Defects in oxygen supply to skeletal muscle of prediabetic ZDF rats

Abstract

In humans, prediabetes is characterized by marked increases in plasma insulin and near normal blood glucose levels as well as microvascular dysfunction of unknown origin. Using the extensor digitorum longus muscle of 7-wk inbred male Zucker diabetic fatty rats fed a high-fat diet as a model of prediabetes, we tested the hypothesis that hyperinsulinemia contributes to impaired O(2) delivery in skeletal muscle. Using in vivo video microscopy, we determined that the total O(2) supply to capillaries in the extensor digitorum longus muscle of prediabetic rats was reduced to 64% of controls with a lower O(2) supply rate per capillary and higher O(2) extraction resulting in a decreased O(2) saturation at the venous end of the capillary network. These findings suggest a lower average tissue Po(2) in prediabetic animals. In addition, we determined that insulin, at concentrations measured in humans and Zucker diabetic fatty rats with prediabetes, inhibited the O(2)-dependent release of ATP from rat red blood cells (RBCs). This inability to release ATP could contribute to the impaired O(2) delivery observed in rats with prediabetes, especially in light of the finding that the endothelium-dependent relaxation of resistance arteries from these animals is not different from controls and is not altered by insulin. Computational modeling confirmed a significant 8.3-mmHg decrease in average tissue Po(2) as well as an increase in the heterogeneity of tissue Po(2), implicating a failure of a regulatory system for O(2) supply. The finding that insulin attenuates the O(2)-dependent release of ATP from RBCs suggests that this defect in RBC physiology could contribute to a failure in the regulation of O(2) supply to meet the demand in skeletal muscle in prediabetes.

Fig. 1.

In situ capillary data for control (n = 6) and prediabetes (n = 6) groups showing red blood cell (RBC) velocity, RBC supply rate, RBC O₂ saturation at the arteriolar and venular end of the capillary bed and the density of perfused capillaries. Box and whisker plots show median, upper and lower quartiles, and maximum and minimum values. *P < 0.05, different from control.

Fig. 2.

The average O₂ supply rate was calculated for the arteriolar and venular end of the capillary bed as the product of RBC supply rate × O₂ saturation for both control and prediabetes group. The O₂ extraction ratio was calculated for the control and prediabetes group from the O₂ saturation gradient from arteriolar (S_art) to venular (S_ven) end divided by the arteriolar end O₂ saturation, i.e., (S_art − S_ven)/S_art. Box and whisker plots show median, upper and lower quartiles, and maximum and minimum values. So₂, oxyhemoglobin saturation. *P < 0.05, different from control.

Fig. 3.

Effect of low O₂ on ATP release from washed rat RBCs of control (n = 6) and prediabetic (n = 6) Zucker diabetic fatty rats. RBCs were exposed to 15% O₂, 6% CO₂, balance N₂ (P = 105.9 ± 1.3 mmHg), in a tonometer for 30 min, and ATP was measured. The gas was then changed to 0% O₂, 6% CO₂, balance N₂ (Po₂ = 11.4 ± 1.3 mmHg), and ATP was measured after 10 min. The percent increase in ATP release from baseline (15% O₂) is reported. White bars, washed RBCs in the absence of insulin; black bars, RBCs incubated with insulin (1 nM) for 30 min before ATP determinations. Values are means ± SE. *P < 0.05, different from control.

Fig. 4.

Measurement of amounts of α-subunit of the heterotrimeric G protein, G_i2, in RBC membranes of control (n = 7) and prediabetic (n = 7) Zucker diabetic fatty rats. Values represent the ratio of G_iα₂ to β-actin in the same samples. NS, not statically significant.

Fig. 5.

Effect of prediabetes on acetylcholine-induced relaxation of isolated resistance arteries (200–300 μm) in the presence and absence of insulin. Vessels were incubated with insulin (1 nM) or its vehicle (saline) for 30 min before administration of acetylcholine (10 μM) to phenylephrine-contracted vessels. White bars represent control rats (n = 11), and black represent prediabetic rats (n = 7). Values are means ± SE. *P < 0.05, different from phenylephrine-induced contraction.

Fig. 6.

Color contour plots of calculated tissue Po₂ at the venular exit of the capillary bed (z = 300 μm). Control Po₂ in this plane (A) is 46.6 ± 0.6 mmHg (mean ± SD). Decreased O₂ supply rate and capillary density in prediabetes resulted in a lower exit plane Po₂ of 37.9 ± 1.0 mmHg (B). Black circles indicate RBC-perfused capillaries delivering O₂ to the tissue (19 in control and 14 in prediabetes). Color spectrum scale bars (right) represent tissue Po₂ levels (in mmHg).

Fig. 7.

Calculated Po₂ distributions for tissue volume surrounding array of parallel capillaries. Relative to control (red line), prediabetes (blue line) results in a shift of the Po₂ distribution to the left (decreased mean Po₂) and broadening (increased heterogeneity) of the Po₂ distribution curve. In prediabetes, increasing the entrance O₂ saturation and RBC supply rate to control levels (case 1, dashed green line) does not fully restore the Po₂ distribution to control. A further increase in RBC supply rate to twice control levels (case 2, dashed orange line) is required to restore tissue oxygenation.