Retinol kinetics in unsupplemented and vitamin A-retinoic acid supplemented neonatal rats: a preliminary model

Vitamin A (VA) metabolism in neonates is virtually uncharacterized. Our objective was to develop a compartmental model of VA metabolism in unsupplemented and VA-supplemented neonatal rats. On postnatal day 4, pups (n = 3/time) received 11,12-[³H]retinol orally, in either oil (control) or VA combined with retinoic acid (VARA) [VA (∼6 mg/kg body weight) + 10% retinoic acid]. Plasma and tissues were collected at 14 time points up to 14 days after dose administration. VARA supplementation rapidly, but transiently, increased total retinol mass in plasma, liver, and lung. It decreased the peak fraction of the dose in plasma. A multi-compartmental model developed to fit plasma [³H]retinol data predicted more extensive recycling of retinol between plasma and tissues in neonates compared with that reported in adults (144 vs. 12–13 times). In VARA pups, the recycling number for retinol between plasma and tissues (100 times) and the time that retinol spent in plasma were both lower compared with controls; VARA also stimulated the uptake of plasma VA into extravascular tissues. A VARA perturbation model indicated that the effect of VARA in stimulating VA uptake into tissues in neonates is both dramatic and transient.     

Vitamin A combined with retinoic acid increases retinol uptake and lung retinyl ester formation in a synergistic manner in neonatal rats

Vitamin A (VA) is stored in tissues predominantly as retinyl esters (REs), which provide substrate for the production of bioactive retinoids. Retinoic acid (RA), a principal metabolite, has been shown to induce postnatal lung development. To better understand lung RE storage, we compared VA (given as retinyl palmitate), RA, and a nutrient-metabolite combination, VARA, given orally on postnatal days 5-7, for their ability to increase lung RE in neonatal rats. VARA increased lung RE significantly [ approximately 14, 2.4, 2.1, and <1 nmol/g for VARA, VA, RA, and control (C), respectively; P < 0.001]; the increase by VARA was more than additive compared with the effects of VA and RA alone. Lung histology and morphometry were unchanged. In a 6 h metabolic study, providing [(3)H]retinol with VARA, compared with VA or C, increased the uptake of newly absorbed (3)H by 3-fold, indicating that VARA stimulated the uptake of [(3)H]retinol and its retention as [(3)H]RE in neonatal lungs. After cessation of VARA, lung RE remained increased for 9 d afterward, through the period of alveolar development. In conclusion, VARA, a 10:1 nutrient-metabolite combination, increased lung RE significantly compared with VA alone and could be a promising therapeutic option for enhancing the delivery of VA to the lungs.     

Compartmental modeling of whole-body vitamin A kinetics in unsupplemented and vitamin A-retinoic acid-supplemented neonatal rats

Little is known about the contribution of different tissues to whole-body vitamin A (VA) kinetics in neonates. Here, we have used model-based compartmental analysis of tissue tracer kinetic data from unsupplemented (control) and VA-retinoic acid (VARA)-supplemented neonatal rats to determine VA kinetics in specific tissues under control and supplemented conditions. First, compartmental models for retinol kinetics were developed for individual tissues, and then an integrated compartmental model incorporating all tissues was developed for both groups. The models predicted that 52% of chylomicron (CM) retinyl ester was cleared by liver in control pups versus 22% in VARA-treated pups, whereas about 51% of VA was predicted to be extrahepatic in 4- to 6-day-old unsupplemented neonatal rats. VARA increased CM retinyl ester uptake by lung, carcass, and intestine; decreased the release into plasma of retinol that had been cleared by liver and lung as CM retinyl esters; stimulated the uptake of retinol from plasma holo-retinol binding protein into carcass; and decreased the retinol turnover out of the liver. Overall, neonatal VA trafficking differed from that previously described for adult animals, with a larger contribution of extrahepatic tissues to CM clearance, especially after VA supplementation, and a significant amount of VA distributed in extrahepatic tissues.     

