Limitations to the assessment of reperfusion injury with radiolabeled 2-deoxyglucose.
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Animals↗
Biomedical subjects
T Doenst
Publications and source records attributed to T Doenst.
Variability of the lumped constant for [18F]2-deoxy-2-fluoroglucose and the experimental isolated rat heart model: clinical perspectives for the measurement of myocardial tissue viability in humans.
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Animals↗
alpha-adrenergic stimulation mediates glucose uptake through phosphatidylinositol 3-kinase in rat heart.
We examined whether insulin and catecholamines share common pathways for their stimulating effects on glucose uptake. We perfused isolated working rat hearts with Krebs-Henseleit buffer containing [2-3H]glucose (5 mmol/L, 0.05 microCi/mL) and sodium oleate (0.4 mmol/L). In the absence or presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (3 micromol/L), we added insulin (1 mU/mL), epinephrine (1 micromol/L), phenylephrine (100 micromol/L) plus propranolol (10 micromol/L, selective alpha-adrenergic stimulation), or isoproterenol (1 micromol/L) plus phentolamine (10 micromol/L, selective beta-adrenergic stimulation) to the perfusate. Cardiac power was found to be stable in all groups (between 8.07+/-0.68 and 10.7+/-0. 88 mW) and increased (25% to 47%) with addition of epinephrine, but not with selective alpha- and beta-adrenergic stimulation. Insulin and epinephrine, as well as selective alpha- and beta-receptor stimulation, increased glucose uptake (the following values are in micromol/[min. g dry weight]: basal, 1.19+/-0.13; insulin, 3.89+/-0.36; epinephrine, 3.46+/-0.27; alpha-stimulation, 4.08+/-0.40; and beta-stimulation, 3.72+/-0.34). Wortmannin completely inhibited insulin-stimulated and selective alpha-stimulated glucose uptake, but it did not affect the epinephrine-stimulated or selective beta-stimulated glucose uptake. Sequential addition of insulin and epinephrine or insulin and alpha-selective stimulation showed additive effects on glucose uptake in both cases. Wortmannin further blocked the effects of insulin on glycogen synthesis. We conclude that alpha-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K-dependent pathway. However, the additive effects of alpha-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, potentiation, or inhibition by wortmannin of another intermediate of the alpha-adrenergic signaling cascade. The stimulating effects of both the alpha- and the beta-adrenergic pathways on glucose uptake are independent of changes in cardiac performance.
Adrenergic Agents↗
Ischemia-stimulated glucose uptake does not require catecholamines in rat heart.
The authors tested the hypothesis that ischemia stimulates glucose uptake in rat heart independent of the insulin signaling pathway and independent of endogenous catecholamines. Isolated working rat hearts were perfused with Krebs-Henseleit buffer containing [2-3H]glucose (5 mmol/l, 0.05 muCi/ml) and Na-oleate (0.4 mmol/l) with or without the phosphatidylinositol 3-kinase inhibitor wortmannin (3 mumol/l). Insulin (1 mU/ml) was added to the perfusate in the middle of the experiments or the hearts were subjected to 30 min of low-flow ischemia (75% reduction in coronary flow) followed by 15 min of reperfusion. In a separate group, hearts were subjected to ischemia and reperfusion in the presence of propranolol (10 mumol/l) plus phentolamine (10 mumol/l). Cardiac power was stable but decreased (-75%) during ischemia. Both insulin and ischemia increased glucose uptake (P < 0.01). Glucose uptake returned to pre-ischemic values during reperfusion. Wortmannin completely inhibited insulin-stimulated glucose uptake and glycogen synthesis, but did not affect the ischemia-stimulated glucose uptake or glycogen resynthesis during reperfusion. Full adrenergic blockade did not abolish the ischemia-stimulated glucose uptake. The authors conclude that: (1) insulin and ischemia increase glucose uptake through different mechanisms; (2) ischemia-stimulated glucose uptake is not catecholamine mediated: and (3) glycogen resynthesis during reperfusion is independent of PI3-K.
