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Department of Intensive Care Medicine, Burn Unit and Center for Experimental Surgery and Anesthesiology (F.W., M.M., G.V.d.B.), Laboratory for Experimental Medicine and Endocrinology (A.-P.G., E.V.H., W.C., C.M.), Catholic University of Leuven, B-3000 Leuven, Belgium
Address all correspondence and requests for reprints to: Frank Weekers, M.D., Departement of Intensive Care Medicine, University Hospitals of Leuven, Herestraat 49, B-3000 Leuven, Belgium. E-mail: Frank.Weekers{at}uz.kuleuven.ac.be.
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Abstract |
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Introduction |
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Hyperglycemia and insulin resistance are uniformly present in the critically ill (3). No scientifically supported treatment guidelines were available until very recently. We have been able to show that intensive insulin treatment to maintain blood glucose concentration normal (between 4.4 and 6.1 mmol/liter) in prolonged critically ill patients significantly reduces morbidity and mortality (4). There was a striking reduction in the incidence of severe infections and of lethal multiple organ failure. These findings indicated that intensive insulin therapy may exert its beneficial effects on the outcome of critical illness partially by improving the innate immune response. It is well known that diabetic patients are susceptible to postoperative infections (5) due to changes in humoral and cellular immunity that has been linked to the duration of hyperglycemia. It remains unclear, however, whether acute stress-induced hyperglycemia has similar effects.
An animal model of prolonged critical illness in rabbits has recently been shown to reveal the metabolic and endocrine changes observed in patients with insulin resistance and hyperglycemia, transient stress-induced GH release, and the occurrence of a low T3 syndrome (6). It is hitherto unknown to what extent the changes in glucose metabolism contribute to the altered GH and thyroid status and whether they are detrimental for host defense. In this novel in vivo model of sustained critical illness, we investigated the effect of preventing hyperglycemia with insulin infusion on endocrine, metabolic, and innate immune function.
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Materials and Methods |
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Experiment 1: effects of alloxan in healthy and injured rabbits
In our previously reported animal model of prolonged critical illness (6) (i.e. chronically instrumented, burn-injured, parenterally fed New Zealand White rabbit), insulin resistance and a variable degree of stress hyperglycemia are present. To better mimic the clinical condition of limited endogenous insulin reserve in critically ill adult patients (7) and to allow accurate control of blood glucose at a preset level, the model was optimized. For this purpose, an acute deficit of endogenous insulin secretion was induced by injection of alloxan monohydrate (150 mg/kg; Sigma-Aldrich, Bornem, Belgium) followed by insulin-titrated control of blood glucose. We validated this optimized model controlling for the effect of injury and of alloxan.
In brief, male New Zealand White rabbits were purchased from a local rabbitry and housed individually. They had free access to water and were fed once daily and exposed to artificial light for 14 h per day. After weighing and cannulation of both the carotid artery and jugular vein under general anesthesia [30 mg/kg im ketamine (Merial, Lyon, France) and 0.15 ml/kg im medetomidine (Orion Corp., Espoo, Finland)], animals were fitted into a homemade jacket to secure the position of the catheters. To assure patency of the catheters, lines were filled with heparin (5000 IU/ml; Rhône-Poulenc Rorer, Brussels, Belgium). Two days later, after recovery of anesthesia, animals were reweighed and blood samples for baseline biochemical and blood gas analysis (ABL 700 analyzer; Radiometer Medical A/S, Copenhagen, Denmark) were taken via the arterial line.
The rabbits were then randomized to one of three experimental groups. In group 1 (n = 6), a slow (over 2–3 min) iv bolus injection of 150 mg/kg alloxan was given, followed by a 15% burn injury on both flanks, under anesthesia, as described above (6). Group 2 (n = 5) received a NaCl 0.9% injection (same volume as alloxan) followed by burn injury under anesthesia as described for group 1. Group 3 (n = 5) received only a slow iv bolus injection of alloxan under general anesthesia, but no injury was imposed. In all three groups, iv total parenteral nutrition (TPN) was started 2 h after the injection of alloxan or saline. All iv infusions were weighed before and after administration, thus allowing accurate determination of the infused quantity. Parenteral nutrition was prepared daily in the hospital pharmacy under laminar airflow conditions. The infusion bags contained 103 ml of glucose 20%, 37.5 ml Aminoplasmal L 10 (Braun, Melsungen, Germany), and 34 ml Intralipid 20% (Fresenius Kabi, Stockholm, Sweden); 150 ml sterile water was added. Thus, bags with 325 ml solution contained 150 kCal and 0.6 g nitrogen. Of all calories, 56% were delivered as carbohydrates and 44% as fat. Protein intake equaled 1.25 g amino acids/kg·d. There were no vitamins or trace elements added. TPN was continuously administered (24/24 h) via a volumetric pump. The animals had free access to water, whereas oral caloric intake was denied. TPN bags were replaced daily. To assure free movement of the rabbits without twisting of the catheter and infusion lines, a swivel device was incorporated.
