Stress-induced diabetes, a hallmark of burn, trauma, and critical illness, worsens clinical outcomes by increasing morbidity and mortality.9–11
Despite insulin resistance and hyperglycemia being well-documented pathophysiologic responses in severely burned and critically ill patients, the underlying mechanisms are poorly understood. Our study is the first to identify cellular and molecular alterations in burned patients allowing development of novel treatments to attenuate hyperglycemia and insulin resistance.
Burn injury induces a vast inflammatory response, which leads to cellular stress responses in many tissues—even those not directly burned. Recently, ER stress and the UPR were identified as central intracellular stress signaling pathways associated with inflammation.13,15
More importantly, in a model of type 2 diabetes, ER stress and UPR have been shown to be causally linked with hyperglycemia and insulin resistance,12,16
It, therefore, seems that ER stress and UPR are central for the development of insulin resistance and hyperglycemia postburn. We have recently reported in animal models that burn induces marked ER stress and UPR leading to impaired insulin receptor (IR) signaling via activation of JNK resulting in insulin resistance and hyperglycemia.18,23
The aim of the present study was to determine whether ER stress and UPR are present in humans who suffered a severe burn injury. It is well documented that glucose kinetics are almost always abnormal in burn patients, with glucose utilization occurring predominantly via inefficient anaerobic mechanisms as characterized by increased lactate production, which accounts for increased glucose consumption.4,24–26
Plasma insulin levels are markedly increased over a prolonged period after a severe burn injury along with increased glucose levels.3,5,6
The fact that the rate of basal glucose production is elevated despite elevated plasma insulin levels indicates insulin resistance, because under normal conditions elevated serum insulin would lower the rate of glucose production.4,27,28
As mentioned previously, the importance of hyperglycemia and insulin resistance is that both worsen the outcomes of severely burned patients.9–11,29
It is well known that many stress hormones can increase glucose levels, including glucagon, glucocorticoid, adrenalin, and so on. All these hormones are significantly increased by severe burn injury. There is thus the possibility that insulin increased secondary to the dramatically increased glucose and that the glucose level still remains high because of a relatively insufficient insulin concentration.
In this study, we showed for the first time that a trauma, severe burn in our case, induces systemic ER stress and UPR in humans. We determined the genomic changes in peripheral blood leukocytes, fat, and muscle postburn and compared these changes to the same tissues from normal, healthy, nonburned volunteers. Genomic analysis revealed that burn injury activated ER stress and UPR in blood, fat, and muscle for up to 1 year postburn. The genomic expression pattern was not identical in blood, fat, and muscle, indicating some tissue-specific responses, but in all 3 tissues, we found that major components of the ER stress and UPR were activated. These findings indicate that ER stress and UPR is not limited to one tissue type but is present in many, if not all, organs and tissues postburn.
The ER is recognized as the site of protein synthesis and folding of secreted and membrane-bound proteins.15
Protein folding is essential for cell function and cell survival, and the presence of an excess of misfolded proteins results in the activation of signaling pathways to restore homeostasis.15
Calcium depletion from ER stores leads to increased unfolded and/or misfolded proteins in the ER lumen. This results in the activation of the UPR.15
The accumulation of unfolded protein is detected by the cell via
3-key ER transmembrane receptors, protein kinase RNA (PKR)-like ER kinase (PERK), IRE-1, and ATF6. In resting cells, all 3 ER receptors are maintained in an inactive state. On accumulation of unfolded proteins, GRP78/BiP (an ER chaperone) dissociates from the receptors, which leads to their activation and triggers the UPR. The UPR is a prosurvival response that reduces the accumulation of unfolded proteins and restores normal ER function.15
However, if protein aggregation is persistent and the stress cannot be resolved, signaling switches from prosurvival to proapoptotic and protein synthesis decreases and catabolism occurs. In the present study, we were able to show that burn injury induces proapoptotic and calcium signaling pathways, indicating the enormous stress response postburn.18
The link between calcium signaling and ER stress seems important and will require further elucidation in future studies. We have shown that there appears an association between the two.
