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Am J Physiol Heart Circ Physiol 275: H2181-H2190, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 6, H2181-H2190, December 1998

beta -Adrenergic stimulation induces transient imbalance between myocardial substrate uptake and metabolism in vivo

Ya Xu1, Li Lu1, Peili Zhu1, and Gregory G. Schwartz1,2,3

1 Cardiovascular Research Institute and 2 Department of Medicine, University of California, San Francisco; and 3 Cardiology Section, Department of Veterans Affairs Medical Center, California 94121

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

At steady state, a balance is expected between net myocardial uptake of the principal exogenous carbon substrates and the rate at which these substrates are metabolized. Such a balance is present when the sum of the oxygen extraction ratios (OERs) for glucose, lactate, and free fatty acids (FFA) is near unity. We have previously observed that systemic administration of the beta -adrenergic agonist isoproterenol (Iso) induces a state of excess myocardial substrate uptake relative to the rate of substrate metabolism, reflected by a sum of OERs significantly >1.0. This occurs in conjunction with an Iso-stimulated increase in circulating insulin levels. The goal of the present study was to determine whether this excess substrate uptake depends on the effects of insulin and time. In open-chest anesthetized pigs, myocardial blood flow, substrate uptake, and oxygen consumption were measured at baseline and during systemic administration of Iso (0.08 µg · kg-1 · min-1 iv) under the following conditions: group 1 (n = 10), normal endogenous insulin release; group 2 (n = 10), inhibition of endogenous insulin release with somatostatin; group 3 (n = 7), at 45 and 90 min Iso; group 4 (n = 7), at 45 and 90 min Iso, with exogenous insulin given during the latter measurement. In group 1, plasma insulin rose fivefold with Iso while the sum of the OERs for glucose, lactate, and FFA increased from 0.92 ± 0.21 at baseline to 1.57 ± 0.17 with Iso (P < 0.01). In group 2, somatostatin blunted the increase in insulin with Iso and there was no significant change in the sum of OERs between baseline and Iso. In group 3, the sum of OERs increased from 0.95 ± 0.11 at baseline to 1.69 ± 0.20 at 45 min Iso (P < 0.01), similar to the response of group 1. However, the state of excess substrate uptake was transient; by 90 min Iso the sum of OERs declined to 0.69 ± 0.21 (P < 0.05 vs. 45 min Iso). In group 4, excess substrate uptake could not be sustained at 90 min Iso despite administration of exogenous insulin. Systemic beta -adrenergic stimulation causes a transient condition of myocardial substrate uptake in excess of metabolism. Increased plasma insulin is necessary to produce this condition, but a high insulin level does not prolong the condition.

pig; isoproterenol; insulin; energy metabolism; glucose; fatty acids

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

UNDER STEADY-STATE conditions, a balance should exist between the rate at which exogenous carbon substrates are taken up by the myocardium and the rate at which these substrates are metabolized. For each substrate, there is a stoichiometric relation between moles of substrate oxidized and moles of oxygen consumed. For example, complete oxidation of 1 mole of glucose requires 6 moles of oxygen; similarly, complete oxidation of 1 mole of lactate or free fatty acids (FFA) requires 3 and ~23.5 moles of oxygen, respectively. The balance between substrate uptake and metabolism can be estimated by calculating oxygen extraction ratios (OERs) for these principal exogenous substrates. The OER for each substrate is the fraction of total myocardial oxygen consumption (MVO2) that could be accounted for if the net uptake of that substrate were fully directed to oxidative metabolism (15). If the net myocardial uptake of glucose, lactate, and FFA accounted precisely for the simultaneous rate of MVO2, then the sum of the OERs for these substrates would equal 1.0. Glucose uptake for anaerobic glycolysis has no net effect on the sum of OERs, because each mole of glucose uptake so metabolized results in the formation of 2 moles of lactate, thereby reducing net lactate uptake by 2 moles. Thus an increase in the OER of glucose due to anaerobic glycolysis is offset by a simultaneous decrease in the OER of lactate.

A sum of OERs exceeding 1.0 implies that myocardial uptake of exogenous substrates exceeds the simultaneous rate of substrate oxidation, and implies net intracellular accumulation of substrate, principally in the form of glycogen and/or triglyceride. Conversely, a sum of OERs <1.0 implies that myocardial uptake of exogenous substrates is insufficient to account for the simultaneous rate of substrate oxidation, and implies net depletion of intracellular substrate stores. Clearly, a state of imbalance between net substrate uptake and oxidation cannot be sustained indefinitely. However, the conditions that may lead to a temporary imbalance are poorly defined.

