(A) Hepatic MTP protein levels. (B) Hepatic MTTP mRNA levels. (C) Hepatic MTTP mRNA levels. HepG2 cells were transduced with control LacZ or FoxO1 vector or FoxO1 plus CA-Akt vector (MOI, 25 pfu/cell), followed by the determination of MTTP mRNA levels after 24-h incubation in the presence or absence of insulin (100 nM). (D) FoxO1 subcellular distribution. FoxO1 vector-transduced HepG2 cells were incubated with control or CA-Akt vector for 24 h, followed by immunoblot analysis of FoxO1 protein levels in cytosolic and nuclear fractions (E). (F) Hepatic MTTP mRNA levels. HepG2 cells were treated with PI3K activator IRS-1 (Y608) peptide (IRS-1, 1 μM) or inhibitor LY294002 (LY, 10 μM) in the presence and absence of insulin for 24 h. (G) FoxO1 induction of MTP promoter activity. HepG2 cells were cotransfected with 2 μg each pGH11 and pCMV-LacZ vectors in the presence of Adv–FoxO1-ADA vector at doses ranging from 50 to 400 pfu/cell, followed by luciferase activity assay after 24-h incubation. Likewise, HepG2 cells were cotransfected with pGH11 and pCMV-LacZ plasmids together with FoxO1 (H) or FoxO1-ADA (I) vector at a fixed dose (MOI, 100 pfu/cell) in the presence and absence of insulin for the determination of luciferase activity. (J) FoxO1 subcellular distribution. FoxO1 vector-treated HepG2 cells were incubated in the absence or presence of insulin for 30 min, followed by immunoblot analysis of FoxO1 in cytosolic and nuclear fractions (K). (L) Insulin inhibition of MTP promoter activity. FoxO1 vector-transduced HepG2 cells were transfected with MTP promoter–directed reporter system in the presence or absence of insulin. Cells were assayed for luciferase activity at different times. Data represent 3–5 experiments. *P < 0.05, ^#P < 0.001 versus controls.

(A) EMSA for assaying FoxO1 binding to DNA. HepG2 cells were transduced with FoxO1 vector at an MOI of 200 pfu/cell. Cells were lysed for the preparation of nuclear protein extracts 24 h after transduction. Aliquots of FoxO1 protein extracts (5 μg) were incubated with a biotin-labeled DNA probe, followed by chemiluminescent EMSA. DNA probe was derived from a 27-bp DNA covering the consensus IRE (–499/–473 nt) of the mouse MTP promoter, shown in lanes 1–4. A mutant DNA with an altered IRE motif was used as a control, shown in lanes 5–8. In addition, ChIP assay was used to assay for FoxO1 binding to DNA in cells. HepG2 cells were transfected with pGH11 in the presence of FoxO1 vector at an MOI of 100 pfu/cell in triplicate. After 24-h incubation, cells were cross-linked with 1% formaldehyde, followed by ChIP assay using rabbit anti-FoxO1 antibody (lanes 1 and 2) or preimmune IgG as a control (lanes 3 and 4). Immunoprecipitates were subjected to immunoblot assay using anti-FoxO1 antibody (B), and to PCR analysis using a pair of primers flanking the IRE sequence in the MTP promoter (C). As a positive control, aliquots of input DNA samples (1 μl) were used in PCR analysis. As a negative control, the immunoprecipitates were subjected to PCR analysis using a pair of off-target primers flanking a distal region (–3,528/–3,045 nt) devoid of the consensus IRE motif at 3 kb upstream of the MTP promoter.

Liver tissues collected from sacrificed mice in 6-month old FoxO1^S253A transgenic (n = 6) and control groups (n = 6) were used for the preparation of total RNA. Hepatic mRNA levels of SREBP-1c (A), FAS (B), ACC (C), ACOX-1 (D), PPARα (E), and HNF4α (F) were determined using real-time quantitative RT-PCR assay. *P < 0.05 versus control.

