Lactate oxidation is enhanced by dichloroacetate, an inhibitor of the pyruvate dehydrogenase kinase in neurons but not in astrocytes, suggesting that.
J. Inker. Metab. Dis. 19 (1996) 432-442 © SSIEM and Kluwer Academic Publishers. Printed in the Netherlands
Metabolic fuel utilization and pyruvate
oxidation during the postnatal period J. M. Medina*, A. Tabernero, J. A. Tovar and J. Martin-Barrientos
Departamento de Bioquimica Biologia Molecular, Facultad de Farmada, Universidad de Salamanca, Salamanca, Spain
* Correspondence: Edificio Departamental, Avenida del Campo Charro sin, 37007 Salamanca, Spain
Summary: The transplacental supply of nutrients is interrupted at birth, which diverts maternal metabolism to lactation. After birth, energy homeostasis is rapidly
regained through milk nutrients which supply the newborn with the fatty acids and ketone bodies required for neonatal development. However, immediately after birth and before the onset of suckling there is a time lapse in which the newborn under
goes a unique kind of starvation. During this period glucose is scarce and ketone
bodies are not available owing to the delay in ketogenesis. Under these circumstances, the newborn is supplied with another metabolic fuel, lactate, >yhich is utilized as a source of energy and carbon skeletons. Neonatal rat lung, heart, liver and brain utilize
lactate for energy production and lipogenesis. Lactate is also utilized by the brain of human babies with type I glycogenosis. Both rat neurons and astrocytes in primary culture actively use lactate as an oxidizable substrate and as a precursor of phos pholipids and sterols. Lactate oxidation is enhanced by dichloroacetate, an inhibitor of the pyruvate dehydrogenase kinase in neurons but not in astrocytes, suggesting that the pyruvate dehydrogenase is regulated differently in each type of cell. Despite the low activity of this enzyme in newborn brain, pyruvate decarboxylation is the main fate of glucose in both neurons and astrocytes. The occurrence of a yeast-like pyruvate decarboxylase activity in neonatal brain may explain these results.
During gestation most fetal requirements are met by the mother, who supplies the fetus with the nutrients necessary for development. The transplacental supply of nutrients is interrupted
by delivery, which diverts maternal metabolism to lactation. Thus, the fuel supply is rapidly restored by milk nutrients which supply the newborn with the energy and carbon skeletons required for neonatal development. However, immediately after birth and before the onset of suckling there is a time lapse in which the newborn undergoes a unique kind of starvation. This period, the so-called presuckling period, is probably due to the time delay until the obligatory change in utilization of metabolic substrates from the womb to the mammary glands is achieved. During this presuckling period, the newborn has to depend on its own reserves in a unique period of stress and vulnerability. For a review see Medina et al (1992). 432
Pyruvate
oxidation
in
the
newborn
433
0.4
0.0 J 0
4
8
12
24
48
72
96
hours pi p.
hours pip.
Figure 1 Time courses of blood glucose (•; mg/ml), free fatty acids (□; mmol/L) and ketone bodies (A; mmol/L) in human and rat newborns during the first 4 days after birth. (Exton 1972; Medina et al 1992)
METABOLIC FUEL UTILIZATION DURING THE POSTNATAL PERIOD
During late gestation the fetus accumulates a substantial amount of glycogen in different
tissues. However, most liver glycogen is rapidly depleted within the first hours after
delivery (Shelley and Neligan 1966). Despite the high rate of liver glycogenolysis, normal glycaemia is not regained immediately (Figure 1), suggesting that glucose is actively
utilized during the early neonatal period. However, the decrease in glucose availability is balanced by the rise in plasma free fatty acids (Figure 1), which supply energy to most neonatal tissues. In addition, the occurrence of an active ketogenesis at term provides the
brain with ketone bodies (Figure 1) as an alternative substrate for glucose (Williamson and
Buckley 1973). However, substrate supply is interrupted during the presuckling period. Thus, in the human baby, free fatty acids are released from adipose tissue but ketone-body synthesis is delayed owing to a lack of carnitine which is supplied with the milk (see Medina et al 1992). In the case of the rat, the lack of adipose tissue at birth prevents free
fatty acids from becoming available until the onset of lactation (Cuezva et al 1980)
because they come from milk triacylglycerols. During this lag period, the newborn has to live on its own reserves and increase the efficiency of its metabolic machinery in order to survive this untimely period of starvation. Under these circumstances, lactate may play an J. Inker. Metab. Dis. 19 (1996)
Medina et al. Human
Human -o-
•&
4.0 lactate
(mmol/L)
60
6 0
Time (m£n)
Time (min) Rat —r
220
Human
-a-
180 -J
y>\ ^
-
200
■ 180
3-hydroxyl)utyrate (nmol/L)
160 -l\ 140 ■ T
■\
120- \ ■ 160
100 - \ - 140 80-
-4- 120
>
60-1
6 0
6 0
Time (min)
Time (min)
Figure 2 Time courses of blood glucose, lactate, free fatty acids and 3-hydroxybutyrate concentra tions during the first 2h after delivery in human (□) and rat (•) newborns. (Persson and Tunell 1971; Juanes et al 1986)
important role as an alternative substrate. In fact, lactate accumulates in fetal blood during the perinatal period and is rapidly removed (Figure 2) before the onset of suckling takes place, i.e. during the presuckling period (Persson and Tunell 1971; Juanes et al 1986). This underlines the importance of lactate as a metabolic fuel for the newborn immediately after delivery. Indeed, during the presuckling period, glycaemia is very low and the plasma concentrations of other relevant substrates are negligible (Figure 2). Consequently, lactate may play an important role as a source of energy and carbon skeletons for neonatal tissues in these circumstances.
