Circulating glucose, insulin and ketone bodies and enzymes of ketone body utilization in brain mitochondria from suckling rats treated with high L-thyroxine doses

The neo-T4 syndrome was induced by subcutaneous administration of a total dose of (150 micrograms) L-thyroxine (T4) to rats from their first day of live. Neo-T4 animals and their controls were sacrificed at 2, 4, 8, 11, 14, 22 and 25 days of age. A decrease in body weight was observed from the second day of life, and a decrease in brain weight from the eighth day of life in the neo-T4 animals. Blood glucose and plasma insulin levels were decreased from 2nd day through 22nd day of life. Total plasma ketone bodies and beta-OH butyrate levels increased in the neo-T4 animals with respect to controls. until 8th day, although acetoacetate increased only until 4th day. The activity of key enzymes in the ketone bodies utilization pathway (3-hydroxybutyrate dehydrogenase, 3-oxoacid CoA-transferase and acetoacetyl-CoA thiolase) were also measured in the animals brain. We found an activation of 3-hydroxybutyrate dehydrogenase until 11th day and 3-oxoacid CoA-transferase until 14th day, but no change in acetoacetyl CoA-thiolase was observed. Ketone bodies play a key role as energy substrates and precursors of brain lipids during the period of intense growth and myelination of the CNS. Considering the alterations described in this paper it seems that neo-T4 syndrome could be an interesting model for studying metabolism of those substances in brain.     

Factors influencing the utilization of ketone bodies by mouse adipose tissue

Factors influencing the utilization of ketone bodies by mouse adipose tissue in vitro were studied. Epididymal fat pads can oxidize DL-Beta-hydroxybutyrate-3-(14)C and acetoacetate-3-(14)C to (14)CO(2) as well as convert these compounds to fatty acid-(14)C. An increased output of (14)CO(2) from Beta-hydroxybutyrate-3-(14)C was noted in response to glucose plus insulin, succinate, oxaloacetate, L-asparate, and L-malate. Fatty acid synthesis from Beta-hydroxybutyrate was enhanced by glucose plus insulin, L-aspartate, L-malate, oxaloacetate, and citrate. Nicotinamide stimulated the oxidation of Beta-hydroxybutyrate but not of acetoacetate to CO(2), and did not affect fatty acid synthesis from either ketone body. Nicotinamide increased NAD(+) and NADP(+) levels in epididymal fat pads without affecting the concentration of NADH and NADPH. "Superlipogenesis" caused by fasting the mice for 48 hr and re-feeding them for 24 hr sharply enhanced CO(2) output and lipogenesis from Beta-hydroxybutyrate. The activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, NADP-malic dehydrogenase, and citrate cleavage enzyme from mouse adipose tissue were increased during "superlipogenesis." Free fatty acid release by epididymal fat pads in vitro was slightly increased by Beta-hydroxybutyrate. The relationship of ketone body metabolism and lipogenesis in adipose tissue is discussed.     

Circadian rhythm of ketone bodies in the blood of fasting rats

SPF male Wistar rats were kept under standard conditions with a light: dark schedule of 12:12 h. The total ketone body concentration was determined in the blood, and the non-esterified fatty acid level in the serum, of fed rats and of animals which had fasted 24 and 48 h. The amount of ketone bodies in fed rats rose in the second half of the light period and fell with the onset of the dark period. After a 24 h fast, the amount of ketone bodies in the blood rose, but the basic characteristics of the curve and the rhythm remained the same as in fed animals. After a 48 h fast, the mean ketone body concentration was decoupled, a significant phase shift occurred and the rhythm was lost. No relationship between the oscillations of the total ketone body concentration in the blood and the oscillations of the serum non-esterified fatty acid level was found.     

