Abstract
Current pharmacological therapies for treating obesity are of limited efficacy. Genetic ablation or loss of function of AMP-activated protein kinase alpha 1 (AMPKα1) in steroidogenic factor 1 (SF1) neurons of the ventromedial nucleus of the hypothalamus (VMH) induces feeding-independent resistance to obesity due to sympathetic activation of brown adipose tissue (BAT) thermogenesis. Here, we show that body weight of obese mice can be reduced by intravenous injection of small extracellular vesicles (sEVs) delivering a plasmid encoding an AMPKα1 dominant negative mutant (AMPKα1-DN) targeted to VMH-SF1 neurons. The beneficial effect of SF1-AMPKα1-DN-loaded sEVs is feeding-independent and involves sympathetic nerve activation and increased UCP1-dependent thermogenesis in BAT. Our results underscore the potential of sEVs to specifically target AMPK in hypothalamic neurons and introduce a broader strategy to manipulate body weight and reduce obesity.
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All additional data that support the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.
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Acknowledgements
The research leading to these results has received funding from the Xunta de Galicia (R.N. grant no. 2016-PG057, M.L. grant no. 2016-PG068); EuroNanoMed III (R.A. and M.L. grant no. EURONANOMED2019-050/ENAMEP); Junta de Andalucía (M.T.-S. grant no. P12-FQM-01943); Ministerio de Ciencia y Universidades cofunded by the FEDER Program of EU (C.D. grant no. BFU2017-87721; M.T.-S. grant no. BFU2014-57581-P, R.N. grant no. BFU2015-70664R; M.L. grant no. RTI2018–101840-B-I00 and grant no. BFU2015-70454-REDT/Adipoplast); USA National Institutes of Health (K.R. grant no. HL084207); the German Research Foundation (DFG) (grant nos. MI1242/3-2 to J.M. and OE723/2-1 to R.O.); the USA Department of Veterans Affairs (K.R. grant no. I01BX004249); The University of Iowa Fraternal Order of Eagles Diabetes Research Center (K.R.); European Research Council ERC (C.G.-C. STG grant AstroNeuroCrosstalk no. 757393); Atresmedia Corporación (R.N. and M.L. grant no. 2017-PO004) and ‘la Caixa’ Foundation (grant no. ID 100010434), under grant agreement no. LCF/PR/HR19/52160022 (M.L.). E.M. was the recipient of a Predoctoral Fellowship of the Nanofar Erasmus Mundus Program. I.G.-G. is the recipient of a fellowship from Alexander von Humboldt Foundation (ref. 3.3, ESP-1206916-HFST-P) and European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie actions (grant no. 842080, H2020-MSCA-IF-2018). T.S. is the recipient of a research contract from the Miguel Servet Program (CPII17/00027, Instituto de Salud Carlos III). The CiMUS is supported by the Xunta de Galicia (grant no. 2016-2019, ED431G/05). CIBER de Fisiopatología de la Obesidad y Nutrición is an initiative of ISCIII. INSERM U1063 is supported by the Institut National de Santé et Recherche Médicale and Université d’Angers. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper. We thank S. Recoquillon and M. Wertheimer for technical assistance. The TEM analysis was performed in the Service Commun d’Imageries et d’Analyses Microscopiques of University of Angers. We thank SCAHU staff (Université d’Angers) for taking care of animals. The PET–CT analysis was performed in the Molecular Imaging Unit of the Department of Nuclear Medicine of USC.
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E.M., N.R.V.D., I.G.-G., M.R.-G., F.R.-P., V.R.-L., C.C., J.C. and B.P. performed the in vivo experiments, analytical methods and collected and analysed the data. E.M., L.P., G.H., P.M. and L.V. generated and validated the sEVs. F.R.-P., J.R. and M.T.-S. analysed testis and adrenal function. D.A.M. and K.R. performed and analysed the SNA studies. R.I.-R. and T.S. performed the NMR studies. A.G.-N. and F.V. performed the experiments with brown adipocytes. N.R.V.D., I.G.-G. and C.G.-C. performed the immunohistochemistry studies. R.O. and J.M. provided the ucp1 null mice. E.M., N.R.V.D., I.G.-G., A.V., F.V., C.D., R.N., C.G.-C., M.T.-S., M.C.M., J.M., K.R., R.A. and M.L. analysed, interpreted and discussed the data. E.M., M.C.M., R.A. and M.L. developed the hypothesis and conceived and designed the experiments. E.M. and M.L. made the figures and wrote the paper. All authors revised and edited the paper. R.A. and M.L. jointly supervised this work, secured funding, coordinated the project and served as guarantors. M.L. is the lead contact for this study.
