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AKT Signalling
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 91. POSTBURN HEPATIC INSULIN RESISTANCE IS DUE TO ALTERED JNK/IRS-1 ACTIVATION LEADING TO IMPAIRED P13K/AKT SIGNALLING. G. G. Gauglitz, S. C. Halder, G. Kulp, F. N. Williams, D. N. Herndon, M. G. Jeschke; Shriners Hospitals for Children, Galveston, TX
90. ROLE OF P38 MAPK SIGNALING IN BURN-INDUCED INTESTINAL BARRIER BREAKDOWN. T. W. Costantini, C. Y. Peterson, L. M. Kroll, W. H. Loomis, J. G. Putnam, B. P. Eliceiri, A. Baird, V. Bansal, R. Coimbra; University of California San Diego School of Medicine, San Diego, CA Background: Severe burn injury results in intestinal barrier breakdown, which may lead to the generation of a systemic inflammatory response and distant organ injury. Intestinal barrier integrity is regulated, in part, by the tight junction protein myosin light chain kinase (MLCK). Activation of MLCK is known to increase tight junction breakdown through phosphorylation of myosin light chain (MLC). Previous studies in cell culture have shown that activation of p38 MAPK plays an important role in modulating intestinal barrier function. Therefore, we sought to further define the role of intestinal p38 MAPK signaling in an animal model of cutaneous burn. We hypothesize that (1) severe burn upregulates p38 MAPK activation and results in increased intestinal permeability via augmented expression of MLCK, and (2) inhibition of p38 MAPK will prevent the burn-induced increased in MLCK expression resulting in improved intestinal barrier integrity. Methods: Male balb/c mice were subjected to a 30% total body surface area (TBSA) full thickness steam burn. Immediately following injury, animals were randomized to receive an intraperitoneal injection of a p38 MAPK inhibitor (SB203580, 25mg/kg) or vehicle. An in vivo intestinal permeability assay was performed by measuring circulating 4 kDa FITC-Dextran after injection into an isolated segment of ileum. Expression of phosphorylated p38 MAPK, total p38 MAPK, MLCK, and phosphorylated MLC from intestinal extracts was assessed by immunoblotting. Results: Severe thermal injury resulted in a significant increase in intestinal permeability, which was associated with activation of p38 MAPK, increased expression of MLCK, and increased phosphorylation of MLC. Treatment with SB203580 significantly attenuated burn-induced intestinal permeability (203 g/ml vs. 94.5 g/ml, p ⬍ 0.05). Inhibition of p38 MAPK following burn decreased expression of intestinal MLCK by 73% (p ⬍ 0.02), resulting in decreased phosphorylation of MLC. Conclusion: p38 MAPK plays an important role in regulating burn-induced intestinal permeability through activation of MLCK. Therefore, inhibition of p38 MAPK may be an important therapeutic target aimed at attenuating intestinal barrier breakdown by preventing the burn-induced activation of tight junction proteins.
Introduction: Insulin resistance with its associated hyperglycemia represents one of the most significant contributors to mortality in burned and critically ill patients. While the metabolic pathophysiology is defined by increased glucose production with a decreased glucose uptake, the molecular mechanisms underlying insulin resistance are not well defined. The purpose of the present study was to determine the molecular mechanisms underlying hepatic insulin resistance. Materials and Methods: Rats received a 60% TBSA thermal injury and a laparatomy was performed at 24, 72 and 192 h post-burn. Liver was harvested before and 1 min after insulin injection (1 IU/kg) into the portal vein. PKR-like ER-Kinase (PERK), Inositol Requiring Enzyme (IRE)-1, activating transcription factor 6 (ATF-6), c-Jun N-terminal Kinase (JNK), Insulin Receptor Substrate (IRS)-1, Phosphatidylinositol 3-Kinase (PI3K), Protein Kinsae B (Akt), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Caveolin-1, phophorylated Caveolin-1 (pY14)were determined by Western blotting. Glucose-6-Phosphatase (G-6-P), Glucose Transporter (GLUT)-2 and ⫺4 were determined by real-time RT-PCR techniques at similar time points. Before insulin injections, serum was collected to determine fasting glucose and insulin levels by standard ELISA techniques. Results: Insulin resistance indicated by increased fasting serum glucose and insulin levels occurred starting 24 h post burn. Severe burn injury resulted in a profound activation of ER stress pathways, reflected by the increased accumulation of phospho-PERK, phospho-IRE-1 and ATF-6 leading to an elevation of phospho-JNK and serine phosphorylation of IRS-1. Insulin administration caused a significant increase in tyrosine phosphorylation of IRS-1 leading to activation of the PI3K/Akt pathway in normal liver. Post burn tyrosine phosphorylation of IRS-1 was significantly impaired (P⬍0.05) associated