Expression Of Retinol-binding Protein Research Articles

Bovine hepatic and adipose retinol-binding protein gene expression and relationship with tumor necrosis factor-α

Retinol-binding protein (RBP) is the main transport system for retinol in circulation, is a relatively small protein with one binding site for retinol in the all-trans form, and is bound to transthyretin. The objectives of this study were to characterize the temporal pattern of bovine hepatic mRNA expression of RBP during the periparturient period and to determine if a relationship exists between the expression of RBP and that of tumor necrosis factor (TNF)-α in dairy cows. In experiment 1, we assessed hepatic mRNA expression of RBP during the periparturient period. Liver tissues were sampled from periparturient dairy cows (n=9) at −21, −4, +1, +7, and +21 relative to parturition and frozen in liquid N2. Total RNA was extracted from each tissue sample and cDNA was generated. Gene expressions of RBP and β-actin (as a housekeeping gene) were measured as relative quantity using reverse transcription-PCR. Data were analyzed using cycle threshold values, adjusted to β-actin, and significance was determined at P<0.05. Serum samples (−21, −4, +1, +7, and +21 relative to parturition) were analyzed for retinol concentration using a standard HPLC-based method. Cows had variable expression of hepatic RBP and serum retinol over the transition period, with a decline near parturition and a rebound toward prepartum levels later in lactation. In experiment 2, liver and visceral (intestinal) adipose tissues were sampled from dairy cows (n=28) at slaughter. Expression of RBP and TNF-α was detected in all samples and variations among cows were highly significant for both genes. Across tissues, expression of RBP was positively correlated with that of TNF-α (r=0.60). Within adipose tissue, expression of RBP and TNF-α was weakly correlated (r=0.23), whereas in hepatic tissue, expression was strongly correlated (r=0.62). In experiment 3, late-lactation dairy Holstein cows were blocked by parity and feed intake, and randomly assigned to control, recombinant bovine (rb)TNF challenge, or pair-fed control treatment (n=5/treatment). Cows were injected with either rbTNF (subcutaneous injection of 2µg/kg of body weight in saline) or sterile saline (control and pair-fed control animals) once daily for 7d. Liver biopsy was performed on d 7 and samples were processed for expression of RBP and TNF-α. Although TNF challenge caused an upregulation of hepatic TNF-α expression, as expected, it did not alter hepatic RBP expression. Overall, the temporal pattern of hepatic RBP gene expression during the periparturient period followed, to a great extent, that of plasma retinol. Although a strong positive correlation was previously detected between bovine hepatic RBP and TNF-α transcripts, rbTNF challenge did not cause alter RBP expression. These observations collectively imply that regulation of RBP at the transcription level is influenced by physiological state but may be independent from that of transthyretin, which is altered by proinflammatory stimuli (such as TNF-α) via induction of transcription factor nuclear factor-interleukin 6.

Eradication of hepatitis C virus subgenomic replicon by interferon results in aberrant retinol-related protein expression.

Hepatitis C virus (HCV) infection induces several changes in hepatocytes, such as oxidative stress, steatosis, and hepatocarcinogenesis. Although considerable progress has been made during recent years, the mechanisms underlying these functions remain unclear. We employed proteomic techniques in HCV replicon-harboring cells to determine the effects of HCV replication on host-cell protein expression. We examined two-dimensional electrophoresis (2-DE) and mass spectrometry to compare and identify differentially expressed proteins between HCV subgenomic replicon-harboring cells and their "cured" cells. One of the identified proteins was confirmed using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Full-length HCV genome RNA replicating and cured cells were also assessed using ELISA. Replicon-harboring cells showed higher expression of retinal dehydrogenase 1 (RALDH-1), which converts retinol to retinoic acid, and the cured cells showed higher expression of retinol-binding protein (RBP), which transports retinol from the liver to target tissues. The alteration in RBP expression was also confirmed by ELISA and Western blot analysis. We conclude that protein expression profiling demonstrated that HCV replicon eradication affected retinol-related protein expression.

Nitrofen interferes with trophoblastic expression of retinol-binding protein and transthyretin during lung morphogenesis in the nitrofen-induced congenital diaphragmatic hernia model

Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression. Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 μg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 μg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group. Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary hypoplasia in CDH.

