PFKFB3, a key glucose metabolic enzyme regulated by pathogen recognition receptor TLR4 in liver cells.

AIMS: Toll-like receptor 4 (TLR4) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3) are involved in the progress of inflammation and glucose metabolism. Here, we aimed to assess the relationship between TLR4 and PFKFB3 in liver cells.
METHODS: We detected the expression of TLR4 and PFKFB3 in both normal liver cell lines and liver cancer cell lines. Then, a small interfering RNA (siRNA) was used to knock down the expression of TLR4 and analyze the expression of PFKFB3 in the HL-7702 cell line. Further, following stimulation of the HL-7702 cell line with free fatty acids (FFA) or insulin, we observed the expression of TLR4 and PFKFB3, respectively.
RESULTS: Knocking down siRNA-mediated TLR4 significantly reduced PFKFB3 expression at the mRNA and protein level. Furthermore, activating TLR4 with FFA dramatically increased PFKFB3 expression. Insulin increased the expression of TLR4 and PFKFB3, which could be inhibited by TLR siRNA.
CONCLUSION: These findings suggest that PFKFB3 expression is regulated via the TLR4-PFKFB3 axis, which might be a bridge linking fat and glucose metabolism.

Introduction

The Warburg effect takes place in most malignant tumors. With the Warburg effect, cancer cells turn to anaerobic glycolysis, instead of aerobic oxidation, to produce energy for rapid proliferation and macromolecular synthesis. For example, lactate, the side product of anaerobic glycolysis, is secreted by MCT1 (monocarboxylate transporters 1) or MCT4 (monocarboxylate transporters 4) into the cancer microenvironment to maintain acidity, allowing immune escape.[1] Lactate can also be taken in by some cancer cells as energy sources.

PFKFB3 encodes 6-phosphofructo-2-kinase/fructose-2, and 6-biphosphatase 3 enzyme (PFK-2/FBPase-2) in humans. PFK-2/FBPase-2 is a bi-functional enzyme that controls glycolytic flux via fructose 2,6-bisphosphate (F-2,6-P). F-2,6-P is a potent allosteric activator of 6-phosphofructokinase-1 (PFK-1) that can trigger aerobic oxidation for glucose metabolism. Recent studies have reported the pivotal role of PFKFB3 in the regulation of high-fat diet (HFD)-induced inflammation, overnutrition-associated inflammatory response in adipose tissues and intestines, and insulin resistance.[2]

Metabolic syndrome was first termed by Haller in 1977 to describe the associations among central obesity, blood pressure, fasting glucose level, triglyceride level, and high-density lipoprotein cholesterol level.[3] The mechanisms involving obesity, diabetes mellitus, and other diseases have not yet been unveiled.[4] Metabolic syndrome is always accompanied with chronic low-grade inflammation, which has been widely accepted and proved by research in vivo and in vitro.[5,6] Pro-inflammatory cytokines or inflammatory biomarkers, such as interleukin-6 (IL-6) and C-reactive protein, are elevated in patients with metabolic syndrome,[7,8] whereas anti-inflammation cytokines decrease. Thus, it is suggested that chronic inflammation is essential for all patterns of metabolic syndrome, including type 2 diabetes mellitus and obesity.[9,10]

Toll-like receptor 4 (TLR4), a pathogen recognition receptor of the TLR family, can recognize and bind to its exogenous ligands (bacteria cell wall, lipopolysaccharide, virus DNA/RNA) and endogenous ligands (fatty acids) to initiate innate immune responses.[11,12] In individuals with central obesity, high-level fatty acids always boost chronic inflammation via activating TLR4 and its downstream signaling pathways to promote cytokine secretion and regulate cells function.[13,14]

The relationship between TLR4 and PFKFB3 was first discovered though research on leukocytes. Stimulating TLR4 can upregulate expression of PFKFB3.[15] In 2011, Díaz-Guerra and colleagues reported that the exogenous agonist of TLR4, lipopolysaccharides (LPS), also increase PFKFB3 expression and promote ATP generation.[16] As we known, free fatty acids (FFA) are an endogenous ligand of TLR4 and can promote chronic inflammation response as LPS. Therefore, this discovery suggests that lipo-metabolism and pathogen recognition receptor pathways also interact with glucose metabolism.

