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Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Attenuates the Action of Insulin in Hepatocytes

Signal Transduction Laboratory (F.F., N.A., A.H., P.X.), Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, and Department of Medicine, University of Adelaide, Adelaide, South Australia 5005, Australia; and Signal Transduction Laboratory (L.W., P.X.), Centenary Institute, Faculty of Medicine, The University of Sydney, Sydney, New South Wales 2042, Australia

Address all correspondence and requests for reprints to: Pu Xia, M.D., Signal Transduction Laboratory, Centenary Institute, Locked Bag 6, Newtown, New South Wales 2042, Australia. E-mail: p.xia{at}centenary.org.au.

	Abstract

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TNF-like weak inducer of apoptosis (TWEAK), a relatively new member of the TNF superfamily, is an important immune/inflammatory regulator that has different functional properties from that of other members of this superfamily. We report herein that TWEAK induces cellular insulin resistance in both human hepatocellular carcinoma cell lines (Huh7 and HepG2) and primary rat hepatocytes by inhibiting both early insulin receptor (IR) signaling events and the downstream actions of insulin. TWEAK profoundly inhibited insulin-induced Akt phosphorylation in both a concentration- and time-dependent manner. This inhibitory effect occurred via mechanisms that involved the TWEAK receptor Fn14 and the activation of the canonical and noncanonical nuclear factor-B signaling pathways. Furthermore, TWEAK significantly inhibited IRβ autophosphorylation and IR substrate-1 activation, with concomitant increases in serine phosphorylation of IR substrate-1. Moreover, insulin-induced reduction of gluconeogenic enzyme gene expression and increases in glycogen synthesis in hepatocytes were significantly attenuated by TWEAK treatment. Therefore, these findings not only reveal a novel pathophysiological function of TWEAK/Fn14 but also uncover a new player that may contribute to the development of cellular insulin resistance in hepatocytes.

	Introduction

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INSULIN RESISTANCE IS a fundamental pathogenic event in the development of type 2 diabetes (T2D). Although the precise mechanisms underlying insulin resistance remain to be elucidated, substantial evidence indicates a causative link for chronic, low-grade inflammation (reviewed in Refs. 1 and 2). Numerous epidemiological and clinical-based studies have consistently shown a strong correlation between insulin resistance and elevated production of various proinflammatory cytokines, such as TNF, IL-1, and IL-6 (3, 4, 5). For instance, TNF, being the first molecular link between inflammation and insulin resistance, was found to be significantly elevated in the circulation in animal models of insulin resistance and in obese humans (6, 7). Administration of recombinant TNF to cultured cells or to whole animals impaired insulin action (3), whereas neutralization of TNF activity in rodent models that lack functional TNF or its receptors improved insulin sensitivity and overcame insulin resistance induced by a high-fat diet (8). Furthermore, it has become increasingly clear that various inflammatory networks and metabolic pathways often overlap with interdependent cross-talk occurring at cellular and systemic levels, supporting the notion of a direct role for inflammation in the development of insulin resistance.

TNF-like weak inducer of apoptosis (TWEAK, also known as Apo3L or TNFSF12) is a relatively new member of the TNF superfamily of cytokines that function both as type II transmembrane proteins and as cleaved soluble molecules (9). Its mRNA is broadly expressed in a variety of tissues and most major organs, including the heart, brain, liver, skeletal muscle, and adipose and hematopoietic tissues (10). TWEAK specifically binds with high affinity to a receptor known as Fn14 (fibroblast growth factor-inducible 14-kDa protein) and now named the TWEAK receptor (11). The receptor Fn14, as a distant relative of the TNF receptor superfamily, is not activated by other known homotrimers of the TNF superfamily, whereas TWEAK does not bind to any other known TNF receptor superfamily members (10). Therefore, it is not surprising that TWEAK possesses various biological functions that differ from the action of TNF. For instance, TWEAK has been shown to suppress the production of interferon- and IL-12, attenuating the innate response and its transition to adaptive immunity (12). In contrast, TNF promotes the innate inflammatory response by stimulating the cytokine production. Although TNF is a strong inducer of NF-B activation that occurs in a rapid / transient manner through the canonical pathway, TWEAK promotes a prolonged nuclear factor (NF)-B activation via both canonical and noncanonical mechanisms (9, 13). In addition to its role in the immune and inflammatory responses, TWEAK appears to play an important role in regulation of cell survival, proliferation, and migration (reviewed in Ref. 10). The TWEAK/Fn14 pathway is also involved in the process of liver regeneration (14, 15). In the present study, we demonstrate that TWEAK is capable of inhibiting both insulin receptor signaling events and the downstream actions of insulin in human hepatocellular carcinoma cell lines (Huh7 and HepG2) and primary rat hepatocytes. These findings thus suggest a novel function for TWEAK/Fn14 and also uncover a new mediator that may contribute to the development of cellular insulin resistance in hepatocytes.

