Reduced synthesis of NO causes marked alterations in myocardial substrate metabolism in conscious dogs

Fabio A. Recchia1, Juan Carlos Osorio1, Margaret P. Chandler2, Xiaobin Xu1, Ashish R. Panchal2, Gary D. Lopaschuk3, Thomas H. Hintze1, and William C. Stanley2

1 Department of Physiology, New York Medical College, Valhalla, New York 10595; 2 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106; and 3 Heritage Medical Research Centre, University of Alberta, Edmonton, Canada T6G 2S2


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test whether the acute reduction of nitric oxide (NO) synthesis causes changes in cardiac substrate metabolism and in the activity of key enzymes of fatty acid and glucose oxidation, we blocked NOS by giving Nomega -nitro-L-arginine methyl ester (L-NAME; 35 mg/kg iv two times) to nine chronically instrumented dogs. [3H]oleate, [14C]glucose, and [13C]lactate were infused to measure the rate of cardiac substrate uptake and oxidation. Glyceraldehyde-3-phosphate dehydrogenase, acetyl-CoA carboxylase, and malonyl-CoA decarboxylase activities were measured in myocardial biopsies. In eight control dogs, ANG II was infused (20-40 ng · kg-1 · min-1) to mimic the hemodynamic effects of L-NAME. After L-NAME, significant changes occurred for fatty acid oxidation (from 9.8 ± 0.8 to 7.1 ± 1.2 µmol/min), glucose uptake (from 12.9 ± 5.5 to 45.0 ± 14.2 µmol/min), and oxidation (from 4.4 ± 1.2 to 19.9 ± 2.3 µmol/min). ANG caused only a significantly lower increase in glucose oxidation. Lactate uptake increased by more than twofold in both groups. The enzyme activities did not differ significantly between the two groups. In conclusion, the acute inhibition of NO synthesis causes marked metabolic alterations that do not involve key rate-controlling enzymes of fatty acid oxidation nor glyceraldehyde-3-phosphate dehydrogenase.

fatty acids; glucose; lactate; oxidation; heart


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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UNDER AEROBIC CONDITIONS, the heart derives most of the energy necessary for its contractile function from fatty acid oxidation (18, 20, 26). After an overnight fast, carbohydrate oxidation accounts for only 0-20% of total cardiac oxygen consumption, but in some pathological states, such as hypertrophic cardiomyopathy and heart failure, myocardial utilization of fatty acids can diminish significantly (18, 25, 28, 47-48). Studies in humans (33), pigs (16), dogs (22), and rats (2) indicate that the type of oxidized substrate may influence the efficiency of normal hearts. Moreover, several studies have emphasized the contribution of metabolic alterations to the mechanisms of cardiac dysfunction in pathological conditions. For instance, it has been shown that the pharmacological inhibition of fatty acid utilization to favor carbohydrate oxidation can attenuate electrophysiological alterations and improve performance in ischemic or failing hearts (27, 30, 41).

Despite the abundance of studies on this topic, the complex regulation of cardiac substrate metabolism, in health and disease, is only partially understood. Among the numerous factors involved in this regulation, an important role is likely played by nitric oxide (NO) tonically synthesized in cardiac tissue by the constitutive form of NO synthase (eNOS). We found that the fall in cardiac NO production occurring during end-stage heart failure was temporally associated with a decrease in free fatty acid (FFA) uptake and a marked increase in glucose uptake and respiratory quotient (28). Similarly, acute inhibition of nitric oxide synthase (NOS) in normal hearts caused a shift toward carbohydrate utilization that was completely reversed by the administration of exogenous NO (29). Further and strong evidence indicating the involvement of NO in cardiac substrate metabolism derives from in vitro studies. Tada et al. (40) found that baseline glucose uptake is markedly elevated in isolated hearts from eNOS knockout mice. This study and another by Depre et al. (4) showed that cGMP, the second messenger of NO, inhibits glucose uptake in isolated hearts. Despite this experimental evidence, the exact role of NO in the control of myocardial substrate utilization and the underlying mechanisms remain undetermined. To date, the only enzyme of carbohydrate metabolism known to be inhibited by pharmacological or pathological concentrations of NO is the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which catalyzes a key reaction in the glycolytic pathway (23, 49). On the other hand, the rate of FFA oxidation is limited by the activity of carnitine palmitoyltransferase I, which is necessary for the transfer of long-chain FFA into mitochondria (20). This enzyme is reversibly inhibited by cytosolic malonyl-CoA, which is synthesized and degraded, respectively, by acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD; see Refs. 1, 14, 31). No studies have explored possible interactions between NO and the activity of ACC and MCD.