But all these studies focused only on the role of TLR4 in leukocytes. The liver is the most important organ accomplishing glucose and fat metabolism.[17,18] We conducted this study to investigate the correlation between TLR4 and PFKFB3, with or without FFA and insulin stimulation, in liver cells.

Materials and methods

Cell line and culture

Human liver cancer cell lines HepG2 and QSG-7701, and the normal human cell line HL-7702 (Shanghai Cell Bank, Chinese Academy of Sciences, Shanghai, China) were preserved in our laboratory. All cells were cultured in RPMI 1640 medium. The medium (Cyclone GE Healthcare Life Sciences, South Logan, UT, USA) was supplemented with 10% fetal bovine serum (FBS; Clark Bioscience, Houston, TX, USA). The cells were incubated at 37°C in a humidified atmosphere with 5% CO2. FFA (0.5 mmol/l) was added to the culture medium of HL-7702 and maintained for 72 h with or without small interfering RNA (siRNA). Then, TLR4 and PFKFB3 expression were detected using western blotting. The main component of FFA is palmitic acid (Sigma, St. Louis, MO, USA).

Western blotting

Protein samples were treated using whole-cell extracts prepared by lysing 1 × 106 cells in radio immunoprecipitation assay (RIPA) lysis buffer, which contained phosphatase inhibitor, protease inhibitor, and 1 mmol/l phenylmethylsulfonyl fluoride (PMSF; KeyGEN Bio TECH, Nanjing, China). Samples containing equal amounts of protein were boiled in denaturing buffer and then separated by 10% SDS-PAGE. After that, the samples were transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated with the indicated antibodies at a concentration of 1:1000 at 4°C overnight, followed by incubation with secondary antibody for 1 h at room temperature. The immune-reactive bands were visualized using Beyo ECL Plus (Beyotime, Bejing, China). Image J software was severed to analyze the results of Western Blot. TLR4 antibodies (sc-52962, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at a concentration of 100 µg/ml.

RNA isolation and purification, and first-strand cDNA synthesis

Total RNA was isolated from 1.5 × 106 cells using TRIzol and quantified by a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Total RNA was treated with RNase-free DNase to remove residual genomic DNA. First strand cDNA was synthesized with 1 μg RNA using an oligo-dT primer and avian myeloblastosis virus (AMV) reverse transcriptase.

Relative real-time polymerase chain reaction

The relative expression of TLR4 and PFKFB3 were analyzed using a ViiATM 7 Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA). First-strand cDNA was amplified in a reaction mixture of 20 μl PCR: 10 μl 2xSYBR green PCR master mix, 0.4 μl 50 × ROX, 0.4 μl of each specific primer sets, and ddH2O were added to 20 μl PCR, 40 amplification cycles with annealing at 55°C. Primer sequences for human TLR4 and PFKFB3 were as follows: TLR4, Forward: 5′-GATTAGCATACTTAGACTACTACCTCCATG-3′, and reverse, 5′-GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA-3′. PFKFB3, Forward: 5′-ATTGCGGTTTTCGATGCCAC-3′, and reverse, 5′-GCCACAACTGTAGGGTCGT-3′. β-actin, forward: 5′-AGCGAGCATCCCCCAAAGTT-3′, and reverse, 5′-GGGCACGAAGGCTCATCATT-3′). TLR4 and PFKFB3 expression were calculated using the 2 –ΔΔCt method and normalized to β-actin expression.

RNA interference assay

The cells were transfected with various siRNAs directed against TLR4 and negative control (NC) siRNA with a random sequence was used as a control. The siRNA sequences were as follows: TLR4-siRNA#1 sense: GGGCUUAGAACAACUAGAATT, anti-sense: UUCUAGUUGUUCUAAGCCCTT; TLR4-siRNA#2: sense: CCCACAUUGAAACUCAAAUTT, anti-sense: AUUUGAGUUUCAAUGUGGGTT. For transfection, HL-7702 cells were planted into six-well plates. When cells were 80–90% confluent, the TLR4-siRNA or negative siRNA were transfected into cells using Lipofectamine 2000 (Invitrogen life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s protocol.

