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Abstract
The subcellular distribution of glucose transporter in rat hepatocytes was studied in the absence and presence of insulin by measuring glucose-sensitive cytochalasin B binding sites and immunoreactivities to antibodies specific to hepatocyte glucose transporter. Total hepatocyte membranes bound cytochalasin B at a class of glucose-sensitive sites with a Kd of l.6×l0-6M. and a Bt of 6.8 pmol/mg protein. The glucose-sensitive cytochalasin B binding sites were found in various subcellular membrane fractions with a relative abundance of 47% in a plasma membrane-nuclei-mitochondria-enriched fraction(PM/NM). 29% in a lysosome-enriched fraction(LYSO). 16% in a Golgi-enriched. high density microsomal fraction(HDM) and 8% in the low density microsome fraction(LDM). Relative abundances of two well known plasma membrane markers. 5'-nucleotidase and cell surface carbohydrate label, on the other hand, were found to he 48~50% in PM/NM. 41~43% in LYSO. 6~8% in HDM and l~2% in LDM.
Insulin treatment of intact hepatocytes did not induce any significant changes in the subcellular distributions of the glucose-sensitive. cytochalasin B binding activities, the immunoreactivities to the transporter specific antibodies, or the two cell surface membrane markers. These findings indicate that as much as 15% of the total hepatocyte glucose transporters occur in organelle(s) other than the pasma membrane, most likely representing an intracellular storage pool. which is not decreased by insulin. It is concluded that the rat hepatocyte lacks the insulin-mediated, glucose transporter translocation mechanism, thus would be a valuable experimental system in which one can study the celluar and molecular basis of this deficiency.
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