	Materials and Methods

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Reagents
Recombinant human TWEAK was purchased from R&D Systems (Minneapolis, MN) and insulin was from Sigma-Aldrich (St. Louis, MO). Antibodies to Akt, phosphorylated Akt (Ser 473), insulin receptor (IR) substrate-1 (IRS-1), and phosphorylated IRS-1 (Ser 312 and Ser 616) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against IRβ, p85, and phosphotyrosine (4G10) were provided by Upstate Biotechnology (Lake Placid, NY) and anti-Fn14 antibody (ITEM-2) was from eBioscience (San Diego, CA). Antibodies against inhibitor of B (IB) and p52 were from Santa Cruz Biotechnology (Santa Cruz, CA). MG132 and Bay 11-7082 were purchased from Biomol (Plymouth Meeting, PA).

Cell culture
HepG2 and Huh7 human hepatocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA) and grown in DMEM supplemented with 10% fetal calf serum (HyClone, Logan, UT) at 37 C in an atmosphere containing 5% CO₂. Primary hepatocytes were isolated from Wistar rats by in situ perfusion of collagenase as previously described (16). Purity of the isolated hepatocytes was determined by FACS analysis, showing 96–99% CK18-positive and 0.3–0.8% ED-2-positive cells. Hepatocytes were resuspended in DMEM and plated in six-well plates. Culture medium was replaced after 4 h with DMEM supplemented with 10% fetal calf serum. After an additional 6-h culture, cells were serum starved overnight followed by the indicated treatment. Cell viability was assessed by dimethylthiazol-tetrazolium (Promega, Madison, WI) dye conversion at 490 nm following the manufacturer’s instructions.

Immunoblot and immunoprecipitation assays
Immunoblot assays were performed routinely as described previously (17). For immunoprecipitation experiments, cell lysates containing equal amounts of proteins were immunoprecipitated with anti-IRβ or anti-IRS-1 antibodies for 2 h at 4 C. The immune complexes were precipitated by incubation with protein A-Sepharose CL-4B beads (Bio-Rad, Hercules, CA) for another 2 h. After a series of washes, the immunoprecipitates were separated by SDS-PAGE. Subsequent immunoblot assays were performed using antibodies to IRβ, IRS-1, p85, and 4G10, respectively. Blots were detected by using enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Piscataway, NJ) and quantified using ImageQuant Software.

RT-PCR and quantitative real-time PCR
Total RNA was isolated from hepatocytes after the indicated treatment using TriZol (Invitrogen, Carlsbad, CA). First-strand cDNA was synthesized from 1 µg total RNA using Omniscript RT Kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were amplified on a PTC-100 programmable thermal controller (MJ Research) with an internal control, cyclophilin. The primers used to amplify included the following: PEPCK, 5'-AGG CGG CTG AAG AAG TAT GA-3' (forward) and 5'-ACG TAG GGT GAA TCC GTC AG-3' (reverse); G6Pase, 5'-TAC GTC CTC TTC CCC ATC TG-3' (forward) and 5'-TCC CTG GTC CAG TCT CAC AA-3' (reverse); Fn14, 5'-CCA AGC TCC TCC AAC CAC AA-3' (forward) and 5'-TGG GGC CTA GTG TCA AGT CT-3' (reverse); and cyclophilin, 5'-GGC AAA TGC TGG ACC CAA CAC AA-3' (forward) and 5'-CTA GGC ATG GGA GGG AAC AAG GA-3' (reverse). The amplified products were visualized by electrophoresis on 1.5% agarose gels stained with ethidium bromide and photographed by a gel documentation system (Uvitec). For transcript quantification purposes, the real-time PCR assay was performed using Rotor Gene-3000 (Corbett Research). One microliter of the reverse transcriptase product was used in a 25-µl volume reaction containing 200 µM of each dNTP, 5 pmol each primer, 1x PCR buffer II, 1.5 mM MgCl₂, 0.2x SYBR Green I (Molecular Probes, Eugene, OR), and 1.25 U AmpliTaq Gold (Applied Biosystems, Foster City, CA). Variability in the initial quantities of cDNA was normalized to the internal control, cyclophilin. A negative control was included in each set of experiments. Melting-curve analysis was performed to enhance specificity of amplification reaction, and the Rotorgene software was used to compare amplification in the experimental samples during the log-linear phase to the standard curve.

