Increased aerobic glycolysis through β2 stimulation is a common mechanism involved in lactate formation during shock states.
Traditionally, hyperlactatemia in critically ill patients, particularly those in shock, has been interpreted as a marker of secondary anaerobic metabolism due to an inadequate oxygen supply inducing cellular distress (1). In contrast, lactate is an important intermediate in the process of wound repair and regeneration (2). It now appears that increased lactate production as a result of anoxia or dysoxia is often the exception, rather than the rule (2). Numerous studies have demonstrated that epinephrine, via β2 stimulation, increases cyclic adenosine monophosphate production, thereby inducing stimulation of glycogenolysis and glycolysis (adenosine triphosphate [ATP] production) and activation of the Na+K+-ATPase pump (3-7). This activation will, in turn, consume ATP, thereby producing adenosine diphosphate. Generated adenosine diphosphate, via phosphofructokinase stimulation, will reactivate glycolysis and hence generate more pyruvate and, consequently, more lactate. Muscle tissue, which represents approximately 40% of total body cell mass, is particularly implicated in this mechanism, and it is noteworthy that more than 99% of muscle adrenergic receptors are β2 receptors (8). In human septic shock, we demonstrated that muscle was a net producer of lactate, and that this could be totally inhibited by ouabain, thus confirming a Na+K+-ATPase-dependent mechanism yet being clearly independent of tissue hypoxia (9). The significance of hyperlactatemia during septic shock remains one of the most debated issues in acid-base physiology. Indeed, the initial reaction to a state of shock or to a severe insult, regardless of etiology, is catecholamine secretion (10). It therefore seems logical that at least a proportion of hyperlactatemia in low cardiac output states, such as that observed during hypovolemic, hemorrhagic or cardiogenic shock, would be due to a Na+K+-ATPase pump-dependent mechanism (5, 11).
Therefore, the goals of our present investigation were (1) to demonstrate from the muscle-arterial blood lactate gradient that muscle produces lactate in shock states; (2) to determine using microdialysis and selective and nonselective pharmacological modulation of muscle lactate metabolism whether muscle lactate production is linked to β2 adrenergic stimulation and Na+K+-ATPase activity in different models of experimental shock.
MATERIALS AND METHODS
This study was approved by the Nancy Institutional Committee on Animal Care and Use, and all animals were treated in compliance with the principles of laboratory animal care of the National Institutes of Health.
Animal preparation and monitoring
Male Wistar rats weighing 300 to 350 g were housed in groups of four under standard conditions at a temperature of 22°C (±1°C) and a 12-h light-dark cycle. They had free access to standard food pellets and tap water. Rats were anesthetized with thiopental sodium (30 mg/kg, i.p.). Animals were placed on a water-heated, temperature-controlled surgical board. Body temperature was monitored with an electronic rectal probe (Lauda, Königshofen, Germany) and kept constant at 37°C throughout the experiment. Animals then underwent tracheotomy and were subsequently ventilated with a volume-controlled rodent ventilator. The right jugular vein and carotid artery were cannulated for infusion, blood withdrawal, and arterial pressure monitoring. Abdominal aortic blood flow (asurrogate of cardiac output) and right femoral blood flow were measured with appropriately-sized perivascular probes connected to an ultrasonic flowmeter (T206, Transonic Systems, Ithaca, NY).
Three different models were used to mimic different clinical situations of shock. The anesthetized fixed pressure model of hemorrhagic shock, resuscitated endotoxic shock, and peritonitis induced by cecal ligation and puncture (CLP). In this model, the animals did not receive volume resuscitation nor antibiotics to mimic the unresuscitated, low cardiac output state associated with the early phase of septic shock.
Hemorrhagic shock was induced by withdrawing blood from the arterial catheter to achieve a MAP of 50 mmHg during a 5-min period. Blood was withdrawn/reinfused to maintain a MAP (MAP) of 50 mmHg during a 2-h period.
Endotoxic shock was induced by administration of 15 mg/kg of endotoxin (Escherichia coli O127:B8, Sigma, St. Quentin Fallavier, France) infused intravenously from T0 to T20 min. All animals received saline as a continuous infusion (8 mL/h) from T0 to T120 min.
