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 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.

Retinol dehydrogenase 11 is essential for the maintenance of retinol homeostasis in liver and testis in mice.

Retinol dehydrogenase 11 (RDH11) is a microsomal short-chain dehydrogenase/reductase that recognizes all-trans- and cis-retinoids as substrates and prefers NADPH as a cofactor. Previous work has suggested that RDH11 contributes to the oxidation of 11-cis-retinol to 11-cis-retinaldehyde during the visual cycle in the eye's retinal pigment epithelium. However, the role of RDH11 in metabolism of all-trans-retinoids remains obscure. Here, we report that microsomes isolated from the testes and livers of Rdh11-/- mice fed a regular diet exhibited a 3- and 1.7-fold lower rate of all-trans-retinaldehyde conversion to all-trans-retinol, respectively, than the microsomes of WT littermates. Testes and livers of Rdh11-/- mice fed a vitamin A-deficient diet had ∼35% lower levels of all-trans-retinol than those of WT mice. Furthermore, the conversion of ß-carotene to retinol via retinaldehyde as an intermediate appeared to be impaired in the testes of Rdh11-/-/retinol-binding protein 4-/-(Rbp4-/-) mice, which lack circulating holo RBP4 and rely on dietary supplementation with ß-carotene for maintenance of their retinoid stores. Together, these results indicate that in mouse testis and liver, RDH11 functions as an all-trans-retinaldehyde reductase essential for the maintenance of physiological levels of all-trans-retinol under reduced vitamin A availability.

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.

Hepatic retinoid stores are required for normal liver regeneration.

Preliminary studies of liver regeneration induced by partial hepatectomy (PHE) identified a substantial depletion of hepatic retinoid stores, by greater than 70%, in regenerating livers of wild-type C57Bl/6J mice. To understand this, we compared responses of wild-type and lecithin:retinol acyltransferase (Lrat)-deficient mice, which totally lack hepatic retinoid stores, to PHE. The Lrat-deficient livers showed delayed regeneration in the first 24 h after PHE. At 12 h after PHE, we observed significantly less mRNA expression for growth factors and cytokines implicated in regulating the priming phase of liver regeneration, specifically for Hgf and Tgfα, but not Tgfß. Compared with wild-type mice, the changes in mRNA levels for p21 and cyclins E1, B1, and A2 mRNAs and for hepatocellular BrdU incorporation and mitoses were delayed (i.e., shifted to later times) in regenerating Lrat(-/-) livers. Concentrations of all-trans-retinoic acid were significantly lower in the livers of Lrat(-/-) mice following PHE, and this was accompanied by diminished expression of known retinoid-responsive genes. At later times after PHE, the rate of liver weight restoration for Lrat(-/-) mice was parallel to that of wild-type mice, although additional biochemical differences were observed. Thus, hepatic retinoid stores are required for maintaining expression of signaling molecules that regulate cell proliferation and differentiation immediately after hepatic injury, accounting for the delayed restoration of liver mass in Lrat(-/-) mice.

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.

Actin fence therapy with exogenous V12Rac1 protects against acute lung injury.

High mortality in acute lung injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF-α receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-α-induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss, and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally (i.n.) as a noncovalent construct with a cell-permeable peptide, enhanced alveolar filamentous actin (F-actin) and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from i.n. instillation of LPS or of Pseudomonas aeruginosa. We propose a potentially new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, and therefore blocking ALI.

Hepatic stellate cells are an important cellular site for ß-carotene conversion to retinoid.

Hepatic stellate cells (HSCs) are responsible for storing 90-95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary ß-carotene, but the ability of HSCs to metabolize ß-carotene in situ has not been explored. To gain understanding of this, we investigated whether ß-carotene-15,15'-monooxygenase (Bcmo1) and ß-carotene-9',10'-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary ß-carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12'-carotenal levels of Bcmo1-deficient mice. Although apo-10'-carotenal, like apo-12'-carotenal, was readily detectable in livers and serum from both wild type and Bcmo1-deficient mice, we were unable to detect either apo-8'- or apo-14'-carotenals in livers or serum from the two strains. We further observed that hepatic triglyceride levels were significantly elevated in livers of Bcmo1-deficient mice fed a ß-carotene-containing diet compared to mice receiving no ß-carotene. Collectively, our data establish that HSCs are an important cellular site for ß-carotene accumulation and metabolism within the liver.

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.

Hepatic stellate cell activation: A source for bioactive lipids.

