Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis.

BACKGROUND: Hepatic stellate cells (HSCs) are a major fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. With increasing interest in developing antifibrotic therapies, there is a need for cell lines that preserve the in vivo phenotype of human HSCs to elucidate pathways of human hepatic fibrosis. We established and characterised two human HSC cell lines termed LX-1 and LX-2, and compared their features with those of primary human stellate cells. METHODS AND RESULTS: LX-1 and LX-2 were generated by either SV40 T antigen immortalisation (LX-1) or spontaneous immortalisation in low serum conditions (LX-2). Both lines express alpha smooth muscle actin, vimentin, and glial fibrillary acid protein, as visualised by immunocytochemistry. Similar to primary HSCs, both lines express key receptors regulating hepatic fibrosis, including platelet derived growth factor receptor beta (betaPDGF-R), obese receptor long form (Ob-RL), and discoidin domain receptor 2 (DDR2), and also proteins involved in matrix remodelling; matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-2, and MT1-MMP, as determined by western analyses. LX-2 have reduced expression of TIMP-1. LX-2, but not LX-1, proliferate in response to PDGF. Both lines express mRNAs for alpha1(I) procollagen and HSP47. Transforming growth factor beta1 stimulation increased their alpha1(I) procollagen mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction. LX-2, but not LX-1, cells are highly transfectable. Both lines had a retinoid phenotype typical of stellate cells. Microarray analyses showed strong similarity in gene expression between primary HSCs and either LX-1 (98.4%) or LX-2 (98.7%), with expression of multiple neuronal genes. CONCLUSIONS: LX-1 and LX-2 human HSC lines provide valuable new tools in the study of liver disease. Both lines retain key features of HSCs. Two unique advantages of LX-2 are their viability in serum free media and high transfectability.

Subclinical vitamin A deficiency in Israeli-Bedouin toddlers.

OBJECTIVE: This study was designed to estimate the prevalence of and evaluate risk factors for subclinical vitamin A deficiency in Arab-Bedouin children at age 18 months, followed from birth. DESIGN: Community-based, prospective, cohort study conducted in Rahat, a large Arab-Bedouin township, located near the city of Beer Sheva in the Negev region of southern Israel. SUBJECTS: Healthy Bedouin infants (n=117) from the township, born at Soroka University Medical Center (SUMC) in Beer Sheva, were randomly recruited at birth. Enrollment was restricted to well infants born weighing >2500 g at birth. RESULTS: More than 15% of the children had serum retinol concentrations below 0.7 micromol/l. Male sex (odds ratio (OR) 4.17 [1.14-15.32], P=0.031), stunting at age 12 months (OR 10.09 [2.00-50.97], P=0.05) and warm season at age 18 months (OR 6.20 [1.36-28.28], P=0.018) were associated with vitamin A deficiency. Maternal education decreased the risk of vitamin A deficiency (OR 0.81 [0.68-0.95], P=0.011). CONCLUSIONS: Study results indicate a significant vitamin A deficiency problem among Bedouin children. Deficiency may be prevented by increasing dietary intake of vitamin A, especially during the warm season. Other interventions include preventing and controlling diarrheal diseases in order to avert nutritional stunting, and providing nutritional education to women of childbearing age. SPONSORSHIP: This study received financial support from the National Institute of Allergy and Infectious Diseases (AI-26497), the US-Israel Bi-national Science Foundation (BSF 90-00257), and the National Academy of Sciences/Institute of Medicine (AID/ANE 0158-G-SS-9035-00).

Cellular metabolism and actions of 13-cis-retinoic acid.

