FoxO transcription factors are required for hepatic HDL cholesterol clearance

Abstract

Insulin resistance and type 2 diabetes are associated with low levels of high-density lipoprotein cholesterol (HDL-C). The insulin-repressible FoxO transcription factors are potential mediators of the effect of insulin on HDL-C. FoxOs mediate a substantial portion of insulin-regulated transcription, and poor FoxO repression is thought to contribute to the excessive glucose production in diabetes. In this work, we show that mice with liver-specific triple FoxO knockout (L-FoxO1,3,4), which are known to have reduced hepatic glucose production, also have increased HDL-C. This was associated with decreased expression of the HDL-C clearance factors scavenger receptor class B type I (SR-BI) and hepatic lipase and defective selective uptake of HDL cholesteryl ester by the liver. The phenotype could be rescued by re-expression of SR-BI. These findings demonstrate that hepatic FoxOs are required for cholesterol homeostasis and HDL-mediated reverse cholesterol transport to the liver.

Keywords: Cholesterol; Insulin signaling; Lipoproteins; Metabolism.

Figure 1. Plasma cholesterol profiles of chow-fed and WTD-fed L-FoxO1,3,4 mice.

(A) Total plasma cholesterol in chow-fed L-FoxO1,3,4 mice and littermate controls after a 5-hour fast (n = 5). Data are presented as the mean ± SEM. (B) Cholesterol levels in plasma fractionated by FPLC in the same mice as in A. (C) Western blot of plasma apoA-I and apoE from pooled pairs of fractionated plasma obtained from FPLC in B. (D) Total plasma cholesterol levels in WTD-fed L-FoxO1,3,4 mice and littermate controls after a 5-hour fast (n = 5). (E) Cholesterol levels in plasma fractionated by FPLC in the same mice as in D. (F) Western blot of plasma apoA-I and apoE from pooled pairs of fractionated plasma obtained from FPLC in E. Independent FPLC cholesterol profiles for both chow-fed and WTD-fed mice yielded qualitatively identical results. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by Student’s t test.

Figure 2. Defects in HDL metabolism genes due to ablation of hepatic FoxOs.

(A and B) Relative hepatic gene expression by qPCR in (A) chow-fed L-FoxO1,3,4 mice and littermate controls (n = 4–5) and (B) WTD-fed L-FoxO1,3,4 mice and littermate controls (n = 6–7). (C) Representative Western blot of hepatic SR-BI expression in both chow-fed and WTD-fed L-FoxO1,3,4 mice and littermate controls. SR-BI/actin denotes relative SR-BI expression levels by densitometric scanning (n = 4/group total). (D) Relative plasma HL activity in WTD-fed L-FoxO1,3,4 mice and littermate controls (n = 5–7). *P < 0.05, **P < 0.01, and ****P < 0.0001; ^§§P < 0.01 between chow-fed L-FoxO1,3,4 mice and littermate controls; ^††P < 0.01 between WTD-fed L-FoxO1,3,4 mice and littermate controls by Student’s t test. Data are presented as the mean ± SEM.

Figure 3. Plasma decay kinetics of ¹²⁵I-TC–/[³H]CEt-WT-HDL in chow-fed L-FoxO1,3,4 mice.

(A and B) ¹²⁵I-TC–/[³H]CEt-WT-HDL was injected i.v. into chow-fed L-FoxO1,3,4 mice and littermate controls. Thereafter, over a 24-hour period, blood samples were harvested periodically, and plasma was analyzed for ¹²⁵I-TC (crosses) and [³H]CEt (circles). The y axis represents the fraction of the tracer in plasma (percentage). Shown is a trace from a representative mouse from each genotype, with (A) control and (B) L-FoxO1,3,4 mice. The experiment was performed in 7 control and 5 L-FoxO1,3,4 mice.

Figure 4. Plasma FCRs and tissue tracer uptake rates for ¹²⁵I-TC–/[³H]CEt-WT-HDL in chow-fed L-FoxO1,3,4 mice.

¹²⁵I-TC–/[³H]CEt-WT-HDL was injected i.v. into chow-fed L-FoxO1,3,4 mice and littermate controls. (A) During the subsequent 24-hour period, blood was harvested periodically to determine the plasma decay of both tracers. ¹²⁵I-TC (¹²⁵I) and [³H]CEt ([³H]) were analyzed, and plasma FCRs for ¹²⁵I-TC and [³H]CEt were calculated. The difference in plasma FCRs between [³H]CEt and ¹²⁵I-TC was calculated. Twenty-four hours after tracer injection, the animals were euthanized, and tissues were analyzed for both tracers. (B) Liver, (C) adrenal gland, and (D) kidney organ FCRs for ¹²⁵I-TC (¹²⁵I) and [³H]CEt ([³H]). The difference in organ FCRs between [³H]CEt and ¹²⁵I-TC ([³H]CEt – ¹²⁵I-TC) was calculated. All calculations were done as described in Methods. n = 7 control mice; n = 5 L-FoxO1,3,4 mice. An independent experiment yielded qualitatively identical results. **P < 0.01, ***P < 0.001, and ****P ≤ 0.0001, by Student’s t test. No significant differences between genotypes were detected in adrenal glands. Data are presented as the mean ± SEM.

