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Interaction between CHOP and FoxO6 promotes hepatic lipid accumulation

Abstract Background & Aims Endoplasmic reticulum (ER) stress is one of the major causes of hepatic insulin resistance through increasing de novo lipogenesis. Forkhead box O6 (FoxO6) is a transcription factor mediating insulin signalling to glucose and lipid metabolism, therefore, dysregulated FoxO6 is involved in hepatic insulin resistance. In this study, we elucidated the role of FoxO6 in ER stress‐induced hepatic lipogenesis. Methods Hepatic ER stress responses and lipogenesis were monitored in mice overexpressed with constitutively active FoxO6 allele and FoxO6‐null mice. In the in vitro study, HepG2 cells overexpressing constitutively active FoxO6 were treated with palmitate, and then alterations in ER stress and lipid metabolism were measured. Results FoxO6 activation induced hepatic lipogenesis and the expression of ER stress‐inducible genes. The expression and transcriptional activity of peroxisome proliferator‐activated receptor γ (PPARγ) were significantly increased in constitutively active FoxO6 allele. Interestingly, we found that the active FoxO6 physically interacted with C/EBP homologous protein (CHOP), an ER stress‐inducible transcription factor, which was responsible for PPARγ expression. Palmitate treatment caused the expression of ER stress‐inducible genes, which was deteriorated by FoxO6 activation in HepG2 cells. Palmitate‐induced ER stress led to PPARγ expression through interactions between CHOP and FoxO6 corresponding to findings in the in vivo study. On the other hand, the expression of PPARα and β‐oxidation were decreased in constitutively active FoxO6 allele which implied that lipid catabolism is also regulated by FoxO6. Conclusion Our data present significant evidence demonstrating that CHOP and FoxO6 interact to induce hepatic lipid accumulation through PPARγ expression during ER stress.


Forkhead box O (FoxO) transcription factors induce target genes
involved in the regulation of cellular metabolic pathways. The FoxO subfamily of proteins, including FoxO1, FoxO3, FoxO4 and FoxO6, are characterized by a highly conserved, winged-helix DNA-binding motif. Furthermore, FoxO proteins act as Akt substrates to mediate the inhibitory effect of insulin (or IGF-1) on key genes involved in cell survival, proliferation, differentiation, oxidative stress and metabolism in mammals. 1 For instance, the phosphorylation of FoxO by Akt, in response to insulin or other growth factors, allows FoxO to be translocated from the nucleus to the cytoplasm. [1][2][3] Suppressed growth factor signalling activates FoxO because of the Akt-induced FoxO inhibition, whereas elevated certain fatty acids, such as palmitate, activate FoxO via a distinct mechanism involving the c-Jun N-terminal kinase (JNK) pathway. 4 Endoplasmic reticulum (ER) stress has been associated with the JNK pathway through IRE1-mediated activation of JNK signalling. 5,6 JNK phosphorylates the insulin receptor substrate (IRS) proteins and limits the activation of phosphatidylinositol-3-kinase (PI3K)/Akt signalling in response to insulin.
The function of FoxO6 in hepatic lipid metabolism and its possible contribution to hypertriglyceridemia in type 2 diabetes remain unclear. Although increased ER stress results in insulin resistance, the molecular mechanism by which ER stress causes aberrant insulin responses has not been completely elucidated. Martinez et al 7 investigated the nutrient-regulated cellular localization and activity of FoxO1 in β-cells and found that the gene encoding the nutrient-regulated C/EBP homologous protein (CHOP) was a potential transcriptional target of FoxO1. However, both CHOP and FoxO3 may regulate apoptosis. 8 Here we report a similar mechanism for ER stress that acts via the protein kinase IRE-1 and PERK, which overrides the insulin-mediated inhibition of FoxO6 activity. ER stress has been suggested to be a crucial common factor in hepatic lipogenesis, liver-specific inflammation and insulin resistance. 9,10 ER stress also induces serine phosphorylation of IRS-1 via the JNK pathway, inhibits insulin response in cultured liver cells, 6,11 enhances lipogenesis, affects hepatic steatosis and influences insulin resistance. 12 However, these inferences were drawn from studies conducted in genetically obese or prolonged chronic high-fat feeding models, 6,13 which have not provided reasonable insights into the effect of ER stress on de novo lipogenesis or lipid influx. The unfolded protein response (UPR) is induced by the accumulation of unfolded protein aggregates or excessive protein trafficking. 14,15 Arsenic can activate the UPR, which is initiated by IRE-1, PERK and ATF6. 16 ATF4, ATF6 and XBP1 regulate the transcription of several genes such as CHOP10, which is one of the genes highly expressed during ER stress. 15 However, C/EBPβ initially induces the expression of peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα, which then form a positive feedback loop by activating each other's expression and eventually contribute to the induction and maintenance of expression of adipocyte-specific genes. 17,18 To investigate the potential relationship between FoxO6 and other ER stress-related molecules in HepG2 cells, CHOP expression was examined. CHOP plays a critical role in apoptosis induced by ER stress. 19 In the present study, we investigated the role of FoxO6 in upregulating PPARγ expression through ER stress-induced CHOP activation in the liver and HepG2 cells to achieve a better understanding of the molecular mechanisms involved in hepatic lipogenesis.

