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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Obesity is a calorie-excessive state associated with high risk of diabetes, atherosclerosis, and certain types of tumors. Obesity may induce inflammation and insulin resistance (IR). We found that the expression of interferon (IFN) regulatory factor 9 (IRF9), a major transcription factor mediating IFN responses, was lower in livers of obese mice than in those of their lean counterparts. Furthermore, whole-body IRF9 knockout (KO) mice were more obese and had aggravated IR, hepatic steatosis, and inflammation after chronic high-fat diet feeding. In contrast, adenoviral-mediated hepatic IRF9 overexpression in both diet-induced and genetically (ob/ob) obese mice showed markedly improved hepatic insulin sensitivity and attenuated hepatic steatosis and inflammation. We further employed a yeast two-hybrid screening system to investigate the interactions between IRF9 and its cofactors. Importantly, we identified that IRF9 interacts with peroxisome proliferator-activated receptor alpha (PPAR-α), an important metabolism-associated nuclear receptor, to activate PPAR-α target genes. In addition, liver-specific PPAR-α overexpression rescued insulin sensitivity and ameliorated hepatic steatosis and inflammation in IRF9 KO mice. Conclusion: IRF9 attenuates hepatic IR, steatosis, and inflammation through interaction with PPAR-α. (Hepatology 2013;58:603–616)

Abbreviations
Abs

antibodies

ACC

acetyl-CoA carboxylase

Akt

protein kinase B

ALP

alkaline phosphatase

ALT

alanine transaminase

AMP

adenosine monophosphate

AMPK

AMP-activated protein kinase

AP-1

activator protein 1

AST

aspartate transaminase

β-HB

β-hydroxybutyrate

CoA

coenzyme A

Co-IP

coimmunoprecipitation

DBD

DNA-binding domain

DGAT

diglyceride acyltransferase

FFA

free fatty acid

GFP

green fluorescent protein

GST

glutathione S-transferase

HDL

high-density lipoprotein

H&E

hematoxylin and eosin

HFD

high-fat diet

HNF4

hepatic nuclear factor 4

HOMA-IR

homeostasis model assessment of insulin resistance

IAD

IRF association domain

IF

immunofluorescent

IFN

interferon

IgG

immunoglobulin G

IKKβ

inhibitor of nuclear factor kappa B kinase beta subunit

IL

interleukin

IP

immunoprecipitation

IPGTT

intraperitoneal glucose tolerance test

IPITT

intraperitoneal insulin tolerance test

IR

insulin resistance

IRs

insulin receptors

IRF9

IFN regulatory factor 9

IRSs

IR substrates

JNK1

Jun N-terminal kinase 1

KCs

Kupffer cells

KO

knockout

LCAD

long-chain acyl-CoA dehydrogenase

LDL

low-density lipoprotein

MCP-1

monocyte chemoattractant protein 1

MGL

macrophage galactose-type C-type lectin

mRNA

messenger RNA

NC

normal chow

NEFA

nonesterified fatty acid

NFκB

nuclear factor kappa B

NRs

nuclear receptors

PCR

polymerase chain reaction

PPAR

peroxisome proliferator-activated receptor

SEM

standard error of the mean

TC

total cholesterol

TG

triglyceride

TNF-α

tumor necrosis factor alpha

UCP

uncoupling protein

WAT

white adipose tissue

WT

wild type.

Metabolic disorders, including obesity, nonalcoholic fatty liver disease, metabolic syndrome, and diabetes, are global public health issues and are increasingly severe as the result of an aging population, urbanization, and associated lifestyle changes.[1, 2] Obesity is recognized as a chronic low-grade systemic inflammatory state.[3] In obesity, the inhibitor of nuclear factor kappa B kinase beta subunit/nuclear factor kappa B (IKKβ/NFκB) and Jun N-terminal kinase 1/activator protein 1 (JNK1/AP-1) pathways are activated in multiple tissues.[4] Consequently, inflammatory cells infiltrate into adipose tissue. M1-like macrophages secrete proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-α] and interleukin [IL]-1β), which impair insulin action.[5, 6] Additionally, ectopic lipid accumulation[7] and endoplasmic reticulum stress[8] may contribute to insulin resistance (IR). Nuclear receptors (NRs) and their cofactors play essential roles in glucose and lipid metabolism and insulin sensitivity, among which peroxisome proliferator-activated receptors (PPARs)[9] and PPAR-γ coactivator 1 alpha[10] have been intensively studied. However, the underlying mechanisms of obesity-related metabolic disorders still remain elusive.

Interferon (IFN) regulatory factors (IRFs) are a family of nine transcription factors (IRF1-IRF9) in mammals.[11] IRFs are involved in cytosolic pattern-recognition receptor- and Toll-like receptor-mediated signal transduction and regulate type I IFN expression.[12] IRFs play central roles in gene-expression regulation in innate immunity and immune cell differentiation.[13] IRFs were also involved in malignant transformation through regulation of cell growth and apoptosis.[14] Moreover, we newly observed that cardiovascular diseases, such as cardiac hypertrophic, can be regulated by IRF family members.[15] Besides the above-mentioned factors, metabolic roles of IRFs have also emerged. IRF3 was reported to regulate metabolism-related NRs, such as liver X receptor and retinoid X receptor alpha.[16, 17] Another group found that IRFs regulate adipogenesis and adipocyte lipid metabolism.[18, 19] However, the roles of IRFs in hepatic and whole-body metabolism are unclear.