Increased retinol‐binding protein (RBP) 4 and anti‐RBP4 antibody in alopecia areata

Neither the underlying pathogenesis of alopecia areata (AA) nor the molecular mechanisms leading to hair loss have been fully elucidated. To compare the protein profiles of sera obtained from patients with AA with those from healthy controls. Protein profiles of sera obtained from subjects with AA and healthy controls were compared using proteomics techniques. Serum levels of the identified protein were quantified by specific enzyme-linked immunosorbent assay (ELISA). The relative serum reactivities of the recombinant human protein were compared between patients with AA and healthy controls using Western blots and double indirect immunofluorescence. The upregulated expression of retinol-binding protein (RBP) 4 was identified, and RBP4 ELISA demonstrated significantly increased serum levels of RBP4 among subjects with AA when compared with healthy controls. Western blots using recombinant human RBP4 and the sera from both groups presented serum reactivity of antihuman recombinant RBP4 IgG antibodies in 10/15 subjects with AA (67%) and 2/15 healthy controls (13%). Double indirect immunofluorescence demonstrated merged fluorescence signals of serum anti-RBP4 IgG antibodies and monoclonal antibodies to RBP4 in subjects with AA on the outer root sheath and companion layer. Our data demonstrate that AA is associated with increased serum levels of RBP4 and positive IgG immunoreactivity against recombinant human RBP4. These results suggest that the major components for the retinoic acid biosynthesis pathway may be crucially involved in the pathogenic process of AA.

Depot-specific expression of retinol-binding protein 4 in human adipose tissue and their relationship with obesity and insulin resistance

To explore the depot-specific mRNA and protein expression of retinol-binding protein 4 (RBP4) in human subcutaneous and omental adipose tissue and investigate their relationship with the serum RBP4 levels, obesity and insulin resistance. A total of 41 subjects with normal glucose regulation were recruited. Among them, there were 29 cases with normal body mass index (BMI) and 12 cases with BMI ≥ 24 kg/m(2). All were prepared to undergone a selective abdominal surgery. The levels of serum RBP4 and fasting insulin (FINS) were measured by ELISA (enzyme-linked immunosorbent assay) and chemiluminescence ELISA kit respectively. Fasting plasma glucose (FPG) was tested by glucose oxidase and the home model insulin resistance index (HOMA-IR) calculated. Real-time PCR (RT-PCR) and Western blot were employed to assess the relative mRNA and protein expression of RBP4 in subcutaneous and omental adipose tissues. The mRNA and protein expression of RBP4 from different fat depots were compared and their correlations with BMI, the serum RBP4 concentrations and HOMA-IR analyzed. (1) The serum concentrations of RBP4, FINS and HOMA-IR were significantly higher in overweight and obesity group than those in the normal BMI group [RBP4: (39.61 ± 1.57) mg/L vs (33.40 ± 1.28) mg/L, P < 0.01; FINS: (8.82 ± 3.79) mU/L vs (6.43 ± 4.38) mU/L, P < 0.05; HOMA-IR: 1.91 ± 0.85 vs 1.36 ± 0.72, P < 0.05]. (2) The expression levels of RBP4 mRNA and protein were significantly higher in omental adipose tissues than those in subcutaneous adipose tissue [mRNA: (5.88 ± 2.37) vs (2.07 ± 1.66), P < 0.01; protein: (0.76 ± 0.18 vs 0.15 ± 0.07, P < 0.05] and these depots difference in both groups (P < 0.05). Moreover, the overweight patients had the higher expression levels of RBP4 mRNA and protein in omental adipose tissue than normal BMI group (mRNA: 7.52 ± 0.58 vs 4.37 ± 0.45, P < 0.01; protein: 0.92 ± 0.23 vs 0.57 ± 0.13, P < 0.05). (3) The expressions of both RBP4 mRNA and protein in the omental adipose tissue were positively correlated with BMI, waist circumstance, serum concentrations of RBP4, FINS and HOMA-IR. The expression of was negatively correlated with HOMA-IR (r = -0.635, P < 0.05) in subcutaneous adipose tissue. No correlations were found between the expressions of RBP4 mRNA and protein in subcutaneous adipose tissue with BMI, waist circumstance, serum RBP4 and FINS concentrations. There is depot-specific expression of RBP4 in human subcutaneous and omental adipose tissues. A high expression of RBP4 in omental adipose tissue and an elevated level of serum RBP4 may contribute to the pathogenesis of obesity and IR.

