Retinyl Esters Research Articles

Probiotic modulation of perfluorobutanesulfonate toxicity in zebrafish: Disturbances in retinoid metabolism and visual physiology

Perfluorobutanesulfonate (PFBS), an aquatic pollutant of emerging concern, is found to disturb gut microbiota, retinoid metabolism and visual signaling in teleosts, while probiotic supplementation can shape gut microbial community to improve retinoid absorption. However, it remains unknown whether probiotic bacteria can modulate the toxicities of PFBS on retinoid metabolism and visual physiology. In the present study, adult zebrafish were exposed for 28 days to 0, 10 and 100 μg/L PFBS, with or without dietary administration of probiotic Lactobacillus rhamnosus. Interaction between PFBS and probiotic was examined regarding retinoid dynamics (intestine, liver and eye) and visual stimuli transmission. PFBS single exposures remarkably inhibited the absorption of retinyl ester in female intestines, which were, however, restored by probiotic to normal status. Although coexposure scenarios markedly increased the hepatic storage of retinyl ester in females, mobilization of retinol was reduced in livers by single or combined exposures regardless of sex. In the eyes, transport and catalytic conversion of retinol to retinal and retinoic acid were interrupted by PFBS alone, which were efficiently antagonized by probiotic presumably through an indirect action. In response to the availability of retinal chromophore, transcriptions of opsins and arrestin genes were altered adaptively to control visual perception and termination. Neurotransmission across retina circuitry was changed accordingly, centering on epinephrine and norepinephrine. In summary, the present study found the efficient modulation of probiotic on retinoid metabolic disorders of PFBS pollution, which subsequently impacted visual signaling. A future work is warranted to provide mechanistic clues in retinoid interaction.

Validation of a Method for Estimating Vitamin A Absorption Efficiency Based on Compartmental Analysis of Post-Prandial Plasma Retinyl Ester Kinetics

Abstract Objectives Because good methods are not available to estimate vitamin A (VA) absorption, we evaluated an approach based on modeling retinyl ester response to an oral VA dose. Methods We generated data for 12 theoretical subjects, assigning values for VA absorption, stores, and kinetic parameters; we used WinSAAM (Simulation, Analysis and Modeling software) to simulate data (without and with 5% average error) for plasma chylomicron retinyl esters (RE) and retinol versus time (30 min to 8 h or 56 d, respectively) after ingestion of labeled VA; next we fit data to a previously-published 9-component model for VA metabolism to obtain “known” values for VA absorption. Then RE data only were modeled for each subject using a robust (n = 16 times) vs truncated sampling schedule (n = 10) and model-predicted absorptions were compared to known values. Areas under the plasma RE response curves (AUCs) were also calculated and compared to known absorption values. Results Known values for VA absorption based on modeling all data with error ranged from 54 – 92% (mean, 72%), VA stores from 160 – 1775 μmol, and chylomicron t1/2 from 6 – 12 min. Using the full sampling scheme for RE, the ratio of model-predicted to known absorption ranged from 0.927 – 1.06 (mean, 0.997); using the truncated scheme, the ratio was 0.814 – 1.13 (mean, 0.973). AUCs were not significantly correlated with known values for VA absorption (R2 = 0.112; P > 0.05), presumably because absorption and chylomicron catabolism are occurring simultaneously. Conclusions By modeling chylomicron RE tracer data after ingestion of labeled VA, absorption efficiency was estimated accurately using error-free data; using data with 5% error, estimates were within 10% of known values (full sampling) or within 20% (truncated). If subjects, after an overnight fast, consume a breakfast containing some fat and a known amount of VA, then no tracer is required to estimate VA absorption using this modeling approach. By incorporating a population-based design, the method could be used in children. Funding Sources Supported by Bill & Melinda Gates Foundation (Project Number OPP1115464) and HarvestPlus (BH183438).

