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Cardiovascular Research 2002 55(3):642-659; doi:10.1016/S0008-6363(02)00468-6
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Copyright © 2002, European Society of Cardiology

Ischemic preconditioning protection against stunning in conscious diabetic sheep: role of glucose, insulin, sarcolemmal and mitochondrial KATP channels

Hector F del Valle*, Elena C Lascano and Jorge A Negroni

Department of Physiology, Pharmacology and Biochemistry, Favaloro University, Buenos Aires, Argentina

delvalle{at}favaloro.edu.ar

* Corresponding author. Favaloro University, Solis 453, Buenos Aires 1078, Argentina. Tel.: +54-11-4378-1185/1187; fax: +54-11-4381-0323

Received 25 October 2001; accepted 8 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Introduction: Sarcolemmal and mitochondrial ATP-sensitive potassium (KATP) channels have been postulated to participate in preconditioning protection against infarction and stunning. However, these structures appear to be altered in diabetes and thus, it would be possible that preconditioning does not develop in diabetic hearts. Objective: The purpose of this study was to know whether early (EP) and late (LP) ischemic preconditioning against stunning develop in conscious diabetic (D) sheep and whether diabetes affects KATP channel function. Methods: Male castrated sheep received alloxan monohydrate (1 g) and were ascribed to three experimental groups: control [DC, 12 min of ischemia (i) followed by 2 h of reperfusion (r)], early preconditioning (DEP, six 5 min i–5 min r periods were performed 45 min before the 12 min i) and late preconditioning (DLP, same as DEP except that the preconditioning stimulus was performed 24 h before the 12 min i). Regional mechanics during reperfusion was evaluated by wall thickening fraction (%WTH) and expressed as percentage of basal values (100%), and KATP channel behavior was indirectly assessed by monophasic action potential duration (MAPD) in relation to its sensitivity to glibenclamide blockade (0.1 and 0.4 mg/kg). The results were compared to those obtained in normal (N) sheep. The effects of sarcolemmal and mitochondrial KATP channel blockade on recovery from stunning were assessed by administration of glibenclamide (0.1 and 0.4 mg/kg) and 5-hydroxydecanoate (5-HD, 5 mg/kg i.v.) and/or diazoxide (10 µg/kg/min over 90 min). Whether acute hyperglycemia (H) in normal animals and insulin (I) treatment in diabetic sheep affected preconditioning protection and KATP channel behavior were also evaluated. Results: Results expressed as mean % recovery of %WTH showed that preconditioning protected against stunning in normal sheep (NC=65±3.5, NLP=82±6**, NEP=76±4*, *P<0.05 and **P<0.01 against NC) while this did not occur in diabetic ones, where DLP (58±7.6) afforded a similar recovery to DC (54±5) and DEP worsened instead of improving mechanical function (37±9, P<0.01 against DC). Acute hyperglycemia did not affect preconditioning development (NEPH=72±3 and NLPH=80±4) and insulin treatment reverted the lack of early and late preconditioning protection in diabetic hearts (DEPI=72±4* and DLPI=76±3*, P<0.05 against DC). Sarcolemmal KATP channel behavior appeared altered in diabetic hearts as shown by MAPD in normal sheep (276+10 ms) compared to diabetic ones (365+9 ms, P<0.05) and by the sensitivity to glibenclamide [0.1 mg/kg completely blocked KATP channels in diabetic (P<0.05) but not in normal hearts]. Insulin also restored MAPD in diabetic heart. Mitochondrial KATP channels appeared not to account for the reported results in diabetes, since glibenclamide (%WTH=40±4, P<0.01 vs. NC), but not 5HD nor diazoxide affected myocardial functional recovery during reperfusion. Conclusions: Sarcolemmal KATP channel dysfunction due to the lack of insulin affords a primary approach to explain the absence of preconditioning protection against stunning in diabetic sheep hearts.

KEYWORDS Diabetes; Preconditioning; Stunning; K-ATP channel


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Several years ago an important endogenous cardioprotective mechanism against infarction was termed ischemic preconditioning [1]. The phenomenon called the attention of cardiovascular researchers as the most important mechanism of cardioprotection described up to the present. Although the first works pointed out protection against infarction [1–3], later, many authors described preconditioning protection against arrhythmias [4,5] and both systolic [6,7] and diastolic [8] stunning.

Ischemic preconditioning has two well recognized phases: early preconditioning (which appears immediately after the stimulus and disappears within 3 h) and delayed or late preconditioning (which appears 12 to 24 h after the stimulus and remains for at least 48 to 72 h) [9].

Even though ischemic preconditioning protection has been studied in a great variety of experimental models, the phenomenon has almost always been described in ‘normal’ hearts and there is relatively little experience in pathologic hearts (e.g., diabetic, hypertrophic) which are at high risk of suffering from acute cardiovascular events. Although some reports have studied classic or early ischemic preconditioning protection against infarction in diabetic [10–13] or hypertrophied [14,15] hearts, the afforded cardioprotection has been controversial. However, there are almost no reports in which early and late preconditioning protection against stunning or infarction have been studied in a large pathologic animal model. Thus, we undertook to study both early and late preconditioning protection against stunning in a sheep model of diabetes mellitus.

