Lecithin:retinol Acyltransferase Research Articles

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

Abstract 6971: The impact of lecithin retinol acyltransferase in squamous cell carcinoma: Tissue formation, cell mechanics, and gene transcription

Abstract Squamous cell carcinoma, along with several other cancers, has been found to lack expression of lecithin retinol acyltransferase (LRAT), a protein which catalyzes retinol esterification and thereby mediates retinoic acid biosynthesis. When expression of LRAT was reintroduced in squamous cell carcinoma, the protein promoted cell differentiation in addition to retinoid status normalization—whether this effect was dependent on the known enzymatic activity of LRAT in retinol esterification, versus another function, has not yet been established. Cell elasticity differences have also been reported between squamous cell carcinoma and normal keratinocytes. In this study, the role of LRAT expression and activity was evaluated in human squamous cell carcinoma cell line SCC13. LRAT was integrated into the genome of SCC13 via retroviral transduction, both as wild-type LRAT and LRAT with a cysteine-to-serine mutation at the active site for retinol esterification (C161-S). In these transformed cell lines, LRAT promoted formation of a cohesive sheet of tissue which remained intact after its removal from the tissue culture plastic surface, as evaluated at two weeks post-confluence. SCC13 alone produced tissue which easily fragmented during this scraping assay. In an evaluation of cell elasticity with shear modulation force microscopy, relative cell modulus measurements of pre-confluent SCC13 with LRAT expression were found to be significantly greater and consistent with the cell modulus of human keratinocytes (DO33). These effects persisted with mutation of the retinol esterification active site, suggesting that LRAT possesses an additional function which has yet to be described. Variation in transcription levels of multiple genes—urokinase-type plasminogen activator, plasminogen activator inhibitor 1, tissue inhibitor of metalloproteinases 1, and kallikrein 5—was also found based on LRAT expression and level of confluence. These findings suggest that the presence of LRAT influences tissue formation, cell elasticity, and gene transcription in SCC13. Citation Format: Emily Peterson, Kuan-Che Feng, Jay Gao, Miriam Rafailovich, Marcia Simon. The impact of lecithin retinol acyltransferase in squamous cell carcinoma: Tissue formation, cell mechanics, and gene transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6971.

290 Vitamin A and Beta-Carotene Supplementation on Growth Performance, Immune System, and Mammary Gland Development in Finishing Pigs

Abstract A total of 224 finishing pigs were randomly assigned to split-sex pens (n = 4 pigs per pen). Each pen was assigned to one of four diets: control (7,656 IU vitamin A/kg), control diet supplemented with vitamin A (4.36 ppm; Rovimix A 1000, DSM, NJ, US), control diet supplemented with beta-carotene (163.28 ppm; Rovimix β-Carotene 10%, DSM, NJ, US), or control supplemented with oxidized beta-carotene (40 ppm; 10% active ingredient, Avivagen, Ottawa, ON, Canada). Pigs and feeder weights were recorded at the start of the study (d 0) and end of each phase (days 21, 42, and 63). A subset of gilts was randomly selected and a blood sample for vitamin A (retinol) analysis was collected on days 0, 18, 39, 60, and 63. The same gilts were vaccinated against Lawsonia intracellularis and porcine circovirus. Antibodies against each vaccination were measured on days 18, 39, and 60. At the end of the study, the subset of gilts was euthanized to obtain a liver sample for vitamin A (retinyl palmitate is converted to retinol for measurement) analysis and a jejunum sample to measure the mRNA abundance of genes involved in vitamin A metabolism (alcohol dehydrogenase class 1, lecithin retinol acyltransferase phosphatidylcholine-retinol O-acyltransferase, retinol binding protein, and beta-carotene oxygenase 1). Mammary tissue from the second and fourth right anterior mammary glands were collected to assess gland development via immunohistochemical staining for T cells, B cells, and macrophages (CD3, CD20, and IBA1, respectively) and DNA extraction. Vaccine antibodies measures, vitamin A analysis, and mammary immunohistochemical staining was completed at Iowa State University Veterinarian Diagnostic Laboratory (Ames, IA). Data were analyzed in SAS 9.4 (Statistical Analysis System, Cary, NC) via GLIMMIX procedure. Pen was the experimental unit and fixed effects were diet and time. Gilts were nested within pen when appropriate to account for more than one gilt per pen. Oxidized beta-carotene supplementation increased (P = 0.02) average daily gain across phases; however, there were no differences (P = 0.18) in the body weight of pigs. There was no effect (P > 0.05) of diet on plasma or hepatic retinol, IgG or IgM concentrations, immune cell presence, or DNA in mammary tissue. Supplemented vitamin A tended (P = 0.05) to increase the expression of retinol binding protein in the jejunum, but other genes were not affected (P > 0.05) by dietary treatments. A diet by time interaction (P = 0.04) was noted for circovirus sample-to-positive ratios and titer levels, where vitamin A supplementation had better vaccine responses at the end of the study. Thus, pigs supplemented with oxidized beta-carotene had an improved average daily gain over vitamin A supplemented pigs, but pigs supplemented with vitamin A appeared to have a more robust immune system.

