Effects of diabetes and insulin on ketone bodies metabolism in heart

This work investigates the effect of alloxan-induced short-term diabetes (24 h) on D-3-hydroxybutyrate metabolism at physiological and non-physiological concentrations of the ketone body in the isolated non-working perfused rat heart. Also the effect of insulin (2 mU.ml⁻¹) on D-3-hydroxybutyrate metabolism was investigated in hearts from normal and diabetic rats. The rates of D-3-hydroxybutyrate utilization and oxidation and of acetoacetate production were proportional to D-3-hydroxybutyrate concentration. The utilization of D-3-hydroxybutyrate showed saturation kinetics in hearts from normal and diabetic rats, in the presence and absence of insulin. Acute short-term diabetes augmented D-3-hydroxybutyrate utilization and oxidation at 1.25 and 2.5 mM DL-3-HB, with no significant effect at higher concentrations, but increased acetoacetate production at all investigated concentrations. In hearts from normal rats, insulin enhanced D-3-hydroxybutyrate utilization and oxidation at 2.5, 5, and 10 mM DL-3-HB, but no effect was observed at the lowest (1.25 mM) and highest (16 mM) DL-3-HB concentrations. Insulin had no effect on D-3-hydroxybutyrate metabolism in hearts from diabetic rats. No significant effect of insulin on the rate of acetoacetate production in normal and diabetic states was observed.     

Rapid inhibition by intragastric triolein of the re-activation of glucose utilization and lipogenesis in the mammary gland during the starved-refed transition in lactating rats. Evidence for a direct effect of oral lipid on mammary tissue.

1. Oral administration of triacylglycerol (triolein) to starved/chow-refed lactating rats suppressed the lipogenic switch-on in the mammary gland in vivo. 2. A time-course study revealed that triolein, administered at 30 min after the onset of refeeding, had no influence on lipogenic rate in the mammary gland between 30 and 60 min, but markedly decreased it between 60 and 90 min. Glucose uptake by the mammary gland (arteriovenous difference) increased by 30 min of refeeding, as did lactate production. Between 30 and 90 min glucose uptake remained high in the control animals, but glucose uptake and net C3-unit uptake were decreased in the triolein-loaded animals by 90 min. 3. Triolein increased [glucose 6-phosphate] in the gland and simultaneously decreased [fructose 1,6-bisphosphate], indicative of a decrease in phosphofructokinase activity. This cross-over occurred at 60 min, i.e. immediately before the inhibition of lipogenesis, and by 90 min had reached 'starved' values. 4. Triolein had no effect on plasma [insulin] nor on whole-blood [glucose], [lactate] or [3-hydroxybutyrate]; a small increase in [acetoacetate] was observed. 5. Infusion of the lipoprotein lipase inhibitor, Triton WR1339, abolished the suppression of mammary-gland lipogenesis by triolein and the increase in the [glucose 6-phosphate]/[fructose 1,6-bisphosphate] ratio, suggesting a direct influence of dietary lipid on mammary-gland glucose utilization and phosphofructokinase activity.     

Ketone body utilization in duodenum. Differential effect of fasting on lipogenesis from acetoacetate and 3-hydroxybutyrate

The effect of fasting and refeeding on oxidation, lipogenesis and amino acid synthesis from ketone bodies has been studied in neonatal chick duodenal mucosa. Oxidation and amino acid synthesis were higher from acetoacetate and were stimulated by fasting from both 3-hydroxybutyrate and acetoacetate. On the contrary, lipogenesis was always higher from 3-hydroxybutyrate and fasting reduced lipogenesis rate from acetoacetate (by 66%) but not from 3-hydroxybutyrate. Results suggests the existence of a cytosolic fast-dependent acetoacetyl-CoA synthetase in chick duodenal mucosa which is involved in phospholipid synthesis.     

Fuel utilization in colonocytes of the rat.

