Inactivation of hepatic Foxo1 by insulin signaling is required for adaptive nutrient homeostasis and endocrine growth regulation

Abstract

The forkhead transcription factor Foxo1 regulates expression of genes involved in stress resistance and metabolism. To assess the contribution of Foxo1 to metabolic dysregulation during hepatic insulin resistance, we disrupted Foxo1 expression in the liver of mice lacking hepatic Irs1 and Irs2 (DKO mice). DKO mice were small and developed diabetes; analysis of the DKO-liver transcriptome identified perturbed expression of growth and metabolic genes, including increased Ppargc1a and Igfbp1, and decreased glucokinase, Srebp1c, Ghr, and Igf1. Liver-specific deletion of Foxo1 in DKO mice resulted in significant normalization of the DKO-liver transcriptome and partial restoration of the response to fasting and feeding, near normal blood glucose and insulin concentrations, and normalization of body size. These results demonstrate that constitutively active Foxo1 significantly contributes to hyperglycemia during severe hepatic insulin resistance, and that the Irs1/2 --> PI3K --> Akt --> Foxo1 branch of insulin signaling is largely responsible for hepatic insulin-regulated glucose homeostasis and somatic growth.

Figure 1. Hepatic deficiency of Irs1 and Irs2 results in insulin resistance and diabetes

(A, B) Irs1 or Irs2 mRNA levels in the liver of 6-week old control (Irs1^L/L, Irs2^L/L, 12^L/L for floxed Irs1, Irs2, or both, respectively) and liver-specific knockout mice for Irs1 (LKO1), Irs2 (LKO2), and both (DKO) were analyzed by real-time PCR (n=3). (C) Immunoblot analysis of Irs1 and Irs2 proteins in the liver of 6-week old control and knockout mice. (D–F) CNTR indicates the combined floxed gene control samples. (D) Quantitative immunoblot analysis of insulin signaling in the liver extracts of 8-week old CNTR and liver-specific knockout mice (n=3) injected via the vena cava with saline (−) or 5 units of insulin (+) for 4 min. Glucose (E) and insulin tolerance tests (F) of 8-week old male mice (n=8–12). Individual areas under curves were analyzed by ANOVA; groups that share a vertical bar at the final time point did not significantly differ. All other between-group comparisons in (E–F) were significant with p< 0.05.

Figure 2. Hyperglycemia in DKO-mice can be rescued by Irs1 or Irs2 re-expression or knockdown or knockout of Foxo1

(A) Immunoblot analysis of liver extracts from 12-week old floxed control (CNTR) and DKO-mice treated with adenovirus expressing control green fluorescent protein (Ad-GFP) or Irs1 (Ad-Irs1) or Irs2 (Ad-Irs2). (B) Immunoblot analysis of Foxo1 knockdown in the liver extracts of 12-week old CNTR and DKO-mice by adenovirus-mediated siRNA against Foxo1 (Ad-siFoxo1) or GFP (Ad-siGFP). (C) Fasting (6 hours) blood glucose levels in 12-week old mice treated with adenoviruses expressing GFP, Irs1, or Irs2, or siRNA against Foxo1 or control GFP (n=4). (D) Immunoblot analysis of Irs1, Irs2 and Foxo1 in liver extracts of 8-week old floxed control (CNTR) and liver-specific Irs1:Irs2:Foxo1 triple knockout (TKO) mice. Actin is used as a loading control. (E) Analysis of insulin signaling in 8-week old floxed control (CNTR), TKO and DKO-liver extracts using phospho-specific and total protein antibodies.

Figure 3. Gene expression in the liver of control and knockout mice

(A–C) The normalized expression of liver genes was analyzed in fasted (16 hours) and fed (4 hours) 6-week old control (CNTR), DKO, LKO1, LKO2 or TKO-mice (n=2–8) using Affymetrix GeneChips. Liver genes that were changed significantly (FDR<0.05) were further analyzed for either a positive (+) or negative (−) correlation with principal component 1 (PC1), PC2 and PC3 using the NIA Array Analysis Tool. Data were presented as the average normalized expression (log2 scale) of gene clusters positively correlated (●) and negatively correlated () with principal component PC1 (A), PC2 (B) or PC3 (C), respectively. The error bars represent the standard deviation. (D–F) Gene expression was independently confirmed by real-time PCR in the liver of 6-week old control (CNTR) and knockout mice (n=3) fasted for 16 hours (−) or fasted and then allowed access to food for 4 hours (+). (D) Expression of genes involved in metabolism, including Gck, Pck1, G6pc and Cpt1a. (E) Expression of genes involved in gene regulation, including Ppargc1a, Fgf21, Srebp1c and Onecut1. (F) Expression of genes involved in animal growth, including Ghr, Igf1, Igfbp1, and Igfals. Data are presented as relative expression of the gene of interest over β-actin (mean ± s.e.m.). * p<0.05 vs CNTR mice under the same feeding condition by Student’s t test.