Subacute sepsis was induced by CLP, as previously described (12). Briefly, rats were anesthetized with ketamine (150 mg/kg, i.p., with additional doses given when necessary) and a 3- to 4-cm abdominal incision was performed to expose the cecum. This was subsequently ligated and punctured once with a 21-gauge needle and a small amount of feces extruded. The bowel was returned into the abdomen and the abdominal cavity closed in two layers. All animals were resuscitated with 5mL/100 g body weight of isotonic sodium chloride solution administered subcutaneously after completion of surgery. The animals were carefully observed postsurgery. After 18 hours, the rats were prepared, anesthetized with thiopental sodium (30 mg/kg, i.p.) and monitored. After initial measurements were taken (T0), animals received 15 mL/h hydroxyethyl starch (Voluven; Fresenius FAG, Bad Hombourg, Melsungen, Germany) during 20 min, and this was subsequently reduced to 8 mL/h.
For the control group, eight rats were sham-operated and monitored (peritonitis control group), and eight rats were used as a control for endotoxin and hemorrhagic shock (control group) and were monitored as described in the chapter "Animal preparation and monitoring."
Commercially available microdialysis probes (CMA/20; CMA/Microdialysis, Stockholm, Sweden) were used for the study. The length of the microdialysis membrane (20 mm) and a very slow perfusion flow rate (0.3 μL/min) guarantee 100% recovery (i.e., uptake of molecules from the interstitial space) of molecules up to 20 kd in size, thus providing true tissue concentrations (13). A 1-h equilibration period was allowed after insertion of the probe. Because changes in the concentration of a metabolite in the interstitial space are determined by the equilibrium between local production, uptake, and removal by the tissue flow, it is essential to obtain information on local tissue blood flow. Changes in tissue blood flow at the probe site were determined using the ethanol dilution technique based on Fick principle (14). Ethanol, which is not significantly metabolized in skeletal muscle, was used to assess the effects on local tissue blood flow of propranolol, a nonselective β blocker; ICI-118551, a selective β2 blocker; and ouabain, a selective inhibitor of the Na+K+-ATPase. All products were obtained from Sigma (St. Louis, Mo). A decrease in the ratio of ethanol concentration in the dialysate to the original ethanol concentration in the perfusate is indicative of changes in blood flow and vice versa. For purposes of simplicity, the term "ethanol ratio" is used to describe this relationship. To minimize evaporation, the dialysate was collected into sealed 250-μL glass tubes in a refrigerated (4°C) fraction collector. Dialysate fractions were collected every 20 min.
Experiment 1: Measurement of muscle-blood lactate gradient (n = 16 rats)
The gradient between the dialysate and blood concentration of lactate was measured using a 0.3-μL/min perfusate flow, with a positive gradient indicating muscle lactate production.
Experiment 2: Modulation of lactate formation
Three probes were inserted in each rat, one in the left thigh and two in the right thigh, and perfused at a flow rate of 1 μL/min with free lactated Ringer solution (147 mM Na+, 4 mM K+, 2.20 mM Ca2+, 156 mM Cl−) and ethanol (50 mM). One probe was perfused with free lactated Ringer alone, whereas the other two were randomly perfused with propranolol (10−4 M), ouabain (10−5 M), or ICI-118551 (10−8 M). These concentrations of propranolol and ICI-118551 have been shown in previous microdialysis experiments to effectively and selectively block their respective designated receptors, whereas higher concentrations would lead to a mixed blockade of more than one receptor. Eight rats were used in each group.
Analytical sampling procedures
Arterial blood samples were collected in fluoride-oxalate-containing tubes. Lactate was measured by an enzymatic-colorimetric method adapted to the Wako automatic analyser (Biochem Systems, Paris, France). Normal blood values are less than 2 mM.
Arterial blood samples were immediately deproteinized by the addition of iced perchloric acid (1 M) and analyzed. Pyruvate was measured by the enzymatic-UV method. The normal range of values in blood is 40 to 68 μM.
Analytical ranges were as follows: 0 to 10,000 μM for lactate and 0 to 300 μM for pyruvate. Run-to-run precision, expressed as the coefficient of variation, was 1.5% for lactate and 5.9% for pyruvate.
The concentration of ethanol in both perfusate (inflow) and dialysate (outflow) was determined using a standard enzymatic assay.
Plasma epinephrine concentration was measured using a high-performance liquid chromatography-electrochemical detection technique.
Results are expressed as mean ± SEM. ANOVA for repeated measurement was performed over time to evaluate within-group differences using Newman-Keuls test for post hoc analysis. For nonparametric data, a Friedman test was used. Comparisons between the local lactate or pyruvate responses to the different drugs were performed over time, with a two-factor ANOVA. When the relevant F values were significant at the 5% level, further pair-wise comparisons were made using Dunnett test for the effect of time and with the Bonferroni correction for the effects of treatment at specific times. For statistical evaluation of changes in the ethanol ratio variations over time, one-factor ANOVA for repeated measurements was used. Differences were considered statistically significant at P < 0.05. A software package was used for all statistical analysis (PRISM 4; GraphPad Software, San Diego, Calif).