RNA interference
Huh7 cells were transfected with small interfering RNA (siRNA) specifically targeted against the p65 subunit of NF-B or a scrambled control siRNA (purchased from Santa Cruz Biotechnology). siRNA were transfected into the cells by using TranPass R2 reagents (BioLabs) according to the manufacturer’s protocol. For assessing the efficacy of siRNA, levels of p65 expression were determined by RT-PCR and/or Western blot analysis after 48 h siRNA transfection.

Luciferase reporter assays
NF-B transcriptional activity was assessed by a reporter gene assay as described previously (18). We used an NF-B reporter construct (pLuc-IgK) that comprised an NF-B response element upstream of the firefly luciferase and a Renilla luciferase plasmid (pRL) constructed with the cytomegalovirus promoter serving as an internal control. All transfections were performed using TranPass R2 reagents as described above. Cell extracts were prepared 24 h after transfection and the indicated treatment, and reporter gene activity was determined by a dual-luciferase assay system (Promega) and normalized relative to Renilla luciferase activity.

Analysis of glycogen synthesis
Primary rat hepatocytes were seeded onto collagen-coated six-well tissue culture plates at 0.5 x 10⁶ cells per well. After 24 h incubation, cells were pretreated for 2 h with TWEAK (100 ng/ml) in serum-free low-glucose DMEM. Cells were then labeled with 1.5 µCi [¹⁴C]D-glucose (300 mCi/mmol·liter) and treated with 10 nmol/liter insulin. After 4 h, the reaction was terminated by washing cells three times with ice-cold PBS. Cells were solubilized with 0.3 ml 10 N KOH, and glycogen was isolated and analyzed as described previously (19). Briefly, cold glycogen carrier (4 mg) was added to the lysates, and samples were boiled for 30 min. Glycogen was precipitated with 2 vol ethanol overnight on ice. Precipitated glycogen was collected by centrifugation, washed with 60% ethanol, and resuspended in 0.5 ml water. The ¹⁴C-labeled glycogen was then quantified by scintillation counting and normalized to total protein levels.

Statistical analysis
Unpaired Student’s t tests were used for comparison between two groups. For multiple comparisons, results were analyzed by ANOVA followed by the Dunnet’s test. A value of P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TWEAK inhibits insulin-induced Akt phosphorylation in hepatocytes
Because Akt activation has been documented as being one of the crucial signaling events underlying the action of insulin (20), we first examined the effect of TWEAK on insulin-induced Akt phosphorylation at Ser 473 in two human hepatoma cell lines, Huh7 and HepG2. Cells were serum starved overnight and then exposed to TWEAK (0–150 ng/ml) for 12 h followed by a 10-min treatment with insulin (10 nmol/liter). As expected, administration of insulin induced a significant increase in Akt phosphorylation in both Huh7 and HepG2 cells (Fig. 1A). In the presence of TWEAK, insulin-induced Akt phosphorylation was profoundly attenuated in a concentration-dependent manner (Fig. 1A). Although a small suppression was seen at a concentration of 25 ng/ml, significant inhibition was observed at concentrations of more than 50 ng/ml TWEAK (P < 0.05). At the highest concentration (150 ng/ml) used, TWEAK almost completely abolished insulin-induced Akt phosphorylation in both Huh7 and HepG2 cells. Although Huh7 and HepG2 cell lines are considered as a useful model to investigate various aspects of insulin action, primary hepatocytes remain a more physiologically relevant in vitro model. As shown in Fig. 1B, treatment of primary rat hepatocytes with TWEAK also resulted in a dose-dependent inhibition of insulin-induced Akt phosphorylation, an effect that was similar to that observed in Huh7 and HepG2 cell lines. There were no detectable changes in cell viability after TWEAK treatment in either primary hepatocytes or Huh7 and HepG2 cells, compared with untreated cells (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Effect of TWEAK on insulin-induced Akt phosphorylation. A and B, Huh7 and HepG2 cells (A) and primary rat hepatocytes (B) were pretreated for 16 h with increasing concentrations of TWEAK; C, Huh7 cells were pretreated with 100 ng/ml TWEAK for 0–24 h. Cells were then stimulated with 10 nmol/liter insulin for 10 min. Cell lysates were separated by SDS-PAGE and subjected to Western blot analysis with anti-phospho-Akt (Ser 473) or anti-Akt antibodies. The histograms represent band intensities that were normalized to total Akt levels and expressed as the mean ± SD; n 3. *, P < 0.05; , P < 0.01, TWEAK-treated vs. insulin alone. pAKT, Phosphorylated Akt; Twk, TWEAK.

