Acetoacetate, d-3-hydroxybutyrate and glucose utilization by capillaries isolated from developing rat brain

Isolated cerebral capillaries from developing rats utilize glucose as well as ketone bodies essentially for oxidative metabolism. However, CO₂ production from [U-¹⁴C]glucose was significantly greater than from ketone bodies (except at 5 mM). Ketone body utilization (in the presence of 5 mM glucose in the incubation medium) was concentration-dependent (up to 5 mM). Lipid synthesis from ketone bodies was comparable to that from glucose up to 1 mM. At concentrations 1 mM, acetoacetate incorporation into total lipids and fatty acids was higher than other substrates, however, this difference was statistically significant only at 5 mM. Incorporation of substrates into sterols was very low (> 1 pmol/h/mg protein).     

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

CEREBRAL BLOOD FLOW AND CEREBRAL METABOLIC RATES FOR OXYGEN, GLUCOSE, AND KETONE BODIES IN NEWBORN DOGS

Cerebral blood flow (CBF) and the cerebral metabolic rates for oxygen, glucose, acetoacetate, β-hydroxybutyrate and lactate were measured in 1- to 5-day old Beagle dogs under nitrous oxide anesthesia. CBF was determined by ¹³³Xe washout with mechanically integrated blood samples withdrawn simultaneously from a femoral artery and from the posterior one-third of the superior sagittal sinus. CBF and CMRO₂ in normocapnia (PaCO₂ 40 × 1 mm Hg) were 48 × 5 ml/100 g/min and 2.15 ml/100 g/min, respectively. There was a positive, linear relationship between CBF and PaCO₂, calculated for PaCO₂ values ranging from 26 to 70 mm Hg. Induced hypocapnia (PaCO₂ 31 × 1 mm Hg) or hypercapnia (PaCO₂ 58 × 2 mm Hg) did not alter the CMRO₂. Glucose and acetoacetate were taken up by the brain at all PaCO₂ levels examined; however, the cerebral uptake of glucose always exceeded the combined uptake of ketone bodies by more than a factor of ten. The cerebral metabolic rate for glucose (94.6 × 3.6 μmol/100 g/min) more than accounted for overall cerebral oxygen consumption, and yielded an oxygen:glucose ratio (mol:mol) of 5.1. Thus, as in adult animals, PaCO₂ is an important regulator of cerebral blood flow in puppies, and glucose is the major substrate for oxidative energy production in the immature brain. The oxidation of ketone bodies by the newborn dog brain accounts for not more than 6% of the in vivo cerebral oxygen consumption.     

β-Hydroxybutyrate is the preferred substrate for GABA and glutamate synthesis while glucose is indispensable during depolarization in cultured GABAergic neurons

The ketogenic diet has multiple beneficial effects not only in treatment of epilepsy, but also in that of glucose transporter 1 deficiency, cancer, Parkinson’s disease, obesity and pain. Thus, there is an increasing interest in understanding the mechanism behind this metabolic therapy. Patients on a ketogenic diet reach high plasma levels of ketone bodies, which are used by the brain as energy substrates. The interaction between glucose and ketone bodies is complex and there is still controversy as to what extent it affects the homeostasis of the neurotransmitters glutamate, aspartate and GABA. The present study was conducted to study this metabolic interaction in cultured GABAergic neurons exposed to different combinations of ¹³C-labeled and unlabeled glucose and β-hydroxybutyrate. Depolarization was induced and the incorporation of ¹³C into glutamate, GABA and aspartate was analyzed. The presence of β-hydroxybutyrate together with glucose did not affect the total GABA content but did, however, decrease the aspartate content to a lower value than when either glucose or β-hydroxybutyrate was employed alone. When combinations of the two substrates were used ¹³C-atoms from β-hydroxybutyrate were found in all three amino acids to a greater extent than ¹³C-atoms from glucose, but only the ¹³C contribution from [1,6-¹³C]glucose increased upon depolarization. In conclusion, β-hydroxybutyrate was preferred over glucose as substrate for amino acid synthesis but the total content of aspartate decreased when both substrates were present. Furthermore only the use of glucose increased upon depolarization.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Interrelations between C4 Ketogenesis, C5 Ketogenesis, and Anaplerosis in the Perfused Rat Liver

