Post-translational dysregulation of glucose uptake during exhaustive cycling exercise in vastus lateralis muscle of healthy homozygous carriers of the ACE deletion allele

Abstract

Homozygous carriers of the deletion allele in the gene for angiotensin-converting enzyme (ACE-DD) demonstrate an elevated risk to develop inactivity-related type II diabetes and show an overshoot of blood glucose concentration with enduring exercise compared to insertion allele carriers. We hypothesized that ACE-DD genotypes exhibit a perturbed activity of signaling processes governing capillary-dependent glucose uptake in vastus lateralis muscle during exhaustive cycling exercise, which is associated with the aerobic fitness state. 27 healthy, male white Caucasian subjects (26.8 ± 1.1 years; BMI 23.6 +/- 0.6 kg m^-2) were characterized for their aerobic fitness based on a threshold of 50 ml O₂ min^-1 kg^-1 and the ACE-I/D genotype. Subjects completed a session of exhaustive one-legged exercise in the fasted state under concomitant measurement of cardiorespiratory function. Capillary blood and biopsies were collected before, and ½ and 8 h after exercise to quantify glucose and lipid metabolism-related compounds (lipoproteins, total cholesterol, ketones) in blood, the phosphorylation of 45 signaling proteins, muscle glycogen and capillaries. Effects of aerobic fitness, ACE-I/D genotype, and exercise were assessed with analysis of variance (ANOVA) under the hypothesis of a dominant effect of the insertion allele. Exertion with one-legged exercise manifested in a reduction of glycogen concentration ½ h after exercise (-0.046 mg glycogen mg^-1 protein). Blood glucose concentration rose immediately after exercise in association with the ACE-I/D genotype (ACE-DD: +26%, ACE-ID/II: +6%) and independent of the fitness state (p = 0.452). Variability in total cholesterol was associated with exercise and fitness. In fit subjects, the phosphorylation levels of glucose uptake-regulating kinases [AKT-pT308 (+156%), SRC-pY419, p38α-pT180/T182, HCK-pY411], as well as cytokine/angiotensin 1-7 signaling factors [(STAT5A-pY694, STAT5B-pY699, FYN-pY420, EGFR-pY1086] were higher in angiotensin converting enzyme I-allele carriers than ACE-DD genotypes after exercise. Conversely, the AKT-S473 phosphorylation level (+117%) and angiotensin 2's blood concentration (+191%) were higher in ACE-DD genotypes. AKT-S473 phosphorylation levels post-exercise correlated to anatomical parameters of muscle performance and metabolic parameters (p < 0.05 and │r│>0.70). The observations identify reciprocal alterations of S473 and T308 phosphorylation of AKT as gatekeeper of a post-translational dysregulation of transcapillary glucose uptake in ACE-DD genotypes which may be targeted in personalized approaches to mitigate type II diabetes.

Keywords: angiotensin; diabetes; exercise; genotype; signalling.

FIGURE 1

Metabolic changes with one-legged exercise. Box-Whisker plots (box: first and third quartile, whisker minima and maxima, line: median, x: mean, circles: non overlapping data points) for the fractional changes for blood metabolites and respiration exchange ratio immediately post one-legged exercise, and muscle glycogen concentration 30 min post one-legged exercise combined (A) and split for aerobic fitness state (B). *, **, ***p < 0.05, <0.01, <0.005, respectively, for significant post vs. pre differences. ANOVA for the repeated factor of exercise and the factor of aerobic fitness. N = 27.

FIGURE 2

Exercise-induced alterations of glucose metabolism related compounds per genotype. Box-Whisker plots of the differences in the concentration of blood glucose (A) and muscle glycogen (B) immediately and 30 min, respectively, after one-legged exercise in carriers (ACE-ID/II) and non-carriers (ACE-DD) of the ACE I-allele. *, p < 0.05 for post vs. pre differences between genotypes. ANOVA for the factor ACE-I/D genotype. N = 27.

FIGURE 3

Exercise-induced alterations of glucose metabolism related compounds per genotype and fitness state. Box-Whisker plots of the differences in the concentration of blood glucose (A) and muscle glycogen (B) immediately and ½ hour, respectively, after one-legged exercise in association with the ACE I-allele (i.e., genotype) and fitness state. ^{†, ††, †††} p < 0.05, <0.01 < 0.001 for exercise-induced changes. *, p < 0.05 for differences between genotypes. Repeated measures ANOVA for the factor ACE-I/D genotype x exercise. N = 27.

FIGURE 4

Quantification of protein phosphorylation. (A) Sketch of the assessed phosphorylation of signaling proteins being involved in GLUT-4 mediated glucose import as induced by contraction and insulin, angiotensin and cytokine signaling. Abbreviation: AT1, angiotensin receptor 1; CHO, glucose. (B–E) Images depicting the detection of the phosphorylation content for vastus lateralis muscle samples from an ACE-DD (B,C) and ACE-II (D,E) genotype ½ hrs post exercise in pairs of filters of the phospho-kinase arrays. Each phosphorylation content is assessed as measurement from a pair of spots. The position of the spot pairs for AKT-pS473 and AKT-pT308 is indicated. Strongest signals correspond to the reference signals used to ‘standardize’ the assessed counts.

FIGURE 5

Phosphotransferases demonstrating ACE-I/D genotype differences post exercise. Box-Whisker plots of the (referenced) values for the phosphorylation of glucose metabolism related phosphotransferases in the fit carriers (ACE-ID/II) and non-carriers of the ACE I-allele (ACE-DD) as average of values ½ and 8 h after one-legged exercise. (A) AKT-pT308, (B) p38α-pT180/Y182, (C) SRC-pY419, (D) HCK-pY411, (E) STAT5A-pY694, (F) STAT5B-pY699, (G) EGFR-pY1086, (H) GSK3α/β-pS21/S9. +, * and ***, p < 0.10, <0.05, <0.001 for differences vs. ACE-DD genotype. ANOVA for the factor ACE-I/D genotype.

FIGURE 6

Phosphotransferase demonstrating ACE-I/D genotype related ‘counter-regulation’ post exercise. Box-Whisker plots of the (referenced) values for the phosphorylation of glucose metabolism related AKT-pS473 in the fit carriers (ACE-ID/II) and non-carriers of the ACE I-allele (ACE-DD) ½ - 8 h after one-legged exercise. ***, p < 0.001 for differences vs. ACE-DD genotype. ANOVA for the factor ACE-I/D genotype.