burns 41 (2015) 1076–1085
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Selective decontamination of the digestive tract ameliorates severe burn-induced insulin resistance in rats Jun Li a,b,1, Liang Zhu c,1, Ming Xu a,1, Juntao Han b, Xiaozhi Bai b, Xuekang Yang b, Huayu Zhu b, Jie Xu a,d, Xing Zhang a,d, Yangfan Gong b, Dahai Hu b,*, Feng Gao a,d,* a
Department of Physiology, School of Basic Medical Sciences, Fourth Military Medical University, Xi’an, China Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China c Department of Medical Education, Fourth Military Medical University, Xi’an, China d Department of Aerospace Medicine, Fourth Military Medical University, Xi’an, China b
article info
abstract
Article history:
Background: Severe burns often initiate the prevalence of hyperglycemia and insulin resis-
Received 7 July 2014
tance, significantly contributing to adverse clinical outcomes. However, there are limited
Received in revised form
treatment options. This study was designed to investigate the role and the underlying
22 December 2014
mechanisms of oral antibiotics to selectively decontaminate the digestive tract (SDD) on
Accepted 25 December 2014
burn-induced insulin resistance. Materials and methods: Rats were subjected to 40% of total body surface area full-thickness burn or sham operation with or without SDD treatment. Translocation of FITC-labeled LPS
Keywords:
was measured at 4 h after burn. Furthermore, the effect of SDD on post-burn quantity of
Burn
gram-negative bacteria in gut was investigated. Serum or muscle LPS and proinflammatory
Insulin resistance
cytokines were measured. Intraperitoneal glucose tolerance test and insulin tolerance test
SDD
were used to determine the status of systemic insulin resistance. Furthermore, intracellular
Proinflammatory cytokine
insulin signaling (IRS-1 and Akt) and proinflammatory related kinases (JNK and IKKb) were
LPS
assessed by western blot. Results: Burn increased the translocation of LPS from gut 4 h after injury. SDD treatment effectively inhibited post-burn overgrowth of gram-negative enteric bacilli in gut. In addition, severe burns caused significant increases in the LPS and proinflammatory cytokines levels, activation of proinflammatory related kinases, and systemic insulin resistance as well. But SDD treatment could significantly attenuate burn-induced insulin resistance and improve the whole-body responsiveness to insulin, which was associated with the inhibition of gut-derived LPS, cytokines, proinflammatory related kinases JNK and IKKb, as well as activation of IRS-1 and Akt. Conclusions: SDD appeared to have an effect on proinflammatory signaling cascades and further reduced severe burn-induced insulin resistance. # 2015 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding authors at: Department of Burns and Cutaneous Surgery, Xijing Hospital; Department of Physiology and Department of Aerospace Medicine, Fourth Military Medical University, Xi’an 710032, China. Tel.: +86 29 84775297/+86 29 84776423; fax: +86 29 83246270. E-mail addresses: [email protected] (D. Hu), [email protected] (F. Gao). 1 These authors were co-first authors and contributed equally to this work. http://dx.doi.org/10.1016/j.burns.2014.12.018 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.
burns 41 (2015) 1076–1085
1.
Introduction
Insulin resistance and hyperglycemia are frequent phenomena in severe burn patients, which have been recognized as the hallmarks of burn-induced diabetes [1,2]. More recently, studies have showed the close relationship between adverse clinical outcomes and impaired glucose metabolism in these patients, who have a significantly higher incidence of bacteremia or fungemia infection, metabolic disorder and mortality [3]. Conversely, controlling hyperglycemia or alleviating insulin resistance is associated with improved outcomes [4]. Insulin resistance can be defined as a state of reduced response of peripheral target tissues to insulin stimulation [5]. It often occurs in chronic diseases, such as type 2 diabetes and obesity, and normally takes months or years to develop [6]. However, recent findings have revealed that acute insulin resistance exists in critically ill like burns, characterized by a rapid onset of uncontrolled hyperglycemia within hours or days [7]. Until now, the precise mechanisms involved in it are poorly understood, thus lacking effective treatments and prevention strategies. Severe burn injury usually induces a distinct systemic inflammatory response that has been well described previously [8,9]. Proinflammatory cytokines are the primary mediators of this inflammatory reaction. The levels of various cytokines such as interleukin 6 (IL-6) [10] and tumor necrosis factor a (TNF-a) [11] changes in early stage and have been used as markers of the severity of burns. Emerging data indicate that some cytokines play an important role in regulating glucose metabolism, and the excessive activation of proinflammatory related pathways may represent a fundamental step in the development of insulin resistance [12,13]. In addition, alterations in cytokine levels often occur prior to metabolic abnormalities in severe burns [9]. Whatever the mechanisms involve, it has now been demonstrated that cytokines are able to negatively influence insulin signaling in insulin-responsive tissues [14]. Early modulating cytokines releasing and proinflammatory related kinases activation may represent a feasible strategy for acute insulin resistance and subsequent hyperglycemia. Bacterial and endotoxin (lipopolysaccharide, LPS) invasion from the digestive tract are closely related to an increased incidence of sepsis and multiple organ failure in burned patients [15,16]. Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents, and promotes a generalized inflammatory state that may lead, via cytokinemia, to organ damage [17]. Selective decontamination of the digestive tract (SDD) is conducted by oral administration of non-absorbable, small-spectrum antibiotics to eliminate gram-negative bacillus from the digestive tract, and it has been widely used to prevent gut-derived infection in critically ill patients, such as pancreatitis, liver transplantation, and trauma. Many randomized controlled trials of SDD have been published. Although some of the evidence supporting the use of SDD, the opponents still rely on historical arguments against its use, such as the bacterial resistance and the absence of influence on mortality [18]. The predominant concern was that use of SDD will promote the development of antimicrobial-resistant pathogens. Recently, a meta-analysis
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detected no relationship between the use of SDD and the antimicrobial resistance. However, the authors acknowledged the need for research over a longer term [19]. Despite the controversies, SDD has showed beneficial effect on mortality in patients with severe burns [20,21]. Moreover, SDD applied either before or after burn injury in rodents showed remarkable cardioprotection by suppression of myocardial inflammation [22]. Given gut-derived pathogens are one of the primary mechanisms for systemic inflammatory responses in burns, SDD application would be expected to attenuate proinflammatory cytokines release and subsequent intercellular signaling cascade activation, and thus improve insulin signaling. However, the efficacy of SDD on insulin resistance after severe burns has not been evaluated yet. In this study, we hypothesized that gut-derived endotoxin could mediate excessive inflammatory reaction and then provoke insulin resistance, but the application of SDD would reduce endotoxin translocation from the gastrointestinal tract, inhibit systematic proinflammatory cytokines releasing and subsequent intercellular signaling cascade, which ultimately ameliorate acute insulin resistance.
2.
Materials and methods
2.1.
Animals
Sprague-Dawley male rats (200–250 g) were obtained from the Center of Experimental Animals of the Fourth Military Medical University. Rats were housed in an environmentally controlled room with a 12 h light/dark cycle, with free access to regular rat chow and water. All procedures in this study were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University, in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
2.2.
Burn procedure and SDD therapy
Rats were anesthetized with 2% pentobarbital (40 mg/kg i.p.). After clipping of the hair, a 40% total body surface area (TBSA) full thickness third degree burn injury was produced by immersing the back of trunk in 100 8C water for 12 s. Weight and age matched rats were immersed in room temperature water as sham group. All rats received Lactated Ringer resuscitation after burn injury (4 ml/kg/% burn). Oral antibiotic therapy for SDD was carried out as described previously [22]. Briefly, water mixture (final volume 3 ml) of polymyxin E (15 mg), tobramycin (6 mg), and 5-flucytosin (100 mg) (Sigma-Aldrich, St Louis, MI, U.S.) were given 4 h after burn by oral gavage and twice daily afterward for 5 days. Animals given the same volume of water at the same time intervals were set as vehicle treatment.
