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Anesth Analg 2008; 106:135-142
© 2008 International Anesthesia Research Society
doi: 10.1213/01.ane.0000297299.91527.74
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ANESTHETIC PHARMACOLOGY

Anesthesia's Effects on Plasma Glucose and Insulin and Cardiac Hexokinase at Similar Hemodynamics and Without Major Surgical Stress in Fed Rats

Coert J. Zuurbier, PhD, Peter J. M. Keijzers, MD, Anneke Koeman, Harry B. Van Wezel, PhD, MD, and Markus W. Hollmann, PhD, MD

From the Department of Anesthesiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Address correspondence and reprint requests to Dr. C. J. Zuurbier, Department of Anaesthesiology, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Address e-mail to c.j.zuurbier{at}amc.uva.nl.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: Recent evidence suggests that hexokinase mitochondria association attenuates cell death, and that plasma glucose and insulin concentrations can influence clinical outcome. In the present study, we examined how different anesthetics per se affect these variables of glucose metabolism, i.e., under similar hemodynamic conditions and in the absence of major surgical stress.

METHODS: In fed rats, the effects of pentobarbital (PENTO), isoflurane (ISO), sevoflurane (SEVO), ketamine-medetomidine-atropine (KMA), and sufentanil-propofol-morphine (SPM) on the cardiac cellular localization of hexokinase (HK) and levels of plasma glucose and insulin were determined and compared with values obtained in nonanesthetized animals (control). The role of mitochondrial and sarcolemmal KATP-channels and {alpha}2-adrenergic receptor in ISO-induced hyperglycemia was also evaluated.

RESULTS: Mean arterial blood pressure was similar among the different anesthetic strategies. PENTO (5.3 ± 0.2 mM) and SPM (5.1 ± 0.2 mM) had no significant effect on plasma glucose when compared with control (5.6 ± 0.1 mM). All other anesthetics induced hyperglycemia: 7.4 ± 0.2 mM (SEVO), 9.9 ± 0.3 mM (ISO), and 14.8 ± 1.0 mM (KMA). Insulin concentrations were increased with PENTO (2.13 ± 0.13 ng/mL) when compared with control (0.59 ± 0.22 ng/mL), but were unaffected by the other anesthetics. Inhibition of the mitochondrial KATP channel (5-hydroxydecanoate acid) or the {alpha}2-adrenergic receptor (yohimbine) did not prevent ISO-induced hyperglycemia. Only the nonspecific KATP channel inhibitor glibenclamide was able to prevent hyperglycemia by ISO. Cytoslic HK relative to total HK increased in the following sequence: control (35.5% ± 2.1%), SEVO (35.5% ± 2.7%), ISO (36.6% ± 1.7%), PENTO (41.2% ± 2.0%; P = 0.082 versus control), SPM (43.0% ± 1.8%; P = 0.039 versus control), and KMA (46.6 ± 2.3%; P = 0.002 versus control).

CONCLUSIONS: Volatile anesthetics and KMA induce hyperglycemia, which can be explained, at least partly, by impaired glucose-induced insulin release. The data indicate that the inhibition of insulin release by ISO is mediated by sarcolemmal KATP channel activation. The use of PENTO and SPM is not associated with hyperglycemia. SPM and KMA reduce the antiapoptotic association of HK with mitochondria.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Numerous studies have demonstrated associations between plasma glucose concentrations and poor clinical outcome or cell death: an increased plasma glucose concentration is usually associated with decreased clinical outcome during critical illness1 or in patients with acute myocardial infarction.2 Aggressive perioperative control of glucose is expected to become an important component of good clinical practice.3,4 It is commonly believed that hyperglycemia is a result of stress signals caused by pain and surgical interventions. However, anesthesia per se (without surgical stress) may also increase stress signals such as catecholamines and cortisol5 or may more directly manipulate glucose homeostasis by affecting pancreatic insulin release.6–8 Several studies have indicated that volatile anesthetics and ketamine-xylazine induce hyperglycemia.7–15 Such data are currently lacking for the clinically relevant sufentanil-propofol-morphine (SPM) regimen. Moreover, in studies comparing the effects of anesthesia, the presence of surgical stress or varying levels of depth of anesthesia have often confounded the results.7–15 This hinders an unequivocal examination of the effects that anesthetics per se have on glucose and insulin concentrations. The first goal of the present study was therefore to perform a comprehensive examination of the effects of different anesthetic regimens on plasma glucose and insulin levels under similar hemodynamic conditions in the absence of major surgical stress. The five different anesthetic regimens examined were specifically chosen as being relevant for both clinical interventions [sevoflurane (SEVO), isoflurane (ISO), SPM], and experimental, animal, studies [pentobarbital (PENTO), ketamine-medetomidine].