Retinoids (vitamin A and its derivatives) are potent substances for regulating the expression of many different genes within the body. The gene regulatory activities of retinoids are mediated primarily by the all-trans and 9-cis isomers of retinoic acid. Although 13-cis-retinoic acid (isotretinoin) does not have the potent gene regulatory activity of the other two isomers, it is an effective pharmacologic agent for treating a variety of dermatologic conditions. Because 13-cis-retinoic acid is also a naturally occurring retinoid that is present in the circulation, question is raised as to the biochemical mechanism(s) responsible for its pharmacologic efficacy. Some of this efficacy likely arises from the ability of 13-cis-retinoic acid to undergo isomerization to the significantly more active all-trans and 9-cis isomers; however, this does not account for all of the pharmacologic effects observed upon use of this retinoid. Some recent studies suggest that 13-cis-retinoic acid may act by inhibiting the actions of enzymes that are needed to metabolize steroids, while other recent studies indicate that 13-cis-retinoic acid acts through membrane receptors present on the surface of cells. At the present, it is not possible to rule out still other possible biochemical actions of 13-cis-retinoic acid in the body. It is clear, however, that if we are to fully understand the basis for the clinical efficacy of 13-cis-retinoic acid, a better understanding of such biochemical actions is needed.

Preservation of light signaling to the suprachiasmatic nucleus in vitamin A-deficient mice.

To investigate the role of retinal-based pigments (opsins) in circadian photoreception in mice, animals mutated in plasma retinol binding protein were placed on a vitamin A-free diet and tested for photic induction of gene expression in the suprachiasmatic nucleus. After 10 months on the vitamin A-free diet, the majority of mice contained no detectable retinal in their eyes. These mice demonstrated fully intact photic signaling to the suprachiasmatic nucleus as measured by acute mPer mRNA induction in the suprachiasmatic nucleus in response to bright or dim light. The data suggest that a non-opsin pigment is the primary circadian photoreceptor in the mouse.

Expression and characterization of a murine enzyme able to cleave beta-carotene. The formation of retinoids.

Because animals are not able to synthesize retinoids de novo, ultimately they must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (beta-carotene 15,15'-dioxygenase) has been characterized primarily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encoding a mouse homolog of this enzyme. When the cDNA was expressed in either Escherichia coli or Chinese hamster ovary cells, expression conferred upon bacterial and Chinese hamster ovary cell homogenates the ability to cleave beta-carotene to retinal. Several lines of evidence obtained upon kinetic analyses of the recombinant enzyme suggested that carotene cleavage enzyme interacts with other proteins present within cell or tissue homogenates. This was confirmed by pull-down experiments upon incubation of recombinant enzyme with tissue 12,000 x g supernatants. Matrix-assisted laser desorption ionization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate dehydrogenase associates with recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain reaction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understanding the biochemistry of carotene cleavage to retinoids.

Gender differences in autoantibodies to oxidative DNA base damage in cigarette smokers.

Oxidative DNA damage and antibodies to that damage have been implicated in lung, breast, and colorectal cancer. In this observational validation study, the relationship between anti-5-hydroxymethyl-2'-deoxyuridine (HMdU) autoantibody (aAb) and plasma micronutrients was assessed in 140 heavy smokers by ELISA. Anti-HMdU aAbs were 50% higher in women after adjustment for cigarettes/day (CPD; P = 0.002), although men smoked more and had higher plasma cotinine levels. The women reported taking more vitamin C (P < 0.005) and had higher plasma levels of alpha-carotene and beta-carotene (P < 0.001) and cryptoxanthin (P < 0.01) than men. Neither CPD nor cotinine was associated with aAb titers. Anti-HMdU aAbs were associated inversely with alpha-tocopherol (P = 0.10), retinol (P = 0.06), and age (P = 0.04) in women but not in men. In contrast to the men, women 50 years of age (P = 0.05). Given the same duration of exposure, women had higher anti-HMdU aAbs and also reached peak levels at a lower cumulative smoking exposure (30 years) compared with male smokers (40 years). Subjects smoked an average of 28.9 +/- 0.81 CPD and initiated smoking at 17.2 +/- 0.33 (SE) years of age. Therefore, smokers who reported smoking for 30 years were typically <50 years old. Women

Studies of vitamin A metabolism in mouse model systems.