Arterial blood samples for determination of whole-blood glucose, hemoglobin, sodium, potassium, lactate, and ionized calcium concentrations and blood gases were taken 2 and 8 h after alloxan or NaCl 0.9% injection and then twice daily on d 1, 3, 4, 5, and 8. An additional blood sample was taken at 4 and 6 h after injection of alloxan/saline to control whole-blood glucose concentrations. When whole-blood glucose concentration reached 22.2 mmol/liter after recovery of anesthesia, a continuous iv insulin infusion (Actrapid 100 IU/ml; Novo Nordisk, Bagsvaerd, Denmark) via a SE 200 B infusion pump (Vial Medical, Brezins, France) was started to control blood glucose levels, aiming for the level observed before the experiment (on average 8.3 mmol/liter). In the evening of d 7 after injury, insulin infusion was stopped to evaluate the hyperglycemic response 12 h later.
Experiment 2: effects of controlling stress hyperglycemia with insulin in burn-injured, parenterally fed rabbits
In a second experiment, we evaluated the metabolic, endocrine, and immunological effects of controlling blood glucose at a high vs. a normal level with exogenous insulin in the above described optimized model. Animals were catheterized under general anesthesia, as described above. Forty-eight hours later (time point from here on labeled d -3), all rabbits received alloxan (iv injection of 150 mg/kg), followed 2 h later by the initiation of parenteral nutrition. For determination of whole-blood glucose levels, arterial blood was sampled at 2 h and 8 h after alloxan injection and then twice daily on d -2 and -1. When blood glucose concentrations reached 22.2 mmol/liter, rabbits were considered to be insulin deficient and an iv insulin infusion was started to control blood glucose levels around 11 mmol/liter. Seventy-two hours after alloxan injection (d 0), after randomization (sealed envelopes) into a hyperglycemic and a normoglycemic group, parenteral nutrition was interrupted and, under deep general anesthesia supplemented with a local paravertebral block and after shaving both flanks, a 15% full-thickness burn injury was imposed. In the normoglycemic group, blood glucose levels were controlled between 3.3 and 6.1 mmol/liter by iv infusion of insulin. In the hyperglycemic group, insulin was infused only if blood glucose levels exceeded 13.8 mmol/liter in which case blood glucose concentrations were controlled between 13.8 and 16.6 mmol/liter. After a 2-h recovery period during which the animals received supplemental oxygen, they were returned to their cages and the parenteral nutrition infusion was restarted. In the evening, a supplemental dose of a major analgesic drug (1 mg piritramide im; Janssen-Cilag, Beerse, Belgium) was given to all animals. Blood glucose levels were further kept within the predetermined narrow limits for the two study groups by frequent blood glucose monitoring and titration of insulin.
Twice daily, at 0730 h (±1 h) and at 1930 h (±1 h), whole-blood analysis was done. Additional blood samples were taken whenever blood glucose levels were unstable, to allow for adjustment of the insulin dose. In addition, 2 ml of blood was collected on d -3, -2, 0, 1, 2, 3, 5, 7, and 9 and centrifuged for 10 min at 13,000 rpm. The plasma was stored at -80 C until further analysis of biochemical and hormonal (GH and thyroid hormones) concentrations. Furthermore, on d 1 and 3 after injury, 5 ml of arterial blood was withdrawn for determination of macrophage function. Also, plasma concentrations of triglycerides, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were determined on d -3, 1, 3, and 9.
Animals surviving the 12-d experimental period were weighed and then euthanized by iv injection of sodium pentothal (Nembutal, 60 mg/ml; Sanofi Winthrop, Morrisville, PA). Organs were harvested, and heart, lungs, liver, spleen, and kidney were weighed. Tissue samples were taken from left and right ventricles, lung, liver, spleen, kidney, ileum, muscle, intact skin, and burned skin for determination of dry weight and water content. Water content was determined by weighing the samples before and after 24 h of lyophilization (Alfa I-5 Chriss, Aichach, Germany).