Diabetes mellitus is a disorder of carbohydrate, lipid and protein metabolism that affects many organs. In addition to contractile abnormalities, this disease causes disturbances in the function of cardiac subcellular organelles, including the sarcolemma, sarcoplasmic reticulum and mitochondria [16]. This pathology is also associated [16] with several abnormalities in energy metabolism, depressed Na+–Ca2+ and Na+–H+ exchange activities, decreased Na+–K+ pump and sarcoplasmic reticulum Ca2+ pump activities, and elevated antioxidant defenses. All or many of the mentioned alterations might explain the reported differences in response to ischemic injury in diabetic vs. normal hearts [17,18]. Diabetes also alters the function of vascular and myocardial ATP-dependent potassium channels (KATP channels) [19–21] and in addition, channel density appears to be diminished in diabetic hearts [20,22]. Since many authors have identified the KATP channels as major contributors to preconditioning protection against infarction and stunning [23], it is possible that the cardioprotective action afforded by this phenomenon is reduced in diabetic hearts. Glucose and insulin have been mentioned to have both deleterious and beneficial effects on cardiovascular function [24–27] during ischemia/reperfusion events and both hyperglycemia and insulin have been described to have an effect on KATP channels [28–30]. Whether hyperglycemia and hypoinsulinemia in diabetic hearts affect preconditioning protection against stunning and whether these metabolic alterations exert a particular effect on KATP channel function is completely unknown. Experiments in anesthetized dogs have shown that acute hyperglycemia abolishes ischemic preconditioning against infarction [31–33]; however, it is unknown whether acute hyperglycemia might exert the same effect in a conscious animal model undergoing a reversible ischemic episode (stunning). Of particular clinical interest is the assessment of whether insulin treatment is able to restore preconditioning protection in conscious animals since many authors have shown that insulin protects against ischemia–reperfusion events in diabetic hearts and that insulin re-instated preconditioning protection in "in vitro" and isolated heart preparations [24,34,35]. Recent clinical findings suggest that insulin might restitute preconditioning protection in type II diabetic patients but until now this fact has not been experimentally proved [36].

Since the development of large animal models with co-incident cardiovascular pathology has been recommended for the study of ischemic preconditioning [37] and due to the fact that many authors have mentioned preconditioning as a ‘healthy heart phenomenon’ [11,38,39]; the purpose of this work was to assess whether early and late preconditioning protection against postischemic myocardial dysfunction (stunning) could be obtained in diabetic conscious sheep, and whether KATP channels, glucose and insulin play a role in the diabetic heart response to ischemia–reperfusion events.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Animal treatment
One hundred and thirty six male castrated Hampshire Down sheep aged 7 to 9 months, weighing 29 to 35 kg were used and treated according to the Guide for the Care and Use Laboratory Animals, published by the US National Institute of Health (NIH publication No. 85-23, revised 1996). On arrival to the animal house, they were deparasitised with ivermectine, and vaccinated against tetanus, anthrax, gas gangrene and clostridial enterotoxemia. Adequate health condition was assessed by professional veterinary staff through clinical examination and laboratory tests. During the days before and after surgery the animals were familiarised with the animal house personnel and the laboratory environment.

2.2 Diabetic conscious sheep model
Five weeks before instrumentation a diabetic state was induced by alloxan monohydrate (Sigma, St. Louis, MO, USA) infused at a total dose of 1 g (28±3 mg/kg). The drug was previously dissolved in 10 ml sterile saline (290 mOsm/l) just prior to use and was administered over 1 min via a jugular vein to sheep that had been fasted for the previous 24 h, similarly to the procedure performed in dogs [40]. Nearly 55% of the alloxan treated sheep never developed diabetes, not even transiently. Of the remaining 45% diabetic animals nearly 15% died the week following drug infusion due to diabetic ketoacidosis. A similar animal loss in the alloxan-induced diabetic model has been described in rabbits [41].

To ensure diabetic state maintenance, venous blood samples were taken in the fasted state on two consecutive days before alloxan injection and twice a week after drug infusion. Fig. 1 shows the behavior of blood glucose, creatinine, cholesterol levels and body weight throughout the study and the altered curve of tolerance to glucose in diabetic sheep, while Tables 1 and 2Go show the characterization of the diabetic metabolic state. To make the comparisons between the diabetic and normal metabolic states, a group of six non-diabetic animals was used.


Figure 1
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  		Fig. 1 Left upper panel shows glycemic levels in normal (n = 6) and diabetic animals (n = 5). Note the increase in blood glucose levels after alloxan injection and their maintenance over 4 weeks. Oral glucose tolerance curves did not show changes in their slope (right upper panel) although diabetic sheep did not return to their basal values after 2.5 h, a finding consistent with those previously reported in sheep (see text). Noteworthy, although the alloxan dose (1 g) resulted in diabetes development, it did not cause a toxic effect nor a decompensated pathology as shown in the lower panel. An instability of biochemical and weight measurements was seen during the first 15 days but afterwards they remained stable. #: P<0.0001 against normal sheep (t test). Data are mean±S.E. Seric creatinine was expressed in µg/10 ml to be better visualized.

 

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  		Table 1 Blood biochemical profile in normal and diabetic sheep

 

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  		Table 2 Body weight, plasma pH and urinary biochemical profile in normal and diabetic sheep

 
After 4 weeks of diabetic state stability the animals underwent surgical instrumentation before preconditioning studies in the conscious unsedated state.

2.3 Biochemical assays
Glucose (enzymatic UV method, hexokinase), triglycerides (GPO-PAP), total cholesterol (CHOD-PAP enzymatic color test), HDL (direct enzymatic test without precipitation), LDL (BM Hitachi-1730860), creatinine (color kinetic method), total proteins and albumin (Biuret method) were automatically determined by a Hitachi 912 Automatic Analyzer (Boehringer Mannheim Systems). Glycated hemoglobin was evaluated by a colorimetric procedure [42] in an IMX analyzer (Abbot Laboratories, Argentina), plasma pH was determined in a ABL 510 Radiometer Copenhagen (Denmark). For insulinemia assessment we employed microparticle enzyme immunoassay (MEIA) technology by using an AXSYM system (Abbot Laboratories). To evaluate ketonuria, urinary pH, urinary proteins and glycosuria a Multistix 10 SG (Bayer, Argentina) was used.

To perform the glucose tolerance test, glucose (1 g/kg) was slowly infused over 1 min through a venous catheter in the relaxed, conscious sheep. Blood samples were taken before (at –10, –5 and 0 min) and after glucose injection at 10, 30, 60, 90, 120 and 150 min (see Fig. 1). The procedure was similar to that mentioned in dogs [40] and sheep [43], and the obtained results were very similar to those reported by McCandless et al. [43].