Renal effects of microwave ablation compared with radiofrequency, cryotherapy and surgical resection at different volumes of the liver treated

Renal changes after microwave tissue ablation (MTA) were compared with those following hepatic resection, cryotherapy (CRYO), and radiofrequency ablation (RFA). Structural damage producing renal impairment has been assessed directly by examining tissue specimens and by serum analysis for two sensitive biomarkers, retinol binding protein (RBP) and the heat shock protein 70 (HSP-70) for each modality at different ablation volumes. Live rats underwent MTA, surgical resection, CRYO or RFA of 15, 33 or 66% of total hepatic volume. Urine and tissue samples were collected at the time of death. Percentage of tubules with casts and glomerular damage, tissue expression of HSP-70 and urine RBP were evaluated and compared. Behaviour of the animals was also assessed by means of five different parameters and combined to produce a response score. All RFA and CRYO rats undergoing 66% died and these animals had >60% of damaged tubuli and 8% of altered glomeruli. No animals treated by MTA or surgical resection died. Cut-off values (those predicting fatal treatments) could be identified for levels of HSP-70 and RBP. Large volume MTA is associated with a significant reduced renal damage and is well tolerated compared with RFA and CRYO.

Correlation of retinol binding protein 4 expression in maternal serum and subcutaneous adipose tissue with insulin resistance in gestational diabetes mellitus.

To investigate the expression of retinol binding protein 4 (RBP-4) in maternal serum and subcutaneous adipose tissue and its relationship with insulin resistance (IR) in gestational diabetes mellitus (GDM). From May 2008 to April 2009, 62 pregnant women who underwent elective cesarean section in the Department of Obstetrics, Affiliated Hospital of Qingdao University Medical College, were recruited, including 32 with GDM (GDM group) and 30 with normal glucose tolerance test (control group). Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum concentrations of RBP-4 and radio immunoassay to measure the serum levels of fasting insulin (FINS). Fasting plasma glucose (FPG) was tested by glucose oxidase, and the Home model insulin resistance index (HOMA-IR) was calculated. Reverse transcription PCR (RT-PCR) and Western blot were applied to investigate the expression of RBP-4 mRNA and protein in subcutaneous adipose tissue. The correlations between the expression of RBP-4 mRNA and protein in subcutaneous adipose tissue and the serum RBP-4 concentrations and HOMA-IR were analyzed. (1) The serum concentrations of RBP-4, FINS, FPG, HOMA-IR in GDM group were significantly higher compared with the control group [(27.0 +/- 1.2) mg/L vs. (19.4 +/- 1.8) mg/L, (12.1 +/- 1.4) mU/L vs. (8.3 +/- 0.8) mU/L, (5.3 +/- 0.9) mmol/L vs. (4.1 +/- 0.6) mmol/L, 2.5 +/- 0.2 vs. 1.5 +/- 0.1, P < 0.05, respectively]. (2) The expression of RBP-4 mRNA and protein in subcutaneous adipose tissue in the GDM group were significantly higher than that of the control group (0.76 +/- 0.12 vs. 0.53 +/- 0.06, 0.74 +/- 0.09 vs 0.54 +/- 0.06, P < 0.05). (3) In the GDM group, the expression of both RBP-4 mRNA and protein in the subcutaneous adipose tissue were positively correlated with HOMA-IR (r = 0.575 and 0.851, P < 0.05). The serum concentration of RBP-4 were also positively correlated with HOMA-I (r = 0.635, P < 0.05). No correlations was found between the expressions of RBP-4 mRNA and protein in subcutaneous adipose tissue with the serume RBP-4 concentrations. High expression of RBP-4 mRNA in subcutaneous adipose tissue and the elevation of serum RBP-4 levels in GDM women may contribute to IR.

