Transcription factor 4 (TCF-4) was recently identified as a candidate gene for the cause of type 2 diabetes, although the mechanisms have not been fully elucidated. In the present study, we demonstrated that the TCF-4 transgene in macrophages aggravated high-fat diet (HFD)-induced insulin resistance and chronic inflammation, characterized by the elevation of proinflammatory cytokines in the blood, liver and white adipose tissue, as well as a proinflammatory profile of immune cells in visceral fats in mice. Mechanistically, TCF-4 functioned as a co-activator of p65 to amplify the saturated free fatty acid (FFA)-stimulated promoter activity, mRNA transcription and secretion of proinflammatory cytokines in primary macrophages. Blockage of p65 with a specific interfering RNA or inhibitor could prevent TCF-4-enhanced expression of proinflammatory cytokines in FFA/lipopolysaccharide-treated primary macrophages. The p65 inhibitor could abolish macrophage TCF-4 transgene-aggravated systemic inflammation, glucose intolerance and insulin resistance in HFD-treated mice. In addition, we demonstrated that the mRNA expression of TCF-4 in the peripheral blood monocytes from humans was positively correlated to the levels of interleukin (IL)-1β, tumour necrosis factor α, IL-6 and fasting plasma glucose. In summary, we identified TCF-4 as a co-activator of p65 in the potentiation of proinflammatory cytokine production in macrophages and aggravation of HFD-induced chronic inflammation and insulin resistance in mice.

CLINICAL PERSPECTIVES

  • TCF7L2 polymorphisms have a strong association with the risk of type 2 diabetes, with the mechanisms not yet being elucidated.

  • In the present study we identified that TCF-4 functions as a co-activator of p65 both to potentiate proinflammatory cytokine production in macrophages and to aggravate HFD-induced chronic inflammation and insulin resistance in mice.

  • Targeting of TCF-4 or p65 in macrophages represents a potential strategy for the management of chronic inflammation-associated diseases.

INTRODUCTION

Obesity and diabetes are common diseases worldwide [1,2], with chronic inflammation and insulin resistance being pivotal features of these abnormalities [1]. Emerging studies reveal that chronic inflammation is a major inducer of insulin resistance [36]. White adipose tissue is the key source of proinflammatory cytokines in obesity [4,6,7]. In particular, the visceral fats display a proinflammatory profile of immune cells in response to the treatment of a high-fat diet (HFD). The increased infiltration of CD11c+ macrophages [4,8], CD8+ T-cells [9], B-cells [10,11] and neutrophils [12] is causally linked to chronic inflammation and insulin resistance in mice, whereas CD4+ T-cells and CD4+CD25+Foxp3+ T-cells (Treg) do the opposite [13,14].

Macrophages are the most prominent innate immune cells with high plasticity [8]. In genetically or HFD-induced obese mouse models, macrophages are classically activated, characterized by overexpression of a series of proinflammatory cytokines [e.g. interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6] and activation of multiple inflammatory signalling pathways [e.g. nuclear factor κ light chain enhancer of activated B-cells (NF-κB) and c-Jun N-terminal kinase (JNK)] [4,6,8]. The macrophage-derived proinflammatory cytokines are important intervention factors for insulin signalling [3,15]. Therefore, the process of macrophage polarization represents an efficient strategy to regulate chronic inflammation and insulin sensitivity.

Transcription factor 4 (TCF-4), encoded by transcription factor 7-like 2 (TCF7L2), is a terminal effector downstream of Wnt signalling [16]. As a result of the consensus motif for the TCF-4-binding site (A/TA/TCAAAG) in the promoter region, c-myc [17], cyclin D1 [18], c-jun [19] and the matrix metalloproteinase MMP7 [20] are considered to be the targets of the Wnt signalling pathway. Through regulation of its targets, TCF-4 plays an important role in cell cycle progression, apoptosis, proliferation and migration in normal or cancer cells. Recently, emerging evidence indicates that TCF7L2 polymorphisms are strongly associated with the risk of type 2 diabetes in different ethnic populations [2124]. Molecular mechanisms underlying this association are, however, far from understood at this time.

TCF-4 knockout mice die shortly after birth, associated with a lack of proliferative compartments in the prospective crypt regions between the intestinal villi [25]. The tissue-specific expression and roles of TCF-4 were investigated recently, with controversial observations. Lyssenko et al. [26] and Savic et al. [27] suggest deleterious effects of TCF-4 in pancreatic β-cells in contrast to multiple lines of work by Shu et al. [28,29]. Very little is known about how TCF-4 expression is regulated during the development of diabetes. TCF-4 expression in adipose tissue is decreased in obese individuals with type 2 diabetes [30], whereas TCF-4 mRNA is increased in islets isolated from the Zucker diabetic fatty rat [31]. These studies indicate that expression of TCF-4 is independently regulated in different tissues or cells.

The NF-κB pathway was activated in adipose tissue-derived macrophages (ATMs) from obese animal models and humans [4,32,33]. NF-κB controlled the transcriptional specificity via the assembly of homodimers or heterodimers of five different NF-κB proteins (p65, p50, c-Rel, p105 and p100) [34]. In response to proinflammatory stimulation, the I-κB kinase (IKK) was activated to phosphorylate I-κB protein and suppress I-κB-mediated p65 degradation [34]. Recently, it has been reported that TCF-4 could cooperate with p65 to regulate the expression of proinflammatory cytokines in chondrocytes [35], indicating a proinflammatory role of TCF-4 in other cell types, such as macrophages.

Therefore, we presumed that TCF-4 might regulate macrophage activation via p65 and play a role in the progression of insulin resistance. In the present study, we set up a mouse model with a macrophage-specific transgene of TCF-4 and investigated the roles of macrophage TCF-4 in in vivo chronic inflammation and insulin sensitivity.