2.4 Surgical procedures
As previously described [8,44,45], after sedation with acepromazine maleate (0.3 mg/kg), anesthesia was induced with thiopental sodium (20 mg/kg). Following intubation and connection to mechanical ventilation (Neumovent 910, Córdoba, Argentina), anesthesia was maintained with 3% enflurane carried in oxygen and fentanyl citrate 0.1 mg total dose. A sterile thoracotomy was performed at the fifth intercostal space. After pericardiotomy a solid-state pressure microtransducer (Konisberg P7, Pasadena, CA, USA) was inserted in the left ventricular cavity through a stab wound at the apical dimple. Tygon fluid-filled catheters were inserted in the mammary internal vein (for drug infusion) and in the left ventricle (for later calibration of the pressure microtransducer). The left anterior descending coronary artery was dissected free from adjacent tissue just distal to the second or third diagonal branch, and a pneumatic cuff occluder was positioned around it. To obtain left ventricular wall thickness (WTH), a pair of piezoelectric crystals (5 Mhz) was placed within the zone to be rendered ischemic. A pair of steel multifilament wires were sutured to the right ventricular surface for electrocardiographic recordings. All wires and catheters were tunnelled subcutaneously to emerge between the scapulae, and the thoracotomy was closed without pericardial closure. Cefalomicin 1 g/day i.m. was administered during 3 days after surgery. The venous and ventricular catheters were flushed daily with sodium heparin (5000 U) diluted in saline solution.

2.5 Experimental protocol
One week after surgery, the animals were studied standing in a cage. The fluid filled ventricular catheter was connected to a pressure transducer (DT-XX, Viggo-Spectramed, Oxnard, CA, USA) previously calibrated using a transducer calibration system (Xcaliber, Viggo-Spectramed). The zero pressure point was set approximately at the level of the right atrium, and the signal generated by the Konigsberg transducer was adjusted to match that of a Statham transducer. The ultrasonic pair of crystals was connected to a sonomicrometer (Triton, San Diego, CA, USA) and calibrated in mm using the internal calibration. The electrocardiogram was recorded with an ECG Gould transducer. At each acquisition time all signals were digitized at 4 ms interval during 15 s using a personal computer equipped with an A/D converter (National Instruments Lab-PC, Austin, TX, USA) and software developed in our laboratory.

To assess differences in the response to ischemia–reperfusion events between healthy and pathologic hearts, experiments were performed in non-diabetic (normal) and diabetic sheep. The animals were randomly ascribed to different experimental groups (Fig. 2): (1) normal control ischemia (NC, n = 9): after 20 min of basal recordings the sheep underwent 12 min of complete left ventricular regional ischemia followed by 120 min of reperfusion; (2) diabetic control ischemia (DC, n = 7): same as in (1); (3) normal early preconditioning (NEP, n = 8): six 5 min ischemia/5 min reperfusion periods were performed 45 min before the 12 min ischemia; (4) diabetic early preconditioning (DEP, n = 6): same as in (3); (5) normal late preconditioning (NLP, n = 7): the same as the early preconditioning protocol except that the preconditioning stimulus was performed 24 h before the sustained 12 min ischemia; and (6) diabetic late preconditioning (DLP, n = 6): same as in (5). To evaluate the effects of glucose levels on myocardial functional recovery we studied normal sheep with acute hyperglycemia during early (NEPH) and late preconditioning (NLPH) [glucose 0.5 g/kg (25% in water solution) in bolus followed by an infusion of 0.15 g/min (5% in water solution)]. Insulin effect on diabetic sheep was assessed after 2 weeks of insulin treatment (crystalline insulin 10 U in fasting condition followed by insulin NPH 20 U/day twice a day) during early (DEPI) and late (DLPI) preconditioning protocols (n = 4 in each group).


Figure 2
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  		Fig. 2 Schematic diagram illustrating experimental protocols performed in normal and diabetic sheep. Control ischemia, glibenclamide, 5-hydroxydecanoate (5HD), diazoxide treatment and early (EP) and late (LP) preconditioning protocols were performed in normal and diabetic sheep while hyperglycemia and insulin treatment were only done in normal sheep and diabetic sheep, respectively.

 
Previous studies have mentioned sarcolemmal KATP channels to be involved in myocardial protection against stunning while mitochondrial KATP channels appeared to participate in cardioprotection against infarction [46]. To evaluate the involvement of both KATP channels in stunned myocardium in conscious sheep we performed experiments with glibenclamide (0.1 and 0.4 mg/kg in bolus infused 30 min before control ischemia in healthy (NG) and diabetic animals (DG) (n = 6 in each group) and with diazoxide (NX, n = 5) and 5-hydroxydecanoate in normal (N5HD, n = 3) and diabetic (D5HD, n = 3) sheep. Diazoxide was infused at a rate of 10 µg/kg/min in a continuous infusion over 90 min starting 80 min before ischemia because it was the minimal dose without hemodynamic effect (preliminar experiments in sheep), while 5HD was injected at a dose of 5 mg/kg 10 min before ischemia. The effectiveness of these drug doses have been previously validated in stunning and preconditioning studies [7,44–46]. Although glibenclamide blocks both sarcolemmal and mitochondrial KATP channels, mitochondrial structures are not inhibited "in vivo" by the drug [47] making the drug specific for sarcolemmal channels in "in vivo" studies. Regarding the study of mitochondrial KATP channels; we use 5HD, a well known mitochondrial KATP channel blocker, because it has been extensively employed in assessing mitochondrial KATP channel involvement in preconditioning protection [7,23,46,47]. However, it should be mentioned that this drug has been reported not to be as specific as was thought [23,47,48], and in addition, it might be not active in "in vivo" experiments [23,47], as demonstrated by Takano et al. in conscious rabbits where SHD was ineffective to block preconditioning protection [7]. Thus, we also decided to use diazoxide to study mitochondrial KATP channel involvement in sheep heart response to ischemia/reperfusion events, because diazoxide was previously employed to study mitochondrial KATP channel protection against infarction [33] and stunning [7] and is active "in vivo" [47,48].

The signals of 15 to 25 consecutive steady beats were recorded at each acquisition time: basal (after stabilization of left ventricular pressure and dimensions), preischemia (immediately before ischemia), ischemia (at 12 min of the ischemic period) and reperfusion every 10 min during the first hour and every 20 min during the second hour.

