The serine protease prostasin regulates hepatic insulin sensitivity by modulating TLR4 signalling - PubMed (original) (raw)

Manabu Hayata 1, Teruhiko Mizumoto 1, Yoshikazu Miyasato 2, Yutaka Kakizoe 2, Jun Morinaga 2, Tomoaki Onoue 2, Rika Yamazoe 2, Miki Ueda 2, Masataka Adachi 2, Taku Miyoshi 2, Naoki Shiraishi 2, Wataru Ogawa 3, Kazuki Fukuda 4, Tatsuya Kondo 4, Takeshi Matsumura 4, Eiichi Araki 4, Kimio Tomita 2, Kenichiro Kitamura 2

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Free PMC article

The serine protease prostasin regulates hepatic insulin sensitivity by modulating TLR4 signalling

Kohei Uchimura et al. Nat Commun. 2014.

Free PMC article

Abstract

The effects of high-fat diet (HFD) and postprandial endotoxemia on the development of type 2 diabetes are not fully understood. Here we show that the serine protease prostasin (PRSS8) regulates hepatic insulin sensitivity by modulating Toll-like receptor 4 (TLR4)-mediated signalling. HFD triggers the suppression of PRSS8 expression by inducing endoplasmic reticulum (ER) stress and increases the TLR4 level in the liver. PRSS8 releases the ectodomain of TLR4 by cleaving it, which results in a reduction in the full-length form and reduces the activation of TLR4. Liver-specific PRSS8 knockout (LKO) mice develop insulin resistance associated with the increase in hepatic TLR4. Restoration of PRSS8 expression in livers of HFD, LKO and db/db mice decreases the TLR4 level and ameliorates insulin resistance. These results identify a novel physiological role for PRSS8 in the liver and provide new insight into the development of diabetes resulting from HFD or metabolic endotoxemia.

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Figures

Figure 1

Figure 1. HFD-induced ER stress contributes to insulin resistance via a reduction in hepatic PRSS8.

(a) Blood glucose and serum insulin levels under fasting and refeeding conditions in mice fed a ND or a HFD; (_n_=6–8 mice per group). Values are shown as the mean±s.d.; *P<0.05 and **P<0.01 versus ND (one-way analysis of variance (ANOVA)). (b) Western blotting analysis of PRSS8 in the livers of ND and HFD mice under fasting and refeeding conditions. A representative western blot image is shown (_n_=4 mice per group). (c) Blood glucose levels during GTT (2 g kg−1) or PTT (2 g kg−1) and the total area under the curve for the PTT in ND and HFD mice treated with vehicle or PBA (20 mM) (n_=10 mice per group). Values are shown as the mean±s.d.; *P<0.05, **P<0.01 and ***P<0.001 for HFD–PBA versus HFD–vehicle (two-way ANOVA). †††_P<0.001 versus ND–vehicle (one-way ANOVA). (d) Western blotting for insulin-stimulated Akt phosphorylation and the level of PRSS8 and ER stress markers (pPERK and peIF-2α) in livers from ND and HFD mice treated with vehicle or PBA. Representative western blot images are shown (_n_=4 mice per group). (e) Blood glucose levels during a GTT or PTT and AUC for a PTT in ND and HFD mice infected with an adenovirus carrying LacZ (Ad-LacZ) or human PRSSS8 (Ad-hPRSS8) (n_=10 mice per group). Values are shown as the mean±s.d.; **P<0.01 and ***P<0.001 for HFD-Ad-hPRSS8 versus HFD-Ad-LacZ (two-way ANOVA). †††_P<0.001 versus ND-Ad-LacZ (one-way ANOVA). (f) Western blotting for the insulin-stimulated phosphorylation of Akt and PRSS8 level in livers from ND and HFD mice infected with Ad-LacZ or Ad-hPRSS8. Representative western blot images are shown (_n_=4 mice per group).

Figure 2

Figure 2. LKO mice have glucose intolerance and hepatic insulin resistance.

(a) Blood glucose and serum insulin levels under fasting and refeeding conditions in Flox and LKO mice (_n_=10 mice per group). Values are shown as the mean±s.d.; *P<0.05 and **P<0.01 versus Flox (one-way ANOVA). (b) mRNA expression of PEPCK, G6Pase, FBPase and glucokinase (GK) in the liver under fasting and refeeding conditions (_n_=7–9 mice per group). Values are shown as the mean±s.d.; *P<0.05 and **P<0.01 versus Flox (one-way ANOVA). (c) Blood glucose levels during a GTT, (insulin 1.5 U kg−1) insulin tolerance test and PTT (_n_=10 mice per group). Values are shown as the mean±s.d.; *P<0.05, **P<0.01 and ***P<0.001 versus Flox (two-way ANOVA). (d) Glucose infusion rate, hepatic glucose production and glucose Rd in the hyperinsulinemic–euglycemic clamp study (_n_=5 mice per group). Values are shown as the mean±s.d.; *P<0.05 and **P<0.01 versus Flox (one-way ANOVA). (e) Western blotting for the insulin (1 U kg−1)-stimulated phosphorylation of IRS-1 (Ser636/639), IRS-2, Akt and GSK-3β in the liver. Representative western blot images are shown (_n_=4 mice per group).

