Endothelial cells actively concentrate insulin during its transendothelial transport

Abstract

Objective: We examined insulins uptake and transendothelial transport by endothelial cells in order to: (i) ascertain whether insulin accumulates within the cells to concentrations greater than in the media; (ii) compare trans endothelial insulin transport to that of inulin (using the latter as a tracer for passive transport or leaked); and; (iii) determine whether insulins transported depended on insulin action.

Methods: Using 125I-insulin at physiologic concentrations we measured both the uptake and trans endothelial transport of insulin by bovine aortic endothelial cells and measured cell volume using tritiated 3-O-methylglucose.

Results: Bovine aortic endothelial cells accumulate insulin to > five-fold above the media concentrations and the trans endothelial transport of insulin, but not inulin, is saturable and requires intact PI-3-kinase and MEK signaling.

Conclusion: The insulin receptor and downstream signaling from the receptor regulates endothelial insulin transport. Insulin is accumulated against a concentration gradient by the endothelial cell. We suggest that insulin uptake is rate limiting for insulin trans endothelial transport.

Keywords: endothelium; insulin receptor; insulin transport.

Figure 1

Panel A. 80pM ¹²⁵I-insulin cellular uptake, n = 3. Panel B. ³H-3-MG cellular uptake was used as a measure of intracellular volume in order to calculate the intracellular concentration of ¹²⁵I-insulin and thus the rate of uptake, n = 3.

Figure 2

Panel A. 80pM ¹²⁵I-insulin uptake into BAECs (black bars) +/− 1µM cold unlabeled insulin (white bars) at 30min, * p <0.05, n = 9. Panel B. 80pM ¹²⁵I-insulin uptake into BAECs (black bars) +/− IGF-IR antibody (grey bars) at 30min.

Figure 3

Panel A. Transport of heat-denatured 400pM ¹²⁵I-insulin (black bar) with or without 1 µM cold unlabeled insulin (white bar). Panel B. Movement of 80nM ³H-inulin (black bar)(+ 80µM unlabeled inulin) (white bar) which is expected to travel by a non-receptor mediated pathway. Panel C. Decrease in TEER with media only (black bar) incubation for one hour and one hour incubation with 1 µM insulin (white bar). Panel D. 400pM ¹²⁵I-insulin trans-endothelial transport is inhibited by addition of excess (1µM) cold unlabeled insulin at 30min. n = 5–8. * p = <0.05.

Figure 4

Panel A. ³H-inulin trans-endothelial transport. Panel B. TEER change and Panel C. 400pM ¹²⁵I-insulin transport. Black bars are in the absence of inhibitors, wortmannin indicated by grey bars, and PD98059 by white bars in each panel. n = 6 * p < 0.05