Effects of methylmalonate and propionate on uptake of glucose and ketone bodies in vitro by brain of developing rats

Methylmalonate (MMA) and propionate effects on glucose and ketone body uptake in vitro by brain of fed and 30-hour-fasted 15-day-old rats were studied. In some experiments cerebrum prisms were incubated in the presence of glucose and either MMA or propionate in Krebs-Ringer bicarbonate buffer, pH 7.0. In others, the incubation medium contained beta-hydroxybutyrate (HBA) or acetoacetate (AcAc) instead of glucose. We verified that MMA increased glucose uptake by brain of fasting animals, whereas propionate had no effect. In addition, MMA diminished HBA but not AcAc incorporation into brain prisms, whereas propionate provoked a diminished utilization of both ketone bodies by brain. The in vitro effect of MMA and propionate on brain and liver beta-hydroxybutyrate dehydrogenase activity was also investigated. It was shown that MMA but not propionate significantly inhibited this activity. Rats were also injected subcutaneously three times with a MMA buffered solution, and the in vivo effects of MMA on the above-mentioned parameters assessed. Results from these experiments confirmed the previously found in vitro MMA effects. Methylmalonic acidemic patients accumulate primarily methylmalonate and secondarily propionate and other metabolites in their tissues at levels comparable to those we used in our assays. Most patients who survive early stages of the disease show a variable degree of neuromotor delay. Since glucose and sometimes ketones are the vital substrates for brain metabolism, it is possible that our findings may contribute to a certain extent to an understanding of the biochemical basis of mental retardation in these patients.     

The utilization of ketone bodies by the interscapular brown adipose tissue of the rat

The activities of 3-oxo acid-CoA transferase (EC 2.8.3.5, 13-15 micromol/min per g) and acetoacetyl-CoA thiolase (EC 2.3.1.9, 18-21 micromol/min per g) in interscapular brown adipose tissue of the rat are comparable to the activities reported for heart and kidney. The incorporation of D-3-hydroxy[3-14C]butyrate into lipid in vivo was about 30-fold higher in interscapular brown adipose tissue than in white adipose tissue of virgin rats. In lactating rats, the mammary gland was the major site of ketone body incorporation into lipid and incorporation of D-3-hydroxy-[3-14C]butyrate into lipid in brown adipose tissue was lower than in virgin rats. After an oral load of medium chain triacylglycerol, which inhibits lipogenesis in lactating mammary gland, the incorporation of ketone bodies into lipid was decreased in mammary gland but increased in brown adipose tissue. The rate of oxidation of D-3-hydroxy[3-14C]butyrate by brown adipose tissue slices in vitro was higher than the rate of incorporation into lipid.     

The estimation of rates of utilization of glucose and ketone bodies in the brain of the suckling rat using compartmental analysis of isotopic data

The brains of 18-day-old rats utilize glucose and ketone bodies. The rates of acetyl-CoA formation from these substrates and of glycolysis were determined in vivo from the labelling of intermediary metabolites after intraperitoneal injection of d-[2-(14)C]glucose, l(+)-[3-(14)C]- and l(+)-[U-(14)C]-lactate and d(-)-3-hydroxy[(14)C]butyrate. Compartmental analysis was used in calculating rates to allow for the rapid exchange of blood and brain lactate, the presence in brain of at least two pools each of glucose and lactate, and the incomplete equilibration of oxaloacetate with aspartate and of 2-oxoglutarate with glutamate. Results were as follows. 1. Glucose and ketone bodies labelled identical pools of tricarboxylate-cycle metabolites, and were in every way alternative substrates. 2. The combined rate of oxidation of acetyl-CoA derived from pyruvate (and hence glucose) and ketone bodies was 1.05mumol/min per g. 3. Ketone bodies contributed 0.11-0.53mumol/min per g in proportion to their concentration in blood, with a mean rate of 0.30mumol/min per g at 1.24mm. 4. Pyruvate and ketone bodies were converted into lipid at 0.018 and 0.008mumol/min per g respectively. 5. Glycolysis, at 0.48mumol/min per g, was more rapid in most rats than pyruvate utilization by oxidation and lipid synthesis, resulting in a net output of lactate from brain to blood. 6. Rates of formation of brain glutamate, glutamine and aspartate were also measured. Further information on the derivation of the models has been deposited as Supplementary Publication SUP 50034 (18 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

