GLUCOSAMINE ADMINISTRATION IMPROVES SURVIVAL RATE AFTER... : Shock (original) (raw)
INTRODUCTION
Despite the advances in traffic and occupational safety, trauma remains the major cause of death in people younger than 35 years (1). Early deaths are caused by severe hemorrhage and central nervous system injuries, whereas late deaths are caused by septic complications and multiple-organ dysfunction syndrome (2-5). Hemorrhage also remains as the primary cause of death on the battlefield in conventional warfare (6). The early treatment of hemorrhagic shock includes surgical intervention and restoration of the intravascular volume by means of crystalloid infusion and blood transfusion. Although the early restoration of intravascular volume improves cardiac output and helps to maintain the mean arterial pressure (MAP), the massive resuscitation before the surgical control of the ongoing bleeding may result in further blood loss and higher mortality rate (7-10). Because the modern combat environment can lead to significant delays in evacuation, there is considerable interest in the development of new, small-volume resuscitation strategies designed to improve the battlefield survival and to minimize subsequent complications (11, 12). The availability of such strategies would also clearly be of value in the treatment of hemorrhage in noncombat situations.
Stress-induced hyperglycemia is a common occurrence after severe injury. This may be beneficial either by ensuring adequate energy supply or by facilitating the mobilization of interstitial fluid reserves by increasing osmolarity (13-15). However, the increased levels of glucose also facilitate the flux through the hexosamine biosynthesis pathway. The primary product of this pathway, uridine 5′-diphosphate-_N_-acetylglucosamine (UDP-GlcNAc), serves as the substrate for the addition of _O_-linked _N_-acetylglucosamine (_O_-GlcNAc) to cytosolic and nuclear proteins. In addition to being sensitive to insulin and glucose, the levels of UDP-GlcNAc and _O_-GlcNAc can also be increased by glucosamine (16-20). Interestingly, the increased levels of _O_-GlcNAc have recently been shown associated with improved cell survival after stress (21). We recently reported that glucosamine treatment improved functional recovery in isolated perfused hearts after ischemia-reperfusion, which seemed mediated via an increase in the _O_-GlcNAc levels (22). We also found that glucosamine treatment during resuscitation after trauma-hemorrhage in rats in vivo improved the cardiac function and the tissue perfusion, reduced the serum levels of proinflammatory cytokines, and increased the _O_-GlcNAc levels in the heart and the brain (23). Therefore, the goal of this study was to demonstrate whether bolus i.v. administration of glucosamine improves the survival rate after severe trauma-hemorrhage in the absence of resuscitation.
MATERIALS AND METHODS
Animals
All animal experiments were approved by the University of Alabama at Birmingham Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, 1996).
Reagents
All reagents and solutions were purchased from Thermo Fisher Scientific, (Waltham, Mass), or from Sigma-Aldrich, (St. Louis, Mo), unless indicated otherwise.
Trauma-hemorrhage model
We used a modified version of a volume-controlled hemorrhage model described by Macias et al. (11) that in the absence of any intervention exhibited 0% survival within 35 min after the end of the hemorrhage period.
Male adult Sprague Dawley rats (weight, 285 ± 18 g; Taconic Farm, Hudson, NY) were fasted for 24 h before the experiment to deplete the endogenous glycogen stores, thereby minimizing the variability in metabolic status between animals. Water was given ad libitum. After isoflurane prenarcosis, the animals were placed on a metal plate combined with heating pad (Allegiance K-MOD 107 heat therapy system; Cardinal Health Inc., Dublin, Ohio) and kept in a supine position under 1.5% vol/vol of isoflurane anesthesia with 1 L/min oxygen flow during the remainder of the experiment. The two femoral arteries were cannulated by using PE-50 catheters for blood pressure monitoring and blood withdrawal, then the right femoral vein was catheterized for drug administration. A 5-cm-long midline laparotomy was performed to induce a soft tissue damage imitating "trauma" before hemorrhage. A thermoprobe was left in the abdomen to monitor the central body temperature for the whole duration of the experiment by means of a digital thermometer (Thermalert TH5; Physitemp Instruments, Clifton, NJ). The left femoral artery was connected to a blood pressure analyzer (Digi-Med, Louisville, Ky) for continuous measurement of MAP, pulse pressure, and heart rate. Heparin was not administered at anytime during the procedures.
