Increased ketone utilization by the kidney reduces renal lactate uptake but does not affect tubular sodium reabsorption.

The purpose of the present study was to assess whether a moderate increase in ketonemia interferes with renal uptake of energy-providing substrates, and whether it may promote natriuresis similar to that occurring spontaneously during the early phase of starvation. To this end, the sodium salt of D(-)3-hydroxybutyrate (3OHB) was infused to 10 anesthetized dogs at a rate of 20 mumol/kg.min-1 over 135 minutes. To allow comparison, an equivalent amount of sodium was infused as sodium bicarbonate to 10 other dogs (to induce an alkalinization similar to that resulting from the utilization of the sodium salt of 3OHB), while 10 additional dogs received an equimolar infusion of NaCl to provide reference values. Before 3OHB or bicarbonate infusion, lactate represented the major fuel taken up by the kidney (28 +/- 3 mumol/100 g kidney.min-1 on average). While small but significant amounts of 3OHB were taken up by the kidney in control conditions (0.7 +/- 0.1 mumol/100 g.min-1; P < .05), there was no significant uptake of free fatty acids (FFA) or glucose. Upon 3OHB infusion, which increased plasma 3OHB levels to 1.7 mmol/L, the renal uptake of this substrate increased to 25 +/- 3 mumol/100 g.min-1, in proportion to renal 3OHB availability (r = .73, P < .001). Despite an increase in arterial lactate levels that occurred during 3OHB infusion, renal lactate uptake and the extraction ratio of this substrate were decreased (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Stabilization of left ventricular function with D-(-)-3-hydroxybutyrate after coronary occlusion in the intact dog.

The D-(-) isomer or natural form of 3-hydroxybutyrate (D-(-)-3OHB) is a readily used energy substrate. Studies on anesthetized intact dogs in our laboratory have demonstrated that raising the arterial level of D-(-)-3OHB to 1 mM enhances the ketone uptake not only by the normal, but also by the acutely ischemic myocardium, though at a lower rate. Whether this moderate rise in arterial D-(-)-3OHB does modify the time course of left ventricular (LV) function during acute regional ischemia remains unsettled. In the present study, 13 anesthetized intact dogs with occluded left anterior descending (LAD) coronary artery (balloon catheter) were infused with D-(-)-3OHB as the L-(+)-arginine salt at a rate of 20 mumol/kg/min i.v. for 90 min, starting 40 min after the LAD occlusion. Arterial D-(-)-3OHB rose to 1.1 mM. Arterial pH was not modified. By comparison with the decline observed in 13 saline-treated ischemic dogs, the ketone treatment significantly stabilized the time course of LV peak positive dP/dt and output per minute. This effect was not attributable to the simultaneous infusion of arginine since it was not observed with equimolar infusions of this amino acid alone in eight additional ischemic dogs.

In vivo effect of the D-(-) isomer or natural form of 3-hydroxybutyrate on initial release of lactate dehydrogenase from the acutely ischaemic myocardium.

D-(-)-3-hydroxybutyrate, the isomer found in the circulation and in the urine of diabetic patients, generally is believed to be the physiologically important form of 3-hydroxybutyrate [10]. Little is known concerning the effects of an elevated plasma level of the D-(-) isomer of 3-hydroxybutyrate upon the acutely ischaemic heart. Using anaesthetized intact dogs with a balloon catheter inserted into the proximal part of the left anterior descending coronary artery (LAD), we have recently demonstrated that a 1 mM ketonaemia induced with the arginine salt of D-(-)-3-hydroxybutyric acid reduces the uptake of non-esterified fatty acids (NEFA) in the myocardial area distal to the inflated balloon [4]. The question arises as to whether the concomitant increase in ketone uptake in this area could be detrimental to the acutely ischaemic myocardium. Indeed, a previous study on isolated coronary ligated hearts from normal rats has shown that the rate of release of lactate dehydrogenase (LDH) during the first 90 min of ischaemia can be enhanced by replacing glucose (11 mM) in the perfusion fluid with either albumin-bound palmitate (0.9 mM) or sodium DL-3-hydroxybutyrate (10 mM) as the sole energy substrate [11]. This would suggest that the ketone might be as deleterious as its metabolic precursors for membrane integrity in the acutely ischaemic myocardium. In the present report, we examine the effect of arginine D-(-)-3-hydroxybutyrate on LDH release from ischaemic myocardium in our in vivo preparation. The dogs were treated with lidocaine in order to minimize the frequency and, hence, the adverse metabolic effects of ectopic beats.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of the prolonged stimulatory effect of corticotrophin on pregnenolone production by guinea-pig adrenocortical mitochondria.