Figure 5. Uptake of ¹²⁵I-TC–/[³H]CEt-WT-HDL by hepatocytes isolated from chow-fed L-FoxO1,3,4 mice.

(A and B) Hepatocytes from chow-fed L-FoxO1,3,4 mice and littermate controls were incubated (37°C, 2 hours) in medium containing ¹²⁵I-TC–/[³H]CEt-WT-HDL (10, 20, 40, or 100 μg HDL protein/ml). Finally, cells were harvested, and apparent HDL particle uptake was analyzed as outlined in Methods. Values represent the mean of (A) n = 3 (control) and (B) n = 3 (L-FoxO1,3,4) independent determinations. An independent similar experiment yielded qualitatively identical results. Where no error bars are visible, the SEM was smaller than the symbol. (C) Western blots of SR-BI in primary hepatocytes from L-FoxO1,3,4 mice and littermate controls. Representative bands are shown. Two independent experiments yielded qualitatively identical results. (D) Relative Scarb1 and Lipc expression by qPCR in hepatocytes from chow-fed L-FoxO1,3,4 mice and littermate controls at different stages of the cell isolation procedure (see Methods). (A and B) ^§§§P < 0.001 and ^§§§§P < 0.0001, comparing [³H]CEt between L-FoxO1,3,4 and control hepatocytes; ***P < 0.001 and ****P < 0.0001, comparing [³H]CEt – ¹²⁵I-TC between L-FoxO1,3,4 and control hepatocytes; and ^†P < 0.05 and ^††P < 0.01, comparing ¹²⁵I-TC between L-FoxO1,3,4 and control hepatocytes (Student’s t test). (D) ^¶¶P < 0.01, ^¶¶¶P < 0.001, and ^¶¶¶¶P < 0.0001, comparing gene expression between L-FoxO1,3,4 and control hepatocytes at each stage; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001, comparing gene expression between control hepatocytes at baseline (before filtering) and control hepatocytes at each stage; and ^‡‡P < 0.01 and ^‡‡‡‡P < 0.0001, comparing gene expression between L-FoxO1,3,4 hepatocytes at baseline (before filtering) and L-FoxO1,3,4 hepatocytes at each stage (2-way ANOVA, followed by Tukey’s post hoc test). All data are presented as the mean ± SEM.

Figure 6. Ad.SR-BI in WTD-fed L-FoxO1,3,4 mice.

(A) Western blot of hepatic SR-BI expression in WTD-fed L-FoxO1,3,4 mice and littermate controls transduced with Ad.SR-BI or Ad.GFP control virus. SR-BI/actin denotes relative SR-BI expression levels by densitometric scanning (n = 2–4). (B) Cholesterol levels in plasma fractionated by FPLC in the same mice as in A. (C) Western blots of plasma apoA-I and apoE in fractionated plasma obtained from FPLC in B. Lanes a, b, and c correspond to pooled fractions 30–33, 34–39, and 40–45, respectively.

Figure 7. Acute knockdown via Cre adeno-associated virus in chow-fed hepatic FoxO–floxed mice.

(A–D) Chow-fed, adult Foxo1^fl/fl, Foxo3^fl/fl, and Foxo4^fl/Y control mice transduced with adeno-associated virus (serotype 8) expressing Cre recombinase driven by the hepatocyte-specific Tbg promoter (AAV8.Tbg.Cre) or control virus (AAV.GFP) (n = 5). For comparison, we also included a group of traditional L-FoxO1,3,4–knockout mice that were not transduced with virus, but rather had FoxO deficiency since birth (n = 4). Four weeks after virus transduction, mice were fasted for five hours, and then plasma and livers were collected. (A) Cholesterol levels in plasma fractionated by FPLC 4 weeks after virus transduction. (B) Relative hepatic gene expression by qPCR. (C) Representative Western blot of hepatic SR-BI expression. SR-BI/actin denotes relative SR-BI expression levels by densitometric scanning. *P < 0.05 and **P < 0.01 versus AAV.GFP mice. No significant difference was detected between AAV8.Tbg.Cre mice and L-FoxO1,3,4 mice. (D) Correlation between Scarb1 or Lipc mRNA and FoxO1 mRNA. The P values in B and C were calculated by 1-way ANOVA, followed by a post hoc t test using the pooled SD, without Bonferroni’s correction. Data are presented as the mean ± SEM.