Cellular Mechanisms of Brain Energy Metabolism

Key Concepts

The activity of the human brain depends on a sufficient and tightly regulated supply of glucose, oxygen, and other substances and removal of the by-products of metabolism of these substances The directions of delivery in turn depend on the concentrations and pressures of the substances in the vascular and tissue compartments.
Except for extreme exertions, concentrations and pressures of key metabolites in brain tissue are maintained within narrow limits, the needs of changing brain function being met by changes of flux rather than concentration or pressure. Current evidence suggests that this regulation is accomplished without a rigid association between the changes of oxygen consumption, glucose combustion, and blood flow associated with variable brain function. The claim that increases of blood flow occur simply to satisfy the demands for oxygen and glucose during neuronal excitation must therefore be regarded as one dimensional.
Energy budget estimates suggest that most of the brain’s demand for energy turnover reflects the steady-state magnitude of graded membrane depolarization rather than action potential generation and propagation. The increased energy turnover is required to maintain the graded depolarization of neuronal membranes associated with changing sodium and potassium conductance. Conversely, it is not the case that increased energy turnover is required to sustain the instances of membrane hyperpolarization caused by decreased conductance of sodium or increased conductance of potassium or chloride.
Glucose, pyruvate, and lactate occupy single tissue compartments with uniform concentrations, but the pathways and enzymes responsible for glycolysis and oxidative phosphorylation are unevenly distributed within and among the main cell populations.
The bulk of current knowledge of cerebral metabolic rates, volumes of cells, and distribution of glycolytic and oxidative activities in vitro and in vivo in rodents and humans implies that cell bodies and extensions in the form of terminals, synapses, and end-feet differ substantially.
Substantial pyruvate and lactate generation and accumulation occur when the less oxidative compartments are activated more than the more oxidative neuronal sites. In the early stages of activation, there is demand for glutamate removal of a magnitude that exceeds the low oxidative capacity of the neuropil and astrocytes. Although the resulting pyruvate and lactate accumulation is influenced by lactate exchange across the blood-brain barrier, the accumulated pyruvate and lactate pools are available for common use at the sites of oxidative phosphorylation in neurons and astrocytes. Astrocytes, and to a lesser extent distal parts of the neurons, appear to contribute more pyruvate and lactate to the common pools than do the proximal parts of neurons, which in turn extract more pyruvate and lactate from the common pool.
The increase in blood flow appears to be coupled to the rate of glycolysis rather than oxygen consumption. There is increasing evidence that the putative mechanism underlying the flow-glycolysis coupling is a calcium ion–mediated astrocytic response, aided and possibly initiated by a signal arising from lactate accumulation. The physiologic reason for the glycolytic mediation of blood flow activation is not clear because flow has a moderate effect on glucose delivery. Instead, it now appears more likely that the increased glycolysis is responsible for the increase of blood flow as mediated by the signaling role of lactate, rather than the direct demand for increased blood flow. This sequence of events inverts the flow-metabolism coupling sequence from the conventional flow-oxygen-glucose series to the revised glucose-flow-oxygen steps.

Brain Work Supports Information Processing

Theoretical considerations and experimental results indicate that the human brain operates close to the highest rates of energy turnover and information exchange achievable by known biologic systems. The close coupling of neuronal function to glucose and oxygen metabolism is well described. In contrast, the processes that consume the most energy in activated cells under different conditions, the regional and activation-dependent differences in the stoichiometry of oxygen and glucose utilization, and the mechanisms that supply energy for the work of the brain are less well understood. This means that explanations of the changes in the energy budget under different circumstances of localized and general brain activity must frequently be modified as new evidence accumulates.

Combustion of glucose yields most of the energy required by the brain. About 90% of the glucose is metabolized to carbon dioxide, and oxidative metabolism of glucose in turn provides 99.5% of the brain’s energy budget under normal resting circumstances. In special circumstances (e.g., lactation, diabetes, starvation), nonoxidative glucose metabolism or oxidative metabolism of the monocarboxylic acids lactate, β-hydroxybutyrate, acetoacetate, and acetate cover varying fractions of the brain’s energy turnover. The normal steady-state energy yield from these sources is supplied to brain tissue in the chemical form of 5 to 10 μmol adenosine triphosphate (ATP) for every gram of brain tissue every minute. In part, this depends on the degree of mitochondrial uncoupling; the average value is 7.5 μmol/g per minute.

The brain weighs about 1% to 2% of total human body weight, but it normally consumes at least 10% of the body’s glucose and oxygen supplies and receives 10% of the cardiac output of resting healthy awake humans. Because whole-body glucose and oxygen turnover each increase by as much as a factor of 10 during strenuous exercise, fractions of blood flow and the amounts of glucose and oxygen supplied to the brain decrease accordingly during the highest rates of physical exertion (i.e., the absolute amounts supplied to the brain are proportional to brain and body weights). Thus brain energy turnover is near the highest level achievable for the body as a whole. This finding implies that the regulatory mechanisms of brain metabolism may have fundamental differences from the regulatory mechanisms responsible for the wide range of energy metabolic rates for the body as a whole.

The brain’s work may be considered to be the mechanics of the transfer, processing, and exchange of information. Information generally is the term used to describe the restriction of a system’s degrees of freedom when a certain order is imposed on it. The restriction is described as a decline in entropy, which is a function of the number of different states that the system can occupy. Thus for n different states, each state is said to contain log ₂ ( n ) bits of information. A bit is the information necessary to complete a single binary operation, such as turning a switch on or off. If one point of contact between two cells in the brain (a synapse) is considered a simple on-off device (switch), the human CNS may have as many as 10 ¹⁵ switches. This allows the brain to occupy at least (2 ¹⁰ ) ¹⁵ different states and thus hold 10 ¹⁵ bits of information. Changes in information occur when the on-off switches are activated or deactivated in different combinations.

The toggle of a switch may be considered a binary operation and incurs a cost to the system’s energy status that qualifies as work. Two kinds of work are involved: the work of the mechanism that physically operates the switch and the work of the mechanism that determines the position that the switch comes to occupy. The switch’s mechanical operation probably accounts for most of the work and is most likely unrelated to the cost of the knowledge that the information imparts to the system.