Aberrant expression of retinol-binding protein, osteopontin and fibroblast growth factor 7 in the porcine uterine endometrium of pregnant recipients carrying embryos produced by somatic cell nuclear transfer

The technique of somatic cell nuclear transfer (NT) is a useful tool to produce cloned animals for various purposes, but the efficiency to generate cloned animals using this technique is still very low. To improve the low efficiency in production of cloned pigs it is critical to understand the reprogramming process during development of cloned embryos, but it is also essential to understand the uterine function interacting with the transferred cloned embryos during implantation and placentation. Thus, to understand the uterine responsiveness to NT cloned embryos during pregnancy, we investigated expression of retinol-binding protein (RBP), osteopontin (OPN) and fibroblast growth factor 7 (FGF7), which play important roles in implantation and/or maintenance of pregnancy as a transport protein, an extracellular matrix protein and a growth factor, respectively, in the uterine endometrium in pigs. The uterine tissue samples were obtained by C-section from pigs with NT cloned normal (NT-normal) embryos and NT cloned abnormal (NT-abnormal) embryos and pigs with non-NT (Non-NT) embryos at term. Immunoblot analysis showed that expression of RBP and FGF7 decreased in the uterine endometrium of recipient gilts carrying NT embryos than in the endometrium of gilts carrying Non-NT embryos. Levels of OPN protein of 70 and 45 kDa were not different in between the uterine endometrium of gilts carrying Non-NT and NT-normal embryos, but in the uterine endometrium of gilts carrying NT-abnormal embryos 70 and 45 kDa OPN proteins increased compared to those in the endometrium of gilts carrying Non-NT embryos. Immunohistochemistry results showed that RBP expression was lower in the endometrial glandular epithelial cells, while OPN expression was higher in the endometrial luminal epithelial cells of the uterus of gilts carrying NT embryos than in the uterus of gilts carrying Non-NT embryos. Results of this study showed that maternal uterine genes were aberrantly expressed in the uterine endometrium of gilts carrying NT cloned embryos in varying degrees depending on the normality of the developing embryos. These results indicate that abnormal maternal–fetal interactions of the uterus carrying the developing NT cloned embryos may cause problems in development of cloned embryos.

Plasma Levels and Adipose Tissue Messenger Ribonucleic Acid Expression of Retinol-Binding Protein 4 Are Reduced during Calorie Restriction in Obese Subjects but Are Not Related to Diet-Induced Changes in Insulin Sensitivity

Retinol-binding protein 4 (RBP4) may play a role in the development of insulin resistance. We investigated whether RBP4 adipose tissue mRNA expression and plasma level are related to insulin sensitivity during a diet-induced weight loss. Obese women followed a dietary intervention composed of a 4-wk very low-calorie diet (VLCD), a 2-month low-calorie diet, and 3-4 months of a weight maintenance (WM) phase. Clinical investigation was performed before and at the end of each phase. Insulin sensitivity was assessed with the euglycemic hyperinsulinemic clamp. Adipose tissue mRNA and plasma levels of RBP4 were determined using reverse transcription-quantitative PCR and ELISA, respectively. Weight and fat mass decreased during VLCD and were stabilized during WM. Glucose disposal rate increased during VLCD and remained elevated thereafter. Plasma levels of RBP4 decreased after VLCD and, although increasing at subsequent phases, remained lower than prediet values. Adipose tissue mRNA levels were diminished after VLCD, and increased during low-calorie diet and WM to reach basal values. Basal RBP4 levels or diet-induced variations of RBP4 were not different in lean women and two groups of obese women with high- and low-insulin sensitivity. Severe calorie restriction promotes a reduction in adipose tissue and plasma levels of RBP4. The study does not bring evidence for a role for RBP4 in the regulation of diet-induced changes in insulin sensitivity.

Human binucleate hepatocytes: Are they a defence during chronic liver diseases?

Binucleate cells are commonly found in various human organs including liver, salivary glands and endometrium, but their functional advantage remains unknown. The increased occurrence of binucleate hepatocytes during the necro-inflammation stage of progressive chronic hepatitis and its end-stage of cirrhosis, but their absence in hepatocellular carcinoma (HCC), has led us to hypothesise that they may be an index of the severity of hepatic illness rather than the result of errors occurring during the course of the cell cycle. This hypothesis is supported by the immunohistochemical analysis of retinol-binding protein expression, and the different life cycles of hepatitis B virus in mononucleate and binucleate hepatocytes. If founded, this hypothesis would add to our understanding of the relationship between binucleate hepatocytes and the evolution of chronic liver disease, and promises the ideation of new criteria for identifying potential HCC patients.

