Acute Insulin Resistance Mediated by Advanced Glycation Endproducts in Severely Burned Rats Xing Zhang, PhD1; Jie Xu, MD1; Xiaoqing Cai, MD1; Lele Ji, PhD1; Jia Li, MD, PhD1; Bing Cao, MD1; Jun Li, MD2; Dahai Hu, MD, PhD2; Yan Li, MD, PhD3; Haichang Wang, MD, PhD3; Lize Xiong, MD, PhD4; Ruiping Xiao, MD, PhD5; Feng Gao, MD, PhD1,3
Objective: Hyperglycemia often occurs in severe burns; however, the underlying mechanisms and importance of managing postburn hyperglycemia are not well recognized. This study was designed to investigate the dynamic changes of postburn hyperglycemia and the underlying mechanisms and to evaluate whether early glycemic control is beneficial in severe burns. Design: Prospective, randomized experimental study. Setting: Animal research laboratory. Subjects: Sprague-Dawley rats. Interventions: Anesthetized rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive vehicle, insulin, and a soluble form of receptor for advanced glycation endproducts treatments. An in Department of Physiology, School of Basic Medical Sciences, The Fourth Military Medical University, Xi’an, China. 2 Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. 3 Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. 4 Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. 5 Institute of Molecular Medicine, Peking University, Beijing, China. Drs. Zhang, Xu, and Cai contributed equally to this study. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ ccmjournal). This work was supported by grants for article research from the National Basic Research Program of China (Nos. 2013CB531204, 2012CB518000), the State Key Program of National Natural Science Foundation of China (No. 81030005), and the National Natural Science Foundation of China (Nos. 81170184, 81270301). Drs. Gao, Zhang, Cai, Ji, Jia Li, Cao, Jun Li, Hu, Yan Li, Wang, Xiong, and Xiao received support for article research from the National Basic Research Program of China (No. 2013CB531204), the State Key Program of National Natural Science Foundation of China (No. 81030005), and the National Natural Science Foundation of China (Nos. 81170184, 81270301). Dr. Xu received support for article research. For information regarding this article, E-mail: [email protected]; xiaor@ pku.edu.cn Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000314 1
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vitro study was performed on cultured H9C2 cells subjected to vehicle or carboxymethyllysine treatment. Measurements and Main Results: We found that blood glucose change presented a distinct pattern with two occurrences of hyperglycemia at 0.5- and 3-hour postburn, respectively. Acute insulin resistance evidenced by impaired insulin signaling and glucose uptake occurred at 3-hour postburn, which was associated with the second hyperglycemia and positively correlated with mortality. Mechanistically, we found that serum carboxymethyllysine, a dominant species of advanced glycation endproducts, increased within 1-hour postburn, preceding the occurrence of insulin resistance. More importantly, treatment of animals with soluble form of receptor for advanced glycation endproducts, blockade of advanced glycation endproducts signaling, alleviated severe burn– induced insulin resistance. In addition, early hyperglycemic control with insulin not only reduced serum carboxymethyllysine but also blunted postburn insulin resistance and reduced mortality. Conclusions: These findings suggest that severe burn–induced insulin resistance is partly at least mediated by serum advanced glycation endproducts and positively correlated with mortality. Early glycemic control with insulin or inhibition of advanced glycation endproducts with soluble form of receptor for advanced glycation endproducts ameliorates postburn insulin resistance. (Crit Care Med 2014; 42:e472–e480) Key Words: acute insulin resistance; advanced glycation endproducts; burn injury; carboxymethyllysine; insulin; soluble form of receptor for advanced glycation endproducts
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yperglycemia often develops in critical illness, especially following trauma, burn, stroke, and surgery. This trauma-induced hyperglycemia has long been believed to be an adaptive stress response and was initially thought to be a beneficial adaptation or paraphenomenon essential to provide fuel for vital organs and thus better left undisturbed (1). However, recent studies have suggested that hyperglycemia is potentially toxic (2–5) and is identified as an independent risk factor for adverse outcomes in critical illness (4–7). Several randomized controlled trials of strict glucose control, especially the June 2014 • Volume 42 • Number 6
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landmark study by van den Berghe et al (2), have shown the benefits of blood glucose control with intensive insulin treatment in critically ill patients, while subsequent studies (8–12) had mixed or even conflicting results. For example, the Normoglycaemia in Intensive Care Evaluation and Survival Using Glucose Algorithm Regulation study (11) does not support intensive insulin treatment, but it only demonstrated that “strict” glucose control may be more harmful than conventional glucose control. Uncontrolled hyperglycemia is still regarded as undesirable in critically ill patients. One of the major underlying reasons for these controversies over glycemic control in critical illness is that the development of hyperglycemia and its intrinsic nature under these pathological conditions are not fully understood. Severe burn injury is one of the most serious forms of critical illness that are characterized by stress, hyperglycemia, inflammation, and hypermetabolic response. Studies have demonstrated that the postburn hyperglycemia is associated with injuryinduced insulin resistance possibly resulted from increased concentrations of hormones (e.g., glucocorticoids, growth hormone, and progesterone) or inflammatory cytokines (tumor necrosis factor -α) that oppose insulin release or the action of insulin on peripheral tissues (13). A prospective randomized study has suggested that glycemic control with intensive insulin treatment reduced the mortality in severely burned patients (14), whereas it has been found that it is often difficult to control the blood glucose. Impaired insulin sensitivity has been suggested to contribute to the persistent hyperglycemia and thus the adverse outcomes of burn injuries. However, the intrinsic nature and characteristics of burn-induced hyperglycemia and the mechanism underlying postburn insulin resistance are unclear. Advanced glycation endproducts (AGEs) formed in hyperglycemic environments are modifications of proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars. Carboxymethyllysine (CML) is a principal AGE that accumulates in vivo (15, 16). Studies have shown that AGEs contribute to the pathophysiology of multiple chronic diseases, including diabetes via the engagement of cellular receptors for AGEs (RAGE) (17, 18). Recently, AGEs were also reported to be involved in acute diseases, such as acute nerve injury and myocardial ischemic injury (19–21). It is suggestive that AGEs may be involved in severe burn–induced hyperglycemia and insulin resistance. In this study, we found that severe burn–induced blood glucose change presented a distinct pattern, and more importantly, early postburn hyperglycemia induced a rapid formation of AGEs which contributed to acute insulin resistance (AIR) and a more prominent hyperglycemia. Early glycemic control with insulin or inhibition of AGEs blunted postburn AIR in rats.
