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Dietary control of peripheral adipose storage capacity through membrane lipid remodelling

Abstract

Genetic and dietary cues are known drivers of obesity, yet how they converge at the molecular level is incompletely understood. Here we show that PPARγ supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, an endoplasmic reticulum (ER)-resident O-acyltransferase that selectively enriches diet-derived omega-6 polyunsaturated fatty acids (n-6 PUFAs) in the membrane lipidome. In mice fed a high-fat diet, lowering membrane n-6 PUFA levels through genetic or dietary interventions results in aberrant adipose triglyceride (TG) turnover, ectopic fat deposition and insulin resistance. Additionally, we detail a non-canonical adaptive response in ‘lipodystrophic’ Lpcat3–/– adipose tissues that engages a futile lipid cycle to increase metabolic rate and offset lipid overflow to ectopic sites. Live-cell imaging, lipidomics and molecular dynamics simulations reveal that adipocyte LPCAT3 activity enriches n-6 arachidonate in the phosphatidylethanolamine (PE)-dense ER–lipid droplet interface. Functionally, this localized PE remodelling optimizes TG storage by driving the formation of large droplets that exhibit greater resistance to adipose TG lipase activity. These findings highlight the PPARγ–LPCAT3 axis as a mechanistic link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

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Fig. 1: Dietary n-6 PUFAs enhance WAT plasticity through the PPARγ–LPCAT3 axis.
Fig. 2: Membrane n-6 PUFA deficiency in WAT predisposes mice to partial lipodystrophy.
Fig. 3: Aberrant TG turnover and adaptive TG–FA cycling in Lpcat3AKO iWAT during DIO.
Fig. 4: A distinct molecular signature in obese Lpcat3AKO iWAT tracks with TG–FA cycling.
Fig. 5: Adaptive TG–FA cycling in iWAT protects Lpcat3AKO mice against DIO.
Fig. 6: LPCAT3 catalytic activity drives efficient TG storage in adipocytes.
Fig. 7: Adipocyte LPCAT3 activity promotes efficient TG storage by converging on LD budding size.

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Data availability

Source data for all figures are provided with the paper. RNA-seq and sNuc-seq datasets generated for this paper are available in the NCBI Gene Expression Omnibus repository under accession code GSE218943. Reads were aligned to the mm10 (GRCm38) mouse reference genome. All source data and input files for MD simulations can be found at Zenodo94. All unique biological materials used are readily available from the authors. Source data are provided with this paper.

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Acknowledgements

This article is dedicated to the memory of T. C. P. M. Kemper, a beloved friend and enduring source of inspiration. We are grateful to J. Sandhu, C. Priest and B. Clifford for technical assistance. We thank all current and former members of the Tontonoz and Tarling–Vallim labs for valuable discussions and for sharing reagents. PET–CT was performed at the Crump Institute Preclinical Imaging Technology Center, with the assistance of S. Xu and M. Tamboline. Oxylipin analysis was performed at the RIKEN Center for Integrative Medical Sciences with the assistance of M. Honda. TMT labelling was performed at the Pasarow Mass Spectrometry laboratory with the assistance of W. Cohn and J. P. Whitelegge. RNA-seq and sNuc-seq were performed at the Technology Center for Genomics & Bioinformatics. This work was supported by a postdoctoral fellowship from the American Diabetes Association (1-19-PDF-039 to M.J.T.); the Japan Society for the Promotion of Science abroad and the Osamu Hayaishi Memorial Scholarship for Study Abroad (to Y.S.); the National Institutes of Health (R01DK129276 to P.T.; HL139725 to S.G.Y.); the Leducq Foundation (19CVD04 to P.T. and S.G.Y.); the Swiss National Science Foundation (grants 310030_219264 to S.V.); the European Research Council under European Union’s Horizon 2020 Research and Innovation Programme (grant agreement no. 803952, to S.V.) and grants of the Swiss National Supercomputing Centre (CSCS) under project ID s1131 and s11876.