Assays
Arterial blood was immediately analyzed on a blood gas analyzer to quantitate whole-blood pH, pO2, pCO2, hemoglobin, lactate, glucose, sodium, potassium, bicarbonate, and ionized calcium concentrations. Plasma concentrations of lipids (triglycerides, total cholesterol, and LDL and HDL cholesterol) were determined within 2 h after sampling on d -3, 1, 3, and 9. Analysis of lipids was done using the Roche Modular with Roche reagents (Roche Diagnostics, Mannheim, Germany). LDL cholesterol concentrations were calculated with the Friedewald formula (8).
Plasma rabbit GH (rGH) concentrations were measured using a specific rabbit RIA (reagents purchased from Dr. A. Parlow, National Hormone and Peptide Program, Torrance, CA) as described previously (6). All samples were analyzed in duplicate within a single assay run. The detection limit was 1 µg/liter and the within-assay coefficient of variation was 2.3%. All samples contained detectable plasma concentrations.
Total plasma T4 and T3 concentrations were determined in duplicate by a specific RIA (Immunotech, Marseille, France) on d -3, d 0, and on d 3. The sensitivity for T3 was 0.2 nmol/liter and for T4 was 6 nmol/liter.
Peripheral blood mononuclear cells (PBMCs) were used to assess macrophage function. Five milliliters of blood was withdrawn on d 1 and d 3 after injury. Blood was sampled (BD Vacutainer K2E tubes, Plymouth, UK) and taken immediately to the lab for analysis. PBMCs were separated from whole blood by density gradient with Lymphoprep (Axis-Shield PoCAS, Oslo, Norway). In summary, blood was diluted in the same volume of RPMI 1640 medium (Life Technologies, Inc., Paisley, Scotland, UK) and antibiotics (5 µg/ml of geneticin, Life Technologies, Rockville, MD, and mercaptoethanol, UCB, Brussels, Belgium). Then, the solution was laid over 15 ml of Lymphoprep and centrifuged for 25 min at 300 x g. After centrifugation, the layer containing PBMCs was harvested and washed with medium twice. Cells were resuspended in medium RPMI 1640 plus antibiotics until a concentration of 106 cells/ml was attained and divided in three groups. All manipulations were made on ice. The first group was analyzed at 0 h (fresh samples). The other two groups were analyzed after 24 h of incubation at 37 C with 10% fetal calf serum (Life Technologies) in the presence or absence of lipopolysaccharide (10 µl/ml; Sigma-Aldrich). All three groups were tested for their capacity of chemotaxis, phagocytosis, and oxidative burst.
For chemotaxis evaluation, the ability of binding casein was measured as described previously (9). Briefly, 100 µl of the cell suspension was brought into a FACS tube and added to 50 µl of a standardized solution of fluorescein-labeled casein (Molecular Probes Europe, Leiden, The Netherlands). After incubation of 85 min at 4 C, cells were washed and dissolved in 400 µl of PBS and 1% paraformaldehyde. Cells were then analyzed by flow cytometry (FACSort, Becton Dickinson, Erembodegen, Belgium). Chemotaxis capacity was expressed as percentage of fluorescein-positive cells.
Phagocytosis capacity was evaluated by measurement of fluorescein-labeled particles of zymosan (Zymosan A BioParticles, Saccharomyces cerevisiae, Molecular Probes, Eugene, OR) (9). The particles were brought to 1 ml of a suspension of cells containing 106 cells (100 particles per cell). Cells were incubated for 80 min at 37 C and then washed with PBS. Reminiscent red blood cells were lysed in the presence of ammonium chloride. Cells were resuspended and fixed in 400 µl of PBS and 1% paraformaldehyde. Cells were then ready for analysis in the FACSort. Phagocytosis was expressed as percentage of fluorescein-positive cells.
Evaluation of oxidative burst capacity was assessed using an available commercial kit by Orpegen Pharma (Heidelberg, Germany) called BURTTEST. The test was carried out following the instructions given by the manufacturer. FACSort was used to access the cells that had produced reactive oxygen radicals. Results were expressed as percentage of fluorescein-positive cells.
