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. 2018 May 15;9(1):1916.
doi: 10.1038/s41467-018-04363-w.

Tip60-mediated lipin 1 acetylation and ER translocation determine triacylglycerol synthesis rate

Terytty Yang Li  1 , Lintao Song  1 , Yu Sun  1 , Jingyi Li  1 , Cong Yi  2 , Sin Man Lam  3 , Dijin Xu  4 , Linkang Zhou  4 , Xiaotong Li  1 , Ying Yang  1 , Chen-Song Zhang  1 , Changchuan Xie  1 , Xi Huang  1 , Guanghou Shui  3 , Shu-Yong Lin  1 , Karen Reue  5 , Sheng-Cai Lin  6
Affiliations

Tip60-mediated lipin 1 acetylation and ER translocation determine triacylglycerol synthesis rate

Terytty Yang Li et al. Nat Commun. .

Abstract

Obesity is characterized by excessive fatty acid conversion to triacylglycerols (TAGs) in adipose tissues. However, how signaling networks sense fatty acids and connect to the stimulation of lipid synthesis remains elusive. Here, we show that homozygous knock-in mice carrying a point mutation at the Ser86 phosphorylation site of acetyltransferase Tip60 (Tip60 SA/SA ) display remarkably reduced body fat mass, and Tip60 SA/SA females fail to nurture pups to adulthood due to severely reduced milk TAGs. Mechanistically, fatty acids stimulate Tip60-dependent acetylation and endoplasmic reticulum translocation of phosphatidic acid phosphatase lipin 1 to generate diacylglycerol for TAG synthesis, which is repressed by deacetylase Sirt1. Inhibition of Tip60 activity strongly blocks fatty acid-induced TAG synthesis while Sirt1 suppression leads to increased adiposity. Genetic analysis of loss-of-function mutants in Saccharomyces cerevisiae reveals a requirement of ESA1, yeast ortholog of Tip60, in TAG accumulation. These findings uncover a conserved mechanism linking fatty acid sensing to fat synthesis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Tip60SA/SA mice demonstrate lean phenotypes and are resistant to obesity-associated metabolic disorders. a Schematic diagram of the strategy for Tip60S86A knock-in mice. b Body weights of wild-type (WT), Tip60+/SA and Tip60SA/SA mice fed with a normal chow diet (ND) (male, n = 20 per group) or high-fat diet (HFD, starting at 6 weeks of age) (male, n = 24 per group). c Weight gains of ND or HFD mice from b. d Representative photographs of WT and Tip60SA/SA HFD mice at 18 weeks of age. e Weights of different tissues from 18-week-old WT and Tip60SA/SA ND or HFD mice (male, n = 16 per group). iWAT inguinal white adipose tissue, eWAT epididymal WAT, BAT brown adipose tissue. f Percentages of the weights of different tissues as compared to their corresponding body weight. g Representative sections of iWAT, eWAT, BAT and liver from HFD 18-week-old male mice. Scale bar, 100 μm. h–k Levels of liver triacylglycerol (TAG) (h), plasma TAG (i), plasma glucose (j) and plasma insulin (k) in WT and Tip60SA/SA ND (male, n = 8 per group) or HFD (male, n = 12 per group) mice fasted for 6 h. l, m Glucose tolerance test (GTT) (male, n = 12 per group) (l) and insulin tolerance test (ITT) (male, n = 10 per group) (m) in WT and Tip60SA/SA ND mice. Inset graphs show area under curve (AUC). n, o GTT (male, n = 10 per group) (n) and ITT (male, n = 12 per group) (o) in WT and Tip60SA/SA HFD mice. p The adjusted means of AUCs of o analyzed by ANCOVA, using body weight as the covariate. q Energy expenditure (EE) of WT and Tip60SA/SA mice fed HFD for 1 week (male, n = 6 per group), before the mice diverge too much in body weight. For EE, data were collected for 3 consecutive days, expressed as adjusted means based on body weight to the power 0.75. EE is estimated with Weir equation (16.3 × VO2 + 4.57 × VCO2). Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in b, c, e, f, h–l and q or by two-tailed unpaired Student’s t-test in AUC graphs of l–o. *P < 0.05; **P < 0.01
Fig. 2
Fig. 2