Hemorrhage was induced by withdrawal of 55% of the calculated total blood volume (total blood volume [mL] = body weight [g] × 0.061) (23) for 25 min using a syringe pump (Harvard Instruments, Holliston, Mass). At the end of the hemorrhage, the rats were divided randomly into two groups: glucosamine- and mannitol-treated (control) animals. In the glucosamine group (n = 17), 2.5 mL of 150mM glucosamine solution (Fluka) was administered i.v. for 10 min. In case of the mannitol-treated rats (n = 15), 2.5 mL of 300mM mannitol was injected for the same duration of time. The pH level of the glucosamine solution (osmolarity, 320 mOsm) was adjusted to 7.36 with NaOH; an equal dose of NaCl was added to the mannitol solution to achieve a similar osmolarity and sodium ion concentration. The dose of glucosamine administered in this study was the same as that previously shown to increase the tissue _O_-GlcNAc levels and to improve the recovery after trauma-hemorrhage and full resuscitation (23).
During and after the hemorrhage period, no additional treatment was given. Blood samples were taken from the right femoral artery before and after the hemorrhage and at the end of the experiment. The animals were observed for 2 h after treatment or until death (apnea duration > 1 min) (11). At the end of the experiment, the rats were killed by means of i.v. injection of concentrated potassium chloride solution. Then, different organs (heart, brain, liver, and abdominal muscle) were harvested for the assessment of _O_-GlcNAc levels using Western immunoblot.
In the animals that survived for 2 h after hemorrhage, there were no differences in the _O_-GlcNAc levels in the tissues examined. Because _O_-glycosylation is a rapid process, an additional group of animals were subjected to trauma-hemorrhage followed by glucosamine (n = 6) or mannitol (n = 5) treatment, as described previously, but were killed 30 min after treatment when the survival rate was 100%. To compare our results observed after trauma-hemorrhage with nonoperated controls, a separate group of animals (n = 5) were killed after 24 h of food deprivation without hypovolemic shock. Blood samples were collected before and after the hemorrhage and at the end of the experiment. Heart, brain, liver, and abdominal muscle samples were harvested for the assessment of _O_-GlcNAc levels.
High-performance liquid chromatography measurements
The tissue adenosine triphosphate (ATP) levels were determined as previously described (24). Briefly, frozen tissue powder was homogenized in 0.3M perchloric acid, centrifuged for 10 min under a temperature of 4°C at 14,000 g; then, the supernatant was mixed with 1:4 trioctylamine:1,1,2-trichlorotrifluoroethane. The mixture was centrifuged for 5 min under a temperature of 4°C; then, the aqueous phase was loaded onto a strong anion exchange column (Partisil 10 SAX; Whatman, Brentford, Middlesex, UK) with 262-nm wavelength of detection. Data were analyzed and quantified as area under the curve by using the System Gold Nouveau software (Beckman Coulter, Fullerton, Calif).
The serum glucosamine levels were measured as previously described (25). Briefly, 0.1 mL of serum was added to 0.4 mL acetonitrile to precipitate the serum proteins. After vortex mixing (duration, 1 min) and centrifugation (duration, 3 min at 10,000 g), the supernatant was mixed with 0.2 mL of methanol and was dried under vacuum. The samples were derivatized with 0.2 mL of 88 mg/mL 1-naphthyl isothiocyanate; then, the reaction was stopped by using 0.4 mL of 1.5% acetic acid. The excess derivatizing reagent was partitioned into an organic phase by the addition of 1.0 mL of chloroform. The aqueous layer was purified by means of an anion exchange cartridge (Alltech, Columbia, Md); and the eluted solution was loaded onto ODS2 analytical column (Waters Corporation, Milford, Mass). The samples were run with an isocratic system. The mobile phase contained acetonitrile-water-acetic acid-triethylamine (dilution ratio, 4.5:95.5:0.1:0.05; pH value, 4.5) with a constant flow rate of 0.9 mL/min. The UV detector wavelength was set to 254 nm and a column heater was applied to maintain 41°C temperature. Glucosamine standard was run to establish molar reference values; then, d-galactosamine was added to each sample as an internal standard. The areas under the curve were calculated by using the System Gold Nouveau software provided for the Beckman Coulter high-performance liquid chromatography system.
The serum glucosamine and the tissue ATP levels were determined 30 min after treatment, at the time when the _O_-glycosylation levels were shown to be at the point of significant increase in the glucosamine-treated group.
Serum cytokine levels
The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-10 in serum samples collected 30 min after treatment were determined by means of BioSource sandwich enzyme-linked immunosorbent assay kits (BioSource, Camarillo, Calif).