Hepatic retinoid stores are required for normal liver regeneration

Preliminary studies of liver regeneration induced by partial hepatectomy (PHE) identified a substantial depletion of hepatic retinoid stores, by greater than 70%, in regenerating livers of wild-type C57Bl/6J mice. To understand this, we compared responses of wild-type and lecithin:retinol acyltransferase (Lrat)-deficient mice, which totally lack hepatic retinoid stores, to PHE. The Lrat-deficient livers showed delayed regeneration in the first 24 h after PHE. At 12 h after PHE, we observed significantly less mRNA expression for growth factors and cytokines implicated in regulating the priming phase of liver regeneration, specifically for Hgf and Tgfα, but not Tgfβ. Compared with wild-type mice, the changes in mRNA levels for p21 and cyclins E1, B1, and A2 mRNAs and for hepatocellular BrdU incorporation and mitoses were delayed (i.e., shifted to later times) in regenerating Lrat^−/− livers. Concentrations of all-trans-retinoic acid were significantly lower in the livers of Lrat^−/− mice following PHE, and this was accompanied by diminished expression of known retinoid-responsive genes. At later times after PHE, the rate of liver weight restoration for Lrat^−/− mice was parallel to that of wild-type mice, although additional biochemical differences were observed. Thus, hepatic retinoid stores are required for maintaining expression of signaling molecules that regulate cell proliferation and differentiation immediately after hepatic injury, accounting for the delayed restoration of liver mass in Lrat^−/− mice.     

Acidic retinoids synergize with vitamin A to enhance retinol uptake and STRA6, LRAT, and CYP26B1 expression in neonatal lung

Vitamin A (VA) is essential for fetal lung development and postnatal lung maturation. VA is stored mainly as retinyl esters (REs), which may be mobilized for production of retinoic acid (RA). This study was designed 1) to evaluate several acidic retinoids for their potential to increase RE in the lungs of VA-supplemented neonatal rats, and 2) to determine the expression of retinoid homeostatic genes related to retinol uptake, esterification, and catabolism as possible mechanisms. When neonatal rats were treated with VA combined with any one of several acidic retinoids (RA, 9-cis-RA, or Am580, a stable analog of RA), lung RE increased ∼5–7 times more than after an equal amount of VA alone. Retinol uptake and esterification during the period of absorption correlated with increased expression of both STRA6 (retinol-binding protein receptor) and LRAT (retinol esterification), while a reduction in RE after 12 h in Am580-treated, VA-supplemented rats correlated with a strong and persistent increase in CYP26B1 (RA hydroxylase). We conclude that neonatal lung RE can be increased synergistically by VA combined with both natural and synthetic acidic retinoids, concomitant with induction of the dyad of STRA6 and LRAT. However, the pronounced and prolonged induction of CYP26B1 by Am580 may counteract lung RE accumulation after the absorption process is completed.     

Diet-dependent retinoid effects on liver gene expression include stellate and inflammation markers and parallel effects of the nuclear repressor Shp

For mice, a maternal vitamin A (VA)-deficient diet initiated from midgestation (GVAD) produces serum retinol deficiency in mature offspring. We hypothesize that the effects of GVAD arise from preweaning developmental changes. We compare the effect of this GVAD protocol in combination with a postweaning high-fat diet (HFD) or high-carbohydrate diet (LF12). Each is compared to an equivalent VA-sufficient combination. GVAD extensively decreased serum retinol and liver retinol, retinyl esters, and retinoid homeostasis genes (Lrat, Cyp26b1 and Cyp26a1). These suppressions were each more effective with LF12 than with HFD. Postweaning initiation of VA deficiency with LF12 depleted liver retinoids, but serum retinol was unaffected. Liver retinoid depletion, therefore, precedes serum attenuation. Maternal LF12 decreased the obesity response to the HFD, which was further decreased by GVAD. LF12 fed to the mother and offspring extensively stimulated genes marking stellate activation (Col1a1, Timp2 and Cyp1b1) and novel inflammation markers (Ly6d, Trem2 and Nupr1). The GVAD with LF12 diet combination suppressed these responses. GVAD in combination with the HFD increased these same clusters. A further set of expression differences on the HFD when compared to a high-carbohydrate diet was prevented when GVAD was combined with HFD. Most of these GVAD gene changes match published effects from deletion of Nr0b2/Shp, a retinoid-responsive, nuclear co-repressor that modulates metabolic homeostasis. The stellate and inflammatory increases seen with the high-carbohydrate LF12 diet may represent postprandial responses. They depend on retinol and Shp, but the regulation reverses with an HFD.     

Retinol-binding protein 4 is expressed in chondrocytes of developing mouse long bones: implications for a local role in formation of the secondary ossification center

Retinol-binding protein 4 (Rbp4) is the major carrier of retinol in the bloodstream, a retinoid whose metabolites influence osteogenesis, chondrogenesis and adipogenesis. Rbp4 is mainly produced in the liver where it mobilizes hepatic retinol stores to supply other tissues. However, Rbp4 is also expressed in several extrahepatic tissues, including limbs, where its role is largely unknown. This study aimed to identify the cellular localization of Rbp4 to gain insight into its involvement in limb development and bone growth. Using immunohistochemistry, we discovered that Rbp4 was present in a variety of locations in developing embryonic and postnatal mouse hindlimbs. Rbp4 was present in a restricted population of epiphyseal chondrocytes and perichondral cells correlating to the future region of secondary ossification. With the onset of secondary ossification, Rbp4 was detected in chondrocytes of the resting zone and in chondrocytes that bordered invading cartilage canals and the expanding front of ossification. Rbp4 was less abundant in proliferating chondrocytes involved in primary ossification. Our data implicate the involvement of chondrocytic Rbp4 in bone growth, particularly in the formation of the secondary ossification center of the limb.     