2.3.
Translocation of FITC-labeled LPS after burn
Rats were orally gavaged with FITC-labeled LPS (Sigma, St Louis, MO, USA) 3 mg/kg dissolved in deionized water
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immediately after burn or sham operation. Both portal and systemic blood were collected at 4 h after burn injury. Serum was separated by centrifugation at 4500 rpm for 3 min at 4 8C. FITC fluorescence was measured by using a fluorescence spectrophotometer at an excitation of 485 nm and an emission of 530 nm. The concentration of FITC in plasma was expressed as mg/ml of plasma.
2.4. Analysis of gram-negative bacteria in cecum after SDD therapy Rats were randomly divided into burn with vehicle and burn plus SDD groups. The animals were sacrificed before burn injury and at 1, 3 and 5 days post-bum (n = 6 rats per group at each time point). The cecum was excised under aseptic conditions, and the cecal content weighing 0.1 g was collected, homogenized, and serially diluted with saline. The homogenates from cecum were inoculated into MacConkey agar designed to selectively grow gram-negative bacteria. After 24 h of incubation at 37 8C, colonies were identified and counted. The bacterial counts were expressed as the log10 of the total number of viable bacteria per gram weight of cecal content.
2.5. Experimental protocols for post-burn insulin resistance Rats were randomized into four groups: (1) sham with vehicle (3 ml water), (2) sham plus SDD, (3) burn with vehicle, and (4) burn plus SDD. Animals were sacrificed at 1, 3, 5 and 7 days post-burn (n = 6 rats per group at each time point). Blood samples and skeletal muscles were collected for further analysis. In addition, insulin resistance was evaluated at 3 days post-burn by intraperitoneal glucose tolerance test or insulin tolerance test, respectively.
2.6.
Serum endotoxin (LPS) determination
Blood samples from each group were collected, and serum was separated by centrifugation at 4500 rpm for 3 min at 4 8C and stored at 70 8C until analyzed. LPS was measured using a commercially available kit following the manufacturer’s instructions (Hycult Biotech, The Netherlands). Results were expressed as endotoxin units (EU) per ml (EU/ml).
2.7.
Serum proinflammatory cytokines determination
Serum cytokines IL-1b, IL-6 and TNF-a were determined by the rat Bio-Plex suspension array (Bio-Rad, Hercules, CA, U.S.) at 1, 3, 5, or 7 d after burn or sham operations. In brief, blood samples from each group were collected, and serum was separated by centrifugation at 4500 rpm for 3 min at 4 8C and stored at 70 8C until analyzed. The supernatant was collected and incubated with microbeads labeled with specific antibodies to one of the mentioned cytokines for 30 min. After washing with PBS, samples were then incubated with the detection antibody cocktail, followed by another wash step. Collecting the beads by centrifugation, the samples were incubated with streptavidin–phycoerythrin for 10 min, washed, and the concentration of each cytokine was determined by optical density, which was measured in a microplate
reader (TECAN Austria GmbH, Grodig, Austria) set to 450 nm wave lengths.
2.8.
Assay of proinflammatory cytokines in muscles
0.1–0.3 g frozen skeletal muscles were homogenized in RIPA buffer (0.625% sodium deoxycholate, 0.625% Nonidet P-40, 6.25 mM sodium phosphate, and 1 mM EDTA at pH 7.4) containing 10 mg/ml of a protease inhibitor cocktail (SigmaAldrich, St. Louis, MO). Homogenates were centrifuged at 12,000 g for 10 min at 4 8C, the supernatant was saved, and protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a reference. The protein levels of IL-1b, IL-6, and TNF-a in muscles were measured in duplicate by using commercially available rat ELISA kits (DuoSet ELISA, R&D Systems, Minneapolis, MN). The assays were carried out according to the manufacturer’s instructions. Cytokine data of muscle are expressed as pg of cytokine per mg of total protein.
2.9.
Intraperitoneal glucose tolerance test (IPGTT)
Previous reports had indicated that insulin resistance was clearly observed at 2–7 days after burn injury [23,24]. Thus, we chose 3 days as the time point for analysis of insulin resistance. In brief, IPGTT were performed following an overnight fast period. The animals were administered an intraperitoneal injection of glucose (2 g/kg body weight). The blood glucose was measured from tail-tip blood at 0, 30, 60, 90 and 120 min after the glucose challenge by using a Glucometer (Abbott Laboratories, MediSense Products, Bedford, MA, USA). The area under the curve (AUC) for glucose was determined by the trapezoidal rule with the following formula as described previously [25]: AUC = 0.25 G0 + 0.5 G30 + 0.5 G60 + 0.5 G90 + 0.25 G120, where G0, G30, G60, G90, and G120 were blood glucose levels at each time point.
2.10.
Insulin tolerance test (ITT)
In a paralleled experiment, whole body insulin sensitivity was assessed by performing ITT following an overnight fasting period. Rats were administered a hypodermic injection of insulin (0.5 U/kg body weight). Whole blood glucose levels were determined at 0, 30, 60, 90, and 120 min after the insulin injection by tail clippings. The AUC for ITT were calculated to evaluate insulin sensitivity.
2.11.
Western blot analysis
All antibodies were purchased from Sigma (St. Louis, MO, U.S.). Insulin signaling pathways (IRS-1, phospho-serine307, phospho-tyrosine of IRS-1, Akt, and phospho-Akt), and proinflammatory related kinases, c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-kB kinase subunit b (IKKb) were investigated in muscle lysates by western blot. Briefly, muscles were homogenized in ice-cold lysis buffer containing (in mmol/l): 20 Tris–HCl, 50 NaCl, 50 NaF, 50 Na4P2O7, 250 sucrose, 2 Na3VO4, 1 DTT and 1% protease inhibitor cocktail. The homogenates were centrifuged at 12,000 g for 10 min, and the supernatants were collected. Protein concentration
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was determined by BCA protein assay (BCA, modified Lowry, Bradford, U.S.). The protein samples (50–120 mg protein per lane) were separated by electrophoresis with 8–12% SDS-PAGE gels, transferred to a polyvinylidene difluoride (PVDF) membrane with a semi-dry blotting apparatus (Bio-Rad Laboratories, Hercules, CA, U.S.), and blocked with 5% nonfat milk for 1 h at room temperature. The immunoblots were incubated with primary antibodies overnight at 4 8C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ECL plus reagent and the results were caputured by LabImage version 2.7.1 (Kapelan GmbH, Halle, Germany). Then, immunoblots were stripped with a strip buffer at 50 8C for 30 min and re-probed. GAPDH was used as the internal loading control. Densitometry analysis was performed with Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
2.12.
Statistical analyses
All data were presented as mean SEM. Statistical significance (P < 0.05) for each variable was estimated by analysis of variance (ANOVA) followed by a Tukey’s post hoc analysis. All statistical analyses were performed by using Statistical Package for Social Sciences 11.0 software (SPSS, Chicago, IL, USA).
3.
Results
3.1.
Effect of burn on FITC-labeled LPS translocation
3.2.
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Effect of SDD on cecal gram-negative bacteria level
To further identify the possible source of LPS and the effect of SDD on gut gram-negative bacteria level after burn, cecal gram-negative bacterial cultures were carried out as shown in Fig. 2. Burn injury resulted in overgrowth of gram-negative enteric bacilli in the cecum on post-bum days 1, 3 and 5. However, animals receiving SDD therapy exhibited lower growth level of gram-negative enteric bacilli throughout the experiment after burn injury.
3.3.