Measurements of left ventricular regional (percent wall thickening fraction [%WTH]) and global function (end systolic pressure [Pes], end diastolic pressure [Pd], the maximum [P'max] value of the time derivative of left ventricular pressure [P'] and heart rate [HR]) were performed to assess whether early and late preconditioning elicited any effect on left ventricular functional recovery from stunning. A 12 min ischemic period was used because this short-term regional ischemia induced considerable deterioration of myocardial function without myocyte death and afforded complete recovery of function [44,45]. The latter is of major importance since myocardial stunning must be studied only in conditions of full reversibility [49].

To study KATP channel function in healthy and diabetic sheep open chest experimental protocols were performed. Sarcolemmal KATP channels were indirectly studied by measuring monophasic action potential duration (MAPD) and by assessing their response to the blocking effect of two different doses of glibenclamide. Monophasic action potentials (MAPs) [44,45] were measured in a group of 37 open chest sheep. An Ag/AgCI suction bipolar electrode was placed on the epicardium within the zone to be rendered ischemic. Control recordings were taken in five healthy and five diabetic sheep during basal, preischemia, ischemia (at 2 and 12 min occlusion), and at 2 min of reperfusion. Of the remaining 20 sheep, five healthy and five diabetic were treated with glibenclamide 0.4 mg/kg, and five healthy and five diabetic with glibenclamide 0.1 mg/kg. The effects of hyperglycemia (n = 4) and insulin (n = 3) on MAPD was also evaluated. All drugs and glucose were infused at the same times employed in the mechanical study of stunning and all experimental recordings were acquired as in control MAP recordings. The effect of diazoxide on MAPs was not evaluated due to the reported absence of drug effect on sarcolemmal structures [47].

We have to mention that although MAPD has become controversial as a reliable marker of sarcolemmal KATP channel function. Many "ex vivo" and "in vivo" experiments have found a tight correlation between MAPD and KATP channel function in normal [44,45,50,51] and diabetic hearts [29,52–54] and in addition, MAPD has been accepted as a marker to analyse KATP channel involvement in stunning [44,45,52] and preconditioning in dogs [55], rabbits [48] and pigs [56]. Controversies might have arisen from differences in the type of preconditioning end point (infarction, stunning, arrhythmias) assessed when analysing MAPD; almost all works have studied infarction [47], a particular situation in which sarcolemmal KATP channel appears not to play the main role nor to be directly involved [46]. In our experience, MAPD measurement seems to be a reliable approach to study sarcolemmal KATP channel "in vivo" [44,45].

2.6 Data analysis
End diastole was defined to occur at the onset of the rapid upstroke of the digitally obtained P'. End-systole was defined as the time point where P'min reached 10% of its minimal value. This point was similar to that obtained in preliminary experiments where the end-systolic point was defined as the maximal value of the pressure/diameter ratio [8,44,45]. End ejection was established to occur at P'min.

Percent (%) regional wall thickening (WTH) was calculated as:


Formula

where WTHe is maximum ejective wall thickness between end-systole and end-ejection, and WTHd is end-diastolic wall thickness.

At each acquisition time, Pes, Pd, HR, P'max and %WTH were calculated from each recorded beat and the average of processed beats was the value assigned to the corresponding acquisition time. The value assigned to reperfusion for global hemodynamic variables was the mean integral of the first, second, third and fourth half hours, whereas regional parameters were assessed at 10, 20, 30, 40, 50, 60, 80, 100 and 120 min of the reperfusion period. %WTH was referred to its basal value considered as 100%. MAPD was determined at a repolarization of 50% (MAPD50) and 90% (MAPD90) of maximal plateau amplitude [44,45]. The way in which mechanical recovery and electrophysiologic parameters were assessed has been previously validated in stunning and preconditioning studies [44–46,51,55–57].

2.7 Exclusion criteria
Animals were excluded when they had poor quality of signals or when regional wall thickening was less than 15%. If ventricular tachycardia (VT) appeared at the onset of reperfusion, an intravenous bolus of lidocaine (2 mg/kg) was used to revert the arrhythmia; when VT evolved to ventricular fibrillation (VF), cardioversion was done with one electric shock of 200 to 300 J (Rhomicron defibrilator, Model 790, Argentina). The animal continued the experiment and recordings were acquired over the reperfusion period for hemodynamic and functional measurements when arrhythmia reversion occurred, but was not included if more than one electric shock was needed. The validity for including sheep with reverted arrhythmia and one electric shock was previously stated [44,45]. If after three consecutive electric shocks VF was not reverted, the animal was sacrificed with an overdose of thiopental sodium followed by an overdose of potassium chloride.

2.8 Statistical analysis
Values were expressed as mean±S.E. We used the corresponding analysis of variance (ANOVA) test (see figures and tables) to compare global hemodynamics throughout the protocol, the protection afforded by early and late preconditioning, MAPD and alterations in the biochemical profile between diabetic and healthy control groups. When statistical differences resulted in P<0.05, a post hoc analysis using a Scheffé test was performed. A ‘t’ test was used to compare glycemic levels (Fig. 1).


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
The total number of sheep to perform our study was 136 (48 healthy controls and 88 with alloxan injection). All healthy controls underwent surgery, four of them were discarded due to loss of signals (two), malfunctioning occluder (one) and intractable arrhythmia during the onset of reperfusion (one). Of the 88 alloxan injected sheep, 48 never developed diabetes (55%) and from the remaining 40, four of them died because of diabetic ketoacidosis. Thus 36 diabetic sheep underwent surgical instrumentation; of these, two were discarded (one died during surgery and one lost the signals). Results of 78 animals (healthy control, n = 44; diabetic, n = 34) are thus reported for hemodynamic and mechanical studies. Healthy (n = 19) and diabetic (n = 18) open chest animals were included for MAP measurements and then instrumented for mechanical assessment.

3.1 Characterization of diabetic state
The biochemical profile of blood protein, lipids, insulin and glucose contents is shown in Table 1 while the urine profile, plasma pH and body weight changes are shown in Table 2. The diabetic state altered all the measured parameters and our results are completely in accordance with those reported by McCandless et al. [43] and Wilkinson [58] regarding the characterization of diabetes in ruminants and specially in sheep. Even though diabetes highly affected metabolism, this did not result in an unbalanced state as shown by global hemodynamics (Table 3), regional function (Table 4) and the stabilization in glycemia, cholesterol, creatinine and body weight; all these parameters remained stable before and at the time of the experiments (Fig. 1). After 2 weeks of insulin treatment, diabetic sheep had partially restored their blood and urine biochemical profile (Tables 1 and 2Go).


