Liver governs adipose remodelling via extracellular vesicles in response to lipid overload

Figures

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  Fig. 1. The liver responds to acute lipid overload first in mice. a Body weight of mice. b–c Percentages of liver b, iWAT c and eWAT c weight relative to the whole-body weight of HFD-fed mice at different time points. d H&E staining of liver, iWAT and eWAT from HFD-fed mice at different time points. (Scale bar: 50 μm). e TG content in the liver. f Expression of genes related to fatty-acid transport, lipogenesis and fatty-acid oxidation in the liver of HFD-fed mice at the indicated times. g TG contents in iWAT and eWAT. h–i Expression of genes related to fatty-acid transport, lipogenesis and fatty-acid oxidation in iWAT and eWAT of HFD-fed mice at the indicated times. j Quantification of adipocyte number in iWAT and eWAT from HFD-fed mice at different time points. k Expression of genes related to adipogensis in the WATs of HFD-fed mice at the indicated times. Six-week-old C57BL/6J mice were fed a HFD for 0 h, 6 h, 12 h, 24 h, 48 h and 1 week (n = 6 mice per group). iWAT: inguinal white adipose tissue. eWAT: epididymal white adipose tissue. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t test. Source data are provided as a Source Data file. See also…

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  Fig. 2. Hepatocytes remodel adipocytes via EVs after lipid overload. a Hepatocyte-conditioned medium (Chow-CM or HFD-CM) prepared from primary hepatocytes from mice fed either a normal chow diet or a HFD that were incubated in EV-free medium for 48 h was added to 3T3-L1 preadipocytes. Adipocytes that differentiated from 3T3-L1 preadipocytes at day 6 after MDI induction were stained with ORO. ORO-positive staining in 3T3-L1 preadipocytes was quantified. (n = 3 biologically independent samples, scale bar, 50 μm). b Expression of lipogenesis-related genes in 3T3-L1 preadipocytes treated with Chow-CM and HFD-CM. (n = 3 biologically independent samples). c Electron microscopy analysis of EVs secreted by primary hepatocytes from mice fed a normal chow diet. (Scale bar, 200 nm). d The EV-related protein markers CD63, CD81, TSG101 and Syntenin-1 were measured by western blotting. These blots are representative of three independent replicated experiments, each containing three samples. e EV production by primary hepatocytes from mice fed a normal chow diet or HFD. (n = 6 biologically independent samples). f Hepatocyte-derived EVs (Chow-EV or HFD-EV) were added to 3T3-L1 preadipocytes. GW4869 (10…

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  Fig. 3. Liver-driven adipose remodelling is mediated by liver Ggpps. a Ggpps protein levels in livers from HFD-fed (12 h) C57BL/6J mice following the time-dependent FFA incubation of primary hepatocytes. The FFA mixture included oleic acid and palmitic acid at a ratio of 2:1 dissolved in BSA. (n = 3 mice per group). b Fluorescence microscopy of primary hepatocytes treated with a scrambled control (Scramble) or a Ggpps siRNA (siGgpps). (Scale bar, 5 μm). c EV production by WT and Ggpps-knockdown primary hepatocytes. (n = 6 biologically independent samples). d Whole-body fat distribution in WT and liver-specific Ggpps knockout mice (LKO) fed a normal chow diet or a HFD, as visualised by NMR. e Calculation of eWAT and iWAT mass in the mice from d. (n = 6 mice per group). f Comparison of eWAT and iWAT mass in WT and LKO mice fed a normal chow diet or a HFD. (n = 6 mice per group, scale bar, 5 mm). g Fat index (percentage of fat pad weight relative to the whole-body weight) of iWAT and eWAT in the mice from f. (n = 6 mice per group). h H&E staining of iWAT and eWAT from mice in g (Scale bar, 50 μm). i Quantification of the diameters of adipocytes from iWAT and eWAT collected from WT and LKO…

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  Fig. 4. Ggpps regulates hepatocyte-derived EV secretion. a EV production by primary hepatocytes from WT and liver-specific Ggpps knockout mice (LKO) fed a normal chow diet or a HFD. (n = 6 mice per group). b CD63, TSG101 and Calnexin protein levels in parental cells and EVs from WT and LKO mice fed a normal chow diet or a HFD. c Membrane-associated Rab27A (lipid-soluble protein with geranylgeranylation in the detergent phase, down) and cytoplasm-associated Rab27A (water-soluble protein with no geranylgeranylation in the aqueous phase, up) were obtained by Triton X-114 extraction and analysed by immunoblotting. (n = 3 biologically independent samples). d Membrane-associated Rab5 (lipid-soluble protein with geranylgeranylation in the detergent phase, down) and cytoplasm-associated Rab5 (water-soluble protein with no geranylgeranylation in the aqueous phase, up) were obtained by Triton X-114 extraction and analysed by immunoblotting. (n = 3 biologically independent samples). e Fluorescence microscopy of primary hepatocytes co-infected with empty vector or Ggpps vector and WT or mutant Rab27A and stained with anti-CD63 antibodies and DAPI (nucleus, blue). (Scale bar, 5 μm). f EV production…