MATERIALS AND METHODS Rat Burn Model and Experiment Design Male Sprague-Dawley rats weighing 180–220 g were used. The experiments were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Fourth Military Medical University Committee on Animal Care. To investigate postburn insulin resistance, 72 rats were randomly and evenly assigned to subgroups of six time Critical Care Medicine
points at 0, 0.5, 1, 3, 6, and 12 hours after burn or sham burn (n = 6 per subgroup). To determine the effects of soluble form of receptor for advanced glycation endproducts (sRAGE) and insulin on postburn insulin resistance, another 18 rats were randomly and evenly assigned to groups of burn + vehicle, burn + sRAGE, and burn + insulin (n = 6 per group). It should be noted that more than 14 rats for each group were used in the examination of blood glucose and survival. The number of animals used in other experiments is indicated elsewhere. Rats were anesthetized by continuous 1.5–3% isoflurane in 100% oxygen using a face cone/mask. The rats in both sham and burn groups were shaved on the dorsal and ventral surface of the trunk. The rats in burn group received a 40% total burn surface area (135 cm2) by immersing the dorsum in 95°C heated water for 15 seconds and ventral surface for 8 seconds as described previously (22). Lactated Ringer’s solution (10 mL/ kg body weight) was intraperitoneally administered immediately after burns for resuscitation. After burns, the rats were housed in a temperature-controlled environment and given free access to water within 12-hour postburn. A weight- and time-matched sham group was treated in the same manner as the burn group; however, the sham group was immersed in lukewarm water. Determination of Blood Variables Blood glucose levels were measured using a glucose meter (Life-Scan, Milpitas, CA) at indicated time points. Serum insulin, glucagon, and cortisol levels were measured using commercial radioimmunoassay kits (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer’s instructions. Serum sRAGE and CML levels were measured using commercial detection kits (Abcam, Cambridge, MA; and Cell Biolabs, San Diego, CA) according to the manufacturer’s instructions. Glucose Tolerance Test and Insulin Tolerance Test Rats were administered an intraperitoneal injection of glucose (2 g/kg) or a hypodermic injection of insulin (0.5 U/kg). Whole blood glucose levels were determined at 0, 15, 30, 60, 90, and 120 minutes after the glucose challenge for the glucose tolerance test and at 0, 30, 60, 90, 120, and 240 minutes for the insulin tolerance test using tail clippings. Measurement of Cardiac Glucose Uptake Using MicroPET/CT In Vivo To visualize cardiac glucose uptake using microPET/CT imaging, each rat was IV injected with an average of 1 mCi of [18F]-fluorodeoxyglucose and then left for 30 minutes to allow the uptake of [18F]-fluorodeoxyglucose. After that, the animals were anesthetized using isoflurane, and the PET/CT images were acquired using a nanoScan PET/CT scanner (Mediso, Budapest, Hungary). Exogenous insulin (10 U/kg body weight) was intraperitoneally injected 20 minutes before imaging. Cell Culture H9C2 cells were cultured in Dulbecco’s-modified Eagle’s medium (Gibco, Frederick, MD) supplemented with 10% www.ccmjournal.org
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fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA) at 37°C under 5% Co2. Western Blot Analysis Proteins were extracted from hearts, skeletal muscles, or H9C2 cells. The phosphorylated protein kinase B (PKB/Akt) (Thr473) and phosphorylated glycogen synthase kinase 3β (GSK-3β) (Ser-9) were probed with specific antibodies (Cell Signaling, Santa Cruz, CA) as reported in our previous study (23). The pAkt or pGSK-3β immunoblots were then stripped with strip buffer at 50°C for 30 minutes and reblotted for total Akt or GSK-3β. Statistical Analysis Statistical significance was evaluated using Student t test for comparisons between two independent samples and Pearson simple correlation for the correlation calculation. The survival data were estimated using the Kaplan-Meier method and compared using the log-rank test. The results were expressed as means ± sem unless noted otherwise. A p value of less than 0.05 was considered statistically significant.
RESULTS Dynamic Change of Postburn Hyperglycemia and Its Correlation With Mortality Intermittent hyperglycemia presented in burned rats within 12-hour postburn. The first hyperglycemia (117.0 ± 3.1 vs 135.3 ± 6.2 mg/dL) appeared at 0.5 hour (H1) and the second hyperglycemia (109.0 ± 2.6 vs 180.7 ± 17.9 mg/dL) appeared at 3-hour (H2) postburn (n = 15 sham and 31 burned rats) (Fig. 1A). Following the intermittent hyperglycemia, 9 of 31 (29%) burned rats died at 5–10 hours after burns. More importantly, we found that the blood glucose level at 3 hours was associated with the prognosis of the burned rats: the mortality (50%, n = 16) in rats with severe hyperglycemia (blood glucose > 144 mg/dL) at 3 hours was higher than the mortality (7%, n = 15) of animals with mild hyperglycemia (blood glucose < 144 mg/dL) at 3 hours within 12-hour postburn (p 144 mg/dL, n = 16) had a lower survival rate than those with mild hyperglycemia at H2 (blood glucose < 144 mg/dL, n = 15) (p < 0.05). Serum glucagon (C) and cortisol (D), two stress hormones, increased significantly following severe burns (n = 6 for each subgroup). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01.
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Figure 2. Blunted insulin signaling at 3 hr following severe burns in rats. A, Serum insulin levels displayed an approximately five-fold increase within 3-hour postburn and an approximately 2.3-fold increase at 6- and 12-hour postburn (n = 6 for each subgroup). B, Representative Western blot results of the following insulin signal molecules in heart and skeletal muscle tissues of burned rats and sham rats: total Akt, phosphorylated AktSer-473, total GSK-3β, and phosphorylated GSK-3βSer-9. The statistical results of pAkt/Akt ratio in myocardium (C) and skeletal muscle (D) (n = 6 for each subgroup). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01.