Animals↗
Insulin improves functional and metabolic recovery of reperfused working rat heart.
BACKGROUND: Glucose, insulin, and potassium solution improves left ventricular function in refractory pump failure. Direct effects of insulin on the heart cannot be determined in vivo. We hypothesized that insulin has a direct positive inotropic effect on the reperfused heart. METHODS: Isolated working rat hearts were perfused with buffer containing glucose (5 mmol/L) plus oleate (1.2 mmol/L). Hearts were subjected to 15 minutes of ischemia and reperfused with or without insulin (100 microU/mL) for 40 minutes. Epinephrine (1 micromol/L) was added for the last 20 minutes. RESULTS: Hearts recovered 51.1% of preischemic cardiac power in the absence and 76.4% in the presence of insulin (p < 0.05). Whereas oleate oxidation remained unchanged, glucose uptake and oxidation increased during reperfusion with epinephrine (p < 0.01). This increase was significantly greater when hearts were reperfused in the presence of insulin (p < 0.01). Insulin also prevented an epinephrine-induced glycogen breakdown during reperfusion (p < 0.05). CONCLUSIONS: Insulin has a direct positive inotropic effect on postischemic rat heart. This effect is additive to epinephrine and occurs without delay. Increased rates of glucose oxidation and net glycogen synthesis are more protracted.
Animals↗
Protection of acutely ischemic myocardium by controlled reperfusion.
The goal of revascularization after acute occlusion of a coronary artery is the return of contractile function and the reduction of mortality. Although reperfusion of ischemic myocardium is a prerequisite for return of function, it may, in itself, cause further injury. Controlled blood cardioplegic reperfusion reduces this "reperfusion injury" and provides maximal myocardial protection. In this article, we review recent advances in surgically controlled reperfusion and speculate on future prospects for myocardial protective techniques in patients with acute coronary artery occlusion.
Cardiopulmonary Bypass↗
Myocardial glucose uptake measured with fluorodeoxyglucose: a proposed method to account for variable lumped constants.
UNLABELLED: Quantitative assessment of myocardial glucose uptake by the glucose tracer analog 2-deoxy-2-[18F]fluoro-D-glucose (FDG) depends on a correction factor (lumped constant [LC]), which may vary. We propose that this variability is caused by different affinities of FDG and glucose for membrane transport and phosphorylation and can be predicted from the time course of FDG retention. We therefore measured the LC under steady-state metabolic conditions and compared the results with values predicted from the tracer retention alone. METHODS: We measured rates of myocardial glucose uptake by tracer ([2-3H]glucose) and tracer analog methods (FDG) in isolated working Sprague-Dawley rat hearts perfused with Krebs buffer and glucose, or glucose plus insulin or beta-hydroxybutyrate. In separate experiments, we established the theoretical upper and lower limits for the LC (Rt and Rp), which are determined by the relative rates of FDG and glucose membrane transport (Rt, 1.73 +/- 0.22) and the relative rates of FDG and glucose phosphorylation (Rp, 0.15 +/- 0.04). RESULTS: The LC was decreased in the presence of insulin or beta-hydroxybutyrate or both (from 1.14 +/- 0.3 to 0.58 +/- 0.16 [insulin], to 0.75 +/- 0.17 [beta-hydroxybutyrate] or to 0.53 +/- 0.17 [both], P < 0.05). The time-activity curves of FDG retention reflected these changes. Combining the upper and lower limits for the LC with the ratio between unidirectional and steady-state FDG uptake rates allowed the prediction of individual LCs, which agreed well with the actually measured values (r = 0.96, P < 0.001). CONCLUSION: The LC is not a constant but is a predictable quotient. As a result of the fixed relation between tracer and tracee for both membrane transport and phosphorylation, the quotient can be determined from the FDG time-activity curve and true rates of myocardial glucose uptake can be measured.