IRF9, an IRF family member, has previously been characterized as mediating innate immunity by activating IFN-mediated transcription.[20-22] In the present study, we discovered a protective role for IRF9 against metabolic disorders. IRF9 knockout (KO) mice fed a high-fat diet (HFD) had higher levels of obesity-induced inflammation, lower insulin sensitivity, and more severe hepatic steatosis than did wild-type (WT) controls, whereas liver-specific IRF9 overexpression ameliorated these phenotypes in both diet-induced and genetically (ob/ob) obese mice. Furthermore, we determined that IRF9 up-regulated the expression of PPAR-α target genes. These results suggest that IRF9 improves hepatic lipid metabolism and insulin sensitivity.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information
Mice and Diets

All protocols were approved by the animal care and use committee of Renmin Hospital of Wuhan University (Wuhan, China). IRF9 KO mice were kindly provided by Dr. Tadatsugu Taniguchi (Department of Immunology, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, Tokyo, Japan). Ob/ob mice were purchased from The Jackson Laboratory (stock no.: 000632; The Jackson Laboratory, Bar Harbor, ME). Nine-week-old female and 8-week-old male ob/ob mice were used. Eight-week-old male C57BL/6 mice and IRF9 KO mice were fed with either normal chow (NC; protein, 18.3%; fat, 10.2%; carbohydrates, 71.5%; D12450B; Research Diets, Inc., New Brunswick, NJ) or an HFD (protein, 18.1%; fat, 61.6%; carbohydrates, 20.3%; D12492; Research Diets) ad libitum for up to 26 weeks. Detailed protocols for animal experiments were described in the Supporting Materials.

Recombinant Adenoviral Vectors and In Vivo Adenovirus-Mediated Gene Transfer

To overexpress IRF9 and PPAR-α, we used replication-defective adenoviral vectors encompassing the entire coding region of Flag-tagged IRF9 (obtained from OriGene Technologies, Inc., Rockville, MD) and Flag-tagged PPAR-α (ordered from Seajet Scientific Inc., Beijing, China) under the control of the cytomegalovirus promoter. A similar adenoviral vector encoding green fluorescent protein (GFP) was used as a control. Adenovirus was injected through the jugular vein. Please find the animal perform procedures in the Supporting Materials.

Yeast Two-Hybrid Analysis

For yeast two-hybrid screening, we used a Matchmaker Gold Y2H system according to the manufacturer's instruction (Clontech Laboratories, Inc., Mountain View, CA). The bait vector, pGAKT7-IRF9, was constructed by cloning the encoding region of IRF9 gene of human into pGAKT7 to create an in-frame fusion with the Gal4 DNA-binding domain. pGAKT7-IRF9 was transformed into yeast strain Y2H Gold on SD/–Trp according to a standard polyethylene glycol/single-stranded DNA/lithium acetate procedure. The Y2H Gold [pGADT7-IRF9] strain was mated with the Y187 (Mate & Plate Library; Clontech) strain by mixing 4-5 mL of Bait Strain and 1 mL of Library Strain in 45 mL of 2×YPDA liquid medium and incubating at 30°C for 20-24 hours, slowly shaking (30-50 rpm). Then, we centrifuged to pellet cells and discarded the supernatant. Pelleted cells were then resuspended in 10 mL of 0.5× YPDA/Kan liquid medium. Dilutions (100 µL of 1/10) were plated onto SD/–Leu/–Trp minimal media double dropouts to select for mated colonies. Plates were incubated at 30°C for 5 days.

Statistical Analysis

Data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed with the Student two-tailed t test or one-way analysis of variance. P < 0.05 was considered statistically significant.

Methods for histological analysis, serum examination, western blotting, and real-time polymerase chain reaction (PCR) analysis are described in the Supporting Materials and have been detailed previously.[23] Methods for plasmid construction, immunoprecipitation (IP), glutathione S-transferase (GST) pull-down assay, and confocal microscopy are also included in the Supporting Materials.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information
Diet-Induced and Genetically Obese Mice Have Lower Hepatic IRF9 Levels Than Normal Controls

To investigate whether IRF9 is involved in metabolic diseases, we used HFD-induced and genetic (ob/ob) obesity models. We stained liver section slides with antibodies (Abs) against hepatic nuclear factor 4 (HNF4), a molecular marker of hepatocytes, and IRF9. Almost all IRF9 was localized in HNF4-positive cells, which indicates that IRF9 was mainly expressed in hepatocytes, rather than other types of cells, in the liver (Fig. 1A,C). We calculated the proportion of IRF9-positive hepatocytes. We observed that hepatocytes expressed IRF9 decreased after 26 weeks of HFD (Fig. 1B). Consistently, the proportion of IRF9-expressing cells in livers of ob/ob mice was lower than in lean mice (Fig. 1D). Messenger RNA (mRNA) and protein expression levels of IRF9 were significantly lower in livers of the HFD-fed obese mice than in NC controls (Fig. 1E,F). In agreement with these results, ob/ob mice also had lower IRF9 expression levels than lean mice (Fig. 1E,F). All these data indicate that IRF9 expression in the liver is down-regulated in obesity, which suggests an important role for IRF9 in metabolic disorders.

image

Figure 1. IRF9 expression decreases in livers of obese mice. (A and C) Representative IF images of liver section slides, which were stained with Abs against HNF4 (green) and IRF9 (red). 4',6-diamidino-2-phenylindole (DAPI; blue) was used to show nuclei. Scale bar indicates 50 μm. (B and D) Representative IF expression of IRF9 in livers of C57BL/6 mice (WT) and lean, ob/ob mice. For (A) to (D), WT mice were on the NC diet or HFD feeding for 26 weeks. Ob/ob mice were fed with the chow diet for 9 weeks. Each group contained four sections for staining analysis. Scale bar indicates 20 μm. Quantitative analysis of percents of IRF9-positive cells in liver is also shown. (E) Real-time PCR analysis of mRNA levels of IRF9 in liver; n = 6-12 per group. (F) Protein expression of IRF9 in liver was detected by western blotting. Quantification of protein expression levels was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. All values are shown as mean ± SEM; n = 3 per group. All statistical significance is indicated and compared to the WT NC group: **P < 0.01, compared to the lean group; ##P < 0.01.