Over the past several years, discoveries from mouse genetics have had direct impact on our understanding of vitamin A metabolism. Although the metabolism of vitamin A in the mouse does have some special features (for example very large stores of liver and pulmonary retinyl esters), the ability to construct knockout and transgenic mouse models has yielded an impressive amount of information directly relevant to understanding the general principles of vitamin A transport, storage and degradation. We discuss below the metabolism of vitamin A through a number of genetically engineered mouse strains with alterations in genes that affect this metabolism. The novelty of this experimental approach is evidenced by the fact that the oldest of these strains was first reported only eight years ago.1)

Transthyretin, thyroxine, and retinol-binding protein in human cerebrospinal fluid: effect of lead exposure.

Transthyretin (TTR), synthesized by the choroid plexus, is proposed to have a role in transport of thyroid hormones in the brain. Our previous studies in animals suggest that sequestration of lead (Pb) in the choroid plexus may lead to a marked decrease in TTR levels in the cerebrospinal fluid (CSF). The objectives of this study were to establish in humans whether TTR and thyroxine (T(4)) are correlated in the CSF, and whether CSF levels of Pb are associated with those of TTR, T(4), and/or retinol-binding protein (RBP). Eighty-two paired CSF and blood/serum samples were collected from patients undergoing clinical diagnosis of CSF chemistry. Results showed that the mean value of CSF concentrations for TTR was 3.33 +/- 1.60 microg/mg of CSF proteins (mean +/- SD, n = 82), for total T(4) (TT(4)) was 1.56 +/- 1.68 ng/mg (n = 82), for RBP was 0.34 +/- 0.19 microg/mg (n = 82), and for Pb was 0.53 +/- 0.69 microg/dl (n = 61 for those above the detection limit). Linear regression analyses revealed that CSF TTR levels were positively associated with those of CSF TT(4) (r = 0.33, p < 0.005). CSF TTR concentrations, however, were inversely associated with CSF Pb concentrations (r = -0.29, p < 0.05). There was an inverse, albeit weak, correlation between CSF TT(4) and CSF Pb concentrations (r = -0.22, p = 0.09). The concentrations of TTR, TT(4), and Pb in the CSF did not vary as the function of their levels in blood or serum, but RBP concentrations in the CSF did correlate to those of serum (r = 0.39, p < 0.0005). Unlike TTR, CSF RBP concentrations were not influenced by PB: These human data are consistent with our earlier observations in animals, which suggest that TTR is required for thyroxine transport in the CSF and that Pb exposure is likely associated with diminished TTR levels in the CSF.

Retinol binding protein as a surrogate measure for serum retinol: studies in vitamin A-deficient children from the Republic of the Marshall Islands.

BACKGROUND: Serum retinol is transported by retinol binding protein (RBP), which has one high-affinity binding site for retinol; consequently, the molar ratio of retinol to RBP in the circulation is approximately 1 to 1. In vitamin A deficiency (VAD), both serum retinol and RBP decline. However, the retinol-RBP relation has not been well studied in populations with a high incidence of severe VAD. OBJECTIVE: The purpose of this study was to determine whether RBP is a good surrogate for serum retinol at the very low retinol concentrations encountered in VAD. DESIGN: The stoichiometric relation between retinol and RBP was studied in 239 Marshallese children: 65 with severe VAD (< or = 0.35 micromol retinol/L), 94 with moderate VAD (0.36-0.70 micromol retinol/L), and 80 with vitamin A sufficiency (> 0.70 micromol retinol/L). RESULTS: Excellent correlation between retinol and RBP (r = 0.94) was observed across all retinol concentrations. Severe VAD was predicted with 96% sensitivity and 91% specificity on the basis of an RBP cutoff of < or = 0.48 micromol/L, whereas moderate VAD was predicted with 87% sensitivity and 98% specificity on the basis of an RBP cutoff of < or = 0.70 micromol/L. CONCLUSIONS: The use of RBP results in the classification of essentially the same children with VAD as does retinol, and RBP is an excellent surrogate for serum retinol. Considering the relative ease of measuring RBP with immunodiagnostic kits compared with that of serum retinol by HPLC, the use of RBP concentrations to assess VAD may be particularly advantageous in field settings. Consequently, measuring RBP concentrations may be a practical alternative to measuring serum retinol in population surveys assessing the prevalence of VAD.

Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice.