The first aim of the present study was to determine the metabolic fate of the three main cardiac substrates, i.e., FFA, lactate, and glucose, before and after inhibition of NO synthesis in conscious dogs. In our previous studies (28-29), we measured net substrate uptake by the heart, but we could not determine the rate of FFA and carbohydrate oxidation before and after acute NOS blockade, and we did not explore the biochemical mechanisms involved in the observed metabolic changes. Moreover, the arterial concentration of FFA fell markedly after NOS inhibition, and this phenomenon itself could have stimulated cardiac carbohydrate oxidation. In the present study, we used three different isotopic tracers to track the main cardiac substrates, while preventing marked oscillations of FFA levels by infusion of a triglyceride emulsion plus heparin. Our second aim was to determine whether inhibition of NO synthesis caused changes in the concentration of some intermediate products of substrate metabolism and in the activity of myocardial GAPDH, ACC, and MCD. GAPDH was selected among the numerous enzymes of glucose metabolism to test whether physiological concentrations of endogenously produced NO can exert a tonic inhibition on this enzyme. On the other hand, ACC and MCD are the key enzymes known to regulate the rate of FFA oxidation by controlling the cytosolic concentration of malonyl-CoA. We tested the hypothesis that NO influences the activity of one or both of these enzymes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
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Surgical instrumentation. Seventeen adult, male, mongrel dogs (25-27 kg) were sedated with acepromazine maleate (1 mg/kg im), anesthetized with pentobarbital sodium (25 mg/kg iv), and ventilated with room air. A thoracotomy was performed in the left fifth intercostal space. One catheter was placed in the descending thoracic aorta, and a second catheter was placed in the coronary sinus with the tip leading away from the right atrium. A solid-state pressure gauge (P6.5; Konigsberg Instruments) was inserted in the left ventricle through the apex. A Doppler flow transducer (Craig Hartley) was placed around the left circumflex coronary artery (LcxCBF), and a pair of 3-MHz piezoelectric crystals was fixed on opposing endocardial surfaces at the base of the left ventricle. A human, screw-type, unipolar myocardial pacing lead was placed on the left ventricular (LV) wall. Wires and catheters were run subcutaneously to the intrascapular region, the chest was closed in layers, and the pneumothorax was reduced. Antibiotics were given after surgery, and the dogs were allowed to recover fully. After 7-10 days, dogs were trained to lie quietly on the laboratory table. The protocols were approved by the Institutional Animal Care and Use Committee of the New York Medical College and conform to the guiding principles for the care and use of laboratory animals published by the National Institutes of Health. We have described these methods previously (28, 29).

Hemodynamic recordings. The aortic catheter was attached to a P23ID strain-gauge transducer for measurement of aortic pressure. LV pressure was measured using the solid-state pressure gauge. The first derivative of LV pressure (LV dP/dt) was obtained using an operational amplifier (National Semiconductor LM 324). Coronary blood flow (CBF) was measured with a pulsed Doppler flowmeter (model 100; Triton Technology). LV diameter was measured by connecting the implanted piezoelectric crystals to an ultrasonic dimension gauge. All signals were recorded on an eight-channel direct-writing oscillograph (model RS 3800; Gould). The analog signals were also stored in computer memory through an analog-digital interface (National Instruments), at a sampling rate of 250 Hz. By using a custom-made program, we measured end-systolic and end-diastolic diameters at the left upper corner and the right lower corner of the pressure-diameter loop, respectively. We found that, at the pacing rate used in our experiments, these diameters corresponded to the minimum and maximum measured during the cardiac cycle. Fractional shortening was calculated as (LV end-diastolic - LV end-systolic diameter)/LV end-diastolic diameter × 100 (28). Pressure-diameter loop areas, proportional to LV work, were also calculated.

Total and labeled metabolite measurements. Blood gases were measured in a blood gas analyzer (Instruments Laboratory). Oxygen content was measured by a hemoglobin analyzer (CO-Oximeter; Instruments Laboratory). Total FFA concentration was determined spectrophotometrically in plasma after centrifugation of EDTA-treated blood samples using a colorimetric assay (NEFA C kit from Wako Pure Chemical Industries). Total glucose and lactate were measured in blood deproteinized with ice-cold 1 M perchloric acid (1:2 vol/vol) using spectrophotometric enzymatic assays (10, 11).

The following three isotopically labeled substrates were infused into the dogs: [9,10-3H]oleate, [U-14C]glucose, and L-[1-13C]lactate. Plasma [3H]oleate concentration was measured by extracting fatty acids from 1 ml of plasma in 4 ml of heptane-isopropanol (3:7) and counting the radioactivity of the organic phase with a liquid scintillation counter (model LS 6500; Beckman). 3H2O concentration was measured by distilling 2 ml of plasma in custom-made, modified Hickman stills (Kontes Glass; Custom Shop). We developed this method and tested it to exclude possible contamination of the distillate with [14C]- or [3H]oleate. Blood samples for [14C]glucose measurements were first deproteinized in ice-cold 1 M perchloric acid (1:2 vol/vol). The acidity of the extract was then neutralized with K2CO3, and the neutral solution was run through ion-exchange resin columns (Dowex 1 and 50) to separate secondary labeled [14C]lactate, -pyruvate, and -alanine. Total glucose concentration and 14C activity were then measured in the eluate to calculate [14C]glucose specific activity. 14CO2 concentration in blood was determined using the method of Gertz et al. (6) and Wisneski et al. (45). Briefly, 3-ml blood samples were placed in a sealed flask with a center well containing 800 µl of 1 M NaOH. The blood was acidified with 1 ml of concentrated lactic acid (98%), and the flask was placed on a rotating table at room temperature for 3 h. All of the CO2 so displaced from the blood combined with solution contained in a well inside the flask to form bicarbonate. The newly formed solution of bicarbonate was then collected to count the activity of 14C (6, 45). The isotopic enrichment of lactate with [13C]lactate was measured by gas chromatography-mass spectrometry in plasma samples deproteinized with sulfosalicylic acid, as previously described (24).

