Effects Of Lactate And Carbon Monoxide Interactions On Neuroprotection And Neuropreservation Part 2

Fatty acid β-oxidation pathway

Lipids (classified into five major subcategories – fatty acids, triglycerides, sphingolipids, sterol lipids, and phospholipids) are important to normal CNS function and development and serve as bioactive molecules, energy substrates, acting as building blocks or a combination of these. 

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Fatty acid imbalance affects CNS development and function. Fatty acids are the essential component of all lipids and consist of a carbon chain that ends in a carboxylic acid functional group (subclassified as short-chain fatty acids (2–4 carbons), medium-chain fatty acids (6–12 carbons), long-chain fatty acids (14–18 carbons), and very long-chain fatty acids with 18+ carbons.) If saturated, all of the carbons have hydrogen atoms and single bonds only exist between carbons whereas unsaturated fatty acids have carbon chains where a double bond has been introduced (mono- versus polyunsaturated defines whether there is more than one double bond.) Fatty acid β-oxidation results in the formation of acetyl-CoA as noted in the equation: β-oxidation of fatty acids->->->->->- >->->Acetyl-CoA (Figure 1).

Mitochondrial β-oxidation of fatty acids results in energy – a repeating sequence of four reactions catalyzed by acyl-CoA dehydrogenase, enoyl-CoA thiolase, hydroxy acyl-CoA dehydrogenase, and ketoacyl-CoA thiolase. 

Acetyl-CoA results in a fatty acyl molecule that is two carbons shorter –the reaction cycle repeats (Figure 1). With an odd number of carbon atoms, propionyl-CoA is the final product. The acetyl-CoA enters the tricarboxylic acid cycle and pyruvate and, possibly, lactate may be generated. Propionyl-CoA is converted into succinyl-CoA (substrate for gluconeogenesis via oxaloacetate formation). 

Pyruvate and, possibly, lactate may be generated.46-50 Cytoplasmic fatty acyl CoA is converted to fatty acyl carnitine by carnitine acyl transferase I, an enzyme of the inner leaflet of the outer mitochondrial membrane. Fatty acylcarnitine is then transported by an antiport in exchange for free carnitine to the inner surface of the inner mitochondrial membrane. 

There carnitine acyl transferase II reverses the process, producing fatty acyl-CoA and carnitine. This shuttle mechanism is required only for longer-chain fatty acids as follows:

Medium- and short-chain fatty acids are carnitine-independent. They cross the mitochondrial membranes and are activated in the mitochondrion. In the astrocyte and neuron mitochondria and cytosol, the four steps of β-oxidation include dehydrogenation of the fatty acyl-CoA to make a trans double bond between α and β carbon (requires short, medium, and long chain acyl-CoA dehydrogenases, electron removed transferred to flavin adenine dinucleotide), hydration of the double bond, dehydrogenation of the β-hydroxyl group to a ketone (electron removed transferred to NAD+ ), acylation (addition of CoA and production of acetyl-CoA.

Urea cycle

While the urea cycle and the tricarboxylic acid cycle (TCA) are independent cycles, they are linked. Fumarate that is produced in the cycle is an intermediate in the TCA cycle, metabolized to malate, and then to pyruvate and lactate (Figure 2). Urea cycle - Citrulline->->->argininosuccinate->->- >arginine and fumarate TCA cycle - Fumarate->->->malate.51-54

Pentose phosphate pathway

Taking place in the cytosol, the pentose phosphate pathway is anabolic more than catabolic, generates reducing equivalents, produces ribose 5-phosphate (needed for the synthesis of nucleotides and nucleic acids) and erythrose 4-phosphate (needed for the synthesis of aromatic amino acids), and is linked to glycolysis. During the oxidative phase (irreversible), glucose 6-phosphate is metabolized to ribulose 5-phosphate. The latter can be metabolized to fructose 6-phosphate during the nonoxidative phase (reversible.)

Oxidative phosphorylation

Cellular respiration depends on glycolysis (cytosol) which results in pyruvate, pyruvate transformation into acetylCoA (mitochondria), the citric acid cycle (acetyl-CoA is modified in the mitochondria to produce energy precursors), and oxidative phosphorylation (or electron transport-linked phosphorylation) – process where electron transport from the energy precursors from the citric acid cycle leads to the phosphorylation of adenosine diphosphate producing ATP (occurs in the mitochondria). 

Uncoupling between glycolysis and oxidative phosphorylation involves the partitioning between pyruvate (the primary substrate for glucose-driven oxidative phosphorylation) and lactate. In the mitochondria, succinate is reduced to fumarate by Complex II. 

