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. 2015 Apr 15;34(8):1042-55.
doi: 10.15252/embj.201489897. Epub 2015 Feb 19.

The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC-1β

Hidetoshi Fujita  1 , Naoko Yagishita  2 , Satoko Aratani  1 , Tomoko Saito-Fujita  3 , Saori Morota  4 , Yoshihisa Yamano  2 , Magnus J Hansson  5 , Masato Inazu  6 , Hiroko Kokuba  6 , Katsuko Sudo  7 , Eiichi Sato  8 , Ko-Ichi Kawahara  9 , Fukami Nakajima  6 , Daisuke Hasegawa  2 , Itsuro Higuchi  10 , Tomoo Sato  2 , Natsumi Araya  2 , Chie Usui  11 , Kenya Nishioka  12 , Yu Nakatani  13 , Ikuro Maruyama  14 , Masahiko Usui  6 , Naomi Hara  4 , Hiroyuki Uchino  4 , Eskil Elmer  5 , Kusuki Nishioka  6 , Toshihiro Nakajima  15
Affiliations

The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC-1β

Hidetoshi Fujita et al. EMBO J. .

Abstract

Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post-neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER-resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild-type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue-specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator-activated receptor coactivator (PGC)-1β, and Syvn1 mutants showed upregulation of PGC-1β target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS-102 abolished the negative regulation of PGC-1β by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post-translational regulator of PGC-1β and a potential therapeutic target in obesity treatment.

Keywords: PGC‐1β; endoplasmic reticulum; mitochondria; obesity; synoviolin.

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Figures

Figure 1
Figure 1
Body weight and WAT in post-neonatal Syvn1 mutant mice

  1. A Schematic depiction of Syvn1 gene targeting strategy. Homologous recombination resulted in exons 2–12 being flanked by loxP sites; deletion was achieved by Tam-induced Cre recombinase-mediated excision.

  2. B PCR products amplified from genomic DNA isolated from tails on day 7 after Tam administration.

  3. C Real-time PCR analysis of adipocyte mRNA from control (Control) and CAG-Cre-ER;Syvn1flox/flox (KO) mice on day 7 after Tam administration.

  4. D Western blotting of spleen proteins on day 7 after Tam administration.

  5. E,F Changes in body weight. *P < 0.05, **P < 0.01 for Tam-treated CAG-Cre-ER;Syvn1flox/flox mice versus vehicle-treated CAG-Cre-ER;Syvn1flox/flox mice, Tam- or vehicle-treated Syvn1flox/flox mice, Tam- or vehicle-treated C57BL/6J mice (analysis of variance with Tukey–Kramer post hoc analysis).

  6. G Average daily food intake measured after Tam injection.

  7. H–J Fat accumulation in post-neonatal Syvn1 KO mice on 7 day after Tam administration. Subcutaneous adipose (H), epididymal adipose (I), and mesentery adipose (J) tissues are shown (Control, n = 8; KO, n = 3). Mesentery fat is shown by black arrows.

Data information: All data are expressed as mean ± SD. Data were analyzed with the Student's t-test. *P < 0.05, **P < 0.01. Source data are available online for this figure.
Figure 2
Figure 2
Changes in body weight and WAT in post-neonatal Syvn1 knockout (KO) and genetically obese (ob/ob and db/db) mice Compound mutants of Syvn1 and ob/ob or db/db were generated as described in Materials and Methods.

  1. A SYVN1 expression in the WAT of ob/+, ob/ob, db/+, and db/db mice.

  2. B,C Changes in body weight in Tam-treated mice. CAG-Cre-ER;Syvn1flox/flox;ob/ob (KO;ob/ob) and CAG-Cre-ER;Syvn1flox/flox;db/db (KO;db/db) mice, red line; Syvn1flox/flox;ob/ob, Syvn1flox/+;ob/ob, and Syvn1+/+;ob/ob (Control;ob/ob) and Syvn1flox/flox;db/db, Syvn1flox/+;db/db, and Syvn1+/+;db/db (Control;db/db) mice, blue line.

  3. D,E Subcutaneous fat accumulation in CAG-Cre-ER;Syvn1flox/flox;ob/ob and CAG-Cre-ER;Syvn1flox/flox;db/db mice after post-neonatal Syvn1 knockout. Histological analysis of adipose tissue from Control;ob/ob and Control;db/db mice (left), and CAG-Cre-ER;Syvn1flox/flox;ob/ob and CAG-Cre-ER;Syvn1flox/flox;db/db mice (right) after Tam administration. Subcutaneous fat is shown by white arrows.

  4. F,G Average daily food intake measured after Tam injection in CAG-Cre-ER;Syvn1flox/flox;ob/ob (D) and CAG-Cre-ER;Syvn1flox/flox;db/db (G) mice.

