ANGIOTENSIN-(1-7) RECRUITS MUSCLE MICROVASCULATURE AND ENHANCES INSULIN’S METABOLIC ACTION VIA MAS RECEPTOR

Abstract

Angiotensin (Ang)-(1-7), an endogenous ligand for the G protein-coupled receptor Mas, exerts both vasodilatory and insulin sensitizing effects. In skeletal muscle relaxation of pre-capillary arterioles recruits microvasculature and increases the endothelial surface area available for nutrient and hormone exchanges.

To assess whether Ang-(1-7) recruits microvasculature and enhances insulin action in muscle, overnight-fasted adult rats received an intravenous infusion of Ang-(1-7) (0, 10 or 100 ng/kg/min) for 150 min with or without a simultaneous infusion of the Mas inhibitor A-779 and a superimposition of a euglycemic insulin clamp (3 mU/kg/min) from 30 to 150 min. Hindlimb muscle microvascular blood volume (MBV), microvascular flow velocity (MFV) and microvascular blood flow (MBF) were determined. Myographic changes in tension were measured on pre-constricted distal saphenous artery.

Ang-(1-7) dose-dependently relaxed the saphenous artery (p<0.05) ex vivo. This effect was potentiated by insulin (p<0.01) and abolished by either endothelium denudement or Mas inhibition. Systemic infusion of Ang-(1-7) rapidly increased muscle MBV and MBF (p<0.05, each) without altering MFV. Insulin infusion alone increased muscle MBV by 60–70% (p<0.05). Adding insulin to the Ang-(1-7) infusion further increased muscle MBV and MBF (~2.5 fold, p<0.01). These were associated with a significant increase in insulin-mediated glucose disposal and muscle Akt and ERK1/2 phosphorylation. A-779 pre-treatment blunted Ang-(1-7)’s microvascular and insulin-sensitizing effects.

We conclude that Ang-(1-7) by activating Mas recruits muscle microvasculature and enhances insulin’s metabolic action. These effects may contribute to the cardiovascular protective responses associated with Mas activation and explain Ang-(1-7)’s insulin sensitizing action.

INTRODUCTION

Muscle accounts for 70–80% of insulin-stimulated whole body glucose disposal during insulin infusion. A rate-limiting step in muscle insulin action is the delivery of insulin to the muscle interstitium which contributes up to 40% of insulin’s metabolic action ¹. This process is critically regulated by the skeletal muscle microvasculature which not only regulates the delivery of nutrients and insulin to the muscle microcirculation but also provides the endothelial surface area for their exchanges between plasma and muscle interstitium ^2–4. Insulin also actively regulates its own delivery in muscle by increasing muscle microvascular blood volume (MBV) and stimulating its own trans-endothelial transport ⁴.

The renin-angiotensin system (RAS) critically regulates vascular tone, tissue perfusion and water balance. Angiotensin (Ang) II, the main RAS component, exerts a potent vasoconstrictive action via its type 1 receptors (AT₁R) but also a vasodilatory effect via its type 2 receptors (AT₂R) ^{5, 6}. In addition, the RAS actively regulates insulin action ^7–9. Prior evidence mainly focused on the antagonistic action of Ang II on insulin action via AT₁R-mediated NADPH oxidase/oxidative stress pathway ^{10, 11} and we have recently demonstrated that Ang II also regulates insulin action via its effects on muscle microvascular perfusion. While basal AT₁R activity restricts muscle microvascular perfusion and decreases muscle insulin delivery and action, AT₂R activation potently recruits muscle microvasculature and rescues insulin action during systemic lipid infusion via increased insulin delivery in rodents ^12–14. In mildly hypertensive humans acute AT₁R blockade with irbesartan increases insulin-induced skin microvascular perfusion ¹⁵. Interestingly in healthy humans Ang II has been shown to enhance insulin-stimulated whole body glucose disposal but impair insulin-induced skin capillary recruitment ¹⁶.