CD8 + T cell memory is sustained in mice by hepatic stellate cells.

Long-lasting immunological memory is the ultimate goal of vaccination. Homeostatic maintenance of memory CD8 + cytotoxic T cells (MemCD8TCs) is thought to be mediated by IL-15/IL-15R heterodimer (15HD)-expressing myeloid cells. Nonmyeloid hepatic stellate cells (HSCs) also express 15HD, but their role in maintaining MemCD8TC homeostasis is unknown. We engineered a genetically engineered mouse in which IL-15R complementary DNA (cDNA) had been inserted in-frame with lecithin-retinol acyltransferase gene and bred onto an IL-15R-KO (15R-KO) genetic background (L15R) that expressed IL-15R in HSCs at normal levels, but not in other liver cells. Outside of the liver of L15R mice, IL-15R expression was found in a number of organs, but not in dendritic cells and macrophages. The low IL-15R expression in the bone marrow (BM) of L15R mice was eliminated by the reconstitution of lethally-irradiated L15R mice with 15R-KO BM to generate L15RC mice. Because MemCD8TC maintenance is mediated by 15HD, not empty IL-15R, 15HD content in L15R mice was determined and found for liver, lung, kidney, and heart. L15R and L15RC mice developed and maintained long-lasting, systemic antigen-specific MemCD8TCs that were efficacious against tumor growth and Listeria monocytogenes infection in an antigen-specific manner. Among the four organs with 15HD content, liver-associated MemCD8TCs were different from those found in the lung, kidney, and heart in two ways: (1) they were quantitatively the most numerous, and (2) they appeared uniquely in the form of clusters in a specialized structure, sinusoidal niches of the liver. The liver, the largest organ of the body, is endowed with the capability of effectuating long-lasting functional cytotoxic T cell memory.

Lipid-induced alteration in retinoic acid signaling leads to mitochondrial dysfunction in HepG2 and Huh7 cells.

A surfeit of mitochondrial reactive oxygen species (ROS) and inflammation serve as obligatory mediators of lipid-associated hepatocellular maladies. While retinoid homeostasis is essential in restoring systemic energy balance, its role in hepatic mitochondrial function remains elusive. The role of lecithin-retinol acyltransferase (LRAT) in maintenance of retinoid homeostasis is appreciated earlier; however, its role in modulating retinoic acid (RA) bioavailability upon lipid-imposition is unexplored. We identified LRAT overexpression in high-fat diet (HFD)-fed rats and palmitate-treated hepatoma cells. Elevation in LRAT expression depletes RA production and deregulates RA signaling. This altered RA metabolism enhances fat accumulation, accompanied by inflammation that leads to impaired mitochondrial function through enhanced ROS generation. Hence, LRAT inhibition could be a novel approach preventing lipid-induced mitochondrial dysfunction in hepatoma cells.

PNPLA2 mobilizes retinyl esters from retinosomes and promotes the generation of 11-cis-retinal in the visual cycle.

Retinosomes are intracellular lipid bodies found in the retinal pigment epithelium (RPE). They contain retinyl esters (REs) and are thought to be involved in visual chromophore regeneration during dark adaptation and in case of chromophore depletion. However, key enzymes in chromophore regeneration, retinoid isomerase (RPE65), and lecithin:retinol acyltransferase (LRAT) are located in the endoplasmic reticulum (ER). The mechanism and the enzyme responsible for mobilizing REs from retinosomes remained unknown. Our study demonstrates that patatin-like phospholipase domain containing 2 (PNPLA2) mobilizes all-trans-REs from retinosomes. The absence of PNPLA2 in mouse eyes leads to a significant accumulation of lipid droplets in RPE cells, declined electroretinography (ERG) response, and delayed dark adaptation compared with those of WT control mouse. Our work suggests a function of PNPLA2 as an RE hydrolase in the RPE, mobilizing REs from lipid bodies and functioning as an essential component of the visual cycle.