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E.M., M.C.M., R.A. and M.L. declare that the research described in this paper is patent pending: European Patent Application EP21382763.7 entitled ‘Populations of small extracellular vesicles for use in the treatment of obesity’, European Patent Office (EPO). The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Characterization of SF1-AMPKα1-DN loaded neuronal-targeted dendritic cell-derived sEVs.
a, Western blotting using antibodies against Lamp2b in native and Lamp2b-RVG sEVs. b, Quantification of Lamp2b levels in native (n = 4 samples) and Lamp2b-RVG (n = 5 samples) sEVs in % of native control; P = 0.00031. c, Western blotting using antibodies against GRP94 in Jaws II cells (lane 1), unmodified native sEVs (lane 2) and Lamp2b-RVG sEVs (lane 3). d, Circular representation of the SF1-AMPKα1-DN encoding plasmid. e, Example of curve obtained by nanoparticle tracking analysis of a sample of native (left panel) and SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs (right panel). The graph represents concentration of sEVs (particles/mL) according to the size (nm). f, Electron microscopy image of SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs showing specific round shape and average size of ~70 nm vesicles. g, Agarose gel electrophoresis of native (lane 1), Lamp2b-RVG (lane 2), SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs (lane 3) and negative control H2O (lane 4) of AMPK and GAPDH. h, Agarose gel electrophoresis of SF1-AMPKα1-DN loaded Lamp2b-RVG sEVs treated with DNAse (lane 1), DNAse + Triton X-100 0.2% (lane 2) and Triton X-100 0.2% (lane 3) of AMPK and GAPDH. i, Quantification of pACCα/ACCα in primary astrocytes treated for 24 h with native and Lamp2b-RVG (n = 6 samples/group) sEVs. j, Quantification of pACCα/ACCα in Neuro2A cells treated for 24 h with native and Lamp2b-RVG (n = 3 samples per group) sEVs. Data expressed as mean ± SEM. ***P < 0.001 vs. Control. Statistical significance was assessed by two-sided Student’s t-test.
Extended Data Fig. 2 Control of hypothalamic nuclei dissections.
Quantification of Pomc, Sf1 and Hcrt/orexin mRNA levels in ARC, VMH and LHA dissections [Pomc: ARC n = 20 mice, VMH n = 20 mice, LHA n = 19 mice; Sf1: ARC n = 19 mice, VMH n = 19 mice, LHA n = 19 mice; Hcrt: ARC n = 20 mice, VMH n = 20 mice, LHA n = 19 mice; box plots indicate median (middle line), 25th, 75th percentile (box) and 10th-90th percentiles (whiskers; minima and maxima, respectively)]. Data expressed as mean ± SEM. ***P < 0.001 vs. Pomc ARC, Sf1 VMH and Hcrt LHA. Statistical significance was assessed by two-sided ANOVA.
Extended Data Fig. 3 Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on hypothalamic AMPK activity in DIO mice.
a, Representative confocal images depicting GFAP (green), pACCα (magenta) and merged reactivity in brain sections (control n = 8 fields, 4 mice/group; SF1-AMPKα1-DN n = 8 fields, 4 mice/group) after 24 h of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded sEVs; scale bars represent 20 µm. b, Representative confocal images depicting Iba1 (green), pACCα (magenta) and merged reactivity in brain sections after 24 h of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded sEVs; scale bars represent 20 µm. c, Negative controls for pACCα and SF1 double immunofluorescence. Representative confocal images depicting DAPI (blue), Alexa594, with or without SF1 (red), Alexa 488 with or without pACCα (green) and merged reactivity in brain sections; scale bars represent 20 µm. The experiments were repeated 3 times. d, Quantification of pACCα fluorescence in ARC, DMH and PVH (quantification per field; ARC control n = 12 fields, 3 mice/group; SF1-AMPKα1-DN n = 8 fields, 2 mice/group; DMH control n = 12 fields, 3 mice/group; SF1-AMPKα1-DN n = 12 fields, 3 mice/group; PVH control n = 4 fields, 1 mice/group; SF1-AMPKα1-DN n = 12 fields, 3 mice/group) after 24 h of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded sEVs. Data expressed as mean ± SEM. Statistical significance was assessed by two-sided Student’s t-test.
Extended Data Fig. 4 Effect of systemic treatment with SF1-AMPKα1-DN loaded sEVs on central and peripheral tissues in DIO mice.
