AJP - Gastrointestinal and Liver Physiology

Malo, A.; Krüger, B.; Seyhun, E.; Schäfer, C.; Hoffmann, R. T.; Göke, B.; Kubisch, C. H.

doi: 10.1152/ajpgi.00423.2009pmid: 20671193

Abstract Endoplasmic reticulum (ER) stress leads to accumulation of un- or misfolded proteins inside the ER and initiates the unfolded protein response (UPR). Several UPR components are physiologically involved in pancreatic development and are pathophysiologically activated during acute pancreatitis. However, the exact role of ER stress in exocrine pancreatic acini is mainly unclear. The present study examined the effects of tauroursodeoxycholic acid (TUDCA), a known ER chaperone, on acinar function and UPR components. Isolated rat pancreatic acini were stimulated by increasing concentrations of cholecystokinin (CCK-8) with or without preincubation of TUDCA. UPR components were analyzed, including chaperone binding protein (BiP), protein kinase-like ER kinase (PERK), X-box binding protein (XBP)-1, c-Jun NH 2 -terminal kinase (JNK), CCAAT/enhancer binding protein homologues protein (CHOP), caspase 3 activation, and apoptosis. In addition, TUDCA effects were measured on amylase secretion, calcium signaling, trypsin, and cathepsin B activation. TUDCA preincubation led to a significant increase in amylase secretion after CCK-8 stimulation, a 50% reduction of intracellular trypsin activation, and reduced cathepsin B activity, although the effects for cathepsin B were not statistical significant. Furthermore, TUDCA prevented the CCK-8-induced BiP upregulation, diminished PERK and JNK phosphorylation, and prohibited the expression of CHOP, caspase 3 activation and apoptosis. XBP-1 splicing was not altered. ER stress response mechanisms are activated in pancreatic inflammation. Chemical chaperones enhance enzyme secretion of pancreatic acini, reduce ER stress responses, and attenuate ER stress-associated apoptosis. These data hint new perspectives for an employment of chemical chaperones in the therapy of acute pancreatitis. exocrine pancreatic acini endoplasmic reticulum stress chemical chaperone TUDCA Copyright © 2010 the American Physiological Society

Hajnal, Andras; Kovacs, Peter; Ahmed, Tamer; Meirelles, Katia; Lynch, Christopher J.; Cooney, Robert N.

doi: 10.1152/ajpgi.00070.2010pmid: 20634436

Abstract Roux-en-Y gastric bypass surgery (GBS) is the most effective treatment for morbid obesity. GBS is a restrictive malabsorptive procedure, but many patients also report altered taste preferences. This study investigated the effects of GBS or a sham operation (SH) on body weight, glucose tolerance, and behavioral and neuronal taste functions in the obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-1 receptors and lean controls (LETO). OLETF-GBS rats lost body weight (−26%) and demonstrated improved glucose tolerance. They also expressed a reduction in 24-h two-bottle preference for sucrose (0.3 and 1.0 M) and decreased 10-s lick responses for sucrose (0.3 through 1.5 M) compared with OLETF-SH or LETO-GBS. A similar effect was noted for other sweet compounds but not for salty, sour, or bitter tastants. In lean rats, GBS did not alter responses to any stimulus tested. Extracellular recordings from 170 taste-responsive neurons of the pontine parabrachial nucleus revealed a rightward shift in concentration responses to oral sucrose in obese compared with lean rats (OLETF-SH vs. LETO-SH): overall increased response magnitudes (above 0.9 M), and maximum responses occurring at higher concentrations (+0.46 M). These effects were reversed by GBS, and neural responses in OLETF-GBS were statistically not different from those in any LETO groups. These findings confirm obesity-related alterations in taste functions and demonstrate the ability of GBS to alleviate these impairments. Furthermore, the beneficial effects of GBS appear to be independent of CCK-1 receptor signaling. An understanding of the underlying mechanisms for reduced preferences for sweet taste could help in developing less invasive treatments for obesity. overeating dietary obesity surgical weight reduction insulin resistance palatability gustatory coding cholecystokinin-1 receptor Copyright © 2010 the American Physiological Society