Calculations. Myocardial oxygen consumption (MVO2) was calculated by multiplying the arterial-coronary sinus difference in oxygen content by CBF. The rate of FFA uptake (µmol/min) was calculated as: CPF × [FFA]a × ([3H]oleatea - [3H]oleatecs)/[3H]oleatea, where CPF is the coronary plasma flow [taken as CBF × (1 - hematocrit)], [FFA]a is the arterial FFA concentration (µmol/ml), and [3H]oleate is the concentration of [3H]oleate in arterial (a) and coronary sinus (cs) plasma expressed as dpm per milliliter (12). The rate of FFA oxidation (µmol/min) was calculated as: CPF × ([3H2O]cs - [3H2O]a)/([3H]oleatea/[FFA]cs), where [3H2O] was the concentration of [3H]water in the plasma expressed as dpm per milliliter (12). The rate of glucose uptake was calculated as the arterial-coronary sinus concentration difference times the CBF, and the rate of glucose oxidation was calculated from [14C]glucose as: CBF × ([14CO2]cs - [14CO2]a)/([14C]glucosea/[glucose]a) as previously described (7, 45). The rate of lactate uptake (µmol/min) was calculated as: [a] × CBF × ([13C]lactate Ea [a] - [13C]lactate Ecs [cs])/([13C]lactate Ea [a]) where [13C]lactate E is the fraction of the lactate in the blood that is enriched with [1-13C]lactate, as determined by gas chromatography-mass spectrometry analysis and corrected for background enrichment; [a] and [cs] are the concentrations of lactate (µmol/ml) in arterial and coronary sinus blood, respectively (7, 24). It is known that the tracer-measured lactate uptake matches total lactate oxidation by the heart, as previously demonstrated in studies where ~100% of [1-14C]lactate tracer taken up by the heart is immediately decarboxylated and released in the venous circulation as 14CO2 (7). The lactate uptakes measured with [U-13C]lactate and [1-14C]lactate are equivalent (24). [13C]lactate tracer-measured lactate output was calculated as the difference between tracer-measured lactate uptake and the net lactate uptake arterial-coronary sinus difference of total lactate times CBF (46). The output quantifies the rate of nonoxidative glycolysis of endogenous and exogenous glucose. In all of the calculations described above, CBF was assumed as double the mean flow measured in the LcxCBF (28, 29). The percentage of the MVO2 resulting from the oxidation of glucose, lactate, and FFA was calculated by multiplying the respective rates of oxidation (measured with [14C]glucose, [13C]lactate, and [3H]oleate) times 6.0, 3.0, and 24.5 µmol O2 /µmol substrate, respectively, and dividing it by the MVO2 (expressed in µmol/min). For lactate it was assumed that 100% of the [13C]lactate taken up by the heart is oxidized to CO2 (7).

Enzyme activities and metabolic intermediates. To measure the activities of each of the enzymes GAPDH, ACC, and MCD, ~200 mg of frozen cardiac tissue were used. GAPDH activity was assessed in the reverse direction using the procedure described by Molina y Vedia et al. (23) provided by Boehringer Mannheim (assay instruction 5178) with a homogenization procedure modified from Knight et al. (15).

ACC activity was determined in the presence and absence of maximally stimulating citrate (10 mM) as described by Dyck et al. (5). Briefly, 200 mg of tissue were homogenized and dialyzed. Dialysate (25 µl) was reacted in 60 mM Tris acetate, pH 7.5, 1 mg/ml BSA, 1.32 µM beta -mercaptoethanol, 2.12 mM ATP, 1.06 mM acetyl-CoA, 5.0 mM Mg acetate, 18.2 mM NaHCO3, and ±10 mM magnesium citrate. Samples were run for 0, 1, 2, and 4 min and stopped with perchloric acid (1 M) to be assayed for malonyl-CoA concentration as described for tissue samples below. MCD activity was assayed by using the method of Dyck et al. (5), which measures the conversion of [14C]malonyl-CoA to acetyl-CoA. Briefly, tissue homogenates were incubated in 210 µl reaction mixture (0.1 M Tris, pH 8, 0.5 mM dithiothreitol, and 1 mM malonyl-CoA) for 10 min at 37°C, in the presence or absence of NaF (50 mM) and NaPPi (sodium pyrophosphate, 5 mM). The reaction was stopped with perchloric acid, neutralized with 10 µl of 2.2 M KHCO3 (pH 10), and centrifuged at 10,000 g for 5 min to remove precipitated proteins. The incubation of the heart sample with malonyl-CoA allowed for the conversion of malonyl-CoA to acetyl-CoA, which was reacted with [14C]oxaloacetate (0.17 µCi/ml) to produce [14C]citrate. All reactions were in the presence of N-ethylmaleimide, which removes excess CoA remaining in the latter stages of the reaction. Unreacted [14C]oxaloacetate was removed from the reaction mixture by the addition of sodium glutamate (6.8 mM) and aspartate aminotransferase (0.533 µU/µl), followed by a 20-min incubation at room temperature to transaminate unreacted [14C]oxaloacetate back to [14C]aspartate. [14C]aspartate was removed with Dowex AG 50W-8X resin (100-200 mesh), and the solution was counted for [14C]citrate. The amount of acetyl-CoA produced by MCD was quantified by comparison with acetyl-CoA standard curves that had been subjected to the identical assay conditions.