Western immunoblots
Tissue lysates were centrifuged at 14,000 g for 10 min. The supernatant was collected and protein concentration was measured using DC protein assay kit (Bio-Rad Laboratories, Hercules, Calif). The proteins were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to polyvinylidene difluoride membrane (Millipore Corporation, Billerica, Mass). The protein load was confirmed by using SYPRO Ruby (Bio-Rad) staining. The membranes were incubated with a 1:5000 dilution of anti-_O_-GlcNAc antibody CTD 110.6 (a kind gift from Mary Ann Accavitti, University of Alabama at Birmingham Epitope Recognition and Immunodetection Facility) in 1% casein/phosphate-buffered saline solution (PBS; Pierce Biotechnology, Rockford, Ill) for 2 h. After washing three times for 10 min in PBS, the membrane was incubated with a 1:10000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Ig) M (Calbiochem, San Diego, Calif) in 1% casein/PBS (Pierce Biotechnology) for 1 h. The membranes were visualized with enhanced chemiluminescence (SuperSignal West Pico; Pierce Biotechnology), the signal was detected by using UVP BioChemi System (Upland, Calif), and the densitometry was performed using LabWorks analysis software (UVP). The membranes were stripped and the blots containing the proteins from the heart and the muscle were probed with 1:2000 dilution of anti-calsequestrin antibody (Abcam Inc., Cambridge, Mass) and then incubated with 1:2000 goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, Calif) for another hour. The membranes containing brain and liver proteins were incubated with 1:5000 anti-β-actin antibody (Sigma-Aldrich), followed by a 1-h-long incubation period with 1:10000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Cell Signaling Technology, Inc., Danvers, Mass). Then, the membranes were developed with enhanced chemiluminescence as described previously. After densitometry, the raw densities were normalized to the density of calsequestrin or β-actin bands.
Blood gas analysis, serum enzyme, glucose, and electrolyte measurements
The blood gas parameters from arterial samples were analyzed by using an Instrumentation Laboratory IL1640 (Lexington, Mass) commercial blood gas analyzer. Serum enzymes (alkaline phosphatase, alanine aminotransferase, total bilirubin, amylase), creatinine, serum urea nitrogen, glucose, electrolyte levels (natrium, kalium, calcium, phosphate) and protein levels (albumin, globulin, total protein) were determined by the Veterinary Pathology Laboratory (Animal Resource Program, University of Alabama at Birmingham) using a VetScan comprehensive diagnostic profile reagent rotor and chemistry analyzer (Abaxis, Union City, Calif). Serum lactate was detected by means of a method based on enzymatic determination and reflectance photometry using an Accutrend lactate analyzer (F. Hoffmann, La Roche Ltd., Basel, Switzerland).
Data analysis
All statistical analyses were performed using GraphPad Prism 4 software (GraphPad, San Diego, Calif). The survival data were compared using log-rank test and were presented as survival percentage. Other statistical comparisons were performed using Student t test, one-way analysis of variance (ANOVA) with Bonferroni post hoc test, two-way ANOVA with Bonferroni post hoc test, or repeated two-way ANOVA where appropriate. Data are expressed as mean ± SEM. The statistically significant differences between groups or time points were defined as P < 0.05.
RESULTS
Effect of glucosamine on the survival rate
A total of 37 animals were subjected to trauma-hemorrhage; 5 rats died before the end of the blood withdrawal and were excluded from the experiment. Of the remaining 32 animals, 17 were treated with glucosamine and 15 treated with mannitol; 8 of the 17 animals in the glucosamine group and 3 of the 15 rats in the control group survived for 2 h after treatment (Fig. 1). The difference in the overall survival rate between the glucosamine- and the mannitol-treated rats was statistically significant (survival rate after 2 h, 47% vs. 20%, respectively; P < 0.05).
Percent survival over time for glucosamine- and mannitol-treated rats (P < 0.05, log-rank test). Zero (0) minute indicates the start of the drug administration.
Effect of glucosamine on MAP, heart rate, and pulse pressure
The MAP, the heart rate, and the pulse pressure for glucosamine- and mannitol-treated groups throughout the protocol are shown in Figure 2. There were no significant differences between the groups in any of the parameters before treatment. For 18 min after treatment, the MAP was significantly higher in the glucosamine group. Similar results were observed when comparing only those animals in the groups of animals that survived the 2-h follow-up period (data not shown). There were no significant differences in the heart rate or the pulse pressure between the glucosamine- and the mannitol-treated rats after hemorrhage.
Time course of (A) MAP, (B) heart rate, and (C) pulse pressure. Data points indicate means; error bars, SEM. *P < 0.05 versus control.
Serum glucosamine levels
Thirty minutes after treatment with glucosamine, the serum glucosamine concentration was 2.6 mM ± 0.5 mM. In the mannitol-treated group and in the nonoperated control group, the serum glucosamine levels were undetectable (data not shown).
Effect of glucosamine on tissue ATP levels
In Figure 3, it can be seen that hemorrhagic shock had no effect on ATP levels in heart, brain, or abdominal muscle; however, there was a marked decrease in ATP levels in liver. The tissue ATP levels were not altered by glucosamine treatment in any organ (Fig. 3, A-D).