Animals↗
Profound underestimation of glucose uptake by [18F]2-deoxy-2-fluoroglucose in reperfused rat heart muscle.
BACKGROUND: [18F]2-deoxy-2-fluoroglucose (FDG) is widely used as a tracer for glucose uptake in ischemic heart muscle. We tested the effects of low-flow ischemia and reperfusion on the ratio of tracer/tracee (lumped constant, LC). METHODS AND RESULTS: Isolated working rat hearts were perfused with Krebs-Henseleit buffer containing only glucose 5 mmol/L (group 1) or glucose 5 mmol/L plus oleate 0.4 mmol/L (group 2, fed; group 3, fasted). Dynamic glucose uptake was measured simultaneously with [2-3H]glucose and with FDG. After 20 minutes, coronary flow was reduced by 75% for 30 minutes before it was returned to control conditions for the final 20 minutes. Hexokinase activity in the cytosolic and mitochondrial fractions and tissue metabolites were determined. Rates of glucose uptake were highest when glucose was the only substrate. Glucose uptake, FDG uptake, and the LC increased during ischemia only in group 3. There was no change of these parameters during ischemia in groups 1 and 2. FDG uptake decreased significantly with reperfusion in groups 2 and 3, and there was a striking fall in the LC (from >1.0 to <0.2, P<.001). The fall in the LC was associated with a significant increase in intracellular free glucose. Neither ischemia nor reperfusion affected the kinetic properties of hexokinase. CONCLUSIONS: FDG profoundly underestimates glucose uptake during reperfusion in the presence of fatty acids. In the fasted state, however, FDG overestimates glucose uptake during ischemia. The results indicate limitations in the use of FDG to quantify myocardial glucose uptake in human heart.
Animals↗
Complexities underlying the quantitative determination of myocardial glucose uptake with 2-deoxyglucose.
The quantitative determination of glucose uptake by using 2-deoxy-D-glucose (2-DG) is based on the assumption that 2-deoxyglucose-6-phosphate (2-DG6P) cannot be further metabolized and requires the lumped constant (LC) to equate the kinetic differences in uptake between 2-DG and glucose. We tested whether insulin or epinephrine affect the LC, and quantitated the incorporation of 2-DG6P into glycogen in the isolated working rat heart. Hearts were perfused for 35 min at near physiological workload with Krebs-Henseleit buffer containing glucose (5 mmol/l) plus oleate (0.4 mmol/l, Group 1) with either insulin (1 mU/ml, Group 2), or epinephrine (1 micromol/l, Group 3). In all groups [2-3H] glucose and [U-14C]2-DG (10 microCi each) were present in the perfusate for the first 30 min. In order to estimate the quantitative relationship of glucose and 2-DG uptake and glycogen synthesis from glucose and 2-DG, we perfused hearts with equimolar amounts of glucose and 2-DG (5 mmol each) and either [18F]2-deoxy-2-fluoroglucose plus [2-3H]glucose or [U-14C]glucose plus [1,2-3H]2-DG as tracers. All hearts were freeze-clamped for determination of 2-DG accumulation, glycogen, and tracer activity in glycogen. Glucose and 2-DG uptake were similar in the absence of insulin (LC 1.27+/-0.09). In the presence of insulin, 2-DG underestimated glucose uptake (LC 0.61+/-0.02). Epinephrine did not affect the tracer/tracee ratio (LC 1.31+/-0.09). Incorporation of [U-14C]2-DG into glycogen occurred in all groups (Group 15.38+/-0. 65%, Group 25.72+/-0.59%, Group 32.70+/-0.16% of total tracer uptake.) When equimolar amounts of glucose and 2-DG were present, 2-DG uptake, measured by dynamic assessment of FDG accumulation, significantly decreased over 30 min while glucose uptake remained unchanged. The hearts perfused with [U-14C]glucose and [1,2-3H]2-DG synthesized 39.5+/-7.1 micromol glycogen/g dry/30 min. 2-DG contributed 4.2+/-1.4%. We conclude that insulin and epinephrine have differential effects on the LC, and 2-DG6P is a substrate for glycogen synthesis.