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  		Table 3 Hemodynamic values of left ventricular global function throughout the protocol in diabetic and healthy sheep

 

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  		Table 4 Comparison of %WTH (real values) between normal and diabetic sheep groups in basal conditions (before starting the protocols)

 
3.2 Hemodynamic data
Hemodynamic data in control and preconditioned sheep groups are summarized in Table 3. Although a significant rise in Pd was observed during ischemia, it returned immediately to its preischemic value during reperfusion. HR, P'max and Pes remained unchanged throughout the experiment and there were no differences among experimental groups. NEPH and NLPH showed hemodynamic values similar to NEP and NLP, also DLPI and DEPI were similar to DEP and DLP. Neither the infusion of 5HD nor diazoxide and glibenclamide affected hemodynamic parameters as shown in Table 5.


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  		Table 5 Hemodynamic values of left ventricular global function throughout the protocol in diabetic and healthy sheep treated with glibenclamide, 5HD and diazoxide

 
3.3 Left ventricular regional function behavior during ischemia and reperfusion
Table 4 shows the regional mechanical parameter (%WTH) at the start of the protocol (basal condition) in all sheep groups. The data show that regional function among groups was similar. This result and the hemodynamic values at the start of the protocol (Table 3) seem to indicate that the stable diabetic state after 4 weeks of inducing the pathology did not have an effect on cardiovascular function and supports the assumption that our model was a model of compensated diabetes.

Figs. 3 and 4Go show the mechanical recovery during reperfusion in normal and diabetic sheep when control ischemia and preconditioning protocols were performed. Fig. 3A shows that diabetic hearts exhibited less improvement in functional recovery during reperfusion when compared to healthy sheep while Fig. 3B shows whether glibenclamide worsened mechanical recovery and Fig. 3C whether diazoxide and 5HD affected functional recovery from stunning. Blockade of sarcolemmal KATP channels exerted a deleterious effects (Fig. 3B) while the activation of mitochondrial structures by diazoxide or their blockade by 5HD did not affected regional mechanical recovery during reperfusion (Fig. 3C). Glibenclamide exerted a more serious mechanical impairment when infused to diabetic sheep (Fig. 3D) which might reflect sarcolemmal KATP dysfunction (see action potential behaviors in Fig. 7A and B). Fig. 4 shows that although ischemic preconditioning exerted a protective effect against stunning during reperfusion in healthy sheep (panel A), its protection was not present in diabetic animals (panel B). Noteworthy, while late ischemic preconditioning afforded a mechanical recovery similar to that obtained in diabetic control, the early preconditioning protocol worsened myocardial recovery during reperfusion. There appeared to be a ‘cumulative ischemic damage’ caused by the brief preconditioning periods, contrary to the reported action of these brief ischemia/reperfusion intervals as a trigger stimulus to elicit classic preconditioning protection in healthy hearts (Fig. 5A). The effects of acute hyperglycemia (glycemic levels=237±19 mg/dl, insulinemia=3.6±0.5 µU/ml) and insulin treatment on early and late ischemic preconditioning showed that hyperglycemia per se was not sufficient to explain the absence of antistunning preconditioning protection in diabetic sheep (Fig. 4C). Interestingly, insulin treatment resulted not only in restoration of early and late preconditioning protection against stunning (Fig. 4D) but also in avoiding the ‘cumulative ischemic damage’ induced by triggering episodes (Fig. 5B).


Figure 3
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  		Fig. 3 Recovery of wall thickening fraction (%WTH) during reperfusion after a 12 min control ischemia in normal and diabetic sheep. NC: Normal control; DC: diabetic control; NG: normal sheep treated with glibenclamide 0.1 mg/kg; NX: normal sheep treated with diazoxide; N5HD: normal sheep treated with 5-hydroxydecanoate and DG: diabetic sheep undergoing glibenclamide 0.1 mg/kg. The figure shows that diabetes impaired functional recovery from stunning (panel A). When glibenclamide, 5HD and diazoxide effects were assessed, glibenclamide worsened mechanical recovery in both normal control (panel B) and diabetic control (panel D) sheep. Notoriously, diabetic hearts were shown to be more sensitive to sulfonylurea effects which might reflect an altered sarcolemmal KATP channel behavior (see discussion). The infusion of diazoxide and 5HD did not affect mechanical recovery from stunning. In panel A: *: P<0.05 against normal group; in panel B: #: P<0.05 vs. NG; in panel C: P<0.05 vs DG (Two-way ANOVA for repeated measures followed by Scheffé). Data are mean±S.E.

 

Figure 4
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  		Fig. 4 Recovery of wall thickening fraction (%WTH) during reperfusion for control, early and late preconditioning protocols. NC: Normal control; NLP: normal late preconditioning; NEP: normal early preconditioning; DC: diabetic control; DLP: diabetic late preconditioning; DEP: diabetic early preconditioning; NEPH: normal early preconditioning protocol subjected to acute hyperglycemia; NLPH: normal late preconditioning protocol subjected to acute hyperglycemia; DEPI: diabetic early preconditioning after 2 weeks insulin treatment; DLPI: diabetic late preconditioning after 2 weeks insulin treatment. Panel A shows results obtained in normal sheep while panel B shows those obtained in diabetic ones. Noteworthy, early preconditioning was shown to impair regional functional recovery from stunning in diabetic sheep. Acute hyperglycemia in normal sheep (panel C) did not alter preconditioning development. The most important finding was that insulin treatment in diabetic animals (panel D) was able to restore preconditioning protection against stunning. The latter results indicate that the lack of insulin and not hyperglycemia per se is the causative pathologic explanation for the absence of protection in diabetic sheep. The relationship between insulin, KATP channels and preconditioning is treated in Discussion. In panel A: #: P<0.01 NC vs. NEP and NLP; **: P<0.05 NC vs. NEP and P<0. 01 vs. NLP; ##: P<0.01 NC vs. NLP. In panels B and D *P<0.05 and # P<0.01 DEP vs. DC and DLP, and DC vs. DEPI and DLPI. (Two-way ANOVA for repeated measures followed by Scheffé). Data are mean±S.E.

