Large yellow croaker juveniles were bought from a commercial fish farm (Ningbo, China). After 1 week of acclimation, the fish were randomly divided into two groups equally, which were respectively injected with dimethyl sulfoxide (DMSO) (Solarbio, China) or Tunicamycin (TM) (Sigma, USA) (3 μg/g fish). Fish were anesthetized with MS-222 and collected at 24 h after injection. The intestines of the fish were immediately collected and frozen in liquid nitrogen, and then stored at -80°C for further analysis.

Intestinal cells were isolated from healthy large yellow croaker and cultured in Dulbecco’s Modified Eagle Medium/Ham’s F12 (DMEM/F12) medium (Biological Industries, Israel) with 15% fetal bovine serum (FBS) (Biological Industries) and penicillin and streptomycin (Solarbio) in 5% CO₂ atmosphere at 27°C. Intestinal cells were seeded into 6-well plates and cultured overnight. Cells were treated with 1 μM TM for different time points to explore the effects of ER stress on intestinal lipid metabolism and inflammation in vitro. Then cells were harvested for further analysis.

The TG content was analyzed according to a previous study (20). After TM treatment for 12 h, cells were incubated with BODIPY 493/503 (Sigma) for 10 min, washed with phosphate buffer solution (PBS, Solarbio) three times, and then immediately observed lipid droplets in the cells with a fluorescent microscope (Nikon, Japan), according to the methods in a previous study (21).

The total RNA of the intestines and isolated intestinal cells was prepared using the TRIzol reagent (Takara, Japan), and cDNA was synthesized using the PrimeScript™ RT reagent kit (Takara), according to the method in our previous study (22). qRT-PCR was performed using SYBR Premix Ex Taq (Takara) and Roche LightCycler^® 96 system (USA). The specific primer sequences of genes were designed according to published sequences in NCBI and listed in Table 1 . In the present study, we found that the most stable reference gene was βactin across all the samples rather than 18S rRNA or gapdh, according to geNorm analysis (23). Thus, βactin was used as a reference gene. The relative mRNA expression of related genes was calculated using the comparative CT method (24).

The total protein of the sample was extracted by adding RIPA lysis (Solarbio), and nuclear protein was collected using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA), according to the method in our previous study (25). The protein concentration was measured by a BCA Protein Assay Kit (Beyotime, China) for adjustments. The protein was separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V for 1 h and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA) at 100 V for 1 h. Then the PVDF membranes were blocked with 5% skimmed milk at room temperature for 1 h, followed by overnight incubation at 4°C with specific primary antibodies, including glucose-regulated protein 78 (GRP78) (3117, CST, USA), X-box-binding protein 1 s (XBP1s) (12782, CST), ATF6 (bs-1634R, Bioss, China), phosphorylated double-stranded RNA-dependent protein kinase-like ER kinase (p-PERK) (Thr982) (137056, Absin, China), acetyl-CoA carboxylase (ACC) (3662, CST), diacylglycerol acyltransferase 1 (DGAT1) (Gensript, China), cleavage of sterol-regulatory element binding protein 1 c (SREBP1c) (WL02093, Wanleibio, China), fatty acid translocase (FAT/CD36) (Gensript), microsomal triglyceride transfer protein (MTP) (ab186446, Abcam, England), apolipoprotein B 48 (APOB48) (Gensript), GTPase-activating protein SEC13 (SEC13) (sc-514308, Santa Cruz, USA), secretion associated Ras related GTPase 1B (SAR1B) (ab155278, Abcam), acyl-CoA oxidase (ACO) (ab184032,Abcam), carnitine palmitoyl transferase 1 α (CPT1α) (15184-1-AP, Proteintech, USA), peroxisome proliferator-activated receptor-γ coactlvator-1α (PGC1α) (ab118102, Abcam), peroxisome proliferator-activated receptor α (PPARα) (117362, Absin), extracellular-regulated kinase 1/2 (ERK1/2) (4695, CST), p-ERK1/2 (Thr202/Tyr204) (4370, CST), p38 mitogen-activated protein kinase (p38) (8690, CST), p-p38 (Thr180/Tyr182) (9215, CST), NF-kappaB p65 (p65) (8242, CST), interleukin-1 β (IL-1β) (Gensript), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (R001, Goodhere, China), and Histone H3 (ab1791, Abcam). Then the protein bands were incubated with HRP-conjugated secondary antibody (A0208, Beyotime) for 1 h at room temperature. The immunoreactive protein was detected using ELC reagent (Beyotime).