Statistics
All data are expressed as mean ± SD, unless specified otherwise. Comparisons between the different groups were done with ANOVA and Fisher’s protected least significant difference post hoc testing or with unpaired Student’s t test, when appropriate. Within-group changes were tested by paired t test followed by Bonferroni corrections for multiple comparisons. Data not distributed normally were analyzed by nonparametric methods (Kruskal Wallis, Mann-Whitney U test, and Wilcoxon).
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Results |
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All animals developed hyperglycemia within 2 h after the induction of anesthesia (Fig. 1
, upper panel). In the burn-injury only group, there was a progressive rise in whole-blood glucose concentrations after injury, necessitating insulin infusion in all rabbits after 4–6 h. In the alloxan-only-treated animals, there was a significant decrease in blood glucose concentrations at 4, 6, and 8 h after alloxan injection, as compared with the two other groups, followed by a progressive increase in blood glucose concentrations necessitating insulin infusion between 26 and 48 h after alloxan injection. Blood glucose levels in the alloxan-treated burn-injured animals followed a time course on d 0 in between the two other groups. All rabbits needed insulin 12–72 h after injection of alloxan.
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Insulin requirement was 1.9 ± 0.3 IU/kg per survived day in the alloxan-only group, 1.3 ± 0.4 IU/kg per survived day in the alloxan-treated burn-injured animals and 4.0 ± 4.0 IU/kg per survived day in the burn-injured only group (P = 0.05). In rabbits surviving the 8-d experimental period, insulin requirement was 1.8 ± 0.2 IU/kg per survived day in the alloxan-only group, 1.3 ± 0.4 IU/kg per survived day in the alloxan-treated burn-injured animals, and 2.3 ± 0.8 IU/kg per survived day in the burn-injured only group (P = NS).
Overall, burn injury, but not alloxan, induced changes in the other studied biochemical markers
Plasma sodium concentrations (Fig. 1
, middle panel) at baseline were identical in the three groups. During the day of injury and during the next day, plasma sodium concentrations were lower in both burn-injured groups as compared with the alloxan-only group, which mirrored the changes in blood glucose concentrations. Thereafter there was a progressive increase in sodium in these two groups, with levels indistinguishable from the alloxan-only group on d 8.
Plasma lactate concentration (Fig. 1, lower panel) increased transiently but significantly in both burn-injured groups at 2 and 8 h post anesthesia, again paralleling the changes in blood glucose concentrations.
Hemoglobin increased significantly at 2 h after the burn injury (from 11.2 ± 1.0 g/dl to 14.1 ± 1.1 g/dl in the alloxan-treated burn-injured animals; P < 0.0001 and from 12.0 ± 1.1 g/dl to 15.6 ± 2.6 g/dl in the burn-injured only group; P = 0.01), whereas there was no change in the alloxan-only group (from 12.2 ± 0.5 g/dl to 10.8 ± 1.6 g/dl; P = NS). Thereafter, there was a similar decline in hemoglobin concentrations in all three groups.
Ionized calcium concentrations at baseline were 1.54 ± 0.28, 1.73 ± 0.06, and 1.43 ± 0.06 mmol/liter in alloxan-treated burn-injured, in burn-injured only, and the alloxan-treated only animals, respectively. Anesthesia induced a significant decrease in ionized calcium on d 0 in all three groups (–31 ± 14% in the burn-injured alloxan-treated animals, P = 0.02 vs. baseline; -35 ± 31% in the burn-injured only group, P = 0.04 vs. baseline; and -25 ± 11% in the alloxan-treated only group, P = 0.04 vs. baseline). Ionized calcium quickly normalized thereafter in the alloxan-only-treated group, whereas it remained lower on d 1 in burn-injured animals (for both, P = 0.04 vs. baseline).
Burn injury also induced a transient, partially respiratory-compensated, metabolic acidosis with low serum bicarbonate concentrations [bicarbonate = 15 ± 4.3 mmol/liter in the alloxan-treated burn-injured group and 13 ± 2.3 mmol/liter in the burn-injured only group; P = 0.002 and P = 0.01 vs. baseline (23 ± 3 mmol/liter), whereas levels were not different from baseline in the alloxan-treated only group] and low arterial pCO2 [pCO2 = 27 ± 4 mm Hg in the alloxan-treated burn-injured group and 23 ± 5 mm Hg in the burn-injured only group; P = 0.01 and P = 0.03 vs. baseline (35 ± 5 mmol/liter), whereas pCO2 did not change in the alloxan-treated only group] at the end of d 0 after burn injury. Thereafter, there was a gradual normalization of both parameters toward d 8.
