Research Paper
Brown adipose tissue transcriptome unveils an important role of the Beta-alanine/alamandine receptor, MrgD, in metabolism

https://doi.org/10.1016/j.jnutbio.2023.109268Get rights and content

Abstract

Alamandine is a recently described heptapeptide component of the renin-angiotensin system (RAS), and its effects are mediated by the receptor Mas-related G protein-coupled receptor D (MrgD) RAS represents an important link between obesity and its consequences by directly modulating the thermogenesis and brown adipose tissue (BAT) function. The alamandine/MrgD metabolic effects and signaling remain unexplored. In this context, the main goal of the present study was to assess the metabolic consequences of MrgD genetic ablation in C57BL6/J mice by evaluating brown adipose tissue RNA sequencing. The main results showed that MrgD-KO mice have diminished brown adipose tissue and that a high-glucose diet (HG) decreased both circulating alamandine levels and MrgD expression in BAT from wild-type mice (WT). BAT transcriptome reveals that MrgD-KO HG mice regulated 45 genes, while WT HG mice regulated 1,148 genes. MrgD-KO mice fed a standard diet (ST) compared with WT ST mice regulated 476 genes, of which 445 genes were downregulated. BAT uses the MrgD receptor to display a normal pattern of gene expression and to respond, like WT mice, to an HG diet. In conclusion, the MrgD signaling is important for the metabolic regulation and manutention of BAT functionality.

Graphical abstract

The alamandine/ Mas-related G protein-coupled receptor D (MrgD) axis is important for the regulation of metabolism and maintains the functionality of BAT. MrgD-KO mice fed a standard diet (ST) exhibited decreased brown adipose tissue (BAT) weight and increased body weight and adiposity. MrgD-KO mice showed resistance to glucose intolerance induced by a high glucose diet (HG). WT HG mice exhibited decreased MrgD expression in BAT and decreased circulating alamandine levels. MrgD receptor absence caused a profound depletion of transcripts in BAT; of 476 regulated genes, 445 were downregulated. After the HG diet, WT HG mice regulated 1,148 genes in BAT, while MrgD-KO HG mice regulated only 45 genes. This result showed that MrgD expression is central for a predictable response to the HG diet in BAT. RNA sequencing analysis showed that the main targets of MrgD signaling are AMPK, extracellular matrix (ECM) components, ion channels, ribosomal and mitochondrial genes. MrgD deletion caused significant alterations in the size, functionality, and gene expression of BAT, impairing its response to a caloric challenge. We suggest that this is mediated by decreased AMPK signaling in BAT exhausting the brown adipocyte precursor pool in MrgD-KO mice. In addition, we suggest that AMPK has a central role in MrgD signaling by interacting with all the regulated groups of genes. Green dots and outlines represent downregulated genes, and red dots and outlines represent upregulated genes.

Image, graphical abstract
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Introduction

The renin-angiotensin system (RAS) is an endocrine system that provides a powerful mechanism for blood pressure control and electrolyte homeostasis. The RAS is composed of two main arms: the vasoconstrictor axis, with the angiotensin-converting enzyme (ACE)/angiotensin (Ang) II/AT1 receptor (AT1R), and the counter regulatory axis, with angiotensin-converting enzyme 2 (ACE2)/Ang-(1–7)/Mas receptor. Ang-(1–7) effects often oppose the AngII effects and fulfill important roles for an organism by providing vasodilatory, antifibrotic, cardioprotective, antihypertrophic, antithrombotic, and antioxidant activities [1].

Alamandine is a newly described RAS peptide. It is formed by the action of a decarboxylase on Ang-(1–7) or by the catalytic action of ACE2 on alatensin Ang-A (Ala1-AngII). Alamandine (Ala-Arg-Val-Tyr-Ile-His-Pro) and Ang-(1–7) (Asp-Arg-Val-Tyr-Ile-His-Pro) share high similarities in terms of their structures, and the difference is the presence of alanine in the place of aspartic acid at the amino terminus [2]. In addition to the structure, some effects of alamandine and Ang-(1–7) are similar; e.g., (1) alamandine treatment produces a long-lasting effect of lowering blood pressure in hypertensive rats, and (2) alamandine decreases the deposition of collagen I, III and fibronectin in isoproterenol-treated rats [2]. (3) The microinjection of alamandine in brain areas related to blood pressure control, such as in the caudal ventrolateral medulla and in the rostral ventrolateral medulla of rats, generates depressor and pressor effects on blood pressure, respectively [2,3]. Moreover, (4) alamandine exhibits a vasodilatory effect on the aortic rings of rats and mice. Despite the similarities, the effects of alamandine are mediated by a different receptor, Mas-related G protein-coupled receptor D (MrgD) [2].