The postulated prolonged stimulatory influence of ACTH on the adrenocortical mitochondrial synthesis of pregnenolone in response to ACTH was studied in adrenal mitochondria isolated from control guinea-pigs and from animals treated s.c. with 100 micrograms ACTH(1-24) twice daily on the day before the animals were killed. The animals from both groups were injected with 100 micrograms ACTH s.c. 30 min before killing. The mitochondrial production of pregnenolone (expressed in nmol per mg mitochondrial protein after 10-min incubation) increased from 1.52 +/- 0.46 (S.E.M.) in the control group to 4.50 +/- 0.59 for mitochondria from ACTH-treated animals, despite a similar free cholesterol content in the mitochondria, even when determined after a previous in-vivo treatment with aminoglutethimide to block further metabolism of cholesterol into pregnenolone. In addition, in the presence of an excess of exogenous cholesterol (100 mumol/l), the production of pregnenolone remained higher for mitochondria from ACTH-treated animals. In contrast, when the calcium concentration in the incubation medium was raised to 1 mmol/l, with subsequent enhancement in pregnenolone synthesis, the mitochondrial pregnenolone production became similar for both groups (8.28 +/- 1.11 nmol in the ACTH-treated group and 9.55 +/- 1.90 nmol in the control group), even in the presence of 100 mumol cholesterol/l (13.5 +/- 1.80 nmol in ACTH-treated animals and 14.8 +/- 1.93 nmol in controls). Cycloheximide treatment administered on the day before the animals were killed was without any effect on pregnenolone production in control animals (3.51 +/- 0.43 nmol before and 3.65 +/- 0.63 nmol after cycloheximide treatment).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of the D(-)isomer of 3-hydroxybutyrate on cardiac non-esterified fatty acid uptake and oxygen demand induced by norepinephrine in the intact dog.

The effects of ketosis on the norepinephrine-induced high rates of cardiac uptake of non-esterified fatty acids (NEFA = free fatty acids = FFA) and oxygen consumption were studied in anesthetized intact dogs. After a control infusion of norepinephrine (500 ng/kg.min into the left ventricle), the D(-) isomer or natural form of 3-hydroxybutyrate was infused intravenously as the arginine salt at rates of 20 mumol/kg.min in group A (10 dogs) and 80 mumol/kg.min in group B (10 dogs) and a second norepinephrine infusion was superimposed on the ketone treatment. At the time the effects of the second catecholamine infusion were measured, the arterial 3-hydroxybutyrate concentration averaged 1.2 +/- 0.1 mM in group A and 8.3 +/- 0.4 mM in group B, and the cardiac uptake of the ketone amounted to 17.4 +/- 0.6 and 35.8 +/- 5.3 mumol/min.100 g, respectively. Relative to the control norepinephrine infusion, the arterial NEFA concentration was reduced to 88 +/- 4% in group A and to 62 +/- 8% in group B, but the cardiac uptake of NEFA was significantly more depressed, to 65 +/- 7% in group A and to 35 +/- 8% in group B. These changes were not observed in ten non-ketotic animals under repeated norepinephrine infusion. Thus, ketosis inhibited the norepinephrine-stimulated uptake of NEFA, presumably through (1) a lowered availability of NEFA from arterial blood, attributable to a reduction of extracardiac lipolysis, and (2) competition of 3-hydroxybutyrate with NEFA for metabolism by the myocardium in the face of still high arterial NEFA concentrations, 1.7 +/- 0.1 mM in group A and 1.1 +/- 0.2 mM in group B. In both groups, the lowering of the contribution of NEFA to cardiac metabolism was associated with a reduction of the estimated oxygen demand per beat (ratio of cardiac oxygen consumption/min to the pressure-rate product), while the pressure response to norepinephrine was not modified. There was no evidence for abnormal cardiac function.