Potassium increased to 4.9 ± 0.3 mmol/liter in the alloxan-treated burn-injured group and to 5.4 ± 0.5 mmol/liter in the burn-injured only group (P = 0.07 and P = 0.02 vs. baseline). Hemoglobin, pH, and pO2 showed only burn-injury-related changes with no differences between alloxan-treated and saline-treated burn-injured animals and followed the changes we described previously (6).
Experiment 2: effects of controlling stress hyperglycemia with insulin in burn-injured parenterally fed rabbits
A total of 63 rabbits were used for this experiment. Although this experiment was not designed to study survival (the lesion has no potential to heal and the model is thus inevitably lethal), there was a trend for a higher mortality in the normoglycemic group (P = 0.09 with 
2 for survival on the last day). The Kaplan-Meier survival curves (Fig. 2) show the substantial loss of animals before randomization. Immediately after randomization, more animals were lost in the normoglycemic group than in the hyperglycemic group. Thereafter, survival curves evolved in parallel.
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Blood glucose control
All 30 animals developed severe hyperglycemia after alloxan injection. After injury, blood glucose levels in the two study groups were separated, as determined by the study protocol (Fig. 3). The normoglycemic group required more than three times as much insulin per survival day, as compared with the hyperglycemic group (Fig. 3).
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, upper panel). In line with this observation, animals in the hyperglycemic group were more acidotic than those in the normoglycemic group (Fig. 4, middle panel). Controlling blood glucose concentrations also prevented the significant decrease in natremia associated with hyperglycemia on d 2 and 3 (Fig. 4, lower panel).
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Inflammation and function of macrophages
Within 24 h after burn injury, C-reactive protein (CRP) was significantly elevated in both study groups as compared with baseline (Fig. 6). At d 3 after injury, there was a further increase in CRP only in the hyperglycemic group.
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, upper panel).
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Effect on body weight
Before alloxan injection, body weight was 2868 ± 284 g in the hyperglycemic group and 2838 ± 237 g in the normoglycemic group (P = NS). Of animals surviving the 12 experimental days, only the hyperglycemic rabbits lost 14% of body weight, weighing 2470 ± 181 g on the last day as compared with 2908 ± 128 g for the normoglycemic animals (P = 0.003). The weight of heart, lungs, liver, spleen, and kidney, corrected for the difference in body weight, was not different between the two groups. Furthermore, water content of lungs, left and right ventricle, liver, spleen, gut, kidney, muscle and skin was equal (data not shown).
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Discussion |
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We have previously reported an animal model that reveals many of the metabolic and endocrine changes seen in prolonged critically ill patients. In this model, which is intrinsically lethal as the burn injury cannot heal without surgical treatment, insulin resistance and a variable degree of stress hyperglycemia was documented. To better mimic the clinical condition of limited endogenous insulin reserve in critically ill adult patients (7) and to allow accurate control of blood glucose at a preset level, avoiding interindividual differences in insulin secretion capacity during stress, we replaced endogenous insulin secretion by exogenous insulin infusion after alloxan injection. Alloxan was preferred to streptozotocin because the latter induces irreversible defects in lymphoid cells (14). In healthy rabbits treated with alloxan only, the reduced blood glucose concentrations during the first 8 h after alloxan injection, despite infusion of parenteral nutrition, is in agreement with the original early reports from Dunn et al. (15, 16) on alloxan-induced diabetes in the rat. A transient drop in blood glucose is most likely explained by lysis of ß-cells induced by alloxan, evoking an acute release of endogenous insulin. As expected, burn injury and the accompanying insulin resistance attenuated the early fall in blood glucose concentrations during the first 24 h. During this experiment, insulin was infused whenever blood glucose concentrations exceeded 22.2 mmol/liter, aiming at a blood glucose level of 8.3 mmol/liter. Hence, identical blood glucose concentrations were achieved in the three groups between d 1 and 5. There was indeed no significant difference in blood glucose concentrations between burn-injured rabbits, whether or not they had been treated with alloxan, except that all burn-injured rabbits required exogenous insulin early after the burn, whereas this was delayed in alloxan-treated burn-injured rabbits. When we analyzed the insulin requirement in rabbits surviving the 8-d postinjury period, no differences between the study groups could be detected. At first glance this might be strange. However, in the burn-injured only group, initially high infusion rates of insulin were necessary to control blood glucose concentrations, whereas in the alloxan-treated groups, no insulin was needed during the hours after burn injury. After the day of injury, insulin requirement gradually decreased in the burn-injured only group but increased in the alloxan-treated burn-injured animals resulting in an equal amount of exogenous insulin given over the total study period.