MATERIALS AND METHODS

Cell culture and reagents

Primary macrophages were grown in Dulbecco's Modified Eagle Medium supplemented with 10% FBS at 37°C in a humidified 5% CO2 atmosphere. A saturated free fatty acid (FFA) (C18:0, S4751, Sigma) was dissolved in ethanol at a high concentration and then diluted with 5% FFA BSA (Sigma) to obtain a 200 mM stock solution for cell treatment. JSH-23 (J4455, Sigma) was dissolved in DMSO to obtain a 40 mM stock solution for further cell treatment.

Animals and diets

The cDNA of mouse tcf7l2 gene (NM_009333, from OriGene cDNA ORF clone: MR207325L1) was subcloned into a transgenic construct that contained the human CD11b promoter to drive myeloid cell-specific gene expression [36,37]. The transgenic construct was microinjected into C57BL/6 embryos according to standard protocols and the founders were crossed with the wild-type (WT) C57BL/6 strain. The line with the highest expression of TCF-4 protein in macrophages was selected for further study. The ob/ob obese mice were purchased from Jackson Laboratory. Mice aged 6 weeks were fed with either a chow diet (CD; D12450J, 10 kcal% fat) or an HFD (D12492, 60 kcal% fat; both from Research Diets). All the mice were housed in a pathogen-free environment at the Third Military Medical University, and all experimental procedures were approved by the Institutional Animal Care and Use Committee.

Collection and isolation of human peripheral blood monocytes

All the experiments involving humans were approved by the ethics committee in the Chengdu Military General Hospital and informed consent was obtained from all participants. Human blood samples were obtained from the male volunteers (age=38.2±5.3 years, n=20). The fasting plasma glucose and insulin levels were determined. All the steps in the blood monocyte preparation were carried out at room temperature. The content of the blood-collecting tubes from the same donor were pooled and mixed 1:1 (v/v) with pre-warmed (room temperature) 1×PBS (without Ca2+, Mg2+). The blood:PBS mixture was layered on to Lymphoprep (Axis-Shield) and subsequently centrifuged at 800 g for 30 min at room temperature (slow acceleration, no brake). The ring-shape interphase (peripheral blood monocytes or PBMCs) was collected with a Pasteur pipette into a new 50-ml tube, diluted up to 50 ml with pre-warmed 1×PBS (without Ca2+, Mg2+) and centrifuged at 300 g for 10 min at room temperature (fast acceleration and brake). The total cell number and the number of living cells were determined using Trypan Blue exclusion. Then, the monocytes were subjected to RNA extraction, reverse transcription and real-time PCR (RT-PCR) assays of TCF-4, IL-1β, TNF-α and IL-6.

Measurement of the HOMA-IR index

Mice were fasted overnight (for about 12 h) and then the blood was harvested via the tail vein. The levels of plasma glucose and insulin were measured. The HOMA-IR index {[fasting glucose level (mmol/l)×fasting insulin level (mIU/l)]/22.5]}. The HOMA-IR index was used to evaluate the grade of insulin resistance.

Glucose tolerance test and insulin tolerance test

For the glucose tolerance test (GTT), male mice aged 18 weeks (on a CD or HFD for 12 weeks, starting at age 6 weeks) were fasted for 12 h during the dark cycle. The tail vein was cut and the baseline blood glucose levels were measured using a glucometer and strips. The mice were then injected intraperitoneally with glucose solution at 1.5 g/kg body weight, and the blood glucose levels were monitored at 30, 60 and 120 min post-glucose injection.

For the insulin tolerance test (ITT), male mice aged 19 weeks (1 week after the GTT) were fasted for 4 h during the light cycle, and the baseline blood glucose levels were measured via the tail vein. The recombinant human insulin (NOVO Nordisk, Inc.) was then injected intraperitoneally into the CD-fed and HFD-fed mice at 1.0 and 1.5 IU/kg body weight, respectively. The blood glucose levels were measured at 30, 60 and 120 min after insulin administration.

Hyperinsulinaemic–euglycaemic clamp studies

Hyperinsulinaemic–euglycaemic clamp experiments were conducted as previously described [4,6]. Overnight-fasted male WT and transgenic mice treated with a CD or HFD for 12 or 24 weeks were infused with human insulin at a rate of 2.5 munits/kg per min, coupled with variable infusion rates of 20% glucose to maintain the blood glucose concentration at 5.5 mM for 45 min, and the glucose infusion rate was recorded at 5, 15, 25, 35 and 45 min.

ELISA

The ELISA kits TNF-α (#MTA00B), IL-6 (#M6000B) and IL-1β (#MLB00C) were purchased from R&D Systems. The detailed procedures of measurement were as described in our previous reports [4].

Quantitative RT-PCR

Total RNA from livers, adipose tissues, monocytes or macrophages was extracted using the TRIzol reagent from Invitrogen. The reverse transcription and RT-PCR was performed as described previously [4,38]. All the primers are available on request.

Western blotting

Cell proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) and quantified with a BCA kit (Beyotime). Immunoblotting was performed as previously described. The primary antibodies included: anti-TCF-4 (#2565), anti-phospho-IKK (#2697), anti-I-κBα (#4812), anti-GAPDH (#2118, all from Cell Signaling), anti-p65 (sc-109, Santa Cruz), and anti-phospho-p65 (#3033), anti-JNK (#9252) and anti-phospho-JNK (#4668, all from Cell Signaling). The filter was incubated with Enhanced Chemiluminescence Substrate (PerkinElmer) for 1 min and the signals were captured with a Bio-Rad ChemiDoc MP System.