Effect of SDD on serum LPS concentrations
Burn with vehicle group exhibited significant elevation of LPS throughout the observation period, which peaked at the 5th day post-burn and then gradually decreased at 7th day (Fig. 3). A marked reduction of LPS was found in the serum of burn plus SDD group, indicating that SDD treatment efficiently attenuated burn-related increases in serum LPS concentrations.
3.4.
Effect of SDD on serum proinflammatory cytokines
To determine whether SDD treatment after burn injury altered systematic proinflammatory cytokines, serum from each group were prepared at 1, 3, 5, or 7 days post-burn, and the cytokines were detected afterward. As showed in Fig. 4, burn injury significantly elevated levels of serum IL-1b, IL-6 and TNF-a in burn with vehicle group. On the contrary, the treatment of SDD remarkably reduced burn injury induced IL1b, IL-6 and TNF-a elevation in burn plus SDD group.
A FITC-labeled LPS tracing experiment was used to clarify if burn injury would enhance gut-derived LPS translocation. As showed in Fig. 1, marked elevations of plasma FITC-labeled LPS in both the portal and systemic circulation were found in burn rats compared with those in sham burn group. These data indicated that burn injury increased the gut permeability and translocation of LPS directly from gut.
Skeletal muscles have been identified as the major tissue in glucose metabolism, accounting for 75% of whole-body insulin-stimulated glucose uptake. To further evaluate the
Fig. 1 – Effect of burn on FITC-labeled LPS translocation. Rats were gavaged with FITC-labeled LPS immediately after burn or sham operation. Both portal and systemic blood samples were collected at 4 h post-burn. After injury, the levels of serum FITC-labeled LPS in the portal and systemic circulation are both elevated compared with sham groups. Data are expressed as mean W SEM (in mg/ml). n = 6 rats for each time point. * P < 0.05 vs. sham group.
Fig. 2 – Effect of SDD on cecal gram-negative bacteria level after burn injury. Rats were treated with or without SDD after burn injury. Samples of cecum were collected and inoculated into MacConkey agar to selectively grow gramnegative bacteria before burn injury and at 1, 3 and 5 days post-bum. Data are expressed as mean W SEM (in log10 CFU/g). n = 6 rats for each time point. * P < 0.05 vs. preburn bacterial counts, # P < 0.05 vs. burn group.
3.5. Effect of SDD on proinflammatory cytokines in muscles
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Fig. 3 – Effect of SDD on serum LPS after burn injury. Rats were treated with or without SDD after burn or sham injury. Serum LPS concentrations were detected at 1, 3, 5, or 7 days after burn. After injury, the levels of serum LPS are elevated, but efficiently inhibited by SDD treatment. Data are expressed as mean W SEM (in EU/ml). n = 6 rats for each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
effect of SDD on burn-induced inflammation in skeletal muscles, local protein levels of cytokines were detected at 1, 3, 5, or 7 days after burn injury. The results in Fig. 5 showed that the proinflammatory cytokines such as IL-1b, IL6, as well as TNF-a were all significantly higher in burn with vehicle group. All the tendencies were inhibited by SDD treatment. This finding implied that SDD treatment could alleviate burn-induced local inflammation reaction in the skeletal muscles.
3.6.
Detection of insulin resistance after burn injury
To evaluate the effect of SDD treatment on the glucose metabolism after burn injury, IPGTT and ITT were performed. The time-plasma glucose concentration curves of IPGTT were illustrated in Fig. 6A. The blood glucose levels in burn with vehicle group were significantly increased at 30, 60, 90 and 120 min compared with that in the sham groups, while SDD treatment significantly blunted the hyperglycemia response. Similarly, The AUC values of IPGTT in burn group increased significantly compared to control groups, but SDD treatment significantly reduced AUC values compared to burn group (Fig. 6B). Meanwhile, in ITT, obviously retarded insulin response after burn is observed as evidenced by greater blood glucose levels at 30, 60, 90 and 120 min in burn with vehicle group (Fig. 6C), indicating that severe burn decreased the bioactivity of insulin to lower glucose in rats. However, SDD treatment markedly improved glucose metabolism, as evidenced by decreased blood glucose level in burn with SDD group. Coincidently, the AUC values of ITT in burn with SDD group were also lower than that in burn group (Fig. 6D). These findings demonstrated that SDD treatment significantly improved burn-induced insulin resistance, which in turn alleviated hyperglycemia.
Fig. 4 – Effect of SDD on serum cytokines after burn injury. Rats were treated with or without SDD after burn or sham injury. Serum IL-1b (A), IL-6 (B) and TNF-a (C) levels were measured by the rat Bio-Plex suspension array at 1, 3, 5 or 7 days after burn. Serum cytokines levels are all increased post-burn, but significantly inhibited by SDD treatment. Data are expressed as mean W SEM (in pg/ml). n = 6 rats for each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
3.7.
Role of SDD on IRS-1 and Akt phosphorylation
To further explore the role of SDD on key intercellular insulin signaling, phosphoralted IRS-1 and Akt in the skeletal muscles were evaluated by western blot. As shown in Fig. 7A–C,
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elevated phospho-serine307 of IRS-1 and decreased tyrosine phosphorylation of IRS-1 in skeletal muscles exhibited in burn with the vehicle group compared with those in the sham groups. SDD treatment can significantly decrease serine307 phosphorylation levels and increase tyrosine phosphorylation of IRS-1 in skeletal muscles after burn. Coincidently, a decrease in phosphorylation of Akt was found in burn with vehicle group, which was attenuated by SDD treatment (Fig. 7D). Considering that the activated insulin signaling is crucial for the translocation of glucose transporter, these results indicate that SDD treatment ameliorates intercellular insulin signaling impaired by burn injury.
3.8.
Role of SDD on JNK and IKKb activation
Proinflammatory kinases JNK and IKKb were further evaluated in skeletal muscles by western blot to illustrate the potential mechanisms behind the role of SDD on insulin resistance after burn injury. Our data revealed that burn injury markedly increased phosphorylated JNK (Fig. 8A) and IKKb (Fig. 8B) in burn with vehicle group compared with that in sham groups, while SDD significantly decreased phosphorylation of JNK and IKKb. This finding implied that SDD treatment depresses burn injury induced inflammatory signaling activation.
4.
