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

Abstract

PINK1/Parkin-mediated ubiquitin-dependent mitophagy is a critical negative regulatory machinery for browning in the inguinal white adipose tissue (iWAT). However, the precise regulatory mechanism underlying PINK1/Parkin-mediated mitophagy during browning of iWAT remains largely unknown. Here we report that PNPLA7, an Endoplasmic Reticulum and mitochondria-associated membrane (MAM) protein, inhibits browning of iWAT by promoting PINK1/Parkin-mediated mitophagy upon cold challenge or β3-adrenergic receptor agonist treatment. With genetic manipulation in mice, we show that adipose tissue overexpressing PNPLA7 induces mitophagy, abolishes iWAT browning and interrupts adaptive thermogenesis. Conversely, conditional ablation of PNPLA7 in adipose tissue promotes browning of iWAT, resulting in enhanced adaptive thermogenesis. Mechanistically, PNPLA7 interacts with Parkin to promote mitochondrial recruitment of Parkin for mitophagy activation and mitochondria degradation by disrupting PKA-induced phosphorylation of Parkin under cold challenge. Taken together, our findings suggest that PNPLA7 is a critical regulator of mitophagy that resists cold-induced browning of iWAT, thus providing a direct mechanistic link between mitophagy and browning of iWAT.

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 PCR analysis of Pnpla7 and Ucp1 mRNA expression in differentiated primary SVF-derived adipocytes that were stimulated with vehicle (Ctrl), or 0.2 μM norepinephrine (NE) for 2 days (n = 4/group) (f), 0.1 μM CL 316,243 (CL) for 24 h (n = 4/group)(g), 10 μM isoproterenol (ISO) for 4 h (n = 4/group) (h), 500 μM dibutyryl-cAMP (cAMP) for 6 h (n = 4/group) (i), 1 μM triiodothyronine (T3) for 20 h (n = 3/group) (j), 1 μM rosiglitazone (Rosi) for 2 days (n = 4/group) (k). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). RT: room temperature. Source data are provided as a Source Data file.

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 Pnpla7^Tg 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 Pnpla7^Tg mice. (RT: n = 5/group; cold: +/+: n = 8; Pnpla7^Tg: 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 Pnpla7^Tg 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 Pnpla7^Tg male mice (n = 3 biological replicates, Scale bar = 100 μm). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). h, i Regression-based analysis of absolute oxygen consumption (h) and energy expenditure (i) against body mass from 8-week-old control and Pnpla7^AKO male mice (n = 5/group). Oxygen consumption and energy expenditure as dependent variable, genotype as fixed variable, and body mass as covariate. (Two-sided analysis of covariance). j, k Adiposity index (j) and fat mass (k) to body weight ratio analysis of Flox and Pnpla7^AKO male mice (RT: n = 5/group; cold: Flox: n = 9; AKO: n = 10; saline: n = 5/group; CL 316,243: Flox: n = 6; AKO: n = 5). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). l TAG levels of iWAT harvested from Flox and Pnpla7^AKO male mice (RT: n = 5/group; cold: n = 6/group; saline: n = 5/group; CL 316,243: n = 5/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). m, n Representative H&E and UCP1 immunohistochemical staining images (m) as well as representative immunoblot results and densitometry analysis (n) of iWAT from Flox and Pnpla7^AKO male mice (n = 3 biological replicates, scale bar = 100 μm). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). Source data are provided as a Source Data file.

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; Pnpla7^Tg group: n = 17; Cold: +/+ group: n = 20; Pnpla7^Tg group: n = 18) of iWAT from control and Pnpla7^Tg 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 Pnpla7^Tg 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; AKO group: n = 30) of iWAT of Flox and Pnpla7^AKO male mice. Cristae number normalized by each section. Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). g, h Representative TOM20 immunohistochemical staining images (c, n = 3 biological replicates, scale bar = 100 μm) and relative mtDNA content (b, n = 4/group) of the iWAT from Flox and Pnpla7^AKO male mice. Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). i, j Representative Immunoblot results and densitometry analysis of indicated protein levels in iWAT of control and Pnpla7^Tg (i) as well as Flox and Pnpla7^AKO (j) male mice (n = 3/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). k, l Mitochondrial oxygen consumption rate of differentiated mature white and beige adipocytes from control and Pnpla7^Tg (k) as well as control and Pnpla7^AKO (l) male mice (n = 3 biological replicates). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). The stromal vascular fraction (SVF) was isolated from iWAT of 3-week-old male mice and differentiated into mature adipocytes ex vitro. Source data are provided as a Source Data file.

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 mitophagosome number per 100 mitochondria in the cells described in (c). (n = 3 independent experiments). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). e Relative mtDNA content in differentiated mature adipocytes treated with DMSO or CCCP (10 μM) for 24 h (DMSO: WT group: n = 6; KO group: n = 5; CCCP: n = 5/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). f, g Representative Immunoblot results (f) and densitometry analysis (g) of mitochondrial membrane, and OXPHOS protein levels in differentiated mature adipocytes treated with CCCP (10 μM) or Valinomycin (1 μM) for 24 h, respectively (n = 3 biological replicates). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons). Source data are provided as a Source Data file.