Previous data have demonstrated that part of the ISO-induced hyperglycemia is a result of increased endogenous glucose production accompanied by a decreased glucose clearance.9 In that study, however, insulin levels remained low despite increased glucose levels, indicating that ISO impairs glucose-induced insulin release. In the present study, we explored the mechanism behind the impaired insulin release by ISO. In general, the secretion of insulin is under the control of both KATP-dependent and independent pathways (such as {alpha}2-adrenergic signaling), which may both be influenced by mitochondrial metabolism.16 For ketamine-xylazine, it was reported that hyperglycemia was mainly induced through activation of {alpha}2-adrenergic signaling.8 ISO opens mitochondrial KATP channels17 and thereby alters mitochondrial metabolism.18 It is unknown, however, through which pathway ISO may affect insulin secretion and thereby induces hyperglycemia. Thus, as a second goal, we examined the role of KATP-channels (mitochondrial and sarcolemmal) and {alpha}2-adrenergic receptors in ISO-induced hyperglycemia.

Finally, we examined how the different anesthetic regimens affected the localization of hexokinase (HK) within the myocardial cell, the enzyme catalyzing the first committed step of glucose metabolism in the cell. The enzyme is peculiar in that it is the only glycolytic enzyme that can translocate from the cytosol to the mitochondria.19 This makes this enzyme ideally suited to play an important role in cardioprotection, since current knowledge indicates that both mitochondria and glycolysis figure prominently in cardioprotection.20,21 Several studies indicate that increased HK association with mitochondria protects against apoptosis and can be viewed as an antioxidant defense mechanism.22–24 We were the first to report that many cardioprotective interventions all share translocation of HK to the mitochondria within the heart.25 Our third goal was therefore to determine the effect of anesthesia on the subcellular distribution of HK within the heart.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
All procedures were in accordance with and performed with permission from the Animal Ethics Commission of the University of Amsterdam and conformed to the National Institute of Health (USA) guidelines. All experiments were performed with 3- to 4-mo-old male Wistar rats obtained from Charles River (The Netherlands). The animals were kept in cages in groups of 1–5 subjected to a 12-h dark–light cycle, with water and food (69% carbohydrates, 22% protein, and 9% fat) ad libitum. All experiments were performed between 10 am and 4 pm.

General Surgical Procedure
After anesthesia was induced, a tracheotomy was performed and mechanical ventilation was started. The lungs were ventilated with 34% O2 to 66% N2 at an end-tidal pressure of 15 mm Hg using a modified infant ventilator (model MK-78; Medec, Aalst, The Netherlands). Ventilation variables such as inspiratory phase (0.25–0.35) and respiration rate (50–75 breaths/min) were adjusted to keep arterial Pco2 values within physiologic limits (25–50 mm Hg) as checked by taking a blood sample. The body temperature of each rat was kept between 36.5°C and 37.5°C with the use of a heating pad that was thermocontrolled by a temperature probe placed in the rat's rectum. In addition, heat loss was compensated for with the use of a ceramic heating lamp positioned 40–50 cm above the rat. A carotid artery and jugular vein were cannulated with 0.5 x 0.9 mm polyethylene vein catheter (Braun, Melsungen, Germany). The catheters were filled with 0.9% NaCl solution (Baxter, Utrecht, The Netherlands) with heparin (0.25 IU/mL). The carotid artery catheter was fitted to a pressure transducer (Baxter, Dearfield, IL) for continuous monitoring of mean arterial blood pressure and heart rate. After instrumentation, a 20-min stabilization period was allowed before the 30-min experimental protocol (Fig. 1). No fluid was administered during the approximately 80 min that the animals were anesthetized.