| Animal experimental procedures
C57BL/6J male mice aged 6 weeks were purchased from the Jackson Laboratory. The mice received standard rodent chow and water ad libitum and were kept in sterile cages with a 12 hours light/dark cycle.
The C57BL/6J mice were weighed and randomly assigned to two groups; the mice in one group received an intravenous tail injection of an adenoviral vector containing a constitutively active FoxO6 allele (AdV-FoxO6-CA), whereas the mice in the other group received an intravenous tail injection of an AdV-null vector, both at 1.5 × 10 11 plaque forming units (pfu)/kg body weight. The mice were sacrificed 2 weeks after the AdV-FoxO6-CA injection. The mice had fasted for 24 h, and venous blood samples were collected from the tail in order to determine blood glucose levels as previously described.

| Cell culture
HepG2 cells were purchased from American Type Culture Collection (ATCC, Virginia, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Lonza, Walkersville, MD) and were transduced with an Adv-FoxO6-CA vector expressing the constitutively active FoxO6 allele as previously described. 20 The Adv-Empty vector was used as a control. The Adv-FoxO6-siRNA vector expressing the FoxO6-specific siRNA and Adv-Sc-siRNA vector encoding scrambled siRNA have been previously described. 20 The Adv-Akt-CA vector encodes a constitutively active form of Akt, as previously described. 21 All adenoviral vectors were produced in HEK293 cells, as previously described. 22

| Biochemical analysis
Blood samples were collected from each group of mice. Different kits were used according to the manufacturer's instructions to determine the concentrations of the following metabolites in the serum: insulin (Shibayagi, Japan), glucose, non-esterified fatty acids (NEFA) and TG (Shinyang, South Korea).

| Glucose tolerance test
Mice were fasted for 24 hours, followed by an intraperitoneal injection of glucose (2 g/kg). Blood glucose levels were measured before and after the glucose injection using a Glucometer Elite meter (Bayer, IN, USA).
Equal amounts of total protein from each sample were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% acrylamide gels and transferred to PVDF membranes at 80 V for 1.5 hours in a semidry transfer system. The membranes were immediately placed in blocking buffer (5% non-fat milk in 10 mmol/L Tris at pH 7.5, 100 mmol/L NaCl and 0.1% Tween 20). The blot was blocked at room temperature for 30 minutes. The membrane was incubated with a specific primary antibody at 4°C overnight, followed by a horse radish peroxidase-conjugated anti-rabbit antibody at room temperature for 1.5 hours. Labelled antibodies were detected using Westsave TM (Abfromtier, South Korea). Prestained protein markers were used for molecular weight determinations.