Transcription activity assay of NF-κB

The promoter region (−2500/−2000) of the mouse CCL2 gene, containing two well-documented nuclear factors for κ light chain enhancer of activated B-cells (NF-κB or p65)-binding sites (−2374: GGGAATTTCC; −2348: GGGAACTTCC) [39], was subcloned into PGL3-Basic vector (Promega) to form a luciferase reporter gene. This reporter construct was used to evaluate the transcription activity of NF-κB (p65). The relative luciferase activity of the macrophages that were transfected with the reporter gene represented the relative transcription activity of NF-κB (p65).

Chromatin immunoprecipitation assays

This experiment measured the binding between p65 protein and the corresponding binding sites (−2374: GGGAATTTCC; −2348: GGGAACTTCC) in the promoter DNA of the mouse CCL2 gene. In brief, the cultured WT or TCF-4 transgenic PMs (treated with BSA or FFA) were cross-linked with 1% formaldehyde, followed by sonication. The supernatant with equal amounts of protein was immunoprecipitated with 1 μg of mouse p65 (sc-109, Santa Cruz) or rabbit immunoglobulin G (IgG) as control using the chromatin immunoprecipitation (ChIP) kit (#17-10460, Millipore Corp.) according to the manufacture's protocol. The immunoprecipitates were analysed using RT-PCR for detecting the co-immunoprecipitated DNA containing the functional p65-binding sites (−2374: GGGAATTTCC; −2348: GGGAACTTCC). The ChIP primers were designed as: forward: 5′-TGCTTGGCTGCAGGCCCAG-3′, reverse: 5′-AGGATGTTCTTCCCAGCGG-3′. The length of the desired product was 150 bp.

Loss-of-function studies

Mouse endogenous p65 was knocked down by using two mixed commercial siRNAs (sc-29411 and sc-44213, Santa Cruz). The control siRNA was also purchased from Santa Cruz (sc-37007). The endogenous TCF-4 was silenced by OriGene commercial siRNAs (SR414923: si-TCF4-1, RefSeq NM_001142918; si-TCF4-2, RefSeq NM_001142919) for mouse cells. The transfection concentration for the siRNAs was 20 nmol/ml.

Cell transfection and reporter gene assays

The transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, the plasmids (0.4 μg/ml) or the siRNAs (20 nmol/ml) were transfected in the serum-free and antibiotic-free media. After transfection for 6 h, the media were removed and replaced with complete growth media using a different treatment. Then, the luciferase activity of the cell lysate was evaluated according to the manufacturer's instructions (Promega Corp.), and the total protein concentration in each well was measured as an internal control. The relative luciferase activity was displayed to compare the difference between each group.

Cell extraction and sorting

Isolation of peritoneal macrophages (PMs), bone marrow-derived macrophages (BMDMs), adipose stromal–vascular fraction (SVF) and ATMs, as well as immunostaining and FACS, were performed exactly as described in our previous study [4].

Statistical analysis

All the data were expressed as means±S.E.M.s and analysed using either one-way ANOVA or two-tailed unpaired Student's t-test. The group difference was considered statistically significant for P<0.05. For each parameter of all data presented, P<0.05, P<0.01, P<0.005 and values not sharing a common superscript letter differ significantly (P<0.05).

RESULTS

Macrophage TCF-4 potentiates HFD-induced insulin resistance in mice

To observe the role of macrophage TCF-4 in insulin sensitivity in vivo, we employed a macrophage TCF-4 transgenic (Tg) mouse model. The specific expression of exogenous TCF-4 was driven by human CD11b promoter (see Supplementary Figure S1A). The morphology of the PMs was not affected by the TCF-4 transgene (see Supplementary Figure S1B). Using an RT-PCR assay, we verified that TCF-4 mRNA was specifically overexpressed in PMs or BMDMs (see Supplementary Figure S1C). Identical results were obtained in immunoblotting assays (see Supplementary Figure S1D). Although fasting or fed plasma glucose concentration did not differ between the WT and Tg mice regardless of diet (Figure 1A), Tg mice on an HFD showed notable increases in fasting and fed plasma insulin (Figure 1B), as well as the HOMA-IR index (Figure 1C), indicating augmented insulin resistance. For further assessment of systemic insulin sensitivity, the GTT and ITT were performed. Glucose tolerance (Figure 1D) and insulin sensitivity (Figure 1E) were unaltered in CD-fed mice but impaired in HFD-fed Tg mice (Figures 1F and 1G). Furthermore, hyperinsulinaemic − euglycaemic clamp studies showed that the glucose infusion rate was notably decreased in Tg mice relative to the WT ones on an HFD for 12 (Figure 1H) or 24 weeks (Figure 1I). We further investigated the body weight gain of Tg and WT mice, because insulin resistance was usually caused by obesity. Unexpectedly, there was no genotype-caused difference of body weight gain between the WT and Tg mice (see Supplementary Figure S1E). Taken together, these results demonstrated that overexpression of TCF-4 in macrophages aggravated HFD-induced insulin resistance in mice.

Macrophage TCF-4 transgene potentiates HFD-induced insulin resistance in mice

Figure 1
Macrophage TCF-4 transgene potentiates HFD-induced insulin resistance in mice

(A) Fasting (12 h) and fed plasma glucose levels of male mice on a CD or HFD for 12 weeks (n=8). (B) Fasting (12 h) and fed plasma insulin levels of the mice described in (A) (n=8, *P<0.05). (C) The HOMA-IR index of male mice calculated from data in (A) and (B) (n=8, **P<0.01). ND, not detectable. (D, E) (D) GTT and (E) ITT on the 18- to 19-week-old WT or Tg mice on a CD (n=8). (F) GTT and (G) ITT on the WT or Tg mice on an HFD for 12–13 weeks (n=8, *P<0.05, **P<0.01). (H, I). Glucose infusion rates measured by hyperinsulinaemic–euglycaemic clamp studies in male mice on an HFD for (H) 12 weeks and (I) 24 weeks (n=5, **P<0.01). For (A) to (G), the experiments were repeated twice independently. The tests in (H) and (I) were performed once only.