In addition to AT₂R, an important RAS pathway poised to oppose AT₁R is the Ang-(1-7) – Ang converting enzyme-2 (ACE-2) – Mas receptor (Mas) hormonal cascade. Ang-(1-7) is a heptapeptide generated primarily from Ang II by the actions of ACE-2. Similar to Ang II’s action on the AT₂R, Ang-(1-7) exerts potent vasodilatory actions on conduit arteries and microvessels via a nitric oxide (NO)-dependent mechanism ^{17, 18}. Recent evidence suggests that in addition to its vasodilatory action Ang-(1-7) also reduces Ang II- or fructose-induced insulin resistance ^19–21. Transgenic rats that express an Ang-(1-7)-releasing fusion protein have elevated circulating Ang-(1-7) levels with improved glucose tolerance and insulin-stimulated glucose uptake ²². This action does not appear to be mediated via either AT₁R or AT₂R. Rather, Ang-(1-7) acts on a unique G-protein coupled receptor Mas ^{23, 24} which is encoded by the Mas proto-oncogene and expressed constitutively in endothelial cells ²⁵. Murine Mas-deficient aortae lose their Ang-(1-7)-induced relaxation response ²³. Similarly, Ang-(1-7)-mediated relaxation of wild-type mesenteric arteries is equally impaired in both wild-type arteries pretreated with Mas inhibitor A-779 and arteries isolated from Mas-deficient mice ²⁶. However, the mechanisms underlying Ang-(1-7)’s insulin sensitization remain unknown.

Given the critical role that microvasculature plays in regulating muscle insulin action and that Ang-(1-7) has been demonstrated to exert both vasodilatory and insulin-sensitizing actions, it is possible that Ang-(1-7) exerts its insulin sensitizing action via regulating muscle microvascular endothelial surface area. This was examined in the current study and our results indicate that Ang-(1-7) potently recruits muscle microvasculature and increases insulin-stimulated whole body glucose disposal via a Mas-dependent mechanism.

METHODS

Animal Preparations and Experimental Protocols

Overnight-fasted adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 250–380 g were studied under one of the 3 protocols (Figure 1). Prior to the study rats were fed standard laboratory chow and water ad libitum and housed at 22 ± 2°C on a 12 hr light-dark cycle. Rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.; Abbott Laboratories, North Chicago, IL), placed in a supine position on a heating pad to ensure euthermia and intubated to maintain a patent airway. The carotid artery and the jugular vein were cannulated with polyethylene tubing (PE-50, Fisher Scientific, Newark, DE) for arterial blood pressure monitoring, arterial blood sampling and various infusions.

Figure 1.

Experimental protocols. Protocol 1 examines Ang-(1-7)’s effects on muscle microvasculature. Protocol 2 studies whether Ang-(1-7) alters insulin’s microvascular and metabolic actions. Protocol 3 determines whether Mas mediates Ang-(1-7)’s microvascular actions.

After a 30 min baseline period to ensure hemodynamic stability and a stable level of anesthesia, baseline microvascular parameters, including MBV, microvascular flow velocity (MFV) and microvascular blood flow (MBF), were determined on the proximal adductor muscle group (adductor magnus and semimembranosus) of the right hindlimb using contrast-enhanced ultrasound (CEU) and microbubble (Definity, Lantheus Medical Imaging) as the contrast agent, as previously described ^{27, 28}. Rats then received a continuous intravenous infusion of Ang-(1-7) (0, 10 or 100 ng/kg/min) for 150 min (−30 – 120 min) without (Protocol 1) or with (Protocol 2) a hyperinsulinemic euglycemic clamp (3 mU/kg/min) superimposed on the last 120 min (0 – 120 min) (Figure 1). Additional rats were studied in the same fashion as Protocol 2 except all rats received a continuous infusion of A-779 (10 nmol/kg/min), a specific Mas inhibitor ²⁹, which began 30 min prior to starting the Ang-(1-7) infusion (−60 – 120 min) (Protocol 3).

Microvascular parameters were determined at time points indicated in Figure 1 using CEU. Throughout the study, mean arterial blood pressure (MAP) was monitored via a sensor connected to the arterial catheter (Harvard Apparatus, Holliston, MA and ADInstruments, Inc., Colorado Springs, CO). Pentobarbital sodium was infused at a variable rate to maintain steady levels of anesthesia and blood pressure throughout the study. During the insulin clamp, arterial blood glucose was determined every 10 min using an Accu-Chek Advantage glucometer (Roche Diagnostics, Indianapolis, IN), and 30% dextrose was infused at a variable rate to maintain blood glucose within 10% of basal. At the end of each study the rat was sacrificed by a pentobarbital overdose and the gastrocnemius muscle was freeze-clamped in liquid nitrogen for later measurement of protein kinase B (Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation using Western blot analysis.