Systemic administration of a beta -adrenergic agonist produces multiple effects on myocardial substrate metabolism. The utilization of carbon substrates by the myocardium must increase to meet heightened energy demand imposed by increased heart rate and contractility. Simultaneously, the supply of exogenous carbon substrates available to the myocardium is altered. beta -Adrenergic stimulation of glycogenolysis in liver and muscle mobilizes glucose, stimulation of lipolysis in adipose tissue raises circulating FFA concentrations, and stimulation of pancreatic islet cells raises circulating insulin concentration (1, 6, 7, 12, 13). The supply of myocardial carbon substrates may also be altered by beta -adrenergic stimulation of myocardial glycogenolysis and lipolysis, although a concomitant increase in insulin levels would be expected to oppose these effects (21).

We have previously observed that systemic administration of the beta -adrenergic agonist isoproterenol (Iso) results in a state of excess myocardial substrate uptake, reflected by a sum of OERs averaging 1.4 (27). This state occurred in conjunction with a significant stimulation of insulin release, with mean increase in plasma insulin concentration increasing from 9 to 45 µU/ml. However, it is unknown how long such a state of excess substrate uptake can be maintained, or whether elevated insulin levels contribute to this condition.

Accordingly, the goal of the present study was to determine the effects of time and insulin on the unbalanced state of net myocardial substrate accumulation provoked by systemic beta -adrenergic stimulation. The former was investigated with serial measurements of myocardial substrate uptake and oxygen consumption during Iso stimulation. The latter was investigated by suppressing endogenous insulin release with somatostatin during Iso administration, or by administering exogenous insulin along with Iso.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animal Preparation

The investigation conformed to the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. Thirty-eight female Yorkshire-Landrace pigs, weighing 29-40 kg, were studied. The pigs comprised five groups, each described below.

All pigs were premedicated with ketamine-HCl (25 mg/kg im). Anesthesia was induced with alpha -chloralose (80-100 mg/kg iv) and maintained with alpha -chloralose (40 mg · kg-1 · h-1 iv). Pigs were placed on a recirculating hot water blanket, and the lower body was wrapped with an insulated pad. After endotracheal intubation via a tracheotomy, the pigs were mechanically ventilated with an air-oxygen mixture to maintain arterial blood pH between 7.35 and 7.50 and arterial PO2 >200 mmHg. Normal saline was infused rapidly with induction of anesthesia (500 ml iv) and followed by continuous infusion of 150 ml/h.

The chest was opened via a median sternotomy. Fluid-filled catheters were inserted in the aortic arch via a carotid artery, in the left ventricle via its apex, and in the left atrium via its appendage. A 20-gauge Teflon catheter was inserted into the anterior interventricular vein for withdrawal of coronary venous blood samples. In groups 1 and 2 (see below), a 2.5-cm diameter, 2-turn surface coil for in vivo 31P NMR spectroscopy was loosely sutured to the anteroapical free wall of the left ventricle.

Regional Myocardial Blood Flow, Oxygen Consumption, and Substrate Uptake

Transmural myocardial blood flow distribution was determined under each experimental condition by left atrial injection of 3-5 × 106 well-mixed 15-µm-diameter microspheres coated with blue, green, orange, red, or crimson fluorescent dye (Molecular Probes, Eugene, OR). A reference arterial blood sample was withdrawn simultaneously from the carotid artery into a heparinized syringe with a calibrated pump. Postmortem, myocardial tissue samples were excised from the anterior free wall of the left ventricle. Each sample was divided into three equal transmural layers (subendocardium, midmyocardium, and subepicardium) weighing 1.5-2.5 g each. The processing of blood and tissue samples for extraction of microspheres, determination of fluorescence, and calculation of regional blood flow has been described in detail in previous communications from this laboratory (28). Mean transmural blood flow of the anterior left ventricle was calculated as the average of values determined in each of the three transmural layers.

Paired arterial and coronary venous blood samples were withdrawn into iced, heparinized syringes for determination of oxygen content. Hemoglobin-bound oxygen content was measured with a Radiometer (Copenhagen, Denmark) OSM3 hemoximeter and dissolved oxygen content with a Radiometer ABL30 blood gas analyzer. The sum of bound and dissolved oxygen contents was used to compute the coronary arteriovenous oxygen extraction. MVO2 of the anterior left ventricular wall was calculated as the product of coronary arteriovenous oxygen extraction and mean transmural blood flow determined by the fluorescent microsphere technique.