The minimum energy cost of binary operations can be calculated from the decline in entropy, which happens only by means of an energy supply. The value must be compared with the actual cost of implementing the mechanical operation. These comparisons suggest that the cost of mechanical implementation of the binary operations in the brain is at least 9 orders of magnitude greater than the cost of the logical operation, which may be as low as 5 × 10 ⁻²¹ J. In contrast, Laughlin and colleagues and Abshire and Andreou reported that a single binary operation of the blowfly retina requires a minimum energy supply of 2 to 20 × 10 ⁻¹² J. This is 9 orders of magnitude greater, thus suggesting that the cost of the logical operation is negligible.

Hydrolysis of the cell’s energy currency in the shape of ATP yields free energy of about 3 × 10 ⁻² J/μmol. The human brain hydrolyzes 5 to 10 μmol ATP per gram per minute. This suggests that brain tissue has the capacity to perform binary operations at the maximum rate of 10 ¹² binary operations per second per average human brain (i.e., an exchange of 100 gigabytes of information per second). It is generally assumed that both blood flow and the energy metabolic rates of the brain are linked to this information processing, but the precise link has not been established, nor is it known whether this link is important for understanding the brain’s organization. To understand the mechanisms that link brain functions to the brain’s electrochemical work, as measured by physiologic methods, it is important to consider the following:

The type of work carried out in the brain
The coupling factor between work and information processing
The cells that carry out the brain’s work
The mechanism linking the work of these cells to the relative and absolute magnitudes of oxidative and nonoxidative energy metabolism in brain tissue
The mechanism that relates the brain’s blood supply to the magnitudes of the brain’s oxidative and nonoxidative metabolism

Brain Work Maintains Ion Concentrations

Information transfer and processing in the brain take place as a result of propagated changes of membrane potential differences. The electrical membrane potential differences exist when ion concentrations vary across selectively permeable membranes. The selective permeabilities to the major extracellular and intracellular cations (sodium, potassium, calcium) determine the membrane potential difference at a value that is more or less far removed from the equilibrium potential of each of these ions, depending on the permeability of the membranes to each of the ions. However, the membrane permeabilities that collectively determine the potential difference also serve to dissipate the concentration gradients of the ions when the ion concentrations are not maintained at their steady-state value. Energy is needed to support the ion transport that maintains these concentration gradients at all times, including periods of rapidly changing functional activity.

Neurons and other cells in the brain impose their metabolic needs on the brain by being subject to excitatory or inhibitory changes in membrane permeability to sodium, potassium, chloride, and calcium ions. The resulting changes in the intracellular concentration of sodium ions (Na ⁺ ) and the extracellular concentration of potassium ions (K ⁺ ) stimulate the activity of a population of membrane pumps fueled by the breakdown of ATP, known as sodium-potassium adenosine triphosphatase (Na ⁺ ,K ⁺ -ATPase). The increased activity is required to maintain the resting steady-state potential and ion gradients during and after dynamic changes in the potential.

It is not known how much of the ATPase activity that fuels the dynamic transfer and processing of information is necessary to keep the cells intact at the resting steady state, and poised and ready for increased functional activity. Several lines of evidence suggest that the ATPase activity associated with the functional activity of information transfer varies from a value that is no more than twice the absolute minimum required to maintain cellular integrity to a value that is an order of magnitude greater. Whether the actual changes in information transfer are reflected in proportional changes in energy turnover is not known; however, it appears that energy turnover generally is maintained at specific thresholds associated with major categories of functional activity ( Fig. 66.1 ) rather than being coupled to the moment-to-moment fluctuations in brain function, with minimal variation in the energy turnover rate in response to the fluctuating functional contingencies of each category. This description introduces the concept of a “subliminal” fringe of membrane potential difference that is maintained just below the level required to initiate the exchange of nerve impulses among neurons. This means that the actual initiation and regulation of impulse activity can take place with very little change in the energy turnover rate. The small cost of the logical element of a binary operation is consistent with this view when the mechanical part of the operation is poised very close to the all-or-none position of the switch, as well as measurements of brain states’ influence on the generation of impulses among neurons.

Figure 66.1, Relationship between the functional activity of mammalian brain and estimates of cerebral metabolic rates of oxygen ( CMRO 2 ) and glucose ( CMR glc ) .

The impulses that neurons exchange are known as action potentials. The classic “sodium theory” explains both the origin of the membrane potential and the graded or alternating depolarization of cells induced by the presence of sodium, calcium, potassium, and chloride equivalents as free ions in the intracellular and extracellular spaces, as well as the action on, and the action of, specific ion channels in the plasma membranes across which the ions move. The membrane potential differences are diffusion potentials established by the membrane conductances controlled by the channels that determine the ion permeability. The conductances of sodium and potassium associated with the resting membrane potential and the increased conductances associated with excitation above a baseline or a resting or “default” average are matched by the active ion pumping that strives to maintain constant ion concentrations.

The plasma membrane, or P-type, Na ⁺ ,K ⁺ -ATPase is the protein that enables transport of the major ions across the semipermeable membranes. The protein combines with ATP, magnesium (Mg ²⁺ ), Na ⁺ , and K ⁺ to form an enzyme-substrate complex in which the enzyme is phosphorylated and the Mg ²⁺ ions and adenosine diphosphate (ADP) are released. As the phosphorylated enzyme splits into inorganic phosphate (P _i ) and the original enzyme, Na ⁺ and K ⁺ ions are translocated in the appropriate directions, outward for sodium and inward for potassium. The energy released by the hydrolysis of ATP at a rate of, for example, 5 μmol/g per minute is sufficient to transport 15 μmol Na ⁺ per gram per minute and 10 μmol K ⁺ per gram per minute. In the steady state, a chloride flux matches the difference between the sodium and potassium fluxes and thus renders the total ion flux electroneutral. In steady-state conditions, the ion fluxes equal the diffusion rates in the opposite directions. The resulting half-life of sodium in the cells is less than 1 minute under normal circumstances.