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Authors

Contributions

M.J.T., Y.S., S.V. and P.T. conceived the project. M.J.T., Y.S., A.H.B., J.S., L.C., M.C.-G., P.H., K.J.W., M.A., D.A.F. and S.V. were responsible for the methodology. A.H.B. and J.S. contributed equally to the computational analyses. K.J.W., B.S., D.P.P. and D.A.F. performed lipidomic analyses. M.J.T., Y.S., A.H.B., J.S., L.C., K.J.W., M.A., D.P.P., D.A.F. and S.V. conducted formal analyses. M.J.T., Y.S., A.H.B., J.S., L.C., A.F., K.Q., J.P.K., S.D.L., Y.G., X.X., J.G., J.J.M., T.A.W., K.J.W., D.P.P. and M.A. performed the investigation. M.C.-G., P.H., C.P., A.J.L., K.J.W., B.S., A.R., N.M., M.A., B.F.C., S.G.Y., D.A.F., R.Z., S.V. and P.T. provided resources. M.J.T., Y.S., A.H.B., J.S., K.J.W., M.A., S.V. and P.T. curated the data. M.J.T., Y.S. and P.T. wrote the manuscript. S.V. and P.T. supervised the research.

Corresponding author

Correspondence to Peter Tontonoz.

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Extended data

Extended Data Fig. 1 Lpcat3 is an evolutionarily conserved PPARγ target gene.

(a) qPCR analysis of Lpcat1-4 mRNA levels in eWAT and iWAT of 12-week-old NCD-fed wild-type mice (n = 5, 5), and (b) in iWAT from wild-type mice fed low-fat diet (LFD) with or without rosiglitazone (Rosi; 50 mg kg–1) for 14 days (n = 7, 7). (c) Immunoblot analysis of LPCAT3 protein levels in iWAT lysates from (b). Tubulin served as a sample processing control. (d) Immunoblot analysis of LPCAT3 protein levels and known adipogenic markers during the course of 10T1/2 adipocyte differentiation. Calnexin served as a loading control for LPCAT3. (e) Sequence alignment of conserved PPAR and LXR response elements in the Lpcat3 promoter across multiple species. The PAM sequence of sgRNAs targeting Lpcat3-PPRE and LXRE are underlined in red. (f) ChIP-qPCR analysis of PPARγ (or normal rabbit IgG) occupancy at Lpcat3, Plin1, Fabp4, Agpat2, or Aqp7 PPRE elements in wild-type and ΔPPRE 10T1/2 adipocytes (n = 3/group; 100–mm plate per replicate from 3 independent experiments). A region of the 36b4 promoter served as a negative control. (g) PCR analysis of a 700-bp genomic segment flanking the PPRE/LXRE sites in the Lpcat3 promoter region of WT, ΔPPRE, and ΔLXRE clonally-derived cell lines, revealing no major deletions introduced by CRISPR/Cas9 gene editing. Primer anneal sites in the Lpcat3 promoter are shown in orange, indels in green (insertions) or red (deletions). (h) Immunoblot analysis of LPCAT3 protein levels in WT, ΔPPRE, and ΔLXRE 10T1/2 adipocytes differentiated for 4 days. Calnexin served as a loading control for LPCAT3. Data are presented as mean ± SEM. ***P < 0.001 by Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (b); or one-way ANOVA with Tukey’s multiple comparisons test (f).

Source data

Extended Data Fig. 2 Lpcat3AKO mice display no metabolic phenotype under standard laboratory conditions.

(a) Graphical illustration of in vivo Lpcat3AKO strategy. (b) qPCR analysis of Lpcat3 mRNA levels in fat depots and insulin target-tissues of 12-week-old NCD-fed control and Lpcat3AKO mice (n = 4, 4). (c) Lipidomic analysis of PC/PE or TG-FA species in the inguinal adipocyte fraction isolated from of 22-week-old NCD-fed control and Lpcat3AKO mice (n = 5, 5). (d) Body weight (BW) gain curves and composition of 22-week-old NCD-fed control and Lpcat3AKO mice (n = 30, 34). (e) Wet weights of the indicated tissues in NCD-fed control and Lpcat3AKO mice (n = 23, 25). (f) H&E stainings of iBAT and iWAT depots (scale bar, 100 µm). (g) qPCR analysis of iWAT of NCD-fed control and Lpcat3AKO mice (n = 7, 7). (h) 14C-oleate uptake in insulin-target tissues of 18-week-old NCD-fed control and Lpcat3AKO mice (n = 5, 7). Radioactivity (counts per min, CPM) was calculated in the extracted lipids from whole-organs 4 h post-gavage with 14C-Triolein and normalized for g/tissue. (i) Ex vivo lipolysis in dissected iWAT explants from 18-week-old NCD-fed control and Lpcat3AKO mice (n = 4, 4). Secreted glycerol was measured under basal and stimulated (2 µM isoproterenol, ISO) conditions. (j) Ex vivo β-oxidation in crude lysates prepared from iBAT, iWAT, and livers of 18-week-old NCD-fed control and Lpcat3AKO mice (n = 5, 5), assessed by conversion of 14C-palmitic acid to 14CO2. (k) Plasma TGs and cholesterol in 22-week-old NCD-fed control and Lpcat3AKO mice (n = 13, 7). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch’s t-tests with Holm-Sidak’s correction on CLR-transformed values of each lipid class (c); or Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (b,g).