LACTATE AS A METABOLIC FUEL OF NEONATAL TISSUES
The lactate accumulated during late gestation is actively oxidized within the first hours of
extrauterine life (Figure 3) (Medina et al 1980), indicating that neonatal tissues actively utilize blood lactate. It is noteworthy that gluconeogenesis is not yet induced in these J. Inker. Meiab. Dis. 19 (1996)
435
Pyruvate oxidation in the newborn
H65%
To t a l
2 h
1 h
Oh
B Preterm □ Te r m
0%
7 0
2 0
4 0
60
8 0
% of initial lactate body pooi size
Figure 3 Lactate oxidation in vivo in newborn rat immediately after delivery. Newborn rats were injected with 4/rCi L-(U-'"*C]lactate and the radioactive CO^ expired was trapped in KOH and measured by liquid scintillation counting. Initial lactate body pool sizes were 46.7 ^ol for term (21.5 days post coitum) and 22.2 /rmol for preterm newborns (20.5 days post coitum). (From Medina etal 1980)
circumstances (Medina el al 1980; Fernandez et al 1983), a fact consistent with the idea that lactate is utilized directly as a source of energy and carbon skeletons for some neonatal tissues (see Medina et al 1992). Since lactate removal takes place at a very high rate, it is likely that several tissues would be involved in lactate utilization. Accordingly, it has been reported that neonatal lung (Patterson et al 1986), heart (E. Fernandez and J.M. Medina, unpublished results) and liver (Almeida et al 1992) utilize lactate for energy production and/or lipogenic purposes. However, special attention has also been paid to lactate utilization by the brain, probably because this organ has to continue its development even under the starvation occurring during the presuckling period. Lactate utilization by the brain has been reported in fetal (Bolanos and Medina 1993), early newborn (Arizmendi and Medina 1983; Fernandez and Medina 1986; Vicario et al 1991; Vicario and Medina
1992) and suckling rats (Itoh and Quastel 1970), in newborn dogs (Hellmann et al 1982) and in glucose-6-phosphatase-deficient human babies (Femandes et al 1984). It may therefore be suggested that lactate is utilized by a wide range of tissues playing a singular role in the development of the fetus and the newborn. L A C TAT E U T I L I Z AT I O N B Y T H E N E O N ATA L B R A I N
As lactate is actively taken up by the brain during the perinatal period (Cremer 1982), we investigated the possibility that lactate might be utilized by the neonatal brain. Accordingly, lactate oxidation was measured in isolated cells from neonatal brain with increasing concentrations of lactate (Vicario et al 1991). Lactate oxidation approached saturation at lactate concentrations of about 8mmol/L. It is noteworthy that this concentration is the
highest found in newborn rats under physiological circumstances (Figure 2), strongly J. Inker. Metab. Dis. 19 (1996)
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Medina et al.
glutairine
3-hyck-oxybutyrate
glucose
glutamine
3-hydrcixybutyra(e
glucose
lactate
Figure 4 Lactate utilization in rat neurons and astrocytes in primary culture. Cells were incubated
with lOmol/L L-lactate, 5mmoiyL glucose, 2mmol/L 3-hydroxybutyrate or 2mmol/L glutamine and
600-5000dpm/nmol of the radioactive substrate for 1 h and the radioactivity incorporated into CO^ (upper panels) or lipids (lower panels) was measured by liquid scintillation counting. Results arc means
of at least three experiments and are expressed as /imol/h per million cells of substrate incorporated corrected to six carbons, i.e. to glucose equivalents. (J.A. Tovar and J.M. Medina, unpublished)
suggesting that lactate oxidation observed in our experiments may be of physiological relevance. In addition, the rales of lactate oxidation in isolated cells from neonatal brain
were higher than those maximal for glucose or 3-hydroxybutyrate. These results (Vicario et al 1991) point to the importance of lactate as an energy substrate for the brain during the presuckling period.