We investigated the interrelations between C₄ ketogenesis (production of β-hydroxybutyrate + acetoacetate), C₅ ketogenesis (production of β-hydroxypentanoate + β-ketopentanoate), and anaplerosis in isolated rat livers perfused with ¹³C-labeled octanoate, heptanoate, or propionate. Mass isotopomer analysis of C₄ and C₅ ketone bodies and of related acyl-CoA esters reveal that C₄ and C₅ ketogenesis share the same pool of acetyl-CoA. Although the uptake of octanoate and heptanoate by the liver are similar, the rate of C₅ ketogenesis from heptanoate is much lower than the rate of C₄ ketogenesis from octanoate. This results from the channeling of the propionyl moiety of heptanoate into anaplerosis of the citric acid cycle. C₅ ketogenesis from propionate is virtually nil because acetoacyl-CoA thiolase does not favor the formation of β-ketopentanoyl-CoA from propionyl-CoA and acetyl-CoA. Anaplerosis and gluconeogenesis from heptanoate are inhibited by octanoate. The data have implications for the design of diets for the treatment of long chain fatty acid oxidation disorders, such as the triheptanoin-based diet.The regulation of the metabolism of C₄ ketone bodies, i.e. β-hydroxybutyrate (BHB)² and acetoacetate (AcAc) has been extensively investigated in vivo in isolated livers, hepatocytes, and subcellular preparations (for reviews, see Refs. 1–4). In contrast, very little information is available on the metabolism of C₅ ketone bodies, i.e. β-hydroxypentanoate (BHP) and β-ketopentanoate (BKP), which are known in the clinical literature as 3-hydroxyvalerate and 3-ketovalerate (5, 6). The C₅ ketone bodies are formed in liver from the partial oxidation of odd-chain fatty acids (see Fig. 1, center column). C₅ ketogenesis uses the same enzymes of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) cycle as C₄ ketogenesis. The counterpart of HMG-CoA in C₅ ketogenesis is 3-hydroxy-3-ethylglutaryl-CoA (HEG-CoA). We only found one report on the formation of [¹⁴C]HEG-CoA in liver extract incubated with propionyl-CoA and [1-¹⁴C]acetyl-CoA (7).Open in a separate windowFIGURE 1.Scheme of C₄ ketogenesis and C₅ ketogenesis in the liver. Numbers refer to the following enzymes: 3-ketoacyl-CoA thiolase (1), HMG-CoA synthase (2), HMG-CoA lyase (3), and β-hydroxybutyrate dehydrogenase (4). The figure also shows the link between propionyl-CoA and the CAC via anaplerosis.Because odd-chain fatty acids are absent from the diet of non-ruminant mammals, body fluids contain only traces of C₅ ketone bodies. However, C₅ ketone bodies and hydroxyethylglutarate are found in body fluids of patients with disorders of the anaplerotic pathway, propionyl-CoA → methylmalonyl- CoA → succinyl-CoA, such as deficiency in propionyl-CoA carboxylase and methylmalonyl-CoA mutase as well as biotin or vitamin B12 deficiency (5, 6, 8). The formation of C₅ ketone bodies in these pathological states involves either the conversion of propionyl-CoA to BKP-CoA via 3-ketoacyl-CoA thiolase (Fig. 1, reaction 1) or the β-oxidation of odd-chain fatty acids synthesized in these patients (9) using propionyl-CoA as a primer (10).Like their C₄ counterparts, the C₅ ketone bodies are interconverted by mitochondrial BHB dehydrogenase (11). In peripheral tissues, C₅ ketone bodies are converted to propionyl-CoA (which is anaplerotic) + acetyl-CoA via 3-oxoacid-CoA transferase (12) and 3-ketoacyl-CoA thiolase. Peripheral tissues have a high capacity to utilize exogenous C₅ ketone bodies (13), especially heart, kidney, and brain, which have high activities of 3-oxoacid-CoA transferase (14, 15).Our interest in C₅ ketone body metabolism arose from an ongoing clinical trial where patients with long chain fatty acid oxidation disorders are treated with a diet containing triheptanoin (16, 17) instead of the classical treatment with the even-chain triglyceride trioctanoin. These patients suffer from muscle weakness and rhabdomyolysis, manifested by the release of creatine kinase in plasma. It was hypothesized that the accumulation of long chain acyl-CoAs and long chain acylcarnitines results in membrane damage with release of large and small molecules from cells. The leakage of small molecules would deplete intermediates of the citric acid cycle (CAC) which carry acetyl groups as they are oxidized. It was further hypothesized that boosting anaplerosis with a suitable substrate would compensate for the chronic cataplerosis and improve heart and muscle function. The catabolism of heptanoate yields propionyl-CoA, which can be used for anaplerosis in most tissues, and C₅ ketone bodies in liver. C₅ ketone bodies are converted to propionyl-CoA, which can be used for anaplerosis in peripheral tissues. The marked improvement of the patients'' conditions after switching from a trioctanoin- to a triheptanoin-based diet supported the hypothesis.After ingestion of meals containing triheptanoin as the only lipid component, both C₅ ketone bodies and C₄ ketone bodies accumulated in the plasma of patients that have been diagnosed with disorders of long chain fatty acid oxidation (16). This suggested that acetyl groups derived from heptanoate can be used for the synthesis of C₄ and C₅ ketone bodies. Alternatively, the accumulation of C₄ ketone bodies after triheptanoin ingestion might result from the inhibition of the utilization of C₄ ketone bodies in peripheral tissues by C₅ ketone bodies.The aim of the present study was to investigate the interaction between C₄ and C₅ ketogenesis in rat livers perfused with octanoate and/or heptanoate. To gain insight on the fates of the acetyl groups of both fatty acids and on the fate of the propionyl-CoA moiety of heptanoate, we conducted the experiments with a series of labeled substrates: [1-¹³C]octanoate, [8-¹³C]octanoate, [5,6,7-¹³C₃]heptanoate, [1-¹³C]heptanoate, and [¹³C₃]propionate. The outcome of the propionyl-CoA moiety of [5,6,7-¹³C₃]heptanoate and [¹³C₃]propionate was traced by measurements of anaplerosis and glucose labeling by mass isotopomer³ analysis (18). In previous studies on the metabolism of odd-chain fatty acids in liver or hepatocytes (19, 20), ketone bodies were assayed with BHB dehydrogenase. This assay does not differentiate C₄ from C₅ ketone bodies. In the present study we used gas chromatography-mass spectrometry to specifically assay C₄ and C₅ ketone bodies (13).     