Tissue ATP levels of (A) heart, (B) liver, (C) brain, and (D) abdominal muscle extracts 30 min after treatment, compared with the nonoperated control. Data are expressed as mean ± SEM. *P < 0.05 versus nonoperated control.
Effect of glucosamine on cytokine release
The serum levels determined 30 min after the treatment of TNF-α and IL-10 were slightly increased compared with nonoperated controls; this finding was not altered by the glucosamine treatment (Fig. 4, A-B). The serum IL-6 levels remained undetectable during the experiment.
Serum cytokine levels of (A) TNF-α and (B) IL-10 30 min after treatment, compared with the nonoperated control. Data are expressed as mean ± SEM.
Effect of glucosamine on heart, brain, liver, and muscle protein _O_-GlcNAc levels
Initially, the tissue samples were harvested 2 h after the end of hemorrhage or when the death of the animal was confirmed; however, the comparison of the tissue _O_-GlcNAc levels at different time points did not show significant differences between the groups (data not shown). Therefore, 11 additional animals were subjected to the same trauma-hemorrhage procedure followed by mannitol (n = 5) or glucosamine (n = 6) treatment. The animals were killed and serum, heart, brain, liver and muscle were collected 30 min after treatment, shortly after the period when the increase in MAP was significantly higher in the glucosamine-treated group (Fig. 2). Glucosamine treatment significantly increased the protein _O_-GlcNAc in heart, brain, and liver samples compared with the mannitol-treated animals (Fig. 5, A-C); however, the _O_-GlcNAc levels in abdominal muscle were not significantly increased (Fig. 5D). CTD 110.6 is specific for the _O_-GlcNAc moiety attached to Ser/Thr residues of proteins, but not for specific proteins; therefore, this finding is consistent with that of a number of other studies demonstrating that immunoblots show multiple bands, indicating the wide variety of proteins modified by _O_-GlcNAc in response to glucosamine treatment.
CTD 110.6 immunoblots of (A) heart, (B) brain, (C) liver and (D) abdominal muscle extracts from glucosamine- and mannitol-treated rats 30 min after injection are shown on the left side. Bar charts on the right side represent the mean of related area densities ± SEM of the mean raw densities assessed from the heart, brain, liver and muscle. Mean raw densities were normalized to calsequestrin staining in the heart and the muscle and β-actin staining in the brain and the liver. *P < 0.05 vs control.
To determine the effect of hemorrhagic shock on protein _O_-glycosylation levels and how this response was modified by glucosamine treatment, we measured the _O_-GlcNAc levels in heart and brain from nonoperated animals and from animals at 30 min and 2 h after hemorrhage (Figs. 6 and 7). Two-way ANOVA with Bonferroni post hoc analysis indicated a significant difference in the glucosamine group compared with the mannitol group at 30 min in both heart and brain, consistent with the single time point comparisons in Figure 5, A and B. However, despite the apparent increase between the _O_-GlcNAc level in nonoperated rats and the _O_-GlcNAc level after 30 min of treatment in rats administered with glucosamine, particularly in the brain, there was no significant time effect on _O_-GlcNAc levels in heart or brain in either mannitol- or glucosamine-treated groups. This was presumably caused by the small number of animals surviving at 2 h because 2-way ANOVA, excluding the 2 h data points, indicated a significant time and treatment effect in the brain.
The CTD 110.6 immunoblots from the heart of (A) mannitol- and (B) glucosamine-treated animals at 0 min (nonoperated)-, 30 min-, and 120-min time points. C, Mean raw densities from immunoblots normalized to calsequestrin as a loading control. Data are expressed as mean ± SEM. *P < 0.05 versus mannitol at same time point.
The CTD 110.6 immunoblots from the brain of (A) mannitol- and B) glucosamine-treated animals at 0 min (nonoperated)-, 30 min-, and 120-min time points. C, Mean raw densities from immunoblots normalized to β-actin as a loading control. *P < 0.05 versus mannitol at same time point.
Effect of glucosamine on arterial blood gas and serum biochemical parameters
The Pao2 and the oxygen saturation did not change significantly over the time because of the 100% oxygen inhalation. However, Paco2, acid base status (pH), base excess (BE), and serum bicarbonate decreased in both groups after trauma-hemorrhage compared with the baseline, indicating the severity of the shock (Table 1). There was also a marked increase in serum lactate levels, consistent with the development of severe metabolic acidosis. Hematocrit was also significantly decreased by the end of the hemorrhage and showed a further decrease 30 min after glucosamine or mannitol treatment. The serum potassium and phosphate levels were significantly increased at the end of the hemorrhage in both groups, parallel with the severity of shock. The blood glucose levels and the serum sodium levels were not significantly affected by the trauma-hemorrhage protocol.