Borthakur, Alip; Anbazhagan, Arivarasu N.; Kumar, Anoop; Raheja, Geetu; Singh, Varsha; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K.

doi: 10.1152/ajpgi.00279.2010pmid: 20671196

Abstract The major short-chain fatty acid (SCFA) butyrate is produced in the colonic lumen by bacterial fermentation of dietary fiber. Butyrate serves as primary fuel for the colonocytes and also ameliorates mucosal inflammation. Disturbed energy homeostasis seen in inflamed mucosa of inflammatory bowel disease patients has been attributed to impaired absorption of butyrate. Since sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) has recently been shown to play a role in Na + -coupled transport of monocarboxylates, including SCFA, such as luminal butyrate, we examined the effects of proinflammatory TNF-α on SMCT1 expression and function and potential anti-inflammatory role of probiotic Lactobacillus species in counteracting the TNF-α effects. Rat intestinal epithelial cell (IEC)-6 or human intestinal Caco-2 cells were treated with TNF-α in the presence or absence of Lactobacilli culture supernatants (CS). TNF-α treatments for 24 h dose-dependently inhibited SMCT1-mediated, Na + -dependent butyrate uptake and SMCT1 mRNA expression in IEC-6 cells and SMCT1 promoter activity in Caco-2 cells. CS of L. plantarum (LP) stimulated Na + -dependent butyrate uptake (2.5-fold, P < 0.05), SMCT1 mRNA expression, and promoter activity. Furthermore, preincubating the cells with LP-CS followed by coincubation with TNF-α significantly attenuated the inhibitory effects of TNF-α on SMCT1 function, expression, and promoter activity. In vivo, oral administration of live LP enhanced SMCT1 mRNA expression in the colonic and ileal tissues of C57BL/6 mice after 24 h. Efficacy of LP or their secreted soluble factors to stimulate SMCT1 expression and function and to counteract the inhibitory effects of TNF-α on butyrate absorption could have potential therapeutic value. intestinal epithelial cell-6 SLC5A8 short-chain fatty acid inflammatory bowel disease Copyright © 2010 the American Physiological Society

Masyuk, Anatoliy I.; Huang, Bing Q.; Ward, Christopher J.; Gradilone, Sergio A.; Banales, Jesus M.; Masyuk, Tatyana V.; Radtke, Brynn; Splinter, Patrick L.; LaRusso, Nicholas F.

doi: 10.1152/ajpgi.00093.2010pmid: 20634433

Abstract Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic “saucer-shaped” morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia. bile cholangiocytes Copyright © 2010 the American Physiological Society

Annaba, Fadi; Ma, Ke; Kumar, Pradeep; Dudeja, Amish K.; Kineman, Rhonda D.; Shneider, Benjamin L.; Saksena, Seema; Gill, Ravinder K.; Alrefai, Waddah A.