Glucose and ketone bodies production in hepatocytes isolated from fetuses at term--I. Effect of maternal fasting

The effect produced by maternal fasting on glucose and ketone bodies production has been studied in hepatocytes isolated from fetal rat. Maternal fasting produces a decrease in the weight of fetal liver. Maternal fasting produces a decrease in glucose production, both from endogenous substrates and adding lactate (10 mM) to the incubation medium. Maternal fasting produces an increase in ketone bodies production, both from endogenous substrates and adding acetate (5 mM) to the incubation medium.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

The metabolism of lipids in mouse pancreatic islets. The oxidation of fatty acids and ketone bodies.

The rate of oxidation of 14C-labelled fatty acids and of ketone bodies was measured in isolated pancreatic islets of obese-hyperglycaemic mice (ob/ob). The following main observations were made. 1. Octanoate, palmitate and oleate were all converted into CO2 by the pancreatic islets. Octanoate was oxidized with the highest rate followed by palmitate and oleate. 2. The rate of oxidation of 0.7 mM-palmitate was 3.1 pmol/h per mug drug weight. This was decreased by 50% in the presence of 16.7 mM-glucose. The rate of palmitate oxidation was also inhibited by 2-bromostearate. The palmitate oxidation showed a concentration-dependent increase, which was most marked between 0.25 and 1.0 mM. 3. Octanoate (5 mM) had no effect on the rate of oxidation of 3.3 mM- glucose. 4. Acetoacetate (5 mM) and D-3-hydroxybutyrate (5 mM) were oxidized at rates of 5.9 and 5.4 pmol/h per mug dry weight respectively. These rates were less than 10% of those found in kidney-cortex slices. The magnitude of the oxidation rates found for fatty acids and for ketone bodies suggest that these substrates represent important metabolic fuels for the pancreatic B-cells.     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Hexose metabolism in pancreatic islets

Summary In rat pancreatic islets, a rise in extracellular D-glucose concentration is known to cause a greater increase in the oxidation of D-[6-¹⁴C]glucose than utilization of D-[5-³H]glucose. In the present study, such a preferential stimulation of acetyl residue oxidation relative to glycolytic flux was mimicked by nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate, 3-phenylpyruvate, L-leucine, 2-ketoisocaproate, D-fructose and ketone bodies. The preferential stimulation of D-[6-¹⁴C]glucose oxidation by these nutrients was observed at all hexose concentrations (0.5, 6.0 and 16.7 mM), coincided with an unaltered rate of D-[3,4-¹⁴C]glucose oxidation, was impaired in the absence of extracellular Ca²⁺, and failed to be affected by NH₄ ⁺. Although the ratio between D-[6-¹⁴C]glucose oxidation and, D-[5-³H]glucose utilization in islets exposed to other nutrient secretagogues could be affected by factors such as isotopic dilution and mitochondrial redox state, the present data afford strong support to the view that the preferential stimulation of oxidative events in the Krebs cycle of nutrient-stimulated islets is linked to the activation of key mitochondrial dehydrogenases, e.g. 2-ketoglutarate dehydrogenase. The latter activation might result from the mitochondrial accumulation of Ca²⁺, as attributable not solely to stimulation of Ca²⁺ inflow into the islet cells but also to an increase in ATP availability.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

