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The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1

Nature volume 524, pages 243–246 (2015)Cite this article

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Abstract

Abnormal accumulation of triglycerides in the liver, caused in part by increased de novo lipogenesis, results in non-alcoholic fatty liver disease and insulin resistance1,2. Sterol regulatory element-binding protein 1 (SREBP1), an important transcriptional regulator of lipogenesis, is synthesized as an inactive precursor that binds to the endoplasmic reticulum (ER). In response to insulin signalling, SREBP1 is transported from the ER to the Golgi in a COPII-dependent manner, processed by proteases in the Golgi, and then shuttled to the nucleus to induce lipogenic gene expression3,4,5; however, the mechanisms underlying enhanced SREBP1 activity in insulin-resistant obesity and diabetes remain unclear. Here we show in mice that CREB regulated transcription coactivator 2 (CRTC2)6 functions as a mediator of mTOR7 signalling to modulate COPII-dependent SREBP1 processing. CRTC2 competes with Sec23A, a subunit of the COPII complex8, to interact with Sec31A, another COPII subunit, thus disrupting SREBP1 transport. During feeding, mTOR phosphorylates CRTC2 and attenuates its inhibitory effect on COPII-dependent SREBP1 maturation. As hepatic overexpression of an mTOR-defective CRTC2 mutant in obese mice improved the lipogenic program and insulin sensitivity, these results demonstrate how the transcriptional coactivator CRTC2 regulates mTOR-mediated lipid homeostasis in the fed state and in obesity.

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Figure 1: Accumulation of hepatic lipids and mature SREBP1 in Crtc2−/− mice.
Figure 2: CRTC2 attenuates SREBP1 processing by competing with Sec23A for binding to Sec31A.
Figure 3: mTOR phosphorylates CRTC2 and promotes COPII-dependent SREBP1 activation.
Figure 4: Dysregulated SREBP1 signalling in obese mice is improved by overexpression of mTOR-defective CRTC2.

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Acknowledgements

We thank M. Montminy, W. E. Balch, L. Yu, S. Fu, P. Li, J. Wu and D. Li for discussion, and X. Liu and P. Li for technical help with lipogenesis analyses and clamp studies, respectively. This work was supported by grants from 1000 Talents Program for Young Scholars and the National Science Foundation of China (31322027, 31321003 and 31471127) to Y.W.

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Author notes

  1. Jinbo Han and Erwei Li: These authors contributed equally to this work.

Authors and Affiliations

  1. MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Jinbo Han, Erwei Li, Liqun Chen, Yuanyuan Zhang, Fangchao Wei & Yiguo Wang

  2. Proteomics Facility, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Jieyuan Liu & Haiteng Deng

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  1. Jinbo Han
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Contributions

J.H., E.L. and Y.W. designed the study and analysed the data. J.H., E.L., L.C., Y.Z., F.W. and Y.W. performed the experiments. J.L. and H.D. carried out mass spectrometry analysis. J.H. and Y.W. wrote the paper. All authors reviewed and commented on the manuscript.

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Correspondence to Yiguo Wang.

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Extended data figures and tables

Extended Data Figure 1 Profiling of cholesterol, gene expression, protein and circulating insulin levels in Crtc2+/+ and Crtc2−/− mice.

a–d, Hepatic cholesterol levels (a), qPCR results for expression of genes involved in lipogenic regulation, lipid transport, fatty acid oxidation and triglyceride synthesis (b), plasma insulin level (c), and immunoblots showing hepatic amounts of full-length, inactive SREBP2 (flSREBP2) and cleaved, active SREBP2 (nSREBP2), phospho-CREB (pCREB), CREB, and subunits of the COPII complex (Sec12, Sar1, Sec23A, Sec24A, Sec13 and Sec31A) (d) in Crtc2+/+ and Crtc2−/− mice fed with a regular diet (RD) or a high-fat diet (HFD) for 18 weeks. Data are shown as mean ± s.e.m. *P < 0.01, n = 10.