Figure 126
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  		Figure 1. Experimental protocol. Composite protocol used to study the effects of anesthetics, KATP channels, and {alpha}2-adrenergic receptors in the intact rat. Following instrumentation and stabilization, all groups underwent a 30-min experimental protocol with measurements of mean arterial blood pressure, heart rate, glucose, and insulin. Specific measurements and interventions, denoted by the numbered arrows, are as follows: 1: intraperitioneal administration of yohimbine; 2: determination of glucose; 3: IV administration of 5-hydroxydecanoate (10 mg/kg) or diazoxide (10 mg/kg) or glibenclamide (1 mg/kg); 4: isoflurane (1.25%), in the IIH-subgroup only; 5: insulin.

 

Anesthetics and Plasma Glucose and Insulin Concentrations
One nonanesthetized group and five different anesthetic regimens were studied: 1) quickly decapitated, nonanesthetized (CONTROL; n = 10), 2) PENTO; n = 9, 3) ISO; n = 7, 4) SEVO; n = 7, 5) ketamine-medetomidine-atropine (KMA; n = 6), and 6) SPM; n = 6. Each group was only treated with the type of anesthesia examined for that group, during induction of anesthesia, the instrumentation period, and the subsequent experimental protocol. PENTO was administered intraperitoneally (IP) at 90 mg/kg as induction dose, followed by 2/5 of the induction dose at 30 min after induction, and 1/5 of the induction dose every 30 min thereafter. The induction of anesthesia with the volatile ISO or SEVO was started by placement of the animal in a container with 3%–4% ISO or 4%–5% SEVO, whereas maintenance of anesthesia was provided by continuous inhalation of 1.5% ISO or 2.5% SEVO. The KMA regimen was chosen because it is one of the recommended regimens for studies in small animals.26,27 After IP induction by ketamine (90 mg/kg)-medetomidine (0.01 mg/kg)-atropine (KMA) (0.18 mg/kg), maintenance was provided by continuous IV administration of 50 mg · kg–1 · h–1 ketamine and 0.05 mg · kg–1 · h–1 atropine. The SPM regimen was chosen because this combination is frequently used in clinical practice, especially during cardiac surgery.28 Morphine is added to this regimen because of its reported cardioprotective effects29 and it facilitates smooth, easy recovery after surgery. Induction was started by IP injection of sufentanil (43 mg/kg) and morphine (0.3 mg/kg), followed by an IV bolus of propofol (11 mg/kg). Anesthesia was maintained by continuous IV administration of 10 mg · kg–1 · h–1 propofol and 7.5 mg · kg–1 · h–1 sufentanil.

KATP-Channels/{alpha}2-Adrenergic Receptors
To further explore the mechanisms causing hyperglycemia by volatile anesthetics, ISO-induced hyperglycemia was evaluated with the use of several drugs that manipulate the KATP-channels and {alpha}2-adrenergic receptors. Diazoxide (10 mg/kg) and 5-hydroxydecanoate (5HD; 10 mg/kg) were used as specific mitochondrial KATP-channel opener and inhibitor, respectively, and glibenclamide (1 mg/kg) as a nonspecific KATP-channel inhibitor. Delivery and solubilization of these drugs follow previous reports.30,31 In short, diazoxide and 5HD were dissolved in NaOH (1 mM): NaCl (1:2) and administered IV through the tail vein at the start of the experimental protocol (Fig. 1) as 0.3 mL/kg, flushed with NaCl to a total IV volume of 1 mL/kg. Glibenclamide (1 mg/kg) was dissolved in NaOH:ethanol: PEG:NaCl (0.1:0.1:0.1:2.7) and administered IV as 1.5 mL/kg, flushed with NaCl to a total IV volume of 2 mL/kg. Yohimbine (6 mg/kg) was used to specifically inhibit {alpha}2-adrenergic receptors. Yohimbine was dissolved in H2O (1 mg/mL) and administered IP at the start of anesthesia (60 min before start experimental protocol) as 4 mg/kg as reported by Saha et al.8 To obtain maximal inhibition, an additional bolus of 2 mg/kg was given at the start of the experimental protocol (Fig. 1).

