Despite remarkable advances in wound healing, mortality and morbidity of severe burn patients (30%–40% total body surface area) remain poor because of the inflammatory and hypermetabolic responses (1–4). Burn-induced inflammation and hypermetabolism are characterized by increased protein turnover and degradation to meet metabolic demands, loss of lean body mass, compromised function of essential organs, hyperglycemia, and insulin resistance (5).
Burn-induced hepatic damage and dysfunction worsen morbidity and delay recovery (6). Previous research has shown that severe burn impairs hepatocyte structure and function and leads to cell death, by both apoptosis and necrosis (7–9). In other disease models, liver damage has been linked to insulin resistance and inflammation (10). The liver may be influencing survival and recovery by modulating the systemic responses to thermal injury; therefore, it is crucial that we decipher the hepatic molecular mechanisms.
The source of hepatic damage postburn is likely the endoplasmic reticulum (ER). Endoplasmic reticulum stress is induced when there is an excess of unfolded proteins and is elevated in rodent models of severe burn injury (8). Hepatic ER stress is an important mediator of insulin resistance, inflammation, and apoptosis (11–13). In vitro and in vivo studies by Ozcan et al. (11) demonstrated that ER stress mediates peripheral insulin resistance and type 2 diabetes at the molecular, cellular, and organism levels. Endoplasmic reticulum stress leads to apoptosis through either IRE1α activation and/or calcium release (14, 15).
c-Jun N-terminal kinase (JNK) proteins are activated downstream of ER stress (16). After phosphorylation of IRE1 and activation of TRAF2 (16), activated JNK leads to serine phosphorylation of IRS1, which reduces insulin receptor signaling (11). Increased JNK activation by treatment with palmitic acid, a saturated fatty acid, leads to insulin resistance in primary mouse hepatocytes (17). JNK inhibition 1 h after smoke inhalation improved mouse survival by preventing inflammatory cell infiltration, cytokine release, and airway apoptosis (18). Specifically, JNK activates proapoptotic Bim and inactivates antiapoptotic Bcl2 proteins (19). In the context of burn injury, rapid JNK activation has been observed in the liver and cardiac tissue (13, 20, 21). Activation in the cardiac tissue could be replicated by treatment with α1-adrenergic agonism, but the consequences of JNK phosphorylation were not explored (20). In summary, JNK proteins have been linked to insulin resistance, inflammation, and apoptosis in other disease settings; however, JNK’s role in burn has not been thoroughly defined.
The functions of JNK1, JNK2, and JNK3 differ; thus, it is critical to study them separately. JNK1 phosphorylates c-Jun and has been associated with increasing insulin sensitivity (22, 23). Inhibitor studies in models of obesity and nonalcoholic fatty liver disease (NAFLD) have revealed that JNK1 mediates development of obesity, insulin resistance, steatosis, hepatitis, inflammation, apoptosis, and liver injury (23, 24). Hepatic-specific knockdown of JNK1 reduces serum insulin and glucose in obese mice but increases glucose intolerance and insulin resistance in a model of NAFLD (25, 26). Less information is available regarding JNK2’s role. JNK2 may actually block c-Jun phosphorylation (22). JNK2−/− NAFLD studies suggest a role in mediating insulin resistance and steatohepatitis (23). JNK3 is primarily found in the brain and is involved in ischemic apoptosis (27).
The objective of this study was to examine JNK2’s role in mediating hypermetabolism, inflammation, and apoptosis after burn.
MATERIALS AND METHODS
The study protocol was approved by the Institutional Animal Care and Use Committee of Texas Medical Branch at Galveston. The Guide for the Care and Use of Laboratory Animals (1996) of the National Academy Press was met. C57BL/6 wild-type mice and JNK2 knockout mice (20–30 g) were purchased from Harlan (Houston, Tex) and housed for 1 week before the experiments.
Reverse transcription–polymerase chain reaction (PCR) was performed to confirm the genotype of the animals. Total RNA was isolated from liver samples, quantified, and reverse transcribed. Wild-type JNK2 was identified with primers 5′-GGA GCC CGA TAG TAT CGA GTT ACC-3′ and 5′-GTT AGA CAA TCC CAG AGG TTG TGT G-3′. Mutant (knockout) JNK2 was identified with primers 5′-GGA GCC CGA TAG TAT CGA GTT ACC -3′ and 5′-CCA GCT CAT TCC TCC ACT CAT G-3′ (Jackson Laboratory, Bar Harbor, Me). Cycling parameters included one cycle of 94°C for 5 min then 35 cycles of 94°C for 30 s, 52°C for 1 min, and 72°C for 1 min followed by one cycle of 72°C for 5 min.