The latter enters the Krebs cycle and pyruvate (and then lactate via lactate dehydrogenase (LDH)) can be produced.58-60 Nitric oxide production also inhibits the mitochondrial respiratory chain at cytochrome c oxidase in astrocytes and the majority of glucose consumed is released as lactate.61

Citric acid cycle

Oxidation of acetyl-CoA to CO2 by the citric acid cycle is the central process in energy production. The process also results in the formation of intermediates which can be converted primarily to fatty acids, glucose derivatives, γ-aminobutyric acid (GABA), glutamate, glutamine, and aspartate. 

The balance between the two biochemical processes related to the cycle (anaplerosis and cataplerosis) is essential to normal CNS physiology.62 Pyruvate carboxylation is important for the net synthesis of glutamate, GABA, and aspartate (anaplerosis) and is associated with cerebral activity - flux through pyruvate carboxylase is higher in the awake state compared to deep phenobarbital anesthesia.63 

However, the brain cannot fully metabolize 4- and 5-carbon molecules – they must be removed by cataplerosis, a process that may be linked to biosynthetic processes resulting in the synthesis of fatty acids and amino acids and gluconeogenesis. 

Glutamate seems to be the molecule connecting anaplerosis and cataplerosis in the Krebs cycle. 

Cytosolic malic enzyme and phosphoenolpyruvate carboxykinase in astrocytes convert malate and oxaloacetate into lactate which leaves the brain (blood or periventricular system.) Efflux of lactate allows for ongoing pyruvate carboxylation.64-66

Movement of lactate in the brain Lactate shuttles

The lactate shuttle hypothesis describes the movement of lactate within and between cells and is based on the observation that lactate is formed and utilized continuously in diverse cells under both anaerobic and aerobic conditions. 

Interconversion of lactate and pyruvate occurs via lactate dehydrogenase and is produced at sites with high rates of glycolysis and glycogenolysis. Lactate plays an important role as a shuttle and can be shuttled to adjacent or remote sites and can be used as a gluconeogenic precursor or substrate for oxidation. 

The hypothesis also addresses the role of lactate in lipolytic control, redox signaling, and gene expression and affirms the role of lactate in cell signaling, endocrine/autocrine/paracrine link between oxidative and glycolytic metabolism, and the delivery of gluconeogenic and oxidative substrates – L-lactate is considered the link between aerobic and glycolytic pathways (substrate for mitochondrial respiration and a product of glycolysis.).

Glycolytic lactate producer cells provide lactate that is taken up by the blood and mitochondrial reticulum (oxidative lactate consumer cells.)67,68 The anion is metabolically available under aerobic conditions and is important in a complicated feedback loop – lactate production is promoted with decreased ATP levels which leads to the cells ability to promote ATP homeostasis. 

Redox state, concentration gradient, and/ or pH gradient are known to drive these shuttles.6 Cell-to-cell lactate interactions are important to CNS function. There is a normal physiological function and the relationship between astrocytes and neurons – the astrocyte-neuron lactate shuttle. 

An increase in synaptic activity or low glucose conditions results in the transfer of lactate from astrocytes to neurons which sustains neuronal oxidative metabolism. 

Astrocytes express MCT4 (a low affinity transporter for lactate suggesting that lactate produced by glycolysis is exported.) Neurons express MCT2 (a high-affinity transporter for lactate.).

The hypothesis is that astrocytes produce lactate which is then taken up by neurons and oxidized for energy.69-71 There are other CNS cell-to-cell lactate shuttles as well and these include microglia and their preferential utilization of lactate under pathologic conditions, the activity of lactate shuttles in traumatic brain injury and neurodegenerative diseases, and the astrocyte–microglia lactate shuttle.6,15,72,73.

Cell-to-cell, when coupled to intracellular, lactate shuttles allow for the simultaneous presence of glycolytic (i.e., glucose to lactate) and oxidative (i.e., lactate to pyruvate to acetyl-CoA) pathways. This intracellular lactate pathway – lactate exchange and conversion into pyruvate maintaining the redox balance in the cytosol and mitochondria – is thought to exist between cytosol and mitochondria and between cytosol and peroxisomes.15,74

Glymphatic system

About 60% to 68% of the brain's total water content is within the intracellular space and 32–40% in the extracellular space (12% to 20% in the interstitial fluid (ISF), 10% in the cerebrospinal fluid (CSF), 10% in the blood.) The CSF, formed by the choroid plexuses (no BBB), depends on the osmotic and hydrostatic movement of fluid, crystalloids, and colloids from plasma into the stroma. 