The methods for determination of glucose, lactate, and FFA concentration in paired arterial and coronary venous blood samples have been described in previous publications from this laboratory (26, 27). Glucose concentration was determined by the hexokinase-glucose-6-phosphate dehydrogenase-coupled enzymatic method (2). Lactate was determined by an enzymatic spectrophotometric method. Plasma FFA concentration was determined by gas chromatography using a modification of the method of Ko and Royer (14). Blood FFA concentration was then computed by multiplying the plasma concentration by (1 - hematocrit). Substrate uptake of the anterior left ventricular wall was calculated as the product of the coronary arteriovenous concentration difference and mean transmural myocardial blood flow determined by the fluorescent microsphere technique. The OERs of glucose, lactate, and FFA were calculated as (glucose uptake × 6)/MVO2, (lactate uptake × 3)/MVO2, and (FFA uptake × 23.5)/MVO2. Substrate uptake and MVO2 were expressed in micromoles per gram wet weight per minute. Arterial plasma insulin and glucagon concentrations were determined by radioimmunoassay.

31P NMR Spectroscopy (Groups 1 and 2)

Spectroscopy was performed at 1.5 tesla using an imaging/spectroscopy system (Siemens Medical Systems, Erlangen, Germany). The magnet was shimmed on cardiac water protons to an average line width of 20 Hz. The radio frequency pulse amplitude was adjusted to obtain maximal signal intensity from phosphocreatine (PCr). Phosphorus spectra were obtained from 256 acquisitions, with cardiac gating to end diastole and respiratory gating to end-expiration. The mean repetition time was 6.7 s. The methods of spectral processing and analysis have been described previously (26, 27). The PCr-to-ATP ratio was computed using the beta -ATP resonance area.

Experimental Protocols

Five groups of pigs were studied. The experimental protocol for each group is schematized in Fig. 1.


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  		Fig. 1.   Experimental protocol. Arrows indicate times of measurements. SRIF, somatostatin (somatotropin release-inhibiting factor); Iso, isoproterenol. Shaded areas indicate periods of Iso infusion.

Groups 1 and 2: Effects of endogenous insulin release on myocardial substrate uptake during beta -adrenergic stimulation. Pigs in both groups (n = 10 each) underwent four sets of measurements: baseline, vehicle or somatostatin without Iso, vehicle or somatostatin with Iso, and recovery. After baseline measurements of hemodynamics and 31P spectroscopy, injection of microspheres, and withdrawal of blood samples for determination of plasma insulin and glucagon, MVO2, and substrate uptake, alternate pigs were assigned to group 1 (vehicle) or group 2 (somatostatin). In group 2, 5 mg of somatostatin (Bachem, Torrance, CA) were mixed in 250-ml normal saline with 625 mg of endotoxin-free bovine serum albumen (Intergen, Purchase, NY) and 1.0 mg aprotinin (ICN, Costa Mesa, CA). Somatostatin was administered intravenously as a 100-µg bolus, followed by an infusion of 0.8 µg · kg-1 · min-1. In group 1, an equivalent volume of vehicle (albumen and aprotinin in normal saline) was infused. After 45 min of somatostatin (or vehicle) treatment, the second set of measurements was performed. Next, an infusion of Iso was begun (0.08 µg · kg-1 · min-1 iv) while infusion of vehicle (group 1) or somatostatin (group 2) was continued. After 45 min of combined Iso and somatostatin (or vehicle) treatment, the third set of measurements was performed. Infusions of Iso and somatostatin (or vehicle) were then discontinued, and the fourth (recovery) set of measurements was performed 45 min later.

Groups 3 and 4: Effect of time and exogenous insulin on myocardial substrate uptake during beta -adrenergic stimulation. Pigs in both groups (n = 7 each) underwent four sets of measurements: baseline, Iso 45 min, Iso 90 min, and recovery. After baseline measurements of hemodynamics, injection of microspheres, and collection of blood samples for determination of insulin, MVO2, and substrate uptake, an infusion of Iso was begun at 0.08 µg · kg-1 · min-1 iv. After 45 min of Iso, the second set of measurements was performed in both groups. At 60 min of Iso, pigs were then assigned to group 3 (continued Iso without exogenous insulin) or group 4 (continued Iso with insulin 100 mU · kg-1 · h-1 iv). During insulin infusion in group 4, arterial blood glucose was monitored every 5-10 min (Glucometer; McGaw, Irvine, CA). The results were used to adjust the rate of an intravenous infusion of 10% dextrose to maintain arterial blood glucose at or near the level observed in the same pig during Iso without insulin. The third set of measurements was performed after 90 min Iso treatment in both groups 3 and 4. Insulin and/or Iso infusions were then discontinued, and a fourth and final set of measurements was performed 45 min later.

Group 5: Effect of exogenous insulin on myocardial substrate uptake in the absence of beta -adrenergic stimulation. Under baseline conditions, pigs (n = 4) underwent measurements of hemodynamics, injection of microspheres, and withdrawal of blood samples for determination of insulin, MVO2, and substrate uptake. An infusion of insulin was then begun at 30 or 150 mU · kg-1 · h-1 iv in alternate pigs. Simultaneously, an infusion of 10% dextrose was begun at a rate of 2 ml · kg-1 · h-1 and adjusted every 5 min thereafter to maintain blood glucose near its baseline level in each pig. After 45 min of insulin treatment, a second set of measurements was performed. The insulin dose was then changed to 150 or 30 mU · kg-1 · h-1 in alternate pigs, and a third set of measurements was performed after an additional 45 min.

Statistical Analysis

Comparisons between baseline and subsequent values of a variable within a group were performed using one-way analysis of variance for repeated measures, followed by Tukey's multiple-comparison procedure. The statistical significance of a difference in a variable between groups 1 and 2 or between groups 3 and 4 at a specified time point was made using an unpaired Student's t-test. Statistical analysis was performed using True Epistat software (Epistat Services, Richardson, TX).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Groups 1 and 2

At baseline, there were no significant differences between groups 1 and 2 in hemodynamics, circulating hormone and substrate concentrations, myocardial blood flow, MVO2, substrate uptake, OERs, or high-energy phosphates (Tables 1 and 2, Figs. 2 and 3). Infusion of vehicle (group 1) or somatostatin (group 2) without Iso had no significant effect on any measured variable.

                              
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  		Table 1.   Hemodynamics and myocardial blood flow and oxygen consumption: groups 1 and 2 

                              
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  		Table 2.   Hormone concentrations, substrate concentrations, and oxygen extraction ratios: groups 1 and 2 


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  		Fig. 2.   Myocardial substrate uptake (groups 1 and 2): uptake of glucose (A), free fatty acids (FFA; B), and lactate (C). Group 1, normal insulin release. Group 2, insulin release inhibited by SRIF. Data are means ± SE; n = 10 in each group. * P < 0.05 vs. baseline in same group.


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  		Fig. 3.   Sum of oxygen extraction ratios (OERs) for glucose, FFA, and lactate (groups 1 and 2). OER for a substrate is fraction of myocardial oxygen consumption that could be accounted for if entire uptake of that substrate were oxidized. Net balance between substrate uptake and metabolism would be reflected by a sum of OERs for the principal exogenous substrates (glucose, FFA, and lactate) congruent  1.0. A sum of OERs > 1.0 indicates myocardial substrate uptake in excess of metabolism and implies net myocardial substrate accumulation. Data are means ± SE; n = 10 in each group. * P < 0.05 vs. baseline in same group and vs. 1.0. ddager  P < 0.05 vs. group 1 under same condition.

In both groups, infusion of Iso increased heart rate, decreased diastolic and mean aortic pressure, and increased MVO2 and transmural myocardial blood flow. There were no significant differences between groups in the responses of these variables to Iso. In group 1, the PCr-to-ATP ratio was 2.3 ± 0.1 at baseline and 2.6 ± 0.2 during Iso (P = NS); in group 2, the PCr-to-ATP ratio was 2.4 ± 0.1 under both conditions.

As expected, Iso stimulated insulin release in group 1, with mean plasma insulin concentration rising from 9 ± 2 µU/ml at baseline to 44 ± 6 µU/ml during Iso (P < 0.001). In contrast, somatostatin effectively suppressed insulin release during Iso in group 2, with plasma insulin concentrations of 6 ± 1 µU/ml at baseline and 10 ± 2 µU/ml during Iso (P = NS between the two measurements, P < 0.05 vs. response of group 1). In group 1, there was a nonsignificant trend toward increased plasma glucagon concentration during Iso. The relative change in glucagon (mean 42% increase with Iso) was much less than the change in insulin (mean 389% increase). In group 2, there was minimal change in glucagon during Iso.