The Occurrence of AIR Following Severe Burns The serum insulin level rose rapidly after burns from baseline of 30 to 145 mU/L at 0.5 hour (n = 6 for each subgroup) (Fig. 2A). Meanwhile, both myocardial and skeletal muscle Akt and GSK-3β phosphorylation levels transiently increased at 0.5- and 1-hour postburn but sustainably suppressed after 3-hour postburn (n = 6 for each subgroup) (Fig. 2, B–D; Fig. S1, Supplemental Digital Content 1, http://links.lww.com/ CCM/A961). More importantly, micro PET/CT study showed that in consistent with the dynamic changes of myocardial and skeletal muscle Akt phosphorylation, cardiac glucose uptake increased at 1 hour and then decreased at 3 hours following burns (Fig. 3A). Upon exogenous insulin stimulation (10 U/kg, injected 20 min before detection), cardiac glucose uptake increased at 1-hour postburn. However, this effect was largely blunted at 3- and 6-hour postburn (Fig. 3A). These results, together with the second rise of blood glucose level at 3-hour postburn, suggest that postburn insulin resistance developed and was most severe at 3 hours following burns. We next intraperitoneally administrated the animals with glucose or insulin at 3- and 6-hour postburn, respectively, Critical Care Medicine
to provide additional information about the whole-body responsiveness to insulin. As shown in Figure 3B, severe burns decreased the ability of insulin to lower glucose in burned rats at both 3- and 6-hour postburn (n = 6 for each group, p < 0.05). Similarly, following intraperitoneal glucose injection, glucose clearance was depressed at both 3 and 6 hours in burned rats (n = 6 for each group, p < 0.05) (Fig. 3C). These results clearly indicate that severe burn triggers systemic insulin resistance. Rapid AGEs Production Following Burns Contributes to Postburn AIR Serum CML level was markedly increased at 0.5 hour (11.6 ± 0.7 vs 14.6 ± 0.6 ng/mL) (p < 0.05) and peaked at 1-hour (10.3 ± 0.9 vs 18.6 ± 1.3 ng/mL) (p < 0.01) postburn (n = 6 for each subgroup) (Fig. 4A). More importantly, blockade of AGEs-RAGE interaction with sRAGE (known as a decoy of RAGE, 3 μg/kg, IV) immediately after burns not only resulted in decreased serum CML level at 1-hour postburn (n = 6 for each group, p < 0.01) (Fig. S2, Supplemental Digital Content 1, http://links. lww.com/CCM/A961) but also significantly increased the survival rate of severely burned rats (71% in 31 untreated burned www.ccmjournal.org
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Figure 3. Postburn insulin resistance in severely burned rats. A, Typical images of basal and exogenous insulin-stimulated (10 U/kg, IP injected 20 min before detection) cardiac glucose uptake measured by microPET/CT before and at 1, 3, and 6 hr after burns. B, Insulin tolerance test at 3- and 6-hour postburn (n = 6 for each group). C, Intraperitoneal glucose tolerance test at 3- and 6-hour postburn (n = 6 for each group). Data are expressed as mean ± sem. *p < 0.05 versus sham.
rats and 93% in 27 sRAGE-treated rats, p < 0.05) (Fig. S3, Supplemental Digital Content 1, http://links.lww.com/CCM/ A961). Interestingly, sRAGE treatment did not affect the first immediate hyperglycemia at 0.5-hour postburn but blunted the second hyperglycemia at 3-hour postburn (n = 15 for each group) (Fig. 4B), indicating that sRAGE alleviates burn injury– induced systemic insulin resistance. Indeed, sRAGE treatment increased Akt phosphorylation levels at 3-hour postburn in both myocardial and skeletal muscles of the severely burned rats (n = 6 for each group, p < 0.05) (Fig. 4C). Furthermore, improvement of insulin sensitivity by sRAGE was also manifested by glucose tolerance test. sRAGE improved the glucose tolerance at 3-hour postburn (n = 6 for each group, p < 0.05) (Fig. 4D). Taken together, our results suggest that the rapid increase in AGEs formation contributes, at least in part, to burn-induced AIR and hyperglycemia. To further determine whether AGEs have a direct role in the development of AIR, we next examined whether exogenous methylglyoxal (MG), an AGE precursor which binds to proteins and forms CML, can induce insulin resistance (24). MG administration (50 mg/kg, IV) increased serum CML levels by 11% at 1.5 hours after MG treatment (p < 0.05) and resulted in significant glucose intolerance as assessed by glucose tolerance test in rats without burns (p < 0.05) (n = 6 for each group) (Fig. 5A). e476
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Concomitantly, Akt phosphorylation in both myocardial and skeletal muscles was blunted (n = 6 for each group, p < 0.05) (Fig. 5B) at 2 hours after MG treatment. Furthermore, the cellular experiments performed on H9C2 cells showed that both basal and insulin-induced (100 nM for 30 min) Akt phosphorylation were suppressed following CML-modified bovine serum albumin challenge (10 μg/mL for 30 min) (n = 6, p < 0.05) (Fig. 5C), suggesting that AGEs can directly suppress insulin signaling. Early Glycemic Control With Insulin Blunted Postburn AIR via Inhibiting AGEs Production Previous studies showed that hyperglycemia is a crucial factor for the formation of AGEs in vivo, and we therefore examined the effect of glycemic control with insulin on AGEs formation and AIR following severe burns. Profoundly, early insulin treatment (2.5 U/kg, subcutaneous injection immediately after burns) prevented the occurrence of not only the first hyperglycemia at 0.5-hour postburn but also the second hyperglycemia at 3 hours and thereafter within 12 hours following burns (n = 15 for each group) (Fig. 4C). As expected, the serum CML concentrations at 1-hour postburn decreased significantly compared with those in the untreated burned rats (n = 6 for each group, p < 0.01) (Fig. 6A) together with alleviated postburn AIR at 3-hour postburn as assessed by glucose June 2014 • Volume 42 • Number 6
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Figure 4. Elevated serum advanced glycation endproducts (AGEs) production contributed to the development of postburn insulin resistance. A, Serum carboxymethyllysine (CML), a principal AGEs in vivo, increased at 0.5 hr and peaked at 1-hour postburn (n = 6 for each subgroup). B, Blood glucose after early soluble form of receptor for advanced glycation endproducts (sRAGE) (3 μg/kg, IV, immediately after burns) or insulin (2.5 U/kg, subcutaneous injection immediately after burns) treatment in burned rats (n = 15 for each group). C, Treatment with sRAGE increased Akt phosphorylation levels at 3-hour postburn in both cardiac and skeletal muscle tissues of burned rats (n = 6 for each group). D, Treatment with sRAGE improved postburn insulin sensitivity as assessed by intraperitoneal glucose tolerance test at 3-hour postburn (n = 6 for each group). *p < 0.05 versus sham. #p < 0.05 versus burn + vehicle (3 hr). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01.
tolerance test (n = 6 for each group, p < 0.05) (Fig. 6B). Importantly, the survival rate of rats treated with insulin (n = 43) was higher than that in vehicle-treated group (n = 31) within 12-hour postburn (95% vs 71%, p < 0.01) (Fig. S3, Supplemental Digital Content 1, http://links.lww.com/CCM/A961). These data indicate that early insulin treatment exerts protective effect against burn injury via, at least in part, preventing burn-induced AGEs formation.