Male C57BL/6J mice (9 weeks old) were stratified by body weight and randomly assigned to 2 groups (n = 8), which were intravenously injected with Adv–FoxO1-RNAi or Adv-null vectors at 1.5 × 10¹¹ pfu/kg body weight. Three days after vector administration, mice were fasted for 24 h, followed by the determination of fasting blood glucose (A), plasma insulin (B), plasma cholesterol (C), plasma TG (D), and plasma NEFA levels (E). Mice were intravenously injected with 500 mg/kg of tyloxapol on day 5 after a 5-h fast. (F) Plasma TG levels were determined at different times. The relative rates of hepatic VLDL production, defined as mg of TG produced per kg of body weight per unit of time, were calculated from F based on the slopes of plasma TG profiles. (G) The mean VLDL production rates were compared between control and FoxO1-RNAi groups. (H) Aliquots of plasma (20 μg protein) 80 min after tyloxapol injection were analyzed by semiquantitative western blot assay using anti-apoB antibody for the determination of plasma apoB secretion. Mice were sacrificed after an overnight fast on day 10. (I) Liver tissues were collected for the determination of hepatic TG content. Aliquots of liver tissue were used for the preparation of hepatic protein extracts, which were subjected to semiquantitative immunoblot assay for the determination of hepatic FoxO1 (J) and MTP (K) protein levels in control and FoxO1-RNAi groups, using actin protein as an internal control. *P < 0.05 versus control.

Male db/db mice at 3 months of age were stratified by body weight and randomly assigned to 2 groups (n = 8), which were intravenously injected with FoxO1-RNAi or scrambled RNAi vector at 1.5 × 10¹¹ pfu/kg body weight. Three days after vector administration, mice were fasted for 24 h for the determination of fasting blood glucose (A) and plasma insulin levels (B). Mice were intravenously injected with 500 mg/kg of tyloxapol on day 5 after a 5-h fast. (C) Plasma TG levels were determined at different times. (D) The relative rates of hepatic VLDL production were calculated based on the slopes of plasma TG profiles from C and compared between control scrambled RNAi and FoxO1-RNAi groups. (E) Aliquots of plasma (20 μg protein) 80 min after tyloxapol injection were analyzed by semiquantitative western blot assay using anti-apoB antibody for the determination of plasma apoB secretion. Fasting plasma levels of TG (F), NEFA (G), and cholesterol (H) were determined at day 3 after vector administration. (I) Body weight was determined on day 7. Mice were sacrificed on day 7, and liver tissues were collected for the determination of hepatic protein levels of FoxO1 (J) and MTP (K) as well as hepatic TG content (L). *P < 0.05 versus control.

Insulin inhibits FoxO1 activity via Akt/PKB-dependent phosphorylation, resulting in FoxO1 nuclear exclusion. This effect is instrumental for liver to curb hepatic glucose and VLDL-TG production and limit postprandial glucose and lipid excursion. Loss of insulin inhibition of FoxO1 activity in insulin-resistant livers results in excessive production of both glucose and VLDL-TG, contributing to the dual pathogenesis of hyperglycemia and hypertriglyceridemia in diabetes.

(A) HepG2 cells in 6-well microplates were cotransfected with pCVM5-LacZ vector plus wild-type or mutant MTP promoter–directed luciferase reporter systems in the presence of Adv-null vector (–) or Adv-FoxO1 vector (+) at a fixed dose (MOI, 200 pfu/cell). After 24-h incubation, the luciferase activities were normalized to β-gal activity and then determined and compared between control and FoxO1 conditions. Data were obtained from 8 experiments. (B) Response of wild-type and mutant MTP promoters to insulin. HepG2 cells were transfected with test plasmids using pCMV-LacZ as a control and transduced with either Adv-null or Adv-FoxO1 vector in the presence and absence of insulin (100 nM). The relative luciferase activity for each construct was determined after 24-h incubation. Data were obtained from 4–9 experiments. *P < 0.001 versus control.

