Foxo1 mediates insulin action on apoC-III and triglyceride metabolism (original) (raw)
Effects of Foxo1 on hepatic apoC-III expression. Previous studies indicated that hepatic apoC-III expression is negatively regulated by insulin (12, 13), but the underlying mechanism is unknown. To study the molecular mechanism underlying this inhibitory effect of insulin on hepatic apoC-III expression, we examined the effect of Foxo1 on hepatic apoC-III expression in cultured hepatocytes. Using an adenovirus-mediated gene delivery system, we transferred the wild- type Foxo1 cDNA to cultured rat hepatocytes and determined the intracellular apoC-III mRNA levels following 24 hours of Foxo1 transgene expression. Adenovirus-mediated production of Foxo1 significantly stimulated endogenous apoC-III expression (Figure 1A), which correlated with Foxo1 expression in hepatocytes (Figure 1B). In contrast, the level of apoC-III expression in control vector–transduced hepatocytes remained unchanged. To demonstrate that elevated hepatic apoC-III production in Foxo1 vector–transduced hepatocytes is attributable to the specific effect of Foxo1, we determined the expression level of glucokinase (GK), a glycolytic enzyme that is not regulated by Foxo1 (22), following Foxo1 expression in hepatocytes. As shown in Figure 1C, Foxo1 production did not affect GK expression, as reflected by the lack of changes in the relative level of GK mRNA in Foxo1 vector– versus control vector–transduced cells. These results demonstrate that Foxo1 stimulates hepatic apoC-III expression in cultured primary hepatocytes.
Effects of Foxo1 on hepatic apoC-III expression. Rat primary hepatocytes were transduced with Foxo1 or LacZ vector at an MOI of 50 PFU/cell or mock-transduced with PBS. After 24 hours of transduction, the intracellular levels of apoC-III (A), Foxo1 (B), and GK (C) mRNA were determined by real-time RT-PCR using β-actin mRNA as control. The effect of Foxo1 on hepatic apoC-III expression in response to insulin was assayed in HepG2 cells. Cells were transduced with Foxo1, Foxo1-ADA, or control LacZ vector (50 PFU/cell) in the absence or presence of insulin at different concentrations. Twenty-four hours after transduction, cells were collected for determination of the intracellular levels of apoC-III mRNA induced by Foxo1 (D) and Foxo1-ADA (E). *P < 0.05, **P < 0.005; significantly different from controls. NS, not significant by ANOVA. Data were from 3 independent experiments.
To study whether Foxo1 is an effective mediator of insulin in regulating hepatic apoC-III production, we transferred Foxo1 cDNA into HepG2 cells. Unlike primary hepatocytes, HepG2 cells express little Foxo1, so that the effect of Foxo1 on hepatic apoC-III expression would have to be ascribed to exogenous Foxo1. Here, we studied the endogenous expression level of apoC-III induced by Foxo1 transduction in the presence and absence of insulin. As shown in Figure 1D, adenovirus-mediated Foxo1 production significantly stimulated apoC-III mRNA expression, but the stimulatory effect was inhibited by insulin in a concentration-dependent manner. Insulin appeared to play a dominant role in inhibiting apoC-III expression, as the inhibitory effect of insulin was detected in the presence of Foxo1. To corroborate this finding, we expressed a constitutively active Foxo1 allele, Foxo1-ADA, in HepG2 cells. Foxo1-ADA contains 3 amino acid substitutions at three conserved phosphorylation sites and is unable to undergo insulin-dependent phosphorylation (17). As a result, Foxo1-ADA is associated with constitutive _trans_-activation of target gene expression (17). Indeed, adenovirus-mediated production of Foxo1-ADA resulted in a 3-fold induction of hepatic apoC-III expression in HepG2 cells. Unlike its wild-type counterpart, Foxo1-ADA–mediated stimulation was no longer subject to insulin inhibition, as the elevated apoC-III mRNA levels in Foxo1-ADA vector–transduced cells remained unchanged irrespective of the addition of insulin into culture media (Figure 1E).