TWEAK inhibits insulin-induced dose-dependent Akt phosphorylation
Because the signaling and hence the physiological effects of insulin are largely governed by its concentration, a phenomenon that is especially relevant in insulin-resistant states, we then examined the effects of TWEAK on AKT phosphorylation across variable insulin concentrations. Figure 2 shows that insulin increased Akt phosphorylation at 1 nmol/liter in Huh7 cells and reached a maximum 6-fold increase at 10–100 nmol/liter. Pretreatment of cells with TWEAK resulted in a significant inhibition of Akt phosphorylation to levels similar for all insulin concentrations examined (Fig. 2). This indicates a potent inhibitory effect of TWEAK on insulin signaling at both physiological and supraphysiological concentrations. Serving as a control, TWEAK alone had no effect on Akt phosphorylation in the absence of insulin. In addition, a mild level of basal Akt phosphorylation was detectable, which was not affected by TWEAK (Fig. 2), suggesting a specific inhibitory effect on insulin signaling in Huh7 cells.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Effect of TWEAK on dose-dependent insulin signaling. Huh7 cells were pretreated for 12 h with or without TWEAK (Twk) (100 ng/ml) and stimulated for 10 min with insulin at the indicated concentrations. Levels of phosphorylated Akt (pAkt) were determined and quantified as described in Fig. 1. n = 3. *, P < 0.01, TWEAK-treated vs. insulin alone.

View larger version (45K): [in this window] [in a new window]   FIG. 3. Effect of TWEAK on IR and IRS-1 activation. Huh7 cells were pretreated for 12 h with or without TWEAK (100 ng/ml) and stimulated with insulin (10 nmol/liter) for 10 min. Cell lysates were normalized to equal amounts of protein and subjected to immunoprecipitation (IP) assays for IRβ and IRS-1 with the indicated antibodies, as described in Materials and Methods. A, Tyrosine phosphorylation (pY) of IRβ (upper blot) and total levels of IRβ (lower blot); B, tyrosine phosphorylation (pY) of IRS-1 (upper blot), association of IRS-1 with p85 subunit (middle blot), and total levels of IRS-1 (lower blot); C, levels of serine phosphorylation of IRS-1 at Ser 312 (upper blot) and Ser-616 (middle blot) and total levels of IRS-1 (lower blot) in Huh7 cells after treatment with TWEAK (100 ng/ml) for the indicated time period. The histograms represent band intensities that were normalized to total levels of IRβ or IRS-1. Data are expressed as mean ± SD derived from at least three separate experiments. *, P < 0.01, TWEAK-treated vs. insulin alone (A and B); *, P < 0.01, TWEAK-treated vs. nil (C). Twk, TWEAK.

View larger version (22K): [in this window] [in a new window]   FIG. 4. Effect of TWEAK on insulin-regulated PEPCK and G6Pase expression and glycogen synthesis in hepatocytes. Huh7 or HepG2 cells were serum starved overnight before pretreatment for 2 h with or without TWEAK (100 ng/ml) and subsequent treatment with insulin (10 nmol/liter) for 8 h. A and B, The mRNA levels of PEPCK and G6Pase were determined by RT-PCR (A), quantified by real-time PCR analysis (B), and normalized against cyclophilin (Cycl); C, primary rat hepatocytes were pretreated with TWEAK (100 ng/ml) for 2 h followed by treatment with [14C]D-glucose and insulin (10 nmol/liter). After 4 h, glycogen formation was then examined as described in Materials and Methods. Data are expressed as mean ± SD derived from three independent experiments. *, P < 0.01, TWEAK-treated vs. insulin alone.

View larger version (25K): [in this window] [in a new window]   FIG. 5. Effect of Fn14 on TWEAK-induced suppression of insulin action. A, Levels of Fn14 mRNA expression were determined by RT-PCR in freshly isolated primary hepatocytes (PHC) and Huh7 and HepG2 cells treated with or without TWEAK for 4 h; B, Huh7 cells were preincubated with or without anti-Fn14 blocking antibodies (Fn14), an isotype-matched irrelevant antibody (M2) or mouse antiserum (As) for 1 h before TWEAK (100 ng/ml) treatment. Cells were then stimulated with insulin (10 nmol/liter) for 10 min. Levels of phosphorylated Akt (pAkt) were determined and quantified as described in Fig. 1. Data are expressed as mean ± SD derived from three separate experiments. *, P < 0.01, anti-Fn14 antibodies treated cells vs. the control.