The actual ion concentrations and the permeability of the membrane to the ions together determine the membrane potential difference. The average membrane potential approaches the potassium equilibrium potential. Intuitively, it makes sense that the potassium ion, which has greater permeability in the membrane, has the major influence on the sign and magnitude of the membrane potential difference. In the steady state, the apparent average potassium and sodium ion permeabilities, or the membrane permeability–surface area products, can be calculated from the potassium and sodium ion transport fluxes. The average ion concentrations vary among cells and tissue; typical values are listed in Table 66.1 . Using the typical values of the average steady-state membrane potential difference, the potassium and sodium fluxes calculated from measured ATP turnover rates, and the 3:2 ratio between the net sodium and potassium fluxes in the steady state, the average permeability–surface area products of sodium, potassium, and chloride ions can be calculated by means of Goldman’s flux equation ( Table 66.2 ). ^, The calculation ignores the calcium ion flux, which represents only a small fraction of the total energy requirement, although it is of major functional significance.

TABLE 66.1

Ion Concentrations in Nerve Cells

Data from Erecinska M, Silver I. ATP and brain function. J Cereb Blood Flow Metab. 1989;9:2–19; and McCormick DA. Membrane properties and neurotransmitter actions. In: Shepherd G, ed. The Synaptic Organization of the Brain. 3rd ed. Oxford University Press; 1990;199:32–66.

Variable	Unit	Sodium Ion		Potassium Ion		Chloride Ion
Variable	Unit	Erecinska	McCormick	Erecinska	McCormick	McCormick
Equilibrium potential	mV	+41	+40	−84	−100	−75
Intracellular concentration	mM	27	30	80	140	8
Extracellular concentration	mM	133	150	3	3	130

TABLE 66.2

Ion Movements Across Nerve Cell Membranes ^a

From Gjedde A. The energy cost of neuronal depolarization. In: Gulyas B, Ottoson D, Roland PE, eds. Functional Organization of the Human Visual Cortex. Pergamon; 1993:291–306.

Variable	Unit	Ion
Variable	Unit	Sodium	Potassium	Chloride
Transmembrane leakage	μmol/g/min	15	10	5
PS product at −65 mV	mL/g/min	0.038	0.404	0.549
PS product at −55 mV	mL/g/min	0.044	0.285	0.246

PS, Permeability-surface area.

a Assuming that 50% of the turnover of adenosine triphosphate is dedicated to ion transport, as calculated from the concentrations listed in Table 66.1 (McCormick). To estimate chloride permeability, it was necessary to use a simplified form of the equation.

Brain Energy Metabolism Enables Brain Work

The exact mechanism that links the work performed by brain tissue and the rate of metabolism under steady-state conditions is unclear. When concentrations of metabolites by definition do not change in the steady state, it is hard to conceive of feedback signals that can provide the link. Noradrenaline and the transmitter vasoactive intestinal polypeptide (VIP), when mediated by accumulation of cyclic adenosine monophosphate (cAMP), both promote glycogenolysis and mobilize glycogen in the cerebral cortex. Activation of metabotropic receptors also increases oxidative energy metabolism in brain tissue slices. This suggests that monoaminergic neuromodulation regulates both neuron excitability and the energy metabolic rates of glycolysis and oxidative phosphorylation. Because intracortical VIP neurons have a narrow field of action, whereas there is wide intracortical expansion of noradrenergic fibers, the two systems may serve to focus the modulation: VIP regulates the energy metabolism locally, within individual cortical columns, and norepinephrine exerts an effect across multiple columns.

Definition of Brain Activity Levels

Brain functions vary, and brain activity varies with these functions. Internal brain states regulate sensory perception, sensorimotor coordination, and learning, as reflected in different patterns of cortical synchrony. However, in the presence of distinct brain states, it is unclear that the work and the energy turnover rates change in direct proportion to the functional activity. The definition of a baseline functional activity and the issue of the brain’s normal or average activity are debated. Recent descriptions of brain functional activity distinguish between activation, default, resting, and baseline states, but the definitions of these states are not yet universally understood or accepted.

The issue hinges on the definitions of baseline and average functional activity in relation to the different rates of metabolism in these states. Work by Shulman and colleagues implies that steady-state functional activity is related to rates of release of the excitatory neurotransmitter glutamate and the linearly correlated rates of energy metabolism. ^, In this work, the highest rates of glutamate release and energy metabolism are about twice the average rates observed in the resting awake or “default” state of these brains, which in turn are twice the rates observed in apparently unconscious but otherwise intact brains. These rates turn out to be about twice the rates associated with a completely inactive but intact baseline state, which in turn are about twice the rates associated with the state of absent ion transport. This state is not compatible with survival because the necessary ion gradients gradually dissolve.

Brain energy metabolism probably doubles for every categorical increment in functional activity, although within each category, energy metabolism may be fairly constant (see Fig. 66.1 ). By categorical increment is meant a change from one fundamental state of functional activity to the next. These stages are illustrated in Fig. 66.1 and include the states of no ion transport (stage 0 or metabolic baseline), ion transport but no functional activity (stage 1 or functional baseline), low functional activity without consciousness (stage 2), normal functional activity with consciousness (stage 3 or default activity), and high functional activity associated with the highest level of physiologic activation (stage 4). Activity above stage 4 may be pathologic. Panel B shows that the metabolic or work rates associated with these stages can be described by the following simple formula:

CMR (S) = CMR (0) 2^{S}

where CMR is the cerebral metabolic rate, CMR(0) is the cerebral metabolic rate of stage 0, and S is one of the five functional stages (0–4) defined earlier. The equation has an interesting similarity to the formula (see earlier) for a system that holds S bits of information. It probably shows that distinct internal brain states dynamically regulate cortical membrane potential synchrony during behavior and define specific levels of cortical processing.

Description of Brain Activity Stages

Metabolic and Nonfunctional Baselines (Stages 0 and 1)

It is necessary to distinguish between metabolic and nonfunctional baselines. The metabolic baseline is the absence of ion transport; this is not consistent with continued cellular integrity. In contrast, the nonfunctional baseline in this context is the state of absent functional activity. A rule of thumb derived from recent experiments with living intact mammalian brains estimates that only about one-fourth of the normal energy turnover maintains the minimal ion transport required in the absence of functional activity, as seen with isoelectricity on the electroencephalogram.

It is likely that maintenance of the membrane potential makes the major contribution to brain energy use at the nonfunctional baseline (stage 1). The fraction of metabolism of isolated brain tissue associated with the transport of sodium and potassium is approximately half of the metabolism of the low functional state of isolated brain tissue. About half of the nonfunctional baseline remains when ion transport in isolated nervous tissue is blocked completely by inactivation of Na ⁺ ,K ⁺ -ATPase.