Transthyretin (TTR) acts physiologically in the transport of retinol in the circulation. We previously reported the generation and partial characterization of TTR-deficient (TTR(-)) mice. TTR(-) mice have very low circulating levels of retinol and its specific transport protein, retinol-binding protein (RBP). We have examined the biochemical basis for the low plasma retinol-RBP levels. Cultured primary hepatocytes isolated from wild type (WT) and TTR(-) mice accumulated RBP in their media to an identical degree, suggesting that RBP was being secreted from the hepatocytes at the same rate. In vivo experiments support this conclusion. For the first 11 h after complete nephrectomy, the levels retinol and RBP rose in the circulations of WT and TTR(-) mice at nearly identical rates. However, human retinol-RBP injected intravenously was more rapidly cleared from the circulation (t(12) = 0.5 h for TTR(-) versus t(12) >6 h for WT) and accumulated faster in the kidneys of TTR(-) compared with WT mice. The rate of infiltration of the retinol-RBP complex from the circulation to tissue interstitial fluids was identical in both strains. Taken together, these data indicate that low circulating retinol-RBP levels in TTR(-) mice arise from increased renal filtration of the retinol-RBP complex.

Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.

Cellular retinol-binding protein, type I (CRBP-I) and type II (CRBP-II) are the only members of the fatty acid-binding protein (FABP) family that process intracellular retinol. Heart and skeletal muscle take up postprandial retinol but express little or no CRBP-I or CRBP-II. We have identified an intracellular retinol-binding protein in these tissues. The 134-amino acid protein is encoded by a cDNA that is expressed primarily in heart, muscle and adipose tissue. It shares 57 and 56% sequence identity with CRBP-I and CRBP-II, respectively, but less than 40% with other members of the FABP family. In situ hybridization demonstrates that the protein is expressed at least as early as day 10 in developing heart and muscle tissue of the embryonic mouse. Fluorescence titrations of purified recombinant protein with retinol isomers indicates binding to all-trans-, 13-cis-, and 9-cis-retinol, with respective K(d) values of 109, 83, and 130 nm. Retinoic acids (all-trans-, 13-cis-, and 9-cis-), retinals (all-trans-, 13-cis-, and 9-cis-), fatty acids (laurate, myristate, palmitate, oleate, linoleate, arachidonate, and docosahexanoate), or fatty alcohols (palmityl, petrosenlinyl, and ricinolenyl) fail to bind. The distinct tissue expression pattern and binding specificity suggest that we have identified a novel FABP family member, cellular retinol-binding protein, type III.

2,3,7,8-tetrachlorodibenzo-p-dioxin increases serum and kidney retinoic acid levels and kidney retinol esterification in the rat.

Halogenatedorganic environmental contaminants such as dioxins are well-known to affect tissue levels of retinoids. To further investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid homeostasis, adult male Sprague-Dawley rats were killed 1-112 days after a single oral dose of 10 microg TCDD/kg body wt. Additional groups of rats were killed three days after a single oral dose of 0.1, 1, 10, or 100 microg TCDD/kg body wt. Serum and renal retinoic acid levels were measured, as were levels of serum retinol-binding protein (RBP) in liver, kidneys, and serum. Hepatic and renal formation as well as hepatic hydrolysis of retinyl esters were determined, together with hepatic and renal retinoid levels. In addition, one of the retinyl ester hydrolase (REH) activities was investigated in isolated hepatocytes and hepatic stellate cells from rats killed 7 days after a single oral dose of 10 microg TCDD/kg body wt. No increased hepatic REH activity that could explain the decreased hepatic retinyl ester levels following TCDD treatment was found. In the liver, TCDD increased protein levels, but not mRNA levels, of RBP. A causal relationship is suggested for the increased renal lecithin:retinol acyltransferase (LRAT) activity and increased renal retinyl ester levels in TCDD-treated rats. Importantly, TCDD was shown to substantially increase serum and renal levels of retinoic acid. The ability of TCDD to cause increased tissue retinoic acid levels suggests that TCDD may alter the transcription of retinoic acid-responsive genes.

Hand-grip muscle strength, lean body mass, and plasma proteins as markers of nutritional status in patients with chronic renal failure close to start of dialysis therapy.