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Extended Data Fig. 3 Lpcat3AKO does not affect adipocyte and whole-body glucose homeostasis.

(a) Immunoblot analysis of phospho-Akt/PKB and rpS6 in iWAT of 12-week-old NCD-fed control and Lpcat3AKO mice after i.p. injection with insulin (1 U kg–1). Total Akt/PKB and rpS6 served as sample processing controls. (b) Coronal, sagittal, and transverse views of 12-week-old NCD-fed control and Lpcat3AKO mice by positron emission tomography–computed tomography (PET–CT) imaging 1 h post-injection with 18F-FDG and insulin (1 U kg–1). (c) 18F-FDG uptake (× 106 pCi g–1 tissue) in the indicated insulin-target tissues (n = 4, 4). (d) glucose tolerance tests (GTT; 1.5 g kg–1) and (e) insulin tolerance tests (ITT; 0.75 U kg–1) were performed on 18-week-old NCD-fed control and Lpcat3AKO mice (n = 10, 9). (f) Respiratory exchange ratios (RER) of 18-week-old NCD-fed control and Lpcat3AKO mice were monitored over a period of 48 h in Oxymax metabolic cages (n = 16, 15). Data are presented as mean ± SEM.

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Extended Data Fig. 4 Metabolic consequences of membrane n-6 PUFA deficiency in WAT during DIO.

(a) GS–MS analysis of dietary fatty acid profiles and presented as n-6/n-3 PUFA ratio, FA species (% of total), or FA class (sfa:mufa:pufa; n = 3, 3, 3). (b) Body weight (BW) gain curves of 18-week-old control and Lpcat3AKO mice fed either HFD (n = 42, 40), HFDsfa (n = 39, 38), or HFDn-3 (n = 40, 37) for 10 weeks. (c) Average daily food intake of singly-housed control and Lpcat3AKO mice fed the indicated HFDs, recorded during the last 5 weeks of HFD-feeding (n = 5–6/group). (d) EchoMRI analysis of lean mass in control and Lpcat3AKO mice fed HFD (n = 42, 40), HFDsfa (n = 39, 38), or HFDn-3 (n = 40, 37) for 10 weeks. (e) PUFA-derived oxylipin species were analysed in the inguinal adipocyte fraction from control and Lpcat3AKO mice fed the indicated HFDs for 10 weeks (n = 4/group). (f-i) Plasma (f) TG, (g) cholesterol, (h) ALT, and (i) AST levels in control and Lpcat3AKO mice fed the indicated HFDs for 10 weeks (n = 10–13/group). (j) VLDL secretion in control and Lpcat3AKO mice fed HFD (n = 5, 5), HFDsfa (n = 6, 6), or HFDn-3 (n = 6, 7) for 6 weeks. Animals were fasted for 12 h before injection with the LPL inhibitor poloxamer-407 (1 g kg–1; i.p.). Plasma TGs were measured in blood collected retro-orbitally at the indicated times post-injection. (k) Ex vivo hepatic β-oxidation rates in control and Lpcat3AKO mice fed the indicated HFDs for 6 weeks, as assessed by the conversion of 14C-palmitic acid (PA) to 14CO2 (n = 3–4/group). Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001 by two-way RM ANOVA with Sidak’s multiple comparisons test (b); ***P < 0.001 vs. the HFD-fed control group; #P < 0.05, vs. HFDsfa; same genotype, by two-way ANOVA with Tukey’s multiple comparisons test (d); or **P < 0.01, ***P < 0.001 by Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (f,h,i).

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Extended Data Fig. 5 Lipolytic dysregulation in Lpcat3AKO mice during DIO.