In incubated colonocytes isolated from rat colons, the rates of utilization O2, glucose or glutamine were linear with respect to time for over 30 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 30 min. Glutamine, n-butyrate or ketone bodies were the only substrates that caused increases in O2 consumption by isolated incubated colonocytes. The maximum activity of hexokinase in colonic mucosa is similar to that of 6-phosphofructokinase. Starvation of the donor animal decreased the activities of hexokinase and 6-phosphofructokinase, whereas it increased those of glucose-6-phosphatase and fructose-bisphosphatase. Isolated incubated colonocytes utilized glucose at about 6.8 mumol/min per g dry wt., with lactate accounting for 83% of glucose removed. These rates were not affected by the addition of glutamine, acetoacetate or n-butyrate, and starvation of the donor animal. Isolated incubated colonocytes utilized glutamine at about 5.5 mumol/min per g dry wt., which is about 21% of the maximum activity of glutaminase. The major end-products of glutamine metabolism were glutamate, aspartate, alanine and ammonia. Starvation of the donor animal decreased the rate of glutamine utilization by colonocytes, which is accompanied by a decrease in glutamate formation and in the maximum activity of glutaminase. Isolated incubated colonocytes utilized acetoacetate at about 3.5 mumol/min per g dry wt. This rate was not markedly affected by addition of glucose or by starvation of the donor animal. When colonocytes were incubated with n-butyrate, both acetoacetate and 3-hydroxybutyrate were formed, with the latter accounting for only about 19% of total ketones produced.     

Stimulation by 3-hydroxybutyrate of pyruvate carboxylation in mitochondria from rat liver

Isolated rat liver mitochondria incubated in the presence of 3-hydroxybutyrate display a markedly increased rate of pyruvate carboxylation as measured by malate and citrate production from pyruvate. The stimulation was demonstrable both with exogenously added pyruvate, even at saturating concentration, and with pyruvate intramitochondrially generated from alanine. The concentration of DL-3-hydroxybutyrate required for half-maximal stimulation amounted to about 1.5 mM. The intramitochondrial ATP/ADP ratio as well as the matrix acetyl-CoA level was found to remain unchanged by 3-hydroxybutyrate exposure, which, however, lowered the absolute intramitochondrial contents of the respective adenine nucleotides. The effects of 3-hydroxybutyrate were diminished by the concomitant addition of acetoacetate. Moreover, a direct relationship between mitochondrial reduction by proline and the rate of pyruvate carboxylation was observed. The results seem to indicate that the mitochondrial oxidation--reduction state might be involved in the expression of the 3-hydroxybutyrate effect. As to the physiological relevance of the findings, 3-hydroxybutyrate could be shown to activate pyruvate carboxylation in isolated hepatocytes.     

Pseudoketogenesis in the perfused rat heart

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.     

Enhanced ketone body uptake by perfused skeletal muscle in trained rats

Training effect on exercise-induced hyperketonemia was investigated in normal post-absorptive rats subjected to running exercise on a treadmill. Furthermore, rat hindlimb-muscle perfusion was performed to elucidate the mechanism of the training effect. A medium intensity prolonged exercise (running at 15 m/min for 90 min) caused a greater increase in plasma 3-hydroxybutyrate than in acetoacetate both during and after the exercise. Training with medium-intensity exercise (15 m/min) for 90 min 3 times per week for 14 wks or 28 wks caused 1) a reduction of the increase in plasma ketone body (mainly 3-hydroxybutyrate), free fatty acids and glucagon induced by the exercise, and 2) an increase in ketone body (mainly acetoacetate) uptake by perfused skeletal muscle. The present study demonstrates that the reduction of exercise-induced hyperketonemia by prolonged training is caused by increased ketone body utilization in skeletal muscle, and suggested that inhibition of hepatic ketogenesis might also participate in this reduction.     

A simplified model of hypoxic injury in primary cultured rat hepatocytes

Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.     

The effects of ketone bodies,bicarbonate, and calcium on hepatic mitochondrial ketogenesis

The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 mm calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH₂ via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD⁺ from NADH₂ during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis.     