Statistical analysis

All experiments were repeated three times to assure repeatability and reliability. Data were presented as mean ± standard deviation (SD). SAS 8.0 software was used for statistical analysis. Before comparison, we performed normality test and variance homogeneity analysis. For multiple testing among groups, one-way ANOVA was performed and Bonferroni correction was used. p < 0.05 was considered statistically significant.

Results

TLR4 is expressed in several liver cell lines

HepG2, QSG-7701, and HL-7702 cell lines were studied to evaluate the expression of TLR4 in liver cells. HepG2 and QSF-7701 were derived from cancer liver cell lines, and HL-7702 from normal liver cell lines. Both TLR4 and PFKFB3 were analyzed by western blotting. Whole protein extraction was prepared according to the classic western blotting protocol. As illustrated in Figure 1, both TLR4 and PFKFB3 were expressed in HepG2, QSG-7701, and HL-7702 cell lines. We found that the relative expression of TLR4 and PFKFB3 in HL-7702 was significantly higher than in HepG2 and QSG-7701. Therefore, we selected cell line HL-7702 for subsequent experiments.

Figure 1.

The expression properties of TLR4 and PFKFB3 in liver cell lines. Western blot analysis of TLR4 and PFKFB3 protein were performed in liver cancer cell line HepG2 and normal liver cell lines QSG-7701 and HL-7702. Both TLR4 and PFKFB3 protein are expressed in all three cell lines.

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PFKFB3, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3; TLR4, toll-like receptor 4 .

Decreasing TLR4 expression reduced PFKFB3 expression

TLR4 has been recognized as an innate immunity receptor. Exogenous LPS and endogenous FFA are ligands binding to TLR4 to activate nuclear factor kappa B (NF-κB) and other signal pathways. In this experiment, we knocked down TLR4 to investigate its influence on PFKFB3, a key enzyme regulating glucometabolic expression. siRNA1 and siRNA2 targeting TLR4 were transfected into cells as experiment groups, respectively; nonsense DNA sequences were employed as a negative control. qPCR was performed to detect the corresponding interferential efficiency.

As shown in Figure 2, compared with blank or NC groups, both siRNA1 and siRNA2 decreased TLR4 expression by over 40% at the mRNA level and 60% at the protein level (p < 0.05 and p < 0.01). As TLR4 expression level decreased, the expression of PFKFB3 was sequentially down-regulated, and its expression at mRNA level was significantly lower than that of the blank control or isotope control. The expression of TLR4 and PFKFB3 at the protein level had a similar alteration in the siRNA1 group and siRNA2 group. These results implied that expression of PFKFB3 was influenced by TLR4, or that TLR4 might regulate PFKFB3 by some signaling pathway. The results also suggested that the TLR4–PFKFB3 axis could be a linkage between innate immunity and glycose metabolism or lipo-metabolism, specifically in liver cells.

Figure 2.

siRNA mediated knockdown of TLR4 significantly reduced mRNA and protein level of PFKFB3.

(A) Western blot analysis showed that whereas TLR4 protein level was decreased, the PFKFB3 protein level was also down-regulated in the liver cell line HL-7702. (B) Western blot analysis was translated into relative expression level for TLR4 and PFKFB3 as in (A). (C) Quantitative PCR was performed to investigate the TLR4 mRNA level in siRNA-transfected cell lines. (D) PFKFB3 mRNA level was also down-regulated whereas TLR4 was decreased using siRNA technology. *p < 0.05, **p < 0.01.

NC, negative control; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; siRNA, small interfering RNA; TLR4, toll-like receptor 4 .

FFA induced TLR4 and PFKFB3 expression

LPS and FFA are exogenous and endogenous ligands of TLR4, respectively. Higher FFA concentration can stimulate innate immune cells. As described above, FFA might affect glycometabolism via TLR4. In order to verify that TLR4 and PFKFB3 are pivotal molecules involved in both glycose metabolism and lipo-metabolism, we added FFA (0.5 mmol/l) to the culture medium of HL-7702. The mixture was maintained for 72 h with or without siRNA. Then, we detected TLR4 and PFKFB3 expression using western blotting.

As illustrated in Figure 3, FFA could mildly up-regulate the expression of TLR4. After ligand bound to the receptor, internalization was triggered immediately, moving receptor from cell membrane to cell cytoplasm. FFA increased TLR4 expression, implying that some regenerative feedback may be involved in this process.