To gain insight into the participation of neurons and astrocytes in lactate metabolism, rat neurons and astrocytes from primary culture were used to measure the rates of lactate utilization (Vicario et al 1993). When the rates of lactate utilization were measured under
optimal conditions and were compared to those of other important metabolic substrates for the neonatal brain it was observed (Figure 4) that in astrocytes the rate of lactate oxidation
was 3-fold higher than that of glucose, 2-fold higher than that of 3-hydroxybutyrate, and 1.5-fold higher than that of glutamine. In the case of neurons, the rate of glutamine oxidation was about 2-fold that of lactate but the latter was 5-fold higher than that of glucose and 1.5-fold higher than that of 3-hydroxybutyrate (Figure 4). It is important to note that in J. Inker. Metab. Dis. 19 (1996)
Pyruvate oxidation in the newborn
437
order to compare the rates of substrate utilization shown in Figure 4 properly, they are corrected to six carbons, i.e. to glucose.
In addition, lactate was utilized by both neurons and astrocytes not only as a source of energy but also as an excellent precursor of lipids (Vicario et al 1993). Thus, the rates of
lipogenesis from lactate in both neurons and astrocytes were higher than those from glucose 3-hydroxybutyrate and glutamine (Figure 4). The importance of lactate as a lipogenic substrate prompted us to investigate the incorporation of lactate into lipid species (Tabemero et al 1993). Accordingly, washed chloroform-methanol extracts of neurons and astrocytes previously incubated with labelled substrates were chromatographed on an HPLC column and the identified fractions were counted for radioactivity. Lactate was preferentially incorporated into saponifiable fractions, suggesting that it is mainly incorporated into the fatty acid moiety of phospholipid. However, sterol synthesis was much higher in neurons than in astrocytes, resulting in a much lower ratio of phospholipid/sterol synthesis in neurons. Moreover, in both types of cells the main phospholipid synthesized from lactate was phosphatidylcholine, and the main sterols synthesized were lanosterol in neurons and desmosterol in astrocytes (Tabemero et al 1993). This is in agreement with the lipid composition of the neonatal brain and confirms the physiological role played by lactate in brain development. This notion may be important because blood lactate utilization takes place during a period in which neural development occurs (Fernandez and Medina 1986; Vicario and Medina 1992; Bolanos and Medina 1993).
The rates of glutamine oxidation were similar in both types of cells, but lactate was preferentially oxidized by astrocytes. Moreover, lactate is the best lipogenic substrate for both neurons and astrocytes (Figure 4). Consequently, it could be suggested that lactate is the main substrate for the brain during the early neonatal period, being utilized not only as a source of energy but also to supply the brain with carbon skeletons for the synthesis of cerebral stmctures.
Lactate inhibits glucose utilization (Fem^dez and Medina 1986; Vicario and Medina
1992), suggesting that during the presuckling period it is utilized as the main fuel, reserving glucose for specific destinations such as oxidation by the pentose phosphate pathway or glycerogenesis. Likewise, hypoglycaemia increases the rate of entry of lactate into the brain,
highlighting the importance of lactate as a substrate for the brain during the hypoglycaemic postnatal period (Hellmann et al 1982). Lactate utilization during the presuckling period is presumably not inhibited by ketone bodies because at the physiological con
centrations occurring during the presuckling period (Figure 2) the presence of 3-hydroxy-
butyrate does not affect lactate metabolism (Vicario and Medina 1992). On the other hand,
3-hydroxybutyrate concentrations similar to those found in plasma during the suckling period (Figure 1) result in the inhibition of lactate utilization (Vicario and Medina 1992),
indicating that once the onset of suckling has taken place, ketone bodies become the major fuel for brain development. Under these circumstances lactate would be used as the major gluconeogenic substrate (Femandez et al 1983). P Y R U VAT E O X I D AT I O N A N D L A C TAT E M E TA B O L I S M I N T H E B R A I N
The limiting step in lactate metabolism is the pyruvate dehydrogenase complex (PDH; EC 1.2.4.1+EC 2.3.1.12+EC 1.8.1.4)-catalysed reaction, which is responsible for the transy. Inher. Metab, Dis. 19 (1996)
Medina et al.