The enhancement of pregnenolone production as the main mechanism of the prolonged stimulatory effect of ACTH on cortisol production by guinea-pig adrenocortical cells.

The prolonged stimulatory influence of corticotropin (ACTH) on the adrenocortical steroidogenic response to ACTH was studied in guinea-pig adrenocortical cells harvested from control and ACTH-treated animals (ACTH1-24, 50 micrograms s.c. twice daily on the day preceding the in vitro experiment). The maximal capacity to produce cortisol in response to ACTH (by 10(5) cells and 2 h incubation) was increased from 341.8 +/- 36.3 ng (control group) to 663.3 +/- 37.6 ng for cells obtained from guinea-pigs treated in vivo with ACTH. In the presence of trilostane, added to the cells in order to block the conversion of pregnenolone to cortisol, the net maximal output of pregnenolone and 17-hydroxypregnenolone in response to ACTH was significantly increased in adrenocortical cells from ACTH-treated animals (449.5 +/- 35.8 ng pregnenolone and 85.7 +/- 10.5 ng 17-hydroxypregnenolone vs 269.1 +/- 36.3 ng pregnenolone and 43.7 +/- 8.51 ng 17-hydroxypregnenolone for cells from control guinea-pigs). It appeared therefore that the total production of pregnenolone (as estimated by the sum of pregnenolone and 17-hydroxypregnenolone produced by the cells incubated with trilostane) nearly reached the level of the maximal production of cortisol in response to ACTH and was also significantly enhanced for cells from ACTH-treated animals (532.2 +/- 38.4 ng vs 312.8 +/- 40.0 ng for cells from control group). By contrast, no effect was documented on 17 alpha-hydroxylase activity since 17 alpha-hydroxylation index was similar for both types of adrenocortical cells (16.3 +/- 2.05% for ACTH-treated animals and 14.2 +/- 2.83% for control group). It was concluded therefore that the prolonged stimulatory influence of ACTH on pregnenolone production is the main mechanism of the enhancement of cortisol synthesis by guinea-pig adrenocortical cells previously stimulated by ACTH.

On the specificity of the inhibitory effect of trilostane and aminoglutethimide on adrenocortical steroidogenesis in guinea pig.

The effect of trilostane and aminoglutethimide on steroidogenesis was studied on isolated guinea pig adrenocortical cells in order to verify whether, in addition to the inhibitory influence of trilostane on 3beta-hydroxysteroid dehydrogenase and isomerase and the inhibition by aminoglutethimide of pregnenolone formation, these inhibitors may also affect other enzymatic steps of cortisol synthesis. While trilostane completely abolished cortisol production in response to ACTH with concomitant enhancement in pregnenolone and 17-hydroxypregnenolone formation, the conversion of progesterone, 17-hydroxyprogesterone and 11-deoxycortisol into cortisol was not affected by the presence of the inhibitor. Contrasting with this specific inhibitory effect of trilostane, aminoglutethimide inhibited not only pregnenolone formation but also several other enzymatic steps involved in the conversion of this precursor of steroidogenesis into cortisol. Among these additional effects of aminoglutethimide on steroidogenesis, the inhibition of 11 beta-hydroxylation was clearly demonstrated.

The prolonged stimulatory effect of ACTH on 11 beta-hydroxylation, and its contribution to the steroidogenic potency of adrenocortical cells.