Blood gas and serum biochemical parameters of mannitol and glucosmaine treated animals at 0 min (baseline), at the end of the hemorrhage and 30 min after the treatment
Despite the effect of glucosamine on survival rates, there were no significant differences between the glucosamine- and the mannitol-treated group in any of these parameters. There were also no differences between the glucosamine- and the mannitol-treated rats that survived the 2-h posthemorrhage period (data not shown).
DISCUSSION
Hemorrhage remains the primary cause of death on the battlefield in conventional warfare and is also the major cause of death in people younger than 35 years (1, 6). Because most combat deaths occur before the casualties reach a medical facility, there is considerable interest in the development of small-volume resuscitation strategies that increase the survival time during hypotension (12). We have previously shown that the addition of glucosamine during resuscitation after trauma-hemorrhage in the rat improved cardiac function and organ perfusion (23). In this study, we demonstrated for the first time that a bolus of glucosamine administered during hemorrhage significantly improved survival 2 h after the end of hemorrhage. Glucosamine administration also resulted in a significantly higher mean arterial pressure for 18 minutes following treatment and consistent with our earlier study (23), glucosamine also increased protein _O_-GlcNAc levels in the heart, brain, and liver extracts.
The model of volume-controlled hemorrhage used in this study was based on the model described by Macias et al. (11) who reported that in the absence of any intervention, all animals died within 35 min after the end of hemorrhage, whereas treatment with drag-reducing polymer solution significantly improved the 2-h survival rate. In this study, we found that with glucosamine treatment, the 2-h survival rate was 47%; this rate was significantly higher than that of treatment used in the iso-osmotic mannitol control group. The higher survival rate in our control group (i.e., 20%) relative to the survival rate of the control group in the study by Macias et al. is likely caused by the higher osmolarity of this solution. However, because both the mannitol and the glucosamine solutions were of similar osmolarity, the improved survival in the glucosamine group is likely a direct effect of glucosamine.
Parallel with the progression of hypovolemic shock, a metabolic acidosis develops as indicated by increased serum lactate accompanied by decreased pH, Pao2, BE, and HCO3− (26). Although improvements in metabolic acidosis are typically associated with improved outcome after hypovolemic shock, this is not necessarily the case. For example, Torres et al. (27) demonstrated that in a rat model of hypovolemic shock, the arterial K+ levels showed the clearest distinction between survivors and nonsurvivors. Consistent with the findings of Torres et al., we found that in response to hemorrhage, there were significant increases in serum lactate and K+ levels, whereas BE and HCO3− showed a marked decrease parallel with the progression of the shock. Thirty minutes after treatment, there were no significant differences between the mannitol- and the glucosamine-treated groups in any of these parameters, suggesting that the improved survival in the glucosamine group cannot be attributed to an early reduction in metabolic acidosis or the severity of shock at that time. However, we cannot rule out the possibility that glucosamine treatment may have attenuated the development of metabolic acidosis at a later time point.
Glucosamine treatment resulted in a significantly increased MAP for a short duration immediately after treatment. It is possible that this transitory increase in MAP was related to a short-term, vasoactive effect of glucosamine or could be caused by glucosamine on the neuroendocrine response. Yang et al. (23) reported that glucosamine treatment during resuscitation improved cardiac performance; thus, the increase in MAP observed in this study with glucosamine could also be a consequence of increased cardiac contractility. Because the increase in MAP shortly after treatment was the only physiological parameter that was different between the two groups, it is tempting to suggest that this may contribute to the subsequent improved survival, possibly by improving the perfusion to critical organ systems at crucial time early during hemorrhagic shock. However, we cannot attribute a direct cause-and-effect relationship between the early increase in the MAP and survival; the precise mechanisms underlying the effect of glucosamine on both MAP and survival clearly require further study.
The maintenance of tissue bioenergetic status, particularly ATP levels, could contribute to improved survival; therefore, we determined the ATP levels 30 min after treatment, which was a time just before the animals in the mannitol-treated group started to drop out. Consistent with previous reports, hypovolemic shock resulted in significant loss of ATP in liver compared with nonoperated controls, but there were no changes in ATP levels in the heart, brain, and abdominal muscle (28, 29). However, glucosamine treatment had no effect on ATP levels in any organs examined, suggesting that that the improved survival in this group was not related to the improved bioenergetics. These results are consistent with recent reports demonstrating in both cardiomyocytes and the whole heart that glucosamine attenuates ischemia/reperfusion injury without increasing ATP content (22, 30).
