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Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy

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Abstract

Nonalcoholic fatty liver disease (NAFLD) is related to elevated cytoplasmic calcium signaling in hepatocytes, which may be mediated by store-operated calcium channel (SOCC) and inositol triphosphate receptor (IP3R). However, the regulatory effect of calcium signaling on lipid accumulation and degeneration in hepatocytes and the underlying molecular mechanism remain unknown. Autophagy inhibition promotes lipid accumulation and steatosis in hepatocytes. However, the association between elevated calcium signaling and autophagy inhibition in hepatocytes and its effect on hepatocyte fatty lesions remain unclear. Here, we established a mouse hepatocyte fatty gradient model using oleic acid. SOCC and IP3R channel opening and cytoplasmic calcium levels gradually increased with the hepatocyte pimelosis degree, whereas autophagy gradually decreased. We also established an optimal oleic acid (OOA) hepatocyte model, observing significantly increased SOCC and IP3R channel opening and calcium influx alongside significantly decreased autophagy and aggravated cellular fatty lesion. Calcium channel blockers (CCBs) and calcium channel gene silencing reagents (CCGSRs), respectively, reversed these effects, indicating that elevated cytoplasmic calcium signaling promotes NAFLD occurrence and the development by inhibiting hepatocyte autophagy. In the OOA model, upregulated extracellular regulated protein kinases 1/2 (ERK1/2), which can be regulated by SOCC and IP3R proteins transient receptor potential canonical 1 (TRPC1)/IP3R with elevated cytoplasmic calcium signaling, over-inhibited forkhead/winged helix O (FOXO) signaling and over-activated mammalian target of rapamycin complex 1 (mTORC1) signaling. Over-inhibited FOXO signaling significantly downregulated autophagy-related gene 12, which inhibits autophagosome maturation, while over-activated mTORC1 signaling over-inactivated Unc-51 like autophagy activating kinase 1, which inhibits preautophagosome formation. CCBs and CCGSRs recovered autophagy by significantly downregulating ERK1/2 to block abnormal changes in FOXO and mTORC1 signaling. Our findings indicate that upregulated SOCC and IP3R channels and subsequent elevated cytoplasmic calcium signaling in hepatocyte fatty lesions inhibits hepatocyte autophagy through (TRPC1/IP3R)/ERK/(FOXO/mTORC1) signaling pathways, causes lipid accumulation and degeneration in hepatocytes, and promotes NAFLD occurrence and development.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

2-APB:

2-Aminoethoxydiphenyl borate

ATG:

Autophagy-related gene

BECLIN1:

Yeast ATG6 homolog

CCB:

Calcium channel blocker

CCGSR:

Calcium channel gene silencing reagent

DMEM:

Dulbecco's Modified Eagle Medium

ECL:

Electrochemiluminescence

EP tubes:

Eppendorf micro test tubes

ER:

Endoplasmic reticulum

ERK1/2:

Extracellular regulated protein kinases 1/2

FBS:

Fetal bovine serum

FOXO:

Forkhead/winged helix O

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GOA:

Gradient oleic acid

HCC:

Hepatocellular carcinoma

IP3R:

Inositol triphosphate receptor

LC3B:

Microtubule-associated protein 1- light chain 3 B

MAPK:

Mitogen-activated protein kinase

mTORC1:

Mammalian target of rapamycin complex 1

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAFLD:

Nonalcoholic fatty liver disease

NASH:

Nonalcoholic steatohepatitis

OD:

Optical density

OOA:

Optimal oleic acid

ORAI1:

Calcium release-activated calcium channel protein 1

p:

Phosphorylated

PBS:

Phosphate-buffered saline

RT:

Reverse transcription

SOCC:

Store-operated calcium channel

STIM1:

Stromal interaction molecule 1

t:

Total

TBST:

Tris-buffered saline Tween

TG:

Triglyceride

TRPC1:

Transient receptor potential canonical 1

TSC1/2:

Tuberous sclerosis 1/2

ULK1:

Unc-51 like autophagy activating kinase 1

VHP:

Verapamil HCl

VPS:

Vacuolar protein sorting

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Acknowledgements

We thank Elsevier Language Editing Services for their linguistic assistance during the preparation of this manuscript.

Funding

This work was supported by Department of Health of Zhejiang Province, China [Grant Number 2018KY553]; the Young and Middle-Aged Research Innovation Foundation of the Zhejiang Chinese Medical University, China [Grant Number KC201921]; and the Teacher Professional Development Project Foundation of University Visiting Scholars, China [Grant Number FX2019021].

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Authors and Affiliations

  1. College of Life Science/Institute of Molecular Medicine, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China

    Lin Zhang, Yifan Zhang, Xiaobing Dou, Hui Chai & Qianyu Qian

  2. College of Life Science, Zhejiang University, Hangzhou, 310058, Zhejiang, China

    Yuanqing Jiang

  3. Institute of Molecular Medicine, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China

    Songtao Li

  4. Department of General Practice, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China

    Miaojuan Wang

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  1. Lin Zhang
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Contributions

LZ and MW developed the idea and obtained the fund. LZ designed the study. LZ and YZ executed the experiments, analyzed the data, and drafted the manuscript. LZ and YJ wrote the manuscript. XD, SL, and HC provided experimental resources and critically evaluated the manuscript. QQ provided experimental resources and support.

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Correspondence to Miaojuan Wang.

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Zhang, L., Zhang, Y., Jiang, Y. et al. Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy. Mol Cell Biochem 476, 3163–3175 (2021). https://doi.org/10.1007/s11010-021-04150-0

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