David Z. Gerhart

Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.

Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats: A quantitative electron microscopic immunogold study

Transcellular transport of energy substrates across the vascular endothelial cells of the brain is accomplished by integral membrane carrier proteins, such as the glucose transporter GLUT1 and the monocarboxylic acid transporter MCT1. The abundance of these proteins may vary depending on age and nutritional status. In this study we compared the expression of MCT1 in cerebral cortex of suckling and adult rats to determine whether the former, which use considerably more monocarboxylates such as lactate and ketone bodies as fuel than do older rats, correspondingly express more MCT1 than adults. Using electron microscopic immunogold methods, we found that 17-day old suckling rat pups had 25 times more MCT1 labeling in the membranes of capillary endothelial cells than adults. This transporter was nearly equally distributed in luminal and abluminal membranes with less than 10% of the immunogold particles in the endothelial cytoplasmic compartment. The suckling rats also had 15 times more immunogold particles associated with pericyte membranes and 19 times heavier labeling of membranes associated with astrocytic end feet adjacent to microvessels. Neuropil and choroid plexus were lightly labeled. Some MCT1-positive astrocyte and neuron cell bodies were observed, suggesting active synthesis of MCT1 by these cells. The potential for regulation of expression of MCTs by dietary or other factors may have important consequences for the progression and treatment of cerebrovascular disorders and other diseases.

Expression of the monocarboxylate transporter MCT2 by rat brain glia

The nucleotide sequence of the rat monocarboxylate transporter MCT2 was determined from brain‐derived cDNA. A polyclonal antibody was raised in chickens against the carboxyl terminal end of the deduced amino acid sequence and affinity purified. The MCT2 antibody identified a 46‐kDa band on immunoblots and labeled kidney, skeletal muscle, and stomach consistent with the reported cellular expression for this transporter. Light microscopic immunocytochemistry indicated that the MCT2 transporter was abundant in glial limiting membranes, ependymocytes, and neuropil, particularly in the lacunosum molecular layer of hippocampus and the molecular layer of cerebellum. Labeled astrocytes were commonly observed in white matter. The distribution of this transporter differed in several respects from that previously reported for MCT1. MCT2 was abundantly distributed in astrocyte foot processes and was usually not detected in other cells of the cerebrovasculature, including vascular smooth muscle cells, pericytes, and endothelium. In addition, the granular layer of cerebellum, which showed little MCT1 labeling, exhibited MCT2 labeling of cellular processes in the neuropil surrounding the granule and Purkinje cells. The results lend support to the concept that astrocytes play a significant role in cerebral energy metabolism by transporting lactate and other monocarboxylates. GLIA 22:272–281, 1998.

Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain

Precise localization of glucose transport proteins in the brain has proved difficult, especially at the ultrastructural level. This has limited further insights into their cellular specificity, subcellular distribution, and function. In the present study, preembedding ultrastructural immunocytochemistry was used to localize the major brain glucose transporters, GLUTs 1 and 3, in vibratome sections of rat brain. Our results support the view that, besides being present in endothelial cells of central nervous system (CNS) blood vessels, GLUT 1 is present in astrocytes. GLUT 1 was detected in astrocytic end feet around blood vessels, and in astrocytic cell bodies and processes in both gray and white matter. GLUT 3, the neuronal glucose transporter, was located primarily in pre‐ and postsynaptic nerve endings and in small neuronal processes. This study: (1) affirms that GLUT 3 is neuron‐specific, (2) shows that GLUT 1 is not normally expressed in detectable quantities by neurons, (3) suggests that glucose is readily available for synaptic energy metabolism based on the high concentration of GLUT 3 in membranes of synaptic terminals, and (4) demonstrates significant intracellular and mitochondrial localization of glucose transport proteins. J. Neurosci. Res. 49:617–626, 1997.

Cultured human and canine endothelial cells from brain microvessels.

Cultures of endothelial cells (EC) derived from human (autopsy) and canine brain microvessels were characterized with respect to growth, morphology, and biochemical features. The endothelial nature of these cells was confirmed by analyses of angiotensin-converting enzyme activity, Factor VIII-related antigen, and ultrastructure. Human EC required coated substrates and tumor-conditioned medium to achieve rapid growth, and cells derived from human microvessels were morphologically diverse. In contrast, canine EC grew rapidly on plastic substrates and produced colonies of uniform morphology. Morphological variations of EC were associated with the use of heparin-containing medium and with the use of a commercially-prepared basement membrane extract (Matrigel). Lectin histochemistry demonstrated that human EC lack the abundant alpha-galactose residues characteristic of canine EC membranes and organelles and that canine EC lack the alpha-N-acetylgalactosamine residues which are associated with human EC. The lectin Ricinus communis agglutinin I may be useful for distinguishing canine EC from pericytes. Gel electrophoresis of membrane proteins revealed protein bands present in human EC at Mr 210,000 and 37,000-39,500 which were not present in canine EC. These proteins may be related to the presence of junctional complexes in cultures of human EC.

Distribution of monocarboxylate transporters MCT1 and MCT2 in rat retina.