Endocrine disrupters with (anti)estrogenic and (anti)androgenic modes of action affecting reproductive biology of Xenopus laevis: I. Effects on sex steroid levels and biomarker expression

Adult Xenopus laevis were exposed in vivo to ethinylestradiol, tamoxifen, methyldihydrotestosterone and flutamide as (anti)estrogenic and (anti)androgenic compounds, respectively, for four weeks at a concentration of 10 − 8 M and to Lambro river water, a polluted river from Italy. Effects of the treatments were analysed by mRNA expression of retinol-binding protein (RBP), transferrin (TF), transthyretin (TTR) and vitellogenin (VTG) in the liver of male and female X. laevis, to analyse the potential of these genes to detect endocrine disrupting compounds (EDC) with different modes of action. In addition, plasma VTG and sex steroid levels, estradiol-17β (E 2) and testosterone (T), were analysed. Sex steroids were depressed by ethinylestradiol in both sexes whereas tamoxifen increased E 2 in females. The induction of VTG protein plasma levels was more pronounced at the protein level compared to hepatic VTG mRNA expression in response to estrogenic treatment but VTG mRNA expression detected both, estrogenic and antiestrogenic EDC. The mRNA expression of TF was decreased by estrogenic and increased by antiestrogenic treatment while TTR mRNA expression was down-regulated and RBP mRNA up-regulated by estrogenic exposure. The other treatments did not affect the mRNA expression of the examined genes.

Effect of systemic progesterone concentration on the expression of progesterone-responsive genes in the bovine endometrium during the early luteal phase

Increasing evidence indicates an association between the concentration of systemic progesterone during the early luteal phase of the oestrous cycle and embryo survival rate in cattle. We examined the relationship between the concentration of systemic progesterone on Days 4 to 8 post-ovulation and expression of progesterone receptor (PGR), oestrogen receptor +/- (ESR1) and retinol-binding protein (RBP) mRNA in the bovine endometrium. Heifers were blood sampled from the day of ovulation (Day 0) to Day 8 post-ovulation. On Day 4, animals were divided into low progesterone control (LC) and high progesterone control (HC) groups based on their plasma progesterone concentrations. Half of each group was supplemented with exogenous progesterone resulting in two further groups, low progesterone supplemented (LS) and high progesterone supplemented (HS). Endometrial tissues were recovered from all groups on Day 6 or Day 8 and gene expression was analysed following Northern blotting. Increasing progesterone concentrations were associated with decreased PGR and ESR1 expression. Duration-dependent effects of progesterone supplementation on ESR1 were evident and there was an effect of systemic progesterone concentrations between Day 0 and Day 4 on the expression of RBP at Days 6 and 8. Such progesterone-responsive changes in uterine gene expression are likely to affect embryo development.

Pathways of Vitamin A Delivery to the Embryo: Insights from a New Tunable Model of Embryonic Vitamin A Deficiency

Circulating retinoids (vitamin A and its derivatives) are found predominantly as retinol bound to retinol-binding protein (RBP), which transports retinol from liver stores to target tissues, or as retinyl ester incorporated in lipoproteins of dietary origin. The transport of retinoids from maternal to fetal circulation is poorly understood, especially under conditions of inadequate dietary vitamin A intake. Here we present RBP-/- mice as a tunable model of embryonic vitamin A deficiency. This model has enabled us to analyze metabolic links between maternal nutrition and retinoid delivery to the fetus. Our data show that retinol-RBP is the primary contributor to fetal development, whereas retinyl ester are largely responsible for accumulation of fetal retinoid stores. Furthermore, these studies indicate the importance of embryonic RBP in distributing vitamin A to certain developing tissues under restrictive diets. We also show differences among developing tissues in their dependency on the embryonic retinol-RBP pathway. Finally, we demonstrate that accumulation of embryonic vitamin A stores does not depend on the expression of RBP in the fetal liver.

Retinol-binding protein gene is highly expressed in higher-order association areas of the primate neocortex.