Fig. 5 – Effect of SDD on cytokines in muscles after burn injury. Rats were treated with or without SDD after burn or sham injury. Level of IL-1b (A), IL-6 (B) and TNF-a (C) in muscles were detected by using ELISA at 1,3,5, or 7 days after burn injury. The results showed that elevation of cytokines in post-burn muscles were all significantly inhibited by SDD treatment. Data are expressed as mean W SEM. n = 6 rats for each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
Discussion
Insulin resistance increases morbidity and mortality in patients after severe burn injury, with limited treatment options available. Moreover, the underlying mechanism has not been well understood [26,27]. In this study, we have observed that burn injury caused overgrowth of cecal gramnegative bacteria, increase of LPS translocation, significant elevations of proinflammatory cytokines, impairment of glucose metabolism and insulin resistance. SDD therapy efficiently attenuated insulin resistance with the improved intercellular insulin signaling pathway, as evidenced by activation of IRS-1 and Akt. The possible mechanisms might be related to the inhibition of the gut-derived LPS, proinflammatory cytokines releasing, and activation of proinflammatory kinases, JNK and IKKb. Our results showed that serum concentrations of proinflammatory cytokines were significantly elevated in early stage of burn, which was consistent with previous studies [28,29]. Furthermore, in skeletal muscles, the production of the cytokines was also elevated, indicating enhanced local inflammation. Many studies have confirmed that the monocytes and macrophages activity are enhanced in burns, and they are responsible for the production of proinflammatory cytokines [30]. Macrophages are highly susceptible to endotoxin by virtue of their CD14 and Toll-like receptor proteins. Severe burn often leads to damage of intestinal barrier function and increases the intestinal mucosa permeability. These damages trigger bacterial and endotoxin translocation, intestinal infection, and further lead to elevation of endotoxin levels in systemic circulations and various other organs [31]. In patients, endotoxin could translocate across the gastrointestinal tract barrier within 1 h of burn injury [32]. In the FITCconjugated LPS tracing experiment, we also confirmed that
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Fig. 6 – Post-burn insulin resistance in rats after SDD treatment. (A) Intraperitoneal Glucose Tolerance Test. Glucose (2 g/kg of body weight) were injected intraperitoneally into the rats fasted overnight at 3 days after burn. Blood glucose was measured at 0, 30, 60, 90, and 120 min after glucose injection by using a glucometer. (B) The glucose area under the curve (AUC) values for IPGTT. (C) Insulin tolerance test. Rats were administered a hypodermic injection of insulin (0.5 U/kg body weight). Blood glucose levels were determined at 0, 30, 60, 90, and 120 min using tail clippings. (D) The glucose area under the curve (AUC) values for ITT. The results indicated that SDD treatment could significantly attenuate burn-induced insulin resistance and improve the whole-body responsiveness to insulin. Data are expressed as mean W SEM. n = 6 rats per group at each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
burn increased the translocation of LPS from gut quickly. The gut-derived LPS had been suggested as a key factor to promote secretion of proinflammatory cytokines by activating Toll-like receptor 4 [33]. Exposure to LPS induces a systemic inflammatory response that involves many interconnected cellular and plasma mediators. Our results also showed that serum concentrations of cytokines were quickly elevated in early stage of burn, which was parallel to increased circulating LPS. However, the maximal level of serum LPS was not parallel with the peak of serum cytokines on the 5th day after burn. The unexpected association between plasma LPS and proinflammatory cytokines in the late stage of burn could be explained by endotoxin tolerance or hyporesponsiveness. A second high dose of LPS challenge was reported to induce reduced cytokine production and releasing by monocytes/macrophages in vivo and in vitro [34–36]. Thus attenuated cytokines response with maximal LPS releasing in our finding is speculated to represent a tolerance of cells after an initial endotoxin stimulus, which might contribute to limiting inflammatory damage [37]. However, the detailed mechanisms of this phenomenon are still unclear. Previous study indicated that hemorrhagic shock led to significantly elevated LPS-stimulated proinflammatory cytokines release in macrophages. In contrast, by SDD therapy, cytokines released in macrophages stimulated by LPS were
significantly suppressed [38]. Similarly, our study proved that SDD treatment efficiently inhibited post-bum overgrowth of gram-negative bacteria in gut and the elevation of serum LPS. In addition, both increased circulating and intramuscular levels of IL-1b, IL-6 and TNF-a were efficiently inhibited, indicating that SDD was a feasible strategy for prevention of the systemic immune activation secondary to translocation of endotoxin. Systemic or target tissue inflammation may be an important contributor to type 2 diabetes and obesity associated insulin resistance [39]. The action of proinflammatory cytokines like TNF-a or IL-1b to promote serine phosphorylation of IRS-1 might provide a common mechanism for insulin resistance observed during chronic obesity. IL-1b was reported to reduce insulin-stimulated glucose uptake by decreasing IRS-1 protein abundance, insulin-stimulated phosphorylation of IRS-1, and insulin-induced membrane translocation of glucose transporter [40]. It has also been shown that IL-6 can inhibit tyrosine phosphorylation of IRS-1 in mice [41]. In addition, TNF-a has been proposed as a link between obesity and insulin resistance since obese mice lacking TNF-a show protection against developing insulin resistance [42]. In our study, increased proinflammatory cytokines were accompanied by the impaired intercellular insulin signaling, and positively associated with insulin resistance, which implied
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Fig. 7 – Phosphorylation of IRS-1 and Akt in muscles after SDD treatment. Phosphorylation of IRS-1 and Akt in muscles were measured by western blot at 3 days after burn. The results indicated that early SDD treatment could significantly decreased serine307 phosphorylation levels, increased tyrosine phosphorylation of IRS-1 after burn. Besides, the significant decrease in phosphorylation of Akt in burned animals was also attenuated by SDD treatment. (A) Representative gels depicting expression of phospho-ser307 and phosphorylated tyrosine of IRS-1, total IRS-1, phosphorylated Akt and total Akt in muscles. GAPDH was used as the loading control. (B) Phospho-ser307 of IRS-1 (normalized to total IRS-1). (C) Phosphotyrosine of IRS-1 (normalized to total IRS-1). (D) Phospho-Akt (normalized to total Akt). Data are expressed as mean W SEM. n = 6 animals per group. * P < 0.05 vs. sham groups, ** P < 0.05 vs. burn group.
that the rapid raise of cytokines might play an important role in triggering burn-induced insulin resistance. Pharmacological agents with anti-inflammatory actions have been found to protect experimental animals and human subjects from dietinduced insulin resistance [43]. Similarly, in the present study, SDD exerted its effect on insulin resistance, at least in part, by inhibiting the proinflammatory cytokines and subsequent intercellular signaling pathways. At the molecular level, there are several key proteins in the insulin/glucose regulatory pathway cascade, including IRS-1 and Akt. In the current study, we have demonstrated that burn injury resulted in increased phosphorylation of IRS-1 at serine307 residue as well as a decrease in tyrosine phosphorylation of IRS-1, limiting the ability of IRS-1 protein to activate its downstream pathway, and thereby reduced phosphorylation of Akt. Inflammatory pathways, including
JNK and IKKb activated by proinflammatory cytokines, can crosstalk with insulin signaling via the IRS-PI3K-Akt pathway [44]. JNK is reported to phosphorylate IRS-1 serine307 residue, which is located on its C-terminal side of the phospho-tyrosine-binding domain, thereby to inhibit insulin signaling [45]. IKKb is also capable of phosphorylating serine307 residues on IRS-1 proteins, and thus leads to insulin signaling block [46]. Moreover, phosphorylation on serine residues also increases IRS-1 protein degradation, contributing to the establishment of insulin resistance [47]. In this study, SDD efficiently blocked post-burn activation of JNK and IKKb with improved insulin signaling. Based on the above, it can be speculated that the molecular mechanisms underlying SDD reducing insulin resistance were probably associated with inhibition of proinflammatory kinase JNK and IKKb activation.
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signaling cascades may serve as a convenient and effective way to attenuate burn-induced insulin resistance.
Source of funding This study was supported by the funding of China Postdoctoral Science Foundation (Nos. 2013M542501; 2012M521785), and the National Natural Science Foundation of China (No. 81270301).
Conflicts of interest statement The authors have declared that no conflict of interest exists.
references
Fig. 8 – Phosphorylation of JNK and IKKb in muscles after SDD treatment. Phosphorylation of JNK and IKKb in skeletal muscles were measured by western blot at 3 days after burn injury. The results indicated that SDD treatment significantly attenuated post-burn phosphorylation of JNK and IKKb in muscles. (A) Western blot results of JNK (normalized to total JNK), (B) Western blot results of IKKb (normalized to total IKKb). Data are expressed as mean W SEM. n = 6 animals per group. * P < 0.05 vs. sham groups, ** P < 0.05 vs. burn group.
5.