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IRF9 Deficiency Aggravates Obesity and Impairs Glucose Metabolism

To fully understand the effect of IRF9 on metabolism, we utilized IRF9 KO mice. After consuming an HFD, although there was no significant difference in food consumption between the two genotypes (Supporting Fig. 1A), IRF9 KO mice were more obese (Fig. 2A) and displayed lower insulin sensitivity than WT controls. IRF9 KO mice also had higher fasting blood glucose and insulin levels and a higher homeostasis model assessment of insulin resistance (HOMA-IR) index than WT controls (Fig. 2B). During fasting, the liver generates glucose to stabilize serum glucose level; after feeding, insulin increases and gluconeogenesis slows down correspondingly. We found that although IRF9 KO mice had a higher serum insulin level, gluconeogenic gene expression, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was still higher in IRF9 KO livers than in WT ones (Supporting Fig. 1B). We also performed intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (IPITTs), both of which revealed compromised insulin sensitivity and glucose regulatory functions in IRF9 KO mice, as compared to WT mice (Fig. 2C,D). Insulin regulates organ function in an endocrine manner. Upon insulin binding, insulin receptors (IRs) display increased kinase activity against intracellular adaptors, such as insulin receptor substrates (IRSs), which relay signals to downstream pathways.[24] Western blotting determined that levels of tyrosine phosphorylation of IRS1 and serine phosphorylation of protein kinase B (Akt) were lower in livers of IRF9 KO mice than in WT mice, indicating down-regulation of the insulin-signaling pathway (Fig. 2E).

image

Figure 2. IRF9 deficiency aggravates obesity and impairs glucose metabolism. (A) Comparison of body-weight gains in WT or IRF9 KO mice on the 26-week NC diet or HFD feeding; n = 28-39 per group. (B) Fasting blood glucose levels were detected at an interval of 4 weeks from 0 to 24 weeks feeding duration in WT or KO mice with food deprivation for 6 hours. Serum fasting insulin levels were determined by enzyme-linked immunosorbent assay every 8 weeks. HOMA-IR index was calculated as [FBG (mmol/L) × FIns(mIU/L)]/22.5; n = 4-8 per group at each time point. (C) An IPGTT (1 g/kg body weight) was performed on WT and KO mice in both the NC and HFD groups with 24-week diet feeding. The corresponding area under the curve (AUC) of blood glucose levels in each group was calculated; n = 12-16 per group. (D) An IPITT (0.75 units/kg body weight) was performed on WT and KO mice in both the NC and the HFD groups at the 25th week of food administered. The corresponding area under the curve (AUC) of blood glucose levels in each group was calculated; n = 13-17 per group. (E) Immunoblot analysis established systemic IR in KO mice and indicated marked inhibition of the phosphorylation of IRS1-tyr608 and Akt in insulin target organs. Quantification of phosphorylated protein expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); n = 4 per group. All statistical significance is indicated and compared to the WT HFD group: *P < 0.05; **P < 0.01, compared to the WT NC group; #P < 0.05; ##P < 0.01. All values are expressed as mean ± SEM.

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Metabolic disorders involve a series of systemic changes. With continuous HFD feeding, metabolic dysfunction became increasingly significant in IRF9 KO mice. Triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), free fatty acid (FFA), and β-hydroxybutyrate (β-HB) levels were higher in sera of IRF9 KO mice, whereas the level of high-density lipoprotein (HDL) was lower (Table 1). All these data indicate catabolism insufficiency and energy overabundance in IRF9 KO mice, compared to WT mice.

Table 1. Serum Lipid, Hormone, and Cytokine Levels in WT and KO Mice With a 24-Week Diet Treatment
ParametersWT NCKO NCWT HFDKO HFD
  1. Data are expressed as mean ± SEM (n = 5-8 mice per group).

  2. a

    P < 0.05 versus WT NC.

  3. b

    P < 0.01 versus WT NC.

  4. c

    P < 0.01 versus WT HFD.

Lipid contents    
TG (mg/dL)39.96 ± 1.7143.50 ± 1.7549.82 ± 2.28b69.67 ± 2.46c
TC (mg/dL)130.79 ± 5.67144.82 ± 5.52251.55 ± 11.69b322.62 ± 12.05c
HDL (mg/dL)86.52 ± 3.7478.67 ± 2.4773.15 ± 3.38b46.29 ± 1.43c
LDL (mg/dL)17.33 ± 0.7219.34 ± 0.6024.18 ± 1.09b39.62 ± 1.26c
FFA (mmol/L)0.74 ± 0.030.92 ± 0.03a1.09 ± 0.04b2.44 ± 0.07c
β-HB (mmol/L)0.23 ± 0.030.29 ± 0.030.89 ± 0.07b1.28 ± 0.09c
Hormones and cytokines    
Leptin (pg/mL)5,173.10 ± 225.025,520.90 ± 223.0612,265.00 ± 570.63b17,324.00 ± 433.83c
Resistin (pg/mL)891.67 ± 38.50924.62 ± 31.871,292.20 ± 59.10b1,663.50 ± 55.35c
Adiponectin (μg/mL)1.10 ± 0.041.03 ± 0.040.57 ± 0.02b0.22 ± 0.01c
IL-1β (pg/mL)38.66 ± 1.6444.20 ± 1.2157.21 ± 2.50b78.29 ± 2.59c
IL-4 (pg/mL)21.73 ± 0.9021.61 ± 0.6924.34 ± 1.0445.64 ± 1.46c
IL-6 (pg/mL)0.71 ± 0.020.98 ± 0.03a1.20 ± 0.04b3.14 ± 0.09c
TNF-α (pg/mL)2.79 ± 0.123.11 ± 0.11a3.63 ± 0.14b4.98 ± 0.16c
MCP-1 (pg/mL)23.40 ± 0.9927.58 ± 0.9329.40 ± 1.31b47.28 ± 1.60c
IRF9 Deficiency Aggravates Hepatic Steatosis and Inflammation

