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Vitamin A, via retinoic acid (RA), is a critical micronutrient. Normally, plasma concentrations are tightly regulated. Concentrations of vitamin A metabolites (13cis-RA, atRA) and relationships between RBP4 and retinoids have never been fully evaluated in adult patients with CKD. We measured retinoid and RBP4 concentrations in plasma and urine from 55 adult patients with CKD and 21 matched healthy subjects. RBP4 and retinol levels were increased approximately twofold in patients with CKD, with a negative correlation between plasma retinol and eGFR (p = 0.006) and plasma RBP4 and eGFR (p = 0.0007). RBP4 renal clearance was higher in patients with CKD than healthy subjects but not associated with eGFR. Circulating concentrations of atRA increased and concentrations of 13cis-RA decreased in subjects with CKD with no change in RA-to-retinol ratio. Increases in circulating retinol, RBP4, and atRA may be due to increased hepatic RBP4 synthesis, retinyl ester hydrolysis, and/or hepatic secretion of RBP4-retinol.
© 2016 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.
Multiphoton excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in subcellular structures called retinosomes mediate regeneration of the visual chromophore, 11-cis-retinal, by the visual cycle. But harmful fluorescent condensation products of retinoids also occur in the retina. We report that in wild-type mice, excitation with a wavelength of ∼730 nm identified retinosomes in the retinal pigment epithelium, and excitation with a wavelength of ∼910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older wild-type mice and mice with defective clearance of all-trans-retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age-related macular degeneration and other defects in retinoid metabolism.
OBJECTIVE - Several transcription factors are essential to pancreatic islet β-cell development, proliferation, and activity, including MafA and MafB. However, MafA and MafB are distinct from others in regard to temporal and islet cell expression pattern, with β-cells affected by MafB only during development and exclusively by MafA in the adult. Our aim was to define the functional relationship between these closely related activators to the β-cell.
RESEARCH DESIGN AND METHODS - The distribution of MafA and MafB in the β-cell population was determined immunohistochemically at various developmental and perinatal stages in mice. To identify genes regulated by MafB, microarray profiling was performed on wild-type and MafB(-/-) pancreata at embryonic day 18.5, with candidates evaluated by quantitative RT-PCR and in situ hybridization. The potential role of MafA in the expression of verified targets was next analyzed in adult islets of a pancreas-wide MafA mutant (termed MafA(ΔPanc)).
RESULTS - MafB was produced in a larger fraction of β-cells than MafA during development and found to regulate potential effectors of glucose sensing, hormone processing, vesicle formation, and insulin secretion. Notably, expression from many of these genes was compromised in MafA(ΔPanc) islets, suggesting that MafA is required to sustain expression in adults.
CONCLUSIONS - Our results provide insight into the sequential manner by which MafA and MafB regulate islet β-cell formation and maturation.
In a previous study, we used differential proteomics to identify retinal proteins whose steady-state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady-state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina-retinal recycling and generation of retinoic acid-we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11-cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology.
(c) 2010 Wiley Periodicals, Inc.
BACKGROUND - Chronic ethanol consumption disrupts glucose homeostasis and is associated with the development of insulin resistance. While adipose tissue and skeletal muscle are the two major organs utilizing glucose in response to insulin, the relative contribution of these two tissues to impaired glucose homeostasis during chronic ethanol feeding is not known. As other models of insulin resistance, such as obesity, are characterized by an infiltration of macrophages into adipose tissue, as well as changes in the expression of adipocytokines that play a central role in the regulation of insulin sensitivity, we hypothesized that chronic ethanol-induced insulin resistance would be associated with increased macrophage infiltration into adipose tissue and changes in the expression of adipocytokines by adipose tissue.
METHODS - Male Wistar rats were fed a liquid diet containing ethanol as 36% of calories or pair-fed a control diet for 4 weeks. The effects of chronic ethanol feeding on insulin-stimulated glucose utilization were studied using the hyperinsulinemic-euglycemic clamp technique, coupled with the use of isotopic tracers. Further, macrophage infiltration into adipose tissue and expression of adipocytokines were also assessed after chronic ethanol feeding.
