Vstřebávání insulinu přes luminální membránu krysího proximálního kanálku in vivo a in vitro

Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro.

journal article - other

en

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 ~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 B2-glycoprotein.

Článek popisuje vstřebávání inzulínu přes luminální membránu ledvinného krysího proximálního kanálku in vivo studované pomocí konfokální mikroskopie a porovnává s výsledky získanými in vitro na buňkách krysího proximálního kanálku (WKPT) pomocí fluorescenční mikroskopie a průtokové cytometrie. Podpovrchové ledvinné kanálky byly pozorovány in vivo v reálném čase v optickém řezu pomocí konfokálního laserového (633 nm) rastrovacího mikroskopu. Byly vybírány oblasti obsahující proximální i distální kanálky. Inzulín obarvený fluorescenčním barvivem Cy5 byl aplikován ve dvou dávkách (s časovým odstupem 140 min) do pravé krční žíly a fluorescenční signál (o vlnových délkách 650-670 nm) byl zaznamenáván digitální video-kamerou. Fluorescence byla detekována téměř okamžitě na kartáčovém lemu buněk proximálního kanálku a během 30-40 min byla přítomna uvnitř buněk. Jako míra vstřebávání byl zvolen poměr přírůstku fluorescenčního signálu po druhé a po první dávce. Poměr 2,11 +/- 0,26 naznačuje stimulační efekt inzulínu na mechanizmy jeho vstřebávání na kartáčovém lemu, což bylo potvrzeno in vitro. Tento efekt nenastal v případě podání infuze obsahující lysine před podáním první dávky inzulínu (poměr 1,03 +/- 0,07).

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 ~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 B2-glycoprotein.

in vivo konfokální mikroskopie, fluorescence, ledviny

intravital confocal microscopy; fluorescence; kidney

2009

04.04.2009

0363-6127

AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY

296

5

F1227–F1237

10