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 2

SNAP23 deficiency induces adipocyte apoptotic cell death.

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SNAP23 deficiency induces adipocyte apoptotic cell death. (A) Subcutaneo...
(A) Subcutaneous adipose tissue SVCs from 3-week-old WT and KO mice were isolated and differentiated into adipocytes, and SNAP23 mRNA levels were determined on day 6. Data represent the mean ± SEM from 3 independent experiments. (B) Cell morphology was examined on days 3, 6, 12, and 15 following differentiation, with medium changes every 2 days for the first 6 days and then every 4 days thereafter. Images are representative of 5 independent SVC isolations. Original magnification, x20. (C) mRNA from WT and KO on days 6 and 12 of differentiation was subjected to qRT-PCR for perilipin expression. Data represent the mean ± SEM from 3 independent experiments. (D) Control (NM shRNA), SNAP23-specific shRNA (SNAP23 shRNA), and shRNA-resistant human SNAP23 cDNA–transfected SNAP23 shRNA (SNAP23 shRNA/hSNAP23) 3T3L1 adipocytes were differentiated for 12 days under NR conditions and immunoblotted for SNAP23 and actin. Immunoblots are representative of 3 independently performed experiments. (E) The adipocytes differentiated for 12 days were maintained under NR conditions, with a medium change every 2 days, or under ND conditions, with a medium change every 4 days after differentiation for 6 days. Cell extracts were then immunoblotted for PARP, caspase 3, and actin. Immunoblots are representative of 3 independent experiments. (F) The adipocytes differentiated for 12 days and maintained under NR or ND conditions for 6 hours were subjected to PI and DAPI labeling. Scale bars: 70 μm. (G) Quantification of PI-positive nuclei was determined by counting approximately 400 cells from 3 independent determinations. Data represent the mean ± SEM from 3 independent experiments. *P < 0.05, by Student’s t test (A and C) and ****P < 0.0001, by ANOVA with Dunnett’s post hoc test (G).