In response to severe injury, proinflammatory and anti-inflammatory cytokines are released (5); the differences in cytokine levels could be a contributing factor to improved survival. However, although we observed the slightly increased levels of the proinflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 30 min after treatment, glucosamine had no effect on these levels. This is counter to our recent study showing that glucosamine attenuated TNF-α and IL-6 levels after trauma-hemorrhage, followed by resuscitation (23). One reason for this discrepancy may be that the time between treatment and cytokine measurements was insufficient to allow for significant cytokine release (31). Furthermore, it has been reported that cytokine release is increased primarily after delayed resuscitation, whereas an immediate resuscitation or nonresuscitation had no significant effect on the serum cytokines levels (32). Because the animals in this study were not subject to delayed resuscitation, this may also account for cytokine levels remaining in a relatively low range.
Although the increase in _O_-GlcNAc levels in the heart, liver, and brain does not demonstrate a causal relationship between survival and increased _O_-GlcNAc levels, it is consistent with the notion that elevated _O_-GlcNAc levels are associated with improved survival (21). Surprisingly, there were no significant differences in the protein _O_-GlcNAc levels between the glucosamine- and the mannitol-treated groups in those animals that survived for 2 h or those that died at similar time points during the original experiment. Because the turnover of _O_-GlcNAc on proteins is relatively rapid, it is possible that 2 h after treatment, the _O_-GlcNAc levels in the glucosamine-treated group started to return to baseline levels. Indeed, from the data in Figures 6 and 7, it seems that this may be the case. However, the apparent lack of difference observed at 2 h may also reflect the small number of surviving animals at that time, resulting in insufficient statistical power to determine whether a difference exists between the glucosamine and the mannitol groups. Thus, further studies are needed to better understand the impact of hemorrhagic shock on the regulation of tissue _O_-GlcNAc levels and how this regulation is affected by glucosamine treatment.
These results are also consistent with those of our recent study in the isolated perfused heart where glucosamine increased the _O_-GlcNAc levels and improved the functional recovery after ischemia/reperfusion; in addition, the inhibition of _O_-GlcNAc formation with alloxan blocked this protection (22). However, the nonspecific toxic effects of alloxan precluded its use in this study. The mechanism(s) underlying the protection associated with increased _O_-GlcNAc levels remains to be determined; however, it is increasingly recognized that _O_-glycosylation plays an important role in regulating signal transduction pathways, including mitogen-activated protein kinase pathway (33, 34). Interestingly, the estrogen receptor is subject to modification by _O_-GlcNAc (35), and estrogen treatment significantly improves the recovery after trauma-hemorrhage (36). The increased _O_-GlcNAc levels have also been shown to increase the expression of heat shock proteins 70 and 40 (21).
Small-volume fluid resuscitation strategies are preferred for the treatment of hemorrhagic shock in combat operations (12) or in the prehospital phase before surgical intervention, when large-volume resuscitation promotes further blood loss (7-10). The dose of glucosamine used in this study would translate to 150 to 250 mL/70 kg of body weight or less if a more concentrated solution was used and this volume could be easily transported by a first responder (37). However, if the protective effect of glucosamine is indeed mediated primarily by the increase in _O_-GlcNAc, an alternative to glucosamine infusion would be the pharmacological approaches designed to increase the _O_-GlcNAc levels, such as the inhibition of _O_-GlcNAcase (21, 34, 38). It should be noted that a limitation with regard to the potential translation of these studies to humans is the fact that the animals were anesthetized and received 100% O2 throughout the study. In some models of trauma-hemorrhage, anesthesia is removed during the hemorrhage period; however, we chose to maintain anesthesia and O2 to be consistent with earlier studies using a similar model (11) and to reduce the variability between groups. Furthermore, the increased fraction of inspired oxygen is reported to have no significant effect on the heart rate or cardiac output under hypovolemic conditions (39). It should also be noted that the animals were fasted for 24 h before the experiment to empty the liver glycogen pools. Although a 24 h period of food deprivation can attenuate the restitution of blood volume after hemorrhage, it does not alter significantly the responses of arterial blood pressure, heart rate, cardiac output, or total peripheral resistance (40). Nevertheless, it is possible that glucosamine treatment may be less effective under normal feeding conditions.
In conclusion, we have demonstrated for the first time that the administration of a relatively small volume of 150 mM glucosamine improved the survival rate and significantly increased the MAP after severe trauma-hemorrhage without resuscitation in rats. This protective effect could be related to the increased level of protein _O_-GlcNAc modification in heart, liver, and brain. These data suggest that glucosamine administration could be useful in the treatment of hemorrhagic shock, especially in special conditions, such as in a battlefield or in a rural area, when a prompt surgical intervention and large volume of resuscitation fluids are not feasible or are unavailable.