(a) 14C-oleate uptake in insulin-target tissues from 13-week-old control and Lpcat3AKO mice fed HFD for 5 weeks (n = 7, 8). Radioactivity (counts per min, CPM) was calculated in the extracted lipids from whole-organs 4 h post-gavage with 14C-Triolein and normalized for g/tissue. (b) Ex vivo β-oxidation in crude lysates prepared from iWAT, iBAT, or liver lysates of 13-week-old control and Lpcat3AKO mice fed HFD for 5 weeks, as assessed by the conversion of 14C-palmitic acid (PA) to 14CO2 (n = 4, 4). (c) Ex vivo lipolysis in freshly dissected iWAT explants from 13-week-old control and Lpcat3AKO mice fed HFD for 5 weeks (n = 3, 3). Secreted glycerol was determined under basal and stimulated (2 µM isoproterenol, ISO) lipolytic states. (d) 14C-oleate uptake in insulin-target tissues from 18-week-old control and Lpcat3AKO mice fed HFD for 10 weeks (n = 9, 8). (e,f) 14C-oleate uptake and turnover in (e) iBAT and (f) livers from 18-week-old control and Lpcat3AKO mice fed HFD for 10 weeks. Radioactivity (counts per min, CPM) was calculated in extracted lipids from whole-organs 4 h (n = 9, 8); 4 days (n = 6, 6); and 20 days (n = 7, 7) post-gavage with 14C-Triolein and normalized for g/tissue. (g) UMAP projection of 28,414 sequenced iWAT nuclei or split by genotype (13,466 and 14,948 nuclei for 18-week-old control vs. Lpcat3AKO mice fed HFD for 10 weeks). (h) Violin plots (clusters as columns, genes as rows) of cluster-specific markers. (i) Heatmap of normalized expression values of pan-adipogenic markers in inguinal adipocytes and the SVC fraction. Alternatively activated macrophages (AAMs), lipid-scavenging macrophages (LSMs), adipose stem and progenitor cells (ASPCs). Data are presented as mean ± SEM. ***P < 0.001 by Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (b-d).

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Extended Data Fig. 6 The Lpcat3AKO iWAT molecular signature is enriched for lipid cycling.

(a) Gene ontology (GO) terms and circos plots of upregulated genes in iWAT of 18-week-old control and Lpcat3AKO mice fed HFD for 10 weeks. GO terms were determined on the top 250 Nnat or Kcnc2 co-expressing iWAT gene transcripts. (b) Violin plots of representative markers for UCP1-dependent and -independent futile cycles.

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Extended Data Fig. 7 The non-canonical adaptive Lpcat3AKO response to DIO is WAT-selective.

(a-c) qPCR analysis of pan-adipogenic, DNL, lipid cycling, thermogenic, and ER stress-associated gene expression in (a) iWAT, (b) eWAT, and (c) iBAT of 18-week-old control and Lpcat3AKO mice fed HFD for 10 weeks (n = 6, 6; ND, not detected). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (a-c).

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Extended Data Fig. 8 Dietary n-6 PUFA restriction or Cidec–/– mirrors Lpcat3AKO adaptation to DIO.

(a) Heatmap of the top 70 differentially expressed genes (35 up or down) in iWAT of 18-week-old control and Lpcat3AKO mice fed the indicated HFDs for 10 weeks (n = 4/group). (b,c) GSEA analyses of the top 500 upregulated genes in (b) iWAT of HFDn-3 or HFDsfa-fed control mice vs. HFD-fed controls or (c) in eWAT of ob/ob Cidec–/– mice vs. ob/ob controls75. The ranked list metric is the product of log2 (fold-change)* –log10 (adjusted P value) from the differentially expressed genes in iWAT from HFD-fed Lpcat3AKO mice vs. controls (NES). (d) Correlation matrix depicting the association between Lpcat3AKO markers and clinical traits in METSIM95. Node colour and size reflect the correlation directionality and p value. Correlations were analysed by midweight bicorrelation coefficient or corrected p value using the R package WGCNA. (e) Body weight (BW) gain curves and (f) composition of 28-week-old control and Lpcat3AKO female mice fed HFD for 20 weeks (n = 14, 13). (g) Wet weight of the indicated tissues in HFD-fed control and Lpcat3AKO female mice (n = 14, 13). (h) Liver wet weights (% of BW; n = 14, 13). Data are presented as mean ± SEM. ***P < 0.001 by two-way RM ANOVA with Sidak’s multiple comparisons test (e); Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (f,g); or Welch’s t-test (h).

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Extended Data Fig. 9 Characterization of Lpcat3-null 10T1/2 adipocytes.