RESULTS - Hyperinsulinemic-euglycemic clamp studies revealed that chronic ethanol feeding to rats decreased whole-body glucose utilization and decreased insulin-mediated suppression of hepatic glucose production. Chronic ethanol feeding decreased glucose uptake in epididymal, subcutaneous, and omental adipose tissue during the hyperinsulinemic-euglycemic clamp, but had no effect on glucose disposal in skeletal muscle. Chronic ethanol feeding increased the infiltration of macrophages into epididymal adipose tissue and changed the expression of mRNA for adipocytokines: expression of mRNA for monocyte chemoattractant protein 1, tumor necrosis factor alpha, and interleukin-6 were increased, while expression of mRNA for retinol binding protein 4 and adiponectin were decreased in epididymal adipose tissue.
CONCLUSIONS - These data demonstrate that chronic ethanol feeding results in the development of insulin resistance, associated with impaired insulin-mediated suppression of hepatic glucose production and decreased insulin-stimulated glucose uptake into adipose tissue. Chronic ethanol-induced insulin resistance was associated with increased macrophage infiltration into adipose tissue, as well as changes in the expression of adipocytokines by adipose tissue.
Our goal is to decipher which DNA sequences are required for tissue-specific expression of epididymal genes. At least 6 epididymis-specific lipocalin genes are known. These are differently regulated and regionalized in the epididymis. Lipocalin 5 (Lcn5 or mE-RABP) and Lipocalin 8 (Lcn8 or mEP17) are homologous genes belonging to the epididymis-specific lipocalin gene cluster. Both the 5 kb promoter fragment of the Lcn5 gene and the 5.3 kb promoter fragment of the Lcn8 gene can direct transgene expression in the epididymis (Lcn5 to the distal caput and Lcn8 to the initial segment), indicating that these promoter fragments contain important cis-regulatory element(s) for epididymis-specific gene expression. To define further the fragments regulating gene expression, the Lcn5 promoter was examined in transgenic mice and immortalized epididymal cell lines. After serial deletion, the 1.8 kb promoter fragment of the Lcn5 gene was sufficient for tissue-specific and region-specific gene expression in transgenic mice. Transient transfection analysis revealed that a transcription factor forkhead box A2 (Foxa2) interacts with androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 kb and 1.3 kb and that Foxa2 expression inhibits androgen-dependent induction of the Lcn5 promoter activity. Immunohistochemistry indicated a restricted expression of Foxa2 in the epididymis where endogenous Lcn5 gene expression is suppressed and that the Foxa2 inhibition of the Lcn5 promoter is consistent with the lack of expression of Lcn5 in the corpus and cauda. Our approach provides a basic strategy for further analysis of the epididymal lipocalin gene regulation and flexible control of epididymal function.
Murine epididymal retinoic acid-binding protein [or lipocalin 5 (Lcn5)] is synthesized and secreted by the principal cells of the mouse middle/distal caput epididymidis. A 5-kb promoter fragment of the Lcn5 gene can dictate androgen-dependent and epididymis region-specific gene expression in transgenic mice. Here, we reported that the 1.8-kb Lcn5 promoter confers epididymis region-specific gene expression in transgenic mice. To decipher the mechanism that directs transcription, 14 chimeric constructs that sequentially removed 100 bp of 1.8-kb Lcn5 promoter were generated and transfected into epididymal cells and nonepididymal cells. Transient transfection analysis revealed that 1.3 kb promoter fragment gave the strongest response to androgens. Between the 1.2-kb to 1.3-kb region, two androgen receptor (AR) binding sites were identified. Adjacent to AR binding sites, a Foxa2 [Fox (Forkhead box) subclass A] binding site was confirmed by gel shift assay. Similar Foxa binding sites were also found on the promoters of human and rat Lcn5, indicating the Foxa binding site is conserved among species. We previously reported that among the three members of Foxa family, Foxa1 and Foxa3 were absent in the epididymis whereas Foxa2 was detected in epididymal principal cells. Here, we report that Foxa2 displays a region-specific expression pattern along the epididymis: no staining observed in initial segment, light staining in proximal caput, gradiently heavier staining in middle and distal caput, and strongest staining in corpus and cauda, regions with little or no expression of Lcn5. In transient transfection experiments, Foxa2 expression inhibits AR induction of the Lcn5 promoter, which is consistent with the lack of expression of Lcn5 in the corpus and cauda. We conclude that Foxa2 functions as a repressor that restricts AR regulation of Lcn5 to a segment-specific pattern in the epididymis.
