Glucagon-like peptide-1 (GLP-1) induces M2 polarization of human macrophages via STAT3 activation

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Abstract

It is known that glucagon-like peptide-1 (GLP-1) is a hormone secreted postprandially from the L-cells of the small intestine and regulates glucose homeostasis. GLP-1 is now used for the treatment of diabetes because of its beneficial role against insulin resistance. The GLP-1 receptor (GLP-1R) is expressed on many cell types, including macrophages, and GLP-1 suppresses the development of atherosclerosis by inhibiting macrophage function. However, there have so far been few studies that have investigated the significance of GLP-1/GLP-1R signaling in macrophage activation. In the present study, we examined the effect of GLP-1 and exenatide, a GLP-1R agonist, on human monocyte-derived macrophage (HMDM) activation. We found that GLP-1 induced signal transducer and activator of transcription 3 (STAT3) activation. Silencing of GLP-1R suppressed the GLP-1-induced STAT3 activation. In addition, alternatively activated (M2) macrophage-related molecules, such as IL-10, CD163, and CD204 in HMDM, were significantly upregulated by GLP-1. Furthermore, the co-culture of 3T3-L1 adipocytes with GLP-1-treated RAW 264.7 macrophages increased the secretion of adiponectin compared to co-culture of the 3T3-L1 adipocytes with untreated RAW 264.7 macrophages. Our results demonstrate that GLP-1 induces macrophage polarization toward the M2 phenotype, which may contribute to the protective effects of GLP-1 against diabetes and cardiovascular diseases.

Highlights

► We examined the effect of GLP-1 on macrophage activation in this study. ► GLP-1 induces STAT3 activation in human macrophages. ► GLP-1 induces macrophages differentiation toward the M2 phenotype. ► GLP-1-treated macrophages increase adiponectin secretion from adipocytes.

Introduction

The incretin hormone, glucagon-like peptide 1 (GLP-1), is released from intestinal L cells following meal ingestion. GLP-1 stimulates insulin secretion and decreases glucagon secretion, reduces gastric emptying, and promotes satiety in a glucose-dependent manner [1]. The physiological relevance and pharmacology of GLP-1 have been researched extensively, with a major focus on its incretin actions, and its application in the treatment of type-2 diabetes. However, the administration of GLP-1 is not effective as a treatment for diabetes, because the protein is rapidly degraded by dipeptidyl peptidase-4 (DPP-4). Thus, GLP-1 receptor agonists that are resistant to DPP-4 and DPP-4 inhibitors are currently being used for the treatment of type 2 diabetes [2]. In addition, GLP-1 receptors are abundantly expressed in many cell types other than pancreatic islet cells, including gastrointestinal cells, and neural cells [1]. It was recently reported that GLP-1 receptor agonists (exenatide or exendin-4) reduced monocyte/macrophage accumulation in the arterial wall by inhibiting the inflammatory response in macrophages and attenuated atherosclerosis [2]. Therefore GLP-1 has been suggested to be involved in macrophage activation and polarization.

Macrophages are broadly classified into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages), according to their roles. M1 macrophages are potent effecter cells that kill microorganisms and produce primarily proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-6, and IL-12 [3]. In contrast, M2 macrophages reduce these inflammatory and adaptive Th1 responses by producing anti-inflammatory factors (IL-10, TGF-β and IL-1 receptor antagonist), and promote angiogenesis, tissue remodeling, and repair [3]. Macrophages are plastic cells, because they can switch from an activated M1 state back to M2, and vice versa, upon the induction of specific signals [3]. The signal transducer and activator of transcription 3 (STAT3) signaling in macrophages is well known to be involved in the regulation of immune responses in murine models [4], [5], and STAT3 activation is essential for macrophage differentiation toward the M2 phenotype [6].

It has also been reported that adipose tissue macrophages (ATMs) from lean mice express many genes characteristic of M2 macrophages, which protect adipocytes from inflammation, whereas diet-induced obesity led to a shift in the activation state to an M1 pro-inflammatory state that contributes to insulin resistance [7], [8], [9], thus suggesting that macrophage activation may thus play an important role in the functions of adipocytes.

To examine the effect of GLP-1 on macrophage activation and function, we performed an in vitro study using human macrophages. In the present study, we mainly focused on STAT3 signaling in macrophages, and we demonstrated that GLP-1 induced macrophage differentiation into the M2 phenotype via STAT3 activation. In addition, the effect of GLP-1 on cell–cell interactions between macrophages and adipocytes was also investigated, and we found that GLP-1-stimulated macrophages had enhanced adiponectin secretion from adipocytes. These findings suggest a new mechanism involved in the protective role of GLP-1R agonists and DPP-4 inhibitors against diabetes and cardiovascular disease.

Section snippets

Chemicals

Human GLP-1 was purchased from PeproTech (NJ, USA). Exenatide was purchased from GenScript (NJ, USA). All other chemicals were of the best grade available from commercial sources.

Cells and cell culture conditions

Peripheral blood mononuclear cells were obtained from healthy volunteer donors. Informed written consent was obtained from all healthy donors. CD14+ monocytes were purified from peripheral blood mononuclear cells by positive selection via magnetic-activated cell sorting technology (Miltenyi Biotec, Bergisch Gladbach,

Effect of GLP-1 and exenatide on STAT3 activation in HMDM

To identify the effects of GLP-1 on macrophage activation, we first measured the effects of GLP-1 and exenatide, a GLP-1R agonist, on the activation of STAT3, which plays an important role in macrophage differentiation toward the M2 phenotype in human monocyte-derived macrophages (HMDM). We observed that both GLP-1 and exenatide induced STAT3 activation in a dose-dependent manner (Fig. 1). Since STAT3 activation is a key event for macrophage activation toward the M2 phenotype, it has been

Discussion

Macrophages are broadly classified into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages), according to their roles. It is well known that M1 macrophage polarization is associated with inflammation and tissue destruction, whereas the M2 macrophage phenotype is associated with anti-inflammatory effects, wound repair and angiogenesis [17].

In the present study, we revealed that GLP-1 induces the activation of STAT3, which is essential for

Acknowledgments

We thank Mrs. Emi Kiyota, Mr. Takenobu Nakagawa and Ms. Yui Hayashida for their technical assistance. This study was supported in part by Grants-in-Aid for Scientific Research (No. 23790747 to Y.F., No. 23790407 to Y.K. and No. 20390113 to M.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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These authors contributed equally to this work.

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