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Liver-derived extracellular vesicles improve whole-body glycaemic control via inter-organ communication

Nature Metabolism volume 6, pages 254–272 (2024)Cite this article

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Abstract

Small extracellular vesicles (EVs) are signalling messengers that regulate inter-tissue communication through delivery of their molecular cargo. Here, we show that liver-derived EVs are acute regulators of whole-body glycaemic control in mice. Liver EV secretion into the circulation is increased in response to hyperglycaemia, resulting in increased glucose effectiveness and insulin secretion through direct inter-organ EV signalling to skeletal muscle and the pancreas, respectively. This acute blood glucose lowering effect occurs in healthy and obese mice with non-alcoholic fatty liver disease, despite marked remodelling of the liver-derived EV proteome in obese mice. The EV-mediated blood glucose lowering effects were recapitulated by administration of liver EVs derived from humans with or without progressive non-alcoholic fatty liver disease, suggesting broad functional conservation of liver EV signalling and potential therapeutic utility. Taken together, this work reveals a mechanism whereby liver EVs act on peripheral tissues via endocrine signalling to restore euglycaemia in the postprandial state.

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Fig. 1: Confirmation of NAFL and NASH and validation of small EV isolation secreted from the livers of mice.
Fig. 2: The liver EV proteome in mice with NAFL and NASH.
Fig. 3: Liver-secreted EVs improve glycaemic control.
Fig. 4: Liver-secreted EVs do not regulate insulin sensitivity.
Fig. 5: Liver EVs improve glycaemic control through enhanced GE.
Fig. 6: Liver EVs activate cell signalling associated with calcium metabolism.
Fig. 7: Liver-secreted EVs directly stimulate GSIS in primary murine islets.
Fig. 8: Human liver-secreted EVs improve glycaemic control.

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Data availability

All data are available within the manuscript, extended data, source data files and supplementary files. The analysis in this study used standard techniques and protocols without generating new unique reagents or custom code. The proteomic and phosphoproteomic data generated in this study have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/cgi/GetDataset) via the PRIDE database84, under the identifiers PXD037924, PXD036400 and PXD043137. These data are also provided as supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank the team at the Ian Holmes Imaging Centre and Melbourne Mass Spectrometry and Proteomics facility of the Bio21 Institute at the University of Melbourne for instrument support, the infrastructure within the Melbourne Murine Metabolic Phenotyping Platform for calorimetry assessment, the Melbourne Histology Platform at the University of Melbourne, M. Shambrook (La Trobe University) for instrument support for the NTA, D. Johnson for technical assistance with running the imaging flow cytometry, and D. Keating (Flinders University) and M. Kebede (University of Sydney) for helpful discussions. P.M.M. was supported by post-doctoral fellowships through the Natural Sciences and Engineering Research Council of Canada (PDF-516731-2018) and Canadian Institutes of Health Research (application no. 430145). This work was supported by a National Health and Medical Research Council (NHMRC) Ideas grant (M.J.W., P.M.M., P.R.B., grant no. APP1184309), NHMRC Investigator grants (P.M.M., grant no. APP2018187; B.J.P., grant no. APP2009642), a Diabetes Australia Research Trust grant (P.M.M.), a Medicine, Dentistry and Health Sciences Early Career Researcher grant (P.M.M., grant no. ECR1782020) and Department of Anatomy and Physiology seed grant (P.M.M.). Graphical illustrations were created with BioRender.com.