All animals in this subgroup were anesthetized with PENTO (90 mg/kg), to allow stress-free administration of drugs before administration of ISO (1.25%). The following groups were studied: 1) diazoxide without ISO (dia; n = 6), 2) 5-HD with ISO (n = 5), 3) glibenclamide + ISO (n = 4), 4) glibenclamide without ISO (gli; n = 5), 5) yohimbine with ISO (yoh + iso; n = 4), and 6) yohimbine without ISO (yoh; n = 4). See Figure 1 for timing of protocols. Diazoxide, 5-HD, glibenclamide and yohimbine were obtained from Sigma (St. Louis, MO).

Determination of Plasma Glucose and Insulin
Glucose concentrations in arterial blood (carotid) were determined (Distronic Freestyle glucose strips) at the beginning of the stabilization period and at the end (t = 30 min) of the experimental protocol (Fig. 1). Insulin was determined in plasma with the use of an ELISA assay (Mercodia, Sweden). The plasma for insulin determination was obtained from 1 mL blood, taken from the thorax cavity after excising of the heart at the end of the experimental protocol using heparinized syringes. The blood was centrifuged and the supernatant (plasma) stored at –80°C until analysis.

Cardiac Hexokinase Activity
The excised heart was immediately rinsed and minced in 2 x 8 mL ice-cold homogenization medium (0.25 M sucrose, 0.02 M HEPES, 1 mM β-mercaptoethanol, pH 7.4) and homogenized (Potter S, Sartorius) on ice. Part of the homogenate was immediately centrifuged at 18,000g for 10 min at 4°C. The supernatants were recovered and represented the soluble or cytosolic fraction, whereas the part of the homogenate that was not centrifuged represented the total whole-cell fraction. Both fractions were quickly frozen at –80°C until determination of enzyme activity.

For measurement of whole-cell enzyme activities, the homogenate was treated with 0.5% Triton X-100 and sonicated for 5 s, followed by centrifugation in an Eppendorf microcentrifuge (18,000g; 15 s). Whole-cell and cytosolic HK activity were measured spectrophotometrically with glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides), glucose, ATP and NAD+, in the presence of rotenone (1 mM) to inhibit the mitochondrial respiration chain.

Since HK distribution may be influenced by the time-consuming procedure of mitochondria purification, we chose to rapidly separate the mitochondria from the cytosol and to measure HK activity in the cytosolic fraction instead of in a purified mitochondrial fraction. Changes in cytosolic HK activity relative to whole-cell activities are used as indices of HK redistribution with the disappearance of HK from the cytosol as index of increased HK association with the mitochondrial fraction.25

Statistics
All data are presented as means ± sem. ANOVA with Dunnett's post hoc tests was used to compare group means at identical times, and Student's t-test were used to compare within group or within treatment differences. P values <0.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemodynamic data and body weights are summarized in Table 1. Body weight was similar among the groups. Mean arterial blood pressure and heart rate for treatment groups, at the end of the protocol when the heart was excised, did not differ significantly among groups, with the only exception being a slower heart rate for the KMA group.


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  		Table 1. Body Weight and Hemodynamics Measured at End of Experiment for all Five Anesthetic Regimens

 

Anesthetics and Blood Glucose and Insulin Concentrations
The glucose and insulin concentrations reported were all determined at the end of the protocol (30 min, Fig. 1). Glucose determined at the beginning of the protocol was not significantly different from the values determined at the end of the protocol. The nonanesthetized, control animals displayed normoglycemia (5.6 ± 0.1 mM) (Fig. 2A). Anesthesia with PENTO or SPM did not affect glucose concentrations when compared with control. Significant hyperglycemia was observed with the volatile anesthetics ISO (9.9 ± 0.3 mM) and SEVO (7.4 ± 0.2 mM). The largest increase in plasma glucose was shown with KMA anesthesia (14.8 ± 1.0 mM). Converse results were observed for the plasma insulin concentrations (Fig. 2B). Only PENTO (2.13 ± 0.13 ng/mL) increased insulin significantly compared with control (0.59 ± 0.22 ng/mL). All the other anesthetic strategies were without effect on insulin. The absence of increased insulin concentrations during anesthesia-induced hyperglycemia suggests that pancreatic insulin release was inhibited.