Free CoA and short-chain CoA esters were extracted from ~100 mg of powdered heart tissue as described previously (31) and assayed using HPLC separation and ultraviolet detection as previously described by Corkey (3).

Protocol. Experiments were conducted in conscious dogs placed on the laboratory table after overnight fasting. In all of the 17 dogs, hemodynamics were recorded, and the isotopic tracers [9,10-3H]oleate, [U-14C]glucose, and L-[1-13C]lactate were infused continuously for the entire duration of the experiment through a peripheral vein. [3H]oleate bound to albumin was infused at a rate of 0.7 µCi/min. The bolus-continuous infusion method (7, 46) was used for [14C]glucose (20 µCi + 0.3 µCi/min) and for [13C]lactate (600 µmol + 700 µmol/h). Heart rate was maintained constant throughout the experiment by pacing the heart at 130 ± 2 beats/min. After 40 min of tracer infusion, control paired blood samples were withdrawn from the aorta and coronary sinus. At this point, dogs were divided into two groups. Nine dogs received a bolus of 35 mg/kg of Nomega -nitro-L-arginine methyl ester (L-NAME) intravenously. Immediately after L-NAME administration, a bolus of 5,000 IU of heparin was given intravenously, and an infusion of 10% Intralipid plus 125 IU/ml heparin was started at the rate of 0.006 ml · kg-1 · min-1 and continued until the end of the experiment. This low infusion rate was chosen since we found that, at higher rates, the cocktail of Intralipid plus heparin caused an increase in arterial FFA concentration that sometimes exceeded the control values. Paired arterial and coronary sinus blood samples were withdrawn at 30, 60, 90, and 120 min after L-NAME. A second bolus of 35 mg/kg of L-NAME was given immediately after collection of blood samples at 90 min. A second group of eight dogs (control group) underwent a continuous intravenous infusion of synthetic (Sigma) human ANG II at the rate of 20-40 ng · kg-1 · min-1, started immediately after withdrawal of control blood samples. The rate of ANG II infusion was adjusted initially to obtain a mean arterial pressure (MAP) of ~150 mmHg, matching the hypertension caused by NOS inhibition, and then was maintained constant during the remaining part of the experiment. Heparin and Intralipid were infused, and blood samples were collected following the same protocol described for the group receiving L-NAME. After withdrawal of the last blood samples, all of the dogs were anesthetized with 30 mg/kg pentobarbital sodium intravenously, intubated, and ventilated. The fifth intercostal space was opened rapidly to harvest a tissue sample from the heart. A large (~10 g) biopsy was obtained by punching a hole through the LV anterior free wall with a stainless steel "cork borer" (2 cm ID) while the heart was beating. The harvested tissue was immediately freeze-clamped with tongs precooled in liquid nitrogen. The total time from anesthesia to tissue harvesting was <3 min, and we found that, during this time, the dog was not hypotensive. The time from tissue harvesting to clamping between tongs was ~3-4 s. This approach was established by previous studies that combined measurements of metabolic changes in vivo with enzyme assays in vitro (15, 32, 35).

Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed by employing commercially available software (Sigma Stat 2.01). Changes in hemodynamics and in rates of substrate uptake and oxidation over time were tested by one-way ANOVA for repeated measurements followed by Dunnett's test. Tukey's test was also used to obtain multiple comparisons. Changes resulting from L-NAME and ANG II infusion were compared and tested for differences by using two-way ANOVA followed by Tukey's test.


    RESULTS
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Hemodynamics. As shown in Fig. 1, comparable changes occurred in the main hemodynamic parameters after L-NAME and during ANG II infusion. Additional hemodynamic data are reported in Table 1. As described in previous studies, mean blood flow in the LcxCBF, MAP, and LV systolic pressure (LVSP) increased significantly after NOS inhibition (Fig. 1). By infusing ANG II, it was possible to mimic with a good approximation changes in LcxCBF and MAP caused by L-NAME. However, LVSP gradually declined after 30 min of NOS blockade, and, at 90 and 120 min, it was significantly lower than LVSP measured in dogs with ANG II infusion. Despite the fact that LV end-diastolic diameter did not change significantly after L-NAME or during ANG II infusion (Table 1), dp/dtmax (Fig. 1), fractional shortening of LV diameter, and pressure-diameter area (Table 1) were significantly lower in dogs with NOS blockade compared with the control group.