 

Figure 7
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  		Fig. 7 Action potential behavior (percent change in MAPD90 from its basal values considered as 100%) during ischemia and reperfusion in normal control (NC) and diabetic control (DC) sheep. Panels A and B show changes in MAPD during ischemia and reperfusion in normal and diabetic hearts before and after glibenclamide (NG: normal plus glibenclamide and DG: diabetic plus glibenclamide) infusion. The figures show MAPD during preischemia (0 min), ischemia (at 2 and 12 min) and reperfusion (2 min). Panel A: Glibenclamide at low doses (0.1 mg/kg) completely blocked action potential shortening during ischemia in diabetic hearts while the same did not occur in normal animals (*P<0.05 NG vs. DG; **P<0.05 NC vs. DC; #P<0.01 DC vs. DG). This might be explained on the basis of a differential function of KATP channels during ischemia in both groups. Panel B: When glibenclamide 0.4 mg/kg was used, there were no differences in drug effects between normal and diabetic animals (**P<0.05 NC vs. DC; #P<0.01 NC vs. NG and DC vs. DG. Panel C: Hyperglycemia (NH: normal sheep undergoing acute hyperglycemia) did not exert a notorious effect on APD although there was a tendency to have an intermediate response between normal and diabetic behavior (**P<0.05 NC vs. DC). Panel D: Insulin (DI: diabetic sheep undergoing 2 weeks insulin treatment) recovered MAPD almost completely to that obtained in healthy normal sheep (**P<0.05 NC vs. DC). Two-way ANOVA for repeated measures followed by Scheffé was used for statistical comparisons. Data are mean±S.E.

 

Figure 5
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  		Fig. 5 Early ischemic preconditioning periods in healthy control and in diabetic sheep. DEP: Diabetic early preconditioning; NEP: normal early preconditioning; DEPI: diabetic early preconditioning protocol after 2 weeks insulin treatment. The figure shows the progressive decay in wall thickening fraction during brief ischemia and reperfusion episodes. There appears to be a ‘cumulative deleterious effect’ in diabetic animals. While normal sheep quickly recovered their function at 15, 10 and 5 min before the prolonged ischemia, diabetic hearts were not able to improve their motility. Insulin treatment completely avoided these effects. *: P<0.05 and #: P<0.01 NEP vs. DEP (Two-way ANOVA for repeated measures followed by Scheffé). Data are mean±S.E.

 
The mechanical behavior occurred during reperfusion in the absence of global functional changes (Tables 3 and 5Go) which indicates that the ischemic area was small enough (less than 20% of the total left ventricular mass, data not shown) to be well compensated by the rest of the ventricular mass and that regional measurements were not influenced by global changes.

3.4 KATP channel function in healthy and diabetic sheep
As previously stated KATP function was indirectly assessed by measuring MAPD and the response to glibenclamide in healthy normal and diabetic animals. Fig. 6 shows MAP recordings and Fig. 7A and B the response of MAPD when glibenclamide was administered to block KATP channels. MAPD was longer in diabetic sheep heart (MAPD 90=345±10 ms, P<0.05) when compared to the healthy control (MAPD 90=282±8 ms) before ischemia and during reperfusion (MAPD 90=365±9 ms vs. MAPD 90=276±10 ms, P<0.05) (Fig. 6). During ischemia, when MAPD diminishes as a consequence of KATP channel activation, MAPD in diabetic hearts (MAPD 90=126±8 ms) shown to be shorter (P<0.05) than in non diabetic ones (MAPD 90=156±10 ms) (Figs. 6 and 7Go). The response to glibenclamide blockade differed notoriously between healthy and diabetic sheep; while 0.4 mg/kg completely blocked MAP shortening during ischemia in both groups (Fig. 7B), 0.1 mg/kg had a 100% blocking effect on MAP shortening in diabetic animals but did not completely block MAP shortening in normal sheep (Fig. 7A). This last result and the previous one regarding differences in MAPD between groups during ischemia might reflect an altered KATP channel behavior in diabetic hearts.


Figure 6
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  		Fig. 6 Monophasic action potential recordings from the epicardial surface using an Ag AgCl suction bipolar electrode. The figure shows action potential duration during preischemia, ischemia (at 12 min) and reperfusion (at 2 min) in normal and diabetic hearts and in acute hyperglycemic and insulin treated hearts. Note that action potential duration in diabetic hearts was longer during preischemia and early reperfusion while during ischemia it showed a greater shortening compared to normal hearts. Hyperglycemic recordings show an intermediate duration between normal and diabetic hearts. Insulin treatment resulted in an almost complete recovery of MAPD making it practically identical to those obtained in normal animals. It is probable that the hormone acted on sarcolemmal KATP channels restoring their function (see Discussion). Horizontal axis represents Time in MS.

 
When the effects of acute hyperglycemia and insulin on MAPD were explored (Fig. 6 and Fig. 7C and D), MAPD in hyperglicemia treated sheep during preischemia (MAPD 90=296±4 ms), ischemia (MAPD 90=136±5 ms) and reperfusion (MAPD 90=294±4 ms) showed an intermediate behavior between diabetic and healthy normal sheep (Fig. 6 and Fig. 7C) while insulin-treated sheep (Fig. 6 and Fig. 7D) almost resembled the same behavior in MAP changes seen in control recordings (MAPD 90: preischemia=284±6 ms, ischemia=150±4 ms, reperfusion=290±5 ms). The infusion of 5HD did not affect MAPD in open chest sheep (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The present work is the first to study the effects of early and late ischemic preconditioning in a diabetic conscious animal model. The main findings regarding ischemia–reperfusion events in the diabetic sheep heart were: (a) diabetes resulted in less mechanical recovery from stunning after a sustained reversible ischemia; (b) early and late preconditioning did not protect the heart against stunning; (c) early preconditioning stimuli had a ‘cumulative deleterious effect’ on left ventricular regional function accounting for a lower mechanical recovery during reperfusion after sustained ischemia; (d) sarcolemmal KATP channel dysfunction in the diabetic heart might provide an explanation to the mentioned results as shown by the differences in MAPD and in the sensitivity to glibenclamide blockade with respect to normal hearts; (e) insulin treatment completely reverted action potential changes, restored preconditioning development and avoided the ‘cumulative deleterious effect’ of triggering episodes reinforcing its role as a cardioprotective hormone; (f) mitochondrial KATP channel and hyperglycemia appeared not to account for the reported findings.