Burn injury clearly induced insulin resistance, as well as transient lactic acidosis and hyponatremia in the parenterally fed rabbit. Alloxan adequately removed endogenous insulin reserve and did not affect circulating levels of hemoglobin, calcium, bicarbonate, sodium, potassium, lactate, or blood gases in burn-injured rabbits. Major toxicity of alloxan in our model was thus excluded. As a limited number of animals was used in this study, the possibility of a type II error should be considered.
The main finding of our study was that prevention of hyperglycemia with insulin infusion after trauma significantly improved innate immunity in the fed rabbit model. Indeed, 3 d after injury, the surviving rabbits allocated to the normoglycemic group had a better phagocytosis capacity as compared with the hyperglycemic group, with a trend already after 1 d. Furthermore, the capacity of phagocytotic cells to generate an oxidative burst 24 h after incubation and hereby kill bacteria also improved with glycemic control early after injury, whereas chemotaxis was not affected.
Infection remains one of the major causes of morbidity and mortality in intensive-care patients. During the initial response to infection, monocytes are activated into macrophages for phagocytosis and intracellular killing of bacteria and to generate inflammatory cytokines, such as TNF-
, IL-1, and IL-6 (17). The latter play a role in host defense by attracting activated neutrophils to the site of infection. During later stages of sepsis, however, deactivation of monocytes and granulocytes has been reported (18), referred to as immunoparalysis, and this presumably relates to insufficient control of severe infections. Stephan et al. (19) indeed observed that monocytes from critically ill patients developing nosocomial infections (mainly bacteremia) during intensive care had a significantly reduced phagocytosis capacity and impaired bacterial killing as compared with cells from patients who did not develop infection. Our data indicate that aggravation of insulin resistance and hyperglycemia, which occurs during severe infection, may play a role.
In diabetes, an increased risk of infection is present. This has been related to impaired neutrophil function, which has been studied both in vitro (20) and in vivo and was linked to lack of glycemic control (21, 22). Both acute (23) and more prolonged hyperglycemia have been associated with immunological changes in diabetes. Glycosylation of different proteins may in part be responsible for the effects on immune function (20, 24). Controlling hyperglycemia in diabetes has been associated with improved immune function and outcome. Rassias et al. (25) showed that even short time administration of insulin to tightly control blood glucose concentrations in diabetics improved the phagocytotic capacity of monocytes, an effect that persisted as long as the treatment was given.
Only recently, the negative effects of even moderate increases in blood glucose concentrations in nondiabetic critically ill patients have been appreciated (4). This study revealed that normalization of stress-induced hyperglycemia with intensive insulin therapy prevented serious infections and sepsis-associated organ failure and death, although the mechanisms of action remain unclear. There are very few studies investigating the effect of stress-induced hyperglycemia on immune function in nondiabetic models. Glucose administration (a large bolus in a fasted state) has been shown to negatively influence metabolic and inflammatory changes in an endotoxin shock model (26). In septic dogs (27), a protease inhibitor improved both Escherichia coli clearance (associated with improving in vitro phagocytosis) and blood glucose concentrations, but no causal relationship between both was suggested. Kwoun et al. (28) have observed immune changes induced by glucose infusion in postoperative rats. In contrast to our current findings and to the human diabetes literature, these authors reported hyperglycemia-induced improvement of phagocytosis in alveolar macrophages. The divergence between these findings and the results from our study might be accounted for by the species studied (rats vs. rabbits), the method of inducing hyperglycemia (infusing different amounts of glucose vs. different amounts of insulin), the timing (short-term study of <24 h vs. sustained critical condition for several days), and the difference in studied cells (alveolar vs. peripheral macrophages).