DISCUSSION We have made the following novel findings in this study. First, severe burn induced AIR that was positively correlated with mortality. Second, there was a rapid increase in serum AGEs production following severe burns, which contributed to the development of AIR. Third, early insulin treatment alleviated AIR and increased survival, at least in part, via suppressing serum AGEs production in response to severe burn in rats. Numerous studies have documented hyperglycemia following burn, trauma, myocardial infarction, and some surgeries. However, whether hyperglycemia represents a beneficial physiological stress response or a detrimental effect of poor prognosis is still in debate. This study demonstrated a distinct pattern of blood glucose changes following severe burns. The first Critical Care Medicine
hyperglycemia occurred as early as 0.5-hour postburn, and this immediate elevated blood glucose is what is generally believed to be a compensatory mechanism of stress, as evidenced by the increased release of stress hormones, such as cortisol and glucagon. The second occurrence of hyperglycemia at 3 hours following burns was positively correlated with mortality of the severely burned rats. Notably, in mild burn, only initial hyperglycemia appeared without the secondary hyperglycemia response (data not shown), indicating that hyperglycemic response in acute illness is a normal physiological defense mechanism, whereas in severe illness conditions, the excessive and prolonged increase in blood glucose leads to organ damage, resulting in increased mortality (25). Thus, targeted glycemic control could potentially improve clinical outcomes. More importantly, we found, for the first time, that the second occurrence of hyperglycemia that is detrimental is mainly attributable to insulin resistance as evidenced by restrained activation of insulin signaling of Akt and GSK-3β at 3-hour postburn. In addition, during this period of time, the occurrence of hyperinsulinemia, decreased cardiac glucose uptake, and blunted insulin sensitivity further indicated the development of insulin resistance. This rapid onset of insulin resistance, that is, AIR, occurred as early as 3 hours following severe burns, which presented a dynamic model of change, being most www.ccmjournal.org
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valuable evidence than observational studies. Compared with insulin resistance developed chronically in diabetics, the mechanism underlying AIR in acute injuries and critical illness is poorly understood. The development of AIR is complex and might involve multiple causative factors and intracellular signaling pathways (13). In this study, we found a rapid and transient increase in the production of serum AGEs. More importantly, we found that AGEs formation contributed to the development of AIR following severe burns. It has been shown that AGEs are produced in response to hyperglycemia and oxidative stress (17) and disturb the actions of insulin in multiple cell types, including adipocytes, myocytes, and hepatocytes Figure 5. Advanced glycation endproducts (AGEs) inhibited Akt phosphorylation both in vivo and in vitro. A, Methylglyoxal (MG, an AGE precursor) treatment (50 mg/kg, IV) increased serum carboxymethyllysine (CML) (26), marking an important at 1.5 hr and decreased insulin sensitivity as assessed by intraperitoneal glucose tolerance test at 2 hr after MG role of AGEs in the pathogentreatment in rats without burns (n = 6 for each group). B, MG treatment decreased Akt phosphorylation at 2 hr after esis of insulin resistance. In this MG treatment in both cardiac and skeletal muscles of rats without burns (n = 6 for each group). C, CML-bovine serum albumin (BSA) challenge (10 μg/mL for 30 min) decreased both basal and insulin-stimulated (100 nM for regard, Unoki-Kubota et al (27) 30 min) Akt phosphorylation in H9C2 cells (n = 6). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01. provided evidence that the inhibition of AGEs formation improved insulin sensitivity in obese and type 2 diabetic mice. severe at 3-hour postburn and partially reversed at 6-hour In this study, there is a 116% increase in blood glucose concenpostburn. The absence of the AIR-related secondary hyperglytrations within 1-hour postburn, accompanied with increased cemia response following mild burns (data not shown) further circulating CML levels postburn, consistent with a previous indicates that AIR is predicative of increased severity and poor outcomes of acute illness/injuries. These findings are based on notion that acute hyperglycemia can induce AGEs formation dynamic changes of several response variables over a longer in healthy volunteers (28). Furthermore, we have shown that time span and thus provide more reliable information and blocking the AGEs-RAGE interaction with sRAGE diminished the blood glucose level and attenuated AIR. Our data not only define AGEs-RAGE signaling cascade as a crucial mechanism underlying severe burn–induced insulin resistance and resultant hyperglycemia and poor clinical outcome but also argue against the conventional knowledge that AGEs are only the products of chronic and sustained elevated blood glucose. The role of AGEs in triggering AIR is illustrated in Figure 7. Taken together, these results suggest that strategies Figure 6. Early glycemic control with insulin inhibited advanced glycation endproducts production and blunted aimed at limiting AGEs accupostburn acute insulin resistance. A, Insulin treatment (2.5 U/kg, subcutaneous injection immediately after burns) decreased serum carboxymethyllysine (CML) concentrations at 1-hour postburn (n = 6 for each group). mulation or activity may bear B, Insulin treatment improved insulin sensitivity at 3-hour postburn as assessed by intraperitoneal glucose important clinical implications tolerance test (n = 6 for each group). *p < 0.05 versus sham. #p < 0.05 versus burn + vehicle (3 hr). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01. in the context of severe burn. e478
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Figure 7. Schematic figure illustrating the time-course relationship among postburn hyperglycemia, advanced glycation endproducts (AGEs) production, and insulin sensitivity. The first occurrence of postburn hyperglycemia resulting from elevated glucose release leads to serum AGEs production. The elevated AGEs contribute to the development of acute insulin resistance (AIR). Glycemic control with early insulin treatment or scavenging serum AGEs with soluble form of receptor for advanced glycation endproducts (sRAGE) significantly blunts postburn AIR.
This study provided further substantial evidence that timely glycemic control with early insulin treatment significantly reduced AGEs production and ameliorated AIR following severe burns. Immediate insulin administration in severely burned rats resulted in prevention of the first occurrence of hyperglycemia. In addition to lowering blood glucose, insulin also exerts antioxidative (29) and anti-inflammatory (30) actions, which together may contribute to the reduction of AGEs generation. The reduced AGEs production prevented AGEs-mediated AIR, which was substantiated by the absence of the second occurrence of hyperglycemia.
CONCLUSIONS This study demonstrates that severe burn–induced AIR is mediated at least in part by elevated production of AGEs and that AIR is positively correlated with the mortality of severely burned rats. Early hyperglycemic control with insulin or inhibition of AGEs with sRAGE significantly ameliorated AIR and increased animal survival following severe burns.