Figure 3

Figure 3. ER stress-mediated PRSS8 modulates hepatic TLR4 expression.

(a) The expression of TLR4 was analysed by western blotting for protein levels and real-time PCR for mRNA levels in livers from Flox and LKO mice under fasting and refeeding conditions (_n_=4 mice per group). Values are shown as the mean±s.d.; *P<0.05 and **P<0.01 versus Flox (one-way ANOVA). (b) Protein level of TLR4 in livers from ND and HFD mice treated with Ad-LacZ or Ad-hPRSS8 (upper panel) and vehicle or PBA (lower panel) under fasting conditions were analysed by western blotting. Representative western blot images are shown (_n_=4 mice per group). (c) Western blotting for TLR4, PRSS8 and CHOP in HepG2 cells treated with and without tunicamycin in the presence of siRNA for Control or PRSS8 and pcDNA3.1 alone or pcDNA3.1-hPRSS8.

Figure 4

Figure 4. LKO mice and PRSS8-depleted HepG2 cells show an excessive response to LPS.

(a) Time course of serum AST, ALT and LDH levels after intraperitoneal LPS injection (80 mg kg−1) (_n_=8 mice per group). *P<0.05 versus Flox (one-way ANOVA). Values are shown as the mean±s.d. (b) LPS-induced mRNA expression of proinflammatory cytokines in the livers of Flox and LKO mice 6 h after intraperitoneal injection (_n_=8 mice per group). **P<0.01 and ***P<0.001 versus Flox (one-way ANOVA). Values are shown as the mean±s.d. (c) HepG2 cells were transfected with Control siRNA or PRSS8 siRNA. The LPS-induced mRNA expression of proinflammatory cytokines was determined 12 h after treatment (_n_=6 per group). ***P<0.001 versus Control siRNA (one-way ANOVA). Values are shown as the mean±s.d.

Figure 5

Figure 5. Restoration of hepatic PRSS8 expression ameliorates the insulin resistance and LPS-induced inflammatory response in LKO mice.

(a) Blood glucose levels during a GTT and PTT and area under the curve for the PTT (_n_=10 mice per group). *P<0.05, **P<0.01 and ***P<0.001 for LKO-Ad-PRSS8 versus LKO-Ad-LacZ (two-way ANOVA). Values are shown as the mean±s.d. (b) Western blotting for the insulin-stimulated phosphorylation of Akt and level of TLR4 and PRSS8 in the liver (_n_=4 mice per group). (c) Serum AST, ALT and LDH levels after LPS (80 mg kg−1) injection in LKO mice receiving Ad-LacZ or Ad-hPRSS8 (_n_=8 mice per group). **P<0.01 versus LKO-Ad-LacZ (one-way ANOVA). Values are shown as the mean±s.d. (d) mRNA expression of proinflammatory cytokines 6 h after intraperitoneal LPS injection (_n_=8 mice per group). *P<0.05 and **P<0.01 versus LKO-Ad-LacZ (one-way ANOVA). Values are shown as the mean±s.d.

Figure 6

Figure 6. PRSS8 decreases the level of TLR4 at the plasma membrane via proteolytic shedding.

(a) Western blotting for TLR4 and PRSS8 in HepG2 cells transfected with Control siRNA and PRSS8 siRNA. (b) Western blotting for TLR4 and PRSS8 in HepG2 cells transfected with pcDNA3.1 or pcDNA3.1-hPRSS8. (c) Lys and Arg residues conserved between humans and mice are highlighted in black with white lettering. These residues are located within the extracellular domain and are ~30 kDa upstream from the carboxy terminus. Site-directed mutagenesis was conducted by replacing these residues with Ala (K560A/K561A, K595A or R598A). (d) Wild-type or mutant human TLR4 was transfected with or without PRSS8 into HEK293 cells, and the level of TLR4 and PRSS8 was determined by western blotting. (e) Immunoprecipitated wild-type or K560/561A mutant HA-tagged TLR4 was incubated with or without recombinant human PRSS8 and subjected to immunoblotting using an anti-HA antibody. (f) HepG2 cells transfected with Control, PRSS8 or TLR4 siRNA or PRSS8 and TLR4 siRNA were pretreated with LPS (50 μg ml−1) and then insulin (500 nM)-stimulated Akt phosphorylation was determined by western blotting.