Measurement of muscle-blood lactate gradient (experiment 1)
When using a low flow rate (0.3 μL/min), muscle lactate concentrations were consistently higher than arterial levels, with a mean gradient of 2.5 ± 0.3 mM in endotoxic shock, 2.1± 0.2 mM in peritonitis group, and 0.9 ± 0.2 mM in hemorrhagic shock. By contrast, there was no gradient in the control group (0.1 ± 0.1 mM; P < 0.05 for all groups; Table 1).
Muscle pyruvate concentrations were also always higher than arterial levels, with a mean gradient of 260 ± 40, 210 ± 30, and 90 ± 10 μM (P < 0.05 for all groups). There was no gradient in the control group.
Systemic and regional hemodynamics (experiment 2)
During endotoxin infusion, heart rate increased from 430 ± 25 to 460 ± 20 beats per minute (P < 0.05), whereas MAP decreased from 125 ± 15 to 78 ± 8 mmHg (P < 0.01). Aortic (Fig. 1) and femoral blood flow (from 12 ± 2 to 18 ± 3 mL/min) increased (P < 0.01).
When compared with the control group, at baseline, MAP (125 ± 14 to 90 ± 17 mmHg), aortic (Fig. 1), and femoral blood flow (11 ± 3 to 7 ± 3 mL/min) significantly decreased (P < 0.01). Heart rate increased from 420 ± 25 to 450 ± 20 beats per minute (P < 0.05). All variables returned to baseline values during volume resuscitation (P < 0.01).
Hemorrhagic shock group
Heart rate increased from 420 ± 21 to 480 ± 30 beats per minute, whereas MAP (128 ± 15 to 44 ± 5 mmHg), aortic (Fig. 1), and femoral blood flow (13 ± 4 to 3 ± 2 mL/min) significantly decreased (P < 0.01).
Plasma catecholamines, glucose, lactate, and pyruvate concentrations (experiment 2)
Epinephrine levels were elevated in all shock groups. (Fig. 2). From T0 to T120 min, blood glucose levels did not change in the control group (5.9 ± 1.0 - 6.05 ± 1.0 mM). Blood glucose levels increased from 6.1 ± 1 to 8.8 ± 1.2, 6.0 ± 1 to 7.7 ± 1.1, and 6.2 ± 1 to 6.8 ± 1.0 mM in the endotoxic, peritonitis, and hemorrhagic groups, respectively (P < 0.01). Nevertheless, the elevation in blood glucose levels was more marked in the septic groups than in the hemorrhagic shock group (P < 0.05).
All the models induced hypotension and marked hyperlactatemia at T120 min (P < 0.01 vs. T0 and vs. control group; 5.1 ± 3.4 mM in endotoxic shock, 9.2 ± 5.6 mM in hemorrhagic shock, and 4.9 ± 2.3 mM in peritonitis). In the peritonitis group, lactate decreased to 2.8 ± 2.1 mM at the end of the experiment. The lactate-to-pyruvate (L/P) ratio at T120 increased from 15 ± 5 to 25 ± 5 and 14 ± 5 to 45 ± 8 in the endotoxic and hemorrhagic groups, respectively. At T0 before volume resuscitation, the L/P ratio in the peritonitis group was increased when compared with the peritonitis control group (15 ± 5 to 29 ± 5; P < 0.05). The L/P ratio decreased from 29± 5 to 20 ± 7 in the peritonitis group at T120 min.
Effects of propranolol, ICI-118551, and ouabain on ethanol ratio and muscle lactate production (experiment 2)
During hemorrhagic shock, the ethanol ratio increased markedly, indicating a decrease in blood flow surrounding the probe (P < 0.01). Endotoxin administration and subsequent fluid resuscitation were associated with a slight decrease in ethanol ratio (P < 0.05), indicating an improvement in blood flow due to vascular loading. In the peritonitis model, volume resuscitation was associated with a marked decrease in ethanol ratio (P < 0.01), indicating an increase in local perfusion due to an increase in MAP and cardiac output (Fig. 3).
