Concerning the decreased D-3-hydroxybutyrate dehydrogenase activity in the liver and heart of hyperthyroid rats

Summary Whereas in rat liver mitochondria the hyperthyroid state causes an increase both in fatty acid unsaturation and in the Ea of D-3-hydroxybutyrate dehydrogenase and a decrease in phase transition temperature, in hyperthyroid rat heart mitochondria these changes are negligible. D-3-hydroxybutyrate dehydrogenase in both the liver and the heart mitochondria of hyperthyroid rats is reduced by about 35% [l2] but this reduction is not due to changes in membrane fluidity in either tissue. Hypothyroidism, on the other hand, affects BDH activity in neither heart nor liver.Abbreviations BDH D-3-hydroxybutyrate dehydrogenase - PTU 6n-propyl-2-thiouracil - T₃,3,3 5-L-triiodothyronine - Tm temperature phase transition - Ea apparent activation energy     

The effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria

Effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria is described. State 3 respiration is inhibited by acetoacetate, while state 4 respiration is not inhibited, though cytochrome c reduction was decreased. Acetoacetate is also non-competitive inhibitor of 3-hydroxybutyrateoxidase and 3-hydroxybutyrate dehydrogenase activity in frozen-thawed mitochondria. The results are discussed in terms of the thermodynamic hypothesis and control strength method.     

Ketone body and fatty acid metabolism in sheep tissues. 3-Hydroxybutyrate dehydrogenase, a cytoplasmic enzyme in sheep liver and kidney

1. 3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) activities in sheep kidney cortex, rumen epithelium, skeletal muscle, brain, heart and liver were 177, 41, 38, 33, 27 and 17μmol/h per g of tissue respectively, and in rat liver and kidney cortex the values were 1150 and 170 respectively. 2. In sheep liver and kidney cortex the 3-hydroxybutyrate dehydrogenase was located predominantly in the cytosol fractions. In contrast, the enzyme was found in the mitochondria in rat liver and kidney cortex. 3. Laurate, myristate, palmitate and stearate were not oxidized by sheep liver mitochondria, whereas the l-carnitine esters were oxidized at appreciable rates. The free acids were readily oxidized by rat liver mitochondria. 4. During oxidation of palmitoyl-l-carnitine by sheep liver mitochondria, acetoacetate production accounted for 63% of the oxygen uptake. No 3-hydroxybutyrate was formed, even after 10min anaerobic incubation, except when sheep liver cytosol was added. With rat liver mitochondria, half of the preformed acetoacetate was converted into 3-hydroxybutyrate after anaerobic incubation. 5. Measurement of ketone bodies by using specific enzymic methods (Williamson, Mellanby & Krebs, 1962) showed that blood of normal sheep and cattle has a high [3-hydroxybutyrate]/[acetoacetate] ratio, in contrast with that of non-ruminants (rats and pigeons). This ratio in the blood of lambs was similar to that of non-ruminants. The ratio in sheep blood decreased on starvation and rose again on re-feeding. 6. The physiological implications of the low activity of 3-hydroxybutyrate dehydrogenase in sheep liver and the fact that it is found in the cytoplasm in sheep liver and kidney cortex are discussed.     

Regulators of 3-hydroxybutyrate dehydrogenase activity in rat liver mitochondria

Effects of numerous organic acids on the 3-hydroxybutyrate dehydrogenase activity were studied in isolated rat liver mitochondria with nonspecific permeability. Amino acids, most of citric acid cycle intermediates, lactate, maleate, acetate, glycerol-3-phosphate, urea, palmitate, and phosphoenolpyruvate plus ADP were shown to modify the enzyme activity insignificantly. The inhibitory effect of pyruvate seems to be a result of the concomitant cytosolic lactate dehydrogenase activity, and the effect of oxaloacetate is that of the mitochondrial matrix malate dehydrogenase activity. Malonate proves to be a competitive inhibitor of the 3-hydroxybutyrate dehydrogenase activity, enzyme affinity for malonate being the same irrespective of the source or purification of the preparation.     