Conclusion
In summary, the present study suggests that endotoxin absorbed from the intestinal tract promotes proinflammatory cytokines releasing and subsequent signaling pathway activation, which is associated with severe burn-induced insulin resistance. The application of SDD to eliminate the gut endotoxin absorption and subsequent proinflammatory
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Available online at www.sciencedirect.com
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Selective decontamination of the digestive tract ameliorates severe burn-induced insulin resistance in rats Jun Li a,b,1, Liang Zhu c,1, Ming Xu a,1, Juntao Han b, Xiaozhi Bai b, Xuekang Yang b, Huayu Zhu b, Jie Xu a,d, Xing Zhang a,d, Yangfan Gong b, Dahai Hu b,*, Feng Gao a,d,* a
Department of Physiology, School of Basic Medical Sciences, Fourth Military Medical University, Xi’an, China Department of Burns and Cutaneous Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, China c Department of Medical Education, Fourth Military Medical University, Xi’an, China d Department of Aerospace Medicine, Fourth Military Medical University, Xi’an, China b
article info
abstract
Article history:
Background: Severe burns often initiate the prevalence of hyperglycemia and insulin resis-
Received 7 July 2014
tance, significantly contributing to adverse clinical outcomes. However, there are limited
Received in revised form
treatment options. This study was designed to investigate the role and the underlying
22 December 2014
mechanisms of oral antibiotics to selectively decontaminate the digestive tract (SDD) on
Accepted 25 December 2014
burn-induced insulin resistance. Materials and methods: Rats were subjected to 40% of total body surface area full-thickness burn or sham operation with or without SDD treatment. Translocation of FITC-labeled LPS
Keywords:
was measured at 4 h after burn. Furthermore, the effect of SDD on post-burn quantity of
Burn
gram-negative bacteria in gut was investigated. Serum or muscle LPS and proinflammatory
Insulin resistance
cytokines were measured. Intraperitoneal glucose tolerance test and insulin tolerance test
SDD
were used to determine the status of systemic insulin resistance. Furthermore, intracellular
Proinflammatory cytokine
insulin signaling (IRS-1 and Akt) and proinflammatory related kinases (JNK and IKKb) were
LPS
assessed by western blot. Results: Burn increased the translocation of LPS from gut 4 h after injury. SDD treatment effectively inhibited post-burn overgrowth of gram-negative enteric bacilli in gut. In addition, severe burns caused significant increases in the LPS and proinflammatory cytokines levels, activation of proinflammatory related kinases, and systemic insulin resistance as well. But SDD treatment could significantly attenuate burn-induced insulin resistance and improve the whole-body responsiveness to insulin, which was associated with the inhibition of gut-derived LPS, cytokines, proinflammatory related kinases JNK and IKKb, as well as activation of IRS-1 and Akt. Conclusions: SDD appeared to have an effect on proinflammatory signaling cascades and further reduced severe burn-induced insulin resistance. # 2015 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding authors at: Department of Burns and Cutaneous Surgery, Xijing Hospital; Department of Physiology and Department of Aerospace Medicine, Fourth Military Medical University, Xi’an 710032, China. Tel.: +86 29 84775297/+86 29 84776423; fax: +86 29 83246270. E-mail addresses: [email protected] (D. Hu), [email protected] (F. Gao). 1 These authors were co-first authors and contributed equally to this work. http://dx.doi.org/10.1016/j.burns.2014.12.018 0305-4179/# 2015 Elsevier Ltd and ISBI. All rights reserved.
burns 41 (2015) 1076–1085
1.
Introduction
Insulin resistance and hyperglycemia are frequent phenomena in severe burn patients, which have been recognized as the hallmarks of burn-induced diabetes [1,2]. More recently, studies have showed the close relationship between adverse clinical outcomes and impaired glucose metabolism in these patients, who have a significantly higher incidence of bacteremia or fungemia infection, metabolic disorder and mortality [3]. Conversely, controlling hyperglycemia or alleviating insulin resistance is associated with improved outcomes [4]. Insulin resistance can be defined as a state of reduced response of peripheral target tissues to insulin stimulation [5]. It often occurs in chronic diseases, such as type 2 diabetes and obesity, and normally takes months or years to develop [6]. However, recent findings have revealed that acute insulin resistance exists in critically ill like burns, characterized by a rapid onset of uncontrolled hyperglycemia within hours or days [7]. Until now, the precise mechanisms involved in it are poorly understood, thus lacking effective treatments and prevention strategies. Severe burn injury usually induces a distinct systemic inflammatory response that has been well described previously [8,9]. Proinflammatory cytokines are the primary mediators of this inflammatory reaction. The levels of various cytokines such as interleukin 6 (IL-6) [10] and tumor necrosis factor a (TNF-a) [11] changes in early stage and have been used as markers of the severity of burns. Emerging data indicate that some cytokines play an important role in regulating glucose metabolism, and the excessive activation of proinflammatory related pathways may represent a fundamental step in the development of insulin resistance [12,13]. In addition, alterations in cytokine levels often occur prior to metabolic abnormalities in severe burns [9]. Whatever the mechanisms involve, it has now been demonstrated that cytokines are able to negatively influence insulin signaling in insulin-responsive tissues [14]. Early modulating cytokines releasing and proinflammatory related kinases activation may represent a feasible strategy for acute insulin resistance and subsequent hyperglycemia. Bacterial and endotoxin (lipopolysaccharide, LPS) invasion from the digestive tract are closely related to an increased incidence of sepsis and multiple organ failure in burned patients [15,16]. Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents, and promotes a generalized inflammatory state that may lead, via cytokinemia, to organ damage [17]. Selective decontamination of the digestive tract (SDD) is conducted by oral administration of non-absorbable, small-spectrum antibiotics to eliminate gram-negative bacillus from the digestive tract, and it has been widely used to prevent gut-derived infection in critically ill patients, such as pancreatitis, liver transplantation, and trauma. Many randomized controlled trials of SDD have been published. Although some of the evidence supporting the use of SDD, the opponents still rely on historical arguments against its use, such as the bacterial resistance and the absence of influence on mortality [18]. The predominant concern was that use of SDD will promote the development of antimicrobial-resistant pathogens. Recently, a meta-analysis
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detected no relationship between the use of SDD and the antimicrobial resistance. However, the authors acknowledged the need for research over a longer term [19]. Despite the controversies, SDD has showed beneficial effect on mortality in patients with severe burns [20,21]. Moreover, SDD applied either before or after burn injury in rodents showed remarkable cardioprotection by suppression of myocardial inflammation [22]. Given gut-derived pathogens are one of the primary mechanisms for systemic inflammatory responses in burns, SDD application would be expected to attenuate proinflammatory cytokines release and subsequent intercellular signaling cascade activation, and thus improve insulin signaling. However, the efficacy of SDD on insulin resistance after severe burns has not been evaluated yet. In this study, we hypothesized that gut-derived endotoxin could mediate excessive inflammatory reaction and then provoke insulin resistance, but the application of SDD would reduce endotoxin translocation from the gastrointestinal tract, inhibit systematic proinflammatory cytokines releasing and subsequent intercellular signaling cascade, which ultimately ameliorate acute insulin resistance.
2.
Materials and methods
2.1.
Animals
Sprague-Dawley male rats (200–250 g) were obtained from the Center of Experimental Animals of the Fourth Military Medical University. Rats were housed in an environmentally controlled room with a 12 h light/dark cycle, with free access to regular rat chow and water. All procedures in this study were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University, in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
2.2.
Burn procedure and SDD therapy
Rats were anesthetized with 2% pentobarbital (40 mg/kg i.p.). After clipping of the hair, a 40% total body surface area (TBSA) full thickness third degree burn injury was produced by immersing the back of trunk in 100 8C water for 12 s. Weight and age matched rats were immersed in room temperature water as sham group. All rats received Lactated Ringer resuscitation after burn injury (4 ml/kg/% burn). Oral antibiotic therapy for SDD was carried out as described previously [22]. Briefly, water mixture (final volume 3 ml) of polymyxin E (15 mg), tobramycin (6 mg), and 5-flucytosin (100 mg) (Sigma-Aldrich, St Louis, MI, U.S.) were given 4 h after burn by oral gavage and twice daily afterward for 5 days. Animals given the same volume of water at the same time intervals were set as vehicle treatment.