| Hepatic lipid content
Liver tissues or cells (20 mg) were homogenized in 400 µL of HPLCgrade acetone. After an overnight incubation with agitation at room temperature, 50 µL aliquots of acetone-extracted lipid suspensions were used to determine triglyceride concentrations via the Infinity triglyceride reagent (Thermo Electron). Hepatic lipid content was defined as milligrams of triglyceride per gram of total liver proteins, as described earlier.

| RNA isolation and real-time quantitative reverse transcriptase PCR (qRT-PCR)
RNA was isolated from liver tissue or cells using the RNeasy Mini Kit  Table S1. All primers were purchased from IDT (Coralville, IA).

| Chromatin immunoprecipitation (ChIP) assay
ChIP was used to study the interaction between  (1 µg/mL), and pepstatin A (1 µg/mL). Aliquots of cell extracts were centrifuged at 12,000g at 4°C for 15 min, incubated overnight at 4°C with the required antibody, and then incubated overnight at 4°C in a 50% protein A agarose slurry. After washing the immunoprecipitates three times with IP buffer, immunoprecipitated proteins were analysed by SDS-PAGE, and western blotting analysis was performed as described above.

| Histological analysis
The Oil red O staining was performed as previously described with optimal cutting temperature of frozen tissues to visualize lipid accumulation.

| Statistical analyses
All data are expressed as mean ± SEM. Treatments were compared by one-way ANOVA followed by Dunnett's post hoc test. p-values < 0.05 were considered statistically significant.

| FoxO6 induces hyperinsulinemia and hyperlipidemia
Obesity generally accompanies alterations in glucose tolerance and the blood lipid profile, which are closely associated with hepatic steatosis. To examine whether FoxO6-induced lipid accumulation occurs in the liver, we measured triglyceride content in liver homogenates.
Male mice developed hyperinsulinemia and hypertriglyceridemia after injection with a constitutively active FoxO6-CA allele, and consequently showed high levels of fasting glucose, insulin and plasma TG levels ( Figure 1A-C). Plasma NEFA levels showed no change ( Figure 1D). However, glucose tolerance was impaired in FoxO6-CA mice compared with empty virus-injected mice ( Figure 1E).

| FoxO6 upregulates lipid accumulation
To  Figure 2D). FoxO6 is a transcription factor that is negatively regulated by Akt during insulin signalling. FoxO6 dephosphorylation enhances its stability and activity, thereby stimulating gluconeogenesis and hyperlipidemia. PPARγ expression was suppressed by various concentrations of Akt vector ( Figure S1A). In addition, immunoprecipitation results showed that the interaction between FoxO6 and PPARγ was induced in Akt overexpressed cells ( Figure S1B). We analysed FoxO6-stimulated PPARγ activation in HepG2 cells. FoxO6 was found to bind PPARγ promoter, as determined by a chromatin immunoprecipitation assay ( Figure S2). Furthermore, FoxO6 increased transcriptional activity of PPARγ using PPRE/PPARγ luciferase assay with the empty vector and FoxO6-CA-transduced HepG2 cells ( Figure S2B). However, in the immunoprecipitation study, an interaction between FoxO6 and CHOP was demonstrated in the liver. As shown in Figure 2E, the interaction between CHOP and FoxO6 was increased in FoxO6-CA livers. We examined insulin signalling in the livers of FoxO6-CA-injected mice. As shown in Figure 2F, treatment with FoxO6-CA suppressed insulin signalling.

