Angiotensin-(1-7) Participates in Enhanced Skeletal Muscle Insulin Sensitivity After a Bout of Exercise

Abstract

A single bout of exercise increases subsequent insulin-stimulated glucose uptake in skeletal muscle; however, it is unknown whether angiotensin-(1-7) (Ang-(1-7)), a vasoactive peptide of the renin-angiotensin system, participates in this process. The aim of this study was to investigate the possible involvement of Ang-(1-7) in enhanced skeletal muscle insulin sensitivity after an exercise session. Male Wistar rats were forced to swim for 2.5 hours. Two hours after exercise, insulin tolerance tests and 2-deoxyglucose uptake in isolated soleus muscle were assessed in the absence or presence of the selective Mas receptor (MasR, Ang-(1-7) receptor) antagonist A779. Ang II and Ang-(1-7) levels were quantified in plasma and soleus muscle by HPLC. The protein abundance of angiotensin-converting enzyme (ACE), ACE2, Ang II type 1 receptor (AT₁R), and MasR was measured in soleus muscle by Western blot. Prior exercise enhanced insulin tolerance and insulin-mediated 2-deoxyglucose disposal in soleus muscle. Interestingly, these insulin-sensitizing effects were abolished by A779. After exercise, the Ang-(1-7)/Ang II ratio decreased in plasma, whereas it increased in muscle. In addition, exercise reduced ACE expression, but it did not change the protein abundance of AT₁R, ACE2, and MasR. These results suggest that Ang-(1-7) acting through MasR participates in enhanced insulin sensitivity of skeletal muscle after a bout of exercise.

Keywords: angiotensin-(1-7); exercise; glucose uptake; insulin sensitivity; renin-angiotensin system; skeletal muscle.

Figure 1.

Ang-(1-7) participates in enhanced skeletal muscle insulin sensitivity after a bout of exercise. (A) Experimental design. All rats were trained to swim 10 minutes/day for 2 consecutive days. On day 3, under nonfasting conditions, sedentary rats remained in their cages, whereas exercised rats were forced to swim for 2.5 hours. After the first 30 minutes of exercise, extra weight (corresponding to 2% of the body weight) was placed at the base of the tail of each rat; the load was removed at the end of the exercise protocol. All rats had free access to water and food restriction during and 4 hours after exercise. Insulin tolerance tests, 2-deoxyglucose uptake assays, and Western blot (WB) experiments were performed 2 hours postexercise. Angiotensins were quantified before and after exercise (0, 1, 2, and 4 hours). In both insulin tolerance tests and 2-deoxyglucose uptake assays, A779 (a selective Mas receptor [MasR, angiotensin 1-7 [Ang-(1-7)] receptor] antagonist) was administered or hatched 15 minutes before insulin delivery or 2-deoxyglucose incubation, respectively. (B) Effect of insulin administration (0.25 U/kg, IP) on blood glucose levels (decrements, ∇) from sedentary and 2-hours postexercised rats. Some rats were pretreated with deionized water (1 mL/kg, IP) or A779 (100 μg/kg, IP) 15 minutes before insulin delivery. (C) Areas under the curve (AUC) of the insulin tolerance tests performed in sedentary and previously exercised rats. (D) 2-Deoxyglucose uptake (measured as 2-deoxyglucose-6-phosphate [2-DG6P]) stimulated by saline (10 μL/mL) or insulin (0.06 μM) in isolated soleus muscles from sedentary and 2 hours postexercised rats. Some muscles were incubated with deionized water (10 μl/mL) or A779 (1 μM) 15 minutes before adding 2-deoxyglucose (8 mM). The tissues were hatched for 30 minutes. (E) Increments (∆) of 2-deoxyglucose uptake stimulated by insulin or insulin + A779 in soleus muscles from sedentary and previously exercised rats. Results are expressed as the mean ± SEM of 6 to 11 rats/assays per group. Two-way ANOVA followed by the SNK post hoc test was used to analyze the data. *P < 0.05 vs. sedentary; ^#P < 0.05 vs. postexercised + A779; ^&P < 0.05 vs. respective saline; ^$P < 0.05 vs. respective saline + A779; ^%P < 0.05 vs. insulin + A779, postexercised; ^/P < 0.05 vs. insulin, sedentary.

Figure 2

. Exercise downregulates the ACE/Ang II/AT ₁ R axis of RAS in rat skeletal muscle. Angiotensin II (Ang II) and Ang-(1-7) levels in (A) plasma and (B) soleus muscle from sedentary (Sed) and postexercised rats (0, 1, 2, and 4 hours PEx). Representative immunoblots and densitometry analysis of protein expression of (C) angiotensin-converting enzyme (ACE), (D) Ang II type 1 receptor (AT₁R), (E) ACE2, and (F) Mas Receptor (MasR) in soleus muscle from sedentary and 2-hour postexercised rats. β-actin was used as load control. Western blot data are expressed as the fold induction of the protein/actin relationship in units of relative density. All results represent the mean ± SEM of 4 to 6 rats per group. Two-way ANOVA followed by the SNK post hoc test was used to analyze angiotensin data. The unpaired Student t test was used to compare Western blot results. *P < 0.05 vs. corresponding group of Ang-(1-7); ^#P < 0.05 vs. Ang II, Sed; ^&P < 0.05 vs. Ang-(1-7), Sed; ^$P < 0.05 vs. sedentary.

Figure 3.

Proposed mechanism for the participation of angiotensin 1-7 (Ang-(1-7)) in enhanced skeletal muscle insulin sensitivity after a bout of exercise. Sympathetic nervous system (SNS) and renin-angiotensin system (RAS) are activated during exercise. Sympathetic discharge in both kidney and adipose tissue stimulates the release of renin into the systemic circulation. As result, the plasma levels of Ang II increase, whereas those of Ang-(1-7) decrease. After exercise, circulating concentrations of Ang II and Ang-(1-7) (through Ang II type 2 receptor [AT₂R] and Mas receptor [MasR], respectively) could facilitate transendothelial insulin transport to the skeletal muscle interstitium by increasing insulin signal transduction. On the other hand, the renin produced during exercise or in vitro contractions travels to the skeletal muscle and stimulates the local synthesis of angiotensin peptides. Because exercise decreases muscle ACE expression, the main enzyme for Ang II generation and Ang-(1-7) degradation, the synthesis of Ang II is reduced while that of Ang-(1-7) is raised by alternate pathways in muscle. It is feasible that the local elevation of Ang-(1-7) through MasR amplifies proximal (IRS-1, PI3K, Akt) or distal (AS160) insulin signaling. As a result, Ang-(1-7) enhances insulin-stimulated glucose uptake in skeletal muscle. Thus, Ang-(1-7), through microvascular and metabolic actions, could improve skeletal muscle insulin sensitivity after a bout of exercise. Kallikrein-Kinin system (KKS) also is upregulated during exercise; however, evidence suggests that bradykinin (BK), a vasoactive protein of KKS, does not participate in this process. ACE, angiotensin-converting enzyme; Akt, protein kinase B; Ang, angiotensin; Aog, angiotensinogen; AS160, TBC1 domain family member 4; GLUT4, glucose transporter 4; IR, insulin receptor; IRS, insulin receptor substrate; NEP, neutral endopeptidase; PI3K, phosphatidylinositol 3-kinase; (P)RR, (pro)renin receptor. Dotted lines indicate the proposed mechanisms.