Dissociation of the glucose and lipid regulatory functions of FoxO1 by targeted knockin of acetylation-defective alleles in mice

Abstract

FoxO1 integrates multiple metabolic pathways. Nutrient levels modulate FoxO1 acetylation, but the functional consequences of this posttranslational modification are unclear. To answer this question, we generated mice bearing alleles that encode constitutively acetylated and acetylation-defective FoxO1 proteins. Homozygosity for an allele mimicking constitutive acetylation (Foxo1(KQ/KQ)) results in embryonic lethality due to cardiac and angiogenesis defects. In contrast, mice homozygous for a constitutively deacetylated Foxo1 allele (Foxo1(KR/KR)) display a unique metabolic phenotype of impaired insulin action on hepatic glucose metabolism but decreased plasma lipid levels and low respiratory quotient that are consistent with a state of preferential lipid usage. Moreover, Foxo1(KR/KR) mice show a dissociation between weight gain and insulin resistance in predisposing conditions (high fat diet, diabetes, and insulin receptor mutations), possibly due to decreased cytokine production in adipose tissue. Thus, acetylation inactivates FoxO1 during nutrient excess whereas deacetylation selectively potentiates FoxO1 activity, protecting against excessive catabolism during nutrient deprivation.

Figure 1. FoxO1 acetylation and localization

(A) Immunoblot analysis of FoxO1 acetyl-lysine content in epididymal adipose tissue extracts from mice subjected to fasting and re-refeeding. (B) Fluorescence microscopy of primary mouse hepatocytes transduced with FoxO1-GFP-WT, -KR or -KQ adenoviruses and maintained in serum-free medium (upper panel) or incubated with insulin (lower panel). We show representative images. (C–D) Immunoblotting analysis of FoxO1 content in nuclear and cytosolic fractions isolated from epididymal adipose tissue extracts of fasted (C) or refed (D) wild type (WT) or Foxo1^KR/KR (KR) animals. We used nucleophosmin (NPM) or tubulin as markers of cellular fractions in (C) and Hdac1 or βactin in (D).

Figure 2. Embryonic lethality in mice homozygous for the Foxo1^KQ/KQ mutation

(A–B) Microphotographs of Foxo1^+/+ and Foxo1^KQ/KQ embryos at E10.5 and E11.5. (C–D) Microphotographs of E9.5 embryos showing first (I) and second branchial arch (II), as well as atrioventricular canal (AVC) and ventricle (V). Arrow in D indicates the distended AVC in the mutant. (E–F) Histological sections of E9.5 embryos across the AVC (arrows) and ventricle. (G–L) Whole-mount PECAM-1 immunostaining with details of the aortic arch arteries in the head (arrowheads in I, missing in J) and the dorsal aorta (arrows) in the tail region (K–L) of a Foxo1^+/+ (G, I, and K) and Foxo1^KQ/KQ embryo (H, J, and L). Asterisks in k and l mark somites which are smaller in the mutant. (M–N) Histological sections through the first branchial arch showing dorsal aortae (arrows in M) and the first branchial arch artery (arrowheads in M). (M′-N′) Sections through more posterior regions showing paired dorsal aortae (arrows that are distended in the mutant (N′) compared with wild type (M′). (O–P) Vascular plexus in the yolk sac in Foxo1^KQ/KQ and wild type embryos shown by immunostaining for PECAM.

Figure 3. Metabolic data in Foxo1^KR/KR mice

(A) HOMA-IR values calculated from the data in Table S2. (B) Pyruvate tolerance tests in WT and Foxo1^KR/KR mice. (C) Glucose infusion rates, (D) Endogenous glucose production under hyperinsulinemic conditions, and (E) Glycolysis during euglycemic hyperinsulinemic clamps. (F) Determination of TG content in lipoprotein fractions isolated by FPLC analysis. (G) Basal and isoproterenol-stimulated glycerol release from primary cultures of epididymal fat pads. n= ≥ 6 mice per group and each experiment. *= P<0.05; ** =P<0.01 by 2-way ANOVA. Data are presented as means ± SEM.

Figure 4. Gene expression analysis

(A)Hepatic expression of genes involved in glucose and (B, C) lipid metabolism in 2-month-old male mice. (D) Adipose expression of fatty acid and TG synthetic genes in the same cohort. n = ≥ 6 for each genotype. *=P<0.05, **=P<0.01 by Student’s t-test. Data are presented as means ± SEM.

Figure 5. Characterization of Foxo1^KR/KR mice in dietary and genetic models of insulin resistance

(A) Body weight curves, (B) body composition, and (C) intraperitoneal glucose tolerance tests in WT and Foxo1^KR/KR mice during high fat feeding. (D) Body weight, (E) 16-hr fasting glucose levels, (F) insulin levels in random fed, 16-hr fasted, and 4-hr re-fed, (G) glucose tolerance tests in male and (H) female db/db and Foxo1^KR/KR;db/db mice. n=6–12 for each genotype and each experiment. *=P<0.05, **=P<0.01 by 2-way ANOVA. Data are presented as means ± SEM.

Figure 6. Energy homeostasis studies

(A) Food intake, (B) body weight, (C) energy expenditure as measured by CO₂ produced (VCO₂), (D) respiratory exchange ratio (CO₂ produced/O₂ consumed) (RER), (E) locomotor activity, and (F) metabolic efficiency plots in 2 month old WT and Foxo1^KR/KR mice maintained on standard chow diet. n=8 for each genotype and each experiment. *=P<0.05 by 2-way ANOVA. Data are presented as means ± SEM.