NF-B activation is involved in TWEAK-induced insulin resistance in hepatocytes

View larger version (20K): [in this window] [in a new window]   FIG. 6. TWEAK-induced NF-B activation. Huh7 cells were serum starved overnight and treated with TWEAK (100 ng/ml) for the indicated time periods. A and B, After the treatment, cells were harvested and cell lysates were subjected to Western blot analysis using anti-IB (A) and anti-p52 (B) antibodies, respectively. The histograms represent band intensities that were normalized to β-actin levels. C, Huh7 cells were transfected with an NF-B reporter (pLuc-IgK), and a control vector containing Renilla luciferase (pRL). After 16 h, cells were treated with or without TWEAK (100 ng/ml) for another 8 h. The reporter gene activity was then analyzed by the dual-luciferase assay system and normalized relative to Renilla luciferase activity as described in the Materials and Methods. Data are expressed as mean ± SD derived from at least three separate experiments. *, P < 0.05; , P < 0.01, TWEAK-treated vs. nil.

View larger version (34K): [in this window] [in a new window]   FIG. 7. Effect of NF-B on TWEAK-induced inhibition of insulin action. A, Insulin-induced Akt phosphorylation was assessed in Huh7 cells after the indicated treatment in the presence or absence of Bay 11-7082 (5 µM), MG132 (0.5 µM), or vehicle alone; B, Huh7 cells were transfected with p65 or scrambled control siRNAs. After 36 h, cells were pretreated for 2 h with TWEAK followed by insulin (10 nmol/liter) stimulation, and cell lysates were subjected to Western blot assays using antibodies against phospho-Akt (pAkt) (Ser 473), total Akt, or p65. The histograms represent band intensities that were normalized to total Akt levels. Data are expressed as mean ± SD derived from three separate experiments. *, P < 0.01, Bay 11-7082 or MG132-treated vs. vehicle alone (A); *, P < 0.01, p65 siRNA transfected cells vs. the control (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

An important physiological function of insulin in the liver is the regulation of hepatic gluconeogenesis and glycogen synthesis, which is critical for the maintenance of normal glucose homeostasis. Dysfunction of this process is a common characteristic of insulin resistance and T2D (25). The activation of the phosphatidylinositol 3-kinase/Akt pathway that regulates the expression of the key gluconeogenic enzymes PEPCK and G6Pase is regarded as a central signaling mechanism underlying insulin’s ability to regulate the glucose production in the liver (22, 26). Herein we report that TWEAK profoundly inhibited both the proximal and distal signaling events of insulin and affected PEPCK and G6Pase expression in Huh7 and HepG2 hepatic cell lines. TWEAK was also capable of attenuating insulin-mediated signaling events in primary hepatocytes, as evidenced by the dramatic inhibition of insulin-induced Akt phosphorylation in a concentration-dependent manner (Fig. 1). Moreover, insulin-induced glycogen synthesis was also greatly attenuated by TWEAK in primary hepatocytes (Fig. 4C), suggesting that hepatocytes may serve as a physiological target for the inhibitory effect of TWEAK on the actions of insulin. In addition, the finding that TWEAK inhibited cellular signaling induced by insulin at either physiological or supraphysiological concentrations, reveals a potent inhibitory effect on insulin action under both normal and hyperinsulinemic conditions.