After 45-min Iso infusion, arterial glucose concentration increased nonsignificantly from baseline in both groups 1 and 2, and did not differ significantly between groups. Myocardial glucose uptake increased significantly from baseline in both groups (from 0.19 ± 0.05 to 0.63 ± 0.19 µmol · g-1 · min-1 in group 1 and from 0.17 ± 0.07 to 0.49 ± 0.14 µmol · g-1 · min-1 in group 2, both P < 0.05). Despite the fourfold higher insulin concentration in group 1 during Iso, the difference between groups in glucose uptake during Iso was not significant.

After the 45-min Iso infusion, arterial FFA concentration approximately doubled in both groups, presumably reflecting Iso stimulation of lipolysis in adipose tissue. The nearly equal FFA levels in groups 1 and 2 during Iso indicate that the higher insulin level of group 1 was not sufficient to suppress lipolysis in adipose tissue. In group 1, myocardial FFA uptake increased sevenfold with Iso (P < 0.001 vs. baseline). In group 2, the increase in FFA uptake with Iso was of smaller magnitude and did not achieve statistical significance (P = 0.09 vs. baseline). The difference between the two groups in the response of FFA uptake to Iso was of borderline statistical significance (P = 0.06).

Arterial lactate concentration rose slightly after 45 min of Iso in both groups, without a significant difference between groups. There were no significant changes in myocardial lactate uptake in either group 1 or group 2 during any of the four conditions.

Table 2 and Fig. 3 show the individual OERs of each substrate and the sum of OERs, respectively. In group 1, the sum of OERs increased significantly from 0.92 ± 0.21 at baseline to 1.57 ± 0.17 after 45 min Iso. The latter value was significantly greater than unity. The rise in the sum of OERs was due predominantly to a large increase in the OER of FFA. These data indicate that the combined uptake of glucose, FFA, and lactate after 45 min Iso in group 1 was 57% greater than the rate at which these substrates could be metabolized, based on the simultaneous MVO2 and accounting for anaerobic glycolysis. As a result, there was significant net myocardial substrate accumulation.

In group 2, the sum of OERs was 0.90 ± 0.15 at baseline and 1.12 ± 0.16 after 45 min of Iso. The latter value was not significantly different from baseline or from unity. The difference between groups 1 and 2 in the sum of OERs during Iso was significant (P = 0.05). Thus inhibition of insulin release with somatostatin in group 2 prevented a state of excess myocardial substrate uptake during Iso stimulation.

During the recovery period, insulin levels returned to baseline in group 1 and remained unchanged in group 2. In both groups, the uptake and OER of each substrate and the sum of OERs for all three substrates returned to levels not significantly different from baseline.

Groups 3 and 4

At baseline, there were no significant differences between groups 3 and 4 in any measured variable (Tables 3 and 4, Figs. 4 and 5). Iso increased myocardial blood flow and MVO2 in groups 3 and 4 to a somewhatgreater extent than in groups 1 and 2, possibly because of a longer anesthetic time in groups 1 and 2 due to preparations for NMR spectroscopy. Otherwise, infusion of Iso for 45 min elicited increases in heart rate and circulating concentrations of insulin, glucose, and FFA that were similar to those observed in group 1. Myocardial uptake of glucose and FFA increased significantly at 45 min of Iso. The sum of OERs at 45 min Iso rose significantly to 1.69 ± 0.20 in group 3 and to 1.45 ± 0.16 in group 4, both values significantly greater than baseline (0.95 ± 0.11 and 1.01 ± 0.07, respectively) and significantly greater than unity. Thus 45 min of Iso treatment induced a state of net myocardial substrate accumulation in groups 3 and 4, just as it did in group 1.

                              
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  		Table 3.   Hemodynamics and myocardial blood flow and oxygen consumption: groups 3 and 4 

                              
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  		Table 4.   Insulin, substrate concentrations, and oxygen extraction ratios: groups 3 and 4 


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  		Fig. 4.   Myocardial substrate uptake (groups 3 and 4): uptake of glucose (A), FFA (B), and lactate (C). Group 3, without exogenous insulin. Group 4, exogenous insulin infusion at Iso 90 min. Data are means ± SE; n = 7 in each group. * P < 0.05 vs. baseline in same group. ddager  P < 0.05 vs. group 3 under same condition.