Dynamics of vitamin A uptake, storage, and utilization in vocal fold mucosa

ObjectiveExtrahepatic vitamin A is housed within organ-specific stellate cells that support local tissue function. These cells have been reported in the vocal fold mucosa (VFM) of the larynx; however, it is unknown how vitamin A reaches and is disseminated among VFM target cells, how VFM storage and utilization vary as a function of total body stores, and how these parameters change in the context of pathology. Therefore, in this study, we investigated fundamental VFM vitamin A uptake and metabolism. MethodsUsing cadaveric tissue and serum from human donors representing the full continuum of clinical vitamin A status, we established a concentration range and analyzed the impact of biologic and clinical covariates on VFM vitamin A. We additionally conducted immunodetection of vitamin A-associated markers and pharmacokinetic profiling of orally dosed α-retinyl ester (a chylomicron tracer) in rats. ResultsSerum vitamin A was a significant predictor of human VFM concentrations, suggesting that VFM stores may be rapidly metabolized in situ and replenished from the circulatory pool. On a vitamin A-sufficient background, dosed α-vitamin A was detected in rat VFM in both ester and alcohol forms, showing that, in addition to plasma retinol and local stellate cell stores, VFM can access and process postprandial retinyl esters from circulating chylomicra. Both α forms were rapidly depleted, confirming the high metabolic demand for vitamin A within VFM. ConclusionThis thorough physiological analysis validates VFM as an extrahepatic vitamin A repository and characterizes its unique uptake, storage, and utilization phenotype.

Lysosomal acid lipase is the major acid retinyl ester hydrolase in cultured human hepatic stellate cells but not essential for retinyl ester degradation

Vitamin A is stored as retinyl esters (REs) in lipid droplets of hepatic stellate cells (HSCs). To date, two different pathways are known to facilitate the breakdown of REs: (i) Hydrolysis of REs by neutral lipases, and (ii) whole lipid droplet degradation in autolysosomes by acid hydrolysis.In this study, we evaluated the contribution of neutral and acid RE hydrolases to the breakdown of REs in human HSCs. (R)-Bromoenol lactone (R-BEL), inhibitor of adipose triglyceride lipase (ATGL) and patatin-like phospholipase domain-containing 3 (PNPLA3), the hormone-sensitive lipase (HSL) inhibitor 76-0079, as well as the serine-hydrolase inhibitor Orlistat reduced neutral RE hydrolase activity of LX-2 cell-lysates between 20 and 50%. Interestingly, in pulse-chase experiments, R-BEL, 76-0079, as well as Orlistat exerted little to no effect on cellular RE breakdown of LX-2 cells as well as primary human HSCs. In contrast, Lalistat2, a specific lysosomal acid lipase (LAL) inhibitor, virtually blunted acid in vitro RE hydrolase activity of LX-2 cells. Accordingly, HSCs isolated from LAL-deficient mice showed RE accumulation and were virtually devoid of acidic RE hydrolase activity. In pulse-chase experiments however, LAL-deficient HSCs, similar to LX-2 cells and primary human HSCs, were not defective in degrading REs.In summary, results demonstrate that ATGL, PNPLA3, and HSL contribute to neutral RE hydrolysis of human HSCs. LAL is the major acid RE hydrolase in HSCs. Yet, LAL is not limiting for RE degradation under serum-starvation. Together, results suggest that RE breakdown of HSCs is facilitated by (a) so far unknown, non-Orlistat inhibitable RE-hydrolase(s).

Characterization of an Acyl‐CoA:retinol Acyltransferase (ARAT) Activity in the Hepatopancreas of the American Lobster (Homarus americanus)

Vitamin A (retinol) is a necessary nutrient in higher animals. In mammals, the pathways by which retinol is transported and metabolized are well described. One important reaction in these pathways is the conversion of retinol into retinyl esters, which occurs in several tissues, including the small intestine, liver, and eye. Little is known about the conversion of retinol into retinyl esters in invertebrates. We have identified an acyl‐CoA:retinol acyltransferase (ARAT) activity in the hepatopancreas of the American lobster (Homarus americanus). This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax values for both retinol and acyl‐CoA.Support or Funding InformationCentre College

Postnatal loss of Cyp26a1 and Cyp26b1 in mice causes impaired retinoid homeostasis in multiple organs