The inhibitory effect of TWEAK appears to initially occur at the levels of the IR and IRS-1, because TWEAK induced a significant inhibition of both IR autophosphorylation and IRS-1 activation. Furthermore, TWEAK was also capable of inducing serine phosphorylation of IRS-1 at Ser 312 and Ser 616, which has been reported to convert IRS-1 into an inhibitor of the intrinsic IR activity (23). These findings suggest a direct inhibitory effect of TWEAK on IR and/or IRS. However, it is noted that the time course of inhibition on Akt phosphorylation (Fig. 1C) was not closely correlated with serine phosphorylation of IRS-1 induced by TWEAK, suggesting an additional mechanism underlying the inhibitory effect of TWEAK on insulin action. Indeed, the production of IL-6 and suppressor of cytokine signaling 3 (SOCS3) has been suggested as one of important mechanisms for TNF-induced insulin resistance (1, 19). It has been acknowledged that insulin resistance and/or T2D are often associated with chronic, low-grade systemic inflammation that involves multiple cytokine networks and multiple regulatory processes (2). In this regard, it is unlikely that an individual cytokine can function in an isolated manner to inhibit insulin signaling in vivo. Although TWEAK has been shown to be up-regulated by interferon- (27), TWEAK has also been reported to stimulate the production of IL-6, IL-8, and other proinflammatory molecules (10). In addition, TWEAK is also able to potentiate the proinflammatory activities of TNF and IL-1 (28), suggesting a regulatory role for TWEAK in the cytokine network systems. Interestingly, besides its own proinflammatory activities, TWEAK has been recently shown to attenuate the transition from innate response to adaptive immunity (12), suggesting a potential role for TWEAK in guiding the process and outcomes of immune and inflammatory responses. As such, TWEAK may serve as an intriguing intervention target for the protection against the development of insulin resistance by modulating inflammatory responses and regulating the cytokine networks.

Although various signaling pathways and networks, including IB kinase (IKK), c-Jun N-terminal kinase (JNK), p38, protein kinase C (PKC), and others, have been documented to mediate inflammation-associated insulin resistance, the transcription factor NF-B, a master regulator of gene expression for many inflammation-related molecules, has been suggested as a key player in this pathogenic event (29, 30). Shoelson and colleagues (24) reported that NF-B was activated in the liver of obese subjects, causing both hepatic and systemic insulin resistance in an animal model. Moreover, inhibition of the NF-B pathway by genetic means or by high-dose aspirin has been shown to improve insulin responsiveness in both obese mice (29) and in human diabetics (31). In the current study, we found that TWEAK was capable of activating both the canonical and noncanonical NF-B pathways in hepatocytes, as manifested by the rapid degradation of IB (Fig. 6A) and the prolonged induction of p100 processing to p52 (Fig. 6B), which occurred at 30 min and 5–24 h, respectively, after TWEAK stimulation. The noncanonical pathway has been suggested to play an important role in constitutive NF-B activation (32), which is often associated with chronic inflammation. For instance, the processing of p100 to p52 has been reported in mature B cells, native T cells, differentiated macrophages, and dendritic cells, along with a variety of tumor cells with constitutive NF-B activation (32, 33). The finding that TWEAK activated the canonical NF-B pathway as well as inducing p100 processing to p52 suggests that TWEAK is a potent activator of NF-B in hepatocytes. Notably, inhibition of NF-B activity by either chemical inhibitors (Bay 11-7082 and MG132) or siRNA against p65 almost completely abrogated the inhibitory effect of TWEAK on insulin-induced Akt phosphorylation, indicating a critical role for NF-B in mediating TWEAK-induced inhibition of insulin action in hepatocytes. These observations not only further support a role for NF-B in mediating hepatic insulin resistance but also provide a mechanistic explanation for the TWEAK-induced suppression of insulin action in hepatocytes. As the inhibitors Bay 11-7082 and MG132 or p65 siRNA are unable to differentiate between the canonical and noncanonical NF-B pathways, it is not clear which of the respective pathways are more important for TWEAK-mediated inhibition. This requires further investigation.

Although the precise mechanism by which TWEAK suppresses the action of insulin in hepatocytes remains undefined, the current study has provided evidence illustrating a critical role for the TWEAK receptor Fn14. We were able to confirm Fn14 mRNA expression in both the hepatocellular carcinoma cell lines (Huh7 and HepG2) and the primary hepatocytes, as previously reported by Feng et al. (14). It is noted that the expression levels of Fn14 in Huh7 and HepG2 cell lines were significantly higher than the primary hepatocytes, despite TWEAK being equally as effective at attenuating the signaling properties of insulin in all the cell types examined. This could be attributed to the natural properties of the Fn14 receptor that has a reported affinity constant (K_d) of about 2.4 nmol/liter (11). Indeed, the inhibitory effect of TWEAK was evident at concentrations as low as 25–50 ng/ml (1.4–2.7 nmol/liter), which fall well within the range of the receptor K_d value, suggesting a possible physiological role for the TWEAK/Fn14 system in the regulation of insulin action in hepatocytes. This notion was further supported by the finding that TWEAK-induced inhibition of insulin-dependent Akt phosphorylation was diminished by the blocking antibody against Fn14. Interestingly, it has been reported that the expression levels of TWEAK/Fn14 were increased during the process of liver regeneration after hepatic injury (14, 15). More recently, Chacon et al. (34) have detected the expression of TWEAK/Fn14 in human sc adipose tissue. Despite there being no significant difference in the expression levels of TWEAK mRNA between lean and obese subjects (n = 40), elevated expression levels of TWEAK/Fn14 were detected in morbidly obese patients (34), suggesting that this cytokine-receptor system may contribute to aggravating the inflammatory and insulin-resistant state observed in extreme obesity. It will be of interest to investigate whether TWEAK has a similar inhibitory effect on insulin action in adipocytes, similar to that observed in liver cells. Additional studies are imperatively required to clarify the in vivo role of TWEAK/Fn14 in the development of insulin resistance and T2D, particularly in a clinical setting. This could ultimately allow the creation of a new strategy targeting the TWEAK/Fn14 pathway for the prevention and treatment of insulin resistance and T2D.