A 30% total body surface area burn was induced as previously described (8). Briefly, animals were anesthetized (80 mg/kg ketamine, 7.5 mg/kg xylazine), shaved along the dorsum, and administered saline subcutaneously along the spinal cord (0.9%, 1 mL). Mice were placed in a mold exposing a defined area of skin on their back and then lowered into 96°C to 98°C water for 10 s to induce a full-thickness scald burn. Lactated Ringer’s solution (2 mL) was administered for resuscitation and buprenorphine (0.1 mg/kg) for analgesia. Similar to burned mice, sham mice were treated with anesthesia, saline, Ringer’s solution, and analgesia but were not lowered into a water bath.
Thermal injury was induced in wild-type and JNK2−/− mice at day 0. Animals were killed 1, 3, and 5 days after burn, and liver and serum samples were collected. There were five to six mice per group per time point.
Serum protein analysis: multiplex
Serum concentrations of 11 cytokines and hormones were measured using a Linco multiplex bead array system (St Charles, Mo) and MiraiBio software package (Hitachi, San Francisco, Calif). These included insulin, leptin, glucagon, GLP-1, monocyte chemoattractant protein 1, interleukin 6 (IL-6), total plasminogen activator inhibitor 1, resistin, amylin, peptide YY, and pancreatic polypeptide.
Hepatic protein analysis: Western blotting
Immediately after the animals were killed, liver tissue was snap frozen in liquid nitrogen and stored at −80°C. To isolate protein, approximately 100 mg of tissue was homogenized in a RIPA lysis buffer (containing protease and phosphatase inhibitors) and centrifuged. Protein concentration was quantified using a Pierce BCA assay kit (Thermo Scientific, Waltham, Mass). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were used to analyze 30 μg of each protein sample. Band intensities were analyzed with ImageJ software, with β-actin as the loading control.
The antibody against mouse ATF6 was purchased from Imgenex (San Diego, Calif). Anti-GRP78/Bip antibody was purchased from Abcam (Cambridge, Mass). β-Actin, Bcl2, FoxO1 (forkhead box O 1), and phosphorylated (p) FoxO1 were purchased from Cell Signaling (Beverly, Mass).
Gene expression: quantitative real-time PCR
Total RNA was isolated from liver tissue following manufacturer’s instructions (RNeasy Mini Kit; Qiagen, Hilden, Germany), quantified and reverse transcribed (Applied Biosystems, San Diego, Calif). Real-time PCR for XBP1spliced, Pdia3, Dnajb9, IL-6, and albumin expression was performed on cDNA with the housekeeping gene rRNA 18S.
Hepatic apoptosis: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
Hepatic apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). To prepare samples, liver tissue was isolated, formalin-fixed, paraffin-embedded, and sectioned at 5 μm each. We used a DeadEnd TUNEL fluorometric system from Promega (Madison, Wis). Briefly, the DeadEnd fluorometric TUNEL system detects fragmented DNA of apoptotic cells by incorporating fluorescein-12-dUTP (green) at the 3′-OH ends of the DNA strands using the enzyme terminal deoxynucleotidyl transferase (recombinant), which forms a polymeric tail. We used propidium iodide (red) as a nuclear counterstain. We then imaged the staining with a confocal microscope (Carl Zeiss Microscopy, Jena, Germany) and quantified apoptotic cells per centimeter squared of tissue.
Liver injury: aspartate aminotransferase and alanine aminotransferase activity assays
Liver injury was assessed by quantifying serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). BioVision AST and ALT activity assay kits were used (San Diego, Calif).
Data are presented as mean ± SEM. Comparisons between groups and time points were made with a two-way analysis of variance and Bonferroni correction was performed when differences between means were found (GraphPad Prism 5, version 5.0a, San Diego, Calif). Differences were considered significant when P < 0.05.
Figure 1 confirms the genotype of JNK2−/− and wild-type mice. No mortality was observed in wild-type or JNK2−/− mice. In the majority of parameters measured, no significant differences were found between sham wild-type and sham JNK2−/− mice. Trends that were observed are described below.
Endoplasmic reticulum chaperones were activated after burn injury in the liver of wild-type and JNK2−/− (Fig. 2, A and B). Western blots of Bip showed a slight increase, and blots of ATF6 demonstrated a significant increase at day 1 in both strains (P < 0.001 wild-type burn and P < 0.05 JNK2−/− burn vs. sham). Downstream targets of ER stress, spliced XBP1 and Dnajb9, were significantly elevated 5 days after burn in wild-type mice (P < 0.05 vs. sham) but not in JNK2−/− mice (Fig. 2, C and E). Pdia3 transcription was moderately—but not significantly—elevated in wild-type mice but not in JNK2−/− mice (Fig. 2D). Interestingly, the basal levels of downstream ER stress markers appeared slightly reduced in sham JNK2−/− mice compared with sham wild-type mice.