Animals↗
F-18 FDG uptake in transplanted heart.
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Deoxyglucose↗
Unloaded heart in vivo replicates fetal gene expression of cardiac hypertrophy.
The cardiac response to increased work includes a reactivation of fetal genes. The response to a decrease in cardiac work is not known. Such information is of clinical interest, because mechanical unloading can improve the functional capacity of the failing heart. We compared here the patterns of gene expression in unloaded rat heart with those in hypertrophied rat heart. Both conditions induced a re-expression of growth factors and proto-oncogenes, and a downregulation of the 'adult' isoforms, but not of the 'fetal' isoforms, of proteins regulating myocardial energetics. Therefore, opposite changes in cardiac workload in vivo induce similar patterns of gene response. Reactivation of fetal genes may underlie the functional improvement of an unloaded failing heart.
Anastomosis, Surgical↗
Clinical experience with blood cardioplegia.
Since its initial description, blood cardioplegia has become the preferred tool to arrest the heart for open heart surgery in both children and adults. This shift from crystalloid-type cardioplegia to blood cardioplegia occurred because experimental and clinical studies demonstrated superior protection of the arrested myocardium by blood cardioplegia. In this article we review and describe the different strategies and protocols which have evolved for the use of blood cardioplegia under different conditions. The protocols differ in the composition of the crystalloid component of the solution, the direction of delivery (antegrade, retrograde, or both), and/or the temperature of the solution. In order to maxime myocardial protection, specific protocols for the administration of blood cardioplegia have been designed for specific clinical conditions. We further define the indications for the use of the different protocols. Our own clinical experience with blood cardioplegia is corroborated with the concepts and results described in the literature. We conclude that blood cardioplegia is 1.) an effective way to safely arrest the myocardium for cardiac operations, 2.) affords excellent myocardial protection, and 3.) allows for quick recovery of contractile function upon reperfusion.
Adult↗
Insulin does not change the intracellular distribution of hexokinase in rat heart.
Preliminary evidence has suggested that hexokinase in rat heart changes its kinetic properties in response to insulin through translocation to the outer mitochondrial membrane. We reexamined this hypothesis in light of tracer kinetic evidence to the contrary. Our methods were as follows. Working rat hearts were perfused with Krebs-Henseleit buffer containing glucose (5 mmol/l) and sodium oleate (0.4 mmol/l). Dynamic glucose uptake was measured with [2-3H]glucose and with 2-deoxy-2-[18F]fluoroglucose (2-[18F]DG). Hexokinase activity was determined in the cytosolic and mitochondrial fractions. Our results are as follows. Uptake of glucose and uptake of 2-[18F]DG were parallel. Insulin (1 mU/ml) increased glucose uptake threefold but had no effect on 2-[18F]DG uptake. The tracer-to-tracee ratio decreased significantly. The Michaelis-Menten constant of hexokinase for 2-deoxyglucose was up to 10 times higher than for glucose. There was no difference in maximal reaction velocity. Two-thirds of hexokinase was bound to mitochondria. Insulin neither caused translocation nor changed Michaelis-Menten constant or maximal reaction velocity. In conclusion, the insulin-induced changes in the tracer-to-tracee ratio are due to a shift of the rate-limiting step for glucose uptake from transport to phosphorylation by hexokinase. Insulin does not affect the intracellular distribution or the kinetics of hexokinase.
Animals↗
Energy provision from glycogen, glucose, and fatty acids on adrenergic stimulation of isolated working rat hearts.