Genetic dissection of retinoid esterification and accumulation in the liver and adipose tissue

Approximately 80–90% of all retinoids in the body are stored as retinyl esters (REs) in the liver. Adipose tissue also contributes significantly to RE storage. The present studies, employing genetic and nutritional interventions, explored factors that are responsible for regulating RE accumulation in the liver and adipose tissue and how these influence levels of retinoic acid (RA) and RA-responsive gene expression. Our data establish that acyl-CoA:retinol acyltransferase (ARAT) activity is not involved in RE synthesis in the liver, even when mice are nutritionally stressed by feeding a 25-fold excess retinol diet or upon ablation of cellular retinol-binding protein type I (CRBPI), which is proposed to limit retinol availability to ARATs. Unlike the liver, where lecithin:retinol acyltransferase (LRAT) is responsible for all RE synthesis, this is not true for adipose tissue where Lrat-deficient mice display significantly elevated RE concentrations. However, when CrbpI is also absent, RE levels resemble wild-type levels, suggesting a role for CrbpI in RE accumulation in adipose tissue. Although expression of several RA-responsive genes is elevated in Lrat-deficient liver, employing a sensitive liquid chromatography tandem mass spectrometry protocol and contrary to what has been assumed for many years, we did not detect elevated concentrations of all-trans-RA. The elevated RA-responsive gene expression was associated with elevated hepatic triglyceride levels and decreased expression of Pparδ and its downstream Pdk4 target, suggesting a role for RA in these processes in vivo.     

Perinatal Exposure to Low-Dose Methoxychlor Impairs Testicular Development in C57BL/6 Mice

Methoxychlor (MXC), an organochlorine pesticide, has adverse effects on male reproduction at toxicological doses. Humans and wild animals are exposed to MXC mostly through contaminated dietary intake. Higher concentrations of MXC have been found in human milk, raising the demand for the risk assessment of offspring after maternal exposure to low doses of MXC. In this study, pregnant mice (F0) were given intraperitoneal daily evening injections of 1 mg/kg/d MXC during their gestational (embryonic day 0.5, E0.5) and lactational periods (postnatal day 21.5, P21.5), and the F1 males were assessed. F1 testes were collected at P0.5, P21.5 and P45.5. Maternal exposure to MXC disturbed the testicular development. Serum testosterone levels decreased, whereas estradiol levels increased. To understand the molecular mechanisms of exposure to MXC in male reproduction, the F1 testes were examined for changes in the expression of steroidogenesis- and spermatogenesis- related genes. RT-PCR analysis demonstrated that MXC significantly decreased Cyp11a1 and increased Cyp19a1; furthermore, it downregulated certain spermatogenic genes (Dazl, Boll, Rarg, Stra8 and Cyclin-a1). In summary, perinatal exposure to low-dose MXC disturbs the testicular development in mice. This animal study of exposure to low-dose MXC in F1 males suggests similar dysfunctional effects on male reproduction in humans.     

Retinol-binding protein 2 (RBP2): biology and pathobiology

Abstract

Retinol-binding protein 2 (RBP2; originally cellular retinol-binding protein, type II (CRBPII)) is a 16?kDa cytosolic protein that in the adult is localized predominantly to absorptive cells of the proximal small intestine. It is well established that RBP2 plays a central role in facilitating uptake of dietary retinoid, retinoid metabolism in enterocytes, and retinoid actions locally within the intestine. Studies of mice lacking Rbp2 establish that Rbp2 is not required in times of dietary retinoid-sufficiency. However, in times of dietary retinoid-insufficiency, the complete lack of Rbp2 gives rise to perinatal lethality owing to RBP2 absence in both placental (maternal) and neonatal tissues. Moreover, when maintained on a high-fat diet, Rbp2-knockout mice develop obesity, glucose intolerance and a fatty liver. Unexpectedly, recent investigations have demonstrated that RBP2 binds long-chain 2-monoacylglycerols (2-MAGs), including the canonical endocannabinoid 2-arachidonoylglycerol, with very high affinity, equivalent to that of retinol binding. Crystallographic studies establish that 2-MAGs bind to a site within RBP2 that fully overlaps with the retinol binding site. When challenged orally with fat, mucosal levels of 2-MAGs in Rbp2 null mice are significantly greater than those of matched controls establishing that RBP2 is a physiologically relevant MAG-binding protein. The rise in MAG levels is accompanied by elevations in circulating levels of the hormone glucose-dependent insulinotropic polypeptide (GIP). It is not understood how retinoid and/or MAG binding to RBP2 affects the functions of this protein, nor is it presently understood how these contribute to the metabolic and hormonal phenotypes observed for Rbp2-deficient mice.     