(a) TIDE analysis (left) and Western blot analysis of LPCAT3 protein levels (right) validating Lpcat3-KO in 10T1/2 clonal cell lines transfected with sgRNAs targeting either exon 1 (L3_E1) or exon 3 (L3_E3). Calnexin served as a loading control (b) qPCR analysis of adipogenic markers in Lpcat3KO adipocytes expressing LPCAT3WT or LPCAT3H374A and differentiated for 6 days (n = 4, 4). (c) Shotgun lipidomic analysis of a wild-type 10T1/2 clonal cell line differentiated in the presence of 0.1% DMSO (veh ctrl) or the LPCAT3 inhibitor (R)-HTS-3 (10 µM) for 5 days. (d) Shotgun lipidomic analysis, (e) TG content (n = 3 wells/group), and (f) Oil red-O staining of Lpcat3KO adipocytes reconstituted with GFP Ctrl or the indicated GFP-tagged LPCAT3 fusion proteins (day 7 of differentiation). Data are presented as mean ± SEM. ***P < 0.001 by Welch’s t-tests with FDR correction using the Benjamini, Krieger, and Yekutieli procedure (b); or one-way ANOVA with Tukey’s multiple comparisons test (e).

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Extended Data Fig. 10 LPCAT3 activity does not affect gross membrane lipid-packing indices.

(a) Shotgun lipidomic analysis of PC composition in ER or LD–ER-enriched fractions from Lpcat3KO adipocytes expressing Flag-LPCAT3WT or Flag-LPCAT3H374A and differentiated for 6 days. Data are presented as the distribution of PC species (% of total PC) within each sample. (b) Schematic depiction of the trilayer model system containing a bulk oil phase flanked by phospholipid monolayers, solvated with water (left), and top/side views of trilayers exhibiting LPDs that expose the oil core to the solvent (right). (c) Deep/shallow LPDs in model bilayers and LD-like trilayer systems (± 3–5% surface tension, ST). Each replicate corresponds to a block-averaged LPD value derived from individual MD simulation trajectories (n = 3/group). WT and Lpcat3KO compositions reflect complex (left) and simplified (right) lipidomic approximations (Supplementary Table 6). (d,e) Immunoblot analysis of Lpcat3KO adipocytes expressing Flag-LPCAT3WT or Flag-LPCAT3H374A and treated with (d) ± isoproterenol (ISO, 10 µM), or (e) ± insulin (100 nM) for 15 min. Actin and Calnexin served as sample processing controls. (f) Tandem mass tag (TMT) labelling on 8,000g LDs isolated from Lpcat3KO adipocytes expressing Flag-LPCAT3WT or Flag-LPCAT3H374A (day 6 of differentiation). TMT data were overlaid with Uniprot mouse proteome for annotated LD localization. (g) Top/side snapshots from CG–MD simulations of an ER–LD neck system. Bilayer/monolayers consist of 50% PUFA (cyan) and 50% MUFA (white) phospholipids, with a triolein (TO; orange) core. (h) Electron microscopy analysis of Lpcat3KO adipocytes expressing Flag-LPCAT3WT or Flag-LPCAT3H374A (day 3 of differentiation). (i) Lipolysis in Lpcat3KO and Lpcat3/AtglDKO adipocytes expressing Flag-LPCAT3WT or Flag-LPCAT3H374A. Secreted glycerol was measured after ISO (10 µM) treatment for 2 h, normalized to intracellular TG stores (n = 3 wells/group). Where indicated, cells were differentiated in the presence of 0.1% DMSO (veh ctrl) or Atglistatin (ATGLi; 40 µM) for 6 days. Data are presented as mean ± SEM. Protein abundance ratios were compared across groups using Student’s t-test (P < 0.01) while controlling for false-discovery rate with Benjamini-Hochberg procedure (f); or ***P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test (i).

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Supplementary information

Supplementary Information

Supplementary Data Fig. 1.

Reporting Summary

Supplementary Tables 1–8

Supplementary Tables 1–8.

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Source Data Figs. 1 and 5–7

Figs. 1 and 5–7 uncropped images.

Source Data Figs. 1–3 and 5–7

Graph data for Figs. 1–3 and 5–7.

Source Data Extended Data Figs. 1, 3, 9 and 10

Source Data Extended Data Figs. 1, 3, 9 and 10 uncropped images.

Source Data Extended Data Figs. 1–5 and 7–10

Graph data for Extended Figs. 1–5 and 7–10.

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Tol, M.J., Shimanaka, Y., Bedard, A.H. et al. Dietary control of peripheral adipose storage capacity through membrane lipid remodelling. Nat Metab 7, 1424–1442 (2025). https://doi.org/10.1038/s42255-025-01320-y

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