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Authors and Affiliations

  1. Department of Anatomy and Physiology, School of Biomedical Sciences, Faculty of Medicine Dentistry and Health Sciences, University of Melbourne, Melbourne, Victoria, Australia

    Paula M. Miotto, Stacey N. Keenan, William De Nardo, Cait A. Beddows, Gio Fidelito, Garron T. Dodd, Benjamin L. Parker & Matthew J. Watt

  2. St. Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia

    Chieh-Hsin Yang & Kim Loh

  3. Department of Biochemistry and Chemistry, School of Agriculture, Biomedicine and Environment, La Trobe University, Bundoora, Victoria, Australia

    Andrew F. Hill

  4. Institute for Health and Sport, Victoria University, Footscray, Victoria, Australia

    Andrew F. Hill

  5. Centre for Obesity Research and Education, Department of Surgery, Monash University, Melbourne, Victoria, Australia

    Paul R. Burton

  6. Department of Medicine, University of Melbourne, Fitzroy, Victoria, Australia

    Kim Loh

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  1. Paula M. Miotto
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Contributions

P.M.M. and M.J.W. conceptualized the study design and funded the research. P.M.M., C.-H.Y., S.N.K., W.D.N., C.A.B., G.F., G.T.D. and B.L.P. performed the experiments. P.M.M., C.-H.Y., S.N.K., G.F. and B.L.P. analysed the data. G.T.D., A.F.H., B.L.P. and K.L. provided the resources and guided the experimental design. P.R.B. provided the human samples. P.M.M. and M.J.W. wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Matthew J. Watt.

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Competing interests

M.J.W. received the research funding from Gilead Sciences and CSL, as well as honoraria from Gilead Sciences. The other authors declare no competing interests.

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Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Characteristics of diet-induced glucose intolerance and extracellular vesicle purity.

(A) Body weight over 20 weeks consumption of control (n = 30), NAFL (n = 15), and NASH diet (n = 15) across 1 cohort. *, significant from Control; #, significant from NASH. P < 0.001. (B) Liver and epididymal (EWAT) tissue mass in response to Control (n = 29), NAFL (n = 14), or NASH (n = 15 liver, n = 13 EWAT) across 1 cohort. *, significant from Control; #, significant from NASH. P < 0.001. (C-D) Blood glucose levels during an oral glucose tolerance test (2 g/kg) (C) and the corresponding glucose area under the curve (AUC; D) of mice fed different dietary interventions (n = 15 control, n = 9 NAFL, n = 13 NASH) across 1 cohort. *, significant from Control; #, significant from NASH. P < 0.001. (E) Serum insulin during the oral glucose tolerance test (n = 5 control, n = 9 NAFL, n = 9 NASH) across 1 experiment. *, significant from Control; #, significant from NASH. P = 0.01. (F) Steatosis score in Control (n = 8), NAFL (n = 10), and NASH (n = 10) livers derived from histopathology across 1 cohort. P = 0.1. (G) Lobular inflammation score in Control (n = 8), NAFL (n = 10), and NASH (n = 10) livers derived from histopathology across 1 cohort. P = 0.017. (H) Hepatocyte ballooning score in Control, NAFL, and NASH livers derived from histopathology (n = 8 control, n = 10 NAFL, n = 10 NASH) across 1 cohort. *, significant from Control; #, significant from NAFL. P < 0.001. (I) ATP / ADP ratio in liver slices and whole liver from Control, NAFL, and NASH mice (n = 6/group for liver slices, n = 9 for whole liver pooled from Control, NAFL, and NASH mice) across 1 experiment. P = 0.07. (J) Endotoxin levels in the circulation and in extracellular vesicle (EV) preparations (each bar represents an individual biological sample) across 1 experiment. Data are expressed as mean ± SEM. Each datapoint represents an independent biological sample. Data was analysed using a repeated measures two-way ANOVA (A, C, E) or one-way ANOVA (B (EWAT), D, F, H, I) followed by a Student Newman Keuls post-hoc, or Kruskal Wallis one-way ANOVA on ranks (B (liver), G) followed by a Dunn’s test.

Source data

Extended Data Fig. 2 Pathways analysis of the liver extracellular vesicle proteome in response to NAFL or NASH.

(A-B) Top 20 biological processes identified using pathways enrichment analysis (Metascape.com) in NAFL relative to control conditions (A), and in NASH relative to control conditions (B). n = 10/group. P values indicated within figure. (C-D) Pathway enrichment analysis of disease functions and toxicology using Ingenuity Pathways Analysis in NAFL (C) and NASH (D) compared to healthy control mice. n = 10/group. P < 0.001. Data represented as z-score or -log10 P-value, derived from proteomics analysis using unpaired two-tailed t-test.

