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Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development. [ABSTRACT FROM PUBLISHER]

Dietary carote-noids like β-carotene are converted within the body either to retinoid, via β-carotene-15,15′-dioxygenase (BCO1), or to β-apo-carotenoids, via β-carotene-9′,10′-oxygenase 2. Some β-apo-carotenoids are po-tent antagonists of retinoic acid receptor (RAR)-mediated transcrip-tional regulation, which is required to ensure normal heart develop-ment and functions. We established liquid chromatography tandem mass spectrometery methods for measuring concentrations of 10 β-apo-carotenoids in mouse plasma, liver, and heart and assessed how these are influenced by Bco1 deficiency and β-carotene intake. Sur-prisingly, Bco1-/- mice had an increase in heart levels of retinol, nonesterified fatty acids, and ceramides and a decrease in heart triglycerides. These lipid changes were accompanied by elevations in levels of genes important to retinoid metabolism, specifically retinol dehydrogenase 10 and retinol-binding protein 4, as well as genes involved in lipid metabolism, including peroxisome proliferator-acti-vated receptor-7, lipoprotein lipase, Cd36, stearoyl-CoA desaturase 1, and fatty acid synthase. We also obtained evidence of compromised heart function, as assessed by two-dimensional echocardiography, in Bco1-/- mice. However, the total absence of Bco1 did not substan-tially affect β-apo-carotenoid concentrations in the heart. β-Carotene administration to matched Bco1-/- and wild-type mice elevated total β-apo-carotenal levels in the heart, liver, and plasma and total β-apo-carotenoic acid levels in the liver. Thus, BCO1 modulates heart metabolism and function, possibly by altering levels of cofactors required for the actions of nuclear hormone receptors. [ABSTRACT FROM AUTHOR]

Abstract: Retinoids (vitamin A and its metabolites) are essential micronutrients that regulate many cellular processes. Greater than 70% of the body's retinoid reserves are stored in the liver as retinyl ester (RE). Chronic alcohol consumption induces depletion of hepatic retinoid stores, and the extent of this has been correlated with advancing stages of alcoholic liver disease. The goal of this study was to analyze the mechanisms responsible for depletion of hepatic RE stores by alcohol consumption. A change in the fatty-acyl composition of RE in alcohol-fed mice was observed within two weeks after the start of alcohol consumption. Specifically, alcohol-feeding was associated with a significant decline in hepatic retinyl palmitate levels; however, total RE levels were maintained by a compensatory increase in levels of usually minor RE species, particularly retinyl oleate. Our data suggests that alcohol feeding initially stimulates a futile cycle of RE hydrolysis and synthesis, and that the change in RE acyl composition is associated with a change in the acyl composition of hepatic phosphatidylcholine. The alcohol-induced change in RE acyl composition was specific to the liver, and was not seen in lung or white adipose tissue. This shift in hepatic RE fatty acyl composition is a sensitive indicator of alcohol consumption and may be an early biomarker for events associated with the development of alcoholic liver disease. [Copyright &y& Elsevier]

Abstract: Alcohol, a major cause of human cardiomyopathy, decreases cardiac contractility in both animals and man. However, key features of alcohol-related human heart disease are not consistently reproduced in animal models. Accordingly, we studied cardiac histology, contractile function, cardiomyocyte long chain fatty acid (LCFA) uptake, and gene expression in male C57BL/6J mice consuming 0, 10, 14, or 18% ethanol in drinking water for 3months. At sacrifice, all EtOH groups had mildly decreased body and increased heart weights, dose-dependent increases in cardiac triglycerides and a marked increase in cardiac fatty acid ethyl esters. [3H]-oleic acid uptake kinetics demonstrated increased facilitated cardiomyocyte LCFA uptake, associated with increased expression of genes encoding the LCFA transporters CD36 and Slc27a1 (FATP1) in EtOH-fed animals. Although SCD-1 expression was increased, lipidomic analysis did not indicate significantly increased de novo LCFA synthesis. By echocardiography, ejection fraction (EF) and the related fractional shortening (FS) of left ventricular diameter during systole were reduced and negatively correlated with cardiac triglycerides. Expression of myocardial PGC-1α and multiple downstream target genes in the oxidative phosphorylation pathway, including several in the electron transport and ATP synthase complexes of the inner mitochondrial membrane, were down-regulated. Cardiac ATP was correspondingly reduced. The data suggest that decreased expression of PGC-1α and its target genes result in decreased cardiac ATP levels, which may explain the decrease in myocardial contractile function caused by chronic EtOH intake. This model recapitulates important features of human alcoholic cardiomyopathy and illustrates a potentially important pathophysiologic link between cardiac lipid metabolism and function. [Copyright &y& Elsevier]

Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice.ATLOLpLmRNAand activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicronTGuptake intoBATand lung.ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL inBATmodulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the "gatekeeper" for tissue lipid distribution. [ABSTRACT FROM AUTHOR]

Hepatic stellate cell (HSC) lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT) mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS) based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP) mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFPHSCs have: (i) increased expression of typical markers of HSC activation; (ii) decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT); (iii) decreased triglyceride levels; (iv) increased expression of genes associated with lipid catabolism; and (v) an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1. Conclusion: Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury. [ABSTRACT FROM AUTHOR]

Abstract: 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 β-carotene, but the ability of HSCs to metabolize β-carotene in situ has not been explored. To gain understanding of this, we investigated whether β-carotene-15,15′-monooxygensase (Bcmo1) and β-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 β-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 β-carotene-containing diet compared to mice receiving no β-carotene. Collectively, our data establish that HSCs are an important cellular site for β-carotene accumulation and metabolism within the liver. [ABSTRACT FROM AUTHOR]

Retinoids are absolutely required for normal growth and development during the postnatal period. We studied the delivery of retinoids to milk, availing of mouse models modified for proteins thought to be essential for this process. Milk retinyl esters were markedly altered in mice lacking the enzyme lecithin:retinol acyltransferase (Lrat-/-), indicating that this enzyme is normally responsible for the majority of retinyl esters incorporated into milk and not an acyl-CoA dependent enzyme, as proposed in the literature. Unlike wild-type milk, much of the retinoid in Lrat-/- milk is unesterified retinol, not retinyl ester. The composition of the residual retinyl ester present in Lrat-/- milk was altered from predominantly retinyl palmitate and stearate to retinyl oleate and medium chain retinyl esters. This was accompanied by increased palmitate and decreased oleate in Lrat-/- milk triglycerides. In other studies, we investigated the role of retinol-binding protein in retinoid delivery for milk formation. We found that Rbp-/- mice maintain milk retinoid concentrations similar to those in matched wild-type mice. This appears to arise due to greater postprandial delivery of retinoid, a lipoprotein lipase (LPL)-dependent pathway. Importantly, LPL also acts to assure delivery of long-chain fatty acids (LCFA) to milk. The fatty acid transporter CD36 also facilitated LCFA but not retinoid incorporation into milk. Our data show that compensatory pathways for the delivery of retinoids ensure their optimal delivery and that LRAT is the most important enzyme for milk retinyl ester formation. [ABSTRACT FROM AUTHOR]

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2–3-fuld) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3–4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these chylomicron retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase. [ABSTRACT FROM AUTHOR]

Retinoids are required for normal embryonic development. Both embryonic retinoid deficiency and excess result in congenital malformations. There is little understanding of the physiology underlying retinoid transfer from the maternal circulation to the embryo. We now report studies that explore this process using retinol-binding protein-deficient (RBP-/-) mice and mice that express human RBP on the RBP-/- background. Our studies establish that dietary retinoid, bound to lipoproteins, can serve as an important source for meeting tissue retinoid requirements during embryogenesis. Indeed, retinyl ester concentrations in the circulations of pregnant RBP-/- mice are significantly elevated over those observed in wildtype mice, suggesting that lipoprotein retinyl esters may compensate for the absence of retinol-RBP during pregnancy. We also demonstrate, contrary to earlier proposals, that maternal RBP does not cross the placenta and cannot enter the fetal circulation. Overall, our data indicate that both retinol-RBP and retinyl esters bound to lipoproteins are able to provide sufficient retinoid to the embryo to allow for normal embryonic development. [ABSTRACT FROM AUTHOR]