The evaluation of the supplementation of vitamin A, beta-carotene, and oxidized beta-carotene in prepubertal gilts.

Finishing pigs (N = 224; 28.66 ± 1.90 kg bodyweight) were randomly assigned across 56 pens of either four barrows or gilts, and assigned to one of four diets: control (7,656 IU vitamin A/kg), control supplemented with vitamin A (4.36 ppm, Rovimix A 1000, DSM, Parsippany, NJ, USA), control supplemented with beta-carotene (163.28 ppm, Rovimix β-Carotene 10%, DSM, Parsippany), or control supplemented with oxidized beta-carotene (40 ppm; 10% active ingredient, Avivagen, Ottawa, ON, Canada). Pigs and feeder weights were obtained at the start of the study (d 0), and end of each phase (d 21, 42, and 63). A subset of gilts had a blood sample taken via jugular venipuncture on d 0, a blood sample and vaccinations of Lawsonia intracellularis and porcine circovirus type 2 (PCV2) on d 18, a blood sample and booster vaccination of PCV2 on d 39, a blood sample on day 60, and a final blood sample on day 63. Gilts were euthanized at the end of the study to obtain a liver (entire right lobe) and a jejunum sample (15.24 cm at 10% of length). Additionally, the second and fourth right anterior mammary were collected to assess anterior mammary tissues. Data were analyzed in SAS 9.4 (Statistical Analysis System, Cary, NC) via GLIMMIX procedure. Oxidized beta-carotene supplementation increased (P = 0.02) ADG across phases over vitamin A supplementation, although there were no differences (P = 0.18) in the body weight of pigs. There was no effect (P > 0.05) of diet on plasma or hepatic retinol, IgG or IgM levels, or immune cell presence in developing mammary tissue. Supplemented vitamin A tended (P = 0.05) to increase the mRNA abundance of retinol binding protein in the jejunum, but other mRNA abundance for genes (alcohol dehydrogenase class 1, lecithin retinol acyltransferase phosphatidylcholine-retinol O-acyltransferase, and beta-carotene oxygenase 1) were not affected (P > 0.05) by dietary treatments. A diet by time interaction (P = 0.04) was noted for the circovirus S/P ratio, where vitamin A supplementation had the best ratio compared to other diets. Analyzed titer levels for the circovirus vaccine had an interaction (P < 0.01) for diet by time, where vitamin A supplementation had the highest titer at the end of the study. Thus, pigs supplemented with oxidized beta-carotene had an improved ADG over vitamin A supplemented pigs, but pigs supplemented with vitamin A seemed to have an improved immune status.

Evaluation of saturated and unsaturated fat with vitamin A or beta-carotene supplementation in nursery pigs.

One hundred and fifty-two nursery pigs (PIC, Hendersonville, TN) were randomly assigned to mix sex pens and one of six dietary treatments in a 3 × 2 factorial. Diets included no added fat, 3% added choice white grease, or 3% added soy oil with either a supplemented vitamin A (for a total of 11,640 IU vitamin A/kg, Rovimix A 1000, DSM, Parsippany, NJ, US) or beta-carotene (for a total of 8,708 IU vitamin A/kg equivalent, Rovimix β-Carotene 10%, DSM). Pigs were given a 3-d adaptation period upon arrival. Pigs were weighed at the start of the study and at the end of each phase. A blood sample was taken from one pig per pen at the start and end of the study. Tissues were collected from eight pigs at the start of the study and six pigs per treatment at the end of the study. Data were analyzed via the GLIMMIX procedure in SAS 9.4 (SAS Inst., Cary, NC). Pen was the experimental unit, and repeated measures were used for growth performance and blood parameters. There was no fat by supplement interaction (P > 0.05) on body weight (BW), but there was a tendency (P = 0.054) for heavier BWs when soy oil was added to diets. There was no difference (P > 0.05) in average daily feed intake or average daily gain (ADG). There was an improved gain:feed (P = 0.02) when pigs were fed choice white grease over no added fat. There were time differences (P < 0.05) for plasma vitamins A (retinol), D (25 hydroxy vitamin D3), and E (alpha-tocopherol). Vitamin A and D values were higher at the end of the study, whereas vitamin E values were lower at the end of the study. The choice white grease diets had the highest (P < 0.05) plasma vitamins D and E (6.74 ng/mL and 2.87 ppm, respectively). Pigs supplemented with vitamin A had higher (P < 0.05) hepatic vitamin A than pigs supplemented with beta-carotene (19.9 vs. 15.6 ppm, respectively). There were no differences (P < 0.05) between immunoglobulins G and M or mRNA abundance of select genes (retinol binding protein 2, alcohol dehydrogenase class 1, lecithin retinol acyltransferase phosphatidylcholine-retinol O-acyltransferase, and beta-carotene oxygenase 1). In conclusion, fat inclusion level and type, with either vitamin A or beta-carotene supplementation, did not affect the overall nursery pig growth performance. The addition of fat resulted in an increase in ADG and BW. Diets with choice white grease had the highest plasma vitamins D and E, and supplemental vitamin A increased hepatic vitamin A.

