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Suppression of Rho-kinase 1 is responsible for insulin regulation of the AMPK/SREBP-1c pathway in skeletal muscle cells exposed to palmitate

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Abstract

Aims

Clinical and experimental data suggest that early insulin therapy could reduce lipotoxicity in subjects and animal models with type 2 diabetes mellitus. However, the underlying mechanisms need to be clarified. Sterol regulatory element-binding protein 1c (SREBP-1c), which is negatively regulated by AMP-activated protein kinase (AMPK), plays a critical role in lipotoxicity and insulin resistance in skeletal muscle cells. Here, we investigated the effect and molecular mechanism of insulin intervention on the AMPK/SREBP-1c pathway in skeletal muscle cells with chronic exposure to palmitic acid (PA).

Methods

Male C57BL/6 mice were fed with a high-fat diet for 12 weeks and were then treated with insulin, AMPK inhibitor, or metformin. L6 myotubes incubated with palmitic acid (PA) were treated with insulin or metformin. Dominant-negative AMPKα2 (DN-AMPKα2) lentivirus, AMPKα2 siRNA, or Rho-kinase 1 (ROCK1) siRNA were transfected into PA-treated L6 myotubes.

Results

We found that the ability of PA to stimulate SREBP-1c and inhibit AMPK was reversed by insulin in L6 cells. Moreover, DN-AMPKα2 lentivirus and AMPKα2 siRNA were transfected into PA-treated L6 myotubes, and the decrease in SREBP-1c expression caused by insulin was blocked by AMPK inhibition independent of the phosphatidylinositol-4,5-biphosphate-3-kinase (PI3K)/AKT pathway. The serine/threonine kinase Rho-kinase (ROCK) 1, a downstream effector of the small G protein RhoA, was activated by PA. Interestingly, knockdown of ROCK1 by siRNA blocked the downregulation of AMPK phosphorylation under PA-treated L6 myotubes, which indicated that ROCK1 mediated the effect of insulin action on AMPK.

Conclusions

Our study indicated that insulin reduced lipotoxicity via ROCK1 and then improved AMPK/SREBP-1c signaling in skeletal muscle under PA-induced insulin resistance.

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Acknowledgements

This study was supported by grants from National Natural Science Foundation of China Grant Award (81570736, 81270906, 81570737, 81370947, 81500612, 81500630, 81400832, 81300651, 81600632, 81600637), the Project of National Key Clinical Division, the China Postdoctoral Science Foundation (2012M521050), Jiangsu Province’s Key Discipline of Medicine (XK201105), Jiangsu Province’s Key Provincial Talents Program (RC2011011), Jiangsu Key Laboratory for Molecular Medicine (BM2007208), Jiangsu Province’s Project of Standardized Diagnosis and Treatment of Key Diseases (2015604), Jiangsu Postdoctoral Science Foundation, the Key Project of Nanjing Clinical Medical Science, Nanjing Outstanding Youth Fund Projects (JQX13010), Nanjing Science and Technology Development Projects (2013ZD005), and Medical and Health Research Projects of Nanjing Health Bureau (YKK14055, YKK11092), 2016 China Diabetes Young Scientific Talent Research Project.

Author contributions

Yan Bi and Dalong Zhu contributed to the study design, data interpretation, and final approval of the version to be published. Sunyinyan Tang and Wenjun Wu wrote the main manuscript text and contributed to the acquisition of data and approval of the final version. Wenjuan Tang, Zhijuan Ge, and Ting Hong prepared Figures 1–4 and contributed to acquisition of data. All authors reviewed the manuscript.

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    Authors and Affiliations

    1. Department of Endocrinology, Drum Tower Hospital Affiliated to the Nanjing University Medical School, Nanjing, Jiangsu, People’s Republic of China

      Sunyinyan Tang, Wenjuan Tang, Zhijuan Ge, Hongdong Wang, Ting Hong, Dalong Zhu & Yan Bi

    2. Department of Endocrinology, Wuxi People’s Hospital of Nanjing Medical University, Wuxi, Jiangsu, People’s Republic of China

      Wenjun Wu

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    Conflict of interest

    Sunyinyan Tang, Wenjun Wu, Wenjuan Tang, Zhijuan Ge, Ting Hong, Dalong Zhu, and Yan Bi declare that they have no competing financial interests.

    Ethical standards

    All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5).

    Statement of human and animal rights

    All animal procedures were performed according to the National Institutes of Health guidelines and approved by the animal care committee of Drum Tower Hospital affiliated with Nanjing University Medical School (Nanjing, China).

    Informed consent

    Our study is not clinical trial, so there is no informed consent obtained from individual participants included in the study.

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    Managed by Massimo Porta.

    The authors Sunyinyan Tang and Wenjun Wu contributed equally to this work.

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    Tang, S., Wu, W., Tang, W. et al. Suppression of Rho-kinase 1 is responsible for insulin regulation of the AMPK/SREBP-1c pathway in skeletal muscle cells exposed to palmitate. Acta Diabetol 54, 635–644 (2017). https://doi.org/10.1007/s00592-017-0976-z

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