Hepatic steatosis is an important manifestation of metabolic dysfunction and IR. We found that livers of IRF9 KO mice were larger than those from WT mice after 26 weeks of an HFD because of cellular lipid accumulation, as determined by hematoxylin and eosin (H&E) and Oil Red O staining (Fig. 3A-C). Considering that steatohepatitis devastates liver integrity and function, we tested hepatic function in mice. Alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) levels were all significantly higher in HFD-fed IRF9 KO mouse serum than in WT mouse serum, indicating poorer hepatic function in IRF9 KO mice (Supporting Fig. 2A). IRF9 KO mice also had higher hepatic TG, TC, and FFA levels (Fig. 3D). Quantitative real-time PCR demonstrated that expression levels of genes related to cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A [CoA] reductase and LDL receptor), lipogenesis (e.g., diglyceride acyltransferase [DGAT]1 and DGAT2), fatty acid synthesis (e.g., sterol response element-binding protein 1c, acetyl-CoA carboxylase [ACC]-α, fatty acid synthase, and stearoyl-CoA desaturase 1), and uptake (e.g., CD36, fatty-acid–binding protein 1 and fatty-acid–transporting protein 1) were higher, whereas expression of genes regulating cholesterol output, lipolysis (e.g., adipose triacylglycerol lipase), and fatty acid oxidation (e.g., PPAR-α, long-chain acyl-CoA dehydrogenase [LCAD], and uncoupling protein [UCP]3) were lower in livers of IRF9 KO mice than in livers of WT mice (Fig. 3E). Adenosine monophosphate (AMP)-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, stimulates catabolism in response to low adenosine triphosphate levels.[25] In livers of IRF9 KO mice, lower levels of phosphorylated AMPK and ACC2 indicated a compromised AMPK-signaling pathway (Supporting Fig. 2B).

image

Figure 3. IRF9 deficiency aggravates hepatic steatosis and exacerbates global and local inflammation in mice. (A) Macroscopic pictures of livers in the WT and KO mice fed with the HFD. (B) Quantification of liver weight and the ratio to body weight; n = 28-39 per group. (C) Representative liver sections stained with H&E (upper panel) or Oil Red O (lower panel) in WT or KO mice with 26 weeks of NC or HFD feeding. Scale bar indicates 100 μm. (D) Levels of TG, TC, and NEFA were extracted from liver tissue in WT with KO mice upon the HFD; n = 5-13 per group. (E) mRNA levels of genes related to lipid metabolism were examined by real-time PCR; n = 6-12 per group. (F) mRNA levels of pro-inflammatory and anti-inflammatory markers in liver were measured by real-time PCR; n = 9–12 for each group. All values are expressed as mean ± SEM. Statistical significance is indicated and compared to the WT HFD group: *P < 0.05; **P < 0.01, compared to the WT NC group; ##P < 0.01.

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To rule out the possibility that hepatic phenotype of IRF9 KO mice was secondary to changes in white adipose tissue (WAT), we studied the effects of IRF9 in WAT. Real-time PCR results showed that the expression of genes of adipogenesis, lipogenesis, and lipid catabolism in IRF9 KO WAT was comparable to that in WT mice (Supporting Fig. 3). Through H&E staining of WAT sections, we did not observe any significant difference in adipocyte size between these two genotypes either (data not shown). Therefore, the liver, rather than WAT, is more likely to be the ringleader of the metabolic disorders developed in IRF9 KO mice.

Considering that inflammation is intimately related to metabolic disorders, we further tested hepatic inflammation. Immunofluorescent (IF) staining of inflammatory markers (e.g., 7/4, CD45, and CD68) indicated more hepatic inflammatory cell infiltration in IRF9 KO mice (data not shown) than in WT mice. Meanwhile, real-time PCR demonstrated Kupffer cell (KC) activation and M1 macrophage polarization in IRF9 KO livers. Levels of proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and monocyte chemoattractant protein 1 [MCP-1]) were higher, whereas those of anti-inflammatory markers (e.g., IL-10, macrophage galactose-type C-type lectin [MGL]1, and MGL2) were lower in livers of IRF9 KO mice (Fig. 3F). Adipokines are important regulators of adipose inflammation and insulin sensitivity.[26] Serum levels of leptin and resistin were higher and that of adiponectin was lower in IRF9 KO mice, as compared to WT controls. Furthermore, levels of proinflammatory cytokines were higher, in the circulation of IRF9 KO mice (Table 1). All these factors contribute to IR and metabolic dysfunction. In line with results in the liver, more proinflammatory factors and fewer anti-inflammatory factors were also detected in serum of IRF9 KO mice than in WT mice. All the findings described above illustrate that IRF9 deficiency aggravates hepatic steatosis and enhances local and global inflammation in mice.

Hepatic IRF9 Overexpression Attenuates Diet-Induced Hepatic Steatosis, IR, and Inflammation

To determine the in vivo function of IRF9 on hepatic lipid metabolism and insulin sensitivity, we used adenovirus infection, a well-established method, to overexpress IRF9 in mouse liver. The adenovirus-mediated gene-transfer approach acutely delivers genes to the liver without confounding developmental effects that commonly occur during chronic overexpression.[27, 28] After 20 weeks of HFD feeding, the mice were injected with an IRF9-expressing adenovirus through the jugular vein. Four weeks after adenovirus injection, the protein expression level of IRF9 had a more than four-fold increase in the liver, but remained unchanged in WAT and skeletal muscle (Fig. 4A). IF staining of HNF4 and IRF9 confirmed the elevation of IRF9 expression in hepatocytes, rather than in other types of cells (Fig. 4B). Four weeks after adenovirus injection, mice with IRF9 overexpression had lower liver weight than those WT mice injected with an adenovirus-expressing GFP as a control (Fig. 4C). H&E and Oil Red O staining revealed lower hepatic lipid accumulation in livers with IRF9 overexpressed (Fig. 4D). Hepatic TG, TC, and nonesterified fatty acid (NEFA) contents were also lower in IRF9-overexpressing mice than in control mice (Supporting Fig. 4A). IRF9-injected mice displayed lower ALT, AST, and ALP levels (Supporting Fig. 4B). All these factors indicate that IRF9 promotes hepatic lipid metabolism and protects liver function. IRF9-overexpressing mice displayed lower fasting serum glucose and insulin levels when on an HFD than did control animals (Fig. 4E). Both the IPGTT and IPITT showed improved glucose regulation in IRF9-overexpressing mice (Fig. 4F and 4G). Consistent with these results, the insulin-signaling pathway was up-regulated in IRF9-overexpressing livers, compared to control livers, as measured by immunoblotting (Fig. 4H). Moreover, liver-specific IRF9 overexpression ameliorated obesity-induced inflammation in the liver. Decreased proinflammatory markers (e.g., F4/80, CD11c, TNF-α, IL-1β, IL-6, and MCP-1) and increased anti-inflammatory markers (e.g., IL-10, arignase 1, mannose receptor, C type 1, MGL1, and MGL2) were detected by real-time PCR and indicate a shift in the balance to M2-like macrophages (Supporting Fig. 4C).

