Ching-Ying Huang

Anti-apoptotic PI3K/Akt signaling by sodium/glucose transporter 1 reduces epithelial barrier damage and bacterial translocation in intestinal ischemia

Intestinal ischemia/reperfusion (I/R) causes mucosal barrier damage and bacterial translocation (BT), leading to septic complications. Previous in vitro studies showed that activation of sodium/glucose transporter 1 (SGLT1) prevented the epithelial apoptosis and permeability rise induced by microbial products. Our aim was to investigate whether luminal glucose uptake by SGLT1 protects against ischemia-induced epithelial cell death and barrier dysfunction, and to explore the glucose-mediated cellular survival pathways in vivo. Rat jejunum was luminally instilled with either vehicle, a pancaspase inhibitor ZVAD, or glucose prior to I/R challenge (occlusion of the superior mesenteric artery for 20 min and reperfusion for 60 min). Histopathology and apoptosis in the jejunum were examined by TUNEL staining and caspase-3 cleavage. Intestinal permeability was evaluated using in vivo assays measuring luminal-to-blood passage of fluorescein-dextran and portal drainage of enterally administered gadodiamide by magnetic resonance imaging. BT was determined by culturing liver and spleen homogenates. Immunofluorescent analysis and kinase assay were used to study PI3K/Akt signaling pathways. Intestinal I/R caused enterocyte apoptosis and villous destruction. Intestinal infusion with ZVAD decreased the I/R-triggered gut permeability rise and BT, suggesting that the barrier damage was partly dependent on cell apoptosis. Enteral instillation of glucose attenuated the epithelial apoptosis, barrier damage, and mucosal inflammation caused by I/R. Phloridzin (a SGLT1 inhibitor) reduced the protective effect of glucose in a dose-dependent manner. Enteral glucose increased the mucosal Akt kinase activity as evidenced by the augmented phosphorylation of exogenous GSK3. Enhanced membrane translocation and phosphorylation of Akt in epithelial cells were associated with elevated phosphorylation of mTOR, Bad, and FoxO1/3a following glucose uptake. Inhibition of PI3K/Akt signaling by LY294002 and wortmannin partially blocked the glucose-mediated rescue of cell apoptosis and barrier damage. In conclusion, SGLT1 glucose uptake alleviated I/R-induced barrier dysfunction and BT, partly by inhibiting epithelial apoptosis via activation of PI3K/Akt signaling.

SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalis-induced apoptosis

Infection with Giardia duodenalis is one of the most common causes of waterborne diarrheal disease worldwide. Mechanisms of pathogenesis and host response in giardiasis remain incompletely understood. Previous studies have shown that exposure to G. duodenalis products induce apoptosis in enterocytes. We recently discovered that sodium-dependent glucose cotransporter (SGLT)-1-mediated glucose uptake modulates enterocytic cell death induced by bacterial lipopolysaccharide. The aim of this study was to examine whether enhanced epithelial SGLT-1 activity may constitute a novel mechanism of host defense against G. duodenalis-induced apoptosis. SGLT-1-transfected Caco-2 cells were exposed to G. duodenalis products in low (5mM) or high (25mM) glucose media. In low glucose environments, G. duodenalis-induced caspase-3 activation and DNA fragmentation in these cells. These apoptotic phenomena were abolished in the presence of high glucose. A soluble proteolytic fraction of G. duodenalis was found to upregulate SGLT-1-mediated glucose uptake in a dose- and time-dependent manner, in association with increased apical SGLT-1 expression on epithelial cells. Kinetic analysis showed that this phenomenon resulted from an increase in the maximal rate of sugar transport (V(max)) by SGLT-1, with no change in the affinity constant (K(m)). The addition of phloridzin (a competitive inhibitor for glucose binding to SGLT-1) abolished the anti-apoptotic effects exerted by high glucose. Together, the findings indicate that SGLT-1-dependent glucose uptake may represent a novel epithelial cell rescue mechanism against G. duodenalis-induced apoptosis.

Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion

Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47phox and p67phox, as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative respiratory bursts and through promotion of epithelial barrier integrity for luminal confinement of enteric bacteria.

