Six- and 9-week-old male or female C57BLKS/J-lean (wild type [WT]) and C57BLKS/J-db/db mice were obtained from SLC (Hamamatsu, Japan). The purchased stains of mice were maintained under condition of a 12-hour light/dark cycle, 23°C±1°C and 50%±5% relative humidity, specific pathogen-free environment. The mice were sacrificed after 1 week.

Whole PAR2 knockout (KO) mice (homozygote, PAR2^−/−; strain: B6.Cg-F2rl1^tm1Mslb/J), 8-week-old, were provided by Dr. Yu (Pusan National University, YangSan, Korea) [24]. The male mice (n=6 per group) were fed with water vehicle and normal chow diet or 60% high-fat diet (HFD) for total 4 months period. Genotyping of mice was performed according to the Jackson Laboratory’s protocol with minor modifications (Jackson Laboratory, Bar Harbor, ME, USA). DNA was extracted from tail samples using the QuickExtract^TM DNA Extraction Solution 1.0 (Epicentre, Madison, WI, USA). Standard polymerase chain reaction (PCR) amplification of cDNA was carried out with nTaq (Mg²⁺ plus) DNA polymerase (Enzynomics, Daejeon, Korea) and specific primers. The primer sequences for genotyping PAR2 KO mice were as follows: mutant, 5′-GCC AGA GGC CAC TTG TGT AG -3′; WT forward, 5′-TCA AAG ACT GCT GGT GGT TG-3′; WT reverse, 5′-GGT CCA ACA GTA AGG CTG CT-3′. PCR products were separated on a 1.5% agarose gel, visualized, and photographed under ultraviolet light. The animal protocol for this study was approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC-2019-2197; PNU-IACUC-2019-2280) under guideline of ethics and animal care.

The chemicals for Western blotting were purchased from Amersham (Bucks, UK). RNAzol^TM B was purchased from TEL-TEST Inc. (Friendwood, TX, USA). Primary antibodies against FoxO1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transcription factor IIB (TFIIB), tyrosine reside phosphorylation of insulin receptor substrate (p-IRS (Tyr632)), p-IRS (Ser307), phosphorylation-serine/threonine protein kinase (p-Akt), total Akt, peroxisome proliferator-activated receptor-alpha (PPARα), PPARβ, PPARγ, β-arrestin, phosphorylation-AMP-activated protein kinase (p-AMPK), AMPK, sterol regulatory element-binding protein 1c (SREBP-1c), and PAR2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against p-FoxO1 (Thr24) and p-FoxO1 (Ser256) were manufactured by Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies, both anti-rabbit and anti-mouse immunoglobulin G (IgG)-horseradish peroxidase-conjugated antibodies, were purchased from Amersham. Horseradish peroxidase-conjugated donkey anti-sheep/goat IgG was obtained from Serotec (Oxford, UK). Polyvinylidene difluoride (PVDF) transfer membranes were obtained from manufacturer Millipore Corporation (Bedford, MA, USA).

Frozen mouse liver tissues (20 mg) were washed in phosphate buffered saline (PBS) buffer and then were subject to a Dounce tissue grinder (Wheaton Manufacturers, Millville, NJ, USA). Solution A (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES] pH 7.9, 10 mM KCl, 0.1 mM ethylenediamine tetraacetic acid [EDTA], 0.1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA], 0.5 mM phenylmethylsulfonyl fluoride [PMSF], and 1 mM dithiothreitol [DTT]) was then added to the tissues (2.5 g/mL). Tissues were stressed and homogenized with five hits of a pestle after addition of NP-40 (0.5%) and then further homogenized with five more strokes. Liver homogenates were transferred to 1.5 mL Eppendorf tubes and centrifuged using a microcentrifuge (Beckman, Brea, CA, USA) for 5 minutes at 12,000 rpm.

Supernatants is majorly comprised of cytoplasmic constituents. To have nuclear pellets, 400 μL of solution C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM of each of EDTA, EGTA, PMSF, and DTT) was added to the supernatants. Tube contents were blended thoroughly and set on a small rotatory shaker for 15 minutes. Finally, they were centrifuged for 10 minutes at 12,000 rpm in a microcentrifuge. The supernatants, which contained nuclear proteins, were transferred carefully to 1.5 mL new tubes, and kept at −80°C until required for Western blotting. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Sigma, St. Louis, MO, USA).

