Anuradha Rao

Bile acid transporters

In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na+ taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTα-OSTβ. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers.

The organic solute transporter α-β, Ostα-Ostβ, is essential for intestinal bile acid transport and homeostasis

The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostα-Ostβ exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostα-Ostβ have not been investigated. To determine the role of Ostα-Ostβ in intestinal bile acid absorption, the Ostα gene was disrupted by homologous recombination in mice. Ostα−/− mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostα−/− vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostα−/−Mrp3−/− mice. The bile acid pool size was significantly reduced (>65%) in Ostα−/− mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostα−/− mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostα-Ostβ is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.

Vitamin D receptor and p21/WAF1 are targets of genistein and 1,25-dihydroxyvitamin D3 in human prostate cancer cells.

We investigated mechanisms by which genistein and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] act synergistically to inhibit the growth of the human prostate cancer cell line LNCaP. We demonstrate that 1,25(OH)2D3 and genistein cooperate to up-regulate the vitamin D receptor protein by increasing the stability of the vitamin D receptor. Genistein and 1,25(OH)2D3 also cooperate to up-regulate the levels of p21/WAF1 (p21). Small interfering RNA-mediated knockdown of p21 expression showed that p21 is essential for significant growth inhibition of LNCaP cells in response to either compound or their combination. We conclude that one mechanism of synergism between genistein and 1,25(OH)2D3 is through genistein modulation of vitamin D signaling.

Getting the mOST from OST: Role of organic solute transporter, OSTα-OSTβ, in bile acid and steroid metabolism

The organic solute transporter (OST)(alpha)-OST(beta) is an unusual heteromeric carrier expressed in a variety of tissues including the small intestine, colon, liver, biliary tract, kidney, and adrenal gland. In polarized epithelial cells, OSTalpha-OSTbeta protein is localized on the basolateral membrane and functions in the export or uptake of bile acids and steroids. This article reviews recent results including studies of knockout mouse models that provide new insights to the role of OSTalpha-OSTbeta in the compartmentalization and metabolism of these important lipids.

Yin Yang 1 regulates the transcriptional activity of androgen receptor

The multifunctional protein Yin Yang 1 (YY1) has an important role in epigenetic regulation of gene expression. YY1 is highly expressed in various types of cancers, including prostate cancer. Currently, the mechanism underlying the functional role of YY1 in prostate tumorigenesis remains unclear. In this report, we investigated the functional interplay between YY1 and androgen receptor (AR), and the effect of YY1 on AR-mediated transcription. We found that YY1 physically interacts with AR both in a cell-free system and in cultured cells. YY1 is required for the optimal transcriptional activity of AR in promoting the transcription of the prostate-specific antigen (PSA) promoter. However, ectopic YY1 expression in LNCaP cells did not further enhance the reporter driven by the PSA promoter, suggesting that an optimal level of YY1 is already established in prostate tumor cells. Consistently, YY1 depletion in LNCaP cells reduced endogenous PSA levels, but overexpressed YY1 did not significantly increase PSA expression. We also observed that YY1–AR interaction is essential to YY1-mediated transcription activity of AR and YY1 is a necessary component in the complex binding to the androgen response element. Thus, our study demonstrates that YY1 interacts with AR and regulates its transcriptional activity.

Analysis of G/A polymorphism in the androgen response element I of the PSA gene and its interactions with the androgen receptor polymorphisms

OBJECTIVES To compare the in vitro functional activity of the prostate-specific antigen (PSA) androgen response element (ARE) I alleles alone or in combination with androgen receptor (AR) polymorphisms and to determine the association of ARE I alleles with serum PSA in men without clinical prostatic disease. Data are conflicting regarding the association of PSA promoter alleles with serum PSA in men. METHODS In vitro functional analyses of ARE I and AR polymorphisms were conducted by luciferase reporter assays in LNCaP and PC-3 cells. Associations among serum PSA, ARE I, and AR genotypes were determined by genotyping 109 white and 71 African-American men determined to be free of clinical prostatic disease. RESULTS We found no significant difference in the androgen responsiveness of the two alleles when cells were transfected with PSA promoter reporter constructs differing only in the ARE I single nucleotide polymorphism and treated with varying doses of androgen. The response to androgens of the ARE I alleles co-transfected with AR expression vectors of 9, 21, and 29 CAG repeat lengths were identical. No individual or combined effects of the ARE I genotype and the AR genotype on serum PSA were noted. CONCLUSIONS Our data indicate that ARE I polymorphisms, alone or in combination with AR polymorphisms, have no functional effect on the activity of the PSA promoter in vitro and in vivo.

Interaction of Macrolide Antibiotics with Intestinally Expressed Human and Rat Organic Anion-Transporting Polypeptides

The macrolide antibiotics azithromycin and clarithromycin are large molecular weight compounds that exhibit moderate to excellent oral bioavailability in preclinical species and humans. Previous concomitant dosing studies in rats using rifamycin SV, a general organic anion-transporting polypeptide (OATP) inhibitor, suggested that the high oral absorption of azithromycin and clarithromycin may be caused by facilitative uptake by intestinal Oatps. In this study, we used OATP/Oatp-expressing cells to investigate the interaction of macrolides with rat Oatp1a5, human OATP1A2, and human/rat OATP2B1/Oatp2b1. These experiments showed that azithromycin and clarithromycin were potent inhibitors of rat Oatp1a5-mediated taurocholate uptake with apparent inhibitor constant (Ki) values of 3.3 and 2.4 μM, respectively. The macrolides functioned as noncompetitive inhibitors but were not transport substrates for rat Oatp1a5, as assessed by direct uptake measurements of radiolabeled azithromycin and clarithromycin. cis-Inhibition and direct uptake studies further showed that azithromycin and clarithromycin were only very weak inhibitors and not substrates for human OATP1A2 and human/rat OATP2B1/Oatp2b1. In summary, these results indicate that the macrolides azithromycin and clarithromycin potently inhibit rat Oatp1a5 but do not significantly interact with OATP1A2 and OATP2B1/Oatp2b1. These intestinally expressed OATP/Oatp(s) are not responsible for the postulated facilitative uptake of azithromycin and clarithromycin, and alternative facilitative pathways must exist for their intestinal absorption.

