**2. Metabolic sources and principal metabolic pathways for brain energy metabolism**

There are two barriers protecting the brain from toxic metabolites: the blood-brain barrier and the blood-CSF barrier formed by epithelial cells of the choroid plexus. These barriers protect the brain from possible changes in the concentration of blood metabolites. In the same way, they work as selective filters to allow for controlled delivery of metabolic substrates into the brain. These metabolic substrates are mainly glucose, ketone bodies and some metabolic intermediaries such as lactate and pyruvate.

#### **2.1 Use of glucose in brain**

Glucose is an essential energy source for the adult human brain. According to *in vivo* studies using labeled 2-deoxyglucose or 2-fluorodeoxyglucose there is a correlation between brain function and brain metabolism (Sokoloff et al., 1977). Glucose is rich in potential energy and thus, it is a good metabolic fuel. In any tissue glucose can follow several metabolic pathways. In brain, glucose is almost entirely oxidized through sequential glycolysis and the tricarboxylic cycle (TCA) associated with oxidative phosphorylation. Glucose can also be stored as the polysaccharide, glycogen. Glucose is not only an excellent energetic fuel, it is also a remarkably versatile precursor supplying metabolic intermediaries for biosynthetic reactions. In brain most of these intermediaries serve to synthesize neurotransmitters and gliotransmitters, as well as other molecules of biological significance. Glucose oxidation via glycolysis provides metabolic intermediaries besides producing ATP. Glucose can be oxidized through an alternative pathway: the pentose phosphate pathway (PPP). Glucose oxidation through PPP provides 5-carbon monosaccharides for nucleic acid synthesis and NADPH for reductive biosynthetic processes and for mantainance of the redox balance in the cell.

#### **2.1.1 Glycolysis**

In glycolysis, a molecule of glucose is oxidized to two molecules of pyruvate. This oxidation occurs through ten enzyme-catalyzed reactions (Figure 1). The first step in glycolysis is phosphorylation of glucose at C-6, by hexokinase, to yield glucose-6-phosphate. Most of the

transmission is an adequate supply of nutrients and oxygen from blood. Neurotransmitters stimulate neurovascular messenger production in astrocytes and neurons. These molecules induce the local constriction and dilation of smooth muscle around the neighboring

There have been several reports of metabolic impairment in a variety of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and Parkinson's disease, among others (Mosconi et al., 2005, Leenders et al., 1986, Kuwert et al., 1993, Hattingen et al., 2009). Because brain is an expensive organ in energetic terms, disruptions in energy production may affect neuronal transmission and thus, neuronal survival. Moreover, deregulation of energy metabolism could be implicated in an increased production of oxidative species. Indeed, oxidative damage has been proposed in such

**2. Metabolic sources and principal metabolic pathways for brain energy** 

There are two barriers protecting the brain from toxic metabolites: the blood-brain barrier and the blood-CSF barrier formed by epithelial cells of the choroid plexus. These barriers protect the brain from possible changes in the concentration of blood metabolites. In the same way, they work as selective filters to allow for controlled delivery of metabolic substrates into the brain. These metabolic substrates are mainly glucose, ketone bodies and

Glucose is an essential energy source for the adult human brain. According to *in vivo* studies using labeled 2-deoxyglucose or 2-fluorodeoxyglucose there is a correlation between brain function and brain metabolism (Sokoloff et al., 1977). Glucose is rich in potential energy and thus, it is a good metabolic fuel. In any tissue glucose can follow several metabolic pathways. In brain, glucose is almost entirely oxidized through sequential glycolysis and the tricarboxylic cycle (TCA) associated with oxidative phosphorylation. Glucose can also be stored as the polysaccharide, glycogen. Glucose is not only an excellent energetic fuel, it is also a remarkably versatile precursor supplying metabolic intermediaries for biosynthetic reactions. In brain most of these intermediaries serve to synthesize neurotransmitters and gliotransmitters, as well as other molecules of biological significance. Glucose oxidation via glycolysis provides metabolic intermediaries besides producing ATP. Glucose can be oxidized through an alternative pathway: the pentose phosphate pathway (PPP). Glucose oxidation through PPP provides 5-carbon monosaccharides for nucleic acid synthesis and NADPH for reductive

In glycolysis, a molecule of glucose is oxidized to two molecules of pyruvate. This oxidation occurs through ten enzyme-catalyzed reactions (Figure 1). The first step in glycolysis is phosphorylation of glucose at C-6, by hexokinase, to yield glucose-6-phosphate. Most of the

arterioles (Attwell et al., 2010).

**metabolism** 

**2.1 Use of glucose in brain** 

**2.1.1 Glycolysis** 

disorders (Mazziotta et al., 1987, Navarro et al., 2009).

some metabolic intermediaries such as lactate and pyruvate.

biosynthetic processes and for mantainance of the redox balance in the cell.

glucose-6-phosphate is degraded to pyruvate. But glucose-6-phosphate can be used to generate glucose-1-phosphate, which in turn is used to synthesize glycogen (see below) or to generate NADPH and ribose-5-phosphate through PPP (see below). Myo-inositol is also synthesized from glucose-6-phosphate and serves as a precursor for phosphatidylinositide signaling molecules. The first step in glycolysis and the next nine enzymatic reactions occur in all brain cells (Lowry & Passonneau, 1964).