The study conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (publication No. 85-23, revised 1996). The study protocols were approved by the Animal Care and Use Committee of the University of Virginia.

Determination of protein phosphorylation

Muscle Akt and ERK1/2 phosphorylation was determined by Western blot analysis as described previously ^{13, 30–32}. Primary antibodies against phospho-Akt (Ser⁴⁷³), total Akt, phospho-ERK (Thr²⁰²/Tyr²⁰⁴) and total ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). All blots were developed using enhanced chemiluminescence (GE Healthcare Bio-Sciences Corp, Piscataway, NJ). Autoradiographic films were scanned densitometrically and quantified using ImageQuant 3.3 software. Both the total and phosphospecific densities were quantified and the ratios of phosphospecific to total protein density calculated.

Assessment of Ang-(1-7)’s vasorelaxant effect on pre-constricted small arteries

The distal saphenous artery (≤ 250 μm) was dissected, cleaned of adhering connective tissues and cut into segments of ~2 mm in length. Each segment was mounted in a Multi Myograph System (Danish Myo Technology, Aarhus, Denmark). The organ chamber was filled with 6 mL physiological salt solution buffer (NaCl 130 mM, KCl 4.7 mM, CaCl₂ 1.6 mM, MgSO₄ 1.17 mM, KH₂PO₄ 1.18 mM, NaHCO₃ 14.9 mM, EDTA 0.026 mM, glucose 5.5 mM; pH 7.4), which was constantly bubbled with 95% O₂ – 5% CO₂ and maintained at 37°C. Each ring was stretched initially to 5 mN and then allowed to stabilize at baseline tone. In some arteries, the endothelium was mechanically removed by rubbing the luminal surface of the ring with human hair. Functional removal of the endothelium was verified by the lack of relaxation response to acetylcholine. After pre-constriction of the arterial ring with 2 μM phenylephrine, Ang-(1-7) and/or insulin at various concentrations were added into the chamber. For the Mas inhibitor study, pre-constricted arterial rings were incubated with A-779 (20 μM) for 30 min before Ang-(1-7) was added. Changes in arterial tone were recorded.

Statistical Analysis

All data are presented as mean ± SEM. Statistical analyses were performed with SigmaStat 11.0 software (Systat Software, Inc), using either Student’s t-test or ANOVA with post-hoc analysis as appropriate. A p-value of < 0.05 was considered statistically significant.

RESULTS

Ang-(1-7) induces arterial relaxation via Mas in endothelium

Prior evidence has strongly suggested a direct vasodilatory action of Ang-(1-7) on microvessels, likely via a Mas-mediated, NO-dependent pathway ^{18, 33}. Likewise, insulin also exerts potent vasodilatory action via increased NO production ^{34, 35}. As such, we first examined whether Ang-(1-7) and insulin could potentiate each other’s vasodilatory actions in vitro (Figure 2). Using phenylephrine pre-constricted distal saphenous arteries, we found that Ang-(1-7) and insulin each induced dose-dependent vasorelaxation (p<0.05). Interestingly, insulin at high physiologic concentrations (1 nM) did not induce significant vasorelaxation but it markedly potentiated Ang-(1-7)’s vasodilatory effect (by up to 40%, p<0.01). Both endothelium denudement and pre-treatment with Mas inhibitor A-779 completely abolished Ang-(1-7)-induced vasorelaxation.

Figure 2.

Ang-(1-7) induces vasodilation via endothelium- and Mas-dependent mechanisms. Rat distal saphenous artery was isolated, pre-constricted with phenylephrine, and then incubated with various concentrations of Ang-(1-7) and/or insulin. A and B: Effect of Ang-(1-7) on intact or endothelium denuded arterial rings. n=6, * p<0.05. C and D: Effect of insulin. n=6, * p<0.05 vs. control. E and F: Effect of Ang-(1-7) in the presence of insulin (1 nM). n=6, #p<0.01 vs. control. G and H: Effect of Mas inhibition on Ang-(1-7)-induced vasodilation. n=6, * p<0.05. B, D, F and H: representative myographic tracings.