Hepatic stellate cells (HSCs) are non-parenchymal liver cells that characteristically contain multiple retinoid (vitamin A)-containing lipid droplets. In this study, we addressed the metabolic fate of non-retinoid lipids originating from lipid droplet loss during HSCs activation. UPLC/MS/MS and qRT-PCR were used to monitor the lipid composition and mRNA expression of selected genes regulating lipid metabolism in freshly isolated, overnight-, 3- and 7-day cultures or primary mouse HSCs. A preferential accumulation of specific C20-C24 fatty acid species, especially arachidonic (C20:4) and docosahexaenoic acids (C22:6), was revealed in culture-activated HSCs along with an upregulation of transcription of fatty acid desaturases (Scd1, Scd2) and elongases (Elovl5, Elovl6). This was accompanied with an enrichment of activated HSCs with 36:4 and 38:4 phosphatidylcholine species containing polyunsaturated fatty acids and associated accumulation of selective lipid mediators, including endocannabinoids and related N-acylethanolamides, as well as ceramides. An increase in 2-arachidonoylglycerol and N-arachydonoylethanolamide concentrations was observed along with an upregulation of Daglα mRNA expression in HSCs during culture activation. N-palmitoylethanolamide was identified as the most abundant endocannabinoid-like species in activated HSCs. An increase in total ceramide levels and enrichment with N-palmitoyl (C16:0), N-tetracosenoyl (C24:1), N-tetracosanoyl (C24:0) and N-docosanoyl (C22:0) ceramides was detected in activated HSC cultures and was preceded by increased mRNA expression of ceramide synthesizing enzymes (CerS2, CerS5 and Smpd1). Our data suggest an active redistribution of non-retinoid lipids in HSCs underlying the formation of low abundance, highly bioactive lipid species that may affect signaling during HSC activation, as well as extracellularly within the liver.

The Potential Protective Effect of Oligoribonucleotides-d-Mannitol Complexes against Thioacetamide-Induced Hepatotoxicity in Mice.

This study investigated the potential hepatoprotective effect of oligoribonucleotides-d-mannitol complexes (ORNs-d-M) against thioacetamide (TAA)-induced hepatotoxicity in mice. The hepatoprotective activity of ORNs-d-M was evaluated in thioacetamide (TAA)-treated C57BL/6J. Results indicate that treatment with ORNs-d-M displayed a protective effect at the TAA-induced liver injury. Treatment with ORNs-d-M, starting at 0 h after the administration of TAA, decreased TAA-elevated serum alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (GGT). Activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx), and levels of glutathione (GSH), were enhanced with ORNs-d-M administration, while the hepatic oxidative biomarkers (TBA-reactive substances, protein carbonyl derivatives, protein-SH group) and myeloperoxidase (MPO) activity were reduced. Furthermore, genetic analysis has shown that the ORNs-d-M decreases the expression of mRNA pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6), profibrogenic cytokine-transforming growth factor ß1 (TGF-ß1), as well as the principal protein of the extracellular matrix-collagen I. The present study demonstrates that ORNs-d-M exerts a protective effect against TAA-induced liver injury, which may be associated with its anti-inflammatory effects, inhibition of overexpression of mRNA cytokines, and direct effects on the metabolism of the toxin.

Hepatic Detoxification of Bisphenol A is Retinoid-Dependent.

Bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl) propane) is a widely used industrial chemical. The extensive distribution of BPA in the environment poses risks to humans. However, the molecular mechanisms underlying BPA toxicity as well as its effective detoxification and elimination are not well understood. We have investigated specifically for BPA the notion raised in the literature that the optimal sensing, detoxification, and elimination of xenobiotics requires retinoid (natural derivatives and synthetic analogs of vitamin A) actions. The objective of the study was to explore how retinoids, both those stored in the liver and those originating from recent oral intake, help maintain an optimal xenobiotic detoxification response, affecting mRNA expression and activities of elements of xenobiotic detoxification system upon BPA administration to mice. Wild-type and mice lacking hepatic retinoid stores (Lrat-/-) were acutely treated with BPA (50 mg/kg body weight), with or without oral supplementation with retinyl acetate. Hepatic mRNA expression levels of the genes encoding nuclear receptors and their downstream targets involved in xenobiotic biotransformation, phase I and phase II enzyme activities, and levels of oxidative damage to cellular proteins and lipids in hepatic microsomes, mitochondria and cytosol, were assessed. BPA treatment induced hepatic activities needed for its detoxification and elimination in wild-type mice. However, BPA failed to induce these activities in the livers of Lrat-/- mice. Oral supplementation with retinyl acetate restored phase I and phase II enzyme activities, but accelerated BPA-induced oxidative damage through enhancement of non-mitochondrial ROS production. Thus, the activities of the enzymes involved in the hepatic elimination of BPA require hepatic retinoid stores. The extent of hepatic damage that arises from acute BPA intoxication is directly affected by retinoid administration during the period of BPA exposure and hepatic retinoid stores that have accumulated over the lifetime of the organism.

Antitumor Activity of Alloy and Core-Shell-Type Bimetallic AgAu Nanoparticles.