a, Quantification of pACCα/ACCα levels in cortex, thalamus and cerebellum after 28-day of intravenous injection with control (non-loaded; cortex n = 7 mice; thalamus n = 7 mice; cerebellum n = 6 mice) or SF1-AMPKα1-DN loaded (cortex n = 7 mice; thalamus n = 7 mice; cerebellum n = 7 mice) sEVs. b, Quantification of pACCα/ACCα levels in liver, adrenal gland, testis, BAT, heart and skeletal muscle after 28-day of intravenous injection with control (non-loaded, liver n = 7 mice; adrenal n = 7 mice; testis n = 6 mice; BAT n = 7 mice; heart n = 7 mice; skeletal muscle n = 6 mice) or SF1-AMPKα1-DN loaded (liver n = 7 mice; adrenal n = 7 mice; testis n = 7 mice; BAT n = 7 mice; heart n = 7 mice; skeletal muscle n = 7 mice) sEVs. c, Quantification of pACCα/ACCα levels in brown adipocytes after 24 h of incubation with control (non-loaded) or SF1-AMPKα1-DN loaded (n = 5 samples/group) sEVs. d, Quantification of circulating testosterone levels after 28-day of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded (n = 7 mice/group) sEVs. e, Quantification of mRNA levels of steroidogenic enzymes (STAR, p450scc and 17β-HSD3) in testis after 28-day of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded (n = 7 mice/group) sEVs. f, Quantification of circulating CORT levels after 28-day of intravenous injection with control (non-loaded, n = 7 mice) or SF1-AMPKα1-DN loaded (n = 6 mice) sEVs. g, Quantification of mRNA levels of P450scc and STAR in adrenals after 28-day of intravenous injection with control (non-loaded; P450scc n = 5 mice; STAR n = 6 mice) or SF1-AMPKα1-DN loaded (P450scc n = 5 mice/group; STAR n = 5 mice/group) sEVs. h, Quantification of circulating LH levels after 28-day of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded (n = 6 mice/group) sEVs. i, Quantification of mRNA levels of LH β subunit in pituitary after 28-day of intravenous injection with control (non-loaded, n = 6 mice) or SF1-AMPKα1-DN loaded (n = 7 mice) sEVs. a-i, j, Quantification of BAT UCP1 levels at 1, 2, 3 and 7 days after a single injection in the tail vein of control (non-loaded; day 1 n = 6 mice; day 2 n = 5 mice; day 3 n = 5 mice; day 7 n = 5 mice) or SF1-AMPKα1-DN sEVs (day 1 n = 4 mice; day 2 n = 4 mice; day 3 n = 5 mice; day 7 n = 5 mice) at day 0; day 1 P = 0.023; day 2 P = 0.0054. Data expressed as mean ± SEM. *P < 0.05 and **P < 0.01 vs. Control. Statistical significance was assessed by two-sided Student’s t-test.
Extended Data Fig. 5 Effect of systemic treatment with SF1-AMPKα1-DN sEVs on circulating and hemodynamic parameters in DIO mice.
a-i, Quantification of circulating leptin (a; control n = 12 mice, SF1-AMPKα1-DN n = 13 mice), GDF15 (b; control n = 7 mice, SF1-AMPKα1-DN n = 8 mice), IL-6 (c; control n = 6 mice, SF1-AMPKα1-DN n = 5 mice), IP-10 (d; control n = 6 mice, SF1-AMPKα1-DN n = 5 mice), triglycerides (e; control n = 7 mice, SF1-AMPKα1-DN n = 6 mice), cholesterol (f; control n = 7 mice, SF1-AMPKα1-DN n = 6 mice), NEFA (g; control n = 7 mice, SF1-AMPKα1-DN n = 6 mice, P = 0,0142), AST (h; control n = 8 mice, SF1-AMPKα1-DN n = 8 mice) and ALT (i; control n = 8 mice, SF1-AMPKα1-DN n = 8 mice) after 28-day of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded sEVs. j-m, Quantification of heart rate (j, control n = 6 mice, SF1-AMPKα1-DN n = 6 mice), systolic arterial pressure (k; control n = 6 mice, SF1-AMPKα1-DN n = 6 mice), diastolic arterial pressure (l; n control n = 6 mice, SF1-AMPKα1-DN n = 6 mice) and mean arterial pressure (m; n = 6 mice/group) after 24 h of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded sEVs. Data expressed as mean ± SEM. Statistical significance was assessed by two-sided Student’s t-test.
Extended Data Fig. 6 Effect of systemic treatment with SF1-AMPKα 1-DN sEVs on BAT and skeletal muscle thermogenic markers in DIO mice.
a, Quantification of UCP1 protein levels in the BAT after 28-day of intravenous injection with control (non-loaded) or SF1-AMPKα1-DN loaded (n = 7 mice/group) sEVs; P = 0.029. b, Quantification of mRNA levels of thermogenic markers (Ucp3, Gpd2, Pparɣ, Sln, Ryr1, Atp2a2) in skeletal muscle after 28-day of intravenous injection with control (non-loaded; Ucp3 n = 7 mice; Gpd2 n = 7 mice; Pparγ n = 7 mice; Sln n = 6 mice; Ryr n = 7 mice; Atp2a2 n = 6 mice) or SF1-AMPKα1-DN loaded (Ucp3 n = 7 mice; Gpd2 n = 7 mice; Pparγ n = 7 mice; Sln n = 6 mice; Ryr n = 7 mice; Atp2a2 n = 6 mice) sEVs. Data expressed as mean ± SEM. *, **P < 0.05 vs. Control. Statistical significance was assessed by two-sided Student´s t-test.
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Milbank, E., Dragano, N.R.V., González-García, I. et al. Small extracellular vesicle-mediated targeting of hypothalamic AMPKα1 corrects obesity through BAT activation. Nat Metab 3, 1415–1431 (2021). https://doi.org/10.1038/s42255-021-00467-8
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DOI: https://doi.org/10.1038/s42255-021-00467-8
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