Ang-(1-7) recruits skeletal muscle microvasculature

To examine the effect of Ang-(1-7) on the pre-capillary arterioles which control muscle microvascular perfusion ^{4, 36}, we next used CEU to measure three important indices of the muscle microcirculation i.e. MBV, MFV and MBF before and during a continuous infusion of Ang-(1-7) at various concentrations. As shown in Figure 3, Ang-(1-7) dose-dependently increased muscle microvascular recruitment by increasing MBV within the first 30 min and this effect lasted for the entire 150 min infusion period while MFV was not affected. The increase of MBV led to a maximum increase in muscle MBF (by ~80%, p<0.05). Neither MAP nor blood glucose levels changed during Ang-(1-7) infusion (Tables 1 and 2).

Figure 3.

Effects of Ang-(1-7) on muscle microvascular recruitment. Each rat received a continuous intravenous infusion of Ang-(1-7) or saline (Control) for 150 min (−30 – 120 min). Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min. A: MBV; B: MFV; C: MBF. n=5 each. * p<0.05 vs control.

Table 1.

Blood glucose levels (mg/dL).

Treatment	−60 min	−30 min	0 min	30 min	60 min	90 min	120 min
Protocol 1
Control	n/a	109±4.18	109±3.77	109±5.15	108±6.73	109±5.43	106±6.66
Ang-(1-7)_10	n/a	112±4.22	110±5.45	110±6.26	108±4.95	109±5.32	108±5.86
Ang-(1-7)_100	n/a	114±4.22	114±5.45	113±6.26	113±4.95	111±5.32	110±5.86
Protocol 2
Control	n/a	112±1.94	110±1.66	109±2.8	108±2.57	111±2.74	110±2.23
Ang-(1-7)_10	n/a	113±1.41	111±1.92	110±2.43	110±2.3	109±2.71	110±1.73
Ang-(1-7)_100	n/a	111±2.07	110±2.53	105±2.4	107±2.54	108±2.6	109±3.11
Protocol 3
Control	105±1.69	106±1.82	106±2.09	97±2.16	104±2.13	108±2.21	108±2.93
Ang-(1-7)_10	108±3.7	109±3.84	110±4.28	105±3.7	103±5.36	109±3.2	110±3.76
Ang-(1-7)_100	105±2.72	106±2.91	105±3.2	99±3.63	102±3.58	101±3.35	109±3.99

Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min.

Table 2.

Mean arterial pressure (mmHg).

Treatment	−60 min	−30 min	0 min	30 min	60 min	90 min	120 min
Protocol 1
Control	n/a	120±5.07	120±6.11	121±2.49	116±4.76	119±6.43	120±5.22
Ang-(1-7)_10	n/a	116±8.71	117±5.15	116±4.72	115±2.17	112±7.3	112±13.54
Ang-(1-7)_100	n/a	121±8.71	120±5.15	117±4.72	120±2.17	116±7.3	113±13.54
Protocol 2
Control	n/a	116±2.59	112±3.33	112±2.21	119±1.88	107±3.14	112±2.85
Ang-(1-7)_10	n/a	111±3.28	112±0.94	107±2.93	106±1.76	109±1.73	108±2.66
Ang-(1-7)_100	n/a	114±4.56	114±1.16	110±2.75	108±1.95	111±1.88	113±2.92
Protocol 3
Control	113±2.47	112±2.47	113±2.3	116±2.88	115±1.31	115±3.35	112±2.76
Ang-(1-7)_10	113±8.93	113±7.57	109±7.67	113±7.67	109±7.66	108±3.7	107±4.42
Ang-(1-7)_100	124±1.96	121±1.74	119±3.71	119±3.75	117±1.96	119±4.29	115±1.52

Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min.