The acc, scd1, dgat1, dgat2, il-1β, il-6, tnfα, and cox2 promoter fragments of large yellow croaker were cloned, and then they were constructed into the PGL3-basic vector to assemble reporter plasmids, respectively, using a ClonExpress II One Step Cloning Kit (Vazyme Biotech, China), according to previously described methods (26). The XBP1, CHOP, ATF4, and ATF6 CDS fragments were cloned and constructed into a PCS2+ vector to assemble expression plasmids, respectively. According to the manufacturer’s instructions, all plasmids for transfection were prepared by using the EasyPure HiPure Plasimid MinPrep Kit (TransGen Biotech, China).

HEK293T cells were cultured in DMEM high glucose medium (Biological Industries) with 10% FBS (Biological Industries) and penicillin and streptomycin (Solarbio) in 5% CO₂ atmosphere at 37°C. To determine the effects of UPR sensors on the promoter activities of lipid synthesis and proinflammatory genes, HEK293T cells were cotransfected with reporter plasmid, expression plasmid, and phRL-CMV plasmid using Lipofectamine 2000 (Invitrogen, USA). After transfection for 24 h, cells were harvested and measured for luciferase activity using a Dual-Luciferase Reporter Assay Kit (TransGen Biotech), according to the manufacturer’s instructions.

Statistical evaluations were analyzed with independent sample t-test or one-way analysis of variance followed by Tukey’s multiple-range test. The analysis was carried out using the SPSS 17.0 software (IBM, USA). The results were presented as mean ± standard deviation. P < 0.05 was considered statistically significant.

To investigate the role of ER stress in the regulation of lipid homeostasis and inflammation in the intestine, we injected TM into large yellow croaker. Compared with the control group, the mRNA expression of ER stress-related genes including grp78, xbp1s, atf4, atf6, and chop was significantly increased in the intestine after TM injection (P < 0.05) ( Figure 1A ). Besides, the protein levels of GRP78, XBP1s, and p-PERK were significantly upregulated (P < 0.05), while the protein level of ATF6 was not significantly changed in the TM group (P > 0.05) ( Figure 1B ). These results indicated that TM injection induced ER stress in the intestine of large yellow croaker in vivo.

Compared with the control group, TM injection significantly increased the TG content in the intestine (P < 0.05) ( Figure 2A ). Lipid homeostasis in the intestine is maintained through multiple pathways, including fatty acid uptake, lipid synthesis, lipid secretion, and fatty acid β-oxidation. In terms of lipid uptake, the results showed that the mRNA levels of cd36, fatp4, and fabp3 in the intestine were significantly downregulated in the TM group (P < 0.05) ( Figure 2B ). TM injection significantly increased the mRNA expression of genes related lipid synthesis, including srebp1c, scd1, acc1, acc2, dgat1, dgat2, and adrp (P < 0.05) ( Figure 2C ). Compared with the control group, TM injection significantly increased mRNA levels of mtp, sar1b, sec13, sec31, sec23, and sec24 (P < 0.05), while the m RNA level of apob was significantly decreased in the TM group (P < 0.05) ( Figure 2D ). We also found that the mRNA levels of pparα, cpt1α, and aco were increased after TM injection (P < 0.05) ( Figure 2E ). Compared with the control group, the protein levels of ACC, SREBP1c, APOB48, CPT1α, and PGC1α were significantly upregulated (P < 0.05) ( Figure 2F ). Collectively, these results indicated that ER stress disturbed lipid metabolism leading to abnormal lipid accumulation in the intestine of large yellow croaker.

TM injection significantly promoted mRNA expression of proinflammatory genes in the intestine, including il-1β, il-6, il-8, cox2, and tnfα (P < 0.05) compared with the control group ( Figure 3A ). Compared with the control group, the protein levels of IL-1β and nuclear p65 were significantly increased (P < 0.05), while the protein levels of p-ERK1/2/ERK1/2, p-p38/p38, and total p65 were not remarkably different in the TM group (P > 0.05) ( Figure 3B ). The results above indicated that ER stress augmented inflammatory response in the intestine of large yellow croaker.