The glymphatic system, integral to CNS fluid balance and removal of waste, has glial-dependent perivascular channels formed by astroglial cells, is dependent on aquaporin (AQP) 4 water channels on astrocyte endfeet, has peri-venous clearance and peri-arterial influx pathways, is connected to the lymphatic system associated with the dura, cranial nerves, and vessels which are located at skull exits, and allows continuous ISF and CSF removal of potentially harmful molecular products. 

Activity is increased during general anesthesia and sleep and is decreased with stroke, diabetes mellitus, Alzheimer's disease, and aging.75-82 CNS lactate (higher while awake than during sleep and associated with reduction with cerebral glycolysis), a biomarker of the sleep-wake cycle, is exported as lactate by the glymphatic system – brain lactate concentration is inversely correlated with glymphatic-lymphatic clearance and resulting lactate/pyruvate ratio (and NADH/NAD redox potential) – and decreases rapidly with the onset of sleep. 

Elimination of lactate during sleep combined with the lack of elimination during wakefulness results in an increase of redox state with wakefulness (relative to sleep.)83-85

ELIMINATION OF LACTATE FROM THE CENTRAL NERVOUS SYSTEM

Maintaining a normal lactate level in the CNS depends on its utilization versus its accumulation. While the resting brain releases lactate (release from various brain structures differs under basal conditions), this increases incrementally with activation of the brain.86 

Total lactate clearance is determined by its metabolism in the CNS, excretion via capillaries, or excretion through the CSF – clearance systems can be via CSF, ISF within the CNS, and blood.86-88 Three major models of lactate elimination via brain fluids have been hypothesized: 1) meningeal lymphatics,89 the glymphatic system,83,90, and the intramural periarterial system.91,92 The lymphatic system is a part of the vascular system. 

Peripherally, plasma is filtered through the vascular endothelium of capillaries in the extracellular space with over 80% being reabsorbed back into venous vessels and the remainder in the extracellular fluid or ISF. 

ISF is then filtered back into the lymphatics which return it into the venous system. In the CNS, the volume of brain fluid is fixed, there is BBB restriction of fluid filtration from plasma into the brain, and there is no common agreement about ISF formation in the brain. 

The three models of ISF formation that have been proposed include production by brain metabolism, cerebral capillary secretion of solutes, and ISF is a fraction of recycled CSF (flows from choroid plexus into subarachnoid space and then into perivascular space where CSF merges with ISF that has been generated by cerebral capillaries) – lymphatic communication with brain fluids affects the clearance of lactate as well as that of waste products, bacteria, viruses, toxins, etc.93-95 

The extensive meningeal lymphatic vessel network that helps with macromolecular clearance and immune cell trafficking in the CNS also communicates with the glymphatic system, a system that allows CNS perfusion by ISF and CSF and complements intramural, periarterial clearance.96-98

EXCESS OR TOO LITTLE LACTATE

Maintenance of lactate levels is necessary for normal health and well-being – an excess or relatively too little can result in changes in body metabolism. Fluctuations are not usually due to low oxygen levels as hyperlactatemia can occur with normal tissue perfusion/oxygenation and relative hypolactatemia with poor tissue perfusion/oxygenation. 

Although lactate (the major gluconeogenic precursor, a signaling molecule, and a major energy source) production occurs normally during rest, it is increased with sepsis, pancreatitis, trauma, heart failure, etc.68,99-102 The resulting lactic acidosis, overproduction or underutilization of lactic acid with the body not being able to adjust to these changes, is associated with a buildup of acid and an imbalance of the body's pH level. 

Elevated levels are used as a biomarker for risk and therapy and high levels are associated with an increased risk of death independent of organ failure and shock.103-105 Cellular metabolic reprogramming, metabolic inflexibility, and a decrease in cellular proliferation occur.106-111 Effects on the cardiovascular system can result in hyporesponsiveness of the vascular system to vasopressors and reduced cardiac contractility. NAD+ /NADH ratio, production of reactive oxygen species, regulation of genes, the release of vascular endothelial growth factor/interleukin-1/ transforming growth factor-β, and activation of Sirtuins are affected.101,109-112 Severe lactic acidosis has been suggested as a prerequisite of brain infarction from complete ischemia. 

This may be due to elevated brain glucose metabolism to lactate and its effect on astrocytes as excessive lactate is considered to be detrimental to the CNS and can affect recovery from brain trauma, anoxia, epilepsy, and ischemia.102 However, metabolism and cellular interactions determine if lactate is a waste product or a useful substrate. 