with an inactivation of signaling molecules acting downstream of IRS-1 (P⬍0.05). Conclusion: We found that dysregulation of signaling cascades deriving from ER stress may explain, at least partially, the metabolic abnormalities regarding insulin action following severe burn. The ER stress signaling cascades found in this study to be responsible for hepatic metabolic dysregulation following severe burn and inflammatory response pathways converge at the JNK/IRS-1/PI3K/Akt hub. Therefore, targeting of these molecules may provide a viable and efficacious alternative for the management of patients post burn. 92. UTILIZING PHAGE DISPLAY TECHNOLOGY TO IDENTIFY PEPTIDE SEQUENCES TARGETING THE BURN INJURED INTESTINAL BARRIER. T. W. Costantini, C. Y. Peterson, J. G. Putnam, R. Sawada, W. H. Loomis, B. P. Eliceiri, A. Baird, V. Bansal, R. Coimbra; University of California San Diego School of Medicine, San Diego, CA Background: Severe burn injury results in intestinal barrier dysfunction which may be responsible for significant morbidity and mortality. Therefore, it would be ideal to directly target the gut with biotherapeutics to either prevent barrier breakdown, or rapidly promote barrier restoration. Phage display is a molecular technique used to identify peptides that can home drugs, proteins, and particles to specific cell populations within tissues. We postulated that mining a peptide library that was displayed on phage would identify peptide sequences that bind and internalize into the gut epithelium. Using this technique, we hoped to identify a platform for the delivery of biotherapeutics capable of modulating intestinal dysfunction following severe burn. Methods: Two hours following 30% total burn surface area (TBSA) steam burn, male balb/c mice were sacrificed and the distal ileum harvested. The intestinal mucosa was isolated and incubated in complete DMEM culture media with 10 12 phage of a phage library (NEB12) containing 10 9 different peptide sequences.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 91. POSTBURN HEPATIC INSULIN RESISTANCE IS DUE TO ALTERED JNK/IRS-1 ACTIVATION LEADING TO IMPAIRED P13K/AKT SIGNALLING. G. G. Gauglitz, S. C. Halder, G. Kulp, F. N. Williams, D. N. Herndon, M. G. Jeschke; Shriners Hospitals for Children, Galveston, TX
90. ROLE OF P38 MAPK SIGNALING IN BURN-INDUCED INTESTINAL BARRIER BREAKDOWN. T. W. Costantini, C. Y. Peterson, L. M. Kroll, W. H. Loomis, J. G. Putnam, B. P. Eliceiri, A. Baird, V. Bansal, R. Coimbra; University of California San Diego School of Medicine, San Diego, CA Background: Severe burn injury results in intestinal barrier breakdown, which may lead to the generation of a systemic inflammatory response and distant organ injury. Intestinal barrier integrity is regulated, in part, by the tight junction protein myosin light chain kinase (MLCK). Activation of MLCK is known to increase tight junction breakdown through phosphorylation of myosin light chain (MLC). Previous studies in cell culture have shown that activation of p38 MAPK plays an important role in modulating intestinal barrier function. Therefore, we sought to further define the role of intestinal p38 MAPK signaling in an animal model of cutaneous burn. We hypothesize that (1) severe burn upregulates p38 MAPK activation and results in increased intestinal permeability via augmented expression of MLCK, and (2) inhibition of p38 MAPK will prevent the burn-induced increased in MLCK expression resulting in improved intestinal barrier integrity. Methods: Male balb/c mice were subjected to a 30% total body surface area (TBSA) full thickness steam burn. Immediately following injury, animals were randomized to receive an intraperitoneal injection of a p38 MAPK inhibitor (SB203580, 25mg/kg) or vehicle. An in vivo intestinal permeability assay was performed by measuring circulating 4 kDa FITC-Dextran after injection into an isolated segment of ileum. Expression of phosphorylated p38 MAPK, total p38 MAPK, MLCK, and phosphorylated MLC from intestinal extracts was assessed by immunoblotting. Results: Severe thermal injury resulted in a significant increase in intestinal permeability, which was associated with activation of p38 MAPK, increased expression of MLCK, and increased phosphorylation of MLC. Treatment with SB203580 significantly attenuated burn-induced intestinal permeability (203 g/ml vs. 94.5 g/ml, p ⬍ 0.05). Inhibition of p38 MAPK following burn decreased expression of intestinal MLCK by 73% (p ⬍ 0.02), resulting in decreased phosphorylation of MLC. Conclusion: p38 MAPK plays an important role in regulating burn-induced intestinal permeability through activation of MLCK. Therefore, inhibition of p38 MAPK may be an important therapeutic target aimed at attenuating intestinal barrier breakdown by preventing the burn-induced activation of tight junction proteins.