The interconversion and disposal of ketone bodies in untreated and injured post-absorptive rats

[3-(14)C]Acetoacetate and beta-hydroxy[3-(14)C]butyrate were used to investigate the kinetics of ketone body metabolism in rats 3h after bilateral hind-limb ischaemia and in controls, both groups being in the post-absorptive state and in a 20 degrees C environment. Calculations were carried out as described by Heath & Barton (1973) and the following conclusions were reached. 1. In both injured and control rats, the rates of irreversible disposal (extrahepatic utilization) of beta-hydroxybutyrate and acetoacetate were proportional within experimental error to their blood concentrations up to at least 0.4mm (the maximum found in these rats), implying that they were determined, via these concentrations, by the rates of production by the liver. 2. Conversion of blood beta-hydroxybutyrate into blood acetoacetate took place mainly in the liver, but the reverse process occurred mainly in extrahepatic tissues. 3. The ;metabolic clearance rate' (the volume of blood which, if completely cleared of substrate in unit time, would give a disposal rate equal to that in the whole animal) was calculated for beta-hydroxybutyrate and acetoacetate. Comparison with the cardiac output showed that in control rats the proportion of circulating beta-hydroxybutyrate extracted was lower than that of acetoacetate, clearance of which appeared almost complete. After injury both metabolic clearance rates decreased, probably because of the lower cardiac output. 4. After injury, because the average blood concentrations of ketone bodies, especially acetoacetate, were higher, the mean total rate of disposal also increased. Assuming complete oxidation, the mean contribution of ketone bodies to the whole body O(2) consumption rose from 7 to 15%.     

The differential effect of insulin in vivo on the peripheral utilization of glucose and ketone bodies in the rat.

This in vivo study assessed the immediate effects of insulin on glucose and ketone body utilization in the fed, fasted, and diabetic ketoacidotic rat. The experimental design consisted of the functional removal of the liver (the site of glucose and ketone body production) and the pancreas from the anesthetized animals. This surgical procedure permitted the assessment of the effect of exogenously administered insulin on the rate of both glucose and ketone body utilization by peripheral tissues. Insulin exerted hypoglycemic activity in all three metabolic states studied. This hypoglycemic activity contrasted to the lack of demonstrable effect of this hormone on ketone body uptake by peripheral tissues. It was concluded that in the rat, the immediate effect of insulin, i.e. within 30 minutes, was to exert hypoglycemic activity without simultaneous hypoketoniemic activity.     

Effect of hyperphenylalaninaemia on lipid synthesis from ketone bodies by rat brain.

The effect of hyperphenylalaninaemia on the metabolism of ketone bodies in vivo and in vitro by developing rat brain was investigated. The incorporation in vivo of [14C]acetoacetate into cerebral lipids was decreased by both chronic (for 3 days) and acute (for 6h) hyperphenylalaninaemia induced by injecting phenylalanine into 1-week-old rats. In studies in vitro it was observed that the incorporation of the radioactivity from [14C]acetoacetate and 3-hydroxy[14C]butyrate into cerebral lipids was inhibited by phenyl-pyruvate, but not by phenylalanine. Phenylpyruvate also inhibited the incorporation of 3H from 3H2O into lipids by brain slices metabolizing either 3-hydroxybutyrate or acetoacetate in the presence of glucose. These findings suggest that the decrease in the incorporation in vivo of [14C]acetoacetate into cerebral lipids in hyperphenylalaninaemic rats is most likely caused by phenylpyruvate and not by phenylalanine. Phenylpyruvate as well as phenylalanine had no inhibitory effects on ketone-body-catabolizing enzymes, namely 3-hydroxybutyrate dehydrogenase, 3-oxo acid CoA-transferase and acetoacetyl-CoA thiolase, in rat brain. Phenylpyruvate but not phenylalanine inhibited the activity of the 2-oxoglutarate dehydrogenase complex from rat and human brain. These findings suggest that the metabolism of ketone bodies is impaired in brains of untreated phenylketonuric patients, and in turn may contribute to the diminution of mental development and function associated with phenylketonuria.