While acute and chronic pathologies alter brain metabolism resulting in changes in lactate concentration, lactate is a preferred fuel over glucose in brain preparations.32,112,113 When there is an increase in blood lactate in exercising humans (or when exogenous lactate is supplied), lactate is the major gluconeogenic precursor and substitutes for glucose as an energy substrate – cerebral lactate uptake increases. 

Improved cognitive function in brain-injured rats given intravenous lactate therapy and rapid increases of lactate MCT protein expression in rat brains following traumatic brain injury suggest that lactate metabolism in humans following traumatic brain injury is significant to recovery. The roles of lactate in normal cerebral metabolism and with CNS pathology are being redefined. 

Lactate supports hepatic and renal gluconeogenesis which is important to brain metabolism, production by the body indirectly supplies glucose to the injured brain, isotopically labeled lactate is directly consumed and used by the injured human brain, and exogenous lactate infusion augments cerebral substrate supply when glycolysis is affected.113-116 Lactate changes, reflective of overall metabolic health, are affected by heme-oxygenase (and CO) and affect the health of the CNS.

CARBON MONOXIDE EFFECTS ON LACTATE IN THE CENTRAL NERVOUS SYSTEM

CNS extracellular lactate concentration increases with neuropathology as well as CNS activation - both result in significant release of lactate.117 The latter, which can be associated with excitotoxicity, may result in neuronal damage and death as excitotoxicity is a major component of CNS pathology.118-122 L-lactate (protects neurons against excitotoxicity, a regulator and metabolite in the CNS, regulates the expression of synaptic plasticity and neuroprotection genes in cortical neurons, promotes angiogenesis/immune escape/cell migration, serves as a glucose-sparing substrate, regulates cerebral blood flow, and plays an important role in learning and memory)123-127 acts as a signaling molecule in pathological states,123-125 is neuroprotective, and effects are directed at neurotransmission. 

CO has been found to modulate L-lactate levels in astrocytes providing evidence that the heme oxygenase and L-lactate neuroprotective systems interact.8 The relationship between CNS L-lactate and the heme oxygenase/CO system is complex. The cytoprotective properties of the heme oxygenases are attributed to the production of CO. 

Endogenous production originates from heme metabolism (at least 86%) and heme-independent sources which include iron ascorbate-catalyzed lipid peroxidation of microsomal lipids and phospholipids, photo-oxidation of organic compounds, auto- and enzymatic oxidation of phenols, and reduction of cytochrome b5. 

Heme oxygenase isozymes catalyze the first and rate-limiting step in the degradation of heme to CO, iron, and biliverdin. Neuroprotective effects of CO are at metabolic (metabolic support, regulation of local blood flow, glycogen synthesis/ storage, formation of the neuro-glial-vascular unit and glial-vascular interface), molecular (regulation of pH, water transport/ homeostasis, neurotransmitter homeostasis, ion homeostasis), organ (effects on the lymphatic system and BBB), cellular/network (synaptic plasticity, synaptogenesis/maintenance/elimination, neurogenesis, neuronal development/guidance, defining architecture of CNS), and systemic levels (affects sleep, regulates energy balance, important to chemosensing) levels. Interactions between CNS L-lactate and the heme oxygenase/CO systems affecting neuroprotection can occur at multiple levels.

Metabolic

Metabolic support

Energy metabolism supports cerebral function, has moment-to-moment dynamic ranges, involves essential functional and metabolic interactions between neurons and astrocytes, and is compartmentalized. 

Neurons and astrocytes are dependent on each other for metabolic support of the CNS. Formation of lactate by astrocytes is a response to neuronal stimulation – lactate supplements CNS energy provision during euglycemia, may contribute to an increase in ATP reserves (brain prioritizes regulation of its own ATP concentration), and is a primary tricarboxylic acid cycle substrate. 

The rate of oxidative metabolism in astrocytes can increase as much as the rate of neuronal metabolism in response to sensory stimulation. In this milieu, a standing lactate gradient exists with higher lactate concentration in astrocytes than neurons and neuronal lactate may be extruded to nearby neurons with lower lactate concentrations or the extracellular space. 

Astrocytic K+ uptake, but not astrocytic Na+-coupled glutamate uptake, is important for the establishment of a neuron-astrocyte metabolic partnership. A decrease in the oxygen-glucose index – accompanied by stimulation of pyruvate formation (and, therefore, lactate formation) and astrocyte respiration – is also seen with neuronal activation.3,60,65,128,129 Astrocytes are targets for treating neurological disorders as neurons are outnumbered by non-neuronal cells (i.e., astrocytes) and astrocytes contain glycogen which can be transformed into L-lactate (see above). 