The fuel of respiration of rat kidney cortex

1. In kidney-cortex slices from the well-fed rat, glucose (5mm) supplied 25–30% of the respiratory fuel; in the starved state, the corresponding value was 10%. These results are based on measurements of the net uptake of glucose and of the specific radioactivity of labelled carbon dioxide formed in the presence of [U-¹⁴C]-glucose. 2. Added acetoacetate (5mm) or butyrate (10mm) provided up to 80%, and added oleate (2mm) up to 50% of the fuel of respiration. The oxidation of endogenous substrates was suppressed correspondingly. 3. More [U-¹⁴C]oleate was removed by the tissue than could be oxidized by the amount of oxygen taken up; less than 25% of the oleate removed was converted into respiratory carbon dioxide and about two-thirds was incorporated into the tissue lipids. The rate of oleate incorporation into the neutral-lipid fraction was calculated to be equivalent to the rate of oxidation of endogenous fat, which provided the chief remaining fuel. 4. The contribution of endogenous substrates to the respiration (50%) in the presence of added oleate is taken to reflect either a high turnover rate of the endogenous neutral lipids (approx. half-life 2·5hr.) or a raised rate of lipolysis caused by the experimental conditions in vitro. 5. Added l-α-glycerophosphate (2·5mm) increased oleate incorporation into the neutral-lipid fraction by up to 40% (i.e. caused a net synthesis of triglyceride). 6. Lactate (2·5mm) added as sole substrate supplied 30% of the respiratory fuel, but with added oleate (2mm) lactate was converted quantitatively into glucose. Oleate stimulated the rate of gluconeogenesis from lactate by 45%. 7. The oxidation of both long-chain and short-chain even-numbered fatty acids was accompanied by ketone-body formation. Ketone-body synthesis from oleate, but not from butyrate, increased six- to seven-fold after 48hr. of starvation. The maximum rates of renal ketogenesis (80μmoles/hr./g. dry wt., with butyrate) were about 20% of the maximum rates observed in the liver (on a weight-for-weight basis) and accounted for, at most, 35% of the fatty acid removed. 8. dl-Carnitine (1·0mm) had no effect on the rates of uptake of acetate, butyrate or oleate or on the rate of radioactive carbon dioxide formation from [U-¹⁴C]oleate, but increased ketone-body formation from oleate by more than 100%. Ketone-body formation from butyrate was not increased. 9. There is evidence supporting the assumption that there are cells in which gluconeogenesis and ketogenesis occur together, characterized by equal labelling of [U-¹⁴C]oleate and the ketone bodies formed, and other cells that oxidize fat and do not form ketone bodies. 10. Inhibitory effects of unlabelled acetoacetate on the oxidation of [1-¹⁴C]butyrate and of unlabelled butyrate on [4-¹⁴C]acetoacetate oxidation show that fatty acids and ketone bodies compete as fuels on the basis of their relative concentrations. 11. The pathway of ketogenesis in renal cortex must differ from that of the liver, as β-hydroxy-β-methylglutaryl-CoA synthetase is virtually absent from the kidney. In contrast with the liver the kidney possesses 3-oxo acid CoA-transferase (EC 2.8.3.5), and the ready reversibility of this reaction and that of thiolase (EC 2.3.1.9) provide a mechanism for ketone-body formation from acetyl-CoA. This mechanism may apply to extrahepatic tissues generally, with the possible exception of the epithelium of the rumen and intestines.     

INTERACTION OF LEUCINE, GLUCOSE, AND KETONE METABOLISM IN RAT BRAIN IN VITRO

Brain cortex slices from fed, 48 h and 120 h fasted rats were incubated and ¹⁴CO₂ was measured from (a) [U-¹⁴C]glucose (5 mm ) either alone or in the presence of l -lcucine (0.1 or 1 mm ), and (b) [U-¹⁴C]leucine or [l-¹⁴C]leucine at 0.1 or 1 mm with or without glucose (5 mm ). In other experiments, sodium dl -3-hydroxybutyrate (3-OHB) or acetoacetate (AcAc) at 1 or 5 mm were added in the above incubation mixture. The rate of conversion of [U¹⁴C]glucose to CO₂ was decreased 20% by leucine at 1 mm and 30–50% by 3-OHB at 1 or 5 mm but not by leucine at 0.1 mm . The effects of 3-OHB and of leucine (1 mm ) were not additive. The effects of leucine were similar in the fed and fasted rats. The rate of conversion of [U-¹⁴C]leucine or [l-^,4C]leucine to ¹⁴CO₂ at 0.1 mm and 1.0 mm was increased by glucose (35%) in the fed or fasted rats. Ketone bodies in the absence of glucose had no effect on leucine oxidation. However, the stimulatory effect of glucose on the rate of conversion of leucine to CO₂ was inhibited by 3-OHB at 5 mm . These results suggest that (a) leucine in increased concentrations (1 mm ) may reduce glucose oxidation by brain cortex while itself becoming an oxidative fuel for brain, and (b) leucine oxidation by brain may be influenced by the prevailing glucose and ketone concentrations.     