Data information: Data were analyzed by the Student's t-test and are expressed as mean ± SD. *P < 0.05, **P < 0.01. Source data are available online for this figure.
Figure 3
Figure 3
Effect of adipose tissue-specific Syvn1 deletion on body weight and fat mass

  1. A PCR products amplified from genomic DNA isolated from WAT, liver, tail, and muscle of Adipoq-Cre;Syvn1flox/flox (KO) and Syvn1flox/flox mice (Control).

  2. B Western blotting of WAT proteins 15 weeks after birth.

  3. C,D Changes in body weight after weaning. Adipoq-Cre;Syvn1flox/flox mice (KO), red line; control (Syvn1flox/flox and Syvn1flox/+) mice, blue line. Data were analyzed with the Student's t-test and represent the mean ± SD. *P < 0.05. (D) Control and KO mice 15 weeks after birth.

  4. E,F Reduction in subcutaneous fat (E) and epididymal adipose tissue (F) in KO mice 15 weeks after birth. Data were analyzed with the Student's t-test and represent the mean ± SD (Control, n = 10; KO, n = 8). *P < 0.05, **P < 0.01.

Source data are available online for this figure.
Figure 4
Figure 4
PGC-1β is a substrate of SYVN1

  1. A GST and GST-tagged SYVN1 lacking the transmembrane domain (GST-SYVN1ΔTM) were incubated with HEK 293T whole-cell extracts expressing HA-PPARα, HA-PPARγ, HA-PGC-1α, or HA-PGC-1β.

  2. B,C Interaction of SYVN1 with PGC-1β. (B) Schematic representation of SYVN1. TM, transmembrane domain; SyU, SYVN1 unique domain. (B, C) An in vitro binding assay was performed with HA-PGC-1β and GST or GST-tagged SYVN1 deletion mutants.

  3. D,E SYVN1 interacts with PGC-1β in vivo. (D) Whole-cell extracts from HEK 293T cells transfected with HA-PGC-1β and/or SYVN1/FLAG expression plasmids were immunoprecipitated with anti-FLAG antibody. *Immunoprecipitated IgG heavy chain. (E) Whole-cell extracts from HEK 293 cells were immunoprecipitated with anti-SYVN1 antibody. Western blotting was performed using anti-PGC-1β antibody. *Non-specific band.

  4. F HEK 293T cells were transfected with HA-PGC-1β (green) and/or SYVN1/FLAG, SYVN1 R266A, R267A/FLAG (red) expression plasmids. After 24 h, cells were immunolabeled with anti-HA and anti-FLAG antibodies.

  5. G In vitro ubiquitination assay was performed with maltose-binding protein (MBP)-tagged SYVN1ΔTM-His, GST-PGC-1β (aa 1–367), E1 and E2 enzymes, and HA-Ub. Lower panel shows the loading control.

  6. H Whole-cell extracts from HEK 293T cells transfected with HA-PGC-1β, Ub/FLAG, and WT or mutant SYVN1 (SYVN1 WT or 3S, respectively) expression plasmids were immunoprecipitated with anti-HA antibody. Lower panels show the loading controls.

  7. I Cell extracts from adipose tissues were obtained from control and CAG-Cre-ER;Syvn1flox/flox mice 7 days after Tam administration. Western blotting was performed using an anti-PGC-1β antibody. Representative data from three sets of control and CAG-Cre-ER;Syvn1flox/flox mice are shown.

  8. J Skin fibroblasts from CAG-Cre-ER;Syvn1flox/flox mice were cultured, and 48 h after Tam or vehicle treatment and addition of 10 μM MG-132 for 2 h, cell lysates were collected for Western blotting.

  9. K HEK 293 cells were transiently transfected with control or Syvn1 siRNA. After 48 h, cells were incubated with 10 μM MG-132 for 2 h. Western blotting was performed with anti-SYVN1 and anti-PGC-1β antibodies.

  10. L Measurement of PGC-1β half-life. Mouse embryonic fibroblasts derived from Syvn1 knockout mice were transfected with HA-PGC-1β and SYVN1/FLAG or SYVN1ΔSyU/FLAG; 48 h later, cells were treated with 40 μM cycloheximide for the indicated times, and immunoblotting was performed with anti-PGC-1β antibody. Intensity of bands was measured (right panel). Each experiment was performed at least three times.

Source data are available online for this figure.
Figure 5
Figure 5
Regulation of PGC-1β function by SYVN1

  1. A–C HEK 293 cells (1 × 105 cells) were transiently co-transfected with a reporter plasmid containing PPAR binding sites (PPRE X3-TK-luc) and expression constructs or siRNAs (A). For WT and mutant SYVN1 overexpression, 50 or 100 μg SYVN1 expression vector was co-transfected (B, C); 16 h later, cells were treated with vehicle or 10 μM Wy-14643 for 6 h. Each experiment was performed at least three times.