4.1 Animal model
The healthy sheep model to study ischemia–reperfusion events has been fully justified in previous works from our laboratory [8,44,45]. Ischemic preconditioning in the diabetic heart was therefore studied in sheep because diabetes develops in this species [58] and because the sheep is a very docile animal which remains conscious and calm without sedation throughout the whole experiment.

Alloxan, at the employed dose, did not result in altered kidney function. The small urinary protein content could be ascribed to both an early diabetic-induced nephritis (sheep have a high tendency to be nephritic and diabetes could have accelerated the process) or to an increase in urine protein content which is normal in sheep [43]. Furthermore, the urinary biochemical profile was done 4 weeks after induction of diabetes, which is beyond the period of acute renal toxicity that might be associated with administration of alloxan [32], and our dose was three times lower than the minimal one shown to exert renal toxicity in sheep [43]. The maintenance of seric creatinine levels confirms our assumption.

One limitation of our model would be the type and the duration of the pathology; type I diabetes (or insulin dependent) although it might result in a similar pattern of clinical and pathological manifestations as type II diabetes, presents specific events in the development of the illness that make it different from type II. However, preconditioning protection has been proved to develop in type II [10] (including human cardiomyocytes [59]) and in type I diabetic hearts [11–13]. Regarding the duration of diabetes in sheep, it was similar to the time reported in rat [11,13] and dog [32,33] hearts. Nevertheless, the duration of the pathology seems not to be determinant in the experimental findings since hearts subjected to 8 weeks of diabetes have shown confronting results in the same animal model [10,11]. Since the third week of diabetes induction, the metabolic state was stable (Fig. 1) and all sheep showed mild symptoms of established diabetes (polyuria, polyphagia, polydipsia). Regarding the metabolic profile (Tables 1 and 2Go), it was completely in accordance to that previously reported in diabetic sheep [43,58] and dogs [33,60]. Thus, we think that within reasonable limits our model accurately resembles the pattern of type I diabetes.

4.2 Ischemic preconditioning in diabetic hearts and the effects of hyperglycemia and insulinemia
The lack of mechanical recovery from stunning and the absence of early and late preconditioning protection against stunning during diabetes in sheep are completely in accordance with works that have shown that diabetic hearts are more sensitive to ischemic injury (stunning) [17] and those that did not find preconditioning protection against infarction [11,31–33] and arrhythmias [61] in isolated rat hearts and anesthetized dogs with diabetic pathology. Nevertheless, some studies have mentioned that diabetic hearts are less sensitive to ischemia [18] whereas others have found early preconditioning protection against infarction [10,13] and endothelial dysfunction [12]. The conflicting data might be ascribed to differences in the experimental model, the species subject to study and the type and duration of the diabetic state.

Regarding the involvement of hyperglycemia and/or hypoinsulinemia on mechanical recovery, it is almost completely unknown whether one or both of them affect preconditioning development in diabetic hearts. Kersten and co-workers in diabetic anesthetized dogs mentioned hyperglycemia per se as a possible explanation for the absence of early preconditioning protection against infarction [31–33]. Our work is the first in evaluating hyperglycemia effects on early and late preconditioning protection development against stunning in a conscious diabetic large model and in contrast to Kersten's results we did not find that hyperglycemia per se was sufficient to explain the lack of preconditioning. This apparent contradiction might be explained on the basis of two different mechanisms of protection against stunning and infarction [46]. Regarding insulin effects, experimental findings have shown that chronic insulin therapy prevents or gradually reverts all of the diabetic-related changes in myocardial function, biochemistry and structure [16]. Of particular interest are experimental works that have shown that insulin infusion increased cardiac contractility improving regional function [24] and restored preconditioning protection [24,34,35]. Clinically, insulin was advocated as a therapy for ischemia reperfusion injury nearly 40 years ago, when Sodi-Pallares et al. [62] infused a glucose–insulin–potassium solution. Noteworthy, recent reports suggest that insulin might restore preconditioning protection in diabetic patients [36]. This assumption is now experimentally confirmed, for the first time, by our findings in a large mammal model of diabetes. Insulin might exert its role in restoring preconditioning protection due to its effects on sarcolemmal KATP channels (see below).

4.3 Lack of recovery from stunning and absence of preconditioning protection in diabetes: role of sarcolemmal KATP channels
Our findings reinforce for the first time in an ‘in vivo’ large mammal model the altered behavior of KATP channel function reported in ‘in vitro’ experiments in diabetic rat [19,20,29,53] and mouse [54] hearts, where MAPD reflects sarcolemmal KATP alteration. Interestingly, MAPD behavior under the effects of insulin and hyperglycemia are also in accordance with those previously reported in cardiomyocytes [28–30].

Sarcolemmal KATP channels are important structures present in many tissues and are of particular interest in the cardiovascular system where they have been suggested to play a cardioprotective role during ischemic episodes [23]. Sarcolemmal KATP channels, which are normally closed by high ATP concentration, open during ischemia when ATP generation diminishes, increasing the outward potassium current and reducing MAPD [50,51]. Consequently, it has been speculated that their cardioprotective action might be explained by action potential shortening, decreasing the time of Ca2+ influx through Ca2+ voltage dependent channels and avoiding the deleterious effects of Ca2+ overload [50,51]. This mechanism appears to be implicated in sarcolemmal KATP channel protection against stunning and arrhythmias [44,45,50,51]. More recently, these channels have been reported to be involved in the development of myocardial preconditioning [46,48,55,56], and specifically they appear to participate in its anti-stunning effect [46].