Male FoxO1^S253A transgenic mice (n = 6) and control littermates (n = 6) at 6 months of age were fasted for 5 h, followed by intravenous injection of tyloxapol at 500 mg/kg body weight per mouse to inhibit plasma VLDL clearance. (A) Aliquots of tail vein blood were taken at different times for the determination of plasma TG levels. (B) The relative rates of VLDL secretion are defined by the slopes of linear increases of plasma TG as a function of time following intravenous injection of tyloxapol. (C) Aliquots of plasma (20 μg protein) obtained from mice at 80 min after tyloxapol injection were analyzed by semiquantitative immunoblot assay using anti-apoB antibody for the determination of plasma apoB secretion. Mice were sacrificed at 6 months of age, and liver tissues were subjected to semiquantitative immunoblot analysis for the determination of hepatic MTP protein abundance (D), and separately to real-time quantitative RT-PCR for the determination of hepatic MTTP mRNA levels (E). *P < 0.05 versus control. In addition, liver tissues were subjected to ChIP analysis using preimmune rabbit serum (lanes 1 and 2) and rabbit anti-FoxO1 antibody (lanes 3 and 4). The resulting immunoprecipitates were analyzed by PCR (F) and immunoblot (G) assays as described in Figure 3.

HepG2 cells in 6-well plates were transduced with either control or FoxO1-ADA vector at an MOI of 100 pfu/cell. (A) After 24-h incubation, conditioned media were collected for the determination of TG levels. Cells were collected for the determination of apoB100 mRNA abundance by real-time quantitative RT-PCR assay using β-actin mRNA as control (B), and for determination of apoB100 protein levels by semiquantitative immunoblot assay using anti-apoB100 and anti-actin antibodies (C). In addition, HepG2 cells pretransduced with Adv–FoxO1-ADA and Adv-null vectors were pulse-labeled with 200 μCi/ml of L-[³⁵S]-methionine for 1 h, followed by a 30-min chase with 10 mM unlabeled methionine. Conditioned medium and cell lysates were subjected to electrophoresis on 5% SDS-polyacrylamide gels, which were dried prior to autoradiography. Gel slices corresponding to apoB100 protein bands were excised from gels with the aid of autoradiogram for the determination of radioactive content of apoB100 proteins that were secreted into conditioned medium (D) and present in cells (E). Data were from 3 experiments. *P < 0.05; **P < 0.001 versus control.

Liver tissues collected from sacrificed C57BL/6J mice in control and RNAi treatment groups (n = 8 per group) were used for the preparation of total RNA. Hepatic mRNA levels of SREBP-1c (A), FAS (B), ACC (C), ACOX-1 (D), PPARα (E), and HNF4α (F) were determined using real-time quantitative RT-PCR. *P < 0.05 versus control.

Male FoxO1^S253A transgenic mice at 4 months of age were stratified by body weight and randomly assigned to 2 groups (n = 8), which were intravenously injected with FoxO1-RNAi or scrambled RNAi vector at 1.5 × 10¹¹ pfu/kg body weight. (A) Fasting blood glucose levels. (B) Hepatic FoxO1 protein levels. (C) Fasting plasma insulin levels. (D) Plasma TG levels after intravenous administration of tyloxapol. (E) Hepatic VLDL production rates. (F) Plasma apoB secretion. Aliquots of plasma (20 μg protein) 80 min after tyloxapol injection were analyzed by semiquantitative western blot assay using anti-apoB antibody. (G) Hepatic MTP protein levels. (H) Fasting plasma TG levels. (I) Hepatic TG levels. (J) Fasting plasma NEFA levels. (K) Fasting plasma cholesterol levels. (L) Body weight. All data were obtained between 4 and 8 days from mice fasted for 24 h after vector administration. *P < 0.05 versus control.