Fig. 1. Glycolysis. The first step in glycolysis is phosphorylation of glucose to glucose-6 phosphate by hexokinase, an enzyme inhibited by the same glucose-6-phosphate. Glucose must be phosphorylated to glucose-6-phosphate for it to enter glycolysis. The most important step in the control of glycolytic flux is a reaction catalyzed by phosphofructokinase (PFK). PFK is inhibited by compounds that accumulate when energy status is high (ATP, citrate) and it is activated by products of functional metabolic activity (AMP, ADP). PFK1 in neurons is not activated by fructose 2,6-bisphosphate (Fructose-2,6-P2) because the enzyme that catalyzes F2,6BP production is constantly degraded.

Major sites for the control of glycolytic flux are hexokinase (EC 2.7.1.1), phosphofructokinase 1 (PFK1, EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40; Lowry & Passoneau, 1964). Hexokinase catalyzes the transfer of a phosphate group from one

Brain Energy Metabolism in Health and Disease 335

synthase kinase 3 (EC 2.7.11.26). The abnormal activation of glycogen synthase is responsible for abnormal glycogen deposits in neurons, a feature observed in Lafora disease. Lafora disease is an autosomal recessive form of epilepsy which has been associated with mutations in malin and laforin genes. The malin-laforin complex induces the degradation of both PTG (regulatory subunit of protein phosphatase 1 that activates glycogen synthase) by dephosphorylation, and glycogen synthase. Interaction of malin and laforin is a regulated process that is modulated by the AMP-activated protein kinase (Solaz-Fuster et al., 2008).

Fig. 2. Glycogen metabolism. Glycogen is synthesized in astrocytes by glycogen synthase (GS). Glycogenolysis is mediated by glycogen phosphorylase (GP). Both GS and GP are

phosphatase-1 and inhibited by GS kinase-3 (GSK-3). Both GS and protein phosphatase-1 interact with glycogen via protein targeting to glycogen (PTG). In neurons, GS is present in a highly phosphorylated, inactive state and, together with PTG, is targeted for proteasomedependent degradation by the laforin-malin complex. Glycogenolysis in astrocytes depends on GP, which is activated by phosphorylation by several kinases, including the cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) in response to several neurochemical signals. Glycogenolysis in astrocytes leads to the production of lactate, which

regulated in opposite directions by phosphorylation. GS is activated by protein

serves as an energy substrate for oxidative metabolism in the active neurons.

molecule of ATP to glucose (Figure 1). Type-I hexokinase is the predominant isoenzyme in brain (Wilson, 1995). This reaction is irreversible under intracellular conditions. Type-I hexokinase is normally saturated with substrate and strongly inhibited by its product, glucose-6-phosphate (Wilson, 2003). Type-I hexokinase binds to the outer mitochondrial membrane via a hydrophobic N-terminal sequence (Wilson, 1997). Type-I hexokinase interacts with porin (also named voltage-dependent anion channel) which is a membrane protein located at the outer mitochondrial membrane. When type-I hexokinase binds to mitochondria, the Ki for glucose-6-phosphate is increased and the Km for ATP is reduced, suggesting that the bound form is more active (Wilson, 2003). This association occurs alongside changes in the cellular metabolic state. It has also been proposed that type-I hexokinase can bind to microtubules in order to increase their activity. Indeed, under mitogenic stimulus, mitochondria and type-I hexokinase colocalization decreases, while tubulin and type-I hexokinase colocalization increases in glial cells (Sanchez-Alvarez et al., 2004). These changes in localization are accompanied by an increase in glucose transporters (GLUTs) and hexokinase expression (Sanchez-Alvarez et al., 2004). Therefore, hexokinase and GLUTs should be key factors in the regulation of glycolytic flux capacity.

All three isotypes of PFK1 (muscle [M], liver [L] and brain [B]) have been observed by immunohistochemical analysis in neurons and astrocytes. The three isoenzymes differ in their allosteric properties and their distribution in different brain cells. These differences might be important for regulation of brain glycolysis in the different cellular compartments. Regulation of PFK activity is a major control point for glycolysis (Passoneau & Lowry, 1964). PFK1 is inhibited by compounds that accumulate when energy status is high (ATP, citrate) and is activated by products of functional metabolic activity (AMP, ADP). The powerful allosteric activator of PFK1 is fructose 2,6-bisphosphate, which is produced by the enzymatic reaction catalyzed by phosphofructokinase 2 (PFK2 or PFKB, EC 2.7.1.105). PFKB3 is the main isoform expressed in brain. However, PFKB3 has been described as being constantly degraded via the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 (Herrero-Mendez et al., 2009). Therefore, glycolysis in neurons is not activated. Indeed, when mitochondrial respiration is inhibited, astrocytes upregulate their glycolytic rates through fructose 2,6-bisphosphate production, while neurons die quickly. Thus glucose must be oxidized through other metabolic pathways, such as PPP, in neurons (see below).