Retinoic acid (RA), the active metabolite of vitamin A (retinol), is essential to various biological processes such as maintenance of epithelia and regulation of cell division, proliferation and differentiation. Cyp26a1 and Cyp26b1 enzymes catalyze hydroxylation of RA and are believed to be responsible for RA clearance in all chordates. Global knockout of either Cyp26a1 or Cyp26b1 is embryonic lethal, demonstrating a critical role of Cyp26 enzymes in regulating RA signaling during fetal development. However, whether Cyp26 activity is required in postnatal life to maintain vitamin A homeostasis is still not clear. We hypothesized that postnatal loss of Cyp26a1 and Cyp26b1 would decrease RA clearance, and consequently alter tissue retinoid concentrations and cause retinoid toxicity. A tamoxifen‐inducible Cre‐loxP system was used to induce a conditional global knockout of Cyp26a1 and Cyp26b1 (double‐knockout; DKO). The knock‐out was induced in female and male mice with tamoxifen injections at 21 days postpartum and then these animals were monitored (body weight and condition) biweekly for 8 weeks. Animals were euthanized if they received a body condition score ≤ 2 or had lost > 15% of their pre‐tamoxifen injection body weight. Tissues were harvested for histological analysis and retinoid measurements. All WT animals were healthy and euthanized after the 8‐week follow‐up but 45% of male and 41% of female DKO animals were euthanized before the completion of the 8‐week follow‐up according to the criteria described above. Compared to WT mice, DKO animals showed significant decrease in weight gain. DKO mice had moderate to severe blepharitis and dermatitis with skin thickening and inflammation. Spleens of DKO mice were significantly enlarged and showed extramedullary hematopoiesis. Cell proliferation in the epidermis and in the epithelium of nonglandular stomach was increased in DKO animals. In general, retinoid homeostasis was altered in DKO animals. In comparison to WT animals, skin, spleen and liver RA concentrations were significantly increased in DKO mice while tissue retinol and retinyl ester concentrations were unchanged. In contrast, serum retinol and retinol binding protein 4 (RBP4) concentrations were significantly decreased in DKO mice with no change in RA concentrations. When DKO animals were dosed with exogenous RA (1mg/kg; i.p.), animals showed signs of toxicity and the exposure to RA was significantly greater in DKO animals than in WT animals. Collectively these data show that Cyp26a1 and Cyp26b1 are required for maintaining vitamin A homeostasis and retinoid signaling also in postnatal life.

Dissecting the Functional Role of Palmitoylation on RPE65 Protein

RPE65, also known as retinoid isomerase, is a key player in the visual cycle of retina. Interaction with membranes is necessary for the catalytic function of RPE65, even though it lacks any membrane binding motifs. It has been proposed that palmitoylation of RPE65 controls its membrane association. However, several other studies have challenged the palmitoylation of RPE65 and suggested different modes of membrane association such as electrostatic or hydrophobic interactions. To date, the structural characterization of RPE65-membrane interaction is still rather limited. To gain insight into the mechanism of RPE65 membrane association, we re-examined the palmitoylation status of RPE65 and determined its functional significance. Our findings demonstrate that RPE65 is a palmitoylated protein using modern palmitoylation detection assays in combination with mass spectrometric techniques, but this is not universal (about 25%). We mapped the two palmitoylation sites at residues C112 and C146. Inhibition of palmitoylation partly abolished RPE65-membrane association, furthermore, C112 mutants were significantly impeded in membrane association. In a non-RPE heterologous visual cycle system, the extent of RPE65 palmitoylation was modulated by the presence of active LRAT which clearly suggests that palmitate shuttles between RPE65 and LRAT and thus regulates RPE65 palmitoylation dynamics. Isomerase assays show that C146 mutants are still active, ∼20-50% lower than the wild type RPE65, which indicates that C146 might contribute in the efficient release of bound palmitate derived from substrate, all-trans retinyl ester. In contrast, C112 mutants do not show any isomerization activity and thus are catalytically inactive. The absence of RPE65 on membrane correlates with the loss in the activity of C112 mutants. We propose that the origin and role of palmitoylation at C112 and C146 are different in nature, a point which needs further investigation in detail and currently ongoing in the laboratory.

Post-natal all-trans-retinoic acid biosynthesis.

