The human hepatoma cell line HepG2 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). PA (Sigma-Aldrich, St. Louis, MO, USA) was used to induce hepatic steatosis in cultured hepatocytes. To examine the protective effect of dulaglutide on PA-induced steatosis, cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide (MedChemExpress, Monmouth Junction, NJ, USA) for 24 hours. To investigate whether the beneficial effects of dulaglutide in hepatic steatosis are mediated by GLP-1R, cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide and 100 nM exendin (9-39) (Sigma-Aldrich) for 24 hours. The cells were cultured in an incubator at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

To measure total intracellular triglyceride (TG) content, lipids were extracted from the cultured cells and total TG content was determined by enzymatic assays (TR0100, Sigma-Aldrich) and normalized to protein concentration. The protein concentration was measured using the Bradford assay (Bio-Rad, Munich, Germany). HepG2 cells were seeded in 6-well plates and stained using the Oil red O method. Briefly, after treatment, cells were washed three times with phosphate-buffered saline (PBS) and fixed for 30 minutes with 10% formalin. After fixation, cells were washed again with PBS, stained with Oil-Red O (Sigma-Aldrich) for 1 hour, and then rinsed with distilled water. The cells were observed under a light microscope (Olympus, Tokyo, Japan) with a 400× magnification and images were photographed. For quantitative analysis of cellular lipid, 1 mL isopropanol (Sigma-Aldrich) was added to the stained culture plate and absorbance of the extracted dye was recorded at 540 nm.

To silence the target protein, small interfering RNA (siRNA) duplexes targeting FAM3A and negative control siRNA were purchased from Bioneer (Daejeon, Korea), and the cells were transiently transfected with synthetic siRNAs using the Lipofectamine RNAi MAX transfection reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. After 24 hours of transfection, the cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM dulaglutide for 24 hours. The cells were then harvested for further analysis, and knockdown efficiency of the siRNAs was determined by Western blotting.

Total RNA was isolated from cultured HepG2 cells using the TRIzol reagent (Invitrogen). The RNA was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Gene expression analysis was performed by quantitative real time polymerase chain reaction using LightCycler 480 SYBR Green (Roche, Lewis, UK). Primer sequences used for the detection of genes are shown in Supplemental Table S1. The expression levels of the target genes were normalized to the housekeeping control gene (18S rRNA) and relative quantification of the target gene mRNA was performed using the 2^−ΔΔCt method.

Total protein extracts were isolated from the HepG2 cells. The cells were lysed with RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Rockford, IL, USA) on ice. Then, protein concentrations were measured and equal amounts of the protein samples (15 μg) were dissolved in 4× lithium dodecyl sulfate sample buffer and 10× reducing sample agent (Invitrogen) and denatured at 95°C for 10 minutes. The samples were loaded on a 4% to 12% Bis-Tris NuPAGE gel (Thermo Fisher Scientific). The separated proteins were transferred from the gel to a polyvinylidene difluoride membrane by using the iBlot2 PVDF stack (Invitrogen). The membranes were blocked with 5% bovine serum albumin in tris-buffered saline and tween 20 (TBST) for 1 hour at room temperature and probed at 4°C overnight with the following primary antibodies: anti-sirtuin 1 (SIRT1) (sc-15404, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-phospho-AMPK (#2531, Cell Signaling Technology), anti-AMPK (#2532, Cell Signaling Technology), anti-perilipin 1 (PLIN1) (sc-67164, Santa Cruz Biotechnology Inc.), anti-adipose differentiation-related protein (ADFP) (ab52355, Abcam, Cambridge, MA, USA), anti-adipose triglyceride lipase (ATGL) (#2138, Cell Signaling Technology), anti-phospho-hormone-sensitive lipase (p-HSL) (#4126, Cell Signaling Technology), anti-phospho-acetyl-CoA carboxylase (p-ACC) (#3661, Cell Signaling Technology), anti-peroxisome proliferator-activated receptor alpha (PPARα) (ab8934, Abcam), anti-FAM3A (SAB1102488, Sigma-Aldrich), anti-GLP-1R (sc-66911, Santa Cruz Biotechnology Inc.), anti-Ca²⁺/calmodulin-dependent protein kinase kinaseβ (CaMKKβ) (sc-271674, Santa Cruz Biotechnology Inc.), and anti-β-actin (#4967, Cell Signaling Technology). The membranes were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). The density of the bands was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Quantitative data are expressed as the mean±standard error. The statistical analysis was performed using SPSS version 12.0 software (SPSS Inc., Chicago, IL, USA) and sigma plot software. The data were analyzed using Student’s t test or one-way analysis of variance, and multiple comparisons among the experimental groups were investigated with the Bonferroni correction test. P values <0.05 were considered to be statistically significant. All the analyses were performed in triplicate.