Figure 1
Macrophage TCF-4 transgene potentiates HFD-induced insulin resistance in mice

(A) Fasting (12 h) and fed plasma glucose levels of male mice on a CD or HFD for 12 weeks (n=8). (B) Fasting (12 h) and fed plasma insulin levels of the mice described in (A) (n=8, *P<0.05). (C) The HOMA-IR index of male mice calculated from data in (A) and (B) (n=8, **P<0.01). ND, not detectable. (D, E) (D) GTT and (E) ITT on the 18- to 19-week-old WT or Tg mice on a CD (n=8). (F) GTT and (G) ITT on the WT or Tg mice on an HFD for 12–13 weeks (n=8, *P<0.05, **P<0.01). (H, I). Glucose infusion rates measured by hyperinsulinaemic–euglycaemic clamp studies in male mice on an HFD for (H) 12 weeks and (I) 24 weeks (n=5, **P<0.01). For (A) to (G), the experiments were repeated twice independently. The tests in (H) and (I) were performed once only.

Macrophage TCF-4 aggravates HFD-induced chronic inflammation in mice

Chronic inflammation is a major contributor to glucose intolerance and insulin resistance [15]. Therefore, we further investigated the role of macrophage TCF-4 in proinflammatory cytokine production in mice. We demonstrated that macrophage TCF-4 did not influence the levels of proinflammatory cytokines (IL-1β, TNF-α and IL-6) in the plasma, liver or white adipose tissue of the mice on a CD (Figures 2A–2C). However, in response to the HFD treatment, macrophage TCF-4 transgene largely amplified the levels of those cytokines in the plasma (Figure 2A), liver (Figure 2B) and white adipose tissue (Figure 2C). Consistent with this, the histological staining indicated that the macrophage TCF-4 transgene obviously potentiated the infiltration of immune cells in the liver (see Supplementary Figure S2A) and epididymal fats (see Supplementary Figure S2B) of the HFD-treated mice. For further verification of the proinflammatory function of macrophage TCF-4, we treated the WT and Tg mice with lipopolysaccharide (LPS), which is increased in the plasma in obesity [40]. We demonstrated that the Tg mice displayed higher levels of IL-1β, TNF-α and IL-6 in the plasma (see Supplementary Figure S2C), liver (see Supplementary Figure S2D) and white adipose tissue (see Supplementary Figure S2E) than the WT mice. These findings indicated that macrophage TCF-4 could aggravate HFD-induced chronic inflammation in mice.

Macrophage TCF-4 amplifies HFD-induced chronic inflammation in mice

Figure 2
Macrophage TCF-4 amplifies HFD-induced chronic inflammation in mice

(A) IL-1β, TNF-α and IL-6 levels in the plasma from the WT or Tg mice on a CD or HFD for 12 weeks (n=5, *P<0.05, **P<0.01). ND, not detectable. (B) Expression of mRNA of IL-1β, TNF-α and IL-6 in the liver of the mice as described in (A) (n=5, *P<0.05, **P<0.01). (C) Levels of mRNA of IL-1β, TNF-α and IL-6 in the epididymal fat (eFat) of the mice described in (A) (n=5, *P<0.05). Those experiments in (A), (B) and (C) were repeated three times.

Figure 2
Macrophage TCF-4 amplifies HFD-induced chronic inflammation in mice

(A) IL-1β, TNF-α and IL-6 levels in the plasma from the WT or Tg mice on a CD or HFD for 12 weeks (n=5, *P<0.05, **P<0.01). ND, not detectable. (B) Expression of mRNA of IL-1β, TNF-α and IL-6 in the liver of the mice as described in (A) (n=5, *P<0.05, **P<0.01). (C) Levels of mRNA of IL-1β, TNF-α and IL-6 in the epididymal fat (eFat) of the mice described in (A) (n=5, *P<0.05). Those experiments in (A), (B) and (C) were repeated three times.

TCF-4 potentiates saturated FFA/LPS-stimulated proinflammatory cytokine production in macrophages

To decipher the proinflammatory mechanism of macrophage TCF-4 in HFD-treated mice, we observed the roles of FFA and LPS, two well-documented factors induced by HFD treatment [40,41], in the proinflammatory cytokine production of primary macrophages. As shown in Figure 3A, the TCF-4 transgene notably amplified FFA-induced mRNA levels of IL-1β, TNF-α and IL-6 in the cultured PMs (Figure 3A). We confirmed these findings further by measuring the proinflammatory cytokine levels in the supernatant of the cultured PMs (Figure 3B). Likewise, when treated with LPS, the TCF-4 transgenic macrophages expressed significantly higher mRNA and protein levels of proinflammatory cytokines (IL-1β, TNF-α and IL-6) than the WT macrophages (see Supplementary Figures S3A and S3B). For further validation of the proinflammatory activation of macrophages in Tg mice, we isolated the SVF of the epididymal fat pads and performed FACS analysis. On a CD, no significant differences were observed in the total number of SVF cells (Figure 3C), F4/80+ cells (macrophages; Figure 3D), CD45R/B220+ cells (B-cells; see Supplementary Figure S3F) and Gr1+ cells (neutrophils; see Supplementary Figure S3G) between the two genotypes. On an HFD, the total number of SVF cells (Figure 3C), F4/80+ cells (macrophages; Figure 3D), CD45R/B220+ cells (B-cells; see Supplementary Figure S3F) and Gr1+ cells (neutrophils; see Supplementary Figure S3G) were notably higher in Tg mice than in controls. It is noteworthy that the percentages of CD11c+ cells in the F4/80+ population (Figure 3E) and CD3+CD4−CD8+ cells (CD8+ T-cells) in the SVF population (see Supplementary Figure S3C) were significantly increased, whereas the percentages of the CD3+CD4+CD8− cells (CD4+ T-cells; see Supplementary Figure S3D) and CD4+CD25+Foxp3+ (Treg) cells (see Supplementary Figure S3E) in the SVF population were largely decreased in Tg mice relative to the WT ones on an HFD for 12 weeks. These results displayed a proinflammatory profile of immune cells (e.g. M1 macrophages) in white adipose tissue of HFD-fed Tg mice. However, how the proinflammatory cytokines were regulated by TCF-4 in macrophages needs further elucidation.