Extended Data Figure 2 Validation of Srebp1 knockdown in mice.

a–c, Hepatic triglycerides (TGs) (a), immunoblots (b) and qPCR results for lipogenic gene expression (c) showing the effect of Srebp1 RNAi in liver extracts of fed mice. Data are shown as mean ± s.e.m. *P < 0.01, n = 10. US, unspecific.

Extended Data Figure 3 Effect of CRTC2 and its mutants on gluconeogenic and lipogenic gene expression.

a, Cellular localization of CRTC2 and its mutants CRTC2(ΔTAD) (amino acids 1–630) and CRTC2(ΔTAD/AA) (amino acids 1–630 with double alanine mutations at Ser171 and Ser275) in mouse primary hepatocytes. FSK, forskolin. Scale bar, 10 μm. b, Effect of CRTC2 and its mutants on Cre-luc activity in mouse primary hepatocytes treated with or without 10 μM FSK for 4 h. Data are shown as mean ± s.e.m. *P < 0.01, n = 6. c, d, Effect of CRTC2 and its mutants on lipogenic gene (Fasn, Scd1, Acaca) expression (c) and plasma insulin level (d) in fed mice. Data are shown as mean ± s.e.m. *P < 0.01, n = 8. NS, no significant statistical difference.

Extended Data Figure 4 Association of CRTC2 with Sec31A.

a, Immunostaining showing relative co-localization of CRTC2 with an endoplasmic reticulum (ER) marker (KDEL) in CRL-2189 cells. Scale bar, 10 μm. b, c, Strategy to purify CRTC2-interacting proteins (b), and the peptides identified from Sec31A and Sec13 (c) by MS analysis of immunoprecipitates prepared with anti-CRTC2 antibody from CRL-2189 ER fractions. d, Co-immunoprecipitation assay showing amounts of Flag-tagged Sec13 or Sec31A recovered from immunoprecipitates of endogenous CRTC2 in HEK293T cells. e, Deletion analysis of regions in CRTC2 required for the CRTC2–Sec31A interaction. Interaction-competent CRTC2 peptides are indicated by (+) in each schematic. f, g, Cellular localization of the tryptophan-to-alanine mutant of CRTC2 (W143A) and its effect on Cre-luc activity in HEK293T cells. Scale bars, 10 μm. Data are shown as mean ± s.e.m. *P < 0.01, n = 6. NS, no significant statistical difference. h, Co-immunoprecipitation assay showing amounts of Flag-tagged CRTC2 and YFP-tagged Sec23A recovered from immunoprecipitates of HA-tagged Sec31A in HEK293T cells.

Extended Data Figure 5 Modulation of COPII-dependent SREBP1 activity by CRTC2.

a, Immunostaining showing the effect of Crtc2 RNAi on the cellular localization of Sec31A. Scale bars, 10 μm. b, c, Effect of wild-type (WT) and Sec31A-interaction-defective (W143A) CRTC2 on lipogenic gene expression (b) and plasma insulin level (c) in fed mice. Data are shown as mean ± s.e.m. *P < 0.01, n = 8. NS, no significant statistical difference.

Extended Data Figure 6 Characterization of CRTC2 phosphorylation site(s) by mTOR.