Tip60 plays an essential role in fatty acid-induced TAG synthesis. a Survival rates of Tip60SA/SA litters nursed by WT or Tip60SA/SA females (n = 8 litters for each group), and WT litters nursed by WT or Tip60SA/SA females (n = 7 litters for each group). b Representative BODIPY staining (n = 3 experiments) of milk fat globules (green) from the mammary glands of early lactating (day 2 postpartum) WT and Tip60SA/SA females. Scale bar, 50 μm. c Level of TAG in milk collected from WT and Tip60SA/SA females (day 2 postpartum, n = 5 per group). d Representative images of mammary glands from WT and Tip60SA/SA females at day 2 postpartum (n = 3 experiments), visualized by H&E staining. The WT mouse (left) has numerous fat globules in the apical regions of mammary epithelial cells and in the ductal lumens, but were rare in the Tip60SA/SA mouse (right). Scale bars, 50 μm. e Lentivirus-mediated knockdown of Tip60 in 3T3-L1 adipocytes. TCL total cell lysate. f TAG and phosphatidylcholine (PC) synthesis rates of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNAs targeting Tip60 with 3H-labeled oleic acid (OA) treatment (n = 4 experiments). g TAG and PC synthesis rates of 3T3-L1 adipocytes treated with DMSO (ctrl) or Tip60 inhibitor MG149 (n = 4 experiments). h Rates of diacylglycerol (DAG) and TAG synthesis from 3H-OA in eWAT explants isolated from 6-week-old WT and Tip60SA/SA mice (n = 4 individuals per group). i Fatty acid oxidation rates of control (ctrl) or Tip60 knockdown or MG149-treated 3T3-L1 adipocytes or adipocytes derived from WT or Tip60SA/SA MEFs (day 8) (n = 4 experiments). Error bars denote SEM. Statistical analysis was performed by two-tailed unpaired Student’s t-test in c, g, h and middle and right bar graph of i, by one-way ANONA followed by Tukey in f and left bar graph of i. *P < 0.05; **P < 0.01; N.S. not significant. Uncropped blots can be found in Supplementary Fig. 6
Fig. 3
Fig. 3
Tip60 regulates lipid biosynthesis by directly interacting with and acetylating lipin 1. a The TAG biosynthesis pathway in mammalian cells. b Association of Flag-tagged enzymes in TAG synthesis pathway with Myc-tagged Tip60. Protein extracts of HEK293T after transfection were immunoprecipitated with antibody to Flag and immunoblotted as indicated. c Lipin 1 was acetylated by Tip60 in vitro. Bacterially expressed His-lipin 1 was incubated with His-Tip60 in the presence or absence of acetyl-CoA (Ac-CoA) and immunoblotted as indicated. d Tip60-dependent acetylation of lipin 1. 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNAs targeting Tip60 were treated with or without OA for 3 h. Acetylated proteins were immunoprecipitated with antibody to acetylated lysine, followed by immunoblotting. e Tip60 inhibitor MG149 abrogates the acetylation of lipin1 mediated by Tip60. Flag-tagged lipin 1 co-transfected with or without Tip60 in HEK293T cells treated or untreated with MG149 for 3 h were immunoprecipitated by antibody to Flag and immunoblotted as indicated. f Reduced acetyltransferase activity of Tip60-S86A towards lipin 1. Flag-tagged Tip60 or its S86A mutant expressed in HEK293 cells was immunoprecipitated with antibody to Flag. In vitro acetylation assays were performed using purified His-lipin 1 as substrate (n = 4 experiments). g Tip60-dependent acetylation of lipin 1 in vivo. eWAT from WT or Tip60SA/SA ND or HFD mice was immunoprecipitated by using antibody to acetylated lysine or Tip60, followed by immunoblotting. Each lane represents a different individual (n = 3 experiments). h Biosynthesis of phospholipids and neutral lipids from glycerol-3-phosphate (G3P) in mammalian cells. i Total levels of PA (left) and individual PA species (right) normalized to cellular protein in eWAT from 6-week-old WT and Tip60SA/SA mice. The molecular species of PA are indicated as total number of carbons/number of double bonds (n = 3 individuals for each group). j Total levels of DAG normalized to cellular protein in eWAT from 6-week-old WT and Tip60SA/SA mice (n = 3 individuals for each group). k, l Identification of acetylation sites on lipin 1 targeted by Tip60. WT-lipin 1 or its lysine-to-arginine (KR) mutants were co-expressed with or without Tip60 in HEK293T cells, immunoprecipitated by antibody to Flag and immunoblotted as indicated (k). His-tagged WT-lipin 1 or its KR mutants were incubated with Tip60 in the presence or absence of Ac-CoA and immunoblotted as indicated (l). m Sequence alignment of the residues flanking Lys425 across different species. Arrowheads point to the Lys425 residue corresponding to human lipin 1. Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in g or by two-tailed unpaired Student’s t-test in f, i and j. *P < 0.05; ** P < 0.01. Uncropped blots can be found in Supplementary Fig. 6