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Fig. 1.   Hemodynamic changes after administration of Nomega -nitro-L-arginine methyl ester (L-NAME; n = 9) and during infusion of ANG II (n = 8). Time 0 = control. A: mean coronary blood flow in the left circumflex coronary artery (LcxCBF). B: mean arterial pressure (MAP). C: left ventricular systolic pressure (LVSP). D: maximum derivative of left ventricular pressure (dP/dtmax). Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. corresponding time point.


                              
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Table 1.   Changes in some indexes of left ventricular performance after administration of L-NAME and during infusion of ANG II

Cardiac metabolism. The continuous infusion of Intralipid plus heparin could not prevent a transient decrease in arterial FFA concentration after L-NAME (Fig. 2). However, FFA concentration did not fall below 0.45 ± 0.05 mmol/l, and the values at 120 min did not differ significantly from control. A significant decrease in circulating FFA was observed also at 90 and 120 min of ANG II infusion. Arterial glucose concentration did not change significantly in either of the two groups, whereas lactate increased significantly in dogs receiving ANG II and L-NAME, with a significant difference between the two groups at 90 min (Fig. 2). The arterial specific radioactivity of [3H]oleate and [14C]glucose (radioactive/total substrate in dpm/mmol), and the lactate enrichment with [13C]lactate (fraction of lactate in the blood that is enriched with [1-13C]lactate) are shown in Fig. 3 as a function of time. These plots demonstrate that there was isotopic steady state and no significant differences between the two groups for [14C]glucose specific activity and for [13C]lactate enrichment from 30 to 120 min of the protocol. There was an increase in the specific activity of [3H]oleate over time, which was more rapid in the L-NAME group. As expected, this increase corresponded to the time points at which the plasma concentration of total FFA fell significantly (see Fig. 2). As shown in Fig. 4, NOS inhibition and ANG II infusion caused almost superimposable increases in MVO2. Despite matched levels of oxygen consumption, changes in cardiac oxidation of FFA and glucose evolved differently in the two groups of dogs (Fig. 4). Between 30 and 60 min after L-NAME, FFA oxidation fell significantly by ~28% compared with control, reaching the minimum value at 60 min. This decrease was only transient, since FFA oxidation returned to control values at 90 min, but it fell again at 120 min after administration of the second dose of L-NAME. Cardiac oxidation of glucose showed a marked and stable augmentation, and a significant difference was found at 30 and 60 min between the 400% increase and the 270% increase occurring after L-NAME and during ANG II infusion, respectively. Total cardiac lactate uptake increased significantly by ~130% in both groups (Fig. 4). Changes in FFA and glucose oxidation were not paralleled by significant changes in uptake, except for a significant increase in glucose uptake at 60 min after L-NAME (Fig. 5). Also, cardiac production of lactate (as measured with [13C]lactate tracer) did not change significantly in either of the two groups (Fig. 5). The sum of the oxidation of glucose, lactate, and FFA varied between 104 and 121% of the measured MVO2 and did not change significantly over the time course of the protocol, and no significant differences were found between the two groups (Table 2).


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Fig. 2.   Changes in the arterial concentration of free fatty acid (FFA; A), glucose (B), and lactate (C) after administration of L-NAME (n = 9) and ANG II (n = 8). Time 0 = control. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. corresponding time point.



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Fig. 3.   Changes in the arterial specific radioactivity (SA) of [3H]oleate and [14C]glucose and of lactate enrichment with [13C]lactate after administration of L-NAME and ANG II; n = 9 for FFA and glucose and n = 7 for lactate in the L-NAME group; n = 8 for the three substrates in the ANG II group. #P < 0.05 for [3H]oleate in L-NAME vs. ANG II.



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Fig. 4.   Changes in the myocardial O2 uptake (MVO2, A) and in the rates of cardiac oxidation of FFA (B) and glucose (C) and total uptake of lactate (D) after administration of L-NAME and ANG II; n = 9 for FFA and glucose, and n = 7 for lactate in the L-NAME group; n = 8 for the three substrates in the ANG II group. Time 0 = control. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. corresponding time point.



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Fig. 5.   Changes in the rate of cardiac uptake of FFA (A) and glucose (B) and output of lactate (C) after administration of L-NAME and ANG II; n = 9 for FFA and glucose, and n = 7 for lactate in the L-NAME group; n = 8 for the three substrates in the ANG II group. Time 0 = control. Data are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. corresponding time point.