a, Immunoblots showing co-immunoprecipitation of CRTC2 and Sec23A with Sec31A in mouse primary hepatocytes in response to insulin and/or torin1 treatment. Mouse primary hepatocytes were incubated with 250 nM torin1 or control vehicle for 1 h before 30 min insulin (100 nM) stimulation. Phospho-S6K (pS6K), total S6K, phospho-AKT (pAKT), total AKT and phospho-CRTC2 (Ser136) levels are also indicated. b, Immunoblots showing co-immunoprecipitation of CRTC2 and Sec23A with Sec31A in mouse primary hepatocytes in response to amino acids and/or torin1 treatment. Mouse primary hepatocytes incubated with amino-acid-free MEM for 3 h were exposed to 250 nM torin1 or control vehicle for another 1 h, then treated with amino acids for 30 min. c, Phospho-peptides of Flag-tagged CRTC2 identified by MS analysis of immunoprecipitates prepared with anti-Flag from HEK293T cells treated with 100 nM rapamycin for 1 h (Rap+) or not (Rap−). Serine 136 was phosphorylated (Yes) in the absence of Rap treatment (Rap−) and dephosphorylated (No) in the presence of Rap (Rap+). d, Co-immunoprecipitation assay showing the association between Flag-tagged CRTC2 and Myc-tagged mTOR in HEK293T cells. e, Effect of the mTOR inhibitors Rap and torin1 on CRTC2 phosphorylation. Mouse primary hepatocytes were pretreated with vehicle (Veh), 100 nM Rap, or 250 nM torin1 for 1 h before 100 nM insulin stimulation for 30 min. f, g, Cellular localization of the phosphorylation-defective CRTC2 mutant (S136A) (f) and its effect on Cre-luc activity (g) in mouse primary hepatocytes. Scale bars, 10 μm. Data are shown as mean ± s.e.m. *P < 0.01, n = 6. NS, no significant statistical difference.

Extended Data Figure 7 Effect of CRTC2(S136A) on SREBP1 maturation, lipin1 localization and circulating insulin level.

a, b, Effect of wild-type or CRTC2(S136A) on lipogenic gene expression in liver (a) and plasma insulin level (b) of fasted (3 h) and refed (1 h after 3 h fasting) mice. c–e, Effect of CRTC2(S136A) and torin1 treatment on SREBP1 maturation (c), hepatic triglycerides (d) and lipin1 localization in mouse primary hepatocytes (e). Torin1 (20 mg kg−1) was intraperitoneally injected 6 h before livers were harvested. For lipin1 localization, mouse primary hepatocytes were treated with vehicle (Torin1−) or 250 nM torin1 (Torin1+) for 4 h. Scale bars, 10 μm. Data are shown as mean ± s.e.m. *P < 0.01, n = 8. NS, no significant statistical difference.

Extended Data Figure 8 Enhanced SREBP1 activation, triglyceride levels and branched-chain amino acid levels in obese mice.

a–c, Immunoblots showing relative amounts and/or phosphorylation status of SREBP1, SREBP2, SCAP, mTOR, S6K, CRTC2, AKT and COPII subunits in fed lean and db/db mice (a), ob/ob mice (b), and relative amounts of SREBP2, SCAP and COPII subunits in HFD-fed mice (c). d–g, Hepatic triglyceride amounts and branched-chain amino acid (BCAA) levels in liver extracts from lean, db/db, ob/ob and HFD-fed mice in the fed state. Data are shown as mean ± s.e.m. *P < 0.01, n = 10.

Extended Data Figure 9 Improved insulin sensitivity in HFD-fed mice in the presence of CRTC2(ΔTAD/S136A).

a–k, Effect of the mTOR-defective mutant CRTC2(ΔTAD/S136A) on metabolic parameters (a), including body weight, relative fat mass, food intake, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, plasma cholesterol, plasma triglycerides, plasma insulin and blood glucose; energy expenditure (b); lipogenic gene expression (c); glucose tolerance (d); insulin tolerance (e); pyruvate tolerance (f); hepatic glucose production (HGP; g); insulin-stimulated glucose disposal rate (IS-DGR; h); percentage of free fatty acid (FFA) suppression (i); pAKT level in skeletal muscle (j); and pAKT level in epididymal white adipose tissue (k) from mice fed on a HFD for 18 weeks. Data are shown as mean ± s.e.m. *P < 0.01, **P < 0.05, n = 8 (a–f), n = 6 (g–i).

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Han, J., Li, E., Chen, L. et al. The CREB coactivator CRTC2 controls hepatic lipid metabolism by regulating SREBP1. Nature 524, 243–246 (2015). https://doi.org/10.1038/nature14557

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