Generation of the autacoid all-trans-retinoic acid (ATRA) from retinol (vitamin A) relies on a complex metabolon that includes retinol binding-proteins and enzymes from the short-chain dehydrogenase/reductase and aldehyde dehydrogenase gene families. Serum retinol binding-protein delivers all-trans-retinol (vitamin A) from blood to cells through two membrane receptors, Stra6 and Rbpr2. Stra6 and Rbpr2 convey retinol to cellular retinol binding-protein type 1 (Crbp1). Holo-Crbp1 delivers retinol to lecithin: retinol acyl transferase (Lrat) for esterification and storage. Lrat channels retinol directly into its active site from holo-Crbp1 by protein-protein interaction. The ratio apo-Crbp1/holo-Crbp1 directs flux of retinol into and out of retinyl esters, through regulating esterification vs ester hydrolysis. Multiple retinol dehydrogenases (Rdh1, Rdh10, Dhrs9, Rdhe2, Rdhe2s) channel retinol from holo-Crbp1 to generate retinal for ATRA biosynthesis. β-Carotene oxidase type 1 generates retinal from carotenoids, delivered by the scavenger receptor-B1. Retinal reductases (Dhrs3, Dhrs4, Rdh11) reduce retinal into retinol, thereby restraining ATRA biosynthesis. Retinal dehydrogenases (Raldh1, 2, 3) dehydrogenate retinal irreversibly into ATRA. ATRA regulates its own concentrations by inducing Lrat and ATRA degradative enzymes. ATRA exhibits hormesis. Its effects relate to its concentration as an inverted J-shaped curve, transitioning from beneficial in the "goldilocks" zone to toxicity, as concentrations increase. Hormesis has distorted understanding physiological effects of ATRA post-nataly using chow-diet fed, ATRA-dosed animal models. Cancer, immune deficiency and metabolic abnormalities result from mutations and/or insufficiency in Crbp1 and retinoid metabolizing enzymes.

PNPLA3-A Potential Therapeutic Target for Personalized Treatment of Chronic Liver Disease.

Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a lipid droplet-associated protein that has been shown to have hydrolase activity toward triglycerides and retinyl esters. The first evidence of PNPLA3 being associated with fatty liver disease was revealed by a genome-wide association study (GWAS) of Hispanic, African American, and European American individuals in the Dallas Heart Study back in 2008. Since then, numerous GWAS reports have shown that PNPLA3 rs738409[G] (148M) variant is associated with hepatic triglyceride accumulation (steatosis), inflammation, fibrosis, cirrhosis, and even hepatocellular carcinoma regardless of etiologies including alcohol- or obesity-related and others. The frequency of PNPLA3(148M) variant ranges from 17% in African Americans, 23% in European Americans, to 49% in Hispanics in the Dallas Heart Study. Due to high prevalence of obesity and alcohol consumption in modern societies, the PNPLA3(148M) gene variant and environment interaction poses a serious concern for public health, especially chronic liver diseases including alcohol-related liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD). Therefore, PNPLA3(148M) variant is a potential therapeutic target for chronic liver disease in the rs738409 allele carriers. Currently, there is no approved drug specifically targeting the PNPLA3(148M) variant yet. With additional mechanistic studies, novel therapeutic strategies are expected to be developed for the treatment of the PNPLA3(148M) variant-associated chronic liver diseases in the near future.

Farnesoid X receptor and bile acids regulate vitamin A storage

The nuclear receptor Farnesoid X Receptor (FXR) is activated by bile acids and controls multiple metabolic processes, including bile acid, lipid, carbohydrate, amino acid and energy metabolism. Vitamin A is needed for proper metabolic and immune control and requires bile acids for efficient intestinal absorption and storage in the liver. Here, we analyzed whether FXR regulates vitamin A metabolism. Compared to control animals, FXR-null mice showed strongly reduced (>90%) hepatic levels of retinol and retinyl palmitate and a significant reduction in lecithin retinol acyltransferase (LRAT), the enzyme responsible for hepatic vitamin A storage. Hepatic reintroduction of FXR in FXR-null mice induced vitamin A storage in the liver. Hepatic vitamin A levels were normal in intestine-specific FXR-null mice. Obeticholic acid (OCA, 3 weeks) treatment rapidly reduced (>60%) hepatic retinyl palmitate levels in mice, concurrent with strongly increased retinol levels (>5-fold). Similar, but milder effects were observed in cholic acid (12 weeks)-treated mice. OCA did not change hepatic LRAT protein levels, but strongly reduced all enzymes involved in hepatic retinyl ester hydrolysis, involving mostly post-transcriptional mechanisms. In conclusion, vitamin A metabolism in the mouse liver heavily depends on the FXR and FXR-targeted therapies may be prone to cause vitamin A-related pathologies.