We studied 115 patients (69 men, 46 women) with chronic renal failure (CRF) aged younger than 70 years close to the start of dialysis therapy to assess the prevalence of malnutrition and study the relationship between various nutritional parameters in these patients. Nutritional status was classified by means of subjective global assessment. Anthropometric measurements (AMs) were performed, and hand-grip strength (HGS) was measured using the Harpenden dynamometer. Body composition, including lean body mass (LBM), was evaluated by dual-energy x-ray absorptiometry (DXA), and LBM was also estimated by means of AMs and creatinine kinetics (CK). The mean age of the patients was 52 +/- 12 years, and creatinine clearance was 9 +/- 3 mL/min. Malnutrition was seen in 53 patients (48%). As expected, malnourished patients differed in several aspects from well-nourished patients. LBM (estimated by all methods), fat mass (FM), HGS, creatinine clearance, and transthyretin and vitamin A levels were less in malnourished patients, whereas serum albumin levels did not differ. Estimates of LBM by means of DXA, AMs, and CK correlated well with each other. Although DXA and AMs gave similar mean values, LBM was an average of 8 kg less estimated by means of CK, and Bland-Altman plots showed the best agreement between AMs and DXA. HGS showed a strong correlation to LBM (regardless of method) in both men and women. Serum albumin level was not related to HGS or LBM, whereas significant correlations were found between serum albumin level and albumin losses in urine, C-reactive protein (CRP) level, and creatinine clearance. Multiple logistic regression showed that low HGS, low percentage of FM, female sex, and high serum CRP levels were independent factors associated with malnutrition, whereas serum albumin level and percentage of LBM did not reach statistical significance. In conclusion, the present study shows a high prevalence of malnutrition in predialysis patients with CRF and suggests that HGS is a reliable, inexpensive, and easy-to-perform nutritional parameter in patients with CRF. Conversely, serum albumin level seems to be a poor nutritional marker in patients with advanced CRF.

Predictors of postprandial triacylglycerol response in children: the Columbia University Biomarkers Study.

BACKGROUND: Predictors of postprandial lipemia have not been explored in children. OBJECTIVE: Our objective was to determine whether the postprandial triacylglycerol response is associated with low HDL-cholesterol and high fasting triacylglycerol concentrations and family history of early-onset ischemic heart disease (IHD) in children. DESIGN: We administered a standardized fat load (52.5 g fat/m(2)) to 60 children (mean age: 14.0 y), 20 with and 40 without a family history of early-onset IHD, and to 29 mothers, all recruited from families enrolled in the Columbia University Biomarkers Study. Plasma lipid and retinyl palmitate concentrations were measured in the fasting state and 3, 6, and 8 h after the oral fat load. RESULTS: In children, postprandial lipemia, as indicated by the incremental area under the triacylglycerol response curve, was associated with elevated fasting triacylglycerol concentrations (>/=1.13 mmol/L; P: < 0.01), with low fasting HDL-cholesterol concentrations (

Retinol and retinol-binding protein: gut integrity and circulating immunoglobulins.

Vitamin A (retinol) is required to maintain immunity and epithelial turnover and is a key micronutrient needed for combating infection. Vitamin A actions on the immune system are diverse and cannot be accounted for by a single effect or mechanism. The actions of retinol in maintaining gut integrity in humans and immunoglobulin levels in mice was investigated. For 30 children, performance on the lactulose/mannitol test, a test commonly used to assess intestinal barrier function, was inversely correlated (P=.012) with serum retinol concentrations. Thus, children with lower serum retinol, and presumably poorer vitamin A nutritional status, are more likely to have impaired intestinal integrity. Knockout mice that have impairments in plasma retinol transport have circulating immunoglobulin levels that are half those observed in matched wild type mice. No differences were observed in B and T cell populations present in spleen, thymus, and bone marrow.

9-cis-retinoids: biosynthesis of 9-cis-retinoic acid.