doi: 10.1152/ajpgi.00139.2010pmid: 20651004

Abstract Increased intestinal bile acid absorption and expansion of the bile acid pool has been implicated in the hypercholesterolemia associated with diabetes mellitus. However, the molecular basis of the increase in bile acid absorption in diabetes mellitus is not fully understood. The ileal apical Na + -dependent bile acid transporter (ASBT) is primarily responsible for active reabsorption of the majority of bile acids. Current studies were designed to investigate the modulation of ASBT function and expression in streptozotocin (STZ)-induced diabetes mellitus in rats and to examine the effect of insulin on rat ASBT promoter by insulin. Diabetes mellitus was induced in Sprague-Dawley rats by intraperitoneal injection of low doses of STZ (20 mg/kg body wt) on five consecutive days. Human insulin (10 U/day) was given to a group of diabetic rats for 3 days before euthanasia. RNA and protein were extracted from mucosa isolated from the small intestine and ASBT expression was assessed by real-time quantitative RT-PCR and Western blotting. Our data showed that ASBT mRNA and protein expression were significantly elevated in diabetic rats. Insulin treatment of diabetic rats reversed the increase in ASBT protein expression to control levels. Consistently, ileal Na + -dependent 3 Htaurocholic uptake in isolated intestinal epithelial cells was significantly increased in diabetic rats. In vitro studies utilizing intestinal epithelial Caco-2 cells demonstrated that ASBT expression and promoter activity were significantly decreased by insulin. These studies demonstrated that insulin directly influences ASBT expression and promoter activity and that ASBT function and expression are increased in rats with STZ-induced diabetes mellitus. The increase in ASBT expression may contribute to disturbances in cholesterol homeostasis associated with diabetes mellitus. hypercholesterolemia insulinopenia enterohepatic circulation Footnotes Copyright © 2010 the American Physiological Society

Castro, Rui E.; Ferreira, Duarte M. S.; Zhang, Xiaoxiao; Borralho, Pedro M.; Sarver, Aaron L.; Zeng, Yan; Steer, Clifford J.; Kren, Betsy T.; Rodrigues, Cecília M. P.

doi: 10.1152/ajpgi.00216.2010pmid: 20689055

Abstract New gene regulation study tools such as microRNA (miRNA or miR) analysis may provide unique insights into the remarkable ability of the liver to regenerate. In addition, we have previously shown that ursodeoxycholic acid (UDCA) modulates mRNA levels during liver regeneration. Bile acids are also homeotrophic sensors of functional hepatic capacity. The present study was designed to determine whether miRNAs are modulated in rats following 70% partial hepatectomy (PH) and elucidate the role of UDCA in regulating miRNA expression during liver regeneration (LR). Total RNA was isolated from livers harvested at 3–72 h following 70% PH or sham operations, from both 0.4% (wt/wt) UDCA and control diet-fed animals. By using a custom microarray platform we found that several miRNAs are significantly altered after PH by >1.5-fold, including some previously described as modulators of cell proliferation, differentiation, and death. In particular, expression of miR-21 was increased after PH. Functional modulation of miR-21 in primary rat hepatocytes increased cell proliferation and viability. Importantly, UDCA was a strong inducer of miR-21 both during LR and in cultured HepG2 cells. In fact, UDCA feeding appeared to induce a sustained increase of proliferative miRNAs observed at early time points after PH. In conclusion, miRNAs, in particular miR-21, may play a significant role in modulating proliferation and cell cycle progression genes after PH. miR-21 is additionally induced by UDCA in both regenerating rat liver and in vitro, which may represent a new mechanism behind UDCA biological functions. bile acids liver regeneration miR-21 miRNAs Copyright © 2010 the American Physiological Society

Scharl, Michael; Rudenko, Ivan; McCole, Declan F.

