Fig. 3: Activated BAT-derived EVs (Cold-BDEVs) enhances hepatic gluconeogenesis.
a–d 4 × 106 primary hepatocytes were cocultured with equal proteins of RT-BDEVs or Cold-BDEVs (32 μg) for 48 hours. a Schematic diagram. b Glucose output assay of hepatocytes (n = 6 biological replicates, from 2 independent experiments). c Relative mRNA expression of gluconeogenic genes in hepatocytes (n = 7 biological replicates, from 2 independent experiments). d Western blotting and densitometry analysis of phosphorylated and total AKT in hepatocytes (n = 6 biological replicates, from 2 independent experiments). e–l Eight-week-old male C57BL6/J mice were administered 80 μg equal protein of RT-BDEVs or Cold-BDEVs once daily for a total of 3 injections, and subsequent studies were performed 12 hours after the last injection. The mice were fasted for 4 h before sacrifice. e Schematic diagram. f Glucose levels during i.p. pyruvate tolerance tests (PTTs) (n = 6 each group, from 2 independent experiment). g Area under the curve of PTT, as indicated in Fig. 3f (n = 6 each group, from 2 independent experiment). h Relative mRNA expression of gluconeogenic genes in the liver (n = 8 each group, from 2 independent experiments). i Western blotting and densitometry analysis of phosphorylated and total AKT, FOXO1 and GSK3β in the liver. (n = 6 each group, from 2 independent experiment). Food intake j, serum insulin k and serum glucagon l levels in BDEV treated mice (n = 8 each group, from 2 independent experiments). m–o Cold-exposed eight-week-old male C57BL6/J mice were injected in situ with either GW4869 or control vehicle at 0, 24, and 48 h (for a total of 3 injections) and subsequent studies were performed 4 h after the last injection. The mice were fasted for 4 h before sacrifice. m Schematic diagram. n Western blotting and densitometry analysis of phosphorylated and total AKT in the liver (n = 6 each group, from 2 independent experiments). o Relative mRNA expression of gluconeogenic genes in the liver (n = 9 each group, from 3 independent experiments). Data presented as mean ± sem. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test f and others performed two-sided unpaired t-test. Source data are provided as a Source Data file.