Low Functional State (Stage 2)

The unconscious baseline can be thought of as a state of lowered but not absent functional activity in which higher cognitive activity is not present. The state exists in conditions of severed cortical connections, coma, vegetative and minimally conscious states, or anesthesia. In these conditions the metabolic rate is close to 50% of the normal resting awake or default average. Here, work is associated with lowered but not absent depolarization of neuronal membranes, and the rate of work is about half of the work imposed by the average degree of depolarization existing in the resting awake or default condition.

Normal (Default) and Physiologically Elevated Functional States (Stages 3 and 4)

The normal or default metabolic stage refers to the awake and normally functioning mammalian brain. This stage has been studied more comprehensively in awake humans than in other mammals. Stage 3 metabolism refers more accurately to the cerebral cortex of a resting awake human, whereas whole-brain values tend to represent a mixture of stage 2 and stage 3 metabolic states. Recent steady-state values for the human brain are listed in Table 66.3 , together with the estimated steady-state turnover rates of ATP, pyruvate, and lactate.

TABLE 66.3

Average Properties of Human Whole Brain and Cerebral Cortex

Data modified from Kuwabara H, Ohta S, Brust P, et al. Density of perfused capillaries in living human brain during functional activation. Prog Brain Res. 1992;91:209–215; Vafaee MS, Meyer E, Marrett S, et al. Frequency-dependent changes in cerebral metabolic rate of oxygen during activation of human visual cortex. J Cereb Blood Flow Metab. 1999;19:272–277; and Gjedde A, Johannsen P, Cold GE, et al. Cerebral metabolic response to low blood flow: possible role of cytochrome oxidase inhibition. J Cereb Blood Flow Metab. 2005;25:1183–1196.

Variable (Unit)	Whole Brain	Cerebral Cortex
CMR _glc (μmol/g/min)	0.25	0.30
CMRO ₂ (μmol/g/min)	1.40	1.60
CBF (mL/g/min)	0.43	0.50
OGI (ratio)	5.6	5.3
ATP turnover (J _ATP , μmol/g/min)	6.25	7.5
Pyruvate turnover (J _pyr , μmol/g/min)	0.5	0.6
Lactate efflux (J _lact , μmol/g/min)	0.035	0.07
LGI (ratio)	−0.14	−0.23

ATP, Adenosine triphosphate; CBF, cerebral blood flow; CMR _glc , cerebral metabolic rate of glucose; CMRO ₂ , cerebral metabolic rate of oxygen; LGI, lactate-glucose index; OGI, oxygen-glucose index.

The whole-brain molar oxygen-to-glucose ratio or index (OGI) is slightly less than 6, thus indicating that about 90% of the metabolized glucose is fully oxidized. At the normal (default) stage, the total glucose consumption of the human cerebral cortex is about 30 μmol/hg per minute, with an OGI of about 5.5. The 10% nonoxidative metabolism of glucose leads to a rate of lactate production of about 5 to 7 μmol/hg per minute. This lactate flux is about 25% of the maximum transport capacity (T _max ) of the type 1 monocarboxylic acid transporter (MCT1, see later) in the blood-brain barrier at a tissue lactate concentration of about 1.5 mM (see Table 66.3 ). The corresponding ATP turnover in humans is unknown because it depends on the average degree of uncoupling of oxidative metabolism in mitochondria. ^, In the absence of lactate production and uncoupling in mitochondria, the theoretical upper limit of ATP generation is 38 mol per molecule of glucose. However, with lactate production and uncoupling in mitochondria, the average gain is less than 30 mol per molecule of glucose. ^, Brain tissue metabolite stores are listed in Table 66.4 ; an oxidative phosphorylation rate of 7.5 μmol/hg per minute represents less than 1 minute’s worth of ATP turnover in the human brain.

TABLE 66.4

Average Metabolites in Human Brain

Data from Olesen J. Total CO ₂ , lactate, and pyruvate in brain biopsies taken after freezing the tissue in situ. Acta Neurol Scand. 1970;46:141–148; and Roth K, Weiner MW. Determination of cytosolic ADP and AMP concentrations and the free energy of ATP hydrolysis in human muscle and brain tissues with 31P nuclear magnetic resonance spectroscopy. Magn Reson Med. 1991;22:505–511.
From Prichard J, Rothman D, Novotny E, et al. Lactate rise detected by 1H NMR in human visual cortex during physiologic stimulation. Proc Natl Acad Sci U S A. 1991;88:5829–5831; and Sappey-Marinier D, Calabrese G, Fein G, et al. Effect of photic stimulation on human visual cortex lactate and phosphates using 1H and 31P magnetic resonance spectroscopy. J Cereb Blood Flow Metab. 1992;12:584–592.

Metabolite	Cytosol		Glycolytic Equivalents ^a
Metabolite	Concentration (mM)	Content (μmol/g)	ATP (μmol/g)	Lactate (μmol/g)
PC	5.0	4.0	4.0
Glycogen	3.0	2.4	3.6	3.6
Glucose	1.2	1.0	2.0	2.0
ATP	2.2	1.7	1.7
ADP	1.2 × 10 ⁻²	1.0 × 10 ⁻²	5.0 × 10 ⁻³
AMP	7.1 × 10 ⁻⁵	5.6 × 10 ⁻⁵
Pyruvate	0.16	0.13		0.1
Lactate	2.9 (0.75 ^b )	2.3 (0.6†)		2.3
T otal			11.3	8.0

ADP, Adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; PC, phosphocreatine.

a The “glycolytic equivalent” is the ATP reserve that each metabolite would represent in the case of complete depletion.

b MR spectroscopic measurements generally yield lower values of lactate in vivo (0.5–1 mM), but corresponding pyruvate values are not reported, and the determination is indirect.

Attwell and colleagues evaluated the energy demands imposed by the different mechanisms that contribute to functional activity, commonly known as the brain’s energy “budget.” ^, The main components are the requirements for ion homeostasis and impulse generation. The budget also includes processes such as biosynthesis during functional activity in vivo and neurotransmitter vesicle formation, fusion, and release. In the primate brain, almost all of the energy is spent on the restoration of ion gradients through the action of Na ⁺ ,K ⁺ -ATPase. According to the budget, 90% of the energy turnover is devoted to “synaptic” activity and hence maintenance of membrane potential associated with functional activity in the brain. Eighty percent of the turnover occurs in neurons and about 15% in glial cells.

