PA, tunicamycin (Tuni), exendin-4, tauroursodeoxycholic acid (TUDCA), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to fetuin-A, inositol-requiring enzyme-1α (IRE1α), CCAAT/enhancer binding homologous protein (CHOP), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). SEPP1 and phosphor-IRE1α (P-IRE1α) antibodies were purchased from Abcam (Cambridge, MA, USA). PKR-like endoplasmic reticulum kinase (PERK), phosphor-PERK (P-PERK), activating transcription factor 6 (ATF6), X-box binding protein 1 (XBP-1) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The HepG2 human hepatoma cell line (ATCC, Manassas, VA, USA) was cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). At approximately 85% confluence in six-well plates, cells were exposed to the ER stress inducers PA and Tuni. After 24 hours, cultured cells were treated with 100 nM exendin-4, 200 µM TUDCA, and 1 mM AICAR, after which they were incubated for 24 hours.

For gene knockout, the cells were transfected with 10 nM small interfering RNA (siRNA) of AMP-activated protein kinase (AMPK) and scrambled control siRNA (Santa Cruz) using the lipofectamine RNAiMAX reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After incubation for 24 hours, the transfected cells were treated with exendin-4 (100 nM) for 24 hours.

Total RNA was isolated from the cells using Trizol reagent (Invitrogen) to measure the messenger RNA (mRNA) levels of the SEPP1 and fetuin-A genes. Total RNA (2 µg) was reverse-transcribed to complementary DNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). After cDNA synthesis, quantitative real-time PCR was performed using SYBR green (Roche, Lewis, UK) and specific primers (Bioneer Co., Daejeon, Korea) according to the manufacturers' instructions. To normalize the expression of the target genes, the expression of β-actin (Actb) was used as an endogenous control in the comparative Ct method (2-delta delta Ct).

Cells were lysed in ice-cold RIPA buffer (Cell Signaling Technology) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails (Sigma-Aldrich). The lysates were incubated on ice for 30 minutes, and then centrifuged at 13,000 rpm for 20 minutes at 4℃. The protein concentration of the supernatant was quantified using the Bradford protein assay (Bio-Rad Protein Assay, BioRad, Hercules, CA, USA) with bovine serum albumin standard (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein (20 µg) were electrophoresed on 4% to 12% Bis-Tris Nupage gels (Invitrogen) and transferred to polyvinylidene difluoride membranes using the iBlot Dry Blotting System (Invitrogen). After transfer, the membranes were blocked in 5% bovine serum albumin /Tris buffered saline with Tween-20 buffer for 1 hour, and then incubated overnight at 4℃ with antibodies to SEPP1, fetuin-A, IRE1α, P-IRE1α, PERK, P-PERK, ATF6, XBP-1, CHOP, and β-actin followed by incubation with horseradish peroxidase conjugated secondary antibodies. Immunoreactive bands were visualized with enhanced chemiluminescence Western blotting detection reagents (GE Healthcare, Chalfont St. Giles, UK).

All statistical analyses were performed using PASW Statistics version 17 (SPSS Inc., Chicago, IL, USA). The data are presented as the mean±standard error. A P value less than 0.05 was considered statistically significant.

The expression of the SEPP1 and fetuin-A genes was higher in cells treated with PA alone than in the untreated controls, and significantly decreased with exendin-4 treatment in cells that did and did not undergo PA treatment (Fig. 1A). In addition, the expression of proteins exhibited a trend similar to that of their respective protein transcripts (Fig. 1B).

PA, a saturated fatty acid, disrupts ER homeostasis [17], potentially leading to diabetes and hepatic steatosis. To evaluate the effect of exendin-4 on PA-induced ER stress in hepatocyte cells, we examined the effect of exendin-4 on the expression of the ER stress markers IRE1α, PERK, ATF6, and CHOP. Cells exposed to PA displayed higher P-IRE1α, P-PERK, ATF6, and CHOP protein levels, whereas PA-induced increases in the ER stress marker protein levels were reversed in cells treated with exendin-4 (Fig. 2). These results suggest that exendin-4 reduces the expression of PA-induced increases in SEPP1 and fetuin-A, and that exendin-4 improves PA-induced ER stress.

To examine whether increased expression of SEPP1 and fetuin-A secondary to PA treatment is associated with ER stress, HepG2 cells were pretreated with Tuni, an ER stress inducer, followed by the addition of TUDCA, an ER stress inhibitor, or exendin-4. As shown in Fig. 3, the expression of SEPP1 and fetuin-A mRNA as well as XBP-1, a marker for ER stress, was significantly higher in cells treated with Tuni than in the untreated controls. In contrast, supplementation of TUDCA in cells exposed to Tuni completely abolished the effect of Tuni on the expression of these genes. Interestingly, the expression of XBP-1, SEPP1, and fetuin-A in cells treated with exendin-4 exhibited levels similar to those in cells treated with TUDCA. These data suggest that exendin-4 has a protective effect against ER stress, and that exendin-4 attenuates the expression of the SEPP1 and fetuin-A genes by relieving ER stress.

The AMPK activator AICAR can inhibit fatty acid-induced ER stress [18]. SIRT1-AMPK signaling induces a potent pro-tective effect of exendin-4 against fatty liver disease [19]. In this study, we have demonstrated that AICAR downregulates the expression of the SEPP1 and fetuin-A genes (Fig. 4A). We also examined whether the inhibitory effect of exendin-4 on the expression of ER stress-induced SEPP1 and fetuin-A is mediated by AMPK. The expression of AMPK increased in HepG2 cells treated with exendin-4 [19]. However, when the expression of AMPK was inhibited by AMPK siRNA, the expression of SEPP1 and fetuin-A in cells treated with exendin-4 did not decrease (Fig. 4B-D). These results suggest that exen-din-4 inhibits expression of ER stress-induced SEPP1 and fetuin-A via stimulation of AMPK. Activation of AMPK may mediate an inhibitory effect of exendin-4 on ER stress-induced hepatokines, such as SEPP1 and fetuin-A.