Figure 226
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  		Figure 2. Blood glucose (A) and blood insulin (B) measured at the end of the experimental protocol for the control (nonanesthetized) and the five anesthetic protocols used. *P < 0.05 versus control (ANOVA, Dunnett's post hoc tests).

 

The Role of KATP-Channels and {alpha}2-Adrenergic Receptors in ISO-Induced Hyperglycemia
The administration of 1.25% ISO after initial PENTO anesthesia increased plasma glucose concentrations (7.5 ± 0.4 mM) significantly (Fig. 3A), albeit to a lesser extent than when 1.5% ISO was used alone (Fig. 2A). Opening of the mitochondrial KATP channel with diazoxide was without effect on plasma glucose. In addition, inhibition of these channels with 5HD did not prevent hyperglycemia by ISO. In contrast, the nonspecific KATP channel inhibitor glibenclamide significantly reduced plasma glucose concentrations from 5.4 ± 0.3 mM to 2.6 ± 0.1 mM. Importantly, in the presence of glibenclamide, ISO was unable to significantly increase plasma glucose concentrations (3.2 ± 0.4 mM). Yohimbine significantly reduced plasma glucose concentrations (from 5.1 ± 0.3 mM to 4.0 ± 0.1 mM), but did not prevent the ISO-induced hyperglycemia (5.8 ± 0.4 mM). Glibenclamide increased plasma insulin concentrations significantly above the already increased concentrations observed with PENTO anesthesia alone (Fig. 3B).


Figure 326
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  		Figure 3. The effect of KATP channels and {alpha}2-adrenergic receptor on the isoflurane-induced hyperglycemia in pentobarbital-anesthetized animals. Blood glucose (A) was measured before the start of the experimental protocol (filled bars, see Fig. 1 for timing), i.e., before application of drugs and isoflurane, respectively, and at the end of the experimental protocol (open bars). Insulin levels (B) were measured at end of experimental protocol. Dia = diazoxide (10 mg/kg), Glib = glibenclamide (1 mg/kg), 5HD = 5-hydroxydecanoate (10 mg/kg), and Yoh = yohimbine (6 mg/kg). {dagger}P < 0.05 versus baseline value within group (paired Student's t-test), #P < 0.05 versus value at end exp. protocol with same treatment but without iso administration (unpaired Student's t-test), *P < 0.05 versus Pento (ANOVA, Dunnett's post hoc tests).

 

Anesthetics and Cellular Translocation of Cardiac HK
The amount of cytosolic HK activity normalized to total whole-cell activity is depicted in Figure 4. The volatile anesthetics, known to be cardioprotective, demonstrated the lowest levels of cytosolic HK (35.5% ± 2.7% and 36.6% ± 1.7% for SEVO and ISO, respectively), with levels similar to the nonanesthetized, control condition (35.5% ± 2.1%). The cardioprotective neutral anesthetic PENTO showed a nonsignificant trend (P = 0.082) of increased level of cytosolic HK. Finally, an increase of cytosolic HK (equal to solubilization of HK from mitochondria) was observed with the SPM (43.0% ± 1.8%; P = 0.039) and KMA combination (46.6% ± 2.2%; P = 0.002).


Figure 426
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  		Figure 4. Anesthetics effects on the cardiac cellular distribution of hexokinase (HK). The cellular distribution is given by HK activity measured in the cytosolic fraction relative to the total, whole-cell HK activity. *P < 0.05 versus control (ANOVA, Dunnett's post hoc tests).