Previous studies from our group have shown that Foxa1 is expressed in the prostate and interacts with the androgen receptor (AR) to regulate prostate-specific genes such as prostate-specific antigen (PSA) and probasin (PB). We report here that Foxa2 but not Foxa1 is expressed in the epididymis. Further, Foxa2 interacts with the AR to regulate the mouse epididymal retinoic acid binding protein (mE-RABP) gene, an epididymis-specific gene. Binding of Foxa2 to the mE-RABP promoter was confirmed by gel-shift and chromatin immunoprecipitation (ChIP) assays. Overexpression of Foxa2 suppresses androgen activation of the mE-RABP promoter while overexpression of Foxa2 with prostate-specific promoters activates gene expression in an androgen-independent manner. GST pull-down assays determined that both Foxa1 and Foxa2 physically interact with the DNA binding domain of the AR. The interaction between Foxa proteins and AR was further confirmed by gel-shift assays where Foxa protein was recruited to AR binding oligomers even when Foxa binding sites were not present, and AR was recruited to Foxa binding oligomers even in the absence of an AR binding site. Given that Foxa1 and Foxa2 proteins are expressed differentially in the prostate and epididymis, these data suggest that the Foxa proteins have distinct effects on AR-regulated genes in different male reproductive accessory organs.
Mammalian spermatozoa undergo several modification and finally acquire the ability to fertilize during epididymal transit. One of the distinct features of the epididymis is that it displays a highly regionalized pattern of gene expression. This tissue-, region-, and cell-specific pattern of gene expression is critical for the maintenance of a fully functional epididymis. One would hypothesize that disrupting this process provides an ideal approach to male contraception, since it would not interfere with testicular endocrine output or sperm production. To achieve this purpose, we studied a cluster of epididymis-specific lipocalin genes for understanding the specific mechanisms involved in the control of gene expression in the epididymis. We have identified six epididymis-specific lipocalin genes that are differently regulated and regionalized in the epididymis. Lipocalin 5 [Lcn5 or epididymal retinoic acid-binding protein (E-RABP)] is a member of this epididymis-specific lipocalin gene cluster, which binds hydrophobic molecules such as retinoic acid. We have previously shown that the 5kb promoter fragment of the Lcn5 gene confers both androgen-dependent regulation and epididymis-specific gene expression in transgenic mice whereas 0.6 kb promoter fragment does not. To further narrow down the important cis-regulatory elements that regulate gene expression in the epididymis, we studied the Lcn5 promoter in both transgenic mice and immortalized epididymal cells. We have found that 1.8kb promoter fragment of the Lcn5 gene was sufficient for tissue- and region-specific expression in transgenic mice, and that a transcription factor Forkhead box A2 (Foxa2) interacts with the androgen receptor and binds to the 100 bp fragment of the Lcn5 promoter between 1.2 and 1.3 kb. Our finding provides a framework for further analysis of the epididymal lipocalin gene regulation and modulated control of epididymis-specific expression.
Immunohistochemical and biochemical approaches were utilized to compare the expression of microsomal triglyceride transfer protein (MTP) and cellular retinol binding protein II (CRBPII) with the expression of apolipoprotein (apo)B and apoA-I along the entire length of the small intestine in mice. MTP is expressed in villus-associated enterocytes along the length of the small intestine. Maximal expression occurs within the first 20% of the intestine and decreases to less than 3% of maximum in the distal third of the intestine. The expression of CRBPII is nearly identical with that of MTP. Peak expression of apoB and apoA-I occurs in the first 25% of the intestine; however, expression in the most distal segments of the intestine is 10%-15% of maximum expression. In mice fed a Western diet for 3 weeks the expression of MTP and CRBPII was elevated in the distal regions of the intestine, whereas the expression patterns for apoB and apoA-I were similar to those found in mice on control diets. We conclude that the patterns of expression, as well as the regulation of MTP and CRBPII, are similar. However, the expression and regulation of these two proteins differ from those of apoB and apoA-I. In particular, the expression of MTP is not coordinated with the expression of apoB, even though the two proteins are essential for the assembly and secretion of chylomicrons.