Ang-(1-7) enhances insulin’s vascular and metabolic actions

Knowing that Ang-(1-7) could indeed recruit muscle microvasculature and we and others have previously demonstrated that microvascular recruitment regulates insulin delivery and action in muscle ^1,4, we next examined whether Ang-(1-7) could modulate insulin’s microvascular and metabolic actions in vivo. Similar to prior reports, insulin infusion alone increased both muscle MBV and MBF by ~60–70% (p<0.05) without affecting MFV (Figure 4). Administration of Ang-(1-7) prior to the initiation of insulin infusion recruited significantly more microvasculature than insulin alone. This was associated with ~30% increase (p<0.05) in insulin-mediated whole body glucose disposal (Figure 5). Neither MAP nor blood glucose levels changed during the entire procedure (Tables 1 and 2).

Figure 4.

Effects of Ang-(1-7) on muscle microvascular recruitment during insulin clamp. Each rat received a continuous intravenous infusion of Ang-(1-7) or saline (Control) for 150 min (−30 – 120 min) with an insulin clamp (3 mU/kg/min) superimposed on the last 120 min. Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min. A: MBV; B: MFV; C: MBF. n=5 each. * p<0.05 vs baseline (−30 min). # p<0.01 vs insulin alone.

Figure 5.

Ang-(1-7) dose-dependently augments insulin-stimulated whole body glucose disposal. Each rat received a continuous intravenous infusion of Ang-(1-7) or saline (Control) for 150 min with an insulin clamp (3 mU/kg/min) superimposed on the last 120 min. Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min. A: Time course of glucose infusion rate (GIR) during insulin clamp; B: GIR area under the curve during insulin clamp. n=5 each. * p<0.05 vs control.

Ang-(1-7)’s in vivo effects are Mas-dependent

Ang-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas and our ex vivo myographic studies showed that Ang-(1-7)’s vasodilatory effect is indeed Mas dependent. We therefore tested whether Ang-(1-7)’s in vivo effects on the muscle microvasculature and insulin’s metabolic action are also mediated by Mas. As shown in Figure 6, inhibition of Mas with its specific antagonist A-779 abolished Ang-(1-7)-induced microvascular recruitment without affecting insulin-mediated increase in muscle MBV, evidenced by a lack of increase in MBV and MBF 30 min after Ang-(1-7) infusion (time 0 min) but a significant increase in MBV and MBF 30 min after beginning the insulin infusion (time 30 to 150 min). Interestingly, there is a tendency for Ang-(1-7), at 100 ng/kg/min, to synergize with insulin after 30 min to increase MBV and MBF, despite the presence of A-779 (p<0.05 at 120 min for MBV). These were coupled with a loss of the previously seen Ang-(1-7) enhancement of insulin-mediated glucose disposal (Figure 7). Neither MAP nor blood glucose levels changed during the entire procedure (Tables 1 and 2).

Figure 6.

Effects of Mas inhibition on Ang-(1-7) and insulin’s microvascular actions. Each rat received a continuous intravenous infusion of A-779 (10 nmol/kg/min) 30 min before Ang-(1-7) or saline infusion and 60 min before insulin clamp (3 mU/kg/min). Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min. A: MBV; B: MFV; C: MBF. n=5 each. * p<0.05 vs baseline (−60 min), # p<0.05 vs insulin alone.

Figure 7.

Mas inhibition abolishes Ang-(1-7)-mediated enhancement in insulin’s metabolic action. Each rat received a continuous intravenous infusion of A-779 (10 nmol/kg/min) 30 min before Ang-(1-7) or saline infusion and 60 min before insulin clamp (3 mU/kg/min). Ang-(1-7)_10: Ang-(1-7) at 10 ng/kg/min. Ang-(1-7)_100: Ang-(1-7) at 100 ng/kg/min. A: Time course of glucose infusion rate (GIR) during insulin clamp; B: GIR area under the curve during insulin clamp. n=5 each.

Effects of Ang-(1-7) on muscle Akt and ERK1/2 phosphorylation

Since Ang-(1-7) increased insulin’s metabolic action via a Mas-dependent signaling pathway, we measured the phosphorylation of muscle Akt and ERK1/2, two key signal intermediates in the insulin signaling pathways, in rats studied under all 3 protocols. As shown in Figure 8, insulin infusion significantly increased muscle Akt and ERK1/2 phosphorylation. While Ang-(1-7) infusion alone did not affect either Akt or ERK1/2 phosphorylation, it did further enhance insulin-mediated phosphorylation of these two proteins in a dose dependent fashion. A-779 pre-treatment completely abolished Ang-(1-7)’s effect without affecting insulin’s actions on either Akt or ERK1/2 phosphorylation.

