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 31, 2018; First published August 13, 2018
Citation Information: J Clin Invest. 2018;128(9):3941-3956. https://doi.org/10.1172/JCI99217.
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Categories: 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 6

ATG7 deficiency suppresses macroautophagy but does not induce BAX protein levels or cell death.

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ATG7 deficiency suppresses macroautophagy but does not induce BAX protei...
(A) ATG9 shRNA and ATG7 shRNA NIH3T3 cells under NR conditions were treated with 20 μg/ml CHX, and at the indicated time points, cell extracts were prepared and immunoblotted for BAX and actin. Immunoblot is representative of 3 independent experiments. (B) NM shRNA– and ATG7 shRNA–knockdown NIH3T3 cells were maintained under NR or ND conditions for 2 hours. Cell extracts were prepared and immunoblotted for ATG7, actin, SNAP23, and BAX. Immunoblots for ATG7 and actin are from the same samples run on parallel gels. Immunoblots are representative of 3 independent experiments on 2 cell lines each. (C) NM shRNA– and ATG7 shRNA–knockdown NIH3T3 cells were then maintained under ND conditions for 2 hours in the absence and presence of lysosomotropic agents. Cell extracts were immunoblotted for LC3 and actin. (D) Net LC3II flux was calculated as the difference between LC3II protein levels in the presence and absence of the lysosomotropic agents as described in Methods. Data represent the average, with the SEM determined from 3 independent experiments. (E) Cells were then maintained under NR or ND conditions for 6 hours and subjected to PI and DAPI labeling followed by visualization under fluorescence microscopy. Scale bars: 100 μm. (F) Quantification of the PI-positive nuclei was determined by counting 500 cells. Data represent the mean ± SEM of 3 independent experiments. **P < 0.01, by Student’s t test (D) and NS, by ANOVA with Tukey’s post hoc test (F).