Human brain oxygen consumption may increase to as much as 300 μmol/hg per minute under some physiologic circumstances, accompanied by increased glucose consumption to as much as 50 μmol/hg per minute. ^, These increases are based on MR spectroscopic measurements of the total oxidative metabolism of pyruvate, which may increase to as much as 100 μmol/g per minute in normal human cerebral cortex.

Metabolism Depends On Delivery Of Substrates To The Brain

Glucose Transport

Glucose is the source of energy and enters brain tissue, including neurons and astrocytes, by means of members of the glucose transporter (GLUT) family of membrane-spanning proteins. In brain tissue the important members of this family are the GLUT1 and GLUT3 proteins. The 55-kD GLUT1 protein resides exclusively in the capillary endothelium that constitutes the blood-brain barrier, whereas the alternative 45-kD GLUT1 protein resides in glial cells, the choroid plexus, and the ependyma. ^, The GLUT3 protein occupies the plasma membranes of neurons. Transport of glucose is nonlinearly proportional to the gradient across the different membranes, but the transport is held to be so avid that the glucose concentration is close to the same substantial fraction of plasma glucose everywhere in brain tissue.

The transport capacity of the GLUT1 protein in the blood-brain barrier is known in some detail, and it has been demonstrated that glucose delivery is rarely rate limiting for brain glucose metabolism ( Fig. 66.2 ). ^, ^, ^,

Figure 66.2, Distribution of key transporters in mammalian brain as a schematic representation of the cellular localization of glucose transporters (GLUTs) and monocarboxylate transporters (MCTs) in mammalian brain.

Blood-brain glucose transport can become rate limiting in pronounced hypoglycemia, and it is in principle possible that it also could be rate limiting in conditions of extreme glycolytic activity unless blood flow keeps pace with glucose demand. In the brains of very active rats, Silver and Erecinska found slight decreases in the extracellular glucose concentration, as determined by means of a glucose-sensitive microelectrode placed in brain tissue.

Monocarboxylate Transport

The monocarboxylic acids pyruvate and lactate and the ketone bodies acetoacetate and β-hydroxybutyrate cross brain tissue membranes by facilitative proton-dependent transport catalyzed by the MCT family of 14 membrane-spanning proteins. In brain tissue, the important transporters are MCT1, MCT2, and MCT4. The low-affinity MCT1 and MCT4 transporters dominate the membranes of the capillary endothelium and astrocytes (with MCT4 found only in astrocytes), whereas the high-affinity MCT2 transporter is specific to neurons, particularly the glutamatergic synapses and postsynaptic densities.

The members of the MCT family are near-equilibrium proton symporters and as such are influenced by cell pH such that symport declines when pH rises. In view of the surface area of neurons and glia, it is probable that exchange of pyruvate and lactate among the compartments of the brain is at near equilibrium in steady-state conditions, as it is held to be for glucose. The MCT2 protein has fivefold higher affinity (0.7 mM) for pyruvate and lactate than the MCT1 protein (3.4 mM), and 50-fold higher affinity than MCT4 (30 mM), ^, ^, ^, ^, thus indicating that the MCT2 protein achieves greater occupancy by lactate at normal concentrations than do the MCT1 and MCT4 proteins ( Table 66.5 ). For this reason, it is likely that lactate’s preference for transport by MCT proteins is determined by the lactate concentration in the brain. Therefore because of the low affinity, the glial MCT1 and MCT4 proteins continue to increase lactate transport at higher concentrations. The lower affinities mean that the transporter turnover numbers are higher, which makes the approach to a new steady state faster, other factors being equal. None of the isoforms restrict the exchange of pyruvate at the normal low concentration, but the high affinity of the MCT2 isoform may restrict the exchange of lactate between neurons and the extracellular space at higher concentrations. The restriction depends on the T _max of MCT2, which may increase in common with glutamate receptors during activation.

TABLE 66.5

Estimated Saturability of Transporters and Enzymes of Pyruvate and Lactate

Transporter or Enzyme	Pyruvate				Lactate
	K _Mapp (mM)	V _max (mmol/hg/min)	Saturation		K _Mapp (mM)	V _max (mmol/hg/min)	Saturation
	K _Mapp (mM)	V _max (mmol/hg/min)	0.1 mM	0.5 mM	K _Mapp (mM)	V _max (mmol/hg/min)	1 mM	5 mM
MCT4	30		0.003	0.016	30		0.032	0.143
MCT1 (astrocyte)	3.5	2	0.028	0.135	3.5	2	0.222	0.588
MCT1 (BBB)		0.02				0.02
MCT2	0.7	0.04	0.125	0.417	0.7	0.04	0.588	0.877
mMCT	0.5	0.3	0.167	0.50	0.5	0.3	0.677	0.909
LDH(c)	0.08	20,000	0.556	0.862	1.5/8.6	2000	0.40/0.104	0.769/0.368
LDH(s)	0.03	10,000	0.769	0.943	1.7/7.8	4000	0.37/0.114	0.746/0.391

BBB, Blood-brain barrier; LDH(c), lactate dehydrogenase in cell bodies; LDH(s), lactate dehydrogenase in synaptosome; MCT, monocarboxylate transporter; mMCT, mitochondrial monocarboxylate transporter.

The T _max of the MCT1 protein at the blood-brain barrier is 20 μmol/hg per minute, with a Michaelis constant for lactate of about 3 to 5 mM, which is higher than the normal lactate content of brain (see Table 66.4 ). In brain tissue, the combined transport capacities of the MCT1, MCT4, and MCT2 proteins amount to as much as 2.5 mmol/hg per minute, or 100-fold greater than the blood-brain barrier transport capacity. Because the T _max and half-saturation concentrations or Michaelis constants ( K _t ) of the MCT proteins are about the same for pyruvate and lactate, efflux of lactate and pyruvate across the blood-brain barrier under normal conditions appears to be the rate-limiting step in the lactate and pyruvate tissue distributions. For the same reason, export of pyruvate to the circulation is no more than 10% of that of lactate and thus may be ignored in the greater perspective of brain energy metabolism. The transport of lactate across neuronal membranes is close to saturation because of the high affinity of the MCT2 protein, which implies that the net transport, as well as the direction of the net transport of lactate or pyruvate across neuronal membranes, depends on the clearance of the substrates from the neurons and the interstitium.