Figure 8.

Effect of Ang-(1-7) on muscle Akt and ERK1/2 phosphorylation. A: Akt phosphorylation. B: ERK1/2 phosphorylation. n=4 each. * p<0.05 vs baseline [no insulin or A-779], # p<0.05 vs insulin alone.

DISCUSSION

In the current study we provide experimental evidence using both ex vivo arterial myographic and in vivo animal study approaches that Ang-(1-7) potently recruits muscle microvasculature and enhances insulin’s metabolic action and these actions are mediated by Mas in the endothelium.

Prior studies have confirmed that Ang-(1-7) exerts a potent vasodilatory action in various arterial beds ^{17, 18, 23, 37, 38}. Ang-(1-7) counteracts the vasoconstrictor actions of Ang II and lowers blood pressure in transgenic (mRen2)27 hypertensive rats ³⁹ and in spontaneously hypertensive rats ⁴⁰. However, in the current study we did not observe a significant change in the mean arterial blood pressure during Ang-(1-7) infusion, likely secondary to the fact that our animals were not hypertensive and/or that the RAS was not up-regulated. Indeed, even chronic infusion of Ang-(1-7) failed to produce a significant change in blood pressure in the presence of high dietary salt intake and in normal rats. Ang-(1-7) likely is more important in the regulation of regional as opposed to systemic vascular resistance. Acute infusion of Ang-(1-7) leads to a significant change in regional blood flow distribution with a decrease in total peripheral resistance in Wistar rats ⁴¹. Similarly, expression of an Ang-(1-7)-producing fusion protein in rats elevates plasma Ang-(1-7) levels and reduces total peripheral resistance, which is associated with increased stroke volume and cardiac index ⁴². Although we did not assess cardiac parameters and peripheral vascular resistance, our findings that Ang-(1-7) significantly increased microvascular recruitment suggests that Ang-(1-7) is able to vasodilate muscle pre-capillary arterioles which control microvascular perfusion and are major contributors to total peripheral resistance.

The increased muscle microvascular recruitment seen with Ang-(1-7) infusion provides a physiological explanation for numerous study findings that Ang-(1-7) administration improves insulin sensitivity and action, as muscle microvasculature plays a critical role in the regulation of muscle insulin action by providing the endothelial exchange surface area and actively regulating insulin delivery to the muscle interstitium. Indeed, it is the insulin concentrations in the muscle interstitium, not in plasma, that best correlate with insulin’s muscle metabolic action and whole body glucose disposal ⁴³. A recent study demonstrates that in rats Ang-(1-7) by itself does not promote glucose transport but significantly increases insulin-stimulated glucose disposal ⁴⁴. This lends further support to our study findings. We and others have previously demonstrated that factors that are able to recruit microvasculature, such as low intensity exercise ^27,28, AT₁R blockade ^13,14, adiponectin ⁴⁵, ranolazine ³², and GLP-1 ⁴⁶, all significantly increase muscle insulin delivery and action. That Ang-(1-7)-induced microvascular recruitment is associated with increased whole body glucose disposal and muscle Akt and ERK1/2 phosphorylation during insulin clamp is certainly consistent with increased muscle delivery and action of insulin.

Our observations that the combination of Ang-(1-7) and insulin (1 nM) exerted an enhanced vasodilatory action compared to Ang-(1-7) or insulin alone ex vivo and that the MBV increase in vivo was also greater with combined insulin plus Ang-(1-7) infusion compared to either agent alone, suggest that Ang-(1-7) and insulin exert additive or synergistic vasodilatory actions on the pre-capillary arterioles. This is not surprising as insulin ^{34, 35} and Ang-(1-7) ^{17, 18} each engenders vasorelaxation via a NO-dependent pathway and high NO production, as seen in the case of GLP-1 and losartan, can increase MBV by 2–3-fold ^{13, 14, 46}.