To examine the protective effect of dulaglutide on hepatic steatosis, HepG2 cells were treated with a saturated fatty acid, PA. PA significantly increased intracellular lipid accumulation (Fig. 1A-C), which was revealed by Oil red O staining and measurement of TG content. In addition, PA dramatically increased the expression of lipid droplet binding proteins, PLIN1 and PLIN2, which help prevent lipolysis, compared with that in control cells (Fig. 1D, E). However, the mRNA expression of PLIN2 was not significantly affected by PA. In contrast, dulaglutide inhibited TG accumulation and the mRNA and protein expression of PLIN1 and protein expression of PLIN2 in cells pretreated with PA. No significant difference in the expression of PLIN2 mRNA was observed between the cells treated with PA and PA+dulaglutide. These results suggest that dulaglutide has a protective effect against PA-induced hepatic steatosis.

The liver is an important site for lipid metabolism, and hepatic lipid homeostasis is maintained through the balance of synthesis, degradation, and secretion of lipid [19]. We confirmed the change in the mRNA expression of lipogenic genes, including sterol regulatory-binding protein 1 (SREBP-1), ACC, and fatty acid synthase (FAS), the TG synthesis enzyme genes, including diacylglycerol acyltransferase (DGAT) 1 and 2, and key factor genes associated with TG secretion, including microsomal triglyceride transfer protein (MTP) and apolipoprotein B (ApoB). PA increased the expression of SREBP-1, ACC, FAS, and DGAT2 and decreased the expression of MTP and ApoB compared with that in control cells, but no significant change was observed in the expression of DGAT1 (Fig. 2). In contrast, treatment with dulaglutide significantly decreased the expression of SREBP-1, ACC, FAS, and DGAT2 and increased the expression of MTP and ApoB compared with that in the PA-treated cells. We examined whether dulaglutide regulates lipolysis and fatty acid oxidation in PA-treated cells. PA reduced the expression of p-HSL, but not ATGL, which are key enzymes involved in intracellular degradation of TG, and the expression of fatty acid oxidation-associated factors such as SIRT1, p-AMPK, p-ACC, and PPARα, compared with that in control cells (Fig. 3). Meanwhile, treatment with dulaglutide significantly increased the expression of ATGL, p-HSL, SIRT1, p-AMPK, and PPARα proteins compared with that in the PA-treated cells. Therefore, these results suggest that the beneficial effect of dulaglutide against PA-induced hepatic steatosis might be mediated by increasing lipid catabolism and decreasing lipid anabolism.

An increase in the expression of FAM3A in the liver is associated with improvement in hyperglycemia, insulin resistance, and fatty liver [15]. As shown in Fig. 4A, the expression of FAM3A was decreased in cells treated with PA compared with that in control cells, and increased in cells treated with PA+dulaglutide compared to the cells treated with PA alone. To investigate whether the regulation of FAM3A expression by dulaglutide is mediated by the GLP-1R, HepG2 cells pre-treated with PA were treated with dulaglutide in the absence or presence of exendin (9-39), a GLP-1R antagonist. The protein expression of GLP-1R, FAM3A, SIRT1, p-AMPK, p-ACC, and PPARα was decreased in cells treated with PA compared to the untreated control, and was increased in cell treated with PA+dulaglutide compared to the cells treated with PA alone (Fig. 4B). However, in the presence of exendin (9-39), the increase in GLP-1R, FAM3A, SIRT1, p-AMPK, p-ACC, and PPARα expression in cells treated with PA+dulaglutide was attenuated. Therefore, these results suggest that dulaglutide regulates the expression of FAM3A and fatty acid oxidation-associated factors via a GLP-1R dependent pathway.

To clarity the role of FAM3A in dulaglutide-regulated lipid metabolism in PA-induced hepatic steatosis, HepG2 cells were transfected with the FAM3A siRNA. Inhibition of FAM3A by the siRNA resulted in an increase in TG accumulation; however, treatment with dulaglutide did not decrease TG accumulation in PA-treated cells transfected with FAM3A siRNA (Fig. 5A, Supplemental Fig. S1). In addition, the expression of SIRT1, p-AMPK, p-ACC, and PPARα was decreased in cells transfected with FAM3A siRNA, but dulaglutide treatment did not increase the expression of these proteins in cells transfected with FAM3A siRNA (Fig. 5B). These results suggest that inhibition of TG accumulation by dulaglutide in PA-treated cells is mediated by FAM3A, and that FAM3A expression increased by dulaglutide is involved in the regulation of the expression of SIRT1/AMPK signaling, which is associated with fatty acid oxidation.