An inappropriate response of monocytes and neutrophils contributes to adverse outcome of critical illness, but it is equally presumed that an overexuberant release of cytokines into the systemic circulation has a central role in microvascular injury and multiple organ failure (17). Interestingly, improved capacity of monocytes to clear and kill bacteria in our study was associated with a faster resolution of the injury-induced inflammation, as indicated by CRP measurements. CRP levels were equally elevated in both groups during the first day after injury but failed to increase further in the normoglycemic group on d 3 as they did in the hyperglycemic group. This antiinflammatory effect of intensive insulin therapy, coinciding with improved innate immune function, is striking and in line with our observations in the human study (29). CRP is released from the liver as an acute-phase protein. Although, theoretically, the suppression of the acute-phase CRP response with intensive insulin could be a secondary phenomenon to an effect on innate immunity and prevention of infections, a direct antiinflammatory effect may be involved, particularly in view of the short time frame in which it occurred in our study. In rat hepatoma cells, insulin has been shown to inhibit cytokine-induced transcription of acute-phase proteins (30). However, in the current study, the direct hepatic effects of insulin cannot with certainty be distinguished from its effects on glycemic control, as both occurred concomitantly. In patients with type II diabetes, treatment with insulin, but not improved glycemic control per se, has been shown to reduce circulating CRP (31), which is in favor of a direct effect of insulin on the hepatic acute-phase response.
Insulin is indeed emerging as a molecule with strong antiinflammatory properties, suppressing the generation of a range of early proinflammatory substances including TNF-, macrophage migration inhibitory factor, superoxide anions, and intranuclear nuclear factor 
B (32, 33).
A recent study, however, showed that short-term hyperinsulinemia per se can also induce proinflammatory responses in normoglycemic healthy volunteers (34). In addition, hyperglycemia has also been shown to exert direct proinflammatory effects in nondiabetic rats (28).
Insulin infusion after burn injury, titrated to prevent hyperglycemia, also significantly prevented the development of hyponatremia. In addition, lactic acidosis, frequently observed after severe trauma, was shorter lasting in the normoglycemic group as compared with the hyperglycemic group. A direct effect of prevention of hyperglycemia as well as improved cardiac output (36), mitochondrial function (37), and/or microcirculation (38) may play a role.
Strict glycemic control prevented the loss of body weight up to 9 d after injury in this intrinsically lethal model of prolonged severe illness. Because food intake was comparable and water content of the different tissues was identical, this suggests that intensive insulin therapy may have had a protein-sparing effect (39).
Critical illness is known to be associated with low concentrations of LDL and HDL cholesterol (40) and elevated levels of triglycerides. The increasing concentrations of triglycerides and LDL and total cholesterol with time observed in the sick rabbits may in part be related to the parenteral nutrition and the cytokine release (41). Although no significant effect of glycemic control on the studied lipids was observed, the development of stress-induced diabetes could theoretically play a role (42).
We observed that intensive insulin therapy resulted in a faster resolution of rGH hypersecretion in response to stress. This may be an indirect effect, related to the shorter lasting inflammatory response in the normoglycemic group. Alternatively, as in type I diabetes (43, 44), better glycemic control may improve GH responsiveness, and through IGF-I-mediated feedback inhibition, GH hypersecretion may be suppressed. Finally, insulin per se may have had an attenuating effect on ghrelin release (45) or a more proximal effect on the hypothalamo-pituitary axis (46), which may in turn limit GH secretion. However, a thorough analysis of GH secretion requires repeated sampling, which was not feasible in the current study. Unlike in an acute, experimental model of starvation (47), insulin infusion did not affect the low T3 syndrome in the critically ill model. The severity of the insult, the duration of the protocol, and the experimental conditions may explain this difference.
Although there was a trend for a higher mortality in the normoglycemic group, the difference did not reach statistical significance. The limited size of the study as well as the study protocol (no intervention that allows healing of the burn injury and limited study episode with killing the survivors at the end) precludes any conclusion regarding mortality.
In conclusion, strict glycemic control with insulin improves innate immune function and prevents excessive inflammation and acidosis in this lethal model of prolonged, burn-injury-induced severe illness. Whether these findings offer an explanation for the beneficial effects observed with intensive insulin therapy in critically ill patients remains to be established.
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Acknowledgments |
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Footnotes |
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Abbreviations: CRP, C-reactive protein; PBMC, peripheral blood mononuclear cell; rGH, rabbit GH; TPN, total parenteral nutrition.
Received June 4, 2003.
Accepted for publication August 20, 2003.
