SNAP23 regulates BAX-dependent adipocyte programmed cell death independently of canonical macroautophagy
Daorong Feng, … , Richard N. Kitsis, Jeffrey E. Pessin
Daorong Feng, … , Richard N. Kitsis, Jeffrey E. Pessin
Published August 13, 2018
Citation Information: J Clin Invest. 2018;128(9):3941-3956. https://doi.org/10.1172/JCI99217.
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Research Article Cell biology Metabolism

SNAP23 regulates BAX-dependent adipocyte programmed cell death independently of canonical macroautophagy

Abstract

The t-SNARE protein SNAP23 conventionally functions as a component of the cellular machinery required for intracellular transport vesicle fusion with target membranes and has been implicated in the regulation of fasting glucose levels, BMI, and type 2 diabetes. Surprisingly, we observed that adipocyte-specific KO of SNAP23 in mice resulted in a temporal development of severe generalized lipodystrophy associated with adipose tissue inflammation, insulin resistance, hyperglycemia, liver steatosis, and early death. This resulted from adipocyte cell death associated with an inhibition of macroautophagy and lysosomal degradation of the proapoptotic regulator BAX, with increased BAX activation. BAX colocalized with LC3-positive autophagic vacuoles and was increased upon treatment with lysosome inhibitors. Moreover, BAX deficiency suppressed the lipodystrophic phenotype in the adipocyte-specific SNAP23-KO mice and prevented cell death. In addition, ATG9 deficiency phenocopied SNAP23 deficiency, whereas ATG7 deficiency had no effect on BAX protein levels, BAX activation, or apoptotic cell death. These data demonstrate a role for SNAP23 in the control of macroautophagy and programmed cell death through an ATG9-dependent, but ATG7-independent, pathway regulating BAX protein levels and BAX activation.

Authors

Daorong Feng, Dulguun Amgalan, Rajat Singh, Jianwen Wei, Jennifer Wen, Tszki Peter Wei, Timothy E. McGraw, Richard N. Kitsis, Jeffrey E. Pessin

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Figure 5

ATG9 deficiency increases BAX protein levels and induces cell death.

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ATG9 deficiency increases BAX protein levels and induces cell death. (A)...
(A) Cell extracts from NM shRNA, SNAP23 shRNA, and ATG9 shRNA NIH3T3 cells maintained under NR conditions were immunoblotted for BAX, actin, ATG9, and SNAP23. The immunoblots for ATG9 and SNAP23 are from the same samples run on parallel gels. Immunoblots are representative of 3 independent experiments on 2 cell lines each. (B) BAX protein levels were quantified. Data represent the mean ± SEM. (C) NM shRNA, ATG9 shRNA, and rescued ATG9 shRNA/hATG9 cells were maintained under NR or ND conditions for 6 hours. The cells were labeled with PI and DAPI and visualized by fluorescence microscopy. Scale bars: 200μm. (D) Quantification of the PI-positive nuclei was determined by counting 500 cells from 3 independent determinations per genotype. Data represent the mean ± SEM of 3 independent experiments for each genotype. (E) Independent control and ATG9 single-guide RNA (sgRNA) NIH3T3 cells were generated and transfected with nonspecific or BAX-specific siRNA. Cells were then placed under ND conditions for 2 hours in the presence and absence of lysosomotropic agents. Cell extracts were immunoblotted for the indicated proteins. Relatively lighter and darker exposures for the LC3 immunoblot are shown. Immunoblot is representative of 3 independent experiments. (F) Nonspecific siRNA and BAX siRNA–knockdown cells in the context of the control NM shRNA– and ATG9 shRNA–knockdown cells were placed in ND conditions for 6 hours. Cells were then subjected to PI and DAPI labeling and visualized by fluorescence microscopy. Scale bars: 200 μm. (G) Quantification of PI-positive nuclei was determined by counting 500 cells from 3 independent determinations per genotype. Data represent the mean ± SEM. *P < 0.05 and ****P < 0.0001, by ANOVA with Dunnett’s or Tukey’s post hoc test.