Escape of Insulin from Muscle Capillaries
Key to the maintenance of homeostasis in a complex, multicellular organism is inter-organ communication by means of molecular signals that are delivered via the circulation. However, to gain access to the target tissue, these molecules must be able to traverse the tightly adherent endothelial cells characteristic of the microcirculation in many tissues. This may be a minor problem for small molecule hormones such as cortisol or estrogen, but it is not clear how proteins hormones, such as insulin and glucagon cross the endothelial cell barrier. Indeed, the mechanism by which insulin leaves the bloodstream to bind to skeletal muscle membranes is a topic of considerable controversy. Some reports suggest that the process is dependent on the insulin receptor and therefore saturable. In contrast, other studies demonstrate normal glucose homeostasis in the absence of endothelial insulin receptor expression, and still others demonstrate a failure to saturate trans endothelial insulin export. Attempts to clarify the mechanism of insulin efflux across the endothelium suffer from the lack of a method to accurately measure the process in vivo without perturbing the tissue being studied. To address this problem, Vanderbilt Basic Sciences investigator David Wasserman and his laboratory developed a new approach using quantitative intravital microscopy. They began by synthesizing human INS-647, a probe comprising human insulin bearing Alexa Fluor 647 at the N-terminus of the B chain. This fluorescent probe retained the biological activity of human insulin and exhibited excellent stability and optical properties for use in confocal laser microscopy. They next developed an in vivo mouse gastrocnemius muscle preparation that enabled visualization of INS-647 in the tissue capillaries, which were outlined by a dextran-based fluorescent dye. Finally, they devised a computational algorithm to analyze real-time fluorescent signals acquired from the muscle preparation following INS-647 infusion. Data collected from this model revealed that INS-647 exited the capillaries by fluid phase transport. The process was not saturable, and could not be blocked by an insulin receptor antagonist. These findings are an important first step to understanding how aberrant efflux from the capillaries can contribute to insulin resistance in diabetes, and the approach developed by the Wasserman lab can readily be applied to the study of transcapillary efflux of other molecules in the future. The work is published in the Journal of Clinical Investigation [I. M. Williams, et al. J. Clin. Invest., (2018) published online Jan. 8, DOI: 10.1172/JCI94053].