To elucidate the molecular mechanism of the Foxo1-mediated stimulatory effect on hepatic apoC-III expression, we constructed an apoC-III promoter–directed luciferase reporter system in plasmid pHD317, in which the firefly luciferase cDNA was directed by the human APOC3 promoter (Figure 2A). We then transfected pHD317 into HepG2 cells in the presence and absence of Foxo1 expression using a β-gal–expressing plasmid as an internal standard for the normalization of transfection efficiency. In the absence of Foxo1 expression, transfection with pHD317 resulted in only basal luciferase expression. However, cotransfection with pHD317 and the Foxo1-expressing plasmid pCMV5-Foxo1 resulted in a greater than 4-fold induction of luciferase activity (Figure 2B). To confirm that this stimulatory effect on luciferase reporter expression was via a Foxo1-dependent mechanism, we determined the luciferase expression level following coexpression of Foxo1 and its dominant-negative mutant Foxo1-Ø256. Foxo1-Ø256, containing only the amino DNA binding domain of Foxo1, binds to Foxo1-target promoters in a competitive manner and interferes with Foxo1 function in a dominant-negative manner (17, 22). Consistent with its role, Foxo1-Ø256 was shown to counteract the effect of Foxo1 and reduce the luciferase reporter expression to basal levels (Figure 2B). These results suggest that Foxo1 might act directly on the APOC3 promoter in stimulating hepatic apoC-III production.
Effects of Foxo1 on the human APOC3 promoter activity. (A) The APOC-III promoter–directed luciferase reporter system. The wild-type and mutant IRE sequences are underlined. (B) Foxo1-mediated induction of the APOC3 promoter activity. HepG2 cells were transfected by pHD317 together with Foxo1 construct, or with both Foxo1 and Foxo1-Ø256 constructs. For each construct, 1 μg of DNA for each construct was used in transfection. For normalization of transfection efficiency, 1 μg pCMV5-LacZ DNA was included for normalization of transfection efficiency. (C) The APOC3 promoter variants in the luciferase reporter system. (D) Responses of APOC3 promoter variants to Foxo1 production. HepG2 cells were transfected with individual test plasmids in the absence (–) or presence (+) of pCMV5-Foxo1. The relative luciferase activity, after normalizing to β-gal activity, was compared between basal (–) and Foxo1-inducible (+) conditions. (E) Responses of wild-type and mutant APOC3 promoters to insulin. Test plasmids were transduced into HepG2 cells in the presence and absence of pCMV5-Foxo1 transfection in culture media, either supplemented with or without insulin (30 nM). The relative luciferase activity in transduced cells was determined using β-gal activity as control. *P < 0.001 vs. controls.
Characterization of Foxo1 -target site in the human APOC3 promoter. To characterize the Foxo1-target site in the APOC3 promoter, we constructed different versions of the human APOC3 promoter by progressively deleting portions of its upstream region. As shown in Figure 2C, the resulting mutant APOC3 promoters were subcloned into the luciferase reporter system, and the transcriptional activity of each mutant promoter in response to Foxo1 production was tested in HepG2 cells. As shown in Figure 2D, deletions up to –498 nt in the APOC3 promoter produced little effect on the responsiveness of the mutant promoters to Foxo1 production, as the transcriptional activity of the mutant promoters was induced to approximately the same level as that of their wild-type counterparts. However, further deletion up to –403 nt in the promoter completely abolished the responsiveness of the mutant promoter to Foxo1 induction, as evidenced by the lack of changes in the transcriptional activity of the mutant promoter in the presence and absence of Foxo1 production in cells. Thus, the Foxo1 target site was confined within a small nucleotide region between –498 and –403 in the human APOC3 promoter. Consistent with this observation, this DNA region contains a putative insulin response element (IRE). To confirm this finding, we generated 2 mutant promoter variants, one of which contained a small deletion (–498 to –403) and the other harbored 2 nucleotide substitutions (–A458C and –A460G) within the IRE DNA. The resulting mutant promoters were tested in HepG2 cells for their abilities to respond to Foxo1 induction using the luciferase reporter assay system. As shown in Figure 2D, both deletion and alterations of the IRE DNA sequence abrogated the responsiveness of the mutant promoters to Foxo1, as the transcriptional activity of the APOC3 promoter variants remained unchanged in the presence and absence of Foxo1 production in cells.
To address whether the resulting promoter variants are associated with the loss of insulin responsiveness, we transfected pHD322 and pHD334 together with the Foxo1-expressing plasmid pCMV5-Foxo1 to HepG2 cells in the presence and absence of insulin in culture media, followed by the determination of luciferase activity in the cells after 24 hours of incubation. As shown in Figure 2E, the intracellular level of luciferase activity remained at basal levels regardless of the presence or absence of insulin. As a positive control, cotransfection of HepG2 cells with both pHD317 and pCMV5-Foxo1 plasmids resulted in about a 4-fold induction of luciferase activity, which was suppressed to a basal level by the addition of insulin into culture media.