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formation of intracellular reactive oxygen species. Diabetes 2006; 55:1289–1299 25. Jeschke MG, Gauglitz GG, Finnerty CC, et al: Survivors versus nonsurvivors postburn: Differences in inflammatory and hypermetabolic trajectories. Ann Surg 2013; 259:814–823 26. Puddu A, Viviani GL: Advanced glycation endproducts and diabetes. Beyond vascular complications. Endocr Metab Immune Disord Drug Targets 2011; 11:132–140 27. Unoki-Kubota H, Yamagishi S, Takeuchi M, et al: Pyridoxamine, an inhibitor of advanced glycation end product (AGE) formation ameliorates insulin resistance in obese, type 2 diabetic mice. Protein Pept Lett 2010; 17:1177–1181 28. Schiekofer S, Andrassy M, Chen J, et al: Acute hyperglycemia causes intracellular formation of CML and activation of ras, p42/44 MAPK, and nuclear factor kappaB in PBMCs. Diabetes 2003; 52:621–633 29. Ji L, Fu F, Zhang L, et al: Insulin attenuates myocardial ischemia/ reperfusion injury via reducing oxidative/nitrative stress. Am J Physiol Endocrinol Metab 2010; 298:E871–880 30. Li J, Zhang H, Wu F, et al: Insulin inhibits tumor necrosis factor-alpha induction in myocardial ischemia/reperfusion: Role of Akt and endothelial nitric oxide synthase phosphorylation. Crit Care Med 2008; 36:1551–1558
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Objective: Hyperglycemia often occurs in severe burns; however, the underlying mechanisms and importance of managing postburn hyperglycemia are not well recognized. This study was designed to investigate the dynamic changes of postburn hyperglycemia and the underlying mechanisms and to evaluate whether early glycemic control is beneficial in severe burns. Design: Prospective, randomized experimental study. Setting: Animal research laboratory. Subjects: Sprague-Dawley rats. Interventions: Anesthetized rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive vehicle, insulin, and a soluble form of receptor for advanced glycation endproducts treatments. An in Department of Physiology, School of Basic Medical Sciences, The Fourth Military Medical University, Xi’an, China. 2 Department of Burns and Cutaneous Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. 3 Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. 4 Department of Anesthesiology, Xijing Hospital, The Fourth Military Medical University, Xi’an, China. 5 Institute of Molecular Medicine, Peking University, Beijing, China. Drs. Zhang, Xu, and Cai contributed equally to this study. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ ccmjournal). This work was supported by grants for article research from the National Basic Research Program of China (Nos. 2013CB531204, 2012CB518000), the State Key Program of National Natural Science Foundation of China (No. 81030005), and the National Natural Science Foundation of China (Nos. 81170184, 81270301). Drs. Gao, Zhang, Cai, Ji, Jia Li, Cao, Jun Li, Hu, Yan Li, Wang, Xiong, and Xiao received support for article research from the National Basic Research Program of China (No. 2013CB531204), the State Key Program of National Natural Science Foundation of China (No. 81030005), and the National Natural Science Foundation of China (Nos. 81170184, 81270301). Dr. Xu received support for article research. For information regarding this article, E-mail: [email protected]; xiaor@ pku.edu.cn Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000314 1
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vitro study was performed on cultured H9C2 cells subjected to vehicle or carboxymethyllysine treatment. Measurements and Main Results: We found that blood glucose change presented a distinct pattern with two occurrences of hyperglycemia at 0.5- and 3-hour postburn, respectively. Acute insulin resistance evidenced by impaired insulin signaling and glucose uptake occurred at 3-hour postburn, which was associated with the second hyperglycemia and positively correlated with mortality. Mechanistically, we found that serum carboxymethyllysine, a dominant species of advanced glycation endproducts, increased within 1-hour postburn, preceding the occurrence of insulin resistance. More importantly, treatment of animals with soluble form of receptor for advanced glycation endproducts, blockade of advanced glycation endproducts signaling, alleviated severe burn– induced insulin resistance. In addition, early hyperglycemic control with insulin not only reduced serum carboxymethyllysine but also blunted postburn insulin resistance and reduced mortality. Conclusions: These findings suggest that severe burn–induced insulin resistance is partly at least mediated by serum advanced glycation endproducts and positively correlated with mortality. Early glycemic control with insulin or inhibition of advanced glycation endproducts with soluble form of receptor for advanced glycation endproducts ameliorates postburn insulin resistance. (Crit Care Med 2014; 42:e472–e480) Key Words: acute insulin resistance; advanced glycation endproducts; burn injury; carboxymethyllysine; insulin; soluble form of receptor for advanced glycation endproducts
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yperglycemia often develops in critical illness, especially following trauma, burn, stroke, and surgery. This trauma-induced hyperglycemia has long been believed to be an adaptive stress response and was initially thought to be a beneficial adaptation or paraphenomenon essential to provide fuel for vital organs and thus better left undisturbed (1). However, recent studies have suggested that hyperglycemia is potentially toxic (2–5) and is identified as an independent risk factor for adverse outcomes in critical illness (4–7). Several randomized controlled trials of strict glucose control, especially the June 2014 • Volume 42 • Number 6
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landmark study by van den Berghe et al (2), have shown the benefits of blood glucose control with intensive insulin treatment in critically ill patients, while subsequent studies (8–12) had mixed or even conflicting results. For example, the Normoglycaemia in Intensive Care Evaluation and Survival Using Glucose Algorithm Regulation study (11) does not support intensive insulin treatment, but it only demonstrated that “strict” glucose control may be more harmful than conventional glucose control. Uncontrolled hyperglycemia is still regarded as undesirable in critically ill patients. One of the major underlying reasons for these controversies over glycemic control in critical illness is that the development of hyperglycemia and its intrinsic nature under these pathological conditions are not fully understood. Severe burn injury is one of the most serious forms of critical illness that are characterized by stress, hyperglycemia, inflammation, and hypermetabolic response. Studies have demonstrated that the postburn hyperglycemia is associated with injuryinduced insulin resistance possibly resulted from increased concentrations of hormones (e.g., glucocorticoids, growth hormone, and progesterone) or inflammatory cytokines (tumor necrosis factor -α) that oppose insulin release or the action of insulin on peripheral tissues (13). A prospective randomized study has suggested that glycemic control with intensive insulin treatment reduced the mortality in severely burned patients (14), whereas it has been found that it is often difficult to control the blood glucose. Impaired insulin sensitivity has been suggested to contribute to the persistent hyperglycemia and thus the adverse outcomes of burn injuries. However, the intrinsic nature and characteristics of burn-induced hyperglycemia and the mechanism underlying postburn insulin resistance are unclear. Advanced glycation endproducts (AGEs) formed in hyperglycemic environments are modifications of proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars. Carboxymethyllysine (CML) is a principal AGE that accumulates in vivo (15, 16). Studies have shown that AGEs contribute to the pathophysiology of multiple chronic diseases, including diabetes via the engagement of cellular receptors for AGEs (RAGE) (17, 18). Recently, AGEs were also reported to be involved in acute diseases, such as acute nerve injury and myocardial ischemic injury (19–21). It is suggestive that AGEs may be involved in severe burn–induced hyperglycemia and insulin resistance. In this study, we found that severe burn–induced blood glucose change presented a distinct pattern, and more importantly, early postburn hyperglycemia induced a rapid formation of AGEs which contributed to acute insulin resistance (AIR) and a more prominent hyperglycemia. Early glycemic control with insulin or inhibition of AGEs blunted postburn AIR in rats.
