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.     

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.     

CrbpI modulates glucose homeostasis and pancreas 9-cis-retinoic acid concentrations

Cellular retinol-binding protein type I (CrbpI), encoded by Rpb1, serves as a chaperone of retinol homeostasis, but its physiological effects remain incompletely understood. We show here that the Rbp1(-/-) mouse has disrupted retinoid homeostasis in multiple tissues, with abnormally high 9-cis-retinoic acid (9cRA), a pancreas autacoid that attenuates glucose-stimulated insulin secretion. The Rbp1(-/-) pancreas has increased retinol and intense ectopic expression of Rpb2 mRNA, which encodes CrbpII: both would contribute to increased β-cell 9cRA biosynthesis. 9cRA in Rbp1(-/-) pancreas resists postprandial and glucose-induced decreases. Rbp1(-/-) mice have defective islet expression of genes involved in glucose sensing and insulin secretion, as well as islet α-cell infiltration, which contribute to reduced glucose-stimulated insulin secretion, high glucagon secretion, an abnormally high rate of gluconeogenesis, and hyperglycemia. A diet rich in vitamin A (as in a standard chow diet) increases pancreas 9cRA and impairs glucose tolerance. Crbp1 attenuates the negative impact of vitamin A (retinol) on glucose tolerance, regardless of the dietary retinol content. Rbp1(-/-) mice have an increased rate of fatty acid oxidation and resist obesity when fed a high-fat diet. Thus, glucose homeostasis and energy metabolism rely on Rbp1 expression and its moderation of pancreas retinol and of the autacoid 9cRA.     

Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss‐of‐function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 75–92, 2016     

Understanding the physiological role of retinol-binding protein in vitamin A metabolism using transgenic and knockout mouse models

Retinoids (vitamin A and its derivatives) play an essential role in many biological functions. However mammals are incapable of de novo synthesis of vitamin A and must acquire it from the diet. In the intestine, dietary retinoids are incorporated in chylomicrons as retinyl esters, along with other dietary lipids. The majority of dietary retinoid is cleared by and stored within the liver. To meet vitamin A requirements of tissues, the liver secretes retinol (vitamin A alcohol) into the circulation bound to its sole specific carrier protein, retinol-binding protein (RBP). The single known function of this protein is to transport retinol from the hepatic stores to target tissues. Over the last few years, the generation of knockout and transgenic mouse models has significantly contributed to our understanding of RBP function in the metabolism of vitamin A. We discuss below the role of RBP in maintaining normal vision and a steady flux of retinol throughout the body in times of need.     

Downregulation of STRA6 in Adipocytes and Adipose Stromovascular Fraction in Obesity and Effects of Adipocyte-Specific STRA6 Knockdown In Vivo

To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6^−/− mice. Adipose-Stra6^−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6^−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6^−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.     

In vitro and in vivo characterization of retinoid synthesis from beta-carotene

Retinoids are indispensable for the health of mammals, which cannot synthesize retinoids de novo. Retinoids are derived from dietary provitamin A carotenoids, like β-carotene, through the actions of β-carotene-15,15′-monooxygenase (BCMO1). As the substrates for retinoid-metabolizing enzymes are water insoluble, they must be transported intracellularly bound to cellular retinol-binding proteins. Our studies suggest that cellular retinol-binding protein, type I (RBP1) acts as an intracellular sensor of retinoid status that, when present as apo-RBP1, stimulates BCMO1 activity and the conversion of carotenoids to retinoids. Cellular retinol-binding protein, type II (RBP2), which is 56% identical to RBP1 does not influence BCMO1 activity. Studies of mice lacking BCMO1 demonstrate that BCMO1 is responsible for metabolically limiting the amount of intact β-carotene that can be absorbed by mice from their diet. Our studies provide new insights into the regulation of BCMO1 activity and the physiological role of BCMO1 in living organisms.     

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.     

Transgenic Mice Overexpressing Serum Retinol-Binding Protein Develop Progressive Retinal Degeneration through a Retinoid-Independent Mechanism

Serum retinol-binding protein 4 (RBP4) is the sole specific transport protein for retinol in the blood, but it is also an adipokine with retinol-independent, proinflammatory activity associated with obesity, insulin resistance, type 2 diabetes, and cardiovascular disease. Moreover, two separate studies reported that patients with proliferative diabetic retinopathy have increased serum RBP4 levels compared to patients with mild or no retinopathy, yet the effect of increased levels of RBP4 on the retina has not been studied. Here we show that transgenic mice overexpressing RBP4 (RBP4-Tg mice) develop progressive retinal degeneration, characterized by photoreceptor ribbon synapse deficiency and subsequent bipolar cell loss. Ocular retinoid and bisretinoid levels are normal in RBP4-Tg mice, demonstrating that a retinoid-independent mechanism underlies retinal degeneration. Increased expression of pro-interleukin-18 (pro-IL-18) mRNA and activated IL-18 protein and early-onset microglia activation in the retina suggest that retinal degeneration is driven by a proinflammatory mechanism. Neither chronic systemic metabolic disease nor other retinal insults are required for RBP4 elevation to promote retinal neurodegeneration, since RBP4-Tg mice do not have coincident retinal vascular pathology, obesity, dyslipidemia, or hyperglycemia. These findings suggest that elevation of serum RBP4 levels could be a risk factor for retinal damage and vision loss in nondiabetic as well as diabetic patients.     

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.     