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  		Fig. 5.   Sum of OERs for glucose, FFA, and lactate (groups 3 and 4). Data are means ± SE; n = 7 in each group. * P < 0.05 vs. baseline in same group and vs. 1.0. ddager  P < 0.05 vs. Iso 45 min in group 3.

The measurements at 90 min Iso in group 3 were performed without administration of exogenous insulin to determine time-dependent changes in the response to Iso. Although there were no significant changes in circulating insulin, arterial substrate concentrations, or MVO2 between 45 and 90 min of Iso treatment, there were significant changes in myocardial substrate uptake. Myocardial glucose and FFA uptake at 90 min Iso declined to less than one-half their respective values at 45 min Iso. Lactate uptake remained approximately unchanged. As a result, the sum of OERs declined significantly from 1.69 ± 0.20 at 45 min Iso to 0.69 ± 0.21 at 90 min Iso. Thus the state of excess myocardial substrate uptake relative to substrate metabolism persisted for at least 45 min, but less than 90 min, of Iso treatment.

In group 4, measurements at 90 min Iso were made with concomitant administration of exogenous insulin to determine whether such treatment could prolong or enhance the state of excess myocardial substrate uptake. Plasma insulin rose from 25 ± 4 µU/ml at 45 min Iso to 123 ± 15 µU/ml at 90 min Iso, while arterial blood glucose concentration was maintained at a nearly constant level. Despite the increase in insulin, myocardial glucose uptake decreased to less than one-third its level at 45 min Iso (P < 0.05). Thus a high level of insulin failed to stimulate myocardial glucose uptake after 90 min of Iso treatment. Compared with the measurements at 45 min Iso without exogenous insulin, measurements at 90 min Iso with exogenous insulin treatment demonstrated no significant change in myocardial FFA uptake, but a significant increase in myocardial lactate uptake. Overall, the sum of OERs in group 4 declined from 1.45 ± 0.16 at 45 min Iso to 1.09 ± 0.17 at 90 min Iso, the latter not significantly different from baseline (1.01 ± 0.07) or from unity. Thus a high level of insulin did not sustain the state of net myocardial substrate accumulation through 90 min of Iso treatment.

Group 5

Because myocardial glucose uptake did not appear to be sensitive to insulin during Iso stimulation in groups 1-4, additional studies were undertaken in group 5 to determine whether myocardial glucose uptake was sensitive to insulin in the absence of Iso, but under the same conditions of anesthesia and surgery as the other groups. Plasma insulin levels in group 5 increased from a baseline of 6 ± 2 µU/ml to 45 ± 24 µU/ml with low-dose insulin and to 184 ± 22 µU/ml with high-dose insulin (Table 5). Thus the range of insulin levels achieved in group 5 spanned the range of levels obtained in groups 1-4. In group 5, insulin had no discernible effect on myocardial blood flow or MVO2. Myocardial glucose uptake increased significantly between baseline and low-dose insulin measurements, and again between low-dose and high-dose insulin measurements. Myocardial glucose uptake with high-dose insulin in group 5 (0.7 ± 0.2 µmol · g-1 · min-1) was approximately equal to the maximum observed in groups 1-4 during Iso stimulation (0.5-0.8 µmol · g-1 · min-1).

                              
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  		Table 5.   Responses to insulin in the absence of isoproterenol: group 5 

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

New Findings of This Study

This study demonstrates that systemic beta -adrenergic stimulation produces a transient state of myocardial substrate uptake in excess of metabolism, implying net myocardial substrate accumulation. This state persists for at least 45 min, but less than 90 min. An elevated plasma insulin concentration is a necessary condition to establish the state of excess myocardial substrate uptake; when insulin release was suppressed with somatostatin, the sum of OERs did not increase significantly during beta -adrenergic stimulation and did not significantly exceed 1.0. The predominant effect of an elevated insulin level during beta -adrenergic stimulation is to augment the uptake of exogenous FFA by the myocardium. Despite its effects in the early phase of the response to beta -adrenergic stimulation, increased insulin is not a sufficient condition to prolong the duration of the response. Surprisingly, myocardial glucose uptake during beta -adrenergic stimulation does not depend on insulin levels, although glucose uptake in the absence of beta -adrenergic stimulation is highly dependent on insulin.