MATERIALS AND METHODS Rat Burn Model and Experiment Design Male Sprague-Dawley rats weighing 180–220 g were used. The experiments were performed according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Fourth Military Medical University Committee on Animal Care. To investigate postburn insulin resistance, 72 rats were randomly and evenly assigned to subgroups of six time Critical Care Medicine
points at 0, 0.5, 1, 3, 6, and 12 hours after burn or sham burn (n = 6 per subgroup). To determine the effects of soluble form of receptor for advanced glycation endproducts (sRAGE) and insulin on postburn insulin resistance, another 18 rats were randomly and evenly assigned to groups of burn + vehicle, burn + sRAGE, and burn + insulin (n = 6 per group). It should be noted that more than 14 rats for each group were used in the examination of blood glucose and survival. The number of animals used in other experiments is indicated elsewhere. Rats were anesthetized by continuous 1.5–3% isoflurane in 100% oxygen using a face cone/mask. The rats in both sham and burn groups were shaved on the dorsal and ventral surface of the trunk. The rats in burn group received a 40% total burn surface area (135 cm2) by immersing the dorsum in 95°C heated water for 15 seconds and ventral surface for 8 seconds as described previously (22). Lactated Ringer’s solution (10 mL/ kg body weight) was intraperitoneally administered immediately after burns for resuscitation. After burns, the rats were housed in a temperature-controlled environment and given free access to water within 12-hour postburn. A weight- and time-matched sham group was treated in the same manner as the burn group; however, the sham group was immersed in lukewarm water. Determination of Blood Variables Blood glucose levels were measured using a glucose meter (Life-Scan, Milpitas, CA) at indicated time points. Serum insulin, glucagon, and cortisol levels were measured using commercial radioimmunoassay kits (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer’s instructions. Serum sRAGE and CML levels were measured using commercial detection kits (Abcam, Cambridge, MA; and Cell Biolabs, San Diego, CA) according to the manufacturer’s instructions. Glucose Tolerance Test and Insulin Tolerance Test Rats were administered an intraperitoneal injection of glucose (2 g/kg) or a hypodermic injection of insulin (0.5 U/kg). Whole blood glucose levels were determined at 0, 15, 30, 60, 90, and 120 minutes after the glucose challenge for the glucose tolerance test and at 0, 30, 60, 90, 120, and 240 minutes for the insulin tolerance test using tail clippings. Measurement of Cardiac Glucose Uptake Using MicroPET/CT In Vivo To visualize cardiac glucose uptake using microPET/CT imaging, each rat was IV injected with an average of 1 mCi of [18F]-fluorodeoxyglucose and then left for 30 minutes to allow the uptake of [18F]-fluorodeoxyglucose. After that, the animals were anesthetized using isoflurane, and the PET/CT images were acquired using a nanoScan PET/CT scanner (Mediso, Budapest, Hungary). Exogenous insulin (10 U/kg body weight) was intraperitoneally injected 20 minutes before imaging. Cell Culture H9C2 cells were cultured in Dulbecco’s-modified Eagle’s medium (Gibco, Frederick, MD) supplemented with 10% www.ccmjournal.org
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fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA) at 37°C under 5% Co2. Western Blot Analysis Proteins were extracted from hearts, skeletal muscles, or H9C2 cells. The phosphorylated protein kinase B (PKB/Akt) (Thr473) and phosphorylated glycogen synthase kinase 3β (GSK-3β) (Ser-9) were probed with specific antibodies (Cell Signaling, Santa Cruz, CA) as reported in our previous study (23). The pAkt or pGSK-3β immunoblots were then stripped with strip buffer at 50°C for 30 minutes and reblotted for total Akt or GSK-3β. Statistical Analysis Statistical significance was evaluated using Student t test for comparisons between two independent samples and Pearson simple correlation for the correlation calculation. The survival data were estimated using the Kaplan-Meier method and compared using the log-rank test. The results were expressed as means ± sem unless noted otherwise. A p value of less than 0.05 was considered statistically significant.
RESULTS Dynamic Change of Postburn Hyperglycemia and Its Correlation With Mortality Intermittent hyperglycemia presented in burned rats within 12-hour postburn. The first hyperglycemia (117.0 ± 3.1 vs 135.3 ± 6.2 mg/dL) appeared at 0.5 hour (H1) and the second hyperglycemia (109.0 ± 2.6 vs 180.7 ± 17.9 mg/dL) appeared at 3-hour (H2) postburn (n = 15 sham and 31 burned rats) (Fig. 1A). Following the intermittent hyperglycemia, 9 of 31 (29%) burned rats died at 5–10 hours after burns. More importantly, we found that the blood glucose level at 3 hours was associated with the prognosis of the burned rats: the mortality (50%, n = 16) in rats with severe hyperglycemia (blood glucose > 144 mg/dL) at 3 hours was higher than the mortality (7%, n = 15) of animals with mild hyperglycemia (blood glucose < 144 mg/dL) at 3 hours within 12-hour postburn (p 144 mg/dL, n = 16) had a lower survival rate than those with mild hyperglycemia at H2 (blood glucose < 144 mg/dL, n = 15) (p < 0.05). Serum glucagon (C) and cortisol (D), two stress hormones, increased significantly following severe burns (n = 6 for each subgroup). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01.
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Figure 2. Blunted insulin signaling at 3 hr following severe burns in rats. A, Serum insulin levels displayed an approximately five-fold increase within 3-hour postburn and an approximately 2.3-fold increase at 6- and 12-hour postburn (n = 6 for each subgroup). B, Representative Western blot results of the following insulin signal molecules in heart and skeletal muscle tissues of burned rats and sham rats: total Akt, phosphorylated AktSer-473, total GSK-3β, and phosphorylated GSK-3βSer-9. The statistical results of pAkt/Akt ratio in myocardium (C) and skeletal muscle (D) (n = 6 for each subgroup). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01.