Figure 3.

FFAs induce TLR4 and PFKFB3 expression. (A) TLR4 and PFKFB3 protein expression were detected using Western blot. When FFA was added into the culture medium of the liver cell line HL-7702, both TLR4 and PFKFB3 protein expression were elevated. For the stable targeted-TLR4 siRNA (including siRNA1 and siRNA2) transfected cell line of HL-7702, when FFA was added, both TLR4 and PFKFB3 protein expression were elevated. However, the level did not reach to the level in the original cell line. (B) TLR4 and PFKFB3 protein expression in various conditions were compared. The relative expression of the two proteins was quantified by scanning the western blot figures (*p < 0.05).

FFA, free fatty acids; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; siRNA, small interfering RNA; TLR4, toll-like receptor 4 .

Under stimulation by FFA, PFKFB3 expression increased significantly. SiRNA targeting TLR4, either siRNA1 or siRNA2, can effectively block this effect, proving that knocking down TLR4 could inhibit PFKFB3 expression. In other words, FFA might regulate PFKFB3 expression, and then influence glucose metabolism.

Influence of insulin onTLR4 and PFKFB3 expression was blocked by TLR4 siRNA

Hyperinsulinemia is a hallmark of metabolic syndrome, usually accompanied with hyperlipidemia or hyperglycemia. So, investigating the regulation of TLR4 in PFKFB3 at different insulin levels can help understand the metabolic mechanisms involved.

In complete HL-7702 medium, we supplemented insulin of 0 to 8 μmol/l. After 72 h, TLR4 and PFKFB3 expression profiles were analyzed using western blotting. Column diagrams were plotted based on relative expression amount.

Figure 4A shows that TLR4 expression increased gradually as the insulin concentration rose from 0 to 8 μmol/l. This result proved that insulin regulates not only blood glucose, but also pathogen recognition receptor TLR4. As we know, TLR4 is a key innate immune receptor, so this result indicates that hyperinsulinemia is one cause for chronic inflammation response.

Figure 4.

Insulin can enhance TLR4 and PFKFB3 expression. Insulin was added into the culture medium of the cells. (A) TLR4 expression was enhanced as the concentration of insulin increased. PFKFB3 expression is also evidently promoted as the insulin added increased. (B) TLR4 and PFKFB3 expression were significantly promoted as insulin increased. (C) In the stable TLR4-targeted siRNA-transfected cell line HL-7702, only a very tiny promotion of TLR4 expression was observed as insulin increased. However, PFKFB3 expression was enhanced, as the concentration of insulin added was over 2 µmol/l. (D) Expression of TLR4 and PFKFB3 presented as histograms acquired by scanning the western blot results in (C).

NC, negative control; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; siRNA, small interfering RNA; TLR4, toll-like receptor 4 .

We used siRNA1 to diminish TLR4 expression, and then added insulin to the culture medium for 72 h. Figure 4B shows that insulin can also induce TLR4 expression, although its mRNA has been partly resolved by transcriptional regulation. Simultaneously, PFKFB3 can also be induced by insulin. PFKFB3 expression gradually elevated as insulin concentration and TLR4 expression increased. TLR4 siRNA1 and siRNA2 could inhibit the effect of insulin

Discussion

In this study, we proved that FFA could upregulate TLR4 expression and promote PFKFB3 expression, a process that could be blocked by TLR4 interference. Insulin increased TLR4 and PFKFB3 expression in a concentration-dependent manner. After TLR4 was knocked down, PFKFB3 expression was significantly lower than that of the control.

TLR4, a member of the TLR family, is a transmembrane protein composed of extracellular domain, transmembrane domain, and intracellular domain. Exogenous ligand LPS and endogenous ligand FFA bind to and activate TLR4’s extracellular domain.[11,12] NF-κB is an important signal of TLR4 to transfer activation signal to cytoplasm and nucleus.[19,20] In macrophages, LPS induce PFKFB3 expression through increasing the expression of adenosine.[21] Since LPS and FFA act as TLR4 ligands, we speculated that FFA can upregulate PFKFB3 expression through the TLR4pathway. This study proved that FFA can increase expression of both TLR4 and PFKFB3, and this effect can be blocked significantly by two TLR4-targeted siRNA with different sequences.

