Insulin entry into muscle involves a saturable process in the vascular endothelium

Abstract

Aims/hypothesis: Insulin's rate of entry into skeletal muscle appears to be the rate-limiting step for muscle insulin action and is slowed by insulin resistance. Despite its obvious importance, uncertainty remains as to whether the transport of insulin from plasma to muscle interstitium is a passive diffusional process or a saturable transport process regulated by the insulin receptor.

Methods: To address this, here we directly measured the rate of (125)I-labelled insulin uptake by rat hindlimb muscle and examined how that is affected by adding unlabelled insulin at high concentrations. We used mono-iodinated [(125)I]Tyr(A14)-labelled insulin and short (5 min) exposure times, combined with trichloroacetic acid precipitation, to trace intact bioactive insulin.

Results: Compared with saline, high concentrations of unlabelled insulin delivered either continuously (insulin clamp) or as a single bolus, significantly raised plasma (125)I-labelled insulin, slowed the movement of (125)I-labelled insulin from plasma into liver, spleen and heart (p < 0.05, for each) but increased kidney (125)I-labelled insulin uptake. High concentrations of unlabelled insulin delivered either continuously (insulin clamp), or as a single bolus, significantly decreased skeletal muscle (125)I-labelled insulin clearance (p < 0.01 for each). Increasing muscle perfusion by electrical stimulation did not prevent the inhibitory effect of unlabelled insulin on muscle (125)I-labelled insulin clearance.

Conclusions/interpretation: These results indicate that insulin's trans-endothelial movement within muscle is a saturable process, which is likely to involve the insulin receptor. Current findings, together with other recent reports, suggest that trans-endothelial insulin transport may be an important site at which muscle insulin action is modulated in clinical and pathological settings.

Figure 1

Outline of protocol #1 -the insulin clamp (upper), and protocol #2 -insulin bolus (lower). For the control studies saline was substituted for the insulin infusion (upper panel) and for the insulin bolus (lower panel).

Figure 2

The fraction of total ¹²⁵I present as intact ¹²⁵I insulin in blood (white bars) and muscle (grey bars) 5 min after tracer injection in the insulin-treated (insulin clamp and bolus) groups vs. controls. A greater fraction of ¹²⁵I present in the muscle was not TCA precipitable relative to the blood (p = <0.05, for each of the four experimental groups).

Figure 3

¹²⁵I-Insulin in various tissues at 5 min after tracer injection accompanied by either an insulin bolus (grey bars) or saline (white bars) (n = 3 each). Note plasma counts are higher when unlabeled insulin is given (insert). The uptake of ¹²⁵I insulin in the kidney is greatly increased relative to other tissues particularly when insulin treatment is given, and there is a relative decrease in insulin uptake in liver, spleen, and heart, with insulin bolus treatment. Amounts are in counts (dpm) per gram dry tissue. In the kidney > 95% of the ¹²⁵I-insulin was intact in both insulin, and saline treated animals.

Figure 4

A. Insulin clearance in insulin clamp vs. control animals separated by limbs exposed to exercise vs those that were not exercised. Differences between insulin clamp and controls were significant, but differences between exercised and non-exercised limbs were not. B. Comparison of insulin clearance between insulin clamp and control animals using all limbs combined (exercised and non-exercised). There was a significant reduction in insulin clearance in the insulin clamp treated animals.

Figure 5

Insulin clearance in insulin bolus vs. control animals separated by limbs exposed to exercise vs those that were not exercised. Differences between insulin bolus and controls (bolus exercise vs control exercise p=0.0013, bolus no exercise vs control no exercise p=0.0067) were significant as were differences between the insulin bolus exercised vs non-exercised limbs (p =0.023). There was not a significant difference between the control exercise and non exercise limbs (p = 0.44).

Figure 6

A: ³H-inulin uptake in blood, skeletal muscle, liver and kidney at 5 min after tracer injection accompanied by either unlabeled inulin (grey bars) or saline (white bars) (n = 6–8 each). Amounts are in counts (dpm) per gram dry tissue. B: ³H-inulin clearance in skeletal muscle, liver and kidney. There was no significant effect of unlabeled inulin (grey bars) in any tissue surveyed.