice are significantly greater than those of matched controls establishing that RBP2 is a physiologically relevant MAG-binding protein. The rise in MAG levels is accompanied by elevations in circulating levels of the hormone glucose-dependent insulinotropic polypeptide (GIP). It is not understood how retinoid and/or MAG binding to RBP2 affects the functions of this protein, nor is it presently understood how these contribute to the metabolic and hormonal phenotypes observed for Rbp2-deficient mice.

Retinol-binding protein 2 (RBP2) binds monoacylglycerols and modulates gut endocrine signaling and body weight.

Expressed in the small intestine, retinol-binding protein 2 (RBP2) facilitates dietary retinoid absorption. Rbp2-deficient (Rbp2-/- ) mice fed a chow diet exhibit by 6-7 months-of-age higher body weights, impaired glucose metabolism, and greater hepatic triglyceride levels compared to controls. These phenotypes are also observed when young Rbp2-/- mice are fed a high fat diet. Retinoids do not account for the phenotypes. Rather, RBP2 is a previously unidentified monoacylglycerol (MAG)-binding protein, interacting with the endocannabinoid 2-arachidonoylglycerol (2-AG) and other MAGs with affinities comparable to retinol. X-ray crystallographic studies show that MAGs bind in the retinol binding pocket. When challenged with an oil gavage, Rbp2-/- mice show elevated mucosal levels of 2-MAGs. This is accompanied by significantly elevated blood levels of the gut hormone GIP (glucose-dependent insulinotropic polypeptide). Thus, RBP2, in addition to facilitating dietary retinoid absorption, modulates MAG metabolism and likely signaling, playing a heretofore unknown role in systemic energy balance.

Macrophage MerTK Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is emerging as a leading cause of chronic liver disease. However, therapeutic options are limited by incomplete understanding of the mechanisms of NASH fibrosis, which is mediated by activation of hepatic stellate cells (HSCs). In humans, human genetic studies have shown that hypomorphic variations in MERTK, encoding the macrophage c-mer tyrosine kinase (MerTK) receptor, provide protection against liver fibrosis, but the mechanisms remain unknown. We now show that holo- or myeloid-specific Mertk targeting in NASH mice decreases liver fibrosis, congruent with the human genetic data. Furthermore, ADAM metallopeptidase domain 17 (ADAM17)-mediated MerTK cleavage in liver macrophages decreases during steatosis to NASH transition, and mice with a cleavage-resistant MerTK mutant have increased NASH fibrosis. Macrophage MerTK promotes an ERK-TGFß1 pathway that activates HSCs and induces liver fibrosis. These data provide insights into the role of liver macrophages in NASH fibrosis and provide a plausible mechanism underlying MERTK as a genetic risk factor for NASH fibrosis.

Constitutive androstane receptor mediates PCB-induced disruption of retinoid homeostasis.

Environmental exposure to polychlorinated biphenyls (PCBs) is associated with an increased risk of incidence of metabolic disease, however the molecular mechanisms underlying this phenomenon are not fully understood. Our study provides new insights into molecular interactions between PCBs and retinoids (vitamin A and its metabolites) by defining a role for constitutive androstane receptor (CAR) in the disruption of retinoid homeostasis by non-coplanar 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153). Administration of four weekly 50 mg/kg doses of PCB153 to C57BL/6 male mice resulted in a significant decline in the tissue concentrations of retinyl esters, retinol and all-trans-retinoic acid (atRA), while no decline in hepatic and adipose tissue retinoid levels were detected in Car-null littermates. Our data imply that disrupted retinoid homeostasis occurs as a consequence of PCB153-induced activation of CAR, and raise the possibility that CAR signaling can affect atRA homeostasis in vivo. A strong correlation between the changes in retinoid metabolism and extensive upregulation of hepatic CAR-driven Cyp2b10 expression implicates this CYP isoform as contributing to retinoid homeostasis disruption via atRA oxidation during PCB153 exposure. In response to PCB153-induced CAR activation and disruption of retinoid homeostasis, expression of hepatic Pepck, Cd36 and adipose tissue Pparγ, Cd36, Adipoq, and Rbp4 were altered; however, this was reversed by administration of exogenous dietary retinoids (300 IU daily for 4 weeks). Our study establishes that PCB153 exposure enables a significant disruption of retinoid homeostasis in a CAR-dependent manner. We propose that this contributes to the obesogenic properties of PCB153 and may contribute to the predisposition to the metabolic disease.

Design, Synthesis, and Preclinical Efficacy of Novel Nonretinoid Antagonists of Retinol-Binding Protein 4 in the Mouse Model of Hepatic Steatosis.

Retinol-binding protein 4 (RBP4) serves as a transporter for all- trans-retinol (1) in the blood, and it has been proposed to act as an adipokine. Elevated plasma levels of the protein have been linked to diabetes, obesity, cardiovascular diseases, and nonalcoholic fatty liver disease (NAFLD). Recently, adipocyte-specific overexpression of RBP4 was reported to cause hepatic steatosis in mice. We previously identified an orally bioavailable RBP4 antagonist that significantly lowered RBP4 serum levels in Abca4-/- knockout mice with concomitant normalization of complement system protein expression and reduction of bisretinoid formation within the retinal pigment epithelium. We describe herein the discovery of novel RBP4 antagonists 48 and 59, which reduce serum RBP4 levels by >80% in mice upon acute oral dosing. Furthermore, 59 demonstrated efficacy in the transgenic adi-hRBP4 murine model of hepatic steatosis, suggesting that RBP4 antagonists may also have therapeutic utility for the treatment of NAFLD.