The mechanism of the prolonged stimulatory influence of corticotropin (ACTH) on the capacity of adrenocortical cells to produce cortisol in response to ACTH and more specifically the role of 11 beta-hydroxylation, was studied on guinea-pig adrenocortical cells dispersed from control and ACTH-treated animals. As a result of the previous in vivo exposure to ACTH, the net maximal production of glucocorticoids in response to ACTH (by 10(5) cells and 2 h incubation) increased from 660 +/- 33.9 ng (control group) to 1105 +/- 117.9 ng for cells from ACTH-treated animals (P less than 0.001), whereas the apparent affinity of the steroidogenic response remained unchanged. In addition there occurred an increased conversion of exogenous pregnenolone into cortisol by cells from ACTH-treated animals, indicating a prolonged stimulatory influence of ACTH on the post-pregnenolone pathway of cortisol biosynthesis. The activity of 11 beta-hydroxylation step was therefore examined by incubating the adrenocortical cells from control and ACTH-treated animals in the presence of increasing amounts of 11-deoxycortisol. The maximal capacity of 11-deoxycortisol conversion into cortisol was increased as a result of the in vivo exposure to ACTH, averaging 3423 +/- 211 ng cortisol formed from 5 micrograms 11-deoxycortisol by 10(5) cells from ACTH-treated animals vs 2074 +/- 185 ng for cells from control guinea-pigs (P less than 0.001). However, the conversion of lower amounts of 11-deoxycortisol into cortisol, reproducing quantitatively the maximal effect of ACTH on cortisol biosynthesis, was only barely increased in cells from ACTH-treated animals (P greater than 0.05). Therefore it was concluded that ACTH increases in a lasting way not only the overall steroidogenic capacity of adrenocortical cells but also the maximal efficiency of 11 beta-hydroxylation. Since the latter effect cannot account quantitatively for the magnitude of the lasting effect of ACTH on the maximal capacity of adrenocortical cells to produce cortisol in response to ACTH, it appears that the prolonged influence of ACTH on cortisol biosynthesis should also involve a stimulatory influence of the peptide on earlier step(s) of steroidogenesis.

The stimulatory effect of corticotropin on cortisol biosynthetic pathway in guinea-pig adrenocortical cells.

The mechanism of the prolonged stimulatory effect of corticotropin (ACTH) on adrenocortical synthesis of cortisol was studied in guinea-pig adrenocortical cells harvested from control animals and from guinea-pigs submitted 24 h before the sacrifice to a prolonged ether anesthesia in an attempt to induce a release of endogenous ACTH. As a result of this in vivo exposure to endogenous ACTH, the maximal capacity to produce glucocorticoids (by 1 X 10(5) cells incubated during 2 h) in response to ACTH increased from 579 +/- 111 ng (control group) to 915 +/- 143 ng for cells from treated animals, whereas the apparent affinity of the steroidogenic response to ACTH remained unchanged. This hyper-reactivity of cells from anesthetized animals was also evident in the presence of dibutyryl cyclic AMP. Moreover, there was increased conversion of exogenous pregnenolone into cortisol by cells from previously anesthetized animals. It was therefore concluded that ACTH increases in a lasting way the activity of steroidogenic pathway leading to cortisol synthesis by adrenocortical cells at sites distal to cyclic AMP generation. Besides an obvious increase of formation of pregnenolone in response to ACTH, it seems that this ACTH-induced enhancement in the capacity of the steroidogenic response to ACTH also implies a prolonged stimulatory influence of the peptide on the post-pregnenolone steroidogenic pathway leading to cortisol synthesis.

Release of cyclic adenosine 3',5'-monophosphate in the coronary venous blood during an intracoronary infusion of adenosine.

