PNPLA7 mediates Parkin-mitochondrial recruitment in adipose tissue for mitophagy and inhibits browning

Figures

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  Fig. 1. PNPLA7 is downregulated in adipose tissues during browning. a Representative Immunoblot results of the indicated proteins in BAT, iWAT, and eWAT of 10-week-old WT C57BL/6J male mice (n = 3/group). b Quantitative PCR analysis of Pnpla7 and Ucp1 mRNA expression in iWAT and BAT of 10-week-old WT C57BL/6 J male mice (RT: n = 4; Cold: n = 5). RT: room temperature. Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). c Representative Immunoblot results and densitometry analysis of the indicated proteins in iWAT and BAT of 10-week-old WT C57BL/6 J male mice (n = 3/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). d Quantitative PCR analysis of Pnpla7 and Ucp1 mRNA expression in iWAT and BAT of 10-week-old WT C57BL/6 J male mice (Ctrl: n = 4; CL316,243: n = 5). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). e Representative Immunoblot results and densitometry analysis of the indicated proteins in iWAT and BAT of 10-week-old WT C57BL/6J male mice (n = 3/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). f–k Quantitative…

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  Fig. 2. PNPLA7 inhibits browning of iWAT in response to cold exposure. a, b Regression-based analysis of absolute oxygen consumption (a) and energy expenditure (b) against body mass from 8-week-old control and Pnpla7Tg male mice. (n = 6/group). Oxygen consumption and energy expenditure as dependent variable, genotype as fixed variable, and body mass as covariate. (Two-sided analysis of covariance). c, d Adiposity index (c) and fat mass to body weight ratio (d) analysis of control and Pnpla7Tg mice. (RT: n = 5/group; cold: +/+: n = 8; Pnpla7Tg: n = 9; saline: n = 8/group; CL 316,243: n = 9/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). e TAG levels of iWAT obtained from control and Pnpla7Tg male mice (RT: n = 5/group; cold: n = 5/group; saline: n = 4/group; CL 316,243: n = 5/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). f, g Representative H&E and UCP1 immunohistochemical staining images (f) as well as representative immunoblot results and densitometry analysis (g) of iWAT from control and Pnpla7Tg male mice (n = 3 biological replicates, Scale bar = 100 μm). Data are presented as mean ±…

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  Fig. 3. PNPLA7 decreases mitochondrial content and mitochondrial oxidative respiration in the adipose tissue. a, b Representative TEM images showing mitochondria (a, n = 3 biological replicates, scale bar = 500 nm) as well as quantification of mitochondrial number per section and cristae number per mitochondrion (b, RT: +/+ group: n = 19; Pnpla7Tg group: n = 17; Cold: +/+ group: n = 20; Pnpla7Tg group: n = 18) of iWAT from control and Pnpla7Tg male mice. The white arrow highlights the mitophagosome. Cristae number normalized by each section. Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). c, d Representative TOM20 immunohistochemical staining images (c, n = 3 biological replicates, scale bar = 100 μm) and relative mtDNA content (b, n = 4/group) of iWAT from control and Pnpla7Tg male mice. Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). e, f Representative TEM images showing mitochondria (e, n = 3 biological replicates, scale bar = 500 nm) as well as quantification of mitochondrial number per section and cristae number per mitochondrion (RT: Flox group: n = 15; AKO group: n = 24; Cold: Flox group: n = 16;…

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  Fig. 4. Ablation of PNPLA7 decreases mitophagy in WAT adipocytes. Stromal vascular fraction (SVF) was isolated from iWAT of 3-week-old control (+/+) or PNPLA7 knockout (KO) male mice and differentiated into mature adipocytes ex vitro. a Representative fluorescence images of cells stained with mito-Keima in differentiated adipocytes. Differentiated adipocytes were infected with mito-Keima lentivirus and then treated with DMSO or CCCP (10 μM) for 12 h (n = 3 independent experiments). The fluorescence image obtained by confocal microscopy after excitation at 488-nm and 568-nm are shown in green and red in the same cell, respectively. Scale bar = 20 μm. b Quantification of the relative ratio of fluorescence intensity (568 nm:488 nm) of the cells described in (a) (DMSO group: WT: n = 24; KO: n = 23; CCCP group: WT: n = 22; KO: n = 26). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). c Representative TEM images showing mitophagosome in differentiated mature adipocytes treated with DMSO or CCCP (10 μM) for 12 h (n = 3 independent experiments). The white box highlights the mitochondria and mitophagosome. Scale bar = 500 nm. d Quantification of…

