Glucose metabolism and astrocyte–neuron interactions in the neonatal brain

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Review

Glucose metabolism and astrocyte–neuron interactions in the neonatal brain Q1 Eva Brekke a,b, Tora Sund Morken c,d, Ursula Sonnewald a,* a

Department of Neuroscience, Norwegian University of Science and Technology, Trondheim N-7489, Norway Department of Pediatrics, Division of Pediatrics, Obstetrics and Women’s Health, Nordland Hospital Trust, Bodo, Norway Department of Laboratory Medicine, Children’s and Women’s Health, Norwegian University of Science and Technology (NTNU), Trondheim N-7489, Norway d Department of Ophthalmology, St. Olav’s Hospital HF, Trondheim, Norway b c

A R T I C L E

I N F O

Article history: Received 11 November 2014 Received in revised form 7 February 2015 Accepted 9 February 2015 Available online Keywords: Glycolysis Neonatal Rat Pentose phosphate pathway Glutamate Pyruvate carboxylation

A B S T R A C T

Glucose is essentially the sole fuel for the adult brain and the mapping of its metabolism has been extensive in the adult but not in the neonatal brain, which is believed to rely mainly on ketone bodies for energy supply. However, glucose is absolutely indispensable for normal development and recent studies have shed light on glycolysis, the pentose phosphate pathway and metabolic interactions between astrocytes and neurons in the 7-day-old rat brain. Appropriately 13C labeled glucose was used to distinguish between glycolysis and the pentose phosphate pathway during development. Experiments using 13C labeled acetate provided insight into the GABA–glutamate–glutamine cycle between astrocytes and neurons. It could be shown that in the neonatal brain the part of this cycle that transfers glutamine from astrocytes to neurons is operating efficiently while, in contrast, little glutamate is shuttled from neurons to astrocytes. This lack of glutamate for glutamine synthesis is compensated for by anaplerosis via increased pyruvate carboxylation relative to that in the adult brain. Furthermore, compared to adults, relatively more glucose is prioritized to the pentose phosphate pathway than glycolysis and pyruvate dehydrogenase activity. The reported developmental differences in glucose metabolism and neurotransmitter synthesis may determine the ability of the brain at various ages to resist excitotoxic insults such as hypoxia-ischemia. © 2015 Published by Elsevier Ltd.

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1. Introduction Glucose metabolism has been thoroughly studied in the adult brain. However, much less is known about neonatal glucose and energy metabolism, although some aspects have been published such as ontogenesis of transporters (Vannucci and Simpson, 2003), enzyme levels and enzyme activities (Baquer et al., 1977; Booth et al., 1980). Knowledge about brain energy metabolism is essential in order to design appropriate treatment for neonatal illnesses such as hypoxic ischemic brain injury. In the latter, magnetic resonance spectroscopy (MRS) markers of hypoxia-ischemia such as lactate and N-acetylaspartate (NAA) levels have prognostic value for outcome (Thayyil et al., 2010) and measurements of mitochondrial function (NAA) and ATP generation following neonatal hypoxia-ischemia have received increasing attention (Hagberg et al., 2014). A particular type of spectroscopy, 13C MRS, provides unique insight in this context, because it does not only give a static picture but also allows

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* Corresponding author. INM, Medical Faculty, NTNU, MTFS, N-7489 Trondheim, Norway. Tel.: +47 73 59 0492; fax: +47 55 13 50. E-mail address: [email protected] (U. Sonnewald).

investigation of metabolic pathways after application of 13 Clabeled glucose or other substrates. In this way it is possible to link enzyme activities and the changes in energy levels and metabolites. Not only glycolysis and tricarboxylic acid (TCA) cycle metabolism but also other pathways fueled by glucose such as the pentose phosphate pathway (PPP) can be investigated with appropriately 13C labeled glucose. The use of 13C labeled acetate is of particular interest since it is converted to acetyl CoA in astrocytes but not neurons (for references see McKenna et al., 2012) and is thus able to give information about astrocyte–neuronal interactions. This has been done extensively in adult animals (explained in Sonnewald and Kondziella, 2003) but is also applicable in the neonate. We have conducted studies investigating intermediary metabolism in the neonatal rat using 13C labeled substrates in combination with 13C MRS (Brekke et al., 2014; Morken et al., 2013, 2014). Fig. 1 illustrates the pathways analyzed with 13C labeled substrates while Figs. 2–4 summarize changes in key transporters, metabolites, enzyme activities and activities of metabolic pathways in relation to glucose and energy metabolism during postnatal brain development. The aim of this review is to provide an overview over neonatal glucose and acetate metabolism and metabolic interactions between astrocytes and neurons and compare it to that of the adult under non-pathological conditions.

http://dx.doi.org/10.1016/j.neuint.2015.02.002 0197-0186/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Eva Brekke, Tora Sund Morken, Ursula Sonnewald, Glucose metabolism and astrocyte–neuron interactions in the neonatal brain, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.02.002

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2

Metabolic pathways and cellular compartmentaon GLUT3

Glucose CO 2 NADPH

Glycogen

PPP

GLUT1

GLUT1

Glucose

Ribulose-5-P

Glucose-6-P

Astrocyte

Glucose

Glucose

Fructose-6-P

GLYCOLYSIS

GLYCOLYSIS

Neuron

Glyceraldehyde-3-P

Glucose CO 2 NADPH

Glucose-6-P

PPP Ribulose-5-P

Fructose-6-P

Alanine Pyruvate LDH

PDH

Lactate

GLUT1

Glyceraldehyde-3-P LDH

Lactate

Oxaloacetate Aspartate

Alanine

TCA Citrate CYCLE

Blood

PC

PDH

Acetyl CoA

Oxaloacetate Glutamine

Glutamate

PAG

MCT

α-Ketoglutarate Succinate

Glutamate

Glutamine

K+

Glu

Acetate Ketone bodies

TCA Citrate CYCLE

Aspartate Glutamine

Lactate Acetate Ketone bodies

Pyruvate

PYRUVATE CARBOXYLATION

α-Ketoglutarate Succinate

Glu

Glucose

Ketone bodies

Acetyl CoA

GS

GLUTAMATE GLUTAMINE CYCLE Glutamate + + 3Na

Glutamate

GS

K

+ Glutamate

Glutamate + + Ca Na

Glutamate + + 3Na

S LA G 1, LT G

+ + Ca Na

T

NMDAR

1 2 3 4 5

AMPAR

Fig. 1. Illustration of the pathways studied and compartmentation of metabolism in neurons and astrocytes. Associated pathways, metabolites, enzyme activities and pathway activities are marked with corresponding colors in Figs. 1–4. Abbreviations: AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; GLAST, glutamate aspartate transporter; Glu, glutamate; GLT1, glutamate transporter 1; GLUT, glucose transporter; GS, glutamine synthetase; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; NMDAR, N-methyl-D-aspartate receptor; PAG, phosphate activated glutaminase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PPP, pentose phosphate pathway.