TCF-4 potentiates saturated FFA-stimulated proinflammatory cytokine production in macrophages and HFD-induced macrophage polarization in white adipose tissue

Figure 3
TCF-4 potentiates saturated FFA-stimulated proinflammatory cytokine production in macrophages and HFD-induced macrophage polarization in white adipose tissue

(A) Levels of mRNA of IL-1β, TNF-α and IL-6 in the isolated primary WT or Tg macrophages that were treated with BSA (5%) or FFA (200 μM) for 24 h (n=5, *P<0.05). (B) Supernatant cytokine levels of the isolated primary WT or Tg macrophages that were treated with BSA (5%) or FFA (200 μM) for 24 h (n=5, *P<0.05, **P<0.01). (C) Total cell number of the SVF in epididymal fat (eFat) pads isolated from age-matched male mice on a CD or HFD for 12 weeks (n=8, *P<0.05). (D) F4/80+ macrophage number in eFat (n=8, *P<0.05). (E) Percentage of CD11c+ cells in F4/80+ cell population in eFat (n=8, *P<0.05). The experiments in (A) and (B) were repeated three times and the tests in (C), (D) and (E) were performed twice independently.

Figure 3
TCF-4 potentiates saturated FFA-stimulated proinflammatory cytokine production in macrophages and HFD-induced macrophage polarization in white adipose tissue

(A) Levels of mRNA of IL-1β, TNF-α and IL-6 in the isolated primary WT or Tg macrophages that were treated with BSA (5%) or FFA (200 μM) for 24 h (n=5, *P<0.05). (B) Supernatant cytokine levels of the isolated primary WT or Tg macrophages that were treated with BSA (5%) or FFA (200 μM) for 24 h (n=5, *P<0.05, **P<0.01). (C) Total cell number of the SVF in epididymal fat (eFat) pads isolated from age-matched male mice on a CD or HFD for 12 weeks (n=8, *P<0.05). (D) F4/80+ macrophage number in eFat (n=8, *P<0.05). (E) Percentage of CD11c+ cells in F4/80+ cell population in eFat (n=8, *P<0.05). The experiments in (A) and (B) were repeated three times and the tests in (C), (D) and (E) were performed twice independently.

TCF-4 co-activates NF-κB (p65) in macrophages

To investigate the mechanism linking TCF-4 to proinflammatory cytokine production, the potential inflammatory pathways downstream of TCF-4 were studied. NF-κB and JNK are the terminal pathways in response to inflammatory stimulation [42]. FFA treatment largely stimulated the phosphorylation of JNK, whereas the TCF-4 transgene did not affect it significantly, regardless of BSA or FFA treatment (see Supplementary Figures S4A and S4B). It is noteworthy that we found that TCF-4 could notably amplify FFA-induced phosphorylation of NF-κB (p65), with the levels of I-κBα, IKK and its phosphorylated derivatives not being altered significantly (Figures 4A and 4B). These results indicated that TCF-4 stimulated p65 phosphorylation not through the classic upstream member IKK or I-κBα. Indeed, we demonstrated that TCF-4 could directly interact with p65 (see Supplementary Figure S4C). The TCF-4 transgene could largely potentiate the protein–protein interaction between TCF-4 and p65 in the FFA-treated primary macrophages (see Supplementary Figure S4D). Simultaneously, the FFA-induced transcription activity of NF-κB (p65) was largely enhanced in Tg macrophages relative to the WT ones (Figure 4C). Meanwhile, the binding activity between NF-κB (p65) and the promoter DNA of CCL2 was also notably potentiated in response to FFA treatment (Figure 4D). Those findings indicated that TCF-4 might function as a co-activator of NF-κB (p65) in macrophages. To verify this notion, we silenced TCF-4 expression in primary macrophages with siRNAs (see Supplementary Figure S4E), and found that TCF-4 silence attenuated the interaction between TCF-4 and p65 in response to FFA treatment (see Supplementary Figure S4F). Meanwhile, the FFA-induced transcription activity of NF-κB was also largely prevented by TCF-4 silence (see Supplementary Figure S4G).