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  Fig. 5. Hepatic EVs mediate adipose remodelling by Ggpps. a Primary hepatocyte-derived EVs uptake in 3T3-L1 adipocytes after 5 h of co-culture. (Scale bar, 50 μm). b EVs produced by primary hepatocytes isolated from WT mice fed a normal chow diet were injected via tail vein into WT and LKO mice fed a normal chow diet or a HFD. Empty liposomes were used as controls. Red fluorescence levels in the liver, iWAT and eWAT were acquired after 6 h with an IVIS Spectrum system. c EVs produced by primary hepatocytes isolated from mice fed a normal chow diet (Chow-EV) or a HFD (HFD-EV) were injected via tail vein every 3 days for 6 weeks into 8-week-old mice fed a normal chow diet. Comparison of iWAT and eWAT in the mice after treatment with EVs. The percentages of iWAT and eWAT weight relative to the whole-body weight of mice treated with Chow-EV and HFD-EV were calculated. (n = 4 mice per group). d Expression of genes related to lipogenesis in iWAT and eWAT of mice treated with Chow-EV and HFD-EV in c. (n = 4 mice per group). e Hepatic EVs produced from WT and LKO mice fed a HFD (HFD-WT EV and HFD-LKO EV, respectively) were added to 3T3-L1 preadipocytes. Adipocytes that differentiated from 3T3-L1…

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  Fig. 6. MiRNAs in hepatic EVs mediate liver-driven adipose remodelling. a The fold change of several miRNAs under HFD-EVs compared with Chow-EVs. b The relative miRNAs levels in miRNA knockout adipocytes that received WT, miR-122−/−, let-7e-5p−/−, miR-31-5p−/− or miR-210-3p−/− EVs derived from primary hepatocytes. c EVs from primary hepatocytes isolated from Chow- or HFD-fed WT mice and transfected with miRNA inhibitors (Chow-EV, HFD-EV, HFD-EV-WT, HFD-EV-miR-210-3p−/−, HFD-EV-let-7e-5p−/−, HFD-EV-miR-31-5p−/−) were added to 3T3-L1 preadipocytes. Adipocytes that differentiated from 3T3-L1 preadipocytes at day 8 after MDI induction were stained with ORO. (Scale bar, 50 μm). d Expression of genes related to adipogenesis and lipogenesis in the 3T3-L1 preadipocytes from c. e EVs from primary hepatocytes isolated from HFD-fed WT or LKO mice and transfected with various miRNA mimics (HFD-WT EV, HFD-LKO EV, HFD-LKO EV-mimic-NC, HFD-LKO EV-miR-210-3p-mimic, HFD-LKO EV-let-7e-5p-mimic, HFD-LKO EV-miR-31-5p-mimic) were added to 3T3-L1 preadipocytes. Adipocytes that differentiated from 3T3-L1 preadipocytes at day 8 after MDI induction were stained with ORO. (Scale bar, 50 μm). f Expression of genes…

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  Fig. 7. Ggpps mRNA levels and MiRNA levels of EVs in mice and patients. a Linear regression analysis between liver TG count and Ggpps mRNA levels in patients (n = 23 samples). b–f The levels of let-7e-5p, miR-210-3p, miR-23b-3p, miR-690 and miR-31-5p among the plasma EVs of mice fed a normal chow diet and a HFD. The relative values refer to the fold change of each miRNA in HFD-EVs compared with that in Chow-EVs. (n = 6 mice per group). g–j Linear regression analysis between BMI and let-7e-5p, miR-210-3p, miR-31-5p and miR-23b-3p levels in plasma EVs from patients (n = 28 samples). k The relative expression levels of miR-122, let-7e-5p, miR-210-3p and miR-31-5p in the livers of patients. The relative values refer to the fold change of let-7e-5p, miR-210-3p and miR-31-5p levels compared to miR-122 levels (n = 6 samples). l–o Let-7e-5p, miR-210-3p, miR-31-5p and miR-23b-3p levels in the plasma EVs of NAFLD patients with a BMI < 30 or > 30. The relative values refer to the fold change of each miRNA in EVs from patients with BMI > 30 patients compared with that in EVs from patients with BMI < 30. (BMI < 30: n = 6 samples; BMI > 30: n = 10 samples). Data are presented as the mean ± SD. *P <…

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  Fig. 8. Schematic for the contribution of hepatic EVs to adipose remodelling. Induced Ggpps expression in hepatocytes by acute and chronic HFD consumption gives rise to hepatocyte-derived EV secretion through Rab27A geranylgeranylation, which remodels adipose tissue via adipogenesis and lipogenesis. Hepatocyte-derived EVs containing miRNAs enhance lipid deposition in adipocytes by increasing lipogenesis and inhibiting lipid oxidation through the let-7e-5p-Pgc1α axis.