Characterization of Transendothelial Insulin Efflux as a Barrier to Insulin Action in Vivo
Authors: Williams Ian M , Valenzuela Francisco A , Mezo Adam R , Young Jamey D , Wells K Sam , Wasserman David H 
Published: November 2023

Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space surrounding the myocyte. The continuous endothelium of skeletal muscle (SkM) capillaries restricts access of circulating macromolecules, such as insulin, to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understanding insulin action and insulin resistance. Previous studies have produced conflicting results as to whether insulin diffuses through interendothelial junctions or is transported by a receptor‐mediated process. One reason for the uncertainty regarding transendothelial insulin transport is the lack of methodology available to directly study this process in vivo. Therefore, we developed an intravital microscopy technique to measure the rate of transendothelial insulin efflux.First, we labeled the N‐terminal phenylalanine of the B chain of human insulin with AlexaFluor647 (ins‐647). Unlike commercially available fluorescent insulin conjugates, ins‐647 maintains full receptor‐binding capacity and glucose lowering bioactivity. Next, we developed a method to expose the gastrocnemius muscle for optical imaging. This preparation requires minimal manipulation of the gastrocnemius and does not induce any significant inflammatory response, as determined by the absence of albumin leakage and leukocyte adhesion to capillaries. Ins‐647 imaging is performed by initially infusing a 2 megadalton rhodamine‐labeled dextran tracer to demarcate the vascular space. Subsequently a 4U/kg bolus of ins‐647 is injected and imaged over 30 minutes using 3D confocal microscopy. Images are then analyzed with a custom vascular segmentation algorithm to generate profiles of ins‐647 intensity as a function of time and distance from capillaries. To determine the mechanism of transendothelial insulin efflux, we fit imaging data to mathematical models describing either diffusion or Michaelis‐Menten kinetics. Using an F‐test, we found that the Michaelis‐Menten model was a statistically better fit for the ins‐647 efflux kinetics data. This indicates that insulin crosses the endothelium by a saturable transport mechanism. To then test whether transendothelial insulin transport is mediated by the insulin receptor, we infused an insulin receptor antagonist (S961). While the antagonist effectively blocked the glucose‐lowering effect of insulin, S961 had no effect on insulin kinetics, indicating that the insulin receptor does not mediate transendothelial insulin transport. In summary, our data suggest that insulin transport is accomplished by a saturable transport mechanism that does not involve the insulin receptor. Furthermore, our intravital microscopy technique can readily be adapted to study other aspects of the SkM microcirculation such as blood flow, inflammation, and the transport of other labeled compounds.Support or Funding InformationThis work was funded by an individual graduate NRSA award (1F31DK109594), an institutional training grant (5T32DK007563‐27), and R01DK054 (DHW). Gracious support was also provided by Lilly Research Laboratories and the Vanderbilt Mouse Metabolic Phenotyping Center.