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SMCT1 is a sodium-coupled (Na+-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, β-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, α-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na+-coupled and electrogenic. The Michaelis constant is 1.4 ± 0.1 mm for β-d-hydroxybutyrate, 0.21 ± 0.04 mm for acetoacetate and 0.21 ± 0.03 mm for α-ketoisocaproate. The Na+ : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na+-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na+-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na+ gradient and hence, contribute to the maintenance of the energy status and function of neurons.
Glucose is the major energy substrate used by the brain (Pellerin and Magistretti 2004). The metabolism of glucose in the brain is compartmentalized. Even though the metabolic utilization of glucose in the brain is increased during intense neuronal activity, uptake of glucose in astrocytes, rather than in neurons, accounts for most of this activity-associated glucose utilization (Pellerin and Magistretti 2004). Astrocytes convert glucose into l-lactate, a monocarboxylate, and release it into the extracellular medium to be taken up subsequently by neurons. Thus, the energy needs of the active neurons are not met directly by glucose oxidation but rather, by oxidation of l-lactate supplied by astrocytes. l-Lactate is therefore the primary metabolic fuel for neurons under normal physiological conditions. Normal levels of l-lactate in blood are quite significant (1–1.2 mm) and this l-lactate in the circulation is also available for neurons, in addition to the l-lactate generated by astrocytes.
Under circumstances when glucose availability is limited, such as during the suckling period in mammals (Hawkins et al. 1971; Cremer 1982), prolonged starvation and uncontrolled diabetes (Gjedde and Crone 1975; Hawkins et al. 1986), neurons must rely on substrates other than l-lactate to fulfil their high energy demands and sustain normal function. To preserve normal brain function, the body begins to mobilize its fat stores. Fatty acids released from the adipose tissue then undergo β-oxidation in the liver, which produces the ketone bodies β-d-hydroxybutyrate (β-D-HB) and acetoacetate for subsequent use as metabolic substrates by the neurons (Mitchell et al. 1995; Pellerin and Magistretti 2004). The concentration of ketone bodies in the circulation is usually very low (about 0.3 mm) and, under normal circumstances, these substrates are of little physiological relevance as a source of energy in the brain. However, during conditions such as pregnancy, starvation and uncontrolled diabetes, the concentration of these substrates in the blood increases substantially (approximately 10 mm) (Laffel 1999). Recent evidence suggests that astrocytes may also serve as a significant source of ketone bodies for the support of brain function (Guzman and Blazquez 2004). Similar to the interaction between the astrocytes and neurons in l-lactate production and utilization, ketone bodies are also shuttled between astrocytes and neurons (Guzman and Blazquez 2004).
l-Lactate and ketone bodies enter the brain across the endothelial cell layer of the blood–brain barrier via monocarboxylate transporters (MCTs) (Pellerin et al. 2005; Pierre and Pellerin 2005). MCTs belong to the solute-linked carrier gene family, SLC16 (Halestrap and Meredith 2004). Three MCT isoforms (MCT1, MCT2 and MCT4) are expressed in the brain. MCT1 and MCT4 represent low-affinity transporters and are expressed in astrocytes whereas MCT2, a high-affinity transporter, is expressed primarily in neurons (Pellerin et al. 2005; Pierre and Pellerin 2005). MCT1 is also expressed in the blood–brain barrier, where it plays a role in the transfer of blood-borne monocarboxylates into the brain, and its expression is subject to regulation under certain physiological and pathological conditions (Gerhart et al. 1997; Leino et al. 2001). Thus, the existing view of the metabolic utilization of l-lactate and ketone bodies by the brain is as follows. l-Lactate and ketone bodies present in the circulation are transported into the brain across the blood–brain barrier via MCT1, and neurons then take up these metabolites via MCT2. Astrocytes take up glucose and fatty acids, metabolize them to l-lactate and ketone bodies, respectively, and release these metabolites into the extracellular medium via MCT1 and MCT4; neurons take up this astrocyte-derived l-lactate and ketone bodies via MCT2. Therefore, the current belief is that MCT2 is the principal transporter responsible for meeting the energy needs of the neurons by providing l-lactate and ketone bodies under various physiological and pathological conditions.
Recently, we identified an Na+-coupled transporter for the transport of several monocarboxylates, including l-lactate (Ganapathy et al. 2005; Gupta et al. 2006). This transporter, known as SMCT (SLC5A8) (Sodium-coupled MonoCarboxylate Transporter), belongs to the solute-linked carrier gene family SLC5. In the light of the most recent studies in our laboratory that have identified a second isoform of SMCT, SLC5A12 (Srinivas et al. 2005), we refer to the SMCT described herein as SMCT1. The present study was undertaken to investigate the handling of ketone bodies (β-D-HB and acetoacetate) by SMCT1, and to determine the expression pattern of the transporter in the brain.
