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TP53INP2 regulates adiposity by activating β-catenin through autophagy-dependent sequestration of GSK3β

Nature Cell Biology volume 20, pages 443–454 (2018)Cite this article

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Abstract

Excessive fat accumulation is a major risk factor for the development of type 2 diabetes mellitus and other common conditions, including cardiovascular disease and certain types of cancer. Here, we identify a mechanism that regulates adiposity based on the activator of autophagy TP53INP2. We report that TP53INP2 is a negative regulator of adipogenesis in human and mouse preadipocytes. In keeping with this, TP53INP2 ablation in mice caused enhanced adiposity, which was characterized by greater cellularity of subcutaneous adipose tissue and increased expression of master adipogenic genes. TP53INP2 modulates adipogenesis through autophagy-dependent sequestration of GSK3β into late endosomes. GSK3β sequestration was also dependent on ESCRT activity. As a result, TP53INP2 promotes greater β-catenin levels and induces the transcriptional activity of TCF/LEF transcription factors. These results demonstrate a link between autophagy, sequestration of GSK3β into late endosomes and inhibition of adipogenesis in vivo.

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Fig. 1: TP53INP2 regulates adipogenesis.
Fig. 2: TP53INP2 loss of function promotes adipose hyperplasia.
Fig. 3: TP53INP2 loss of function promotes adiposity.
Fig. 4: TP53INP2 increases WNT signalling.
Fig. 5: TP53INP2 enhances β-catenin levels.
Fig. 6: TP53INP2 facilitates the sequestration of GSK3β into a late-endosomal compartment in an ESCRT-dependent manner.
Fig. 7: Autophagy activity is necessary for the TP53INP2-dependent sequestration of GSK3β.

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Acknowledgements

We thank D. Rossell (Department of Statistics, University of Warwick, Coventry, UK) and C. Stephan-Otto Attolini (Biostatistics and Bioinformatics Unit, Institute for Research in Biomedicine, Barcelona, Spain) for their support with the transcriptomic studies. We also thank L. Bardia (Advanced Digital Microscopy Facility, Institute for Research in Biomedicine, Barcelona, Spain), J. Comas (Cytometry and Genomics Facility, University of Barcelona, Spain), N. Plana, V. Hernández and J. Manuel Seco for their technological assistance. A.Z. is a recipient of an ICREA 'Academia' (Generalitat de Catalunya), M.R. is a researcher hired by CIBERDEM (Instituto de Salud Carlos III) and A.S.-P. is a recipient of a predoctoral fellowship from the Universitat de Barcelona. This study was supported by research grants from the MINECO (SAF2013-40987R), grant 2014SGR48 from the Generalitat de Catalunya, CIBERDEM ('Instituto de Salud Carlos III') and INTERREG IV-B-SUDOE-FEDER (DIOMED, SOE1/P1/E178). IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). IRB Barcelona is a member of CERCA.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, Spain

    Montserrat Romero, Alba Sabaté-Pérez, Víctor A. Francis, Ignacio Castrillón-Rodriguez, Ángels Díaz-Ramos, Manuela Sánchez-Feutrie, Manuel Palacín & Antonio Zorzano

  2. Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain

    Montserrat Romero, Alba Sabaté-Pérez, Víctor A. Francis, Ignacio Castrillón-Rodriguez, Ángels Díaz-Ramos, Manuela Sánchez-Feutrie, Manuel Palacín & Antonio Zorzano

  3. Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain

    Montserrat Romero, Alba Sabaté-Pérez, Víctor A. Francis, Ignacio Castrillón-Rodriguez, Ángels Díaz-Ramos, Manuela Sánchez-Feutrie, Xavier Durán, Joan Vendrell & Antonio Zorzano

  4. Department of Endocrinology, Hospital Joan XXIII, Rovira i Virgili University, Tarragona, Spain

    Xavier Durán & Joan Vendrell

  5. Institut d’Investigació Sanitaria Pere Virgili (IISPV), Tarragona, Spain

    Xavier Durán & Joan Vendrell

  6. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain

    Manuel Palacín

  7. Department of Diabetes, Endocrinology and Nutrition, Institut d’Investigació Biomèdica de Girona (IdIBGi), Hospital of Girona ‘Dr Josep Trueta’, Girona, Spain