Pyruvate kinase is expressed in neurons and astrocytes. But apparently, in brain, the reaction catalyzed by pyruvate kinase does not appear to be a major control point in glycolysis (Lowry & Passoneau, 1964).

#### **2.1.2 Glycogen metabolism**

Glucose can be stored in the form of glycogen in brain. Glycogen is synthesized from glucose-6-phosphate via glucose-1-phosphate and UDPglucose. Glycogen stores may only sustain brain metabolic turnover for a few minutes at most. Glycogen metabolism is regulated by two enzyme reactions, catalyzed by glycogen synthase and glycogen phosphorylase (Figure 2). Most, if not all, glycogen is localized in astrocytes (Vilchez et al., 2007). However, neuronal cells express enzymatic machinery for glycogen synthesis. Glycogen synthase is expressed in cultured neurons in a highly phosphorylated (inactive) state (Vilchez et al., 2007). Glycogen synthase phosphorylation is achieved by glycogen

molecule of ATP to glucose (Figure 1). Type-I hexokinase is the predominant isoenzyme in brain (Wilson, 1995). This reaction is irreversible under intracellular conditions. Type-I hexokinase is normally saturated with substrate and strongly inhibited by its product, glucose-6-phosphate (Wilson, 2003). Type-I hexokinase binds to the outer mitochondrial membrane via a hydrophobic N-terminal sequence (Wilson, 1997). Type-I hexokinase interacts with porin (also named voltage-dependent anion channel) which is a membrane protein located at the outer mitochondrial membrane. When type-I hexokinase binds to mitochondria, the Ki for glucose-6-phosphate is increased and the Km for ATP is reduced, suggesting that the bound form is more active (Wilson, 2003). This association occurs alongside changes in the cellular metabolic state. It has also been proposed that type-I hexokinase can bind to microtubules in order to increase their activity. Indeed, under mitogenic stimulus, mitochondria and type-I hexokinase colocalization decreases, while tubulin and type-I hexokinase colocalization increases in glial cells (Sanchez-Alvarez et al., 2004). These changes in localization are accompanied by an increase in glucose transporters (GLUTs) and hexokinase expression (Sanchez-Alvarez et al., 2004). Therefore, hexokinase

and GLUTs should be key factors in the regulation of glycolytic flux capacity.

oxidized through other metabolic pathways, such as PPP, in neurons (see below).

glycolysis (Lowry & Passoneau, 1964).

**2.1.2 Glycogen metabolism** 

Pyruvate kinase is expressed in neurons and astrocytes. But apparently, in brain, the reaction catalyzed by pyruvate kinase does not appear to be a major control point in

Glucose can be stored in the form of glycogen in brain. Glycogen is synthesized from glucose-6-phosphate via glucose-1-phosphate and UDPglucose. Glycogen stores may only sustain brain metabolic turnover for a few minutes at most. Glycogen metabolism is regulated by two enzyme reactions, catalyzed by glycogen synthase and glycogen phosphorylase (Figure 2). Most, if not all, glycogen is localized in astrocytes (Vilchez et al., 2007). However, neuronal cells express enzymatic machinery for glycogen synthesis. Glycogen synthase is expressed in cultured neurons in a highly phosphorylated (inactive) state (Vilchez et al., 2007). Glycogen synthase phosphorylation is achieved by glycogen

All three isotypes of PFK1 (muscle [M], liver [L] and brain [B]) have been observed by immunohistochemical analysis in neurons and astrocytes. The three isoenzymes differ in their allosteric properties and their distribution in different brain cells. These differences might be important for regulation of brain glycolysis in the different cellular compartments. Regulation of PFK activity is a major control point for glycolysis (Passoneau & Lowry, 1964). PFK1 is inhibited by compounds that accumulate when energy status is high (ATP, citrate) and is activated by products of functional metabolic activity (AMP, ADP). The powerful allosteric activator of PFK1 is fructose 2,6-bisphosphate, which is produced by the enzymatic reaction catalyzed by phosphofructokinase 2 (PFK2 or PFKB, EC 2.7.1.105). PFKB3 is the main isoform expressed in brain. However, PFKB3 has been described as being constantly degraded via the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 (Herrero-Mendez et al., 2009). Therefore, glycolysis in neurons is not activated. Indeed, when mitochondrial respiration is inhibited, astrocytes upregulate their glycolytic rates through fructose 2,6-bisphosphate production, while neurons die quickly. Thus glucose must be synthase kinase 3 (EC 2.7.11.26). The abnormal activation of glycogen synthase is responsible for abnormal glycogen deposits in neurons, a feature observed in Lafora disease. Lafora disease is an autosomal recessive form of epilepsy which has been associated with mutations in malin and laforin genes. The malin-laforin complex induces the degradation of both PTG (regulatory subunit of protein phosphatase 1 that activates glycogen synthase) by dephosphorylation, and glycogen synthase. Interaction of malin and laforin is a regulated process that is modulated by the AMP-activated protein kinase (Solaz-Fuster et al., 2008).