 


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this study may be summarized as follows: 1) at similar levels of hemodynamics and without major surgical stress, volatile anesthetics, and ketamine-medetomidine, but not PENTO or SPM, produce hyperglycemia, and 2) the ISO-induced hyperglycemia is at least partly mediated by pancreatic sarcolemmal KATP channel activation, and 3) ketamine-medetomidine and SPM reduce HK binding to mitochondria. To our knowledge, this is the first study directly comparing the hyperglycemia-inducing effects of anesthetics at similar hemodynamic levels. Interestingly, the anesthetic strategy of SPM does not affect either plasma glucose or insulin concentrations, and may be the anesthetic strategy of choice when hyperglycemia needs to be prevented. This is in concert with our own observations, where SPM was without effect on plasma glucose concentrations in coronary artery bypass graft patients before and during the early phase of surgery.28

There is indirect evidence that volatile anesthesia may indeed increase blood glucose when compared with epidural anesthesia in humans, although confounding factors, such as surgical stress and differences in hemodynamic conditions, were present in these studies.7,9 Considering that hyperglycemia is currently viewed as a risk factor for poor clinical outcome,1 our data would suggest that barbiturates and opioid-propofol combinations are less harmful than volatile anesthetics. This is in contrast, however, with studies reporting improved clinical outcome with volatile anesthetics when compared with, for example, remifentanil-propofol.32 Although plasma glucose concentrations were not described, the study of De Hert et al.32 suggests that cardioprotective actions of volatile anesthetics may overrule possible adverse effects of hyperglycemia. It is also possible that hyperglycemia did not occur with SEVO in that study because the patients were fasted and/or only low concentrations of SEVO were used. The effects of volatile anesthetics on plasma glucose concentrations are dependent on age, nutritional status, and the concentration used.8,10–12,15,33,34 Interestingly, the KATP channels figure prominently in both the hyperglycemia and the cardioprotective effects of volatile anesthetics. Our data suggest that the opening of the sarcolemmal KATP channels contributes to the possible detrimental hyperglycemia effects, whereas the opening of the mitochondrial KATP channels probably contributes to the cardioprotective actions of the volatile anesthetics.35 It will therefore be of interest to directly compare within one study the hyperglycemic effects of anesthetics with their cardioprotective action.

Insulin secretion is under the control of both KATP-dependent and independent pathways (such as {alpha}2-adrenergic signaling), which are both influenced by mitochondrial metabolism.16 The observation that ketamine in combination with the {alpha}2 agonist (medetomidine) produces severe hyperglycemia is in support of recent reports.8,11,12 Saha et al.8 demonstrated that the {alpha}2 agonist xylazine caused hyperglycemia through impaired insulin secretion, since inhibition with yohimbine ({alpha}2 antagonist) prevented hyperglycemia. Ketamine alone was without effect on blood glucose. In our study, we observed the lowest insulin concentrations with medetomidine. These data indicate that the clinical use of {alpha}2-agonists (e.g., medetomidine, clonidine) should be carefully evaluated in terms of occurrence of hyperglycemia.

Although yohimbine also decreased plasma glucose concentrations in our study, it did not prevent the ISO-induced increases in blood glucose, demonstrating that hyperglycemia with ISO is not mediated by {alpha}2 activation. In addition, manipulation of the mitoKATP channel with 5HD and diazoxide negated a significant role for these channels in ISO-induced hyperglycemia. Diazoxide and 5HD were without a significant effect on plasma glucose or insulin concentrations in our study, measured 30 min after drug administration. This is in agreement with data showing that these channels have minimal effects on plasma glucose and insulin concentrations, especially when the effects are being studied more than 15 min after IV administration.36 The present study is, thus, the first to demonstrate that ISO produces hyperglycemia through activation of sarcolemmal KATP channels.