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Extended Data Fig. 3 Glucose tolerance testing in mice in response to extracellular vesicles derived from various sources and glucose-stimulated extracellular vesicle secretion.

(A-C) The GTT glucose area under the curve (AUC) in control (A, n = 8 saline, n = 9 NAFL EV, n = 7 NASH EV), NAFL (B, n = 8/group), or NASH (C, n = 8 saline, n = 9 Control EV) mice in response to saline or liver EVs across 1 cohort. P < 0.001. *, significant from Saline. (D) Blood glucose and AUC during GTT in female mice in response to saline or liver EV (n = 3/group) across 1 cohort. *, significant from NAFL EV injection. P < 0.001, P = 0.02 (inset). (E-F) Blood glucose (E, P = 0.001) and serum insulin (F, P < 0.001) during GTT in control mice that received i.v injection of saline or liver EV (n = 6 saline, n = 4 NAFL EV) across 1 cohort. *, significant from NAFL EV injection. (G-I) Blood glucose levels and the AUC (inset) of Control mice injected intraperitoneally with saline or empty liposomes (G, n = 5/group, P < 0.001 (main effect: Time), P = 0.91 (inset)), adipose-derived EVs (H, n = 7 saline, n = 6 adipose EV, P < 0.001 (main effect: Time), P = 0.76 (inset)), or serum derived EVs (I, n = 4 saline, n = 6 serum EV, P < 0.001 (main effect: Time), P = 0.603 (inset)) across 2 cohorts. (J) Serum insulin during an oral GTT in response to administration of saline (n = 6), adipose-derived EVs (n = 5), or serum EVs (n = 5) across 1 experiment. P < 0.001 (main effect: Time). (K-L) Nanoparticle-tracking analysis of liver EVs from liver slices in response to high glucose (20 mM), in the absence or presence of (K, P = 0.994) saline (n = 6), glucagon (n = 5), or norepinephrine (n = 5), and (L, P = 0.22) saline (n = 6) or insulin (n = 5) across 1 experiment for each comparison. (M) Volcano plot comparison of the liver EV proteome secreted by healthy liver slices in response to high glucose (20 mM, n = 6) relative to low glucose (2 mM, n = 5) across 1 experiment. (N-Q) Gating strategy for imaging flow cytometry in Control mice (n = 4/group) that were fasted and subjected to an oral gavage of water (vehicle) or glucose. The unfocused bright field signals were excluded using Gradient RMS feature (N). Particles with relative area smaller than the speed beads were gated by the discriminating aspect ratio and area of particles (previously optimised) (O). CD63-PE-Cy7 and CD9-APC positive particles were gated (P). ASGPR positive particles were plotted and gated based on the isotype control (Q). 1 independent experiment. Data are expressed as mean ± SEM. Each datapoint represents an independent biological sample. Data was analysed using a one-way ANOVA (A, K (calculated from AUC), repeated measures two-way ANOVA (D, E, F, G, H, I, J) with Student Newman Keuls post-hoc, or unpaired two-tailed t-test (B, C, D (inset), G (inset), H (inset), I (inset), L (calculated from AUC), M).

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Extended Data Fig. 4 Glucose tolerance testing in mice in response to extracellular vesicles derived from various treatments, tissue insulin sensitivity in isolated tissue, and blood glucose and insulin levels during a hyperinsulinemic-euglycemic clamp.