To study the molecular interaction between Foxo1 and the APOC3 promoter, we produced Foxo1 protein from a coupled in vitro transcription-translation system using pHD334, in which Foxo1 cDNA is under the control of the T7 promoter. After verification of Foxo1 by immunoblot analysis using anti-Foxo1 antibody, aliquots of the translation products were applied in an electrophoretic mobility shift assay (EMSA) using a radioactively labeled DNA probe from the APOC3 IRE (27 bp). As shown in Figure 3A, migration of this DNA fragment was retarded in the presence of Foxo1, resulting in a shifted DNA band. Inclusion of anti-Foxo1 antibody in the reaction mixture resulted in a supershifted DNA band, suggesting that this shifted DNA band is specific for Foxo1. In response to the addition of 50 M excess of nonlabeled cognate DNA to the reaction mixture, both DNA retardation and supershift were abolished. As a positive control, a previously characterized IRE DNA (28 bp) from phosphoenolpyruvate carboxykinase (PEPCK) promoter was applied to EMSA (23), and similar results were produced (Figure 3B). As a negative control, the mutant APOC3 IRE containing 2 nucleotide substitutions (Figure 2A) was applied to EMSA and no DNA retardation was detected in the presence of Foxo1, correlating with the inability of the mutant APOC3 promoter to respond to Foxo1 stimulation (Figure 2D). Using a similar assay, we and others have previously shown that Foxo1 is capable of binding to DNA fragments corresponding to the IRE sequences that are present in the promoter of Foxo1-targeted genes, including insulin-like growth factor binding protein 1 (IGFBP-1), PEPCK, and glucose-6-phosphatase (G6Pase) (18, 22, 24, 25).
Molecular interaction between Foxo1 and the APOC3 promoter. Molecular association between Foxo1 and the APOC3 promoter was analyzed by EMSA and ChIP. Aliquots of Foxo1 protein from linked in vitro transcription-translation products (5 μg) were incubated with 2.5 μl of radioactively labeled DNA corresponding to –467/–440 nt in the human APOC3 promoter (WT-IRE) (A), a mutant APOC3 IRE (mt-IRE) containing 2 substitutions, of –A458C and –A460G, and a control PEPCK IRE DNA (B), followed by electrophoresis through 8% nondenaturing polyacrylamide gels for 30 minutes. Lane 1, DNA probe alone. Lane 2, DNA probe + Foxo1 protein lysates. Lane 3, DNA probe + Foxo1 protein lysates + anti-Foxo1 antibody (1 μg). Lane 4, DNA probe + Foxo1 protein lysates + nonlabeled competitor DNA at a molar concentration of 50-fold excess. Free, shifted, and supershifted DNA bands were visualized by autoradiography. For ChIP assay, HepG2 cells were transduced with Foxo1 vector at an MOI of 50 PFU/cell. Cells were harvested 24 hours later and subjected to ChIP using PBS as a negative control (lane 5), control IgG (lane 6), and anti-Foxo1 antibody (lane 7). The coimmunoprecipitated chromatin DNA was analyzed by immunoblot (C) using anti-Foxo1 antibody and PCR (D) using the primers that correspond to –655/–20 nt of the APOC3 promoter.
To correlate this finding with the ability of Foxo1 to bind the APOC3 promoter in living cells, we performed chromatin immunoprecipitation (ChIP) on Foxo1-expressing HepG2 cells. The rationale is that if Foxo1 binds to the APOC3 promoter, then the resulting interaction between Foxo1 and the APOC3 promoter DNA should be detectable through immunoprecipitation of the DNA-protein complex using anti-Foxo1 antibody. HepG2 cells transduced with Foxo1 vector were divided into 3 aliquots and, which were subjected to ChIP analysis using rabbit anti-Foxo1 antibody, control IgG, or PBS buffer (as a negative control), respectively. The resulting immunoprecipitates were studied by immunoblot and PCR analyses. As expected, Foxo1 was detected only in the products that were immunoprecipitated with anti-Foxo1 antibody, not with control IgG or PBS (Figure 3C). Furthermore, a small DNA fragment (676 bp) corresponding to the proximal region (–675/+1) of the APOC3 promoter was detected in the immunoprecipitates by anti-Foxo1 antibody, as analyzed by PCR (Figure 3D). In contrast, no DNA products were amplified in control immunoprecipitates by the same PCR assay. These results demonstrate a direct protein-DNA interaction between Foxo1 and the APOC3 promoter in cells.