Homogenized samples were boiled for 5 minutes with a gel-loading buffer (125 mM Tris-Cl, 4% SDS, 10% 2-mercaptoethanol, pH 6.8, and 0.2% bromophenol blue) at a ratio of 1:1. The same amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using 6% to 17% separating running gels and then proteins were transferred to PVDF membranes at 15 V for 1 hour using a semi-dry transfer method. Immediately, the membrane was immersed in a blocking buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween-20 containing 1% non-fat milk. Membranes were blocked for 1 hour at room temperature, and the membranes were incubated and proteins were probed with the appropriate specific primary antibody at 25°C for 1 hour, subsequently followed by horseradish peroxidase-conjugated secondary antibody at 25°C for another 1 hour. Antibody labeling was detected using enhanced chemiluminescence in accordance to the manufacturer’s instructions. Molecular weights were determined using pre-stained lane protein markers.

Serum glucose concentration was measured using kits from Bioassay Systems (Hayward, CA, USA). Specific kits were used, following the manufacturer’s protocols, to measure the concentrations of insulin (Shibayagi, Japan).

Cytosol or nuclear extract fractions were immunoprecipitated in a immunoprecipitation (IP) buffer comprised of 40 mM Tris-HCl (pH 7.6), 120 mM NaCl, 20 mM β-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF, 5 mM EDTA, 0.1% NP-40, leupeptin (2 μg/mL), aprotinin (1 μg/mL), and pepstatin A (1 μg/mL). Aliquots of cytosol or nuclear extracts were than pre-cleared by using 50% protein A agarose for 30 minutes at 4°C, centrifuged at 12,000 ×g at 4°C for 15 minutes, incubated overnight at 4°C with the appropriate antibody, and then incubated overnight at 4°C with a 50% protein A agarose slurry. After washing the immunoprecipitates three times with an IP buffer, immunoprecipitated proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); Western blotting analysis was performed as described above.

Liver tissues were thoroughly homogenized in PBS. TGs were extracted with a 1:2 methanol-chloroform solution by incubating at room temperature for 2 hours. After filtering out impurities using a filter, the TG liquid solvent was evaporated. The TG levels were assessed using a kit (Bioassay Systems).

RNA isolation from mouse liver tissues (20 mg) was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis was performed to quantify mRNA levels using the SYBR Green and CFX Connect System (Bio-Rad Laboratories Inc., Hercules, CA, USA). All primers were designed and purchased from Integrated DNA Technologies (Coralville, IA, USA). The primers sequence used are listed in Supplemental Table S1.

Analysis of variance (ANOVA) was used in order to determine the statistical significance of the differences between the groups. Fisher’s protected least significant difference post hoc test was used to test the significant difference between group means. P values <0.05 were cutoff to indicate statistical significance.

Obesity changes blood lipid levels, which are highly related with hepatic steatosis [25]. Liver weight increased in diabetic mice (Fig. 1A), and blood lipid profiles were investigated in db/db mice. Glucose level significantly elevated glucose levels in mice (Fig. 1B). Furthermore, hyperinsulinemia significantly elevated insulin in the serum, compared to age-matched animals (Fig. 1C). Our data suggest that obesity induced hyperinsulemia and hyperglycemia in diabetic mice.

To confirm potential changes hepatic lipid accumulation in obesity, we measured the TG concentration in liver tissues. The contents of hepatic TG were significantly increased in age-matched male db/db mice (Fig. 2A). In addition, female db/db mice had increase in hepatic TG content, while male db/db mice showed no increasing tendency (Supplemental Fig. S1). Therefore, we further focus on male db/db mice and investigated its mechanism of hepatic steatosis.