The Occurrence of AIR Following Severe Burns The serum insulin level rose rapidly after burns from baseline of 30 to 145 mU/L at 0.5 hour (n = 6 for each subgroup) (Fig. 2A). Meanwhile, both myocardial and skeletal muscle Akt and GSK-3β phosphorylation levels transiently increased at 0.5- and 1-hour postburn but sustainably suppressed after 3-hour postburn (n = 6 for each subgroup) (Fig. 2, B–D; Fig. S1, Supplemental Digital Content 1, http://links.lww.com/ CCM/A961). More importantly, micro PET/CT study showed that in consistent with the dynamic changes of myocardial and skeletal muscle Akt phosphorylation, cardiac glucose uptake increased at 1 hour and then decreased at 3 hours following burns (Fig. 3A). Upon exogenous insulin stimulation (10 U/kg, injected 20 min before detection), cardiac glucose uptake increased at 1-hour postburn. However, this effect was largely blunted at 3- and 6-hour postburn (Fig. 3A). These results, together with the second rise of blood glucose level at 3-hour postburn, suggest that postburn insulin resistance developed and was most severe at 3 hours following burns. We next intraperitoneally administrated the animals with glucose or insulin at 3- and 6-hour postburn, respectively, Critical Care Medicine
to provide additional information about the whole-body responsiveness to insulin. As shown in Figure 3B, severe burns decreased the ability of insulin to lower glucose in burned rats at both 3- and 6-hour postburn (n = 6 for each group, p < 0.05). Similarly, following intraperitoneal glucose injection, glucose clearance was depressed at both 3 and 6 hours in burned rats (n = 6 for each group, p < 0.05) (Fig. 3C). These results clearly indicate that severe burn triggers systemic insulin resistance. Rapid AGEs Production Following Burns Contributes to Postburn AIR Serum CML level was markedly increased at 0.5 hour (11.6 ± 0.7 vs 14.6 ± 0.6 ng/mL) (p < 0.05) and peaked at 1-hour (10.3 ± 0.9 vs 18.6 ± 1.3 ng/mL) (p < 0.01) postburn (n = 6 for each subgroup) (Fig. 4A). More importantly, blockade of AGEs-RAGE interaction with sRAGE (known as a decoy of RAGE, 3 μg/kg, IV) immediately after burns not only resulted in decreased serum CML level at 1-hour postburn (n = 6 for each group, p < 0.01) (Fig. S2, Supplemental Digital Content 1, http://links. lww.com/CCM/A961) but also significantly increased the survival rate of severely burned rats (71% in 31 untreated burned www.ccmjournal.org
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Figure 3. Postburn insulin resistance in severely burned rats. A, Typical images of basal and exogenous insulin-stimulated (10 U/kg, IP injected 20 min before detection) cardiac glucose uptake measured by microPET/CT before and at 1, 3, and 6 hr after burns. B, Insulin tolerance test at 3- and 6-hour postburn (n = 6 for each group). C, Intraperitoneal glucose tolerance test at 3- and 6-hour postburn (n = 6 for each group). Data are expressed as mean ± sem. *p < 0.05 versus sham.
rats and 93% in 27 sRAGE-treated rats, p < 0.05) (Fig. S3, Supplemental Digital Content 1, http://links.lww.com/CCM/ A961). Interestingly, sRAGE treatment did not affect the first immediate hyperglycemia at 0.5-hour postburn but blunted the second hyperglycemia at 3-hour postburn (n = 15 for each group) (Fig. 4B), indicating that sRAGE alleviates burn injury– induced systemic insulin resistance. Indeed, sRAGE treatment increased Akt phosphorylation levels at 3-hour postburn in both myocardial and skeletal muscles of the severely burned rats (n = 6 for each group, p < 0.05) (Fig. 4C). Furthermore, improvement of insulin sensitivity by sRAGE was also manifested by glucose tolerance test. sRAGE improved the glucose tolerance at 3-hour postburn (n = 6 for each group, p < 0.05) (Fig. 4D). Taken together, our results suggest that the rapid increase in AGEs formation contributes, at least in part, to burn-induced AIR and hyperglycemia. To further determine whether AGEs have a direct role in the development of AIR, we next examined whether exogenous methylglyoxal (MG), an AGE precursor which binds to proteins and forms CML, can induce insulin resistance (24). MG administration (50 mg/kg, IV) increased serum CML levels by 11% at 1.5 hours after MG treatment (p < 0.05) and resulted in significant glucose intolerance as assessed by glucose tolerance test in rats without burns (p < 0.05) (n = 6 for each group) (Fig. 5A). e476
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Concomitantly, Akt phosphorylation in both myocardial and skeletal muscles was blunted (n = 6 for each group, p < 0.05) (Fig. 5B) at 2 hours after MG treatment. Furthermore, the cellular experiments performed on H9C2 cells showed that both basal and insulin-induced (100 nM for 30 min) Akt phosphorylation were suppressed following CML-modified bovine serum albumin challenge (10 μg/mL for 30 min) (n = 6, p < 0.05) (Fig. 5C), suggesting that AGEs can directly suppress insulin signaling. Early Glycemic Control With Insulin Blunted Postburn AIR via Inhibiting AGEs Production Previous studies showed that hyperglycemia is a crucial factor for the formation of AGEs in vivo, and we therefore examined the effect of glycemic control with insulin on AGEs formation and AIR following severe burns. Profoundly, early insulin treatment (2.5 U/kg, subcutaneous injection immediately after burns) prevented the occurrence of not only the first hyperglycemia at 0.5-hour postburn but also the second hyperglycemia at 3 hours and thereafter within 12 hours following burns (n = 15 for each group) (Fig. 4C). As expected, the serum CML concentrations at 1-hour postburn decreased significantly compared with those in the untreated burned rats (n = 6 for each group, p < 0.01) (Fig. 6A) together with alleviated postburn AIR at 3-hour postburn as assessed by glucose June 2014 • Volume 42 • Number 6
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Figure 4. Elevated serum advanced glycation endproducts (AGEs) production contributed to the development of postburn insulin resistance. A, Serum carboxymethyllysine (CML), a principal AGEs in vivo, increased at 0.5 hr and peaked at 1-hour postburn (n = 6 for each subgroup). B, Blood glucose after early soluble form of receptor for advanced glycation endproducts (sRAGE) (3 μg/kg, IV, immediately after burns) or insulin (2.5 U/kg, subcutaneous injection immediately after burns) treatment in burned rats (n = 15 for each group). C, Treatment with sRAGE increased Akt phosphorylation levels at 3-hour postburn in both cardiac and skeletal muscle tissues of burned rats (n = 6 for each group). D, Treatment with sRAGE improved postburn insulin sensitivity as assessed by intraperitoneal glucose tolerance test at 3-hour postburn (n = 6 for each group). *p < 0.05 versus sham. #p < 0.05 versus burn + vehicle (3 hr). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01.
tolerance test (n = 6 for each group, p < 0.05) (Fig. 6B). Importantly, the survival rate of rats treated with insulin (n = 43) was higher than that in vehicle-treated group (n = 31) within 12-hour postburn (95% vs 71%, p < 0.01) (Fig. S3, Supplemental Digital Content 1, http://links.lww.com/CCM/A961). These data indicate that early insulin treatment exerts protective effect against burn injury via, at least in part, preventing burn-induced AGEs formation.