Effects of Foxo1 on apoC-III and TG metabolism in vivo. To examine the effect of Foxo1 on hepatic apoC-III production and plasma TG metabolism in vivo, we transferred Foxo1 cDNA into liver of mice. CD-1 mice at 12 weeks of age were stratified by body weight and randomly assigned to 1 of 3 groups to receive intravenous injections of 1.5 × 1011 PFU/kg of Foxo1 vector, LacZ vector, or vector buffer, respectively, as described (22). This approach has been shown to result in transduction of hepatocytes predominantly in liver, with little transduction in extrahepatic tissues (26). Animals treated with Foxo1 vector exhibited significantly elevated fasting plasma TG levels 2 days after vector administration (Figure 4A). In contrast, fasting plasma TG levels in control vector–treated animals remained unchanged. To assess their relative abilities to tolerate fat, we challenged the mice with an oral bolus of olive oil, followed by the determination of plasma TG profiles. As shown in Figure 4B, plasma TG concentrations in the Foxo1 group were markedly elevated and remained at a significantly higher level even after 3 hours of olive oil administration (380 ± 25 mg/dl vs. 110 ± 8 mg/dl in mock-treated control mice; P < 0.05 by ANOVA). In contrast, plasma TG excursion in control vector–treated animals was not significantly different from that in mock-treated controls. In addition, we determined the relative levels of plasma apoC-III by semi-quantitative immunoblot analysis. Consistent with elevated plasma TG levels in Foxo1 vector–treated mice, elevated Foxo1 production was associated with significantly increased plasma apoC-III levels (Figure 4C).
Effect of Foxo1 on hepatic apoC-III and plasma TG metabolism in vivo. CD-1 mice (12 weeks old) were stratified by body weight to ensure a similar mean body weight per group (31 ± 1.4 g, n = 6). The groups were Foxo1 vector–treated, LacZ vector–treated, or mock-treated. (A) Fasting plasma TG levels. Fasting plasma TG levels were determined on day 3 of hepatic Foxo1 production following an overnight fast. (B) Fat tolerance test. Plasma TG profiles in response to an oral bolus of olive oil were determined on day 4 after vector administration. (C) Plasma apoC-III levels. Mice were sacrificed after 1 week of hepatic Foxo1 production. Blood samples were collected for determination of the relative plasma apoC-III levels using a semi-quantitative immunoblot assay. A typical immunoblot is shown at the bottom of the panel. (D) TG levels in VLDL, LDL/IDL, and HDL fractions. Plasma (400 μl) pooled from individual mice at day 7 after vector administration was subjected to gel filtration column chromatography. Fifty fractions (200 μl per fraction) were eluted for determination of TG and cholesterol levels. (E) Plasma LPL activity. Post-heparin sera were obtained from individual mice on day 5 after vector administration and used for the determination of plasma LPL activity. (F) Cholesterol levels in VLDL, LDL/IDL, and HDL fractions, as described in D. *P < 0.05 by ANOVA.
The effect of hepatic Foxo1 production on plasma TG metabolism was further illustrated following fractionation of plasma lipoproteins by gel filtration chromatography. As shown in Figure 4D, in accordance with their elevated fasting plasma TG levels, Foxo1 vector–treated mice displayed significantly increased VLDL-TG profile compared with control mice. However, the fractional concentrations of TG and cholesterol in HDL and LDL/IDL peaks in Foxo1 vector–treated mice were not significantly different from those of controls (Figure 4F). To study the effect of hepatic Foxo1 production on plasma LPL activity, we heparinized the animals by intravenous injection of heparin and took sample aliquots of tail vein blood to test the level of LPL activity. As shown in Figure 4E, no significant differences were detected among different groups of animals, indicating that the observed increase in plasma VLDL-TG levels in Foxo1 vector–treated mice was not due to reduced systemic LPL levels. When total plasma cholesterol levels were determined, relatively higher cholesterol levels were detected in Foxo1 vector–treated-treated mice. However, the differences in total plasma cholesterol levels between Foxo1 vector–treated (201 ± 14 mg/dl) and control mice (178 ± 22 mg/dl in mock-treated or 199 ± 28 mg/dl in LacZ vector–treated group) did not reach statistical significance (P > 0.05 by ANOVA).