An intriguing result is the strong, coordinated downregulation of caspases in muscle. It does not seem to fit with the hypothesis of induced stress/apoptosis in burn patients. We hypothesize that this is a compensatory mechanisms to attenuate the vast muscle catabolism that occurs postburn and persists for 3 years. We hypothesize that the body adapts to the stress and tries to preserve as much muscle mass as possible, thus decreasing caspase activation.
It is currently unknown by which exact mechanisms ER stress and UPR are induced, but several studies suggest that proinflammatory cytokines (IL-6, IL-8, MCP-1, or TNF), metabolic stress (hypermetabolism), and stress hormones (cortisol, catecholamines) activate ER stress and UPR.14
In the present study, and in a large clinical study from our group, we showed that a burn injury is associated with dramatically increased proinflammatory cytokines, hypermetabolism, and release of stress hormones, all of which could activate ER stress and UPR postburn.3
The mechanisms by which ER stress and UPR induces insulin resistance was recently identified by Ozcan and colleagues.12
They showed in a type 2 diabetic mouse model that ER stress leads to suppression of insulin receptor signaling through hyperactivation of JNK and subsequent serine phosphorylation of IRS-1.12
The authors concluded that ER stress is a central feature of peripheral insulin resistance and type 2 diabetes at the molecular, cellular, and organismal levels.12
Phosphorylation of JNK leads to serine phosphorylation of the insulin receptor inhibiting the essential tyrosine phosphorylation of the insulin receptor and PI3K signaling resulting not only in insulin resistance and hyperglycemia but also in increased cell stress and apoptosis.30
In the present study, we have shown on both a transcriptome and protein level that burn activates proinflammatory and metabolic mediators, key indicators of ER stress/UPR, and JNK, the interactions of which result in vast insulin resistance and hyperglycemia. We furthermore confirmed that burn decreases prosurvival PI3K/Akt signaling, and consequently alters apoptosis and proapoptotic calcium signaling pathways. These results confirm our recent study in burned rats demonstrating that a severe burn causes ER stress and UPR and gross alterations in ER calcium with increased cytosolic calcium concentrations.18
Increased cytosolic calcium induces mitochondrial dysfunction and damage, leading to apoptosis and tissue dysfunction. Here, we demonstrate the relationship between systemic postburn inflammation and stress-induced diabetes with alterations in tissue-specific molecular signaling pathways related to ER stress, UPR, insulin signaling, and insulin resistance.
Limitations of this study need to be mentioned and are severalfold. This study provides a link between ER stress and insulin resistance; however, we lack direct evidence. However, this study was setup to determine whether ER stress and UPR occurs in humans after burn injury, and we feel that we have shown that these responses do occur. This will allow further studies to determine cause-effect relationships and potential therapeutic targets for severely burned patients. In addition, we believe that this study may have implications for other forms of trauma patients as similar responses occur in these patients. Thus, this study showing the presence of ER stress and UPR for up to 1 year posttrauma has importance as it may open new therapeutic windows. New therapeutic options to be tested in in vitro and in vivo models may include established clinical perturbations or novel agents such as chemical chaperones. Another limitation is associated with the use of GeneChip analysis. We found 455 genes in peripheral blood leucocytes, 360 genes in fat, and 448 genes in muscle that were significantly changed. This is a high number of genes, because we used a lower cutoff and higher P value for discovery purposes to screen for novel genes. However, this discovery genomics is associated with false detection and may be responsible for an “artificial” enrichment of deregulated genes within the selected pathways.
In summary, we showed that a severe thermal injury induces vast insulin resistance and hyperglycemia, which our data indicate are associated with the systemic induction of ER/SR stress and the UPR. Furthermore, we demonstrated that ER stress and UPR occurs in peripheral blood leukocytes, fat, and muscle, and also that alterations of these pathways persist for up to 1 year postburn after a severe burn injury. ER stress and UPR alters JNK, PI3K/Akt, calcium, and apoptosis signaling pathways leading to insulin resistance and cellular dysfunction postburn. We suggest that ER stress and UPR is induced by inflammation, hypermetabolism, and release of stress hormones as reported in this study. This study is the first to describe these pathologic phenomena in severely traumatized humans.