Both pyruvate and lactate are transported into mitochondria by a specific mitochondrial monocarboxylate transporter (mMCT). The exact nature of the mMCT protein is not known, but the bulk of the evidence suggests that it is related to the high-affinity MCT2 transporter, although identity with the MCT1 protein is also suggested. ^, Kinship of the mMCT protein to MCT2 rather than the MCT1 protein is supported by the magnitude of the Michaelis constant of mMCT, which is 0.5 μmol/g, or higher than pyruvate’s concentration of 0.1 to 0.2 μmol/g in the cytosol. The T _max of 0.3 mmol/hg per minute depends on mitochondrial density, ^, but in any case is fivefold higher than the average flux of pyruvate in human cerebral cortex (60 μmol/hg per minute). Thus, on average, the rate of pyruvate entry into mitochondria can rise fivefold as the protein approaches saturation.

It is possible that the mMCT protein may become rate limiting for oxidative metabolism in the brain, as in the heart. However, it is more commonly held that the mitochondrial pyruvate concentration saturates the flux-generating pyruvate dehydrogenase (PDH) complex, thus rendering the pyruvate flux independent of the pyruvate concentration and instead a function of PDH and mitochondrial activity.

Distribution of Enzymes and Transporters

Despite the fact that the presence of mitochondria in astrocytes is well documented, astrocytes have a limited capacity to upregulate the oxidation of glucose on increased uptake. One of the reasons for this limited capacity is that in astrocytes, PDH, the rate-limiting step for the entry of pyruvate in the tricarboxylic acid (TCA) cycle, is close to saturation under basal metabolic conditions. The activity of the PDH is regulated by phosphorylation at several phosphorylation sites, resulting in decreased activity. Such a profile favors aerobic glycolysis, namely the production of lactate in the presence of normal oxygen tension. Accordingly, it was shown that dephosphorylation of PDH by dichloroacetate results in a marked increase in oxidative use of glucose by astrocytes. In contrast, neurons cannot upregulate glycolysis and hence do express high oxidative activity. The reason for this lack of ability to increase glycolysis is the fact that the enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-3 (Pfkfb3), a key positive modulator of glycolysis in neurons, is constantly subject to proteasomal degradation.

An analysis of the transcriptome of genes expressed in acutely isolated neurons and astrocytes by RNA sequencing has confirmed the metabolic complementarity between these two cell types. Thus, Pfkfb3 is enriched in astrocytes, and the isoform of pyruvate kinase, PKM2, is exclusively expressed in astrocytes, whereas PKM1 is present only in neurons. The PKM2 isoform can upregulate the glycolytic flux in response to increased energy demands, whereas PKM1 does not have this property. Overall, this selective expression of glucose metabolism genes is configured to drive glucose processing in astrocytes toward aerobic glycolysis and toward oxidative phosphorylation in neurons.

Oxygen Delivery

Oxygen delivery from blood to brain tissue is limited by its binding to hemoglobin. Other factors, such as specific resistance at the endothelium of brain capillaries, may also influence oxygen delivery. A significant fraction of the oxygen transported to brain tissue is extracted during the passage of blood in microvessels in the brain. On average, 40% of the oxygen in blood is extracted, but it may increase to as much as 60%. It appears that oxygen is delivered to the tissue entirely by diffusion and that the large extraction lowers the pressure gradient responsible for diffusion of oxygen. It is possible to calculate the loss of oxygen from the blood that flows through capillaries and hence the decline in oxygen partial pressure, which depends on the extraction fraction. Elevation of blood flow that exceeds the increment in oxygen consumption counters the decline in the pressure gradient, as described by simple one-dimensional models of oxygen diffusion to brain tissue. These models explain the nonlinear relationship between changes in blood flow and changes in oxygen consumption, which ensures that the disproportionately elevated blood flow delivers more oxygen during functional activation. The effect is to maintain mitochondrial oxygen tension relatively constant.

With typical values of the physiologic variables in the equations, the estimated normal distribution of oxygen pressure in the vasculature and tissue compartments is shown in Fig. 66.3 . Quantitative considerations predict that delivery fails when oxygen extraction reaches 60%, in which case extraction cannot increase further because the low capillary oxygen pressure restricts the diffusion. The neurovascular coupling responsible for the relationship between blood flow, oxygen delivery, and oxygen consumption has been the subject of research for the past 30 years, but no firm conclusions have yet emerged.

Figure 66.3, Compartment model of oxygen tensions in brain.

Homeostatic Mechanisms Maintain Constant Adenosine Triphosphate

ATP is the energy “currency” of brain cells that links the energy-using and energy-producing processes. This section describes the mechanisms that maintain a constant ATP concentration regardless of the rate of its expenditure. Generally speaking, these processes are glycolysis, or the breakdown of glucose to pyruvate, and oxidative phosphorylation, or the breakdown of pyruvate to carbon dioxide and the reduction of oxygen to water.

At steady state, the normal stoichiometric relationships between the main substrate fluxes are given by the following equations, which ignore the negligible alternative cytosolic glucose and oxygen sinks but do include a degree of uncoupling of mitochondrial activity from ATP production:

J_{pyr} = 2 J_{glc}

J_{ATP} = β (J_{pyr} + 6 J_{O 2})

and

J_{lact} = J_{pyr} - \frac{1}{3} J_{O 2}

where J _ATP is ATP production, β is coupling efficiency (e.g., 75%), J _glc is glucose consumption, J _pyr is the pyruvate generation rate, and J _lact is the lactate production and efflux rate. These relationships apply only to the steady state, in which there are no changes in substrate concentrations in the brain, and glucose and oxygen do not enter other pathways. The formulation of lactate efflux applies to the tissue as a whole, under the assumption that pyruvate and lactate as monocarboxylic acids are not subject to compartmentation. Thus the accumulation of lactate is a simple function of the pyruvate concentration and lactate exchange across the blood-brain barrier. Under non–steady-state circumstances, the glucose, glycogen, pyruvate, and lactate concentrations change in complex ways (see later).