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 Pnpla7^Tg 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 Pnpla7^Tg (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 down strategy of Parkin in iWAT of Pnpla7^Tg mice and littermate control. 4-week-old control (+/+) and Pnpla7^Tg (Tg) mice were injected with recombinant adeno-associated virus of AAV-Adipo-shscramble or AAV-Adipo-shParkin into the iWAT. After 28 days injection, the mice were exposed to 6 °C for additional 7 days. These mice were fed with normal chow diet. The mouse picture was created in BioRender. Ji, X. (2025) https://BioRender.com/v1sk48t. i, j The iWAT weight ratio (i) and relative mtDNA content (j) of control (+/+) and Pnpla7^Tg (Tg) mice with or without Parkin knockdown as described in (h). (shScramble: +/+ group: n = 6; Pnpla7^Tg group: n = 5; shParkin: n = 6/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons; ns indicates no statistical significance). k Representative H&E, UCP1, and TOM20 immunohistochemical staining images of iWAT sections from control (+/+) and Pnpla7^Tg (Tg) mice with or without Parkin knockdown as described in (h). (n = 3/group). Scale bar = 100 μm. l Representative Immunoblot results and densitometry analysis of the indicated proteins in iWAT control (+/+) and Pnpla7^Tg (Tg) mice with or without Parkin knockdown as described in (h). (n = 3/group). Data are presented as mean ± SEM. (Two-tailed Student’s t test for 2-group comparisons; ns indicates no statistical significance). Source data are provided as a Source Data file.

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 Representative immunofluorescence image of EGFP-PNPLA7 colocalization with mitochondria in 3T3-L1 cells. EGFP-PNPLA7 localization to mitochondria was analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). e, f Representative co-immunoprecipitation Immunoblot data of endogenous Parkin with PNPLA7 in iWAT. Pnpla7^Tg mice iWAT total lysates were immunoprecipitated with anti-Parkin (e) or anti-Flag (f) antibodies, respectively. Calnexin was used as a protein loading control. IgG, immunoglobulin G. (n = 3 biological replicates). g Schematic diagram of construction of PNPLA7 truncations. Full length of PNPLA7(1-1352aa), N-terminal region (1-941aa), Transmembrane domain (36-1352aa), and C-terminal region (942-1352aa). All truncations carry HA-tag at the carboxyl terminus. h, i Representative co-immunoprecipitation Immunoblot data of Flag-Parkin with different truncations of HA-PNPLA7 in HEK293T cells. HEK293T cells were transfected with Flag-Parkin and different truncations of HA-PNPLA7 for 48 h. Cell total lysates were immunoprecipitated with anti-HA (h) or anti-Flag (i) antibodies, respectively. HA-vector was used as a negative control of immunoprecipitation. Calnexin was used as a protein loading control. (n = 3 biological replicates). j Mitochondrial translocation of Parkin in PNPLA7-depleted mature adipocytes overexpressing different PNPLA7 truncations. SVF from iWAT of PNPLA7 KO mice was differentiated into adipocytes ex vitro. Immunofluorescence images of EGFP-Parkin (green) and mCherry-PNPLA7 (red) were analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). Source data are provided as a Source Data file.

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 Pnpla7^Tg (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 Pnpla7^Tg (d) as well as control and Pnpla7^AKO (e) mice. 10-week-old control (+/+) and Pnpla7^Tg (Tg) mice were injected with saline or CL 316,243 (1 mg/kg) via tail vein for 30 min. Co-immunoprecipitated endogenous phosphorylated Parkin (P-Parkin) protein was detected by anti-Parkin antibody. The input protein level of Parkin, PNPLA7, p-PKA substrate, and calnexin were detected by the indicated antibodies, respectively. IgG immunoglobulin G. (n = 3 biological replicates). f Representative co-immunoprecipitation Immunoblot data of endogenous p-Parkin with p-PKA substrate in adipocytes of control and PNPLA7 KO mice. The SVF was isolated from iWAT of 3-week-old wild-type (+/+) and PNPLA7 KO mice and differentiated into adipocytes ex vitro. Then adipocytes were incubated with saline or CL 316,243 (1 μM) for 30 min, with or without pretreatment with H89 (1 μM) for 1 h. Co-immunoprecipitated endogenous phosphorylated Parkin (P-Parkin) protein was detected by anti-Parkin antibody. The input protein level of Parkin, PNPLA7, p-PKA substrate, and calnexin were detected by the indicated antibodies, respectively. (n = 3 biological replicates). g A schematic depicting the role of PNPLA7 in inhibiting browning of iWAT. This Illustration created in BioRender. Ji, X. (2025) https://BioRender.com/5c8g75r. Source data are provided as a Source Data file.