2.3.
Translocation of FITC-labeled LPS after burn
Rats were orally gavaged with FITC-labeled LPS (Sigma, St Louis, MO, USA) 3 mg/kg dissolved in deionized water
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immediately after burn or sham operation. Both portal and systemic blood were collected at 4 h after burn injury. Serum was separated by centrifugation at 4500 rpm for 3 min at 4 8C. FITC fluorescence was measured by using a fluorescence spectrophotometer at an excitation of 485 nm and an emission of 530 nm. The concentration of FITC in plasma was expressed as mg/ml of plasma.
2.4. Analysis of gram-negative bacteria in cecum after SDD therapy Rats were randomly divided into burn with vehicle and burn plus SDD groups. The animals were sacrificed before burn injury and at 1, 3 and 5 days post-bum (n = 6 rats per group at each time point). The cecum was excised under aseptic conditions, and the cecal content weighing 0.1 g was collected, homogenized, and serially diluted with saline. The homogenates from cecum were inoculated into MacConkey agar designed to selectively grow gram-negative bacteria. After 24 h of incubation at 37 8C, colonies were identified and counted. The bacterial counts were expressed as the log10 of the total number of viable bacteria per gram weight of cecal content.
2.5. Experimental protocols for post-burn insulin resistance Rats were randomized into four groups: (1) sham with vehicle (3 ml water), (2) sham plus SDD, (3) burn with vehicle, and (4) burn plus SDD. Animals were sacrificed at 1, 3, 5 and 7 days post-burn (n = 6 rats per group at each time point). Blood samples and skeletal muscles were collected for further analysis. In addition, insulin resistance was evaluated at 3 days post-burn by intraperitoneal glucose tolerance test or insulin tolerance test, respectively.
2.6.
Serum endotoxin (LPS) determination
Blood samples from each group were collected, and serum was separated by centrifugation at 4500 rpm for 3 min at 4 8C and stored at 70 8C until analyzed. LPS was measured using a commercially available kit following the manufacturer’s instructions (Hycult Biotech, The Netherlands). Results were expressed as endotoxin units (EU) per ml (EU/ml).
2.7.
Serum proinflammatory cytokines determination
Serum cytokines IL-1b, IL-6 and TNF-a were determined by the rat Bio-Plex suspension array (Bio-Rad, Hercules, CA, U.S.) at 1, 3, 5, or 7 d after burn or sham operations. In brief, blood samples from each group were collected, and serum was separated by centrifugation at 4500 rpm for 3 min at 4 8C and stored at 70 8C until analyzed. The supernatant was collected and incubated with microbeads labeled with specific antibodies to one of the mentioned cytokines for 30 min. After washing with PBS, samples were then incubated with the detection antibody cocktail, followed by another wash step. Collecting the beads by centrifugation, the samples were incubated with streptavidin–phycoerythrin for 10 min, washed, and the concentration of each cytokine was determined by optical density, which was measured in a microplate
reader (TECAN Austria GmbH, Grodig, Austria) set to 450 nm wave lengths.
2.8.
Assay of proinflammatory cytokines in muscles
0.1–0.3 g frozen skeletal muscles were homogenized in RIPA buffer (0.625% sodium deoxycholate, 0.625% Nonidet P-40, 6.25 mM sodium phosphate, and 1 mM EDTA at pH 7.4) containing 10 mg/ml of a protease inhibitor cocktail (SigmaAldrich, St. Louis, MO). Homogenates were centrifuged at 12,000 g for 10 min at 4 8C, the supernatant was saved, and protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a reference. The protein levels of IL-1b, IL-6, and TNF-a in muscles were measured in duplicate by using commercially available rat ELISA kits (DuoSet ELISA, R&D Systems, Minneapolis, MN). The assays were carried out according to the manufacturer’s instructions. Cytokine data of muscle are expressed as pg of cytokine per mg of total protein.
2.9.
Intraperitoneal glucose tolerance test (IPGTT)
Previous reports had indicated that insulin resistance was clearly observed at 2–7 days after burn injury [23,24]. Thus, we chose 3 days as the time point for analysis of insulin resistance. In brief, IPGTT were performed following an overnight fast period. The animals were administered an intraperitoneal injection of glucose (2 g/kg body weight). The blood glucose was measured from tail-tip blood at 0, 30, 60, 90 and 120 min after the glucose challenge by using a Glucometer (Abbott Laboratories, MediSense Products, Bedford, MA, USA). The area under the curve (AUC) for glucose was determined by the trapezoidal rule with the following formula as described previously [25]: AUC = 0.25 G0 + 0.5 G30 + 0.5 G60 + 0.5 G90 + 0.25 G120, where G0, G30, G60, G90, and G120 were blood glucose levels at each time point.
2.10.
Insulin tolerance test (ITT)
In a paralleled experiment, whole body insulin sensitivity was assessed by performing ITT following an overnight fasting period. Rats were administered a hypodermic injection of insulin (0.5 U/kg body weight). Whole blood glucose levels were determined at 0, 30, 60, 90, and 120 min after the insulin injection by tail clippings. The AUC for ITT were calculated to evaluate insulin sensitivity.
2.11.
Western blot analysis
All antibodies were purchased from Sigma (St. Louis, MO, U.S.). Insulin signaling pathways (IRS-1, phospho-serine307, phospho-tyrosine of IRS-1, Akt, and phospho-Akt), and proinflammatory related kinases, c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-kB kinase subunit b (IKKb) were investigated in muscle lysates by western blot. Briefly, muscles were homogenized in ice-cold lysis buffer containing (in mmol/l): 20 Tris–HCl, 50 NaCl, 50 NaF, 50 Na4P2O7, 250 sucrose, 2 Na3VO4, 1 DTT and 1% protease inhibitor cocktail. The homogenates were centrifuged at 12,000 g for 10 min, and the supernatants were collected. Protein concentration
burns 41 (2015) 1076–1085
was determined by BCA protein assay (BCA, modified Lowry, Bradford, U.S.). The protein samples (50–120 mg protein per lane) were separated by electrophoresis with 8–12% SDS-PAGE gels, transferred to a polyvinylidene difluoride (PVDF) membrane with a semi-dry blotting apparatus (Bio-Rad Laboratories, Hercules, CA, U.S.), and blocked with 5% nonfat milk for 1 h at room temperature. The immunoblots were incubated with primary antibodies overnight at 4 8C followed by incubation with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ECL plus reagent and the results were caputured by LabImage version 2.7.1 (Kapelan GmbH, Halle, Germany). Then, immunoblots were stripped with a strip buffer at 50 8C for 30 min and re-probed. GAPDH was used as the internal loading control. Densitometry analysis was performed with Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
2.12.
Statistical analyses
All data were presented as mean SEM. Statistical significance (P < 0.05) for each variable was estimated by analysis of variance (ANOVA) followed by a Tukey’s post hoc analysis. All statistical analyses were performed by using Statistical Package for Social Sciences 11.0 software (SPSS, Chicago, IL, USA).
3.
Results
3.1.
Effect of burn on FITC-labeled LPS translocation
3.2.
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Effect of SDD on cecal gram-negative bacteria level
To further identify the possible source of LPS and the effect of SDD on gut gram-negative bacteria level after burn, cecal gram-negative bacterial cultures were carried out as shown in Fig. 2. Burn injury resulted in overgrowth of gram-negative enteric bacilli in the cecum on post-bum days 1, 3 and 5. However, animals receiving SDD therapy exhibited lower growth level of gram-negative enteric bacilli throughout the experiment after burn injury.
3.3.