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2. Fuel for the brain It is generally accepted that the neonatal cerebral metabolic rate is much lower than that of the adult in gray matter while it might be higher in white matter (references in McKenna et al., 2012). However, the levels of high-energy phosphates such as phosphocreatine and ATP are quite similar in neonatal and adult brain (Lowry et al., 1964) (see Fig. 2). A low cerebral metabolic rate of glucose in neonatal gray matter is thought to account for the ability of the brain at this age to survive prolonged periods of hypoxia which are lethal in adults (Duffy et al., 1975; Lowry et al., 1964; McKenna et al., 2012; Rice et al., 1981; Thurston and McDougal, 1969). Under normal conditions, glucose is essentially the sole fuel for the adult brain (references in McKenna et al., 2012). In contrast, the neonatal brain uses also other energy sources (McKenna et al., 2012) such as ketone bodies. 2.1. Glucose It has been estimated that glucose supplies approximately 63% of the necessary energy for the brain of a 7-day-old (P7) rat (Vannucci et al., 1989). The contribution of glucose to the cerebral metabolic rate may be lower. A more recent study suggests that glucose accounts for 63% of the cerebral metabolic rate (Vannucci et al., 1994a)

under completely aerobic conditions, while metabolism of ketone bodies may supply at least 30% of the total energy in the suckling rat, and 48% of the energy demand of the 16-day-old rat. In fact, the glucose utilization may be as low as 12% of adult values at P7 and 28% at P14 (Vannucci et al., 1994b) (see Fig. 4). Nonetheless, the availability of glucose in the blood is largely similar between post-natal day 4 and adult age (Movassat et al., 1997). The low glucose utilization coincides with a low number of glucose transporters (GLUT) (Vannucci, 1994; Vannucci and Simpson, 2003) (see Fig. 2). GLUT1 is the transporter responsible for glucose transport over the blood–brain barrier (BBB), but is also present on glia, whereas GLUT3 is predominantly expressed on neurons (Vannucci, 1994). It has been suggested that the expression of glucose transporters is the rate limiting factor for glucose utilization in early postnatal development (Vannucci et al., 1994b), contrasting the adult brain where transport across the BBB is not rate limiting under normal conditions (Paulson, 2002). However, following intraperitoneal (i.p.) injection of [1-13C]glucose the % 13C enrichment in glucose in brain stayed constant around 25% between 5 and 45 minutes past injection in P7 rats (Morken et al., 2013), suggesting that the transport capacity is sufficient to reach equilibrium between the [1-13C]glucose in the blood and in the brain certainly after 5 minutes, possibly earlier. In line with low glucose utilization, glycolysis is also lower in the neonatal brain compared to the adult (see Fig. 4). Activity of all

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Amounts of key metabolites and transporters (% of adult values) Glucose (Plasma) #

%

se

co

(a)

e

os

(a)

(a) (a)

100

lu

G

Cell

GLUT3

c lu

G

Glycogen

% 100

50

(c)

(d)

(d)

GLUT1 50

Age P0 P7 P14 P21 P28

Adult

Glycogen %

GLUT1 (In Cortex)

%

100

(b)

(b)

P0 P7 P14 P21 P28

Age

P0 P7 P14 P21 P28

P0 P7 P14 P21 P28

Blood

Adult

Adult

Adult

Lactate ##

%

Alanine

%

(b) Age

Age

(d)

Glucose

(b) (b)

(c)

50

50

(b) (b) (b)

Adult

(d)

100

(b) 50

Age P0 P7 P14 P21 P28

(b)

(b)

(b)

100

Glucose (Brain)

%

GLUT3 (In Cortex)

MCT-1

%

(Vascular endothelium)

Lactate Acetate Ketone bodies

(f)

(c)

1200

(d)

100

900

(d)

200

(e)

600

50

MCT

300 100

(e)

100

Age P0 P7 P14 P21 P28

(f) P0 P7 P14 P21 P28

Adult

Age

Adult

Age P0 P7 P14 P21 P28

Lactate Alanine

Aspartate

%

Acetyl CoA

Aspartate

50

(e)

Succinate

%

Phosphocreatine

%

Age P0 P7 P14 P21 P28

ATP

%

%

Glutamate

(c) (e)

100

(d)

(e)

100

(e)

(d)

100

GABA

% (e)

100

(d)

(c)

Adult

GABA

Glutamate

(d) 100

200

200

50

Citrate

α-Ketoglutarate Succinate

Adult

(e)

(e)

Glutamine ATP

Age P0 P7 P14 P21 P28

100

Ketone bodies Acetate

Oxaloacetate

(e)

100

Glutamine

%

Pyruvate

Adult

50

50

(e)

50

(e)

1 2 3 4 5 6

Q4

Adult

Age

Age

Age P0 P7 P14 P21 P28

P0 P7 P14 P21 P28

Adult

P0 P7 P14 P21 P28

Adult

Age P0 P7 P14 P21 P28

Adult

Age P0 P7 P14 P21 P28

Adult

Fig. 2. Amounts of glucose transporters, MCT1 transporter and metabolites related to glucose metabolism in development. Values are given in percentage of adult values (100%) at various postnatal ages given in postnatal days. Measurements of error, where present, are given as SEM in percentage of adult values. Colored panels show metabolites related to the metabolic pathways marked in corresponding colors in Fig. 1. The color legend is repeated in Fig. 4. #, Plasma glucose was measured in non-fasting animals; ##, the amounts of lactate are affected by the time it takes to stop metabolism through freezing. The size of the head influences this, thus the numbers should be interpreted carefully. For experimental procedures, see the respective references: a, Movassat et al. (1997); b, Vannucci (1994); c, Thurston and McDougal (1969); d, Lowry et al. (1964); e, Morken et al. (2013); f, Leino et al. (1999). Abbreviations: GLUT, glucose transporter; MCT, monocarboxylate transporter; P0/7/14/21/28, postnatal day 0/7/14/21/28.

