Mode of Transcapillary Transport of Insulin and Insulin Analog NN304 in Dog Hindlimb

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Abstract
A defect in transcapillary transport of insulin in skeletal muscle and adipose tissue has been proposed to play a role in the insulin resistance that leads to type 2 diabetes, yet the mechanism of insulin transfer across the capillary endothelium from plasma to interstitium continues to be debated. This study examined in vivo the interstitial appearance of insulin in hindlimb using the fatty acid acylated insulin analog LysB29-tetradecanoyl des-(B30) human insulin, or NN304, as a marker for insulin transport. If the insulin transport were a saturable process, then “swamping” the capillary endothelial insulin receptors with native insulin would suppress the subsequent appearance in interstitial fluid of the insulin analog NN304. This analog binds to insulin receptors with an affinity of ∼50% of native insulin. Experimental conditions established a physiologic NN304 dose in the absence or presence of pharmacologic and saturating concentrations of regular human insulin. Euglycemic clamps were performed in dogs under inhalant anesthesia with deep hindlimb lymphatic sampling, representative of skeletal muscle interstitial fluid (ISF). In group 1 (n = 8), NN304 alone was infused (3.6 pmol · min−1 · kg−1) from 60 to 360 min. In group 2 (n = 6), starting at time 0, human insulin was infused at a pharmacologic dose (60 pmol · min−1 · kg−1) with the addition of NN304 infusion (3.6 pmol · min−1 · kg−1) from 60 to 360 min. In group 3 (n = 4), the human insulin infusion was increased to a saturating dose (120 pmol · min−1 · kg−1). Pharmacologic insulin infusion (group 2) established steady-state human insulin concentrations of 6,300 ± 510 pmol/l in plasma and 5,300 ± 540 pmol/l in ISF. Saturating insulin infusion (group 3) achieved steady-state human insulin concentrations of 22,000 ± 1,800 pmol/l in plasma and 19,000 ± 1,500 pmol/l in ISF. Total (bound and unbound) NN304 plasma concentrations rose from a steady state of 1,900 ± 110 (group 1) to 2,400 ± 200 pmol/l (group 2) and 3,100 ± 580 pmol/l (group 3), consistent with a competition-driven decline in NN304 clearance from plasma as the human insulin level increased (P < 0.05 by ANOVA). Steady-state interstitial NN304 concentrations also rose with increasing human insulin levels but did not achieve significance in comparison with analog alone (162 ± 15 vs. 196 ± 22 and 241 ± 53 pmol/l for group 1 versus groups 2 and 3, respectively; P = 0.20), yet the steady-state plasma:ISF ratio for NN304 remained essentially unchanged in the absence and presence of elevated human insulin levels (12.6 ± 1.2 vs. 12.4 ± 0.5 and 13.1 ± 1.5 for group 1 versus groups 2 and 3, respectively; P = 0.93). Last, NN304 rate of appearance in interstitial fluid (i.e., half-time to steady state) was similar between groups; mean half-time of 92 ± 4 min (NS between groups). In conclusion, appearance of the insulin analog NN304 in skeletal muscle interstitial fluid was constant whether in the absence or presence of human insulin concentrations sufficient to saturate the endothelial insulin receptors. These findings support the hypothesis, provided that the mechanism of insulin and NN304 transcapillary transport is similar, that transcapillary transport of insulin in skeletal muscle occurs primarily via a nonsaturable process such as passive diffusion via a paracellular or transcellular route.

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