Glucose transporter localization in brain using light and electron immunocytochemistry

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).