Clamping Skeletal Muscle PO2 Eliminates Hyperinsulinemic Microvascular Blood Flow Response
Authors: Wells Brenda N. , Russell McEvoy Gaylene M. , Shogan Hamza , Kiley Meghan E. , Fraser Graham M. 
Published: May 2022

Objective

To determine if insulin mediated hyperemia is partially dependent on local muscle oxygen concentration.

Hypothesis

The blood flow responses to insulin are partially mediated by the local metabolic demand reflecting the increased oxygen consumption required to uptake and convert glucose into glycogen in skeletal muscle.

Methods

16 male Sprague Dawley rats (161g ‐ 204g) were anesthetized with sodium pentobarbital, the carotid artery and jugular vein were then cannulated for blood pressure monitoring and fluid resuscitation. Following cannulation, the extensor digitorum longus (EDL) was isolated and reflected onto an inverted microscope. Intravital video microscopy sequences of the EDL microcirculation were recorded during baseline and following hyperinsulinemic euglycemia or sham in all experiments. The muscle was reflected over a glass stage insert during Experiment 1a and 1b which allowed for direct observation of microvascular blood flow response to systemic insulin infusion in Experiment 1a. Experiment 1b consisted of a sham hyperinsulinemic euglycemic clamp which involved infusing saline at similar rates as insulin and glucose in standard clamp procedures. In Experiment 2, the EDL was reflected over a gas exchange chamber and microvascular capillary blood flow was recorded during sequential changes (7%‐12%‐2%‐7%) of oxygen concentration [O2] within the chamber during baseline and euglycemic conditions. The gas exchange chamber is fitted into the stage of an inverted microscope that interfaces directly with the EDL via a gas permeable polydimethylsiloxane membrane. By adjusting the concentration of gases flowing through the gas exchange chamber we have direct control of the [O2] within the overlying muscle. Hemodynamic measurements including capillary red blood cell (RBC) velocity, supply rate (SR), hematocrit, and oxygen saturation (SO2) were taken from in focus capillaries in the EDL during each experiment and analyzed offline using custom MATLAB software. All animal protocols were approved by Memorial University’s Institutional Animal Care Committee.

Results

In Experiment 1a, the hyperinsulinemic euglycemic clamp caused a significant increase in SR from 8.9 ± 3.1 to 14.2 ± 4.9 cells/s at baseline to euglycemia (p = 0.01; N = 6, 677 capillaries at baseline and 552 at clamp), while no significant SR variation was detected after performing a sham clamp in Experiment 1b (11.0 ± 1.6 cells/sec at baseline and 11.4 ± 3.7 cells/sec at sham clamp; N = 6, 837 capillaries at baseline and 724 at sham clamp). In Experiment 2 using the gas exchange chamber, SR significantly decreased at 12% O2 (p = 0.01, p < 0.03) and significantly increased at 2% O2 (p = 0.01, p < 0.04), compared to baseline 7% O2, under both baseline and euglycemic conditions respectively (N = 6, 313 capillaries at baseline and 195 at clamp). SR responses to oxygen oscillations during euglycemia were not different to those at baseline for each O2 concentration (p > 0.9). SO2 significantly increased at 12% O2 and significantly decreased at 2% O2 under both baseline and euglycemic conditions (p < 0.05).

Conclusions

Our saturation measurements demonstrate the effectiveness of using a gas exchange device to manipulate local oxygen concentrations in the muscle. Our SR results suggest the increase in blood flow observed in response to insulin is eliminated if the tissue oxygen microenvironment is fixed at a given [O2].