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.

Emergence of highly profibrotic and proinflammatory Lrat+Fbln2+ HSC subpopulation in alcoholic hepatitis.

Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in a mouse AH model by single-cell RNA sequencing (scRNA-seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy-1 cell surface antigen [Thy1] and fibulin 2 [Fbln2]) markers and vitamin A (VitA) storage. Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl 4 , and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FACS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-Fbln2- activated HSCs were VitA-depleted (vs. ~13% in BDL and CCl 4 ). Supervised clustering identified a subset co-expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44-fold, 17-fold, and 1.3-fold in AH, BDL, and CCl 4 . Lrat+Fbln2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2- cells, but 2-4-times repressed HSC-selective genes. AH activated HSCs had up-regulated inflammatory (chemokine [C-X-C motif] ligand 2 [Cxcl2], chemokine [C-C motif] ligand 2), antimicrobial (Il-33, Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl 4 . Computational deconvolution of AH versus normal human bulk-liver RNA-sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co-expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat-Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH. A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.

Specialized neurons in the right habenula mediate response to aversive olfactory cues.

Hemispheric specializations are well studied at the functional level but less is known about the underlying neural mechanisms. We identified a small cluster of cholinergic neurons in the dorsal habenula (dHb) of zebrafish, defined by their expression of the lecithin retinol acyltransferase domain containing 2 a (lratd2a) gene and their efferent connections with a subregion of the ventral interpeduncular nucleus (vIPN). The lratd2a-expressing neurons in the right dHb are innervated by a subset of mitral cells from both the left and right olfactory bulb and are activated upon exposure to the odorant cadaverine that is repellent to adult zebrafish. Using an intersectional strategy to drive expression of the botulinum neurotoxin specifically in these neurons, we find that adults no longer show aversion to cadaverine. Mutants with left-isomerized dHb that lack these neurons are also less repelled by cadaverine and their behavioral response to alarm substance, a potent aversive cue, is diminished. However, mutants in which both dHb have right identity appear more reactive to alarm substance. The results implicate an asymmetric dHb-vIPN neural circuit in the processing of repulsive olfactory cues and in modulating the resultant behavioral response.

Vitamin A Transporters in Visual Function: A Mini Review on Membrane Receptors for Dietary Vitamin A Uptake, Storage, and Transport to the Eye.

Vitamins are essential compounds obtained through diet that are necessary for normal development and function in an organism. One of the most important vitamins for human physiology is vitamin A, a group of retinoid compounds and carotenoids, which generally function as a mediator for cell growth, differentiation, immunity, and embryonic development, as well as serving as a key component in the phototransduction cycle in the vertebrate retina. For humans, vitamin A is obtained through the diet, where provitamin A carotenoids such as β-carotene from plants or preformed vitamin A such as retinyl esters from animal sources are absorbed into the body via the small intestine and converted into all-trans retinol within the intestinal enterocytes. Specifically, once absorbed, carotenoids are cleaved by carotenoid cleavage oxygenases (CCOs), such as Beta-carotene 15,15’-monooxygenase (BCO1), to produce all-trans retinal that subsequently gets converted into all-trans retinol. CRBP2 bound retinol is then converted into retinyl esters (REs) by the enzyme lecithin retinol acyltransferase (LRAT) in the endoplasmic reticulum, which is then packaged into chylomicrons and sent into the bloodstream for storage in hepatic stellate cells in the liver or for functional use in peripheral tissues such as the retina. All-trans retinol also travels through the bloodstream bound to retinol binding protein 4 (RBP4), where it enters cells with the assistance of the transmembrane transporters, stimulated by retinoic acid 6 (STRA6) in peripheral tissues or retinol binding protein 4 receptor 2 (RBPR2) in systemic tissues (e.g., in the retina and the liver, respectively). Much is known about the intake, metabolism, storage, and function of vitamin A compounds, especially with regard to its impact on eye development and visual function in the retinoid cycle. However, there is much to learn about the role of vitamin A as a transcription factor in development and cell growth, as well as how peripheral cells signal hepatocytes to secrete all-trans retinol into the blood for peripheral cell use. This article aims to review literature regarding the major known pathways of vitamin A intake from dietary sources into hepatocytes, vitamin A excretion by hepatocytes, as well as vitamin A usage within the retinoid cycle in the RPE and retina to provide insight on future directions of novel membrane transporters for vitamin A in retinal cell physiology and visual function.

