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SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis

Acta Pharmacologica Sinica volume 44, pages 1161–1174 (2023)Cite this article

Abstract

Gliflozins are known as SGLT2 inhibitors, which are used to treat diabetic patients by inhibiting glucose reabsorption in kidney proximal tubules. Recent studies show that gliflozins may exert other effects independent of SGLT2 pathways. In this study we investigated their effects on skeletal muscle cell viability and paracrine function, which were crucial for promoting revascularization in diabetic hindlimb ischemia (HLI). We showed that treatment with empagliflozin (0.1−40 μM) dose-dependently increased high glucose (25 mM)-impaired viability of skeletal muscle C2C12 cells. Canagliflozin, dapagliflozin, ertugliflozin, ipragliflozin and tofogliflozin exerted similar protective effects on skeletal muscle cells cultured under the hyperglycemic condition. Transcriptomic analysis revealed an enrichment of pathways related to ferroptosis in empagliflozin-treated C2C12 cells. We further demonstrated that empagliflozin and other gliflozins (10 μM) restored GPX4 expression in high glucose-treated C2C12 cells, thereby suppressing ferroptosis and promoting cell viability. Empagliflozin (10 μM) also markedly enhanced the proliferation and migration of blood vessel-forming cells by promoting paracrine function of skeletal muscle C2C12 cells. In diabetic HLI mice, injection of empagliflozin into the gastrocnemius muscle of the left hindlimb (10 mg/kg, every 3 days for 21 days) significantly enhanced revascularization and blood perfusion recovery. Collectively, these results reveal a novel effect of empagliflozin, a clinical hypoglycemic gliflozin drug, in inhibiting ferroptosis and enhancing skeletal muscle cell survival and paracrine function under hyperglycemic condition via restoring the expression of GPX4. This study highlights the potential of intramuscular injection of empagliflozin for treating diabetic HLI.

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Fig. 1: Gliflozins promote skeletal muscle cells survival under hyperglycemia.
Fig. 2: Empagliflozin inhibits ferroptosis in skeletal muscle cells under hyperglycemia.
Fig. 3: Empagliflozin inhibits hyperglycemia-induced ferroptosis by enhancing GPX4 expression.
Fig. 4: Empagliflozin inhibits ferroptosis under hyperglycemia through GPX4.
Fig. 5: Gliflozins inhibit ferroptosis under hyperglycemia through GPX4.
Fig. 6: Empagliflozin promotes blood vessel-forming cells proliferation and migration by enhancing skeletal muscle cells paracrine function in a GPX4-dependent manner.
Fig. 7: Empagliflozin enhances revascularization in diabetic HLI mice by inhibiting ferroptosis through GPX4.
Fig. 8: Schematic diagram showing the mechanism of intramuscularly-injected empagliflozin in enhancing therapeutic angiogenesis.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31871367 and 81872273), the Natural Science Foundation of Chongqing (CSTB2022NSCQ-MSX0611 and CSTB2022NSCQ-MSX0612).

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Authors and Affiliations

  1. Key Laboratory for Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China

    Jing-xuan Han, Lai-liu Luo, Yi-cheng Wang, Vivi Kasim & Shou-rong Wu

  2. The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing, 400044, China

    Jing-xuan Han, Lai-liu Luo, Yi-cheng Wang, Vivi Kasim & Shou-rong Wu

  3. State and Local Joint Engineering Laboratory for Vascular Implants, College of Bioengineering, Chongqing University, Chongqing, 400044, China

    Yi-cheng Wang, Vivi Kasim & Shou-rong Wu

  4. Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, 305-8566, Japan

    Makoto Miyagishi

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  1. Jing-xuan Han
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Contributions

SRW and VK arranged the research, designed the experiments, examined and construed the data, provided financial support and guided all the experiments. JXH did literature research, performed the cellular and animal experiments, examined and construed the data, prepared the figures and wrote the manuscript. LLL performed part of the cellular and animal experiments, examined and construed the data, and assisted in writing the manuscript. YCW performed part of the cellular and animal experiments. MM designed, constructed, and examined the shRNA expression vectors.

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Correspondence to Vivi Kasim or Shou-rong Wu.

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All authors declare no competing interests. The results of this study have been patented in China (No. ZL202010701077.7).

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Han, Jx., Luo, Ll., Wang, Yc. et al. SGLT2 inhibitor empagliflozin promotes revascularization in diabetic mouse hindlimb ischemia by inhibiting ferroptosis. Acta Pharmacol Sin 44, 1161–1174 (2023). https://doi.org/10.1038/s41401-022-01031-0

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