(A-D) Gating strategy for imaging flow cytometry in NAFL mice (n = 3 fasted, n = 4 glucose gavage) that were fasted and subjected to an oral gavage of water (vehicle) or glucose. The unfocused bright field signals were excluded using Gradient RMS feature (A). Particles with relative area smaller than the speed beads were gated by the discriminating aspect ratio and area of particles (previously optimised) (B). CD63-PE-Cy7 and CD9-APC positive particles were gated (C). ASGPR positive particles were plotted and gated based on the isotype control (D). 1 independent experiment. (E) Glucose AUC during an oral GTT in Control (n = 6/group) and NAFL mice (n = 5/group) in response to sonicated liver EVs across 1 cohort. P < 0.001 (main effect). (F-G) Glucose AUC during an oral GTT in Control (F, n = 9 saline, n = 10 NAFL EV, n = 9 ‘shaved EV) or NAFL (G, n = 5 saline, n = 3 Control EV, n = 6 ‘shaved’ EV) mice that received saline or ‘shaved’ liver EVs across 2 cohorts (control) and 1 cohort (NAFL). P < 0.001. *, significant from Saline; #, significant from Control EV. (H) Representative cryo-electron microscopy of ‘shaved’ liver EV preparation. 1 experiment. (I) Nanoparticle tracking analysis of ‘shaved’ liver EV. 1 experiment, n = 4 biological replicates graphed as an average. (J-K) Blood glucose levels and AUC (inset) during an oral GTT (J), and serum insulin (K) of mice that received ICV of saline/liver EVs during 1 experiment (n = 9/group). P < 0.001 (main effect: time). (L-N) Glucose uptake in isolated soleus muscle (L, n = 3 saline, n = 4 Control EV, P = 0.61, basal and insulin-stimulated samples are paired from the same animal within each treatment), white adipose tissue (M, n = 7 saline/basal, n = 6 saline/insulin, n = 7 Control EV/basal, n = 6 Control EV/insulin, P = 0.73), and brown adipose tissue (N, n = 7/group, P = 0.932) collected from NAFL mice and treated ex vivo with saline or liver EVs (10 μg/ml) obtained from healthy Control mice 1 hr prior to and during the experiment. 1 cohort across tissues. Basal and insulin-stimulated samples from adipose are paired from the same animal across both treatments, with exception of 1 mouse for the EWAT comparison (that is, basal saline vs. basal EV). (O) Blood glucose levels at baseline (that is, fasted state) and during a hyperinsulinemic-euglycemic clamp in Control mice (n = 7/group) following intraperitoneal injection of NAFL EV 1 hr prior to the experiment. P < 0.001 (main effect). 1 independent cohort. (P) Serum insulin at baseline (that is, fasted state) and during a hyperinsulinemic-euglycemic clamp in Control mice (n = 6 saline, 9 NAFL EV) following intraperitoneal injection of NAFL EV 1 hr prior to the experiment. P < 0.001 (main effect). 1 independent cohort. Data are expressed as mean ± SEM. Each datapoint represents an independent biological sample. Data was analysed using a one-way ANOVA (F, G), two-way ANOVA (E, L, M, N) or repeated measures two-way ANOVA (J, K, O, P) with Student Newman Keuls post-hoc analyses, or unpaired two-tailed t-test (J (inset)). *, significant main effect compared to basal.

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Extended Data Fig. 5 Effect of liver extracellular vesicles in regulating glucose effectiveness.