Comparison between the enzymatic activity, structure and substrate binding of mouse and human lecithin retinol acyltransferase

Lecithin retinol acyltransferase (LRAT) is involved in the visual cycle where it catalyzes the formation of all-trans retinyl ester. The mouse animal model has been widely used to study LRAT. Primary sequence alignment shows 80% identity and 90% similarity between human and mouse LRAT. However, human LRAT has a proline at position 173 (hLRAT (P173)) while an arginine can be found at this position for the mouse protein (mLRAT (R173)). Moreover, residue 173 is important for the human protein since a substitution mutation of this residue to a leucine (P173L-hLRAT) caused night blindness in a patient. The present study was thus undertaken to determine whether mouse and human LRAT have a similar enzymatic activity, structure and substrate binding affinity using a truncated form of LRAT (tLRAT). The enzymatic activity and binding affinity to the substrate, all-trans retinol, of mtLRAT (R173) were found to be 2.7- and 3.9-fold lower, respectively, than that of htLRAT (P173). Moreover, the enzymatic activity of P173L-htLRAT is 6.3-fold lower compared to that of htLRAT (P173). Furthermore, a significant difference was observed between the intrinsic fluorescence emission as well as between the circular dichroism spectra of mtLRAT (R173) and htLRAT (P173). In addition, mtLRAT proteins are less thermostable than htLRAT proteins, which suggests that structural differences exist between the mouse and human proteins. Altogether, these data strongly suggest that the much lower catalytic activity of mtLRAT (R173) compared to that of htLRAT (P173) mostly results from differences between their structure, predominantly revealed by their dissimilar thermal stability, as well as their efficiency to bind all-trans retinol. Therefore, conclusions regarding the behavior of human LRAT based on measurements performed with mouse LRAT must be made with caution. Also, the much lower enzymatic activity of P173L-htLRAT compared to that of htLRAT (P173) might explain the night blindness of a patient carrying this mutation.

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.

South African preschool children habitually consuming sheep liver and exposed to vitamin A supplementation and fortification have hypervitaminotic A liver stores: a cohort study

In some regions, multiple vitamin A (VA) interventions occur in the same target groups, which may lead to excessive stores. Retinol isotope dilution (RID) is a more sensitive technique than serum retinol to measure VA status. We evaluated VA status before and after a high-dose supplement in preschool children living in a region in South Africa with habitual liver consumption and exposed to VA supplementation and fortification. After baseline blood samples, subjects (46.7±8.4 mo; n=94) were administered 1.0 μmol [14,15]-13C2-retinyl acetate to estimate total liver retinol reserves by RID with a follow-up 14-d blood sample. Liver intake was assessed with a frequency questionnaire. In line with current practice, a routine 200,000 IU VA capsule was administered after the RID test. RID was repeated 1 mo later. Serum retinyl esters were evaluated using ultra-performance liquid chromatography. At baseline, 63.6% of these children had hypervitaminosis A defined as total liver retinol reserves ≥1.0 μmol/g liver, which increased to 71.6% after supplementation (1.13±0.43 to 1.29±0.46 μmol/g; P<0.001). Total serum VA as retinyl esters was elevated in 4.8% and 6.1% of children before and after supplementation. The odds of having hypervitaminosis A at baseline were higher in children consuming liver ≥1/mo (ratio 3.70 [95% CI: 1.08, 12.6]) and in children receiving 2 (4.28 [1.03, 17.9]) or 3 (6.45 [0.64, 65.41]) supplements in the past 12 mo. Total body stores decreased after the supplement in children in the highest quartile at baseline compared with children with lower stores, who showed an increase (P=0.007). In children, such as this cohort in South Africa, with adequate VA intake through diet, and overlapping VA fortification and supplementation, preschool VA capsule distribution should be re-evaluated. This trial was registered at https://clinicaltrials.gov/ct2/show/NCT02915731 as NCT02915731.