Identification of SRY‐box 30 as an age‐related essential gatekeeper for male germ‐cell meiosis and differentiation

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY‐box 30 (Sox30) is an age‐related and essential regulator of meiosis in the postnatal testis. Sox30‐null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ‐cell differentiation and irregular Leydig cell proliferation. In aged Sox30‐null mice, the observed testicular impairments were more severe. Furthermore, the germ‐cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a “fail‐safe” mechanism for Sox30 to facilitate germ‐cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30‐null testes. Re‐expression of Sox30 in Sox30‐null mice successfully restored germ‐cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age‐associated for germ‐cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ‐cell meiosis and differentiation in the postnatal testis.     

Accumulation of retinol in the liver after prolonged hyporetinolemia in the vitamin A-sufficient rat

We assessed the effects of prolonged reduction of plasma retinol concentrations (hyporetinolemia) on the distribution of tissue vitamin A (VA) and of its active compounds using a model of continuous recombinant human interleukin-6 (rhIL-6) infusion via osmotic minipumps in VA-sufficient male rats. Plasma retinol and retinol-binding protein (RBP) concentrations remained decreased and lower in rhIL-6-treated rats compared with controls from 7.5 h throughout 7 days of infusion (P < 0.001). This reduction was accompanied by a 68% increase in hepatic retinol concentration by 7 days (P < 0.05). Hepatic and renal retinyl palmitate and retinoic acid concentrations did not change, and renal megalin content remained unchanged; hepatic RBP concentrations were 41% lower in rhIL-6-treated rats compared with controls (P < 0.05). These results indicate that instead of being lost, retinol accumulated in the liver during inflammation and that hyporetinolemia was attributable to a decrease in the availability of hepatic RBP. A plausible consequence of the effect of rhIL-6-induced hyporetinolemia is that by 7 days tissues that are dependent on plasma retinol may become deprived of VA. These results have important implications in understanding the mechanism by which measles infection induces hyporetinolemia and VA deficiency of extrahepatic tissues.     

Liver Retinol Transporter and Receptor for Serum Retinol-binding Protein (RBP4)

Vitamin A (retinol) is absorbed in the small intestine, stored in liver, and secreted into circulation bound to serum retinol-binding protein (RBP4). Circulating retinol may be taken up by extrahepatic tissues or recycled back to liver multiple times before it is finally metabolized or degraded. Liver exhibits high affinity binding sites for RBP4, but specific receptors have not been identified. The only known high affinity receptor for RBP4, Stra6, is not expressed in the liver. Here we report discovery of RBP4 receptor-2 (RBPR2), a novel retinol transporter expressed primarily in liver and intestine and induced in adipose tissue of obese mice. RBPR2 is structurally related to Stra6 and highly conserved in vertebrates, including humans. Expression of RBPR2 in cultured cells confers high affinity RBP4 binding and retinol transport, and RBPR2 knockdown reduces RBP4 binding/retinol transport. RBPR2 expression is suppressed by retinol and retinoic acid and correlates inversely with liver retinol stores in vivo. We conclude that RBPR2 is a novel retinol transporter that potentially regulates retinol homeostasis in liver and other tissues. In addition, expression of RBPR2 in liver and fat suggests a possible role in mediating established metabolic actions of RBP4 in those tissues.     

Ontogeny of rdh9 (Crad3) expression: Ablation causes changes in retinoid and steroid metabolizing enzymes,but RXR and androgen signaling seem normal