    José María Moreno-Navarrete & José Manuel Fernández-Real

  8. Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), ISCIII, Madrid, Spain

    José María Moreno-Navarrete & José Manuel Fernández-Real

  9. Department of Molecular and Clinical Medicine, The Lundberg Laboratory for Diabetes Research, Sahlgrenska Academy at the University of Gothenburg, Gothenburg, Sweden

    Birgit Gustafson, Ann Hammarstedt & Ulf Smith

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  1. Montserrat Romero
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Contributions

M.R. conceived and performed the experiments and wrote the manuscript. V.A.F. conceived and performed the experiments. A.S.-P., I.C.-R. A.D.-R., X.D., B.G., A.H., M.S.-F. and J.M.M.-N. performed the experiments. J.V., J.M.F.-R. and U.S. revised the experimental data and contributed to the discussion. M.P. contributed to the discussion. A.Z. directed the research, revised the experimental data and wrote the manuscript.

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Correspondence to Antonio Zorzano.

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Supplementary Figure 1 TP53INP2 regulates adipogenesis.

Panels a to h: Control (C) and TP53INP2 knockdown (KD) 3T3-L1 cells. (a) TP53INP2 mRNA (n = 4 independent experiments) and protein (n = 3 independent experiments) expression. Data are mean ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test (*P < 0.0001); (b) Distribution of adipocyte cell area at day 8 of differentiation. Data are mean ± SEM. Statistical significance was calculated using two-way ANOVA (*P = 0.0005); (c) Triglycerides content. Data are mean ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test (*P < 0.0001); Panels d and e: GLUT4 (d) and perilipin (e) protein quantification in adipocytes. Data are mean ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test (GLUT4: *P = 0.0004 and Perilipin: *P < 0.0001); (f) Insulin-stimulated 2-deoxy-glucose uptake. Data are mean ± SEM (n = 6 independent experiments). Statistical significance was calculated using two-way ANOVA followed by Sidak’s test (*P = 0.0080); Panels g and h: PPARγ (g) and C/EBPα (h) protein quantification during differentiation. Data are mean ± SEM. Statistical significance was calculated using two-way ANOVA followed by Sidak’s test (PPARγ: *P < 0.0001; C/EBPα: *P = 0.0001 and #P < 0.0001); Panels i to n: Control (-) and TP53INP2- (+) overexpressing 3T3-L1CAR∆1 cells. (i) TP53INP2 mRNA and protein expression. Data are mean ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test (*P < 0.0001); (j) Triglycerides content. Data are mean ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0034); Panels k and l: GLUT-4 (k) and perilipin (l) protein quantification in adipocytes. Data are mean ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test (GLUT4: *P = 0.0125 and Perilipin: *P < 0.0496); Panels m and n: PPARγ (m) and C/EBPα (n) protein quantification during differentiation. Data are mean ± SEM. Statistical significance was calculated using two-way ANOVA followed by Sidak’s test (PPARγ: *P = 0.0049, #P = 0.0004 and §P < 0.0001; C/EBPα: *P = 0.0029 and #P = 0.0060); o) Control and TP53INP2-overexpressing human preadipocytes. PPARγ protein expression during human adipogenesis. Data are mean ± SEM. Statistical significance was calculated using two-way ANOVA followed by Sidak’s test (*P = 0.0269, #P = 0.0207 and §P < 0.0473). All unprocessed blots are provided in Supplementary Figure 8.

Supplementary Figure 2 TP53INP2 loss-of-function promotes adipose hyperplasia.

Panels a, b and c: TP53INP2 mRNA levels in human subcutaneous and visceral adipose tissue are inversely proportional to BMI. The sample size of the study 1 is n = 24 samples of scWAT (a) and n = 38 samples of vWAT (b). In the study 2, the sample size is n = 35 samples of scWAT. Statistical significance was calculated using a Spearman correlation analysis (r = -0.439 and P = 0.0051 (a); r = -0.567 and P = 0.0002 (b); r = -0.348 and P = 0.04 (c); d) Experimental design for the generation of TP53INP2 KOUbc mice: two-month-old mice expressing CreTP53INP2loxP/loxP and their non-expressing Cre-ERT2TP53INP2loxP/loxP control littermates were fed a tamoxifen diet. Experimental data were collected 2 months after starting the tamoxifen diet; Panels e to j: Control and TP53INP2 KOUbc four-month-old mice. Data represent means ± SEM. (e) TP53INP2 mRNA levels in ingWAT (n = 4 control and n = 5 KOUbc mice) and vWAT (n = 5 control and n = 7 KOUbc mice). Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0002 and #P < 0.0001); (f) TP53INP2 protein expression in ingWAT (n = 3) and pgWAT (n = 3) depots; (g) TP53INP2 mRNA levels in other tissues (n = 3 samples for each tissue). Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0139; #P = 0.0031; §P ≤ 0.0001 and &P = 0.0004); Panels h and i: Tissues weigh of females (h) (Brain, kidney and heart: n = 10 control and n = 15 KOUbc mice, and Gastroc, Quads and Tib: n = 5 control and n = 9 KOUbc mice) and males (i) (n = 4 control and n = 7 KOUbc mice). Data represent means ± SEM. Statistical significance was calculated using an unpaired two-tailed t-test; j) Fsp27, Perilipin, Leptin, Resistin, LPL and VEGF mRNA levels in adipose tissue. Data represent means ± SEM (n = 4 control and n = 6 KOUbc mice). Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0113; #P = 0.0320; §P < 0.0271; &P = 0.0452; °P = 0.0465 and +P < 0.0107). All unprocessed blots are provided in Supplementary Figure 8.

Supplementary Figure 3 TP53INP2 loss-of-function promotes adiposity.

Panels a to d: Control and TP53INP2 KOMyf5 mice. Data represent means ± SEM. (a) TP53INP2 mRNA levels in white adipose tissue depots of control (asWAT: n = 6; rWAT: n = 8; ingWAT: n = 6 and pgWAT: n = 4) and TP53INP2 KOMyf5 mice (asWAT: n = 6; rWAT: n = 8; ingWAT: n = 7 and pgWAT: n = 4). Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0020 and #P = 0.0001); (b) Body weight of control (n = 7) and TP53INP2 KOMyf5 (n = 9) mice; (c) Weight of asWAT (n = 7 control and n = 8 KOMyf5 mice), rWAT (n = 7 control and n = 8 KOMyf5 mice), ingWAT (n = 7 control and n = 10 KOMyf5 mice) and pgWAT (n = 7 control and n = 10 KOMyf5 mice); (d) Adipocyte area of ingWAT (n = 4 control and KOMyf5 mice) and pgWAT (n = 5 control and n = 6 KOMyf5 mice); e) Plasma insulin levels in control (n = 6) and TP53INP2 KOUbc (n = 6) four-month-old mice; f) Plasma insulin levels in control (n = 5) and TP53INP2 KOUbc (n = 5) eight-month-old mice; g) Plasma insulin levels in control (n = 5) and TP53INP2 KOUbc (n = 9) mice fed with high fat diet; Panels h to l: Control and TP53INP2 KOUbc four-month-old mice. (h) TNFα and IL-1β mRNA levels in ingWAT. Data are means ± SEM (TNFα: n = 5 control and KOUbc mice, respectively; IL-1β: n = 4 control and n = 8 KOUbc mice). Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0052 and #P = 0.0486); (i) Oxygen consumption of control (n = 6) and TP53INP2 KOUbc (n = 6) mice. Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0241); (j) Locomotor activity of control (n = 6) and TP53INP2 KOUbc (n = 6) mice; Panels k and l: Food (k) and water intake (l) of control (n = 6 females and n = 4 males) and TP53INP2 KOUbc (n = 6 females and n = 8 males) mice.

Supplementary Figure 4 TP53INP2 increases WNT signaling.

a) TOP-Flash and FOP-Flash reporter activity in control and TP53INP2-overexpressing HEK293T cells, and effects of LiCl. Data are means ± SEM of n = 3 independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test (*P = 0.0002 indicates effects caused by TP53INP2 overexpression and #P < 0.0001 indicates effects caused by LiCl; b) TOP-Flash and FOP-Flash reporter activity in control HEK293T cells and cells overexpressing wild-type, L36A/L40A or E97K/D98K mutant forms of TP53INP2, in the presence or absence of LiCl. Data are means ± SEM of n = 3 independent experiments. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test (*P < 0.0001 indicates effects caused by TP53INP2 overexpression, and #P < 0.0001, §P = 0.0061, &P = 0.0004 indicates effects caused by LiCl).

Supplementary Figure 5 TP53INP2 enhances β-catenin levels.

a) Protein expression of total and active form of β-catenin in control and TP53INP2 KD 3T3-L1 preadipocytes. Data represent n = 3 independent experiments; b) Quantification of total and active forms of β-catenin in control and TP53INP2 KD 3T3-L1 preadipocytes. Statistical significance was calculated using an unpaired two-tailed t-test (β-catenin: *P = 0.0022 and Activeβ-catenin: *P = 0.0002); c) β-catenin mRNA levels in control and KD preadipocytes. Statistical significance was calculated using an unpaired two-tailed t-test (P = 0.6934); d) β-catenin protein quantification in ingWAT and pgWAT depots from control and TP53INP2 KOUbc mice. Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0094 and #P = 0.0073); Panels e and f: Quantification of total and active forms of β-catenin in control and TP53INP2-overexpressing 3T3-L1 CAR∆1 preadipocytes (n = 3 independent experiments) (panel e) or human preadipocytes (n = 3 independent experiments) (panel f). Statistical significance was calculated using an unpaired two-tailed t-test (3T3-L1 CAR∆1 preadipocytes: *P = 0.0016 and *P = 0.0041 for β-catenin and Activeβ-catenin, respectively; Human preadipocytes: *P = 0.0006 and *P = 0.0027 for β-catenin and Activeβ-catenin, respectively); Panels g and h: 3T3-L1 adipocytes differentiated in the presence or absence of WNT3a (n = 3 independent experiments). (g) Representative images at day 8 of differentiation; (h) Protein expression of total and active form of β-catenin, PPARγ and C/EBPα during the differentiation process. All unprocessed blots are provided in Supplementary Figure 8.

Supplementary Figure 6 TP53INP2 facilitates the sequestration of GSK3β into a late endosomal compartment in an ESCRT-dependent manner.

a) Immunofluorescence of TP53INP2 (blue) and endogenous CD63 (cyan) in the presence or absence of Rab5Q79L in Hela cells (n = 3 independent experiments). Scale bar, 8 μm; Panels b and c: β-catenin protein quantification in the presence of wild-type TP53INP2 (b) or mutant form W35/I38A (c). Data are means ± SEM (n = 3 independent experiments). Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0002 control vs TP53INP2-overexpressing cells); d) Cellular localization of GSK3β-RFP in the presence or absence of TP53INP2 in Rab5S34N-overexpressing HeLa cells (n = 3 independent experiments). Confocal images show TP53INP2 (cyan), EGFP-Rab5S34N (red), RFP-GSK3β (green) and DAPI (blue). Scale bar, 8 μm; e) Immunofluorescence staining shows endogenous CD63 (red), endogenous GSK3β (green) and DAPI (blue) in control and TP53INP2 KD 3T3-L1 cells (n = 3 independent experiments). Insets are enlargements of the outlined areas. White arrows show colocalization between endogenous CD63 and GSK3β. Scale bar, 8 μm and 1 μm (insets); f) Immunofluorescence staining shows endogenous EEA1 (green) and DAPI (blue) in control and TP53INP2 KD 3T3-L1 cells (n = 3 independent experiments). Scale bar, 10 μm; Panels g to i: HRS KD, VPS24 KD and control NIH-3T3 cells. (g) Protein expression of HRS and VPS24; (h) Protein expression of β-catenin (n = 5 independent experiments); (i) Quantification of β-catenin. Data represent the mean ± SEM of n = 5 independent experiments. Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0159 indicates effects caused by TP53INP2). All unprocessed blots are provided in Supplementary Figure 8.

Supplementary Figure 7 Autophagy activity is necessary for the TP53INP2-dependent sequestration of GSK3β.

a) LC3 protein abundance in 3T3-L1 cells upon incubation with increasing concentrations of chloroquine; b) Quantification of C/EBPα protein expression in chloroquine treated-3T3-L1 cells at 48h and 72h of adipogenic differentiation. Statistical significance was calculated using one-way ANOVA followed by Tukey’s test (*P = 0.0055 and #P = 0.0065 indicates effects caused by CQ treatment); Panels c and d: Atg5+/+ and Atg5-/- MEF cells. (c) β-catenin protein abundance in the presence or absence of TP53INP2; (d) β-catenin protein quantification. Data are means ± SEM of n = 3 independent experiments from control and TP53INP2-overexpressing cells. Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0079 control vs TP53INP2-overexpressing cells); Panels e and f: Atg3+/+ and Atg3-/- MEF cells. (e) Protein expression of β-catenin in the presence or absence of TP53INP2; (f) β-catenin protein quantification. Data are means ± SEM of n = 6 independent experiments from control and TP53INP2-overexpressing cells. Statistical significance was calculated using an unpaired two-tailed t-test (*P = 0.0001 control vs TP53INP2-overexpressing cells; g) Immunofluorescence of endogenous LC3-II in Rab5Q79L-overexpressing Atg5+/+ and Atg5-/- MEFs in the presence of TP53INP2. Scale bar, 8 μm. Data represent n = 3 independent experiments. Confocal images show TP53INP2 (cyan), DsRed-Rab5Q79L (red), GSK3β-GFP (green) and LC3II (blue). All unprocessed blots are provided in Supplementary Figure 8.

Supplementary Figure 8 Unprocessed images of blots.

a) Blots corresponding to the Fig. 1c; b) Blots corresponding to the Fig. 1f; c) Blots corresponding to the Fig. 1h; d) Blots corresponding to the Fig. 1k; e) Blots corresponding to the Fig. 1m. f) Blots corresponding to the Fig. 5a; g) Blots corresponding to the Fig. 5b; h) Blots corresponding to the Fig. 5c; i) Blots corresponding to the Fig. 5d; j) Blots corresponding to the Fig. 5f; k) Blots corresponding to the Fig. 5j; l) Blots corresponding to the Fig. 6c. m) Blots corresponding to the figure S1a; n) Blots corresponding to the figure S1i; o) Blots corresponding to the figure S2f; p) Blots corresponding to the figure S5a; q) Blots corresponding to the figure S5h. r) Blots corresponding to the figure S6g; s) Blots corresponding to the figure S6h; t) Blots corresponding to the figure S7a; v) Blots corresponding to the figure S7e; w) Blots corresponding to the figure S7c.

Supplementary information

Supplementary Information

Supplementary Figures 1–8 and Supplementary Table legends.

Life Sciences Reporting Summary

Supplementary Table 1

List of Wnt pathway-related genes regulated in TP53INP2 KD 3T3-L1 pre-adipocytes.

Supplementary Table 2

List of Wnt pathway-related genes regulated in ingWAT from TP53INP2 KOUbc mice.

Supplementary Table 3

List of Wnt pathway-related genes regulated in pgWAT from TP53INP2 KOUbc mice.

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Romero, M., Sabaté-Pérez, A., Francis, V.A. et al. TP53INP2 regulates adiposity by activating β-catenin through autophagy-dependent sequestration of GSK3β. Nat Cell Biol 20, 443–454 (2018). https://doi.org/10.1038/s41556-018-0072-9

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