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 3

SNAP23 deficiency suppresses adipocyte macroautophagy.

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SNAP23 deficiency suppresses adipocyte macroautophagy. (A) Snap23fl/fl (...
(A) Snap23fl/fl (WT) and Adipoq-Cre Snap23fl/fl (KO) mice were crossed with GFP-LC3–transgenic mice as described in Methods. At 3 weeks of age, subcutaneous SVCs were isolated and differentiated into adipocytes for 6 days under NR conditions. The cells were then maintained in NR (DMEM with 10% FBS) or subjected to ND (RPMI-aa medium) conditions for 2 hours in the absence and presence of the lysosomotropic agents NH4Cl and leupeptin. The cells were then fixed and visualized for GFP (green) and DAPI (blue) by fluorescence microscopy. Scale bars: 15 μm. (B) The average number of GFP-LC3 puncta, with the SEM, was quantified in approximately 400 cells from 3 independent experiments. (C) NM shRNA, SNAP23 shRNA, and rescued SNAP23 shRNA/hSNAP23 3T3L1 adipocytes were differentiated under NR conditions for 8 days. The cells were then maintained in NR conditions or subjected to ND conditions for 4 hours in the absence and presence of the lysosomotropic agents NH4Cl and leupeptin. Cell extracts were immunoblotted for LC3, actin, and SNAP23. Immunoblot is representative of 5 independent experiments. (D) Net LC3II flux was calculated from 3 independent experiments. (E) Mouse NIH3T3 fibroblasts were transfected with NM shRNA or SNAP23 shRNA lentiviruses and subsequently transfected with the dual mCherryGFPLC3 reporter as described in Methods. Cells were then maintained under NR conditions or ND conditions for 5 hours and subjected to fluorescence microscopy. Scale bars: 15 μm. (F) The average number of red puncta per cell, with the SEM, was quantified in 500 individual cells from 3 independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001, by ANOVA with Tukey’s post hoc test.