Increased local levels of CO may increase astrocyte L-lactate formation and, therefore, affect the expression of neurologic diseases.8 Studies are needed to further evaluate the effects of CO on astrocyte lactate formation (and on metabolic support) and the interplay between CO, astrocyte lactate formation, metabolic support, and neurologic diseases.

Regulation of local blood flow

Neurovascular coupling results in a local increase in blood flow to match energy demands and occurs physiologically and in disease at the capillary and arteriole levels (differ mechanistically.).

At the capillary level, this is dependent on calcium signaling (calcium concentration changes in response to neuronal activity.) Arteriole dilation is dependent on Nmethyl-D-aspartate (NMDA) receptor activation and nitric oxide synthesis.130 CO and lactate affect calcium signaling and NMDA receptors and, therefore, potentially local CNS blood flow and the latter may be dependent on CO concentration. 

CO is known to be an endogenous vascular modulator functioning as both a vasoconstrictor and vasodilator and is highly protective of the vasculature. The vasoconstrictor action may be associated with the generation of reactive oxygen species.131 In the CNS, the gasotransmitter dilates cerebral arterioles. Vasodilatory stimuli (seizures, hypoxia, adenosine diphosphate, glutamatergic stimulation) result in an increase in CO. CO binds to smooth muscle cell Ca2+-activated K+ channel BKCa) channel-bound heme which leads to an increase in Ca2+ sparks to-BKCa coupling (also binds directly to the BKCa channel at different locations) resulting in vasodilation. Inhibition of heme oxygenase affects the dilation of the vasculature to these stimuli. 

Apoptosis and oxidant-generating pathways are also inhibited.132,133 Lactate may act as a dose-dependent regulator of cerebral microcirculation by affecting the hyperemic response, which may be related to a cytosolic redox impairment, and optimization of blood flow may interact with nitric oxide and arachidonic acid metabolites released by neuronal and glial cells during neural activity.109,134,135 As both CO and lactate affect calcium channels and NMDA, the relationship to each other, calcium channels/NMDA, and CNS local blood flow are important questions that have not been addressed. Further studies are needed.

Glycogen synthesis and storage

A polymer of glucose, glycogen is present in the CNS at low concentrations relative to the liver and skeletal muscle and, in developed brains in mammals under normal conditions, is located predominantly in astrocytes (has been found in embryonic neurons.) Synthesis is a multi-step process, is tightly regulated, and glucose transport across the BBB is needed. 

Glycogen synthase and the glycogen branching enzymes elongate glucose chains and introduce branch points. Astrocytes depleted of glycogen are not as successful at supporting neurons as are those not depleted of glycogen.136-138 Astrocyte glycogen metabolism is important for the functioning of the CNS. The substance is too large to be released and travel between cells, but studies have shown that astrocytes release lactate into the media.1,3,7,8,19,22 Modulated by several factors, neuronal activity signals result in astrocyte release of lactate – formed predominantly in astrocytes from glucose or glycogen and transferred from astrocytes to neurons to match the neuronal energetic needs and to provide signals that modulate neuronal functions and important to CNS health and disease. CO modulates L-lactate levels (and, therefore, glycogen metabolism) in astrocytes and is being further studied.3,8,139,140.

Formation of neuro-glial-vascular unit and glial-vascular interface

Composed of neurons, astrocytes, microglial cells, endothelial cells, pericytes, smooth muscle cells, and circulating blood cells, the neuro-glial-vascular unit interacts to control synaptic communication, BBB function, local blood supply, neuronal development, and surveillance/immune function. 

Models (Buxton-Want model of vascular dynamics, Hodgkin-Huxley formulation of neuronal membrane excitability, biophysical model of metabolic pathways) of the neuro-glial-vascular unit describe neural dynamics and propound that the energy inside of the neuron is dynamic and depends on neural activity – lactate in astrocytes can fuel neuronal activity – and local vasculature. 

The astrocyte-neuron lactate shuttle is predicted by these models – temporal dynamics of tissue lactate, tissue glucose, and oxygen consumption, and BOLD signal reported in human studies correctly predicted, transfer of lactate from astrocytes to neurons noted in response to activity, dynamics of extracellular lactate and oxygen as observed in vivo in rats correctly predicted, neuronal oxidative metabolism increased first upon activation with a subsequent delayed astrocytic glycolysis increase.141,142 This requires MCT. 

Nitric oxide has been shown to down-regulate MCT 1. The effect of CO on MCTs and, therefore, lactate effects on the neuro-glial-vascular unit have not been studied.

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