doi: 10.1152/ajpgi.00106.2010pmid: 20689057

Abstract The Crohn's disease candidate gene, protein tyrosine phosphatase nonreceptor type 2 (PTPN2), has been shown to regulate epidermal growth factor (EGF)-induced phosphatidylinositol 3-kinase (PI3K) activation in fibroblasts. In intestinal epithelial cells (IECs), EGF-induced EGF receptor (EGFR) activation and recruitment of PI3K play a key role in regulating many cellular functions including Ca 2+ -dependent Cl − secretion. Moreover, EGFR also serves as a conduit for signaling by other non-growth factor receptor ligands such as the proinflammatory cytokine, IFN-γ. Here we investigated a possible role for PTPN2 in the regulation of EGFR signaling and Ca 2+ -dependent Cl − secretion in IECs. PTPN2 knockdown enhanced EGF-induced EGFR tyrosine phosphorylation in T 84 cells. In particular, PTPN2 knockdown promoted EGF-induced phosphorylation of EGFR residues Tyr-992 and Tyr-1068 and led subsequently to increased association of the catalytic PI3K subunit, p110, with EGFR and elevated phosphorylation of the downstream marker, Akt. As a functional consequence, loss of PTPN2 potentiated EGF-induced inhibition of carbachol-stimulated Ca 2+ -dependent Cl − secretion. In contrast, PTPN2 knockdown affected neither IFN-γ-induced EGFR transactivation nor EGF- or IFN-γ-induced phosphorylation of ERK1/2. In summary, our data establish a role for PTPN2 in the regulation of EGFR signaling in IECs in response to EGF but not IFN-γ. Knockdown of PTPN2 directs EGFR signaling toward increased PI3K activation and increased suppression of epithelial chloride secretory responses. Moreover, our findings suggest that PTPN2 dysfunction in IECs leads to altered control of intestinal epithelial functions regulated by EGFR. EGF receptor phosphatidylinositol 3′-kinase IFN-γ carbachol Copyright © 2010 the American Physiological Society

Lissner, S.; Nold, L.; Hsieh, C.-J.; Turner, J. R.; Gregor, M.; Graeve, L.; Lamprecht, G.

doi: 10.1152/ajpgi.00191.2010pmid: 20634435

Abstract The Cl/HCO 3 exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity. Cl/HCO 3 exchange PDZK1 E3KARP NHERF IKEPP Copyright © 2010 the American Physiological Society

King, Adrienne L.; Swain, Telisha M.; Dickinson, Dale A.; Lesort, Mathieu J.; Bailey, Shannon M.

doi: 10.1152/ajpgi.00246.2010pmid: 20651005

Abstract Chronic ethanol consumption increases mitochondrial oxidative stress and sensitivity to form the mitochondrial permeability transition pore (MPTP). The mechanism responsible for increased MPTP sensitivity in ethanol-exposed mitochondria and its relation to mitochondrial Ca 2+ handling is unknown. Herein, we investigated whether increased sensitivity to MPTP induction in liver mitochondria from ethanol-fed rats compared with controls is related to an ethanol-dependent change in mitochondrial Ca 2+ accumulation. Liver mitochondria were isolated from control and ethanol-fed rats, and Ca 2+ -mediated induction of the MPTP and mitochondrial Ca 2+ retention capacity were measured. Levels of proposed MPTP proteins as well as select pro- and antiapoptotic proteins were measured along with gene expression. We observed increased steatosis and TUNEL-stained nuclei in liver of ethanol-fed rats compared with controls. Liver mitochondria from ethanol-fed rats had increased levels of proapoptotic Bax protein and reduced Ca 2+ retention capacity compared with control mitochondria. We observed increased cyclophilin D (Cyp D) gene expression in liver and protein in mitochondria from ethanol-fed animals compared with controls, whereas there was no change in the adenine nucleotide translocase and voltage-dependent anion channel. Together, these results suggest that enhanced sensitivity to Ca 2+ -mediated MPTP induction may be due, in part, to higher Cyp D levels in liver mitochondria from ethanol-fed rats. Therefore, therapeutic strategies aimed at normalizing Cyp D levels may be beneficial in preventing ethanol-dependent mitochondrial dysfunction and liver injury. mitochondria alcohol steatosis calcium bioenergetics Copyright © 2010 the American Physiological Society

Steenbergen, Rineke H. G.; Joyce, Michael A.; Lund, Garry; Lewis, Jamie; Chen, Ran; Barsby, Nicola; Zhu, Lin Fu; Tyrrell, D. Lorne J.; Kneteman, Norman M.

doi: 10.1152/ajpgi.00200.2010pmid: 20651006

Abstract Although multiple determinants for hepatitis C virus (HCV) infection are known, it remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ∼70–80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins. apolipoprotein B low-density lipoprotein xenotransplantation immunodeficient mouse Flavivirus Footnotes Copyright © 2010 the American Physiological Society