Metabolism of S-3-hydroxybutyrate in the perfused rat liver

The metabolism of millimolar concentrations of S-3-hydroxybutyrate (the unnatural enantiomer) has been studied in perfused livers from fed and starved rats. Protocols were designed to test whether S-3-hydroxybutyrate is metabolized in the cytosol or in the mitochondria via a racemase, a dehydrogenase, or a ligase. Our data show that only a minor fraction of S-3-hydroxybutyrate metabolism could occur via L-3-hydroxyacid dehydrogenase. Most of the metabolism of S-3-hydroxybutyrate proceeds via mitochondrial activation. In rat liver, S-3-hydroxybutyrate is converted to physiological ketone bodies (i.e., R-3-hydroxybutyrate, acetoacetate, acetone), lipids, and CO2. Carbons from S-3-hydroxybutyrate are transferred from the mitochondria to the cytosol mostly via citrate and the citrate cleavage pathway.     

Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.     

Ketone-body metabolism in hyperthyroid rats: reduced activity of D-3-hydroxybutyrate dehydrogenase in both liver and heart and of succinyl-coenzyme A: 3-oxoacid coenzyme A-transferase in heart

The specific activity of D-3-hydroxybutyrate dehydrogenase is reduced by about a third in liver and heart mitochondria of hyperthyroid rats. State 3 respiration is also reduced in isolated mitochondria from the same animals when DL-3-hydroxybutyrate is the substrate. Determination of the kinetic parameters of the membrane-bound D-3-hydroxybutyrate dehydrogenase in liver of hyperthyroid rats reveals a decreased in maximal velocity (Vmax). The Michaelis and dissociation constants of NAD+ and D-3-hydroxybutyrate are also significantly influenced, thus indicating that both the affinity and the binding of this enzyme toward its substrates are affected. In hyperthyroid rats a significant ketone-body increase is found in both liver and heart: in blood, an almost doubled concentration can be measured. At the same time, in heart mitochondria of these animals the activity of succinyl-coenzyme A: 3-oxoacid coenzyme A-transferase is significantly reduced. The decrease in both D-3-hydroxybutyrate dehydrogenase and 3-oxoacid coenzyme A-transferase associated with the increase in ketone bodies supports the suggestion that there is a lower utilization of these compounds by peripheral tissues. In the blood of hyperthyroid rats a higher D-3-hydroxybutyrate/acteoacetate ratio is also found, probably resulting from a selective utilization of the two compounds in this pathological state.     

Studies on the metabolism of glycolyl-CoA

Glycolyl-CoA can be formed during the course of the beta-oxidation by rat liver mitochondria of 4-hydroxybutyrate. The existence of this beta-oxidation has been previously supported by the occurrence of 4-hydroxybutyrate and its beta-oxidation catabolites in urine from patients with 4-hydroxybutyric aciduria, an inborn error of gamma-aminobutyric acid metabolism due to the deficiency of succinic semialdehyde dehydrogenase. The characteristics of the mitochondrial beta-oxidation of 4-hydroxybutyrate were, in rat liver, compared with those of the mitochondrial beta-oxidation of butyrate. The inhibition by malonate of the oxidation of 4-hydroxybutyrate was about twofold weaker than that of oxidation of butyrate, whereas both oxidations were abolished by preincubating the mitochondria with 1 mM valproic acid, a known inhibitor of mitochondrial beta-oxidation. Mitochondria from rat kidney cortex were demonstrated to catalyse, as previously shown for hepatic mitochondria, the carnitine-dependent oxidation of 12-hydroxylauroyl-CoA-omega-Hydroxymonocarboxylyl-CoAs are thus concluded to be precursors of glycolyl-CoA also in rat kidney cortex. In addition, 3-hydroxypyruvate was found to be a precursor of glycolyl-CoA, since it was oxidized by bovine heart pyruvate dehydrogenase with a cofactor requirement similar to that of pyruvate oxidation. Glycolyl-CoA was a substrate of carnitine acetyltransferase (pigeon breast muscle). Pig heart citrate synthase was capable of catalyzing the condensation of glycolyl-CoA with oxaloacetate. The product of this reaction induced low NADH production rates dependent on the addition of porcine heart aconitase and isocitrate dehydrogenase.     