Transport of lactic acid and other monocarboxylates such as pyruvate and the ketone bodies through cellular membranes is facilitated by specific transport proteins. We used chicken polyclonal antibodies to the monocarboxylate transporters-1 and -2 to determine their cellular and subcellular distributions in rat retina, and we compared these distributions to those of the glucose transporters-1 and -3. Monocarboxylate transporter-1 was most highly expressed by the apical processes of retinal pigment epithelium that surround the outer segments of the photoreceptor cells. In contrast to glucose transporter-1, monocarboxylate transporter-1 was not detected on the basal membranes of pigment epithelium. The luminal and abluminal endothelial plasma membranes in retina also exhibited heavy labeling by antibody to monocarboxylate transporter-1. In addition, this transporter was associated with the Müller cell microvilli, the plasma membranes of the rod inner segments, and all retinal layers between the inner and external limiting membranes. Monocarboxylate transporter-2 was found to be abundantly expressed on the inner (basal) plasma membrane of Müller cells and by glial cell processes surrounding retinal microvessels. This transporter was also present in the plexiform and nuclear layers but was not detected beyond the external limiting membrane. Recent studies have shown that lactic acid transport is of particular importance at endothelial and epithelial barriers where membranes of adjoining cells are linked by tight junctions. Our results suggest that monocarboxylate transporter-1 functions to transport lactate between the retina and the blood, both at the retinal endothelium and the pigment epithelium. The location of monocarboxylate transporter-2 on glial foot processes surrounding retinal vessels suggests that this transporter is also important in blood-retinal lactate exchange. In addition, the abundance of these transporters in Müller cells and synaptic (plexiform) layers suggests that they function in lactate exchange between neurons and glia, supporting the notion that lactate plays a key role in neural metabolism.

Expression of large amino acid transporter LAT1 in rat brain endothelium

The expression of the large amino acid transporter, LAT1, was investigated in brain of adult Long-Evans rats. The LAT1 transcript was readily detected in brain microvessels and choroid plexus by reverse transcription polymerase chain reaction analysis using three different gene specific primer pairs. A polyclonal affinity purified antibody against the N-terminus of LAT1 was generated in chickens and used in immunoblot and immunocytochemical analyses of brain tissue sections of adult rats. On immunoblots, the antibody detected a peptide-inhibitable 45 kDa band in a rat brain microvessel membrane preparation. It also identified the same protein band in membrane preparations of different brain structures, as well as in heart and testis, whereas the protein was absent or only faintly detectable in muscle, kidney, and liver. In brain sections, the antibody intensely labeled the luminal and abluminal membranes of brain microvessel endothelial cells in all brain areas examined including cerebral cortex, cerebellum, hippocampus, and in gray and white matter regions. These results suggest that LAT1 is involved in transcellular transport and may play an important role in large, neutral amino acid transfer across the blood–brain barrier.

Expression of monocarboxylate transporter MCT1 in normal and neoplastic human CNS tissues.

Expression of monocarboxylate transporter MCT1 was studied in archival tissues from human CNS using antibodies to the carboxyl-terminal end of MCT1. Sections of neocortex, hippocampus and cerebellum of brains from 10 adult autopsy patients who died from other than CNS disease, and from archival surgical biopsy specimens of 83 primary CNS and eight non-CNS tumors were studied. MCT1 immunoreactivity was present in microvessels and, ependymocytes of normal CNS tissues similar to that reported for MCT1 expression in rat brains. MCT1 immunoreactivity was strongest in ependymomas, hemangioblastomas and high grade glial neoplasms, and weakest in low grade gliomas. Increased MCT1 expression in high grade glial neoplasms may provide a potential therapeutic target for treatment of some CNS neoplasms.

GLUT1 and GLUT3 gene expression in gerbil brain following brief ischemia: an in situ hybridization study

GLUT1 and GLUT3 mRNAs in normal and post-ischemic gerbil brains were examined qualitatively and semi-quantitatively using in situ hybridization in conjunction with image analysis. Coronal brain sections at the level of the anterior hippocampus were prepared three hours, one day, and three days after animals were subjected to six min of ischemia. The sections were hybridized with vector- and PCR-generated RNA probes labeled with 35S. Microscopic evaluation of hybridized brain sections coated with autoradiographic emulsion indicated that GLUT1 mRNA was associated with brain microvessels, choroid plexus, and some ependymal cells. GLUT1 mRNA was not observed in neurons, except that one day following ischemia, this mRNA was induced in neurons of the dentate gyrus. GLUT3 mRNA was detected only in neurons. Image analysis of film autoradiograms revealed that both the GLUT1 and GLUT3 messages increased following ischemia but returned nearly to control levels by day three. In the CA1 region of the hippocampus the increase in GLUT3 mRNA was not statistically significant, and by day three the level had fallen significantly below the control, coinciding with the degeneration of the CA1 neurons. Our results suggest that the brain possesses mechanisms for induction and up-regulation of glucose transporter gene expression.

Characterization of the blood-brain barrier: glycoconjugate receptors of 14 lectins in canine brain, cultured endothelial cells, and blotted membrane proteins

The avidin-biotinylated peroxidase complex (ABC) method was used to detect binding of 14 lectins in tissue, cultured cells, and nitrocellulose blots. When applied to frozen sections of canine cerebral cortex and pituitary and evaluated by light microscopy, these lectins produced distinct staining patterns as determined by their individual carbohydrate specificities. Major saccharide residues detected in the endothelium of these cerebral tissues include alpha- and beta-galactose, alpha-mannose and/or alpha-glucose, and N-acetylglucosamine. Application to cells cultured from the canine cerebral endothelium gave staining results similar to those of microvessels in tissue. Thus, these characteristics of intact capillaries are retained in cultured cells and define fundamental properties of the blood-brain interface. Visual comparison of these staining patterns to those obtained for electrophoretic blots of solubilized membrane proteins identified multiple glycoprotein receptors and illustrated the vast quantity and variety of surface carbohydrate residues and the complexity of the cerebral endothelial cell glycocalyx. This carbohydrate-rich layer, which extends into the capillary lumen, may be of significant importance to the unique function of the blood-brain barrier.