Our results show that MAPD greatly differs between diabetic and healthy sheep hearts (Figs. 6 and 7Go). This finding coincides with other results that have been described in ‘in vitro’ rat and mouse heart tissues or cells [19,20,54] where these differences were explained on the bases of sarcolemmal K+ altered currents [19,20,52,53]. An altered sensitivity to variations in ATP levels [53] and changes in the physical structure [19] of the channel, due to changes in the transcription or expression of channel proteins [30,53] explain this altered KATP channel behavior in diabetes. Whatever the involved mechanism, both during ischemia and during early reperfusion (when activated KATP channels shorten MAPD, protecting the heart against ischemia–reperfusion injury [51]), action potential lengthening (specially at the start of reperfusion [51]) might result in Ca2+ overload. Data from our laboratory have demonstrated this mechanism in sheep [45]. Thus, it could be assumed that KATP channel dysfunction causes an inadequate Ca2+ handling during ischemia and reperfusion which finally determines the lack of functional recovery from stunning, the ‘cumulative ischemic damage’ during triggering episodes and the absence of early and late preconditioning protection in diabetic sheep.

The attribution of mechanical results obtained in diabetic sheep to a KATP channel altered behavior appears to be reinforced by the different vasodilatory response due to sarcolemmal KATP channel dysfunction in diabetic patients [21]. Moreover, the reduction in K+ outward current together with the decrease in KATP channel density [20,22] described in hearts from experimental diabetic animals might afford an explanation to the prolongation of the QT interval in diabetic patients [34]. In addition, it has been speculated that the different response of sarcolemmal KATP channels to glibenclamide blockade in diabetic hearts could be attributed to an altered configuration of the sulfonylurea receptor [53]. Since sulfonylureas, mainly due to KATP channel blockade, have been shown to have deleterious cardiovascular actions [44,45,63], the greater sensitivity to glibenclamide blockade seen in our model might be a plausible explanation to the high mortality due to cardiovascular events observed in diabetic patients treated with these compounds [62]. Noteworthy, the deleterious effect of glibenclamide on myocardial recovery from stunning in sheep has been mentioned to be caused by Ca2+ overload due to KATP channel blockade [45]. No changes in myocardial perfusion nor in glycolitic metabolism could accounted for this result in sheep [44,45].

Although the previous explanation reinforces the role of sarcolemmal structures, we have to keep in mind that mitochondrial KATP channels have been also postulated to be involved in preconditioning protection [23,47]. Interestingly these structures appear to be involved in the anti-infarct effect in normal [23,47] and diabetic hearts [33] while their participation in preconditioning protection against stunning is controversial [7,46]. Furthermore, their antistunning effect has been discarded in both isolated rabbit and in situ sheep hearts [46]. This finding appears to be reinforced by the detrimental effect of glibenclamide and the lack of effect of SHD and diazoxide seen in our model and by Takano et al. who have shown that 5-hydroxydecanoate was unable to abolish late preconditioning protection against stunning in conscious rabbits [7]. In contrast, Kersten et al., who studied preconditioning against infarction in anesthetized diabetic dogs, have found that mitochondrial KATP channels are the structures responsible in affording cardioprotection [33]. Thus, this appears to be enough evidence to consider that the antistunning effect is afforded by the sarcolemmal KATP channel [46], while the anti-infarct protection is mediated by the mitochondrial KATP channel [33,47]. A recent report in human diabetic heart tissues mentioned mitochondrial KATP channel as a mechanism of preconditioning protection against cell death but not against heart dysfunction [64].

Whether hyperglycemia and insulinemia play a role in mediating structural changes in KATP channels determining their altered behavior is difficult to understand. Hyperglycemia appears to explain the lack of preconditioning effect against infarction [31,32] because it affects mitochondrial KATP channels functioning in diabetic dogs [33]. In our sheep model, hyperglycemia seemed not to affect preconditioning protection against stunning (Fig. 4C). However, in ventricular myocytes acute hyperglycemia lenghtens MAPD slowing intracellular Ca2+ removal [28], probably accounting for less functional recovery during reperfusion. In our model MAPD tended to be longer in hyperglycemia although it did not reach statistical significance. However, acute hyperglycemia does not necessarily resemble what happens in diabetes where the heart is exposed to a high glycemic level for a prolonged period. Thus, the effects of hyperglycemia on altered sarcolemmal KATP channel behavior in diabetic sheep cannot be completely discarded and have to be further evaluated.

Previous findings have shown that insulin restores, in part, sarcolemmal K+ currents [29,30] and thus, it is possible that the lack of insulin determines the differential KATP channel behavior in diabetic sheep. This appears to be the case since diabetic sheep treated with insulin during 2 weeks almost completely reverted action potential lengthening, which means that insulin restores sarcolemmal KATP response to ischemia–reperfusion events. It is also interesting to note that insulin has been shown to improve the function of cytoskeleton structures in diabetic myocytes [29] and that sarcolemmal KATP channel function is tightly associated to cytoskeleton function [65]. Thus, insulin could have a dual action on the restitution of KATP channel function: (a) by directly acting on it, and/or (b) by improving cytoskeleton function. In addition, insulin regulates the synthesis of KATP channel constitutive proteins [53]. However, these insulin effects might not be the only ones involved in restoring preconditioning development in diabetic hearts since insulin is also cardioprotective by a mechanism independent of KATP channels [66].


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
KATP channel dysfunction in diabetic hearts could afford a physiopathologic approach to the development of diabetic cardiomyopathy and allows to establish a rational explanation to the high cardiovascular risk observed in these patients. Whether hyperglycemia and/or hypoinsulinemia determines KATP channel dysfunction or whether they affect any other metabolic pathway that explains the lower functional recovery from stunning and the lack of preconditioning development in diabetic hearts is an issue that deserves further research.

Since KATP channels are cardioprotectors, the higher sensitivity of the diabetic heart to glibenclamide due to sarcolemmal KATP channel dysfunction might afford an explanation to the reported high mortality due to cardiovascular events in diabetic patients treated with sulfonylureas.

Time for primary review 32 days.


   Acknowledgements
 
We thank Julio Martinez and Fabián Gauna for surgical and technical help. Animal care provided by veterinarians Maria I. Besansón, Pedro Iguain and Marta Tealdo and veterinary assistants: Juan Mansilla, Juan Ocampo and Osvaldo Sosa is gratefully acknowledged. The authors specially thank Dr. Karina B. Orofino for her technical collaboration in drawing tables and figures.


   References
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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