Though prior studies have reported that Ang-(1-7) stimulated muscle Akt phosphorylation and this was completely inhibited by the co-administration of Mas inhibitor A-779 ^{47, 48}, Ang-(1-7) alone did not increase muscle Akt phosphorylation in the current study. This discrepancy likely resulted from the difference in timing of muscle sample collection for Akt phosphorylation analysis (5 min in prior reports vs. 150 min in the current study). Similarly, Ang-(1-7) did not alter ERK1/2 phosphorylation. It is certainly possible that Ang-(1-7) could acutely phosphorylate muscle Akt and ERK1/2 but this effect is not sustained. The lack of Ang-(1-7) enhancement of insulin-mediated Akt and ERK1/2 phosphorylation during A-779 infusion is consistent with the findings that Mas inhibition blunts Ang-(1-7)-mediated microvascular recruitment and enhancement in insulin-stimulated whole body glucose disposal. Thus, our study results strongly suggest that Ang-(1-7) recruits muscle microvasculature and increases microvascular endothelial surface area which lead to increased muscle delivery thus interstitial concentrations and action of insulin. Though the Western blot results should be interpreted with caution as we did not separate myocytes from other cell types such as endothelial cells, vascular smooth muscle cells, and fibroblasts, our results most likely reflect the findings in myocytes given its dominance in number in muscle.

Although expression of Mas in endothelial cells is well-documented and Mas has been repeatedly shown to mediate Ang-(1-7)’s vasodilatory actions, several studies have suggested that Ang-(1-7) may exert vasorelaxation via AT₂R activation ^{49, 50} or by antagonizing AT₁R ^{51, 52}. While we have previously reported that basal AT₁R tone restricts and basal AT₂R activity enhances muscle MBV ¹², it is unlikely that Ang-(1-7) acted directly on the AT₁R to exert its biological effects in our study due to its extremely low affinity for AT₁R ⁵³. It remains possible that Ang-(1-7) regulates AT₁R function via Mas which is a physiological antagonist of AT₁R ⁵⁴. Our finding that pre-treatment with Mas antagonist A-779 blocks both Ang-(1-7)’s vasodilatory actions on isolated arterioles ex vivo and precapillary arterioles in vivo confirms that Mas plays a key role in this process. However, our observations that Mas inhibition completely abolished the Ang-(1-7)-mediated increase in MBV and its potentiation on insulin’s microvascular action at 10 but not at 100 ng/kg/min are somewhat puzzling. This may reflect that microvessels undergo a non-specific loss of tension mediated by NO ¹⁸ or non-Mas-mediated effects of Ang-(1-7) at high concentrations. Indeed, Ang-(1-7) is capable of binding to AT₂R directly though at much lower affinity than Ang II ⁵³. Further study is warranted.

Perspectives.

Muscle microvasculature plays a critical role in the regulation of muscle delivery of nutrients and hormones including insulin by providing endothelial exchange surface area. Ang-(1-7) has been shown to cause arterial dilation and increase insulin sensitivity. Our current results demonstrate that acute administration of Ang-(1-7) potently recruits muscle microvasculature via its receptor Mas in the endothelium which expands the microvascular exchange surface area and thus enhances insulin’s metabolic action in muscle. These effects may contribute to the cardiovascular protective responses associated with Mas activation and explain the insulin sensitizing action of Ang-(1-7).

Novelty and Significance.

What Is New?

• Ang-(1-7) potently recruits microvasculature to increase endothelial exchange surface area in muscle.

• Ang-(1-7) and insulin exerts additive/synergistic actions on the pre-capillary arterioles.

• Ang-(1-7) enhances insulin’s metabolic actions in muscle.

• Mas inhibition abolishes Ang-(1-7)’s microvascular and insulin-sensitizing effects.

What Is Relevant?

• Mas plays a pivotal role in Ang-(1-7)-mediated microvascular recruitment and insulin sensitization.

• This may contribute to the cardiovascular protective responses associated with Mas activation and explain Ang-(1-7)’s insulin sensitizing action.

Summary

• Ang-(1-7) by activating Mas recruits muscle microvasculature and enhances insulin’s metabolic action.