Figure 1. Brown adipocytes secrete exosomes.

(a) Upper lane: Expression of CD63–GFP fusion protein in murine brown adipocytes. Representative bright field (left) and fluorescence image (right) are shown, (scale bar, 10 μm). Lower lane: electron microscopy images of exosomes in supernatant of brown adipocytes, (left scale bar, 120 nm, right scale bar, 40 nm, arrow heads indicate exosomes). (b) Qualitative western blot of exosome marker protein CD63 and Hsp70 expression in exosomes released from BAT (left panel) and in BAT of mice (right panel). The tissue was treated ex vivo with 10 μM norepinephrine (NE) or without NE (wt), protein isolation buffer served as vehicle control. Western blotting of tubulin and cytochrome c (cytC) are shown as loading control and cellular marker (n=1). (c,d) ELISA quantification of CD63-positive particles released from cells per mg protein before and after cAMP (200 μM) treatment (c) and released per mg tissue before and after cold exposure (4 °C for 7 days) (d). 3T3-L1, 3T3-L1 differentiated white adipocytes; BA, brown adipocytes; BAT, brown adipose tissue; beige, 12 h 1 μM NE treated or untreated (Control) differentiated white adipocytes; C₂C₁₂, muscle cells; exo, exosomes; HepG2, liver cells; PRE, brown pre-adipocytes; WATg, gonadal white adipose tissue; WATi, inguinal white adipose tissue. Data are presented as mean±s.e.m. (unpaired, two-tailed t-test, *P<0.05, **P<0.01, ***P<0.001, n=3 for cells, n=6 for mice).