                              
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Table 2.   Ratio between calculated and measured MVO2

Enzyme activities and metabolic products in cardiac tissue. The activities of GAPDH, ACC, and MCD and the concentrations of malonyl-CoA, free CoA, and acetyl-CoA are reported in Table 3. No significant differences were found between the two groups, except for the levels of free CoA and acetyl-CoA, which resulted in higher levels in L-NAME-treated hearts.

                              
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Table 3.   Changes in the activity of key enzymes of glucose and FFA metabolism and in the concentration of free CoA and short-chain CoA esters in left ventricular tissue


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The first aim of the present study was to measure changes in the metabolism of cardiac substrates after inhibition of NO synthesis. Our results show that an acute reduction of NO production causes a reduction in FFA oxidation and an increase in glucose oxidation by cardiac muscle. The function of NO as a metabolic modulator has been the object of intense investigations. Endogenous NO, likely generated by the microvascular endothelium in which eNOS is preponderant, inhibits oxygen consumption in heart and other organs (17, 19, 39). We now provide evidence that NO plays an important role in the complex control that directs cardiac metabolism toward preferential oxidation of FFA. A notorious effect of acute NOS blockade is the marked elevation of arterial blood pressure with a consequent increase in LV afterload. This effect was present also in our study and could have been itself a cause of cardiac metabolic alterations. To control for this, we used for comparison a control group of dogs in which arterial blood pressure was elevated by a continuous infusion of ANG II to match the increase in cardiac workload and MVO2. This comparison proved that the reduction in FFA oxidation in the L-NAME group was specifically associated with NOS inhibition. On the other hand, myocardial glucose oxidation increased during ANG II infusion, but to a significantly lesser extent compared with the increase observed after L-NAME. Finally, values of lactate uptake/oxidation in the groups were almost overlapping, indicating that this phenomenon was not specifically related to NO availability, but, more in general, to elevations in cardiac workload.

The second aim of our study was to test whether alterations in myocardial substrate utilization, after NOS inhibition, were the result of changes in the activity of some key enzymes involved in FFA and carbohydrate metabolism. ACC, MCD, and GAPDH activities in L-NAME- and ANG II-treated hearts were not significantly different. And no significant difference was found between groups in the myocardial concentrations of malonyl-CoA, the physiological inhibitor of FFA transport in mitochondria. Therefore, the metabolic alterations caused by NOS inhibition likely did not involve these key enzymes regulating FFA and glucose utilization. On the other hand, tissue acetyl-CoA content was about twofold higher in the L-NAME group, which is consistent with studies in swine where the rate of myocardial carbohydrate oxidation was increased with either dichloroacetate (35) or dobutamine (11). Finally, the increased content of free CoA by ~150% was consistent with a reduced rate of FFA oxidation and a consequent fall in the synthesis of long-chain fatty acyl-CoA and the release of free CoA after L-NAME.

The present findings are in agreement with the conclusions that we drew from our previous studies in conscious dogs in which we blocked NOS synthesis (28, 29) and reversed metabolic changes by infusing an NO donor (29). An important difference, however, consists in the separate measurements of oxidation and uptake allowed, in the present case, by the use of isotopes. To our knowledge, this is the first study in which cardiac metabolism was explored by employing, simultaneously, three different isotopes labeling the three main substrates consumed by the heart. To rule out possible artifacts resulting from isotope infusion and blood sampling, we performed pilot experiments in conscious dogs not subjected to any pharmacological intervention and did not observe any significant changes in substrate oxidation over a period of 3 h (data not shown). The differentiation between uptake and oxidation revealed that these two events did not necessarily vary in parallel after NOS inhibition. FFA uptake, for instance, did not change significantly after L-NAME, despite a decrease in oxidation by almost 30% that occurred at 60 and 120 min. In our previous study, we hypothesized a similar dissociation between FFA uptake and oxidation based on the calculation of cardiac respiratory quotient. However, in that study, FFA uptake fell markedly at 60 min after administration of NOS inhibitor. Such a phenomenon was likely because of a significant drop in the arterial concentration of FFA that we observed after nitro-L-arginine (NLA) administration. To avoid the confounding effects of fluctuations in FFA availability that could have specifically altered myocardial metabolism, in the present set of experiments we tried to clamp the level of circulating FFA by continuously infusing Intralipid plus heparin. Although we did not obtain a complete clamp, FFA concentration remained well above the low value of 0.25 mmol/l that we observed in our earlier studies with NOS inhibition (28, 29). The decrease in FFA oxidation found in the present investigation cannot be explained simply by a fall in plasma fatty acid levels and uptake. In fact, arterial FFA concentration was constantly lower than control from 30 to 90 min after the first bolus of L-NAME, even though FFA oxidation was significantly reduced at 60 min only. Furthermore, at 120 min, after the second bolus of L-NAME, FFA concentration increased and was not different from control. Nonetheless, oxidation surprisingly fell again by ~30% despite the fact that hemodynamics did not change further. We have no data to explain this phenomenon. It seems that the depression of FFA oxidation was reversible and occurred only at high concentrations of NOS inhibitor. Unfortunately, we could collect a large cardiac biopsy only at the end of the experiment, and our measurement of enzyme activities could not follow the entire dynamics of metabolic change.