Is there Ca2+(Sr2+)-3-hydroxybutyrate symport in rat-liver mitochondria? A reappraisal

The observation in this laboratory that respiration and Sr2+ import were stimulated by the addition of 3-hydroxybutyrate to suspensions of N-ethylmaleimide-treated mitochondria respiring in state 6, after the addition of Sr2+, in a sucrose medium containing choline as substrate, led to the proposal by Moyle and Mitchell [(1977) FEBS Lett. 84, 135-140] that there is a Ca2+(Sr2+)-3-hydroxybutyrate symporter in rat liver mitochondria. However, experiments described in the present paper support a different interpretation. Under the conditions of the experiments by Moyle and Mitchell, the rate of respiration and the poise of Sr2+ accumulation are mainly limited, not by delta mu H+, but by lack of respiratory substrate. Even though N-ethylmaleimide is a potent inhibitor of 3-hydroxybutyrate dehydrogenase, we have found that, somewhat surprisingly, under the special conditions of these experiments, sufficient 3-hydroxybutyrate dehydrogenase activity remains available to account for the 3-hydroxybutyrate-dependent respiratory stimulation and Sr2+ import.     

Modified oscillation behavior and decreased D-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria

One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d-3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d-3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa₃ content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b, c₁, and c content, and the P:O ratio for mitochondria oxidizing d-3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d-3-hydroxybutyrate oxidation by intact mitochondria to their normal values.     

Expression of R-3-hydroxybutyrate dehydrogenase, a ketone body converting enzyme in heart and liver mitochondria of ruminant and non-ruminant mammals.

1. The properties of rat liver and bovine heart R-3-hydroxybutyrate dehydrogenase (BDH) have been extensively studied in the past 20 years, but little is known concerning the biogenesis and the regulation of this dehydrogenase over different species. 2. In addition, controversial results were often reported concerning the activity, the level and the subcellular location of this enzyme in ruminants. 3. BDH activity found in liver and kidney mitochondria from ruminants (cow and sheep) is low, while it is much higher in rat. 4. However, the enzyme activity is detected in microsomes and in cytosol of liver and of kidney cells from ruminants. These activities are not correlated to ketonaemia level. 5. Although low BDH activity is detected in liver mitochondria from ruminants; the bovine liver BDH gene seems to be translated since BDH can be immunodetected by using an antiserum raised against bovine heart BDH. 6. Beside this, the good cross-reactivity between heart BDH and liver BDH suggests their high level of homology in ruminants.     

Enzymes of l-(+)-3-hydroxybutyrate metabolism in the rat

The three enzymes required for the production and utilization of l-(+)-3-hydroxybutyrate were sought in various tissues of the rat. All tissues examined contained substantial amounts of (No. 1) l-(+)-3-hydroxybutyryl CoA dehydrogenase (EC 1.1.1.35). The specific activity of (No. 2) l-(+)-3-hydroxybutyryl CoA deacylase (EC 3.1.2) was highest in liver (3.8 mU/mg in mitochondrial matrix (1 U = 1 μmol/min). Brain, heart, and skeletal muscle contained < 20% of this activity. The chromatography of liver mitochondrial “matrix” preparations on DEAE-cellulose resolved the deacylase into two peaks. Peak I hydrolyzed 2- or 3- carbon acylCoA esters more efficiently than l-(+)-3-hydroxybutyrate CoA, while Peak II activity was highest using l-(+)-3-hydroxybutyryl CoA. The K_m(app) for Peak II deacylase with l-(+)-3-hydroxybutyryl CoA was 19 μm. Acyl CoA synthetase (EC 6.2.1.2) (No. 3) was assayed with sorbate (sorboyl CoA ligase) or l-(+)-3-hydroxybutyrate (l-(+)-3-hydroxybutyryl CoA ligase). The highest specific activity for l-(+)-3-hydroxybutyryl CoA ligase was associated with brain mitochondria (8.3 mU/mg). In the “matrix” fraction of rat liver mitochondria the activities of these two acyl CoA synthetases were distinguished chromatographically and by their stability at various pH values. Heart and skeletal muscle mitochondria contained <10% of the liver activities of both ligases. These data implicate the liver as a site of l-(+)-3-hydroxybutyrate production.     