	Acknowledgments

 
We are grateful to Dr. Grigori Rychkov for providing primary rat hepatocytes.

	Footnotes

 
This work was supported by a Career Development Fellowship from National Heart Foundation of Australia and grants from the National Health and Medical Research Council of Australia to P.X.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 3, 2008

Abbreviations: Fn14, Fibroblast growth factor-inducible 14-kDa protein; G6Pase, glucose-6-phosphatase; IB, inhibitor of B; IR, insulin receptor; IRS-1, insulin receptor substrate-1; NF, nuclear factor; PEPCK, phosphoenolpyruvate carboxykinase; T2D, type 2 diabetes; TWEAK, TNF-like weak inducer of apoptosis.

Received August 13, 2007.

Accepted for publication December 21, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wellen KE, Hotamisligil GS 2005 Inflammation, stress, and diabetes. J Clin Invest 115:1111–1119[CrossRef][Medline]
2. Shoelson SE, Lee J, Goldfine AB 2006 Inflammation and insulin resistance. J Clin Invest 116:1793–1801[CrossRef][Medline]
3. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM 1995 Increased adipose tissue expression of tumor necrosis factor- in human obesity and insulin resistance. J Clin Invest 95:2409–2415[Medline]
4. Pickup JC, Chusney GD, Thomas SM, Burt D 2000 Plasma interleukin-6, tumour necrosis factor and blood cytokine production in type 2 diabetes. Life Sci 67:291–300[CrossRef][Medline]
5. Pickup JC Crook MA 1998 Is type II diabetes mellitus a disease of the innate immune system? Diabetologia 41:1241–1248[CrossRef][Medline]
6. Hotamisligil GS, Shargill NS, Spiegelman BM 1993 Adipose expression of tumor necrosis factor-: direct role in obesity-linked insulin resistance. Science 259:87–91[Abstract/Free Full Text]
7. Kern PA, Saghizadeh M, Ong JM, Bosch RJ, Deem R, Simsolo RB 1995 The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest 95:2111–2119[Medline]
8. Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS 1997 Protection from obesity-induced insulin resistance in mice lacking TNF- function. Nature 389:610–614[CrossRef][Medline]
9. Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C, Garcia I, Browning JL 1997 TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly induces apoptosis. J Biol Chem 272:32401–32410[Abstract/Free Full Text]
10. Wiley SR, Winkles JA 2003 TWEAK, a member of the TNF superfamily, is a multifunctional cytokine that binds the TweakR/Fn14 receptor. Cytokine Growth Factor Rev 14:241–249[CrossRef][Medline]
11. Wiley SR, Cassiano L, Lofton T, Davis-Smith T, Winkles JA, Lindner V, Liu H, Daniel TO, Smith CA, Fanslow WC 2001 A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis. Immunity 15:837–846[CrossRef][Medline]
12. Maecker HE, Varfolomeev E, Kischkel F, Lawrence D, LeBlanc H, Lee W, Hurst S, Danilenko D, Li J, Filvaroff E, Yang B, Daniel D, Ashkenazi A 2005 TWEAK attenuates the transition from innate to adaptive immunity. Cell 123:931–944[CrossRef][Medline]
13. Saitoh T, Nakayama M, Nakano H, Yagita H, Yamamoto N, Yamaoka S 2003 TWEAK induces NF-B2 p100 processing and long lasting NF-B activation. J Biol Chem 278:36005–36012[Abstract/Free Full Text]
14. Feng SL, Guo Y, Factor VM, Thorgeirsson SS, Bell DW, Testa JR, Peifley KA, Winkles JA 2000 The Fn14 immediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas. Am J Pathol 156:1253–1261[Abstract/Free Full Text]
15. Jakubowski A, Ambrose C, Parr M, Lincecum JM, Wang MZ, Zheng TZ, Browning B, Michaelson JS, Baetscher M, Wang B, Bissell DM, Burkly LC 2005 TWEAK induces liver progenitor cell proliferation. J Clin Invest 115:2330–2340[CrossRef][Medline]