Magnitude of Excess Substrate Uptake

The magnitude of excess myocardial substrate uptake demonstrated in this study is substantial. Let us assume that during 45 min of Iso stimulation actual MVO2 is 10 µmol · g-1 · min-1 and the sum of OERs is 1.5. Complete oxidation of the substrate taken up by the myocardium during Iso would therefore require an MVO2 of 15 µmol · g-1 · min-1. The difference of 5 mmol · g-1 · min-1 represents the oxidative potential of the excess myocardial substrate uptake. Over 45 min, the accumulated excess substrate would have an oxidative potential of 225 µmol O2/g myocardium. Excess substrate is stored as glycogen and/or triglyceride. If the accumulated excess substrate were stored entirely as glycogen, myocardial glycogen content (in glucosyl equivalents) would increase by 225/6 or 37.5 µmol/g (because the oxidative potential of 1 mol of glucose is 6 mol of O2). Given that the nominal glycogen content of porcine myocardium is ~40 µmol glucosyl equivalents/g (31), this would represent a doubling of myocardial glycogen content. Alternatively, if we assume that the substrate accumulated during 45 min of Iso stimulation were stored entirely as triglyceride, myocardial triglyceride content would increase by 225/23.5 or 9.6 µmol FFA equivalents/g (because the oxidative potential of 1 mol FFA is ~23.5 mol of O2). Given that the nominal triglyceride content of porcine myocardium is ~4 µmol FFA equivalents/g wet wt (22), this would represent a tripling of myocardial triglyceride content. Viewed in this context, it is not surprising that the state of Iso-induced excess substrate uptake was transient and was no longer present after 90 min.

Effects of Insulin and Iso on Myocardial Substrate Uptake

In all groups, FFA became the predominant exogenous substrate during Iso stimulation. This was most likely due to increased circulating FFA levels resulting from Iso-mediated lipolysis. In addition, myocardial malonyl CoA, an inhibitor of FFA metabolism, has been shown to decrease in response to catecholamine stimulation (9). FFA uptake during early Iso stimulation was greater in group 1 (with elevated insulin levels) than in group 2 (with inhibition of insulin release). This difference is unlikely to be due to a direct effect of insulin on FFA uptake, because group 5 demonstrated no increase in FFA uptake when mean plasma insulin was increased from 6 to 45 µU/ml while arterial FFA concentration remained constant. More likely, insulin antagonized Iso-stimulated myocardial lipolysis (18, 30), thus reducing intracellular formation of FFA and increasing uptake of exogenous FFA.

The apparent insensitivity of myocardial glucose uptake to insulin during Iso stimulation (group 1 vs. group 2, group 4 vs. group 3) may be due to effects of substrate competition, accelerated glycogenolysis, beta -adrenergic inactivation of the glucose transporter GLUT-4, and/or constraint by the maximum rate of myocardial glucose transport. Substrate competition (25), implying reciprocal changes in utilization of FFA and glucose, is likely to have occurred during Iso because a high rate of FFA utilization is expected to increase concentrations of glycolytic inhibitors such as citrate, acetyl CoA, and NADH (24), thereby limiting the ability of insulin to stimulate glucose uptake. beta -Adrenoceptor activation stimulates myocardial glycogenolysis (5, 16), thereby increasing the concentration of glucose-6-phosphate (5), an inhibitor of hexokinase. Reduced hexokinase flux may become rate limiting for glucose transport and prevent further, insulin-mediated increases in myocardial glucose uptake (4, 33). In isolated cardiomyocytes (29) and adipocytes (23, 32), beta -adrenergic stimulation has been shown to render GLUT-4 nonfunctional for glucose transport, even after insulin-mediated translocation to the plasma membrane (17, 32). If a similar effect occurs in vivo, it may have contributed to the absence of incremental insulin-stimulated glucose uptake during Iso in these experiments. In isolated hearts perfused with glucose as the sole substrate, the maximum rate of glucose uptake generally does not exceed 1 µmol · g-1 · min-1, even under conditions of high cardiac workload and metabolic demand (5). Because myocardial glucose uptake approached this level during Iso in the current experiments even when insulin levels were low (group 2), there may have been limited potential for further, insulin-mediated increases in glucose uptake. There was no significant difference in plasma glucagon concentration between groups 1 and 2; therefore, the insensitivity of myocardial glucose uptake to insulin during Iso was not likely due to a countervailing effect of higher glucagon in group 1.