The neocortex consists of histochemically, connectionally, and functionally distinguishable areas. Recently, molecular biological techniques have enabled us to find rare types of genes expressed in specific neocortical areas. We previously reported occ1 gene as preferentially expressed in the primary visual cortex (V1), using the differential display method. Here, by differential display, we found selective and strong expression of the serum retinol-binding protein (RBP) gene, in higher-order association areas. In V1, RBP mRNA was expressed only in the superficial part of layer II, but its expression increased, involving deeper layers, along the visual pathway. In visual association areas such as TE, RBP mRNA was strongly expressed in both supra- and infragranular layers. In primary auditory and somatosensory areas, as in V1, RBP expression was low, and restricted to the upper part of the supragranular layers. The laminar pattern of RBP expression is in marked contrast with that of occ1; and in early visual areas where both genes are expressed, these occur in distinct sublayers within the supragranular layers. In neonatal monkeys, the area-specific expression pattern of RBP was less distinct, suggesting that the characteristic expression of RBP in higher-order association areas is mainly established postnatally.

Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary.

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.

Long-Term Marginal Intakes of Zinc and Retinol Affect Retinol Homeostasis without Compromising Circulating Levels during Lactation in Rats

Marginal zinc or vitamin A intake is more common than previously thought in industrialized and developing countries, with pregnant and lactating women believed to be particularly at risk. However, the lack of sensitive indicators of zinc and vitamin A status precludes accurate assessment of marginal nutriture. Concurrent deficiencies in zinc and vitamin A intake often coexist, and the interaction between zinc deficiency and vitamin A metabolism may confound results from epidemiologic or intervention studies. To investigate effects of a maternal diet chronically restricted in zinc or vitamin A intake on indices of vitamin A metabolism, we fed rats a control diet (C) or a diet marginal in zinc (ZD), marginal in vitamin A (AD), marginal in both (DD) or pair-fed to DD (PF), preconception through lactation. Plasma retinol (ROH) was greater and retinol binding protein (RBP) was lower in rats fed ZD, AD and DD compared with those fed C. Hepatic cellular retinol binding protein (CRBP) expression was greater than controls in rats fed ZD and AD and lower in those fed DD, whereas RBP expression was greater in the DD- and PF-fed groups compared with rats C. Mammary gland CRBP and RBP expression were not affected by the diets. Milk ROH was lower in rats fed AD, and milk RBP was lower in those fed ZD and DD compared with rats fed C. In summary, chronic, marginal intake of zinc or vitamin A resulted in alterations in tissue retinol metabolism and milk retinol levels without decreasing plasma zinc, retinol or ROH:RBP during lactation. These observations are of concern because these parameters, which are commonly used to assess zinc and vitamin A status, may lead to misassessment of marginal zinc or vitamin A nutriture in some human populations.

Prenatal Ethanol Consumption Increases Retinol and Cellular Retinol-Binding Protein Expression in the Rat Fetal Snout

Fetal exposure to ethanol disrupts normal craniofacial development, resulting in characteristic features of fetal alcohol syndrome (FAS). One mechanism that could result in some anomalies of this syndrome is through ethanol disrupting the regulatory role played by vitamin A in fetal development, thereby inducing morphological alterations which manifest as FAS. This work begins to explore a possible interaction of ethanol with vitamin A in craniofacial development. Retinoid levels and the expression of cellular retinol-binding protein (CRBP) and retinoic acid receptor (RAR) mRNA were determined in snouts of 20-day fetuses exposed to ethanol throughout gestation, compared to controls. Snout retinol and retinyl palmitate levels were elevated in fetuses of ethanol-treated rats, but retinoic acid levels were unaffected. The expression of CRBP mRNA, as determined by Northern analysis, was greater in snouts of fetuses exposed to ethanol, but there was no change in RAR α, β, γ or retinoid X receptor β mRNA. These results demonstrate that prenatal ethanol consumption can alter certain markers of vitamin A metabolism and function in the fetal snout.

