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Insulin exits skeletal muscle capillaries by fluid-phase transport
Ian M. Williams, … , K. Sam Wells, David H. Wasserman
Ian M. Williams, … , K. Sam Wells, David H. Wasserman
Published January 8, 2018
Citation Information: J Clin Invest. 2018;128(2):699-714. https://doi.org/10.1172/JCI94053.
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Research Article Metabolism Vascular biology

Insulin exits skeletal muscle capillaries by fluid-phase transport

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Abstract

Before insulin can stimulate myocytes to take up glucose, it must first move from the circulation to the interstitial space. The continuous endothelium of skeletal muscle (SkM) capillaries restricts insulin’s access to myocytes. The mechanism by which insulin crosses this continuous endothelium is critical to understand insulin action and insulin resistance; however, methodological obstacles have limited understanding of endothelial insulin transport in vivo. Here, we present an intravital microscopy technique to measure the rate of insulin efflux across the endothelium of SkM capillaries. This method involves development of a fully bioactive, fluorescent insulin probe, a gastrocnemius preparation for intravital microscopy, an automated vascular segmentation algorithm, and the use of mathematical models to estimate endothelial transport parameters. We combined direct visualization of insulin efflux from SkM capillaries with modeling of insulin efflux kinetics to identify fluid-phase transport as the major mode of transendothelial insulin efflux in mice. Model-independent experiments demonstrating that insulin movement is neither saturable nor affected by insulin receptor antagonism supported this result. Our finding that insulin enters the SkM interstitium by fluid-phase transport may have implications in the pathophysiology of SkM insulin resistance as well as in the treatment of diabetes with various insulin analogs.

Authors

Ian M. Williams, Francisco A. Valenzuela, Steven D. Kahl, Doraiswami Ramkrishna, Adam R. Mezo, Jamey D. Young, K. Sam Wells, David H. Wasserman

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Figure 1

Synthesis and characterization of a novel fluorescent insulin probe.

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Synthesis and characterization of a novel fluorescent insulin probe.
(A)...
(A) Schematic depicting the chemical synthesis of INS-647. (B) Analytical RP-HPLC trace of INS-647. (C) Mass spectrum of INS-647. The labeled peaks indicate the 3 most abundant ions, (M+5H)5+, (M+6H)6+, and (M+7H)7+. (D–G) Bioactivity comparison of unlabeled insulin (n = 4) and INS-647 (n = 4) by hyperinsulinemic-euglycemic clamp. (D) Arterial glucose levels were clamped at approximately 150 mg/dl by variable venous infusion of 50% glucose. (E) The glucose infusion rate required to maintain euglycemia during hyperinsulinemia, a measure of insulin sensitivity. (F and G) Basal and insulin-stimulated rates of peripheral glucose disposal (Rd) and endogenous glucose production (endoRa) as determined by steady-state plasma levels of 3[3H]glucose. Groups were compared using unpaired Student’s t test. Boc-OSu, (butyloxycarbonyl)succinimide ester; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide; m/z, mass/charge; NHS-AF647, Alexa Fluor 647–NHS ester; t-Boc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid; TIPS, triisopropylsilane.

Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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