Figure 4. Deletion of hepatic Foxo1 (TKO-mice) reverses the diabetes and growth retardation in DKO-mice

(A, B) Body length and bone mineral density by dual energy X-ray absorptiometry were measured for 3-month old floxed control (CNTR), DKO, and TKO-mice (n=4–9). Values were compared by Kruskal-Wallis (K-W) test and Dunn’s procedure with tabled p values for small n. * p<0.05 vs CNTR. (C) Growth curves of control, DKO and TKO-mice. Data (n=6–22 per group at each point) were analyzed by ANOVA and the analysis showed a significant difference between DKO-mice and the other groups with p<0.05. (D) Hepatic glycogen content was measured in 6-week old mice (n=4–6) that were refed for 4 hours after overnight fasting. The K-W test demonstrated a significant difference in data location for one or more of the groups (p< 0.01), but post-procedures did not identify significant between-group differences. (E) Signal transduction analysis in the liver of 6-week old control and knockout mice that were either fasted overnight (−) or refed for 4 hours after fasting (+). (F) Insulin resistance was determined by HOMA2 approximation using fasting blood glucose and insulin data collected from 7–8 weeks old mice (n=5–9). LKOF=Foxo1 liver-specific knockout mice. Data were analyzed by ANOVA and Scheffe post-procedure for comparison of all groups. * p<0.05 vs all other groups. (G, H) Insulin (G) and glucose tolerance tests (H) were performed in 6–8 weeks old liver-specific knockout (n=8–13) and pooled floxed control (n=26) mice. Individual areas under curves were analyzed by ANOVA; groups that share a vertical bar at the final time point did not significantly differ. All other between-group comparisons in (G, H) were significant with p< 0.05.

Figure 5. Lipid parameters and gene expression in DKO and TKO-mice

(A–C) Serum free fatty acids (FFA), triglyceride and cholesterol levels (mean ± s.e.m.) were measured after blood samples were collected from overnight fasted 8-week old control, DKO, and TKO-mice (n=6–10), respectively. Bars indicate interquartile ranges (1st to 3rd quartile) and horizontal lines inside the bars represent median values. Data were analyzed by ANOVA for comparison of all groups. * p<0.05 vs CNTR. (D) Serum total cholseterol, HDL-cholesterol, and LDL/VLDL-cholesterol levels were measured in the overnight fasted 4-month old control, DKO, and TKO-mice (n=6–8) using a commercial assay kit. * p<0.05 between CNTR and DKO or TKO-mice. (E) Hepatic triglyceride content was determined in 7-week old mice (n=3–15) that were fasted overnight. Bars indicate interquartile ranges (1st to 3rd quartile) and horizontal lines inside the bars represent median values. The K-W statistic analysis did not reach significance. (F) Triglyceride secretion was analyzed in 3-month old control, DKO, and TKO-mice (n=6–8) that were fasted for 4 hours and then injected with Triton WR1339. Serum triglyceride concentrations were measured at 0, 1, and 2 hours after the injection. At the 0 time point, the serum triglyceride levels in all groups of mice were below 10 mg/dl. * p<0.05 between CNTR and DKO or TKO-mice. (G) Normalized expression of top 10% significantly changed genes involved in cholesterol homeostasis and lipid synthesis was analyzed by Ingenuity Pathway Analysis software. Bars indicate interquartile ranges (1st to 3rd quartile) of normalized expression values and horizontal lines inside the bars represent median expression values. The gene set includes Abca1 [ATP-binding cassette, sub-family A (ABC1), member 1], Acyl (ATP citrate lyase), Cyp51a1 (cytochrome P450, family 51, subfamily A, polypeptide 1), Dhcr24 (24-dehydrocholesterol reductase), Dhcr7 (7-dehydrocholesterol reductase), Fasn (fatty acid synthase), Fdft1 (farnesyl-diphosphate farnesyltransferase 1), Fdps (farnesyl diphosphate synthase), Gck (Glucokinase), Hmgcr (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), Idi1 (isopentenyl-diphosphate delta isomerase 1), Ldlr (low density lipoprotein receptor), Mvk (mevalonate kinase), Nsdhl [NAD(P) dependent steroid dehydrogenase-like], Nudt7 [nudix (nucleoside diphosphate linked moiety X)-type motif 7], Pcsk9 (proprotein convertase subtilisin/kexin type 9), Sc4mol (sterol-C4-methyl oxidase-like), Sqle (squalene epoxidase), Thrsp (thyroid hormone responsive SPOT14 homolog). * p<0.05 between DKO and CNTR or TKO-mice (n=2–8). (H) Apolipoprotein levels were analyzed by immunoblotting analysis of fasted serum from control, DKO, and TKO-mice (n=2) using specific antibodies.

Figure 6. A working model for an integrative role of Foxo1 in the insulin and growth hormone signaling pathways

Insulin can inhibit Foxo1 transcriptional activity through Irs1/2→PI3K→Akt pathway to phosphorylate Foxo1. As a transcription factor, Foxo1 can activate one set of genes including Ppargc1a, Pck1, G6pc, and Igfbp1 and directly or indirectly suppress another set of genes including Ghr, Igf1, Onecut1, and Gck. Through regulation of Ghr gene expression, Foxo1 also influences expression of growth hormone-regulated genes including Igf1, Igfals and Onecut1. Moreover, in response to insulin and growth hormone signals, Onecut1 can impact on nutrient metabolism through activation of Foxa2 and Gck gene expression and suppression of Pck1 gene expression. Fonts in bold indicate protein molecules and fonts in italic indicate mRNA molecules. Arrows indicate activation and blunted lines represent inhibition. Solid lines or arrows indicate reported links in the literature and dotted lines or arrows indicate implicated links from this current study.