Retinoids function through conformational alterations of ligand-dependent nuclear transcription factors, the retinoic acid receptors, and retinoid X receptors. 9-cis-Retinoic acid is a known biological ligand for retinoid X receptors, but its synthesis pathway in vivo is largely unknown. Recently, we identified a cis-retinol dehydrogenase (cRDH) that oxidizes 9-cis-retinol to 9-cis-retinal. Since both the expression of cRDH mRNA and its substrate are found in liver, we studied 9-cis-retinol metabolism and 9-cis-retinoic acid biosynthesis in two hepatic-derived cell types, Hep G2 hepatoma cells and HSC-T6 stellate cells. Both cell lines accumulate similar amounts of 9-cis-retinol provided in the medium. However, Hep G2 cells preferentially incorporate all-trans-retinol when equimolar concentrations of all-trans- and 9-cis-retinol were provided. In contrast, HSC-T6 cells did not exhibit a preference between all-trans- and 9-cis-retinol under the same conditions. Esterification of 9-cis-retinol occurred in both cell types, likely by acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase. In vitro enzyme assays demonstrated that both cell types can hydrolyze 9-cis-retinyl esters via retinyl ester hydrolase(s). In Hep G2 cells, 9-cis-retinoic acid synthesis was strongly inhibited by high concentrations of 9-cis-retinol, which may explain the low levels of 9-cis-retinol in liver of mice. Cell homogenates of Hep G2 can convert all-trans-retinol to 9-cis-retinal, suggesting that the free form of all-trans-retinol may be used as a source for 9-cis-retinol and, thus, 9-cis-retinoic acid synthesis. Our studies provide the basis for identification of additional pathways for the generation of 9-cis-retinoic acid in specialized tissues.

Evidence for independent control at the mRNA and protein levels of cellular retinol binding protein 1 in rat Sertoli cells.

Cellular retinol binding protein 1 (CRBP1) is the cytosolic carrier for retinol. It is expressed in many tissues, but the concentrations vary considerably. In Sertoli cells from immature rat testis, CRBP1 is highly expressed. The results of the present study show that regulation of CRBP1 expression at the protein level appears to be independent of regulation at the mRNA level. In Sertoli cells from prepubertal 19-day-old rats, CRBP1 mRNA is strongly induced for up to 72 h by the presence of serum factors. In contrast, treatment of the cells with cAMP analogues led to a rapid reduction in mRNA to quantities less than 5% of control values. However, the changes in CRBP1 mRNA did not lead to similar changes in the concentration of CRBP1 protein during 72 h of observation. Similarly, treatment of cells from 32- and 44-day-old rats with serum led to increased CRBP1 mRNA, whereas cAMP treatment resulted in a decrease in CRBP1 mRNA. Again, no changes were observed in the concentration of CRBP1 protein. Furthermore, co-incubation of Sertoli cells from 19-day-old rats with purified pachytene spermatocytes or round spermatids resulted in an increase in mRNA for CRBP1. However, comparable changes in CRBP1 protein concentrations were not observed. Neither cAMP nor serum changed the fraction of CRBP1 mRNA that was associated with polysomes. As a possible explanation for some of the results, pulse-chase experiments showed that the rate of CRBP1 degradation in cultured Sertoli cells is decreased by cAMP. It is proposed that these changes at the level of protein turnover contribute to the maintenance of stable concentrations of CRBP1 even when the corresponding mRNA concentrations vary markedly.

An immortalized rat liver stellate cell line (HSC-T6): a new cell model for the study of retinoid metabolism in vitro.