To correlate the changes in plasma TG metabolism with the alterations in hepatic apoC-III production, we sacrificed animals after 1 week of hepatic Foxo1 production and determined the relative Foxo1 and apoC-III mRNA levels by real-time RT-PCR using β-actin mRNA as a control. As shown in Figure 5A, hepatic apoC-III mRNA expression was significantly increased in response to Foxo1 production in liver, which correlated with increased plasma apoC-III levels (Figure 4C) and elevated hepatic Foxo1 expression in Foxo1 vector–treated mice (Figure 5B). In addition, we studied the expression of apoA-1 and apoA-IV in response to hepatic Foxo1 production. These 2 apolipoprotein genes are clustered along with the apoC-III gene at the same locus on chromosome 11q23 (27). apoA-1 is mainly produced in liver and present as an exchangeable moiety of HDL and TG-rich particles, whereas apoA-IV is predominantly expressed in intestine and to a lesser extent in liver (28). In response to hepatic Foxo1 production, hepatic apoA-1 expression was reduced (Figure 5C), whereas the expression level of apoA-IV remained unchanged (Figure 5D). Whether this observed reduction of hepatic apoA-I expression is due to the effect of Foxo1 or secondary to altered TG metabolism remains to be determined. In addition, we determined the body weight of mice before and 1 week after hepatic Foxo1 cDNA delivery. No significant differences in body weight changes were detected among different groups of mice.
Hepatic mRNA abundance in Foxo1 vector–treated mice. Total hepatic RNA was prepared for the determination of hepatic mRNA levels of apoC-III (A), Foxo1 (B), apoA-1 (C), and apoA-IV (D) using real-time RT-PCR. Hepatic protein extracts were prepared for immunoblot analysis of Foxo1 protein levels in Foxo1 vector– vs. control vector–treated mice using β-tubulin as control, as shown at the bottom of B. Values shown in the y axes are normalized to mock-treated controls. *P < 0.05 by ANOVA; **P < 0.001 by ANOVA.
Plasma TG metabolism in Foxo1S253A transgenic mice. Foxo1S253A transgenic mice bear a constitutive Foxo1 mutant allele under the control of the transthyretin promoter and express Foxo1S253A mainly in liver (29). To study the effect of hepatic Foxo1S253A transgene expression on TG metabolism, we determined plasma TG levels in Foxo1S253A transgenic mice. Compared with wild-type control littermates, Foxo1S253A transgenic mice exhibited significantly elevated plasma TG levels (Figure 6A), correlating with their relatively higher plasma apoC-III levels (Figure 6B). To investigate the impact of transgenic Foxo1S253A expression on postprandial TG metabolism, plasma TG profiles were determined in response to an oral bolus of olive oil (Figure 6C). Plasma TG levels in Foxo1S253A transgenic mice were markedly elevated (196 ± 28 mg/dl vs. 103 ± 9 mg/dl in control mice, P < 0.01) 2 hours after olive oil administration and remained at a relatively higher level (153 ± 38 mg/dl vs. 75 ± 7 mg/dl in control mice, P < 0.01) even after 4 hours after fat tolerance. In contrast, plasma TG levels in control littermates were only moderately raised in response to the same bolus of olive oil and returned to normal levels within 4 hours. To determine whether the impaired postprandial TG excursion was due to reduced plasma LPL activity, we subjected aliquots of tail vein blood from heparinized mice to an LPL activity assay. As shown in Figure 6D, relatively higher levels of plasma LPL activity were detected in Foxo1S253A transgenic mice compared with control littermates, but the difference between these 2 groups was statistically insignificant. When total plasma cholesterol levels were measured, Foxo1S253A transgenic mice exhibited significantly higher plasma cholesterol levels (136 ± 7 mg/dl vs. 109 ± 5 mg/dl in wild-type control littermates, P < 0.05 by ANOVA).
Plasma TG metabolism in Foxo1 transgenic mice (4 months old). Foxo1S253A transgenic mice (n = 8) and control littermates (n = 8) were studied for fasting plasma TG levels (A), plasma apoC-III levels (B), plasma TG profiles in response to fat tolerance test (C), and plasma LPL activity (D). Sera (500 μl) pooled from individual mice were fractionated by fast-performance liquid chromatography through 2 consecutive Tricorn High-Performance Superose S-6 10/300GL Columns and 70 fractions (400 μl per fraction) were collected and assayed for TG (E) and cholesterol levels (F). *P < 0.01 by ANOVA.