ACKNOWLEDGMENTS
We thank Dr. Irshad H. Chaudry (Director of the Center for Surgical Research, University of Alabama at Birmingham) for insightful input and suggestions, Mary Ann Accavitti from the University of Alabama at Birmingham Epitope Recognition and Immunodetection Core for the production of the CTD 110.6 monoclonal antibody, Dr. Boglárka Laczy (Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham) for help in immunostaining and editing, and Julie G. Erwin (Veterinary Pathology Laboratory, Animal Resource Program, University of Alabama at Birmingham) for the measurement of serum biochemical parameters.
REFERENCES
1. Anderson RN, Minino MA, Fingerhut LA, Warner M, Heinen M: Deaths: injuries, 2001. Natl Vital Stat Rep 52:1-86, 2004.
2. Sauaia A, Moore FA, Moore EE, Moser KS, Brennan R, Read RA, Pons PT: Epidemiology of trauma deaths: a reassessment. J Trauma 38:185-193, 2004.
3. Peng R, Chang C, Gilmore D, Bongard F: Epidemiology of immediate and early trauma deaths at an urban level I trauma center. Am Surg 64:950-954, 2004.
4. Acosta JA, Yang JC, Winchell RJ, Simons RK, Fortlage DA, Hollingsworth-Fridlund P, Hoyt DB: Lethal injuries and time to death in a level I trauma center. J Am Coll Surg 186:528-533, 1998.
5. Keel M, Trentz O: Pathophysiology of polytrauma. Injury 36:691-709, 2005.
6. Alam HB, Koustova E, Rhee P: Combat casualty care research: from bench to the battlefield. World J Surg 29(Suppl 1):7-11, 2005.
7. Shah KJ, Chiu WC, Scalea TM, Carlson DE: Detrimental effects of rapid fluid resuscitation on hepatocellular function and survival after hemorrhagic shock. Shock 18:242-247, 2002.
8. Handrigan MT, Bentley TB, Oliver JD, Tabaku LS, Burge JR, Atkins JL: Choice of fluid influences outcome in prolonged hypotensive resuscitation after hemorrhage in awake rats. Shock 23:337-343, 2005.
9. Riddez L, Drobin D, Sjostrand F, Svensen C, Hahn RG: Lower dose of hypertonic saline Dextran reduces the risk of lethal rebleeding in uncontrolled hemorrhagic shock in rats. Shock 17:337-382, 2002.
10. Bickell WH, Wall MJ Jr, Pepe PE, Martin RR, Ginger VF, Allen MK, Mattox KL: Immediate versus delayed fluid resuscitation for hypotensive patients with penetrating torso injuries. N Engl J Med 331:1105-1109, 1994.
11. Macias CA, Kameneva MV, Tenhunen JJ, Puyana JC, Fink MP: Survival in rat model of lethal hemorrhagic shock is prolonged following resuscitation with a small volume of a solution containing drag-reducing polymer derived from aloe vera. Shock 22:151-156, 2004.
12. Dubick MA, Atkins JL: Small-volume fluid resuscitation for the far-forward combat environment: current concepts. J Trauma 54(5 Suppl):43-45, 2003.
13. Mizock BA: Alterations in fuel metabolism in critical illness: hyperglycaemia. Best Pract Res Clin Endocrinol Metab 15:533-551, 2001.
14. Jarhult J: Osmotic fluid transfer from tissue to blood during hemorrhagic hypotension. Acta Physiol Scand 89:213-226, 1973.
15. Ware J, Ljungqvist O, Norberg KA, Nylander G: Osmolar changes in haemorrhage: the effects of an altered nutritional status. Acta Chir Scand 148:641-646, 1982.
16. Wells L, Vosseller K, Hart GW: Glycosylation of nucleocytoplasmic proteins: signal transduction and _O_-GlcNAc. Science 291:2376-2378, 2001.
17. Wells L, Gao Y, Mahoney JA, Vosseller K, Chen C, Rosen A, Hart GW: Dynamic _O_-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-_N_-acetylglucosaminidase, _O_-GlcNAcase. J Biol Chem 277:1755-1761, 2002.
18. Whelan SA, Hart GW: Proteomic approaches to analyze the dynamic relationships between nucleocytoplasmic protein glycosylation and phosphorylation. Circ Res 93:1047-1058, 2003.
19. Hart GW, Haltiwanger RS, Holt GD, Kelly WG: Glycosylation in the nucleus and cytoplasm. Annu Rev Biochem 58:841-874, 1989.
