To directly determine the role of FoxO1 in DA neurons-directed metabolic homoeostasis, we generated DA-specific FoxO1 knockout mice (KO, FoxO1 KODAT) by crossing DATIREScre with FoxO1loxP/loxP (FoxO1F/F) mice41415. Littermate mice homozygous for the floxed FoxO1 allele (FoxO1F/F) and without the DATIREScre allele served as controls (WT, wild-type). The tdTomato reporter mice16 and co-immunostaining with antibody against TH-verified DATIREScre activity specifically in DA neurons as reported (Supplementary Fig. 1a–c)1517. Immunofluorescence showed robust FoxO1 signal in virtually all DA neurons and the elimination of that signal in the KO mice (Fig. 1a). Furthermore, allele-specific PCR confirmed FoxO1 locus rearrangement (delta band), consistent with the deletion of FoxO1 in DA neurons including substantia nigra (SN) and midbrain containing the ventral tegmental area (VTA)18 but not in other brain regions such as hypothalamus, cortex, cerebellum and peripheral tissues including adrenal glands, BATs, muscles, livers or in WT mice (Fig. 1b and Supplementary Fig. 1d). In addition, FoxO1 protein levels of the KO mice were significantly decreased in VTA and SN but not in other CNS areas (Fig. 1c,d, and Supplementary Fig. 7) or peripheral
