D. Z. Gerhart

A polyclonal affinity-purified antibody to the carboxyl-terminal end of the rat monocarboxylate transporter 1 (MCT1) was generated in chickens and used in immunocytochemical studies of brain tissue sections from adult and suckling rats. The antibody identified a 48-kDa band on immunoblots and stained tissue sections of heart, cecum, kidney, and skeletal muscle, consistent with the reported molecular mass and cellular expression for this transporter. In tissue sections from adult brains, the antibody labeled brain microvessel endothelial cells, ependymocytes, glial-limiting membranes, and neuropil. In brain sections from 3- to 14-day-old rats, microvessels were much more strongly labeled and neuropil was weakly labeled compared with sections from adults. Immunoelectron microscopy indicated that labeling was present on both luminal and abluminal endothelial cell plasma membranes. These results suggest that MCT1 may play an important role in the passage of lactate and other monocarboxylates across the blood-brain barrier and that suckling rats may be especially dependent on this transporter to supply energy substrates to the brain.

Abstract A polyclonal antibody to a synthetic 13 amino acidpeptide found at the carboxyl‐terminal end of the glucose transporter protein was raised in rabbit and used in light and electron immunocytochemical studies of human and canine brain. This antibody identified a broad band of polypeptide of average M r 55,000 on immunoblots (immunogold‐silver stains) of electrophoresed membrane proteins from human red blood cells. A similar polypeptide band (M r 45,000–60,000) was identified on immunoblots of microvessel membrane proteins isolated from canine cerebrum, suggesting that this antibody is a useful tool for studying the distribution and abundance of the glucose transporter protein in mammalian nervous tissue. Peroxidase antiperoxidase stains of cerebrum using this antibody demonstrated that transporters are abundant in the intima pia, in the endothelium of blood vessels in the subarachnoid space, and in the endothelium of arterioles, venules, and capillaries of gray and white matter. In cerebellum, reaction product was localized in the vessels of the subarachnoid space and in microvessels of the molecular layer, the granular layer, and the white matter. However, transporters were not found in the intima pia of cerebellum. In medulla oblongata, transporters were found in the intima pia, the endothelium of some subarachnoid vessels, and the microvessels of gray and white matter. In pituitary, microvessels in adenohypophysis contained no reaction product, but the antigen was detected in some microvessels in neurohypophysis. Electron microscopy of cerebral cortex using a protein A‐gold technique demonstrated that glucose transporters are equally abundant on the luminal and abluminal membranes of microvessel endothelial cells. When thin sections were stained by floating grids on top of antibody and reagents (single‐sided staining), 84% of the gold particles counted were associated with endothelial plasma membranes and tight junctions, 9% were associated with endothelial cytoplasm, vesicles, or plasmalemma invaginations, and 6% were associated with nonendothelial structures (basement membrane, pericyte, smooth muscle cell, or vessel lumen).

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.

To elucidate glucose transport mechanisms in brain and to demonstrate the cellular expression of the brain-type glucose transporter (GLUT3), antisera to a synthetic peptide corresponding to the C terminus were prepared and used as probes for this isoform of the facilitative glucose transporter family. Immunocytochemistry of frozen sections of dog and rat brain demonstrated GLUT3 antigen in pyramidal cell bodies and processes, in microvessels, and in intima pia or glia limitans. Immunoanalysis of Western blots identified a protein (Mr, 45,000) that was present in both neuron/neuropil and microvessel fractions. The presence of the GLUT3 message in brain was confirmed by Northern blot analysis and by amplifying and partially sequencing GLUT3 cDNA by PCR. These findings demonstrate a neuron glucose transporter in tissue and suggest that GLUT3 may play an important role in brain metabolism under physiological and pathophysiological conditions.