Hyperinsulinemia and insulin resistance are commonly found in diabetes mellitus and metabolic syndrome, and even cancer.[22,23] Epidemiologic and experimental evidence has proven that hyperinsulinemia promotes colon carcinogenesis,[24,25] and colonic epithelial cell growth in vitro. Insulin level has also shown close correlation with other cancers.[26] The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP), such as the PI3K/Akt signaling pathway, plays an important role in metabolic diseases.[27] PFKFB3 may participate in ISP, generating a positive feedback loop.[28] With the Warburg effect, cancer cells take in more glucose than normal to elevate aerobic glycolysis. PFKFB3, a key enzyme regulating glucose metabolism, promotes aerobic glycolysis through cooperation with F2,6BP, an allosteric activator of glycolytic enzyme phosphofructokinase-1 (PFK1).[29] Inhibitors targeting PFKFB3 have been found to suppress aerobic glycolysis, decrease glucose uptake, and induce cancer cell autophagy.[30,31]

Our study proved that high-level insulin could induce expression of PFKFB3. After TLR4 was blocked by siRNA, this induction was curbed, indicating that TLR4 participates in this process. In patients with obesity or hyperinsulinemia, does high-level FFA exert a synergistic effect on chronic inflammation process? In order to answer this question, we tested how TLR4 regulates glucose metabolism in normal liver cells under the effect of insulin or FFA. We found that TLR4 induced abnormal expression of PFKFB3, a process in energy metabolism.[32] Some studies have confirmed the close correlation between inflammation and cancer.[33] Some inflammatory mediators can improve glucose metabolism. For example, in human hepatoma HepG2 cells, the IL-6/STAT3 pathway promotes glycolysis through inducing the expression of PFKFB3.[34] Furthermore, TLR4 has been found to promote the proliferation of hepatocellular carcinoma (HCC) cells.[35] Most importantly, the COX-2/PGE2/STAT3 positive feedback loop in HCC cells can be provoked by TLR4 activation.[36] This evidence indicates that TLR4 works together with PFKFB3 through STAT3. A study on drug resistance of breast cancer cells has found that PFKFB3 overexpression boosts lactate production. High-level lactate can activate TLR4 signaling to promote cell viability.[37] In short, TLR4 is closely related to the survival of cancer cells. So we hypothesized that TLR4 may be a new anti-tumor target, which will require further experiments to verify.

Metabolic syndrome is often accompanied by insulin resistance, hyperinsulinemia, and dyslipidemia.[32,38] The liver is a target organ of insulin, and also a main organ responsible for gluconeogenesis.[39] To maintain glucose metabolism and lipid metabolism, the liver is indispensable.[17] Insulin resistance in the liver could result in glycol-lipid metabolic disorders, high blood glucose level, and liver steatosis, all contributing to diabetes and other metabolic diseases.[39,40] TLR4 is a critical mediator for obesity-related low-grade inflammation and insulin resistance. Mice deficient in hepatocyte TLR4 exhibit elevated insulin sensitivity, and hepatic steatosis that can be ameliorated with HFD.[6] A previous study found that insulin-stimulated lactate production was correlated with Fru-2,6-P2 level and PFK-2 activity. Insulin regulates transcription of the PFKFB3 gene in the HT29 colon adenocarcinoma cell line.[41] In the present study, we demonstrated that insulin could enhance intracellular TLR4 protein expression in liver cells HL-7702, a process which, in turn, raises PFKFB3 expression. These effects are blocked by siRNA targeting TLR4. It is noteworthy that FBS components may affect the oxidative modifications of FFA. Therefore, using serum starvation may yield more reliable experimental results. Furthermore, the mechanism whereby insulin upregulates TLR4 and PFKFB3 deserves further study.

The function of TLR4 in glucose metabolism has been investigated only rarely. Duran et al. reported that overexpression of PFKFB3 increased the expression of key gluconeogenic and lipogenic enzyme genes.[42] Our study is the first to confirm that TLR4 could regulate PFKFB3 expression in liver cells. Based on our data, TLR4 might be the bridge that links hyperlipidemia with hyperglycemia. As an exogenous ligand of TLR4, FFA binds to TLR4 and is expressed in the liver cell membrane. This expression then up-regulates PFKFB3, a key enzyme in glucose metabolism.