L-Threonine induces heat shock protein expression and decreases apoptosis in heat-stressed intestinal epithelial cells.

OBJECTIVES: Osmotically acting amino acids can be cytoprotective following injury. As threonine (THR) induces osmotic cell swelling, our aim was to investigate the potential for THR to induce cellular protection in intestinal epithelial cells and evaluate possible mechanisms of protection. METHODS: Cells treated with a range of THR doses were evaluated following heat stress (HS) injury. Alpha-aminoisobutyric acid (AIB), a non-metabolizable amino acid analog, was used as an osmotic control. MTS assays were used to assess cell survival. Heat shock protein (HSP) expression and cleaved caspase-3 (CC3) were evaluated via Western blot. Cell morphology and cell size were analyzed via microscopy. RESULT: Following HS, THR treatment increased cell viability in a dose dependent manner vs. non-THR treated cells (CT). The non-metabolized amino acid analogue, AIB, also increased cell survival in heat-stressed cells versus HS controls. HSP70 and HSP25 expression increased with THR and AIB treatment versus HS controls. THR also increased HSP25 in non-stressed cells. Microscopic evaluation revealed both THR and AIB preserved the structural integrity of the actin cytoskeleton in heat-stressed cells versus HS controls. THR, but not AIB, enhanced nuclear translocation of HSP25 during HS. This nuclear translocation was associated with a 60% decrease in apoptosis in heat-stressed cells with THR. No antiapoptotic effect was observed with AIB. CONCLUSIONS: This is the first demonstration that THR increases HSP70 and HSP 25 and protects cells from HS. THR's mechanism of protection may involve cytoskeletal stabilization, HSP up-regulation and nuclear translocation, and decreased apoptosis. THR's protection appears to involve both cell-swelling-dependent and -independent processes.

Fibronectin-integrin signaling is required for L-glutamine's protection against gut injury.

BACKGROUND: Extracellular matrix (ECM) stabilization and fibronectin (FN)-Integrin signaling can mediate cellular protection. L-glutamine (GLN) is known to prevent apoptosis after injury. However, it is currently unknown if ECM stabilization and FN-Integrin osmosensing pathways are related to GLN's cell protective mechanism in the intestine. METHODS: IEC-6 cells were treated with GLN with or without FN siRNA, integrin inhibitor GRGDSP, control peptide GRGESP or ERK1/2 inhibitors PD98059 and UO126 under basal and stressed conditions. Cell survival measured via MTS assay. Phosphorylated and/or total levels of cleaved caspase-3, cleaved PARP, Bax, Bcl-2, heat shock proteins (HSPs), ERK1/2 and transcription factor HSF-1 assessed via Western blotting. Cell size and F-actin morphology quantified by confocal fluorescence microscopy and intracellular GLN concentration by LC-MS/MS. RESULTS: GLN's prevention of FN degradation after hyperthermia attenuated apoptosis. Additionally, inhibition of FN-Integrin interaction by GRGDSP and ERK1/2 kinase inhibition by PD98059 inhibited GLN's protective effect. GRGDSP attenuated GLN-mediated increases in ERK1/2 phosphorylation and HSF-1 levels. PD98059 and GRGDSP also decreased HSP levels after GLN treatment. Finally, GRGDSP attenuated GLN-mediated increases in cell area size and disrupted F-actin assembly, but had no effect on intracellular GLN concentrations. CONCLUSION: Taken together, this data suggests that prevention of FN degradation and the FN-Integrin signaling play a key role in GLN-mediated cellular protection. GLN's signaling via the FN-Integrin pathway is associated with HSP induction via ERK1/2 and HSF-1 activation leading to reduced apoptosis after gut injury.

Glutamine prevents apoptosis in intestinal epithelial cells and induces differential protective pathways in heat and oxidant injury models.

BACKGROUND: Glutamine (GLN) can decrease mortality and length of hospital stay in the critically ill. GLN protects via enhancing protective heat shock proteins (HSPs) in heat stress (HS). GLN's effect on HSPs in oxidant injury and apoptosis remains to be elucidated. The purpose of this study was to determine if GLN protects via decreasing apoptosis during both heat and oxidative stress. METHODS: IEC-18 cells were treated (15 minutes) with 0 mM GLN (control cells [CTs]) or 8 mM GLN and exposed to either lethal injury (44°C for 50 minutes or 4 mM H(2)O(2) for 30 minutes) or nonlethal injury (43°C for 45 minutes or 600 µM H(2)O(2) for 30 minutes). Survival was determined via MTS assay. Injured groups were normalized to noninjured controls. HSPs and cleaved caspase-3 (CC3), a key mediator for apoptosis, were evaluated via Western blot following a 3-hour recovery. RESULTS: MTS assays showed GLN increased survival 4- to 5-fold (P < .001 vs HS CT or H(2)O(2)). Western blot showed GLN increased all 3 HSPs in HS (P < .001 vs HS CTs) but only HSP32 during oxidant injury (P < .02 vs H(2)O(2) only). GLN decreased CC3 in both injuries (P < .03 vs non-GLN-treated cells). CONCLUSIONS: GLN protects intestinal cells from both heat and oxidant injury. HSP25, 32, and 70 levels increased with GLN during HS, but in oxidant injury, only HSP32 increased, suggesting GLN's mechanism of protection may vary in different models of injury. In both injuries, GLN lowered the expression of CC3, indicating prevention of apoptosis may be a key mechanism by which GLN protects.