Fig. 2. Glycogen metabolism. Glycogen is synthesized in astrocytes by glycogen synthase (GS). Glycogenolysis is mediated by glycogen phosphorylase (GP). Both GS and GP are regulated in opposite directions by phosphorylation. GS is activated by protein phosphatase-1 and inhibited by GS kinase-3 (GSK-3). Both GS and protein phosphatase-1 interact with glycogen via protein targeting to glycogen (PTG). In neurons, GS is present in a highly phosphorylated, inactive state and, together with PTG, is targeted for proteasomedependent degradation by the laforin-malin complex. Glycogenolysis in astrocytes depends on GP, which is activated by phosphorylation by several kinases, including the cyclic adenosine monophosphate (cAMP)-activated protein kinase A (PKA) in response to several neurochemical signals. Glycogenolysis in astrocytes leads to the production of lactate, which serves as an energy substrate for oxidative metabolism in the active neurons.

Brain Energy Metabolism in Health and Disease 337

purpose of the TCA is the complete oxidation of acetyl carbons from acetyl coenzyme A (Acetyl-CoA) with the subsequent formation of NADH and FADH2 (Figure 4). The TCA cycle is closely linked to oxidative phosphorylation. In oxidative phosphorylation NADH and FADH2 are reoxidized. NADH and FADH2 electrons are transferred from electron

Fig. 3. The pentose phosphate pathway and glutathione. In the oxidative branch of the pentose phosphate pathway, two NADPH are generated per glucose-6-phosphate. The only limiting reaction of the pathway is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), which is allosterically activated by NADP+. G6PDH catalyzes glucose-6-

decarboxylation of 6-phosphogluconate, a reaction catalyzed by glucose-6-

intermediates glyceraldehyde-3-phosphate and fructose-6-phosphate.

phosphate oxidation and the production of the first NADPH. This NADPH is oxidized and thus glutathione is reduced. The second NADPH is generated through the oxidative

donors to consecutive electron acceptors, in which the last electron acceptor is oxygen (oxygen is reduced to H2O, Figure 4). The energy released by electrons flowing through this electron transport chain is used to transport protons across the inner mitochondrial membrane, generating a potential energy (as an electrochemical gradient). This energy is

phosphogluconate dehydrogenase. The nonoxidative branch of the pentose phosphate pathway provides a reversible link with glycolysis by regenerating the two glycolytic

On the other hand, astrocytic glycogen is good for the brain. During synaptic activity, breakdown of astrocytic glycogen is stimulated by neurotransmitters such as glutamate and norepinephrine (Walls et al., 2008). Adrenergic agents cause protein kinase A (PKA) activation in astrocytes through metabotropic receptor binding. Glycogenolysis in astrocytes depends on glycogen phosphorylase, which is activated by phosphorylation of several kinases, including PKA (Benarroch, 2010). Glycogenolysis produces glucose-6-phosphate. It has been proposed that glucose-6-phosphate is used to produce metabolic substrates such as lactate, which can be exported from astrocytes to neuronal cells (Benarroch, 2010; Walls et al., 2008). It has also been proposed that glucose-6-phosphate from glycogen in astrocytes inhibits glycolysis (by hexokinase inhibition) and thus, glucose consumption is favored by neurons (DiNuzzo et al., 2011). In any case, glycogen is important for brain. Indeed glycogen metabolism in astrocytes only occurs in mature astrocytic cells.

#### **2.1.3 Pentose phosphate pathway**

The pentose phosphate pathway (PPP) generates NADPH and 5-carbon carbohydrates. PPP has two distinct phases (Figure 3). The first is the oxidative phase, in which NADPH is generated, and in the second phase, non-oxidative synthesis of 5-carbon carbohydrates occurs. Even if this pathway involves oxidation of glucose, its primary role is anabolic rather than catabolic. Glucose-6-phosphate dehydrogenase is the regulatory enzyme in PPP. It is regulated by the NADPH:NADP+ ratio and is allosterically stimulated by nicotinamide adenine dinucleotide phosphate (NADP+). The ratio of NADPH:NADP+ is normally high. Thus, this makes the cytosol a highly-reducing environment.

NADP+ is a coenzyme used in anabolic reactions, such as lipid and nucleic acid synthesis, which require NADPH as a reducing agent. Therefore, the PPP has a more prominent function in developing brain compared to adult brain due to lipogenesis and myelin formation during development. Astrocytes have a glucose-6-phosphate dehydrogenase Ki 10-times lower than glioma cells because PPP is an indicator of cellular biosynthesis, while glycolysis indicates cellular proliferation. However, adult brain slices from several age groups have shown similar PPP capacities. Thus, adult brain has a high PPP capacity. NADPH could be used for neurotransmitter and gliotransmitter turnover and to metabolize aldehydes and peroxides produced by monoamine oxidase, among other enzymes. In this way, NADPH is used to regenerate glutathione (GSH) from glutathione disulfide, which is a product of peroxide scavenging (Baquer et al., 1988).

As was mentioned previously, neurons are unable to increase glycolysis due to the lack of PFKB3. PFKB3 is constantly degraded in neuronal cells and thus glucose should be directed mainly to the PPP to generate NADPH and regenerate GSH (Bolaños et al., 2010). Recently, it has been demonstrated that glucose metabolism and glycogen utilization is impaired in astrocytes with a chronic GSH deficit (Lavoie et al., 2011). So, PPP activity is important in other brain cells besides neurons.