We postulated that glycogen is a significant energy substrate compared with fatty acids and glucose in response to adrenergic stimulation of working rat hearts. Oxidation rates were determined at 1-min intervals by release of 3H2O from [9,10-(3)H]oleate (0.4 mM, 1% albumin) and 14CO2 from exogenous [U-14C]glucose (5 mM) or, by a pulse-chase method, from [14C]glycogen. We estimated the 14C enrichment of glycogen metabolized at each time point to determine true rates of glycogen use. Based on the pattern of glycogen enrichment over time, glycogenolysis did not exhibit a high degree of preference for newly synthesized glycogen. Epinephrine (1 microM) increased contractile performance 86% but did not stimulate oleate oxidation. The increased energy demand was supplied by carbohydrates, initially by a burst of glycogenolysis (contributing 35% to total ATP synthesis for 5 min) and followed by delayed increase in the use of exogenous glucose (eventually contributing 29% to ATP synthesis). On the basis of the release of 14CO2 and [14C]lactate specifically from glucose or glycogen, we found that a larger portion of glycogen was oxidized compared with exogenous glucose, augmenting the yield of ATP from glycogen. Thus the heart responds to an acute increase in energy demand by selective oxidation of glycogen.
Adenosine Triphosphate↗
Ischemic preconditioning in rat heart: no correlation between glycogen content and return of function.
We tested the hypothesis that glycogen levels at the beginning of ischemia affect lactate production during ischemia and postischemic contractile function. Isolated working rat hearts were perfused at physiological workload with bicarbonate buffer containing glucose (10 mmol/L). Hearts were subjected to four different preconditioning protocols, and cardiac function was assessed on reperfusion. Ischemic preconditioning was induced by either one cycle of 5 min ischemia followed by 5, 10, or 20 min of reperfusion (PC5/5, PC5/10, PC5/20), or three cycles of 5 min ischemia followed by 5 min of reperfusion (PC3 x 5/5). All hearts were subjected to 15 min total, global ischemia, followed by 30 min of reperfusion. We measured lactate release, timed the return of aortic flow, compared postischemic to preischemic power, and determined tissue metabolites at selected time points. Compared with preischemic function, cardiac power during reperfusion improved in groups PC5/10 and PC5/20, but was not different from control in groups PC5/5 and PC3 x 5/5. There was no correlation between preischemic glycogen levels and recovery of function during reperfusion. There was also no correlation between glycogen breakdown (or resynthesis) and recovery of function. Lactate accumulation during ischemia was lowest in group PC5/20 and highest in the group with three cycles of preconditioning (PC3 x 5/5). Lactate release during reperfusion was significantly higher in the groups with low recovery of power than in the groups with high recovery of power. In glucose-perfused rat heart recovery of function is independent from both pre- and postischemic myocardial glycogen content over a wide range of glycogen levels. The ability to utilize lactate during reperfusion is an indicator for postischemic return of contractile function.
Animals↗
Substrate metabolism as a determinant for postischemic functional recovery of the heart.
The mammalian myocardium meets its high energy needs through the oxidation of a variety of substrates, chiefly fatty acids. This review examines the hypothesis that efficient energy transfer in the heart occurs through a series of moiety-conserved cycles, which makes the heart an obligatory "omnivore." Ischemia results in a transformation of efficient metabolic cycles to less-efficient linear pathways. Substrate metabolism during reperfusion requires the replenishment of depleted cycles and is a major determinant for the return of contractile function. Although there is growing recognition of the concept that regulation of substrate flux through metabolic pathways is shared by several of the pathway enzymes it is apparent that glucose oxidation and glycogen resynthesis promote the return of normal contractile function in the postischemic heart. This concept is supported by clinical observations on the beneficial effects of a solution containing glucose, insulin, and potassium (GIK) for treatment of refractory left ventricular contractile failure after hypothermic ischemic arrest during cardiac surgery.
Animals↗
Fasting, lactate, and insulin improve ischemia tolerance in rat heart: a comparison with ischemic preconditioning.