  2. D Representative electron micrographs of siRNA-treated cells. An increase in mitochondrial volume can be seen as the large cytoplasmic area in 3T3 L1 cells (a mitochondrion is shown by the white arrow). Magnification, 5,000×. The number of mitochondria in the area (500 pixel × 500 pixel) was measured (right panel). Each experiment was performed at least three times.

  3. E Total RNA was isolated from adipose tissue 15 weeks after birth, and mRNA expression was measured by real-time PCR relative to 18s rRNA level, with the average for control mice set to 1 (n = 3).

  4. F Cell extracts from adipose tissue were obtained from control and Adipoq-Cre;Syvn1flox/flox (KO) mice 15 weeks after birth. Western blotting was performed using anti-medium chain acyl-coenzyme A dehydrogenase (MCAD) antibody.

  5. G Representative electron micrographs of adipose tissue of control (left) and CAG-Cre-ER;Syvn1flox/flox (right) mice. An increase in mitochondrial volume can be seen as an enlargement in cytoplasmic area (M, mitochondria). Magnification, 10,000×.

  6. H Mitochondrial respiration was measured in WAT 7 days after Tam administration (the value for control mice was set to 100%) (n = 3).

  7. I Basal energy expenditure of control and CAG-Cre-ER;Syvn1flox/flox mice 7 days after Tam administration.

Data information: Data were analyzed with the Student's t-test (A–E, I) or Mann–Whitney U-test (H) and represent the mean ± SD. *P < 0.05, **P < 0.01. Source data are available online for this figure.
Figure 6
Figure 6
Effect of SYVN1 inhibition on diet-induced weight gain or genetic obesity

  1. A HEK 293 cells were transfected with FLAG-PGC-1β plasmid and immunoprecipitated with anti-FLAG antibody. An in vitro ubiquitination assay was performed with GST-SYVN1ΔTM, immunoprecipitated FLAG-PGC-1β, E1 and E2 enzymes, and HA-Ub in the presence or absence of LS-102. Each experiment was performed at least three times.

  2. B HEK 293 cells were transiently transfected with PPRE X3-TK-luc and PPARα expression plasmid; 16 h later, cells were treated with vehicle or 10 μM Wy-14643 for 6 h. **P < 0.01. Each experiment was performed at least three times.

  3. C,D Body weight (C) and average food intake (D) were measured daily for C57BL/6J mice treated once a day with DMSO or 50 mg/kg LS-102. *P < 0.05 for C57BL/6J LS-102-treated versus C57BL/6J DMSO-treated mice.

  4. E Epididymal adipose tissue of C57BL/6J mice treated with DMSO (left) or LS-102 (right) on day 57 (n = 4).

  5. F Hematoxylin and eosin staining of epididymal adipose tissue of C57BL/6J mice treated with DMSO (left) or LS-102 (right) (lipid droplets are shown by black arrows). Magnification, 400×.

  6. G Representative electron micrographs of adipose tissue from DMSO- (left) and LS-102-treated (right) mice on day 57. An increase in mitochondrial volume can be seen as enlarged cytoplasmic areas (a mitochondrion is shown by the white arrow). Magnification, 10,000×. The number of mitochondria in the area (2,000 pixel × 2,000 pixel) was measured (right panel).

  7. H–J Obese db/db mice were treated daily with DMSO or LS-102, and body weight (H, I) and average daily food intake (J) were measured (Control, n = 3; LS-102, n = 8).

  8. K Blood glucose was measured on days 13 and 27 (Control, n = 3; LS-102, n = 8).

Data information: All data were analyzed with the Student's t-test and are shown as mean ± SD. *P < 0.05, **P < 0.01. Source data are available online for this figure.
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References

    1. Abella V, Scotece M, Conde J, Lopez V, Lazzaro V, Pino J, Gomez-Reino JJ, Gualillo O. Adipokines, Metabolic Syndrome and Rheumatic Diseases. J Immunol Res. 2014;2014:343746. - PMC - PubMed
    1. Amano T, Yamasaki S, Yagishita N, Tsuchimochi K, Shin H, Kawahara K, Aratani S, Fujita H, Zhang L, Ikeda R, Fujii R, Miura N, Komiya S, Nishioka K, Maruyama I, Fukamizu A, Nakajima T. Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy. Genes Dev. 2003;17:2436–2449. - PMC - PubMed
    1. Andersson U, Scarpulla RC. Pgc-1-related coactivator, a novel, serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells. Mol Cell Biol. 2001;21:3738–3749. - PMC - PubMed
    1. Aratani S, Fujii R, Oishi T, Fujita H, Amano T, Ohshima T, Hagiwara M, Fukamizu A, Nakajima T. Dual roles of RNA helicase A in CREB-dependent transcription. Mol Cell Biol. 2001;21:4460–4469. - PMC - PubMed
    1. Bernasconi R, Galli C, Calanca V, Nakajima T, Molinari M. Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates. J Cell Biol. 2010;188:223–235. - PMC - PubMed

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