#### **2.1.4 Tricarboxylic acid cycle and oxidative phosphorylation**

The tricarboxylic acid (TCA) cycle, also called the citric acid cycle or Krebs cycle, includes eight enzyme-catalysed chemical reactions, which are of central importance in all living cells. The

On the other hand, astrocytic glycogen is good for the brain. During synaptic activity, breakdown of astrocytic glycogen is stimulated by neurotransmitters such as glutamate and norepinephrine (Walls et al., 2008). Adrenergic agents cause protein kinase A (PKA) activation in astrocytes through metabotropic receptor binding. Glycogenolysis in astrocytes depends on glycogen phosphorylase, which is activated by phosphorylation of several kinases, including PKA (Benarroch, 2010). Glycogenolysis produces glucose-6-phosphate. It has been proposed that glucose-6-phosphate is used to produce metabolic substrates such as lactate, which can be exported from astrocytes to neuronal cells (Benarroch, 2010; Walls et al., 2008). It has also been proposed that glucose-6-phosphate from glycogen in astrocytes inhibits glycolysis (by hexokinase inhibition) and thus, glucose consumption is favored by neurons (DiNuzzo et al., 2011). In any case, glycogen is important for brain. Indeed

The pentose phosphate pathway (PPP) generates NADPH and 5-carbon carbohydrates. PPP has two distinct phases (Figure 3). The first is the oxidative phase, in which NADPH is generated, and in the second phase, non-oxidative synthesis of 5-carbon carbohydrates occurs. Even if this pathway involves oxidation of glucose, its primary role is anabolic rather than catabolic. Glucose-6-phosphate dehydrogenase is the regulatory enzyme in PPP. It is regulated by the NADPH:NADP+ ratio and is allosterically stimulated by nicotinamide adenine dinucleotide phosphate (NADP+). The ratio of NADPH:NADP+ is normally high.

NADP+ is a coenzyme used in anabolic reactions, such as lipid and nucleic acid synthesis, which require NADPH as a reducing agent. Therefore, the PPP has a more prominent function in developing brain compared to adult brain due to lipogenesis and myelin formation during development. Astrocytes have a glucose-6-phosphate dehydrogenase Ki 10-times lower than glioma cells because PPP is an indicator of cellular biosynthesis, while glycolysis indicates cellular proliferation. However, adult brain slices from several age groups have shown similar PPP capacities. Thus, adult brain has a high PPP capacity. NADPH could be used for neurotransmitter and gliotransmitter turnover and to metabolize aldehydes and peroxides produced by monoamine oxidase, among other enzymes. In this way, NADPH is used to regenerate glutathione (GSH) from glutathione disulfide, which is a

As was mentioned previously, neurons are unable to increase glycolysis due to the lack of PFKB3. PFKB3 is constantly degraded in neuronal cells and thus glucose should be directed mainly to the PPP to generate NADPH and regenerate GSH (Bolaños et al., 2010). Recently, it has been demonstrated that glucose metabolism and glycogen utilization is impaired in astrocytes with a chronic GSH deficit (Lavoie et al., 2011). So, PPP activity is important in

The tricarboxylic acid (TCA) cycle, also called the citric acid cycle or Krebs cycle, includes eight enzyme-catalysed chemical reactions, which are of central importance in all living cells. The

glycogen metabolism in astrocytes only occurs in mature astrocytic cells.

Thus, this makes the cytosol a highly-reducing environment.

product of peroxide scavenging (Baquer et al., 1988).

**2.1.4 Tricarboxylic acid cycle and oxidative phosphorylation** 

other brain cells besides neurons.

**2.1.3 Pentose phosphate pathway** 

purpose of the TCA is the complete oxidation of acetyl carbons from acetyl coenzyme A (Acetyl-CoA) with the subsequent formation of NADH and FADH2 (Figure 4). The TCA cycle is closely linked to oxidative phosphorylation. In oxidative phosphorylation NADH and FADH2 are reoxidized. NADH and FADH2 electrons are transferred from electron

Fig. 3. The pentose phosphate pathway and glutathione. In the oxidative branch of the pentose phosphate pathway, two NADPH are generated per glucose-6-phosphate. The only limiting reaction of the pathway is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), which is allosterically activated by NADP+. G6PDH catalyzes glucose-6 phosphate oxidation and the production of the first NADPH. This NADPH is oxidized and thus glutathione is reduced. The second NADPH is generated through the oxidative decarboxylation of 6-phosphogluconate, a reaction catalyzed by glucose-6 phosphogluconate dehydrogenase. The nonoxidative branch of the pentose phosphate pathway provides a reversible link with glycolysis by regenerating the two glycolytic intermediates glyceraldehyde-3-phosphate and fructose-6-phosphate.

donors to consecutive electron acceptors, in which the last electron acceptor is oxygen (oxygen is reduced to H2O, Figure 4). The energy released by electrons flowing through this electron transport chain is used to transport protons across the inner mitochondrial membrane, generating a potential energy (as an electrochemical gradient). This energy is

