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

Abstract

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 fluoresceindextran 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.

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

“…5,47 Here, we showed that rats preconditioned to a hypoxic environment displayed a significant reduction in bacterial counts in extraintestinal organs, associated with decreases in transepithelial macromolecular flux and mucosal histopathology on I/R challenge. The attenuation of BT may be attributed to higher barrier integrity and/or stronger antimicrobial effects of phagocytes.…”

“…45,46 Magnetic Resonance Imaging (MRI)-Based Intestinal Permeability Assay To assess the intestinal permeability in vivo, Gadodiamide (Gd)-containing contrast solution (Omniscan; GE Healthcare, USA) was instilled into the lumen of a ligated intestinal sac (10 cm) to a final concentration of 0.25 M immediately after the release of the artery clamp in I/R rats, and the signal intensity of this reagent in the plasma was quantified as described previously. 5,47 The intestinal sac was created 1 cm proximal to the cecum by thread ligature and a PE-10 tube was intubated to one end of the sac for instillation of Gd solution. In sham-operated animals, the Gd probe was injected into the intestinal sac after mock manipulation.…”

“…The plasma Gd concentration was calculated from the standard curve. 5,47 Neutrophil Respiratory Burst Activity Fresh heparinized blood was used for the measurement of neutrophil respiratory burst activity as previously described. 48 A sample of whole blood (20 ml) was diluted with 980 ml of PBS.…”

“…[1][2][3] Intestinal I/R may trigger mucosal barrier damage and enteric bacterial translocation (BT), leading to development of septic complications. 4,5 The intestine contains over 100 trillion commensal bacteria that are normally restricted to the lumen. 6,7 Germ-free rodents subjected to mesenteric I/R exhibited less intestinal and lung histopathology, and showed higher survival rates than conventionally raised animals, underscoring the role of enteric bacteria in the pathogenesis of I/R-induced local and remote organ dysfunction.…”

Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide

“…Primary antibodies included mouse antihuman PCNA (1:2,000), phospho (p)-and total (t)-IkB (1:2,000), p-and t-ERK1/2 (1:4,000), p-and t-JNK (1:2,000), p-and t-Akt (1:2,000), p-PKCz (Thr410; 1:2,000), p-PKCz (Thr560; 1:5,000; Abcam Epitomics), t-PKCz (1:2,000), p-tyrosine (1:2,000; EMD Millipore), CD14 (1:50 for immunofluorescence and 1:2,000 for Western blot; R&D Systems), TLR4 (1:100 for immunofluorescence and 1:2,000 for Western blot; Santa Cruz Biotechnology), TLR2 (1:2,000; Santa Cruz Biotechnology), MyD88 (1:2,000), bromodeoxyuridine (BrdUrd; 1:200; Abcam), mouse IgG1 and IgG2a isotype controls (R&D Systems), rat IgG2a isotype controls (Abcam), and b-actin (1:10,000; Sigma; refs. 19,31). The secondary antibodies used were goat anti-mouse IgG conjugated to horseradish peroxidase (1:2,000), and to Alexa 594 or 488 (1:1,000; ThermoFisher).…”