The Mas receptor mediates modulation of insulin signaling by angiotensin-(1–7)

https://doi.org/10.1016/j.regpep.2012.04.001Get rights and content

Abstract

Angiotensin (Ang)-(1–7) stimulates proteins belonging to the insulin signaling pathway and ameliorates the Ang II negative effects at this level. However, up to date, receptors involved and mechanisms behind these observations remain unknown. Accordingly, in the present study, we explored the in vivo effects of antagonism of the Ang-(1–7) specific Mas receptor on insulin signal transduction in rat insulin-target tissues. We evaluated the acute modulation of insulin-stimulated phosphorylation of Akt, GSK-3β (Glycogen synthase kinase-3β) and AS160 (Akt substrate of 160 kDa) by Ang-(1–7) and/or Ang II in the presence and absence of the selective Mas receptor antagonist A-779 in insulin-target tissues of normal rats. Also using A-779, we determined whether the Mas receptor mediates the improvement of insulin sensitivity exerted by chronic Ang-(1–7) treatment in fructose-fed rats (FFR), a model of insulin resistance, dyslipidemia and mild hypertension. The two major findings of the present work are as follows; 1) Ang-(1–7) attenuates acute Ang II-mediated inhibition of insulin signaling components in normal rats via a Mas receptor-dependent mechanism; and 2). The Mas receptor appears to be involved in beneficial effects of Ang-(1–7) on the phosphorylation of crucial insulin signaling mediators (Akt, GSK-3β and AS160), in liver, skeletal muscle and adipose tissue of FFR. These results shed light into the mechanism by which Ang-(1–7) exerts its positive physiological modulation of insulin actions in classical metabolic tissues and reinforces the central role of Akt in these effects.

Highlights

► Our data highlight mechanisms of modulation of insulin signaling by Ang-(1-7). ► Ang-(1-7) reverts acute in vivo Ang II-mediated inhibition of insulin signaling. ► Ang-(1-7) restores insulin signaling in fructose-fed rats. ► These actions of Ang-(1-7) disappear in presence of a Mas receptor antagonist. ► Our data shows that Ang-(1-7) modulates insulin action through the Mas receptor.

Introduction

Alterations within the renin-angiotensin system (RAS) are an important contributor to the development of insulin resistance [1], [2], [3]. The RAS is classically conceived as a coordinated hormonal cascade in the control of cardiovascular, renal, and adrenal functions, mainly through the actions of angiotensin (Ang) II [4]. Advances in cell and molecular biology have led to the recognition of other active fragments of RAS metabolism, such as Ang III, Ang IV, and Ang-(1–7), the angiotensin-converting enzyme (ACE) 2, an homolog of classic ACE that forms Ang-(1–7) directly from Ang II and indirectly from Ang I [5], and the Ang-(1–7) specific G protein-coupled receptor Mas [6]. In general terms, the ACE2/Ang-(1–7)/Mas receptor axis opposes the vascular and proliferative effects of Ang II [7].

A large body of evidence indicates that Ang II plays a critical role in the etiology of insulin resistance [8]. The mechanism behind this deleterious effect appears to be related to a negative modulation exerted by Ang II through AT1 receptor (AT1R) on several steps of the insulin signaling cascade, including insulin-induced phosphorylation of the insulin receptor, insulin receptor substrate-1, and activation of Akt mediated by a mechanism that involves phosphatidylinositol 3-kinase (PI3K) [8]. Accordingly, clinical trials have shown that inhibition of ACE or selective AT1R blockade reduces the development of type 2 diabetes in patients with essential hypertension [9], [10]. In line with reports in humans, improvement of insulin sensitivity [1], [11], [12], [13] along with an enhancement in the response to insulin at various steps of the insulin signaling cascade [14], [15], [16], has been detected in various animal models of insulin resistance and/or type 2 diabetes as a consequence of reduction of Ang II formation or inhibition of its actions. This evidence clearly indicates that the signaling crosstalk between insulin and Ang II has significant physiological relevance. In contrast, Ang-(1–7), through its specific G protein-coupled receptor Mas induces responses that oppose those of Ang II, including antihypertensive, antihypertrophic, antifibrotic and antithrombotic properties [5], [7]. Considering that inhibition of ACE or chronic blockade of AT1R is associated with increased levels of circulating Ang-(1–7), this hormone could be involved in the beneficial effects of antihypertensive therapy [17], [18], [19].

