A Sensitive Reporter Mouse Model to Study Adipocyte‐Derived Extracellular Vesicles In Vivo

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  FIGURE 1. The CD9‐EGFP‐based EV reporter system for labelling adipocyte‐derived EVs. (A) Adipocyte‐specific Cre expression in CD9truc‐EGFP reporter mice was achieved via AAV‐mediated gene delivery of Cre recombinase driven by a short human adiponectin (hAdipoq) promoter. In the presence of Cre, the double‐floxed oriented CD9truc‐EGFP transgene is recombined and activated, leading to the expression of EGFP‐labelled CD9‐positive EVs driven by a constitutively active CAG promoter. (B) Protein expression of CD9truc‐EGFP in rAAV9‐hAdipoq‐Cre/CD9truc‐EGFP mice (+) was detected in adipose tissues, that is, interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT), and in the heart, compared to wild‐type tissues (−). Full blots are shown in Figure S6A–C. (C) EGFP staining of tissues from rAAV9‐hAdipoq‐Cre/CD9truc‐EGFP mice showed vague signals in adipose tissues and off‐target effects in the liver, heart and pancreas compared to wild‐type tissues. The scalebar represents 50 µm. (D) CD9truc‐EGFP protein expression was not detected in PEG‐precipitated plasma samples from chow‐fed rAAV9‐hAdipoq‐Cre/CD9truc‐EGFP mice receiving different…

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  FIGURE 2. In vitro characterization of the CD63‐NanoLuc and sec‐NanoLuc system in transiently transfected HEK293 cells. (A) Fluorescence microscopy of EGFP confirmed successful transfection with CD63‐NanoLuc and sec‐NanoLuc constructs. (B) Human CD63 and HA‐tag protein expression was detected in cells and cell‐conditioned medium from CD63‐NanoLuc and sec‐NanoLuc expressing cells. Full blots are shown in Figure S6I–J. (C) Luciferase activity (RLU/s) was detected in fractions 8–10 (containing EVs) and fraction > 16 (containing proteins) of CD63‐NanoLuc (purple, n = 3) and sec‐NanoLuc (green, n = 3) cell‐conditioned medium separated by size‐exclusion chromatography, respectively. Error bars represent mean ± SD (no statistical testing was performed). (D) In sec‐NanoLuc conditioned medium (green, n = 3), the luciferase activity (RLU/s) (normalized to Milli‐Q treated sample) was abolished by Proteinase K in contrast to CD63‐NanoLuc conditioned medium (purple, n = 3), indicating that the systems can be used to study EV and protein secretion. A paired t‐test was performed with error bars representing mean ± SD. (E) ALIX and Flotillin‐1 protein expression were detected in cells and cell‐conditioned…

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  FIGURE 3. The CD63‐NanoLuc and sec‐NanoLuc mouse model. (A) The CD63‐NanoLuc and sec‐NanoLuc models were generated by AAV delivery to an adipocyte‐specific Cre‐expressing mouse line (Adipoq‐Cre). In the presence of adiponectin‐driven Cre, the double‐floxed oriented viral CD63‐NanoLuc and sec‐NanoLuc transgenes were recombined and expressed under a ubiquitous promoter (CAG or EFS), leading to the labelling of adipocyte‐derived CD63‐positive EVs or secreted protein by Nanoluciferase (NanoLuc) and hemagglutinin (HA)‐tag. (B) Adipose tissue‐specific HA‐tag protein expression was detected in both models (+) compared to wild‐type tissues (−). Full blots are shown in Figure S6N–O. (C) The HA‐tag protein expression was strongest in the adipose tissue of CD63‐NanoLuc mice compared to sec‐NanoLuc mice, while both had strong signals in the kidneys. Vague HA‐tag expression was also detected in the liver, small intestine and pancreas compared to wild‐type tissues. The scalebar represents 50 µm. (D) Human CD63 protein expression was determined between tissues with (Anti‐CD63) or without (Background) primary antibody targeting human CD63. Human CD63 protein expression was strong in adipose tissues of the…

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  FIGURE 4. A 2‐week diet experiment using the CD63‐NanoLuc and sec‐NanoLuc models. (A) Three weeks post AAV delivery, CD63‐NanoLuc and sec‐NanoLuc mice were put on a chow diet or high‐fat diet (HFD) for 2 weeks before sacrifice. The body weight (BW) and heart weight (HW) were measured before and after the diet experiment. (B) Tissues with significantly higher luciferase activities in CD63‐NanoLuc mice on HFD than the chow diet did not have markedly higher protein expression of human CD63. The presence of human CD63 was determined between tissues with (Anti‐hCD63) or without (Background) primary antibody targeting human CD63. The scalebar represents 50 µm. (C) No difference was observed in HA‐tag protein expression between diets of CD63‐NanoLuc mice. The scalebar represents 50 µm. (D) CD63‐NanoLuc mice had significantly higher accumulated luciferase activity in HFD‐fed mice (blue, n = 12) compared to chow diet‐fed mice (orange, n = 13), in contrast to no significant difference between chow diet (green, n = 10) and HFD (pink, n = 10) among sec‐NanoLuc mice. Mann–Whitney tests were performed with error bars representing the median and IQR. (E) Heatmap luciferase activity in tissues and body…