Commensal bacterial endocytosis in epithelial cells is dependent on myosin light chain kinase-activated brush border fanning by interferon-γ.

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.

Magnetic Resonance Imaging Detects Intestinal Barrier Dysfunction in a Rat Model of Acute Mesenteric Ischemia/Reperfusion Injury

Objectives:To develop an in vivo intestinal permeability assay applying magnetic resonance imaging (MRI) to monitor real-time gut barrier defects in animal models of acute mesenteric ischemia/reperfusion (I/R) insult. Materials and Methods:Twenty Wistar rats were divided to 2 groups for I/R challenge or sham controls. I/R rats received occlusion of superior mesenteric artery for 20 minutes and reperfusion for 1 hour. Sham-operation controls received laparotomy without manipulation of artery. To assess gut permeability, a 10-cm jejunal sac was created distal to the ligament of Treitz in both groups of rats after laparotomy, and a contrast agent (gadodiamide) was injected into the lumen of the ligated intestinal sac. The signals produced by gadodamide in the liver, kidney, and plasma before and after the start of reperfusion were examined by 1.5 Tesla MRI (GE Signa Excite), and the increment of signal-to-noise ratio (SNR) in these organs that parallels the luminal-to-serosal flux rate of the probe was used as an indicator of gut permeability. At the end of procedures, jejunal tissues and mucosal scrapings were collected for histologic examination and Western blotting for epithelial tight junctional proteins. Moreover, liver and spleen homogenates were cultured on fresh blood agar plates to measure the bacterial colony-forming units per gram of tissue. Results:In I/R rats, disrupted villous structure and decreased epithelial tight junctional expression were seen in the jejunum associated with massive enteric bacterial translocation to the liver and spleen. The SNR in the liver of I/R rats was higher than sham controls (2.65 ± 0.56 vs. 0.65 ± 0.26, P < 0.01) at 15 minutes postreperfusion. Elevation of SNR in the kidney was also found in I/R rats compared with sham controls (11.61 ± 2.07 vs. 3.06 ± 1.15, P < 0.05). The plasma gadodiamide concentration in I/R rats was significantly increased compared with sham controls (0.220 ± 0.044 vs. 0.006 ± 0.004 mM, P < 0.01) at 15 minutes postreperfusion. Conclusions:This novel MRI-based intestinal permeability assay has shown a significant increase in the signal intensity in liver, kidney, and plasma samples that correlated with mucosal barrier defects in experimental models of acute mesenteric I/R.

Pathophysiological mechanisms of death resistance in colorectal carcinoma

Colon cancers develop adaptive mechanisms to survive under extreme conditions and display hallmarks of unlimited proliferation and resistance to cell death. The deregulation of cell death is a key factor that contributes to chemoresistance in tumors. In a physiological context, balance between cell proliferation and death, and protection against cell damage are fundamental processes for maintaining gut epithelial homeostasis. The mechanisms underlying anti-death cytoprotection and tumor resistance often bear common pathways, and although distinguishing them would be a challenge, it would also provide an opportunity to develop advanced anti-cancer therapeutics. This review will outline cell death pathways (i.e., apoptosis, necrosis, and necroptosis), and discuss cytoprotective strategies in normal intestinal epithelium and death resistance mechanisms of colon tumor. In colorectal cancers, the intracellular mechanisms of death resistance include the direct alteration of apoptotic and necroptotic machinery and the upstream events modulating death effectors such as tumor suppressor gene inactivation and pro-survival signaling pathways. The autocrine, paracrine and exogenous factors within a tumor microenvironment can also instigate resistance against apoptotic and necroptotic cell death in colon cancers through changes in receptor signaling or transporter uptake. The roles of cyclooxygenase-2/prostaglandin E2, growth factors, glucose, and bacterial lipopolysaccharides in colorectal cancer will be highlighted. Targeting anti-death pathways in the colon cancer tissue might be a promising approach outside of anti-proliferation and anti-angiogenesis strategies for developing novel drugs to treat refractory tumors.

Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut

Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria‐derived septic complications. Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive. A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes. Pyruvate suppressed epithelial cell death in an ATP‐independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut. Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti‐necroptotic role of glycolytic pyruvate under ischaemic stress.