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- Materials and methods
These studies establish, for the first time, the functional identity of human SMCT1 as a Na+-coupled transporter not only for l-lactate but also for the ketone bodies β-D-HB and acetoacetate. Similarly to the transport of l-lactate via SMCT1, the transport of the ketone bodies via the transporter is also Na+-dependent and electrogenic. The Na+ : substrate stoichiometry of 2 : 1 for the ketone bodies suggests that the transporter can mediate the concentrative uptake of these metabolic intermediates into cells very effectively coupled to the sodium motive force. These findings are of biological significance because SMCT1 is the only transporter, thus far known, that can transport l-lactate and ketone bodies into cells in a Na+-coupled manner. This has significant physiological and pathological implications for the survival and function of tissues which depend on these metabolites for generation of energy. Arguably, the most important of such tissues is the brain. In the brain, neurons are the major users of metabolic energy and therefore, they would benefit the most from such an energy-coupled active uptake system for these metabolic intermediates. Our studies show for the first time that SMCT1 is expressed in the brain in a neuron-specific manner. Therefore, it is most likely that SMCT1 plays a significant role in the energetics of neurons in the central nervous system. MCT2, which is expressed in neurons, is also capable of transporting l-lactate and ketone bodies by an H+-coupled and electroneutral mechanism. MCT2 possesses higher affinities for l-lactate and ketone bodies compared with MCT1 and MCT4. The Michaelis constants (Km) for l-lactate and β-D-HB for transport via MCT2 are approximately 0.7 mm and 1.2 mm, respectively (Lin et al. 1998; Broer et al. 1999). SMCT1 has a Km value of about 0.2 mm for l-lactate and about 1.5 mm for β-D-HB (Miyauchi et al. 2004; present study). Therefore, we speculate that SMCT1 and MCT2 may complement each other in the maintenance of the energy status of the neurons by providing l-lactate and β-D-HB via two different mechanisms. Further work is needed to assess precisely the relative contributions of MCT2 and SMCT1 to the provision of these energy substrates to neurons under various physiological and pathological conditions.
The finding that α-ketoisocaproate is a transportable substrate for SMCT1 may be relevant to the handling of this keto acid in the brain. Astrocytes are capable of glutamate synthesis for subsequent conversion into glutamine that is then released into the extracellular medium. The released glutamine is taken up by neurons where it serves as an immediate precursor for the generation of glutamate. Synthesis of glutamate within astrocytes occurs via transamination between leucine and α-ketoglutarate, a process which results in the generation of α-ketoisocaproate. This keto acid is then released from the astrocytes for subsequent uptake into neurons for conversion back into leucine. A Na+-coupled transport system has been described for this keto acid in neuroblastoma cells (Bachowska-Mac et al. 1997), but the molecular identity of the transport system remains unknown. Interestingly, other α-keto acids that arise from transamination of valine and isoleucine are able to compete with α-ketoisocaproate for uptake into these cells, whereas β-hydroxybutyrate and lactate are without any effect. Our studies show that SMCT1 expressed in neurons can recognize α-ketoisocaproate as well as β-hydroxybutyrate and lactate. The substrate selectivity of the transport system described in neuroblastoma cells thus seems to be different from that of SMCT1. However, it has to be noted that, under the experimental conditions used for uptake measurements, the rate of conversion of the transported α-ketoisocaproate into leucine seems to be very fast in these cells compared with the rate of its entry (Bachowska-Mac et al. 1997), which might make interpretation of the data difficult. Further work is needed to determine whether SMCT1 is indeed responsible for the uptake of α-ketoisocaproate reported in these cells.
The relevance of ketone bodies to neuronal function has become increasingly apparent in recent years. Ketone bodies are an important source of metabolic energy for the brain in the neonatal period when fat-rich and ketogenic breast milk constitutes the primary caloric source in the diet (Morris 2005). In addition to the physiological significance of a ketogenic diet in the neonatal period, such diets appear to have a therapeutic potential in the management of epilepsy (Swink et al. 1997; Greene et al. 2003; Stafstrom and Bough 2003). Furthermore, ketone bodies have been shown to offer protection to neurons against various insults, such as hypoxia (Masuda et al. 2005) and excitotoxicity (Massieu et al. 2003), as well as in neurodegenerative disorders such as Parkinson's disease (Tieu et al. 2003). Ketone bodies also support synaptic function during development (Izumi et al. 1998) and improve cognitive function in patients with Alzheimer's disease (Reger et al. 2004). Such biological and therapeutic roles of ketone bodies are undoubtedly related to the ability of these metabolites to serve as an important energy source for the neurons. SMCT1, expressed in neurons, may play a critical role in the efficient entry of these metabolites into neurons for subsequent oxidation to produce the metabolic energy.