We performed a peritoneal glucose tolerance test, but there were no differences evident between groups (data not shown). Serum insulin was significantly elevated 5 days after burn in wild-type mice (P < 0.05, day 5 vs. sham) but not in JNK2−/− (Fig. 3A). Sham JNK2−/− mice displayed a slight (nonsignificant) increase in insulin compared with sham wild-type mice. Serum leptin decreased in both strains at 3 and 5 days after burn (P < 0.01, Fig. 3B). Interestingly, resistin did decrease in both strains but returned to sham levels later in JNK2−/− than in wild-type mice, at day 5 instead of day 3 (Fig. 3C). The other hormones measured were not significantly different between groups (data not shown).
Inflammatory cytokine IL-6 increased (nonsignificantly) in the serum of both wild-type and JNK2−/− (Fig. 4A). On the other hand, IL-6 gene expression in the liver peaked only in wild-type mice (P < 0.01, day 5 wild-type vs. JNK2−/− and vs. sham); IL-6 mRNA levels did not increase in JNK2−/− (Fig. 4B). The other cytokines and adipokines measured did not display significant differences between groups (data not shown).
TUNEL staining indicated that hepatic apoptosis was prevented in JNK2−/− (Fig. 5, A and B). In hepatic tissue of wild-type mice collected 5 days after burn, TUNEL positive staining increased significantly (P < 0.01 vs. sham). No TUNEL staining was detected in hepatic tissue of JNK2−/− 5 days after burn. Consistent with this, prosurvival (antiapoptotic) signaling in the liver was higher when JNK2 is knocked out. In wild-type mice, FoxO1 phosphorylation significantly decreased 3 days after burn (Fig. 5C). In JNK2−/−, there was no significant change in FoxO1 phosphorylation after burn injury; however, lower FoxO1 levels were observed in sham JNK2−/− than sham wild-type mice. In JNK2 knockout mice, Bcl2 levels were increased at day 1 (P < 0.05) compared with wild-type mice (Fig. 5D).
Liver injury was examined by measuring AST and ALT activities in the serum before and 1 and 3 days after burn (Fig. 6). Both AST and ALT activities peaked on day 1 in both strains. Knockout of JNK2 lowers day 3 AST activity and day 1 ALT activity.
Liver function was evaluated by measuring albumin production in the liver. At 5 days after burn, albumin synthesis in the liver (indicated by mRNA expression, Fig. 6C) was reduced in JNK2−/− compared with wild-type mice (P < 0.05).
The overall objective of this study was to examine the role of JNK2 activation in the hypermetabolic, inflammatory, and apoptotic response after a severe thermal injury. We hypothesized that JNK2 contributes predominantly to the apoptotic response after burn injury downstream of ER stress. This study indicates that JNK2 may influence hypermetabolism, does contribute to inflammation, and is a central mediator of hepatic apoptosis after a severe burn.
First, we confirmed the genotype of the JNK2−/− and confirmed that JNK2 activation is downstream of chaperone activation during the hepatic unfolded protein response. Figure 1 confirms the knockout genotype, similar to that used in a study by Singh et al. (23), who were studying NAFLD. Figure 2 indicates increased hepatic ER stress (upstream chaperone activation and downstream effector transcription) after thermal injury in wild-type mice. The burned JNK2−/− mice also demonstrated ER chaperone activation compared with shams. Consistent with this, ER stress has previously been described upstream of JNK2 activation in obesity and ER stress studies (11, 16). In contrast, transcription of downstream effectors of ER stress (XBP1s, Pdia3, and Dnajb9) was unaffected by burn injury in the JNK2−/− mice. Previous models of the ER stress response illustrate XBP1s and JNK2 activation in parallel (28); however, our results strongly indicate that JNK2 activation is upstream of XBP1 splicing. These data suggest that the ER stress pathway is interrupted by JNK2 knockout.
Our second aim was to compare the metabolic response to a severe burn in wild-type and JNK2−/− (Fig. 3). Primary mouse hepatocytes treated with palmitic acid exhibit sustained JNK activation and insulin resistance (17). In JNK2 knockout mice fed a high-fat diet, obesity and insulin resistance remained (23). On the other hand, knockdown of JNK2 using antisense oligonucleotides prevented insulin resistance (23). As shown in Figure 3, burned JNK2 knockout mice fail to exhibit elevated insulin after burn injury relative to sham JNK2 knockouts, indicating that JNK2 may contribute to hyperinsulinemia in the postburn stress response. On the other hand, insulin levels increased slightly (but not significantly) in JNK2 knockout shams compared with wild-type shams. An alternative interpretation of these data is that the JNK2 knockouts do not exhibit postburn hyperinsulinemia because they start with higher insulin levels; this is difficult to be certain of, however, given that the elevation with JNK2−/− was not significant.