A key physiological modulator of FFA oxidation is cytosolic malonyl-CoA, the concentration of which is largely determined by the activities of ACC and MCD. None of these factors changed significantly in L-NAME-treated dogs compared with the control group. Based on these data, it is only possible to speculate that NO is one of the regulatory factors of the FFA oxidation pathway, but its action is exerted at a site of regulation that remains unknown. Alternatively, it is possible that NO reversibly modulates the activities of ACC and/or MCD and/or other enzymes of the FFA metabolic pathway in vivo, but this function is lost in tissue homogenates.

After L-NAME (60 min), a significant increase in glucose oxidation was paralleled by a significant increase in uptake. This finding is consistent with our previous studies documenting a marked increase in glucose uptake associated with diminished NO synthesis in failing hearts (28), in normal hearts treated with NLA (28-29), and in hearts isolated from eNOS knockout mice (40). It is also consistent with a decrease in glucose uptake found in isolated hearts after infusion of 8-bromo-cGMP (8-BrcGMP; see Refs. 4 and 40), a putative second messenger of NO. Endogenous NO likely inhibits glucose uptake via a mechanism that remains undetermined. Glucose entry in cells is strongly dependent on the incorporation of specific transporters in the cell membrane (34). Depre et al. (4) hypothesized a direct inhibition by NO, via the second messenger cGMP, on glucose transporters in myocytes. In support of this hypothetical mechanism, Tada et al. (40) found that 1-H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one, an inhibitor of cGMP synthesis, markedly stimulates glucose uptake in isolated mouse hearts. Specific experiments at the cellular level will be necessary to elucidate the possible modulatory action of cGMP or NO on the function of glucose transporters, specifically on the translocation and kinetics of GLUT-1 and GLUT-4. Interestingly, in our experiments, the effect of NOS inhibition on cardiac glucose uptake was transient, limited to one time point and not so pronounced as observed in isolated hearts. This could be because of the incomplete inhibition of NO synthesis by pharmacological blockade of NOS compared with the complete shut off in eNOS knockouts. It also could be due in part to the competition by other substrates available in intact animal preparations. In the present study, however, glucose oxidation was constantly higher than control after NOS inhibition, although it tended to decrease over time so that no significant differences vs. the ANG II group were found at 90 and 120 min. It is noteworthy that, even if less pronounced, there was a significant increase in glucose oxidation also during ANG II infusion. This could have been simply because of the increased metabolic demand, but we cannot exclude additional mechanisms, such as an ANG II-mediated elevation of intracellular pH (9) with consequent phosphofructokinase activation.

Glucose oxidation includes the following two phases: the first one, cytosolic, consists of the glycolytic pathway with pyruvate as the end product; the second one leads to complete oxidation of pyruvate in mitochondria. We tested the hypothesis that physiological concentrations of NO exert a tonic inhibition on GAPDH, a key enzyme of the glycolytic pathway. This hypothesis was based on the previous findings that pharmacological or pathological concentrations of NO inhibit GAPDH in vivo and in vitro (23, 49). We did not find significant differences between the two groups. Therefore, it is likely that GAPDH function is unaffected by changes in the physiological concentration of NO. As for the enzymes regulating FFA oxidation, an alternative hypothesis is that the action of physiological amounts of NO on GAPDH is rapidly reversible in vivo; thus, it was lost in tissue homogenates, in which spontaneous production of NO did not occur, exogenous NO was not added, and tissutal scavengers could have rapidly neutralized NO bound to thiol groups. Another important limiting step shared by glucose and lactate oxidation is the irreversible dehydrogenation of pyruvate by the pyruvate dehydrogenase enzyme complex in mitochondria (38). We did not test this enzyme, but similar increases in lactate oxidation in both groups suggested that the activity of pyruvate dehydrogenase was not stimulated exclusively by reduced availability of NO but rather by generic elevations in ventricular afterload. Other investigators have found reduced glycolysis, but no changes in pyruvate dehydrogenase activity, in isolated hearts during infusion of 8-BrcGMP (4). It is likely, therefore, that the inhibitory action exerted by NO on myocardial glucose metabolism is limited to the transmembrane transport and, perhaps, to one or more enzymes of glycolysis other than GAPDH. For instance, it has been shown that NO can inhibit phosphofructokinase in pancreatic islets (42). The release of tonic inhibition of myocardial phosphofructokinase after NOS blockade could thus be a candidate for the mechanism of stimulated glucose metabolism. Future studies specifically exploring the role of NO in the control of the glycolytic pathway, in well-controlled conditions in vitro, will be necessary to answer these open questions. It is also important to note that the [13C]lactate tracer-measured output of lactate, an index of anaerobic glycolysis, did not change significantly during the experiments, indicating a preserved match between oxygen demand and supply.

