Dysfunction of Organic Anion Transporting Polypeptide 1a1 Alters Intestinal Bacteria and Bile Acid Metabolism in Mice

Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in liver and is able to transport bile acids (BAs) in vitro. Male Oatp1a1-null mice have increased concentrations of taurodeoxycholic acid (TDCA), a secondary BA generated by intestinal bacteria, in both serum and livers. Therefore, in the present study, BA concentrations and intestinal bacteria in wild-type (WT) and Oatp1a1-null mice were quantified to investigate whether the increase of secondary BAs in Oatp1a1-null mice is due to alterations in intestinal bacteria. The data demonstrate that Oatp1a1-null mice : (1) have similar bile flow and BA concentrations in bile as WT mice; (2) have a markedly different BA composition in the intestinal contents, with a decrease in conjugated BAs and an increase in unconjugated BAs; (3) have BAs in the feces that are more deconjugated, desulfated, 7-dehydroxylated, 3-epimerized, and oxidized, but less 7-epimerized; (4) have 10-fold more bacteria in the small intestine, and 2-fold more bacteria in the large intestine which is majorly due to a 200% increase in Bacteroides and a 30% reduction in Firmicutes; and (5) have a different urinary excretion of bacteria-related metabolites than WT mice. In conclusion, the present study for the first time established that lack of a liver transporter (Oatp1a1) markedly alters the intestinal environment in mice, namely the bacteria composition

Expression of hepatocytic- and biliary-specific transcription factors in regenerating bile ducts during hepatocyte-to-biliary epithelial cell transdifferentiation

Background\ud Under compromised biliary regeneration, transdifferentiation of hepatocytes into biliary epithelial cells (BEC) has been previously observed in rats, upon exposure to BEC-specific toxicant methylene dianiline (DAPM) followed by bile duct ligation (BDL), and in patients with chronic biliary liver disease. However, mechanisms promoting such transdifferentiation are not fully understood. In the present study, acquisition of biliary specific transcription factors by hepatocytes leading to reprogramming of BEC-specific cellular profile was investigated as a potential mechanism of transdifferentiation in two different models of compromised biliary regeneration in rats.\ud \ud Results\ud In addition to previously examined DAPM + BDL model, an experimental model resembling chronic biliary damage was established by repeated administration of DAPM. Hepatocyte to BEC transdifferentiation was tracked using dipetidyl dipeptidase IV (DDPIV) chimeric rats that normally carry DPPIV only in hepatocytes. Following DAPM treatment, ~20% BEC population turned DPPIV-positive, indicating that they are derived from DPPIV-positive hepatocytes. New ductules emerging after DAPM + BDL and repeated DAPM exposure expressed hepatocyte-associated transcription factor hepatocyte nuclear factor (HNF) 4α and biliary specific transcription factor HNF1β. In addition, periportal hepatocytes expressed biliary marker CK19 suggesting periportal hepatocytes as a potential source of transdifferentiating cells. Although TGFβ1 was induced, there was no considerable reduction in periportal HNF6 expression, as observed during embryonic biliary development.\ud \ud Conclusions\ud Taken together, these findings indicate that gradual loss of HNF4α and acquisition of HNF1β by hepatocytes, as well as increase in TGFβ1 expression in periportal region, appear to be the underlying mechanisms of hepatocyte-to-BEC transdifferentiation

Temporal Dynamics of Spontaneous Ca2+ Transients, ERBB4, vGLUT1, GAD1, Connexin, and Pannexin Genes in Early Stages of Human Stem Cell Neurodifferentiation