Pancreatic lipase-related protein 2 is responsible for the increased hepatic retinyl ester hydrolase activity in vitamin A-deficient mice.

The formation and hydrolysis of hepatic retinyl esters (RE) is a key process inmaintaining serum retinol homeostasis. During vitamin A deficiency, the activity of RE hydrolases (REH) in liver increases to cope with the reduced dietary vitamin A intake. However, it remains unclear which REH is the main enzyme responsible for RE hydrolysis in the liver under physiological conditions. Our previous studies have shown that pancreatic lipase-related protein 2 (PLRP2) is conditionally expressed in the liver and may be involved in the hydrolysis of hepatic RE. In the current study, we generated Plrp2-/- mice using transcription activator-like effector nuclease technology to investigate the role of PLRP2 in the metabolism of hepatic RE. Compared with the mice fed normal diet, the hepatic REH activity of wild-type (WT) mice fed vitamin A-deficient diet (VAD) increased significantly, while this activity did not increase in Plrp2-/- mice fed VAD. Plrp2-/- mice showed higher residual RE content in liver and lower serum retinol level, compared with WT mice fed VAD. Hepatic metabolic profiling from 1 H NMR-based metabolomics suggested that Plrp2-/- mice were more sensitive to VAD. Docking analysis and enzyme activity assay revealed that retinyl palmitate was the substrate with higher affinity for PLRP2. Our results indicate that Plrp2 can be activated in the liver and is responsible for the increased REH activity in the liver of mice fed VAD.

Hepatocyte-specific deletion of lysosomal acid lipase leads to cholesteryl ester but not triglyceride or retinyl ester accumulation

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl ester (CE) and retinyl ester (RE) and triglyceride (TG). Mice globally lacking LAL accumulate CE most prominently in the liver. The severity of the CE accumulation phenotype progresses with age and is accompanied by hepatomegaly and hepatic cholesterol crystal deposition. In contrast, hepatic TG accumulation is much less pronounced in these mice, and hepatic RE levels are even decreased. To dissect the functional role of LAL for neutral lipid ester mobilization in the liver, we generated mice specifically lacking LAL in hepatocytes (hep-LAL-ko). On a standard chow diet, hep-LAL-ko mice exhibited increased hepatic CE accumulation but unaltered TG and RE levels. Feeding the hep-LAL-ko mice a vitamin A excess/high-fat diet (VitA/HFD) further increased hepatic cholesterol levels, but hepatic TG and RE levels in these mice were lower than in control mice. Performing in vitro activity assays with lysosome-enriched fractions from livers of mice globally lacking LAL, we detected residual acid hydrolytic activities against TG and RE. Interestingly, this non-LAL acid TG hydrolytic activity was elevated in lysosome-enriched fractions from livers of hep-LAL-ko mice upon VitA/HFD feeding. In conclusion, the neutral lipid ester phenotype in livers from hep-LAL-ko mice indicates that LAL is limiting for CE turnover, but not for TG and RE turnovers. Furthermore, in vitro hydrolase activity assays revealed the existence of non-LAL acid hydrolytic activities for TG and RE. The corresponding acid lipase(s) catalyzing these reactions remains to be identified.

The Effect of Chronic High Dose Vitamin a Supplementation on Lipid Metabolism in Adipose Tissue (P02-013-19)