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glycolytic enzymes, lactate dehydrogenase as well as the enzymes related to TCA cycle metabolism increase manifold between birth and adult age in rats (Baquer et al., 1977; Booth et al., 1980) (see Fig. 3). The low enzyme activity is reflected in the low percent enrichment with [3-13C]lactate and [3-13C]alanine (Table 1) as well as low 13C labeling of TCA cycle derived metabolites after injection of

[1-13C]glucose in the P7 rat compared to the adult rat brain (Table 1) (Morken et al., 2013) and the P10 compared to the P30 rat brain (Chowdhury et al., 2007). It should be noted that the experimental methods used in Morken et al. (2013) and Chowdhury et al. (2007) differ (i.p. bolus injection versus continuous infusion; 15–30 minutes labeling time versus 2–3 hours), and cannot be directly compared.

Please cite this article in press as: Eva Brekke, Tora Sund Morken, Ursula Sonnewald, Glucose metabolism and astrocyte–neuron interactions in the neonatal brain, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.02.002

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4

Acvies of key enzymes (% of adult values unless specified) %

Phosphofructokinase-1 (PFK-1)

%

(g)

100

100

Hexokinase % (g)

(g) (g) 50

50

(g)(g) (g)

(g)

(g) (g)(g)

(g) (g)(g)

(g) (g)

50

Age

Age

Age P0 P7 P14 P21 P28

100

(g)

Glucose-6-phosphate dehydrogenase (G6PD)

P0 P7 P14 P21 P28

Adult

P0 P7 P14 P21 P28

Adult

Adult

Glucose Hexokinase

CO 2 NADPH

Ribulose-5-P

Glucose-6-P G6PD

Lactate Dehydrogenase (LDH)

% 100

Fructose-6-P

(h)

(h)

PFK-1

(h)

Fructose-1,6-bisphosphate

(h)

50

(h) (h)

Glyceraldehyde-3-P Age

P0 P7 P14 P21 P28

%

Pyruvate Dehydrogenase (PDH)

Adult

(h)

100

Pyruvate Carboxylase (PC)

%

Lactate

LDH

(h)

(h) Age

Acetyl CoA

(i)

P0 P7 P14 P21 P28

Adult

(i) (i)

(i)

CS

Oxaloacetate

(i)

Citrate Synthase (CS)

Age P0 P7 P14 P21 P28

%

Citrate

Adult

100

GABA transaminase (GABA-t - % of P28)

%

α-Ketoglutarate Succinate

(k)

Age P0 P7 P14 P21 P28

GABA

Age P0 P7 P14 P21 P28

Glutamic Acid Decarboxylase (GAD - % of P28)

50

%

GS

PAG %

Phosphate Activated Glutaminase (PAG - % of P28)

Glutamine

(k)

100

(k)

(k)

(k)

(k) (k)

50

Age

50

(k) (k) (k)

P0 P7 P14 P21 P28

Glutamate

(k) (k) (k)

100

(k)

GAD

Glutamine Synthetase (GS - % of P28)

(k)

100

Adult

GABA-t

(k)

%

(h) (h) (h)

Succinate semialdehyde

(k)

50

(h)

(h) (h)

50

(k)

100

1 2 3 4

(h) (h)(h)

PDH

PC

(i) 50

(h)

50

(i)

100

Pyruvate

P0 P7 P14 P21 P28

Age

Age P0 P7 P14 P21 P28

Fig. 3. Development of activities of key enzymes related to glucose metabolism and astrocyte–neuron interactions. Values are given in percentage of adult values (100%) or in % of values at postnatal day 28 at various postnatal ages (given in postnatal days). Colored panels show metabolites related to the metabolic pathways marked in corresponding colors in Fig. 1. The color legend is repeated in Fig. 4. For experimental procedures, see the respective references: g, Baquer et al. (1977); h, Booth et al. (1980); i, Wilbur and Patel (1974); k, Larsson et al. (1985).

Please cite this article in press as: Eva Brekke, Tora Sund Morken, Ursula Sonnewald, Glucose metabolism and astrocyte–neuron interactions in the neonatal brain, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.02.002

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Acvies of metabolic pathways (% of adult values) %

Glucose utilization Glycolysis and related metabolites

PPP

%

(c,m)

100

The pentose phosphate pathway

(m) (m) 50

400

(c) (m) (m) (c) (m)

Age Adult

(g)

P0 P7 P14 P21 P28

Adult

CO 2 NADPH (g)

100

Glucose-6-P

Ribulose-5-P

PPP Glycolysis + PPP

%

(g) 400

Fructose-6-P

(g)

(e) Lactate (e) Glutamate

300 200

Age P0 P7 P14 P21 P28

Adult

(n)

100

Age

Glyceraldehyde-3-P

Pyruvate carboxylation Pyruvate dehydrogenation

P0 P7 P14 P21 P28

400

(e) Glutamine

300

%

Pyruvate

200

(o)

Acetate Acetyl CoA

Age P0 P7 P14 P21 P28

Acetate vs glucose utilization

800

(e) Glutamate 100

Adult

Adult

(e) Glutamine

600

Ketone bodies

Oxaloacetate

400

(e) Glutamate

200 100

Glutamate Glutamine

(g) 50

%

Glutmate-glutamine cycle

(g)

100

Age

Glucose

Glycolysis

TCA cycle and related metabolites (g)

200

P0 P7 P14 P21 P28

%

Pyruvate carboxylation

(g)

300

(e) Age P0 P7 P14 P21 P28

Citrate α-Ketoglutarate Succinate

%

Adult

Transfer from glutamatergic neurons to astrocytes (e)

100

TCA cycle

%

Glutamate

Glutamate

50

(g)

100

(e) Age

(g)

P0 P7 P14 P21 P28

Adult

(g)

50

(g)

%

Age P0 P7 P14 P21 P28

Adult

Glutamine

Transfer from astrocytes to glutamatergic neurons

Glutamine

200

(e)

Astrocyte

Neuron

(e)

100

Age P0 P7 P14 P21 P28

1 2 3 4 5

Q5

Adult

Fig. 4. Development of activities of metabolic pathways related to glucose metabolism. Values are given in percentage of adult values (100%) at various postnatal ages given in postnatal days. Measurements of error, where present, are given as SEM in percentage of adult values. Colored panels show metabolites related to the metabolic pathways marked in corresponding colors in Fig. 1 and are also given in the legend. For experimental procedures, see the respective references: c, Thurston and McDougal (1969); e, Morken et al. (2013); g, Baquer et al. (1977); m, Vannucci et al. (1994b); n, Ben-Yoseph et al. (1996); o, Kanamatsu and Tsukada (1999). Abbreviations: PPP, pentose phosphate pathway; P0/7/14/21/28, postnatal day 0/7/14/21/28.

