A high yield preparation of brush border membrane vesicles from organ-cultured embryonic chick jejunum: demonstration of insulin sensitivity of Na(+)-dependent D-glucose transport.
Although embryonic chick small intestinal segments provide a very limited amount of tissue for preparation of enterocyte brush border membrane vesicles (BBMV), we were able to develop a procedure for isolation of BBMV from cultured 20-d-old embryonic chick jejunum in high yield by modifying a divalent cation precipitation method. Total yield of the brush border marker enzyme alkaline phosphatase in the vesicle fraction as compared to the crude homogenate was approximately 40%, and the specific activity of the enzyme was increased 25-fold on the average. The brush border membrane vesicle fraction was only contaminated with other cellular organelles (basolateral membranes, mitochondria, lysosomes or endoplasmic reticulum) to a minor extent. Functional integrity of the brush border vesicles was indicated by Na+ gradient-driven electrogenic D-glucose transport leading to concentrative transfer (overshoot) of the sugar into an osmotically active intravesicular space. When jejuna were cultured for 48 h in the presence of 10(-6) mol/L insulin, the initial rate of Na(+)-dependent D-glucose uptake by brush border membrane vesicles as well as Na(+)-dependent [3H]phlorizin binding to brush border membranes was approximately twice as high as in vesicles from untreated controls. This strongly suggests that insulin could enhance intestinal absorption of D-glucose by increasing the intrinsic activity of the Na(+)-dependent D-glucose transport system at the luminal membrane of enterocytes.
Debiec
H
H
Department of Biochemistry and Experimental Medicine, Child's Health Center, Warsaw, Poland.
Cross
H S
HS
Peterlik
M
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002458:Cell Fractionation; D002642:Chick Embryo; D005947:Glucose / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007583:Jejunum / Q000196:embryology* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D009924:Organ Culture Techniques; D010695:Phlorhizin / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology*
D007328:Insulin; D012964:Sodium; D010695:Phlorhizin; D000469:Alkaline Phosphatase; D005947:Glucose