ObjectivesThe objective of this study was to assess the impact of high-dose vitamin A (VA) on lipid metabolism. Previously, VA has been shown to enhance fat mobilisation, leading to a reduction in body fat. We hypothesise that hypervitaminosis A will increase expression of genes associated with lipid catabolism. MethodsTo induce chronic hypervitaminosis A, two groups of pigs (n = 8) were fed a commercial diet. The treatment group was additionally dosed, daily, with an oral supplement of retinyl propionate of 10,000 μg/KgBW for 17 weeks. To assess the impact of VA on lipid metabolism, a microarray analysis was performed to identify gene expression in adipose tissue. Differentially expressed transcripts and pathways were identified using Genespring and mapped to human orthologues for Ingenuity Pathway Analysis (IPA); gene fold changes were confirmed using qRT-PCR. Additionally, an untargeted UPLC-MS lipidomic analysis was carried out in serum samples to identify changes in lipd classes and their metabolites. ResultsIn dosed animals, significant increases in plasma retinol (0.66 μmol/L) and liver retinyl ester concentrations (11.98 μmol/g both P < 0.001), as well as an increase in serum NEFA of 92.84 μmol/L (P = 0.001) were observed. Gene expression fold changes in subcutaneous adipose tissue were related to mitochondrial dysfunction and lipid metabolism, including increased expression of MT-CYTB (↑4.78x, P < 0.05) and ATP5A1 (↑3.13x, P < 0.05). Metabolomics confirmed changes in lipids and their metabolites relevant to adipose tissue in blood (P = 0.05), namely a decrease in triacylglyceride concentration and increases in acyl carnitine and cardiolipin concentrations. ConclusionsAn integrated pathway is suggested to explain the role of vitamin A in leading to increased lipolysis, β-oxidation and oxidative phosphorylation, but when in excess, markers of mitochondrial dysfunction were observed. Funding SourcesFunded by the Bill and Melinda Gates foundation.

Hormone-sensitive lipase is a retinyl ester hydrolase in human and rat quiescent hepatic stellate cells

Hepatic stellate cells (HSC) store vitamin A as retinyl esters and control circulating retinol levels. Upon liver injury, quiescent (q)HSC lose their vitamin A and transdifferentiate to myofibroblasts, e.g. activated (a)HSC, which promote fibrosis by producing excessive extracellular matrix. Adipose triglyceride lipase/patatin-like phospholipase domain-containing protein 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3) have so far been shown to mobilize retinol from retinyl esters in HSC. Here, we studied the putative role of hormone-sensitive lipase (HSL/LIPE) in HSC, as it is the major retinyl ester hydrolase (REH) in adipose tissue.Lipe/HSL expression was analyzed in rat liver and primary human and rat qHSC and culture-activated aHSC. Retinyl hydrolysis was analyzed after Isoproterenol-mediated phosphorylation/activation of HSL.Primary human HSC contain 2.5-fold higher LIPE mRNA levels compared to hepatocytes. Healthy rat liver contains significant mRNA and protein levels of HSL/Lipe, which predominates in qHSC and cells of the portal tree. Q-PCR comparison indicates that Lipe mRNA levels in qHSC are dominant over Pnpla2 and Pnpla3. HSL is mostly phosphorylated/activated in qHSC and partly colocalizes with vitamin A-containing lipid droplets. Lipe/HSL and Pnpla3 expression is rapidly lost during HSC culture-activation, while Pnpla2 expression is maintained. HSL super-activation by isoproterenol accelerates loss of lipid droplets and retinyl palmitate from HSC, which coincided with a small, but significant reduction in HSC proliferation and suppression of Collagen1A1 mRNA and protein levels.In conclusion, HSL participates in vitamin A metabolism in qHSC. Equivalent activities of ATGL and ADPN provide the healthy liver with multiple routes to control circulating retinol levels.

357 Deciphering biosynthesis of all-trans-retinoic acid from retinaldehyde and the regulation of retinoid homeostasis in skin

All-trans-retinoic acid (atRA), the biologically active metabolite of vitamin A, regulates numerous physiological processes in the skin and controls proliferation and differentiation of keratinocytes. The concentration of atRA in skin is tightly regulated and involves multiple enzymes responsible for its degradation and biosynthesis. One of the key intermediates in the biosynthesis of atRA is all-trans-retinaldehyde (RAL). RAL can be irreversibly oxidized to atRA by aldehyde dehydrogenases or reversibly reduced to retinol by retinaldehyde reductases. Retinol can then be converted to retinyl esters that become stored in cells. Hence, the control of retinaldehyde levels is essential in the regulation of atRA concentration and, therefore, of its availability to modulate retinoid-signaling pathways. The clearance of atRA is predominantly mediated by hydroxylations catalyzed by cytochrome P450 family 26 enzymes (CYP26). In this study, we have compared the effects of RAL and atRA on the expression of the enzymes involved in atRA metabolism and biosynthesis, as well as on differentiation markers in keratinocyte cultures and in reconstructed human epidermis (RHE). Expression of differentiation markers and CYP26s were similarly modulated by atRA and RAL via endogenous atRA synthesis, whereas expression of the enzymes possibly involved in the biosynthesis of atRA from RAL such as the retinaldehyde reductase AKR1B10 or aldehyde dehydrogenases were differentially modulated by RAL and atRA. These preliminary results may lead to a novel therapeutic strategy for controlling the endogenous concentration of atRA, and provide insights into the mechanisms underlying atRA homeostasis in the skin.