image

Figure 4. Hepatic IRF9 overexpression improves metabolism in diet-induced obese mice. (A) Immunoblot showed IRF9 expression in livers, epididymal fat pads, and gastrocnemius muscles 4 weeks after jugular vein injection of adenovirus containing IRF9 complementary DNA or GFP sequence control. (B) Representative IF images of liver section slides, which were stained with Abs against HNF4 (green) and IRF9 (red). 4',6-diamidino-2-phenylindole (DAPI; blue) was used to show nuclei. Scale bar indicates 50 μm. (C) Body weight, liver weight, and the liver/body-weight ratio of WT mice fed with HFD were examined. These mice were either injected with IRF9 adenovirus or GFP adenovirus control; n = 12-13 per group. (D) Representative liver sections stained with H&E (top panel) and Oil Red O (lower panel) of IRF9 overexpressed and vector control mice fed with HFD. (E) Fasting blood glucose levels were detected with food deprivation for 6 hours. Serum fasting insulin levels were determined by enzyme-linked immunosorbent assay. HOMA-IR index was also calculated; n = 6 per group. (F) An IPGTT (1 g/kg body weight) was performed on HFD-fed WT mice injected with IRF9 adenovirus or vector control virus. Corresponding area under the curve (AUC) of blood glucose levels in each group was calculated; n = 12-13 per group. (G) An IPITT (0.75 units/kg body weight) was also performed. Corresponding AUC of blood glucose levels in each group was calculated; n = 12-13 per group. (H) Immunoblot analysis indicated reverse IR in mice with IRF9 liver-specific overexpression and displayed marked up-regulation of phosphorylation of IRS1-tyr608 and Akt in the liver. Quantification of phosphorylated protein expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); n = 4 per group. All values are expressed as mean ± SEM. Statistical significance is indicated and compared to the GFP adenovirus-injected group: *P < 0.05; **P < 0.01.

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Hepatic IRF9 Overexpression Attenuates Hepatic Steatosis, IR, and Inflammation in ob/ob Mice

To rule out any potential effect of unidentified components of the HFD on our results, we used a genetic obesity model to assess the metabolic role of IRF9. We fed NC to leptin-deficient (ob/ob) mice, which spontaneously develop obesity. As with the dietary model described above, we injected male ob/ob mice with IRF9 adenovirus through the jugular vein for liver-specific IRF9 overexpression (Fig. 5A,B). Four weeks later, hepatic lipid depots were greatly reduced in the IRF9-overexpressing mice, compared to the GFP adenovirus-injected controls (Fig. 5C,D). IRF9-overepressing ob/ob mice had lower fasting serum glucose and insulin levels and lower hepatic TG, TC, and NEFA content than did control mice (Fig. 5E and Supporting Fig. 5A). Liver function was also protected from IRF9 overexpression (Supporting Fig. 5B). IPGTT and IPITT results demonstrated improved glucose tolerance and reduced IR in IRF9-overexpressing mice, compared to control mice (Fig. 5F,G). Phosphorylation of key insulin-signaling molecules, such as IRS1 and Akt, was elevated after IRF9 overexpression (Fig. 5H). Down-regulated proinflammatory factors and up-regulated anti-inflammatory factors were also observed in IRF9-overexpressing mice (Supporting Fig. 5C). Therefore, using dietary and genetic obesity models, we have now determined that IRF9 attenuates obesity-induced hepatic steatosis, IR, and inflammation.

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Figure 5. Hepatic IRF9 overexpression improves metabolism in ob/ob mice. (A) Immunoblot showed IRF9 expression in livers, epididymal fat pads, and gastrocnemius muscles 4 weeks after jugular vein injection of adenovirus containing IRF9 complementary DNA or GFP sequence control. (B) Representative IF images of liver section slides, which were stained with Abs against HNF4 (green) and IRF9 (red). 4',6-diamidino-2-phenylindole (DAPI; blue) was used to show nuclei. Scale bar indicates 50 μm. (C) Body weight, liver weight, and the liver/body-weight ratio of ob/ob mice injected with IRF9 or GFP adenovirus were examined; n = 12 per group. (D) Representative liver sections stained with H&E (top panel) and Oil Red O (lower panel) of IRF9 overexpressed and GFP control ob/ob mice. (E) Fasting blood glucose levels were detected with food deprivation for 6 hours. Serum fasting insulin levels were determined by enzyme-linked immunosorbent assay. HOMA-IR index was also calculated; n = 6 per group. (F) An IPGTT (1 g/kg body weight) was performed on ob/ob mice. Corresponding area under the curve (AUC) of blood glucose levels in each group was calculated; n = 12 per group. (G) An IPITT (0.75 units/kg body weight) was also performed. Corresponding AUC of blood glucose levels in each group was calculated; n = 12 per group. (H) Immunoblotting analysis of phosphorylation of IRS1-tyr608 and Akt in livers. Quantification of phosphorylated protein expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); n = 4 per group. All values are expressed as mean ± SEM. Statistical significance is indicated and compared to the GFP adenovirus-injected group: *P < 0.05; **P < 0.01.