The Lrat-/- Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for Retinitis Pigmentosa.

Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat−/− and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays. Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat−/− animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat−/− animals. Lrat−/− animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat−/− animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat−/− rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.

Loss of Hepatic Vitamin A Reserve Lowers Cardiac Function and Prevents Cardiac Calcium Overload Under Stress Conditions

Vitamin A is an essential nutrient for both cardiac development and repair following injury. Vitamin A is primarily stored in the liver by the lecithin retinol acyltransferase (LRAT) enzyme and the liver secretes 30-40% of its vitamin A stores following myocardial injury. Previously, we found that mice lacking LRAT (LRAT KO), and thus lacking hepatic Vitamin A stores, preserve cardiac function following myocardial infarction compared to wild type (WT) control mice. Our finding contradicts other reports that suggest vitamin A is needed for cardiac repair. A gene microarray of left and right ventricular tissue identified changes in multiple calcium and ion channels in LRAT KO mice. All of our findings led us to hypothesize that LRAT KO mice have impaired cardiac calcium handling and that therefore the heart may be protected from calcium overload during an ischemic event. Hearts from LRAT KO and WT mice were perfused in the Langendorff mode to measure systolic and diastolic parameters. Heart function was determined at baseline, at high calcium levels (4 mM), and during adrenergic stimulation with dobutamine (300 nM). At baseline, LRAT KO and WT mice showed no significant differences in any of the cardiac function parameters. However, when stressed with dobutamine LRAT KO hearts had a 34% decrease in diastolic pressure (KO 4329±591 mmHg vs. WT 6103±508 mmHg, n=6-7, p<0.01) and a 25% reduction in systolic pressure (KO 8674±1365 mmHg vs. WT 11103±872 mmHg, n=6-7, p<0.05). Coronary flow, heart rate, rate pressure product and developed pressure where not significantly altered. When stressed with 4 mM extracellular calcium, we found a strong impairment in all contractile function parameters in LRAT KO compared to WT hearts. Most affected were diastolic relaxation (ndP/dt) with a decrease of 43% (KO 2819±281 mmHg/svs. WT 4347±383 mmHg/s, n=6-7,p<0.01) and contractility (pdP/dt) with a 40% reduction (KO 5013±444 mmHg/s vs. WT 7507±790 mmHg/s, n=6-7, p<0.05). Western blot analysis of cardiac tissue identified a reduction in calmodulin (p<0.05) and calsequestrin 2 in the LRAT null hearts supporting that blunted calcium handling is occurring in these hearts. In summary, hearts from mice lacking vitamin A stores are unable to increase cardiac function under stress conditions. This finding is more pronounced with high calcium stress than with adrenergic stimulation. These results support our hypothesis that LRAT KO mice are potentially protected from calcium overload during ischemic injury.

Challenges with the Preparation of a Soluble and Active Form of LRAT

Lecithin retinol acyltransferase (LRAT) is a membrane protein that transforms all-trans retinol into all-trans retinyl ester. LRAT mutations lead to vision loss. A high activity is obtained with truncated LRAT (tLRAT; LRAT 30-196) purified in the presence of sodium dodecyl sulfate (SDS) whereas its natural mutations lead to a loss of its activity. Moreover, our data show that tLRAT is not properly structured on the basis of our NMR analyses. Structural information of LRAT must however be obtained to better understand the mechanism of its enzymatic activity.

Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse.

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.

LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis from β-carotene

AbstractThere is increasing recognition that dietary lipids can affect the expression of genes encoding their metabolizing enzymes, transporters, and binding proteins. This mechanism plays a pivotal role in controlling tissue homeostasis of these compounds and avoiding diseases. The regulation of retinoid biosynthesis from β-carotene (BC) is a classic example for such an interaction. The intestine-specific homeodomain transcription factor (ISX) controls the activity of the vitamin A-forming enzyme β-carotene oxygenase-1 in intestinal enterocytes in response to increasing concentration of the vitamin A metabolite retinoic acid. However, it is unclear how cells control the concentration of the signaling molecule in this negative-feedback loop. We demonstrate in mice that the sequestration of retinyl esters by the enzyme lecithin:retinol acyltransferase (LRAT) is central for this process. Using genetic and pharmacological approaches in mice, we observed that in LRAT deficiency, the transcription factor ISX became hypersensitive to dietary vitamin A and suppressed retinoid biosynthesis. The dysregulation of the pathway resulted in BC accumulation and vitamin A deficiency of extrahepatic tissues. Pharmacological inhibition of retinoid signaling and genetic depletion of the Isx gene restored retinoid biosynthesis in enterocytes. We provide evidence that the catalytic activity of LRAT coordinates the negative-feedback regulation of intestinal retinoid biosynthesis and maintains optimal retinoid levels in the body.

Liver fibrosis is driven by protease‐activated receptor‐1 expressed by hepatic stellate cells in experimental chronic liver injury

BackgroundBlood coagulation protease activity is proposed to drive hepatic fibrosis through activation of protease‐activated receptors (PARs). Whole‐body PAR‐1 deficiency reduces experimental hepatic fibrosis, and in vitro studies suggest a potential contribution by PAR‐1 expressed by hepatic stellate cells. However, owing to a lack of specific tools, the cell‐specific role of PAR‐1 in experimental hepatic fibrosis has never been formally investigated. Using a novel mouse expressing a conditional PAR‐1 allele, we tested the hypothesis that PAR‐1 expressed by hepatic stellate cells contributes to hepatic fibrosis. MethodsPAR‐1flox/flox mice were crossed with mice expressing Cre recombinase controlled by the lecithin retinol acyltransferase (LRAT) promoter, which induces recombination in hepatic stellate cells. Male PAR‐1flox/flox/LRATCre and PAR‐1flox/flox mice were challenged twice weekly with carbon tetrachloride (CCl4, 1 mL/kg i.p.) for 6 weeks to induce liver fibrosis. ResultsPAR‐1 mRNA levels were reduced (>95%) in hepatic stellate cells isolated from PAR‐1flox/flox/LRATCre mice. Hepatic stellate cell activation was evident in CCl4‐challenged PAR‐1flox/flox mice, indicated by increased α‐smooth muscle actin labeling and induction of several profibrogenic genes. CCl4‐challenged PAR‐1flox/flox mice displayed robust hepatic collagen deposition, indicated by picrosirius red staining and type I collagen immunolabeling. Notably, stellate cell activation and collagen deposition were significantly reduced (>30%) in PAR‐1flox/flox/LRATCre mice. Importantly, the reduction in liver fibrosis was not a consequence of reduced acute CCl4 hepatotoxicity in PAR‐1flox/flox/LRATCre mice. ConclusionsThe results constitute the first direct experimental evidence that PAR‐1 expressed by stellate cells directly promotes their profibrogenic phenotype and hepatic fibrosis in vivo.

Vitamin A Rich Diet Diminishes Early Urothelial Carcinogenesis by Altering Retinoic Acid Signaling.

Urinary bladder cancer is one of the leading malignancies worldwide, with the highest recurrence rates. A diet rich in vitamin A has proven to lower the risk of cancer, yet the molecular mechanisms underlying this effect are unknown. We found that vitamin A decreased urothelial atypia and apoptosis during early bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Vitamin A did not alter urothelial cell desquamation, differentiation, or proliferation rate. Genes like Wnt5a, involved in retinoic acid signaling, and transcription factors Pparg, Ppara, Rxra, and Hoxa5 were downregulated, while Sox9 and Stra6 were upregulated in early urothelial carcinogenesis. When a vitamin A rich diet was provided during BBN treatment, none of these genes was up- or downregulated; only Lrat and Neurod1 were upregulated. The lecithin retinol acyltransferase (LRAT) enzyme that produces all-trans retinyl esters was translocated from the cytoplasm to the nuclei in urothelial cells as a consequence of BBN treatment regardless of vitamin A rich diet. A vitamin A-rich diet altered retinoic acid signaling, decreased atypia and apoptosis of urothelial cells, and consequently diminished early urothelial carcinogenesis.