Figure 7

Figure 7. Reduction in hepatic TLR4 improves insulin resistance in HFD and LKO mice.

(a) Blood glucose levels during a GTT and PTT, and the AUC for the PTT in ND and HFD mice transfected with Control siRNA or TLR4 siRNA (n_=8 mice per group). Values shown are the mean±s.d.; *P<0.05, **P<0.01 and ***P<0.001 for HFD-TLR4 KD versus HFD-Control KD (two-way ANOVA). ††_P<0.01 and †††P<0.001 versus ND-Control KD (one-way ANOVA). (b) Western blotting for the insulin-stimulated Akt phosphorylation and TLR4 protein levels in livers from ND and HFD mice transfected with Control siRNA and TLR4 siRNA. Representative western blot images are shown (_n_=4 mice per group). (c) Blood glucose levels during a GTT and PTT, and the AUC for the PTT in ND mice infected with an adenovirus carrying LacZ (Ad-LacZ) or mouse TLR4 (Ad-mTLR4) (_n_=8 mice per group). Values are shown as the mean±s.d.; *P<0.05, **P<0.01 and ***P<0.001 versus ND-Ad-LacZ (two-way ANOVA). (d) Western blotting for the insulin-stimulated Akt phosphorylation and TLR4 protein levels in livers from ND mice infected with Ad-LacZ or Ad-mTLR4. Representative western blot images are shown (_n_=4 mice per group). (e) Blood glucose levels during a GTT and PTT, and the area under the curve for the PTT in LKO mice transfected with Control, TLR4, MYD88 or TLR2 siRNA (n_=6–8 mice per group). Values are shown as the mean±s.d. *P<0.05, **P<0.01 and ***P<0.001 for LKO-TLR4 KD versus LKO-Control KD (two-way ANOVA). #P<0.05, ##P<0.01 and ###P<0.001 for LKO-MYD88 KD versus LKO-Control KD (two-way ANOVA). †††_P<0.001 versus Flox-Control KD (one-way ANOVA). (f) Western blotting for insulin-stimulated Akt phosphorylation and the TLR4, MYD88 and TLR2 protein level in livers from LKO mice transfected with Control, TLR4, MYD88 and TLR2 siRNA. Representative western blot images are shown (_n_=3 mice per group).

Figure 8

Figure 8. Restoration of hepatic PRSS8 levels improves insulin resistance in db/db mice.

(a) The levels of PRSS8 and TLR4 in livers from db/m and db/db mice under fasting and refeeding conditions were analysed by western blotting. Representative western blot images are shown (_n_=4 mice per group). (b) Blood glucose levels during a GTT and PTT, and the AUC for the PTT in db/m and db/db mice infected with Ad-LacZ or Ad-hPRSS8 (_n_=10 mice per group). Values are shown as the mean±s.d.; *P<0.05, **P<0.01 and ***P<0.001 for _db/db_-Ad-hPRSS8 versus db/db_-Ad-LacZ (two-way ANOVA). †††_P<0.001 versus db/m-Ad-LacZ (one-way ANOVA). (c) Western blotting for insulin-stimulated Akt phosphorylation and the level of TLR4 and PRSS8 in livers from db/m and db/db mice infected with Ad-LacZ or Ad-hPRSS8. Representative western blot images are shown (_n_=4 mice per group). (d) Blood glucose levels during a GTT and PTT, and the AUC for the PTT in db/m and db/db mice transfected with Control or TLR4 siRNA (_n_=8 mice per group). Values are shown as the mean±s.d.; *P<0.05, **P<0.01, and ***P<0.001 for _db/db_-TLR4 KD versus db/db_-Control KD (two-way ANOVA). †††_P<0.001 versus _db/m_-Control KD (one-way ANOVA). (e) Western blotting for insulin-stimulated Akt phosphorylation and the TLR4 protein level in livers from db/m and db/db mice transfected with Control or TLR4 siRNA. Representative western blot images are shown (_n_=4 mice per group).

Figure 9

Figure 9. The serum PRSS8 levels are negatively correlated with BMI and HOMA-IR in healthy human subjects (_n_=153).

(a) Western blotting analysis of serum PRSS8. A representative western blot image is shown (_n_=4 mice per group). (b) Correlation between serum PRSS8 levels and BMI. (c) Correlation between serum PRSS8 levels and HOMA-IR. (d) Comparison of the serum PRSS8 level in healthy human subjects with a HOMA-IR <1.6 (_n_=111) and a HOMA-IR ≥1.6 (_n_=42). Statistical comparisons were made with the Mann–Whitney _U_-test. Values are shown as the mean±s.d.

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