TCF-4 enhances NF-κB (p65) activity in macrophages

Figure 4
TCF-4 enhances NF-κB (p65) activity in macrophages

(A) Immunoblotting assays of p65, phosphorylated p65 (p-p65), I-κBα and phosphorylated IKK (p-IKK) in the primary WT or Tg macrophages stimulated with BSA (5%) or FFA (200 μM) for 24 h. These experiments were performed three times and representative images are displayed. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (B) The statistical results of the immunoblotting assays in (A) (n=3, *P<0.05, **P<0.01). (C) TCF-4 amplifies FFA-stimulated transcription activity of NF-κB. A luciferase reporter construct containing mouse CCL2 promoter, harbouring two NF-κB-binding elements, was transfected (0.4 μg/ml) into the WT or Tg macrophages for 5 h. Then, the macrophages were treated with BSA (5%) or FFA (200 μM) for 24 h and harvested for a luciferase activity assay (n=3, **P<0.01). (D) TCF-4 enhances FFA-induced, promoter-binding activity of NF-κB. The primary WT or Tg macrophages were treated with BSA (5%) or FFA (200 μM) for 24 h, and then harvested to evaluate the binding activity between p65 and the CCL2 promoter DNA using a ChIP assay (n=3, *P<0.05, **P<0.01). The tests in (C) and (D) were performed twice.

Figure 4
TCF-4 enhances NF-κB (p65) activity in macrophages

(A) Immunoblotting assays of p65, phosphorylated p65 (p-p65), I-κBα and phosphorylated IKK (p-IKK) in the primary WT or Tg macrophages stimulated with BSA (5%) or FFA (200 μM) for 24 h. These experiments were performed three times and representative images are displayed. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (B) The statistical results of the immunoblotting assays in (A) (n=3, *P<0.05, **P<0.01). (C) TCF-4 amplifies FFA-stimulated transcription activity of NF-κB. A luciferase reporter construct containing mouse CCL2 promoter, harbouring two NF-κB-binding elements, was transfected (0.4 μg/ml) into the WT or Tg macrophages for 5 h. Then, the macrophages were treated with BSA (5%) or FFA (200 μM) for 24 h and harvested for a luciferase activity assay (n=3, **P<0.01). (D) TCF-4 enhances FFA-induced, promoter-binding activity of NF-κB. The primary WT or Tg macrophages were treated with BSA (5%) or FFA (200 μM) for 24 h, and then harvested to evaluate the binding activity between p65 and the CCL2 promoter DNA using a ChIP assay (n=3, *P<0.05, **P<0.01). The tests in (C) and (D) were performed twice.

TCF-4 potentiates p65-dependent proinflammatory cytokine production in macrophages

To verify the role of p65 in TCF-4-mediated proinflammatory cytokine production, the experiments for p65 blockage were performed in macrophages. By interference of RNA, p65 expression was successfully silenced in primary macrophages (see Supplementary Figure S5A). Via the treatment with a well-known p65 inhibitor JSH-23 [43,44], FFA-induced p65 phosphorylation was largely prevented (see Supplementary Figure S5B). As expected, FFA-stimulated mRNA levels of IL-1β (Figure 5A), TNF-α (Figure 5B) and IL-6 (Figure 5C) were notably amplified by the TCF-4 transgene, and this effect was abolished by the knockdown or inactivation of p65. In addition, we also demonstrated that TCF-4 did not potentiate proinflammatory cytokine expression in basal conditions. However, the blockage of p65 with siRNA or JSH-23 could significantly prevent the induction of TCF-4 on proinflammatory cytokines in LPS-treated macrophages (see Supplementary Figures S5C–S5E). These results indicated that TCF-4 potentiated p65-dependent proinflammatory cytokine production in macrophages.

TCF-4 potentiates p65-dependent proinflammatory cytokine production in macrophages

Figure 5
TCF-4 potentiates p65-dependent proinflammatory cytokine production in macrophages

The WT or Tg PMs were treated with a scrambled siRNA (siNC, 20 nmol/ml), a mouse p65-specific siRNA (si-p65, 20 nmol/ml) or a p65 inhibitor (JSH-23, 40 μM) for 12 h, and then treated with BSA (5%) or FFA (200 μM) for 24 h before collection for an RT-PCR assay of (A) IL-1β, (B) TNF-α and (C) IL-6. Values not sharing a common superscript letter differ significantly (n=5, P<0.05), and the tests were performed twice.

Figure 5
TCF-4 potentiates p65-dependent proinflammatory cytokine production in macrophages

The WT or Tg PMs were treated with a scrambled siRNA (siNC, 20 nmol/ml), a mouse p65-specific siRNA (si-p65, 20 nmol/ml) or a p65 inhibitor (JSH-23, 40 μM) for 12 h, and then treated with BSA (5%) or FFA (200 μM) for 24 h before collection for an RT-PCR assay of (A) IL-1β, (B) TNF-α and (C) IL-6. Values not sharing a common superscript letter differ significantly (n=5, P<0.05), and the tests were performed twice.

Macrophage TCF-4 aggravates HFD-induced chronic inflammation and insulin resistance in mice in a p65-dependent manner

To verify the role of p65 in macrophage TCF-4-regulated chronic inflammation and insulin sensitivity in vivo, the activity of p65 was inhibited by JSH-23 in an HFD-treated mouse model. We demonstrated that the macrophage TCF-4 transgene-augmented levels of plasma IL-1β (Figure 6A), TNF-α (Figure 6B) and IL-6 (Figure 6C) in HFD-treated mice were abolished by treatment with JSH-23. Simultaneously, JSH-23 treatment prevented the macrophage TCF-4 transgene-amplified HOMA-IR index (Figure 6D). We also demonstrated that macrophage TCF-4-aggravated glucose intolerance (Figure 6E) and insulin resistance (Figure 6F) in HFD-treated mice were prevented by additional administration of JSH-23. Finally, we verified that macrophage TCF-4 transgene-suppressed glucose infusion rates in HFD-fed mice were rescued by JSH-23 treatment (Figure 6G). However, under a CD, treatment with JSH-23 had no effects on the production of proinflammatory cytokines in plasma (see Supplementary Figures S6A and S6B). Likewise, JSH-23 treatment did not affect the glucose or insulin tolerance in CD-treated mice (see Supplementary Figures S6C and S6D).