The effect of adenosine on the release of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in the coronary venous blood was examined in 30 anaesthetized intact dogs. Adenosine was infused into the left anterior descending coronary artery (LAD) at a rate of 150 nmol . min-1 and coronary venous blood was sampled in the great cardiac vein (GCV), near the outflow site of the veins accompanying the LAD. Significant coronary veno-arterial differences in cyclic AMP plasma level were observed before and during the infusion. The myocardial blood flow supplied by the LAD increased from 56 +/- 3 to 237 +/- 23 ml . min-1 per 100 g myocardium and the rate of cyclic AMP release in the venous blood near the origin of the GCV increased from 15.2 +/- 3.0 to 104.8 +/- 29.5 pmol . min-1 per 100 g myocardium. The adenosine-induced release of cyclic AMP did not result from an action of the substance on the platelets in the coronary circulation since adenosine concentration comparable to that achieved in the LAD did not modify the cyclic AMP plasma level of arterial blood in vitro. It is concluded that the released nucleotide originated essentially in cardiac structures, i.e. the myocardial and/or coronary cells. The results support in vitro studies suggesting that cyclic AMP is involved in the metabolic regulation of coronary blood flow by adenosine.

Adenosine-induced release of cyclic adenosine 3',5'-monophosphate from the left ventricle in the anaesthetized intact dog.

1. The influence of adenosine on the release of cyclic adenosine 3',5'-monophosphate (cyclic AMP) from the heart was examined in twenty-two anaesthetized intact dogs. The animals were pre-treated with propranolol and vagotomized. The rate of nucleotide release from the left ventricle was determined as the product of the left ventricular myocardial plasma flow and the coronary veno-arterial difference in plasma nucleotide concentration. 2. Adenosine was infused into the left ventricle during three successive 15 min periods at rates of 25, 50 and 100 n-mole/kg. min, respectively. The mean blood pressure in the ascending aorta was prevented from falling by inflating a balloon placed into the thoracic aorta. The measurements were performed before the infusion and at the 10th min of each infusion period. The values given are means +/- S.E. 3. During the 45 min infusion of adenosine at increasing rate, the cyclic AMP concentration in arterial plasma increased from 7.0 +/- 0.3 to 14.0 +/- 0.9 p-mole/ml. The nucleotide was released from the left ventricle at rates increasing from 48.2 +/- 7.1 to 206.5 +/- 56.2 p-mole/100 g. min while the left ventricular myocardial blood flow increased from 127 +/- 6 to 399 +/- 33 ml./100 g.min. The oxygen consumption of the left ventricle was not modified. 4. When adenosine was infused at a rate of 100 n-mol/kg.min, the thorax was opened and the apex of the heart and the left atrial appendage were removed for nucleotide assay. The cardiac cyclic AMP concentration did not differ from that observed in control dogs. 5. The results suggest that cyclic AMP is likely to be involved in the membrane and cellular events underlying the relaxant effect of adenosine on the coronary smooth muscle. The lack of change in cardiac cyclic AMP concentration, as determined by whole tissue extractions, is consistent with a study by others showing that, under normoxic conditions, cyclic AMP is released from a small, compartmentalized fraction of the cyclic AMP content of the heart. The elevation of the plasma nucleotide concentration could result from adenosine effects on various cell systems or organs, in addition to the observed release from the heart.

Isoprenaline-induced release of cyclic adenosine 3',5'-monophosphate by the left ventricle in the anaesthetized intact dog.

The ability of the heart to release cyclic adenosine 3',5'-monophosphate (cyclic AMP) in the coronary venous blood was examined in closed-chest anaesthetized dogs before and during an infusion of isoprenaline. Before the infusion, no difference was observed between the cyclic AMP plasma levels in the aortic and coronary sinus blood. During the infusion, significant coronary veno-arterial differences in nucleotide concentration were observed despite a considerable elevation of the cyclic AMP plasma level in the aortic blood.

Cyclic 3',5'-adenosine monophosphate level and adenylate cyclase activity in human blood platelets during storage in ACD solution.

The level of cyclic 3',5'-adenosine monophosphate (cAMP) in human platelets and the activity of platelet adenylate cyclase in response to prostaglandin E1 stimulation do not change during two days storage at room temperature in ACD solution. However, the level of cyclic AMP is lower in platelets stored in ACD solution than in platelets from blood anticoagulated by ethylenediamine tetra-acetic acid.