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Glucose is also used to synthesize glycogen, the concentration of which is roughly similar in the adult and 10-day-old rat brain (see Fig. 2). However, during ischemia, glycogen is rapidly depleted in the adult rat brain with a maximum utilization rate five

times that of the rat at P10 (Lowry et al., 1964). Nonetheless, glycogenolysis contributes to a significant proportion of the energy provided during hypoxia in the neonatal brain. Taking the generally lower cerebral metabolic rate into account, the contribution of

Please cite this article in press as: Eva Brekke, Tora Sund Morken, Ursula Sonnewald, Glucose metabolism and astrocyte–neuron interactions in the neonatal brain, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.02.002

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1 2 3 4

Table 1 Mapping of energy metabolism in the rat brain after injection of a combination of [1-13C]glucose and [1,2-13C]acetate or infusion of [1,6-13C]glucose or [2-13C]acetate. Percent of 13C labeled metabolites of lactate (lac), alanine (ala), glutamate (glu) and glutamine (gln) compared to total amounts of metabolites. Injection of

5 6 7 8 9 10

[3P7 P7 P9 Adult* Infusion of

11 12 13 14 15 16 17

[1-13C]glucose 13C]lac

5.5 ± 0.3% 4.3 ± 0.8% 4.1 ± 0.4% 10.4 ± 0.8%

13C]ala

25 ± 0.6% 40 ± 4.1%

Reference

[4-13C]glu

[4-13C]gln

[4,5-13C]glu

[4,5-13C]gln

5.3 ± 0.3% 9.7 ± 1.0% 8.5 ± 0.5% 9.4 ± 0.7%

4.3 ± 0.4% 2.0 ± 0.3% 1.7 ± 0.3% 7.7 ± 0.6%

0.5 ± 0.1% 0.4 ± 0.1% 0.4 ± 0.1% 4.4 ± 0.5%

4.1 ± 0.3% 3.9 ± 0.1% 3.5 ± 0.2% 3.1 ± 0.2%

10.7 ± 0.7% 8.7 ± 0.7% 9.7 ± 0.4% 14.4 ± 1.4%

[1,6-13C]glucose [3-

P10# P30

[1,2-13C]acetate [3-13C]ala

Morken et al. (2013) Morken et al. (2014) Morken et al. (2014) Morken et al. (2013)

[2-13C]acetate [4-13C]glu

[4-13C]gln

[4-13C]glu

[4-13C]gln

27.4 ± 2.9% 39.6 ± 2.1%

13.5 ± 2.1% 26.4 ± 1.2%

6.7 ± 0.7% 6.6 ± 0.6%

14.3 ± 1.4% 11.3 ± 1.2%

Chowdhury et al. (2007) Chowdhury et al. (2007)

For experimental details see the references. Briefly: For experiments by Morken et al. rats were injected with a solution containing [1-13C]glucose and [1,2-13C]acetate and euthanized 30 or 15(*) min later. For experiments by Chowdhury et al. (2007) rats were given a continuous infusion of labeled [1,6-13C]glucose for 150 or 120(#) minutes or of [2-13C]acetate for 120 minutes. 13C Labeled lactate (lac), alanine (ala), [4-13C]glutamate (glu) and [4-13C]glutamine (gln) were labeled from 13C labeled glucose and [4,5-13C]glutamate and [4,5-13C]glutamine from [1,2-13C]acetate. Results are given as mean ± SEM.

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glycogenolysis is in fact similar in the neonatal and adult brain (Vannucci and Vannucci, 1980). The increase in glucose consumption in the neonatal rat has been shown to correlate with the acquisition of neurological competence and new functions (Cremer, 1982; Nehlig, 2004). This applies also to the neonatal human brain since local increases in the glucose metabolic rate follow the functional maturation of specific brain regions (Takahashi et al., 1999). Thus there is a great potential for studying glucose metabolism in the developing brain in order to understand brain development. 2.2. Ketone bodies and acetate During lactation and consumption of high-fat milk, ketone bodies derived from lipids cover a substantial part of the energy need of the neonatal brain (Cremer, 1982; Dombrowski et al., 1989; Nehlig, 2004). These nutrients enter the brain through monocarboxylate transporters (MCT) which are more abundant in the neonatal than the adult brain (Leino et al., 1999) (see Fig. 2). Acetate shares the carboxylic acid group with ketone bodies and also enters the brain via MCTs (Rae et al., 2012). Thus, the study of acetate and its metabolism may serve as a proxy of ketone body metabolism in neonatal brain. Once taken up into the cell, ketone bodies are converted to acetyl CoA via β-oxidation. 13C labeled acetate can similarly be converted to 13C labeled acetyl CoA and be metabolized in the TCA cycle to give 13C labeling in compounds like glutamate, GABA and glutamine. [1,2-13C]acetate injection led to substantial labeling of these metabolites, which reached similar 13 C per cent enrichments in the neonatal brain compared to the adult brain (Table 1). In the adult, transport of [1,2-13C]acetate over the BBB (via MCT1) must be slower than metabolism of acetate since [1,2-13C]acetate is present in serum (Melo et al., 2005; Melø et al., 2006) but not often in the brain. However, in the neonatal brain, its transport over the BBB exceeds intracellular metabolism, evidenced by the presence of [1,2-13C]acetate in the brain of P7 rats (Morken et al., 2013, 2014). This may be related to the high expression of MCT-1 in the BBB of the neonatal brain, which decreases after weaning (Vannucci and Simpson, 2003). The dissimilarity in glucose and acetate metabolism might be due to differences in transport capacity. In addition, there appear to be developmental differences between expressions of different TCA cycle related enzymes. Indeed, pyruvate dehydrogenase (PDH) is reported to have a slower increase in activity than citrate synthase. At P7 PDH has only ~15%, while citrate synthase is already at ~30% of adult activity (Booth et al., 1980) (see Fig. 3). The low PDH activity could be a key factor for the low aerobic metabolism