Under normal circumstances, brain energy metabolism maintains an approximately constant ATP concentration. Observations in the heart and brain suggest that 2- to 10-fold variations in work rate can be sustained with minimal change of ATP. Thus the processes that maintain this metabolite must be sensitive (directly or indirectly) to increased ATP use by feedback or feed-forward mechanisms such as monoaminergic and glutamatergic activation of metabotropic receptors.

Several mechanisms potentially explain the remarkable ability of brain tissue to vary energy turnover, blood flow, and metabolic rates many-fold with little change in ATP concentration. The true ADP concentration is much more difficult to ascertain, but it is likely that it undergoes some increase during elevations of metabolism. Enzymes and transporters are among the proteins that subserve the nonequilibrium and near-equilibrium reactions that could contribute to these mechanisms. Near-equilibrium reactions buffer minute changes in the relevant substrates, but flux-generating and flux-directing nonequilibrium reactions adjust the magnitude and direction of metabolism dictated by extrinsic regulators. The main targets of extrinsic regulators include the nonequilibrium hexokinase I (HK-I) and phosphofructokinase-1 (PFK-1) reactions of glycolysis and the nonequilibrium PDH, citrate synthase, and oxoglutarate dehydrogenase reactions of oxidative phosphorylation in mitochondria.

Hydrolysis of Phosphocreatine

Creatine kinase (CK) occupies a pivotal role in early buffering of the ATP concentration. This cytosolic enzyme has tissue-specific isoforms, including the brain-predominant subtype BB-CK and the Mi-CK isoform bound to the inner mitochondrial membrane. The cytosolic CK reaction is near equilibrium in living human brain. The reaction regenerates ATP by transfer of a high-energy phosphate bond from phosphocreatine (PC) to ADP. When the near equilibrium of the cytoplasmic CK reaction is perturbed, it buffers any increase in ADP by increased phosphorylation of ADP. The cytoplasmic PC is replenished by the Mi-CK, which in turn is regenerated by hydrolysis of ATP synthesized in mitochondria. The advantage is that PC diffuses an order of magnitude faster through the cytosol than do the adenine nucleotides. Yet under conditions of high metabolic activity, ATP homeostasis may be limited by the speed of the CK transphosphorylation reaction in mitochondria.

Glycolysis

Glycolysis is defined as the breakdown of glucose to pyruvate under normal aerobic conditions. The overall reaction is the conversion of one part glucose to two parts pyruvate at the expense of the oxidized form of nicotinamide adenine dinucleotide (NAD ⁺ ), which is converted to the reduced form of NAD (NADH) and ADP, which in turn is phosphorylated to ATP. The gains in NADH and ATP are important because these metabolites block glycolysis when they appear in excess. Some pyruvate is always converted to lactate, a process that regenerates NAD ⁺ and contributes to the glycolytic flux. The magnitude and direction of this conversion by the near-equilibrium lactate dehydrogenase (LDH) depends on the relative net losses of lactate or pyruvate. The formation of lactate from pyruvate at normal oxygen tension sometimes is known as the Warburg effect when it occurs in tumor cells, and by extension in brain.

In the brain the rate of glycolysis is regulated by the nonequilibrium reactions catalyzed by the enzymes HK-I and PFK-1. Most of the hexokinase protein in brain tissue is coupled to a mitochondrial protein complex consisting of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), and the mitochondrial translocator protein (TSPO), spanning the two mitochondrial membranes. As little as 15% is present in soluble form in the cytosol, but the distribution is variable and depends on numerous factors that are not fully understood. The functional significance of the soluble and bound forms of the enzyme is also uncertain.

Because glucose is normally the preferred substrate for brain metabolism, control of glycolysis is integral to understanding how increased energy production and use are linked. This regulation also explains the circumstances in which brain metabolism in general or metabolism in a separate cellular compartment such as neurons or glial cells has a preference for ketone bodies, lactate, or acetate when these substrates are present in excess (lactation, starvation, physical exertion, and possibly neuronal excitation).

The main reactions and their time constants are listed in Table 66.6 , and their interactions are shown in schematic form in Fig. 66.4 . The time constants dictate the half-times of change—that is, the time that it takes the system to reach the halfway point of a new steady state. It is apparent from Table 66.6 that glycolysis responds to change with time constants on the order of milliseconds, whereas oxidative metabolism responds with time constants of seconds or minutes. Hence, oxidative metabolism responds to any stimulus with a certain delay, compared with glycolysis.

TABLE 66.6

Selected Brain Metabolic Reactions and Metabolite Transporters

Reaction or Transporter	Equilibrium Status	Activity (μmol/g ⁺¹ /min ⁺¹ )	Substrate Concentration (μmol/g ⁺¹ )
Reaction or Transporter	Equilibrium Status	Activity (μmol/g ⁺¹ /min ⁺¹ )	Substrate Concentration (μmol/g ⁺¹ )	Time Constant (s)
HK-I	Flux generating	0.3	2	400
PFK-1		0.3	0.1	20
PDH		0.3	0.01	2
GLUT1	Flux limiting	2	5	150
MCT (BBB)		0.2	0.1	30
mMCT (mitochondria)		3	0.1	3
GLUT3	Near equilibrium	1000	1	60
LDH1-5 (lactate)		2000	1	30
MCT1-MCT4 (cell membranes)		1000	0.1	6
LDH1-LDH5 (pyruvate)		2000	0.1	3

BBB, Blood-brain barrier; GLUT, glucose transporter; HK, hexokinase; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; mMCT, mitochondrial monocarboxylate transporter; PDH, pyruvate dehydrogenase; PFK, phosphofructokinase.

Figure 66.4, Simplified illustration of the main metabolic pathways active in mammalian brain.

The important regulators of HK-I and PFK-1 are phosphate, magnesium, citrate, ammonium, and hydrogen ions; adenosine monophosphate; and PC. Changes in Mg ²⁺ ions accompany increased ATP turnover and increased CK activity. Although 2 mol of ATP is consumed per mole of glucose metabolized during the first stage of glycolysis, 4 mol of ATP is generated during the second stage of glycolysis, for a net return of 1 mol of ATP and two hydrogen ion equivalents per mole of pyruvate synthesized.