We have previously demonstrated that Ang-(1–7) reverses insulin resistance in rats fed a high-fructose diet [20]. This effect could be ascribed to the capability of Ang-(1–7) to induce the activation of Akt in insulin‐target tissues [21], [22] and also to counteract the inhibitory effects of Ang II on insulin-induced Akt phosphorylation [22]. In line with the finding that Ang-(1–7) has metabolic actions, Santos et al. reported that genetic deletion of the specific Ang-(1–7) Mas receptor leads to a metabolic syndrome-like state in mice [23]. More recently, it was reported that rats overexpressing Ang-(1–7) display improved glucose and lipid metabolism [24], and that ACE2 gene therapy improves glycemic control in diabetic mice, through a mechanism mediated by Ang-(1–7) acting through its specific receptor Mas [25]. Despite this available information, the mechanism behind the amelioration of insulin resistance induced by Ang-(1–7) through its specific receptor Mas in FFR is intriguing and deserves further exploration.

Accordingly, in the current study, we explored the effects of simultaneous acute injection of different combinations of Ang-(1–7) and/or Ang II in the presence and absence of the selective Mas receptor antagonist A-779 on insulin-stimulated phosphorylation of Akt, GSK-3β (Glycogen synthase kinase-3β) and AS160 (Akt substrate of 160 kDa) in insulin-target tissues of normal animals. In addition, by the use of A-779, we determined the role of the Mas receptor in the improvement of insulin sensitivity exerted by chronic Ang-(1–7) treatment in FFR, a model of insulin resistance, dyslipidemia and mild hypertension.

Section snippets

Reagents

The peptides Ang II, Ang-(1–7) and [7-D-Ala-Ang-(1–7)] (A-779) were purchased from Bachem Americas, Inc. (Torrance, CA, USA.). The reagents and apparatus for SDS-PAGE and immunoblotting were obtained from Bio-Rad (Hercules, CA, USA). The phospho-Akt (Ser473) rabbit polyclonal antibody that detects endogenous levels of Akt only when phosphorylated at Ser473 (anti-p-Akt-Ser473; 9271), the rabbit polyclonal Akt antibody that detects endogenous levels of total Akt1, Akt2 and Akt3 proteins

Protocol 1: in vivo interactions between Ang II, Ang-(1–7) and insulin on the phosphorylation of Akt, GSK-3β and AS160 in liver, muscle and adipose tissue

As shown in Fig. 1, upper panel, insulin (8 pmol/kg) stimulated the phosphorylation of Akt in liver, adipose tissue and skeletal muscle. The acute administration of Ang II alone (8 pmol/kg) did not stimulate phosphorylation of Akt at Ser473. On the other hand, Ang-(1–7) induced the phosphorylation of Akt to a similar extent of that attained after stimulation with insulin.

Insulin-induced phosphorylation of Akt was blunted in the presence of Ang II (Fig. 1, upper panel). The simultaneous

Discussion

One of the major finding of the current study is that the beneficial effects exerted by Ang-(1–7) on crucial insulin signaling mediators in vivo, disappeared in the presence of an antagonist of the Ang-(1–7) specific Mas receptor. By acute administration of mixtures of hormones we determined that Ang II attenuates the insulin-stimulated phosphorylation of Akt, GSK-3β and AS160 in metabolic tissues (liver, adipose tissue and skeletal muscle). This result agrees with our previously reported

Acknowledgments

This study was partially supported by grants from the National Research Council of Argentina (CONICET) through PIP 114-200801-00374, the University of Buenos Aires (UBACyT, B051, B080 and 20020100100207). A. Carranza, F. P. Dominici, M.C. Muñoz, C. Taira are researchers at CONICET. J. F. Giani is a post-doctoral research fellow from CONICET.

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