FoxO1 is majorly suppressed by Akt during insulin signaling activation. FoxO1 shuttling from the nucleus to cytoplasmic fraction by Akt, inhibiting its action as its role as a transcription factor [26]. We found that FoxO1 phosphorylation level was reduced in the liver of diabetic db/db mice (Fig. 2B). In addition, the nuclear protein levels of PPARβ were found to be decreased in the liver of diabetic mice in comparison to that of the normal mice. In contrast, nuclear PPARγ levels were notably increased. However, lipogenic gene SREBP-1c level was increased in db/db mice (Fig. 2C). Based on the IP experiments, the physical association between FoxO1 and PPARγ was shown to be induced in db/db mice liver (Fig. 2D). Furthermore, the modulatory action of FoxO1 as a regulator of lipogenic genes was explored in association with the obesity. Diabetic mice exhibited remarkably higher mRNA levels of PPARγ, whereas the transcripts of β-oxidation genes, such as PPARα and PPARβ, did not alter (Fig. 2E). The expression levels of target lipogenic genes of PPARγ, such as fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase 1 (SCD1), were correlated to PPARγ expression levels and increased in db/db mice in comparison to those of control db⁺ mice (Fig. 2F). In contrast, the expression levels of PPARα target genes remained unchanged in db/db mouse livers (Fig. 2G). These data collectively indicate that in the liver of db/db mice, the greater interaction between FoxO1 and PPARγ was induced and resulted in upregulated lipid accumulation in liver of db/db mice.

As fasting glucose contents are majorly modulated by insulin in the liver, we investigated insulin signaling in the livers of db/db mice. As demonstrated in Fig. 3, insulin resistance marker, p-IRS1 (Ser307) was notably upregulated in age-matched db/db mice, whereas the concentration of Ser473-phosphorylated Akt were found to be decreased in db/db mice (Fig. 3A). AMPK is widely recognized to be a key regulator of fatty acid and glucose homeostasis. We next investigated whether obesity modulates AMPK. As shown in Fig 3B, obesity mice significantly decreased the phosphorylation of AMPK.

PAR2 plays a critical role in inflammatory response and steatosis in liver [27]. Up to now, there found four isoforms of PARs, PAR1-4. Particularly, PAR2 becomes activated by trypsin-like proteases including tryptase. In addition, PAR2 signaling becomes initiated by coagulation factors VIIα and Xα in TF expression.

Our previously reported PAR2 expression becomes upregulated in the liver of FoxO6-transgenic mice compare with another PAR family [28]. To find whether PAR2 acts a regulatory role in insulin resistance in vivo, we studied PAR2 and β-arrestin expression in db/db mice. PAR2 and β-arrestin expression was increased in db/db mice (Fig. 4A). In the IP experiments, PAR2 and β-arrestin were observed to interact in db/db mice: PAR2 and β-arrestin strongly interacted with each other in db/db mice (Fig. 4B). Furthermore, we found weak interactions between β-arrestin and Akt in db⁺ mice (Fig. 4C).

Blood lipid profiles were investigated in HFD-feeding PAR2-KO mice. Hyperglycemia was significantly elevated glucose level in HFD-mice (Fig. 5A). Otherwise, glucose level no changed in HFD-PAR2-KO compare with HFD groups. However, HFD-induced hypertriglyceridemia significantly elevated, as compared with WT chow. However, TG levels reduced in HFD-feeding PAR2-KO compared with HFD-feeding mice (Fig. 5B). Otherwise, no differences were found in the levels of non-esterified fatty acids (NEFA) (Fig. 5C).

HFD-mediated insulin resistance condition was notably promoted and positively associated with the pathogenesis of hyperlipidemia. We investigated PAR2 pathway during hepatic steatosis in HFD-fed PAR2-KO mice. As shown in Fig. 6, we investigated whether PAR2 modulate lipid accumulation in the livers of HFD-feeding mice. This result that FoxO1 activation was induced in HFD-feeding groups. However, nuclear protein levels of PPARγ level was increased in HFD-feeding animals. On the other hand, PPARγ protein expression was reduced HFD-PAR2-KO mice compared with HFD-feeding groups (Fig. 6A). Moreover, to confirm potential PAR2-regulated changes in hepatic lipid accumulation, we measured the TG concentration in liver tissue. This result that the contents of hepatic TG were significantly reduced in HFD-feeding PAR2-KO mice compared with HFD-feeding groups (Fig. 6B). However, PPARγ and its target genes, such as FASN, ACC, and SCD1, which was additively increased HFD-feeding groups, and PPARγ, FASN, and SCD1 was reduced in HFD-feeding PAR2-KO compared with HFD-feeding groups (Fig. 6C). These results showed that PAR2 promoted stimulatory effect on lipid accumulation in HFD-mediated groups.