Fig. 4
Fig. 4
Sirt1 deacetylates lipin 1 and represses the synthesis of DAG and TAG. a Sirtuins inhibitor nicotinamide (NAM) increases the amount of acetylated lipin 1. Lysates of HEK293T cells transfected with HA-lipin 1 and/or Tip60 treated with trichostatin A (TSA) (a class I and II HDAC inhibitor) or NAM for 6 h were immunoprecipitated with antibody to HA, followed by immunoblotting. b Deacetylation of lipin 1 by co-expression with Sirtuins. Lysates of HEK293T cells transfected with HA-lipin 1, Tip60 and Flag-tagged Sirt1–7 as indicated were immunoprecipitated with antibody to HA, followed by immunoblotting. c In vitro deacetylation of lipin 1 by Sirt1. Acetylated-lipin1 were incubated with Flag-tagged WT-Sirt1 or the inactive H363Y mutant (expressed and immunoprecipitated from HEK293T cells) in the presence or absence of NAD+ for 1 h, followed by immunoblotting. d Knockdown of Sirt1 increases the amount of acetylated lipin 1. 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNAs targeting Sirt1 were treated with or without OA for 3 h. Acetylated proteins were immunoprecipitated with antibody to acetylated lysine. e Increased lipin 1 acetylation by Sirt1 inhibitor EX527. 3T3-L1 adipocytes were treated with or without NAM, EX527 or OA for 3 h and analyzed as in d. f, g Knockdown of Sirt1 by shRNAs (f) (n = 4 experiments) or inhibition of Sirt1 activity by EX527 (g) (n = 4 experiments) promotes DAG and TAG synthesis in 3T3-L1 adipocytes. h TAG, DAG and PC synthesis rates of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNA targeting Lpin1 with or without EX527 treatment (n = 4 experiments). i TAG/DAG synthesis rates of WT or Tip60SA/SA MEF-derived adipocytes expressing control shRNA (ctrl) or shRNA targeting Lpin1. Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in f, h, i or by two-tailed unpaired Student’s t-test in g. *P < 0.05, **P < 0.01. Uncropped blots can be found in Supplementary Fig. 6
Fig. 5
Fig. 5
Tip60-mediated lipin 1 acetylation leads to lipin 1 translocation from cytosol to ER membranes. a Attenuated translocation of lipin 1 from cytosol to ER membranes after Tip60 depletion. Representative images (n = 3 replicate experiments) of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNA targeting Tip60 (Tip60 KD) treated with BSA or OA for 2 h. Calnexin is an ER/microsomal marker. Mander’s overlap coefficients between lipin 1 and Calnexin were graphed (n = 30 for each group). Scale bar, 10 μm. b Depletion of Tip60 impairs lipin 1 ER localization. Western blotting of proteins in subcellular fractions of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNAs targeting Tip60 treated with or without OA for 3 h. Calnexin, microsomal (Mic) marker; β-tubulin, cytosol (Cyt) marker. c Impairment of lipin 1 ER localization in adipocytes derived from Tip60SA/SA MEFs. Adipocytes (differentiation day 8) derived from WT or Tip60SA/SA MEFs were treated with OA for 1–3 h and analyzed by fractionation, followed by western blotting. d Depletion of Sirt1 promotes lipin 1 ER localization. e Knockdown of Lpin1 in 3T3-L1 adipocytes. Western blotting of proteins of 3T3-L1 adipocytes expressing control shRNA (ctrl) or shRNA targeting Lpin1. f, g Acetylation-dependent ER localization of lipin 1 and DAG/TAG synthesis. Western blotting of proteins (f) or DAG, TAG and PC synthesis rates (g) of 3T3-L1 adipocytes expressing shRNA targeting Lpin1 reconstituted with vector, WT-lipin 1 or 2KR-lipin 1 after OA treatment (n = 4 experiments). Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in a, g. **P < 0.01; N.S. not significant. Uncropped blots can be found in Supplementary Fig. 6