of glucose, while allowing for a relatively higher aerobic metabolism of ketone bodies and acetate. Consequently, the percent of acetate metabolized compared to glucose was much higher in the P7 than in the adult rat brain (Morken et al., 2013) (see Fig. 4). Acetate is predominantly utilized in astrocytes, also in the neonatal brain. This is evidenced by metabolism of acetate leading to higher labeling of glutamine (Table 1) (Morken et al., 2013, 2014), which is synthesized in astrocytes and not in neurons (Norenberg and Martinez-Hernandez, 1979), whereas glutamate is mainly synthesized and located in neurons (Storm-Mathisen et al., 1983). This preferential conversion of acetate to acetyl CoA in astrocytes allows for investigation of metabolic interactions between astrocytes and neurons also in the neonatal brain using [1,2-13C]acetate.

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3. Glucose is essential at all developmental stages due to its role in the pentose phosphate pathway (PPP) Even if the neonatal brain relies on alternative energy sources, glucose is indispensable to the brain from the very beginning and throughout life. This is partly because glucose is necessary to fuel the PPP, which is imperative to supply the cells with pentoses for DNA/RNA synthesis as well as the regeneration of the reducing equivalent NADPH. The latter is used in lipid synthesis and is also a vital cofactor for one of the major cellular defenses against oxidative stress, the glutathione system. Under physiological conditions, the PPP has been shown to have a low activity in the adult brain compared to glycolysis, comprising only 2–5% of the total glucose consumption (Ben-Yoseph et al., 1995; Gaitonde et al., 1983; Hostetler and Landau, 1967). However, the PPP related enzymes have a large extra capacity, and flux through this pathway has been demonstrated to increase during oxidative stress (Bartnik et al., 2005; Ben-Yoseph et al., 1995; Dusick et al., 2007). In contrast to the enzymes necessary for glycolysis and the TCA cycle, the maximum activity of the enzymes related to the PPP remains remarkably stable from birth until adult age with the exception of transaldolase and transketolase (Baquer et al., 1977) (see also Fig. 3). However, studies using [1-14C] and [6-14C] labeled glucose have shown that the basal PPP activity is high at birth and declines with age (Baquer et al., 1977) (see Fig. 4). In agreement with this, the basal PPP activity accounts for 5–15% of glucose metabolism in the P7 rat brain under physiological conditions (Brekke et al., 2014; Morken et al., 2013). This indicates that not only is the enzyme activity higher at birth than in adult life, but also a larger part of the available glucose is used in the PPP in the neonate compared to the adult (Table 2).

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Table 2 Metabolic ratios in per cent for PPP/GM and PC/PDH ratios for glutamate and glutamine in the adult and neonatal (P7) rat brains.

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PPP/GM (%)

PC/PDH (%) Glu

Gln

11–15 4.9 ± 0.6

9±1 9±1 4 – 16

109 ± 22 82 ± 7 27 – 31

5 6 7 8 9 10 11 12 13 14 15

P7 P7 Adult Adult Adult Adult

2.9 4.5 ± 1 2.3

Morken et¬ al. (2013) Brekke et¬ al. (2014) Kanamatsu and Tsukada (1999) Hostetler and Landau (1967) Ben-Yoseph et¬ al. (1996) Gaitonde et¬ al. (1983)

For details on calculations see Brekke et al., 2014 and for experimental procedures the references in the right hand column. Abbreviations: GM, glucose metabolism; PC pyruvate carboxylation; PDH, pyruvate dehydrogenase; PPP, pentose phosphate pathway.

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Even though approximately 30% of the carbons necessary for lipid synthesis is provided by ketone bodies (Nehlig, 2004), high activity of the PPP is also essential for lipid synthesis and also to fill the need for a large amount of NADPH. In fact, the total fatty acid content increases fourfold from P5 until adulthood (Patel and Clark, 1980). Lipid synthesis is necessary for myelination, which starts around P7–P10 in the neonatal rat. If this was the major driving force, one might expect the PPP to be low in the newborn rat brain, and peak around myelination. Instead it is actually higher at birth than at P7– P10 (Baquer et al., 1977) (see Fig. 4). However, myelination is not the only process that requires lipid synthesis in the developing brain. Indeed the rapid growth that occurs during the first weeks (Bandeira et al., 2009) will also require considerable amounts of nucleotides and lipids. Part of this growth is due to the extensive gliogenesis that completely changes the cellular distribution from almost only neurons at birth to approximately 50% astrocytes in the adult rat cortex (Nedergaard et al., 2003). 4. Neurotransmitter homeostasis in the neonatal brain and the role of pyruvate carboxylase (PC) The TCA cycle is intimately involved in the synthesis of glutamate and aspartate, the main excitatory amino acid neurotransmitters, and GABA, the main inhibitory amino acid neurotransmitter. In humans, glutamate levels double from gestational week 32 until term, and continue to increase during the first year of life (Kreis et al., 2002). Similarly, glutamate levels are half of the adult values in P7 rats, while GABA levels are only slightly lower (Chowdhury et al., 2007; Morken et al., 2013) (see Fig. 2). Thus, there is a need for substantial de novo synthesis of neurotransmitters, in particular of glutamate. To avoid depleting the TCA cycle of metabolites, it must be replenished through an anaplerotic processes (Sonnewald, 2014). Carboxylation of pyruvate via pyruvate carboxylase (PC) accomplishes this in the brain (Kanamatsu and Tsukada, 1999; Mohler et al., 1974; Patel, 1974) by generating new oxaloacetate. PC is only present in astrocytes, and consequently anaplerosis occurs solely in astrocytes (for references see Sonnewald and Rae, 2010). Thus, the study of metabolism via PC gives insight into astrocyte function and interactions between neurons and astrocytes. As mentioned, at birth, there are fewer astrocytes compared to the adult brain, and the major period of gliogenesis occurs during the first postnatal weeks, making astrocytes the most numerous cell type in the CNS (Bandeira et al., 2009; Nedergaard et al., 2003). Together with low PC activity in astrocytes at birth (Larsson et al., 1985; Wilbur and Patel, 1974) (see Fig. 3), these findings support the notion that the neonatal glial compartment may have a lower capacity for metabolic support of neurons compared to later in life. However, the low capacity coincides with low metabolic activity of neurons and indeed there are reports showing that the activity of TCA cycle related enzymes in neurons is very low in the neonate, indicating