Nanoparticles (NPs) of noble metals, namely gold and silver, remain promising anticancer agents capable of enhancing current surgery- and chemotherapeutic-based approaches in cancer treatment. Bimetallic AgAu composition can be used as a more effective agent due to the synergetic effect. Among the physicochemical parameters affecting gold and silver nanoparticle biological activity, a primary concern relates to their size, shape, composition, charge, etc. However, the impact of metal components/composition as well as metal topological distribution within NPs is incompletely characterized and remains to be further elucidated and clarified. In the present work, we tested a series of colloidal solutions of AgAu NPs of alloy and core-shell type for an antitumor activity depending on metal molar ratios (Ag:Au = 1:1; 1:3; 3:1) and topological distribution of gold and silver within NPs (AucoreAgshell; AgcoreAushell). The efficacy at which an administration of the gold and silver NPs inhibits mouse Lewis lung carcinoma (LLC) growth in vivo was compared. The data suggest that in vivo antitumor activity of the studied NPs strongly depends on gold and silver interaction arising from their ordered topological distribution. NPs with Ag core covered by Au shell were the most effective among the NPs tested towards LLC tumor growth and metastasizing inhibition. Our data show that among the NPs tested in this study, AgcoreAushell NPs may serve as a suitable anticancerous prototype.

Retinoid-xenobiotic interactions: the Ying and the Yang.

The literature provides compelling evidence pointing to tight metabolic interactions between retinoids and xenobiotics. These are extensive and important for understanding xenobiotic actions in the body. Within the body, retinoids affect xenobiotic metabolism and actions and conversely, xenobiotics affect retinoid metabolism and actions. This article summarizes data that establish the importance of retinoid-dependent metabolic pathways for sustaining the body's responses to xenobiotic exposure, including the roles of all-trans- and 9-cis-retinoic acid for protecting mammals from harmful xenobiotic effects and for ensuring xenobiotic elimination from the body. This review will also consider molecular mechanisms underlying xenobiotic toxicity focusing on how this may contribute to retinoid deficiency and disruption of normal retinoid homeostasis. Special attention is paid to xenobiotic molecular targets (nuclear receptors, regulatory proteins, enzymes, and transporters) which affect retinoid metabolism and signaling.

Retinoids modulate thioacetamide-induced acute hepatotoxicity.

The literature indicates that retinoids can influence the metabolism and actions of xenobiotics and conversely that xenobiotics can influence the metabolism and actions of retinoids. We were interested in understanding the degree to which hepatic retinoid stores, accumulated over a lifetime, affect xenobiotic metabolism, and actions. To investigate this, we induced liver injury through administration of the hepatotoxin thioacetamide (TAA) to chow fed wild type (WT) mice and lecithin:retinol acyltransferase-deficient (Lrat(-/-)) mice that are genetically unable to accumulate hepatic retinoid stores. Within 48 h of TAA-treatment, WT mice develop liver injury as evidenced by focal necrotic areas and increases in serum ALT activity and myeloperoxidase activity in hepatic parenchyma. Simultaneously, features of hepatic encephalopathy develop, as evidenced by a 25% increase in blood ammonia and a threefold reduction of blood glucose levels. This is accompanied by reduced hepatic glutathione, and increased thiobarbituric acid reactive substances, protein carbonyl and sulfhydryl groups, and increased cytochrome P450-catalyzed hydroxylation activity and flavin-containing monooxygenase activity in microsomes prepared from WT liver. Strikingly, none of these TAA-induced effects were observed for matched Lrat(-/-) mice. To confirm that TAA hepatotoxicity depends on retinoid availability, we administered, over 48 h, four oral doses of 3000 IU retinyl acetate each to the mice. This led to the development of hepatotoxicity in Lrat(-/-) mice that was similar in extent to that observed in WT mice. Our findings establish that endogenous hepatic retinoid stores can modulate the toxicity of TAA in mice.

Tryptophan-Assisted Synthesis Reduces Bimetallic Gold/Silver Nanoparticle Cytotoxicity and Improves Biological Activity.

Aiming to reduce the potential in vivo hepato-and nephrotoxicity of Ag/Au bimetallic nanoparticles (NPs) stabilized by sodium dodecyl sulphate (SDS), an approach involving a simultaneous reduction of silver nitrate and tetrachlorauratic acid using tryptophan (Trp) as a reducing/stabilizing agent was applied during NP synthesis. The obtained Ag/Au/Trp NPs (5-15 nm sized) were able to form stable aggregates with an average size of 370-450 nm and were potentially less toxic than Ag/Au/SDS in relation to a mouse model system based on clinical biochemical parameters and oxidative damage product estimation. Ag/Au/Trp NPs were shown to exhibit anticancer activity in relation to a Lewis lung carcinoma model. The data generated from the present study support the fact that the use of tryptophan in NP synthesis is effective in attenuating the potential hepatotoxicity and nephrotoxicity of NPs during their in vivo application.