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References |
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. Nat Med 3:678–681[CrossRef][Medline]
B and stimulates IB in mononuclear cells in obese subjects: evidence for an anti-inflammatory effect. J Clin Endocrinol Metab 7:3257–3265
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 Authors/Task Force Members, L. Ryden, E. Standl, M. Bartnik, G. V. d. Berghe, J. Betteridge, M.-J. de Boer, F. Cosentino, B. Jonsson, M. Laakso, et al. Guidelines on diabetes, pre-diabetes, and cardiovascular diseases: full text: The Task Force on Diabetes and Cardiovascular Diseases of the European Society of Cardiology (ESC) and of the European Association for the Study of Diabetes (EASD) Eur. Heart J. Suppl., June 1, 2007; 9(suppl_C): C3 - C74. [Full Text] [PDF]
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 Authors/Task Force Members, L. Ryden, E. Standl, M. Bartnik, G. Van den Berghe, J. Betteridge, M.-J. de Boer, F. Cosentino, B. Jonsson, M. Laakso, et al. Guidelines on diabetes, pre-diabetes, and cardiovascular diseases: executive summary: The Task Force on Diabetes and Cardiovascular Diseases of the European Society of Cardiology (ESC) and of the European Association for the Study of Diabetes (EASD) Eur. Heart J., January 1, 2007; 28(1): 88 - 136. [Full Text] [PDF]
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 E. A. Oral and M. Pietropaolo Metabolism Lessons for Survival: When Adults and Children Are Not Alike. J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3763 - 3765. [Full Text] [PDF]
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I. Vanhorebeek, R. P. Peeters, S. Vander Perre, I. Jans, P. J. Wouters, K. Skogstrand, T. K. Hansen, R. Bouillon, and G. Van den Berghe Cortisol Response to Critical Illness: Effect of Intensive Insulin Therapy J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3803 - 3813. [Abstract] [Full Text] [PDF]
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 B. Ellger, Y. Debaveye, I. Vanhorebeek, L. Langouche, A. Giulietti, E. Van Etten, P. Herijgers, C. Mathieu, and G. Van den Berghe Survival benefits of intensive insulin therapy in critical illness: impact of maintaining normoglycemia versus glycemia-independent actions of insulin. Diabetes, April 1, 2006; 55(4): 1096 - 1105. [Abstract] [Full Text] [PDF]
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 G. Van den Berghe, A. Wilmer, G. Hermans, W. Meersseman, P. J. Wouters, I. Milants, E. Van Wijngaerden, H. Bobbaers, and R. Bouillon Intensive Insulin Therapy in the Medical ICU N. Engl. J. Med., February 2, 2006; 354(5): 449 - 461. [Abstract] [Full Text] [PDF]
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 L. Langouche, I. Vanhorebeek, and G. Van den Berghe Glycaemic control in trauma patients, is there a role? Trauma, January 1, 2006; 8(1): 13 - 19. [Abstract] [PDF]
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 R. Lattermann, G. Belohlavek, S. Wittmann, B. Fuchtmeier, and M. Gruber The Anticatabolic Effect of Neuraxial Blockade After Hip Surgery Anesth. Analg., October 1, 2005; 101(4): 1202 - 1208. [Abstract] [Full Text] [PDF]
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 G. Van den Berghe Tight Blood Glucose Control With Insulin in "Real-Life" Intensive Care Mayo Clin. Proc., August 1, 2004; 79(8): 977 - 978. [PDF]
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 G. Van den Berghe and R. Bouillon Optimal Control of Glycemia Among Critically Ill Patients JAMA, March 10, 2004; 291(10): 1198 - 1199. [Full Text] [PDF]
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), in burn-injured only rabbits (
), and in alloxan-treated only rabbits (
). Pre denotes the time before alloxan or NaCl 0.9% administration. When whole-blood glucose concentration exceeded 22.2 mmol/liter, insulin infusion was started. On d 7, insulin infusion was stopped to evaluate the hyperglycemic response and whole-blood concentrations were determined 12 h later. Data represent means ± SEM *, P < 0.05 between alloxan-treated only and alloxan-treated burn-injured animals; 
, P < 0.02 between burn-injured only and alloxan-treated only arm; +, P < 0.05 in all groups vs. preinjury values.
and open bar) and in hyperglycemic (
, P < 0.01 (difference between day of injury and d 2 or 3).