Involvement of intestinal uptake transporters in the absorption of azithromycin and clarithromycin in the rat.

Macrolide antibiotics azithromycin (AZI) and clarithromycin (CLARI) are large molecular weight compounds and are substrates for apically polarized efflux transporters such as P-glycoprotein, which can potentially restrict intestinal absorption. However, despite these undesired physicochemical and biopharmaceutical properties, AZI and CLARI exhibit moderate to excellent p.o. bioavailability in preclinical species and humans. Intestinal uptake transporters, such as organic anion transporting polypeptides (OATPs), can facilitate the uptake of drugs that are substrates and hence increase p.o. absorption. The present study was designed to determine whether the intestinal Oatps are involved in absorption of these macrolides. AZI or CLARI was dosed p.o. to Sprague-Dawley rats after p.o. administration with vehicle or rifamycin SV (RIF), an OATP inhibitor. The p.o. exposures of AZI and CLARI were reduced 65 and 45%, respectively, when coadministered with an optimized RIF regimen. The p.o. RIF had no affect on the total blood clearance of these macrolides and most likely did not cause induction of metabolizing enzymes and/or transporters. Therefore, the results suggest that inhibition of an RIF-sensitive uptake transporter such as Oatp along the rat gastrointestinal tract was responsible for reduced p.o. exposure of AZI and CLARI. In addition, AZI and CLARI caused inhibition of taurocholate uptake in rat Oatp1a5-transfected Madin-Darby canine kidney cell monolayers. The in vitro and in vivo results suggest that the intestinal Oatps are involved in the p.o. absorption of AZI and CLARI in the rat.

Impact of murine intestinal apolipoprotein A-IV expression on regional lipid absorption, gene expression, and growth

Apolipoprotein A-IV (apoA-IV) is synthesized by intestinal enterocytes during lipid absorption and secreted into lymph on the surface of nascent chylomicrons. A compelling body of evidence supports a central role of apoA-IV in facilitating intestinal lipid absorption and in regulating satiety, yet a longstanding conundrum is that no abnormalities in fat absorption, feeding behavior, or weight gain were observed in chow-fed apoA-IV knockout (A4KO) mice. Herein we reevaluated the impact of apoA-IV expression in C57BL6 and A4KO mice fed a high-fat diet. Fat balance and lymph cannulation studies found no effect of intestinal apoA-IV gene expression on the efficiency of fatty acid absorption, but gut sac transport studies revealed that apoA-IV differentially modulates lipid transport and the number and size of secreted triglyceride-rich lipoproteins in different anatomic regions of the small bowel. ApoA-IV gene deletion increased expression of other genes involved in chylomicron assembly, impaired the ability of A4KO mice to gain weight and increase adipose tissue mass, and increased the distal gut hormone response to a high-fat diet. Together these findings suggest that apoA-IV may play a unique role in integrating feeding behavior, intestinal lipid absorption, and energy storage.

Mouse organic solute transporter alpha deficiency alters FGF15 expression and bile acid metabolism

BACKGROUND & AIMS Blocking intestinal bile acid (BA) absorption by inhibiting or inactivating the apical sodium-dependent BA transporter (Asbt) classically induces hepatic BA synthesis. In contrast, blocking intestinal BA absorption by inactivating the basolateral BA transporter, organic solute transporter alpha-beta (Ostα-Ostβ) is associated with an altered homeostatic response and decreased hepatic BA synthesis. The aim of this study was to determine the mechanisms underlying this phenotype, including the role of the farnesoid X receptor (FXR) and fibroblast growth factor 15 (FGF15). METHODS BA and cholesterol metabolism, intestinal phenotype, expression of genes important for BA metabolism, and intestinal FGF15 expression were examined in wild type, Ostα(-/-), Fxr(-/-), and Ostα(-/-)Fxr(-/-) mice. RESULTS Inactivation of Ostα was associated with decreases in hepatic cholesterol 7α-hydroxylase (Cyp7a1) expression, BA pool size, and intestinal cholesterol absorption. Ostα(-/-) mice exhibited significant small intestinal changes, including altered ileal villus morphology, and increases in intestinal length and mass. Total ileal FGF15 expression was elevated almost 20-fold in Ostα(-/-) mice as a result of increased villus epithelial cell number and ileocyte FGF15 protein expression. Ostα(-/-)Fxr(-/-) mice exhibited decreased ileal FGF15 expression, restoration of intestinal cholesterol absorption, and increases in hepatic Cyp7a1 expression, fecal BA excretion, and BA pool size. FXR deficiency did not reverse the intestinal morphological changes or compensatory decrease for ileal Asbt expression in Ostα(-/-) mice. CONCLUSIONS These results indicate that signaling via FXR is required for the paradoxical repression of hepatic BA synthesis but not the complex intestinal adaptive changes in Ostα(-/-) mice.