Introduction: Insulin resistance with its associated hyperglycemia represents one of the most significant contributors to mortality in burned and critically ill patients. While the metabolic pathophysiology is defined by increased glucose production with a decreased glucose uptake, the molecular mechanisms underlying insulin resistance are not well defined. The purpose of the present study was to determine the molecular mechanisms underlying hepatic insulin resistance. Materials and Methods: Rats received a 60% TBSA thermal injury and a laparatomy was performed at 24, 72 and 192 h post-burn. Liver was harvested before and 1 min after insulin injection (1 IU/kg) into the portal vein. PKR-like ER-Kinase (PERK), Inositol Requiring Enzyme (IRE)-1, activating transcription factor 6 (ATF-6), c-Jun N-terminal Kinase (JNK), Insulin Receptor Substrate (IRS)-1, Phosphatidylinositol 3-Kinase (PI3K), Protein Kinsae B (Akt), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Caveolin-1, phophorylated Caveolin-1 (pY14)were determined by Western blotting. Glucose-6-Phosphatase (G-6-P), Glucose Transporter (GLUT)-2 and ⫺4 were determined by real-time RT-PCR techniques at similar time points. Before insulin injections, serum was collected to determine fasting glucose and insulin levels by standard ELISA techniques. Results: Insulin resistance indicated by increased fasting serum glucose and insulin levels occurred starting 24 h post burn. Severe burn injury resulted in a profound activation of ER stress pathways, reflected by the increased accumulation of phospho-PERK, phospho-IRE-1 and ATF-6 leading to an elevation of phospho-JNK and serine phosphorylation of IRS-1. Insulin administration caused a significant increase in tyrosine phosphorylation of IRS-1 leading to activation of the PI3K/Akt pathway in normal liver. Post burn tyrosine phosphorylation of IRS-1 was significantly impaired (P⬍0.05) associated with an inactivation of signaling molecules acting downstream of IRS-1 (P⬍0.05). Conclusion: We found that dysregulation of signaling cascades deriving from ER stress may explain, at least partially, the metabolic abnormalities regarding insulin action following severe burn. The ER stress signaling cascades found in this study to be responsible for hepatic metabolic dysregulation following severe burn and inflammatory response pathways converge at the JNK/IRS-1/PI3K/Akt hub. Therefore, targeting of these molecules may provide a viable and efficacious alternative for the management of patients post burn. 92. UTILIZING PHAGE DISPLAY TECHNOLOGY TO IDENTIFY PEPTIDE SEQUENCES TARGETING THE BURN INJURED INTESTINAL BARRIER. T. W. Costantini, C. Y. Peterson, J. G. Putnam, R. Sawada, W. H. Loomis, B. P. Eliceiri, A. Baird, V. Bansal, R. Coimbra; University of California San Diego School of Medicine, San Diego, CA Background: Severe burn injury results in intestinal barrier dysfunction which may be responsible for significant morbidity and mortality. Therefore, it would be ideal to directly target the gut with biotherapeutics to either prevent barrier breakdown, or rapidly promote barrier restoration. Phage display is a molecular technique used to identify peptides that can home drugs, proteins, and particles to specific cell populations within tissues. We postulated that mining a peptide library that was displayed on phage would identify peptide sequences that bind and internalize into the gut epithelium. Using this technique, we hoped to identify a platform for the delivery of biotherapeutics capable of modulating intestinal dysfunction following severe burn. Methods: Two hours following 30% total burn surface area (TBSA) steam burn, male balb/c mice were sacrificed and the distal ileum harvested. The intestinal mucosa was isolated and incubated in complete DMEM culture media with 10 12 phage of a phage library (NEB12) containing 10 9 different peptide sequences.