Unlike FFA and glucose uptake, myocardial lactate uptake did not increase during treatment with Iso alone. Because lactate is both a primary substrate and an end product of anaerobic glycolysis, an increase in the latter will decrease net lactate uptake. There are at least three reasons why anaerobic glycolysis may have increased during Iso. First, relative myocardial ischemia could have developed during Iso because of increased metabolic demand in the face of tachycardia and decreased diastolic coronary perfusion pressure. However, this is unlikely because other evidence of ischemia (such as a decline in subendocardial blood flow, PCr-to-ATP ratio, or coronary venous oxygen content) was absent. Second, increased anaerobic glycolysis may have been driven by Iso-stimulated glycogenolysis. In isolated, perfused hearts, myocardial glycogenolysis and lactate release are both stimulated by beta -adrenergic agonists (5, 9). Third, pyruvate dehydrogenase may be inhibited under conditions of high FFA utilization, resulting in diversion of glycolytic flux to lactate.

Insulin increased myocardial lactate uptake during Iso stimulation. Lactate uptake increased with exogenous insulin treatment in group 4 and lactate uptake tended to be higher in group 1 (insulin release intact) than in group 2 (insulin release inhibited). The most likely explanation for these findings is that higher insulin levels directed more glucose and FFA uptake to myocardial glycogen and triglyceride synthesis, resulting in greater utilization of lactate for immediate myocardial metabolism.

Limitations

It is reasonable to assume that the excess substrate uptake during Iso stimulation resulted in synthesis of additional myocardial glycogen and triglyceride; however, these substances were not measured in the current study and so the relative changes in each pool cannot be determined. It is possible that additional transient effects of Iso stimulation occurred within a shorter time frame than that examined in this study. For example, Goodwin et al. (10) found that when epinephrine was added to the perfusate of isolated rat hearts, there was a transient burst of glycogenolysis that contributed 35% to total ATP synthesis for 5 min. However, this effect is quantitatively small compared with a 50% excess of substrate uptake versus metabolism over 45 min. In addition to the three principal exogenous carbon substrates measured in this study, there is modest myocardial uptake of ketone bodies and minimal uptake of pyruvate and amino acids for energy metabolism (3, 15). Generally, these substrates contribute less than 10% to the sum of OERs (15). If uptake of these secondary substrates had been measured, the magnitude of excess myocardial substrate uptake during Iso would likely have been found to be even greater than that reported. Because the fractional transmyocardial extraction of glucose was low (~0.05), calculation of glucose uptake by the Fick method was subject to increased experimental variability. Nonetheless, we demonstrated significant changes in glucose uptake with Iso stimulation. Alternative techniques for measuring glucose uptake using a tracer analog such as 18F-labeled 2-deoxy-D-glucose have been shown to be accurate only under steady-state conditions (11).

Implications

A substantial transient imbalance between myocardial substrate uptake and oxidation may be produced by systemic beta -adrenergic stimulation in vivo, due to the concomitant elevation of plasma insulin and FFA levels. Effects of time and insulin must therefore be considered in the design, interpretation, and comparison of experimental studies of myocardial substrate and energy metabolism in response to catecholamine stimulation.

Excessive myocardial FFA uptake and accumulation of fatty acyl intermediates may be detrimental to cardiac function in both normoxic and ischemic hearts (19). Clinically relevant doses of inotropic agents such as dobutamine elevate plasma insulin and FFA levels and increase myocardial FFA uptake to a similar extent as observed in the current study (20). The magnitude of net myocardial substrate accumulation demonstrated in this study and the likelihood that a substantial portion of this accumulation was in the form of lipid raise the possibility that significant lipid accumulation occurs in the human heart as a consequence of inotropic stimulation with catecholamines.

    ACKNOWLEDGEMENTS

The authors thank Dr. Judith Wisneski for helpful advice and support of the substrate assays, Maria Mayr for performing the substrate assays, Joshua Cohen for assistance in conducting the experimental protocol, Dr. Michael Weiner for advice in the use of NMR spectroscopy, Sean Steinman for constructing the NMR coil and assisting in performing NMR measurements, and Dr. Clifford Greyson for valuable suggestions for the manuscript.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-49944 (to G. G. Schwartz) and the Department of Veterans Affairs Medical Research Service. Li Lu is a Postdoctoral Fellow of the American Heart Association, California Affiliate.

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. §1734 solely to indicate this fact.

Address for reprint requests: G. G. Schwartz, Cardiology Section (111C), VA Medical Center, 4150 Clement St., San Francisco, CA 94121.

Received 21 April 1998; accepted in final form 12 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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