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Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Published on: June 23, 2013

Adipocyte-Derived Exosomes Carrying Sonic Hedgehog Mediate M1 Macrophage Polarization-Induced Insulin Resistance via

Ming Song¹, Lu Han^1,2, Fang-Fang Chen¹

¹The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China.

Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology

|August 1, 2018

Abstract

Background/Aims:

Adipocyte-derived exosomes (ADEs) stimulate the activation of macrophages and contribute to the development of insulin resistance. Sonic Hedgehog (Shh) is an exosome-carrying protein and stimulates macrophages to secrete inflammatory cytokines. However, the impact of ADEs carrying Shh on the pro-inflammatory activation of macrophages and consequently, adipocyte insulin resistance is unclear.

Methods:

3T3-L1 adipocytes were cultured with high glucose and insulin to imitate the pathogeny of insulin resistance. ADEs were isolated from conditioned media of 3T3-L1 adipocytes via differential ultracentrifugation. We explored the role of ADEs carrying Shh in the polarization of macrophages by flow cytometry. Western blot and electrophoretic mobility shift assay (EMSA) were performed to determine the activation of Shh-mediated signalling pathways. The effects of ADE-treated macrophages on adipocyte insulin signalling were studied by Western blot.

Results:

We found that circulating Shh-positive exosomes were increased in type 2 diabetes patients. High glucose and insulin increased the secretion of Shh-positive ADEs. The ADEs carrying Shh induced pro-inflammatory or M1 polarization of bone marrow-derived macrophages (BMDM) and RAW 264.7 macrophages. Inhibitors of Ptch and PI3K blocked the M1 polarization induced by ADEs, which suggests that ADEs carrying Shh mediated M1 macrophage polarization through the Ptch/PI3K signalling pathway. ADE-treated RAW 264.7 macrophages were subsequently used to assess the effect on insulin signalling in adipocytes. Using a co-culture assay, we showed that both ADE-treated macrophages and exosomes from these macrophages could decrease the expression of insulin-resistant substrate-1 (IRS-1) and hormone-sensitive lipase (HSL) in adipocytes. Inhibitors of Ptch and PI3K blocked the down-regulation of IRS-1 and HSL induced by ADE-treated macrophages.

Conclusion:

Together, these data indicate that ADEs carrying Shh induce the M1 polarization of macrophages, which contributes to insulin resistance in adipocytes through the Ptch/PI3K pathway.

Insights

Adipocyte-derived exosomes (ADEs) carrying Sonic Hedgehog (Shh) promote M1 macrophage polarization, contributing to insulin resistance in adipocytes via the Ptch/PI3K pathway. This highlights a novel mechanism in type 2 diabetes pathogenesis.

Area of Science:

Metabolic disease research
Cell signaling pathways
Exosome biology

Background:

Adipocyte-derived exosomes (ADEs) are implicated in macrophage activation and insulin resistance.
Sonic Hedgehog (Shh), a protein found in exosomes, stimulates inflammatory cytokine secretion from macrophages.
The specific role of Shh-carrying ADEs in macrophage activation and subsequent insulin resistance remains unclear.

Purpose of the Study:

To investigate the impact of ADEs carrying Shh on macrophage polarization.
To elucidate the signaling pathways involved in Shh-mediated macrophage activation.
To determine how ADE-activated macrophages influence insulin signaling in adipocytes.

Main Methods:

3T3-L1 adipocytes were cultured under conditions mimicking insulin resistance.
ADEs were isolated using differential ultracentrifugation.
Macrophage polarization was assessed by flow cytometry; signaling pathways were analyzed via Western blot and EMSA.

Main Results:

Shh-positive exosomes were elevated in type 2 diabetes patients and increased with high glucose/insulin treatment.
ADEs carrying Shh induced M1 polarization in macrophages through the Ptch/PI3K pathway.
ADEs and ADE-treated macrophages reduced insulin signaling (IRS-1, HSL) in adipocytes, a process blocked by Ptch/PI3K inhibitors.

Conclusions:

ADEs carrying Shh drive M1 macrophage polarization.
This M1 polarization contributes to adipocyte insulin resistance via the Ptch/PI3K pathway.
These findings reveal a novel mechanism linking ADEs, Shh, and insulin resistance.

Read full analysis

Keywords:

Exosome Insulin resistance Macrophage polarization Sonic hedgehog

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