Contribution of glucose and gluconeogenic substrates to insulin-stimulated glycogen synthesis in cultured fetal hepatocytes

The influence of medium composition on basal and insulin-stimulated glycogenesis was studied in cultured 17-day-old rat fetal hepatocytes, which contain no glycogen at the time of transplantation. Continuous-labeling ¹⁴C-glucose experiments were used to determine both glycogen content and glycogen labeling. The specific activity of glucose units in the newly formed glycogen (a) was compared to that of the medium glucose (b): the ratio a/b expresses the contribution of medium glucose to glycogen formation. In standard medium (5.5 mM glucose), this ratio averaged 0.60. Variations of glucose concentration in the medium from 1 to 40 mM were accompanied by a progressive increase in both glycogen content and the ratio a/b (up to 0.80). Supplementation of standard medium with fructose, galactose, glycerol, or lactate-pyruvate decreased the hepatocyte glucose uptake from the medium. Galactose (1 to 5 mM) or lactate-pyruvate (5 mM) enhanced the glycogen content whereas glycerol or fructose (1 to 5 mM) had no effect. The ratio a/b, not modified by glycerol or lactate-pyruvate, was decreased to 0.45 by fructose (5 mM). Galactose at concentrations as low as 1 to 2 mM brought the ratio down to 0.30, indicating that it is a superior precursor of glycogen as compared to glucose. When the hepatocytes were grown in the presence of 10 nM insulin, the glycogen content was constantly higher than in the absence of the hormone (2-fold stimulation). Also the amplitude of the glycogenic effect of insulin was similar whatever the modifications of the medium, whereas ratio a/b and glucose uptake were hardly increased by insulin. Thus several substrates can contribute to glycogen formation (especially galactose) in cultured fetal hepatocytes and the essential effect of insulin is a stimulation of the final step of the glycogenosynthetic pathway.     

The metabolic impact of β‐hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity

Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β‐hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β‐hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β‐hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β‐hydroxybutyrate was present in these neurons. In addition, the NMDA receptor‐induced calcium responses in the neurons were diminished in the presence of β‐hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β‐hydroxybutyrate augmented transmitter release induced by the K_ATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of K_ATP channels in the effects of ketone bodies on transmitter release.

     

Ketone-body utilization and lipid synthesis by developing rat brain—a comparison between in vivo and in vitro experiments

The distribution of ketone bodies between oxidation and lipid synthesis was analysed in homogenates of developing rat brain. The capacity for lipid synthesis of homogenized or minced brain preparations was compared with rates of lipid synthesis in vivo, assessed by incorporation of ³H from ³H₂O into fatty acids and cholesterol. Brain homogenates of suckling rats (but not those of adults) incorporated label from [3-¹⁴C]ketone bodies into lipids, but this process was slow as compared to ¹⁴CO₂ production (< 5%) and much slower than the total rate of ketone-body utilization (< 0.5%). Study of ³H₂O incorporation demonstrated that the rates of lipogenesis and cholesterogenesis are at least one order of magnitude higher in vivo than in vitro. Maximal rates of ³H incorporation into fatty acids (3 μmol/g brain . h) and into cholesterol (0.6 μmol/g brain . h) were found during the third postnatal week. Adult rats still incorporated ³H into brain fatty acids at an appreciable rate (1 μmol/g brain . h), whereas cholesterogenesis was very low. It is concluded that in vitro measurements of lipid synthesis severely underestimate the rates that occur in developing rat brain in vivo. The high rate of ³H incorporation into lipids by developing and adult rat brain as compared to the amounts of these lipids present in the brain suggests an important contribution of endogenous lipid synthesis during brain development and an appreciable rate of fatty acid turnover during brain growth, but also in the adult brain.     

Metformin inhibition of mTORC1 activation,DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser⁷⁹ and Raptor at Ser⁷⁹², was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05–0.1 mM) that were 10–100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the α₁ and α₂ catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032