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. 2023 Dec;28(6):234.
doi: 10.3892/mmr.2023.13121. Epub 2023 Nov 3.

IGF‑1 inhibits palmitic acid‑induced mitochondrial apoptosis in macrophages

Wanying Tang #  1 , Ming Zhang #  2 , Yu Wang  2 , Dan Ma  3 , Mi Hu  1 , Yangkai Zhang  1 , Huiling Lin  1 , Weiwei Jiang  4 , Yuxin Ouyang  1 , Liping Jiang  5 , Pingping He  6 , Guojun Zhao  2 , Xinping Ouyang  1
Affiliations

IGF‑1 inhibits palmitic acid‑induced mitochondrial apoptosis in macrophages

Wanying Tang et al. Mol Med Rep. 2023 Dec.

Abstract

Insulin growth factor‑1 (IGF‑1) is an endocrine regulator that plays an important role in normal growth and development. IGF‑1 mediated effects may result in protecting macrophages from immunometabolic response. However, it is unclear whether IGF‑1 has a protective effect on fatty acid‑induced macrophages damage. In the present study, THP‑1 cells were differentiated into macrophages and stimulated with palmitic acid (PA) in the absence or presence of IGF‑1. Macrophages apoptosis was measured by Cell Counting Kit‑8 assay, flow cytometry, Hoechst 33342 staining and western blotting. The mitochondrial damage was evaluated using JC‑1 staining and mitochondrial reactive oxygen species detection. The activation of mitophagy was assessed using immunofluorescence and western blotting. As a result, IGF‑1 significantly restored the survival rate in macrophages, while the apoptosis was inhibited through mitochondrial pathway. In addition, IGF‑1 protected the mitochondrial damage induced by PA. Furthermore, PA induced mitophagy via phosphatase and tensin homolog‑induced putative kinase protein 1/Parkin, which was reversed by IGF‑1. Taken together, the present study demonstrated the protective effect of IGF‑1 on PA‑induced mitochondrial apoptosis in macrophages, which might provide a potential therapeutic strategy for treatment of lipotoxicity.

Keywords: apoptosis; insulin growth factor; macrophages; mitochondria; palmitic acid.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1.
Figure 1.
CCK8 activity assay. (A) Dose-dependent effect of PA on cell viability. (B) Effect of IGF-1 on PA-induced macrophage survival. Mean ± SD. n=3. *P<0. 05, **P<0. 01 vs. control; ##P<0. 01 vs. PA group. PA, palmitic acid; IGF-1, insulin growth factor-1.
Figure 2.
Figure 2.
Effect of IGF-1 on PA-induced apoptosis of macrophages. (A) Hoechst 33342 staining was performed to observe apoptotic cells. (B) Quantitative analysis of Hoechst 33342 staining (C) Annexin V/PI staining to detect cell apoptosis. (D) Quantitative analysis of Annexin V/PI staining for total apoptosis (E) Quantitative analysis of Annexin V/PI staining for advanced apoptosis. (F) Quantitative analysis of Annexin V/PI staining for early apoptosis. Mean ± SD. n=3. *P<0. 05, **P<0. 01, ***P<0. 001 vs. control group; #P<0. 05, ##P<0. 01, ###P<0. 001 vs. PA group. IGF-1, insulin growth factor-1; PA, palmitic acid.
Figure 3.
Figure 3.
Effect of IGF-1 on PA-induced expression of caspase-3 in macrophages. (A) Caspase-3 activity detection. (B) Quantitative analysis of caspase-3 activity. (C) Western blotting detection of c-caspase-3 protein expression. (D) Quantitative analysis of caspase-3 protein expression. Mean ± SD. n=3. ***P<0. 001 vs. control group; #P<0. 05, ##P<0. 01 vs. PA group. IGF-1, insulin growth factor-1; PA, palmitic acid.
Figure 4.
Figure 4.
The effect of IGF-1 on PA-induced expression of Bcl-2 and Bax in macrophages. (A) Western blotting detection of Bcl-2, Bax protein expression. (B-D) Quantitative analysis of Bax, Bcl-2, and Bcl-2/Bax ratio. Mean ± SD. n=3. ***P<0.001 vs. control group; ##P<0. 01, ###P<0. 001 vs. PA group. IGF-1, insulin growth factor-1; PA, palmitic acid.
Figure 5.
Figure 5.
Effect of IGF-1 on PA-induced mitochondrial function in macrophages. (A) JC-1 staining to show changes in mitochondrial membrane potential. (B) Quantitative analysis of JC-1 staining. (C) Fluorescence intensity of mitochondrial ROS observed by flow cytometry. (D) Quantitative analysis of mitochondrial ROS. (E) Western blotting detection of protein expression of cytochrome c. (F) Quantitative analysis of cytochrome c protein expression. Mean ± SD. n=3. **P<0. 01, ***P<0. 001 vs. control group; #P<0. 05, ###P<0. 001 vs. PA. IGF-1, insulin growth factor-1; PA, palmitic acid; ROS, reactive oxygen species.
Figure 6.
Figure 6.
IGF-1 diminished PA-induced mitophagy in macrophages. (A) Colocalization of mitochondria and LC3. (B) The colocalization of the mitochondria and LC3 was quantitatively analyzed using the Pearson correlation coefficient. (C) Colocalization of mitochondria and lysosomes. (D) The colocalization of the mitochondria and lysosomes was quantitatively analyzed using the Pearson correlation coefficient. (E) Western blotting analysis of mitophagy-related proteins. (F and G) Quantitative analysis of PINK1 and Parkin protein expression. Mean ± SD. n=3. *P<0. 05, **P<0. 01, ***P<0. 001 vs. control group; #P<0. 05, ##P<0. 01 vs. PA. IGF-1, insulin growth factor-1; PA, palmitic acid.
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