Effect of SDD on serum LPS concentrations
Burn with vehicle group exhibited significant elevation of LPS throughout the observation period, which peaked at the 5th day post-burn and then gradually decreased at 7th day (Fig. 3). A marked reduction of LPS was found in the serum of burn plus SDD group, indicating that SDD treatment efficiently attenuated burn-related increases in serum LPS concentrations.
3.4.
Effect of SDD on serum proinflammatory cytokines
To determine whether SDD treatment after burn injury altered systematic proinflammatory cytokines, serum from each group were prepared at 1, 3, 5, or 7 days post-burn, and the cytokines were detected afterward. As showed in Fig. 4, burn injury significantly elevated levels of serum IL-1b, IL-6 and TNF-a in burn with vehicle group. On the contrary, the treatment of SDD remarkably reduced burn injury induced IL1b, IL-6 and TNF-a elevation in burn plus SDD group.
A FITC-labeled LPS tracing experiment was used to clarify if burn injury would enhance gut-derived LPS translocation. As showed in Fig. 1, marked elevations of plasma FITC-labeled LPS in both the portal and systemic circulation were found in burn rats compared with those in sham burn group. These data indicated that burn injury increased the gut permeability and translocation of LPS directly from gut.
Skeletal muscles have been identified as the major tissue in glucose metabolism, accounting for 75% of whole-body insulin-stimulated glucose uptake. To further evaluate the
Fig. 1 – Effect of burn on FITC-labeled LPS translocation. Rats were gavaged with FITC-labeled LPS immediately after burn or sham operation. Both portal and systemic blood samples were collected at 4 h post-burn. After injury, the levels of serum FITC-labeled LPS in the portal and systemic circulation are both elevated compared with sham groups. Data are expressed as mean W SEM (in mg/ml). n = 6 rats for each time point. * P < 0.05 vs. sham group.
Fig. 2 – Effect of SDD on cecal gram-negative bacteria level after burn injury. Rats were treated with or without SDD after burn injury. Samples of cecum were collected and inoculated into MacConkey agar to selectively grow gramnegative bacteria before burn injury and at 1, 3 and 5 days post-bum. Data are expressed as mean W SEM (in log10 CFU/g). n = 6 rats for each time point. * P < 0.05 vs. preburn bacterial counts, # P < 0.05 vs. burn group.
3.5. Effect of SDD on proinflammatory cytokines in muscles
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Fig. 3 – Effect of SDD on serum LPS after burn injury. Rats were treated with or without SDD after burn or sham injury. Serum LPS concentrations were detected at 1, 3, 5, or 7 days after burn. After injury, the levels of serum LPS are elevated, but efficiently inhibited by SDD treatment. Data are expressed as mean W SEM (in EU/ml). n = 6 rats for each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
effect of SDD on burn-induced inflammation in skeletal muscles, local protein levels of cytokines were detected at 1, 3, 5, or 7 days after burn injury. The results in Fig. 5 showed that the proinflammatory cytokines such as IL-1b, IL6, as well as TNF-a were all significantly higher in burn with vehicle group. All the tendencies were inhibited by SDD treatment. This finding implied that SDD treatment could alleviate burn-induced local inflammation reaction in the skeletal muscles.
3.6.
Detection of insulin resistance after burn injury
To evaluate the effect of SDD treatment on the glucose metabolism after burn injury, IPGTT and ITT were performed. The time-plasma glucose concentration curves of IPGTT were illustrated in Fig. 6A. The blood glucose levels in burn with vehicle group were significantly increased at 30, 60, 90 and 120 min compared with that in the sham groups, while SDD treatment significantly blunted the hyperglycemia response. Similarly, The AUC values of IPGTT in burn group increased significantly compared to control groups, but SDD treatment significantly reduced AUC values compared to burn group (Fig. 6B). Meanwhile, in ITT, obviously retarded insulin response after burn is observed as evidenced by greater blood glucose levels at 30, 60, 90 and 120 min in burn with vehicle group (Fig. 6C), indicating that severe burn decreased the bioactivity of insulin to lower glucose in rats. However, SDD treatment markedly improved glucose metabolism, as evidenced by decreased blood glucose level in burn with SDD group. Coincidently, the AUC values of ITT in burn with SDD group were also lower than that in burn group (Fig. 6D). These findings demonstrated that SDD treatment significantly improved burn-induced insulin resistance, which in turn alleviated hyperglycemia.
Fig. 4 – Effect of SDD on serum cytokines after burn injury. Rats were treated with or without SDD after burn or sham injury. Serum IL-1b (A), IL-6 (B) and TNF-a (C) levels were measured by the rat Bio-Plex suspension array at 1, 3, 5 or 7 days after burn. Serum cytokines levels are all increased post-burn, but significantly inhibited by SDD treatment. Data are expressed as mean W SEM (in pg/ml). n = 6 rats for each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
3.7.
Role of SDD on IRS-1 and Akt phosphorylation
To further explore the role of SDD on key intercellular insulin signaling, phosphoralted IRS-1 and Akt in the skeletal muscles were evaluated by western blot. As shown in Fig. 7A–C,
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elevated phospho-serine307 of IRS-1 and decreased tyrosine phosphorylation of IRS-1 in skeletal muscles exhibited in burn with the vehicle group compared with those in the sham groups. SDD treatment can significantly decrease serine307 phosphorylation levels and increase tyrosine phosphorylation of IRS-1 in skeletal muscles after burn. Coincidently, a decrease in phosphorylation of Akt was found in burn with vehicle group, which was attenuated by SDD treatment (Fig. 7D). Considering that the activated insulin signaling is crucial for the translocation of glucose transporter, these results indicate that SDD treatment ameliorates intercellular insulin signaling impaired by burn injury.
3.8.
Role of SDD on JNK and IKKb activation
Proinflammatory kinases JNK and IKKb were further evaluated in skeletal muscles by western blot to illustrate the potential mechanisms behind the role of SDD on insulin resistance after burn injury. Our data revealed that burn injury markedly increased phosphorylated JNK (Fig. 8A) and IKKb (Fig. 8B) in burn with vehicle group compared with that in sham groups, while SDD significantly decreased phosphorylation of JNK and IKKb. This finding implied that SDD treatment depresses burn injury induced inflammatory signaling activation.
4.
Fig. 5 – Effect of SDD on cytokines in muscles after burn injury. Rats were treated with or without SDD after burn or sham injury. Level of IL-1b (A), IL-6 (B) and TNF-a (C) in muscles were detected by using ELISA at 1,3,5, or 7 days after burn injury. The results showed that elevation of cytokines in post-burn muscles were all significantly inhibited by SDD treatment. Data are expressed as mean W SEM. n = 6 rats for each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
Discussion
Insulin resistance increases morbidity and mortality in patients after severe burn injury, with limited treatment options available. Moreover, the underlying mechanism has not been well understood [26,27]. In this study, we have observed that burn injury caused overgrowth of cecal gramnegative bacteria, increase of LPS translocation, significant elevations of proinflammatory cytokines, impairment of glucose metabolism and insulin resistance. SDD therapy efficiently attenuated insulin resistance with the improved intercellular insulin signaling pathway, as evidenced by activation of IRS-1 and Akt. The possible mechanisms might be related to the inhibition of the gut-derived LPS, proinflammatory cytokines releasing, and activation of proinflammatory kinases, JNK and IKKb. Our results showed that serum concentrations of proinflammatory cytokines were significantly elevated in early stage of burn, which was consistent with previous studies [28,29]. Furthermore, in skeletal muscles, the production of the cytokines was also elevated, indicating enhanced local inflammation. Many studies have confirmed that the monocytes and macrophages activity are enhanced in burns, and they are responsible for the production of proinflammatory cytokines [30]. Macrophages are highly susceptible to endotoxin by virtue of their CD14 and Toll-like receptor proteins. Severe burn often leads to damage of intestinal barrier function and increases the intestinal mucosa permeability. These damages trigger bacterial and endotoxin translocation, intestinal infection, and further lead to elevation of endotoxin levels in systemic circulations and various other organs [31]. In patients, endotoxin could translocate across the gastrointestinal tract barrier within 1 h of burn injury [32]. In the FITCconjugated LPS tracing experiment, we also confirmed that
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Fig. 6 – Post-burn insulin resistance in rats after SDD treatment. (A) Intraperitoneal Glucose Tolerance Test. Glucose (2 g/kg of body weight) were injected intraperitoneally into the rats fasted overnight at 3 days after burn. Blood glucose was measured at 0, 30, 60, 90, and 120 min after glucose injection by using a glucometer. (B) The glucose area under the curve (AUC) values for IPGTT. (C) Insulin tolerance test. Rats were administered a hypodermic injection of insulin (0.5 U/kg body weight). Blood glucose levels were determined at 0, 30, 60, 90, and 120 min using tail clippings. (D) The glucose area under the curve (AUC) values for ITT. The results indicated that SDD treatment could significantly attenuate burn-induced insulin resistance and improve the whole-body responsiveness to insulin. Data are expressed as mean W SEM. n = 6 rats per group at each time point. * P < 0.05 vs. sham groups, # P < 0.05 vs. burn group.