Hepatocytes and hepatic stellate cells play important roles in retinoid storage and metabolism. Hepatocytes process postprandial retinyl esters and are responsible for secretion of retinol bound to retinol-binding protein (RBP) to maintain plasma retinol levels. Stellate cells are the body's major cellular storage sites for retinoid. We have characterized and utilized an immortalized rat stellate cell line, HSC-T6 cells, to facilitate study of the cellular aspects of hepatic retinoid processing. For comparison, we also carried out parallel studies in Hepa-1 hepatocytes. Like activated primary stellate cells, HSC-T6 express myogenic and neural crest cytoskeletal filaments. HSC-T6 cells take up and esterify retinol in a time- and concentration-dependent manner. Supplementation of HSC-T6 culture medium with free fatty acids (up to 300 micrometer) does not affect retinol uptake but does enhance retinol esterification up to 10-fold. RT-PCR analysis indicates that HSC-T6 cells express all 6 retinoid nuclear receptors (RARalpha, -beta, -gamma, and RXRalpha, -beta, -gamma) and like primary stellate cells, HSC-T6 stellate cells express cellular retinol-binding protein, type I (CRBP) but fail to express either retinol-binding protein (RBP) or transthyretin (TTR). Addition of retinol (10(-8)-10(-5) m) or all-trans-retinoic acid (10(-10)-10(-6) m) rapidly up-regulates CRBP expression. Using RAR-specific agonists and antagonists and an RXR-specific agonist, we show that members of the RAR-receptor family modulate HSC-T6 CRBP expression.Thus, HSC-T6 cells display the same retinoid-related phenotype as primary stellate cells in culture and will be a useful tool for study of hepatic retinoid storage and metabolism.

Hydrolysis of retinyl esters by pancreatic triglyceride lipase.

Previously [van Bennekum, A. M., et al. (1999) Biochemistry 38, 4150-4156] we showed that carboxyl ester lipase (CEL)-deficient (CELKO) mice have normal levels of pancreatic, bile salt-dependent retinyl ester hydrolase (REH) activity. In the present study, we further investigated this non-CEL REH activity in pancreas homogenates of CELKO and wild-type (WT) mice, and rats. REH activity was detected in both the presence and absence of tri- and dihydroxy bile salts in rats, WT mice, and CELKO mice. In contrast, pancreatic cholesteryl ester hydrolase (CEH) activity was only detected in the presence of trihydroxy bile salts and only in rats and WT mice, consistent with CEL-mediated cholesteryl ester hydrolysis. Enzyme assays of pancreatic triglyceride lipase (PTL) showed that there was a colipase-stimulated REH activity in rat and mouse (WT and CELKO) pancreas, consistent with hydrolysis of retinyl ester (RE) by PTL. Pancreatic enzyme activities related to either CEL or PTL were separated using DEAE-chromatography. In both rats and mice (WT and CELKO), REH activity could be attributed mainly to PTL, and to a much smaller extent to CEL. Finally, purified human PTL exhibited similar enzymatic characteristics for triglyceride hydrolysis as well as for retinyl ester hydrolysis, indicating that RE is a substrate for PTL in vivo. Altogether, these studies clearly show that PTL is the major pancreatic REH activity in mice, as well as in rats.

Nicotine enhances the biosynthesis and secretion of transthyretin from the choroid plexus in rats: implications for beta-amyloid formation.

Epidemiological studies indicated that cigarette smoking protects against the development of several neurodegenerative disorders, including Alzheimer's disease (AD). However, the molecular mechanism(s) underlying this is poorly understood. To gain insight into these protective effects, we used differential display PCR (DD-PCR) to amplify RNA from various brain regions of rats self-administering (SA) nicotine compared with yoked-saline controls. We found that the transthyretin (TTR) gene, whose product has been shown to bind to amyloid beta (Abeta) protein and prevent Abeta aggregation, was more abundantly expressed ( approximately 1.5- to 2.0-fold) in the brainstem and hippocampus (areas containing choroid plexus) of nicotine SA rats. Subsequently, quantitative reverse transcription-PCR analysis confirmed these DD-PCR findings and demonstrated that nicotine increased TTR mRNA levels in these regions in a time- and dose-dependent manner. Significantly higher TTR protein concentrations were also detected in the ventricular CSF of nicotine-treated rats. In contrast, no differences either in plasma TTR or in CSF and plasma retinol-binding protein were detected. Immunohistochemical analysis showed that immunoreactive TTR was 41.5% lower in the choroid plexus of nicotine-treated rats compared with the saline controls. On the basis of these data, we speculate that the protective effects of nicotine on the development of AD may be attributable, in part, to the increased biosynthesis and secretion of TTR from the choroid plexus. These findings also point toward new approaches that may take advantage of the potentially novel therapeutic effects of nicotinic agonists in patients with AD.