Matthijs Lopes-Cardozo

Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex

Abstract: Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3‐14C]acetoacetate or [2‐14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, d‐3‐hydroxybu‐tyrate, and 14CO2 and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only ∼20% in the case of cultured astrocytes. This indicates that the rate at which 14C‐labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.

Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures.

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

SummaryThe synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratioper se does not affect the activity of citrate synthase in thisin vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis.The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed.Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation.The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.

Cellular location of glutamine synthetase and lactate dehydrogenase in oligodendrocyte-enriched cultures from rat brain.

Abstract: Glial cells were isolated from 1‐week‐old rat brain and cultured in a serum‐free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside‐positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement‐mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol3‐phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double‐label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.

Preferential utilization of ketone bodies for the synthesis of myelin cholesterol in vivo

1. The distribution of radioactivity among lipid classes of myelin and other subcellular brain fractions of young rats (18-21 days) was determined after in vivo injection of (3-(14)C-labelled ketone bodies, [U-(14)C] glucose or [2-(14)C] glucose. 2. The incorporation ratios (sterol/fatty acids) were 0.67, 1.48, 0.25, 0.62 and 0.54 for whole brain, myelin, mitochondria, microsomes and synaptosomes, respectively, with (3-(14)C)-labelled ketone bodies as substrate and 0.37, 0.89, 0.19, 0.34 and 0.29 with [U-(14)C] glucose as substrate. These data show that, both in whole brain and in subcellular brain fractions, acetyl groups derived from ketone bodies are used for sterol synthesis to a large extent than acetyl groups originating from glucose. 3. The specific radioactivity of cholesterol is much higher in myelin than in whole brain or in the other brain fractions, particularly after administration of labelled ketone bodies as substrate. 4. The incorporation patterns of acetoacetate and D-3-hydroxybutyrate were very similar, indicating that both ketone bodies contribute acetyl groups for lipid synthesis via the same metabolic route. 5. Our data suggest that a direct metabolic path from ketone bodies towards cholesterol exists - possibly via acetoacetyl-CoA formation in the cytosol of brain cells - and that this process is most active in oligodendrocytes.

Characterization of three arylsulfatases in semen: seminolipid sulfohydrolase activity is present in seminal plasma

Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.

Inhibition of lipolysis in bovine adipose tissue by butyrate and β-hydroxybutyrate

In a previous paper it was shown that butyrate and DL-β-hydroxybutyrate, at a concentration of 10 mM, inhibit lipolysis in bovine adipose tissue in vitro. This inhibition was observed for basal lipolysis as well as for lipolysis stimulated by noradrenalin. Acetate, propionate and acetoacetate were shown to have no effect on these processes. In this paper it will be shown that the inhibition of lipolysis by β-hydroxybutyrate, contrary to the effect of butyrate, may be important in dairy cows in vivo. Both optical isomers of the ketone body exhibit the inhibitory effect. It is concluded that the inhibition of lipolysis of β-hydroxybutyrate occurs through an inhibitory effect on the formation of CAMP* in the fat cell.

Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes: a study with fluorescent- and radio-labelled ceramide analogues

In most cell types sphingomyelin is synthesized predominantly in the cis‐medial compartments of the Golgi stacks whereas the contribution of the plasma membrane is much lower. The aim of this study was to assess the contribution of both compartments to the synthesis of sphingomyelin in myelinating cells. Therefore, oligodendrocytes from rat spinal cord were incubated in culture with fluorescently‐ or radiolabelled ceramides, and the effects of a block in the vesicular flow (monensin, brefeldin A, low temperature) on surface synthesis of sphingomyelin were evaluated. The results indicate that ≈50% of the sphingomyelin synthase is present at the plasma and myelin membranes of oligodendrocytes.

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain.

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5-8-day old rats. Cultures containing 60-80% oligodendrocytes were incubated for 16h with [3-(14)C]acetoacetate, d-[3-(14)C]3-hydroxybutyrate, [U-(14)C]glucose, l-[U-(14)C]glutamine and [1-(14)C]pyruvate or [2-(14)C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO(2) was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.

Interferon-γ induced ia antigen expression on cultured neuroglial cells and brain macrophages from rat spinal cord and cerebrum

The inducibility of major histocompatibility complex class II (Ia) antigens on glial cells of the brain suggests that neuroglia have immunoregulatory functions within the central nervous system (CNS), i.e., recognition and presentation of antigens. The aim of the present study was to investigate rat recombinant-interferon-gamma (r-IFN-gamma) induced Ia antigen expression in rat cerebral cultures containing type-1 astrocytes and macrophages, and in rat spinal cord cultures enriched in type-2 astrocytes or oligodendrocytes. We compared induction of Ia antigen expression in glial cell cultures derived from Lewis rats, which are very susceptible to experimental allergic encephalomyelitis (EAE), with those from Wistar rats, which are but modestly EAE susceptible. After 5 days in culture we found in Wistar rat type-1 astrocyte-enriched cultures that Ia antigens were expressed by 19% of the astrocytes, whereas we found that in Lewis rat type-1 astrocyte cultures a considerably higher number of astrocytes expressed Ia antigens (53%). However, no significant difference were found in Ia antigen expression between type-2 astrocytes derived from Wistar rat spinal cord (49%) and Lewis rat type-2 astrocytes (56%). In contrast, in oligodendrocyte-enriched cell cultures derived from either Lewis or Wistar rats no Ia antigen expression was found. Interestingly, we found in type-1 astrocyte-enriched cerebral cultures a large number (approx. 46% of the cells) of brain macrophages (amoeboid microglia), all expressing Ia antigens after treatment with r-IFN-gamma.