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  Fig. 5. PNPLA7 enhances Parkin mitochondrial recruitment and E3 ligase activity. a–c Representative Immunoblot results and densitometry analysis of the indicated proteins in differentiated mature inguinal adipocytes (a), total lysate (b), mitochondrial and cytosolic fraction (c) of iWAT isolated from control and Pnpla7Tg mice (n = 3 biological replicates). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). d, e Colocalization of Parkin with mitochondria (d) and quantification of the mitochondrial EGFP-Parkin positive cells (e) in differentiated adipocytes with or without PNPLA7 overexpression. Mitochondria were stained with Mito-tracker (red). EGFP-Parkin translocation to mitochondria was analyzed using confocal microscopy. (n = 3 biological replicates, scale bar = 20 μm). Data are presented as mean ± SEM. (Two-tailed Student’s t-test for 2-group comparisons). f, g Representative Immunoblot results of the indicated proteins in differentiated mature adipocytes obtained from control and Pnpla7Tg (f) as well as control and PNPLA7 knockout (g) male mice. (n = 3 biological replicates). h Illustration of the Adeno-Associated Virus (AAV)-mediated knocking…

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  Fig. 6. PNPLA7 is a MAM protein and interacts with Parkin to facilitate mitochondrial translocation of Parkin. a Representative Immunoblot results of PNPLA7 protein levels in the indicated organelle fractions isolated from 3T3-L1 cells. H: homogenate, Mp: pure mitochondrial fraction, Mc: crude mitochondrial fraction, ER: the endoplasmic reticulum, MAM: mitochondria-associated membrane, Cyto: cytosol. Organelle fractions were verified by testing the presence of specific markers: Calnexin for ER and MAMs, FACL-4 for MAMs, COX4 for mitochondrial, tubulin for cytosol. Representative results from 3 independent experiments were shown. b Representative immunofluorescence image of EGFP-PNPLA7 colocalization with ER in 3T3-L1 cells. EGFP-PNPLA7 localization to ER was analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). c Representative immunofluorescence image of mCherry-PNPLA7 colocalization with mitochondria-associated membrane in 3T3-L1 cells. Mitochondria-associated membranes were stained for FACL-4 (green), mCherry-PNPLA7 localization to mitochondria-associated membrane was analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). d…

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  Fig. 7. PNPLA7 promotes Parkin mitochondrial translocation by inhibiting PKA-mediated phosphorylation of Parkin. a, b Representative co-immunoprecipitation Immunoblot data of endogenous Parkin with PNPLA7 in iWAT (a) and inguinal adipocytes (b) of wild-type mice. Calnexin was used as a protein loading control. IgG: immunoglobulin G. (n = 3 biological replicates). c Stromal vascular fraction (SVF) was isolated from iWAT of 3-week-old control (+/+) and Pnpla7Tg (Tg) and PNPLA7 knockout (KO) male mice and differentiated into mature adipocytes ex vivo. Mitochondrial translocation of Parkin was analyzed in adipocytes with PNPLA7 overexpression or knockout. Immunofluorescence imaging of EGFP-Parkin (green) and Mito-Red (red) was analyzed by confocal microscopy and quantification of Parkin colocalization with the mitochondria analyzed by Image J. Scale bar = 20 μm. (n = 3 independent experiments) Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). d, e Co-immunoprecipitation Immunoblot data of endogenous p-Parkin with p-PKA substrate in iWAT of control and Pnpla7Tg (d) as well as control and Pnpla7AKO (e) mice. 10-week-old control (+/+) and Pnpla7Tg (Tg)…