DISCUSSION We have made the following novel findings in this study. First, severe burn induced AIR that was positively correlated with mortality. Second, there was a rapid increase in serum AGEs production following severe burns, which contributed to the development of AIR. Third, early insulin treatment alleviated AIR and increased survival, at least in part, via suppressing serum AGEs production in response to severe burn in rats. Numerous studies have documented hyperglycemia following burn, trauma, myocardial infarction, and some surgeries. However, whether hyperglycemia represents a beneficial physiological stress response or a detrimental effect of poor prognosis is still in debate. This study demonstrated a distinct pattern of blood glucose changes following severe burns. The first Critical Care Medicine
hyperglycemia occurred as early as 0.5-hour postburn, and this immediate elevated blood glucose is what is generally believed to be a compensatory mechanism of stress, as evidenced by the increased release of stress hormones, such as cortisol and glucagon. The second occurrence of hyperglycemia at 3 hours following burns was positively correlated with mortality of the severely burned rats. Notably, in mild burn, only initial hyperglycemia appeared without the secondary hyperglycemia response (data not shown), indicating that hyperglycemic response in acute illness is a normal physiological defense mechanism, whereas in severe illness conditions, the excessive and prolonged increase in blood glucose leads to organ damage, resulting in increased mortality (25). Thus, targeted glycemic control could potentially improve clinical outcomes. More importantly, we found, for the first time, that the second occurrence of hyperglycemia that is detrimental is mainly attributable to insulin resistance as evidenced by restrained activation of insulin signaling of Akt and GSK-3β at 3-hour postburn. In addition, during this period of time, the occurrence of hyperinsulinemia, decreased cardiac glucose uptake, and blunted insulin sensitivity further indicated the development of insulin resistance. This rapid onset of insulin resistance, that is, AIR, occurred as early as 3 hours following severe burns, which presented a dynamic model of change, being most www.ccmjournal.org
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valuable evidence than observational studies. Compared with insulin resistance developed chronically in diabetics, the mechanism underlying AIR in acute injuries and critical illness is poorly understood. The development of AIR is complex and might involve multiple causative factors and intracellular signaling pathways (13). In this study, we found a rapid and transient increase in the production of serum AGEs. More importantly, we found that AGEs formation contributed to the development of AIR following severe burns. It has been shown that AGEs are produced in response to hyperglycemia and oxidative stress (17) and disturb the actions of insulin in multiple cell types, including adipocytes, myocytes, and hepatocytes Figure 5. Advanced glycation endproducts (AGEs) inhibited Akt phosphorylation both in vivo and in vitro. A, Methylglyoxal (MG, an AGE precursor) treatment (50 mg/kg, IV) increased serum carboxymethyllysine (CML) (26), marking an important at 1.5 hr and decreased insulin sensitivity as assessed by intraperitoneal glucose tolerance test at 2 hr after MG role of AGEs in the pathogentreatment in rats without burns (n = 6 for each group). B, MG treatment decreased Akt phosphorylation at 2 hr after esis of insulin resistance. In this MG treatment in both cardiac and skeletal muscles of rats without burns (n = 6 for each group). C, CML-bovine serum albumin (BSA) challenge (10 μg/mL for 30 min) decreased both basal and insulin-stimulated (100 nM for regard, Unoki-Kubota et al (27) 30 min) Akt phosphorylation in H9C2 cells (n = 6). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01. provided evidence that the inhibition of AGEs formation improved insulin sensitivity in obese and type 2 diabetic mice. severe at 3-hour postburn and partially reversed at 6-hour In this study, there is a 116% increase in blood glucose concenpostburn. The absence of the AIR-related secondary hyperglytrations within 1-hour postburn, accompanied with increased cemia response following mild burns (data not shown) further circulating CML levels postburn, consistent with a previous indicates that AIR is predicative of increased severity and poor outcomes of acute illness/injuries. These findings are based on notion that acute hyperglycemia can induce AGEs formation dynamic changes of several response variables over a longer in healthy volunteers (28). Furthermore, we have shown that time span and thus provide more reliable information and blocking the AGEs-RAGE interaction with sRAGE diminished the blood glucose level and attenuated AIR. Our data not only define AGEs-RAGE signaling cascade as a crucial mechanism underlying severe burn–induced insulin resistance and resultant hyperglycemia and poor clinical outcome but also argue against the conventional knowledge that AGEs are only the products of chronic and sustained elevated blood glucose. The role of AGEs in triggering AIR is illustrated in Figure 7. Taken together, these results suggest that strategies Figure 6. Early glycemic control with insulin inhibited advanced glycation endproducts production and blunted aimed at limiting AGEs accupostburn acute insulin resistance. A, Insulin treatment (2.5 U/kg, subcutaneous injection immediately after burns) decreased serum carboxymethyllysine (CML) concentrations at 1-hour postburn (n = 6 for each group). mulation or activity may bear B, Insulin treatment improved insulin sensitivity at 3-hour postburn as assessed by intraperitoneal glucose important clinical implications tolerance test (n = 6 for each group). *p < 0.05 versus sham. #p < 0.05 versus burn + vehicle (3 hr). Data are expressed as mean ± sem. *p < 0.05 and **p < 0.01. in the context of severe burn. e478
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Figure 7. Schematic figure illustrating the time-course relationship among postburn hyperglycemia, advanced glycation endproducts (AGEs) production, and insulin sensitivity. The first occurrence of postburn hyperglycemia resulting from elevated glucose release leads to serum AGEs production. The elevated AGEs contribute to the development of acute insulin resistance (AIR). Glycemic control with early insulin treatment or scavenging serum AGEs with soluble form of receptor for advanced glycation endproducts (sRAGE) significantly blunts postburn AIR.
This study provided further substantial evidence that timely glycemic control with early insulin treatment significantly reduced AGEs production and ameliorated AIR following severe burns. Immediate insulin administration in severely burned rats resulted in prevention of the first occurrence of hyperglycemia. In addition to lowering blood glucose, insulin also exerts antioxidative (29) and anti-inflammatory (30) actions, which together may contribute to the reduction of AGEs generation. The reduced AGEs production prevented AGEs-mediated AIR, which was substantiated by the absence of the second occurrence of hyperglycemia.
CONCLUSIONS This study demonstrates that severe burn–induced AIR is mediated at least in part by elevated production of AGEs and that AIR is positively correlated with the mortality of severely burned rats. Early hyperglycemic control with insulin or inhibition of AGEs with sRAGE significantly ameliorated AIR and increased animal survival following severe burns.
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June 2014 • Volume 42 • Number 6