We found no increased plasma glucose concentration with PENTO anesthesia in our study, which was probably related to the observed increased plasma insulin concentration. Saha et al.8 and Brown et al.14 also demonstrated that hyperglycemia did not occur with PENTO; however, insulin was not reported in these studies. Lee et al.12 did not observe increased insulin concentrations with PENTO anesthesia in mice as compared to the nonanesthetized control animals. It cannot be excluded that insulin was not increased with PENTO in that study, because their control animals were already severely hyperglycemic (12.6 mM). In contrast to opening of sarcolemmal KATP channels by ISO anesthesia, and therefore inhibiting insulin release, our data suggest inhibition of KATP channels by barbiturate anesthesia and thus activating insulin release. This is in agreement with Ashcroft and Ashcroft,37 who reported that barbiturates inhibit KATP channels in cellular studies. Kozlowski and Ashford38 also demonstrated that barbiturates partially inhibit the sarcolemmal KATP channels in insulin secretion cells. However, in both studies the effects of barbiturates on insulin were not reported. To the best of our knowledge, our study is the first to demonstrate that barbiturate in the intact organism does not inhibit insulin release as observed for volatile anesthetics and {alpha}2 agonists, but rather augment plasma insulin concentrations. The inhibition of the sarcolemmal KATP channels stimulates insulin release, and it is thought that the increased plasma insulin concentrations prevent hyperglycemia from occurring with PENTO anesthesia.

The effects of anesthetics on plasma glucose and insulin reported in this study related to the nonfasted condition. It is likely that fasting will affect these changes.8,12,15 Saha et al.8 showed that 18 h fasting in the rats mitigated the ISO and ketamine/xylazine-induced hyperglycemia; however, hyperglycemia was still observed. Pomplun et al.15 showed that after overnight fasting the relative increase in plasma glucose with anesthesia was even increased when compared with the fed condition. Similarly, after overnight fasting in humans, ISO anesthesia was still associated with hyperglycemia.9 Further studies are necessary to examine to what extent the anesthetic effects on plasma glucose and insulin reported in his study are affected by the duration of fasting. It is anticipated that, in general, these anesthetic effects will still be present, albeit attenuated, in the fasted condition.

Older literature reported the solubilization of HK with anesthesia in the brain,39 and suggested the effect to be largely mediated by the rate of energy metabolism, i.e., the stage of anesthesia.40 Although we also observed solubilization of HK with certain anesthetics, we are the first to report that ISO or SEVO do not result in HK solubilization in the heart. It seems unlikely that the effect can be ascribed to various depths of anesthesia, since the hemodynamics was similar among the different anesthetic regimens in our study. One likely explanation is that volatile anesthetics differently affect survival kinases that have as a final target, among others, the cellular distribution of HK.24,41,42 It was shown that HK association with mitochondria (preventing HK solubilization into the cytosol) is increased by inhibition of glycogen synthase kinase 3β.42 One study41 demonstrated that the protective effect of ISO was mediated by inhibition of glycogen synthase kinase 3β through activation of survival kinases such as phosphatidylinositol 3-kinase and protein kinase B. This result, together with the results of the present study, may therefore suggest that HK binding to mitochondria may contribute to ISO-induced protective cellular signaling. Interestingly, when arranging the anesthetics in accordance with the observed degree of HK binding to mitochondria, the obtained sequence of SEVO, ISO, PENTO, SPM, and ketamine-medetomidine is remarkably similar to the sequence obtained when arranged according to reported cardioprotective potential of the different anesthetics,32,43,44 indicating that mitochondrial HK binding may contribute to the cardioprotective potential of the different anesthetics. Further studies directly examining the cardioprotective effects of anesthetics with mitochondrial HK binding will be necessary to definitely prove that there is such a correlation between mitochondrial HK and the cardioprotective potential of anesthetics.

In conclusion, during similar hemodynamic conditions and without major surgical stress, anesthesia per se is a major determinant of plasma glucose and insulin concentrations. PENTO and SPM keep glucose in the normoglycemic range, whereas glucose concentrations are increasingly elevated in the order of SEVO, ISO, and ketamine-medetomidine. For ISO, hyperglycemia is, at least partly, a result of impaired insulin release due to sarcolemmal KATP-channel activation. These results indicate that the choice of anesthetic may be an important determinant of whether, and to what degree, hyperglycemia develops. Finally, ketamine-medetomidine and SPM reduce the antiapoptotic binding of HK with the mitochondria within the heart.


   Footnotes
 
Accepted for publication September 19, 2007.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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E. Gurel, K. M. Smeele, O. Eerbeek, A. Koeman, C. Demirci, M. W. Hollmann, and C. J. Zuurbier
Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion
J Appl Physiol, June 1, 2009; 106(6): 1909 - 1916.
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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press