We tested the hypothesis that improved ischemia tolerance in an isolated working rat heart preparation can be achieved by interventions other than ischemic preconditioning. Hearts were perfused at near-physiological workload with bicarbonate buffer containing glucose (10 mM). A preischemic period of 25 min was followed by 15 min of global ischemia and 30 min of reperfusion under preischemic conditions. Hearts came from either fed or fasted animals (groups 1 and 2). In group 3 lactate (10 mM) and insulin (10 mU/ml) were added to the perfusate of fasted animals. In group 4 hearts from fed animals were perfused with glucose (10 mM) and were ischemically preconditioned by one cycle of ischemia between 10 and 15 min of the preischemic perfusion. Cardiac power and glucose uptake were measured continuously to assess functional and metabolic recovery. In addition, we measured the time to return of aortic flow. Glucose metabolites and the ratio of latent of free citrate synthase activity (citrate synthase ratio, a marker for the structural integrity of mitochondria) were determined at selected time points. Groups 2, 3, and 4 recovered significantly faster than group 1, whereas recovery of power showed an improvement in groups 3 and 4 only. In addition, there was an early increase in glucose uptake during reperfusion in these two groups, suggesting an early need for glucose substrate. Glycogen levels decreased with ischemia in all groups and returned to preischemic levels in groups 2, 3, and 4. The citrate synthase ratio was low in the control group and preserved in the groups showing improved functional recovery. We conclude that metabolic interventions may be as effective as ischemic preconditioning in protecting the heart from ischemic injury.
Animals↗
Fundamental limitations of [18F]2-deoxy-2-fluoro-D-glucose for assessing myocardial glucose uptake.
BACKGROUND: The glucose tracer analog [18F]2-deoxy-2-fluoro-D-glucose (FDG) is widely used for assessing regional myocardial glucose metabolism in vivo. The reproducibility of this method has recently been questioned because of a discordant affinity of hexokinase for its substrates glucose and 2-deoxyglucose. We therefore compared rates of glucose utilization simultaneously with tissue time-activity curves of FDG uptake before and after changes in the physiological environment of the heart. METHODS AND RESULTS: Isolated working rat hearts were perfused for 60 minutes with recirculating Krebs buffer containing glucose (10 mmol/L), FDG (1 microCi/mL), [2-3H]glucose (0.05 microCi/mL), and [U-14C]2-deoxyglucose (2-DG; 0.025 microCi/mL). Myocardial glucose uptake was measured by tracer ([2-3H]glucose) and tracer analog methods (FDG and 2-DG) before and after the addition of either insulin (1 mU/mL), epinephrine (1 mumol/L), lactate (40 mmol/L), or D,L-beta-hydroxybutyrate (40 mmol/L) at 30 minutes of perfusion and after acute changes in cardiac workload. Under steady-state conditions, myocardial rates of glucose utilization as measured by tritiated water (3H2O) production from metabolism of [2-3H]glucose, FDG uptake, and 2-DG retention were linearly related. The addition of competing substrates decreased glucose utilization immediately. The addition of insulin increased the rate of glucose utilization as measured by the glucose tracer but not as measured by the tracer analogs. The ratio of 3H2O release/myocardial FDG uptake increased by 111% after the addition of insulin, by 428% after the addition of lactate, and by 232% after the addition of beta-hydroxybutyrate. Epinephrine increased rates of glucose utilization and contractile performance, whereas there was no increase in glucose uptake with a comparable increase in workload alone. There was no change in the relation between the glucose tracer and the tracer analog either with epinephrine or with acute changes in workload. CONCLUSIONS: The uptake and retention of FDG in heart muscle is linearly related to glucose utilization only under steady-state conditions. Addition of insulin or of competing substrates changes the relation between uptake of the glucose tracer and FDG. These observations preclude the determination of absolute rates of myocardial glucose uptake by the tracer analog method under non-steady-state conditions.
Animals↗