Ouabain, propranolol, and ICI-118551 were associated with an increase in ethanol ratio when compared with the Ringer-perfused probe, indicating a decrease in local blood flow (P < 0.01). Despite this decrease in blood flow, all drugs decreased muscle lactate and pyruvate concentrations (P < 0.01). Pyruvate decreased in the same proportion to lactate; thus, the L/P ratio did not change.
The present study demonstrates that, in shock states, a significant portion of muscle lactate formation is dependent on β2 activation through epinephrine stimulation of the Na+K+-ATPase pump, irrespective of the underlying shock mechanism.
Effect of selective blockers on local blood flow and muscle lactate production
Muscle tissue accounts for 40% of body cell mass and physiologically produces 25% of total body lactate (380 mmol/d). During shock states, Daniel et al. (15) demonstrated in a hypokinetic model of shock that a portion of lactate originates from muscle. In the present study, muscle lactate and pyruvate concentrations were always higher than arterial lactate and pyruvate concentrations, irrespective of shock mechanism. Our findings lend support to the notion of muscle lactate production during shock. Nevertheless, muscle lactate and pyruvate concentrations were higher in the septic models. In a population of shock patients, we found that septic patients exhibit higher lactate and pyruvate level concentrations than patients with cardiogenic shock (16).
Competition studies with selective antagonists have demonstrated that β-adrenergic receptors of human skeletal muscle are exclusively of the β2 subtype (8). To demonstrate that muscle lactate production in shock states is due to an increase in Na+K+-ATPase activity under β2 stimulation induced by endogenous epinephrine, three specific antagonists were used: a selective inhibitor of the Na+K+-ATPase pump (ouabain), a nonselective β blocker (propranolol), and a selective β2 blocker (ICI-118551). As expected, the drugs decreased blood flow adjacent to the microdialysis probes via adrenergic blockade for propranolol and ICI-118551, and intracellular calcium mediated vasoconstriction for ouabain (17) (Fig. 4). Despite this decrease in flow, microdialysate lactate and pyruvate significantly decreased in all models of shock. This demonstrates that lactate production during shock states is related, at least in part, to increased Na+K+-ATPase activity under β2 stimulation induced by elevated endogenous epinephrine. Nevertheless, the agents did not totally abolish overproduction of lactate and pyruvate, thus underscoring other mechanisms behind formation of lactate.
The basis for aerobic lactate production during shock is not straightforward. Accelerated aerobic glycolysis is defined as a state in which the rate of glycolysis exceeds the oxidative capacity of the mitochondria and is present during states associated with an increase in endogenous/exogenous catecholamines in conditions where there is sufficient oxygen available for the mitochondria to carry out oxidation of the pyruvate produced. Clearly, the Na+K+-ATPase pump that is situated in the plasma membrane of virtually all animal cells is the major mechanism for maintaining the low intracellular Na+ and high intracellular K+ concentrations required for a multitude of cellular functions (18). Moreover, stimulating the pump is a necessary part of the mechanism required for conversion of muscle glycogen to lactate so it can leave the cells and be used elsewhere. Epinephrine-induced lactate production serves as the main metabolic precursor for the liver via the Cori cycle. Indeed, the lactate shuttle theory postulates that aerobic glycolysis confers increased metabolic flexibility by allowing tissues to share a common source for oxygenation or gluconeogenesis (2). The elevated blood lactate concentrations observed in shock states may therefore represent a key protective metabolic event by favoring lactate, rather than glucose oxidation in tissues where oxygen is available, and preserving glucose use for nonaerobic ATP resynthesis through glycolysis in tissues where oxidative metabolism is limited. The L/P ratio reflects the cytoplasmic accumulation of reducing equivalents. Such a high L/P ratio as observed in the present study may be associated with a large interorgan flux of reducing equivalents (lactate, nicotinamide adenine dinucleotide) for ATP synthesis. Therefore, this seemingly high and unnecessary rate of aerobic glycolysis under epinephrine stimulation may provide some assistance to such organs as the heart, wounded tissue, and brain to sustain specific processes that require a high rate of cytoplasmic ATP (20, 21). We recently demonstrated that plasma lactate depletion using selective β2 blocker was associated to a decrease in myocardial efficiency and early death (22).
During shock states, including low-output situations, muscle lactate and, by extension, hyperlactatemia is, in large part, related to the stimulation of muscle β2 receptors and thus independent of tissue hypoxia. The concept of lactate as merely a metabolic waste product has now evolved toward lactate being viewed as an energetic shuttle (23, 24). Thus, in most clinical critical care situations, hyperlactatemia must be mainly perceived as an adaptive response to a shock state and not as a marker of tissue hypoxia.
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