Macrophage TCF-4 aggravates HFD-induced chronic inflammation and insulin resistance in a p65-dependent manner

Figure 6
Macrophage TCF-4 aggravates HFD-induced chronic inflammation and insulin resistance in a p65-dependent manner

(A–C) Macrophage TCF-4 enhances proinflammatory cytokine production in the plasma of the mice on an HFD. The 6-week-old male WT or Tg mice were treated with an HFD for 10 weeks and administered a p65 inhibitor (JSH-23, 3 mg/kg, orally in 0.5% sodium carboxymethylcellulose) for an additional 2 weeks. Then, the blood was collected via the tail vein for the assay of (A) IL-1β, (B) TNF-α and (C) IL-6 levels, using an ELISA (n=8, **P<0.01). (D) The HOMA-IR index of male mice described above in (A–C) (n=8, **P<0.01). (E) The GTT on the WT or Tg mice as described above in (A–C). Values not sharing a common superscript letter differ significantly (n=8, P<0.05). (F) ITT on the WT or Tg mice as described above in (A–C). Values not sharing a common superscript letter differ significantly (n=8, P<0.05). (G) Glucose infusion rates measured by hyperinsulinaemic–euglycaemic clamp studies in male mice described above in (A–C) (n=5, *P<0.05). From (A) to (F), the tests were performed twice. The test in (G) was performed once.

Figure 6
Macrophage TCF-4 aggravates HFD-induced chronic inflammation and insulin resistance in a p65-dependent manner

(A–C) Macrophage TCF-4 enhances proinflammatory cytokine production in the plasma of the mice on an HFD. The 6-week-old male WT or Tg mice were treated with an HFD for 10 weeks and administered a p65 inhibitor (JSH-23, 3 mg/kg, orally in 0.5% sodium carboxymethylcellulose) for an additional 2 weeks. Then, the blood was collected via the tail vein for the assay of (A) IL-1β, (B) TNF-α and (C) IL-6 levels, using an ELISA (n=8, **P<0.01). (D) The HOMA-IR index of male mice described above in (A–C) (n=8, **P<0.01). (E) The GTT on the WT or Tg mice as described above in (A–C). Values not sharing a common superscript letter differ significantly (n=8, P<0.05). (F) ITT on the WT or Tg mice as described above in (A–C). Values not sharing a common superscript letter differ significantly (n=8, P<0.05). (G) Glucose infusion rates measured by hyperinsulinaemic–euglycaemic clamp studies in male mice described above in (A–C) (n=5, *P<0.05). From (A) to (F), the tests were performed twice. The test in (G) was performed once.

In human peripheral blood monocytes, TCF-4 expression positively correlates with the levels of proinflammatory cytokines

The aforementioned results demonstrated an important role of TCF-4 in FFA/LPS-induced proinflammatory cytokine production. To correlate this finding to physiological conditions, we isolated peripheral blood monocytes (PBMCs) from age-matched human donors and analysed the correlation between the mRNA levels of IL-1β, TNF-α or IL-6 and TCF-4. We found that the IL-1β (Figure 7A), TNF-α (Figure 7B) and IL-6 (Figure 7C) mRNA expression levels in PBMCs were positively correlated with the TCF-4 mRNA levels. It is noteworthy that the TCF-4 mRNA levels were also positively correlated with the levels of fasting plasma glucose (Figure 7D). Therefore, we presumed that the expression of TCF-4 in monocytes/macrophages might be induced by obese conditions or obesity-associated factors such as LPS and saturated FFA. We isolated PBMCs and ATMs from the male C57BL/6 mice on a CD or HFD and measured the TCF-4 mRNA levels. As expected, we demonstrated that the expression levels of TCF-4 were notably increased in PBMCs and ATMs from the HFD-fed mice relative to the CD-fed ones (see Supplementary Figure S7A). Likewise, the ob/ob obese mice relative to WT lean littermates displayed significant increases in TCF-4 mRNA levels in the PBMCs and ATMs (see Supplementary Figure S7B). In in vitro tests, we also demonstrated that treatment with FFA or LPS could largely induce TCF-4 expression in mRNA (see Supplementary Figures S7C and S7D) and protein (see Supplementary Figures S7E and S7F) levels in the primary PMs. These findings indicated that obesity-induced increases of TCF-4 in monocytes/macrophages might potentiate systemic inflammation and insulin resistance.

In human PBMCs, TCF-4 expression positively correlates with the levels of IL-1β, TNF-α, IL-6 and fasting plasma glucose

Figure 7
In human PBMCs, TCF-4 expression positively correlates with the levels of IL-1β, TNF-α, IL-6 and fasting plasma glucose

(A) Correlation of TCF-4 levels with IL-1β expression in PBMCs (n=20; Pearson's correlation: r=0.69, P<0.05). (B) Correlation of TCF-4 expression with TNF-α levels in PBMCs (n=20; Pearson's correlation: r=0.68, P<0.05). (C) Correlation of TCF-4 expression with IL-6 levels in PBMCs (n=20; Pearson's correlation: r=0.65, P<0.05). (D) Correlation of TCF-4 expression in PBMCs with fasting (overnight) plasma glucose levels (n=20; Pearson's correlation: r=0.64, P<0.05. The measurements were taken twice for (A–D).