The localization of RAS components is important in determining the local effects of each peptide. All RAS components, including the MrgD receptor, are expressed in a wide variety of tissues, including white and brown adipose tissues [4], [5], [6], [7], [8], [9]. Although the effects of alamandine on metabolism have not yet been elucidated, it has been known for a long time that the RAS exerts an important influence on metabolism. AngII is well known as a peptide that mediates the metabolic and vascular consequences of obesity. The over activity of AngII, through AT1R, triggers a vicious cycle that reinforces obesity symptoms, e.g., dyslipidemia and glucose intolerance [10], [11], [12], [13]. On the other hand, Ang-(1–7) represents a protective peptide for metabolism, improving glycemic, thermogenic, and adiposity parameters in obese animals [14], [15], [16], [17].

Adipose tissues are closely associated with obesity. White adipose tissue (WAT) is a large endocrine organ and the main site of lipid storage. Brown adipose tissue (BAT) is responsible for adaptive thermogenesis and has great anti-obesity potential [18]. Although obesity is a multifactorial disease, the energy source of obesity is the imbalance between energy intake and energy expenditure. BAT activity increases energy expenditure by utilizing lipids and glucose to generate heat. This ability makes BAT a promising target against obesity [19], [20], [21], [22].

In addition to adrenergic stimulation, BAT can also be activated by other systems, e.g., by the RAS. ACE2/Ang-(1-7)/Mas signaling is closely associated with adipose tissue browning and increased thermogenic activity. In high-lipid diet-induced obesity, treatment with ACE2 increased O2 consumption, improved insulin and glucose signaling, increased BAT mass, and increased thermogenic activity [23], and treatment with Ang-(1–7) induced significant improvement in glucose levels and thermogenesis. In these animals, increased expression of UCP1, beta-adrenergic receptor, PKA, AMPK and PRDM16 was observed [24]. Ang-(1–7) through Mas increases thermogenic activity by converting white adipocytes into beige thermogenic active adipocytes [17].

Based on these multiple effects of RAS components on adipose tissue, we hypothesize that alamandine may also exert metabolic actions by mediating effects through different signaling pathways. Therefore, we evaluated the consequences of the genetic deletion of the alamandine receptor MrgD on the metabolism of mice and BAT function.

Section snippets

Animals

MrgD receptor-knockout mice (MrgD-KO) and C57BL6/J wild-type mice (WT), aged approximately 8 weeks old and 5 d old, were obtained from the transgenic animal facilities of the Hypertension Laboratory at Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil. MrgD-KO mice and WT mice were purchased from the Mutant Mouse Regional Resource Center (National Institutes of Health, RRID: MMRRC_036050-UNC). All the mice had free access to water and food. The animals were maintained under

Absence of MrgD leads to a BAT reduction independent of age

Our first results showed robust BAT atrophy in MrgD-KO mice compared with WT mice at 5 d and 8 weeks of age (Fig. 1A–B). Therefore, we studied the effects of a high-glucose (HG) diet on these mice. All mice started the experiment at 8 weeks of age, and the duration of HG diet intake was 8 weeks. Again, robust BAT atrophy was identified in MrgD-KO mice fed a standard diet (ST) at 16 weeks of age (Fig. 1C). These results indicated that the BAT reduction observed in the absence of MrgD is

Discussion

The main findings of the present study show that in the absence of MrgD, white adiposity was increased at a young age, and BAT was reduced and exhibited an altered gene expression pattern. Moreover, we show that BAT uses the MrgD receptor to display a normal pattern of gene expression and to respond, like wild-type mice, to a high glucose diet.

The RAS connects obesity with its metabolic consequences. This linkage can be harmful or beneficial depending on the peptide and the activated receptor.

Author Contributions

G.C.C. and R.A.S.S. designed the research; G.C.C. performed experiments and analyzed data; G.C.C, M.B and R.A.S.S. interpreted results of experiments; G.C.C. drafted manuscript; G.C.C, S.H.S.S, M.B and R.A.S.S. edited and revised manuscript; G.C.C. and R.A.S.S. approved final version of manuscript.

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by Alexander von Humboldt Foundation, National Council for Scientific and Technological Development (CNPq), Novogene and Angitec.

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