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that the neuronal demand for metabolic support from astrocytes may be low and that the contribution of PC from glia to neurons is sufficient (Baquer et al., 1977; Booth et al., 1980; Wilbur and Patel, 1974). Indeed, a much larger part of the available glucose is channeled into the pyruvate carboxylase pathway in the P7 rat than in the adult (Table 2) (Fig. 4). In fact, for every glucose molecule that is not used for the PPP, half is metabolized via pyruvate dehydrogenase, and the other half is used for anaplerosis (pyruvate carboxylation) (Brekke et al., 2014; Morken et al., 2013). Since anaplerosis occurs in astrocytes only, it is possible to calculate the relative glucose utilization in astrocytes compared to neurons at the level of pyruvate using results from experiments with [1,2-13C]glucose injection. This was calculated to be 34 ± 3% of glucose metabolism at the level of pyruvate in the P7 brain (based on numbers from table 3 in Brekke et al., 2014; Morken et al., 2013). This is in surprisingly good accordance with results from the adult brain based on a corresponding equation using [U-13C]glucose. Qu et al. (2000) found that astrocytes metabolize maximally 34% of glucose at the level of pyruvate in the adult brain, and similar results have been reported by Hassel et al. (1995). This could indicate that both astrocytes and neurons have lower glucose metabolism at P7 compared to adult animals. However, the distribution between nonneuronal cells and neurons changes considerably from P7 until adult age, since the number of astrocytes increases dramatically in this period to outnumber neuronal cells at adult age (Bandeira et al., 2009). This may suggest that each astrocyte has a higher metabolic rate relative to each neuron in the neonatal brain compared to later in life. In order to synthesize de novo glutamate, an anaplerotic substrate must be transferred from astrocytes to neurons, since the latter do not contain PC. In contrast to glutamate, glutamine can safely be released to the extracellular milieu without interacting with receptors. When glutamine is released into the synapse it is taken up by high affinity transporters on neurons (Varoqui et al., 2000) where it is converted to glutamate by phosphate activated glutaminase (Hogstad et al., 1988). In adults it has been established that after release in neurotransmission, glutamate is taken up via specialized transporters mainly on astrocytes (Danbolt, 2001; Danbolt et al., 1992). In astrocytes, glutamate is either rapidly converted to glutamine via the enzyme glutamine synthetase (GS), exclusively localized in astrocytes (Norenberg and Martinez-Hernandez, 1979), or converted to α-ketoglutarate, thereby entering the TCA cycle (McKenna et al., 1996). This is termed the glutamate–glutamine cycle (references in McKenna et al., 2012), which is necessary to ensure precise neural signaling, preserve carbon atoms for neuronal glutamate synthesis and limit excitotoxicity by way of excessive glutamate receptor stimulation (Danbolt, 2001). To which extent the processes mentioned earlier function in the neonatal brain has not yet been fully unraveled. In rats the number of glutamine transporters on astrocytes at P7 is similar to adult levels but increases over the next 7 days and peaks at P14 with twice the number found on adult astrocytes (Boulland et al., 2003). Neuronal glutamine transporters are also present from late gestation (Weiss et al., 2003), setting the stage for transport of glutamine from astrocytes to neurons and the subsequent utilization of glutamine for glutamate synthesis. Indeed, the transport of glutamine from astrocytes to glutamatergic neurons is higher in the neonatal than the adult brain (Morken et al., 2013, 2014). However, the reciprocal transfer of glutamate from neurons to astrocytes was very low at P7 compared to adults (see Fig. 4). This could be (1) because the astrocytes do not take up the glutamate, (2) because it is not converted into glutamine in the astrocytes or (3) because only a small amount of glutamate is released from the neurons at this stage. In support of (1) is the finding that expression of astrocytic glutamate transporters (GLT-1 and GLAST) is low

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early in brain development (Danbolt, 2001). This suggests that the ability of astrocytes to take up glutamate from the synapse is limited in the neonatal brain. In support of (2) it has been reported that GS, like PC, is very low at birth, but increases sharply during the first weeks of life (Larsson et al., 1985; Wilbur and Patel, 1974), coinciding with the major period of gliogenesis (Bandeira et al., 2009) (see Fig. 3). In support of (3) is the finding that in the neonatal rat brain the action potentials are smaller in amplitude and the firing rate is lower (McCormick and Prince, 1987) and also that the EEG does not develop an adult pattern until P12 (Snead and Stephens, 1983). Consequently, there may be less excitatory input to depolarize the neurons causing reduced neurotransmitter release. Thus all three might contribute to some extent but this has yet to be fully explored. It has been proposed that both glutamate and GABA are key regulators of neurodevelopment (Danbolt, 2001; Wang and Kriegstein, 2009), and further studies of neurotransmitter metabolism may give valuable insight into brain development under physiological as well as pathological conditions.

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5. Conclusion Even if energy demand in the neonatal brain may largely be met by supply of ketone bodies, glucose is essential because of the need for a functioning pentose phosphate pathway and pyruvate carboxylase activity. Similar to what has been observed in the adult brain, neurons metabolize the larger part of glucose at the level of pyruvate. During the high de novo production of neurotransmitters pyruvate carboxylase activity is prioritized and there is substantial transfer of glutamine from astrocytes to neurons, while the reciprocal transfer of glutamate from neurons to astrocytes is very low. Finally, neonatal metabolism is far from identical to that of adults and there is a great need for further research on neonatal and pediatric models.