To study the effect of Foxo1 transgenic expression on VLDL-TG metabolism, we used gel filtration chromatography to study plasma pooled from Foxo1S253A transgenic mice or control littermates. Similar to adenovirus-mediated Foxo1 production, Foxo1 transgenic expression also significantly elevated plasma VLDL-TG levels (Figure 6E). In contrast to the acute effect of adenovirus-mediated Foxo1 production on cholesterol metabolism, chronic Foxo1 production was associated with increased HDL cholesterol levels (Figure 6F), which accounted for relatively higher plasma cholesterol levels in Foxo1S253A transgenic mice.
Hepatic Foxo1 and apoC-III expression in diabetic mice. To study the physiological significance of Foxo1-mediated regulation of hepatic apoC-III expression, we determined hepatic Foxo1 levels in correlation with apoC-III expression in livers of type 1 and type 2 diabetic mice. These studies involved the use of diabetic NOD and db/db mice, and their respective nonobese nondiabetic (NON) and db/+ control animals. NOD mice are the commonly used genetic model of type 1 diabetes with spontaneous onset of diabetes by 12 weeks of age. Here, 1 group of severely diabetic NOD mice (average blood glucose levels, 554 ± 18 mg/dl, n = 6) and 1 group of age-matched NON mice (122 ± 12 mg/dl, n = 6) were used. In addition, 1 group of diabetic db/db mice (>600 mg/dl, n = 6) and 1 group of their heterozygous littermates (93 ± 8 mg/dl, n = 6) were also used. Using real-time RT-PCR, we determined the hepatic expression levels of Foxo1 and apoC-III mRNA in livers of diabetic NOD and db/db mice and their respective control animals. We detected in liver of both diabetic NOD and db/db mice, compared with controls, a significant increase in hepatic Foxo1 abundance, along with a concomitant elevation in hepatic apoC-III expression (Figure 7, A and B). Consistent with these observations, plasma apoC-III and TG levels were markedly elevated in db/db mice compared with littermate controls (Figure 7, C and D).
Foxo1 and apoC-III expression in livers of NOD and db/db mice. Diabetic NOD (18-week-old) and db/db (6-month-old) mice, together with their respective NON and db/+ controls, were killed. Foxo1 (A) and apoC-III (B) mRNA in liver were determined by real-time RT-PCR using β-actin mRNA as control. The relative levels of plasma TG (C) and apoC-III (D) were determined in diabetic and control mice. Data in A, B, and D are plotted as relative values after normalization to controls. *P < 0.05 by ANOVA (n = 6); **P < 0.001 by ANOVA.
To study the potential alteration in Foxo1 subcellular distribution as a result of insulin deficiency or insulin insufficiency, we examined the localization of Foxo1 in livers of diabetic NOD and db/db mice by immunohistochemistry using anti-Foxo1 antibody. As shown in Figure 8, Foxo1 was immunostained mainly in the cytoplasm of liver cells in nondiabetic mice. In contrast, positive immunostaining was detected predominantly in the nucleus in liver of diabetic NOD and db/db mice. These results are consistent with our previous studies, in which we subjected protein extracts of the nuclear and cytoplasmic fractions of hepatocytes isolated from diabetic db/db and lean littermates to semi-quantitative immunoblot analysis (22). In that study, we found a quantitative (>3-fold) redistribution of Foxo1 from its cytoplasmic to nuclear location as the liver undergoes a shift from normal to insulin-resistant states (22).
Immunohistochemistry. Liver tissues of diabetic NOD, db/db, and control mice were used for immunofluorescent labeling with rabbit anti-Foxo1 antibody (1:400 dilution). Foxo1 was immunostained green using donkey anti-rabbit IgG conjugated with FITC (1:200 dilution) (A, D, G, and J). Nuclei of hepatocytes were stained blue with DAPI (B, E, H, and K). Merged images are shown in C, F, I, and L. Scale bar: 200 μm.