Crad3 (cis-retinol/androgen dehydrogenase 3), a short-chain dehydrogenase/reductase, converts 9-cis-retinol into 9-cis-retinal and 3α-androstanediol into dihydrotestosterone. Crad3 may serve in biosynthesis of 9-cis-retinoic acid, a putative RXR ligand, and/or regeneration of potent androgens. RT-PCR showed that expression of the gene that encodes Crad3, rdh9, begins in liver by e11.5, and in kidney, testis, brain and intestine during e15.5–e16.5. In situ hybridization showed rdh9 expression in embryonic liver, ganglia, small intestine, lung, skin and vertebral cartilage. In adult, in situ hybridization revealed rdh9 expression intensely in hepatocytes, weakly in kidney glomerulus, and intensely in collecting tubules. In intestine, undifferentiated epithelia had greater expression than differentiated epithelia at the distal villus end. Testes expressed rdh9 in spermatogonia, and weakly in Leydig cells. Adult brain expressed rdh9 in the dentate gyrus and CA regions of the hippocampus, the cerebellum Purkinje cells, and the glomerular and mitral cell layers of the olfactory bulb. Rdh9-null mice, backcrossed against C57BL/6J mice, were born in Mendelian frequency, were healthy and fertile, and had normal tissue retinoid and serum dihydrotestosterone levels. Expression of rdh1, a gene that encodes an efficient retinol dehydrogenase, decreased 3- to 8-fold in rdh9-null mice, depending on dietary vitamin A. Microarray analysis and quantitative PCR revealed 2- to 4-fold increases in mRNA of enzymes that catalyze xenobiotic and steroid metabolism, including Cyp2, Cyp3, 11β-hydroxysteroid dehydrogenase type 2, and 17β-hydroxsteroid dehydrogenases types 4 and 5. These data indicate widespread Crad3 function(s) in steroid and/or retinoid metabolism starting mid embryogenesis.     

The STRA6 Receptor Is Essential for Retinol-binding Protein-induced Insulin Resistance but Not for Maintaining Vitamin A Homeostasis in Tissues Other Than the Eye

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.     

The retinol signaling pathway in mouse pluripotent P19 cells

atRA (all-trans-retinoic acid), the active metabolite of retinol (vitamin A), is essential for embryogenesis and maintenance of cellular phenotype in adults. Chemicals that interfere with the metabolism of retinol to atRA, therefore, are a human health concern. During development of a screen for disruptors of this signaling pathway, we investigated whether the mouse pluripotent P19 cell metabolizes retinol to atRA and thus can be used in a cell-based screen for disruptors of the pathway. We found that retinol induced the identical pattern of homeobox gene expression as atRA and its precursor, retinal. Retinol was 160-fold less potent than atRA as an inducer, however. In spite of its lower potency, increased Hoxa1 gene expression was detected 30 min after retinol exposure and increased 40-fold by 2 h. Rdh10 and Aldh1a2/Raldh2, which together convert retinol to atRA in the embryo, were the predominant alcohol and aldehyde dehydrogenases expressed in P19 cells. The cell expressed high mRNA levels of retinol binding proteins, Rbp1 and Rbp4, and the 13,14-dihydroretinol saturase, Retsat. It also expressed all Rar and Rxr isotypes, Crabp1&2, the three Cyp26 genes, and both β-carotene-cleaving genes, Bcmo1 and Bco2. The basal expression levels and retinol responsiveness of 25 pathway-related genes were quantitated by RT-qPCR. A test of the Aldh1a2 inhibitor, citral, showed that the disruption of the pathway was easily detected and quantitated showing that the P19 cell provides an in vitro model system for identifying and exploring the mechanism of action of chemicals that interfere with this critical cellular pathway.     

Targeting of GFP-Cre to the Mouse Cyp11a1 Locus Both Drives Cre Recombinase Expression in Steroidogenic Cells and Permits Generation of Cyp11a1 Knock Out Mice

To permit conditional gene targeting of floxed alleles in steroidogenic cell-types we have generated a transgenic mouse line that expresses Cre Recombinase under the regulation of the endogenous Cytochrome P450 side chain cleavage enzyme (Cyp11a1) promoter. Mice Carrying the Cyp11a1-GC (GFP-Cre) allele express Cre Recombinase in fetal adrenal and testis, and adrenal cortex, testicular Leydig cells (and a small proportion of Sertoli cells), theca cells of the ovary, and the hindbrain in postnatal life. Circulating testosterone concentration is unchanged in Cyp11^+/GC males, suggesting steroidogenesis is unaffected by loss of one allele of Cyp11a1, mice are grossly normal, and Cre Recombinase functions to recombine floxed alleles of both a YFP reporter gene and the Androgen Receptor (AR) in steroidogenic cells of the testis, ovary, adrenal and hindbrain. Additionally, when bred to homozygosity (Cyp11a1^GC/GC), knock-in of GFP-Cre to the endogenous Cyp11a1 locus results in a novel mouse model lacking endogenous Cyp11a1 (P450-SCC) function. This unique dual-purpose model has utility both for those wishing to conditionally target genes within steroidogenic cell types and for studies requiring mice lacking endogenous steroid hormone production.