FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase.

Generation of DA neuron-specific FoxO1 KO (FoxO1 KODAT) mice.(a) DA neuron-specific FoxO1 deletion. Green fluorescence indicates FoxO1. TdTomato (Td, red) was used to visualize DA neurons expressing DAT-cre. DAPI stains nuclei (blue). Scale bar, 50 μm. (b) Allele-specific PCR using different brain areas from FoxO1F/+, FoxO1F/F, and FoxO1 KODAT (DAT-cre; FoxO1F/F). SN, substantia nigra. Hypo., hypothalamus. Cb, cerebellum. (c,d) Immunoblots (c) and relative FoxO1 protein levels (d) from indicated brain regions of FoxO1 KODAT and WT littermates. (e) Brain weight of FoxO1 KODAT and WT littermates. (f) Representative figures and graphs showing the brain width of FoxO1 KODAT and WT littermates. Scale bar, 10 mm. (g–i) Representative figures (g) and graphs showing the number (h) and size (i) of DA neurons of FoxO1 KODAT and WT littermates. Scale bar, 50 μm. The values are mean±s.e.m. (*P<0.05, **P<0.01, Student's t-test).

Metabolic phenotypes of FoxO1 KODAT mice in chow diet.(a) Body weight of male mice on chow diet. Body weight data were combined from three cohorts of mice. (b) GTT and area under curve (AUC) of 24-week old male mice fed chow diet. (c) Plasma insulin levels of male mice during GTT (Not significant from two-way analysis of variance (ANOVA)). (d) ITT and AUC of 24-week-old male mice on chow diet. The results are expressed as mean±s.e.m. (*P<0.05, Student's t-test for bar graphs and two-way ANOVA for comparison of multiple time points).

FoxO1 KODAT mice are resistant to diet-induced obesity.(a) Body weight change (%) after HFD feeding (HFD started at 8 weeks old). Body weight data were combined from 4 cohorts. (b) Body fat and lean mass of male mice at 6 weeks on HFD. (c,d) Serum insulin (c) and leptin (d) levels of male mice at 6 weeks on HFD (Body weight, WT: 38.93±1.75 g, KO: 34.95±1.63 g, P=0.13 for c and WT: 37.87±1.78 g, KO: 33.92±1.50 g, P=0.12 for d, respectively). (e) Blood glucose (fed) level of male mice at 10 weeks on HFD (Body weight, WT: 42.71±2.12 g and KO: 36.18±2.17 g, P=0.04). (f) GTT and AUC of male mice on HFD for 10 weeks (Body weight, WT: 37.94±2.07 g and KO: 32.74±1.31 g, P=0.04). (g) ITT and AUC of male mice on HFD for 10 weeks (Body weight, WT: 39.64±1.96 g and KO: 33.86±1.55 g, P=0.03). The results are expressed as mean±s.e.m. (*P<0.05, Student's t-test for bar graphs and two-way analysis of variance for comparison of multiple time points in line graphs).

Feeding behaviour of FoxO1 KODAT mice.(a) Daily food intake of 8-10 weeks old male mice on chow diet (averaged from 7 days). (b–d) Rebound food intake (b) rebound weight gain (c) and rebound blood glucose (d) of overnight fasted male mice after re-feeding with normal chow. (e) Percentage of sucrose preference in fed and fasted conditions of male mice. 2% sucrose was used for the preference test. (f) Daily HFD consumption of 8-10 weeks old male mice (averaged from 7 days). The results are expressed as mean±s.e.m. (*P<0.05, Student's t-test for bar graphs in a,e–f and two-way analysis of variance for comparison of multiple time points in b–d).

Increased energy expenditure in FoxO1 KODAT mice.(a) Cumulative food intake of male mice fed on HFD for 1 week. (b) Locomotor activity. (c) Temporal changes of O2 consumption. (d) Average O2 consumption. (e) Temporal changes of CO2 production. (f) Average CO2 production. (g) Heat generation between genotypes. (h) Rectal temperature of male and female mice measured at room temperature environment. ♂, male. ♀, female. NS, not significant. Data are expressed as mean±s.e.m. (*P<0.05, Student's t-test for bar graphs in h and two-way analysis of variance for multiple comparisons in a–g).

Increased catecholamines level and iBAT thermogenesis in FoxO1 KODAT mice.(a) Serum norepinephrine levels of WT and KO male and female mice. (b) Dopamine contents measured from the midbrain and SN samples of WT and KO male mice. (c) Gene expression in iBAT of WT and KO male mice. (d) Immunoblots for p-Creb, p-p38 MAPK, UCP1, PGC1α in iBAT. (e) Relative protein levels for p-Creb, p-p38 MAPK, UCP1, PGC1α in iBAT from (d). Normalized to total p38 MAPK or GAPDH. (f) Representative figures of H&E staining and UCP1 staining from iBAT samples of WT and KO mice (n=3). Scale bar, 50 μm. (g) Quantification of mitochondrial DNA contents from iBAT samples of WT and KO mice. ♂, male. ♀, female. Data are expressed as mean±s.e.m. (*P<0.05, **P<0.01, Student's t-test).

FoxO1 directly regulates tyrosine hydroxylase (TH) expression in DA neurons.(a) mRNA level of Th in DA neurons of WT and KO littermates. (b) Immunoblots for TH and phosphorylated TH (p-TH) in SN and midbrain from WT and KO littermates. (c) Relative TH and p-TH protein levels from b. (d) Top, schematic diagram for mouse TH promoter. Bottom, ChIP assays using whole-brain and Neuro2A cells transfected with myc-tagged FoxO1-ADA showing a direct and specific binding of FoxO1 on the proximal region of TH promoter. (e) Top, schematic diagram for luciferase constructs with or without FoxO1 potential binding sites. Bottom left, relative luciferase activity after FoxO1-WT (WT), constitutive active form of FoxO1 (ADA) and dominant-negative form of FoxO1 (DN) overexpression (n=6). Bottom right, immunoblots confirming expression of FoxO1-WT, -ADA, and -DN. (f) Th mRNA expression in Neuro2A cells after FoxO1-WT and -ADA overexpression (n=9). (g) Th mRNA level in Neuro2A cells after FoxO1 knockdown (n=9). The results are expressed as mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001, Student's t-test and one-way analysis of variance for luciferase and quantitative real-time PCR analyses) from more than three independent experiments.