burn increased the translocation of LPS from gut quickly. The gut-derived LPS had been suggested as a key factor to promote secretion of proinflammatory cytokines by activating Toll-like receptor 4 [33]. Exposure to LPS induces a systemic inflammatory response that involves many interconnected cellular and plasma mediators. Our results also showed that serum concentrations of cytokines were quickly elevated in early stage of burn, which was parallel to increased circulating LPS. However, the maximal level of serum LPS was not parallel with the peak of serum cytokines on the 5th day after burn. The unexpected association between plasma LPS and proinflammatory cytokines in the late stage of burn could be explained by endotoxin tolerance or hyporesponsiveness. A second high dose of LPS challenge was reported to induce reduced cytokine production and releasing by monocytes/macrophages in vivo and in vitro [34–36]. Thus attenuated cytokines response with maximal LPS releasing in our finding is speculated to represent a tolerance of cells after an initial endotoxin stimulus, which might contribute to limiting inflammatory damage [37]. However, the detailed mechanisms of this phenomenon are still unclear. Previous study indicated that hemorrhagic shock led to significantly elevated LPS-stimulated proinflammatory cytokines release in macrophages. In contrast, by SDD therapy, cytokines released in macrophages stimulated by LPS were
significantly suppressed [38]. Similarly, our study proved that SDD treatment efficiently inhibited post-bum overgrowth of gram-negative bacteria in gut and the elevation of serum LPS. In addition, both increased circulating and intramuscular levels of IL-1b, IL-6 and TNF-a were efficiently inhibited, indicating that SDD was a feasible strategy for prevention of the systemic immune activation secondary to translocation of endotoxin. Systemic or target tissue inflammation may be an important contributor to type 2 diabetes and obesity associated insulin resistance [39]. The action of proinflammatory cytokines like TNF-a or IL-1b to promote serine phosphorylation of IRS-1 might provide a common mechanism for insulin resistance observed during chronic obesity. IL-1b was reported to reduce insulin-stimulated glucose uptake by decreasing IRS-1 protein abundance, insulin-stimulated phosphorylation of IRS-1, and insulin-induced membrane translocation of glucose transporter [40]. It has also been shown that IL-6 can inhibit tyrosine phosphorylation of IRS-1 in mice [41]. In addition, TNF-a has been proposed as a link between obesity and insulin resistance since obese mice lacking TNF-a show protection against developing insulin resistance [42]. In our study, increased proinflammatory cytokines were accompanied by the impaired intercellular insulin signaling, and positively associated with insulin resistance, which implied
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Fig. 7 – Phosphorylation of IRS-1 and Akt in muscles after SDD treatment. Phosphorylation of IRS-1 and Akt in muscles were measured by western blot at 3 days after burn. The results indicated that early SDD treatment could significantly decreased serine307 phosphorylation levels, increased tyrosine phosphorylation of IRS-1 after burn. Besides, the significant decrease in phosphorylation of Akt in burned animals was also attenuated by SDD treatment. (A) Representative gels depicting expression of phospho-ser307 and phosphorylated tyrosine of IRS-1, total IRS-1, phosphorylated Akt and total Akt in muscles. GAPDH was used as the loading control. (B) Phospho-ser307 of IRS-1 (normalized to total IRS-1). (C) Phosphotyrosine of IRS-1 (normalized to total IRS-1). (D) Phospho-Akt (normalized to total Akt). Data are expressed as mean W SEM. n = 6 animals per group. * P < 0.05 vs. sham groups, ** P < 0.05 vs. burn group.
that the rapid raise of cytokines might play an important role in triggering burn-induced insulin resistance. Pharmacological agents with anti-inflammatory actions have been found to protect experimental animals and human subjects from dietinduced insulin resistance [43]. Similarly, in the present study, SDD exerted its effect on insulin resistance, at least in part, by inhibiting the proinflammatory cytokines and subsequent intercellular signaling pathways. At the molecular level, there are several key proteins in the insulin/glucose regulatory pathway cascade, including IRS-1 and Akt. In the current study, we have demonstrated that burn injury resulted in increased phosphorylation of IRS-1 at serine307 residue as well as a decrease in tyrosine phosphorylation of IRS-1, limiting the ability of IRS-1 protein to activate its downstream pathway, and thereby reduced phosphorylation of Akt. Inflammatory pathways, including
JNK and IKKb activated by proinflammatory cytokines, can crosstalk with insulin signaling via the IRS-PI3K-Akt pathway [44]. JNK is reported to phosphorylate IRS-1 serine307 residue, which is located on its C-terminal side of the phospho-tyrosine-binding domain, thereby to inhibit insulin signaling [45]. IKKb is also capable of phosphorylating serine307 residues on IRS-1 proteins, and thus leads to insulin signaling block [46]. Moreover, phosphorylation on serine residues also increases IRS-1 protein degradation, contributing to the establishment of insulin resistance [47]. In this study, SDD efficiently blocked post-burn activation of JNK and IKKb with improved insulin signaling. Based on the above, it can be speculated that the molecular mechanisms underlying SDD reducing insulin resistance were probably associated with inhibition of proinflammatory kinase JNK and IKKb activation.
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signaling cascades may serve as a convenient and effective way to attenuate burn-induced insulin resistance.
Source of funding This study was supported by the funding of China Postdoctoral Science Foundation (Nos. 2013M542501; 2012M521785), and the National Natural Science Foundation of China (No. 81270301).
Conflicts of interest statement The authors have declared that no conflict of interest exists.
references
Fig. 8 – Phosphorylation of JNK and IKKb in muscles after SDD treatment. Phosphorylation of JNK and IKKb in skeletal muscles were measured by western blot at 3 days after burn injury. The results indicated that SDD treatment significantly attenuated post-burn phosphorylation of JNK and IKKb in muscles. (A) Western blot results of JNK (normalized to total JNK), (B) Western blot results of IKKb (normalized to total IKKb). Data are expressed as mean W SEM. n = 6 animals per group. * P < 0.05 vs. sham groups, ** P < 0.05 vs. burn group.
5.
Conclusion
In summary, the present study suggests that endotoxin absorbed from the intestinal tract promotes proinflammatory cytokines releasing and subsequent signaling pathway activation, which is associated with severe burn-induced insulin resistance. The application of SDD to eliminate the gut endotoxin absorption and subsequent proinflammatory
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