16. Berry MN, Halls HJ, Grivell MB 1992 Techniques for pharmacological and toxicological studies with isolated hepatocyte suspensions. Life Sci 51:1–16[CrossRef][Medline]
17. Wang L, Xing XP, Holmes A, Wadham C, Gamble JR, Vadas MA, Xia P 2005 Activation of the sphingosine kinase-signaling pathway by high glucose mediates the proinflammatory phenotype of endothelial cells. Circ Res 97:891–899[Abstract/Free Full Text]
18. Xia P, Wang L, Moretti PA, Albanese N, Chai F, Pitson SM, D’Andrea RJ, Gamble JR, Vadas MA 2002 Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor- signaling. J Biol Chem 277:7996–8003[Abstract/Free Full Text]
19. Mooney RA, Senn J, Cameron S, Inamdar N, Boivin LM, Shang Y, Furlanetto RW 2001 Suppressors of cytokine signaling-1 and -6 associate with and inhibit the insulin receptor. A potential mechanism for cytokine-mediated insulin resistance. J Biol Chem 276:25889–25893[Abstract/Free Full Text]
20. Taniguchi CM, Emanuelli B, Kahn CR 2006 Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85–96[CrossRef][Medline]
21. Gunton JE, Delhanty PJ, Takahashi S, Baxter RC 2003 Metformin rapidly increases insulin receptor activation in human liver and signals preferentially through insulin-receptor substrate-2. J Clin Endocrinol Metab 88:1323–1332[Abstract/Free Full Text]
22. Barthel A. Schmoll D 2003 Novel concepts in insulin regulation of hepatic gluconeogenesis. Am J Physiol Endocrinol Metab 285:E685–E692
23. Nakayama M, Ishidoh K, Kojima Y, Harada N, Kominami E, Okumura K, Yagita H 2003 Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death. J Immunol 170:341–348[Abstract/Free Full Text]
24. Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson SE 2005 Local and systemic insulin resistance resulting from hepatic activation of IKK-β and NF-B. Nat Med 11:183–190[CrossRef][Medline]
25. Klover PJ, Mooney RA 2004 Hepatocytes: critical for glucose homeostasis. Int J Biochem Cell Biol 36:753–758[CrossRef][Medline]
26. Liao J, BarthelA, Nakatani K, Roth RA 1998 Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene. J Biol Chem 273:27320–27324[Abstract/Free Full Text]
27. Nakayama M, Kayagaki N, Yamaguchi N, Okumura K, Yagita H 2000 Involvement of TWEAK in interferon -stimulated monocyte cytotoxicity. J Exp Med 192:1373–1380[Abstract/Free Full Text]
28. Chicheportiche Y, Chicheportiche R, Sizing I, Thompson J, Benjamin CB, Ambrose C, Dayer JM 2002 Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies. Arthritis Res 4:126–133[CrossRef][Medline]
29. Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI 2001 Prevention of fat-induced insulin resistance by salicylate. J Clin Invest 108:437–446[CrossRef][Medline]
30. Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE 2001 Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkβ. Science [Erratum (2002) 295:277] 293:1673–1677
31. Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI 2002 Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 109:1321–1326[CrossRef][Medline]
32. Karin M, Greten FR 2005 NF-B: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5:749–759[CrossRef][Medline]
33. Senftleben U, Cao Y, Xiao G, Greten FR, Krahn G, Bonizzi G, Chen Y, Hu Y, Fong A, Sun SC, Karin M 2001 Activation by IKK of a second, evolutionary conserved, NF-B signaling pathway. Science 293:1495–1499[Abstract/Free Full Text]
34. Chacon MR, Richart C, Gomez JM, Megia A, Vilarrasa N, Fernandez-Real JM, Garcia-Espana A, Miranda M, Masdevall C, Ricard W, Caubet E, Soler J, Vendrell J 2006 Expression of TWEAK and its receptor Fn14 in human subcutaneous adipose tissue. Relationship with other inflammatory cytokines in obesity. Cytokine 33:129–137[CrossRef][Medline]

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