Brain Energy Metabolism in Health and Disease 339

dehydrogenase complex is regulated by phosphorylation state and Ca2+ concentration among other factors (Huang et al., 1998). Pyruvate dehydrogenase complex immunoreactivity has been observed in neuronal cells (Calingasan et al., 1994). A low pyruvate dehydrogenase complex enzyme activity and immunoreactivity has been described in neurons following ischemia and reperfusion. This could explain the reduced cerebral glucose and oxygen

Although brain Acetyl-CoA is mainly derived from pyruvate, it can also be synthesized form fatty acids, ketone bodies (see below), monocarboxylate acids, such as lactate (see below) and acetate. Fatty acids are mainly oxidized inside the mitochondria through -

The transport of lactate and ketone bodies is accomplished by the monocarboxylate transporters (MCTs). Although BBB cells express MCT1, monocarboxylates cross the BBB with poor efficiency under physiological conditions. Only under extreme conditions, like starvation or prolonged exercise, could these compounds be important exogenous sources of

Normally, when the glycolysis rate exceeds the rate of triose entry into the TCA cycle, pyruvate can be reduced to lactate by lactate dehydrogenase (LDH, EC 1.1.1.27) which catalyzes a reversible reaction (Figure 1). Lactate must be released because local lactate accumulation would be an opposing driving force that would influence many reversible

Extracellular lactate is transported into the cell and is oxidized to pyruvate because LDH catalyzes the reversible interconversion between pyruvate and lactate. Two distinct subunits combine to form the five tetrameric isoenzymes of LDH. The LDH-5 subunit (muscle type, also termed the A or M subunit) has a higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate, while the LDH-1 subunit (heart type, also referred to as the B or H subunit) favors the reaction towards the production of pyruvate. The different catalytic properties of the five isoenzymes of LDH (H4 or LDH-1, H3M or LDH-2, H2M2 or LDH-3, HM3 or LDH-4, and M4 or LDH-5) are in proportion to the ratio of LDH-5 to LDH-1 subunits. It has been demonstrated that neurons contain predominantly LDH-1, while astrocytes express LDH-5 (Bittar et al. 1996). In this way, lactate synthesized within astrocytes and released into the interstitial space in brain may serve as energy fuel for neurons. Astrocytes release lactate at a greater rate than neurons and lactate is preferentially metabolized in neural cells to produce energy and in

oligodendrocytes to synthesize lipids (to make myelin, Sanchez-Abarca et al., 2001).

Glucose is the main energetic fuel for brain. However, under certain conditions the brain can meet its energetic needs using ketone bodies. During ketosis, glucose brain consumption decreases by about 10% per each mM of plasma ketone bodies (LaManna et al., 2009). Ketone bodies are produced from Acetyl-CoA. Lipids are not a major energy source for the

consumption that occurs after cerebral ischemia (Martin et al., 2005).

oxidation to produce Acetyl-CoA, NADH and FADH2.

metabolic fuel for the brain (Quistorff et al. 2008).

**2.2.1 Lactate formation and lactate oxidation** 

**2.2.2 Ketone bodies formation and oxidation** 

NAD+/NADH-coupled redox reactions.

**2.2 Use of lactate and ketone bodies** 

used to synthesize ATP in a enzyme reaction catalyzed by a large enzyme called ATP synthase.

Fig. 4. The tricarboxylic acid cycle (TCA) and oxidative phosphorylation. Pyruvate entry into the cycle is controlled by pyruvate dehydrogenase activity that is inhibited by ATP and NADH. Two other regulatory steps in the cycle are controlled by isocitrate and αketoglutarate dehydrogenase, whose activity is controlled by the levels of high-energy phosphates. All dehydrogenases are stimulated by Ca2+. NADH and FADH2 produced in TCA are reoxidized by the donation of their electron to the electron transport chain, the final goal of which is reduction of oxygen to water. Electron flux is responsible for the necessary energy to drive ATP synthesis.

#### **2.1.4.1 Formation of Acetyl-CoA**

Acetyl-CoA is produced mainly from pyruvate, the product of glycolysis. Pyruvate enters the mitochondrion and it is oxidized via an enzyme reaction catalyzed by the pyruvate dehydrogenase complex. This is composed of three different enzymes that catalyse seven reactions in order to oxidize pyruvate to Acetyl-CoA. Pyruvate dehydrogenase complex Km for Acetyl-CoA is approximately 0.05 mM. which is similar to pyruvate concentrations in brain. The enzyme complex also requires a variety of substrates and cofactors: pyruvate, NAD+, thiamine pyrophosphate, FAD, lipoic acid and coenzyme A. Activity of pyruvate

used to synthesize ATP in a enzyme reaction catalyzed by a large enzyme called ATP

Fig. 4. The tricarboxylic acid cycle (TCA) and oxidative phosphorylation. Pyruvate entry into the cycle is controlled by pyruvate dehydrogenase activity that is inhibited by ATP and