(A) Glucose area under the curve (AUC) during an oral glucose tolerance test in streptozotocin (STZ) treated mice that received an intraperitoneal injection of saline or Control liver EVs (n = 4 control saline, n = 6/group STZ) across 1 cohort. *, significant from Control + saline; #, significant from STZ + saline. P = 0.001. (B-D) Whole body calorimetry and activity in Control mice over 2 hours in response to saline or liver EVs. Fat oxidation (B, P = 0.58), energy expenditure (C, P = 0.04), and physical activity (D, P = 0.54). n = 4/group. 1 experiment. *, significant from Saline. (E) Serum insulin in STZ mice in response to saline or liver EVs, followed by gavage with vehicle (n = 8 saline/H2O) or glucose (n = 8 saline/high glucose (HG), n = 7 liver control EV/HG) 1 hr later across 2 cohorts. P = 0.245. (F) Plasma membrane CAV3 content in STZ mice in response to saline or liver EVs followed by oral gavage with vehicle (n = 8 saline/H2O) or glucose (n = 8 saline/HG, n = 9 control EV + HG) 1 hr later across 2 cohorts. P = 0.918. (G) Pathways enrichment analysis for phosphosites regulated in soleus muscle from Control mice following high glucose (HG + saline, n = 5) treatment relative to low glucose (LG + saline, n = 4) in the presence of saline. q < 0.05. (H) Pathways enrichment analysis for phosphosites regulated in soleus muscle from Control mice following high glucose treatment and liver EVs (HG + EV, n = 5) relative to HG and saline (HG + saline, n = 5). q < 0.05. (I) Glucose uptake between no drug, dantrolene (5 μM), and KN-62 (10 μM) under LG (2 μM, n = 5 no drug, n = 3 dantrolene, n = 3 KN-62) and HG (20 μM, n = 7 no drug, n = 5 dantrolene, n = 8 KN-62) conditions in soleus muscle. P < 0.001 main effect: condition. Same values reassessed from Figs. 6E and F. Data are expressed as mean ± SEM. Each data point represents an independent biological sample. Data was analysed using one-way ANOVA (A, F) and two- way repeated measures ANOVA (E) with Student Newman Keuls post-hoc, or unpaired two-tailed t-test (B, C, D, G, H).

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Extended Data Fig. 6 Effect of liver extracellular vesicles on insulin secretion and cellular insulin content.

(A-E) Glucose-stimulated insulin secretion from isolated healthy murine islets in the presence or absence of NAFL liver EVs (10 μg/ml), when treated with tolbutamide (A, n = 10 saline/2.8 mM or 20 mM, n = 12 NAFL EV/2.8 mM, n = 11 NAFL EV/20 mM, P < 0.001 (main effect)), diazoxide (B, n = 4 saline/group, n = 6 NAFL EV/group, P = 0.364), arginine (C, n = 6/group, P < 0.001 (main effect)), alanine (D, n = 8 saline/group, n = 11 NAFL EV/group, P < 0.008), or exendin 4 (E, n = 5/group, P = 0.019) across 2 experiments involving isolations from 9 mice. *, significant from low glucose (2.8 mM) within same condition (that is, saline or liver EV) or significant main effect of high glucose (20 mM); #, significant from saline within 20 mM glucose condition. (F-J) Islet insulin content in response to tolbutamide (F, n = 5 saline/2.8 mM, n = 4 saline/20 mM, n = 5/group NAFL EV, P = 0.820), diazoxide (G, n = 6/group, P = 0.004 (main effect)), arginine (H, n = 6 saline/2.8 mM, n = 5 saline/20 mM, n = 6/group NAFL EV, P = 0.744), alanine (I, n = 9 saline/2.8 mM, n = 11 saline/20 mM, n = 11/group NAFL EV, P = 0.543), and exendin 4 (J, n = 5/group, P = 0.720). Data are expressed as mean ± SEM. Data was analysed using two-way repeated measures ANOVA (A, B, C, D, E, H, J) or two-way ANOVA (F, G, I) with Student Newman Keuls post-hoc analyses.

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Extended Data Table 1 Patient clinical variables
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Extended Data Table 2 Primer sequences
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Supplementary information

Reporting Summary

Supplementary Tables 1–5

Supplementary Tables 1–5: Table 1: Liver EV proteomic analysis. NAFL versus control. Table 2: Liver EV proteomic analysis. NASH versus control. Table 3: Liver EV proteomic analysis. HG versus LG stimulation. Table 4: Skeletal muscle phosphoproteome. HG versus LG. Table 5: Skeletal muscle phosphoproteome. HG EV versus saline.

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Miotto, P.M., Yang, CH., Keenan, S.N. et al. Liver-derived extracellular vesicles improve whole-body glycaemic control via inter-organ communication. Nat Metab 6, 254–272 (2024). https://doi.org/10.1038/s42255-023-00971-z

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