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IRF9 Interacts With PPAR-α to Activate PPAR-α Target Genes

Transcription factors usually recruit cofactors to facilitate downstream gene expression. To investigate how IRF9 improves hepatic metabolism, we employed a yeast two-hybrid screening system and used IRF9 as bait to identify IRF9-interacting proteins in a human liver library. One of the candidate IRF9 interactors was PPAR-α; the prey clone encoded the N-terminal 254 residues of PPAR-α (data not shown). We confirmed the interaction between IRF9 and PPAR-α in HepG2 cells, a human hepatocellular carcinoma cell line, with coimmunoprecipitation (Co-IP). We found that IRF9 Co-IPed with PPAR-α, but not control immunoglobulin G (IgG), in HepG2 cells and vice versa (Fig. 6A). Additionally, a GST pull-down assay also confirmed the interaction between IRF9 and PPAR-α (Fig. 6B). To rule out the possibility that the interaction was newly formed during the Co-IP or GST pull down, we performed IF to identify IRF9 and PPAR-α localization. We found that IRF9 and PPAR-α colocalized predominantly in the nucleus (Fig. 6C). To map the PPAR-α-interacting region of IRF9, a series of IRF9 deletion mutants were generated. Neither the IRF9 N-terminal DNA-binding domain (DBD) nor the C-terminal IRF association domain (IAD) associated with PPAR-α; only the less-conserved IRF9 intermediate region interacted with PPAR-α (Fig. 6D). We also generated a series of PPAR-α deletion mutants. The mapping demonstrated that the DNA-binding domain (C domain), the hinge region (D domain), and the ligand-binding domain (E/F domain) of PPAR-α were all able to interact with IRF9 (Fig. 6E), and only the N-terminal A/B domain was not.

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Figure 6. IRF9 interacts with PPAR-α. (A) Co-IP of IRF9 and PPAR-α is shown. Cell lysates from HepG2 cells transfected with HA-tagged IRF9 and FLAG-tagged PPAR-α were prepared. These lysates were subjected to IP with HA Ab or IgG and analyzed by immunoblotting using HA or FLAG Abs. From the other side, these lysates were subjected to IP with FLAG Ab, then went through immunoblotting using HA or FLAG Abs. (B) GST pull-down assays of enhanced GFP (EGFP)-Myc-tagged PPAR-α with GST or GST-IRF9 are indicated. (C) Colocalization of IRF9 and PPAR-α in the nucleus is shown. pCherry-IRF9 and pEGFP-PPAR-α were transfected into primary mouse hepatocytes, and indirect IF analysis was performed. Cells were visualized by confocal microscopy, and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). (D) Left panel: schematic representation of four kinds of IRF9 deletion mutants. Right panel: mapping the PPAR-α-binding region of IRF9. Cell lysates from HEK293T cells transfected with FLAG-tagged PPAR-α and EGFP-Myc-tagged IRF9 deletion mutants were IPed with anti-FLAG, followed by immunoblotting with anti-FLAG and anti-Myc. (E) Left panel: schematic representation of four kinds of PPAR-α deletion mutants. Right panel: mapping the IRF9-binding region of PPAR-α. HEK293T cells were transfected with EGFP-Myc-tagged IRF9 and FLAG-tagged PPAR-α deletion mutants.

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We next sought to determine why IRF9 binds to PPAR-α. As shown earlier, we found that mRNA levels of PPAR-α target genes (e.g., acyl-CoA oxidase, carnitine palmitoyltransferase II, medium-chainacyl-CoA dehydrogenase, LCAD, UCP2, UCP3, fibroblast growth factor 21, pyruvate dehydrogenase lipoamide kinase isozyme 4, and phosphoenolpyruvate carboxykinase 1) were universally lower in livers of IRF9 KO mice than in controls (Fig. 3E). We found that PPAR-α target genes were activated in primary mouse hepatocytes transfected with WT IRF9 plasmids (Supporting Fig. 6A). To confirm the activation of PPAR-α target genes through IRF9-PPAR-α interaction, we constructed a mutant IRF9 plasmid in which the PPAR-α-interaction domain was deleted. Expression of PPAR-α target genes did not change in cells transfected with mutant IRF9 plasmids (Supporting Fig. 6B). When we further overexpressed IRF9 specifically in the liver, we observed up-regulation of PPAR-α target genes in livers of both diet-induced and genetically obese mice (Supporting Fig. 6C,D). Taken together, these results suggest that IRF9 activates PPAR-α target gene expression by interacting with PPAR-α.

Hepatic PPAR-α Overexpression Rescues Insulin Sensitivity and Ameliorates Hepatic Steatosis and Inflammation in IRF9 KO Mice

As expected, primary mouse hepatocytes trasfected with PPAR-α had markedly higher levels of its target genes than those transfected with GFP controls (Supporting Fig. 7A). To determine the sufficiency of PPAR-α in mediating the metabolic functions of IRF9, we overexpressed PPAR-α specifically in livers of WT mice and IRF9 KO mice. We injected mice with PPAR-α adenovirus through the jugular vein. Four weeks later, PPAR-α and its target genes were significant increased in the liver (Supporting Fig. 7B,C). After 24 weeks of HFD feeding, IRF9 KO mice displayed aggravated hepatic steatosis, IR, and inflammation, as described earlier. However, after PPAR-α was overexpressed, IRF9 KO mice displayed reduced liver weight (Fig. 7A). H&E and Oil Red O staining confirmed less hepatic lipid accumulation (Fig. 7B). Lower hepatic lipid content and preserved liver function indicated attenuated steatohepatitis (Supporting Fig. 7D,E). Fasting serum glucose and insulin levels and the HOMA-IR index in PPAR-α-overexpressed IRF9 KO mice were similar to those of GFP adenovirus-infected controls (Fig. 7C). Similar results were obtained with IPGTTs and IPITTs (Fig. 7D and 7E). Insulin signaling was also up-regulated upon PPAR-α overexpression (Fig. 7F). Measurement of inflammation- related genes by real-time PCR indicated a shifting macrophage population from M1 to M2 (Supporting Fig. 7F,G). Thus, we demonstrated that liver-specific PPAR-α overexpression rescues insulin sensitivity and ameliorates hepatic steatosis and inflammation in IRF9 KO mice.