438
Figure 5 Regulation of pyruvate dehydrogenase complex by phosphorylation/dephosphorylation mechanism. PDH, pyruvate dehydrogenase complex; DCA, dichloroacetate
formation of pyruvate into acetyl-CoA (for a review, see Wieland 1983). This enzyme has a very sophisticated regulation through a phosphorylation/dephosphorylation mechanism (Figure 5). Dephosphorylated enzyme is the active form that is inactivated by a phos phorylation catalysed by PDH kinase (EC 2.1.7.99), while activation by dephosphorylation is catalysed by a specific phosphatase (PDH phosphatase, EC 3.1.3.43). The activity of the main enzyme is controlled by the activity of kinase and phosphatase. Thus, high ratios of ATP/ADP, acetyl-CoA/CoA-SH or NADH/NAD activate the kinase and hence inhibit the PDH. This may be prevented by pyruvate, TPP or calcium ions. These latter also activate the phosphatase, together increasing the activity of the main enzyme (Figure 5). Since pyruvate activates the PDH by inhibiting PDH kinase, we considered the possibility that the activity of PDH might be maximal in the presence of high concentrations of lactate such as those found in the blood of newborns. To test this, the rates of lactate oxidation
were measured in the presence or the absence of dichloroacetate (DCA). In addition to the natural effectors, the drug dichloroacetate strongly inhibits PDH kinase and is commonly used to test the extent of PDH phosphorylation. This tool has been used by us (J. A. Tovar and J.M. Medina, unpublished) to test the regulation of the PDH in neurons and astrocytes in primary culture. Lactate oxidation was found to increase by about 40% in the presence of DCA, suggesting that the phosphorylation/dephosphorylation mechanism is operative in neurons. In astrocytes, however, the effect of DCA was not significant, suggesting that the astrocyte PDH is poorly regulated by this mechanism. In addition, these results suggest that the activity of the PDH is not maximal in rat neurons in primary culture despite the high concentrations of lactate used in these experiments (lOmmol/L; see also Figure 4). In the light of these results, we became interested in measuring the rate of pyruvate decarboxylation in cultured neurons and astrocytes in situ. For this we used glucose radioactively labelled at carbons 3 and 4 because these carbons are specifically decarboxylated during pyruvate decarboxylation. The rate of pyruvate decarboxylation was compared with other destinations of glucose carbons, such as the pentose phosphate pathway measured with glucose labelled at carbons 1 or 6 - or the tricarboxylic acid cycle and
J. Inker. Metab. Dis. 19 (1996)
Pyruvate oxidation in the newborn ASTROCYTES
Figure 6 Glucose utilization by neurons and astrocytes from primary culture. Pyruvate decarboxy
lation: [2,3-'''C]glucose incorporated into COj, equivalent to glucose decarboxylated in the pyruvate
decarboxylation reactions. PPP: [l-'''C]glucose minus [6-"*]glucose incorporated into COj, equivalent
to glucose decarboxylated in the pentose phosphate pathway. TAG: [6-'"*]glucose incorporated into
COj, equivalent to glucose decarboxylated in the tricarboxylic acid cycle. Lipogenesis: glucose incorporated into lipids (white portion, [2,3-"'C]glucose incorporated, equivalent to glucose incor porated into glycerol-bome phospholipids; hatched portion, [6-"*C]glucose incorporated, equivalent to glucose incorporated into fatty acid-bome phospholipids)
lipogenesis - measured with glucose labelled at carbon 6. Under these circumstances, the rate of pyruvate decarboxylation was similar in both types of cells (Figure 6). In addition, more than 50% of the total glucose was consumed by pyruvate decarboxylation as com
ft
pared with about 25% by pentose phosphate shunt or about 10% in the tricarboxylic acid cycle (Figure 6). The most important conclusion that can be drawn from these results is that pyruvate decarboxylation is a very important process in both neurons and astrocytes. This contrasts with the low activity of the PDH in the brain immediately after delivery (Malloch et al 1986). These results seem paradoxical because the activity of the enzyme is very small while the rate of decarboxylation at the level of the reaction catalysed by the enzyme is quite high. Therefore, we considered the possibility that pyruvate might be decarboxylated by an alternative pathway in addition to the PDH-catalysed reaction. The possible occurrence of such an alternative pathway was first proposed by Lefresne and colleagues (1978) and later by Patel and Clark (1980), who suggested that the alternative pathway might be extramitochondrial because it was still operative in the presence of «-cyano-4-hydroxycinnamate (a-CN), a potent inhibitor of mitochondrial pyruvate transporter. Under our experimental conditions, a-CN strongly inhibited lactate and glucose oxidation in neurons, although about 40% of the rate of lipogenesis from lactate or glucose remained in the presence of the drug. The effects of a-CN in astrocytes were considerably lower regarding the rates of both oxidation and lipogenesis (A. Tabemero and J.M. Medina, unpublished). This finding suggested that a considerable portion of pyruvate decarboxylation remains when
J. Inher. Metab. Dis. 19 (1996)
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Medina et al.