In conditions such as failure or pathological hypertrophy, the utilization of glucose by cardiac muscle increases (20, 25, 28, 47, 48). NO production is impaired in both of these pathological states (21, 28). In the light of the present findings, it is possible to hypothesize that NO plays a role in the metabolic changes typical of diseased myocardium. Understanding this role assumes a particular importance if, as speculated by some authors on the basis of recent data, the performance of the failing heart may be affected by the type of substrate oxidized (30). Furthermore, solid evidence supports the concept that, during cardiac ischemia, the stimulation of glucose, rather than FFA oxidation, is advantageous in preserving function and facilitating recovery of the ischemic myocardium (20, 27, 41). On the other hand, evidence also exists that the recovery of stunned myocardium is potentiated by oxidation of FFA (43). Like failure and hypertrophy, cardiac ischemia and stunning are pathological conditions in which the synthesis of NO is altered (8). In these cases, the changed availability of NO might be in part responsible for metabolic alterations and functional consequences.

Limitations of the study. A number of limitations should be pointed out. Our study was aimed at measuring changes in cardiac metabolism in conscious animals. The heart was therefore studied under optimal conditions of perfusion, oxygenation, and supply of the entire range of physiological substrates. This represented a remarkable advantage relative to isolated heart preparations. A disadvantage consisted of the impossibility to control simultaneously numerous variables, including substrate concentration, cardiac load, and neurohumoral factors that might have been responsible for part of the myocardial metabolic alteration. For instance, the switch in substrate oxidation could have been the result of the release of insulin triggered in pancreatic beta -cells by the arginine analog L-NAME (44). This was not likely the case, however, since in our previous study (29) we found that arterial insulin levels were rather decreased after NOS inhibition. The hemodynamic changes occurring after L-NAME were in part mimicked, in the control group, by infusion of ANG II, since we found this vasopressor well tolerated by conscious dogs, easy to titrate, and rapidly reversible. We could indeed reproduce the increase in MAP resulting from NOS blockade, although ANG II enhanced or preserved cardiac contractility, as indicated by the differences in dP/dtmax, LV diameter fractional shortening, and pressure-diameter areas between the two groups. We did not measure ventricular volume and aortic flow, but cardiac output was likely unchanged (13). However, despite these differences in hemodynamics, the groups presented a similar increase in oxygen consumption, i.e., in metabolic demand. Coronary flow increased in both groups in response to the augmented metabolic demand. Another limitation regards the calculated MVO2 that systematically exceeded the measured MVO2 (Table 2). This suggests that there was an overestimation in the rate of oxidation of one or more of the substrates for both groups. This could be because of a greater fractional extraction and oxidation of oleate compared with other fatty acids in plasma or because of equilibration between intracellular lactate and pyruvate and overestimation of exogenous lactate oxidation with the [13C]lactate tracer (37, 38). Even with this limitation, the calculated ratio did not change significantly over time, despite marked alterations in the rate of substrate oxidation. A limitation regarding the calculation of glucose uptake was based on measurements of the arteriovenous difference in the total concentration. The low sensitivity of this method could explain the large SEs reported for glucose uptake. The best alternative would have been the use of two different isotopic tracers to label glucose, one to measure uptake and the other to measure oxidation. This method would also have allowed precise estimates of glycolytic flux. Unfortunately, we already infused all of the three isotopes universally used to label cardiac substrates. Finally, the negative findings relative to enzyme activities could have been caused simply by the techniques used and may not necessarily reflect regulatory processes that occurred in vivo. Unfortunately, we could adopt only the method presently available; metabolic changes observed in vivo were correlated with enzyme activities tested in biopsied tissue ex vivo, under nonphysiological conditions. We measured maximal activities of the enzymes in the presence of saturating concentrations of substrate, and we cannot exclude that the affinity for the substrate, indicated by the Michaelis-Menter constant (Km), was significantly different between the two groups. Further studies in vitro will be necessary to test the effects of NO/cGMP on the Km of purified enzymes.

In conclusion, physiological levels of endogenous NO play an important role in the complex mechanism of regulation of myocardial substrate oxidation. After an overnight fast, the heart uses mainly FFA, but acute inhibition of NO synthesis strongly stimulates glucose oxidation by cardiac muscle with a simultaneous decrease in FFA oxidation. However, in bioptic tissue, we could not detect changes in the activities of key enzymes of substrate metabolism, such as GAPDH, MCD, and ACC, to explain the metabolic alterations observed in vivo.


    ACKNOWLEDGEMENTS

This study was supported by the National Heart, Lung, and Blood Institute Grant RO1 HL-62573 (F. A. Recchia), in part by PO1 HL-43023 (T. H. Hintze), RO1 HL-58653, and HL-64848 (W. C. Stanley), and by a grant from the Canadian Institutes of Health (G. D. Lopaschuk).


    FOOTNOTES

Address for reprint requests and other correspondence: F. A. Recchia, Dept of Physiology, BSB 622, New York Medical College, Valhalla, NY 10595 (E-mail: fabio_recchia{at}nymc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 June 2001; accepted in final form 30 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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