Figure 7
In human PBMCs, TCF-4 expression positively correlates with the levels of IL-1β, TNF-α, IL-6 and fasting plasma glucose

(A) Correlation of TCF-4 levels with IL-1β expression in PBMCs (n=20; Pearson's correlation: r=0.69, P<0.05). (B) Correlation of TCF-4 expression with TNF-α levels in PBMCs (n=20; Pearson's correlation: r=0.68, P<0.05). (C) Correlation of TCF-4 expression with IL-6 levels in PBMCs (n=20; Pearson's correlation: r=0.65, P<0.05). (D) Correlation of TCF-4 expression in PBMCs with fasting (overnight) plasma glucose levels (n=20; Pearson's correlation: r=0.64, P<0.05. The measurements were taken twice for (A–D).

DISCUSSION

Previous studies revealed that TCF-4 variations were intensely associated with the occurrence and progression of type 2 diabetes, although the mechanisms are not fully understood [2124]. In the present study, we were the first to identify TCF-4 as a co-activator of p65 in potentiating the production of proinflammatory cytokines in macrophages and aggravating an HFD-induced chronic inflammation and insulin resistance in mice (Figure 8). Our findings provide promising targets for the management of chronic inflammation and insulin resistance, the pathophysiological basis of obesity-related diseases such as type 2 diabetes [45].

Proposed mechanisms for macrophage TCF-4-regulated chronic inflammation and insulin resistance

Figure 8
Proposed mechanisms for macrophage TCF-4-regulated chronic inflammation and insulin resistance

Chronic over-nutrition/obesity increases circulating levels of saturated FFAs and LPS, both of which stimulate TCF-4 expression in macrophages. TCF-4 co-activates p65 to promote proinflammatory cytokine production in macrophages and to aggravate chronic inflammation and insulin resistance in mice.

Figure 8
Proposed mechanisms for macrophage TCF-4-regulated chronic inflammation and insulin resistance

Chronic over-nutrition/obesity increases circulating levels of saturated FFAs and LPS, both of which stimulate TCF-4 expression in macrophages. TCF-4 co-activates p65 to promote proinflammatory cytokine production in macrophages and to aggravate chronic inflammation and insulin resistance in mice.

In the present study, we demonstrated that TCF-4 functioned as a co-activator of p65 to amplify the expression of proinflammatory cytokines. The direct protein–protein interaction between TCF-4 and p65 was consistent with a previous report [35]. TCF-4 was associated with β-catenin [46] in the nucleus, whereas NF-κB was associated with five different subunits as homodimers or heterodimers [42]. Therefore, we presumed that the TCF-4/p65 complex found in the present study might include other co-factors such as β-catenin, p50, c-Rel, p105 and p100. However, the detailed mechanisms of protein–protein interaction need to be investigated in future studies. Even so, we still managed to uncover a novel role for TCF-4 in regulating inflammation in addition to mediating Wnt signal.

Proinflammatory cytokines impaired insulin signalling in different cascades [47,48]. We demonstrated that the TCF-4/p65 complex amplified the production of multiple proinflammatory cytokines simultaneously. Therefore, it is hard to distinguish which cytokine downstream of TCF-4 contributed the most to the impairment of insulin signalling. Blockage of each cytokine needs to be carried out to evaluate its unique role in insulin resistance. Those findings would uncover more efficient targets for the prevention and therapy of insulin resistance-associated diseases.

Wnt signalling-mediated TCF-4 activation was closely associated with multiple tumours [16,49]. It is of interest in the present study that we revealed macrophage TCF-4 being causally linked to the development of insulin resistance in obesity. According to an epidemiological investigation, obesity positively correlated with type 2 diabetes [50] and multiple cancers (e.g. colorectal, breast and prostatic cancers) [5153]. We presumed that the obesity-related chronic inflammation and insulin resistance might be the common pathophysiological basis of these diseases. In particular, for patients with diabetes and cancer, blockage of the Wnt and/or NF-κB signals might be a promising strategy. However, this presumption must be validated in future studies.

Taken together, we identified TCF-4 as a co-activator of p65 in the regulation of proinflammatory cytokine production in macrophages and the aggravation of diet-induced chronic inflammation, as well as insulin resistance, in mice. Our findings could represent potential targets for chronic inflammation- and insulin resistance-associated diseases.

AUTHOR CONTRIBUTION

Hongming Miao and Wei Zheng designed the experiments. Xia Kang, Along Hou, Rui Wang and Hongming Miao performed the experiments and analysed the data. Da Liu, Wei Xiang, Qingyun Xie, Bo Zhang, Lixia Gan and Wei Zheng discussed the data. Hongming Miao wrote the manuscript, had full access to all the data and took full responsibility for their integrity. Xia Kang, Along Hou and Rui Wang contributed equally to this work.

The Department of Orthopaedics at Chengdu Military General Hospital and the Department of Biochemistry and Molecular Biology at Third Military Medical University contributed equally to this work.

FUNDING

The present research was supported in part by the National Natural Science Foundation of China [81302136] to H. Miao and by the Foundation of Science and Technology Department of Sichuan Province [2014JY0009] to W. Zheng.

Abbreviations

     
  • ATM

    adipose tissue macrophage

  •  
  • BMDM

    bone marrow-derived macrophage

  •  
  • CD

    chow diet

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • GTT

    glucose tolerance test

  •  
  • HFD

    high-fat diet

  •  
  • IKK

    I-κB kinase

  •  
  • ITT

    insulin tolerance test

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • NF-κB

    nuclear factor κ light chain enhancer of activated B-cells

  •  
  • PBMC

    peripheral blood monocyte

  •  
  • PM

    peritoneal macrophage

  •  
  • RIPA

    radioimmunoprecipitation assay

  •  
  • SVF

    stromal–vascular fraction

  •  
  • TCF-4

    transcription factor 4

  •  
  • TCF7L2

    transcription factor 7-like 2

  •  
  • WT

    wild type

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Supplementary data