AMP
*
Pi
AT P
Figure 7 Pathways of pyruvate decarboxyiation in rat brain. PDH, pyruvate dehydrogenase complex (EC 1.2.4.1 + EC 2.3.1.12 + EC! .8.1.4); CS, citrate synthase (EC 4,1.3.7); CL, ATP-citrate lyase (EC
4.1.3.6); ALDH, aldehyde dehydrogenase (EC 1.2.1.5); PDC, pyruvate decarboxylase (EC 4.1.1.1) the transport of pyruvate across the mitochondrial membrane is inhibited. Consequently, these results are consistent with the idea of the occurrence of an extramitochondrial path way for pyruvate decarboxylation (Figure 7). In agreement with this, we believe we have found an enzyme activity that catalyses transformation of pyruvate into acetaldehyde in the cytosol of rat brain. The putative enzyme has similar characteristics to those of yeast pyruvate decarboxylase (EC 4.1.1.1). Accordingly, the enzyme is heat-resistant, it shows a high for pyruvate, and is inhibitable by glyoxylate (Hubner et al 1978; Kuo et al 1986). Likewise, the activity of this enzyme in neonatal rat brain is about double that found in the adult (M.A. Alonso, J. Martm-Barrientos and J.M. Medina, unpublished), suggesting that the enzyme plays an important role in brain development. Indeed, during the perinatal period, i.e. when the activity of pyruvate dehydrogenase is very low, putative pyruvate decarboxylase may transform pyruvate into acetyl-CoA in the cytosol by a path way that includes aldehyde dehydrogenase (EC 1.2.1.5) and acetate thiokinase (EC 6.2.1.1) (Figure 7), which are also present in rat brain (Buckley and Williamson 1973; Hassinen et al 1974). The acetyl-CoA produced would presumably be used as a lipid precursor. In conclusion, our results suggest that the brain shows a high capacity for lactate utilization because of the occurrence of an alternative pathway for pyruvate decarboxylation in addition to the PDH-calalysed reaction. This may play an important role during the early postnatal
J. Jnher. Metab. Dis. 19 (1996)
Pyruvate oxidation in the newborn
441
period in which other putative substrates, such as glucose and ketone bodies, are not freely available. However, this pathway may be also used in the adult brain as a component of the metabolic collaboration between neurons and astrocytes. Since brain glycogen is confined to astrocytes, which lack glucose-6-phosphatase activity, it has been suggested that glycogen carbons are transferred to the neurons in the form of lactate (Dringen et al 1993). The high capacity of neurons for lactate metabolism may have been designed to take over lactate
from astrocyte glycogen. Likewise, lactate utilization by neurons and astrocytes may play an important role in the recovery from hypoxic-ischaemic episodes. Thus, lactate is accumulated when the tissue undergoes an ischaemic-hypoxic episode, such as an impaired delivery of oxygen and substrates to the cells. This increase in lactic acid concentrations may be a consequence of a reduction in the activity of the PDH (Lundgren et al 1990)
coupled with increases in the lactate dehydrogenase M-subunit synthesis (Tholey et al 1991) detected after ischaemic/hypoxic episodes. Consequently, the high capacity for lactate utilization may help the brain to recover once oxygen becomes available. Finally, the possible importance of the putative brain pyruvate decarboxylase in lactic acidosis should be stressed because the occurrence of such an enzyme must be taken into account to explain
the morbidity of these diseases. Whether the deficit of such enzyme activity may result in the development of an unidentified lactic acidosis remains to be elucidated.
ACKNOWLEDGEMENTS
This work was supported by grants from CICYT, FISSS, and the Fundacion Ramon Areces,
Spain. A. Tabemero was a recipient of a fellowship from the FISSS, Spain. J.A. Tovar was a recipient of a fellowship from the Agencia de Cooperacion Iberoamericana, Spain. REFERENCES
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