Acetyl-CoA is produced mainly from pyruvate, the product of glycolysis. Pyruvate enters the mitochondrion and it is oxidized via an enzyme reaction catalyzed by the pyruvate dehydrogenase complex. This is composed of three different enzymes that catalyse seven reactions in order to oxidize pyruvate to Acetyl-CoA. Pyruvate dehydrogenase complex Km for Acetyl-CoA is approximately 0.05 mM. which is similar to pyruvate concentrations in brain. The enzyme complex also requires a variety of substrates and cofactors: pyruvate, NAD+, thiamine pyrophosphate, FAD, lipoic acid and coenzyme A. Activity of pyruvate

NADH. Two other regulatory steps in the cycle are controlled by isocitrate and αketoglutarate dehydrogenase, whose activity is controlled by the levels of high-energy phosphates. All dehydrogenases are stimulated by Ca2+. NADH and FADH2 produced in TCA are reoxidized by the donation of their electron to the electron transport chain, the final goal of which is reduction of oxygen to water. Electron flux is responsible for the necessary

energy to drive ATP synthesis.

**2.1.4.1 Formation of Acetyl-CoA** 

synthase.

dehydrogenase complex is regulated by phosphorylation state and Ca2+ concentration among other factors (Huang et al., 1998). Pyruvate dehydrogenase complex immunoreactivity has been observed in neuronal cells (Calingasan et al., 1994). A low pyruvate dehydrogenase complex enzyme activity and immunoreactivity has been described in neurons following ischemia and reperfusion. This could explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia (Martin et al., 2005).

Although brain Acetyl-CoA is mainly derived from pyruvate, it can also be synthesized form fatty acids, ketone bodies (see below), monocarboxylate acids, such as lactate (see below) and acetate. Fatty acids are mainly oxidized inside the mitochondria through oxidation to produce Acetyl-CoA, NADH and FADH2.

### **2.2 Use of lactate and ketone bodies**

The transport of lactate and ketone bodies is accomplished by the monocarboxylate transporters (MCTs). Although BBB cells express MCT1, monocarboxylates cross the BBB with poor efficiency under physiological conditions. Only under extreme conditions, like starvation or prolonged exercise, could these compounds be important exogenous sources of metabolic fuel for the brain (Quistorff et al. 2008).

### **2.2.1 Lactate formation and lactate oxidation**

Normally, when the glycolysis rate exceeds the rate of triose entry into the TCA cycle, pyruvate can be reduced to lactate by lactate dehydrogenase (LDH, EC 1.1.1.27) which catalyzes a reversible reaction (Figure 1). Lactate must be released because local lactate accumulation would be an opposing driving force that would influence many reversible NAD+/NADH-coupled redox reactions.

Extracellular lactate is transported into the cell and is oxidized to pyruvate because LDH catalyzes the reversible interconversion between pyruvate and lactate. Two distinct subunits combine to form the five tetrameric isoenzymes of LDH. The LDH-5 subunit (muscle type, also termed the A or M subunit) has a higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate, while the LDH-1 subunit (heart type, also referred to as the B or H subunit) favors the reaction towards the production of pyruvate. The different catalytic properties of the five isoenzymes of LDH (H4 or LDH-1, H3M or LDH-2, H2M2 or LDH-3, HM3 or LDH-4, and M4 or LDH-5) are in proportion to the ratio of LDH-5 to LDH-1 subunits. It has been demonstrated that neurons contain predominantly LDH-1, while astrocytes express LDH-5 (Bittar et al. 1996). In this way, lactate synthesized within astrocytes and released into the interstitial space in brain may serve as energy fuel for neurons. Astrocytes release lactate at a greater rate than neurons and lactate is preferentially metabolized in neural cells to produce energy and in oligodendrocytes to synthesize lipids (to make myelin, Sanchez-Abarca et al., 2001).

#### **2.2.2 Ketone bodies formation and oxidation**

Glucose is the main energetic fuel for brain. However, under certain conditions the brain can meet its energetic needs using ketone bodies. During ketosis, glucose brain consumption decreases by about 10% per each mM of plasma ketone bodies (LaManna et al., 2009). Ketone bodies are produced from Acetyl-CoA. Lipids are not a major energy source for the

Brain Energy Metabolism in Health and Disease 341

metabolism with blood flow should thus exist. An increase in extracellular K+, adenosine, and lactate and related changes in pH are all a consequence of increased neuronal activity. All of them have been considered mediators of neurovascular coupling because they have vasoactive effects (Villringer & Dirnagl, 1995). Astroglial cells seem to be protagonists in the coupling of neuron metabolism and blood flow. Astrocytes, like neurons, are organized into networks, although glial cells are organized as a syncytium, linked to each other by gap junctions (Giaume et al., 2010). Neuronal cells exchange information through chemical and electrical synapses. Astrocyte gap junctions are regulated by extra- and intracelullar signals. Astrocytes release molecules that induce local constriction or dilation of smooth muscle cells surrounding endothelial cells forming arterioles (Attwell et al., 2010). Astrocytes also release molecules in response to neuronal activation. These molecules can modulate synaptic transmission and neuronal excitability (a concept referred to as the tripartite synapse, Perea & Araque, 2010). So, during brain activation, blood flow is increased due to several interactions between brain cells. We will discuss below the metabolic pathways that function during brain activation in neuronal and astroglial cells. We will then discuss the

neuroglial interation and molecular mechanisms that modulate the interaction.

neurotransmission consumes most of the energy expended in the brain.