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References Bandeira, F., Lent, R., Herculano-Houzel, S., 2009. Changing numbers of neuronal and non-neuronal cells underlie postnatal brain growth in the rat. Proc. Natl. Acad. Sci. U.S.A. 106, 14108–14113. Baquer, N.Z., Hothersall, J.S., McLean, P., Greenbaum, A.L., 1977. Aspects of carbohydrate metabolism in developing brain. Dev. Med. Child Neurol. 19, 81–104. Bartnik, B.L., Sutton, R.L., Fukushima, M., Harris, N.G., Hovda, D.A., Lee, S.M., 2005. Upregulation of pentose phosphate pathway and preservation of tricarboxylic acid cycle flux after experimental brain injury. J. Neurotrauma 22, 1052–1065. Ben-Yoseph, O., Camp, D.M., Robinson, T.E., Ross, B.D., 1995. Dynamic measurements of cerebral pentose phosphate pathway activity in vivo using [1,6-13C2,6,62H2]glucose and microdialysis. J. Neurochem. 64, 1336–1342. Booth, R.F., Patel, T.B., Clark, J.B., 1980. The development of enzymes of energy metabolism in the brain of a precocial (guinea pig) and non-precocial (rat) species. J. Neurochem. 34, 17–25. Boulland, J.-L., Rafiki, A., Levy, L.M., Storm-Mathisen, J., Chaudhry, F.A., 2003. Highly differential expression of SN1, a bidirectional glutamine transporter, in astroglia and endothelium in the developing rat brain. Glia 41, 260–275. Brekke, E.M., Morken, T.S., Wideroe, M., Haberg, A.K., Brubakk, A.M., Sonnewald, U., 2014. The pentose phosphate pathway and pyruvate carboxylation after neonatal hypoxic-ischemic brain injury. J. Cereb. Blood Flow Metab. 34, 724–734. Chowdhury, G.M., Patel, A.B., Mason, G.F., Rothman, D.L., Behar, K.L., 2007. Glutamatergic and GABAergic neurotransmitter cycling and energy metabolism in rat cerebral cortex during postnatal development. J. Cereb. Blood Flow Metab. 27, 1895–1907. Cremer, J.E., 1982. Substrate utilization and brain development. J. Cereb. Blood Flow Metab. 2, 394–407. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Danbolt, N.C., Storm-Mathisen, J., Kanner, B.I., 1992. An [Na+ + K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience 51, 295–310. Dombrowski, G.J., Jr., Swiatek, K.R., Chao, K.L., 1989. Lactate, 3-hydroxybutyrate, and glucose as substrates for the early postnatal rat brain. Neurochem. Res. 14, 667–675. Duffy, T.E., Kohle, S.J., Vannucci, R.C., 1975. Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J. Neurochem. 24, 271–276.

