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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 Article has an altmetric score of 5

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

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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 4

SNAP23 deficiency increases BAX protein levels by inhibiting BAX protein degradation.

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SNAP23 deficiency increases BAX protein levels by inhibiting BAX protein...
(A) Duplicate NM shRNA and SNAP23 shRNA 3T3L1 adipocytes 8 days after differentiation were immunoblotted for BAX, BAK, and actin. The actin immunoblot is from the same samples run on parallel gels. Immunoblots shown are representative of 4 independently performed experiments. (B) BAX mRNA levels were quantified by qRT-PCR from 4 independent determinations. Data represent the mean ± SEM. (C) Control NM shRNA differentiated adipocytes were treated with 5 μM MG132 or 20 nM bafilomycin A1 for the indicated durations. Cell extracts were prepared and immunoblotted for BAX protein and actin. Immunoblots are representative of 6 independently performed experiments. (D) NM shRNA and SNAP23 shRNA cells under NR conditions were treated with 20 μg/ml CHX and at the time points indicated were immunoblotted for BAX and actin. The NM shRNA BAX immunoblot is from the same samples run on parallel gels. Immunoblot is representative of 3 independent experiments. (E) In the context of the SNAP23 shRNA, the NIH3T3 cells were transfected with nonspecific siRNA or BAX-specific siRNA. Cells were then subjected to ND conditions for 2 hours in the presence and absence of lysosomotropic agents and immunoblotted for the indicated proteins. A relatively lighter and darker exposure for the LC3 immunoblot is 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 SNAP23 shRNA cells were subjected to ND conditions for 6 hours. The cells were then labeled for PI and DAPI. 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 < 0.01, by ANOVA with Tukey’s post hoc test.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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