20. Virkamaki A, Yki-Jarvinen H: Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart. Diabetes 48:1101-1107, 1999.
21. Zachara NE, O‘Donnell N, Cheung WD, Mercer JJ, Marth JD, Hart GW: Dynamic _O_-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J Biol Chem 279:30133-30142, 2004.
22. Liu J, Pang Y, Chang T, Bounelis P, Chatham JC, Marchase RB: Increased hexosamine biosynthesis and protein _O_-GlcNAc levels associated with myocardial protection against calcium paradox and ischemia. J Mol Cell Cardiol 40:302-312, 2006.
23. Yang S, Zou L, Bounelis P, Chaudry I, Chatham JC, Marchase RB: Glucosamine administration during resuscitation improves organ function following trauma-hemorrhage. Shock 25:600-607, 2006.
24. Robinson KA, Weinstein ML, Lindenmayer GE, Buse MG: Effects of diabetes and hyperglycemia on the hexosamine synthesis pathway in rat muscle and liver. Diabetes 44:1438-1446, 1995.
25. Aghazadeh-Habashi A, Sattari S, Pasutto F, Jamali F: High performance liquid chromatographic determination of glucosamine in rat plasma. J Pharm Pharm Sci 5:176-180, 2002.
26. Dunham CM, Siegel JH, Weireter L, Fabian M, Goodarzi S, Guadalupi P, Gettings L, Linberg SE, Vary TC: Oxygen debt and metabolic acidemia as quantitative predictors of mortality and the severity of the ischemic insult in hemorrhagic shock. Crit Care Med 19:231-243, 1991.
27. Torres LN, Torres-Filho IP, Barbee RW, Tiba MH, Ward KR, Pittman RN: Systemic responses to prolonged hemorrhagic hypotension. Am J Physiol Heart Circ Physiol 286:H1811-H1820, 2004.
28. Keller ME, Aihara R, LaMorte WW, Hirsch EF: Organ-specific changes in high-energy phosphates after hemorrhagic shock and resuscitation in the rat. JAm Coll Surg 196:685-690, 2003.
29. Shahani R, Marshall JG, Rubin BB, Li RK, Walker PM, Lindsay TF: Role of TNF-α in myocardial dysfunction after hemorrhagic shock and lower-torso ischemia. Am J Physiol Heart Circ Physiol 278:H942-H950, 2000.
30. Champattanachai V, Marchase RB, Chatham JC: Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated _O_-GlcNAc. Am J Physiol Cell Physiol 292:C178-C187, 2006.
31. Lee CC, Chang IJ, Yen ZS, Hsu CY, Chen SY, Su CP, Chiang WC, Chen SC, Chen WJ: Effect of different resuscitation fluids on cytokine response in a rat model of hemorrhagic shock. Shock 24:177-181, 2005.
32. Lee CC, Chang IJ, Yen ZS, Hsu CY, Chen SY, Su CP, Chiang WC, Chen SC, Chen WJ: Delayed fluid resuscitation in hemorrhagic shock induces proinflammatory cytokine response. Ann Emerg Med 49:37-44, 2006.
33. Kneass ZT, Marchase RB: Neutrophils exhibit rapid agonist-induced increases in protein-associated _O_-GlcNAc. J Biol Chem 279:45759-45765, 2004.
34. Kneass ZT, Marchase RB: Protein _O_-GlcNAc modulates motility-associated signaling intermediates in neutrophils. J Biol Chem 280:14579-14585, 2005.
35. Cheng X, Hart GW: Alternative _O_-glycosylation/_O_-phosphorylation of serine-16 in murine estrogen receptor β. J Biol Chem 276:10570-10575, 2001.
36. Jarrar D, Wang P, Knöferl MW, Kuebler JF, Cioffi WG, Bland KI, Chaudry IH: Insight into the mechanism by which estradiol improves organ functions after trauma-hemorrhage. Surgery 128:246-252, 2000.
37. Nadler SB, Hidalgo JU, Bloch T: Prediction of blood volume in normal human adults. Surgery 51:224-232, 1961.
38. Nagy T, Champattanachai V, Marchase RB, Chatham JC: Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated _O_-linked _N_-acetylglucosamine. Am J Physiol Cell Physiol 290:C57-C65, 2006.
39. Gunther RA, Reynoso R, Talken L: Heart rate and cardiac output do not improve with supplemental oxygen following hemorrhage in the rat. FASEB J 20:A1383, 2006.
40. Darlington DN, Jones RO, Marzella L, Gann DS: Changes in regional vascular resistance and blood volume after hemorrhage in fed and fasted awake rats. J Appl Physiol 78:2025-2032, 1995.
Keywords:
Hexosamine biosynthesis pathway; mean arterial pressure; _O_-glycosylation; organ function
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