Glucose-6-phosphate occupies a pivotal position in glycolysis where it links a number of reactions, including the main glycolytic pathway itself, the pentose phosphate pathway, the glycogenesis pathway, and the important hexosamine and nucleotide sugar pathways of glycosaminoglycan and glycoprotein biosynthesis that require glucose-6-phosphate as precursor (UDP-Glc, uridine diphosphate glucose; UDP-GlcNAc, uridine diphosphate N -acetyl-glucosamine). These pathways are responsible for the fates of glucose-6-phosphate, but the relative contributions are not known with certainty, although they are likely to be of minor quantitative importance.

An important step in glycolysis is the reaction catalyzed by the triose phosphate dehydrogenase step. The enzyme transfers hydrogen from glyceraldehyde phosphate to the intermediate NAD ⁺ to form NADH, which plays a key role in the integration of glycolysis with oxidative metabolism because it influences the ratio between pyruvate and lactate in the cytosol and the metabolic rate of mitochondria. In turn, this ratio determines the direction and net flux of the reaction between pyruvate and lactate and the competition between glucose and lactate as sources of pyruvate when lactate is available in excess.

Lactate Synthesis

Pyruvate directly participates in at least three primary reactions in brain tissue. It can be reduced by conversion to lactate, transported into mitochondria, or removed from cells by the MCT in the cell membranes and in the endothelium of brain capillaries. The last of these processes usually is ignored because of pyruvate’s low concentration relative to lactate. In mitochondria, pyruvate is the substrate for the PDH-complex reactions and, in some cells, the pyruvate carboxylase reaction.

As a near-equilibrium reaction, LDH buffers changes in the concentration of pyruvate because the LDH reaction is strongly balanced toward lactate. Synthesis of 1 mol of lactate from 1 mol of pyruvate removes one cytosolic reducing equivalent, with a net yield of only 2 mol of ATP per mole of glucose converted to lactic acid.

LDH is a tetrameric protein composed of one or more of two subunits, A and B, or M (for muscle type) and H (for heart type), respectively. The combinations yield five normal variants or isozymes (A ₄ = LDH5, A ₃ B = LDH4, A ₂ B ₂ = LDH3, AB ₃ = LDH2, and B ₄ = LDH1). The brain has all of the isozymes of LDH, but the “muscle” isozymes LDH4 and LDH5 predominate, particularly in astrocytes. Synaptic terminals have a little more of the “heart” isozyme LDH1 than cytosol in general. The distribution of LDH isozymes in the cytosol of both neurons and astrocytes and synaptic terminals is shown in Fig. 66.5 .

Figure 66.5, Schematic illustrating the possible role of lactate dehydrogenase (LDH) isozymes and monocarboxylate transporters (MCTs) in the trafficking of substrates between astrocytes and neurons in brain and the distribution of LDH activities in mammalian brain.

Both LDH4 and LDH5 have medium affinities for pyruvate and lactate that are close to the normal tissue concentrations of these metabolites, similar to those of less oxidative tissues such as liver and some muscle cells. The relatively intermediate affinities for pyruvate and lactate are reflected in the measured lactate-pyruvate concentration ratio of 10:15. Because the near-equilibrium LDH reaction is easily saturated at higher pyruvate and lactate concentrations, these higher concentrations tend to prevent net conversion from pyruvate to lactate or vice versa. The observation that the affinity of LDH for pyruvate is 10-fold higher than the affinity of mMCT for pyruvate (see Table 66.5 ) suggests that pyruvate prefers entry into mitochondria at higher pyruvate concentrations.

The entities controlling the steady-state lactate-to-pyruvate ratio, in addition to the inherent affinities of the enzyme for the two substrates, which must have the same ratio for all the subtypes present in a cell for thermodynamic reasons, include both pH and the NAD ⁺ :NADH ratio according to the following relationship:

\frac{[Lact]}{[Pyr]} = K_{eq} \frac{[NADH] [H^{+}]}{[{NAD}^{+}]}

where K _eq is the equilibrium constant of the LDH reaction. The ratio between the concentrations of lactate and pyruvate is also the ratio between their affinities. At steady state, the ratio must be the same everywhere, given the near-equilibrium and facilitated diffusion nature of the proton symporters of lactate and pyruvate. The constant ratio implies that the different inherent kinetic properties of LDH exist to accommodate differences of the NAD ⁺ :NADH ratios in different parts of the tissue.

The affinity of an enzyme for a substrate is a function of, among other factors, the turnover number of the enzyme, thus indicating that the velocity of the reaction is related to the affinity constant. A higher affinity constant (i.e., a lower affinity) will result in a higher turnover number and hence a higher reaction velocity, other factors being equal. The cytosolic NAD ⁺ :NADH ratio is an indicator of the oxidation status of the tissue, which is low when the ratio is lower than normal (<1000) and high when it is higher than normal (>1000).

It is a common claim that the relatively low affinity of LDH1 for pyruvate makes this enzyme particularly useful to a tissue with high oxidative capacity because it allows rapid buildup of pyruvate, whereas LDH5 is more effective at buffering the increase in pyruvate in a tissue with lower oxidative capacity because of its relatively high affinity for pyruvate. There is evidence that more LDH1 and the messenger RNA (mRNA) for the subtype are found in synaptic terminals than in neuronal and astrocytic cytosol in general, which in turn appears to possess particularly abundant LDH4 and LDH5 subtypes and their corresponding mRNA. ^, For practical purposes, however, it is more useful to regard the properties of the LDH subtypes as being regulated to match the prevailing NAD ⁺ :NADH ratios during transient events of activation or deactivation of the tissue, as discussed later. Interesting to note, LDH has been shown to be a potential molecular target for epilepsy treatment. Indeed, decrease of lactate production evoked by patch-pipette injection into an astrocyte of an inhibitor of LDH results in a hyperpolarization of neighboring neurons. Based on this in vitro observation, the authors developed a novel set of antiepileptic drugs that target LDH. ^,

Oxidative Phosphorylation

At steady state, brain metabolism has a respiratory quotient of unity, consistent with the oxidation of glucose and with the integration of glycolysis and oxidative metabolism. The net effect of the metabolism of pyruvate to CO ₂ is to provide the electron chain complexes with the nicotinamide (NADH) and flavin (FADH ₂ ) adenine dinucleotides necessary for electron transport and oxygen metabolism. This subsection presents the primary regulatory steps for oxidative metabolism in mitochondria.

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