| Effect of FoxO6 on palmitate-induced ER stress in HepG2 cells
The transcriptional activities of FoxO family proteins have been previously demonstrated to increase when insulin levels were reduced. 2 We examined the expression of ER stress genes in FoxO6-CA-transduced HepG2 cells. Treatment of cells with ER stress activators (brefeldin, tunicamycin and thapsigargin) decreased FoxO6 phosphorylation ( Figure S3). These data collectively indicate that ER stress induces FoxO6 activation. To examine the hypothesis that FoxO6 targets and transactivates ER stress genes, we conducted  Figure 3A). However, combining FoxO6-CA with palmitate treatment further decreased PPARα level ( Figure 3A). This effect was accompanied by increased expression of PERK, IRE, XBP1 and CHOP mRNAs ( Figure 3C). These observations support the idea that FoxO6 regulates transactivation of PPARγ, and thus contributes to the regulation of palmitate. As shown in Figure 3B, palmitate treatment induced a remarkable shift of FoxO6 from the cytoplasm to the nucleus as determined by immunostaining. However, FoxO6 increased cellular TG levels in combination with palmitate compared to cells treated with FoxO6-CA alone ( Figure 3D). These results indicated that FoxO6 or palmitate have enhanced stimulatory effects on lipid accumulation.

| Effect of CHOP loss-of-function on PPARγ level and activity by palmitate-mediated FoxO6 in HepG2 cells
In order to determine the roles of CHOP and FoxO6, HepG2 cells were treated with CHOP-siRNA, FoxO6-CA and palmitate. As a result, palmitate enhanced FoxO6, CHOP and PPARγ expression ( Figure 4). In addition, palmitate treatment decreased nuclear PPARα level. These results indicated that treatment with palmitate had an enhanced stimulatory effect on the level of CHOP, FoxO6 and PPARγ. However, treatment with CHOP-siRNA and palmitate suppressed FoxO6 and PPARγ levels compared with palmitate treatment alone. CHOP-siRNA treatment also reduced PPARγ and FoxO6 levels ( Figure 4A). To examine palmitate-induced PPARγ activity in HepG2 cells, the PPRE/ PPARγ-luciferase assay was performed and revealed that palmitate was associated with increased activity of PPARγ. However, in the absence of CHOP, PPARγ activity induced by palmitate was decreased ( Figure 4B). Palmitate-mediated FoxO6 was found to be associated with increased PPARγ activity in CHOP-siRNA-transduced cells, compared to the empty vector in HepG2 cells.

| Changes in the expression of ER stress genes in FoxO6-KO liver
To characterize the role of FoxO6 in glucose metabolism, Calabuig-Navarro et al 23  FoxO6-KO and WT littermates on a high fat diet. We also measured ER stress levels, and observed significant differences in the expression of ER stress genes in the liver of FoxO6-KO mice (vs WT littermates) via western blotting ( Figure 5A), as well as qPCR ( Figure 5B). We also measured levels of lipogenesis genes by western blotting and qPCR. These results showed a significant decrease in FoxO6, CHOP and PPARγ protein levels in FoxO6-KO mice vs WT littermates ( Figure 5A). However, FoxO6-KO increased PPARα level in liver tissues ( Figure 5A). Furthermore, compared with control littermates, FoxO6-KO mice showed increased expression of β-oxidation-associated genes. The mRNA levels of lipogenesis genes such as PPARγ, FASN and SCD1 were also decreased in the FoxO6-KO liver ( Figure 5B). Collectively, these data underscore the importance of the β-oxidation pathway in FoxO6-induced lipogenesis in liver cells, especially under excessive ER stress.