Intestinal Foxo1 and apoC-III expression. In addition to its hepatic expression, apoC-III is expressed in intestine. However, little is known about its regulation in response to insulin. Given the fact that intestine is an insulin- sensitive organ, intestinal apoC-III might be governed by insulin in a Foxo1-dependent mechanism. To test this hypothesis, we delivered Foxo1 cDNA to the human intestinal Caco-2 cells and determined the level of apoC-III mRNA expression in the absence and presence of insulin at different concentrations in culture media. As shown in Figure 9A, adenovirus-mediated Foxo1 production significantly increased intestinal apoC-III expression in the absence of insulin. In the presence of insulin, Foxo1-mediated stimulation of apoC-III expression in Caco-2 cells was suppressed in an insulin concentration–dependent manner.
Effects of Foxo1 on intestinal apoC-III expression. (A) Foxo1-dependent regulation of apoC-III expression in Caco-2 cells. Cells were transduced with Foxo1 vector (MOI, 500 PFU/cell) in the absence and presence of insulin at indicated concentrations. After 24 hours, cells were harvested for the determination of endogenous apoC-III mRNA expression by real- time RT-PCR. The products of real-time RT-PCR were analyzed on 0.7% agarose gels and visualized under UV lights after ethidium bromide staining (below A). (B) Immunoblot. Foxo1 vector–transduced Caco-2 cells were subjected to ChIP analysis using anti-Foxo1 antibody, control sheep IgG, or PBS. Immunoprecipitates were studied by immunoblot analysis. (C) PCR analysis of coimmunoprecipitated DNA by ChIP. (D) Foxo1 and apoC-III expression in liver versus intestine. RNA (1 μg) isolated from liver or intestine of lean C57BL/6J mice (n = 3) was analyzed by RT-PCR for Foxo1, apoC-III, and β-actin mRNA abundance. RT-PCR products were resolved on a 0.7% agarose gel and visualized under UV -light after staining with ethidium bromide. (E) Intestinal apoC-III mRNA levels. (F) Intestinal Foxo1 mRNA levels. The relative levels of apoC-III and Foxo1 mRNA in intestine of diabetic NOD and db/db versus nondiabetic control mice were determined by real-time RT-PCR using β-actin mRNA as control. *P < 0.05 and **P < 0.001 vs. controls.
To corroborate these results, we examined the potential interaction between Foxo1 protein and APOC3 promoter in Caco-2 cells. Caco-2 cells were transduced by Foxo1 vector. After 24 hours of transduction, cells were subjected to ChIP analysis using anti-Foxo1 antibody or control IgG, or they were mock-treated identically following the ChIP protocol. As shown in Figure 9B, Foxo1 protein was detected in the products that were immunoprecipitated by anti-Foxo1 antibody, but not in control IgG- or mock-immunoprecipitated products. When analyzed by PCR, a specific DNA fragment (675 bp) corresponding to the nucleotide region (–675/+1) of the APOC3 promoter was amplified from the DNA products that were coimmunoprecipitated by anti-Foxo1 antibody (Figure 9C). In contrast, no specific DNA was produced from control IgG or mock-immunoprecipitated products in the same PCR assay. Thus, similar to its action in hepatocytes, Foxo1 appeared to associate with the APOC3 promoter in stimulating apoC-III expression in enterocytes.
To address the physiological significance of these findings, we studied intestinal Foxo1 production in correlation with apoC-III expression in intestine. Total RNA from intestine and liver of normal C56BL/6J mice (12 weeks old) was prepared and subjected to RT-PCR analysis. As shown in Figure 9D, Foxo1 was expressed in both liver and intestine, but its intestinal expression level in intestine was significantly lower than it was in liver. Likewise, a similar expression pattern was detected for apoC-III expression, with the liver being a major source of apoC-III production, which was consistent with the data in the literature (30).
To study the alterations in intestinal apoC-III expression in response to insulin deficiency or insulin resistance, we determined the expression level of apoC-III mRNA relative to that of β-actin mRNA in intestine of diabetic NOD and db/db mice and then compared it to that in NON and db/+ controls, respectively. As shown in Figure 9E, the relative apoC-III mRNA abundance in intestine of both NOD and db/db mice was significantly increased, in comparison with nondiabetic controls. To study whether Foxo1 contributes to elevated apoC-III expression in intestine of diabetic NOD and db/db mice, we determined intestinal Foxo1 mRNA abundance relative to that of β-actin mRNA. In comparison with nondiabetic controls, intestinal Foxo1 expression was significantly increased in insulin-deficient and insulin-resistant animals (Figure 9F). These results indicate that insulin deficiency and insulin insufficiency are invariably associated with elevated Foxo1 production in intestine, which might act as a contributing factor for increased intestinal apoC-III expression in diabetic NOD and db/db mice.