Retinol-binding protein-deficient mice: biochemical basis for impaired vision

We reported previously that mice lacking plasma retinol-binding protein (RBP) are phenotypically normal except that they display impaired vision at the time of weaning. This visual defect is associated with greatly diminished eyecup levels of retinaldehyde and is reversible if the mutants are maintained for several months on a vitamin A-sufficient diet. Here we provide a biochemical basis for the visual phenotype of RBP-deficient mice. This phenotype does not result from inadequate milk total retinol levels since these are not different for RBP-deficient and wild-type mice. The eye, unlike all other tissues that have been examined, takes up dietary retinol very poorly. Moreover, compared to other tissues, the eye displays a strong preference for retinol uptake when retinol is delivered bound to RBP. The poor uptake of dietary retinol by the eye coupled with its marked ability to take up retinol from RBP, we propose, provides a basis for the impaired vision observed in weanling RBP-deficient mice. Further study of the mutants suggests that the impaired vision is reversible because the eyes of mutant mice slowly acquire sufficient retinol from the low levels of retinol present in their circulation either bound to albumin or present in lipoprotein fractions. Thus, the eye is unlike other tissues in the body in that it shows a very marked preference for acquiring retinol needed to support vision from the retinol-RBP complex and is unable to meet adequately its retinol need through uptake of recently absorbed dietary retinol. This provides an explanation for the impaired vision phenotype of RBP-deficient mice.     

Muscle expression of human retinol-binding protein (RBP). Suppression of the visual defect of RBP knockout mice

Mice lacking retinol-binding protein (RBP) have low circulating retinol levels. They have severe visual defects due to a low content of retinol or retinyl esters in the eye. A transgenic mouse strain that expresses human RBP under the control of the muscle creatine kinase promoter in the null background was generated. The exogenous protein bound retinol and transthyretin in the circulation and effectively delivered retinol to the eye. Thus, RBP expressed from an ectopic source suppresses the visual phenotype, and retinoids accumulate in the eye. No human RBP was found in the retinal pigment epithelium of the transgenic mice, indicating that retinol uptake by the eye does not entail endocytosis of the carrier RBP.     

Retinol-binding protein 4 downregulation during osteogenesis and its localization to non-endocytic vesicles in human cranial suture mesenchymal cells suggest a novel tissue function

Craniosynostosis is a developmental disorder of the skull arising from premature bony fusion of cranial sutures, the sites of skull bone growth. In a recent gene microarray study, we demonstrated that retinol-binding protein 4 (RBP4) was the most highly downregulated gene in suture tissue during the pathological process of premature bony fusion. To gain insight into the function of RBP4 in cranial sutures, we analysed primary cells cultured from human cranial suture mesenchyme. These cells express RBP4 but not CRBP1, cellular retinol-binding protein 1, the typical cytoplasmic retinol storage protein. Using flow cytometry, we showed that suture mesenchymal cells express the RBP4 receptor, STRA6, on the cell surface. In a cell culture model of cranial osteogenesis, we found that RBP4 was significantly downregulated during mineralization, analogous to its decrease in pathological suture fusion. We found that cranial suture cells do not secrete detectable levels of RBP4, suggesting that it acts in a cell-autonomous manner. High-resolution confocal microscopy with a panel of antibody markers of cytoplasmic organelles demonstrated that RBP4 was present in several hundred cytoplasmic vesicles of about 300 nm in diameter which, in large part, were conspicuously distinct from the ER, the Golgi and endosomes of the endocytic pathway. We speculate that in suture mesenchymal cells, endogenous RBP4 receives retinol from STRA6 and the RBP4-retinol complex is stored in vesicles until needed for conversion to retinoic acid in the process of osteogenesis. This study extends the role of RBP4 beyond that of a serum transporter of retinol and implicates a broader role in osteogenesis.     

Analysis of the effect of a novel therapeutic for type 2 diabetes on the proteome of a muscle cell line

Elevated serum retinol‐binding protein (RBP) concentration has been implicated in the development of insulin resistance and type 2 diabetes. Two series of small molecules have been designed to lower serum levels by reducing secretion of the transthyretin–RBP complex from the liver and enhancing RBP clearance through the kidney. These small molecules were seen to improve glucose and insulin tolerance tests and to reduce body weight gain in mice rendered diabetic through a high fat diet. A proteomics study was conducted to better understand the effects of these compounds in muscle cells, muscle being the primary site for energy expenditure. One lead compound, RTC‐15, is seen to have a significant effect on proteins involved in fat and glucose metabolism. This could indicate that the compound is having a direct effect on muscle tissue to improve energy homeostasis as well as a whole body effect on circulating RBP levels. This newly characterized group of antidiabetic compounds may prove useful in the treatment and prevention of insulin resistance and obesity.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased neonatal mortality in mice lacking cellular retinol-binding protein II

Cellular retinol-binding protein II (CRBP II) is a member of the cellular retinol-binding protein family, which is expressed primarily in the small intestine. To investigate the physiological role of CRBP II, the gene encoding CRBP II was inactivated. The saturable component of intestinal retinol uptake is impaired in CRBP II(-/-) mice. The knockout mice, while maintained on a vitamin A-enriched diet, have reduced (40%) hepatic vitamin A stores but grow and reproduce normally. However, reducing maternal dietary vitamin A to marginal levels during the latter half of gestation results in 100% mortality/litter within 24 h after birth in the CRBP II(-/-) line but no mortality in the wild type line. The neonatal mortality in heterozygote offspring of CRBP II(-/-) dams (79 +/- 21% deaths/litter) was increased as compared with the neonatal mortality in heterozygote offspring of wild type dams (29 +/- 25% deaths per litter, p < 0.05). Maternal CRBP II was localized by immunostaining in the placenta at 18 days postcoitum as well as in the small intestine. These studies suggest that both fetal as well as maternal CRBP II are required to ensure adequate delivery of vitamin A to the developing fetus when dietary vitamin A is limiting.     

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein

As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.     

The molecular basis of retinoid absorption: a genetic dissection

The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.     

Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.

A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.