Dusick, J.R., Glenn, T.C., Lee, W.N., Vespa, P.M., Kelly, D.F., Lee, S.M., et al., 2007. Increased pentose phosphate pathway flux after clinical traumatic brain injury: a [1,2-13C2]glucose labeling study in humans. J. Cereb. Blood Flow Metab. 27, 1593–1602. Gaitonde, M.K., Evison, E., Evans, G.M., 1983. The rate of utilization of glucose via hexosemonophosphate shunt in brain. J. Neurochem. 41, 1253–1260. Hagberg, H., Mallard, C., Rousset, C.I., Thornton, C., 2014. Mitochondria: hub of injury responses in the developing brain. Lancet Neurol. 13, 217–232. Hassel, B., Sonnewald, U., Fonnum, F., 1995. Glial-neuronal interactions as studied by cerebral metabolism of [2-13C]acetate and [1-13C]glucose: an ex vivo 13C NMR spectroscopic study. J. Neurochem. 64, 2773–2782. Hogstad, S., Svenneby, G., Torgner, I.A., Kvamme, E., Hertz, L., Schousboe, A., 1988. Glutaminase in neurons and astrocytes cultured from mouse brain: kinetic properties and effects of phosphate, glutamate, and ammonia. Neurochem. Res. 13, 383–388. Hostetler, K.Y., Landau, B.R., 1967. Estimation of the pentose cycle contribution to glucose metabolism in tissue in vivo. Biochemistry 6, 2961–2964. Kanamatsu, T., Tsukada, Y., 1999. Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo 13C NMR spectroscopic study of rats after administering [2-13C]] glucose with or without ammonium acetate. Brain Res. 841, 11–19. Kreis, R., Hofmann, L., Kuhlmann, B., Boesch, C., Bossi, E., Huppi, P.S., 2002. Brain metabolite composition during early human brain development as measured by quantitative in vivo 1H magnetic resonance spectroscopy. Magn. Reson. Med. 48, 949–958. Larsson, O.M., Drejer, J., Kvamme, E., Svenneby, G., Hertz, L., Schousboe, A., 1985. Ontogenetic development of glutamate and GABA metabolizing enzymes in cultured cerebral cortex interneurons and in cerebral cortex in vivo. Int. J. Dev. Neurosci. 3, 177–185. Leino, R.L., Gerhart, D.Z., Drewes, L.R., 1999. Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats: a quantitative electron microscopic immunogold study. Brain Res. Dev. Brain Res. 113, 47–54. Lowry, O.H., Passonneau, J.V., Hasselberger, F.X., Schulz, D.W., 1964. Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain. J. Biol. Chem. 239, 18–30. McCormick, D.A., Prince, D.A., 1987. Post-natal development of electrophysiological properties of rat cerebral cortical pyramidal neurones. J. Physiol. 393, 743–762. McKenna, M., Gruetter, R., U., S., S., W.H., A., S., 2012. Energy metabolism of the brain. In: Brady, S.G.J., Siegel, G., Albers, R.W., Price, D.L. (Eds.), Basic Neurochemistry: Principles of Molecular, Cellular, and Medical Neurobiology, eighth ed. Elsevier Academic Press, Oxford, UK, pp. 200–231. McKenna, M.C., Sonnewald, U., Huang, X., Stevenson, J., Zielke, H.R., 1996. Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J. Neurochem. 66, 386–393. Melo, T.M., Nehlig, A., Sonnewald, U., 2005. Metabolism is normal in astrocytes in chronically epileptic rats: a (13)C NMR study of neuronal-glial interactions in a model of temporal lobe epilepsy. J. Cereb. Blood Flow Metab. 25, 1254–1264. Melø, T.M., Nehlig, A., Sonnewald, U., 2006. Neuronal-glial interactions in rats fed a ketogenic diet. Neurochem. Int. 48, 498–507. Mohler, H., Patel, A.J., Balazs, R., 1974. Metabolic compartmentation in the brain: metabolism of a tricarboxylic acid cycle intermediate, (1,4-14C)succinate, after intracerebral administration. J. Neurochem. 23, 1281–1289. Morken, T.S., Brekke, E., Haberg, A., Wideroe, M., Brubakk, A.M., Sonnewald, U., 2013. Neuron-astrocyte interactions, pyruvate carboxylation and the pentose phosphate pathway in the neonatal rat brain. Neurochem. Res. 39 (3), 556–569. Morken, T.S., Brekke, E., Haberg, A., Wideroe, M., Brubakk, A.M., Sonnewald, U., 2014. Altered astrocyte-neuronal interactions after hypoxia-ischemia in the neonatal brain in female and male rats. Stroke 45, 2777–2785. Movassat, J., Saulnier, C., Portha, B., 1997. Insulin administration enhances growth of the beta-cell mass in streptozotocin-treated newborn rats. Diabetes 46 (9), 1445–1452. Nedergaard, M., Ransom, B., Goldman, S.A., 2003. New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci. 26, 523–530. Nehlig, A., 2004. Brain uptake and metabolism of ketone bodies in animal models. Prostaglandins Leukot. Essent. Fatty Acids 70, 265–275. Norenberg, M.D., Martinez-Hernandez, A., 1979. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303–310. Patel, M.S., 1974. The relative significance of CO2-fixing enzymes in the metabolism of rat brain. J. Neurochem. 22, 717–724. Patel, T.B., Clark, J.B., 1980. Comparison of the development of the fatty acid content and composition of the brain of a precocial species (guinea pig) and a nonprecocial species (rat). J. Neurochem. 35, 149–154. Paulson, O.B., 2002. Blood-brain barrier, brain metabolism and cerebral blood flow. Eur. Neuropsychopharmacol. 12, 495–501. Qu, H., Haberg, A., Haraldseth, O., Unsgard, G., Sonnewald, U., 2000. 13)C MR spectroscopy study of lactate as substrate for rat brain. Dev. Neurosci. 22, 429–436. Rae, C., Fekete, A.D., Kashem, M.A., Nasrallah, F.A., Broer, S., 2012. Metabolism, compartmentation, transport and production of acetate in the cortical brain tissue slice. Neurochem. Res. 37, 2541–2553. Rice, J.E., 3rd, Vannucci, R.C., Brierley, J.B., 1981. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann. Neurol. 9, 131–141. Snead, O.C., 3rd, Stephens, H.I., 1983. Ontogeny of cortical and subcortical electroencephalographic events in unrestrained neonatal and infant rats. Exp. Neurol. 82, 249–269.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Sonnewald, U., 2014. Glutamate synthesis has to be matched by its degradation – where do all the carbons go? J. Neurochem. 131, 399–406. Sonnewald, U., Kondziella, D., 2003. Neuronal glial interaction in different neurological diseases studied by ex vivo 13C NMR spectroscopy. NMR Biomed. 16, 424–429. Sonnewald, U., Rae, C., 2010. Pyruvate carboxylation in different model systems studied by 13C MRS. Neurochem. Res. 35, 1916–1921. Storm-Mathisen, J., Leknes, A.K., Bore, A.T., Vaaland, J.L., Edminson, P., Haug, F.M., et al., 1983. First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301, 517–520. Takahashi, T., Shirane, R., Sato, S., Yoshimoto, T., 1999. Developmental changes of cerebral blood flow and oxygen metabolism in children. AJNR Am. J. Neuroradiol. 20, 917–922. Thayyil, S., Chandrasekaran, M., Taylor, A., Bainbridge, A., Cady, E.B., Chong, W.K., et al., 2010. Cerebral magnetic resonance biomarkers in neonatal encephalopathy: a meta-analysis. Pediatrics 125, e382–e395. Thurston, J.H., McDougal, D.B., Jr., 1969. Effect of ischemia on metabolism of the brain of the newborn mouse. Am. J. Physiol. 216, 348–352. Vannucci, R.C., Christensen, M.A., Stein, D.T., 1989. Regional cerebral glucose utilization in the immature rat: effect of hypoxia-ischemia. Pediatr. Res. 26, 208–214. Vannucci, R.C., Yager, J.Y., Vannucci, S.J., 1994a. Cerebral glucose and energy utilization during the evolution of hypoxic-ischemic brain damage in the immature rat. J. Cereb. Blood Flow Metab. 14, 279–288. Vannucci, S.J., 1994. Developmental expression of GLUT1 and GLUT3 glucose transporters in rat brain. J. Neurochem. 62, 240–246.

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Vannucci, S.J., Simpson, I.A., 2003. Developmental switch in brain nutrient transporter expression in the rat. Am. J. Physiol. Endocrinol. Metab. 285, E1127–E1134. Vannucci, S.J., Vannucci, R.C., 1980. Glycogen metabolism in neonatal rat brain during anoxia and recovery. J. Neurochem. 34, 1100–1105. Vannucci, S.J., Seaman, L.B., Brucklacher, R.M., Vannucci, R.C., 1994b. Glucose transport in developing rat brain: glucose transporter proteins, rate constants and cerebral glucose utilization. Mol. Cell. Biochem. 140, 177–184. Varoqui, H., Zhu, H., Yao, D., Ming, H., Erickson, J.D., 2000. Cloning and functional identification of a neuronal glutamine transporter. J. Biol. Chem. 275, 4049– 4054. Wang, D.D., Kriegstein, A.R., 2009. Defining the role of GABA in cortical development. J. Physiol. 587, 1873–1879. Weiss, M.D., Derazi, S., Rossignol, C., Varoqui, H., Erickson, J.D., Kilberg, M.S., et al., 2003. Ontogeny of the neutral amino acid transporter SAT1/ATA1 in rat brain. Brain Res. Dev. Brain Res. 143, 151–159. Wilbur, D.O., Patel, M.S., 1974. Development of mitochondrial pyruvate metabolism in rat brain. J. Neurochem. 22, 709–715.

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Uncited reference Hawkins, R.A., Williamson, D.H., Krebs, H.A., 1971. Ketone-body utilization by adult and suckling rat brain in vivo. Biochem. J. 122, 13–18.

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