Electrical stimulation opens Na+ channels in neuronal populations. An increase in intracellular Na+ concentration activates the Na+/K+-ATPase at its Na+-sensitive intracellular site. The activation of Na+/K+-ATPase is accompanied by a decrease in the ATP/ADP ratio and glycolysis activation (Figure 6, probably via allosteric activation of PFK1 by ADP). Glycolysis activation is accompanied by a Ca2+ increase and an increase in pyruvate and NADH/NAD+ concentrations. TCA and oxidative phosphorylation are also

In a chemical synapse, neurotransmitters released from the presynaptic neuron bind to ionotropic receptors in the postsynaptic membrane increasing the open probability of these ion channels. The ion flow can produce membrane depolarization (excitatory action potentials) or hyperpolarization (inhibitory action potentials). Excitatory action potentials are produced by the sequential opening of voltage-sensitive Na+ channels, followed by the delayed opening of K+ channels. This permits movement of positive charges from and into the neuronal cytosol. According to the biophysical properties of neuronal membranes, a theoretical minimum positive charge transfer (carried by Na+) was predicted per propagating action potential of 121 nC/cm2. This was corroborated by Alle and colleagues (2009) who calculated that charging of membrane capacitance requires 153 nC/cm2 only 1.3 times more than the theoretical minimum. Action potentials are thus energy-efficient, minimizing their contribution to activity-dependent metabolism. But human brain has billions of neurons and each neuron maintains a large number of synaptic connections to other neurons. Even though action potentials are energy-efficient, this is consistent with the fact that neuronal activity accounts for 80% of brain energy consumption (Sibson et al., 1998). Most of the energy consumed in the brain is attributable to restoration of the membrane resting potential following depolarization. This is accomplished by the Na+/K+- ATPase (EC 3.6.1.3, Attwell & Laughlin, 2001). Other energy-consuming processes are neurotransmitter recycling and axonal and dendritic transport (Ames, 2000). Because glutamate synapses represent at least 80% of cortical synapses, glutamate-mediated

**3.1 Neuron metabolism during activity periods** 

brain. Astrocytes are able to oxidize fatty acids and ketone bodies, while neurons and oligodendrocytes can only use ketone bodies.

Ketone bodies are synthesized mainly in the liver. Ketone body synthesis is activated after exhaustion of cellular carbohydrate stores (glycogen). Fatty acids are broken down via oxidation to Acetyl-CoA, NADH and FADH2. Normally, Acetyl-CoA is completely oxidized via the TCA cycle. However, if the amount of Acetyl-CoA generated in fatty-acid oxidation increases disproportionately, the processing capacity of the TCA cycle will start to drop due to low levels of intermediates. Acetyl-CoA will then be used instead for biosynthesis of acetoacetate, -hydroxybutyrate and acetone (ketone bodies) through four enzymatic reactions (Figure 4). The utilization of ketones bodies is controlled by leptin through AMPK inhibition in the hypothalamic region (Narishima et al., 2011).

During starvation, during chronic feeding with high fat/low carbohydrate or in pathophysiological conditions as diabetes, glucose stores are depleted and ketone body synthesis is stimulated. Under these conditions, the concentration of ketone bodies in blood increases and MCT expression increases at the blood brain barrier. Within the brain, ketone bodies may be used because all brain cells express MCTs and because the enzymatic reactions of ketone body synthesis are reversible (except for spontaneous decarboxylation of acetoacetate to acetone). Thus brain cells obtain Acetyl-CoA from ketone bodies and Acetyl-CoA may be oxidized for those cells through the TCA cycle. The starving brain also extracts fatty acids from blood. Astrocytes can degrade fatty acids by -oxidation to provide neurons with ketone bodies (Edmond, 1992). Moreover, ketone body oxidation must be important during the first postnatal period. Knockout animals for the ketolytic enzyme succinyl-CoA: 3-oxoacid CoA-transferase exhibit normal prenatal development, but develop ketoacidosis, hypoglycemia, and reduced plasma lactate concentrations within the first 48 h of birth (Cotter et al., 2011).

A ketogenic diet has been proposed for treatment of epilepsy. This diet is a strict high fat, low protein, low carbohydrate diet, and is anticonvulsant in many drug-resistant epileptic children. The diet is also effective in mice. The mechanism by which this diet controls intractable seizures is unknown. The diet does not affect behavorial performance or synaptic plasticity. Ketone bodies not only function as an energy substrate, but also as intermediates for the synthesis of lipids and neurotransmitters. Because of this, a link has been proposed between ketone bodies, brain function and polyunsaturated fatty acids (Pifferi et al., 2008). Another proposed mechanism to explain the neuroprotective effect of the ketogenic diet is via mitochondrial improvement by scavenging of reactive species and regulation of gene expression to increase neuronal survival (Beskow et al., 2008). Ketone bodies are also proposed as protectors from neurotoxicity in other pathologies, such as Parkinson's disease and Alzheimer's disease (Kashiwaya et al., 2000).