Fig. 6
Fig. 6
Lipin 1 acetylation senses fatty acid abundance by its reciprocal association with Sirt1 and Tip60. a Lipin 1 dephosphorylation promotes its acetylation by Tip60. Flag-tagged WT-lipin 1 or 2KR-lipin1 or 16SA-lipin 1 was co-expressed with or without Tip60 in HEK293 cells, immunoprecipitated by antibody to Flag, followed by immunoblotting. b Dephosphorylation of lipin 1 increases its acetylation by Tip60. HEK293 cells transfected with vector, WT-lipin 1 or 2KR-lipin 1 or 16SA-lipin 1 (phosphorylation-defective) were treated with or without OA for 3 h, immunoprecipitated with antibody to Flag and immunoblotted as indicated. c Dephosphorylation of lipin 1 attenuates its association with Sirt1. HEK293T cells were transfected with Flag-lipin 1 and Myc-Sirt1, immunoprecipitated with antibody to Flag and immunoblotted as indicated. d Tip60 and Sirt1 compete with each other in the interaction with lipin 1. HEK293T cells were transfected with Flag-lipin 1, HA-Tip60 and varied amounts of Myc-Sirt1, immunoprecipitated with antibody to Flag and immunoblotted as indicated. e Interaction of endogenous Tip60 and Sirt1 with lipin 1. Lysates of 3T3-L1 adipocytes treated with or without OA for 3 h were immunoprecipitated by antibody to lipin 1 or Tip60 and analyzed by immunoblotting. f TAG/DAG production rates of WT or Tip60SA/SA adipocytes expressing shRNA targeting Lpin1 with reintroduction of WT-lipin 1, acetylation-defective (2KR) or acetylated mimetic (2KQ) mutant of lipin 1 (n = 4 experiments). Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in f. *P < 0.05; N.S. not significant. Uncropped blots can be found in Supplementary Fig. 6
Fig. 7
Fig. 7
Essential roles of Tip60 and Tip60-mediated lipin 1 acetylation in TAG synthesis in S. cerevisiae. a Representative images (n = 3 replicate experiments) of BODIPY staining of WT (BY4741) and ESA1 (yeast Tip60) loss-of-function mutant (esa1-1, an auxin-sensitive mutant strain of ESA1) cells grown in synthetic complete (SC) medium. DIC differential interference contrast microscopy images. Scale bar, 2 μm. b Identification of ESA1 as essential gene for TAG accumulation in yeast. TAG was extracted from cells grown in SC medium, separated by thin-layer chromatography (TLC) and quantified (n = 4 experiments). c Decreased lipid droplet (LD) formation in esa1-1 mutant strain. Representative images (n = 3 replicate experiments) of cells expressing chromosomally GFP-tagged fatty acid activation protein 4 (Faa4) (a LD marker protein) were grown in SC medium and analyzed (n = 100 cells quantified for each group). Scale bar, 2 μm. d TAG synthesis pathway in S. cerevisiae. e ESA1 specifically acetylates PAH1 but not other enzymes in the TAG synthesis pathway. Cell lysates of HEK293T cells transfected as indicated were immunoprecipitated with antibody to Flag, followed by immunoblotting. f Identification of acetylation sites on PAH1 (yeast lipin 1) targeted by ESA1. Flag-tagged WT-PAH1 or its KR mutants were co-expressed with or without ESA1 in HEK293T cells, immunoprecipitated by antibody to Flag and immunoblotted as indicated. g Protein expression levels of pah1Δ cells reconstituted with Myc-tagged WT-PAH1 or K496/801R-PAH1. Equal amounts of cell lysates from yeast cells as indicated were analyzed by immunoblotting. h Representative images (n = 3 replicate experiments) of BODIPY staining of WT (BY4741) and PAH1 knockout (pah1∆) cells reconstituted with vector, WT-PAH1 or K496/801R-PAH1. Scale bar, 2 μm. i Representative TLC result showing the TAG levels of WT or PAH1 knockout (pah1Δ) cells reconstituted with vector, WT-PAH1 or K496/801R-PAH1. j Model of Tip60/Sirt1-regulated lipin 1 acetylation and TAG biosynthesis. Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey in b or by two-tailed unpaired Student’s t-test in c. **P < 0.01. Uncropped blots can be found in Supplementary Fig. 6
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