title: Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro.
abstract: We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after approximately 140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 +/- 0.26, mean +/- SE, n = 10), indicating a "priming," or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 +/- 0.07, n = 6; P < 0.01). Cy2- or Cy3-labeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand beta2-glycoprotein.
authors.affiliation: Institute of Physical Engineering, Brno University of Technology, Brno, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Kolman
authors.forename: Angelo
authors.initials: A
authors.lastname: Pica
authors.forename: Nicolas
authors.initials: N
authors.lastname: Carvou
authors.forename: Alan
authors.initials: A
authors.lastname: Boyde
authors.forename: Shamshad
authors.initials: S
authors.lastname: Cockcroft
authors.forename: Andrew
authors.initials: A
authors.lastname: Loesch
authors.forename: Arnold
authors.initials: A
authors.lastname: Pizzey
authors.forename: Mariadelina
authors.initials: M
authors.lastname: Simeoni
authors.forename: Giovambattista
authors.initials: G
authors.lastname: Capasso
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Unwin
mesh_terms: D000818:Animals; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005434:Flow Cytometry; D007004:Hypoglycemic Agents / Q000493:pharmacokinetics*; D066298:In Vitro Techniques; D007328:Insulin / Q000493:pharmacokinetics*; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D011921:Rats, Inbred WKY; D017207:Rats, Sprague-Dawley
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin