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The mitochondrial protein Opa1 promotes adipocyte browning that is dependent on urea cycle metabolites

Nature Metabolism volume 3, pages 1633–1647 (2021)Cite this article

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An Author Correction to this article was published on 11 February 2022

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Abstract

White to brown/beige adipocytes conversion is a possible therapeutic strategy to tackle the current obesity epidemics. While mitochondria are key for energy dissipation in brown fat, it is unknown if they can drive adipocyte browning. Here, we show that the mitochondrial cristae biogenesis protein optic atrophy 1 (Opa1) facilitates cell-autonomous adipocyte browning. In two cohorts of patients with obesity, including weight discordant monozygotic twin pairs, adipose tissue OPA1 levels are reduced. In the mouse, Opa1 overexpression favours white adipose tissue expandability as well as browning, ultimately improving glucose tolerance and insulin sensitivity. Transcriptomics and metabolomics analyses identify the Jumanji family chromatin remodelling protein Kdm3a and urea cycle metabolites, including fumarate, as effectors of Opa1-dependent browning. Mechanistically, the higher cyclic adenosine monophosphate (cAMP) levels in Opa1 pre-adipocytes activate cAMP-responsive element binding protein (CREB), which transcribes urea cycle enzymes. Flux analyses in pre-adipocytes indicate that Opa1-dependent fumarate accumulation depends on the urea cycle. Conversely, adipocyte-specific Opa1 deletion curtails urea cycle and beige differentiation of pre-adipocytes, and is rescued by fumarate supplementation. Thus, the urea cycle links the mitochondrial dynamics protein Opa1 to white adipocyte browning.

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Fig. 1: OPA1 expression is reduced in patients with obesity.
Fig. 2: Opa1tg mice are metabolically healthier on a HFD.
Fig. 3: Opa1 promotes BAT thermogenic activity.
Fig. 4: Opa1 promotes adipocyte Ucp1 transcription via Kdm3a.
Fig. 5: Opa1 promotes adipocyte Ucp1 transcription via urea cycle induction.
Fig. 6: Increased urea cycle flux in Opa1tg pre-adipocytes.
Fig. 7: Opa1 promotes adipocyte Ucp1 transcription via CREB-mediated urea cycle induction.

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Data availability

Further information and reagents are available from the corresponding author on reasonable request. Source data are provided with this paper. All raw sequencing data associated with this manuscript have been deposited in the NCBI SRA Archive under the data submission ID SUB4304286, BioProject ID PRJNA481938. All the remaining data that support the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank F. Caicci and F. Boldrin (EM Facility, Department of Biology, University of Padova) for electron microscopy samples preparation; F. Fuks (ULB, Brussels) for the gift of retroviral shKdm3a and shScramble expression vectors; Y. Capetanaki (Biomedical Research Foundation, Academy of Athens) for the gift of Platinum-E Retroviral Packaging Cell Line; S. Ciciliot (VIMM, Padova) for help with metabolic cages; M. Ghidotti (Department of Biology, University of Padova) for help with animal handling; R. Fabris, L. Busetto, M. Foletto, R. Serra, L. Prevedello, R. Bardini, C. Dal Prà, A. Belligoli, M. Sanna and C. Compagnin (University of Padova) for patient recruitment and adipose tissue biopsies collection; M. Rossato (University of Padova) for FLIR thermometry; and K. Lefkimmiatis and G. Di Benedetto (VIMM) for helpful discussions. The energy expenditure ANCOVA analysis was provided by the NIDDK Mouse Metabolic Phenotyping Centers (MMPC, www.mmpc.org) using their energy expenditure analysis page (http://www.mmpc.org/shared/regression.aspx) supported by grant nos DK076169 and DK115255. This work was supported by EFSD/Novo Nordisk Programme for Diabetes Research in Europe 2016 grant (L.S.), by Fondation Leducq TNE15004 (L.S.) and by Ministero dell’Istruzione, dell’Università e della Ricerca FIRB RBAP11Z3YA_005 and PRIN 2017BF3PXZ grants (L.S.); by PRIN 2010329EKE_005 (R.V.) and NanoBAT H2020-EU.1.3.3 MSCA-RISE-2015 (R.V.); and by Ministry of Education, University and Research (MIUR) Progetto Eccellenza (2018-2022) to the Department of Pharmacological and Biomolecular Sciences, University of Milan (N.M.). Twin research was supported by the Academy of Finland (grant nos 335443, 314383 and 272376) (K.P.); Finnish Medical Foundation (K.P.); Gyllenberg Foundation (K.P.); Novo Nordisk Foundation (grant nos NNF20OC0060547, NNF17OC0027232 and NNF10OC1013354) (K.P.) and Finnish Diabetes Research Foundation (K.P.).

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Authors and Affiliations

  1. Department of Biology, University of Padova, Padova, Italy

    Camilla Bean, Tatiana Varanita, Marta Medaglia, Lena Pernas, Marta Giacomello, Stèphanie Herkenne & Luca Scorrano

  2. Veneto Institute of Molecular Medicine, Padova, Italy

    Camilla Bean, Tatiana Varanita, Marta Medaglia, Lena Pernas, Stèphanie Herkenne & Luca Scorrano

  3. Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy

    Matteo Audano & Nico Mitro

  4. Department of Medicine, University of Padova, Padova, Italy

    Francesca Favaretto, Fabio Stasi, Gabriella Milan & Roberto Vettor

  5. Department of Life Science, University of Trieste, Trieste, Italy

    Marco Gerdol & Alberto Pallavicini

  6. Obesity Research Unit, Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Maheswary Muniandy, Sini Heinonen & Kirsi H. Pietiläinen

  7. Institute for Molecular Medicine Finland, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland

    Emma Cazaly & Miina Ollikainen

  8. Obesity Centre, Abdominal Centre, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

    Kirsi H. Pietiläinen

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  1. Camilla Bean
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Contributions

C.B. and L.S. conceptualized the project, acquired funds and wrote the manuscript. C.B. performed and analysed most of the experiments and prepared figures. T.V. conceived and performed initial in vivo studies. F.F., F.S. and G.M. contributed to human and mouse studies. M. Medaglia performed qRT–PCR experiments. M. Gerdol and A.P. contributed to RNA-seq data analyses. L.P. advised on metabolic studies. M.A. and N.M. performed and analysed metabolomic experiments. M. Giacomello performed and analysed calcium imaging experiments. S. Herkenne provided reagents. K.H.P. and S. Heinonen examined twins and provided expression and clinical data. M. Muniandy, M.O. and E.C. analysed twins expression data. R.V. provided bariatric surgery studies. L.S. supervised the project. All authors edited the manuscript.

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Correspondence to Luca Scorrano.

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Extended data

Extended Data Fig. 1 Opa1 increases thermogenic activity.

(a) Representative Haematoxylin-Eosin stained sections of BAT from HFD-fed Wt and Opa1tg mice (n = 8/genotype). Scale bar: 200 µm. (b,c) Expression fold change (Opa1tg vs. Wt) of thermogenic, adipogenic and mitochondrial gene markers in BAT (b) SAT (c) of HFD-fed Opa1tg (n = 5) and Wt mice (n = 4). Dots represent individual mice, I-shaped boxes mean ± SEM; whiskers indicate the 10th-90th percentile. Two-tailed Mann-Whitney U test in HFD-BAT: p = 0.037 for Opa1, Ucp1, Mfn1, p = 0.019 for CideA, Pparγ2 Pgc1β. Two-tailed Mann-Whitney U test in HFD-SAT: p = 0.03 for Opa1, Ucp1, Pparg2, Pgc1α, Pgc1β.

Source data

Extended Data Fig. 2 Heat production and ANCOVA analysis of heat production in Opa1tg mice.

(a) Plot of ANCOVA predicted mean energy expenditure for total body weight (BW) values in Opa1tg and Wt mice. Data are from experiments in Fig. 3h (n = 7 mice/genotype). Multiple linear regression analysis of the impact of body mass covariate on energy expenditure was calculated using the MMPC Web Tool at the MMPC Energy Expenditure Analysis Page. Overall p value refers to significance of the ANCOVA-adjusted comparison when BW was taken over both Opa1tg and Wt groups. Group p-value refers to significance of the ANCOVA-adjusted comparison when BW was taken over the two individual Opa1tg and Wt groups. (b, c) Average±SEM of heat release normalized by lean mass (LM, b) or fat mass (FM, c) in light and dark (indicated as gray vertical stripes on the graph) cycles recorded in male mice of the indicated genotypes at 24 °C and at the indicated temperature. n = 7 mice/genotype. ***, p = 4.1×10−4 (basal heat production at 24 °C) and 9.7×10-5 (at 18 °C) in one-way repeated measures ANOVA.

Source data

Extended Data Fig. 3 Opa1 promotes browning of white adipocytes.

(a) Expression fold change (SAT vs. VAT) of the indicated BAT specific and mitochondrial genes in Wt mice (n = 4-5). Dots indicate individual mice, center lines mean, whiskers SEM. *, p = 0.019 for Opa1, Ucp1, Pgc1α; p = 0.03 for Cox8b; p = 0.05 for CideA. (b) Electron microscopy (EM) images from SAT and VAT of Wt and Opa1tg mice treated for 5 days with 10 mg/kg CL316,243 (CL), administered i.p. every day (n = 6 independent experiments). Scale bar: 5 µm.

Source data

Extended Data Fig. 4 Analysis of master gene regulators in differentiating Opa1tg preadipocytes.

(a) Heat map of the expression levels of the Opa1-specific downregulated genes subset identified in Fig.4h. Each column corresponds to the indicated sample. The dendrogram clustering on the Y-axis groups genes with similar expression profiles. Gene expression levels are indicated per each row. (b) Upstream regulators for the DEGs dataset in Fig.4h by Upstream Regulator Analysis in IPA. y-axis corresponds to the Log10 (p-Value), and the x-axis displays the z-score activation values. (c) Expression fold change of the indicated genes in Opa1tg pre-adipocytes transduced with shRNAs against Hif1α (shHif1α) or an untargeted control sequence (shUT) and differentiated into brite adipocytes (n = 10 independent experiments). Dots indicate individual experiments, center lines the mean, whiskers SEM. *, p = 0.001 for Hif1α and 0.003 for Ucp1 in a two-tailed Mann-Whitney U test. (d) Selected DEGs identified in Fig. 4i were used to identify functional relationships among selected genes by IPA. (e) Kdm3a expression fold change (brite vs. preadipocytes). Pre-adipocytes of the indicated genotype transduced with the indicated shRNA were differentiated into brite adipocytes. N = 3 independent experiments from 6 pooled Opa1tg or Wt mice/experiment. Dots indicate individual experiments, box represents mean ± SEM, whiskers the 10th-90th percentile.; *p = 0.04, **p = 0.01 in two-tailed sample t-test.

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Extended Data Fig. 5 Opa1 controls fumarate levels in vivo.

(a) Average+SEM of metabolite levels in mature adipocytes from SAT of Wt and Opa1tg mice (n = 3). Metabolites with a significantly different mean concentration in Opa1tg vs Wt adipocytes are highlighted in the inset. * p = 0.049 in a Kruskal-Wallis ANOVA test. (b) Fumarate levels in 40 mg of SAT isolated from Wt (n = 4) and Opa1tg (n = 6) mice. Box represents mean ± SEM, whiskers the 10th-90th percentile. **p = 0.014 in a two-tailed Mann-Whitney U test. (c) Metabolites in (a) and (b) were analyzed using the Pathway Analysis module of MetaboAnalyst tool. Red dots show the top significantly relevant pathways.

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Extended Data Fig. 6 Mass isotopomer distribution in flux analysis experiments.

(a) Mass isotopomer distribution (MID) of fumarate and aspartate in Opa1tg and Wt preadipocytes following [13C3]-pyruvate infusion. (b) Mass isotopomer distribution of IMP, Aspartate, Fumarate, Arginine, Glutamate in Opa1tg and Wt preadipocytes following [13C4,15N]-L-aspartic acid infusion. (c) Mass isotopomer distribution of fumarate and aspartate in Opa1tg preadipocytes transduced with control (shScr) shRNA and against Cps1 (shCps1) following [13C3]-pyruvate infusion. (d) Mass isotopomer distribution of IMP, Aspartate, Fumarate, Arginine, Glutamate in Opa1tg preadipocytes transduced with control (shScr) shRNA and against Cps1 (shCps1) following [13C4,15N]-L-aspartic acid infusion. In all graphs, unlabeled (m + 0) and labeled isotopomers are shown. Bars represents the average value from 2 indicated (dots) independent measurements (n = 4 pooled mice/genotype/experiment).

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Extended Data Fig. 7 Adipocyte Opa1 deletion causes lipoatrophy and metabolic dysfunction.

(a) Equal amounts of protein (20 µg) from brown adipose tissue (BAT), subcutaneous (SAT) and visceral (VAT) white adipose tissues, heart and liver lysates from mice of the indicated genotypes were separated by SDS-PAGE and immunoblotted using the indicated antibodies (n = 7/genotype). (b) Representative photographs of SAT and VAT from control and Opa1ΔAT mice (n = 7/genotype). Scale bar: 1 cm. (c) Box-dot plots of SAT and VAT weights from control and Opa1ΔAT mice (n = 7-8). **, p = 0.009 Opa1ΔAT vs. Wt SAT; p = 0.001 Opa1ΔAT vs. Wt VAT in a two-tailed Mann-Whitney U test. (d) Body composition assessed by EchoMRI in Wt (n = 23) and Opa1ΔAT (n = 20) mice. *, p = 0.04 Opa1ΔAT vs. Wt lean mass; ***, p = 2.2×10−8 Opa1ΔAT vs. Wt fat mass in a two-tailed Mann-Whitney U test. (e) Representative Hematoxylin-Eosin staining of SAT and VAT from control and Opa1ΔAT mice (n = 7/genotype). (f) Box-dot plots of adipocytes area in control and Opa1ΔAT mice. (n > 1000 adipocyte/mouse; 4 mice/genotype). ***, p = 1×10−5 in a two-tailed Mann-Whitney U test between groups. (g,h) Box-dot plots of Serum Adiponectin (g) and Insulin (h) levels measured by ELISA in control and Opa1ΔAT mice (n = 5-8). **, p = 0.01 for Adiponectin and p = 0.004 for Insulin in a two-tailed Mann-Whitney U test. (i) Representative photographs of livers from control and Opa1ΔAT mice (n = 7/genotype). Scale bar: 1 cm. (j) Representative Oil Red O staining of livers from control and Opa1ΔAT mice (n = 7/genotype). Red indicates lipid deposits. Inset is magnified 2X. Bar: 20 µm. (k) Average±SEM of blood glucose levels following an i.p. glucose tolerance test (GTT) performed on control and Opa1ΔAT mice (n = 5-6). **, p < 0.01; *, p < 0.05 in a two-tailed Mann-Whitney U test. (l) Average±SEM of blood glucose levels following an i.p. insulin tolerance test (ITT) performed on control and Opa1ΔAT mice. **, p < 0.01 in a two-tailed Mann-Whitney U test. In panels c, d, g, h dots represent the individual measurements, boxes mean ± SEM, whiskers the 10th-90th percentile.

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Extended Data Fig. 8 Adipocyte Opa1 deletion impairs BAT thermogenic activity.

(a) Top: Gross morphology of representative BAT from Wt and Opa1ΔATmice. Scale bar: 1 cm. Bottom: representative Hematoxylin-Eosin staining of BAT mice of the indicated genotype. Scale bar: 50 µm. (b) Representative fluorescence images of Wt and Opa1ΔAT BAT sections immunostained for UCP1 (red) and with DAPI (blue) (n = 3/genotype). Scale bar: 50 µm. (c) Box-dot plots of UCP1 staining intensity in BAT sections of Wt and Opa1ΔAT mice (n = 27 sections over 3 mice/genotype). Dots represent individual sections, boxes SEM, center line mean, whiskers the 10th-90th percentile. ***, p = 2.6×10−11 in a two-tailed Mann-Whitney U test. (d) Fold change (Opa1ΔAT vs. Wt) of the indicated genes in Wt and Opa1ΔAT BAT (n = 3 mice/genotype). Centre line: mean; whiskers: SEM. *, p = 0.049 for Opa1, Pparγ2, CideA, Tfam, Pgc1α and p = 0.043 for Ucp1 in a Kruskal-Wallis ANOVA test. (e) Representative pseudocolored dorsal view images of IR thermography of conscious male Wt and Opa1ΔATmice. Images were collected at room temperature (RT, 22–23 °C) and 1 h after cold exposure (4 °C). (f) Average±SEM of body skin temperature measured at the indicated times in experiments as in (e) (n = 4 mice/genotype). *, p < 0.05 in a two-tailed Mann-Whitney U test.

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Extended Data Fig. 9 Adipocyte Opa1 deletion impairs preadipocytes browning by reduction in fumarate levels.

(a) Equal amounts of protein (20 µg) from SAT isolated from littermates of the indicated genotype treated where indicated with the beta-3 adrenergic agonist CL-316,243 (CL; 10 mg/kg) for 5 days were separated by SDS-PAGE and immunoblotted using the indicated antibodies. TUB: tubulin. Each lane corresponds to an individual mouse. (b) Electron microscopy (EM) images from SAT of control and Opa1ΔAT mice treated for 5 days with ip injection of 10 mg/kg of CL316,243 (CL) every day. Scale bar: 5 µm. (c) Volcano plot of a metabolomic analysis of SAT from Wt and Opa1ΔAT mice (n = 4). The red dots indicate significantly different metabolites. (d) Metabolites in (c) were analyzed using the Pathway Analysis module of MetaboAnalyst tool. Red dots show the top significantly relevant pathways. (e) Box-dot plots of Opa1 and Ucp1 expression fold change (brite vs. preadipocytes) in Wt and Opa1ΔAT SAT pre-adipocytes differentiated into brite adipocytes. Data are normalized for β-actin gene expression, calculated by ΔΔCT (n = 7). ***, p = 7.9×10−4 for Opa1 and p = 4.1×10−4 for Ucp1 in a two-tailed Mann-Whitney U test. (f) Heat map of hierarchical clustering by Pearson Correlation of metabolomics analysis of brite-differentiated white preadipocytes infected with AdCre or AdGFP as control. Each column represents one independent experiment. (g) Dot plots of Ucp1 relative expression in pre-adipocytes isolated from Opa1flxflx mice and differentiated into brite adipocytes after adenovirus-mediated infection with Cre-GFP or GFP as control. Data are expression fold change normalized for β-actin gene expression, calculated by ΔΔCT (n = 3). *, p < 0.05 Kruskal-Wallis ANOVA test (p = 0.049). In e,g dots represent biologically independent experiments, I-shaped boxes mean ± SEM, whiskers the 10th-90th percentile.

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Extended Data Fig. 10 KDM3a and CPS1 are reduced in WAT of obese individuals.

(a,b) Box-dot plots of quantitative PCR analysis of KDM3A (a) and CPS1 (b) transcripts from abdominal wall fat pad biopsies of 5 lean subjects, 15-17 obese normoglycemic subjects (Obese), and 17-19 obese diabetic patients (Obese-T2DM). Data are normalized for the expression of S18. *, p = 0.01 (Obese vs. Lean) and p = 0.03 (Obese-T2DM vs. Lean) for KDM3A and p = 0.02 for CPS1 in a one way ANAOVA test. (c,d) Pearson correlation analysis of OPA1 with KDM3A (c) and CPS1 expression (d) in lean and individuals with obesity as in (a,b).

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Supplementary information

Reporting Summary

Supplementary Data 1

Lists of differentially expressed genes (FDR-corrected P value threshold is 0.05, FC threshold is |2|) obtained from the comparison of white preadipocytes induced to brite differentiation and white differentiated adipocytes. Pre-adipocytes were isolated from Opa1tg and control mice (n = 4). Sequenced reads, previously trimmed by quality, were aligned to the mouse reference genome (version GRCm38). Differential gene expression analyses were carried out with the CLC Genomics Workbench v.11.

Supplementary Data 2

List of differentially expressed genes (FDR-corrected P value threshold is 0.05, FC threshold is |1.5|) obtained from the comparison of white preadipocytes isolated from Opa1tg and control mice (n = 4). Sequenced reads, previously trimmed by quality, were aligned to the mouse reference genome (version GRCm38). Differential gene expression analyses were carried out with the CLC Genomics Workbench v.11.

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Supplementary Tables 1–3.

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Bean, C., Audano, M., Varanita, T. et al. The mitochondrial protein Opa1 promotes adipocyte browning that is dependent on urea cycle metabolites. Nat Metab 3, 1633–1647 (2021). https://doi.org/10.1038/s42255-021-00497-2

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