A possible mechanism for the anti-ketogenic action of alanine in the rat

1. The anti-ketogenic effect of alanine has been studied in normal starved and diabetic rats by infusing l-alanine for 90min in the presence of somatostatin (10μg/kg body wt. per h) to suppress endogenous insulin and glucagon secretion. 2. Infusion of alanine at 3mmol/kg body wt. per h caused a 70±11% decrease in [3-hydroxybutyrate] and a 58±9% decrease in [acetoacetate] in 48h-starved rats. [Glucose] and [lactate] increased, but [non-esterified fatty acid], [glycerol] and [3-hydroxybutyrate]/[acetoacetate] were unchanged. 3. Infusion of alanine at 1mmol/kg body wt. per h caused similar decreases in [ketone body] (3-hydroxybutyrate plus acetoacetate) in 24h-starved normal and diabetic rats, but no change in other blood metabolites. 4. Alanine [3mmol/kg body wt. per h] caused a 72±9% decrease in the rate of production of ketone bodies and a 57±8% decrease in disappearance rate as assessed by [3-¹⁴C]acetoacetate infusion. Metabolic clearance was unchanged, indicating that the primary effect of alanine was inhibition of hepatic ketogenesis. 5. Aspartate infusion at 6mmol/kg body wt. per h had similar effects on blood ketone-body concentrations in 48h-starved rats. 6. Alanine (3mmol/kg body wt. per h) caused marked increases in hepatic glutamate, aspartate, malate, lactate and citrate, phosphoenolpyruvate, 2-phosphoglycerate and glucose concentrations and highly significant decreases in [3-hydroxybutyrate] and [acetoacetate]. Calculated [oxaloacetate] was increased 75%. 7. Similar changes in hepatic [malate], [aspartate] and [ketone bodies] were found after infusion of 6mmol of aspartate/kg body wt. per h. 8. It is suggested that the anti-ketogenic effect of alanine is secondary to an increase in hepatic oxaloacetate and hence citrate formation with decreased availability of acetyl-CoA for ketogenesis. The reciprocal negative-feedback cycle of alanine and ketone bodies forms an important non-hormonal regulatory system.     

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

(1) d(-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d- and l-lactate, n-butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD⁺ or NADH. With d-3-hydroxybutyrate as the variable reactant (K_{m app} = 0.26 mM) the inhibition constant of methylmalonate (K_is) was 0.09 mm. (3) The rate of utilization of d-3-hydroxybutyrate (78 μm) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μm methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n-octanoate in the absence of added ADP, methylmalonate (1–3 mm) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 mm) although the inhibition of total ketone formation from octanoate was more severe.     

A double-isotope method for the measurement of ketone-body turnover in the rat. Effect of L-alanine.

The synthesis of 4-3H-labelled ketone bodies, and their use along with 14C-labelled ketone-body precursors, is employed using an 'in vivo' rat infusion model to measure ketone-body turnover. The use of two isotopes is necessary to measure ketone-body turnover when ketogenesis may occur from more than one precursor such as glucose and fatty or amino acids. Requirements of isotopic equivalence in terms of metabolic similarity, valid stoichiometry and the lack of differences in the kinetics of relevant enzymes is demonstrated for the 4-3H- and 14C-labelled ketone bodies. The hypoketonaemic effect of L-alanine is shown by two distinct phases after the administration of L-alanine. During the first 12 min after alanine administration ther was a 50% decrease in acetoacetate and a 30% decrease in 3-hydroxybutyrate production, with no significant change in the utilization of either compound. The hypoketonaemic action of alanine during the following 16 min was primarily associated with an uptake of 3-hydroxybutyrate that was somewhat greater than the increase in its production. There were essentially equivalent decreases in production and utilization of acetoacetate, resulting in no significant net change in the level of this ketone body in the blood.     

Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells

Tumors of peripheral tissues contain low levels of succinyl CoA-acetoacetate CoA transferase activity which is not induced in vitro by prolonged cultivation in 2.5 mM DL-3-hydroxybutyrate. Although this enzyme is considered to be the main agent controlling the extent to which ketone bodies serve as metabolic substrates such tumors metabolize D(-)-3-hydroxy[3(14)C]butyrate to 14CO2. Also addition of 3-hydroxybutyrate and/or acetoacetate reduces the amount of 14CO2 produced from D-[U-14C] glucose suggesting a common metabolic intermediate. These observations can be accounted for by the presence of acetoacetyl-CoA synthetase, an enzyme which is able to synthesize acetoacetyl-CoA directly from acetoacetate, ATP and coenzyme A. This is the first demonstration of this enzyme in tumor tissue. The rate of metabolism of acetoacetate by this enzyme is sufficient to account for the production of CO2 from 3-hydroxybutyrate.     

Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm.

1. Diaphragms from 48h-starved rats were incubated in Krebs-Ringer bicarbonate medium at 37degreesC for 30min and then transferred into new medium and incubated for 1, 2 and 3 h. 2. The amount of free amino acids found at the end of each time of incubation was larger than the amount at the beginning of incubation, indicating that in this system proteolysis is prevailing. 3. The diaphragms was releasing mainly alanine and glutamine into the incubation medium. 4. Within the periods of incubation the release and metabolism of free amino acids was proceeding at a constant rate. 5. Addition of sodium DL-3-hydroxybutyrate decreased the tissue content of several amino acids, among which were tyrosine and phenylalanine, suggesting that proteolysis was decreased by ketone bodies. 6. In the presence of glucose (10mM) and branched-chain amino acids (0.5mM), sodium DL-3-hydroxybutyrate at concentrations of 4 or 6 mM resulted in 30% decrease in tissue alanine content and a 20% decline in alanine release. Release of taurine and glutamine was decreased by 19 and 16% respectively with 6 mM-sodium DL-3-hydroxybutyrate. Addition of sodium acetoacetate (1-3mM) also resulted in a 20-35% decrease in tissue content of alanine, glutamine and taurine and in a 15-24% decrease of alanine and glutamine release. Smaller decreases (less than 15%) in the release of glycine, threonine, proline, serine and aspartate were also observed in the presence of sodium DL-3-hydroxybutyrate or sodium acetoacetate. 7. Substitution of pyruvate (1.0mM) for glucose in the presence of acetoacetate restored alanine and glutamine production to control values. In the presence of acetoacetate, pyruvate also increased the tissue content of aspartate by 77% and decreased the tissue content of glutamate by 30%. 8. It is suggested that in diaphragms from starved rats, ketone bodies (a) in the absence of other substrates inhibit protein catabolism and (b) in the presence of glucose and branched-chain amino acids decrease alanine and glutamine production, by inhibiting glycolysis.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part III. Multiresponse kinetic studies

A lipase from Aspergillus niger immobilized by adsorption on microporous, polypropylene hollow fibers was used to effect the continuous hydrolysis of the glycerides of butter oil at 40 degrees C and pH 7.0. The effluent concentrations of 10 different free fatty acid products were measured by highperformancee liquid chromatography (HPLC). Multiresponse nonlinear regression methods were used to fit the data to a multisubstrate rate expression derived from a Ping Pong Bi Bi mechanism in which the rate-controlling step is deacylation of the lipase. Thermal deactivation of the enzyme was also included in the mathematical model of reactor performance. A postulated normal distribution of v(max) with respect to the chain length of the fatty acid (with an additive correction for the degree of unsaturation) was tested for statistical significance. The model is useful for predicting the free fatty acid profile of the lipolyzed butteroil product over a wide range of flow rates.     

Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol.

The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.     

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30-40 mM. The various relevant physical parameters such as w₀ (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.     

The effect of fasting on D-3-hydroxybutyrate metabolism in the perfused rat heart

Summary The effects of fasting for 24 h and 48 h on D-3-hydroxybutyrate utilization and acetoacetate, L-lactate and pyruvate production by the isolated non-working perfused rat heart were investigated over a wide range of DL-3-HB concentrations. D-3-HB utilization is concentration dependent and shows saturation kinetics, D-3-HB oxidation is correlated with D-3-HB concentration. Acetoacetate production is proportional to DL-3-HB concentration. L-lactate production is proportional to DL-3-HB concentration up to 5 mM following a 24h fast and up to 10 mM after a 48h fast, but further increase in DL-3-HB concentration decreases the rate of lactate production. Fasting enhances D-3-HB utilization at 16 mM DL-3-HB by 16% and 25% in 24 h and 48 h fast respectively, but has no significant effect at lower concentration. Fasting has no effect on acetoacetate production. Fasting for 48 h doubled the half-saturation concentration (Ku) without significant change in the maximum rate of utilization (Vu) of D-3-HB.