Spontaneous Ca2+ transients drive stem cell proliferation and neurodifferentiation. Deciphering the relationship between neuronal and glial human genes on one side and spontaneous Ca2+ activity on the other side is essential for our understanding of normal brain development, and for insights into the pathogenesis of neurodegenerative and neurodevelopmental disorders. In the present study, forebrain neurons were derived from human embryonic and induced pluripotent stem cells (hESC-H9 and iPSC-15; 22q11.2 deletion) over a period of 21 days in vitro (DIV). Every 1–2 days, multisite optical imaging technique was applied to detect populations of cells with spontaneous Ca2+ transients. The expression levels of 14 genes of interest were analyzed by quantitative polymerase chain reaction (qPCR) on the same biological samples where physiological recordings were performed. The genes analyzed include: the schizophrenia candidate gene ERBB4, connexin (Cx) genes Cx26, Cx36, Cx43, Cx45, Cx47, pannexin-1 (PNX1), neuronal markers PAX6, vGLUT1, GAD1, TUBB3, glial lineage markers BLBP, GFAP, and housekeeping gene ACTB. We found that Ca2+ signals decrease in amplitude, decrease in duration, and increase in frequency during the first 21 days of human neurodifferentiation. The expression levels of ERBB4, PAX6, GAD1, vGLUT1, BLBP, Cx36, Cx45, and PNX1 were found to be strongly positively correlated with the percentage of cells exhibiting spontaneous Ca2+ transients (“Active Cells”). While expression of BLBP, Cx45, ERBB4, GAD1, PAX6, PNX1, and vGLUT1 were correlated with short-duration and long-amplitude Ca2+ transients, Cx43, TUBB3, and Cx47 were better correlated with long-duration and short-amplitude transients. The expression dynamics of Cx26 was unrelated to any aspect of spontaneous Ca2+ activity. Four genes showed an exponential time course with a distinct onset on a given DIV. The onset of PNX1, ERBB4, and vGLUT1 occurred before, while the onset of Cx36 occurred after the first action potentials were detected in early differentiating human neurons

Concentrations of primary and secondary BAs in the feces of WT and Oatp1a1-null mice.

<p>Feces were collected from WT and Oatp1a1-null mice for 24 hr and dried under vacuum. The concentrations of primary BAs (a, b, and c) and secondary BAs (d, e, and f) in the feces of male WT and Oatp1a1-null mice (n = 5/group) were analyzed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and normalized by fecal dry weight. All data are expressed as mean ± S.E. of five mice in each group. *, statistically significant difference between WT and Oatp1a1-null mice (<i>p</i><0.05).</p

BA composition in the small (a and b) and large (c and d) intestinal contents of mice.

<p>BA concentrations in the intestinal contents of male WT and Oatp1a1-null mice (n = 5/group) were analyzed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). All data are expressed as mean ± S.E. for five mice in each group. *, statistically significant difference between WT and Oatp1a1-null mice (<i>p</i><0.05).</p

Gene accession numbers of the 16 s rRNA gene assessed in this study.

<p>Gene accession numbers of the 16 s rRNA gene assessed in this study.</p

Small and large intestinal bacteria in WT and Oatp1a1-null mice.

<p>Clostridia (a), Bacteroides (b), Lactobacilli (c), and other bacteria (d) in the small and large intestinal contents of WT and Oatp1a1-null mice were quantified using a branched DNA assay (Panomics/Affymetrix, Fremont, CA). All data are expressed as mean ± S.E. of five mice in each group. *, statistically significant difference between WT and Oatp1a1-null mice (<i>p</i><0.05).</p

Oatp1a1-null mice had higher hippuric acid, but lower indole-3-carboxylic acid-glucuronide in urine than WT mice.

<p>Structural elucidations were performed based on accurate mass measurement and MS/MS fragmentations of hippuric acid (a) and indole-3-carboxylic acid-glucuronide (b) in urine of WT and Oatp1a1-null mice. By comparison of the retention time in the same MS/MS chromatograph window between authentic standards and urine samples, hippuric acid (c) and indole-3-carboxylic acid-G (d) were confirmed. The authentic standard indole-3-carboxylic acid-glucuronide (indole-3-carboxylic acid-G) was enzymatically synthesized from indole-3-carboxylic acid.</p

Bacteria-mediated BA metabolism.

<p>(a) Deconjugation, dehydroxylation, oxidation, and epimerization of cholic acid (CA) in the intestine of mice. Black dotted arrows represent for deconjugation, black solid arrows represent for 7-dehydroxylation, grey dotted arrows represent for oxidation, grey solid arrows represent for epimerization; (b) Deconjugation, dehydroxylation, oxidation, and epimerization of chenodeoxycholic acid (CDCA) and muricholic acid (MCA) in the intestine of mice. Black dotted arrows represent for deconjugation, black solid arrows represent for 7-dehydroxylation, grey dotted arrows represent for oxidation, grey solid arrows represent for epimerization; (c) Structures and abbreviations of various BAs. TCA and GCA stand for taurocholic acid and glycocholic acid, respectively.</p