Mamey sapote fruit and carotenoid formulations derived thereof are dietary sources of vitamin A - A comparative randomized cross-over study.

Mamey sapote is a fruit rich in specific keto-carotenoids, namely sapotexanthin and cryptocapsin. Their chemical structure suggests their provitamin A activity, although their absorption and conversion to vitamin A remained to be demonstrated in humans. Besides structure-related factors, the fruit matrix might also hamper absorption and conversion efficiency. Therefore, we monitored carotenoid and vitamin A levels in triacylglycerol-rich lipoprotein (TRL) fractions in plasma of human participants after consumption of fresh sapote and a carotenoid-rich "matrix-free" formulation derived thereof. A randomized 2-way cross-over study was conducted to compare the post-prandial bioavailability of 0.8 mg sapotexanthin and 1.2-1.5 mg cryptocapsin from the above-mentioned test meals. Seven blood samples were drawn over 9.5 h after test meal consumption. Carotenoids and retinoids were quantitated in TRL fractions using HPLC-DAD. Sapotexanthin was absorbed by all participants from all meals, being ca. 36% more bioavailable from the "matrix-free" formulation (AUCmedian = 73.4 nmol∙h/L) than from the fresh fruit (AUCmedian = 54.0 nmol∙h/L; p ≤ 0.001). Cryptocapsin was only absorbed by 4 of 13 participants. The appearance of retinyl esters was observed in all participants independent of the test meal. Although the fruit matrix hampered carotenoid in vivo-bioavailability from sapote, the fruit clearly represents a valuable source of vitamin A for humans.

Comparative Metabonomic Investigations of Schistosoma japonicum From SCID Mice and BALB/c Mice: Clues to Developmental Abnormality of Schistosome in the Immunodeficient Host.

The growth and development of schistosome has been affected in the immunodeficient hosts. But it remains unresolved about the molecular mechanisms involved in the development and reproduction regulation of schistosomes. This study tested and compared the metabolic profiles of the male and female Schistosoma japonicum worms collected from SCID mice and BALB/c mice at 5 weeks post infection using liquid chromatography tandem mass spectrometry (LC-MS/MS) platform, in which the worms from SCID mice were the investigated organisms and the worms from BALB/c mice were used as the controls. There were 1015 ion features in ESI+ mode and 342 ion features in ESI- mode were identified after filtration by false discovery rate. Distinct metabolic profiles were found to clearly differentiate both male and female worms in SCID mice from those in BALB/c mice using multivariate modeling methods including the Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). There were more differential metabolites in female worms than in male worms between SCID mice and BALB/c mice. And common and uniquely perturbed metabolites and pathways were identified among male and female worms from SCID mice when compared with BALB/c mice. The enriched metabolite sets of the differential metabolites in male worms between SCID mice and BALB/c mice included bile acid biosynthesis, taurine and hypotaurine metabolism, sphingolipid metabolism, retinol metabolism, purine metabolism, etc. And the enriched metabolite sets of differential metabolites in female worms included retinol metabolism, alpha linolenic acid and linoleic acid metabolism, purine metabolism, sphingolipid metabolism, glutamate metabolism, etc. Further detection and comparison in transcript abundance of genes of the perturbed retinol metabolism and its associated meiosis process in worms identified clues suggesting accumulated retinyl ester and perturbed meiotic process. These findings suggested an association between the schistosome with retarded growth and development in SCID mice and their perturbed metabolites and metabolic pathways, and provided a new insight into the growth and development regulation of S. japonicum worms from the metabolic level, which indicated great clues for discovery of drugs or vaccines against the parasites and disease with more researches.