Inhibition of pacing-induced coronary dilation by aminophylline.

In closed-chest anaesthetized dogs, aminophylline was infused into the left circumflex coronary artery at a rate of 250 mug/min. When the heart rate was lower than 100 beats/min, infusion did not modify the left circumflex coronary blood flow and resistance. When the heart rate was elevated to an average of 153 +/- 6 beats/min by right atrial pacing, infusion inhibited the increase in left circumflex coronary blood flow and decrease in coronary resistance. The pacing-induced increment in coronary blood flow was reduced by an average of 64% and the flow deficit was associated with a lowering of the coronary sinus blood oxygen tension. Since aminophylline is known to inhibit the coronary dilator action of exogenous adenosine, the results support the concept that adenosine resulting from the breakdown of adenine nucleotides is a mediator of the metabolic regulation of coronary blood flow.

Rate of uptake of endogenous cortisol by the left ventricle of the anaesthetized dog during ventilation with ambient air and during hypoxia.

1. The rate of uptake of endogenous cortisol by the left ventricle was determined in eight dogs as the product of the left ventricular myocardial plasma flow and the coronary arteriovenous difference in plasma cortisol concentration. The arteriovenous difference in hormone concentration in resting skeletal muscle (M. gracilis) was also measured. The values given are means +/- S.E.2. During ventilation with ambient air (arterial P(O2): 85 +/- 2 mm Hg), the rate of cortisol uptake by the left ventricle averaged 382 +/- 92 ng/100 g.min. The arteriovenous difference in hormone concentration in the vessels of the M. gracilis was more than twice that in the coronary vessels. Assuming a value of 3-4 ml./100 g.min for the blood flow in this muscle, the corresponding rate of cortisol uptake would be in the range of 25-33 ng/100 g.min.3. During hypoxia (arterial P(O2): 35 +/- 1 mm Hg), the rate of cortisol uptake by the left ventricle was not significant, averaging 26 +/- 113 ng/100 g.min. By contrast, the skeletal muscle still extracted significant amounts of cortisol from the plasma. It is inferred that the suppression of a significant uptake of the hormone by the left ventricle was related, at least in part, to the changes in the mechanical and associated metabolic activity of the heart muscle elicited by the lowering of the arterial P(O2).4. Hypoxia depressed the net uptake of cortisol by the left ventricle rather than the uptake mechanism itself. Indeed, bi-directional movements of cortisol between the plasma and the heart muscle, with rates of release up to 1150 ng/100 g.min, were observed.

Left ventricular contractility and developed tension in the intact dog submitted to an intracoronary infusion of adenosine.

1. The effect of adenosine on left ventricular contractility and developed tension was studied in the anaesthetized intact dog. Normal and propranolol-treated animals were used. Adenosine was infused through a catheter into one of the two main branches of the left coronary artery, usually the circumflex branch. The rate of infusion was 150 x 10(-9)M/min. The infusion was maintained during 50 min.2. In both series of animals, no change was observed in the heart rate, aortic pressure, left ventricular end-diastolic pressure, time from onset of left ventricular contraction to peak dP/dt, peak dP/dt and left ventricular tension-time index. It is concluded that a regional increase in adenosine concentration in the left ventricular wall has no inotropic effect when the adrenergic mechanisms are normal or depressed.3. The myocardial blood flow response to adenosine was determined at the 10th, 30th and 50th min of the infusion by the radioactive inert gas method. At the 10th min of the infusion, the myocardial blood flow averaged three times the control value in both series of dogs. Thereafter, the flow response remained stable in the normal dogs but declined at the 50th min of the infusion in the propranolol-treated animals. It is suggested that autoregulation of the coronary circulation in response to overperfusion of the myocardium at constant cardiac work may be enhanced at the lower myocardial oxygen requirements of the propranolol-treated dogs while, in the normal animals, it was insufficient to overcome the potent coronary dilator action of adenosine.