The influence of fasting on chicken liver metabolites, enzymes and mitochondrial respiration

Chicken liver mitochondria were isolated in relatively pure form as indicated by electron microscopy and marker enzyme assay. The rate of respiration, respiratory control index and ADP/O ratios with several different substrates indicated that chicken liver mitochondria are more uncoupled than rat liver mitochondria. Chickens have ten-fold higher malate concentrations in liver than do rats, 2-oxoglutarate was also more abundant in chicken livers. Fasted birds had a five-fold increase in beta-hydroxybutyrate as compared with fed birds; whereas malate and lactate concentrations decreased. Fasted birds had increased levels of isocitrate dehydrogenase (NADP dependent) and lactate dehydrogenase in the cytosol, and increased malate dehydrogenase (NAD dependent), isocitrate dehydrogenase (NADP dependent) and malic enzyme activities in the mitochondria.     

Diurnal changes in succinate and D-3-hydroxybutyrate dehydrogenase activities of rat liver mitochondria after chronic alcohol consumption and withdrawal

1. The succinate dehydrogenase (SDH) and D-3-hydroxybutyrate dehydrogenase (HBDH) activities were measured over a 24-hr period in rat liver mitochondria after chronic alcohol ingestion and withdrawal. 2. The diurnal patterns of both the enzyme activities were shown to change after alcohol consumption, with 64-66% decrease in the daily mean levels. 3. The diurnal rhythms of the SDH and HBDH activities are partially restored 24-72 hr after alcohol withdrawal. 4. There was no correlation between changes in both the enzyme activities and the NAD+/NADH ratio of liver mitochondria from control, ethanol-fed and withdrawn rats over the day.     

Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats.

Heart mitochondria from chronically diabetic rats ('diabetic mitochondria'), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats ('normal mitochondria'). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.     

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.     

Evidence for the existence of enzymatic variants of beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria.

A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at ?20°. The brain dehydrogenase was found to have a larger K_m (apparent) for the 3-acetylpyridine analog of NAD⁺, and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD⁺ and β-hydroxybutyrate. The K_m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.     

Enzyme activities of isolated hepatic peroxisomes from genetically lean and obese male mice.

Hepatic peroxisomes have been isolated on isopycnic sucrose gradients from white mice [HA(ICR)] and lean and obese (C57BL/6J) mice. Nearly all of the catalase activity was in the peroxisomal fraction. The activity for β-oxidation of palmitoyl-CoA was about threefold higher per milligram of protein in the isolated peroxisomal fraction or per gram of liver from the obese mouse compared to its lean littermates. Glycerol-3-phosphate dehydrogenase activity also was higher in the peroxisomes and cytoplasm of the obese mouse. The matrix enzymes of the organelles, catalase and urate oxidase of the peroxisome and glutamate dehydrogenase of the mitochondria, had similar activities per gram of liver from either lean or obese mice. Membrane components, NADPH: cytochrome c reductase of the microsomes and β-hydroxybutyrate dehydrogenase of the mitochondria, had lower activities in the obese mouse in inverse proportion to the larger size of the liver.     

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.