Developmental expression, tissue distribution and hormonal regulation of fish (Sparus aurata) serum retinol-binding protein

Retinol-binding protein (RBP) is the specific carrier of retinol in vertebrates and forms a 1:1 complex with transthyretin (TTR). A cDNA encoding serum RBP was cloned from liver and 7-day larvae of the marine fish Sparus aurata. The mature protein is 176 amino acids long and shows sequence identity of 77–78%, 56%, 63% and 62% with rainbow trout, Xenopus, chicken and human RBP, respectively. Northern blot analysis of hepatic RBP revealed two transcripts: a major one of approximately 1.4–1.5 kb and a minor of approximately 0.7 kb. Distribution of RBP mRNA in various tissues was studied by RT-PCR and showed high expression in liver and skin, and low expression in brain, kidney and gill filament (20–35% of the level in liver). RBP expression in intestine, pyloric caeca, muscle and pituitary was estimated to be approximately 7–14% of the level in liver. The ontogeny of RBP expression in S. aurata was examined in unfertilized eggs, embryos and larvae by using RT-PCR followed by hybridization with a specific probe. RBP transcript was found in all larval stages studied. Very low levels of RBP mRNA were detected in unfertilized eggs and in embryos 8 h after fertilization with a gradual increase at 12 h and 15–16 h post-fertilization. A single injection of estradiol-17β to S. aurata immature, bisexual fish or to adult males reduced steady-state levels of hepatic RBP by 37 and 25%, respectively. The same treatment induced vitellogenin expression. The present data suggest that in fish, liver is the main site of RBP synthesis, but that RBP may have an important function in fish skin. RBP is expressed early in embryonic development and in fish its expression can be down regulated by estrogen.

Retinol binding protein expression is induced in HepG2 cells by zinc deficiency

Zinc (Zn) deficiency is often associated with low plasma vitamin A (retinol) concentrations. It has been suggested that the reduction in plasma retinol is secondary to reduced liver retinol binding protein (RBP) synthesis. In the present study, RBP expression was determined in HepG2 cells cultured in either Zn adequate media or chelated media containing varying concentrations of Zn. Levels of RBP mRNA increased in a time- and Zn concentration-dependent manner such that 0.5 μM Zn-treated cells exhibited a >7.5-fold increase while cells treated with 15 μM Zn were increased 2.9-fold at 72 h compared to controls. RBP protein also progressively increased by 72 h to levels >8-fold and 3-fold higher than controls, in 0.5 μM and 15 μM Zn-treated cells, respectively. The increase in RBP occurred without any change in DNA concentration between groups through 72 h. The Zn deficiency-induced elevations in RBP transcript levels could be reversed within 24–48 h of repletion in Zn adequate media. Thus, the reductions in plasma retinol observed in Zn deficiency are in part a direct consequence of the deficiency.

Immunolocalization of retinol-binding protein, cellular retinoic acid-binding protein I and retinoid X receptor beta in the porcine reproductive tract during the oestrous cycle.

Retinoid-binding proteins and nuclear receptors are expressed in the reproductive tissues of different species and their expression is hormonally regulated. In the present study, we demonstrated immunocytochemically the temporal and spatial localization of retinol-binding protein (RBP), cellular retinoic acid-binding protein I (CRABPI) and retinoid X receptor beta (RXRbeta) in porcine ovary, oviduct and uterus during the oestrous cycle. RBP and CRABPI were localized in the cytoplasm, whereas RXRbeta occurred in the nucleus. RBP was not detected in either the ovary or the oviduct at any stage of the oestrous cycle. CRABPI was present in luteal cells of the ovary only during dioestrus and in glandular and ciliated cells of the oviduct during oestrus. In the ovary, RXRbeta was always present in granulosa cells and germinal epithelium, with highest levels observed during oestrus. In the uterus, RXRbeta was present throughout the cycle in both the endometrium and the myometrium. However, changes in RXRbeta were observed in the endometrium, with highest levels observed during dioestrus. RBP and CRABPI could be observed in the endometrium only during dioestrus. The results show that the occurrence of retinoid-binding proteins and nuclear receptors in individual tissues of the reproductive tract are strongly dependent on the stage of the oestrous cycle. In the oviduct, the expression of CRABPI seems to be dependent on oestrogen, whereas in the uterus the expression of RBP and CRABPI is influenced by progesterone. The association of expression in different sections of the reproductive tissues investigated shows that the presence of specific proteins involved in retinoid metabolism was dependent on events associated with ovulation, the migration of the oocyte through the oviduct and the possible implantation of the blastocyst into the uterus.