To further investigate that ER stress induced by TM mediated lipid metabolism and inflammation in the intestine of large yellow croaker, the intestinal cells were isolated from large yellow croaker and incubated with 1 μM TM at different time points. The results showed that TM treatment significantly increased the mRNA levels of grp78, xbp1s, atf4, atf6, and chop compared with the control group (P < 0.05) ( Figure 4A ). Meanwhile, the protein levels of GRP78, XBP1s, and p-PERK were significantly higher than those in the control group (P < 0.05), while the protein level of ATF6 was not remarkably different (P > 0.05) ( Figure 4B ). The results suggested that TM incubation could induce ER stress in the isolated intestinal cells of large yellow croaker in vitro.

We next examined the effect of ER stress on lipid metabolism in the intestinal cells. Compared with the control group, TM treatment significantly increased the level of TG (P < 0.05) ( Figure 5A ) and the number and size of lipid droplets in cells ( Figure 5B ). In terms of lipid uptake, TM treatment significantly increased the mRNA level of cd36 (P < 0.05) and decreased the mRNA levels of fatp1, fabp2, and fabp3 (P < 0.05) ( Figure 5C ). The mRNA levels of lipid synthesis, including srebp1c, scd1, acc1, acc2, dgat2, and adrp, were significantly increased after TM treatment (P < 0.05) ( Figure 5D ). Compared with the control group, TM treatment significantly upregulated the gene levels of apob and sec23, while the mRNA levels of mtp, sar1b, sec13, sec31, sec23, and sec24 were not remarkably different (P > 0.05) ( Figure 5E ). For fatty acid β-oxidation, the gene levels of pparα and aco were significantly upregulated (P < 0.05) ( Figure 5F ). Western bolt results showed that the protein levels of SREBP1c and ACO were significantly upregulated, and the protein level of CD36 was downregulated (P < 0.05) ( Figure 5G ). In agreement with TM injection in vivo, these results indicated that ER stress boosted the lipid synthesis leading to abnormal TG accumulation in cells.

We have established that ER stress disrupted lipid metabolism in the intestine and isolated the intestinal cells of large yellow croaker. To further investigate the regulatory mechanism, we cotransfected the reporter plasmids of lipid synthesis promoters and expression plasmids of UPR sensors into HEK293T cells. Compared with HEK293T cells transfected with PCS2+ plasmid, cells transfected with XBP1s, CHOP, or ATF4 expression plasmid showed significantly higher luciferase activity of the acc promoter of large yellow croaker (P < 0.05) ( Figure 6A ). Similarly, the promoter activity of scd1 in cells that transfected with UPR sensor plasmid was significantly higher than those transfected with PCS2+ plasmid (P < 0.05) ( Figure 6B ). Overexpression of XBP1s, CHOP, or ATF4 significantly promoted the promoter activity of dgat1 (P < 0.05) ( Figure 6C ). Overexpression of CHOP or ATF4 significantly upregulated the promoter activity of dgat2 compared with the control group (P < 0.05) ( Figure 6D ).

We next examined the effect of ER stress on inflammatory response in the intestinal cells. TM treatment significantly upregulated the mRNA expression of proinflammatory genes, including il-1β, il-6, cox2, and tnfα (P < 0.05) ( Figure 7A ). Western blot results showed that TM treatment significantly increased the protein levels of p-p38/p38, total p65, IL-1β, and nuclear p65 (P < 0.05), while the protein level of p-ERK1/2/ERK1/2 was not remarkably different (P > 0.05) ( Figure 7B ). Collectively, these results indicated that ER stress induced inflammation in the isolated intestinal cells of large yellow croaker.

Next, we further studied the mechanism of UPR sensors on the regulating transcriptional activity of proinflammatory genes. Compared with HEK293T cells transfected with PCS2+ plasmid, cells transfected with ATF4 expression plasmid showed significantly higher luciferase activity of the il-1β promoter of large yellow croaker (P < 0.05) ( Figure 8A ). Overexpression of CHOP or ATF4 expression plasmid significantly increased the promoter activity of tnfα compared with the control group (P < 0.05) ( Figure 8B ). Similarly, XBP1s or ATF6 could significantly upregulate the promoter activity of il-6 (P < 0.05) ( Figure 8C ). Compared with the control group, HEK293 cells transfected with XBP1s, CHOP, or ATF6 expression plasmid showed significantly high promoter activity of cox2 (P < 0.05) ( Figure 8D ).