| PPARα deficiency increases liver lipid accumulation
To

| D ISCUSS I ON
Our study characterized FoxO6 as a novel transcription factor that independently mediates hepatic lipogenesis during ER stress. We demonstrated crosstalk between FoxO6, CHOP, and ER stressinduced hepatic lipogenesis. The lipotoxicity of non-alcoholic fatty liver disease (NAFLD) is caused by lipid oversupply that directly influences the balance between ER homoeostasis and ER stress. 24 Thus far, little was known about FoxO6 (a member of the FoxO family) and its regulatory role in ER stress-induced lipogenesis. Our data suggests that ER stress induces FoxO6 activation ( Figure S3).
Consequently, constitutive FoxO6 expression is observed in cells transfected with inactivated CHOP, as shown by ER stress-induced CHOP-mediated regulation of FoxO6 expression (Figure 4). When ER homoeostasis was disturbed, unfolded proteins accumulate in the ER lumen, which consequently activates the UPR through dissociation of immunoglobulin protein/78 kDa glucose-regulated protein (BiP/GRP78) from PERK, IRE1α and ATF6α. 25,26 Paradoxically, under these conditions, translation of ATF4 mRNA was selectively enhanced, which induced transcription of CHOP. 27 ER stress induction increased the CHOP response to a greater extent than the induction of DNA damage. 15 Miyazaki et al 28  This effect, which helped suppress TG overproduction and attenuate steatosis in the insulin-resistant liver, contributed to the increase in free fatty acid and glucose levels in plasma in FoxO6-CA-injected mice (Figure 1). In addition, FoxO6 was associated with elevated lipogenesis, contributing to increased fat accumulation in the liver of FoxO6-CA-injected mice (Figure 1). To investigate F I G U R E 5 Effect of FoxO6 depletion on lipid metabolism and ER stress in FoxO6-KO mice. Mice were fed a high fat diet at 4 weeks of age for 12 weeks (n = 6). (A) Nuclear proteins were subjected to semiquantitative immunoblot analysis for FoxO6, ATF6, CHOP, PPARγ, SREBP-1c and PPARα, and TFIIB was used as a loading control for the nuclear fractions. (B) Expression levels of FoxO6, ER stress genes (ATF6, IRE1, PERK, CHOP and XBP1), β-oxidation genes (PPARα, CPT1 and Acox) and lipogenesis genes (PPARγ, SREBP-1c, Acoca, Fasn and SCD1) were analysed by qPCR. Results were normalized to β-actin mRNA levels. *P < .05, **P < .01 and ***P < .001 vs wild-type control. NS, not significant. (C) Possible mechanism for how FoxO6 activates ER stress in lipogenesis the underlying mechanism, we showed that hepatic expression of PPARγ, SREBP-1c, FAS and ACC genes was significantly upregulated, and the level of IRS/Akt protein decreased in the liver of FoxO6-CA-injected mice ( Figure 2). Fatty liver is a common secondary disease in obesity, 30  Nuclear protein expression of FoxO6, PPARα and cytosolic protein expression of CPT1α and ACOX1 were measured by western blotting. TFIIB and β-actin were used as the respective loading controls. (B) Gene expression levels of β-oxidation and lipogenesis targets were measured by qPCR. * P < .05, ** P < .01, *** P < .001 vs normal. # P < .05, ## P < .01 vs FoxO6-CA. (C) HepG2 cells were treated with PPARα-siRNA (20 nmol/L) or FoxO6-CA vector for 48 h. TGs were quantified in the liver cells. ** P < .01 vs normal. # P < .05 vs FoxO6-CA stimulate hepatic steatosis, the molecular relationship between PPARγ and FoxO6 in the liver remained unclear. Our data showed that FoxO6 directly binds to the PPARγ promoter region, and this binding was strongly increased during hepatic steatosis, possibly due the significant increase in PPARγ expression after FoxO6-CA treatment. However, FoxO6-CA significantly increased liver lipid accumulation and decreased the expression of PPARα and β-oxidation-associated genes ( Figure 6). These data also suggest that the FoxO6-mediated increase in PPARγ activity at least partially contributed to the increase in hepatic lipid accumulation.
In summary, lipid accumulation during ER stress and suppressed IRS/Akt signalling induces FoxO6 activation, which consequently results in hepatic lipogenesis gene expressions. Under ER stress, FoxO6 interacted with CHOP, upregulated PPARγ and inhibited PPARα in the mouse liver and HepG2 cells ( Figure 5C).
These results provide significant insights into the molecular and cellular basis of the association between FoxO6 and CHOP, suggesting these as novel molecular targets for potential hepatic lipogenesis therapeutics.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.