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Figure 7. Hepatic PPAR-α overexpression rescues deregulated metabolism in IRF9 KO mice. (A) Body weight, liver weight, and the liver/body-weight ratio of HFD-fed mice injected with PPAR-α or vector adenovirus were examined; n = 12 per group. (B) Representative liver sections stained with H&E (top panel) and Oil Red O (lower panel) of PPAR-α overexpressed and GFP control mice. (C) Fasting blood glucose levels were detected with food deprivation for 6 hours. Serum fasting insulin levels were determined by enzyme-linked immunosorbent assay. HOMA-IR index was also calculated; n = 6-7 per group. (D) and (E) An IPGTT (1 g/kg body weight) and an IPITT (0.75 units/kg body weight) were performed; n = 12 per group. Corresponding area under the curve (AUC) of blood glucose levels in each group was calculated; n = 12 per group. (F) Immunoblot analysis of phosphorylation of IRS1-tyr608 and Akt signaling in livers. Quantification of phosphorylated protein expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH); n = 4 per group. All values are expressed as mean ± SEM. Statistical significance is indicated and compared to the GFP adenovirus-injected WT group: *P < 0.05, compared to the PPAR-α adenovirus-injected WT group; +P < 0.05, compared to the GFP adenovirus-injected IRF9 KO group; #P < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

 IRF9 KO mice have a relatively normal physical appearance, but are susceptible to virus infection because of the crucial role of IRF9 in mediating type I IFN responses.[21, 29] Therefore, most studies on IRF9 have been focused on its involvement in innate immunity and oncogenesis.[11] However, whether IRF9 is involved in the regulation of metabolism is unclear. In the present study, we, for the first time, demonstrated a critical role for IRF9 in hepatic lipid homeostasis. IRF9 expression was lower in livers of both diet-induced and genetic obesity models. On an HFD, IRF9 KO mice exhibited more-severe obesity, hepatic steatosis, IR, and inflammation. When IRF9 was specifically overexpressed in the liver, diet-induced and genetically obese mice displayed attenuated hepatic steatosis, IR, and inflammation, which indicate that IRF9 has an antidiabetic role.

Eguchi et al. identified IRFs to have potential roles in adipogenesis and adipose biology by high-throughput DNase hypersensitivity analysis.[18] This group further reported that IRF4 expression was nutritionally regulated in adipocytes. After feeding, IRF4 was down-regulated by insulin by effects of FoxO1 in WAT.[19] In the present study, we investigated the metabolic effects of another IRF family member (IRF9), which has ubiquitous distribution, rather than IRF4, the expression of which is highly restricted to adipose tissue and immune cells. In our study, obese mice displayed lower IRF9 expression in the liver than that of lean mice. Still, the mechanism by which IRF9 expression is down-regulated during obesity remains to be elucidated.

IRF9 KO mice showed higher levels of hepatic cholesterol and fatty acid synthesis, fatty acid uptake and lipogenesis, and lower levels of hepatic cholesterol output, lipolysis, and fatty acid oxidation, which all lead to hepatic lipid overload. All these factors indicate that IRF9 functions for hepatic lipid clearance and against hepatic steatosis. We further identified an interaction between IRF9 and PPAR-α and observed that PPAR-α target genes were significantly activated upon IRF9 overexpression. Because PPAR-α promotes lipid catabolism by increasing fatty acid uptake and oxidation in the liver and other organs,[30] PPAR-α mediates at least part of the antihepatic steatosis function of IRF9. PPARs are a family of NRs that initiate transactivation of target genes through ligand binding, corepressor removal, and coactivator recruitment.[30] Our results implicate IRF9 as a novel cofactor of PPAR-α, which is involved in the regulation of PPAR-α transactivation.

The present study demonstrated that hepatic insulin sensitivity in IRF9 KO mice was impaired, but was rescued, by liver-specific PPAR-α overexpression. It seems paradoxical given that PPAR-α-deficient mice were protected from HFD-induced IR, as reported by Guerre Millo et al.[31] Additionally, according to Koo et al., PPAR-α impairs liver insulin signaling by activating TRB3, which inhibits Akt activation.[32] Therefore, PPAR-α-mediated enhancement of insulin signaling, in the context of the current study, might be attributed to its lipid-clearing functions and the associated prevention of inflammation.[33]

Obesity-induced inflammation, as proposed by Gregor and Hotamisligil, originates from signals within metabolic cells, followed by metabolic tissue reconstruction to an inflammatory state.[3] Activation of IKK-β/NF-κB and JNK1/AP-1 pathways contributes to IR.[34-37] Cytokines (e.g., TNF-α and IL-6) also induce hepatic lipogenesis and increase hepatic TG accumulation.[38, 39] Thus, obesity and inflammation form a vicious cycle. Unlike the situation in adipose tissue, macrophage infiltration plays a secondary role in the liver during obesity; instead, liver-resident macrophage-like KCs become activated.[40] On an HFD, IRF9 KO livers displayed increased obesity-induced inflammation and M1-type polarization of KCs, both of which contribute to compromised insulin activities.

We employed IRF9 global KO mice to study the metabolic roles of IRF9 and found a poor hepatic metabolic phenotype. After overexpressing IRF9 specifically in the liver, nearly all the devastating metabolic effects of IRF9 deficiency were mitigated. This phenomenon reflects the importance of IRF9 in the liver to regulate glucose and lipid metabolism. Probably resulting from the short period of IRF9 overexpression using the adenovirus injection method and the preexistence of endogenous IRF9, the metabolic changes during IRF9 overexpression were, although statistically significant, not as drastic as those during IRF9 deficiency. Despite all these factors, IRF9 was vividly shown to relieve hepatic lipid overabundance and the development of hepatic steatosis in our obesity models.

In mammals, the IRF family consists of nine members that share similar structures. Different IRFs have overlapping targets and functions.[12] Some may wonder whether other IRFs compensate for the loss of IRF9 in IRF9 KO mice. Through deletion mutant plasmid construction and IP mapping, we identified that the less-conserved intermediate region of IRF9, rather than the well-conserved DBD or IAD, interacts with PPAR-α. Therefore, the regulation of PPAR-α transactivation could be uniquely attributed to IRF9, rather than other IRF family members.