Next, we evaluated the influence of JNK2 on inflammation. After smoke inhalation, administration of a JNK inhibitor prevented the influx of inflammatory cells and cytokine release and significantly prolonged animal survival (18). In our analysis, the serum cytokine levels did not differ between wild-type and JNK2−/−; however; the peak in synthesis of IL-6 in the liver was entirely prevented after JNK2 knockout (Fig. 4). These data suggest that JNK2 has a strong influence on postburn IL-6 production in the liver but not on IL-6 production in other tissues, given that serum IL-6 does not differ between strains. This study focused on the liver postburn, but we are eager to study IL-6 production in adipose and muscle tissue in future research.
Finally, we examined hepatic apoptosis, injury, and function (Figs. 5 and 6). Apoptosis has been noted after severe burn injury in several studies in the liver, heart, and gastrointestinal tract (7, 8, 29). After JNK2 knockout, we observed a remarkable elimination of TUNEL-positive hepatocytes (Fig. 5, A and B). JNK induces apoptosis when the PI3K pathway is inhibited, either by stress or inhibitor (30). Knockout of JNK2 in a mouse model of LPS-induced toxic liver injury also resulted in significant reduction of apoptosis (31). JNK protein kinases have been implicated in deactivation of Bcl2, a mitochondrial antiapoptotic protein (19). After burn injury, Bcl2 activation is elevated in wild-type mice but even more so in JNK2 knockout mice (Fig. 5D).
The transcription factor FoxO1 is a key regulator of cell survival and mediator of insulin action. Dephosphorylation of FoxO1 activates and shuttles it from the cytoplasm to the nucleus where it upregulates proapoptotic and gluconeogenic genes (32). Our data indicate that, after burn injury in wild-type mice, phosphorylated FoxO1 decreased significantly compared with sham mice; however, phosphorylated FoxO1 did not change significantly after burn in JNK2 knockouts (Fig. 5C). This suggests that JNK2 may be mediating apoptosis through FoxO1 dephosphorylation.
Caspase 3 activity was not affected by knockout of JNK2 in this model (data not shown). Instead, JNK2 may contribute to apoptosis through a mitochondrial pathway. We have previously described severe mitochondrial swelling and damage after burn (33). Specifically, JNK2 may contribute to hepatocellular injury and death via mitochondrial permeability transition, as observed in an ischemia/reperfusion study by Theruvath et al. (34). This would be interesting to confirm in a follow-up study.
Liver structure and function are damaged after burn-induced hepatic apoptosis (7, 8). Hepatic damage has been linked to insulin resistance in the setting of streptozotocin-induced diabetes and to inflammation in the setting of acute-phase inflammation (10). In our model of burn injury, both AST and ALT were elevated in serum of burned wild-type mice (Fig. 6, A and B). Knockout of JNK2 lowers day 3 AST activity and day 1 ALT activity, indicating reduced liver damage. JNK2 knockout in the toxic liver injury model produced the same reduction in ALT activity (31). In a mouse model of burn, we previously demonstrated that liver-secreted albumin decreased, indicating liver dysfunction (8). In this study, albumin synthesis in the liver (indicated by mRNA expression, Fig. 6C) is reduced in JNK2−/− compared with wild-type mice.
A natural follow-up study to this one will investigate the role of JNK1 in severe burn injury. Interestingly, in the obesity study of Singh et al. (23), knockout or knockdown of JNK1 indicated that the two JNK isoforms have distinct effects on hepatocyte cell death and inflammation. Another interesting follow-up study would be to investigate the relationship between hepatic ER stress and mitochondrial dysfunction and to test a novel mitochondria-targeted antioxidant peptide, SS-31, which has been shown to reduce postburn ER stress and apoptosis in skeletal muscle (35).
To summarize, JNK2 may influence hypermetabolism, does contribute to inflammation, and is a central mediator of hepatic apoptosis after a severe burn. JNK2 may be an important therapeutic target to interrupt and prevent hepatic apoptosis and damage. If we alleviate hepatic damage, we may be able to maintain proper hepatic function, which could improve insulin sensitivity and prevent inflammation. Any effort to reduce hypermetabolism and inflammation will no doubt improve morbidity and mortality of severely burned patients.
The authors acknowledge the SRI Genomics Core Facility for genotyping the samples.
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