Our study reveals the versatility of IRF9 and broadens our view toward the IRF family, which, as the name implies, was renowned for mediating immune responses. We now have successfully suggested a key role for IRF9 in metabolic function independent of its effect on immunity. However, uncovering the metabolic role of IRF9 in the liver is only the tip of the iceberg. There are many more unanswered questions, such as the tissue specificity of IRF function, interactions among IRFs and multiple cofactors, and influence of one IRF family member on the other family members. Investigating the mechanisms of IRF-mediated metabolic regulation will undoubtedly shed new light on treatment for obesity and diabetes.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

The authors thank Dr. Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan) for providing the IRF9 knockout mice. The authors also appreciate the RIKEN BRC for shipping IRF9 knockout mice through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep26368-sup-0001-suppinfo.doc59KSupplementary Information
hep26368-sup-0002-supptables.doc163K

Supporting table 1. Serum biochemical and cytokine, hormone analysis and liver function analysis kits.

Supporting table 2. Primers for Real-time PCR detection.

Supporting table 3. Antibodies for immunoblot analyses.

Supporting table 4. The primers for making constructs.

hep26368-sup-0003-suppfig1.tif151KSupporting Figure 1. IRF9 deficiency does not affect food intake but enhances hepatic gluconeogenesis. (A) Energy of food intake by wild-type and IRF9 KO mice fed with the normal chow or HFD was calculated every week. n=28-39 per group. (B) For all the statistical significance is indicated and compared with the WT HFD group, **p < 0.01.
hep26368-sup-0004-suppfig2.tif278KSupporting Figure 2. IRF9 deficiency aggravates the hepatic steatosis upon HFD feeding. (A) Liver function was examined by measuring the levels of alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) in the serum by reagent kits, n=7 per group. (B) Immunoblot analysis indicated the inhibition of AMPK signaling in liver extracts from IRF9 KO mice. Protein expression levels were quantified and normalized to loading control GAPDH. All values are expressed as the mean ± SEM. The statistical significance is indicated and compared with the WT HFD group,**p<0.01, ***p < 0.001; compared with the WT NC group, ##p < 0.01.
hep26368-sup-0005-suppfig3.tif157KSupporting Figure 3. IRF9 deficiency does not affect lipid metabolism in WAT. Relative mRNA levels of representative adipogenesis associated genes (PPARγ, C/EBPα, C/EBPδ, pref-1 and Cyclin D1), and lipogenic genes (GPAT, FAS and SREBP-1c) and genes responsible for fatty acid oxidation (PPARα, ACOX and CPT-1α) and lipolyisis (HSL and ATGL) in epididymal fat extracts from IRF9 KO and WT mice. n=6-12 per group. All values are expressed as the mean ± SEM.
hep26368-sup-0006-suppfig4.tif466KSupporting Figure 4. Hepatic IRF9 overexpression improves metabolism in diet-induced obese mice. (A) The levels of TG, cholesterol and NEFA were extracted in liver tissue from HFD group between WT with KO mice. n= 5 each group. (B) Liver function was examined by measuring the levels of ALT, AST and ALP in the serum by reagent kits, n=6 per group. (C) The mRNA levels of proinflammatory and anti-inflammatory markers in liver were measured by real-time PCR. n=12 for each group. All values are expressed as mean ± SEM. The statistical significance is indicated and compared with the GFP adenovirus injected group, *p < 0.05, **p < 0.01.
hep26368-sup-0007-suppfig5.tif476KSupporting Figure 5. Hepatic IRF9 overexpression improves metabolism in ob/ob mice. (A) The levels of TG, cholesterol and NEFA were extracted in liver tissue from HFD group between WT with KO mice. n= 5 each group. (B) Liver function was examined by measuring the levels of ALT, AST and ALP in the serum by reagent kits, n=7 per group. (C) The mRNA levels of proinflammatory and anti-inflammatory markers in liver were measured by real-time PCR. n=12 for each group. All values are expressed as mean ± SEM. The statistical significance is indicated and compared with the GFP adenovirus injected group, *p < 0.05, **p < 0.01.
hep26368-sup-0008-suppfig6.tif433KSupporting Figure 6. IRF9 interacts with PPARα to activate PPARα target genes. (A) The mRNA levels of PPARα target genes in the primary mouse hepatocytes transfected with GFP or IRF9 plasmid are examined by real-time PCR. (B) The mRNA levels of PPARα target genes in the primary mouse hepatocytes transfected with GFP or mutant IRF9 plasmid of which the PPARα-interaction domain was deleted. (C) The mRNA levels of PPARα target genes in the liver of WT mice fed with HFD injected with adenovirus containing GFP or IRF9. (D) The mRNA levels of PPARα target genes in the liver of ob/ob mice injected with adenovirus containing GFP or IRF9. In (A) to (D), values are presented as the mean ± SEM,and statistical significance is indicated and compared with the Ad-GFP group, *p < 0.05, **p < 0.01.
hep26368-sup-0009-suppfig7.tif901KSupporting Figure 7. Hepatic PPARα overexpression rescues deregulated metabolism in IRF9 KO mice. (A) The mRNA levels of PPARα target genes in primary mouse hepatocytes infected with adenovirus expressing IRF9 or vector controls. (B) Immunoblot showed the PPARα expression in livers four weeks after jugular vein injection of adenovirus within PPARα cDNA or GFP sequence control. (C) The mRNA levels of PPARα and its target genes were determined by real-time PCR. (D) The levels of TG, cholesterol and NEFA were extracted in liver tissue from HFD group between WT with KO mice. n= 5 for each group. (E) Liver function was examined by measuring the levels of ALT, AST and ALP in the serum by reagent kits, n=7-9 per group. (F and G) The mRNA levels of proinflammatory and anti-inflammatory markers in livers of WT and IRF9 KO mice were measured by real-time PCR. n=9-12 for each group. All values are expressed as mean ± SEM. The statistical significance is indicated and compared with the GFP adenovirus injected WT group, *p < 0.05; compared with PPARα adenovirus injected WT group, +p<0.05; compared with the GFP adenovirus injected IRF9 KO group, #p< 0.05.

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